The GATA1-HS2 Enhancer Allows Persistent and Position-Independent Expression of a β-globin Transgene

Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a β-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine β-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and β-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of β-globin gene transfer by reducing expression variegation and epigenetic silencing

Triggering CD40 on endothelial cells contributes to tumor growth

Inflammatory cells can either promote or inhibit tumor growth. Here we studied whether CD40, a key molecule for adaptive immune response, has any role in mammary carcinogenesis of BALB/NeuT transgenic tumor-prone mice. We transferred the HER2/neu oncogene into CD40-null background to obtain the CD40-KO/NeuT strain. CD40-KO/NeuT mice showed delayed tumor onset and reduced tumor multiplicity. BM (BM) transplantation experiments excluded a role of BM-derived cells in the reduced tumorigenicity associated with CD40 deficiency. Rather, CD40 expressed by endothelial cells (ECs) takes part to the angiogenic process. Accordingly, large vessels, well organized around the tumor lobular structures, characterize BALB/NeuT tumors, whereas tiny numerous vessels with scarce extracellular matrix are dispersed in the parenchyma of poorly organized CD40-KO/NeuT tumors

3D printing fabrication of Ethylene-Vinyl Acetate (EVA) based intravaginal rings for antifungal therapy

Vulvovaginal candidiasis is a vaginal infection that affects women of reproductive age. Nowadays, the high administration frequency of conventional antifungal formulations, and recurrences negatively impact patients’ well-being. In this context, intravaginal rings (IVRs) offer the possibility of controlled local drug delivery with one single application, thus possibly increasing patient compliance. This project aimed to fabricate 3D printed IVRs to highlight the potential application of these medical devices for antifungal therapy, as well as emphasize the employment of 3DP as alternative manufacturing tool. Ethylene-Vinyl Acetate copolymer was chosen as matrix, and the antifungal efficacy of bifonazole and clotrimazole loaded in the IVRs was compared. The resulting medical devices were characterized using Fourier Transformed Infrared spectroscopy, the thermal behavior was investigated with Thermogravimetric Analysis and Differential Scanning Calorimetry, proving the stability of the incorporated drugs. In addition, the drug release profile was evaluated in a vaginal fluid simulant pH 4.2, at 37 °C, showing a sustained release over a week. The compressive strength of the IVRs was investigated, confirming that the mechanical properties comply with the already commercialized devices. To evaluate the antifungal activity, an in vitro time-kill assay was performed against Candida albicans for 7 days, exhibiting a complete growth inhibition after 4 days for the 3D printed IVRs. Overall, this work represents a step forward in the production of 3D printed IVRs potentially able to exert antifungal activity with one single application

Organization and nucleotide sequence of the genes for ribosomal protein S2 and elongation factor Ts in Spirulina platensis.

A 6.5 kb region from the genome of the cyanobacterium Spirulina platensis was cloned using as a probe the Escherichia coli gene for ribosomal protein S2. Sequence analysis revealed, in this region, the presence of the gene for ribosomal protein S2 and part of the gene for the elongation factor Ts (EF-Ts). The arrangement rpsB-spacer-tsf resembles that reported for E. coli. The deduced amino acid sequences of the platensis S2 and EF-Ts show significant homology with the E. coli counterparts

Characterization of the str operon genes from Spirulina platensis and their evolutionary relationship to those of other prokaryotes.

A 5.3 kb DNA segment containing the str operon (ca. 4.5 kb) of the cyanobacterium Spirulina platensis has been sequenced. The str operon includes the structural genes rpsL (ribosomal protein S12), rpsG (ribosomal protein S7), fus (translation elongation factor EF-G) and tuf (translation elongation factor EF-Tu). From the nucleotide sequence of this operon, the primary structures of the four gene products have been derived and compared with the available corresponding structures from eubacteria, archaebacteria and chloroplasts. Extensive homologies were found in almost all cases and in the order S12 greater than EF-Tu greater than EF-G greater than S7; the largest homologies were generally found between the cyanobacterial proteins and the corresponding chloroplast gene products. Overall codon usage in S. platensis was found to be rather unbiased

A Fluorinated Analogue of Marine Bisindole Alkaloid 2,2-Bis(6-bromo-1H-indol-3-yl)ethanamine as Potential Anti-Biofilm Agent and Antibiotic Adjuvant Against Staphylococcus aureus

Methicillin resistant Staphylococcus aureus (MRSA) infections represent a major global healthcare problem. Therapeutic options are often limited by the ability of MRSA strains to grow as biofilms on medical devices, where antibiotic persistence and resistance is positively selected, leading to recurrent and chronic implant-associated infections. One strategy to circumvent these problems is the co-administration of adjuvants, which may prolong the efficacy of antibiotic treatments, by broadening their spectrum and lowering the required dosage. The marine bisindole alkaloid 2,2-bis(6-bromo-1H-indol-3-yl)ethanamine (1) and its fluorinated analogue (2) were tested for their potential use as antibiotic adjuvants and antibiofilm agents against S. aureus CH 10850 (MRSA) and S. aureus ATCC 29213 (MSSA). Both compounds showed antimicrobial activity and bisindole 2 enabled 256-fold reduction (ΣFICs = 0.5) in the minimum inhibitory concentration (MIC) of oxacillin for the clinical MRSA strain. In addition, these molecules inhibited biofilm formation of S. aureus strains, and compound 2 showed greater eradicating activity on preformed biofilm compared to 1. None of the tested molecules exerted a viable but non-culturable cells (VBNC) inducing effect at their MIC values. Moreover, both compounds exhibited no hemolytic activity and a good stability in plasma, indicating a non-toxic profile, hence, in particular compound 2, a potential for in vivo applications to restore antibiotic treatment against MRSA infections

Quercetin Loaded Monolaurate Sugar Esters-Based Niosomes: Sustained Release and Mutual Antioxidant-Hepatoprotective Interplay

Flavonoids possess different interesting biological properties, including antibacterial, antiviral, anti-inflammatory and antioxidant activities. However, unfortunately, these molecules present different bottlenecks, such as low aqueous solubility, photo and oxidative degradability, high first-pass effect, poor intestinal absorption and, hence, low systemic bioavailability. A variety of delivery systems have been developed to circumvent these drawbacks, and among them, in this work niosomes have been selected to encapsulate the hepatoprotective natural flavonoid quercetin. The aim of this study was to prepare nanosized quercetin-loaded niosomes, formulated with different monolaurate sugar esters (i.e., sorbitan C12; glucose C12; trehalose C12; sucrose C12) that act as non-ionic surfactants and with cholesterol as stabilizer (1:1 and 2:1 ratio). Niosomes were characterized under the physicochemical, thermal and morphological points of view. Moreover, after the analyses of the in vitro biocompatibility and the drug-release profile, the hepatoprotective activity of the selected niosomes was evaluated in vivo, using the carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. Furthermore, the levels of glutathione and glutathione peroxidase (GSH and GPX) were measured. Based on results, the best formulation selected was glucose laurate/cholesterol at molar ratio of 1:1, presenting spherical shape and a particle size (PS) of 161 ± 4.6 nm, with a drug encapsulation efficiency (EE%) as high as 83.6 ± 3.7% and sustained quercetin release. These niosomes showed higher hepatoprotective effect compared to free quercetin in vivo, measuring serum biomarker enzymes (i.e., alanine and aspartate transaminases (ALT and AST)) and serum biochemical parameters (i.e., alkaline phosphatase (ALP) and total proteins), while following the histopathological investigation. This study confirms the ability of quercetin loaded niosomes to reverse CCl4 intoxication and to carry out an antioxidant effect

Correction: mTrop1/Epcam Knockout Mice Develop Congenital Tufting Enteropathy through Dysregulation of Intestinal E-cadherin/β-catenin.

Congenital tufting enteropathy (CTE) is a life-threatening hereditary disease that is characterized by enteric mucosa tufting degeneration and early onset, severe diarrhea. Loss-of-function mutations of the human EPCAM gene (TROP1, TACSTD1) have been indicated as the cause of CTE. However, loss of mTrop1/Epcam in mice appeared to lead to death in utero, due to placental malformation. This and indications of residual Trop-1/EpCAM expression in cases of CTE cast doubt on the role of mTrop1/Epcam in this disease. The aim of this study was to determine the role of TROP1/EPCAM in CTE and to generate an animal model of this disease for molecular investigation and therapy development. Using a rigorous gene-trapping approach, we obtained mTrop1/Epcam -null (knockout) mice. These were born alive, but failed to thrive, and died soon after birth because of hemorrhagic diarrhea. The intestine from the mTrop1/Epcam knockout mice showed intestinal tufts, villous atrophy and colon crypt hyperplasia, as in human CTE. No structural defects were detected in other organs. These results are consistent with TROP1/EPCAM loss being the cause of CTE, thus providing a viable animal model for this disease, and a benchmark for its pathogenetic course. In the affected enteric mucosa, E-cadherin and β-catenin were shown to be dysregulated, leading to disorganized transition from crypts to villi, with progressive loss of membrane localization and increasing intracellular accumulation, thus unraveling an essential role for Trop-1/EpCAM in the maintenance of intestinal architecture and functionality.Supporting information is available for this article

Incorporation of PEGylated δ-decalactone into lipid bilayers: thermodynamic study and chimeric liposomes development

Liposomes have been on the market as drug delivery systems for over 25 years. Their success comes from the ability to carry toxic drug molecules to the appropriate site of action through passive accumulation, thus reducing their severe side effects. However, the need for enhanced circulation time and site and time specific drug delivery, turned research focus on other systems, such as polymers. In this context, novel composites that combine the flexibility of polymeric nanosystems with the properties of liposomes gained a lot of interest. In the present work a mixed/chimeric liposomal system, composed of phospholipids and block copolymers, was developed and evaluated in regards with its feasibility as a drug delivery system. These innovative nano-platforms combine advantages from both classes of biomaterials. Thermal analysis was performed in order to offers an insight into the interactions between these materials and consequently into their physicochemical characteristics. In addition, colloidal stability was assessed by monitoring z-potential and size distribution over time. Finally, their suitability as carriers for biomedical applications was evaluated by carrying out in vitro toxicity studies