Translocation of non-lytic antimicrobial peptides and bacteria penetrating peptides across the inner membrane of the bacterial envelope.

The increase in multidrug-resistant pathogenic bacteria has become a problem worldwide. Currently there is a strong focus on the development of novel antimicrobials, including antimicrobial peptides (AMP) and antimicrobial antisense agents. While the majority of AMP have membrane activity and kill bacteria through membrane disruption, non-lytic AMP are non-membrane active, internalize and have intracellular targets. Antimicrobial antisense agents such as peptide nucleic acids (PNA) and phosphorodiamidate morpholino oligomers (PMO), show great promise as novel antibacterial agents, killing bacteria by inhibiting translation of essential target gene transcripts. However, naked PNA and PMO are unable to translocate across the cell envelope of bacteria, to reach their target in the cytosol, and are conjugated to bacteria penetrating peptides (BPP) for cytosolic delivery. Here, we discuss how non-lytic AMP and BPP-PMO/PNA conjugates translocate across the cytoplasmic membrane via receptor-mediated transport, such as the cytoplasmic membrane transporters SbmA, MdtM/YjiL, and/or YgdD, or via a less well described autonomous process.

A modified Delphi approach to determine current treatment advances for the development of a resuscitation program for maternal cardiac arrest.

OBJECTIVE: Maternal cardiac arrest is a rare and complex process requiring pregnancy-specific responses and techniques. The goals of this study were to (1) identify, evaluate, and determine the most current best practices to treat this patient population and (2) establish a standardized set of guidelines to serve as a foundation for a future educational simulation-based curriculum. STUDY DESIGN: We used a three-step modified Delphi process to achieve consensus. Twenty-two healthcare experts from across North America agreed to participate in the expert panel. In round 1, 12 pregnancy-specific best practice statements were distributed to the expert panel. Panelists anonymously ranked these using a 7-point Likert scale and provided feedback. Round 2 consisted of a face-to-face consensus meeting where statements that had not already achieved consensus were discussed and then subsequently voted upon by the panelists. RESULTS: Through two rounds, we achieved consensus on nine evidence-based pregnancy-specific techniques to optimize response to maternal cardiac arrest. Round one resulted in one of the 12 best practice statements achieving consensus. Round two resulted in six of the remaining 12 gaining consensus. Best practice techniques involved use of point-of care ultrasound, resuscitative cesarean delivery, cardiopulmonary resuscitation techniques, and the use of extracorporeal cardiopulmonary resuscitation. CONCLUSION: The results of this study provide the foundation to develop an optimal, long-term strategy to treat cardiac arrest in pregnancy. We propose these nine priorities for standard practice, curricula, and guidelines to treat maternal cardiac arrest and hope they serve as a foundation for a future educational curriculum.

Antisense inhibition of the Escherichia coli NrdAB aerobic ribonucleotide reductase is bactericidal due to induction of DNA strand breaks.

BACKGROUND: Antisense peptide nucleic acids (PNAs) constitute an alternative to traditional antibiotics, by their ability to silence essential genes. OBJECTIVES: To evaluate the antibacterial effects of antisense PNA-peptide conjugates that target the gene encoding the alpha subunit (NrdA) of the Escherichia coli ribonucleotide reductase (RNR). METHODS: Bacterial susceptibility of a series of NrdA-targeting PNAs was studied by MIC determination and time-kill analysis. Western-blot analysis, gene complementation and synergy with hydroxyurea were employed to determine the efficiency of NrdA-PNA antisense treatment. The effect on chromosome replication was addressed by determining the DNA synthesis rate, by flow cytometry analysis, by quantitative PCR and by fluorescence microscopy. The use of DNA repair mutants provided insight into the bactericidal action of NrdA-PNA. RESULTS: Treatment with NrdA-PNA specifically inhibited growth of E. coli, as well as NrdA protein translation at 4 µM. Also, the DNA synthesis rate was reduced, preventing completion of chromosome replication and resulting in formation of double-stranded DNA breaks and cell death. CONCLUSIONS: These data present subunits of the NrdAB RNR as a target for future antisense microbial agents and provide insight into the bacterial physiological response to RNR-targeting antimicrobials.

Effective Cellular Delivery of Antisense Peptide Nucleic Acid by Conjugation to Guanidinylated Diaminobutanoic Acid-Based Peptide Dendrons.

A series of amino- and guanidino-terminating 3- and 4-generation 2,4-diaminobutanoic acid (Dab) dendrons have been robustly synthesized on a solid phase and characterized as cellular delivery agents in antisense peptide nucleic acid (PNA) conjugates in the pLuc705 HeLa cell splice switching system. The dendron-PNA conjugates exhibited splice correction activity at one digit micromolar concentrations, and guanidino-terminating dendrons were significantly more effective than analogous amine terminating ones. Furthermore, introduction of lipophilic groups such as phenyl, alkyl, or fatty acids increased efficacy, but also increased cellular toxicity. Fluorescence microscopy analyses supported an endosomal uptake mechanism and furthermore predominantly showed colocalization with late endosomes and lysosomes. The robust solid phase synthesis should make such Dab-dendrons a useful platform for further in vitro as well as in vivo optimization.

Antibacterial Peptide Nucleic Acid-Antimicrobial Peptide (PNA-AMP) Conjugates: Antisense Targeting of Fatty Acid Biosynthesis.

Antisense peptide nucleic acid (PNA) oligomers constitute a novel class of potential antibiotics that inhibit bacterial growth via specific knockdown of essential gene expression. However, discovery of efficient, nontoxic delivery vehicles for such PNA oligomers has remained a challenge. In the present study we show that antimicrobial peptides (AMPs) with an intracellular mode of action can be efficient vehicles for bacterial delivery of an antibacterial PNA targeting the essential acpP gene. The results demonstrate that buforin 2-A (BF2-A), drosocin, oncocin 10, Pep-1-K, KLW-9,13-a, (P59→W59)-Tat48-60, BF-2A-RXR, and drosocin-RXR are capable of transporting PNA effectively into E. coli (MICs of 1-4 µM). Importantly, presence of the inner-membrane peptide transporter SbmA was not required for antibacterial activity of PNA-AMP conjugates containing Pep-1-K, KLW-9,13-a, or drosocin-RXR (MICs of 2-4 µM).

Detection and interpretation of 8-oxodG and 8-oxoGua in urine, plasma and cerebrospinal fluid.

BACKGROUND: DNA and RNA oxidations have been linked to diseases such as cancer, arteriosclerosis, neurodegeneration and diabetes. The prototype base modification studied is the 8-hydroxylation of guanine. DNA integrity is maintained by elaborate repair systems and RNA integrity is less studied but relies mainly on degradation. SCOPE OF REVIEW: DNA and RNA oxidations are measured by very similar techniques. The scope of this review is to highlight the preferred methods of measurement of oxidized nucleic acid metabolites, to highlight novel findings particularly in RNA oxidation, and to present the interpretation of the measurements. MAJOR CONCLUSIONS: Tissue levels are snap-shots of the level in a specific organ or cell system and reflect the balance between formation rate and elimination rate (repair), and must be interpreted as such. Urinary excretion is a global measure of oxidative stress in an organism and is therefore best suited for situations or diseases where large parts or the entire organism is stressed by oxidation. It represents the body average rate by which either RNA or DNA is oxidized and is interpreted as oxidative stress. Oxidations of RNA and DNA precursors have been demonstrated and the quantitative importance is debated. GENERAL SIGNIFICANCE: Careful experimental designs and appropriate choice of methodology are paramount for correct testing of hypotheses related to oxidative stress, and pitfalls are plentiful. There is accumulating evidence that DNA oxidation is associated with disease, particularly cancer, and recent evidence points at an association between RNA oxidation and neurodegenerative diseases and diabetes. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Cellular Antisense Activity of PNA-Oligo(bicycloguanidinium) Conjugates Forming Self-Assembled Nanoaggregates.

A series of peptide nucleic acid-oligo(bicycloguanidinium) (PNA-BGn ) conjugates were synthesized and characterized in terms of cellular antisense activity by using the pLuc750HeLa cell splice correction assay. PNA-BG4 conjugates exhibited low micromolar antisense activity, and their cellular activity required the presence of a hydrophobic silyl terminal protecting group on the oligo(BG) ligand and a minimum of four guanidinium units. Surprisingly, a nonlinear dose-response with an activity threshold around 3-4 µM, indicative of large cooperativity, was observed. Supported by light scattering and electron microscopy analyses, we propose that the activity, and thus cellular delivery, of these lipo-PNA-BG4 conjugates is dependent on self-assembled nanoaggregates. Finally, cellular activity was enhanced by the presence of serum. Therefore we conclude that the lipo-BG-PNA conjugates exhibit an unexpected mechanism for cell delivery and are of interest for further in vivo studies.

Accuracy of the Adverse Outcome Index: An Obstetrical Quality Measure.

BACKGROUND: In obstetrics, a nationally accepted set of quality indicators for patient safety was not available in the United States until the development of a set of 10 adverse outcome measures-the Adverse Outcome Index (AOI). The National Perinatal Information Center (NPIC) developed hospital discharge data-based algorithms combined with a small set of supplemental patient data for calculation of the AOI. A study was conducted to determine the specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) of the AOI by using the National Perinatal Information Center (NPIC) algorithm. METHODS: A retrospective chart review of 4,252 obstetrical and neonatal charts from 2003 through 2007 was performed. NPIC definitions were compared with the "gold standard"-chart review. RESULTS: A total of 229 deliveries among the 4,000 randomly selected charts had at least one adverse outcome, reflecting an AOI of 5.7%. For detection of the 10 adverse outcomes within the AOI, the overall sensitivity of the AOI was 81.7%, specificity was 98.2%, PPV was 86.3%, and NPV was 97.4%. The Kappa value for agreement between the coded charts and the chart review was 0.82 (standard deviation=0.01, 95% confidence interval [CI]=0.80-0.85), which is considered very good. DISCUSSION: The AOI is highly reliant on accurate coding and provider documentation and requires validation with manual chart review. Concurrent chart review improves the accuracy of the AOI. Caution is advised when using the AOI as an exclusive measure of assessing obstetric quality because it may be heavily influenced by a single outcome measure; perineal laceration rates represented twice the frequency of all other outcomes combined. The AOI should be modified to better measure preventable adverse events and include a means of accounting for preexisting conditions.

Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B.

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B.

Cross-catalytic peptide nucleic acid (PNA) replication based on templated ligation.

We report the first PNA self-replicating system based on template directed cross-catalytic ligation, a process analogous to biological replication. Using two template PNAs and four pentameric precursor PNAs, all four possible carbodiimide assisted amide ligation products were detected and identified by HPLC and MALDI-TOF analysis. We conclude that the two template complementary reaction products are generated via cross-catalysis, while the other two self-complementary (and in principle auto-catalytic) products are formed via intra-complex coupling between the two sets of complementary PNA precursors. Cross-catalytic product formation followed product inhibited kinetics, but approximately two replication rounds were observed. Analogous but less efficient replication was found for a similar tetrameric system. These results demonstrate that simpler nucleobase replication systems than natural oligonucleotides are feasible, thereby strengthening the foundation for the discussion of a possible role for PNA (like) genetic material in the prebiotic evolution of life and lay the ground for further studies into evolution of such potentially prebiotic systems.

Adaptation of the World Health Organization (WHO) Safe Surgery Checklist for Use With Cesarean Sections: Implementation and Outcomes With the Safe Cesarean Section Checklist.

Introduction The World Health Organization (WHO) Safe Surgery Checklist significantly decreases morbidity and mortality in regular operating room cases. However, significant differences in workflow and processes exist between regular operating room cases and cesarean sections performed on the labor and delivery unit. The aim of this study is to adapt the WHO Safe Surgery Checklist for the labor and delivery unit and cesarean sections to improve communication and patient safety. Methods A multidisciplinary team consisting of all major stakeholders reviewed and revised the WHO Safe Surgery Checklist making it more applicable to cesarean section operations. The new Safe Cesarean Section Checklist was tested and then integrated into the electronic medical record and utilized on the labor and delivery unit. A specific cesarean section safety attitudes questionnaire was developed, validated, and administered prior to and one year after implementation. Results Usage of the Safe Cesarean Section Checklist was greater than 95% after initial implementation. Significant improvements were reported by the staff on the cesarean section attitudes questionnaire for several key areas including the feeling that all necessary information was available at the beginning of the procedure, decreases in communication breakdowns and delays, and fewer issues related to not knowing who was in charge during the procedure. Discussion Implementation of the Safe Cesarean Section Checklist was successfully adopted by the staff, and improvements in staff perceptions of several key safety issues on our unit were demonstrated. Additional studies should be undertaken to determine if clinical outcomes from this intervention are comparable to those seen with the use of the WHO Safe Surgery Checklist.

Polymyxins with Potent Antibacterial Activity against Colistin-Resistant Pathogens: Fine-Tuning Hydrophobicity with Unnatural Amino Acids.

In view of the increased prevalence of antimicrobial resistance among human pathogens, antibiotics against multidrug-resistant (MDR) bacteria are in urgent demand. In particular, the rapidly emerging resistance to last-resort antibiotic colistin, used for severe Gram-negative MDR infections, is critical. Here, a series of polymyxins containing unnatural amino acids were explored, and some analogues exhibited excellent antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Hydrophobicity of the compounds within this series (as measured by retention in reversed-phase analytical HPLC) exhibited a discernible correlation with their antimicrobial activity. This trend was particularly pronounced for colistin-resistant pathogens. The most active compounds demonstrated competitive activity against a panel of Gram-negative pathogens, while exhibiting low in vitro cytotoxicity. Importantly, most of these hits also retained (or even had increased) potency against colistin-susceptible strains. These findings infer that fine-tuning hydrophobicity may enable the design of polymyxin analogues with favorable activity profiles.

Maternal pulse pressure at admission is a risk factor for fetal heart rate changes after initial dosing of a labor epidural: a retrospective cohort study.

OBJECTIVE: To examine low maternal admission pulse pressure (PP) as a risk factor for new onset postepidural fetal heart rate (FHR) abnormalities. STUDY DESIGN: Retrospective cohort study of nulliparous, singleton, vertex-presenting women admitted to labor and delivery after 37 0/7 weeks that received an epidural during labor. Women with a low admission PP were compared with those with a normal admission PP. The primary outcome was new onset FHR abnormalities defined as recurrent late or prolonged FHR decelerations in the first hour after initial dosing of a labor epidural. RESULTS: New onset FHR abnormalities, defined as recurrent late decelerations and/or prolonged decelerations, occurred in 6% of subjects in the normal PP cohort compared with 27% in the low PP cohort (odds ratio, 5.6; 95% confidence interval, 2.1-14.3; P < .001). A multivariate logistic regression analysis generated an adjusted odds ratio of 28.9 (95% confidence interval, 3.7-221.4; P < .001). CONCLUSION: New onset FHR abnormalities after initial labor epidural dosing occur more frequently in women with a low admission PP than those with a normal admission pulse. Admission PP appears to be a novel predictor of new onset postepidural FHR abnormalities.

Peptide nucleic acid-zirconium coordination nanoparticles.

Ideal drug carriers feature a high loading capacity to minimize the exposure of patients with excessive, inactive carrier materials. The highest imaginable loading capacity could be achieved by nanocarriers, which are assembled from the therapeutic cargo molecules themselves. Here, we describe peptide nucleic acid (PNA)-based zirconium (Zr) coordination nanoparticles which exhibit very high PNA loading of [Formula: see text] w/w. This metal-organic hybrid nanomaterial class extends the enormous compound space of coordination polymers towards bioactive oligonucleotide linkers. The architecture of single- or double-stranded PNAs was systematically varied to identify design criteria for the coordination driven self-assembly with Zr(IV) nodes at room temperature. Aromatic carboxylic acid functions, serving as Lewis bases, and a two-step synthesis process with preformation of [Formula: see text] turned out to be decisive for successful nanoparticle assembly. Confocal laser scanning microscopy confirmed that the PNA-Zr nanoparticles are readily internalized by cells. PNA-Zr nanoparticles, coated with a cationic lipopeptide, successfully delivered an antisense PNA sequence for splicing correction of the [Formula: see text]-globin intron mutation IVS2-705 into a functional reporter cell line and mediated splice-switching via interaction with the endogenous mRNA splicing machinery. The presented PNA-Zr nanoparticles represent a bioactive platform with high design flexibility and extraordinary PNA loading capacity, where the nucleic acid constitutes an integral part of the material, instead of being loaded into passive delivery systems.

Validation of a Simulation-Based Resuscitation Curriculum for Maternal Cardiac Arrest.

OBJECTIVE: To assess the knowledge, skills, and self-efficacy of health care participants completing a simulation-based blended learning training curriculum on managing maternal medical emergencies and maternal cardiac arrest (Obstetric Life Support). METHODS: A formative assessment of the Obstetric Life Support curriculum was performed with a prehospital cohort comprising emergency medical services professionals and a hospital-based cohort comprising health care professionals who work primarily in hospital or urgent care settings and respond to maternal medical emergencies. The training consisted of self-guided precourse work and an instructor-led simulation course using a customized low-fidelity simulator. Baseline and postcourse assessments included multiple-choice cognitive test, self-efficacy questionnaire, and graded Megacode assessment of the team leader. Megacode scores and pass rates were analyzed descriptively. Pre- and post-self-confidence assessments were compared with an exact binomial test, and cognitive scores were compared with generalized linear mixed models. RESULTS: The training was offered to 88 participants between December 2019 and November 2021. Eighty-five participants consented to participation; 77 participants completed the training over eight sessions. At baseline, fewer than half of participants were able to achieve a passing score on the cognitive assessment as determined by the expert panel. After the course, mean cognitive assessment scores improved by 13 points, from 69.4% at baseline to 82.4% after the course (95% CI 10.9-15.1, P <.001). Megacode scores averaged 90.7±6.4%. The Megacode pass rate was 96.1%. There were significant improvements in participant self-efficacy, and the majority of participants (92.6%) agreed or strongly agreed that the course met its educational objectives. CONCLUSION: After completing a simulation-based blended learning program focused on managing maternal cardiac arrest using a customized low-fidelity simulator, most participants achieved a defensible passing Megacode score and significantly improved their knowledge, skills, and self-efficacy.

Nanomolar cellular antisense activity of peptide nucleic acid (PNA) cholic acid ("umbrella") and cholesterol conjugates delivered by cationic lipids.

Limited cellular uptake and low bioavailability of peptide nucleic acids (PNAs) have restricted widespread use of PNAs as antisense/antigene agents for cells in culture and not least for in vivo applications. We now report the synthesis and cellular antisense activity in cultured HeLa pLuc705 cells of cholesterol and cholic acid ("umbrella") derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 µM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs, conjugated to cholesterol through an ester hemisuccinate linker or to cholic acid, exhibited low nanomolar activity (EC(50) ∼ 25 nM). Excellent sequence specificity was retained, as mismatch PNA conjugates did not show any significant antisense activity. Furthermore, we show that increasing the transfection volume improved transfection efficiency, suggesting that accumulation (condensation) of the PNA/lipid complex on the cellular surface is part of the uptake mechanism. These results provide a novel, simple method for very efficient cellular delivery of PNA oligomers, especially using PNA-cholic acid conjugates which, in contrast to PNA-cholesterol conjugates, exhibit sufficient water solubility. The results also question the generality of using cholic acid "umbrella" derivatives as a delivery modality for antisense oligomers.

Mobile health evaluation methods: the Text4baby case study.

Mobile phones have been shown effective in several public health domains. However, there are few evaluations of the effectiveness of mobile health in health promotion. Also, although many studies have referenced behavioral theory, none appears to have explicitly tested theoretical assumptions or demonstrated mechanisms of change. More robust evaluation models that incorporate theory and measurement of behavioral mediators are needed. As in all public health programs, mobile health operates within a social ecological context. For example, organizational- and individual-level programs seek to influence health and health care practices and individual health behaviors. New programs such as Text4baby demonstrate how theory and explicit testing of mediators can be incorporated in evaluations. There are challenges and opportunities facing mHealth evaluations given the nature of the mobile channel. Mobile communication is ubiquitous, available at all times and places, and thus experimental control is often difficult. Natural experiments using variation in dosage of mHealth and place- or location-based designs may increase experimental control. Text4baby is a text messaging program that provides prenatal care messages to pregnant women and new mothers. It uses a partnership model with health care facilities often serving as local implementation partners. The authors review a case example of the evaluation of Text4baby at Madigan Army Medical Center. Participants were randomized to usual prenatal care plus text messaging or usual care alone. The evaluation has a theoretical model of behavior change and measures mediators as well as behavioral outcomes. Results will inform how behavioral theory works within mobile health programs.

Maintaining access to maternal fetal medicine care by telemedicine during a global pandemic.

OBJECTIVE: This study aims to compare a conventional medical treatment model with a telehealth platform for Maternal Fetal Medicine (MFM) outpatient care during the global novel coronavirus pandemic. METHODS: In this study, we described the process of converting our MFM clinic from a conventional medical treatment model to a telemedicine platform. We compared clinical productivity between the two models. Outcomes were analysed using standard statistical tests. RESULTS: We suffered three symptomatic COVID-19 infections among our clinical providers and staff prior to the conversion, compared with none after the conversion. We had a significant decrease in patient visits following the conversion (53.35 visits per day versus 40.3 visits per day, p < 0.0001). However, our average daily patient visits per full-time equivalent (FTE) were only marginally reduced (11.1 visit per FTE versus 7.6 visits per FTE, p < 0.0001), resulting in a relative decrease in adjusted work relative value units (6987 versus 5440). There was an increase in more basic follow-up ultrasound procedures, complexity (current procedural technology [CPT] code 76816 (10.7% versus 19.5%, relative risk [RR] 1.81, 95% CI 1.60-2.05, p < 0.0001)) over comprehensive follow-up ultrasound procedures, CPT code 76805 (17.2% versus 7.8%, RR 0.46, 95% CI 0.39-0.53, p < 0.0001) after conversion. Despite similar proportions of new consults, there was an increase in the proportion of follow-up visits and medical decision-making complexity evaluation and management CPT codes (e.g. 99214/99215) after the conversion (17.2% versus 24.6%, RR 1.43, 95% CI 1.26-163, p < 0.0001). There were no differences between amniocentesis procedures performed between the two time periods (0.3% versus 0.2%, p = 0.5805). CONCLUSION: The rapid conversion of an MFM platform from convention medical treatment to telemedicine platform in response to the novel coronavirus pandemic resulted in protection of healthcare personnel and MFM patients, with only a modest decrease in clinical productivity during the initial roll-out. Due to the ongoing threat from the novel coronavirus-19, an MFM telemedicine platform is a practicable and innovative solution and merits the continued support of CMS and health care administrators.

Effects of LPS Composition in Escherichia coli on Antibacterial Activity and Bacterial Uptake of Antisense Peptide-PNA Conjugates.

The physical and chemical properties of the outer membrane of Gram-negative bacteria including Escherichia coli have a significant impact on the antibacterial activity and uptake of antibiotics, including antimicrobial peptides and antisense peptide-peptide nucleic acid (PNA) conjugates. Using a defined subset of E. coli lipopolysaccharide (LPS) and envelope mutants, components of the LPS-core, which provide differential susceptibility toward a panel of bacterial penetrating peptide (BPP)-PNA conjugates, were identified. Deleting the outer core of the LPS and perturbing the inner core only sensitized the bacteria toward (KFF)3K-PNA conjugates, but not toward conjugates carrying arginine-based BPPs. Interestingly, the chemical composition of the outer LPS core as such, rather than overall hydrophobicity or surface charge, appears to determine the susceptibility to different BPP-PNA conjugates thereby clearly demonstrating the complexity and specificity of the interaction with the LPS/outer membrane. Notably, mutants with outer membrane changes conferring polymyxin resistance did not show resistance toward the BPP-PNA conjugates, thereby eliminating one possible route of resistance for these molecules. Finally, envelope weakening, through deletion of membrane proteins such as OmpA as well as some proteins previously identified as involved in cationic antimicrobial peptide uptake, did not significantly influence BPP-PNA conjugate activity.

Antisense Peptide Nucleic Acid-Diaminobutanoic Acid Dendron Conjugates with SbmA-Independent Antimicrobial Activity against Gram-Negative Bacteria.

Precision antisense antibacterial agents may be developed into novel antibiotics in the fight against multidrug-resistant Gram-negative bacteria. In this study, a series of diaminobutanoic acid (DAB) dendrons are presented as novel carriers for the delivery of antisense antibacterial peptide nucleic acids (PNAs). The dendron-PNA conjugates targeting the essential acpP gene exhibit specific antisense antimicrobial bactericidal activity against Escherichia coli and Klebsiella pneumoniae at one-digit micromolar concentrations, while showing low toxicity to human cells. One compound selected from a structure-activity relationship series showed high stability in mouse and human serum (t1/2 ≫ 24 h) as well as in vivo activity against a multidrug-resistant, extended spectrum beta-lactamase-producing E. coli in a murine peritonitis model. The compound was also well tolerated in mice upon i.v. administration up to a dose of 20 mg/kg, and in vivo fluorescence imaging indicated clearance via renal excretion with slight accumulation in the kidneys and liver. Thus, DAB-based dendrons constitute a promising new chemistry platform for development of effective delivery agents for antibacterial drugs with possible in vivo use.