Alkaline phosphatase in nasal secretion of cattle: biochemical and molecular characterisation

BACKGROUND: Nasal secretion (NS) was investigated as a source of information regarding the mucosal and systemic immune status of cattle challenged by respiratory disease. A method for the collection of substantial volumes (~12 ml) of NS from cattle was developed to establish a reference range of analytes that are present in the NS of healthy cattle. Biochemical profiles of NS from a group of 38 healthy Holstein-Friesian cows revealed high alkaline phosphatase (AP) activity of up to 2392 IU/L. The character and source of the high activity of AP in bovine NS was investigated. RESULTS: Histochemical analysis confirmed the localization of the AP enzyme activity to epithelial cells and serous glands of the nasal respiratory mucosa. Analysis of mRNA levels from nasal mucosa by end point RT-PCR and PCR product sequencing confirmed that the AP was locally produced and is identical at the nucleotide level to the non-specific AP splice variant found in bovine liver, bone and kidney. Analysis by isoelectric focussing confirmed that AP was produced locally at a high level in nasal epithelium demonstrating that AP from nasal secretion and nasal mucosa had similar pI bands, though differing from those of the liver, kidney, bone and intestine, suggesting different post-translational modification (PTM) of AP in these tissues. CONCLUSIONS: A nasal isozyme of AP has been identified that is present at a high activity in NS, resulting from local production and showing distinctive PTM and may be active in NS as an anti-endotoxin mediator

Modulating Integrin αIIbβ3 Activity through Mutagenesis of Allosterically Regulated Intersubunit Contacts

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.biochem.9b00430.Integrin αIIbβ3, a transmembrane heterodimer, mediates platelet aggregation when it switches from an inactive to an active ligand-binding conformation following platelet stimulation. Central to regulating αIIbβ3 activity is the interaction between the αIIb and β3 extracellular stalks, which form a tight heterodimer in the inactive state and dissociate in the active state. Here, we demonstrate that alanine replacements of sensitive positions in the heterodimer stalk interface destabilize the inactive conformation sufficiently to cause constitutive αIIbβ3 activation. To determine the structural basis for this effect, we performed a structural bioinformatics analysis and found that perturbing intersubunit contacts with favorable interaction geometry through substitutions to alanine quantitatively accounted for the degree of constitutive αIIbβ3 activation. This mutational study directly assesses the relationship between favorable interaction geometry at mutation-sensitive positions and the functional activity of those mutants, giving rise to a simple model that highlights the importance of interaction geometry in contributing to the stability between protein–protein interactions.NIH P01 HL40387NIH R35 GM122603National Science Foundation 1709506National Science Foundation 165011

Further evidence for the involvement of EFL1 in a Shwachman-Diamond-like syndrome and expansion of the phenotypic features.

Recent evidence has implicated EFL1 in a phenotype overlapping Shwachman-Diamond syndrome (SDS), with the functional interplay between EFL1 and the previously known causative gene SBDS accounting for the similarity in clinical features. Relatively little is known about the phenotypes associated with pathogenic variants in the EFL1 gene, but the initial indication was that phenotypes may be more severe, when compared with SDS. We report a pediatric patient who presented with a metaphyseal dysplasia and was found to have biallelic variants in EFL1 on reanalysis of trio whole-exome sequencing data. The variant had not been initially reported because of the research laboratory's focus on de novo variants. Subsequent phenotyping revealed variability in her manifestations. Although her metaphyseal abnormalities were more severe than in the original reported cohort with EFL1 variants, the bone marrow abnormalities were generally mild, and there was equivocal evidence for pancreatic insufficiency. Despite the limited number of reported patients, variants in EFL1 appear to cause a broader spectrum of symptoms that overlap with those seen in SDS. Our report adds to the evidence of EFL1 being associated with an SDS-like phenotype and provides information adding to our understanding of the phenotypic variability of this disorder. Our report also highlights the value of exome data reanalysis when a diagnosis is not initially apparent

Arthropod Borne Disease: The Leading Cause of Fever in Pregnancy on the Thai-Burmese Border

Fever during pregnancy can be harmful for the mother and the infant. In resource poor settings health workers have very few field-based tests that help them identify the cause of infection. This study examined the causes of fever in pregnant women using laboratory support that is typically unavailable to most women living in the tropics. On the Thai-Burmese border there has been a great reduction in malaria in the last 20 years. However malaria remained the leading cause of fever in pregnancy in this study conducted between 2004 and 2006. Urinary tract infection was also a common cause of fever as it is in resource rich countries. Other diseases transmitted by mosquitoes (dengue), ticks (scrub and murine typhus), or rodents (leptospirosis) were common. Scrub and murine typhus were associated with stillbirth and low birth weight. Microscopy remains the most useful tool in the field for the diagnosis of fever in pregnant women. Leptospirosis, dengue and rickettsial infections require improved field-based diagnostic tools to ensure that women receive appropriate antibiotic therapy

The Wnt co-receptor PTK7/Otk and Its Homolog Otk-2 in neurogenesis and patterning

Wnt signaling is a highly conserved metazoan pathway that plays a crucial role in cell fate determination and morphogenesis during development. Wnt ligands can induce disparate cellular responses. The exact mechanism behind these different outcomes is not fully understood but may be due to interactions with different receptors on the cell membrane. PTK7/Otk is a transmembrane receptor that is implicated in various developmental and physiological processes including cell polarity, cell migration, and invasion. Here, we examine two roles of Otk-1 and Otk-2 in patterning and neurogenesis. We find that Otk-1 is a positive regulator of signaling and Otk-2 functions as its inhibitor. We propose that PTK7/Otk functions in signaling, cell migration, and polarity contributing to the diversity of cellular responses seen in Wnt-mediated processes

A Planetary Microlensing Event with an Unusually Red Source Star: MOA-2011-BLG-291

We present the analysis of planetary microlensing event MOA-2011-BLG-291, which has a mass ratio of $q=(3.8\pm0.7)\times10^{-4}$ and a source star that is redder (or brighter) than the bulge main sequence. This event is located at a low Galactic latitude in the survey area that is currently planned for NASA's WFIRST exoplanet microlensing survey. This unusual color for a microlensed source star implies that we cannot assume that the source star is in the Galactic bulge. The favored interpretation is that the source star is a lower main sequence star at a distance of $D_S=4.9\pm1.3\,$kpc in the Galactic disk. However, the source could also be a turn-off star on the far side of the bulge or a sub-giant in the far side of the Galactic disk if it experiences significantly more reddening than the bulge red clump stars. However, these possibilities have only a small effect on our mass estimates for the host star and planet. We find host star and planet masses of $M_{\rm host} =0.15^{+0.27}_{-0.10}M_\odot$ and $m_p=18^{+34}_{-12}M_\oplus$ from a Bayesian analysis with a standard Galactic model under the assumption that the planet hosting probability does not depend on the host mass or distance. However, if we attempt to measure the host and planet masses with host star brightness measurements from high angular resolution follow-up imaging, the implied masses will be sensitive to the host star distance. The WFIRST exoplanet microlensing survey is expected to use this method to determine the masses for many of the planetary systems that it discovers, so this issue has important design implications for the WFIRST exoplanet microlensing survey

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal

UroMark-a urinary biomarker assay for the detection of bladder cancer.

BACKGROUND: Bladder cancer (BC) is one of the most common cancers in the western world and ranks as the most expensive to manage, due to the need for cystoscopic examination. BC shows frequent changes in DNA methylation, and several studies have shown the potential utility of urinary biomarkers by detecting epigenetic alterations in voided urine. The aim of this study is to develop a targeted bisulfite next-generation sequencing assay to diagnose BC from urine with high sensitivity and specificity. RESULTS: We defined a 150 CpG loci biomarker panel from a cohort of 86 muscle-invasive bladder cancers and 30 normal urothelium. Based on this panel, we developed the UroMark assay, a next-generation bisulphite sequencing assay and analysis pipeline for the detection of bladder cancer from urinary sediment DNA. The 150 loci UroMark assay was validated in an independent cohort (n = 274, non-cancer (n = 167) and bladder cancer (n = 107)) voided urine samples with an AUC of 97%. The UroMark classifier sensitivity of 98%, specificity of 97% and NPV of 97% for the detection of primary BC was compared to non-BC urine. CONCLUSIONS: Epigenetic urinary biomarkers for detection of BC have the potential to revolutionise the management of this disease. In this proof of concept study, we show the development and utility of a novel high-throughput, next-generation sequencing-based biomarker for the detection of BC-specific epigenetic alterations in urine