Accurate Quantification and Imaging of Cellular Uptake Using Single-Particle Surface-Enhanced Raman Scattering.

Understanding the uptake, distribution, and stability of gold nanoparticles (NPs) in cells is of fundamental importance in nanoparticle sensors and therapeutic development. Single nanoparticle imaging with surface-enhanced Raman spectroscopy (SERS) measurements in cells is complicated by aggregation-dependent SERS signals, particle inhomogeneity, and limited single-particle brightness. In this work, we assess the single-particle SERS signals of various gold nanoparticle shapes and the role of silica encapsulation on SERS signals to develop a quantitative probe for single-particle level Raman imaging in living cells. We observe that silica-encapsulated gap-enhanced Raman tags (GERTs) provide an optimized probe that can be quantifiable per voxel in SERS maps of cells. This approach is validated by single-particle inductively coupled mass spectrometry (spICP-MS) measurements of NPs in cell lysate post-imaging. spICP-MS also provides a means of measuring the tag stability. This analytical approach can be used not only to quantitatively assess nanoparticle uptake on the cellular level (as in previous digital SERS methods) but also to reliably image the subcellular distribution and to assess the stability of NPs in cells.

Metal enrichment in ice-melt water and uptake by chironomids as possible legacy of World War One in the Italian Alps.

Relics of World War One (WW1) were buried in alpine glaciers around 100 years ago. Today, these are emerging from the ice due to widespread glacier retreat, and are in direct contact with glacial meltwater-fed streams. To address a possible emergent contamination, we quantified major and trace elements (M-TEs) by mass spectrometry in water and larvae of Diamesa zernyi from three glacial streams fed by glaciers differently impacted by the Italian Austro-Hungarian war, in the Adamello-Presanella mountain range (Italian Alps): Lares and Presena, the two main battlefields, and Amola, 8 km from the front. M-TEs in stream water were interpreted using the crustal enrichment factor (EFc) while larval uptake was quantified by adopting the bioaccumulation factor (BAF). Despite low M-TEs concentrations in the water, in a range between 1 ng L-1 (Ag, Ta) and 1-2 mg L-1 (Al, Fe, Mg), low to moderate enrichments (10 ≥ EFc≥ 6) were observed for Sb and U in Presena and for Ag, As, Bi, Cd, Li, Mo, Pb, Sb and U in Lares. In addition, M-TE mass concentrations in larvae were up to ninety thousand times higher than in water, from 20 to 50 ng g-1 dry weight (d.w.; for Bi, Sb, Ta, Tl) to 1-4 mg g-1 d.w. (for Al, Fe, Na, and Mg). Larvae from Lares accumulated the largest amount of metals and metalloids, including those mostly used in the manufacture of artillery shells (As, Cu, Ni, Pb, Sb; BAFs from 375 to about 11,500). This was expected as most of the WW1 battles in this mountain range were fought on the Lares glacier, where the greatest number of war relics are emerging. These results provide preliminary evidence of water contamination and bioaccumulation of metals and metalloids by glacial fauna as a possible legacy of WW1 in the Alps.

Dissipation of transmembrane potassium gradient is the main cause of cerebral ischemia-induced depolarization in astrocytes and neurons.

Membrane potential (VM) depolarization occurs immediately following cerebral ischemia and is devastating for the astrocyte homeostasis and neuronal signaling. Previously, an excessive release of extracellular K+ and glutamate has been shown to underlie an ischemia-induced VM depolarization. Ischemic insults should impair membrane ion channels and disrupt the physiological ion gradients. However, their respective contribution to ischemia-induced neuronal and glial depolarization and loss of neuronal excitability are unanswered questions. A short-term oxygen-glucose deprivation (OGD) was used for the purpose of examining the acute effect of ischemic conditions on ion channel activity and physiological K+ gradient in neurons and glial cells. We show that a 30 min OGD treatment exerted no measurable damage to the function of membrane ion channels in neurons, astrocytes, and NG2 glia. As a result of the resilience of membrane ion channels, neuronal spikes last twice as long as our previously reported 15 min time window. In the electrophysiological analysis, a 30 min OGD-induced dissipation of transmembrane K+ gradient contributed differently in brain cell depolarization: severe in astrocytes and neurons, and undetectable in NG2 glia. The discrete cellular responses to OGD corresponded to a total loss of 69% of the intracellular K+ contents in hippocampal slices as measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A major brain cell depolarization mechanism identified here is important for our understanding of cerebral ischemia pathology. Additionally, further understanding of the resilient response of NG2 glia to ischemia-induced intracellular K+ loss and depolarization should facilitate the development of future stroke therapy.

Trace Element Analysis of Borrelia burgdorferi by Inductively Coupled Plasma-Sector Field Mass Spectrometry.

Transition metal ions play important structural, regulatory, and catalytic roles in all biological systems by serving as cofactors for proteins. Due to their relatively low levels in the cell compared to abundant metal ions such as potassium and magnesium, transition metals are often considered micronutrients and referred to as trace elements. Manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) are the most prevalent transition metals in the Lyme disease spirochete Borrelia burgdorferi. Here, we describe a method for the accurate measurement of these trace elements in B. burgdorferi utilizing inductively coupled plasma-sector field mass spectrometry (ICP-SFMS).

Uptake of bright fluorophore core-silica shell nanoparticles by biological systems.

Nanoparticles are used in a variety of consumer applications. Silica nanoparticles in particular are common, including as a component of foods. There are concerns that ingested nano-silica particles can cross the intestinal epithelium, enter the circulation, and accumulate in tissues and organs. Thus, tracking these particles is of interest, and fluorescence spectroscopic methods are well-suited for this purpose. However, nanosilica is not fluorescent. In this article, we focus on core-silica shell nanoparticles, using fluorescent Rhodamine 6G, Rhodamine 800, or CdSe/CdS/ZnS quantum dots as the core. These stable fluorophore/silica nanoparticles had surface characteristics similar to those of commercial silica particles. Thus, they were used as model particles to examine internalization by cultured cells, including an epithelial cell line relevant to the gastrointestinal tract. Finally, these particles were administered to mice by gavage, and their presence in various organs, including stomach, small intestine, cecum, colon, kidney, lung, brain, and spleen, was examined. By combining confocal fluorescence microscopy with inductively coupled plasma mass spectrometry, the presence of nanoparticles, rather than their dissolved form, was established in liver tissues.

A novel iron- and copper-binding protein in the Lyme disease spirochaete.

Iron and copper are transition metals that can be toxic to cells due to their abilities to react with peroxide to generate hydroxyl radical. Ferritins and metallothioneins are known to sequester intracellular iron and copper respectively. The Lyme disease pathogen Borrelia burgdorferi does not require iron, but its genome encodes a ferritin-like Dps (DNA-binding protein from starved bacteria) molecule, which has been shown to be important for the spirochaete's persistence in the tick and subsequent transmission to a new host. Here, we show that the carboxyl-terminal cysteine-rich (CCR) domain of this protein functions as a copper-binding metallothionein. This novel fusion between Dps and metallothionein is unique to and conserved in all Borrelia species. We term this molecule BicA for Borrelia iron- and copper-binding protein A. An isogenic mutant lacking BicA had significantly reduced levels of iron and copper and was more sensitive to iron and copper toxicity than its parental strain. Supplementation of the medium with iron or copper rendered the spirochaete more susceptible to peroxide killing. These data suggest that an important function of BicA is to detoxify excess iron and copper the spirochaete may encounter during its natural life cycle through a tick vector and a vertebrate host.

Impaired myocardial perfusion reserve and fibrosis in Friedreich ataxia: a mitochondrial cardiomyopathy with metabolic syndrome.

AIMS: Cardiomyopathy produces significant mortality in patients with Friedreich ataxia (FA), a genetic disorder that produces intra-mitochondrial iron accumulation. We sought to test the hypothesis that abnormal myocardial perfusion reserve and fibrosis represent early manifestations of cardiomyopathy. METHODS AND RESULTS: Twenty-six patients with genetically proven FA ages 36 ± 12 years without cardiomyopathy and eight controls underwent cardiac magnetic resonance with adenosine. Precontrast imaging for myocardial iron estimation was performed. Myocardial perfusion reserve index (MPRI) was quantified using the normalized upslope of myocardial enhancement during vasodilator stress vs. rest. Left ventricular (LV) mass and volumes were computed from short-axis cine images. Serologies included lipids, and platelets were isolated for iron quantification using inductively coupled plasma mass spectrometry. Left ventricular ejection fraction and mass averaged 64.1 ± 8.3% and 62.7 ± 16.7 g/m², respectively, indicating preserved systolic function and absence of significant hypertrophy. Myocardial perfusion reserve index quantification revealed significantly lower endocardial-to-epicardial perfusion reserve in patients vs. controls (0.80 ± 0.18 vs. 1.22 ± 0.36, P = 0.01). Lower MPRI was predicted by increased number of metabolic syndrome (met-S) features (P < 0.01). Worse concentric remodelling occurred with increased GAA repeat length (r = 0.64, P < 0.001). Peripheral platelet iron measurement showed no distinction between patients and controls (5.4 ± 8.5 × 10⁻7 vs. 5.5 ± 2.9 × 10⁻7 ng/platelet, P = 0.88), nor did myocardial T2* measures. CONCLUSIONS: Patients with FA have abnormal myocardial perfusion reserve that parallels met-S severity. Impaired perfusion reserve and fibrosis occur in the absence of significant hypertrophy and prior to clinical heart failure, providing potential therapeutic targets for stage B cardiomyopathy in FA and related myocardial diseases.