MRC2020: improvements to Ximdisp and the MRC image-processing programs.

The great success of single-particle electron cryo-microscopy (cryoEM) during the last decade has involved the development of powerful new computer programs and packages that guide the user along a recommended processing workflow, in which the wisdom and choices made by the developers help everyone, especially new users, to obtain excellent results. The ability to carry out novel, non-standard or unusual combinations of image-processing steps is sometimes compromised by the convenience of a standard procedure. Some of the older programs were written with great flexibility and are still very valuable. Among these, the original MRC image-processing programs for structure determination by 2D crystal and helical processing alongside general-purpose utility programs such as Ximdisp, label, imedit and twofile are still available. This work describes an updated version of the MRC software package (MRC2020) that is freely available from CCP-EM. It includes new features and improvements such as extensions to the MRC format that retain the versatility of the package and make it particularly useful for testing novel computational procedures in cryoEM.

Profiling the expression of fatty acid-binding proteins and fatty acid transporters in mouse microglia and assessing their role in docosahexaenoic acid-d5 uptake.

While the processes governing docosahexaenoic acid (DHA) trafficking across the blood-brain barrier have been elucidated, factors governing DHA uptake into microglia, an essential step for this fatty acid to exert its anti-inflammatory effects, are unknown. This study assessed the mRNA and protein expression of fatty acid-binding proteins (FABPs) and fatty acid transport proteins (FATPs) in mouse BV-2 cells and their mRNA expression in primary mouse microglia. The microglial uptake of DHA-d5, a surrogate of DHA, was assessed by LC-MS/MS following interventions including temperature reduction, silencing of various FABP isoforms, competition with DHA, and metabolic inhibition. It was found that DHA-d5 uptake at 4°C was 39.6% lower than at 37°C, suggesting that microglial uptake of DHA-d5 likely involves passive and/or active uptake mechanisms. Of all FABP and FATP isoforms probed, only FABP3, FABP4, FABP5, FATP1, and FATP4 were expressed at both the mRNA and protein level. Silencing of FABP3, FABP4, and FABP5 resulted in no change in cellular DHA-d5 uptake, nor did concomitant DHA administration or the presence of 0.1% sodium azide/50 mM 2-deoxy-D-glucose. This study is the first to identify the presence of FABPs and FATPs in mouse microglia, albeit these proteins are not involved in the microglial uptake of DHA-d5.

Secondary non-invasive prenatal screening for fetal trisomy: an effectiveness study in a public health setting.

OBJECTIVE: To evaluate the effectiveness of secondary screening using non-invasive prenatal testing (NIPT) in a routine NHS setting including test performance, turn-around times (TATs) and no-call (failure to obtain result) rates. To examine the influence of maternal and fetal characteristics on test performance. DESIGN: Retrospective cohort. SETTING: London teaching hospital. SAMPLE: A total of 8651 pregnancies undergoing screening for fetal trisomy using NIPT provided by an NHS cell-free DNA screening laboratory - the SAFE laboratory. METHODS: Screening test evaluation and TATs. Univariate and multivariate logistic regression analysis to identify significant predictors of no-call results and reported by low fetal fraction (<2%), very high fetal fraction (>40%) and processing failure. MAIN OUTCOME MEASURES: Test performance, TATs and no-call rates, factors affecting no-call results. RESULTS: Average TAT was 4.0 days (95% CI 4.0-4.2 days). Test sensitivities for trisomies 21 and 13/18 were 98.9% (95% CI 95.9-99.9%) and 90.4% (95% CI 80.0-96.8%), respectively. The overall no-call rate was 32/8651 (0.37%, 95% CI 0.26-0.52%). The overall risk of a no-call result was influenced by gestational age, dichorionic twin pregnancy, history of malignancy and pregnancies affected by trisomy 13/18, but not by maternal weight or use of low-molecular-weight heparin. CONCLUSIONS: High-throughput NIPT can be effectively embedded into a public health NHS setting. TATs of 4 days and no-calls of <0.5% were well within clinically desirable tolerances. Gestational age, maternal weight, assisted reproductive techniques, use of low-molecular-weight heparin and past history of malignancy did not have major impacts on test no-call rates and should not constitute reasons for withholding the option of NIPT from women. TWEETABLE ABSTRACT: Turn-around times of 4 days, no-call (test failure) rates of 0.37% and highly accurate NIPT can be successfully embedded in the NHS.

Modeling salinization and recovery of road salt-impacted lakes in temperate regions based on long-term monitoring of Lake George, New York (USA) and its drainage basin.

Road salt mitigates winter highway icing but accumulates in watershed soils and receiving waters, affecting soil chemistry and physical, biological, and ecological processes. Despite efforts to reduce salt loading in watersheds, accumulated cations and Cl- continue to impact tributaries and lakes, and the recovery process is not well understood. Lake George, New York (USA) is typical of many temperate lakes at risk for elevated Cl- concentrations from winter deicing; the lake salt concentration increased by ~3.4% year-1 since 1980. Here, we evaluated the ionic composition in Finkle Brook, a major watershed draining to Lake George, studied intermittently since 1970 and typical of other salt-impacted Lake George tributaries. Salt loading in the Lake George basin since the 1940s displaced cations from exchange sites in basin soils; these desorbed cations follow a simple ion-exchange model, with lower sodium and higher calcium, magnesium and potassium fluxes in runoff. Reduced salt application in the Finkle Brook watershed during the low-snow winter of 2015-2016 led to a 30-40% decline of Cl- and base cations in the tributary, implying a Cl- soil half-life of 1-2 years. We developed a conceptual model that describes cation behavior in runoff from a watershed that received road salt loading over a long period of time, and then recovery following reduced salt loading. Next, we developed a dynamic model estimating time to steady-state for Cl- in Lake George with road salt loading starting in 1940, calibrating the model with tributary runoff and lake chemistry data from 1970 and 1980, respectively, and forecasting Cl- concentrations in Lake George based on various scenarios of salt loading and soil retention of Cl-. Our Lake George models are readily adaptable to other temperate lakes with drainage basins where road salt is applied during freezing conditions and paved roads cover a portion of the watershed.

Cerebral [18 F]T807/AV1451 retention pattern in clinically probable CTE resembles pathognomonic distribution of CTE tauopathy.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disorder most commonly associated with repetitive traumatic brain injury (TBI) and characterized by the presence of neurofibrillary tangles of tau protein, known as a tauopathy. Currently, the diagnosis of CTE can only be definitively established postmortem. However, a new positron emission tomography (PET) ligand, [18F]T807/AV1451, may provide the antemortem detection of tau aggregates, and thus various tauopathies, including CTE. Our goal was to examine [18F]T807/AV1451 retention in athletes with neuropsychiatric symptoms associated with a history of multiple concussions. Here we report a 39-year-old retired National Football League player who suffered 22 concussions and manifested progressive neuropsychiatric symptoms. Emotional lability and irritability were the chief complaints. Serial neuropsychological exams revealed a decline in executive functioning, processing speed and fine motor skills. Naming was below average but other cognitive functions were preserved. Structural analysis of longitudinally acquired magenetic resonance imaging scans revealed cortical thinning in the left frontal and lateral temporal areas, as well as volume loss in the basal ganglia. PET with [18F]florbetapir was negative for amyloidosis. The [18F]T807/AV1451 PET showed multifocal areas of retention at the cortical gray matter-white matter junction, a distribution considered pathognomonic for CTE. [18F]T807/AV1451 standard uptake value (SUV) analysis showed increased uptake (SUVr⩾1.1) in bilateral cingulate, occipital, and orbitofrontal cortices, and several temporal areas. Although definitive identification of the neuropathological underpinnings basis for [18F]T807/AV1451 retention requires postmortem correlation, our data suggest that [18F]T807/AV1451 tauopathy imaging may be a promising tool to detect and diagnose CTE-related tauopathy in living subjects.

Adverse events associated with chloramphenicol use in dogs: a retrospective study (2007-2013).

Chloramphenicol is a broad spectrum antibiotic that has been increasingly utilised since the emergence of methicillin-resistant staphylococcal infections. Due to toxicities in humans, use of the drug has been limited. In dogs, gastrointestinal signs are common adverse events described, and bone marrow suppression is possible. The aim of this study was to evaluate the adverse events associated with chloramphenicol in dogs seen by one specialty practice from January 2007 through June 2013. The database was searched for all dogs prescribed chloramphenicol during the time period. Dosage, length of treatment, age and body weight of the dogs were recorded as well as any adverse events that occurred during treatment. A total of 105 cases were evaluated. Thirty-nine dogs experienced at least one adverse event while on the medication. The most commonly noted were gastrointestinal signs and hindlimb weakness. The mean body weight for dogs with hindlimb weakness was 35.3 kg, which was significant. Resolution was documented in 54 per cent of cases when the drug was discontinued. Methicillin-resistant Staphylococcus pseudintermedius on bacterial culture was listed as the reason for chloramphenicol use in 76 per cent of the cases. Based on this information, further prospective studies are recommended to evaluate the reproducibility of this report.

Deep sequencing analysis of defective genomes of parainfluenza virus 5 and their role in interferon induction.

Preparations of parainfluenza virus 5 (PIV5) that are potent activators of the interferon (IFN) induction cascade were generated by high-multiplicity passage in order to accumulate defective interfering virus genomes (DIs). Nucleocapsid RNA from these virus preparations was extracted and subjected to deep sequencing. Sequencing data were analyzed using methods designed to detect internal deletion and "copyback" DIs in order to identify and characterize the different DIs present and to approximately quantify the ratio of defective to nondefective genomes. Trailer copybacks dominated the DI populations in IFN-inducing preparations of both the PIV5 wild type (wt) and PIV5-VΔC (a recombinant virus that does not encode a functional V protein). Although the PIV5 V protein is an efficient inhibitor of the IFN induction cascade, we show that nondefective PIV5 wt is unable to prevent activation of the IFN response by coinfecting copyback DIs due to the interfering effects of copyback DIs on nondefective virus protein expression. As a result, copyback DIs are able to very rapidly activate the IFN induction cascade prior to the expression of detectable levels of V protein by coinfecting nondefective virus.

Heterocellular induction of interferon by negative-sense RNA viruses.

The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN). To counter this, most, if not all, RNA viruses encode antagonists of the IFN system. Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus. We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells.

Practical methods for the measurement of logP for surfactants.

Hydrophobicity is a commonly used parameter in quantitative structure activity relationships. The ability to determine the octanol-water partition coefficient (logP) empirically for non-ionizing, non-surfactant type chemicals using traditional stir-flask methods has been successful and well documented. In comparison the ability to measure logP for surfactants is considered impractical due to their amphiphilic nature, which gives them a tendency to form micelles and reside at the octanol-water interface. In this study we have shown that working with compounds below their critical micelle concentrations (CMC), at the experimental concentrations, it is possible to obtain experimental logP values for a series of sulphobetaines using the stir-flask method coupled with reversed phase-high performance liquid chromatography (RP-HPLC). Until now the ability to verify calculated logP values for surfactants has been limited. Measuring logP as described here can now be applied to other surfactants to validate existing and new modifications to the fragment method.

Cue-conditioned alcohol seeking in rats following abstinence: involvement of metabotropic glutamate 5 receptors.

BACKGROUND AND PURPOSE: The current study was designed to: (i) examine whether functional interactions occur between receptors known to regulate alcohol self-administration; and (ii) characterize relapse to alcohol seeking following abstinence. EXPERIMENTAL APPROACH: The selective cannabinoid CB(1) receptor antagonist SR141716A (0.03-1.0 mg.kg(-1) i.p.) resulted in a dose-dependent reduction in ethanol self-administration in ethanol-preferring Indiana-preferring rats. SR141716A was then co-administered with either the selective glutamate metabotropic glutamate 5 (mGlu(5)) receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) or the selective adenosine A(2A) receptor antagonist SCH58261. KEY RESULTS: When administered at individually sub-threshold doses, a combination of SR141716A (0.1 mg.kg(-1)) and SCH58261 (0.5 mg.kg(-1) i.p.) produced a reduction (28%) in ethanol self-administration. Combinations of threshold doses of SR141716A (0.3 mg.kg(-1)) and SCH58261 (2.0 mg.kg(-1), i.p.) caused an essentially additive reduction (68%) in alcohol self-administration. A combination of individually sub-threshold doses of CB(1) and mGlu(5) receptor antagonists did not affect alcohol self-administration; however, combined threshold doses of SR141716A (0.3 mg.kg(-1)) and MTEP (1.0 mg.kg(-1) i.p.) did reduce ethanol self-administration markedly (80%). Cue-conditioned alcohol seeking was attenuated by pretreatment with MTEP (1.0 mg.kg(-1)) co-administered with SR141716A (0.3 mg.kg(-1) i.p.). In contrast, SCH58261 (2.0 mg.kg(-1)) co-administered with SR141716A (0.3 mg.kg(-1) i.p.) did not reduce cue-conditioned alcohol seeking. CONCLUSIONS AND IMPLICATIONS: Adenosine A(2A) and cannabinoid CB(1) receptors regulated alcohol self-administration additively, but combined low-dose antagonism of these receptors did not prevent cue-conditioned alcohol seeking after abstinence. In contrast, combined low-dose antagonism of mGlu(5) and CB(1) receptors did prevent relapse-like alcohol seeking after abstinence, suggesting a prominent role for mGlu(5) receptors in this paradigm.

A case study of delayed HIV-1 seroconversion highlights the need for Combo assays.

This report describes a case in which a patient took at least four months to seroconvert to anti-HIV positivity. A concomitant CMV infection probably contributed to the profound immune suppression observed. It is essential that fourth generation HIV antigen/antibody combo assays be used to ensure that such cases are not missed.

The representation of anaesthesia in children's literature.

Hospital admission for an operation can be a frightening and bewildering experience for a child. Effective communication is a prerequisite for all those who anaesthetise children, and good pre-operative preparation reduces anxiety and improves the child's ability to cope. Books are familiar to children and their use in providing information is an established practice in paediatric nursing. By searching web-based bookshops, we identified 19 books whose subject was admission to hospital for an operation. These books were analysed according to the accuracy of their description of the anaesthetic element of the surgical experience. Seven of these books can be recommended as useful descriptions of the key elements involved in general anaesthesia. These books provide a simple, effective and cheap resource to help children and their parents prepare for anaesthesia. They may also provide a useful tool for trainee anaesthetists by giving an insight into the child's level of understanding.

Reversible left hemispheric ischemia secondary to carotid compression in Eagle syndrome: surgical and CT angiographic correlation.

Symptoms of ipsilateral carotid artery compression secondary to an elongated styloid process or calcified stylohyoid ligament may be seen in Eagle syndrome. The patient will typically experience cervicofacial pain due to stimulation of the arterial nervous plexus. In addition, symptoms directly attributable to compression of the carotid artery may be seen, including visual symptoms and syncope. We report here the case of a patient who developed symptoms consistent with left hemispheric ischemia within 15 seconds of turning his head to the left. These symptoms were completely reversible on returning the head to the neutral position. No long-term sequelae were detected clinically or radiographically.

Metachronous, multicentric giant cell tumor of the sphenoid bone with histologic, CT, MR imaging, and positron-emission tomography/CT correlation.

Giant cell tumor (GCT) of the sphenoid bone is a relatively rare entity and metachronous multicentric GCT of the sphenoid is even rarer; we are aware of only 3 previous cases in the literature. We describe here a tumor of the sphenoid bone that was identified 15 years after multiple resections of a GCT of the left inferior pubic ramus. Correlation is made between the histopathologic findings, MR imaging of the brain, CT of the head, and fusion positron-emission tomography (PET)/CT scan performed with fluorine-18 fluoro-2-deoxy-D-glucose (18F-FDG). This report is the first to describe the appearance of a GCT of the sphenoid bone on a fusion PET/CT examination. High metabolic activity in the base of the skull adjacent to the middle cranial fossa was demonstrated in a fashion similar to that of the known pelvic lesion. This case also demonstrates that the increased metabolic activity seen in a GCT of the sphenoid bone may be partially obscured by the adjacent physiologic high metabolic activity of the brain.

Inflammatory myofibroblastic tumor of the orbit with associated enhancement of the meninges and multiple cranial nerves.

Inflammatory myofibroblastic tumor (IMT), Tolosa-Hunt syndrome (THS), and idiopathic hypertrophic pachymeningitis (IHP) seem to be part of a spectrum of disorders that have diverse locations but similar histologic and imaging findings. We report a case of a 50-year-old man presenting with multiple progressive cranial nerves palsies with leptomeningeal cranial nerve enhancement on MRI (II, V1-V3, and X), orbital and infraorbital masses, prominence within the left cavernous sinus, and diffuse dural enhancement. Biopsies of the orbital lesion and infraorbital nerve revealed IMT. The patient's lesions, symptoms, and dural enhancement quickly improved with steroid administration and nearly resolved over multiple subsequent scans over the next few months. This case illustrates a rare case of pseudotumor mimicking a more aggressive appearance that would usually portend a case of malignancy. There is a potential association of IMT, THS, and IHP, which may have existed in a concomitant fashion in this patient. The case also describes the unique finding of enhancement of the cisternal segments of multiple cranial nerves (simulating leptomeningeal malignant involvement), which may be related to inflammatory perineural edema or ischemic neuropathy.

Reversible findings of restricted diffusion in 5-fluorouracil neurotoxicity.

A 35-year-old woman presented with neurotoxicity correlated to an i.v. regimen of 5-fluorouracil as episodes of acute confusional state and abnormalities of symmetrically restricted diffusion in the periventricular white matter and corpus callosum. On discontinuing the medication, the areas of severely restricted diffusion had entirely resolved, with minimal residual T2 signal abnormality. In this case, immediate discontinuation of the chemotherapeutic agent apparently reversed the patient's symptoms and findings on MRI. The scant information available in the published literature regarding this phenomenon is reviewed with regard to 5-fluorouracil.

Genetic interdependence of adenosine and dopamine receptors: evidence from receptor knockout mice.

Dopamine and adenosine receptors are known to share a considerable overlap in their regional distribution, being especially rich in the basal ganglia. Dopamine and adenosine receptors have been demonstrated to exhibit a parallel distribution on certain neuronal populations, and even when not directly co-localized, relationships (both antagonistic and synergistic) have been described. This study was designed to investigate dopaminergic and purinergic systems in mice with ablations of individual dopamine or adenosine receptors. In situ hybridization histochemistry and autoradiography was used to examine the level of mRNA and protein expression of specific receptors and transporters in dopaminergic pathways. Expression of the mRNA encoding the dopamine D2 receptor was elevated in the caudate putamen of D1, D3 and A2A receptor knockout mice; this was mirrored by an increase in D2 receptor protein in D1 and D3 receptor knockout mice, but not in A2A knockout mice. Dopamine D1 receptor binding was decreased in the caudate putamen, nucleus accumbens, olfactory tubercle and ventral pallidum of D2 receptor knockout mice. In substantia nigra pars compacta, dopamine transporter mRNA expression was dramatically decreased in D3 receptor knockout mice, but elevated in A2A receptor knockout mice. All dopamine receptor knockout mice examined exhibited increased A2A receptor binding in the caudate putamen, nucleus accumbens and olfactory tubercle. These data are consistent with the existence of functional interactions between dopaminergic and purinergic systems in these reward and motor-related brain regions.

The fragile X tremor ataxia syndrome in the differential diagnosis of multiple system atrophy: data from the EMSA Study Group.

The recent identification of fragile X-associated tremor ataxia syndrome (FXTAS) associated with premutations in the FMR1 gene and the possibility of clinical overlap with multiple system atrophy (MSA) has raised important questions, such as whether genetic testing for FXTAS should be performed routinely in MSA and whether positive cases might affect the specificity of current MSA diagnostic criteria. We genotyped 507 patients with clinically diagnosed or pathologically proven MSA for FMR1 repeat length. Among the 426 clinically diagnosed cases, we identified four patients carrying FMR1 premutations (0.94%). Within the subgroup of patients with probable MSA-C, three of 76 patients (3.95%) carried premutations. We identified no premutation carriers among 81 patients with pathologically proven MSA and only one carrier among 622 controls (0.16%). Our results suggest that, with proper application of current diagnostic criteria, FXTAS is very unlikely to be confused with MSA. However, slowly progressive disease or predominant tremor are useful red flags and should prompt the consideration of FXTAS. On the basis of our data, the EMSA Study Group does not recommend routine FMR1 genotyping in typical MSA patients.