Cinchona Alkaloid Polymers Demonstrate Highly Efficient Gene Delivery Dependent on Stereochemistry, Methoxy Substitution, and Length.

Nucleic acid delivery with cationic polymers is a promising alternative to expensive viral-based methods; however, it often suffers from a lower performance. Herein, we present a highly efficient delivery system based on cinchona alkaloid natural products copolymerized with 2-hydroxyethyl acrylate. Cinchona alkaloids are an attractive monomer class for gene delivery applications, given their ability to bind to DNA via both electrostatics and intercalation. To uncover the structure-activity profile of the system, four structurally similar cinchona alkaloids were incorporated into polymers: quinine, quinidine, cinchonine, and cinchonidine. These polymers differed in the chain length, the presence or absence of a pendant methoxy group, and stereochemistry, all of which were found to alter gene delivery performance and the ways in which the polymers overcome biological barriers to transfection. Longer polymers that contained the methoxy-bearing cinchona alkaloids (i.e., quinine and quinidine) were found to have the best performance. These polymers exhibited the tightest DNA binding, largest and most abundant DNA-polymer complexes, and best endosomal escape thanks to their increased buffering capacity and closest nuclear proximity of the payload. Overall, this work highlights the remarkable efficiency of polymer systems that incorporate cinchona alkaloid natural products while demonstrating the profound impact that small structural changes can have on overcoming biological hurdles associated with gene delivery.

Topical Solution for Retinal Delivery: Bevacizumab and Ranibizumab Eye Drops in Anti-Aggregation Formula (AAF) in Rabbits.

PURPOSE: Wet age-related macular degeneration (AMD) is a blinding retinal disease. Monthly intravitreal anti-VEGF antibody injections of bevacizumab (off-label) and ranibizumab (FDA approved) are the standard of care. Antibody aggregation may interfere with ocular absorption/distribution. This study assessed topical delivery of dilute antibodies to the posterior segment of rabbit eyes using a novel anti-aggregation formula (AAF). METHODS: Bevacizumab, or biosimilar ranibizumab was diluted to 5 mg/ml in AAF. All rabbits were dosed twice daily. Substudy 1 rabbits (bevacizumab, 100 µl eye drops): Group 1 (bevacizumab/AAF, n = 6); Group 2 (bevacizumab/PBS, n = 7) and Vehicle control (AAF, n = 1). Substudy 2 rabbits (ranibizumab biosimilar/AAF, 50 µl eye drops): (ranibizumab biosimilar/AAF, n = 8). At 14.5 days, serum was drawn from rabbits. Aqueous, vitreous and retina samples were recovered from eyes and placed into AAF aliquots. Tissue analyzed using AAF as diluent. RESULTS: Bevacizumab in AAF permeated/accumulated in rabbit aqueous, vitreous and retina 10 times more, than when diluted in PBS. AAF/0.1% hyaluronic acid eye drops, dosed twice daily, provided mean tissue concentrations (ng/g) in retina (29.50), aqueous (12.34), vitreous (3.46), and serum (0.28 ng/ml). Additionally, the highest concentration (ng/g) of ranibizumab biosimilar was present in the retina (18.0), followed by aqueous (7.82) and vitreous (1.47). Serum concentration was negligible (< 0.04 ng/ml). No irritation was observed throughout the studies. CONCLUSIONS: Bevacizumab and ranibizumab, in an AAF diluent eye drop, can be delivered to the retina, by the twice daily dosing of a low concentration mAb formulation. This may prove to be an adjunct to intravitreal injections.

Disentangling direct and indirect effects of extreme events on coastal wetland communities.

One of the primary ways in which climate change will impact coastal freshwater wetlands is through changes in the frequency, intensity, timing and distribution of extreme weather events. Disentangling the direct and indirect mechanisms of population- and community-level responses to extreme events is vital to predicting how species composition of coastal wetlands will change under future conditions. We extended static structural equation modelling approaches to incorporate system dynamics in a multi-year multispecies occupancy model to quantify the effects of extreme weather events on a coastal freshwater wetland system. We used data from an 8-year study (2009-2016) on St. Marks National Wildlife Refuge in Florida, USA, to quantify species-specific and community-level changes in amphibian and fish occupancy associated with two flooding events in 2012 and 2013. We examine how physical changes to the landscape, including potential changes in salinity and increased wetland connectivity, may have contributed to or exacerbated the effects of these extreme weather events on the biota of isolated coastal wetlands. We provide evidence that the primary effects of flooding on the amphibian community were through indirect mechanisms via changes in the composition of the sympatric fish community that may have had lethal (i.e. through direct predation) or non-lethal (i.e. through direct or indirect competitive interactions) effects. In addition, we have shown that amphibian species differed in their sensitivity to direct flooding effects and indirect changes in the fish community and wetland-specific conductance, which led to variable responses across the community. These effects led to the overall decline in amphibian species richness from 2009 to 2016, suggesting that wetland-breeding amphibian communities on St. Marks National Wildlife Refuge may not be resilient to predicted changes in coastal disturbance regimes because of climate change. Understanding both direct and indirect effects, as well as species interactions, is important for predicting the effects of a changing climate on individual species, communities and ecosystems.

MRSA-induced endothelial permeability and acute lung injury are attenuated by FTY720 S-phosphonate.

Disruption of the lung endothelial barrier is a hallmark of acute respiratory distress syndrome (ARDS), for which no effective pharmacologic treatments exist. Prior work has demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine-1-phosphate (S1P) and FTY720, exhibits potent endothelial cell (EC) barrier protective properties. In this study, we investigated the in vitro and in vivo efficacy of Tys against methicillin-resistant Staphylococcus aureus (MRSA), a frequent bacterial cause of ARDS. Tys-protected human lung EC from barrier disruption induced by heat-killed MRSA (HK-MRSA) or staphylococcal α-toxin and attenuated MRSA-induced cytoskeletal changes associated with barrier disruption, including actin stress fiber formation and loss of peripheral VE-cadherin and cortactin. Tys-inhibited Rho and myosin light chain (MLC) activation after MRSA and blocked MRSA-induced NF-κB activation and release of the proinflammatory cytokines, IL-6 and IL-8. In vivo, intratracheal administration of live MRSA in mice caused significant vascular leakage and leukocyte infiltration into the alveolar space. Pre- or posttreatment with Tys attenuated MRSA-induced lung permeability and levels of alveolar neutrophils. Posttreatment with Tys significantly reduced levels of bronchoalveolar lavage (BAL) VCAM-1 and plasma IL-6 and KC induced by MRSA. Dynamic intravital imaging of mouse lungs demonstrated Tys attenuation of HK-MRSA-induced interstitial edema and neutrophil infiltration into lung tissue. Tys did not directly inhibit MRSA growth or viability in vitro. In conclusion, Tys inhibits lung EC barrier disruption and proinflammatory signaling induced by MRSA in vitro and attenuates acute lung injury induced by MRSA in vivo. These results support the potential utility of Tys as a novel ARDS therapeutic strategy.

Concentration-associated pathology of alkali burn in a mouse model using anterior segment optical coherence tomography with angiography.

Pathological features of alkali concentration-associated burn were studied using non-invasive anterior segment optical coherence tomography (AS-OCT) and OCT angiography (OCTA). Alkali burn was induced in C57BL/6J mice (n = 20) by placing filter paper soaked in 0.1, 0.25, 0.5, and 1 M NaOH for 30s on the right eye (left eye control). Longitudinal imaging was performed with AS-OCT/OCTA and fluorescein angiography over 14 days, after which eyes were enucleated at 7 and 14 days for histology and immunofluorescence. Concentration-associated corneal swelling was maximal at 0.5M, increasing linearly in a concentration-dependent fashion at 0.1, 0.25, and 0.5 M NaOH, to levels of 50%, 100%, and 175% of control, respectively. At 0.1M, corneal swelling and surface erosions were prominent, while at 0.25M, deep tissue damage, limbal neovascularization, and stromal haze were evident at 7 days. At 0.5M and 1M, severe exacerbation of the corneal swelling, angle closure, Descemet's membrane detachment, hyphema, and profuse central neovascularization were noted as early as day 3, which further progressed to inflammation, fibrosis, and opacity by day 7. We conclude that alkali concentration-dependent burn intensity biomarkers can be assessed by non-invasive AS-OCT/OCTA, distinguishing between mild, moderate, and severe ocular injury, with potential relevance toward clinical utilization in human eyes.

Early-stage attenuation of phase-amplitude coupling in the hippocampus and medial prefrontal cortex in a transgenic rat model of Alzheimer's disease.

Alzheimer's disease (AD) is pathologically characterized by amyloid-ß peptide (Aß) accumulation, neurofibrillary tangle formation, and neurodegeneration. Preclinical studies on neuronal impairments associated with progressive amyloidosis have demonstrated some Aß-dependent neuronal dysfunction including modulation of gamma-aminobutyric acid-ergic signaling. The present work focuses on the early stage of disease progression and uses TgF344-AD rats that recapitulate a broad repertoire of AD-like pathologies to investigate the neuronal network functioning using simultaneous intracranial recordings from the hippocampus (HPC) and the medial prefrontal cortex (mPFC), followed by pathological analyses of gamma-aminobutyric acid (GABAA ) receptor subunits α1, α5, and δ, and glutamic acid decarboxylases (GAD65 and GAD67). Concomitant to amyloid deposition and tau hyperphosphorylation, low-gamma band power was strongly attenuated in the HPC and mPFC of TgF344-AD rats in comparison to those in non-transgenic littermates. In addition, the phase-amplitude coupling of the neuronal networks in both areas was impaired, evidenced by decreased modulation of theta band phase on gamma band amplitude in TgF344-AD animals. Finally, the gamma coherence between HPC and mPFC was attenuated as well. These results demonstrate significant neuronal network dysfunction at an early stage of AD-like pathology. This network dysfunction precedes the onset of cognitive deficits and is likely driven by Aß and tau pathologies. This article is part of the Special Issue "Vascular Dementia".

Cortical Actin Dynamics in Endothelial Permeability.

The pulmonary endothelial cell forms a critical semi-permeable barrier between the vascular and interstitial space. As part of the blood-gas barrier in the lung, the endothelium plays a key role in normal physiologic function and pathologic disease. Changes in endothelial cell shape, defined by its plasma membrane, determine barrier integrity. A number of key cytoskeletal regulatory and effector proteins including non-muscle myosin light chain kinase, cortactin, and Arp 2/3 mediate actin rearrangements to form cortical and membrane associated structures in response to barrier enhancing stimuli. These actin formations support and interact with junctional complexes and exert forces to protrude the lipid membrane to and close gaps between individual cells. The current knowledge of these cytoskeletal processes and regulatory proteins are the subject of this review. In addition, we explore novel advancements in cellular imaging that are poised to shed light on the complex nature of pulmonary endothelial permeability.

TRPC6 channel translocation into phagosomal membrane augments phagosomal function.

Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H(+) into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.

Species interactions and the effects of climate variability on a wetland amphibian metacommunity.

Disentangling the role that multiple interacting factors have on species responses to shifting climate poses a significant challenge. However, our ability to do so is of utmost importance to predict the effects of climate change on species distributions. We examined how populations of three species of wetland-breeding amphibians, which varied in life history requirements, responded to a six-year period of extremely variable precipitation. This interval was punctuated by both extensive drought and heavy precipitation and flooding, providing a natural experiment to measure community responses to environmental perturbations. We estimated occurrence dynamics using a discrete hidden Markov modeling approach that incorporated information regarding habitat state and predator-prey interactions. This approach allowed us to measure how metapopulation dynamics of each amphibian species was affected by interactions among weather, wetland hydroperiod, and co-occurrence with fish predators. The pig frog, a generalist, proved most resistant to perturbations, with both colonization and persistence being unaffected by seasonal variation in precipitation or co-occurrence with fishes. The ornate chorus frog, an ephemeral wetland specialist, responded positively to periods of drought owing to increased persistence and colonization rates during periods of low-rainfall. Low probabilities of occurrence of the ornate chorus frog in long-duration wetlands were driven by interactions with predators due to low colonization rates when fishes were present. The mole salamander was most sensitive to shifts in water availability. In our study area, this species never occurred in short-duration wetlands and persistence probabilities decreased during periods of drought. At the same time, negative effects occurred with extreme precipitation because flooding facilitated colonization of fishes to isolated wetlands and mole salamanders did not colonize wetlands once fishes were present. We demonstrate that the effects of changes in water availability depend on interactions with predators and wetland type and are influenced by the life history of each of our species. The dynamic species occurrence modeling approach we used offers promise for other systems when the goal is to disentangle the complex interactions that determine species responses to environmental variability.

Life history plasticity does not confer resilience to environmental change in the mole salamander (Ambystoma talpoideum).

Plasticity in life history strategies can be advantageous for species that occupy spatially or temporally variable environments. We examined how phenotypic plasticity influences responses of the mole salamander, Ambystoma talpoideum, to disturbance events at the St. Marks National Wildlife Refuge (SMNWR), FL, USA from 2009 to 2014. We observed periods of extensive drought early in the study, in contrast to high rainfall and expansive flooding events in later years. Flooding facilitated colonization of predatory fishes to isolated wetlands across the refuge. We employed multistate occupancy models to determine how this natural experiment influenced the occurrence of aquatic larvae and paedomorphic adults and what implications this may have for the population. We found that, in terms of occurrence, responses to environmental variation differed between larvae and paedomorphs, but plasticity (i.e. the ability to metamorphose rather than remain in aquatic environment) was not sufficient to buffer populations from declining as a result of environmental perturbations. Drought and fish presence negatively influenced occurrence dynamics of larval and paedomorphic mole salamanders and, consequently, contributed to observed short-term declines of this species. Overall occurrence of larval salamanders decreased from 0.611 in 2009 to 0.075 in 2014 and paedomorph occurrence decreased from 0.311 in 2009 to 0.121 in 2014. Although variation in selection pressures has likely maintained this polyphenism previously, our results suggest that continued changes in environmental variability and the persistence of fish in isolated wetlands could lead to a loss of paedomorphosis in the SMNWR population and, ultimately, impact regional persistence in the future.

scyllo-Inositol promotes robust mutant Huntingtin protein degradation.

Huntington disease is characterized by neuronal aggregates and inclusions containing polyglutamine-expanded huntingtin protein and peptide fragments (polyQ-Htt). We have used an established cell-based assay employing a PC12 cell line overexpressing truncated exon 1 of Htt with a 103-residue polyQ expansion that yields polyQ-Htt aggregates to investigate the fate of polyQ-Htt-drug complexes. scyllo-Inositol is an endogenous inositol stereoisomer known to inhibit accumulation and toxicity of the amyloid-ß peptide and α-synuclein. In light of these properties, we investigated the effect of scyllo-inositol on polyQ-Htt accumulation. We show that scyllo-inositol lowered the number of visible polyQ-Htt aggregates and robustly decreased polyQ-Htt protein abundance without concomitant cellular toxicity. We found that scyllo-inositol-induced polyQ-Htt reduction was by rescue of degradation pathways mediated by the lysosome and by the proteasome but not autophagosomes. The rescue of degradation pathways was not a direct result of scyllo-inositol on the lysosome or proteasome but due to scyllo-inositol-induced reduction in mutant polyQ-Htt protein levels.

Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function.

CFTR regulates phagosome acidification in macrophages and alters bactericidal activity.

Acidification of phagosomes has been proposed to have a key role in the microbicidal function of phagocytes. Here, we show that in alveolar macrophages the cystic fibrosis transmembrane conductance regulator Cl- channel (CFTR) participates in phagosomal pH control and has bacterial killing capacity. Alveolar macrophages from Cftr-/- mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria. Lysosomes from CFTR-null macrophages failed to acidify, although they retained normal fusogenic capacity with nascent phagosomes. We hypothesize that CFTR contributes to lysosomal acidification and that in its absence phagolysosomes acidify poorly, thus providing an environment conducive to bacterial replication.

Cyclohexanehexol inhibitors of Abeta aggregation prevent and reverse Alzheimer phenotype in a mouse model.

When given orally to a transgenic mouse model of Alzheimer disease, cyclohexanehexol stereoisomers inhibit aggregation of amyloid beta peptide (Abeta) into high-molecular-weight oligomers in the brain and ameliorate several Alzheimer disease-like phenotypes in these mice, including impaired cognition, altered synaptic physiology, cerebral Abeta pathology and accelerated mortality. These therapeutic effects, which occur regardless of whether the compounds are given before or well after the onset of the Alzheimer disease-like phenotype, support the idea that the accumulation of Abeta oligomers has a central role in the pathogenesis of Alzheimer disease.

Amyloid-ß-dependent compromise of microvascular structure and function in a model of Alzheimer's disease.

The majority of patients with Alzheimer's disease have cerebral amyloid angiopathy, thus showing deposition of amyloid-ß peptides in the walls of leptomeningeal and cortical arterioles. These deposits are believed to result from impaired clearance of parenchymal amyloid-ß peptides. In the current work, we examined the changes in cortical microvascular structure and function in situ in TgCRND8, a transgenic mouse model of Alzheimer's disease. In contrast to venules, cortical arterioles were shown to increase in tortuosity and decrease in calibre with amyloid-ß peptide accumulation. These structural changes were accompanied by progressive functional compromise, reflected in higher dispersion of microvascular network transit times, elongation of the transit times, and impaired microvascular reactivity to hypercapnia in the transgenic mice. Moreover, inhibition of amyloid-ß peptide oligomerization and fibrillization via post-weaning administration of scyllo-inositol, a naturally occurring stereoisomer of myo-inositol, rescued both structural and functional impairment of the cortical microvasculature in this Alzheimer's disease model. These results demonstrate that microvascular impairment is directly correlated with amyloid-ß accumulation and highlight the importance of targeting cerebrovascular amyloid angiopathy clearance for effective diagnosis, monitoring of disease progression and treatment of Alzheimer's disease.

Prevalence of the metabolic syndrome in children with psoriatic disease.

Adults with psoriasis have a greater risk of developing metabolic syndrome (MetS) and cardiovascular disease (CVD), but few studies have investigated the prevalence of MetS and other risk factors for CVD in children with psoriasis. In an assessor-blinded study, 20 children ages 9-17 years with a current or previously documented history of psoriasis involving 5% or more of their body surface area or psoriatic arthritis were compared with a cohort of age- and sex-matched controls with benign nevi, warts, or acne. MetS, our primary endpoint, was defined by the presence of abnormal values in at least three of the following measures: triglycerides, high-density lipoprotein cholesterol (HDL-C), fasting blood glucose (FBG), waist circumference, and blood pressure. Secondary endpoints included high-sensitivity C-reactive protein (hs-CRP), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C). Thirty percent (6/20) of children with psoriasis met the criteria for MetS, compared with 5% (1/20) of the control group (p < 0.05). Subjects with psoriasis had higher mean FBG (91.1 mg/dL) than the control group (82.9 mg/dL) (p = 0.01). There were no statistically significant differences in the other four components of MetS, BMI, BMI percentile, hs-CRP, TC, or LDL-C. The results of this trial demonstrate that children with psoriasis have higher rates of MetS than age- and sex-matched controls. It may therefore be important to evaluate children with psoriasis for components of MetS to prevent future CVD morbidity and mortality.

Enhancing pDNA Delivery with Hydroquinine Polymers by Modulating Structure and Composition.

Quinine is a promising natural product building block for polymer-based nucleic acid delivery vehicles as its structure enables DNA binding through both intercalation and electrostatic interactions. However, studies exploring the potential of quinine-based polymers for nucleic acid delivery applications (transfection) are limited. In this work, we used a hydroquinine-functionalized monomer, HQ, with 2-hydroxyethyl acrylate to create a family of seven polymers (HQ-X, X = mole percentage of HQ), with mole percentages of HQ ranging from 12 to 100%. We developed a flow cytometer-based assay for studying the polymer-pDNA complexes (polyplex particles) directly and demonstrate that polymer composition and monomer structure influence polyplex characteristics such as the pDNA loading and the extent of adsorption of serum proteins on polyplex particles. Biological delivery experiments revealed that maximum transgene expression, outperforming commercial controls, was achieved with HQ-25 and HQ-35 as these two variants sustained gene expression over 96 h. HQ-44, HQ-60, and HQ-100 were not successful in inducing transgene expression, despite being able to deliver pDNA into the cells, highlighting that the release of pDNA is likely the bottleneck in transfection for polymers with higher HQ content. Using confocal imaging, we quantified the extent of colocalization between pDNA and lysosomes, proving the remarkable endosomal escape capabilities of the HQ-X polymers. Overall, this study demonstrates the advantages of HQ-X polymers as well as provides guiding principles for improving the monomer structure and polymer composition, supporting the development of the next generation of polymer-based nucleic acid delivery vehicles harnessing the power of natural products.

MET Receptor Tyrosine Kinase Inhibition Reduces Interferon-Gamma (IFN-γ)-Stimulated PD-L1 Expression through the STAT3 Pathway in Melanoma Cells.

Melanoma is the leading cause of death from cutaneous malignancy. While targeted therapy and immunotherapy with checkpoint inhibitors have significantly decreased the mortality rate of this disease, advanced melanoma remains a therapeutic challenge. Here, we confirmed that interferon-gamma (IFN-γ)-induced PD-L1 expression in melanoma cell lines. This increased expression was down-regulated by the reduction in phosphorylated STAT3 signaling via MET tyrosine kinase inhibitor treatment. Furthermore, immunoprecipitation and confocal immunofluorescence microscopy analysis reveals MET and PD-L1 protein-protein interaction and colocalization on the cell surface membrane of melanoma cells. Together, these findings demonstrate that the IFN-γ-induced PD-L1 expression in melanoma cells is negatively regulated by MET inhibition through the JAK/STAT3 signaling pathway and establish the colocalization and interaction between an RTK and a checkpoint protein in melanoma cells.

Combinatorial Polycation Synthesis and Causal Machine Learning Reveal Divergent Polymer Design Rules for Effective pDNA and Ribonucleoprotein Delivery.

The development of polymers that can replace engineered viral vectors in clinical gene therapy has proven elusive despite the vast portfolios of multifunctional polymers generated by advances in polymer synthesis. Functional delivery of payloads such as plasmids (pDNA) and ribonucleoproteins (RNP) to various cellular populations and tissue types requires design precision. Herein, we systematically screen a combinatorially designed library of 43 well-defined polymers, ultimately identifying a lead polycationic vehicle (P38) for efficient pDNA delivery. Further, we demonstrate the versatility of P38 in codelivering spCas9 RNP and pDNA payloads to mediate homology-directed repair as well as in facilitating efficient pDNA delivery in ARPE-19 cells. P38 achieves nuclear import of pDNA and eludes lysosomal processing far more effectively than a structural analogue that does not deliver pDNA as efficiently. To reveal the physicochemical drivers of P38's gene delivery performance, SHapley Additive exPlanations (SHAP) are computed for nine polyplex features, and a causal model is applied to evaluate the average treatment effect of the most important features selected by SHAP. Our machine learning interpretability and causal inference approach derives structure-function relationships underlying delivery efficiency, polyplex uptake, and cellular viability and probes the overlap in polymer design criteria between RNP and pDNA payloads. Together, combinatorial polymer synthesis, parallelized biological screening, and machine learning establish that pDNA delivery demands careful tuning of polycation protonation equilibria while RNP payloads are delivered most efficaciously by polymers that deprotonate cooperatively via hydrophobic interactions. These payload-specific design guidelines will inform further design of bespoke polymers for specific therapeutic contexts.

A cortactin CTTN coding SNP contributes to lung vascular permeability and inflammatory disease severity in African descent subjects.

The cortactin gene (CTTN), encoding an actin-binding protein critically involved in cytoskeletal dynamics and endothelial cell (EC) barrier integrity, contains single nucleotide polymorphisms (SNPs) associated with severe asthma in Black patients. As loss of lung EC integrity is a major driver of mortality in the Acute Respiratory Distress Syndrome (ARDS), sepsis, and the acute chest syndrome (ACS), we speculated CTTN SNPs that alter EC barrier function will associate with clinical outcomes from these types of conditions in Black patients. In case-control studies, evaluation of a nonsynonymous CTTN coding SNP Ser484Asn (rs56162978, G/A) in a severe sepsis cohort (725 Black subjects) revealed significant association with increased risk of sepsis mortality. In a separate cohort of sickle cell disease (SCD) subjects with and without ACS (177 SCD Black subjects), significantly increased risk of ACS and increased ACS severity (need for mechanical ventilation) was observed in carriers of the A allele. Human lung EC expressing the cortactin S484N transgene exhibited: (i) delayed EC barrier recovery following thrombin-induced permeability; (ii) reduced levels of critical Tyr486 cortactin phosphorylation; (iii) inhibited binding to the cytoskeletal regulator, nmMLCK; and (iv) attenuated EC barrier-promoting lamellipodia dynamics and biophysical responses. ARDS-challenged Cttn+/- heterozygous mice exhibited increased lung vascular permeability (compared to wild-type mice) which was significantly attenuated by IV delivery of liposomes encargoed with CTTN WT transgene but not by CTTN S484N transgene. In summary, these studies suggest that the CTTN S484N coding SNP contributes to severity of inflammatory injury in Black patients, potentially via delayed vascular barrier restoration.