Microscopic defect dynamics during a brittle-to-ductile transition.

Deformation of all materials necessitates the collective propagation of various microscopic defects. On Earth, fracturing gives way to crystal-plastic deformation with increasing depth resulting in a "brittle-to-ductile" transition (BDT) region that is key for estimating the integrated strength of tectonic plates, constraining the earthquake cycle, and utilizing deep geothermal resources. Here, we show that the crossing of a BDT in marble during deformation experiments in the laboratory is accompanied by systematic increase in the frequency of acoustic emissions suggesting a profound change in the mean size and propagation velocity of the active defects. We further identify dominant classes of emitted waveforms using unsupervised learning methods and show that their relative activity systematically changes as the rocks cross the brittle-ductile transition. As pressure increases, long-period signals are suppressed and short-period signals become dominant. At higher pressures, signals frequently come in avalanche-like patterns. We propose that these classes of waveforms correlate with individual dominant defect types. Complex mixed-mode events indicate that interactions between the defects are common over the whole pressure range, in agreement with postmortem microstructural observations. Our measurements provide unique, real-time data of microscale dynamics over a broad range of pressures (10 to 200 MPa) and can inform micromechanical models for semi-brittle deformation.

Nonviral In Vivo Delivery of CRISPR-Cas9 Using Protein-Agnostic, High-Loading Porous Silicon and Polymer Nanoparticles.

The complexity of CRISPR machinery is a challenge to its application for nonviral in vivo therapeutic gene editing. Here, we demonstrate that proteins, regardless of size or charge, efficiently load into porous silicon nanoparticles (PSiNPs). Optimizing the loading strategy yields formulations that are ultrahigh loading─>40% cargo by volume─and highly active. Further tuning of a polymeric coating on the loaded PSiNPs yields nanocomposites that achieve colloidal stability under cryopreservation, endosome escape, and gene editing efficiencies twice that of the commercial standard Lipofectamine CRISPRMAX. In a mouse model of arthritis, PSiNPs edit cells in both the cartilage and synovium of knee joints, and achieve 60% reduction in expression of the therapeutically relevant MMP13 gene. Administered intramuscularly, they are active over a broad dose range, with the highest tested dose yielding nearly 100% muscle fiber editing at the injection site. The nanocomposite PSiNPs are also amenable to systemic delivery. Administered intravenously in a model that mimics muscular dystrophy, they edit sites of inflamed muscle. Collectively, the results demonstrate that the PSiNP nanocomposites are a versatile system that can achieve high loading of diverse cargoes and can be applied for gene editing in both local and systemic delivery applications.

Injury to the tunica media initiates atherogenesis in the presence of hyperlipidemia.

Background and aims: Fatty streaks initiating the formation of atheromatous plaque appear in the tunica intima. The tunica media is not known to be a nidus for lipid accumulation initiating atherogenesis. We assessed changes to the tunica media in response to a micro-injury produced in the pig aorta. In addition, we assessed human carotid endarterectomy plaques for indication of atheroma initiation in the tunica media. Methods: Three healthy landrace female pigs underwent laparotomy to inject autologous blood and create micro-hematomas at 6 sites within the tunica media of the infrarenal abdominal aorta. These pigs were fed a high-fat diet (HFD) for 4-12 weeks. Post-mortem aortas from all pigs, including a control group of healthy pigs, were serially stained to detect lipid deposits, vasa vasora (VV), immune cell infiltration and inflammatory markers, as well as changes to the vascular smooth muscle cell (vSMC) compartment. Moreover, 25 human carotid endarterectomy (CEA) specimens were evaluated for their lipid composition in the tunica media and intima. Results: High lipid clusters, VV density, and immune cell infiltrates were consistently observed at 5 out of 6 injection sites under prolonged hyperlipidemia. The hyperlipidemic diet also affected the vSMC compartment in the tunica media adjacent to the tunica adventitia, which correlated with VV invasion and immune cell infiltration. Analysis of human carotid specimens post-CEA indicated that 32% of patients had significantly greater atheroma in the tunica media than in the arterial intima. Conclusion: The arterial intima is not the only site for atherosclerosis initiation. We show that injury to the media can trigger atherogenesis.

Inositol Polyphosphate-5-Phosphatase K (Inpp5k) Enhances Sprouting of Corticospinal Tract Axons after CNS Trauma.

Failure of CNS neurons to mount a significant growth response after trauma contributes to chronic functional deficits after spinal cord injury. Activator and repressor screening of embryonic cortical neurons and retinal ganglion cells in vitro and transcriptional profiling of developing CNS neurons harvested in vivo have identified several candidates that stimulate robust axon growth in vitro and in vivo Building on these studies, we sought to identify novel axon growth activators induced in the complex adult CNS environment in vivo We transcriptionally profiled intact sprouting adult corticospinal neurons (CSNs) after contralateral pyramidotomy (PyX) in nogo receptor-1 knock-out mice and found that intact CSNs were enriched in genes in the 3-phosphoinositide degradation pathway, including six 5-phosphatases. We explored whether inositol polyphosphate-5-phosphatase K (Inpp5k) could enhance corticospinal tract (CST) axon growth in preclinical models of acute and chronic CNS trauma. Overexpression of Inpp5k in intact adult CSNs in male and female mice enhanced the sprouting of intact CST terminals after PyX and cortical stroke and sprouting of CST axons after acute and chronic severe thoracic spinal contusion. We show that Inpp5k stimulates axon growth in part by elevating the density of active cofilin in labile growth cones, thus stimulating actin polymerization and enhancing microtubule protrusion into distal filopodia. We identify Inpp5k as a novel CST growth activator capable of driving compensatory axon growth in multiple complex CNS injury environments and underscores the veracity of using in vivo transcriptional screening to identify the next generation of cell-autonomous factors capable of repairing the damaged CNS.SIGNIFICANCE STATEMENT Neurologic recovery is limited after spinal cord injury as CNS neurons are incapable of self-repair post-trauma. In vitro screening strategies exploit the intrinsically high growth capacity of embryonic CNS neurons to identify novel axon growth activators. While promising candidates have been shown to stimulate axon growth in vivo, concomitant functional recovery remains incomplete. We identified Inpp5k as a novel axon growth activator using transcriptional profiling of intact adult corticospinal tract (CST) neurons that had initiated a growth response after pyramidotomy in plasticity sensitized nogo receptor-1-null mice. Here, we show that Inpp5k overexpression can stimulate CST axon growth after pyramidotomy, stroke, and acute and chronic contusion injuries. These data support in vivo screening approaches to identify novel axon growth activators.

Are chest X-rays valuable for patients presenting to emergency departments with acute abdominal pain?

BACKGROUND: Emergency department patients presenting with acute abdominal pain are often prescribed a chest X-ray; however, the value of chest X-rays in acute abdominal pain is poorly understood. The aim of this study was to assess the value of chest X-rays in acute abdominal pain. METHODS: A retrospective analysis of 944 chest X-rays performed for acute abdominal pain was conducted. Patient clinical information, radiology reports, and findings of other diagnostic investigations were also collected. MedCal® software was used to calculate diagnostic performance of chest X-rays. A Chi-Square test was used to assess the association between positive chest X-ray findings and both age and gender. RESULTS: Of the 944 chest X-rays identified as satisfying inclusion factors, only 10 cases (approximately 1%) demonstrated pathology that was likely to be the cause of the abdominal pain. Further analysis demonstrated the following performance metrics at 95%CI: sensitivity (12.8; 8.78-17.72); specificity (100; 98.4-100); positive predictive value (100%); negative predictive value (52.76; 51.54-53.98); accuracy (55.82; 51.17-60.40). CONCLUSION: Chest X-ray has limited sensitivity and diagnostic value in patients presenting to the emergency department with abdominal pain and does not appear to be a useful diagnostic investigation for abdominal pain.

FOXO Transcription Factors Are Required for Normal Somatotrope Function and Growth.

Understanding the molecular mechanisms underlying pituitary organogenesis and function is essential for improving therapeutics and molecular diagnoses for hypopituitarism. We previously found that deletion of the forkhead factor, Foxo1, in the pituitary gland early in development delays somatotrope differentiation. While these mice grow normally, they have reduced growth hormone expression and free serum insulin-like growth factor-1 (IGF1) levels, suggesting a defect in somatotrope function. FOXO factors show functional redundancy in other tissues, so we deleted both Foxo1 and its closely related family member, Foxo3, from the primordial pituitary. We find that this results in a significant reduction in growth. Consistent with this, male and female mice in which both genes have been deleted in the pituitary gland (dKO) exhibit reduced pituitary growth hormone expression and serum IGF1 levels. Expression of the somatotrope differentiation factor, Neurod4, is reduced in these mice. This suggests a mechanism underlying proper somatotrope function is the regulation of Neurod4 expression by FOXO factors. Additionally, dKO mice have reduced Lhb expression and females also have reduced Fshb and Prl expression. These studies reveal FOXO transcription factors as important regulators of pituitary gland function.

Do referral guidelines recommend chest x-rays for patients with abdominal pain? A review.

BACKGROUND: Chest X-rays (CXRs) are often requested for patients who present to emergency with abdominal pain, but its benefit to patient management remains unclear. Several guidelines have been developed to ensure that imaging investigations have the highest diagnostic impact. This study aims to compare referral guidelines to establish their recommendations and circumstances for recommendations regarding CXRs for patients with abdominal pain. METHODS: A systematic search of the literature was performed using Medline (via OVID), PubMed, Google, and Google Scholar. Referral guidelines were included if they provided recommendations for imaging of abdominal pain, were based on published evidence, and were broadly utilised. Data related to their recommendations for CXR for abdominal pain was recorded and analysed. RESULTS: Three guidelines supported the use of CXRs in the case of suspected perforation. Two guidelines included CXR for patients presenting with blunt abdominal trauma and severe abdominal pain requiring admission. One of the guidelines included use of CXRs for patients presenting with suspected small bowel obstruction, cholecystitis, and penetrating trauma. Two guidelines recorded no circumstances where the use of CXRs were recommended. CONCLUSION: Published evidence-based guidelines allow for the use of CXRs for patients presenting with abdominal pain in very limited circumstances.

Parkinson's disease and microRNAs - Lessons from model organisms and human studies.

Parkinson's disease (PD) is a progressive, age-associated neurodegenerative disorder that affects an estimated 10 million people worldwide. PD is characterized by proteinaceous, cytoplasmic inclusions containing α-synuclein, called Lewy Bodies, which form in dopaminergic neurons in an age-dependent manner, and are associated with the emergence of characteristic PD symptoms such as resting tremor, rigidity, slow movements and postural instability. Although considerable progress has been made in recent years in identifying genetic and environmental factors that are associated with PD, early diagnosis and therapeutic options remain severely lacking. Recently, microRNAs (miRNAs) have emerged as novel therapeutic targets in various diseases, such as cancer and neurodegenerative diseases. MiRNAs have been shown to play roles in various aging and neurodegenerative disease models across phyla. More recently, studies have identified specific roles for miRNAs and their targets in the pathogenesis and progression of PD in several model organisms. Here, we discuss the evolving field of miRNAs, their association with PD, and the outlook for the future.

Therapeutic MK2 inhibition blocks pathological vascular smooth muscle cell phenotype switch.

Vascular procedures, such as stenting, angioplasty, and bypass grafting, often fail due to intimal hyperplasia (IH), wherein contractile vascular smooth muscle cells (VSMCs) dedifferentiate to synthetic VSMCs, which are highly proliferative, migratory, and fibrotic. Previous studies suggest MAPK-activated protein kinase 2 (MK2) inhibition may limit VSMC proliferation and IH, although the molecular mechanism underlying the observation remains unclear. We demonstrated here that MK2 inhibition blocked the molecular program of contractile to synthetic dedifferentiation and mitigated IH development. Molecular markers of the VSMC contractile phenotype were sustained over time in culture in rat primary VSMCs treated with potent, long-lasting MK2 inhibitory peptide nanopolyplexes (MK2i-NPs), a result supported in human saphenous vein specimens cultured ex vivo. RNA-Seq of MK2i-NP-treated primary human VSMCs revealed programmatic switching toward a contractile VSMC gene expression profile, increasing expression of antiinflammatory and contractile-associated genes while lowering expression of proinflammatory, promigratory, and synthetic phenotype-associated genes. Finally, these results were confirmed using an in vivo rabbit vein graft model where brief, intraoperative treatment with MK2i-NPs decreased IH and synthetic phenotype markers while preserving contractile proteins. These results support further development of MK2i-NPs as a therapy for blocking VSMC phenotype switch and IH associated with cardiovascular procedures.

A Monoclonal Antibody for Malaria Prevention.

BACKGROUND: Additional interventions are needed to reduce the morbidity and mortality caused by malaria. METHODS: We conducted a two-part, phase 1 clinical trial to assess the safety and pharmacokinetics of CIS43LS, an antimalarial monoclonal antibody with an extended half-life, and its efficacy against infection with Plasmodium falciparum. Part A of the trial assessed the safety, initial side-effect profile, and pharmacokinetics of CIS43LS in healthy adults who had never had malaria. Participants received CIS43LS subcutaneously or intravenously at one of three escalating dose levels. A subgroup of participants from Part A continued to Part B, and some received a second CIS43LS infusion. Additional participants were enrolled in Part B and received CIS43LS intravenously. To assess the protective efficacy of CIS43LS, some participants underwent controlled human malaria infection in which they were exposed to mosquitoes carrying P. falciparum sporozoites 4 to 36 weeks after administration of CIS43LS. RESULTS: A total of 25 participants received CIS43LS at a dose of 5 mg per kilogram of body weight, 20 mg per kilogram, or 40 mg per kilogram, and 4 of the 25 participants received a second dose (20 mg per kilogram regardless of initial dose). No safety concerns were identified. We observed dose-dependent increases in CIS43LS serum concentrations, with a half-life of 56 days. None of the 9 participants who received CIS43LS, as compared with 5 of 6 control participants who did not receive CIS43LS, had parasitemia according to polymerase-chain-reaction testing through 21 days after controlled human malaria infection. Two participants who received 40 mg per kilogram of CIS43LS and underwent controlled human malaria infection approximately 36 weeks later had no parasitemia, with serum concentrations of CIS43LS of 46 and 57 µg per milliliter at the time of controlled human malaria infection. CONCLUSIONS: Among adults who had never had malaria infection or vaccination, administration of the long-acting monoclonal antibody CIS43LS prevented malaria after controlled infection. (Funded by the National Institute of Allergy and Infectious Diseases; VRC 612 ClinicalTrials.gov number, NCT04206332.).

Stress distribution in the walls of major arteries: implications for atherogenesis.

BACKGROUND: There is a correlation between the sites of atheroma development and stress points in the arterial system. Generally, pulse pressure results in stresses acting on the vascular vessel, including longitudinal stress, radial or normal stress, tangential stress or hoop stress and shear stress. This paper explores the relationship between arterial wall shear stress and pulsatile blood pressure with the aim of furthering the understanding of atherogenesis and plaque progression. METHODS: We computed the magnitude of the shear stresses within the carotid bifurcation geometry of a patient and calculated the increase in shear stress levels that would occur when the blood pressure and pulse pressures rise during exertion. We also determined in which layer of the artery wall the maximum shear stress is located, and computed the shear stress at different levels within the media. We used the theory of laminate analysis, (Classical Laminate Plate Theory), to analyse the stress distribution on the carotid artery wall. Computational Fluid Dynamics (CFD) analysis was used on anatomy based on a CT angiogram of the carotid bifurcation of a patient with a 90% stenosis on the right side and 10% on the left. The pulsatile non-Newtonian blood flow with a resting blood pressure of 120/80 mmHg and an exertion pressure of 200/100 mmHg was simulated and the resultant forces were transferred to an ANSYS Composite PrepPost (ACP) model for wall shear stress analysis. A multilayer elastic, anisotropic, and inhomogeneous arterial wall (intima, internal elastic lamina, media, external elastic lamina, and adventitial layers) was modelled and the shear stress magnitudes and change over time between the layers was calculated. RESULTS: Shear stress in the individual composite layers is far greater than that acting on the endothelium (less than 5 Pa). At rest, the maximum variation of shear stress in the arterial wall occurs in the intima (138 Pa) and adventitia (135 Pa). The medial layer has the lowest variation of shear stress. Under severe exertion, the maximum shear stress magnitude in the intimal layer and the adjacent medial layer is near the ultimate stress level. The maximum/minimum shear stress ratios during the cardiac cycle vary most widely in the innermost part of the media, adjacent to the intima, with a four-fold ratio increase. This compares with a less than two-fold increase in all the other layers including the intima and adventitia, making the inner media the most vulnerable layer to mechanical injury. CONCLUSIONS: This study showed that the magnitude of exertion-induced shear stress approaches the ultimate stress limit in the intima and the immediate adjacent medial layer. The variation in stress is maximal in the inner layer of the media. These findings correlate the site of atheroma development with the most vulnerable site for injury in the media and emphasise the impact of pulse pressure. Further biological studies are required to ascertain whether this leads to injury that initiates atheroma that then precipitates an injury/healing cycle.

Modifying Cell Membranes with Anionic Polymer Amphiphiles Potentiates Intracellular Delivery of Cationic Peptides.

Rapid, facile, and noncovalent cell membrane modification with alkyl-grafted anionic polymers was sought as an approach to enhance intracellular delivery and bioactivity of cationic peptides. We synthesized a library of acrylic acid-based copolymers containing varying amounts of an amine-reactive pentafluorophenyl acrylate monomer followed by postpolymerization modification with a series of alkyl amines to afford precise control over the length and density of aliphatic alkyl side chains. This synthetic strategy enabled systematic investigation of the effect of the polymer structure on membrane binding, potentiation of peptide cell uptake, pH-dependent disruption of lipid bilayers for endosome escape, and intracellular bioavailability. A subset of these polymers exhibited pKa of ∼6.8, which facilitated stable membrane association at physiological pH and rapid, pH-dependent endosomal disruption upon endocytosis as quantified in Galectin-8-YFP reporter cells. Cationic cell penetrating peptide (CPP) uptake was enhanced up to 15-fold in vascular smooth muscle cells in vitro when peptide treatment was preceded by a 30-min pretreatment with lead candidate polymers. We also designed and implemented a new and highly sensitive assay for measuring the intracellular bioavailability of CPPs based on the NanoLuciferase (NanoLuc) technology previously developed for measuring intracellular protein-protein interactions. Using this split luciferase class of assay, polymer pretreatment enhanced intracellular delivery of the CPP-modified HiBiT peptide up to 30-fold relative to CPP-HiBiT without polymer pretreatment (p < 0.05). The overall structural analyses show that polymers containing 50:50 or 70:30 molar ratios of carboxyl groups to alkyl side chains of 6-8 carbons maximized peptide uptake, pH-dependent membrane disruption, and intracellular bioavailability and that this potentiation effect was maximized by pairing with CPPs with high cationic charge density. These results demonstrate a rapid, mild method for polymer modification of cell surfaces to potentiate intracellular delivery, endosome escape, and bioactivity of cationic peptides.

Interaction of RSC Chromatin Remodeling Complex with Nucleosomes Is Modulated by H3 K14 Acetylation and H2B SUMOylation In Vivo.

Chromatin remodeling complexes are multi-subunit nucleosome translocases that reorganize chromatin in the context of DNA replication, repair, and transcription. To understand how these complexes find their target sites on chromatin, we use genetically encoded photo-cross-linker amino acids to map the footprint of Sth1, the catalytic subunit of the RSC complex, on nucleosomes in living yeast. We find that H3 K14 acetylation induces the interaction of the Sth1 bromodomain with the H3 tail and mediates the interaction of RSC with neighboring nucleosomes rather than recruiting it to chromatin. RSC preferentially resides on H2B SUMOylated nucleosomes in vivo and shows a moderately enhanced affinity due to this modification in vitro. Furthermore, RSC is not ejected from chromatin in mitosis, but changes its mode of nucleosome binding. Our in vivo analyses show that RSC recruitment to specific chromatin targets involves multiple histone modifications likely in combination with histone variants and transcription factors.

Minimally invasive periareolar approach to repair sinus venosus atrial septal defect with partial anomalous pulmonary venous connection.

BACKGROUND AND AIM: Atrial septal defects with anomalous venous connections are commonly repaired via sternotomy, requiring careful baffle reconstruction to redirect pulmonary venous return and ensure a durable result. The cosmetically appealing periareolar incision may provide an esthetically superior alternative to the anterolateral minithoracotomy incision used in minimally invasive cardiac surgery. METHODS: We describe a patient with a sinus venosus atrial septal defect and partial anomalous pulmonary venous connection who underwent successful minimally invasive, endoscopic repair with apical vein translocation and autologous pericardial baffle reconstruction through a periareolar approach. RESULTS: Post-operative echocardiography demonstrated excellent results with no residual shunt and a widely patent baffle and preserved biventricular function. At 1-year post-op, our patient has had a greatly improved quality of life and an excellent cosmetic result with normal nipple-areolar sensation. CONCLUSIONS: We believe that periareolar approaches should be considered for all adult patients with simple and complex atrial septal defects.

Testosterone Modulates Status-Specific Patterns of Cooperation in a Social Network.

Stable cooperation requires plasticity whereby individuals are able to express competitive or cooperative behaviors depending on social context. To date, however, the physiological mechanisms that underlie behavioral variation in cooperative systems are poorly understood. We studied hormone-mediated behavior in the wire-tailed manakin (Pipra filicauda), a gregarious songbird whose cooperative partnerships and competition for status are both crucial for fitness. We used automated telemetry to monitor >36,000 cooperative interactions among male manakins over three field seasons, and we examined how circulating testosterone affects cooperation using >500 hormone samples. Observational data show that in nonterritorial floater males, high testosterone is associated with increased cooperative behaviors and subsequent ascension to territorial status. In territory-holding males, however, both observational and experimental evidence demonstrate that high testosterone antagonizes cooperation. Moreover, circulating testosterone explains significant variation (2%-8%) in social behavior within each status class. Collectively, our findings show that the hormonal control of cooperation depends on a male's social status. We propose that the status-dependent reorganization of hormone-regulatory pathways can facilitate stable cooperative partnerships and thus provide direct fitness benefits for males.

An anionic, endosome-escaping polymer to potentiate intracellular delivery of cationic peptides, biomacromolecules, and nanoparticles.

Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.

Implementing reasonable adjustments for -disabled people in healthcare services.

Disabled people are one of the groups in society with the greatest health needs, yet they experience some of the most significant barriers to accessing healthcare services. This article describes examples of how three healthcare services have met the Equality Act 2010 duty to make reasonable adjustments for disabled people, so that they are not disadvantaged in accessing these services. Each of these services identified disabled patients, and considered and recorded the specific reasonable adjustments that were required. In doing so, they took time to fully understand the needs of the individual from their perspective. The services collaborated and coordinated the provision of reasonably adjusted care by communicating effectively with other health and social care providers, working together as a team, and treating disabled people as individuals.

Applied Bioengineering in Tissue Reconstruction, Replacement, and Regeneration.

IMPACT STATEMENT: The use of autologous tissue in the reconstruction of tissue defects has been the gold standard. However, current standards still face many limitations and complications. Improving patient outcomes and quality of life by addressing these barriers remain imperative. This article provides historical perspective, covers the major limitations of current standards of care, and reviews recent advances and future prospects in applied bioengineering in the context of tissue reconstruction, replacement, and regeneration.

Gal8 Visualization of Endosome Disruption Predicts Carrier-Mediated Biologic Drug Intracellular Bioavailability.

Endolysosome entrapment is one of the key barriers to the therapeutic use of biologic drugs that act intracellularly. The screening of prospective nanoscale endosome-disrupting delivery technologies is currently limited by methods that are indirect and cumbersome. Here, we statistically validate Galectin 8 (Gal8) intracellular tracking as a superior approach that is direct, quantitative, and predictive of therapeutic cargo intracellular bioactivity through in vitro high-throughput screening and in vivo validation. Gal8 is a cytosolically dispersed protein that, when endosomes are disrupted, redistributes by binding to glycosylation moieties selectively located on the inner face of endosomal membranes. The quantitative redistribution of a Gal8 fluorescent fusion protein from the cytosol into endosomes is demonstrated as a real-time, live-cell assessment of endosomal integrity that does not require labeling or modification of either the carrier or the biologic drug and that allows quantitative distinction between closely related, endosome-disruptive drug carriers. Through screening two families of siRNA polymeric carrier compositions at varying dosages, we show that Gal8 endosomal recruitment correlates strongly ( r = 0.95 and p < 10-4) with intracellular siRNA bioactivity. Through this screen, we gathered insights into how composition and molecular weight affect endosome disruption activity of poly[(ethylene glycol)- b-[(2-(dimethylamino)ethyl methacrylate)- co-(butyl methacrylate)]] [PEG-(DMAEMA- co-BMA)] siRNA delivery systems. Additional studies showed that Gal8 recruitment predicts intracellular bioactivity better than current standard methods such as Lysotracker colocalization ( r = 0.35, not significant), pH-dependent hemolysis (not significant), or cellular uptake ( r = 0.73 and p < 10-3). Importantly, the Gal8 recruitment method is also amenable to fully objective high-throughput screening using automated image acquisition and quantitative image analysis, with a robust estimated Z' of 0.6 (whereas assays with Z' > 0 have high-throughput screening utility). Finally, we also provide measurements of in vivo endosomal disruption based on Gal8 visualization ( p < 0.03) of a nanocarrier formulation confirmed to produce significant cytosolic delivery and bioactivity of siRNA within tumors ( p < 0.02). In sum, this report establishes the utility of Gal8 subcellular tracking for the rapid optimization and high-throughput screening of the endosome disruption potency of intracellular delivery technologies.