bluuebunny/crossref_metadata_2025 huggingface dataset:
Laminin‐332 deficient mice present defects during tooth enamel development (LB547)
Authors: Wazen Rima , Moffatt Pierre , Senior Robert , Adair‐Kirk Tracy , Nanci Antonio
Published: June 2021

The epithelial ameloblasts are separated from the maturing enamel surface by an atypical basal lamina (BL) that is enriched in laminin (Lm)‐332. This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BLs and exerts effects on various epithelial cell processes including cell adhesion. Mouse models that lack expression of individual Lm‐332 chains die shortly after birth. However, recently a viable mouse model of Lm‐332 deficiency has been developed which expresses a doxycycline‐controllable human Lm γ2 transgene under the cytokeratin 14 promoter on the Lm γ2 knockout background (PLoS One 7(9):e45546). To investigate the role of Lm‐332 in enamel development, we examined the upper and lower maxillae from ~8‐week‐old wt and Lm‐332 deficient mice. At the light microscope level, incisors from Lm‐332 deficient mice showed severe structural changes at the level of the enamel organ. In decalcified samples, the organic matrix of forming enamel was altered and residual matrix was found throughout the maturation stage, suggesting Lm‐332 is required for both formation and mineralization of enamel. CT‐scans and scanning electron microscope analyses showed that Lm‐332 deficient mice exhibited hypomineralized enamel layer and occlusal surface wear in molars. The alterations in enamel formation observed in this mouse model are similar to those in teeth of patient with junctional epidermolysis bullosa due to Lm‐332 mutations. These mice will be an invaluable tool to study the role of Lm‐332 in enamel formation and better understand the role of the atypical BL found at cell‐enamel interfaces.

Grant Funding Source: Supported by CIHR, NIH/NHLBI P01HL029594, Shriners of North America
Centrosomal proteins are reorganized in fused trophoblastic cells. (LB57)
Authors: Omata Waka , Gonzalez Isabel M. , Ackerman William E. , Vandre Dale D. , Robinson John M.
Published: June 2021

Cell‐cell fusion is a cardinal event in the formation of multinucleate syncytia and is part of the normal biology of the syncytiotrophoblast (STB) layer of the human placenta. We have found numerous differences between non‐fused and fused trophoblastic cells in culture.

We have now compared centrosomal proteins and microtubules in non‐fused and fused BeWo cells. In non‐fused BeWo cells, the centrosomal proteins, pericentrin and gamma‐tubulin, were present as one or two perinuclear dot‐like structures. Following forskolin‐induced cell fusion, the immunofluorescence labeling pattern for pericentrin and gamma‐tubulin was dramatically altered and appeared to increase in amount and had a “cloud‐like” distribution. Microtubule nucleation experiments in which microtubules regrow following depolymerization with nocodazole were conducted. In non‐fused cells, microtubules regrow from the single microtubule organization center (MTOC). However, in fused cells there were multiple sites of regrowth corresponding to pericentrin and gamma‐tubulin positive‐foci. Examination of human placental tissue revealed that centrosomal proteins in the STB were distributed in a manner similar to that found in fused BeWo cells while mononuclear cells within the placenta have MTOCs similar to those in non‐fused BeWo cells. Thus the cell culture model recapitulates the novel situation in the tissue.

Grant Funding Source: Supported by NIH RO1HD058084
Serotonin 5‐HT2 receptor activation prevents the development of allergic asthma: Physiology and functional selectivity (LB575)
Authors: Nichols Charles , Nau Felix , Saravia Jordy , Alhert Terry , Cormier Stephania
Published: June 2021

Activation of serotonin 5‐HT2A receptors has potent anti‐inflammatory effects in multiple tissues including the nd vasculature and gut. Much of these effects in whole body are through blockade of TNF‐α mediated inflammation. We have found that activation of serotonin 5‐HT2 receptors in the lung with the agonist (R)‐DOI completely blocks the development of allergic asthma in the mouse ovalbumin model. At doses of 0.01 mg/kg inhaled, airways hyperresponsiveness, lung inflammation, mucus overproduction, and eosinophilia are blocked. The molecular and cellular mechanisms underlying these effects are currently under investigation. Structure/activity studies indicate that only phenethylamine 5‐HT2 receptor agonists closely related in structure to (R)‐DOI (e.g. R‐DOM and R‐DOB) are effective in preventing the development of asthma in our model. Phenethylamine agonists more dissimilar in structure are less effective, and indoleamine agonists like lysergic acid diethylamide are ineffective. Our results indicate that phenethylamines similar to (R)‐DOI potentially represent a novel small molecule therapeutic class for the treatment of asthma as well other inflammatory diseases.

Grant Funding Source: Supported by NIHR21HL095961, the American Asthma Foundation, and the Heffter Research Institute
Bio‐distribution of superparagmetic iron oxide nanoparticles (SPION’s) in the pig (LB578)
Authors: Shortt Christine , Edge Deirdre , Teughels Stephanie , Roskin Eric , Verbiest Joeri , Gobbo Oliviero , Prina‐Mello Adriele , Volkov Yuri , Markos Farouk
Published: June 2021

Manufactured nanomaterials have been shown to be beneficial in an array of applications, including diagnosis and targeted abolition of tumour cells. The biocompatibility and bio‐distribution of these particles is immensely important for targeted drug delivery and imaging. Therefore the aim of this study was to investigate qualitative bio‐distribution of SPION’s in the pig.

Female landrace pigs (20‐25 kg; n=16) were sedated i.m. with ketamine (14 mg/kg) and xylazine (2.7 mg/kg) and maintained on a continuous infusion of sodium pentobarbital (induction 30 mg/kg; maintenance 6 mg/kg/h i.v.). Dimercaptosuccinic acid (DMSA) coated nanoparticles (0.5, or 2 mg/kg respectively) were injected i.v. Blood samples were collected for a 5 hour period and stored for determination of iron using the particle electron paramagnetic resonance (pEPR) technique. Organs were harvested post mortem for MRI (1.5T) and histological evaluation.

OD15 (2 mg/kg) has a half‐life of ~15 minutes in the circulation. Both doses of MF66 (0.5 and 2 mg/kg) were below detection. SPION’s accumulation was observed in the liver with MRI scans. Using Prussian blue staining, SPION’s were observed in the liver and spleen at higher doses and in the lungs at both doses. Our results suggest that further modifications may be required to enhance the biocompatibility of these particles in order to utilize them as diagnostic and therapeutic agents.

Grant Funding Source: Framework Programme 7
Gas chromatography‐mass spectrometry identification of anticancer phytochemicals in Aframomum danielli (LB579)
Authors: Rotimi Solomon , Bisi‐Adeniyi Tolulope , Adelani Isaacson , Adegbite Oluwatobi , Adedeji Oluwabunmi , Rotimi Oluwakemi
Published: June 2021

Aframomum danielli is one of the African spices used in folklore medicine for the management of several diseases. This study identified the phytochemical components present in the n‐hexane seed extract of the A. danielli by gas chromatography‐mass spectrometry (GC‐MS) analysis and also evaluated the anti‐cancer potential of the identified phytochemicals by performing molecular docking against human Vascular Endothelial Growth Factor (VEGF) using Molegro Virtual Docker. The GC‐MS analysis identified the presence of phytochemical components caryophyllene (RT: 18.479), humulene (RT: 19.189), 2‐butanone (RT: 22.976), benzenesulfonamide (RT: 31.651) and 2‐pyridine acetic acid (RT: 32.446). 2‐Butanone was the strongest binding ligand (‐65.744 kcal/mol) while caryophyllene was the weakest bind lingand (‐56.311 kcal/mol). These compounds showed relative strong docking to VEGF with docking energies comparable to an anticancer drug, bevacizumab (‐77.883 kcal/mol). This in silico molecular docking study has shown that these phytochemical components could be responsible for anti‐cancer properties of A. danielli.
Activation of CYP3A4‐mediated letrozole metabolism by ketoconazole (LB593)
Authors: Black Stephen , Thurman Beth , Harrelson John
Published: June 2021

The objective was to investigate the effect of ketoconazole, an imidazole‐containing inhibitor of CYP enzymes, on the metabolism of letrozole, a triazole‐containing inhibitor of CYP19. The inhibition of cytochrome P450 enzymes by drugs that contain nitrogen heterocycles, for example, imidazole‐ and triazole‐containing agents, is well documented and involves the ligation of the nitrogen heterocycle to the heme iron. Letrozole metabolism was evaluated in microsomal and recombinant systems by measuring the formation of 4,4'‐hydroxymethylene‐dibenzonitrile, the major metabolite. A 12.3% increase in the formation of 4,4'‐hydroxymethylene‐dibenzonitrile was observed in the presence of ketoconazole (1 µM), a well‐known and potent inhibitor of CYP3A4 and CYP51, at letrozole concentrations within therapeutic plasma concentrations (e.g., 0.5 µM). In microsomal incubations the effect increased with ketoconazole concentration (0.005 µM to 10 µM) up to 26% and was observed in three different sources of human liver microsomes (50 and 150 donor‐pools from BD Gentest and a 50‐donor pool from Celsis). The activation is mediated through CYP3A. Concentration‐dependent activation was also observed in experiments with recombinant CYP3A4 (14.6 ± 3.6 and 25.6 ± 4.0% activation with 0.1 and 1 µM ketoconazole, respectively). Azamulin (1 µM), the CYP3A‐specific inhibitor, eliminated the activation effect in microsomal and recombinant systems. Ketoconazole metabolism in these systems did not generate peaks that coeluted with 4,4'‐hydroxymethylene‐dibenzonitrile, as detected by HPLC‐fluorescence. The surprising results reported here indicate that the nitrogen heterocycle ketoconazole can act as a heterotropic activator of CYP3A4‐mediated letrozole metabolism, suggesting that drugs that contain nitrogen heterocycles can have effects that extend beyond the normally expected CYP inhibition.
All‐trans retinoic acid promotes fatty acid oxidation in human hepatoma (HepG2) cells (LB612)
Authors: Tripathy Sasmita , Arnold Samuel , Chapman John , Goodlett David , Isoherranen Nina
Published: June 2021

It has recently emerged that retinoic acid (RA) acts as a ligand for peroxisome proliferator activated receptor (PPAR), a master nuclear receptor of lipid metabolism, in addition to the retinoic acid receptor. The role of all‐trans‐RA (atRA) on regulating lipid metabolism is, however, not clearly understood. In the present study, we examined the effect of atRA on fatty acid oxidation (FAO) in human hepatoma (HepG2) cells. HepG2 cells were cultured in presence or absence of atRA for 24 or 72 hrs, after which, cells were harvested to isolate RNA/protein to check gene/protein expression. Proteomics analysis performed on isolated protein after 72hrs of incubation with atRA showed that atRA significantly increased expression of key enzymes involved in FAO pathway such as acyl CoA dehydrogenase, long chain fatty acyl CoA ligase 3 and 4, delta (3, 5)‐delta (2, 4)‐dienoyl‐CoA isomerase and mitochondrial ATP synthase subunit alpha. Additionally, treatment with atRA significantly increased the mRNA of PPARδ, PPARα and its co‐activator PGC1α, and PPARα/δ target genes: CPT1α, PDK4 and FGF19, which all have role in FAO. Collectively, these results indicate that atRA promotes FAO via PPARα/δ induction in a dose dependent manner. Thus, our findings improve our understanding of RA role in lipid metabolism and signaling in liver.

Grant Funding Source: Supported by NIH R01 GM081569 and GM081569‐S1
8’‐glycinil β‐carboline inhibits nitric oxide formation but not NF‐kB‐mediated inflammatory responses in macrophages (LB614)
Authors: Grodzki Ana , Gorin Fredric , Lein Pamela
Published: June 2021

Neuropathic pain arising secondarily from an acute nerve injury is frequently associated with a protracted inflammatory response. While multiple inflammatory molecules, signaling pathways and neurochemical alterations contribute to chronic neuropathic pain, an essential role for nitric oxide generated by peripheral macrophages and microglia in the dorsal root ganglia and spinal cord has been reported. Specifically, the non‐selective nitric oxide synthase (NOS) inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME) was reported to reduce thermal hyperalgesia in inflammatory pain models and selective blockers for constitutive NOS and inducible NOS have been shown to reduce inflammatory response and neuropathic pain. Here, as part of a screen for novel therapeutics for neuropathic pain, we studied the effects of a novel hydrophilic 8’‐glycinyl 6‐chloro beta carboline (8’‐Gly carb) on nitric oxide formation and NF‐kB activation in macrophages. Our data show that 8’‐Gly carb inhibits constitutive and inducible nitric oxide formation and interferes with NF‐kB‐mediated reporter. However, 8’‐Gly carb does not affect NF‐kB p65 subunit translocation to the nuclei or expression of pro‐inflammatory cytokines TNF and IL‐1β. Moreover, 8’‐Gly carb does not inhibit Erk 1/2 or P38 MAP kinase activity. Since 8’‐Gly carb significantly inhibits constitutive and inducible nitric oxide formation without interfering with downstream effects of NF‐kB, 8’‐Gly carb holds promise as an agent that might be used to reduce neuropathic pain in the face of chronic inflammation.

Grant Funding Source: Supported by NIH/NIEHS ES017592
Impact of fatty acids on endothelial cell Cx43 expression (LB653)
Authors: Zucker Noah , Kurjiaka David
Published: June 2021

Increases in the prevalence of obesity worldwide have prompted concerns of an upswing in atherosclerotic diseases. The dyslipidemia that characterizes obesity includes elevated circulating free fatty acids that contribute to atherosclerosis by compromising endothelial cell function. While there are many aspects of endothelial cell function, we focused on connexin 43 (Cx43) as hypertension and diabetes increase endothelial Cx43 expression. Since the epidemiological evidence argues for structure‐based differences in the atherosclerotic potential of fatty acids (cis protect vs trans promote), we evaluated the impact of fatty acid structure on endothelial cell Cx43 expression. First, we quantified endothelial cell (mouse bEnd.3: ATCC) responses to 4 fatty acids [saturated (steric) or unsaturated (oleic {cis}, linoleic {omega‐6}, and eladic acid {trans})]. After 24 hours at 0.3 to 300 μM, there was little change in endothelial cell Cx43 expression. However, treatment with 30 μM linoleic or oleic acid generated a biphasic response: Cx43 expression increased early (~3 hrs) and decreased later (~12 hrs). This time‐dependent response may reflect differences in the underlying mechanisms (prostaglandin synthesis vs receptor activation). Thus, preliminary data argue that endothelial Cx43 expression is altered by fatty acids although evidence for structure dependent difference is lacking.

Grant Funding Source: Supported by Student Summer Scholars at GVSU
The role of wall shear stress on cerebral aneurysm development via P2X4 ion channel, a sensor for shear stress mechanotransduction, in vascular endothelial cells (LB669)
Authors: Fukuda Miyuki , Fukuda Shunichi , Ando Joji , Hasegawa Koji
Published: June 2021

[Objective] Our study using an animal model of experimentally induced cerebral aneurysms suggests that perception of an excessive increase in wall shear stress (WSS) by endothelial cells initiates the arterial wall degeneration caused by expression of various enzymes, leading to aneurysm development. Our computational fluid dynamics analysis also demonstrated that the magnitude of WSS is enhanced at the site where human aneurysms develop. In this study, we examine the role of WSS on cerebral aneurysm formation, using a shear sensor, P2X4 purinoceptor deficient mice (P2X4 KO). [Methods] P2X4 KO (n = 19) and wild types (n = 21) were used for aneurysm‐inducing surgery as described before. Aneurysm as defined here refers to an outward bulging detected by light microscopy (more than 10 um). Damage to internal elastic lamina as early aneurysm changes was classified into 3 grades according to microscopic pathological changes. [Result] The grade of damage in P2X4 KO group was significantly lower than that in wild type group (p = 0.027). Moreover, the incidence of induced aneurysm formation in P2X4 KO group was significantly below that in wild type group (p = 0.018). [Conclusion] The data suggest that blockage of a shear‐sensor, P2X4 ion channels, causes the suppression of WSS mechanotransduction, leading to aneurysm formation.
A novel function of TIMP1/CD63 signaling in the regulation of tumor metabolism (LB68)
Authors: Jung Young‐Suk
Published: June 2021

A distinctive feature of cancer cells that sets them apart from normal non‐transformed cells is a unique metabolic profile. In the 1920s, Otto Warburg recognized that carcinogenesis was accompanied by a striking shift to glycolysis as the major energy‐generating pathway. Nearly a century later, it is still unclear if this “glycolytic switch” is an epiphenomenon or a mechanistic determinant for malignancy. During the course of studying tissue inhibitor of metalloproteinase (TIMP)‐1 signaling, we found that TIMP‐1 upregulates the rate of the aerobic glycolysis as evidenced by increased glucose uptake and lactate production. TIMP‐1 upregulated hexokinaseII RNA level, known to be the rate‐limiting step of glycolysis. Interestingly, TIMP‐1‐induced glycolysis was accompanied with reduced mitochondrial respiration as shown by reduced cytochrome c oxidase activity and a low rate of oxygen consumption. ShRNA‐mediated knockdown of the tetraspanin CD63, a cell surface binding partner of TIMP‐1, reversed the TIMP‐1‐mediated glycolytic switch. Interestingly, TIMP‐1 activation of the CD63 signaling complex resulted in constitutive activation of HIF1α and protected breast epithelial cells from hypoxia‐induced cell death. In contrast, TIMP‐1‐stimulated cells rapidly underwent cell death upon glucose withdrawal. Taken together, the present study demonstrates a novel function of TIMP‐1 in the regulation of tumor metabolism in a CD63‐dependent manner.
Leucine‐Rich Repeat Containing 10 (LRRC10) protein associates with and regulates the cardiac Cav1.2 L‐type calcium channels (LB693)
Authors: August Benjamin , Reynolds Courtney , Balijepalli Ravi
Published: June 2021

Leucine‐rich repeat containing 10 (LRRC10) is a cardiac‐specific protein expressed in embryonic and adult hearts and plays critical roles in cardiac development and function. Recently we have demonstrated that the Lrrc10‐null (Lrrc10‐/‐) mice develop dilated cardiomyopathy and ventricular myocytes from Lrrc10‐/‐ mice exhibit reduced L‐type Ca2+ channel (LTCC) current (ICa,L) density. However, the precise role of LRRC10 protein in the regulation of LTCC function in the cardiomyocytes is unknown. To investigate the regulatory role of LRRC10 on Cav1.2 channel function, we co‐expressed the Myc tagged LRRC10 (LRRC10‐Myc), heamagglutinin tagged Cav1.2 (Cav1.2‐HA) and auxiliary Cavβ2CN4 subunit in HEK293 cells and performed co‐immunoprecipitation (co‐IP) on lysates using either anti‐HA, anti‐Myc antibody or control IgG. Western blot analysis demonstrated that Cav1.2 and LRRC10 associated with one another. Also, the auxiliary Cavβ2C subunit and LRRC10 did not co‐IP with one another suggesting that the LRRC10 may directly interact with the Cav1.2 subunit. A single point mutation H150A or triple point mutations Y104A, W127A and H150A mutation in LRRC10 disrupted the LRRC10 protein association with Cav1.2. Co‐IP analysis using mouse ventricular homogenates demonstrated that LRRC10 and Cav1.2 subunit are associated with one another. Additionally, double immunogold labeling and electron microscopy analysis revealed that LRRC10 and Cav1.2 proteins are co‐localized at T‐tubules in the ventricular myocytes. Finally, whole‐cell patch clamp experiments performed in ventricular myocytes from Lrrc10‐/‐ mice demonstrated a significant reduction in the ICa,L density (‐2.5 0.2 pA/pF) and compared to WT myocytes (‐6 0.6 pA/pF). Furthermore, the inactivation of the ICa,L in Lrrc10‐/‐ myocytes was significantly delayed. We conclude that LRRC10 protein is a novel and essential regulator of the LTCC function in ventricular myocytes.

Grant Funding Source: R01HL105713
Altered cardiovascular and baroreflex response to air jet stress in dexamethasone programed rat (LB738)
Authors: Prestipino Louise , Dissanayake Hasthi , Polson Jaimie
Published: June 2021

Raised gestational glucocorticoid may program hypertension in the offspring, although the data are inconsistent. The pathophysiological mechanisms may be due to altered autonomic responses to stress. We compared the cardiovascular and autonomic responses to air jet stress in rats programed by maternal injection of dexamethasone (DEX, 0.2 mg/kg) at gestational days E15 and E16 with control rats. At 6 months, offspring were implanted with a telemetry blood pressure (BP) transmitter. BP, heart rate (HR), and spontaneous baroreflex gain (sBRG) were recorded at rest and during exposure to a series of puffs of air directed at the face. There was a trend towards a higher resting mean BP in DEX rats than controls (97.6±4.9 v 84.1±2.5 mmHg, n=6, P=0.06), although there was no difference in systolic BP (109.7±5.5 v 104.7±1.3 mmHg, P=0.45). This was accompanied by a lower heart rate (260±9 v 302±13 bpm, P<0.05) and sBRG (1.5±0.2 v 2.3±0.2 ms/mmHg, P<0.05) in DEX rats. Air jet stress increased BP and HR in both groups, however the BP increase was greater in DEX (initial increase 22.2±3.7 vs 8.3±3.9 mmHg, P<0.05). Stress reduced sBRG in control but not DEX rats (by 0.86±0.14 vs 0.01±0.3 ms/mmHg). Our results show that DEX programmed rats have altered cardiovascular and baroreflex responses to airjet stress.
Ketogenesis prevents diet‐induced fatty liver injury and hyperglycemia through coenzyme A recycling (LB769)
Authors: Cotter David , Ercal Baris , Leid Jamison , D'Avignon D. Andre , Graham Mark , Dietzen Dennis , Brunt Elizabeth , Crawford Peter
Published: June 2021

Nonalcoholic fatty liver disease (NAFLD) spectrum disorders affect approximately one billion individuals worldwide. The mechanisms driving progressive steatohepatitis remain incompletely defined. Here we use a murine model of mitochondrial 3‐hydroxymethylglutaryl CoA synthase (HMGCS2)‐deficiency (using anti‐sense oligonucleotides) to demonstrate a critical role for ketogenesis in prevention of diet‐induced steatohepatitis. Adult‐onset loss of HMGCS2 caused mild hyperglycemia and increased hepatic gluconeogenesis from pyruvate in chow‐fed mice. High‐fat diet (HFD) feeding of ketogenesis insufficient mice caused extensive hepatocyte injury and inflammation, decreased glycemia, deranged hepatic tricarboxylic acid (TCA) cycle intermediate concentrations, and impaired hepatic gluconeogenesis due to fatty acid‐induced sequestration of coenzyme A (CoASH). CoASH precursor supplementation (pantothenic acid and cysteine) normalized TCA intermediates and gluconeogenesis in ketogenesis insufficient livers. These findings identify ketogenesis as a critical regulator of hepatic glucose metabolism and TCA cycle function via coordination of CoASH homeostasis in the absorptive state and indicate that ketogenesis modulates fatty liver disease.

Grant Funding Source: HG00668702, DK091538, DK020579, DK056341, HL007873
AMP‐activated protein kinase (AMPK) α1 is required for brown adipogenesis (LB843)
Authors: Yang Qiyuan , Liang Xingwei , Fu Xing , Rogers Carl , Zhu Meijun , Du Min
Published: June 2021

Brown adipose tissue (BAT) burns fatty acids to generate heat, holding the promise to treat obesity and type 2 diabetes. However, mechanisms regulating brown adipocyte formation remain poorly defined. AMP‐activated protein kinase (AMPK) is a key regulator of energy metabolism. We hypothesized that AMPK regulates the development of brown fat, an important organ involved in energy catabolism. To test, we employed AMPKα1 floxed mice which were crossed with ER‐Cre mice to conditionally knockout AMPKα1. AMPKα1 knockout was induced by 4‐hydrotamoxifen. Abrogation of AMPKα1 blunted the brown adipogenic differentiation of stromal vascular cells isolated from brown fat. Correspondingly, the expression of UCP‐1 and Prdm16, two markers of brown adipogenesis, was pronouncedly suppressed during differentiation (around 4 fold and 3 fold reduction respectively, P = 0.03). Moreover, adipogenic progenitor cells were separated from brown stromal vascular cells by FACS using surface markers (sca1+/CD34+/CD45‐). Consistently, the absence of AMPKα1 blocked brown adipogenesis in purified progenitors. Taken together, these data suggest that the AMPKα1 is required for brown adipogenesis (The work was funded by NIH R01HD067449).

Grant Funding Source: Supported by NIH R01HD067449
Aldosterone transcriptionally regulates large conductance K+ channel β1 (BKβ1)‐ subunit specific mRNA abundance in rat distal colon (LB845)
Authors: Sreekumar Shruthi , Rajendran Vazhaikkurichi
Published: June 2021

Background: Large conductance K+ (BK) channels localized on the apical membranes mediate cAMP and aldosterone (“aldo”) activated K+ secretion in rat distal colon. BK channels composed of α (BKα) and β (BKβ) subunits. Four different BKβ (BKβ1, BKβ2, BKβ3 and BKβ4) isoforms have been shown expressed in tissue specific pattern. Recent studies have shown “aldo” transcriptionally up‐regulates BKα‐subunit. The identity of BKβ isoform and its regulation by “aldo” is not known in rat distal colon. Aim:This study was aimed: 1) to identify the BKβ isoform; and 2) to demonstrate whether BKβ subunit also transcriptionally regulated by “aldo” in rat distal colon. Methods: “Aldo” rats (Sprague‐Dawley, 200 ‐ 220 g) were produced by feeding Na‐freed diet ad libitum for 6 ‐ 7 days. Total RNA was purified from epithelial cells isolated form rat proximal and distal colon. BKβ isoform specific fragments were amplified using isoform specific primers and polymerase chain reaction (PCR). Quantitative RT‐PCR (RT‐qPCR) analyses were performed to quantify the expression level of BKβ.isoform in “aldo” rat distal colon. Results: PCR amplified BKβ1, but not BKβ2, BKβ3 and BKβ4 isoform specific fragments in both proximal and distal colon. RT‐qPCR analyses revealed that similar to BKα‐subunit, BKβ1‐subunit specific mRNA expression also enhanced by 3.5‐fold in “aldo” rat distal colon. The P‐values for BKβ1 subunit was less than 0.05 thus indicating a significant change at a 95% confidence. BKβ1‐subunit specific mRNA level is not altered in “aldo” proximal colon. Conclusions: Rat colonic BK channel consists of BKβ1 isoform; and 2) “Aldo” transcriptionally up regulates both BKα‐ and BKβ1‐subunits in distal, but not proximal colon.

Grant Funding Source: West Virgina University Intramural award
Brain‐derived neurotrophic factor causes chloride gradient collapse in supraoptic neurons inducing vasopressin‐mediated blood pressure increase (LB847)
Authors: Choe Katrina , Han Su , Gaub Perrine , Shell Brent , Knapp Blayne , Barker Phillip , Brown Colin , Voisin Daniel , Cunningham J. , Bourque Charles
Published: June 2021

Vasopressin (VP), an antidiuretic hormone, is also a potent vasoconstrictor and has been found at high levels in salt‐sensitive hypertensives. Hypertension normally reduces VP secretion by MNCs in the SON, by GABA‐dependent synaptic inhibition of their firing activities. However, in rats under chronic hypernatremia, GABAA receptor‐mediated synaptic inhibition is abolished in SON neurons due to a depolarization in chloride reversal potential (ECl), suggesting that continued VP secretion despite BP elevations may underlie the development of salt‐dependent hypertension. In order to test this hypothesis, we measured the mean arterial pressure (MAP) by radio telemetry in freely moving rats made chronically hypernatremic by 2% saline drinking. MAP of these rats rose steadily rise during the course of the 7‐day SL treatment (basal BP: 97.7±1.9 mmHg; day 7: 113.5±2.1 mmHg; n=10; P<0.05), however remained stable in euhydrated rats over the same time period (basal BP: 98.3±2.5 mmHg; day 7: 100.6±2.2 mmHg; n=6; P=0.50). Furthermore, the SL‐mediated increase in MAP was significantly reduced by continuous systemic infusion of a VP receptor type 1 (V1R) antagonist (αday 7; +8.8±2.5 mmHg; n=6) when compared to SL controls (α15.1±1.0 mmHg; n=10; P<0.05). These findings confirm our hypothesis that circulating VP contributes to increases in BP during chronic hypernatremia. Brain‐derived neurotrophic factor (BDNF) is a high‐affinity TrkB agonist previously shown to be responsible for chloride gradient collapse. In order to investigate this possibility in the SON, we induced a specific knockdown of this protein in SL rats in vivo by stereotaxic injections of adeno‐associated virus (AAV) containing BDNF‐shRNA into the SON. ECl measurements via Gramicidin perforated patch‐clamp revealed that this treatment significantly hyperpolarized the ECl of SON neurons (‐56.2±5.4 mV; n=10) when compared to values measured in SL rats injected with control AAV containing a scrambled sequence of shRNA (‐40.1±2.4 mV; n=7; p<0.05). These results strongly suggest that BDNF is the TrkB agonist responsible for producing the chloride gradient collapse in SON neurons under chronic hypernatremia, a phenomenon which potentially underlie the development of salt‐sensitive hypertension. Funding source: Canadian Institutes of Health Research.

Grant Funding Source: CIHR
Lubiprostone (a prostone) slows the in vitro growth of colon adenosarcoma cells with low [NAD+] 15‐OH PGDH (LB856)
Authors: Cuppoletti John , Malinowska Danuta , Chakrabarti Jayati , Tewari Kirti , Ueno Ryuji
Published: June 2021

Objective. Lubiprostone is a bicyclic fatty acid (prostone) which is used widely for treatment of chronic idiopathic constipation (CIC), opioid‐induced constipation (OIC) and constipation‐associated irritable bowel syndrome (IBS‐C). [NAD+]‐15‐OH PGDH (PGDH), which synthesizes endogenous prostones from prostaglandins, is a suppressor of human gastrointestinal cancers and genetic therapy with PGDH slowed the growth of colon tumor xenographs. The present study was to determine whether lubiprostone, acting as a prostone, would slow growth of colon cancer cells which express low PGDH and whether transfection of low PGDH colon cancer cells with PGDH would ablate the effects of lubiprostone on the cancer cells.

Methods. LS174T (colon adenosarcoma) cells that contain low PGDH were grown in Eagle’s MEM/10% FBS, transfected with human PGDH cDNA in pCMV6‐Entry vector or vector alone using Lipofectamine and selected using G418 (200 µg/ml). PGDH:GAPDH levels were determined by densitometry of western blots. LS174T cells, non‐transfected and transfected with hPGDH or vector alone (mock) were grown in 35mm petri dishes (0.5x104 cells/ dish) to 40% confluence and then treated with either 0.1% EtOH‐0.01% DMSO (vehicle) or 100 nM lubiprostone. Every 24 h media were changed and fresh vehicle or lubiprostone added. DMSO and lubiprostone were from Sucampo. Cell growth was monitored by directly counting the cells in a randomly chosen field using a cell cytometer at 0, 24, 48, 72 and 96 h. Similar studies were carried out with HT‐29 colon cancer cells.

Results. LS174 T cell growth was slowed or stopped by 100 nM lubiprostone. Transfection of LS174T cells with PGDH, but not mock transfection, prevented lubiprostone inhibition of cell growth. Similar results were found with H‐T29 colon cancer cells.

Conclusions. These results demonstrate that lubiprostone will slow or prevent the growth of colon cancer cells which contain low 15‐OH PGDH. These results provide a rationale for further study in clinical trials.
The cold never bothered me anyway: RiAFP (LB95)
Authors: Arp Allison , Edwards Kristina , Hamann Bailey , Lesher Jordanne , Parfitt Kim , Varga Krizstina
Published: June 2021

Antifreeze proteins (AFP) exist in a wide variety of cold adapted organisms including fishes, bacteria, and insects. This unusual molecule inhibits the growth of ice crystals by irreversibly adsorbing to the flat surface of existing ice particles with a complex mechanism. The protein warps the smooth surface of ice to prevent the addition of other water molecules to the crystal, allowing the organism to survive in subzero temperatures. Longhorn beetle (Rhagium inquisitor) AFP (RiAFP) is a hyperactive protein that binds to basal, as well as prism, ice surfaces. This multiple surface attachment results in a 10‐fold higher activity than AFP in fishes. RiAFP forms a compressed [[Unsupported Character ‐ Symbol Font β]]‐solenoid architecture consisting of two parallel 6‐ and 7‐stranded [[Unsupported Character ‐ Symbol Font β]]‐sheets. Two cysteines involved in a disulfide bridge link the [[Unsupported Character ‐ Symbol Font β]]‐sandwich fold. RiAFP has an exceptionally flat and large ice‐binding surface lined with a regular array of Thr residues that are spaced at the same distance as oxygen atoms in the ice surface, creating a surface complementarity to ice. AFP are of particular interest to researchers because of their many potential uses in cryopreservation, such as tissue storage and plant genetic engineering. The Central SMART Team (Students Modeling A Research Topic) modeled RiAFP using 3D printing technology.

Grant Funding Source: Supported by grants from the NIH‐SEPA and NIH‐CTSA.
Associations between menopause, cardiac remodeling, and diastolic function: the CARDIA study
Authors: Ying Wendy , Post Wendy S. , Michos Erin D. , Subramanya Vinita , Ndumele Chiadi E. , Ouyang Pamela , Ambale-Venkatesh Bharath , Doria De Vasconcellos Henrique , Nwabuo Chike C. , Schreiner Pamela J. , Lewis Cora E. , Reis Jared , Lloyd-Jones Donald , Sidney Stephen , Lima Joao A.C. , Vaidya Dhananjay
Published: June 2021

Objectives:

Heart failure with preserved ejection fraction (HFpEF) affects more women than men. Menopause may influence HFpEF development in women. We assessed cross-sectional and longitudinal associations between menopause and echocardiographic measures of left ventricular (LV) function and cardiac remodeling.

Methods:

We studied 1,723 women with available echo data from at least two of: year 5 (Y5) (1990-1991), Y25 (2010-2011), or Y30 (2015-2016) in the Coronary Artery Risk Development in Young Adults study. Cardiac structure and function were measured using 2D and Doppler echocardiography. Cross-sectional associations between menopausal status and repeated echo measures at Y25 and Y30 were analyzed using linear mixed models. Two-segmented models were used to compare longitudinal changes in echocardiographic measures in the premenopausal period to changes in the postmenopausal period.

Results:

Mean ± SD age (years) at enrollment was 27 ± 3 in those with menopause by Y25, 25 ± 3 in those with menopause between Y25 and Y30, and 21 ± 3 in those premenopausal at Y30. There were no significant differences in race, body mass index, systolic blood pressure, or diabetes between the groups. Postmenopausal women had higher early diastolic mitral inflow (E) to annular (e’) velocity ratio than premenopausal after adjusting for demographics and risk factors (P < 0.05). Menopause was associated with relative increases in the rates of change in LV mass and left atrial volume, even after adjustment. Change in E/e’ ratio was similar before and after menopause.

Conclusions:

Menopause is associated cross-sectionally with worse diastolic function and longitudinally with adverse LV and left atrial remodeling. This may contribute to the increased HFpEF risk in postmenopausal women.

Video Summary:

http://links.lww.com/MENO/A787.
NOVEL PRPH2/RDS MUTATION IDENTIFIED IN A FAMILY WITH VARYING CLINICAL MANIFESTATIONS: A CASE REPORT
Authors: Tauscher Robert G. , Rahmani Safa , Szymaniak Brittany M. , Jampol Lee M. , Mirza Rukhsana G.
Published: June 2021

Purpose:

To present the case of a family with a novel PRPH2/RDS mutation.

Methods:

A case report of a 44-year-old woman and her immediate family, including the father and a sister who shared her PRPH2/RDS mutation.

Results:

A 44-year-old woman presented with examination findings consistent with a butterfly-type pattern dystrophy. A sister had a similar butterfly-type dystrophy, whereas their father had a severe cone–rod dystrophy. Genetic testing revealed the same novel PRPH2/RDS mutation in all three affected individuals, suggesting that this single mutation can produce at least two disparate retinal disease phenotypes.

Conclusion:

This case describes a novel p.Y225X nonsense mutation in the PRPH2/RDS gene and demonstrates that it is both pathologic and capable of significant phenotypic variability.
Twelve-Year Incidence of Open-angle Glaucoma: The Thessaloniki Eye Study
Authors: Founti Panayiota , Coleman Anne L. , Wilson M. Roy , Yu Fei , Harris Alon , Pappas Theofanis , Anastasopoulos Eleftherios , Koskosas Archimidis , Salonikiou Angeliki , Keskini Christina , Malamas Angelakis , Kilintzis Vassilis , Raptou Anastasia , Tzoanou Grigoria , Topouzis Fotis
Published: June 2021

Objective:

The objective of this study was to determine the 12-year incidence of open-angle glaucoma (OAG), with further classification into primary open-angle glaucoma (POAG) and pseudoexfoliative glaucoma (PEXG), in an elderly White population.

Design:

A longitudinal, population-based study in urban Northern Greece.

Participant:

Surviving cohort of the 2554 Thessaloniki Eye Study subjects 60 years and above who had the baseline examination.

Methods:

The surviving cohort was re-examined 12 years after baseline, using the same methodology and the same standard operating procedures as in the baseline examination. The definitions of glaucoma and pseudoexfoliation were consistent throughout the study. The 12-year incidences of OAG, POAG, and PEXG with corresponding 95% confidence intervals (CIs) were calculated for the whole study population, consisting of clinic-visit and home-visit participants. The population at risk was defined as those who did not meet the study criteria for the diagnosis of glaucoma in either eye at baseline.

Main Outcome Measures:

Twelve-year incidence of OAG, with further classification into POAG and PEXG.

Results:

Of 1468 eligible subjects in the surviving cohort, 1092 were examined (participation rate 74%). Mean age at baseline was 68.9±4.6 years. Mean follow-up time was 11.6±1.6 years. The 12-year incidence of OAG was 4.4% (95% CI: 3.3-5.8); 0.37% per year. In the overall population the incidence of POAG and PEXG was 2.1% (95% CI: 1.3-3.2) and 2.3% (95% CI: 1.5-3.4), respectively. The corresponding incidence proportions were 2.9 (95% CI: 1.8-4.3) in those without PEX and 8.9 (95% CI: 5.8-12.9) in those with PEX at baseline and/or incidence. The latter was strongly associated with higher odds for incident glaucoma (odds ratio=3.34, 95% CI: 1.83-6.08, P<0.001). Of all incident OAG cases, 11.1% (95% CI: 4.4-24) had baseline intraocular pressure >21 mm Hg.

Conclusions:

The incidence of OAG was similar or higher compared with other White populations. The incidence of glaucoma in those with PEX was higher compared with the incidence of glaucoma in those without PEX.
Pulmonary hypertension in interstitial lung disease: screening, diagnosis and treatment
Authors: Behr Jürgen , Nathan Steven D.
Published: June 2021

Purpose of review

Pulmonary vascular disease resulting in pulmonary hypertension in the context of interstitial lung disease (PH-ILD) is a common complication that presents many challenges in clinical practice. Despite recent advances, the pathogenetic interplay between parenchymal and vascular disease in ILD is not fully understood. This review provides an overview of the current knowledge and recent advances in the field.

Recent findings

Clinical trials employing the phosphodiesterase-5-inhibitor sildenafil delivered negative results whereas riociguat showed harmful effects in the PH-ILD population. More recently, inhaled treprostinil showed positive effects on the primary endpoint (six-min walk-distance) in the largest prospective randomized placebo-controlled trial to date in this patient population. Additionally, a pilot trial of ambulatory inhaled nitric oxide suggests beneficial effects based on the novel endpoint of actigraphy.

In view of these novel developments this review provides an overview of the status quo of screening, diagnosis and management of pulmonary vascular disease and PH in patients with ILD.
Colonic Transit Time and Fecal Impaction in Children and Adolescents With Cystic Fibrosis-associated Constipation
Authors: de Sillos Marcela Duarte , Chiba Sonia Mayumi , Soares Ana Cristina Fontenele , Gomes Clóvis Eduardo Tadeu , de Morais Mauro Batista
Published: June 2021

Background:

Constipation is prevalent in pediatric cystic fibrosis (CF) patients and colonic motility has not been studied in this population. In this study, we aimed to evaluate the total and segmental colonic transit time in children and adolescents with CF based on the presence of constipation and radiological fecal impaction.

Methods:

In this case series, all patients aged 3 to 20 years of a CF reference center were invited to participate. CF-associated constipation was diagnosed based on the European Society for Paediatric Gastroenterology Hepatology and Nutrition criteria. Total and segmental colonic transit time was determined using radiopaque markers. Fecal impaction on plain abdominal radiography was assessed based on the Barr score.

Results:

Of the 43 eligible patients, 34 (79%) agreed to participate. Constipation was found in 44.1% of children and adolescents, predominantly in girls. The total colonic transit time (medians of 42 and 24 hours, respectively, P = 0.028) and the segmental right colon transit time (medians of 8 and 2 hours, respectively, P = 0.012) were significantly longer in CF-associated constipation group than in the group of patients without constipation. The frequency of radiological fecal impaction was similar in patients with (50.0%) and without (64.2%) CF-associated constipation (P = 0.70). There was no relationship between radiological fecal impaction and the total and segmental colonic transit time.

Conclusions:

Children and adolescents with CF-associated constipation had a longer total and segmental right colon transit time. Colonic transit time was similar in patients with and without radiological fecal impaction.
Sir Frederick Edward Warner. 31 March 1910—3 July 2010
Authors: Garside John
Published: June 2021

Frederick Edward (‘Ned’) Warner's childhood home was a London County Council flat in north London. He won a scholarship to Bancroft's School, where he was successful in both academic and sporting activities and was awarded an Exhibition to read chemistry at University College London. Following this chemistry degree he took a diploma in chemical engineering. Sport, debates and left-wing politics dominated his student years. Warner was at the forefront in developing health, safety, risk assessment and environmental policies, particularly in their implementation to chemical process plant; early professional experience in several chemical manufacturing companies and extensive wartime work associated with acid manufacture had driven this commitment. He was a leading figure in the creation of the consulting company Cremer and Warner, and was appointed court expert to the Court of Enquiry following the Flixborough explosion in 1974. As treasurer of the Scientific Committee on Problems of the Environment, he chaired three of its major projects: Environmental Consequences of Nuclear War; Pathways of Artificial Radionucleotides; and Radiation from Nuclear Test Explosions. His expertise in the environmental effects of radiation put him in a position to lead the first international team to Chernobyl after the reactor meltdown in 1986. Warner served on many governmental, professional and academic bodies, particularly the Institution of Chemical Engineers, of which he was president in 1966–1967. He was knighted in 1968 for services to chemical engineering, was elected to the Royal Society in 1976 and was a founder fellow of the Fellowship of Engineering (now the Royal Academy of Engineering). He died in 2010, aged 100.
Role of amylopectin synthesis in
Toxoplasma gondii
and its implication in vaccine development against toxoplasmosis
Authors: Lyu Congcong , Yang Xuke , Yang Jichao , Hou Lun , Zhou Yanqin , Zhao Junlong , Shen Bang
Published: June 2021

Toxoplasma gondii
is a ubiquitous pathogen infecting one-third of the global population. A significant fraction of toxoplasmosis cases is caused by reactivation of existing chronic infections. The encysted bradyzoites during chronic infection accumulate high levels of amylopectin that is barely present in fast-replicating tachyzoites. However, the physiological significance of amylopectin is not fully understood. Here, we identified a starch synthase (SS) that is required for amylopectin synthesis in
T. gondii
. Genetic ablation of SS abolished amylopectin production, reduced tachyzoite proliferation, and impaired the recrudescence of bradyzoites to tachyzoites. Disruption of the parasite Ca
2+
-dependent protein kinase 2 (CDPK2) was previously shown to cause massive amylopectin accumulation and bradyzoite death. Therefore, the
Δcdpk2
mutant is thought to be a vaccine candidate. Notably, deleting SS in a
Δcdpk2
mutant completely abolished starch accrual and restored cyst formation as well as virulence in mice. Together these results suggest that regulated amylopectin production is critical for the optimal growth, development and virulence of
Toxoplasma
. Not least, our data underscore a potential drawback of the
Δcdpk2
mutant as a vaccine candidate as it may regain full virulence by mutating amylopectin synthesis genes like SS.
Microanatomical and secretory characterization of the salivary gland of the
Rhodnius prolixus
(Hemiptera, Reduviidae, Triatominae), a main vector of Chagas disease
Authors: Borella Marfil Anhê Ana Carolina , Maia Godoy Raquel Soares , Nacif-Pimenta Rafael , Barbosa Wagner Faria , Lacerda Marcus Vinicius , Monteiro Wuelton Marcelo , Costa Secundino Nágila Francinete , Paolucci Pimenta Paulo Filemon
Published: June 2021

Rhodnius prolixus
is the principal vector of
Trypanosoma cruzi
, the aetiological agent of Chagas disease in American countries. This insect is haematophagous during all life cycles and, to antagonize its haemostatic, inflammatory and immune systems, it secretes saliva while feeding on the vertebrate host's blood. Here, we investigated characteristic changes of the salivary glands (SG) that occur during insect development. Two pairs of lobules and ducts comprise the SG of
R. prolixus
. The organ's size increases over time, but the microanatomical structures are preserved during insect development. Both lobules have a single layer epithelium formed by binucleated cells, which surrounds the saliva reservoir. The principal lobule presents higher polysaccharide and total protein contents than the accessory lobe. A network of external muscle layers is responsible for organ contraction and saliva release. Apocrine, merocrine and holocrine secretion types occur in the secretory epithelium. Dopamine, serotonin and tyrosine-hydroxylase are neural-related molecules that regulate SG function both during and after feeding.