Development of Antiplasmodial Peptide-Drug Conjugates Using a Human Protein-Derived Cell-Penetrating Peptide with Selectivity for Infected Cells.

Malaria continues to impose a global health burden. Drug-resistant parasites have emerged to each introduced small-molecule therapy, highlighting the need for novel treatment approaches for the future eradication of malaria. Herein, targeted drug delivery with peptide-drug conjugates (PDCs) was investigated as an alternative antimalarial therapy, inspired by the success of emerging antibody-drug conjugates utilized in cancer treatment. A synthetic peptide derived from an innate human defense molecule was conjugated to the antimalarial drug primaquine (PQ) to produce PDCs with low micromolar potency toward Plasmodium falciparum in vitro. A suite of PDCs with different design features was developed to identify optimal conjugation site and investigate linker length, hydrophilicity, and cleavability. Conjugation within a flexible spacer region of the peptide, with a cleavable linker to liberate the PQ cargo, was important to retain activity of the peptide and drug.

Modified horseshoe crab peptides target and kill bacteria inside host cells.

Bacteria that occupy an intracellular niche can evade extracellular host immune responses and antimicrobial molecules. In addition to classic intracellular pathogens, other bacteria including uropathogenic Escherichia coli (UPEC) can adopt both extracellular and intracellular lifestyles. UPEC intracellular survival and replication complicates treatment, as many therapeutic molecules do not effectively reach all components of the infection cycle. In this study, we explored cell-penetrating antimicrobial peptides from distinct structural classes as alternative molecules for targeting bacteria. We identified two ß-hairpin peptides from the horseshoe crab, tachyplesin I and polyphemusin I, with broad antimicrobial activity toward a panel of pathogenic and non-pathogenic bacteria in planktonic form. Peptide analogs [I11A]tachyplesin I and [I11S]tachyplesin I maintained activity toward bacteria, but were less toxic to mammalian cells than native tachyplesin I. This important increase in therapeutic window allowed treatment with higher concentrations of [I11A]tachyplesin I and [I11S]tachyplesin I, to significantly reduce intramacrophage survival of UPEC in an in vitro infection model. Mechanistic studies using bacterial cells, model membranes and cell membrane extracts, suggest that tachyplesin I and polyphemusin I peptides kill UPEC by selectively binding and disrupting bacterial cell membranes. Moreover, treatment of UPEC with sublethal peptide concentrations increased zinc toxicity and enhanced innate macrophage antimicrobial pathways. In summary, our combined data show that cell-penetrating peptides are attractive alternatives to traditional small molecule antibiotics for treating UPEC infection, and that optimization of native peptide sequences can deliver effective antimicrobials for targeting bacteria in extracellular and intracellular environments.

Characterization of a highly stable zwitterionic hydrophilic interaction chromatography stationary phase based on hybrid organic-inorganic particles.

We have characterized a sulfobetaine stationary phase based on 1.7 µm ethylene-bridged hybrid organic-inorganic particles, which is intended for use in hydrophilic interaction chromatography. The efficiency of a column packed with this material was determined as a function of flow rate, demonstrating a minimum reduced plate height of 2.4. The batch-to-batch reproducibility was assessed using the separation of a mixture of acids, bases, and neutrals. We compared the retention and selectivity of the hybrid sulfobetaine stationary phase to that of several benchmark materials. The hybrid sulfobetaine material gave strong retention for polar neutrals and high selectivity for methyl groups, hydroxy groups, and configurational isomers. Large differences in cation and anion retention were observed among the columns. We characterized the acid and base stability of the hybrid sulfobetaine stationary phase, using accelerated tests at pH 1.3 and 11.0, both at 70°C. The results support a recommended pH range of 2-10. We also investigated the performance of columns packed with this material for metal-sensitive analytes, comparing conventional stainless steel column hardware to hardware that incorporates hybrid surface technology to mitigate interactions with metal surfaces. Compared to the conventional columns, the hybrid surface technology columns showed a greatly improved peak shape.

"Pink-tinted glasses": looking and affirmation in Gentleman Jack.

This article examines how the 2019 period drama Gentleman Jack generates its affirmative project of lesbian televisual representation. GJ turns negative realities of gender and sexuality nonconformity in the nineteenth century to productive, affirmative use through what we view as a highly effective strategy of visually engaging viewers as witnesses to and coconspirators in Lister's journey through Lister's fourth wall breaks and the camera's prioritization of her perspective. We analyze GJ's various overlooked characters and reinscribed power hierarchies, which we argue results from both prioritizing Lister's perspective and fans' tendencies to view Lister through "pink-tinted glasses" as a result of cathexis with Lister. Through close reading of scenes illustrating social censorship, Lister's nonconforming gender performance, the labor behind Lister's wealth, and marriage's promise of happiness, we assert that the series misses subtle opportunities to critique the hierarchies on which Lister's exceptionalism relies. Pulling from Janet Lea's reception analysis, this article further asserts that fans interpret GJ politically as well as for entertainment, and the elements to which they most strongly cathect reflect what fans most desire from LGBTQ + representational politics. Ultimately, we argue that if these continue to be the priorities of LGBTQ + fans and activist organizations today, the characters overlooked in Lister's pursuit of her own happiness offer audiences the opportunity to reflect on who may be sidelined or undercut in contemporary pursuits of LGBTQ + affirmation.

Antimicrobial and Anticancer Properties of Synthetic Peptides Derived from the Wasp Parachartergus fraternus.

Agelaia-MPI and protonectin are antimicrobial peptides isolated from the wasp Parachartergus fraternus that show antimicrobial and neuroactive activities. Previously, two analogues of these peptides, neuroVAL and protonectin-F, were designed to reduce nonspecific toxicity and improve potency. Here, the three-dimensional structures of neuroVAL, protonectin and protonectin-F were determined by using circular dichroism and NMR spectroscopy. Antibacterial, antifungal, cytotoxic and hemolytic activities were tested for the parent peptides and analogues. All peptides showed moderate antimicrobial activity against Gram-positive bacteria, with agelaia-MPI being the most active. Protonectin and protonectin-F were found to be toxic to cancerous and noncancerous cell lines. Internalization experiments revealed that these peptides accumulate inside both cell types. By contrast, neuroVAL was nontoxic to all tested cells and was able to enter cells without accumulating. In summary, neuroVAL has potential as a nontoxic cell-penetrating peptide, while protonectin-F needs further modification to realize its potential as an antitumor peptide.

Characterization of a highly stable mixed-mode reversed-phase/weak anion-exchange stationary phase based on hybrid organic/inorganic particles.

We have characterized Atlantis ethylene-bridged hybrid C18 anion-exchange, a mixed-mode reversed-phase/weak anion-exchange stationary phase designed to give greater retention for anions (e.g., ionized acids) compared to conventional reversed-phase materials. The retention and selectivity of this stationary phase were compared to that of three benchmark materials, using a mixture of six polar compounds that includes an acid, two bases, and three neutrals. The compatibility of the ethylene-bridged hybrid C18 anion-exchange material with 100% aqueous mobile phases was also evaluated. We investigated the batch-to-batch reproducibility of the ethylene-bridged hybrid C18 anion-exchange stationary phase for 27 batches across three different particle sizes (1.7, 2.5, and 5 µm) and found it to be comparable to that of one of the most reproducible C18 stationary phases. We also characterized the acid and base stability of the ethylene-bridged hybrid C18 anion-exchange stationary phase and the results show it to be usable over a wide pH range, from 2 to 10. The extended upper pH limit relative to silica-based reversed-phase/weak anion-exchange materials is enabled by the use of ethylene-bridged hybrid organic/inorganic particles. The improved base stability allows Atlantis ethylene-bridged hybrid C18 anion-exchange to be used with a wider range of mobile phase pH values, opening up a greater range of selectivity options.

Cyclotide Structures Revealed by NMR, with a Little Help from X-ray Crystallography.

This review highlights the predominant role that NMR has had in determining the structures of cyclotides, a fascinating class of macrocyclic peptides found in plants. Cyclotides contain a cystine knot, a compact structural motif that is constrained by three disulfide bonds and able to resist chemical and biological degradation. Their resistance to proteolytic degradation has made cyclotides appealing as drug leads. Herein, we examine the developments that led to the identification and conclusive determination of the disulfide connectivity of cyclotides and describe in detail the structural features of exemplar cyclotides. We also review the role that X-ray crystallography has played in resolving cyclotide structures and describe how racemic crystallography opened up the possibility of obtaining previously inaccessible X-ray structures of cyclotides.

Trogocytosis by Entamoeba histolytica contributes to cell killing and tissue invasion.

Entamoeba histolytica is the causative agent of amoebiasis, a potentially fatal diarrhoeal disease in the developing world. The parasite was named "histolytica" for its ability to destroy host tissues, which is probably driven by direct killing of human cells. The mechanism of human cell killing has been unclear, although the accepted model was that the parasites use secreted toxic effectors to kill cells before ingestion. Here we report the discovery that amoebae kill by ingesting distinct pieces of living human cells, resulting in intracellular calcium elevation and eventual cell death. After cell killing, amoebae detach and cease ingestion. Ingestion of human cell fragments is required for cell killing, and also contributes to invasion of intestinal tissue. The internalization of fragments of living human cells is reminiscent of trogocytosis (from Greek trogo, nibble) observed between immune cells, but amoebic trogocytosis differs because it results in death. The ingestion of live cell material and the rejection of corpses illuminate a stark contrast to the established model of dead cell clearance in multicellular organisms. These findings change the model for tissue destruction in amoebiasis and suggest an ancient origin of trogocytosis as a form of intercellular exchange.

Gating modifier toxins isolated from spider venom: Modulation of voltage-gated sodium channels and the role of lipid membranes.

Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures and modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here, we examined whether there is a relationship among spider GMT amphipathicity, membrane binding, and potency or selectivity for voltage-gated sodium (NaV) channels. We used NMR spectroscopy and in silico calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to NaV channels. Electrophysiology was used to quantify GMT activity on NaV1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of NaV channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for NaVs. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple NaVs.

Characterization of Tachyplesin Peptides and Their Cyclized Analogues to Improve Antimicrobial and Anticancer Properties.

Tachyplesin I, II and III are host defense peptides from horseshoe crab species with antimicrobial and anticancer activities. They have an amphipathic ß-hairpin structure, are highly positively-charged and differ by only one or two amino acid residues. In this study, we compared the structure and activity of the three tachyplesin peptides alongside their backbone cyclized analogues. We assessed the peptide structures using nuclear magnetic resonance (NMR) spectroscopy, then compared the activity against bacteria (both in the planktonic and biofilm forms) and a panel of cancerous cells. The importance of peptide-lipid interactions was examined using surface plasmon resonance and fluorescence spectroscopy methodologies. Our studies showed that tachyplesin peptides and their cyclic analogues were most potent against Gram-negative bacteria and melanoma cell lines, and showed a preference for binding to negatively-charged lipid membranes. Backbone cyclization did not improve potency, but improved peptide stability in human serum and reduced toxicity toward human red blood cells. Peptide-lipid binding affinity, orientation within the membrane, and ability to disrupt lipid bilayers differed between the cyclized peptide and the parent counterpart. We show that tachyplesin peptides and cyclized analogues have similarly potent antimicrobial and anticancer properties, but that backbone cyclization improves their stability and therapeutic potential.

Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes Is a Prerequisite for Its Inhibition of Human Voltage-gated Sodium Channel NaV1.7.

ProTx-II is a disulfide-rich peptide toxin from tarantula venom able to inhibit the human voltage-gated sodium channel 1.7 (hNaV1.7), a channel reported to be involved in nociception, and thus it might have potential as a pain therapeutic. ProTx-II acts by binding to the membrane-embedded voltage sensor domain of hNaV1.7, but the precise peptide channel-binding site and the importance of membrane binding on the inhibitory activity of ProTx-II remain unknown. In this study, we examined the structure and membrane-binding properties of ProTx-II and several analogues using NMR spectroscopy, surface plasmon resonance, fluorescence spectroscopy, and molecular dynamics simulations. Our results show a direct correlation between ProTx-II membrane binding affinity and its potency as an hNaV1.7 channel inhibitor. The data support a model whereby a hydrophobic patch on the ProTx-II surface anchors the molecule at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. This is the first study to demonstrate that binding of ProTx-II to the lipid membrane is directly linked to its potency as an hNaV1.7 channel inhibitor.

Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNaV1.7.

The human voltage-gated sodium channel sub-type 1.7 (hNaV1.7) is emerging as an attractive target for the development of potent and sub-type selective novel analgesics with increased potency and fewer side effects than existing therapeutics. HwTx-IV, a spider derived peptide toxin, inhibits hNaV1.7 with high potency and is therefore of great interest as an analgesic lead. In the current study we examined whether engineering a HwTx-IV analogue with increased ability to bind to lipid membranes would improve its inhibitory potency at hNaV1.7. This hypothesis was explored by comparing HwTx-IV and two analogues [E1PyrE]HwTx-IV (mHwTx-IV) and [E1G,E4G,F6W,Y30W]HwTx-IV (gHwTx-IV) on their membrane-binding affinity and hNaV1.7 inhibitory potency using a range of biophysical techniques including computational analysis, NMR spectroscopy, surface plasmon resonance, and fluorescence spectroscopy. HwTx-IV and mHwTx-IV exhibited weak affinity for lipid membranes, whereas gHwTx-IV showed improved affinity for the model membranes studied. In addition, activity assays using SH-SY5Y neuroblastoma cells expressing hNaV1.7 showed that gHwTx-IV has increased activity at hNaV1.7 compared to HwTx-IV. Based on these results we hypothesize that an increase in the affinity of HwTx-IV for lipid membranes is accompanied by improved inhibitory potency at hNaV1.7 and that increasing the affinity of gating modifier toxins to lipid bilayers is a strategy that may be useful for improving their potency at hNaV1.7.

Development of cell-penetrating peptide-based drug leads to inhibit MDMX:p53 and MDM2:p53 interactions.

The transcription factor p53 has a tumor suppressor role in leading damaged cells to apoptosis. Its activity is regulated/inhibited in healthy cells by the proteins MDM2 and MDMX. Overexpression of MDM2 and/or MDMX in cancer cells inactivates p53, facilitating tumor development. A 12-mer dual inhibitor peptide (pDI) was previously reported to be able to target and inhibit MDMX:p53 and MDM2:p53 interactions with nanomolar potency in vitro. With the aim of improving its cellular inhibitory activity, we produced a series of constrained pDI analogs featuring lactam staples that stabilize the bioactive helical conformation and fused them with a cell-penetrating peptide to increase cytosol delivery. We compared pDI and its analogs on their inhibitory potency, toxicity, and ability to enter cancer cells. Overall, the results show that these analogs keep their nanomolar affinity for MDM2 and MDMX and are highly active against cancer cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 853-863, 2016.

Mutator suppression and escape from replication error-induced extinction in yeast.

Cells rely on a network of conserved pathways to govern DNA replication fidelity. Loss of polymerase proofreading or mismatch repair elevates spontaneous mutation and facilitates cellular adaptation. However, double mutants are inviable, suggesting that extreme mutation rates exceed an error threshold. Here we combine alleles that affect DNA polymerase δ (Pol δ) proofreading and mismatch repair to define the maximal error rate in haploid yeast and to characterize genetic suppressors of mutator phenotypes. We show that populations tolerate mutation rates 1,000-fold above wild-type levels but collapse when the rate exceeds 10⁻³ inactivating mutations per gene per cell division. Variants that escape this error-induced extinction (eex) rapidly emerge from mutator clones. One-third of the escape mutants result from second-site changes in Pol δ that suppress the proofreading-deficient phenotype, while two-thirds are extragenic. The structural locations of the Pol δ changes suggest multiple antimutator mechanisms. Our studies reveal the transient nature of eukaryotic mutators and show that mutator phenotypes are readily suppressed by genetic adaptation. This has implications for the role of mutator phenotypes in cancer.

Preparation and investigation of two-component silica-modified hydrophobic layer for minimizing retention loss of reversed-phase chromatographic columns using fully aqueous mobile phases.

Chromatographers often employ fully aqueous mobile phases to retain highly polar compounds in reversed-phase liquid chromatography (RPLC). However, when the flow rate is interrupted, either accidentally or intentionally, a substantial loss in retention occurs due to the spontaneous dewetting of water from the hydrophobic surface of conventional RPLC-C18 particles. Previous studies have shown that maintaining a low C18 surface coverage (approximately 1.5 µmol/m2) can mitigate water dewetting by increasing chain disorder, facilitating the intercalation of water clusters between the C18-bonded chains, and keeping the mesopores wetted. In this research, we explore the potential and additional benefits of using two-component surface bonding materials (C8/C18 and PhenylHexyl (PhHx)/C18) at a constant and low total surface coverage of 1.51 ± 0.15 µmol/m2. We synthesized seven one- and two-component modified silica particles with a volume average particle size of 5.22 µm and an average mesopore size of 104 Å. The surface coverage was increased from 0 to 0.54, 1.00, and to 1.66 µmol2 for C8 chains and from 0 to 0.52, 0.70, and to 1.65 µmol2 for PhHx ligands. To prevent interactions between water and any unreacted silanols, all seven derivatized particles were heavily endcapped with trimethylsilane (TMS) reagent. The fraction of the surface area remaining in contact with water was determined by measuring the retention times of weakly (thiourea) and strongly (thymine) retained compounds at intervals of 1, 2, 4, 8, 16, 32, and 64 minutes following the cessation of flow. Two distinct column temperatures, 24°C and 60°C, were employed in the experiments. Retention losses were found to be minimized in the presence of a small quantity of C8 chains (less than 40% of the total surface coverage). Additionally, it is essential to consider substantial fractions of PhHx chains, as long as the presence of the PhHx ligand does not significantly impact retention and selectivity. Combining mixed RPLC bondings with a low total surface coverage of approximately 1.5 µmol/m2 emerges as a viable solution for further minimizing retention loss in standard C18-bonded RPLC columns, particularly within the surface coverage range of 2.5-3.0 µmol/m2.

Quitting intentions and behaviours among young Australian e-cigarette users.

BACKGROUND AND AIMS: With the prevalence of e-cigarette use among Australian youth increasing significantly in recent years, greater attention is being paid to encouraging and supporting cessation. However, research to inform such efforts is lacking. The present study sought to (i) measure desire to quit e-cigarette use and actual quitting attempts among young Australians and (ii) explore correlates of quitting-related cognitions and behaviours. DESIGN, SETTING AND PARTICIPANTS: This was a cross-sectional on-line survey conducted in Australia. The participants were 14-25-year-old e-cigarette users (n = 602; 53% women). MEASUREMENTS: Desire to quit vaping and attempts to quit vaping were the primary dependent variables. The independent variables included several individual (e.g. harm perceptions, perceived appeal of vapes), social (descriptive norms) and environmental (e.g. ease of e-cigarette access) factors. FINDINGS: A majority of respondents (61%) expressed a desire to quit vaping, and just over half (55%) had made a quit attempt. Finding vapes easy to access was associated with both a lack of desire [odds ratio (OR) = 0.71] and attempts to quit (OR = 0.77), while self-reported addiction to vaping (OR = 1.42 and OR = 3.11) and perceiving vaping to be associated with mental health risks (OR = 1.30 and OR = 1.40) were positively correlated with these variables. Perceiving that vaping is common among people of one's age (OR = 0.82) and finding vapes appealing (OR = 0.55) were associated with a lack of desire to quit, while perceiving vaping to have physical health risks was positively associated with quitting desire (OR = 1.58). School-based education on vaping was associated with reporting an attempt/s to quit (OR = 0.47). CONCLUSIONS: This survey of young Australian e-cigarette users suggests a high level of desire to quit using e-cigarettes and attempts to quit. Increasing knowledge regarding the physical and mental health risks associated with e-cigarette use may assist with promoting quitting-related intentions. Changing social norms, reducing the accessibility of e-cigarettes and reducing the appeal of the products also constitute potential means of increasing the desire to quit.

Repurposing a plant peptide cyclase for targeted lysine acylation.

Transpeptidases are powerful tools for protein engineering but are largely restricted to acting at protein backbone termini. Alternative enzymatic approaches for internal protein labelling require bulky recognition motifs or non-proteinogenic reaction partners, potentially restricting which proteins can be modified or the types of modification that can be installed. Here we report a strategy for labelling lysine side chain ε-amines by repurposing an engineered asparaginyl ligase, which naturally catalyses peptide head-to-tail cyclization, for versatile isopeptide ligations that are compatible with peptidic substrates. We find that internal lysines with an adjacent leucine residue mimic the conventional N-terminal glycine-leucine substrate. This dipeptide motif enables efficient intra- or intermolecular ligation through internal lysine side chains, minimally leaving an asparagine C-terminally linked to the lysine side chain via an isopeptide bond. The versatility of this approach is demonstrated by the chemoenzymatic synthesis of peptides with non-native C terminus-to-side chain topology and the conjugation of chemically modified peptides to recombinant proteins.

Deciphering the structure and mechanism of action of computer-designed mastoparan peptides.

Mastoparans are cationic peptides with multifunctional pharmacological properties. Mastoparan-R1 and mastoparan-R4 were computationally designed based on native mastoparan-L from wasps and have improved therapeutic potential for the control of bacterial infections. Here, we evaluated whether these peptides maintain their activity against Escherichia coli strains under a range of salt concentrations. We found that mastoparan-R1 and mastoparan-R4 preserved their activity under the conditions tested, including having antibacterial activities at physiological salt concentrations. The overall structure of the peptides was investigated using circular dichroism spectroscopy in a range of solvents. No significant changes in secondary structure were observed (random coil in aqueous solutions and α-helix in hydrophobic and anionic environments). The three-dimensional structures of mastoparan-R1 and mastoparan-R4 were elucidated through nuclear magnetic resonance spectroscopy, revealing amphipathic α-helical segments for Leu3-Ile13 (mastoparan-R1) and Leu3-Ile14 (mastoparan-R4). Possible membrane-association mechanisms for mastoparan-R1 and mastoparan-R4 were investigated through surface plasmon resonance and leakage studies with synthetic POPC and POPC/POPG (4:1) lipid bilayers. Mastoparan-L had the highest affinity for both membrane systems, whereas the two analogs had weaker association, but improved selectivity for lysing anionic membranes. This finding was also supported by molecular dynamics simulations, in which mastoparan-R1 and mastoparan-R4 were found to have greater interactions with bacteria-like membranes compared with model mammalian membranes. Despite having a few differences in their functional and structural profiles, the mastoparan-R1 analog stood out with the highest activity, greater bacteriostatic potential, and selectivity for lysing anionic membranes. This study reinforces the potential of mastoparan-R1 as a drug candidate.

Effect of the leptin receptor Q223R polymorphism on the host transcriptome following infection with Entamoeba histolytica.

Resistance to amebiasis is associated with a polymorphism in the leptin receptor. Previous studies demonstrated that humans with the ancestral Q223 leptin receptor allele were nearly four times less likely to be infected with Entamoeba histolytica than those carrying the mutant R223 allele. We hypothesized that the Q223 allele protected against E. histolytica via STAT3-mediated transcription of genes required for mucosal immunity. To test this, mice containing the humanized LEPR Q or R allele at codon 223 were intracecally infected with E. histolytica. Susceptibility to amebiasis was assessed, and cecal tissues were analyzed for changes in gene expression. By 72 h postchallenge, all Q223 mice had cleared E. histolytica, whereas 39% of 223R mice were infected. Thirty-seven genes were differentially expressed in response to infection at 72 h, including proinflammatory genes (CXCL2, S100A8/9, PLA2G7, ITBG2, and MMP9) and functions pertaining to the movement and activity of immune cells. A comparison at 12 h postchallenge of infected Q223 versus R223 mice identified a subset of differentially expressed genes, many of which were closely linked to leptin signaling. Further analyses indicated that the Q223 gene expression pattern was consistent with a suppressed apoptotic response to infection, while 223R showed increased cellular proliferation and recruitment. These studies are the first to illuminate the downstream effects of leptin receptor polymorphisms on intestinal infection by E. histolytica. As such, they are important for the insight that they provide into this previously uncharacterized mechanism of mucosal immunity.

The circular bacteriocin enterocin NKR-5-3B has an improved stability profile over nisin.

Bacteriocins are a large family of bacterial peptides that have antimicrobial activity and potential applications as clinical antibiotics or food preservatives. Circular bacteriocins are a unique class of these biomolecules distinguished by a seamless circular topology, and are widely assumed to be ultra-stable based on this constraining structural feature. However, without quantitative studies of their susceptibility to defined thermal, chemical, and enzymatic conditions, their stability characteristics remain poorly understood, limiting their translational development. Here, we produced the circular bacteriocin enterocin NKR-5-3B (Ent53B) in mg/L quantities using a heterologous Lactococcus expression system, and characterized its thermal stability by NMR, chemical stability by circular dichroism and analytical HPLC, and enzymatic stability by analytical HPLC. We demonstrate that Ent53B is ultra-stable, resistant to temperatures approaching boiling, acidic (pH 2.6) and alkaline (pH 9.0) conditions, the chaotropic agent 6 M urea, and following incubation with a range of proteases (i.e., trypsin, chymotrypsin, pepsin, and papain), conditions under which most peptides and proteins degrade. Ent53B is stable across a broader range of pH conditions and proteases than nisin, the most widely used bacteriocin in food manufacturing. Antimicrobial assays showed that differences in stability correlated with differences in bactericidal activity. Overall, this study provides quantitative support for circular bacteriocins being an ultra-stable class of peptide molecules, suggesting easier handling and distribution options available to them in practical applications as antimicrobial agents.