Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the (conductive) P2 380 intermediate decays with τ ≈ 40 ms and 200 ms after pulsed excitation. The vibrational changes between the closed and the conductive states were analyzed in the X-H stretching region (X = O, S, N), comprising vibrational changes of water molecules, sulfhydryl groups of cysteine side chains and changes of the amide A of the protein backbone. The O-H stretching vibrations of ¿dangling¿ water molecules were detected in two different states of the protein using H2 18O exchange. Uncoupling experiments with a 1:1 mixture of H2O:D2O provided the natural uncoupled frequencies of the four O-H (and O-D) stretches of these water molecules, each with a very weakly hydrogen-bonded O-H group (3639 and 3628 cm−1) and with the other O-H group medium (3440 cm−1) to moderately strongly (3300 cm−1) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the ¿dangling¿ water molecules within the protein, providing a better understanding of their functional relevance in CaChR1

NRP2 transcriptionally regulates its downstream effector WDFY1.

Neuropilins (NRPs) are cell surface glycoproteins that often act as co-receptors for plexins and VEGF family receptors. Neuropilin-2 (NRP2), a family member of NRPs, was shown to regulate autophagy and endocytic trafficking in cancer cells, a function distinctly different from its role as a co-receptor. WD Repeat and FYVE domain containing 1 (WDFY1)-protein acts downstream of NRP2 for this function. Our results indicated that NRP2 maintains an optimum concentration of WDFY1 by negatively regulating its expression. Since increased expression of WDFY1 reduces the endocytic activity, maintenance of WDFY1 level is crucial in metastatic cancer cells to sustain high endocytic activity, essential for promotion of oncogenic activation and cancer cell survival. Here, we have delineated the underlying molecular mechanism of WDFY1 synthesis by NRP2. Our results indicated that NRP2 inhibits WDFY1 transcription by preventing the nuclear localization of a transcription factor, Fetal ALZ50-reactive clone 1 (FAC1). Our finding is novel as transcriptional regulation of a gene by NRP2 axis has not been reported previously. Regulation of WDFY1 transcription by NRP2 axis is a critical event in maintaining metastatic phenotype in cancer cells. Thus, inhibiting NRP2 or hyper-activating WDFY1 can be an effective strategy to induce cell death in metastatic cancer

The inhomogeneous ISM toward PKS1830-211 SW - A detailed view on molecular gas at a look-back time of 7.5 Gyr

Based on measurements with the Effelsberg 100-m telescope, a multi-line study of molecular species is presented toward the south-western source of the gravitational lens system PKS 1830-211, which is by far the best known target to study molecular gas in absorption at intermediate redshift. Determining line parameters and optical depths and performing Large Velocity Gradient radiative transfer calculations, the aims of this study are (1) to evaluate physical parameters of the absorbing foreground gas at z~0.89, in particular its homogeneity, and (2) to monitor the spectroscopic time variability caused by fluctuations of the z~2.5 background continuum source. We find, that the gas is quite inhomogeneous with n(H2)~2 x 10^3 cm^-3 for most molecular species but with higher values for H2CO and lower ones for SO. Measuring the CS J=1-0 transition during a time interval of more than a decade, from 2001 to 2012, the peak absorption depth of the line remains approximately constant, while the line shape undergoes notable variations. Covering the time between 1996 and 2013, CS, HCO+, and CH3OH data indicate maximal integrated optical depths in ~2001 and 2011/2012. This is compatible with a ~10 yr periodicity, which, however, needs confirmation by substantially longer time monitoring. Comparing molecular abundances with those of different types of Galactic and nearby extragalactic clouds we find that the observed cloud complex does not correspond to one particular type but to a variety of cloud types with more diffuse and denser components as can be expected for an observed region with a transverse linear scale of several parsec and a likely larger depth along the line-of-sight. A tentative detection of Galactic absorption in the c-C3H2 1(10)-1(01) line at 18.343 GHz is also reported.Comment: Accepted for publication in A&A, 11 pages, 10 figures, 4 table

The inhomogeneous ISM toward PKS 1830–211 SW: A detailed view of molecular gas at a look-back time of 7.5 Gyr

Based on measurements with the Effelsberg 100-m telescope, a multiline study of molecular species is presented toward the southwestern source of the gravitational lens system PKS 1830–211, which is by far the best known target for studying molecular gas in absorption at intermediate redshift. Determining line parameters and optical depths and performing large velocity gradient radiative transfer calculations, the aims of this study are (1) to evaluate physical parameters of the absorbing foreground gas at z ~ 0.89, in particular its homogeneity; and (2) to monitor the spectroscopic time variability caused by fluctuations in the z ~ 2.5 background continuum source. We find, that the gas is quite inhomogeneous with n(H2) ~ 2 × 103 cm-3 for most molecular species but with higher values for H2CO and lower ones for SO. Measuring the CS J = 1 ← 0 transition during a time interval of more than a decade, from 2001 to 2012, the peak absorption depth of the line remains approximately constant, while the line shape undergoes notable variations. Covering the time between 1996 and 2013, CS, HCO+, and CH3OH data indicate maximum integrated optical depths in ~2001 and 2011/2012. This is compatible with a ~10 yr periodicity, which, however, needs confirmation by substantially longer time monitoring. Comparing molecular abundances with those of different types of Galactic and nearby extragalactic clouds we find that the observed cloud complex does not correspond to one particular type but to a variety of cloud types with more diffuse and denser components as can be expected for an observed region with a transverse linear scale of several parsec and a likely greater depth along the line of sight. A tentative detection of Galactic absorption in the c-C3H2 110−101 line at 18.343 GHz is also reported

Validation of core competencies during residency training in anaesthesiology

Background and goal: Curriculum development for residency training is increasingly challenging in times of financial restrictions and time limitations. Several countries have adopted the CanMEDS framework for medical education as a model into their curricula of specialty training. The purpose of the present study was to validate the competency goals, as derived from CanMEDS, of the Department of Anaesthesiology and Intensive Care Medicine of the Berlin Charité University Medical Centre, by conducting a staff survey. These goals for the qualification of specialists stipulate demonstrable competencies in seven areas: expert medical action, efficient collaboration in a team, communications with patients and family, management and organisation, lifelong learning, professional behaviour, and advocacy of good health. We had previously developed a catalogue of curriculum items based on these seven core competencies. In order to evaluate the validity of this catalogue, we surveyed anaesthetists at our department in regard to their perception of the importance of each of these items. In addition to the descriptive acquisition of data, it was intended to assess the results of the survey to ascertain whether there were differences in the evaluation of these objectives by specialists and registrars

First 230 GHz VLBI Fringes on 3C 279 using the APEX Telescope

We report about a 230 GHz very long baseline interferometry (VLBI) fringe finder observation of blazar 3C 279 with the APEX telescope in Chile, the phased submillimeter array (SMA), and the SMT of the Arizona Radio Observatory (ARO). We installed VLBI equipment and measured the APEX station position to 1 cm accuracy (1 sigma). We then observed 3C 279 on 2012 May 7 in a 5 hour 230 GHz VLBI track with baseline lengths of 2800 M$\lambda$ to 7200 M$\lambda$ and a finest fringe spacing of 28.6 micro-arcseconds. Fringes were detected on all baselines with SNRs of 12 to 55 in 420 s. The correlated flux density on the longest baseline was ~0.3 Jy/beam, out of a total flux density of 19.8 Jy. Visibility data suggest an emission region <38 uas in size, and at least two components, possibly polarized. We find a lower limit of the brightness temperature of the inner jet region of about 10^10 K. Lastly, we find an upper limit of 20% on the linear polarization fraction at a fringe spacing of ~38 uas. With APEX the angular resolution of 230 GHz VLBI improves to 28.6 uas. This allows one to resolve the last-photon ring around the Galactic Center black hole event horizon, expected to be 40 uas in diameter, and probe radio jet launching at unprecedented resolution, down to a few gravitational radii in galaxies like M 87. To probe the structure in the inner parsecs of 3C 279 in detail, follow-up observations with APEX and five other mm-VLBI stations have been conducted (March 2013) and are being analyzed.Comment: accepted for publication in A&

The end of star formation in Chamaeleon I? A LABOCA census of starless and protostellar cores

Context. Chamaeleon I is the most active region in terms of star formation in the Chamaeleon molecular cloud complex. Although it is one of the nearest low-mass star forming regions, its population of prestellar and protostellar cores is not known and a controversy exists concerning its history of star formation. Aims. Our goal is to search for prestellar and protostellar cores and characterize the earliest stages of star formation in this cloud. Methods. We used the bolometer array LABOCA at the APEX telescope to map the cloud in dust continuum emission at 870 μm with a high sensitivity. This deep, unbiased survey was performed based on an extinction map derived from 2MASS data. The 870 μm map is compared with the extinction map and C18O observations, and decomposed with a multiresolution algorithm. The extracted sources are analysed by carefully taking into account the spatial filtering inherent in the data reduction process. A search for associations with young stellar objects is performed using Spitzer data and the SIMBAD database. Results. Most of the detected 870 μm emission is distributed in five filaments. We identify 59 starless cores, one candidate first hydrostatic core, and 21 sources associated with more evolved young stellar objects. The estimated 90% completeness limit of our survey is 0.22 M⊙ for the starless cores. The latter are only found above a visual extinction threshold of 5 mag. They are less dense than those detected in other nearby molecular clouds by a factor of a few on average, maybe because of the better sensitivity of our survey. The core mass distribution is consistent with the IMF at the high-mass end but is overpopulated at the low-mass end. In addition, at most 17% of the cores have a mass larger than the critical Bonnor-Ebert mass. Both results suggest that a large fraction of the starless cores may not be prestellar in nature. Based on the census of prestellar cores, Class 0 protostars, and more evolved young stellar objects, we conclude that the star formation rate has decreased with time in this cloud. Conclusions. The low fraction of candidate prestellar cores among the population of starless cores, the small number of Class 0 protostars, the high global star formation efficiency, the decrease of the star formation rate with time, and the low mass per unit length of the detected filaments all suggest that we may be witnessing the end of the star formation process in Chamaeleon I

A rotating molecular jet in Orion

We present CO(2-1), $^{13}$CO(2-1), CO(6-5), CO(7-6), and SO(6$_5-5_4$) line observations made with the {\it IRAM 30 m} and {\it APEX} radiotelescopes and the {\it Submillimeter Array} toward the highly collimated and extended southwest lobe of the bipolar outflow {\it Ori-S6} located in the Orion South region. We report, for all these lines, the detection of velocity asymmetries about the flow axis, with velocity differences roughly on the order of 1 km s$^{-1}$ over distances of about 5000 AU, 4 km s$^{-1}$ over distances of about 2000 AU, and close to the source of between 7 and 11 km s$^{-1}$ over smaller scales of about 1000 AU. We interpret these velocity differences as a signature of rotation but also discuss some alternatives which we recognize as unlikely in view of the asymmetries' large downstream continuation. This rotation across the {\it Ori-S6} outflow is observed out to (projected) distances beyond 2.5 $\times$ 10$^4$ AU from the flow's presumed origin. Comparison of our large-scale and small-scale observations suggests the rotational velocity to decline not faster than 1/R with distance R from the axis; in the innermost few arcsecs an increase of rotational velocity with R is even indicated. The magnetic field lines threading the inner rotating CO shell may well be anchored in a disk of radius $\sim$ 50 AU; the field lines further out need a more extended rotating base.Comment: Accepted by Astronomy and Astrophysic

Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy-resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer