Development and validation of a generic methyltransferase enzymatic assay based on an SAH riboswitch.

Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) - the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAM. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z' > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1 µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.

Stent graft-induced new entry tear after endoluminal grafting for aortic dissection repaired with open interposition graft.

Thoracic endovascular aortic repair is a successful treatment strategy for type B aortic dissections, with low morbidity and mortality compared with the gold standard of open repair. Questions still remain regarding its long-term durability and complication rate. There is a growing awareness of new entry tears induced by the stent graft, a potentially lethal complication. We report the case of a 74-year-old woman with a complicated retrograde type A aortic dissection treated with endovascular stent graft coverage. She required open surgical conversion after she developed a symptomatic, new entry tear induced by the stent graft.

Ascending aortic injuries following blunt trauma.

BACKGROUND: The diagnosis and the management of traumatic thoracic aortic injuries have undergone significant changes due to new technology and improved prehospital care. Most of the discussions have focused on descending aortic injuries. In this review, we discuss the recent management of ascending aortic injuries. METHODS: We found 5 cohort studies on traumatic aortic injuries and 11 case reports describing ascending aortic injuries between 1998 to the present through Medline research. RESULTS: Among case reports, 78.9% of cases were caused by motor vehicle accidents (MVA). 42.1% of patients underwent emergent open repair and the operative mortality was 12.5%. 36.8% underwent delayed repair. Associated injuries occurred in 84.2% of patients. Aortic valve injury was concurrent in 26.3% of patients. The incidence of ascending aortic injury ranged 1.9-20% in cohort studies. CONCLUSIONS: Traumatic injuries to the ascending aorta are relatively uncommon among survivors following blunt trauma. Aortography has been replaced by computed tomography and echocardiography as a diagnostic tool. Open repair, either emergent or delayed, remains the treatment of choice.

Outcomes in Sutured Versus Sutureless Wound Closure in Pediatric Cataract Surgery.

PURPOSE: To analyze the postoperative course, specifically postoperative complications, of pediatric patients who underwent cataract surgery by a single surgeon. The type of wound closure was compared to provide an evidence-based approach to surgical technique in pediatric cataract surgery. METHODS: This retrospective study analyzed pediatric patients who underwent cataract extraction by a single surgeon from 2014 to 2020. Excluded from the study were patients with postoperative follow-up of less than 1 month. The primary outcome compared postoperative complications between patients with sutured and sutureless wound closure. Other outcomes analyzed included intraocular pressure, mean corrected distance visual acuity, and incidence of procedure needed to remove posterior capsule opacification. RESULTS: The study comprised 116 eyes with sufficient follow-up; 86 had sutureless wound closure and 30 had sutured wound closure. An intraocular lens was placed in 52% of eyes in the sutureless group and in 80% of eyes in the sutured group. There was no statistically significant difference between preoperative and postoperative intraocular pressure between groups. The best corrected distance visual acuity was better in the sutured group at 6 months but not at the most recent follow-up visit. No cases of endophthalmitis were found in either group. There was no statistically significant difference between the incidence of retinal detachments and iris prolapse. CONCLUSIONS: The incidence of endophthalmitis, retinal detachment, and iris prolapse was similar between pediatric patients who underwent cataract removal with sutureless versus sutured wound closure. Therefore, it may be reasonable to avoid the suture after pediatric cataract surgery. [J Pediatr Ophthalmol Strabismus. 2023;60(2):147-151.].

Endovascular management of acute aortic dissections.

INTRODUCTION: Acute aortic dissection (AAD) is one of the most common aortic emergencies that vascular specialists are asked to manage. Traditional surgical interventions for cases complicated by malperfusion have resulted in significant morbidity and mortality. With increasing availability of thoracic endografts, endovascular interventions for complicated AAD have become more acceptable. We reviewed our experience with endovascular treatment of AAD since January 2005. METHODS: Medical records of patients admitted for AAD from January 1, 2005, to December 31, 2008, were entered into our vascular registry and analyzed for risk factors, extent of dissection, type of management, fate of the false lumen, complications, and survival. There were 249 admissions for aortic dissections during the study period. Our study group included 28 patients with complicated AAD who underwent endovascular intervention. RESULTS: During the study interval, 28 patients (16 male) underwent 44 procedures. The average age was 54 years. Risk factors differed from the typical atherosclerotic patient and were dominated by an 89.3% incidence of hypertension. Five patients (17.9%) presented with a history of recent cocaine use. The average length of stay was 25.1 days (range, 1-196 days). Stanford type B dissections were present in all but one patient. Twenty-six thoracic endografts were placed in 25 patients. Eight patients required multiple procedures in addition to a thoracic endograft. Morbidity occurred in 17 (60.7%) patients, with renal insufficiency occurring in 11 patients (39.3%) and one requiring permanent dialysis. Four neurologic events occurred: three strokes (10.7%) and one patient (3.6%) with temporary paraplegia. Three patients (10.7%) died in the periprocedural period, with ruptured dissection in one and pericardial tamponade in another. Eight of 10 computed tomography scans (80%) available for review in follow-up showed complete thrombosis of the thoracic false lumen. CONCLUSIONS: Complicated AAD remains a challenging problem, with significant morbidity and mortality rates. However, our early experience with endovascular management offers a favorable reduction in mortality from historic controls.

Regionalized autosomal STR profiles among Armenian groups suggest disparate genetic influences.

The archeology and ethnology of Armenia suggest that this region has acted as a crossroads for human migrations from Europe and the Middle East since at least the Neolithic. Near continual foreign influx has, in turn, led to the supposition that the gene pools of geographically separated Armenian populations may have diverged as differing historical influences potentially left distinct genetic traces in the various regions of the Armenian plateau. In this study, we seek to address whether any evidence for such genetic regional partitioning in Armenians exists by analyzing, for the first time, 15 autosomal short tandem repeat (STR) loci in 404 Armenians from four geographically well-characterized collections (Ararat Valley, Gardman, Sasun, and Lake Van) that represent distinct communities from across Historical Armenia. In addition, to determine whether genetic differences among these four Armenian populations are the result of differential affinities to populations of known historical influence in Armenia, we utilize 27 biogeographically targeted reference populations for phylogenetic and admixture analyses. From these examinations, we find that while close genetic affiliations exist between the two easternmost Armenian groups analyzed, Ararat Valley and Gardman, the remaining two populations display substantial distinctions. In particular, Sasun is distinguished by evidence for genetic contributions from Turkey, while a stronger Balkan component is detected in Lake Van, potentially suggestive of remnant genetic influences from ancient Greek and Phrygian populations in this region.

Development of a High-Throughput Assay to Identify Inhibitors of ENPP1.

The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP2/GMP2 Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP2/GMP2 Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z' value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2'3'cGAMP compared with 2'3'c-diGMP and showed no activity with 3'3'c-diAMP. The Transcreener AMP2/GMP2 Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.

RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors.

Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 µM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.

Pseudoaneurysm of mitral-aortic intervalvular fibrosa: two case reports and review of literature.

The aim of this study was to identify the echocardiographic characteristics of pseudoaneurysm of the mitral-aortic intervalvular fibrosa, which is a rare and life-threatening complication of infective endocarditis. We have demonstrated the difference in clinical presentation and management of acute and chronic types of this pseudoaneurysm, together with a review of literature of the topic. We present two cases, one acute and the other an example of a chronic pseudoaneurysm of the mitral-aortic intervalvular fibrosa. The abscess may enlarge rapidly and rupture, resulting in haemorrhage with a catastrophic outcome. Rarely, the pseudoaneurysm will undergo a subclinical course, thicken and organize into a chronic aneurysm. Transoesophageal echocardiogram demonstrates a false lumen below the aortic valve annulus at the mitral-aortic intervalvular fibrosa with marked pulsatility with systolic expansion and diastolic collapse. The successful management of acute pseudoaneurysm necessitates extensive resection and replacement of the infected areas around the pseudoaneurysm. In chronic pseudoaneurysm, there is structural integrity around the calcified pseudoaneurysm, potentially minimizing the need for an extirpative surgery. Pseudoaneurysm of the mitral-aortic intervalvular fibrosa is a rare complication of infective endocarditis, but delay in diagnosis can lead to devastating outcome.

High-Throughput Screening Assays for Cancer Immunotherapy Targets: Ectonucleotidases CD39 and CD73.

Production of adenosine in the extracellular tumor microenvironment elicits strong immunosuppression and is associated with tumor progression. Thus, targeting adenosine-generating ectonucleotidases is a potential strategy to stimulate and prolong antitumor immunity. Because the reaction products of ectonucleotidases differ by a single phosphate group, selective detection in an assay format that is compatible with high-throughput screening (HTS) has been elusive. We report the development of biochemical assays capable of measuring the activity of ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; also known as CD39) and ecto-5'-nucleotidase (CD73). Both assays leverage the Transcreener HTS Assay platform, which facilitates selective immunodetection of nucleotides with homogenous fluorescent readouts, fluorescence polarization or time-resolved fluorescence energy transfer. The Transcreener AMP2 Assay was used to measure CD39 activity, allowing detection of adenosine monophosphate (AMP) production (Z' > 0.6) with subnanomolar amounts of CD39, allowing IC50 determination for tool compounds, consistent with previously reported values. To detect the production of adenosine by CD73, the Transcreener ADP2 Assay was coupled with adenosine kinase (AK); conversion of adenosine to AMP and adenosine diphosphate (ADP) by AK allows detection with ADP2 antibody. The Transcreener AMP2 Assay was used to screen a 1280 Library of Pharmacologically Active Compounds (LOPAC) library and a 1600-compound subset of a ChemBridge diversity library for CD39 inhibitors, allowing the identification of nine and eight candidate compounds from each library, respectively. The Transcreener ADP2 Assay was used to screen 1600 compounds from the ChemBridge diversity library for CD73 inhibitors and identified 14 potential candidates. HTS-compatible assays for ectonucleotidase activity may allow identification of purinergic signaling pathway inhibitors important for tumor-specific immune responses during tumor pathogenesis.

Insights on human evolution: an analysis of Alu insertion polymorphisms.

We analyzed the genetic profile of 563 individuals from 12 geographically targeted human populations from Europe, Asia and Africa using 27 human-specific polymorphic Alu insertions. Phylogenetic analyses indicated a clear correspondence between genetic profiles and historical patterns of gene flow and genetic drift. Sub-Saharan African populations (Benin, Cameroon, Kenya and Rwanda) formed a visibly differentiated cluster, indicating the role of the Sahara desert as a strong natural barrier to gene flow. Moreover, a higher than expected genetic affinity between populations from Europe, North Africa and Asia was detected, probably reflecting the homogenizing effects of bidirectional migratory processes between Eurasia and North Africa during the Plio-Pleistocene and Neolithic periods or the insensitivity of these markers in discriminating between these groups. The Ami aborigines of Formosa present a distinctive degree of genetic uniqueness from all the other groups, consistent with a pattern of isolation by distance, small population size and, accordingly, substantial genetic drift. We further tested all 27 Alu loci for their potential usefulness as ancestry informative markers (AIMs). On the basis of differences between weighted allelic frequencies (delta-values) and F(ST) values, we propose that 11 of the 27 Alu elements could be useful as part of the current AIM panels to assess phylogenetic relationships.

Characterization and optimization of a red-shifted fluorescence polarization ADP detection assay.

ATP depletion and ADP formation are generic detection methods used for the identification of kinase and other ATP-utilizing enzyme inhibitors in high-throughput screening campaigns. However, the most widely used nucleotide detection approaches require high ATP consumption rates or involve the use of coupling enzymes, which can complicate the selection of lead compounds. As an alternative, we have developed the Transcreener (BellBrook Labs, Madison, WI) platform, which relies on the direct immunodetection of nucleotides. Here we describe the development of antibodies with >100-fold selectivity for ADP versus ATP, which enable robust detection of initial velocity rates (Z' > 0.7 at 10% substrate consumption) at ATP concentrations ranging from 0.1 microM to 1,000 microM in a competitive fluorescence polarization (FP) immunoassay. Competitive binding experiments indicate similar affinities for other nucleotide diphosphates, including 2' -deoxy ADP, GDP, and UDP. The antibody-tracer complex and the red-shifted, ratiometric FP signal are stable for at least 24 h at room temperature, providing suitable conditions for high-throughput screening. A method for calculating a kinase ATP Km with this FP immunoassay is also presented. The Transcreener ADP assay provides a simple, generic assay platform for inhibitor screening and selectivity profiling that can be used for any ADP-generating enzyme.

Evaluating PI3 kinase isoforms using Transcreener ADP assays.

Development of drugs targeting lipid kinases has been delayed by the lack of robust screening assays. Methods are needed that can accommodate the presentation of different acceptor substrates in the optimal lipid environment. The Transcreener ADP Assay relies on homogeneous immunodetection of adenosine diphosphate (ADP), using either fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET) as a signal output. Detection of ADP--the invariant product of all kinase reactions--provides complete flexibility for varying lipid substrate parameters. The authors used this assay to optimize dispersal methods for C8 and C16 phosphatidylinositol 4,5 bisphosphate substrates and to assess the effects of chain length on the activity and inhibition of phosphoinositide-3-kinase (PI3K) isoforms. The nonphysiological C8 substrate supported the highest activity. Known inhibitors were profiled using both the FP- and TR-FRET-based assays, and there was excellent concordance (r(2)=0.93) in the IC(50) values. The overall rank order of inhibitors was the same using the C8 and C16 substrates, except for minor deviations. Adenosine triphosphate (ATP) hydrolysis in the absence of substrate was detected with the PI3Kalpha isoform, and inhibitors affected PI3Kalpha intrinsic ATP hydrolysis activity similarly to lipid phosphorylation.

CT diagnosis of isolated anomalous origin of the RCA arising from the main pulmonary artery.

Anomalies of the coronary arteries can be benign or life threatening. The prevalence of these anomalies is reported to be approximately 0.3% to 1%, however, this may be an underestimation as conventional angiography may not allow correct identification of these abnormalities. Morphologic variations can arise in the origin, course, or termination of coronary arteries. These variations may be related to other congenital abnormalities or isolated. Some anomalies can lead to myocardial ischemia and have been implicated in episodes of sudden death in young adults. Noninvasive imaging modalities such as multidetector computerized tomography provide an efficient method of evaluating coronary artery anomalies by allowing more complete visualization of chest, mediastinal, and vascular structures. We describe a case of anomalous origin of the right coronary artery originating from the main pulmonary trunk demonstrated by computerized tomography in a patient with exertional ischemia. In the past, this diagnosis has only been made by angiography, echocardiography, and at autopsy.

Early readmission of low-risk patients after coronary surgery.

BACKGROUND: Early readmission after coronary artery bypass grafting (CABG) is an expensive adverse outcome. Although the perioperative experience of high-risk CABG patients has been studied extensively, little attention has been paid to low-risk CABG patients. The primary goal of this study was to identify the preoperative characteristics and to define risk predictors of readmission and preventive factors for readmission in low-risk isolated-CABG patients. METHODS: We identified 2157 patients who underwent CABG between January 2000 and December 2005 at Washington Hospital Center, Washington, DC, and defined as low risk patients who had a Parsonnet bedside risk score lower than the 25th percentile. Patients who were rehospitalized within 30 days after surgery were compared with those who were not rehospitalized during this period. RESULTS: The overall readmission rate for this study cohort was 6.3%. Compared with non-readmitted patients, early-readmitted patients were more likely to have diabetes mellitus (27.94% versus 20.88%, P = .05) and less likely to have hypertension (42.65% versus 51.36%, P = .05). Blood product transfusion (P < .01), postoperative length of intensive care unit stay (P = .01), and length of hospital stay (P = .05) were all significantly increased in the readmitted patients. The use of beta-blockers (P = .03) and angiotensin-converting enzyme inhibitors (P = .04) was significantly lower at discharge in this group of patients; however, multivariate regression analysis demonstrated diabetes (odds ratio, 1.59; 95% confidence interval, 1.08-2.42) to be the only independent predictor of early readmission. CONCLUSIONS: For low-risk CABG patients, diabetes mellitus is the risk predictor of early readmission. Early discharge was not associated with early readmission.

High-Throughput Assay for RhoGEFs Based on the Transcreener® GDP Assay.

We describe a high-throughput screening (HTS)-compatible method for detecting GTPase exchange factor (GEF) activity based on stimulation of GDP formation by Rho GTPases. The method is based on the fact that GDP dissociation is the rate-limiting step in the Rho GTPase catalytic cycle, so by accelerating its release a GEF causes an increase in the steady-state rate of GDP formation. The Transcreener® GDP GTPase Assay, a fluorescence polarization immunoassay (FPIA), is used to detect GDP formation in a homogeneous format.

A High-Throughput Method for Measuring Drug Residence Time Using the Transcreener ADP Assay.

Analysis of drug-target residence times during drug development can result in improved efficacy, increased therapeutic window, and reduced side effects. Residence time can be estimated as the reciprocal of the dissociation rate ( koff) of an inhibitor from its target. The traditional methods for measuring koff require synthesis of labeled ligands or low-throughput label-free methods. To provide an alternative that is better suited to an automated high-throughput screening (HTS) environment, we adapted a classic "jump dilution" catalytic assay method for determination of koff values for kinase inhibitor drugs. We used the Transcreener ADP2 Kinase assay as a universal, homogenous method to monitor the recovery of kinase activity as the drugs dissociated from preformed inhibitor-kinase complexes. We measured residence times for several drugs that bind the epidermal growth factor receptor (EGFR), ABL1, and Aurora kinases and found that the rank ordering of inhibitor koff values correlated with literature values determined using ligand binding assays. Moreover, very similar results were obtained using the Transcreener assay with fluorescence polarization (FP), fluorescence intensity (FI), and time-resolved Förster resonance energy transfer (TR-FRET) detection modes. This HTS-compatible, generic assay method should facilitate the use of residence time as a parameter for compound prioritization and optimization early in kinase drug discovery programs.

Analysis of ligand-dependent recruitment of coactivator peptides to estrogen receptor using fluorescence polarization.

Ligand-dependent recruitment of coactivators to estrogen receptor (ER) plays an important role in transcriptional activation of target genes. Agonist-bound ER has been shown to adopt a favorable conformation for interaction with the LXXLL motifs of the coactivator proteins. To further examine the affinity and ligand dependence of the ER-coactivator interaction, several fluorescently tagged short peptides bearing an LXXLL motif (LXXLL peptide) from either natural coactivator sequences or random phage display sequences were used with purified ERalpha or ERbeta in an in vitro high-throughput fluorescence polarization assay. In the presence of saturating amounts of ligand, several LXXLL peptides bound to ERalpha and ERbeta with affinity ranging from 20-500 nm. The random phage display LXXLL peptides exhibited a higher affinity for ER than the natural single-LXXLL coactivator sequences tested. These studies indicated that ER agonists, such as 17beta-estradiol or estrone, promoted the interaction of ER with the coactivator peptides, whereas antagonists such as 4-hydroxytamoxifen or ICI-182,780 did not. Different LXXLL peptides demonstrated different affinities for ER depending on which ligand was bound to the receptor, suggesting that the peptides were recognizing different receptor conformations. Using the information obtained from direct measurement of the affinity of the ER-LXXLL peptide interaction, the dose dependency (EC50) of various ligands to either promote or disrupt this interaction was also determined. Interaction of ER with the LXXLL peptide was observed with ligands such as 17beta-estradiol, estriol, estrone, and genistein but not with ICI-182,780, 4-hydroxytamoxifen, clomiphene, or tamoxifen, resulting in distinct EC50 values for each ligand and correlating well with the ligand biological function as an agonist or antagonist. Ligand-dependent recruitment of the LXXLL peptide to ERbeta was observed in the presence of the ERbeta-selective agonist diarylpropionitrile, but not the ERalpha-selective ligand propyl pyrazole triol. This assay could be used to classify unknown ligands as agonists, antagonists, or partial modulators, based on either the receptor-coactivator peptide affinities or the dose dependency of this interaction in comparison with known compounds.