Cloning and molecular characterization of the HgiCI restriction/modification system from Herpetosiphon giganteus Hpg9 reveals high similarity to BanI.

The genes coding for the GGYRCC specific restriction/modification system HgiCI from Herpetosiphon giganteus Hpg9 have been cloned in Escherichia coli in three steps. As an initial step, the methyltransferase gene could be obtained after heterologous in vitro selection of a plasmid gene bank by cleavage with the isoschizomeric restriction endonuclease BanI. The adjacent endonuclease gene was cloned following Southern blot analysis of flanking genomic regions. The two genes code for polypeptides of 420 amino acids (M.HgiCI) and 345 amino acids (R.HgiCI). Establishing a functional endonuclease gene could only be achieved using a tightly regulated expression system or by methylation of the genomic DNA prior to transformation of the endonuclease gene. The methyltransferase M.HgiCI shows significant similarities to the family of 5-methylcytidine methyltransferases. Striking similarities could be found with both the isoschizomeric endonuclease and methyltransferase of the BanI restriction/modification system from Bacillus aneurinolyticus.     

Recurrent mutation pressure does not explain the prevalence of the marker (X) syndrome

Summary In order to test the hypothesis that the high prevalence of the mar(X) syndrome is caused by a high mutation rate in male germ cells only, the fraction of new mutants among mothers of probands in 112 informative families has been examined by segregation analysis among their brothers and sisters. The estimated fraction of new mutants among these mothers is much lower than expected if a stable equilibrium existed between an unusually high mutation rate and a selective disadvantage of mentally retarded, male and female mar(X) carriers. Hence, the above-mentioned hypothesis could not be confirmed.     

The membraneous nitrite reductase involved in the electron transport of Wolinella succinogenes

Wolinella succinogenes grown with nitrate as terminal electron acceptor contains two nitrite reductases as measured with the donor viologen radical, one in the cytoplasm and the other integrated in the cytoplasmic membrane. The fumarate-grown bacteria contain only the membraneous species.The isolated membraneous enzyme consists of a single polypeptide chain (M _r 63,000) carrying 4 hemeC groups and probably an iron-sulphur cluster as prosthetic groups. The enzyme amounts to about 1% of the total membrane protein.The isolated enzyme catalyses the reduction of nitrite to ammonium without accumulation of significant amounts of intermediates or alternative products. The Michaelis constant for nitrite was 0.1 mM and the turnover number of the hemeC 1.5 · 10⁵ electrons per min at 37°C.The viologen-reactive site of the enzyme in the membrane is oriented towards the cytoplasm. When the isolated enzyme is incorporated into liposomes, the viologen-as well as the nitrite-reactive site is exposed to thooutside.The cytoplasmic membrane contains a second hemeC protein (M _r 22,000) which may represent a cytochrome c.Abbreviations NQNO 2-(n-nonyl)-4-hydroxyquinoline-N-oxide - MES 2-(N-morpholino)ethanesulfonate - MOPS 3-(N-morpholino)propanesulfonate - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - MK menaquinone     

Identification of a urine metabolite constellation characteristic for kidney allograft rejection

Introduction

Allograft rejection is still an important complication after kidney transplantation. Currently, monitoring of these patients mostly relies on the measurement of serum creatinine and clinical evaluation. The gold standard for diagnosing allograft rejection, i.e. performing a renal biopsy is invasive and expensive. So far no adequate biomarkers are available for routine use.

Objectives

We aimed to develop a urine metabolite constellation that is characteristic for acute renal allograft rejection.

Methods

NMR-Spectroscopy was applied to a training cohort of transplant recipients with and without acute rejection.

Results

We obtained a metabolite constellation of four metabolites that shows promising performance to detect renal allograft rejection in the cohorts used (AUC of 0.72 and 0.74, respectively).

Conclusion

A metabolite constellation was defined with the potential for further development of an in-vitro diagnostic test that can support physicians in their clinical assessment of a kidney transplant patient.

     

Soil properties and sediment accretion modulate methane fluxes from restored wetlands

Wetlands are the largest source of methane (CH₄) globally, yet our understanding of how process‐level controls scale to ecosystem fluxes remains limited. It is particularly uncertain how variable soil properties influence ecosystem CH₄ emissions on annual time scales. We measured ecosystem carbon dioxide (CO₂) and CH₄ fluxes by eddy covariance from two wetlands recently restored on peat and alluvium soils within the Sacramento–San Joaquin Delta of California. Annual CH₄ fluxes from the alluvium wetland were significantly lower than the peat site for multiple years following restoration, but these differences were not explained by variation in dominant climate drivers or productivity across wetlands. Soil iron (Fe) concentrations were significantly higher in alluvium soils, and alluvium CH₄ fluxes were decoupled from plant processes compared with the peat site, as expected when Fe reduction inhibits CH₄ production in the rhizosphere. Soil carbon content and CO₂ uptake rates did not vary across wetlands and, thus, could also be ruled out as drivers of initial CH₄ flux differences. Differences in wetland CH₄ fluxes across soil types were transient; alluvium wetland fluxes were similar to peat wetland fluxes 3 years after restoration. Changing alluvium CH₄ emissions with time could not be explained by an empirical model based on dominant CH₄ flux biophysical drivers, suggesting that other factors, not measured by our eddy covariance towers, were responsible for these changes. Recently accreted alluvium soils were less acidic and contained more reduced Fe compared with the pre‐restoration parent soils, suggesting that CH₄ emissions increased as conditions became more favorable to methanogenesis within wetland sediments. This study suggests that alluvium soil properties, likely Fe content, are capable of inhibiting ecosystem‐scale wetland CH₄ flux, but these effects appear to be transient without continued input of alluvium to wetland sediments.     

The β2 Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes

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Decreased E‐Cadherin in MCF7 Human Breast Cancer Cells Forming Multicellular Spheroids Exposed to Simulated Microgravity

MCF7 human breast cancer cells were cultured under normal gravity (1 g) and on a random positioning machine (RPM) preventing sedimentation. After 2 weeks, adherent 1 g‐control and adherent RPM cells (AD) as well as multicellular spheroids (MCS) were harvested. AD and MCS had been exposed to the RPM in the same culture flask. In a subsequent proteome analysis, the majority of the proteins detected showed similar label‐free quantification (LFQ) scores in each of the respective subpopulations, but in both AD or MCS cultures, proteins were also found whose LFQs deviated at least twofold from their counterparts in the 1 g‐control cells. They included the cell junction protein E‐cadherin, which was diminished in MCS cells, where proteins of the E‐cadherin autodegradation pathway were enhanced and c‐Src (proto‐oncogene tyrosine‐protein kinase c‐Src) was detected. Spheroid formation was prevented by inhibition of c‐Src but promoted by antibodies blocking E‐cadherin activity. An interaction analysis of the detected proteins that are involved in forming and regulating junctions or adhesion complexes and in E‐cadherin autodegradation indicated connections between the two protein groups. This suggests that the balance of proteins that up‐ or downregulate E‐cadherin mediates the tendency of MCF7 cells to form MCS during RPM exposure.     

Effects of feeding high-quality hay with graded amounts of concentrate on feed intake,performance and blood metabolites of cows in early lactation

Dairy cows are commonly fed energy-dense diets with high proportions of concentrate feedstuffs to meet the increased energy needs of early lactation. However, feeding large amounts of concentrates may cause rumen acidosis and impact cow health. The hypothesis tested was that the energy supply and metabolic health of early-lactation Simmental cows can be maintained when high-quality hay rich in water-soluble carbohydrates (WSC) and crude protein (CP) is fed, despite the proportion of concentrates in the diet being reduced or even excluded. Twenty-four Simmental cows were allocated to one of four feeding groups beginning 10 d before the expected calving date, until 28 d thereafter. The feeding groups were 60CH (60% conventional fibre-rich hay plus 40% concentrate feed), 60HQH (60% high-quality hay plus 40% concentrate feed), 75HQH (75% high-quality hay plus 25% concentrate feed) and 100HQH (100% high-quality hay). The fibre-rich hay and high-quality hay differed in WSC content (110 g vs. 198 g of dry matter (DM)), neutral detergent fibre (646 g vs. 423 g of DM) and CP (65 g vs. 223 g of DM). Individual feed intake and milk production were monitored daily, and blood samples were collected weekly. Dry matter intake (DMI) and milk yield increased post partum, but 4 weeks post partum, the DMI of cows fed 100HQH only reached a daily mean DMI of 18.6 kg, whereas the DMI of the other groups averaged 21.9 kg (p < 0.046). The negative energy balance was less pronounced in cows fed 75HQH since they showed similar milk yields to the cows fed 60CH and 100HQH, but their energy intake was higher. Concentrations of milk components were similar across rations 60CH, 60HQH and 75HQH, as were most of blood parameters. Cows fed 100HQH responded to the energy deficit post partum with a higher ratio of non-esterified fatty acids to cholesterol and a higher concentration of ß-hydroxybutyrate (significant in comparison to cows fed 75HQH, p < 0.05). In conclusion, feeding high-quality hay with a WSC content of 20% in DM has the potential to decrease the proportion of concentrates in dairy cow feeding in early lactation, but cannot fully replace their supplementation due to a limited rumen capacity for forage intake.     

Transmission of molecularly undetectable circulating parasite clones leads to high infection complexity in mosquitoes post feeding

Plasmodium falciparum malaria infections often comprise multiple distinct parasite clones. Few datasets have directly assessed infection complexity in humans and mosquitoes they infect. Examining parasites using molecular tools may provide insights into the selective transmissibility of isolates. Using capillary electrophoresis genotyping and next generation amplicon sequencing, we analysed complexity of parasite infections in human blood and in the midguts of mosquitoes that became infected in membrane feeding experiments using the same blood material in two West African settings. Median numbers of clones in humans and mosquitoes were higher in samples from Burkina Faso (4.5, interquartile range 2–8 for humans; and 2, interquartile range 1–3 for mosquitoes) than in The Gambia (2, interquartile range 1–3 and 1, interquartile range 1–3, for humans and mosquitoes, respectively). Whilst the median number of clones was commonly higher in human blood samples, not all transmitted alleles were detectable in the human peripheral blood. In both study sample sets, additional parasite alleles were identified in mosquitoes compared with the matched human samples (10–88.9% of all clones/feeding assay, n?=?73 feeding assays). The results are likely due to preferential amplification of the most abundant clones in peripheral blood but confirm the presence of low density clones that produce transmissible sexual stage parasites.