Chemical Genetics in C. elegans Identifies Anticancer Mycotoxins Chaetocin and Chetomin as Potent Inducers of a Nuclear Metal Homeostasis Response.

C. elegans numr-1/2 (nuclear-localized metal-responsive) is an identical gene pair encoding a nuclear protein previously shown to be activated by cadmium and disruption of the integrator RNA metabolism complex. We took a chemical genetic approach to further characterize regulation of this novel metal response by screening 41,716 compounds and extracts for numr-1p::GFP activation. The most potent activator was chaetocin, a fungal 3,6-epidithiodiketopiperazine (ETP) with promising anticancer activity. Chaetocin activates numr-1/2 strongly in the alimentary canal but is distinct from metal exposure, because it represses canonical cadmium-responsive metallothionine genes. Chaetocin has diverse targets in cancer cells including thioredoxin reductase, histone lysine methyltransferase, and acetyltransferase p300/CBP; further work is needed to identify the mechanism in C. elegans as genetic disruption and RNAi screening of homologues did not induce numr-1/2 in the alimentary canal and chaetocin did not affect markers of integrator dysfunction. We demonstrate that disulfides in chaetocin and chetomin, a dimeric ETP analog, are required to induce numr-1/2. ETP monomer gliotoxin, despite possessing a disulfide linkage, had almost no effect on numr-1/2, suggesting a dimer requirement. Chetomin inhibits C. elegans growth at low micromolar levels, and loss of numr-1/2 increases sensitivity; C. elegans and Chaetomiaceae fungi inhabit similar environments raising the possibility that numr-1/2 functions as a defense mechanism. There is no direct orthologue of numr-1/2 in humans, but RNaseq suggests that chaetocin affects expression of cellular processes linked to stress response and metal homeostasis in colorectal cancer cells. Our results reveal interactions between metal response gene regulation and ETPs and identify a potential mechanism of resistance to this versatile class of preclinical compounds.

Current status and progress of laparoscopic inguinal hernia repair: A review.

After 30 years of development, laparoscopic inguinal hernia repair (LIHR) has become the main method for treating adult inguinal hernia. LIHR is more standardized, the approach of single-port laparoscopic hernioplasty, the advantages of robotic inguinal hernioplasty, the application of new patches and the selection of surgical methods for different populations have become the focus and difficulty of current research. This article summarized the research progress of LIHR in recent years. Different keywords and phrases including inguinal hernia, LIHR, transabdominal laparoscopic preperitoneal hernia repair, and total extraperitoneal hernia repair were used to search the PubMed, China National Knowledge Infrastructure, and Web of Science databases for related original and review articles that serve the aim of this article well, which was to perform a nonsystematic review of the development, progress, and current status of LIHR.

Molecular characterization of ethyl carbamate toxicity in Caenorhabditis elegans.

Ethyl carbamate is a common contaminant prevalent in fermented food with probable carcinogenic effects in animals. To date, other toxicological properties of ethyl carbamate are not well characterized. Using the genetic model Caenorhabditis elegans, we found that chronic exposure to ethyl carbamate during larval development impedes growth while exposure during adulthood inhibits reproduction, shortens lifespan, and promotes degeneration to dopaminergic neurons. Through whole-transcriptome RNA-sequencing, we found that ethyl carbamate invokes a widespread transcriptomic response inducing the differential expression of > 4,000 genes by at least 2-fold. Functional analysis of RNA-sequencing data revealed that up-regulated genes enrich to various neuron regulatory processes and xenobiotic defense. Gene expression analysis confirms that various genes encoding antioxidant enzymes and those functioning within phase I and II detoxification responses along with ABC transporters are highly up-regulated after ethyl carbamate exposure, suggesting the onset of oxidative stress. Overall, these findings report new toxicological properties of chronic ethyl carbamate exposure and provide new insights on its effects on transcriptome regulation in the C. elegans model.

Magnetic hydrogel with long in situ retention time for self-regulating temperature hyperthermia.

Aim: Magnetic hydrogels (MHGs) have been proposed to avoid the redistribution and loss of magnetic nanoparticles (MNPs) when administrated by intratumoral injection. However, the requirement of complex cooling systems and temperature monitoring systems still hinder the clinical application of MHGs. This study investigates the feasibility of developing an MHG to realize the self-regulation of hyperthermia temperature. Methods: The MHG was developed by dispersing the MNPs with self-regulating temperature property into the temperature-sensitive hydrogel through physical crosslinking. The MHG's gelation temperature was tested by measuring the storage modulus and loss modulus on a rotational rheometer. The biocompatibility of the MHG and MNPs was characterized by CCK-8 assay against HaCaT cells. The in vivo magnetic heating property was examined through monitoring the temperature in the MHG on mice back upon the application of the alternating magnetic field (400 ± 5 Oe, 100 ± 5 kHz) every week for successive six weeks. Results: The gelation temperature of the MHG falls in 28.4°C-37.4°C. At in vivo applied concentration of 80 mg/mL, the MHG exhibits over 80% cell viability after 72 h, significantly higher than 50% cell viability of the MNPs (p<0.001). The MHG's stable magnetic hyperthermia temperatures in vivo are in the range of 43.4°C-43.8°C. Conclusions: The developed MHG can be injected using a syringe and will solidify upon body temperature. The biocompatibility is improved after the MNPs being made into MHG. The MHG can self-regulate the temperature for six weeks, exhibiting application potential for self-regulating temperature hyperthermia.

Dehydration stress alters the mitogen-activated-protein kinase signaling and chaperone stress response in Xenopus laevis.

In arid conditions, the African Clawed frog Xenopus laevis enters a state of estivation dormancy as an adaptive survival strategy. Under estivation, X. laevis experience severe dehydration stress as 25-35% of total body water is lost. Dehydration in X. laevis can lead to periods of hypoxia due to elevated blood viscosity that impedes tissue perfusion. To understand how X. laevis survives under such stress, we studied the regulation pattern of key mitogen-activated protein kinases (MAPK) and their downstream transcription factors, along with several heat shock proteins in the oxygen sensitive brain and heart tissue of X. laevis under dehydration stress. Our study revealed that the activation phosphorylation residues of MAPK including JNK and MSK and their downstream transcription factors c-Jun and ATF2 are significantly decreased in the heart under dehydration. Given that JNK, c-Jun, and ATF2 are known positive regulators of apoptosis, this regulatory pattern suggest that a state of pro-survival signals may be established in the dehydrated heart. In support of this, protein levels of HSP60, a pro-apoptotic mitochondrial chaperone, was also downregulated in the heart in response to dehydration stress. In the brain tissue, most proteins remain unchanged with the exception of the apoptosis regulating p53 transcription factor, which showed a significant decrease in its activating phosphorylation residue under dehydration. Overall, our study revealed that in the Xenopus brain and heart, a specific suppression pattern of MAPK, transcription factors, and HSP takes place to potentially establish a state of pro-survival under dehydration stress.

RNA processing errors triggered by cadmium and integrator complex disruption are signals for environmental stress.

BACKGROUND: Adaptive responses to stress are essential for cell and organismal survival. In metazoans, little is known about the impact of environmental stress on RNA homeostasis. RESULTS: By studying the regulation of a cadmium-induced gene named numr-1 in Caenorhabditis elegans, we discovered that disruption of RNA processing acts as a signal for environmental stress. We find that NUMR-1 contains motifs common to RNA splicing factors and influences RNA splicing in vivo. A genome-wide screen reveals that numr-1 is strongly and specifically induced by silencing of genes that function in basal RNA metabolism including subunits of the metazoan integrator complex. Human integrator processes snRNAs for functioning with splicing factors, and we find that silencing of C. elegans integrator subunits disrupts snRNA processing, causes aberrant pre-mRNA splicing, and induces the heat shock response. Cadmium, which also strongly induces numr-1, has similar effects on RNA and the heat shock response. Lastly, we find that heat shock factor-1 is required for full numr-1 induction by cadmium. CONCLUSION: Our results are consistent with a model in which disruption of integrator processing of RNA acts as a molecular damage signal initiating an adaptive stress response mediated by heat shock factor-1. When numr-1 is induced via this pathway in C. elegans, its function in RNA metabolism may allow it to mitigate further damage and thereby promote tolerance to cadmium.

Onion-structure bionic hydrogel capsules based on chitosan for regulating doxorubicin release.

Inspired by the multilayer structure of onion, herein, a novel strategy to prepare bionic multilayer hydrogel capsules for inhibiting burst release of doxorubicin (DOX) is reported. The bionic multilayer hydrogel capsules are prepared by the ionotropic crosslinking method. Compared with monolayer hydrogel capsules, the multilayer hydrogel capsules can largely homogenize the distribution of DOX and suppress the concentration gradient of DOX between the outermost hydrogel layer and external environment and the dense cuticular membranes of capsules can restrict the migration and diffusion of DOX. As a result, a significant inhibition of the burst release of DOX can be achieved. Moreover, the bionic multilayer hydrogel capsules demonstrate pH sensitivity and good biocompatibility to human epidermal keratinocyte (HaCaT) cells. This work opens up a new horizon in the burst release of biomaterial-based hydrogels for drug delivery system.

The Medication Risk of Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis in Asians: The Major Drug Causality and Comparison With the US FDA Label.

Specific ethnic genetic backgrounds are associated with the risk of Stevens-Johnson syndrome / toxic epidermal necrolysis (SJS/TEN) especially in Asians. However, there have been no large cohort, multiple-country epidemiological studies of medication risk related to SJS/TEN in Asian populations. Thus, we analyzed the registration databases from multiple Asian countries who were treated during 1998-2017. A total 1,028 SJS/TEN cases were identified with the algorithm of drug causality for epidermal necrolysis. Furthermore, those medications labeled by the US Food and Drug Administration (FDA) as carrying a risk of SJS/TEN were also compared with the common causes of SJS/TEN in Asian countries. Oxcarbazepine, sulfasalazine, COX-II inhibitors, and strontium ranelate were identified as new potential causes. In addition to sulfa drugs and beta-lactam antibiotics, quinolones were also a common cause. Only one acetaminophen-induced SJS was identified, while several medications (e.g., oseltamivir, terbinafine, isotretinoin, and sorafenib) labeled as carrying a risk of SJS/TEN by the FDA were not found to have caused any of the cases in the Asian countries investigated in this study.

Injectable and body temperature sensitive hydrogels based on chitosan and hyaluronic acid for pH sensitive drug release.

Hydrogels based on chitosan/hyaluronic acid/ß-sodium glycerophosphate demonstrate injectability, body temperature sensitivity, pH sensitive drug release and adhesion to cancer cell. The drug (doxorubicin) loaded hydrogel precursor solutions are injectable and turn to hydrogels when the temperature is increased to body temperature. The acidic condition (pH 4.00) can trigger the release of drug and the cancer cell (Hela) can adhere to the surface of the hydrogels, which will be beneficial for tumor site-specific administration of drug. The mechanical strength, the gelation temperature, and the drug release behavior can be tuned by varying hyaluronic acid content. The mechanisms were characterized using dynamic mechanical analysis, Fourier transform infrared spectroscopy, scanning electron microscopy and fluorescence microscopy. The carboxyl group in hyaluronic acid can form the hydrogen bondings with the protonated amine in chitosan, which promotes the increase of mechanical strength of the hydrogels and depresses the initial burst release of drug from the hydrogel.

Regulation of the insulin-Akt signaling pathway and glycolysis during dehydration stress in the African clawed frog Xenopus laevis.

Estivation is an adaptive stress response utilized by some amphibians during periods of drought in the summer season. In this study, we examine the regulation of the insulin signaling cascade and glycolysis pathway in the African clawed frog Xenopus laevis during the dehydration stress induced state of estivation. We show that in the brain and heart of X. laevis, dehydration reduces the phosphorylation of the insulin growth factor-1 receptor (IGF-1R), and this is followed by similar reductions in the phosphorylation of the Akt and mechanistic target of rapamycin (mTOR) kinase. Interestingly, phosphorylation levels of IGF-1R and mTOR were not affected in the kidney, and phosphorylation levels of P70S6K and the ribosomal S6 protein were elevated during dehydration stress. Animals under estivation are also susceptible to periods of hypoxia, suggesting that glycolysis may also be affected. We observed that protein levels of many glycolytic enzymes remained unchanged during dehydration; however, the hypoxia response factor-1 alpha (HIF-1α) protein was elevated by greater than twofold in the heart during dehydration. Overall, we provide evidence that shows that the insulin signaling pathway in X. laevis is regulated in a tissue-specific manner during dehydration stress and suggests an important role for this signaling cascade in mediating the estivation response.

Micro/nano-particle decorated metal wire for cutting soft matter.

To cut soft materials such as biological tissues with minimal damage and reduced positional error is highly desired in medical surgery and biomechanics. After years of natural selection and evolution, mosquitoes have acquired the ability to insert their proboscises into human skin with astonishingly tiny forces. This can be associated with the unique structure of their proboscises, with micro/nano sawteeth, and the distinctive insertion manner: high frequency reciprocating saw cutting. Inspired by these, this communication describes the successful implantation of metal oxide particles onto molybdenum wire surfaces through a sol-calcination process, to form a biomimetic sawblade with a high density of micro/nano saw teeth, where the acidification is essential in terms of generating active anchoring sites on the wire. When used as a sawblade in conjunction with reciprocating action to cut the viscoelastic gel, both the cut-in force and cut-in displacement could be decreased substantially. The cutting speed and frequency of reciprocating action are important operating parameters influencing cut-in force.

Post-translational regulation of PTEN catalytic function and protein stability in the hibernating 13-lined ground squirrel.

BACKGROUND: The insulin signaling pathway functions as a major regulator of many metabolic and cellular functions, and has been shown to be reversibly suppressed in many species during hibernation. This study characterized the regulation of PTEN phosphatase, a negative regulator of the insulin receptor network, over the torpor-arousal cycle of hibernation in the skeletal muscle of Ictidomys tridecemlineatus. METHODS: Western blotting and RT-PCR were used to analyze post-translational and transcriptional regulations of PTEN respectively. Enzymatic activities were determined by the malachite green assay, while protein stability was assessed the using pulse-proteolysis method. RESULTS: During torpor, the ratio of non-phosphorylated PTEN (S380/T382/T383) was significantly elevated by 1.4-fold during late torpor compared with euthermic controls; this was coupled with an increase in substrate affinity for PIP3 (by 56%) in late torpor. Two proteolytic cleavage PEST motifs were identified in the C-terminus that overlapped with the phosphorylation sites of PTEN; pulse-proteolysis analysis of PTEN protein showed a decrease in protein stability during late torpor (Cm of urea decreased by 21%). Furthermore, the increase in PTEN activity observed was correlated with a decrease in PDK-1 phosphorylation by 32%, suggesting a downstream effect of PTEN activation during torpor. Transcriptional analysis showed that mRNA expression of pten and pdk-1 remain unchanged during hibernation, suggesting post-translation modification as the primary regulatory mechanism of PTEN function. CONCLUSION: Phosphorylation plays an important role in the regulation of PTEN enzymatic activity and protein stability. GENERAL SIGNIFICANCE: Activation of PTEN during torpor can regulate insulin signaling during periods of low energy state.

Regulation of the PI3K/AKT Pathway and Fuel Utilization During Primate Torpor in the Gray Mouse Lemur, Microcebus murinus.

Gray mouse lemurs (Microcebus murinus) from Madagascar present an excellent model for studies of torpor regulation in a primate species. In the present study, we analyzed the response of the insulin signaling pathway as well as controls on carbohydrate sparing in six different tissues of torpid versus aroused gray mouse lemurs. We found that the relative level of phospho-insulin receptor substrate (IRS-1) was significantly increased in muscle, whereas the level of phospho-insulin receptor (IR) was decreased in white adipose tissue (WAT) of torpid animals, both suggesting an inhibition of insulin/insulin-like growth factor-1 (IGF-1) signaling during torpor in these tissues. By contrast, the level of phospho-IR was increased in the liver. Interestingly, muscle, WAT, and liver occupy central roles in whole body homeostasis and each displays regulatory controls operating at the plasma membrane. Changes in other tissues included an increase in phospho-glycogen synthase kinase 3α (GSK3α) and decrease in phospho-ribosomal protein S6 (RPS6) in the heart, and a decrease in phospho-mammalian target of rapamycin (mTOR) in the kidney. Pyruvate dehydrogenase (PDH) that gates carbohydrate entry into mitochondria is inhibited via phosphorylation by pyruvate dehydrogenase kinase (e.g., PDK4). In the skeletal muscle, the protein expression of PDK4 and phosphorylated PDH at Ser 300 was increased, suggesting inhibition during torpor. In contrast, there were no changes in levels of PDH expression and phosphorylation in other tissues comparing torpid and aroused animals. Information gained from these studies highlight the molecular controls that help to regulate metabolic rate depression and balance energetics during primate torpor.

Transcriptional Activation of p53 during Cold Induced Torpor in the 13-Lined Ground Squirrel Ictidomys tridecemlineatus.

The transcription factor p53 is located at the centre of multiple pathways relating the cellular response to stress. Commonly known as a tumor suppressor, it is responsible for initiating diverse actions to protect the integrity of the genome, ranging from cell cycle arrest to apoptosis. This study investigated the regulation of p53 protein in hibernating 13-lined ground squirrel Ictidomys tridecemlineatus during multiple stages of the torpor-arousal cycle. Transcript and protein levels of p53 were both elevated in the skeletal muscle during early and late torpor stages of the hibernation cycle. Nuclear localization of p53 was also increased during late torpor, and this is associated with an increase in its DNA binding activity and expression of p53 transcriptional targets p21CIP, gadd45α, and 14-3-3σ. The increase in p53 transcriptional activity appears to be independent of its phosphorylation at Ser-15, Ser-46, and Ser-392, consistent with an absence of checkpoint kinase activation during torpor. Sequence analysis revealed unique amino acid substitutions in the ground squirrel p53 protein, which may contribute to an increase in protein stability compared to nonhibernators. Overall, the study results provided evidences for a potential role of p53 in the protection of the skeletal muscle during torpor.

Dehydration mediated microRNA response in the African clawed frog Xenopus laevis.

Exposure to various environmental stresses induces metabolic rate depression in many animal species, an adaptation that conserves energy until the environment is again conducive to normal life. The African clawed frog, Xenopus laevis, is periodically subjected to arid summers in South Africa, and utilizes entry into the hypometabolic state of estivation as a mechanism of long term survival. During estivation, frogs must typically deal with substantial dehydration as their ponds dry out and X. laevis can endure >30% loss of its body water. We hypothesize that microRNAs play a vital role in establishing a reversible hypometabolic state and responding to dehydration stress that is associated with amphibian estivation. The present study analyzes the effects of whole body dehydration on microRNA expression in three tissues of X. laevis. Compared to controls, levels of miR-1, miR-125b, and miR-16-1 decreased to 37±6, 64±8, and 80±4% of control levels during dehydration in liver. By contrast, miR-210, miR-34a and miR-21 were significantly elevated by 3.05±0.45, 2.11±0.08, and 1.36±0.05-fold, respectively, in the liver. In kidney tissue, miR-29b, miR-21, and miR-203 were elevated by 1.40±0.09, 1.31±0.05, and 2.17±0.31-fold, respectively, in response to dehydration whereas miR-203 and miR-34a were elevated in ventral skin by 1.35±0.05 and 1.74±0.12-fold, respectively. Bioinformatic analysis of the differentially expressed microRNAs suggests that these are mainly involved in two processes: (1) expression of solute carrier proteins, and (2) regulation of mitogen-activated protein kinase signaling. This study is the first report that shows a tissue specific mode of microRNA expression during amphibian dehydration, providing evidence for microRNAs as crucial regulators of metabolic depression.

Regulation of the mTOR signaling network in hibernating thirteen-lined ground squirrels.

For many small mammals, survival over the winter months is a serious challenge because of low environmental temperatures and limited food availability. The solution for many species, such as thirteen-lined ground squirrels (Ictidomys tridecemlineatus), is hibernation, an altered physiological state characterized by seasonal heterothermy and entry into long periods of torpor that are interspersed with short arousals back to euthermia. During torpor, metabolic rate is strongly reduced to achieve major energy savings, and a coordinated depression of non-essential ATP-expensive functions such as protein synthesis takes place. This study examines the mammalian target of rapamycin (mTOR) signaling pathway, a crucial component of the insulin receptor network, over six stages of the torpor-arousal cycle of hibernation. Immunoblots showed that the phosphorylation state of mTOR(Ser2448) was strongly reduced in skeletal muscle (by 55%) during late torpor but increased by 200% during early arousal compared with euthermia. However, the phosphorylation state of this residue remained relatively constant in cardiac muscle during torpor but was enhanced during entrance into torpor and early arousal from torpor stages (by 2.9- and 3.2-fold, respectively). Phosphorylation states of upstream regulators of mTOR, p-Akt(Thr473) and p-TSC2(Thr1462), were also suppressed in skeletal muscle by 55 and 51%, respectively, during late torpor, as were selected downstream substrates--p-4E-BP1(Thr46) and p-S6(Ser235) contents dropped by 74 and 41%, respectively. Overall, the results indicate suppressed mTOR signaling in skeletal muscle, but not cardiac muscle, during torpor. By contrast, activation of mTOR and other components of the mTORC1 complex (p-PRAS40(Thr246) and GßL) occurred during early arousal in both skeletal and cardiac muscle.

Pattern of cellular quiescence over the hibernation cycle in liver of thirteen-lined ground squirrels.

Mammalian hibernation is a state of seasonal heterothermy that consists of long periods of torpor at low body temperature interspersed with short periods of arousal back to euthermia. Entry into the hypometabolic state of torpor is achieved via a strong coordinated reduction of energy expenditures, particularly ATP-expensive activities, such as transcription and translation. The present study analyzes the status of the cell cycle during hibernation in liver of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) and examines the expression levels of cell cycle regulators over six stages of the torpor-arousal cycle. Immunoblot analysis showed that the protein expression levels of cyclin D1 and cyclin E were reduced in liver (by 31% and 48%, respectively) during long-term torpor; however, during arousal from torpor, PCR analysis showed an upregulation of cyclin D1 transcript levels of about 1.5-fold. Protein expression of cyclin A and cyclin B1 were also elevated (1.57-fold and 2.44-fold, respectively) during early arousal from torpor, and in addition, cyclin A2 transcript levels increased by about 1.8-fold during arousal. Protein levels of two CKIs, p15(INK4b) and p21(CIP1), each increased by about 1.4-fold during torpor, and transcript levels of p15(INK4b) also rose by 1.7-2.1 fold during torpor, whereas p21(CIP1) transcripts increased by 1.5-1.7-fold. A reduction in cyclin D and E protein expression coupled with upregulation of p15(INK4b) and p21(CIP1) inhibitors during torpor reflects markers of cell cycle arrest during hibernation. Elevated expression of cyclin A and B protein expression during arousal stages of torpor suggests cell cycle progression is reversibly arrested during torpor and is reinitiated upon return to euthermia.

The B56gamma3 regulatory subunit of protein phosphatase 2A (PP2A) regulates S phase-specific nuclear accumulation of PP2A and the G1 to S transition.

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a scaffold subunit (A), a catalytic subunit (C), and a variable regulatory subunit (B). The regulatory B subunits determine the substrate specificity and subcellular localization of the PP2A holoenzyme. Here, we demonstrate that the subcellular localization of the B56gamma3 regulatory subunit is regulated in a cell cycle-specific manner. Notably, B56gamma3 becomes enriched in the nucleus at the G(1)/S border and in S phase. The S phase-specific nuclear enrichment of B56gamma3 is accompanied by increases of nuclear A and C subunits and nuclear PP2A activity. Overexpression of B56gamma3 promotes nuclear localization of the A and C subunits, whereas silencing both B56gamma2 and B56gamma3 blocks the S phase-specific increase in the nuclear localization and activity of PP2A. In NIH3T3 cells, B56gamma3 overexpression reduces p27 phosphorylation at Thr-187, concomitantly elevates p27 protein levels, delays the G(1) to S transition, and retards cell proliferation. Consistently, knockdown of endogenous B56gamma3 expression reduces p27 protein levels and increases cell proliferation in HeLa cells. These findings demonstrate that the dynamic nuclear distribution of the B56gamma3 regulatory subunit controls nuclear PP2A activity, which regulates cell cycle controllers, such as p27, to restrain cell cycle progression, and may be responsible for the tumor suppressor function of PP2A.

Self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: effects of positional difference of glutamic acids on side chain ionization constant and intra- and inter-peptide interactions deduced from NMR and gel electrophoresis measurements.

Two glutamic acid-rich fusion peptide analogs of influenza hemagglutinin were synthesized to study the organization of the charged peptides in the membranous media. Fluorescence and gel electrophoresis experiments suggested a loose association between the monomers in the vesicles. A model was built which showed that a positional difference of 3, 7 and 4, 8 results in the exposure of Glu3 and Glu7 side chains to the apolar lipidic core. Supportive results include: first, pK(a) values of two pH units higher than reference value in aqueous medium for Glu3 and Glu7 CgammaH, whereas the deviation of pK(a) from the reference value for Glu4 and Glu8 CgammaH is substantially smaller; second, Hill coefficients of titration shift of these protons indicate anti-cooperativity for Glu3 and Glu7 side chain protons but less so for Glu4 and Glu8, implying a strong electrostatic interaction between Glu3 and Glu7 possibly resulting from their localization in an apolar environment; third, positive and larger titration shift for NH of Glu3 is observed compared to that of Glu4, suggesting stronger hydrogen bond between the NH and the carboxylic group of Glu3 than that of Glu4, consistent with higher degree of exposure to hydrophobic medium for the side chain of Glu3.