The discovery of archaea: from observed anomaly to consequential restructuring of the phylogenetic tree.

Observational and experimental discoveries of new factual entities such as objects, systems, or processes, are major contributors to some advances in the life sciences. Yet, whereas discovery of theories was extensively deliberated by philosophers of science, very little philosophical attention was paid to the discovery of factual entities. This paper examines historical and philosophical aspects of the experimental discovery by Carl Woese of archaea, prokaryotes that comprise one of the three principal domains of the phylogenetic tree. Borrowing Kuhn's terminology, this discovery of a major biological entity was made during a 'normal science' project of building molecular taxonomy for prokaryotes. Unexpectedly, however, an observed anomaly instigated the discovery of archaea. Substantiation of the existence of the new archaeal entity and consequent reconstruction of the phylogenetic tree prompted replacement of a long-held model of a prokarya and eukarya bipartite tree of life by a new model of a tripartite tree comprising of bacteria, archaea, and eukarya. This paper explores the history and philosophical implications of the progression of Woese's project from normal science to anomaly-instigated model-changing discovery. It is also shown that the consequential discoveries of RNA splicing and of ribozymes were similarly prompted by unexpected irregularities during normal science activities. It is thus submitted that some discoveries of factual biological entities are triggered by unforeseen observational or experimental anomalies.

Clinical presentation, diagnostics, treatment, and outcome of goats diagnosed with presumptive cerebrospinal nematodiasis at a veterinary teaching hospital.

This retrospective study describes clinical presentation, diagnostic approach, treatment, and outcome for goats with presumptive cerebrospinal nematodiasis. A presumptive diagnosis was made based on neurologic signs, results of cerebrospinal fluid analysis, and response to treatment. Six goats were identified that met inclusion criteria. Cerebrospinal fluid analysis revealed eosinophilic pleocytosis (total nucleated cell count: 12 to 430/µL, 33 to 89% eosinophils). All 6 goats were treated with fenbendazole and anti-inflammatory drugs (NSAIDs ± corticosteroids) and 4 received physical rehabilitation therapy. At discharge or follow-up, all 6 goats were ambulatory and had minimal neurologic deficits. Key clinical message: In goats, cerebrospinal nematodiasis caused by Parelaphostrongylus tenuis is often a presumptive diagnosis based on neurologic signs, shared habitat with white-tailed deer, eosinophilic pleocytosis, and response to anthelmintic therapy. Presumptive cases in goats have many similarities to confirmed cases in camelids. Further study is indicated to characterize the clinical signs and optimize the diagnosis and treatment of goats infected with P. tenuis.

Présentation clinique, diagnostic, traitement et devenir des chèvres diagnostiquées avec une nématodose cérébro-spinale présumée dans un hôpital d'enseignement vétérinaire. Cette étude rétrospective décrit la présentation clinique, l'approche diagnostique, le traitement et les résultats pour des chèvres atteintes de nématodose cérébro-spinale présumée. Un diagnostic présomptif a été posé sur la base des signes neurologiques, des résultats de l'analyse du liquide céphalo-rachidien et de la réponse au traitement. Six chèvres ont été identifiées qui répondaient aux critères d'inclusion. L'analyse du liquide céphalo-rachidien a révélé une pléocytose éosinophile (nombre total de cellules nucléées : 12 à 430/µL, 33 à 89 % d'éosinophiles). Les six chèvres ont été traitées avec du fenbendazole et des anti-inflammatoires (AINS ± corticostéroïdes) et quatre ont reçu une thérapie de réadaptation physique. À la sortie ou au suivi, les six chèvres étaient ambulatoires et présentaient des déficits neurologiques minimes.Message clinique clé :Chez les chèvres, la nématodose cérébro-spinale causée par Parelaphostrongylus tenuis est souvent un diagnostic présomptif basé sur des signes neurologiques, un habitat partagé avec des cerfs de Virginie, une pléocytose éosinophile et une réponse à un traitement anthelminthique. Les cas présumés chez les chèvres présentent de nombreuses similitudes avec les cas confirmés chez les camélidés. Une étude plus approfondie est indiquée pour caractériser les signes cliniques et optimiser le diagnostic et le traitement des chèvres infectées par P. tenuis.(Traduit par Dr Serge Messier).

Differential effects of single fatty acids and fatty acid mixtures on the phosphoinositide 3-kinase/Akt/eNOS pathway in endothelial cells.

SCOPE: Dietary fat composition is an important modulator of vascular function. Non-esterified fatty acids (NEFA) enriched in saturated fatty acids (SFA) are thought to reduce vascular reactivity by attenuating insulin signalling via vasodilator pathways (phosphoinositide 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS)) and enhancing signalling via pro-inflammatory pathways. METHODS: To examine the effects of fatty acids on these pathways, human aortic endothelial cells were incubated with single fatty acids, and mixtures of these fatty acids to mimic typical NEFA composition and concentrations achieved in our previous human study. RNA was extracted to determine gene expression using real-time RT-PCR and cell lysates prepared to assess protein phosphorylation by Western blotting. RESULTS: Oleic acid (OA, 100 µM) was shown to down regulate expression of the insulin receptor, PTEN and a PI3K catalytic (p110ß) and regulatory (p85α) subunit compared to palmitic, linoleic and stearic acids (P < 0.04), and promote greater eNOS phosphorylation at Ser1177. Both concentration and composition of the SFA and SFA plus n-3 polyunsaturated fatty acids (PUFA) mixtures had significant effects on genes involved in the PI3K/Akt pathway. Greater up-regulation was found with 800 than 400 µM concentration (respective of concentrations in insulin resistant and normal individuals), whereas greater down-regulation was evident with SFA plus n-3 PUFA than SFA mixture alone. CONCLUSION: Our findings provide novel insights into the modulation of the PI3K/Akt/eNOS pathway by single fatty acids and fatty acid mixtures. In particular, OA appears to promote signalling via this pathway, with further work required to determine the primary molecular site(s) of action.

Question-driven stepwise experimental discoveries in biochemistry: two case studies.

Philosophers of science diverge on the question what drives the growth of scientific knowledge. Most of the twentieth century was dominated by the notion that theories propel that growth whereas experiments play secondary roles of operating within the theoretical framework or testing theoretical predictions. New experimentalism, a school of thought pioneered by Ian Hacking in the early 1980s, challenged this view by arguing that theory-free exploratory experimentation may in many cases effectively probe nature and potentially spawn higher evidence-based theories. Because theories are often powerless to envisage workings of complex biological systems, theory-independent experimentation is common in the life sciences. Some such experiments are triggered by compelling observation, others are prompted by innovative techniques or instruments, whereas different investigations query big data to identify regularities and underlying organizing principles. A distinct fourth type of experiments is motivated by a major question. Here I describe two question-guided experimental discoveries in biochemistry: the cyclic adenosine monophosphate mediator of hormone action and the ubiquitin-mediated system of protein degradation. Lacking underlying theories, antecedent data bases, or new techniques, the sole guides of the two discoveries were respective substantial questions. Both research projects were similarly instigated by theory-free exploratory experimentation and continued in alternating phases of results-based interim working hypotheses, their examination by experiment, provisional hypotheses again, and so on. These two cases designate theory-free, question-guided, stepwise biochemical investigations as a distinct subtype of the new experimentalism mode of scientific enquiry.

Ontologically simple theories do not indicate the true nature of complex biological systems: three test cases.

A longstanding philosophical premise perceives simplicity as a desirable attribute of scientific theories. One of several raised justifications for this notion is that simple theories are more likely to indicate the true makeup of natural systems. Qualitatively parsimonious hypotheses and theories keep to a minimum the number of different postulated entities within a system. Formulation of such ontologically simple working hypotheses proved to be useful in the experimental probing of narrowly defined bio systems. It is less certain, however, whether qualitatively parsimonious theories are effective indicators of the true nature of complex biological systems. This paper assesses the success of ontologically simple theories in envisaging the makeup of three complex systems in bacteriology, immunology, and molecular biology. Evidence shows that parsimonious theories completely misconstrued the actual ontologically complex constitutions of the three examined systems. Since evolution and selective pressures typically produce ontologically intricate rather than simple bio systems, qualitatively parsimonious theories are mostly inapt indicators of the true nature of complex biological systems.

Regulation of Early Steps of GPVI Signal Transduction by Phosphatases: A Systems Biology Approach.

We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation.

Ibrutinib Inhibits Platelet Integrin αIIbß3 Outside-In Signaling and Thrombus Stability But Not Adhesion to Collagen.

OBJECTIVE: Ibrutinib is an irreversible Bruton tyrosine kinase inhibitor approved for treatment of Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, and mantle cell lymphoma that increases the risk of bleeding among patients. Platelets from ibrutinib-treated patients exhibit deficiencies in collagen-evoked signaling in suspension; however, the significance of this observation and how it relates to bleeding risk is unclear, as platelets encounter immobile collagen in vivo. We sought to clarify the effects of ibrutinib on platelet function to better understand the mechanism underlying bleeding risk. APPROACH AND RESULTS: By comparing signaling in suspension and during adhesion to immobilized ligands, we found that the collagen signaling deficiency caused by ibrutinib is milder during adhesion to immobilized collagen. We also found that platelets in whole blood treated with ibrutinib adhered to collagen under arterial shear but formed unstable thrombi, suggesting that the collagen signaling deficiency caused by ibrutinib may not be the predominant cause of bleeding in vivo. However, clot retraction and signaling evoked by platelet adhesion to immobilized fibrinogen were also inhibited by ibrutinib, indicating that integrin αIIbß3 outside-in signaling is also effected in addition to GPVI signaling. When ibrutinib was combined with the P2Y12 inhibitor, cangrelor, thrombus formation under arterial shear was inhibited additively. CONCLUSIONS: These findings suggest that (1) ibrutinib causes GPVI and integrin αIIbß3 platelet signaling deficiencies that result in formation of unstable thrombi and may contribute toward bleeding observed in vivo and (2) combining ibrutinib with P2Y12 antagonists, which also inhibit thrombus stability, may have a detrimental effect on hemostasis.

EFFECTS OF MOUTHING AND INTERLOCUTOR PRESENCE ON MOVEMENTS OF VISIBLE VS. NON-VISIBLE ARTICULATORS.

Speakers take into account what information a conversation partner requires in a given context in order to best understand an utterance. Despite growing evidence showing that movements of visible articulators such as the lips are augmented in mouthed speech relative to vocalized speech, little to date has been done comparing this effect in visible vs. non-visible articulators. In addition, no studies have examined whether interlocutor engagement differentially impacts these. Building on a basic present/not-present design, we investigated whether presence of audible speech information and/or an interlocutor affect the movements of the lips and the tongue. Participants were asked to a) speak or b) mouth three target syllables in interlocutor-present and interlocutor-not-present conditions, while lip and tongue movements were recorded using video and ultrasound imaging. Results show that lip protrusion was greater in mouthed conditions compared to vocalized ones and tongue movements were either attenuated (/wa/) or unaffected (/ri/, /ra/) by these same conditions, indicating differential effects for the visible and non-visible articulators in the absence of an auditory signal. A significant interaction between the social engagement and vocalizing conditions in reference to lip aperture showed that participants produced smaller lip apertures when vocalizing alone, as compared to when in the presence of an interlocutor. However, measures of lip protrusion failed to find an effect of social engagement. We conclude that speakers make use of both auditory and visual modalities in the presence of an interlocutor, and that when acoustic information is unavailable, compensatory increases are made in the visual domain. Our findings shed new light on the multimodal nature of speech, and pose new questions about differential adaptations made by visible and non-visible articulators in different speech conditions.

Les locuteurs prennent en compte l'information qu'un partenaire de conversation nécessite pour mieux comprendre une expression. Malgré l'évidence grandissante que les mouvements d'articulateurs visibles (comme les lèvres) sont augmentés dans l'articulation silencieuse par rapport à l'articulation vocalisée, peux d'études ont comparé cet effet dans les articulateurs visibles contre les articulateurs non visibles. De plus, aucune étude n'a examiné si l'engagement de l'interlocuteur changera ces résultats. En élaborant un conception d'expérience présent/non présent, nous avons testé si la présence d'information audible et/ou d'un interlocuteur affecte les mouvements des lèvres et de la langue. Les participants ont parlé trois syllabes, avec et sans production audible, dans chacune des conditions interlocuteur-présent et interlocuteur-non présent. Les mouvements des lèvres et de la langue étaient enregistrés avec la vidéo et l'échographie. Nos résultats montrent que la protubérance des lèvres était plus grande dans les conditions non audibles par rapport à ceux audibles et que les mouvements de la langue étaient atténués (/wa/) ou non affectés (/ri/, /ra/) par ces mêmes conditions, indiquant les effets différents pour les articulateurs visibles et non-visibles dans l'absence d'un signal auditif. Une interaction significative entre les conditions d'engagement sociale et d'audibilité de vocalisation avec référence à la fermeture orale a montré que les participants ont produit des fermetures plus étroites dans les conditions de vocalisation audible, interlocuteur-non présent (par rapport à la condition interlocuteur-présent). Cependant, les mesures de protubérance des lèvres n'étaient pas affectées par condition d'engagement sociale. Nous concluons que les locuteurs utilisent à la fois les modalités auditives et visuelles dans la présence d'un interlocuteur, et lorsque l'information acoustique n'est pas disponible, les augmentations compensatoires sont réalisés dans le domain visuel. Nos résultats soulignent encore le caractère multimodal de discours, et posent des nouvelles questions au sujet des adaptations différentielles faites par les articulateurs visibles et non visibles dans les différentes conditions de parole.

Detection of electrographic seizures by critical care providers using color density spectral array after cardiac arrest is feasible.

OBJECTIVE: To determine the accuracy and reliability of electroencephalographic seizure detection by critical care providers using color density spectral array electroencephalography. DESIGN: Tutorial and questionnaire. SUBJECTS: Critical care providers (attending physicians, fellow trainees, and nurses). INTERVENTIONS: A standardized powerpoint color density spectral array tutorial followed by classification of 200 color density spectral array images as displaying seizures or not displaying seizures. MEASUREMENTS AND MAIN RESULTS: Using conventional electroencephalography recordings obtained from patients who underwent electroencephalography monitoring after cardiac arrest, we created 100 color density spectral array images, 30% of which displayed seizures. The gold standard for seizure category was electroencephalographer determination from the full montage conventional electroencephalography. Participants did not have access to the conventional electroencephalography tracings. After completing a standardized color density spectral array tutorial, images were presented to participants in duplicate and in random order. Twenty critical care physicians (12 attendings and eight fellows) and 19 critical care nurses classified the color density spectral array images as having any seizure(s) or no seizures. The 39 critical care providers had a color density spectral array seizure detection sensitivity of 70% (95% CI, 67-73%), specificity of 68% (95% CI, 67-70%), positive predictive value of 46%, and negative predictive value of 86%. The sensitivity of color density spectral array detection of status epilepticus was 72% (95% CI, 69-74%). CONCLUSION: Determining which post-cardiac arrest patients experience electrographic seizures by critical care providers is feasible after a brief training. There is moderate sensitivity for seizure and status epilepticus detection and a high negative predictive value.

Health of common bottlenose dolphins ( Tursiops truncatus ) in Barataria Bay, Louisiana, following the deepwater horizon oil spill.

The oil spill resulting from the explosion of the Deepwater Horizon drilling platform initiated immediate concern for marine wildlife, including common bottlenose dolphins in sensitive coastal habitats. To evaluate potential sublethal effects on dolphins, health assessments were conducted in Barataria Bay, Louisiana, an area that received heavy and prolonged oiling, and in a reference site, Sarasota Bay, Florida, where oil was not observed. Dolphins were temporarily captured, received a veterinary examination, and were then released. Dolphins sampled in Barataria Bay showed evidence of hypoadrenocorticism, consistent with adrenal toxicity as previously reported for laboratory mammals exposed to oil. Barataria Bay dolphins were 5 times more likely to have moderate-severe lung disease, generally characterized by significant alveolar interstitial syndrome, lung masses, and pulmonary consolidation. Of 29 dolphins evaluated from Barataria Bay, 48% were given a guarded or worse prognosis, and 17% were considered poor or grave, indicating that they were not expected to survive. Disease conditions in Barataria Bay dolphins were significantly greater in prevalence and severity than those in Sarasota Bay dolphins, as well as those previously reported in other wild dolphin populations. Many disease conditions observed in Barataria Bay dolphins are uncommon but consistent with petroleum hydrocarbon exposure and toxicity.

Customization of Advia 120 thresholds for canine erythrocyte volume and hemoglobin concentration, and effects on morphology flagging results.

This study sought to develop customized morphology flagging thresholds for canine erythrocyte volume and hemoglobin concentration [Hgb] on the ADVIA 120 hematology analyzer; compare automated morphology flagging with results of microscopic blood smear evaluation; and examine effects of customized thresholds on morphology flagging results. Customized thresholds were determined using data from 52 clinically healthy dogs. Blood smear evaluation and automated morphology flagging results were correlated with mean cell volume (MCV) and cellular hemoglobin concentration mean (CHCM) in 26 dogs. Customized thresholds were applied retroactively to complete blood (cell) count (CBC) data from 5 groups of dogs, including a reference sample group, clinical cases, and animals with experimentally induced iron deficiency anemia. Automated morphology flagging correlated more highly with MCV or CHCM than did blood smear evaluation; correlation with MCV was highest using customized thresholds. Customized morphology flagging thresholds resulted in more sensitive detection of microcytosis, macrocytosis, and hypochromasia than default thresholds.

Adaptation des seuils d'Advia 120 pour le volume d'érythrocytes et la concentration d'hémoglobines des chiens et les effets sur le signalement des résultats de la morphologie. Cette étude a cherché à développer des seuils de signalement adaptés à la morphologie pour le volume des érythrocytes et la concentration d'hémoglobines [Hgb] des chiens sur l'analyseur d'hématologie ADVIA 120; à comparer le signalement automatique de la morphologie avec les résultats de l'évaluation microscopique des frottis sanguins; et à examiner les effets des seuils adaptés sur les résultats de signalement de la morphologie. Des seuils adaptés ont été déterminés à l'aide de données de 52 chiens cliniquement en santé. L'évaluation des frottis sanguins et les résultats de signalement automatiques de la morphologie ont été corrélés avec le volume cellulaire moyen (VCM) et la concentration d'hémoglobines cellulaires moyennes (CHCM) chez 26 chiens. Les seuils adaptés ont été appliqués rétroactivement à des données d'hémogramme provenant de 5 groupes de chiens, y compris un groupe de référence, de cas cliniques et d'animaux avec une anémie ferriprive induite expérimentalement. Il y avait une corrélation supérieure du signalement automatique de la morphologie avec le VCM ou le CHCM par rapport à l'évaluation des frottis sanguins; la corrélation avec le VCM était supérieure en utilisant des seuils adaptés. Des seuils de signalement adaptés à la morphologie ont produit une détection plus sensible de la microcytose, de la macrocytose et de l'hypochromasie que les seuils par défaut.(Traduit par Isabelle Vallières).

A model to create an efficient and equitable admission policy for patients arriving to the cardiothoracic ICU.

OBJECTIVE: To develop queuing and simulation-based models to understand the relationship between ICU bed availability and operating room schedule to maximize the use of critical care resources and minimize case cancellation while providing equity to patients and surgeons. DESIGN: Retrospective analysis of 6-month unit admission data from a cohort of cardiothoracic surgical patients, to create queuing and simulation-based models of ICU bed flow. Three different admission policies (current admission policy, shortest-processing-time policy, and a dynamic policy) were then analyzed using simulation models, representing 10 yr worth of potential admissions. Important output data consisted of the "average waiting time," a proxy for unit efficiency, and the "maximum waiting time," a surrogate for patient equity. SETTING: A cardiothoracic surgical ICU in a tertiary center in New York, NY. PATIENTS: Six hundred thirty consecutive cardiothoracic surgical patients admitted to the cardiothoracic surgical ICU. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Although the shortest-processing-time admission policy performs best in terms of unit efficiency (0.4612 days), it did so at expense of patient equity prolonging surgical waiting time by as much as 21 days. The current policy gives the greatest equity but causes inefficiency in unit bed-flow (0.5033 days). The dynamic policy performs at a level (0.4997 days) 8.3% below that of the shortest-processing-time in average waiting time; however, it balances this with greater patient equity (maximum waiting time could be shortened by 4 days compared to the current policy). CONCLUSIONS: Queuing theory and computer simulation can be used to model case flow through a cardiothoracic operating room and ICU. A dynamic admission policy that looks at current waiting time and expected ICU length of stay allows for increased equity between patients with only minimum losses of efficiency. This dynamic admission policy would seem to be a superior in maximizing case-flow. These results may be generalized to other surgical ICUs.

Comparison of osmolality and refractometric readings of Hispaniolan Amazon parrot (Amazona ventralis) urine.

To evaluate the relationship between osmolality and specific gravity of urine samples from clinically normal adult parrots and to determine a formula to convert urine specific gravity (USG) measured on a reference scale to a more accurate USG value for an avian species, urine samples were collected opportunistically from a colony of Hispaniolan Amazon parrots (Amazona ventralis). Samples were analyzed by using a veterinary refractometer, and specific gravity was measured on both canine and feline scales. Osmolality was measured by vapor pressure osmometry. Specific gravity and osmolality measurements were highly correlated (r = 0.96). The linear relationship between refractivity measurements on a reference scale and osmolality was determined. An equation was calculated to allow specific gravity results from a medical refractometer to be converted to specific gravity values of Hispaniolan Amazon parrots: USGHAp = 0.201 +0.798(USGref). Use of the reference-canine scale to approximate the osmolality of parrot urine leads to an overestimation of the true osmolality of the sample. In addition, this error increases as the concentration of urine increases. Compared with the human-canine scale, the feline scale provides a closer approximation to urine osmolality of Hispaniolan Amazon parrots but still results in overestimation of osmolality.

Bone marrow iron scoring in healthy and clinically ill dogs with and without evidence of iron-restricted erythropoiesis.

BACKGROUND: There are few reports in dogs that have evaluated the utility of semi-quantitative scoring of bone marrow iron stores in conjunction with reticulocyte hemoglobin (CHr) to identify iron-restricted erythropoiesis due to absolute iron deficiency or iron sequestration. OBJECTIVES: An established system for scoring iron stores in human bone marrow samples was applied to dogs. The objectives were to evaluate interobserver agreement (Κω ), determine marrow iron scores in dogs without detectable hematologic abnormalities, and assess combined interpretation of iron scores and CHr to evaluate for iron-restricted erythropoiesis. METHODS: Four blinded observers independently scored iron in 139 Prussian blue-stained canine marrow samples from 0 (none) to 6 (very heavy), including healthy controls (n = 12), clinically ill dogs with (n = 100) and without (n = 16) detectable hematologic abnormalities, and dogs with experimental nutritional iron deficiency (n = 11). Additional medical record data were available for 118 dogs to evaluate for other evidence of iron deficiency (abnormal CHr, RBC indices, serum iron variables, external blood loss, or nutritional deficiencies). RESULTS: Mean Κω was 0.69 (substantial agreement) for all samples but was 0.44 (moderate agreement) for samples with iron scores <3, indicating distinguishing scores 0-2 may not be reliable. Dogs without detectable hematologic abnormalities had scores from 3-5. Dogs with scores <3 and decreased CHr often had more indicators of iron deficiency vs dogs only having low iron scores or low CHr. CONCLUSIONS: Evaluation of dogs with marrow iron score <3 for external blood loss or nutritional deficiencies is likely clinically worthwhile, particularly if there is also decreased CHr.

Silica and barium: Comparison of microscopic appearance and characterization of effects of silica on cytomorphology.

BACKGROUND: Silica from plastic red top sample collection tubes and barium cause recognized artifacts in slide preparations for microscopic examination. OBJECTIVES: The objectives of this study were to evaluate and directly compare the microscopic appearance of silica and barium particles and various slide preparation techniques (e.g., use of coverslips, oil immersion, and different stains). A secondary objective of this study was to evaluate the effects of silica particles on cellular morphology after mechanical trauma with cytocentrifugation. METHODS: Fluid samples (deionized water, pleural effusion, peritoneal effusion, cerebrospinal fluid, and urine) were collected and evaluated in silica- and non-silica-containing tubes. Barium was added to silica and non-silica samples. Direct and cytocentrifuge preparations were compared to evaluate the effect of silica particles on cellular morphology. Preparations were stained with Wright-Giemsa, rhodizonic acid disodium salt, Alizarin Red, Grocott's methenamine silver, and Prussian blue. RESULTS: Silica and barium particles were identifiable via light microscopy with and without polarized light, although silica particles diminished with immersion oil. Barium particles retained their structure and diminished less under oil. Cytoseal mounting medium for coverslip placement resulted in diminished refractility of silica and some barium particles. Silica particles with mechanical interaction during cytocentrifugation resulted in disrupted cellular morphology with many lysed cells. Silica and barium particles were negative for all special stains tested. CONCLUSIONS: Silica from plastic red top tubes adversely affects cell morphology in cytocentrifuge preparations, potentially affecting manual differential cell counts and compromising diagnostic interpretation. Samples intended for microscopic evaluation should not be collected in silica-containing tubes.

Quadruplex structures of muscle gene promoter sequences enhance in vivo MyoD-dependent gene expression.

Gene promoters are enriched in guanine clusters that potentially fold into quadruplex structures. Such quadruplexes were implicated in the regulation of gene expression, plausibly by interacting with transcription factors. We showed previously that homodimers of the myogenic transcription factor MyoD bound in vitro most tightly bimolecular quadruplexes of promoter sequences of muscle-specific genes. By contrast, MyoD-E47 heterodimers formed tighter complexes with d(CANNTG) E-box motifs that govern muscle gene expression. Here, we show that DNA quadruplexes enhance in vivo MyoD and E-box-driven expression of a firefly luciferase (FL) reporter gene. HEK293 cells were transfected with FL expressing p4RTK-FL vector alone or together with MyoD expressing pEMSV-MyoD plasmid, with quadruplexes of alpha7 integrin or sarcomeric mitochondrial creatine kinase (sMtCK) muscle gene promoters or with a combination thereof. Whereas MyoD elevated by approximately 10-fold the levels of FL mRNA and protein, the DNA quadruplexes by themselves did not affect FL expression. However, together with MyoD, quadruplex DNA increased by approximately 35-fold the amounts of FL mRNA and protein. Without affecting its expression, DNA quadruplexes bound MyoD in the cells. Based on these results, we propose models for the regulation of muscle gene transcription by direct interaction of MyoD with promoter quadruplex structures.

The quadruplex r(CGG)n destabilizing cationic porphyrin TMPyP4 cooperates with hnRNPs to increase the translation efficiency of fragile X premutation mRNA.

The 5' untranslated region of the FMR1 gene which normally includes 4-55 d(CGG) repeats expands to > 55-200 repeats in carriers of fragile X syndrome premutation. Although the levels of premutation FMR1 mRNA in carrier cells are 5-10-fold higher than normal, the amount of the product FMR protein is unchanged or reduced. We demonstrated previously that premutation r(CGG)(n) tracts formed quadruplex structures that impeded translation and lowered the efficiency of protein synthesis. Normal translation could be restored in vivo by the quadruplex r(CGG)(n) destabilizing action of CBF-A and hnRNP A2 proteins. Here we report that the quadruplex-interacting cationic porphyrin TMPyP4 by itself and in cooperation with CBF-A or hnRNP A2 also unfolded quadruplex r(CGG)(n) and increased the efficiency of translation of 5'-(CGG)(99) containing reporter firefly (FL) mRNA. TMPyP4 destabilized in vitro a (CGG)(33) intramolecular quadruplex structure and enhanced the translation of 5'-(CGG)(99)-FL mRNA in a rabbit reticulocyte lysate and in HEK293 cells. The efficiency of translation of (CGG)(99)-FL mRNA was additively increased in cells exposed to TMPyP4 together with CBF-A. Whereas low doses of TMPyP4, CBF-A or hnRNP A2 by themselves did not affect the in vivo utilization of (CGG)(99)-FL mRNA, introduction of TMPyP4 together with either protein synergistically augmented its translation efficiency.

Scenario-robust pre-disaster planning for multiple relief items.

The increasing vulnerability of the population from frequent disasters requires quick and effective responses to provide the required relief through effective humanitarian supply chain distribution networks. We develop scenario-robust optimization models for stocking multiple disaster relief items at strategic facility locations for disaster response. Our models improve the robustness of solutions by easing the difficult, and usually impossible, task of providing exact probability distributions for uncertain parameters in a stochastic programming model. Our models allow decision makers to specify uncertainty parameters (i.e., point and probability estimates) based on their degrees of knowledge, using distribution-free uncertainty sets in the form of ranges. The applicability of our generalized approach is illustrated via a case study of hurricane preparedness in the Southeastern United States. In addition, we conduct simulation studies to show the effectiveness of our approach when conditions deviate from the model assumptions.

Comparison of iron staining and scoring methods on canine bone marrow aspirates.

BACKGROUND: Insufficient iron for erythropoiesis can occur in multiple conditions, including absolute iron deficiency, which is often caused by chronic external hemorrhage in dogs. Distinguishing this from other causes of iron-restricted erythropoiesis allows appropriate intervention. Decreased marrow iron assessed by Prussian blue staining is a method to diagnose absolute iron deficiency, but scoring systems for marrow iron are not validated in dogs. OBJECTIVES: Our objectives were to (a) evaluate the technical performance of two bone marrow iron scoring systems used in human medicine and (b) examine the effects of destaining and restaining on iron stores after Wright-stained marrow slides are destained and restained with a Prussian blue stain. METHODS: Two marrow aspirate slides were included from each of 12 ill dogs in which marrow was collected during clinical evaluation. One slide was directly stained with Prussian blue. The other was first stained with Wright stain, then destained and restained with Prussian blue. Three blinded observers scored the presence of iron in each of the 24 randomized slides using the Gale (scale 0-6) and sideroblast methods (percentage score). Slides were then re-randomized and rescored. RESULTS: For the Gale method, interobserver agreement was fair, and intraobserver agreement was substantial to perfect. There was less agreement using the sideroblast method, with a significant observer effect. Iron scores were significantly lower in destained slides compared with those stained directly. CONCLUSIONS: Interobserver and intraobserver agreements were acceptable for the Gale method, but the sideroblast method should be used cautiously. A destaining procedure before Prussian blue staining could decrease marrow iron scores.

Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration.

The lipid products of phosphoinositide 3-kinase (PI3K) are involved in many cellular responses such as proliferation, migration, and survival. Disregulation of PI3K-activated pathways is implicated in different diseases including cancer and diabetes. Among the three classes of PI3Ks, class I is the best characterized, whereas class II has received increasing attention only recently and the precise role of these isoforms is unclear. Similarly, the role of phosphatidylinositol-3-phosphate (PtdIns-3-P) as an intracellular second messenger is only just beginning to be appreciated. Here, we show that lysophosphatidic acid (LPA) stimulates the production of PtdIns-3-P through activation of a class II PI3K (PI3K-C2beta). Both PtdIns-3-P and PI3K-C2beta are involved in LPA-mediated cell migration. This study is the first identification of PtdIns-3-P and PI3K-C2beta as downstream effectors in LPA signaling and demonstration of an intracellular role for a class II PI3K. Defining this novel PI3K-C2beta-PtdIns-3-P signaling pathway may help clarify the process of cell migration and may shed new light on PI3K-mediated intracellular events.