Rhodamines with a Chloronicotinic Acid Fragment for Live Cell Superresolution STED Microscopy*.

Formylation of 2,6-dichloro-5-R-nicotinic acids at C-4 followed by condensation with 3-hydroxy-N,N-dimethylaniline gave analogs of the popular TAMRA fluorescent dye with a 2,6-dichloro-5-R-nicotinic acid residues (R=H, F). The following reaction with thioglycolic acid is selective, involves only one chlorine atom at the carbon between pyridine nitrogen and the carboxylic acid group and affords new rhodamine dyes absorbing at 564/ 573 nm and emitting at 584/ 597 nm (R=H/ F, in aq. PBS). Conjugates of the dyes with "small molecules" provided specific labeling (covalent and non-covalent) of organelles as well as of components of the cytoskeleton in living cells and were combined with fluorescent probes prepared from 610CP and SiR dyes and applied in two-color STED microscopy with a 775 nm STED laser.

Green-Emitting Rhodamine Dyes for Vital Labeling of Cell Organelles Using STED Super-Resolution Microscopy.

Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell-permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green-emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super-resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining.

Photoactivatable Rhodamine Spiroamides and Diazoketones Decorated with "Universal Hydrophilizer" or Hydroxyl Groups.

Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers applicable in multicolor super-resolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is often limited by aggregation, unspecific adhesion, and low reactivity caused by insufficient solubility and precipitation from aqueous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels decorated with a reactive group: attachment of 3-sulfo-l-alanyl-beta-alanine dipeptide (a "universal hydrophilizer") or allylic hydroxylation in photosensitive rhodamine diazoketones (and spiroamides). The super-resolution images of tubulin and keratin filaments in fixed and living cells exemplify the performance of "blinking" spiroamides derived from N, N, N', N'-tetramethyl rhodamine.

"Reduced" Coumarin Dyes with an O-Phosphorylated 2,2-Dimethyl-4-(hydroxymethyl)-1,2,3,4-tetrahydroquinoline Fragment: Synthesis, Spectra, and STED Microscopy.

Large Stokes-shift coumarin dyes with an O-phosphorylated 4-(hydroxymethyl)-2,2-dimethyl-1,2,3,4-tetrahydroquinoline fragment emitting in the blue, green, and red regions of the visible spectrum were synthesized. For this purpose, N-substituted and O-protected 1,2-dihydro-7-hydroxy-2,2,4-trimethylquinoline was oxidized with SeO2 to the corresponding α,ß-unsaturated aldehyde and then reduced with NaBH4 in a "one-pot" fashion to yield N-substituted and 7-O-protected 4-(hydroxymethyl)-7-hydroxy-2,2-dimethyl-1,2,3,4-tetrahydroquinoline as a common precursor to all the coumarin dyes reported here. The photophysical properties of the new dyes ("reduced coumarins") and 1,2-dihydroquinoline analogues (formal precursors) with a trisubstituted C=C bond were compared. The "reduced coumarins" were found to be more photoresistant and brighter than their 1,2-dihydroquinoline counterparts. Free carboxylate analogues, as well as their antibody conjugates (obtained from N-hydroxysuccinimidyl esters) were also prepared. All studied conjugates with secondary antibodies afforded high specificity and were suitable for fluorescence microscopy. The red-emitting coumarin dye bearing a betaine fragment at the C-3-position showed excellent performance in stimulation emission depletion (STED) microscopy.

Carboxylated Photoswitchable Diarylethenes for Biolabeling and Super-Resolution RESOLFT Microscopy.

Reversibly photoswitchable 1,2-bis(2-ethyl-6-phenyl-1-benzothiophene-1,1-dioxide-3-yl)perfluorocyclopentenes (EBT) having fluorescent "closed" forms were decorated with four or eight carboxylic groups and attached to antibodies. Low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and good photophysical properties were demonstrated. Alternating light pulses of UV and blue light induce numerous reversible photochemical transformations between two stables states with distinct structures. Using relatively low light intensities, EBTs were applied in biology-related super-resolution microscopy based on the reversible saturable (switchable) optical linear fluorescence transitions (RESOLFT) and demonstrated optical resolution of 75 nm.

Discovery of the novel HLA-B*39:198 allele, a variant of HLA-B*39:01:01, in a Russian individual.

One nucleotide substitution in codon 336 of HLA-B*39:01:01:01 results in the novel allele, HLA- B*39:198.

Characterization of the novel HLA-C*07:402:02 allele detected in a potential hematopoietic stem cell donor.

The novel HLA-C*07:402:02 allele was characterized using next-generation sequencing technology.

4-Trifluoromethyl-substituted coumarins with large Stokes shifts: synthesis, bioconjugates, and their use in super-resolution fluorescence microscopy.

Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetero)aryl- or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413-480 and 527-668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.

The identification of three novel HLA-C alleles: HLA-C*01:218, HLA-C*03:550 and HLA-C*05:01:60.

Three novel HLA-C alleles HLA-C*01:218, -C*03:550 and -C*05:01:60 alleles detected during routine next generation sequencing.

Characterization of the novel HLA-B*35:547 allele detected in a potential hematopoietic stem cell donor.

The novel HLA-B*35:547 allele was characterized using next generation sequencing technology.

Two novel HLA-DQB1 alleles identified in potential hematopoietic stem cell donors: DQB1*03:01:50 and -DQB1*03:453.

HLA-DQB1*03:01:50 and -DQB1*03:453, two novel alleles detected by next generation sequencing.

The detection of three novel HLA-DQB1 alleles: HLA-DQB1*02:186, -DQB1*06:02:49 and -DQB1*06:391.

Three novel HLA-DQB1 alleles HLA-DQB1*02:186, -DQB1*06:02:49, and -DQB1*06:391 alleles detected during routine next generation sequencing.

The detection of three novel HLA-A alleles: HLA-A*02:1037, -A*02:1038 and -A*02:1039.

Three novel HLA-A alleles HLA-A*02:1037, -A*02:1038 and -A*02:1039 alleles detected during routine next generation sequencing.

Characterization of the novel alleles: HLA-A*31:212, HLA-B*50:01:20 and HLA-C*03:593.

Three novel HLA alleles HLA-A*31:212, HLA-B*50:01:20 and HLA-C*03:593 alleles detected during routine next generation sequencing.

Identification and characterization of the novel HLA-B*39:189 allele by next-generation sequencing.

The novel HLA-B*39:189 allele was characterized using next generation sequencing technology.

Identification of the novel HLA-C*12:349 allele in a potential hematopoietic stem cell donor.

The novel HLA-C*12:349 allele was characterized using next generation sequencing technology.

Phosphorylated 3-heteroarylcoumarins and their use in fluorescence microscopy and nanoscopy.

Photostable and bright fluorescent dyes with large Stokes shifts are widely used as markers in far-field optical microscopy, but the variety of useful dyes is limited. The present study introduces new 3-heteroaryl coumarins decorated with a primary phosphate group (OP(O)(OH)(2)) attached to C-4 in 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused with the coumarin fluorophore. The general synthetic route is based on the Suzuki reaction of 3-bromocoumarines with hetarylboronic acids followed by oxidation of the methyl group at the C=C bond with SeO(2) (to an aldehyde), reduction with NaBH(4) (to an alcohol), and conversion into a primary phosphate. The 4 position in the coumarin system may be unsubstituted or bear a methyl group. Phosphorylated coumarins were found to have high fluorescence quantum yields in the free state and after conjugation with proteins (in aqueous buffers). In super-resolution light microscopy with stimulated emission depletion (STED), the new coumarin dyes provide an optical resolution of 40-60 nm with a low background signal. Due to their large Stokes shifts and high photostability, phosphorylated coumarins enable to combine multilabel imaging (using one detector and several excitation sources) with diffraction unlimited optical resolution.

The novel HLA-DQB1 allele, HLA-DQB1*04:72, detected in a potential hematopoietic stem cell donor.

Identification of the novel HLA-DQB1*04:72 allele that differs from DQB1*04:02:01:06 by two nucleotide substitutions.

The novel HLA-DQB1*03:445 allele was identified in two unrelated bone marrow donors from Russia.

HLA-DQB1*03:445 has one nucleotide change from HLA-DQB1*03:03:02:01 at nucleotide 692.

The novel HLA-C*06:327 allele was identified in three unrelated bone marrow donors.

HLA-C*06:327 has one non-synonymous nucleotide change from C*06:02:01:01 at codon 222.