Increased microRNA-323-3p in IL-22/IL-17-producing T cells and asthma: a role in the regulation of the TGF-ß pathway and IL-22 production.

BACKGROUND: IL-22- and IL-17-producing T cells have important roles in allergic diseases. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and modulate numerous biological processes. Little is known about the functions of miRNAs in IL-22/IL-17-producing T cells. MATERIAL AND METHODS: IL-22- and IL-17-positive T cells were sorted from human peripheral blood mononuclear cells (PBMCs) by intracellular staining and dual-secretion assay. miRNA expression profiles were detected with TaqMan array microfluidic cards. T cells were transfected with miRNA mimics. Gene expression was analyzed using RT-qPCR and/or enzyme-linked immunosorbent assay in T-cell subsets and PBMCs from patients with asthma and atopic dermatitis. RESULTS: The increased expression of miR-323-3p and noncoding RNA nc886 and reduced expression of miR-93, miR-181a, miR-26a, and miR-874 were detected in IL-22-producing T cells. The pathway analysis of the putative targets suggested that these differentially expressed miRNAs could impact the proliferation, differentiation, and effector functions of T cells. Further analyses showed the highest expression for miR-323-3p in IL-22- and IL-17-double-positive T cells and its capacity to suppress multiple genes from the transforming growth factor-ß pathway and the production of IL-22 in T cells. An increased expression of miR-323-3p in PBMCs from patients with asthma and reverse correlation between miR-323-3p levels and IL-22 production in PBMCs cultured in T-cell growth conditions was observed. CONCLUSIONS: Our data suggest that miR-323-3p acts in a negative feedback loop to control the production of IL-22 in IL-22/IL-17-producing T cells and might thus impact the T-cell responses in asthma.

Human rhinoviruses enter and induce proliferation of B lymphocytes.

BACKGROUND: Human rhinoviruses (HRVs) are one of the main causes of virus-induced asthma exacerbations. Infiltration of B lymphocytes into the subepithelial tissue of the lungs has been demonstrated during rhinovirus infection in allergic individuals. However, the mechanisms through which HRVs modulate the immune responses of monocytes and lymphocytes are not yet well described. OBJECTIVE: To study the dynamics of virus uptake by monocytes and lymphocytes, and the ability of HRVs to induce the activation of in vitro-cultured human peripheral blood mononuclear cells. METHODS: Flow cytometry was used for the enumeration and characterization of lymphocytes. Proliferation was estimated using 3 H-thymidine or CFSE labeling and ICAM-1 blocking. We used bead-based multiplex assays and quantitative PCR for cytokine quantification. HRV accumulation and replication inside the B lymphocytes was detected by a combination of in situ hybridization (ISH), immunofluorescence, and PCR for positive-strand and negative-strand viral RNA. Cell images were acquired with imaging flow cytometry. RESULTS: By means of imaging flow cytometry, we demonstrate a strong and quick binding of HRV types 16 and 1B to monocytes, and slower interaction of these HRVs with CD4+ T cells, CD8+ T cells, and CD19+ B cells. Importantly, we show that HRVs induce the proliferation of B cells, while the addition of anti-ICAM-1 antibody partially reduces this proliferation for HRV16. We prove with ISH that HRVs can enter B cells, form their viral replication centers, and the newly formed virions are able to infect HeLa cells. In addition, we demonstrate that similar to epithelial cells, HRVs induce the production of pro-inflammatory cytokines in PBMCs. CONCLUSION: Our results demonstrate for the first time that HRVs enter and form viral replication centers in B lymphocytes and induce the proliferation of B cells. Newly formed virions have the capacity to infect other cells (HeLa). These findings indicate that the regulation of human rhinovirus-induced B-cell responses could be a novel approach to develop therapeutics to treat the virus-induced exacerbation of asthma.

Spontaneous emission of a nanoscopic emitter in a strongly scattering disordered medium.

Fluorescence lifetimes of nitrogen-vacancy color centers in individual diamond nanocrystals were measured at the interface between a glass substrate and a strongly scattering medium. Comparison of the results with values recorded from the same nanocrystals at the glass-air interface revealed fluctuations of fluorescence lifetimes in the scattering medium. After discussing a range of possible systematic effects, we attribute the observed lengthening of the lifetimes to the reduction of the local density of states. Our approach is very promising for exploring the strong threedimensional localization of light directly on the microscopic scale.

Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins.

Fluorescent proteins with long emission wavelengths are particularly attractive for deep tissue two-photon microscopy. Surprisingly, little is known about their two-photon absorption (2PA) properties. We present absolute 2PA spectra of a number of orange and red fluorescent proteins, including DsRed2, mRFP, TagRFP, and several mFruit proteins, in a wide range of excitation wavelengths (640-1400 nm). To evaluate 2PA cross section (sigma(2)), we use a new method relying only on the optical properties of the intact mature chromophore. In the tuning range of a mode-locked Ti:sapphire laser, 700-1000 nm, TagRFP possesses the highest two-photon cross section, sigma(2) = 315 GM, and brightness, sigma(2)phi = 130 GM, where phi is the fluorescence quantum yield. At longer wavelengths, 1000-1100 nm, tdTomato has the largest values, sigma(2) = 216 GM and sigma(2)phi = 120 GM, per protein chain. Compared to the benchmark EGFP, these proteins present 3-4 times improvement in two-photon brightness.

Resonance enhancement of two-photon absorption in fluorescent proteins.

We measure two-photon absorption (2PA) spectra of wild-type green fluorescent protein, cyan fluorescent protein, and monomeric red fluorescent protein in absolute cross section values in a wide spectral range (lambda2PA = 550 - 1300 nm), and find, for the first time to our knowledge, a new S0 --> Sn 2PA transition in all three proteins in the short-wavelength region. This transition is strongly resonantly enhanced, showing 2PA cross section values of approximately 20-160 GM, which are at least 2-4 times higher than those measured in the lowest energy (S0 --> S1) transition of corresponding proteins. We also show that the change of permanent dipole moment upon S0 --> S1 excitation (|Deltamu10|) can be deduced from 2PA cross section, providing a new tool for fast evaluation of |Deltamu10| in physiological conditions.

U82, a novel snoRNA identified from the fifth intron of human and mouse nucleolin gene.

A novel snoRNA, designated as U82, was found from the sequence analysis of the 5th intron of human and mouse nucleolin gene. The snoRNA U82 has characteristic boxes C, D and D' and 11 nucleotides (nt) antisense complementarity to the 18S rRNA. Presumably U82 functions as a guide in the methylation of residue A1678 in human 18S rRNA. Northern blot analysis with various oligodeoxynucleotide probes showed that human and mouse U82 is expressed as RNA variants with length of 70 (+/- 1) and 67 (+/- 1) nt in HeLa and mouse C127 cells. Most probably, the 70 nt variant of U82 is encoded by nucleolin gene 5th intron. The 67 nt variant of U82 could be a transcript of another gene, the genomic locus of which remains unknown.

Long- and Short-Range Electrostatic Fields in GFP Mutants: Implications for Spectral Tuning.

The majority of protein functions are governed by their internal local electrostatics. Quantitative information about these interactions can shed light on how proteins work and allow for improving/altering their performance. Green fluorescent protein (GFP) and its mutation variants provide unique optical windows for interrogation of internal electric fields, thanks to the intrinsic fluorophore group formed inside them. Here we use an all-optical method, based on the independent measurements of transition frequency and one- and two-photon absorption cross sections in a number of GFP mutants to evaluate these internal electric fields. Two physical models based on the quadratic Stark effect, either with or without taking into account structural (bond-length) changes of the chromophore in varying field, allow us to separately evaluate the long-range and the total effective (short- and long-range) fields. Both types of the field quantitatively agree with the results of independent molecular dynamic simulations, justifying our method of measurement.

A novel snoRNA (U73) is encoded within the introns of the human and mouse ribosomal protein S3a genes.

The mouse ribosomal protein S3a-encoding gene (mRPS3a) was cloned and sequenced in this study. mRPS3a shares identical exon/intron structure with its human counterpart. Both genes are split to six exons and exhibit remarkable conservation of the promoter region (68.8% identity in the 250 bp upstream of cap site) and coding region (the proteins differ in two amino acids). mRPS3a displays many features common to other r-protein genes, including the CpG-island at 5'-end of the gene, cap site within an oligopyrimidine tract and no consensus TATA or CAAT boxes. However, mRPS3a represents a rare subclass of r-protein genes that possess a long coding sequence in the first exon. Comparison of human and mouse S3a genes revealed sequence fragments with striking similarity within introns 3 and 4. Here we demonstrate that these sequences encode for a novel small nucleolar RNA (snoRNA) designated U73. U73 contains C, D and D' boxes and a 12-nucleotide antisense complementarity to the 28S ribosomal RNA. These features place U73 into the family of intron-encoded antisense snoRNAs that guide site-specific 2'-O-ribose methylation of pre-rRNA. We propose that U73 is involved in methylation of the G1739 residue of the human 28S rRNA. In addition, we present the mapping of human ribosomal protein S3a gene (hRPS3a) and internally nested U73 gene to the human chromosome 4q31.2-3.

Human ribosomal protein S3a: cloning of the cDNA and primary structure of the protein.

The amino acid (aa) sequence of human ribosomal protein S3a (hRPS3a) was deduced partially from the nucleotide sequence of the corresponding cDNA and confirmed by direct aa sequencing from the N terminus of the purified hRPS3a protein. The cDNA clone was isolated from a cDNA expression library in the pEX vector using antibodies. The hRPS3a protein has 263 aa and its calculated M(r) is 29 813.

All-Optical Sensing of the Components of the Internal Local Electric Field in Proteins.

Here, we present a new all-optical method of interrogation of the internal electric field vector inside proteins. The method is based on experimental evaluation of the permanent dipole moment change upon excitation and the pure electronic transition frequency of a fluorophore embedded in a protein matrix. The permanent dipole moment change can be obtained from two-photon absorption measurements. In addition, permanent dipole moment change, tensor of polarizability change, and transition frequency for the free chromophore should be calculated quantum-mechanically. This allows obtaining the components of the electric field by considering the second-order Stark shift. We use the fluorescent protein mCherry as an example to demonstrate the applicability of the method.

Describing two-photon absorptivity of fluorescent proteins with a new vibronic coupling mechanism.

Fluorescent proteins (FPs) are widely used in two-photon microscopy as genetically encoded probes. Understanding the physical basics of their two-photon absorption (2PA) properties is therefore crucial for creation of two-photon brighter mutants. On the other hand, it can give us better insight into molecular interactions of the FP chromophore with a complex protein environment. It is known that, compared to the one-photon absorption spectrum, where the pure electronic transition is the strongest, the 2PA spectrum of a number of FPs is dominated by a vibronic transition. The physical mechanism of such intensity redistribution is not understood. Here, we present a new physical model that explains this effect through the "Herzberg-Teller"-type vibronic coupling of the difference between the permanent dipole moments in the ground and excited states (Δµ) to the bond-length-alternating coordinate. This model also enables us to quantitatively describe a large variability of the 2PA peak intensity in a series of red FPs with the same chromophore through the interference between the "Herzberg-Teller" and Franck-Condon terms.

Near-infrared two-photon absorption in phthalocyanines: enhancement of lowest gerade-gerade transition by symmetrical electron-accepting substitution.

This paper presents, to the best of our knowledge, the first study of two-photon absorption (2PA) spectra of a number of symmetrically substituted phthalocyanines in the excitation wavelength region from lambda(ex)=800 to 1600 nm. The selected molecules vary by position of substitution (alpha or beta), number of substituent groups (4, 8, or 16), and presence or absence of metal (Zn or Al) in the center. For all phthalocyanines we find a moderately strong (sigma(2) approximately 100-200 GM), pure electronic, gerade-gerade (g-g) 2PA transition, which shows up as a well-resolved relatively narrow peak in the energy region between Q and B bands (lambda(ex)=870-1100 nm). In metallophthalocyanines (MPcs) this lowest g-g transition is followed by the onset of other higher-frequency 2PA transitions. In some metal-free phthalocyanines (H(2)Pcs) we also reveal a second, broader 2PA transition at slightly higher frequency. In both MPcs and H(2)Pcs, we find a strong monotonic increase of integrated strength of the lowest g-g transition as a function of electron-accepting ability of peripheral substituents, expressed as their aggregated Hammett constant. By using few essential states models (three states for MPcs and four states for H(2)Pcs) we demonstrate the primary role of excited-state transition dipole moment in this effect.

Cooperative activation of transcription by autoimmune regulator AIRE and CBP.

Autoimmune regulator (AIRE) is a transcriptional regulator that is believed to control the expression of tissue-specific genes in the thymus. Mutated AIRE is responsible for onset of the hereditary autoimmune disease APECED. AIRE is able to form nuclear bodies (NBs) and interacts with the ubiquitous transcriptional coactivator CBP. In this paper, we show that CBP and AIRE synergistically activate transcription on different promoter reporters whereas AIRE gene mutation R257X, found in APECED patients, interferes with this coactivation effect. Furthermore, the overexpression of AIRE and CBP collaboratively enhance endogenous IFNbeta mRNA expression. The immunohistochemical studies suggest that CBP, depending on the balance of nuclear proteins, is a component of AIRE NBs. We also show that AIRE NBs are devoid of active chromatin and, therefore, not sites of transcription. In addition, we demonstrate by 3D analyses that AIRE and CBP, when colocalizing, are located spatially differently within AIRE NBs. In conclusion, our data suggest that AIRE activates transcription of the target genes, i.e., autoantigens in collaboration with CBP and that this activation occurs outside of AIRE NBs.

Holographic interferometry of ultrafast transients by photochemical hole burning.

A novel approach for interferometric analysis of ultrafast transient processes as short as 10(-13)-10(-14) sec is suggested. The method of holographic storage of ultrafast time-space images in spectrally selective hole-burning materials is used. It is shown that differential interferometric comparison of time-space images, which are permanently stored, provides an effective means to resolve ultrafast transient signals in space and in the spectral domain.

Single femtosecond exposure recording of an image hologram by spectral hole burning in an unstable tautomer of a phthalocyanine derivative.

We demonstrate, for what we believe is the first time, recording of a femtosecond image hologram by illumination with a single pair of high-intensity femtosecond pulses in a broad inhomogeneous bandwidth spectral hole-burning material consisting of a polymer film doped with anthraceno-phthalocyanine dye molecules. High efficiency of spectral hole burning is achieved by preillumination of the sample at a low temperature to convert the dye molecules into an unstable tautomer form.

Dendrimer molecules with record large two-photon absorption cross section.

We report what is to our knowledge a record high value for an intrinsic two-photon absorption (TPA) cross section, sigma(2) = 11 x 10(-47)> cm>(4)> s photon(-1) molecule(-1), measured with femtosecond pulses in a new dendrimer molecule comprising 29 repeat units of 4, 4(?)-bis(diphenylamino)stilbene chromophore. We measure the dependence of TPA on excitation wavelength in three consecutive generations of the dendrimer and show that the maximum sigma(2) value increases faster than the total number of stilbene chromophores. This result indicates that it is possible to obtain even larger sigma(2) values in higher generations of this dendrimer family.

Noncollinear parametric generation in LiIO(3) and beta-barium borate by frequency-doubled femtosecond Ti:sapphire laser pulses.

In LiIO(3) and BBO crystals the wave-matching conditions for femtosecond noncollinear parametric light generation at lambda = 390 nm pumping wavelength are investigated. In the LiIO(3) crystal simultaneous phase- and group-velocity-matching angles are determined. Parametric generation occurred at 0.45-2.9-mu;m wavelengths by pumping with the second harmonic of 150-fs Ti:sapphire laser pulses and is in qualitative agreement with calculated directions in both crystals.

Second-harmonic generation of amplified femtosecond Ti:sapphire laser pulses.

We study theoretically and experimentally second-harmonic generation (SHG) of 150-fs-duration amplified Ti:sapphire laser pulses at a wavelength of 780 nm in the nonlinear crystal KDP of different lengths and at different power densities as high as 150 GW cm(-2). The experimentally observed SHG conversion efficiency does not exceed 50%. It is shown theoretically that one possible process limiting the SHG efficiency at low as well as at high intensities is the modulation of the phase of the fundamental wave. In addition, continuum generation is observed at high intensities and can decrease the SHG efficiency.

Stimulated Raman scattering in hydrogen by frequency-doubled amplified femtosecond Ti:sapphire laser pulses.

We present results on stimulated Raman scattering in hydrogen excited by 200-fs-duration 1-kHz repetitionrate and 0.6-W average-power Ti:sapphire (780-nm) and 0.2-W average-power second-harmonic (390-nm) laser pulses with linear and circular polarizations. Spectral and temporal measurements of the vibrational and rotational components at Stokes and anti-Stokes frequencies in the wavelength range of 289-1154 nm are reported. We observe threefold shortening of the first vibrational Stokes component pulses, excited by 400-fsduration second-harmonic pulses.