Novel gene-intergenic fusion involving ubiquitin E3 ligase UBE3C causes distal hereditary motor neuropathy.

Distal hereditary motor neuropathies (dHMNs) are a group of inherited diseases involving the progressive, length-dependent axonal degeneration of the lower motor neurons. There are currently 29 reported causative genes and four disease loci implicated in dHMN. Despite the high genetic heterogeneity, mutations in the known genes account for less than 20% of dHMN cases, with the mutations identified predominantly being point mutations or indels. We have expanded the spectrum of dHMN mutations with the identification of a 1.35 Mb complex structural variation (SV) causing a form of autosomal dominant dHMN (DHMN1 OMIM %182906). Given the complex nature of SV mutations and the importance of studying pathogenic mechanisms in a neuronal setting, we generated a patient-derived DHMN1 motor neuron model harbouring the 1.35 Mb complex insertion. The DHMN1 complex insertion creates a duplicated copy of the first 10 exons of the ubiquitin-protein E3 ligase gene (UBE3C) and forms a novel gene-intergenic fusion sense transcript by incorporating a terminal pseudo-exon from intergenic sequence within the DHMN1 locus. The UBE3C intergenic fusion (UBE3C-IF) transcript does not undergo nonsense-mediated decay and results in a significant reduction of wild-type full-length UBE3C (UBE3C-WT) protein levels in DHMN1 iPSC-derived motor neurons. An engineered transgenic Caenorhabditis elegans model expressing the UBE3C-IF transcript in GABA-ergic motor neurons shows neuronal synaptic transmission deficits. Furthermore, the transgenic animals are susceptible to heat stress, which may implicate defective protein homeostasis underlying DHMN1 pathogenesis. Identification of the novel UBE3C-IF gene-intergenic fusion transcript in motor neurons highlights a potential new disease mechanism underlying axonal and motor neuron degeneration. These complementary models serve as a powerful paradigm for studying the DHMN1 complex SV and an invaluable tool for defining therapeutic targets for DHMN1.

Transcriptome analysis of islets from diabetes-resistant and diabetes-prone obese mice reveals novel gene regulatory networks involved in beta-cell compensation and failure.

The mechanisms underpinning beta-cell compensation for obesity-associated insulin resistance and beta-cell failure in type 2 diabetes remain poorly understood. We used a large-scale strategy to determine the time-dependent transcriptomic changes in islets of diabetes-prone db/db and diabetes-resistant ob/ob mice at 6 and 16 weeks of age. Differentially expressed genes were subjected to cluster, gene ontology, pathway and gene set enrichment analyses. A distinctive gene expression pattern was observed in 16 week db/db islets in comparison to the other groups with alterations in transcriptional regulators of islet cell identity, upregulation of glucose/lipid metabolism, and various stress response genes, and downregulation of specific amino acid transport and metabolism genes. In contrast, ob/ob islets displayed a coordinated downregulation of metabolic and stress response genes at 6 weeks of age, suggestive of a preemptive reconfiguration in these islets to lower the threshold of metabolic activation in response to increased insulin demand thereby preserving beta-cell function and preventing cellular stress. In addition, amino acid transport and metabolism genes were upregulated in ob/ob islets, suggesting an important role of glutamate metabolism in beta-cell compensation. Gene set enrichment analysis of differentially expressed genes identified the enrichment of binding motifs for transcription factors, FOXO4, NFATC1, and MAZ. siRNA-mediated knockdown of these genes in MIN6 cells altered cell death, insulin secretion, and stress gene expression. In conclusion, these data revealed novel gene regulatory networks involved in beta-cell compensation and failure. Preemptive metabolic reconfiguration in diabetes-resistant islets may dampen metabolic activation and cellular stress during obesity.

Vascular transcriptome landscape of Trail-/- mice: Implications and therapeutic strategies for diabetic vascular disease.

Circulating plasma TRAIL levels are suppressed in patients with cardiovascular and diabetic diseases. To identify novel targets in vascular metabolic diseases, genome-wide transcriptome of aortic tissue from Trail-/- versus Trail+/+ mice were interrogated. We found 861 genes differentially expressed with TRAIL deletion. Gene enrichment analyses showed many of these genes were related to inflammation, cell-to-cell cytoskeletal interactions, and transcriptional modulation. We identified vascular protective and pathological gene clusters, with Ifi205 as the most significantly reduced vascular protective gene, whereas Glut1, the most significantly increased pathological gene with TRAIL deletion. We hypothesized that therapeutic targets could be devised from such integrated analysis and validated our findings from vascular tissues of diabetic mice. From the differentially expressed gene targets, enriched transcription factor (TF) and microRNA binding motifs were identified. The top two TFs were Elk1 and Sp1, with enrichment to eight gene targets common to both. miR-520d-3p and miR-377-3p were the top enriched microRNAs with TRAIL deletion; with four overlapping genes enriched for both microRNAs. Our findings offer an alternate in silico approach for therapeutic target identification and present a deeper understanding of gene signatures and pathways altered with TRAIL suppression in the vasculature.

Identification of miR-34 regulatory networks in settings of disease and antimiR-therapy: Implications for treating cardiac pathology and other diseases.

Expression of the miR-34 family (miR-34a, -34b, -34c) is elevated in settings of heart disease, and inhibition with antimiR-34a/antimiR-34 has emerged as a promising therapeutic strategy. Under chronic cardiac disease settings, targeting the entire miR-34 family is more effective than targeting miR-34a alone. The identification of transcription factor (TF)-miRNA regulatory networks has added complexity to understanding the therapeutic potential of miRNA-based therapies. Here, we sought to determine whether antimiR-34 targets secondary miRNAs via TFs which could contribute to antimiR-34-mediated protection. Using miRNA-Seq we identified differentially regulated miRNAs in hearts from mice with cardiac pathology due to transverse aortic constriction (TAC), and focused on miRNAs which were also regulated by antimiR-34. Two clusters of stress-responsive miRNAs were classified as "pathological" and "cardioprotective," respectively. Using ChIPBase we identified 45 TF binding sites on the promoters of "pathological" and "cardioprotective" miRNAs, and 5 represented direct targets of miR-34, with the capacity to regulate other miRNAs. Knockdown studies in a cardiomyoblast cell line demonstrated that expression of 2 "pathological" miRNAs (let-7e, miR-31) was regulated by one of the identified TFs. Furthermore, by qPCR we confirmed that expression of let-7e and miR-31 was lower in hearts from antimiR-34 treated TAC mice; this may explain why targeting the entire miR-34 family is more effective than targeting miR-34a alone. Finally, we showed that Acsl4 (a common target of miR-34, let-7e and miR-31) was increased in hearts from TAC antimiR-34 treated mice. In summary, antimiR-34 regulates the expression of other miRNAs and this has implications for drug development.

Sex differences in response to miRNA-34a therapy in mouse models of cardiac disease: identification of sex-, disease- and treatment-regulated miRNAs.

KEY POINTS: MicroRNA (miRNA)-based therapies are in development for numerous diseases, including heart disease. Currently, very limited basic information is available on the regulation of specific miRNAs in male and female hearts in settings of disease. The identification of sex-specific miRNA signatures has implications for translation into the clinic and suggests the need for customised therapy. In the present study, we found that a miRNA-based treatment inhibiting miRNA-34a (miR-34a) was more effective in females in a setting of moderate dilated cardiomyopathy than in males. Furthermore, the treatment showed little benefit for either sex in a setting of more severe dilated cardiomyopathy associated with atrial fibrillation. The results highlight the importance of understanding the effect of miRNA-based therapies in cardiac disease settings in males and females. ABSTRACT: MicroRNA (miRNA)-34a (miR-34a) is elevated in the diseased heart in mice and humans. Previous studies have shown that inhibiting miR-34a in male mice in settings of pathological cardiac hypertrophy or ischaemia protects the heart against progression to heart failure. Whether inhibition of miR-34a protects the female heart is unknown. Furthermore, the therapeutic potential of silencing miR-34a in settings of dilated cardiomyopathy (DCM) and atrial fibrillation (AF) has not been assessed previously. In the present study, we examined the effect of silencing miR-34a in males and females in (1) a model of moderate DCM and (2) a model of severe DCM with AF. The cardiac disease models were administered with a locked nucleic acid-modified oligonucleotide (LNA-antimiR-34a) at 6-7 weeks of age when the models display cardiac dysfunction and conduction abnormalities. Cardiac function and morphology were measured 6 weeks after treatment. In the present study, we show that inhibition of miR-34a provides more protection in the DCM model in females than males. Disease prevention in LNA-antimiR-34a treated DCM female mice was characterized by attenuated heart enlargement and lung congestion, lower expression of cardiac stress genes (B-type natriuretic peptide, collagen gene expression), less cardiac fibrosis and better cardiac function. There was no evidence of significant protection in the severe DCM and AF model in either sex. Sex- and treatment-dependent regulation of miRNAs was also identified in the diseased heart, and may explain the differential response of males and females. These studies highlight the importance of examining the impact of miRNA-based drugs in both sexes and under different disease conditions.

KCa1.1, a calcium-activated potassium channel subunit alpha 1, is targeted by miR-17-5p and modulates cell migration in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive, locally invasive, cancer elicited by asbestos exposure and almost invariably a fatal diagnosis. To date, we are one of the leading laboratory that compared microRNA expression profiles in MPM and normal mesothelium samples in order to identify dysregulated microRNAs with functional roles in mesothelioma. We interrogated a significant collection of MPM tumors and normal pleural samples in our biobank in search for novel therapeutic targets. METHODS: Utilizing mRNA-microRNA correlations based on differential gene expression using Gene Set Enrichment Analysis (GSEA), we systematically combined publicly available gene expression datasets with our own MPM data in order to identify candidate targets for MPM therapy. RESULTS: We identified enrichment of target binding sites for the miR-17 and miR-30 families in both MPM tumors and cell lines. RT-qPCR revealed that members of both families were significantly downregulated in MPM tumors and cell lines. Interestingly, lower expression of miR-17-5p (P = 0.022) and miR-20a-5p (P = 0.026) was clearly associated with epithelioid histology. We interrogated the predicted targets of these differentially expressed microRNA families in MPM cell lines, and identified KCa1.1, a calcium-activated potassium channel subunit alpha 1 encoded by the KCNMA1 gene, as a target of miR-17-5p. KCa1.1 was overexpressed in MPM cells compared to the (normal) mesothelial line MeT-5A, and was also upregulated in patient tumor samples compared to normal mesothelium. Transfection of MPM cells with a miR-17-5p mimic or KCNMA1-specific siRNAs reduced mRNA expression of KCa1.1 and inhibited MPM cell migration. Similarly, treatment with paxilline, a small molecule inhibitor of KCa1.1, resulted in suppression of MPM cell migration. CONCLUSION: These functional data implicating KCa1.1 in MPM cell migration support our integrative approach using MPM gene expression datasets to identify novel and potentially druggable targets.

Chronic high-fat diet in fathers programs ß-cell dysfunction in female rat offspring.

The global prevalence of obesity is increasing across most ages in both sexes. This is contributing to the early emergence of type 2 diabetes and its related epidemic. Having either parent obese is an independent risk factor for childhood obesity. Although the detrimental impacts of diet-induced maternal obesity on adiposity and metabolism in offspring are well established, the extent of any contribution of obese fathers is unclear, particularly the role of non-genetic factors in the causal pathway. Here we show that paternal high-fat-diet (HFD) exposure programs ß-cell 'dysfunction' in rat F(1) female offspring. Chronic HFD consumption in Sprague-Dawley fathers induced increased body weight, adiposity, impaired glucose tolerance and insulin sensitivity. Relative to controls, their female offspring had an early onset of impaired insulin secretion and glucose tolerance that worsened with time, and normal adiposity. Paternal HFD altered the expression of 642 pancreatic islet genes in adult female offspring (P < 0.01); genes belonged to 13 functional clusters, including cation and ATP binding, cytoskeleton and intracellular transport. Broader pathway analysis of 2,492 genes differentially expressed (P < 0.05) demonstrated involvement of calcium-, MAPK- and Wnt-signalling pathways, apoptosis and the cell cycle. Hypomethylation of the Il13ra2 gene, which showed the highest fold difference in expression (1.76-fold increase), was demonstrated. This is the first report in mammals of non-genetic, intergenerational transmission of metabolic sequelae of a HFD from father to offspring.

Paternal high-fat diet consumption induces common changes in the transcriptomes of retroperitoneal adipose and pancreatic islet tissues in female rat offspring.

We previously showed that paternal high-fat diet (HFD) consumption programs ß-cell dysfunction in female rat offspring, together with transcriptome alterations in islets. Here we investigated the retroperitoneal white adipose tissue (RpWAT) transcriptome using gene and pathway enrichment and pathway analysis to determine whether commonly affected network topologies exist between these two metabolically related tissues. In RpWAT, 5108 genes were differentially expressed due to a paternal HFD; the top 5 significantly enriched networks identified by pathway analysis in offspring of HFD fathers compared with those of fathers fed control diet were: mitochondrial and cellular response to stress, telomerase signaling, cell death and survival, cell cycle, cellular growth and proliferation, and cancer. A total of 187 adipose olfactory receptor genes were down-regulated. Interrogation against the islet transcriptome identified specific gene networks and pathways, including olfactory receptor genes that were similarly affected in both tissues (411 common genes, P<0.05). In particular, we highlight a common molecular network, cell cycle and cancer, with the same hub gene, Myc, suggesting early onset developmental changes that persist, shared responses to programmed systemic factors, or crosstalk between tissues. Thus, paternal HFD consumption triggers unique gene signatures, consistent with premature aging and chronic degenerative disorders, in both RpWAT and pancreatic islets of daughters.

Therapeutic silencing of miR-652 restores heart function and attenuates adverse remodeling in a setting of established pathological hypertrophy.

Expression of microRNA-652 (miR-652) increases in the diseased heart, decreases in a setting of cardioprotection, and is inversely correlated with heart function. The aim of this study was to assess the therapeutic potential of inhibiting miR-652 in a mouse model with established pathological hypertrophy and cardiac dysfunction due to pressure overload. Mice were subjected to a sham operation or transverse aortic constriction (TAC) for 4 wk to induce hypertrophy and cardiac dysfunction, followed by administration of a locked nucleic acid (LNA)-antimiR-652 (miR-652 inhibitor) or LNA control. Cardiac function was assessed before and 8 wk post-treatment. Expression of miR-652 increased in hearts subjected to TAC compared to sham surgery (2.9-fold), and this was suppressed by ∼95% in LNA-antimiR-652-treated TAC mice. Inhibition of miR-652 improved cardiac function in TAC mice (fractional shortening:29±1% at 4 wk post-TAC compared to 35±1% post-treatment) and attenuated cardiac hypertrophy. Improvement in heart function was associated with reduced cardiac fibrosis, less apoptosis and B-type natriuretic peptide gene expression, and preserved angiogenesis. Mechanistically, we identified Jagged1 (a Notch1 ligand) as a novel direct target of miR-652. In summary, these studies provide the first evidence that silencing of miR-652 protects the heart against pathological remodeling and improves heart function.

Therapeutic inhibition of the miR-34 family attenuates pathological cardiac remodeling and improves heart function.

MicroRNAs are dysregulated in a setting of heart disease and have emerged as promising therapeutic targets. MicroRNA-34 family members (miR-34a, -34b, and -34c) are up-regulated in the heart in response to stress. In this study, we assessed whether inhibition of the miR-34 family using an s.c.-delivered seed-targeting 8-mer locked nucleic acid (LNA)-modified antimiR (LNA-antimiR-34) can provide therapeutic benefit in mice with preexisting pathological cardiac remodeling and dysfunction due to myocardial infarction (MI) or pressure overload via transverse aortic constriction (TAC). An additional cohort of mice subjected to MI was given LNA-antimiR-34a (15-mer) to inhibit miR-34a alone as a comparison for LNA-antimiR-34. LNA-antimiR-34 (8-mer) efficiently silenced all three miR-34 family members in both cardiac stress models and attenuated cardiac remodeling and atrial enlargement. In contrast, inhibition of miR-34a alone with LNA-antimiR-34a (15-mer) provided no benefit in the MI model. In mice subjected to pressure overload, LNA-antimiR-34 improved systolic function and attenuated lung congestion, associated with reduced cardiac fibrosis, increased angiogenesis, increased Akt activity, decreased atrial natriuretic peptide gene expression, and maintenance of sarcoplasmic reticulum Ca(2+) ATPase gene expression. Improved outcome in LNA-antimiR-34-treated MI and TAC mice was accompanied by up-regulation of several direct miR-34 targets, including vascular endothelial growth factors, vinculin, protein O-fucosyltranferase 1, Notch1, and semaphorin 4B. Our results provide evidence that silencing of the entire miR-34 family can protect the heart against pathological cardiac remodeling and improve function. Furthermore, these data underscore the utility of seed-targeting 8-mer LNA-antimiRs in the development of new therapeutic approaches for pharmacologic inhibition of disease-implicated miRNA seed families.

Phage Therapy in Korea: A Prescribers' Survey of Attitudes Amongst Korean Infectious Diseases Specialists Towards Phage Therapy.

BACKGROUND: Concerns about the rise in antimicrobial resistance have led to renewed interest in phage therapy worldwide, but perceptions among relevant medical professionals in Korea remain largely unknown. MATERIALS AND METHODS: We conducted a semi-quantitative online survey to evaluate the Korean infectious disease specialists' perception of phage therapy. RESULTS: We sent out the link to the questionnaire to 380 subjects and received 91 replies, with 90/91 respondents identifying as Korean infectious diseases specialists or trainees. Ten out of 91 (11.0%) respondents scored themselves as well-informed about phage therapy. The majority (93.4%) of respondents would consider using phage therapy if the safety of the phage formulation is guaranteed, and 80% of respondents would consider participating in clinical trials with phage therapy given adequate support. The biggest concern was uncertainty about safety (73.6%) and efficacy (65.9%). Acinetobacter baumannii was ranked as a high priority for phage therapy research, as were bone and joint infections. CONCLUSION: Korean infectious diseases specialists are receptive to phage therapy, but a better understanding of safety, efficacy and clinical trials are warranted to progress phage therapy within the Korean healthcare system.

Refractory Pseudomonas aeruginosa infections treated with phage PASA16: A compassionate use case series.

BACKGROUND: A growing number of compassionate phage therapy cases were reported in the last decade, with a limited number of clinical trials conducted and few unsuccessful clinical trials reported. There is only a little evidence on the role of phages in refractory infections. Our objective here was to present the largest compassionate-use single-organism/phage case series in 16 patients with non-resolving Pseudomonas aeruginosa infections. METHODS: We summarized clinical phage microbiology susceptibility data, administration protocol, clinical data, and outcomes of all cases treated with PASA16 phage. In all intravenous phage administrations, PASA16 phage was manufactured and provided pro bono by Adaptive Phage Therapeutics. PASA16 was administered intravenously, locally to infection site, or by topical use to 16 patients, with data available for 15 patients, mainly with osteoarticular and foreign-device-associated infections. FINDINGS: A few minor side effects were noted, including elevated liver function enzymes and a transient reduction in white blood cell count. Good clinical outcome was documented in 13 out of 15 patients (86.6%). Two clinical failures were reported. The minimum therapy duration was 8 days with a once- to twice-daily regimen. CONCLUSIONS: PASA16 with antibiotics was found to be relatively successful in patients for whom traditional treatment approaches have failed previously. Such pre-phase-1 cohorts can outline potential clinical protocols and facilitate the design of future trials. FUNDING: The study was funded in part by The Israeli Science Foundation IPMP (ISF_1349/20), Rosetrees Trust (A2232), United States-Israel Binational Science Foundation (2017123), and the Milgrom Family Support Program.

Influence of atrial fibrillation on microRNA expression profiles in left and right atria from patients with valvular heart disease.

Chronic atrial fibrillation (AF) is a complication associated with the dilated atria of patients with valvular heart disease and contributes to worsened pathology. We examined microRNA (miRNA) expression profiles in right and left atrial appendage tissue from valvular heart disease (VHD) patients. Right atrial (RA) appendage from patients undergoing coronary artery bypass grafting and left atrial (LA) appendage from healthy hearts, not used for transplant, were used as controls. There was no detectable effect of chronic AF on miRNA expression in LA tissue, but miRNA expression in RA was strongly influenced by AF, with 47 miRNAs (15 higher, 32 lower) showing differential expression between the AF and control sinus rhythm groups. VHD induced different changes in miRNA expression in LA compared with RA. Fifty-three (12 higher, 41 lower) miRNAs were altered by VHD in LA, compared with 5 (4 higher, 1 lower) in RA tissue. miRNA profiles also differed between VHD-LA and VHD-RA (13 higher, 26 lower). We conclude that VHD and AF influence miRNA expression patterns in LA and RA, but these are affected differently by disease progression and by the development of AF. These findings provide new insights into the progression of VHD.

Cell cycle sensing of oxidative stress in Saccharomyces cerevisiae by oxidation of a specific cysteine residue in the transcription factor Swi6p.

Yeast cells begin to bud and enter the S phase when growth conditions are favorable during the G(1) phase. When subjected to some oxidative stresses, cells delay entry at G(1), allowing repair of cellular damage. Hence, oxidative stress sensing is coordinated with the regulation of cell cycle. We identified a novel function of the cell cycle regulator of Saccharomyces cerevisiae, Swi6p, as a redox sensor through its cysteine residue at position 404. When alanine was substituted at this position, the resultant mutant, C404A, was sensitive to several reactive oxygen species and oxidants including linoleic acid hydroperoxide, the superoxide anion, and diamide. This mutant lost the ability to arrest in G(1) phase upon treatment with lipid hydroperoxide. The Cys-404 residue of Swi6p in wild-type cells was oxidized to a sulfenic acid when cells were subjected to linoleic acid hydroperoxide. Mutation of Cys-404 to Ala abolished the down-regulation of expression of the G(1) cyclin genes CLN1, CLN2, PCL1, and PCL2 that occurred when cells of the wild type were exposed to the lipid hydroperoxide. In conclusion, oxidative stress signaling for cell cycle regulation occurs through oxidation of the G(1)/S-specific transcription factor Swi6p and consequently leads to suppression of the expression of G(1) cyclins and a delay in cells entering the cell cycle.

Phosphoinositide 3-kinase (PI3K(p110alpha)) directly regulates key components of the Z-disc and cardiac structure.

Maintenance of cardiac structure and Z-disc signaling are key factors responsible for protecting the heart in a setting of stress, but how these processes are regulated is not well defined. We recently demonstrated that PI3K(p110α) protects the heart against myocardial infarction. The aim of this study was to determine whether PI3K(p110α) directly regulates components of the Z-disc and cardiac structure. To address this question, a unique three-dimensional virtual muscle model was applied to gene expression data from transgenic mice with increased or decreased PI3K(p110α) activity under basal conditions (sham) and in a setting of myocardial infarction to display the location of structural proteins. Key findings from this analysis were then validated experimentally. The three-dimensional virtual muscle model visually highlighted reciprocally regulated transcripts associated with PI3K activation that encoded key components of the Z-disc and costamere, including melusin. Studies were performed to assess whether PI3K and melusin interact in the heart. Here, we identify a novel melusin-PI3K interaction that generates lipid kinase activity. The direct impact of PI3K(p110α) on myocyte structure was assessed by treating neonatal rat ventricular myocytes with PI3K(p110α) inhibitors and examining the myofiber morphology of hearts from PI3K transgenic mice. Results demonstrate that PI3K is critical for myofiber maturation and Z-disc alignment. In summary, PI3K regulates the expression of genes essential for cardiac structure and Z-disc signaling, interacts with melusin, and is critical for Z-disc alignment.

A microRNA guide for clinicians and basic scientists: background and experimental techniques.

MicroRNAs (miRNAs) are short non-coding RNA molecules that are approximately 22 nucleotides in length. In the last 10 years, miRNA research and discovery has advanced at a rapid rate. This review provides a brief overview of the discovery and biology of miRNAs, and summarises some of the experimental techniques used for isolation, detection, target prediction, and regulation of miRNAs. We also outline experimental workflows for investigators new to the field, and discuss the diagnostic and therapeutic application of miRNAs.

Data to Power Precision Phage Therapy: A Look at the Phage Directory-Phage Australia Partnership.

Phage Directory has recently partnered with Phage Australia to help optimize Australia's data-centric standardized approach to personalized phage therapy. Here is a behind-the-scenes look at the genesis of Phage Australia, how the Phage Directory-Phage Australia partnership started, and what it is working toward.

Two plus One: Combination Therapy Tri-systems Involving Two Membrane-Disrupting Antimicrobial Macromolecules and Antibiotics.

The escalating issue of multidrug-resistant (MDR) bacteria indicates the urgent need for new and effective strategies to combat this global health challenge. Here, we describe a new combinatorial approach that can be put forward for experimental therapy application against MDR bacteria. Specifically, we have developed a tri-system that includes the coadministration of two different membrane-disrupting-type antimicrobial agents─a synthetic antimicrobial polymer P and an antimicrobial peptide (AMP) colistin methanesulfonate (Col)─in conjunction with an antibiotic [doxycycline (Dox), rifampicin (Rif), or azithromycin (Azi)]. Traditionally, the administration of membrane-disrupting antimicrobial agents causes toxicity, but, in comparison, we demonstrated synergy and biocompatibility using this combinatorial approach. Checkerboard assays showed the occurrence of synergistic interactions in Col-Dox-P, Col-Rif-P, and Col-Azi-P tri-systems against wild-type and MDR Pseudomonas aeruginosa, with the Col-Dox-P system being the most effective. The ability to synergize thus enables the use of a lower dosage in combinations compared to the standalone agents. The tri-systems not only demonstrated bacteriostatic activity but were also bactericidal. For example, the Col-Dox-P system (at 8, 4, and 8 µg mL-1, respectively) and the Col-Rif-P system (at 4, 8, and 16 µg mL-1, respectively) were able to kill >99.999% of planktonic P. aeruginosa cells within 3 h of treatment. More importantly, an improvement of the therapeutic/selectivity index was achieved via combination therapy. Taking the Col-Dox-P system as an example, its biocompatibility with murine embryonic fibroblast cells was found to be comparable to that of polymer P alone despite the synergistic enhancement in antimicrobial activity of the combination. This resulted in a significant increase in selectivity by 16-fold for the Col-Dox-P combination system compared to P alone. Furthermore, the broad applicability of this tri-system strategy was demonstrated via the successful application of the AMP melittin in place of Col or P. Overall, this study sheds new insights on the application of membrane-disrupting antimicrobial agents in combination therapy and their potential for safer clinical use. Additionally, the information gathered in this study could inform the development of future combination therapy systems involving the simultaneous employment of multiple AMPs with antibiotics.