bluuebunny/crossref_metadata_2025 huggingface dataset:
Etomidate and Propofol Binding Sites on GABAA Receptors: Overlapping but not Identical?
Authors: Li Guo‐Dong , Olsen Richard W
Published: June 2021

γ‐Aminobutyric acid type A (GABAA) receptors, the major inhibitory neurotransmitter system in the mammalian brain, are an important target for general anesthetics including etomidate and propofol. Amino acid residues αM236 (TM1) and βM286 (TM3) were identified as directly contributing to a single anesthetic binding site with [3H]azietomidate photolabeling, and this binding site is probably located at the α‐β subunit interface in the transmembrane domain (Li et al., J. Neurosci. (2006) 26:11599). The validity and specificity of [3H]azietomidate photolabeling in GABAA receptors were also demonstrated in that paper, establishing a new approach to study binding sites for other anesthetics like propofol. Etomidate and propofol have similar anesthetic and pharmacologic actions, and they probably share a binding pocket in GABAA receptors according to reported electrophysiology data. Here, we studied the relationship of their sites in GABAA receptors using [3H]azietomidate photolabeling. We found that propofol produced a concentration‐dependent inhibition of [3H]azietomidate photolabeling. However, only half of the total labeling was inhibited at saturating propofol concentrations. This finding brings up an interesting idea: although propofol and etomidate may share a binding pocket in GABAA receptors, their exact binding sites may be partly different. Supported by NIH grant P01 GM 58448 to K.W. Miller.
Dominant FGF Autocrine Signaling in Gefitinib‐Resistant Non‐Small Cell Lung Cancer Cells
Authors: Heasley Lynn E , Marek Lindsay , Fritzsche Alexa , Helton Wallace R , Nemenoff Raphael A , McDermott Lee A , Helfrich Barbara , Bunn Paul A.
Published: June 2021

The frequent intrinsic resistance of non‐small cell lung cancers (NSCLC) to EGF receptor (EGFR)‐specific tyrosine kinase inhibitors (TKIs) such as gefitinib limits their effectiveness as therapeutics. We tested the hypothesis that autocrine growth signaling through one or more distinct receptor tyrosine kinases occurs in NSCLC cells. Gene expression array data indicated frequent co‐expression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. Notably, FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein were frequently co‐expressed. Specific silencing of FGF2 or expression of a dominant‐negative FGFR1 construct reduced proliferation and anchorage‐independent growth of H226 cells that secrete high levels of FGF2 and express both FGFR1 and FGFR2. Moreover, a TKI (RO4383596) that targets FGFRs inhibited basal ERK signaling as a measure of FGFR signaling as well as anchorage‐independent growth of NSCLC cell lines that co‐express FGF2 or FGF9 and FGFRs. By contrast, gefitinib‐sensitive NSCLC cell lines expressed little or no FGF2, FGF9, FGFR1 IIIc or FGFR2 IIIc. In addition, RO4383596 exerted no effect on basal ERK activity or anchorage‐independent growth of these cell lines. Thus, we propose that a growth factor autocrine loop involving FGF2, FGF9 and their respective high‐affinity FGFRs is dominant in gefitinib‐resistant NSCLC.
THE ROLE OF PPT1 DURING EMBRYONIC NEURAL DEVELOPMENT IN DROSOPHILA MELANOGASTER
Authors: Denefrio Cassandra , Chu‐LaGraff Quynh
Published: June 2021

Palmitoyl‐protein thioesterase 1 (PPT1) is a lysosomal enzymatic protein which acts in the depalmitoylation of cysteine modified proteins. Mutations in PPT1 results in the pediatric neurological disease called Infantile Ceroid Lipofuscinosis (INCL). To determine whether PPT1 is essential in neural development, we investigate the role of the homologous protein, Ppt1, in Drosophila melanogaster. Previously Ppt1 mutant flies were examined for potential neural defects using a number of neural and axon‐specific antibodies. Results indicate that Ppt1 mutant embryos exhibit misguided or absent axons, along with abnormal development of the central and peripheral nervous systems. It has been proposed that these phenotypes may be due to abnormal Ppt1 lysosomal function or perhaps, abnormal Ppt1 function outside the lysosome, such as in synaptic networks or extralysosomal substrate, relating to growth cone guidance during nervous system development. We will investigate whether Drosophila Ppt1 mutants display axon guidance and fasciculation defects by using three mutant fly strains: Df(1)446‐20, Ppt1A179T, and Ppt1S77F stained with three neuronal antibodies which specifically target axon proteins, ROBO, FasII, and 22C10.
IL‐1RI‐dependent Hematopoietic Stem Cell Proliferation in Support of Inflammatory Granulopoiesis
Authors: Ueda Yoshihiro , Cain Derek , Kondo Motonari , Kelsoe Garnett
Published: June 2021

Normally, steady‐state granulopoiesis generates adequate numbers of bone marrow (BM) and peripheral neutrophils. Infection and inflammation mobilize these neutrophil pools and trigger a distinct hematopoietic program of increased granulopoiesis necessary to support the reactive neutrophilia elicited by microbial infections. Whereas inflammatory granulopoiesis can be defined by its requirement for the C/EBPbeta transcription factor, the cellular cues that activate this special granulopoietic pathway are unknown. We demonstrate that adjuvant‐induced, reactive neutrophilias are the consequence of IL‐1R signals that induce hematopoietic stem cell (HSC) proliferation and preferential differentiation into granulocyte progenitors. We propose that the switch from steady‐state to inflammatory granulopoiesis occurs prior to granulocyte lineage commitment through IL‐1RI signaling.
The transcription of tryptase gene in human mast cells is regulated by mi transcription factor
Authors: Lee Sun‐Hee , Lee Ji‐Eun , Kim Bum‐Tae , Lee Young‐Mi , Moon Woo‐Sung , Kim Dae‐Ki
Published: June 2021

Mast cells play a central role in initiation and development of allergic diseases through release of various mediators. Tryptase may be a key mediator in mast cell‐mediated inflammatory reactions. In the present study, we investigated whether the transcription of tryptase gene in human mast cells was involved in mi transcription factor (MITF). Human mast cells express strongly MITF‐A, which is one of MITF isoforms. We observed that overexpression of MITF‐A elevates the production of tryptase in the immature human mast cell line HMC‐1, while the introduction of specific siRNA against MITF attenuated their expression. The level of granules in mast cells also was increased by overexpression of MITF‐A. Moreover, the transfection of MITF‐A into Jurkat cells (MITF‐negative) increased significantly the luciferase activity. Using mutant constructs of tryptase promoter, we identified that two E‐box motifs associate with MITF‐A to transactivate tryptase gene. An experiment by EMSA also showed that these E‐box are major motifs binding to MITF for tryptase transcription. These data suggest that MITF might play a role in regulating the transcription of tryptase gene in human mast cells.

Supported by the Korea Research Foundation Grant (KRF‐2006‐E00103).
Role of p38/ERK Pathway in IL‐12 Restoration of T Cell IL‐2/IFN‐γ Production Following Alcohol (EtOH) Intoxication and Injury
Authors: Li Xiaoling , Schwacha Martin G , Chaudry Irshad H , Choudhry Mashkoor A
Published: June 2021

The mechanism by which EtOH potentiates post burn pathogenesis remains unknown. We have shown that EtOH intoxication prior to burn injury potentiates the suppression of mesenteric lymph node (MLN) T cell IL‐2/IFN‐γ and enhances bacterial translocation. The suppression in T cell was accompanied with a decrease in p38 and ERK activation. Since we also found a decrease in IL‐12, this study examined whether IL‐12 restitution restores T cell IL‐2/IFN‐γ following EtOH and burn injury and whether p38/ERK pathway is involved in IL‐12 restoration of IL‐2/IFN‐γ. Male rats (250g) were gavaged with 5 ml of 20% EtOH 4h prior to ~12.5% total body surface area burn or sham injury. Rats were sacrificed on day one after injury. MLN T cells were isolated, stimulated with anti‐CD3 in the absence or presence of recombinant (r) IL‐12 (10ng/ml) for 5 min and lysed for p38 and ERK1/2 measurements. In some experiments, T cells were cultured for 48h with or without the inhibitors of p38 (10μM SB203580) or ERK (50μM PD98059). Results indicate that the incubation of T cells with rIL‐12 prevents the suppression of T cell IL‐2/IFN‐γ following EtOH and burn injury. Furthermore, IL‐12 normalizes both p38 and ERK activation in T cells, but results obtained using p38 and ERK inhibitors indicate that the restoration of ERK and not p38 is critical to IL‐12‐mediated restoration of T cell IL‐2/IFN‐γ production following EtOH and burn injury. (R01AA015731–01A2)
Role of C‐type lectins and activation receptors in the transmission of HIV‐1 from B cells to CD4+ T cells
Authors: Wasil Laura Rose , Rappocciolo Giovanna , Piazza Paolo , Rinaldo Charles
Published: June 2021

Objective: Based on our previous findings that DC‐SIGN‐expressing B cells could transmit HIV‐1 to T cells (PLoS Pathog. 2:e70, 2006), we determined the relative role of other B cell surface markers in this process.

Methods: B cells were activated with T‐dependent and T‐independent ligands CD40L, BAFF, CpG DNA, PHA, and LPS, and monitored for expression of C type lectins and activation markers by flow cytometry. Mabs specific for lectins or cell surface receptors were used to block transmission of HIV‐1 to T cells.

Results: B cells stimulated with CD40L, BAFF or LPS had enhanced expression of C type lectins and activation receptors, and transmitted HIV‐1 to T cells. In contrast, B cells stimulated with CpG DNA and PHA were activated but did not transmit HIV‐1 to T cells. Blocking DC‐SIGN with mAb prior to loading with HIV‐1 inhibited virus transmission to T cells.

Conclusions: B cells can be activated by both T‐dependent and T‐independent ligands to express C type lectins. This activation state determines their ability to transmit HIV‐1 to T cells, extending our previous results that DC‐SIGN is needed for B cell mediated transmission of HIV‐1 to T cells. (AI35041, CA82053, AI41870)
CD8 T cell responses to live, viral Yellow fever vaccine in humans
Authors: Akondy Rama , Miller Joseph , Monson Nathan , Wu Hong , Quyyumi Farah , Mulligan Mark , Teuwen Dirk , Altman John , Ahmed Rafi
Published: June 2021

Yellow Fever virus 17D (YFV‐17D) is a live attenuated vaccine that generates long‐lasting protective immunity. It provides an invaluable model to study CD8 T cell differentiation in a primary, acute viral infection in humans. Using a comprehensive peptide library spanning the viral polyprotein, we report an analysis of the YFV‐17D specific primary CD8 T cell response in vaccinated subjects. The largest proteins NS3, NS5 and Env accounted for the majority of the response. Identification of an immunodominant HLA‐0201 restricted CD8 T cell epitope enabled longitudinal phenotypic and functional analysis of antigen specific CD8 T cells in response to YFV‐17D. Effector CD8s seen as early as 11 days post vaccination till as late as 21 days upregulated proliferation (Ki‐67) and activation (HLA‐DR, CD38) markers and acquired cytolytic molecules like granzyme B. These markers were downregulated by day 30 after which a contraction of tetramer specific cells was seen. In short‐term in vitro stimulations of PBMCs with peptide, antigen experienced CD8 T cells were capable of producing cytokines like IFNγ, TNFα and IL‐2, chemokines like Mip‐1β and showed degranulation indicating that the vaccine elicits a polyfunctional CD8 response similar to vaccinia, another successful live viral vaccine. Evaluating the quality of the response may reveal the immune mechanisms responsible for an effective vaccine.
The effect of all‐trans retinoic acid (ATRA) and mesenchymal stem cells (MSCs) transplantation on increased expression of Foxp3 and IL‐10
Authors: Ju Sun‐Young , Cho Kyung‐Ah , Jung Yun‐Jae , Cho Su‐Jin , Ryu Kyung‐Ha , Seoh Ju‐Young , Woo So‐Youn
Published: June 2021

PURPOSE: It has been known that MSCs have the capability of regenerating organs, as well as modulating immune function. Recent studies suggest that the vitamin A metabolite, ATRA is capable of promoting anti‐inflammatory Treg cell differentiation. With this, we analyzed expressional changes of CD4+CD25+Foxp3+Tregs with mice injected MSCs and/or ATRA.

METHOD: MSCs (1×106) prepared from C57BL/6 mice bone marrow were adoptively transferred into syngenic mice via a tail vein. After one day, the MSCs injected mice were I.P. immunized with OVA (100 μg/mouse) and Alum (1 mg). After immunization, ATRA (10 mg/kg) was I.P. injected everyday. Two weeks after postimmunization, we obtained spleen cells and measured Treg cells related markers of CD4, CD25, Foxp3 and cytokine pofiles of IL‐10, IL‐4, IL‐12 and IFN‐r by flow cytometry analysis.

RESULTS: Compared to the group that did not undergo MSCs transplantation, the group that received transplantation showed to have a high expression of Foxp3 within the spleen cells, and more so with ATRA‐treatment. Also, expressions of IL‐4 and IL‐10 were higher in the group with that received transplantation and more so in the ATRA‐treated group.

CONCLUSION: MSCs and ATRA treatment might have immunomodulatory effet in immunized mice through the up‐regulation of Foxp3 and IL‐10 expression.
Modification of a food frequency questionnaire for use in African Americans in the rural South
Authors: Shikany James , Bovell‐Benjamin Adelia , Dawkins Norma , Hubbell Anne , Pace Ralphenia
Published: June 2021

Experimental and anecdotal data suggest that dietary practices of African Americans in the rural South differ from those of other groups and regions of the US. Existing instruments that seek to assess long‐term dietary intake, such as food frequency questionnaires (FFQs), may not be sensitive to the dietary practices of African Americans in the rural South. This study used focus groups in African‐American men and women in Macon County, AL, to generate information about dietary practices, food preferences, and food preparation methods in this population to modify an existing FFQ – the Diet History Questionnaire (DHQ) from the National Cancer Institute. Based on the completed focus groups, examples of foods to be added to the modified DHQ include pig ears, pork neck bones, pork skin, purple hull peas, fried okra, succotash, cornbread in buttermilk, and fried cornbread. Examples of foods to be deleted include bagels, English muffins, low‐fat processed meats, soy‐based foods, egg substitutes, and sorbets and ices. Following modification of the DHQ, we will assess the validity of the modified DHQ by comparing nutrient intakes calculated from the modified DHQ to those calculated from multiple 3‐day food records and 24‐hour dietary recalls. We hypothesize that the modified FFQ created in this study will better reflect true dietary intake in this population, facilitating future epidemiologic studies of diet and disease risk.

(Supported by NIH grant 2 U54 CA118948‐03)
Development of Ethanol‐induced Chronic Gastric Ulcer Model and the Effect of Protein Sources and their Hydrolysates on the Model of adult rats
Authors: Lee Sun‐Hye , Mo Jung‐Min , Park Mi‐Na , Lee Yeon‐Sook
Published: June 2021

This study was aimed to establish chronic gastric ulcer model and compare the healing effects of protein sources and their hydrolysates using Sprague‐Dawley(SD) adult rats. We administrated 50 to 80% ethanol for inducing acute ulcer and then 10 to 20% ethanol to maintain gastric ulcer for 3 to 11 days. From the result, chronic gastric ulcer model was developed with oral administration of 70% ethanol followed by 15% ethanol solution(15 ml EtOH/dl D.W.) for 5 days; apparent gastric lesions were not observed after 7 days. With this model, the rats were divided into 5 groups and fed with AIN 93M diet containing various protein sources; 20% of casein, whey protein (WP), whey protein hydrolysate(WPH), gluten protein(GP), and gluten protein hydrolysate(GPH), respectively, for 5 days. Weight gains were significantly low and the severe lesions in gastric tissue were observed in GP and GPH groups. However, there was no significant difference in gastric acid(pH) and glutathione level as an antioxidant among groups. The results indicate that chronic gastric ulcer can be maintained with continuous provision of 15% ethanol for 5 days after the oral feeding of 70% ethanol for once. Whey protein may have positive impacts on ethanol‐induced chronic gastric ulcer while gluten may not. Besides, protein hydrolysates are more desirable for healing chronic gastric ulcer than intact proteins. The details of biochemical data will be discussed.
Conditional cash and in‐kind transfers improve the quality of the household diet in rural Mexico
Authors: Leroy Jef L , Gadsden Paola , Gutiérrez Juan Pablo , Rodríguez Sonia , González‐Cossío Teresa
Published: June 2021

The objective of this study was to evaluate the differential impact of conditional cash versus in‐kind transfers on the monetary value (MV) and nutrient composition (NC) of household food consumption. Transfers were part of a government program. The study was conducted in poor rural communities in Southern Mexico.

A total of 208 communities were randomly allocated to one of four groups: a monthly food basked (FB) with or without nutrition and health education, a cash transfer equivalent to the cost of the FB (about 13 USD/month) with education or the control group. In each community 33 households were randomly selected to be surveyed at baseline. The follow‐up survey was conducted in the same households 2 years later. A double difference model with household fixed effects was used to estimate program impact.

The program significantly improved the household diet (P<0.05). The largest impacts were found in the MV of animal source foods, fruits and vegetables and in consumption of micronutrients (Zn, vitamins C and A). There were no differences between intervention groups in MV but the impact on NC was significantly larger in the FB group (P<0.05), due to the consumption of a micronutrient fortified milk. The results show that household dietary quality can be effectively improved through a conditional transfer program. Studies of household food consumption data benefit from analyzing both the MV and NC.
Effect of obesity and physical activity on D‐dimer – A Randomized controlled study in Adolescents
Authors: Balagopal Prabhakaran , Mann Karl J , Sweeten Shawn , George Donald
Published: June 2021

We studied the effects of obesity and physical activity on D‐dimer (DD), a marker of increased turnover of intravascular cross‐linked fibrin, in children and its relationship with fibrinolytic factors, fibrinogen & fibrinogen fractional synthetic rate (FSR) in 21 children (15 obese and 6 lean;Tanner stage, IV–V). Obese subjects underwent a 3‐month randomized controlled physical activity‐based intervention. New data show that the higher DD levels in obese vs. lean controls (112.3±11.8 vs. 63.7±12.9 ng/mL; p=0.02) were reduced after the intervention (p<0.01). We previously reported that fibrinogen levels and FSR decreased (p<0.01), while fibrinolytic factors were unaffected by the intervention. Parallel reductions in DD and fibrinogen levels and the FSR of fibrinogen after intervention indicated a putative decrease in the production and availability of fibrinogen for fibrin formation, thus ‘indirectly’ lowering DD, the primary degradation product of fibrin. The lack of change in fibrinolysis reflects a lack of degradation of preexistent fibrin, but may also argue against generation of new fibrin from fibrinogen representing a potentially beneficial change in coagulation function. Therefore, it appears that despite depressed fibrinolysis, the balance between coagulation and fibrinolysis was maintained after intervention suggesting a state of overall lower atherogenesity.
Dairy Modulation of Oxidative and Inflammatory Stress in Overweight and Obese Subjects
Authors: Zemel Michael B , Sun Xiaocun , Wilson Beth
Published: June 2021

We recently found calcitriol to increase both oxidative and inflammatory stress; moreover, inhibition of calcitriol with high Ca diets decreased both adipose tissue and systemic oxidative and inflammatory stress in obese mice, while dairy exerted a greater effect. However, these findings may be confounded by concomitant changes in adiposity. Consequently, we have now evaluated the acute effects of a dairy‐rich diet on oxidative and inflammatory stress in overweight and obese subjects in the absence of adiposity changes. 20 subjects (10 obese, 10 overweight) participated in a blinded randomized crossover study of dairy vs. soy supplemented eucaloric diets. The dairy supplement resulted in significant suppression of oxidative stress (plasma MDA, 22%; 8 isoprostane F2α, 12%, p<0.0005) and reduced inflammatory markers (TNFα, 15%, p<0.002; IL6, 13%, p<0.01; MCP‐1, 10%, p<0.0006) and increased adiponectin (20%, p<0.002), while the soy exerted no significant effect. These effects were evident by day 7 of treatment and increased in magnitude at the end of the 28‐day treatment periods. Greater effects were noted in obese vs. overweight subjects for oxidative, but not inflammatory, markers. These data demonstrate that high Ca dairy foods produce significant and substantial suppression of the oxidative and inflammatory stress associated with overweight and obesity. Supported by the National Dairy Council.
Dermytol, A Novel Compound For The Prevention Of Melanoma Skin Cancer Tested For Efficacy In Mice
Authors: Evans Malkanthi , Lloyd Jonathan , Guthrie Najla
Published: June 2021

A novel proprietary compound, trade named Dermytol™ has been shown to reduce melanoma tumor cell growth in male mice. Six Male C57BL mice (3–4 weeks old) were treated by oral gavage with 6.0% Dermytol™ w/v 0.2ml Canola oil. Mice in a control group were administered Canola oil. In a second treatment group of 6 male C57BL mice, a topical application of 6.0% Dermytol™ w/v in a cream base was applied to a shaved 1cm2 area of the right hind flank. Control group mice for this treatment received an application of a placebo cream base. Both treatments were applied for 7 consecutive days followed by subcutaneous injection in the right hind flank of B16‐F1 malignant melanoma tumor cells (2.0x105) in 100μl saline. Oral and topical Dermytol™ treatments were continued for 25 days after tumor cell injection. Results showed that orally treated mice showed an average decrease in tumor volume of 44.5%. Tumor volume was decreased by 61.2% in topically treated mice. This pronounced decrease in tumor volume for both applications indicates that Dermytol™ is effective at inhibiting the proliferation of B16‐F1 melanoma tumor cells and that a topical application of the compound produces a more potent effect.

Source: KGK Synergize, Inc.
Rabdosia rubescens inhibition of emergence of androgen‐independent prostate cancer
Authors: Seeram Navindra P. , Zhang Yanjun , Moro Aune , Lee Ru‐Po , Rettig Matthew , Heber David
Published: June 2021

Isobolographic analysis of the antiproliferative actions on LNCaP cells in vitro of a mixture of five TCM botanicals and Saw palmetto (Adams et al, 2006) demonstrated that Rabdosia rubescens (RR; Chinese: dongling cao) was the most potent botanical. The major active phytochemical in RR is oridonin (a kaurene diterpene). We compared the tumor inhibitory effects on LAPC‐4 xenografts in SCID mice in the intact and castrate conditions of RR extract (RRE) compared to pure oridonin at equivalent concentrations of oridonin. In intact mice, RRE was more potent than purified oridonin at the equivalent concentration found in RRE and was biologically equivalent to the 5X concentrated oridonin. In the castrate studies, the effects of RRE in reducing tumor growth were greater than the 5X concentration of oridonin. The maximum concentrations in the blood were reached two hours after oral gavage for both RRE and oridonin at equivalent doses were the same. These results clearly demonstrate that the greater potency of RRE by comparsion to oridonin is not due to increased bioavailabilty but presumably due to constituents other than oridonin in RRE. Studies to explore differences in mechanism of action of oridonin and RRE are underway.
Inhibition of cholesterol esterification in the control of leukemia proliferation in vitro
Authors: Gasperi‐Campani Anna , Batetta Barbara , Baiocchi Daniela , Putzolu Marirosa , Roncuzzi Laura , Dessi Sandra
Published: June 2021

On the basis of recent evidence indicating a plausible link between cholesterol esterification, MDR1 expression and rate of growth in different models of human proliferative diseases, the aim of this study was to investigate the association of MDR1 and cholesterol esterification with the growth in leukemia cells. Lymphoid leukemic cells isolated from patients with acute or chronic lymphocytic leukemia and CEM cells were treated with two inhibitors of cholesterol esterification: SaH 58‐035 and progesterone. Results showed a 60% average reduction of proliferation rate in leukemia cells treated with each cholesterol esterification inhibitor. This effect was not caused by cell toxicity, as cell morphology and viability were unaffected. In addition, it appears that changes in the cholesterol biosynthetic pathway are not responsible for the antiproliferative effect observed, being cholesterol synthesis unaltered. The inhibition of cell proliferation was associated with a reduction of ACAT and MDR1 mRNA expression and conversely with an up‐regulation of p‐53 protein expression. ACAT inhibitors, by reducing cellular cholesterol esters and MDR1 gene expression and through these pathways the proliferation rate of leukemia cells, may warrant consideration as innovative targeted anticancer agents.
Performance of Cancer/Testis Antigens and EEF1A2 mRNAs measured by qRT‐PCR as tissue biomarkers for NSCLC
Authors: Franklin Wilbur A , Haney Jerry , Sugita Michio , Mitchell John D , Weyant Michael J , Bemis Lynne , Gu Jesse , Feng Zideng
Published: June 2021

Candidate genes were cloned into TOPO TA plasmids (Invitrogen) to serve as standards in a qRT‐PCR assay in which copy numbers for the candidate biomarker genes were normalized to 106 copies of β‐actin. A panel of biomarkers was selected to directly test sensitivity and specificity in frozen tissue samples from 103 NSCLC, 60 matched non‐malignant tissue samples from the same resected lung specimens and 18 normal lung tissue samples from never smokers. Mean copy numbers/106 copies β‐actin are shown below.

Performance of individual markers and combinations of markers was analyzed by ROC. Maximum AUC for an individual marker was obtained for MAGE A (0.712 tumor vs adjacent lung and 0.784 tumor vs never smoker lung). AUC for the combined five gene panel was 0.885 for tumor versus adjacent lung, 0.957 for tumor vs never smoker lung and 0.945 for adjacent lung vs never smoker lung. A logistic model using percentiles had an AUC of 0.814 for tumor vs adjacent lung. We conclude that rigorous marker by marker testing in tumor tissue may yield candidate RNA biomarkers that can be assembled into a highly sensitive and specific panel.
Type‐specific reproducibility of the Roche Linear Array HPV Genotyping Test
Authors: Steinau Martin , Swan David C , Unger Elizabeth R
Published: June 2021

Type‐specific detection of Human Papillomavirus (HPV) is increasingly important. The impact of sampling, extraction and assay characteristics on HPV results is increasingly recognized. Inter‐assay comparability studies have been performed, but the robustness of type‐specific results has not been emphasized, nor has the degree of intra‐assay reproducibility been addressed.

The objective was to assess the general and type specific reproducibility of the Linear Array HPV Genotyping Test (Roche Molecular Diagnostics, Alameda, CA). Extracts of 276 cervical samples from two ongoing epidemiologic HPV studies were retested while blinded to prior results. The testing involved 5 different reagent lots and three technologists. Concordance for HPV detection (sample positive versus negative for any of the 37 types) was high (98.2%, kappa = 0.959). Type‐specific concordance for individual HPV types was also high (99.4%, kappa = 0.915), and most samples (83.0%) showed complete concordance for all types. Reliability of this HPV test is important as type‐specific assays are considered for clinical management and post‐vaccination surveillance.
Salmonella evades host innate immunity via AvrA mediated inhibition of cytokine production and pro‐apoptotic pathways
Authors: Wu Huixia , Jones Rheinallt , Luo Liping , Neish Andrew S
Published: June 2021

The enteric pathogen Salmonella typhimurium suppresses host innate immune responses via secretion of preformed effector proteins into host cells during invasion. One of these proteins, AvrA, has potent immunosuppressive and anti‐apoptotic effects in mammalian cultured cells and in a Drosophila animal model via inhibition of the JNK pathway. To study the role of AvrA in a murine model of natural infection, wild type Salmonella harboring AvrA or an isogenic AvrA null mutant Salmonella were orally administered to mice pretreated with streptomycin and infectious phenotypes compared. Infection with the AvrA null Salmonella caused increased levels of apoptosis in the cecal epithelium and in lymphoid resident monocytic cells at 6h post infection. Additionally, the AvrA null Salmonella induced increased serum cytokine levels, including KC, IL‐1beta and TNFalpha. Furthermore, AvrA null Salmonella caused increased hepatosplenomegaly by 7 days post infection. This coincided with increased bacterial load in the liver, spleen and mesenteric lymph node, and more severe weight loss. We conclude that Salmonella evades host innate immunity and gains systemic access/dissemination via AvrA mediated inhibition of cytokine production and apoptosis. Research funding provided by NIH AI 64462.
Drug effects on degraded on non‐degraded performance on the 5‐choice serial‐reaction‐time task (5‐CSRTT) in rats
Authors: Koffarnus Mikhail N. , Katz Jonathan L.
Published: June 2021

The 5‐CSRTT is commonly used as a pre‐clinical model of attention. While some drugs improve performance on this task, effects are generally small and statistically unreliable. In the current study, we degraded performance by increasing the number of correct responses required for reinforcement from 1 (FR 1) to FR 3 or FR 10. Compared to FR 1, correct responses (%) were lower under FR 10 and higher under FR 3, and response latency was slightly greater in the FR 10 group. Under FR 3 and FR 10 there was less responding during trials than under FR 1. None of the drugs tested significantly increased percent correct under FR 1, but dose‐dependent increases were observed in the FR 3 and/or FR 10 groups with d‐amphetamine (0.56–1.7 mg/kg), methylphenidate (3.0 mg/kg), and nicotine (1.0 mg/kg) at doses that did not significantly increase omissions. With d‐amphetamine and methylphenidate, improved performance was obtained at doses that did not alter response latency. Inactive drugs included atomoxetine (0.3–10.0 mg/kg), morphine (0.1–10), pentobarbital (1.0–17), desipramine (0.3–10), nortriptyline (0.3–10), clonidine (0.001–0.03), and selegiline (0.3–30). The data suggest that added response requirements may render the 5‐CSRTT more sensitive to some drugs that are in clinical use for attention deficit disorder. Supported by the NIDA IRP.
Impact of Gαz on axon growth and synaptic transmission
Authors: Hultman1 Rainbo C , Casey Patrick J
Published: June 2021

Heterotrimeric G proteins such as Gz play essential roles in cell biology. Gz is differentially expressed in certain brain regions and neuroendocrine cells, and has been shown to couple to endogenous D2 dopamine, 2A adrenergic, and E prostanoid receptors. Furthermore, Gz‐null mice are insensitive to catecholaminergic uptake inhibitor antidepressants. In order to further understand the neurobiology of Gz, its mechanism of action in neurons was characterized. Differential centrifugation was used to separate synaptic membrane components to characterize their Gαz levels. Interestingly, while some Gz was found at the pre‐synaptic plasma membrane, a larger pool of Gz co‐localized with synaptic vesicles. Regulation of synaptic vesicle loading, docking, or release could account for Gz‐null mouse phenotypes, and suggest Gz as a possible target for treatment of monoamine‐related disorders such as schizophrenia, ADHD, and depression.

Activated Gz has also been shown to interact with Rap1GAP, a GTPase activating protein for the monomeric G‐protein Rap1. Brain‐derived neurotrophin factor (BDNF), an important regulator of normal neuron growth, signals through Rap1. We show that BDNF‐stimulated axon growth in cortical neurons is inhibited by constitutively active Gαz, suggesting Gz as an intriguing target for further study.

This work was supported by Grant GM55717 from the National Institutes of Health
(2R,3R)‐2‐(3′,4′‐dihydroxybenzyl)‐3‐(3″,4″‐dimethoxybenzyl)butyrolactone(PP‐6) suppresses fMLP‐induced superoxide production by inhibiting fMLP‐receptor binding in human neutrophils
Authors: Liao Chang‐Hui , Chen Ih‐Sheng , Huang Yi‐Jia , Tseng Ching‐Ping , Day Yuan‐Ji , Lin Yin‐Chou
Published: June 2021

This study investigated the mechanism underlying the inhibiting effect of PP‐6, a lignan from Piper philippinum, on superoxide anion production induced by the chemotactic peptide fMLP in human neutrophils. Human neutrophils were stimulated with fMLP (1 μM), PMA (100 nM) or LTB4 (1 μM) and induced superoxide anion release. PP‐6 inhibited fMLP‐induced superoxide anion production in a concentration‐dependent manner with an IC50 value of 0.3±0.1 μM. Intracellular signaling caused by fMLP, PMA or LTB4 were evaluated. PP‐6 inhibited fMLP‐induced intracellular calcium mobilization and ERK, Akt and p38 phosphorylation. Moreover, PP‐6 inhibited fMLP‐induced Mac‐1 expression without affecting this caused by LTB4 or PMA. PP‐6 did not increase cAMP level in human neutrophils. PP‐6 did not inhibit superoxide anion production by NaF, the target of the inhibitory action of PP‐6 appears to be a component of the signal transduction pathway upstream of G‐protein. PP‐6 inhibited FITC‐fMLP binding to neutrophils in a concentration‐dependent manner with an IC50 of 1.5±0.2 μM. PP‐6 did not bring a parallel shift in the concentration response of fMLP‐induced superoxide anion. Additionally, the inhibiting effect of PP‐6 on fMLP induced superoxide anion was reversed when PP‐6 was washed out. These experimental results suggest that PP‐6 exerts non‐competitive and reversible antagonistic effect on fMLP receptor.
Biomechanical Stress in VSM Cells Leads to Vascular Remodeling and Hypertension in Profilin 1 Transgenic Mouse Model
Authors: Hessein‐Hassona Mohamed Darwish , Abouelnaga Zeinb A. , Alhaj Mazin A. , Wani Altaf , Moustafa‐Bayoumi Moustafa , Hassanain Hamdy
Published: June 2021

Actin polymerization is dynamically regulated and is essential for vascular smooth muscle contraction. Profilin 1 promotes actin assembly from G‐actin into F‐actin. We overexpressed the cDNA of human profilin 1 in the blood vessels of transgenic mice. Our results showed significant increase in F/G‐actin ratio in VSMCs in profilin 1 mice (301 ± 4%) compared to controls (153 ± 5%; P<0.0001) and increase in the medial thickness (138 ±4 μm) compared to control (90 ±3 μm; P<0.05). Western blotting confirmed the activation of the hypertrophic signaling in the aortas of profilin 1 mice. Phospho‐ERK1/2 was significantly higher in profilin 1 than control (512.3% and 361.7%, P<0.05). Profilin 1 mice had significant increase in phospho‐JNK compared to control (371.4% and 346%, P<0.05). There was a significant increase in ROCK II kinase in aorta of profilin 1 mice compared to control (>400%, P<0.05). RT‐PCR analysis confirmed the activation of Rac 1, eNOS and significant changes in antioxidant signaling. Monitoring of blood pressure showed significant increase in systolic and mean arterial blood pressure of profilin 1 mice starting at age 6 month (~ 25 mmHg, P<0.05). Isometeric tension analysis in mesenteric arteries showed significant differences in response to vasoactive drugs. These results suggest the regulation of blood pressure is directly related to F/G actin ratio in VSMCs. Startup fund.
1‐Oleoyl‐2‐acetyl‐glycerol (OAG) dependent Ca2+ influx is inhibited by dominant negative TRPC6 in human microvascular endothelial cells
Authors: Hamdollah‐Zadeh Maryam Akram , Glass Cathrine Ann , Bates David Owen
Published: June 2021

Ca2+ signaling in endothelial cells plays a key role in maintaining the barrier function between blood vessel and tissues. The canonical transient receptor potential (TRPC) ion channel are considered important in Ca2+ signal generation, but their roles in human microvascular endothelial cells (HMVECs) remain elusive. We previously showed that VEGF, acting through DAG could increase cation entry into HMVECs through a store independent process consistent with TRPC6 activation. To assess the response of HMVECs expressing endogenous, overexpressed and dominant‐negative TRPC6 channels to OAG, a structural analog of DAG, VEGF and other agents, adenoviruses were generated that encoded the DN‐TRPC6 or WT TRPC6 cDNAs (courtesy of Wiloliam Cole, University of Calgary). Calcium concentration was assessed in 20μM Fura‐2AM‐loaded HMVEC. Fluorescence intensity of the dye at 340nm excitation to that at 380nm (Rnorm) was measured. OAG (200uM) induced an increase in Rnorm ‐ indicating increased calcium ‐ in control transduced and WT TRPC6, but the OAG mediated increase in calcium was blocked by the dominant negative isoform of TRPC6 adenovirus. (p<0.05, Student Newman Keuls Post‐hoc ANOVA). In HMVEC cells, natively expressed TRPC6s are involved in DAG mediated calcium entry. DN‐TRPC6 may block this calcium entry either through inhibition of TRPC6 homodimers, or by blocking functional TRPC heterodimers. The effect of VEGF on DN‐TRPC6 HMVECs will also be presented.
Vasopressin V2 receptor (V2R)‐mediated potentiation of platelet activating factor (PAF)‐induced increase in rat mesentery venular microvessel permeability
Authors: Lenz J. F. , Laporte R. , Schteingart C. D. , Rivière P. J.‐M. , Curry F. E.
Published: June 2021

We tested the hypothesis that V2R activation potentiates the increase in microvessel permeability induced by PAF, an inflammatory mediator involved in sepsis. Individual venular microvessels in rat mesentery were perfused with 10mg/ml albumin in Ringer to measure hydraulic conductivity (Lp) by the modified Landis technique. The vessels were exposed to PAF (10nM), which induced an Lp increase peaking in 5–10min followed by return toward baseline values. The same vessels were then exposed to the selective V2R agonist desmopressin (dDAVP) for 40min followed by a second exposure to PAF in the presence of dDAVP. Peak PAF‐induced Lp significantly increased at dDAVP concentrations of 30pM (2.1 fold ±0.4 SEM, n=8) and 100pM (2.8 fold ±0.9 SEM, n=5) relative to peak Lp with PAF alone. In contrast, 10pM dDAVP did not increase PAF‐induced peak Lp. In control experiments, a second exposure to PAF alone did not cause an increase in peak Lp relative to the first PAF exposure. Also, dDAVP alone had no effect on baseline Lp. In further experiments, the V2R antagonist AdAc‐D‐Tyr(Et)‐Phe‐Val‐Asn‐Abu‐Pro‐Arg‐Arg‐NH2 (86nM) abolished potentiation by dDAVP (30pM) of PAF‐induced peak Lp (n=6). These results show that V2R activation potentiates PAF‐induced increase in vascular permeability, suggesting that V2R activation may exacerbate vascular leak syndrome in conditions such as sepsis. Supported by Ferring Research Institute Inc.
Epithelial‐Mesenchymal Transition Is Not Critical In The Pathogenesis of The Chronic Lung Disease of The Premature Infant
Authors: Rehan Virender , Torday John
Published: June 2021

Although Epithelial‐Mesenchymal Transition (EMT) in response to lung injury has been recently reported in the adult lung, its role in the Chronic Lung Disease (CLD) of the premature infant is not known. We examined whether fetal rat alveolar type II cells (ATII) undergo EMT in response to cytokines that are known to induce EMT of the adult ATII. Fetal rat (Sprague Dawley) lung ATII at embryonic day (e)19 were maintained in culture with and without TGFβ 1 (2.5 ng/ml), TNFα (1 ng/ml), and TGFβ 1 (2.5 ng/ml) + TNFα (1 ng/ml) for up to 10 days. EMT was determined by the expression of well established markers of the alveolar epithelial (Surfactant Protein C, CTP:cholinephosphate cytidylyltransferase α, and aquaporin‐5) and mesenchymal (α Smooth Muscle Actin and calponin) phenotypes on days 1, 6, and 10 at mRNA and protein levels along with the functional assessment of ATII function by their capacity to synthesize saturated phosphatidylcholine. There was no evidence of either spontaneous or cytokine‐stimulated EMT up to 10 days in culture in e19 rat ATII cells. Further, on cytokine stimulation, there was a dramatic increase in bone morphogenetic protein‐7 expression, a transcription factor that is known to block EMT. We conclude that premature ATII cells are resistant to EMT and unlike the pathogenesis of pulmonary fibrosis in the adult, EMT is not a prominent feature of CLD in the premature infant. Funding‐NIH HL75405 & HL55268).
Influence of Extracellular Matrix Proteins on Volume‐Sensitive Osmolyte Anion Channels (VSOACs) in C2C12 Cells
Authors: Neveux Iva , Doe Jinger , Leblanc Normand , Valencik Maria L
Published: June 2021

Browe & Baumgarten (J. Gen. Physiol. 122:689–702, 2003) demonstrated that mechanical stretch of β1 integrin receptors led to activation of a stretch‐sensitive outwardly rectifying Cl− current in ventricular myocytes. The purpose of this study was to determine whether extracellular matrix (ECM) composition modulated the VSOACs elicited by hypotonic medium (HYPO; 215 mosm) in skeletal muscle derived C2C12 cells. Cells grown on Laminin (Lm) or Fibronectin (Fn) were compared to cells grown in the absence of a defined ECM (NM). Cl− currents were recorded in whole‐cell voltage clamped cells. Basal membrane currents in isotonic media (300 mosm) were larger in cells grown on Lm (12X) or Fn (3X) vs NM. Membrane currents recorded in HYPO were larger on Lm (1.5X) or Fn (2.2X) vs NM. These currents displayed outward rectification, reversed at ECl and inactivated at potentials > +60 mV. Additionally, the tamoxifen‐sensitive VSOACs activated more quickly on Fn and Lm. Pre‐incubation of cells with RGD blocking peptide, prior to growth on Fn, nearly returned HYPO membrane currents to NM levels. Therefore, ECM composition profoundly influences the biophysical properties and mechanisms of onset of VSOACs.
Susceptibility to renal injury is increased in a rat model of intrauterine growth restriction.
Authors: Ojeda Norma Beatriz , Daniela Grigore , Royals Thomas P , Alexander Barbara T
Published: June 2021

Several studies report an association between intrauterine growth restriction (IUGR) and increased risk to develop disease. Our laboratory has developed a unique rat model of IUGR induced‐hypertension in which testosterone plays a critical role in blood pressure regulation. Whether IUGR is a factor that increases renal disease is unclear. Thus, to test the hypothesis that IUGR exhibit a greater susceptibility to renal injury following renal insult, rats were exposed to 1 week high salt diet (HSD) prior to 3 weeks reduced renal mass (RRM) by 5/6 nephrectomy. Intact IUGR RRM rats exhibited a significant increase in mean arterial pressure (MAP), proteinuria, and glomerulosclerosis (GS) compared with intact control RRM; CTX ameliorated these effects (Table). Thus, these results suggest that IUGR increases susceptibility to renal injury induced by high salt diet and a reduction in renal mass; and that testosterone plays an important role in mediating the enhanced susceptibility to a renal insult in male IUGR rats.
Human natriuretic hormones, xanthurenic acid 8‐O‐β‐D‐glucoside and xanthurenic acid 8‐O‐sulfate, new potent natriuretic and non‐kaliuretic tryptophan derivatives
Authors: Cain Christopher D. , Shankel Stewart W. , Schroeder Frank C. , Mitchnick Mark , Schmertzler Michael , Bricker Neal S.
Published: June 2021

For decades, researchers have searched for a non‐peptidic natriuretic hormone that acts as a definitive modulator of renal sodium excretion. We present herein the demonstration of natriuretic activity of two such recently identified and synthesized molecules isolated from human urine (PNAS Nov. 6, 2007 104: 17873–17878). These molecules are xanthurenic acid 8‐O‐β‐D‐glucoside (termed “NH”) and xanthurenic acid 8‐O‐sulfate (termed “NH‐1”). Substantial natriuresis occurred in rats following administration of synthesized NH and NH‐1 administered intravenously, intra‐arterially, and orally. With the first two routes, natriuresis began during or shortly after the infusion and persisted for one to two hours. Kaliuresis was small or non‐existent. With oral administration, natriuresis was delayed in onset but generally prolonged. When acetazolamide was infused along with the natriuretic hormones, natriuresis was enhanced, but the typical acetazolamide‐induced kaliuresis was blocked. We believe that these two natriuretic compounds may represent a new class of hormone and could have important therapeutic uses in the diagnosis and/or treatment of hypertension and fluid disorders. This research was funded by Naturon Pharmaceutical Corporation.
The effect of morphological variation on the sensitivity of lateral line neuromasts
Authors: Van Trump William Jackson , McHenry Matthew J.
Published: June 2021

Fish detect water flow with specialized structures of the lateral line system called neuromasts. Neuromasts are comprised of a cluster of hair cells that project kinocillia into a gelatinous cupula that is deflected by flow. The morphology of neuromasts dictate how fluid forces are transduced into mechanical stimuli by the hair cells. Analytical models predict that changes in morphology likely have a large effect on the mechanics of flow sensation. Therefore, we studied how differences in neuromast morphology in larval zebrafish (D. rerio) influence their sensitivity. This was achieved by morphometric analysis and mathematical modeling. In over 350 neuromasts that were imaged in 30 larvae, we recorded the dimensions of the cupula and kinocilia. We found that the shape and size of the neuromasts varied in ways that are predicted to greatly influence their sensitivity. Furthermore, this variation did not generally correlate with body position. One hypothesis for this high degree of variability is that observable neuromast morphology is the result of random wear and tear on the cupula rather than a consistently maintained form. One example of this is that total neuromast height may be constrained by the sloughing of cupular material from the end of the structure during free‐swimming activity. An investigation of pre‐hatch larvae supported this idea. It was found that the neuromasts of pre‐hatched individuals, which are insulated from wear, were longer than those of post‐hatch fish. Neuromasts in certain locations, such as those of the orbital canals, were consistently smaller than the body average suggesting that certain areas have developmentally ascribed differences in sensitivity but that in general the system must deal with a wide range of rapidly changing neuromast sensitivities.