Proximal humerus fractures in COVID-19 lockdown: the experience of three orthopedics and traumatology departments in the first ten weeks of the Italian epidemic.

INTRODUCTION: Coronavirus disease (CoVID-19) is causing millions of deaths worldwide and the crisis of the global healthcare system.  Aim Of The Study: evaluate the preliminary impact of CoVID-19 in three Italian Orthopedics and Traumatology Departments in the first 10 weeks of the national lockdown. We focused on proximal humerus fractures, analyzing data and results in comparison with the same period of 2019. MATERIALS AND METHODS: From February 22nd to May 3rd 2020, 55 patients were admitted to our departments for promixal humerus fractures. Our cohort of patients is composed by 13 males (23.6%) and 42 females (76.4%), with an average age of 73.8 ± 11.7 years (range 44 - 94). Trauma occurred at home in 43 cases (78.2%), by the roadside in 10 cases (18.2%), in a retirement home in 1 case (1.8%), and at work in 1 case (1.8%). We proposed surgical treatment in 15/55 cases, but 4 patients refused hospitalization, mainly because of the risk of contracting n-CoV19 infection. RESULTS: We noticed a decrease in proximal humerus fractures compared to 2019 (-37.5%). Particularly, we observed a significant drop in traumas occurred on the road and at work respectively 23.9% and 3.4%% in 2019, and 18.2% and 1.8% in 2020, probably due to the consequences of the national lockdown. Sports traumas had a reset during the pandemic (6 cases in 2019, 0 in 2020). As consequence, surgical treatment had a decrease due to the reduction in number of fractures, indications and patient's consent. CONCLUSION: The incidence of proximal humerus fractures had a significant reduction during CoVID-19 spread. We assume that the reasons of this reduction are to be found in the national lockdown (since March 10th, 2020) and Ministerial Decrees that limited the access to the E.R. only in case of severe traumas in order to avoid CoVID-19 spread.

Fractures around Trochanteric Nails: The "Vergilius Classification System".

INTRODUCTION: The fractures that occurred around trochanteric nails (perinail fractures, PNFs) are becoming a huge challenge for the orthopaedic surgeon. Although presenting some specific critical issues (i.e., patients' outcomes and treatment strategies), these fractures are commonly described within peri-implant ones and their treatment was based on periprosthetic fracture recommendations. The knowledge gap about PNFs leads us to convene a research group with the aim to propose a specific classification system to guide the orthopaedic surgeon in the management of these fractures. MATERIALS AND METHODS: A steering committee, identified by two Italian associations of orthopaedic surgeons, conducted a comprehensive literature review on PNFs to identify the unmet needs about this topic. Subsequently, a panel of experts was involved in a consensus meeting proposing a specific classification system and formulated treatment statements for PNFs. Results and Discussion. The research group considered four PNF main characteristics for the classification proposal: (1) fracture localization, (2) fracture morphology, (3) fracture fragmentation, and (3) healing status of the previous fracture. An alphanumeric code was included to identify each characteristic, allowing to describe up to 54 categories of PNFs, using a 3- to 4-digit code. The proposal of the consensus-based classification reporting the most relevant aspects for PNF treatment might be a useful tool to guide the orthopaedic surgeon in the appropriate management of these fractures.

The orthopaedic and traumatology scenario during Covid-19 outbreak in Italy: chronicles of a silent war.

BACKGROUND: From February 21, the day of hospitalisation in ICU of the first diagnosed case of Covid-19, the social situation and the hospitals' organisation throughout Italy dramatically changed. METHODS: The CIO (Club Italiano dell'Osteosintesi) is an Italian society devoted to the study of traumatology that counts members spread in public and private hospitals throughout the country. Fifteen members of the CIO, Chairmen of 15 Orthopaedic and Trauma Units of level 1 or 2 trauma centres in Italy, have been involved in the study. They were asked to record data about surgical, outpatients clinics and ER activity from the 23rd of February to the 4th of April 2020. The data collected were compared with the data of the same timeframe of the previous year (2019). RESULTS: Comparing with last year, overall outpatient activity reduced up to 75%, overall Emergency Room (ER) trauma consultations up to 71%, elective surgical activity reduced up to 100% within two weeks and trauma surgery excluding femoral neck fractures up to 50%. The surgical treatment of femoral neck fractures showed a stable reduction from 15 to 20% without a significant variation during the timeframe. CONCLUSIONS: Covid-19 outbreak showed a tremendous impact on all orthopaedic trauma activities throughout the country except for the surgical treatment of femoral neck fractures, which, although reduced, did not change in percentage within the analysed timeframe.

Clearly Detectable, Kinetically Restricted Solid-Solid Phase Transition in cis-Ceramide Monolayers.

Sphingosine [(2 S,3 R,4 E)-2-amino-4-octadecene-1,3-diol] is the most common sphingoid base in mammals. Ceramides are N-acyl sphingosines. Numerous small variations on this canonical structure are known, including the 1-deoxy, the 4,5-dihydro, and many others. However, whenever there is a Δ4 double bond, it adopts the trans (or E) configuration. We synthesized a ceramide containing 4 Z-sphingosine and palmitic acid ( cis-pCer) and studied its behavior in the form of monolayers extended on an air-water interface. cis-pCer acted very differently from the trans isomer in that, upon lateral compression of the monolayer, a solid-solid transition was clearly observed at a mean molecular area ≤44 Å2·molecule-1, whose characteristics depended on the rate of compression. The solid-solid transition, as well as states of domain coexistence, could be imaged by atomic force microscopy and by Brewster-angle microscopy. Atomistic molecular dynamics simulations provided results compatible with the experimentally observed differences between the cis and trans isomers. The data can help in the exploration of other solid-solid transitions in lipids, both in vitro and in vivo, that have gone up to now undetected because of their less obvious change in surface properties along the transition, as compared to cis-pCer.

Direct visualization of the lateral structure of giant vesicles composed of pseudo-binary mixtures of sulfatide, asialo-GM1 and GM1 with POPC.

We compared the lateral structure of giant unilamellar vesicles (GUVs) composed of three pseudo binary mixtures of different glycosphingolipid (GSL), i.e. sulfatide, asialo-GM1 or GM1, with POPC. These sphingolipids possess similar hydrophobic residues but differ in the size and charge of their polar head group. Fluorescence microscopy experiments using LAURDAN and DiIC18 show coexistence of micron sized domains in a molar fraction range that depends on the nature of the GSLs. In all cases, experiments with LAURDAN show that the membrane lateral structure resembles the coexistence of solid ordered and liquid disordered phases. Notably, the overall extent of hydration measured by LAURDAN between the solid ordered and liquid disordered membrane regions show marked similarities and are independent of the size of the GSL polar head group. In addition, the maximum amount of GSL incorporated in the POPC bilayer exhibits a strong dependence on the size of the GSL polar head group following the order sulfatide>asialo-GM1>GM1. This observation is in full harmony with previous experiments and theoretical predictions for mixtures of these GSL with glycerophospholipids. Finally, compared with previous results reported in GUVs composed of mixtures of POPC with the sphingolipids cerebroside and ceramide, we observed distinctive curvature effects at particular molar fraction regimes in the different mixtures. This suggests a pronounced effect of these GSL on the spontaneous curvature of the bilayer. This observation may be relevant in a biological context, particularly in connection with the highly curved structures found in neural cells.

PEEK radiolucent plate for distal radius fractures: multicentre clinical results at 12 months follow up.

INTRODUCTION: Open reduction and internal fixation (ORIF) with plate and screws represents the recommended treatment for unstable intra-articular distal radius fractures. Although significant progresses in surgical technique have been made, anatomical reconstruction of radio-carpal articular surface still represent a difficult task, especially in multifragmentary fractures. Available PEEK reinforced-carbon fiber composite radiolucent devices allow both an easier and more careful assessment of intra-operative reduction of the articular surface of distal radius and prompt correction of any residual step deformity. MATERIALS AND METHODS: We retrospectively reviewed clinical and radiological multicentre results of 71 consecutive AO B and C fracture pattern of distal radius treated using the same PEEK reinforced-carbon fiber composite radiolucent plate. RESULTS: Three patients lost at final follow up and 4 cases with incomplete radiological documentations were excluded from the study. 64 patients (38 females, 26 males) were available and formed the basis of this report. Fracture types included 9 patients with 23-B, 13 patients 23-B2,15 patients with 23-B3,10 patients with 23-C1, 7 patients with 23-C2 and 10 patients with 23-C3. Mean Modified Mayo wrist Score was on average 38.11 (SD 10.1; range 24-75, 95%CI 34.7-41.4), 67.22 (SD 9.6, range 50-90, 95%CI 64-70.4), 90.54 (SD 6.3, range 75-100, 95%CI 88.4-92.6) at one, two and twelve months of follow-up, respectively. A statistically significant difference was found between mean scores at different follow-up periods (p = 0.001). We noted 1 case of distal screw fixation aseptic loosening at 5 months post surgical intervention. CONCLUSIONS: PEEK reinforced-carbon fiber composite radiolucent plate represents a useful device for treatment of complex distal radius fractures in the adult population. It possesses unique biomechanical properties and allows for an easier anatomical reduction during surgical intervention.

The many faces (and phases) of ceramide and sphingomyelin I - single lipids.

Ceramides, the simplest kind of two-chained sphingolipids, contain a single hydroxyl group in position 1 of the sphingoid base. Sphingomyelins further contain a phosphocholine group at the OH of position 1 of ceramide. Ceramides and sphingomyelins show a variety of species depending on the fatty acyl chain length, hydroxylation, and unsaturation. Because of the relatively high transition temperature of sphingomyelin compared to lecithin and, particularly, of ceramides with 16:0-18:0 saturated chains, a widespread idea on their functional importance refers to formation of rather solid domains enriched in sphingomyelin and ceramide. Frequently, and especially in the cell biology field, these are generally (and erroneously) assumed to occur irrespective on the type of N-acyl chain in these lipids. This is because most studies indicating such condensed ordered domains employed sphingolipids with acyl chains with 16 carbons while scarce attention has been focused on the influence of the N-acyl chain on their surface properties. However, abundant evidence has shown that variations of the N-acyl chain length in ceramides and sphingomyelins markedly affect their phase state, interfacial elasticity, surface topography, electrostatics and miscibility and that, even the usually conceived "condensed" sphingolipids and many of their mixtures, may exhibit liquid-like expanded states. This review is a summarized overview of our work and of related others on some facts regarding membranes composed of single molecular species of ceramide and sphingomyelin. A second part is dedicated to discuss the miscibility properties between species of sphingolipids that differ in N-acyl and oligosaccharide chains.

The many faces (and phases) of ceramide and sphingomyelin II - binary mixtures.

A rather widespread idea on the functional importance of sphingolipids in cell membranes refers to the occurrence of ordered domains enriched in sphingomyelin and ceramide that are largely assumed to exist irrespective of the type of N-acyl chain in the sphingolipid. Ceramides and sphingomyelins are the simplest kind of two-chained sphingolipids and show a variety of species, depending on the fatty acyl chain length, hydroxylation, and unsaturation. Abundant evidences have shown that variations of the N-acyl chain length in ceramides and sphingomyelins markedly affect their phase state, interfacial elasticity, surface topography, electrostatics, and miscibility, and that even the usually conceived "condensed" sphingolipids and many of their mixtures may exhibit liquid-like expanded states. Their lateral miscibility properties are subtlety regulated by those chemical differences. Even between ceramides with different acyl chain length, their partial miscibility is responsible for a rich two-dimensional structural variety that impacts on the membrane properties at the mesoscale level. In this review, we will discuss the miscibility properties of ceramide, sphingomyelin, and glycosphingolipids that differ in their N-acyl or oligosaccharide chains. This work is a second part that accompanies a previous overview of the properties of membranes formed by pure ceramides or sphingomyelins, which is also included in this Special Issue.

Cavity Orientation Regulated by Mixture Composition and Clustering of Amphiphilic Cyclodextrins in Phospholipid Monolayers.

Artificial supramolecular-hierarchical structures that emulate nature represent an overcoming alternative for the design of new drug delivery systems. Thermodynamic and topographic properties of films formed by a monoacylated amphiphilic ß-cyclodextrin (ßCD-C16) with the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at the air/water interface were studied. ßCD-C16 formed stable mixed films with POPC at several proportions when spread together at the air/water interface. The orientation of ßCD-C16 cavity at the interface depends on its mole fraction in the film as reveled by the analysis of partial mean molecular areas as a function of composition. Furthermore, ßCD-C16 was able to penetrate POPC preformed films in a broad range of initial surface pressures, including that near the collapse pressure of the phospholipid. These results demonstrated the strong tendency of ßCD-C16 to be inserted into this lipid matrix commonly used in liposome formulations. Topography studies show that ßCD-C16 segregate from POPC forming clusters enriched in ßCD-C16. Segregation of ßCD-C16 was especially noticeable when ßCD-C16 were incorporated by themselves into a preformed POPC matrix leading to ordered and highly birefringent structures that suggest the formation of hierarchical stacked ßCD-C16 arrangement at the interface.

Development of a Nonionic Azobenzene Amphiphile for Remote Photocontrol of a Model Biomembrane.

We report the synthesis and characterization of a simple nonionic azoamphiphile, C12OazoE3OH, which behaves as an optically controlled molecule alone and in a biomembrane environment. First, Langmuir monolayer and Brewster angle microscopy (BAM) experiments showed that pure C12OazoE3OH enriched in the (E) isomer was able to form solidlike mesophase even at low surface pressure associated with supramolecular organization of the azobenzene derivative at the interface. On the other hand, pure C12OazoE3OH enriched in the (Z) isomer formed a less solidlike monolayer due to the bent geometry around the azobenzene moiety. Second, C12OazoE3OH is well-mixed in a biological membrane model, Lipoid s75 (up to 20%mol), and photoisomerization among the lipids proceeded smoothly depending on light conditions. It is proposed that the cross-sectional area of the hydroxyl triethylenglycol head of C12OazoE3OH inhibits azobenzenes H-aggregation in the model membrane; thus, the tails conformation change due to photoisomerization is transferred efficiently to the lipid membrane. We showed that the lipid membrane effectively senses the azobenzene geometrical change photomodulating some properties, like compressibility modulus, transition temperature, and morphology. In addition, photomodulation proceeds with a color change from yellow to orange, providing the possibility to externally monitor the system. Finally, Gibbs monolayers showed that C12OazoE3OH is able to penetrate the highly packing biomembrane model; thus, C12OazoE3OH might be used as photoswitchable molecular probe in real systems.

Sialoganglioside Micelles for Enhanced Paclitaxel Solubility: In Vitro Characterization.

Efficiency of mono-sialogangliosides to load Paclitaxel (Ptx) has recently been found to depend on the structure of the polysaccharide chain. In this study, we demonstrated that incorporation of only one more sialic acid into the ganglioside molecule, independently of its position, causes a 4-fold increase in Ptx-loading capacity, the maximum being at a 5:1 molar ratio (di-sialoganglioside/Paclitaxel, GD/Ptx). These complexes are stable in solution for at least 3 months, and over 90% of Ptx remains loaded in the micelles after extreme stress conditions such as high-speed centrifugation, lyophilization, or freeze-thaw cycles. Ganglioside micelles protect 50% of the initially loaded Ptx from alkaline hydrolysis after 24 h at pH 10. Dynamic light scattering studies revealed that GD micelles increase their size from 9 to 12 nm when loaded with Ptx. Transmission electron microscopy shows a homogeneous population of spherical micelles either with or without Ptx. In vitro biological activity was similar to that of the free drug. These results provide further options of self-assembled nanostructures of di- and tri-sialogangliosides with a higher loading capacity.

Interfacial stabilization of the antitumoral drug Paclitaxel in monolayers of GM1 and GD1a gangliosides.

Molecular interactions between the anti-cancer agent Paclitaxel (Ptx), and two gangliosides with different sialic acid content, GM1 and GD1a, were investigated using the Langmuir film balance technique. Ptx showed interfacial activity reducing the air/water surface tension by 18 mN·m(-1). However, the drug was able to insert into preformed ganglioside monolayers at much higher surface pressures, indicating a preferential interaction of Ptx with GM1 and GD1a. Compression isotherms of binary mixtures of Ptx and GM1 or GD1a also indicated non-ideal mixed monolayers in which the drug became stabilized at the interface in the presence of gangliosides. Ptx reached much higher surface pressure values in the mixed monolayers than those sustained in pure Ptx, although partial desorption of the drug from the interface into the subphase was also observed at high Ptx contents. The mean molecular area of the mixtures showed condensation, mainly in the case of GD1a, whereas Ptx induced a decrease in the compressibility of monolayers when mixed with either GM1 or GD1a. Additionally, Brewster angle microscopy analysis indicated that higher amounts of Ptx are present at the mixed ganglioside/Ptx interface when compared to pure drug monolayers. Finally, GD1a micelles increased in size in the presence of Ptx, whereas GM1 micelles kept their diameter, according to dynamic light scattering measurements, which could be explained by the different properties of ganglioside monolayers. The results obtained on ganglioside-Ptx interactions allowed interpreting the different Ptx loading capacity of GM1 and GD1a, enabling them to act as potential drug carriers.

Zn(2+)-dependent surface behavior of diacylglycerol pyrophosphate and its mixtures with phosphatidic acid at different pHs.

Diacylglycerol pyrophosphate (DGPP) is a minor lipid that attenuates the phosphatidic acid (PA) signal, and also DGPP itself would be a signaling lipid. Diacylglycerol pyrophosphate is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol that was shown to respond to changes of pH, thus affecting the surface organization of DGPP and their interaction with PA. In this work, we have investigated how the presence of Zn(2+) modulates the surface organization of DGPP and its interaction with PA at acidic and basic pHs. Both lipids formed expanded monolayers at pHs 5 and 8. At pH 5, monolayers formed by DGPP became stiffer when Zn(2+)was added to the subphase, while the surface potential decreased. At this pH, Zn(2+) induced a phase transition from an expanded to a condensed-phase state in monolayers formed by PA. Conversely, at pH 8 the effects induced by the presence of Zn(2+) on the surface behaviors of the pure lipids were smaller. Thus, the interaction of the bivalent cation with both lipids was modulated by pH and by the ionization state of the polar head groups. Mixed monolayers of PA and DGPP showed a non-ideal behavior and were not affected by the presence of Zn(2+) at pH 8. This could be explained considering that when mixed, the lipids formed a closely packed monolayer that could not be further modified by the cation. Our results indicate that DGPP and PA exhibit expanded- and condensed-phase states depending on pH, on the proportion of each lipid in the film and on the presence of Zn(2+). This may have implications for a possible role of DGPP as a signaling lipid molecule.

N-Acyl Chain in Ceramide and Sphingomyelin Determines Their Mixing Behavior, Phase State, and Surface Topography in Langmuir Films.

Sphingolipids are membrane lipids composed by a long chain aminediol base, usually sphingosine, with a N-linked fatty acyl chain whose quality depends on the membrane type. The effect of length and unsaturation of the N-acyl chain on the mixing behavior of different sphingolipids has scarcely been studied, and in this work this issue is addressed employing Langmuir monolayers at the air-water interface, in order to assess the surface mixing in binary mixtures of different species of sphingomyelins and ceramides. The dependence on the monolayer composition of the mean molecular area, perpendicular dipole moment, domain segregation, and surface topography, as well as the film elasticity and optical thickness were studied. The results indicate that composition-dependent favorable interactions among sphingomyelin and ceramide occur as a consequence of complementary lateral packing and increased acyl chain ordering; the phase state of the components appears as a major factor determining miscibility among sphingomyelins and ceramides even in cases where the lipids have a considerable hydrocarbon chain length mismatch.

Distinctive interactions of oleic acid covered magnetic nanoparticles with saturated and unsaturated phospholipids in Langmuir monolayers.

The growing number of innovations in nanomedicine and nanobiotechnology are posing new challenges in understanding the full spectrum of interactions between nanomateriales and biomolecules at nano-biointerfaces. Although considerable achievements have been accomplished by in vivo applications, many issues regarding the molecular nature of these interactions are far from being well-understood. In this work, we evaluate the interaction of hydrophobic magnetic nanoparticles (MNP) covered with a single layer of oleic acid with saturated and unsaturated phospholipids found in biomembranes through the use of Langmuir monolayers. We find distinctive interactions among the MNP with saturated and unsaturated phospholipids that are reflected by both, the compression isotherms and the surface topography of the films. The interaction between MNP and saturated lipids causes a noticeable reduction of the mean molecular area in the interfacial plane, while the interaction with unsaturated lipids promotes area expansion compared to the ideally mixed films. Moreover, when liquid expanded and liquid condensed phases of the phospholipid(s) coexist, the MNP preferably partition to the liquid-expanded phase, thus hindering the coalescence of the condensed domains with increasing surface pressure. In consequence organizational information on long-range order is attained. These results evidence the existence of a sensitive composition-dependent surface regulation given by phospholipid-nanoparticle interactions which enhance the biophysical relevance of understanding nanoparticle surface functionalization in relation to its interactions in biointerfaces constituted by defined types of biomolecules.

Sticholysin I-membrane interaction: an interplay between the presence of sphingomyelin and membrane fluidity.

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.

Surface behavior of sphingomyelins with very long chain polyunsaturated fatty acids and effects of their conversion to ceramides.

Molecular species of sphingomyelin (SM) with nonhydroxy (n) and 2-hydroxy (h) very long chain polyunsaturated fatty acids (n- and h-28:4, 30:5, and 32:5) abound in rat spermatogenic cells and spermatozoa. These SMs are located on the sperm head, where they are converted to the corresponding ceramides (Cer) after the completion of the acrosomal reaction, as induced in vitro. The aim of this study was to look into the surface properties of these unique SM species and how these properties change by the SM → Cer conversion. After isolation by HPLC, these SMs were organized in Langmuir films and studied alone, in combination with different proportions of Cer, and during their conversion to Cer by sphingomyelinase. Compression isotherms for all six SMs under study were compatible with a liquid-expanded (LE) state and showed large molecular areas. Only the longest SMs (n-32:5 and h-32:5 SM) underwent a phase transition upon cooling. Interestingly, the abundant h-28:4 Cer exhibited a highly compressible liquid-condensed (LC) phase compatible with a high conformational freedom of Cer molecules but with the characteristic low diffusional properties of the LC phase. In mixed films of h-28:4 SM/h-28:4 Cer, the components showed favorable mixing in the LE phase. The monolayer exhibited h-28:4 Cer-rich domains both in premixed films and when formed by the action of sphingomyelinase on pure h-28:4 SM films. Whereas the SMs from sperm behaved in a way similar to that of shorter acylated SMs, the corresponding Cers showed atypical rheological properties that may be relevant to the membrane structural rearrangements that take place on the sperm head after the completion of the acrosomal reaction.

Boundary region between coexisting lipid phases as initial binding sites for Escherichia coli alpha-hemolysin: a real-time study.

α-Hemolysin (HlyA) is a protein toxin, a member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action of this toxin seems to involve three stages that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane. Since the influence of phase segregation on HlyA binding and insertion in lipid membranes is not clearly understood, we explored at the meso- and nanoscale-both in situ and in real-time-the interaction of HlyA with lipid monolayers and bilayers. Our results demonstrate that HlyA could insert into monolayers of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/16:0SM/Cho) and DOPC/24:1SM/Cho. The time course for HlyA insertion was similar in both lipidic mixtures. HlyA insertion into DOPC/16:0SM/Cho monolayers, visualized by Brewster-angle microscopy (BAM), suggest an integration of the toxin into both the liquid-ordered and liquid-expanded phases. Atomic-force-microscopy imaging reported that phase boundaries favor the initial binding of the toxin, whereas after a longer time period the HlyA becomes localized into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho. Our AFM images, however, showed that the HlyA interaction does not appear to match the general strategy described for other invasive proteins. We discuss these results in terms of the mechanism of action of HlyA.

Atypical surface behavior of ceramides with nonhydroxy and 2-hydroxy very long-chain (C28-C32) PUFAs.

Unique species of ceramide (Cer) with very-long-chain polyunsaturated fatty acid (VLCPUFA), mainly 28-32 carbon atoms, 4-5 double bonds, in nonhydroxy and 2-hydroxy forms (n-V Cer and h-V Cer, respectively), are generated in rat spermatozoa from the corresponding sphingomyelins during the acrosomal reaction. The aim of this study was to determine the properties of these sperm-distinctive ceramides in Langmuir monolayers. Individual Cer species were isolated by HPLC and subjected to analysis of surface pressure, surface potential, and Brewster angle microscopy (BAM) as a function of molecular packing. In comparison with known species of Cer, n-V Cer and h-V Cer species showed much larger mean molecular areas and increased molecular dipole moments in liquid expanded phases, which suggest bending and partial hydration of the double bonded portion of the VLCPUFA. The presence of the 2-hydoxyl group induced a closer molecular packing in h-V Cer than in their chain-matched n-V Cer. In addition, all these Cer species showed liquid-expanded to liquid-condensed transitions at room temperature. Existence of domain segregation was confirmed by BAM. Additionally, thermodynamic analysis suggests a phase transition close to the physiological temperature for VLCPUFA-Cers if organized as bulk dispersions.