Thermodynamics for the Self-Assembly of Alkylated Peptides.

Self-assembling peptides form aggregates with various nanostructures such as spheres, sheets, and fibers and have potential applications in nanomedicine and drug delivery. The alkylation of peptides is a promising strategy for controlling the self-assembly of peptides. In this study, we investigated the thermodynamic properties associated with the aggregation of alkyl-chain-modified self-assembling peptides. The tripeptide sequence, KYF, which has been reported to form fibrous aggregates via self-assembly, was modified with various fatty acids at the N-terminus. The fibrous morphology of the aggregates was observed by transmission electron microscopy and atomic force microscopy. Thioflavin T fluorescence and circular dichroism spectroscopy revealed the formation of ß-sheet structures. The critical micelle concentration and its temperature dependence were determined to obtain the thermodynamic parameters for aggregation. The results showed that the aggregation was an entropy-driven process at low temperatures, whereas it was enthalpy-driven at high temperatures. The negative heat capacity changes for aggregation suggested that hydrophobic interactions were the major driving force for self-assembly. Other entropic and enthalpic interactions were also contributed in part to the self-assembly. We individually identified the contributions of the peptide and alkyl chain moiety to the self-assembly. These contributions can be explained by the theoretical values for the self-assembly of each component. The results of this study provide fundamental insights into the design of self-associating peptides.

Improvement of Thermal Stability of Amphipathic Peptide-Phospholipid Nanodiscs via Lateral Association of α-Helices by Disulfide Cross-Linking.

Amphipathic α-helical peptides have been reported to form discoidal particles or nanodiscs with phospholipids, in which a lipid bilayer patch is encircled by peptides. Peptide-based nanodiscs have broad applicability because of their ease of preparation, size flexibility, and structural plasticity. We previously revealed that the nanodiscs formed by apolipoprotein-A-I-derived peptide 18A showed temperature-dependent structural destabilization above the gel-to-liquid-crystalline phase transition temperature of the lipid bilayer. It has been suggested that this destabilization is due to the migration of peptides bound to the edge of the discs to the bilayer surface. In this study, we designed a peptide that could stabilize nanodisc structures against the phase transition of lipid bilayers by disulfide cross-linking of peptides. An 18A-dimer cross-linked by a proline residue, 37pA (Ac-18A-P-18A-CONH2), also showed thermal destabilization of nanodiscs like 18A. However, cross-linking the sides of the two α-helices of the cysteine-substituted analogue 37pA-C2 with disulfide bonds led to the formation of nanodiscs that were more stable to temperature changes. This stabilizing effect was mainly due to the formation of a cyclic 37pA-C2 monomer by intramolecular disulfide cross-linking. These results suggest that the lateral association of two α-helices, which is the basis of the double-belt structure, is an important factor for the implementation of stable nanodiscs. The results of this study will help in development of more stable nanoparticles with membrane proteins in the future.

Near-field thermophotovotaic devices with surrounding non-contact reflectors for efficient photon recycling.

Near-field thermophotovoltaic (TPV) power generation has been attracting increasing attention as a promising approach for efficient conversion of heat into electricity with high output power density. Here, we numerically investigate near-field TPV devices with surrounding reflectors for efficient recycling of low-energy photons, which do not contribute to the power generation. We reveal that the conversion efficiency of a near-field TPV system can be drastically increased by introducing a pair of reflectors above and below the system, especially when the two mirrors are not in contact with the emitter and absorber. In addition, we investigate the influence of non-perfect photon recycling on the TPV efficiency and reveal that near-field TPV systems are more robust against the decrease of the reflectivity of the reflectors than the far-field TPV systems.

Treatment with Histone Deacetylase Inhibitor Attenuates Peripheral Inflammation-Induced Cognitive Dysfunction and Microglial Activation: The Effect of SAHA as a Peripheral HDAC Inhibitor.

It has been demonstrated that peripheral inflammation induces cognitive dysfunction. Several histone deacetylase (HDAC) inhibitors ameliorate cognitive dysfunction in animal models of not only peripheral inflammation but also Alzheimer's disease. However, it is not clear which HDAC expressed in the central nervous system or peripheral tissues is involved in the therapeutic effect of HDAC inhibition on cognitive dysfunction. Hence, the present study investigated the effect of peripheral HDAC inhibition on peripheral inflammation-induced cognitive dysfunction. Suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor that is mainly distributed in peripheral tissues after intraperitoneal administration, was found to prevent peripheral inflammation-induced cognitive dysfunction. Moreover, pretreatment with SAHA dramatically increased mRNA expression of interleukin-10, an anti-inflammatory cytokine, in peripheral and central tissues and attenuated peripheral inflammation-induced microglial activation in the CA3 region of the hippocampus. Minocycline, a macrophage/microglia inhibitor, also ameliorated cognitive dysfunction. Furthermore, as a result of treatment with liposomal clodronate, depletion of peripheral macrophages partially ameliorated the peripheral inflammation-evoked cognitive dysfunction. Taken together, these findings demonstrate that inhibition of peripheral HDAC plays a critical role in preventing cognitive dysfunction induced by peripheral inflammation via the regulation of anti-inflammatory cytokine production and the inhibition of microglial functions in the hippocampus. Thus, these findings could provide support for inhibition of peripheral HDAC as a novel therapeutic strategy for inflammation-induced cognitive dysfunction.

Amphipathic Peptide-Phospholipid Nanofibers: Phospholipid Specificity and Dependence on Concentration and Temperature.

The design of nanoassemblies is an important part of the development of new materials for applications in nanomedicine and biosensors. In our previous study, cysteine substitution of the apolipoprotein A-I-derived peptide 18A at residue 11, 18A[A11C], bound to 1-palmitoyl-2-oleoylphosphatidylcholine to form fibrous aggregates at a lipid-to-peptide molar ratio of ≤2 and a fiber diameter of 10-20 nm. However, the mechanisms underlying the lipid-peptide interactions that enable nanofiber formation remain unclear. Here, we evaluated the phospholipid specificity, concentration dependence, and temperature dependence of the formation of 18A[A11C]-lipid nanofibers. Nanofibers were found to form in the presence of specific phospholipids and have a constant lipid/peptide stoichiometry of 1.2 ± 0.2. Moreover, an increase in the length of the acyl chain in phosphatidylcholines was found to increase the structural stability of the nanofibers. These results indicate that specific molecular interactions between peptides and both the headgroups and acyl chains of phospholipids are involved in nanofiber formation. Furthermore, the formation and disassembly of the nanofibers were reversibly controlled by changes in temperature and concentration. The results of the present study provide an insight into the creation of nanoassembling structures.

One-Chip Near-Field Thermophotovoltaic Device Integrating a Thin-Film Thermal Emitter and Photovoltaic Cell.

Thermal radiation transfer between two objects separated by a subwavelength gap (near-field thermal radiation transfer) can be orders of magnitude larger than that in free space, which is attracting increasing attention with respect to both fundamental nanoscience and its potential for high-power-density and high-efficiency conversion of heat to electricity in thermophotovoltaic (TPV) systems. However, the realization of near-field thermal radiation transfer in TPV systems involves significant challenges because it requires a subwavelength gap and large temperature difference between the emitter and the PV cell while minimizing the heat transfer that does not contribute to the photocurrent generation. To overcome these challenges, here we demonstrate a one-chip near-field TPV device consisting of a thin-film Si emitter and InGaAs PV cell with an intermediate Si substrate, which enables the suppression of the heat transfer due to sub-bandgap radiation by free carriers and surface modes. Through the one-chip integration and thermal isolation using Si process technologies, we realize a deep subwavelength gap (<150 nm) between the emitter and the intermediate substrate without using any external positioners while maintaining a large temperature difference (>700 K). Compared to the equivalent device operating in the far-field regime, we achieve 10-fold enhancement of the photocurrent in the PV cell without degrading the open-circuit voltage and fill factor, demonstrating the potential of our one-chip device for the future applications of near-field thermal radiation transfer.

Intrinsic 2D Ferromagnetism in V5Se8 Epitaxial Thin Films.

The discoveries of intrinsic ferromagnetism in atomically thin van der Waals crystals have opened a new research field enabling fundamental studies on magnetism at two-dimensional (2D) limit as well as development of magnetic van der Waals heterostructures. Currently, a variety of 2D ferromagnetism has been explored mainly by mechanically exfoliating "originally ferromagnetic (FM)" van der Waals crystals, while a bottom-up approach by thin-film growth technique has demonstrated emergent 2D ferromagnetism in a variety of "originally non-FM" van der Waals materials. Here we demonstrate that V5Se8 epitaxial thin films grown by molecular-beam epitaxy exhibit emergent 2D ferromagnetism with intrinsic spin polarization of the V 3d electrons despite that the bulk counterpart is "originally antiferromagnetic". Moreover, thickness-dependence measurements reveal that this newly developed 2D ferromagnet could be classified as an itinerant 2D Heisenberg ferromagnet with weak magnetic anisotropy, broadening a lineup of 2D magnets to those potentially beneficial for future spintronics applications.

Biophysical Parameters of the Sec14 Phospholipid Exchange Cycle.

Sec14, the major yeast phosphatidylcholine (PC)/phosphatidylinositol (PI) transfer protein (PITP), coordinates PC and PI metabolism to facilitate an appropriate and essential lipid signaling environment for membrane trafficking from trans-Golgi membranes. The Sec14 PI/PC exchange cycle is essential for its essential biological activity, but fundamental aspects of how this PITP executes its lipid transfer cycle remain unknown. To address some of these outstanding issues, we applied time-resolved small-angle neutron scattering for the determination of protein-mediated intervesicular movement of deuterated and hydrogenated phospholipids in vitro. Quantitative analysis by small-angle neutron scattering revealed that Sec14 PI- and PC-exchange activities were sensitive to both the lipid composition and curvature of membranes. Moreover, we report that these two parameters regulate lipid exchange activity via distinct mechanisms. Increased membrane curvature promoted both membrane binding and lipid exchange properties of Sec14, indicating that this PITP preferentially acts on the membrane site with a convexly curved face. This biophysical property likely constitutes part of a mechanism by which spatial specificity of Sec14 function is determined in cells. Finally, wild-type Sec14, but not a mixture of Sec14 proteins specifically deficient in either PC- or PI-binding activity, was able to effect a net transfer of PI or PC down opposing concentration gradients in vitro.

Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation.

Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7M serine and 0.3M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays.

Characterization of human ATP-binding cassette protein subfamily D reconstituted into proteoliposomes.

In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1‒3 are located on peroxisomal membrane and play an important role in the transportation of various fatty acid-CoA derivatives, including very long chain fatty acid-CoA, into peroxisomes. ABCD4 is located on lysosomal membrane and is suggested to be involved in the transport of vitamin B12 from lysosomes to the cytosol. However, the precise transport mechanism by which these ABC transporters facilitate the import or export of substrate has yet to be well elucidated. In this study, the overexpression of human ABCD1‒4 in the methylotrophic yeast Pichia pastoris and a purification procedure were developed. The detergent-solubilized proteins were reconstituted into liposomes. ABCD1‒4 displayed stable ATPase activity, which was inhibited by AlF3. Furthermore, ABCD1‒4 were found to possess an equal levels of acyl-CoA thioesterase activity. Proteoliposomes is expected to be an aid in the further biochemical characterization of ABCD transporters.

Veratridine binding to a transmembrane helix of sodium channel Nav1.4 determined by solid-state NMR.

The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Nav1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Nav1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Nav1.4. Here, we determine the NMR structure of a selectively 13C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By 13C NMR, we obtain NMR structural constraints as 13C chemical shifts and the 1H-2H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the 1H-2H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel NavRh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Nav1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6.

The effects of 3% diquafosol sodium eye drop application on meibomian gland and ocular surface alterations in the Cu, Zn-superoxide dismutase-1 (Sod1) knockout mice.

PURPOSE: The purpose of the study is to investigate the effect of 3% diquafosol sodium eye drops on meibomian gland and ocular surface alterations in the superoxide dismutase-1 (Sod1 -/- ) mice in comparison to the wild-type mouse. METHODS: Three percent diquafosol sodium eye drop was instilled to 20 eyes of 10 50-week-old male Sod1 -/- mice and 22 eyes of 11 C57BL/6 strain 50-week-old wild-type (WT) male mice six times a day for 2 weeks. Aqueous tear secretion quantity was measured with phenol red-impregnated cotton threads without anesthesia. Tear film stability and corneal epithelial damage were assessed by fluorescein and lissamine green staining. We also performed oil red O (ORO) lipid staining to evaluate the lipid changes in the meibomian glands. Meibomian gland specimens underwent hematoxylin and eosin staining to examine histopathological changes and meibomian gland acinar unit density after sacrifice. Immunohistochemistry staining was performed using cytokeratin 4, cytokeratin 13, and transglutaminase-1 antibodies. Quantitative real-time polymerase chain reaction for cytokeratin 4, cytokeratin 13, and transglutaminase-1 mRNA expression was also performed. RESULTS: The aqueous tear quantity, the mean tear film breakup time, and the number of lipid droplets significantly improved in the Sod1 -/- mice with treatment. The mean meibomian acinar unit density did not change in the Sod1 -/- mice and WT mice after treatment. Application of 3% diquafosol sodium eye drop significantly decreased the corneal fluorescein and lissamine green staining scores in the Sod1 -/- mice after 2 weeks. We showed a notable increase in cytokeratin 4, cytokeratin 13 immunohistochemistry staining, and cytokeratin 4, cytokeratin 13 mRNA expressions with a marked decrease in immunohistochemistry staining and significant decline in mRNA expression of transglutaminase-1 after 3% diquafosol sodium treatment. CONCLUSION: Topical application of 3% diquafosol sodium eye drop improved the number of lipid droplets, tear stability, and tear production which in turn appeared to have a favorable effect on the ocular surface epithelium. Three percent diquafosol sodium eye drop may be a potential treatment for age-related meibomian gland and dry eye disease based on the observations of the current study.

Membrane-Spanning Sequences in Endoplasmic Reticulum Proteins Promote Phospholipid Flip-Flop.

The mechanism whereby phospholipids rapidly flip-flop in the endoplasmic reticulum (ER) membrane remains unknown. We previously demonstrated that the presence of a hydrophilic residue in the center of the model transmembrane peptide sequence effectively promoted phospholipid flip-flop and that hydrophilic residues composed 4.5% of the central regions of the membrane-spanning sequences of human ER membrane proteins predicted by SOSUI software. We hypothesized that ER proteins with hydrophilic residues might play a critical role in promoting flip-flop. Here, we evaluated the flip rate of fluorescently labeled lipids in vesicles containing each of the 11 synthetic peptides of membrane-spanning sequences, using a dithionite-quenching assay. Although the flippase activities of nine peptides were unexpectedly low, the peptides based on the EDEM1 and SPAST proteins showed enhanced flippase activity with three different fluorescently labeled lipids. The substitution of hydrophobic Ala with His or Arg in the central region of the EDEM1 or SPAST peptides, respectively, attenuated their ability to flip phospholipids. Interestingly, substituting Ala with Arg or His at a location outside of the central region of EDEM1 or SPAST, respectively, also affected the enhancement of flip-flop. These results indicated that both Arg and His are important for the ability of these two peptides to increase the flip rates. The EDEM1 peptide exhibited high activity at significantly low peptide concentrations, suggesting that the same side positioning of Arg and His in α-helix structure is critical for the flip-flop promotion and that the EDEM1 protein is a candidate flippase in the ER.

pH-dependent promotion of phospholipid flip-flop by the KcsA potassium channel.

KcsA is a pH-dependent potassium channel that is activated at acidic pH. The channel undergoes global conformational changes upon activation. We hypothesized that the open-close conformational changes of the transmem brane region could promote the flip-flop of phosphoiipids. Based on this hypothesis, we measured the flip-flop ofNBD-labeled phospholipids in KcsA-incorporated proteoliposomes. Both flip and flop rates of ~NBD-PC were significantly enhanced in the presence of KcsA and were several times higher at pH 4.0 than at pH 7.4, suggesting that KcsA promotes the phospholipid flip in a conformation-dependent manner. Phospholipids were nonselectively flipped with respect to the glycerophospholipid structure. In the active state of KcsA channel,tetrabutylammonium locks the channel in the open conformation at acidic pH; however, it did not alter the fliprate of CGNBD-PC. Thus, the open-close transition of the transmembrane region did not affect the flip-flop of phospholipids. In addition, the KcsA mutant that lacked anN-terminal amphipathic helix (MO-helix) was found to show reduced ability to fl ip C6NBD-phospholipids at acidic pH. The closed conformation is stabilized in the absence of MO-heli x, and thus the attenuated flip could be explained by the reduced prevalence of the open conformation.These results suggest that the open conformation of KcsA can disturb the bilayer integrity and facilitate the flip-flop of phospholipids.

Energetics of the Mixing of Phospholipids in Bilayers Determined Using Vesicle Solubilization.

Here, we report an experimental approach for determining the change in the free energy and the enthalpy that accompanies the mixing of the anionic phosphatidylglycerol and the zwitterionic phosphatidylcholine. The enthalpy change originates in the thermal changes of disrupting lipid bilayer vesicles titrated into a surfactant micelle solution and is monitored using isothermal titration calorimetry. The difference in the solubilization enthalpies between pure and mixed lipid vesicles yields the lipid mixing enthalpy. The Gibbs free energy changes are estimated by determining the thermodynamic equilibrium constants of forming a molecular complex between phospholipids and methyl-ß-cyclodextrin. We provide direct experimental evidence that mixing of the anionic lipid and the zwitterionic lipid is explained well by the entropic term of the electrostatic free energy of a charged surface in the Gouy-Chapman model. The present strategy enables us to determine the precise energetics of lipid-lipid interactions in near-native environments such as liposomes without any chemical modification to lipid molecules.

Kinetic Analysis of the Methyl-ß-cyclodextrin-Mediated Intervesicular Transfer of Pyrene-Labeled Phospholipids.

Methyl-ß-cyclodextrin (MßCD) can transfer phospholipids between vesicles, and its transfer ability has been utilized for the preparation of asymmetric vesicle and lipid incorporation into culture cells. Nevertheless, a detailed kinetic analysis of the MßCD-mediated phospholipid transfer has not yet been carried out. We performed real-time monitoring of intervesicular lipid transfer by means of the fluorescence of pyrene-labeled phospholipids. Intermolecular excimer formation of the pyrene-labeled lipids in a membrane strongly depends on the local concentration of the fluorophore and decreases when the pyrene-labeled lipids are transferred from donor (fluorophore-containing) vesicles to acceptor (fluorophore-free) vesicles. We monitored the fluorescence intensity of the pyrene monomer and excimer simultaneously and found that the excimer/monomer ratio decreased in the presence of MßCD, pointing to MßCD-mediated lipid transfer. The transfer rate depended on the MßCD concentration but not on the lipid concentration, suggesting that dissociation from the membrane via extraction by MßCD is the rate-limiting step of the lipid transfer. Calibration of the excimer/monomer ratio to the molar fraction of the pyrene-labeled lipids enabled us to evaluate the dissociation rate constant correctly. From the temperature dependence of the transfer, we obtained the thermodynamic activation parameters, which revealed that the extraction of phosphatidylcholine by MßCD from membranes is less enthalpically unfavorable than that of phosphatidylglycerol and phosphatidylinositol.

Self-reproduction of nanoparticles through synergistic self-assembly.

We describe a self-reproduction mechanism of nanometer-sized particles (i.e., nanodiscs) through chemical ligation of the precursors and self-assembly of the building blocks. The ligation reaction was accelerated on lipid bilayer surfaces, and the products spontaneously assembled into nanodiscs with lipid molecules. With the increase in the number of nanodiscs, a rapid proliferation of the nanodiscs occurred through the spatial rearrangements of the molecules between the pre-existing nanodiscs and the unreacted materials, rather than template- or complex-enhanced ligation of the precursors. The subsequent process of surface-enhanced ligation of integrated precursors matured the nanoparticles into identical copies of the pre-existing assembly. Our study showed that the synergistic self-assembly mechanism probably underlie the self-replication principles for heterogeneous multimolecular systems.

High Membrane Curvature Enhances Binding, Conformational Changes, and Fibrillation of Amyloid-ß on Lipid Bilayer Surfaces.

Aggregation of the amyloid-ß (Aß) protein and the formation of toxic aggregates are the possible pathogenic pathways in Alzheimer's disease. Accumulating evidence suggests that lipid membranes play key roles in protein aggregation, although the intermolecular forces that drive the interactions between Aß-(1-40) and the membranes vary in different membrane systems. Here, we observed that a high positive curvature of lipid vesicles with diameters of ∼30 nm enhanced the association of Aß with anionic phosphatidylglycerol membranes in the liquid-crystalline phase and with zwitterionic phosphatidylcholine membranes in the gel phase. The binding modes of Aß to these membranes differ in terms of the location of the protein on the membrane and of the protein secondary structure. The fibrillation of Aß was accelerated in the presence of the vesicles and at high protein-to-lipid ratios. Under these conditions, the protein accumulated on the surfaces, as demonstrated by a high (10(7) M(-1)) binding constant. Our findings suggest that packing defects on membranes with high curvatures, such as the intraluminal vesicles in multivesicular bodies and the exosomes, might result in the accumulation of toxic protein aggregates.

Mechanism for the antibacterial action of epigallocatechin gallate (EGCg) on Bacillus subtilis.

Catechins are a class of polyphenols and have high anti-bacterial activity against various microorganisms. Here, we report the mechanism for antibacterial activity of epigallocatechin gallate (EGCg) against Gram-positive bacteria Bacillus subtilis, which is highly sensitive to EGCg. Transmission electron microscope analysis revealed that deposits containing EGCg were found throughout the cell envelope from the outermost surface to the outer surface of cytoplasmic membrane. Aggregating forms of proteins and EGCg were identified as spots that disappeared or showed markedly decreased intensity after the treatment with EGCg compared to the control by two-dimensional electrophoresis. Among the identified proteins included 4 cell surface proteins, such as oligopeptide ABC transporter binding lipoprotein, glucose phosphotransferase system transporter protein, phosphate ABC transporter substrate-binding protein, and penicillin-binding protein 5. Observations of glucose uptake of cells and cell shape B. subtilis after the treatment with EGCg suggested that EGCg inhibits the major functions of these proteins, leading to growth inhibition of B. subtilis.

Technical improvement of total pharyngo-laryngo-esophagectomy for esophageal cancer and head and neck cancer.

PURPOSE: Total pharyngo-laryngo-esophagectomy (PLE) is highly invasive, and the subsequent reconstruction is difficult. The purpose of this study was to clarify the techniques that can decrease the surgical stress and allow for safe reconstruction after this operation. METHODS: The surgical method and clinical outcomes of total PLE were reviewed in 12 patients with either cervicothoracic esophageal cancer or double cancer of the esophagus and pharynx. Microscopic venous anastomosis was principally performed, and arterial anastomosis was added, if needed. RESULTS: A narrow gastric tube was used in ten patients, including two patients who underwent free jejunal interposition, while the colon was used as the main reconstructed organ in two other patients. Staged operations were performed in three high-risk patients. All six patients treated after 2010 were able to undergo thoracoscopic and/or laparoscopic surgery. No critical postoperative complications developed, although minor anastomotic leakage developed in two patients who were successfully treated conservatively. CONCLUSION: When performing PLE, it is important to decrease the surgical stress and ensure a reliable reconstruction by adopting techniques that are appropriate for each case, such as thoracoscopic and laparoscopic surgery, staged operations, microvascular anastomosis, and muscular flaps.