Fatty acid-related modulations of membrane fluidity in cells: detection and implications

Abstract

Metabolic homeostasis of fatty acids is complex and well-regulated in all organisms. The biosynthesis of saturated fatty acids (SFA) in mammals provides substrates for β-oxidation and ATP production. Monounsaturated fatty acids (MUFA) are products of desaturases that introduce a methylene group in cis geometry in SFA. Polyunsaturated fatty acids (n-6 and n-3 PUFA) are products of elongation and desaturation of the essential linoleic acid and α-linolenic acid, respectively. The liver processes dietary fatty acids and exports them in lipoproteins for distribution and storage in peripheral tissues. The three types of fatty acids are integrated in membrane phospholipids and determine their biophysical properties and functions. This study was aimed at investigating effects of fatty acids on membrane biophysical properties under varying nutritional and pathological conditions, by integrating lipidomic analysis of membrane phospholipids with functional two-photon microscopy (fTPM) of cellular membranes. This approach was applied to two case studies: first, pancreatic beta-cells, to investigate hormetic and detrimental effects of lipids. Second, red blood cells extracted from a genetic mouse model defective in lipoproteins, to understand the role of lipids in hepatic diseases and metabolic syndrome and their effect on circulating cells.     

Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE)

One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.     

Cryopreservation of the Mediterranean mussel (Mytilus galloprovincialis) spermatozoa

The effects of cryoprotectants, cooling rate and freezing on the mussel Mytilus galloprovincialis sperm were evaluated. At the end of each step of the experimental protocol, motility and fertilization ability of sperm were analyzed, compared to fresh semen. Five cryoprotectants were tested in their toxicity level: dimethylsulfoxide, ethylene glycol, 1-2 propylene glycol at 5%, 7%, 10%, 15% and 20% concentration; glycerol and methanol at concentration of 5%, 7% and 10%. The incubation times were 10, 20 and 30 min at 20 ± 1 °C. Only dimethylsulfoxide, ethylene glycol and 1-2 propylene glycol at 5%, 7% and 10% were chosen for the following pre-freezing step. Five adaptation/chilling rates were analyzed: 10 min at 20 ± 1, −2, −1, −0.5 and −0.25 °C/min and the last one was used for testing the best freezing procedure among seven gradients. Particularly, two rapid rates, three slow rates and two double step rates were conducted.Thawing results showed that M. galloprovincialis sperm are very sensitive to rapid pre-freezing and freezing protocols and only a slow procedure assured good motility and fertilization percentages.     

Ribonuclear inclusions as biomarker of myotonic dystrophy type 2, even in improperly frozen or defrozen skeletal muscle biopsies

Myotonic dystrophy type 2 (DM2) is a dominantly inherited disorder caused by a CCTG repeat expansion in intron 1 of ZNF9 gene. The size and the somatic instability of DM2 expansion complicate the molecular diagnosis of DM2. In situ hybridization represents a rapid and sensitive method to obtain a definitive diagnosis in few hours, since it allows the direct visualization of the mutant mRNA foci on skeletal muscle sections. This approach makes the muscle biopsy an important tool for definitive diagnosis of DM2. Consequently, a rapid freezing at ultra cold temperature and a good storage of muscle specimens are essential to avoid morphologic alterations and nucleic acids degradation. However incorrect freezing or thawing may accidentally occur. In this work we report that fluorescence in situ hybridization may be applied on improperly frozen or inappropriately stored muscle biopsies since foci of mutant mRNA are well preserved and can still be detected in muscle sections no more useful for histopathological evaluation.Key words: myotonic dystrophy type 2, defrozen muscle biopsy, fluorescence, in situ hybridization, ribonuclear inclusions.Myotonic dystrophy type 2 (DM2) is a neuromuscular disorder due to the unstable (CCTG)n repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene on chromosome 3q21.3 (Liquori et al. 2001). Mutant ZNF9 pre-mRNA is spliced and polyadenylated, and the mRNA is exported to the cytoplasm where normal levels of ZNF9 protein expression occur (Botta et al., 2006; Margolis et al. 2006); however, the expanded repeats remain in cell nuclei as ribonuclear inclusions (Liquori et al. 2001). The DM2 ribonuclear inclusions contain only the CCUG repeat sequence derived from intron 1 but with no detectable flanking intronic RNA (Margolis et al. 2006). CCUG-containing mutant mRNAs form double-stranded hairpin loop structures that bind specific RNA-binding proteins such as muscle-blind-like proteins (MBNLs) that colocalize with ribonuclear inclusions in myonuclei (Mankodi et al., 2001; Fardaei et al., 2002). Sequestration of these proteins which are regulators of alternative splicing, alters the splicing of several pre-mRNA (reviewed by Osborne and Thornton, 2006) such as the insulin receptor (IR) and the chloride channel (ClC1) (Savkur et al., 2004; Charlet et al., 2002; Mankodi et al., 2002). Alterations in IR splicing leads to insulin insensitivity and predisposition to diabetes (Savkur et al. 2004) and alterations in ClC1 splicing results in electrical myotonia (Charlet et al., 2002; Mankodi et al., 2002). Conventional Southern blot analysis is not adequate for a definitive molecular diagnosis in DM2 due to the extremely large size and somatic instability of the expansion mutation (Liquori et al., 2001; Bachinski et al., 2003). The extraordinary somatic instability complicates the analysis of genotype-phenotype correlations including those in the effect of the gender of transmitting parents and anticipation. The copy number of DM2 CCTG is below 30 in phenotypically normal individuals and up 11.000 in patients (Day and Ranum, 2005). A complex genotyping diagnostic procedure is now commonly used consisting of a three-step molecular protocol (Day et al., 2003; Udd et al., 2003). However, a more practical tool to obtain a definitive diagnosis in few hours is represented by in situ hybridization which detects ribonuclear inclusions in cell nuclei of muscle fibers (Cardani et al., 2004; Sallinen et al., 2004). This approach makes muscle biopsy an essential tool for DM2 diagnosis. For this reason muscle specimens should be sent fresh, for rapid freezing, from the operating room to the pathology laboratory.To avoid RNA degradation, biopsies require special precautions with handling of material, such as immediate freezing of fresh tissues, because retrospective genetic analysis is impaired by conventional tissue processing techniques. However, many small hospitals are ill-equipped for snap freezing which requires access to liquid nitrogen or dry ice; thus, frequently outside hospitals provide specimens that are obscured with freeze artefacts because they either were submitted incorrectly or were improperly frozen, at the point of origin prior to shipment. Moreover, an accidental tissue thawing and refreezing may occur (for example power failure of the freezer) causing severe tissue damages and possible RNA degradation.Here we report our experience on DM2 muscle biopsies improperly preserved: these were no more useful for a histopathological analysis since they showed evident morphologic artefacts, but they proved to be still suitable for diagnosis by fluorescence in situ hybridization (FISH) since ribonuclear inclusions were preserved and still detectable on muscle sections.     

The beneficial use of ultramicronized palmitoylethanolamide as add-on therapy to Tapentadol in the treatment of low back pain: a pilot study comparing prospective and retrospective observational arms

Background

This pilot study was designed to compare the efficacy of ultramicronized palmitoylethanolamide (um-PEA) as add-on therapy to tapentadol (TP) with TP therapy only in patients suffering from chronic low back pain (LBP).

Methods

This pilot observational study consists in two arms: the prospective arm and the retrospective one. In the prospective arm patients consecutively selected received um-PEA as add-on therapy to TP for 6 months; in the retrospective arm patients were treated with TP only for 6 months. Pain intensity and neuropathic component were evaluated at baseline, during and after 6 months. The degree of disability and TP dosage assumption were evaluated at baseline and after 6 months.

Results

Statistical analysis performed with generalized linear mixed model on 55 patients (30 in the prospective group and 25 in the retrospective group) demonstrated that um-PEA as add-on treatment to TP in patients with chronic LBP, in comparison to TP alone, led to a significantly higher reduction in pain intensity, in the neuropathic component, the degree of disability and TP dosage assumption. No serious side effects were observed.

Conclusion

Overall, the present findings suggest that um-PEA may be an innovative therapeutic intervention as add-on therapy to TP for the management of chronic LBP with a neuropathic component, as well as to improve patient quality of life. Additionally, this combination treatment allowed a reduction in TP dose over time and did not show any serious side effects.

     

Cloning,expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel,Mytilus galloprovincialis

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32?kDa. Moreover, the determined kinetic parameters for the CO₂ hydration reaction were k_cat?=??4.2?×?10⁵?s^?1 and k_cat/K_m of 3.5?×?10⁷?M^?1 ×s^?1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50?kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.     

Effects of cooling and freezing on the motility of Ostrea edulis (L., 1758) spermatozoa after thawing

The aim of this work was to evaluate the effects of temperature, cryoprotectant agents and freezing curves on sperm motility of Ostrea edulis. All phases of cryopreservation were studied (evaluation of semen motility pattern, choice of cryoprotectants and freezing rates) to restore after thawing the motility characteristics distinctive of fresh semen.To assess the temperature effects on sperm motility, semen was activated using four different temperatures (25, 18, 10 and 3 °C). Sperm aliquots were maintained inactive at these temperatures for 1 and 3 h, then activated with FSW at same temperature of conservation. Sperm was activated and incubated to 3 °C with dimethylsulfoxide (Me₂SO), ethylene glycol (EG), 1–2 propylene glycol (PG) (5%, 7%, 10% and 15% final concentrations), glycerol (GlOH; 5%, 10% and 15% final concentrations) and methanol (MetOH; 4% and 10% final concentrations) for 10, 20 and 30 min. A first evaluation of freezing rates was made by testing four freezing curves: −1, −3, −6 and −10 °C/min. Then, an optimization was made by testing four freezing curves: −2.5, −3.0, −3.5 and −4 °C/min.The selected temperature for short term conservation has been 3 °C, because only this temperature has allowed good sperm motility conservation after 3 h of dry-storage; this is a time sufficient to conduct cryopreservation procedures. The sperm showed a particular sensitivity to GlOH and PG to all tested concentrations and to 15% Me₂SO. EG and MetOH to all concentrations and Me₂SO to concentrations lower than 15% have not shown significant toxic effects. The freezing rate −3 °C/min using 15% EG has shown an highest percentage of RVF (rapid, vigorous and forward) spermatozoa (class 3, about 75% of fresh semen) and an highest sperm motility duration.