Improved risk stratification of nasopharyngeal cancer by targeted sequencing of Epstein-Barr virus DNA in post-treatment plasma.

BACKGROUND: Quantitative measurement of plasma Epstein-Barr virus (EBV) DNA by real-time PCR at the end of primary treatment is a robust prognostic marker for nasopharyngeal carcinoma (NPC) patients. However, up to 40% of patients who would later develop disease recurrence had undetectable post-treatment plasma EBV DNA. Targeted sequencing for the entire EBV genome potentially allows a more comprehensive and unbiased detection of plasma EBV DNA and enables the use of other parameters such as fragment size as biomarkers. Hence, we explored if plasma EBV DNA sequencing might allow more accurate prognostication of NPC patients. PATIENTS AND METHODS: Plasma samples collected from 769 patients with stage IIB-IVB NPC at 6-8 weeks after radiotherapy were analysed using targeted sequencing for EBV DNA. RESULTS: The sensitivities of the PCR-based analysis, at a cut-off of any detectable levels of plasma EBV DNA, for prediction of local and distant recurrences were 42.3% and 85.3%, respectively. The sequencing-based analysis (involving quantitation and size profiling) achieved better performance for both local and distant recurrences than PCR. Using a cut-off of the proportion of plasma EBV DNA deduced by sequencing at 0.01%, the sensitivities of the sequencing-based analysis for local and distant recurrences were 88.5% and 97.1%, with the resultant negative predictive values of 99.1% and 99.4%, respectively. Among patients with undetectable EBV DNA on quantitative PCR, sequencing could further define a subgroup that enjoyed superior survival outcomes based on the proportion of plasma EBV DNA, with a 5-year progression-free survival (PFS) approaching 90%. On multivariate analysis, sequencing-based quantitative level of plasma EBV DNA was the independent prognostic factor with the highest hazard ratio for prediction of overall survival and PFS. CONCLUSION: NPC prognostication using post-treatment plasma EBV DNA could be enhanced through sequencing.

Observation of High Transformer Ratio of Shaped Bunch Generated by an Emittance-Exchange Beam Line.

Collinear wakefield acceleration has been long established as a method capable of generating ultrahigh acceleration gradients. Because of the success on this front, recently, more efforts have shifted towards developing methods to raise the transformer ratio (TR). This figure of merit is defined as the ratio of the peak acceleration field behind the drive bunch to the peak deceleration field inside the drive bunch. TR is always less than 2 for temporally symmetric drive bunch distributions and therefore recent efforts have focused on generating asymmetric distributions to overcome this limitation. In this Letter, we report on using the emittance-exchange method to generate a shaped drive bunch to experimentally demonstrate a TR≈5 in a dielectric wakefield accelerator.

Precision Control of the Electron Longitudinal Bunch Shape Using an Emittance-Exchange Beam Line.

We report on the experimental generation of relativistic electron bunches with a tunable longitudinal bunch shape. A longitudinal bunch-shaping (LBS) beam line, consisting of a transverse mask followed by a transverse-to-longitudinal emittance exchange (EEX) beam line, is used to tailor the longitudinal bunch shape (or current profile) of the electron bunch. The mask shapes the bunch's horizontal profile, and the EEX beam line converts it to a corresponding longitudinal profile. The Argonne wakefield accelerator rf photoinjector delivers electron bunches into a LBS beam line to generate a variety of longitudinal bunch shapes. The quality of the longitudinal bunch shape is limited by various perturbations in the exchange process. We develop a simple method, based on the incident slope of the bunch, to significantly suppress the perturbations.

Interaction of an Ultrarelativistic Electron Bunch Train with a W-Band Accelerating Structure: High Power and High Gradient.

Electron beam interaction with high frequency structures (beyond microwave regime) has a great impact on future high energy frontier machines. We report on the generation of multimegawatt pulsed rf power at 91 GHz in a planar metallic accelerating structure driven by an ultrarelativistic electron bunch train. This slow-wave wakefield device can also be used for high gradient acceleration of electrons with a stable rf phase and amplitude which are controlled by manipulation of the bunch train. To achieve precise control of the rf pulse properties, a two-beam wakefield interferometry method was developed in which the rf pulse, due to the interference of the wakefields from the two bunches, was measured as a function of bunch separation. Measurements of the energy change of a trailing electron bunch as a function of the bunch separation confirmed the interferometry method.

Development and characterization of Rift Valley fever virus-like particles.

Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV) and spread by arthropod vectors. RVF is currently prevalent in Africa and the Arabian Peninsula, and causes substantial economic losses. Furthermore, this disease poses a serious threat to animal and human health in regions worldwide, making it a serious public health concern. However, RVFV vaccines for human use are still unavailable, and hence there is an urgent need for novel efficient vaccines against RVFV. Vaccine preparation techniques have become a crucial factor in developing new vaccines. In the current study, the N and G protein genes of RVFV were inserted into the pFastBacDual baculovirus expression vector downstream of the pP10 and pPH promoters. The resultant recombinant vector, pFastBacDual-S-M, was transfected into Sf9 insect cells by lipofection. The recombinant baculovirus, named rBac-N-G, was retrieved and infected into Sf9 insect cells to generate RVFV virus-like particles (VLPs). Using polyclonal antibodies against RVFV proteins in immunofluorescence and western blot analyses, we positively identified the presence of the RVFV proteins in VLP preparations. Sucrose density gradient centrifugation and transmission electron microscopy revealed that the morphology of the RVFV VLPs was consistent with previous reports of RVFV virions. This study describes a technique for efficient production of RVFV VLPs, and has laid the foundation for future VLP-based RVFV vaccines.

Experimental demonstration of energy-chirp compensation by a tunable dielectric-based structure.

A tunable energy-chirp compensator was used to remove a correlated energy chirp from the 60-MeV beam at the Brookhaven National Laboratory Accelerator Test Facility. The compensator operates through the interaction of the wakefield of the electron bunch with itself and consists of a planar structure comprised of two alumina bars with copper-plated backs separated by an adjustable beam aperture. By changing the gap size, the correlated energy chirp of the electron bunch was completely removed. Calculations show that this device, properly scaled to account for the electron bunch charge and length, can be used to remove residual correlated energy spread at the end of the linacs used for free-electron lasers. The experimental results are shown to be in good agreement with numerical simulations. Application of this technique can significantly simplify linac design and improve free-electron lasers performance.

Subpicosecond bunch train production for a tunable mJ level THz source.

A strong energy modulation in an electron bunch passing through a dielectric-lined waveguide was recently demonstrated in Antipov et al., Phys. Rev. Lett. 108, 144801 (2012). In this Letter, we demonstrate a successful conversion of this energy modulation into a beam density modulation, and the formation of a series of microbunches with a subpicosecond periodicity by means of magnetic optics (chicane). A strong coherent transition radiation signal produced by the microbunches is obtained and the tunability of its carrier frequency in the 0.68-0.9 THz range by regulating the energy chirp in the incoming electron bunch is demonstrated using infrared interferometry. A tabletop, compact, tunable, and narrowband source of intense THz radiation based on this technology is proposed.

Experimental observation of energy modulation in electron beams passing through terahertz dielectric wakefield structures.

We report the observation of a strong wakefield induced energy modulation in an energy-chirped electron bunch passing through a dielectric-lined waveguide. This modulation can be effectively converted into a spatial modulation forming microbunches with a periodicity of 0.5-1 ps and, hence, capable of driving coherent terahertz radiation. The experimental results agree well with theoretical predictions.

Surface-emission studies in a high-field RF gun based on measurements of field emission and Schottky-enabled photoemission.

We report on investigations into the fundamental surface emission parameters, the geometric field enhancement factor (ß) and the work function (φ), by making both field emission and Schottky-enabled photoemission measurements. The measurements were performed on a copper surface in the Tsinghua University S-band RF gun in two separate experiments. Fitting our data to the models for each experiment indicate that the traditionally assumed high value of ß(≈50-500) does not provide a plausible explanation of the data, but incorporating a low value of φ at some sites does. In addition, direct measurements of the surface conducted after the experiment show that ß is on the order of a few, consistent with our understanding of the electron emission measurements. Thus we conclude that the dominant source of electron emission in high gradient RF cavities is due to low φ sites, as opposed to the conventionally assumed high ß sites. The origin of low φ at these sites is unclear and should be the subject of further investigation.

Emittance-exchange-based high harmonic generation scheme for a short-wavelength free electron laser.

Generation of short-wavelength radiation by a free-electron laser using up-frequency conversion of an electron bunch density modulation is currently an area of active research. We propose a new scheme for producing the longitudinal electron bunch density modulation similar to the recently proposed echo-enabled harmonic generation but based on an emittance exchange beam line and a multislit mask. Beam line analysis and start-to-end simulation are presented.

Experimental demonstration of wakefield acceleration in a tunable dielectric loaded accelerating structure.

We report on a collinear wakefield experiment using the first tunable dielectric loaded accelerating structure. By introducing an extra layer of nonlinear ferroelectric, which has a dielectric constant sensitive to temperature and dc bias, the frequency of a dielectric loaded accelerating structure can be tuned. During the experiment, the energy of a witness bunch at a fixed delay with respect to the drive beam was measured while the temperature of the structure was scanned over a 50 °C range. The energy change corresponded to a change of more than half of the nominal structure wavelength.

Differential localization of GABAA receptor subunits within the substantia nigra of the human brain: an immunohistochemical study.

Gamma-aminobutyric acid(A) (GABA(A)) receptors (GABA(A)R) are inhibitory heteropentameric chloride ion channels comprising a variety of subunits and are localized at postsynaptic sites within the central nervous system. In this study we present the first detailed immunohistochemical investigation on the regional, cellular, and subcellular localisation of alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits of the GABA(A)R in the human substantia nigra (SN). The SN comprises two major regions, the SN pars compacta (SNc) consisting of dopaminergic projection neurons, and the SN pars reticulata (SNr) consisting of GABAergic parvalbumin-positive projection neurons. The results of our single- and double-labeling studies demonstrate that in the SNr GABA(A) receptors contain alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and are localized in a weblike network over the cell soma, dendrites, and spines of SNr parvalbumin-positive nonpigmented neurons. By contrast, GABA(A)Rs on the SNc dopaminergic pigmented neurons contain predominantly alpha(3) and gamma(2) subunits; however there is GABA(A)R heterogeneity in the SNc, with a small subpopulation (6.5%) of pigmented SNc neurons additionally containing alpha(1) and beta(2,3) GABA(A)R subunits. Also, in the SNr, parvalbumin-positive terminals are adjacent to GABA(A)R on the soma and proximal dendrites of SNr neurons, whereas linear arrangements of substance P-positive terminals are adjacent to GABA(A) receptors on all regions of the dendritic tree. These results show marked GABA(A)R subunit hetereogeneity in the SN, suggesting that GABA exerts quite different effects on pars compacta and pars reticulata neurons in the human SN via GABA(A) receptors of different subunit configurations.

Rotenone and elevated extracellular potassium concentration induce cell-specific fibrillation of α-synuclein in axons of cholinergic enteric neurons in the guinea-pig ileum.

BACKGROUND: Parkinson's disease is a progressive neurodegenerative disorder that results in the widespread loss of select classes of neurons throughout the nervous system. The pathological hallmarks of Parkinson's disease are Lewy bodies and neurites, of which α-synuclein fibrils are the major component. α-Synuclein aggregation has been reported in the gut of Parkinson's disease patients, even up to a decade before motor symptoms, and similar observations have been made in animal models of disease. However, unlike the central nervous system, the nature of α-synuclein species that form these aggregates and the classes of neurons affected in the gut are unclear. We have previously reported selective expression of α-synuclein in cholinergic neurons in the gut (J Comp Neurol. 2013; 521:657), suggesting they may be particularly vulnerable to degeneration in Parkinson's disease. METHODS: In this study, we used immunohistochemistry to detect α-synuclein oligomers and fibrils via conformation-specific antibodies after rotenone treatment or prolonged exposure to high [K+ ] in ex vivo segments of guinea-pig ileum maintained in organotypic culture. KEY RESULTS: Rotenone and prolonged raising of [K+ ] caused accumulation of α-synuclein fibrils in the axons of cholinergic enteric neurons. This took place in a time- and, in the case of rotenone, concentration-dependent manner. Rotenone also caused selective necrosis, indicated by increased cellular autofluorescence, of cholinergic enteric neurons, labeled by ChAT-immunoreactivity, also in a concentration-dependent manner. CONCLUSIONS & INFERENCES: To our knowledge, this is the first report of rotenone causing selective loss of a neurochemical class in the enteric nervous system. Cholinergic enteric neurons may be particularly susceptible to Lewy pathology and degeneration in Parkinson's disease.

SUMO-1 marks subdomains within glial cytoplasmic inclusions of multiple system atrophy.

Conjugation of the small ubiquitin-like modifier, SUMO-1, to target proteins is linked to the regulation of multiple cellular pathways, including nucleocytoplasmic trafficking, cell cycle progression, the ubiquitin-proteasome system and apoptosis. Recently, the accumulation of SUMOylated proteins in pathological neuronal intranuclear aggregates has been found in several neurodegenerative diseases. The aim of our study was to examine SUMO-1 in the alpha-synucleinopathy diseases, Multiple System Atrophy (MSA) and Dementia with Lewy Bodies (DLB). We conducted anti-SUMO-1 immunostaining of fixed brain tissue sections and smears of unfixed brain tissue homogenates of DLB and MSA cases. We found that oligodendroglial cytoplasmic inclusions, the alpha-synuclein-positive cytoplasmic aggregates that characterize MSA, exhibit robust punctate SUMO-1 immunostaining, marking discrete submicron-sized subdomains within the inclusion bodies. Lewy bodies in smears of DLB tissue homogenates showed similar SUMO-1-positive structures, although these were not detected in fixed tissue. In cell culture experiments, we found that the nuclear and perinuclear accumulation of SUMO-1 aggregates could be induced in glioma cells by chemical inhibition of proteasomal protein degradation.

Alpha B-crystallin is a major component of glial cytoplasmic inclusions in multiple system atrophy.

Multiple system atrophy (MSA) is characterized by the formation of oligodendroglial cytoplasmic inclusions (GCIs) consisting of alpha-synuclein filaments. AlphaB-crystallin, a small chaperone protein that binds to unfolded proteins and inhibits aggregation, has been documented in GCIs. We investigated the relative abundance and speciation of alphaB-crystallin in GCIs in MSA brains. We also examined the influence of alphaB-crystallin on the formation of cytoplasmic inclusions in cultured glial cells. Immunohistochemistry and confocal microscopy revealed alphaB-crystallin is a prominent component of GCIs, more abundant than in Lewy bodies in Lewy body dementia. One- and two-dimensional gel electrophoresis and mass spectrometric analysis of GCIs immunopurified from MSA brains indicated that alphaB-crystallin is a major protein component with multiple post-translationally modified species. In cultured C6 glioma cells treated with the proteasomal inhibitor, lactacystin, to induce accumulation of ubiquitinated proteins, a subset of cells showed increased cytoplasmic staining for alphaB-crystallin. Proteasome-inhibited cells transfected with GFP-tagged alpha-synuclein resulted in ubiquitin- and alphaB-crystallin-positive aggregates resembling GCIs in MSA brains. Our results indicate that alphaB-crystallin is a major chaperone in MSA, and suggest a role of the protein in the formation of inclusion bodies in glial cells.

Proteasomal activation mediates down-regulation of inositol 1,4,5-trisphosphate receptor and calcium mobilization in rat pancreatic islets.

Inositol 1,4,5-trisphosphate receptor (IP3R) protein levels in isolated rat pancreatic islets were investigated in response to carbachol (CCh) and sulfated cholecystokinin 26-33 amide stimulation. Within 2 h, CCh reduced IP3R-I protein levels by 22% and IP3R-II and -III levels to 65% or more below basal. Sulfated cholecystokinin 26-33 amide decreased the levels of IP3R-I, -II, and -III by 34%, 60%, and 66% below basal, respectively. The effect of CCh was concentration- and time-dependent, with a persistent decline in IP3R levels for up to 6 h after the onset of stimulation. CCh-pretreated islets also showed an inhibition of glucose-stimulated insulin secretion. Proteasome inhibition completely blocked the down-regulatory effects of CCh on IP3Rs and significantly increased the insulin secretory response to glucose stimulation in the presence of CCH: Islet stimulation by glucose, alpha-ketoisocaproic acid, and tolbutamide completely protected IP3Rs against the down-regulatory effects of CCH: 2-deoxyglucose and 3-O-methyl glucose failed to affect CCh-induced IP3R down-regulation. The protective effects of glucose on IP3R down-regulation were completely inhibited by the Ca(2+) channel-blocking agent nimodipine. Intracellular Ca(2+) ([Ca(2+)](i)) levels in Fura-2 (fluorescent Ca(2+) indicator)-loaded islets, in the absence of extracellular Ca(2+), increased in response to glucose stimulation; but in islets pretreated with CCh, glucose did not increase [Ca(2+)](i) above basal levels. However, in islets pretreated with CCh and the proteasomal inhibitor MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinyl-H), the glucose-stimulated increase in [Ca(2+)](i) was significantly higher than the change observed for glucose-stimulated [Ca(2+)](i) in the absence of MG-132. The results suggest that muscarinic receptor stimulation modulates IP3R protein levels in islets through a proteasomal activation pathway, and that down-regulation of IP3Rs has a profound effect on Ca(2+) mobilization in islets that may relate to insulin secretory responsiveness.

Galanin immunoreactive neurons in the human hypothalamus: colocalization with vasopressin-containing neurons.

Galanin (GA) is a recently described neuropeptide that has been demonstrated to be widely distributed in the hypothalamus of experimental animals. So far there is no immunohistochemical description of GA in the human hypothalamus and, in particular, no studies of the colocalization of this neuropeptide with other transmitter candidates in the human hypothalamus. We have now investigated this question immunohistochemically by using human brains fixed by vascular perfusion within 24 hours of death. Nerve cell bodies and fibers stained for GA were observed throughout the hypothalamus. Major populations of GA-ir cell bodies were found in the suprachiasmatic, intermediate, supraoptic, paraventricular, arcuate, tuberomammillary, and supramammillary nuclei. Scattered positive neurons were found in the periventricular preoptic area, the posterior hypothalamic nucleus, the lateral hypothalamic area, and zona incerta. A few positive cells were located in the dorsomedial and ventromedial hypothalamic nuclei. The number of GA-ir neurons estimated from three brains was 11,100 +/- 2,400 for the intermediate nucleus, 57,800 +/- 9,100 for the supraoptic nucleus and 47,400 +/- 13,900 for the paraventricular nucleus. GA-ir fibers were widely distributed in the hypothalamus. They were more dense in the periventricular and medial hypothalamic zones, whereas the lateral tuberal nuclei and the dorsolateral part of the supraoptic nucleus contained sparse positive fibers. The mammillary complex contained almost no GA-ir fibers. In the ventromedial tuberal region, GA-ir axons formed bundles travelling down in the infundibular stem. In the median eminence the vascular plexus was wrapped by GA-ir fiber networks. The coexistence of GA with arginine vasopressin (AVP), oxytocin (OXY), and tyrosine hydroxylase (TH) was examined in the supraoptic, paraventricular, and suprachiasmatic nuclei in adjacent paraffin sections. Neurons containing both GA and AVP were very common in the supraoptic nucleus and also occurred in the paraventricular and suprachiasmatic nuclei. The supraoptic and paraventricular nuclei also contained some neurons immunoreactive for both GA and OXY. Neurons positive for GA and TH were rare. The topographic distribution of GA-ir neuronal structures in the hypothalamus and the colocalization of GA, principally with AVP and to a lesser extent with OXY, in some hypothalamic nuclei constitute anatomical evidence that this neuropeptide may be involved in the regulation of endocrine, autonomic, and behavioural homeostatic responses.

Nitric oxide-synthesising neurons in the central subnucleus of the nucleus tractus solitarius provide a major innervation of the rostral nucleus ambiguus in the rabbit.

We describe an intramedullary nitric oxide synthase (NOS) neural pathway that projects from the nucleus tractus solitarius (NTS) to the rostral nucleus ambiguus (NA) in the rabbit. With the use of NADPH diaphorase histochemistry and NOS immunohistochemistry, a compact group of NOS-positive perikarya was identified in the central subnucleus of the NTS dorsomedial to the tractus solitarius and rostral to the obex. A dense network of NOS terminals was seen in the rostral NA. We investigated whether NOS terminals in the NA derive from NOS perikarya in the central NTS and whether the central NOS pathway links esophageal afferents and efferents. In some rabbits, the central NTS was unilaterally lesioned. In others, Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected into the central NTS, or cholera toxin-gold was injected into the NA, or cholera toxin-horseradish peroxidase (HRP) was injected into the wall of the esophagus. The medulla was subsequently processed to demonstrate PHA-L, cholera toxin-gold, HRP, and NOS reactivity. Seven days after the NTS lesion, we observed a marked decrease in the density of NOS terminals in the ipsilateral NA. After injection of PHA-L into the central NTS, a dense group of PHA-L fibres was seen in the rostral NA, principally ipsilaterally. Afferent fibres from the esophagus were found around the NOS cell bodies in the central NTS, and many of these NOS neurons were double labeled with cholera toxin-gold after injection of this tracer into the NA. NOS terminals were found around NA neurons that were retrogradely labelled from the esophagus. We conclude that the NOS neurons in the central NTS act as interneurons in a central pathway connecting esophageal afferents and efferents.

Age-related loss of dorsal vagal neurons in Parkinson's disease.

Older patients who die with Parkinson's disease (PD) have fewer pigmented neurons in the locus coeruleus and fewer substance P-containing neurons in mesopontine tegmental nuclei. We analyzed two other medullary nuclei, the dorsal vagal nucleus and the hypoglossal nucleus, in eight PD patients and six normal controls by counting neurons in serial Nissl stained sections to determine the relationship between age at death and cell loss in these nuclei. PD-related neurodegenerative changes (Lewy bodies and neuronal loss) were present only in the dorsal vagal nucleus (13,637 +/- 1,323 neurons in PD, 24,885 +/- 1,157 in normal controls). Cells in the intermediate rostrocaudal part of the nucleus were most severely affected. There was a significant correlation between loss of vagal neurons and age at death in PD patients. No age-related cell loss was present in the dorsal vagal nucleus of normal brains, or in the hypoglossal nucleus in either PD or normal brains. These results confirm that age-related cell death depends on whether or not there is coexistent PD.