Computational methods to automate the initial interpretation of lower extremity arterial Doppler and duplex carotid ultrasound studies.

BACKGROUND: Lower extremity arterial Doppler (LEAD) and duplex carotid ultrasound studies are used for the initial evaluation of peripheral arterial disease and carotid stenosis. However, intra- and inter-laboratory variability exists between interpreters, and other interpreter responsibilities can delay the timeliness of the report. To address these deficits, we examined whether machine learning algorithms could be used to classify these Doppler ultrasound studies. METHODS: We developed a hierarchical deep learning model to classify aortoiliac, femoropopliteal, and trifurcation disease in LEAD ultrasound studies and a random forest machine learning algorithm to classify the amount of carotid stenosis from duplex carotid ultrasound studies using experienced physician interpretation in an active, credentialed vascular laboratory as the reference standard. Waveforms, pressures, flow velocities, and the presence of plaque were input into a hierarchal neural network. Artificial intelligence was developed to automate the interpretation of these LEAD and carotid duplex ultrasound studies. Statistical analysis was performed using the confusion matrix. RESULTS: We extracted 5761 LEAD ultrasound studies from 2015 to 2017 and 18,650 duplex carotid ultrasound studies from 2016 to 2018 from the Indiana University Health system. The results showed the ability of artificial intelligence algorithms and method, with 97.0% accuracy for predicting normal cases, 88.2% accuracy for aortoiliac disease, 90.1% accuracy for femoropopliteal disease, and 90.5% accuracy for trifurcation disease. For internal carotid artery stenosis, the accuracy was 99.2% for predicting 0% to 49% stenosis, 100% for predicting 50% to 69% stenosis, 100% for predicting >70% stenosis, and 100% for predicting occlusion. For common carotid artery stenosis, the accuracy was 99.9% for predicting 0% to 49% stenosis, 100% for predicting 50% to 99% stenosis, and 100% for predicting occlusion. CONCLUSIONS: The machine learning models using LEAD data, with the collected blood pressure and waveform data, and duplex carotid ultrasound data with the flow velocities and the presence of plaque, showed that novel machine learning models are reliable in differentiating normal from diseased arterial systems and accurate in classifying the extent of vascular disease.

Parker's Solar Wind Model for a Polytropic Gas.

Parker's hydrodynamic isothermal solar wind model is extended to apply for a more realistic polytropic gas flow that can be caused by a variable extended heating of the corona. A compatible theoretical formulation is given and detailed numerical and systematic asymptotic theoretical considerations are presented. The polytropic conditions favor an enhanced conversion of thermal energy in the solar wind into kinetic energy of the outward flow and are hence shown to enhance the acceleration of the solar wind, thus indicating a quicker loss of the solar angular momentum.

Anxiety-associated alternative polyadenylation of the serotonin transporter mRNA confers translational regulation by hnRNPK.

The serotonin transporter (SERT) is a major regulator of serotonergic neurotransmission and anxiety-related behaviors. SERT is expressed in two alternative polyadenylation forms that differ by an evolutionarily conserved element in the 3' untranslated region of its mRNA. Expression of SERT mRNA containing the distal polyadenylation element is associated with decreased anxiety-related behaviors in mice and humans, suggesting that this element has behaviorally relevant modulatory effects on SERT expression. We have identified heterogeneous nuclear ribonucleoprotein K (hnRNPK), a protein known to integrate multiple signal transduction pathways with gene expression, as a SERT distal polyadenylation element binding protein. This interaction is functionally meaningful because genetic manipulation of hnRNPK alters expression of the SERT protein. Furthermore, the trophic factor S100ß induces Src-family kinase-mediated tyrosine phosphorylation of hnRNPK and increased SERT expression. These results identify a previously unknown mechanism of regulated SERT expression and provide a putative mechanism by which the SERT distal polyadenylation element modulates anxiety-related behaviors.

Prevalence and associations of incidental nonvenous duplex findings discovered during lower extremity venous imaging.

OBJECTIVE: Venous duplex imaging defines venous pathology (VP). Unexpected clinically relevant findings are also found but rarely mentioned in the literature. This study aims to define the prevalence of ancillary findings (nonvenous duplex) by study type and venous outcome and subgroup associations with primary study indication and risk factors. METHODS: Our vascular laboratory database was queried for lower extremity venous duplex studies with comments regarding ancillary findings and associated patient demographics, primary study indication, associated conditions, and venous study outcome. RESULTS: There were 52,215 venous studies performed, 48,425 to evaluate for venous occlusion (acute/chronic) and 3790 for venous reflux. Of these studies, 15,810 found VP and 36,405 found no venous disease. There were 875 studies with venous disease that had ancillary duplex findings (5.5%) noted as 559 (3.5%) with prominent lymph node(s) (LN), 179 (1.1%) Baker's cyst (BC), 44 (0.3%) hematoma/mass (HM), 31 (0.2%) arterial aneurysm, and 16 (0.1%) arterial occlusion. There were 3130 studies free of VP with ancillary findings (8.6%) noted as 2258 (6.2%) prominent LN(s), 626 (1.7%) BC, 156 (0.4%) HM, 37 (0.1%) arterial aneurysm, and 22 (0.06%) arterial occlusion. The overall prevalence of ancillary findings was 8.62%. Analysis demonstrated statistically more ancillary findings in venous occlusion (odds ratio [OR], 1.25) studies, which was the largest group at 13 to 1. Studies free of venous disease had more ancillary findings (P < .001) with an OR of 1.88 and similar results were noted for LN(s), BC, and hematoma. Studies with VP favored a finding of aneurysm (OR, 0.52). Subgroup analyses demonstrated that those with prominent LN(s) were statistically older and male and BC statistically older in those with coexistent venous disease. BC subgroup analysis showed that studies free of venous disease were 2.5 times more likely to report pain as the primary study indication (P < .0001). In general, within ancillary subgroups, leg symptoms were statistically more prominent on the side with ancillary pathology and free of venous disease. CONCLUSIONS: Ancillary findings are not uncommon and are more common in studies found free of VP. The most common are LNs, BC and HM and, within subgroups, significant leg symptoms favors the presence of ancillary findings without coexisting venous disease. Ancillary findings should be an integral part of a quality report.

Transcription cofactor GRIP1 differentially affects myeloid cell-driven neuroinflammation and response to IFN-ß therapy.

Macrophages (MФ) and microglia (MG) are critical in the pathogenesis of multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). Glucocorticoids (GCs) and interferon ß (IFN-ß) are frontline treatments for MS, and disrupting each pathway in mice aggravates EAE. Glucocorticoid receptor-interacting protein 1 (GRIP1) facilitates both GR and type I IFN transcriptional actions; hence, we evaluated the role of GRIP1 in neuroinflammation. Surprisingly, myeloid cell-specific loss of GRIP1 dramatically reduced EAE severity, immune cell infiltration of the CNS, and MG activation and demyelination specifically during the neuroinflammatory phase of the disease, yet also blunted therapeutic properties of IFN-ß. MФ/MG transcriptome analyses at the bulk and single-cell levels revealed that GRIP1 deletion attenuated nuclear receptor, inflammatory and, interestingly, type I IFN pathways and promoted the persistence of a homeostatic MG signature. Together, these results uncover the multifaceted function of type I IFN in MS/EAE pathogenesis and therapy, and an unexpectedly permissive role of myeloid cell GRIP1 in neuroinflammation.

Clinical and laboratory characteristics of patients with novel coronavirus disease-2019 infection and deep venous thrombosis.

OBJECTIVE: Early reports suggest that patients with novel coronavirus disease-2019 (COVID-19) infection carry a significant risk of altered coagulation with an increased risk for venous thromboembolic events. This report investigates the relationship of significant COVID-19 infection and deep venous thrombosis (DVT) as reflected in the patient clinical and laboratory characteristics. METHODS: We reviewed the demographics, clinical presentation, laboratory and radiologic evaluations, results of venous duplex imaging and mortality of COVID-19-positive patients (18-89 years) admitted to the Indiana University Academic Health Center. Using oxygen saturation, radiologic findings, and need for advanced respiratory therapies, patients were classified into mild, moderate, or severe categories of COVID-19 infection. A descriptive analysis was performed using univariate and bivariate Fisher's exact and Wilcoxon rank-sum tests to examine the distribution of patient characteristics and compare the DVT outcomes. A multivariable logistic regression model was used to estimate the adjusted odds ratio of experiencing DVT and a receiver operating curve analysis to identify the optimal cutoff for d-dimer to predict DVT in this COVID-19 cohort. Time to the diagnosis of DVT from admission was analyzed using log-rank test and Kaplan-Meier plots. RESULTS: Our study included 71 unique COVID-19-positive patients (mean age, 61 years) categorized as having 3% mild, 14% moderate, and 83% severe infection and evaluated with 107 venous duplex studies. DVT was identified in 47.8% of patients (37% of examinations) at an average of 5.9 days after admission. Patients with DVT were predominantly male (67%; P = .032) with proximal venous involvement (29% upper and 39% in the lower extremities with 55% of the latter demonstrating bilateral involvement). Patients with DVT had a significantly higher mean d-dimer of 5447 ± 7032 ng/mL (P = .0101), and alkaline phosphatase of 110 IU/L (P = .0095) than those without DVT. On multivariable analysis, elevated d-dimer (P = .038) and alkaline phosphatase (P = .021) were associated with risk for DVT, whereas age, sex, elevated C-reactive protein, and ferritin levels were not. A receiver operating curve analysis suggests an optimal d-dimer value of 2450 ng/mL cutoff with 70% sensitivity, 59.5% specificity, and 61% positive predictive value, and 68.8% negative predictive value. CONCLUSIONS: This study suggests that males with severe COVID-19 infection requiring hospitalization are at highest risk for developing DVT. Elevated d-dimers and alkaline phosphatase along with our multivariable model can alert the clinician to the increased risk of DVT requiring early evaluation and aggressive treatment.

Assessment of the duration of protection in Campylobacter jejuni experimental infection in humans.

A human Campylobacter jejuni infection model provided controlled exposure to assess vaccine efficacy and investigate protective immunity for this important diarrheal pathogen. A well-characterized outbreak strain, C. jejuni 81-176, was investigated using a volunteer experimental infection model to evaluate the dose range and duration of protection. Healthy Campylobacter-seronegative adults received C. jejuni strain 81-176 via oral inoculation of 10(5), 10(7), or 10(9) CFU (5 adults/dose), which was followed by clinical and immunological monitoring. Based on dose range clinical outcomes, the 10(9)-CFU dose (n = 31) was used to assess homologous protection at 28 to 49 days (short-term veterans [STV]; n = 8) or 1 year (long-term veterans [LTV]; n = 7) after primary infection. An illness dose effect was observed for naïve subjects (with lower doses, 40 to 60% of the subjects were ill; with the 10(9)-CFU dose, 92% of the subjects were ill) along with complete protection for the STV group and attenuated illness for the LTV group (57%). Partial resistance to colonization was seen in STV (25% of the subjects were not infected; 3-log-lower maximum excretion level). Systemic and mucosal immune responses were robust in naïve subjects irrespective of the dose or the severity of illness. In contrast, in STV there was a lack of circulating antibody-secreting cells (ASC), reflecting the local mucosal effector responses. LTV exhibited comparable ASC responses to primary infection, and anamnestic fecal IgA responses likely contributed to self-resolving illness prior to antibiotic treatment. Campylobacter antigen-dependent production of gamma interferon by peripheral blood mononuclear cells was strongly associated with protection from illness, supporting the hypothesis that TH1 polarization has a primary role in acquired immunity to C. jejuni. This study revealed a C. jejuni dose-related increase in campylobacteriosis rates, evidence of complete short-term protection that waned with time, and immune response patterns associated with protection.

Negative elongation factor complex enables macrophage inflammatory responses by controlling anti-inflammatory gene expression.

Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation, whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report that widespread promoter-proximal RNA polymerase II pausing in resting macrophages is marked by co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation.

Dual Cross-Linking Chromatin Immunoprecipitation Protocol for Next-Generation Sequencing (ChIPseq) in Macrophages.

Macrophages arise from distinct progenitor cell populations throughout development and are one of the most diverse cell types, capable of performing discrete functions, undergoing distinct modes of activation, and infiltrating or residing in numerous niches in the body. In adapting to their environments, macrophages display high levels of plasticity which is associated with profound epigenomic and transcriptional changes. Understanding these changes has been greatly facilitated by the next-generation sequencing (NGS)-based approaches such as RNAseq and chromatin immunoprecipitation (ChIP)seq. Despite the recent advances, obtaining quality ChIPseq data in macrophages for endogenous factors and especially coregulators recruited to DNA indirectly has proved to be extremely challenging. Here, we describe a dual crosslinking protocol for ChIPseq in macrophages that we developed for difficult-to-ChIP transcription factors, coregulators, and their posttranslational modifications. Further, we provide guidance on crucial optimization steps throughout this protocol. Although our experience has been predominantly in murine and human macrophages, we believe our protocols can be modified and optimized to study signal-induced epigenomic changes in any cell type of choice.

Gene-specific mechanisms direct glucocorticoid-receptor-driven repression of inflammatory response genes in macrophages.

The glucocorticoid receptor (GR) potently represses macrophage-elicited inflammation, however, the underlying mechanisms remain obscure. Our genome-wide analysis in mouse macrophages reveals that pro-inflammatory paused genes, activated via global negative elongation factor (NELF) dissociation and RNA Polymerase (Pol)2 release from early elongation arrest, and non-paused genes, induced by de novo Pol2 recruitment, are equally susceptible to acute glucocorticoid repression. Moreover, in both cases the dominant mechanism involves rapid GR tethering to p65 at NF-kB-binding sites. Yet, specifically at paused genes, GR activation triggers widespread promoter accumulation of NELF, with myeloid cell-specific NELF deletion conferring glucocorticoid resistance. Conversely, at non-paused genes, GR attenuates the recruitment of p300 and histone acetylation, leading to a failure to assemble BRD4 and Mediator at promoters and enhancers, ultimately blocking Pol2 initiation. Thus, GR displays no preference for a specific pro-inflammatory gene class; however, it effects repression by targeting distinct temporal events and components of transcriptional machinery.

Glucocorticoid-induced phosphorylation by CDK9 modulates the coactivator functions of transcriptional cofactor GRIP1 in macrophages.

The glucocorticoid (GC) receptor (GR) suppresses inflammation by activating anti-inflammatory and repressing pro-inflammatory genes. GR-interacting protein-1 (GRIP1) is a GR corepressor in macrophages, however, whether GRIP1 mediates GR-activated transcription, and what dictates its coactivator versus corepressor properties is unknown. Here we report that GRIP1 loss in macrophages attenuates glucocorticoid induction of several anti-inflammatory targets, and that GC treatment of quiescent macrophages globally directs GRIP1 toward GR binding sites dominated by palindromic GC response elements (GRE), suggesting a non-redundant GRIP1 function as a GR coactivator. Interestingly, GRIP1 is phosphorylated at an N-terminal serine cluster by cyclin-dependent kinase-9 (CDK9), which is recruited into GC-induced GR:GRIP1:CDK9 hetero-complexes, producing distinct GRE-specific GRIP1 phospho-isoforms. Phosphorylation potentiates GRIP1 coactivator but, remarkably, not its corepressor properties. Consistently, phospho-GRIP1 and CDK9 are not detected at GR transrepression sites near pro-inflammatory genes. Thus, GR restricts actions of its own coregulator via CDK9-mediated phosphorylation to a subset of anti-inflammatory genes.

There goes the neighborhood: Assembly of transcriptional complexes during the regulation of metabolism and inflammation by the glucocorticoid receptor.

Glucocorticoids (GCs), as ligands for the glucocorticoid receptor (GR), represent one of the most effective and frequently used classes of drugs for anti-inflammatory and immunosuppressive therapy. In addition, its role in physiological and pathophysiological processes makes the GR an important research target. The past decades have yielded a wealth of insight into the physiological and pharmacological effects of GCs. Today's era of next generation sequencing techniques is now beginning to elucidate the molecular and genomic circuits underlying GR's cell type-specific actions. This review focuses on the concepts and insights gained from recent studies in two of the most important tissues for GC action: the liver (mediating GR's metabolic effects) and macrophages (as the main target of anti-inflammatory GC therapy). We summarize results obtained from transgenic mouse models, molecular and genome-wide studies to illustrate GR's complex interactions with DNA, chromatin, co-regulators and other transcription factors. Characterizing the cell type-specific transcriptional complexes assembled around GR will pave the road for the development of new anti-inflammatory and metabolic therapies in the future.

Minireview: nuclear receptor coregulators of the p160 family: insights into inflammation and metabolism.

Nuclear receptor coactivators (NCOAs) are multifunctional transcriptional coregulators for a growing number of signal-activated transcription factors. The members of the p160 family (NCOA1/2/3) are increasingly recognized as essential and nonredundant players in a number of physiological processes. In particular, accumulating evidence points to the pivotal roles that these coregulators play in inflammatory and metabolic pathways, both under homeostasis and in disease. Given that chronic inflammation of metabolic tissues ("metainflammation") is a driving force for the widespread epidemic of obesity, insulin resistance, cardiovascular disease, and associated comorbidities, deciphering the role of NCOAs in "normal" vs "pathological" inflammation and in metabolic processes is indeed a subject of extreme biomedical importance. Here, we review the evolving and, at times, contradictory, literature on the pleiotropic functions of NCOA1/2/3 in inflammation and metabolism as related to nuclear receptor actions and beyond. We then briefly discuss the potential utility of NCOAs as predictive markers for disease and/or possible therapeutic targets once a better understanding of their molecular and physiological actions is achieved.

Cataract surgery at the Vancouver Eye Care Centre: do patient indications meet provincial clinical practice guidelines?

BACKGROUND: Cataract surgery, already the most common elective surgical procedure among patients over the age of 65 in North America, is rising in volume, because the population is aging while at the same time holding growing expectations of continued vitality. In meeting the need, there is a public interest in ensuring that cataract surgery is used appropriately. The purpose of this paper was to investigate the indication for cataract surgery performed in adults at a large tertiary care centre in Vancouver and to determine the extent to which patient criteria met the guidelines of the College of Physicians & Surgeons of British Columbia. METHODS: Patients attending the centre were enrolled in the cataract outcome assessment program through systematic sampling that included every patient with a personal health card number ending in 5 or 8 (20% of patients). There were no patient exclusion criteria. Pre-, intra- and postoperative clinical information was collected from patients' medical records. Patients were also sent a pre- and postoperative visual function and quality-of-life questionnaire. RESULTS: A total of 1098 cataract surgery procedures were performed in 896 patients from March 1999 to December 2000. The average age of the patients was 72.9 years; most (69.5%) were women. Just over half of the procedures (620 [56.6%]) were performed as first-eye surgery. In 191 cases (17.9%) the preoperative visual acuity was 20/40 or better in the affected eye. In most cases (82.1%) visual acuity of 20/50 or worse was the indication for surgery. In 89 cases (8.3%) there was not enough information in the chart to justify the surgery. INTERPRETATION: By examining standard information provided in patients' medical records, we were able to determine the indication for cataract surgery in 92% of cases. The most common indication was poor visual acuity.

Cataract surgical outcome at the Vancouver Eye Care Centre: can it be predicted using current data?

BACKGROUND: Patients selected for cataract surgery have an increasingly wider range of coexisting ocular and systemic diseases. The aim of this study was to determine whether preoperative patient characteristics can assist surgeons in identifying patients who are at increased risk of having little or no improvement in visual outcome after cataract surgery. METHODS: We prospectively studied a randomly selected subset (20%) of patients with cataract undergoing cataract extraction at a tertiary centre in Vancouver. There were no patient exclusion criteria. Pre-, intra- and postoperative clinical data were collected from medical records. RESULTS: We studied 1329 cataract surgical procedures from March 1999 to December 2000. Of the 1329, 851 had complete pre- and postoperative visual acuity data. The mean age of the 851 patients was 73.2 (standard deviation 10.6) years, 575 (67.6%) were women, and 472 procedures (55.5%) were first-eye procedures. Preoperatively, the best-corrected visual acuity was 20/40 or better in 146 eyes (17.2%), 20/50 in 173 (20.3%), 20/60 to 20/70 in 248 (29.1%), and 20/80 or worse in 284 (33.4%). Almost half of the patients (419 [49.2%]) had at least one coexisting eye disease in the operated eye. At about 3 months postoperatively, the best-corrected visual acuity had improved in 786 eyes (92.4%), remained the same in 42 (4.9%) and had worsened in 23 (2.7%). Of the 23 eyes with worse visual acuity, half had preoperative visual acuity of 20/40 or better; most of the remaining eyes had preoperative vision of 20/50 to 20/70. Of the 42 eyes with unchanged vision, 22 (52%) had preoperative visual acuity worse than 20/100; the remaining eyes were distributed across the visual acuity range. An increase of 1 year of age increased the odds of having a poor visual acuity outcome by 2% to 4%. Worse preoperative visual acuity predicted an increased likelihood of a poor outcome; this effect increased sharply for eyes with preoperative visual acuity of 20/80 or worse. After adjustment for age and preoperative visual acuity, patients with three or more coexisting eye diseases in the operated eye were 10 to 24 times more likely to have the worst postoperative visual acuity. INTERPRETATION: The presence and number of coexisting ocular diseases, increased age and poor preoperative visual acuity predicted an unchanged or worse visual acuity after cataract surgery.

Prevention of disease in ferrets fed an inactivated whole cell Campylobacter jejuni vaccine.

Ferrets were used to demonstrate the potential of a killed whole cell vaccine prepared from Campylobacter jejuni to protect against disease. C. jejuni strain 81-176 was grown in BHI broth, formalin-fixed, and resuspended in PBS to a concentration of 10(10) cells per ml. This vaccine (CWC) or live organisms were delivered orally with a nasogastric tube into anesthetized animals treated to reduce gastric acidity and intestinal motility. When 5x10(10) CFU of the vaccine strain (Lior serotype 5) or one of two other serotypes, CGL-7 (Lior 4) or BT44 (Lior 9), was used to challenge the ferrets, all of the animals developed a mucoid diarrhea. If the animals had been challenged with 5x10(9) CFU of the homologous strain 1 month before challenge with 10(10) CFU, 80-100% protection against disease was seen. This protection was also obtained after an initial exposure to the 81-176 strain followed by challenge with either of the heterologous strains. CWC was used to see if protection demonstrated with the live organisms could be produced with the non-living preparation. When 10(9) cells of CWC was given as two doses 7 days apart with or without 25mug of a coadministered mucosal adjuvant, LT(R192G), only 40-60% of the animals were protected. If the regimen was changed to four doses given 48h apart, 80% of the animals were free of diarrhea after subsequent challenge. Increasing the number of cells in the four dose regimen to 10(10) cells did not improve protection. Animals given four doses of 10(10) cells combined with LT(R192G) were subsequently challenged with 10(10) cells of the homologous strain or the heterologous strain CGL-7. The CWC protected against both strains. Serum IgG antibody titers determined by ELISA showed little increase following the CWC four dose vaccination regimen, compared to animals given one dose of the live organism. On subsequent challenge, however, both CWC vaccinated and live-challenged ferrets showed comparable antibody titer increases above those obtained following the initial challenge or vaccination. Western blots were used to show that the immunodominant antigen in vaccinated animals was a 45kDa protein, while in ferrets challenged with live organisms the immunodominant antigen was a 62kDa protein. These data show that the CWC can be used to protect against disease caused by Campylobacter. They also show that protection and serum IgG responses do not depend upon the use of the mucosal adjuvant and that cross protection among some of the major serotypes of Campylobacter responsible for human disease is possible.