The new sequencer on the block: comparison of Life Technology’s Proton sequencer to an Illumina HiSeq for whole-exome sequencing

We assessed the performance of the new Life Technologies Proton sequencer by comparing whole-exome sequence data in a Centre d’Etude du Polymorphisme Humain trio (family 1463) to the Illumina HiSeq instrument. To simulate a typical user’s results, we utilized the standard capture, alignment and variant calling methods specific to each platform. We restricted data analysis to include the capture region common to both methods. The Proton produced high quality data at a comparable average depth and read length, and the Ion Reporter variant caller identified 96 % of single nucleotide polymorphisms (SNPs) detected by the HiSeq and GATK pipeline. However, only 40 % of small insertion and deletion variants (indels) were identified by both methods. Usage of the trio structure and segregation of platform-specific alleles supported this result. Further comparison of the trio data with Complete Genomics sequence data and Illumina SNP microarray genotypes documented high concordance and accurate SNP genotyping of both Proton and Illumina platforms. However, our study underscored the problem of accurate detection of indels for both the Proton and HiSeq platforms.     

Pyronaridine–artesunate real-world safety,tolerability, and effectiveness in malaria patients in 5 African countries: A single-arm,open-label,cohort event monitoring study

BackgroundIn Phase II/III randomized controlled clinical trials for the treatment of acute uncomplicated malaria, pyronaridine–artesunate demonstrated high efficacy and a safety profile consistent with that of comparators, except that asymptomatic, mainly mild-to-moderate transient increases in liver aminotransferases were reported for some patients. Hepatic safety, tolerability, and effectiveness have not been previously assessed under real-world conditions in Africa.Methods and findingsThis single-arm, open-label, cohort event monitoring study was conducted at 6 health centers in Cameroon, Democratic Republic of Congo, Gabon, Ivory Coast, and Republic of Congo between June 2017 and April 2019. The trial protocol as closely as possible resembled real-world clinical practice for the treatment of malaria at the centers. Eligible patients were adults or children of either sex, weighing at least 5 kg, with acute uncomplicated malaria who did not have contraindications for pyronaridine–artesunate treatment as per the summary of product characteristics. Patients received fixed-dose pyronaridine–artesunate once daily for 3 days, dosed by body weight, without regard to food intake. A tablet formulation was used in adults and adolescents and a pediatric granule formulation in children and infants under 20 kg body weight. The primary outcome was the hepatic event incidence, defined as the appearance of the clinical signs and symptoms of hepatotoxicity confirmed by a >2× rise in alanine aminotransferase/aspartate aminotransferase (ALT/AST) versus baseline in patients with baseline ALT/AST >2× the upper limit of normal (ULN). As a secondary outcome, this was assessed in patients with ALT/AST >2× ULN prior to treatment versus a matched cohort of patients with normal baseline ALT/AST. The safety population comprised 7,154 patients, of mean age 13.9 years (standard deviation (SD) 14.6), around half of whom were male (3,569 [49.9%]). Patients experienced 8,560 malaria episodes; 158 occurred in patients with baseline ALT/AST elevations >2×ULN. No protocol-defined hepatic events occurred following pyronaridine–artesunate treatment of malaria patients with or without baseline hepatic dysfunction. Thus, no cohort comparison could be undertaken. Also, as postbaseline clinical chemistry was only performed where clinically indicated, postbaseline ALT/AST levels were not systematically assessed for all patients. Adverse events of any cause occurred in 20.8% (1,490/7,154) of patients, most frequently pyrexia (5.1% [366/7,154]) and vomiting (4.2% [303/7,154]). Adjusting for Plasmodium falciparum reinfection, clinical effectiveness at day 28 was 98.6% ([7,369/7,746] 95% confidence interval (CI) 98.3 to 98.9) in the per-protocol population. There was no indication that comorbidities or malnutrition adversely affected outcomes. The key study limitation was that postbaseline clinical biochemistry was only evaluated when clinically indicated.ConclusionsPyronaridine–artesunate had good tolerability and effectiveness in a representative African population under conditions similar to everyday clinical practice. These findings support pyronaridine–artesunate as an operationally useful addition to the management of acute uncomplicated malaria.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03201770","term_id":"NCT03201770"}}NCT03201770.

Gaston Tona Lutete and co-workers report on safety and effectiveness of the antimalarial drug pyronaridine-artesunate in African countries.     

Commensal bacteria augment Staphylococcus aureus infection by inactivation of phagocyte-derived reactive oxygen species

Staphylococcus aureus is a human commensal organism and opportunist pathogen, causing potentially fatal disease. The presence of non-pathogenic microflora or their components, at the point of infection, dramatically increases S. aureus pathogenicity, a process termed augmentation. Augmentation is associated with macrophage interaction but by a hitherto unknown mechanism. Here, we demonstrate a breadth of cross-kingdom microorganisms can augment S. aureus disease and that pathogenesis of Enterococcus faecalis can also be augmented. Co-administration of augmenting material also forms an efficacious vaccine model for S. aureus. In vitro, augmenting material protects S. aureus directly from reactive oxygen species (ROS), which correlates with in vivo studies where augmentation restores full virulence to the ROS-susceptible, attenuated mutant katA ahpC. At the cellular level, augmentation increases bacterial survival within macrophages via amelioration of ROS, leading to proliferation and escape. We have defined the molecular basis for augmentation that represents an important aspect of the initiation of infection.     

Solution Structure of the SGTA Dimerisation Domain and Investigation of Its Interactions with the Ubiquitin-Like Domains of BAG6 and UBL4A

Background

The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates.

Methodology and Principal Findings

SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes.

Significance

This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.     

Using Information from the Electronic Health Record to Improve Measurement of Unemployment in Service Members and Veterans with mTBI and Post-Deployment Stress

Objective

The purpose of this pilot study is 1) to develop an annotation schema and a training set of annotated notes to support the future development of a natural language processing (NLP) system to automatically extract employment information, and 2) to determine if information about employment status, goals and work-related challenges reported by service members and Veterans with mild traumatic brain injury (mTBI) and post-deployment stress can be identified in the Electronic Health Record (EHR).

Design

Retrospective cohort study using data from selected progress notes stored in the EHR.

Setting

Post-deployment Rehabilitation and Evaluation Program (PREP), an in-patient rehabilitation program for Veterans with TBI at the James A. Haley Veterans'' Hospital in Tampa, Florida.

Participants

Service members and Veterans with TBI who participated in the PREP program (N = 60).

Main Outcome Measures

Documentation of employment status, goals, and work-related challenges reported by service members and recorded in the EHR.

Results

Two hundred notes were examined and unique vocational information was found indicating a variety of self-reported employment challenges. Current employment status and future vocational goals along with information about cognitive, physical, and behavioral symptoms that may affect return-to-work were extracted from the EHR. The annotation schema developed for this study provides an excellent tool upon which NLP studies can be developed.

Conclusions

Information related to employment status and vocational history is stored in text notes in the EHR system. Information stored in text does not lend itself to easy extraction or summarization for research and rehabilitation planning purposes. Development of NLP systems to automatically extract text-based employment information provides data that may improve the understanding and measurement of employment in this important cohort.     

Hypothermia Modulates Cytokine Responses After Neonatal Rat Hypoxic-Ischemic Injury and Reduces Brain Damage

While hypothermia (HT) is the standard-of-care for neonates with hypoxic ischemic injury (HII), the mechanisms underlying its neuroprotective effect are poorly understood. We examined ischemic core/penumbra and cytokine/chemokine evolution in a 10-day-old rat pup model of HII. Pups were treated for 24 hr after HII with HT (32℃; n = 18) or normothermia (NT, 35℃; n = 15). Outcomes included magnetic resonance imaging (MRI), neurobehavioral testing, and brain cytokine/chemokine profiling (0, 24, 48, and 72 hr post-HII). Lesion volumes (24 hr) were reduced in HT pups (total 74%, p < .05; penumbra 68%, p < .05; core 85%, p = .19). Lesion volumes rebounded at 72 hr (48 hr post-HT) with no significant differences between NT and HT pups. HT reduced interleukin-1β (IL-1β) at all time points (p < .05); monocyte chemoattractant protein-1 (MCP-1) trended toward being decreased in HT pups (p = .09). The stem cell signaling molecule, stromal cell-derived factor-1 (SDF-1) was not altered by HT. Our data demonstrate that HT reduces total and penumbral lesion volumes (at 24 and 48 hr), potentially by decreasing IL-1β without affecting SDF-1. Disassociation between the increasing trend in HII volumes from 48 to 72 hr post-HII when IL-1β levels remained low suggests that after rewarming, mechanisms unrelated to IL-1β expression are likely to contribute to this delayed increase in injury. Additional studies should be considered to determine what these mechanisms might be and also to explore whether extending the duration or degree of HT might ameliorate this delayed increase in injury.     

Focus on Weed Control: Natural Compounds as Next-Generation Herbicides

Herbicides with new modes of action (MOAs) are badly needed due to the rapidly evolving resistance to commercial herbicides, but a new MOA has not been introduced in over 20 years. The greatest pest management challenge for organic agriculture is the lack of effective natural product herbicides. The structural diversity and evolved biological activity of natural phytotoxins offer opportunities for the development of both directly used natural compounds and synthetic herbicides with new target sites based on the structures of natural phytotoxins. Natural phytotoxins are also a source for the discovery of new herbicide target sites that can serve as the focus of traditional herbicide discovery efforts. There are many examples of strong natural phytotoxins with MOAs other than those used by commercial herbicides, which indicates that there are molecular targets of herbicides that can be added to the current repertoire of commercial herbicide MOAs.The evolutionary forces driving the survival of species include chemical interactions between organisms, which function in positive interactions such as mutualistic and symbiotic relationships and negative interactions such as competitive and parasitic relationships. These processes have led to the emergence of novel secondary metabolic pathways (often through gene duplication), producing a vast array of structurally diverse and biologically active molecules (Moore and Purugganan, 2005; Ober, 2005; Flagel and Wendel, 2009; Jiang et al., 2013). This evolutionary process is similar to a high-throughput screen. However, unlike conventional in vitro screens, which test many compounds on a single biochemical target over a very short period of time, this natural high-throughput process selects molecules based on their whole-organism activities, involving numerous chemical interactions between countless organisms and target sites over millions of years. To date, approximately 200,000 secondary metabolites have been identified (Tulp and Bohlin, 2005), with many more expected to be discovered. Few of these compounds have been examined for phytotoxicity, and the modes or mechanisms of action (MOAs) of even fewer known phytotoxins have been elucidated.The negative chemical interactions between organisms are often characterized using anthropomorphic language, such as chemical warfare, referring to the production of phytotoxins used by plant pathogens to invade their host plants (Maor and Shirasu, 2005), and the novel weapons hypothesis, which is associated with the chemical-based advantage of some invasive plant species over native plant populations (Callaway and Aschehoug, 2000; Callaway and Ridenour, 2004; Callaway and Maron, 2006; Cappuccino and Arnason, 2006; Callaway et al., 2008). While simplistic, this terminology illustrates how these toxin-based interactions exploit biochemical weaknesses between an organism and its host or enemy/competitor to enhance its own survival (Verhoeven et al., 2009). In fact, these interactions can even be multitrophic, such as when exotic plants enhance their invasiveness by promoting the growth of certain native soil pathogens noxious to native plants (Mangla et al., 2008; Barto et al., 2011).As humans evolved from a nomadic hunter-gatherer subsistence existence to an agricultural lifestyle, they learned to utilize certain biologically active secondary metabolites to manage agricultural pests. Indeed, the concept that nature is an excellent source of natural pesticides is captured in the following ancient Lithica poem (circa 400 B.C.): “All the pests that out of earth arise, the earth itself the antidote supplies” (Ibn et al., 1781). Less than a century later, Greek and Roman treatises described practices to control agricultural pests that include the use of essential oils. Similar documents are found in Chinese literature, such as a survey describing plant species used to control plant pests (Yang and Tang, 1988). The mid-20th century ushered in the use of synthetic pesticides, which have revolutionized agriculture. Like pharmaceuticals (Harvey, 1999, 2008; Newman and Cragg, 2012), many pesticides are based on natural compounds. However, natural products have not played a major role in herbicide discovery (Copping and Duke, 2007; Hüter, 2011).     

Mu Opioid Receptor Binding Correlates with Nicotine Dependence and Reward in Smokers

The rewarding effects of nicotine are associated with activation of nicotine receptors. However, there is increasing evidence that the endogenous opioid system is involved in nicotine''s rewarding effects. We employed PET imaging with [¹¹C]carfentanil to test the hypotheses that acute cigarette smoking increases release of endogenous opioids in the human brain and that smokers have an upregulation of mu opioid receptors (MORs) when compared to nonsmokers. We found no significant changes in binding potential (BP_ND) of [¹¹C]carfentanil between the placebo and the active cigarette sessions, nor did we observe differences in MOR binding between smokers and nonsmokers. Interestingly, we showed that in smokers MOR availability in bilateral superior temporal cortices during the placebo condition was negatively correlated with scores on the Fagerström Test for Nicotine Dependence (FTND). Also in smokers, smoking-induced decreases in [¹¹C]carfentanil binding in frontal cortical regions were associated with self-reports of cigarette liking and wanting. Although we did not show differences between smokers and nonsmokers, the negative correlation with FTND corroborates the role of MORs in superior temporal cortices in nicotine addiction and provides preliminary evidence of a role of endogenous opioid signaling in frontal cortex in nicotine reward.     

Tetrahydrocurcumin Protects against Cadmium-Induced Hypertension,Raised Arterial Stiffness and Vascular Remodeling in Mice

Background

Cadmium (Cd) is a nonessential heavy metal, causing oxidative damage to various tissues and associated with hypertension. Tetrahydrocurcumin (THU), a major metabolite of curcumin, has been demonstrated to be an antioxidant, anti-diabetic, anti-hypertensive and anti-inflammatory agent. In this study, we investigated the protective effect of THU against Cd-induced hypertension, raised arterial stiffness and vascular remodeling in mice.

Methods

Male ICR mice received CdCl₂ (100 mg/l) via drinking water for 8 weeks. THU was administered intragastrically at dose of 50 or 100 mg/kg/day concurrently with Cd treatment.

Results

Administration of CdCl₂ significantly increased arterial blood pressure, blunted vascular responses to vasoactive agents, increased aortic stiffness, and induced hypertrophic aortic wall remodeling by increasing number of smooth muscle cells and collagen deposition, decreasing elastin, and increasing matrix metalloproteinase (MMP)-2 and MMP-9 levels in the aortic medial wall. Supplementation with THU significantly decreased blood pressure, improved vascular responsiveness, and reversed the structural and mechanical alterations of the aortas, including collagen and elastin deposition. The reduction on the adverse response of Cd treatment was associated with upregulated eNOS and downregulated iNOS protein expressions, increased nitrate/nitrite level, alleviated oxidative stress and enhanced antioxidant glutathione. Moreover, THU also reduced the accumulation of Cd in the blood and tissues.

Conclusions

Our results suggest that THU ameliorates cadmium-induced hypertension, vascular dysfunction, and arterial stiffness in mice through enhancing NO bioavailability, attenuating oxidative stress, improving vascular remodeling and decreasing Cd accumulation in other tissues. THU has a beneficial effect in moderating the vascular alterations associated with Cd exposure.     

raf RBD and ubiquitin proteins share similar folds, folding rates and mechanisms despite having unrelated amino acid sequences

Recent experimental and theoretical studies in protein folding suggest that the rates and underlying mechanisms by which proteins attain the native state are largely determined by the topological complexity of a specific fold rather than by the fine details of the amino acid sequences. However, such arguments are based upon the examination of a limited number of protein folds. To test this view, we sought to investigate whether proteins belonging to the ubiquitin superfamily display similar folding behavior. To do so, we compared the folding-unfolding transitions of mammalian ubiquitin (mUbi) with those of its close yeast homologue (yUbi), and to those of the structurally related Ras binding domain (RBD) of the serine/threonine kinase raf that displays no apparent sequence homology with the ubiquitin family members. As demonstrated for mUbi [Krantz, B. A., and Sosnick, T. R. (2000) Biochemistry 39, 11696-11701], we show that a two-state transition model with no burst phase intermediate can describe folding of both yUbi and raf RBD. We further demonstrate that (1) all three proteins refold at rates that are within 1 order of magnitude (1800, 1100, and 370 s(-1) for mUbi, raf RBD, and yUbi, respectively), (2) both mUbi and raf RBD display similar refolding heterogeneity, and (3) the folding free energy barriers of both mUbi and raf RBD display a similar temperature dependence and sensitivity to a stabilizing agent or to mutations of a structurally equivalent central core residue. These findings are consistent with the view that rates and mechanisms for protein folding depend mostly on the complexity of the native structure topology rather than on the fine details of the amino acid sequence.