Nebuliinivarianttien vaikutus nebuliini-aktiini-vuorovaikutukseen

Nemaline myopathy (NM) is a rare congenital disorder, the most common of congenital myopathies. It affects primarily the skeletal muscles and it is recognised by nemaline bodies in muscle tissue samples and muscle weakness. Mutation of eleven genes are known to lead to NM and the most frequent disease-causing variants are either recessive NEB variants or dominant ACTA1 variants. Variants in NEB are thought to be well tolerated and only 7% of them are hypothesized to be pathogenic. Over 200 pathogenic NEB-variants have been identified in Helsinki and the majority occurred in patients as a combination of two different variants. The missense variants were speculated to have a modifying effect on pathogenicity by affecting nebulin-actin or nebulin-tropomyosin interactions. Nebulin is a gigantic protein coded by NEB and is one of the largest proteins in vertebrates. It is located in the thin filament of the skeletal muscle sarcomere. Enclosed by terminal regions, nebulin has an extensive repetitive modular region that covers over 90% of the protein. The repetitive zone comprises of 26 modules called super repeats (SR). SRs consist of seven simple repeats. There are seven conserved SDXXYK actin-binding sites at each super repeat, one per simple repeat, and one conserved WLKGIGW tropomyosin-binding site. Due to its enormous size and highly repetitive sequence, nebulin is one of the least studied proteins in vivo, in vitro or in silico. In the NM patient database used for this study, there are 70 families with verified pathogenic mutations and in 30 of them, there were additional missense variants in NEB. These missense variants can be pathogenic modifying factors or have no impact on the phenotype. Seven missense variants were selected to study the effect of these mutations on actin-binding capacity compared to wild-type nebulin using the SR panel constructed previously by Laitila and Lehtonen. Also, due to the differences in actin-binding capacity of SRs compared to each other, one of the aims was to determine whether corresponding mutations in different SRs would have a similar or different effect on actin-binding capacity. For this aim, one missense mutation in the strongly actin-binding SR 1, and one in the weakly actin-binding SR 7 were selected from the NM database, and corresponding variants were created. Also, an in-frame deletion in SR7 found in the ExAC database and the corresponding mutation in SR1 were constructed for this study. The actin-binding strength was determined using actin co-sedimentation assay and actin affinity assay. The results for co-sedimentation assay indicate that missense variants can have an effect on nebulin-actin interactions and, therefore, can be a possible cause for NM. The corresponding mutations had no correlation in their effect on actin-binding strength, just the opposite. S1-m-2 decreased actin-binding strength of SR1 and S7-m-2 had no effect on SR7. Likewise, S7-m-1 and S7-del-1 decreased actin-binding strength of SR7 and corresponding mutations had no effect on SR1. The selected missense mutations found in NM patients in SRs 2 and 4 decreased actin-binding strength, if located at the actin-binding sites and in SR 10 increased the actin-binding strength, if located at the actin-binding site. The change in actin binding strength was defined as significant if the P-value was below 0.005. The more accurate affinity assay was performed as a trial only for S16 and S16-m-1, a variant at a tropomyosin-binding site close to an actin-binding site. It indicated a difference in actin-binding affinity missed by the actin co-sedimentation assay. The results are preliminary, but show big promise and should be optimized and implemented in the future missense mutation affinity studies. In an attempt to understand if the effect missense mutations have on nebulin-actin interaction is based on the change in nebulin structure, the 3D-structure of each produced fusion protein was predicted in silico. Considering that the variants were produced as GST-fusion proteins, the position and effect of GST in them is also a point of interest. In order to predict the structure of these large proteins, a combined approach was implemented using I-TASSER (Iterative Threading ASSEmbly Refinement) software. The software uses ab initio modeling, threading methods and atomic-level structure refinement to build an accurate 3D-model of a protein from sequence. According to the predicted 3D models of the fusion proteins, the GST-part of the proteins folds into a globular structure and acts as a core around which the nebulin fragments fold. The GST does not bind to actin and is positioned on the inside, which indicates minimal effect on nebulin-actin interaction, but may be a reason for an alternative nebulin fragment folding. The accuracy of the default set of programs in software does not give the definitive answer of the possible effect missense mutations can have on structural changes. However, I-TASSER approach for 3D-modeling is promising with further software optimization and can possibly serve as an effective bioinformatic tool in the future.Nemaliinimyopatia (NM) on harvinainen synnynnäinen sairaus, mutta yleisin syntyperäisistä lihastaudeista. Se vaikuttaa ensisijaisesti luustolihaksiin ja se tunnistetaan nemaliinikappaleista lihaskoepaloissa ja lihasheikkoudesta. Tällä hetkellä on tunnistettu mutaatioita 11 geenissä, jotka johtavat NM:aan ja yleisimmät sairautta aiheuttavat variantit ovat joko resessiiviset NEB-variantit tai dominantit ACTA1-variantit. NEB-varianttien uskotaan olevan hyvin siedettyjä ja vain 7% niistä oletetaan olevan patogeenisiä. Helsingissä on tunnistettu yli 200 patogeenistä NEB-varianttia ja suurin osa niistä esiintyi kahden eri variantin yhdistelmänä. Missense-variantteilla on ajateltu olevan modifioiva vaikutus patogeenisyyteen vaikuttamalla nebuliini-aktiini- ja nebuliini-tropomyosiini-vuorovaikutuksiin. Nebuliini on jättikokoinen proteiini, jonka koodaa NEB-geeni, ja se on yksi suurimmista selkärankaisten proteiineista. Se sijaitsee luustolihaksen sarkomeerissä, ohuessa säikeessä. Suurin osa proteiinista, yli 90%, on toistuvaa modulaarista aluetta, jonka päissä sijaitsevat terminaaliset alueet. Toistoalue koostuu 26 moduulista, joita kutsutaan supertoistoiksi (super repeat, SR). SR:t rakentuvat seitsemästä yksinkertaisesta toistosta. Jokaisessa supertoistossa on seitsemän konservoitunutta SDXXYK-aktiinisitoutumiskohtaa, yksi per yksinkertainen toisto, ja yksi konservoitunut WLKGIGW-tropomyosiinisitoutumiskohtaa. Valtavan kokonsa ja hyvin toistuvan sekvenssinsä vuoksi nebuliini on yksi vähiten tutkituista proteiineista in vivo, in vitro tai in silico. Tässä tutkimuksessa käytettiin NM-potilastietokantaa, jossa 70:ssä perheessä on vahvistettu olevan patogeeninen mutaatio ja niistä 30:ssä on lisäksi löydetty missense-variantteja NEB:ssa. Nämä missense-variantit voivat olla patogeenisyyttä muuntelevia tekijöitä tai voi olla, ettei niillä ole vaikutusta fenotyyppiin. Seitsemän missense-varianttia oli valittu ja mutaatioiden vaikutukset nebuliinin aktiinisitomiskykyyn verrattuna villityyppi-nebuliiniin oli tutkittu käyttäen aiemmin Laitilan ja Lehtosen kehittämää SR-paneelia. Lisäksi, toistojaksojen aktiinisitoutumiskyky vaihtelee toisiinsa nähden, joten yhtenä tavoitteista oli selvittää, onko vastaavilla mutaatioilla eri SR:ssa samanlainen vai erilainen vaikutus aktiinin sitomiskykyyn. Tähän tarkoitukseen oli valittu yksi missense-mutaatio vahvasti sitovasta SR 1:stä ja yksi heikosti sitovasta SR7:stä NM-potilastietokannasta, ja rakennettiin vastaavat variantit molemmissa SR:ssa. Lisäksi, perustuen ExAC-tietokannasta löydettyyn in-frame deleetioon SR 7:ssä, tutkimusta varten oli rakennettu SR 7-variantti ja sitä vastaava SR 1-variantti. Aktiinisitoutumisen vahvuus määriteltiin käyttäen aktiinin ko-sedimentaatioanalyysia ja aktiiniaffiniteettianalyysia. Kosedimentaaatio-analyysin tulokset viittaavat siihen, että missense-varianteilla voi olla vaikutus nebuliini-aktiini-vuorovaikutukseen ja siten voi mahdollisesti aiheuttaa NM:aa. Vastaavien mutaatioiden vaikutukset aktiinin sitomisvahvuuteen eivät korreloineet, vaan olivat päinvastaisia. S1-m-2 vähensi SR1:n aktiinin sitomisvahvuutta ja S7-m-2:lla ei ollut siihen vaikutusta. Samalla tavalla S7-m-1 ja S7-del-1 laskivat SR7:n aktiinin sitomisvahvuutta ja vastaavilla mutaatioilla ei ollut vaikutusta. Potilastietokannasta valitut missense-mutaatiot SR2:ssa ja SR4:ssa laskivat aktiinin sitomisvahvuutta, mikäli ne sijaitsivat aktiinin sitoutumiskohdissa ja SR10:ssä aktiinin sitoutumiskohdissa olevat mutaatiot lisäsivät aktiinin sitomisvahvuutta. Aktiinin sitoutumisvahvuuden muutos määriteltiin merkitseväksi, jos sen P-arvo oli alle 0.005. Tarkempi affiniteettianalyysi suoritettiin kokeiluna vain S16:lle ja S16-m-1:lle, tropomyosiinin sitoutumiskohdassa ja lähellä aktiinin sitoutumiskohtaa sijaitsevalle variantille SR16:ssa. Affiniteettianalyysin tulosten mukaan näiden kahden nebuliinifragmentin aktiiniaffiniteetissä on eroa, jota ei havaittu aktiinin kosedimentaatioanalyysissa. Alustavat tulokset näyttävät lupaavilta ja affiniteetinmääritysmenetelmä tulisi optimoida ja käyttää tulevissa missense-mutaatioiden affiniteettitutkimuksissa. Selvittääksemme johtuuko missense-mutaatioiden vaikutus nebuliini-aktiini-vuorovaikutukseen muutoksesta nebuliinin rakenteessa, jokaisen tuotetun GST-nebuliini-fuusioproteiinin 3D-rakenne oli ennustettu in silico. GST:n sijainti ja vaikutus fuusioproteiinin rakenteeseen olivat myös mielenkiinnon kohteena. Mallien ennustamiseen käytettiin I-TASSER (Iterative Threading ASSEmbly Refinement)-ohjelmistoa, jossa yhdistyvät kolme proteiinien mallintamisen lähestymistapaa: ab initio-mallinnus, laskostumisen tunnistusmenetelmät ja atomitason rakenteen tarkennus. Ohjelmisto rakentaa tarkan 3D-mallin aminohapposekvenssistä. Ennustettujen 3D-mallien mukaan, fuusioproteiinien GST-osa laskostuu pallomaiseksi rakenteeksi ja toimii ytimenä, jonka ympärille nebuliinifragmentit laskostuvat. GST ei sido aktiinia ja sijaitsee rakenteen keskustassa, mikä viittaa siihen, että sen vaikutus nebuliini-aktiini-vuorovaikutukseen on hyvin pieni tai olematon. On kuitenkin mahdollista, että GST:n läsnäolo johtaa vaihtoehtoiseen nebuliinifragmenttien laskostumiseen. Ohjelmiston tarkkuus ei oletusohjelmilla anna selkeää vastausta mahdollisista missense-mutaatioiden vaikutuksista proteiinirakenteeseen. Kuitenkin, I-TASSER-lähestymistapa nebuliinivarianttien 3D-mallintamiseen näyttää lupaavalta ohjelmiston edelleenoptimisaatiolla ja se voi mahdollisesti toimia tehokkaana bioinformaattisena työkaluna tulevaisuudessa

A novel satellite mission concept for upper air water vapour, aerosol and cloud observations using integrated path differential absorption LiDAR limb sounding

We propose a new satellite mission to deliver high quality measurements of upper air water vapour. The concept centres around a LiDAR in limb sounding by occultation geometry, designed to operate as a very long path system for differential absorption measurements. We present a preliminary performance analysis with a system sized to send 75 mJ pulses at 25 Hz at four wavelengths close to 935 nm, to up to 5 microsatellites in a counter-rotating orbit, carrying retroreflectors characterized by a reflected beam divergence of roughly twice the emitted laser beam divergence of 15 µrad. This provides water vapour profiles with a vertical sampling of 110 m; preliminary calculations suggest that the system could detect concentrations of less than 5 ppm. A secondary payload of a fairly conventional medium resolution multispectral radiometer allows wide-swath cloud and aerosol imaging. The total weight and power of the system are estimated at 3 tons and 2,700 W respectively. This novel concept presents significant challenges, including the performance of the lasers in space, the tracking between the main spacecraft and the retroreflectors, the refractive effects of turbulence, and the design of the telescopes to achieve a high signal-to-noise ratio for the high precision measurements. The mission concept was conceived at the Alpbach Summer School 2010

Inverse problems in stellar occultation

Remote sensing plays a key role in monitoring the atmosphere, which becomes increasingly important for the Earth's changing environment. The stellar occultation technique is a novel method to monitor vertical distribution of ozone and other trace gases from the troposphere to the upper mesosphere. GOMOS (Global Ozone Monitoring by Occultation of Stars) flying on board the European Space Agency's ENVISAT satellite is the first operational stellar occultation instrument. Satellite measurements are indirect, and therefore inversion methods play a key role in the retrieval of atmospheric parameters. This dissertation is dedicated to inversion methods for stellar occultation measurements and to the optimization of retrievals. Optimization of retrievals by inclusion of a priori information about smoothness of atmospheric profiles is considered. Two methods are developed. One of them –"the target resolution method"– develops the classical Tikhonov regularization. The second method includes smoothness a priori information in the form of Bayesian optimal estimation. The methodology for creating a priori information about smoothness of atmospheric profiles is developed. This dissertation considers optimal selection of measurements based on information theory, aiming at optimal design of future instruments, as well as at improved efficiency of the current data processing. Two optimization problems, both taking the information content of the measurements as a criterion, are defined and discussed. The selecting procedures were developed, compared with each other and existing methodologies and applied to selection of the most informative channels for GOMOS measurements in the UV-Visible wavelength range. The dissertation presents a feasibility study for retrieval of temperature and density profiles from pointing measurements by stellar occultation instruments. This study introduces extra geophysical parameters that can be obtained from GOMOS measurements. The inversion methods developed are applied to GOMOS measurements. However, the methods are formulated in a general form that allows their application beyond the GOMOS mission.reviewe

Turbulent Diffusion and Turbulent Thermal Diffusion of Aerosols in Stratified Atmospheric Flows

The paper analyzes the phenomenon of turbulent thermal diffusion in the Earth atmosphere, its relation to the turbulent diffusion and its potential impact on aerosol distribution. This phenomenon was predicted theoretically more than 10 years ago and detected recently in the laboratory experiments. This effect causes a non-diffusive flux of aerosols in the direction of the heat flux and results in formation of long-living aerosol layers in the vicinity of temperature inversions. We demonstrated that the theory of turbulent thermal diffusion explains the GOMOS aerosol observations near the tropopause (i.e., the observed shape of aerosol vertical profiles with elevated concentrations located almost symmetrically with respect to temperature profile). In combination with the derived expression for the dependence of the turbulent thermal diffusion ratio on the turbulent diffusion, these measurements yield an independent method for determining the coefficient of turbulent diffusion at the tropopause. We evaluated the impact of turbulent thermal diffusion to the lower-troposphere vertical profiles of aerosol concentration by means of numerical dispersion modelling, and found a regular upward forcing of aerosols with coarse particles affected stronger than fine aerosols.Comment: 19 pages, 10 figure

Validation of Copernicus Sentinel-3/OLCI Level 2 Land Integrated Water Vapour product

Validation of the Integrated Water Vapour (IWV) from Sentinel-3 Ocean and Land Colour Instrument (OLCI) was performed as a part of the “ESA/Copernicus Space Component Validation for Land Surface Temperature, Aerosol Optical Depth and Water Vapour Sentinel-3 Products” (LAW) project. High-spatial-resolution IWV observations in the near-infrared spectral region from the OLCI instruments aboard the Sentinel-3A and Sentinel-3B satellites provide continuity with observations from MERIS (Medium Resolution Imaging Spectrometer). The IWV was compared with reference observations from two networks: GNSS (Global Navigation Satellite System) precipitable water vapour from the SuomiNet network and integrated lower tropospheric columns from radio-soundings from the IGRA (Integrated Radiosonde Archive) database. Results for cloud-free matchups over land show a wet bias of 7 %–10 % for OLCI, with a high correlation against the reference observations (0.98 against SuomiNet and 0.90 against IGRA). Both OLCI-A and OLCI-B instruments show almost identical results, apart from an anomaly observed in camera 3 of the OLCI-B instrument, where observed biases are lower than in other cameras in either instrument. The wavelength drift in sensors was investigated, and biases in different cameras were found to be independent of wavelength. Effect of cloud proximity was found to have almost no effect on observed biases, indicating that cloud flagging in the OLCI IWV product is sufficiently reliable. We performed validation of random uncertainty estimates and found them to be consistent with the statistical a posteriori estimates, but somewhat higher

A method for random uncertainties validation and probing the natural variability with application to TROPOMI on board Sentinel-5P total ozone measurements

In this paper, we discuss the method for validation of random uncertainties in the remote sensing measurements based on evaluation of the structure function, i.e., root-mean-square differences as a function of increasing spatiotemporal separation of the measurements. The limit at the zero mismatch provides the experimental estimate of random noise in the data. At the same time, this method allows probing of the natural variability of the measured parameter. As an illustration, we applied this method to the clear-sky total ozone measurements by the TROPOspheric Monitoring Instrument (TROPOMI) on board the Sentinel-5P satellite. We found that the random uncertainties reported by the TROPOMI inversion algorithm, which are in the range 1–2 DU, agree well with the experimental uncertainty estimates by the structure function. Our analysis of the structure function has shown the expected results on total ozone variability: it is significantly smaller in the tropics compared to mid-latitudes. At mid-latitudes, ozone variability is much larger in winter than in summer. The ozone structure function is anisotropic (being larger in the latitudinal direction) at horizontal scales larger than 10–20 km. The structure function rapidly grows with the separation distance. At mid-latitudes in winter, the ozone values can differ by 5 % at separations 300–500 km. The method discussed is a powerful tool in experimental estimates of the random noise in data and studies of natural variability, and it can be used in various applications.Peer reviewe

Ice nucleation by viruses and their potential for cloud glaciation

In order to effectively predict the formation of ice in clouds we need to know which subsets of aerosol particles are effective at nucleating ice, how they are distributed and where they are from. A large proportion of ice-nucleating particles (INPs) in many locations are likely of biological origin, and some INPs are extremely small, being just tens of nanometres in size. The identity and sources of such INPs are not well characterized. Here, we show that several different types of virus particles can nucleate ice, with up to about 1 in 20 million virus particles able to nucleate ice at -20 degrees C. In terms of the impact on cloud glaciation, the ice-nucleating ability (the fraction which are ice nucleation active as a function of temperature) taken together with typical virus particle concentrations in the atmosphere leads to the conclusion that virus particles make a minor contribution to the atmospheric ice-nucleating particle population in the terrestrial-influenced atmosphere. However, they cannot be ruled out as being important in the remote marine atmosphere. It is striking that virus particles have an ice-nucleating activity, and further work should be done to explore other types of viruses for both their ice-nucleating potential and to understand the mechanism by which viruses nucleate ice.Peer reviewe

Aerosol generation during general anesthesia is comparable to coughing : An observational clinical study

Background Intubation, laryngoscopy, and extubation are considered highly aerosol-generating procedures, and additional safety protocols are used during COVID-19 pandemic in these procedures. However, previous studies are mainly experimental and have neither analyzed staff exposure to aerosol generation in the real-life operating room environment nor compared the exposure to aerosol concentrations generated during normal patient care. To assess operational staff exposure to potentially infectious particle generation during general anesthesia, we measured particle concentration and size distribution with patients undergoing surgery with Optical Particle Sizer. Methods A single-center observative multidisciplinary clinical study in Helsinki University Hospital with 39 adult patients who underwent general anesthesia with tracheal intubation. Mean particle concentrations during different anesthesia procedures were statistically compared with cough control data collected from 37 volunteers to assess the differences in particle generation. Results This study measured 25 preoxygenations, 30 mask ventilations, 28 intubations, and 24 extubations. The highest total aerosol concentration of 1153 particles (p)/cm(3) was observed during mask ventilation. Preoxygenations, mask ventilations, and extubations as well as uncomplicated intubations generated mean aerosol concentrations statistically comparable to coughing. It is noteworthy that difficult intubation generated significantly fewer aerosols than either uncomplicated intubation (p = .007) or coughing (p = 0.006). Conclusions Anesthesia induction generates mainly small (Peer reviewe

Harmonized Dataset of Ozone Profiles from Satellite Limb and Occultation Measurements

In this paper, we present a HARMonized dataset of OZone profiles (HARMOZ) based on limb and occultation measurements from Envisat (GOMOS, MIPAS and SCIAMACHY), Odin (OSIRIS, SMR) and SCISAT (ACE-FTS) satellite instruments. These measurements provide high-vertical-resolution ozone profiles covering the altitude range from the upper troposphere up to the mesosphere in years 2001-2012. HARMOZ has been created in the framework of the European Space Agency Climate Change Initiative project. The harmonized dataset consists of original retrieved ozone profiles from each instrument, which are screened for invalid data by the instrument teams. While the original ozone profiles are presented in different units and on different vertical grids, the harmonized dataset is given on a common pressure grid in netCDF (network common data form)-4 format. The pressure grid corresponds to vertical sampling of similar to 1 km below 20 km and 2-3 km above 20 km. The vertical range of the ozone profiles is specific for each instrument, thus all information contained in the original data is preserved. Provided altitude and temperature profiles allow the representation of ozone profiles in number density or mixing ratio on a pressure or altitude vertical grid. Geolocation, uncertainty estimates and vertical resolution are provided for each profile. For each instrument, optional parameters, which are related to the data quality, are also included. For convenience of users, tables of biases between each pair of instruments for each month, as well as bias uncertainties, are provided. These tables characterize the data consistency and can be used in various bias and drift analyses, which are needed, for instance, for combining several datasets to obtain a long-term climate dataset. This user-friendly dataset can be interesting and useful for various analyses and applications, such as data merging, data validation, assimilation and scientific research. The dataset is available at http://www.esa-ozone-cci.org/?q=node/161 or at doi: 10.5270/esa-ozone_cci-limb_occultation_profiles-2001_2012-v_1-201308