A novel high-content immunofluorescence assay as a tool to identify at the single cell level γ-globin inducing compounds

The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ-and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathie

La valutazione neuropsicologica in pazienti affetti da disturbo schizofrenico: correlazione con la psicopatologia e il funzionamento globale.

Scopo dello studio è valutare in un campione di pazienti affetti da un disturbo dello spettro schizofrenico il profilo neuropsicologico e confrontarlo con quello di una popolazione di controllo

Efficacia costi/benifici della psicoterapia cognitivo-comportamentale di gruppo nel DAP rispetto alla terapia farmacologica. Follow-up di 1 anno.

Studio clinico controllato prospettico della durata di un anno, al fine di valutare i costi e i benefici di un intervento cognitivo comportamentale di gruppo verso un intervento farmacologico con paroxetina. Il rapporto costi/benefici \ue8 risultato decisamente a favore della psicoterapia

Multi-Rater Agreement Using the Adapted Fracture Healing Scale (AFHS) for the Assessment of Tubular Bones on Conventional Radiographs : Preliminary Study

Better understanding of the timing of fracture healing may help in cases of interpersonal violence but also of personal identification. The intra- and inter-rater agreement for the adapted fracture healing scale (AFHS) assessing the post-traumatic time interval on radiographs were tested. This is a preliminary study, providing essential information on method reliability for upcoming studies using the AFHS. Five raters (two radiologists, a forensic pathologist, an orthopedist, and an anthropologist) were presented with a test in three parts consisting of 85 radiographs (from 30 adults) of fractures of tubular bones in different stages of healing purposefully selected from more than 1500 radiographs. The raters were firstly asked to assess 15 features describing fracture healing as present, absent, or not assessable. Thereafter, the raters were asked to choose from the AFHS a single-stage best representing the observed healing pattern. The intra- and inter-rater agreement were assessed using single-rating, absolute agreement, two-way mixed-effects intra-class correlation (ICC) coefficients. The intra-rater ICC of radiologist 1 ranged from 0.80 to 0.94. The radiologists' inter-rater ICC ranged from 0.68 to 0.74, while it ranged from -0.01 to 0.90 for the other raters. The good to excellent ICC among the radiologists and forensic anthropologist provides good foundation for the use of the AFHS in forensic cases of trauma dating. The poor to good results for the other physicians indicate that using the AFHS requires training in skeletal anatomy and radiology

Multi‐Rater Agreement Using the Adapted Fracture Healing Scale (AFHS) for the Assessment of Tubular Bones on Conventional Radiographs: Preliminary Study*

Better understanding of the timing of fracture healing may help in cases of interpersonal violence but also of personal identification. The intra- and inter-rater agreement for the adapted fracture healing scale (AFHS) assessing the post-traumatic time interval on radiographs were tested. This is a preliminary study, providing essential information on method reliability for upcoming studies using the AFHS. Five raters (two radiologists, a forensic pathologist, an orthopedist, and an anthropologist) were presented with a test in three parts consisting of 85 radiographs (from 30 adults) of fractures of tubular bones in different stages of healing purposefully selected from more than 1500 radiographs. The raters were firstly asked to assess 15 features describing fracture healing as present, absent, or not assessable. Thereafter, the raters were asked to choose from the AFHS a single-stage best representing the observed healing pattern. The intra- and inter-rater agreement were assessed using single-rating, absolute agreement, two-way mixed-effects intra-class correlation (ICC) coefficients. The intra-rater ICC of radiologist 1 ranged from 0.80 to 0.94. The radiologists' inter-rater ICC ranged from 0.68 to 0.74, while it ranged from -0.01 to 0.90 for the other raters. The good to excellent ICC among the radiologists and forensic anthropologist provides good foundation for the use of the AFHS in forensic cases of trauma dating. The poor to good results for the other physicians indicate that using the AFHS requires training in skeletal anatomy and radiology

IgG and IgG4 to 91 allergenic molecules in early childhood by route of exposure and current and future IgE sensitization: Results from the Multicentre Allergy Study birth cohort

Abstract BACKGROUND: Studies of a limited number of allergens suggested that nonsensitized children produce IgG responses mainly to foodborne allergens, whereas IgE-sensitized children also produce strong IgG responses to the respective airborne molecules. OBJECTIVE: We sought to systematically test the hypothesis that both the route of exposure and IgE sensitization affect IgG responses to a broad array of allergenic molecules in early childhood. METHODS: We examined sera of 148 children participating in the Multicentre Allergy Study, a birth cohort born in 1990. IgG to 91 molecules of 42 sources were tested with the ImmunoCAP Solid-Phase Allergen Chip (ISAC; TFS, Uppsala, Sweden). IgE sensitization at age 2 and 7 years was defined by IgE levels of 0.35 kUA/L or greater to 1 or more of 8 or 9 extracts from common allergenic sources, respectively. RESULTS: The prevalence and geometric mean levels of IgG to allergenic molecules in nonsensitized children were lower at age 2 years than in IgE-sensitized children, and they were extremely heterogeneous: highest for animal food (87% ± 13%; 61 ISAC Standardized Units [ISU], [95% CI, 52.5-71.5 ISU]), intermediate for vegetable food (48% ± 27%; 13 ISU [95% CI, 11.2-16.1 ISU]), and lowest for airborne allergens (24% ± 20%; 3 ISU [95% CI, 2.4-3.4 ISU]; P for trend < .001 [for percentages], P for trend < .001 [for levels]). IgG4 antibodies were infrequent (<5%) and contributed poorly (<3%) to overall IgG antibody levels. IgG responses at age 2 years were slightly more frequent and stronger among children with than in those without IgE sensitization at age 7 years. CONCLUSION: The children's repertoire of IgG antibodies at 2 years of age to a broad array of animal foodborne, vegetable foodborne, and airborne allergenic molecules is profoundly dependent on the route of allergen exposure and the child's IgE sensitization status and only marginally involves the IgG4 isotype

Using Failure Mode, Effect and Criticality Analysis to improve safety in the cancer treatment prescription and administration process

Key points FMECA is a proactive risk assessment tool that has proved useful in identifying potential failures in the process of prescribing and administering cancer treatments, which is a high-risk process. The analysis evidenced 22 failures modes, distributed over the various phases of the process. In order to reduce the likelihood of the cause, or to improve the chances of a failure mode being detected, this work identified a number of recommendations to be adopted in the administration of cancer treatment in an outpatient setting

High-content γ/β globin analysis as readout of siRNA screening in β-K562 confirms HDAC as targets for γ-globin activation.

<p>A) Cells were transfected with a non-targeting oligo (siNTO) as negative control and with a siRNA directed to HDAC3. Two siRNAs were tested, with two technical replicates. C) β-K562 treated with two different HDAC inhibitors: entinostat and dacinostat (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.s003" target="_blank">S3 Fig</a>). A and C) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B and D) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.g001" target="_blank">Fig 1</a>.</p