Clinical Validation of Integrated Nucleic Acid and Protein Detection on an Electrochemical Biosensor Array for Urinary Tract Infection Diagnosis

BACKGROUND: Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management. METHODOLOGY/PRINCIPAL FINDINGS: The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both). CONCLUSION/SIGNIFICANCE: We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics

Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype

BACKGROUND: Tumours contain hypoxic regions that select for an aggressive cell phenotype; tumour hypoxia induces metastasis-associated genes. Treatment refractory patients with metastatic cancer show increased numbers of circulating tumour cells (CTCs), which are also associated with disease progression. The aim of this study was to examine the as yet unknown relationship between hypoxia and CTCs. METHODS: We generated human MDA-MB-231 orthotopic xenografts and, using a new technology, isolated viable human CTCs from murine blood. The CTCs and parental MDA-MB-231 cells were incubated at 21 and 0.2% (hypoxia) oxygen, respectively. Colony formation was assayed and levels of hypoxia- and anoxia-inducible factors were measured. Xenografts generated from CTCs and parental cells were compared. RESULTS: MDA-MB-231 xenografts used to generate CTCs were hypoxic, expressing hypoxia factors: hypoxia-inducible factor1 alpha (HIF1alpha) and glucose transporter protein type 1 (GLUT1), and anoxia-induced factors: activating transcription factor 3 and 4 (ATF3 and ATF4). Parental MDA-MB-231 cells induced ATF3 in hypoxia, whereas CTCs expressed it constitutively. Asparagine synthetase (ASNS) expression was also higher in CTCs. Hypoxia induced ATF4 and the HIF1alpha target gene apelin in CTCs, but not in parental cells. Hypoxia induced lower levels of carbonic anhydrase IX (CAIX), GLUT1 and BCL2/adenovirus E1B 19-KD protein-interacting protein 3 (BNIP3) proteins in CTCs than in parental cells, supporting an altered hypoxia response. In chronic hypoxia, CTCs demonstrated greater colony formation than parental cells. Xenografts generated from CTCs were larger and heavier, and metastasised faster than MDA-MB-231 xenografts. CONCLUSION: CTCs show an altered hypoxia response and an enhanced aggressive phenotype in vitro and in vivo

Novel and Recurrent Mutations of WISP3 in Two Chinese Families with Progressive Pseudorheumatoid Dysplasia

BACKGROUND: The WNT1-inducible signaling pathway protein 3 (WISP3), which belongs to the CCN (cysteine-rich protein 61, connective tissue growth factor, nephroblastoma overexpressed) family, is a secreted cysteine-rich matricellular protein that is involved in chondrogenesis, osteogenesis and tumorigenesis. WISP3 gene mutations are associated with progressive pseudorheumatoid dysplasia (PPD, OMIM208230), an autosomal recessive genetic disease that is characterized by the swelling of multiple joints and disproportionate dwarfism. METHODOLOGY/PRINCIPAL FINDINGS: Four PPD patients from two unrelated Chinese families were recruited for this study. The clinical diagnosis was confirmed by medical history, physical examinations, laboratory results and radiological abnormalities. WISP3 mutations were detected by direct DNA sequence analysis. In total, four different mutations were identified, which consisted of two missense mutations, one deletion and one insertion that spanned exons 3, 5 and 6 of the WISP3 gene. One of the missense mutations (c.342T>G/p.C114W) and a seven-base pair frameshift deletion (c.716_722del/p.E239fs*16) were novel. The other missense mutation (c.1000T>C/p. S334P) and the insertion mutation (c.866_867insA/p.Q289fs*31) had previously been identified in Chinese patients. All four cases had a compound heterozygous status, and their parents were heterozygous carriers of these mutations. CONCLUSIONS/SIGNIFICANCE: The results of our study expand the spectrum of WISP3 mutations that are associated with PPD and further elucidate the function of WISP3

Generation of integration-free neural progenitor cells from cells in human urine

Human neural stem cells hold great promise for research and therapy in neural disease. We describe the generation of integration-free and expandable human neural progenitor cells (NPCs). We combined an episomal system to deliver reprogramming factors with a chemically defined culture medium to reprogram epithelial-like cells from human urine into NPCs (hUiNPCs). These transgene-free hUiNPCs can self-renew and can differentiate into multiple functional neuronal subtypes and glial cells in vitro. Although functional in vivo analysis is still needed, we report that the cells survive and differentiate upon transplant into newborn rat brain.postprin

Optimisation of UAVs‐SfM data collection in aeolian landform morphodynamics : a case study from the Gonghe Basin, China

UAVs‐SfM (Unmanned Aerial Vehicles‐ Structure from Motion) systems can generate high‐resolution 3D topographic models of aeolian landforms. To explore the optimisation of UAVs‐SfM for use in aeolian landform morphodynamics, this study tested flight parameters for two contrasting aeolian landform areas (free dune and blowout) to assess the 3D reconstruction accuracy of the UAVs survey compared with field point measurements using differential RTK‐GPS (Real‐time Kinematic‐Global Positioning System). The results reveal the optimum UAVs‐SfM flight set‐up at the free‐dune site was: flying height = 74 m, camera tilt angle = ‐90°, photo overlap ratio = 85%/70% (heading/sideways). The horizontal/vertical location error was around 0.028~0.055 m and 0.053‐0.069 m respectively, and a point cloud density of 463/m3 was found to generate a clear texture using these flying parameters. For the <20m deep blowout the optimum set‐up with highest accuracy and the lowest cliff texture distortion was: flying height = 74 m combined camera tilt angle = ‐90° and ‐60°, photo overlap ratio = 85%/70% (heading/sideways), and an evenly distributed GCPs (Ground Control Points) density of 42/km2 using these flying parameters. When the depth of the blowouts exceeded 40 m, the optimum flight/survey parameters changed slightly to account for more challenging cliff texture generation: flying height = 80 m (with ‐90° and ‐60°combined camera tilt angle), GCPs density = 63/km2 to generate horizontal and vertical location error of 0.024 m and 0.050 m respectively, and point cloud density of 2597.11/m3. The main external factors that affect the successful 3D reconstruction of aeolian landforms using UAVs‐SfM are the weather conditions, manipulation errors, and instrument system errors. The UAVs‐SfM topographic monitoring results demonstrate that UAVs provide a viable and robust means for aeolian landform morphodynamics monitoring. Importantly, the rapid and high precision 3D reconstruction processes were significantly advanced using the optimal flight parameters reported here

Screening Level of PAHs in Sediment Core from Lake Hongfeng, Southwest China

Using data from a 25-year retrospective of polycyclic aromatic hydrocarbons (PAHs) in sediment core from Lake Hongfeng, Southwest China, their possible sources and potential toxicologic significance were investigated. The total PAH concentrations (16 priority PAHs as proposed by the United States Environmental Protection Agency) in sediments ranged from 2936.1 to 5282.3 ng/g and gradually increased from the analyzed deeper sediments to surface sediments. PAHs were dominated by low molecular-weight components, especially phenanthrene (PHEN) and fluorene (FLU). However, a significantly increased number of high molecular-weight (HMW) PAHs was found in upper segments. The temporal trends of individual PAH species suggest that there may have been a change in energy use from low- to high-temperature combustion, especially after approximately 2001. PAH input to Lake Hongfeng originated mainly from domestic coal combustion and biomass burning, whereas fuel combustion characteristics have also been found in recent years. Sediment-quality assessment implied that potential adverse biologic impact could be a probability for most low-ring PAHs (including naphthalene, acenaphthylene, acenaphthylene, FLU, PHEN, and anthracene). Nevertheless, more concern should be paid to HMW PAHs in the future due to their rapidly increasing trends in upper sediments. Because only one core was analyzed in this study, more work is needed to confirm the sources and toxicity of PAHs in Lake Hongfeng

The effect of agitation speed, enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer’s spent grain

Brewer’s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 ºC to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2 3 full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer’s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo), Brazil. Novozymes ( FAPESP )Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Analysis of the effects of exposure to acute hypoxia on oxidative lesions and tumour progression in a transgenic mouse breast cancer model

<p>Abstract</p> <p>Background</p> <p>Tumour hypoxia is known to be a poor prognostic indicator, predictive of increased risk of metastatic disease and reduced survival. Genomic instability has been proposed as one of the potential mechanisms for hypoxic tumour progression. Both of these features are commonly found in many cancer types, but their relationship and association with tumour progression has not been examined in the same model.</p> <p>Methods</p> <p>To address this issue, we determined the effects of 6 week <it>in vivo </it>acute hypoxic exposure on the levels of mutagenic lipid peroxidation product, malondialdehyde, and 8-oxo-7,8-dihydro-2'-deoxyguanosine DNA (8-oxo-dG) lesions in the transgenic polyomavirus middle T (PyMT) breast cancer mouse model.</p> <p>Results</p> <p>We observed significantly increased plasma lipid peroxidation and 8-oxo-dG lesion levels in the hypoxia-exposed mice. Consumption of malondialdehyde also induced a significant increase in the PyMT tumour DNA lesion levels, however, these increases did not translate into enhanced tumour progression. We further showed that the <it>in vivo </it>exposure to acute hypoxia induced accumulation of F4/80 positive tumour-associated macrophages (TAMs), demonstrating a relationship between hypoxia and macrophages in an experimental model.</p> <p>Conclusion</p> <p>These data suggest that although exposure to acute hypoxia causes an increase in 8-oxo-dG lesions and TAMs in the PyMT tumours, these increases do not translate into significant changes in tumour progression at the primary or metastatic levels in this strong viral oncogene-driven breast cancer model.</p

Involvement of Sulfur in the Biosynthesis of Essential Metabolites in Pathogenic Fungi of Animals, Particularly Aspergillus spp. : Molecular and Therapeutic Implications

Fungal sulfur uptake is required for incorporation into the sidechains of the amino acids cysteine and methionine, and is also essential for the biosynthesis of the antioxidant glutathione (GSH), S-adenosylmethionine (SAM), the key source of methyl groups in cellular transmethylation reactions, and S-adenosylhomocysteine (SAH). Biosynthesis of redox-active gliotoxin in the opportunistic fungal pathogen Aspergillus fumigatus has been elucidated over the past 10 years. Some fungi which produce gliotoxin-like molecular species have undergone unexpected molecular rewiring to accommodate this high-risk biosynthetic process. Specific disruption of gliotoxin biosynthesis, via deletion of gliK, which encodes a γ-glutamyl cyclotransferase, leads to elevated intracellular antioxidant, ergothioneine (EGT), levels, and confirms crosstalk between the biosynthesis of both sulfur-containing moieties. Gliotoxin is ultimately formed by gliotoxin oxidoreductase GliT-mediated oxidation of dithiol gliotoxin (DTG). In fact, DTG is a substrate for both GliT and a bis-thiomethyltransferase, GtmA. GtmA converts DTG to bisdethiobis(methylthio)gliotoxin (BmGT), using 2 mol SAM and resultant SAH must be re-converted to SAM via the action of the Methyl/Met cycle. In the absence of GliT, DTG fluxes via GtmA to BmGT, which results in both SAM depletion and SAH overproduction. Thus, the negative regulation of gliotoxin biosynthesis via GtmA must be counter-balanced by GliT activity to avoid Methyl/Met cycle dysregulation, SAM depletion and trans consequences on global cellular biochemistry in A. fumigatus. DTG also possesses potent Zn2+ chelation properties which positions this sulfur-containing metabolite as a putative component of the Zn2+ homeostasis system within fungi. EGT plays an essential role in high-level redox homeostasis and its presence requires significant consideration in future oxidative stress studies in pathogenic filamentous fungi. In certain filamentous fungi, sulfur is additionally indirectly required for the formation of EGT and the disulfide-bridge containing non-ribosomal peptide, gliotoxin, and related epipolythiodioxopiperazines. Ultimately, interference with emerging sulfur metabolite functionality may represent a new strategy for antifungal drug development

Persistent activation of interlinked type 2 airway epithelial gene networks in sputum-derived cells from aeroallergen-sensitized symptomatic asthmatics

© 2018 The Author(s). Atopic asthma is a persistent disease characterized by intermittent wheeze and progressive loss of lung function. The disease is thought to be driven primarily by chronic aeroallergen-induced type 2-associated inflammation. However, the vast majority of atopics do not develop asthma despite ongoing aeroallergen exposure, suggesting additional mechanisms operate in conjunction with type 2 immunity to drive asthma pathogenesis. We employed RNA-Seq profiling of sputum-derived cells to identify gene networks operative at baseline in house dust mite-sensitized (HDM S ) subjects with/without wheezing history that are characteristic of the ongoing asthmatic state. The expression of type 2 effectors (IL-5, IL-13) was equivalent in both cohorts of subjects. However, in HDM S -wheezers they were associated with upregulation of two coexpression modules comprising multiple type 2- and epithelial-associated genes. The first module was interlinked by the hubs EGFR, ERBB2, CDH1 and IL-13. The second module was associated with CDHR3 and mucociliary clearance genes. Our findings provide new insight into the molecular mechanisms operative at baseline in the airway mucosa in atopic asthmatics undergoing natural aeroallergen exposure, and suggest that susceptibility to asthma amongst these subjects involves complex interactions between type 2- and epithelial-associated gene networks, which are not operative in equivalently sensitized/exposed atopic non-asthmatics