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SUMMARY Multiple salmonellosis outbreaks have been linked to contaminated tomatoes. We investigated a multistate outbreak of Salmonella Typhimurium infections among 190 cases. For hypothesis generation, review of patients' food histories from four restaurant-associated clusters in four states revealed that large tomatoes were the only common food consumed by patients. Two case-control studies were conducted to identify food exposures associated with infections. In a study conducted in nine states illness was significantly associated with eating raw, large, round tomatoes in a restaurant [matched odds ratio (mOR) 3·1, 95% confidence interval (CI) 1·3-7·3]. In a Minnesota study, illness was associated with tomatoes eaten at a restaurant (OR 6·3, mid-P 95% CI 1·05-50·4, P=0·046). State, local and federal regulatory officials traced the source of tomatoes to Ohio tomato fields, a growing area not previously identified in past tomato-associated outbreaks. Because tomatoes are commonly eaten raw, prevention of tomato contamination should include interventions on the farm, during packing, and at restaurants. [ABSTRACT FROM AUTHOR]

Salmonella Newport causes more than an estimated 100,000 infections annually in the United States. In 2002, tomatoes grown and packed on the eastern shore of Virginia contaminated with a pan-susceptible S. Newport strain caused illness in 510 patients in 26 states. In July-November 2005, the same strain caused illness in at least 72 patients in 16 states. We conducted a case-control study during the 2005 outbreak, enrolling 29 cases and 140 matched neighbourhood controls. Infection was associated with eating tomatoes (matched odds ratio 9.7, 95% confidence interval 3.3-34.9). Tomatoes were traced back to the eastern shore of Virginia, where the outbreak strain was isolated from pond water used to irrigate tomato fields. Two multistate outbreaks caused by one rare strain, and identification of that strain in irrigation ponds 2 years apart, suggest persistent contamination of tomato fields. Further efforts are needed to prevent produce contamination on farms and throughout the food supply chain. [ABSTRACT FROM AUTHOR]

C36H28Co1N6O4, triclinic, P1̄ (no. 2), a = 6.3166(4) Å, b = 8.5454(5) Å, c = 14.8075(9) Å, α = 105.157(2)°, β = 94.494(3)°, γ = 101.890(2)°, V = 747.5(2) Å3, Z = 1, Rgt(F) = 0.0332, wRref(F2) = 0.0790, T = 100(2) K.

C37H32O6, monoclinic, P21/c (No. 14), a = 5.5354(7) Å, b = 16.983(2) Å, c = 31.211(4) Å, β = 91.535(8)°, V = 2932.9(7) Å3, Z = 4, Rgt(F) = 0.0584, wRref(F2) = 0.1738, T = 100 K.

C57H51CuOP4, triclinic, P1̄ (no. 2), a = 11.2814(8) Å, b = 13.585(1) Å, c = 15.666(1) Å, α = 88.785(4)°, β = 84.288(3)°, γ = 85.103(3)°, V = 2380.1 Å3, Z = 2, Rgt(F) = 0.0444, wRref(F2) = 0.1159, T = 100 K.

C22H19BrO4, triclinic, P1̅ (no. 2), a = 8.550(2) Å, b = 11.343(2) Å, c = 11.715(2) Å, ɑ = 102.02(3)°, β = 110.47(3)°, γ = 110.84(3)°, V = 920.2 Å3, Z = 2, Rgt(F) = 0.0368, wRref(F2) = 0.099, T = 100 K.

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Drug resistance represents a major problem in cancer treatment. Doxorubicin (adriamycin) is an injectable DNA intercalating drug that halts cancer cell growth by inhibiting topoisomerase 2, but its long-term effectiveness is compromised by onset of resistance. This study demonstrates that expression of the PAR2 gene in human colon adenocarcinoma tissue samples was the highest among 32 different cancer types (n = 10,989), and higher in colon adenocarcinoma tissues (n = 331) than normal colon tissues (n = 308), revealing an association between PAR2 expression and human colon cancer. HT29 cells are a human colorectal adenocarcinoma cell line that is sensitive to the chemotherapeutic drug doxorubicin and also expresses PAR2. We find that PAR2 activation in HT29 cells, either by an endogenous protease agonist (trypsin) or an exogenous peptide agonist (2f-LIGRL-NH 2 ), significantly reduces doxorubicin-induced cell death, reactive oxygen species production, caspase 3/7 activity and cleavage of caspase-8 and caspase-3. Moreover, PAR2-mediated MEK1/2-ERK1/2 pathway induced by 2f-LIGRL-NH 2 leads to upregulated anti-apoptotic MCL-1 and Bcl-xL proteins that promote cellular survival. These findings suggest that activation of PAR2 compromises efficacy of doxorubicin in colon cancer. Further support for this conclusion came from experiments with human colon cancer HT29 cells, either with the PAR2 gene deleted or in the presence of a pharmacological antagonist of PAR2, which showed full restoration of all doxorubicin-mediated effects. Together, these findings reveal a strong link between PAR2 activation and signalling in human colon cancer cells and increased survival against doxorubicin-induced cell death. They support PAR2 antagonism as a possible new strategy for enhancing doxorubicin therapy., (© 2023. The International CCN Society.)

Shorter and more effective treatment regimens as well as new drugs are urgent priorities for reducing the immense global burden of tuberculosis (TB). As treatment of TB currently requires multiple antibiotics with diverse mechanisms of action, any new drug lead requires assessment of potential interactions with existing TB antibiotics. We previously described the discovery of wollamides, a new class of Streptomyces -derived cyclic hexapeptides with antimycobacterial activity. To further assess the value of the wollamide pharmacophore as an antimycobacterial lead, we determined wollamide interactions with first- and second-line TB antibiotics by determining fractional inhibitory combination index and zero interaction potency scores. In vitro two-way and multiway interaction analyses revealed that wollamide B1 synergizes with ethambutol, pretomanid, delamanid, and para-aminosalicylic acid in inhibiting the replication and promoting the killing of phylogenetically diverse clinical and reference strains of the Mycobacterium tuberculosis complex (MTBC). Wollamide B1 antimycobacterial activity was not compromised in multi- and extensively drug-resistant MTBC strains. Moreover, growth-inhibitory antimycobacterial activity of the combination of bedaquiline/pretomanid/linezolid was further enhanced by wollamide B1, and wollamide B1 did not compromise the antimycobacterial activity of the isoniazid/rifampicin/ethambutol combination. Collectively, these findings add new dimensions to the desirable characteristics of the wollamide pharmacophore as an antimycobacterial lead compound. IMPORTANCE Tuberculosis (TB) is an infectious disease that affects millions of people globally, with 1.6 million deaths annually. TB treatment requires combinations of multiple different antibiotics for many months, and toxic side effects can occur. Therefore, shorter, safer, more effective TB therapies are required, and these should ideally also be effective against drug-resistant strains of the bacteria that cause TB. This study shows that wollamide B1, a chemically optimized member of a new class of antibacterial compounds, inhibits the growth of drug-sensitive as well as multidrug-resistant Mycobacterium tuberculosis isolated from TB patients. In combination with TB antibiotics, wollamide B1 synergistically enhances the activity of several antibiotics, including complex drug combinations that are currently used for TB treatment. These new insights expand the catalogue of the desirable characteristics of wollamide B1 as an antimycobacterial lead compound that might inspire the development of improved TB treatments., Competing Interests: The authors declare no conflict of interest.

Complement activation and Rab GTPase trafficking are commonly observed in inflammatory responses. Recruitment of innate immune cells to sites of infection or injury and secretion of inflammatory chemokines are promoted by complement component 5a (C5a) that activates the cell surface protein C5a receptor1 (C5aR1). Persistent activation can lead to a myriad of inflammatory and autoimmune diseases. Here, we demonstrate that the mechanism of C5a induced chemotaxis of human monocyte-derived macrophages (HMDMs) and their secretion of inflammatory chemokines are controlled by Rab5a. We find that C5a activation of the G protein coupled receptor C5aR1 expressed on the surface of HMDMs, recruits β-arrestin2 via Rab5a trafficking, then activates downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling that culminates in chemotaxis and secretion of pro-inflammatory chemokines from HMDMs. High-resolution lattice light-sheet microscopy on live cells showed that C5a activates C5aR1-GFP internalization and colocalization with Rab5a-tdTomato but not with dominant negative mutant Rab5a-S34N-tdTomato in HEK293 cells. We found that Rab5a is significantly upregulated in differentiated HMDMs and internalization of C5aR1 is dependent on Rab5a. Interestingly, while knockdown of Rab5a inhibited C5aR1-mediated Akt phosphorylation, it did not affect C5aR1-mediated ERK1/2 phosphorylation or intracellular calcium mobilization in HMDMs. Functional analysis using transwell migration and µ-slide chemotaxis assays indicated that Rab5a regulates C5a-induced chemotaxis of HMDMs. Further, C5aR1 was found to mediate interaction of Rab5a with β-arrestin2 but not with G proteins in HMDMs. Furthermore, C5a-induced secretion of pro-inflammatory chemokines (CCL2, CCL3) from HMDMs was attenuated by Rab5a or β-arrestin2 knockdown or by pharmacological inhibition with a C5aR1 antagonist or a PI3K inhibitor. These findings reveal a C5a-C5aR1-β-arrestin2-Rab5a-PI3K signaling pathway that regulates chemotaxis and pro-inflammatory chemokine secretion in HMDMs and suggests new ways of selectively modulating C5a-induced inflammatory outputs., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

An alpha helical turn can be reproduced in a cyclic pentapeptide if the first and fifth amino acid sidechains are correctly joined. Here structural studies (CD, NMR, in silico ) reveal why N -methylation at positions not involved in hydrogen bonds disrupts helicity whereas ester bonds can maintain helicity and promote greater cell uptake.

Glucagon-like peptide-1 (GLP-1) lowers blood glucose by inducing insulin but also has other poorly understood properties. Here, we show that hydroxy amino acids (Thr11, Ser14, Ser17, Ser18) in GLP-1(7-36) act in concert to direct cell signaling. Mutating any single residue to alanine removes one hydroxyl group, thereby reducing receptor affinity and cAMP 10-fold, with Ala11 or Ala14 also reducing β-arrestin-2 10-fold, while Ala17 or Ala18 also increases ERK1/2 phosphorylation 5-fold. Multiple alanine mutations more profoundly bias signaling, differentially silencing or restoring one or more signaling properties. Mutating three serines silences only ERK1/2, the first example of such bias. Mutating all four residues silences β-arrestin-2, ERK1/2, and Ca 2+ maintains the ligand and receptor at the membrane but still potently stimulates cAMP and insulin secretion in cells and mice. These novel findings indicate that hydrogen bonding cooperatively controls cell signaling and highlight an important regulatory hydroxyl patch in hormones that activate class B G protein-coupled receptors.

Objective: If non-invasive markers of liver fibrosis were recorded frequently enough in clinical practice, it might be feasible to use them for opportunistic community screening for liver disease. We aimed to determine their current pattern of usage in the national primary care population in Wales., Design: Using the Secure Anonymised Information Linkage (SAIL) Databank at Swansea University (2000-2017), we quantified the frequency of common liver blood tests (aspartate aminotransferase (AST), alanine aminotransferase (ALT), platelet count and albumin) used in fibrosis marker algorithms. We examined measurement variation by age and sex., Results: During the 18-year study period, there were 2 145 178 adult patients with at least one blood test available for analysis. Over the study period, the percentage of SAIL patients receiving an ALT test in each year increased from 2% to 33%, with platelet count and albumin measurement increasing by a similar factor. AST testing, although initially rising, had decreased to 1% by the end of the study. AST and ALT values varied by age and sex, particularly in males with the upper normal range of ALT values decreasing rapidly from 90 U/L at age 30 to 45 U/L by age 80., Conclusion: The reduction in AST testing to only 1% of the adult population limits the use of many non-invasive liver marker algorithms. To enable widespread screening, alternative algorithms for liver fibrosis that do not depend on AST should be developed. Liver fibrosis markers should be modified to include age-specific and sex-specific normal ranges., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

The retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signaling. Mutation of the retromer subunit Vps35 causes late-onset Parkinson’s disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function, we have created a novel series of macrocyclic peptides that bind retromer with high affinity and specificity. Crystal structures show that most of the cyclic peptides bind to Vps29 via a Pro-Leu–containing sequence, structurally mimicking known interactors such as TBC1D5 and blocking their interaction with retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting retromer. Last, tagged peptides can be used to probe the cellular localization of retromer and its functional interactions in cells, providing novel tools for studying retromer function.

N- or C-methylation in natural and synthetic cyclic peptides can increase membrane permeability, but it remains unclear why this happens in some cases but not others. Here we compare three-dimensional structures for cyclic peptides from six families, including isomers differing only in the location of an N- or Cα-methyl substituent. We show that a single methyl group only increases membrane permeability when it connects or expands hydrophobic surface patches. Positional isomers, with the same molecular weight, hydrogen bond donors/acceptors, rotatable bonds, calculated LogP, topological polar surface area, and total hydrophobic surface area, can have different membrane permeabilities that correlate with the size of the largest continuous hydrophobic surface patch. These results illuminate a key local molecular determinant of membrane permeability., (© 2020 Wiley-VCH GmbH.)

In its 33 years, ADDR has published regularly on the po5tential of oral delivery of biologics especially peptides and proteins. In the intervening period, analysis of the preclinical and clinical trial failures of many purported platform technologies has led to reflection on the true status of the field and reigning in of expectations. Oral formulations of semaglutide, octreotide, and salmon calcitonin have completed Phase III trials, with oral semaglutide being approved by the FDA in 2019. The progress made with oral peptide formulations based on traditional permeation enhancers is against a background of low and variable oral bioavailability values of ~1%, leading to a current perception that only potent peptides with a viable cost of synthesis can be realistically considered. Desirable features of candidates should include a large therapeutic index, some stability in the GI tract, a long elimination half-life, and a relatively low clearance rate. Administration in nanoparticle formats have largely disappointed, with few prototypes reaching clinical trials: insufficient particle loading, lack of controlled release, low epithelial particle uptake, and lack of scalable synthesis being the main reasons for discontinuation. Disruptive technologies based on engineered devices promise improvements, but scale-up and toxicology aspects are issues to address. In parallel, medicinal chemists are synthesizing stable hydrophobic macrocyclic candidate peptides of lower molecular weight and with potential for greater oral bioavailability than linear peptides, but perhaps without the same requirement for elaborate drug delivery systems. In summary, while there have been advances in understanding the limitations of peptides for oral delivery, low membrane permeability, metabolism, and high clearance rates continue to hamper progress., Competing Interests: Declaration of competing interest DB and DF act as consultants to Pharma researching oral peptides. Past collaborative research in DB's lab has been funded by Sanofi and Novo-Nordisk. RJM was the scientific founder of Trinity Biosystems, examining CD91-mediated oral delivery and he has also provided scientific guidance to Syntoxin Ltd. on the development of technology to target FcRn receptors., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The introduction of an amide bond linking side chains of the first and fifth amino acids forms a cyclic pentapeptide that optimally stabilizes the smallest known α-helix in water. The origin of the stabilization is unclear. The observed dependence of α-helicity on the solvent and cyclization linker led us to discover a novel long-range n to π* interaction between a main-chain amide oxygen and a uniquely positioned carbonyl group in the linker of cyclic pentapeptides. CD and NMR spectra, NMR and X-ray structures, modelling, and MD simulations reveal that this first example of a synthetically incorporated long-range n to π* CO⋅⋅⋅C γ =Ο interaction uniquely enforces an almost perfect and remarkably stable peptide α-helix in water but not in DMSO. This unusual interaction with a covalent amide bond outside the helical backbone suggests new approaches to synthetically stabilize peptide structures in water., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rule-of-five parameters and membrane permeabilities have been routinely used to guide development of orally bioavailabile drugs. Here we compare enantiomeric pairs of cyclic hexapeptides with identical rule-of-five parameters and membrane permeabilities. For each enantiomeric pair, the isomer with more l- than d-amino acids is much more orally bioavailable in rats, more metabolically stable to rat liver microsomes, and cleared more slowly in vivo.

Protein-protein interactions involve hotspots as small as 4 sequential amino acids. Corresponding tetrapeptides have no structure in water. Here we report linking side chains of amino acids X and Z to form 24 cyclic tetrapeptides, cyclo-[XAAZ]-NH 2 , and stabilise 14-18 membered rings that mimic different kinds of non-regular secondary structures found in protein hotspots. 2D NMR spectra allowed determination of 3D structures for 14 cyclic tetrapeptides in water. Five formed two ( i , i + 3) hydrogen bonds and a beta/gamma ( 6 , 7 ) or beta ( 9 , 19 , 20 ) turn; eight formed one ( i , i + 4) hydrogen bond and twisted into a non-helical ( 13 , 18 , 21 , 22 , 24 ) or helical ( 5 , 17 , 23 ) alpha turn; one was less structured ( 15 ). A beta or gamma turn was favoured for Z = Dab, Orn or Glu due to a χ 1 gauche (+) rotamer, while an alpha turn was favoured for Z = Dap (but not X = Dap) due to a gauche (-) rotamer. Surprisingly, an unstructured peptide ARLARLARL could be twisted into a helix when either a helical or non-helical alpha turn ( 5 , 13 , 17 , 18 , 21-24 ) with Z = Dap was attached to the N-terminus. These structural models provide insights into stability for different turns and twists corresponding to non-regular folds in protein hotspots., (This journal is © The Royal Society of Chemistry 2019.)

Cyclization is an important post-translational modification of peptides and proteins that confers key advantages such as protection from proteolytic degradation, altered solubility, membrane permeability, bioavailability, and especially restricted conformational freedom in water that allows the peptide backbone to adopt the major secondary structure elements found in proteins. Non-ribosomal synthesis in bacteria, fungi, and plants or synthetic chemistry can introduce unnatural amino acids and non-peptidic constraints that modify peptide backbones and side chains to fine-tune cyclic peptide structure. Structures can be potentially altered further upon binding to a protein in biological environments. Here we analyze three-dimensional crystal structures for 211 bioactive cyclic peptides bound to 65 different proteins. The protein-bound cyclic peptides were examined for similarities and differences in bonding modes, for main-chain and side-chain structure, and for the importance of polarity, hydrogen bonds, hydrophobic effects, and water molecules in interactions with proteins. Many protein-bound cyclic peptides show backbone structures like those (strands, sheets, turns, helices, loops, or distorted variations) found at protein-protein binding interfaces. However, the notion of macrocycles simply as privileged scaffolds that primarily project side-chain substituents for complementary interactions with proteins is dispelled here. Unlike small-molecule drugs, the cyclic peptides do not rely mainly upon hydrophobic and van der Waals interactions for protein binding; they also use their main chain and side chains to form polar contacts and hydrogen bonds with proteins. Compared to small-molecule ligands, cyclic peptides can bind across larger, polar, and water-exposed protein surface areas, making many more contacts that can increase affinity, selectivity, biological activity, and ligand-receptor residence time. Cyclic peptides have a greater capacity than small-molecule drugs to modulate protein-protein interfaces that involve large, shallow, dynamic, polar, and water-exposed protein surfaces.

The Kuiper Belt is a distant region of the outer Solar System. On 1 January 2019, the New Horizons spacecraft flew close to (486958) 2014 MU 69 , a cold classical Kuiper Belt object approximately 30 kilometers in diameter. Such objects have never been substantially heated by the Sun and are therefore well preserved since their formation. We describe initial results from these encounter observations. MU 69 is a bilobed contact binary with a flattened shape, discrete geological units, and noticeable albedo heterogeneity. However, there is little surface color or compositional heterogeneity. No evidence for satellites, rings or other dust structures, a gas coma, or solar wind interactions was detected. MU 69 's origin appears consistent with pebble cloud collapse followed by a low-velocity merger of its two lobes., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Wollamides are cyclic hexapeptides, recently isolated from an Australian soil Streptomyces isolate, that exhibit promising in vitro antimycobacterial activity against Mycobacterium bovis Bacille Calmette Guérin without displaying cytotoxicity against a panel of mammalian cells. Here, we report the synthesis and antimycobacterial activity of 36 new synthetic wollamides, collated with all known synthetic and natural wollamides, to reveal structure characteristics responsible for in vitro growth-inhibitory activity against Mycobacterium tuberculosis (H37Rv, H37Ra, CDC1551, HN878, and HN353). The most potent antimycobacterial wollamides were those where residue VI d-Orn (wollamide B) was replaced by d-Arg (wollamide B1) or d-Lys (wollamide B2), with all activity being lost when residue VI was replaced by Gly, l-Arg, or l-Lys (wollamide B3). Substitution of other amino acid residues mainly reduced or ablated antimycobacterial activity. Significantly, whereas wollamide B2 was the most potent in restricting M. tuberculosis in vitro , wollamide B1 restricted M. tuberculosis intracellular burden in infected macrophages. Wollamide B1 synergized with pretomanid (PA-824) in inhibiting M. tuberculosis in vitro growth but did not antagonize prominent first- and second-line tuberculosis antibiotics. Furthermore, wollamide B1 exerted bactericidal activity against nonreplicating M. tuberculosis and impaired growth of multidrug- and extensively drug-resistant clinical isolates. In vivo pharmacokinetic profiles for wollamide B1 in rats and mice encourage further optimization of the wollamide pharmacophore for in vivo bioavailability. Collectively, these observations highlight the potential of the wollamide antimycobacterial pharmacophore., (Copyright © 2019 American Society for Microbiology.)

The integration of genomics and phylogenetics allows new insight into the structure of gene tree discordance, the relationships among gene position, gene history, and rate of evolution, as well as the correspondence of gene function, positive selection, and gene ontology enrichment across lineages. We explore these issues using the tribe Capsiceae (Solanaceae), which is comprised of the genera Lycianthes and Capsicum (peppers). In combining the annotated genomes of Capsicum with newly sequenced transcriptomes of four species of Lycianthes and Capsicum, we develop phylogenies for 6747 genes, and construct a backbone species tree using both concordance and explicit phylogenetic network approaches. We quantify phylogenetic discordance among individual gene trees, measure their rates of synonymous and nonsynonymous substitution, and test whether they were positively selected along any branch of the phylogeny. We then map these genes onto the annotated Capsicum genome and test whether rates of evolution, gene history, and gene ontology vary significantly with gene position. We observed substantial discordance among gene trees. A bifurcating species tree placing Capsicum within a paraphyletic Lycianthes was supported over all phylogenetic networks. Rates of synonymous and nonsynonymous substitution varied 41-fold and 130-fold among genes, respectively, and were significantly lower in pericentromeric regions. We found that results of concordance tree analyses vary depending on the subset of genes used, and that genes within the pericentromeric regions only capture a portion of the observed discordance. We identified 787 genes that have been positively selected throughout the diversification history of Capsiceae, and discuss the importance of these genes as targets for investigation of economically important traits in the domesticated peppers., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Most protein-protein interactions occur inside cells. Peptides can inhibit protein-protein interactions but tend not to enter cells. We systematically compare cell permeability for 8-12 residue model peptides with helix-inducing lactam/hydrocarbon linkers between amino acid sidechains. Cell uptake increases when hydrophobic residues and lactam linkers (i, i + 4) form a contiguous hydrophobic surface patch. Uptake increases further when both hydrophobic and positively charged (but not neutral or negative) residues are clustered into like surface patches. Amphipathicity alone is however insufficient for cell uptake of acyclic sequences. Changing the linker from lactam to hydrocarbon further increases uptake, but also promotes cell lysis. Helicity, positive charge and amphipathicity together promote cell permeability. Most known bioactive helical peptides do not optimally cluster residues for amphipathicity and so are likely unoptimised for cell uptake.

Linked-Read sequencing technology has recently been employed successfully for de novo assembly of human genomes, however, the utility of this technology for complex plant genomes is unproven. We evaluated the technology for this purpose by sequencing the 3.5-gigabase (Gb) diploid pepper ( Capsicum annuum ) genome with a single Linked-Read library. Plant genomes, including pepper, are characterized by long, highly similar repetitive sequences. Accordingly, significant effort is used to ensure that the sequenced plant is highly homozygous and the resulting assembly is a haploid consensus. With a phased assembly approach, we targeted a heterozygous F 1 derived from a wide cross to assess the ability to derive both haplotypes and characterize a pungency gene with a large insertion/deletion. The Supernova software generated a highly ordered, more contiguous sequence assembly than all currently available C. annuum reference genomes. Over 83% of the final assembly was anchored and oriented using four publicly available  de novo linkage maps. A comparison of the annotation of conserved eukaryotic genes indicated the completeness of assembly. The validity of the phased assembly is further demonstrated with the complete recovery of both 2.5-Kb insertion/deletion haplotypes of the PUN1 locus in the F 1 sample that represents pungent and nonpungent peppers, as well as nearly full recovery of the BUSCO2 gene set within each of the two haplotypes. The most contiguous pepper genome assembly to date has been generated which demonstrates that Linked-Read library technology provides a tool to de novo assemble complex highly repetitive heterozygous plant genomes. This technology can provide an opportunity to cost-effectively develop high-quality genome assemblies for other complex plants and compare structural and gene differences through accurate haplotype reconstruction., Competing Interests: D.J., S.W., N.W., V.K., P.S., and D.M.C. have competing commercial interests as employees and stockholders of 10x Genomics, which is a commercial company that provides the Linked-Read technology and analysis software. This does not alter the authors’ adherence to all of the Genome Biology policies on sharing data and materials. The remaining authors declare that they have no competing interests.

A plasmonic cavity is shown to greatly reduce the inhomogeneity of dynamic optical properties such as quantum efficiency and radiative lifetime of InGaN quantum dots. By using an open-top plasmonic cavity structure, which exhibits a large Purcell factor and antenna quantum efficiency, the resulting quantum efficiency distribution for the quantum dots narrows and is no longer limited by the quantum dot inhomogeneity. The standard deviation of the quantum efficiency can be reduced to 2% while maintaining the overall quantum efficiency at 70%, making InGaN quantum dots a viable candidate for high-speed quantum cryptography and random number generation applications.

Incorporating disease resistance into cultivars is a primary focus of modern breeding programs. Resistance to pathogens is often introgressed from landrace or wild individuals with poor fruit quality into commercial-quality cultivars. Sites of multiple disease resistance (MDR) are regions or "hot spots" of the genome with closely linked genes for resistance to different pathogens that could enable rapid incorporation of resistance. An F 2 -derived F 6 recombinant inbred line population from a cross between 'Criollo de Morelos 334' (CMM334) and 'Early Jalapeno' was evaluated in inoculated fruit studies for susceptibility to oomycete and fungal pathogens: Phytophthora capsici, P. nicotianae, Botrytis cinerea, Fusarium oxysporum, F. solani, Sclerotinia sclerotiorum, Alternaria spp., Rhizopus oryzae, R. stolonifer, and Colletotrichum acutatum. All isolates evaluated were virulent on pepper. Significant differences in disease susceptibility were identified among lines for each of the pathogens evaluated. P. capsici was the most virulent pathogen, while R. oryzae and one Sclerotinia isolate were the least virulent. Quantitative trait loci associated with resistance were identified for Alternaria spp. and S. sclerotiorum. Positive correlations in disease incidence were detected between Alternaria spp. and F. oxysporum, F. solani, and C. acutatum, as well as between C. acutatum and Botrytis spp., F. oxysporum, F. solani, and P. capsici. No sites of MDR were identified for pathogens tested; however, positive correlations in disease incidence were detected among pathogens suggesting there may be genetic linkage among resistance genes in CM334 and Early Jalapeno.

Fruits, as an important part of the human diet, have been under strong selection during domestication. In general, continued directed selection has led to varieties having larger fruit with greater shape variation and tremendous increases in fruit mass. Common cultivated peppers ( L.) are found in a wide range of sizes and shapes. Analysis of genetic relatedness and population structure has shown that the large-fruited, nonpungent types have reduced diversity and comprise a highly structured group. To explore this population structure, a statistical method for detecting fixation within subpopulations was applied to a set of 21 pungent and 19 nonpungent lines that represent the pepper breeding germplasm. We have identified 17 blocks within the pepper genome that are conserved among nonpungent large-fruited varieties. To determine if these regions were fixed by selection on fruit size or pungency, quantitative trait loci (QTLs) from seven studies along with capsaicin biosynthesis genes and homologs of organ size regulatory genes were mapped onto the current pepper genome assembly. Of the 17 fixed regions, 14 overlapped with fruit size or shape QTLs. There were seven putative organ size regulators and seven capsaicin biosynthetic genes within these regions. This work defines genomic regions that underly structure within the nonpungent pepper germplasm and QTLs or genes that may have been selected for during the development of large-fruited nonpungent pepper varieties., (Copyright © 2017 Crop Science Society of America.)

The clinical manifestations of scabies infestation vary according to age, making the diagnosis challenging, particularly for primary care providers and dermatologists who do not routinely care for young children. We present seven cases of newborns and infants who developed inflammatory burrows and nodules early in the course, which is not typical of the eruption seen in older children and adults. We review the cutaneous features, differential diagnosis, and treatment recommendations for scabies in different age groups., (© 2017 Wiley Periodicals, Inc.)

The oncogenic transcription factor activator protein-1 (AP-1) is a DNA-binding protein that assembles through dimerization of Fos and Jun protein subunits, their leucine-rich helical sequences entwining into a coiled-coil structure. This study reports on downsizing the proto-oncogene cFos protein (380 residues) to shorter peptides (37-25 residues) modified with helix-inducing constraints to enhance binding to Jun. A crystal structure is reported for a 37-residue Fos-derived peptide (FosW) bound to Jun. This guided iterative downsizing of FosW to shorter peptide sequences that were constrained into stable water-soluble α-helices by connecting amino acid side chains to form cyclic pentapeptide components. Structural integrity in the presence and absence of Jun was assessed by circular dichroism spectroscopy, while the thermodynamics of binding to cFos was measured by isothermal titration calorimetry. A 25-residue constrained peptide, one-third shorter yet 25% more helical than the structurally characterized 37-residue Fos-derived peptide, retained 80% of the binding free energy as a result of preorganization in a Jun-binding helix conformation, with the entropy gain (TΔS = +3.2 kcal/mol) compensating for the enthalpy loss. Attaching a cell-penetrating peptide (TAT 48-57 ) and a nuclear localization signal (SV40) promoted cell uptake, localization to the nucleus, and inhibition of the proliferation of two breast cancer cell lines.

Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer, and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH 2 ) triggered PAR2-mediated calcium release (EC 50 2 μM) but not ERK1/2 phosphorylation (EC 50 > 100 μM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH 2 ) was a more potent calcium-biased agonist (EC 50 40 nM, Ca 2+ ; EC 50 2 μM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH 2 ) was an ERK-biased agonist (EC 50 2 nM, ERK1/2; EC 50 80 nM, Ca 2+ ). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing, and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.

Glucagon-like peptide (GLP-1) is an endogenous hormone that induces insulin secretion from pancreatic islets and modified forms are used to treat diabetes mellitus type 2. Understanding how GLP-1 interacts with its receptor (GLP-1R) can potentially lead to more effective drugs. Modeling and NMR studies of the N-terminus of GLP-1 suggest a β-turn between residues Glu9-Phe12 and a kinked alpha helix between Val16-Gly37. N-terminal turn constraints attenuated binding affinity and activity (compounds 1-8). Lys-Asp (i, i+4) crosslinks in the middle and at the C-terminus increased alpha helicity and cAMP stimulation without much effect on binding affinity or beta-arrestin 2 recruitment (compounds 9-18). Strategic positioning of helix-inducing constraints and amino acid substitutions (Tyr16, Ala22) increased peptide helicity and produced ten-fold higher cAMP potency (compounds 19-28) over GLP-1(7-37)-NH 2 . The most potent cAMP activator (compound 23) was also the most potent inducer of insulin secretion., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The large GTPase dynamin mediates membrane fission during clathrin-mediated endocytosis (CME). The aminopyrimidine compounds were reported to disrupt dynamin localization to the plasma membrane via the PH domain and implicate this mechanism in the inhibition of CME. We have used a computational approach of binding site identification, docking, and interaction energy calculations to design and synthesize a new library of aminopyrimidine analogues targeting site-2 of the pleckstrin homology (PH) domain. The optimized analogues showed low micromolar inhibition against both dynamin I (IC 50 = 10.6 ± 1.3 to 1.6 ± 0.3 μM) and CME (IC 50(CME) = 65.9 ± 7.7 to 3.7 ± 1.1 mM), which makes this series among the more potent inhibitors of dynamin and CME yet reported. In CME and growth inhibition cell-based assays, the data obtained was consistent with dynamin inhibition. CEREP ExpresS profiling identified off-target effects at the cholecystokinin, dopamine D 2 , histamine H 1 and H 2 , melanocortin, melatonin, muscarinic M 1 and M 3 , neurokinin, opioid KOP and serotonin receptors.

Cyclic pentapeptides (e.g. Ac-(cyclo-1,5)-[KAXAD]-NH2 ; X=Ala, 1; Arg, 2) in water adopt one α-helical turn defined by three hydrogen bonds. NMR structure analysis reveals a slight distortion from α-helicity at the C-terminal aspartate caused by torsional restraints imposed by the K(i)-D(i+4) lactam bridge. To investigate this effect on helix nucleation, the more water-soluble 2 was appended to N-, C-, or both termini of a palindromic peptide ARAARAARA (≤5 % helicity), resulting in 67, 92, or 100 % relative α-helicity, as calculated from CD spectra. From the C-terminus of peptides, 2 can nucleate at least six α-helical turns. From the N-terminus, imperfect alignment of the Asp5 backbone amide in 2 reduces helix nucleation, but is corrected by a second unit of 2 separated by 0-9 residues from the first. These cyclic peptides are extremely versatile helix nucleators that can be placed anywhere in 5-25 residue peptides, which correspond to most helix lengths in protein-protein interactions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cyclic peptides and macrocycles have the potential to be membrane permeable and orally bioavailable, despite often not complying with the "rule of five" used in medicinal chemistry to guide the discovery of oral drugs. Here we compare solvent-dependent three-dimensional structures of three cyclic hexapeptides containing d-amino acids, prolines, and intramolecular hydrogen bonds. Conformational rigidity rather than flexibility resulted in higher membrane permeability, metabolic stability and oral bioavailability, consistent with less polar surface exposure to solvent and a reduced entropy penalty for transition between polar and nonpolar environments., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cyclic constraints are incorporated into an 11-residue analogue of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concentrations in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal β-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small molecule agonists of the GLP-1 receptor to treat type 2 diabetes.

We examined multistate outbreaks attributed to raw tomatoes in the United States from 1990 to 2010. We summarized the demographic and epidemiological characteristics of 15 outbreaks resulting in 1959 illnesses, 384 hospitalizations, and three deaths. Most (80%) outbreaks were reported during 2000-2010; 73% occurred May-September. Outbreaks commonly affected adult (median age 34 years) women (median 58% of outbreak cases). All outbreaks were caused by Salmonella [serotypes Newport (n = 6 outbreaks), Braenderup (n = 2), Baildon, Enteritidis, Javiana, Montevideo, Thompson, Typhimurium (n = 1 each); multiple serotypes (n = 1)]. Red, round (69% of outbreaks), Roma (23%), and grape (8%) tomatoes were implicated. Most (93%) outbreaks were associated with tomatoes served predominantly in restaurants. However, traceback investigations suggested that contamination occurred on farms, at packinghouses, or at fresh-cut processing facilities. Government agencies, academia, trade associations, and the fresh tomato industry should consider further efforts to identify interventions to reduce contamination of tomatoes during production and processing.

Nociceptin (orphanin FQ) is a 17-residue neuropeptide hormone with roles in both nociception and analgesia. It is an opioid-like peptide that binds to and activates the G-protein-coupled receptor opioid receptor-like-1 (ORL-1, NOP, orphanin FQ receptor, kappa-type 3 opioid receptor) on central and peripheral nervous tissue, without activating classic delta-, kappa-, or mu-opioid receptors or being inhibited by the classic opioid antagonist naloxone. The three-dimensional structure of ORL-1 was recently published, and the activation mechanism is believed to involve capture by ORL-1 of the high-affinity binding, prohelical C-terminus. This likely anchors the receptor-activating N-terminus of nociception nearby for insertion in the membrane-spanning helices of ORL-1. In search of higher agonist potency, two lysine and two aspartate residues were strategically incorporated into the receptor-binding C-terminus of the nociceptin sequence and two Lys(i)→Asp(i+4) side chain-side chain condensations were used to generate lactam cross-links that constrained nociceptin into a highly stable α-helix in water. A cell-based assay was developed using natively expressed ORL-1 receptors on mouse neuroblastoma cells to measure phosphorylated ERK as a reporter of agonist-induced receptor activation and intracellular signaling. Agonist activity was increased up to 20-fold over native nociceptin using a combination of this helix-inducing strategy and other amino acid modifications. An NMR-derived three-dimensional solution structure is described for a potent ORL-1 agonist derived from nociceptin, along with structure-activity relationships leading to the most potent known α-helical ORL-1 agonist (EC₅₀ 40 pM, pERK, Neuro-2a cells) and antagonist (IC₅₀ 7 nM, pERK, Neuro-2a cells). These α-helix-constrained mimetics of nociceptin(1-17) had enhanced serum stability relative to unconstrained peptide analogues and nociceptin itself, were not cytotoxic, and displayed potent thermal analgesic and antianalgesic properties in rats (ED₅₀ 70 pmol, IC₅₀ 10 nmol, s.c.), suggesting promising uses in vivo for the treatment of pain and other ORL-1-mediated responses., (© 2015 Elsevier Inc. All rights reserved.)

Many proteins exert their biological activities through small exposed surface regions called epitopes that are folded peptides of well-defined three-dimensional structures. Short synthetic peptide sequences corresponding to these bioactive protein surfaces do not form thermodynamically stable protein-like structures in water. However, short peptides can be induced to fold into protein-like bioactive conformations (strands, helices, turns) by cyclization, in conjunction with the use of other molecular constraints, that helps to fine-tune three-dimensional structure. Such constrained cyclic peptides can have protein-like biological activities and potencies, enabling their uses as biological probes and leads to therapeutics, diagnostics and vaccines. This Review highlights examples of cyclic peptides that mimic three-dimensional structures of strand, turn or helical segments of peptides and proteins, and identifies some additional restraints incorporated into natural product cyclic peptides and synthetic macrocyclic peptidomimetics that refine peptide structure and confer biological properties., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR-derived structures, amide H-D exchange rates, and temperature-dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N-methylation., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with N-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no N-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4-17%) approaching that of the heavily N-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for N-methylation or modified amino acids.

Helix-constrained polypeptides have attracted great interest for modulating protein-protein interactions (PPI). It is not known which are the most effective helix-inducing strategies for designing PPI agonists/antagonists. Cyclization linkers (X1-X5) were compared here, using circular dichroism and 2D NMR spectroscopy, for α-helix induction in simple model pentapeptides, Ac-cyclo(1,5)-[X1-Ala-Ala-Ala-X5]-NH2, in water. In this very stringent test of helix induction, a Lys1→Asp5 lactam linker conferred greatest α-helicity, hydrocarbon and triazole linkers induced a mix of α- and 3₁₀-helicity, while thio- and dithioether linkers produced less helicity. The lactam-linked cyclic pentapeptide was also the most effective α-helix nucleator attached to a 13-residue model peptide., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phytophthora capsici is an important pepper (Capsicum annuum) pathogen causing fruit and root rot, and foliar blight in field and greenhouse production. Previously, an F6 recombinant inbred line population was evaluated for fruit rot susceptibility. Continuous variation among lines and partial and isolate-specific resistance were found. In this study, Phytophthora fruit rot resistance was mapped in the same F6 population between Criollo del Morelos 334 (CM334), a landrace from Mexico, and 'Early Jalapeno' using a high-density genetic map. Isolate-specific resistance was mapped independently in 63 of the lines evaluated and the two parents. Heritability of the resistance for each isolate at 3 and 5 days postinoculation (dpi) was high (h(2) = 0.63 to 0.68 and 0.74 to 0.83, respectively). Significant additive and epistatic quantitative trait loci (QTL) were identified for resistance to isolates OP97 and 13709 (3 and 5 dpi) and 12889 (3 dpi only). Mapping of fruit traits showed potential linkage with few disease resistance QTL. The partial fruit rot resistance from CM334 suggests that this may not be an ideal source for fruit rot resistance in pepper.

The actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers.

Dynamin is required for clathrin-mediated endocytosis (CME). Its GTPase activity is stimulated by phospholipid binding to its PH domain, which induces helical oligomerization. We have designed a series of novel pyrimidine-based "Pyrimidyn" compounds that inhibit the lipid-stimulated GTPase activity of full length dynamin I and II with similar potency. The most potent analogue, Pyrimidyn 7, has an IC50 of 1.1 μM for dynamin I and 1.8 μM for dynamin II, making it among the most potent dynamin inhibitors identified to date. We investigated the mechanism of action of the Pyrimidyn compounds in detail by examining the kinetics of Pyrimidyn 7 inhibition of dynamin. The compound competitively inhibits both GTP and phospholipid interactions with dynamin I. While both mechanisms of action have been previously observed separately, this is the first inhibitor series to incorporate both and thereby to target two distinct domains of dynamin. Pyrimidyn 6 and 7 reversibly inhibit CME of both transferrin and EGF in a number of non-neuronal cell lines as well as inhibiting synaptic vesicle endocytosis (SVE) in nerve terminals. Therefore, Pyrimidyn compounds block endocytosis by directly competing with GTP and lipid binding to dynamin, limiting both the recruitment of dynamin to membranes and its activation. This dual mode of action provides an important new tool for molecular dissection of dynamin's role in endocytosis.