bluuebunny/crossref_metadata_2025_split huggingface dataset:
MSC‐Derived Exosomes Deliver Cardioprotective miR‐21
Authors: Haar Lauren , Luther Kristin , Phan Ahn , McGuinness Michael , Zilliox Michael , Wang Yang , Jones W. Keith 
Published: November 2023

Mesenchymal stem cell (MSC) transplantation has been shown to improve outcomes after myocardial infarction (MI) in animal models and in man. Specifically, reduction of infarct size (cardioprotection), increased LV muscle, improved function, and preserved ejection fraction have variously been reported. However, the mechanism of beneficial effect is not well understood. While some data supports transdifferentiation of MSC into myocardial constituent cells, other results support direct cardioprotection against ischemia/reperfusion injury (IRI). Overall, there is a growing consensus that the MSC and their derivatives do not contribute in proportion to the extent of the reparative effects, supporting the concept of paracrine effects of MSC. If paracrine factors can be identified, they might be employed therapeutically and contribute to identification of new therapeutic targets. Several recent results, including ours, support that exosomes, and their microRNA (miR) cargo, underlie at least some of these paracrine effects. Our objective was to test the hypothesis that MSC‐derived exosomes from non‐treated MSC can mediate cardioprotection via specific miR cargo. To test this hypothesis, we identified by next generation RNA sequencing, the miRs present in exosomes isolated from murine MSC. We identified both protective and injurious miRs, and focused upon miR‐21, the most highly expressed miR in MSC derived exosomes. We also identified miRs associated with apoptotic signaling, cell proliferation, angiogenesis, and heat shock response. We report in vitro data that the exosomes and miR‐21 are protective in a dose‐dependent manner against sim I/R. We next showed in vivo that exosomes were cardioprotective, reducing infarct size (normalized for risk region) by 68%, (P≤0.01 vs. control) and that exosomes isolated from MSC having miR‐21 deleted reduced infarct size by only 28% (P=0.039 vs. control), demonstrating a major role for miR‐21. In vivo, the exosomes increased miR‐21 levels in myocardium, resulting in significant depression of four target protein levels, PDCD4, Peli1, FasL, and PTEN. These results support that suppression of cell death is by coordinated reduction of several parallel cell death pathways, and derepression of pro‐survival Akt. In conclusion, our data supports that the cardioprotective aspects of MSC are explained, at least in part, by secretion of exosomes and the action of the miR‐21 cargo. Whether other aspects of MSC action are regulated by other of the miR cargo is under study.Support or Funding InformationNIH R01HL091478
Angiopoietin‐2 levels in the risk stratification and mortality outcome prediction of sepsis associated coagulopathy
Authors: Statz Steve , Walborn Amanda , Williams Mark , Hoppensteadt Debra , Mosier Michael , Rondina Matthew , Fareed Jawed 
Published: November 2023

BackgroundAngiopoietin‐2 (Ang‐2) is a 70 kDa glycoprotein which plays multiple roles in angiogenesis, inflammation, and vascular development. Ang‐2 is upregulated in vascular stress, and circulates at high levels in certain pathologic conditions such as sepsis. Ang‐2 upregulation may be related to the severity of sepsis associated coagulopathy (SAC), and therefore it may have a predictive role in the clinical outcome of this syndrome.AimThe aim of this study is to measure Ang‐2 levels in a defined cohort of patients with sepsis and suspected disseminated intravascular coagulation (DIC), to compare the circulating levels of this biomarker to controls, and to demonstrate its predictive value for the clinical outcome and severity of SAC.MethodsPlasma samples were collected from 102 patients with sepsis and suspected DIC at ICU admission and immediately frozen at −80°C for batch analysis. Control plasma represented 50 normal samples which were commercially obtained from George King (Orland Park, KS). Ang‐2 levels were quantified using a sandwich ELISA method (R&D Systems, Minneapolis, MN, USA). This method employs a monoclonal antibody specific for human Ang‐2. DIC scores in all 102 patients were evaluated using the ISTH scoring algorithm.ResultsTable 1 shows that in comparison to normal levels, the samples collected from all patients showed significantly higher levels of Ang‐2 for all groups (p=0.0005). Table 2 shows that the Ang‐2 levels were also significantly different between the survivors and non‐survivors for 28‐day mortality outcome by the Mann‐Whitney test (p=0.001).ConclusionThis study demonstrates that Ang‐2 levels are significantly upregulated in SAC, and this biomarker can be used to risk stratify the severity of this disease. Ang‐2 level can also be used to differentiate non‐overt and overt DIC. Furthermore, Ang‐2 level at the time of initial diagnosis provides a predictive biomarker for mortality outcome.
Ang‐2 levels in patients with Sepsis and Suspected DIC Broken Down by ISTH DIC Score

Category
Normals
+ Sepsis−DIC
+ Sepsis + Non‐Overt DIC
+ Sepsis + Overt DIC

ISTH DIC Score
N/A
0–2
3–4
≥ 5

n
50
20
57
24

Ang‐2 Mean ± SEM (ng/mL)
1.87 ± 0.15
8.34 ± 2.50
11.53 ± 1.40
29.44 ± 6.27

Ang‐2 Std. Deviation (ng/mL)
1.07
11.19
10.54
30.73

Significant Difference in Ang‐2 Level Between Survivors and Non‐Survivors

Survivors
Non‐Survivors

n
86
15

Ang‐2 Mean ± SEM (ng/mL)
12.54 ± 1.54
30.17 ± 8.62

Ang‐2 Std. Deviation (ng/mL)
14.28
33.39
Minimal effect of aliskiren on renal vascular effects of fat embolism in an acute rat model
Authors: Khalafi Farzad , Elsherbiny Hisham , Vaidyanathan Vaishnavi , Ahmad Yembur , Poisner Alan , Molteni Agostino 
Published: November 2023

IntroductionIn a rat model of fat embolism (FE) induced by injection of triolein (T), a severe inflammatory reaction leads to vasoconstriction and pulmonary fibrosis (1). Lung damage is already evident at 48 hrs, and after partial resolution, a second phase of increasing severity develops up to 10 weeks. (2) The renin angiotensin system (RAS) is involved in the process: administration of captopril and losartan prevents the acute inflammatory response and fibrotic process in the lungs (3) and the direct renin inhibitor aliskiren (ALI) also protects the lungs from vasculitis, and fibrotic change in an early phase (48 hrs) (4). We extended the study of ALI to the kidneys by evaluating the renal arterial response to T treatment in this model of FE.Methods22 Sprague Dawley rats received T (0.2 ml IV, n=18) or saline (n=4). The T treated rats were divided into three groups of 6 rats each and injected IP one hour later with 0.2 ml saline, ALI 50mg/kg or ALI 100mg/kg. Four controls received saline. Rats were killed 48 hrs later; the organs fixed and stained with H&E. Trichrome stain was used for fibrosis evaluation.The vascular evaluation included lumen patency (LP) and media adventitia ratio (MAR), a marker of edema. LP was determined by the ratio of luminal internal diameter of the media vs. its external diameter. MAR was measured by the external medial diameter vs. external adventitia diameter. 10 photos at 400 X were taken by two pathologists unaware of the slide identity. Two photos at 100 X were also taken in each slide for evaluation of fibrosis. In addition to this evaluation, we also divided the arteries into two groups: small caliber with diameter of <150 mm (mostly located in the cortical section) and the larger caliber >150 mm (mostly present in tubuli).ResultsNo significant difference was observed for the LP in any of the treatments, P=0.8669. However there was a significant effect on the MAR when all the arteries were considered, P=0.0007. The statistical difference in the MAR was due to effects on the larger arteries >150 mm with P=0.0033 with no differences in the small arteries. Renal fibrosis was minimal with the same amount in each group, mostly located around the arteries.ConclusionThese findings were different than our observations in the lungs where statistical significance was observed in LP of the T treated rats vs. controls, and the 50 mg ALI treated rats. Fibrosis was severe in the lungs with damage mostly located around the small caliber arteries. The study suggests that the influence of the RAS on renal arteries after FE may be different from those in the lungs or that the course of time changes is different in the two organs. Nevertheless, ALI treatment did show protection against effects of T on the media adventitia ratio implicating the RAS in some effects of FE on the kidney at 48 hrs.Support or Funding InformationMary Katherine Geldmacher Research Foundation, St Louis MO.
De novo Thymidylate Biosynthesis and dUTPase Interact in DNA Synthesis
Authors: Chon James , Gondokusumo Jabez C , Field Martha S , Stover Patrick J 
Published: November 2023

Impaired folate mediated one‐carbon metabolism is implicated in several deleterious health outcomes including neural tube defects, neurodegeneration, and colon cancer. Folate is a cofactor for de novo thymidylate biosynthesis. The de novo thymidylate biosynthesis enzymes serine hydroxymethyl‐transferase (SHMT1), dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and methylene tetrahydrofolate dehydrogenase (MTHFD1) are SUMOylated, translocate to the nucleus, and form a complex at sites of DNA replication and repair. The nuclear de novo thymidylate biosynthesis complex is unique to mammalian cells and does not form in yeast. Impaired de novo thymidylate biosynthesis leads to increased uracil incorporation into DNA in cell culture models and in mice, subsequently leading to repair‐mediated DNA damage and cell death. In this study, we demonstrate that disruption of de novo thymidylate biosynthesis via SHMT1 CRISPR knock‐out in HeLa cells does not increase uracil levels in DNA. This suggests de novo thymidylate biosynthesis may not be a major determinant of uracil incorporation into HeLa cell nuclear DNA. We also show that dUTPase is a key regulator of uracil incorporation in HeLa cells, and that dUTPase physically interacts with SHMT1 and is a major source of dUMP substrate for de novo thymidylate biosynthesis. These data indicate that dUTPase‐catalyzed dUTP degradation and de novo thymidylate biosynthesis complex interact during DNA synthesis and repair to limit uracil accumulation in DNA.Support or Funding InformationNational Institutes of Health Grants DK58144 and HD059120
Involvement of Tenascin‐R in Post‐Stroke Brain Damage
Authors: Nalamolu Koteswara Rao , Klopfenstein Jeffrey D , Pinson David M , Wang David Z , Veeravalli Krishna Kumar , Chelluboina Bharath 
Published: November 2023

Tenascins represent an important class of extracellular matrix proteins that have a clear ability to regulate cell behavior, and display highly restricted dynamic patterns of re‐expression following a vascular injury. Tenascin‐R (TNR) is reported to be involved in regulating the action potential and the conduction of neurons during neuronal excitability. TNR serum levels were upregulated in various CNS diseases and its acute upregulation led to neuronal apoptosis, blood brain barrier disruption and neuroinflammation. Further, the functional recovery was improved in TNR knockout mice subjected to spinal cord injury. The main objective of this study is to elucidate the possible role of TNR in the pathogenesis of brain injury after ischemic stroke. Male Sprague‐Dawley rats were obtained and randomly assigned to various groups. Rats were subjected to a two‐hour middle cerebral artery occlusion procedure followed by reperfusion. Rats were sacrificed at various time points after reperfusion and the ischemic brains were analyzed for TNR expression by immunoblot analysis. To achieve the specific knockdown of TNR, plasmids expressing shRNA specific to TNR and formulated as nanoparticles were administered immediately after reperfusion to the appropriate groups of rats intravenously via tail vein at a dose of 1 mg/kg body weight. We performed TTC staining procedure on the coronal brain sections obtained from various groups to evaluate the extent of brain damage. Our results indicated a significant upregulation of TNR at all the post‐reperfusion time points tested. Further, TNR suppression by gene silencing technology revealed a significant reduction in infarct size indicating TNR's detrimental role in brain damage after ischemic stroke. Our results clearly indicate a possible role for TNR in post‐stroke pathogenesis.Support or Funding InformationWe would like to thank OSF Illinois Neurological Institute for the financial support.
The Changes of the Dopaminergic D1 Cell after Stimulation by STN‐DBS in Parkinson's Disease Rats
Authors: Duan Hubin , Hao Chunyan , Li Hao , Wang Suping , Zhang Ce , Fan Yimin , Liu Yueting , Pei Mingyang , Wang Huan 
Published: November 2023

ObjectiveTo investigate the changes of the dopaminergic D1 cell after stimulated by subthalamic nucleus deep brain stimulation (STN‐DBS) in Parkinson's Disease (PD) rats.Methods55 healthy male SD rats were randomly divided into normal control group (n=15)、 PD group (n=15)、 sham stimulation group (n=15) and stimulation group (n=15). The apomorphine rotation was observed as the symbol of successful PD model. Stimulation and sham stimulation groups were planted in unilateral STN‐DBS. STN‐DBS in stimulation group was powered on and was stimulated consecutive days at the regular time, while Sham stimulation group complied with the same program except power on the STN‐DBS. The latency to find the platform, swimming speed, swimming distance and crossing times were detected with Morris water maze. Immunohistochemistry was used to observe the change and count the dopaminergic D1 receptor cells in hippocampal C1 area and prefrontal cortex.ResultsAfter stimulated by STN‐DBS regularly for 7 days, the symptoms of PD rats were improved significantly after stimulated by STN‐DBS. Compared with PD model group and sham stimulation model group, after trained, not only the escape latency of rats in the DBS stimulated model group was decreased (P < 0.01), but also swimming distance (P < 0.05)in the plate quadrant was increased. The dopaminergic D1 receptor cell of hippocampal C1 area and prefrontal cortex in stimulation group increased by compare with that in PD model group and sham stimulation group, furthermore, which there were statistical significance.ConclusionSTN‐DBS stimulation not only can improve the ability of PD rats' cognition, but also can protect dopaminergic receptor cells in some degree.Support or Funding InformationThis study was supported by the National Natural Science Foundation Youth Fund (Fund No. 30600637), China Postdoctral Foundation(Fund No.2014M561207), the Shanxi Province Basic Research Program Youth Science and Technology Research Fund (Fund No. 2010021034‐4), the Shanxi Scholarship Council of China (Fund No. 2011‐096 and Key Projects No.4), The science and technology research projects of health department of Shanxi Province(Fund No.2016D011119).
Chemical cross‐linking of doxorubicin to synthetic oligonucleotides by formaldehyde
Authors: Medina Juan , Nagesetti Abhignyan , McGoron Anthony J , Leng Fenfei 
Published: November 2023

Anthacycline antibiotics, such as doxorubicin and daunorubicin, are among the most effective anti‐cancer drugs that treat various cancers. Previous research has shown that doxorubicin and daunorubicin were efficiently cross‐linked to double‐stranded (ds) DNA in the presence of formaldehyde in a regioselective and base specific manner. In an effort to further explore the cross‐linking of anthracycline antibiotics to DNA, doxorubicin is crosslinked to a variety of synthetic oligonucleotides including sequence specific ds and single stranded (ss) DNA oligomers. Our results demonstrate that doxorubicin is able to efficiently crosslink to the N2 amino group of guanine for the ds DNA oligomers. Intriguingly, we also discovered that doxorubicin is able to crosslink to ssDNA and ssRNA specifically to guanine residues by formaldehyde. These results suggest that ssDNA and ssRNA may serve as a new delivery system for the treatment of cancer patients by doxorubicin and daunorubicin.Support or Funding Information1R15GM109254‐01A1; R25GM061347
Elevated Circulating CD40‐L in Atrial Fibrillation and Its Modulation by Ablation
Authors: Otto Abigail McKenzie , Fareed Jawed , Statz Stephen , Walborn Amanda , Liles Jeffrey , Hoppensteadt Debra , Rowe Tim , Jabati Sallu , Syed Mushabbar 
Published: November 2023

IntroductionAtrial fibrillation (AF) is currently the most common cardiac arrhythmia encountered in clinical practice and a major cause of morbidity and mortality among adults. Inflammation plays a crucial role in the development and progression of atrial fibrillation. CD40‐Ligand (CD40‐L) is a cellular mediator that is characteristically seen in inflammatory processes. CD40‐L can be expressed on platelets, leading to activation of endothelial cells and the release of cytokines during inflammation. The purpose of this study was to characterize and quantify the levels of the inflammatory biomarker CD40‐L to evaluate its relevance to AF both pre‐ and post‐ablation.MethodsIn a prospective study, plasma samples were collected from 55 AF patients at baseline prior to ablation, and at 1 and 3‐month follow‐up post‐ablation. Normal human plasma samples (n=50) were supplied by George King Biomedical Inc. (Overland Park, KS). ELISA kits were obtained to profile CD40‐Ligand (R&D Systems, Minnesota USA). Statistical analysis was performed using non‐parametric Mann‐Whitney t‐tests.ResultsThere was a significant increase in the mean levels of CD40‐L at pre‐ablation baseline in patients with AF compared to normal (401.0±73.3 pg/mL vs. 53.6±4.74 pg/mL, p=0.0034). CD40‐L levels post‐ablation significantly decreased. At 1 month follow up, CD40‐L levels dropped (401.0±73.3 pg/mL vs. 40.6±6.86 pg/mL, p=<0.0001). At 3 months, the levels of CD40‐L remained decreased (43.3±7.98 pg/mL, p=<0.0001). There was no significant change between levels at 1 month and 3 months (p=0.8573). There was also no significant difference in post‐ablation levels of CD40‐L compared to normal human plasma levels (p=0.2095).ConclusionInflammation plays an important role in the pathogenesis of atrial fibrillation. In our study, patients with atrial fibrillation have highly elevated levels of CD40‐L compared with normal healthy individuals. Contrary to previous reports, our study suggests that elevated levels of CD40‐L may play an important role in the pathogenesis of AF. It is also noted that the levels of this inflammatory mediator are modulated by atrial ablation as a medical treatment for AF.Support or Funding InformationThis project is supported by internal funds of Loyola University Medical Center.
Base unpairing at the flap junction controls the rate of FEN1‐catalyzed cleavage of DNA during replication and repair
Authors: Song Bo , Hingorani Manju M. 
Published: November 2023

Flap Endonuclease 1 (FEN1) recognizes and cleaves 5′ single‐stranded DNA flaps to create nicked duplex products during DNA metabolic processes such as Okazaki fragment maturation, long‐patch base excision repair and telomere maintenance, and therefore has an essential role in genome duplication and stability (1). Defects in human FEN1 are associated with cancer and other genetic diseases (2). Hence, there is considerable interest in hFEN1 as a therapeutic target and diagnostic or prognostic biomarker (3–5), which can be aided by a detailed mechanistic understanding of the enzyme.Previous studies have shown that hFEN1 bends its DNA substrate, and suggest that unpairing of two bases in the duplex beyond the 5′ flap helps position the scissile phosphate in the active site for cleavage (6,7). These studies have largely been performed under equilibrium conditions and in the absence of Mg2+, which is indispensible for catalysis. To study the hFEN1 mechanism under catalytically competent conditions, we utilized transient kinetic approaches and monitored individual events in the millisecond time scale, including DNA binding and bending, base unpairing and cleavage, which elucidated the order of events and rate‐determining step(s) in the reaction. Furthermore, we examined the role of several conserved active‐site residues in the reaction mechanism. A FRET assay revealed that hFEN1 binds and bends the DNA substrate rapidly at a diffusion‐limited rate (109 M−1s−1), indicating that these events occur simultaneously. Lack of Mg2+ and mutations in the active site do not affect this binding/bending rate, thus this initial event does not depend on specific contacts within the catalytic pocket. A 2‐aminopurine fluorescence‐based assay revealed unpairing of base(s) at the flap junction at a much slower rate of 20 s−1. Importantly, quench‐flow measurements revealed that base unpairing limits the cleavage rate to 20 s−1. A subsequent event, likely related to product release, limits the overall steady state turnover rate to 1 s−1. Mutations in active site residues D34, Y40, K93, R100, E160 and D181 exhibit differential effects on base unpairing and cleavage. The results show that base unpairing is a pre‐requisite for flap cleavage, and clarify the contributions of these conserved residues toward an electrostatic environment that stimulates precise cleavage of the 5′ flap. The findings also apply to other 5′‐nucleases that share similar active site residues and a two‐metal ion dependent reaction mechanism (8,9).