bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage migration inhibitory factor mediates the antidepressant actions of voluntary exercise
Authors: MOON HYO YOUL , Kim Se Hyun , Kim Yong Sik , Ryu Sung Ho , Suh Pann‐Ghill
Published: June 2021

Voluntary exercise is known to have effects on various brain functions, including an antidepressant action. However, underlying mechanism of exercise effects on psychological disease is still ambiguous. We identified the candidate target genes for alleviating depression by the analysis of existing microarray data sets of mouse hippocampus mRNA from voluntary exercise and ECS (Electroconvulsive seizure) stimulated experiments. To validate the precise changes of those candidate genes, we conducted 28 day voluntary exercise and 10 day chronic ECS treatment with rats. Reverse transcription‐PCR and real‐time PCR analysis showed 5 genes; BDNF, COX‐2, SLC1A1, NPY and MIF were commonly induced by both ECS and voluntary exercise experiments. Furthermore, immunohistochemistry analysis firmly remarked that the amount of MIF, a proinflammatory cytokine, was also increased by voluntary exercise. We showed that recombinant MIF administration produced antidepressant‐like behavior in animal FST (Forced swim test). Moreover, MIF treatment for 1 hour increased the expression of TPH2, a rate‐limiting enzyme in synthesis of serotonin, and 12 hour treatment increased the amount of BDNF in Neuro‐2a neuroblastoma cell line. These results suggest the new role of MIF in relieving the symptom of depression by exercise, probably via enhancing neurotransmitter and neurotrophic factor in the brain.
Attenuated increases in sweat rate but not skin blood flow to increasing doses of methacholine in aged skin
Authors: Shibasaki Manabu , Inoue Yoshimitsu , Crandall Craig G
Published: June 2021

Sweating and skin blood flow responses to whole‐body heat stress are decreased in aged skin. This study tested the hypothesis that these responses are attenuated to exogenous methacholine (MCh) in aged skin. Two microdialysis membranes were placed in dorsal forearm and lower leg skin in 7 healthy young (22±1) and elderly (63±2) subjects. Local sweat rate (SR) and skin blood flow were simultaneously measured over each microdialysis membrane. After insertion trauma subsided, and while skin temperature was clamped at 34°C, progressively increasing doses of MCh were administered (5 min/dose). Cutaneous vascular conductance (CVC) was not different between groups prior to and across all doses of MCh regardless of site, although forearm CVC was greater relative to lower leg CVC in both groups (both P<0.001). On the other hand, SR in aged skin was lower relative to young skin (significant dose × age interaction), while there was no regional difference between leg and arm responses in either group. These results confirm prior findings that sweat gland function is reduced in aged skin. Conversely, there is no effect of aging on cutaneous vasodilator responsiveness to exogenous methacholine.

Supported by JSPS 21790225 and HL84072
Protective effects of Cordyceps sinensis and exercise training on septic shock
Authors: TENG I‐HSIEN , LEE SHIN‐DA , Cheng Yu‐Jung , Ou Hsiu‐Chung
Published: June 2021

Cordyceps sinensis (CS) has been considered to be an herbal medicine used in various inflammatory diseases, and other studies show the exercise training can decrease septic responses. In this study, we would like to evaluate whether CS and exercise training could reduce the damage in septic mice. 16 male C57BL/6 mice were randomly assigned into 3 groups: sedentary + saline (control group), sedentary + CS, and exercise + saline (training group). Mice were treated with saline or the extract of CS (20 mg/kg) by oral gavage 4 hours before swimming training (60 min/d, 5d/week for 4 weeks). At end of the treatment, all mice were induced endotoxemia by lipopolysaccharide injection (Escherichia coli, B4, 10, 40 mg/kg, intraperitoneal). Blood samples were collected at various times after LPS infusion. We analyzed the count of blood cells, cytokines, biochemical factors (aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen) and lipid peroxidation. Organs were also analyzed by H&E staining and TUNEL assay. Exercise training group had lower basal levels of neutrophils and TNF‐α than control group. Biochemical factors in control group were higher than training group. Histopathological micrographs also show the changes in the organ tissues of control group were more severe than in the exercise training group. Taken together, exercise training attenuates septic responses and protects organs from damage.
Exercise‐induced fatigue and hyperthermia limit muscle force production capacity through peripheral and spinal alterations respectively
Authors: Racinais Sebastien , Matic Ivana , Girard Olivier
Published: June 2021

Objective

To discriminate the mechanisms by which exercise‐induced fatigue and hot environments impair muscle force production alone or in conjunction.

Methods

7 participants (preliminary data at the date of abstract submission) were tested at rest, following 20 min of moderate cycling (100 W) and after incremental cycling to exhaustion in two environmental conditions (CON: 24°C, 24% rH; HOT: 40°C, 40% rH). Electrically evoked potentials (M‐wave and H‐reflex) were obtained at rest and during maximal voluntary contractions (MVC) of the plantar flexors.

Results

Exercise‐induced fatigue significantly altered the indexes of peripheral fatigue (e.g., electrically evoked peak twitch, and quantity of neural drive needed to produce an absolute level of force). Heat exposure significantly decreased the different indexes of spinal modulation (e.g., H‐reflex, V‐wave).

Conclusion

Maximal voluntary force is decreased by both exercise‐induced fatigue (−9%) and heat exposure (−7%). These decrements originate respectively from muscle contractility and spinal modulation, and are cumulative when exercising in hot environment (−15%).
Alcohol consumption promotes insulin resistance via accumulation of proinflammatory responses in the NIDDM murine model
Authors: Matsumoto Hiroshi , Tanaka Maki , Tadeda Kenji , Ishizaka Atsushi , Okazaki Shunichiro , Katada Ryuichi , Mizuo Keisuke , Watanabe Satoshi
Published: June 2021

Alcohol consumption causes fatty liver disease and other organ damages. On the other hand, alcohol consumption is one factor of development of metabolic syndrome. We hypothesized alcoholic fatty liver disease disturbs glucogenesis, resulting in insulin resistance. The Nagoya‐Shibata‐Yasuda (NSY) mouse is an animal model of human Type II (non‐insulin‐dependent) diabetes mellitus (NIDDM) where the onset is age‐dependent, the animals are not severely obese, and both insulin resistance and impaired insulin response to glucose contribute to disease development. In the present study, we clarified the effect of ethanol consumption on the onset age and the pathogenesis of diabetes and to analyze glucogenesis and proinflammatory response in the NSY mice. We used the NSY mice and control C3H/He mice fed Lieber‐DeCarli diet (5% v/v ethanol) for 4 weeks. Insulin resistance was evaluated in overnight‐fasted mice by insulin tolerance ant glucose tolerance tests. Alcohol diet promoted insulin resistance significantly compared as control liquid diet. Insulin resistance in the NSY mice occurred significantly earlier than the C3H/He mice fed alcohol diet. Expression of TNFalpha, IL‐1beta, IL‐6, and other proinflammatory response genes in the liver of the NSY mice were significantly higher than the control mice fed alcohol diet. On the other hand, PGC‐1alpha related to gluconeogensis was deceased by alcohol diet. In conclusion, alcohol consumption induces accumulation of proinflammatory responses in the liver to promote insulin tolerance.
Sodium metaarsenite (KML001) induces a G1 phase arrest in HCT116 colorectal cancer cells
Authors: Hong Yunkyung , Kim Sunmi , Rho Gyu‐jin , Hong Yonggeun
Published: June 2021

Arsenic‐based reagents have been used as medicines, but they can cause chromosomal abnormalities in vitro and in vivo. Recent studies have reported alterations in telomere kinetics caused by the arsenic compound sodium metaarsenite (KML001), suggesting that it may be useful as an anti‐cancer reagent. Here, we investigated the anti‐cancer effects of KML001 on HCT116 cells. The expression of autophagic and apoptotic molecules were measured by RT‐PCR and immunoblotting in HCT116 cells and SW480 treated with KML001. KML001 reduced colorectal cancer cell proliferation in a concentration‐dependent manner, with a significant reduction at 1 μM (p<.05). p53 expression and conversion of LC3I to LC3II were dramatically increased, whereas no significant changes in caspase 3 and Bcl‐1 were observed (p<.05). Compared to the controls, the number of cells in G1 phase decreased 48 h after KML001 treatment, and the expression of several cyclin‐dependent kinase inhibitors was significantly increased (p<.05). In neonatal cardiomyocytes, morphologic signs of atrophy and a reduction in the number of differentiated cells were observed. Finally, a dramatic decrease in beating frequency and the number of embryoid bodies was noted after 18 h of treatment. KML001, which targets telomere kinetics, suppresses the proliferation of HCT116 cells, which might induced by a G1 phase arrest.

Funding: BioGreen21 Program (Code No. 20070401‐034‐006‐009‐02‐00), Rural Development Administration, Korea
Exogenous melatonin leads to the activation of autophagy and cellular senescence in colorectal cancer cells
Authors: Hong Yunkyung , Kim Sunmi , Park Sookyoung , Hong Yonggeun
Published: June 2021

The purpose of the present study was to determine the anti‐cancer effect of melatonin on HCT116 colorectal cancer cells. At 10 μM, melatonin induced a significant reduction in MT‐R1A expression and an increase in RORα expression over time (p<.01). Proliferation was strikingly reduced by 10 μM melatonin in a time‐dependent manner. Bcl‐2 mRNA expression was decreased at 48 h after treatment (p<.01). Furthermore, the induction of cleaved caspase 3, a significant increase in converted LC3II, and a decrease in Bcl‐1 expression were observed in response to melatonin treatment (p<.01). A time‐dependent increase in the number of cells in G1 phase was also caused by melatonin at 10 μM. Cyclin D expression was unchanged, whereas cyclin E expression was significantly reduced by melatonin at 10 μM (p<.01). The level of cyclin‐dependent kinase inhibitors, p16 was also altered in melatonin‐treated HCT116 cells. Treatment with melatonin at a pharmacologic concentration significantly inhibited the overgrowth of HCT116 cells, but activated by stress‐induced senescence as well as autophagy indicating it could be useful chemotherapeutic drug with minimal cytotoxicity.

Funding: BioGreen21 Program (Code No. 20070401‐034‐006‐009‐02‐00), Rural Development Administration, Korea
Macrophage RAGE Signaling Upregulates Early Growth Response‐1 in Acute Hypoxia
Authors: Xu Yunlu , Toure Fatouma , Qu Wu , Song Fei , Shen Xiaoping , Garcia Joel , Schmidt Ann Marie , Yan Shi Fang
Published: June 2021

Receptor for advanced glycation endproduct (RAGE)‐dependent signaling has been implicated in ischemia/reperfusion injury in the multiple organs, such as heart, lung, liver and brain. As macrophages contribute to vascular perturbation and regulation of inflammatory and prothrombotic responses, we tested the hypothesis that upregulation of Egr‐1 in hypoxic macrophages would be mediated by RAGE. THP‐1 cells and peritoneal macrophages (PM) were exposed to hypoxia (0.5% O2). Increased RAGE ligand AGE release was observed in THP‐1 cells minutes of hypoxia vs. normoxia; p< 0.0001 (peak, 10 min). Egr‐1 mRNA or nuclear protein were increased ≈ 31‐fold and ≈ 4‐fold at 15 and 30 min of hypoxia vs. normaxia; p<0.0001. However, upregulation of Egr‐1 in hypoxic THP‐1 cells was blocked by silencing of RAGE, treatment with soluble RAGE, anti‐RAGE or anti‐AGE IgG; p<0.0001 or p<0.001. In parallel, hypoxia‐induced upregulation of Egr‐1 in RAGE−/− PM was strongly diminished compared to RAGE+/+ PM; p<0.0001. In addition, in hypoxic THP‐1 cells, silencing of RAGE blocked activation of ERK1/2 and JNK; p<0.0001; and induction of Egr‐1 was suppressed by inhibitors of ERK1/2 (U0126) and JNK (SP600125), but not by an inhibitor of p38 MAPK (SB203580). In conclusion, these findings delineate a critical role for RAGE‐dependent macrophage signaling via activation of ERK1/2 and JNK MAP kinases in upregulation of Egr‐1 in acute hypoxic stress.
Pyruvate pretreatment protects glial cells from ischemia‐reperfusion injury by stabilizing HIF‐1
Authors: Ryou Myoung‐Gwi , Mallet Robert T , Yang Shaohua
Published: June 2021

Background

During brain ischemia and reperfusion (I/R), substantial amounts of reactive oxygen species (ROS) are produced, damaging brain cells. Pyruvate, an energy substrate and potent antioxidant, inhibits HIF‐1α degradation under normoxic conditions. Recently we reported that pyruvate increased neuroprotective erythropoietin via HIF‐1 pathway. Hypothesis: Pyruvate pretreatment protects glial cells from I/R injury.

Methods

C6 cells maintained in glucose and pyruvate free DMEM were randomly assigned to control or pyruvate groups. 30 min prior to hypoxic stress (0.5% O2), cells were pretreated with 10mM pyruvate followed by 1h hypoxia and 24 h reoxygenation. Cell viability was assessed from LDH release and Calcein AM. ROS generation was evaluated with DCF stain. Glucose oxidase (GOx) (1mU) +/− pyruvate was applied to test the role of pyruvate's antioxidant effect in stabilizing HIF‐1α. Immunoblot was used to determine protein content of HIF‐1α, erythropoietin, and β‐site of APP cleaving enzyme (BACE).

Results

Cell viability was greater in pyruvate than control group. Pyruvate dampened ROS generation and BACE content vs. control. Pyruvate stabilized HIF‐1α in the face of GOx challenge. Similarly, HIF‐1α was increased after I/R in pyruvate vs. control. Erythropoietin content paralleled HIF‐1α in each group.

Conclusion

Pyruvate dampens ROS generation, and augments HIF‐1α, and increased cell viability during I/R. Pyruvate enhancement of HIF‐1 increased erythropoietin and decreased BACE protein content. Therefore, pyruvate pretreatment can protect C6 cells by stabilizing HIF‐1.
Protein translation and degradation signaling in skeletal muscle following total hip arthroplasty with or without an essential amino acid supplement
Authors: Merritt Edward K. , Ferrando Arny A. , Bamman Marcas M.
Published: June 2021

Total hip arthroplasty (THA) and other major orthopedic surgical procedures lead to severe atrophy of skeletal muscles around the affected area. This atrophy can persist two years or longer. As these surgeries are often performed on older patients already suffering from atrophy, further loss of muscle mass can seriously impact quality of life. Attenuation of muscle loss post‐THA is critical for rapid recovery of mobility and function. The purpose of this study was to determine the effects of an essential amino acid supplement (EAA) on signaling pathways regulating muscle mass in THA patients. THA patients were given normal standard of care or an AA (Travasol 10%) during surgery and EAA capsules thrice daily post‐surgery. Muscle samples obtained perioperatively and at discharge from acute care were analyzed via Western blot for markers of protein translation and degradation and compared to matched controls (CON). Patients who did not receive the EAA supplement had significantly lower phosphorylated 4EBP1 perioperatively. Calpastatin levels were significantly lower at discharge in both groups relative to perioperative and CON. Interestingly, atrogin‐1 levels were lower than CON in both THA groups at both time points. No differences were found in other pathways. Based off these markers, the EAA supplement might act through 4EBP1 to modulate protein synthesis, but does not likely have any effect on degradation.
Melatonin prevents muscle atrophy and the alteration of myosin heavy chain in spinal cord injured rats
Authors: Seo Hyeongsun , Park Kanghui , Park Sookyoung , Javaregowda Palaksha Kanive , Hong Yonggeun
Published: June 2021

The purpose of this study was to confirm the behavioral features of spinal cord injury (SCI) and to investigate the effects of melatonin on muscle atrophy and shifts in myosin heavy chain (MHC) in an SCI model. SCIs were surgically induced in eight‐week‐old male Sprague‐Dawley (SD) rats that had been randomly divided into three groups: Control, SCI (n=13), and melatonin‐treated after SCI (SCI+MT, n=13). Melatonin (10 mg/kg body weight) was injected subcutaneously twice a day (07:00 and 19:00) for four weeks, after which the body weight and motor function of the rats were evaluated. The expression of muscle atrophy‐related genes in hindlimb muscles, including MAFbx and MuRF1, and MHC, were also analyzed. The body weight and motor functions of the rats were decreased at the acute phase after SCI. The gene expression of MAFbx and MuRF1 were increased in the hindlimb muscles (p<.05), indicating SCI‐induced muscle atrophy. Melatonin contributed to motor recovery, decreased MAFbx and MuRF1 gene expression, and altered MHC expression in the hindlimb muscles (p<.05). Thus, melatonin may promote functional recovery, prevent muscle atrophy, and alter MHC expression after SCI.

Funding: BioGreen21 Program (Code No. 20070401‐034‐006‐009‐02‐00), Rural Development Administration, Korea
Altered myosin heavy chain phenotype in locomotor and non‐locomotor muscles of mini‐muscle mice bred for high running capacity
Authors: Talmadge Robert J , Garland Theodore
Published: June 2021

Replicated selective breeding of mice for high voluntary wheel running for over 50 generations resulted in four lines of mice that display a high‐running phenotype. Individuals in two of the four lines display a smaller hindlimb muscle mass (mini‐muscle mice), inherited as a Mendelian recessive. In one line (lab designation line 3), all mice now have the mini‐muscle phenotype. Previous studies suggested that mini‐muscle mice also have differences in myosin heavy chain (MyHC) isoform content in hindlimb muscles. This study evaluated whether the changes in MyHC content were restricted to locomotor muscles. Electrophoretic analyses revealed that both locomotor (vastus lateralis, trapezius) and non‐locomotor (lingual) muscles of mini‐muscle mice had greatly reduced 2b MyHC isoform content compared to control mice. The loss in 2b MyHC isoform content in the mini‐muscle mice was compensated by a corresponding increase in 2a and 2x MyHC isoforms. The data suggest that alterations in the MyHC isoform profile in mini‐muscle mice are not restricted to locomotor muscles. Thus, the underlying gene appears to have a general influence on specifying muscle phenotype. NSF IOB‐0543429 to T.G.
SEPSIS DOWNREGUALTES MYOSTATIN mRNA LEVELS WITHOUT ALTERING MYOSTATIN PROTEIN LEVELS IN SKELETAL MUSCLE
Authors: Smith Ira J. , Aversa Zaira , Alamdari Nima , Petkova Victoria , Hasselgren Per‐Olof
Published: June 2021

Myostatin is a negative regulator of muscle mass and has been reported to be upregulated in several conditions characterized by muscle atrophy. The influence of sepsis on myostatin expression and activity is poorly understood. Here, we tested the hypothesis that sepsis upregulates the expression and downstream signaling of myostatin in skeletal muscle. Because sepsis‐induced muscle wasting is at least in part regulated by glucocorticoids, we also determined the influence of glucocorticoids on myostatin expression. Sepsis was induced in rats by cecal ligation and puncture and control rats were sham‐operated. In other experiments, rats were injected intraperitoneally with dexamethasone (10 mg/kg) or corresponding volume of vehicle. Surprisingly, myostatin mRNA levels were reduced and myostatin protein levels were unchanged in muscles from septic rats. Muscle levels of total and phosphorylated Smad2 were not influenced by sepsis, suggesting that myostatin downstream signaling was not altered during sepsis. Similarly, treatment of rats with dexamethasone reduced myostatin mRNA levels and did not alter myostatin protein levels. The results suggest that sepsis and glucocorticoids do not upregulate the expression and activity of myostatin in skeletal muscle. The role of myostatin may vary between different conditions characterized by muscle wasting.
Effect of chronic metformin treatment in a mouse model of Huntington's disease
Authors: Adhihetty Peter J , Iorio Leanne M , Hennessy Thomas , Ho Daniel J , Calingasan Noel , Yang William X , Beal Flint M
Published: June 2021

Huntington's disease (HD) is an autosomal dominant disorder caused by a mutation in the huntingtin gene. HD progression is associated with mitochondrial dysfunction in neural and skeletal muscle tissue. Mutant huntingtin suppresses PGC‐1α, a transcriptional coactivator that regulates genes influencing numerous metabolic programs. The purpose was to determine whether metformin, an AMPK agonist and mitochondrial biogenesis inducer, would delay HD‐associated symptoms by improving mitochondrial content and function. BACHD mice, exhibiting a mutant form of human huntingtin, and wild‐type animals, were given metformin or water (non‐treated) for 12 months and a series of measurements were performed. Our results indicate metformin treatment provided a modest, yet significant, improvement in the behavioural and locomotory deficits in BACHD mice. Metformin treatment in BACHD mice reversed the suppressed expression of mitochondrial‐related genes (i.e. PGC‐1α, NRF‐2, cyt‐c) in soleus, but not gastrocnemius muscle, of non‐treated BACHD mice. However, metformin was incapable of reducing the striatal atrophy, or restore impaired mitochondrial respiratory function, in BACHD mice. Our findings suggest that chronic metformin treatment delays the progression of HD‐associated symptoms in HACHD mice and that the beneficial effects of metformin are fiber and tissue‐specific. This research was supported by NIH.
GENETIC VARIANTS OF MICRO‐RNA 196a2 SEQUENCES CAN PREDICT SURVIVAL OF CHOLANGIOCARCINOMA
Authors: Ko Kwang Hyun , Kim Nam Keun
Published: June 2021

Background

MicroRNAs (mir) are a new class of non‐protein‐coding, small RNAs that has function of tumor suppressors or oncogenes. They participate in diverse biological pathways and function as gene regulators. Only a few studies have been published about the association between microRNA polymorphism and malignant tumor.

Aim

We evaluated that the association between genetic variants of MicroRNA and survival of patients with cholangiocarcinoma. Patients and

Method

We enrolled 94 patients, who were diagnosed with cholangiocarcinoma between Jan.1999 to Nov. 2006 in Bundang CHA hospital, CHA University and obtained blood sample. Using DNA extract from white blood cell, we analyzed mir‐196a2, mir‐149, mir‐499 and mir‐146a expressions by PCR‐RFLP and patients’ survival.

Results

Mean age of patients was 66.9 ± 10.7 yrs old and fifty five was male and thirty nine was female. Median survival was 210 days (18 – 1290 days) after diagnosis of chlangiocarcinoma. According to genetic variant of mir‐196a2, wild type (TT) showed significantly longer survival than in variant type (CT, CC) (P = 0.018), especially in male patients (p=0.011). There was no association between mir‐149mir‐499 and mir‐146a polymorphism and survival of cholangiocarcinoma patients.

Conclusion

These findings suggest that genetic variants of microRNA 196a2 sequence could be important to predict survival of cholangiocarcinoma patients, but these findings need to be validated in larger prospective study.
Effects of Typha latifolia aqueous extracts on livers and kidneys of mice
Authors: Habacha Souhaila , Khmiss Fathia , Maaroufi Khira
Published: June 2021

The aim of this study was to investigate the in vivo effects of roots and flowers extracts of Typha latifolia with an emphasis on liver and kidney of mice and to determine the LD50 of extracts. Animals were divided into three groups. They were respectively orally administrated by roots aqueous extract (ARE), flowers (AFE) and water. On day 14, liver and kidney were removed and weighted for histological study. The results showed that the liver and kidney weights remained uniform during treatments. The results showed that weights were not significantly different from those of control group. LD50 for the ARE was >600mg/Kg body wt and was equal to 200 mg/Kg for AFE. Histological study showed that livers conserve structural features with ARE. However, they undergo structural alteration with AFE: a centrolobular sinusoidal congestion and disruption of the central vein and hepatocellualr necrosis. ARE had no pernicious effect on kidneys. However, AFE caused alteration consisting of inflammatory infiltration and necrosis.

Our results may be in relation with a toxic effect of AFE on liver and kidney while the ARE seems to be a safe extract.
Intrarenal superoxide dismutase normalizes renal vascular resistance in rabbits with pacing induced heart failure
Authors: Clayton Sarah C , Curry Pamela L , Li Yu , Zucker Irving H
Published: June 2021

The cardio‐renal syndrome represents a major complication in heart failure (HF) patients, but the underlying mechanisms are not completely understood. The sympathetic nervous system and the renin‐angiotensin II system are likely to induce oxidative stress in the kidney in chronic HF. We hypothesized that in rabbits with HF reducing oxidant stress with superoxide dismutase (SOD) would improve renal blood flow in HF. In rabbits with HF (verified by echocardiography), renal blood flow was reduced (37.6 ± 1.7 ml/min in sham, vs. 29.2 ± 1.7 ml/min in HF, P=0.001). Unilateral intrarenal infusion of CuZn‐superoxide dismutase conjugated to polyethylene glycol (PEG‐SOD) at a dose of 0.30 U/kg/hr for 7 days tended to increase renal blood flow in HF rabbits (35.9 ± 2.1 ml/min). Renal vascular resistance was increased from sham (1.96 ± 0.10 mm Hg/ml/min, sham, vs. 2.36 ± 0.16 mm Hg/ml/min, HF, P=0.03) which was prevented by PEG‐SOD infusion (1.45 ± 0.14 mm Hg/ml/min, P=0.03 vs. HF alone). We confirmed that PEG‐SOD treatment reduced dihydroethidium staining in the treated kidneys from HF rabbits but not the contralateral, untreated kidneys, suggesting the beneficial effect was limited to the treated kidney and not from a systemic effect. These results suggest the change in renal hemodynamics in HF is due to intrarenal oxidative stress and could implicate a potential therapeutic strategy to improve the cardio‐renal syndrome.
Inhibition of Myeloperoxidase by Phenazine‐Based Bacterial Metabolites: Implications for Cystic Fibrosis
Authors: Hosein Ramie Jamal , Vasu Vihas , Cross Carroll , Eiserich Jason
Published: June 2021

Cystic fibrosis (CF) patients have chronic airway inflammation due to persistent neutrophil influx in response to bacterial infection, most notably Pseudomonas aeruginosa (PA). Neutrophils express myeloperoxidase (MPO), an enzyme that catalyzes the formation of hypochlorous acid to kill bacteria in the lungs of CF patients. Pyocyanin (PYO) and 1‐hydroxyphenazine (1‐HP) are metabolites abundantly produced by PA, known to cause detrimental effects in the lung; however, the effect of PYO and 1‐HP on MPO activity is not known. We hypothesized that PA produce PYO and 1‐HP as a means of inhibiting MPO and incapacitating this important mammalian host defense mechanism. In order to test this hypothesis, we utilized colorimetric assays of MPO activity in the presence or absence of 1‐HP or PYO. Our data indicate that 1‐HP and PYO inhibit both the peroxidase and chlorination activities of MPO. The data suggest that 1‐HP and PYO can modulate MPO activity and presumably decreasing its antibactericidal functions, and thus acting as a mechanism by which PA can thrive in the CF airway. Identifying the effects PYO and 1‐HP have on MPO will influence overall strategies for therapeutic treatments in cystic fibrosis.
Macrophage scavenger receptor MARCO modulates early events in innate immunity against Influenza
Authors: Ghosh Sanjukta , Kobzik Lester
Published: June 2021

We investigated the role of MARCO against infection with influenza A virus (IAV). In vitro infection of MARCO−/− or WT macrophages with IAV showed no significant difference in virus uptake. However, MARCO−/− mice had increased PMNs in the lungs early after infection compared to wild‐type controls accompanied with greater induction of acute inflammatory cytokines investigated at day 2 and lower viral M1 gene expression at day 4, post infection. Experimental enhancement of the early inflammatory response in infected WT mice by aerosol exposure to LPS (200 ng/ml, 20 minutes) 12 h after IAV challenge resulted in much lower viral M1 gene expression in the lungs in 2 days, suggesting that inflammatory cellular influx early during infection helps to suppress viral outgrowth. Notably, the MARCO−/− mice showed greater survival following low dose viral challenge than the WT (67% vs 34%, N=10 per group) without showing appreciable difference in T lymphocyte numbers. We hypothesized that accumulation of oxidized proinflammatory lipoproteins in lungs in absence of its scavenger MARCO, could trigger increased neutrophilia. BAL of infected MARCO−/− animals showed 2‐fold greater oxidized lipoproteins accumulation early after infection. The data suggest that MARCO clearance of inflammatory intermediates during influenza acts to reduce an early inflammatory response, which is beneficial for viral clearance and recovery from influenza. NIH Grant ES011008‐08 ES 00002
Incorporation of content delivery via an online course management system in an introductory physiology course for pharmacy students
Authors: Kleiber Allison , Browning Ryan
Published: June 2021

Due to the substantial amount of content included in the semester‐long introductory physiology course for second‐year pharmacy students, it was desirable to improve efficiency of content delivery. Typical lectures in this physiology course traditionally include both the anatomy and physiology of each system. Students complete anatomy coursework as a prerequisite and thus often find that the physiology component is more difficult to grasp than the anatomy component; however, the more time that is spent discussing anatomy and basic components decreases the amount of time that can be used for the complex concepts. Thus, we sought to allocate more in‐class time to focusing on challenging concepts. The solution was to re‐assign anatomy and basic physiology content to 20‐ to 30‐minute pre‐class lectures recorded using Camtasia Studio and stored in the course management system and available for viewing at any time. Students are required to view a lecture at the beginning of each new topic of physiology prior to the class period for which it is assigned; a short quiz is administered at the beginning of class to ensure student viewing and understanding of the material. Time in class is thus devoted to discussion of the more difficult concepts. The result is a course incorporating online content delivery through a course management system and in‐class content delivery, as well as in‐class active learning. Support: STLCOP
Thermodynamic Examination of the Amber/W Site in the Hepatitis Delta Virus Type III
Authors: Dang Angela , Grover Neena
Published: June 2021

The hepatitis delta virus III (HDVIII) RNA can form two secondary structures, one branched and one unbranched. As editing requires a branched double‐hairpin structure, editing levels are determined by the fraction of RNA that folds into the correct structure and the efficiency with which ADAR (adenosine deaminase) edits. Due to the difference of only five bases in the editing region, the Peruvian and Ecuadorian isolates of the HDVIII form slightly different secondary structures and thus, exhibit different editing levels. Near the Amber/W site, the Peruvian isolate forms a 4x3 and 1x1 internal loop and is edited more efficiently than the Ecuadorian isolate, which forms a 4x2 and 0x1 internal loop. In this study, we examined the difference in the stabilities of the RNA constructs derived from the two isolates. The more efficiently edited Peruvian isolate was predicted to form a more stable secondary structure. Under 1 M KCl and varying concentrations of Mg2+ or Ca2+, the Peruvian Amber/W constructs was 1.5–2 times more stable than the Ecuadorian construct. Only a small increase in RNA stability was observed for the two isolates in the presence of divalent ions in comparison to the stability in 1 M KCl.
Ethanol and both Endogenous and Exogenous Reactive Species Accelerate the Mutation Rate of Hepatitis C Virus RNA
Authors: Seronello Scott , Presleigh Kristen , Barlow Miriam , Choi Jinah
Published: June 2021

Hepatitis C virus (HCV) demonstrates a high level of genetic variability existing as a population of closely related but distinct genetic variants, referred to as quasispecies. Ethanol intake is one of the major cofactors in HCV‐induced pathogenesis and is correlated with increased sequence heterogeneity of HCV RNA in patients, which is likely to promote viral persistence and complicate anti‐HCV therapy. However, the mechanism underlying this interaction between HCV and ethanol is unknown. The purpose of this study was to examine whether ethanol and endogenous and exogenous reactive species can increase the mutation rate of HCV. The NS5A region of Con1 HCV replicon RNA was sequenced after treatment with ethanol, acetaldehyde, exogenous hydrogen peroxide, and L‐buthionine‐S,R‐sulfoximine, which accelerated the mutation rate of HCV RNA over that of the control group within 48 hrs. The dn/ds ratio also increased significantly with the nonsynonymous mutations clustering in the C‐terminal half of NS5A. Half of amino acid substitutions involved serine, threonine, or tyrosine, suggesting possible changes in NS5A phosphorylation. The treatments also induced RNA damage. Therefore, ethanol may synergize with virus‐induced oxidative/nitrosative stress to induce RNA damage that amplifies the error rate of the viral replicase increasing the heterogeneity of HCV RNA.

This work was supported by funds from UC Merced.
Nuclear Bmp4: a novel ROC1 and ROC2 binding partner
Authors: Loos Trina J , Bridgewater Laura C
Published: June 2021

Bone morphogenetic protein 4 (Bmp4) is a well known growth factor that regulates gene expression through the SMAD signaling pathway. Bmp4 is involved in many developmental processes and has been identified as an important factor in several cancers. Due to the observation of Bmp4 in the nuclei of some colon cancer tissues by Madoz‐Gurpide et al. and the recent discovery of a nuclear variant of Bmp2 (nBmp2) in our lab, we hypothesized that a nuclear variant of Bmp4 (nBmp4) also exists. Immunocytochemistry experiments confirmed the presence of Bmp4 in the nuclei of several cell lines, and subsequent analyses determined that nBmp4 is translated from an alternative start codon downstream of the signal peptide sequence, which allows a bipartite nuclear localization signal to direct translocation of nBmp4 to the nucleus. Yeast two‐hybrid experiments were performed in order to identify potential nBmp4 binding partners. Among the identified proteins were regulator of cullins 1 and 2 (ROC1 and ROC2), which are components of SCF E3 ubiquitin ligases. The putative interactions between nBmp4 and ROC1 and ROC2 have been confirmed by co‐immunoprecipitation, and preliminary evidence suggests that nBmp4 also interacts with another member of the SCF complex, Cullin1. These interactions suggest that nBmp4 plays a role in the ubiquitin‐proteasome pathway. Research supported by NIH grant AR48839 and the BYU Cancer Research Center.
Dose dependent effects of dominant‐negative K6W‐ubiquitin: Construction of mini‐genes that encode multiple copies to K6W‐ubiquitin
Authors: Shang Fu , Guo Weimin , Taylor Allen , Bian Qingning
Published: June 2021

Purpose

We previously demonstrated that K6W‐ubiquitin behaves as a dominant negative inhibitor of the ubiquitin proteasome pathway (UPP). The objective of this work is to construct mini‐genes that encode multiple copies K6W‐ubiquitin for effectively inhibiting the function of the UPP for investigating the roles of the UPP in vivo and in vitro.

Methods

Mouse cDNA for ubiquitin was used as a template to generate mini‐genes that encode 1, 2, 4 and 8 copies of K6W‐ubiquitin, respectively. To test the effects of K6W‐ubiquitin on UPP‐mediated degradation, HeLa cells were co‐transfected with plasmids that encode K6W‐ubiquitin and a plasmid that encodes UbG76V GFP. Levels of GFP were monitored under fluorescent microscope and by Western blotting.

Results

Similar to endogenous poly‐ubiquitin, the tandem poly‐K6W‐ubiquitin was processed into mono ubiquitin in the cells. As compared on the basis of same amount DNA, levels of mono‐K6W‐ubiquitin and high mass ubiquitin conjugates were proportional to copies of K6W‐ubiquitin in the mini‐genes. Expression of K6W‐ubiquitin in HeLa cells resulted in a dose‐dependent inhibition of UPP‐mediated degradation of UbG76V GFP and TNFα‐induced degradation of I‐κB.

Conclusions

K6W‐ubiquitin inhibits UPP‐mediated protein degradation in a dose dependent manner. Mini‐genes that encode multiple copies of K6W‐ubiquitin are unique tools for investigating the functions of the UPP.
SBP1 Genetically Interacts With WHI3, a Cell Cycle Control Gene in Saccharyomyces cerevisiae
Authors: Segal Scott , Theede Kelly
Published: June 2021

Proper control of cell cycle is crucial allows cells to grow to the appropriate size in the budding yeast, Saccharyomyces cerevisiae, and to ensure cancer formation does not occur in humans. Although there are a number of cell cycle checkpoints which control passage through the cell cycle, the most critical of these is the G1/S checkpoint, also known as START. Upon passage through the G1/S checkpoint, the cell is committed to replicate its DNA and undergo a round of cell division. In yeast, expression of the cyclin Cln3p, a mammalian cyclin homolog, is necessary to progress through the G1/S checkpoint. Cln3p accumulates to high levels during late G1phase and forms a complex with Cdc28p. The Cln3p/CDC28 complex then enters the nucleus and drives the cell through the G1/S checkpoint.

In order to control Cln3p levels through early and middle G1 phase, such that the cell proceeds through the G1/S checkpoint at the appropriate time, it is thought that Whi3p, an RNA recognition motif (RRM) containing protein, translationally represses the CLN3 mRNA. Recently, a genetic interaction was identified between WHI3 and SBP1. SBP1 encodes a single stranded RNA binding protein Sbp1p which has been shown to work in conjunction with Dhh1p repress global mRNA translation under physiological stress. Therefore, it is possible Whi3p may work in conjunction with Sbp1p to translationally repress the CLN3 mRNA during early G1 phase. In this work, we show that overexpression of Sbp1p has a significant effect in whi3Δ cells. First, overexpression of Sbp1p in whi3Δ cells results in a significant growth defect at 37° C. Second, overexpression of Sbp1p in whi3Δ cells results in increased cell size as compared to whi3Δ cells. Next, we will investigate whether Sbp1p is necessary for CLN3 mRNA translational repression.
High‐resolution mass spectrometry analysis of Mycobacterium tuberculosis lipid virulence metabolites
Authors: Holsclaw Cynthia , Chow Eric D. , Cox Jeffrey S. , Leary Julie A.
Published: June 2021

Mycobacterium tuberculosis (M.tb), infects one‐third of the global population and kills two million people per year. A number of unique lipid metabolites produced by M.tb have been implicated in the virulence of the bacterium, however a comprehensive lipidomic study of differing M.tb lineages is lacking. In this study, we have sought to characterize the cell‐surface lipid profiles of M.tb laboratory strains, the virulent W/Beijing lineage, and the vaccine strain M. bovis BCG, starting with the anti‐virulence lipid S881, a sulfated menaquinone. Cell surface lipids of these strains were extracted and subject to high‐resolution mass spectrometry analysis, and the identities of the virulence lipids were confirmed via exact mass measurements with an error of < 2 ppm. Our results indicate that S881 is present in the H37Rv and CDC1551 laboratory strains, but absent in the related Erdman strain. The W/Beijing strains T85 and HN878 showed qualitatively similar levels of S881 to H37Rv, indicating that other virulence factors may play a larger role in the virulence of these strains. Finally, S881 was found to be present at qualitatively high levels in the M. bovis BCG vaccine strain, consistent with the hypothesis S881 negatively regulates the virulence of the cell. Further studies are currently underway to apply this method to the analysis of other M.tb virulence lipids. This work was supported by NIH # AI51622.
Cholesterol‐depletion in human blood‐derived neutrophils by methyl‐β‐cyclodextrin leads to the formation of neutrophil extracellular traps (1001.5)
Authors: Neumann Ariane , Brogden Graham , Brodesser Susanne , Naim Hassan Y. , von Köckritz‐Blickwede Maren
Published: June 2021

The formation of neutrophil extracellular traps (NETs) as an innate immune defence mechanism has been shown to be the result of a novel cell death process called NETosis. Mechanisms leading to the release of NETs are still not completely understood. The objective of the study was to investigate the role of cholesterol in the formation of NETs.

To this end, primary human neutrophils were treated with different concentrations of methy‐β‐cyclodetxrin (MβCD) to deplete cholesterol from the cell. The effect of MβCD on the lipid composition of the cells was determined using TLC. The identities of lipids separated by TLC were confirmed by MS/MS. Furthermore, the formation of NETs was studied using immunofluorescence microscopy.

Treatment of neutrophils with MβCD revealed distinct changes in the lipid composition. These changes were statistically significant for cholesterol; other lipid classes like phosphatidylethanolamine and sphingomyelin were only slightly affected. Interestingly, neutrophils treated with MβCD showed a dose‐ and time‐dependent formation of NETs. In contrast, the treatment of neutrophils with N‐butyl‐deoxynojirimycin to reduce the levels of glycosphingolipids had no effect on NET formation, thus confirming a cholesterol‐specific effect.

In conclusion, this study shows that MβCD treatment of neutrophils leads to the depletion of cholesterol and thereby facilitates formation of NETs.
Role of ICAM‐1 hypoglycosylation in atherosclerotic plaque formation (1003.1)
Authors: Vallejo Matthew , Scott David , Patel Rakesh
Published: June 2021

N‐glycosylation is an ubiquitous post‐translational modification of proteins secreted from cells or expressed on the cell surface. N‐glycan maturation occurs sequentially moving from high mannose to hybrid to complex glycoforms. We recently showed that that during chronic vascular inflammation that underlies atherosclerosis, endothelial cell surface proteins are hypoglycosylated, characterized by increases in high mannose / hybrid glycoforms. Importantly, these epitopes provide ligands for monocyte rolling and adhesion. The protein(s) or adhesion molecule(s) that harbor high mannose epitopes to mediate monocyte rolling and adhesion was determined in this study. Evidence is provided that that a high mannose glycoform of intercellular adhesion molecule‐1 (ICAM‐1), referred to as HM‐ICAM‐1, is produced in activated endothelial cells. HM‐ICAM‐1 formation was also indicated on human atherosclerotic lesions by proximity ligation assay analysis. Moreover, a role for HM‐ICAM‐1 in mediating monocyte rolling and adhesion was demonstrating in cell culture by expressing ICAM‐1 in Cos‐1 cells in the presence of absence of selective inhibitors of N‐glycan processing (kifunensine or swainsonine) which result in high mannose N‐glycan accumulation. Interestingly, at equal surface expression levels, HM‐ICAM‐1 was more effective at binding monocytes than WT (complex glycosylated ICAM‐1). Finally, we present data showing the effects of mutation of all eight individual N‐glycan binding sites on ICAM‐1 and effects on monocyte rolling and adhesion. The assessment of the specific role of each N‐glycan site in ICAM‐1 may lead to future N‐glycan targeted therapies to modulate atherogenic inflammation.

Grant Funding Source: Supported in part by by NIH T32 HL007457, Mechanisms of Hypertension and Cardiovascular Disease
N‐glycan analysis of IgG by enzymatic digestion with Remove‐iT® Endo S and PNGase F (1004.1)
Authors: McLeod Elizabeth , McClung Colleen , Bielik Alicia , Magnelli Paula , Guthrie Ellen
Published: June 2021

Objective: The objective of this study was to compare the release of N‐glycans from different sources of IgG using a novel endoglycosidase with a chitin‐binding domain tag (Remove‐iT Endo S) and PNGase F. Biotechnology companies are currently developing a growing number of monoclonal IgG antibodies as therapeutic agents. Critical for the structure and biological activity of the molecule is the N‐glycan moiety attached to the asparagine 297 residue in the constant domain of the antibody. There are many variables in cell culture systems that can greatly influence the heterogeneity of the glycans on IgG. For this reason it has become increasingly important to monitor the glycosylation profiles of these biotherapeutics in the production process.

Methods: Remove‐iT Endo S (also known as Endoglycosidase S) was cloned from Streptococcus pyogenes and overexpressed in E. coli as a fusion to the chitin‐binding domain. IgG samples were enzymatically deglycosylated under native conditions using Remove‐iT Endo S and PNGase F Glycerol Free. The deglycosylated IgG samples were then analyzed by SDS‐PAGE gel shift analysis and ESI‐TOF MS to estimate the degree of deglycosylation. In addition, the glycans released from IgG following deglycosylation by both enzymes were analyzed using nano LC‐TOF MS.

Results: Remove‐iT Endo S has a high specificity for removing the N‐glycan moiety of IgG after the first N‐acetylglucosamine (GlcNAc) residue, leaving only the GlcNAc with or without a core fucose residue. However, PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N‐linked glycoproteins.

Conclusion: Remove‐iT Endo S is a more robust enzyme for the method described herein as it completely removes the sugar residues from IgG. Conversely, the PNGase F glycerol free digest does not result in a complete digestion under native conditions.
ArtinM activates CD4+ T cells through recognition of N‐glycans of CD3γ chain (1004.3)
Authors: da Silva Thiago , Souza Maria , Roque Barreira Maria Cristina
Published: June 2021

ArtinM, from Artocarpus heterophyllus, a lectin that binds to the manotriose core of N‐glycans, modulates immunity toward Th1 axis and confers resistance to intracellular pathogens. These properties were credited to the ArtinM binding to TLR2 N‐glycans on innate immune cells, with the consequent production of IL‐12. In this work we investigated the ArtinM effects on murine CD4+ T cells. ArtinM stimulated CD4+ T cells released high levels of IL‐2, IFN‐γ, IL‐6 and IL‐17A and increased the expression of CD25 and CTLA‐4. These effects were inhibited by D‐mannose. To determine the ArtinM glycotarget(s) on CD4+ T cells, specific antibodies for CD3εγ, CD3ε, CD28, CD45 and CD4 were assayed for the inhibition of the responses to ArtinM. Only anti‐CD3εγ antibody prevented the cytokines secretion and the ArtinM binding to CD4+ T cells. Because anti‐CD3ε had no effect on the responses to ArtinM, we inferred that CD3γ glycan(s) is(are) targeted by ArtinM and accounts for its ability to activate CD4+ T cells. By using pharmacological inhibitors of signaling molecules, we verified that PI3K, PTK and p42/44MAPK participate in the cytokine production induced by ArtinM. We conclude that ArtinM (1) activates CD4+ T cells through binding to glycans of CD3γ chain and (2) promotes naive CD4+ T cells differentiation into Th1 and Th17. We postulate that the effects exerted on CD4+ T cells contribute for the immunomodulatory activity of ArtinM.

Grant Funding Source: Supported by FAPESP, CAPES, CNPq, FAEPA, FINEP