bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Heme Synthesis in Ovarian Cancer and Triple Negative Breast Cancer Cells Leads to Downregulation of Glycolysis, Mitochondrial Respiration, and Cell Migration.
Authors: Kaur Pritpal , Nagar Shreya , Bhagwat Madhura , Padmanabhan Sveta , Gaire Bijaya , Vancura Ales
Published: April 2020

Cancer cells undergo a complex rearrangement of metabolic pathways involved in biosynthetic processes to support tumor initiation and progression. Targeting glycolysis and TCA cycle that provide precursors for biosynthetic pathways emerged as a potential therapeutic strategy in some cancers. Heme has a potential to regulate both glycolysis and TCA cycle. Heme serves as a prosthetic group in proteins and enzymes that sense, transport, or use oxygen, such as hemoglobin, myoglobin, cytochrome complexes, catalases, and cyclooxygenases. Heme is also required for ATP production by the mitochondrial electron transport chain (ETC) and oxidative phosphorylation (OXPHOS). The first and rate‐limiting step in heme synthesis is production of 5‐aminolevulinic acid (ALA). In this study, we show that promoting heme synthesis by ALA supplementation in ovarian cancer and triple negative breast cancer cells leads to downregulation of glycolysis and lactate production, mitochondrial respiration, as well as cell migration. The mechanism responsible for reduced glycolysis and cell migration involves destabilization of heme‐regulated transcription factor Bach1.

Support or Funding Information

Supported by NIH GM120710
Relationship between Decreased Oxygenation during Acute Hypoxia and Cognitive Deterioration in Healthy Humans
Authors: Uchida Koji , Baker Sarah E. , Wiggins Chad C. , Senefeld Jonathon W. , Shepherd John R.A. , Trenerry Max R. , Clifton Haider R. , Holmes David R. , Joyner Michael J. , Curry Timothy B.
Published: April 2020

Background

Despite improvements in military aircraft safety, many aviation incidents are attributed to hypoxia. The correlation between moderate hypoxia and cognitive function are well studied but results remain controversial. Comparatively, the effects of severe hypoxia are still not well understood. In the present study, we examined the executive cognitive function and physiological indices under both moderate and severe hypoxia conditions.

Purpose

To examine the relationship between executive function and physiological indices during acute hypoxia exposure.

Methods

Ten young healthy participants (4M/6F, 29±4 years) completed two different levels of hypoxia: 1) 10 minute moderate hypoxia (MH; FiO2= 0.118) and 2) 3 minute severe hypoxia (SH; FiO2= 0.077) following 3 minute baseline (BL; FiO2=0.209). Reaction time and errors of commission (defined as a response to a non‐target stimulus) were evaluated continuously using a custom Go/No‐go test at baseline and throughout hypoxia exposure. In this Go/No‐go test, participants were required to push a key when upper case “A” or “@” appeared but not when lower case “a” appeared on a computer monitor as quick and as accurately as possible. The stimuli were presented one at a time at any one of nine differing locations across the screen. Cerebral oxygenation (rSO2) was continuously monitored with near‐infrared spectroscopy (NIRS) positioned above the left supraorbital ridge. Finger oxygen saturation (SpO2) was continuously assessed using a pulse oximetry. Data were analyzed in every 30 seconds and are presented as mean±SE. Two‐way ANOVA and Bonferroni test for multiple comparisons was used to evaluate significant differences.

Results

Both SpO2 and rSO2 were reduced with SH (SpO2 %, BL: 99.3±0.1% vs. SH: 70.7±1.5%, P<0.01; rSO2 %, BL: 70±4% vs. SH: 58±4%, P<0.01). In SH, correlations of SpO2 and rSO2 were significant to the number of errors during the cognitive performance task (SpO2; R= −0.65, rSO2; R= −0.58, Both P<0.01). SH significantly increased errors of commission of the last 30 seconds in 3 minute hypoxic exposure. (BL: 0.6±0.2 errors vs. SH: 1.7±0.5 errors, P<0.01). SH significantly decreased reaction time over hypoxic exposure (BL: 0.43±0.01 sec vs. SH: 0.41±0.01 sec, P<0.01). Comparatively, MH did not elicit an increase in the number of errors of commission nor a decrease in reaction time (Both, P>0.05).

Conclusion

Executive function deteriorated during severe hypoxia. Oxygenation of the prefrontal lobe which controls inhibition may be related to the increase in commission errors. Devices that monitor physiological indices such as SpO2 or rSO2 might be beneficial for a model to predict executive deterioration.

Support or Funding Information

This project was funded by the Office of Naval Research (ONR), grant number N00014‐18‐D‐7001‐TO1.
Chemical Inhibition of PTEN‐Induced Kinase 1 (PINK1) Degradation Confers Neuroprotection in Culture Models of Parkinson’s Disease
Authors: Chu Charleen T. , Liu Yuan , Verma Manish , Wang Kent Z. Q. , Otero P. Anthony , Chen Bill , Mallampalli Rama
Published: April 2020

PTEN‐induced kinase 1 (PINK1) loss of function is linked to familial recessive Parkinson’s disease and, more recently, sporadic Alzheimer’s disease. After mitochondrial processing and release, PINK1 is rapidly degraded by the proteasome.

Objective

We hypothesized that stabilization of PINK1 by interfering with binding to its SCF ubiquitin ligase complex would elevate PINK1 expression levels and confer neuroprotection.

Methods & Results

After identifying an F‐box protein that interacted with PINK1 and regulated PINK1 expression levels, we used computational binding simulations to screen compounds likely to interfere with this interaction. We identified a small molecule that stabilized PINK1 expression and reduced both cell death and dendritic retraction in several models of Parkinsonian injury in primary cortical neurons and patient‐derived cells.

Conclusions

Our identification of an F‐box protein‐targeted small molecule antagonist capable of elevating endogenous PINK1 levels sets the stage for additional target validation for the potential treatment of neurodegenerative diseases and other disorders characterized by mitochondrial dysfunction.

Support or Funding Information

Grant support: NIH grants AG026389, NS101628 (co‐funded by NINDS/NIA) and NS065789 (C.T.C.), HL096376, HL097376, HL098174, HL081784, P01 HL114453 and Merit Review Award from the United States Department of Veterans Affairs (R. K. M.), HL139860 and HL133184 (B.B.C.), HL142777 and AHA 16SDG27650008 (Y.L.). The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.
A single nucleotide polymorphism in DDAH1 leads to decreased nitric oxide production in neonatal cord blood‐derived stimulated lymphoblastoid cells
Authors: Milton Avante Dionne , Almazroue Hanadi , Jin Yi , Neinast Reid , Zender Gloria , McBride Kim L. , Nelin Leif D. , Trittmann Jennifer K.
Published: April 2020

Bronchopulmonary dysplasia (BPD) is chronic lung disease in preterm infants. Pulmonary hypertension (PH) develops in 25–40% of BPD and is a major contributor to morbidity and mortality in BPD patients. Nitric oxide (NO) is a potent vasodilator and apoptotic mediator made by nitric oxide synthase (NOS). NOS is inhibited by asymmetric dimethylarginine (ADMA). Dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA. Translational studies from our group identified a single nucleotide polymorphism (SNP) in the DDAH1 gene, rs480414, that was associated with a decreased risk for PH in BPD patients. Therefore, we aim to determine if the DDAH1 SNP rs480414 affects DDAH1 function in cell culture. Neonatal cord blood specimens were treated with Epstein‐Barr virus to select for B‐lymphocytes and transform into lymphoblastoid cell lines (LCLs) that were genotyped (SNP or wild‐type) at the rs480414 locus. We tested the hypothesis that in LCLs the SNP rs480414 results in a gain of function mutation in DDAH1, which would lead to greater NO‐mediated apoptosis as compared to DDAH1 wild‐type (WT). LCLs (WT, n=3 and SNP, n=3) were stimulated with phorbol myristate (PMA), IL‐4, and IL‐13 for 48 hours, protein was analyzed by western blot analysis for DDAH1, cleaved and total caspase‐3 and ‐8, and β‐actin. RT‐PCR was performed and expression of DDAH1 and iNOS mRNA was evaluated. Cell media was assayed for concentrations of nitrite using a chemiluminescence NO analyzer. Spectrophotometry was used to measure the conversion of ADMA to L‐citrulline. A standard L‐citrulline curve was used to calculate DDAH activity after normalization to protein concentration. LCLs with the DDAH1 SNP had similar levels of DDAH1 protein expression (n=3, p=0.58), similar mRNA expression (n=3, p=0.1), and similar DDAH activity (p=0.35) compared to DDAH1 WT. We found that LCLs with the DDAH1 SNP had similar iNOS mRNA expression (p=0.6), lower nitrite levels (p=0.04), lower cleaved caspase‐3 levels (p=0.04), and similar cleaved caspase‐8 levels (p=0.2) than DDAH1 WT LCLs. Contrary to our hypothesis, these finding suggests that the DDAH1 SNP rs480414 may result in a loss of function mutation, resulting in lower NO production and lower apoptosis in LCLs. This is the first evidence that the DDAH1 SNP rs480414 has an effect on cell function, however we speculate that pulmonary vascular endothelial cells will likely have a different physiology.

Support or Funding Information

This work was supported by NIH‐K08 HL129080.
Adhesion‐GPCR Gpr116 (ADGRF5) is a Regulator of Urine Acidification
Authors: Zaidman Nathan , Tomilin Viktor , Damarla Mahendra , Tidmore Josephine , Pochynyuk Oleh , Pluznick Jennifer
Published: April 2020

G‐protein coupled receptors (GPCR) are a large and diverse family of integral membrane proteins that recognize a tremendous assortment of extracellular molecules including neurotransmitters, hormones, light and odors. They are a common target of pharmaceutical drug development, and uncovering the function of novel GPCRs in the kidney represents a wealth of untapped therapeutic potential. We previously performed an mRNA screen for novel GPCRs in the kidney to identify promising yet overlooked renal GPCRs. This screen revealed that Gpr116, an adhesion‐class GPCR, is highly expressed in the kidney. To understand the role of Gpr116 in renal physiology, we have taken a multidisciplinary approach. Using transfected HEK293 cells expressing cloned Gpr116, we have validated a monoclonal antibody for Gpr116. In murine kidney, this antibody indicates localization of Gpr116 to intercalated cells (ICs) in the collecting duct of mouse kidneys. This staining is absent in kidneys from targeted knockout (KO) of Gpr116 in renal tubules (Gpr116flox/flox, ksp‐Cre). Additionally, kidney‐specific KO animals have significantly (p<0.02) reduced urine pH (5.66±0.29, n=9) compared to wild‐type (WT; 5.96±0.23, n=15) littermates. Challenging the animals with an acid load (280mM NH4Cl added to drinking water) significantly acidifies WT urine (5.71±0.18, n=14, p<0.03) but does not significantly reduce KO urine pH (5.55±0.20, n=9), suggesting the loss of Gpr116 from α‐ICs is sufficient to maximally acidify urine. KO mice do not have significantly more ICs compared to WT. Interestingly, the loss of acid in the urine is accompanied by a small, but significant (p=0.001), increase in blood pH, and a small, but significant (p<0.05), decrease in pCO2 compared to WT. We conclude that loss of Gpr116 from the nephron causes a primary loss of acid in the urine which results in a mild metabolic alkalosis (“renal tubular alkalosis”) due to reabsorption of HCO3− by α‐ICs. This study establishes a physiologic role of the previously understudied Gpr116 in the murine kidney and demonstrates the scientific potential of future investigations into novel GPCRs.

Support or Funding Information

Research was funded by NIH F32DK116499 (N.A.Z.) and NIH R01DK107726 (J.L.P.).
Calcium‐Dependent Localization of S100A6 in Pulmonary Microvascular Endothelial Cells
Authors: Haldar Barnita , Hamilton Caleb Lucas , Solodushko Viktoriya , Honkanen Richard E. , Scammell Jonathan G. , Cioffi Donna Louise
Published: April 2020

During inflammation, mediators increase calcium influx into endothelial cells through store‐operated calcium entry (SOCE) channels. Increasing calcium influx through the SOCE current ISOC promotes endothelial barrier disruption through the formation of inter‐endothelial cell gaps. Inter‐endothelial cell gap formation leads to increased permeability. Identifying mechanisms of ISOC inhibition may aid in the development of therapeutic strategies against endothelial permeability‐associated vascular inflammation. Our studies indicate FK506‐binding protein 51 (FKBP51), in conjunction with protein phosphatase 5 (PPP5C), inhibit ISOC and calcium entry‐induced inter‐endothelial cell gap formation. Further, we determined that the calcium‐binding protein S100A6 is necessary for the PPP5C‐FKBP51‐mediated inhibition of ISOC in pulmonary microvascular endothelial cells (PMVECs). In PMVECs, S100A6 interacts with the ISOC channel in a calcium‐dependent manner, i.e., following an increase in cytosolic calcium. However, we do not know whether calcium entry specifically through the ISOC channel or through other SOCE channels that provides the calcium source to promote S100A6 interaction with the ISOC channel.

Methods

PMVECs were used for co‐precipitation and immunocytochemistry‐based studies. When cells are treated with rolipram (R) plus thapsigargin (Tg), ISOC and other SOCE channels are activated. However, when cells are treated with Tg alone, ISOC is not activated, but other SOCE channels are activated. The TRPC4 subunit of the ISOC channel was immunoprecipitated and probed for co‐precipitation of S100A6.

Results

Following R/Tg treatment, robust co‐precipitation of S100A6 with TRPC4 was observed, whereas following Tg alone only minor co‐precipitation was seen. Immunocytochemistry revealed that in untreated cells S100A6 localization is principally cytosolic. Following R/Tg treatment, but not Tg alone, a distinct membrane‐delimited pool of S100A6 was observed, particularly at sites of cell‐cell contact. Membrane localization was confirmed by co‐localization with wheat germ agglutinin (WGA).

Conclusion

Overall, our data reveal that calcium entry specifically through the ISOC channel is an important determinant of S100A6 translocation to the plasma membrane promoting interaction of S100A6 with the ISOC channel.

Support or Funding Information

NIH HL
R56HL107778
MLP regulates ENaC Activity in Renal Distal Convoluted Tubule Cells
Authors: Song Chang , Moseley Auriel , Yue Qiang , Al-Khalili Otor , Wynne Brandi , Eaton Douglas
Published: April 2020

The epithelial sodium channel (ENaC) plays an important role in blood pressure regulation through the fine‐tuning of sodium reabsorption in the distal nephron. We previously demonstrated that ENaC is present in distal convoluted tubule (DCT) cells, and the PIP2‐dependent regulation of ENaC is mediated by the myristoylated alanine‐rich protein kinase C substrate (MARCKS)‐like protein‐1 (MLP‐1). MLP‐1 regulation of ENaC is controlled by MLP‐1 phosphorylation of MLP‐1’s PIP2 binding domain. Here we examine the role of MLP‐1 in the function of regulating ENaC in DCT‐15 cells. We also examined whether ENaC interacts with specific domains of MLP‐1. DCT‐15 cells and Cos‐7 cells were used in our study. To further dissect the mechanism of MLP in regulating ENaC in DCT‐15 cells, we constructed various mutants of MLP. WT is the full length wild‐type protein, whereas S3A contains amino acid substitutions in the effector domain to create a molecule that could not be phosphorylated. The S3D mutant was created to mimic constitutive phosphorylation. The GA mutant had an N‐terminal glycine replaced so that it could not be myristoylated. The single‐channel patch‐clamp recordings were used to measure the channel activity of ENaC channels after transfection, and by Mammalian two‐hybrid assay we examined the interaction sites of MLP‐1 and ENaC. S3A construct of MLP has proved to be particularly effective in regulating ENaC function in DCT‐15 cells compared to other variants, given its higher channel activity. MLP‐1 regulates ENaC by changing the open probability of ENaC but not the protein expression or the number of functional channels in DCT‐15 cells. In a two‐hybrid screen, the N‐terminal (but not the C‐terminal) domain of MLP protein associated strongly with the N‐terminus of gamma ENaC and weakly with the N‐terminus of beta ENaC but not with the alpha subunit in cells.

Support or Funding Information

Supported by R01 DK‐110409 to DCE and K01 DK‐115660 and ASN Gottschalk AWARD to BMW
ANGPTL4 Induces TMZ Resistance of Glioblastoma by Promoting Cancer Stemness Enrichment via the EGFR/AKT/4E‐BP1 Cascade
Authors: Tsai Yu-Ting , Ko Chiung-Yuan , Chang Wen-Chang , Hsu Tsung-I
Published: April 2020

Glioblastoma (GBM) is the most aggressive brain tumor, with a strong invasiveness and high tolerance to chemotherapy. With the current standard treatment combining temozolomide (TMZ) and radiotherapy, glioblastoma can be incurable due to drug resistance. The existence of glioma stem‐like cells (GSCs) is considered the major reason for drug resistance. However, the mechanism of GSC enrichment remains unclear. Herein, we found that the expression and secretion of angiopoietin‐like 4 protein (ANGPTL4) were obviously increased in GSCs. The overexpression of ANGPTL4 induced GSC enrichment that was characterized by BMI‐1 and SOX2 expression, resulting in TMZ resistance in GBM. Furthermore, epidermal growth factor (EGFR) phosphorylation induced 4E‐BP1 phosphorylation that was required for ANGPTL4‐induced GSC enrichment. In particular, ANGPTL4 induced 4E‐BP1 phosphorylation by activating PI3K/AKT and ERK cascades for inducing stemness. To elucidate the mechanism contributing to ANGPTL4 upregulation in GSCs, chromatin immunoprecipitation coupled with sequencing (ChIP‐Seq) revealed that specificity protein 4 (Sp4) associates with the promoter region, −979 to −606, and the luciferase reporter assay revealed that Sp4 positively regulates activity of the ANGPTL4 promoter. Moreover, both ANGPTL4 and Sp4 were highly expressed in GBM and resulted in a poor prognosis. Taken together, Sp4‐mediated ANGPTL4 upregulation induces GSC enrichment through the EGFR/AKT/4E‐BP1 cascade.

Support or Funding Information

This research was supported by the Ministry of Science and Technology of Taiwan (MOST 106‐2320‐B‐038 ‐003 ‐MY2, 107‐2320‐B‐038‐001, and 108‐2628‐B‐038‐005‐)
Tracking the ancestral functions of erythropoietin: neuroprotection & mitochondria
Authors: Soliz Jorge , Losantos-Ramos Karen , Suarez Renato , Arias-Reyes Christian , Laouafa Sofien , Furrer Daniela , Joseph Vincent
Published: April 2020

It has long been thought that erythropoietin (Epo) is exclusively involved in erythropoiesis, allowing erythroid progenitor cells to survive and mature through their antiapoptotic action. We now know that Epo in mammals has also other functions in the brain, playing key roles in the development, maintenance, protection, and repair of the nervous system. However, the recombinant human Epo (rhEpo) has neuroprotective effects in orthoptera insects (such as grasshoppers), and this effect appears to be mediated by the cytokine receptor‐like factor 3 (CRLF3), raising interesting questions about the evolutionary origin of the Epo signaling pathway and its role in invertebrate species. Taking into account that: 1) Epo in mammals modulates the mitochondrial oxidative phosphorylation and the production of reactive oxygen species (ROS) in several tissues, including the brain; and 2) that insects appeared during a geological period (Cambrian explosion) in which the atmospheric O2 was increasing and required the implementation of antioxidant systems at the cellular level; here we tested the hypothesis that activation of the “Epo‐like” system in the brain domestic crickets (Acheta domesticus) exposed to 6 % of hypoxia during 5 days, modulates mitochondrial functions for preventing against oxidative damages. To do so, we used our oxygraph‐2K system (OROBOROS) that measures the mitochondrial bioenergetics in saponin‐permeabilized tissue of 2 mg weight. Our preliminary results showed that rhEpo increased the survival of domestic crickets exposed to hypoxia by 20%. We showed also that, in normoxic animals, rhEpo increased the mitochondrial O2 consumption rate (OCR), but in hypoxic animals, rhEPO limited the increase of mitochondrial OCR. In parallel, rhEpo significantly decreased the production of ROS in hypoxia. These preliminary results suggest that rhEpo significantly improves cricket’s survival under hypoxia, by promoting a robust antioxidant effect through mitochondria. Our data also suggest that a neuroprotective “Epo‐like” endogenous molecule evolved during the “Cambrian explosion” from a urbilaterian (common to vertebrates and invertebrates) ancestor.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada
Single Prolonged Stress Provokes Depressive‐like Behavior, Anxiety and Impaired Social Interaction in Females with Potential for Early Invention with Neuropeptide Y
Authors: Nahvi Roxanna J. , Nwokafor Chiso , Sabban Esther
Published: April 2020

Sex contributes to susceptibility of stress‐elicited disorders. Women are at nearly double the risk for developing symptoms of post‐traumatic stress disorder (PTSD), depression, anxiety disorders, and social anxiety or phobia. Despite the higher prevalence of these stress‐triggered disorders, animal models, including the single prolonged stress (SPS) PTSD rodent model, and novel therapies for them are largely understudied in females. Previous studies in males indicate that neuropeptide Y (NPY) has therapeutic potential for stress‐elicited disorders; however none examine its effects in females. Here, we evaluated behavioral manifestations in terms of depressive‐like behavior, anxiety, social interaction, and episodic‐like memory and changes in gene expression in the noradrenergic system in the brain after SPS in females. In addition, we examined the potential therapeutic effects of NPY for these behavioral impairments. Females were exposed to SPS and were immediately infused with intranasal NPY or vehicle in three separate experiments. (1) Stressed females treated with 150μg NPY or vehicle were compared to unstressed controls on forced swim test (FST) and for levels of expression of several genes in the locus coeruleus (LC) 12 days after SPS exposure. (2) We examined stressed females treated with 300μg NPY or vehicle and unstressed controls on the elevated plus maze (EPM) and LC gene expression 7 days after SPS stressors. (3) We evaluated stressed females treated with 600μg NPY or vehicle and unstressed controls on the open field (OF), novel object recognition (NOR), social interaction (SI), and EPM at 7, 13, 19, and 21 days after SPS stressors, respectively. SPS led to increased depressive‐like behavior on the FST and anxiety‐like behavior on the OF and EPM, as well as impaired social interaction. Following FST, the rats displayed elevated tyrosine hydroxylase (TH), CRHR1 and Y1R mRNA levels in the LC, consistent with increased activation of the noradrenergic system. The expression level of these mRNAs was unchanged following EPM, except for Y1R. A higher dose of intranasal NPY tended to have larger anxiolytic effects than the lower dose, however unlike in previously tested males neither dose was significantly different from vehicle. Intranasal NPY did significantly prevent impaired social interaction, as compared to vehicle. There was no effect of SPS or NPY on impairment of memory on the NOR. The results indicate that SPS appropriately elicits PTSD associated behavior and molecular impairments in females. Intranasal NPY has potential for therapeutic intervention with important sex‐specific differences.

Support or Funding Information

U.S. Department of Defense (DOD) Award No. W81XWH‐16‐1‐0016 and New York Medical College (NYMC)/Touro Bridge Funding Program 49‐506.
Effects of clindamycin hydrochloride on reversing LPS induced growth inhibition and TNF‐α secretion in human periodontal ligament cells
Authors: Wang Jianhong , Duan Hubin , Qiao Xiaohong , Li Youliang , Wu Yunxia
Published: April 2020

Objective

To investigate the role of clindamycin hydrochloride on reversing Lipopolysaccharide(LPS)‐induced proliferation inhibition and Tumor necrosis factor‐α(TNF‐α) secretion in human periodontal ligament cells.

Method

Human periodontal ligament cells (HPDLCs) was cultured in vitro and identified by immunohistochemical staining. 4–8 generations HPDLCs were selected for the experiment. Above HPDLCs were divided into control group, LPS group and drug group. In the control group, only Dulbecco's Modified Eagle Medium(DMEM) culture solution was added to each hole. In the LPS group,100 μg/ml LPS was added to DMEM culture solution for each hole.In the drug group, different concentrations(10μg/ml,40μg/ml and 160μg/ml) of clindamycin hydrochloride and 100μg/ml LPS were distributed to different test Wells.MTT and enzymelinkedimmunosorbentassay(ELISA) were used to detect the effects of clindamycin hydrochloride on reversing LPS induced growth inhibition and TNF‐α secretion in human periodontal ligament cells.

Results

MTT results showed that the absorbance of MTT in the LPS group decreased significantly compared with that in the control group. Compared with LPS group, the absorbance of MTT in the drug groups with different concentrations of clindamycin hydrochloride showed a trend of increase. ELISA results showed that the level of TNF‐α in the LPS group increased significantly compared with that in the control group. Compared with LPS group, the level of TNF‐α in the drug groups with different concentrations of clindamycin hydrochloride showed a trend of decrease.

Conclusion

Clindamycin hydrochloride can reverse LPS‐induced proliferation inhibition, which is beneficial to the regeneration of periodontal tissue and the production of new periodontal attachment. It also can significantly inhibit the role of LPS in promoting the secretion of TNF‐α by HPDLCs, thereby reducing the inflammatory response of periodontal tissues, which is beneficial to the treatment of periodontal disease.

Support or Funding Information

This study was supported by Lvliang City Science and Technology Fundation Grant, Lvliang people‘s hospital Fundation Grant and China Postdoctoral Science Fundation Grant (2019T120195)
Plasma Rare Sugar Levels and the Effect on Plasma Glucose Levels in GLUT5‐induced Rats Gavaged with Rare Sugar D‐Sorbose, D‐Tagatose, or D‐Allose
Authors: Kishida Kunihiro , Yoshikawa Kazushi , Taguchi Tadao , Tamaoki Ryo , Iida Tetsuo , Yamada Takako , Toyoda Yukiyasu
Published: April 2020

D‐Sorbose, D‐tagatose, and D‐allose are rare sugars that have been reported to have various beneficial health effects
1,
2). They are contained in Rare Sugar Syrup as functional ingredients
3). It is essential to clarify which transporters are involved in their intestinal absorption to elucidate their physiological effects and also to apply them to “Foods with Function Claims”. We have already reported that D‐allulose, an epimer of fructose, was a substrate of glucose transporter type 5 (GLUT5) in the small intestine
4,
5). GLUT5 is a transporter that mediates intestinal fructose absorption. In the present study, we examined whether D‐sorbose, D‐tagatose, and D‐allose are absorbed via GLUT5 in the small intestine and also compared the effects on plasma glucose levels after gavage administration of these rare sugars. We divided rats into 2 groups. One group was fed high‐fructose diet, and the other group was fed high‐glucose diet for a week. Fructose‐fed rats induced their intestinal GLUT5 expression. Then, we gavaged the rats with 2g/kg BW of D‐sorbose, D‐tagatose, or D‐allose, and collected blood at 0, 30, 60, 90, 120, 180 min from the tail vein into a heparinized tube. Plasma levels of rare sugars and glucose were analyzed by HPLC. When D‐sorbose was gavaged to the rats, the fructose‐fed rats exhibited significantly higher D‐sorbose levels than the glucose‐fed rats at 30, 60, 90, 120 min. A similar result was obtained with D‐tagatose administration experiment. By contrast, D‐allose levels did not differ between the fructose‐fed and glucose‐fed rats when D‐allose was gavaged to the rats. These results suggest that D‐sorbose, D‐tagatose but not D‐allose are absorbed via GLUT5 in the small intestine. The fructose‐fed rats exhibited significantly higher plasma glucose levels only when D‐tagatose was gavaged. As D‐tagatose was reported to be a substrate of gluconeogenesis
6). The results indicate that more D‐tagatose was absorbed in the fructose‐fed, GLUT5‐induced rats, resulting in the higher conversion into glucose than in the glucose‐fed rats.
Tridax procumbens suppresses cellular markers of tumour growth, proliferation and invasion in pancreatic ductal adenocarcinoma cell line
Authors: Samuel Ekundayo , Tosh David , Odunola Oyeronke
Published: April 2020

Pancreatic ductal adenocarcinoma is one of the most fatal solid malignancies, with a 5‐year survival rate of approximately five percent. Hence, there is much interest in developing promising anticancer drugs. To address this issue, we cultured the pancreatic ductal adenocarcinoma cell line (Panc‐1) in supplemented Dulbecco’s Modified Eagle's medium at 37°C in a humidified incubator with 5% CO2. Panc‐1 cells were cultured without or with 20 μg/ml of the dichloromethane fraction (DCMF) of the ethanol leaf extract of Tridax procumbens. Dimethyl sulfoxide was used as control. We examined the expression levels of ductal (cytokeratin 7, peanut agglutinin), mesenchymal (vimentin), cell cycle and proliferation markers (Ki67, p53, p21Cip1/Wap‐1) by immunofluorescence. Confocal images were collected (n≥3) and processed with Image J and Zen 2.3 lite software. Data were analysed using student’s t‐test and results expressed as mean ±standard error of mean with P value <0.05 considered as significant.

We observed a significant reduction (p<0.05) in the expression levels of cytokeratin 7, peanut agglutinin, vimentin and Ki67 proteins in DCMF‐treated Panc‐1 cells as compared to control cells. There was a significant increase (p<0.05) in the expression levels of p53 and p21Cip1/Wap‐1 proteins in DCMF‐treated Panc‐1 cells as compared to the control. In conclusion, overexpression of cytokeratin 7, peanut agglutinin, vimentin and Ki67, and reduced expression of p53 and p21Cip1/Wap‐1 in pancreatic cancer correlate with increased tumour growth, invasion and poor prognosis. Therefore, results from this study suggest that Tridax procumbens could be a potential therapeutic agent in the treatment of pancreatic ductal adenocarcinoma.

Support or Funding Information

We appreciate the support and funding received from the University of Ibadan, Ibadan, Nigeria, Commonwealth Scholarship Commission, United Kingdom and University of Bath, United Kingdom.
Cardiovascular Programming by Preeclampsia Sensitizes Mother Rats to Hemodynamic and Cardiac Autonomic Depressant Effects of Postpartum Endotoxemia
Authors: Abuiessa Salwa A. , El-Gowilly Sahar M. , Helmy Mai M. , El-Mas Mahmoud M.
Published: April 2020

Preeclampsia is a pregnancy related disorder with serious maternal and fetal consequences. Considering that postpartum infection and endotoxemia are important causes of maternal death, we investigated whether preeclamptic maternal programming alters cardiovascular and cardiac autonomic consequences of endotoxemia in rats. Preeclampsia was induced by daily oral administration of 50 mg/kg of L‐NAME for 7 days starting from day 14 of conception. Changes in blood pressure (BP), heart rate (HR), and cardiac sympathovagal balance caused by i.v. administration of lipopolysaccharide (LPS) were assessed in conscious mothers pre‐instrumented with femoral indwelling catheters at 3 weeks (weaning time) and 9 weeks postpartum. At weaning, LPS caused significantly greater falls in BP and rises in HR and LV contractility (dP/dtmax) in PE rats compared with their non‐PE counterparts. Additionally, more apparent reductions in time and frequency domain indices of heart rate variability and shifts in cardiac sympathovagal balance (low‐frequency/high‐frequency ratio, LF/HF) towards parasympathetic dominance were observed after treatment of weaning PE mothers with LPS. Measurement of arterial baroreflex activity by the vasoactive method revealed that LPS attenuated reflex bradycardic responses to phenylephrine in endotoxic PE, but not non‐PE, weaning rats. Remarkably, the intensified cardiovascular effects of endotoxemia in weaning PE rats were found to subside after 9 weeks of delivery. In conclusion, maternal programming by preeclampsia elicits short‐term exacerbation of cardiovascular and autonomic derangements caused by endotoxemia in rats.

Support or Funding Information

Supported by the Science and Technology Development Fund, Egypt (STDF Grants No. 14895)
TNFα Decreases Succinate Dehydrogenase Activity in Motor Neurons
Authors: Yap Jane Q. , Heino Amanda , Brown Alyssa D. , Rana Sabhya Denise , Fogarty Matthew J. , Delmotte Philippe D. , Mantilla Carlos B. , Sieck Gary C.
Published: April 2020

Inflammation triggers a homeostatic response that is mediated by pro‐inflammatory cytokines such as TNFα. We hypothesize that in a motor neuron‐like cell line, NSC‐34, TNFα decreases mitochondrial function, specifically succinate dehydrogenase (SDH) activity, which is a key enzyme in the tricarboxylic acid (TCA) cycle as well as complex II in the electron transport chain (ETC). Experiments were performed on cultured NSC‐34 cells that were differentiated by serum depletion until neurite structures (>50 μm) were observed. The differentiated cells were treated with TNFα (20 ng/ml) for 24 h. To measure the maximum velocity of the SDH reaction (SDHmax), cells were incubated in a solution containing 80 mM succinate and 1.5 mM nitro blue tetrazolium (NBT), the reaction indicator. As the SDH reaction proceeded, images were acquired every 15 s using a 40× objective on an Olympus IX71 inverted microscope. Linearity of the SDH reaction was confirmed across the 8‐min period, and SDHmax was determined and expressed as mM fumarate/L tissue/min. To determine mitochondrial volume density, the cells were loaded with MitoTracker Green and imaged in 3D using a confocal microscope. The cell volume occupied by labeled mitochondria was determined from 3D reconstructions of 1 mm optical slices. In NSC‐34 cells exposed to TNFα, we found that mitochondrial volume density increased, and mitochondria were fragmented as compared to untreated controls. We also found that TNFα exposure decreased SDHmax when normalized for mitochondrial volume density. In other cell types, we found that TNFα‐induces an increase in PGC1a and Drp1 expression and a decrease in Mfn2 expression, all of which affect mitochondrial structure. In related studies, we also found that TNFα exposure decreases O2 consumption per mitochondrion in NSC‐34 cells. We conclude that by reducing the redox potential (SDHmax) of mitochondria, O2 consumption per mitochondrion is reduced, thereby damping ROS formation. The impact of this homeostatic response is offset by mitochondrial biogenesis and an increase in mitochondrial volume density, thereby maintaining overall cellular O2 consumption and ATP production but at reduced cost of ROS formation.

Support or Funding Information

Supported by NIH grants AG44615 and HL105355.
Using ENCODE Project Data As Part Of A Course‐Based Undergraduate Research Experience (CURE): Student Perspectives And Feedback
Authors: Conrad William H.
Published: April 2020

At Lake Forest College, molecular biology is taught as a third‐year upper‐level course. A central learning objective of this course is for students to independently formulate and test hypotheses by evaluating genome‐wide epigenetic data. In this course‐based undergraduate research project, students (1) select high confidence differentially methylated regions (DMRs) present in colorectal cancer samples (2) justify their selection based on literature review (3) predict the effect of DMR methylation on gene expression based on ENCODE project data, (4) predict if the DMR is methylated in HCT‐116 colorectal cancer cells, and (5) test if the DMR is methylated in HCT‐116s and demethylated following decitabine treatment. I will present anonymous survey data of students’ perceptions of the scientific method and their perceptions of themselves as researchers as they engage in research based on big data projects.

Support or Funding Information

Research equipment funded by the Sherman Fairchild Foundation Scientific Equipment Grant. Other funding provided by Lake Forest College Biochemistry and Molecular Biology Program.
The Protective Effects of Ketodiet in Salt‐Sensitive Hypertension
Authors: Golosova Daria , Levchenko Vladislav , Spires Denisha , Staruschenko Alexander , Palygin Oleg
Published: April 2020

Two‐thirds of hypertension cases in the United States are positively correlated with excessive weight gain. The ketogenic diet gained popularity through the weight loss community. It’s a low‐carb high‐fat diet triggering the body to burn fat for energy instead of carbohydrates. Dietary manipulation, ketoacid supplementation can potentially exert a cardiovascular protective effect in chronic renal failure. We hypothesized that ketodiet can have beneficial effects during the development of salt‐induced hypertension. The Dahl Salt Sensitive (SS) rats fed a high salt diet develop salt‐induced hypertension accompanied by kidney injury. To test effect of ketodiet, the Dahl SS rats were given either 4% NaCl diet (HS; Dyets Inc, 113756) or 4% NaCl modified ketodiet (KD; Harland Tekland, TD 190564) with free access to water for 4 weeks. Blood pressure was monitored with telemetry throughout the study, and urine samples were collected in metabolic cages on days 0, 7, 14, and 28. At the end of the protocol animals were sacrificed and blood and kidney tissues were harvested for following analyses. Urine and plasma samples were analyzed to estimate electrolyte and glucose homeostasis. Dahl SS rats fed a KD had a lower blood pressure compared to control rats fed a HS diet. By the end of the experiment mean arterial pressure was 143 ± 4 mmHg vs 156 ± 4 mmHg for Dahl SS rats fed a KD and HS diets, respectively (p<0.05). The KD group has gained less weight (70 ± 8 vs 85 ± 12 g, p<0.05 compared to HS). Furthermore, we observed reduced renal hypertrophy with decreased kidney to body weight ratio (0.94 ± 0.05 vs 1.14 ± 0.06 p<0.05 compared to HS) and smaller kidneys (3.0 ± 0.1 vs 4.0 ± 0.3 g, p<0.05 compared to HS) in the KD animals. The rats on KD experienced hypoglycemia (305 ± 8 vs 364 ± 11 mg/dL, p<0.05 compared to HS) although they had comparably the same as the HS group amount of food consumption (17 ± 2 vs 19 ± 2 g/day, p>0.05). Metabolic cage studies revealed diminished diuresis (0.010 ± 0.001 ml/100g/h vs 0.020 ± 0.002 ml/100g/hr, p<0.05) in KD compared to HS group with no significant changes in electrolyte excretion. Glucose excretion has been reduced in a KD group (1.2 ± 0.5 vs 12.8 ± 4.8, glucose to creatinine ratio, p<0.05 compared to HS). Therefore, we can conclude that prolonged KD provide protective effect on the development of hypertension during HS challenge. Our data revealed that KD during HS challenge resulted in decreased blood pressure, reduced kidney hypertrophy and diminished levels of plasma glucose.
Pharmacological Characterisation of PACAP‐responsive Receptors Reveals Signalling Bias and Probe‐dependent Antagonism
Authors: Tasma Zoe , Hay Debbie , Walker Christopher
Published: April 2020

Pituitary adenylate cyclase‐activating peptide (PACAP) is a neuropeptide which exists in two biologically active forms; PACAP‐27 and PACAP‐38. PACAP is widely expressed in the brain and periphery where it is involved in many physiological processes including pain, inflammation, stress and anxiety. PACAP potently activates three different G protein‐coupled receptors (GPCRs); the PAC1 receptor, the VPAC1 receptor and VPAC2 receptor. The VPAC receptors are co‐receptors for PACAP and vasoactive intestinal peptide (VIP). The PAC1 receptor has several known splice variants in the N‐terminal domain, which include the PAC1n receptor (full N‐terminus) and the PAC1s receptor (21 amino acid N‐terminal deletion). To develop therapeutics targeting the PACAP system, it is essential to understand how these receptors signal and how this signalling is inhibited. Here we pharmacologically characterised the signalling capabilities of PACAP‐responsive receptors and how these responses were blocked by antagonists.

The pharmacology of the human PAC1n, PAC1s, VPAC1, VPAC2 receptors were examined in transfected Cos7 cells. Using concentration‐response curves, the ability of PACAP and related peptides to stimulate cAMP, IP1, pAkt, pERK and pCREB responses were measured. Signalling bias was then quantified. The ability of multiple antagonists to block PACAP‐38, PACAP‐27 or VIP‐induced cAMP responses at PACAP‐responsive receptors was then determined.

PACAP‐responsive receptors stimulated activation of all signalling molecules measured. However, only limited signalling bias was observed. For example, VIP was 4‐fold biased towards cAMP production over IP1 production at the PAC1n receptor. Interestingly, the PAC1n and PAC1s receptors displayed distinct pharmacology. At the PAC1s receptor, VIP was found to be 20‐fold more potent than at the PAC1n receptor. Probe‐ or agonist‐dependent antagonism was observed at the PAC1n and PAC1s receptors. Both VIP and PACAP‐27 were more potently antagonised than PACAP‐38 by the antagonists PACAP6–38 and M65.

The distinct pharmacological profile displayed by the PAC1s receptor, suggests that it can act as a dual receptor for VIP and PACAP under specific conditions. Furthermore, differences in antagonist pharmacology at PACAP‐responsive receptors suggests the effectiveness of blocking a signalling pathway can be influenced by which endogenous agonist is present. These effects have potential implications for the development and effectiveness of drugs targeting the PAC1 receptor.

Support or Funding Information

This research is supported by grants from the Royal Society of New Zealand Marsden Fund (RSNZ) and the Health Research Council of New Zealand (HRC).
Yiqi‐Huoxue Granule suppresses inflammation induced by Lipopolysaccharide in mice by inhibiting the activation of NF‐κB pathway
Authors: WU HONG , Wang Xinzhou , Gao Shuibo , Dai Liping , Wang Zhentao , Han Lihua
Published: April 2020

Objective

To study the effects of Yiqi Huoxue Granule (YQHX) on inflammatory responses and endothelial injury induced by lipopolysaccharide (LPS) in mice.

Methods

Male C57BL/6 mice (8–12 weeks old) were divided into five groups, including control group, LPS group, YQHX group, PDTC (NF‐κB inhibitor) group, and PDTC plus YQHX group. After orally administered with YQHX for 2 weeks or intraperitoneally injected with PDTC for 1 h, LPS was injected into mice for another 6 h, and serums and aortas were collected, respectively. The levels of TNF‐α and IL‐6 in the serum were detected using ELISA. The mRNA expression levels of VCAM‐1, ICAM‐1, PAI‐1 and TF were determined using qRT‐PCR and the protein levels of these factors as well as the phosphorylation levels of NF‐κB pathway were detected using Western blot.

Results

The levels of TNF‐α, IL‐6, VCAM‐1, ICAM‐1, PAI‐1 and TF in LPS group were higher than those in control group (P <0.01), and the phosphorylation levels of P65 and IκB were also increased significantly (P <0.01). Compared with the LPS group, the levels of TNF‐α, IL‐6, VCAM‐1, ICAM‐1, PAI‐1, TF and NF‐κB pathway phosphorylation were decreased in YQHX group, PDTC group and YQHX plus PDTC group (P <0.05, P <0.01). There was no significant difference among groups of PDTC and YQHX plus PDTC (P >0.05).

Conclusion

YQHX suppresses the inflammatory responses and endothelial injuries induced by LPS in mice by inhibiting the activation of NF‐κB pathway.

Support or Funding Information

This work was financially supported by grants from the National Natural Science Foundation of China (Grant No. 81473453 & 81673800)
Remodeling of tight junction protein zonula occludens (ZO)‐1 by glutamine under glucose deprivation in colorectal cancer cells
Authors: Huang Ching-Ying , Chen Ji-Kai
Published: April 2020

Malignant cells exhibit significant metabolic alteration in nutrients to meet the energetic and biosynthetic demands. Dual routes of nutrients uptake (blood supply and luminal source) are unique characteristics of the cancerous colonic cells. Recently, several lines of evidence have suggested that glutamine can support primary cancer cell growth and serve as an alternate energy fuel during distant metastasis when available glucose concentrations are limited. Tight junctions are specialized epithelial structures for the cell‐cell adhesion and interaction. Tumor dissociation and subsequent metastasis in the tumor microenvironment may occur due to the tight junction remodeling. The current study aims to investigate the role of glutamine in colorectal cancer cells under glucose deprivation. Human colorectal cancer Caco‐2 cells were grown in the transwells to confluency and cultured in DMEM with various glutamine concentration (0 – 50 mM) in the absence of glucose. Apical administration of glutamine in Caco‐2 cells displayed a dose‐dependent drop of TER at 24 hours with no changes of the apical‐to‐basolateral FITC‐labeled dextran 4000 flux. Immunofluorescence staining of tight junction protein zonula occludens (ZO)‐1 and claudin‐1 showed lateral undulations and punctate of the structure accompanied with enlargement of cellular and nuclear size in Caco‐2 cells treated with glutamine (50 mM). Quantification of undulations and cell size revealed significant changes of tight junctional morphological after glutamine treatment in Caco‐2 cells. Decreased protein levels of ZO‐1, but not claudin‐1, were found in detergent‐insoluble fractions of cell preparation of Western blotting. The decline of TER and alteration of the tight junctional structure was not influencing the cell viability. However, the addition of glutamate, which is the amino acid produced during the first step in glutamine catabolism, has no impact on TER. In conclusion, these results suggested that the enteral administration of glutamine may play a role in the modulation of tight junction dynamics in colorectal cancer cells.