Viral co-infection, autoimmunity, and CSF HIV antibody profiles in HIV central nervous system escape.

Antiretroviral therapy (ART) suppresses plasma and cerebrospinal fluid (CSF) HIV replication. Neurosymptomatic (NS) CSF escape is a rare exception in which CNS HIV replication occurs in the setting of neurologic impairment. The origins of NS escape are not fully understood. We performed a case-control study of asymptomatic (AS) escape and NS escape subjects with HIV-negative subjects as controls in which we investigated differential immunoreactivity to self-antigens in the CSF of NS escape by employing neuroanatomic CSF immunostaining and massively multiplexed self-antigen serology (PhIP-Seq). Additionally, we utilized pan-viral serology (VirScan) to deeply profile the CSF anti-viral antibody response and metagenomic next-generation sequencing (mNGS) for pathogen detection. We detected Epstein-Barr virus (EBV) DNA more frequently in the CSF of NS escape subjects than in AS escape subjects. Based on immunostaining and PhIP-Seq, there was evidence for increased immunoreactivity against self-antigens in NS escape CSF. Finally, VirScan revealed several immunodominant epitopes that map to the HIV envelope and gag proteins in the CSF of AS and NS escape subjects. Whether these additional inflammatory markers are byproducts of an HIV-driven process or whether they independently contribute to the neuropathogenesis of NS escape will require further study.

Task-evoked pupillary responses track precision-weighted prediction errors and learning rate during interceptive visuomotor actions.

In this study, we examined the relationship between physiological encoding of surprise and the learning of anticipatory eye movements. Active inference portrays perception and action as interconnected inference processes, driven by the imperative to minimise the surprise of sensory observations. To examine this characterisation of oculomotor learning during a hand-eye coordination task, we tested whether anticipatory eye movements were updated in accordance with Bayesian principles and whether trial-by-trial learning rates tracked pupil dilation as a marker of 'surprise'. Forty-four participants completed an interception task in immersive virtual reality that required them to hit bouncing balls that had either expected or unexpected bounce profiles. We recorded anticipatory eye movements known to index participants' beliefs about likely ball bounce trajectories. By fitting a hierarchical Bayesian inference model to the trial-wise trajectories of these predictive eye movements, we were able to estimate each individual's expectations about bounce trajectories, rates of belief updating, and precision-weighted prediction errors. We found that the task-evoked pupil response tracked prediction errors and learning rates but not beliefs about ball bounciness or environmental volatility. These findings are partially consistent with active inference accounts and shed light on how encoding of surprise may shape the control of action.

Integrating central nervous system metagenomics and host response for diagnosis of tuberculosis meningitis and its mimics.

The epidemiology of infectious causes of meningitis in sub-Saharan Africa is not well understood, and a common cause of meningitis in this region, Mycobacterium tuberculosis (TB), is notoriously hard to diagnose. Here we show that integrating cerebrospinal fluid (CSF) metagenomic next-generation sequencing (mNGS) with a host gene expression-based machine learning classifier (MLC) enhances diagnostic accuracy for TB meningitis (TBM) and its mimics. 368 HIV-infected Ugandan adults with subacute meningitis were prospectively enrolled. Total RNA and DNA CSF mNGS libraries were sequenced to identify meningitis pathogens. In parallel, a CSF host transcriptomic MLC to distinguish between TBM and other infections was trained and then evaluated in a blinded fashion on an independent dataset. mNGS identifies an array of infectious TBM mimics (and co-infections), including emerging, treatable, and vaccine-preventable pathogens including Wesselsbron virus, Toxoplasma gondii, Streptococcus pneumoniae, Nocardia brasiliensis, measles virus and cytomegalovirus. By leveraging the specificity of mNGS and the sensitivity of an MLC created from CSF host transcriptomes, the combined assay has high sensitivity (88.9%) and specificity (86.7%) for the detection of TBM and its many mimics. Furthermore, we achieve comparable combined assay performance at sequencing depths more amenable to performing diagnostic mNGS in low resource settings.

A Rare Bird: Diagnosis of Psittacosis Meningitis by Clinical Metagenomic Next-Generation Sequencing.

Psittacosis is a zoonotic infectious disease caused by the transmission of Chlamydia psittaci; it often presents as a pulmonary infection but rarely as disseminated disease. Because diagnoses of psittacosis are often underreported due to infrequent pathogen-specific testing, clinical metagenomic next-generation sequencing may be helpful to diagnose such an uncommon syndrome.

Neurite Orientation Dispersion and Density Imaging for Assessing Acute Inflammation and Lesion Evolution in MS.

BACKGROUND AND PURPOSE: MR imaging is essential for MS diagnosis and management, yet it has limitations in assessing axonal damage and remyelination. Gadolinium-based contrast agents add value by pinpointing acute inflammation and blood-brain barrier leakage, but with drawbacks in safety and cost. Neurite orientation dispersion and density imaging (NODDI) assesses microstructural features of neurites contributing to diffusion imaging signals. This approach may resolve the components of MS pathology, overcoming conventional MR imaging limitations. MATERIALS AND METHODS: Twenty-one subjects with MS underwent serial enhanced MRIs (12.6 ± 9 months apart) including NODDI, whose key metrics are the neurite density and orientation dispersion index. Twenty-one age- and sex-matched healthy controls underwent unenhanced MR imaging with the same protocol. Fifty-eight gadolinium-enhancing and non-gadolinium-enhancing lesions were semiautomatically segmented at baseline and follow-up. Normal-appearing WM masks were generated by subtracting lesions and dirty-appearing WM from the whole WM. RESULTS: The orientation dispersion index was higher in gadolinium-enhancing compared with non-gadolinium-enhancing lesions; logistic regression indicated discrimination, with an area under the curve of 0.73. At follow-up, in the 58 previously enhancing lesions, we identified 2 subgroups based on the neurite density index change across time: Type 1 lesions showed increased neurite density values, whereas type 2 lesions showed decreased values. Type 1 lesions showed greater reduction in size with time compared with type 2 lesions. CONCLUSIONS: NODDI is a promising tool with the potential to detect acute MS inflammation. The observed heterogeneity among lesions may correspond to gradients in severity and clinical recovery after the acute phase.

Visual attention, EEG alpha power and T7-Fz connectivity are implicated in prosthetic hand control and can be optimized through gaze training.

BACKGROUND: Prosthetic hands impose a high cognitive burden on the user that often results in fatigue, frustration and prosthesis rejection. However, efforts to directly measure this burden are sparse and little is known about the mechanisms behind it. There is also a lack of evidence-based training interventions designed to improve prosthesis hand control and reduce the mental effort required to use them. In two experiments, we provide the first direct evaluation of this cognitive burden using measurements of EEG and eye-tracking (Experiment 1), and then explore how a novel visuomotor intervention (gaze training; GT) might alleviate it (Experiment 2). METHODS: In Experiment 1, able-bodied participants (n = 20) lifted and moved a jar, first using their anatomical hand and then using a myoelectric prosthetic hand simulator. In experiment 2, a GT group (n = 12) and a movement training (MT) group (n = 12) trained with the prosthetic hand simulator over three one hour sessions in a picking up coins task, before returning for retention, delayed retention and transfer tests. The GT group received instruction regarding how to use their eyes effectively, while the MT group received movement-related instruction typical in rehabilitation. RESULTS: Experiment 1 revealed that when using the prosthetic hand, participants performed worse, exhibited spatial and temporal disruptions to visual attention, and exhibited a global decrease in EEG alpha power (8-12 Hz), suggesting increased cognitive effort. Experiment 2 showed that GT was the more effective method for expediting prosthesis learning, optimising visual attention, and lowering conscious control - as indexed by reduced T7-Fz connectivity. Whilst the MT group improved performance, they did not reduce hand-focused visual attention and showed increased conscious movement control. The superior benefits of GT transferred to a more complex tea-making task. CONCLUSIONS: These experiments quantify the visual and cortical mechanisms relating to the cognitive burden experienced during prosthetic hand control. They also evidence the efficacy of a GT intervention that alleviated this burden and promoted better learning and transfer, compared to typical rehabilitation instructions. These findings have theoretical and practical implications for prosthesis rehabilitation, the development of emerging prosthesis technologies and for the general understanding of human-tool interactions.

Action observation for sensorimotor learning in surgery.

BACKGROUND: Acquiring new motor skills to learn complex movements and master the use of a diverse range of instruments is fundamental for developing expertise in surgery. Although aspects of skill development occur through trial and error, watching the performance of another individual (action observation) is an increasingly important adjunct for the acquisition of these complex skills before performing a procedure. The aim of this review was to examine the evidence in support of the use of action observation in surgery. METHODS: A narrative review of observational learning for surgical motor skills was undertaken. Searches of PubMed and PsycINFO databases were performed using the terms 'observational learning' OR 'action observation' AND 'motor learning' OR 'skill learning'. RESULTS: Factors such as the structure of physical practice, the skill level of the demonstrator and the use of feedback were all found to be important moderators of the effectiveness of observational learning. In particular, observation of both expert and novice performance, cueing attention to key features of the task, and watching the eye movements of expert surgeons were all found to enhance the effectiveness of observation. It was unclear, however, whether repeated observations were beneficial for skill learning. The evidence suggests that these methods can be employed to enhance surgical training curricula. CONCLUSION: Observational learning is an effective method for learning surgical skills. An improved understanding of observational learning may further inform the refinement and use of these methods in contemporary surgical training curricula.

Optimization of the Promega PowerSeq™ Auto/Y system for efficient integration within a forensic DNA laboratory.

The application of massively parallel sequencing (MPS) is growing in the forensic DNA field, as forensic DNA laboratories are continuously seeking methods to gain information from a limited or degraded forensic sample. However, the laborious nature of current MPS methodologies required for successful library preparation and sequencing leave opportunities for improvement to make MPS a practical option for processing forensic casework. In this study, the Promega PowerSeq™ Auto/Y System Prototype, a MPS laboratory workflow that incorporates multiplex amplification, was selected for optimization with the objectives to introduce automation for relieving manual processing, and to reduce the number of steps recommended by the standard protocol. Successful changes in the optimized workflow included a switch from column-based PCR purification to automatable bead-based purification, adoption of the library preparation procedures by a liquid handling robot platform, and removal of various time-consuming quality checks. All data in this study were found to be concordant with capillary electrophoresis (CE) data and previously-generated MPS results from this workflow. Read abundance and allele balance, metrics related to sample interpretation reliability, were not significantly different when compared to samples processed with the manufacturer's protocol. All the modifications implemented resulted in increased laboratory efficiency, reduced the protocol steps associated with risk of contamination and human error events, and decreased manual processing time by approximately 12h. These findings provide forensic DNA laboratories a more streamlined option when considering implementation of a MPS workflow.

Molecular dynamics simulations of the structure and the morphology of graphene/polymer nanocomposites.

The structure and morphology of three polymer/graphene nanocomposites have been studied using classical molecular dynamics (MD) simulations. The simulations use 10-monomer oligomeric chains of three polymers: polyethylene (PE), polystyrene (PS) and polyvinylidene fluoride (PVDF). The structure of the polymer chains at the graphene surface has been investigated and characterized by pair correlation functions (PCF), g(r), g(θ) and g(r,θ). In addition, the influence of the temperature on the graphene/polymer interactions has been analysed for each of the three polymer/graphene nanocomposite systems. The results indicate that graphene induces order in both the PE and PVDF systems by providing a nucleation site for crystallisation, steering the growth of oligomer crystals according to the orientation of the graphene sheet, whereas the PS system remains disordered in the presence of graphene. The overall results are in line with the findings in a recent quantumchemical study by some of the present authors.

Acute West Nile Virus Meningoencephalitis Diagnosed Via Metagenomic Deep Sequencing of Cerebrospinal Fluid in a Renal Transplant Patient.

Solid organ transplant patients are vulnerable to suffering neurologic complications from a wide array of viral infections and can be sentinels in the population who are first to get serious complications from emerging infections like the recent waves of arboviruses, including West Nile virus, Chikungunya virus, Zika virus, and Dengue virus. The diverse and rapidly changing landscape of possible causes of viral encephalitis poses great challenges for traditional candidate-based infectious disease diagnostics that already fail to identify a causative pathogen in approximately 50% of encephalitis cases. We present the case of a 14-year-old girl on immunosuppression for a renal transplant who presented with acute meningoencephalitis. Traditional diagnostics failed to identify an etiology. RNA extracted from her cerebrospinal fluid was subjected to unbiased metagenomic deep sequencing, enhanced with the use of a Cas9-based technique for host depletion. This analysis identified West Nile virus (WNV). Convalescent serum serologies subsequently confirmed WNV seroconversion. These results support a clear clinical role for metagenomic deep sequencing in the setting of suspected viral encephalitis, especially in the context of the high-risk transplant patient population.

Quiet eye training aids the long-term learning of throwing and catching in children: Preliminary evidence for a predictive control strategy.

Quiet eye training (QET) may be a more effective method for teaching children to catch than traditional training (TT) methods, but it is unclear if the benefits accrued persist in the long term. Thirty children were randomly allocated into a QET or TT group and, while wearing a mobile eye tracker, underwent baseline testing, training and two retention tests over a period of eight weeks, using a validated throw and catch task. During training, movement-related information was provided to both groups, while the QET group received additional instruction to increase the duration of their targeting fixation (QE1) on the wall prior to the throw, and pursuit tracking (QE2) period on the ball prior to catching. In both immediate (R1) and delayed (R2, six weeks later) retention tests, the QET group had a significantly longer QE1 duration and an earlier and longer QE2 duration, compared to the TT group, who revealed no improvements. A performance advantage was also found for the QET compared to the TT group at both R1 and R2, revealing the relatively robust nature of the visuomotor alterations. Regression analyses suggested that only the duration of QE1 predicted variance in catch success post-training, pointing to the importance of a pre-programming visuomotor strategy for successful throw and catch performance.

Alpha-2 macroglobulin in Alzheimer's disease: a marker of neuronal injury through the RCAN1 pathway.

Preclinical changes that precede the onset of symptoms and eventual diagnosis of Alzheimer's disease (AD) are a target for potential preventive interventions. A large body of evidence suggests that inflammation is closely associated with AD pathogenesis and may be a promising target pathway for such interventions. However, little is known about the association between systemic inflammation and preclinical AD pathophysiology. We first examined whether the acute-phase protein, alpha-2 macroglobulin (A2M), a major component of the innate immune system, was associated with cerebrospinal fluid (CSF) markers of neuronal injury in preclinical AD and risk of incident AD in the predictors of cognitive decline among normal individuals (BIOCARD) cohort. We find that A2M concentration in blood is significantly associated with CSF concentrations of the neuronal injury markers, tau and phosphorylated tau, and that higher baseline serum A2M concentration is associated with an almost threefold greater risk of progression to clinical symptoms of AD in men. These findings were replicated in the Alzheimer's Disease Neuroimaging (ADNI) study. Then, utilizing a systems level approach combining large multi-tissue gene expression datasets with mass spectrometry-based proteomic analyses of brain tissue, we identified an A2M gene network that includes regulator of calcineurin (RCAN1), an inhibitor of calcineurin, a well-characterized tau phosphatase. A2M gene and protein expression in the brain were significantly associated with gene and protein expression levels of calcineurin. Collectively these novel findings suggest that A2M is associated with preclinical AD, reflects early neuronal injury in the disease course and may be responsive to tau phosphorylation in the brain through the RCAN1-calcineurin pathway.

Sulfate-reducing bacteria slow intestinal transit in a bismuth-reversible fashion in mice.

BACKGROUND: Hydrogen sulfide (H2 S) serves as a mammalian cell-derived gaseous neurotransmitter. The intestines are exposed to a second source of this gas by sulfate-reducing bacteria (SRB). Bismuth subsalicylate binds H2 S rendering it insoluble. The aim of this study was to test the hypothesis that SRB may slow intestinal transit in a bismuth-reversible fashion. METHODS: Eighty mice were randomized to five groups consisting of Live SRB, Killed SRB, SRB+Bismuth, Bismuth, and Saline. Desulfovibrio vulgaris, a common strain of SRB, was administered by gavage at the dose of 1.0 × 109 cells along with rhodamine, a fluorescent dye. Intestinal transit was measured 50 minutes after gavage by euthanizing the animals, removing the small intestine between the pyloric sphincter and the ileocecal valve and visualizing the distribution of rhodamine across the intestine using an imaging system (IVIS, Perkin-Elmer). Intestinal transit (n=50) was compared using geometric center (1=minimal movement, 100=maximal movement). H2 S concentration (n=30) was also measured when small intestinal luminal content was allowed to generate this gas. KEY RESULTS: The Live SRB group had slower intestinal transit as represented by a geometric center score of 40.2 ± 5.7 when compared to Saline: 73.6 ± 5.7, Killed SRB: 77.9 ± 6.9, SRB+Bismuth: 81.0 ± 2.0, and Bismuth: 73.3 ± 4.2 (P<.0001). Correspondingly, the Live SRB group had the highest luminal H2 S concentration of 4181.0 ± 968.0 ppb compared to 0 ± 0 ppb for the SRB+Bismuth group (P<.0001). CONCLUSIONS & INFERENCES: Live SRB slow intestinal transit in a bismuth-reversible fashion in mice. Our results demonstrate that intestinal transit is slowed by SRB and this effect could be abolished by H2 S-binding bismuth.

Paraorosius, a new genus of leafhopper (Hemiptera: Cicadellidae: Deltocephalinae: Opsiini), with description of a new species from the Middle East.

A new genus, of the leafhopper tribe Opsiini, Paraorosius is proposed to accommodate Orosius minuicus Dlabola, 1979 and, P. hanii sp. n. from the Kingdom of Saudi Arabia. A key to the genera of Opsiini of the Arabian Peninsula is provided.

Rapid flow cytometric measurement of protein inclusions and nuclear trafficking.

Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules.

Clusterin facilitates apoptotic cell clearance and prevents apoptotic cell-induced autoimmune responses.

Clusterin (Clu), an extracellular chaperone, exhibits characteristics of soluble innate immunity receptors, as assessed by its ability to bind some bacteria strains. In this study, we report that Clu also binds specifically to late apoptotic cells but not to live, early apoptotic, or necrotic cells. Histones, which accumulate on blebs during the apoptotic process, represent privileged Clu-binding motifs at the surface of late apoptotic cells. As a consequence, Clu potentiates, both in vitro and in vivo, the phagocytosis of late apoptotic cells by macrophages. Moreover, the increased phagocytosis of late apoptotic cells induced by Clu favors the presentation and cross-presentation of apoptotic cell-associated antigens. Finally, we observed that, in a model of apoptotic cell-induced autoimmunity, and relative to control mice, Clu(-/-) mice develop symptoms of autoimmunity, including the generation of anti-dsDNA antibodies, deposition of immunoglobulins and complement components within kidneys, and splenomegaly. These results identify Clu as a new molecule partner involved in apoptotic cell efferocytosis and suggest a protective role for Clu in inflammation and autoimmune diseases.

Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications.

Next-generation sequencing has generated a need for a broadly applicable method to remove unwanted high-abundance species prior to sequencing. We introduce DASH (Depletion of Abundant Sequences by Hybridization). Sequencing libraries are 'DASHed' with recombinant Cas9 protein complexed with a library of guide RNAs targeting unwanted species for cleavage, thus preventing them from consuming sequencing space. We demonstrate a more than 99 % reduction of mitochondrial rRNA in HeLa cells, and enrichment of pathogen sequences in patient samples. We also demonstrate an application of DASH in cancer. This simple method can be adapted for any sample type and increases sequencing yield without additional cost.

The impacts of larval density and protease inhibition on feeding in medicinal larvae of the greenbottle fly Lucilia sericata.

Larval therapy, the therapeutic use of blowfly larvae to treat chronic wounds, is primarily used in debridement. There are, however, gaps in current knowledge of the optimal clinical application of the therapy and mechanisms of action in the debridement process. Using an artificial assay, two studies were undertaken to investigate these aspects of larval debridement by Lucilia sericata Meigen (Diptera: Calliphoridae); the first studied the effects of the density of larvae on tissue digestion and larval mass, and the second considered the effects on the same parameters of incorporating protease inhibitors into the feeding substrate. The total mass of tissue digested increased with larval density until saturation was observed at 5.0-7.5 larvae/cm(2) . This range was considered optimal as lower doses resulted in the removal of less tissue and higher doses offered no additional tissue removal and appeared to exacerbate competition for feeding. In the second study, increased protease inhibitor concentration led to significant decreases in tissue digestion and larval mass, suggesting that serine proteases, particularly trypsin, may play major roles in larval digestion. Such information is important in elucidating the main constituents that make up larval digestive products and may be significant in the development of new therapies.

Movement specific reinvestment and allocation of attention by older adults during walking.

Older repeat fallers have previously been shown to have a higher propensity to consciously monitor and control their movements (i.e. reinvestment) than non-fallers, yet to direct their attention equally between their limb movements and the external environment during locomotion (Wong et al. in J Am Geriatr Soc 57: 920-922, 2009). Whether increased attention to their movements is a result of falling or originates from a prior inclination to reinvest remains unclear. In order to better understand the interaction between reinvestment and attention during locomotion, this study examined the allocation of attention by older adults who had not fallen but displayed a high or low inclination for reinvestment. Twenty-eight low and twenty-eight high reinvestors were required to perform 30 walking trials. Their allocation of attention during walking was evaluated by asking tone-related attentional focus questions shortly after finishing each walking trial. High reinvestors were found to be more aware of their limb movements and less aware of the external environment. Low reinvestors, on the contrary, were more aware of the surrounding environment and less aware of their movement mechanics. Given that focusing internally to body movements has been proposed to utilise working memory capacity, the ability of high reinvestors to pick up all the environmental information necessary for successful locomotion might be compromised and requires further examination.

Quiet eye training facilitates visuomotor coordination in children with developmental coordination disorder.

INTRODUCTION: Quiet eye training (QET) has been shown to be more effective than traditional training (TT) methods for teaching a throw and catch task to typically developing 8-10 yr old children. The current study aimed to apply the technique to children with developmental coordination disorder (DCD). METHOD: 30 children with DCD were randomly allocated into TT or QET intervention groups. The TT group were taught how to control their arm movements during the throw and catch phases, while the QET group were also taught to fixate a target location on the wall prior to the throw (quiet eye1; QE1), followed by tracking the ball prior to the catch (quiet eye2; QE2). Performance, gaze and motion analysis data were collected at pre/post-training and 6-week retention. RESULTS: The QET group significantly increased QE durations from pre-training to delayed retention (QE1 = +247 ms, QE2 = +19%) whereas the TT group experienced a reduction (QE1 = -74 ms, QE2 = -4%). QET participants showed significant improvement in the quality of their catch attempts and increased elbow flexion at catch compared to the TT group (QET = -28°, TT = -1°). CONCLUSION: QET changed DCD children's ability to focus on a target on the wall prior to the throw, followed by better anticipation and pursuit tracking on the ball, which in turn led to improved catching technique. QET may be an effective adjunct to traditional instructions, for therapists teaching visuomotor skills to children with DCD.