bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chemotherapy Induces Pancreatic Cancer Organoid Immune Evasion by Upregulating Programmed Death Ligand 1
Authors: Holokai Loryn , Chakrabarti Jayati , Chang Julie , Lanfranca Mirna Perusina , Wang Jiang , Frankel Timothy , Ahmad Syed , Zavros Yana 
Published: June 2021

BackgroundPancreatic Ductal Adenocarcinoma (PDAC) has a 5‐year survival rate of only 8%. This is due to the lack of targeted therapies and a poor response to chemotherapeutics. PDAC cells evade the immune response by expressing programmed death ligand 1 (PD‐L1), which is able to inhibit cytotoxic T lymphocyte (CTL) proliferation and effector function. Currently, there are no reliable preclinical models that allow us to test the efficacy of chemotherapeutics or PD‐L1 inhibitors. This evidence warrants the development of a preclinical model whereby the patient's immune response within the tumor microenvironment is tested prior to therapy.HypothesisTreatment of PDAC‐derived organoids (huTPOs) with chemotherapeutics induces the expression of PD‐L1 leading to increased immune evasion and tumor growth.MethodsHuTPOs were derived from resected pancreatic tumors of PDAC patients. Conditioned media was collected from cultured huTPOs (huTPOCM). Peripheral blood mononuclear cells (PBMCs) were extracted from autologous patient blood. Dendritic cells (DCs) were cultured in a cytokine cocktail and sorted by flow cytometry for high HLA‐DR expression. CD8+ CTLs were isolated from patient PBMCs using a CD8+ T Cell Enrichment Kit. DCs were then pulsed with huTPOCM for 24 hours and co‐cultured with autologous isolated CTLs for 72 hours. HuTPOs were co‐cultured with autologous‐pulsed DCs and activated CTLs for 24 hours. The cultures were then treated with the chemotherapeutic agent(s) with which the patient was treated, with or without PD‐L1 inhibitor and apoptosis was analyzed by TUNEL assay. In a separate series of experiments, a 72‐hour time course was performed using huTPOs treated with combinations of chemotherapeutics with which the patients were treated.Results

HuTPOs mimic patient response to chemotherapeutics. HuTPOs exhibited divergent responses to chemotherapeutic agents including FOLFIRNOX (oxaliplatin, leucovorin, irinotecan and 5‐FU) or gemcitabine and abraxane. HuTPO response correlated with the patient's tumor response to similar chemotherapeutic treatment.
HuTPOs express increased PD‐L1 expression following chemotherapy treatment: Following chemotherapy treatment, huTPOs expressed increased PD‐L1 compared to untreated huTPOs. HuTPOs that expressed PD‐L1 were protected from CTL mediated apoptosis in an autologous huTPOs/immune cell co‐culture except when treated with PD‐L1 inhibitor.
ConclusionThe current patient‐derived approach is expected to create a personalized model system that can be used to determine the most efficacious chemotherapeutic treatment as well as response to PD‐L1 inhibitors in PDAC patients.Support or Funding InformationSteven Goldman Memorial Pancreatic Cancer Research Grant (Zavros) 1RO1DK083402This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inactivation of Wnt/β‐catenin signaling pathway by SIRT 6 inhibits tumor growth and proliferation in hepatocellular carcinoma
Authors: Jin Hua , Kim Soo Mi 
Published: June 2021

Sirtuins are a family of NAD+‐dependent protein involved in regulating the lifespan and metabolic homeostasis. Sirtuin 6 (SIRT6) has been reported to have tumor suppressing effect in liver. However, the precise molecular mechanism of SIRT6 in human hepatocellular carcinoma (HCC) has not been clearly understood. Therefore, we have investigated the importance of SIRT6 function in HCC cell lines, HepG2 and SNU449. SIRT6 was highly expressed in human HCC cells. Overexpression of SIRT6 significantly diminished the viability of HCC cells whereas silencing of SIRT6 stimulated the viability of HCC cells. Overexpression of SIRT6 increased expression of cleaved‐PARP and cleaved‐caspase9 and decreased the PARP, caspase9, and caspase3. Knockdown of SIRT6 increased the number and size of colonies. In addition, overexpression of SIRT6 significantly inhibited the invasion and metastasis of HCC cells whereas silencing of SIRT6 increased the invasion and metastasis abilities of HCC cells in a time dependent manner. Moreover, overexpression of SIRT6 inhibited vimentin, UPA, and MMP9 protein levels while silencing of SIRT6 in HCC cells increased the protein levels of vimentin, UPA, and MMP9. P‐β‐catenin levels was increased by overexpression of SIRT6 and was decreased by silencing of SIRT6. These data suggest that SIRT6 inhibits the proliferation, invasion and metastasis of HCC cells and may play as a tumor suppressor in HCC cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion of claudin‐7 disrupts epithelial cell self‐renewal in mouse colon
Authors: Xing Tiaosi , Benderman Lesley Jasmine , Sabu Stephiya , Chen Yanghua 
Published: June 2021

Tight junctions are one of the physical barriers protecting the body from invasion of intestinal lumen pathogens. Disruption of tight junctions has been reported in a variety of intestinal disorders, such as intestinal bowel disease, necrotizing enterocolitis, and colon rectal cancer. Claudin‐7 is one of the tight junction membrane proteins and is essential in maintaining intestinal epithelial integrity. However, the function of claudin‐7 in colonic epithelial homeostasis remains to be determined. Here we report that claudin‐7 is required for maintaining the balance of intestinal epithelial cell proliferation and differentiation. To dissect the role of claudin‐7 in colonic epithelium, we used our global (gCldn7−/−) and intestinal‐specific (cCldn7−/−) claudin‐7 knockout (KO) mice that are generated recently as our model systems. Using the histological analysis, we found that enterocytes and enteroendocrine cells are significantly reduced in postnatal day 4 gCldn7−/− and 2–4 months old cCldn7−/− mouse colons. Although the absolute number of Goblet cells and tuft cells are reduced in claudin‐7 KO colon, the ratio of Goblet cells or tuft cells versus the total number of epithelial cells remains unchanged. These results are confirmed by qRT‐PCR and Western blotting, suggesting that epithelial cell differentiation was significantly altered after claudin‐7 deletion. In addition, the ki67 and PCNA positive proliferative cells are significantly increased in claudin‐7 KO colons and no longer limited to the basal‐lateral crypt region, where epithelial stem cells and fate‐committed progenitor cells are located. Genome‐wide gene‐expression microarray analysis of Cldn7+/+ and gCldn7−/− colons identified a list of gene expression changes related to cell proliferation and differentiation. Notch and Hippo signaling pathway are suppressed after claudin‐7 deletion. These findings suggest that claudin‐7 may have a novel, unidentified function regulating epithelial cell differentiation and self‐renewal in mouse colon.Support or Funding InformationThis study is supported by the National Institute of Health grant DK103166.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
H1/H2 histamine receptor blockade lowers substrate‐dependent mitochondrial H2O2 emission in deep gastrocnemius muscle following a bout of prolonged exercise
Authors: Davidson Laura R. , Wallace Helen Elizabeth , Bell Mackenzie CW. , Brebner Karen , Kane Daniel A. 
Published: June 2021

Prolonged exercise increases oxidative metabolism in skeletal muscle mitochondria, which is facilitated, in part, by increased blood flow and delivery of nutrients and oxygen. Histamine mediates many acute exercise responses, including sustained vasodilation and associated delivery of substrates to muscle. We tested the hypothesis that H1 and H2 histamine receptor blockade would attenuate exercise‐induced changes in skeletal muscle mitochondrial function. Adult male Wistar rats (75–94 days old; 441.2 ± 39.5 g) were randomly assigned to one of three groups (n = 12/group): exercise, in the form of one hour of continuous treadmill running at 20 cm/sec (Group 1); mass‐specific oral gavage with H1 antagonist fexofenadine (7.11 mg/kg) and H2 antagonist ranitidine (3.95 mg/kg) one hour prior to the same exercise protocol (Group 2); control, which received neither antihistamines nor exercise (Group 3). An hour post exercise, mitochondrial function was assessed in saponin‐permeabilized myofibers from deep gastrocnemius (mixed fiber types) and soleus (oxidative) skeletal muscle. A sequential substrate‐inhibitor titration protocol was used to examine multiple mitochondrial metabolic pathways (i.e., malate + octanoyl‐carnitine, ADP, lactate + NAD+, pyruvate, glutamate, succinate, inhibitors of antioxidant defense glutathione peroxidase and thioredoxin). Substrate‐dependent oxygen consumption (JO2; high‐resolution respirometry) was not different between treatments; however, H1 and H2 histamine receptor blockade significantly decreased hydrogen peroxide emission (mH2O2; fluorometric monitoring of Amplex Red oxidation) in deep gastrocnemius fibres (P < .05). Given the widespread and routine use of antihistamines, further research into the effects of these antagonists on mitochondrial function, in the context of exercise, is warranted.Support or Funding InformationThe Natural Sciences and Engineering Research Council of Canada (NSERC), The Nova Scotia Health Research Foundation (NSHRF), and Canada Foundation for Innovation (CFI)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Pathological Mitochondrial Fission Restores DSS Associated Respiratory Impairments in an Intestinal Epithelial Cell Line
Authors: Goudie Luke J. L. , Mancini Nicole , Blote Karen R. , Wang Arthur , McKay Derek M. , Shearer Jane 
Published: June 2021

SignificanceRecent evidence has shown that mitochondrial dynamics and more specifically, mitochondrial fission, play an important role in numerous disease of energy intensive tissues. In particular, mitochondrial fission mediated by the enzyme dynamin related protein‐1 (DRP1) and the receptor fission protein‐1 (Fis1) is associated with reduced ATP production, ROS generation and inflammation. Examination of this interaction and corresponding pathological mitochondrial fission has yet to be examined in the colon in the context of inflammatory bowel disease.ObjectiveTo identify if a common murine colitis agent, dextran sodium sulfate (DSS), induces mitochondrial respiration dysfunction and if the novel peptide (P110), can restore these deficiencies by inhibiting DRP1 and Fis1 mediated fission.DesignThe murine intestinal epithelial cell line, IEC4.1, was separated into control, P110, DSS and DSS + P110. Cell were seeded and allowed to incubate for 24 hrs before receiving their respective treatments and further incubation for another 24hrs.MethodsOptimization of both DSS (1–2w./v.%) and P110 (0.5–1.5 uM) dosage and exposure time was performed with the AlamarBlue cell proliferation assay. Basal, mitochondrial complex and maximal respiratory activity were assessed using the Oxygraph‐2k (OROBOROS Instruments, Austria). Respiratory function of cells under various substrate pathways was also examined.ResultsDSS (2%) significantly reduced cell proliferation compared to control and P110 groups (p<0.05). Administration of P110 in 2% DSS cells dose dependently increased cell proliferation, with 1.5 uM P110 exhibiting cell proliferation similar to control. Analysis of mitochondrial complexes revealed that DSS reduced basal, complex I, II, IV and maximal activity (p<0.05). P110 was found to restore DSS associated respiratory deficiencies in complex I, II and IV (p<0.05).ConclusionThis study highlights novel findings regarding the role of mitochondrial fission in a model system of epithelial dysfunction. Importantly, we show i) evidence that DRP1 and Fis1 mediated fission hold potential; pathological consequences in energetically demanding tissues like the colon and ii) P110 treatment reduces DSS induced damage and its associated respiratory deficiencies. These results suggest mitochondrial dysfunction could be intricately involved in gut barrier deterioration seen in inflammatory bowel disease.Support or Funding InformationCrohn's and Colitis Canada. The authors wish to thank Daria Mochly‐Rosen for donating P110.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deep Proteomic Quantification of Inner Medullary Collecting Duct Protein Phosphorylation: Response to Vasopressin
Authors: Deshpande Venkatesh , Chou Chung‐Lin , Knepper Mark 
Published: June 2021

Progress in protein mass spectrometry instrumentation and labeling methods have led to dramatic increases in the depth of phospho‐peptide identification and quantification. Here we used tandem mass tag (TMT) labeling and LC‐MS/MS for deep identification of phospho‐proteomic responses to the vasopressin analog dDAVP (1 nM for 30 minutes) in inner medullary collecting duct (IMCD) suspensions from rats. We analyzed three pairs of samples (dDAVP vs. vehicle) on different days. The analysis yielded 16 GB of spectral data mapping to 11,658 phospho‐peptides in 1758 distinct proteins. Vehicle vs. vehicle comparisons allowed assessment of the variability of the method and permitted dual criteria to be applied for stringent identification of abundance changes (based on t‐statistic and z‐score). Among all phospho‐peptides quantified, only 93 were increased in abundance, and only 49 were decreased, indicating very selective regulation within the broad range of phospho‐peptides quantified. A substantial number of the increases were in aquaporin‐2 and the UT‐A urea channel, including a previously unidentified phosphorylation site at Ser918 of UT‐A1 (sequence RAS*MITK) that was more than doubled in abundance. Abundance changes in aquaporin‐2 peptides confirmed prior findings. Of interest, phosphorylation changes in Raf1 (increased phosphorylation at Ser259, a kinase inhibitory site) and A‐Raf (decreased phosphorylation of Ser255, a kinase activating site) provide an explanation for findings that vasopressin decreases ERK activation and AQP2 phosphorylation at Ser261 in the collecting duct. Finally, vasopressin significantly increased phosphorylation of protein kinase D1 (Prkd1) at Ser255 and of phosphatidylinositol 4‐kinase beta at Ser551. Both proteins play central roles in regulated exocytosis of ENaC, and we speculate that they could play similar roles in vasopressin‐stimulated exocytotic apical insertion of aquaporin‐2 and UT‐A1.Support or Funding InformationV.D. was a member of the 2017 Biomedical Engineering Student Internship Program at the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Myosins II and VI in Mediating Hormonal Regulation of NHE3 Activity in the Rat Renal Proximal Tubule
Authors: Crajoinas Renato Oliveira , Castelo‐Branco Regiane Cardoso , Polidoro Juliano Zequini , Ralph Donna Lee , Malnic Gerhard , McDonough Alicia Ann , Girardi Adriana Castello Costa 
Published: June 2021

Rapid regulation of NHE3 activity may occur through redistribution of the transporter between the body and the base of the proximal tubule microvilli (PT MV). Myosins are motor proteins that move along actin filaments. The present study tested the hypothesis that myosins II (myo II), which moves cargo away from cell center, and VI (myo VI), which moves cargo toward cell center, may mediate NHE3 redistribution within the MV in response to stimuli. Renal PT NHE3 activity was evaluated in Wistar rats by net bicarbonate reabsorption (JHCO3−) in the presence or absence of 10−10 M angiotensin II (Ang II), 10−4 M forskolin (FSK), a cAMP‐elevating agent, 10−5 M blebbistatin (a specific myo II inhibitor) or 4×10−6 M 2,4,6‐triiodophenol (TIP) (a myo VI inhibitor). As expected, Ang II increased [4.36 ± 0.18 nmol/cm2.s (N = 5)] whereas FSK reduced [1.26 ± 0.10 nmol/cm2.s (N = 5)] JHCO3− compared to CTRL [2.74 ± 0.13 nmol/cm2.s (N = 4)]. Simultaneous tubular perfusion of Ang II with blebbistatin prevent Ang II‐induced NHE3 stimulation [2.92 ± 0.14 nmol/cm2.s (N = 5), P < 0.001]. Simultaneous tubular perfusion of FSK with TIP reduced the inhibition with FSK alone [1.78 ± 0.15 (N = 4) vs. 1.26 ± 0.10 nmol/cm2.s (N = 5), P < 0.05] on JHCO3−. The localization of NHE3 and myosins IIB and VI was assessed by confocal immunofluorescence in Wistar rats kidney after a 30 min pressor dose of Ang II (50 ng/kg/min). NHE3 and myo VI shift from base to body of MV after Ang II; myo IIB remains mostly enriched at the MV base. To assess NHE3 activity in OKP cells, experimental pH recovery points were fitted to simple exponential decay curves (y = A·e(−kt) + y0) in order to predict the rate constant (k) of each recovery curve. FSK reduced NHE3 activity [0.025 ± 0.003 s−1 (N = 4)] compared to CTRL [0.048 ± 0.007 s−1 (N = 5)]. siRNA for myo VI efficiently reduced its expression by 64 %; the inhibitory effect of FSK on NHE3 activity was completely abolished by siRNA myo VI [0.043 ± 0.005 s−1 (N = 4)]. These results provide further evidence for myosin II and VI regulation of NHE3 redistribution in the rat kidney proximal tubule.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
cGMP Stimulates K48‐linked Poly‐ubiquitination of NKCC2 in Rats Thick Ascending Limb
Authors: Ares Gustavo 
Published: June 2021

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter NKCC2. The second messenger cyclic guanosine monophosphate (cGMP) decreases NKCC2 activity by decreasing surface NKCC2 levels. We showed that NKCC2 is constitutively ubiquitinated and degraded by the proteasome, and that cGMP enhances the rate of ubiquitination. Ubiquitination is a post‐translational modification in which the C‐terminus of the ubiquitin is attached to a substrate protein. Poly‐ubiquitination occurs when the C‐terminus of another ubiquitin, is then linked to the previously added ubiquitin, creating a chain. There are 7 lysine (k) in the ubiquitin molecule where poly‐ubiquitination can occur. K48‐linked poly‐ubiquitination on ubiquitin is associated with proteasomal degradation. Therefore, we hypothesized what cGMP stimulates K48‐linked poly‐ubiquitination of NKCC2 in rats TAL. To test our hypothesis we immunoprecipitated K48‐linked poly‐ubiquitin using Tandem Ubiquitin Binding Entities (TUBEs) and western blot against NKCC2 in Sprague‐Dawley rat TAL suspensions. First, we performed a k48‐TUBEs concentration‐response curve against K48‐linked poly‐ubiquitination of NKCC2 to ensure linear relationship. TALs suspension were aliquoted in 4 samples and placed at 37°C for 30 minutes in the presence of the proteasomal inhibitor (MG132 20μM) to stop protein degradation. TALs were lysed at 4°C and excess amount of protein lysate (150 μg) was incubated with increasing concentration of k48‐TUBEs. We obtained a linear increase in K48‐linked poly‐ubiquitination of NKCC2 with increasing concentration of k48‐TUBEs. Next, to study the effect of cGMP on k48‐ubiquitination of NKCC2. The conditions were set to fit within the linear range (80 μg of protein) to ensure complete immunoprecipitation of K48‐linked poly‐ubiquitination of NKCC2. We found that the membrane‐permeant cGMP analogue dibutiryl cGMP (db‐cGMP) stimulates K48‐ubiquitination on NKCC2 in a concentration‐dependent manner (baseline: 100.0%; db‐cGMP 100 μM: 115.3 ± 3.7%; db‐cGMP 250 μM: 127.8 ± 5.2%; db‐cGMP 500 μM: 148.3 ± 18.2% p<0.05). These data indicates that cGMP stimulates K48‐linked poly‐ubiquitination of NKCC2, and suggest a triggering mechanism for NKCC2 degradation by the proteasome. We conclude that cGMP increases K48‐linked poly‐ubiquitination of NKCC2. To our knowledge, this is the first piece of evidence showing that cGMP may trigger a signal for NKCC2 degradation through stimulation of k48‐ubiquitination.Support or Funding InformationAHA Post‐Doctoral (14POST20110009)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic High Potassium Diet Decreases Thiazide‐Sensitive Sodium Excretion in High Fat‐Fed Mice
Authors: Nizar Jonathan , Vo Vianna , Bhalla Vivek 
Published: June 2021

High fat‐fed mice recapitulate several features of the Metabolic Syndrome, including impaired sodium excretion and increased blood pressure. In this model, post‐thiazide sodium excretion is increased compared to low fat‐fed controls, providing evidence that increased Na‐Cl co‐transporter (NCC)‐mediated transport may contribute to both impaired sodium excretion and hypertension in the Metabolic Syndrome. We sought to test whether a chronic, high potassium diet regulates NCC differently in high fat‐fed mice compared to low fat‐fed controls.We fed high fat‐fed mice either a normal (0.7% w/w) or high (5% w/w) potassium diet for 1 week then performed an intraperitoneal injection either vehicle or 30 mg/kg hydrochlorothiazide and collected urine for 4 hours. We then switched the dietary potassium content of both groups and rechallenged the mice with the same drugs. After a washout period, we sacrificed mice for tissue analysis.While on a high potassium diet, post‐thiazide urinary sodium excretion was no different from vehicle and significantly lower than excretion on a normal potassium diet (0.164 +/− 0.024 vs. 0.026 +/− 0.005 μmol/min, normal vs. high potassium diet, respectively; p<0.001). Post‐thiazide potassium excretion was no different on either diet (0.138 +/0 0.017 vs. 0.109 +/− 0.006 μmol/min normal vs. high potassium diet, respective; p = 0.28) and higher than vehicle. Urine flow rate was no different while on either normal or high potassium diet after vehicle or thiazide injection. Membrane preparations of whole kidney lysate blotted for both total and phosphorylated NCC were not significantly different in high fat‐fed mice on a normal vs. high potassium diet. High fat‐fed mice do not demonstrate the high potassium diet‐induced repression of NCC phosphorylation that is well‐documented in low fat‐fed mice, and have detectable, albeit diminished sodium excretion. These data support a role for attenuated repression of the potassium‐NCC axis in engendering impaired sodium excretion and hypertension in this model of Metabolic Syndrome.Support or Funding InformationJMN receives support from the NIH (1K08 DK114567‐01). JMN was previously supported by an American Heart Association Postdoctoral Fellowship (16POST27770003). VB receives support from the NIH (1R01 DK091565).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.