3× multiplexed detection of antibiotic resistant plasmids with single molecule sensitivity.

Bacterial pathogens resistant to antibiotics have become a serious health threat. Those species which have developed resistance against multiple drugs such as the carbapenems, are more lethal as these are last line therapy antibiotics. Current diagnostic tests for these resistance traits are based on singleplex target amplification techniques which can be time consuming and prone to errors. Here, we demonstrate a chip based optofluidic system with single molecule sensitivity for amplification-free, multiplexed detection of plasmids with genes corresponding to antibiotic resistance, within one hour. Rotating disks and microfluidic chips with functionalized polymer monoliths provided the upstream sample preparation steps to selectively extract these plasmids from blood spiked with E. coli DH5α cells. Waveguide-based spatial multiplexing using a multi-mode interference waveguide on an optofluidic chip was used for parallel detection of three different carbapenem resistance genes. These results point the way towards rapid, amplification-free, multiplex analysis of antibiotic-resistant pathogens.

Influence of sociodemographic characteristics on the preferred format of health education delivery in individuals with type 2 diabetes mellitus and or cardiovascular disease: a questionnaire study.

AIM: To examine the influence of sociodemographic factors of interest on preference for a particular health education format among people with type 2 diabetes and/or cardiovascular disease. METHODS: A questionnaire was used to collect information on the influence of six sociodemographic factors of interest on the preference for health education formats in people with type 2 diabetes and/or cardiovascular disease. Chi-squared tests were used to examine the distribution of preferences between groups. The characteristics of the population preferring the online format were then examined in more detail using logistic regression. RESULTS: Responses were received from 1559 participants. Overall the preferred health education format was one-to-one learning from a doctor or nurse (67%). Age, gender, diagnosis and educational level all affected the preferences expressed. The characteristics showing most consistent and significant influence were age and educational level. Overall, 29% ranked the online format highly (scores 1 or 2). This group were more likely to be aged < 65 years (P < 0.001) and to have a higher level of educational attainment (upper secondary education or higher; P < 0.001). CONCLUSIONS: Significant differences between sociodemographic groups exist in preferences for health education formats among people with type 2 diabetes and/or cardiovascular disease. Preferences should be considered when designing educational interventions to ensure they are accessible to the target group and to avoid increases in health inequality.

Educational weight loss interventions in obese and overweight adults with type 2 diabetes: a systematic review and meta-analysis of randomized controlled trials.

AIM: The worldwide prevalence of type 2 diabetes mellitus is increasing, with most individuals with the disease being overweight or obese. Weight loss can reduce disease-related morbidity and mortality and weight losses of 10-15 kg have been shown to reverse type 2 diabetes. This review aimed to determine the effectiveness of community-based educational interventions for weight loss in type 2 diabetes. METHODS: This is a systematic review and meta-analysis of randomized controlled trials (RCT) in obese or overweight adults, aged 18-75 years, with a diagnosis of type 2 diabetes. Primary outcomes were weight and/or BMI. CINAHL, MEDLINE, Embase, Scopus and the Cochrane Central Register of Controlled Trials (CENTRAL) were searched from inception to June 2019. Trials were classified into specified a priori comparisons according to intervention type. A pooled standardized mean difference (SMD) (from baseline to follow-up) and 95% confidence intervals (95% CI) between trial groups (difference-in-difference) were estimated through random-effects meta-analyses using the inverse variance method. Heterogeneity was quantified using I2 and publication bias was explored visually using funnel plots. RESULTS: Some 7383 records were screened; 228 full-text articles were assessed and 49 RCTs (n = 12 461 participants) were included in this review, with 44 being suitable for inclusion into the meta-analysis. Pooled estimates of education combined with low-calorie, low-carbohydrate meal replacements (SMD = -2.48, 95% CI -3.59, -1.49, I2  = 98%) or diets (SMD = -1.25, 95% CI -2.11, -0.39, I2  = 95%) or low-fat meal replacements (SMD = -1.15, 95%CI -2.05, -1.09, I2  = 85%) appeared most effective. CONCLUSION: Low-calorie, low-carbohydrate meal replacements or diets combined with education appear the most promising interventions to achieve the largest weight and BMI reductions in people with type 2 diabetes.

Postauricular Melanocytic Neuroectodermal Tumor of Infancy: A Rare Site of a Rare Tumor-MNTI as a Postauricular Mass with Literature Review.

Melanocytic neuroectodermal tumor of infancy (MNTI) is a rare osteolytic neoplasm of neural crest cell origin. There are less than 500 documented cases, most frequently affecting the maxilla of infants less than 1 year old. We present a unique case of a two-month-old male with a progressive postauricular mass since birth, confirmed to be a MNTI. The lesion required three resections over the course of five months, with rapid recurrence ultimately requiring a craniectomy, highlighting the difficulty in treating these tumors. Histological and radiographic features were reviewed; an updated literature review for identifying and treating these lesions is presented.

Effects of haemolysis, icterus and lipaemia on coagulation tests as performed on Stago STA-Compact-Max analyser.

INTRODUCTION: Haemolysis, icterus and lipaemia (HIL) may affect haemostasis test results. This may be influenced by the level of interfering substance and the reagents and end-point detection system used. METHODS: We assessed the influence of HIL on prothrombin time, activated partial thromboplastin time and fibrinogen assay using a viscosity-based detection analyser. RESULTS: Spontaneous haemolysis that occurred during sample collection and processing had no effect on PT with either a rabbit tissue factor extract or recombinant human tissue factor reagents. In contrast, addition of mechanically haemolysed cells impacted on PT for the highest haemoglobin concentration. For APTTs determined with STA®-Cephascreen® reagent, there was no significant difference between results in haemolysed and nonhaemolysed samples. For the other two reagents studied, APTTs were statistically significantly shorter in haemolysed samples compared with nonhaemolysed samples. This bias was clinically significant only for STA®-PTT Automate. For all three APTT reagents, the impact of haemolysis was sufficient to impact patient management decisions, and in some samples, the effects of lipaemia and icterus were not clinically significant. CONCLUSION: Overall, our results confirm that PT and fibrinogen were not clinically significantly affected by HIL. The APTTs of some haemolysed samples were falsely normal. Haemolysed samples for APTT determination should be rejected.

A critical review of cell culture strategies for modelling intracortical brain implant material reactions.

The capacity to predict in vivo responses to medical devices in humans currently relies greatly on implantation in animal models. Researchers have been striving to develop in vitro techniques that can overcome the limitations associated with in vivo approaches. This review focuses on a critical analysis of the major in vitro strategies being utilized in laboratories around the world to improve understanding of the biological performance of intracortical, brain-implanted microdevices. Of particular interest to the current review are in vitro models for studying cell responses to penetrating intracortical devices and their materials, such as electrode arrays used for brain computer interface (BCI) and deep brain stimulation electrode probes implanted through the cortex. A background on the neural interface challenge is presented, followed by discussion of relevant in vitro culture strategies and their advantages and disadvantages. Future development of 2D culture models that exhibit developmental changes capable of mimicking normal, postnatal development will form the basis for more complex accurate predictive models in the future. Although not within the scope of this review, innovations in 3D scaffold technologies and microfluidic constructs will further improve the utility of in vitro approaches.

The Δ133p53 isoform and its mouse analogue Δ122p53 promote invasion and metastasis involving pro-inflammatory molecules interleukin-6 and CCL2.

A number of naturally occurring isoforms of the tumour suppressor protein p53 have been discovered, which appear to have differing roles in tumour prevention or promotion. We are investigating the tumour-promoting activities of the Δ133p53 isoform using our mouse model of Δ133p53 (Δ122p53). Here, we report that tumours from Δ122p53 homozygous mice show evidence of invasion and metastasis and that Δ122p53 promotes migration though a 3-dimensional collagen matrix. We also show that Δ122p53 and Δ133p53 promote cell migration in scratch wound and Transwell assays, similar to the 'gain-of-function' phenotypes seen with mutant p53. Using the well-defined B16 mouse melanoma metastatic model, we show that Δ122p53 leads to faster generation of lung metastases. The increased migratory phenotypes are dependent on secreted factors, including the cytokine interleukin-6 and the chemokine CCL2. We propose that Δ122p53 (and Δ133p53) acts in a similar manner to 'gain-of-function' mutant p53 proteins to promote migration, invasion and metastasis, which may contribute to poor survival in patients with Δ133p53-expressing tumours.

Pressure-actuated microfluidic devices for electrophoretic separation of pre-term birth biomarkers.

We have developed microfluidic devices with pressure-driven injection for electrophoretic analysis of amino acids, peptides, and proteins. The novelty of our approach lies in the use of an externally actuated on-chip peristaltic pump and closely spaced pneumatic valves that allow well-defined, small-volume sample plugs to be injected and separated by microchip electrophoresis. We fabricated three-layer poly(dimethylsiloxane) (PDMS) microfluidic devices. The fluidic layer had injection and separation channels, and the control layer had an externally actuated on-chip peristaltic pump and four pneumatic valves around the T-intersection to carry out sample injection. An unpatterned PDMS membrane layer was sandwiched between the fluidic and control layers as the actuated component in pumps and valves. Devices with the same peristaltic pump design but different valve spacings (100, 200, 300, and 400 µm) from the injection intersection were fabricated using soft lithographic techniques. Devices were characterized through fluorescent imaging of captured plugs of a fluorescein-labeled amino acid mixture and through microchip electrophoresis separations. A suitable combination of peak height, separation efficiency, and analysis time was obtained with a peristaltic pump actuation rate of 50 ms, an injection time of 30 s, and a 200-µm valve spacing. We demonstrated the injection of samples in different solutions and were able to achieve a 2.4-fold improvement in peak height and a 2.8-fold increase in separation efficiency though sample stacking. A comparison of pressure-driven injection and electrokinetic injection with the same injection time and separation voltage showed a 3.9-fold increase in peak height in pressure-based injection with comparable separation efficiency. Finally, the microchip systems were used to separate biomarkers implicated in pre-term birth. Although these devices have initially been demonstrated as a stand-alone microfluidic separation tool, they have strong potential to be integrated within more complex systems.

Summary of the British Thoracic Society guidelines for advanced diagnostic and therapeutic flexible bronchoscopy in adults.

This new guideline covers the rapidly advancing field of interventional bronchoscopy using flexible bronchoscopy. It includes the use of more complex diagnostic procedures such as endobronchial ultrasound, interventions for the relief of central airway obstruction due to malignancy and the recent development of endobronchial therapies for chronic obstructive pulmonary disease and asthma. The guideline aims to help all those who undertake flexible bronchoscopy to understand more about this important area. It also aims to inform respiratory physicians and other specialists dealing with lung cancer of the procedures possible in the management and palliation of central airway obstruction. The guideline covers transbronchial needle aspiration and endobronchial ultrasound-guided transbronchial needle aspiration, electrocautery/diathermy, argon plasma coagulation and thermal laser, cryotherapy, cryoextraction, photodynamic therapy, brachytherapy, tracheobronchial stenting, electromagnetic navigation bronchoscopy, endobronchial valves for emphysema and bronchial thermoplasty for asthma.

The investigation of a prolonged APTT with specific clotting factor assays is unnecessary if an APTT with Actin FS is normal.

INTRODUCTION: An isolated prolongation to the activated partial thromboplastin time (APTT) can be caused by the presence of the lupus anticoagulant or an intrinsic or contact factor deficiency, of which only deficiencies of factors VIII, IX or XI are associated with bleeding. Our local protocol states that further investigation of a prolonged APTT by specific assays of FVIII, FIX and FXI should only be undertaken where the APTT with one reagent (Synthasil) is more than 3 s prolonged, and further investigation by an APTT with a second reagent (Actin FS) is also prolonged, unless there is a history of bleeding in the patient, in which case assays are indicated irrespective of the APTT. METHODS: We retrospectively reviewed the results of all APTTs performed over a 36-month period to evaluate whether strictly applying our protocol would reduce the number of unnecessary clotting factor assays performed, without leaving patients with potentially significant bleeding disorders undiagnosed. RESULTS: Of a total number of 587 samples tested for coagulation factors VIII, IX and XI, only 117 samples yielded an abnormal result. Thus, 80% of all the assays requested in the 3-year period audited gave a result within the reference range for factors VIII, FIX and XI. Three quarters of the abnormal results revealed mild FXI deficiency. CONCLUSION: This review has demonstrated that no significant coagulation factor deficiency would be left undiagnosed if the protocol was followed. This would have considerably reduced the cost and time spent performing these assays.

Genotoxic stress-induced nuclear localization of oncoprotein YB-1 in the absence of proteolytic processing.

Y-box-binding protein 1 (YB-1) is an oncogenic transcription factor whose overexpression and nuclear localization is associated with tumor progression and drug resistance. Transcriptional activation of YB-1 in response to genotoxic stress is believed to occur in the cytoplasm through sequence-specific endoproteolytic cleavage by the 20S Proteasome, followed by nuclear translocation of cleaved YB-1. To study the proteolysis model, we developed a two-step affinity purification of endogenous YB-1 protein species and characterized the products using mass spectrometry. Whereas full-length YB-1 was readily identified, the smaller protein band thought to be activated YB-1 was identified as hnRNP A1. An antibody specific for YB-1 was generated, which revealed only one YB-1 species, even after genotoxic stress-induced nuclear YB-1 translocation. These findings warrant re-evaluation of the mechanism of YB-1 nuclear translocation and transcriptional activation. The relationship between nuclear YB-1 and tumor progression may also have to re-evaluated in some cases.

Compact vibration isolation and suspension for Australian International Gravitational Observatory: performance in a 72 m Fabry Perot cavity.

This paper describes the first demonstration of vibration isolation and suspension systems, which have been developed with view to application in the proposed Australian International Gravitational Observatory. In order to achieve optimal performance at low frequencies new components and techniques have been combined to create a compact advanced vibration isolator structure. The design includes two stages of horizontal preisolation and one stage of vertical preisolation with resonant frequencies approximately 100 mHz. The nested structure facilitates a compact design and enables horizontal preisolation stages to be configured to create a superspring configuration, where active feedback can enable performance close to the limit set by seismic tilt coupling. The preisolation stages are combined with multistage three-dimensional (3D) pendulums. Two isolators suspending mirror test masses have been developed to form a 72 m optical cavity with finesse approximately 700 in order to test their performance. The suitability of the isolators for use in suspended optical cavities is demonstrated through their ease of locking, long term stability, and low residual motion. An accompanying paper presents the local control system and shows how simple upgrades can substantially improve residual motion performance.

Wide variation in thrombin generation in patients with atrial fibrillation and therapeutic International Normalized Ratio is not due to inflammation.

Atrial fibrillation (AF) is a common cardiac arrhythmia with a 5-20% annual risk of stroke. Warfarin reduces this risk by at least 60%. Despite adequate anticoagulation within the target International Normalized Ratio (INR) range of 2.0-3.0, some patients still experience thrombotic and bleeding events. It is now possible to assess the intensity of anticoagulation with automated thrombin generation (TG) tests, such as the calibrated automated thrombogram (CAT). These tests were compared and an inverse relationship was found between the INR and CAT in 143 elderly AF patients. There was equally good correlation between the concentration of factors II, VII, IX and X and the INR and TG parameters. The peak thrombin was most strongly associated with the concentration of prothrombin fragment 1 + 2 in plasma. There was wide variability in TG parameters in patients with identical INR values, sometimes up to a fourfold difference. This TG variability in individuals with the same INR is not due to inflammation, at least when the latter is measured as the concentration of factor VIII coagulant activity, von Willebrand factor antigen, high sensitivity C-reactive protein and fibrinogen. It was concluded that, although the TG and INR were closely correlated there was wide variability in peak thrombin and endogenous thrombin potential in patients within the INR therapeutic range, the cause of which remains unclear.

The use of ecarin chromogenic assay and prothrombinase induced clotting time in the monitoring of lepirudin for the treatment of heparin-induced thrombocytopenia.

Lepirudin (r-hirudin) is one of the two alternative anticoagulants licensed to treat patients with heparin-induced thrombocytopenia (HIT). Manufacturer's guidelines state that lepirudin should be monitored using the activated partial thromboplastin time (APTT) ratio. However, several studies have demonstrated a plateau effect of higher concentrations of lepirudin on APTT ratios and variable results when comparing different APTT reagents. This study compares APTT ratios (using two different APTT reagents) with two other commercially available methods for directly quantifying plasma lepirudin levels: ecarin chromogenic assay and prothrombinase-induced clotting time in 95 samples from five patients receiving lepirudin anticoagulation for HIT.

Planar thin film device for capillary electrophoresis.

Hollow tubular microfluidic channels were fabricated on quartz substrates using sacrificial layer, planar micromachining processes. The channels were created using a bottom-up fabrication technique, namely patterning a photoresist/aluminum sacrificial layer and depositing SiO(2) over the substrate. The photoresist/aluminum layer was removed by etching first with HCl/HNO(3), followed by etching in Nano-Strip, a more stable form of piranha (H(2)SO(4)/H(2)O(2)) stripper. Rapid separation of fluorescently labeled amino acids was performed on a device made with these channels. The fabrication process presented here provides unique control over channel composition and geometry. Future work should allow the fabrication of highly complex and precise devices with integrated analytical capabilities essential for the development of micro-total analysis systems.

The PIVKA II test: the first reliable coagulation test for autopsy investigations.

To date there is no routinely used reliable diagnostic test that can be performed in the post-mortem period to investigate whether a deceased had a coagulation disorder. This paper describes a series of experiments to assess the use of an antigen-based method to investigate the vitamin K-dependent factor II function in the deceased. It illustrates that by using this approach the functional status of factor II can be investigated in the post-mortem period. The abnormal proteins that are investigated by this method appear to remain stable for at least 72 h and potentially up to at least 7 days. The method that is illustrated could thus be reliably used in the post-mortem period to identify a natural or drug-induced factor II abnormality. The potential for other protein components of the coagulation cascade to be investigated by similar antigen-based methodology is suggested.

Structural and functional imaging with carbon nanotube AFM probes.

Atomic force microscopy (AFM) has great potential as a tool for structural biology, a field in which there is increasing demand to characterize larger and more complex biomolecular systems. However, the poorly characterized silicon and silicon nitride probe tips currently employed in AFM limit its biological applications. Carbon nanotubes represent ideal AFM tip materials due to their small diameter, high aspect ratio, large Young's modulus, mechanical robustness, well-defined structure, and unique chemical properties. Nanotube probes were first fabricated by manual assembly, but more recent methods based on chemical vapor deposition provide higher resolution probes and are geared towards mass production, including recent developments that enable quantitative preparation of individual single-walled carbon nanotube tips [J. Phys. Chem. B 105 (2001) 743]. The high-resolution imaging capabilities of these nanotube AFM probes have been demonstrated on gold nanoparticles and well-characterized biomolecules such as IgG and GroES. Using the nanotube probes, new biological structures have been investigated in the areas of amyloid-beta protein aggregation and chromatin remodeling, and new biotechnologies have been developed such as AFM-based haplotyping. In addition to measuring topography, chemically functionalized AFM probes can measure the spatial arrangement of chemical functional groups in a sample. However, standard silicon and silicon nitride tips, once functionalized, do not yield sufficient resolution to allow combined structural and functional imaging of biomolecules. The unique end-group chemistry of carbon nanotubes, which can be arbitrarily modified by established chemical methods, has been exploited for chemical force microscopy, allowing single-molecule measurements with well-defined functionalized tips.