Hypersensitivity drug reactions (HDRs) are the adverse effect of pharmaceuticals that clinically resemble allergy. HDRs account for approximately 1/6 of drug-induced adverse effects, and include immune-mediated ("allergic") and non-immune-mediated ("pseudo allergic") reactions. In recent years, the severe and unpredicted drug adverse events clearly indicate that the immune system can be a critical target of drugs. Enhanced prediction in preclinical safety evaluation is, therefore, crucial. Nowadays, there are no validated in vitro or in vivo methods to screen the sensitizing potential of drugs in the pre-clinical phase. The problem of non-predictability of immunologically-based hypersensitivity reactions is related to the lack of appropriate experimental models rather than to the lack of -understanding of the adverse phenomenon. We recently established experimental conditions and markers to correctly identify drug associated with in vivo hypersensitivity reactions using THP-1 cells and IL-8 production, CD86 and CD54 expression. The proposed in vitro method benefits from a rationalistic approach with the idea that allergenic drugs share with chemical allergens common mechanisms of cell activation. This assay can be easily incorporated into drug development for hazard identification of drugs, which may have the potential to cause in vivo hypersensitivity reactions. The purpose of this review is to assess the state of the art of in vitro models to assess the allergenic potential of drugs based on the activation of dendritic cells.
• Type I reactions: immediate-type reactions mediate by immunoglobulin E (IgE) antibodies. Drug IgE complex bind to mast cells with release of histamine and inflammatory mediators, resulting in anaphylaxis, urticarial, angioedema, bronchospasm. Examples of drugs include penicillin and cephalosporins.
• Type II reactions: cytotoxic reactions mediated by drug-specific immunoglobulin G (IgG) or immunoglobulin M (IgM) antibodies. Specific IgG or IgM antibodies are directed at drug-hapten coated cells, resulting in anemia, cytopenia, thrombocytopenia. Examples of drugs includes hydralazine, methyldopa and procainamide.
• Type III: immune-complex reactions. Tissue deposition of drug-antibody complexes result in complement activation and inflammation (i.e., serum sickness, vasculitis, fever, rash, arthralgia, lupus). Examples of drugs include penicillin, sulphonamides, hydralazine and procainamide.
• Type IV reactions: delayed-type hypersensitivity reactions mediated by cellular immune mechanisms. MHC presentation of drugs to T cells results in the release of cytokine and inflammatory mediators, which recruit inflammatory cells (i.e., contact sensitivity, skin rashes, organ-tissue damage). Type IV reactions can be divided in subcategories, with the activation and recruitment of monocytes, eosinophils, CD4+ or CD8+ T cells, and neutrophils (Pichler, 2003a; Riedl and Casillas, 2003; Warrington and Silviu-Dan, 2011). Examples of drugs include neomycin, penicillin and benzocaine.
In vivo tests like patch, prick and intra-cutaneous tests often do not yield positive reactions for the diagnosis of drug allergy and lacks optimal sensitivity that still remains a major problem in daily clinical practice (Sachs et al., 2002). For these reasons, in vitro stimulation could be required as a complementary diagnostic test, as emerged from some studies (Torres et al., 2003; Romano et al., 2004; Sachs et al., 2004).
Nowadays there are no validated in vivo or in vitro methods for assessing the sensitizing potential of a drug during the pre-clinical phase, although the important adverse reactions directly linked to immune-mediated hypersensitivity and autoimmunity reactions. Available in vitro tests mainly refer to the effector phase of immediate-type drug allergic reactions, such as the CAST-ELISA® (Kubota et al., 1997), which is based on the presence of specific IgE antibodies and the BASO-Test® (Pâris-Köhler et al., 2000), based on the activation of basophils like.
Currently, the popliteal lymph node assay (PLNA), or its modifications, can be used in research studies for the identification of drugs, which may be potential allergens, or may cause autoimmunogenic reactions (Warbrick et al., 2001). However, no reliable models or general strategy and assays (including the PLNA) are at present available (or validated) and requested by regulatory agencies. PLNA appears to be very useful for the assessment of the potential of drug to initiate an immune response. The simplest, the primary PLNA, measures popliteal lymph node hyperplasia after subcutaneous injection of a chemical into the footpad of a mouse or rat. The PLN-index is obtained with the ratio of weight or cell number of the draining lymph node of the chemical-treated animals over the vehicle-treated animals (Pieters, 2001). With the primary PLNA, the involvement of specific T cells cannot be assessed; therefore, in a previously sensitized animal, a secondary PLNA must be performed by measuring the PLN index of a chemical. In this assay, purified and irradiated T cells from sensitized syngeneic donors are injected subcutaneously into the footpad of naïve acceptor mice 1 day before injection of the chemical or its active metabolite. Finally, the modified PLNA, defined reporter antigens TNP-OVA (T cell-dependent antigen) and TNP-Ficoll (T cell-independent antigen) are used to distinguish between sensitizing and non-sensitizing (IgG1-response or not to TNP-Ficoll, respectively) drugs (Albers et al., 1997). The primary PLNA is particularly suitable as preclinical screening assay, but it cannot distinguish between strong irritants and sensitizing compounds. Also the more complicated modified PLNA may be used as screening assay, and it has additional advantages over the primary PLNA including (a) the parameters measured (antibody production) are immunologically more relevant than lymph node weight or cell number; (b) the immune response can be measured without knowing the nature of the neo-antigens and it can discriminate between sensitizing, non-sensitizing and complete innocent chemicals. In any case, some of the compounds known to cause immune adverse effects in humans, however, failed to induce a positive PLNA response, leading to refinements of the technique to include pretreatment with enzyme inducers, depletion of CD4+ T cells or additional endpoints such as histological examination, lymphocyte subset analysis and cytokine fingerprinting (Ravel and Descotes, 2005).
It is well known that immunological adverse drug reactions are rare (Gruchalla, 2001; Pichler, 2003b) but anyway they are generally able to cause a lot of discomfort to patients and may indeed be really dangerous. The withdrawal from the market of drug is also an important economic issue due to the extremely high costs associated with the development of a drug (Pieters, 2007). The development of alternative in vitro assays to detect the sensitization potential during the development phase of a drug would increase safety and possibly reduces the risk of market withdrawal (Corti et al., 2015).
Traditional drugs have low molecular weights (<1000 Da) and as a such they are too small to be "seen" by T cells. Therefore, low molecular weight compounds have first to bind to a protein before they will become visible to T cells (Weltzien et al., 1996; Pichler, 2002). In addition, drugs may alter protein structure, the process of antigen presentation, or may, by causing cellular or organ damage, release auto-antigens (e.g., DNA or histones) for which no tolerance exists (Pieters, 2007), favoring the development of hypersensitivity reactions.
This review will focus on the state of the art of available in vitro models to assess the potential of drugs to induce hypersensitivity for diagnostic purposes and on the potential in vitro test for the pre-clinical assessment.
The cells involved and mediators released during the different phases of hypersensitivity reactions can be assessed using in vitro diagnostic tests. The methods used for the diagnosis of HDR depend on the mechanism involved and the kinetic of the reaction. As shown in Figure 1, in vitro diagnostic tests can be divided in test able to identify the drugs but only at the resolution of the hypersensitivity reaction, and in in vitro assays, which allow to determining the HDR risks before drug administration.
Figure 1. Classification of in vitro models able to assess drug-inducing hypersensitivity reactions. In vitro diagnostic tests can be divided in test able to identify the culprit drugs at the resolution of the reaction, and methods that allow to determining the individual risk of HDR before drug administration. On the other hand, some in vitro methods may be used in the pre-clinical phase of drug development for hazard identification of potential to induce hypersensitivity reactions.
Is important to mention that in vitro tests for the identification of non-immediate reactions (NIR) are not commercially available and therefore standardization is not possible (Mayorga et al., 2016).
The human leukocyte antigen (HLA) system is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. HLA genes are highly polymorphic, which means that they have many different alleles, allowing them to fine-tune the adaptive immune system. HLA genotyping is based on reverse sequence-specific oligonucleotide-polymerase chain reaction using DNA from peripheral blood.
Pharmacogenetic testing is not widely used in routine clinical practice to optimize drug choice or clinical management (Philipps, 2006). This gap between scientific knowledge and clinical application may be explained by the fact that the successful incorporation of a pharmacogenetic test into routine practice requires a combination of high-level evidence that can be generalized to diverse clinical setting, wide-spread availability of cost-effective and reliable laboratory tests, and effective strategies incorporate testing into routine clinical practice (Mallal et al., 2008).
Several clinical studies, as reported in the review by Mayorga et al. (2016), correlate hypersensitivity reaction caused by pharmaceuticals with the presence of different HLA allele. In particular HLA-B*57:01 has been found to be associated with abacavir hypersensitivity; for carbamazepine, the most important association has been established with HLA-B*15:02 and HLA-A31:01, while HLA-B*58:01 allele has been associated with allopurinol hypersensitivity.
Prospective HLA-allele screening may be widely useful, but there are some controversial points like the cost-effectiveness of the test, that depend on several estimates that vary among populations, the health care setting, and also the availability of appropriate laboratory assays (Martin et al., 2006; Hammond et al., 2007).
The LTT measures the in vitro proliferative response of T cells that emerge from the clonal expansion of naïve T cell, after drug exposure (Nyfeler and Pichler, 1997; Lanzavecchia and Sallusto, 2000). The proliferative response of lymphocytes is measured by the incorporation of 3H-thymidine during DNA synthesis or by carboxyfluorescein diacetate succimidyl ester (CFSE). The most widely studied drugs are betalactams (Mayorga et al., 2016). The LTT limits are represented by the use of radioactivity, which limits the application of the method to specific research laboratories, it is a quite long procedure, the sensitivity is quite low (60–70%), and it depends upon the conditions employed (Nyfeler and Pichler, 1997; Romano et al., 1997; Beeler and Pichler, 2007).
ELISpot is a technique used to determine the number of cells able to produce cytokines and cytotoxic markers after their activation by the drug or its metabolites (Sullivan et al., 2015). Clinical studies report the use of IFN-γ ELISpot to diagnose non-immediate reactions to betalactames; granzyme B and granulysin ELISpot for evaluating severe cutaneous reactions induced by amoxicillin, ciprofloxacin, carbamazepine, sulphonamides, allopurinol, mefenamic acid, oxipurinol, and lamotrigine (Zawodniak et al., 2010; Porebski et al., 2013). One advantage of this in vitro test is that drug-reactive T cells remain detectable for long time after the reaction and could be appropriate for high throughput screening but to improve the accuracy of the test, two or more cytokines determination could be necessary.
After drug stimulation, T-cells express or up-regulate a number of surface molecules and produce different inflammatory mediators. Cytokine expression and secretion can be evaluated by several methods such as reverse transcription polymerase chain reaction (RT-PCR), flow cytometric analysis, and enzyme linked immunosorbent assay (ELISA). RT-PCR is used to measure cytokine at the transcriptional levels, while flow cytometric analysis are used to study intracellular cytokines and cell surface markers. Finally, ELISA is used to measure the amount of secreted cytokines in cell culture supernatants (Khalil et al., 2008).
Sachs et al. demonstrated that accumulation of eosinophils following IL-5 secretion, and to a lesser extent also with IL-10 and IFN-γ, from drug-specific stimulated T cells is a characteristic histological feature of drug-induced skin eruption. In vitro determination of drug-specific IL-5 secretion by peripheral blood mononuclear cells may be relevant for the detection of in drug-induced maculopapular exanthems (Sachs et al., 2002). CD69 is up-regulated after 48–72 h, and its determination by flow cytometry correlates with LTT for betalactames, sulphamethoxazole and carbamazepine HDR (Beeler et al., 2008). CD69 may be used for the evaluation of non-immediate reactions.
These in vitro tests represent important tools for diagnosis. They are, however, used mainly as research methods rather than as routine procedures. It must be also taken into consideration the timing of sample collection, which is critical as mediators can be secreted in transitory peaks with variants in the maintenance of detectable levels (Mayorga et al., 2006), and chemokines and cytokines can be degraded by protease (Niwa et al., 2000).
Other in vitro tests have been proposed for the study of drug allergy reactions. Among these the cellular allergen stimulation test (CAST-ELISA®) for the measurement of leukotrienes after peripheral blood leukocyte stimulation, basophil histamine release tests and a basophil activation test (BASO-Test®) can be mentioned. CAST-ELISA® is commercially available but it has not been sufficiently evaluated to recommend as a standard investigation outside the context of prospective studies. Basophil activation markers using fluorescence activated cell sorter analysis are currently being evaluated for certain type of drug allergic reactions but there seems to be no evidence currently of any advantage of these tests over skin testing (Mirakian et al., 2009).
None of the in vitro methods mentioned above are, however, useful in preclinical safety assessment. It will important to have in vitro methods to screen drugs for their potential to induce hypersensitivity reactions. In the last decade an incredible progress has been made in the development of non-animal tests to assess contact hypersensitivity, and some tests have been formally validated. Methods based on the use of dendritic cells and the T cell priming are discussed below.
Chemicals can elicit T-cell-mediated diseases, including adverse drug reactions. The T cell priming assay (TCPA) was developed primarily for the identification of contact allergens within the integrated EU project SENS-IT-IV. This assay allows the detection of chemical-specific T cells in naive human peripheral T-cell population by measurement of proliferation and, at the single cell level, of IFN-γ production in CD4+ and CD8+ T cells. This assay may be a valuable, highly specific element in an integrated testing strategy for the predictive in vitro identification of contact allergens and possibly drugs that cause T cell-mediated adverse drug reactions (Dietz et al., 2010; Martin et al., 2010; Richter et al., 2013).
The MUSST is an in vitro method proposed to assess skin sensitization. Dendritic cell activation following exposure to sensitizers was modeled in the U937 human myeloid cell line by measuring the induction of the expression of CD86 by flow cytometry after 48 h of chemical treatment. A test substance is predicted to have a dendritic cell activating potential indicative of being a sensitizer when CD86 induction exceeds the threshold of 1.5-fold with respect to vehicle treated cells at any tested concentration showing a cell viability ≥70 % in at least two independent experiments (Urbisch et al., 2015).
In the modified version of the MUSST (mMUSST), a test substance is predicted to have a dendritic cell activating potential when CD86 induction exceeds a threshold of 1.2-fold (Bauch et al., 2012). Among more than 145 substances available from these two in vitro assays, also some drug sensitizers, namely benzocaine, hydroquinone, p-benzoquinone and diphenylclopropenone were tested and resulted correctly classified with the MUSST (Bauch et al., 2012; Natsch et al., 2013; Urbisch et al., 2015).
The h-CLAT quantifies changes in CD86 and CD54 expression in the human THP-1 cell line following 24 h exposure to the test chemical. The changes in surface marker expression are measured by flow cytometry. Cytotoxicity measurement is conducted concurrently to assess whether up-regulation of surface maker expression occurs at sub-cytotoxic concentrations. The prediction model use the relative fluorescence intensity of surface markers compared to solvent control to discriminate between sensitizers and non-sensitizers. Among the 166 substances tested in this in vitro assay, the allergenic drugs benzocaine, clofibrate, pyridine, hydroquinone, p-benzoquinone and diphenylclopropenone resulted correctly classified (Nukada et al., 2012; Takenouchi et al., 2013; Urbisch et al., 2015).
Overall, even if the number of drugs tested is very limited, data suggests that pharmaceuticals may share with chemical allergens a common mechanism of action that activates dendritic cells, and support the possibility to use these in vitro methods also for the identification of drugs potentially associated with hypersensitivity reactions.
As mentioned above, most drugs are small molecules and are by themselves, not immunogenic. During the haptenization process, these small molecules bind to carrier proteins to form a complete immunogenic complex (Chang and Gershwin, 2010). The hypersensitivity reaction then requires the activation and maturation of dendritic cells (DCs), which will then drive the activation of specific T cells (Martin, 2012). DCs are antigen-presenting cells (APC) that play a central role in the initiation and regulation of adaptive immune responses. Following the contact with antigens, DCs undergo a process of maturation associated with the expression of several co-stimulatory molecules on the membrane such as CD80, CD86 and CD40, various adhesion molecules (CD2, CD11a, CD54, CD58), and secrete different cytokines, including IL-1β and IL-8 (Quah and O'Neill, 2005). Once activated, DCs migrate into the regional lymph node or in the spleen, where they present antigen to specific T lymphocytes, through MHC class II molecules (Ryan et al., 2007) and co-stimulatory adhesion molecules expressed on both DC (i.e., CD86) and T cell (i.e., CD28) to ensure the necessary contact to achieve full T-cell activation. Following stimulation, a clone of T cells is produce with the ability to react to the antigen, resulting in the clinical manifestation of HDR.
Within the European project SENS-IT-IV, we have previously established an in vitro method able to identify contact and respiratory allergens based on the use of the human THP-1 cell line (the same cell line used in the h-CLAT) and IL-8 release: the THP-1 activation assay (Mitjans et al., 2008, 2010). IL-8 is a potent chemotactic peptide for neutrophils as well as for T lymphocytes, basophils, and NK cells. In parallel to IL-8 production, several of the proposed in vitro methods, including the ones mentioned above, are based on DC and CD86 alone or in combination with CD54 expression for the identification of chemical sensitizers, due to their roles in antigen presentation and T cell activation.
Based on the notion that drug sensitizers and chemical sensitizers share the same mode of action, we recently investigated the possibility to use the THP-1 activation assay developed for skin and respiratory sensitizers, for the in vitro identification of pharmaceuticals, which may be associated with in vivo drug hypersensitivity reactions (Corti et al., 2015). It is well known that allergen drugs share with chemical allergens common mechanisms of cell activation and for reason we propose the THP-1 activation assay also for the hazard identification of immune-mediated hypersensitivity reactions induced by pharmaceuticals. Drugs were selected on the basis of clear in vivo immune-adverse reactions reported in literature, post-marketing data or labeling information, and on the commercial availability as pure drugs. Clonidine, ofloxacine, procainamide, streptozotocin, sulfamethoxazole which have been associated with a relatively high incidence of immune-mediated hypersensitivity reactions (Weaver et al., 2005), methyl salicylate and probenecid, which have been reported to cause irritant or allergic contact dermatitis and anaphylactic reactions (Corti et al., 2015), have been tested. We developed a strategy based on IL-8 production, CD86 and/or CD54 expression in THP-1 cells useful for the in vitro identification of drug sensitizers (see Figure 2). There are some important differences with the previous mentioned h-CLAT. First, the method we use to calculate CD86 and CD54 expression is different from h-CLAT protocol: in h-CLAT only the MFI is considered, while we also include the percentage of positive cells. The second point is the concentration tested. In fact, we observed that quite often a CV75 couldn't be reached with drugs compared to chemicals, meaning that drugs are less cytotoxic. The test we developed allowed the correct identification of all the selected drugs tested, including sulfamethoxazole, probenecid and procainamide for which metabolism is needed. Penicillin G, another drug frequently associated with hypersensitivity reactions (Siegel and Coleman, 1957), was previously tested, and found to be able to induced a dose-related release of IL-8 following 48 h of exposure (Mitjans et al., 2008).
Figure 2. The THP-1 activation assay tier approach. Following 24 h of THP-1 chemical/drug treatment, the effect on IL-8 release and CD86 expression are investigated. If positive (statistically significant release of IL-8 at any concentration and/or a SI ≥ 1.5 for CD86), the chemical/drug will be considered as sensitizer. If negative, in order to exclude any activation, IL-8 release and CD86 expression at 48 h (statistically significant release of IL-8 at any concentration and/or a SI ≥ 1.5 for CD86) or CD54 expression at 24 h (SI ≥ 2.0) or alternatively IL-8 mRNA expression (2−ΔΔCT > 3.0) at 3 h should be assessed. Only if negative results were obtained in all parameters, the chemical/drug will be considered as non-sensitizer.
Exposure of THP-1 cells to sensitizing drugs results in most cases in dose related release of IL-8 and increase in CD86 expression, with some differences among drugs, markers and times of exposure. As shown in Table 1, the combination of both IL-8 and CD86 expression allows the identification of all drugs tested. The use of IL-8 mRNA expression at 3 h or CD54 expression at 24 h may offer an alternative to the 48 h exposure and increase our confidence in the negativity of a drug (see Corti et al., 2015). The expression of IL-8 mRNA at 3 h is based on previous observation we made on chemical allergens failing to induce the release of IL-8: all chemicals sensitizers tested including pro-hapten induced IL-8 mRNA at 3 h (Galbiati et al., 2012).
Table 1. Time of IL-8 release and CD86 expression of the selected drugs in THP-1 assay.
Using streptozotocin as reference drug to study the mechanisms of action, we could demonstrate a key role for p38 mitogen-activated protein kinase (p38 MAPK) and PKC-β activation in streptozotocin-induced IL-8 release and CD86 expression (Corti et al., 2015), confirming previous results obtained with chemical allergens (Mitjans et al., 2008; Corsini et al., 2014).
Evidence indicates that oxidative stress is involved in chemical-induced skin allergic and inflammatory diseases (Okayama, 2005; Byamba et al., 2010). Chemical-induced oxidation of the cell surface thiols appears to be one of the triggers of DC maturation, resulting in intracellular redox imbalance that generate stress-related signal (Figure 3). The Keap1/Nrf2-signaling pathway is dedicated to the detection of electrophilic stress in cells leading to the up-regulation of genes involved in protection or neutralization of chemicals reactive species (Natsch and Emter, 2007). It has been shown in human monocyte-derived dendritic cells that chemical sensitizers induced oxidative stress measured by the glutathione GSH/GSSG ratio, as a redox marker (Mizuashi et al., 2005). The reduction of the glutathione GSH/GSSG ratio was accompanied by CD86 up-regulation and p38 MAPK activation, suggesting that the electrophilic properties of chemicals sensitizers may be perceived by DCs as a danger signal leading to DC maturation (Sasaki and Aiba, 2007). Engagement of certain Toll like receptors (TLR1, 2, and 4) leads to mitochondrial translocation of the signal adaptor TRAF6. At the mitochondria, TRAF6 interacts with ECSIT, a protein implicated in the assembly of complex I, leading to its ubiquitylation, which results in increased ROS production. Proteins that are reversibly modulated by ROS are of high interest. In this context, protein kinases and phosphatases, which act co-ordinately in the regulation of signal transduction through the phosphorylation and dephosphorylation of target proteins, have been described to be key elements in ROS-mediated signaling events. In particular, PKC isoforms have been shown to contain a unique structural feature that is susceptible to oxidative modification (Cosentino-Gomes et al., 2012). The high levels of cysteine residues render the regulatory domain susceptible to redox regulation (Gopalakrishna and Jaken, 2000; Giorgi et al., 2010). Currently, evidence supports the direct activation of different PKC isoforms by ROS generation; in particular the β isoform is able to induce ROS generation through mitochondrial damage (Pinton et al., 2007).
Figure 3. Role of ROS in chemical allergen-induced DC activation.
To summarize the drugs tested in the preclinical available in vitro methods and known to be able to induce hypersensitivity reactions, a list is reported in Table 2.
Table 2. Drugs known to induce hypersensitivity and resulted positive in DC-based in vitro methods.
Develop a new pharmaceutical entity has many possibilities of failure and is a very expensive process. Methods and/or models to assess the hazard of hypersensitivity reactions by pharmaceuticals are not yet validated (or requested on a routine basis). The assessment of hypersensitivity of pharmaceuticals would benefit from a rationalistic approach using clear and linear test methods that are based on immunological knowledge and read out parameters. There are many in vitro tests that can help in diagnosis and identification of the pharmaceuticals able to cause a hypersensitivity reaction, and among of these, we established, using the THP-1 cell line as surrogate of dendritic cells, experimental conditions and markers to correctly identify drug sensitizers. The THP-1 cells assay can be easily incorporated into drug development to identify drugs that may have the potential to cause systemic hypersensitivity reactions.
VG wrote the manuscript; AP, and EK performed the experiments depicted in Figure 3; EC supervised the overall project and finalized the final version of the review. All authors have read this review and gave their agreement for submission.
The reviewer NV and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.
Beeler, A., and Pichler, W. J. (2007). "In vitro tests of T-cell mediated drug hypersensitivity," in Drug Hypersensitivity, ed W. J. Pichler (Basel: Karger), 380–390.
Hammond, E., Almeida, C. A., Mamotte, C., Nolan, D., Philipps, E., Schollaardt, T. A., et al. (2007). External quality assessment of HLA-B*5701 reporting: an international multicentre survey. Antivir. Ther. 12, 1027–1032.
Mayorga, C., Celik, G., Rouzaire, P., Whitaker, P., Bonadonna, P., Cernadas, J. R., et al. (2016). In vitro tests for drug hypersensitivity reactions. An ENDA/EAACI drug allergy interest group position paper. Allergy. doi: 10.1111/all.12886. [Epub ahead of print].
Pichler, W. J. (2003a). Delayed drug hypersensitivity reactions. Ann. Intern. Med. 139, 683–693.
Riedl, M. A., and Casillas, A. M. (2003). Adverse drug reactions: types and treatment options. Am. Fam. Physician 68, 1781–1790.
Romano, A., Torres, M. J., Quaratino, D., Di Fonso, M., Perrone, M. R., Viola, M., et al. (1997). Diagnostic evaluation of delayed hypersensitivity to systematically administered drugs. Allergy 27, 175–181.
Warbrick, E. V., Dearman, R. J., and Kimer, I. (2001). Prediction of drug allergenicity: possible use of the local lymph node assay. Curr. Opin. Drug Discov. Devel. 4, 60–65.
Copyright © 2016 Galbiati, Papale, Kummer and Corsini. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


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Abolition News Network – ANN – Presents Underground Railroad
Above; Harriet Tubman leading slaves to Freedom. Photo from PhotoBucket.com
Steve Edison, of the Abolition News Network, spoke on Thursday, January 21, 2016 at Friendship Village Sunset Hills. The Abolition News Network, ANN, is modeled on CNN, the cable news network. ANN does news interviews of historic figures related to the abolition movement in the United States before the American Civil War. Other topics related to abolition and slavery covered by ANN include:
• The transatlantic slave trade
• Slavery
• The underground railroad
• Indentured servants
• Opinions on slavery by religious leaders
• Opinions on slavery by literary figures
• The Constitution and slavery
• The Fugitive Slave Act of 1850
• Dread Scott
On Thursday, Steve presented on the underground railroad featuring three prominent figures from the time:
• John Parker, underground railroad conductor
• Levi Coffee, underground railroad station master
• Eliza, underground railroad passenger, that is, an escaped slave
See three videos below from YouTube on the Underground Railroad
The interviews of these historic figures are available as videos on YouTube. The videos are below for your convenience.
John Parker, underground railroad conductor
Underground railroad conductor, John P. Parker, an iron foundry worker by day ferried a skiff every night across the Ohio River at Ripley, 50 miles southeast of Cincinnati, from 1846 to 1860, carrying escaped slaves to the home of underground railroad stationmaster John Rankin which was the beginning of the Underground Railroad to Canada. He tells a reporter how he enjoys outwitting dumb slave catchers and bounty hunters and never got caught. His home is now a museum on Front Street in Ripley Ohio. This is also where Eliza in Uncle Toms Cabin crossed the Ohio River from Dover, Kentucky in the winter of 1838 in the ice. See our video on the Real Eliza.
Levi Coffin, station master
Levi Coffin, President Underground Railroad
The Real Eliza Story of "Uncle Tom's Cabin"
The story of the real slave, Eliza, that escaped across the Ohio River from Kentucky in 1838, fictionalized in the book, "Uncle Tom's Cabin" by Harriet Beecher Stowe.
More about Steve and ANN
Steve Edison is the founder of the Abolition News Network, a nonprofit, and he has been producing the video interviews for about 20 years. He does the research, the writing, and the production work. He works at a pace of approximately 3 videos per month.
For more information on Abolition News Network please visit: http://abolitionnewsnetwork.org or call Steve Edison at 314-426-7519.
Rev Moses Dickson of the African Methodist Church, St. Louis, MO, Dec 15, 1872
Moses Dickson is the namesake of the Father Dickson Cemetery on Sappington Road, just south of Big Bend Road and north of the Sappington House.
With this video, Abolition News Network introduces a series of videos on the subject of the viewpoints that major Christian denominations took on the abolition of slavery in the United States from the 18th century up to the Civil War as told by their ministers. Discussions include not only their personal viewpoint on keeping slaves but what the Bible says by chapter and verse on the subject, and what they feel that God's position is on slaves and slavery which they find is considerably buried in the Holy Scriptures.
January 23, 2016 in SCHS at Friendship Village.
The Great Pyramids of St Louis by Mark Leach
St Louis State Hospital by Amanda Hunyar
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Proposal to repeal Utah's price gouging protections stalls in Senate committee
Bill is one of 3 before Legislature that would address price gouging.
(Rick Bowmer | AP file photo) A Utahn wears a mask to protect against COVID-19 as he displays the toilet paper he purchased at a grocery store, March 17, 2020, in Salt Lake City. The Utah Department of Consumer Protection has fielded several complaints about high costs for toilet paper as the pandemic wears on. At least three proposed bills before the Legislature would change Utah's price gouging law.
By Taylor Stevens
| Jan. 26, 2021, 1:00 p.m.
| Updated: 5:26 p.m.
After the coronavirus pandemic hit the state last spring, nearly 1,000 Utahns filed complaints alleging higher-than-average costs for everything from toilet paper to hand sanitizer and food staples.
Now, state lawmakers are poised to consider two bills that would reduce the strength of those consumer protections — and another that would abolish Utah's price gouging act altogether.
Sen. Jake Anderegg, R-Lehi, acknowledged during a hearing Monday on his bill that there are disadvantages to a full repeal of the act, which would remove sanctions for anyone who charges high prices for necessities during an emergency.
But Anderegg believes there are more pros than cons to the effort, arguing that the free market will right itself without the "heavy hand" of government and that the current law has been used "like a club" on businesses that saw costs increase during the pandemic and then became the subject of complaints when they raised their prices as a result.
"I truly believe that the economy adjusts and can adjust as fast as people make decisions to adjust," Anderegg told his colleagues on Monday. "It can go slow. It can go really fast. And to that end, I think it is the better policy. Even though there are pros and cons to the existing statute and there are pros and cons to this repealer, I think the pros to this repealer outweigh the cons by and large."
The Senate Business and Labor Committee ultimately declined to take action on SB74 on Monday, as several legislators questioned the ramifications of a repeal and two members of the public spoke against it.
Sen. Karen Mayne, D-West Valley City, called the effort to abolish the price gouging act "spooky" and described it as a "knee-jerk" reaction that required more study.
"Things are going to get caught up in this that we don't want to just because of, 'Well let's get rid of this; buyer beware, free market takes care of everything,'" she said. "Well if that takes care of everything, why are we here to make laws and regulations for things?"
Stephen Foxley, who spoke on behalf of the insurance company Regence BlueCross BlueShield, said during public comment that he hoped the state would keep the price gouging statute on the books in order to hold "bad actors" to account — like a provider he said has been charging $1,600 for a single COVID-19 test when the national cost is around $130.
"I am a free market person," he said. "I understand that argument. But sometimes there are market failures. And in this case, the federal government created a market failure that allowed a certain provider to come out and charge that $1,600."
Utah's price gouging act has been on the books since 2005 but was never used before the coronavirus pandemic. During the act's first test, the state has fielded nearly 1,000 complaints — though not all actually represent an instance of illegal gouging.
For one thing, the law applies only to goods or services that are deemed "necessary for consumption or use as a direct result of events giving rise to a state of emergency." The statute doesn't define what is "necessary," so it's up to a judge to decide.
The Department of Consumer Protection, which is tasked with considering complaints, also can't look solely at a listed price when deciding whether a company or individual has violated the statute. Instead, investigators find baseline costs to compare the price to — including whether the seller sold the good or service in the 30 days before the emergency and at what cost and whether there were wholesale price increases in the cost of obtaining the good or providing the service.
Overall, the Division of Consumer Protection has filed 15 total actions for price gouging and has recovered nearly $43,800 that was charged in excess of what statute allows. The state has also issued around $1,900 in fines, according to a spokesman with the division.
Those fines suggest the statute "may be working," said Sen. Curt Bramble, R-Provo, in relaying those statistics on Monday — though he said that doesn't mean the act isn't "open for amendment."
Anderegg, however, argued that the fact that so few of the complaints were substantiated could show the law is flawed, and noted that there was likely a cost for businesses to defend against false claims.
While the committee didn't advance his bill Monday, Anderegg said he thinks it deserves further consideration along with two other bills aimed at addressing the state's price gouging statute.
One of those comes from Rep. Rex Shipp, whose proposal would tweak several provisions in the price gouging act, including the thresholds that constitute violations.
His bill, HB157, would allow someone who hadn't sold an item in the 30 days preceding an emergency declaration to charge twice their total cost for obtaining the good or providing the service. The state's price gouging statute currently allows such a seller to charge just 30% above those costs.
The bill also takes into consideration the price of obtaining a good or service in the markup a seller can charge, such as "the cost of shipping that could be included in that acquisition cost," Shipp, R-Cedar City, said in a recent interview.
Under his proposal, those costs "wouldn't affect their margin they could get on the product," he said.
But while Shipp believes there need to be tweaks to the statute, he said he doesn't support efforts to repeal the price gouging protections altogether.
"I feel like there still needs to probably be some sort of control so people don't price gouge in those types of situations, so there's some limits on their margin that they can do there," he said. "I believe in a free market but sometimes people might take advantage of people in a certain situation."
Sen. Lincoln Fillmore, R-South Jordan, is also running a bill to tweak the state's price gouging protections, and his bill has several of the same elements as Shipp's — including a consideration of all acquisition and distribution costs in determining an acceptable markup.
His bill would increase the threshold for a violation of the act by a person who had not previously sold a good or service prior to the emergency declaration. Under his proposal, that would rise from 30% to 40%.
SB86 would also make it harder for the Division of Consumer Protection to investigate alleged violations, allowing the agency to do so only if it has received three or more "credible reports" about a seller and if the person appears to have profited in an aggregate amount of at least $1,000.
tstevens@sltrib.comFollow @tstevensmedia
Senate committee recommends bill to 'raise the bar' on use of gang enhancement penalties
President Joe Biden starts process to restore Utah's national monuments
Utah Senate approves bill requiring certification for K-9 police dogs
Utah Senate panel advances bill to regulate government use of facial recognition technology
Senate President Stuart Adams wants to lower Utah's vaccination age from 70 to 65


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Don't forget Nan Britton, mistress of Warren G. Harding, who was 31 years younger than WGH! DNA tests confirmed in 2015 that her daughter Elizabeth was Harding's child.


Winners of 'Scottish Best Tunnel Growers' award, our organic produce has been certified by the Scottish Organic Proudcers Association.
All of our plants are grown from seed by ourselves ensuring that we can deliver the highest quality organic produce to your door. We are a family run business, that has been 'growing our own' since 1985.
Thank you for choosing Bellfield for your organic delivery, we hope you enjoy it.
Organic produce that has been certified by the Scottish Organic Producers Association or other approved UK certification body. This means that the produce has been grown without the use of chemical fertilisers, pesticides, herbicides and other artificial treatments. Essential minerals and vitamins are preserved and the produce is therefore extremely good for you and your family.
Fresh produce, grown by us, picked by us. Straight to your door the next day, FRESH, HEALTHY and VERY TASTY.


text:
Thicket spinoso del Madagascar è una ecoregione globale che fa parte della lista Global 200 delle ecoregioni prioritarie per la conservazione definita dal WWF.. Appartiene al bioma Deserti e macchia xerofila della regione afrotropicale. Interessa l'area del Madagascar meridionale e sud-occidentale.
Lo stato di conservazione è considerato in pericolo critico.

La regione è caratterizzata da un elevato livello di endemismo.

Territorio
La regione si estende su un'area di circa 124.000 km² lungo la costa del meridionale e sud-occidentale dell'isola del Madagascar, dal fiume Tsiribihina a nord-ovest fino alle pendici occidentali dei monti Anosyennes a sud.est. Il territorio ha una larghezza che varia da circa 200 km. nella sua massima estensione, a poche decine di chilometri nella parte più stretta.

Il clima della regione è caldo semi-arido con una piovosità media inferiore ai 500 mm all'anno.

Stati
L'ecoregione interessa un solo paese:
 .

Ecoregioni
L'ecoregione è composta da 2 ecoregioni terrestri:

 AT1311 - Foresta spinosa del Madagascar
 AT1312 - Foreste succulente del Madagascar

Flora

Molta della flora nell'ecoregione endemica. Con una piovosità media inferiore ai 500 mm all'anno gli alberi si adattano bene al clima secco e crescono alti e privi di rami con lunghe spine affilate, o anche corti, densamente ramificati, con tronchi gonfi. La pianta tipica edemica è costituita da una famiglia di alberi spinosi delle Didiereaceae che include Didierea madagascariensis detto l'albero di polpo e Didierea trollii. Un'altra pianta endemica e il pachypodium detto 'piede di elefante' per la sua forma.

L'area presenta una grande varietà di piante spinose e succulente tra cui Alluaudia ascendens che può raggiungere una altezza di 15 metri, oltre ad aloe Kalanchoe ed euforbia. La flora del suolo è scarsa ed è costituita da ciuffi isolati di un'erba endemica, Humbertochloa bambusiuscula.

Fauna

Anche la fauna dell'ecoregione è distintiva e comprende quattro mammiferi strettamente endemici: il lemure dalla coda ad anelli (Lemur catta), il lepilemure dai piedi bianchi (Lepilemur leucopus), il sifaka di Verreaux (Propithecus verreauxi) e la mangusta di Grandidier (Galidictis grandidieri).

Gli uccelli endemici includono il cua di Verreaux (Coua verreauxi), il cua corridore (Coua cursor), il vanga di Lefresnaye (Xenopirostris xenopirostris) e la ghiandaia terricola codalunga (Uratelornis chimaera).

I rettili endemici includono la tartaruga ragno (Pyxis arachnoides), la testuggine raggiata (Geochelone radiata) e il Chalarodon madagascariensis.

Popolazione
La foresta spinosa di Mikea, lungo la costa sud-occidentale della regione, è abitata dai Mikea, uno degli ultimo popoli di cacciatori-raccoglitori rimasti sulla Terra.

Conservazione
Lo stato di conservazione della regione è considerato in pericolo. Le principali minacce alla biodiversità provengono dalla rimozione degli habitat su piccola scala ma diffusa, principalmente per la produzione di legna da ardere e carbone. Le minacce secondarie sono rappresentate dall'agricoltura di sussistenza, dal pascolo del bestiame e dalle specie invasive. Nonostante alcuni recenti miglioramenti, il sistema di aree protette del Madagascar non comprende ancora tutti i siti critici del paese.

Nella regione vi sono comunque alcune aree protette:
 Parco nazionale di Tsimanampetsotsa;
 Parco nazionale di Andohahela;
 Parco nazionale di Zombitse-Vohibasia;
 Parco nazionale di Kirindy Mitea;
 Riserva naturale di Berenty;
 Riserva speciale di Bezaha Mahafaly;
 Riserva speciale di Andranomena.

Note

Bibliografia

Voci correlate
 Lista delle ecoregioni Global 200

Collegamenti esterni

Global 200

redpajama_set_name: RedPajamaWikipedia


text:
The Future Of Odaiba Marine Park
Video shows 2009 Japan Swimming Federation's Tokyo Marathon Swimming Championships in Odaiba Marine Park, Tokyo Bay, Japan.
Courtesy of the Japan Times.
This is the Olympic year where the fastest and most competitive open water swimming event – the Olympic 10K Marathon Swim – will be held on August 5th (25 women) and August 6th (25 men) in Odaiba Marine Park along Tokyo Bay in Japan.
Tokyo Governor Yuriko Koike told the Japan Times that the construction and preparation of all the Olympic facilities and venues are on schedule. This is not surprising given the Japanese ability to host major international events from the FIFA World Cup to the Rugby World Cup.
The Governor said, "After various adjustments including budget cuts, we've managed to build gorgeous venues based on well-thought-out designs that can remain in use after the Olympics."
"While I am sure that the Governor was not referring to the use of Odaiba Marine Park for swimming, it would be great if the Tokyo metropolitan government were able to clean up Tokyo Bay to make coastal waters swimmable for future generations of Japanese – but that is going to be a difficult challenge and probably not in the top priorities for the Tokyo government moving forward," observed Steven Munatones.
2008 Beijing Olympic 10K Marathon Swim Medalists:
Gold: Larisa Ilchenko (Russia) 1:59:27
Silver: Keri-Anne Payne (Great Britain) 1:59:29
Bronze: Cassandra Patten (Great Britain) 1:59:31
Gold: Maarten van der Weijden (Netherlands) 1:51:51
Silver: David Davies (Great Britain) 1:51:53
Bronze: Thomas Lurz (Germany) 1:51:53
2012 London Olympic 10K Marathon Swim Medalists:
Gold: Éva Risztov (Hungary) 1:57:38
Silver: Haley Anderson (USA) 1:57:38
Bronze: Martina Grimaldi (Italy) 1:57:41
Gold: Oussama Mellouli (Tunisia) – 1:49:55
Silver: Thomas Lurz (Germany) – 1:49:58
Bronze: Richard Weinberger (Canada) – 1:50:00
2016 Rio Olympic 10K Marathon Swim Medalists:
Gold: Sharon van Rouwendaal (Netherlands) 1:56:32
Silver: Rachele Bruni (Italy) 1:56:49
Bronze: Poliana Okimoto (Brazil) 1:56:51
Gold: Ferry Weertman (Netherlands) 1:52:59
Silver: Spyridon Gianniotis (Greece) 1:52:59
Bronze: Marc-Antoine Olivier (France) 1:53:02
2020 Tokyo Olympic 10K Marathon Swim Pre-race Medal Favorites:*
* Xin Xin (China) 1:54:47
* Haley Anderson (USA) 1:54:48
* Rachele Bruni (Italy) 1:54:49
* Lara Grangeon (France) 1:54:50
* Ana Marcela Cunha (Brazil) 1:54:50
* Ashley Twichell (USA) 1:54:50
* Kareena Lee (Australia) 1:54:50
* Sharon van Rouwendaal (Netherlands) 1:54:51
* Florian Wellbrock (Germany) 1:47:55
* Marc-Antoine Olivier (France) 1:47:56
* Rob Muffels (Germany) 1:47:57
* Kristóf Rasovszky (Hungary) 1:47:59
* Jordan Wilimovsky (USA) 1:48:01
* Gregorio Paltrinieri (Italy) 1:48:01
* Ferry Weertman (Netherlands) 1:48:01
* Pre-race predictions for the 2020 Olympic 10K Marathon Swim are based on 2019 Olympic qualification times at the FINA World Championships, ability to swim well in very warm, very flat water, and proven performances in high-pressure competitions.


text:
<?php

namespace Behat\Behat\Formatter;

use Behat\Behat\Event\SuiteEvent;

/**
 * Snippets formatter.
 *
 * @author Konstantin Kudryashov <ever.zet@gmail.com>
 */
class SnippetsFormatter extends ProgressFormatter
{
    /**
     * {@inheritdoc}
     */
    protected function getDefaultParameters()
    {
        return array();
    }

    /**
     * Returns an array of event names this subscriber wants to listen to.
     *
     * The array keys are event names and the value can be:
     *
     *  * The method name to call (priority defaults to 0)
     *  * An array composed of the method name to call and the priority
     *  * An array of arrays composed of the method names to call and respective
     *    priorities, or 0 if unset
     *
     * For instance:
     *
     *  * array('eventName' => 'methodName')
     *  * array('eventName' => array('methodName', $priority))
     *  * array('eventName' => array(array('methodName1', $priority), array('methodName2'))
     *
     * @return array The event names to listen to
     */
    public static function getSubscribedEvents()
    {
        return array('afterSuite' => 'afterSuite');
    }

    /**
     * Listens to "suite.after" event.
     *
     * @param SuiteEvent $event
     *
     * @uses printUndefinedStepsSnippets()
     */
    public function afterSuite(SuiteEvent $event)
    {
        $logger = $event->getLogger();

        $this->writeln();
        $this->printSnippets($logger);
    }
}

redpajama_set_name: RedPajamaGithub


I'll throw in the Flight to Mars to whoever buys the next print.
That Munk One Adelaide poster is way, way underrated. The print is just sharp.
Yeah I had no idea it was that cool looking in person. Surprised this hasn't taken off honestly.
Funny that one caught my eye too. Very cool looking and reasonably priced. Too bad I don't have space for posters.
is the MIA poster S/N?
Damn right JR. I got some close up pics of it in the portfolio if you shoot me your email.
Pre- MIA bump for the 2 year anniversary.
I'm interested in buying the Ames Vegas. pm me.
received my poster in perfect condition. buy from this guy, he's a great guy!!!!
^ thanks for the plug. I've had some bites but everyone around here is getting cold feet. Man up!
$20 drop on all posters till tomorrow.
Got a lot of beer drinking to do next weekend and don't feel like dipping into funds!
Gonna throw some up on the Bay and see what happens.


text:
using namespace std;
using namespace GLnexus;

static auto console = spdlog::create<spdlog::sinks::null_sink_st>("test_cli_null");
static int nr_threads = 2;
static string DB_DIR = "/tmp/cli";
static string DB_PATH = DB_DIR + "/DB";

TEST_CASE("cli") {
    SECTION("Everything") {
        Status s;

        // setup database directory
        REQUIRE(system(("rm -rf " + DB_DIR).c_str()) == 0);
        REQUIRE(system(("mkdir -p " + DB_DIR).c_str()) == 0);

        string basedir = "test/data/cli";
        string exemplar_gvcf = basedir + "/" + "F1.gvcf.gz";
        vector<pair<string,size_t>> contigs;
        s = cli::utils::db_init(console, DB_PATH, exemplar_gvcf, contigs);
        REQUIRE(s.ok());
        REQUIRE(contigs.size() >= 1);

        // DB directory already exists -- make sure we get an error
        s = cli::utils::db_init(console, DB_PATH, exemplar_gvcf, contigs);
        REQUIRE(s.bad());

        vector<string> gvcfs;
        for (auto fname : {"F1.gvcf.gz", "F2.gvcf.gz", "F3.gvcf.gz", "F4.gvcf.gz"}) {
            gvcfs.push_back(basedir + "/" + fname);
        }

        // empty range, so that all records will be loaded into the database
        vector<range> ranges;
        s = cli::utils::db_bulk_load(console, 0, nr_threads, gvcfs, DB_PATH, ranges, contigs);
        REQUIRE(s.ok());
        REQUIRE(contigs.size() >= 1);

        // construct ranges that cover the entire contig set
        ranges.clear();
        for (int rid=0; rid < contigs.size(); rid++) {
            size_t len = contigs[rid].second;
            ranges.push_back(range(rid, 0, len));
        }

        discovered_alleles dsals;
        unsigned sample_count;
        s = cli::utils::discover_alleles(console, 0, nr_threads, DB_PATH, ranges, contigs, dsals, sample_count);
        REQUIRE(s.ok());

        string filename = DB_DIR + "/dsals.yml";
        s = cli::utils::yaml_write_discovered_alleles_to_file(dsals, contigs, sample_count, filename);
        REQUIRE(s.ok());

        // load_config: once from hardcoded preset, then from file
        GLnexus::unifier_config unifier_cfg;
        GLnexus::genotyper_config genotyper_cfg;
        string config_preset = "gatk";
        string config_txt, config_crc32c, config_crc32c2;
        s = cli::utils::load_config(console, config_preset, unifier_cfg, genotyper_cfg, config_txt, config_crc32c);
        REQUIRE(s.ok());
        REQUIRE(config_crc32c == "1926883223");

        YAML::Emitter em;
        em << YAML::BeginMap
        << YAML::Key << "unifier_config" << YAML::Value;
        REQUIRE(unifier_cfg.yaml(em).ok());
        em << YAML::Key << "genotyper_config" << YAML::Value;
        REQUIRE(genotyper_cfg.yaml(em).ok());
        em << YAML::EndMap;
        ofstream outfile(DB_DIR + "/config.yml");
        outfile << em.c_str();
        outfile.close();
        s = cli::utils::load_config(console, DB_DIR + "/config.yml", unifier_cfg, genotyper_cfg, config_txt, config_crc32c2);
        REQUIRE(s.ok());
        REQUIRE(config_crc32c == config_crc32c2);

        unifier_cfg.min_AQ1 = 0;
        unifier_cfg.min_AQ2 = 0;

        // unify sites
        vector<GLnexus::unified_site> sites;
        unifier_stats stats;
        s = cli::utils::unify_sites(console, unifier_cfg,
                                    contigs, dsals, sample_count, sites, stats);
        filename = DB_DIR + "/sites.yml";
        s = cli::utils::write_unified_sites_to_file(sites, contigs, filename);
        REQUIRE(s.ok());

        filename = DB_DIR + "/results.bcf";
        s = cli::utils::genotype(console, 0, nr_threads, DB_PATH, genotyper_cfg, sites, {}, filename);
        REQUIRE(s.ok());
    }

    SECTION("read contigs") {
        Status s;

        vector<pair<string,size_t>> contigs;
        s = cli::utils::db_get_contigs(console, DB_PATH, contigs);
        REQUIRE(s.ok());

        // check that the contigs are correct
        // ##contig=<ID=1,length=3000>
        // ##contig=<ID=2,length=4000>
        // ##contig=<ID=3,length=1000>
        // ##contig=<ID=4,length=4000>
        REQUIRE(contigs[0].first == "1");
        REQUIRE(contigs[0].second == 3000);
        REQUIRE(contigs[1].first == "2");
        REQUIRE(contigs[1].second == 4000);
        REQUIRE(contigs[2].first == "3");
        REQUIRE(contigs[2].second == 1000);
        REQUIRE(contigs[3].first == "4");
        REQUIRE(contigs[3].second == 4000);
    }

    SECTION("describe config presets") {
        cout << cli::utils::describe_config_presets();
    }
}


COE's Marla Morris, Ph.D. performs an original composition titled "Sound Colors" at Georgia Southern University's Performing Arts Center.
Introduced by Department Chair Kent Rittschof, Ph.D., Morris opened by reading an excerpt from her most recent book Curriculum Studies Guidebooks: Volume 2 Concepts and Theoretical Frameworks.
Morris explained that art was both an emotional and thought-filled process.
She also expressed a problematic tendency of American culture to place a low value on the arts.
"Artists in general are not supported in American culture as they are in Europe because they are not valued the way businessmen are," she added.
Morris poses for a photo with her students.
A complete video of "Educating the Inner Ear" is available for streaming on the College of Education's YouTube account. View "Educating the Inner Ear" here.


text:
Health and Wealth
Global Journalist
Health & Wealth Desk
Missouri Health Talks
Missouri Environment
Off the Clock
Radio Friends
Views of the News
Show Me The State
The Obvious Question
True/False Podcast
Access Missouri
Shortage in Rich Land
Heartland, Missouri
Life on the Spectrum
My Life, My Town
Overlooked and Underserved
Mizzou at a Crossroads
Columbia's Payphone Mystery
An Uncertain Road
Sustaining Gift
Working at KBIA
Reports & Public File
Mentoring program helps first-year teachers survive - and thrive
By Dale Singer • Mar 27, 2019
This article first appeared in the St. Louis Beacon, June 8, 2010 - Shaire Strong-Duncan changed her mind about her original career choice, probation and parole, because she decided she didn't want to carry a gun.
Instead, she went into a profession where she was presented with a survival kit of a bottle of water, a granola bar, Post-It notes, pencils, hand sanitizer and a box of Kleenex -- and those got used a lot.
Strong-Duncan just completed her first year as a teacher in the St. Louis Public Schools, with 10 seven-grade students at Big Picture Middle School. She was also part of the first class of teacher interns in the St. Louis Plan, a program designed to help newcomers survive their debut in the classroom with a boost from mentors who can impart techniques to cope with what can be a tough transition.
{C}
With support from Rochelle Lewis, a 30-year classroom veteran, she made it through -- but not without some rough moments teaching students at an age that isn't always the easiest to get along with.
"They were the worst kids in the school," Strong-Duncan said of her class at the start of the year. "They were very disrespectful. They were rude. I'd never seen students like this before. They were just not motivated."
Rude awakenings like that contribute to the high turnover of first-year teachers -- a number estimated as high as 50 percent or more nationwide. To help keep qualified teachers in the classroom -- and help weed out those who may have chosen the wrong career path -- the city schools instituted the St. Louis Plan last fall, modeling it after a similar mentoring program in Toledo, Ohio, that began nearly 30 years ago.
Besides matching rookie teachers with experienced ones, the St. Louis Plan also has introduced an evaluation system that is used with both new teachers and experienced ones whose performance in the classroom has been judged unacceptable. Twelve consultant teachers act as mentors to newcomers across the district. Strong-Duncan is one of 24 teachers to complete the first year.
Cheryl Ward, one of the managers of the St. Louis Plan and a veteran educator herself, said two groups who have not always supported such efforts have helped to make the plan work -- principals and the teachers union, Local 420 of the American Federation of Teachers.
"We didn't know how principals would take this," Ward said. "Principals are in charge of their schools. This program has changed the whole dynamics of the St. Louis Public Schools' setup."
And as far as the union's role, Ward said they actually brought the plan to the district.
Two survival kits
One of the keys to making the St. Louis Plan work, Ward says, is that the mentors are educators whose heart is in the classroom. They are not looking to move up the district's ladder into the principal's office. They know too well what a first-year teacher will face because they deal with the same situations every day.
"If you want to be an administrator," she said, "you aren't part of the St. Louis Plan. We want teachers, people who have been on the battlefield. One of the biggest insults I ever got was when I was an instructional coordinator and someone said 'Cheryl, you've been out of the classroom too long.'"
That's not a problem that Lewis had. As a special education teacher for three decades, she was eager to join the St. Louis Plan and help newbies like Strong-Duncan -- and arm them with things they would need to survive, like the kit she presented on the day before classes were about to begin.
"The very next day she was going to have students," Lewis recalled.
"She had the agenda," Strong-Duncan added. "She was already focused. All I had to was follow through."
Building on the survival idea, Strong-Duncan made her own survival kits for her students -- a Ziploc bag containing a pencil with an eraser, a pen and five Life Savers. The eraser, she said, would show the students that everyone makes mistakes that can be changed, but the pen emphasized that some things are set in concrete.
As for the Life Savers -- they represented the fact that the world is an open place, and we're all in this together.
As the school year progressed, Lewis would show up in Strong-Duncan's classroom for observations, formal and informal. At times, she had to hold her tongue about what she thought was the best way to solve whatever problems came up. Seventh-grade girls often require conflict resolution as much as they need academic instruction, and Strong-Duncan's mediation efforts helped cool a sometimes heated atmosphere.
"The main thing was her interaction with the girls," Lewis said. "She pointed out to the girls whenever their behavior had escalated the problems."
Another key that Lewis taught was to make sure the parents are involved in whatever issues came up, including home visits by the teacher if necessary.
"You have to help us work with your child," Strong-Duncan said of the role that parents have to play. "There is no way this can work without parents, teachers and administrators always being together. Without that, there will always be a missing link."
Lewis said Strong-Duncan "fell right into that" part of the job, even with the caveat that she can't let the students' problems become all-consuming.
"A lot of times you can get overly concerned with things we can't deal with as teachers," Lewis said. "You have to keep things in perspective, figuring out what you can do and what you can't do."
"Every day is not going to be peaches and cream," Strong-Duncan echoed. "You have to let it go and come back tomorrow."
Strong-Duncan said she would use writing prompts to get her students to express their feelings and develop their verbal skills, often asking them to write to music, from classical to jazz to R&B. Because her class will be looped, Strong-Duncan will have the same students again this fall as eighth-graders, so she will be able to help them build on the progress they have made.
Real life experience
She will also be able to build on her experiences. One value of the St. Louis Plan, she said, is that she and her rookie colleagues have seen the difference between what they were taught in school and what they were up against when their own students filed in to the classroom.
"They tell you how to group your students, but they don't tell you what roles they might play, and what role I play as a teacher," Strong-Duncan said. "I have come to realize that these kids are not motivated, and if I were to come in with low energy, complacent, not motivated myself, all of that would come down to the students.
"Book knowledge is fine, but when you get into the classroom, it's a whole different ballgame."
Lewis also tried to stress that teachers have to use their successes as the basis for progress.
"Don't think about what you're doing wrong," she said. "Think about one thing you are doing right, and build on that."
Above all, Lewis said, she tried to communicate a passion for teaching for students -- a passion she feels so strongly, she is leaving the mentoring program after just one year to get back into the classroom herself.
Strong-Duncan seemed to have learned that lesson well and reveled in the progress her students made.
"When you are passionate about something," she said, "and you know it's what you are meant to do, you don't concentrate on monetary rewards. Overcoming obstacles is the best reward ever. Money comes and goes, but to see these students being able to own their own education, that's what makes me feel good."
And she obviously feels good about the mentoring she received.
"It's non-threatening," she said. "Being a first-year teacher, I didn't feel comfortable talking with my principal about behavior problems or lesson plans. I didn't want my principal to know I couldn't control my class. Ms. Lewis was able to relieve some of that stress."
To Ward, such interaction is exactly how the St. Louis Plan is designed to work.
"We need teachers to grow every year," she said. "I will never get to the point where I will know it all. We're here to keep them.
"With the relationship between these two, and in life in general, relationships build things. People do a lot because of that."
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On my first drive up to Whistler along BC's Sea to Sky highway I totally missed this stop off. Well actually, that's a bit of a lie, I saw the stop off marked by heaps of cars crammed into the car park, but didn't stop as I didn't realise what was there. This winter we went back – without the crowds of cars – and explored Murrin Lake Park.
Murrin Lake Provincial Park is a great place for climbing, hiking and snowshoeing (in the winter). Thanks to Browning Lake there's also swimming to be done come summer, and kayaking and fishing.
Climbers will enjoyed the various walls at Murrin Lake Provincial Park named the Shaman, Sugarloaf, Bog Wall, Leviticus, Petrifying Wall and The Brunser.
Technically it's open from May 1st to October 15, but we visited outside of these dates. However, if you visit during the times when the park is officially open you'll have to go early: it gets busy and there isn't a lot of parking space.
Getting to Murrin Lake Park is super easy. From Vancouver it's a 45 minute drive up the Sea to Sky highway (route 99) north. About 9.7km after Squamish you'll see a sign informing you that Murrin Lake Provincial Park is coming up on the left hand side.
There are a couple of different options you can take if you want to hike in Murrin Lake Provincial Park. The best of the trails is probably the Murrin Park Loop Trail; there's more information about it below.
The trail to the lookout is well worth doing whenever you visit Murrin Lake Park. It's not a particularly long trail, but it is pretty steep in parts and involves a few uneven steps.
It's pretty well signposted too so you shouldn't get lost. Once you reach the top of the lookout you'll come out at a view of Howe Sound and the windy Hwy 99 that you've just driven along. It's a gorgeous view!
From the car park, follow the path alongside Browning Lake and head up a signposted trail for the Quercus Viewpoint.
The path gets fairly steep and then descends to a junction. Here you can go either way, but going to the right is the way we went.
You'll climb up some steep steps, pass rock climbing areas and then come to another junction.
Go straight here towards the viewpoint. The trail comes out of the forest and climbs up onto the rock where there are beautiful views of Howe Sound. You can also see Browning Lake (where you started).
Careful climbing the rock to the viewpoint as it can get very slippy!
When you're done soaking up the views, head back off the rock and return to the junction. This time take the other path and continue the Loop Trail. You'll come down through the forest and more climbing areas, over a bridge and down a hill between two rock faces. Here there's another viewpoint and bench looking towards Howe Sound.
As you continue hiking down, head left at the next junction to return to Browning Lake.
You can lengthen the walk by walking around the lake but it's not the best trail! It runs straight along the road where cars zoom past so I'd say skip this and relax on the beach.


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Cocaine in blood of 5-year-old in Cyprus, father arrested
by Athens Bureau
A 45-year-old man was arrested and detained by the police in Cyprus after cocaine was found in the body of one of his minor children, aged five.
According to police officers, on May 21, two foreigners asked a chemist in Limassol for tests to be performed on their minor children, where cocaine was found in the body of a child.
The chemist urged the parents to file a complaint with the Police, which they did not do.
The case came to the attention of the Police on Monday, May 24.
The father was arrested and detained while an arrest warrant was issued against the mother.
According to police spokesman Christos Andreou, she escaped abroad on Sunday with the children.
A case of possession, use and supply of a controlled Class A drug, as well as abuse of a child under 16 years of age, is being examined against the 45-year-old.
READ MORE: British model Stephanie Dubois has died after receiving the AstraZeneca in Cyprus.
Tags: child abuse, cocaine, Cyprus, narcotics
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Dendias: Greece is a bridge builder between Europe and Africa
Athens Bureau May 26, 2021
ATHENS AIRPORT: Two women traveling from Brazil caught smuggling over 13kg's of cocaine!
Two women arrested at Athens IA over 13 kilos of cocaine
Bob Menendez: I will not approve the sale of F-16s, Turkey must stop threatening Greece
Cyprus faces same issues as Ukraine for Leopard tanks as Germany stalls on deliveries
Peloponnese wine producers to hold festival in Cyprus
Nikos Vertis: "I wouldn't be ashamed to say I was gay but I don't even have 1% homosexual tendency in me"


Blue Cross and Blue Shield of North Carolina (BCBSNC) recently learned of a nationwide scheme involving letters from "RED Medical Billing." These letters appear to be collection notices, claiming customers owe a past-due amount to Blue Cross and Blue Shield (BCBS). Sample letters and envelopes are attached.
No specific BCBS plan is mentioned, and these letters are not coming from, or on behalf of, any BCBS plan.
The letters have been mailed to individuals with names and addresses easily obtained from any phone directory. And because the letters do not contain any private health information, we do not believe this scheme is related to any data breach.
If you are contacted about this by BCBSNC members, please ask them to not respond to the letter, and to notify the U.S. Postal Inspection Service. Members can go here to find the closest office, or to report a mail crime. Postal inspectors will likely want to see the letter and envelope in question; however, if a member chooses not to contact the U.S. Postal Inspection Service, the letter can be discarded.
BCBSNC has also reported this as mail fraud to the U.S. Postal Inspection Service. We will continue to monitor this situation, and will update this article as more information becomes available.


REDWOOD CITY, Calif. — March 27, 2013 — Equinix, Inc. (Nasdaq: EQIX), the global interconnection and data center company, today announced availability of NetApp Private Storage for AWS in Equinix International Business Exchange (IBX ®) data centers where AWS Direct Connect is deployed including the United States and Asia. The new solution enables organizations to leverage on-demand cloud compute services while retaining full control of their data; data remains secure on a private NetApp storage array.
NetApp Private Storage for AWS allows organizations to balance private and public cloud resources to best meet their business needs. This enables companies to maintain control of their data, comply with the most stringent privacy and security standards, and deliver sub-5ms performance.
Customers with heavy storage and compute needs can now combine NetApp Private Storage with AWS Direct Connect, to gain all the cost and agility benefits of public cloud while meeting rigorous compliance and security standards, retaining control over data, and maintaining high-levels of application performance.
When located within an Equinix IBX data center, NetApp Private Storage for AWS enables sub-5ms response times on data traveling between the hosted NetApp array and AWS. Other data center providers claiming a "direct connection" to public cloud services use long-haul circuits for the connection, which introduces additional latency and impairs performance.
Companies can use NetApp Private Storage for AWS for a wide range of applications, such as disaster recovery, development and test, burst, tiered data back-up and recovery, big data analytics, and more.
NetApp Private Storage for AWS has been deployed and tested in Equinix data centers located in Silicon Valley and Ashburn, VA. Demo platforms will be made available for testing and validation by Equinix customers in Singapore, Silicon Valley, and Ashburn IBXs.


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