bluuebunny/crossref_metadata_2025 huggingface dataset:
Insertion of a functional copy of six2 into the mouse genome to examine prenatal morphogenetic factors leading to chronic renal failure in the adult
Authors: Chang Cara , Stoytcheva Zoia , Marh Joel , Dee James , Fogelgren Ben , Lozanoff Scott , Moisyadi Stefan
Published: June 2021

Suboptimal kidney development resulting in a decreased nephron number can have lifelong consequences and is associated with a significant risk of hypertension and end‐stage renal disease. The adult Br heterozygous mouse displays the symptoms of suboptimal kidney development along with a reduced six2 expression during embryonic renal morphogenesis. The purpose of this study was to determine whether a functional copy of six2 could be inserted into the Br mouse genome in an effort to rescue the mutant phenotype. A 26 KB six2 gene BAC fragment was isolated and processed by restriction digestion and pulse field gel electrophoresis. The resultant DNA fragment was then cloned into the mGENIE‐3‐BAC transposon vector via the Gibson Assembly method. The mGENIE‐3‐BAC‐six2 construct was subsequently confirmed by colony PCR, restriction digestion; the construct was also tested for functionality and expression in human cell lines and transgenic mice. Results from quantitative RT‐PCR demonstrated that a functional copy can be inserted into the human genome successfully, resulting in elevated transcription of mouse six2. Further experiments are being conducted to determine whether the adult CRF phenotype can be rescued prenatally by achieving an increased number of functional nephrons in adult transgenic mice.

Grant Funding Source: R01‐DK‐064752 (SL), 5P20RR024206 and R01‐GM‐083158–01A1 (SM)
Study on the neurogenic mechanisms of traumatic brain injury
Authors: Duan Hubin , Fan Yimin , Hao Chunyan , Li Shuzhen , Wang Suping , Wang Hongqin , Kuai Dong , Lian Shizhong , Xue Naizhao
Published: June 2021

Backgroud and purpose

In recent years, the mechanism of neurogenic inflammation in traumatic brain injury (TBI) gradually attracted people's attention. The purpose of research is to explore the specific mechanisms of traumatic brain edema caused by TBI.

Methods

40 male Wistar rats were randomly divided into control group, mild trauma group, moderate trauma and severe trauma group. Discharge frequency of the thalamic ventral posterior medial nucleus (VPM) were recorded after TBI.The expression of neuropeptide Y (NPY), substance P (SP), aquaporin‐4 (AQP4) and nuclear transcription factor‐kappa B (NF‐¦ÊB) were detected by immunohistochemical, Western blot, RT‐PCR and ELISA.

Results

With the increase of trauma degree, the more obvious cerebral edema, the more visible neurotransmitters release and inflammatory cell infiltration between the synapses, the more gene and protein expression of SP, NPY, NF‐¦ÊB, and AQP4. There are positive correlations between the above gene and protein expression; There are also positive correlations between SP and the firing frequency of VPM.(P <0.05).

Conclusion

The more severe damage of TBI, the more gene and protein expression of above neuropeptides. Neurogenic inflammation and neurogenic mechanisms play an important role in traumatic brain injury.
Regulation of the DNA‐binding protein CFP1 by ERK1/2
Authors: Klein Aileen M , Earnest Svetlana , Cobb Melanie H
Published: June 2021

CFP1 (CXXC finger protein 1) was identified in our lab as an ERK2 interactor through a yeast two‐hybrid screen. CFP1 binds to the maintenance cytosine methyltransferase DNMT1 and is required for DNA methylation in an embryonic model. Methylation of DNA in promoter regions is critical for transcriptional silencing throughout development and is often dysregulated in cancer. ERK2 is required for KRAS‐driven aberrant gene silencing via promoter methylation by DNMT1, but ERK2 substrates that may mediate this effect have not been identified. I propose that CFP1 is the major ERK2 substrate that directs DNA methylation by promoting the targeting or activity of DNMT1. ERK2‐bound mononucleosomes are enriched for CFP1. Active ERK2 can phosphorylate CFP1 on multiple sites in vitro. Current work is focused on determining the requirement for CFP1 in KRAS‐driven gene silencing, and dissecting the relationship between promoter methylation and the presence of active ERK2 at DNA methylation sites guided by ERK2 ChIP‐sequencing data from a lung cancer model. If ERK1/2 targeting of CFP1 helps to drive directed DNMT1 activity and tumor suppressor gene silencing, it would cement a clear mechanistic linkage between a common activating mutation in cancer and conserved downstream epigenetic effects that promote development of the disease.

This work was supported by NIGMS Training Grant T32 GM008203 and by NIH R37 DK34128.
Mortal Kombat: modeling amyloid fibrils and health implications
Authors: Smaoui Mohamed , Poitevin Frederic , Delarue Marc , Koehl Patrice , Orland Henri , Waldispuhl Jerome
Published: June 2021

Amyloid proteins aggregate into polymorphic fibrils that damage tissues of the brain, nerves, and heart. Experimental and computational studies investigated the structural basis and the nucleation of short fibrils, but the prediction and the precise quantification of the stability of larger aggregates remain elusive. We established a complete classification of fibril shapes and developed a tool, called CreateFibril, to automatically build these complex polymorphic modular structures. CreateFibril constructed HET‐s, Abeta, and Amylin fibrils up to 17nm in length, and utilized a novel dipolar solvent model that captured the effect of dipole‐dipole interactions between water and very large molecular systems to assess their aqueous stability. Our results validated experimental data for HET‐s and Abeta, and suggested novel findings for Amylin. In particular, we predicted the correct structural parameters for the one and three predominant HET‐s protofilaments, revealed and structurally characterized all known Abeta polymorphic fibrils including structures recently classified as wrapped fibrils, and elucidated the predominant Amylin fibrils and asserted that native Amylin is more stable than its amyloid form. CreateFibril and a database of all stable polymorphic fibril models we tested along with their structural energy landscapes are available on http://amyloid.cs.mcgill.ca.
Development of LCMS method for monitoring the effects of meat feasting, fasting, and exercise on urinary 3MH levels
Authors: Fenstermacher Scott , Cleary Kim , Hayes Erik , King Dan
Published: June 2021

LCMS is a proven effective tool for automated quantitation of biological samples. This study describes an LCMS assay for time course studies of metabolites of interest to muscle physiology researchers. The primary advantage to the LCMS approach is the ability to monitor multiple metabolites simultaneously. 3‐methyl histidine (3MH), a skeletal muscle protein breakdown product, was chosen for these method development studies. To account for varying degrees of hydration of the urine samples, 3MH levels are normalized both against the ubiquitous creatinine and the darkness of the sample. Initially, a meat fast and feast were conducted to determine the time required for urinary 3MH levels to reach a baseline. Results suggest that a 24hr meat fast is sufficient prior to intervention and sample collection. To evaluate the method's ability to observe changes in urinary metabolite levels in response to exercise, 3MH levels were monitored in 8 subjects following aerobic (70% HR max, 45min), interval (4 intervals of 3min at 90–95% and 70% HR max), and resistance training (2 sets to fatigue on 8 exercise machines). The exercise that stimulated the greatest 3MH excretion varied among the subjects.
ATPase coupling in the processive RNA helicase NS3 from hepatitis C virus
Authors: Bradley Michael John , Henn Arnon , Ding Steve , Pyle Anna , De La Cruz Enrique
Published: June 2021

Hepatitis C virus (HCV) NS3 motor protein contains both RNA helicase and serine protease activities that are vital to HCV replication in infected cells. NS3 belongs to the DExD/H‐box family of RNA helicases, which recognize short structured stretches of RNA and utilize ATP to remodel RNA and RNA‐protein complex structures. In contrast, NS3 is hypothesized to processively unwind the + and ‐ strands of the entire HCV RNA genome during viral replication, which likely requires that NS3 ATPase activity is tightly coupled to helicase translocation. To characterize the coupling of NS3 ATPase and translocation activities, we utilized equilibrium binding, steady‐state and transient kinetic biophysical methods. We compared a series of RNA substrates and found that RNA length affects both the concentration‐dependence and maximal rate of the RNA‐stimulated ATPase activity. Using transient kinetics, we demonstrated that the steady‐state rate of ATPase cycling is partially limited by a nucleotide‐dependent isomerization of the NS3‐RNA complex. On shorter RNAs, translocating NS3 protomers fall off the 5′ end of the nucleic acid track, and undergo this isomerization upon rebinding the ssRNA in order to restart productive translocation and rapid RNA‐stimulated ATPase. NS3 with an intact protease domain engages more productively with the RNA lattice, which increases apparent processivity of the ssRNA translocase.
LC‐MS plasma biomarkers associated with weight loss over 24 months following Roux‐en‐Y gastric bypass surgery
Authors: Bowers Jeremiah , LeGreve Tracy , Gletsu‐Miller Nana
Published: June 2021

Objective

To observe global changes in plasma metabolic profiles of patients following Roux‐en‐Y gastric bypass (RYGB) surgery.

Method

Plasma global metabolite profiles from patients that were monitored before 6 months (n=13) and 24 months (n=5) following RYGB were determined using high performance liquid chromatography‐mass spectrometry (HPLC‐MS). Fat mass and dietary intake were assessed using air displacement plethysmography and 3‐day dietary records. Data analysis included repeated measures analysis of variance and Pearson correlations, (p < 0.05).

Results

During 6 months following surgery levels of 3‐indoleacetic acid and proline betaine increased while (9R,13R)‐1a,1b‐dihomo‐jasmonic acid, acetoacetic acid, L‐valine, and choline decreased. Over 24 months, DL‐2‐aminooctanoic acid increased. No cross‐sectional associations were found between metabolites and fat mass, dietary fat, dietary carbohydrate, dietary protein, or daily caloric intake.

Conclusions

Changes in plasma metabolic profiles following RYGB suggest a metabolic response unrelated to changes in adiposity or macronutrient intake. The impact of specific foods will be investigated further.
Relationship between Inflammatory cytokines and bone mineral density in posmenopausal women with hormone replacement therapy.
Authors: Carvalho Cristina , Gonçalves Giovana , Bonetti Tatiana , Massad‐Costa Ana Maria , da Silva Ismael Guerreiro
Published: June 2021

Introduction

Bone formation and resorption are influenced by hormonal and inflammatory processes. Osteoporosis is a systemic disease condition with low bone mineral density (BMD), deterioration of the skeletal micro architecture and increased bone fragility highly prevalent at menopausal women.

Aim

We evaluated the relationships among inflammatory markers, hormone replacement therapy (E2) and bone mineral density (BMD) in healthy postmenopausal women and determined the contribution of inflammatory markers in BMD after 1‐year hormone replacement.

Materials and Methods

This preliminary analysis included 9 women evaluated pre and 1‐year pos E2 replacement. lumbar spine (LS) BMD were measured by dual energy x‐ray absorptiometry (DEXA) and serum inflammatory markers interleukin (IL)‐8, IL‐ 1β, IL‐6, IL‐10, IL‐12 and tumor necrosis factor‐alpha (TNF‐α) were measured at baseline and 1‐year post E2 replacement by flow cytometry using the Human Inflammation Kit BD Cytometric Bead Array (CBA). Statistical significance was analyzed by SPSS software and p<0.05 was considered statistically significant.

Results

The Peason correlation analysis showed a significant negative correlation between IL‐1 β and BMD at baseline (r=− 0.803;p=0.009) and 1‐year (r=−0.768;p=0.016).

Conclusions

IL‐ 1β is known for its role as inflammation mediator and recent researchers has shown involvement with ovarian processes. Our results suggest that increases in IL‐1β levels are related to decrease in BMD. Thun, modifying inflammatory markers, even in healthy postmenopausal women, may possibly reduce bone loss.
Cordyceps scarabaeicola inhibits inflammatory mediators by suppressing NF‐κB activation
Authors: Kim Tae‐Woong , Yoon Deok‐Hyo , Cho Jae‐Youl , Sung Gi‐Ho
Published: June 2021

Cordyceps is a parasitic fungus and has long been used to treat illnesses, promote longevity, increase athletic power, and anticancer. Ethanol extract of Cordyceps scarabaeicola was fractionated with hexane, ethyl acetate, buthanol and water. Buthanol fraction from C. scarabaeicola (CSBF) showed the best anti‐inflammatory effect. CSBF decreased the levels of iNOS protein and mRNA in a concentration‐dependent manner. The level of COX‐2 protein was suppressed in a dose‐dependent manner by cotreatment with CSBF. CSBF suppressed the COX‐2 mRNA levels dose dependently. And the increases of TNF‐α levels were significantly decreased in a concentration‐dependent manner by treatment with CSBF. CSBF suppresses the activation of Akt in a dose‐dependent manner. We found that NF‐κB activation by LPS was inhibited by CSBF and the increases of intracellular ROS and p38 activation were strongly inhibited by CSBF. Also CSBF suppresses the activation of ERK1/2 in a dose‐dependent manner. In summary, our findings demonstrated. CSBF inhibited signaling cascade of proinflammatory gene expression by suppressing NF‐κB activation, probably as a result of scavenging intracellular ROS. This inhibitory effect has important implications for the development of anti‐inflammatory drugs and strategies to prevent pathological inflammation. Supported by a grant from RDA(No. PJ00924101), Korea.
Antioxidant and antigenotoxic properties of water extract of sulfur fed duck
Authors: Park Eunju , Choi Mi‐Joo , Seo Bo‐Young
Published: June 2021

Duck fed sulfur has been traditionally used in alternative medicine as a detoxifying agent in Korea. This study was carried out to investigate the antioxidant and antigenotoxic properties of hot water extract of sulfur fed duck (SFD). The antioxidant activities of SFD were measured by DPPH radical scavenging activity, oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAC) values. Non cellular antioxidant activities increased in dose dependent manners, indicating the DPPH radical scavenging activity of 86.6% at 1000 μg/mL and ORAC value of 2.8±0.6 mM trolox equivalent (TE) at 50 μg/mL of SFD. The AAPH− induced oxidative stress in HepG2 cells was significantly inhibited by 17.1, 22.1, and 29.4% at the concentrations of 1, 5, and 10 μg/mL of SFD, respectively. Human leukocyte DNA damage induced by 200 μM of hydrogen peroxide, 200 μM of 4‐hydroxynonenal (HNE), or 250 μM of ferric nitrilotriacetate (FeNTA) was effectively protected by the pre‐treatment of SFD at the concentrations of 1–50 μg/mL. Our data provide evidence that support the usage of sulfur fed duck in traditional medicine in terms of antioxidant and antigenotoxic effect.
Comparative hemmorhagic studies on dabigatran, apixaban and rivaroxaban in a rat‐tail bleeding model
Authors: Jeske Walter , Escalante Vicki , Walenga Jeanine M , Hoppensteadt Debra , Fareed Jawed , Raake Wolfram
Published: June 2021

The new oral anticoagulant drugs include dabigatran (D), apixaban (A) and (R) rivaroxaban which have been recently approved for various indications. These agents produce their anticoagulant effects by inhibiting either Factor IIa or FXa. These drugs exhibit distinct pharmacologic profiles. Bleeding represents a major complication with the use of these agents. This study was designed to compare the bleeding profile of the agents in a standardized rat tail bleeding model. Powdered forms of active D, A and R were commercially obtained and a working solution was prepared at 100 ug/ml. Groups of rats were administered saline or each of the agents at 100 ug/kg IV. Bleeding measurements were made 5 minutes after the administration. Blood samples were drawn at the end of bleeding time for ex vivo analysis. In comparison to saline treated animals (300 + 60 sec), the D group exhibited a markedly high bleeding time (2250 + 240 sec), A did not show any significant increase (320 + 80 sec), whereas R exhibited a moderate increase (1110 + 130 sec). Ex vivo analysis of blood did not detect any D or R; however, varying levels of A were measurable. These studies show that at an equivalent dose of 100 ug/kg D produced a markedly stronger bleeding response, R produced a lowered response and A did not show any bleeding effect. Ex vivo results indicate that the stronger bleeding effects with D and R may relate to non‐plasmatic mechanisms.
Regulation of Cytochrome P450 3A4 in Non‐Alcoholic Fatty Liver Disease by Fibroblast Growth Factor 21
Authors: Woolsey Sarah Joy , Gong Inna , Kim Richard , Levstik Mark , Beaton Melanie , Tirona Rommel
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease affecting approximately one third of adults. For a majority of NAFLD patients, the associated metabolic syndrome necessitates drug therapy for management of diabetes, dyslipidemia and hypertension. Interestingly, while many drugs used by patients with NAFLD are eliminated from the body by hepatic drug metabolism, little is known regarding the effect of steatosis on hepatic drug metabolism and drug response. We have previously shown that Cytochrome P450 (CYP) 3A4 metabolic activity and expression are reduced in patients with NAFLD as well as in mouse and in vitro models of hepatic steatosis. Here, we investigated the role of the lipid‐ and glucose‐modulating hormone, Fibroblast Growth Factor 21 (FGF21), in the molecular mechanism regulating CYP3A4 expression in NAFLD. We confirmed that levels of circulating FGF21 are elevated in NAFLD patients. Using a cultured, human hepatoma cell (Huh7) model, we found forced steatosis caused a 4‐fold induction of FGF21 expression and a 50% decrease in CYP3A4 mRNA. Chemical inhibition of the canonical MAPK/ERK phosphorylation cascade triggered by FGF21, resulted in increased CYP3A4 expression. FGF21 treatment was associated with decreased nuclear localization of the CYP3A4 gene‐regulating transcription factors PXR, CAR. Moreover, transcription factor binding to the CYP3A4 promoter was reduced with FGF21 treatment. We conclude that FGF21 is involved in decreased hepatic drug metabolism in NAFLD. These findings are expected to provide the basis for further studies aimed at optimizing pharmacotherapy in NAFLD.
Stimulation of PAR2 during OIR reduces endothelial cell death and provokes rapid revascularization
Authors: Sitaras Nicholas , Joyal Jean‐Sebastien , Rivera Jose Carlos , Shao Zhuo , Sapieha Przemyslaw , Chemtob Sylvain
Published: June 2021

In the retina, a highly regulated vascular meshwork irrigates neurons and supplies necessary metabolic requirements. Sustained vessel loss, as in Retinopathy of Prematurity (ROP), prompts revascularization despite initial suppression of vaso‐survival cues such as VEGF. Thus, other forces are likely involved. Importantly, we have previously demonstrated the substantial role of Protease‐activated receptor 2 (PAR2), a GPCR, in the developing retina. The exact mechanism by which PAR‐2 modulates angiogenic cues in ROP remains elusive.

Expression and localization of PAR2 was determined by Western blot and immunohistochemistry in mouse whole retina. Appraisal of retinal vasculature and expression of pro‐ and anti‐angiogenic cues was assessed following intravitreal administration of PAR2 agonist peptide (SLIGRL) or lentiviral shRNA knockdown of PAR2 (LV shPAR2) in mice exposed to a murine oxygen‐induced retinopathy model (75% O2 from P7‐P12).

Our results demonstrate a pronounced expression of PAR2 preferentially in RGC. PAR2 sharply increases during the initial phases of vaso‐obliteration (VO) and during the neovascularization (NV) phase. Activation of PAR2 with SLIGRL in vivo reduced Sema3A while increasing VEGF. Importantly, SLIGRL treatment in vivo prior to and following oxygen exposure significantly decreased VO and NV. Treatment with LV shPAR2 increased VO while augmenting NV.

This study underscores the importance of the coupling between neurons and vessels and shows that RGC‐specific PAR2 plays a pivotal role in modulating angiogenesis in a pathological context.
Crystal structure of active Beta‐arrestin1 bound to phosphorylated carboxy‐terminus of a G protein‐coupled receptor
Authors: Shukla Arun K. , Manglik Aashish , Kruse Andrew , Reis Rosana , Tseng Wei Chou , Staus Dean P. , Hilger Daniel , Uysal Serdar , Huang LiYin , Peduch Marcin , Tripathi‐Shukla Prachi , Koide Akiko , Koide Shohei , Weis William I. , Kossiakoff Anthony K. , Kobilka Brian K. , Lefkowitz Robert J.
Published: June 2021

The functions of the ubiquitous family of G‐protein coupled receptors (GPCRs), are mediated and critically modulated by beta arrestins. Beta arrestins bind to phosphorylated receptors and lead to receptor desensitization. Beta arrestins activated by their interaction with phosphorylated receptors can also mediate receptor endocytosis and G protein independent signaling by serving as adaptors linking receptors to numerous signaling pathways. Here, we present a 2.6 Å crystal structure of β‐arrestin1 in complex with the fully phosphorylated carboxyl terminus of the V2 vasopressin receptor (V2R) and a synthetic antibody fragment that selectively recognizes the activated conformation of β‐arrestin1. The overall structure of activated β‐arrestin1 reflects a range of conformational changes compared to the inactive structures. The two domains of β‐arrestin1 undergo a substantial twisting relative to one another. The carboxyl terminus of the V2R makes extensive contacts with the N‐domain and displaces the β‐Arrestin1 carboxyl terminal beta strand. A major rearrangement of the “lariat loop” is also observed which leads to disruption of the polar core and relives the constrains which otherwise keep β‐arrestin 1 in a basal conformation. The structure reveals, for the first time at atomic detail, how interaction with GPCR attached phosphates activate β‐arrestins and starts to provide missing links to completely understand the molecular mechanism for GPCR activation and regulation.
Metformin modulates hyperglycemia‐induced endothelial dysfunction through SIRT1
Authors: Arunachalam Gnanapragasam , Samuel Samson M , Ding Hong , Triggle Christopher R
Published: June 2021

Endothelial dysfunction, detected at an early stage in the development of diabetes‐related microvascular disease, is associated with accelerated endothelial senescence. Hyperglycemia‐induced endothelial senescence is an important contributing factor that promotes the development of ageing‐associated cardiovascular events. Clinical data indicate that the hypoglycemic agent, metformin, has an endothelial protective action. Our data with mouse microvascular endothelial cells cultured in high glucose (HG, 40mM), versus normal glucose (NG, 11mM), indicate a significant reduction in SIRT1 protein, an increase in FoxO‐1 and p53 acetylation along with an increase in the p21 levels and senescence‐associated beta‐galactosidase activity. Metformin treatment significantly abolishes the reduction of SIRT1 level and modulates the SIRT1 downstream targets FoxO‐1, p53/p21. We also investigated the role of small noncoding RNAs, miRNAs, as molecular targets that regulate SIRT1 expression. Our data demonstrates that hyperglycemia‐induced downregulation of SIRT1 plays a crucial role in diabetes‐induced endothelial senescence and, furthermore, the endothelial protective effect of metformin against HG‐induced endothelial dysfunction is, at least in part, due to its effects on SIRT1 expression and/or activity.

This project was funded by grant # NPRP 4–910‐3–244 & NPRP 08–165‐3–054
Detection of the hyponitrite species (HO‐N=N‐O) in denitrification: Reactivity of NO with the heme Fe‐Cu center of cytochrome caa3 and the heme Fe –Fe center of Nitric oxide reductase
Authors: Varotsis Constantinos , Pinakoulaki Eftychia , Ohta Takehiro , Kitagawa Teizo
Published: June 2021

Nitric oxide reductase (NOR) is the third of the four enzymes of bacterial denitrification that is responsible for the catalytic formation of laughing gas (N2O). Here we report on the formation of the hyponitrite (HO‐N=N‐O−) species (νN‐N=1332 cm−1) in the heme b3 Fe/non‐heme FeB dinuclear center of NOR from Paracoccus denitrificans. With the identification of the hyponitrite species the mechanism for the 2e−/2H+ reduction of NO to N2O can be described with more certainty.

This work was financially supported by the Cyprus Research Promotion Foundation grant TEXNOLOGIA/THEPIS/0609(BE)/05.
EXERCISE TRAINING RESTORES IIMPAIRMENT ON CARDIOVASCULAR AUTONOMIC MODULATION IN AGED OVX RATS
Authors: Dias Danielle Silva , Bernardes Nathalia , Brito Janaina , Machi Jacqueline , Barboza Catarina , Rodrigues Bruno , Irigoyen Maria Cláudia
Published: June 2021

The aim was to evaluate the impact of exercise training on hemodynamic parameters and autonomic control in old ovariectomized rats. Female old OVX rats (~18 wks. of age) were divided into 3 groups (n=8): old control (OC), old sedentary ovariectomized (OVX) and old trained ovariectomized (OVXt). The ovariectomy was performed by bilateral ovaries removal. The exercise training was performed on a treadmill for 8 weeks. Arterial pressure (AP) and heart rate (HR) signals were directly recorded in conscious rats. Assessment of autonomic modulation was performed by spectral analysis. The OVX group (130±5.05mmHg) showed increased AP compared to OC group (116 ± 2.56mmHg). Exercise training induced resting bradycardia (344± 7 bpm) compared to OC (375±14bpm) and OVX groups (384±10bbpm). The ovariectomy induced reduction in standard deviation (SD) of pulse interval (OVX:3.80 ± 1.08 vs. OC:8.82±1.96ms2) and RMSSD (OVX:3.15± 1.17 vs. OC:11.41±2.76ms). In addition, there was an increase in the SD of systolic AP in the OVX group (7.41±1.69mmHg2) compared to OC group (4.14±1.29mmHg2). Exercise training attenuated the changes in cardiac and vascular autonomic modulation (SD‐PI: 11.25±8.9ms2, RMSSD:11.48±8 ms and SD‐SAP:4.82±3.12 mmHg2). These data suggests that exercise training reduced AP and HR associated with attenuation of cardiovascular autonomic modulation in aged rats submitted to ovarian hormones deprivation.
Effects of High‐Fructose Consumption on Endothelial Progenitor Cell Function
Authors: Kertowidjojo Elizabeth Calista , Wolin Michael , Hintze Thomas H , Eisenberg Leonard , Eisenberg Carol
Published: June 2021

Many studies have shown a causal relationship between high fructose diet and endothelial dysfunction. This dysfunction is likely a result of increased oxidative stress and subsequent decrease of nitric oxide (NO) bioavailability. We hypothesize that one of the factors contributing to endothelial dysfunction during fructose feeding (FF, isocaloric 60% fructose diet for 49 days) is impaired endothelial progenitor cell (EPC) proliferation, as EPC growth is known to be greatly affected by oxidative stress and NO imbalance. EPCs isolated from FF dogs (N=6) displayed impaired growth (32.8 ± 4.7 FF compared to 73.3 ± 4.1 control cells/mm2 after 2 weeks of culture). Proliferation of FF cells was partially rescued by treatment with AT1R blocker Losartan, suggesting the involvement of Angiotensin II. Scavenging of superoxide by Tiron also improved FF EPC growth, indicating oxidative stress as the cause of dysfunction. Finally, addition of Apocynin produced a similar rescue, indicating NADPH oxidase as the source of elevated oxidative stress. Increased oxidative stress in FF EPCs was confirmed by measurement of H2O2 level, revealing significantly higher concentration in FF cultures (40 ± 1 μM in control and 70 ± 4 μM in FF). H2O2 level was reduced to control with the addition of Losartan, Apocynin, OTC (a glutathione precursor), or the NO donor, SNAP. Supported by PO43023.
Small molecule ACE2 activator, Diminazene aceturate (DIZE) attenuates MI‐induced cardiac pathophysiology
Authors: Qi Yanfei , Shenoy Vinayak , Zhang Juan , Katovich Michael , Raizada Mohan
Published: June 2021

Angiotensin‐converting enzyme 2 (ACE2) plays a critical role in maintaining cardiac homoeostasis during myocardial infarction (MI). We hypothesize that DIZE would produce beneficial outcomes in the cardiac pathophysiology induced by ischemia. DIZE treatment (15 mg/kg/day, s.c.) was initiated two days before MI surgery and continued throughout the study. MI rats showed a significant decrease in fractional shortening (FS, %) (control: 54.2 ± 2.6; DIZE alone: 52.5±2.9; MI: 32.3±2.3), increases in left ventricular end diastolic pressure (LVEDP, mmHg) (control: 3.0±0.7; DIZE alone: 2.6 ±0.8; MI: 14.2±2.6) and ventricular hypertrophy (VH, mg/mm tibia length) (control: 26.5±1.5; DIZE alone: 26.9 ± 1.4; MI: 33.4± 1.1). DIZE treatment significantly prevented each of these impairments in the MI rats (FS 44.29 ± 1.55 %, LVEDP 5.7 ± 2.8 mmHg, VH 26.5 ± 0.7 mg/mm). DIZE treatment also resulted in a significant increase in the cardiac ACE2 activity and its mRNA levels while AT1R and ACE mRNA levels were decreased. DIZE treatment caused an increase in cardiac progenitor cells and prevented the infiltration of macrophages into the peri‐infarct area of the ischemic heart. Collectively, our observations indicate that activation of endogenous ACE2 improves cardiac function and attenuates left ventricular remodeling post‐MI. Thus, DIZE treatment may represent a promising therapeutic strategy for MI.
Modulation and adherence of intestinal mucus by commensal bacteria and the pathogen C. difficile
Authors: Engevik Melinda , Ganesh Bhanu , Morra Christina , Luk Berkley , Versalovic James
Published: June 2021

Gut pathogens initially encounter the host at the gastrointestinal (GI) mucus layer making its regulation a crucial aspect of gut health. Few studies have addressed bacteria‐mucus adherence or mucus oligosaccharide utilization by commensal or pathogenic bacteria. To address this gap in knowledge, the interaction between mucus (human stool, HT‐29‐MTX cells and human ileal enteroids) and commensal bacteria (B. thetaiotaomicron, C. coccoides, C. butryicum, B. producta, A. muciniphila, F. prausnitzii, R. torques, L. reuteri) or the pathogen C. difficile was examined. In vitro cultures in tryptone‐yeast extract media revealed that C. difficile and B. producta were capable of using multiple types of oligosaccharides (fucose, mannose, galactose, GlcNAc, GalNAC, sialic acid) as a sole carbon source, while other commensal bacteria exhibited specific oligosaccharide preferences. Commensal bacteria had enhanced adherence to mucus from human stool compared to HT‐29‐MTX mucus. Additionally, FITC‐labeled oligosaccharide specific lectins demonstrated that all tested commensal bacteria were capable of altering mucus oligosaccharide composition. Select commensal species were also able to stimulate mucus secretion. In contrast, the pathogen C. difficile adhered to multiple oligosaccharides in human stool mucus, but had limited adherence to HT‐29‐MTX mucus. C. difficile did not stimulate mucus secretion in HT‐29‐MTX cells and decreased secretion in enteroids. Together this data demonstrates that commensal and pathogenic bacteria adhere directly to GI mucus, but this interaction is mucin type and composition dependent. Manipulation or adherence to intestinal mucus may represent a method of bacterial‐host modulation. T32DK07644
Protein Quality Control In Alveolar Type 2 Cells: The Proteasome Is Essential For Control Of Aggregation‐Prone SP‐C Mutations
Authors: Hawkins A , Gonzales L , Guttentag S , Mulugeta S , Beers M
Published: June 2021

Mutations in the distal C‐terminus of the Surfactant Protein C (SP‐C) proprotein (BRICHOS domain) result in aggregation‐prone isoforms and are associated with interstitial lung disease (ILD). The ubiquitin proteasome system (UPS) and macroautophagy (autophagy) are the main quality control mechanisms for aberrant proteins. The extent to which these proteostatic pathways are utilized by human alveolar type II cells (hAT2) is unknown. Treatment of primary hAT2 with rapamycin resulted in inhibition of S6 phosphorylation, conversion of LC3I/II, and quenching of vesicular EGFP‐LC3‐mCherry fluorescence consistent with the presence of inducible autophagy. Using GFP‐U1 and chymotrypsin‐like activity as readouts, hAT2 exhibited a high degree of basal proteasomal function which was inhibited by MG132 but also resulted in accumulation of LC3 and p62 suggesting cross‐talk between the pathways. Compared to wild type SP‐C, EGFP tagged clinical SP‐C BRICHOS mutants were expressed at significantly lower levels in transfected hAT2. UPS inhibition by MG132 caused accumulation of BRICHOS mutants while autophagy inhibition with 3‐methyladenine had minimal effects on mutant SP‐C levels. Exposure of hAT2 to cigarette smoke (CS) condensate inhibited proteasomal activity, induced markers of both ER stress (GRP78) and autophagy inhibition (p62), and caused cytosolic aggregation of BRICHOS mutants. These results indicate that the UPS represents a primary protein quality control system for the distal lung epithelia. The inhibitory effect of CS exposure on both systems supports a 2‐hit model of ILD of a susceptible epithelial cell phenotype plus exogenous cellular stress.
TAZ Activation Drives Fibroproliferation in Spheroid Culture
Authors: Jorgenson Amy , Choi Kyoung , Sicard Delphine , Tschumperlin Daniel
Published: June 2021

The transcriptional co‐activator TAZ plays a critical role in lung alveolarization (Mitani et al. Am J Respir Crit Care Med. 2009). We recently observed enhanced TAZ expression in the nuclei of fibroblasts present in lung tissue from patients with idiopathic pulmonary fibrosis, suggesting that TAZ is reactivated during fibrosis. To test the role of TAZ in driving a fibroproliferative response, we developed scaffold‐free cultures in which fibroblasts were grown in 3D hanging drop spheroids. This spheroid model removes the influence of the artificial 2D cell culture environment on cell growth. NIH3T3 fibroblasts were stably transduced with doxycycline (dox)‐inducible constructs expressing nuclear‐localizing mutant TAZ4SA or empty vector (courtesy of X. Varelas, Boston University). Spheroids were initiated by seeding 500 cells in hanging drops, and were subsequently fed growth media supplemented with 0, 1, 10, or 100 ng/mL dox every other day up to 15 days. Spheroid growth was tracked by measuring 2D projected area under light microscopy. Control NIH3T3 fibroblast spheroids remained stable in culture, but neither increased nor decreased in size significantly. In contrast, TAZ4SA‐transduced spheroids grew over time, and by day 15 spheroids treated with 100 ng/ml dox had a 2D projected area ~2.2‐fold larger than those grown in the absence of dox (p=0.0054). Intermediate growth levels were observed in 1 and 10 ng/ml doxycycline treated spheroids. Our results demonstrate the utility of the spheroid culture model for studying fibroproliferation, and document a prominent role for TAZ in promoting fibroblast spheroid growth.

*Supported by NIH grants RO1 HL092961 and NIH R21 HL113796.
Identification of Novel Interferon Stimulated Genes in Differentiated Hepatocytes
Authors: Contreras Deisy , Ignatius Irudayam Joseph , Songyang Ren , Nguyen Quoclinh , French Samuel , Funari Vincent , Arumugaswami Vaithilingaraja
Published: June 2021

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long term survival of engrafting cells in the body, not only do the cells have to execute liver‐specific functions but also withstand metabolic stressors and invading pathogens. The cellular innate immune system orchestrated by the interferon pathway provides the first line of defense against infectious agents. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN‐induced genes during hepatic differentiation of pluripotent stem cells. We derived endodermal cells, hepatoblast and immature hepatocytes from hESC using stage‐specific growth factor cocktails. The cells were stimulated with IFN‐α and subjected to type I IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that IFN‐α treatment activated JAK‐STAT pathway in differentiating cells. Unbiased RNA sequencing analysis indicated stage specific expression of classical and novel IFN‐stimulated genes demonstrating an intact innate immune response. Reverse transcription‐qPCR confirmed the IFN‐induced expression of novel ISGs including RASGRP3, CLMP, PPM1J, and TRANK1. Infection of these hESC‐derived hepatic cells with Hepatitis C virus also induced the expression of ISGs including the classical and novel genes – LAMP3, ETV7, and TRANK1 further confirming intact innate immunity. hESC‐derived hepatic cells contain an intact innate system.
The Molecular Tool‐Kit of Viral Hemorrhagic Fevers
Authors: Ollmann Saphire Erica , Bornholdt Zach , Fusco Marnie , Abelson Dafna , Hashiguchi Takao , Lee Jeffrey , Kawaoka Yoshihiro , Dye John , Crowe James
Published: June 2021

Ebola and Marburg viruses cause hemorrhagic fever with up to 90% lethality, and their genomes encode just seven genes. The few gene products encoded, their “tool‐kit”, are leveraged into a greater array of functions, by remodeling or rearranging of the 3D protein structures. The surface glycoprotein adopts a massive, heavily glycosylated form on the viral surface, but is remodeled into a minimal receptor‐binding core once inside the endosome. As a result, binding of many potential antibodies is lost as their epitopes are stripped form the virus. Crystal structures of potently neutralizing antibodies, however, reveal how one remains bound and reaches into the cryptic receptor‐binding site. Others likely neutralize by locking the GP structure in place. Additional protein transformation takes place at the other end of the virus life cycle, assembly and budding of nascent virions. Through multiple crystal structures, biochemistry and cellular microscopy, we illustrated that the matrix protein VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly‐shaped VP40 dimer trafficks to the cellular membrane. A distinct, linear hexamer structure is critical for assembly and release of the virion. A third structure of VP40, an RNA‐binding ring, regulates viral transcription inside infected cells.
Comparison of LOCI Digoxin Assay and iDigoxin Assay for Rapid Detection of Convallatoxin, the Active Glycoside of the Lily of the Valley Plant
Authors: Everett Jamie , Kojima Yumi , Dasgupta Amitava
Published: June 2021

Objective

Lily of the Valley is a poisonous plant, used as an herbal remedy, with toxic effects due to convallatoxin, a cardiac glycoside. It has been reported that the LOCI digoxin assay, a luminescent oxygen channeling based digoxin immunoassay, has sufficient cross reactivity with Lily of the Valley extract, and the specific toxic component, convallatoxin. Yet, cross reactivity by the iDigoxin immunoassay, a chemiluminescent microparticle immunoassay, has not been reported in the literature. In this study, we evaluate iDigoxin immunoassay's ability to detect Lily of the Valley and convallatoxin, and compare these findings to those measured by the LOCI digoxin assay.

Materials and Methods

Aliquots of drug free and digoxin serum pools were supplemented with microliter amounts of Lily of the Valley extract or nanogram quantities of convallatoxin. Digoxin concentrations were measured using the iDigoxin immunoassay applied to the ARCHITECT i2000SR analyzer (Cabbott Laboratories), and the LOCI digoxin assay applied to the Vista 1500 analyzer (Siemens Diagnostics).

Results

The iDigoxin assay measured significantly higher apparent digoxin concentrations than the LOCI digoxin assay in all supplemented aliquots. Also, the LOCI digoxin assay demonstrated bi‐directional interference with aliquots of a digoxin pool supplemented with various amounts of convallatoxin. However, with the iDigoxin assay all values were falsely elevated, indicating positive interference only.

Conclusions

The iDigoxin assay can be used for rapid detection of convallatoxin and Lily of the Valley in human serum. Also, the iDigoxin assay is more sensitive than the LOCI digoxin assay in convallatoxin detection.
Mechanism of Action in NaHS‐Induced Increase in Aqueous Humor Outflow
Authors: Robinson Jenaye , Okoro Esther , Mitchell Leah , Nguyen Olivia , Opere Catherine , Ohia Sunny , Njie‐Mbye Ya Fatou
Published: June 2021

Previous studies from our laboratory demonstrate that the hydrogen sulfide (H2S) donor, sodium hydrosulfide (NaHS) can increase aqueous humor (AH) outflow facility in porcine trabecular meshwork tissues, ex vivo. In the present study, we investigated the mechanism/s that underlie the NaHS‐induced increase in AH outflow facility

Methods

Porcine ocular anterior segments explants were perfused with Dulbecco's Modified Eagle's Medium maintained at 37° C, 5% CO2 and constant pressure of 7.35 mmHg. Once outflow was stable (~3 hours), explants were exposed to the adenylyl cyclase inhibitor SQ22536 (100 µM), enzyme inhibitors for H2S biosynthesis [proparglyglycine (PAG, 1 mM); aminooxyacetic acid (AOA, 30 µM)], and prostanoids (flurbiprofen, 3 µM), 30 minutes prior to application of NaHS (10 µM). Outflow was then monitored for an additional 4 hours, and vehicle control (0.1% saline) was run in parallel.

Results

The NaHS‐induced increase in AH outflow was attenuated by AOA and PAG by 79% and 56%, respectively. In the presence of flurbiprofen (3 µM), effects of NaHS on outflow was significantly (p<0.02) inhibited from [130.6 ± 10.51% of basal] to [102.9 ± 2.88% of basal]. Furthermore, the adenylyl cyclase inhibitor, SQ22536 completely blocked (p <0.05) the NaHS‐induced increase in outflow facility.

Conclusion

We conclude that the ability of NaHS to increase AH outflow in porcine trabecular meshwork is dependent upon the intramural generation of H2S and prostanoids, and on the integrity of the adenylyl cyclase pathway.
Molecular Mechanisms of Electroacupuncture‐Induced Cardioprotection
Authors: Roessler Anne , Ren Xiaoping , Luther Kristin , Jones W Keith
Published: June 2021

A non‐ischemic, nociceptive stimulus such as electroacupunture (EA) in distant tissues can result in the most powerful cardioprotection reported – an 80% decrease in infarct size – called remote preconditioning of trauma (RPCT). It is known that the mechanisms underlying ischemic preconditioning, remote ischemic preconditioning, and RPCT are similar. Whether the downstream genes implicated in other cardioprotective stimuli are also involved in RPCT remains to be determined and is the objective of this study. Specifically, this study will address the hypothesis that electroacupuncture (EA) as a remote nociceptive stimulus is cardioprotective and increases NF‐κB dependent expression of heat shock proteins in the heart. Activation of heat shock proteins (HSPs) by the transcription factor NF‐κB is necessary for late IPC. Preliminary data supports that NF‐κB is required for protection against myocardial infarction 24h after RPCT. qRT‐PCR and Western Blotting were used to assess the changes in gene and protein levels following EA in a mouse model. Previous studies indicate that EA reduces infarct size after occlusion of the left coronary artery. We show that EA of skin results in significant increases in levels of cardiac mRNA levels of HSP90 and DNAJs 3 hours post‐stimulus. Protein accumulation and kinetics are under investigation and confirmatory studies with knockout mice are planned. Future studies will further address the molecular mechanisms of cardioprotection associated with EA therapy. Understanding the mechanism of RPTC via nociception would facilitate the development of non‐invasive therapies, which might be used to precondition patients against ischemia/reperfusion injury by mimicking remote cardioprotection.
Deletion of Soluble Epoxide Hydrolase Modulates Coronary Reactive Hyperemia in Isolated Mouse Heart
Authors: Hanif Ahmad , Zeldin Darryl , Nayeem Mohammed
Published: June 2021

Coronary reactive hyperemia (RH) is impaired in hypertrophic hearts. Epoxyeicosatrienoic acids (EETs) exert cardioprotective effects in ischemia / reperfusion injury. Soluble epoxide hydrolase (sEH) is the main enzyme responsible for EETs breakdown. EETs level is increased in sEH knock‐out mice (sEH‐/‐). We hypothesized that in the absence of sEH, isolated mouse heart exhibits increased RH through increased generation of EETs in response to no‐flow ischemia compared to wild type (sEH+/+). Coronary flow (CF) in isolated sEH+/+ and sEH‐/‐ mouse heart was measured using Langendorff system. Perfused isolated heart was exposed to 15 second ischemia and RH was assessed. Following ischemia, flow repayment volume (RV) was significantly higher in sEH‐/‐ compared to sEH+/+ (7.6 ± 0.5 in sEH‐/‐ vs. 5.9 ± 0.7 in sEH+/+, p<0.05). Moreover, sEH‐/‐ exhibited significantly higher repayment to debt ratio when compared to sEH+/+ hearts (2.1 ± 0.2 in sEH‐/‐ vs. 1.5 ± 0.1 in sEH+/+, p<0.05). Upstream of EETs; the increased in RV in sEH‐/‐ was significantly decreased by SCH‐58261 (a selective adenosine A2A antagonist, 1.0 mM) (6.8 ± 0.8 in sEH‐/‐ without SCH‐58261 vs. 3.5 ± 0.3 with SCH‐58261, p<0.05). Also, downstream of EETs; T‐0070907 (a selective PPARg antagonist, 10.0 mM) significantly decreased RV in sEH‐/‐ (7.7 ± 1.2 in sEH‐/‐ without T‐0070907 vs. 5.4 ± 0.8 with T‐0070907, p<0.05).

Our results demonstrate that deletion of sEH increases RH compared to WT and that the enhanced RH is significantly decreased by SCH‐58261 and T‐0070907. These data suggest that targeting or silencing sEH may have a beneficial effect on myocardial recovery from ischemia. Supported by HL‐114559 to MAN and z01‐ES025034 to DCZ
The Role of Cross‐links Between Endoplasmic Reticulum Stress, Oxidative stress and Mitochondrial dysfunction in Cardiomyocytes and H9c2 Cells under Hyperglycemia
Authors: HAFEZ GAYE , TUNCAY ERKAN , TOY Aysegul , TURAN Belma
Published: June 2021

Under physiological condition, endoplasmic reticulum (ER) is responsible basically from protein synthesis and secretion as well as intracellular Ca2+ signalling while sarcoplasmic reticulum (SR) plays basical role in excitation‐contraction coupling of cardiomyocytes.A change or differientiation in ER related functions can induced by ER stress via affecting signaling pathways. Various studies showed that the cross‐relations between ER stress, increased ROS/RNS, and mitochondrial (MitC) dysfunction induced cardiomyocyte dysfunction via affecting intracellular free Ca2+and Zn2+ homeostasis. Therefore, we aimed to investigate the cross‐links in cardiomyocytes and H9c2 cells under hyperglycemia, ER‐stress induced by tunicamycin (TM) and increased cytosolic Zn2+due to increased ROS/RNS. Freshly isolated ventricular cardiomyocytes from male rats under hypergycemia and increased cytosolic Zn2+ conditions exibited significant increases in the levels of ER stress chaperon proteins such as GRP78 and calregulin while only hyperglycemia and ER‐stress induced these changes in the levels of calnexin and Gadd153. We also show the levels of those proteins in H9c2 cells after incubation of TM for 18 h. And also, our data show ER and MitC interaction, the levels of PML and some proteins associated with apoptosis as Bcl‐2 and Bax are examined. Furthermore, these conditions induced marked changes in the membrane potential of MitC. These data with functional data demonstrate that increased ROS/RNS observed under these conditions in cardiomyocytes and H9c2 cells underlie both ER stress and MitC dysfunction thereby inducing cardiac dysfunction in diabetic subjects (Supported by TUBITAK‐SBAG 113S466).