Gender-different effect of Src family kinases antagonism on photophobia and trigeminal ganglion activity.

BACKGROUND: Src family kinases (SFKs) contribute to migraine pathogenesis, yet its role in regulating photophobia behaviour, one of the most common forms of migraine, remains unknown. Here, we addressed whether SFKs antagonism alleviates photophobia behavior and explored the underlying mechanism involving hypothalamus and trigeminal ganglion activity, as measured by the alteration of neuropeptide levels and transcriptome respectively. METHODS: A rapid-onset and injury-free mouse model of photophobia was developed following intranasal injection of the TRPA1 activator, umbellulone. The role of SFKs antagonism on light aversion was assessed by the total time the mouse stays in the light and transition times between the dark and light compartments. To gain insight to the preventive mechanism of SFKs antagonism, hypothalamic neuropeptides levels were assessed using enzyme linked immunofluorescent assay and trigeminal ganglion activity were assessed using RNA-sequencing and qPCR analysis. RESULTS: SFKs antagonism by a clinically relevant SFKs inhibitor saracatinib reduced the total time in light and transition times in male mice, but not in females, suggesting SFKs play a crucial role in photophobia progressing and exhibit a male-only effect. SFKs antagonism had no effect on hypothalamic calcitonin gene-related peptide and pituitary adenylate cyclase-activating polypeptide levels of all mice investigated, suggesting the gender-different effect of saracatinib on light aversion appears to be independent of these hypothalamic neuropeptide levels. In trigeminal ganglion of male mice, photophobia is associated with profound alteration of differentially expressed genes, part of which were reversed by SFKs antagonism. Subsequent qPCR analysis showed SFKs antagonism displayed gender-different modulation of expression in some candidate genes, particularly noteworthy those encoding ion channels (trpm3, Scn8a), ATPase signaling (crebbp, Atp5α1) and kinase receptors (Zmynd8, Akt1). CONCLUSIONS: In conclusion, our data revealed that SFKs antagonism reduced photophobia processing in male mice and exhibited gender-different modulation of trigeminal ganglion activity, primarily manifesting as alterations in the transcriptome profile. These findings underscore the potential of SFKs antagonism for allieving photophobia in males, highlighting its value in the emerging field of precision medicine.

The discovery and structural basis of two distinct state-dependent inhibitors of BamA.

BamA is the central component of the essential ß-barrel assembly machine (BAM), a conserved multi-subunit complex that dynamically inserts and folds ß-barrel proteins into the outer membrane of Gram-negative bacteria. Despite recent advances in our mechanistic and structural understanding of BamA, there are few potent and selective tool molecules that can bind to and modulate BamA activity. Here, we explored in vitro selection methods and different BamA/BAM protein formulations to discover peptide macrocycles that kill Escherichia coli by targeting extreme conformational states of BamA. Our studies show that Peptide Targeting BamA-1 (PTB1) targets an extracellular divalent cation-dependent binding site and locks BamA into a closed lateral gate conformation. By contrast, PTB2 targets a luminal binding site and traps BamA into an open lateral gate conformation. Our results will inform future antibiotic discovery efforts targeting BamA and provide a template to prospectively discover modulators of other dynamic integral membrane proteins.

NeuroBooster Array: A Genome-Wide Genotyping Platform to Study Neurological Disorders Across Diverse Populations.

BACKGROUND: Commercial genome-wide genotyping arrays have historically neglected coverage of genetic variation across populations. OBJECTIVE: We aimed to create a multi-ancestry genome-wide array that would include a wide range of neuro-specific genetic content to facilitate genetic research in neurological disorders across multiple ancestral groups, fostering diversity and inclusivity in research studies. METHODS: We developed the Illumina NeuroBooster Array (NBA), a custom high-throughput and cost-effective platform on a backbone of 1,914,934 variants from the Infinium Global Diversity Array and added custom content comprising 95,273 variants associated with more than 70 neurological conditions or traits, and we further tested its performance on more than 2000 patient samples. This novel platform includes approximately 10,000 tagging variants to facilitate imputation and analyses of neurodegenerative disease-related genome-wide association study loci across diverse populations. RESULTS: In this article, we describe NBA's potential as an efficient means for researchers to assess known and novel disease genetic associations in a multi-ancestry framework. The NBA can identify rare genetic variants and accurately impute more than 15 million common variants across populations. Apart from enabling sample prioritization for further whole-genome sequencing studies, we envisage that NBA will play a pivotal role in recruitment for interventional studies in the precision medicine space. CONCLUSIONS: From a broader perspective, the NBA serves as a promising means to foster collaborative research endeavors in the field of neurological disorders worldwide. Ultimately, this carefully designed tool is poised to make a substantial contribution to uncovering the genetic etiology underlying these debilitating conditions. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Enhancing Prehospital Care During the Conflict in Ukraine: NATO's Role in Global Health Engagement.

INTRODUCTION: The conflict in Ukraine, ongoing since 2014 and escalating with the Russian invasion in 2022, has unveiled profound challenges in prehospital care essential for the survival and recovery of warfighters and civilians alike, necessitating a detailed examination of the current medical response mechanisms and their effectiveness. MATERIALS AND METHODS: This study provides an overview of these challenges and examines how these critical vulnerabilities have impacted the delivery of medical care in war-torn regions. It also explores the role of NATO and its member states in addressing these challenges, focusing on the efforts to standardize prehospital care, enhance training, and foster interoperability among medical services. Furthermore, it explores the role of global heath engagement through NGOs in addressing these prehospital care gaps within the Ukrainian conflict zone, drawing from direct observations, expert testimonials, and secondary data. RESULTS: Findings reveal significant enhancements in prehospital care through improved training, interoperability, and logistics management, despite ongoing challenges in medical infrastructure and extended evacuation times, which continue to impact the quality of care. CONCLUSIONS: The study underscores the critical role of international collaboration and standardized protocols in bolstering prehospital medical responses in conflict settings, highlighting the need for continuous adaptation and support to mitigate the complexities of modern warfare. The insights gained from the Ukraine conflict offer valuable lessons for future military and humanitarian medical responses in similar conflict settings.

Advancing Military Medical Planning in Large Scale Combat Operations: Insights From Computer Simulation and Experimentation in NATO's Vigorous Warrior Exercise 2024.

INTRODUCTION: The ongoing conflict in Ukraine from Russian invasion presents a critical challenge to medical planning in the context of multi-domain battle against a peer adversary deploying conventional weapon systems. The potential escalation of preventable morbidity and mortality, reaching a scale unprecedented since World War II, underscores the paramount importance of effective phases of care from Point of Injury (PoI)/Point of Wounding (PoW) or Point of Exposure (PoE) to Role 1 (R1) and Role 2 (R2) echelons of care.The NATO Vigorous Warrior (VW) Live Exercise (LIVEX) serves as a strategic platform for NATO and its partners, providing an opportunity to challenge operational concepts, experiment, innovate life-saving systems, and foster best practices across the Alliance. MATERIALS AND METHODS: This study delineates the strategic application of the VW LIVEX platform for the adaptation of the computational simulation software Simulation for the Assessment and Optimization of Medical Disaster Management (SIMEDIS) within the context of Large-Scale Combat Operations (LSCO). The SIMEDIS computer simulator plays a pivotal role by furnishing real-time insights into the evolving injury patterns of patients, employing an all-hazards approach. This simulator facilitates the examination of temporal shifts in medical timelines and the ramifications of resource scarcity against both morbidity and mortality outcomes. The VW LIVEX provides a unique opportunity for systematic validation to evaluate the results of the computer simulator in a realistic setting and identify gaps for future concepts of operations. RESULTS: We report the process and methodologies to be evaluated at the VW LIVEX in far forward and retrospective medical support operations. Using the SIMEDIS simulator, we can define battlefield scenarios for varied situations including artillery, drone strikes, and Chemical, Biological, Radiological, Nuclear, and explosive (CBRNe) attacks. Casualty health progressions versus time are dependent on each threat. Mortality is computed based on the concepts found in Tactical Combat Casualty Care (TCCC) of "self-aid"/"buddy-aid" factoring in the application or absence of definitive traumatic hemorrhage control and on the distribution policy of victims to medical treatment facilities through appropriate Command and Control (C2) ("Scoop and Run" versus "Stay and Play"). The number of medical supplies available along with the number of transport resources and personnel are set and are scalable, with their effect on both morbidity and mortality quantified.Concept of Medical Operations can be optimized and interoperability enhanced when shared data are provided to C2 for prospective medical planning with retrospective data. The SIMEDIS simulator determines best practices of medical management for a myriad of injury types and tactical/operational situations relevant to policy making and battlefield medical planning for LSCO. CONCLUSIONS: The VW LIVEX provides a Concept Development and Experimentation platform for SIMEDIS refinement and conclusive insights into medical planning to reduce preventable morbidity and mortality. Recommending further iterations of similar methodologies at other NATO LIVEXs for validation is crucial, as is information sharing across the Alliance and partners to ensure best practice standards are met.

A comparative study on surface-engineered nanoceria using a catechol copolymer design: colloidal stability vs. antioxidant activity.

Nanoceria (NC) are widely studied as potent nanozyme antioxidants, featuring unique multifunctional, self-regenerative, and high-throughput enzymatic functions. However, bare NC are reported to show poor colloidal stability in biological media. Despite this, the nexus between colloidal stability and antioxidant activity has rarely been assessed. Here, a library of three copolymeric stabilising agents was synthesised, each consisting of hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) brushes (P(OEGMA)) and a novel catechol anchoring block, and used for surface engineering of NC. The colloidal stability of the surface-engineered NC was assessed in phosphate buffered saline (PBS) by monitoring their precipitation via UV-Vis spectrophotometry, and their catalase (CAT)- and superoxide dismutase (SOD)-like activities were analysed using fluorospectrophotometry. The obtained results indicate that P(OEGMA) coating improves colloidal stability of NC over 48 h, highlighting the stable attachment of catechol functionalities to the surface of NC. In addition, X-ray photoelectron spectroscopy (XPS) indicates that the catechol functionalities lead to an increase in Ce3+/Ce4+ ratio and the concentration of oxygen vacancies, depending on the number of catechol units. Altogether, surface engineering of NC optimally results in an increase in CAT- and SOD-like activities by, respectively, 41% (=57.7% H2O2 elimination) and 78% (=78.0% O2˙- elimination) relative to bare NC, signifying a positive correlation between colloidal stability and antioxidant activity of the NC nanozymes.

Blood Cancer Network Ireland (BCNI) and National Cancer Registry Ireland (NCRI) collaboration: challenges and utility of an Enhanced Blood Cancer Outcomes Registry (EBCOR) pilot.

BACKGROUND: The Blood Cancer Network Ireland and National Cancer Registry Ireland worked to create an Enhanced Blood Cancer Outcomes Registry (EBCOR). Enhanced data in acute myeloid leukaemia (AML) included an extensive data dictionary, bespoke software and longitudinal follow-up. AIMS: To demonstrate the utility of the database, we applied the data to examine a clinically relevant question: Charlson comorbidity index (CCI) usefulness in predicting AML patients' survival. METHODS: A software designer and consultant haematologists in Cork University Hospital worked together to standardise a data dictionary, train registrars and populate a database. One hundred and forty-one AML patients underwent enhanced data registration. Comorbidities identified by chart review were used to examine the capability of the CCI and age at diagnosis to predict mortality using Kaplan-Meier curves, Cox regression and receiver operating characteristic curves. RESULTS: In regression analysis, a dose-response relationship was observed; patients in the highest CCI tertile displayed a greater risk (HR = 4.90; 95% CI 2.79-8.63) of mortality compared to subjects in tertile 2 (HR = 2.74; 95% CI 1.64-4.57) and tertile 1 (reference). This relationship was attenuated in an analysis which adjusted for age at diagnosis. The area under the curve (AUC) for the CCI was 0.76 (95% CI 0.68-0.84) while the AUC for age at diagnosis was 0.84 (95% CI 0.78-0.90). CONCLUSIONS: Results suggest that the CCI provides no additional prognostic information beyond that obtained from age alone at AML diagnosis and that an EBCOR can provide a rich database for cancer outcomes research, including predictive models and resource allocation.

Visual search strategies and game knowledge in junior Australian rules football players: testing potential in talent identification and development.

This study explored video-based decision-making and eye-movement behavior as a complementary method to assess the decision-making skills and knowledge of elite junior Australian Rules (AR) Football players. Performance was measured twice over an 18-month period. This approach tested a practical and reliable assessment of decision-making and game knowledge that does not contribute to physical training load. N = 59 participants were categorized based on their training age groups, U14 (N = 38, Mage 13.37 ± 0.47) and U16 (N = 21, Mage 14.80 ± 0.39). Participants watched 14 brief video clips and provided action choices while wearing eye-movement recording glasses that captured visual search patterns (e.g., fixations). Decision accuracy and speed of decision-making were also recorded. Participants with accurate decisions made significantly faster decisions compared to less skilled players (p < 0.001). Further, skilled participants had significantly fewer fixations of shorter duration compared to less skilled participants at both the initial and follow-up testing sessions (p < 0.0001). This suggests that eye-movement characteristics, remain a relatively stable measure over moderate periods of time. With the ability to differentiate between more and less skilled decision-makers, this proof-of-concept study proposes that examining eye movements in relation to decision-making and game knowledge is a viable tool for Talent Identification and Development (TID) to complement current measures. We provide a platform for further development and research in the quest for efficient and effective talent identification processes.

Potent activity of polymyxin B is associated with long-lived super-stoichiometric accumulation mediated by weak-affinity binding to lipid A.

Polymyxins are gram-negative antibiotics that target lipid A, the conserved membrane anchor of lipopolysaccharide in the outer membrane. Despite their clinical importance, the molecular mechanisms underpinning polymyxin activity remain unresolved. Here, we use surface plasmon resonance to kinetically interrogate interactions between polymyxins and lipid A and derive a phenomenological model. Our analyses suggest a lipid A-catalyzed, three-state mechanism for polymyxins: transient binding, membrane insertion, and super-stoichiometric cluster accumulation with a long residence time. Accumulation also occurs for brevicidine, another lipid A-targeting antibacterial molecule. Lipid A modifications that impart polymyxin resistance and a non-bactericidal polymyxin derivative exhibit binding that does not evolve into long-lived species. We propose that transient binding to lipid A permeabilizes the outer membrane and cluster accumulation enables the bactericidal activity of polymyxins. These findings could establish a blueprint for discovery of lipid A-targeting antibiotics and provide a generalizable approach to study interactions with the gram-negative outer membrane.

Validation and Comparison of Common Thoracolumbar Injury Classification Treatment Algorithms and a Novel Modification.

BACKGROUND AND OBJECTIVES: The most common thoracolumbar trauma classification systems are the Thoracolumbar Injury Classification and Severity Score (TLICS) and the Thoracolumbar AO Spine Injury Score (TL AOSIS). Predictive accuracy of treatment recommendations is a historical limitation. Our objective was to validate and compare TLICS, TL AOSIS, and a modified TLICS (mTLICS) that awards 2 points for the presence of fractured vertebral body height loss >50% and/or spinal canal stenosis >50% at the fracture site. METHODS: The medical records of adult patients with acute, traumatic thoracolumbar injuries at an urban, Level 1 trauma center were retrospectively reviewed. TLICS, mTLICS, and TL AOSIS scores were calculated for 476 patients using computed tomography, MRI, and the documented neurological examination. Treatment recommendations were compared with treatment received. Standard validity measures were calculated. RESULTS: Treatment recommendations matched actual treatments in 95.6% (455/476) of patients for mTLICS, 91.3% (435/476) for TLICS, and 92.6% (441/476) for TL AOSIS. The differences between the accuracy of mTLICS and TLICS (95.6% vs 91.3%, P < .001) and between mTLICS and TL AOSIS (95.6% vs 91.3%, P = .003) were significant. The sensitivity of mTLICS was higher than that of TLICS (96.3% vs 81.3%, P < .001), and the sensitivity of TL AOSIS was higher than that of TLICS (92.5% vs 81.3%, P < .001). The specificity of mTLICS was equal to that of TLICS (95.3%) and higher than that of TL AOSIS (95.3% vs 92.7%, P = .02). The modifier led to substantial outperformance of mTLICS over TLICS due to 38 patients (20 of whom received surgery) moving from a TLICS score of <4 to a mTLICS score equal to 4. CONCLUSION: All systems performed well. The mTLICS had improved sensitivity and accuracy compared with TLICS and higher accuracy and specificity than TL AOSIS. The sensitivity of TL AOSIS was higher than that of TLICS. Prospective, multi-institutional reliability and validity studies of this mTLICS are needed for adoption.

Deciphering the role of a SINE-VNTR-Alu retrotransposon polymorphism as a biomarker of Parkinson's disease progression.

SINE-VNTR-Alu (SVA) retrotransposons are transposable elements which represent a source of genetic variation. We previously demonstrated that the presence/absence of a human-specific SVA, termed SVA_67, correlated with the progression of Parkinson's disease (PD). In the present study, we demonstrate that SVA_67 acts as expression quantitative trait loci, thereby exhibiting a strong regulatory effect across the genome using whole genome and transcriptomic data from the Parkinson's progression markers initiative cohort. We further show that SVA_67 is polymorphic for its variable number tandem repeat domain which correlates with both regulatory properties in a luciferase reporter gene assay in vitro and differential expression of multiple genes in vivo. Additionally, this variation's utility as a biomarker is reflected in a correlation with a number of PD progression markers. These experiments highlight the plethora of transcriptomic and phenotypic changes associated with SVA_67 polymorphism which should be considered when investigating the missing heritability of neurodegenerative diseases.

Potency-Enhanced Peptidomimetic VHL Ligands with Improved Oral Bioavailability.

The von Hippel-Lindau (VHL) protein plays a pivotal role in regulating the hypoxic stress response and has been extensively studied and utilized in the targeted protein degradation field, particularly in the context of bivalent degraders. In this study, we present a comprehensive peptidomimetic structure-activity relationship (SAR) approach, combined with cellular NanoBRET target engagement assays to enhance the existing VHL ligands. Through systematic modifications of the molecule, we identified the 1,2,3-triazole group as an optimal substitute of the left-hand side amide bond that yields 10-fold higher binding activity. Moreover, incorporating conformationally constrained alterations on the methylthiazole benzylamine moiety led to the development of highly potent VHL ligands with picomolar binding affinity and significantly improved oral bioavailability. We anticipate that our optimized VHL ligand, GNE7599, will serve as a valuable tool compound for investigating the VHL pathway and advancing the field of targeted protein degradation.

Providing insight into the mechanism of action of cationic lipidated oligomers using metabolomics.

The increasing resistance of clinically relevant microbes against current commercially available antimicrobials underpins the urgent need for alternative and novel treatment strategies. Cationic lipidated oligomers (CLOs) are innovative alternatives to antimicrobial peptides and have reported antimicrobial potential. An understanding of their antimicrobial mechanism of action is required to rationally design future treatment strategies for CLOs, either in monotherapy or synergistic combinations. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of one CLO, C12-o-(BG-D)-10, which we have previously shown to be effective against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. The metabolomes of MRSA ATCC 43300 at 1, 3, and 6 h following treatment with C12-o-(BG-D)-10 (48 µg/mL, i.e., 3× MIC) were compared to those of the untreated controls. Our findings reveal that the studied CLO, C12-o-(BG-D)-10, disorganized the bacterial membrane as the first step toward its antimicrobial effect, as evidenced by marked perturbations in the bacterial membrane lipids and peptidoglycan biosynthesis observed at early time points, i.e., 1 and 3 h. Central carbon metabolism and the biosynthesis of DNA, RNA, and arginine were also vigorously perturbed, mainly at early time points. Moreover, bacterial cells were under osmotic and oxidative stress across all time points, as evident by perturbations of trehalose biosynthesis and pentose phosphate shunt. Overall, this metabolomics study has, for the first time, revealed that the antimicrobial action of C12-o-(BG-D)-10 may potentially stem from the dysregulation of multiple metabolic pathways.IMPORTANCEAntimicrobial resistance poses a significant challenge to healthcare systems worldwide. Novel anti-infective therapeutics are urgently needed to combat drug-resistant microorganisms. Cationic lipidated oligomers (CLOs) show promise as new antibacterial agents against Gram-positive pathogens like methicillin-resistant Staphylococcus aureus (MRSA). Understanding their molecular mechanism(s) of antimicrobial action may help design synergistic CLO treatments along with monotherapy. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of CLOs against MRSA. The results of our study indicate that the CLO, C12-o-(BG-D)-10, had a notable impact on the biosynthesis and organization of the bacterial cell envelope. C12-o-(BG-D)-10 also inhibits arginine, histidine, central carbon metabolism, and trehalose production, adding to its antibacterial characteristics. This work illuminates the unique mechanism of action of C12-o-(BG-D)-10 and opens an avenue to design innovative antibacterial oligomers/polymers for future clinical applications.

Controlling nanoparticle-induced endothelial leakiness with the protein corona.

Understanding nanoparticle-cell interaction is essential for advancing research in nanomedicine and nanotoxicology. Apart from the transcytotic pathway mediated by cellular recognition and energetics, nanoparticles (including nanomedicines) may harness the paracellular route for their transport by inducing endothelial leakiness at cadherin junctions. This phenomenon, termed as NanoEL, is correlated with the physicochemical properties of the nanoparticles in close association with cellular signalling, membrane mechanics, as well as cytoskeletal remodelling. However, nanoparticles in biological systems are transformed by the ubiquitous protein corona and yet the potential effect of the protein corona on NanoEL remains unclear. Using confocal fluorescence microscopy, biolayer interferometry, transwell, toxicity, and molecular inhibition assays, complemented by molecular docking, here we reveal the minimal to significant effects of the anionic human serum albumin and fibrinogen, the charge neutral immunoglobulin G as well as the cationic lysozyme on negating gold nanoparticle-induced endothelial leakiness in vitro and in vivo. This study suggests that nanoparticle-cadherin interaction and hence the extent of NanoEL may be partially controlled by pre-exposing the nanoparticles to plasma proteins of specific charge and topology to facilitate their biomedical applications.

Forest, climate, and policy literature lacks acknowledgement of environmental justice, diversity, equity, and inclusion.

Forests boast essential resources and potential to mitigate climate change, meriting the development of conservation policies on all governmental scales. Ecosystem services provided by forests, including biodiversity, air quality, and food and fuel production, make forests valuable assets for climate-vulnerable communities that often lack the means to cope with ecosystem service degradation resulting from climate change. Historically, these vulnerable communities are previously marginalized and socio-economically limited, and climate change augments already-existing injustices. Policy discussions around managing forests and carbon, therefore, must consider environmental justice as well as diversity, equity, and inclusion to better meet the needs of all constituents. Using R, we perform a review of forest, climate, and policy peer-reviewed literature published between 2018 and 2021 for prevalence of topics related to diversity, equity, inclusion, and justice (DEIJ). We select DEIJ terms a priori and a posteriori based on our understanding of DEIJ and common considerations of the literature. Out of 2891 unique articles, 15.7% of literature mentioned at least one DEIJ term in the title, keyword list, or abstract. We identify which journals have published DEIJ literature more often in the context of forest, climate, and policy, and we perform a co-occurrence analysis of additional common themes. Concepts such as ecosystem services and economics appeared often in the literature, as well as REDD+ as a specifically mentioned policy. We call for increased consideration of DEIJ in forest, climate, and policy discussions and literature, as vulnerable communities historically have been excluded from and victimized by the conversations held among large, economically motivated entities.

Lipid sulfoxide polymers as potential inhalable drug delivery platforms with differential albumin binding affinity.

Inhalable nanomedicines are increasingly being developed to optimise the pharmaceutical treatment of respiratory diseases. Large lipid-based nanosystems at the forefront of the inhalable nanomedicines development pipeline, though, have a number of limitations. The objective of this study was, therefore, to investigate the utility of novel small lipidated sulfoxide polymers based on poly(2-(methylsulfinyl)ethyl acrylate) (PMSEA) as inhalable drug delivery platforms with tuneable membrane permeability imparted by differential albumin binding kinetics. Linear PMSEA (5 kDa) was used as a hydrophilic polymer backbone with excellent anti-fouling and stealth properties compared to poly(ethylene glycol). Terminal lipids comprising single (1C2, 1C12) or double (2C12) chain diglycerides were installed to provide differing affinities for albumin and, by extension, albumin trafficking pathways in the lungs. Albumin binding kinetics, cytotoxicity, lung mucus penetration and cellular uptake and permeability through key cellular barriers in the lungs were examined in vitro. The polymers showed good mucus penetration and no cytotoxicity over 24 h at up to 1 mg ml-1. While 1C2-showed no interaction with albumin, 1C12-PMSEA and 2C12-PMSEA bound albumin with KD values of approximately 76 and 10 µM, respectively. Despite binding to albumin, 2C12-PMSEA showed reduced cell uptake and membrane permeability compared to the smaller polymers and the presence of albumin had little effect on cell uptake and membrane permeability. While PMSEA strongly shielded these lipids from albumin, the data suggest that there is scope to tune the lipid component of these systems to control membrane permeability and cellular interactions in the lungs to tailor drug disposition in the lungs.

Impact of conjugation to different lipids on the lymphatic uptake and biodistribution of brush PEG polymers.

Delivery to peripheral lymphatics can be achieved following interstitial administration of nano-sized delivery systems (nanoparticles, liposomes, dendrimers etc) or molecules that hitchhike on endogenous nano-sized carriers (such as albumin). The published work concerning the hitchhiking approach has mostly focussed on the lymphatic uptake of vaccines conjugated directly to albumin binding moieties (ABMs such as lipids, Evans blue dye derivatives or peptides) and their subsequent trafficking into draining lymph nodes. The mechanisms underpinning access and transport of these constructs into lymph fluid, including potential interaction with other endogenous nanocarriers such as lipoproteins, have largely been ignored. Recently, we described a series of brush polyethylene glycol (PEG) polymers containing end terminal short-chain or medium-chain hydrocarbon tails (1C2 or 1C12, respectively), cholesterol moiety (Cho), or medium-chain or long-chain diacylglycerols (2C12 or 2C18, respectively). We evaluated the association of these materials with albumin and lipoprotein in rat plasma, and their intravenous (IV) and subcutaneous (SC) pharmacokinetic profiles. Here we fully detail the association of this suite of polymers with albumin and lipoproteins in rat lymph, which is expected to facilitate lymph transport of the materials from the SC injection site. Additionally, we characterise the thoracic lymph uptake, tissue and lymph node biodistribution of the lipidated brush PEG polymers following SC administration to thoracic lymph cannulated rats. All polymers had moderate lymphatic uptake in rats following SC dosing with the lymph uptake higher for 1C2-PEG, 2C12-PEG and 2C18-PEG (5.8%, 5.9% and 6.7% dose in lymph, respectively) compared with 1C12-PEG and Cho-PEG (both 1.5% dose in lymph). The enhanced lymph uptake of 1C2-PEG, 2C12-PEG and 2C18-PEG appeared related to their association profile with different lipoproteins. The five polymers displayed different biodistribution patterns in major organs and tissues in mice. All polymers reached immune cells deep within the inguinal lymph nodes of mice following SC dosing. The ability to access these immune cells suggests the potential of the polymers as platforms for the delivery of vaccines and immunotherapies. Future studies will focus on evaluating the lymphatic targeting and therapeutic potential of drug or vaccine-loaded polymers in pre-clinical disease models.

Exploring SVA Insertion Polymorphisms in Shaping Differential Gene Expressions in the Central Nervous System.

Transposable elements (TEs) are repetitive elements which make up around 45% of the human genome. A class of TEs, known as SINE-VNTR-Alu (SVA), demonstrate the capacity to mobilise throughout the genome, resulting in SVA polymorphisms for their presence or absence within the population. Although studies have previously highlighted the involvement of TEs within neurodegenerative diseases, such as Parkinson's disease and amyotrophic lateral sclerosis (ALS), the exact mechanism has yet to be identified. In this study, we used whole-genome sequencing and RNA sequencing data of ALS patients and healthy controls from the New York Genome Centre ALS Consortium to elucidate the influence of reference SVA elements on gene expressions genome-wide within central nervous system (CNS) tissues. To investigate this, we applied a matrix expression quantitative trait loci analysis and demonstrate that reference SVA insertion polymorphisms can significantly modulate the expression of numerous genes, preferentially in the trans position and in a tissue-specific manner. We also highlight that SVAs significantly regulate mitochondrial genes as well as genes within the HLA and MAPT loci, previously associated within neurodegenerative diseases. In conclusion, this study continues to bring to light the effects of polymorphic SVAs on gene regulation and further highlights the importance of TEs within disease pathology.

Steroid-free combination of 5-azacytidine and venetoclax for the treatment of multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy that, despite an unprecedented increase in overall survival, lacks truly risk-adapted or targeted treatments. A proportion of patients with MM depend on BCL-2 for survival, and, recently, the BCL-2 antagonist venetoclax has shown clinical efficacy and safety in t(11;14) and BCL-2 overexpressing MM. However, only a small proportion of MM patients rely on BCL-2 (approx. 20%), and there is a need to broaden the patient population outside of t(11;14) that can be treated with venetoclax. Therefore, we took an unbiased screening approach and screened epigenetic modifiers to enhance venetoclax sensitivity in 2 non-BCL-2 dependent MM cell lines. The demethylase inhibitor 5-azacytidine was one of the lead hits from the screen, and the enhanced cell killing of the combination was confirmed in additional MM cell lines. Using dynamic BH3 profiling and immunoprecipitations, we identified the potential mechanism of synergy is due to increased NOXA expression, through the integrated stress response. Knockdown of PMAIP1 or PKR partially rescues cell death of the venetoclax and 5-azacytidine combination treatment. The addition of a steroid to the combination treatment did not enhance the cell death, and, interestingly, we found enhanced death of the immune cells with steroid addition, suggesting that a steroid-sparing regimen may be more beneficial in MM. Lastly, we show for the first time in primary MM patient samples that 5-azacytidine enhances the response to venetoclax ex vivo across diverse anti-apoptotic dependencies (BCL-2 or MCL-1) and diverse cytogenetic backgrounds. Overall, our data identify 5-azacytidine and venetoclax as an effective treatment combination, which could be a tolerable steroid-sparing regimen, particularly for elderly MM patients.

Rare and common genetic determinants of mitochondrial function determine severity but not risk of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving selective vulnerability of energy-intensive motor neurons (MNs). It has been unclear whether mitochondrial function is an upstream driver or a downstream modifier of neurotoxicity. We separated upstream genetic determinants of mitochondrial function, including genetic variation within the mitochondrial genome or autosomes; from downstream changeable factors including mitochondrial DNA copy number (mtCN). Across three cohorts including 6,437 ALS patients, we discovered that a set of mitochondrial haplotypes, chosen because they are linked to measurements of mitochondrial function, are a determinant of ALS survival following disease onset, but do not modify ALS risk. One particular haplotype appeared to be neuroprotective and was significantly over-represented in two cohorts of long-surviving ALS patients. Causal inference for mitochondrial function was achievable using mitochondrial haplotypes, but not autosomal SNPs in traditional Mendelian randomization (MR). Furthermore, rare loss-of-function genetic variants within, and reduced MN expression of, ACADM and DNA2 lead to ∼50 % shorter ALS survival; both proteins are implicated in mitochondrial function. Both mtCN and cellular vulnerability are linked to DNA2 function in ALS patient-derived neurons. Finally, MtCN responds dynamically to the onset of ALS independently of mitochondrial haplotype, and is correlated with disease severity. We conclude that, based on the genetic measures we have employed, mitochondrial function is a therapeutic target for amelioration of disease severity but not prevention of ALS.