The upper cretaceousLacazina limestone in the Basco-Cantabrian and Iberian basins of northern Spain: Cold-water grain associations in warm-water environments

Summary The Upper Santonian to Lower CampanianLacazina Limestone consists of massive, often amalgamated beds of packstones and grainstones which were deposited in a shallow marine environment. The most abundant skeletal components are miliolid foraminifera, echinoderm, bivalve and bryozoan fragments, peloids and sparse red algae. Small, solitary corals only occur sporadically. Hermatypic corals, sponges and green algae are missing. The series which reaches thicknesses between 60 m and 160 m, was sampled at intervals of 0.5 m at five localities. The petrographic features throughout the series are mainly a product of changing depositional energy. The limestones are well cemented. Diagenesis is characterized by a transition from marine phreatic to burial cementation. Syntaxial and blocky calcite cements predominate over early acicular to bladed and microgranular cements. The faunal association within theLacazina Limestone exhibits features typical for temperate water i.e.foramol carbonates. On the other hand, oxygen (δ¹⁸O =-1.7 to −6.3 ‰ PDB) and carbon (δ¹³C to 3.0‰ PDB) isotope values of diagentically unaltered bivalve shells indicate warm surface waters corresponding better to the palaeogeographical situation of theLacazina Limestone. Nutrient-surplus is proposed as a limiting factor preventing the development of reefs and finally ofchlorozoan sediments. In the sense of sequence stratigraphy, theLacazina Limestone is interpreted to contain transgressive and highstand systems tracts. This interpretation fits well into theHaq-Vail-curve. The series shows no visible high-frequency cyclicity in the field. Several cycles were found by means of principle component analysis and spectral analysis. Their relationships to the Milankovitch spectrum are discussed. The ammonite fauna of the unit and of preceding sediments (late Coniacian to early Campanian) is described and some inoceramids are figured. They permit—for the first time—the exact dating of theLacazina Limestone in the Basco-Cantabrian Basin (BCB) and throw light on a prominent faunal change at the Coniacian/Santonian boundary. The Cenomanian to Coniacian ammonite faunas which were dominated by endemic Tethyan pseudoceratitic faunas are replaced by cosmopolitan species dominated by Madagascan elements. This drastic change permits speculations about the installation of a new oceanic current system in the Santonian.     

Assessing sub-seafloor microbial activity by combined stable isotope probing with deuterated water and 13C-bicarbonate

Sub‐seafloor sediments are populated by large numbers of microbial cells but not much is known about their metabolic activities, growth rates and carbon assimilation pathways. Here we introduce a new method enabling the sensitive detection of microbial lipid production and the distinction of auto‐ and heterotrophic carbon assimilation. Application of this approach to anoxic sediments from a Swedish fjord allowed to compare the activity of different functional groups, the growth and turnover times of the bacterial and archaeal communities. The assay involves dual stable isotope probing (SIP) with deuterated water (D₂O) and ¹³C_DIC (d issolved i norganic c arbon). Culture experiments confirmed that the D content in newly synthesized lipids is in equilibrium with the D content in labelled water, independent of whether the culture grew hetero‐ or autotrophically. The ratio of ¹³C_DIC to D₂O incorporation enables distinction between these two carbon pathways in studies of microbial cultures and in environmental communities. Furthermore, D₂O‐SIP is sufficiently sensitive to detect the formation of few hundred cells per day in a gram of sediment. In anoxic sediments from a Swedish fjord, we found that > 99% of newly formed lipids were attributed to predominantly heterotrophic bacteria. The production rate of bacterial lipids was highest in the top 5 cm and decreased 60‐fold below this depth while the production rate of archaeal lipids was rather low throughout the top meter of seabed. The contrasting patterns in the rates of archaeal and bacterial lipid formation indicate that the factors controlling the presence of these two lipid groups must differ fundamentally.     

Genetics of osteopontin in patients with chronic kidney disease: The German Chronic Kidney Disease study

Osteopontin (OPN), encoded by SPP1, is a phosphorylated glycoprotein predominantly synthesized in kidney tissue. Increased OPN mRNA and protein expression correlates with proteinuria, reduced creatinine clearance, and kidney fibrosis in animal models of kidney disease. But its genetic underpinnings are incompletely understood. We therefore conducted a genome-wide association study (GWAS) of OPN in a European chronic kidney disease (CKD) population. Using data from participants of the German Chronic Kidney Disease (GCKD) study (N = 4,897), a GWAS (minor allele frequency [MAF]≥1%) and aggregated variant testing (AVT, MAF<1%) of ELISA-quantified serum OPN, adjusted for age, sex, estimated glomerular filtration rate (eGFR), and urinary albumin-to-creatinine ratio (UACR) was conducted. In the project, GCKD participants had a mean age of 60 years (SD 12), median eGFR of 46 mL/min/1.73m² (p25: 37, p75: 57) and median UACR of 50 mg/g (p25: 9, p75: 383). GWAS revealed 3 loci (p<5.0E-08), two of which replicated in the population-based Young Finns Study (YFS) cohort (p<1.67E-03): rs10011284, upstream of SPP1 encoding the OPN protein and related to OPN production, and rs4253311, mapping into KLKB1 encoding prekallikrein (PK), which is processed to kallikrein (KAL) implicated through the kinin-kallikrein system (KKS) in blood pressure control, inflammation, blood coagulation, cancer, and cardiovascular disease. The SPP1 gene was also identified by AVT (p = 2.5E-8), comprising 7 splice-site and missense variants. Among others, downstream analyses revealed colocalization of the OPN association signal at SPP1 with expression in pancreas tissue, and at KLKB1 with various plasma proteins in trans, and with phenotypes (bone disorder, deep venous thrombosis) in human tissue. In summary, this GWAS of OPN levels revealed two replicated associations. The KLKB1 locus connects the function of OPN with PK, suggestive of possible further post-translation processing of OPN. Further studies are needed to elucidate the complex role of OPN within human (patho)physiology.     

Persistent Autoantibody-Production by Intermediates between Short-and Long-Lived Plasma Cells in Inflamed Lymph Nodes of Experimental Epidermolysis Bullosa Acquisita

Autoantibodies are believed to be maintained by either the continuous generation of short-lived plasma cells in secondary lymphoid tissues or by long-lived plasma cells localized in bone marrow and spleen. Here, we show in a mouse model for the autoimmune blistering skin disease epidermolysis bullosa acquisita (EBA) that chronic autoantibody production can also be maintained in inflamed lymph nodes, by plasma cells exhibiting intermediate lifetimes. After EBA induction by immunization with a mCOL7c-GST-fusion protein, antigen-specific plasma cells and CD4 T cells were analyzed. Plasma cells were maintained for months in stable numbers in the draining lymph nodes, but not in spleen and bone marrow. In contrast, localization of mCOL7c-GST -specific CD4 T cells was not restricted to lymph nodes, indicating that availability of T cell help does not limit plasma cell localization to this site. BrdU-incorporation studies indicated that pathogenic mCOL7c- and non-pathogenic GST-specific plasma cells resemble intermediates between short-and long-lived plasma cells with half-lives of about 7 weeks. Immunization with mCOL7c-GST also yielded considerable numbers of plasma cells neither specific for mCOL7c- nor GST. These bystander-activated plasma cells exhibited much shorter half-lives and higher population turnover, suggesting that plasma cell lifetimes were only partly determined by the lymph node environment but also by the mode of activation. These results indicate that inflamed lymph nodes can harbor pathogenic plasma cells exhibiting distinct properties and hence may resemble a so far neglected site for chronic autoantibody production.     

Novel Cultivation-Based Approach To Understanding the Miscellaneous Crenarchaeotic Group (MCG) Archaea from Sedimentary Ecosystems

The uncultured miscellaneous crenarchaeotic group (MCG) archaea comprise one of the most abundant microbial groups in the Earth''s subsurface environment. However, very little information is available regarding the lifestyle, physiology, and factors controlling the distribution of members of this group. We established a novel method using both cultivation and molecular techniques, including a pre-PCR propidium monoazide treatment, to investigate viable members of the MCG in vitro. Enrichment cultures prepared from estuarine sediment were provided with one of a variety of carbon substrates or cultivation conditions and incubated for 3 weeks. Compared with the samples from time zero, there was an order-of-magnitude increase in the number of MCG 16S rRNA genes in almost all cultures, indicating that MCG archaea are amenable to in vitro cultivation. None of the tested substrates or conditions significantly stimulated growth of MCG archaea more than the basal medium alone; however, glycerol (0.02%) had a significantly inhibitory effect (P < 0.05). Diversity analysis of populations resulting from four culture treatments (basal medium, addition of amino acids, H₂-CO₂ as the gas phase, or initial aerobic conditions) revealed that the majority of viable MCG archaea were affiliated with the MCG-8 and MCG-4 clusters. There were no significant differences in MCG diversity between these treatments, also indicating that some members of MCG-4 and MCG-8 are tolerant of initially oxic conditions. The methods outlined here will be useful for further investigation of MCG archaea and comparison of substrates and cultivation conditions that influence their growth in vitro.     

TRAP assists membrane protein topogenesis at the mammalian ER membrane

Membrane protein insertion and topogenesis generally occur at the Sec61 translocon in the endoplasmic reticulum membrane. During this process, membrane spanning segments may adopt two distinct orientations with either their N- or C-terminus translocated into the ER lumen. While different topogenic determinants in membrane proteins, such as flanking charges, polypeptide folding, and hydrophobicity, have been identified, it is not well understood how the translocon and/or associated components decode them. Here we present evidence that the translocon-associated protein (TRAP) complex is involved in membrane protein topogenesis in vivo. Small interfering RNA (siRNA)-mediated silencing of the TRAP complex in HeLa cells enhanced the topology effect of mutating the flanking charges of a signal-anchor, but not of increasing signal hydrophobicity. The results suggest a role of the TRAP complex in moderating the ‘positive-inside’ rule.     

Verbal Memory Deficits Are Correlated with Prefrontal Hypometabolism in 18FDG PET of Recreational MDMA Users

Introduction

3,4-Methylenedioxymethamphetamine (MDMA, “ecstasy”) is a recreational club drug with supposed neurotoxic effects selectively on the serotonin system. MDMA users consistently exhibit memory dysfunction but there is an ongoing debate if these deficits are induced mainly by alterations in the prefrontal or mediotemporal cortex, especially the hippocampus. Thus, we investigated the relation of verbal memory deficits with alterations of regional cerebral brain glucose metabolism (rMRGlu) in recreational MDMA users.

Methods

Brain glucose metabolism in rest was assessed using 2-deoxy-2-(¹⁸F)fluoro-D-glucose positron emission tomography (¹⁸FDG PET) in 19 male recreational users of MDMA and 19 male drug-naïve controls. ¹⁸FDG PET data were correlated with memory performance assessed with a German version of the Rey Auditory Verbal Learning Test.

Results

As previously shown, MDMA users showed significant impairment in verbal declarative memory performance. PET scans revealed significantly decreased rMRGlu in the bilateral dorsolateral prefrontal and inferior parietal cortex, bilateral thalamus, right hippocampus, right precuneus, right cerebellum, and pons (at the level of raphe nuclei) of MDMA users. Among MDMA users, learning and recall were positively correlated with rMRGlu predominantly in bilateral frontal and parietal brain regions, while recognition was additionally related to rMRGlu in the right mediotemporal and bihemispheric lateral temporal cortex. Moreover, cumulative lifetime dose of MDMA was negatively correlated with rMRGlu in the left dorsolateral and bilateral orbital and medial PFC, left inferior parietal and right lateral temporal cortex.

Conclusions

Verbal learning and recall deficits of recreational MDMA users are correlated with glucose hypometabolism in prefrontal and parietal cortex, while word recognition was additionally correlated with mediotemporal hypometabolism. We conclude that memory deficits of MDMA users arise from combined fronto-parieto-mediotemporal dysfunction.