Clinicopathologic significance of DNA mismatch repair protein status in endometrial cancer.

OBJECTIVE: The prognostic implications of DNA mismatch repair protein (MMRP) have not been determined in endometrial cancer. Therefore, in this study, we aimed to evaluate the clinicopathologic characteristics of DNA MMRP deficiency in endometrial cancer. MATERIALS AND METHODS: We examined the MMRP status of 206 patients with endometrial carcinomas, using immunohistochemistry, and analyzed their clinicopathologic factors and survival outcomes stratified by MMRP status using the Kaplan-Meier method and Cox regression analysis. RESULTS: Forty-three cases were deficient for at least one MMRP (20.9%). Loss of MLH1 was the most common (13.1%), followed by MSH6 (7.8%). MMRP deficiency was significantly associated with lympho-vascular space invasion, deep myometrial invasion, and adjuvant treatment (P = 0.032, 0.041, and 0.047, respectively). MMRP-deficient patients had a better overall survival (OS), particularly at advanced cancer stages (III/IV) (100% vs. 73.7%, P = 0.170) or if they had received adjuvant treatment (100% vs. 86.7%, P = 0.087). CONCLUSION: Although MMRP deficiency was associated with unfavorable prognostic risk factors in endometrial cancer, we found a trend in favor of OS in MMRP-deficient patients. More studies are needed to confirm its prognostic implication.

Interactions of HIV gp41's membrane-proximal external region and transmembrane domain with phospholipid membranes from 31P NMR.

HIV-1 entry into cells requires coordinated changes of the conformation and dynamics of both the fusion protein, gp41, and the lipids in the cell membrane and virus envelope. Commonly proposed features of membrane deformation during fusion include high membrane curvature, lipid disorder, and membrane surface dehydration. The virus envelope and target cell membrane contain a diverse set of phospholipids and cholesterol. To dissect how different lipids interact with gp41 to contribute to membrane fusion, here we use 31P solid-state NMR spectroscopy to investigate the curvature, dynamics, and hydration of POPE, POPC and POPS membranes, with and without cholesterol, in the presence of a peptide comprising the membrane proximal external region (MPER) and transmembrane domain (TMD) of gp41. Static 31P NMR spectra indicate that the MPER-TMD induces strong negative Gaussian curvature (NGC) to the POPE membrane but little curvature to POPC and POPC:POPS membranes. The NGC manifests as an isotropic peak in the static NMR spectra, whose intensity increases with the peptide concentration. Cholesterol inhibits the NGC formation and stabilizes the lamellar phase. Relative intensities of magic-angle spinning 31P cross-polarization and direct-polarization spectra indicate that all three phospholipids become more mobile upon peptide binding. Finally, 2D 1H-31P correlation spectra show that the MPER-TMD enhances water 1H polarization transfer to the lipids, indicating that the membrane surfaces become more hydrated. These results suggest that POPE is an essential component of the high-curvature fusion site, and lipid dynamic disorder is a general feature of membrane restructuring during fusion.

Cholesterol Interaction with the Trimeric HIV Fusion Protein gp41 in Lipid Bilayers Investigated by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations.

HIV-1 entry into cells is mediated by the fusion protein gp41. Cholesterol plays an important role in this virus-cell fusion, but molecular structural information about cholesterol-gp41 interaction is so far absent. Here, we present experimental and computational data about cholesterol complexation with gp41 in lipid bilayers. We focus on the C-terminal region of the protein, which comprises a membrane-proximal external region (MPER) and the transmembrane domain (TMD). We measured peptide-cholesterol contacts in virus-mimetic lipid bilayers using solid-state NMR spectroscopy, and augmented these experimental data with all-atom molecular dynamics simulations. 2D 19F NMR spectra show correlation peaks between MPER residues and the cholesterol isooctyl tail, indicating that cholesterol is in molecular contact with the MPER-TMD trimer. 19F-13C distance measurements between the peptide and 13C-labeled cholesterol show that C17 on the D ring and C9 at the intersection of B and C rings are ~7.0 Å from the F673 side-chain 4-19F. At high peptide concentrations in the membrane, the 19F-13C distance data indicate three cholesterol molecules bound near F673 in each trimer. Mutation of a cholesterol recognition amino acid consensus motif did not change these distances, indicating that cholesterol binding does not require this sequence motif. Molecular dynamics simulations further identify two hotspots for cholesterol interactions. Taken together, these experimental data and simulations indicate that the helix-turn-helix conformation of the MPER-TMD is responsible for sequestering cholesterol. We propose that this gp41-cholesterol interaction mediates virus-cell fusion by recruiting gp41 to the boundary of the liquid-disordered and liquid-ordered phases to incur membrane curvature.

Two-dimensional 19F-13C correlation NMR for 19F resonance assignment of fluorinated proteins.

19F solid-state NMR is an excellent approach for measuring long-range distances for structure determination and for studying molecular motion. For multi-fluorinated proteins, assignment of 19F chemical shifts has been traditionally carried out using mutagenesis. Here we show 2D 19F-13C correlation experiments that allow efficient assignment of the 19F chemical shifts. We have compared several rotational-echo double-resonance-based pulse sequences and 19F-13C cross polarization (CP) for 2D 19F-13C correlation. We found that direct transferred-echo double-resonance (TEDOR) transfer from 19F to 13C and vice versa outperforms out-and-back coherence transfer schemes. 19F detection gives twofold higher sensitivity over 13C detection for the 2D correlation experiment. At MAS frequencies of 25-35 kHz, double-quantum 19F-13C CP has higher coherence transfer efficiencies than zero-quantum CP. The most efficient TEDOR transfer experiment has higher sensitivity than the most efficient double-quantum CP experiment. We demonstrate these 2D 19F-13C correlation experiments on the model compounds t-Boc-4F-phenylalanine and GB1. Application of the 2D 19F-13C TEDOR correlation experiment to the tetrameric influenza BM2 transmembrane peptide shows intermolecular 13C-19F cross peaks that indicate that the BM2 tetramers cluster in the lipid bilayer in an antiparallel fashion. This clustering may be relevant for the virus budding function of this protein.

Correction to: PD-L1 expression on stromal tumor-infiltrating lymphocytes is a favorable prognostic factor in ovarian serous carcinoma.

Following publication of the original article [1], the authors opted to correct the incorrect e-mail address of the corresponding author from 82-55-360-1850chungsu1982@gmail.com to chungsu1982@gmail.com , grant in the Acknowledgements section and Author details. Below are the updated version of the Acknowledgements and Author details.

PD-L1 expression on stromal tumor-infiltrating lymphocytes is a favorable prognostic factor in ovarian serous carcinoma.

BACKGROUND: PD-L1 expression levels determined by immunostaining are known to be related to the survival rate and prognosis of patients with various types of cancers, as well as to the therapeutic response to immune checkpoint inhibitors. Recently, the U.S. Food and Drug Administration approved an immune checkpoint inhibitor for the treatment of non-small cell lung cancer along with the clones used for PD-L1 immunostaining to predict the resulting response. In this study, we performed PD-L1 immunostaining of tissue microarrays from ovarian epithelial cancer using SP263, an approved clone, and examined the effect of PD-L1 expression on survival rate and prognosis. METHODS: Tissue microarrays were constructed from ovarian epithelial cancer tissues of 248 patients and PD-L1 immunostaining was performed using the SP263 clone. PD-L1 expression levels in tumor cells, intraepithelial tumor-infiltrating lymphocytes, and stromal tumor-infiltrating lymphocytes were evaluated, and the effect of PD-L1 expression on survival and prognosis was analyzed. RESULTS: PD-L1 was detected in tumor cells as well as intraepithelial tumor-infiltrating lymphocytes and stromal tumor-infiltrating lymphocytes. It was most frequently expressed in stromal tumor-infiltrating lymphocytes. The Kaplan-Meier curve analysis and log rank test showed that only high stromal PD-L1 expression was associated with increased overall survival in ovarian serous carcinoma. Multivariate and univariate Cox regression analyses revealed that stromal PD-L1 expression was an independent prognostic factor, especially in ovarian serous carcinoma. CONCLUSIONS: PD-L1 immunostaining using SP263 was observed in tumor cells as well as intraepithelial and stromal tumor-infiltrating lymphocytes. PD-L1-expressing stromal tumor-infiltrating lymphocytes were associated with an increased overall survival rate and may serve as a favorable prognostic factor in ovarian cancer, particularly serous carcinoma.

Elucidating Relayed Proton Transfer through a His-Trp-His Triad of a Transmembrane Proton Channel by Solid-State NMR.

Proton transfer through membrane-bound ion channels is mediated by both water and polar residues of proteins, but the detailed molecular mechanism is challenging to determine. The tetrameric influenza A and B virus M2 proteins form canonical proton channels that use an HxxxW motif for proton selectivity and gating. The BM2 channel also contains a second histidine (His), H27, equidistant from the gating tryptophan, which leads to a symmetric H19xxxW23xxxH27 motif. The proton-dissociation constants (pKa's) of H19 in BM2 were found to be much lower than the pKa's of H37 in AM2. To determine if the lower pKa's result from H27-facilitated proton dissociation of H19, we have now investigated a H27A mutant of BM2 using solid-state NMR. 15N NMR spectra indicate that removal of the second histidine converted the protonation and tautomeric equilibria of H19 to be similar to the H37 behavior in AM2, indicating that the peripheral H27 is indeed the origin of the low pKa's of H19 in wild-type BM2. Measured interhelical distances between W23 sidechains indicate that the pore constriction at W23 increases with the H19 tetrad charge but is independent of the H27A mutation. These results indicate that H27 both accelerates proton dissociation from H19 to increase the inward proton conductance and causes the small reverse conductance of BM2. The proton relay between H19 and H27 is likely mediated by the intervening gating tryptophan through cation-π interactions. This relayed proton transfer may exist in other ion channels and has implications for the design of imidazole-based synthetic proton channels.

Stromal tumor-infiltrating lymphocytes evaluated on H&E-stained slides are an independent prognostic factor in epithelial ovarian cancer and ovarian serous carcinoma.

Studies on tumor-infiltrating lymphocytes (TILs) in epithelial ovarian cancer (EOC) have focused on the clinical significance of inflammatory cells of specific subtypes that are identifiable using immunohistochemistry. However, the subtypes of inflammatory cells that reportedly affect patient survival and prognosis have differed from study to study, and few studies have examined TILs using hematoxylin and eosin (H&E) staining. Therefore, the present study aimed to identify the clinical importance of general stromal TILs in EOC by using H&E staining to apply breast cancer recommendations from the International TILs Working Group 2014 on breast cancer using H&E staining. Stromal TILs in 256 EOC cases from Pusan National University Hospital and 475 cases of high-grade serous carcinoma from The Cancer Genome Atlas dataset were assessed. Stromal TILs were evaluated using H&E-stained slides according to the breast cancer recommendations of the International Working Group 2014, and patients were classified into low and high stromal TIL groups according to their stromal TIL values. The associations of these groups with clinicopathologic factors were assessed, and it was confirmed that group membership correlated with survival and prognosis. According to the χ2 assessment, the stromal TIL group was associated with tumor grade. Furthermore, the stromal TIL group was associated with overall survival according to Kaplan-Meier analysis with the log-rank test. Finally, the stromal TIL group was an independent prognostic factor according to univariate and multivariate Cox regression analyses. In cases of EOC, the evaluation of general stromal TILs on H&E-stained slides could be used to predict prognosis.

Oligomeric Structure and Three-Dimensional Fold of the HIV gp41 Membrane-Proximal External Region and Transmembrane Domain in Phospholipid Bilayers.

The HIV-1 glycoprotein, gp41, mediates fusion of the virus lipid envelope with the target cell membrane during virus entry into cells. Despite extensive studies of this protein, inconsistent and contradictory structural information abounds in the literature about the C-terminal membrane-interacting region of gp41. This C-terminal region contains the membrane-proximal external region (MPER), which harbors the epitopes for four broadly neutralizing antibodies, and the transmembrane domain (TMD), which anchors the protein to the virus lipid envelope. Due to the difficulty of crystallizing and solubilizing the MPER-TMD, most structural studies of this functionally important domain were carried out using truncated peptides either in the absence of membrane-mimetic solvents or bound to detergents and lipid bicelles. To determine the structural architecture of the MPER-TMD in the native environment of lipid membranes, we have now carried out a solid-state NMR study of the full MPER-TMD segment bound to cholesterol-containing phospholipid bilayers. 13C chemical shifts indicate that the majority of the peptide is α-helical, except for the C-terminus of the TMD, which has moderate ß-sheet character. Intermolecular 19F-19F distance measurements of singly fluorinated peptides indicate that the MPER-TMD is trimerized in the virus-envelope mimetic lipid membrane. Intramolecular 13C-19F distance measurements indicate the presence of a turn between the MPER helix and the TMD helix. This is supported by lipid-peptide and water-peptide 2D 1H-13C correlation spectra, which indicate that the MPER binds to the membrane surface whereas the TMD spans the bilayer. Together, these data indicate that full-length MPER-TMD assembles into a trimeric helix-turn-helix structure in lipid membranes. We propose that the turn between the MPER and TMD may be important for inducing membrane defects in concert with negative-curvature lipid components such as cholesterol and phosphatidylethanolamine, while the surface-bound MPER helix may interact with N-terminal segments of the protein during late stages of membrane fusion.

Conformation and Trimer Association of the Transmembrane Domain of the Parainfluenza Virus Fusion Protein in Lipid Bilayers from Solid-State NMR: Insights into the Sequence Determinants of Trimer Structure and Fusion Activity.

Enveloped viruses enter cells by using their fusion proteins to merge the virus lipid envelope and the cell membrane. While crystal structures of the water-soluble ectodomains of many viral fusion proteins have been determined, the structure and assembly of the C-terminal transmembrane domain (TMD) remains poorly understood. Here we use solid-state NMR to determine the backbone conformation and oligomeric structure of the TMD of the parainfluenza virus 5 fusion protein. 13C chemical shifts indicate that the central leucine-rich segment of the TMD is α-helical in POPC/cholesterol membranes and POPE membranes, while the Ile- and Val-rich termini shift to the ß-strand conformation in the POPE membrane. Importantly, lipid mixing assays indicate that the TMD is more fusogenic in the POPE membrane than in the POPC/cholesterol membrane, indicating that the ß-strand conformation is important for fusion by inducing membrane curvature. Incorporation of para-fluorinated Phe at three positions of the α-helical core allowed us to measure interhelical distances using 19F spin diffusion NMR. The data indicate that, at peptide:lipid molar ratios of ~1:15, the TMD forms a trimeric helical bundle with inter-helical distances of 8.2-8.4Å for L493F and L504F and 10.5Å for L500F. These data provide high-resolution evidence of trimer formation of a viral fusion protein TMD in phospholipid bilayers, and indicate that the parainfluenza virus 5 fusion protein TMD harbors two functions: the central α-helical core is the trimerization unit of the protein, while the two termini are responsible for inducing membrane curvature by transitioning to a ß-sheet conformation.

Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR.

The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined the cholesterol-binding site of the M2 protein in phospholipid bilayers using solid-state NMR spectroscopy. Chain-fluorinated cholesterol was used to measure cholesterol proximity to M2 while sterol-deuterated cholesterol was used to measure bound-cholesterol orientation in lipid bilayers. Carbon-fluorine distance measurements show that at a cholesterol concentration of 17 mol%, two cholesterol molecules bind each M2 tetramer. Cholesterol binds the C-terminal transmembrane (TM) residues, near an amphipathic helix, without requiring a cholesterol recognition sequence motif. Deuterium NMR spectra indicate that bound cholesterol is approximately parallel to the bilayer normal, with the rough face of the sterol rings apposed to methyl-rich TM residues. The distance- and orientation-restrained cholesterol-binding site structure shows that cholesterol is stabilized by hydrophobic interactions with the TM helix and polar and aromatic interactions with neighboring amphipathic helices. At the 1:2 binding stoichiometry, lipid 31P spectra show an isotropic peak indicative of high membrane curvature. This M2-cholesterol complex structure, together with previously observed M2 localization at phase boundaries, suggests that cholesterol mediates M2 clustering to the neck of the budding virus to cause the necessary curvature for membrane scission. The solid-state NMR approach developed here is generally applicable for elucidating the structural basis of cholesterol's effects on membrane protein function.

Effect of Red Ginseng on Genotoxicity and Health-Related Quality of Life after Adjuvant Chemotherapy in Patients with Epithelial Ovarian Cancer: A Randomized, Double Blind, Placebo-Controlled Trial.

We evaluated the effect of red ginseng on toxicity, health-related quality of life (HRQL) and survival after adjuvant chemotherapy in patients with epithelial ovarian cancer (EOC). A total of 30 patients with EOC were randomly assigned to placebo (n = 15) and red ginseng groups (n = 15). All patients took placebo or red ginseng (3000 mg/day) for three months. Then, we compared changes of genotoxicity, HRQL and survival between the two groups. As a result, red ginseng reduced micronuclei yield in comparison with placebo despite no difference of binucleated cells index. Although red ginseng increased serum levels of alanine aminotransferase and aspartate aminotransferase significantly, they were within the normal value. Moreover, there were no differences in adverse events between placebo and red ginseng groups. In terms of HRQL, red ginseng was associated with improved emotional functioning and decreased symptoms of fatigue, nausea and vomiting, and dyspnea, reduced anxiety and interference affecting life and improved daytime somnolence. However, there was no effect of red ginseng on prognosis of EOC. Conclusively, red ginseng may be safe and effective to reduce genotoxicity and improve HRQL despite no benefit of survival in patients with EOC who received chemotherapy.

Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy.

The influenza M2 protein forms an acid-activated proton channel that is essential for virus replication. The transmembrane H37 selects for protons under low external pH while W41 ensures proton conduction only from the N terminus to the C terminus and prevents reverse current under low internal pH. Here, we address the molecular basis for this asymmetric conduction by investigating the structure and dynamics of a mutant channel, W41F, which permits reverse current under low internal pH. Solid-state NMR experiments show that W41F M2 retains the pH-dependent α-helical conformations and tetrameric structure of the wild-type (WT) channel but has significantly altered protonation and tautomeric equilibria at H37. At high pH, the H37 structure is shifted toward the π tautomer and less cationic tetrads, consistent with faster forward deprotonation to the C terminus. At low pH, the mutant channel contains more cationic tetrads than the WT channel, consistent with faster reverse protonation from the C terminus. 15N NMR spectra allow the extraction of four H37 pKas and show that the pKas are more clustered in the mutant channel compared to WT M2. Moreover, binding of the antiviral drug, amantadine, at the N-terminal pore at low pH did not convert all histidines to the neutral state, as seen in WT M2, but left half of all histidines cationic, unambiguously demonstrating C-terminal protonation of H37 in the mutant. These results indicate that asymmetric conduction in WT M2 is due to W41 inhibition of C-terminal acid activation by H37. When Trp is replaced by Phe, protons can be transferred to H37 bidirectionally with distinct rate constants.

Differentiation Potential of Mesenchymal Stem Cells Is Related to Their Intrinsic Mechanical Properties.

PURPOSE: The differentiation properties of stem cells are not yet fully understood due to their close association with multiple environmental and extrinsic factors. This study investigates the differentiation properties of mesenchymal stem cells (MSCs) and correlates them with their intrinsic mechanical properties. METHODS: A total of 3 different types of MSCs, namely bone marrow-derived MSCs (BMSCs), umbilical cord-derived MSCs (UCSCs), and adipose-derived MSCs (ADSCs) were evaluated. These 3 MSCs were individually differentiated into adipocytes and osteoblasts for 3 weeks. The mechanical properties of the MSCs and differentiated cells were determined by atomic force microscopy. RESULTS: ADSCs showed the greatest ability to differentiate into adipocytes, followed by BMSCs and UCSCs. While UCSCs differentiated readily into osteoblasts, BMSCs and ADSCs were less likely to undergo this differentiation. UCSCs were the "hardest" cells, while ADSCs were the "softest." The cells differentiated from "hard" MSCs were stiffer than the cells differentiated from "soft" MSCs, irrespective of lineage specification. CONCLUSIONS: The differentiation ability of MSCs and the mechanical properties of the differentiated cells were closely linked. However, there were no significant correlations regarding changes in the mechanical properties between the nuclear region and the cytoplasm during differentiation.

Selective cytotoxic effect of non-thermal micro-DBD plasma.

Non-thermal plasma has been extensively researched as a new cancer treatment technology. We investigated the selective cytotoxic effects of non-thermal micro-dielectric barrier discharge (micro-DBD) plasma in cervical cancer cells. Two human cervical cancer cell lines (HeLa and SiHa) and one human fibroblast (HFB) cell line were treated with micro-DBD plasma. All cells underwent apoptotic death induced by plasma in a dose-dependent manner. The plasma showed selective inhibition of cell proliferation in cervical cancer cells compared to HFBs. The selective effects of the plasma were also observed between the different cervical cancer cell lines. Plasma treatment significantly inhibited the proliferation of SiHa cells in comparison to HeLa cells. The changes in gene expression were significant in the cervical cancer cells in comparison to HFBs. Among the cancer cells, apoptosis-related genes were significantly enriched in SiHa cells. These changes were consistent with the differential cytotoxic effects observed in different cell lines.

The Influenza M2 Ectodomain Regulates the Conformational Equilibria of the Transmembrane Proton Channel: Insights from Solid-State Nuclear Magnetic Resonance.

The influenza M2 protein is the target of the amantadine family of antiviral drugs, and its transmembrane (TM) domain structure and dynamics have been extensively studied. However, little is known about the structure of the highly conserved N-terminal ectodomain, which contains epitopes targeted by influenza vaccines. In this study, we synthesized an M2 construct containing the N-terminal ectodomain and the TM domain, to understand the site-specific conformation and dynamics of the ectodomain and to investigate the effect of the ectodomain on the TM structure. We incorporated (13)C- and (15)N-labeled residues into both domains and measured their chemical shifts and line widths using solid-state nuclear magnetic resonance. The data indicate that the entire ectodomain is unstructured and dynamic, but the motion is slower for residues closer to the TM domain. (13)C line shapes indicate that this ecto-TM construct undergoes fast uniaxial rotational diffusion, like the isolated TM peptide, but drug binding increases the motional rates of the TM helix while slowing the local motion of the ectodomain residues that are close to the TM domain. Moreover, (13)C and (15)N chemical shifts indicate that the ectodomain shifts the conformational equilibria of the TM residues toward the drug-bound state even in the absence of amantadine, thus providing a molecular structural basis for the lower inhibitory concentration of full-length M2 compared to that of the ectodomain-truncated M2. We propose that this conformational selection may result from electrostatic repulsion between negatively charged ectodomain residues in the tetrameric protein. Together with the recent study of the M2 cytoplasmic domain, these results show that intrinsically disordered extramembrane domains in membrane proteins can regulate the functionally relevant conformation and dynamics of the structurally ordered TM domains.

Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain.

Solid-state NMR-based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly (13)C-labeled. A strategy to meet this challenge is chemical ligation combined with site-specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water-soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra-membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly (13)C-labeled full-length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid-phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post-AH cytoplasmic residues exhibit random-coil chemical shifts, low bond order parameters, and a surface-bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus-mimetic membrane, indicating that the structure and dynamics of the post-AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium-sized membrane proteins to provide site-specific structural constraints, which complement the information obtained from uniformly (13)C, (15)N-labeled proteins.

Rectouterine fistula after laparoscopic ultrasound-guided radiofrequency ablation of a uterine fibroid.

In the conservative management of uterine fibroids is radiofrequency ablation (RFA) considered to be one of the safe, effective and minimal invasive approaches in selected women who desire to retain their uterus. Few studies were conducted on its adverse outcomes and most of the reported complications were minor events such as pain, discharge, adhesion which didn't require any intervention. However, although safe and effective, the RFA of a uterine myoma can be the cause for severe complications such as penetration and burn injuries of pelvic organs. In general, a rectouterine fistula is one of the rarest complications but can lead to serious adverse outcomes. Herein, to our knowledge, we report the first case involving a rectouterine fistula after laparoscopic ultrasound-guided RFA of a uterine myoma with pelvic endometriosis. In addition, we provide a brief review of the relevant literature.

p53 negatively regulates Pin1 expression under ER stress.

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development of many diseases. A previous study indicated that the apoptotic regulator p53 is significantly increased in response to ER stress and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood. Here, we investigated whether p53 contributes to the impairment of Pin1 signaling under ER stress. We found that treatment with thapsigargin, a stimulator of p53 expression and an inducer of ER stress, decreased Pin1 expression in HCT116 cells. Also, we identified functional p53 response elements (p53REs) in the Pin1 promoter. Overexpression of p53 significantly decreased Pin1 expression in HCT116 cells while abolition of p53 gene expression induced Pin1 expression. Pin1 expression was significantly increased by treatment with the p53 inhibitor pifithrin-α or down-regulation of p53 expression. Taken together, ER stress decreased Pin1 expression through p53 activation, and this mechanism may be associated with ER stress-induced cell death. These data reported here support the importance of Pin1 as a potential target molecule mediating tumor development.

A 2H solid-state NMR study of lipid clustering by cationic antimicrobial and cell-penetrating peptides in model bacterial membranes.

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use (2)H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the ß-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. (2)H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct (2)H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.