Clinical code sets are vital to research using routinely-collected electronic healthcare data. Existing code set engineering methods pose significant limitations when considering reproducible research. To improve the transparency and reusability of research, these code sets must abide by FAIR principles; this is not currently happening. We propose 'term sets', an equivalent alternative to code sets that are findable, accessible, interoperable and reusable.

We present a technical platform that allows us to monitor and measure cortex and membrane dynamics during bleb-based chemotaxis. Using D. discoideum cells expressing LifeAct-GFP and crawling under agarose containing RITC-dextran, we were able to simultaneously visualize the actin cortex and the cell membrane throughout bleb formation. Using these images, we then applied edge detect to generate points on the cell boundary with coordinates in a coordinate plane. Then we fitted these points to a curve with known x and y coordinate functions. The result was to parameterize the cell outline. With the parameterization, we demonstrate how to compute data for geometric features such as cell area, bleb area and edge curvature. This allows us to collect vital data for the analysis of blebbing.

Bacteriophages are the most abundant members of the microbiota and have the potential to shape gut bacterial communities. Changes to bacteriophage composition are associated with disease, but how phages impact mammalian health remains unclear. We noted an induction of host immunity when experimentally treating bacterially driven cancer, leading us to test whether bacteriophages alter immune responses. Treating germ-free mice with bacteriophages leads to immune cell expansion in the gut. Lactobacillus, Escherichia, and Bacteroides bacteriophages and phage DNA stimulated IFN-γ via the nucleotide-sensing receptor TLR9. The resultant immune responses were both phage and bacteria specific. Additionally, increasing bacteriophage levels exacerbated colitis via TLR9 and IFN-γ. Similarly, ulcerative colitis (UC) patients responsive to fecal microbiota transplantation (FMT) have reduced phages compared to non-responders, and mucosal IFN-γ positively correlates with bacteriophage levels. Bacteriophages from active UC patients induced more IFN-γ compared to healthy individuals. Collectively, these results indicate that bacteriophages can alter mucosal immunity to impact mammalian health.

Breastmilk contains a complex community of bacteria that may help seed the infant gut microbiota. The composition and determinants of milk microbiota are poorly understood. Among 393 mother-infant dyads from the CHILD cohort, we found that milk microbiota at 3-4 months postpartum was dominated by inversely correlated Proteobacteria and Firmicutes, and exhibited discrete compositional patterns. Milk microbiota composition and diversity were associated with maternal factors (BMI, parity, and mode of delivery), breastfeeding practices, and other milk components in a sex-specific manner. Causal modeling identified mode of breastfeeding as a key determinant of milk microbiota composition. Specifically, providing pumped breastmilk was consistently associated with multiple microbiota parameters including enrichment of potential pathogens and depletion of bifidobacteria. Further, these data support the retrograde inoculation hypothesis, whereby the infant oral cavity impacts the milk microbiota. Collectively, these results identify features and determinants of human milk microbiota composition, with potential implications for infant health and development.

We applied a yeast-two-hybrid (Y2H) analysis to screen for ubiquitin variant (UbV) inhibitors of a human deubiquitinase (DUB), ubiquitin-specific protease 2 (USP2). The Y2H screen used USP2 as the bait and a prey library consisting of UbVs randomized at four specific positions, which were known to interact with USP2 from phage display analysis. The screen yielded numerous UbVs that bound to USP2 both as a Y2H interaction in vivo and as purified proteins in vitro. The Y2H-derived UbVs inhibited the catalytic activity of USP2 in vitro with nanomolar-range potencies, and they bound and inhibited USP2 in human cells. Mutational and structural analysis showed that potent and selective inhibition could be achieved by just two substitutions in a UbV, which exhibited improved hydrophobic and hydrophilic contacts compared to the wild-type ubiquitin interaction with USP2. Our results establish Y2H as an effective platform for the development of UbV inhibitors of DUBs in vivo, providing an alternative strategy for the analysis of DUBs that are recalcitrant to phage display and other in vitro methods.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Given the comments of Dr Elisabeth Bik regarding this article “… the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated”, the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Relapse of minimal residual disease (MRD) is a major problem after conventional chemotherapy in patients with acute myeloid leukemia (AML). The bone marrow stroma can protect AML cells from insults of chemotherapy, partly contributing to AML relapse. Arsenic trioxide (ATO) is the main component of arsenical traditional Chinese medicines and has been widely used for the treatment of hematologic malignancies particularly acute promyelocytic leukemia over the past three decades. ATO acts through a direct arsenic binding to cysteine residues in zinc fingers located in promyelocytic leukemia protein (PML), thus killing the leukemia stem cells (LSCs). Our prior study has demonstrated that adhesion to stroma cells could render AML cells resistant to ATO but the detailed mechanism remains to be explored. Here, we report that the adhesion-induced resistance to ATO is related to the up-regulation of myeloid cell leukemia-1 (Mcl-1). Homoharringtonine (HHT) can potentiate the anti-leukemia effects of ATO on adhered AML cells by suppressing Mcl-1 through glycogen synthase kinase-3β (GSK3β). Furthermore, a potentiating effect of HHT on ATO was also observed in primary AML cells and AML xenografted tumors. Thus, these data indicate that HHT could enhance ATO anti-leukemia activity both in vitro and in vivo.

Vaccination programs are implemented in poultry farms to limit outbreaks and spread of infectious bronchitis virus (IBV), which is a substantial economic burden in the poultry industry. Immune correlates, used to predict vaccine efficacy, have proved difficult to find for IBV-vaccine-induced protection. To find correlates of IBV-vaccine-induced protection, hence, we employed a flow cytometric assay to quantify peripheral leucocyte subsets and expression of cell surface markers of six different non-vaccinated and vaccinated Major Histocompatibility Complex (MHC) haplotypes. Non-vaccinated and vaccinated MHC haplotypes presented differential leucocyte composition and IBV viral load. A strong effect of MHC-B, but not vaccination, on several leucocyte subsets resulted in positive correlations with IBV viral load based on MHC haplotype ranking. In addition, a strong effect of MHC-B and vaccination on monocyte MHC-II expression showed that animals with highest monocyte MHC-II expression had weakest vaccine-induced protection. In conclusion, we found several interesting MHC-B related immune correlates of protection and that flow cytometric analysis can be employed to study correlates of IBV-vaccine-induced protection.

pH-responsive peptides are promising therapeutic molecules that can specifically target the plasma membrane in the acidified extracellular medium that bathes cells in tumors. We designed the acidity-triggered rational membrane (ATRAM) peptide to have a pH-responsive membrane interaction. At physiological pH, ATRAM binds to the membrane surface in a largely unstructured conformation, while in acidic conditions it inserts into lipid bilayers forming a transmembrane helix. However, the molecular mechanism ATRAM uses to target and insert into tumor cells remains poorly understood. Here, we determined that ATRAM inserts into cancer cells with a preferential membrane orientation, where the C-terminus of the peptide traverses the plasma membrane and explores the cytoplasm. Using biophysical techniques, we determined that the membrane interaction of ATRAM is contingent on the concentration of the peptide. Kinetic studies showed that membrane insertion occurs in at least three steps, where only the first step was affected by the membrane density of ATRAM. These observations, combined with membrane binding and leakage data, indicate that the interaction of ATRAM with lipid membranes is dependent on its oligomerization state. SPECT/CT imaging in mice revealed that ATRAM accumulates in the blood pool, where it has a prolonged circulation time (> 4 h). Since fast peptide clearance and degradation in circulation are major problems for clinical development, we studied the mechanism ATRAM uses to remain in the blood stream. Using binding and transfer assays, we determined that ATRAM binds reversibly to human serum albumin. We propose that ATRAM uses albumin as a carrier in the blood stream to evade clearance and proteolysis before interacting with the plasma membrane of cancer cells. We also show that ATRAM is able to be deliver liposomes to cells in a pH dependent way. Our data highlight the potential of ATRAM as a specific therapeutic agent for diseases that lead to acidic tissues, including cancer.

N-myc downstream regulated gene 1 (NDRG1) is an intriguing metastasis suppressor protein, which plays an important role in suppressing multiple oncogenic signaling pathways. Interestingly, multiple isoforms of NDRG1 have been identified, although the molecular mechanisms involved in their generation remains elusive. Herein, we demonstrate the role of two mechanisms involving autophagic and proteasomal machinery as part of an intricate system to generate different NDRG1 isoforms. Examining multiple pancreatic cancer cell-types using immunoblotting demonstrated three major isoforms of NDRG1 at approximately 41-, 46- and 47-kDa. The top NDRG1 band at 47-kDa was shown to be processed by the proteasome, followed by autophagic metabolism of the middle NDRG1 band at 46-kDa. The role of the proteasomal and autophagic pathways in NDRG1 processing was further confirmed by co-localization analysis of confocal images using PSMD9 and LC3 as classical markers of these respective pathways. All NDRG1 isoforms were demonstrated to be, at least in part, phosphorylated forms of the protein. Inhibition of two well-characterized upstream kinases of NDRG1, namely GSK3β and SGK1, resulted in decreased levels of the top NDRG1 band. Studies demonstrated that inhibition of GSK3β decreased levels of the top 47-kDa NDRG1 band, independent of its kinase activity, and this effect was not mediated via the proteasomal pathway. In contrast, the decrease in the top NDRG1 band at 47-kDa after SGK1 inhibition, was due to suppression of its kinase activity. Overall, these studies elucidated the complex and intricate regulatory pathways involving both proteasomal and autophagic processing of the metastasis suppressor protein, NDRG1.

Increasing evidence indicates that, first, the sympathetic nervous system interacts extensively with both vasculature and skeletal muscle fibers near neuromuscular junctions (NMJs) and, second, its neurotransmitter, noradrenaline, influences myofiber molecular composition and function and motor innervation. Since sympathomimetic agents have been reported to improve NMJ transmission, we examined whether two in clinical use, salbutamol and clenbuterol, affect the motor axon terminal via extracellular Ca2+ and molecular targets, such as TRPV1 and P/Q- and N-type voltage-activated Ca2+ channels. Electrophysiological recordings in ex-vivo preparations of peroneal nerves and lumbricalis muscles from young adult mice focused on spontaneous miniature end-plate potentials and singly and repetitively evoked end-plate potentials. Adding one dose of salbutamol or clenbuterol to the nerve/muscle preparation or repeatedly administering salbutamol to a mouse for 4 weeks increased spontaneous and evoked synaptic vesicle release but induced a steep decline in EPP amplitude in response to repetitive nerve stimulation. These effects were mediated primarily by ω-agatoxin IVA-sensitive P/Q-type and secondarily by ω-conotoxin GVIA-sensitive N-type Ca2+ channels. Presynaptic arvanil-sensitive TRPV1 channels seem to regulate Ca2+ at the motor neuron terminal at rest, while putative presynaptic β-adrenergic receptors may mediate sympathomimetic and catecholamine effects on presynaptic Ca2+ channels during NMJ activation.

The abnormal increase in vascular smooth muscle cell (VSMC) proliferation and migration are critical events in the pathogenesis of cardiovascular diseases (CVDs) including restenosis and atherosclerosis. The dried roots of Scutellaria baicalensis Georgi (common name: Huangqin in China) have been confirmed to possess beneficial effects on CVD by clinical and modern pharmacological studies. Flavonoids in Huangqin exert anti-proliferative and anti-migratory effects. Similar to Huangqin, Scutellaria strigillosa Hemsley (SSH) has been used to clear heat and damp and is especially rich in flavonoids including wogonin, wogonoside, baicalein, and baicalin. However, there have been few of reports about pharmacological activities of SSH.

Notch signalling regulates neural stem cell (NSC) proliferation, differentiation and survival for the correct development and functioning of the central nervous system. Overactive Notch2 signalling has been associated with poor prognosis of aggressive brain tumours, such as glioblastoma multiforme (GBM). We recently reported that constitutive expression of the Notch2 intracellular domain (N2ICD) enhances proliferation and gliogenesis in NSCs. Here, we investigated the mechanism by which Notch2 promotes resistance to apoptosis of NSCs to cytotoxic insults. We performed ex vivo studies using NSC cultures from transgenic mice constitutively expressing N2ICD. These NSCs expressed increased levels of pro-survival factors and lack an apoptotic response to the topoisomerase inhibitor etoposide, not showing neither mitochondrial damage nor caspase activation. Interestingly, Notch2 signalling also regulated chemoresistance of human GBM cells to etoposide. We also identified a signalling crosstalk with FGF signalling pathway involved in this resistance to apoptosis of NSCs. Aberrant Notch2 expression enhances fibroblast growth factor receptor-1 (FGFR1) activity to specifically target the AKT-GSK3 signalling pathway to block apoptosis. These results have implications for understanding molecular changes involved in both tumorigenesis and therapy resistance.

Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) is a promising treatment strategy for Duchenne muscular dystrophy (DMD). The most significant limitation of these clinically used compounds is their lack of delivery systems that target muscles; thus, cell-penetrating peptides are being developed to enhance uptake into muscles. Recently, we reported that uptake of peptide-conjugated PMOs into myofibers was mediated by scavenger receptor class A (SR-A), which binds negatively charged ligands. However, the mechanism by which the naked PMOs are taken up into fibers is poorly understood. In this study, we found that PMO uptake and exon-skipping efficiency were promoted in dystrophin-deficient myotubes via endocytosis through a caveolin-dependent pathway. Interestingly, SR-A1 was upregulated and localized in juxtaposition with caveolin-3 in these myotubes and promoted PMO-induced exon skipping. SR-A1 was also upregulated in the skeletal muscle of mdx52 mice and mediated PMO uptake. In addition, PMOs with neutral backbones had negative zeta potentials owing to their nucleobase compositions and interacted with SR-A1. In conclusion, PMOs with negative zeta potential were taken up into dystrophin-deficient skeletal muscle by upregulated SR-A1. Therefore, the development of a drug delivery system targeting SR-A1 could lead to highly efficient exon-skipping therapies for DMD.

We report on the variable venom composition of a population of the Caucasus viper (Vipera kaznakovi) in Northeastern Turkey. We applied a combination of venom gland transcriptomics, de-complexing bottom-up and top-down venomics. In contrast to sole bottom-up venomics approaches and gel or chromatography based venom comparison, our combined approach enables a faster and more detailed comparison of venom proteomes from multiple individuals. In total, we identified peptides and proteins from 15 toxin families, including snake venom metalloproteinases (svMP; 37.8%), phospholipases A2 (PLA2; 19.0%), snake venom serine proteinases (svSP; 11.5%), C-type lectins (CTL; 6.9%) and cysteine-rich secretory proteins (CRISP; 5.0%), in addition to several low abundant toxin families. Furthermore, we identified intraspecies variations of the venom composition of V. kaznakovi, and find these were mainly driven by the age of the animals, with lower svSP abundance detected in juveniles. On the proteoform level, several small molecular weight toxins between 5 and 8 kDa in size, as well as PLA2s, drove the differences observed between juvenile and adult individuals. This study provides novel insights into the venom variability of V. kaznakovi and highlights the utility of intact mass profiling for fast and detailed comparison of snake venom. BIOLOGICAL SIGNIFICANCE: Population level and ontogenetic venom variation (e.g. diet, habitat, sex or age) can result in a loss of antivenom efficacy against snakebites from wide ranging snake populations. The current state of the art for the analysis of snake venoms are de-complexing bottom-up proteomics approaches. While useful, these have the significant drawback of being time-consuming and following costly protocols, and consequently are often applied to pooled venom samples. To overcome these shortcomings and to enable rapid and detailed profiling of large numbers of individual venom samples, we integrated an intact protein analysis workflow into a transcriptomics-guided bottom-up approach. The application of this workflow to snake individuals of a local population of V. kaznakovi revealed intraspecies variations in venom composition, which are primarily explained by the age of the animals, and highlighted svSP abundance to be one of the molecular drivers for the compositional differences observed.

Legionellosis is a severe pneumonic infection caused by inhaling bacteria of the genus Legionella. Most cases reported in the USA and Europe are associated with the species Legionella pneumophila. This Gram-negative bacterium can survive within a wide spectrum of temperatures, and be transmitted via aerosols from multiple aquatic sources: fountains, thermal spas and other water systems. Although the PCR is one of the most popular methods to verify its presence in environmental or clinical samples, the direct application of this technique to ambient air samples is unusual because of the scarce material in the specimens. Here, we have developed a two-PCR assay, carried out over the V3 and V5 hypervariable regions of the 16S rRNA gene, to detect specifically the pathogenic bacteria Legionella pneumophila in outdoor air samples with low concentration of DNA. The application of this protocol does not require culture and retrieves quick results to activate the corresponding public alerts to prevent legionellosis outbreaks.

There exist two major base excision DNA repair (BER) pathways, namely single-nucleotide or "short-patch" (SP-BER), and "long-patch" BER (LP-BER). Both pathways appear to be involved in the repair of small base lesions such as uracil, abasic sites and oxidized bases. In addition to DNA polymerase β (Polβ) as the main BER enzyme for repair synthesis, there is evidence for a minor role for DNA polymerase lambda (Polλ) in BER. In this study we explore the potential contribution of Polλ to both SP- and LP-BER in cell-free extracts. We measured BER activity in extracts of mouse embryonic fibroblasts using substrates with either a single uracil or the chemically stable abasic site analog tetrahydrofuran residue. The addition of purified Polλ complemented the pronounced BER deficiency of POLB-null cell extracts as efficiently as did Polβ itself. We have developed a new approach for determining the relative contributions of SP- and LP-BER pathways, exploiting mass-labeled nucleotides to distinguish single- and multinucleotide repair patches. Using this method, we found that uracil repair in wild-type and in Polβ-deficient cell extracts supplemented with Polλ was ∼80% SP-BER. The results show that recombinant Polλ can contribute to both SP- and LP-BER. However, endogenous Polλ, which is present at a level ˜50% that of Polβ in mouse embryonic fibroblasts, appears to make little contribution to BER in extracts. Thus Polλ in cells appears to be under some constraint, perhaps sequestered in a complex with other proteins, or post-translationally modified in a way that limits its ability to participate effectively in BER.

Hastelloy X (HX) is a Ni-based superalloy which is employed to produce gas turbine and gas-cooled reactor sectors due to its outstanding oxidation resistance and high tensile strength at high temperatures. This alloy can be processed by laser powder bed fusion (LPBF) fabricating complex geometries in a single step. However, post-processing thermal treatments must be applied to generate a suitable microstructure for high-temperature applications. The investigation reports the microstructure evolution of LPBF HX samples under specific post-processing treatments. A hot isostatic pressing (HIP) treatment can close the internal cracks and reduce the residual porosity (less than 0.1%). Moreover, the HIP-triggered recrystallization generated equiaxed grains, while the slow cooling rate generated a film of intergranular carbides (Mo-rich M₆C and Cr-rich M23C₆) and intragranular carbides (Mo-rich M₆C carbides). Therefore, a solution annealing was performed to dissolve the film of carbides which may reduce the ductility. The post solution annealed material consisted of equiaxed grains with ASTM grain size number mainly 4.5-5.5 and inter/intragranular Mo-rich M₆C carbides. The microstructure is highly comparable with solution annealed wrought HX alloy. Finally, after simulating short thermal exposure at 745 °C for 6 h, a significant formation of Cr-rich M23C₆ carbides was observed strengthening the LPBF HX alloy.

In oral language, syntactic structure is cued in part by phrasal metrical hierarchies of acoustic stress patterns. For example, many children's texts use prosodic phrasing comprising tightly integrated hierarchies of metre and syntax to highlight the phonological and syntactic structure of language. Children with developmental language disorders (DLDs) are relatively insensitive to acoustic stress. Here, we disrupted the coincidence of metrical and syntactic boundaries as cued by stress patterns in children's texts so that metrical and/or syntactic phrasing conflicted. We tested three groups of children: children with DLD, age-matched typically developing controls (AMC) and younger language-matched controls (YLC). Children with DLDs and younger, language-matched controls were poor at spotting both metrical and syntactic disruptions. The data are interpreted within a prosodic phrasing hypothesis of DLD based on impaired acoustic processing of speech rhythm.

Marek's disease virus (MDV) is the causative agent of Marek's disease (MD), a complex pathology of chickens characterized by paralysis, immunosuppression, and T-cell lymphomagenesis. MD is controlled in poultry production via vaccines administered in ovo or at hatch, and these confer protection against lymphoma formation, but not superinfection by MDV field strains. Despite vaccine-induced humoral and cell-mediated immune responses, mechanisms eliciting systemic protection remain unclear. Here we report the contents of serum exosomes to assess their possible roles as indicators of systemic immunity, and alternatively, tumor formation. We examined the RNA and protein content of serum exosomes from CVI988 (Rispens)-vaccinated and protected chickens (VEX), and unvaccinated tumor-bearing chickens (TEX), via deep-sequencing and mass spectrometry, respectively. Bioinformatic analyses of microRNAs (miRNAs) and predicted miRNA targets indicated a greater abundance of tumor suppressor miRNAs in VEX compared to TEX. Conversely, oncomiRs originating from cellular (miRs 106a-363) and MDV miRNA clusters were more abundant in TEX compared to VEX. Most notably, mRNAs mapping to the entire MDV genome were identified in VEX, while mRNAs mapping to the repeats flanking the unique long (IRL/TRL) were identified in TEX. These data suggest that long-term systemic vaccine-induced immune responses may be mediated at the level of VEX which transfer viral mRNAs to antigen presenting cells systemically. Proteomic analyses of these exosomes suggested potential biomarkers for VEX and TEX. These data provide important putative insight into MDV-mediated immune suppression and vaccine responses, as well as potential serum biomarkers for MD protection and susceptibility.