bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evans Blue Dye Interferes with Gating for Certain Fluorochrome‐Conjugated Antibodies Using Flow Cytometry in the Ischemic Rat Heart
Authors: Teng Jiajing , De Jeronimo Diaz Cesar , Flynn Ryan A. , Lin Matthew , Rogers Connie J. , Korzick Donna H. 
Published: May 2022

BackgroundAcute myocardial infarction (AMI) is the leading cause of death in the U.S. Understanding the immune response to AMI has potential therapeutic impact. In particular, the differentiation of monocytes into macrophages post‐AMI is of interest given their role in inflammatory cytokine production and tissue repair. Recent studies suggest that impaired macrophage (Mϕ) polarization may promote a proinflammatory phenotype in the heart post‐AMI. Flow cytometry is a gold standard technique to assess immune cell populations including M1 and M2 Mϕ subtypes, using a wide range of fluorochrome‐conjugated antibodies (FC‐Abs). Evans Blue (EB) dye is widely used to assess the size of AMI, but the feasibility of the combining approaches due to spectral overlap is uncertain. Thus, the objective of this study was to determine whether the cardiac viability dye EB can be used simultaneously with FC‐Abs to quantify M1 and M2 Mϕ in cardiac tissue post‐AMI.MethodsImmune cells were isolated from ischemic rat hearts (31 min ischemia/48 hr reperfusion) with or without EB perfusion and stained with FC‐Abs (1 μg/1 × 106 cells) to the following cell surface markers: FITC‐CD45 (experiment 1) or AF750‐CD45 (experiment 2) to detect leukocytes; Pacific Blue‐CD11b, AF700‐CD68 and PE‐CD206 (experiment 1) or Pacific Blue‐CD11b, AF488‐CD68 and PE‐CD206 (experiment 2) to quantify M1 and M2 Mϕ. A total of 500,000‐1,000,000 events were acquired using a BD LSR‐Fortessa and were analyzed and plotted using FlowJo software v10.ResultsOverlap of EB (emission peak at 680 nm) was detected in the AF700‐CD68 channel (emission peak at 720nm) and the AF750‐CD45 channel (emission peak at 775nm), interfering with gating within these two channels. In experiment 1, the M1 (CD45+CD11b+CD68+) population differed between samples with EB (2.5% of CD45+CD11b+ population, sample size n=2) vs without EB (68.5%, n=2). However, in experiment 2, the M1 population was similar between samples with EB (54.3% of CD45+CD11b+ population, n=2) and without EB (48.7%, n=1). The M2 (CD45+CD11b+CD206+) population did not differ in experiment 1 or 2.ConclusionOur data suggest that the EB perfusion interferes with fluorochromes with emission peaks close to 680nm. Thus, caution must be taken when selecting FC‐Abs for flow cytometry when combined with EB perfusion or other related dyes. Our next steps will be to confirm these observations in a larger sample size.
Reduced Angiotensin II Type 2 Receptor Expression Is Associated with Gastric Cancer Progression and Enhanced Gastric Cancer Cell Migration/Invasion
Authors: Sandoval‐Borquez Alejandra M. , Wichman Ignacio , Carrasco‐Avino Gonzalo , Corvalan Alejandro , Lavandero Sergio , Quest Andrew F. G. 
Published: May 2022

IntroductionGastric cancer (GC) is one of the leading causes of death due to cancer worldwide. Most cases have a poor prognosis due to non‐curable surgical resection and the limited efficacy of chemotherapy. Angiotensin II Type 2 Receptor (AT2R), an effector protein of the renin angiotensin system (RAS), is reportedly deregulated in some human cancers and implicated in the regulation of several cancer‐related cellular processes. However, our current understanding of the role of this protein in GC is very limited. The objective of this study was to evaluate the expression of AT2R during GC progression and determine the effect of receptor activation and overexpression on GC cell migration and invasion.HypothesisAT2R acts as a tumor suppressor in GC by reducing cell migration and invasion.MethodsWe used the data published in the The Cancer Genome Atlas (TCGA) to evaluate in GC the expression of AGTR2, the gene that encodes AT2R. In addition, we determined the expression of AT2R in precancerous gastric lesions, advanced GC and non‐tumor adjacent tissue samples by immunohistochemistry. Association analysis of the expression of AGTR2 and AT2R with clinicopathological features and survival curves was also performed. Besides, the effects of AT2R on cell migration, transmigration, invasion and colony formation were evaluated using human GC cells. Finally, we determined the effect of AT2R activation in an intraperitoneal carcinomatosis xenograft mouse model.ResultsWe observed that AGTR2 and AT2R expression was downregulated in GC patients (Fig. 1A), and this decreased expression correlated with depth of tumor invasion, metastasis and poor overall survival (Fig 1B). Moreover, loss of AT2R expression was associated with disease progression from pre‐neoplasic lesions to GC (Fig, 1C). On the other hand, AT2R activation and overexpression reduced migration (Fig. 2A, B), transendothelial migration, invasion and colony formation of AGS and Hs 746T GC cells. Finally, AT2R activation increased mouse survival and decreased tumor formation in an in vivo model (not shown).ConclusionsAT2R was downregulated to a significant extent in GC patients and shown to prevent GC cell migration and invasion, suggesting a role in preventing the progression of GC lesions.
Systematic characterization of enzyme activity on ENPP1 deficiency disease phenotype
Authors: Ansh Anenya J. , Stabach Paul R. , Carpenter Thomas O. , Ferreira Carlos R. , Braddock Demetrios 
Published: May 2022

ENPP1 codes for a type 2 transmembrane glycoprotein in the ectonucleotide pyrophosphatase/phosphodiesterase protein family. The enzyme’s catalytic activity hydrolyzes extracellular ATP to generate pyrophosphate and adenosine monophosphate (AMP), thereby contributing to downstream purinergic signaling pathways. Pathogenic variants in ENPP1 are associated with diseases such as Type 2 Diabetes, Generalized Arterial Calcification of Infancy (GACI), Ossification of the Posterior Longitudinal Ligament (OPLL), Autosomal Recessive Hypophosphatemic Rickets Type 2 (ARHR2), Cole Disease, and early onset osteoporosis. The human phenotypes associated with these diseases are varied, and include life threatening large arterial calcifications, phosphate wasting rickets due to FGF23 elevation, cutaneous hypopigmentation and punctate keratosis, ankylosis and ossification of spinal ligaments, premature osteoporosis, as well as insulin resistance and cardiovascular disease.To determine whether the above disease phenotypes correlate with ENPP1 enzyme activity we quantitated enzyme velocities of ENPP1 variants identified in 42 ENPP1‐deficient patients followed at the NIH, together with an additional 22 variants reported in the literature. We simultaneously evaluated catalytic velocity of all variants in triplicate, enabling a direct comparison of enzyme activity across disease phenotypes. Cole disease is induced by variants in the SMB domains of ENPP1, and variants associated with the Autosomal Dominant form reduced enzyme velocity to a greater extent than the Autosomal Recessive form – between 32% to 8% of wild type (WT) vs. 33% of WT levels, respectively. Further supporting the presence of a gene dose effect, monoallelic variants in ENPP1 associated with elevation of FGF23, phosphate wasting, and early onset osteoporosis, occurred in patients with variants that reduced enzyme velocity below 50% of WT, while the polymorphisms associated with insulin resistance and cardiovascular disease reduced enzyme velocity to 73% of WT levels. Additionally, variants associated with life‐threatening vascular calcifications in GACI, which only occur in autosomal recessive biallelic ENPP1 deficient patients, reduced ENPP1 enzyme activity over a surprisingly wide range, from 25% to 100% of WT levels. Finally, disease severity in GACI correlated with the more severely damaging ENPP1 variant rather than the average of both variant allele products, suggesting that the effects of biallelic ENPP1 variants on in vivo enzyme activity are dominated by the more damaging ENPP1 allele. In summary, comparison of enzyme velocities among various ENPP1 deficient disease phenotypes supports the notion that specific variants determine these variable phenotypes, and are sometimes, but not always, related to enzyme activity. ENPP1 gene dose and dominance of the severe isoform in biallelic disease are notable mechanisms for determination of phenotype in some forms of ENPP1 deficiency.
Abscisic Acid Controlled Redox Proteome of Arabidopsis and its Regulation by Heterotrimeric G‐proteins
Authors: Smythers Amanda L. , McConnell Evan , Pandey Sona , Hicks Leslie M. 
Published: May 2022

Reactive oxygen species (ROS) are highly efficient, membrane permeable cell signals that are essential for cellular regulation. These signals can mark macromolecules for degradation, act as a retrograde signal for transcriptional regulation, as well as directly modulate enzymatic activity via the reversible oxidation of cysteine thiols. Although this reversible cysteine oxidation is an essential regulatory mechanism, the resulting modifications are labile and infrequent compared to the global proteome, making them challenging to maintain ex vivo and detect reproducibly. In our lab, we have optimized an approach for oxidized resin assisted capture (OxRAC) that enables thorough, reproducible analysis of the full reversible cysteine redoxome. By employing iodoacetamide as a blocking reagent during cell lysis, we ensure that cysteines reduced in vivoare excluded before non‐specifically reducing reversibly oxidized cysteines for enrichment and analysis via label free quantitative LC‐MS/MS.In this work, our optimized OxRAC platform was implemented to probe the dependence of heterotrimeric G‐proteins on reversible cysteine oxidation for the mediation of abscisic acid (ABA) signaling pathways in Arabidopsis thaliana. By cross comparing the wildtype Col0plants with the Gβ protein (AGB1) null mutant, agb1, we were able to quantify 6,891 unique oxidized cysteine‐containing peptides and reveal 923 significant changes in oxidation following ABA treatment. Divergent pathways, including primary metabolism, ROS response, translation, and photosynthesis, exhibited both ABA and G‐protein dependent redox changes, many of which occurred on proteins not previously linked to either ABA or G‐proteins. Together, these data uncover a complex network of reversible oxidations that allow ABA and G‐proteins to rapidly adjust cellular signaling to adapt to changing environments and suggest that a functional G‐protein complex is required to maintain intracellular redox homeostasis and fully execute plant stress responses.
Long‐Term Cardioprotective Potential of Exogenous Ubiquitin in Myocardial Ischemia/Reperfusion Injury
Authors: Shook Paige , Dalal Suman , Singh Mahipal , Singh Krishna 
Published: May 2022

BackgroundCoronary heart disease (CHD) is a leading cause of death worldwide. Myocardial ischemia/reperfusion (I/R) injury is a clinical manifestation of CHD. Timely reperfusion of coronary vessels in an ischemic injury is essential to limit the infarct size. However, reperfusion itself can induce cardiac cell death, and lead to increased adverse remodeling after an I/R injury. Previously, our lab has shown that pretreatment with ubiquitin (UB; a highly evolutionarily conserved protein) decreases inflammatory response and preserves heart function 3 days post‐I/R. The objective of this study is to investigate the long‐term cardioprotective potential of UB post‐I/R. To enhance the clinical relevance, UB treatment was started at the time of reperfusion. It is hypothesized that UB treatment has long‐term cardioprotective potential, improves cardiac function, and attenuates cardiac remodeling post injury.MethodsC57BL/6 male mice underwent ligation of the left anterior descending coronary artery (LCA) for 45 min. Mice were then treated with UB (1 µg/g body weight, i.p. injection) or saline at time of reperfusion of LCA. Sham animals underwent the same surgery without the ligation of LCA. Mice were treated with UB or saline twice/day (i.p. injections) for 3 days followed by treatment with UB (1 μg/g/h)or saline using micro‐osmotic pumps for 28 days. Heart function was measured at 3, 7, 14 and 28 days post‐I/R using echocardiography. Mice were sacrificed at 28 days post‐I/R. Left ventricular (LV) tissue was used to measure remodeling parameters and serum samples were used to measure a panel of 23 cytokines/chemokines using multiplex assay.ResultsI/R+saline group exhibited a significant reduction in heart function as observed by decreased percent fractional shortening (%FS) and percent ejection fraction (%EF) vs sham (p<0.05) at 3, 7, 14 and 28 days post‐I/R. However, I/R+UB group showed significantly higher %FS and %EF vs I/R+saline (p<0.05) throughout the observation period. Increases in fibrosis (measured by trichrome staining), myocyte apoptosis (measured using TUNEL assay), and hypertrophy (measured using WGA staining) were significantly lower in I/R+UB vs I/R+saline (p<0.05). Expression of MMP‐2 and TIMP‐4 was lower, while expression of MMP‐9 (active) was significantly higher in I/R+UB vs I/R+saline (p<0.05). Serum levels of IL‐6, IL‐2 and G‐CSF were significantly lower in I/R+UB vs I/R+saline (p<0.05).ConclusionPost‐ischemic UB treatment improves heart function, and associates with decreased myocardial remodeling parameters (fibrosis, apoptosis, and hypertrophy) as well as decreased serum cytokines/chemokines levels.
Developing a Pipeline for Personalized Diagnoses of Glut1 Deficiency Syndrome
Authors: Griffith Laiken , Donohue Katherine , Abul‐Khoudoud Maya , Gentry Matthew 
Published: May 2022

Glut1 deficiency syndrome (Glut1DS) is a brain metabolic disorder, caused by the impairment of glucose transport in the brain. Glut1DS occurs when there is a mutation in the SLC2A1 gene, which encodes for the protein glucose transporter protein type 1. Around 500 people worldwide suffer from this disease, and most deal with symptoms such as seizures, movement disorders, and in more severe cases, microcephaly. A common treatment for Glut1DS is the ketogenic diet, which provides the brain with an alternate energy source. Patients suffering from Glut1DS have been shown to respond more positively to this treatment when they are able to start it early in life.Unfortunately, due to the variety of phenotypes associated with Glut1DS, there is often a large gap between onset of symptoms and diagnosis. This is problematic because a delay in diagnosis means a delay in treatment, leading to more severe disease progression. In order to bridge the gap, our lab applied a suite of bioinformatics techniques to characterize novel mutations and help patients achieve faster diagnoses. Because Glut1DS patients with different mutations experience varying loss of function of the Glut1 transporter, linking mutation type to phenotype could help patients receive a faster diagnosis, allowing them to start treatment earlier.In order to do this, we analyzed and classified different mutations in the SLC2A1 gene based on predicted pathogenicity. By comparing these data to known Glut1DS patient phenotypes, we were able to able to define five mutation classes based on severity. This type of analysis can be used to provide a faster diagnosis and personalized treatment plans for patients based on their mutation class. As personalized medicine for rare genetic diseases progresses, a database of mutations and severity class could help patients start life‐changing treatment much earlier. Additionally, this model for pipeline development could be expanded to assist in the diagnosis of other rare genetic diseases.
Investigating a Ligand‐receptor Role for Glutathione in Angiogenesis
Authors: Mackay Ryan P. , Delgadillo Luisa F. , Rashdan Nabil A. , Pattillo Christopher B. 
Published: May 2022

IntroductionIn addition to its classic role in redox chemistry, glutathione (GSH) also acts as an allosteric regulator of the calcium‐sensing receptor (CaSR). CaSR expression has been found in a variety of cell types including vascular smooth muscle and endothelial cells (ECs), where its activity has been linked to EC adhesion and migration. Although a relationship between GSH and CaSR has been discovered, no link has been established between GSH‐CaSR interaction and regulation of blood vessel development. We hypothesize that a novel mechanism for GSH regulation of blood vessel development occurs through an extracellular interaction with CaSR as a part of normal physiology, with potential extensions to pathological angiogenesis.MethodsMatrigel tube formation and under agarose migration assays were used to measure EC activity in primary human aortic endothelial cells (HAECs). Fura‐2 AM was used with cells from an hTERT immortalized aortic endothelial cell line (tHAECs) to measure changes in intracellular calcium. Cell surface expression assay (CelLISA) and flow cytometry were used to measure surface CaSR expression. Endothelial and endothelial‐mesenchymal transition (EndoMT) markers were measured using real‐time PCR.ResultsExtracellular application of GSH increased endothelial tube length and branching in tube formation assay, while treating cells with selective CaSR inhibitor (NPS‐2143) abrogated these effects, indicating a role for CaSR in vascular modeling. Extracellular GSH influenced EC migration as measured by under agarose migration assay. GSH or a CaSR agonist (R‐568) reduced surface CaSR expression, with little/no change in intracellular calcium. RT‐PCR results suggest that extracellular GSH alters endothelial phenotype along the endothelial‐mesenchymal spectrum, promoting endothelial differentiation.ConclusionIn addition to its classic antioxidant role, GSH appears to act extracellularly to influence EC activity. GSH caused significant increases in EC branching as measured by the tube formation assay. With the use of a selective CaSR inhibitor, we were able to confirm the contribution of CaSR to this effect. Additionally, GSH influenced EC migration in the under agarose assay. These results could be explained by GSH‐CaSR‐mediated alterations in endothelial differentiation. These results suggest a role for GSH in vascular development and angiogenesis, through a ligand interaction with the CaSR G‐protein coupled receptor, which represents a novel mechanism of action for physiological and pathological blood vessel development.
In Vitro Evaluation Of The Antimicrobial Activity Of Monolaurin Against Borrelia burgdorferi,The Lyme Disease Spirochete
Authors: Bhalla Shubhang , Trinh Benjamin , Barragan Jose , Cervantes Jorge 
Published: May 2022

In this study, we evaluated the antimicrobial activity of monolaurin against Borrelia burgdorferi, the Lyme disease spirochete.B. burgdorferipossesses adaptive mechanisms that allow it to evade retrieval by host immune response. Novel therapeutic modalities to address Lyme disease complications are widely being sought. Monolaurin, a glycerol monoester of lauric acid derived from coconut oil, has demonstrated a wide‐range of antimicrobial activity through emulsification.We hypothesize that monolaurin will reduce the viability of B. burgdorferi in a dose response manner.B. burgdorferistrain B31 was cultured under serially diluted monolaurin conditions. Resazurin assay was utilized to assess bacterial viability and establish an approximate minimum inhibitory concentration (MIC). A live‐dead assay was conducted to evaluate monolaurin’s antimicrobial activity.ResultsWe observed a MIC value of monolaurin against B. burgdorferi to reside between 75 µg/mL and 150 µg/mL. The live‐dead assay narrowed the antimicrobial range from 75 µg/mL to 125 µg/mL. There was a trend of the proportion of dead spirochetes with increased monolaurin concentrations that was statistically significant (p‐value<0.0001, Two‐way ANOVA).Our findings support the use of monolaurin as an antibacterial agent against B. burgdorferi. Further studies in vivo are required to establish its pharmacodynamics so it could become available as a treatment modality for Lyme disease.
Loop gain is Increased and the Arousal Threshold is Decreased in Caucasian Compared to African American Males with Obstructive Sleep Apnea
Authors: Puri Shipra , Kissane Dylan , Panza Gino , Lin Ho‐Sheng , Mateika Jason H. 
Published: May 2022

ObjectiveRace and ethnicity may impact the prevalence and treatment of obstructive sleep apnea (OSA). However, only a few studies have made direct comparisons of endotypic characteristics between African Americans and Caucasians. The comparisons completed have focused primarily on differences in anatomical traits (i.e. craniofacial morphology) that may exist between races. The present study was designed to explore if other endotypic traits [(i.e. loop gain (LG) and the arousal threshold (AT)] that influence sleep apnea differ in African American compared to Caucasian males with OSA.Methods26 African Americans [age = 34.9 ± 11.9 years; body mass index = 28.1 ± 4.4 kg/m2; apnea hypopnea index (AHI) = 37.7 ± 21.1 events/hour] and 26 Caucasian male participants (age = 31.2 ± 8.2 years; body mass index = 29.6 ± 3.6 kg/m2; AHI = 41.7 ± 16.5 events/hour) with untreated OSA completed a baseline nocturnal polysomnogram. Thereafter, sleep staging, respiratory events, and arousals were scored according to standard criteria. A MATLAB software program was used to quantify LG and the AT from the ventilatory and sleep measures obtained during non‐rapid eye movement (NREM) sleep throughout the night. LG at the natural frequency (LGn) of obstructive events and at a frequency of 1 cycle/min (LG1, i.e., 60 events/hour) were computed. Data was compared using Student t‐test and is presented as mean ± SD.ResultsAge (p = 0.19), BMI (p = 0.20), and the AHI (p = 0.44) was similar between the two groups. LG was significantly higher in Caucasians compared to African Americans (LGn: 0.44 ± 0.07 vs. 0.40 ± 0.06, p = 0.037; LG1: 0.61 ± 0.12 vs. 0.54 ± 0.12, p = 0.039). In contrast, the AT was significantly lower in Caucasians compared to African Americans (1.22 ± 0.17 vs. 1.38 ± 0.27, p = 0.009). The difference in the AT corresponded to an increased arousal index in Caucasians compared to African Americans (74.99 ± 5.36 vs. 51.13 ± 19.34 arousals/hour, p = 0.029). Furthermore, a subgroup analysis of participants with OSA and co‐existing hypertension also showed that Caucasian males (n = 11) had a higher LG (LGn: 0.45 ± 0.06 vs. 0.39 ± 0.07, p = 0.05; LG1: 0.62 ± 0.12 vs. 0.48 ± 0.09, p = 0.007) and lower AT (1.16 ± 0.12 vs. 1.37 ± 0.27, p = 0.033) than the African American participants (n =10).ConclusionDespite similar anthropometric measures and a similar apnea hypopnea index, loop gain and the arousal threshold were greater in Caucasian compared to African American males with OSA. Our results suggest that these endotypic characteristics may have a more prevalent role in initiating sleep disordered breathing in Caucasians compared to African Americans. Likewise, our findings suggest that Caucasians may be more responsive to therapies that target loop gain and the arousal threshold.