bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary Restriction and Toremifene on PhIP induced Carcinogenesis in rats
Authors: Canene‐Adams Kirstie , Sfanos Karen S. , De Marzo Angelo M.
Published: June 2021

Toremifene inhibits estrogen receptor (ER) alpha which is found in prostate stroma and epithelium, recurring prostate cancer, and metastasis. Exposure to 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), the cooked meat heterocyclic amine, results in binding to ER alpha and cancer. Our hypothesis is the estrogenic effects of PhIP are responsible for carcinogenicity and blocking ERα via toremifene would prevent the effects of PhIP. We provided PhIP (70mg/kg) with or without toremifene (~8.6 mg/kg) in AIN93G diets to male Fischer rats for 20 weeks and then AIN93G diet for 32 weeks before collecting prostates, seminal vesicles, testes, skin, and intestines. Toremifene + PhIP rats consumed 37% less calories than PhIP rats thus a 37% dietary restricted (DR) + PhIP group was used as a control. Decreases in prostate and seminal vesicle weights, histological changes, and lack of mature sperm in the testes’ tubules were seen with toremifene. Ventral prostates showed decreased androgen receptor levels with toremifene, while testes showed no positivity. CK5 increased with toremifene, whereas PhIP had the opposite effect. PhIP rats had the greatest incidences of skin, intestinal, and prostate carcinomas. While some reductions were seen with DR the greatest effects were in rats given toremifene. We conclude that in the rat toremifene has marked anti‐androgenic and anticancer effects.
Phase angle in cancer predicts Quality of Life and prognosis
Authors: Almeida Ana Isabel , Ferreira Catarina , Grillo Isabel Monteiro , Camilo Maria , Ravasco Paula
Published: June 2021

Rationale

Phase angle (PA) in cancer has been suggested as a potential prognostic disease indicator &, as a consequence, is likely to predict patients’ well‐being & Quality of Life (QoL), a gold standard for any clinical intervention. We aimed to assess the value of PA in predicting cancer patients’ QoL.

Methods

51 ambulatory cancer pts were evaluated. PA was assessed by bioelectrical impedance analysis (Biodynamics 450®) & compared with age/sex reference percentiles. QoL was assessed by the European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire. Comparisons were made using non‐parametric tests.

Results

PA<5th percentile was found in 16% of pts. Poorer SRHS was associated with PA<5th percentile (p<0.005); no statistically significant difference was found for global QoL score (p=0.07). PA<5th percentile did predict worse physical, role, emotional & social (p<0.05). Anorexia & insomnia were worse in pts with PA<5th percentile (p<0.05).

Conclusion

PA<5th percentile did predict poorer SRHS & anticipated impaired functional scores & worse symptoms. Thus, the PA 5th percentile cut‐off may allow the identification of pts with worse QoL dimensions, SRHS & symptoms, factors associated with poorer nutritional status & intake, all proven to be improved by individualised nutritional intervention, corroborating the need for adjuvant nutrition as therapy. No research support.
Weight Loss and other Adverse 0utcomes for Head and Neck Cancer Patients Undergoing Treatment
Authors: Hertroijs Dorijn , Platek Mary , Singh Anurag
Published: June 2021

Purpose

To describe the prevalence of weight loss, dysphagia and esophageal stricture in patients with squamous cell carcinoma of the head and neck (SCCHN) undergoing concurrent chemoradiation (CCRT) and to examine pretreatment weight status as a predictor of local regional progression (LRP).

Methods

Retrospective review of 140 SCCHN patients definitively treated with CCRT from 2004 through 2010 at Roswell Park Cancer Institute. Patients were offered a percutaneous endoscopic gastrostomy (PEG) tube. Nutrition follow‐up was by referral only. Patient information was collected from the electronic medical record.

Results

Approximately 93% had dysphagia, 19% had esophageal stricture and 22% with strictures required dilatation. Median pretreatment ideal body weight percentage (IBW%) was significantly different between patients without LRP (118%) and with LRP (n=18) (101.5%) (p=0.02), but was not an independent predictor of LRP when tested in a multivariate regression model. Both groups suffered weight loss of 9% during treatment despite nutrition support.

Conclusions

Pretreatment weight status, a very crude indicator of nutrition status, was not an independent predictor of LRP but may have prognostic value. Next steps include validated malnutrition screening and the testing of evidence based nutrition protocols to minimize weight loss and manage dysphagia.

Research support: Nutritional Cancer Institute
Bioelectrical impedance analysis (BIA) and phase angle: how relevant in cancer?
Authors: Almeida Ana Isabel , Ferreira Catarina , Grillo Isabel Monteiro , Camilo Maria , Ravasco Paula
Published: June 2021

We aim to characterise body composition (BC), concomitantly with phase angle (PA) & explore potential associations with histological aggressiveness & stage. We included 51 pts with solid tumours. BMI was calculated & categorised according to WHO's criteria; %body fat mass (%FM) & PA were assessed by BIA (Biodynamics 450®); %FM & PA were compared with age/sex reference values: percentage intervals & percentiles, respectively. Stages III/IV & moderately/poorly differentiated cancers were prevalent: 55% & 53%, respectively. By BMI, 61% pts were overweight/obese vs 2% underweight. Excessive FM was prevalent (67%) & also found in pts with normal BMI. PA<5th percentile prevalence was of 16%. The prevalence of stage III/IV & moderate/low differentiated cancers was similar in normal BMI/FM as it was in obesity/high FM. Regarding PA, 86% & 83% pts with PA<5th percentile had advanced & aggressive cancers, respectively vs pts with PA>;5th percentile: 54% & 60% had advanced & aggressive cancers, respectively. Excessive FM by BIA was prevalent & underestimated by BMI. Advanced stage & aggressive cancers, indicators of worse disease status, were related with a lower PA. BC analysis & PA determination, both simple & quick for routine use, seem to bear a high clinical relevance. These preliminary results support the continuation of this study for additional analyses. No research support.
Decorin expression is regulated by LPS and sodium salicylate in 3T3‐L1 Adipocytes
Authors: Ward Meliza , Ajuwon Kolapo M
Published: June 2021

Obesity is caused by an increase in adipose tissue mass. Extracellular matrix (ECM) within adipose tissue can impact adipose tissue function. Obesity is characterized as a state of low‐grade, chronic inflammation. Small leucine‐rich‐proteoglycans (SLRPs) are ECM proteins that are play important roles in regulation of inflammation. We determined whether inflammation regulates the expression of SLRPs in adipocytes and preadipocytes. 3T3‐L1 preadipocytes and adipocytes were treated with varying concentrations of lipopolysaccharide (LPS). We show a dose‐dependent increase in decorin transcript expression with LPS treatment in both preadipocytes and adipocytes. Additionally, LPS treatment at 100ng/ml increased protein levels of decorin in differentiated 3T3‐L1 adipocytes. Sodium salicylate is an anti‐inflammatory compound that inhibits NFêB activation. Sodium salicylate (10mM) treatment led to a suppression of decorin transcript abundance compared to untreated cells (p<0.05). Sodium salicylate also partially suppressed LPS‐induced decorin expression. This data suggests that the expression of decorin is influenced by inflammation and decorin may be a potential target of obesity‐related inflammation.
The Effects of High Dietary Iron and Radiation Exposure on Markers of Oxidative Stress and Immune Status of Rats
Authors: Morgan Jennifer L. L. , Crucian Brian E. , Sams Clarence F. , Smith Scott M. , Zwart Sara R.
Published: June 2021

During space flight astronauts are exposed to increased iron stores and radiation. These can independently cause oxidative damage leading to negative health effects including heart disease, cataracts, and cancer. We investigated the combined effects of high dietary iron (650 mg iron/kg diet vs. 45 mg iron/kg diet for controls) and radiation exposure (0.375 Gy of Cs‐137 every other day for 16 days, a total of 3 Gy) on markers of oxidative stress and immune system variables in Sprague‐Dawley rats (n=8/group). Oxidative stress was affected by both treatments, evidenced by increased liver glutathione peroxidase (P < 0.01) and serum catalase (P < 0.01). Effects of iron intake and radiation on the white blood cell count interacted significantly (P < 0.05), with iron increasing the cell count only in the non‐irradiated group. The T‐cell CD4:CD8 ratio was decreased by radiation (P < 0.0001). When various leukocyte subsets from treated rats were stimulated with mitogens, their cytokine production profiles were altered. Serum iron concentration was negatively correlated with monocyte production of cytokines after LPS stimulation. Both dietary iron and radiation, alone and interactively, contributed to increased oxidative stress and resulted in overall immune system alterations, which could contribute negatively to astronaut health during space flight.

Grant Funding Source: Funded by the NASA Human Research Program.
Immunomodulatory effect of Cnidium officinale Makino extract in murine peritoneal macrophages and splenocytes
Authors: de la Cruz Joseph Flores , Lee Woo Sik , Hwang Seong Gu
Published: June 2021

Over the past years, there has been growing interest in the field of herbal medicine research for plant‐derived health promoting products. Cnidium officinale Makino (COM) called “Chun Gung” in Korea is one of the fundamental plants that have been used as a traditional medicinal plant in Asia for a long time. Current study investigates the biological effect and action mechanism of the ethanol extract from COM on the release of inflammatory mediators in murine peritoneal macrophages and splenocytes. Our results showed that COM (up to 0.40 mg/ml) dose‐dependently increased splenocyte proliferation with or without mitogen stimulation. In RT‐PCR analysis, COM inhibited the production of proinflammatory mediators; TNF‐α, IL‐1β, IL‐6, and nitric oxide (NO) in LPS and Con A stimulated macrophages and splenocytes. On the other hand, IL‐2 and INF‐γ expression was significantly increased by COM treatment. Flow Cytometry Analysis (FACS) demonstrated a dose dependent increase in the T cell and B cell population after COM treatment. The present results indicate that COM extract showed evidences of immunomodulating activity through stimulated immune cell growth, modulating Th1/Th2 cytokine production and anti‐inflammatory effect which may have potential use as an immunotherapeutic agent.
Fish oil supplementation ameliorates leukopenia and thrombocytopenia and modulates nitric oxide and TNF‐α production during the acute phase of Trypanosoma cruzi infection
Authors: Lovo‐Martins Maria Isabel , Malvezi Aparecida Donizette , Silva Rosiane Valeriano da , Zanluqui Nagela Ghabdan , Tatakihara Vera Lúcia Hideko , de Oliveira Ana Paula Ligeiro , Peron Jean PS , Camara Neils Olsen Saraiva , Fritsche Kevin L , Pinge‐Filho Phileno
Published: June 2021

Omega‐3 polyunsaturated fatty acids (n‐3 PUFA) supplementation has been reported to reduce inflammation and in some circumstances diminish infectious disease resistance in rodents. We investigated whether supplementation with fish oil, a source of n‐3 PUFA, impaired host resistance against Trypanosoma cruzi, the causal agent of Chagas’ disease. C57BL/6 mice were supplemented (0.6% v/w) by gavage with phosphate buffer saline (PBS), corn oil (CO) or fish oil (FO) for 15 days prior to T. cruzi challenge and throughout the course of the infection. In this study, we observed an increase in spleen weight and cellularity in the infected‐FO supplemented mice. Importantly, FO supplementation in infected mice improved platelets and leukocytes counts, decreased heart parasitism, decreased nitric oxide production in plasma and cardiac tissue, and increased TNF‐α production by spleen cells stimulated with parasite antigen. These data suggest the potential of FO supplementation to attenuate the clinical course of T. cruzi infection and further studies are warranted.
Homocysteine Levels are Associated with HIV Disease Progression in HIV+ Adults in Miami
Authors: Martinez Sabrina S , Campa Adriana , Li Yinghui , Boyer Jacqueline Hernandez , Williams Shawn , Barr Shakira , Nagi Gagandeep , Greer Pedro , Gracia Deborah , Baum Marianna K
Published: June 2021

Objective

To examine the relationship of homocysteine levels to alcohol AUDIT score and HIV disease progression in HIV+ adults.

Methods

After consenting 235 participants, blood was drawn for homocysteine. Hyperhomocysteinemia was defined as >;12μM/L. CD4 count and viral load were obtained from medical records. Questionnaires on demographics, antiretroviral therapy (ART) and the Alcohol Use Disorders Identification Test (AUDIT) were completed.

Results

Mean age was 44.9±8.1 years, mean CD4 count was 499.1±341.3 cells/μL and 66.0% were male. The prevalence of hyperhomocysteninemia was 23.4% (N=56). Homocysteine levels were correlated with viral load (r=0.146, p=0.03) and AUDIT score (r=0.167, p=0.011). In multivariate linear regression, viral load remained significant after controlling for AUDIT score, age and gender (β=.188, p=0.006). Higher homocysteine were seen in those with CD4 cell count <200 cells/μL (11.2 vs. 9.8 μM/L, p=0.024) and those not currently on ART (10.4 vs. 9.4 μM/L, p=0.049).

Conclusions

In this HIV+ cohort, homocysteine was associated with HIV disease progression. Further studies on mechanisms through which homocysteine contributes to HIV disease progression are needed.

Funded by NIAAA
Myeloid expression of co‐inhibitory receptor ligand, B7‐H1, as a novel biomarker/mediator of septic progression/ morbidity
Authors: Huang Xin , Chen Yaping , Chung Chun‐Shiang , Monaghan Sean F. , Ayala Alfred
Published: June 2021

Recent studies indicate programmed cell death receptor (PD)‐1 plays a significant role in the development of immune suppression associated with sepsis. However, while PD‐1 is reported to interact with two cell surface ligands B7‐H1 and B7‐DC, it is not clear what their role to septic morbidity may be. Here we determine if B7‐H1 contributes to the pathogenesis of sepsis. We report that B7‐H1 is up‐regulated extensively on various immune cells during sepsis and B7‐H1 gene deficiency protected mice from the lethality of sepsis. In terms of histological development of multiple organ damage and inflammatory cytokine levels in circulation or at infectious site, B7‐H1 deficient mice showed a reduction in these indices when compared with WT mice. However, B7‐H1 gene deficiency did not alter bacterial burden when compared to WT mice. In addition, we found that, during sepsis, while there were no marked differences affecting ex vivo macrophage cytokine productive capacity between PD‐1 and B7‐H1 gene deficient mice; preservation of ex vivo macrophage phagocytic function was only seen in septic PD‐1 knockout mice cells. Finally, higher % B7‐H1+ neutrophils in peripheral blood was correlated with lethal outcome. Together, these results indicate B7‐H1 expression contributes to septic morbidity and suggest B7‐H1 on neutrophils could be used as a biomarker to indicate the severity of sepsis.
Role of Nox4 in modulating atherosclerosis in diabetes
Authors: Khandelwal Alok R , Chen Lili , Qin Zhexue , Hou Xiuyun , Xiao Jennifer , Sadoshima Junichi , Tong Xiaoyong
Published: June 2021

Elevated levels of reactive oxygen species (ROS) in the vascular wall play a key role in the development of atherosclerosis. Diabetes is associated with accelerated atherosclerosis and leads to increased mortality rates in patients with vascular complications. NADPH oxidases (Nox) are major ROS generating enzymes in the vasculature. Though the roles of other components of Nox in atherosclerosis are well studied, Nox4 as the most abundant Nox component in endothelium and its roles in atherosclerosis are not well understood. The objective of this study was to elucidate the roles of endothelial Nox4 in diabetes‐induced atherosclerosis. ApoE−/− and EDN ApoE−/− (overexpressing an endothelial dominant negative form of Nox4, P437H) mice at 10 wks of age were administered of either vehicle or streptozotocin (STZ). At 20 weeks of age STZ significantly increased the Oil Red O staining of aortic root lesions in EDN ApoE−/− group compared to the littermate ApoE−/− group, correlating with an increase in vascular cell adhesion molecule‐1 levels in endothelial cells from EDN. Our data indicates that downregulation of Nox4 in the endothelium accelerates atherosclerosis in diabetes, suggesting that Nox4 normally attenuates atherosclerosis in diabetes and the mechanism may be due to maintenance of endothelial homeostasis including inhibition of inflammatory factors.
Gene Sequencing Aids in Differentiation of von Willebrand Disease Type 2B from Platelet‐Type vWD
Authors: Desai Diana Shirish , Lyon Elaine , Rodgers George , Jama Mohamed , Wallentine Steven , Smock Kristi
Published: June 2021

An elderly female with a positive bleeding history (personal and family) and prior but undocumented diagnosis of von Willebrand disease (vWD) presented for surgery. Testing showed mild thrombocytopenia, decreased ristocetin cofactor activity to von Willebrand factor antigen ratio, and absent high‐molecular‐weight multimers, suggesting a qualitative (type 2) subtype. Low‐dose ristocetin‐induced platelet aggregation demonstrated abnormally increased aggregation, consistent with either type 2B vWD or platelet‐type vWD (PT‐vWD). Differentiation between these subtypes guides therapy. Optimal therapy for type 2B vWD consists of vWF replacement, while platelet transfusions are used to treat PT‐vWD. Traditional options for differentiation include vWF/platelet binding assays or specialized platelet aggregation studies. However, these subspecialized tests have limited availability and sample stability. Gene sequencing performed in our laboratory allowed for detection of 1 of 4 known mutations in GP1BA, (c.763A>;G, p.Met255Val, using standard nomenclature) responsible for PT‐VWD. This finding confirmed her diagnosis and appropriate therapy and facilitates diagnosis for affected family members in this disorder with an autosomal dominant inheritance pattern. Gene sequencing is a useful tool to aid in diagnosis and subtyping of type 2 vWD. kristi.smock@aruplab.com
Redox regulation of cysteine‐674 of SERCA 2 is critical for growth factor‐and ischemia‐induced angiogenesis
Authors: Thompson Melissa Danielle , Mei Yu , Hou Xiuyun , Silver Marcy , Bolotina Victoria M. , Cohen Richard A. , Tong Xiaoyong
Published: June 2021

The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) is key to Ca2+ homeostasis and is regulated by post‐translational modification of the cysteine (C) 674 thiol on which glutathione adducts stimulate Ca2+ uptake activity and endothelial cell (EC) angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced by femoral artery ligation irreversibly oxidizes EC SERCA. To determine the role of thiol oxidation, we used a SERCA2 C674S heterozygote knock‐in (SKI) mouse lacking the key thiol. Following hind limb ischemia, SKI animals had impaired blood flow recovery. Cultured SKI microvascular EC showed impaired migration and decreased tube formation. Fura‐2 Ca2+ signaling studies revealed lower Ca2+ stores and decreased VEGF‐ and NO‐induced Ca2+ influx. While VEGF decreased ionomycin‐releasable stores in WT EC, it failed to do so in SKI. Also, hypoxia‐induced expression of pro‐angiogenic genes (VEGF, VEGF receptor and eNOS) was decreased in SKI EC. Taken together, our data indicate that impairing normal redox regulation of the C674 thiol alters EC Ca2+ homeostasis and angiogenic gene expression, suggesting mechanisms by which irreversible SERCA oxidation contributes to worsened angiogenesis during ischemia.
Epigenetic regulation of miR‐122 by PPARgammar and hepatitis B virus X protein in hepatocellular carcinoma cells
Authors: Song Kyoung‐sub , Han Chang , Wu Tong
Published: June 2021

MiR‐122, the most abundant miRNA in liver, has been implicated in several liver diseases including hepatocellular carcinoma (HCC) and hepatitis C and B viral infection. This study aimed to explore epigenetic regulation of miR‐122 in human hepatocellular carcinoma (HCC) cells and to examine the effect of hepatitis C virus (HCV) and hepatitis B virus (HBV). We performed microRNA microarray analysis and identified miR‐122 as the most up‐regulated miRNA (6‐fold) in human hepatocellular cancer cells treated with 5'aza‐2'deoxycytidine (5‐Aza‐CdR, DNA methylation inhibitor) and 4‐phenylbutyric acid (PBA, histone deacetylation inhibitor). Real‐time PCR analysis verified significant upregulation of miR‐122 by 5'aza and PBA in HCC cells, and to a lesser extent in primary hepatocytes. Peroxisome proliferator activated receptor‐gamma (PPARγ) and retinoid X receptor alpha (RXRα) complex was found to be associated with the DR1 and DR2 consensus site in the miR‐122 gene promoter which enhanced miR‐122 gene transcription. 5‐Aza‐CdR and PBA treatment increased the association of PPARγ/RXRα, but decreased the association of its co‐repressors (N‐CoR and SMRT), with the miR‐122 DR1 and DR2 motifs. The aforementioned DNA‐protein complex also contains SUV39H1, a H3K9 histone methyl transferase, which downregulates miR‐122 expression. Our findings establish a novel role of the PPARγ binding complex for epigenetic regulation of miR‐122 in human HCC cells. Moreover, we show that hepatitis B virus X protein (HBX) binds PPARγ and inhibits the transcription of miR‐122, whereas hepatitis C viral particles exhibited no significant effect; these findings provide mechanistic insight into reduction of miR‐122 in patients with HBV but not with HCV infection.
Glypican 3 increases IRS4 phosphorylation and stimulates Hepatocellular Carcinoma cell proliferation
Authors: Ganapathy Ekambaram , Khachatoorian Ronik , French Samuel W
Published: June 2021

Hepatocellular carcinoma (HCC) is a common malignancy worldwide. Glypican 3 (GPC3) is one of the most over‐expressed proteins in HCC and is used as a diagnostic marker. Role of GPC3 in cell growth regulation is demonstrated by overgrowth and organ dysgenesis associated with altered gene expression; however the mechanism is not well characterized. To evaluate the effect of altering GPC3 levels, we used shRNA to examine the molecular and biologic effects of GPC3 suppression in HCC cells in vitro. Also, a mammalian expression vector expressing a Flag‐GPC3 fusion protein was used to understand the role of elevated levels of GPC3. Proteomic mass spectrometry was used to identify the cellular proteins in complex with Flag‐GPC3. We identified Insulin Receptor Substrate 4 (IRS4) as a GPC3 interaction partner. Over expression of GPC3 increased cell proliferation and IRS4 phosphorylation in HCC cells, while knock‐down of GPC3 inhibited cell proliferation and decreased IRS4 phosphorylation in HCC cells. These changes were not seen in 293 cells. IRS4 is associated with HCC tumor growth and cell proliferation. IRS4 is activated by tyrosine phosphorylation and mediates multiple signals following activation of insulin receptors by insulin like growth factor‐I (IGF‐I). Our results indicate that GPC3 modulates IRS4 phosphorylation to enhance HCC cell proliferation.
A Rare Presentation of Intravascular Lymphoma in an HIV patient
Authors: Solomon Sanjeev , Dalal Rutul , Dunlap Shariff , Kurukumbi Mohankumar
Published: June 2021

Background

Intravascular lymphoma (IVL) is an extremely rare form of non‐Hodgkin lymphoma characterized by almost exclusive growth of neoplastic lymphocytes within the blood vessel lumen. IVL is morphologically characterized in most instances by large cells with B‐cell lineage. There is a paucity of literature describing the association between HIV and intravascular lymphoma.

Case Presentation

43‐year‐old female with HIV presented with weakness involving her left arm and leg. Physical examination showed decreased strength in the left lower extremity and impaired sensory perception in the right lower extremity. Neuroimaging revealed a lesion in the spinal cord at the level of C5/6 and diffuse leptomeningeal and patchy enhancement in the left frontal lobe. CSF analysis was significant for positive JC virus PCR. The patient was considered to have progressive multifocal leukoencephalopathy; however, the clinical and neuroimaging findings did not support this diagnosis. Leptomeningeal biopsy showed focal infiltrations of large atypical lymphocytes confined within blood vessels with immunohistochemistry being positive for CD3. The morphological findings were consistent with intravascular diffuse large B cell lymphoma.

Conclusion

This case highlights a rare occurrence of HIV infection and intravascular lymphoma and its inherent atypical presentation and diagnostic challenges.
Depletion of PINK1 mediates mitochondrial dynamics in chronic MPP+ toxicity
Authors: Zhu Jianhui , Dadga Ruben K , Chu Charleen
Published: June 2021

Mitochondrial dysfunction is implicated in sporadic and familial Parkinson disease (PD); however, the regulation of mitochondrial function during PD‐related cellular stress remains unclear. We previously found that low dose chronic MPP+ treatment over 2 weeks caused both fragmentation and enlargement of mitochondria accompanied by suppression of mtDNA encoded protein synthesis. Since the mitochondrial kinase PTEN‐induced kinase 1 (PINK1) may regulate the mitochondrial fission/fusion machinery, we hypothesized that alterations in endogenous PINK1 function contributes to chronic MPP+ induced mitochondrial dysfunction. We found that chronic MPP+ treatment caused a significant decrease in PINK1 expression, which is disproportionately greater than the reduction in other mitochondrial proteins by mitophagy. Both mitochondrial fission and fusion proteins DRP1 and MFN2 were also decreased. Over‐expressing Wt‐PINK restored levels of DRP1, MFN2 and mitochondrial transcription factor A involved in mitochondrial biogenesis. The mechanism underlying PINK1 depletion during chronic MPP+ treatment involved HSP90α phosphorylation through an ERK1/2‐dependent mechanism. Preventing ERK1/2 activation or overexpression of PINK1 ameliorated the mitochondrial fission and fusion protein imbalance in MPP+‐treated cells. In sum, depletion of endogenous PINK1 protein is implicated in chronic MPP+ induced mitochondrial dysfunction. Given that mutations in the PINK1 gene are linked to autosomal recessive parkinsonism, these data support PINK1 loss of function as a possible point of convergence for neurotoxic and genetic causes of PD.

Supported in part by NIH grants NS065789 and AG026389.
Endogenous LRRK2 dimerizes and translocates to novel membrane compartments during monocyte activation
Authors: Schapansky Jason , LaVoie Matthew J
Published: June 2021

Mutations in leucine‐rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD), and recent evidence indicates LRRK2 also plays a significant role in monocyte immune cell function. Our previous research indicated that membrane‐associated LRRK2 dimers are likely the more physiologically relevant species, but the function and action of this post‐translational modification was unknown. Here, we demonstrate activation of macrophage or microglial cell lines results in LRRK2 phosphorylation and dimerization, which then coincided with iNOS expression and nitrate production. This activation also resulted in localization to a novel membrane compartment different from basal membrane LRRK2. Disruption of LRRK2 kinase activity, or dimerization with 2BP, prevented iNOS expression and activity. This research shows that, for the first time, acute changes in dimerization and membrane localization of endogenous LRRK2 for cellular function. This project was funded by the Michael J. Fox Foundation and NIH #NS072604.
Effect of fetal cells of the nervous tissue during postresuscitational encephalopathy in rats of different sex and ages
Authors: Aitbayeva Zhaina
Published: June 2021

The aim of the study was to examine the comparative aspect of the influence of the intraperitoneal injection of fetal cells in the nervous tissue during postresuscitational encephalopathy in rats of different sex and age. For the purposes of investigating the qualitative and quantitative composition of phospholipids lipid extraction of the cerebral cortex was produced based on the method of J. Falchion (Folch J. et al., 1957) and fractionation of phospholipids (PL) based on the thin layer chromatography on silica gel. The study revealed that the intraperitoneal injection of a suspension of fetal neural cells accelerates the rate of replenishment of neurological deficit and increases survival reanimated males of all ages.

The introduction of fetal nerve cells improves the spectral composition of phospholipids in brain homogenate lively males and worsens at reanimated females. Repeated injection of cells of fetal neural tissue leads in all experimental groups to the adverse redistribution of fractions of phospholipids.
Investigation of the utility of the Simport‐Matrix Chaperones for Avian Influenza Virus sample storage and transportation
Authors: Echevarria Martha , Ferro Pamela , Vuong Christine , Metz Katy , Hogan Mike , Lupiani Blanca
Published: June 2021

Due to the transmission of avian influenza virus (AIV) H5N1 from birds to humans, an increase in surveillance studies (SS) occurred. SS are important for discovering potential reservoirs or sources of new strains of AIV that could be a threat to human and animal health. SS depend on sample handling and maintenance of samples at low temperature. Sampling strategies for AIV consists of collecting oropharyngeal, tracheal, or cloacal swabs and placing them in viral transport media maintaining them on ice in the field and during shipping. We evaluate a sample collection system in which maintenance of the cold‐chain is not critical to the preservation of sample in the field during sample collection and transport. We hypothesized that the Simport Matrix‐Chaperone (SMC) system is useful for the storage and transportation of AIV samples. A H5N3 AIV was tested using 3 different matrices: MicroCrystals (MC), MicroSpheres (MS), and Elastomer. Samples reached complete dehydration after 24 h of storage at room temperature. After 3 d of room temperature storage, the samples were retrieved from the matrices and tested for viability of the virus by virus isolation and presence of AIV matrix gene by real‐time RT‐PCR. The AIV matrix gene was detected in all 3 matrices and was isolated from the MS and MC. The SMC system could provide a simple system for collection and transport of samples by reducing the need for maintenance of the cold chain.
End Binding protein 3 regulates calcium signaling and permeability of the endothelial barrier
Authors: Geyer Melissa , Gu Lianzhi , Vogel Stephen M. , Malik Asrar B. , Komarova Yulia A.
Published: June 2021

End binding (EBs) proteins bind growing microtubule (MT) plus ends and promote MT growth. We have recently shown that Vascular Endothelial (VE)‐cadherin outside‐in signaling regulates phosphorylation of EB3 to suppress MT persistent growth and thus allowing for the assembly of adherens junctions (AJs). Depletion of EB3 has no effect on either MT dynamics or formation of (AJs) in confluent endothelial monolayers. However, depletion of EB3 significantly attenuates cell shape change and permeability increase in cells subjected to pro‐inflammatory mediator thrombin. EB3 interacts with inositol 1,4,5‐thriphosphate receptor type 3 (IP3R3) and facilitates agonist‐induced clustering of IP3R3 and IP3‐dependent release of calcium from intracellular stores. Cell permeant variant of IP3R3‐derivative peptide containing EB3 consensus motif reduces the interaction between EB3 and IP3R3 and mitigates increased endothelial permeability response. Furthermore, infusion of this peptide in ex vivo murine lung preparation attenuates increase in lung vascular liquid permeability (Kf,c) in response to activation of the Protease‐Activated Receptor‐1 (PAR‐1). Intravenous injection of this peptide prevents lung injury and reduces the lethality of polymicrobial sepsis in mice. These data indicate that EB3 regulates calcium signaling in endothelium and facilitates pulmonary vascular leak in disease states.

This work was supported by NIH grants R01 HL103922 and Giles F. Filley Memorial Award to Y. K.; R01 HL 45638 to A.B. M.; T32 HL07829–17 support to M. G.
Heterogeneity of ATP‐Sensitive K+ Channels in Cardiac Myocytes: Enrichment at the Intercalated Disk
Authors: Hong Miyoun , Bao L , Kefaloyianni E , Agullo‐Pascual E , Chkourko H , Foster M , Taskin E , Reid D A , Rothenberg E , Delmar M , Coetzee W A
Published: June 2021

Ventricular KATP channels link intracellular energy metabolism to membrane excitability and contractility. We identified plakoglobin (PG) and plakophilin‐2 (PKP2) as KATP channel associated proteins and investigated whether the association of KATP channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte. Co‐immunoprecipitation experiments confirmed physical interaction between KATP channels and PKP2 and PG in rat heart. Immunolocalization experiments demonstrated that KATP channel subunits are expressed at a higher density at the intercalated disk (ICD) in hearts, where they colocalized with PKP2 and PG. Super‐resolution microscopy demonstrate that KATP channels are clustered within nanometer distances from junctional proteins. The local KATP channel density was larger at the cell end when compared to local currents recorded from the cell's center. The KATP channel unitary conductance, block by MgATP and activation by MgADP did not differ between these two locations. Whole‐cell KATP channel current density was ~40% smaller in myocytes from mice haploinsufficient for PKP2. Experiments with excised patches demonstrated that the regional heterogeneity of KATP channels was absent in the PKP2 deficient mice, but the KATP channel unitary conductance and nucleotide sensitivities remained unaltered. Our data demonstrate heterogeneity of KATP channel distribution within a cardiac myocyte. The higher KATP channel density at the ICD implies a possible role at the intercellular junctions during cardiac ischemia. Funding from NIH HL085820.
Angiotensin II receptor synergism with prostaglandin E2 receptors
Authors: Palazzo Maria C. , Breyer Richard M
Published: June 2021

Upon intravenous infusion prostaglandin E2 (PGE2) acts as a systemic vasodepressor in mice and humans. Under certain circumstances however PGE2 may act as a vasopressor. Subthreshold concentrations of vasoconstrictors such as potassium chloride or phenylephrine enhance PGE2‐mediated vasoconstriction. Here we demonstrate using wire myography that angiotensin II (Ang II), a principal effector of the renin‐angiotensin‐ aldosterone‐system, potentiates PGE2‐mediated vasoconstriction in mouse femoral arteries. Using knockout mice and pharmacological inhibitors, we have determined that this potentiation effect is mediated via the angiotensin AT1 receptor and the PGE2 EP3 receptor. Y‐27632, a Rho kinase inhibitor, blocked the Ang II‐evoked potentiation of EP3 action suggesting a role for the Rho‐ROCK pathway in the observed potentiation. Our data support the hypothesis that angiotensin II synergizes with PGE2 to induce vasoconstriction. The significance on blood pressure homeostasis and detailed mechanism of this interaction remains to be determined.

This work was supported by a grant from the NIH NIDDK DK37097(RMB)
Investigation of the D1‐D2 dopamine receptor heteromer reveals a complex signaling mechanism not limited to Gq protein activation
Authors: Chun Lani Sunil , Free R Benjamin , Doyle Trevor B. , Huang Xi‐Ping , Sibley David R.
Published: June 2021

The D1 (D1R) and D2 (D2R) dopamine receptors modulate cAMP levels via activation of Gs/olf and Gi/o proteins, but are also are proposed to form hetero‐oligomers that, when stimulated, initiate Gq‐mediated activation of phospholipase C (PLC) and Ca2+mobilization. In addition, the compound SKF83959 has been proposed to be a D1‐D2 heteromer‐selective agonist and has been used as a functional probe of the heteromer in vivo. We investigated several mechanistic and pharmacological aspects of the proposed D1‐D2 heteromer and showed that concurrent stimulation of the D1R and D2R were required to elicit Ca2+ mobilization. Additionally, both the long and short isoforms of the D2R were able to form functional heteromers with the D1R. However, in contrast to previous reports, SKF83959 partially inhibited the D1‐D2‐mediated Ca2+response and non‐selectively exhibited high affinity for a variety of GPCRs. Finally, while overexpression of Gαq did boost the D1‐D2 mediated Ca2+ response, inhibiting the function of the Gi or Gs proteins, using pertussis toxin or cholera toxin, respectively, drastically reduced the Ca2+ response, as did sequestration of Gβγ subunits using catalytically inactive GRK2 mutants. These data indicate that the mechanism of D1‐D2 mediated Ca2+mobilization cannot be solely attributed to Gq protein activation and that additional downstream, crosstalk pathways are involved. Supported by the NIH IRP.
Cross‐talk between beta and alpha2 adrenergic receptors in sympathetic neurons relies on protein kinase A and spinophilin
Authors: Cottingham Christopher , Lu Roujian , Wang Qin
Published: June 2021

Physiological overlap between β and α2 adrenergic receptors (ARs) in the sympathetic nervous system raises the possibility that adrenergic therapeutics targeting one AR may influence the function of other ARs. The present study is therefore aimed at investigating whether and through what mechanism clinically‐relevant βAR ligands can influence α2AAR responses in noradrenergic sympathetic neurons. Our previous work has established the dendritic scaffolding protein spinophilin (Sp) as a key regulator of α2AAR responses. As well, we have shown that protein kinase A (PKA) phosphorylation of Sp prevents its interaction with the α2AAR, providing a potential avenue for crosstalk with the Gs‐coupled, PKA‐activating βARs. In the present study, we show that βAR agonists accelerate α2AR agonist‐mediated α2AAR internalization and enhance α2AAR MAP kinase signaling response sensitivity in primary sympathetic neurons. Importantly, these effects occur in a PKA‐dependent fashion. We further show that agonist‐mediated α2AAR responses are accelerated/enhanced in Sp‐null neurons and that addition of βAR agonists has no additional effect in the Sp‐null system. Mechanistically, we show that agonist‐mediated α2AAR/Sp interaction is attenuated by the addition of a βAR agonist. Our findings underscore the potential importance of AR cross‐talk to adrenergic pharmacology. Work supported by NIMH (MH081917).
CB1 Cannabinoid Receptor Juxtamembrane C‐terminal Phosphorylation in NT18TG2 Cells
Authors: Eldeeb Khalil , Cowsik Sudha M. , Howlett Allyn C.
Published: June 2021

CB1 receptors primarily stimulate the G protein family of Gi/o. The aim of the present study is to investigate the effect of phosphorylation of the juxtamembrane C‐terminal domain of the CB1 receptor on individual Gi proteins activation in N18TG2 neuroblastoma cell membranes using an antibody‐capture[35S]‐GTPγS scintillation proximity assay (SPA). C‐terminal peptides were incubated with N18TG2 membranes in a reaction mix including [35S]‐GTPγS, NaCl, dithiothreitol and GDP for 1 h at 30 °C then terminated by solubilization of the membrane proteins, followed by incubation with specific antibodies. In N18TG2 membranes, the unphosphorylated peptide (Arg401 to Glt420) was able to stimulate [35S]‐GTPγS binding in a dose‐dependent fashion (30–300μM). When applied at 100μM the unphosphorylated peptide was able to stimulate [35S]‐GTPγS binding mediated by Gi1: Emax = 18%, Gi2: Emax = 34%, Gi3: Emax = 25%, Go: Emax =18% over basal, respectively. We investigated the effect of phosphorylation of the three Ser residues (Ser402, Ser411, and Ser415) (100μM) and found that phosphorylation of Ser402 was able to stimulate [35S]‐GTPγS binding mediated by Gi3: Emax = 33%, Go: Emax = 27% over basal. Phosphorylation of Ser415, was able to stimulate [35S]‐GTPγS binding mediated by Gi3: Emax = 38%, Go: Emax =29% over basal. Phosphorylation of Ser411 was able to stimulate [35S]‐GTPγS binding mediated by Gi3: Emax = 31%, Go: Emax =33.6% over basal. The order of potency for Go activation was pSer411>;pSer415>;pSer402>;unphosphorylated and for Gi3 activation was pSer415>;pSer411>;pSer402>;unphosphorylated. In conclusion, these data show that the 8th helix C‐terminal peptides were able to stimulate G protein activation in N18TG2 cell membranes, and the activation could be augmented by phosphorylation of Ser residues.
Role of Spinal Cord Endogenous Opioid Peptides in the Antinociceptive Effect of Hyperbaric Oxygen (HBO2)
Authors: Heeman Jacqueline , Zhang Yangmiao , Shirachi Donald Y. , Quock Raymond M.
Published: June 2021

Earlier research demonstrated that HBO2 treatment produces antinociception in an animal model of acute pain (Chung et al., J. Pain 11:847, 2010). We have previously demonstrated that intracerebroventricular (i.c.v.) pretreatment with opioid antagonists can reduce the magnitude of HBO2–induced antinociception in mice. The purpose of this study was to examine the role of opioid mechanisms in the spinal cord in the acute antinociceptive effect of HBO2 in mice. Male NIH Swiss Mice, 18–22 g, were exposed to HBO2 (100% oxygen @ 3.5 ATA) and, after 5 min, responsiveness to HBO2‐induced antinociception was assessed using the acetic acid‐induced abdominal constriction test. Different groups of mice were pretreated by intrathecal (i.t.) injection with the following: normal rabbit serum (NRS); and rabbit antisera (AS) against the rat methionine‐enkephalin (ME), dynorphin1–17 (DYN) and β‐endorphin (βEP). NRS did not produce significant antinociception in control mice. In animals pretreated with NRS (control), HBO2 produced a 59% antinociceptive response. In mice pretreated with DYN AS, ME AS and βEP AS, HBO2 produced 27%, 52% and 46% antinociceptive responses, respectively. In conclusion, the dramatic decrease in the antinociceptive effect in mice pretreated with DYN AS suggests that DYN is the primary endogenous opioid peptide in the spinal cord that mediates HBO2‐induced antinociception. (This research was supported by NIH Grant AT‐007222, the Allen I. White Distinguished Professorship at Washington State University and an institutional Summer Undergraduate Research Fellowship (SURF) Program from ASPET).
Inflammatory signaling as a therapeutic target for the treatment of breast cancer‐induced bone pain.
Authors: Sayers Nicole Marie , Symons‐Liguori Ashley M , Lozano‐Ondoua Alysia N , Vanderah Todd W
Published: June 2021

Many common cancers, including breast, prostate, lung and kidney, have a high propensity for bone metastasis; for breast cancer, 70% of malignancies will metastasize to bone, resulting in debilitating pain and bone destruction in 75–95% of patients. Despite the prevalence of bone pain in cancer patients, the specific causes and therapeutic targets for managing bone pain are poorly understood. Breast cancer metastasis to bone results in a proinflammatory shift in the bone‐tumor microenvironment. Here we hypothesize that breast cancer‐induced bone metastasis elicits pain by the actions of pro‐inflammatory mediators at their receptors on nociceptive neurons in the bone and periosteum.

In the following work, we utilize an in vivo model of breast cancer‐induced bone pain that results in both spontaneous and evoked pain behaviors. Initial studies determined that the expression of proinflammatory cytokines (tumor necrosis factor‐α, interleukin‐6) and chemokines (chemokine C‐C motif ligands 2 and 3) are enhanced in tumor‐bearing bone, but not the contralateral bone or serum. mRNA quantitation of bone marrow extrudate corroborated these findings. The attenuation of bone‐derived inflammatory mediators by cannabinoid‐receptor 2 agonists, which produce inflammatory suppression of immune cells, was associated with decreases in evoked and spontaneous pain behaviors.

Furthermore, we demonstrate in vitro that inflammatory mediators are constitutively produced by a murine breast cancer cell line. Cannabinoid receptor‐2 agonists attenuated not only the production of cytokines and chemokines by cancer cells, but also attenuated interleukin‐6 or tumor necrosis factor α‐induced proliferation. The results of these data suggest that tumor‐derived inflammatory mediators may drive bone cancer pain. Targeting the inflammatory component of metastatic bone pain provides a novel therapy for pain management in breast cancer‐induced bone pain.
Blockade of extracellular regulated kinase 1/2 (ERK1/2) alters antinociception and tolerance to DAMGO, but not to fentanyl
Authors: Bobeck Erin , Ingram Susan , Morgan Michael
Published: June 2021

Ligand‐biased signaling from the mu‐opioid receptor (MOP) has been shown for a variety of downstream signaling proteins. One known difference is that ERK1/2 is activated in a β‐arrestin dependent manner following fentanyl, but not morphine administration. Although in vitro studies show differences in ERK1/2 activation depending on the opioid, little has been done to evaluate the role of ERK1/2 on antinociception and tolerance to agonists besides morphine. The purpose of this study was to examine the role of ERK1/2 on antinociception and tolerance to fentanyl and DAMGO injected directly into the ventrolateral periaqueductal gray (vlPAG)—an important structure in pain modulation. Administration of the ERK1/2 inhibitor U0126 (100 ng/0.5 μL) had no effect on fentanyl antinociception. However, a single microinjection of U0126 caused a decrease in DAMGO‐induced antinociception. Furthermore, co‐administration of U0126 with the opioid caused a reversal of tolerance to DAMGO, but not fentanyl. Taken together, these data show that ERK1/2 activation is important for antinociceptive properties of DAMGO, but not for fentanyl. This study adds to the growing body of evidence that different signaling pathways may be causing antinociception and tolerance to different MOP agonists. Funding by NIH grant DA 015498.
Multifunctional nanoparticles for detection, quantification, and treatment of endothelial erosions in experimental atherosclerosis
Authors: Pan Hua , Allen John S , Yanaba Noriko , Schlesinger Paul H , Wickline Samuel A
Published: June 2021

Acute atherothrombotic occlusion in heart attack and stroke implies disruption of the vascular endothelial barrier that exposes a highly procoagulant intimal milieu. However, the evolution, severity, and pathophysiological consequences of vascular barrier damage in advanced atherosclerotic plaque remain unknown, in part because quantifiable methods and experimental models are lacking for its in vivo assessment. Here, we report a multifunctional perfluorocarbon (PFC) nanoparticle (NP) for delineation of endothelial erosions as well as targeted antiinflammatory drug delivery to the erosion site. Detection and quantification (MRS and fluorescence imaging, shown in Figure) of endothelial erosions were evaluated in ApoE−/− mice fed a high fat diet for 4 – 7 months. Endothelial barrier disruption was apparent from massive and rapid passive permeation and intimal trapping of PFC nanoparticles (~250nm diameter) that normally do not penetrate intact endothelial barriers after in vivo circulation for 2 hours. RT‐PCR (CCL2, Npy) and immunohistological (I‐kB protein)results suggested that treatment (6 doses, i.v. every other day) PFC nanoparticles loaded with NF‐kB inhibitors (NEMO binding domain peptides) in 4 month fed ApoE−/− mice downregulated inflammation in aortas. Accordingly, PFC NP may be useful for defining disease stage and for following therapy to heal unstable plaques.