In vivo evaluation of antimyotonic efficacy of β-adrenergic drugs in a rat model of myotonia

AbstractThe sodium channel blocker mexiletine is considered the first-line drug in myotonic syndromes, a group of muscle disorders characterized by membrane over-excitability. We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, block voltage-gated sodium channels in a manner reminiscent to mexiletine, whereas salbutamol and nadolol do not. We now developed a pharmacological rat model of myotonia congenita to perform in vivo preclinical test of antimyotonic drugs. Myotonia was induced by i.p. injection of 30 mg/kg of anthracene-9-carboxylic acid (9-AC), a muscle chloride channel blocker, and evaluated by measuring the time of righting reflex (TRR). The TRR was prolonged from <0.5 s in control conditions to a maximum of ∼4 s, thirty minutes after 9-AC injection, then gradually recovered in a few hours. Oral administration of mexiletine twenty minutes after 9-AC injection significantly hampered the TRR prolongation, with an half-maximum efficient dose (ED50) of 12 mg/kg. Both propranolol and clenbuterol produced a dose-dependent antimyotonic effect similar to mexiletine, with ED50 values close to 20 mg/kg. Antimyotonic effects of 40 mg/kg mexiletine and propranolol lasted for 2 h. We also demonstrated, using patch-clamp methods, that both propranolol enantiomers exerted a similar block of skeletal muscle hNav1.4 channels expressed in HEK293 cells. The two enantiomers (15 mg/kg) also showed a similar antimyotonic activity in vivo in the myotonic rat. Among the drugs tested, the R(+)-enantiomer of propranolol may merit further investigation in humans, because it exerts antimyotonic effect in the rat model, while lacking of significant activity on the β-adrenergic pathway. This study provides a new and useful in vivo preclinical model of myotonia congenita in order to individuate the most promising antimyotonic drugs to be tested in humans

Molecular Insights into the Local Anesthetic Receptor within Voltage-Gated Sodium Channels Using Hydroxylated Analogs of Mexiletine

We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, directly blocked voltage-gated sodium channels, whereas salbutamol and nadolol did not (Desaphy et al., 2003), suggesting the presence of two hydroxyl groups on the aromatic moiety of the drugs as a molecular requisite for impeding sodium channel block. To verify such an hypothesis, we synthesized five new mexiletine analogs by adding one or two hydroxyl groups to the aryloxy moiety of the sodium channel blocker and tested these compounds on hNav1.4 channels expressed in HEK293 cells. Concentration–response relationships were constructed using 25-ms-long depolarizing pulses at −30 mV applied from an holding potential of −120 mV at 0.1 Hz (tonic block) and 10 Hz (use-dependent block) stimulation frequencies. The half-maximum inhibitory concentrations (IC50) were linearly correlated to drug lipophilicity: the less lipophilic the drug, minor was the block. The same compounds were also tested on F1586C and Y1593C hNav1.4 channel mutants, to gain further information on the molecular interactions of mexiletine with its receptor within the sodium channel pore. In particular, replacement of Phe1586 and Tyr1593 by non-aromatic cysteine residues may help in the understanding of the role of π–π or π–cation interactions in mexiletine binding. Alteration of tonic block suggests that the aryloxy moiety of mexiletine may interact either directly or indirectly with Phe1586 in the closed sodium channel to produce low-affinity binding block, and that this interaction depends on the electrostatic potential of the drug aromatic tail. Alteration of use-dependent block suggests that addition of hydroxyl groups to the aryloxy moiety may modify high-affinity binding of the drug amine terminal to Phe1586 through cooperativity between the two pharmacophores, this effect being mainly related to drug lipophilicity. Mutation of Tyr1593 further impaired such cooperativity. In conclusion, these results confirm our former hypothesis by showing that the presence of hydroxyl groups to the aryloxy moiety of mexiletine greatly reduced sodium channel block, and provide molecular insights into the intimate interaction of local anesthetics with their receptor

Cationic liposomal vectors incorporating a bolaamphiphile for oligonucleotide antimicrobials

Antibacterial resistance has become a serious crisis for world health over the last few decades, so that new therapeutic approaches are strongly needed to face the threat of resistant infections. Transcription factor decoys (TFD) are a promising new class of antimicrobial oligonucleotides with proven in vivo activity when combined with a bolaamphiphilic cationic molecule, 12-bis-THA. These two molecular species form stable nanoplexes which, however, present very scarce colloidal stability in physiological media, which poses the challenge of drug formulation and delivery. In this work, we reformulated the 12-bis-THA/TFD nanoplexes in a liposomal carrier, which retains the ability to protect the oligonucleotide therapeutic from degradation and deliver it across the bacterial cell wall. We performed a physical-chemical study to investigate how the incorporation of 12-bis-THA and TFD affects the structure of POPC- and POPC/DOPE liposomes. Analysis was performed using dynamic light scattering (DLS), ζ-potential measurements, small-angle x-ray scattering (SAXS), and steady-state fluorescence spectroscopy to better understand the structure of the liposomal formulations containing the 12-bis-THA/TFD complexes. Oligonucleotide delivery to model Escherichia coli bacteria was assessed by means of confocal scanning laser microscopy (CLSM), evidencing the requirement of a fusogenic helper lipid for transfection. Preliminary biological assessments suggested the necessity of further development by modulation of 12-bis-THA concentration in order to optimize its therapeutic index, i.e. the ratio of antibacterial activity to the observed cytotoxicity. In summary, POPC/DOPE/12-bis-THA liposomes appear as promising formulations for TFD delivery

Relevance of rosette patterns in variants of papillary thyroid carcinoma

IntroductionThe detection of rosette‐like clusters (RLC) of follicular cells in thyroid carcinoma has been reported mostly in the columnar cell variant of papillary thyroid carcinoma (PTC). Despite the fact that diagnosing variants of PTC is no longer encouraged by The Bethesda System for Reporting Thyroid Cytopathology, the identification of cytomorphological features such as RLC linked with these tumours might help reduce possible misinterpretation in thyroid fine needle aspiration (FNA) cytology. We accordingly investigated the potential correlation of architectural patterns including RLC with PTC variants.MethodsWe analysed 225 thyroid FNA cytology cases diagnosed as suspicious for malignancy (SFM) and positive for malignancy (M) over a 1‐year time where all samples had corresponding histology. We also included 150 benign lesions from the same period. The presence of RLC vs similar appearing solid clusters, papillary structures and microfollicles were evaluated. We also performed immunocytochemistry and molecular testing for BRAFV600E.ResultsWe included 100 (44.4%) SFM favouring PTC and 125 (55.6%) M cases with cyto‐histological correlation. On histology, all SFM and M cases showed malignancy including 140 (62.2%) classic PTC and 85 (37.8%) PTC variants. The cytomorphological patterns in all FNA samples included solid (74%), papillary (89%), microfollicular (70%), and pseudo‐RLC morphology (25.7%). We identified only pseudo‐RLC in 33 FNA specimens from PTC variant cases that included tall cell variant (42.4%), hobnail variant (21.2%) and miscellaneous variants (36.3%) of PTC. No definitive RLC were detected in our series. Immunocytochemistry and BRAFV600E were not specifically linked with an RLC pattern.ConclusionsThese findings demonstrate that in our dataset the architectural pattern of RLC was not recognised within PTC variants. However, we did identify a pseudo‐RLC pattern that was observed in association with tall cell variant and hobnail variant cases of PTC.The findings discussed in the present study demonstrate that certain architectural patterns such as rosette‐like clusters (RLC) may be recognized more frequently in specific PTC variants that may be helpful in reducing their misinterpretation. In the series presented, RLCs were observed mostly in association with tall cell and hobnail variants of PTC.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163372/2/cyt12885_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163372/1/cyt12885.pd

Type D personality impairs Quality of Life, coping and short-term psychological outcome in patients attending an outpatient intensive program of cardiac rehabilitation

Background: Type D personality represents a risk factor for adverse outcome and impaired Quality of Life (QoL) in CHD patients. Only few studies investigated Type D patients following cardiac rehabilitation (CR). No study investigated Type D personality in Italian patients attending a CR program of 4 weeks. The aims of the study were a) to verify the presence of Type D personality among patients attending an Italian CR program; b) to investigate psychological health status, QoL and coping style of CR patients and c) to test the influence of Type D personality on CR patients outcome. Methods: Data from 59 patients attending an outpatient intensive program of 4 weeks of CR were collected at admission, and 1 month after discharge, using a set of self-report questionnaires. Variables were measured using CBAH, DS-14, Q-LES-Q and Brief COPE Scale. Results: The percentage of Type D personality found in the study sample was 39%. At admission Type D patients showed a significant lower level of psychological health status and QoL satisfaction compared to non Type D patients (p<0.05). After CR a significant percentage of Type D patients, despite an overall improvement, continued to show a clinically relevant psychological impairment in terms of anxiety (p=0.003), depressive mood (p=0.001), impairment in psychophysical well-being (p=0.002), perceived psychophysical stress (p=0.002), interpersonal difficulties (p<0.001), and social anxiety (p=0.045). Type D personality was also found to be associated with a significant greater use of maladaptive coping strategies (p<0.05). Conclusions: Type D personality played a significant clinically relevant role on psychological health outcome in CR. Type D personality patients reported a significant higher level of psychological impairment, in terms of anxiety, depressive mood, impairment in psychophysical wellbeing, perceived psychophysical stress, interpersonal difficulties, social anxiety, and a significant lower QoL, prior and after CR. Type D personality seemed also to be associated with maladaptive coping strategies. Importance of assessment for Type D personality is warranted in CR setting, as additional interventions seem required to enhance the outcome of these patients defined in letterature at high-risk

Development and validation of a framework for the assessment of school curricula on the presence of evolutionary concepts (FACE)

Evolution is a key concept of biology, fundamental to understand the world and address important societal problems, but research studies show that it is still not widely understood and accepted. Several factors are known to influence evolution acceptance and understanding, but little information is available regarding the impacts of the curriculum on these aspects. Very few curricula have been examined to assess the coverage of biological evolution. The available studies do not allow comparative analyses, due to the different methodologies employed by the authors. However, such an analysis would be useful for research purposes and for the development of appropriate educational policies to address the problem of a lack of evolution acceptance in some countries. In this paper we describe the steps through which we developed a valid and reliable instrument for curricula analysis known as FACE: “Framework to Assess the Coverage of biological Evolution by school curricula.” This framework was developed based on the “Understanding Evolution Conceptual Framework” (UECF). After an initial pilot study, our framework was reformulated based on identified issues and experts’ opinions. To generate validity and reliability evidence in support of the framework, it was applied to four European countries’ curricula. For each country, a team of a minimum of two national and two foreign coders worked independently to assess the curriculum using this framework for content analysis. Reliability evidence was estimated using Krippendorf's alpha and resulted in appropriate values for coding the examined curricula. Some issues that coders faced during the analysis were discussed and, to ensure better reliability for future researchers, additional guidelines and one extra category were included in the framework. The final version of the framework includes six categories and 34 subcategories. FACE is a useful tool for the analysis and the comparison of curricula and school textbooks regarding the coverage of evolution, and such results can guide curricula development.info:eu-repo/semantics/publishedVersio

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Circulating Biomarkers and Resistance to Endocrine Therapy in Metastatic Breast Cancers: Correlative Results from AZD9496 Oral SERD Phase I Trial.

PURPOSE: Common resistance mechanisms to endocrine therapy (ET) in estrogen receptor (ER)-positive metastatic breast cancers include, among others, ER loss and acquired activating mutations in the ligand-binding domain of the ER gene (ESR1LBDm). ESR1 mutational mediated resistance may be overcome by selective ER degraders (SERD). During the first-in-human study of oral SERD AZD9496, early changes in circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) were explored as potential noninvasive tools, alongside paired tumor biopsies, to assess pharmacodynamics and early efficacy. EXPERIMENTAL DESIGN: CTC were enumerated/phenotyped for ER and Ki67 using CellSearch in serial blood draws. ctDNA was assessed for the most common ESR1LBDm by droplet digital PCR (BioRad). RESULTS: Before starting AZD9496, 11 of 43 (25%) patients had ≥5 CTC/7.5 mL whole blood (WB), none of whom underwent reduction to <5 CTC/7.5 mL WB on C1D15. Five of 11 patients had baseline CTC-ER+, two of whom had CTC-ER+ reduction. CTC-Ki67 status did not change appreciably. Patients with ≥5 CTC/7.5 mL WB before treatment had worse progression-free survival (PFS) than patients with <5 CTC (P = 0.0003). Fourteen of 45 (31%) patients had ESR1LBDm + ctDNA at baseline, five of whom had ≥2 unique mutations. Baseline ESR1LBDm status was not prognostic. Patients with persistently elevated CTC and/or ESR1LBDm + ctDNA at C1D15 had worse PFS than patients who did not (P = 0.0007). CONCLUSIONS: Elevated CTC at baseline was a strong prognostic factor in this cohort. Early on-treatment changes were observed in CTC-ER+ and ESR1LBDm + ctDNA, but not in overall CTC number. Integrating multiple biomarkers in prospective trials may improve outcome prediction and ET resistance mechanisms' identification over a single biomarker