Loss of Pde1 function acts as an evolutionary gateway to penicillin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen and rising resistance to ß-lactam antibiotics, such as penicillin, is a significant threat to global public health. Mutations occurring in the penicillin-binding proteins (PBPs) can confer high-level penicillin resistance but other poorly understood genetic factors are also important. Here, we combined strictly controlled laboratory experiments and population analyses to identify a new penicillin resistance pathway that is independent of PBP modification. Initial laboratory selection experiments identified high-frequency pde1 mutations conferring S. pneumoniae penicillin resistance. The importance of variation at the pde1 locus was confirmed in natural and clinical populations in an analysis of >7,200 S. pneumoniae genomes. The pde1 mutations identified by these approaches reduce the hydrolytic activity of the Pde1 enzyme in bacterial cells and thereby elevate levels of cyclic-di-adenosine monophosphate and penicillin resistance. Our results reveal rapid de novo loss of function mutations in pde1 as an evolutionary gateway conferring low-level penicillin resistance. This relatively simple genomic change allows cells to persist in populations on an adaptive evolutionary pathway to acquire further genetic changes and high-level penicillin resistance.

Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: A new approach to prevent and treat bacterial infections.

The increase in emerging drug resistant Gram-negative bacterial infections is a global concern. In addition, there is growing recognition that compromising the microbiota through the use of broad-spectrum antibiotics can impact long term patient outcomes. Therefore, there is the need to develop new bactericidal strategies to combat Gram-negative infections that would address these specific issues. In this study, we report and characterize one such approach, an antibody-drug conjugate (ADC) that combines (i) targeting the surface of a specific pathogenic organism through a monoclonal antibody with (ii) the high killing activity of an antimicrobial peptide. We focused on a major pathogenic Gram-negative bacterium associated with antibacterial resistance: Pseudomonas aeruginosa. To target this organism, we designed an ADC by fusing an antimicrobial peptide to the C-terminal end of the VH and/or VL-chain of a monoclonal antibody, VSX, that targets the core of P. aeruginosa lipopolysaccharide. This ADC demonstrates appropriately minimal levels of toxicity against mammalian cells, rapidly kills P. aeruginosa strains, and protects mice from P. aeruginosa lung infection when administered therapeutically. Furthermore, we found that the ADC was synergistic with several classes of antibiotics. This approach described in this study might result in a broadly useful strategy for targeting specific pathogenic microorganisms without further augmenting antibiotic resistance.

Reinforcement-Learning-Based Robust Resource Management for Multi-Radio Systems.

The advent of the Internet of Things (IoT) has triggered an increased demand for sensing devices with multiple integrated wireless transceivers. These platforms often support the advantageous use of multiple radio technologies to exploit their differing characteristics. Intelligent radio selection techniques allow these systems to become highly adaptive, ensuring more robust and reliable communications under dynamic channel conditions. In this paper, we focus on the wireless links between devices equipped by deployed operating personnel and intermediary access-point infrastructure. We use multi-radio platforms and wireless devices with multiple and diverse transceiver technologies to produce robust and reliable links through the adaptive control of available transceivers. In this work, the term 'robust' refers to communications that can be maintained despite changes in the environmental and radio conditions, i.e., during periods of interference caused by non-cooperative actors or multi-path or fading conditions in the physical environment. In this paper, a multi-objective reinforcement learning (MORL) framework is applied to address a multi-radio selection and power control problem. We propose independent reward functions to manage the trade-off between the conflicting objectives of minimised power consumption and maximised bit rate. We also adopt an adaptive exploration strategy for learning a robust behaviour policy and compare its online performance to conventional methods. An extension to the multi-objective state-action-reward-state-action (SARSA) algorithm is proposed to implement this adaptive exploration strategy. When applying adaptive exploration to the extended multi-objective SARSA algorithm, we achieve a 20% increase in the F1 score in comparison to one with decayed exploration policies.

Embryo Loss and Moral Status.

There is a significant debate over the moral status of human embryos. This debate has important implications for practices like abortion and IVF. Some argue that embryos have the same moral status as infants, children, and adults. However, critics claim that the frequency of pregnancy loss/miscarriage/spontaneous abortion shows a moral inconsistency in this view. One line of criticism is that those who know the facts about pregnancy loss and nevertheless attempt to conceive children are willing to sacrifice embryos lost for the healthy children they ultimately have. I respond to this criticism and argue that on the most plausible accounts of well-being, these embryos are not made worse off and thus not "sacrifices." I then make some more general remarks about what people's typical views about pregnancy loss show about their views toward the moral status of embryos.

Characterization of an Antibody Recognizing the Conserved Inner Core of Pseudomonas aeruginosa Lipopolysaccharides.

Bacterial infections are a growing public health threat with carbapenem-resistant Pseudomonas aeruginosa being classified as a Priority 1 critical threat by the World Health Organization. Antibody-based therapeutics can serve as an alternative and in some cases supplement antibiotics for the treatment of bacterial infections. The glycans covering the bacterial cell surface have been proposed as intriguing targets for binding by antibodies; however, antibodies that can engage with high affinity and specificity with glycans are much less common compared to antibodies that engage with protein antigens. In this study, we sought to characterize an antibody that targets a conserved glycan epitope on the surface of Pseudomonas. First, we characterized the breadth of binding of VSX, demonstrating that the VSX is specific to Pseudomonas but can bind across multiple serotypes of the organism. Next, we provide insight into how VSX engages with its target epitope, using a combination of biolayer interferometry and nuclear magnetic resonance, and verify our results using site-directed mutagenesis experiments. We demonstrate that the antibody, with limited somatic hypermutation of the complementarity-determining regions (CDRs) and with a characteristic set of arginines within the CDRs, specifically targets the conserved inner core of Pseudomonas lipopolysaccharides. Our results provide important additional context to antibody-glycan contacts and provide insight useful for the construction of vaccines and therapeutics against Pseudomonas aeruginosa, an important human pathogen.

Why Would the Pope Have A Private Audience with Anesthesiologists? The Origin of the "Doctrine of Double Effect" and Its Application to Pain Control.

Sunday February 24, 1957 was a pivotal day in the history of anesthesiology and pain medicine. The leader of the Roman Catholic Church, Pope Pius XII met with anesthesiologists attending an international symposium sponsored by the Italian Society of Anesthesiologists entitled, "Anesthesia and the Human Personality". The purpose of this audience was to seek clarification about the use of opioids at the end of life to reduce suffering. Three questions had been formulated from the previous year's Italian Congress of Anesthesiologists and sent to the Holy See on this specific issue. The Pope responded during this audience remarking that there was no moral obligation to withhold pain medication that could elevate suffering. He further remarked that the suppression of consciousness that can occur with opioids was consistent with the spirit of the Christian gospels. Finally, he also stated that it was not morally objectionable to administer opioids even if it might shorten life. The moral philosophy behind these answers is the doctrine of double effect. In essence, administering medications to relieve pain, the primary effect, may also hasten death, the unintended secondary effect. In seeking answers to these questions, the Italian anesthesiologists were at the forefront of a larger and ongoing debate. As new therapies are developed that may have unintended consequences, when it is morally permissible to use them?

Biological Evaluation of DNA Biomarkers in a Chemically Defined and Site-Specific Manner.

As described elsewhere in this Special Issue on biomarkers, much progress has been made in the detection of modified DNA within organisms at endogenous and exogenous levels of exposure to chemical species, including putative carcinogens and chemotherapeutic agents. Advances in the detection of damaged or unnatural bases have been able to provide correlations to support or refute hypotheses between the level of exposure to oxidative, alkylative, and other stresses, and the resulting DNA damage (lesion formation). However, such stresses can form a plethora of modified nucleobases, and it is therefore difficult to determine the individual contribution of a particular modification to alter a cell's genetic fate, as measured in the form of toxicity by stalled replication past the damage, by subsequent mutation, and by lesion repair. Chemical incorporation of a modification at a specific site within a vector (site-specific mutagenesis) has been a useful tool to deconvolute what types of damage quantified in biologically relevant systems may lead to toxicity and/or mutagenicity, thereby allowing researchers to focus on the most relevant biomarkers that may impact human health. Here, we will review a sampling of the DNA modifications that have been studied by shuttle vector techniques.

Antibody-Bactericidal Macrocyclic Peptide Conjugates To Target Gram-Negative Bacteria.

To combat antimicrobial infections, new active molecules are needed. Antimicrobial peptides, ever abundant in nature, are a fertile starting point to develop new antimicrobial agents but suffer from low stability, low specificity, and off-target toxicity. These drawbacks have limited their development. To overcome some of these limitations, we developed antibody-bactericidal macrocyclic peptide conjugates (ABCs), in which the antibody directs the bioactive macrocyclic peptide to the targeted Gram-negative bacteria. We used cysteine SN Ar chemistry to synthesize and systematically study a library of large (>30-mer) macrocyclic antimicrobial peptides (mAMPs) to discover variants with extended proteolytic stability in human serum and low hemolytic activity while maintaining bioactivity. We then conjugated, by using sortase A, these bioactive variants onto an Escherichia coli targeted monoclonal antibody. We found that these ABCs had minimized hemolytic activity and were able to kill E. coli at nanomolar concentrations. Our findings suggest macrocyclic peptides if fused to antibodies may facilitate the discovery of new agents to treat bacterial infections.

Characterization of Byproducts from Chemical Syntheses of Oligonucleotides Containing 1-Methyladenine and 3-Methylcytosine.

Oligonucleotides serve as important tools for biological, chemical, and medical research. The preparation of oligonucleotides through automated solid-phase synthesis is well-established. However, identification of byproducts generated from DNA synthesis, especially from oligonucleotides containing site-specific modifications, is sometimes challenging. Typical high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis methods alone are not sufficient for characterizing unexpected byproducts, especially for those having identical or very similar molecular weight (MW) to the products. We used a rigorous quality control procedure to characterize byproducts generated during oligonucleotide syntheses: (1) purify oligonucleotides by different HPLC systems; (2) determine exact MW by high-resolution MS; (3) locate modification position by MS/MS or exonuclease digestion with matrix-assisted laser desorption ionization-time of flight analysis; and (4) conduct, where applicable, enzymatic assays. We applied these steps to characterize byproducts in the syntheses of oligonucleotides containing biologically important methyl DNA adducts 1-methyladenine (m1A) and 3-methylcytosine (m3C). In m1A synthesis, we differentiated a regioisomeric byproduct 6-methyladenine, which possesses a MW identical to uncharged m1A. As for m3C, we identified a deamination byproduct 3-methyluracil, which is only 1 Da greater than uncharged m3C in the ∼4900 Da context. The detection of these byproducts would be very challenging if the abovementioned procedure was not adopted.

Intrinsic mutagenic properties of 5-chlorocytosine: A mechanistic connection between chronic inflammation and cancer.

During chronic inflammation, neutrophil-secreted hypochlorous acid can damage nearby cells inducing the genomic accumulation of 5-chlorocytosine (5ClC), a known inflammation biomarker. Although 5ClC has been shown to promote epigenetic changes, it has been unknown heretofore if 5ClC directly perpetrates a mutagenic outcome within the cell. The present work shows that 5ClC is intrinsically mutagenic, both in vitro and, at a level of a single molecule per cell, in vivo. Using biochemical and genetic approaches, we have quantified the mutagenic and toxic properties of 5ClC, showing that this lesion caused C→T transitions at frequencies ranging from 3-9% depending on the polymerase traversing the lesion. X-ray crystallographic studies provided a molecular basis for the mutagenicity of 5ClC; a snapshot of human polymerase ß replicating across a primed 5ClC-containing template uncovered 5ClC engaged in a nascent base pair with an incoming dATP analog. Accommodation of the chlorine substituent in the template major groove enabled a unique interaction between 5ClC and the incoming dATP, which would facilitate mutagenic lesion bypass. The type of mutation induced by 5ClC, the C→T transition, has been previously shown to occur in substantial amounts both in tissues under inflammatory stress and in the genomes of many inflammation-associated cancers. In fact, many sequence-specific mutational signatures uncovered in sequenced cancer genomes feature C→T mutations. Therefore, the mutagenic ability of 5ClC documented in the present study may constitute a direct functional link between chronic inflammation and the genetic changes that enable and promote malignant transformation.

A broadly neutralizing human monoclonal antibody is effective against H7N9.

Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (-24 h) or double-dose (-12 h, +48 h) administration (P < 0.001). A single dose of VIS410 at 50 mg/kg (-12 h) combined with oseltamivir at 50 mg/kg (-12 h, twice daily for 7 d) in C57BL/6 mice infected with A/Shanghai 2/2013 (H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains.

The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond.

The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli "adaptive response" protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1-8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins.

Next-generation sequencing reveals the biological significance of the N(2),3-ethenoguanine lesion in vivo.

Etheno DNA adducts are a prevalent type of DNA damage caused by vinyl chloride (VC) exposure and oxidative stress. Etheno adducts are mutagenic and may contribute to the initiation of several pathologies; thus, elucidating the pathways by which they induce cellular transformation is critical. Although N(2),3-ethenoguanine (N(2),3-εG) is the most abundant etheno adduct, its biological consequences have not been well characterized in cells due to its labile glycosidic bond. Here, a stabilized 2'-fluoro-2'-deoxyribose analog of N(2),3-εG was used to quantify directly its genotoxicity and mutagenicity. A multiplex method involving next-generation sequencing enabled a large-scale in vivo analysis, in which both N(2),3-εG and its isomer 1,N(2)-ethenoguanine (1,N(2)-εG) were evaluated in various repair and replication backgrounds. We found that N(2),3-εG potently induces G to A transitions, the same mutation previously observed in VC-associated tumors. By contrast, 1,N(2)-εG induces various substitutions and frameshifts. We also found that N(2),3-εG is the only etheno lesion that cannot be repaired by AlkB, which partially explains its persistence. Both εG lesions are strong replication blocks and DinB, a translesion polymerase, facilitates the mutagenic bypass of both lesions. Collectively, our results indicate that N(2),3-εG is a biologically important lesion and may have a functional role in VC-induced or inflammation-driven carcinogenesis.

Heparin octasaccharide decoy liposomes inhibit replication of multiple viruses.

Heparan sulfate (HS) is a ubiquitous glycosaminoglycan that serves as a cellular attachment site for a number of significant human pathogens, including respiratory syncytial virus (RSV), human parainfluenza virus 3 (hPIV3), and herpes simplex virus (HSV). Decoy receptors can target pathogens by binding to the receptor pocket on viral attachment proteins, acting as 'molecular sinks' and preventing the pathogen from binding to susceptible host cells. Decoy receptors functionalized with HS could bind to pathogens and prevent infection, so we generated decoy liposomes displaying HS-octasaccharide (HS-octa). These decoy liposomes significantly inhibited RSV, hPIV3, and HSV infectivity in vitro to a greater degree than the original HS-octa building block. The degree of inhibition correlated with the density of HS-octa displayed on the liposome surface. Decoy liposomes with HS-octa inhibited infection of viruses to a greater extent than either full-length heparin or HS-octa alone. Decoy liposomes were effective when added prior to infection or following the initial infection of cells in vitro. By targeting the well-conserved receptor-binding sites of HS-binding viruses, decoy liposomes functionalized with HS-octa are a promising therapeutic antiviral agent and illustrate the utility of the liposome delivery platform.

Mechanism of repair of acrolein- and malondialdehyde-derived exocyclic guanine adducts by the α-ketoglutarate/Fe(II) dioxygenase AlkB.

The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M1dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M1dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M1dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH4. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.

A chemical genetics analysis of the roles of bypass polymerase DinB and DNA repair protein AlkB in processing N2-alkylguanine lesions in vivo.

DinB, the E. coli translesion synthesis polymerase, has been shown to bypass several N2-alkylguanine adducts in vitro, including N2-furfurylguanine, the structural analog of the DNA adduct formed by the antibacterial agent nitrofurazone. Recently, it was demonstrated that the Fe(II)- and α-ketoglutarate-dependent dioxygenase AlkB, a DNA repair enzyme, can dealkylate in vitro a series of N2-alkyguanines, including N2-furfurylguanine. The present study explored, head to head, the in vivo relative contributions of these two DNA maintenance pathways (replicative bypass vs. repair) as they processed a series of structurally varied, biologically relevant N2-alkylguanine lesions: N2-furfurylguanine (FF), 2-tetrahydrofuran-2-yl-methylguanine (HF), 2-methylguanine, and 2-ethylguanine. Each lesion was chemically synthesized and incorporated site-specifically into an M13 bacteriophage genome, which was then replicated in E. coli cells deficient or proficient for DinB and AlkB (4 strains in total). Biochemical tools were employed to analyze the relative replication efficiencies of the phage (a measure of the bypass efficiency of each lesion) and the base composition at the lesion site after replication (a measure of the mutagenesis profile of each lesion). The main findings were: 1) Among the lesions studied, the bulky FF and HF lesions proved to be strong replication blocks when introduced site-specifically on a single-stranded vector in DinB deficient cells. This toxic effect disappeared in the strains expressing physiological levels of DinB. 2) AlkB is known to repair N2-alkylguanine lesions in vitro; however, the presence of AlkB showed no relief from the replication blocks induced by FF and HF in vivo. 3) The mutagenic properties of the entire series of N2-alkyguanines adducts were investigated in vivo for the first time. None of the adducts were mutagenic under the conditions evaluated, regardless of the DinB or AlkB cellular status. Taken together, the data indicated that the cellular pathway to combat bulky N2-alkylguanine DNA adducts was DinB-dependent lesion bypass.

Removal of N-alkyl modifications from N(2)-alkylguanine and N(4)-alkylcytosine in DNA by the adaptive response protein AlkB.

The AlkB enzyme is an Fe(II)- and α-ketoglutarate-dependent dioxygenase that repairs DNA alkyl lesions by a direct reversal of damage mechanism as part of the adaptive response in E. coli. The reported substrate scope of AlkB includes simple DNA alkyl adducts, such as 1-methyladenine, 3-methylcytosine, 3-ethylcytosine, 1-methylguanine, 3-methylthymine, and N(6)-methyladenine, as well as more complex DNA adducts, such as 1,N(6)-ethenoadenine, 3,N(4)-ethenocytosine, and 1,N(6)-ethanoadenine. Previous studies have revealed, in a piecemeal way, that AlkB has an impressive repertoire of substrates. The present study makes two additions to this list, showing that alkyl adducts on the N(2) position of guanine and N(4) position of cytosine are also substrates for AlkB. Using high resolution ESI-TOF mass spectrometry, we show that AlkB has the biochemical capability to repair in vitro N(2)-methylguanine, N(2)-ethylguanine, N(2)-furan-2-yl-methylguanine, N(2)-tetrahydrofuran-2-yl-methylguanine, and N(4)-methylcytosine in ssDNA but not in dsDNA. When viewed together with previous work, the experimental data herein demonstrate that AlkB is able to repair all simple N-alkyl adducts occurring at the Watson-Crick base pairing interface of the four DNA bases, confirming AlkB as a versatile gatekeeper of genomic integrity under alkylation stress.

ß-Peptide coatings by surface-initiated polymerization.

Homopolymers of ß-lactams can be grown by surface-initiated polymerization. These surface-linked ß-peptides are living polymers with the potential to be utilized as tunable, protease-resistant interfaces in multiphase structural composites where the characteristics of the interface influence bulk properties.