Fabrication and testing of microfluidic devices for blood cell separation

This paper was presented at the 2nd Micro and Nano Flows Conference (MNF2009), which was held at Brunel University, West London, UK. The conference was organised by Brunel University and supported by the Institution of Mechanical Engineers, IPEM, the Italian Union of Thermofluid dynamics, the Process Intensification Network, HEXAG - the Heat Exchange Action Group and the Institute of Mathematics and its Applications.Blood separation is a strategic preliminary step in preparation to on-chip biological analysis. Two microfluidic devices for on-chip blood separation are presented. Both devices will be integrated to form the separation module of a Lab on Chip for non-invasive prenatal diagnosis. In the first device, a blood plasma separator, the separation of blood cells from plasma is made possible in microchannels by bio-physical effects such as an axial migration effect and Zweifach-Fung bifurcation law. Behaviour of mussel and human blood suspensions were studied alongside the effect of different geometries. The second device aims to separate fetal nucleated red blood cells based on their magnetic susceptibility. Biocompatible materials are used in the manufacturing of both devices.The authors acknowledge the financial support of the Engineering and Physical Science Research Council (EPSRC) through the funding of the Grand Challenge Project ‘3DMintegration’, reference EP/C534212/1. This work has also been supported by the EPSRC through a Doctoral Training Account (DTA) and has been performed at the Microsystems Engineering Centre (MISEC), Heriot-Watt University, Edinburgh. We thank Tim Ryan and Phil Summersgill, Epigem Ltd. for the fabrication of the blood plasma chips. The fabrication work was carried out in the Fluence Microfluidics Application Centre supported by the DTI and the OneNE Regional Development Agency as part of the UK's MNT Network

Identification of novel macrolides with antibacterial, anti-inflammatory and type I and III IFN-augmenting activity in airway epithelium

BACKGROUND: Exacerbations of asthma and COPD are triggered by rhinoviruses. Uncontrolled inflammatory pathways, pathogenic bacterial burden and impaired antiviral immunity are thought to be important factors in disease severity and duration. Macrolides including azithromycin are often used to treat the above diseases, but exhibit variable levels of efficacy. Inhaled corticosteroids are also readily used in treatment, but may lack specificity. Ideally, new treatment alternatives should suppress unwanted inflammation, but spare beneficial antiviral immunity. METHODS: In the present study, we screened 225 novel macrolides and tested them for enhanced antiviral activity against rhinovirus, as well as anti-inflammatory activity and activity against Gram-positive and Gram-negative bacteria. Primary bronchial epithelial cells were grown from 10 asthmatic individuals and the effects of macrolides on rhinovirus replication were also examined. Another 30 structurally similar macrolides were also examined. RESULTS: The oleandomycin derivative Mac5, compared with azithromycin, showed superior induction (up to 5-fold, EC50 = 5-11 μM) of rhinovirus-induced type I IFNβ, type III IFNλ1 and type III IFNλ2/3 mRNA and the IFN-stimulated genes viperin and MxA, yet had no effect on IL-6 and IL-8 mRNA. Mac5 also suppressed rhinovirus replication at 48 h, proving antiviral activity. Mac5 showed antibacterial activity against Gram-positive Streptococcus pneumoniae; however, it did not have any antibacterial properties compared with azithromycin when used against Gram-negative Escherichia coli (as a model organism) and also the respiratory pathogens Pseudomonas aeruginosa and non-typeable Haemophilus influenzae. Further non-toxic Mac5 derivatives were identified with various anti-inflammatory, antiviral and antibacterial activities. CONCLUSIONS: The data support the idea that macrolides have antiviral properties through a mechanism that is yet to be ascertained. We also provide evidence that macrolides can be developed with anti-inflammatory, antibacterial and antiviral activity and show surprising versatility depending on the clinical need

Cell salvage and donor blood transfusion during cesarean section: A pragmatic, multicentre randomised controlled trial (SALVO)

BACKGROUND: Excessive haemorrhage at cesarean section requires donor (allogeneic) blood transfusion. Cell salvage may reduce this requirement. METHODS AND FINDINGS: We conducted a pragmatic randomised controlled trial (at 26 obstetric units; participants recruited from 4 June 2013 to 17 April 2016) of routine cell salvage use (intervention) versus current standard of care without routine salvage use (control) in cesarean section among women at risk of haemorrhage. Randomisation was stratified, using random permuted blocks of variable sizes. In an intention-to-treat analysis, we used multivariable models, adjusting for stratification variables and prognostic factors identified a priori, to compare rates of donor blood transfusion (primary outcome) and fetomaternal haemorrhage ≥2 ml in RhD-negative women with RhD-positive babies (a secondary outcome) between groups. Among 3,028 women randomised (2,990 analysed), 95.6% of 1,498 assigned to intervention had cell salvage deployed (50.8% had salvaged blood returned; mean 259.9 ml) versus 3.9% of 1,492 assigned to control. Donor blood transfusion rate was 3.5% in the control group versus 2.5% in the intervention group (adjusted odds ratio [OR] 0.65, 95% confidence interval [CI] 0.42 to 1.01, p = 0.056; adjusted risk difference -1.03, 95% CI -2.13 to 0.06). In a planned subgroup analysis, the transfusion rate was 4.6% in women assigned to control versus 3.0% in the intervention group among emergency cesareans (adjusted OR 0.58, 95% CI 0.34 to 0.99), whereas it was 2.2% versus 1.8% among elective cesareans (adjusted OR 0.83, 95% CI 0.38 to 1.83) (interaction p = 0.46). No case of amniotic fluid embolism was observed. The rate of fetomaternal haemorrhage was higher with the intervention (10.5% in the control group versus 25.6% in the intervention group, adjusted OR 5.63, 95% CI 1.43 to 22.14, p = 0.013). We are unable to comment on long-term antibody sensitisation effects. CONCLUSIONS: The overall reduction observed in donor blood transfusion associated with the routine use of cell salvage during cesarean section was not statistically significant. TRIAL REGISTRATION: This trial was prospectively registered on ISRCTN as trial number 66118656 and can be viewed on http://www.isrctn.com/ISRCTN66118656

The Two-Domain LysX Protein of Mycobacterium tuberculosis Is Required for Production of Lysinylated Phosphatidylglycerol and Resistance to Cationic Antimicrobial Peptides

The well-recognized phospholipids (PLs) of Mycobacterium tuberculosis (Mtb) include several acidic species such as phosphatidylglycerol (PG), cardiolipin, phosphatidylinositol and its mannoside derivatives, in addition to a single basic species, phosphatidylethanolamine. Here we demonstrate that an additional basic PL, lysinylated PG (L-PG), is a component of the PLs of Mtb H37Rv and that the lysX gene encoding the two-domain lysyl-transferase (mprF)-lysyl-tRNA synthetase (lysU) protein is responsible for L-PG production. The Mtb lysX mutant is sensitive to cationic antibiotics and peptides, shows increased association with lysosome-associated membrane protein–positive vesicles, and it exhibits altered membrane potential compared to wild type. A lysX complementing strain expressing the intact lysX gene, but not one expressing mprF alone, restored the production of L-PG and rescued the lysX mutant phenotypes, indicating that the expression of both proteins is required for LysX function. The lysX mutant also showed defective growth in mouse and guinea pig lungs and showed reduced pathology relative to wild type, indicating that LysX activity is required for full virulence. Together, our results suggest that LysX-mediated production of L-PG is necessary for the maintenance of optimal membrane integrity and for survival of the pathogen upon infection

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency–Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research

Monitoring antimalarial drug resistance in India via sentinel sites: outcomes and risk factors for treatment failure, 2009–2010

OBJECTIVE: To describe India’s National Antimalarial Drug Resistance Monitoring System, measure the efficacy of first-line malaria treatments, and determine risk factors for treatment failure. METHODS: In 2009–2010, prospective studies with 28 days of follow-up were conducted at 25 sentinel sites. Patients infected with Plasmodium falciparum were given artesunate plus sulfadoxine-pyrimethamine (AS+SP); those infected with P. vivax were given chloroquine. Polymerase chain reaction was used to distinguish post-treatment reinfection from treatment failure. Isolates of P. falciparum were checked for dhfr and dhps mutations. FINDINGS: Overall, 1664 patients were enrolled. Kaplan–Meier survival analysis showed an efficacy of 98.8% for AS+SP. Most patients with P. falciparum parasitaemia cleared their parasitaemias within 24 hours of treatment initiation, but six, including four with treatment failure, remained parasitaemic after 72 hours. Double mutants in dhfr were found in 68.4% of the genotyped isolates. Triple or quadruple mutants in dhfr and mutations in dhps were rare. A daily dose of artesunate of < 3 mg per kg of body weight, age of less than 5 years, and fever at enrolment were associated with an increased risk of treatment failure. Chloroquine remained 100% efficacious and generally cleared P. vivax parasitaemias within 48 hours. Vomiting (seen in 47 patients) was the most common adverse event. CONCLUSION: India’s National Antimalarial Drug Resistance Monitoring System provides wide coverage. The first-line antimalarials used in the country remain safe and efficacious. The treatment of malaria in young children and the relative benefits of age- and weight-based dosing need further exploration

Epitope-specific airway-resident CD4+ T-cell dynamics during experimental human RSV infection

Background: Respiratory syncytial virus (RSV) is an important cause of acute pulmonary disease and one of the last remaining major infections of childhood for which there is no vaccine. CD4+ T-cells play a key role in antiviral immunity, but they have been little studied in the human lung. Methods: Healthy adult volunteers were inoculated intranasally with RSV A Memphis 37. CD4+ T-cells in blood and lower airway were analysed by flow cytometry and immunohistochemistry. Bronchial soluble mediators were measured using quantitative PCR and MesoScale Discovery. Epitope mapping was performed by IFN-γ ELISpot screening, confirmed by in vitro MHC binding. Results: Activated CD4+ T-cell frequencies in bronchoalveolar lavage correlated strongly with local CXCL10 levels. Thirty-nine epitopes were identified, predominantly towards the 3’ end of the viral genome. Five novel MHC-II tetramers were made using an immunodominant F-EFY epitope restricted to HLA-DR4, -DR9 and -DR11 (combined allelic frequency: 15% in Europeans) and G- DDF restricted to HLA-DPA1*01:03/DPB1*02:01 and -DPA1*01:03/DPB1*04:01 (allelic frequency: 55%). Tetramer labelling revealed enrichment of resident memory CD4+ T-cells (TRM) cells in the lower airway; these TRM displayed progressive differentiation, down-regulation of co- stimulatory molecules and elevated CXCR3 expression as infection evolved. Conclusion: Human infection challenge provides a unique opportunity to study the breadth of specificity and dynamics of RSV-specific T-cell responses in the target organ, allowing the precise investigation of TRM recognising novel viral antigens over time. The new tools that we describe enable precise tracking of RSV-specific CD4+ cells, potentially accelerating the development of effective vaccines

Fundamental investigation of dry electrical discharge machining (DEDM) by optical emission spectroscopy and its numerical interpretation

Dry electrical discharge machining (DEDM) has been developed as an alternative manufacturing process to the traditional EDM in liquid dielectric media. The absence of the liquid dielectric allows DEDM to be performed by simpler and environmentally friendlier machines. The erosion in DEDM mainly occurs due to the bombardment of the workpiece electrode surface by charged particles produced by micro electric discharges. Thus, the understanding of the fundamental properties of the micro plasma is necessary to explain the erosion mechanisms in this process. Optical emission spectroscopy of DEDM single discharges and its numerical interpretation by emission spectra simulation are developed in the present work. The hypothesis of plasmas in local thermal equilibrium (LTE) is developed, whereas the formation of an electron beam in non-LTE plasmas is also considered and briefly introduced. The simulations show that large amount of different ionic species is produced from the anode workpiece material, and the estimated electron temperature profile is peaking at the plasma centre. Moreover, hot anode spots formed on the workpiece surface due to the plasma-material interactions seem to be considerably smaller than the total plasma diameter and the respective eroded crater. These characteristics indicate that DEDM produces discharges similar to anode dominated vacuum arcs, which present properties very different from EDM discharges in liquid dielectric.ISSN:0268-3768ISSN:1433-301