High-throughput multi-antigen microfluidic fluorescence immunoassays

Here we describe the development of a high-throughput multi-antigen microfluidic fluorescence immunoassay system. A 100-chamber polydimethylsiloxane (PDMS) chip performs up to 5 tests for each of 10 samples. In this particular study system, the specificity of detection was demonstrated, and calibration curves were produced for C-reactive protein (CRP), prostate-specific antigen (PSA), ferritin, and vascular endothelial growth factor (VEGF). The measurements show sensitivity at and below clinically normal levels (with a signal-to-noise ratio >8 at as low as 10 pM antigen concentration). The chip uses 100 nL per sample for all tests. The developed system is an important step toward derivative immunoassay applications in scientific research and "point-of-care" testing in medicine.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

Stimulation by auxin of phospholipase A in membrane vesicles from an auxin-sensitive tissue is mediated by an auxin receptor

Microsomal vesicles were prepared from zucchini (Cucurbita pepo L.) hypocotyls containing radioactive phosphatidylethanolamine or phosphatidylcholine, and these lipids were used as substrates by phospholipase A which is activated by auxins. Phospholipase D and phospholipase C hydrolysed the same substrates but were not influenced by auxin. Phospholipase A was activated by the auxins indolyl-3-acetic acid, 2,4-dichlorophenoxyacetic acid and, to a lesser extent, by -naphthaleneacetic acid whereas the weak auxins 2,3-dichlorophenoxyacetic acid and -naphthaleneacetic acid were almost inactive. This hormone specificity was also found in growth tests with etiolated zucchini hypocotyls. Phospholipase A activation by auxin was blocked by a polyclonal antibody against the maize auxin-binding protein. We propose that phospholipase A activation is a primary reaction in the signal transduction leading from hormone-binding to the growth response.Abbreviations IAA indolyl-3-acetic acid - 2,3-D, 2,4-D 2,3- and 2,4-dichlorophenoxyacetic acid - -NAA; -NAA - and -naphthaleneacetic acid This work was supported by the Deutsche Forchungsgemeinschaft. We thank D. Klämbt (Botanical Institute, University of Bonn, FRG) for a generous gift of polyclonal antibody (IgG fraction) against auxin-binding protein and U. Kutschera (Botanical Institute, University of Bonn, FRG) for advice with the growth tests.     

Evaluating the effectiveness of retention forestry to enhance biodiversity in production forests of Central Europe using an interdisciplinary,multi‐scale approach

Retention forestry, which retains a portion of the original stand at the time of harvesting to maintain continuity of structural and compositional diversity, has been originally developed to mitigate the impacts of clear‐cutting. Retention of habitat trees and deadwood has since become common practice also in continuous‐cover forests of Central Europe. While the use of retention in these forests is plausible, the evidence base for its application is lacking, trade‐offs have not been quantified, it is not clear what support it receives from forest owners and other stakeholders and how it is best integrated into forest management practices. The Research Training Group ConFoBi (Conservation of Forest Biodiversity in Multiple‐use Landscapes of Central Europe) focusses on the effectiveness of retention forestry, combining ecological studies on forest biodiversity with social and economic studies of biodiversity conservation across multiple spatial scales. The aim of ConFoBi is to assess whether and how structural retention measures are appropriate for the conservation of forest biodiversity in uneven‐aged and selectively harvested continuous‐cover forests of temperate Europe. The study design is based on a pool of 135 plots (1 ha) distributed along gradients of forest connectivity and structure. The main objectives are (a) to investigate the effects of structural elements and landscape context on multiple taxa, including different trophic and functional groups, to evaluate the effectiveness of retention practices for biodiversity conservation; (b) to analyze how forest biodiversity conservation is perceived and practiced, and what costs and benefits it creates; and (c) to identify how biodiversity conservation can be effectively integrated in multi‐functional forest management. ConFoBi will quantify retention levels required across the landscape, as well as the socio‐economic prerequisites for their implementation by forest owners and managers. ConFoBi's research results will provide an evidence base for integrating biodiversity conservation into forest management in temperate forests.     

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.     

Notch1 control of oligodendrocyte differentiation in the spinal cord

We have selectively inhibited Notch1 signaling in oligodendrocyte precursors (OPCs) using the Cre/loxP system in transgenic mice to investigate the role of Notch1 in oligodendrocyte (OL) development and differentiation. Early development of OPCs appeared normal in the spinal cord. However, at embryonic day 17.5, premature OL differentiation was observed and ectopic immature OLs were present in the gray matter. At birth, OL apoptosis was strongly increased in Notch1 mutant animals. Premature OL differentiation was also observed in the cerebrum, indicating that Notch1 is required for the correct spatial and temporal regulation of OL differentiation in various regions of the central nervous system. These findings establish a widespread function of Notch1 in the late steps of mammalian OPC development in vivo.     

Genomic sequencing reveals the structure of the Kcnk6 and map3k11 genes and their close vicinity to the sipa1 gene on mouse chromosome 19

In this report we present the analysis of two overlapping mouse cosmid clones that contain the entire Kcnk6, Map3k11 and Pcnxl3 genes, as well as part of the Sipa1 gene. The sequence and genomic organisation of the Kcnk6 and Map3k11 genes are described in detail. Sipa1 and Map3k11, which have independently been mapped with low resolution to the centromeric region of mouse chromosome 19, are shown here to lie close to each other and to the Kcnk6 gene, which has not previously been mapped. This gene cluster maps to the vicinity of the Dancer (Dc) mutation, which involves inner ear abnormalities and circling phenotypes. Since potassium channels have been implicated in deafness disorders, we have analysed the Kcnk6 gene, which encodes a two-P domain potassium channel, in the Dc mutant. No Dc-causing mutation in the Kcnk6 coding region could be identified. However, we detected a polymorphism in the Kcnk6 gene that leads to a C-terminal extension of the encoded protein by eight amino acids.     

Could life have arisen in the primitive atmosphere?

Summary A recently proposed model for the origin of prebiotic progenitors of life in particles suspended in a primitive, specially organized atmosphere is considered critically. It is concluded that the physical and chemical framework of the new hypothesis conflicts with the conditions necessary for the evolution of the progenitors of life in the atmosphere of the early Earth. Therefore this model seems not to be a reasonable alternative to the Oparin thesis.     

Radioimmunological and biological measurement of prostaglandins in rabbit urine : Decrease of PGE2 excretion at high NaCl intake

The estimation of prostaglandin (PG) E₂ and of PGF_2α by radioimmunoassay is described in detail. PGE₂ was measured after conversion to either PGB₂ or PGF_2α and the results compared to bioassay. The methods were used to follow the excretion of PGE₂ and PGF_2α after salt loading in rabbits. A marked reduction of PGE₂ levels was observed at high NaCl intake, while PGF_2α excretion remained unchanged.     

The Role of Adipocytes and Adipocyte‐Like Cells in the Severity of COVID‐19 Infections

Coronavirus disease‐2019 (COVID‐19), caused by the highly pathogenic severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), demonstrates high morbidity and mortality caused by development of a severe acute respiratory syndrome connected with extensive pulmonary fibrosis. In this Perspective, we argue that adipocytes and adipocyte‐like cells, such as pulmonary lipofibroblasts, may play an important role in the pathogenic response to SARS‐CoV‐2. Expression of angiotensin‐converting enzyme 2 (the functional receptor for SARS‐CoV) is upregulated in adipocytes of patients with obesity and diabetes, which turns adipose tissue into a potential target and viral reservoir. This may explain why obesity and diabetes are potential comorbidities for COVID‐19 infections. Similar to the recently established adipocyte‐myofibroblast transition, pulmonary lipofibroblasts located in the alveolar interstitium and closely related to classical adipocytes demonstrate the ability to transdifferentiate into myofibroblasts that play an integral part of pulmonary fibrosis. This may significantly increase the severity of the local response to SARS‐CoV‐2 in the lung. To reduce the severity and mortality associated with COVID‐19, we propose to probe for the clinical response to thiazolidinediones, peroxisome proliferator activated receptor γ agonists that are well‐known antidiabetic drugs. Thiazolidinediones are able to stabilize lipofibroblasts in their “inactive” state, preventing the transition to myofibroblasts and thereby reducing the development of pulmonary fibrosis and stimulating its resolution.