Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging.

Live-cell RNA imaging with high spatial and temporal resolution remains a major challenge. Here we report the development of RhoBAST:SpyRho, a fluorescent light-up aptamer (FLAP) system ideally suited for visualizing RNAs in live or fixed cells with various advanced fluorescence microscopy modalities. Overcoming problems associated with low cell permeability, brightness, fluorogenicity, and signal-to-background ratio of previous fluorophores, we design a novel probe, SpyRho (Spirocyclic Rhodamine), which tightly binds to the RhoBAST aptamer. High brightness and fluorogenicity is achieved by shifting the equilibrium between spirolactam and quinoid. With its high affinity and fast ligand exchange, RhoBAST:SpyRho is a superb system for both super-resolution SMLM and STED imaging. Its excellent performance in SMLM and the first reported super-resolved STED images of specifically labeled RNA in live mammalian cells represent significant advances over other FLAPs. The versatility of RhoBAST:SpyRho is further demonstrated by imaging endogenous chromosomal loci and proteins.

Avidity-based bright and photostable light-up aptamers for single-molecule mRNA imaging.

Fluorescent light-up aptamers (FLAPs) have emerged as valuable tools to visualize RNAs, but are mostly limited by their poor brightness, low photostability, and high fluorescence background in live cells. Exploiting the avidity concept, here we present two of the brightest FLAPs with the strongest aptamer-dye interaction, high fluorogenicity, and remarkable photostability. They consist of dimeric fluorophore-binding aptamers (biRhoBAST and biSiRA) embedded in an RNA scaffold and their bivalent fluorophore ligands (bivalent tetramethylrhodamine TMR2 and silicon rhodamine SiR2). Red fluorescent biRhoBAST-TMR2 and near-infrared fluorescent biSiRA-SiR2 are orthogonal to each other, facilitating simultaneous visualization of two different RNA species in live cells. One copy of biRhoBAST allows for simple and robust mRNA imaging with strikingly higher signal-to-background ratios than other FLAPs. Moreover, eight biRhoBAST repeats enable single-molecule mRNA imaging and tracking with minimal perturbation of their localization, translation, and degradation, demonstrating the potential of avidity-enhanced FLAPs for imaging RNA dynamics.

Periarticular calcifications containing giant pseudo-crystals of francolite in skeletal fluorosis from 1,1-difluoroethane "huffing".

Inhalant use disorder is a psychiatric condition characterized by repeated deliberate inhalation from among a broad range of household and industrial chemical products with the intention of producing psychoactive effects. In addition to acute intoxication, prolonged inhalation of fluorinated compounds can cause skeletal fluorosis (SF). We report a young woman referred for hypophosphatasemia and carrying a heterozygous ALPL gene variant (c.457T>C, p.Trp153Arg) associated with hypophosphatasia, the heritable metabolic bone disease featuring impaired skeletal mineralization, who instead suffered from SF. Manifestations of her SF included recurrent articular pain, axial osteosclerosis, elevated bone mineral density, maxillary exostoses, and multifocal periarticular calcifications. SF was suspected when a long history was discovered of 'huffing' a computer cleaner containing 1,1-difluoroethane. Investigation revealed markedly elevated serum and urine levels of F-. Histopathology and imaging techniques including backscattered electron mode scanning electron microscopy, X-ray microtomography, energy dispersive and wavelength dispersive X-ray emission microanalysis, and polarized light microscopy revealed that her periarticular calcifications were dystrophic deposition of giant pseudo-crystals of francolite, a carbonate-rich fluorapatite. Identifying unusual circumstances of F- exposure is key for diagnosing non-endemic SF. Increased awareness of the disorder can be lifesaving.

Aptamer-based proximity labeling guides covalent RNA modification.

We describe the development of a proximity-induced bio-orthogonal inverse electron demand Diels-Alder reaction that exploits the high-affinity interaction between a dienophile-modified RhoBAST aptamer and its tetramethyl rhodamine methyltetrazine substrate. We applied this concept for covalent RNA labeling in proof-of-principle experiments.

Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics.

Overcoming limitations of previous fluorescent light-up RNA aptamers for super-resolution imaging, we present RhoBAST, an aptamer that binds a fluorogenic rhodamine dye with fast association and dissociation kinetics. Its intermittent fluorescence emission enables single-molecule localization microscopy with a resolution not limited by photobleaching. We use RhoBAST to image subcellular structures of RNA in live and fixed cells with about 10-nm localization precision and a high signal-to-noise ratio.

Synthesis, Magnetic Properties, and Catalytic Properties of a Nickel(II)-Dependent Biomimetic of Metallohydrolases.

A dinickel(II) complex of the ligand 1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol (HL1) has been prepared and characterized to generate a functional model for nickel(II) phosphoesterase enzymes. The complex, [Ni2(L1)(µ-OAc)(H2O)2](ClO4)2·H2O, was characterized by microanalysis, X-ray crystallography, UV-visible, and IR absorption spectroscopy and solid state magnetic susceptibility measurements. Susceptibility studies show that the complex is antiferromagnetically coupled with the best fit parameters J = -27.4 cm-1, g = 2.29, D = 28.4 cm-1, comparable to corresponding values measured for the analogous dicobalt(II) complex [Co2(L1)(µ-OAc)](ClO4)2·0.5 H2O (J = -14.9 cm-1 and g = 2.16). Catalytic measurements with the diNi(II) complex using the substrate bis(2,4-dinitrophenyl)phosphate (BDNPP) demonstrated activity toward hydrolysis of the phosphoester substrate with K m ~10 mM, and k cat ~0.025 s-1. The combination of structural and catalytic studies suggests that the likely mechanism involves a nucleophilic attack on the substrate by a terminal nucleophilic hydroxido moiety.

Metallohydrolase biomimetics with catalytic and structural flexibility.

The structural and functional properties of zinc(ii) complexes of two nitrogen rich polydentate ligands, HTPDP = 1,3-bis(bis-pyridin-2-ylmethylamino)propan-2-ol and HTPPNOL = N,N,N'-tris-(2-pyridylmethyl)-1,3-diaminopropan-2-ol, are compared. HTPDP is a hepta-dentate ligand with four pyridyl groups attached to a 1,3-diaminopropan-2-ol backbone while HTPPNOL contains only three pyridyl groups. In reactions with Zn(ClO4)2, HTPDP forms a dinuclear zinc compound [Zn2(TPDP)(OAc)](ClO4)2, 1. On the other hand, mononuclear [Zn(HTPPNOL)](ClO4)2, 2, and tetranuclear [Zn4(TPPNOL)2(OAc)3](ClO4)3, 3, complexes were isolated with the ligand HTPPNOL. Kinetic measurements with the substrate bis(2,4-dinitrophenyl)phosphate (BDNPP) revealed that compound 1 (kcat = 31.4 × 10-3 min-1) is more reactive than 3 (kcat = 7.7 × 10-3 min-1) at pH = 8.5, whilst the mononuclear compound 2 is inactive. Compound 1 displays a typical steady-state kinetic behaviour, while compound 3 exhibits steady-state behaviour only ∼120 s after starting the reaction, preceded by a burst-phase. 31P NMR studies confirm that 1 can promote the hydrolysis of both ester bonds in BDNPP, generating the monoester DNPP and inorganic phosphate in the process. In contrast, DNPP is not a substrate for 3. The crystal structure of the complex formed by 3 and DNPP reveals the formation of a tetranuclear zinc complex [Zn4(TPPNOL)2(DNPP)2](ClO4)2, 4, in which the phosphate moiety of DNPP adopts an unusual tridentate µ-η1:η1:η1 coordination mode.

Clinical case of the month: a 22-year-old man with AIDS presenting with shortness of breath and an oral lesion.

Since the development of combination antiretroviral therapy (cART), the incidence and mortality associated with Kaposi sarcoma (KS) have been reduced, although not eliminated. Clinical presentations of KS range from simple skin involvement to disseminated disease, including involvement of the oral cavity and viscera, which portends a more ominous prognosis. Multiple case reports and data from clinical trials indicate that administration of systemic corticosteroids may aggravate KS. We present a case of disseminated KS following administration of prednisone for presumed immune reconstitution inflammatory syndrome (IRIS) associated with fungal pneumonia in an HIV-infected individual. The discussion that follows outlines the pathophysiology and clinical presentations associated with KS and existing data for the role of corticosteroids in promoting KS progression.

An unexpected silver lining to Katrina: elimination of inter-campus transfer delay in STEMI care.

BACKGROUND: Timely primary percutaneous coronary intervention (PCI) is recommended for ST-elevation myocardial infarction (STEMI), with increasing focus being paid to the time required for inter-hospital transfer. An overlooked barrier to timely care is inter-campus transfer when the emergency department (ED) and cardiac catheterization lab (CCL) are in separate buildings. In August 2005, Hurricane Katrina closed one campus of the Medical Center of Louisiana at New Orleans (MCLNO), forcing the ED and CCL to move into one building. We studied the impact of that closure on door-to-balloon times (DTB). METHODS: DTB for all STEMI patients between 1/04 and 6/11 were analyzed (as reported in MCLNO's Core Measures Report). National recommendations (i.e. single-pager activation, CCL ready within 30 minutes, etc.) were implemented prior to 2004; the consolidation of clinical services under one roof merely eliminated inter-campus transfer. RESULTS: In 2004-5, 28 patients presented with STEMI with a DTB of 156.5 +/- 62.6 minutes. From 2006-11, 97 patients presented with STEMI with a DTB of 90.7 +/- 54.3 minutes. The percentage of patients treated < or = 90 minutes increased from 10.3% to 67.0%. CONCLUSIONS: Along with other measures to reduce DTB, attention needs to be focused on inter-campus transfer. Hospital design should include attention to this element of delay in care. The closure of one campus allowed for significant system improvement at MCLNO.