Controlled addition of small amounts of oxygen with membranes

Summary In some fermentations micro-aerobic conditions are desirable. The controlled addition of trace amounts of oxygen may be achieved by bubble free oxygen supply using silicon membrane tube, operated in dead-end mode. The small oxygen uptake rate is accurately determined with this self-constructable device.     

Preeclampsia and Blood Pressure Trajectory during Pregnancy in Relation to Vitamin D Status

Every tenth pregnancy is affected by hypertension, one of the most common complications and leading causes of maternal death worldwide. Hypertensive disorders in pregnancy include pregnancy-induced hypertension and preeclampsia. The pathophysiology of the development of hypertension in pregnancy is unknown, but studies suggest an association with vitamin D status, measured as 25-hydroxyvitamin D (25(OH)D). The aim of this study was to investigate the association between gestational 25(OH)D concentration and preeclampsia, pregnancy-induced hypertension and blood pressure trajectory. This cohort study included 2000 women. Blood was collected at the first (T1) and third (T3) trimester (mean gestational weeks 10.8 and 33.4). Blood pressure at gestational weeks 10, 25, 32 and 37 as well as symptoms of preeclampsia and pregnancy-induced hypertension were retrieved from medical records. Serum 25(OH)D concentrations (LC-MS/MS) in T1 was not significantly associated with preeclampsia. However, both 25(OH)D in T3 and change in 25(OH)D from T1 to T3 were significantly and negatively associated with preeclampsia. Women with a change in 25(OH)D concentration of ≥30 nmol/L had an odds ratio of 0.22 (p = 0.002) for preeclampsia. T1 25(OH)D was positively related to T1 systolic (β = 0.03, p = 0.022) and T1 diastolic blood pressure (β = 0.02, p = 0.016), and to systolic (β = 0.02, p = 0.02) blood pressure trajectory during pregnancy, in adjusted analyses. There was no association between 25(OH)D and pregnancy-induced hypertension in adjusted analysis. In conclusion, an increase in 25(OH)D concentration during pregnancy of at least 30 nmol/L, regardless of vitamin D status in T1, was associated with a lower odds ratio for preeclampsia. Vitamin D status was significantly and positively associated with T1 blood pressure and gestational systolic blood pressure trajectory but not with pregnancy-induced hypertension.     

Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.Subject terms: Environmental microbiology, Sequencing, Microbiology     

Differential effects of scavenger receptor BI deficiency on lipid metabolism in cells of the arterial wall and in the liver

Scavenger receptor class B, type I (SRBI) is a key regulator of high density lipoprotein (HDL) metabolism. It facilitates the efflux of cholesterol from cells in peripheral tissues to HDL and mediates the selective uptake of cholesteryl esters from HDL in the liver. We investigated the effects of SRBI deficiency in the arterial wall and in the liver using SRBI-deficient mice and wild-type littermates fed a Western-type diet. The SRBI-deficient mice showed massive accumulation of cholesterol-rich HDL in the circulation, reflecting impaired delivery to the liver. Strikingly, SRBI deficiency did not alter hepatic cholesterol (ester) content nor did it affect the expression of key regulators of hepatic cholesterol homeostasis, including HMG-CoA reductase, the low density lipoprotein receptor, and cholesterol 7alpha-hydroxylase. However, a approximately 40% reduction in biliary cholesterol content was observed, and the expression of ABCG8 and ABCG5, ATP half-transporters implicated in the transport of sterols from the liver to the bile, was attenuated by 70 and 35%, respectively. In contrast to the situation in the liver, SRBI deficiency did result in lipid deposition in the aorta and atherosclerosis. Vascular mRNA analysis showed increased expression of inflammatory markers as well as of genes involved in cellular cholesterol homeostasis. Our data show that, although hepatic cholesterol homeostasis is maintained upon feeding a Western-type diet, SRBI deficiency is associated with de-regulation of cholesterol homeostasis in the arterial wall that results in an increased susceptibility to atherosclerosis.     

Accuracy and consistency in application of a probabilistic approach to reporting breast fine needle aspiration

OBJECTIVE: To determine the application of a probabilistic/categorical approach for reporting breast fine needle aspiration (FNA) and its dependence on the cytopathologist's level of experience. STUDY DESIGN: All breast surgical specimens that had preoperative breast FNA at our institution during a 3-year period were identified. The cytologic results were reported as 1 of 6 categories: positive, suspicious, atypical, epithelial proliferative, unremarkable and nondiagnostic, according to well-defined criteria. Five cytopathologists were responsible for all cytology sing-out during the study period. The histologic and cytologic diagnoses were correlated. RESULTS: A total of 297 cases were identified. Overall, there were no false positive cases (positive predictive value [PPV] = 100%). Two false negative cases (negative predictive value [NPV] = 96%) were due to sampling error. This indicates that the PPV and NPV for each of the 5 pathologists were also all 100% except for the 1 pathologist who had two false negative cases due to sampling errors. The probability of finding carcinoma on histology for suspicious and atypical cytologic categories ranged from 67% to 100% and 8% to 31%, respectively, for the individual pathologists. Fifteen cases were signed out by > or = 2 pathologists. The involvement of consultants was significantly associated with diagnosis (P = .02). Ten of the 15 cases were in the suspicious (5) or atypical (5) category. CONCLUSION: The probabilistic approach with defined diagnostic criteria is an accurate method and can be consistently applied in reporting breast FNA. Although use of the indeterminate (suspicious and atypical) categories is variable, a definite and considerable difference in the probability of carcinoma between these 2 categories was observed for all pathologists. The involvement of consultants did not move the cases out of these indeterminate categories.     

Stabilization of the soluble,cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1

The envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 has evolved a structure that is minimally immunogenic while retaining its natural function of receptor-mediated virus-cell fusion. The Env complex is trimeric; its six individual subunits (three gp120 and three gp41 subunits) are associated by relatively weak, noncovalent interactions. The induction of neutralizing antibodies after vaccination with individual Env subunits has proven very difficult, probably because they are inadequate mimics of the native complex. Our hypothesis is that a stable form of the Env complex, perhaps with additional modifications to rationally alter its antigenic structure, may be a better immunogen than the individual subunits. A soluble form of Env, SOS gp140, can be made that has gp120 stably linked to the gp41 ectodomain by an intermolecular disulfide bond. This protein is fully cleaved at the proteolysis site between gp120 and gp41. However, the gp41-gp41 interactions in SOS gp140 are too weak to maintain the protein in a trimeric configuration. Consequently, purified SOS gp140 is a monomer (N. Schülke, M. S. Vesanen, R. W. Sanders, P. Zhu, D. J. Anselma, A. R. Villa, P. W. H. I. Parren, J. M. Binley, K. H. Roux, P. J. Maddon, J. P. Moore, and W. C. Olson, J. Virol. 76:7760-7776, 2002). Here we describe modifications of SOS gp140 that increase its trimer stability. A variant SOS gp140, designated SOSIP gp140, contains an isoleucine-to-proline substitution at position 559 in the N-terminal heptad repeat region of gp41. This protein is fully cleaved, has favorable antigenic properties, and is predominantly trimeric. SOSIP gp140 trimers are noncovalently associated and can be partially purified by gel filtration chromatography. These gp140 trimers are dissociated into monomers by anionic detergents or heat but are relatively resistant to nonionic detergents, high salt concentrations, or exposure to a mildly acidic pH. SOSIP gp140 should be a useful reagent for structural and immunogenicity studies.     

Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins

In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.     

The influence of surface wettability on the adhesion strength of settled spores of the green alga enteromorpha and the diatom amphora

In this paper we report on the effect of surface wettabilityon surface selection and adhesion properties of settled (adhered)spores of the biofouling marine alga Enteromorpha and cellsof the diatom Amphora, through the use of patterned self-assembledmonolayers (SAMs). The SAMs were formed from alkanethiols terminatedwith methyl (CH₃) or hydroxyl (OH) groups, or mixtures of thetwo, creating a discontinuous gradient of wettability as measuredby advancing water contact angle. In the case of Enteromorpha,primary adhesion, as measured by the transition from a motilespore to a settled, sessile organism, is strongly promoted bythe hydrophobic surfaces. On the other hand, adhesion strengthof the settled spores, as measured by resistance to detachmentin a turbulent flow cell, is greatest on a hydrophilic surface.In the case of Amphora, there is little influence of surfacewettability on the primary adhesion of this organism, but motilityis inhibited at contact angles     

Scale-up of stirring as foam disruption (SAFD) to industrial scale

Foam disruption by agitation—the stirring as foam disruption (SAFD) technique—was scaled up to pilot and production scale using Rushton turbines and an up-pumping hydrofoil impeller, the Scaba 3SHP1. The dominating mechanism behind SAFD—foam entrainment—was also demonstrated at production scale. The mechanistic model for SAFD defines a fictitious liquid velocity generated by the (upper) impeller near the dispersion surface, which is correlated with complete foam disruption. This model proved to be scalable, thus enabling the model to be used for the design of SAFD applications. Axial upward pumping impellers appeared to be more effective with respect to SAFD than Rushton turbines, as demonstrated by retrofitting a 12,000 l bioreactor, i.e. the triple Rushton configuration was compared with a mixed impeller configuration from Scaba with a 20% lower ungassed power draw. The retrofitted impeller configuration allowed 10% more broth without risking excessive foaming. In this way a substantial increase in the volumetric productivity of the bioreactor was achieved. Design recommendations for the application of SAFD are given in this paper. Using these recommendations for the design of a 30,000 l scale bioreactor, almost foamless Escherichia coli fermentations were realised. Electronic Publication