A Novel Microfluidic Dielectrophoresis Technology to Enable Rapid Diagnosis of Mycobacteria tuberculosis in Clinical Samples

To achieve the global efforts to end tuberculosis, affordable diagnostics suitable for true point-of-care implementation are required to reach the missing millions. In addition, diagnostics with increased sensitivity and expanded drug susceptibility testing are needed to address drug resistance and to diagnose low-bacterial burden cases. The laboratory-on-a-chip technology described herein used dielectrophoresis to selectively isolate Mycobacterium tuberculosis from sputum samples, purifying the bacterial population ahead of molecular confirmation by multiplex real-time quantitative PCR. After optimization using a panel of 50 characterized sputum samples, the performance of the prototype was assessed against the current gold standards, screening 100 blinded sputum samples using characterized and biobanked sputum provided by Foundation for Innovative New Diagnostics. Concordance with culture diagnosis was 100% for smear-negative samples and 87% for smear-positive samples. Of the smear-positive samples, the high burden sample concordance was 100%. Samples were diagnosed on the basis of visual assessment of the dielectrophoresis array and by multiplex real-time quantitative PCR assay. The results described herein demonstrate the potential of the CAPTURE-XT technology to provide a powerful sample preparation tool that could function as a front-end platform for molecular detection. This versatile tool could equally be applied as a visual detection diagnostic, potentially associated with bacterial identification for low-cost screening or coupled with an expanded PCR assay for genotypic drug susceptibility testing

Intelligent Front-End Sample Preparation Tool Using Acoustic Streaming

We have successfully developed a nucleic acid extraction system based on a microacoustic lysis array coupled to an integrated nucleic acid extraction system all on a single cartridge. The microacoustic lysing array is based on 36{sup o} Y cut lithium niobate, which couples bulk acoustic waves (BAW) into the microchannels. The microchannels were fabricated using Mylar laminates and fused silica to form acoustic-fluidic interface cartridges. The transducer array consists of four active elements directed for cell lysis and one optional BAW element for mixing on the cartridge. The lysis system was modeled using one dimensional (1D) transmission line and two dimensional (2D) FEM models. For input powers required to lyse cells, the flow rate dictated the temperature change across the lysing region. From the computational models, a flow rate of 10 {micro}L/min produced a temperature rise of 23.2 C and only 6.7 C when flowing at 60 {micro}L/min. The measured temperature changes were 5 C less than the model. The computational models also permitted optimization of the acoustic coupling to the microchannel region and revealed the potential impact of thermal effects if not controlled. Using E. coli, we achieved a lysing efficacy of 49.9 {+-} 29.92 % based on a cell viability assay with a 757.2 % increase in ATP release within 20 seconds of acoustic exposure. A bench-top lysing system required 15-20 minutes operating up to 58 Watts to achieve the same level of cell lysis. We demonstrate that active mixing on the cartridge was critical to maximize binding and release of nucleic acid to the magnetic beads. Using a sol-gel silica bead matrix filled microchannel the extraction efficacy was 40%. The cartridge based magnetic bead system had an extraction efficiency of 19.2%. For an electric field based method that used Nafion films, a nucleic acid extraction efficiency of 66.3 % was achieved at 6 volts DC. For the flow rates we tested (10-50 {micro}L/min), the nucleic acid extraction time was 5-10 minutes for a volume of 50 {micro}L. Moreover, a unique feature of this technology is the ability to replace the cartridges for subsequent nucleic acid extractions

Which features of UK farmland are important in retaining territories of the rapidly declining Turtle Dove Streptopelia turtur?

Capsule Turtle Doves continue to show a strong population decline; territories were more likely to be retained in areas with more nesting habitat, and more suitable foraging habitat. Aim To determine which features of farmland in England are important for retaining Turtle Dove territories Methods Fifty-eight grid squares with recent records of territorial Turtle Doves were resurveyed, and squares retaining Turtle Dove territories compared with those from which Turtle Doves had been lost. Results Turtle Dove territories were detected in 48% of squares resurveyed. When correcting for the 70% detection rate of the survey methodology, territories were present in 66% of squares surveyed suggesting a 34% decline over a 2-year period. Established scrub and hedgerows > 4 m tall positively influenced Turtle Dove presence and abundance, as did standing water. Bare ground and fallow had positive effects on Turtle Dove abundance whereas grazed land negatively impacted abundance. Conclusion The positive effects of area of established scrub and volume of large hedgerows are likely to represent a declining density of birds selecting the best quality nest sites. We suggest instead that foraging habitat may be limiting distribution

Cytoskeletal protein kinases: titin and its relations in mechanosensing

Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca2+–calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other “MLCKs”, is not Ca2+–calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation

Genetic variation and exercise-induced muscle damage: implications for athletic performance, injury and ageing.

Prolonged unaccustomed exercise involving muscle lengthening (eccentric) actions can result in ultrastructural muscle disruption, impaired excitation-contraction coupling, inflammation and muscle protein degradation. This process is associated with delayed onset muscle soreness and is referred to as exercise-induced muscle damage. Although a certain amount of muscle damage may be necessary for adaptation to occur, excessive damage or inadequate recovery from exercise-induced muscle damage can increase injury risk, particularly in older individuals, who experience more damage and require longer to recover from muscle damaging exercise than younger adults. Furthermore, it is apparent that inter-individual variation exists in the response to exercise-induced muscle damage, and there is evidence that genetic variability may play a key role. Although this area of research is in its infancy, certain gene variations, or polymorphisms have been associated with exercise-induced muscle damage (i.e. individuals with certain genotypes experience greater muscle damage, and require longer recovery, following strenuous exercise). These polymorphisms include ACTN3 (R577X, rs1815739), TNF (-308 G>A, rs1800629), IL6 (-174 G>C, rs1800795), and IGF2 (ApaI, 17200 G>A, rs680). Knowing how someone is likely to respond to a particular type of exercise could help coaches/practitioners individualise the exercise training of their athletes/patients, thus maximising recovery and adaptation, while reducing overload-associated injury risk. The purpose of this review is to provide a critical analysis of the literature concerning gene polymorphisms associated with exercise-induced muscle damage, both in young and older individuals, and to highlight the potential mechanisms underpinning these associations, thus providing a better understanding of exercise-induced muscle damage

Communicating Information on Eruptions and Their Impacts from the Earliest Times Until the Late Twentieth Century

Volcanoes hold a fascination for human beings and, before they were recorded by literate observers, eruptions were portrayed in art, were recalled in legends and became incorporated into religious practices: being viewed as agents of punishment, bounty or intimidation depending upon their state of activity and the culture involved. In the Middle East the earliest depiction of an eruption is a wall painting dating from the Neolithic at Çatal Hüyük and the earliest record dates from the third millennium BCE. Knowledge of volcanoes increased over time. In some parts of the world knowledge of eruptions was passed down by oral transmission, but as far as written records were concerned, in the first century CE only 9 volcanoes in the Mediterranean region were recognised, together with Mount Cameroon in West Africa. In the next 1000 years the list grew by 17, some 14 of these volcanoes being in Japan. The first recorded eruptions in Indonesia occurred in 1000 and 1006, and volcanoes in newly settled Iceland increased the number to just 48 in 1380 CE. After this the list continued to increase, with important regions such as New Zealand and Hawaii only being added in the past 200 years. Only from 1900 did the rate of growth decline significantly (Simkin et al. 1981: 23; Simkin, 1993 Siebert et al. 2011; Simkin, 1993), but it is sobering to recall that in the twentieth century major eruptions have occurred from volcanoes that were considered inactive or extinct examples including: Mount Lamington - Papua New Guinea, 1951; Mount Arenal - Costa Rica, 1968 and Nyos - Cameroon, 1986. Although there are instances where the human impact of historical eruptions have been compiled - with examples including the 1883 eruption of Krakatau (Simkin and Fiske (1983) and 1943 -1952 eruption of Parícutin (Luhr and Simkin, 1993) - these are exceptions and there remains a significant gap in knowledge about both the short and long-term effects on societies of major eruptions which occurred before the 1980s. Following a broad review the chapter provides a discussion of the ways in which information has been collected, compiled and disseminated from the earliest times until the 1980s in two case study areas: the Azores Islands (Portugal) and southern Italy. In Italy information on eruptions stretches back to prehistoric times and has become progressively better known over more than 2,000 years of written history, yet even here there remain significant gaps in the record even for events that took place between 1900 and 1990. In contrast, located in the middle of the Atlantic, the Azores have been isolated for much of their history and illustrate the difficulties involved in using indigenous sources to compile, not only assessments of impact, but also at a more basic level a complete list of historical events with accurate dates