Real-time imaging of VCAM-1 mRNA in TNF-α activated retinal microvascular endothelial cells using antisense hairpin-DNA functionalized gold nanoparticles.

Vascular cell adhesion molecule 1 (VCAM-1) is an important inflammatory biomarker correlating with retinal disease progression. Thus, detection of VCAM-1 mRNA expression levels at an early disease stage could be an important predictive biomarker to assess the risk of disease progression and monitoring treatment response. We have developed VCAM-1 targeted antisense hairpin DNA-functionalized gold nanoparticles (AS-VCAM-1 hAuNP) for the real time detection of VCAM-1 mRNA expression levels in retinal endothelial cells. The AS-VCAM-1 hAuNP fluorescence enhancement clearly visualized the TNF-α induced cellular VCAM-1 mRNA levels with high signal to noise ratios compared to normal serum treated cells. The scrambled hAuNP probes were minimally detectable under same image acquisition conditions. Intracellular hAuNPs were detected using transmission electron microscopy (TEM) analysis of the intact cells. In addition, the AS-VCAM-1 hAuNP probes exhibited no acute toxicity to the retinal microvascular endothelial cells as measured by live-dead assay.

Size-Dependent Cellular Uptake of DNA Functionalized Gold Nanoparticles.

The extensive use of gold nanoparticles (AuNPs) in nanomedicine, especially for intracellular imaging, photothermal therapy, and drug delivery, has necessitated the study of how functionalized AuNPs engage with living biological interfaces like the mammalian cell. Nanoparticle size, shape, surface charge, and surface functionality can affect the accumulation of functionalized AuNPs in cells. Confocal microscopy, flow cytometry, and inductively coupled plasma mass spectrometry demonstrate that CaSki cells, a human cervical cancer cell line, internalize AuNPs functionalized with hairpin, single stranded, and double stranded DNA differently. Surface charge and DNA conformation are shown to have no effect on the cell-nanoparticle interaction. CaSki cells accumulate small DNA-AuNPs in greater quantities than large DNA-AuNPs, demonstrating that size is the major contributor to cellular uptake properties. These data suggest that DNA-AuNPs can be easily tailored through modulation of size to design functional AuNPs with optimal cellular uptake properties and enhanced performance in nanomedicine applications.

Case report: Aortoesophageal fistula-an extremely rare but life-threatening cardiovascular cause of hematemesis.

Aortoesophageal fistula (AEF) is an extremely rare cardiovascular etiology of hematemesis and upper gastrointestinal bleeding. As such, its recognition and diagnosis are challenging and may be delayed when such patients present to the emergency department (ED). Without timely surgical intervention, AEF is almost always fatal. Awareness of AEF as a possible diagnosis and consequently early identification of these patients presenting to the ED are therefore crucial in optimizing clinical outcomes. We report a 45-year-old male presenting to the ED with the classical triad of an AEF (Chiari's triad)-midthoracic pain or dysphagia, a sentinel episode of minor hematemesis, then massive hematemesis with risk of exsanguination. The case report highlights the importance of considering the differential diagnosis of AEF when evaluating patients presenting to the ED with hematemesis, especially if they have predisposing risk factors such as prior aortic or esophageal surgeries, aortic aneurysms, or thoracic malignancies. Patients suspected of having AEF should be prioritized for early computed tomography angiography to expedite diagnosis and treatment.

Ultrasensitive multiplexed chemiluminescent enzyme-linked immunosorbent assays in 384-well plates.

We have developed an ultrasensitive multiplexed immunoassay using 384-well microtiter plates capable of detecting proteins at subfemtomolar concentrations that requires as little as 2.5 µL of sample. Arrays of up to 4 capture antibodies were patterned on the bottom of the wells of a 384-well plate either by directly printing the capture antibodies or by printing anti-peptide tag anchor antibodies and incubating these arrays with capture antibodies conjugated to the corresponding peptide tags ("customized" assays). Samples were incubated with the antibody arrays and shaken orbitally at 2000 rpm to achieve the greatest sensitivity. Chemiluminescence (CL) from immunocomplexes labeled with horseradish peroxidase was imaged across the entire plate to quantify the amount of protein bound to each antibody spot of the arrays. The 384-well assay had a throughput 5-fold greater than 96-well plates that was achieved from simultaneous imaging of CL in all 384-wells and the use of automated pipettors to allow parallel processing of 384 assays. We developed 4 assays based on the 384-well CL ELISA: a direct print assay for IL-10 (limit of detection (LOD) = 0.075 fM); a customized assay for IL-6 (0.22 fM); a customized pharmacokinetic (PK) assay for measuring adalimumab (7.3 pg/mL); and a customized 4-plex assay for IL-5 (0.1 fM), IL-6 (0.52 fM), IL-10 (0.2 fM), and TNF-α (3.2 fM). The sensitivity and precision of the cytokine assays were comparable to current ultrasensitive protein detection methods in 96-well formats. The PK assay for adalimumab was 650 times more sensitive than a commercially available 96-well plate ELISA. We used the 384-well CL ELISAs to measure endogenous levels of the cytokines in the serum and plasma of healthy humans: the mean concentrations and precision were comparable to those from 96-well immunoassays. This 384-well format with subfemtomolar sensitivity will enable ultrasensitive multiplexed immunoassays to be performed with higher throughput and lower sample volumes than currently possible, a particularly important capability for clinical studies in drug development.

At the heart of the problem: congestive cardiac failure as a cause of ascites: A narrative review.

Heart failure leading to cardiac ascites is an extremely rare and underrecognized entity in clinical practice. Recognizing cardiac ascites can be difficult, especially since patients presenting with ascites may have more than 1 etiology. Various biomarkers are available to aid in the diagnosis of cardiac ascites, though with differing sensitivities and specificities. Such biomarkers include serum albumin, ascitic albumin and protein, as well as serum N-terminal pro-brain natriuretic peptide (NT-proBNP). While serum NT-proBNP is a powerful biomarker in distinguishing the etiology of ascites and monitoring treatment progression, its cost can be prohibitive in low-resource settings. Clinicians practicing under these circumstances may opt to rely on other parameters to manage their patients. We go on further to report a series of 3 patients with cardiac ascites to illustrate how these biomarkers may be employed in the management of this patient population. Clinicians should always keep in mind the differential diagnosis of cardiac failure as a cause of ascites. The resolution of cardiac ascites may serve as a surrogate clinical marker for response to antifailure therapy in lieu of NT-proBNP at resource-scarce centers.

Unilateral live twin tubal ectopic pregnancy presenting at 12 weeks of gestation: A case report.

RATIONALE: Abdominal pain in pregnancy represents a demanding diagnostic challenge in the emergency department (ED) due to the extensive list of differential diagnoses to be considered, coupled with the possibility of each disease having nonclassical, atypical signs and symptoms, resultant from the patient's pregnant state. Additionally, emergency physicians (EPs) face limitations on investigative imaging modalities because of the need to minimize fetal radiation exposure. EPs have to tackle this diagnostic challenge while performing a balancing act to maximize both maternal and fetal outcomes in a time-sensitive manner, becauser any delays in decision-making at the ED may threaten the safety of mother and child. Two common causes of abdominal pain in pregnancy presenting to the ED are acute appendicitis and ectopic pregnancy. The latter is almost always diagnosed by 10 weeks of gestation. Here, we report an extremely rare case of unilateral live spontaneous twin tubal ectopic pregnancy presenting past 12 weeks of gestation, diagnosed after magnetic resonance imaging (MRI) of the abdomen. PATIENT CONCERNS: A 37-year-old gravida 2 para 1 at 12 weeks and 6 days of gestation presented to our ED with a 2-day history of right iliac fossa pain, not associated with vaginal bleeding, fever, diarrhea, and vomiting. On examination, she was tachycardic (pulse rate 124 beats/min) and hypertensive (blood pressure 142/88 mm Hg). There was marked tenderness and guarding at the lower abdomen. DIAGNOSES: Blood investigations were unremarkable, while abdominal ultrasonography found a live twin gestation with foetal heartbeats of 185 and 180 beats/min. MRI of the abdomen revealed an empty uterine cavity; 2 amniotic sacs and fetuses of diameter 10 cm, and a single placenta were noted in the right uterine adnexa. The patient was diagnosed with right live monochorionic diamniotic twin tubal pregnancy. INTERVENTION: Our patient underwent emergency laparoscopic right salpingectomy. OUTCOMES: The operation was successful and her postoperative care remained uneventful up to discharge. LESSONS: Ectopic pregnancy cannot be ruled out based on prior normal antenatal examinations and gestational age of >10 weeks. EPs should not hesitate to order MRI scans for further evaluation if ultrasonography and laboratory findings are equivocal.

Spontaneous self-assembly and disassembly of colloidal gold nanoparticles induced by tetrakis(hydroxymethyl) phosphonium chloride.

Upon reacting with tetrakis(hydroxymethyl) phosphonium chloride, 15 nm citrate gold nanoparticles rapidly assemble into linear chains, followed by slowly disassembling into monodisperse components. This work highlights the first example of (31)P NMR on gold particles of this size and suggests that the phosphonium is oxidized on-particle, contributing to particle disassembly.