Ross Sea Mollusca from the Latitudinal Gradient Program: R/V Italica 2004 Rauschert dredge samples

Information regarding the molluscs in this dataset is based on the Rauschert dredge samples collected during the Latitudinal Gradient Program (LGP) on board the R/V “Italica” in the Ross Sea (Antarctica) in the austral summer 2004. A total of 18 epibenthic dredge deployments/samplings have been performed at four different locations at depths ranging from 84 to 515m by using a Rauschert dredge with a mesh size of 500μm. In total 8,359 specimens have been collected belonging to a total of 161 species. Considering this dataset in terms of occurrences, it corresponds to 505 discrete distributional records (incidence data). Of these, in order of abundance, 5,965 specimens were Gastropoda (accounting for 113 species), 1,323 were Bivalvia (accounting for 36 species), 949 were Aplacophora (accounting for 7 species), 74 specimens were Scaphopoda (3 species), 38 were Monoplacophora (1 species) and, finally, 10 specimens were Polyplacophora (1 species). This data set represents the first large-scale survey of benthic micro-molluscs for the area and provides important information about the distribution of several species, which have been seldom or never recorded before in the Ross Sea. All vouchers are permanently stored at the Italian National Antarctic Museum (MNA), Section of Genoa, enabling future comparison and crosschecking. This material is also currently under study, from a molecular point of view, by the barcoding project “BAMBi” (PNRA 2010/A1.10).     

Methane-Carbon Flow into the Benthic Food Web at Cold Seeps – A Case Study from the Costa Rica Subduction Zone

Cold seep ecosystems can support enormous biomasses of free-living and symbiotic chemoautotrophic organisms that get their energy from the oxidation of methane or sulfide. Most of this biomass derives from animals that are associated with bacterial symbionts, which are able to metabolize the chemical resources provided by the seeping fluids. Often these systems also harbor dense accumulations of non-symbiotic megafauna, which can be relevant in exporting chemosynthetically fixed carbon from seeps to the surrounding deep sea. Here we investigated the carbon sources of lithodid crabs (Paralomis sp.) feeding on thiotrophic bacterial mats at an active mud volcano at the Costa Rica subduction zone. To evaluate the dietary carbon source of the crabs, we compared the microbial community in stomach contents with surface sediments covered by microbial mats. The stomach content analyses revealed a dominance of epsilonproteobacterial 16S rRNA gene sequences related to the free-living and epibiotic sulfur oxidiser Sulfurovum sp. We also found Sulfurovum sp. as well as members of the genera Arcobacter and Sulfurimonas in mat-covered surface sediments where Epsilonproteobacteria were highly abundant constituting 10% of total cells. Furthermore, we detected substantial amounts of bacterial fatty acids such as i-C15∶0 and C17∶1ω6c with stable carbon isotope compositions as low as −53‰ in the stomach and muscle tissue. These results indicate that the white microbial mats at Mound 12 are comprised of Epsilonproteobacteria and that microbial mat-derived carbon provides an important contribution to the crab''s nutrition. In addition, our lipid analyses also suggest that the crabs feed on other ¹³C-depleted organic matter sources, possibly symbiotic megafauna as well as on photosynthetic carbon sources such as sedimentary detritus.     

SERS Properties of Different Sized and Shaped Gold Nanoparticles Biosynthesized under Different Environmental Conditions by Neurospora crassa Extract

Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. It is known that metal nanoparticles, especially gold and silver nanoparticles, exhibit great SERS properties, which make them very attractive for the development of biosensors and biocatalysts. On the other hand, the development of ecofriendly methods for the synthesis of metallic nanostructures has become the focus of research in several countries, and many microorganisms and plants have already been used to biosynthesize metallic nanostructures. However, the majority of these are pathogenic to plants or humans. Here, we report gold nanoparticles with good SERS properties, biosynthesized by Neurospora crassa extract under different environmental conditions, increasing Raman signals up to 40 times using methylene blue as a target molecule. Incubation of tetrachloroauric acid solution with the fungal extract at 60°C and a pH value of a) 3, b) 5.5, and c) 10 resulted in the formation of gold nanoparticles of a) different shapes like triangles, hexagons, pentagons etc. in a broad size range of about 10-200 nm, b) mostly quasi-spheres with some different shapes in a main size range of 6-23 nm, and c) only quasi-spheres of 3-12 nm. Analyses included TEM, HRTEM, and EDS in order to corroborate the shape and the elemental character of the gold nanoparticles, respectively. The results presented here show that these ‘green’ synthesized gold nanoparticles might have potential applicability in the field of biological sensing.     

Podoplanin Immunopositive Lymphatic Vessels at the Implant Interface in a Rat Model of Osteoporotic Fractures

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.     

Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.     

An automated immunoassay for early specificity profiling of antibodies

Antibody-based therapeutics are of great value for the treatment of human diseases. In addition to functional activity, affinity or physico-chemical properties, antibody specificity is considered to be one of the most crucial attributes for safety and efficacy. Consequently, appropriate studies are required before entering clinical trials.

High content protein arrays are widely applied to assess antibody specificity, but this commercial solution can only be applied to final therapeutic antibody candidates because such arrays are expensive and their throughput is limited. A flexible, high-throughput and economical assay that allows specificity testing of IgG or Fab molecules during early discovery is described here. The 384-well microtiter plate assay contains a comprehensive panel of 32 test proteins and uses electrochemiluminescence as readout.

The Protein Panel Profiling (3P) was used to analyze marketed therapeutic antibodies that all showed highly specific binding profiles. Subsequently, 3P was applied to antibody candidates from early discovery and the results compared well with those obtained with a commercially available high content protein chip. Our results suggest that 3P can be applied as an additional filter for lead selection, allowing the identification of favorable antibody candidates in early discovery and thereby increasing the speed and possibility of success in drug development.     

Quantitative Changes in the Sleep EEG at Moderate Altitude (1630 m and 2590 m)

Background

Previous studies have observed an altitude-dependent increase in central apneas and a shift towards lighter sleep at altitudes >4000 m. Whether altitude-dependent changes in the sleep EEG are also prevalent at moderate altitudes of 1600 m and 2600 m remains largely unknown. Furthermore, the relationship between sleep EEG variables and central apneas and oxygen saturation are of great interest to understand the impact of hypoxia at moderate altitude on sleep.

Methods

Fourty-four healthy men (mean age 25.0±5.5 years) underwent polysomnographic recordings during a baseline night at 490 m and four consecutive nights at 1630 m and 2590 m (two nights each) in a randomized cross-over design.

Results

Comparison of sleep EEG power density spectra of frontal (F3A2) and central (C3A2) derivations at altitudes compared to baseline revealed that slow-wave activity (SWA, 0.8–4.6 Hz) in non-REM sleep was reduced in an altitude-dependent manner (∼4% at 1630 m and 15% at 2590 m), while theta activity (4.6–8 Hz) was reduced only at the highest altitude (10% at 2590 m). In addition, spindle peak height and frequency showed a modest increase in the second night at 2590 m. SWA and theta activity were also reduced in REM sleep. Correlations between spectral power and central apnea/hypopnea index (AHI), oxygen desaturation index (ODI), and oxygen saturation revealed that distinct frequency bands were correlated with oxygen saturation (6.4–8 Hz and 13–14.4 Hz) and breathing variables (AHI, ODI; 0.8–4.6 Hz).

Conclusions

The correlation between SWA and AHI/ODI suggests that respiratory disturbances contribute to the reduction in SWA at altitude. Since SWA is a marker of sleep homeostasis, this might be indicative of an inability to efficiently dissipate sleep pressure.     

Population Structure and Evidence for Both Clonality and Recombination among Brazilian Strains of the Subgenus Leishmania (Viannia)

Background/Objectives: Parasites of the subgenus Leishmania (Viannia) cause varying clinical symptoms ranging from cutaneous leishmaniases (CL) with single or few lesions, disseminated CL (DL) with multiple lesions to disfiguring forms of mucocutaneous leishmaniasis (MCL). In this population genetics study, 37 strains of L. (V.) guyanensis, 63 of L. (V.) braziliensis, four of L. (V.) shawi, six of L. (V.) lainsoni, seven of L. (V.) naiffi, one each of L. (V.) utingensis and L. (V.) lindenbergi, and one L. (V.) lainsoni/L. naiffi hybrid from different endemic foci in Brazil were examined for variation at 15 hyper-variable microsatellite markers. Methodology/Principal findings: The multilocus microsatellite profiles obtained for the 120 strains were analysed using both model- and distance-based methods. Significant genetic diversity was observed for all L. (Viannia) strains studied. The two cluster analysis approaches identified two principal genetic groups or populations, one consisting of strains of L. (V.) guyanensis from the Amazon region and the other of strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil. A third group comprised a heterogeneous assembly of species, including other strains of L. braziliensis isolated from the north of Brazil, which were extremely polymorphic. The latter strains seemed to be more closely related to those of L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni, also isolated in northern Brazilian foci. The MLMT approach identified an epidemic clone consisting of 13 strains of L. braziliensis from Minas Gerais, but evidence for recombination was obtained for the populations of L. (V.) braziliensis from the Atlantic coast and for L. (V.) guyanensis. Conclusions/Significance: Different levels of recombination versus clonality seem to occur within the subgenus L. (Viannia). Though clearly departing from panmixia, sporadic, but long-term sustained recombination might explain the tremendous genetic diversity and limited population structure found for such L. (Viannia) strains.