Refining outcome prediction after traumatic brain injury with machine learning algorithms.

Outcome after traumatic brain injury (TBI) is typically assessed using the Glasgow outcome scale extended (GOSE) with levels from 1 (death) to 8 (upper good recovery). Outcome prediction has classically been dichotomized into either dead/alive or favorable/unfavorable outcome. Binary outcome prediction models limit the possibility of detecting subtle yet significant improvements. We set out to explore different machine learning methods with the purpose of mapping their predictions to the full 8 grade scale GOSE following TBI. The models were set up using the variables: age, GCS-motor score, pupillary reaction, and Marshall CT score. For model setup and internal validation, a total of 866 patients could be included. For external validation, a cohort of 369 patients were included from Leuven, Belgium, and a cohort of 573 patients from the US multi-center ProTECT III study. Our findings indicate that proportional odds logistic regression (POLR), random forest regression, and a neural network model achieved accuracy values of 0.3-0.35 when applied to internal data, compared to the random baseline which is 0.125 for eight categories. The models demonstrated satisfactory performance during external validation in the data from Leuven, however, their performance were not satisfactory when applied to the ProTECT III dataset.

VECMAtk: a scalable verification, validation and uncertainty quantification toolkit for scientific simulations.

We present the VECMA toolkit (VECMAtk), a flexible software environment for single and multiscale simulations that introduces directly applicable and reusable procedures for verification, validation (V&V), sensitivity analysis (SA) and uncertainty quantication (UQ). It enables users to verify key aspects of their applications, systematically compare and validate the simulation outputs against observational or benchmark data, and run simulations conveniently on any platform from the desktop to current multi-petascale computers. In this sequel to our paper on VECMAtk which we presented last year [1] we focus on a range of functional and performance improvements that we have introduced, cover newly introduced components, and applications examples from seven different domains such as conflict modelling and environmental sciences. We also present several implemented patterns for UQ/SA and V&V, and guide the reader through one example concerning COVID-19 modelling in detail. This article is part of the theme issue 'Reliability and reproducibility in computational science: implementing verification, validation and uncertainty quantification in silico'.

Large-scale binding affinity calculations on commodity compute clouds.

In recent years, it has become possible to calculate binding affinities of compounds bound to proteins via rapid, accurate, precise and reproducible free energy calculations. This is imperative in drug discovery as well as personalized medicine. This approach is based on molecular dynamics (MD) simulations and draws on sequence and structural information of the protein and compound concerned. Free energies are determined by ensemble averages of many MD replicas, each of which requires hundreds of cores and/or GPU accelerators, which are now available on commodity cloud computing platforms; there are also requirements for initial model building and subsequent data analysis stages. To automate the process, we have developed a workflow known as the binding affinity calculator. In this paper, we focus on the software infrastructure and interfaces that we have developed to automate the overall workflow and execute it on commodity cloud platforms, in order to reliably predict their binding affinities on time scales relevant to the domains of application, and illustrate its application to two free energy methods.

Effects of laboratory salmon louse infection on Arctic char osmoregulation, growth and survival.

High salmon lice (Lepeophtheirus salmonis) infestation levels resulting from intensive salmonid sea-cage aquaculture can threaten populations of wild salmonid hosts. This includes anadromous Arctic char (Salvelinus alpinus), which rely on short migrations into more productive seawater environments to build energy stores for maturation, spawning and over-wintering in freshwater. Elevated salmon lice burdens may limit the benefits of migration by constraining osmoregulation, growth, survival and reproduction. To test for these effects, we simulated anadromous migration in tanks by transferring individually tagged Arctic char smolts (n = 352, averaging 133 g) to seawater where they were infected with salmon lice or left as uninfected controls for 1 month, and then transferring them back to freshwater for 2 months. After the seawater phase, infected post-smolts had a mean of 0.33 (range of 0.09-0.91) mobile lice g-1 fish weight. At this point, specific growth rates (SGRs) dropped in infected compared to control fish (0.1% vs. 1.6% day-1). Higher plasma Na+ and osmolality in infected fish also indicate osmoregulatory impairment. Throughout the study, mortality was 18.2% and 1.7% in infected and control groups, but sexual maturation was low and comparable between groups. Infection intensity correlated positively with mortality rate and plasma Cl-, and correlated negatively with SGR and condition factor (CF). CF dropped (ΔCF < 0) at intensities of >0.09 lice g-1 fish weight, and intensities of >0.3 causing zero or negative SGRs and increased mortality were particularly concerning. If infection intensities reach these levels in the wild, char could be impacted by growth restrictions and increased mortality rates, which potentially cause shorter migration durations, lowered reproductive success and possibly also selection against anadromy. This study provides vital information for conservation practitioners wanting to understand the physiologically derived burden salmon lice can have on Arctic char populations, and can be used to define thresholds in the monitoring and conservation of Arctic char populations affected by aquaculture-driven salmon lice infestations.

Biolayer interferometry predicts ELISA performance of monoclonal antibody pairs for Plasmodium falciparum histidine-rich protein 2.

Predicting antibody pair performance in a sandwich format streamlines development of antibody-based diagnostics and laboratory research tools, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFAs). We have evaluated panels of monoclonal antibodies against the malarial parasite biomarker Plasmodium falciparum histidine rich protein 2 (HRP2), including 9 new monoclonal antibodies, using biolayer interferometry (BLI) and screened antibody pairs in a checkerboard ELISA. This study showed BLI predicts antibody pair ELISA performance for HRP2. Pairs that included capture antibodies with low off-rate constants and detection antibodies with high on-rate constants performed best in an ELISA format.

'Snorkel' lice barrier technology reduced two co- occurring parasites, the salmon louse (Lepeophtheirus salmonis) and the amoebic gill disease causing agent (Neoparamoeba perurans), in commercial salmon sea-cages.

Diverse chemical-free parasite controls are gaining status in Atlantic salmon sea-cage farming. Yet, the intricacies of their use at commercial scale, including effects on co-occurring parasites, are seldom reported. A new salmon lice prevention method involves installing a deep net roof and 'snorkel' lice barrier in cages to shelter salmon from free-living infective larvae which concentrate at shallow depths, and allows salmon to jump and re- inflate their buoyancy-regulating swim bladder by swallowing air. We document use of snorkel cages (10m deep barrier) in commercial farms, where their effects on salmon lice levels, amoebic gill disease (AGD)-related gill scores, the cage environment, fish welfare and farm management practices were compared to standard cages. During an autumn-winter study involving only snorkel cages, high AGD-related gill scores were observed to decline when freshwater was pumped into snorkels, creating a freshwater surface layer for salmon to enter for self-treatment. In a spring-summer study incorporating snorkel and standard cages, snorkel cages were found to reduce new lice infestations by 84%. The deployment of snorkels and intermittent oxygen depletion detected within them in the spring-summer study did not alter fish welfare parameters. Overall, the results suggest snorkel technology has a place in the toolkit of commercial salmon sea-cage farmers co-managing salmon lice and amoebic gill disease outbreaks - two principal parasite issues facing the industry.

Applications of Hairpin DNA-Functionalized Gold Nanoparticles for Imaging mRNA in Living Cells.

Molecular imaging agents are useful for imaging molecular processes in living systems in order to elucidate the function of molecular mediators in health and disease. Here, we demonstrate a technique for the synthesis, characterization, and application of hairpin DNA-functionalized gold nanoparticles (hAuNPs) as fluorescent hybridization probes for imaging mRNA expression and spatiotemporal dynamics in living cells. These imaging probes feature gold colloids linked to fluorophores via engineered oligonucleotides to resemble a molecular beacon in which the gold colloid serves as the fluorescence quencher in a fluorescence resonance energy transfer system. Target-specific hybridization of the hairpin oligonucleotide enables fluorescence de-quenching and subsequent emission with high signal to noise ratios. hAuNPs exhibit high specificity without adverse toxicity or the need for transfection reagents. Furthermore, tunability of hAuNP emission profiles by selection of spectrally distinct fluorophores enables multiplexed mRNA imaging applications. Therefore, hAuNPs are promising tools for imaging gene expression in living cells. As a representative application of this technology, we discuss the design and applications of hAuNP targeted against distinct matrix metalloproteinase enzymes for the multiplexed detection of mRNA expression in live breast cancer cells using flow cytometry and fluorescence microscopy.

Early-stage sea lice recruits on Atlantic salmon are freshwater sensitive.

Sea lice are significant parasites of marine and brackish farmed fishes. Freshwater bathing is a potential control option against numerous sea lice species, although has been viewed as futile against those that are capable of tolerating freshwater for extended periods. By comparing freshwater survival times across host-attached stages of Lepeophtheirus salmonis (Krøyer), a key parasite in Atlantic salmon farming, we show the first attached (copepodid) stage undergoes 96-100% mortality after 1 h in freshwater, whereas later attached stages can tolerate up to 8 days. Thus, regular freshwater bathing methods targeting the more susceptible attached copepodid stage may successfully treat against L. salmonis and potentially other sea lice on fish cultured in marine and brackish waters.

Simple sample processing enhances malaria rapid diagnostic test performance.

Lateral flow immunochromatographic rapid diagnostic tests (RDTs) are the primary form of medical diagnostic used for malaria in underdeveloped nations. Unfortunately, many of these tests do not detect asymptomatic malaria carriers. In order for eradication of the disease to be achieved, this problem must be solved. In this study, we demonstrate enhancement in the performance of six RDT brands when a simple sample-processing step is added to the front of the diagnostic process. Greater than a 4-fold RDT signal enhancement was observed as a result of the sample processing step. This lowered the limit of detection for RDT brands to submicroscopic parasitemias. For the best performing RDTs the limits of detection were found to be as low as 3 parasites per µL. Finally, through individual donor samples, the correlations between donor source, WHO panel detection scores and RDT signal intensities were explored.

Quadruplex priming amplification for the detection of mRNA from surrogate patient samples.

Simple and rapid methods for detecting mRNA biomarkers from patient samples are valuable in settings with limited access to laboratory resources. In this report, we describe the development and evaluation of a self-contained assay to extract and quantify mRNA biomarkers from complex samples using a novel nucleic acid-based molecular sensor called quadruplex priming amplification (QPA). QPA is a simple and robust isothermal nucleic acid amplification method that exploits the stability of the G-quadruplex nucleotide structure to drive spontaneous strand melting from a specific DNA template sequence. Quantification of mRNA was enabled by integrating QPA with a magnetic bead-based extraction method using an mRNA-QPA interface reagent. The assay was found to maintain >90% of the maximum signal over a 4 °C range of operational temperatures (64-68 °C). QPA had a dynamic range spanning four orders of magnitude, with a limit of detection of ~20 pM template molecules using a highly controlled heating and optical system and a limit of detection of ~250 pM using a less optimal water bath and plate reader. These results demonstrate that this integrated approach has potential as a simple and effective mRNA biomarker extraction and detection assay for use in limited resource settings.

First Report of Club Root Caused by Plasmodiophora brassicae on Canola in Australia.

In 2009, a disease survey was conducted in 97 commercial canola (Brassica napus L.) fields in Western Australia by the Department of Agriculture and Food, Western Australia (DAFWA). In about 20% of the fields from the northern agricultural region of Western Australia, small patches were observed where canola plants showed symptoms of stunting and wilting. These plants were collected and roots of affected plants were washed thoroughly and examined for the presence of root disease. Small galls and clublike structures were observed on the secondary roots and sometimes on the main root of the affected plants. Examination of thin free hand sections from the root galls revealed that several cortical cells were enlarged and full of resting spores. The diameter of resting spores ranged between 2.5 and 3.0 µm. Plasmodia and zoosporangia were also observed in the root hairs. The identity of Plasmodiophora brassicae Woronin was confirmed by PCR using a modified method of Cao et al. 2007 (1). DNA from spores and slices of the galls of 14 different samples were extracted using DNeasy plant mini kit (QIAGEN Australia) as per manufacturer's instructions. Samples were disrupted by placing them into MPBIO tube A and placed in the Fast Prep machine at speed of 6 ms-1 for 40 s. This was repeated twice. The species-specific primers TC1F (5'-GTGGTCGAACTTCATTAAATTTGGGCTCTT-3')/TC1R (5'-TTCACCTACGGAACGTATATGTGCATGTGA-3') and TC2F (5'-AAACAACGAGTCAGCTTGAATGCTAGTGTG-3')/TC2R (5'-CTTTAGTTGTGTTTCGGCTAGGATGGTTCG-3') were used (1). The primers TC1F and TC1R failed to produce a PCR product of 548-bp size but using the primers TC2F and TC2R the PCR reaction resulted in a 519- bp fragment. Seven out of 14 samples gave positive results for P. brassicae with primers TC2F and TC2R. This indicates that the P. brassicae pathotype from Western Australia may be different than the one found in Alberta, Canada. However, pathotypes of P. brassicae from brassica vegetables from Australia have been found similar to the populations of P. brassicae present in the United States (2). Pathogenicity of P. brassicae was tested by dipping roots of five 10-day-old canola plants var. Cobbler in a spore suspension (1 × 106 resting spores/ml). Roots of five control plants were dipped in sterile water. Five weeks after inoculation, small galls were observed on the roots of three inoculated plants and the control plants remained symptomless. Resting spores were recovered from the galls developed on the roots of affected plants. Presence of P. brassicae in the affected roots was further confirmed by PCR using the method described above. To our knowledge, this is the first report of club root of canola in Australia. Club root is reported from vegetable brassicas and white mustard (Sinapis alba L.) in Australia. Club root has become a serious disease of canola in Canada since its detection in Alberta in 2006 (3). The resting spores of the fungus can survive for several years in soil, and therefore, this disease could pose a significant threat to canola production in Western Australia. References: (1) Cao et al. Plant Dis. 91:80, 2007. (2) Donald et al. Ann. App. Biol. 148:239, 2006. (3) S. Streklov et al. Can. J. Plant Pathol. 28:467, 2006.

Use of a novel technology for presenting screening measures to detect mild cognitive impairment in elderly patients.

BACKGROUND: Available screening tools for mild cognitive impairment (MCI), often a precursor to Alzheimer's disease, are insensitive or not feasible for administration in a busy primary care setting. Display Enhanced TEsting for Cognitive impairment and Traumatic brain injury (DETECT) addresses these issues by creating an immersive environment for the brief administration of neuropsychological (NP) measures. OBJECTIVE: The aim of this study was to determine if the DETECT cognitive subtests can identify MCI patients as accurately as standard pen and paper NP tests. METHODS: Twenty patients with MCI recruited from a memory disorders clinic and 20 age-matched controls were given both a full battery of NP tests (standard NP) and the DETECT screen. Logistic regression models were used to determine whether individual tests were predictive of group membership (MCI or control). Demographic variables including age, race, education and gender were adjusted as covariates. Selection methods were used to identify subset models that exhibited maximum discrimination between MCI patients and controls for both testing methods. RESULTS: Both the standard NP model (C-index = 0.836) and the DETECT model (C-index = 0.865) showed very good discrimination and were not significantly different (p = 0.7323). CONCLUSION: The DETECT system shows good agreement with standard NP tests and is capable of identifying elderly patients with cognitive impairment.

The DETECT system: portable, reduced-length neuropsychological testing for mild traumatic brain injury via a novel immersive environment.

Undiagnosed mild traumatic brain injury (mTBI) often leads to poor patient management and significant morbidity. The lack of an efficient screening tool is especially apparent in the athletic setting, where repetitive injuries can lead to prolonged disability. We have developed the Display Enhanced Testing for Concussions and mTBI system (DETECT), in order to create a portable immersive environment that could eliminate visual and audio distractions. Neuropsychological tests sensitive to mTBI were modified for use with the system and allow rapid neurological assessment independent of the environment or trained personnel. We evaluated the immersive qualities of the DETECT system in 42 uninjured controls. The system was successful in blocking out external audiovisual stimuli. The neuropsychological test results obtained in a stimulus rich environment were equivalent to those obtained in a controlled quiet environment. The immersive environment, portability, and brevity of the DETECT system allow for real-time cognitive testing in situations previously deemed impractical or unavailable for mTBI patients.

Effects of repeated exposure to JP-8 jet fuel vapor on learning of simple and difficult operant tasks by rats.

Groups of 16 Sprague-Dawley rats each were exposed by whole-body inhalation methods to JP-8 jet fuel at the highest vapor concentration without formation of aerosol (1,000 +/- 10% mg/m3); to 50% of this concentration (500 +/- 10% mg/m3); or to treated room air (70 +/- 81 L/min) for 6 h/d, 5 d/wk, for 6 wk (180 h). Although two subjects died of apparent kidney complications during the study, no other change in the health status of exposed rats was observed, including rate of weight gain. Following a 65-d period of rest, rats were evaluated for their capacity to learn and perform a series of operant tasks. These tasks ranged in difficulty from learning of a simple food-reinforced lever pressing response, to learning a task in which subjects were required to emit up to four-response chains of pressing three different levers (e.g., press levers C, R, L, then C). It was shown that repeated exposure to 1,000 mg/m3 JP-8 vapor induced significant deficits in acquisition or performance of moderately difficult or difficult tasks, but not simple learning tasks, as compared to those animals exposed to 500 mg/m3. Learning/performance of complex tasks by the 500-mg/m3 exposure group generally exceeded the performance of control animals, while learning by the 1,000-mg/m3 group was nearly always inferior to controls, indicating possible "neurobehavioral" hormesis. These findings appear consistent with some previously reported data for operant performance following acute exposure to certain hydrocarbon constituents of JP-8 (i.e., toluene, xylenes). There has, however, been little previously published research demonstrating long-term learning effects for repeated hydrocarbon fuel exposures. Examination of regional brain tissues from vapor-exposed rats indicated significant changes in levels of dopamine in the cerebral cortex and DOPAC in the brainstem, measured as long as 180 d postexposure, as compared to controls.

Serum progesterone levels correlate with decreased cerebral edema after traumatic brain injury in male rats.

Previous animal research suggests that progesterone may have powerful neuroprotective effects in traumatic brain injury (TBI). This experiment tested the hypothesis that progesterone levels correlate with decreased cerebral edema in male rats with bilateral medial frontal cortex injuries. Three groups of male Sprague-Dawley rats were used: injured given progesterone (4 mg/kg), injured given vehicle (oil), and uninjured controls given vehicle. Progesterone or vehicle was administered intraperitoneally at 1, 6, and 24 h postinjury. At 48 h postinjury, the rats were killed, brains extracted, and assayed for edema. Percent difference in water content of the area surrounding the lesion was compared to posterior cortex. A strong inverse relationship was found between serum progesterone levels and percent cerebral edema; the higher the progesterone levels, the lower the percent edema. Both progesterone and oil-treated animals had some edema compared to sham-operated controls. The brains of the injured animals given control solution were higher in water content than either the uninjured group or injured progesterone-treated rats 48 h postinjury. These findings confirm that progesterone significantly decreases cerebral edema after TBI in adult male subjects.

Heme Aggregation inhibitors: antimalarial drugs targeting an essential biomineralization process.

Malaria, resulting from the parasites of the genus Plasmodium, places an untold burden on the global population. As recently as 40 years ago, only 10% of the world's population was at risk from malaria. Today, over 40% of the world's population is at risk. Due to increased parasite resistance to traditional drugs and vector resistance to insecticides, malaria is once again resurgent. An emergent theme from current strategies for the development of new antimalarials is that metal homeostasis within the parasite represents an important drug target. During the intra-erythrocytic phase of its life cycle, the malaria parasite can degrade up to 75% of an infected cell's hemoglobin. While hemoglobin proteolysis yields requisite amino acids, it also releases toxic free heme (Fe(III)PPIX). To balance the metabolic requirements for amino acids against the toxic effects of heme, malaria parasites have evolved a detoxification mechanism which involves the formation of a crystalline heme aggregate known as hemozoin. An overview of the biochemistry of the critical detoxification process will place it in the appropriate context with regards to drug targeting and design. Quinoline-ring antimalarial drugs are effective against the intraerythrocytic stages of pigment-producing parasites. Recent work on the mechanism of these compounds suggests that they prevent the formation of hemozoin. Evidence for such a mechanism is reviewed, especially in the context of the newly reported crystal structure of hemozoin. Additionally, novel drugs, such as the hydroxyxanthones, which have many of the characteristics of the quinolines are currently being investigated. Recent work has also highlighted two classes of inorganic complexes that have interesting antimalarial activity: (1) metal-N(4)O(2) Schiff base complexes and (2) porphyrins. The mechanism of action for these complexes is discussed. The use of these complexes as probes for the elucidation of structure-activity relationships in heme polymerization inhibitor design and the loci of drug resistance is also detailed. As the biochemistry of the complicated interactions between host, parasite, and vector become better understood, the rationale for new antimalarial drug treatments will continue to improve. Clearly, the homeostasis of metal ions is a complicated biochemical process and is not completely understood. For the immediate future, it does, however, provide a clear target for the development of new and improved treatments for malaria.

Metalloporphyrins inhibit beta-hematin (hemozoin) formation.

Metal-substituted protoporphyrin IXs (Cr(III)PPIX (1), Co(III)PPIX (2), Mn(III)PPIX (3), Cu(II)PPIX (4), Mg(II)PPIX (5), Zn(II)PPIX (6), and Sn(IV)PPIX (7)) act as inhibitors to beta-hematin (hemozoin) formation, a critical detoxification biopolymer of malarial parasites. The central metal ion plays a significant role in the efficacy of the metalloprotoporphyrins to inhibit beta-hematin formation. The efficacy of these compounds correlates well with the water exchange rate for the octahedral aqua complexes of the porphyrin's central metal ion. Under these in vitro reaction conditions, metalloporphyrins 5, 6 and 7 are as much as six times more efficacious than the free ligand protoporphyrin IX in preventing beta-hematin formation and four times as efficacious as chloroquine, while metalloporphyrins 3 and 4 are three to four times more effective at preventing beta-hematin formation than the free protoporphyrin IX base. In contrast, the relatively exchange inert metalloporphyrins 1 and 2 are only as efficacious as the free ligand and only two-thirds as effective as chloroquine. Aggregation studies of the heme:MPPIX using UV-Vis and fluorescence spectroscopies are indicative of the formation of pi-pi hetero-metalloporphyrin assemblies. Thus, hemozoin inhibition is likely prevented by the formation of heme:MPPIX complexes through pi-stacking interactions. The ramifications of such hetero-metalloporphyrin assemblies, in the context of the emerging structural picture of hemozoin, are discussed.

Pentobarbital, diazepam and phencyclidine disrupt delayed matching performance: interactions with picrotoxin in pigeons and squirrel monkeys.

The ability of picrotoxin to antagonize selectively the effects of pentobarbital was investigated in pigeons and squirrel monkeys responding under a titrating matching-to-sample schedule of reinforcement. Under the titrating matching-to-sample baseline, the length of the delay changed as a function of the animal's matching accuracy. Picrotoxin (0.03-1mg/kg) failed to alter significantly the matching accuracy of pigeons; however, rate of responding was markedly suppressed at a dose of 1mg/kg. In squirrel monkeys responding under a similar schedule, picrotoxin (0.001-0.3mg/kg) was without significant effect. Selected doses of picrotoxin in both pigeons (0.3 and 0.56mg/kg) and squirrel monkeys (0.1 and 0.3mg/kg) failed to shift the pentobarbital or diazepam dose-response curve for mean delay length to the right. However, in both species, picrotoxin shifted the dose-response curve for pentobarbital on rate of responding to the right. No such shift was observed for the effect of diazepam on rate of responding. In both species, the combination of picrotoxin and phencyclidine shifted the dose-response curves for phencyclidine on rate of responding, but not mean delay, downward and to the left, in an apparent additive manner. Thus, picrotoxin failed to produce a significant pharmacological antagonism of the effects of pentobarbital, diazepam or phencyclidine on matching accuracy. This failure to observe an antagonism of the effects of pentobarbital on matching accuracy, at doses of picrotoxin that antagonized the effects of pentobarbital on rate of responding, suggests that the effects of pentobarbital on matching accuracy and rate of responding are mediated by different receptor sites.

Glucose and leucine uptake by rat dorsal root ganglia is not insulin sensitive.

Dorsal root ganglia from rats were incubated with 3-O-methyl-[14C]glucose, and [3H]leucine in the presence or absence of insulin in order to determine whether insulin influences the uptake of glucose and amino acids by the cells of the ganglion. No effect was detected. A significant proportion (38%) of the uptake of [3H]leucine was shown to be inhibited by ouabain and therefore energy dependent, utilizing Na+K(+)-ATPase. The activity of this enzyme is known to be impaired in dorsal root ganglia in diabetic rats, as is the uptake of amino acids; these phenomena are therefore unlikely to be due to a direct effect of reduced circulating insulin levels. The relevance of these findings to theories as to the causation of diabetic neuropathy is discussed.