bluuebunny/crossref_metadata_2025 huggingface dataset:
Measuring the precise position of transitions in transcription with PRO‐IP‐seq
Authors: Versluis Philip , DeBerardine Michael , Lis John
Published: May 2022

Transcription is a fundamental process for all known organisms. Transcription by RNA Polymerase II (Pol II), the enzyme responsible for mRNA synthesis, is highly regulated by protein factors at every step. In multicellular eukaryotes, Pol II pauses 20‐60 bases downstream of the transcription start site in nearly all genes. This promoter‐proximal pausing is established as an important step in transcription regulation and is dependent on several pausing and elongation factors. The protein complexes DSIF (DRB Sensitivity Inducing Factor) and NELF (Negative Elongation Factor) bind to Pol II early in transcription to induce pausing. Pause release occurs upon phosphorylation of Pol II, DSIF and NELF by the Cdk9, Cyclin T1 heterodimer P‐TEFb (Positive Transcription Elongation Factor b). Although Pol II pausing is a key step in transcription regulation and has been extensively studied, the molecular mechanisms of pause and pause release are incompletely understood. Furthermore, recent studies have implicated controlled termination early in the gene as a mechanism to control transcription. A challenge to studying these key regulatory steps is that they occur in a narrow window: from initiation to Pol II pausing and release occurs in 20‐60 base range. Current approaches to study transcription factor interactions are unable to provide high enough resolution or sensitivity to address precisely where key transitions in transcription occur.

To address this problem, I am developing an assay to measure the exact position of Pol II, when it is bound by a factor of interest. The technique couples our lab’s Precision Run‐on sequencing technique (PRO‐seq), which measures the position of active Pol II with single base‐pair resolution, and an immunoprecipitation enrichment step. Initial PRO‐IP‐seq (Precision Run‐on Immunoprecipitation sequencing) data shows that we can enrich for populations of Pol II bound by specific transcription factors. Utilizing this tool, we will be able to measure genomically where both stable and more transient transcription factors interact with Pol II at near single‐base resolution.
Acute Chemogenetic Inhibition of Caudal NTS Astrocytes Reduced Systemic Blood Pressure in Rats Exposed to Chronic Intermittent Hypoxia‐mimicking Sleep Apnea Syndrome
Authors: Iturriaga Rodrigo , Toledo Camilo , Ortolani Domiziana , Del Rio Rodrigo
Published: May 2022

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea, enhances carotid body (CB) discharges, leading to heightened sympathetic outflow and systemic hypertension. Previously, we found that CBs play a pivotal role in the hyperactivation of central nervous system (CNS) autonomic nuclei following CIH, particularly at the level of the caudal portion of the nucleus of the tractus solitary (NTS). Indeed, increased CB afferent activity during CIH increases the expression of pro‐inflammatory cytokines in the NTS, suggesting that CBs may drive neuroinflammation at key cardiorespiratory regulatory areas. Astrocytes has been implicated in inflammatory processes at the CNS. However, the contribution of NTS astrocytes on the cardiorespiratory abnormalities following CIH has not been studied. Accordingly, we assessed the role of astrocytes residing within the NTS on the maintenance of hypertension following CIH. Male Sprague‐Dawley rats (200 g) were exposed to CIH (5–6% inspired O2 for 20s, followed by room air for 280s, 12 times/h, 8 h/day, for 28 days). Arterial blood pressure (BP) was measured by indwelling telemetry. At 7 days of CIH exposure, rats were anesthetized and an adeno‐associated virus (AAV; 450 nL, 1*10‐12 vg) containing an inhibitory (Gi) Designer Receptor Exclusively Activated by Designer Drugs (DREADD) expressed under the control of the GFAP promoter was bilaterally injected into the caudal portion of the NTS using stereotaxic coordinates (–14.3 mm to bregma). At day 28 of CIH, hemodynamic and respiratory parameters were recorded before and after inhibition of NTS astrocytes with clozapine N‐oxide (CNO, 1mg/kg, ip.). At the end of the experiments rats were transcardially perfused with 4% buffered paraformaldehyde, brains extracted and sectioned to assess astrocyte activation within the NTS. Twenty‐eight days of CIH resulted in a significant 2‐fold increase in NTS astrocyte activation as evidenced by enhanced reactivity of GFAP. Additionally, resting BP was markedly elevated compared to Sham conditions (MABP, 98±2 vs. 84±2 mmHg, CIH vs. Sham; p<0.05). Acute chemogenetic inhibition of NTS astrocytes following 28 days of CIH results in a significant reduction in BP (⁓10 mmHg; p<0.05). In addition, the exacerbated hemodynamic response triggered by acute hypoxic stimulation (FiO2 10%) in rats exposed to CIH for 28 days was also reduced by NTS astrocyte inhibition ΔMABP, 30±2 vs. 15±2 mmHg, pre vs. post CNO; p<0.05). No cardiovascular effects of CNO alone were found in control rats that did not underwent AAV‐DREADD‐Gi injection into the NTS. Taken together, our results support a role for NTS astrocyte activation on the maintenance of hypertension following chronic CIH and suggest that activation of NTS astrocytes may participate in the CB‐mediated cardiovascular reflex response during hypoxic stimulation.
Reconstitution of the calcium‐triggered contractile apparatus of ciliated protozoa
Authors: Honts Jerry E. , Lei Xiangting , Bhamla Saad
Published: May 2022

Protists, notably ciliated protozoa, exhibit a novel form of contraction that is rapidly triggered by calcium ions but does not require ATP hydrolysis. Ultrafast forms of calcium‐triggered contraction had previously been described for the Vorticella stalk and the Spirostomum cell body. In these ciliates, cell structures (stalk or body) contract within a few milliseconds. Spasmin, an EF‐hand calcium binding contractile protein was identified as the key component of the Vorticella spasmoneme, the contractile organelle present within its stalk. In heterotrich ciliates, such as Stentor and Spirostomum, contraction is mediated by branching, spindle‐shaped structures called myonemes, which contain centrin‐like proteins. Bioinformatics analysis of published and recently obtained data revealed the existence of several distinct groups of EF‐hand calcium‐binding contractile proteins in ciliated protozoa. Synthetic genes encoding these calcium‐binding proteins, as well as their putative scaffold proteins, have been engineered for expression in bacteria. In vitro reconstitutions of these contractile systems were then attempted using purified proteins. Tcb2 protein from Tetrahymena thermophila was identified as a major component of contractile gels formed when calcium ions were added to a low ionic strength alkaline extract of the Tetrahymena membrane‐associated cytoskeleton. Purified Tcb2 protein showed calcium‐dependent reversible assembly in microscopic, spectroscopic, and pelleting assays. Tcb2 has also been shown to interact with and drive the collapse of gels formed with either of two other alpha‐helical coiled‐coil cytoskeletal proteins, Epc1 and Fen1. Through proteomic analysis of Spirostomum cytoskeletal preparations, a family of EF‐hand proteins closely related to the calcium‐binding protein centrin have been identified. In addition, a putative centrin‐binding Sfi1‐like scaffold protein has been identified in Spirostomum. Proteins encoding putative components of the Spirostomum ambiguum myoneme contractile systems have been expressed in bacteria and purified. Solutions of these Spirostomum proteins responded to calcium ions in a similar way to that seen for Tetrahymena contractile proteins. Our immediate goal is to engineer Spirostomum contractile proteins to fully reconstitute in vitro the ultrafast myoneme contraction taking place in living Spirostomumcells. These studies should yield insight into the structure, mechanism and regulation of the calcium‐triggered contractile systems present in protistan cells. As a parallel goal, we are working to engineer optically controlled contractile elements built from these proteins, and introduce them into metazoan animal cells and synthetic cells, so that we can manipulate their behaviors through patterned light activation.
Acute Heat Exposure Protects Against Endothelial Ischemia‐reperfusion Injury in Aged Humans
Authors: Hemingway Holden W. , Richey Rauchelle E. , Moore Amy M. , Olivencia‐Yurvati Albert H. , Romero Steven A.
Published: May 2022

Non‐pharmacological therapies that protect against endothelial ischemia‐reperfusion (I/R) injury remain limited in aged adults. Acute heat exposure protects against endothelial I/R injury in young adults, but its efficacy has never been explored in aged adults. Therefore, we tested the hypothesis that acute heat exposure would protect endothelial‐dependent vasodilation following I/R injury in aged adults. Nine (2 men, 69 ± 8 yrs) aged adults were exposed to a thermoneutral control condition or whole‐body passive heating (water‐perfused suit) sufficient to increase body core temperature by 1.2 °C. Experiments were separated by at least 7 days. Heat exposure was always performed first in order to time‐match the thermoneutral control condition. Endothelium‐dependent vasodilation was assessed via flow‐mediated dilation of the brachial artery before (pre‐I/R) and after I/R injury (post‐I/R), which was induced by 20 min of arm ischemia followed by 20 min of reperfusion. Ischemia‐reperfusion injury reduced flow‐mediated dilation following the thermoneutral control condition (pre‐I/R, 4.5 ± 2.9 % vs. post‐I/R, 0.9 ± 2.8 %, P < 0.01), but was well maintained with prior heat exposure (pre‐I/R, 4.4 ± 2.8 % vs. post‐I/R, 3.5 ± 2.8 %, P = 0.5). Taken together, acute heat exposure protects against endothelial I/R injury in aged adults. These results highlight the therapeutic potential of heat therapy to prevent endothelial dysfunction associated with I/R injury in aged adults who are most at risk for an ischemic event.
Evaluating Co‐occurrence as a Criterion for Identification of Undocumented Xenobiotic Exposures in Human Metabolomics
Authors: Singer Grant T. , Liu Ken H. , Lee Choon M. , Miller Gary W. , Li Shuzhao , Go Young‐Mi , Jones Dean P. , Morgan Edward T.
Published: May 2022

High resolution mass spectrometry (HRMS) detects tens of thousands of unknown features in human plasma. Many of these features could be indicative of xenobiotic exposure, but identifying candidates poses a challenge due to low abundance and lack of authentic standards. Here we propose using co‐occurrence of metabolites as a criterion to aid identification of undocumented xenobiotic exposures and co‐exposures in humans. Plasma from 49 volunteer Emory University/Georgia Tech employees in a longitudinal study were analyzed with HRMS, and aligned features were arranged as nodes in networks connected according to feature co‐occurrence and common biotransformation mass differences, marking possible xenobiotic pathways. These networks were compared against curated metabolic profiles of xenobiotic reference material generated on an enzyme‐based biotransformation system (liver S9 fractions). Profile matches, coupled with available MS/MS, suggest confident exposure identification of zolpidem, dextromethorphan, nicotine, citalopram, caffeine and tramadol within the cohort. Further investigation of co‐occurrence networks also facilitated suggestion of possible xenobiotic co‐exposures beyond biotransformation pathways. In this cohort, Zolpidem metabolites co‐occurred with high abundance features having the the masses of melatonin and its metabolites, suggesting co‐exposure due to polypharmacy for a single condition.
Testing the role of mab‐5/Hoxin C. elegansInnate Immunity
Authors: Kywe Christopher N. , Ackley Brian
Published: May 2022

The innate immune system acts as an organism’s first line of defense against invading pathogens. It accomplishes this by using pathogen‐associated molecular patterns (PAMPs) or damage‐associated molecular patterns (DAMPs) to recognize and destroy invaders. While the mechanisms for innate immunity in response to pathogens have been studied, the genetic and environmental influences that govern innate immune development are unknown. In the Ackley lab we use Caenorhabditis elegans,a soil‐dwelling bacterivore nematode, to study the genetics of innate immunity. My project focuses on how mutations in the mab‐5gene affect resistance to bacterial pathogens. mab‐5 is a Hox‐family transcription factor that has primarily been studied in early organismal development, but transcriptome analyses of both gain and loss‐of‐function mutations identified mab‐5 regulation of innate immune genes. I have tested the resistance of four mab‐5mutant strains following exposure to a bacterial pathogen, Staphylococcus epidermidis,and compared survival to animals fed a normal diet of non‐pathogenic Escherichia coli. I have tested the wild‐type strain, N2, two loss‐of‐function mutations, mab‐5(gk670) and mab‐5(e1239), and one gain‐of‐ function mutant, mab‐5(e1750). I have found that the mab‐5(e1750)strain has increased resistance compared to the loss‐of‐function and wild‐type strains during the first week following exposure to S. epidermidis. This is consistent with our observation that increased mab‐5activity leads to increased expression of innate immune genes. To corroborate this observation I am testing another potential gain‐of‐function line, wgIs77,from the ModEncode project, which has an integrated MAB‐5::GFP fusion protein. I am also using a transcriptional mab‐5gfp reporter, muIs16, to monitor when and where mab‐5expression might be occurring in animals after bacterial infection. Together, the results of this project will help illuminate the mechanisms of innate immune pathway activation and the role that transcriptional regulation plays in organismal health.
Structural Analysis of the Roles of CCT and PhLP1 in G Protein β Subunit Folding and Gβγ Dimer Assembly
Authors: Kwon Yujin
Published: May 2022

Defects in G protein signaling are implicated in host of disorders and diseases, which makes them high value therapeutic targets. While the function of the different G protein subunits has been well‐characterized, little is known about their folding and assembly into complexes. The previous work from our lab showed that CCT and its co‐chaperone PhLP1 are required for the folding of the Gβ subunits and their assembly into complexes with Gγ subunits into the Gβγ dimer. To understand the molecular mechanism of Gβ folding by CCT and PhLP1, we have captured the PhLP1‐Gβ1‐CCT complex and determined its structure by high resolution cryo‐electron microscopy. The structure shows Gβ1 in an unexpected position, sitting between the two CCT rings and interacting with the N‐ and C‐ termini of several CCT subunits. In this position, Gβ appears to have reached a near‐native β‐propeller fold. This result confirms the importance of the terminal regions of the CCT subunits in binding β‐propeller proteins, as we recently observed with Gβ‐like mLST8 subunit of the mTOR complex (Cuellar et al. 2019 Nature Commun. 10, 2865). The structure also elucidates the role of PhLP1 in Gβ1 folding. The PhLP1 C‐terminal domain binds to the apical domains of the CCT3 and CCT6 subunits, while its N‐terminal domain extends down to interact with Gβ1 between the CCT rings. The structure explains why the PhLP1 N‐terminal domain is essential in triggering the release of Gβ1 from CCT to interact with Gγ. These findings suggest that a mimetic of the interaction between the N‐terminal domain of PhLP1 and Gβ1 could block Gβγ assembly and interfere with G protein signaling in disease states caused by excessive G protein signaling.
Gitelman‐like syndrome caused by pathogenic variants in mitochondrial DNA
Authors: de Baaij Jeroen H. F. , Viering Daan , Schlingmann Karl P. , Hureaux Marguerite , Nijenhuis Tom , Knoers Nine V. , Vargas Poussou Rosa , Bockenhauer Detlef
Published: May 2022

Gitelman syndrome is the most frequent hereditary salt‐losing tubulopathy characterized by hypokalemic alkalosis and hypomagnesemia. Gitelman syndrome is caused by biallelic pathogenic variants in SLC12A3, encoding the Na+‐Cl‐ cotransporter (NCC) expressed in the distal convoluted tubule. Pathogenic variants in CLCNKB, HNF1B, FXYD2 or KCNJ10 may result in renal phenocopies of Gitelman syndrome, as they can lead to reduced NCC activity. Nevertheless, ±10% of patients with a Gitelman syndrome phenotype remain genetically unsolved.

After identification of mitochondrial DNA (mtDNA) variants in three families with Gitelman syndrome‐like electrolyte abnormalities, 156 families were investigated for variants in MT‐TI and MT‐TF, encoding the transfer RNAs for phenylalanine and isoleucine. Mitochondrial respiratory chain function was assessed in patient fibroblasts. In NCC‐expressing HEK293 cells, mitochondrial dysfunction was induced to assess the effect on thiazide‐sensitive 22Na+ transport.

Genetic investigations revealed four mtDNA variants in 13 families: m.591C>T (n=7), m.616T>C (n=1), m.643A>G (n=1) (all in MT‐TF) and m.4291T>C (n=4, in MT‐TI). Variants were near homoplasmic in affected individuals. Importantly, affected members of six families with an MT‐TF variant additionally suffered from progressive chronic kidney disease. Maximal mitochondrial respiratory capacity was reduced in patient fibroblasts, caused by dysfunction of oxidative phosphorylation complex IV. In vitro pharmacological inhibition of complex IV, mimicking the effect of the mtDNA variants, demonstrated an inhibitory effect on NCC phosphorylation and NCC‐mediated sodium uptake.

Pathogenic mtDNA variants in MT‐TF and MT‐TI can cause a Gitelman syndrome‐like syndrome. Genetic investigation of mtDNA should be considered in patients with unexplained Gitelman syndrome‐like tubulopathies.
A computational analysis of colchicine structural analogs as potential microtubule destabilizing agents
Authors: Rocha De Abreu Isadora , Barkdull Allison , Craddock Travis J. A.
Published: May 2022

Microtubules are important for the formation of the cytoskeleton and mitotic spindles, and they aid in cell movement and intracellular transport. Therefore, they are important for cell survival and cell function. Microtubules are composed of the alpha‐beta tubulin heterodimer protein. During polymerization tubulin proteins experience phases of dynamic instability, which is characterized by microtubule growth and shrinkage at varying rates. Microtubule formation can be inhibited by compounds such as colchicine, a known microtubule destabilizing agent, that binds to tubulin to block polymerization. In this study we investigated one hundred eleven compounds that had a similar structure to colchicine. We used AutoDock Vina to dock each compound including colchicine to tubulin in the same binding site as colchicine. The binding energy of colchicine was ‐10.9 kcal/mol, and the binding energies of the top 10% binding affinity compounds were between ‐9 kcal/mol and ‐7.9 kcal/mol. The pharmacokinetic properties of these drugs, computational binding affinity and similarity to colchicine show promise as novel inhibitors of microtubule polymerization, which will be further explored through laboratory experiments.
Determining Protein D Antibody Suppression by OMP26
Authors: Perdue Janai , Michel Lea , Labbe Natalie , Gleghorn Michael , Jones Seth
Published: May 2022

Acute otitis media (AOM) is one of the most prevalent diseases in young children, and healthcare‐related costs are estimated to be $6 billion in the US each year. AOM is also the most common ailment for which antibiotics are prescribed. Considering all of these challenges and the rising concerns over antibiotic resistant bacteria, it is important to develop a vaccine to protect against AOM. Two proteins, OMP26 and Protein D, are currently leading protein vaccine candidates for one of the main bacterial causes of AOM: nontypeable Haemophilus influenzae (NTHi). On their own, the proteins produce a robust antibody response in mice. However, we recently showed that when the two proteins are combined into a single formulation, Protein D antibodies are suppressed. We first hypothesized that OMP26 and Protein D must physically interact to induce such suppression. However, here, we describe the results of our studies, which employed column chromatography and other biochemistry techniques, which suggest that OMP26 and Protein D do not directly interact. Further studies are underway to determine the mechanism behind Protein D antibody suppression and to assess several experimental strategies to overcome the suppression.
Utilizing mpx:gfp transgenic zebrafish to assess eya3’s potential effects on neutrophils
Authors: Baxter Gloria , Larson Nicole , Tessier Violet , O'Brien Jenean
Published: May 2022

Rhabdomyosarcoma (RMS) is a malignant cancer that develops in children’s muscles. Although RMS is one of the most common soft tissue sarcomas in children (NIH, 2020), there are a limited number of treatment options. As a potential new treatment target, we are investigating the roles of eya3, a transcriptional regulator, in normal muscle development. Previous research has shown a connection between eya3 and the innate immune system (Liu, 2012; Okabe, 2009), sometimes indicating eya3 is involved in innate immune responses, other times demonstrating that eya3 inhibits immune responses (Vartuli, 2018). Innate immune cells include neutrophils, which express mpx or myeloid‐specific peroxidase. To investigate how eya3 affects neutrophil location and/or levels in developing embryos, we utilized mpx:gfp transgenic zebrafish to visualize green fluorescent neutrophils via fluorescent imaging. After injecting eya3 morpholinos into wildtype zebrafish embryos to decrease eya3 expression, we used quantitative PCR (qPCR) to quantify the amount of neutrophils present. While our fluorescent imaging confirmed the presence of green fluorescent neutrophils in the mpx:gfp zebrafish at 96 hours post fertilization (hpf), we have yet to image neutrophils in our experimental group at the same timepoint. Our qPCR results suggest a decrease in mpx expression at 24 hpf with eya3 knockdown. We plan to confirm the potential decrease in neutrophil levels following eya3 knockdown with visualization by fluorescent microscopy. It will be interesting to determine if neutrophil levels are decreased in all locations, or depleted only from some locations in the zebrafish.
Decreased Placental mTOR Signaling And Increased Placental Apoptosis Are Associated With Secondhand Smoke (SHS)‐Induced Intrauterine Growth Restriction (IUGR) In Mice
Authors: Bell Zoe , Kiehl Joe , Clarke Derek , Curtis Katrina , Reynolds Paul R. , Arroyo Juan A.
Published: May 2022

Intrauterine growth restriction (IUGR) is a disease affecting 10% of all pregnancies. IUGR is associated with maternal, fetal, and placental abnormalities, but studies investigating the inducibility of IUGR by secondhand smoke (SHS) are limited. The mTOR pathway regulates protein expression and cell growth. Disrupted mTOR signaling and apoptosis are associated with the development of several obstetric complications including IUGR. We tested the hypothesis that SHS exposure disrupts the mTOR pathway and apoptosis in mice. C57Bl6 mice were exposed to SHS for 4 days from day 14 to day 17 of gestation (dGA). At the time of necropsy (18 dGA) placental and fetal weights were recorded and tissues were immediately frozen for immunoblot analysis. Mice exposed to SHS demonstrated a significant reduction in fetal weight (7.35‐fold; p≤0.0001) and placental weight (1.13‐fold; p≤0.0001) compared to controls. Significant decreases were observed for placental activation of mTOR (2.1‐fold; p<0.03), p70 (1.9‐fold; p,0.03) and 4EBP1 (1.3‐fold (p<0.03) in IUGR placentas compared to controls. Increased placental active caspase 3 (1.1‐fold; p<0.04) and the antiapoptotic placental XIAP protein (2.2‐fold; p<0.03) were also detected during SHS treatment compared to controls. We conclude that decreased mTOR pathway is associated with lessened fetal and placental size during SHS induced IUGR. This decrease was associated with increased caspase 3 and that elevated XIAP protein may signify an attempted protective mechanism that counters increased apoptosis observed in the SHS‐induced IUGR placenta. These studies provide insight into tobacco‐mediated IUGR development and clarify avenues that may be helpful in the alleviation of placental complications.
P2X7 inhibition prevents mitochondrial stress caused by alcohol and e‐cigarette exposure in primary vascular endothelial cells and restores barrier function
Authors: Mekala Naveen K. , Gajghate Sachin , Rom Slava , Reichenbach Nancy , Persidsky Yuri
Published: May 2022

Studies in both human and animal models demonstrated that chronic alcohol/e‐cigarette (e‐Cig) exposure affects mitochondrial function and impairs barrier function in brain microvascular endothelial cells (BMVEC). Identification of the signaling pathways by which chronic alcohol/e‐Cig exposure induces mitochondrial damage in BMVEC is vital for protection of the blood‐brain barrier. We treated primary human BMVEC with ethanol (100 mM) or e‐Cig conditioned medium and showed reduced mitochondrial oxidative phosphorylation (OXPHOS) analyzed by a Seahorse analyzer. Chronic alcohol/e‐Cig treatment significantly reduced the expression of mitochondrial OXPHOS proteins and pyruvate dehydrogenase (a vital enzyme in the TCA cycle) detected by western blot. We observed the cytosolic escape of ATP and its extracellular release due to the disruption of mitochondrial membrane potential after alcohol or e‐Cig treatment of BMVEC. Further, alcohol or e‐Cig treatment resulted in an elevated purinergic receptor (P2X7) and TRPV1 channel expression by qPCR in primary BMVEC. We showed a protective role of the P2X7r antagonist which restored the mitochondrial oxidative phosphorylation levels and prevented ATP release. In summary, we identified potential common pathways of mitochondrial injury caused by alcohol and e‐Cig which allow new protective interventions. We are further investigating the potential link between P2X7 regulatory pathways and mitochondrial health.
Engineering designer biologics in plant cells for oral treatment of inflammatory bowel disease (IBD)
Authors: Xu Jianfeng , Guo Wenzheng , TrejoMartinez Jonathan , Unnold Carmela
Published: May 2022

Inflammatory bowel disease (IBD), including the two most common subtypes: Crohn’s disease (CD) and ulcerative colitis (UC), represents a group of intestinal disorders that cause prolonged inflammation of the digestive tract. The current treatment strategies, including the conventional anti‐inflammatory medications and the new biological drugs targeting the pro‐inflammatory cytokine tumor necrosis factor alpha (TNFα), have limited therapeutic efficacy and adverse drug reactions resulted from systemic administration. Colon‐targeted oral delivery of anti‐TNFα agents is highly desirable for the treatment of IBD, as it improves drugs’ efficacy while reducing systemic toxicity. Plant cell culture has emerged as a safe and cost‐effective bioproduction platform for therapeutic proteins. A unique feature of the plant cells is that they could serve not only as the “bio‐factory,” but also the oral delivery vehicle for recombinant biologics. Recent advances have demonstrated that plant cell walls, made primarily of cellulose microfibrils, can act as an excellent natural capsule for oral delivery of biologic drugs. This project aims to leverage two unique posttranslational modifications – “glycosyl‐phosphatidylinositol (GPI) anchor” and “plant‐specific hydroxyproline (Hyp)‐O‐glycosylation” – to strategically design and engineer novel anti‐TNFα biomolecules in plant cells to develop a new class of oral biologic drugs for the treatment of UC. The designer anti‐TNFα biomolecules consist of three functional domains: a N‐terminal single‐chain fragment variable (scFv) of an anti‐TNFα antibody, a proprietary Hyp‐O‐glycosylation module comprised of tandem repeats of the “Ser‐Pro” motif, or (SP)n (n= 5 to 30), and a C‐terminal GPI anchor. While the GPI anchor “displays” the expressed anti‐TNFα biomolecules at the plant cell surface to presumably create a high local concentration of the biologics, the (SP)n glycomodule stabilizes the protein from degradation during both the bioproduction and oral delivery processes. Designer anti‐TNFα biomolecules consisting of different sizes of the (SP)n glycomodule are investigated for their accumulations in tobacco BY‐2 cells, biological activity, and stability in a simulated gastric fluid, which determines the (SP)20 module as an optimal design for the biomolecules. The therapeutic effectiveness of the orally administrated designer anti‐TNFα biologic (optimal design) in mitigating the UC symptom is assessed in a dextran sulfate sodium (DSS)‐induced colitis mouse model. The immune‐modulatory effects of the anti‐TNFα biologics are determined by histopathological analysis and assay of the inflammatory markers. The research may develops a new platform to produce effective oral biologic drugs for the treatment of UC and other inflammatory diseases of colon.
NMDA Receptor Neuropharmacology: NMDA Receptor‐mediated Calcium Transients are Attenuated by Agmatine in Mouse Spinal Cord Dorsal Horn
Authors: Xie Tongzhen
Published: May 2022

NMDA receptor (NMDAr) plays an important role in synaptic plasticity and one of the targets for chronic pain management is antagonism of the NMDA receptors (NMDAr). Agmatine is an endogenous aminoguanidine that preferentially antagonizes GluN2B‐containing NMDArs. Our previous studies showed that the intrathecal administration of agmatine, an endogenous aminoguanidine that selectively antagonizes GluN2B‐containing NMDArs, reverses the mechanical hypersensitivity without motor side effects. In this study, we demonstrated the spinal modulatory effect of agmatine and other NMDAr antagonists utilizing calcium imaging.

The aim of this study is to use a newly developed NMDAr‐mediated calcium transient assay (Figure 1.) in ex vivo mouse spinal cord slices and examine the effect of agmatine and other NMDAr antagonists in this assay. We hypothesize that agmatine and other NMDAr antagonists can dose‐dependently inhibit NMDAr‐mediated calcium transient in mouse spinal cord dorsal horn. Female and male ICR mice (4‐6 weeks) were perfused before spinal cord extraction and ex vivo spinal slices were incubated with the calcium indicator dye Fluo‐4. Intracellular Ca2+ was visualized by single plane two‐photon microscopy. Time‐lapse of images were acquired and the peak amplitude of fluorescence intensity was analyzed by Student’s t‐test.

We found that NMDAr‐mediated calcium transients were elicited by an NMDA drug mixture containing NMDAr agonists (NMDA, glycine) and AMPA receptor antagonists (NBQX). 5mM agmatine incubation significantly attenuated the NMDAr‐mediated calcium transients compared to the control. Increasing agmatine concentration dose‐dependently attenuates NMDAr‐mediated calcium transients. Other NMDAr antagonists including AP5 and ifenprodil, showed dose‐dependent inhibition of NMDAr‐mediated calcium transients, supporting that the newly developed calcium imaging assay is NMDAr specific. These data suggest that agmatine attenuates NMDAr‐mediated calcium transients in the spinal cord dorsal horn, which is consistent with our previous research demonstrating that agmatine is an effective inhibitor of NMDAr in the spinal cord.
Objectification, Desensitization, and Personification: Medical Student Strategies for Dealing with Human Body in the Anatomy Lab
Authors: Azim Homaira M.
Published: May 2022

Introduction & Purpose

Cadaver labs play a significant role in supporting learners form a professional attitude towards human body. Most medical students have their first authoritative encounter with human body in a cadaver lab where they get a right to manipulate and cut through the body. Dealing with death and dissection of the body may simply be a unique learning experience for some but it can be an emotional roller coaster for others. A few student strategies for dealing with cadaver bodies (such as objectification of the body) are documented in the literature but a full range of such strategies is rarely investigated. This study sought to understand the range of strategies medical students use for dealing with cadaver bodies in the anatomy lab at one medical school.

Methods

Twenty semi‐structured interviews and four focus groups were conducted with a total of 44 students from all four years of undergraduate medical education at Indiana University School of Medicine, in 2019. All sessions were audio‐recorded and later transcribed verbatim. A qualitative thematic analysis was performed by developing an inductive coding scheme which resulted in emergence of salient themes from the data.

Results

The full spectrum of student strategies for dealing with cadaver bodies included: 1) complete, effortless objectification of cadaver’s body as a non‐living thing, 2) actively desensitizing oneself toward cadaver’s body as so to be able to work with it, and 3) paralyzing personification of the cadaver’s body that impedes learning from it let alone dissecting it. Falling within this spectrum were also the so‐called middle ground strategies that were chosen by students attempting to adopt a more balanced behavior between desensitization and personification but still found themselves alternating between those two positions at certain times.

Conclusion

Students employ certain strategies for handling human body in the cadaver lab. These strategies are based on their personal values and/or actively chosen to help them make the most out of their experience working with cadavers in the anatomy lab.

Significance/Implications

Learning to deal with cadaver bodies in a professional manner is an impactful part of medical students’ professional identity formation process. The way students handle those bodies is likely to impact their attitude toward their patients’ bodies in the future as a doctor. Understanding the full range of student strategies for handling bodies in cadaver lab is a critical step for anatomy lab educators to facilitate a healthy, professional, ethical, and at the same time humane relationship between students and their cadaver’s body. Future studies should dig deeper into each of the strategies identified here and explore the best ways to oversee them for an effective medical professional identity formation in medical students.
Sustained Exposure to the SGLT2 Inhibitor Ertugliflozin, But Not NHE‐1 Inhibition By Cariporide, Attenuates Adrenergic Stimulation of Cytosolic CA2+ Levels in Spontaneously Contracting Cardiac Myocytes
Authors: Alam Sifat M. , Gierden Kyle J. , Zhao Tengteng , Lu Sun S. , Dixit Mihir , Babicheva Aleksandra , Yuan Jason , Vallon Volker , Zemljic‐Harpf Alice E.
Published: May 2022

Introduction

Inhibitors of sodium‐glucose cotransporter 2 (SGLT2i) target renal SGLT2 to reduce reabsorption of glucose, facilitate urinary glucose excretion, and lower blood glucose levels in patients with Type II diabetes (T2DM). The drugs, however, also decrease mortality and heart failure (HF) hospitalizations in T2DM patients. Patients with HF exhibit sympathetic overdrive and cytosolic Ca2+ overload in cardiac myocytes. Because SGLT2i harbor cardioprotective effects, we hypothesize that the SGLT2i ertugliflozin (ERTU) will lower cytosolic Ca2+ concentration ([Ca2+]cyt) in metabolically active and contracting cardiac myocytes after adrenergic stimulation.

Methods

ERTU was reconstituted in DMSO based on manufacture’s recommendation. Cardiac myocytes were isolated from 0–2‐day old mice and plated on coated (0.2% v/v gelatin, 1 μg/ml fibronectin, and 1 μg/ml laminin) glass cover slips. After two days, spontaneous contractions were observed, and fetal bovine serum content reduced to limit fibroblast overgrowth. By changing the media daily, spontaneously contracting cardiac myocytes were treated for 72 hours with either 1 µM ERTU, 10 µM cariporide (CARI), ERTU and CARI, or vehicle (0.01% v/v DMSO). After 72 hours, cells were loaded with FURA‐2/AM for 1 hour, a steady baseline [Ca2+]cyt was first recorded, then cells were stimulated with phenylephrine (100 µM), and [Ca2+]cyt was recorded for additional 30 minutes.

Results

ERTU exposure for 72 hours did not alter baseline [Ca2+]cyt in beating cardiac myocytes when compared to vehicle treated controls but reduced the increase in [Ca2+]cyt after adrenergic stimulation with phenylephrine. In contrast, 72‐hour‐long NHE1 inhibition with CARI increased [Ca2+]cyt at baseline when compared to vehicle and ERTU, and further enhanced the increase in [Ca2+]cyt after phenylephrine stimulation. Interestingly, concomitant administration of ERTU and CARI for 72 hours reduced baseline [Ca2+]cyt and inhibited phenylephrine‐mediated increase in [Ca2+]cyt.

Conclusion

Because sustained exposure to the SGLT2i ERTU reduced, but the NHE‐1 inhibitor CARI increased [Ca2+]cyt after acute adrenergic stimulation of metabolically active cardiac myocytes, these data indicate for the first time that ERTU’s beneficial effect on [Ca2+]cyt homeostasis is distinct from NHE1 inhibition. Reduced [Ca2+]cyt levels after sympathetic stimulation may contribute to the cardioprotective effect of SGLT2i. Further research is needed on the molecular mechanisms behind ERTU’s influence on cardiac [Ca2+]cyt.
Chronic Stress Accelerates the Progression of Cerebrovascular Dysfunction with Alzheimer's Disease
Authors: Burrage Emily N. , Prabhu Saina , Frazier James , Simpkins James , Kelley Eric E. , Chantler Paul D.
Published: May 2022

Vascular contributions to cognitive impairment and dementia are a key pathology associated with Alzheimer’s disease (AD). Past work in our lab has shown that chronic stress induces cerebrovascular dysfunction due to a pro‐oxidative environment while epidemiological studies suggest a link between chronic stress and dementia. With this in mind, we tested the hypothesis that chronic stress accelerates the vascular pathology associated with AD potentially via the xanthine oxidase pathway.

Here, we utilized a triple transgenic mouse model of AD (3xTg AD) combined with our unpredicted chronic mild stress paradigm (UCMS) and a xanthine oxidase inhibitor, Febuxostat, to examine middle cerebral artery (MCA) function. Beginning at 4 months of age all mice underwent the UCMS paradigm for 8 weeks total while a subset of mice were given Febuxostat treated water (50mg/L). Mice were either euthanized immediately following the 8 weeks of UCMS (at 6 months‐of‐age) or euthanized 3 months post‐UCMS (at 9 months‐of‐age). Following euthanasia, the MCA’s were removed and positioned in a pressurized myobath. The MCA was exposed to increasing concentrations of acetylcholine (ACh; 10‐9M to 10‐4M), phenylephrine (PE; 10‐9M to 10‐4M), and sodium nitroprusside (SNP; 10‐9M to 10‐4M).

At 6 months‐of‐age, AD mice displayed impaired MCA dilation to ACh compared to WT control mice (8.2±1.4mm to 17.5±2.1mm, respectively; p<0.05). WT and AD mice that underwent UCMS displayed similar response to ACh compared to AD control mice (8.25±0.3mm and 7.125±1mm to 8.2±1.4mm, respectively). Treatment with Febuxostat prevented MCA dysfunction in AD control mice, WT UCMS mice, and AD UCMS mice (8.2±1.4mm vs 17.1±1mm, 8.25±0.3mm vs 18.25±1.1mm, 7.1±1mm vs 17.2±0.9mm, respectively; p<0.05). No differences were found between groups in the PE or SNP response. Interestingly, at 9 months‐of‐age, the majority of these trends persisted with the one exception being a compounded impairment in the AD UCMS group compared to both AD control or WT UCMS mice (3.5±1.2mm vs 8.5±1mm, and 9.3±2.2mm, respectively; p<0.05).

Overall, these data suggest that chronic stress does accelerate the cerebrovascular pathology associated with AD and potentially via a pro‐oxidative state driven by activation of the xanthine oxidase pathway.
Testing the Ability of Borate Transporter Orthologs to Complement Saccharomyces cerevisiae BOR1
Authors: Beltran Jean , McDonnell Alexis , Carr Natalie , Foster Brianna , Thurtle‐Schmidt Bryan
Published: May 2022

In S. cerevisiae, borate resistance is mediated by the borate transport protein Bor1p. In plants, the role of borate transport is more complicated given the organism’s need to prevent borate toxicity while maintaining high enough levels of borate as a micronutrient that supports growth. As a result of these complex needs, the borate transporter family has expanded in plants, with seven borate transporters in Arabidopsis thalianaand four borate transporters in Oryza sativa. In humans, the transporter SLC4A11 has been suggested to transport borate, but this claim remains controversial. To better understand the transport activities of purported borate transporters, we tested the ability of eleven plant transporters and one human transporter to complement a BOR1 deletion in S. cerevisiae. We show that AtBor4, AtBor5, Atbor7, and OsBor3, can each complement yeast BOR1, while the other seven plant transporters do not. Interestingly, mapping these results onto phylogenetic analyses revealing two known clades within the borate transporter family might shed light on their functional and evolutionary differences. Next, we tested whether the ability to complement in yeast could be explained by the identity of the protein’s two domains, the Core and the Gate, by creating chimeric constructs combining the Core and Gate domains of AtBor1 and AtBor4. Our data show that neither chimeric construct complements, and thus the ability to complement arises synergistically from elements present in both domains rather than those that lie within just one protein domain. Lastly, we show that human SLC4A11 does not support borate transport in yeast, supporting claims that its transported substrate is not borate.
Targeted Mutation of Lipid‐Binding Sites in ScBor1 Does Not Prevent Dimerization
Authors: Donoso Valeria , Phillips Haleena , Thurtle‐Schmidt Bryan
Published: May 2022

Many transporters and ion channels form higher‐order structural complexes, but the functional significance of such protein complexes often remains unclear. Additionally, many recent studies have implicated lipid‐binding as a key factor in the formation of multimeric membrane protein complexes. The SLC4 transporters carry bicarbonate across membranes, and are typified by Band 3, the most abundant membrane protein in red blood cells. Structures of multiple SLC4 transporters including Band 3 reveal homodimeric structures, but the role dimerization plays in their function remains unknown. In this study we investigate S. cerevisiaeBor1, a borate transporter homologous to human SLC4 transporters. ScBor1 is predominantly a monomer when purified in detergent, but a recent study reveals a possible site for lipids to bind and support dimerization during lipid reconstitution experiments. To better understand the role of multimerization in SLC4 homolog function, we introduced Arg to Ala mutations in ScBor1 designed to abolish a presumed binding site for phospholipids. After protein purification and lipid reconstitution, we assessed multimerization through both oxidative and glutaraldehyde crosslinking experiments. Unlike previous studies, our data show that dimerization of the wild‐type protein is promoted by both phosphatidylcholine and phosphatidylglycerol, and that the mutated protein retains dimerization to the same extent as the wild‐type protein. Further studies will be required to establish the role that dimerization plays in function in the SLC4 family and its homologs.
Pharmacologic or Genetic PDE4 Inactivation Reduces Obesity and Improves Glucose Handling in Mice
Authors: Irelan Daniel , Boyd Abigail , Aragon Ileana , Fiedler Edward , Richter Wito
Published: May 2022

Purpose

Type 4 cyclic nucleotide phosphodiesterases (PDE4s) comprise a group of four isoenzymes (PDE4A to D) that hydrolyze the second messenger cAMP. Non/PAN‐selective PDE4 inhibitors exert potent anti‐inflammatory properties and are approved for the treatment of COPD and psoriasis, but are also associated with significant side effects, including nausea, emesis and weight loss. Here, we explored the idea that the weight loss associated with PAN‐PDE4 inhibitor use may be pursued as a desirable therapeutic outcome in the treatment of obesity‐related metabolic syndromes. As a first step, we tested whether treatment of mice with a PAN‐PDE4 inhibitor would replicate clinical data and reduce ageing‐ and/or high‐fat diet‐induced obesity in the animals. As each of the four PDE4 subtypes plays unique physiological roles, targeting individual PDE4s is a promising approach to improve the tolerability of PDE4 inhibitor therapy. To this end, we determined the metabolic phenotypes of individual PDE4 subtypes in mice.

Methods

Body and tissue weights, food and water consumption, glucose and insulin tolerance, serum insulin and glucagon levels, and locomotor activity and exercise capacity were assessed in aged mice, as well as in young mice fed a high‐fat diet. To delineate the role of PDE4s, the phenotypes of mice genetically deficient (KO) in each of the four individual PDE4 subtypes, PDE4A to PDE4D, were compared to their wildtype littermates, and mice treated with the PAN‐PDE4 inhibitor Roflumilast were compared to solvent controls.

Results

Treatment with the PAN‐PDE4 inhibitor Roflumilast reduced high‐fat diet‐induced obesity in mice, as reflected by reduced body weight and white fat pads, without lowering food consumption or increasing physical activity (unchanged locomotor activity and exercise capacity) suggesting that the weight‐loss effect of PDE4 inhibition observed in clinical trials is replicated in the mouse. Genetic ablation of PDE4B or PDE4D in mice replicated the effects of PAN‐PDE4 inhibition by reducing body‐ and adipose tissue weights in mice, whereas ablation of PDE4A or PDE4C had no effect. Reduced adiposity as a result of PDE4 inactivation was associated with improved glucose handling and insulin sensitivity, while serum levels of insulin and glucagon were unchanged. These data suggest that PDE4 inactivation does not act via pancreatic hormone release but acts in downstream target tissues to enhance glucose utilization.

Conclusions

Inactivation of PDE4s represents a promising approach to tackle obesity and associated metabolic abnormalities such as elevated blood glucose levels. Targeting PDE4B and/or PDE4D with subtype‐selective PDE4 inhibitors appears sufficient to mediate these therapeutic benefits and may be free of the adverse effects associated with the PAN‐PDE4 inhibitors available to date.