Chelator-Free Radiolabeling of SERRS Nanoparticles for Whole-Body PET and Intraoperative Raman Imaging

A single contrast agent that offers whole-body non-invasive imaging along with the superior sensitivity and spatial resolution of surface-enhanced resonance Raman scattering (SERRS) imaging would allow both pre-operative mapping and intraoperative imaging and thus be highly desirable. We hypothesized that labeling our recently reported ultrabright SERRS nanoparticles with a suitable radiotracer would enable pre-operative identification of regions of interest with whole body imaging that can be rapidly corroborated with a Raman imaging device or handheld Raman scanner in order to provide high precision guidance during surgical procedures. Here we present a straightforward new method that produces radiolabeled SERRS nanoparticles for combined positron emission tomography (PET)-SERRS tumor imaging without requiring the attachment of molecular chelators. We demonstrate the utility of these PET-SERRS nanoparticles in several proof-of-concept studies including lymph node (LN) tracking, intraoperative guidance for LN resection, and cancer imaging after intravenous injection. We anticipate that the radiolabeling method presented herein can be applied generally to nanoparticle substrates of various materials by first coating them with a silica shell and then applying the chelator-free protocol

Differential Allelic Expression in the Human Genome: A Robust Approach To Identify Genetic and Epigenetic Cis-Acting Mechanisms Regulating Gene Expression

The recent development of whole genome association studies has lead to the robust identification of several loci involved in different common human diseases. Interestingly, some of the strongest signals of association observed in these studies arise from non-coding regions located in very large introns or far away from any annotated genes, raising the possibility that these regions are involved in the etiology of the disease through some unidentified regulatory mechanisms. These findings highlight the importance of better understanding the mechanisms leading to inter-individual differences in gene expression in humans. Most of the existing approaches developed to identify common regulatory polymorphisms are based on linkage/association mapping of gene expression to genotypes. However, these methods have some limitations, notably their cost and the requirement of extensive genotyping information from all the individuals studied which limits their applications to a specific cohort or tissue. Here we describe a robust and high-throughput method to directly measure differences in allelic expression for a large number of genes using the Illumina Allele-Specific Expression BeadArray platform and quantitative sequencing of RT-PCR products. We show that this approach allows reliable identification of differences in the relative expression of the two alleles larger than 1.5-fold (i.e., deviations of the allelic ratio larger than 60∶40) and offers several advantages over the mapping of total gene expression, particularly for studying humans or outbred populations. Our analysis of more than 80 individuals for 2,968 SNPs located in 1,380 genes confirms that differential allelic expression is a widespread phenomenon affecting the expression of 20% of human genes and shows that our method successfully captures expression differences resulting from both genetic and epigenetic cis-acting mechanisms

L-Plastin nanobodies perturb matrix degradation, podosome formation, stability and lifetime in THP-1 macrophages

Podosomes are cellular structures acting as degradation ‘hot-spots’ in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages

Population-based Assessment of Aortic-related Outcomes in Aortic Dissection, Intramural Hematoma, and Penetrating Aortic Ulcer

BACKGROUND: The aim of the study was to analyze aortic-related outcomes after diagnosis of aortic dissection (AD), intramural hematoma (IMH), and penetrating aortic ulcer (PAU) from a population-based approach. METHODS: Retrospective review of an incident cohort of AD, IMH, and PAU patients in Olmsted County, Minnesota from 1995 to 2015. Primary end point was aortic death. Secondary end points were subsequent aortic events (aortic intervention, new dissection, or rupture not present at presentation) and first-time diagnosis of an aortic aneurysm. Outcomes were compared with randomly selected population referents matched for age and sex in a 3:1 ratio using Cox proportional hazards regression adjusting for comorbidities. RESULTS: Among 133 patients (77 AD, 21 IMH, and 35 PAU), 57% were males, and mean age was 71.8 years (standard deviation, 14). Median follow-up was 10 years. Of 73 deaths among AD/IMH/PAU patients, 23 (32%) were aortic-related. Estimated freedom from aortic death was 84%, 80%, and 77% at 5, 10, and 15 years. There were no aortic deaths among population referents (adjusted hazard ratio [HR] for aortic death in AD/IMH/PAU, 184.7; 95% confidence interval [95% CI], 10.3–3,299.2; P < 0.001). Fifty (38%) AD/IMH/PAU patients had a subsequent aortic event (aortic intervention, new dissection, or rupture), whereas there were 8 (2%) aortic events among population referents (all elective aneurysm repairs; adjusted HR for any aortic event and aortic intervention in AD/IMH/PAU patients, 33.3; 95% CI, 15.3–72.0; P < 0.001 and 31.5; 95% CI, 14.5–68.4; P < 0.001, respectively). After excluding aortic events/interventions 14 days of diagnosis, AD/IMH/PAU patients remained at increased risk of any aortic event (adjusted HR, 10.8; 95% CI, 3.9–29.8; P < 0.001) and aortic intervention (adjusted HR, 9.6; 95% CI, 3.4–26.8; P < 0.001). Among those subjects with available follow-up imaging, the risk of first-time diagnosis of aortic aneurysm was significantly increased for AD/IMH/PAU patients when compared with population referents (adjusted HR, 10.9; 95% CI, 5.4–21.7; P < 0.001 and 8.3; 95% CI, 4.1–16.7; P < 0.001 for thoracic and abdominal aneurysms, respectively) and remained increased when excluding aneurysms that formed within 14 days of AD/IMH/PAU (adjusted HR, 6.2; 95% CI, 1.8–21.1; P = 0.004 and 2.8; 95% CI, 1.0–7.6; P = 0.040 for thoracic and abdominal aneurysms, respectively). CONCLUSIONS: AD/IMH/PAU patients have a substantial risk of aortic death, any aortic event, aortic intervention, and first-time diagnosis of aortic aneurysm that persists even when the acute phase (≤14 days after diagnosis) is uncomplicated. Advances in postdiagnosis treatment are necessary to improve the prognosis in these patients

Comparative Evaluation of the VERSANT HCV RNA 3.0, QUANTIPLEX HCV RNA 2.0, and COBAS AMPLICOR HCV MONITOR Version 2.0 Assays for Quantification of Hepatitis C Virus RNA in Serum

A comparison of quantitative results expressed in hepatitis C virus (HCV) international units per milliliter, obtained from the VERSANT HCV RNA 3.0 (bDNA-3.0) assay, the QUANTIPLEX HCV RNA 2.0 (bDNA-2.0) assay, and the COBAS AMPLICOR HCV MONITOR version 2.0 (HCM-2.0) test was performed. A total of 168 patient specimens submitted to the Mayo Clinic Molecular Microbiology Laboratory for HCV quantification or HCV genotyping were studied. Of the specimens tested, 97, 88, and 79% yielded quantitative results within the dynamic range of the bDNA-3.0, bDNA-2.0, and HCM-2.0 assays, respectively. Overall, there was substantial agreement between the results generated by all three assays. A total of 15 out of 29 (52%) of the specimens determined to contain viral loads of <31,746 IU/ml by the bDNA-3.0 assay were categorized as containing viral loads within the range of 31,746 to 500,000 IU/ml by the bDNA-2.0 assay. Although substantial agreement was noted between the results generated by the bDNA-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-2.0 assay was noted (P = 0.001). Likewise, although substantial agreement was noted between the results generated by the HCM-2.0 and bDNA-3.0 assays, a bias toward higher viral titer by the bDNA-3.0 assay was noted (P ≤ 0.001). The discrepancy between the HCM-2.0 and bDNA-3.0 results was more pronounced when viral loads were >500,000 IU/ml and resulted in statistically significant differences (P ≤ 0.001) in determining whether viral loads were above or below 800,000 IU/ml of HCV RNA, the proposed threshold value for tailoring the duration of combination therapy. The expression of quantitative values in HCV international units per milliliter was a strength of both the bDNA-3.0 and HCM-2.0 assays