bluuebunny/crossref_metadata_2025_split huggingface dataset:
Remodelling of the atrioventricular canal myocardium determines atrioventricular conduction patterns
Authors: Nanka Ondrej , Vicente‐Steijn Rebecca , Blom Nico A , Jongbloed Monique RM , Sedmera David 
Published: November 2023

AimsBased on our previous study showing accelerated conversion of ventricular activation sequence during hypoxic incubation correlating with increased apoptosis in the atrioventricular canal (AVC), we hypothesized that this slight increase in apoptosis could lead to an accelerated pruning of myocardial atrioventricular connections, which would manifest as earlier appearance of mature apex‐to‐base activation patterns.MethodsTo test this hypothesis, apoptosis was blocked with a caspase inhibitor (zVAD‐fmk) by intrapericardial injection in chick embryos at embryonic day (ED) 4. The epicardial contribution to AVC remodeling was studied by performing epicardial outgrowth inhibitions – microsurgical epicardial ablation at ED3. Ventricular activation was studied by optical mapping and apoptosis rates were quantified by Lysotracker Red and Active Caspase‐3 immunofluorescence staining.ResultsApoptosis measurements and quantification in zVAD treated hearts shows decreased apoptosis in AVC myocardium at ED7. zVAD‐treated and epicardial ablated hearts showed increased frequency of immature ventricular activation patterns. In 5 of the 21 zVAD‐treated hearts we observed activation originating from the right AVC. This pattern was observed in neither control nor epicardial ablation hearts. The epicardial ablation hearts showed activation originating from the left AVC. Ventricular activation time in Base‐to‐Apex activation patterns in zVAD‐treated hearts was significantly longer than in controls.ConclusionAVC maturation requires apoptosis in the AVC myocardium and epicardium‐derived in growth. Disruption of proper AVC morphogenesis can lead to persistence of accessory atrioventricular connections, which form a morphological substrate for ventricular pre‐excitation.Support or Funding InformationSupported by Charles University PRVOUK/PROGRES
Development of the Cardiac Conduction System in the Selected Groups of Reptiles as Determined by HNK‐1 Expression
Authors: Kvasilova Alena , Gregorovicova Martina , Sedmera David 
Published: November 2023

Reptilian hearts show different stages of ventricular septation from its complete absence in the basal squamates to a fully septated heart in crocodiles. We have tested the hypothesis that evolution of the specialized ventricular conduction system is linked to ventricular septation, rather than homeiothermy, as originally postulated. We studied hearts of selected reptilian species (Pogona vitticeps – no ventricular septum, Varanus indicus – partial septum, Crocodylus siamensis – complete septum) at various stages of development to uncover any morphological evidence for specialized conduction pathways. Immunohistochemistry using smooth muscle or sarcomeric actin as myocardial markers and HNK‐1 as a marker of cardiac conduction system showed HNK‐1 expression in the myocardium of the sinus venosus, where the main cardiac pacemaker is located. There was also staining of various intensity in the extracellular matrix of cardiac cushions in all the species examined. There was an HNK‐1 positive ring‐like structure of the developing ventricular septum in the Crocodylus, and increased positivity in the Varanus, while there was no myocardial staining in the ventricle of the Pogona. In all species there was a strong staining in the cardiac nervous fibers and ganglia, and in the epicardium of the Varanus. We conclude that a primordial conduction system is present in reptilian hearts. The embryonic crocodilian hearts possess a morphological preferential conduction pathway similar to one present in the hearts of birds and mammals. Its emergence correlates with completion of ventricular septation.Support or Funding InformationSupported by Ministry of Education PRVOUK P35/LF/5, institutional grant RVO: 67985823 (The Czech Academy of Sciences), and GA CR 16‐02972S.
Functional characterization of stap2b in zebrafish vascular development
Authors: Wang Yi‐Shan , Wu Chang‐Yi 
Published: November 2023

Molecular mechanism of vascular formation is still not fully understood. We previously identified the transcription factors Islet2 (Isl2) and nr2f1b required for specification of the vein and ISV growth mediated by Notch pathway in zebrafish, and identify the potential downstream targets by performing the microarray experiments. In this study, we focus on the activation target signal‐transducing adaptor protein 2b (stap2b) and confirmed this gene expressed in vessels during early development indicating stap2b function in vascular development.stap2 is identified as an adaptor protein that has been showed functions in a variety of cellular signal pathways in immune system, however, there is no description of stap2b functioning in vascular development so far. Our in‐situ hybridization results showed stap2b mRNA expressed in developing vessels, suggesting its roles in vasculization. Knockdown of stap2b by morpholino injection causes vascular defects, suggesting the role of stap2b in controlling ISV and CVP growth. AO staining and TUNEL assay showed that vascular defects do not caused by cell death, but due to the impairment of migration and/or proliferation by examining ISV integrity and ISV cell numbers in Tg(kdrl:mCherry; fli:neGFPy7) fish. We also found a decreased expression of flk, stabilin, enphrinb2, and flt4 in stap2b morphants. Moreover, overexpression of stap2b enhanced the growth of ISV and promotes endothelial cell migration and/or proliferation. These data suggest that stap2b is necessary and sufficient to promote vascular development.We further examine the interaction between stap2b and Notch signals, we showed stap2b is regulated by Notch signals to control ISV growth. In addition, stap2b may interact with BMP signaling to function in CVP formation. Together, we conclude that our microarray results identify stap2b acting downstream of isl2/ nr2f1b pathway. We also showed that stap2b plays an important role for vascular development in zebrafish.
Development of learning progressions through the chemistry curriculum to biochemistry
Authors: Wolfson Adele J. , Mercer Allison M. , Offerdahl Erika G. , Lewis Jennifer E. 
Published: November 2023

Learning progressions describe the stages– not necessarily linear ‐ that students take as they develop more sophisticated ways of knowing a particular concept. Many concepts in biochemistry are introduced in earlier courses, including general chemistry and organic chemistry. We are developing learning progressions for several key concepts in biochemistry informed by interviews with faculty and students and by survey instruments. Faculty interviews and focus groups yielded topics for the progressions, and follow‐up interviews with students after they had completed the surveys allowed us to refine the survey. Students at two different institutions have participated in the survey as pre‐ and/or post‐course assessment. The results will allow us to propose learning progressions related to acid/base equilibria, energy diagrams and kinetics, and thermodynamics of ATP hydrolysis.. We hope to use the progressions to develop teaching materials and to foster discussion among faculty so that articulation from one course to the next is productive.Support or Funding InformationNSF‐DUE‐1503980 (IUSE)
Th1 Effector T cells induce TGFβ mediated Cardiac Fibrosis in Pressure Overload induced Heart Failure by adhering to Alpha 4 Integrin and mediating Fibroblast Transformation
Authors: Nevers Tania 
Published: November 2023

Cardiac fibrosis (CF) is a key factor contributing to the pathogenesis of the deadly syndrome of heart failure (HF). Recent emerging data demonstrate a role for T cells in pro‐fibrotic cardiac repair and healing post‐ischemia. However, very little is known about how T cells directly impact cardiac fibroblasts (CFB) to promote CF and cardiac dysfunction in non‐ischemic HF. Recently, we reported that T cells infiltrated the left ventricle (LV) of the heart in patients with end‐stage non‐ischemic HF, and also in the mouse model of non‐ischemic HF induced by thoracic aortic constriction (TAC). Interestingly, mice deficient in T cells (TCRα−/− mice) are protected from cardiac fibrosis (CF) and cardiac dysfunction. Thus, we hypothesize that T cells activated in such context orchestrate the activation and differentiation of fibroblasts to myofibroblasts. To test this hypothesis, we first co‐cultured adult CFB with T cells isolated from mice 4 weeks post TAC or Sham surgery. TAC but not Sham T cells firmly adhered to CFB and mediated myofibroblasts transformation as demonstrated by increased α‐SMA fluorescent expression. Co‐culture of various CD4 T cell subsets with CFB demonstrated that Th1 effector T cell subset, in contrast to naïve T cells, adhered directly to CFB through alpha 4 integrin and mediated myofibroblasts transformation in a TGFβ dependent manner. Moreover, adoptive transfer (AT) of effector Th1 cells, but not naïve T cells into TCRα−/− mice at 48 hours and 2 weeks post TAC surgery exclusively induced perivascular fibrosis that co‐localized with T cell and leukocyte infiltration and TGFβ expression. AT of Th1 cells also induced LV contractile and systolic dysfunction and a shift in the fetal gene expression of the myosin heavy chain α toβ which occurs in pathological cardiac remodeling. Taken together, these findings identify a novel role for Th1 cells in cardiac fibrosis and function and may provide innovative insights in the treatment of chronic non‐ischemic HF.Support or Funding InformationNIH HL094706
Atrogin‐1 Transgenic (AT1 Tg+) Mice Have Age‐Dependent Cardiac Dysfunction With Atrogin‐1 Mediated Glucocorticoid Receptor (GR) Expression Down Regulation
Authors: Mota Roberto , Parry Traci , Willis Monte 
Published: November 2023

Previous studies have identified the muscle‐specific ubiquitin ligase Atrogin‐1 (MAFbx) as a critical regulator of pathologic and physiologic cardiac hypertrophy. Mechanistically, Atrogin‐1 regulates these processes by ubiquitinating transcription factors (NFAT and FOXO1/3, respectively) to inhibit these processes. However, their role in regulating transcription factors in aging has not previously been described.MethodsTransgenic mice with increased cardiomyocyte‐specific (alpha‐MHC promoter driven) Atrogin‐1 were phenotypically characterized at baseline and up to one year by conscious echocardiography. In parallel, HL‐1 cells were cultured with increasing adenovirus expressing myc‐Atrogin‐1 (Ad.Atrogin1) or GFP control (Ad.GFP) at a multiplicity of infection (MOI) of 0, 10, 20, 50, 100 and 200. Data were subject to statistical analysis with two‐tailed t‐test and/or one‐way ANOVA using GraphPad 7.0 for Mac.ResultsPost‐transduction, HL‐1 cells were lysed and protein was collected. Immunoblots were then performed to identify potential Atrogin‐1 substrates. Conscious echocardiographic analysis of Atrogin‐1 Tg hearts revealed not differences in phenotype up to four months of age. However, by 12 months, Atrogin‐1 hearts exhibited significantly decreased ejection fraction, fractional shortening and left ventricular (LV) mass (p>0.05). This aging phenotype was reminiscent of recent studies where the cardiac GR was deleted, so we next investigated if Atrogin‐1 potentially targeted GR for degradation in the HL‐1 cardiomyocyte‐derived cell line. In HL‐1 cells with increasing Ad.Atrogin‐1 MOI, GR protein expression was significantly decreased in a dose‐dependent manner compared to controls (Ad.GFP) (p<0.05).ConclusionThese results suggest that chronically increasing cardiomyocyte Atrogin‐1 can lead to an age‐dependent decrease in systolic function, mimicking the alterations in GR signaling reported in humans. In the heart, the muscle‐specific ubiquitin ligase Atrogin‐1 may down‐regulate cardiomyocyte GR by mediating its polyubiquitination and degradation by the 26S proteasome. Future studies will delineate the molecular details in which Atrogin‐1 regulated GR levels post‐translationally so that Atrogin‐1's role as a therapeutic target may be evaluated further in aging.Support or Funding InformationSupported by the Leducq Foundation.
Splenectomy‐Induced Diastolic Dysfunction With Normal Ejection Fraction
Authors: Huber Jason S , Ruggiero Sara A , Romanova Nadya , Blois Shauna L , Brunt Keith R , Simpson Jeremy A 
Published: November 2023

BackgroundUnderstudied and often disregarded, the spleen is perceived as redundant, non‐vital or even a vestigial organ. Conversely, epidemiological studies indicate splenectomy (or splenic injury) increase the prevalence of cardiovascular diseases and associated risk factors, suggesting the physiological function of the spleen is not yet realized. Recent investigations have examined the role of the spleen in experimental heart failure, however, the effect of long‐term splenectomy on cardiac function in healthy models remains to be determined. Thus, the aim of this study was to reconcile the observations made in the epidemiological studies by investigating cardiac function and morphology following splenectomy in healthy rodents.MethodsSplenectomized and sham male Wistar rats were assessed at 5, 9, and 18 weeks post‐surgery by echocardiography, invasive hemodynamics, cardiac histology, and thromboelastography. Serum IL‐10 was measured by ELISA and left ventricular free wall was used for the analysis of select molecular markers.ResultsSplenectomy caused progressive left ventricular diastolic dysfunction (i.e., increases in dP/dt min, Tauw, and LVEDP), with no evidence of cardiac decompensation or systolic dysfunction. Left ventricular remodeling (fibrosis, hypertrophy and decreases in capillary to myocyte cross sectional area) were associated with decreases in serum IL‐10 and ventricular BNP expression. Increases in blood coagulability following splenectomy are associated with increased risk of developing thromboembolic pulmonary hypertension. In our model however, we found no evidence of pulmonary arterial hypertension or lung remodeling despite a ~2‐fold increase in coagulability.ConclusionsSplenectomy in healthy rodents caused progressive diastolic dysfunction and significant cardiac remodeling (i.e., fibrosis, hypertrophy and decreased capillary density), demonstrating an underappreciated role of the spleen in cardiac homeostasis. Future studies need to characterize the mechanism(s) by which the spleen contributes to a healthy cardiovascular phenotype.Support or Funding InformationThis work was supported by the J.P. Bickell Foundation (to JA Simpson), the Natural Sciences and Engineering Research Council (to JA Simpson and KR Brunt), and the Ontario Veterinary College Pet Fund (to SL Blois). JA Simpson is a new investigator with the H&SF of Ontario.
Live cell microscopy of intestinal organoid oxygenation
Authors: Dmitriev Ruslan I. , Okkelman Irina A. , Foley Tara , Papkovsky Dmitri B. 
Published: November 2023

Oxygenation of stem cell niche is important metabolic activity indicator for the intestine in normal and diseased states. In vivo, it can be affected by such factors as diet, drugs, metabolites produced by microbiota, and inflammation. We hypothesized that O2 must be also very important for adequate development of organoids and organs‐on‐chip, growing as 3D cultures. We took advantage of cell‐penetrating O2‐sensitive probes and phosphorescence lifetime imaging microscopy (PLIM), which, in contrast to the end‐point hypoxia stains and electrode‐based measurements, provides high‐resolution real‐time visualization of O2. Due to the specialised cultivation conditions for different types of organoids and varied ability for staining with live cell dyes and probes, we optimized the staining parameters with O2‐sensitive probes. Using staining with small molecule Pt‐Glc probe [1] and PLIM technique we analyzed O2 distribution in organoids from small intestine of mouse. We were able to directly measure oxygenation of organoids without their recovery from Matrigel matrix and multiplexed it with number of live fluorescent probes (Hoechst 33342, Cholera Toxin‐Alexa 488, TMRM, CellTox Green, Calcein Green) and immunofluorescent staining of fixed samples. We next analysed the oxygenation of live organoids treated either with mitochondrial uncoupler FCCP or anti‐diabetic drug metformin, inhibiting activity of mitochondrial complex I [2]. We found that even when produced from the same animal, resting organoids displayed highly heterogeneous oxygenation, from 27 to 88 μM (approx. 2.7~8.8% of air saturation). This can be explained by the growth at different depths in Matrigel matrix and limited diffusion of O2 and/or highly variable respiration between organoids. Treatment with FCCP, increasing oxygen consumption, resulted in decrease of organoid oxygenation, confirming that the intestinal epithelia tissue has active mitochondria. In contrast, metformin treatment significantly increased the overall oxygenation of organoids in comparison to non‐treated culture. However, the magnitude of response to metformin varied for samples taken from different animals. This data confirms that when accumulating metformin, intestinal epithelia undergoes inhibition of mitochondrial respiration, which can change its oxygenation and lead to glycolytic switch of metabolism. Altogether, our results point at the importance of analysis of organoid culture oxygenation in tissue engineering, and demonstrate how this information can be translated in studies of molecular mechanisms of diabetes.Support or Funding InformationSupported by Science Foundation Ireland (SFI) grant 13/SIRG/2144.
DDI1 and DDI2‐dependent removal of Replication Termination Factor Domain Containing 1 (RTFDC1) from replication forks is necessary for proper response to replication stress
Authors: Kottemann Molly C , Conti Brooke , Lach Francis P , Smogorzewska Agata 
Published: November 2023

Accurate replication of the genome is a fundamental responsibility for the cycling cell, but DNA replication is itself a source of damage. Replication forks can stall at DNA lesions and physical barriers, in difficult to replicate regions, or due to nucleotide depletion. We have identified DDI1 and DDI2, mammalian homologs of the yeast UBL‐UBA shuttle Ddi1, as participants in DNA damage response due to replication stress. Pulldown of GFP‐DDI2 followed by mass spectrometry identified members of the proteasome and the replisome. Depletion of DDI1/2 caused significant hypersensitivity to replication stress drugs hydroxyurea (HU) and aphidicolin (Aph), and overexpression of either DDI1 or DDI2 rescued the sensitivity. During replication stress, DDI1/2 depletion caused fork restart defects, hyperactivated damage signaling, and increased accumulation of RPA‐coated single stranded DNA. Ultimately, these defects led to severe chromosomal instability and cell death by apoptosis. Using isolation of proteins on nascent DNA (iPOND), we identified proteins that are enriched at the replication fork in DDI1/2 depleted cells. The most enriched protein was Replication Termination Factor Domain Containing 1 (RTFDC1), the mammalian homolog of S. pombe Rtf2. Reducing excess RTFDC1 at the fork by siRNA treatment in the DDI1/2 depleted cells rescued the aberrant phenotypes seen during replication stress response, placing RTFDC1 as the key DDI1/2 target at the replisome. This work establishes a novel fork protection pathway that requires DDI1/2 to remove RTFDC1 from the replisome during replication stress response and we show that this pathway is necessary for maintenance of genome stability.Support or Funding InformationMCK was supported by the Women and Science Fellowship and by the American Cancer Society‐J.T.Tai Postdoctoral Fellowship. This work was supported by the Pershing Square Sohn Prize for Young Investigators in Cancer Research and The Gabrielle H. Reem and Herbert J. Kayden Early‐Career Innovation Award. AS is an HHMI Faculty Scholar.
The Effects of Replication Fork Restart on CAG Repeat Instability in S. pombe
Authors: Gold Michaela , Freon Karine , Su Allen , Lambert Sarah , Freudenreich Catherine 
Published: November 2023

Trinucleotide repeats are a category of repetitive DNA elements that can form secondary structures and cause many diseases. Expansions of CAG repeats in particular can lead to numerous neurodegenerative polyglutamine diseases, including Huntington's disease and some spinocerebellar ataxias. These expansions could occur because the secondary structures the repeats form can stall DNA replicative polymerases, and fork restart may be prone to repeat instability. In order to test this idea, we have used the Schizosaccharomyces pombe fission yeast Replication Fork Barrier (RFB) system to induce fork restart through a CAG repeat tract and study the effects on CAG repeat instability (expansions and contractions). We created constructs with 70 CAG repeats inserted at different locations relative to the RFB. With the repeats either 1.9 kb or 6.7 kb after the RFB, increased CAG repeat instability was detected after RFB induction, indicating that fork restart can be a source of repeat instability.In order to test the mechanism of repeat instability during fork restart, key proteins potentially involved in the process were deleted. In contrast to wild‐type, in cells missing the Rad52 protein, which is involved with homologous recombination (HR) and required for restart in this system, induction of the RFB did not further increase repeat instability. This suggests that HR could be a leading cause of RFB‐induced repeat instability. Msh2 has been shown to bind CAG repeats and cause repeat expansions in mouse models. In S. pombe strains without Msh2, expansions decreased irrespective of RFB status. These results suggest that Msh2 is involved in the CAG expansion mechanism as in vertebrates, but likely not during fork restart, and S. pombe can be used to study this phenomenon. In summary, we have identified two mechanisms of CAG instability in S. pombe: expansions caused by presence of the Msh2 protein unrelated to fork restart, and a second novel mechanism of HR‐dependent fork restart that causes both expansions and contractions of this unstable repeat.Support or Funding InformationThis research was funded by National Institutes of Health grant P01‐GM105473/402373 and the Curie Institute Rothschild‐Yvette Mayent Award to C.H.F., ANR 14‐CE10‐0010‐01 and FRM label funds to S.L., and Tufts University Sandler and Undergraduate Research Funds to C.H.F. and M.A.G..