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Pseudomonas aeruginosa,a major cause of nosocomial infections, has the ability to produce number of factors responsible for its virulence as well as biofilm formation which are regulated by Las and Rhl QS system. The autoinducer OdDHL of las system, play crucial role in pathogenesis of P. aeruginosa. It is involved in induction of factors responsible for the virulence of the organism as well as modulate the host immune responses. In the present study, the effect of KLH OdDHL and BSA OdDHL protein conjugate on various cells in terms of cytotoxicity and phagocytosis was assessed. Concentration of 1µM showed maximum viability of macrophages. Macrophages also showed accelerated phagocytosis, as observed by neutral red assay, which was attributed to the immune-modulatory stimulation provided by the OdDHL conjugate. Other cells like Uroepithelial cells and lymphocytes also showed no significant decrease in cell viability in presence of lower concentrations of conjugated OdDHL. Our Data suggested that interaction of OdDHL conjugated to any of the carrier protein KLH / BSA, with various cells not only improved cell viability but also improved the phagocytic ability of macrophages.

The abstract is available here: https://uscholar.univie.ac.at/o:1631000

Oligonucleotide conjugates constitute a versatile tool for research and bioanalytical purposes. Often, such conjugates are prepared by reaction between a thiol on the protein with a maleimide-modified oligonucleotide. Unlike most other chemical handles the maleimide functionality cannot be introduced directly during the solid-phase oligonucleotide synthesis, and therefore the standard method to introduce the maleimide functionality is to react an amino-modified DNA with a heterobifunctional linker containing an activated ester and a maleimide. Here, we present an alternative method for preparation of maleimide and monobromomaleimide-modified oligonucleotides from the corresponding amine using N-methoxycarbonylmaleimide and N-methoxycarbonylbromomaleimide, respectively. In this method, no additional linker is attached to the oligonucleotide, as the maleimide functionality is formed directly on the existing amine. The maleimide can thereby be positioned close to the oligonucleotide, providing a high degree of control over the final construct. The reaction occurs in 30-60 min under alkaline conditions. Maleimide-modified oligonucleotides prepared in this manner were conjugated to bovine serum albumin, and the reaction shows comparable reactivity to the corresponding oligonucleotide modified using the 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) linker.

The broad host range IncC plasmid family and the integrative mobilizable Salmonella Genomic Island 1 (SGI1) and its derivatives enable the spread of medically-important antibiotic resistance genes among Gram-negative pathogens. Although several aspects of the complex functional interactions between IncC plasmids and SGI1 have been recently deciphered regarding their conjugative transfer and incompatibility, the biological signal resulting in the hijacking of the conjugative plasmid by the integrative mobilizable element remains unknown. Here, we demonstrate that the conjugative entry of IncC/IncA plasmids is detected at an early stage by SGI1 through the transient activation of the SOS response, which induces the expression of the SGI1 master activators SgaDC, shown to play a crucial role in the complex biology between SGI1 and IncC plasmids. Besides, we developed an original tripartite conjugation approach to directly monitor SGI1 mobilization in a time-dependent manner following conjugative entry of IncC plasmids. Finally, we propose an updated biological model of the conjugative mobilization of the chromosomal resistance element SGI1 by IncC plasmids.IMPORTANCEAntimicrobial resistance has become a major public health issue, particularly with the increase in multidrug resistance (MDR) in both animal and human pathogenic bacteria, and with the emergence of resistance to medically important antibiotics. The spread between bacteria of successful mobile genetic elements such as conjugative plasmids and integrative elements conferring multidrug resistance is the main driving force in the dissemination of acquired antibiotic resistances among Gram-negative bacteria. Broad-host range IncC plasmids and their integrative mobilizable SGI1 counterparts contribute to the spread of critically-important resistance genes (e.g., ESBLs, and carbapenemases). A better knowledge of the complex biology of these broad-host range mobile elements will help to understand the dissemination of antimicrobial resistance genes that occurred across γ-proteobacteria borders.

Contains fulltext : 283427.pdf (Publisher’s version ) (Open Access) Hypoxia is a common feature of solid tumors and is associated with increased tumor progression, resistance to therapy and increased metastasis. Hence, tumor hypoxia is a prognostic factor independent of treatment modality. To survive hypoxia, cells activate macroautophagy/autophagy. Paradoxically, in several cancer types, mutations or loss of essential autophagy genes have been reported that are associated with earlier onset of tumor growth. However, to our knowledge, the phenotypic and therapeutic consequences of autophagy deficiency have remained unexplored. In this study, we determined autophagy-defects in head and neck squamous cell carcinoma (HNSCC) and observed that expression of ATG12 (autophagy related 12) was lost in 25%-40% of HNSCC. In line, ATG12 loss is associated with absence of hypoxia, as determined by pimonidazole immunohistochemistry. Hence, ATG12 loss is associated with improved prognosis after therapy in two independent HNSCC cohorts and 7 additional cancer types. In vivo, ATG12 targeting resulted in decreased hypoxia tolerance, increased necrosis and sensitivity of the tumor to therapy, but in vitro ATG12-deficient cells displayed enhanced survival in nutrient-rich culture medium. Besides oxygen, delivery of glucose was hampered in hypoxic regions in vivo, which increases the reliance of cells on other carbon sources (e.g., L-glutamine). We observed decreased intracellular L-glutamine levels in ATG12-deficient cells during hypoxia and increased cell killing after L-glutamine depletion, indicating a central role for ATG12 in maintaining L-glutamine homeostasis. Our results demonstrate that ATG12(low) tumors represent a phenotypically different subtype that, due to the lowered hypoxia tolerance, display a favorable outcome after therapy.Abbreviations: ARCON:accelerated radiotherapy with carbogen and nicotinamide; ATG: autophagy related; BrdUrd: bromodeoxyuridine; CA9/CAIX: carbonic anhydrase 9; HIF1A/HIF1α: hypoxia inducible factor 1 subunit alpha; HNSCC: head and neck squamous cell carcinoma; HPV: human papilloma virus; HR: hazard ratio; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; mRNA: messenger ribonucleic acid; PCR: polymerase chain reaction; SLC2A1/GLUT1: solute carrier family 2 member 1; TCGA: the Cancer Genome Atlas; TME: tumor microenvironment; UTR: untranslated region; VEGF: vascular endothelial growth factor.

In this work, we synthesized folate-conjugated dual-drug loaded double liposomes which are noted for their extremely high target specificity towards pituitary adenomas. It is known that while folate receptors are almost non-existent in normal tissues, they are overexpressed in non-functional pituitary adenomas. Synthesis, characterization and in vitro studies of folate-conjugated dual-drug loaded double liposomes for targeting non-functional pituitary adenomas is the highlight of this study. The size, zeta-potential, polydispersity index, in vitro release studies, stability of the nanoformulation, cytotoxicity and cellular uptake studies have been carried out. It was noted from the results that these are highly targeted liposomal formulation as expressed by the cellular uptake studies and are just sufficiently sized to escape the clearance mechanisms of body. They were also cytocompatible and were stable even after 60 days of shelf life with negligible changes in sizes, zeta potential as well as polydispersity index. The conjugation with folate particles resulted in the high specificity of the formulation to the specific targeted tissue as seen in cellular uptake by primary cell culture of non-functional pituitary adenomas. It may safely be concluded from the results that this approach may be a promising therapy for the future which has low cytotoxicity and high-specificity.

Dicationic bis-quinolinium salts possessing different types of conjugation are presented in which the hetareniums are separated by acetylenic or 1,4-diethynylbenzene spacers. Cross-conjugation is induced by interconnections via the 3-positions of the heteroareniums, and conjugation is realized by 2,2/2,4/4,4-interconnections. We also synthesized mixed cross-conjugated/conjugated 2,3/4,3-interconnected species. The different types of conjugation determine the charge distribution according to the rules of resonance which translate into characteristic C-13 NMR as well as UV-Vis-spectroscopic properties, LUMO geometries, and chemical properties. We found that the triple bond's resonance frequencies are sensitive C-13 NMR spectroscopic indicators for the type of conjugation. Combining cross-conjugated and conjugated structure elements of dicationic bis-quinolinium dyes via triple bonds without additional benzene spacer results in considerable bathochromic shifts of the UV-Vis absorption maxima [Delta lambda(max)approximate to 100 nm]. A charge transfer was calculated from the HOMO of the conjugated into the LUMO of the cross-conjugated partial structure.

Large π-conjugated systems are key in the area of molecular materials. Herein, we prepare via AuI - catalyzed cyclization a series of fully π-conjugated anthracene-fused oligo-BODIPYs. Their structural and optoelectronic properties were studied by several techniques, ranging from X-ray, UV/Vis, and cyclic voltammetry to transient absorption spectroscopy. As a complement, their electronic structures were explored by means of Density Functional Theory (DFT) calculations. Depending on the size and shape of the π-conjugated skeleton, unique features—such as face-to-face supramolecular organization, NIR absorption and fluorescence as well as strong electron accepting character—were noted. All in all, the aforementioned features render them valuable for technological applications, ments We gratefully acknowledge financial support from the Spanish MICINN through Projects PID2020-116490GB-I00 (Porphyrinoids) and TED2021-131255B-C43 (PERSOLAR). We also thank financial support to the Comunidad de Madrid (MAD2D-CM) and MICINN through project “Materiales disruptivos bidimensionales (2D)” within the Plan de recuperación, transformación y resiliencia (Materiales avanzados). IMDEA Nanociencia acknowledges support from the “Severo Ochoa” Programme for Centres of Excellence in R&D (MINECO, Grant SEV2016-0686). J.L. acknowledges MECD, Spain, for a F.P.U. fellowship. European Commission under the Marie Sklodowska-Curie Action Cofund 2015 (EU project 713366-InterTalentum) is acknowledged for the support for G.D.S. Generous allocation of computational time from the Centro de Computación Científica UAM is gratefully acknowledged. Work in Erlangen was supported by the Deutsche Forschungsgemeinschaft (DFG) through SFB 953 project B10 and the Bavarian Collaborative Research Project “Solar Technologies go Hybrid” (SolTech)

IncL/M broad-host-range conjugative plasmids are involved in the global spread of blaOXA-48 and the emergence of blaNDM-1. The aim of this study was to evaluate the transmission potential of plasmids encoding the emergent NDM-1 carbapenemase compared to the pandemic OXA-48. The conjugation rate and fitness cost of IncM2 and IncL plasmids encoding these carbapenemase genes were tested using a variety of host bacteria. Genomic analysis of uropathogenic Escherichia coli SAP1756 revealed that blaNDM-1 was encoded on an IncM2 plasmid, which also harboured blaTEM-1, bleMBL and sul1 and was highly similar to plasmids isolated from the same geographical area. Conjugation experiments demonstrated that NDM-1 and OXA-48-carrying plasmids transfer successfully between different Enterobacterales species, both in vitro and in vivo. Interestingly, E. coli isolates tested as recipients belonging to phylogroups A, B1, D and F were able to receive IncM2 plasmid pSAP1756, while phylogroups B2, C, E and G were not permissive to its acquisition. In general, the IncL OXA-48-carrying plasmids tested transferred at higher rates than IncM2 harbouring NDM-1 and imposed a lower burden to their host, possibly due to the inactivation of the tir fertility inhibition gene and reflecting their worldwide dissemination. IncM2 plasmids carrying blaNDM-1 are considered emergent threats that need continuous monitoring. In addition to sequencing efforts, phenotypic analysis of conjugation rates and fitness cost are effective methods for estimating the pandemic potential of antimicrobial resistance plasmids.

Many pathogens blunt immune responses because they lack immunogenic structural features, which typically results in disease. Here, we show evidence suggesting that pathogen immunogenicity can be chemically enhanced. Using supramolecular host-guest chemistry, we complexed onto the surface of a poorly immunogenic bacterium (Staphylococcus aureus) a TLR7 agonist-based adjuvant. "Adjuvanted" bacteria were readily recognized by macrophages and induced a more pro-inflammatory immunophenotype. Future applications of this concept could yield treatment modalities that bolster the immune system's response to pathogenic microbes.

Interest in nanomedicines has grown rapidly over the past two decades, owing to the promising therapeutic applications they may provide, particularly for the treatment of cancer. Personalised medicine and ‘smart’ actively targeted nanoparticles represent an opportunity to deliver therapies directly to cancer cells and provide sustained drug release, in turn providing overall lower off-target toxicity and increased therapeutic efficacy. However, the successful translation of nanomedicines from encouraging pre-clinical findings to the clinic has, to date, proven arduous. In this review, we will discuss the use of nanomedicines for the treatment of cancer, with a specific focus on the use of polymeric and lipid nanoparticle delivery systems. In particular, we examine approaches exploring the surface functionalisation of nanomedicines to elicit active targeting and therapeutic effects as well as challenges and future directions for nanoparticles in cancer treatment.

Polycyclic aromatic hydrocarbons (PAHs) are widely used in organic electronic devices. The electronic, magnetic, and optical properties of PAHs can be tuned by structural modifications to the aromatic backbone to introduce an inherent distortion from planarity, such as bending or twisting. However, it remains difficult to isolate and control the effects of such distortions. Here, we sought to understand how backbone twisting and bending affect the electronic properties of acenes, as models for larger PAHs. We found that, even when highly distorted from planarity (30° per ring), acenes maintain their aromatic character and π orbital delocalization with minor mixing of the σ and π orbitals. In addition, the energy gap between the HOMO and LUMO decreases with increasing twist, while the gap is hardly affected by bending, since the energy of both orbitals increase to a similar extent. For bent acenes in the triplet state, the spin becomes more localized with increasing bend, whereas twisting produces an evenly distributed spin delocalization. These findings can guide the synthesis of PAHs with tailored properties., A computational study performed on nonplanar acenes demonstrates the different effects of bending and twisting on the effect on their electronic properties.

Plasmids are extrachromosomal segments of DNA that can transfer genes between bacterial cells. Many plasmid genes benefit bacteria but cause harm to human health by granting antibiotic resistance to pathogens. Transfer rate is a key parameter for predicting plasmid dynamics, but observed rates are highly variable, and the effects of selective forces on their evolution are unclear. We apply evolutionary analysis to plasmid conjugation models to investigate selective pressures affecting plasmid transfer rate, emphasizing host versus plasmid control, the costs of plasmid transfer, and the role of recipient cells. Our analyses show that plasmid-determined transfer rates can be predicted with three parameters (host growth rate, plasmid loss rate, and the cost of plasmid transfer on growth) under some conditions. We also show that low-frequency genetic variation in transfer rate can accumulate, facilitating rapid adaptation to changing conditions. Furthermore, reduced transfer rates due to host control have limited effects on plasmid prevalence until low enough to prevent plasmid persistence. These results provide a framework to predict plasmid transfer rate evolution in different environments and demonstrate the limited impact of host mechanisms to control the costs incurred when plasmids are present.

Self‐assembly processes guide disordered molecules or particles into long‐range organized structures due to specific supramolecular interactions among the building entities. Herein, we report a unique evaporation‐induced self‐assembly (EISA) strategy for four different silica nanoparticle systems obtained through peptide functionalization of the particle surface. First, covalent peptide‐silica coupling was investigated in detail, starting with the grafting of a single amino acid (L‐serine) and expanded to specific small peptides (up to four amino acids) and transferred to different particle types (MCM‐48‐type MSNs, solid nanoparticles, and newly developed virus‐like nanoparticles). These materials were investigated regarding their ability to undergo EISA, which was shown to be independent of particle type and amount of peptide anchored to their surface. This EISA‐based approach provides new possibilities for the design of future advanced drug delivery systems, engineered hierarchical sorbents, and nanocatalyst assemblies., Evaporation‐induced self‐assembly of a bioconjugate is reported. The formation of ring‐like structures was independent of different silica particle types and the amount of peptide conjugated to the material. This novel approach paves the way for further production of functional materials for separation, catalysis, and medical applications.

The combination of carbohydrates with BODIPY fluorophores gives rise to a family of BODIPY-carbohydrate hybrids or glyco-BODIPYs, which mutually benefit from the encounter. Thus, from the carbohydrates standpoint, glyco-BODIPYs can be regarded as fluorescent glycoconjugate derivatives with application in imaging techniques, whereas from the fluorophore view the BODIPY-carbohydrate hybrids benefit from the biocompatibility, water-solubility, and reduced toxicity, among others, brought about by the sugar moiety. In this Account we have intended to present the collection of available methods for the synthesis of BODIPY-carbohydrate hybrids, with a focus on the chemical transformations on the BODIPY core., We gratefully acknowledge the Spanish Ministerio de Ciencia e Innovación for financial support, project RTI2018-094862-B-100

Rhizobia are Gram-negative bacteria that are able to establish a nitrogenfixing symbiotic interaction with leguminous plants. Rhizobia genomes usually harbor several plasmids which can be transferred to other organisms by conjugation. Two main mechanisms of the regulation of rhizobial plasmid transfer have been described: quorum sensing (QS) and the rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have recently been described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pRfaLPU83a from Rhizobium favelukesii LPU83, a plasmid that shows a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory system, we show evidence of the functions of these genes in different genomic backgrounds and confirm that homologous proteins from non-closely related organisms have the same functions. These findings set up the basis for a new regulatory circuit of the conjugative transfer of plasmids., Puede accederse a los datos con los que se realizó este trabajo haciendo clic en "Documentos relacionados"., Instituto de Biotecnología y Biología Molecular

In disease, especially cancer, biological barriers impede the delivery of therapeutic agents to effectively reach their intended targets after administration. Developing delivery strategies that understand and take into account the biological environment is critical to improve the efficiency of drug delivery. Nature provides a biological toolkit from which effective drug delivery systems can be developed. In particular, albumin is an attractive delivery carrier with favorable intrinsic properties to overcome intracellular and extracellular delivery barriers in cancer. In a subset of cancers, the RAS family of oncogenes (KRAS, HRAS, NRAS) are the most frequent mutations in cancers and regulate key signaling pathways that drive tumor progression, including hyperactive cell proliferation and metabolism. As a result, drug delivery targeting RAS-driven tumors has been a long-standing challenge in cancer therapy. Looking for solutions inspired by nature, recent observations indicate that extracellular nutrients, including glucose, lipids, and albumin, were found to be actively scavenged by mutant RAS activated cancer cells via macropinocytosis to fulfill the energetic requirements of cancer cells to survive and proliferate. Here, we exploit this mechanism to deliver albumin nanoparticles in cancer cells harboring activating KRAS mutations. We synthesized stable albumin nanoparticles that demonstrate significantly greater uptake in cancer cells with activating mutations of KRAS than monomeric albumin (i.e. dissociated form of clinically used nab-paclitaxel). From pharmacological inhibition and semi-quantitative fluorescent microscopy studies, these nanoparticles exhibit significantly increased uptake in mutant KRAS cancer cells than wild-type KRAS cells by macropinocytosis. Importantly, we demonstrate that their uptake is driven by KRAS. This nanoparticle-based strategy targeting KRAS-driven macropinocytosis is a facile approach towards improved delivery into KRAS-driven cancers. Through our studies with albumin nanoparticles, it was realized that albumin itself potentially possesses excellent pharmacokinetics that could be leveraged to improve drug delivery. To further harness the intrinsic transport properties of albumin yet improve the therapeutic index of current in situ albumin-binding prodrugs, we developed albumin-drug conjugates with a controlled loading that achieved better antitumor efficacy. Model drug monomethyl auristatin E (MMAE) was conjugated ex vivo to Cys34 of albumin via a cathepsin B-sensitive dipeptide linker to ensure that all drugs would be bound specifically to albumin. The resulting albumin-drug conjugate with a drug to albumin ratio (DAR) of 1 (ALDC1) retained the native structure of albumin compared to conjugate with a higher DAR of 3 (ALDC3). ALDC1 exhibited improved drug release and cytotoxicity compared to ALDC3 in vitro. Slower plasma clearance and increased drug exposure over time of ALDC1 were observed compared to ALDC3 and MMAE prodrug. In single dose studies with MIA PaCa2 xenografts, cohorts treated with ALDC1 had the highest amount of MMAE drug in tumor tissues compared to other treatment arms. After multiple dosing, ALDC1 significantly delayed the tumor growth compared to control treatment arms MMAE, MMAE-linker conjugate, and ALDC3. When dosed with the maximum tolerated dose of ALDC1, there was complete eradication of 83.33% of the tumors in the treatment group. Ex vivo conjugated ALDC1 also significantly inhibited tumor growth in an immunocompetent syngeneic mouse model that recapitulates the phenotype and clinical features of human pancreatic cancers. In summary, site-specific loading of drug to albumin at 1:1 ratio allowed the conjugate to maintain the native structure of albumin and its intrinsic properties. By conjugating the drug to albumin prior to administration minimized premature cleavage and instability of the drug in plasma and enabled higher drug accumulation in tumors compared to in situ albumin-binding prodrugs. This strategy to control drug loading ex vivo ensures complete drug binding to the albumin carrier and achieves excellent antitumor efficacy, and it has the potential to greatly improve the outcomes of anticancer therapy. Collectively, these findings suggest that albumin can serve as an effective drug delivery carrier for drug delivery in solid tumors when the intrinsic properties of albumin were carefully utilized.

In recent years, antimicrobial peptides (AMPs) have become a promising alternative to the use of conventional and chemically synthesized antibiotics, especially after the emergence of multidrug-resistant organisms. Thus, this review aims to provide an updated overview of the state-of-the-art for producing antimicrobial peptides fused or conjugated with the elastin-like (ELP) peculiar carriers, and that are mostly intended for biomedical application. The elastin-like biopolymers are thermosensitive proteins with unique properties. Due to the flexibility of their modular structure, their features can be tuned and customized to improve the production of the antimicrobial domain while reducing their toxic effects on the host cells. Both fields of research faced a huge rise in interest in the last decade, as witnessed by the increasing number of publications on these topics, and several recombinant fusion proteins made of these two domains have been already described but they still present a limited variability. Here in, the approaches described to recombinantly fuse and chemically conjugate diverse AMPs with ELPs are reviewed, and the nature of the AMPs and the ELPs used, as well as the main features of the expression and production systems are summarized. This article is protected by copyright. All rights reserved.

The global rise of drug-resistant bacteria is of great concern. Conjugative transfer of antibiotic resistance plasmids contributes to the emerging resistance crisis. Despite substantial progress in understanding the molecular basis of conjugation in vitro, the in vivo dynamics of intra- and interspecies conjugative plasmid transfer are much less understood. In this study, we focused on the streptomycin resistance-encoding mobilizable plasmid pRSF1010SL1344 (P3) of Salmonella enterica serovar Typhimurium strain SL1344. We show that P3 is mobilized by interacting with the conjugation machinery of the conjugative plasmid pCol1B9SL1344 (P2) of SL1344. Thereby, P3 can be transferred into a broad range of relevant environmental and clinical bacterial isolates in vitro and in vivo. Our data suggest that S. Typhimurium persisters in host tissues can serve as P3 reservoirs and foster transfer of both P2 and P3 once they reseed the gut lumen. This adds to our understanding of resistance plasmid transfer in ecologically relevant niches, including the mammalian gut. IMPORTANCE S. Typhimurium is a globally abundant bacterial species that rapidly occupies new niches and survives unstable environmental conditions. As an enteric pathogen, S. Typhimurium interacts with a broad range of bacterial species residing in the mammalian gut. High abundance of bacteria in the gut lumen facilitates conjugation and spread of plasmid-carried antibiotic resistance genes. By studying the transfer dynamics of the P3 plasmid in vitro and in vivo, we illustrate the impact of S. Typhimurium-mediated antibiotic resistance spread via conjugation to relevant environmental and clinical bacterial isolates. Plasmids are among the most critical vehicles driving antibiotic resistance spread. Further understanding of the dynamics and drivers of antibiotic resistance transfer is needed to develop effective solutions for slowing down the emerging threat of multidrug-resistant bacterial pathogens., Journal of Bacteriology, 204 (12), ISSN:0021-9193, ISSN:1098-5530

Conjugation driven by a chromosomally integrated F-plasmid (high frequency of recombination strain) can create bacteria with hybrid chromosomes. Previous studies of interspecies hybrids have focused on hybrids in which a region of donor chromosome replaces an orthologous region of recipient chromosome leaving chromosome size unchanged. Very little is known about hybrids with enlarged chromosomes, the mechanisms of their creation, or their subsequent trajectories of adaptative evolution. We addressed this by selecting 11 interspecies hybrids between Escherichia coli and Salmonella Typhimurium in which genome size was enlarged. In three cases, this occurred by the creation of an F′-plasmid while in the remaining eight, it was due to recombination of donor DNA into the recipient chromosome. Chromosome length increased by up to 33% and was associated in most cases with reduced growth fitness. Two hybrids, in which chromosome length was increased by the addition of 0.97 and 1.3 Mb, respectively, were evolved to study genetic pathways of fitness cost amelioration. In each case, relative fitness rapidly approached one and this was associated with large deletions involving recombination between repetitive DNA sequences. The locations of these repetitive sequences played a major role in determining the architecture of the evolved genotypes. Notably, in ten out of ten independent evolution experiments, deletions removed DNA of both species, creating high-fitness strains with hybrid chromosomes. In conclusion, we found that enlargement of a bacterial chromosome by acquisition of diverged orthologous DNA is followed by a period of rapid evolutionary adjustment frequently creating irreversibly hybrid chromosomes.

Conjugative plasmids often carry virulence and antibiotic-resistant genes. Therefore, understanding the behavior of these extra-chromosomal DNA elements gives insights into their spread. Bacteria frequently replicate slower after plasmids’ entry, an observation inconsistent with the plasmids’ ubiquity in nature. Several hypotheses explain the maintenance of plasmids among bacterial communities. However, the numerous combinations of bacterial species and strains, plasmids, and environments claim a robust elucidatory mechanism of plasmid maintenance. Previous works have shown that donor cells already adapted to the plasmid may use the plasmid as a ‘weapon’ to compete with non-adapted plasmid-free cells. Computer simulations corroborated this hypothesis with a wide range of parameters. Here we show that donor cells benefit from harboring conjugative plasmids even if compensatory mutations in transconjugant cells occur in the plasmid, not on chromosomes. The advantage’s leading causes are as follows: mutations take time to appear, many plasmids remain costly, and re-transfer of mutated plasmids usually occurs in sites distant to the original donors, implying little competition between these cells. Research in previous decades cautioned against uncritical acceptance of the hypothesis that resistance cost helps to preserve antibiotics’ effectiveness. This work gives a new twist to this conclusion by showing that costs help antibiotic-resistant bacteria to compete with plasmid-free cells even if compensatory mutations appear in plasmids.

The typical method for minimizing serum protein adsorption in biological settings and prolonging blood circulation time of nanoparticles, is to anchor hydrophilic polymers (e.g., poly(ethylene glycol), PEG) on the particle surface, which is most often done by covalent attachment (PEGylation). Herein, different PEGylation methods were realised and compared to functionalize mesoporous silica nanoparticles (MSNs). First, reactive groups were installed using post-grafting procedures with different functional silanes. Further, PEGs carrying a functional group and having different chain lengths and termini, were used. The grafting efficacy as well as the structural and physicochemical characteristics of the resulting particles were determined. Finally, the serum protein adsorption behaviour of these functionalized particles was investigated using thermogravimetric analysis. The type of selected coupling method was shown to strongly influence the grafting efficiency as well as the resulting protein adsorption. The results highlight the importance of the right choice of the linking chemistry when aiming at surface functionalization of nanoparticles.

Conjugation drives the horizontal transfer of adaptive traits across prokaryotes. One-fourth of the plasmids encode the functions necessary to conjugate autonomously, the others being eventually mobilizable by conjugation. To understand the evolution of plasmid mobility, we studied plasmid size, gene repertoires, and conjugation-related genes. Plasmid gene repertoires were found to vary rapidly in relation to the evolutionary rate of relaxases, for example, most pairs of plasmids with 95% identical relaxases have fewer than 50% of homologs. Among 249 recent transitions of mobility type, we observed a clear excess of plasmids losing the capacity to conjugate. These transitions are associated with even greater changes in gene repertoires, possibly mediated by transposable elements, including pseudogenization of the conjugation locus, exchange of replicases reducing the problem of incompatibility, and extensive loss of other genes. At the microevolutionary scale of plasmid taxonomy, transitions of mobility type sometimes result in the creation of novel taxonomic units. Interestingly, most transitions from conjugative to mobilizable plasmids seem to be lost in the long term. This suggests a source-sink dynamic, where conjugative plasmids generate nonconjugative plasmids that tend to be poorly adapted and are frequently lost. Still, in some cases, these relaxases seem to have evolved to become efficient at plasmid mobilization in trans, possibly by hijacking multiple conjugative systems. This resulted in specialized relaxases of mobilizable plasmids. In conclusion, the evolution of plasmid mobility is frequent, shapes the patterns of gene flow in bacteria, the dynamics of gene repertoires, and the ecology of plasmids., INCEPTION project (PIA/ANR-16-CONV-0005), Equipe FRM (EQU201903007835), Laboratoire d’Excellence IBEID (ANR-10-LABX-62-IBEID) to E.P.C.R. and C.C. PID2020-117923GB-I00 project funded by Spanish Ministry of Science and Innovation to FdlC and MPG-B.

Acquisition of mobile genetic elements can confer novel traits to bacteria. Some integrative and conjugative elements confer upon members of Bradyrhizobium the capacity to fix nitrogen in symbiosis with legumes. These so-called symbiosis integrative conjugative elements (symICEs) can be extremely large and vary as monopartite and polypartite configurations within chromosomes of related strains. These features are predicted to impose fitness costs and have defied explanation. Here, we show that chromosome architecture is largely conserved despite diversity in genome composition, variations in locations of attachment sites recognized by integrases of symICEs, and differences in large-scale chromosomal changes that occur upon integration. Conversely, many simulated nonnative chromosome–symICE combinations are predicted to result in lethal deletions or disruptions to architecture. Findings suggest that there is compatibility between chromosomes and symICEs. We hypothesize that the size and structural flexibility of symICEs are important for generating combinations that maintain chromosome architecture across a genus of nitrogen-fixing bacteria with diverse and dynamic genomes.

Coiled coils are characterized by an arrangement of two or more alpha-helices into a superhelix and one of few protein motifs where the sequence-to-structure relationship to a large extent have been decoded and understood. The abundance of both natural and de novo designed coil coils provides a rich molecular toolbox for self assembly of elaborate bespoke molecular architectures, nanostructures, and materials. Leveraging on the numerous possibilities to tune both affinities and preferences for polypeptide oligomerization, coiled coils offer unique possibilities to design modular and dynamic assemblies that can respond in a predictable manner to biomolecular interactions and subtle physicochemical cues. In this review, strategies to use coiled coils in design of novel therapeutics and advanced drug delivery systems are discussed. The applications of coiled coils for generating drug carriers and vaccines, and various aspects of using coiled coils for controlling and triggering drug release, and for improving drug targeting and drug uptake are described. The plethora of innovative coiled coil-based molecular systems provide new knowledge and techniques for improving efficacy of existing drugs and can facilitate development of novel therapeutic strategies. (c) 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). 1. Introduction 27 2. The coiled coil motif 27 3. Coiled coils and coiled coil-hybrids for drug delivery and therapeutics 30 3.1. Coiled coils in liposome drug delivery systems 30 3.2. Lipidated coiled coils for assembly of virus-like particles 31 3.3. Coiled coil nanoparticles 31 3.4. Coiled coil nanocarriers 33 3.5. Coiled coil polymer-hybrids 33 3.6. Coiled coil-based hydrogels 36 3.7. Coiled coil inorganic nanoparticle hybrids 37 3.8. Coiled coils combined with cell penetrating peptides 37 3.9. Coiled coils for improved targeting 38 Funding Agencies|Swedish Foundation for Strategic Research (SFF)Swedish Foundation for Strategic Research [FFL15-0026, RMX18-0039]; Swedish Government Strategic Research Area inMaterials Science on Functional Materials at Linkoping University [2009-00971]; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation [KAW 2016.0231]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2017-04475]; Cancer Foundation [17 0534]

The “Warburg effect” provides a novel method for treating cancer cell metabolism. Overexpression of glucose transporter 1 (GLUT1), activation of AMP-activated protein kinase (AMPK), and downregulation of mammalian target of rapamycin (mTOR) have been identified as biomarkers of abnormal cancer cell metabolism. Metformin (MET) is an effective therapy for breast cancer (BC), but its efficacy is largely reliant on the concentration of glucose at the tumor site. We propose a WZB117 (a GLUT1 inhibitor)-OCMC (O-carboxymethyl-chitosan)-MET combo strategy for simultaneous GLUT1 and mTOR targeting for alteration of BC metabolism. WZB117 conjugated polymeric nanoparticles were 225.67 ± 11.5 nm in size, with a PDI of 0.113 ± 0.16, and an encapsulation of 72.78 6.4%. OCMC pH-dependently and selectively releases MET at the tumor site. MET targets the mTOR pathway in cancer cells, and WZB117 targets BCL2 to alter GLUT1 at the cancer site. WZB117-OCMC-MET overcomes the limitations of MET monotherapy by targeting mTOR and BCL2 synergistically. WZB117-OCMC-MET activates AMPK and suppresses mTOR in a Western blot experiment, indicating growth-inhibitory and apoptotic characteristics. AO/EB and the cell cycle enhance cellular internalization as compared to MET alone. WZB117-OCMC-MET affects cancer cells’ metabolism and is a promising BC therapeutic strategy.

Recent advances in peroxidase‐mediated biotin tyramide (BT) signal amplification technology have resulted in high‐resolution and subcellular compartment‐specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H2O2 is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phenoxy radicals react in a sequence‐independent manner with guanosine bases in RNA. In contrast, DNA reactivity with BT cannot be detected by our methods under the same conditions. Remarkably, we show that fluorescein conjugates DNA rapidly and selectively reacts with BT phenoxy radicals, allowing convenient and practical biotinylation of DNA on fluorescein with retention of fluorescence., Enzyme‐catalyzed phenoxy radical biotinylation, an increasingly important tool in biotechnology and molecular biology applications, is here shown to label guanosine bases in RNA, but not in DNA, under defined conditions. Remarkably, common fluorescein conjugates of synthetic DNA oligonucleotides are found to undergo facile biotinylation on fluorescein with retention of fluorescence. This convenient observation points to a variety of practical applications of peroxidase‐catalyzed chemistry for analysis of DNA conjugates in vitro and in cells.

The synthesis of unsubstituted oligo‐para‐phenylenes (OPP) exceeding para‐hexaphenylene—in the literature often referred to as p‐sexiphenyl—has long remained elusive due to their insolubility. We report the first preparation of unsubstituted para‐nonaphenylenes (9PPs) by extending our precursor route to poly‐para‐phenylenes (PPP) to a discrete oligomer. Two geometric isomers of methoxylated syn‐ and anti‐cyclohexadienylenes were synthesized, from which 9PP was obtained via thermal aromatization in thin films. 9PP was characterized via optical, infrared and solid‐state 13C NMR spectroscopy as well as atomic force microscopy and mass spectrometry, and compared to polymeric analogues. Due to the lack of substitution, para‐nonaphenylene, irrespective of the precursor isomer employed, displays pronounced aggregation in the solid state. Intermolecular excitonic coupling leads to formation of H‐type aggregates, red‐shifting emission of the films to greenish. 9PP allows to study the structure–property relationship of para‐phenylene oligomers and polymers, especially since the optical properties of PPP depend on the molecular shape of the precursor., Unsubstituted para ‐nonaphenylenes were prepared via thermal aromatization of cyclohexadienylene precursors in thin films. The greenish fluorescence of the material is caused by pronounced aggregation in the solid state, in which electronic couplings leads to formation of H‐type aggregates. The emission bands of this small‐molecule are similar with respect to its polymer analogue.

The roseobacter group of marine bacteria is characterized by a mosaic distribution of ecologically important phenotypes. These are often encoded on mobile extrachromosomal replicons. So far, conjugation had only been experimentally proven between the two model organisms Phaeobacter inhibens and Dinoroseobacter shibae . Here, we show that two large natural RepABC-type plasmids from D. shibae can be transferred into representatives of all known major Rhodobacterales lineages. Complete genome sequencing of the newly established Phaeobacter inhibens transconjugants confirmed their genomic integrity. The conjugated plasmids were stably maintained as single copy number replicons in the genuine as well as the new host. Co-cultivation of Phaeobacter inhibens and the transconjugants with the dinoflagellate Prorocentrum minimum demonstrated that Phaeobacter inhibens is a probiotic strain that improves the yield and stability of the dinoflagellate culture. The transconjugant carrying the 191 kb plasmid, but not the 126 kb sister plasmid, killed the dinoflagellate in co-culture.

Aim . Detection of SXT elements in cholera vibrios O1 and nonO1/nonO139 serogroups and study of the effectiveness of their conjugative transmission to Escherichia coli cells. Materials and methods . In conjugation experiments, Vibrio cholerae O1 El Tor (3) and V. cholerae nonO1/ nonO139 (3) strains were used as donors. Donor strains, recipients, and transconjugants were tested in realtime PCR for sensitivity to antibiotics and for the presence of drug resistance genes and integrase gene (int). Electrophoresis was carried out on a 0.7% agarose gel with ethidium bromide staining. Results . Resistance to chloramphenicol, trimethoprim/sulfamethoxazole, streptomycin was transmitted in conjugation experiments with a frequency of 2.1 × 10 –9 –7.1 × 10 –9 . The genes int and dfrA1 (resistance to trimethoprim/sulfamethoxazole) were found in most V. cholerae strains, and were stably transmitted to E. coli QD Rif r cells and in reverse crosses of V. cholerae O1 El Tor 5879 Nal r . Conclusion . The detection of the SXT element in V. cholerae strains and its successful horizontal transfer emphasize the need to detect such mobile genetic elements to control the spread of antibiotic resistance in V. cholerae.

N‐Alkylation significantly changes the electronic and optical properties, as well as the reactivity of nitrogen‐containing π‐conjugated molecules. In this study, it is found that treating 5,15‐diazaporphyrins with methyl triflate selectively affords the corresponding N ‐methyl‐5,15‐diazaporphyrinium cations in good yield. N‐Methylation substantially alters the electronic properties and reactivity of diazaporphyrins. The electron‐accepting properties of the N ‐methyl‐5,15‐diazaporphyrinium cations are enhanced due to their lowered LUMO level. Stabilization of the LUMO energy enables regio‐ and stereoselective Diels–Alder reactions of the cationic diazaporphyrin with cyclopentadiene. N‐Methylation also enhances the acidity of the inner NH protons, and thus, allows facile deprotonation to provide nitrogen‐substituted isoporphyrin analogues with only one NH group in the central cavity., ファイル公開：2021-02-26

The primary role of the Cell Wall Integrity Pathway (CWI) in Saccharomyces cerevisiae is monitoring the state of the cell wall in response to general life cycle stresses (growth and mating) and imposed stresses (temperature changes and chemicals). Of the five mechanosensor proteins monitoring cell wall stress, Wsc1p and Mid2p are the most important. We find that WSC1 has a stringent requirement in zygotes and diploids, unlike haploids, and differing from MID2’s role in shmoos. Diploids lacking WSC1 die frequently, independent of mating type. Death is due to loss of cell wall and plasma membrane integrity, which is suppressed by osmotic support. Overexpression of several CWI pathway components suppress wsc1∆ zygotic death, including WSC2, WSC3, and BEM2, as well as the Rho-GAPS, BEM3 and RGD2. Microscopic observations and suppression by BEM2 and BEM3 suggest that wsc1∆ zygotes die during bud emergence. Downstream in the CWI pathway, overexpression of a hyperactive protein kinase C (Pkc1p-R398P) causes growth arrest, and blocks the pheromone response. With moderate levels of Pkc1p-R398P, cells form zygotes and the wsc1∆ defect is suppressed. This work highlights functional differences in the requirement for Wsc1p in diploids Versus haploids and between Mid2p and Wsc1p during mating.

The emergence of the plasmid-borne colistin resistance gene mcr-1 threats public health. IncX4-type plasmids are one of the most epidemiologically successful vehicles for spreading mcr-1 worldwide. Since MCR-1 is known for imposing a fitness cost to its host bacterium, the successful spread of mcr-1-bearing plasmids might be linked to high conjugation frequency, which would enhance the maintenance of the plasmid in the host without antibiotic selection. However, the mechanism of IncX4 plasmids conjugation remains unclear. In this study, we used high-density transposon mutagenesis to identify factors required for IncX4 plasmid transfer and 18 genes were identified, including five with annotations unrelated to conjugation. The Cappable-seq and RNA-seq analysis confirmed that a novel transcriptional regulator gene, pixR, directly regulates the transfer of IncX4 plasmids by binding the promoter of 13 essential transfer genes to increase their transcription. Plasmid invasion and co-culture competition assays revealed that pixR is essential for the spread and persistence of mcr-1-bearing IncX4 plasmids in bacterial populations, and effective conjugation is crucial for alleviating the fitness cost exerted by mcr-1 carriage. The existence of the IncX4-specific pixR gene increases plasmid transmissibility while promoting the invasion and persistence of mcr-1-bearing plasmids in bacterial populations, which helps explain their global prevalence.IMPORTANCEThe spread of clinical important antibiotic resistance genes is frequently related to some epidemic plasmids. However, the underlying molecular mechanisms contributing to the successful spread of these epidemic plasmids remains unclear. The significant of our research indicated that efficient conjugation could promote the invasion and persistence of plasmids within a bacterial population, resulting in the successful dissemination of epidemic plasmids in nature. Our data also highlight the importance of developing plasmid conjugation inhibitors to solve the antibiotic resistance crisis.

Since the discovery of the semiconducting properties of π-conjugated organic oligomers and polymers, many efforts have been devoted to generating structural features that bring about new and outstanding properties for optoelectronics, spintronics and magnetic devices, non-linear optics, or singlet fission processes. In this sense, the play between the resonance and π-electron delocalization phenomena in π-conjugated organic materials is the origin of their semiconducting behavior. For this reason, establishing not only the electron delocalization mechanism and its extension, but also which factors disturb the π-electron density is of utmost importance to enhance the proper performance of the electronic devices and develop ad hoc synthesis for desired application. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Дар фарҳанги «Бурҳони қотеъ» як гурӯҳ вожаҳое, ки дар забони форсӣ-тоҷикӣ аз забони оромӣ маҳфуз мондаанд, дохил шудаанд, ки дар маъхазҳои лексикографӣ ба сифати воҳиди марбут ба луғати «Занд»-у «Позанд» сабт шудаанд. Ин воҳидҳои луғавӣ дар шакли идеограмма нигоҳ дошта шудаанд. Гурӯҳи нисбатан назарраси идеограммаҳое, ки ҳаракату ҳолатро ифода мекунанд, ҳамчун ҷузъи ин воҳидҳои луғавӣ барои таҳлил ба панҷ гурӯҳ ҷудо мешаванд: а) идеограммаҳои феълие, ки самтҳои ҳаракатро ифода мекунанд; б) идеограммаҳои феълие, ки ҳолати шахсро ифода мекунанд; в) идеограммаҳои синонимии феълӣ; г) идеограммаҳои феълии шакли тасрифӣ; д) идеограммаҳои феълии шакли ғайритасрифӣ. Хулоса мешавад, ки идеограммаҳои феълии фарҳанги мавриди назар аз қадимияту ғановати луғавии забони форсӣ-тоҷикӣ гувоҳӣ дода, дар муайян кардани унсурҳои грамматикӣ ва вазифаҳои морфологию синтаксисии онҳо дар як давраи муайяни таърихӣ саҳм гузоштаанд., «Burhoni Kote» dictionary includes a group of words preserved in the Persian-Tajik language from Arameyan which are fixed in lexicographical origins as units appertaining to the vocabulary of «Zand» and «Pozand». The lexical units in question preserved in the term of ideograms. Comparatively significant corpus of ideograms expressing motion and state is divided into five groups for analysis in the composition of the lexical units in question: a) verbal ideograms expressing correction of motion; b) verbal ideograms expressing man`s state; c) synonymical verbal ideograms; d) conjugated verbal ideograms; e) non-conjugated verbal ideograms. The conclusion is made that verbal ideograms of the canvassed dictionary testify to antiquity of the lexical richness of the Tajik-Persian language and promote the elicitation of grammatical elements and their morphologico-syntactical functions in a certain historic period.

First, a site-specific method for covalently and site-specifically attaching two proteins under biocompatible conditions was developed. The method combined the sortase-mediated installation of a peptide containing a cysteine-reactive functionality onto the N-terminus of an antigen binding protein followed by a subsequent chemoselective conjugation. Investigation of the promiscuity of sortase, a cysteine-dependent transpeptidase, for substrates containing cysteine-alkylating reagents suggested good tolerance of the sortase for such electrophiles. Multiple peptides each containing a sortase recognition sequence and a different cysteine-selective electrophile were therefore synthesized via solid-phase peptide synthesis (SPPS). Significant optimization of the sortase reaction was required to yield sufficient product for functional assays. Speeding up the sortase reaction using kinetically enhanced mutants was found to enhance the production of product material, likely by counteracting the concurrent alkylation of the sortase active site by the electrophilic peptide substrate. After successfully attaching cysteine-targeting moieties onto several antigen-binding protein, so-called “nanobodies” using sortase transpeptidation, site-specific conjugation to cysteine-containing proteins was optimized to generate useful yields of products with new functionalities. Conjugation was shown to be unsuccessful without prior purification of the sortase reaction, likely due to the presence of competing crosslinking partners and loss of the transpeptidation product over time in the presence of the sortase. Sortase-mediated electrophile installations were performed on nanobodies (with and without endogenous cysteines) and crosslinked to other nanobodies, GFP, and even intact IgG antibodies to yield bi-specific or mono-specific, fluorescent constructs combining the properties of multiple intact proteins. Final conjugate yields were low after multiple purification steps. The function of both protein entities within all final constructs were confirmed using fluorescence microscopy. In the second part, amber codon suppression was combined with the chemoselective Staudinger-phosphite reaction to enable the selective caging of nanobody active sites with a light-cleavable PEG group. Decaging was envisioned to progress via UV-induced photolysis of an ortho-nitrobenzyl polyethylene glycol (ONB-PEG) functionality, followed by hydrolysis to yield a final aniline. An azido-tyrosine was introduced at the active site of a GFP binding nanobody using amber suppression. To test the activity of the decaged product, TCEP reduction successfully converted the incorporated azide to the corresponding aniline, which was then subjected to a substrate binding assay. Unfortunately, the binding affinity of the decaged nanobody was only partially restored to that of the wild-type nanobody. The azide was subsequently functionalized with a light-cleavable ONB-PEG-phosphite via the Staudinger-phosphite reaction in aqueous buffer. According to a recent literature survey, this represents the first example of a Staudinger-phosphite reaction performed on a nanobody under biological conditions. Caging was shown to prevent GFP-nanobody interaction via a GFP binding assay utilizing the fluorescence-enhancing properties of the chosen nanobody. Cleavage of the ONB group was achieved using UV irradiation, and the resulting decaged construct was shown to possess partially restored GFP binding affinity.

Objectives ‘Integrative and Conjugative Elements’ (ICEs) and ‘Integrative and Mobilizable Elements’ (IMEs) are two classes of mobile genetic elements that are complex to detect and delineate. Therefore, they are yet poorly annotated in bacterial genomes. FirmiData provides to the scientific community of microbiologists and bioinformaticians a reference resource of annotated ICEs and of IMEs from Firmicutes. It illustrates their prevalence and their diversity but also gives information on their organization. FirmiData was designed to assist the scientific community in identifying and annotating these elements by using the sequences of these ICEs and IMEs for the identification of related elements in other genomes of Firmicutes. Therefore, Firmidata meets the needs of the scientific community. Data description Firmidata provides a manually curated annotation of 98 ICEs and 148 IMEs identified in 40 chromosomes of Firmicutes. The delineation at the nucleotide level of almost all of these elements allows for the characterization of the genes they carry.

During conjugation, a conjugative DNA element is transferred from a donor to a recipient cell via a connecting channel. Conjugation has clinical relevance because it is the major route for spreading antibiotic resistance and virulence genes. The conjugation process can be divided into different steps. The initial steps carried out in the donor cell culminate in the transfer of a single DNA strand (ssDNA) of the conjugative element into the recipient cell. However, stable settlement of the conjugative element in the new host requires at least two additional events: conversion of the transferred ssDNA into double-stranded DNA and inhibition of the hosts’ defence mechanisms to prevent degradation of the transferred DNA. The genes involved in this late step are historically referred to as establishment genes. The defence mechanisms of the host must be inactivated rapidly and—importantly—transiently, because prolonged inactivation would make the cell vulnerable to the attack of other foreign DNA, such as those of phages. Therefore, expression of the establishment genes in the recipient cell has to be rapid but transient. Here, we studied regulation of the establishment genes present on the four clades of the pLS20 family of conjugative plasmids harboured by different Bacillus species. Evidence is presented that two fundamentally different mechanisms regulate the establishment genes present on these plasmids. Identification of the regulatory sequences were critical in revealing the establishment regulons. Remarkably, whereas the conjugation genes involved in the early steps of the conjugation process are conserved and are located in a single large operon, the establishment genes are highly variable and organised in multiple operons. We propose that the mosaical distribution of establishment genes in multiple operons is directly related to the variability of defence genes encoded by the host bacterial chromosomes, MINISTRY OF SCIENCE AND INNOVATION OF THE SPANISH GOVERNMENT grants (bio2016-77883-C2-1-P) and (PID2019-108778GB-C21 (AEI/FEDER, EU)) to W.J.J.M., which also funded J.V-C. and A.M-A. A Wellcome Investigator grant (209500) to Jeff Errington supported L.J.W. Institutional grants from the “Fundación Ramón Areces” and “Banco de Santander” supported the Centro de Biología Molecular “Severo Ochoa

Successful strategies for the attachment of oligopeptides to mesoporous silica with pores large enough to load biomolecules should utilize the high surface area of pores to provide an accessible, protective environment. A two-step oligopeptide functionalization strategy is examined here using diazirine-based heterobifunctional linkers. Mesoporous silica nanoparticles (MSNPs) with average pore diameter of ~8 nm and surface area of ~730 m2/g were synthesized and amine-functionalized. Tetrapeptides Gly-Gly-Gly-Gly (GGGG) and Arg-Ser-Ser-Val (RSSV), and a peptide comprised of four copies of RSSV (4RSSV), were covalently attached via their N-terminus to the amine groups on the particle surface by a heterobifunctional linker, sulfo-succinimidyl 6-(4,4′-azipentanamido)hexanoate (sulfo-NHS-LC-diazirine, or SNLD). SNLD consists of an amine-reactive NHS ester group and UV-activable diazirine group, providing precise control over the sequence of attachment steps. Attachment efficiency of RSSV was measured using fluorescein isothiocyanate (FITC)-tagged RSSV (RSSV-FITC). TGA analysis shows similar efficiency (0.29, 0.31 and 0.26 mol peptide/mol amine, respectively) for 4G, RSSV and 4RSSV, suggesting a generalizable method of peptide conjugation. The technique developed here for the conjugation of peptides to MSNPs provides for their attachment in pores and can be translated to selective peptide-based separation and concentration of therapeutics from aqueous process and waste streams.

Plasmid-specific bacteriophages specifically infect bacteria carrying conjugal plasmids. While wastewater has been used as isolation source for such phages, to date, only the distribution and ecology of RNA phages specific to the F plasmid have been described, because they serve as a water quality indicator. Yet, several other plasmid classes have higher clinical and ecological relevance, and the distribution, fate, and ecology of the phages that target them remain uncharacterized. We aimed to (i) provide an experimental platform to quantify the abundance of plasmid-specific phages applicable to several different conjugal plasmid classes, (ii) describe the distribution of such phages in wastewater systems, and (iii) relate their abundance to plasmid abundance and to municipal wastewater treatment processes. We introduced four model conjugal plasmids, belonging to incompatibility groups IncP-1, IncN, IncHI1, or IncF into an avirulent Salmonella enterica strain, for which somatic phages are at low abundance in wastewater. These strains were used in double layer agar assays with waters from contrasting sources. Plasmid-specific phages were common in wastewater but rare in river water. Hospital wastewater contained significantly more IncP-1-, but fewer IncF- and IncN- specific phages than domestic wastewater. This pattern did not match that of plasmid abundance estimated by Inc group targeting high-throughput quantitative PCR. The comparison between influent and effluent of wastewater treatment plants revealed a reduction in phage concentration by ca. 2 log, without significant contribution of primary settling. Overall, the ubiquity of these phages hints at their importance for plasmid ecology, and can provide opportunities in water quality monitoring and in ecological management of mobile resistance genes. Plasmid-specific bacteriophages specifically infect bacteria carrying conjugal plasmids. While wastewater has been used as isolation source for such phages, to date, only the distribution and ecology of RNA phages specific to the F plasmid have been described, because they serve as a water quality indicator. Yet, several other plasmid classes have higher clinical and ecological relevance, and the distribution, fate, and ecology of the phages that target them remain uncharacterized. We aimed to (i) provide an experimental platform to quantify the abundance of plasmid-specific phages applicable to several different conjugal plasmid classes, (ii) describe the distribution of such phages in wastewater systems, and (iii) relate their abundance to plasmid abundance and to municipal wastewater treatment processes. We introduced four model conjugal plasmids, belonging to incompatibility groups IncP-1, IncN, IncHI1, or IncF into an avirulent Salmonella enterica strain, for which somatic phages are at low abundance in wastewater. These strains were used in double layer agar assays with waters from contrasting sources. Plasmid-specific phages were common in wastewater but rare in river water. Hospital wastewater contained significantly more IncP-1-, but fewer IncF- and IncN- specific phages than domestic wastewater. This pattern did not match that of plasmid abundance estimated by Inc group targeting high-throughput quantitative PCR. The comparison between influent and effluent of wastewater treatment plants revealed a reduction in phage concentration by ca. 2 log, without significant contribution of primary settling. Overall, the ubiquity of these phages hints at their importance for plasmid ecology, and can provide opportunities in water quality monitoring and in ecological management of mobile resistance genes.

Aim: Direct analytical comparison of two major drug-linkers in the antibody-drug conjugate (ADC) field was conducted. Methods: Four different analytical methods [AlogP calculation, reverse phase (RP) high-performance liquid chromatography (HPLC; RP-HPLC), size exclusion chromatography HPLC (SEC-HPLC), and differential scanning calorimetry (DSC)] were tested for this comparison. Results: Maytansinoid-based ADCs showed less hydrophobicity than auristatin-based ADCs. Regardless of the drug-linker and drug-to-antibody ratios (DARs), the stability detected by DSC was decreased by conjugation. Conclusions: The cost and time-efficient analytical comparison described in this manuscript may be useful information for an initial characterization of ADCs prior to detailed biological studies.

A popular approach to the calculation of molecular excitation energies is to consider only equilibrium geometries and neglect the effects of thermal motion. Although this static approach is sensible for molecules with distinct potential-energy minima, its adequacy relative to dynamical approaches appears not to have been thoroughly tested. Here, we report a case study investigating how thermal motion accounted for by molecular dynamics simulations influences the optically bright state of astaxanthin, a carotenoid of broad photobiological interest that features 13 conjugated double bonds. Employing several different density functional methods, it is shown that thermal fluctuations in the conjugation result in the Boltzmann-weighted average excitation energies for this state being shifted by up to 0.05 eV relative to those obtained from purely static calculations. Accordingly, it is concluded that the effects of thermal motion on excitation energies of conjugated systems can be quite large even for molecules with distinct potential-energy minima. Funding: Olle Engkvist Foundation [184-568, 204-0183]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2019-03664, 2018-05973]; AForsk [20-570]; Carl Trygger Foundation [CTS20:102]; Linkoping University

Members of the Mesorhizobium genus are soil bacteria that often form nitrogen-fixing symbioses with legumes. Most characterised Mesorhizobium spp. genomes are ~8 Mb in size and harbour extensive pangenomes including large integrative and conjugative elements (ICEs) carrying genes required for symbiosis (ICESyms). Here, we document and compare the conjugative mobilome of 41 complete Mesorhizobium genomes. We delineated 56 ICEs and 24 integrative and mobilizable elements (IMEs) collectively occupying 16 distinct integration sites, along with 24 plasmids. We also demonstrated horizontal transfer of the largest (853,775 bp) documented ICE, the tripartite ICEMspSymAA22. The conjugation systems of all identified ICEs and several plasmids were related to those of the paradigm ICESym ICEMlSymR7A, with each carrying conserved genes for conjugative pilus formation (trb), excision (rdfS), DNA transfer (rlxS) and regulation (fseA). ICESyms have likely evolved from a common ancestor, despite occupying a variety of distinct integration sites and specifying symbiosis with diverse legumes. We found extensive evidence for recombination between ICEs and particularly ICESyms, which all uniquely lack the conjugation entry-exclusion factor gene trbK. Frequent duplication, replacement and pseudogenization of genes for quorum-sensing-mediated activation and antiactivation of ICE transfer suggests ICE transfer regulation is constantly evolving. Pangenome-wide association analysis of the ICE identified genes potentially involved in symbiosis, rhizosphere colonisation and/or adaptation to distinct legume hosts. In summary, the Mesorhizobium genus has accumulated a large and dynamic pangenome that evolves through ongoing horizontal gene transfer of large conjugative elements related to ICEMlSymR7A.

Salmonella enterica and Escherichia coli are important human pathogens that frequently contain plasmids, both large and small, carrying antibiotic resistance genes. Large conjugative plasmids are known to mobilize small Col plasmids, but less is known about the specificity of mobilization. In the current study, six S. enterica and four E. coli strains containing large plasmids were tested for their ability to mobilize three different kanamycin resistance Col plasmids (KanR plasmids). Large conjugative plasmids from five isolates, four S. enterica and one E. coli, were able to mobilize KanR plasmids of various types. Plasmids capable of mobilizing the KanR plasmids were either IncI1 or IncX, while IncI1 and IncX plasmids with no evidence of conjugation had disrupted transfer regions. Conjugative plasmids of similar types mobilized similar KanR plasmids, but not all conjugative plasmid types were capable of mobilizing all of the KanR plasmids. These data describe some of the complexities and specificities of individual small plasmid mobilization.

© 2021 Palencia-Gándara et al., Plasmid conjugation is a major route for the spread of antibiotic resistance genes. Inhibiting conjugation has been proposed as a feasible strategy to stop or delay the propagation of antibiotic resistance genes. Several compounds have been shown to be conjugation inhibitors in vitro, specifically targeting the plasmid horizontal transfer machinery. However, the in vivo efficiency and the applicability of these compounds to clinical and environmental settings remained untested. Here we show that the synthetic fatty acid 2-hexadecynoic acid (2-HDA), when used as a fish food supplement, lowers the conjugation frequency of model plasmids up to 10-fold in controlled water microcosms. When added to the food for mice, 2-HDA diminished the conjugation efficiency 50-fold in controlled plasmid transfer assays carried out in the mouse gut. These results demonstrate the in vivo efficiency of conjugation inhibitors, paving the way for their potential application in clinical and environmental settings., The work performed by the de la Cruz research group was supported by the European Union Seventh Framework Program (FP7-HEALTH-2011-single-stage) “Evolution and Transfer of Antibiotic Resistance” (EvoTAR), grant agreement number 282004. The work performed by C.P.-G. and M.G. was supported by Ph.D. fellowships funded by the University of Cantabria. The work performed by the B.G.-Z. laboratory was supported by The EFFORT project (www.effort-against-amr.eu) FP7-KBBE-2013-7, grant agreement 613754.

Optimization of peptides for therapeutic purposes often includes chemical conjugation or modification with substituents that serve to broaden pharmacology or improve pharmacokinetics. We report a convenient and rapid procedure for one-pot, site-specific conjugation of two cysteine-containing peptides that utilizes a bivalent linker comprising maleimide and iodoacetyl functional groups. Following maleimide-mediated peptide conjugation the linker was converted from an unstable thiosuccinimide to a stable thioether bond suitable for biological study by mild aqueous hydrolysis. The procedure is exemplified by peptide-peptide, peptide-small molecule, and peptide-fatty acid conjugations. The method provides a facile approach to search for enhanced biological outcomes through additive and sustained peptide pharmacology unencumbered by the prospect of chemical rearrangement in the course of biological study.

During infection, pathogen sensing and cytokine signaling by the host induce expression of antimicrobial proteins and specialized post-translational modifications. One such protein is ISG15, a ubiquitin-like protein (UBL) conserved among vertebrates. Similar to ubiquitin, ISG15 covalently conjugates to lysine residues in substrate proteins in a process called ISGylation. Mice deficient for ISGylation or lacking ISG15 are strongly susceptible to many viral pathogens and several intracellular bacterial pathogens. Although ISG15 was the first UBL discovered after ubiquitin, the mechanisms behind its protective activity are poorly understood. Largely, this stems from a lack of knowledge on the ISG15 substrate repertoire. To unravel the antiviral activity of ISG15, early studies used mass spectrometry-based proteomics in combination with ISG15 pulldown. Despite reporting hundreds of ISG15 substrates, these studies were unable to identify the exact sites of modification, impeding a clear understanding of the molecular consequences of protein ISGylation. More recently, a peptide-based enrichment approach revolutionized the study of ubiquitin allowing untargeted discovery of ubiquitin substrates, including knowledge of their exact modification sites. Shared molecular determinants between ISG15 and ubiquitin allowed to take advantage of this technology for proteome-wide mapping of ISG15 substrates and modification sites. In this review, we provide a comprehensive overview of mass spectrometry-based proteomics studies on protein ISGylation. We critically discuss the relevant literature, compare reported substrates and sites and make suggestions for future research.

Metals are widely used in animal feed for their growth-stimulating and antimicrobial effects, yet their use may potentially promote the proliferation of antibiotic resistance through co-selection. We studied the prevalence and associations of metal, antibiotic, and disinfectant resistances of 300 Salmonella Typhimurium isolates from pig meat, pig manure, chicken meat, poultry manure, and human stool from Sichuan, China. Seventy four percent of the 300 Salmonella Typhimurium isolates were considered resistant to Cu, almost 50% to Zn and Cr, over 25% to Mn and Cd, and almost 10% to Co. Most of the isolates carried at least one heavy metal resistance gene (HMRG). The Cr-Zn-Cd-resistance gene czcD was carried by 254 isolates and the Cu-resistance genes pcoR and pcoC by 196 and 179 isolates, respectively. Most of the isolates were resistant to at least one antibiotic and almost 80% were multidrug-resistant. The prevalence of resistance to six antibiotics was higher among the pig meat and manure isolates than among other isolates, and that of streptomycin and ampicillin were highest among the pig meat isolates and that of ciprofloxacin and ofloxacin among the pig manure isolates. From 55 to 79% of the isolates were considered resistant to disinfectants triclosan, trichloroisocyanuric acid, or benzalkonium chloride. The metal resistances and HMRGs were associated with resistance to antibiotics and disinfectants. Especially, Cu-resistance genes were associated with resistance to several antibiotics and disinfectants. The transfer of the Cr-Zn-Cd-resistance gene czcD, Cu-resistance gene pcoC, and Co-Ni-resistance gene cnrA into Escherichia coli and the increased Cu-resistance of the transconjugants implied that the resistance genes were located on conjugative plasmids. Thus, the excessive use of metals and disinfectants as feed additives and in animal care may have the potential to promote antibiotic resistance through co-selection and maintain and promote antibiotic resistance even in the absence of antibiotics.

International audience; Natural peptides isolated from animal venoms generally target cell surface receptors with high affinity and selectivity. On many occasions, some of these receptors are over-expressed in cancer cells. Herein, we identified Lqh-8/6 as a natural peptide analog of chlorotoxin, a proven and useful compound for the diagnosis and treatment of glioma. Lqh-8/6 and two other natural analogues were chemically synthesized for the first time and evaluated for their ability to label, detect and prevent glioma growth in vitro. We demonstrate that a biotinylated version of Lqh-8/6 allows both the labeling of glioma cell lines and the detection of glioma in brain sections of glioma allograft Fisher rats. Lqh-8/6 has intrinsic anti-invasive properties but is non-toxic to glioma cells. To confer anti-tumor properties to Lqh-8/6, we chemically coupled doxorubicin to the glioma-targeting peptide using click chemistry. To this end, we successfully chemically synthesized Lqh-8/6-azide and doxorubicin-alkyne without impairing the toxic nature of doxorubicin. The toxin-drug conjugate efficiently promotes the apoptosis of glioma cells in vitro. This example contributes to the concept that animal venom peptides constitute exquisite warheads for delivering toxic chemical conjugates, a parallel to the popular concept of antibody-drug conjugates for the treatment of cancer.