Bacillus Calmette-Guerin (BCG) induces superior anti-tumour responses by Vδ2+ T cells compared with the aminobisphosphonate drug zoledronic acid.

Vδ2+ T cells can recognize malignantly transformed cells as well as those infected with mycobacteria. This cross-reactivity supports the idea of using mycobacteria to manipulate Vδ2+ T cells in cancer immunotherapy. To date, therapeutic interventions using Vδ2+ T cells in cancer have involved expanding these cells in or ex vivo using zoledronic acid (ZA). Here, we show that the mycobacterium Bacillus Calmette-Guérin (BCG) also causes Vδ2+ T-cell expansion in vitro and that resulting Vδ2+ cell populations are cytotoxic toward tumour cell lines. We show that both ZA and BCG-expanded Vδ2+ cells effectively killed both Daudi and THP-1 cells. THP-1 cell killing by both ZA and BCG-expanded Vδ2+ cells was enhanced by treatment of targets cells with ZA. Although no difference in cytotoxic activity between ZA- and BCG-expanded Vδ2+ cells was observed, BCG-expanded cells degranulated more and produced a more diverse range of cytokines upon tumour cell recognition compared to ZA-expanded cells. ZA-expanded Vδ2+ cells were shown to upregulate exhaustion marker CD57 to a greater extent than BCG-expanded Vδ2+ cells. Furthermore, ZA expansion was associated with upregulation of inhibitory markers PD-1 and TIM3 in a dose-dependent manner whereas PD-1 expression was not increased following expansion using BCG. Intradermal BCG vaccination of rhesus macaques caused in vivo expansion of Vδ2+ cells. In combination with the aforementioned in vitro data, this finding suggests that BCG treatment could induce expansion of Vδ2+ T cells with enhanced anti-tumour potential compared to ZA treatment and that either ZA or BCG could be used intratumourally as a means to potentiate stronger anti-tumour Vδ2+ T-cell responses.

Local Acceleration of Neurofilament Transport at Nodes of Ranvier.

Myelinated axons are constricted at nodes of Ranvier. These constrictions are important physiologically because they increase the speed of saltatory nerve conduction, but they also represent potential bottlenecks for the movement of axonally transported cargoes. One type of cargo are neurofilaments, which are abundant space-filling cytoskeletal polymers that function to increase axon caliber. Neurofilaments move bidirectionally along axons, alternating between rapid movements and prolonged pauses. Strikingly, axon constriction at nodes is accompanied by a reduction in neurofilament number that can be as much as 10-fold in the largest axons. To investigate how neurofilaments navigate these constrictions, we developed a transgenic mouse strain that expresses a photoactivatable fluorescent neurofilament protein in neurons. We used the pulse-escape fluorescence photoactivation technique to analyze neurofilament transport in mature myelinated axons of tibial nerves from male and female mice of this strain ex vivo Fluorescent neurofilaments departed the activated region more rapidly in nodes than in flanking internodes, indicating that neurofilament transport is faster in nodes. By computational modeling, we showed that this nodal acceleration can be explained largely by a local increase in the duty cycle of neurofilament transport (i.e., the proportion of the time that the neurofilaments spend moving). We propose that this transient acceleration functions to maintain a constant neurofilament flux across nodal constrictions, much as the current increases where a river narrows its banks. In this way, neurofilaments are prevented from piling up in the flanking internodes, ensuring a stable neurofilament distribution and uniform axonal morphology across these physiologically important axonal domains.SIGNIFICANCE STATEMENT Myelinated axons are constricted at nodes of Ranvier, resulting in a marked local decrease in neurofilament number. These constrictions are important physiologically because they increase the efficiency of saltatory nerve conduction, but they also represent potential bottlenecks for the axonal transport of neurofilaments, which move along axons in a rapid intermittent manner. Imaging of neurofilament transport in mature myelinated axons ex vivo reveals that neurofilament polymers navigate these nodal axonal constrictions by accelerating transiently, much as the current increases where a river narrows its banks. This local acceleration is necessary to ensure a stable axonal morphology across nodal constrictions, which may explain the vulnerability of nodes of Ranvier to neurofilament accumulations in animal models of neurotoxic neuropathies and neurodegenerative diseases.

Dynamic catch-bonding generates the large stall forces of cytoplasmic dynein.

Cytoplasmic dynein is an important molecular motor involved in the transport of vesicular and macromolecular cargo along microtubules in cells, often in conjunction with kinesin motors. Dynein is larger and more complex than kinesin and the mechanism and regulation of its movement is currently the subject of intense research. While it was believed for a long time that dynein motors are relatively weak in terms of the force they can generate, recent studies have shown that interactions with regulatory proteins confer large stall forces comparable to those of kinesin. This paper reports on a theoretical study which suggests that these large stall forces may be the result of an emergent, ATP-dependent, bistability resulting in a dynamic catch-bonding behavior that can cause the motor to switch between high and low load-force states.

Local regulation of neurofilament transport by myelinating cells.

Axons in the vertebrate nervous system only expand beyond ∼ 1 µm in diameter if they become myelinated. This expansion is due in large part to the accumulation of space-filling cytoskeletal polymers called neurofilaments, which are cargoes of axonal transport. One possible mechanism for this accumulation is a decrease in the rate of neurofilament transport. To test this hypothesis, we used a fluorescence photoactivation pulse-escape technique to compare the kinetics of neurofilament transport in contiguous myelinated and unmyelinated segments of axons in long-term myelinating cocultures established from the dorsal root ganglia of embryonic rats. The myelinated segments contained more neurofilaments and had a larger cross-sectional area than the contiguous unmyelinated segments, and this correlated with a local slowing of neurofilament transport. By computational modeling of the pulse-escape kinetics, we found that this slowing of neurofilament transport could be explained by an increase in the proportion of the time that the neurofilaments spent pausing and that this increase in pausing was sufficient to explain the observed neurofilament accumulation. Thus we propose that myelinating cells can regulate the neurofilament content and morphology of axons locally by modulating the kinetics of neurofilament transport.

The Mobility of Neurofilaments in Mature Myelinated Axons of Adult Mice.

Studies in cultured neurons have shown that neurofilaments are cargoes of axonal transport that move rapidly but intermittently along microtubule tracks. However, the extent to which axonal neurofilaments move in vivo has been controversial. Some researchers have proposed that most axonally transported neurofilaments are deposited into a persistently stationary network and that only a small proportion of axonal neurofilaments are transported in mature axons. Here we use the fluorescence photoactivation pulse-escape technique to test this hypothesis in intact peripheral nerves of adult male hThy1-paGFP-NFM mice, which express low levels of mouse neurofilament protein M tagged with photoactivatable GFP. Neurofilaments were photoactivated in short segments of large, myelinated axons, and the mobility of these fluorescently tagged polymers was determined by analyzing the kinetics of their departure. Our results show that >80% of the fluorescence departed the window within 3 h after activation, indicating a highly mobile neurofilament population. The movement was blocked by glycolytic inhibitors, confirming that it was an active transport process. Thus, we find no evidence for a substantial stationary neurofilament population. By extrapolation of the decay kinetics, we predict that 99% of the neurofilaments would have exited the activation window after 10 h. These data support a dynamic view of the neuronal cytoskeleton in which neurofilaments cycle repeatedly between moving and pausing states throughout their journey along the axon, even in mature myelinated axons. The filaments spend a large proportion of their time pausing, but on a timescale of hours, most of them move.

Optimization of chest CT protocols based on pixel image matrix, kernels and iterative reconstruction levels - A phantom study.

INTRODUCTION: This study investigated the impact of high matrix image reconstruction in combination with different reconstruction kernels and levels of iterative reconstructions on image quality in chest CT. METHODS: An anthropomorphic chest phantom (Kyoto Kagaku Co., Ltd., Kyoto, Japan), and a Catphan® 600 (The Phantom Laboratory, Greenwich, NY, USA) phantom were scanned using a dual source scanner. Standard institutional protocol with 512 × 512 matrix was used as a reference. Reconstructions were performed for 768 × 768 and 1024 × 1024 matrices and all possible combinations of three different kernels and five levels of iterative reconstructions were included. Signal difference to noise ratio (SdNR) and line pairs per cm (lp/cm) were manually measured. A Linear regression model was applied for objective image analysis (SdNR) and inter-and intra-reader agreement was given as Cohen's kappa for the visual image assessment. RESULTS: Matrix size did not have a significant impact on SdNR (p = 0.595). Kernel (p = 0.014) and ADMIRE level (p = 0.001) had a statistically significant impact on SdNR. The spatial resolution ranged from 7 lp/cm to 9 lp/cm. The highest spatial resolution was achieved using kernel Br64 and ADMIRE 1, 2 and 3 in both 768- and 1024-matrices, and with Br59 with ADMIRE 2 and 4 and 768-matrix, all visualizing 9 lp/cm. Both readers scored kernel Br59 highest, and the scoring increased with increasing levels of Iterative Reconstruction. CONCLUSION: Matrix size did not influence image quality, however, the choice of kernel and degree of IR had an impact on objective and visual image quality in 768 - and 1024-matrices, suggesting that increased degree of IR may improve diagnostic image quality in chest CT. IMPLICATIONS FOR PRACTICE: Image quality in CT of the lung may be improved by increasing the level of IR.

Clinical characterisation of a novel paroxysmal dyskinesia in Welsh terrier dogs.

Breed specific paroxysmal dyskinesias are increasingly recognised in veterinary medicine. We aimed to characterise the phenomenology, clinical course and prevalence of a previously unreported paroxysmal dyskinesia in the Welsh terrier breed. Clinical records of five Welsh terriers with paroxysmal episodes were reviewed. Additionally, owners of Welsh terriers were invited to complete a questionnaire with the aim of characterising paroxysmal episodes in the wider breed population. Clinical examinations (n = 5) and diagnostic investigations (n = 3) of affected Welsh terriers were within normal limits, apart from mild-moderate ventriculomegaly on cranial magnetic resonance imaging (n = 3). The survey of Welsh terrier owners revealed episodes consistent with a paroxysmal dyskinesia in 41 (22.8%) of 177 respondents. Median age of onset was 59 months. Episodes were predominantly characterised by sustained hypertonicity with periods of limb flexion, abnormal head and body posture, with preserved consciousness. Episode duration ranged from 30 s to 30 min (median, 3 min 30 s), with frequency varying widely between dogs. Affected dogs demonstrated a stable to improving clinical course in most cases. This study investigated a previously unreported paroxysmal dyskinesia in Welsh terriers. Similar clinical signs within the breed were potentially consistent with an inherited cause, worthy of further investigation.

Molecular Beams with Energies above One Electron Volt.

By using as sources supersonic jets of hydrogen or helium containing small concentrations of heavier molecules we have been able to obtain molecular beams with kinetic energies of the heavy molecules well into the range above I electron volt. A variety of molecules have been successfully accelerated. Intensities of 10(16) to 10(17) heavy molecules per steradian-second have been achieved at these high energies.

Electrospray ionization for mass spectrometry of large biomolecules.

Electrospray ionization has recently emerged as a powerful technique for producing intact ions in vacuo from large and complex species in solution. To an extent greater than has previously been possible with the more familiar "soft" ionization methods, this technique makes the power and elegance of mass spectrometric analysis applicable to the large and fragile polar molecules that play such vital roles in biological systems. The distinguishing features of electrospray spectra for large molecules are coherent sequences of peaks whose component ions are multiply charged, the ions of each peak differing by one charge from those of adjacent neighbors in the sequence. Spectra have been obtained for biopolymers including oligonucleotides and proteins, the latter having molecular weights up to 130,000, with as yet no evidence of an upper limit.

A preliminary mitochondrial genome phylogeny of Orthoptera (Insecta) and approaches to maximizing phylogenetic signal found within mitochondrial genome data.

The phylogenetic utility of mitochondrial genomes (mtgenomes) is examined using the framework of a preliminary phylogeny of Orthoptera. This study presents five newly sequenced genomes from four orthopteran families. While all ensiferan and polyneopteran taxa retain the ancestral gene order, all caeliferan lineages including the newly sequenced caeliferan species contain a tRNA rearrangement from the insect ground plan tRNA(Lys)(K)-tRNA(Asp)(D) swapping to tRNA(Asp) (D)-tRNA(Lys) (K) confirming that this rearrangement is a possible molecular synapomorphy for this suborder. The phylogenetic signal in mtgenomes is rigorously examined under the analytical regimens of parsimony, maximum likelihood and Bayesian inference, along with how gene inclusion/exclusion, data recoding, gap coding, and different partitioning schemes influence the phylogenetic reconstruction. When all available data are analyzed simultaneously, the monophyly of Orthoptera and its two suborders, Caelifera and Ensifera, are consistently recovered in the context of our taxon sampling, regardless of the optimality criteria. When protein-coding genes are analyzed as a single partition, nearly identical topology to the combined analyses is recovered, suggesting that much of the signals of the mtgenome come from the protein-coding genes. Transfer and ribosomal RNAs perform poorly when analyzed individually, but contribute signal when analyzed in combination with the protein-coding genes. Inclusion of third codon position of the protein-coding genes does not negatively affect the phylogenetic reconstruction when all genes are analyzed together, whereas recoding of the protein-coding genes into amino acid sequences introduces artificial resolution. Over-partitioning in a Bayesian framework appears to have a negative effect in achieving convergence. Our findings suggest that the best phylogenetic inferences are made when all available nucleotide data from the mtgenome are analyzed simultaneously, and that the mtgenome data can resolve over a wide time scale from the Permian (approximately 260 MYA) to the Tertiary (approximately 50 MYA).

Axonal neurofilaments exhibit frequent and complex folding behaviors.

Neurofilaments are flexible cytoskeletal polymers that are capable of folding and unfolding between their bouts of bidirectional movement along axons. Here we present a detailed characterization of this behavior in cultured neurons using kymograph analysis with approximately 30 ms temporal resolution. We analyzed 781 filaments ranging from 0.6-42 µm in length. We observed complex behaviors including pinch folds, hairpin folds, orientation changes (flips), and occasional severing and annealing events. On average, the filaments spent approximately 40% of their time in some sort of folded configuration. A small proportion of filaments (4%) moved while folded, but most (96%) moved in an outstretched configuration. Collectively, our observations suggest that motors may interact with neurofilaments at multiple points along their length, but preferentially at their ends. In addition, the prevalence of neurofilament folding and the tendency of neurofilaments to straighten out when they move, suggest that an important function of the movement of these polymers in axons may be to maintain them in an outstretched and longitudinally co-aligned configuration. Thus, neurofilament movement may function as much to organize these polymers as to move them, and this could explain why they spend so much time engaged in apparently unproductive bidirectional movement.

Kymograph analysis with high temporal resolution reveals new features of neurofilament transport kinetics.

We have used kymograph analysis combined with edge detection and an automated computational algorithm to analyze the axonal transport kinetics of neurofilament polymers in cultured neurons at 30 ms temporal resolution. We generated 301 kymographs from 136 movies and analyzed 726 filaments ranging from 0.6 to 42 µm in length, representing ∼37,000 distinct moving and pausing events. We found that the movement is even more intermittent than previously reported and that the filaments undergo frequent, often transient, reversals which suggest that they can engage simultaneously with both anterograde and retrograde motors. Average anterograde and retrograde bout velocities (0.9 and 1.2 µm s-1 , respectively) were faster than previously reported, with maximum sustained bout velocities of up to 6.6 and 7.8 µm s-1 , respectively. Average run lengths (∼1.1 µm) and run times (∼1.4 s) were in the range reported for molecular motor processivity in vitro, suggesting that the runs could represent the individual processive bouts of the neurofilament motors. Notably, we found no decrease in run velocity, run length or run time with increasing filament length, which suggests that either the drag on the moving filaments is negligible or that longer filaments recruit more motors.

The Nurse Who Saw Me.

A mother's long night at her sick newborn's side. What can possibly ease the strain?

Associations Between Anesthetic Variables and Functional Outcome in Dogs With Thoracolumbar Intervertebral Disk Extrusion Undergoing Decompressive Hemilaminectomy.

BACKGROUND: Outcome of acute experimental spinal cord injury is strongly associated with tissue perfusion and oxygenation. Cardiopulmonary depression could affect outcome in dogs undergoing general anesthesia for surgical treatment of thoracolumbar intervertebral disk extrusion (IVDE). HYPOTHESIS/OBJECTIVES: To evaluate the effects of general anesthesia on functional outcome in dogs undergoing surgery to treat thoracolumbar IVDE. ANIMALS: Eighty-four client-owned dogs with acute thoracolumbar IVDE treated by decompressive hemilaminectomy. METHODS: Exploratory, retrospective observational study. Medical records were reviewed for clinical presentation and anesthetic monitoring variables, including duration of anesthesia and surgery, hypotension, bradycardia, temperature, and respiratory parameters. Multivariable regression tree analysis was performed to explore associations between anesthetic variables and functional outcome scores after 6 weeks, as well as return to ambulatory status. RESULTS: Episodes of bradycardia (69%) and hypotension (57%) were frequent. Across all outcome measures, regression tree analysis highlighted functional grade at presentation as the primary determining factor, and among pain perception negative dogs, there was a possible association between increased duration of surgery and poorer outcome. In dogs with intact pain perception, duration of bradycardia, mean body temperature, and mean end-tidal carbon dioxide were highlighted. CONCLUSIONS AND CLINICAL IMPORTANCE: Exploratory statistical methods can facilitate hypothesis-generating studies to inform prospective investigations in veterinary medicine. Although the mechanism is uncertain, increased duration of surgery might be associated with poorer outcome in pain perception negative dogs with thoracolumbar IVDE.

Live-cell imaging of neurofilament transport in cultured neurons.

Neurofilaments, which are the intermediate filaments of nerve cells, are space-filling cytoskeletal polymers that contribute to the growth of axonal caliber. In addition to their structural role, neurofilaments are cargos of axonal transport that move along microtubule tracks in a rapid, intermittent, and bidirectional manner. Though they measure just 10nm in diameter, which is well below the diffraction limit of optical microscopes, these polymers can reach 100 µm or more in length and are often packed densely, just tens of nanometers apart. These properties of neurofilaments present unique challenges for studies on their movement. In this article, we describe several live-cell fluorescence imaging strategies that we have developed to image neurofilament transport in axons of cultured neurons on short and long timescales. Together, these methods form a powerful set of complementary tools with which to study the axonal transport of these unique intracellular cargos.

This house believes the NHS should be privatised - 1st southwest medical debate.

The inaugural southwest medical debate, between Exeter and Plymouth medical schools and respective health services, was held on the 3rd December 2014. Plymouth proposed the motion "This house believes the NHS should be privatised?" In an increasingly political climate, the National Health Service (NHS) has become a constant topic for discussion in the media. On this occasion, all those debating were involved in the medical profession with roles encompassing clinical medicine, education, ethics, economics and policy. By allowing those with knowledge of the NHS to speak, we hoped to spark novel discussions based on evidence and experience.

Treatment planning optimization for multiple arcs stereotactic radiosurgery using a linear accelerator.

PURPOSE: Multiarc stereotactic radiosurgery is a technique used to irradiate an intracranial tumor with minimal damage to the surrounding normal tissue. The purpose of this paper is to present a method for and the results from optimizing three dimensional (3D) treatment dose for multiarc stereotactic radiosurgery. METHODS AND MATERIALS: The normal procedure for a physician-physicist team designing a treatment plan for multiarc stereotactic radiosurgery is the trial-and-error approach of changing the collimator size and the isocenter of radiation by viewing the isodose curves on a two dimensional (2D) computed tomography (CT) or magnetic resonance imaging (MRI) image plane. Not only is this time consuming, but the resulting treatment plan is not optimal in most, if not all, cases. One reason for such nonconformal isodose curves is that the same collimator size is used for all arcs. However, it is very difficult to determine manually the different collimator sizes for different arcs. A derivative free optimization method is used to optimize the collimator size for each arc, as well as the 3D coordinates of the isocenter(s). RESULTS: One spherical and two ellipsoidal artificial tumors, and one actual tumor, were used to show the utilities of the optimization process. The 90% isodose curves resulting from optimization conform very well with the tumor; whereas the 90% isodose curves from the conventional method either do not envelop the entire tumor when the collimator size is too small, or a large volume of normal tissue is also irradiated by the 90% dose when the next larger collimator size is used. CONCLUSIONS: When the collimator size for each arc and the location of the isocenters(s) are optimized in a multiarc stereotactic surgery treatment plan, the 90% isodose curve conforms to the tumor much better than when the same collimator size is used for all arcs.

Ion formation from charged droplets: Roles of geometry, energy, and time.

The formation of ions from the charged droplets produced in the several spray ionization techniques is viewed as an activated rate process involving field-assisted desorption, in accord with the ideas first set forth by Iribame and Thomson. The novel features of the present treatment are particularly relevant to the unique ability of electrospray ionization to transform large molecules in solution to free ions in the gas phase, with extensive multiple charging. These new features stem mainly from the realization that the spacing of charges on a desorbed ion must relate to the spacing of charges on the surface of the droplet whence it came. The consequences of this "rule" can account for the existence of maxima and minima in the number of charges on the ions of a particular species as well as the nature of the distribution of ions among the intervening charge states. They also explain the dependence of charge state on the configuration in solution of the parent molecule of the desorbed ion. In addition, they provide insight into the sequence in time at which ions in the various charge states leave an evaporating droplet.

Surgical principles and polytetrafluoroethylene.

This report describes a 24-month follow-up in 100 consecutive polytetrafluoroethylene (PTFE) arterial grafts. Although initial results were superb, a continued follow-up has showed extremely high closure rates for femoropopliteal and femorotibial grafts. The primary reason for this high attrition rate is thought to be stasis. We believe that PTFE is clearly the best synthetic arterial replacement available, but the material does not approach the autogenous saphenous vein in terms of long-term patency. Therefore, in spite of its many advantages, we do not recommend the elective use of PTFE for peripheral small-vessel bypass.

Use of and cost savings with morphologic criteria and the spot indole test as a routine means of identification of Escherichia coli.

The use of primary isolation plate colonial morphologic criteria (CMC) of a flat, nonmucoid, lactose-fermenting, gram-negative rod on MacConkey agar and the spot indole (SI) test from the sheep blood agar plate was evaluated as a means for identification of Escherichia coli in comparison to kit (Micro-ID, API-20E) and conventional biochemical testing. In this preliminary phase of comparison of accuracy, 427 isolates of E. coli (69.8%) from a total of 612 isolates of lactose-fermenting gram-negative rods were evaluated. Of these E. coli isolates, 357 (83.6%) fit the CMC and were SI positive; 3 (less than 1% error rate) were not E. coli. In the second phase of the evaluation, using CMC and SI alone as a means for identification of E. coli, 472 (57.6%) E. coli isolates from a total of 820 Enterobacteriaceae isolates were assessed. Of these E. coli isolates, 326 could be identified using only CMC and SI (69.1% of the E. coli isolates and 39.8% of all Enterobacteriaceae isolates); 146 (30.9%) required additional biochemical testing because of atypical colonial morphology, because of the investigator's inability to differentiate colony types on both media or lack of isolated colonies on either of the two required media, or because as isolates from sterile body sites they were processed directly to Micro-ID kits. A minimum of 40% savings on Enterobacteriaceae identification schemes without compromising accuracy was calculated. As of November 1983, a direct (labor and materials) cost savings of approximately +200.80 per 100 Enterobacteriaceae identifications was projected.