Genomes of Thaumarchaeota from deep sea sediments reveal specific adaptations of three independently evolved lineages

Marine sediments represent a vast habitat for complex microbiomes. Among these, ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are one of the most common, yet little explored, inhabitants, which seem extraordinarily well adapted to the harsh conditions of the subsurface biosphere. We present 11 metagenome-assembled genomes of the most abundant AOA clades from sediment cores obtained from the Atlantic Mid-Ocean ridge flanks and Pacific abyssal plains. Their phylogenomic placement reveals three independently evolved clades within the order Nitrosopumilales, of which no cultured representative is known yet. In addition to the gene sets for ammonia oxidation and carbon fixation known from other AOA, all genomes encode an extended capacity for the conversion of fermentation products that can be channeled into the central carbon metabolism, as well as uptake of amino acids probably for protein maintenance or as an ammonia source. Two lineages encode an additional (V-type) ATPase and a large repertoire of DNA repair systems that may allow to overcome the challenges of high hydrostatic pressure. We suggest that the adaptive radiation of AOA into marine sediments occurred more than once in evolution and resulted in three distinct lineages with particular adaptations to this extremely energy-limiting and high-pressure environment.Subject terms: Phylogenetics, Metagenomics     

Soluble Endoglin,Transforming Growth Factor-Beta 1 and Soluble Tumor Necrosis Factor Alpha Receptors in Different Clinical Manifestations of Preeclampsia

Background

Despite intensive research, the etiopathogenesis of preeclampsia (PE) remains uncertain. Inflammatory and angiogenic factors are thought to play considerable roles in this disease. The objective of this study was to investigate the association between soluble endoglin (sEng), transforming growth factor beta-1 (TGF-β1) and tumor necrosis factor alpha soluble receptors (sTNF-Rs) and the clinical manifestations of PE.

Methods

Plasma levels of sEng, TGF-β1 and sTNF-Rs were determined by ELISA in 23 non-pregnant, 21 normotensive pregnant and 43 PE women. PE women were stratified into subgroups according to the severity [mild (n = 12) and severe (n = 31)] and onset-time of the disease [early (n = 19) and late (n = 24)].

Results

Pregnancy was associated with higher levels of sEng, sTNF-R1 and sTNF-R2 than the non-pregnant state. Moreover, PE women had higher levels of sEng and sTNF-R1 than normotensive pregnant women. No difference was found in TGF-β1 levels, comparing the three study groups. Late PE had higher levels of sTNF-R1 and sTNF-R2 than early PE. No significant differences were found in sEng and TGF-β1 comparing early and late PE. sEng levels were higher in severe PE than in mild PE and no difference was found for TGF-β1, sTNF-R1 and sTNF-R2 levels. There was a positive correlation among sEng, TNF-R1 and sTNF-2 levels. Logistic regression analysis revealed that primiparity and sEng levels are independently associated with the development of PE. Furthermore, sEng levels are independently associated with the disease severity.

Conclusions

These results suggest that pregnancy is a condition associated with higher levels of anti-angiogenic and pro-inflammatory factors than the non-pregnant state and that PE is associated with an imbalance of these factors in the maternal circulation.     

Influence of Pigment Epithelium-Derived Factor on Outcome after Striatal Cerebral Ischemia in the Mouse

We here suggest that pigment epithelium-derived factor (PEDF) does not have an effect on lesion size, behavioral outcome, cell proliferation, or cell death after striatal ischemia in the mouse. PEDF is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. It influences self-renewal of neural stem cells and proliferation of microglia. We investigated whether intraventricular infusion of PEDF reduces infarct size and cell death, ameliorates behavioral outcome, and influences cell proliferation in the one-hour middle cerebral artery occlusion (MCAO) mouse model of focal cerebral ischemia. C57Bl6/N mice were implanted with PEDF or artificial cerebrospinal fluid (control) osmotic pumps and subjected to 60-minute MCAO 48 hours after pump implantation. They received daily BrdU injections for 7 days after MCAO in order to investigate cell proliferation. Infarct volumes were determined 24 hours after reperfusion using magnetic resonance imaging. We removed the pumps on day 5 and performed behavioral testing between day 7 and 21. Immunohistochemical staining was performed to determine the effect of PEDF on cell proliferation and cell death. Our model produced an ischemic injury confined solely to striatal damage. We detected no reduction in infarct sizes and cell death in PEDF- vs. CSF-infused MCAO mice. Behavioral outcome and cell proliferation did not differ between the groups. However, we cannot exclude that PEDF might work under different conditions in stroke. Further studies will elucidate the effect of PEDF treatment on cell proliferation and behavioral outcome in moderate to severe ischemic injury in the brain.     

Oligomeric State of Purified Transient Receptor Potential Melastatin-1 (TRPM1), a Protein Essential for Dim Light Vision

Transient receptor potential melastatin-1 (TRPM1) is essential for the light-induced depolarization of retinal ON bipolar cells. TRPM1 likely forms a multimeric channel complex, although almost nothing is known about the structure or subunit composition of channels formed by TRPM1 or any of its close relatives. Recombinant TRPM1 was robustly expressed in insect cells, but only a small fraction was localized to the plasma membrane. Similar intracellular localization was observed when TRPM1 was heterologously expressed in mammalian cells. TRPM1 was affinity-purified from Sf9 cells and complexed with amphipol, followed by detergent removal. In blue native gels and size exclusion chromatography, TRPM1 migrated with a mobility consistent with detergent- or amphipol-bound dimers. Cross-linking experiments were also consistent with a dimeric subunit stoichiometry, and cryoelectron microscopy and single particle analysis without symmetry imposition yielded a model with approximate 2-fold symmetrical features. Finally, electron microscopy of TRPM1-antibody complexes revealed a large particle that can accommodate TRPM1 and two antibody molecules. Taken together, these data indicate that purified TRPM1 is mostly dimeric. The three-dimensional structure of TRPM1 dimers is characterized by a small putative transmembrane domain and a larger domain with a hollow cavity. Blue native gels of solubilized mouse retina indicate that TRPM1 is present in two distinct complexes: one similar in size to the recombinant protein and one much larger. Because dimers are likely not functional ion channels, these results suggest that additional partner subunits participate in forming the transduction channel required for dim light vision and the ON pathway.     

Nitric Oxide Inhibits Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia by Increasing the Ubiquitination and Degradation of UbcH10

Nitric oxide (NO) limits formation of neointimal hyperplasia in animal models of arterial injury in large part by inhibiting vascular smooth muscle cell (VSMC) proliferation through cell cycle arrest. The ubiquitin-conjugating enzyme UbcH10 is responsible for ubiquitinating cell cycle proteins for proper exit from mitosis. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing levels of UbcH10. Western blotting and immunofluorescent staining showed that NO reduced UbcH10 levels in a concentration-dependent manner in VSMC harvested from the abdominal aortas of Sprague-Dawley rats. Treatment with NO or siRNA to UbcH10 decreased both UbcH10 levels and VSMC proliferation (P<0.001), while increasing UbcH10 levels by plasmid transfection or angiotensin II stimulation increased VSMC proliferation to 150% (P=0.008) and 212% (P=0.002) of control, respectively. Immunofluorescent staining of balloon-injured rat carotid arteries showed a ~4-fold increase in UbcH10 levels, which was profoundly decreased following treatment with NO. Western blotting of carotid artery lysates showed no UbcH10 in uninjured vessels, a substantial increase in the injury alone group, and a significant decrease in the injury+NO group (~3-fold reduction versus injury alone). Importantly, in vitro and in vivo, a marked increase in polyubiquitinated UbcH10 was observed in the NO-treated VSMC and carotid arteries, respectively, indicating that NO may be decreasing unmodified UbcH10 levels by increasing its ubiquitination. Central to our hypothesis, we report that NO decreases UbcH10 levels in VSMC in vitro and following arterial injury in vivo in association with increasing polyubiquitinated-UbcH10 levels. These changes in UbcH10 levels correlate with VSMC proliferation and neointimal hyperplasia, making UbcH10 a promising therapeutic target for inhibiting this proliferative disease.     

Structural and Functional Characterisation of a Conserved Archaeal RadA Paralog with Antirecombinase Activity

DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. RecA is the sole protein responsible for this reaction in bacteria, whereas there are several Rad51 paralogs that cooperate to catalyse strand exchange in eukaryotes. All archaea have at least one (and as many as four) RadA paralog, but their function remains unclear. Herein, we show that the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions and are not UV inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large archaeal RadC subfamily of archaeal RadA paralogs, functions as an ATPase that binds tightly to single-stranded DNA. However, Sso2452 is not an active recombinase in vitro and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.     

Touch sensation by pectoral fins of the catfish Pimelodus pictus

Mechanosensation is fundamental to many tetrapod limb functions, yet it remains largely uninvestigated in the paired fins of fishes, limb homologues. Here we examine whether membranous fins may function as passive structures for touch sensation. We investigate the pectoral fins of the pictus catfish (Pimelodus pictus), a species that lives in close association with the benthic substrate and whose fins are positioned near its ventral margin. Kinematic analysis shows that the pectoral fins are held partially protracted during routine forward swimming and do not appear to generate propulsive force. Immunohistochemistry reveals that the fins are highly innervated, and we observe putative mechanoreceptors at nerve fibre endings. To test for the ability to sense mechanical perturbations, activity of fin ray nerve fibres was recorded in response to touch and bend stimulation. Both pressure and light surface brushing generated afferent nerve activity. Fin ray nerves also respond to bending of the rays. These data demonstrate for the first time that membranous fins can function as passive mechanosensors. We suggest that touch-sensitive fins may be widespread in fishes that maintain a close association with the bottom substrate.     

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3^R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.     

The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens

For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.