RNA Interference in Mammalia Cells by RNA-3’-PNA Chimeras

The discovery of siRNAs as the mediators of RNA interference has led to an increasing interest in their therapeutic applications. Chemical modifications are introduced into siRNAs to optimize the potency, the stability and the pharmacokinetic properties in vivo. Here, we synthesize and test the effects of RNA-3’-PNA chimeras on siRNA functioning and stability. We demonstrate that the chemical modifications are compatible with the siRNA machinery, because all the PNA-modified siRNAs can efficiently mediate specific gene silencing in mammalian cells. Furthermore, we find that the modification on the sense strand of siRNA results in an increased persistence of the activity, whereas modification on both strands results in enhanced nuclease resistance in serum

Genomic organization and splicing evolution of the doublesex gene, a Drosophila regulator of sexual differentiation, in the dengue and yellow fever mosquito Aedes aegypti

Background: In the model system Drosophila melanogaster, doublesex (dsx) is the double-switch gene at the bottom of the somatic sex determination cascade that determines the differentiation of sexually dimorphic traits. Homologues of dsx are functionally conserved in various dipteran species, including the malaria vector Anopheles gambiae. They show a striking conservation of sex-specific regulation, based on alternative splicing, and of the encoded sex-specific proteins, which are transcriptional regulators of downstream terminal genes that influence sexual differentiation of cells, tissues and organs. Results: In this work, we report on the molecular characterization of the dsx homologue in the dengue and yellow fever vector Aedes aegypti (Aeadsx). Aeadsx produces sex-specific transcripts by alternative splicing, which encode isoforms with a high degree of identity to Anopheles gambiae and Drosophila melanogaster homologues. Interestingly, Aeadsx produces an additional novel female-specific splicing variant. Genomic comparative analyses between the Aedes and Anopheles dsx genes revealed a partial conservation of the exon organization and extensive divergence in the intron lengths. An expression analysis showed that Aeadsx transcripts were present from early stages of development and that sex-specific regulation starts at least from late larval stages. The analysis of the female-specific untranslated region (UTR) led to the identification of putative regulatory cis-elements potentially involved in the sex-specific splicing regulation. The Aedes dsx sex-specific splicing regulation seems to be more complex with the respect of other dipteran species, suggesting slightly novel evolutionary trajectories for its regulation and hence for the recruitment of upstream splicing regulators. Conclusions: This study led to uncover the molecular evolution of Aedes aegypti dsx splicing regulation with the respect of the more closely related Culicidae Anopheles gambiae orthologue. In Aedes aegypti, the dsx gene is sex-specifically regulated and encodes two female-specific and one male-specific isoforms, all sharing a doublesex/mab 3 (DM) domain-containing N-terminus and different C-termini. The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation. Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner. Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs

Eudragit s100 entrapped liposome for curcumin delivery: Anti-oxidative effect in Caco-2 cells

Curcumin is a natural polyphenol with strong antioxidant activity. However, this molecule shows a very poor bioavailability, instability, and rapid metabolism in vivo. In this work curcumin was loaded in Eudragit-coated liposomes to create a gastroresistant carrier, able to protect its load from degradation and free it at the site of absorption in the colon region. Small unilamellar vesicles were prepared and coated with Eudragit by a pH-driven method. The physico-chemical properties of the prepared systems were assessed by light scattering, transmission electron microscopy, infrared spectroscopy, and differential scanning calorimetry. The uptake of vesicles by Caco-2 cells and the anti-oxidant activity in cells were evaluated. The produced vesicles showed dimensions of about forty nanometers that after covering with Eudragit resulted to have micrometric dimensions at acid pH. The experiments showed that at pH > 7.0 the polymeric coating dissolves, releasing the nanometric liposomes and allowing them to enter Caco-2 cells. Delivered curcumin loaded vesicles were then able to decrease significantly ROS levels as induced by H2O2 in Caco-2 cells. The proposed work showed the possibility of realizing effective gastroresistant curcumin liposome formulations for the delivery of antioxidant molecules to Caco-2 cells, potentially applicable to the treatment of pathological conditions related to intestinal oxidative stress. View Full-Tex

Detection of Antibodies against the Acetylcholine Receptor in Patients with Myasthenia Gravis: A Comparison of Two Enzyme Immunoassays and a Fixed Cell-Based Assay

The detection of serum anti-acetylcholine receptor (AChR) antibodies is currently an important tool for diagnosing myasthenia gravis (MG) since they are present in about 85% of MG patients. Many serological tests are now available. Nevertheless, results from these tests can be different in some patients. The aim of this study is to compare the sensitivity of a commercially available fixed cell-based assay (F-CBA) to that of enzyme-linked immunosorbent assay (ELISA) kits for anti-AChR detection in patients with a diagnosis of MG. Overall, 143 patients with a confirmed MG diagnosis were included in the study. The detection and measurement of serum anti-AChR antibodies were performed by three analytical methods, namely, a competitive ELISA (cELISA), an indirect ELISA (iELISA), and an F-CBA, according to the manufacturers' instructions. Anti-AChR antibody titers were positive in 94/143 (66%) using the cELISA, in 75/143 (52%) using the iELISA and in 61/143 (43%) using the F-CBA (adult and/or fetal). Method agreement, evaluated by concordant pairs and Cohen's kappa, was as follows: cELISA-iELISA: 110/143 (77%), k = 0.53 (95%CI 0.40-0.66); cELISA-F-CBA: 108/143 (76%), k = 0.53 (95%CI 0.41-0.66); iELISA-F-CBA: 121/143 (85%), k = 0.70 (95%CI 0.57-0.80). Our findings show that the cELISA has better analytical performance than the iELISA and F-CBA. However, the iELISA and F-CBA show the highest concordance

Preparation of drug-loaded small unilamellar liposomes and evaluation of their potential for the treatment of chronic respiratory diseases

The aim of the present investigation was to evaluate the influence of liposome formulation on the ability of vesicles to penetrate a pathological mucus model obtained from COPD affected patients in order to assess the potential of such vesicles for the treatment of chronic respiratory diseases by inhalation. Therefore, Small Unilamellar Liposomes (PLAIN-LIPOSOMEs), Pluronic® F127- surface modified liposomes (PF-LIPOSOMEs) and PEG 2000PE-surface modified liposomes (PEG-LIPOSOMEs) were prepared using the micelle-to-vesicle transition (MVT) method and beclomethasone dipropionate (BDP) as model drug. The obtained liposomes showed diameters in the range of 40-65 nm, PDI values between 0.25-0.30 and surface electric charge essentially close to zero. The encapsulation efficiency was found to be dependent on the BDP/lipid ratio used and, furthermore, BDP-loaded liposomes were stable in size both at 37°C and at 4°C. All liposomes were not cytotoxic on H441 cell line as assessed by the MTT assay. The liposome uptake was evaluated through a cytofluorimetric assay that showed a non-significant reduction in the internalization of PEG-LIPOSOMEs as compared with PLAIN-LIPOSOMEs. The penetration studies of mucus from COPD patients showed that the PEG-LIPOSOMEs were the most mucuspenetrating vesicles after 27 hours. In addition, PEG- and PF-LIPOSOMEs did not cause any effect on bronchoalveolar lavage fluid proteins after aerosol administration in the mouse. The results highlight that PEG-LIPOSOMEs show the most interesting features in terms of penetration through the pathologic sputum, uptake by airway epithelial cells and safety profile

Successful Heart-Liver Transplant Using Dual-organ Normothermic Perfusion in a Patient With Fontan Failure

Advances in surgical technique and multidisciplinary management have improved long-term survival for patients born with single ventricle physiology. However, patients who have undergone Fontan completion remain at risk for long-term comorbidities associated with the complex hemodynamic changes following the procedure, including Fontan failure and Fontan-associated liver disease.1 Combined heart-liver transplantation (CHLT) is a rare but lifesaving procedure that has been described in the setting of heart and liver failure secondary to Fontan failure.2 As long-term survival continues to improve for Fontan patients, the incidence of Fontan-associated liver disease will increase. Thus, improving CHLT outcomes and access to both organs is a strong priority. Here, we describe a successful CHLT for a patient with chronic ventricular dysfunction and Fontan-associated liver disease. The key innovation in this case was the use of normothermic machine perfusion (NMP) to preserve both the heart and liver grafts. This approach extended the preservation time for the liver while also mitigating the risk of ischemic injury and reducing the time pressure constraints on the heart transplant team. Notably, this stands in contrast to traditional static cold storage (SCS), where metabolic activity is reduced through hypothermia, but the extended cold ischemic time can lead to increased vulnerability to reperfusion injury

Validation of the ONKOTEV Risk Prediction Model for Venous Thromboembolism in Outpatients With Cancer

Importance: The assessment of the risk of venous thromboembolism (VTE) among outpatients with cancer represents an unsolved topic. Current international guidelines recommend primary prophylaxis for patients at intermediate to high risk of VTE, indicated by a Khorana score of 2 or more. A previous prospective study developed the ONKOTEV score, a 4-variable risk assessment model (RAM) consisting of a Khorana score of more than 2, metastatic disease, vascular or lymphatic compression, and previous VTE event. Objective: To validate the ONKOTEV score as a novel RAM to assess the risk of VTE among outpatients with cancer. Design, setting, and participants: ONKOTEV-2 is a noninterventional prognostic study conducted in 3 European centers located in Italy, Germany, and the United Kingdom among a prospective cohort of 425 ambulatory patients with a histologically confirmed diagnosis of a solid tumor who were receiving active treatments. The total study duration was 52 months, with an accrual period of 28 months (from May 1, 2015, to September 30, 2017) and an overall follow up-period of 24 months (data were censored September 30, 2019). Statistical analysis was performed in October 2019. Exposures: The ONKOTEV score was calculated for each patient at baseline by collecting clinical, laboratory, and imaging data from tests performed for routine practice. Each patient was then observed to detect any thromboembolic event throughout the study period. Main outcomes and measures: The primary outcome of the study was the incidence of VTE, including deep vein thrombosis and pulmonary embolism. Results: A total of 425 patients (242 women [56.9%]; median age, 61 years [range, 20-92 years]) were included in the validation cohort of the study. The cumulative incidences for the risk of developing VTE at 6 months were 2.6% (95% CI, 0.7%-6.9%), 9.1% (95% CI, 5.8%-13.2%), 32.3% (95% CI, 21.0%-44.1%), and 19.3% (95% CI, 2.5%-48.0%), respectively, among 425 patients with an ONKOTEV score of 0, 1, 2, and greater than 2 (P < .001). The time-dependent area under the curve at 3, 6, and 12 months was 70.1% (95% CI, 62.1%-78.7%), 72.9% (95% CI, 65.6%-79.1%), and 72.2% (95% CI, 65.2%-77.3%), respectively. Conclusions and relevance: This study suggests that, because the ONKOTEV score has been validated in this independent study population as a novel predictive RAM for cancer-associated thrombosis, it can be adopted into practice and into clinical interventional trials as a decision-making tool for primary prophylaxis