Chromophore Protonation State Controls Photoswitching of the Fluoroprotein asFP595

Fluorescent proteins have been widely used as genetically encodable fusion tags for biological imaging. Recently, a new class of fluorescent proteins was discovered that can be reversibly light-switched between a fluorescent and a non-fluorescent state. Such proteins can not only provide nanoscale resolution in far-field fluorescence optical microscopy much below the diffraction limit, but also hold promise for other nanotechnological applications, such as optical data storage. To systematically exploit the potential of such photoswitchable proteins and to enable rational improvements to their properties requires a detailed understanding of the molecular switching mechanism, which is currently unknown. Here, we have studied the photoswitching mechanism of the reversibly switchable fluoroprotein asFP595 at the atomic level by multiconfigurational ab initio (CASSCF) calculations and QM/MM excited state molecular dynamics simulations with explicit surface hopping. Our simulations explain measured quantum yields and excited state lifetimes, and also predict the structures of the hitherto unknown intermediates and of the irreversibly fluorescent state. Further, we find that the proton distribution in the active site of the asFP595 controls the photochemical conversion pathways of the chromophore in the protein matrix. Accordingly, changes in the protonation state of the chromophore and some proximal amino acids lead to different photochemical states, which all turn out to be essential for the photoswitching mechanism. These photochemical states are (i) a neutral chromophore, which can trans-cis photoisomerize, (ii) an anionic chromophore, which rapidly undergoes radiationless decay after excitation, and (iii) a putative fluorescent zwitterionic chromophore. The overall stability of the different protonation states is controlled by the isomeric state of the chromophore. We finally propose that radiation-induced decarboxylation of the glutamic acid Glu215 blocks the proton transfer pathways that enable the deactivation of the zwitterionic chromophore and thus leads to irreversible fluorescence. We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data. The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins. These insights can guide the rational design and optimization of photoswitchable proteins

Using microbiological sampling to evaluate the efficacy of nasofibroscope disinfection: The tristel trio wipes system in ear–nose–throat (ENT) endoscopy

Disinfection and sterilization are needed for guaranteeing that medical and surgical instruments do not spread contagious microorganisms to patients. The aim of this study was to evaluate the efficacy of a simple manual technique of high-level disinfection (HLD) of flexible fiberoptic nasofibroscopes (FFNs) with wipes impregnated with a chlorine dioxide solution (Tristel Trio Wipes System—TTW) against a conventional automated washer machine (Soluscope ENT, Cimrex 12—AW). FFNs used in 62 patients undergoing endoscopy at an ENT clinic were sampled according to an aseptic procedure. For each nasoendoscopy, microbiological samples were taken at two times: (1) after a patient’s nasoendoscopy and (2) immediately after high-level disinfection. Ten microliters of each prepared sample were inoculated onto specific culture media for the detection of nasopharyngeal flora microorganisms. The microbiological results obtained from 62 post-disinfection samples revealed bacterial growth on two FFNs disinfected with AW, and five FFNs disinfected with TTW, but this difference is not statistically significant. None of the isolates were pathogenic bacteria. Our results are different than the results obtained by two previously published studies on the TTW system. In both studies, sampling was carried out by swabbing the tip and the handle surface of FFNs. This sampling method was the least effective method means of detecting bacteria on a surface. It can be concluded that the two disinfection systems allow providers to obtain a reduction of the saprophytic and pathogenic microbial load

Ultra-flat wideband single-pump Raman-enhanced parametric amplification

We experimentally optimize a single pump fiber optical parametric amplifier in terms of gain spectral bandwidth and gain variation (GV). We find that optimal performance is achieved with the pump tuned to the zero-dispersion wavelength of dispersion stable highly nonlinear fiber (HNLF). We demonstrate further improvement of parametric gain bandwidth and GV by decreasing the HNLF length. We discover that Raman and parametric gain spectra produced by the same pump may be merged together to enhance overall gain bandwidth, while keeping GV low. Consequently, we report an ultra-flat gain of 9.6±0.5 dB over a range of 111 nm (12.8 THz) on one side of the pump. Additionally, we demonstrate amplification of a 60 Gbit/s QPSK signal tuned over a portion of the available bandwidth with OSNR penalty less than 1 dB for Q2 below 14 dB

Reduced AKT/mTOR signaling and protein synthesis dysregulation in a Rett syndrome animal model.

Rett syndrome (RTT) is a neurodevelopmental disorder with no efficient treatment that is caused in the majority of cases by mutations in the gene methyl-CpG binding-protein 2 (MECP2). RTT becomes manifest after a period of apparently normal development and causes growth deceleration, severe psychomotor impairment and mental retardation. Effective animal models for RTT are available and show morphofunctional abnormalities of synaptic connectivity. However, the molecular consequences of MeCP2 disruption leading to neuronal and synaptic alterations are not known. Protein synthesis regulation via the mammalian target of the rapamycin (mTOR) pathway is crucial for synaptic organization, and its disruption is involved in a number of neurodevelopmental diseases. We investigated the phosphorylation of the ribosomal protein (rp) S6, whose activation is highly dependent from mTOR activity. Immunohistochemistry showed that rpS6 phosphorylation is severely affected in neurons across the cortical areas of Mecp2 mutants and that this alteration precedes the severe symptomatic phase of the disease. Moreover, we found a severe defect of the initiation of protein synthesis in the brain of presymptomatic Mecp2 mutant that was not restricted to a specific subset of transcripts. Finally, we provide evidence for a general dysfunction of the Akt/mTOR, but not extracellular-regulated kinase, signaling associated with the disease progression in mutant brains. Our results indicate that defects in the AKT/mTOR pathway are responsible for the altered translational control in Mecp2 mutant neurons and disclosed a novel putative biomarker of the pathological process. Importantly, this study provides a novel context of therapeutic interventions that can be designed to successfully restrain or ameliorate the development of RTT

Mid-infrared optical parametric amplifier using silicon nanophotonic waveguides

All-optical signal processing is envisioned as an approach to dramatically decrease power consumption and speed up performance of next-generation optical telecommunications networks. Nonlinear optical effects, such as four-wave mixing (FWM) and parametric gain, have long been explored to realize all-optical functions in glass fibers. An alternative approach is to employ nanoscale engineering of silicon waveguides to enhance the optical nonlinearities by up to five orders of magnitude, enabling integrated chip-scale all-optical signal processing. Previously, strong two-photon absorption (TPA) of the telecom-band pump has been a fundamental and unavoidable obstacle, limiting parametric gain to values on the order of a few dB. Here we demonstrate a silicon nanophotonic optical parametric amplifier exhibiting gain as large as 25.4 dB, by operating the pump in the mid-IR near one-half the band-gap energy (E~0.55eV, lambda~2200nm), at which parasitic TPA-related absorption vanishes. This gain is high enough to compensate all insertion losses, resulting in 13 dB net off-chip amplification. Furthermore, dispersion engineering dramatically increases the gain bandwidth to more than 220 nm, all realized using an ultra-compact 4 mm silicon chip. Beyond its significant relevance to all-optical signal processing, the broadband parametric gain also facilitates the simultaneous generation of multiple on-chip mid-IR sources through cascaded FWM, covering a 500 nm spectral range. Together, these results provide a foundation for the construction of silicon-based room-temperature mid-IR light sources including tunable chip-scale parametric oscillators, optical frequency combs, and supercontinuum generators

PO-272 Leukemia-associated NPM mutations promote quiescence of hematopoietic stem cells and prevent their functional exhaustion upon oncogene-induced hyper-proliferation

Introduction Acute Myeloid Leukaemia (AML) is a heterogeneous and multi-step disease. The serial acquisition of mutations and the environmental pressure allow one or more clones to expand and contribute to the disease. In particular, 6% of AMLs are characterised by an initial mutation in the DNMT3a gene, followed by mutations in NPM (NPMc) and FLT3 loci (FLT3-ITD). We previously shown that NPMc can drive AML development in mouse model and highly cooperates with FLT3-ITD. Moreover, it has been reported that normal Hematopoietic Stem Cells (HSCs) of elderly people may bear some somatic early AML mutations and this correlate with an increased risk of hematologic diseases suggesting that mutations can shape pre-leukemic HSCs to be more prone to the acquisition of further mutations giving rise to Leukaemia Initiating Cells (LIC). While the ability of FLT3-ITD to drive HSC compartment exhaustion has been already shown, the impact of NPMc on HSCs remains unclear. Material and methods Taking advantage of the extended pre-leukemic phase of our inducible NPMc mouse model, we elucidate the role of NPMc in HSCs by functional and transcriptional analysis. Moreover, to investigate the basis of NPMc and FLT3-ITD cooperation we generate mice carrying both the conditional NPMc transgene and the FLT3-ITD constitutive mutation and, before AML onset, we analyse double mutant HSCs behaviour. Results and discussions We have found that NPMc expression lead to the expansion of the HSC compartment through the enforcement of a stem-cell transcriptional program that increases self-renewal by promoting quiescence. We then investigated how the NPMc dependent quiescence program is linked to its oncogenic function. The expression of NPMc +in the FLT3-ITD background prevents the HSCs exhaustion imposed by FLT3-ITD and restores their repopulating capacity. Accordingly, gene expression analysis revealed a strong dominance of NPMc +with the restoration of the same transcriptional program observed in the NPMc HSCs. These data strongly suggest that NPMc imposes a HSC-specific program that, in combination with the oncogenic signal provided by FLT3-ITD, allows the selection of the LIC and the occurrence of AML. Conclusion In conclusion, enforcement of quiescence might be a critical function for the maintenance of the transformed clone during both the pre-leukemic and the leukemic phase. As consequence, interfering with quiescence key determinants may eradicate the reservoir of quiescent cells responsible for disease recurrence

Estudios sobre anuros de Argentina durante la década 2010-2020: tendencias y vacíos de información

Scientific knowledge may present taxonomic, geographic and thematic biases, which could have negative consequences on conservation decisions, especially in highly threatened groups such as amphibians. This work aimed to explore the scientific production of anurans from Argentina during the 2010-2020 decade, to understand the current state of knowledge and identify possible biases and information gaps. Our results showed that the production of scientific knowledge on anurans from Argentina remained constant during the studied period. Nonetheless, we observed taxonomic, geographic and topic biases. The number of studies was higher in areas with higher human population density and on topics related to morphology, as well as for common species with medium body sizes, general habits, with a broad geographic distribution, and occurring in rural-urban areas. Our results represent a baseline to clarify and evidence the different biases of scientific knowledge about anurans from Argentina.La producción del conocimiento científico puede presentar sesgos taxonómicos, geográficos y temáticos, los que a su vez podrían tener consecuencias negativas al momento de tomar decisiones en conservación, sobre todo en grupos altamente amenazados como son los anfibios. El objetivo de este trabajo fue explorar la producción científica sobre anuros de Argentina durante la década 2010-2020, a fin de establecer el estado actual del conocimiento e identificar posibles sesgos y vacíos de información. Nuestros resultados sugieren que la producción del conocimiento científico en anuros de Argentina se mantuvo constante en el período estudiado. Se observaron sesgos en cuanto a la distribución geográfica de las publicaciones, en las especies más estudiadas y en los temas abordados. El número de estudios fue mayor en provincias con una densidad de su población más alta y en temas relacionados con morfología, así como para especies comunes con tamaños corporales medianos, de hábitos generalistas, con una amplia distribución geográfica, y con ocurrencia en zonas rurales-urbanas. Estos resultados constituyen un punto de partida para esclarecer y evidenciar los diferentes sesgos del conocimiento científico sobre anuros de Argentina.Asociación Herpetológica Argentin