Determination of bending rigidity and tilt modulus of lipid membranes from real-space fluctuation analysis of molecular dynamics simulations.

We have recently developed a novel computational methodology (termed RSF for Real-Space Fluctuations) to quantify the bending rigidity and tilt modulus of lipid membranes from real-space analysis of fluctuations in the tilt and splay degrees of freedom as sampled in molecular dynamics (MD) simulations. In this article, we present a comprehensive study that combines results from the application of the RSF method to a wide range of lipid bilayer systems that encompass membranes of different fluidities and sizes, including lipids with saturated and unsaturated lipid tails, single and multi-component lipid systems, as well as non-standard lipids such as the four-tailed cardiolipin. By comparing the material properties calculated with the RSF method to those obtained from experimental data and from other computational methodologies, we rigorously demonstrate the validity of our approach and show its robustness. This should allow for future applications of even more complex lipidic assemblies, whose material properties are not tractable by other computational techniques. In addition, we discuss the relationship between different definitions of the tilt modulus appearing in current literature to address some important unresolved discrepancies in the field.

Molecular origins of bending rigidity in lipids with isolated and conjugated double bonds: the effect of cholesterol.

We examine the effects of cholesterol (Chol) on the mechanical properties of membranes consisting of 16:0/18:1 POPC lipid and of lipids with conjugated linoleic acid (CLA), cis-9/trans-11 CLA (C9T11) and trans-10/cis-12 CLA (T10C12). Atomistic molecular dynamics (MD) simulations of POPC-Chol and CLA-Chol mixtures at various Chol concentrations are employed within a recently developed and validated computational methodology (Khelashvili et al., 2013) that calculates from MD trajectories the bending rigidity (KC) for these systems. We have found that the addition of 30% Chol stiffens POPC lipid membranes much more significantly (2.3-fold) than it does C9T11 (1.5-fold) or T10C12 (1.75-fold) lipid bilayers. Extensive comparative structural analysis of the simulated mixtures supports a molecular mechanism for the differential effects of cholesterol, whereby the sterol molecules tilt more significantly in CLA membranes where they also insert deeper inside the hydrocarbon core. The observed distinct arrangement of Chol molecules in CLA and POPC bilayers, in turn, is dictated by the interplay between the specific location of the trans double bond in the two CLA lipid isomers and the preferential interaction of the rigid Chol ring with the saturated segments of the lipid tails. The simulations and analysis described in this paper provide novel insights into the specific modes of molecular interaction in bilayers composed of mixtures of Chol and unsaturated lipids that drive emergent macroscopic properties, such as the membrane's bending modulus.

Local recurrence after abdomino-perineal resection.

OBJECTIVE: Local recurrence of rectal cancer is a major cause of morbidity and mortality following curative resection. The published rates vary after abdomino-perineal resection (APR) from 5% to 47%. The aim of this study was to evaluate local recurrence following curative APR for low rectal cancer in our unit. METHOD: The medical notes of patients treated between 1st January 1996 and 31st December 2000 were retrieved. Local recurrence was defined as the presence of tumour within the pelvis confirmed by clinical findings, pathological specimen or radiological reports. A curative resection was defined as excision of tumour in the absence of macroscopic metastatic disease and whose resection margins were greater than 1 mm circumferentially and 10 mm distally. Outcomes and survival were compared using Fisher's exact test and Kaplan-Meier method. RESULTS: Two hundred consecutive cases with a diagnosis of rectal cancer were identified of which 139 underwent a curative resection (69.5%). Of these 40 patients (28%) underwent APR with curative intent. Two patients (5%) developed local recurrence at 18 and 24 months respectively. The overall local recurrence rate for all curative rectal cancer surgery, in the same period was 2.6%. Eleven patients have died in the follow-up period of which nine were cancer-related deaths. CONCLUSION: The local recurrence rates achieved with APR were not significantly different from those achieved with restorative operations. Tumours at the ano-rectal junction should not be dissected off the pelvic floor, but radically excised en bloc with the surrounding levator ani, as a cylinder, as originally described by Miles.

Amino acid deficiency up-regulates specific mRNAs in murine embryonic cells.

The flow of amino acids to both protein and DNA synthesis is particularly important during periods of rapid cell proliferation such as the fetal stages of life. The changes in mRNA levels caused by the different types of growth arrest were studied in F9 embryonal carcinoma cells. The cells were grown in medium deficient in the amino acid lysine or in one containing phosphonoacetyl L-aspartic acid (PALA), which inhibits the incorporation of aspartic acid into pyrimidine nucleotides. A number of mRNAs known to be elevated in growth arrested cells (gas and gadd) were studied by Northern blotting. Samples of RNA from the cells were also compared by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). The results showed that lysine deficiency increased the steady-state levels of a number of mRNAs by 5- to 40-fold. In contrast, the changes in cells treated with PALA were much smaller and less pronounced. Amino acid deficiency induced the mRNAs coding for gadd153 (CHOP-10), gas5, the mouse doublesex-related gene (Dmrt1) and the polyamine modulated factor (PA-1) as well as a number of unidentified expressed sequence tags (EST). These mRNAs were all induced within 24 h of exposure to amino acid deficiency. These very different transcriptional responses may be important in understanding the interactions between protein quantity and quality in different physiologic situations.

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes.

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.

The phase behavior of cationic lipid-DNA complexes.

We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram.

Expression of the growth arrest genes (GAS and GADD) changes during organogenesis in the rat fetus.

Mammalian cells mount an active response to nutrient limitation by overexpressing the growth arrest specific (GAS) and the growth arrest and DNA damage (GADD) genes. During embryogenesis in rats, there are quantitative and temporal differences in GAS and GADD gene expression during the development of the placenta, heart and kidney. Genes associated with the inhibition of DNA synthesis (p53 and GAS1) were predominantly expressed during the early stages of development, whereas those genes associated with inhibition of protein synthesis [GADD153 (also known as CHOP-10 or Ddit3) and C/EBP-beta] were more highly expressed during the later stages. The GADD45 gene was expressed throughout development. There were distinct periods of GAS3 and GAS6 gene expression during the development of the placenta, heart and kidneys, which is consistent with the proposed roles of these genes in cell interactions. These results show that there is a change in the expression of genes associated with the negative regulation of growth as the fetus develops.

Structure, stability, and thermodynamics of lamellar DNA-lipid complexes.

We develop a statistical thermodynamic model for the phase evolution of DNA-cationic lipid complexes in aqueous solution, as a function of the ratios of charged to neutral lipid and charged lipid to DNA. The complexes consist of parallel strands of DNA intercalated in the water layers of lamellar stacks of mixed lipid bilayers, as determined by recent synchrotron x-ray measurements. Elastic deformations of the DNA and the lipid bilayers are neglected, but DNA-induced spatial inhomogeneities in the bilayer charge densities are included. The relevant nonlinear Poisson-Boltzmann equation is solved numerically, including self-consistent treatment of the boundary conditions at the polarized membrane surfaces. For a wide range of lipid compositions, the phase evolution is characterized by three regions of lipid to DNA charge ratio, rho: 1) for low rho, the complexes coexist with excess DNA, and the DNA-DNA spacing in the complex, d, is constant; 2) for intermediate rho, including the isoelectric point rho = 1, all of the lipid and DNA in solution is incorporated into the complex, whose inter-DNA distance d increases linearly with rho; and 3) for high rho, the complexes coexist with excess liposomes (whose lipid composition is different from that in the complex), and their spacing d is nearly, but not completely, independent of rho. These results can be understood in terms of a simple charging model that reflects the competition between counterion entropy and inter-DNA (rho < 1) and interbilayer (rho > 1) repulsions. Finally, our approach and conclusions are compared with theoretical work by others, and with relevant experiments.

Topological defects and the optimum size of DNA condensates.

Under a wide variety of conditions, the addition of condensing agents to dilute solutions of random-coil DNA gives rise to highly compact particles that are toroidal in shape. The size of these condensates is remarkably constant and is largely independent of DNA molecular weight and basepair sequence, and of the nature of condensing agent (e.g., multivalent cation, polymers, or added cosolvent). We show how this optimum size is determined by the interactions between topological defects, which unavoidably strain the circumferentially wound DNA strands in the torus.

Effects of nutrient deprivation and differentiation on the expression of growth-arrest genes (gas and gadd) in F9 embryonal carcinoma cells.

The growth-arrest genes (gas and gadd) are widely expressed during mammalian embryogenesis and may be useful as markers of nutritional stress in the embryo. F9 embryonal carcinoma cells have been used to characterize the effect of serum or amino acid deficiency on growth-arrest gene expression in a differentiating embryonic cell. The differentiation markers, homeobox B2 (HoxB2), collagen type IV and laminin B2, were not induced by growth arrest. Treatment with all-trans retinoic acid (RA) produced a dose-dependent increase in alkaline phosphatase activity, which was unchanged in lysine-deficient medium and reduced in low-serum medium. Low-serum medium also reduced HoxB2 expression. There was a transient 2-6-fold increase in mRNAs for C/EBP-beta, gadd153/CHOP-10 and gas5 genes 24 h after transfer to amino-acid-deficient media. The mRNAs for the gas2 and gas6 genes began to rise slowly by 5-10-fold after a delay of approx. 24 h. The transient increases did not occur in low-serum medium where there was a much smaller and slower increase. Differentiation caused 1-2-fold increases in gas2, gas3 and gas6 mRNA levels. The transient overexpression of gas5, gadd153/CHOP-10 and CCAAT-enhancer-binding protein-beta, and the later expression of gas6 mRNAs in response to amino acid deficiency, were not affected by differentiation. RA treatment increased the expression of gas3 and caused gas2 to be transiently overexpressed in amino-acid-deficient medium. Differentiation in serum-deficient medium did not significantly alter the levels of the growth-arrest gene mRNAs. These results show that in F9 cells the growth-arrest genes are expressed sequentially as a result of nutrient stress.

The growth arrest genes gas5, gas6, and CHOP-10 (gadd153) are expressed in the mouse preimplantation embryo.

The gas and gadd family of genes, known collectively as the growth arrest genes, are associated with the negative control of mammalian cell growth. The steady-state levels of their mRNAs are increased by three to fivefold when exponentially multiplying cells are exposed to a variety of stresses including inadequate nutrition or the removal of serum. Reverse transcription-polymerase chain reaction (RT-PCR) has been used to analyze growth arrest gene expression in the preimplantation mouse embryo. The gas5, gas6, and CHOP-10 (gadd153, Ddit3) genes were expressed from the eight-cell stage onward. The gas2 and gas3 genes associated with apoptosis were not expressed. Embryos were cultured in kSOM medium and a semiquantitative RT-PCR method was used to measure the relative gene expression using beta-actin mRNA as a reference. The ratio of gas5 to beta-actin mRNA was high at the eight-cell stage and fell three to fivefold during development. The decline in the gas5:beta-actin ratio corresponded to the activation of true cell growth (cytokinesis). The gas6:beta-actin ratio was low at the eight-cell stage and increased by twofold as the blastocyst formed. CHOP-10 was expressed at a constant level throughout development. Embryos that had developed in vivo were compared with the equivalent blastocyst-stage embryos cultured in kSOM medium. There were no significant differences in the ratio of CHOP-10, gas5, or gas6 mRNAs relative to beta-actin. These results suggest that these genes are expressed as part of normal early embryonic development. The potential roles of the growth arrest genes are discussed.

Proximal femoral fractures in the elderly: does operative delay for medical reasons affect short-term outcome?

Forty patients over 65 years of age with fractures of the proximal femur, whose operation was delayed more than 24 hours after admission for medical reasons, were studied. Length of post-operative stay on the orthopaedic wards, mobility after one month and need for continuing supervision by the geriatric services, after discharge from the orthopaedic wards, were compared with a matched group in whom there was no delay. There were no significant differences between the results of the two groups, suggesting that, if medical conditions are adequately treated before operative fixation of fractures of the proximal femur in elderly patients, the short-term outcome is not adversely affected.

Anorexia nervosa in the elderly: a case report and review of the literature.

Anorexia nervosa is a rare disorder in the elderly but should be thought of in the differential diagnosis of extreme weight loss, especially if there are accompanying psychological features and normal baseline investigations. Psychiatric opinion should be obtained and treatment, although of unproven value, considered, since improvement may increase mobility and reduce morbidity and mortality.