COVID-19 pandemic-related changes in wellness behavior among older Americans.

BACKGROUND: COVID-19 has taken its toll on citizens in all 50 states of the United States. The United States (U.S.) leads the world with 30,291,863 confirmed reported cases and 549,664 deaths as of March 29, 2021 compared to globally confirmed cases at 127,442,926 and 2,787,915 deaths as of March 29, 2021. The U.S. federal government primarily left the response to the virus to individual states, and each implemented varying measures designed to protect health of citizens and the state's economic well-being. Unintended consequences of the virus and measures to stop its spread may include decreased physical activity and exercise, shifting access and consumption of food, and lower quality-of-life. Therefore, our primary goal was to quantify the impact of COVID-19 on health and well-being by measuring changes in physical activity, mental health-quality of life, food security and nutrition in adults ages 40 and older. We believed shifts in health behaviors would be more prevalent in minorities, less educated, lower socio-economic status, older adults, and those with underlying health conditions, so a secondary goal was to determine the impact of COVID-19 on these sub-populations. METHODS: We conducted an online survey with 9969 adults 40 years and older between 9 August and 15 September 2020 in urban areas across the four U.S. census regions. The survey included questions about demographic variables, pre-existing health conditions, physical activity, access to food, quality-of-life, and nutritional food status and asked participants to respond with information from pre-pandemic and pandemic conditions. We used paired-sample t-tests to detect changes in variables after the start of the pandemic and Cohen's d to determine effect sizes. RESULTS: Our main findings showed a decrease in physical activity since the onset of COVID-19 for minorities and non-minorities. Food security also slightly increased for minorities during the pandemic, but we found no other changes in food security, quality-of-life indicators, or nutritional status of those who responded to this survey. CONCLUSIONS: It is concerning that physical activity declined. Such activity helps maintain physical and mental health, and it is also an important time to socialize for many older adults. In many ways, our data indicate that the older adult population in U.S. cities may be more resilient than expected during the pandemic. However, the pandemic could have negative impacts that we did not detect, either due to the survey instrument or the timing of our survey, so the health and well-being of older adults should continue to be monitored in order to mitigate potential negative impacts.

The ACUTE (Ambulance CPAP: Use, Treatment effect and economics) feasibility study: a pilot randomised controlled trial of prehospital CPAP for acute respiratory failure.

BACKGROUND: Acute respiratory failure (ARF) is a common and life-threatening medical emergency. Standard prehospital management involves controlled oxygen therapy and disease-specific ancillary treatments. Continuous positive airway pressure (CPAP) is a potentially beneficial alternative treatment that could be delivered by emergency medical services. However, it is uncertain whether this treatment could work effectively in United Kingdom National Health Service (NHS) ambulance services and if it represents value for money. METHODS: An individual patient randomised controlled external pilot trial will be conducted comparing prehospital CPAP to standard oxygen therapy for ARF. Adults presenting to ambulance service clinicians will be eligible if they have respiratory distress with peripheral oxygen saturation below British Thoracic Society (BTS) target levels, despite titrated supplemental oxygen. Enrolled patients will be allocated (1:1 simple randomisation) to prehospital CPAP (O_two system) or standard oxygen therapy using identical sealed boxes. Feasibility outcomes will include incidence of recruited eligible patients, number of erroneously recruited patients and proportion of cases adhering to allocation schedule and treatment, followed up at 30 days and with complete data collection. Effectiveness outcomes will comprise survival at 30 days (definitive trial primary end point), endotracheal intubation, admission to critical care, length of hospital stay, visual analogue scale (VAS) dyspnoea score, EQ-5D-5L and health care resource use at 30 days. The cost-effectiveness of CPAP, and of conducting a definitive trial, will be evaluated by updating an existing economic model. The trial aims to recruit 120 patients over 12 months from four regional ambulance hubs within the West Midlands Ambulance Service (WMAS). This sample size will allow estimation of feasibility outcomes with a precision of < 5%. Feasibility and effectiveness outcomes will be reported descriptively for the whole trial population, and each trial arm, together with their 95% confidence intervals. DISCUSSION: This study will determine if it is feasible, acceptable and cost-effective to undertake a full-scale trial comparing CPAP and standard oxygen treatment, delivered by ambulance service clinicians for ARF. This will inform NHS practice and prevent inappropriate prehospital CPAP adoption on the basis of limited evidence and at a potentially substantial cost. TRIAL REGISTRATION: ISRCTN12048261. Registered on 30 August 2017. http://www.isrctn.com/ISRCTN12048261.

A next-generation sequencing-based assay for minimal residual disease assessment in AML patients with FLT3-ITD mutations.

Internal tandem duplications in fms-like tyrosine kinase 3 (FLT3-ITDs) are common in acute myeloid leukemia (AML) and confer a poor prognosis. A sensitive and specific assay for the detection of minimal residual disease (MRD) in FLT3-ITD mutated AML could guide therapy decisions. Existing assays for MRD in FLT3-ITD AML have not been particularly useful because of limited sensitivity. We developed a sensitive and specific MRD assay for FLT3-ITD mutations using next-generation sequencing. The initial validation of this assay was performed by spiking fixed amounts of mutant DNA into wild-type DNA to establish a sensitivity of detection equivalent to ≥1 FLT3-ITD-containing cell in 10 000, with a minimum input of 100 000 cell equivalents of DNA. We subsequently validated the assay in bone marrow samples from patients with FLT3-ITD AML in remission. Finally, we analyzed bone marrow samples from 80 patients with FLT3-ITD relapsed/refractory AML participating in a trial of a novel FLT3 inhibitor, gilteritinib, and demonstrated a relationship between the mutation burden, as detected by the assay, and overall survival. This novel MRD assay is specific and 2 orders of magnitude more sensitive than currently available polymerase chain reaction- or next-generation sequencing-based FLT3-ITD assays. The assay is being prospectively validated in ongoing randomized clinical trials.

Flight behaviour of honey bee (Apis mellifera) workers is altered by initial infections of the fungal parasite Nosema apis.

Honey bees (Apis mellifera) host a wide range of parasites, some being known contributors towards dramatic colony losses as reported over recent years. To counter parasitic threats, honey bees possess effective immune systems. Because immune responses are predicted to cause substantial physiological costs for infected individuals, they are expected to trade off with other life history traits that ultimately affect the performance and fitness of the entire colony. Here, we tested whether the initial onset of an infection negatively impacts the flight behaviour of honey bee workers, which is an energetically demanding behaviour and a key component of foraging activities. To do this, we infected workers with the widespread fungal pathogen Nosema apis, which is recognised and killed by the honey bee immune system. We compared their survival and flight behaviour with non-infected individuals from the same cohort and colony using radio frequency identification tags (RFID). We found that over a time frame of four days post infection, Nosema did not increase mortality but workers quickly altered their flight behaviour and performed more flights of shorter duration. We conclude that parasitic infections influence foraging activities, which could reduce foraging ranges of colonies and impact their ability to provide pollination services.

Pre-hospital Assessment of the Role of Adrenaline: Measuring the Effectiveness of Drug administration In Cardiac arrest (PARAMEDIC-2): Trial protocol.

Despite its use since the 1960s, the safety or effectiveness of adrenaline as a treatment for cardiac arrest has never been comprehensively evaluated in a clinical trial. Although most studies have found that adrenaline increases the chance of return of spontaneous circulation for short periods, many studies found harmful effects on the brain and raise concern that adrenaline may reduce overall survival and/or good neurological outcome. The PARAMEDIC-2 trial seeks to determine if adrenaline is safe and effective in out-of-hospital cardiac arrest. This is a pragmatic, individually randomised, double blind, controlled trial with a parallel economic evaluation. Participants will be eligible if they are in cardiac arrest in the out-of-hospital environment and advanced life support is initiated. Exclusions are cardiac arrest as a result of anaphylaxis or life threatening asthma, and patient known or appearing to be under 16 or pregnant. 8000 participants treated by 5 UK ambulance services will be randomised between December 2014 and August 2017 to adrenaline (intervention) or placebo (control) through opening pre-randomised drug packs. Clinical outcomes are survival to 30 days (primary outcome), hospital discharge, 3, 6 and 12 months, health related quality of life, and neurological and cognitive outcomes (secondary outcomes). Trial registration (ISRCTN73485024).

Cardiac Gene Expression Knockdown Using Small Inhibitory RNA-Loaded Microbubbles and Ultrasound.

RNA interference has potential therapeutic value for cardiac disease, but targeted delivery of interfering RNA is a challenge. Custom designed microbubbles, in conjunction with ultrasound, can deliver small inhibitory RNA to target tissues in vivo. The efficacy of cardiac RNA interference using a microbubble-ultrasound theranostic platform has not been demonstrated in vivo. Therefore, our objective was to test the hypothesis that custom designed microbubbles and ultrasound can mediate effective delivery of small inhibitory RNA to the heart. Microbubble and ultrasound mediated cardiac RNA interference was tested in transgenic mice displaying cardiac-restricted luciferase expression. Luciferase expression was assayed in select tissues of untreated mice (n = 14). Mice received intravenous infusion of cationic microbubbles bearing small inhibitory RNA directed against luciferase (n = 9) or control RNA (n = 8) during intermittent cardiac-directed ultrasound at mechanical index of 1.6. Simultaneous echocardiography in a separate group of mice (n = 3) confirmed microbubble destruction and replenishment during treatment. Three days post treatment, cardiac luciferase messenger RNA and protein levels were significantly lower in ultrasound-treated mice receiving microbubbles loaded with small inhibitory RNA directed against luciferase compared to mice receiving microbubbles bearing control RNA (23±7% and 33±7% of control mice, p<0.01 and p = 0.03, respectively). Passive cavitation detection focused on the heart confirmed that insonification resulted in inertial cavitation. In conclusion, small inhibitory RNA-loaded microbubbles and ultrasound directed at the heart significantly reduced the expression of a reporter gene. Ultrasound-targeted destruction of RNA-loaded microbubbles may be an effective image-guided strategy for therapeutic RNA interference in cardiac disease.

Ultrasound Targeted Microbubble Destruction-Mediated Delivery of a Transcription Factor Decoy Inhibits STAT3 Signaling and Tumor Growth.

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in many cancers where it acts to promote tumor progression. A STAT3-specific transcription factor decoy has been developed to suppress STAT3 downstream signaling, but a delivery strategy is needed to improve clinical translation. Ultrasound-targeted microbubble destruction (UTMD) has been shown to enhance image-guided local delivery of molecular therapeutics to a target site. The objective of this study was to deliver STAT3 decoy to squamous cell carcinoma (SCC) tumors using UTMD to disrupt STAT3 signaling and inhibit tumor growth. Studies performed demonstrated that UTMD treatment with STAT3 decoy-loaded microbubbles inhibited STAT3 signaling in SCC cells in vitro. Studies performed in vivo demonstrated that UTMD treatment with STAT3 decoy-loaded microbubbles induced significant tumor growth inhibition (31-51% reduced tumor volume vs. controls, p < 0.05) in mice bearing SCC tumors. Furthermore, expression of STAT3 downstream target genes (Bcl-xL and cyclin D1) was significantly reduced (34-39%, p < 0.05) in tumors receiving UTMD treatment with STAT3 decoy-loaded microbubbles compared to controls. In addition, the quantity of radiolabeled STAT3 decoy detected in tumors eight hours after treatment was significantly higher with UTMD treatment compared to controls (70-150%, p < 0.05). This study demonstrates that UTMD can increase delivery of a transcription factor decoy to tumors in vivo and that the decoy can inhibit STAT3 signaling and tumor growth. These results suggest that UTMD treatment holds potential for clinical use to increase the concentration of a transcription factor signaling inhibitor in the tumor.

Effective filtering strategies to improve data quality from population-based whole exome sequencing studies.

BACKGROUND: Genotypes generated in next generation sequencing studies contain errors which can significantly impact the power to detect signals in common and rare variant association tests. These genotyping errors are not explicitly filtered by the standard GATK Variant Quality Score Recalibration (VQSR) tool and thus remain a source of errors in whole exome sequencing (WES) projects that follow GATK's recommended best practices. Therefore, additional data filtering methods are required to effectively remove these errors before performing association analyses with complex phenotypes. Here we empirically derive thresholds for genotype and variant filters that, when used in conjunction with the VQSR tool, achieve higher data quality than when using VQSR alone. RESULTS: The detailed filtering strategies improve the concordance of sequenced genotypes with array genotypes from 99.33% to 99.77%; improve the percent of discordant genotypes removed from 10.5% to 69.5%; and improve the Ti/Tv ratio from 2.63 to 2.75. We also demonstrate that managing batch effects by separating samples based on different target capture and sequencing chemistry protocols results in a final data set containing 40.9% more high-quality variants. In addition, imputation is an important component of WES studies and is used to estimate common variant genotypes to generate additional markers for association analyses. As such, we demonstrate filtering methods for imputed data that improve genotype concordance from 79.3% to 99.8% while removing 99.5% of discordant genotypes. CONCLUSIONS: The described filtering methods are advantageous for large population-based WES studies designed to identify common and rare variation associated with complex diseases. Compared to data processed through standard practices, these strategies result in substantially higher quality data for common and rare association analyses.

Ultrasound detection of myocardial ischemic memory using an E-selectin targeting peptide amenable to human application.

Vascular endothelial leukocyte adhesion molecules, such as E-selectin, are acutely upregulated in myocardial ischemia/reperfusion and are thus "ischemic memory" biomarkers for recent cardiac ischemia. We sought to develop an ultrasound molecular imaging agent composed of microbubbles (MBs) targeted to E-selectin to enable the differential diagnosis of myocardial ischemia in patients presenting with chest pain of unclear etiology. Biodegradable polymer MBs were prepared bearing a peptide with specific human E-selectin affinity (MBESEL). Control MBs had scrambled peptide (MBCTL) or nonspecific IgG (MBIgG). MBESEL adhesion to activated rat endothelial cells (ECs) was confirmed in vitro in a flow system and in vivo with intravital microscopy of rat cremaster microcirculation. Ultrasound molecular imaging of recent myocardial ischemia was performed in rats 4 hours after transient (15 minutes) coronary occlusion. MBESEL adhesion was higher to inflamed versus normal ECs in vitro; there was no difference in MBCTL or MBIgG adhesion to inflamed versus normal ECs. There was greater adhesion of MBESEL to inflamed versus noninflamed microcirculation and minimal adhesion of MBCTL or MBIgG under any condition. Ultrasound imaging after injection of MBSEL demonstrated persistent contrast enhancement of the previously ischemic region. Videointensity in postischemic myocardium after MBESEL was higher than that in the nonischemic bed (11.6 ± 2.7 dB vs 3.6 ± 0.8 dB, p < .02) and higher than that after MBCTL (4.0 ± 1.0 dB, p < .03) or MBIgG (1.7 ± 0.1 dB, p < .03). MBs targeted to E-selectin via a short synthetic peptide with human E-selectin binding affinity enables echocardiographic detection of recent ischemia, setting the stage for clinical myocardial ischemic memory imaging to identify acute coronary syndromes.

Ultrasound Detection of Myocardial Ischemic Memory Using an E-Selectin Targeting Peptide Amenable to Human Application.

Vascular endothelial leukocyte adhesion molecules, such as E-selectin, are acutely upregulated in myocardial ischemia/reperfusion and are thus "ischemic memory" biomarkers for recent cardiac ischemia. We sought to develop an ultrasound molecular imaging agent composed of microbubbles (MBs) targeted to E-selectin to enable the differential diagnosis of myocardial ischemia in patients presenting with chest pain of unclear etiology. Biodegradable polymer MBs were prepared bearing a peptide with specific human E-selectin affinity (MBESEL). Control MBs had scrambled peptide (MBCTL) or nonspecific IgG (MBIgG). MBESEL adhesion to activated rat endothelial cells (ECs) was confirmed in vitro in a flow system and in vivo with intravital microscopy of rat cremaster microcirculation. Ultrasound molecular imaging of recent myocardial ischemia was performed in rats 4 hours after transient (15 minutes) coronary occlusion. MBESEL adhesion was higher to inflamed versus normal ECs in vitro; there was no difference in MBCTL or MBIgG adhesion to inflamed versus normal ECs. There was greater adhesion of MBESEL to inflamed versus noninflamed microcirculation and minimal adhesion of MBCTL or MBIgG under any condition. Ultrasound imaging after injection of MBSEL demonstrated persistent contrast enhancement of the previously ischemic region. Videointensity in postischemic myocardium after MBESEL was higher than that in the nonischemic bed (11.6 ± 2.7 dB vs 3.6 ± 0.8 dB, p < .02) and higher than that after MBCTL (4.0 ± 1.0 dB, p < .03) or MBIgG (1.7 ± 0.1 dB, p < .03). MBs targeted to E-selectin via a short synthetic peptide with human E-selectin binding affinity enables echocardiographic detection of recent ischemia, setting the stage for clinical myocardial ischemic memory imaging to identify acute coronary syndromes.

Gain-of-function ADCY5 mutations in familial dyskinesia with facial myokymia.

OBJECTIVE: To identify the cause of childhood onset involuntary paroxysmal choreiform and dystonic movements in 2 unrelated sporadic cases and to investigate the functional effect of missense mutations in adenylyl cyclase 5 (ADCY5) in sporadic and inherited cases of autosomal dominant familial dyskinesia with facial myokymia (FDFM). METHODS: Whole exome sequencing was performed on 2 parent-child trios. The effect of mutations in ADCY5 was studied by measurement of cyclic adenosine monophosphate (cAMP) accumulation under stimulatory and inhibitory conditions. RESULTS: The same de novo mutation (c.1252C>T, p.R418W) in ADCY5 was found in both studied cases. An inherited missense mutation (c.2176G>A, p.A726T) in ADCY5 was previously reported in a family with FDFM. The significant phenotypic overlap with FDFM was recognized in both cases only after discovery of the molecular link. The inherited mutation in the FDFM family and the recurrent de novo mutation affect residues in different protein domains, the first cytoplasmic domain and the first membrane-spanning domain, respectively. Functional studies revealed a statistically significant increase in ß-receptor agonist-stimulated intracellular cAMP consistent with an increase in adenylyl cyclase activity for both mutants relative to wild-type protein, indicative of a gain-of-function effect. INTERPRETATION: FDFM is likely caused by gain-of-function mutations in different domains of ADCY5-the first definitive link between adenylyl cyclase mutation and human disease. We have illustrated the power of hypothesis-free exome sequencing in establishing diagnoses in rare disorders with complex and variable phenotype. Mutations in ADCY5 should be considered in patients with undiagnosed complex movement disorders even in the absence of a family history.

Using an intervening sequence of Faecalibacterium 16S rDNA to identify poultry feces.

This study was designed to identify poultry feces-specific marker(s) within sequences of Faecalibacterium 16S rDNA for detecting poultry fecal pollution in water. Bioinformatics tools were used in the comparative analysis of 7,458 sequences of Faecalibacterium 16S rDNA, reportedly associated with various poultry (chicken and turkey) and animal species. One intervening sequence (IVS) within between the hypervariable region 1 and the conserved region 2, designated as IVS-p, was found to be unique to poultry feces. Based on this sequence, a PCR assay (PCR-p) was developed. The PCR-p produced an amplicon of 132 bp only in the test when fecal or wastewater samples from poultry were used, but not when using fecal or wastewater samples from other sources. The non-poultry sources included feces of beef or dairy cattle, dog, horse, human, domestic or wild geese, seagull, sheep, swine, and wild turkey. These data indicate that IVS-p may prove to be a useful genetic marker for the specific identification of poultry fecal pollution in environmental waterways. Furthermore, results of data mining and PCR assay indicate that the IVS-p may have a broad geographic distribution. This report represents initial evidence of the potential utility of ribosomal intervening sequences as genetic markers for tracking host sources of fecal pollution in waterways.

Ferritin as a novel reporter gene for photoacoustic molecular imaging.

Reporter genes may serve as endogenous contrast agents in the field of photoacoustic (PA) molecular imaging (PMI), enabling greater characterization of detailed cellular processes and disease progression. To demonstrate the feasibility of using ferritin as a reporter gene, human melanoma SK-24 (SK-MEL-24) cells were co-transfected with plasmid expressing human heavy chain ferritin (H-FT) and plasmid expressing enhanced green fluorescent protein (pEGFP-C1) using lipofectamine™ 2000. Nontransfected SK-MEL-24 cells served as a negative control. Fluorescent imaging of GFP confirmed transfection and transgene expression in co-transfected cells. To detect iron accumulation due to ferritin overexpression in SK-MEL-24 cells, a focused high-frequency ultrasonic transducer (60 MHz, f/1.5), synchronized to a pulsed laser (fluence < 5 mJ/cm(2)) was used to scan the PA signal at a wide range NIR wavelengths (850-950 nm). PA signal intensity from H-FT transfected SK-MEL-24 cells was about 5-9 dB higher than nontransfected SK-MEL-24 cells at 850-950 nm. Immunofluorescence and RT-PCR analysis both indicate high levels of ferritin expression in H-FT transfected SK-MEL24 cells, with little ferritin expression in nontransfected SK-MEL-24 cells. In this study, the feasibility of using ferritin as a reporter gene for PMI has been demonstrated in vitro. The use of ferritin as a reporter gene represents a novel concept for PMI using an endogenous contrast agent and may provide various opportunities for molecular imaging and basic science research.

Ultrasound-targeted microbubble destruction to deliver siRNA cancer therapy.

Microbubble contrast agents can specifically deliver nucleic acids to target tissues when exposed to ultrasound treatment parameters that mediate microbubble destruction. In this study, we evaluated whether microbubbles and ultrasound-targeted microbubble destruction (UTMD) could be used to enhance delivery of EGF receptor (EGFR)-directed siRNA to murine squamous cell carcinomas. Custom-designed microbubbles efficiently bound siRNA and mediated RNAse protection. UTMD-mediated delivery of microbubbles loaded with EGFR-directed siRNA to murine squamous carcinoma cells in vitro reduced EGFR expression and EGF-dependent growth, relative to delivery of control siRNA. Similarly, serial UTMD-mediated delivery of EGFR siRNA to squamous cell carcinoma in vivo decreased EGFR expression and increased tumor doubling time, relative to controls receiving EGFR siRNA-loaded microbubbles but not ultrasound or control siRNA-loaded microbubbles and UTMD. Taken together, our results offer a preclinical proof-of-concept for customized microbubbles and UTMD to deliver gene-targeted siRNA for cancer therapy.

Rare copy number variation discovery and cross-disorder comparisons identify risk genes for ADHD.

Attention deficit hyperactivity disorder (ADHD) is a common and persistent condition characterized by developmentally atypical and impairing inattention, hyperactivity, and impulsiveness. We identified de novo and rare copy number variations (CNVs) in 248 unrelated ADHD patients using million-feature genotyping arrays. We found de novo CNVs in 3 of 173 (1.7%) ADHD patients for whom we had DNA from both parents. These CNVs affected brain-expressed genes: DCLK2, SORCS1, SORCS3, and MACROD2. We also detected rare inherited CNVs in 19 of 248 (7.7%) ADHD probands, which were absent in 2357 controls and which either overlapped previously implicated ADHD loci (for example, DRD5 and 15q13 microduplication) or identified new candidate susceptibility genes (ASTN2, CPLX2, ZBBX, and PTPRN2). Among these de novo and rare inherited CNVs, there were also examples of genes (ASTN2, GABRG1, and CNTN5) previously implicated by rare CNVs in other neurodevelopmental conditions including autism spectrum disorder (ASD). To further explore the overlap of risks in ADHD and ASD, we used the same microarrays to test for rare CNVs in an independent, newly collected cohort of 349 unrelated individuals with a primary diagnosis of ASD. Deletions of the neuronal ASTN2 and the ASTN2-intronic TRIM32 genes yielded the strongest association with ADHD and ASD, but numerous other shared candidate genes (such as CHCHD3, MACROD2, and the 16p11.2 region) were also revealed. Our results provide support for a role for rare CNVs in ADHD risk and reinforce evidence for the existence of common underlying susceptibility genes for ADHD, ASD, and other neuropsychiatric disorders.

Detection and monitoring of the multiple inflammatory responses by photoacoustic molecular imaging using selectively targeted gold nanorods.

In vitro cell experiments have been performed to detect and monitor the upregulation of intercellular adhesion molecule-1 (ICAM-1) and E-selectin simultaneously by photoacoustic molecular imaging (PMI). Human umbilical vein endothelial cells (HUVECs) were grown on gelatin-coated glass slides and stimulated with inflammatory cytokines to induce the expression of the inflammatory biomarkers, ICAM-1 and E-selectin. Gold nanorods (GNRs) of aspect ratio (AR) 1:3 with absorption centered at 715 nm conjugated to anti-ICAM-1 antibody and GNRs of AR 1:3.5 with absorption centered at 800 nm conjugated to anti-E-selectin were exposed to HUVECs with different stimulation conditions. A focused high frequency ultrasonic transducer (60 MHz, f/1.5) was used to scan the photoacoustic (PA) signal over the top surface of the cell containing slides. Averaged PA signal intensity from the stimulated cells was about 3 folds higher (~10 dB) compared to the un-stimulated cells for both ICAM-1 and E-selectin. The strong binding of GNRs to the stimulated HUVEC cells was evidenced by fluorescence imaging. Exposure of HUVEC cells to GNRs conjugated to isotype control antibodies confirms a low level non-specific binding. Also, at 0, 2, 6, and 24 hours after inflammatory stimulation, the HUVECs were exposed to GNRs conjugated anti-ICAM-1 antibody and anti-E-selectin antibody. PA intensity at each stage of inflammation compares well with fluorescence imaging and rt-PCR quantification.

Gene therapy of carcinoma using ultrasound-targeted microbubble destruction.

When microbubble contrast agents are loaded with genes and systemically injected, ultrasound-targeted microbubble destruction (UTMD) facilitates focused delivery of genes to target tissues. A mouse model of squamous cell carcinoma was used to test the hypothesis that UTMD would specifically transduce tumor tissue and slow tumor growth when treated with herpes simplex virus thymidine kinase (TK) and ganciclovir. UTMD-mediated delivery of reporter genes resulted in tumor expression of luciferase and green fluorescent protein (GFP) in perivascular areas and individual tumor cells that exceeded expression in control tumors (p=0.02). The doubling time of TK-treated tumors was longer than GFP-treated tumors (p=0.02), and TK-treated tumors displayed increased apoptosis (p=0.04) and more areas of cellular drop-out (p=0.03). These data indicate that UTMD gene therapy can transduce solid tumors and mediate a therapeutic effect. UTMD is a promising nonviral method for targeting gene therapy that may be useful in a spectrum of tumors.

Quantification of plaque neovascularization using contrast ultrasound: a histologic validation.

AIMS: The density of vasa vasorum within atherosclerotic plaque correlates with histologic features of plaque vulnerability in post-mortem studies. Imaging methods to non-invasively detect vasa vasorum are limited. We hypothesized that contrast ultrasound (CUS) can quantify vasa vasorum during atherosclerosis progression. METHODS AND RESULTS: New Zealand white rabbits received a high-fat diet for 3 weeks, and bilateral femoral artery stenosis was induced by balloon injury. Contrast ultrasound femoral imaging was performed at baseline and 2, 4, and 6 weeks post injury to quantify adventitial videointensity. At each imaging time point 10 vessels were sectioned and stained with haematoxylin and eosin and von-Willebrand factor. Adventitial vasa vasorum density was quantified by counting the number of stained microvessels and their total cross-sectional area. Plaque size (per cent lumen area) progressed over time (P < 0.001), as did adventitial vasa vasorum density (P < 0.001). Plateau peak videointensity also progressed, demonstrating a strong linear correlation with histologic vasa vasorum density (P < 0.001). Receiver operating characteristic analysis indicated that a three-fold increase in median adventitial videointensity had a sensitivity of 100% and specificity of 88% for predicting abnormal neovascularization. CONCLUSION: We have histologically validated that CUS quantifies the development of adventitial vasa vasorum associated with atherosclerosis progression. This imaging technique has the potential for characterizing prognostically significant plaque features.

Disruption at the PTCHD1 Locus on Xp22.11 in Autism spectrum disorder and intellectual disability.

Autism is a common neurodevelopmental disorder with a complex mode of inheritance. It is one of the most highly heritable of the complex disorders, although the underlying genetic factors remain largely unknown. Here, we report mutations in the X-chromosome PTCHD1 (patched-related) gene in seven families with autism spectrum disorder (ASD) and in three families with intellectual disability. A 167-kilobase microdeletion spanning exon 1 was found in two brothers, one with ASD and the other with a learning disability and ASD features; a 90-kilobase microdeletion spanning the entire gene was found in three males with intellectual disability in a second family. In 900 probands with ASD and 208 male probands with intellectual disability, we identified seven different missense changes (in eight male probands) that were inherited from unaffected mothers and not found in controls. Two of the ASD individuals with missense changes also carried a de novo deletion at another ASD susceptibility locus (DPYD and DPP6), suggesting complex genetic contributions. In additional males with ASD, we identified deletions in the 5' flanking region of PTCHD1 that disrupted a complex noncoding RNA and potential regulatory elements; equivalent changes were not found in male control individuals. Thus, our systematic screen of PTCHD1 and its 5' flanking regions suggests that this locus is involved in ~1% of individuals with ASD and intellectual disability.