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1.  Characterization of Airborne Molds, Endotoxins, and Glucans in Homes in New Orleans after Hurricanes Katrina and Rita▿  
In August and September 2005, Hurricanes Katrina and Rita caused breeches in the New Orleans, LA, levee system, resulting in catastrophic flooding. The city remained flooded for several weeks, leading to extraordinary mold growth in homes. To characterize the potential risks of mold exposures, we measured airborne molds and markers of molds and bacteria in New Orleans area homes. In October 2005, we collected air samples from 5 mildly water-damaged houses, 15 moderately to heavily water-damaged houses, and 11 outdoor locations. The air filters were analyzed for culturable fungi, spores, (1→3,1→6)-β-d-glucans, and endotoxins. Culturable fungi were significantly higher in the moderately/heavily water-damaged houses (geometric mean = 67,000 CFU/m3) than in the mildly water-damaged houses (geometric mean = 3,700 CFU/m3) (P = 0.02). The predominant molds found were Aspergillus niger, Penicillium spp., Trichoderma, and Paecilomyces. The indoor and outdoor geometric means for endotoxins were 22.3 endotoxin units (EU)/m3 and 10.5 EU/m3, respectively, and for (1→3,1→6)-β-d-glucans were 1.7 μg/m3 and 0.9 μg/m3, respectively. In the moderately/heavily water-damaged houses, the geometric means were 31.3 EU/m3 for endotoxins and 1.8 μg/m3 for (1→3,1→6)-β-d-glucans. Molds, endotoxins, and fungal glucans were detected in the environment after Hurricanes Katrina and Rita in New Orleans at concentrations that have been associated with health effects. The species and concentrations were different from those previously reported for non-water-damaged buildings in the southeastern United States.
PMCID: PMC1828784  PMID: 17209066
2.  RITA can induce cell death in p53-defective cells independently of p53 function via activation of JNK/SAPK and p38 
Cell Death & Disease  2014;5(7):e1318-.
Significant advances have been made in the development of small molecules blocking the p53/MDM2 interaction. The Mdm2 inhibitor Nutlin-3 is restricted to tumors carrying wtp53. In contrast, RITA, a compound that binds p53, has recently been shown also to restore transcriptional functions of mtp53. As more than 50% of solid tumors carry p53 mutations, RITA promises to be a more effective therapeutic strategy than Nutlin-3. We investigated effects of RITA on apoptosis, cell cycle and induction of 45 p53 target genes in a panel of 14 cell lines from different tumor entities with different p53 status as well as primary lymphocytes and fibroblasts. Nine cell strains expressed wtp53, four harbored mtp53, and three were characterized by the loss of p53 protein. A significant induction of cell death upon RITA was observed in 7 of 16 cell lines. The nonmalignant cells in our panel were substantially less sensitive. We found that in contrast to Nultin-3, RITA is capable to induce cell death not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells. Importantly, whereas p53 has a central role for RITA-mediated effects in wtp53 cells, neither p53 nor p63 or p73 were essential for the RITA response in mtp53 or p53-null cells in our panel demonstrating that besides the known p53-dependent action of RITA in wtp53 cells, RITA can induce cell death also independently of p53 in cells harboring defective p53. We identified an important role of both p38 and JNK/SAPK for sensitivity to RITA in these cells leading to a typical caspase- and BAX/BAK-dependent mitochondrial apoptosis. In conclusion, our data demonstrate that RITA can induce apoptosis through p38 and JNK/SAPK not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells, making RITA an interesting tumor-selective drug.
PMCID: PMC4123078  PMID: 25010984
3.  Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents 
Cell Death & Disease  2012;3(4):e294-.
Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3rRITA10 μM to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.
PMCID: PMC3358013  PMID: 22476102
RITA; nutlin-3; p53; p53 activator; drug resistance; radiation
4.  What Is the Best Proximal Anastomosis for the Free Right Internal Thoracic Artery during Bilateral Internal Thoracic Artery Revascularization? A Prospective, Randomized Study 
Objective. Bilateral internal thoracic artery (BITA) grafting provides improved graft patency and potential survival advantage in selected patients as compared to single left internal thoracic artery (LITA) revascularization. The ideal functional BITA configuration remains controversial. Methods. Patients undergoing planned BITA revascularization with greater than 75% stenosis in both the left anterior descending artery (LAD) and in a circumflex branch were prospectively randomized to one of two proximal free right internal thoracic artery (RITA) connections directly off the aorta (Ao) (n = 12) or as a “t” graft off the LITA (t) (n = 12). The LITA was placed to the LAD in all cases, and the RITA was placed to a single lateral wall vessel. Intraoperative transit time flow measurements of all arterial grafts were performed, and RITA fractional flow parameters were compared between the 2 groups. Results. There were no differences in preoperative patient variables between the two groups. Cross-clamp times (91.5 + 15.3 versus 68.0 + 12.5 minutes, P < 0.01) and total cardiopulmonary bypass times (109.0 + 16.2 versus 85.0 + 15.1 minutes, P < 0.01) were shorter in the t group. The Ao group demonstrated significantly higher mean RITA flow (38.3 ± 13.5 versus 22.1 ± 9.5, P < 0.01), mean RITA conductance (flow/mean arterial pressure) (0.45 ± 0.16 versus 0.28 ± 0.11, P < 0.01), RITA fractional flow (0.52 ± 0.15 versus 0.36 ± 0.11, P < 0.01), and RITA fractional conductance (0.51 ± 0.15 versus 0.36 ± 0.11, P < 0.01) than the “t” grafted patients. Thirty-day mortality and wound infection were 0% for each group. Over an average of 42.8 + 6.6 months of followup there were no mortalities in either group. Repeat angiography were performed in 4 patients (33%) in the Ao group and 2 patients in the t group (16%). One occluded RITA graft and one ostial RITA stenosis were detected in the Ao group. Conclusions. Acute flow measurements indicate that the free RITA anastomosed to the aorta provides more acute fractional RITA flow than composite “t” grafting to the LITA. Longer-term angiographic and clinical followup are necessary to determine the consequences of these acute hemodynamic findings.
PMCID: PMC3932723  PMID: 24653860
5.  Make it better but don't change anything 
With massive amounts of data being generated in electronic format, there is a need in basic science laboratories to adopt new methods for tracking and analyzing data. An electronic laboratory notebook (ELN) is not just a replacement for a paper lab notebook, it is a new method of storing and organizing data while maintaining the data entry flexibility and legal recording functions of paper notebooks. Paper notebooks are regarded as highly flexible since the user can configure it to store almost anything that can be written or physically pasted onto the pages. However, data retrieval and data sharing from paper notebooks are labor intensive processes and notebooks can be misplaced, a single point of failure that loses all entries in the volume. Additional features provided by electronic notebooks include searchable indices, data sharing, automatic archiving for security against loss and ease of data duplication. Furthermore, ELNs can be tasked with additional functions not commonly found in paper notebooks such as inventory control. While ELNs have been on the market for some time now, adoption of an ELN in academic basic science laboratories has been lagging. Issues that have restrained development and adoption of ELN in research laboratories are the sheer variety and frequency of changes in protocols with a need for the user to control notebook configuration outside the framework of professional IT staff support. In this commentary, we will look at some of the issues and experiences in academic laboratories that have proved challenging in implementing an electronic lab notebook.
PMCID: PMC2810290  PMID: 20098591
6.  Chk2 mediates RITA-induced apoptosis 
Cell Death and Differentiation  2011;19(6):980-989.
Reactivation of the p53 tumor-suppressor protein by small molecules like Nutlin-3 and RITA (reactivation of p53 and induction of tumor cell apoptosis) is a promising strategy for cancer therapy. The molecular mechanisms involved in the responses to RITA remain enigmatic. Several groups reported the induction of a p53-dependent DNA damage response. Furthermore, the existence of a p53-dependent S-phase checkpoint has been suggested, involving the checkpoint kinase Chk1. We have recently shown synergistic induction of apoptosis by RITA in combination with Nutlin-3, and we observed concomitant Chk2 phosphorylation. Therefore, we investigated whether Chk2 contributes to the cellular responses to RITA. Strikingly, the induction of apoptosis seemed entirely Chk2 dependent. Transcriptional activity of p53 in response to RITA required the presence of Chk2. A partial rescue of apoptosis observed in Noxa knockdown cells emphasized the relevance of p53 transcriptional activity for RITA-induced apoptosis. In addition, we observed an early p53- and Chk2-dependent block of DNA replication upon RITA treatment. Replicating cells seemed more prone to entering RITA-induced apoptosis. Furthermore, the RITA-induced DNA damage response, which was not a secondary effect of apoptosis induction, was strongly attenuated in cells lacking p53 or Chk2. In conclusion, we identified Chk2 as an essential mediator of the cellular responses to RITA.
PMCID: PMC3354051  PMID: 22158418
p53, Chk2; Nutlin-3; RITA; apoptosis
7.  Reduced inclination of cervical spine in a novel notebook screen system - implications for rehabilitation 
Professional working at computer notebooks is associated with high requirements on the body posture in the seated position. By the high continuous static muscle stress resulting from this position at notebooks, professionals frequently working at notebooks for long hours are exposed to an increased risk of musculoskeletal complaints. Especially in subjects with back pain, new notebooks should be evaluated with a focus on rehabilitative issues.
In a field study a new notebook design with adjustable screen was analyzed and compared to standard notebook position.
There are highly significant differences in the visual axis of individuals who are seated in the novel notebook position in comparison to the standard position. Also, differences are present between further alternative notebook positions. Testing of gender and glasses did not reveal influences.
This study demonstrates that notebooks with adjustable screen may be used to improve the posture. Future studies may focus on patients with musculoskeletal diseases.
PMCID: PMC3253038  PMID: 22118159
8.  “You Have No Good Blood in Your Body”. Oral Communication in Sixteenth-Century Physicians’ Medical Practice 
Medical History  2015;59(1):63-82.
In his personal notebooks, the little known Bohemian physician Georg Handsch (1529–c. 1578) recorded, among other things, hundreds of vernacular phrases and expressions he and other physicians used in their oral interaction with patients and families. Based primarily on this extraordinary source, this paper traces the terms, concepts and images to which sixteenth-century physicians resorted when they explained the nature of a patient’s disease and justified their treatment. At the bedside and in the consultation room, Handsch and his fellow physicians attributed most diseases to a local accumulation of impure, putrid or otherwise pathological humours. The latter were commonly said to result, in turn, from an insufficient concoction and assimilation of food and drink in the stomach and the liver or from an obstruction of the humoral flow inside the body and across its borders. By contrast, other notions and explanatory models, which had a prominent place in contemporary learned medical writing, hardly played a role at all in the physicians’ oral communication. Specific disease terms were rarely used, a mere imbalance of the four natural humours in the body was almost never inculpated, and the patient’s personal life-style and other non-naturals did not attract much attention either. These striking differences between the ways in which physicians explained the patients’ diseases in their daily practice and the explanatory models we find in contemporary textbooks, are attributed, above all, to the physicians’ precarious situation in the early modern medical marketplace. Since dissatisfied patients were quick to turn to another healer, physicians had to explain the disease and justify their treatment in a manner that was comprehensible to ordinary lay people and in line with their expectations and beliefs, which, at the time, revolved almost entirely around notions of impurity and evacuation.
PMCID: PMC4304544  PMID: 25498438
Early modern medicine; Oral communication; Humoral pathology; Disease concepts; Medical lay culture; Doctor–patient encounter
9.  Targeting p53 via JNK Pathway: A Novel Role of RITA for Apoptotic Signaling in Multiple Myeloma 
PLoS ONE  2012;7(1):e30215.
The low frequency of p53 alterations e.g., mutations/deletions (∼10%) in multiple myeloma (MM) makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK) signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP) analysis showed that activated c-Jun binds to the activator protein-1 (AP-1) binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA) against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with JNK activators in the treatment of MM.
PMCID: PMC3262803  PMID: 22276160
10.  The p53-Reactivating Small Molecule RITA Induces Senescence in Head and Neck Cancer Cells 
PLoS ONE  2014;9(8):e104821.
TP53 is the most commonly mutated gene in head and neck cancer (HNSCC), with mutations being associated with resistance to conventional therapy. Restoring normal p53 function has previously been investigated via the use of RITA (reactivation of p53 and induction of tumor cell apoptosis), a small molecule that induces a conformational change in p53, leading to activation of its downstream targets. In the current study we found that RITA indeed exerts significant effects in HNSCC cells. However, in this model, we found that a significant outcome of RITA treatment was accelerated senescence. RITA-induced senescence in a variety of p53 backgrounds, including p53 null cells. Also, inhibition of p53 expression did not appear to significantly inhibit RITA-induced senescence. Thus, this phenomenon appears to be partially p53-independent. Additionally, RITA-induced senescence appears to be partially mediated by activation of the DNA damage response and SIRT1 (Silent information regulator T1) inhibition, with a synergistic effect seen by combining either ionizing radiation or SIRT1 inhibition with RITA treatment. These data point toward a novel mechanism of RITA function as well as hint to its possible therapeutic benefit in HNSCC.
PMCID: PMC4132078  PMID: 25119136
11.  From documents to datasets: A MediaWiki-based method of annotating and extracting species observations in century-old field notebooks 
ZooKeys  2012;235-253.
Part diary, part scientific record, biological field notebooks often contain details necessary to understanding the location and environmental conditions existent during collecting events. Despite their clear value for (and recent use in) global change studies, the text-mining outputs from field notebooks have been idiosyncratic to specific research projects, and impossible to discover or re-use. Best practices and workflows for digitization, transcription, extraction, and integration with other sources are nascent or non-existent. In this paper, we demonstrate a workflow to generate structured outputs while also maintaining links to the original texts. The first step in this workflow was to place already digitized and transcribed field notebooks from the University of Colorado Museum of Natural History founder, Junius Henderson, on Wikisource, an open text transcription platform. Next, we created Wikisource templates to document places, dates, and taxa to facilitate annotation and wiki-linking. We then requested help from the public, through social media tools, to take advantage of volunteer efforts and energy. After three notebooks were fully annotated, content was converted into XML and annotations were extracted and cross-walked into Darwin Core compliant record sets. Finally, these recordsets were vetted, to provide valid taxon names, via a process we call “taxonomic referencing.” The result is identification and mobilization of 1,068 observations from three of Henderson’s thirteen notebooks and a publishable Darwin Core record set for use in other analyses. Although challenges remain, this work demonstrates a feasible approach to unlock observations from field notebooks that enhances their discovery and interoperability without losing the narrative context from which those observations are drawn.
“Compose your notes as if you were writing a letter to someone a century in the future.”
Perrine and Patton (2011)
PMCID: PMC3406479  PMID: 22859891
Field notes; notebooks; crowd sourcing; digitization; biodiversity; transcription; text-mining; Darwin Core; Junius Henderson; annotation; taxonomic referencing; natural history; Wikisource; Colorado; species occurrence records
12.  LabTrove: A Lightweight, Web Based, Laboratory “Blog” as a Route towards a Marked Up Record of Work in a Bioscience Research Laboratory 
PLoS ONE  2013;8(7):e67460.
The electronic laboratory notebook (ELN) has the potential to replace the paper notebook with a marked-up digital record that can be searched and shared. However, it is a challenge to achieve these benefits without losing the usability and flexibility of traditional paper notebooks. We investigate a blog-based platform that addresses the issues associated with the development of a flexible system for recording scientific research.
Methodology/Principal Findings
We chose a blog-based approach with the journal characteristics of traditional notebooks in mind, recognizing the potential for linking together procedures, materials, samples, observations, data, and analysis reports. We implemented the LabTrove blog system as a server process written in PHP, using a MySQL database to persist posts and other research objects. We incorporated a metadata framework that is both extensible and flexible while promoting consistency and structure where appropriate. Our experience thus far is that LabTrove is capable of providing a successful electronic laboratory recording system.
LabTrove implements a one-item one-post system, which enables us to uniquely identify each element of the research record, such as data, samples, and protocols. This unique association between a post and a research element affords advantages for monitoring the use of materials and samples and for inspecting research processes. The combination of the one-item one-post system, consistent metadata, and full-text search provides us with a much more effective record than a paper notebook. The LabTrove approach provides a route towards reconciling the tensions and challenges that lie ahead in working towards the long-term goals for ELNs. LabTrove, an electronic laboratory notebook (ELN) system from the Smart Research Framework, based on a blog-type framework with full access control, facilitates the scientific experimental recording requirements for reproducibility, reuse, repurposing, and redeployment.
PMCID: PMC3720848  PMID: 23935832
13.  Seroconverting Blood Donors as a Resource for Characterising and Optimising Recent Infection Testing Algorithms for Incidence Estimation 
PLoS ONE  2011;6(6):e20027.
Biomarker-based cross-sectional incidence estimation requires a Recent Infection Testing Algorithm (RITA) with an adequately large mean recency duration, to achieve reasonable survey counts, and a low false-recent rate, to minimise exposure to further bias and imprecision. Estimating these characteristics requires specimens from individuals with well-known seroconversion dates or confirmed long-standing infection. Specimens with well-known seroconversion dates are typically rare and precious, presenting a bottleneck in the development of RITAs.
The mean recency duration and a ‘false-recent rate’ are estimated from data on seroconverting blood donors. Within an idealised model for the dynamics of false-recent results, blood donor specimens were used to characterise RITAs by a new method that maximises the likelihood of cohort-level recency classifications, rather than modelling individual sojourn times in recency.
For a range of assumptions about the false-recent results (0% to 20% of biomarker response curves failing to reach the threshold distinguishing test-recent and test-non-recent infection), the mean recency duration of the Vironostika-LS ranged from 154 (95% CI: 96–231) to 274 (95% CI: 234–313) days in the South African donor population (n = 282), and from 145 (95% CI: 67–226) to 252 (95% CI: 194–308) days in the American donor population (n = 106). The significance of gender and clade on performance was rejected (p−value = 10%), and utility in incidence estimation appeared comparable to that of a BED-like RITA. Assessment of the Vitros-LS (n = 108) suggested potentially high false-recent rates.
The new method facilitates RITA characterisation using widely available specimens that were previously overlooked, at the cost of possible artefacts. While accuracy and precision are insufficient to provide estimates suitable for incidence surveillance, a low-cost approach for preliminary assessments of new RITAs has been demonstrated. The Vironostika-LS and Vitros-LS warrant further analysis to provide greater precision of estimates.
PMCID: PMC3111407  PMID: 21694760
14.  Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK-1 
Cell Death & Disease  2011;2(5):e160-.
We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.
PMCID: PMC3122121  PMID: 21593792
p53; hypoxia; HIF-1α; DNA damage; CHK-1
15.  RITA (Reactivating p53 and Inducing Tumor Apoptosis) is efficient against TP53abnormal myeloma cells independently of the p53 pathway 
BMC Cancer  2014;14:437.
The aim of this study was to evaluate the efficacy of the p53-reactivating drugs RITA and nutlin3a in killing myeloma cells.
A large cohort of myeloma cell lines (n = 32) and primary cells (n = 21) was used for this study. This cohort contained cell lines with various TP53 statuses and primary cells with various incidences of deletion of chromosome 17. Apoptosis was evaluated using flow cytometry with Apo2.7 staining of the cell lines or via the loss of the myeloma-specific marker CD138 in primary cells. Apoptosis was further confirmed by the appearance of a subG1 peak and the activation of caspases 3 and 9. Activation of the p53 pathway was monitored using immunoblotting via the expression of the p53 target genes p21, Noxa, Bax and DR5. The involvement of p53 was further studied in 4 different p53-silenced cell lines.
Both drugs induced the apoptosis of myeloma cells. The apoptosis that was induced by RITA was not related to the TP53 status of the cell lines or the del17p status of the primary samples (p = 0.52 and p = 0.80, respectively), and RITA did not commonly increase the expression level of p53 or p53 targets (Noxa, p21, Bax or DR5) in sensitive cells. Moreover, silencing of p53 in two TP53mutated cell lines failed to inhibit apoptosis that was induced by RITA, which confirmed that RITA-induced apoptosis in myeloma cells was p53 independent. In contrast, apoptosis induced by nutlin3a was directly linked to the TP53 status of the cell lines and primary samples (p < 0.001 and p = 0.034, respectively) and nutlin3a increased the level of p53 and p53 targets in a p53-dependent manner. Finally, we showed that a nutlin3a-induced DR5 increase (≥1.2-fold increase) was a specific and sensitive marker (p < 0.001) for a weak incidence of 17p deletion within the samples (≤19%).
These data show that RITA, in contrast to nutlin3a, effectively induced apoptosis in a subset of MM cells independently of p53. The findings and could be of interest for patients with a 17p deletion, who are resistant to current therapies.
PMCID: PMC4094448  PMID: 24927749
Myeloma; p53; RITA; Nutlin3a
16.  Small-Molecule Activation of p53 Blocks Hypoxia-Inducible Factor 1α and Vascular Endothelial Growth Factor Expression In Vivo and Leads to Tumor Cell Apoptosis in Normoxia and Hypoxia▿  
Molecular and Cellular Biology  2009;29(8):2243-2253.
The p53 tumor suppressor protein negatively regulates hypoxia-inducible factor 1α (HIF-1α). Here, we show that induction of p53 by the small-molecule RITA (reactivation of p53 and induction of tumor cell apoptosis) [2,5-bis(5-hydroxymethyl-2-thienyl) furan] (NSC-652287) inhibits HIF-1α and vascular endothelial growth factor expression in vivo and induces significant tumor cell apoptosis in normoxia and hypoxia in p53-positive cells. RITA has been proposed to stabilize p53 by inhibiting the p53-HDM2 interaction. However, induction of p53 alone was insufficient to block HIF-1α induced in hypoxia and has previously been shown to require additional stimuli, such as DNA damage. Here, we identify a new mechanism of action for RITA: RITA activates a DNA damage response, resulting in phosphorylation of p53 and γH2AX in vivo. Unlike other DNA damage response-inducing agents, RITA treatment of cells induced a p53-dependent increase in phosphorylation of the α subunit of eukaryotic initiation factor 2, requiring PKR-like endoplasmic reticulum kinase activity, and led to the subsequent downregulation of HIF-1α and p53 target proteins, including HDM2 and p21. Through the identification of a new mechanism of action for RITA, our study uncovers a novel link between the DNA damage response-p53 pathway and the protein translational machinery.
PMCID: PMC2663300  PMID: 19223463
17.  Variability in Skin Prick Test Results Performed by Multiple Operators Depends on the Device Used 
The variability of skin prick test results when carried out by multiple users has not previously been assessed across different devices or between different sites on the body. Such multiuser variability has important implications for clinical practice.
We assessed the variability of measurements from 4 commonly used single-headed skin test devices when used by multiple operators and examined whether the variability in performance was different on the back compared with the forearm.
Eight adult volunteer "operators" were trained in the use of 4 devices: Greer Pick, Quintip, Stallergenes Lancet, and Feather Lancet. Each operator performed a histamine skin prick test with all devices on the backs and forearms of 5 volunteer "receivers." Variability in results was assessed using a multilevel (random effects) regression model.
After controlling for variation between users and receivers, the residual variability or "measurement error" was least for the Stallergenes Lancet, closely followed by the Quintip. The Greer Pick had the greatest variability. There was greater variability in measurements on the arm compared with the back.
The devices using the "puncture" method (Stallergenes Lancet, Quintip) provide less variability in results than those using a "prick" method when carried out by multiple users (Greer Pick and Feather Lancet). Testing on the back also gives less variable results compared with the arm.
PMCID: PMC3651175  PMID: 23282421
device; skin prick test; variability
18.  Mediators between Theoretical and Practical Medieval Knowledge: Medical Notebooks from the Cairo Genizah and their Significance 
Medical History  2013;57(4):487-515.
This article presents a plethora of fragments from the medical notebooks found in the Cairo Genizah that comprise a unique source of historical data for scholarly study and for a better understanding of the ways in which medieval medical knowledge in Egypt was transferred from theory to practice and vice versa. These documents provide the most direct evidence we have for preferred practical medical recipes because they record the choices of medical practitioners in medieval Cairo. Since the language most commonly used in them was Judaeo-Arabic, they were evidently written by Jews. The medical genre in the notebooks was primarily pharmacopoeic, consisting of apparently original recipes for the treatment of various diseases. There are also a few notebooks on materia medica. The subject matter of the Genizah medical notebooks shows that they were mostly of an eclectic nature, i.e. the writers had probably learnt about these treatments and recipes from their teachers, applied them at the hospitals where they worked or copied them from the books they read. Foremost among the subjects dealt with were eye diseases, followed by skin diseases, coughs and colds, dentistry and oral hygiene, and gynaecological conditions. The writers of the Genizah notebooks apparently recorded the practical medical knowledge they wished to preserve for their future use as amateur physicians, students, traditional healers or professional practitioners.
PMCID: PMC3865955  PMID: 24069914
Cairo Genizah; History of Medicine; Jewish; Medieval Middle East; Middle Ages; Notebook
19.  How do the work environment and work safety differ between the dry and wet kitchen foodservice facilities? 
Nutrition Research and Practice  2012;6(4):366-374.
In order to create a worker-friendly environment for institutional foodservice, facilities operating with a dry kitchen system have been recommended. This study was designed to compare the work safety and work environment of foodservice between wet and dry kitchen systems. Data were obtained using questionnaires with a target group of 303 staff at 57 foodservice operations. Dry kitchen facilities were constructed after 2006, which had a higher construction cost and more finishing floors with anti-slip tiles, and in which employees more wore non-slip footwear than wet kitchen (76.7%). The kitchen temperature and muscular pain were the most frequently reported employees' discomfort factors in the two systems, and, in the wet kitchen, "noise of kitchen" was also frequently reported as a discomfort. Dietitian and employees rated the less slippery and slip related incidents in dry kitchens than those of wet kitchen. Fryer area, ware-washing area, and plate waste table were the slippery areas and the causes were different between the functional areas. The risk for current leakage was rated significantly higher in wet kitchens by dietitians. In addition, the ware-washing area was found to be where employees felt the highest risk of electrical shock. Muscular pain (72.2%), arthritis (39.1%), hard-of-hearing (46.6%) and psychological stress (47.0%) were experienced by employees more than once a month, particularly in the wet kitchen. In conclusion, the dry kitchen system was found to be more efficient for food and work safety because of its superior design and well managed practices.
PMCID: PMC3439582  PMID: 22977692
Work safety; dry kitchen; functional area; slipperiness; work environment in foodservice
20.  Rapid identification of high-confidence taxonomic assignments for metagenomic data 
Nucleic Acids Research  2012;40(14):e111.
Determining the taxonomic lineage of DNA sequences is an important step in metagenomic analysis. Short DNA fragments from next-generation sequencing projects and microbes that lack close relatives in reference sequenced genome databases pose significant problems to taxonomic attribution methods. Our new classification algorithm, RITA (Rapid Identification of Taxonomic Assignments), uses the agreement between composition and homology to accurately classify sequences as short as 50 nt in length by assigning them to different classification groups with varying degrees of confidence. RITA is much faster than the hybrid PhymmBL approach when comparable homology search algorithms are used, and achieves slightly better accuracy than PhymmBL on an artificial metagenome. RITA can also incorporate prior knowledge about taxonomic distributions to increase the accuracy of assignments in data sets with varying degrees of taxonomic novelty, and classified sequences with higher precision than the current best rank-flexible classifier. The accuracy on short reads can be increased by exploiting paired-end information, if available, which we demonstrate on a recently published bovine rumen data set. Finally, we develop a variant of RITA that incorporates accelerated homology search techniques, and generate predictions on a set of human gut metagenomes that were previously assigned to different ‘enterotypes’. RITA is freely available in Web server and standalone versions.
PMCID: PMC3413139  PMID: 22532608
21.  Beetles that live with ants (Carabidae, Pseudomorphini, Pseudomorpha Kirby, 1825): A revision of the santarita species group 
ZooKeys  2013;29-54.
The Western Hemisphere genus Pseudomorpha Kirby 1825 was last revised by Notman in 1925 based on only a few known species (22) and paltry few specimens (73); other authors have added an additional six species represented by 53 additional specimens since 1925. Baehr (1997) assigned three species from Australia to this genus, albeit in a new subgenus, Austropseudomorpha Baehr 1997. A recent study of collections from throughout the Americas (1757 specimens) has revealed numerous new species that can be arrayed across 19 species groups based on a suite of attributes, some used by Notman and others newly discovered. A taxonomic revision of the species contained in one of these species groups, santarita, is provided herein, as well as a distributional synopsis of the remaining 18 species groups. New species described herein are as follows, each with its type locality: Pseudomorpha huachineras p. n., Arroyo El Cocono, Sierra Huachinera, Sonora, México; P. patagonia sp. n., Madera Canyon, Santa Rita Mountains, Arizona; P.penablanca sp. n., Peña Blanca Lake, Arizona; P. pima sp. n., Madera Canyon (lower), Santa Rita Mountains, Arizona; P. santacruz sp. n., Madera Canyon, Santa Rita Mountains, Arizona; and P. santarita sp. n., Santa Rita Ranch, Santa Rita Mountains, Arizona.
PMCID: PMC3867105  PMID: 24363599
False-form beetles; new species; new species groups; identification key; distributions; male genitalia; female ovipositor; Hymenoptera: Formicidae
22.  Photoautotrophic Culture of Coffea arabusta Somatic Embryos: Development of a Bioreactor for Large‐scale Plantlet Conversion from Cotyledonary Embryos 
Annals of Botany  2002;90(1):21-29.
Somatic embryos were developed from in vitro‐grown leaf discs of Coffea arabusta in modified Murashige and Skoog medium under 30 µmol m–2 s–1 photosynthetic photon flux (PPF). Cotyledonary stage embryos were selected from the 14‐week‐old cultures and were placed under a high (100 µmol m–2 s–1) PPF for 14 d. These pretreated embryos were grown photoautotrophically in three different types of culture systems: Magenta vessel; RITA‐bioreactor (modified to improve air exchange); and a specially designed temporary root zone immersion bioreactor system (TRI‐bioreactor) with forced ventilation. The aims of the study were to achieve large‐scale embryo‐to‐plantlet conversion, and to optimize growth of plantlets under photoautotrophic conditions. The plantlet conversion percentage was highest (84 %) in the TRI‐bioreactor and lowest in the modified RITA‐bioreactor (20 %). Growth and survival of converted plantlets following 45 d of photoautotrophic culture in each of the three culture systems were studied. Fresh and dry masses of leaves and roots of plantlets developed in the TRI‐bioreactor were significantly greater than those of plantlets developed in the modified RITA‐bioreactor or Magenta vessel. The net photosynthetic rate, chlorophyll fluorescence and chlorophyll contents were also highest in plantlets grown in the TRI‐bioreactor. Normal stomata were observed in leaves of plantlets grown in the TRI‐bioreactor, whereas they could be abnormal in plantlets from the modified RITA‐bioreactor. Survival of the plants after transfer from culture followed a similar pattern and was highest in the group grown in the TRI‐bioreactor, followed by plants grown in the modified RITA‐bioreactor and Magenta vessel. In addition, ex vitro growth of plants transferred from the TRI‐bioreactor was faster than that of plants from the other culture systems.
PMCID: PMC4233855  PMID: 12125769
CO2 enrichment; embryo‐to‐plantlet conversion; ex vitro; forced ventilation; in vitro; stomata; survival percentage
23.  Personal exposures to fine particulate matter and black carbon in households cooking with biomass fuels in rural Ghana 
Environmental research  2013;127:40-48.
To examine cooking practices and 24-h personal and kitchen area exposures to fine particulate matter (PM2.5) and black carbon in cooks using biomass in Ghana.
Researchers administered a detailed survey to 421 households. In a sub-sample of 36 households, researchers collected 24-h integrated PM2.5 samples (personal and kitchen area); in addition, the primary cook was monitored for real-time PM2.5. All filters were also analyzed for black carbon using a multi-wavelength reflectance method. Predictors of PM2.5 exposure were analyzed, including cooking behaviors, fuel, stove and kitchen type, weather, demographic factors and other smoke sources.
The majority of households cooked outdoors (55%; 231/417), used biomass (wood or charcoal) as their primary fuel (99%; 412/413), and cooked on traditional fires (77%, 323/421). In the sub-sample of 29 households with complete, valid exposure monitoring data, the 24-h integrated concentrations of PM2.5 were substantially higher in the kitchen sample (mean 446.8 μg/m3) than in the personal air sample (mean 128.5 μg/m3). Black carbon concentrations followed the same pattern such that concentrations were higher in the kitchen sample (14.5 μg/m3) than in the personal air sample (8.8 μg/m3). Spikes in real-time personal concentrations of PM2.5 accounted for the majority of exposure; the most polluted 5%, or 72 min, of the 24-h monitoring period accounted for 75% of all exposure. Two variables that had some predictive power for personal PM2.5 exposures were primary fuel type and ethnicity, while reported kerosene lantern use was associated with increased personal and kitchen area concentrations of black carbon.
Personal concentrations of PM2.5 exhibited considerable inter-subject variability across kitchen types (enclosed, semi-enclosed, outdoor), and can be elevated even in outdoor cooking settings. Furthermore, personal concentrations of PM2.5 were not associated with kitchen type and were not predicted by kitchen area samples; rather they were driven by spikes in PM2.5 concentrations during cooking. Personal exposures were more enriched with black carbon when compared to kitchen area samples, underscoring the need to explore other sources of incomplete combustion such as roadway emissions, charcoal production and kerosene use.
PMCID: PMC4042308  PMID: 24176411
Biomass; Cooking; Personal exposure; Black carbon; Fine particulate matter
24.  Drug Resistance to Inhibitors of the Human Double Minute-2 E3 Ligase is Mediated by Point Mutations of p53, but can be Overcome with the p53 Targeting Agent RITA 
Molecular cancer therapeutics  2012;11(10):2243-2253.
The human double minute (HDM)-2 E3 ubiquitin ligase plays a key role in p53 turnover, and has been validated pre-clinically as a target in multiple myeloma (MM) and mantle cell lymphoma (MCL). HDM-2 inhibitors are entering clinical trials, and we therefore sought to understand potential mechanisms of resistance in lymphoid models. Wild-type p53 H929 MM and Granta-519 MCL cells resistant to MI-63 or Nutlin were generated by exposing them to increasing drug concentrations. MI-63-resistant H929 and Granta-519 cells were resistant to Nutlin, while Nutlin-resistant cells displayed cross-resistance to MI-63. These cells also showed cross-resistance to bortezomib, doxorubicin, cisplatin, and melphalan, but remained sensitive to the small molecule inhibitor RITA. HDM-2 inhibitor-resistant cells harbored increased p53 levels, but neither genotoxic nor non-genotoxic approaches to activate p53 induced HDM-2 or p21. Resequencing revealed wild-type HDM-2, but mutations were found in the p53 DNA binding and dimerization domains. In resistant cells, RITA induced a G2/M arrest, up-regulation of p53 targets HDM-2, PUMA, and NOXA, and PARP cleavage. Combination regimens with RITA and MI-63 resulted in enhanced cell death compared to RITA alone. These findings support the possibility that p53 mutation could be a primary mechanism of acquired resistance to HDM-2 inhibitors in MCL and MM. Furthermore, they suggest that simultaneous restoration of p53 function and HDM-2 inhibition is a rational strategy for clinical translation.
PMCID: PMC3469746  PMID: 22933706
HDM-2; p53; Multiple Myeloma; Mantle Cell Lymphoma; MI-63; Nutlin; RITA
25.  Targeting MDM2 by the small molecule RITA: towards the development of new multi-target drugs against cancer 
The use of low-molecular-weight, non-peptidic molecules that disrupt the interaction between the p53 tumor suppressor and its negative regulator MDM2 has provided a promising alternative for the treatment of different types of cancer. Among these compounds, RITA (reactivation of p53 and induction of tumor cell apoptosis) has been shown to be effective in the selective induction of apoptosis, and this effect is due to its binding to the p53 tumor suppressor. Since biological systems are highly dynamic and MDM2 may bind to different regions of p53, new alternatives should be explored. On this basis, the computational "blind docking" approach was employed in this study to see whether RITA would bind to MDM2.
It was observed that RITA binds to the MDM2 p53 transactivation domain-binding cleft. Thus, RITA can be used as a lead compound for designing improved "multi-target" drugs. This novel strategy could provide enormous benefits to enable effective anti-cancer strategies.
This study has demonstrated that a single molecule can target at least two different proteins related to the same disease.
PMCID: PMC1243243  PMID: 16174299
multi-target drugs; RITA; cancer treatment; blind docking; MDM2; p53 tumor suppressor

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