bluuebunny/crossref_metadata_2025 huggingface dataset:
Genesis of Antibiotic Resistance XX: CDC (Frieden's) report on AR and executive strategy to mitigate AR pandemic fail to account human migration (immigrants) and Hardy‐Weinberg ‐ Castle Principle§
Authors: Kannan Subburaj , Alvarez Angelica , Carrizales Diana , Carrizales Tania , Jouregui Suzzette , Moody Nadine
Published: November 2023

Human mobility is causing an increase spread of antimicrobial drug–resistant organisms and drug‐resistant infectious diseases. International population movement is an integral component of the globalization process. Current population movement dynamics rapidly and effectively link regions of marked health disparity, and these linkages can be associated with risk for introduction / importation of drug‐resistant infectious diseases to population that are susceptible for diseases such as Tuberculosis and leprosy. The International Labor Organization (ILO) stated that in 2004, an estimated 175 million persons lived permanently outside their country (regions) of birth and that there were 81 million migrant workers excluding refugees globally. They are, however, one of the more likely populations to evolve (in comparison to medical community, underprivileged) because they meet multiple criteria of the HWCP principle (but not all of the conditions). Immigrants meet the condition of small populations. Even though many immigrant populations are large (2015 ‐Syrian ‐ Iraqi refugees in Europe; Approximately 11 million immigrants in United States, originating many Latin American countries including Mexico), they are not as large compared to the native population of the country which they are immigrating to. Systematic analysis of 2014 National Population Projections, as per HWCP, https://www.census.gov/population/projections/data/national/2014 shows that there is a gene flow due to intermixing of ethnic groups (for example a Cuban immigrating to the U.S. has children with a native U.S. citizen). Due to enhanced immunocompetency and innate immunity to diverse pathogens, immigrants tend to have the highest possibility of evolving. We have identified that immigrants fit three of the five categories defining HWCP: Natural selection, small population size, and gene flow. And lastly, immigrants (especially 2nd and 3rd generation) tend to mate with the people of their new home, and therefore, there is a sustained gene flow as defined by HWCP.

Support or Funding Information

Supported by professional development funds by SWTJC to S.Kannan for 2014–2015
Characterization of Heterologously Expressed Putative Cytolysins from “Brachyspira hampsonii “
Authors: Keith Brandon A , Harding John C , Hill Janet E , Loewen Matthew E
Published: November 2023

Cytolysins produced by Brachyspira hyodysenteriae and “Brachyspira hampsonii” strain 30446 are believed to be important virulence factors in the pathology of swine dysentery. To date four putative cytolysin genes termed tlyA, tlyB, tlyC, and hlyA have been identified, all of which are present in both Brachyspira hyodysenteriae and “Brachyspira hampsonii”. While the contributions of tlyB, tlyC, and hlyA to the pathogenesis of swine dysentery are not yet known, tlyA knockout strains of Brachyspira hyodysenteriae have shown decreased virulence in mouse models. In addition, homologues of tlyA are believed to be important virulence factors for human pathogens such as Helicobacter pylori and Mycobacterium tuberculosis, functioning as both cytolysins and contributing to antibiotic resistance through ribosomal RNA methylation. While active hlyA protein has been purified from Brachyspira hyodysenteriae broth cultures, studies of tlyA, tlyB, and tlyC to date have focused on their activity when introduced into Escherichia coli via plasmid. These results were cast into doubt several years later upon the discovery that many lab strains of Escherichia coli exhibit a hemolytic phenotype when foreign genes were introduced. To examine the effects of these putative cytolysins the tlyA and tlyC genes were amplified from “Brachyspira hampsonii” genomic DNA and ligated into expression vectors. The resulting protein products were overexpressed in Escherichia coli and purified by Ni2+ and amylose affinity chromatography. Cytolytic activity of purified recombinant tlyA and tlyC on a sheep erythrocyte suspension was examined, and no cytolysis greater than a phosphate buffered saline control was observed from tlyA at concentrations ranging from 0 to 88 μg/mL or from tlyC at concentrations ranging from 0–34 μg/mL. At the time of writing, attempts are being made to express and purify the α‐toxin from Staphylococcus aureus utilizing the same methodology in order to rule out inactivation of tlyA and tlyC by the purification process. While it is also possible that higher concentrations of tlyA and tlyC are required for hemolysis or that full activity of these proteins require unidentified cofactors or post‐translational modifications not possible in our current Escherichia coli based expression system, the possibility that tlyA and tlyC have been misannotated as cytolysins cannot be ruled out.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada (NSERC) 371364 /2010 to MEL & Alberta Livestock and Meat Association project 2013R054R to JCH, MEL & JEH
Understanding the Phylogenetic Relationship of Bacterial Arginine Kinases
Authors: Stock Katie , Aryal Manish , Fraga Dean
Published: November 2023

Arginine kinases (AKs) are transferase enzymes that are part of the phosphagen kinase family. These enzymes catalyze the transfer of phosphate from ATP to L‐arginine and are important in the energy buffering mechanisms in invertebrates and unicellular organisms. Previous studies have attempted to understand the relationship of the bacterial AKs to other phosphagen ianses. Atleast eight bacterial AKs have been discovered through examining bacterial genome sequences available on‐line. Using phylogenetic analyses it was found previously that the bacterial AKs can be separated into two clusters, cluster A and cluster B. This work will describe a more complete characterization of the bacterial AKs, both biochemically and phylogenetically to better understand their relationships to each other and to other PKs. Our initial results support the observation that there are two ‘forms’ of the bacterial AKs, a kinetically slow form and a kinetically faster form.

Support or Funding Information

College of Wooster
Temporal Dynamics of Apoptotic Activity in Jurkat Cells
Authors: Morris Sean Kyle , Reif Randall
Published: November 2023

Apoptosis, a process in which a cell systematically triggers its own death in response to DNA damage or external stimuli, is widely utilized in the body. Malfunction of the apoptosis process can lead to serious health problems such as cancer. There are several known pathways that execute apoptosis utilizing a family of enzymes called Caspases. The (intrinsic) pathway is the focus of this research, and initiates caspase activity through mitochondrial depolarization.

The goal of this research project was to find suitable initiators of apoptosis in Jurkat T‐Lymphocytes and elucidate their temporal dynamics with respect to caspase activity. Microfluidic devices were fabricated to capture cells and view this process. To identify suitable inducers of apoptosis to use, cells were exposed to several compounds and monitored over six hour time periods using Fluorescence Microscopy. Caspase activation was confirmed with the use of a caspase‐specific fluorogenic probe, L‐bisaspartic acid rhodamine 110.

Anti‐CD95, staurosporine, and hydrogen peroxide were used as inducers of apoptosis resulting in induction rates of 8.88%, 1.66%, and 1.24% with un‐induced rates of 0.59%, 1.88%, and 0.24% respectively. On a microfluidic device, cells were induced with Hydrogen Peroxide and their fluorescence intensity was measured over time. Apoptotic cells with active caspase enzymes consistently reached peak intensity two hours after caspase activation began. Using these results, mitochondrial depolarization will be tracked simultaneously with caspase activity utilizing MitoTracker Red.

Support or Funding Information

This research would not be possible without support from the Department of Chemistry at the University of Mary Washington, as well as a grant from the University of Mary Washington Summer Science Institute.
Mechanism of Transport of Ferric Enterobactin through FepA in Gram Negative Bacteria
Authors: Majumdar Aritri , Trinh Vy , Moore Kyle J. , Smallwood Chuck R. , Newton Salete M.C. , Klebba Phillip E.
Published: November 2023

BACKGROUND AND PURPOSE

Ligand‐gated porin (LGP) receptors of Gram‐negative bacterial outer membrane (OM), typified by FepA, FhuA, BtuB, possess a complex β‐barrel transmembrane channel occluded by a globular N‐terminal domain. The structure poses immediate questions on the mechanism of ligand transport through the receptor protein. Our previous results with in‐vivo fluorescent labeling of engineered Cysteine residues on FepA indicated a ‘Ball and Chain’ mechanism of transport in which the N‐domain is expelled into the inter‐membrane periplasmic space during Ferric enterobactin (FeEnt) ligand passage. This study further investigates whether the N‐domain exits the barrel as a unit or unfolds into the periplasm or forms a ‘transient pore’ within the barrel allowing ligand passage.

METHOD

We constructed a series of double‐cysteine mutants located i) in the N‐domain such that disulphide bonds should not interfere with transport if it exits the channel as a unit ii) in the N‐domain and the β‐barrel such that the cysteines can come close and form double bonds only if the N‐domain exists the channel during ligand uptake. We tested these mutants for ligand uptake capabilities in nutrition tests and conventional radiochemical assays in the presence and absence of a reductant (β‐mercaptoethanol) of disulphide bonds.

RESULTS AND CONCLUSION

Our results suggest that three double‐cysteine mutations G27C/R126C, A33C/E120C and L125C/V141C prevent transport of FeEnt in the absence of β‐mercaptoethanol. The location of these residues on the N‐domain point to a mechanism in which the globular N‐domain undergoes conformational change to allow tight ligand passage.

Support or Funding Information

1R21AI1157187 granted to PEK
Isomerized aspartate residues in rodent eye lens protein act as a marker of biological aging
Authors: Warmack Rebeccah Austin
Published: November 2023

Proteins in biological systems are known to accumulate spontaneous conformational alterations before breakdown and recycling. The racemization and isomerization of aspartic acid in proteins is one such modification that can result in loss of structure and function. Aspartic acid racemization specifically has been studied as a potential measure of chronological aging within low protein turnover mammalian tissues. However, very few of these studies have been optimized for shorter‐lived animals such as rodents, which are increasingly used as genetic and metabolic models of aging. In this study, we have developed a new sensitive approach for the measurement of biological age by taking advantage of the formation of isomerized aspartyl residues of the rodent eye lens. Using the human protein L‐isoaspartate/D‐aspartate methyltransferase (PCMT1) as an analytical reagent, we are able to rapidly and conveniently quantify the levels of L‐isoaspartate within lens protein extracts. We found the levels of isomerized aspartic acid to be about 35 times higher in the extracts of 18 month‐old rats versus two month‐old rats. Importantly, it was observed that the accumulation appeared to plateau in rats of 18 months and older, suggesting the presence of potentially novel mechanisms for the removal of altered proteins that may develop with age.

Support or Funding Information

Ruth L. Kirschstein National Research Service Award GM007185
Role of Janus‐Kinase 3 in Lung Inflammation
Authors: MISHRA JAYSHREE , VERMA RAJ K , YANG XIUDONG , KUMAR NARENDRA
Published: November 2023

Objective

Mucin hyper production is common in inflammatory and allergic lung disease such as bronchial asthma, chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis. Excessive mucin production in the lung leads to obstruction of airways. Hence, it is critical to understand the mechanisms that regulate mucin production under normal physiological conditions and the reason for it's over production during inflammatory lung diseases. Janus kinase 3 (Jak3) is a member of the JAK family of intracellular non‐receptor tyrosine kinase that interacts with receptors for IL‐2, IL‐5, IL‐7, IL‐9, and IL‐15 through the common γ‐chain.2 Though previously Jak3 was thought to be expressed only in hematopoietic tissues, however recent studies have shown that Jak3 is expressed in many organ tissues including lungs of human and mice. Previously, we demonstrated that Jak3 plays an essential role in cytoskeletal remodeling, wound repair, and mucosal homeostasis in intestinal epithelial cells however the role of Jak3 in lung inflammation is not known.

Materials and Methods

Freshly dissected lung tissue from control and Jak3 knockout mice were immunostained for Jak3 ((Invitrogen) as well as different transcription factors like Muc1, Muc2, Hif‐1, Hif‐2 (Novus Biologicals), p(142) β‐catenin (Abcam), p(654) β‐catenin (Santa Cruz Biotechnology), STAT3, pSTAT3, p‐STAT6 (Cell Signaling Technology). Phosphorylation status of these transcription factors in both WT and Jak3 KO mice were assessed through western analysis. Level of pro inflammatory cytokines in the lung tissue as well as the serum sample was measured using Multianylate cytokine assay kit from Qiagen.

Summary of Results

Our results show that lung epithelial cells of mice express Jak3 and knock out of Jak3 resulted in substantial decrease in expression of mucin (Muc1 and Muc2). Absence of Jak3 decreases the expression of transcription factors HIF‐1, HIF‐2, and STAT3. Absence of Jak3 also decreased the tyrosine phosphorylation level of transcription factors such as STAT3, STAT6, and β‐catenin as well as increased expression of pro‐inflammatory cytokines such as IL‐6 and IL‐17A in the lung tissue lysate.

Conclusion

Our data suggest that Jak3 plays an important role in lung inflammation through regulating mucin expression. Further, we will determine how Jak3 regulates different lung epithelial functions such as epithelial barrier function, ion and fluid transport function and the underlying molecular mechanism of it.

Support or Funding Information

Crohn's & Colitis Foundation of America (CCFA Ref # 2188) and NIH (DK081661) to NK

NIH (sub awarded) 1R43GM109528‐01 to NK and JM
Using Anti‐Fungal Bacteria to Contest Snake Fungal Disease
Authors: Malone Katherine
Published: November 2023

Often referred to as indicator species, amphibians and reptiles are abundant and vital in ecosystems across the world. However, across the globe there is a decrease in this abundance due to various factors. Many species are already critically endangered or on the brink of extinction. One of these factors is the spread of various fungal diseases.

Ophidiomvces ophiodiicola, also known as Snake Fungal Disease (SFD), is a new fungi spreading and residing on the skin of snakes. Although SFD does not immediately harm the infected reptile, in combination with other disturbances or stressors SFD could pose a serious threat to the reptile populations infected. The geographic range and the quantity of SFD infections are relatively unknown. Through this research we strive to isolate a residential bacteria from either reptiles’ skin or gut that posses antifungal properties along with estimate the extent of SFD infections. The first phase of this project was to collect skin and fecal samples from 18 species of snakes and lizards from the southeastern and southwestern US. We will then perform DNA extraction and real time PCR to determine if Ophidiomvces ophiodiicola was present on the skin or in the gut of each animal. We will also isolate resident bacterial species from the skin and gut and perform in vitro challenge assays against the snake fungal pathogen to determine the probiotic antifungal effects of resident skin bacteria. Antifungal bacterial species could be used for future probiotic field based treatments of reptiles at risk of infection from Ophidiomvces ophiodiicola.
Novel Brain‐specific Isoforms of Regulator of G protein Signaling 6 (RGS6)
Authors: Ahlers Katelin E , Stewart Adele , Yang Jianqi , Luo Zili , Fisher Rory A
Published: November 2023

RGS proteins act as gatekeepers for cellular responses induced by G protein coupled receptors (GPCRs) by facilitating inactivation of heterotrimeric G protein signaling. By acting as GTPase activating proteins for Gα subunits, an activity bestowed by the semiconserved RGS domain present in all RGS members, RGS proteins determine the magnitude and duration of G protein signaling initiated by GPCRs. RGS6 is a member of the R7 subfamily distinguished by two additional unique domains, DEP/DHEX and GGL, which are important for membrane targeting and protein stability of these members, respectively. RGS6 specific modulation of Gαi/o protein activity has been implicated in regulation of several disease states, particularly in the CNS, including: alcoholism, anxiety/depression, and Parkinson's disease. In addition, RGS6 is unique in that it remains the only RGS7 family member that is known to regulate G protein‐independent pathways promoting pro‐apoptotic and growth suppressive actions in cancer. Potentially key to RGS6's G protein‐independent signaling as well as its modulation of G protein signaling are previously unidentified domains that arise via alternative mRNA splicing. Indeed, when our laboratory first cloned RGS6 from human brain we identified 36 distinct RGS6 isoforms. Recently, using western blot, we identified at least two additional RGS6 isoforms in the brain that are larger (~61 and 69kDa) than the ubiquitously expressed 56kDa form of the protein. The functions for these RGS6 variants and how they arise (either through protein modification or additional RNA splicing) is unknown. Here, we demonstrate that these brain specific isoforms, along with the 56kDa form, are robustly down‐regulated in human glioma, indicating they may be important for the prevention of tumorigenesis in the brain. In addition, utilizing RGS6 specific primers to perform PCR amplification of RGS6 from a human brain cDNA library (Clontech), we have discovered novel RGS6 species not identified in the initial cloning effort. These transcripts include novel exons, some of which appear to encode early stop codons and others which, when included in the final transcript, are predicted to produce proteins around the size of the brain specific isoforms previously identified by western blot. Interestingly, several of the identified exons appear to be shared primarily between humans and other primates. Together, this research will lay the groundwork for future experiments to elucidate the functional significance of RGS6 alternative mRNA splicing in normal brain function as well as pathology.

Support or Funding Information

NIH CA161882
GRK2 Constitutively Downregulates Delta Opioid Receptor Activity in the Periphery
Authors: Doyle Allison P , Gomez Ruben , Henry Michael A , Jeske Nathaniel A
Published: November 2023

Opioids remain the gold standard for prescribed analgesic care. However, systemic opioid receptor agonists carry systemic negative side effects that can contraindicate long‐term administration. In contrast, peripherally restricted opioids would produce significant analgesia while avoiding centralized side effects. Unfortunately, peripheral opioid receptors are analgesically incompetent through an unknown mechanism. Here we report that G Protein‐Coupled Receptor Kinase 2 (GRK2) naively associates with plasma membrane delta opioid receptor (DOR) in peripheral sensory neurons to limit receptor responsiveness. Importantly, bradykinin receptor activation in sensory neurons stimulated GRK2 movement away from plasma membrane DOR and onto the cytosolic scaffolding protein Raf Kinase Inhibitory Protein (RKIP). Protein kinase C (PKC)‐dependent phosphorylation of RKIP dimerized the scaffolding protein to sequester GRK2, restoring full DOR functionality in sensory neurons. Additional results indicate that naive GRK2 association with plasma membrane DOR prevents optimal Gβ subunit association with the receptor, thereby reducing DOR‐mediated inhibition of voltage‐gated Ca+2 channels in sensory neurons responsible for peripheral analgesic competence. Together, these results identify a novel, non‐internalizing role for GRK2 in maintaining peripherally‐expressed DOR in an analgesically incompetent state, thereby providing a mechanism that may be targeted pharmaceutically to increase peripheral opioid analgesic efficacy.

Support or Funding Information

NINDS/RO1 NS082746 (N.A.J.), NIDCR/T32 DE14318 (A.P.D.), and NIDCR/F31 DE025551 (A.P.D.) from NIH funded this work.
Bitter Taste Receptor T2R4 Mediated Inactivation of Rac1 GTPase in Response to Quinine
Authors: Sidhu Crystal , Jaggupilli Appalaraju , Chelikani Prashen , Bhullar Rajinder P
Published: November 2023

Rac1 is a member of the Rho family of low molecular mass GTP binding proteins (GTPases). It regulates the dynamics of actin cytoskeleton by causing membrane ruffling, chemotaxis, and lamellipodia formation. Rac1 has been recently implicated in cancer metastasis and progression. As is the case with other GTPases, Rac1 cycles between the active GTP‐bound form and the inactive GDP‐bound form. This step is facilitated by a variety of Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). T2R4 is a bitter taste receptor that belongs to the GPCR (G protein‐coupled receptor) family of membrane proteins. In addition to mediating bitter taste sensations from the tongue, T2R4 and other T2Rs have been recently found in tissues other than the oral cavity e.g. nasal epithelium, airways, brain, gastrointestinal tract and male reproductive system suggesting a much broader physiological function for these receptors. T2Rs have been implicated in bronchodilation and in vasoconstriction. Quinine is an antimalarial drug and is one of the most bitter tasting compounds known. It is a natural alkaloid with a quinoline ring and a bicyclic quinuclidine. Quinine is a known agonist for T2Rs whereas BCML (Nα,Nα‐Bis(carboxymethyl)‐L‐lysine) acts as an inverse agonist. Since Rac1 is activated via various receptors like tyrosine kinase (RTKs), integrins and GPCRs; we have investigated the potential role of T2R4 in regulating Rac1 activity. In this study, HEK293T cells stably expressing T2R4/Gα16/44 were transiently transfected with HA‐Rac1 followed by treatment with Quinine or Quinine plus BCML for 15 min. After incubation, active Rac1 was pulled‐down from cell lysate using GST‐PAK1 and anti‐Rac1 monoclonal antibody was used in Western blots to quantify amount of active Rac1. The results demonstrated that Quinine treatment resulted in significant (p<0.001) reduction in the amount of active Rac1 whereas in the presence of BCML, Quinine failed to cause any significant change in the amount of active Rac1 when compared to untreated cells. This study is the first to show inhibitory downstream action of a T2R agonist on Rac1 function and further investigation will help in better understanding the extra‐oral physiological roles of T2Rs.

Support or Funding Information

This work is supported by a grant from Research Manitoba and the College of Dentistry.
Computational Analysis of the FLFP Motif in the Cell Survival Mechanism Conferred by PKCδVIII
Authors: Brooks Brady M. , Urbina Kevin A. , Parisot Lorenz , Cole Sharon E. , Shor Audrey C
Published: November 2023

Protein Kinase C δ (PKCδ) is a member of the novel group of serine/threonine kinases. In humans, PKCδ exists as two isoforms with opposite effects; PKCδI is pro‐apoptotic while PKCδVIIII is a cell survival regulator. PKCδVIII varies from the canonical sequence by a 31 amino acid insertion into the hinge region of PKCδI. A highly conserved hydrophobic linear motif, FLFP, was identified in this 31 amino acid variant region that we hypothesize contributes to the pro‐survival properties of PKCδVIII. Our lab has previously demonstrated that PKCδVIII forms complexes with mitogen activated protein kinase 1 (MAPK1) and Cyclin D1. However the exact mechanism of interaction has yet to be elucidated. Work in other model systems has demonstrated MAPK1 and Cyclin D1 to interact with the FLFP motif of other proteins. Thus, it is proposed that the interactions with PKCδVIII occur at the FLFP motif. Computational analyses of the impact site‐directed mutagenesis of the FLFP motif was performed to further investigate these protein complexes. Theoretical structures of PKCδVIII (wild‐type, F339Y, L340T, F341Y, and P342N) were created using I‐TASSER; Theoretical structures of PKCδVIII utilized in docking simulations were created using QUARK. YASARA was utilized to simulate the molecular docking of PKCδVIII to MAPK1 and Cyclin D1. The binding energies resulting from the theoretical docking were analyzed and compared to the wild‐type. Subsequent site‐directed mutagenesis of the FLFP motif has contributed to further clarifying our understanding of this protein complex. The results lend further evidence to support the role of PKCδVIII in promoting cellular survival.
The Coordination of Notch and Integrin Signaling by Resveratrol
Authors: LaFoya Bryce , Albig Allan
Published: November 2023

Integrins are transmembrane proteins which form for cell‐cell and cell‐extracellular matrix connections, allowing environmental signals to be relayed into a cell through their signal transduction capabilities. The Notch signaling pathway has a role in many cellular processes such as differentiation and proliferation. The regulation of this pathway can provide a cell with molecular cues which are used to make cell‐fate decisions. Both αVβ3 integrin and Notch signaling have been implicated in many normal processes of vascular biology such as angiogenesis and arteriovenous development as well as pathological processes such as the development of atherosclerosis and tumor vascularization. Studies on trans‐resveratrol, a stilbene produced in several plant species, have demonstrated beneficial effects on vascular health. Resveratrol is known to regulate two important signaling pathways in endothelial biology: Notch and αVβ3 integrin signaling. Through luciferase reporter assays and western blot analysis we have demonstrated that trans‐resveratrol promotes Notch1 induced transcriptional activation, but does not show the same effect when siRNA is used to knockdown β3 integrin. This data suggests, that resveratrol is able to induce transcription of Notch target genes via an αVβ3 dependent mechanism. Using co‐immunoprecipitation and in vitro kinase assays, we have identified an interaction between Notch1 and SRC kinase, a principle intermediary of integrin signaling. Additionally, using site directed mutagenesis we have identified tyrosine residues within the transcriptionally active portion of Notch1 which are important for Notch activation. Having established these results, we turn our attention towards the importance of the Notch1‐αVβ3 integrin signaling axis in the major cellular hallmarks of atherosclerosis.

Support or Funding Information

Boise State Biomolecular Sciences Ph.D. Program

NIH #1R15GM102852‐01
Histamine Mediates the Response to Light in the Sensory Motor Integration of Gill Lateral Cell Cilia in the Bivalve Mollusc, Crassostrea virginica
Authors: Semple Danellie , Marrett Kimone , Carroll Margaret A , Catapane Edward J
Published: November 2023

Gill lateral cells of Crassostrea virginica are innervated by serotonin and dopamine nerves from their ganglia. Most bivalves have lateral cell cilia that respond to serotonin and dopamine, with serotonin being the neurotransmitter that increases beating rates and dopamine being the neurotransmitter that decreases beating rates. The motor aspects of gill lateral cell innervation have been well studied, but not the sensory side. There is limited information about sensory inputs. Our previous work found that C. virginica can sense and adjust gill lateral cell cilia beating to the presence of food, crab extract and light, as well as a variety of chemical cues including histamine when applied to the mantle rim. Shining light on the mantle rim or applying histamine decreased gill lateral cell cilia beating rates, but did not alter cilia beating rates when applied directly to gill. In many invertebrates histamine is a neurotransmitter involved in photoreception. We hypothesize that in C. virginica, histamine is the neurotransmitter of mantle rim photoreceptors. To test this we used whole animal preparations in which the innervation of the gill from the cerebral and visceral ganglia is intact and tested the actions of a histamine H2 antagonist, famotidine, on the response to light. Cilia beating was measured by stroboscopic microscopy. An eleven lumen, ¼ inch diameter spotlight source was used to stimulate the mantle rim in the vicinity of the siphons. Stimulating the mantle rim with light decreased cilia beating in the gill. Adding the H2 antagonist famotidine caused a statistically significant, dose‐dependent blockage of this action over the range of 10−9 – 10−3. The study further demonstrates the sensory‐motor integration of beating of lateral cilia involving the sensory mantle rim and visceral/cerebral ganglia, supporting the hypothesis that histamine is the sensory neurotransmitter in mantle photoreceptor cells.

Support or Funding Information

This work was supported by grants 2R25GM06003 of the Bridge Program of NIGMS and 0516041071 of NYSDOE.
Regulation of AMPK and Hepatic Metabolism by Folic acid Supplementation in Non‐alcoholic Fatty Liver Disease
Authors: Sid Victoria , Wu Nan , Sarna Lindsei , Shang Yvette , House James , Siow Yaw L. , Karmin O
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) is a worldwide epidemic associated with obesity and type 2 diabetes. Chronic high‐fat diet consumption promotes NAFLD which is characterized by hepatic lipid accumulation, hyperglycemia and hyperlipidemia. Emerging evidence suggests that metabolic aberration during NAFLD may be related to dysregulation of AMP‐activated protein kinase (AMPK). AMPK is an endogenous energy sensor involved in regulating lipid and glucose metabolism, which has been proposed as a potential therapeutic target in NAFLD and its associated metabolic disorders. Folic acid is an essential B vitamin that has been implicated in NAFLD through improvement of liver function. The aim of our study was to investigate the mechanisms by which folic acid supplementation regulated hepatic AMPK and its impact on cholesterol and glucose metabolism. Male C57BL/6J mice were fed a control diet (10% kcals fat), a high‐fat diet (60% kcals fat) or a high‐fat diet supplemented with folic acid for 5 weeks. Mice fed a high‐fat diet exhibited fatty liver, hyperglycemia, elevated activity of hepatic HMG‐CoA reductase and lower AMPK activity. Folic acid supplementation restored AMPK activity, attenuated hepatic lipid accumulation and reduced blood glucose levels. Activation of AMPK by folic acid was mediated, in part, through activation of its upstream kinase LKB1. Folic acid‐induced AMPK activation could attenuate HMG‐CoA reductase activity via AMPK‐dependent phosphorylation of the reductase and hence reduce hepatic cholesterol biosynthesis. Our results suggest that folic acid supplementation can improve cholesterol and glucose metabolism through restoration of AMPK activation in the liver, which may have a therapeutic implication in mitigating hepatic lipid accumulation and hyperglycemia in NAFLD.

Support or Funding Information

This study was supported, in part, by Natural Sciences & Engineering Research Council of Canada and St. Boniface Hospital Research Centre.
Cyclin D1 regulates adipose triglyceride lipase (ATGL) to influence hepatic lipid droplet metabolism and cell proliferation
Authors: Ploeger Jonathan , Kamarajugadda Sushama , Mashek Douglas , Albrecht Jeffrey H
Published: November 2023

Obesity is well‐documented to promote the development of nonalcoholic fatty liver disease including its more advanced stages such as non‐alcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma. Cyclin D1 (D1) is a cell cycle protein that regulates G1/S transition, the rate‐limiting step in cell division. D1 is highly expressed in liver cancer and its overexpression (OE) leads to lipid droplet (LD) accumulation in hepatocytes. Liver‐specific knockdown of adipose triglyceride lipase (ATGL), the rate‐limiting enzyme in triglyceride (TG) catabolism, promotes LD accumulation. In order to characterize a potential link between D1 and ATGL we employed studies in primary hepatocytes and mouse immortalized AML12 cells. As expected, in the absence of mitotic stimuli, D1 OE was sufficient to drive DNA synthesis in primary hepatocytes. However, these effects are abrogated with ATGL. Knockdown of D1 in the presence of mitogens inhibited DNA synthesis and increased ATGL mRNA expression, but knockdown or chemical inhibition of ATGL recovered DNA synthesis. Moreover, cell cycle analysis using flow cytometry confirmed that D1 knockdown increased accumulation of cells in the G0/G1 phase and reduced cells in the S and G2/M phase; these results were reversed with ATGL knockdown or chemical inhibition. Taken together, these data illustrates for the first time that cyclin D1 regulates ATGL to alter hepatic LD metabolism and proliferative capacity in primary hepatocytes and AML12 cells.

Support or Funding Information

This project was supported by NIDDK (DK054921) to JHA and (DK085008) to DGM, NCI T32CA132670 to JMP and the Minnesota Obesity Center (DK050406).
Tracking Diacylglycerol Pools in Budding Yeast
Authors: Ganesan Suriakarthiga , Ponce Maria Laura Sosa , Zaremberg Vanina
Published: November 2023

Many diacylglycerol (DAG) sensing probes have been developed to monitor DAG signalling in mammalian cells. Several C1 domains have been fused to fluorescent proteins that can sense DAG in cellular membranes. The C1 domain of PKCδ is very well characterized and known to bind DAG with high affinity, and has been successfully used as a probe fused to GFP in order to monitor DAG in live cells. We have cloned the C1 domain of PKCδ (C1δ) into a centromeric yeast expression vector under a constitutive promoter for in vivo visualization of DAG pools. Localization of C1δ‐GFP was first monitored in different wild type strains grown under standard conditions. The main localization of this DAG‐binding reporter in actively growing cells was the vacuolar membrane, further confirmed by co‐localization with FM4‐64. This localization is consistent with studies done using similar probes to show that DAG localizes to the membrane of isolated vacuoles from yeast cells. Importantly, no changes in vacuolar morphology of wild type cells due to expression of C1δ‐GFP were detected. In addition to vacuolar membrane localization, the C1δ probe also associated with the plasma membrane of daughter cells and at the bud neck. In contrast to the phosphatidic acid (PA)‐binding reporter GFP‐Spo2051–91, C1δ‐GFP did not show nuclear localization as determined by co‐localization analysis using the nucleolar marker Sik1‐RFP. We then tested if the localization would change in conditions where lipid homeostasis and levels of DAG are known to be altered. For this, we used cells lacking the four enzymes responsible for the final step in the synthesis of triglycerides in yeast, namely DAG acyltransferase Dga1, the transacylase Lro1, and the steryl acyltransferases Are1 and Are2 (4Δ quadruple mutant), which accumulate DAG and have defects in both nuclear and peripheral ER membrane organization. Localization of the C1δ‐GFP probe drastically changed when expressed in 4Δ quadruple mutant cells, clearly revealing its association with the expanded nuclear and cortical ER in addition to the vacuolar membrane. Additionally, the polarized distribution of C1δ‐GFP to the plasma membrane of buds and daughter cells significantly decreased in the quadruple mutant. After validating the use of this probe in yeast, we investigated the localization of DAG pools in response to lysophosphatidylcholine (LysoPC) analogues, as we have determined they induce a 5 times increase in DAG levels. Interestingly, incubation with LysoPC analogues induced a drastic change in the localization of DAG while no differences were observed regarding the distribution of PA. These results further support the use of C1δ‐GFP as a valuable tool to track cytosolic accessible pools of DAG in yeast.

Support or Funding Information

This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to VZ
Cysteinyl Leukotriene 2 Receptor Enhances Angiogenesis, Vascular Permeability and Tumor Metastasis
Authors: Duah Ernest , Kondeti Vinay , Al‐Azzam Nosayba , Adapala Ravi , Patil Prachi , Thodeti Charles , Paruchuri Sailaja
Published: November 2023

Excessive or insufficient angiogenesis can lead to number of diseases including atherosclerosis, diabetic retinopathy, age‐related macular degeneration and cancer. Although long‐standing inflammation secondary to chronic infection or irritation mediated by immune cells has been implicated in cancer progression, the contribution of endothelial cells (EC) in the generation of inflammatory mediators and their effects on angiogenesis is relatively unknown. One of the pro‐inflammatory mediators produced by EC are cysteinyl leukotrienes (Cys‐LTs: LTC4, LTD4 and LTE4), and they mediate their effects through two main receptors, CysLT1R and CysLT2R. Cys‐LTs have been shown to be involved in several human cancers including breast cancer and melanoma. Tumor microenvironment comprises numerous signaling molecules and pathways that influence angiogenic response leading to increased and aberrant vascularization. Further, angiogenesis is required for the supply of nutrients and oxygen for tumor growth and also serves as a route of tumor cells metastasis. Therefore, we investigated the role of CysLTR in angiogenesis, tumor progression and metastasis, using mice lacking CysLT1R (CysLT1RKO) and CysLT2R (CysLT2RKO).

We observed enhanced CysLT2R expression, and not CysLT1R in wild type (WT) tumors and tumor growth as well as angiogenesis were significantly decreased in CysLT2RKO mice compared to WT and CysLT1RKO mice in Lewis lung carcinoma (LLC) tumor model. Further, though few in number, tumor vessels in CysLT2RKO mice showed intact pericyte coverage and reduced permeability with simultaneous reduction in tumor cell metastasis to the lung. Furthermore, we found that activation of CysLT2R, but not CysLT1R increased EC contraction leading to junctional destabilization and permeability in vitro. Importantly, our results show that CysLT2R activates Rho kinase and inhibition of Rho kinase significantly attenuated CysLT2R‐induced EC contraction and permeability. Further, our preliminary results revealed that pericytes express CysLT2R and display calcium influx in response to LTD4. Taken together, our results suggest that CysLT2R promotes leaky blood vessel formation, enhancing tumor growth and metastasis to the lung. Blocking CysLT2R could offer novel CysLT2R‐targeted, therapeutic candidates for the treatment of cancer and other angiogenic disorders.

Support or Funding Information

James Foght Professorship Support, AHA GIA 15GRNT 25670004
Is the site of influenza virus assembly and budding enriched with cholesterol and sphingolipids?
Authors: Kraft Mary L , Yeager Ashley N. , Weber Peter K. , Zimmerberg Joshua
Published: November 2023

The observation that the influenza viral envelope is enriched with cholesterol and sphingolipids contributed to the hypothesis that the influenza virus assembles and bud from cholesterol‐ and sphingolipid‐enriched domains in the plasma membrane. Protein imaging experiments have demonstrated that the clustering of influenza virus proteins in the plasma membrane is sensitive to depletion of cellular cholesterol. However, the precise distributions of cholesterol and sphingolipids in relation to the site of influenza budding remains speculative due to an inability to directly image lipids without the use of potentially perturbing labels. We have used high‐resolution secondary ion mass spectrometry (SIMS), which is performed on a Cameca NanoSIMS 50, to image the distributions of stable isotope‐labeled lipids in the plasma membranes of intact cells with ~100 nm lateral resolution. Here we report using this approach to directly test the long‐standing hypothesis that the influenza virus buds from plasma membrane domains that are enriched with cholesterol and sphingolipids. We imaged metabolically incorporated 15N‐sphingolipids and 18O‐cholesterol and immunolabeled influenza virus envelope proteins on the surfaces of influenza‐infected MDCK cells with high‐resolution SIMS. The sites of virus assembly and budding in the plasma membrane were identified as the regions on the MDCK cell that were enriched with the immunolabeled influenza virus envelope protein, hemagglutinin. High‐resolution SIMS imaging revealed the sites of influenza virus assembly and budding were not enriched with cholesterol and sphingolipids. Although sphingolipid domains were detected in the plasma membranes of these MDCK cells, they had little co‐localization with the hemagglutinin‐rich membrane patches. These findings argue against the hypothesis that the influenza virus buds from cholesterol‐ and sphingolipid‐enriched domains in the plasma membrane.

Support or Funding Information

This research was supported by the U.S. National Science Foundation under CHE 15‐08662.
Autism‐linked Mutations in the Human Serotonin Transporter Induce Distinct Structural Changes that may Account for the Altered Function of the Transporter
Authors: Krout Danielle , Henry L. Keith
Published: November 2023

Background

Physiological studies show that 20–25% of individuals with autism spectrum disorder (ASD) exhibit elevated platelet serotonin (5‐HT) levels suggesting a link between regulation of the serotonergic system and behaviors associated with ASD. Furthermore, multiple ASD‐linked serotonin transporter (SERT) coding variants (ASVs) have been identified which augment 5‐HT transport function through enhanced catalytic activity (Gly56Ala and Lys605Asn) or elevated surface density (Ile425Leu, Phe465Leu, and Leu550Val). We propose that ASV‐induced hyperactivity is a result of altered SERT tertiary structure thereby affecting phosphorylation through the PKG and p38 MAPK signaling pathways and/or modulating plasmalemmal SERT turnover.

Objective

Identify SERT tertiary structure alterations mediated by ASV modifications that contribute to changes in catalytic activity or surface expression.

Results

We utilized the substituted cysteine accessibility method to assess conformational changes by comparing the sensitivity of one native (Cys109) and two engineered cysteine residues (Cys277 and Cys404) to methanethiosulfonate reagents (MTS). Cys277 displayed decreased reactivity in the ASVs Gly56Ala, Ile425Leu, Leu550Val, and Lys605Asn indicating reduced accessibility as a result of ASV‐induced conformational changes. Evaluation of reactivity changes at Cys109 and Cys404 is currently underway. In addition, we are characterizing structural changes in the ASVs after activation of the PKG or p38 MAPK pathways to determine if the functional changes stem from a common mechanism.

Conclusion

The reactivity changes we have observed at Cys277 support alteration of SERT tertiary structure by the ASVs Gly56Ala, Ile425Leu, Leu550Val, and Lys605Asn. In addition, the intracellular position of the Cys277probe in SERT and its decrease in MTS sensitivity suggests an ASV‐induced outward‐facing conformation.
Unraveling the Structural Basis of P‐glycoprotein Transport of Serotonin 5‐HT1B/1D Receptor Agonists: Eletriptan and Sumatriptan
Authors: Nguyen Diana , Wilt Laura , Roberts Audie
Published: November 2023

P‐glycoprotein (Pgp) acts as the gatekeeper at the blood‐brain barrier by preventing the entry of toxic insults; however, drugs that are Pgp substrates cannot penetrate the blood‐brain barrier to treat central nervous system (CNS) disorders and diseases. Serotonin 5‐HT1B/1D receptor agonists are members of the triptan class of drugs that abort migraines during the onset of symptoms and are known Pgp substrates. While members of a drug class are structurally similar, eletriptan (Relpax®, ETT) and sumatriptan (Imitrex®, STT) have large differences in their Pgp efflux ratios. To understand the relationship between drug binding and efflux, ETT and STT were used to probe the structural basis of triptan transport. From ATPase activity assays, ETT stimulated Pgp ATP hydrolysis and was fit to the Michaelis‐Menten equation. In contrast, STT induced very little activation of the Pgp‐coupled hydrolysis. Protein fluorescence quenching was used to determine the binding affinities and the number of binding sites of ETT and STT. Each triptan drug was identified to have a single binding site. To test if these binding sites were overlapping, competition ATPase activity assays were performed. Conformational changes induced by each ETT and STT binding was determined by acrylamide quenching.

Support or Funding Information

NIH ‐1021RR571390, AHA ‐1021RR571388, Pfizer, Inc., University of Georgia, Bioexpression and Fermentation Facility at UGA
A Ten Week, First‐semester Biochemistry Lab Course Studying Bacterial Alkaline Phosphatase
Authors: Witherow D. Scott
Published: November 2023

In a traditional introductory biochemistry lab, students are expected to learn a variety of standard biochemical techniques. One of the challenges in designing a lab curriculum is finding a way to engage students in a way where they not only learn the necessary skills, but see how different aspects of biochemistry work together with one another. This work describes a 10 week laboratory project studying wild‐type and mutant bacterial alkaline phosphatase, where students purifying, quantitate, and perform kinetic assays on wild‐type and selected mutants of the enzyme. Additionally, students also perform vector DNA analysis. In addition to simply learning important techniques, students acquire novel biochemical data in their kinetic analysis of mutant enzyme. The experiments are designed to build on student's work from week to week in a way that encourages students to apply quantitative analysis and reasoning skills, reinforcing traditional textbook biochemical concepts. Students are assessed through lab reports focused on journal style writing, quantitative and conceptual question sheets, and traditional exams. Students also are surveyed to obtain data relating to their perception of the efficacy of the course design and in achievement of learning outcomes.

Support or Funding Information

This work was supported by The University of Tampa Dana Foundation Grant; The University of Tampa David Delo Research Professor Grant; The University of Tampa College of Natural and Health Sciences; and The University of Tampa Department of Chemistry, Biochemistry, and Physics.
Cloning and Characterizing a Beta‐glucanase Gene from Oats: A Structure/Guided Inquiry‐ Based Molecular Biology Laboratory
Authors: Martin Debra J.
Published: November 2023

For the majority of students entering the Molecular Biology laboratory, basic biotechnology (molecular biology) skills are a new concept for them. Previous courses, such as AP or lower division Introductory Biology and Genetics may have introduced them to some of the techniques; but overall, the concept of combining the techniques to actually research a gene by cloning it and characterize it is lacking. Additionally, we want students to learn research skills, like interpreting evidence, reasoning, thinking critically, and conveying ideas and to be able to write like a scientist. Using a structure/guided inquiry laboratory, students develop primers, clone and characterize a beta‐glucanase gene from oat (Avena sativa) (Martin, 2004; Yun et al., 1993, Yun et al., 1993). By the end of the semester long course, the students submit a final paper in the format for submission to the Proceedings of the National Academy of Science (PNAS).