Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds

Conjugation of siRNA to macromolecules such as serum albumin has multiple potential benefits, including enhanced extravasation via albumin-mediated transcytosis across endothelial cells and reduced renal clearance. In attempting to conjugate siRNA to albumin, we used commercially sourced amine-modified siRNA and reacted it with the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group suitable for conjugation to the thiol group of the surface-exposed cysteine residue (Cys 34) within albumin. We found the conjugation of the SMCC-treated siRNA to bovine serum albumin (BSA) to be very inefficient and investigated the cause of the low yield of conjugate. Ultrafiltration with phosphate-buffered saline prior to activation with SMCC dramatically increased the yield of siRNA-albumin conjugate (~15-fold). Communication with the commercial supplier revealed that ammonium acetate buffer was used in a desalting step as part of the siRNA purification process prior to supply, likely resulting in ammonium counterions to the siRNA polyanion, which would interfere with conjugation by consuming the SMCC. After ultrafiltration, a greatly reduced amount of SMCC could be used to affect conjugation, without significant reduction in yield. These data indicate that amine-modified siRNA sourced commercially may require ultrafiltration or dialysis prior to use in conjugation reactions

Geophysical investigation of a badger sett located in a flood embankment on the River Ouse

Ground Penetrating Radar (GPR) and Electrical Resistivity Tomography (ERT) are two nearsurface geophysical methods that are well suited to mapping air-filled cavities (e.g. burrows) in the shallow subsurface. They both have the advantage that they are non-intrusive, so can image the subsurface targets without disturbing them, which is advantageous for detecting, characterising, and monitoring animal burrows. GPR is a very rapid technique that can survey large areas relatively quickly; however, flood embankments often have a clay component that can limit the depth of investigation of GPR surveys, with tunnels beyond the penetration depth of the instrument. ERT surveys are typically slower but are not limited by clay-rich ground. Both techniques are affected by metal objects in the ground, such as sheet piling and wire mesh, which are installed at the stie to prevent animal burrowing. A desk study of badger sett morphology found that: • Badgers rarely dig beyond 10 m from an entrance • The tunnels are 30 cm wide and 20 cm tall • Tunnels rarely go deeper than 2 m below the surface. At the badger sett north of Drax power station, entrances were found within an area extending along 75 m of the flood embankment. The location of the sheet piling was confirmed using the GPR, and all badger tunnel entrances were found to be within this reinforced section of the embankment. However, the entrances at the site's eastern end were very close to the end of the sheet piling, making this area the focus of the geophysical survey. In summary, the results of the geophysical survey (Figure 5) are as follows: • Two areas of extensive tunnelling were found in the GPR data, matching tunnel entrances' locations. • GPR could only detect tunnels in the first three lines, beyond which the tunnels likely continued but were over 1.5 m deep, deeper than the penetration depth of the GPR signal. • Most of the tunnelling seems to be concentrated in the floodplain and embankment toe. • ERT focused on the eastern area but was partially affected by sheet piles, especially the lines perpendicular to the embankment. • Tunnels interpreted from the ERT data head towards the crest of the embankment close to where the sheet pilling ends. • We are unsure if we imaged the full extent of the tunnels or if they continued in to the embankment, but they were beyond the detectability of the instruments at these depths. Furthermore, the proximity of sheet piling at these locations caused significant noise in the data. This report concludes that ERT and GPR could successfully image areas of tunnelling in the embankment northwest of Drax power station. Useable data could be collected despite the sheet piling, which makes interpretation of the data significantly more difficult. While GPR could only find tunnels in the first three lines, it provided some confidence to the ERT interpretation and was able to investigate a much larger area. This highlights the potential benefits of combining ERT and GPR for future badger investigations

Geometrical Effects of Baryon Density Inhomogeneities on Primordial Nucleosynthesis

We discuss effects of fluctuation geometry on primordial nucleosynthesis. For the first time we consider condensed cylinder and cylindrical-shell fluctuation geometries in addition to condensed spheres and spherical shells. We find that a cylindrical shell geometry allows for an appreciably higher baryonic contribution to be the closure density (\Omega_b h_{50}^2 \la 0.2) than that allowed in spherical inhomogeneous or standard homogeneous big bang models. This result, which is contrary to some other recent studies, is due to both geometry and recently revised estimates of the uncertainties in the observationally inferred primordial light-element abundances. We also find that inhomogeneous primordial nucleosynthesis in the cylindrical shell geometry can lead to significant Be and B production. In particular, a primordial beryllium abundance as high as [Be] = 12 + log(Be/H) $\approx -3$ is possible while still satisfying all of the light-element abundance constraints.Comment: Latex, 20 pages + 11 figures(not included). Entire ps file with embedded figures available via anonymous ftp at ftp://genova.mtk.nao.ac.jp/pub/prepri/bbgeomet.ps.g

75%-efficiency blue generation from an intracavity PPKTP frequency doubler

We report on a high-efficiency 461 nm blue light conversion from an external cavity-enhanced second-harmonic generation of a 922 nm diode laser with a quasi-phase-matched KTP crystal (PPKTP). By choosing a long crystal (LC=20 mm) and twice looser focusing (w0=43 $\mu$m) than the "optimal" one, thermal lensing effects due to the blue power absorption are minimized while still maintaining near-optimal conversion efficiency. A stable blue power of 234 mW with a net conversion efficiency of eta=75% at an input mode-matched power of 310 mW is obtained. The intra-cavity measurements of the conversion efficiency and temperature tuning bandwidth yield an accurate value d33(461 nm)=15 pm/V for KTP and provide a stringent validation of some recently published linear and thermo-optic dispersion data of KTP

Outcome following multiple subpial transection in Landau-Kleffner syndrome and related regression

OBJECTIVE: To determine whether multiple subpial transection in the posterior temporal lobe has an impact on long-term outcome in children who have drug-resistant Landau-Kleffner syndrome (LKS) or other "electrical status epilepticus during sleep" (ESES)-related regression. Given the wide variability in outcomes reported in the literature, a secondary aim was to explore predictors of outcome. METHODS: The current study includes a surgery group (n = 14) comprising patients who underwent multiple subpial transection of the posterior temporal lobe and a nonsurgery comparison group (n = 21) comprising patients who underwent presurgical investigations for the procedure, but who did not undergo surgery. Outcomes were assessed utilizing clinical note review as well as direct assessment and questionnaires. RESULTS: The distribution of nonclassical cases was comparable between groups. There were some differences between the surgery and nonsurgery groups at presurgical investigation including laterality of discharges, level of language impairment, and age; therefore, follow-up analyses focused on change over time and predictors of outcome. There were no statistically significant differences between the groups in language, nonverbal ability, adaptive behavior, or quality of life at follow-up. There was no difference in the proportion of patients showing improvement or deterioration in language category over time for either group. Continuing seizures and an earlier age of onset were most predictive of poorer quality of life at long-term follow-up (F2,23  = 26.2, p = <0.001, R(2)  = 0.714). SIGNIFICANCE: Both surgery and nonsurgery groups had similar proportions of classic LKS and ESES-related regression. Because no significant differences were found in the changes observed from baseline to follow-up between the two groups, it is argued that there is insufficient evidence to suggest that multiple subpial transection provides additional benefits over and above the mixed recovery often seen in LKS and related regressive epilepsies

Compact-beam stable-unstable resonator for free-electron laser. Phase 2, Final report

A significant problem in the design of high-energy free-electron lasers (FELs) centers on the technique for outcoupling the output beam. FELs with currently achievable output power usually include a conventional stable resonator with output through a partially transmitting mirror which will not work for arbitrarily high average power. An alternate scheme must be found for high-energy FELs. A high- efficiency grating outcoupler is an attractive possibility, but it is difficult to manufacture. Other suggestions include unstable resonators with an intracavity focus and unstable resonators with an intracavity focus and beam rotation. The intensity distribution at the intracavity focus of a negative-branch unstable resonator has side-lobes that would be scraped off by the faces of the wiggler magnets or by the beam tube through the wiggler. The resulting power loss would be significant. Therefore, it is desirable to develop another type of resonator for use with FELs. The resonator that we have developed is the compact-beam stable-unstable ring resonator. It is a stable resonator in one transverse dimension and an unstable resonator with an intracavity focus in the orthogonal transverse dimension. A scraper mirror outcouples the output beam from one side of the mode only. The resonator can be configured so that it has a small beam waist at the center of the wiggler in the stable direction and has an intracavity focus in the unstable direction. The half- width of the central lobe of the focus is approximately the size of the stable beam waist. In the stable direction, the Gaussian amplitude distribution results in a small loss on the wiggler magnets, or on a beam tube that will fit within the wiggler, if one is used. The beam tube can have an elliptical shape to permit the passage of several side lobes in the unstable dimension. A mode of the CBSUR is a product of the mode of a strip stable resonator with a strip compact-beam negative-branch unstable resonator

Stochasticity in the miR-9/Hes1 oscillatory network can account for clonal heterogeneity in the timing of differentiation.

Recent studies suggest that cells make stochastic choices with respect to differentiation or division. However, the molecular mechanism underlying such stochasticity is unknown. We previously proposed that the timing of vertebrate neuronal differentiation is regulated by molecular oscillations of a transcriptional repressor, HES1, tuned by a post-transcriptional repressor, miR-9. Here, we computationally model the effects of intrinsic noise on the Hes1/miR-9 oscillator as a consequence of low molecular numbers of interacting species, determined experimentally. We report that increased stochasticity spreads the timing of differentiation in a population, such that initially equivalent cells differentiate over a period of time. Surprisingly, inherent stochasticity also increases the robustness of the progenitor state and lessens the impact of unequal, random distribution of molecules at cell division on the temporal spread of differentiation at the population level. This advantageous use of biological noise contrasts with the view that noise needs to be counteracted

Measuring measurement

Measurement connects the world of quantum phenomena to the world of classical events. It plays both a passive role, observing quantum systems, and an active one, preparing quantum states and controlling them. Surprisingly - in the light of the central status of measurement in quantum mechanics - there is no general recipe for designing a detector that measures a given observable. Compounding this, the characterization of existing detectors is typically based on partial calibrations or elaborate models. Thus, experimental specification (i.e. tomography) of a detector is of fundamental and practical importance. Here, we present the realization of quantum detector tomography: we identify the optimal positive-operator-valued measure describing the detector, with no ancillary assumptions. This result completes the triad, state, process, and detector tomography, required to fully specify an experiment. We characterize an avalanche photodiode and a photon number resolving detector capable of detecting up to eight photons. This creates a new set of tools for accurately detecting and preparing non-classical light.Comment: 6 pages, 4 figures,see video abstract at http://www.quantiki.org/video_abstracts/0807244