CCR5 Decorated Rilpivirine Lipid Nanoparticles Build Myeloid Drug Depots Which Sustains Antiretroviral Activities.

Antiretroviral therapy (ART) improves the quality of life for those living with the human immunodeficiency virus type one (HIV-1). However, poor compliance reduces ART effectiveness and leads to immune compromise, viral mutations, and disease co-morbidities. A novel drug formulation is made whereby a lipid nanoparticle (LNP) carrying rilpivirine (RPV) is decorated with the C-C chemokine receptor type 5 (CCR5). This facilitates myeloid drug depot deposition. Particle delivery to viral reservoirs is tracked by positron emission tomography. The CCR5-mediated RPV LNP cell uptake and retention reduce HIV-1 replication in human monocyte-derived macrophages and infected humanized mice. Focused ultrasound allows the decorated LNP to penetrate the blood-brain barrier and reach brain myeloid cells. These findings offer a role for CCR5-targeted therapeutics in antiretroviral delivery to optimize HIV suppression.

Impaired extinction of cocaine seeking in HIV-infected mice is accompanied by peripheral and central immune dysregulation.

Substance use disorders (SUDs) are highly comorbid with HIV infection, necessitating an understanding of the interactive effects of drug exposure and HIV. The relationship between HIV infection and cocaine use disorder is likely bidirectional, with cocaine use directly impacting immune function while HIV infection alters addiction-related behavior. To better characterize the neurobehavioral and immune consequences of HIV infection and cocaine exposure, this study utilizes a humanized mouse model to investigate the outcomes of HIV-1 infection on cocaine-related behaviors in a conditioned place preference (CPP) model, and the interactive effects of cocaine and HIV infection on peripheral and central nervous system inflammation. HIV infection selectively impairs cocaine CPP extinction without effecting reinstatement or cocaine seeking under conflict. Behavioral alterations are accompanied by immune changes in HIV infected mice, including increased prefrontal cortex astrocyte immunoreactivity and brain-region specific effects on microglia number and reactivity. Peripheral immune system changes are observed in human cytokines, including HIV-induced reductions in human TNFα, and cocaine and HIV interactions on GM-CSF levels. Together these data provide new insights into the unique neurobehavioral outcomes of HIV infection and cocaine exposure and how they interact to effect immune responses.

Accelerated Neuroimmune Dysfunction in Aged HIV-1-Infected Humanized Mice.

Disordered immunity, aging, human immunodeficiency virus type one (HIV-1) infection, and responses to antiretroviral therapy are linked. However, how each factor is linked with the other(s) remains incompletely understood. It has been reported that accelerated aging, advanced HIV-1 infection, inflammation, and host genetic factors are associated with host cellular, mitochondrial, and metabolic alterations. However, the underlying mechanism remains elusive. With these questions in mind, we used chronically HIV-1-infected CD34-NSG humanized mice (hu-mice) to model older people living with HIV and uncover associations between HIV-1 infection and aging. Adult humanized mice were infected with HIV-1 at the age of 20 weeks and maintained for another 40 weeks before sacrifice. Animal brains were collected and subjected to transcriptomics, qPCR, and immunofluorescence assays to uncover immune disease-based biomarkers. CD4+ T cell decline was associated with viral level and age. Upregulated C1QA, CD163, and CXCL16 and downregulated LMNA and CLU were identified as age-associated genes tied to HIV-1 infection. Ingenuity pathway analysis affirmed links to innate immune activation, pyroptosis signaling, neuroinflammation, mitochondrial dysfunction, cellular senescence, and neuronal dysfunction. In summary, CD34-NSG humanized mice are identified as a valuable model for studying HIV-1-associated aging. Biomarkers of immune senescence and neuronal signaling are both age- and virus-associated. By exploring the underlying biological mechanisms that are linked to these biomarkers, interventions for next generation HIV-1-infected patients can be realized.

Impaired extinction of cocaine seeking in HIV-infected mice is accompanied by peripheral and central immune dysregulation.

Substance use disorders (SUDs) are highly comorbid with HIV infection, necessitating an understanding of the interactive effects of drug exposure and HIV. The relationship between HIV infection and cocaine use disorder is likely bidirectional, with cocaine use directly impacting immune function while HIV infection alters addiction-related behavior. To better characterize the neurobehavioral and immune consequences of HIV infection and cocaine exposure, this study utilized a humanized mouse model to investigate the outcomes of HIV-1 infection on cocaine-related behaviors in a conditioned place preference (CPP) model, and the interactive effects of cocaine and HIV infection on peripheral and central nervous system inflammation. HIV infection selectively impaired cocaine CPP extinction without effecting reinstatement or cocaine seeking under conflict were observed. Behavioral alterations were accompanied by immune changes in HIV infected mice, including increased prefrontal cortex astrocyte immunoreactivity and brain-region specific effects on microglia number and reactivity. Peripheral immune system changes were observed in both mouse and human cytokines, including HIV-induced reductions in mouse IL-1α and G-CSF and human TNFα, and cocaine induced alterations in mouse GM-CSF. Together these data provide new insights into the unique neurobehavioral outcomes of HIV infection and cocaine exposure and how they interact to effect immune responses.

Impaired extinction of cocaine seeking in HIV-infected mice is accompanied by peripheral and central immune dysregulation.

Substance use disorders (SUDs) are highly comorbid with HIV infection, necessitating an understanding of the interactive effects of drug exposure and HIV. The relationship between progressive HIV infection and cocaine use disorder is likely bidirectional, with cocaine use having direct effects on immune function while HIV infection can alter addiction-related behavior. To better characterized the neurobehavioral and immune consequences of HIV infection and cocaine exposure, this study utilized a humanized mouse model to investigate the outcomes of progressive HIV infection on cocaine-related behaviors in a cocaine conditioned place preference (CPP) model, and the interactive effects of cocaine and HIV infection on peripheral and central nervous system inflammation. HIV infection did not impact the formation of a cocaine CPP, but did result in resistance to extinction of the CPP. No effects of HIV on yohimbine-primed reinstatement or cocaine seeking under conflict were observed. These behavioral alterations were accompanied by immune changes in HIV infected mice, including increased prefrontal cortex astrocyte immunoreactivity and brain-region specific effects on microglia number and reactivity. Peripheral immune system changes were observed in both mouse and human markers. Among other targets, this included HIV-induced reductions in mouse IL-1α and G-CSF and human TNFα and cocaine-induced alterations in human TNFα and mouse GM-CSF such that cocaine exposure increases both cytokines only in the absence of HIV infection. Together these data provide new insights into the unique neurobehavioral processes underlying HIV infection and cocaine use disorders, and further how they interact to effect immune responses.

Humanized Mice for Studies of HIV-1 Persistence and Elimination.

A major roadblock to achieving a cure for human immunodeficiency virus type one (HIV-1) is the persistence of latent viral infections in the cells and tissue compartments of an infected human host. Latent HIV-1 proviral DNA persists in resting memory CD4+ T cells and mononuclear phagocytes (MPs; macrophages, microglia, and dendritic cells). Tissue viral reservoirs of both cell types reside in the gut, lymph nodes, bone marrow, spleen, liver, kidney, skin, adipose tissue, reproductive organs, and brain. However, despite the identification of virus-susceptible cells, several limitations persist in identifying broad latent reservoirs in infected persons. The major limitations include their relatively low abundance, the precise identification of latently infected cells, and the lack of biomarkers for identifying latent cells. While primary MP and CD4+ T cells and transformed cell lines are used to interrogate mechanisms of HIV-1 persistence, they often fail to accurately reflect the host cells and tissue environments that carry latent infections. Given the host specificity of HIV-1, there are few animal models that replicate the natural course of viral infection with any precision. These needs underlie the importance of humanized mouse models as both valuable and cost-effective tools for studying viral latency and subsequently identifying means of eliminating it. In this review, we discuss the advantages and limitations of humanized mice for studies of viral persistence and latency with an eye toward using these models to test antiretroviral and excision therapeutics. The goals of this research are to use the models to address how and under which circumstances HIV-1 latency can be detected and eliminated. Targeting latent reservoirs for an ultimate HIV-1 cure is the task at hand.

CRISPR editing of CCR5 and HIV-1 facilitates viral elimination in antiretroviral drug-suppressed virus-infected humanized mice.

Treatment of HIV-1ADA-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4+ T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals. Highly sensitive nucleic acid nested and droplet digital PCR, RNAscope, and viral outgrowth assays affirmed viral elimination. HIV-1 was not detected in the blood, spleen, lung, kidney, liver, gut, bone marrow, and brain of virus-free animals. Progeny virus from adoptively transferred and CRISPR-treated virus-free mice was neither detected nor recovered. Residual HIV-1 DNA fragments were easily seen in untreated and viral-rebounded animals. No evidence of off-target toxicities was recorded in any of the treated animals. Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments. Taken together, these observations underscore a pivotal role of combinatorial CRISPR gene editing in achieving the elimination of HIV-1 infection.

Animal models for studies of HIV-1 brain reservoirs.

The HIV-1 often evades a robust antiretroviral-mediated immune response, leading to persistent infection within anatomically privileged sites including the CNS. Continuous low-level infection occurs in the presence of effective antiretroviral therapy (ART) in CD4+ T cells and mononuclear phagocytes (MP; monocytes, macrophages, microglia, and dendritic cells). Within the CNS, productive viral infection is found exclusively in microglia and meningeal, perivascular, and choroidal macrophages. MPs serve as the principal viral CNS reservoir. Animal models have been developed to recapitulate natural human HIV-1 infection. These include nonhuman primates, humanized mice, EcoHIV, and transgenic rodent models. These models have been used to study disease pathobiology, antiretroviral and immune modulatory agents, viral reservoirs, and eradication strategies. However, each of these models are limited to specific component(s) of human disease. Indeed, HIV-1 species specificity must drive therapeutic and cure studies. These have been studied in several model systems reflective of latent infections, specifically in MP (myeloid, monocyte, macrophages, microglia, and histiocyte cell) populations. Therefore, additional small animal models that allow productive viral replication to enable viral carriage into the brain and the virus-susceptible MPs are needed. To this end, this review serves to outline animal models currently available to study myeloid brain reservoirs and highlight areas that are lacking and require future research to more effectively study disease-specific events that could be useful for viral eradication studies both in and outside the CNS.

A Link Between Methylglyoxal and Heart Failure During HIV-1 Infection.

Early-onset heart failure (HF) continues to be a major cause of morbidity and mortality in people living with human immunodeficiency virus type one (HIV-1) infection (PLWH), yet the molecular causes for this remain poorly understood. Herein NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ humanized mice (Hu-mice), plasma from PLWH, and autopsied cardiac tissues from deceased HIV seropositive individuals were used to assess if there is a link between the glycolysis byproduct methylglyoxal (MG) and HF in the setting of HIV-1 infection. At five weeks post HIV infection, Hu-mice developed grade III-IV diastolic dysfunction (DD) with an associated two-fold increase in plasma MG. At sixteen-seventeen weeks post infection, cardiac ejection fraction and fractional shortening also declined by 26 and 35%, and plasma MG increased to four-fold higher than uninfected controls. Histopathological and biochemical analyses of cardiac tissues from Hu-mice 17 weeks post-infection affirmed MG increase with a concomitant decrease in expression of the MG-degrading enzyme glyoxalase-1 (Glo1). The endothelial cell marker CD31 was found to be lower, and coronary microvascular leakage and myocardial fibrosis were prominent. Increasing expression of Glo1 in Hu-mice five weeks post-infection using a single dose of an engineered AAV2/9 (1.7 × 1012 virion particles/kg), attenuated the increases in plasma and cardiac MG levels. Increasing Glo1 also blunted microvascular leakage, fibrosis, and HF seen at sixteen weeks post-infection, without changes in plasma viral loads. In plasma from virally suppressed PLWH, MG was also 3.7-fold higher. In autopsied cardiac tissues from seropositive, HIV individuals with low viral log, MG was 4.2-fold higher and Glo1 was 50% lower compared to uninfected controls. These data show for the first time a causal link between accumulation of MG and HF in the setting of HIV infection.

The COVID-19 Pandemic: Reflections of Science, Person, and Challenge in Academic Research Settings.

In spring of 2021, the Society on NeuroImmune Pharmacology (SNIP) organized a virtual workshop on the coronavirus disease 2019 (COVID-19). The daylong event's fourth and final symposium, "Well-being and reflections," offered a glimpse at the pandemic's impact on the lives of our scientists and educators. This manuscript includes a brief summary of the symposium, a transcription of our incoming president Dr. Santosh Kumar's lecture, titled "Intervention and improved well-being of basic science researchers during the COVID-19 era: a case study," and the panel discussion that followed, "Reflection and sharing," featuring Drs. Jean M. Bidlack, Sylvia Fitting, Santhi Gorantla, Maria Cecilia G. Marcondes, Loyda M. Melendez, and Ilker K. Sariyer. The conclusion of this manuscript includes comments from SNIP's president Dr. Sulie L. Chang and our Chief Editor, Dr. Howard E. Gendelman. Drs. Sowmya Yelamanchili and Jeymohan Joseph co-chaired the symposium.

Recovery of Latent HIV-1 from Brain Tissue by Adoptive Cell Transfer in Virally Suppressed Humanized Mice.

Defining the latent human immunodeficiency virus type 1 (HIV-1) burden in the human brain during progressive infection is limited by sample access. Human hematopoietic stem cells (hu-HSCs)-reconstituted humanized mice provide an opportunity for this study. The model mimics, in measure, HIV-1 pathophysiology, transmission, treatment, and elimination in an infected human host. However, to date, brain HIV-1 latency in hu-HSC mice during suppressive antiretroviral therapy (ART) was not studied. To address this need, hu-HSC mice were administered long acting (LA) ART 14 days after HIV-1 infection was established. Animals were maintained under suppressive ART for 3 months, at which time HIV-1 infection was detected at low levels in brain tissue by droplet digital polymerase chain reaction (ddPCR) test on DNA. Notably, adoptive transfer of cells acquired from the hu-HSC mouse brains and placed into naive hu-HSC mice demonstrated viral recovery. These proof-of-concept results demonstrate replication-competent HIV-1 reservoir can be established in hu-HSC mouse brains that persists during long-term ART treatment. Hu-HSC mice-based mouse viral outgrowth assay (hu-MVOA) serves as a sensitive tool to interrogate latent HIV-1 brain reservoirs.

Humanized Mice for Infectious and Neurodegenerative disorders.

Humanized mice model human disease and as such are used commonly for research studies of infectious, degenerative and cancer disorders. Recent models also reflect hematopoiesis, natural immunity, neurobiology, and molecular pathways that influence disease pathobiology. A spectrum of immunodeficient mouse strains permit long-lived human progenitor cell engraftments. The presence of both innate and adaptive immunity enables high levels of human hematolymphoid reconstitution with cell susceptibility to a broad range of microbial infections. These mice also facilitate investigations of human pathobiology, natural disease processes and therapeutic efficacy in a broad spectrum of human disorders. However, a bridge between humans and mice requires a complete understanding of pathogen dose, co-morbidities, disease progression, environment, and genetics which can be mirrored in these mice. These must be considered for understanding of microbial susceptibility, prevention, and disease progression. With known common limitations for access to human tissues, evaluation of metabolic and physiological changes and limitations in large animal numbers, studies in mice prove important in planning human clinical trials. To these ends, this review serves to outline how humanized mice can be used in viral and pharmacologic research emphasizing both current and future studies of viral and neurodegenerative diseases. In all, humanized mouse provides cost-effective, high throughput studies of infection or degeneration in natural pathogen host cells, and the ability to test transmission and eradication of disease.

Human Microglia Extensively Reconstitute in Humanized-BLT Mice With Human Interleukin-34 Transgene and Support HIV-1 Brain Infection.

Humanized bone marrow-liver-thymic (hu-BLT) mice develop a functional immune system in periphery, nevertheless, have a limited reconstitution of human myeloid cells, especially microglia, in CNS. Further, whether bone marrow derived hematopoietic stem and progenitor cells (HSPCs) can enter the brain and differentiate into microglia in adults remains controversial. To close these gaps, in this study we unambiguously demonstrated that human microglia in CNS were extensively reconstituted in adult NOG mice with human interleukin-34 transgene (hIL34 Tg) from circulating CD34+ HSPCs, nonetheless not in hu-BLT NOG mice, providing strong evidence that human CD34+ HSPCs can enter adult brain and differentiate into microglia in CNS in the presence of hIL34. Further, the human microglia in the CNS of hu-BLT-hIL34 NOG mice robustly supported HIV-1 infection reenforcing the notion that microglia are the most important target cells of HIV-1 in CNS and demonstrating its great potential as an in vivo model for studying HIV-1 pathogenesis and evaluating curative therapeutics in both periphery and CNS compartments.

Diagnostics for SARS-CoV-2 infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to nearly every corner of the globe, causing societal instability. The resultant coronavirus disease 2019 (COVID-19) leads to fever, sore throat, cough, chest and muscle pain, dyspnoea, confusion, anosmia, ageusia and headache. These can progress to life-threatening respiratory insufficiency, also affecting the heart, kidney, liver and nervous systems. The diagnosis of SARS-CoV-2 infection is often confused with that of influenza and seasonal upper respiratory tract viral infections. Due to available treatment strategies and required containments, rapid diagnosis is mandated. This Review brings clarity to the rapidly growing body of available and in-development diagnostic tests, including nanomaterial-based tools. It serves as a resource guide for scientists, physicians, students and the public at large.

Pharmacotherapeutics of SARS-CoV-2 Infections.

The COVID-19 pandemic has affected more than 38 million people world-wide by person to person transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therapeutic and preventative strategies for SARS-CoV-2 remains a significant challenge. Within the past several months, effective treatment options have emerged and now include repurposed antivirals, corticosteroids and virus-specific antibodies. The latter has included convalescence plasma and monoclonal antibodies. Complete viral eradication will be achieved through an effective, safe and preventative vaccine. To now provide a comprehensive summary for each of the pharmacotherapeutics and preventative strategies being offered or soon to be developed for SARS-CoV-2.

Efavirenz, atazanavir, and ritonavir disrupt sarcoplasmic reticulum Ca2+ homeostasis in skeletal muscles.

While muscle fatigue, pain and weakness are common co-morbidities in HIV-1 infected people, their underlying cause remain poorly defined. To this end, we evaluated whether the common antiretroviral drugs efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV) could be a contributing factor by pertubating sarcoplasmic reticulum (SR) Ca2+ cycling. In live-cell imaging, EFV (6.0 µM), ATV (6.0 µM), and RTV (3.0 µM) elicited Ca2+ transients and blebbing of the plasma membranes of C2C12 skeletal muscle myotubes. Pretreating C2C12 skeletal muscle myotubes with the SR Ca2+ release channel blocker ryanodine (50 µM), slowed the rate and amplitude of Ca2+ release from and reuptake of Ca2+ into the SR. EFV, ATV and RTV (1 nM - 20 µM) potentiated and then displaced [3H] ryanodine binding to rabbit skeletal muscle ryanodine receptor Ca2+ release channel (RyR1). These drugs at concentrations 0.25-31.2 µM also increased and or decreased the open probability of RyR1 by altering its gating and conductance. ATV (≤5 µM) potentiated and >5µM inhibited the ability of sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA1) to hydrolyze ATP and transport Ca2+. RTV (2.5-31.5 µM) dose-dependently inhibited SERCA1-mediated, ATP-dependent Ca2+ transport. EFV (0.25-31.5 µM) had no measurable effect on SERCA1's ability to hydrolyze ATP and transport Ca2+. These data support the notion that EFV, ATV and RTV could be contributing to skeletal muscle co-morbidities in PLWH by modulating SR Ca2+ homeostasis.

Alzheimer's disease brain-derived extracellular vesicles spread tau pathology in interneurons.

Extracellular vesicles are highly transmissible and play critical roles in the propagation of tau pathology, although the underlying mechanism remains elusive. Here, for the first time, we comprehensively characterized the physicochemical structure and pathogenic function of human brain-derived extracellular vesicles isolated from Alzheimer's disease, prodromal Alzheimer's disease, and non-demented control cases. Alzheimer's disease extracellular vesicles were significantly enriched in epitope-specific tau oligomers in comparison to prodromal Alzheimer's disease or control extracellular vesicles as determined by dot blot and atomic force microscopy. Alzheimer's disease extracellular vesicles were more efficiently internalized by murine cortical neurons, as well as more efficient in transferring and misfolding tau, than prodromal Alzheimer's disease and control extracellular vesicles in vitro. Strikingly, the inoculation of Alzheimer's disease or prodromal Alzheimer's disease extracellular vesicles containing only 300 pg of tau into the outer molecular layer of the dentate gyrus of 18-month-old C57BL/6 mice resulted in the accumulation of abnormally phosphorylated tau throughout the hippocampus by 4.5 months, whereas inoculation of an equal amount of tau from control extracellular vesicles, isolated tau oligomers, or fibrils from the same Alzheimer's disease donor showed little tau pathology. Furthermore, Alzheimer's disease extracellular vesicles induced misfolding of endogenous tau in both oligomeric and sarkosyl-insoluble forms in the hippocampal region. Unexpectedly, phosphorylated tau was primarily accumulated in glutamic acid decarboxylase 67 (GAD67) GABAergic interneurons and, to a lesser extent, glutamate receptor 2/3-positive excitatory mossy cells, showing preferential extracellular vesicle-mediated GABAergic interneuronal tau propagation. Whole-cell patch clamp recordings of CA1 pyramidal cells showed significant reduction in the amplitude of spontaneous inhibitory post-synaptic currents. This was accompanied by reductions in c-fos+ GAD67+ neurons and GAD67+ neuronal puncta surrounding pyramidal neurons in the CA1 region, confirming reduced GABAergic transmission in this region. Our study posits a novel mechanism for the spread of tau in hippocampal GABAergic interneurons via brain-derived extracellular vesicles and their subsequent neuronal dysfunction.

Role of Tunneling Nanotube-like Structures during the Early Events of HIV Infection: Novel Features of Tissue Compartmentalization and Mechanism of HIV Spread.

HIV has become a chronic disease despite the effective use of antiretroviral therapy (ART). However, the mechanisms of tissue colonization, viral evolution, generation of viral reservoirs, and compartmentalization are still a matter of debate due to the challenges involved in examining early events of infection at the cellular and molecular level. Thus, there is still an urgent need to explore these areas to develop effective HIV cure strategies. In this study, we describe the early events of tissue colonization and compartmentalization as well as the role of tunneling nanotube-like structures during viral spread in the presence and absence of effective antiretroviral treatment. To examine these mechanisms, NOD/SCID IL-2 RG-/- humanized mice were either directly infected with HIVADA or with low numbers of HIVADA-infected leukocytes to limit tissue colonization in the presence and absence of TAK779, an effective CCR5 blocker of HIV entry. We identify that viral seeding in tissues occurs early in a tissue- and cell type-specific manner (24-72 h). Reduction in systemic HIV replication by TAK779 treatment did not affect tissue seeding or spreading, despite reduced systemic viral replication. Tissue-associated HIV-infected cells had different properties than cells in the circulation because the virus continues to spread in tissues in a tunneling nanotube-like structure-dependent manner, despite ART. Thus, understanding these mechanisms can provide new approaches to enhance the efficacy of existing ART and HIV infection cure strategies.

Two distinct amphipathic peptide antibiotics with systemic efficacy.

Antimicrobial peptides are important candidates for developing new classes of antibiotics because of their potency against antibiotic-resistant pathogens. Current research focuses on topical applications and it is unclear how to design peptides with systemic efficacy. To address this problem, we designed two potent peptides by combining database-guided discovery with structure-based design. When bound to membranes, these two short peptides with an identical amino acid composition can adopt two distinct amphipathic structures: A classic horizontal helix (horine) and a novel vertical spiral structure (verine). Their horizontal and vertical orientations on membranes were determined by solid-state 15N NMR data. While horine was potent primarily against gram-positive pathogens, verine showed broad-spectrum antimicrobial activity. Both peptides protected greater than 80% mice from infection-caused deaths. Moreover, horine and verine also displayed significant systemic efficacy in different murine models comparable to conventional antibiotics. In addition, they could eliminate resistant pathogens and preformed biofilms. Significantly, the peptides showed no nephrotoxicity to mice after intraperitoneal or intravenous administration for 1 wk. Our study underscores the significance of horine and verine in fighting drug-resistant pathogens.