Computational analysis of anti-HIV-1 antibody neutralization panel data to identify potential functional epitope residues

Advances in single-cell antibody cloning methods have led to the identification of a variety of broadly neutralizing anti–HIV-1 antibodies. We developed a computational tool (Antibody Database) to help identify critical residues on the HIV-1 envelope protein whose natural variation affects antibody activity. Our simplifying assumption was that, for a given antibody, a significant portion of the dispersion of neutralization activity across a panel of HIV-1 strains is due to the amino acid identity or glycosylation state at a small number of specific sites, each acting independently. A model of an antibody’s neutralization IC_(50) was developed in which each site contributes a term to the logarithm of the modeled IC_(50). The analysis program attempts to determine the set of rules that minimizes the sum of the residuals between observed and modeled IC_(50) values. The predictive quality of the identified rules may be assessed in part by whether there is support for rules within individual viral clades. As a test case, we analyzed antibody 8ANC195, an anti-glycoprotein gp120 antibody of unknown specificity. The model for this antibody indicated that several glycosylation sites were critical for neutralization. We evaluated this prediction by measuring neutralization potencies of 8ANC195 against HIV-1 in vitro and in an antibody therapy experiment in humanized mice. These experiments confirmed that 8ANC195 represents a distinct class of glycan-dependent anti–HIV-1 antibody and validated the utility of computational analysis of neutralization panel data

The Impact of Inpatient Boarding on ED Efficiency: A Discrete-Event Simulation Study

In this study, a discrete-event simulation approach was used to model Emergency Department’s (ED) patient flow to investigate the effect of inpatient boarding on the ED efficiency in terms of the National Emergency Department Crowding Scale (NEDOCS) score and the rate of patients who leave without being seen (LWBS). The decision variable in this model was the boarder-released-ratio defined as the ratio of admitted patients whose boarding time is zero to all admitted patients. Our analysis shows that the Overcrowded+ (a NEDOCS score over 100) ratio decreased from 88.4% to 50.4%, and the rate of LWBS patients decreased from 10.8% to 8.4% when the boarder-released-ratio changed from 0% to 100%. These results show that inpatient boarding significantly impacts both the NEDOCS score and the rate of LWBS patient and this analysis provides a quantification of the impact of boarding on emergency department patient crowding

In Vivo Systematic Analysis of Candida albicans Zn2-Cys6 Transcription Factors Mutants for Mice Organ Colonization

The incidence of fungal infections in immuno-compromised patients increased considerably over the last 30 years. New treatments are therefore needed against pathogenic fungi. With Candida albicans as a model, study of host-fungal pathogen interactions might reveal new sources of therapies. Transcription factors (TF) are of interest since they integrate signals from the host environment and participate in an adapted microbial response. TFs of the Zn2-Cys6 class are specific to fungi and are important regulators of fungal metabolism. This work analyzed the importance of the C. albicans Zn2-Cys6 TF for mice kidney colonization. For this purpose, 77 Zn2-Cys6 TF mutants were screened in a systemic mice model of infection by pools of 10 mutants. We developed a simple barcoding strategy to specifically detect each mutant DNA from mice kidney by quantitative PCR. Among the 77 TF mutant strains tested, eight showed a decreased colonization including mutants for orf19.3405, orf19.255, orf19.5133, RGT1, UGA3, orf19.6182, SEF1 and orf19.2646, and four an increased colonization including mutants for orf19.4166, ZFU2, orf19.1685 and UPC2 as compared to the isogenic wild type strain. Our approach was validated by comparable results obtained with the same animal model using a single mutant and the revertant for an ORF (orf19.2646) with still unknown functions. In an attempt to identify putative involvement of such TFs in already known C. albicans virulence mechanisms, we determined their in vitro susceptibility to pH, heat and oxidative stresses, as well as ability to produce hyphae and invade agar. A poor correlation was found between in vitro and in vivo assays, thus suggesting that TFs needed for mice kidney colonization may involve still unknown mechanisms. This large-scale analysis of mice organ colonization by C. albicans can now be extended to other mutant libraries since our in vivo screening strategy can be adapted to any preexisting mutants

Systematics and plastid genome evolution of the cryptically photosynthetic parasitic plant genus Cuscuta (Convolvulaceae)

<p>Abstract</p> <p>Background</p> <p>The genus <it>Cuscuta </it>L. (Convolvulaceae), commonly known as dodders, are epiphytic vines that invade the stems of their host with haustorial feeding structures at the points of contact. Although they lack expanded leaves, some species are noticeably chlorophyllous, especially as seedlings and in maturing fruits. Some species are reported as crop pests of worldwide distribution, whereas others are extremely rare and have local distributions and apparent niche specificity. A strong phylogenetic framework for this large genus is essential to understand the interesting ecological, morphological and molecular phenomena that occur within these parasites in an evolutionary context.</p> <p>Results</p> <p>Here we present a well-supported phylogeny of <it>Cuscuta </it>using sequences of the nuclear ribosomal internal transcribed spacer and plastid <it>rps2</it>, <it>rbcL </it>and <it>matK </it>from representatives across most of the taxonomic diversity of the genus. We use the phylogeny to interpret morphological and plastid genome evolution within the genus. At least three currently recognized taxonomic sections are not monophyletic and subgenus <it>Cuscuta </it>is unequivocally paraphyletic. Plastid genes are extremely variable with regards to evolutionary constraint, with <it>rbcL </it>exhibiting even higher levels of purifying selection in <it>Cuscuta </it>than photosynthetic relatives. Nuclear genome size is highly variable within <it>Cuscuta</it>, particularly within subgenus <it>Grammica</it>, and in some cases may indicate the existence of cryptic species in this large clade of morphologically similar species.</p> <p>Conclusion</p> <p>Some morphological characters traditionally used to define major taxonomic splits within <it>Cuscuta </it>are homoplastic and are of limited use in defining true evolutionary groups. Chloroplast genome evolution seems to have evolved in a punctuated fashion, with episodes of loss involving suites of genes or tRNAs followed by stabilization of gene content in major clades. Nearly all species of <it>Cuscuta </it>retain some photosynthetic ability, most likely for nutrient apportionment to their seeds, while complete loss of photosynthesis and possible loss of the entire chloroplast genome is limited to a single small clade of outcrossing species found primarily in western South America.</p

Progenitor-Derivative Relationships of Hordeum Polyploids (Poaceae, Triticeae) Inferred from Sequences of TOPO6, a Nuclear Low-Copy Gene Region

Polyploidization is a major mechanism of speciation in plants. Within the barley genus Hordeum, approximately half of the taxa are polyploids. While for diploid species a good hypothesis of phylogenetic relationships exists, there is little information available for the polyploids (4×, 6×) of Hordeum. Relationships among all 33 diploid and polyploid Hordeum species were analyzed with the low-copy nuclear marker region TOPO6 for 341 Hordeum individuals and eight outgroup species. PCR products were either directly sequenced or cloned and on average 12 clones per individual were included in phylogenetic analyses. In most diploid Hordeum species TOPO6 is probably a single-copy locus. Most sequences found in polyploid individuals phylogenetically cluster together with sequences derived from diploid species and thus allow the identification of parental taxa of polyploids. Four groups of sequences occurring only in polyploid taxa are interpreted as footprints of extinct diploid taxa, which contributed to allopolyploid evolution. Our analysis identifies three key species involved in the evolution of the American polyploids of the genus. (i) All but one of the American tetraploids have a TOPO6 copy originating from the Central Asian diploid H. roshevitzii, the second copy clustering with different American diploid species. (ii) All hexaploid species from the New World have a copy of an extinct close relative of H. californicum and (iii) possess the TOPO6 sequence pattern of tetraploid H. jubatum, each with an additional copy derived from different American diploids. Tetraploid H. bulbosum is an autopolyploid, while the assumed autopolyploid H. brevisubulatum (4×, 6×) was identified as allopolyploid throughout most of its distribution area. The use of a proof-reading DNA polymerase in PCR reduced the proportion of chimerical sequences in polyploids in comparison to Taq polymerase

Development of a clinical decision model for thyroid nodules

<p>Abstract</p> <p>Background</p> <p>Thyroid nodules represent a common problem brought to medical attention. Four to seven percent of the United States adult population (10–18 million people) has a palpable thyroid nodule, however the majority (>95%) of thyroid nodules are benign. While, fine needle aspiration remains the most cost effective and accurate diagnostic tool for thyroid nodules in current practice, over 20% of patients undergoing FNA of a thyroid nodule have indeterminate cytology (follicular neoplasm) with associated malignancy risk prevalence of 20–30%. These patients require thyroid lobectomy/isthmusectomy purely for the purpose of attaining a definitive diagnosis. Given that the majority (70–80%) of these patients have benign surgical pathology, thyroidectomy in these patients is conducted principally with diagnostic intent. Clinical models predictive of malignancy risk are needed to support treatment decisions in patients with thyroid nodules in order to reduce morbidity associated with unnecessary diagnostic surgery.</p> <p>Methods</p> <p>Data were analyzed from a completed prospective cohort trial conducted over a 4-year period involving 216 patients with thyroid nodules undergoing ultrasound (US), electrical impedance scanning (EIS) and fine needle aspiration cytology (FNA) prior to thyroidectomy. A Bayesian model was designed to predict malignancy in thyroid nodules based on multivariate dependence relationships between independent covariates. Ten-fold cross-validation was performed to estimate classifier error wherein the data set was randomized into ten separate and unique train and test sets consisting of a training set (90% of records) and a test set (10% of records). A receiver-operating-characteristics (ROC) curve of these predictions and area under the curve (AUC) were calculated to determine model robustness for predicting malignancy in thyroid nodules.</p> <p>Results</p> <p>Thyroid nodule size, FNA cytology, US and EIS characteristics were highly predictive of malignancy. Cross validation of the model created with Bayesian Network Analysis effectively predicted malignancy [AUC = 0.88 (95%CI: 0.82–0.94)] in thyroid nodules. The positive and negative predictive values of the model are 83% (95%CI: 76%–91%) and 79% (95%CI: 72%–86%), respectively.</p> <p>Conclusion</p> <p>An integrated predictive decision model using Bayesian inference incorporating readily obtainable thyroid nodule measures is clinically relevant, as it effectively predicts malignancy in thyroid nodules. This model warrants further validation testing in prospective clinical trials.</p

Be careful with triage in emergency departments: interobserver agreement on 1,578 patients in France

<p>Abstract</p> <p>Background</p> <p>For several decades, emergency departments (EDs) utilization has increased, inducing ED overcrowding in many countries. This phenomenon is related partly to an excessive number of nonurgent patients. To resolve ED overcrowding and to decrease nonurgent visits, the most common solution has been to triage the ED patients to identify potentially nonurgent patients, i.e. which could have been dealt with by general practitioner. The objective of this study was to measure agreement among ED health professionals on the urgency of an ED visit, and to determine if the level of agreement is consistent among different sub-groups based on following explicit criteria: age, medical status, type of referral to the ED, investigations performed in the ED, and the discharge from the ED.</p> <p>Methods</p> <p>We conducted a multicentric cross-sectional study to compare agreement between nurses and physicians on categorization of ED visits into urgent or nonurgent. Subgroups stratified by criteria characterizing the ED visit were analyzed in relation to the outcome of the visit.</p> <p>Results</p> <p>Of 1,928 ED patients, 350 were excluded because data were lacking. The overall nurse-physician agreement on categorization was moderate (kappa = 0.43). The levels of agreement within all subgroups were variable and low. The highest agreement concerned three subgroups of complaints: cranial injury (kappa = 0.61), gynaecological (kappa = 0.66) and toxicology complaints (kappa = 1.00). The lowest agreement concerned two subgroups: urinary-nephrology (kappa = 0.09) and hospitalization (kappa = 0.20). When categorization of ED visits into urgent or nonurgent cases was compared to hospitalization, ED physicians had higher sensitivity and specificity than nurses (respectively 94.9% versus 89.5%, and 43.1% versus 30.9%).</p> <p>Conclusions</p> <p>The lack of physician-nurse agreement and the inability to predict hospitalization have important implications for patient safety. When urgency screening is used to determine treatment priority, disagreement might not matter because all patients in the ED are seen and treated. But using assessments as the basis for refusal of care to potential nonurgent patients raises legal, ethical, and safety issues. Managed care organizations should be cautious when applying such criteria to restrict access to EDs.</p

IL-4 Deficiency Is Associated with Mechanical Hypersensitivity in Mice

Interleukin-4 (IL-4) is an anti-inflammatory and analgesic cytokine that induces opioid receptor transcription. We investigated IL-4 knockout (ko) mice to characterize their pain behavior before and after chronic constriction injury (CCI) of the sciatic nerve as a model for neuropathic pain. We investigated opioid responsivity and measured cytokine and opioid receptor gene expression in the peripheral and central nervous system (PNS, CNS) of IL-4 ko mice in comparison with wildtype (wt) mice. Naïve IL-4 ko mice displayed tactile allodynia (wt: 0.45 g; ko: 0.18 g; p<0.001), while responses to heat and cold stimuli and to muscle pressure were not different. No compensatory changes in the gene expression of tumor necrosis factor-alpha (TNF), IL-1β, IL-10, and IL-13 were found in the PNS and CNS of naïve IL-4 ko mice. However, IL-1β gene expression was stronger in the sciatic nerve of IL-4 ko mice (p<0.001) 28 days after CCI and only IL-4 ko mice had elevated IL-10 gene expression (p = 0.014). Remarkably, CCI induced TNF (p<0.01), IL-1β (p<0.05), IL-10 (p<0.05), and IL-13 (p<0.001) gene expression exclusively in the ipsilateral spinal cord of IL-4 ko mice. The compensatory overexpression of the anti-inflammatory and analgesic cytokines IL-10 and IL-13 in the spinal cord of IL-4 ko mice may explain the lack of genotype differences for pain behavior after CCI. Additionally, CCI induced gene expression of μ, κ, and δ opioid receptors in the contralateral cortex and thalamus of IL-4 ko mice, paralleled by fast onset of morphine analgesia, but not in wt mice. We conclude that a lack of IL-4 leads to mechanical sensitivity; the compensatory hyperexpression of analgesic cytokines and opioid receptors after CCI, in turn, protects IL-4 ko mice from enhanced pain behavior after nerve lesion