In Vitro Permeation Screening of a New Formulation of Thiocolchicoside Containing Various Enhancers

Thiocolchicoside, a muscle relaxant agent with anti-inflammatory and analgesic actions, also is used topically for the treatment of muscular spasms and for rheumatologic, orthopedic, and traumatologic disorders. In this study, thiocolchicoside was formulated to use as foam to avoid contact with the afflicted area during the spreading phase. To enhance drug penetration, various enhancers were added to the base formulation. The tested enhancers were ethoxyethylendiglycol (Transcutol), highly purified phosphatidylcholine (Lipoid S20), capsaicin, propylene glycol dipelargonate (DPPG), and glycolysed ethoxylated glycerides (Labrafil M1944 CS). The transdermal absorption of the tested formulations containing enhancers, in comparison with base formulation, was evaluated in vitro through rat skin using standard Franz diffusion cells. Base formulation was found to have a higher permeation profile than the simple aqueous and hydroalcoholic solutions of the drug, meaning that the base formulation by itself enhances the drug permeation. Among the tested formulations, only the formulation containing DPPG/ethanol was found to be statistically different, showing an enhancement factor of 3.58. In the same experimental session, Muscoril ointment, the commercially available pharmaceutical product containing the same thiocolchicoside concentration (0.25%), also was tested. The formulation containing DPPG/ethanol showed a 4.86 times increase of permeability constant in comparison with Muscoril ointment. The formulation containing DPPG/ethanol as an enhancer could be a good candidate for a new topical foam, considering its good characteristics of permeability and compliance

CHM/REP1 Transcript Expression and Loss of Visual Function in Patients Affected by Choroideremia

PURPOSE. To evaluate the disease progression in patients with clinical and genetic diagnoses of choroideremia during a long-term follow-up and to investigate the relationship between pathogenic variants in the CHM/REP1 gene and disease phenotypes. METHODS. We performed a retrospective longitudinal study on 51 affected men by reviewing medical charts at baseline and follow-up visits to extract the following ocular findings: best-corrected visual acuity, Goldmann visual field, optical coherence tomography, microperimetry. Data obtained from the analysis of DNA and mRNA were reevaluated for genetic classification of patients. RESULTS. The longitudinal analysis showed a significant (P < 0.001) worsening of best-corrected visual acuity with a mean rate of 0.011 logMar per year before 50 years and 0.025 logMar per year after 50 years. Similarly, V4e Goldmann visual field area significantly (P ≤ 0.01) decreased at a mean rate of 2.7% per year before 40 years and 5.7% after 40 years. Moreover, we observed a significant (P < 0.05) decrease of macular sensitivity with a mean rate of 5.0% per year and a decrease of mean macular thickness with a mean rate of 0.8% per year. We classified our patients into two groups according to the expression of the CHM/ REP1 gene transcript and observed that mutations leading to mRNA absence are associated with an earlier best-corrected visual acuity and Goldmann visual field loss. CONCLUSIONS. Our analysis of morphological and functional parameters in choroideremia patients showed a slow disease progression, particularly in the first decades of life. Overall, reevaluation of clinical and molecular data suggests exploring the genotype–phenotype relationship based on CHM/REP1 transcript expression. PURPOSE. To evaluate the disease progression in patients with clinical and genetic diagnoses of choroideremia during a long-term follow-up and to investigate the relationship between pathogenic variants in the CHM/REP1 gene and disease phenotypes. METHODS. We performed a retrospective longitudinal study on 51 affected men by reviewing medical charts at baseline and follow-up visits to extract the following ocular findings: best-corrected visual acuity, Goldmann visual field, optical coherence tomography, microperimetry. Data obtained from the analysis of DNA and mRNA were reevaluated for genetic classification of patients. RESULTS. The longitudinal analysis showed a significant (P < 0.001) worsening of best-corrected visual acuity with a mean rate of 0.011 logMar per year before 50 years and 0.025 logMar per year after 50 years. Similarly, V4e Goldmann visual field area significantly (P ≤ 0.01) decreased at a mean rate of 2.7% per year before 40 years and 5.7% after 40 years. Moreover, we observed a significant (P < 0.05) decrease of macular sensitivity with a mean rate of 5.0% per year and a decrease of mean macular thickness with a mean rate of 0.8% per year. We classified our patients into two groups according to the expression of the CHM/ REP1 gene transcript and observed that mutations leading to mRNA absence are associated with an earlier best-corrected visual acuity and Goldmann visual field loss. CONCLUSIONS. Our analysis of morphological and functional parameters in choroideremia patients showed a slow disease progression, particularly in the first decades of life. Overall, reevaluation of clinical and molecular data suggests exploring the genotype–phenotype relationship based on CHM/REP1 transcript expression

Identification and quantification of potential microplastics in shellfish harvested in Sardinia (Italy) by using transillumination stereomicroscopy

Plastics are non-biodegradable polymers made up of different groups of petrochemical materials. Several biotic and abiotic factors can change the density of plastic fragmenting it and originating microplastics (MPs). MPs have been defined as small pieces of plastic less than 5 mm in size. Due to their small size, they are an emerging concern in the marine environment since they can be ingested by aquatic organisms, especially filter-feeding organisms, such as bivalve mollusks. Impacts of MPs exposure have been shown at various levels of biological organization, from cellular to tissue to individual and population levels. For example, oxidative stress and inflammation have been observed in copepods and mussels, obstruction and physical damage of the digestive tract were found in fish and swimming behavior alterations, disruption of foraging and feeding behavior and overall reduced fitness and survival were observed in fish and oysters. In addition, MPs can act as a vector for the transfer of chemicals to marine biota. The aim of the present study was the identification and quantification of potential MPs in shellfish harvested in Sardinia (Italy) by using transillumination stereomicroscopy. Bivalves were collected from 4 of the main production areas located along the Sardinian coast and selected according to the principles of the risk assessment. The results of the present study demonstrated the presence of potential MPs in 70% of the analyzed samples: the presence of MPs in bivalve mollusks may pose a threat to food safety, and there is an urgent need to evaluate the potential risks of MPs to human health

New evidence of pectenotoxins in farmed bivalve molluscs from Sardinia (Italy)

Several planktonic dinoflagellates can produce lipophilic phycotoxins that represent a significant threat to public health as well as to shellfish and fish farming. Poisoning related to some of these toxins is categorised as diarrhetic shellfish poisoning. We analysed 975 shellfish samples from Tortolì in the central-eastern region of Sardinia (Italy) from January 2016 to March 2020, to investigate the prevalence of different lipophilic marine biotoxins in mollusc bivalves. The results highlighted the predominant presence of toxins belonging to the okadaic acid group in all samples with toxin concentrations exceeding legal limits, and revealed the new occurrence of pectenotoxins in oysters and clams with a winter seasonality in recent years. The origin of shellfish toxicity was associated with the same Dinophysis species, mainly D. acuminata. Based on both these results and other precedents, monitoring and recording systems are strongly recommended

Comparison of quantitative HCMV DNAemia in whole blood and leukocytes, and Real-Time PCR in a BMT patient

Background. HCMV is the most opportunistic viral agent in the transplantation of bone marrow and solid organ. Early therapy based on the detection of HCMV pp65 antigen in peripheral blood leukocytes, has led to a significant reduction in the incidence of related HCMV diseases.The pp65 antigenemia is difficult to standardize, while the quantification of HCMV DNA by Real-Time PCR is an alternative diagnostic approach with greater sensitivity and reproducibility, providing important information in the management of infected patients. Objectives. The aim of this study was the comparative analysis of quantitative HCMV DNAemia in leukocytes and in whole blood, with Real-time PCR, in a BMT patient with HCMV post-transplant reactivation, in order to analyze the relationship between levels of viral DNA at different time-points obtained in the two biological matrices. Study Design. The presence of HCMV DNA was detected in whole blood and peripheral blood leukocytes in a patient who underwent allogeneic marrow transplant for Ph1 + acute lymphoblastic leukemia in molecular remission. After 5-6 months, the patient has increased molecular Bcr-Abl (Philadelphia chromosome). It was activated immune reaction by means of tapering (lowering the dose of cyclosporine) that uses the GvL effect to turn negative the Philadelphia chromosome positivity (Bcr/Abl negativity). Later, the patient develops GvHD and cortisone is administered. The persistence of grafts treated with immunosuppressants periodically reactivates HCMV infection. The DNA extraction from whole-blood was performed by automatic extractor QIACUBE (Qiagen), while extraction from leucocytes was performed on a standard number of leukocytes (EXTRAcell- Nanogen).The extracted DNA was amplified by Real-Time Alert Q-PCR (Nanogen).The samples were analyzed weekly for about 5 months from 1 year after transplantation. Results. The patient at 1 year after transplantation, has three HCMV reactivation at 56th, 62th and 67th week. In all reactivations a good overlap values of viraemia in both whole blood leukocytes is shown. Conclusions. Our data confirm that the whole blood is a biological matrix with diagnostic similar performance not only to pp65 antigenaemia, as supported by extensive scientific literature, but also to the DNAemia in leukocytes that has been for a long time considered the elective matrix for evaluating the post-transplant HCMV infection, for pre-emptive therapy particularly in BMT patients

The Insulin Receptor Substrate (IRS)-1 recruits Phosphatidylinositol 3-Kinase to Ret: evidence for a competition between Shc and IRS-1 for the binding to Ret.

Tyrosine 1062 of Ret, which represents an intracytoplasmic docking site for multiple signaling molecules, is essential for Ret-mediated activation of phosphatidylinositol 3-Kinase (PI3-K). PI3-K, in turn, has been implicated in inducing cell survival and neoplastic transformation mediated by Ret. We have examined the mechanisms by which Ret stimulates PI3-K. Here we show that the Insulin Receptor Substrate-1 (IRS-1) is tyrosine phosphorylated and associated with the p85 regulatory subunit of PI3-K in response to Ret activation. IRS-1 coimmunoprecipitates with Ret and co-expression of IRS-1 results in the potentiation of Ret-mediated activation of Akt(PKB), a bona fide effector of PI3-K. The association with the PTB domain of IRS-1 depends on the phosphorylation of tyrosine 1062 of Ret. The deletion of asparagine 1059 (delN1059) and the substitution of leucine 1061 (L1061P), two Ret mutations identified in families affected by congenital megacolon (Hirschsprung's disease), impair the binding of IRS-1 to Ret as well as Ret-mediated Akt(PKB) stimulation. Finally, we show that Shc, which was previously identified as another ligand of Y1062 of Ret, competes with IRS-1 for the binding to Ret pY1062. All together, these findings suggest that IRS-1 is a component of the signaling pathway which leads to Ret-mediated PI3-K activation, a pathway which can be targeted by Hirschsprung-associated Ret mutations. The alternative binding of Shc and IRS-1 to Ret pY1062 can be a system to modulate the activation of different intracellular signaling pathways and to elicit different biological responses following Ret activation

Expression of the RET/PTC1 oncogene impairs the activity of TTF-1 and Pax-8 thyroid transcription factors.

The most frequent genetic alterations described thus far in human papillary thyroid carcinomas are somatic rearrangements of the RET proto-oncogene, which generate the chimeric RET/PTC oncogenes. We recently found that the expression of the RET/PTC1 oncogene blocked the expression of the thyroid-differentiated phenotype in rat thyroid epithelial cell line PC CI 3 (PC). Here, we show that this block occurs at a transcriptional level; indeed, the thyroid-specific thyroglobulin and thyroperoxidase gene promoters were inactive in PC-PTC cells. Specific transcription factors, namely, TTF-1 and Pax-8, regulate the expression of differentiated functions in thyroid cells. Here, we show that Pax-8 is expressed at reduced levels in PC-PTC cells and that its adoptive overexpression is unable to restore the activity of target promoters. In contrast, TTF-1 expression is unaltered in PC-PTC cells; however, by using a synthetic promoter that contains its specific target sequence, we demonstrate that TTF-1 is inactive in PC-PTC cells. We conclude that the RET/PTC1 oncogene alters the expression of the thyroid-differentiated phenotype by at least two different mechanisms, ie., down-regulation of Pax-8 protein and mRNA expression and impaired function of TTF-1 and Pax-8, which occurs at a posttranslational level

Different mutations of the RET gene cause different human tumoral diseases.

The RET gene encodes a tyrosine kinase receptor for neurotrophic molecules. RET is a conceptually valuable example of how different mutations of a single gene may cause different diseases. Gene rearrangements activate the oncogenic potential of RET in human thyroid papillary carcinomas. On the other side, different point mutations activate RET in familial multiple endocrine neoplasia syndromes. Finally, inactivating mutations of RET can be present in Hirschsprung's disease patients. The detailed knowledge of the specific RET mutations responsible for human tumors provides relevant tools for the clinical management of these diseases. Moreover, the recent discovery of the growth factors which in vivo stimulate its signaling may shed new light on the role played by RET in the development and differentiation of the central and peripheral nervous system