Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Carvedilol Prevents Ovariectomy-Induced Myocardial Contractile Dysfunction in Female Rat

Carvedilol has beneficial effects on cardiac function in patients with heart failure but its effect on ovariectomy-induced myocardial contractile dysfunction remains unclear. Estrogen deficiency induces myocardial contractile dysfunction and increases cardiovascular disease risk in postmenopausal women. Our aim was to investigate whether carvedilol, a beta receptor blocker, would prevent ovariectomy-induced myocardial contractile dysfunction. Female rats (8 weeks old) that underwent bilateral ovariectomy were randomly assigned to receive daily treatment with carvedilol (OVX+CAR, 20 mg/kg), placebo (OVX) and SHAM for 58 days. Left ventricle papillary muscle was mounted for isometric tension recordings. The inotropic response to Ca²⁺ (0.62 to 3.75 mM) and isoproterenol (Iso 10⁻⁸ to 10⁻² M) were assessed. Expression of calcium handling proteins was measured by western blot analysis. Carvedilol treatment in the OVX animals: prevented weight gain and slight hypertrophy, restored the reduced positive inotropic responses to Ca²⁺ and isoproterenol, prevented the reduction in SERCA2a expression, abolished the increase in superoxide anion production, normalized the increase in p22^phox expression, and decreased serum angiotensin converting enzyme (ACE) activity. This study demonstrated that myocardial contractile dysfunction and SERCA2a down regulation were prevented by carvedilol treatment. Superoxide anion production and NADPH oxidase seem to be involved in this response.     

Inhibition of Histone Deacetylase Impacts Cancer Stem Cells and Induces Epithelial-Mesenchyme Transition of Head and Neck Cancer

The genome is organized and packed into the nucleus through interactions with core histone proteins. Emerging evidence suggests that tumors are highly responsive to epigenetic alterations that induce chromatin-based events and dynamically influence tumor behavior. We examined chromatin organization in head and neck squamous cell carcinoma (HNSCC) using acetylation levels of histone 3 as a marker of chromatin compaction. Compared to control oral keratinocytes, we found that HNSCC cells are hypoacetylated and that microenvironmental cues (e.g., microvasculature endothelial cells) induce tumor acetylation. Furthermore, we found that chemical inhibition of histone deacetylases (HDAC) reduces the number of cancer stem cells (CSC) and inhibits clonogenic sphere formation. Paradoxically, inhibition of HDAC also induced epithelial-mesenchymal transition (EMT) in HNSCC cells, accumulation of BMI-1, an oncogene associated with tumor aggressiveness, and expression of the vimentin mesenchymal marker. Importantly, we observed co-expression of vimentin and acetylated histone 3 at the invasion front of human HNSCC tumor tissues. Collectively, these findings suggest that environmental cues, such as endothelial cell-secreted factors, modulate tumor plasticity by limiting the population of CSC and inducing EMT. Therefore, inhibition of HDAC may constitute a novel strategy to disrupt the population of CSC in head and neck tumors to create a homogeneous population of cancer cells with biologically defined signatures and predictable behavior.     

Altered Visual and Feet Proprioceptive Feedbacks during Quiet Standing Increase Postural Sway in Patients with Severe Knee Osteoarthritis

Objective

The objective was to investigate how postural control in knee osteoarthritis (KOA) patients, with different structural severities and pain levels, is reorganized under different sensory conditions.

Methods

Forty-two obese patients (BMI range from 30.1 to 48.7 kg*m⁻², age range from 50 to 74 years) with KOA were evaluated. One minute of quiet standing was assessed on a force platform during 4 different sensory conditions, applied 3 times at random: Eyes open (EO) and eyes closed (EC) standing on firm and soft (foam) surfaces (EO-soft and EC-soft). Centre of pressure (Cop) standard deviation, speed, range and Cop mean position in both directions (anterior-posterior and medial-lateral) were extracted from the force platform data. Structural disease severity was assessed from semiflexed standing radiographs and graded by the Kellgren and Lawrence (KL) score. Pain intensity immediately before the measurements was assessed by numeric rating scale (range: 0–10).

Results

The patients were divided into “less severe” (KL 1 and 2, n = 24) and “severe” (KL>2, n = 18) group. The CoP range in the medial-lateral direction was larger in the severe group when compared with the less severe group during EC-soft condition (P<0.01). Positive correlation between pain intensity and postural sway (range in medial-lateral direction) was found during EC condition, indicating that the higher the pain intensity, the less effective is the postural control applied to restore an equilibrium position while standing without visual information.

Conclusion

The results support that: (i) the postural reorganization under manipulation of the different sensory information is worse in obese KOA patients with severe degeneration and/or high pain intensity when compared with less impaired patients, and (ii) higher pain intensity is related to worse body balance in obese KOA patients.     

First attempts to crystallize a non-homogeneous sample of thioredoxin from Litopenaeus vannamei: What to do when you have diffraction data of a protein that is not the target?

The importance of sample homogeneity and purity in protein crystallization is essential to obtain high-quality diffracting crystals. Here, in an attempt to determine the crystal structure of thioredoxin 1 from whiteleg shrimp Litopenaeus vannamei (LvTrx), we inadvertently crystallized the hexameric inorganic pyrophosphatase of Escherichia coli (E-PPase) from a non-homogeneous sample product during the initial over-expression steps and partial purification of LvTrx. The structure determination and identification of the crystallized protein were derived from several clues: the failures in the Molecular Replacement (MR) trials using LvTrx coordinates as a search model, the unit cell parameters and space group determination, and essentially by the use of the program BALBES. After using the previously deposited E-PPase structure (PDB entry 1mjw) as a search model and the correct space group assignation, the MR showed an E-PPase complexed with SO₄^?2 with small changes in the sulfate ion binding region when it compares to previously deposited E-PPases in the PDB. This work stresses the importance of protein purity to avoid the risk of crystallizing a contaminant protein or how pure need to be a protein sample in order to increase the possibility to obtain crystals, but also serves as a reminder that crystallization is by itself a purification process and how the program BALBES can be useful in the crystal structure determination of previously deposited structures in the PDB.     

Sexual development in early- and late-maturing Bos indicus and Bos indicus x Bos taurus crossbred bulls in Brazil

Two experiments were conducted to evaluate sexual development in early- and late-maturing Nelore (Bos indicus) and Canchim (3/8 Bos indicus x 5/8 Bos taurus crossbred) bulls and to determine predictors of sexual precocity, and pubertal and maturity status. In Experiment 1, 12 Nelore bulls where examined from 300 to 900 days of age. Puberty was characterized by an ejaculate containing > or =50 million sperm with > or =10% motile sperm, and maturity by an ejaculate containing > or =70% morphologically normal sperm. In Experiment 2, 28 Canchim bulls where examined from 295 to 488 days of age and puberty was characterized by an ejaculate containing > or =30% motile sperm. In both experiments, bulls were classified as early- or late-maturing based on age at puberty. Early-maturing bulls were younger (P < 0.05) than late-maturing bulls at puberty (527 days versus 673 days in Experiment 1 and 360 days versus 461 days in Experiment 2) and at maturity (660 days versus 768 days in Experiment 1). In general, early-maturing bulls were heavier and had greater scrotal circumference (SC), testes, and testicular vascular cone diameter than late-maturing bulls during the experimental period. Scrotal circumference adjusted for 365 days of age was a good predictor of sexual precocity; minimum yearling SC of 19 and 24 cm for Nelore and Canchim bulls, respectively, had the best predictive values. Early-maturing bulls were lighter and had smaller SC at puberty than late-maturing bulls; therefore, sexual precocity was not related to the attainment of a threshold body weight or testicular size earlier, but to lower thresholds in early-maturing bulls. When predictors of pubertal status were evaluated, SC had the best sensitivity/specificity relationship in Nelore bulls, and high sensitivity and specificity in Canchim bulls. When predictors of sexual maturity were evaluated in Nelore bulls, age, weight, and SC had similar sensitivity, specificity, and predictive values. At puberty, approximately 60% of the sperm present in the ejaculate were morphologically defective. Changes in semen quality after puberty in Nelore bulls were characterized by increased motility and proportion of morphologically normal sperm, with a decrease in the proportion of major sperm defects. In conclusion, early-maturing bulls were more developed in the pre-pubertal period and attained puberty at earlier stages of body and testicular development than late-maturing bulls. Yearling SC could be used to select bulls for sexual precocity and SC was the best predictor of pubertal status. Age, weight, and SC were equally good predictors of sexual maturity in B. indicus bulls.     

Catalase from the white shrimp Penaeus (Litopenaeus) vannamei: molecular cloning and protein detection

Catalase is an antioxidant enzyme that plays a very important role in the protection against oxidative damage by breaking down hydrogen peroxide. It is a very highly conserved enzyme that has been identified from numerous species including bacteria, fungi, plants and animals, but the information about catalase in crustaceans is very limited. A cDNA containing the complete coding sequence for catalase from the shrimp Penaeus (Litopenaeus) vannamei was sequenced and the mRNA was detected by RT-PCR in selected tissues. Catalase was detected in hepatopancreas crude extracts by Western blot analysis with anti-human catalase polyclonal antibodies. The nucleotide sequence is 1692 bp long, including a 72-bp 5′-UTR, a coding sequence of 1515 bp and a 104-bp 3′-UTR. The deduced amino acid sequence corresponds to 505 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases and contains the catalytic residues His71, Asn144, and Tyr354. The predicted protein has a calculated molecular mass of 57 kDa; which coincides with the size of the subunit (55 kDa) and the tetrameric protein (230 kDa) detected in hepatopancreas extracts under native conditions. Catalase mRNA level was higher in hepatopancreas, followed by gills and was not detected in muscle.