Automated 3D Lumbar Intervertebral Disc Segmentation from MRI Data Sets

This paper proposed an automated three-dimensional (3D) lumbar intervertebral disc (IVD) segmentation strategy from Magnetic Resonance Imaging (MRI) data. Starting from two user supplied landmarks, the geometrical parameters of all lumbar vertebral bodies and intervertebral discs are automatically extracted from a mid-sagittal slice using a graphical model based template matching approach. Based on the estimated two-dimensional (2D) geometrical parameters, a 3D variable-radius soft tube model of the lumbar spine column is built by model fitting to the 3D data volume. Taking the geometrical information from the 3D lumbar spine column as constraints and segmentation initialization, the disc segmentation is achieved by a multi-kernel diffeomorphic registration between a 3D template of the disc and the observed MRI data. Experiments on 15 patient data sets showed the robustness and the accuracy of the proposed algorithm.

NonViral Gene Delivery of Growth and Differentiation Factor 6 (GDF6) to whole bovine Intervertebral Disc

Question: Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and de- generation. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM) [1]. Further studies showed that growth factors from the transforming growth factor (TGF) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC) [2]. Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods: Bovine IVD cells were isolated by pronase/collagenase II overnight digestion. After monolayer expansion up to passage 3, cells were transfected with the plasmid pGDF6 (RG211366, Origene, SF) or with green fluorescence protein (GFP) control using the NeonÒ transfection system (Invitrogen, Basel), both equipped with a Cy- tomegalovirus (CMV) promotor to induce over-expression. We tested a range of yet unpublished parameters for each of the primary disc cells to optimize efficiency. To test a non-viral gene therapy applied directly to 3D whole organ culture, bovine IVDs were harvested from fresh tails obtained from the abattoir within 5 h post-mortem [3]. Discs were then pre-incubated for 24 h in high glucose Dulbecco’s Modified Eagle Medium and 5 % fetal calf serum. Each disc was transfected by injection of 5 lg of plasmid GDF6 (Origene, RG211366) into the center by 25G needle and using Hamilton sy- ringe. Electroporation was performed using 2-needle array electrode or tweezertrodes; 8 pulses at 200mv/cm with an interval of 10 ms were applied using ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) (Fig. 1). After transfection discs were cultured for 72 h to allow expression of GFP or GDF6. Discs were then fixed, cryosectioned and analysed by immunofluorescence against GDF6. Results: We successfully transfected bovine NP and AF cells in monolayer culture with the two plasmids using a 1,400 V, 20 ms and 2 pulses with a *25 % efficiency using 0.15 M cells and 3 lg DNA (Fig. 1). Organ IVD culture transfection revealed GFP6 positive staining in the centre of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GFP posi- tive cells. Conclusions: We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgments: This study was supported by the Lindenhof Foundation ‘‘Forschung und Lehre’’ (Project no. 13-02-F). References 1. Roughly PJ (2004) Spine (Phila) 29:2691–2699 2. 3. Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014) Arthritis Res Ther 16:R67 Chan SC, Gantenbein-Ritter B (2012) J Vis Exp 60(60):e3490

Physico-chemical parameters of sediment core PG1972-1 (09-Tik-03) from a Tundra Lake, Northeastern Siberia

Thermokarst lakes in the Siberian Arctic contain sediment archives that can be used for paleoenvironmental inference. Until now, however, there has been no study from the inner Lena River Delta with a focus on diatoms. The objective of this study was to investigate how the diatom community in a thermokarst lake responded to past limnogeological changes and what specific factors drove variations in the diatom assemblage. We analysed fossil diatom species, organic content, grain-size distribution and elemental composition in a sediment core retrieved in 2009 from a shallow thermokarst lake in the Arga Complex, western Lena River Delta. The core contains a 3,000-year record of sediment accumulation. Shifts in the predominantly benthic and epiphytic diatom species composition parallel changes in sediment characteristics. Paleoenvironmental and limnogeological development, inferred from multiple biological and sedimentological variables, are discussed in the context of four diatom zones, and indicate a strong relation between changes in the diatom assemblage and thermokarst processes. We conclude that limnogeological and thermokarst processes such as lake drainage, rather than direct climate forcing, were the main factors that altered the aquatic ecosystem by influencing, for example, habitat availability, hydrochemistry, and water level.

(Table 1) Results of measurements of production characteristics of phytoplankton and some physical parameters of the environment in the northeastern Norwegian Sea in July 1995

From July 4 to 18,1995 surface chlorophyll a concentrations (C_cs) and integral primary production (C_ps) were studied in the northeastern part of the Norwegian Sea (73°42'N; 13°16'E), over a test area where an accident of the nuclear submarine Komsomolets had taken place. It was found that during this interval C_cs decreased by factor of about 3.3 (from 0.78 to 0.24 mg/m**3); average chlorophyll concentration within the photo-synthetic layer (C_cl) decreased by factor of about 3.5 (from 0.97 to 0.28 mg/m**3). The value of C_ps in the water column varied slightly (from 445 to 539 mg C/m**2 per day), since decrease in C_cl was compensated both by 1.5-fold growth of the photosynthetic layer thickness (from 40 to 60 m) and by 2.1-fold increase in its average assimilation number (from 0.58 to 1.20 mg C/mg chl a per hour). Monthly averages of C_ps were obtained from published data on seasonal C_cs changes and on the level of incident solar irradiation. They were found to be less than 100 mg C/m**2 per day in March and October and 100-500 mg C/m**2 per day in April-June. Annual primary production calculated from above values was equal to 105 g C/m**2.

(Table 1) Results of measurements of productive characteristics of phytoplankton and environmental parameters in the Barents Sea in September-October 1997

The described studies were carried out in the eastern part of the sea during the end of the summer seasonal succession from September 1 to October 12, 1997. Concentration of chlorophyll a in the surface layer varied from 0.09 to 1.24 mg/m**3; it tended to increase in the southern regions (<74°N). Primary production in the water column (P_p) varied from 24 to 214 mg C/m**2/day and was on average 91 mg C/m**2/day. The low level of P_p seems to result from combination of physical and chemical environmental factors unfavorable for photosynthesis (e.g. deficiency of nutrients and low values of insolation and temperature) and intensive grazing of phytoplankton by zooplankton. The lower boundary of the photosynthetic layer in open waters was located at depth 60-75 m; irradiance there was 0.1-0.5% of incident irradiance. In deep-water regions (>200 m) the subsurface maximum of chlorophyll occurred in the layer at 20-40 m; usually this maximum resulted in formation of additional maxima of primary production.

(Table 2) Production parameters of phytoplankton and some associated parameters in the area of the Titanic Polygon

Studies were carried out mostly in the area of RMS Titanic wreck site (41°44'N, 49°57'W) located above the continental slope and the south of the Grand Banks of Newfoundland. In a period from 18.06 to 24.09.2001 five surveys of production characteristics of surface phytoplankton were conducted over 5-9 days. Mean values of these characteristics obtained during the surveys were 9.2-11.7 mg C/m**3 per day for primary production (C_phs), 0.102-0.188 mg/m**3 for chlorophyll a (C_chls), and 4.44-7.42 mg C/mg chl. a per hour for assimilation number (AN). The main reason for low C_phs variability was a significant inverse relationship (R=-0.66) between AN and C_chls found over the research area. When cold shelf waters dominated in the area (27.07 to 19.08.2001), C_chls values for the slope region (0.125+/-0.031 µg/l) and for the outer shelf (0.130+/-0.040 µg/l) were similar. During strengthening of influence of warmer slope waters within area (from 29.08 to 13.09.2001), C_chls concentration within surface waters of the outer shelf was 0.152+/-0.039 µg/l and exceeded one for the slope region (0.094+/-0.004 µg/l) by factor 1.6. Against the background of low Cchls values, the High values of integral primary production in the water column (510-1010 mg C/m**2 per day) at low C_chls values measured within the area were determined both by high assimilation activity of phytoplankton and by the deep (30-40 m) maximum of primary production. Main reasons for formation of such a maximum were high chlorophyll concentration within the layer of the deep chlorophyll maximum (up to 0.5-2.5 µg/l) and in the relatively high solar irradiance within this layer varying from 1.4 to 8.6% of subsurface PAR.

Physico-chemical parameters of sediment core PG1975-1 (09-Tik-05) from a Tundra Lake, Northeastern Siberia

The relationships between thermokarst activity, limnogeological processes and climate change in the Siberian Arctic are not well understood. The objective of this paper is to identify the factors controlling the patterns of deposition, using grain size distribution, organic content, elemental composition and mineralogical composition of a 137-cm long sediment core with a maximum age of ~10.9 cal. kyr BP from Lake El'gene-Kyuele in the tundra of northeastern Siberia. Eight fine sand layers are attributed to depositional events associated with thaw slump activity acting upon orthogonally oriented patterns of ice-wedge networks in the ice-rich permafrost on the NW margin of the lake catchment. Sr/Rb ratios, which correspond to the total feldspar and illite content, serve as high-resolution grain size proxies. The Br content relates to the total organic carbon content, and the Fe/Mn ratio reflects the degree of oxidisation. Our results indicate a relationship between repeated phases of fine sand input and retrogressive thaw slumping dependent on hydroclimate variability and orthogonally oriented ice-wedge networks within the catchment.

Age and growth parameters of skates of the genus Bathyraja obtained between 2003-2005 along the Argentine continental shelf

In the last 20 years directed shark and ray fishery has increased alarmingly everywhere in the world. For most species though, no data on growth rate, mortality, fecundity and other life history aspects exist as of now and management of the fishery is therefore insufficient. Also there still exist methodological difficulties in the age determination of elasmobranchs fishes, a fact which complicates the investigation of growth parameters. This study tried to identify the best ageing methods and estimate growth parameters for ten skate species of the genus Bathyraja, all occurring in the southwest Atlantic in depths of 50m and more. 720 samples were collected on board of argentine research vessels in between 2003 and 2005. Crystal violet and a new staining method using potassium permanganate, both applied on sagittal sections of vertebral centra, proved to be most effective in enhancing the banding pattern in most of the species. Thorns were also tested and readings were consistent with the ones made on vertebral sections. Growth parameters could be derived for six species and for the other four estimates could be made. Growth rate as well as infinite length varied between species, with those attaining bigger sizes having lower growth rates. No latitudinal differences in growth rate could be detected but a comparison with samples from other studies showed that total lengths were always reported to be higher around the Malvinas Islands.

Library of medium-resolution Fiber Optic Echelle spectra of F, G, K, and M field dwarfs to giant stars

We present a library of Penn State Fiber Optic Echelle (FOE) observations of a sample of field stars with spectral types F to M and luminosity classes V to I. The spectral coverage is from 3800 to 10000 Å with a nominal resolving power of 12,000. These spectra include many of the spectral lines most widely used as optical and near-infrared indicators of chromospheric activity such as the Balmer lines (Hα to H epsilon), Ca II H & K, the Mg I b triplet, Na I D_1, D_2, He I D_3, and Ca II IRT lines. There are also a large number of photospheric lines, which can also be affected by chromospheric activity, and temperature-sensitive photospheric features such as TiO bands. The spectra have been compiled with the goal of providing a set of standards observed at medium resolution. We have extensively used such data for the study of active chromosphere stars by applying a spectral subtraction technique. However, the data set presented here can also be utilized in a wide variety of ways ranging from radial velocity templates to study of variable stars and stellar population synthesis. This library can also be used for spectral classification purposes and determination of atmospheric parameters (T_eff, log g, [Fe/H]). A digital version of all the fully reduced spectra is available via ftp and the World Wide Web (WWW) in FITS format.

Non-Viral Gene Therapy to Cells of the bovine Intervertebral Disc

Background. Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and degeneration. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM).1 Further studies showed that growth factors from transforming growth factor β (TGFβ) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC).2 Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods. Bovine nucleus pulposus (bNPC) and annulus fibrosus cells (bAFC) were harvested from bovine coccygeal IVD. Primary cells were then electroporized with plasmid GDF6 (Origene, vector RG211366) by optimizing parameters using the Neon Transfection system (Life Technologies, Basel). After transfection, cells were cultured in 2D monolayer or 3D alginate beads for 7, 14 or 21 days. Transfection efficiency of pGDF6 was analyzed by immunohistochemistry and fluorescent microscopy. Cell phenotype was quantified by real-time RT-PCR. To test a non-viral gene therapy applied directly to 3D whole organ culture, coccygeal bovine IVDs were harvested as previously described. Bovine IVDs were transfected by injection of plasmid GDF6 into the center. Electroporation was performed with ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) using 2-needle array electrode or tweezertrodes. 72 h after tranfection discs were fixed and cryosectioned and analyzed by immunofluorescence against GDF6. Results. RT-PCR and immunohistochemistry confirmed up-regulation of GFP and GDF6 in the primary bNPC/bAFC culture. The GFP-tagged GDF6 protein, however, was not visible, possibly due to failure of dimer formation as a result of fusion structure. Organ IVD culture transfection revealed GDF6 positive staining in the center of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GDF6 positive cells. Conclusion. Non-viral transfection is an appealing approach for gene therapy as it fulfills the translational safety aspects of transiency and lacks the toxic effects of viral transduction. We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgements. This project was funded by the Lindenhof Foundation (Funds “Research & Teaching”) Project no. 13-02-F. The imaging part of this study was performed with the facility of the Microscopy Imaging Center (MIC), University of Bern. References. Roughly PJ (2004): Spine (Phila), 29:2691-2699 Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014), Arthritis Research & Therapy, 16:R67

Productive parameters of seawater and epipelagic plankton and mesozooplankton biomass at stations of R/V Akademik Mstislav Keldysh during 1994-1998

Calculated and measured estimations of biomass of small (<3 mm), large (3-30 mm), and total zooplankton were verified (compared). These integral parameters of epipelagic communities were estimated by two methods. We used previously obtained regression equations, which correlate these parameters with water transparency. Measured values of aforesaid parameters were compared with their mean values in waters of different productivity estimated from NASA satellite maps. We compared data collected at fifteen stations in September-December in regions of different productivity in the North Atlantic. In warm regions (to the south of 40°N) measured and calculated values coincide well. In boreal regions in autumn bulk of mesozooplankton descends to deep layers due to seasonal migrations; hence correlation between measured and calculated values is disrupted. It is evident that correlation between water transparency and mesozooplankton biomass (integral index of water productivity) obtained before should be corrected for seasonal variations.