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1.  A general protocol for the crystallization of membrane proteins for X-ray structural investigation 
Nature protocols  2009;4(5):619-637.
Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.
doi:10.1038/nprot.2009.27
PMCID: PMC4075773  PMID: 19360018
2.  Gene-ontology enrichment analysis in two independent family-based samples highlights biologically plausible processes for autism spectrum disorders 
European Journal of Human Genetics  2011;19(10):1082-1089.
Recent genome-wide association studies (GWAS) have implicated a range of genes from discrete biological pathways in the aetiology of autism. However, despite the strong influence of genetic factors, association studies have yet to identify statistically robust, replicated major effect genes or SNPs. We apply the principle of the SNP ratio test methodology described by O'Dushlaine et al to over 2100 families from the Autism Genome Project (AGP). Using a two-stage design we examine association enrichment in 5955 unique gene-ontology classifications across four groupings based on two phenotypic and two ancestral classifications. Based on estimates from simulation we identify excess of association enrichment across all analyses. We observe enrichment in association for sets of genes involved in diverse biological processes, including pyruvate metabolism, transcription factor activation, cell-signalling and cell-cycle regulation. Both genes and processes that show enrichment have previously been examined in autistic disorders and offer biologically plausibility to these findings.
doi:10.1038/ejhg.2011.75
PMCID: PMC3190264  PMID: 21522181
autism; genome-wide association analysis; pathway analysis; family-based association test; gene ontology
3.  Crystal Structure of the Human tRNA m 1A58 Methyltransferase–tRNA3Lys Complex: Refolding of Substrate tRNA Allows Access to the Methylation Target 
Journal of molecular biology  2015;427(24):3862-3876.
Human tRNA3 Lys is the primer for reverse transcription of HIV; the 3′ end is complementary to the primer-binding site on HIV RNA. The complementarity ends at the 18th base, A58, which in tRNA3 Lys is modified to remove Watson–Crick pairing. Motivated to test the role of the modification in terminating the primer-binding sequence and thus limiting run-on transcription, we asked how the modification of RNA could be accomplished. tRNA m1A58 methyltransferase (m1A58 MTase) methylates N1 of A58, which is buried in the TψC-loop of tRNA, from cofactor S-adenosyl-L-methionine. This conserved tRNA modification is essential for stability of initiator tRNA in Saccharomyces cerevisiae. Reported here, three structures of human tRNA m1A58 MTase in complex with human tRNA3 Lys and the product S-adenosyl-L-homocysteine show a dimer of heterodimers in which each heterodimer comprises a catalytic chain, Trm61, and a homologous but noncatalytic chain, Trm6, repurposed as a tRNA-binding subunit that acts in trans; tRNAs bind across the dimer interface such that Trm6 from the opposing heterodimer brings A58 in to the active site of Trm61. T-loop and D-loop are splayed apart showing how A58, normally buried in tRNA, becomes accessible for modification. This result has broad impact on our understanding of the mechanisms of modifying internal sites in folded tRNA. The structures serve as templates for design of inhibitors that could be used to test tRNA m1A58 MTase's impact on retroviral priming and transcription.
Graphical abstract
doi:10.1016/j.jmb.2015.10.005
PMCID: PMC4663122  PMID: 26470919
X-ray; RNA modification; S-adenosyl-L-methionine; 1-methyladenosine; HIV
4.  Cyclophilin D, a target for counteracting skeletal muscle dysfunction in mitochondrial myopathy 
Human Molecular Genetics  2015;24(23):6580-6587.
Muscle weakness and exercise intolerance are hallmark symptoms in mitochondrial disorders. Little is known about the mechanisms leading to impaired skeletal muscle function and ultimately muscle weakness in these patients. In a mouse model of lethal mitochondrial myopathy, the muscle-specific Tfam knock-out (KO) mouse, we previously demonstrated an excessive mitochondrial Ca2+ uptake in isolated muscle fibers that could be inhibited by the cyclophilin D (CypD) inhibitor, cyclosporine A (CsA). Here we show that the Tfam KO mice have increased CypD levels, and we demonstrate that this increase is a common feature in patients with mitochondrial myopathy. We tested the effect of CsA treatment on Tfam KO mice during the transition from a mild to terminal myopathy. CsA treatment counteracted the development of muscle weakness and improved muscle fiber Ca2+ handling. Importantly, CsA treatment prolonged the lifespan of these muscle-specific Tfam KO mice. These results demonstrate that CsA treatment is an efficient therapeutic strategy to slow the development of severe mitochondrial myopathy.
doi:10.1093/hmg/ddv361
PMCID: PMC4634369  PMID: 26374844
5.  Exercise Increases Pyridinoline Cross-linking and Counters the Mechanical Effects of Concurrent Lathyrogenic Treatment 
Bone  2015;81:327-337.
The collagen cross-link profile of bone, associated with bone strength and fracture toughness, is tightly regulated (affecting cross-link quantity, type, lysine hydroxylation and maturity) and may contribute to the improvements in bone quality during exercise. We hypothesized that 1) exercise promotes mature cross-link formation, 2) increased mature cross-linking is accompanied by shifts in lysine hydroxylation, and 3) these changes in collagen cross-link profile have positive effects on mechanical properties. Growing male C57Bl6 mice were treated with 30 min/day of running exercise, 350 mg/kg/day β-aminopropionitrile (BAPN) injected subcutaneously to inhibit enzymatic collagen cross-linking, or both exercise and BAPN, from 5 to 8 weeks of age. Bone collagen cross-linking profile, mechanical properties, morphology, and mineralization were measured from the tibiae. Cross-link measures, including immature, pyridinoline, pyrrole and pentosidine cross-links, ratios reflecting cross-link maturity and hydroxylation, and mineralization were tested for their importance to mechanical properties across 8 week groups through correlation analyses and step-wise linear regressions.
BAPN treatment significantly reduced lysylpyridinoline, pyrrole, hydroxylysinorleucine, and total mature collagen cross-linking, resulting in decreased bone elastic modulus and increased yield strain despite a marginal increase in TMD. Exercise caused a shift toward pyridinoline cross-linking, with increased hydroxylysylpyridinoline and decreased pyrrole cross-linking resulting in total mature cross-linking and estimated tissue level mechanical properties matching sedentary control levels. Exercise superimposed on BAPN treatment increased total mature cross-linking from BAPN to control levels, but did so by increasing pyridinoline, not pyrrole, cross-links. Exercise also counteracted the BAPN effects on modulus and strain, without a change in TMD. Pyrrole cross-linking was the strongest correlate of modulus (r=0.470, p<0.01) and yield strain (r=−0.467, p<0.01). Cross-links with similar levels of telopeptide lysine hydroxylation to pyrrole (lysylpyridinoline and hydroxylysinorleucine) also correlated with modulus and strain to a lesser extent. In conclusion, exercise in growing mice promotes pyridinoline collagen cross-linking in bone, the resulting increase in total mature cross-linking is sufficient to counteract the mechanical effects of concurrent cross-link inhibition, and this responsiveness to loading is a potential means by which exercise might improve bone quality in diseased or otherwise compromised bone.
doi:10.1016/j.bone.2015.07.030
PMCID: PMC4640975  PMID: 26211995
Collagen cross-linking; mechanics; lathyrism; exercise; β-aminopropionitrile; bone quality
6.  Diverse Functional Outcomes of Plasmodium falciparum Ligation of EPCR: Potential Implications for Malarial Pathogenesis 
Cellular microbiology  2015;17(12):1883-1899.
Summary
P. falciparum-infected erythrocytes (IRBC) expressing the domain cassettes (DC) 8 and 13 of the cytoadherent ligand PfEMP1 adhere to the endothelial protein C receptor (EPCR). By interfering with EPCR anti-coagulant and pro-endothelial barrier functions, IRBC adhesion could promote coagulation and vascular permeability that contribute to the pathogenesis of cerebral malaria. In this study, we examined adhesion of DC8- and DC13-expressing parasite lines to endothelial cells from different microvasculature, and the consequences of EPCR engagement on endothelial cell function. We found that IRBC from IT4var19 (DC8) and IT4var07 (DC13) parasite lines adhered to human brain, lung, and dermal endothelial cells under shear stress. However, the relative contribution of EPCR to parasite cytoadherence on the different types of endothelial cell varied. We also observed divergent functional outcomes for DC8 CIDRα1.1 and DC13 CIDRα1.4 domains. IT4var07 CIDRα1.4 inhibited generation of activated protein C (APC) on lung and dermal endothelial cells and blocked the APC-EPCR binding interaction on brain endothelial cells. IT4var19 CIDRα1.1 inhibited thrombin-induced endothelial barrier dysfunction in lung endothelial cells, while IT4var07 CIDRα1.4- inhibited the protective effect of APC on thrombin-induced permeability. Overall, these findings reveal a much greater complexity of how CIDRα1-expressing parasites may modulate malaria pathogenesis through EPCR adhesion.
doi:10.1111/cmi.12479
PMCID: PMC4661070  PMID: 26119044
7.  Plasmodium falciparum adhesion domains linked to severe malaria differ in blockade of endothelial protein C receptor 
Cellular microbiology  2015;17(12):1868-1882.
Summary
Cytoadhesion of Plasmodium falciparum-infected erythrocytes to endothelial protein C receptor (EPCR) is associated with severe malaria. It has been postulated that parasite binding could exacerbate microvascular coagulation and endothelial dysfunction in cerebral malaria by impairing the protein C-EPCR interaction, but the extent of binding inhibition has not been fully determined. Here we expressed the cysteine rich interdomain region (CIDRα1) domain from a variety of DC8 and DC13 P. falciparum erythrocyte membrane 1 (PfEMP1) proteins and show they interact in a distinct manner with EPCR resulting in weak, moderate, and strong inhibition of the APC-EPCR interaction. Overall, there was a positive correlation between CIDRα1-EPCR binding activity and APC blockade activity. In addition, our analysis from a combination of mutagenesis and blocking antibodies finds that an Arg81 (R81) in EPCR plays a pivotal role in CIDRα1 binding, but domains with weak and strong APC blockade activity were distinguished by their sensitivity to inhibition by anti-EPCR mAb 1535, implying subtle differences in their binding footprints. These data reveal a previously unknown functional heterogeneity in the interaction between P. falciparum and EPCR and have major implications for understanding the distinct clinical pathologies of cerebral malaria and developing new treatment strategies.
doi:10.1111/cmi.12478
PMCID: PMC4661071  PMID: 26118955
Plasmodium falciparum; malaria; antigenic variation; adhesion
8.  Truth-telling to the patient, family, and the sexual partner: a rights approach to the role of healthcare providers in adult HIV disclosure in China 
AIDS care  2015;27(0 1):83-89.
Patients’ rights are central in today’s legislation and social policies related to health care, including HIV care, in not only Western countries but around the world. However, given obvious socio-cultural differences it is often asked how or to what extent patients’ rights should be respected in non-Western societies such as China. In this paper, it is argued that the patients’ rights framework is compatible with Chinese culture, and that from the perspective of contemporary patient rights healthcare providers have a duty to disclose truthfully the diagnosis and prognosis to their patients, that the Chinese cultural practice of involving families in care should – with consent from the patient – be promoted out of respect for patients’ rights and well-being, and that healthcare providers should be prepared to address the issue of disclosing a patient’s HIV status to sexual partner(s). Legally, the provider should be permitted to disclose without consent from the patient but not obliged to in all cases. The decision to do this should be taken with trained sensitivity to a range of ethically relevant considerations. Post-disclosure counseling or psychological support should be in place to address the concerns of potentially adverse consequences of provider-initiated disclosure and to maximize the psychosocial and medical benefits of the disclosure. There is an urgent need for healthcare providers to receive training in ethics and disclosure skills. This paper concludes also with some suggestions for improving the centerpiece Chinese legislation, State Council’s “Regulations on AIDS Prevention and Control” (2006), to further safeguard the rights and well-being of HIV patients.
doi:10.1080/09540121.2015.1071772
PMCID: PMC4685610  PMID: 26616129
HIV disclosure; patients’ rights; privacy; cultural differences; family; China; partner notification
9.  Heavy eye syndrome versus sagging eye syndrome in high myopia 
Background
Heavy eye syndrome (HES) presents with esotropia and limited abduction due to superotemporal globe shift relative to the extraocular muscles. Sagging eye syndrome (SES) was originally described in nonmyopic patients exhibiting distance esotropia and cyclovertical strabismus with limited supraduction due to lateral rectus muscle inferodisplacement caused by degeneration of the lateral rectus–superior rectus (LR-SR) band. We hypothesized that SES might also cause strabismus in high myopia.
Methods
Eleven strabismic subjects with high myopia underwent ophthalmological examination and orbital magnetic resonance imaging (MRI) to assess quantitative orbital anatomy.
Results
Of 11 subjects, 5 had HES; 6, SES. Mean axial length in subjects with HES was 32 ± 5 mm; in subjects with SES, 32 ± 6 mm. Average distance esotropia in subjects with HES was 61Δ ± 39Δ; hypotropia was 26Δ ± 21Δ. Average distance esotropia in subjects with SES was 23Δ ± 57Δ; hypertropia was 2Δ ± 2Δ. All 5 subjects with HES had superotemporal globe prolapse; the LR-SR band was thinned in 6 orbits and ruptured in 2. The mean angle between the lateral rectus and superior rectus muscles in HES was 121° ± 7°. In SES the LR-SR band was thinned in 7 orbits and ruptured in 5, with superotemporal soft tissue prolapse. The mean angle between the lateral rectus and superior rectus muscles was 104° ± 11°, significantly less than in HES (P < 0.001).
Conclusions
SES occurs in highly myopic patients who also exhibit less relative globe dislocation than in HES. Unlike HES, SES exhibits superotemporal soft tissue prolapse that may limit superotemporal globe shift. The distinction is important because surgery for HES uniquely requires creation of a surgical connection between the superior rectus and lateral rectus muscles, whereas SES may be treated with conventional surgery. SES can cause strabismus in high axial myopia. Orbital MRI is useful in differentiating SES from HES.
doi:10.1016/j.jaapos.2015.08.012
PMCID: PMC4688571  PMID: 26691027
10.  Development of the gut microbiota and mucosal IgA responses in twins and gnotobiotic mice 
Nature  2016;534(7606):263-266.
Immunoglobulin A (IgA), the major class of antibody secreted by the gut mucosa, is an important contributor to gut barrier function1–3. The repertoire of IgA bound to gut bacteria reflects both T cell-dependent and -independent pathways4,5, plus glycans present on the antibody’s secretory component6. Human gut bacterial taxa targeted by IgA in the setting of intestinal barrier dysfunction are capable of producing intestinal pathology when isolated and transferred to gnotobiotic mice7,8. A complex reorientation of gut immunity occurs as infants transition from passively acquired IgA present in breast milk to host-derived IgA9–11. How IgA responses co-develop with assembly of the microbiota during this period remains poorly understood. Here, we (i) identify a set of age-discriminatory bacterial taxa whose representations define a program of microbiota assembly/maturation during the first 2 postnatal years that is shared across 40 healthy USA twin pairs; (ii) describe a pattern of progression of gut mucosal IgA responses to bacterial members of the microbiota that is highly distinctive for family members (twin pairs) during the first several postnatal months then generalizes across pairs in the second year; and (iii) assess the effects of zygosity, birth mode and breast feeding. Age-associated differences in these IgA responses can be recapitulated in young germ-free mice, colonized with fecal microbiota obtained from two twin pairs at 6 and 18 months of age, and fed a sequence of human diets that simulate the transition from milk feeding to complementary foods. The majority of these responses were robust to diet suggesting that ‘intrinsic’ properties of community members play a dominant role in dictating IgA responses. The approach described can be used to define gut mucosal immune development in health and disease states and help discover ways for repairing or preventing perturbations in this facet of host immunity.
doi:10.1038/nature17940
PMCID: PMC4902178  PMID: 27279225
11.  Demographic and clinical profiles of Plasmodium falciparum and Plasmodium vivax patients at a tertiary care centre in southwestern India 
Malaria Journal  2016;15:569.
Background
Malaria remains an important cause of morbidity and mortality in India. Though many comprehensive studies have been carried out in Africa and Southeast Asia to characterize and examine determinants of Plasmodium falciparum and Plasmodium vivax malaria pathogenesis, fewer have been conducted in India.
Methods
A prospective study of malaria-positive individuals was conducted at Goa Medical College and Hospital (GMC) from 2012 to 2015 to identify demographic, diagnostic and clinical indicators associated with P. falciparum and P. vivax infection on univariate analysis.
Results
Between 2012 and 2015, 74,571 febrile individuals, 6287 (8.4%) of whom were malaria positive, presented to GMC. The total number of malaria cases at GMC increased more than two-fold over four years, with both P. vivax and P. falciparum cases present year-round. Some 1116 malaria-positive individuals (mean age = 27, 91% male), 88.2% of whom were born outside of Goa and 51% of whom were construction workers, were enroled in the study. Of 1088 confirmed malaria-positive patients, 77.0% had P. vivax, 21.0% had P. falciparum and 2.0% had mixed malaria. Patients over 40 years of age and with P. falciparum infection were significantly (p < 0.001) more likely to be hospitalised than younger and P. vivax patients, respectively. While approximately equal percentages of hospitalised P. falciparum (76.6%) and P. vivax (78.9%) cases presented with at least one WHO severity indicator, a greater percentage of P. falciparum inpatients presented with at least two (43.9%, p < 0.05) and at least three (29.9%, p < 0.01) severity features. There were six deaths among the 182 hospitalised malaria positive patients, all of whom had P. falciparum.
Conclusion
During the four year study period at GMC, the number of malaria cases increased substantially and the greatest burden of severe disease was contributed by P. falciparum.
doi:10.1186/s12936-016-1619-5
PMCID: PMC5123287  PMID: 27884146
MESA-ICEMR; Goa; Epidemiology; Diagnostics; Severity; Characteristics; Features
12.  Assessing characteristics of RNA amplification methods for single cell RNA sequencing 
BMC Genomics  2016;17:966.
Background
Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known.
Results
Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5–10 molecules.
Conclusions
Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-3300-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-016-3300-3
PMCID: PMC5122016  PMID: 27881084
Single-cell RNA-sequencing; Biotechnology; Bioinformatics; Genomics
13.  Effects of Pile Driving on the Residency and Movement of Tagged Reef Fish 
PLoS ONE  2016;11(11):e0163638.
The potential effects of pile driving on fish populations and commercial fisheries have received significant attention given the prevalence of pile driving occurring in coastal habitats throughout the world. Behavioral impacts of sound generated from these activities on fish typically have a greater area of influence than physical injury, and may therefore adversely affect a greater portion of the local population. This study used acoustic telemetry to assess the movement, residency, and survival of 15 sheepshead (Archosargus probatocephalus) and 10 grey snapper (Lutjanus griseus) in Port Canaveral, Florida, USA, in response to 35 days of pile driving at a wharf complex. No obvious signs of mortality or injury to tagged fish were evident from the data. Received sound pressure levels from pile strikes on the interior of the wharf, where reef fish primarily occur, were on average 152–157 dB re 1 μPa (peak). No significant decrease in sheepshead daytime residency was observed during pile driving within the central portion of the wharf and area of highest sound exposure, and no major indicators of displacement from the exposure wharf with the onset of pile driving were observed. There was evidence of potential displacement from the exposure wharf that coincided with the start of pile driving observed for 2 out of 4 grey snapper, along with a decrease in daytime residency for a subset of this species with high site fidelity prior to the event. Results indicate that snapper may be more likely to depart an area of pile driving disturbance more readily than sheepshead, but were less at risk for behavioral impact given the lower site fidelity of this species.
doi:10.1371/journal.pone.0163638
PMCID: PMC5120787  PMID: 27880786
14.  K63-Linked Ubiquitination Targets Toxoplasma gondii for Endo-lysosomal Destruction in IFNγ-Stimulated Human Cells 
PLoS Pathogens  2016;12(11):e1006027.
Toxoplasma gondii is the most common protozoan parasitic infection in man. Gamma interferon (IFNγ) activates haematopoietic and non-haematopoietic cells to kill the parasite and mediate host resistance. IFNγ-driven host resistance pathways and parasitic virulence factors are well described in mice, but a detailed understanding of pathways that kill Toxoplasma in human cells is lacking. Here we show, that contrary to the widely held belief that the Toxoplasma vacuole is non-fusogenic, in an immune-stimulated environment, the vacuole of type II Toxoplasma in human cells is able to fuse with the host endo-lysosomal machinery leading to parasite death by acidification. Similar to murine cells, we find that type II, but not type I Toxoplasma vacuoles are targeted by K63-linked ubiquitin in an IFNγ-dependent manner in non-haematopoetic primary-like human endothelial cells. Host defence proteins p62 and NDP52 are subsequently recruited to the type II vacuole in distinct, overlapping microdomains with a loss of IFNγ-dependent restriction in p62 knocked down cells. Autophagy proteins Atg16L1, GABARAP and LC3B are recruited to <10% of parasite vacuoles and show no parasite strain preference, which is consistent with inhibition and enhancement of autophagy showing no effect on parasite replication. We demonstrate that this differs from HeLa human epithelial cells, where type II Toxoplasma are restricted by non-canonical autophagy leading to growth stunting that is independent of lysosomal acidification. In contrast to mouse cells, human vacuoles do not break. In HUVEC, the ubiquitinated vacuoles are targeted for destruction in acidified LAMP1-positive endo-lysosomal compartments. Consequently, parasite death can be prevented by inhibiting host ubiquitination and endosomal acidification. Thus, K63-linked ubiquitin recognition leading to vacuolar endo-lysosomal fusion and acidification is an important, novel virulence-driven Toxoplasma human host defence pathway.
Author Summary
Toxoplasma gondii is an intracellular parasite that can invade nucleated cells of any warm-blooded animal into a compartment known as a parasitophorous vacuole (PV). The production of gamma interferon (IFNγ) drives the restriction and killing of Toxoplasma. It is not fully known how the parasite inside the PV is eliminated in human cells, although its fate depends on the cell type into which it invades. In IFNγ-stimulated epithelial HeLa cells for instance growth of type II parasites is restricted 24h post-infection by employing the cellular autophagy pathway. Distinctly, we show here that in human endothelial cells the parasite is destroyed by fusion of the PV with the cell’s endo-lysosomal pathway as early as 6h post-infection. This process, which is at odds with the normally non-fusogenic nature of the PV, is dependent on IFNγ. Parasite death follows Lysine63-linked ubiquitination of the PV and is specific to type II Toxoplasma. Our results demonstrate for the first time that vacuolar acidification leading to parasite death is central to controlling infection by Toxoplasma in human endothelial cells.
doi:10.1371/journal.ppat.1006027
PMCID: PMC5119857  PMID: 27875583
15.  Bacterial danger sensing 
Journal of molecular biology  2015;427(23):3744-3753.
Here we propose that bacteria detect and respond to threats posed by other bacteria via an innate immune-like process that we term danger sensing. We find support for this contention by reexamining existing literature from the perspective that intermicrobial antagonism, not opportunistic pathogenesis, is the major evolutionary force shaping the defensive behaviors of most bacteria. We conclude that many bacteria possess danger sensing pathways composed of a danger signal receptor and corresponding signal transduction mechanism that regulate pathways important for survival in the presence of the perceived competitor.
Graphical abstract
doi:10.1016/j.jmb.2015.09.018
PMCID: PMC4658316  PMID: 26434507
16.  Coupling of mRNA structure rearrangement to ribosome movement during bypassing of non-coding regions 
Cell  2015;163(5):1267-1280.
SUMMARY
Nearly half of the ribosomes translating a particular bacteriophage T4 mRNA bypass 50 nucleotides in its middle, resuming translation 3’ of this region. How this large-scale, specific hop occurs, and what determines whether a ribosome bypasses, remains unclear. We apply single-molecule fluorescence with zero-mode waveguides to track individual Escherichia coli ribosomes during translation of T4's gene 60 mRNA. Ribosomes that bypass are characterized by a 10- to 20-fold longer pause in a non-canonical rotated state at the take-off codon. During the pause, mRNA secondary structure rearrangements are coupled to ribosome forward movement, facilitated by nascent peptide interactions that disengage the ribosome anticodon-codon interactions for slippage. Close to the landing site, the ribosome then scans the mRNA in search of the optimal base-pairing interactions. Our results provide a mechanistic and conformational framework for bypassing, highlighting a non-canonical ribosomal state to allow for mRNA structure refolding to drive large-scale ribosome movements.
doi:10.1016/j.cell.2015.10.064
PMCID: PMC4813330  PMID: 26590426
17.  Femoral nerve blockade using various concentrations of local anesthetic for knee arthroscopy in the pediatric population 
Journal of Pain Research  2016;9:1073-1079.
Background
Femoral nerve blockade (FNB) provides effective postoperative analgesia in children undergoing arthroscopic knee surgery as evidenced by their opioid-sparing effects and decreased postoperative pain scores. Increasing the local anesthetic concentration in peripheral nerve blockade for adults undergoing orthopedic surgery has been shown to be beneficial, increasing block success rate, and providing a longer duration of analgesia. The effect of increasing the concentration of local anesthetic in extremity blocks in children remains largely unexplored.
Methods
We retrospectively evaluated the effectiveness of FNB using three concentrations of local anesthetic (ropivacaine 0.2%, bupivacaine 0.25%, and ropivacaine 0.5%) in children and adolescents undergoing arthroscopic knee surgery. The primary outcome evaluated was postoperative opioid consumption before discharge. Secondary outcomes included post-anesthesia care unit (PACU) and hospital discharge times, first pain score in PACU, and the incidence of adverse events.
Results
Two hundred and sixty-nine children and adolescents who received a FNB for arthroscopic knee surgery from January 2010 to December 2013 were included for analysis. Local anesthetic used in FNB was ropivacaine 0.2% in 116 (43%) cases, ropivacaine 0.5% in 75 (28%) cases, and bupivacaine 0.25% in 78 (29%) cases. Median postoperative opioid consumption (mg/kg intravenous morphine equivalents) in the ropivacaine 0.5% group was 0 mg/kg (interquartile ranges [IQR]: 0 mg, 0.03 mg/kg) compared to 0.02 mg/kg (IQR: 0, 0.08 mg/kg) in the ropivacaine 0.2% group and 0.01 mg/kg (IQR: 0, 0.08 mg/kg) in the bupivacaine 0.25% group (p=0.009). Median PACU time was shortest in the ropivacaine 0.5% group (47 min; IQR: 36, 68 min) compared to the ropivacaine 0.2% (58 min; IQR: 41, 77) and bupivacaine 0.25% (54 min; IQR: 35, 75 min) groups (p=0.040). Among groups, there were no significant differences in first postoperative pain scores or incidence of nausea and vomiting. No patient in any group experienced a serious adverse event.
Conclusion
The results suggest that ropivacaine 0.5% for FNB offers superior postoperative analgesia in the form of decreased postoperative opioid consumption and earlier PACU/hospital discharge, when compared to ropivacaine 0.2% and bupivacaine 0.25% in the pediatric population.
Level of evidence
III, Retrospective Comparative Study.
doi:10.2147/JPR.S117692
PMCID: PMC5125980  PMID: 27920573
anesthesia; regional; nerve block; pain; postoperative; local anesthetic; child; adolescent
18.  Home peripheral nerve catheters: the first 24 months of experience at a children’s hospital 
Journal of Pain Research  2016;9:1067-1072.
Context
Home peripheral nerve catheters (PNCs) have become common practice for adult patients after major orthopedic surgery. However, use in pediatric patients is a recent application.
Objectives
The purpose of this study was to review the demographics and outcomes of pediatric patients receiving a PNC at our institution.
Methods
This retrospective study included patients from October 2012 through October 2014 undergoing orthopedic procedures with a PNC placed for postoperative pain management.
Results
A total of 118 patients aged 3.2–25.3 years were identified. The types of catheters included femoral (80.5%), interscalene (11.9%), sciatic (5.9%), and supraclavicular (1.7%). The majority of patients were discharged to home on the day of surgery (77.1%). In the postanesthetic care unit, the average pain score was 2.5, the incidence of nausea/emesis was 5.9%, and the need for opioid administration was 50.8%. There were no major complications. Minor complications included a 7.6% rate of early catheter removal with 5.9% of those due to catheter leakage and an unsecure dressing. There was one case of metallic taste in the mouth without other symptoms of local anesthetic toxicity that resolved without further complication.
Conclusion
The implementation of a home PNC program in pediatric patients at our institution has been highly successful with a high rate of ambulatory catheters, low pain scores, low rates of nausea and vomiting, and no serious complications. Minor complications included leaking of the catheter and early discontinuation of the catheter.
doi:10.2147/JPR.S110947
PMCID: PMC5125993  PMID: 27920572
peripheral nerve catheter; pediatric; regional anesthesia
19.  Perception of Sung Speech in Bimodal Cochlear Implant Users 
Trends in Hearing  2016;20:2331216516669329.
Combined use of a hearing aid (HA) and cochlear implant (CI) has been shown to improve CI users’ speech and music performance. However, different hearing devices, test stimuli, and listening tasks may interact and obscure bimodal benefits. In this study, speech and music perception were measured in bimodal listeners for CI-only, HA-only, and CI + HA conditions, using the Sung Speech Corpus, a database of monosyllabic words produced at different fundamental frequencies. Sentence recognition was measured using sung speech in which pitch was held constant or varied across words, as well as for spoken speech. Melodic contour identification (MCI) was measured using sung speech in which the words were held constant or varied across notes. Results showed that sentence recognition was poorer with sung speech relative to spoken, with little difference between sung speech with a constant or variable pitch; mean performance was better with CI-only relative to HA-only, and best with CI + HA. MCI performance was better with constant words versus variable words; mean performance was better with HA-only than with CI-only and was best with CI + HA. Relative to CI-only, a strong bimodal benefit was observed for speech and music perception. Relative to the better ear, bimodal benefits remained strong for sentence recognition but were marginal for MCI. While variations in pitch and timbre may negatively affect CI users’ speech and music perception, bimodal listening may partially compensate for these deficits.
doi:10.1177/2331216516669329
PMCID: PMC5117252  PMID: 27837051
cochlear implants; pitch perception; atypical speech; electro-acoustic stimulation; bimodal perception
20.  Chitinase-3-like 1 protein (CHI3L1) locus influences cerebrospinal fluid levels of YKL-40 
BMC Neurology  2016;16:217.
Background
Alzheimer’s disease (AD) pathology appears several years before clinical symptoms, so identifying ways to detect individuals in the preclinical stage is imperative. The cerebrospinal fluid (CSF) Tau/Aβ42 ratio is currently the best known predictor of AD status and cognitive decline, and the ratio of CSF levels of chitinase-3-like 1 protein (CHI3L1, YKL-40) and amyloid beta (Aβ42) were reported as predictive, but individual variability and group overlap inhibits their utility for individual diagnosis making it necessary to find ways to improve sensitivity of these biomarkers.
Methods
We used linear regression to identify genetic loci associated with CSF YKL-40 levels in 379 individuals (80 cognitively impaired and 299 cognitively normal) from the Charles F and Joanne Knight Alzheimer’s Disease Research Center. We tested correlations between YKL-40 and CSF Tau/Aβ42 ratio, Aβ42, tau, and phosphorylated tau (ptau181). We used studentized residuals from a linear regression model of the log-transformed, standardized protein levels and the additive reference allele counts from the most significant locus to adjust YKL-40 values and tested the differences in correlations with CSF Tau/Aβ42 ratio, Aβ42, tau, and ptau181.
Results
We found that genetic variants on the CH13L1 locus were significantly associated with CSF YKL-40 levels, but not AD risk, age at onset, or disease progression. The most significant variant is a reported expression quantitative trait locus for CHI3L1, the gene which encodes YKL-40, and explained 12.74 % of the variance in CSF YKL-40 in our study. YKL-40 was positively correlated with ptau181 (r = 0.521) and the strength of the correlation significantly increased with the addition of genetic information (r = 0.573, p = 0.006).
Conclusions
CSF YKL-40 levels are likely a biomarker for AD, but we found no evidence that they are an AD endophenotype. YKL-40 levels are highly regulated by genetic variation, and by including genetic information the strength of the correlation between YKL-40 and ptau181 levels is significantly improved. Our results suggest that studies of potential biomarkers may benefit from including genetic information.
Electronic supplementary material
The online version of this article (doi:10.1186/s12883-016-0742-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12883-016-0742-9
PMCID: PMC5105244  PMID: 27832767
CHI3L1; YKL-40; Cerebrospinal fluid; Alzheimer disease
21.  Real-Time Pathogen Detection in the Era of Whole-Genome Sequencing and Big Data: Comparison of k-mer and Site-Based Methods for Inferring the Genetic Distances among Tens of Thousands of Salmonella Samples 
PLoS ONE  2016;11(11):e0166162.
The adoption of whole-genome sequencing within the public health realm for molecular characterization of bacterial pathogens has been followed by an increased emphasis on real-time detection of emerging outbreaks (e.g., food-borne Salmonellosis). In turn, large databases of whole-genome sequence data are being populated. These databases currently contain tens of thousands of samples and are expected to grow to hundreds of thousands within a few years. For these databases to be of optimal use one must be able to quickly interrogate them to accurately determine the genetic distances among a set of samples. Being able to do so is challenging due to both biological (evolutionary diverse samples) and computational (petabytes of sequence data) issues. We evaluated seven measures of genetic distance, which were estimated from either k-mer profiles (Jaccard, Euclidean, Manhattan, Mash Jaccard, and Mash distances) or nucleotide sites (NUCmer and an extended multi-locus sequence typing (MLST) scheme). When analyzing empirical data (whole-genome sequence data from 18,997 Salmonella isolates) there are features (e.g., genomic, assembly, and contamination) that cause distances inferred from k-mer profiles, which treat absent data as informative, to fail to accurately capture the distance between samples when compared to distances inferred from differences in nucleotide sites. Thus, site-based distances, like NUCmer and extended MLST, are superior in performance, but accessing the computing resources necessary to perform them may be challenging when analyzing large databases.
doi:10.1371/journal.pone.0166162
PMCID: PMC5104361  PMID: 27832109
22.  Potent and Selective Modulation of the RhlR Quorum Sensing Receptor using Non-Native Ligands – An Emerging Target for Virulence Control in Pseudomonas aeruginosa 
Pseudomonas aeruginosa uses N-acylated L-homoserine lactone signals and a triumvirate of LuxR-type receptor proteins – LasR, RhlR, and QscR – for quorum sensing (QS). Each of these receptors can contribute to QS activation or repression, and thereby, the control of myriad virulence phenotypes in this pathogen. LasR has traditionally been considered at the top of the QS receptor hierarchy in P. aeruginosa; however, recent reports suggest that RhlR plays a more prominent role in infection than originally predicted, in some circumstances superseding LasR. Herein, we report the characterization of a set of synthetic, small molecule agonists and antagonists of RhlR. Using E. coli reporter strains, we demonstrate that many of these compounds can selectively activate or inhibit RhlR instead of LasR and QscR. Moreover, several molecules maintain their activities in P. aeruginosa at concentrations analogous to native RhlR-signal levels. These compounds represent useful chemical probes to study the role of RhlR in the complex QS circuitry of P. aeruginosa, its direct (and indirect) effects on virulence, and its overall merit as a target for anti-infective therapy.
doi:10.1002/cbic.201500357
PMCID: PMC4648260  PMID: 26460240
N-acylated L-homoserine lactone; LuxR-type receptor; RhlR; virulence; quorum sensing
23.  Creative contributory contests (CCC) to spur innovation in sexual health: Two cases and a guide for implementation 
Sexually transmitted diseases  2015;42(11):625-628.
Sexual health campaigns are often designed “top-down” by public health experts, failing to engage key populations. Using the power of crowdsourcing to shape a “bottom-up” approach, this note describes two creative contributory contests (CCC) to enhance sexual health campaigns. We provide guidance for designing CCCs to improve HIV and other STD testing.
doi:10.1097/OLQ.0000000000000349
PMCID: PMC4610177  PMID: 26462186
HIV; STD; community engagement; contest; innovation
24.  Single-cell systems level analysis of human Toll-Like-Receptor activation defines a chemokine signature in Systemic Lupus Erythematosus 
Background
Activation of Toll-Like Receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in Systemic Lupus Erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has previously not been described.
Objective
To characterize TLR activation across multiple immune cell subsets and individuals, with the goal of establishing a reference framework against which to compare pathological processes.
Methods
Peripheral whole blood samples were stimulated with TLR ligands, and analyzed by mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied seventeen healthy volunteer donors and eight newly diagnosed untreated SLE patients.
Results
Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of NK and T cells selectively induced NF-κB in response to TLR2 ligands. CD14hi monocytes exhibited the most polyfunctional cytokine expression patterns, with over 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared amongst a group of healthy donors, with minimal intra- and inter- individual variability. Furthermore, autoimmune disease altered baseline cytokine production, as newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity.
Conclusion
Mass cytometry analysis defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in inflammatory disease, such as SLE.
doi:10.1016/j.jaci.2015.04.008
PMCID: PMC4640970  PMID: 26037552
Mass cytometry; Toll-Like-Receptors; systemic lupus erythematosus; inflammation; monocytes; MCP1
25.  Acute Myeloid Leukemia with MYC Rearrangement and JAK2 V617F Mutation 
Cancer genetics  2015;208(11):571-574.
Little is known about MYC dysregulation in myeloid malignancies, and we can find no published studies that have evaluated MYC protein expression in primary cases of myelodysplastic syndromes (MDS) or acute myeloid leukemias (AML). We describe the clinical, morphologic, immunophenotypic, cytogenetic, and molecular genetic findings in two MDS/AML cases that contained both MYC rearrangement and JAK2-V617F mutation. We demonstrate MYC protein expression by immunohistochemistry in both patients.
doi:10.1016/j.cancergen.2015.06.004
PMCID: PMC4835801  PMID: 26382622
acute myeloid leukemia; blastic plasmacytoid dendritic cell neoplasm; MYC; JAK2-V617F mutation

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