bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rmi1 is essential for DNA damage response and embryonic development in mice
Authors: Guiraldelli Michel Farchi , Pezza Roberto 
Published: June 2021

Suppression of Crossovers (CO) is critical for maintenance of genome integrity. CO may lead to loss of heterozygosity and CO between low‐copy repeats may account for many human disorders. Eukaryotes possess mechanisms that down regulate the number of CO. Indeed, BLM, the helicase mutated in Bloom syndrome, associates with topoisomerase IIIα and Rmi1 (RecQ‐mediated genome instability 1) to form a complex (the BLM dissolvasome) that efficiently control the number of CO by specifically resolving a key intermediate of recombination, the double Holliday junction (DHJ). To address the requirement of Rmi1 in mammals we generated knockout mice for this protein. Our results show that Rmi1 is required for normal embryonic development. Rmi1−/− knockout embryos die at 8.5 dpc, showing profound developmental defects reminiscent to those described for BLM−/− knockout mice. We obtained mouse embryonic fibroblasts from Rmi1−/− knockout and determined that these cells showed significantly reduced survival rates with respect to Rmi1+/− heterozygote and wild‐type controls. In this work, we will present a detailed characterization of developmental defects observed in Rmi1−/− knockout mice and study the DNA damage response of Rmi1 deficient cells. Our results will define how and when Rmi1 regulates the repair of DNA double‐stand breaks. This is important for understanding the mechanism by which somatic cells eliminate deleterious recombination products which are the root cause of genome instability and losses of heterozygosity.Financial support: OCAST
Suboptimal release of CD34+/CD144+ cells in atherosclerotic patients in response to ischemia: role of plasmatic TGF‐B
Authors: Tejerina Teresa , Gonzalez‐Rocafort Alvaro , Navarro Jorge , Ramajo Marta , Hristov Mihail , Weber Christian , Martinez‐Gonzalez Jose , Gordillo‐Moscoso Antonio , Redondo Santiago 
Published: June 2021

ObjectivesTo determine the number of CD34+/CD144+ cells in coronary artery bypass grafting (CABG) patients, compared to valvular patients and healthy controls, and the apoptotic effect of their plasma on peripheral blood‐derived angiogenic early outgrowth cells (EOCs) from healthy donors.MethodsCD34+/CD144+ cell counts were assessed by cell cytometry from peripheral blood mononuclear cells. EOCs were separated by ficcoll gradient and cultured on fibronectin using microvascular medium. Protein expression in EOCs was determined by Western blot.ResultsWe found a lower number of CD34+/CD144+ cells in CABG patients compared to valvular patients, but a higher number compared to controls. Being bypass or valvular had a strong and independent association with CD34+/CD144+ even after the P was adjusted by the number of comparisons. Plasma from CABG patients decreased apoptosis in cultured EOCs from healthy donors (day 7). This effect was abrogated by the transforming growth factor‐beta 1 (TGF‐b1) blockers SIS3 and SB‐431542. Of note, prior to apoptosis protection, plasma from bypass patients was able to upregulate the expression of both CD34 and CD144 at day 4 of isolation.ConclusionBypass patients, which a high degree of ischemia and previous infarct rate, have a suboptimal release of CD34+/CD144+ despite a TGF‐beta‐mediated protective effect of their plasma on cultured EOCs.
Are parental CHO‐K1 cells appropriate negative controls for engineered CHO cell lines ?: Evidence from label‐free functional assays
Authors: Scibek Jeffery J , Rammohan Aravind , Coldwell Martin , Dodgson Kathryn , Rawlins Philip , Lopez Francisco J 
Published: June 2021

In functional assays, parental cell lines are commonly used as a negative control for cell lines expressing recombinant proteins. Since the apparent primary difference between the two lines is the expression level of the transfected receptor, it is usually assumed that endogenously expressed “background” receptors show similar functionality in both cell lines. Label‐free technologies enable responsiveness testing to a broad variety of endogenous receptors. We have used the Corning® Epic system to empirically test the degree of similarity in the functionality of endogenous receptors in parental CHO‐K1 and a muscarinic receptor‐expressing cell line (CHO‐M).The responsiveness of each cell line to a panel of ~150 receptor agonists (1 μM) was measured on the Epic System. While the two data sets were positively correlated, Bland‐Altman analysis indicated large differences in endogenous receptor responsiveness. Differences were larger when evaluated at later time points. These data indicate that: 1) equivalency of parental to engineered cell lines should be experimentally tested 2) the Epic technology provides an efficient approach for quickly surveying responsiveness to variety of agonists 3) differences between CHO‐K1 and CHO‐M cells extend beyond the expression of the muscarinic receptor and 4) correlation alone is not sufficient to guarantee response equivalency.
The Safety of EXPARELTM Bupivacaine Extended‐release Liposome Injection) Administered by Peripheral Nerve Block in Rabbits and Dogs
Authors: Richard Brigitte M , Newton Paul E , Ott Laura R , Haan Dean , Brubaker Abram N , Cole Phaedra I , Ross Paul E , Rebelatto Marlon C , Nelson Keith G 
Published: June 2021

Bupivacaine solutions have been used for many years by multiple routes with peripheral nerve block (PNB) becoming increasingly popular. A sustained‐release DepoFoam® injection formulation of bupivacaine (EXPAREL, EXP, 15 mg/mL) is currently being investigated for post‐surgical analgesia via PNB. The program focused on showing that this novel formulation is well tolerated and did not produce nerve damage after PNB in rabbits and dogs. The single‐dose toxicology studies of EXP (9, 18, and 30 mg/kg), bupivacaine HCL solution (Bsol, 9 mg/kg) and saline control included clinical pathology, toxicokinetics, organ weight and full histopathology evaluation on Day 3 and Day 15 (2/sex/group/period). The administration of EXP resulted in 2‐ to 3‐fold lower Cmax than Bsol. EXP was well tolerated at doses up to 30 mg/kg. There was no evidence of nerve damage after PNB. The only EXP‐related effect seen was minimal to mild granulomatous inflammation of adipose tissue around nerve roots (7‐8 of 24 animals), in the brachial plexus sites; this low severity effect was considered an expected response to the liposomes, and non adverse. Based on these studies, EXP has a low potential for producing adverse local or systemic effects when given as a single dose. These preclinical toxicology data support the first‐time‐inhuman trial of EXP by this route.Source of Research Support: Pacira Pharmaceuticals, Inc
Structure and Function of p63RhoGEF, a Gαq Activated Effector
Authors: Taylor Veronica G , Boguth Cassandra A , Miller Brittney , Cierpicki Tomasz , Tesmer John J G 
Published: June 2021

RhoA, a key actin cytoskeleton regulator, is activated in response to G protein‐coupled receptors signaling through Gαq. Rho guanine nucleotide exchange factor (RhoGEF) p63RhoGEF, a member of the Dbl GEF family, is directly activated by Gαq. Although the atomic structure of the Gαq‐p63RhoGEF complex is known, the mechanism of activation is not clear, in part because the structure for the basal conformation of p63RhoGEF has not yet been determined. This poster describes our efforts to use nuclear magnetic resonance spectroscopy to determine the solution structure of the p63RhoGEF catalytic core, to map the residues directly interacting with RhoA in the presence and absence of Gαq, and to confirm the multi‐domain interactions of Gαq that were observed in the original crystal structure. The successful completion of this work would be the first example of a heterotrimeric G protein effector characterized in both its basal and activated states, and provide the molecular basis for regulation of an important link between heterotrimeric and small molecular weight G proteins.Support was provided by NIH grants HL071818 and HL086865.
Changes in Glutaminase mRNA Expression in Rat Dorsal Root Ganglion Post Tail Inicision
Authors: Crosby Heith A , Spencer Diana , Miller Kenneth E 
Published: June 2021

Aim of InvestigationGlutamate is a neurotransmitter used by primary sensory neurons of the dorsal root ganglion(DRG). This excitatory neurotransmitter is released from peripheral nerve terminals and spinal synaptic terminals during nociceptive signaling. In this study, we evaluated the changes in Glutaminase (GLS) mRNA at 1, 2, and 4 hours post tail incision in a surgical incision tail model.MethodsSprague Dawley rats were anesthetized and a 20mm incision made in the proximal one third of the tail. Controls were surgical naïve rats. At 1, 2, and 4 hours, rats were asphyxiated, decapitated and sacral DRG were collected. Samples were stored in RNAlater ™ (Ambion). RNA was isolated using the Vantage Total RNA kit ™ (Origene). Real‐Time Quantitative Polymerase Chain Reaction (RT‐qPCR) was performed on sacral DRG samples using iScript One‐Step RT‐PCR Kit with Sybr Green™ (Biorad). Threshold cycle (Ct) and melt data were collected for GLS and beta‐actin (control mRNA).ResultsGLS mRNA in Sacral DRG neurons exhibited both depression and elevation in fold change across sacral levels 1–3 at 1, 2 and 4 hours post incision.ConclusionsThese results provide insight on how GLS mRNA is maintained in sacral dorsal root ganglia and how it responds to tissue injury. From the results of the present study, incisional wounding results in a decrease in GLS mRNA. We hypothesize that GLS mRNA is maintained in a basal pool and that after injury GLS mRNA is rapidly translated thereby decreasing the available reservoir. This, results in an increase in GLS protein observed in our previous immunohistochemisty and Western blot studies.Research Support: NIH Grant AR047410 (KEM) and OSU CHS Intramural Grant
Inflammatory alterations in dual diagnosis patients with schizophrenia and substance use disorders
Authors: Kouassi Edouard , Potvin Stéphane , Stip Emmanuel , Lipp Olivier , Igué Raouf , Bah Ramatoulaye , Gendron Alain 
Published: June 2021

The lifetime prevalence of substance use disorders (SUDs) is high (~50%) in schizophrenia (SCZ). The study objective was to evaluate the inflammatory status of dual diagnosis (DD) patients with SCZ and SUDs. Patients (n = 29) were diagnosed with a schizophrenia‐spectrum disorder and a comorbid SUD (cannabis>alcohol>cocaine) (DSM‐IV criteria). Psychiatric symptoms were evaluated with the Positive and Negative Syndrome Scale and the Calgary Depression Scale for Schizophrenia. Healthy controls (n = 28) consisted of sex‐ and age‐matched volunteers without any known history of SCZ or SUDs. Plasma cytokines were assessed by multiplex immunoassay or by monoplex ELISA for higher sensitivity. Between‐group differences were assessed using ANOVA, and level of significance was set at p < 0.05. Plasma levels of interleukin (IL)‐6, IL‐8, IL‐1 receptor antagonist, and soluble IL‐2 receptor were increased in DD patients, compared to controls, while L‐17 was normal. Both IL‐6 and IL‐8 were associated with the severity of depressive symptoms and with alcohol consumption. These findings indicate that an inflammatory syndrome does exist in DD patients, and it may play a role in psychopathology. Future studies will elucidate the contribution of specific drugs of abuse and their interactions with SCZ in the dysregulation of inflammatory cytokines in DD patients.Supported by CIHR, and by AstraZeneca Pharmaceuticals.
Fluid dynamics in diaphragmatic submesothelial lymphatics
Authors: Negrini Daniela , Bianchin Francesca , Solari Eleonora , Moriondo Andrea 
Published: June 2021

Lymph formation and progression in initial lymphatics is a complex, not completely unveiled process. To clarify it, we studied “in situ” the kinetics of lymph flow in the diaphragmatic submesothelial network of anesthetized, mechanical ventilated rats. In a single lymphatic vessel, intraluminar hydraulic pressure (Plymph) and lymph flow were measured by means of the micropuncture technique combined with the injection of fluorescent dextrans to visualise lymph flow. Measurements of fluorescent front progression and vessels diameter were performed on acquired images to calculate lymph flow (FL) and flow velocity (vL). Data show that FL, vL and flow direction vary considerably with time even in the same vessel (range: from 1 ± 0.3 (1 SE) nL/min to 154.9 ±10.4 nL/min). In addition, in some vessels, flow direction reverts at a frequency (about 11.3 ± 1.6 Hz) not related to the cardiac or respiratory rate. The fact that, in some network segments, flow direction was independent upon the intraluminar Plymph gradient, suggests the existence of an active pumping mechanism coordinating flow kinetics in these vessels.The research has been supported by the Italian Ministry of University (MIUR).
Prolonged Vasoconstriction of Small Resistance Arteries Involves Actin Polymerization Causing Cytoskeletal Remodeling Within the Vascular Smooth Muscle Cells (VSMC)
Authors: Galiñanes Edgar Luis , Martinez‐Lemus Luis 
Published: June 2021

Two groups of isolated, cannulated and pressurized rat cremasteric arterioles with myogenic tone, that were previously transfected with Actin Alexa Flour®488 using electroporation, were imaged hourly with a confocal microscope for 4 hours while incubated at 34C in the absence (control) or presence (treated) of norepinephrine (NE,10‐5.5M) plus angiotensin‐II (Ang,10‐7M).Fluorescence intensity was taken from individual VSMCs using 3D image reconstruction. Over the entire incubation period, treated arterioles (N=6) had a decrease in mean fluorescence of 40% compared to controls (N=4). With removal of the agonists, the mean florescence of our controls returned within 10% of that obtained at myogenic tone while the treated group remained reduced by 30%.Previous reports indicate polymerization of G‐Actin into F‐Actin can decrease fluorescence due to the phenomenon of quenching. Therefore the observation that overall fluorescence at the end of 4hours in the treatment group did not return to control levels implies that actin remained polymerized. Our results are consistent with our hypothesis that prolonged vasoconstriction induces VSMC cytoskeletal remodeling by causing actin polymerization.
TOLL‐LIKE RECEPTOR 2 IS INCREASED IN RENAL ISCHEMIA/REPERFUSION‐INDUCED CARDIAC HYPERTROPHY IN MICE
Authors: Sonoda Mayra Trentin , da Silva Rogerio Cirino , Carneiro‐Ramos Marcela Sorelli 
Published: June 2021

ObjectivesThe innate immune response can exerts innumerous effects in vast number of tissues and organs. It's well established that cytokines and Toll‐like receptors (TLRs) participate of cardiac trophism modulation. The aim of this study was to investigate the role of TLRs in heart after renal ischemia/reperfusion.MethodsC57bl/6J mice were subjected to unilateral 60 minutes kidney ischemia, followed by reperfusion of 5, 8, 15 and 20 days. Levels of urea were measured to confirm renal failure and gene expression were analyzed by real time PCR.ResultsUrea levels were increased in groups 5, 15 and 20 days (p<0.05). The group 15 days showed a significant increase in heart weight/body weight ratio (p<0.05) and ANF mRNA levels (p<0.001), a cardiac hypertrophy marker, when compared to sham group. Cardiac TLR‐2 mRNA levels was increased after 5 days of reperfusion (p<0.05) when compared to sham group. The isoform TLR‐4 has a tendency to increase, but it was not significant.ConclusionFirst, the results indicate that renal ischemia/reperfusion model is able to induce a transient cardiac hypertrophy in mice. Moreover, this hypertrophy has a positive correlation with a transient increase of TLR2 mRNA levels, suggesting that the cascade of activation of TLRs may be involved to cardiac hypertrophy development. Financial Support: FAPESP, CNPq.
Inactivation of Fibrillin‐1 in Vascular Smooth Muscle Cells (VSMCs) but not Endothelial Cells (ECs) replicates Thoracic Aortic Aneurysm (TAA) seen in Marfan Syndrome (MFS)
Authors: Cook Jason R , Carta Luca , del Solar Maria , Ramirez Francesco 
Published: June 2021

IntroductionDissecting TAA in MFS is associated with elevated TGFβ activity secondary to structural or quantitative defects in FBN1. Perinatal lethality of mice with germ line inactivation of Fbn1 has underscored the importance of fibrillin microfibrils in aortic growth and homeostasis without identifying the main tissue compartment contributing to TAA.ObjectiveDetermine the contribution of fibrillin‐1 production by VSMCs and ECs in MFS vascular pathology.MethodA new conditional Fbn1 mouse model was created to allow for the selective study of fibrillin‐1 in unique tissues. Female mice with the conditional allele (Fbn1fl) were crossed with male transgenic mice harboring a germ line null allele (Fbn1mgN) and expressing Cre in either VSMCs (SM22α‐Cre) or ECs (Cdh5‐Cre). The germ line null allele was paired with the conditional allele to increase the probability of tissue specific Cre excision. Tissue specific Cre excision was monitored by crossing the tissue specific mice with RosaLacZ reporter mice.ResultsInactivation of fibrillin‐1 in VSMCs (Fbn1fl/mgN; SM22α‐Cre+), but not ECs, replicates the germ line null phenotype with elastic fiber fragmentation, aortic aneurysm, and death due to dissection by P16.ConclusionsLack of fibrillin‐1 production by VSMCs in the media of the ascending aorta is necessary and sufficient for the development of TAA.
Ventilation‐induced variation in the arterially determined isovolumic contraction period is an excellent indicator of hemorrhagic shock
Authors: van Houwelingen Marc , Merkus Daphne , Hofland Jan , Tenbrinck Robert , Hekkert Maaike te Lintel , van Dijk Geert , Hoeks Arnold P.G. , Duncker Dirk J. 
Published: June 2021

The ventilation‐induced relative variation in pre‐ejection period (ΔPEP) has recently been shown to predict fluid responsiveness in patients in shock. We hypothesized that the ventilatory variation in isovolumic contraction period determined by central arterial pulse wave analysis (ΔAIC) is a more sensitive predictor than the peripherally assessed ΔPEP which is influenced by pulse wave travel time and ventricular electro‐mechanical delay.To test this hypothesis we studied the responses of ΔAIC and ΔPEP to a graded hemorrhage protocol, and compared these to the response of the clinically well known pulse pressure variation (ΔPP), which has a high predictive value for fluid responsiveness.A graded hemorrhage protocol (blood withdrawal up to 20ml/kg) was instituted in 7 anesthetized Yorkshire X Landrace pigs. ΔAIC correlated well with ΔPP (r = 0.86, p<0.001) and ΔPEP (r = 0.84, p<0.001). Moreover, the coefficient of variation of ΔAIC (0.50) was found to be significantly lower than that of ΔPEP (0.94, p=0.04).ConclusionsΔAIC can be used to assess the level of hemorrhagic shock in a swine model and appears to be a more sensitive predictor of fluid responsiveness than ΔPEP.
Insulin selectively increases accessibility of small dextrans in capillaries of rat skeletal muscle: role for glycocalyx
Authors: van Haare Judith , van Teeffelen Jurgen , Roos Sjef , Cleutjens Jack , Vink Hans , Eskens Bart 
Published: June 2021

ObjectiveTo determine the effect of insulin on solute accessibility in skeletal muscle capillaries and the role of the glycocalyx herein.MethodsA bolus of small (40 kDa) dextrans (dex40) and large (2000 kDa) dextrans (dex2000) was i.v. infused in anesthetized rats (control versus hyaluronidase, during rest and 30 minutes after a bolus of insulin (1U/kg); all N=3). 3 minutes after tracer infusion the calf muscle was removed, formaldehyde‐fixed, paraffin‐embedded and sectioned. Images were captured using fluorescence microscopy. For each dextran, the total intensity per mm2 muscle was calculated from the number of tracer filled capillaries multiplied by the average intensity of each tracer per capillary.ResultsTotal intensity of Dex40 did not differ between control and hyaluronidase treated rats (70276 ± 5416 versus 95500 ± 43041) (MEAN ± SEM) in rest, but was increased after insulin in all experiments (control: 103991 ± 7787, hyaluronidase: 190147 ± 47995). Total intensity of Dex2000 did also not differ between control (45744 ± 10730) and hyaluronidase treated rats (31061 ± 2512) at rest. In contrast to Dex40, only an increase after insulin in the hyaluronidase treated rats was observed.ConclusionThese data indicate that insulin facilitates the selective delivery of small‐sized plasma solutes in capillaries of skeletal muscle. The size‐selectivity is lost after enzymatic glycocalyx degradation.Sponsored by Dutch Heart Foundation (NHS2009B56) and Dutch Diabetes Research Foundation (DFN 2006.00.027)
Paracrine Effect of Wnt11 Overexpressing Mesenchymal Stem Cells on Ischemic Injury
Authors: He Zhisong , Yu Bin , Ashraf Muhammad , Xu Meifeng 
Published: June 2021

In this study, we investigated whether Wnt11 enhances bone marrow‐derived mesenchymal stem cell (MSC) mediated cardioprotection via paracrine fashion following acute ischemia.MethodsMSCs were harvested from male rat bone marrow (BM) and transduced with Wnt11 (MSCWnt11). An acute myocardial infarction (MI) model in rats was developed by ligation of the left anterior descending coronary artery (LAD). MSCWnt11 were transplanted into the peri‐infarct region following acute MI. To mimic ischemic injury, cultured cardiomyocytes (CM) isolated from neonatal ventricles were exposed to hypoxia.ResultsELISA studies indicated that the release of Wnt11 (3.45‐fold) as well as transforming growth factor‐β2 (TGFβ2) (1.5‐fold) was significantly increased from MSCWnt11 compared to transduced control MSC (MSCNull). Hypoxia induced apoptosis and cell death was significantly reduced when CM were co‐cultured with MSCWnt11 in a dual chamber system. The cell protection mediated by MSCWnt11 was mimicked by treating CM with conditioned medium (CdM) from MSCWnt11 and abrogated by Wnt11‐ and TGFβ2‐neutralizing antibodies. Furthermore, animals receiving MSCWnt11 showed a significant improvement in cardiac contractile function as assessed by echocardiography. Infarct size measured from infarcted hearts was reduced from 44.6 ± 4.1% in control to 30.1 ± 3.0% in treated hearts (p < 0.05). Similarly, apoptosis of cardiomyocytes evaluated by TUNEL staining was reduced from 9.9 ± 0.71% in control group to 5.30 ± 0.35% in treated group (p < 0.05).ConclusionsTransplantation of MSCWnt11 improved cardiac function. The release of Wnt11 and other factors from MSCWnt11 transplanted in the infarcted left ventricle is more likely responsible for protection of CM at risk. This work was supported by National Institutes of Health grants: HL083236, HL105176 (M.X.) and HL087246 (M.A.).