Rotational multimaterial printing of filaments with subvoxel control.

Helical structures are ubiquitous in nature and impart unique mechanical properties and multifunctionality1. So far, synthetic architectures that mimic these natural systems have been fabricated by winding, twisting and braiding of individual filaments1-7, microfluidics8,9, self-shaping1,10-13 and printing methods14-17. However, those fabrication methods are unable to simultaneously create and pattern multimaterial, helically architected filaments with subvoxel control in arbitrary two-dimensional (2D) and three-dimensional (3D) motifs from a broad range of materials. Towards this goal, both multimaterial18-23 and rotational24 3D printing of architected filaments have recently been reported; however, the integration of these two capabilities has yet to be realized. Here we report a rotational multimaterial 3D printing (RM-3DP) platform that enables subvoxel control over the local orientation of azimuthally heterogeneous architected filaments. By continuously rotating a multimaterial nozzle with a controlled ratio of angular-to-translational velocity, we have created helical filaments with programmable helix angle, layer thickness and interfacial area between several materials within a given cylindrical voxel. Using this integrated method, we have fabricated functional artificial muscles composed of helical dielectric elastomer actuators with high fidelity and individually addressable conductive helical channels embedded within a dielectric elastomer matrix. We have also fabricated hierarchical lattices comprising architected helical struts containing stiff springs within a compliant matrix. Our additive-manufacturing platform opens new avenues to generating multifunctional architected matter in bioinspired motifs.

The renal lineage factor PAX8 controls oncogenic signalling in kidney cancer.

Large-scale human genetic data1-3 have shown that cancer mutations display strong tissue-selectivity, but how this selectivity arises remains unclear. Here, using experimental models, functional genomics and analyses of patient samples, we demonstrate that the lineage transcription factor paired box 8 (PAX8) is required for oncogenic signalling by two common genetic alterations that cause clear cell renal cell carcinoma (ccRCC) in humans: the germline variant rs7948643 at 11q13.3 and somatic inactivation of the von Hippel-Lindau tumour suppressor (VHL)4-6. VHL loss, which is observed in about 90% of ccRCCs, can lead to hypoxia-inducible factor 2α (HIF2A) stabilization6,7. We show that HIF2A is preferentially recruited to PAX8-bound transcriptional enhancers, including a pro-tumorigenic cyclin D1 (CCND1) enhancer that is controlled by PAX8 and HIF2A. The ccRCC-protective allele C at rs7948643 inhibits PAX8 binding at this enhancer and downstream activation of CCND1 expression. Co-option of a PAX8-dependent physiological programme that supports the proliferation of normal renal epithelial cells is also required for MYC expression from the ccRCC metastasis-associated amplicons at 8q21.3-q24.3 (ref. 8). These results demonstrate that transcriptional lineage factors are essential for oncogenic signalling and that they mediate tissue-specific cancer risk associated with somatic and inherited genetic variants.

Controlled flight of a microrobot powered by soft artificial muscles.

Flying insects capable of navigating in highly cluttered natural environments can withstand in-flight collisions because of the combination of their low inertia1 and the resilience of their wings2, exoskeletons1 and muscles. Current insect-scale (less than ten centimetres long and weighing less than five grams) aerial robots3-6 use rigid microscale actuators, which are typically fragile under external impact. Biomimetic artificial muscles7-10 that are capable of large deformation offer a promising alternative for actuation because they can endure the stresses caused by such impacts. However, existing soft actuators11-13 have not yet demonstrated sufficient power density to achieve lift-off, and their actuation nonlinearity and limited bandwidth create further challenges for achieving closed-loop (driven by an input control signal that is adjusted based on sensory feedback) flight control. Here we develop heavier-than-air aerial robots powered by soft artificial muscles that demonstrate open-loop (driven by a predetermined signal without feedback), passively stable (upright during flight) ascending flight as well as closed-loop, hovering flight. The robots are driven by multi-layered dielectric elastomer actuators that weigh 100 milligrams each and have a resonance frequency of 500 hertz and power density of 600 watts per kilogram. To increase the mechanical power output of the actuator and to demonstrate flight control, we present ways to overcome challenges unique to soft actuators, such as nonlinear transduction and dynamic buckling. These robots can sense and withstand collisions with surrounding obstacles and can recover from in-flight collisions by exploiting material robustness and vehicle passive stability. We also fly two micro-aerial vehicles simultaneously in a cluttered environment. They collide with the wall and each other without suffering damage. These robots rely on offboard amplifiers and an external motion-capture system to provide power to the dielectric elastomer actuators and to control their flight. Our work demonstrates how soft actuators can achieve sufficient power density and bandwidth to enable controlled flight, illustrating the potential of developing next-generation agile soft robots.

Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Progress in physiologically based pharmacokinetic-pharmacodynamic models of amino acids in humans.

PURPOSE OF REVIEW: Amino acids are critical to health, serving both as constituents of proteins and in signaling and metabolism. Amino acids are consumed as nutrients, supplements, and nutraceuticals. Much remains to be learned about amino acid function. Physiologically based pharmacokinetic and pharmacodynamic (PBPK-PD) modeling is an emerging tool for studying their complex biology. This review highlights recent PBPK-PD models developed to study amino acid physiology and metabolism and discusses their potential for addressing unresolved questions in the field. RECENT FINDINGS: PBPK-PD models provided several insights. They revealed the interplay between the mechanisms by which leucine governs skeletal muscle protein metabolism in healthy adults. The models also identified optimal dosing regimens of amino acid supplementation to treat sickle-cell disease and recurrent hypoglycemia, and to minimize drug side effects in seizure disorders. Additionally, they characterized the effects of novel anticancer drugs that seek to deprive cancer cells of amino acids. Future models may inform treatment strategies for sarcopenia, characterize distinctions between animal- and plant-based nutrition, and inform nutrient-drug interactions in Parkinson's disease. SUMMARY: PBPK-PD models are powerful tools for studying amino acid physiology and metabolism, with applications to nutrition, pharmacology, and their interplay.

Fidelity, pragmatism and the "grey line" in between-exploring the delivery of a pragmatic physical activity randomised controlled trial-a secondary analysis.

BACKGROUND: Intervention fidelity in health services research has been poor with a reported lack of understanding about what constitutes pragmatic adaptation of interventions and what constitutes failure to maintain intervention fidelity. However, the challenges facing those delivering such interventions have not been thoroughly explored. The aims of this study were to critically explore the challenges in maintaining fidelity experienced by physiotherapy staff and support workers when delivering a complex intervention for older people living with frailty. METHODS: This study is a secondary analysis of data from a process evaluation of a large randomised controlled trial (RCT). The process evaluation employed qualitative methodologies with mixed methods including a variety of data collection methods, including participant observation, semi-structured interviews and documentary analysis. Thematic analysis was used to make sense of the data. RESULTS: Many therapy staff felt ongoing confusion about what was acceptable to adapt and what needed to follow the protocol exactly. We found that some therapy staff were able to embrace the challenges of pragmatically adapting interventions while maintaining intervention fidelity, others stuck rigidly to the protocol and failed to adapt interventions where it was necessary. CONCLUSION: It was clear that the understanding of fidelity and pragmatism was poor. While pragmatic trials are vital to replicate real world clinical practice, further guidance may need to be developed in order to guide the level of adaptation that is acceptable before fidelity is undermined.

Utilising Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to track the oxidation of lignin by an alkaliphilic laccase.

Lignin is a complex heteroaromatic polymer which is one of the most abundant and diverse biopolymers on the planet. It comprises approximately one third of all woody plant matter, making it an attractive candidate as an alternative, renewable feedstock to petrochemicals to produce fine chemicals. However, the inherent complexity of lignin makes it difficult to analyse and characterise using common analytical techniques, proving a hindrance to the utilisation of lignin as a green chemical feedstock. Herein we outline the tracking of lignin degradation by an alkaliphilic laccase in a semi-quantitative manner using a combined chemical analysis approach using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to characterise shifts in chemical diversity and relative abundance of ions, and NMR to highlight changes in the structure of lignin. Specifically, an alkaliphilic laccase was used to degrade an industrially relevant lignin, with compounds such as syringaresinol being almost wholly removed (95%) after 24 hours of treatment. Structural analyses reinforced these findings, indicating a >50% loss of NMR signal relating to ß-ß linkages, of which syringaresinol is representative. Ultimately, this work underlines a combined analytical approach that can be used to gain a broader semi-quantitative understanding of the enzymatic activity of laccases within a complex, non-model mixture.

"Bind, cleave and leave": multiple turnover catalysis of RNA cleavage by bulge-loop inducing supramolecular conjugates.

Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge-loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge-loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.

Blood-Brain Barrier Dysfunction and Exposure to Head Impacts in University Football Players.

OBJECTIVE: To investigate the link between dysfunction of the blood-brain barrier (BBB) and exposure to head impacts in concussed football athletes. DESIGN: This was a prospective, observational pilot study. SETTING: Canadian university football. PARTICIPANTS: The study population consisted of 60 university football players, aged 18 to 25. Athletes who sustained a clinically diagnosed concussion over the course of a single football season were invited to undergo an assessment of BBB leakage. INDEPENDENT VARIABLES: Head impacts detected using impact-sensing helmets were the measured variables. MAIN OUTCOME MEASURES: Clinical diagnosis of concussion and BBB leakage assessed using dynamic contrast-enhanced MRI (DCE-MRI) within 1 week of concussion were the outcome measures. RESULTS: Eight athletes were diagnosed with a concussion throughout the season. These athletes sustained a significantly higher number of head impacts than nonconcussed athletes. Athletes playing in the defensive back position were significantly more likely to sustain a concussion than remain concussion free. Five of the concussed athletes underwent an assessment of BBB leakage. Logistic regression analysis indicated that region-specific BBB leakage in these 5 athletes was best predicted by impacts sustained in all games and practices leading up to the concussion-as opposed to the last preconcussion impact or the impacts sustained during the game when concussion occurred. CONCLUSIONS: These preliminary findings raise the potential for the hypothesis that repeated exposure to head impacts may contribute to the development of BBB pathology. Further research is needed to validate this hypothesis and to test whether BBB pathology plays a role in the sequela of repeated head trauma.

The Role of Denosumab Treatment in Recurrent Giant Cell Bone Tumor of the Orbit.

Giant cell tumor of the bone (GCTB) is a rare primary bone neoplasm, representing about 5% of all primary bone tumors. Most GCTBs are found in the epiphysis of long bones, with only 2% of GCTBs involving the skull. In recent years, the receptor activator of nuclear factor Kappa ligand monoclonal antibody denosumab has been demonstrated as a promising therapeutic option for GCTB; however, this is an evolving field. We present a case of a 57-year-old female with a rare GCTB in the right orbit and sinuses, originally thought to be an aneurysmal bone cyst. Her symptoms included proptosis, intermittent blurry vision, sinus congestion, and frontal headaches. After excision, the tumor recurred within 18 months. Upon repeat excision, a diagnosis of GCTB was made. The patient started denosumab therapy and had no tumor growth over the ensuing 2 years, with stability of symptoms and clinical signs on follow-up.

Exploring Greater Flexibility for Chronic Toxicity Study Designs to Support Human Safety Assessment While Balancing 3Rs Considerations.

Chronic repeated-dose toxicity studies are required to support long-term dosing in late-stage clinical trials, providing data to adequately characterize adverse effects of potential concern for human safety. Different regulatory guidances for the design and duration of chronic toxicity studies are available, with flexibility in approaches often adopted for specific drug modalities. These guidances may provide opportunities to reduce time, cost, compound requirement and animal use within drug development programs if applied more broadly and considered outside their current scopes of use. This article summarizes presentations from a workshop at the 43rd Annual Meeting of the American College of Toxicology (ACT) in November 2022, discussing different approaches for chronic toxicity studies. A recent industry collaboration between the Netherlands Medicines Evaluation Board (MEB) and UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) illustrated current practices and the value of chronic toxicity studies for monoclonal antibodies (mAbs) and evaluated a weight of evidence (WOE) model where a 3-month study rather than a 6-month study might be adequate. Other topics included potential opportunities for single-species chronic toxicity studies for small molecules, peptides and oligonucleotides and whether a 6-month duration non-rodent study can be used more routinely than a 9-month study (similar to ICH S6(R1) for biological products). Also addressed were opportunities to optimize recovery animal use if warranted and whether restriction to one study only (if at all) can be applied more widely within and outside ICH S6(R1).

FIVE YEARS OF PARTULA SNAIL PRE-EXPORT HEALTH SCREENING.

Between 2015 and 2019, a health screening was carried out annually on captive-bred Partula snails prior to export for reintroduction as part of an international effort to repopulate areas of French Polynesia, where the snails were extinct or critically endangered. In total, 129 separate tank populations of 12 different species were screened at ZSL London Zoo. Wet mounts and smears stained with modified Ziehl-Neelsen (MZN) of 535 fecal samples were examined, and 45% contained flagellated protozoa, and 35.5% had MZN-positive oocysts, measuring 3-5 µm in diameter. Smaller (2 µm) presumptive spores, MZN-positive bacilli, ciliated protozoa and nematodes were recorded less frequently. Fecal bacterial culture yielded mixed species, with a clear predominance of Myroides species (88.9% of samples). The MZN-positive oocysts (3-5 µm) were present in 6.5% of impression smears from the apices of 432 snails examined postmortem, plus acid-fast bacilli in a few cases, but no 2 µm spores. Mixed bacteria were cultured from coelomic swabs, with Myroides species again the most common (63.5%). Histologic examination was carried out on 292 snails. Autolysis affected almost 90% of those found dead but only 3.4% of euthanized snails. Histology commonly identified microsporidial sporocysts in the digestive gland and midgut epithelium of all but two species. Intracellular, extracytoplasmic Cryptosporidium-like organisms were also common in the midgut but were only observed when snails were fixed in 10% formalin (2017-2019), not ethanol. There were no clear pathologic changes associated with either organism. Pigmented hemocytic nodules were commonly observed, most frequently in the foot process; these were either age related or evidence of prior chronic inflammatory reaction and of low clinical significance. With no evidence of poor health and no significant organisms found, a total of 4,978 individuals representing 12 species were exported for reintroduction.

Membrane lipids from gut microbiome-associated bacteria as structural and signalling molecules.

Bacteria produce an array of diverse, dynamic and often complex lipid structures, some of which function beyond their typical role in membrane structure. The model organism, E. coli, has three major membrane lipids, which are glycerophosphoglycerol (phosphatidylglycerol), glycerophosphoethanolamine (phosphatidylethanolamine) and cardiolipin. However, it is now appreciated that some bacteria have the capacity to synthesize a range of lipids, including glycerophosphocholines, glycerophosphoinositols, 'phosphorous-free' N-acyl amines, sphingolipids and plasmalogens. In recent years, some of these bacterial lipids have emerged as influential contributors to the microbe-host molecular dialogue. This review outlines our current knowledge of bacterial lipid diversity, with a focus on the membrane lipids of microbiome-associated bacteria that have documented roles as signalling molecules.

Seroconversion and fever are dose-dependent in a nonhuman primate model of inhalational COVID-19.

While evidence exists supporting the potential for aerosol transmission of SARS-CoV-2, the infectious dose by inhalation remains unknown. In the present study, the probability of infection following inhalation of SARS-CoV-2 was dose-dependent in a nonhuman primate model of inhalational COVID-19. The median infectious dose, assessed by seroconversion, was 52 TCID50 (95% CI: 23-363 TCID50), and was significantly lower than the median dose for fever (256 TCID50, 95% CI: 102-603 TCID50), resulting in a group of animals that developed an immune response post-exposure but did not develop fever or other clinical signs of infection. In a subset of these animals, virus was detected in nasopharyngeal and/or oropharyngeal swabs, suggesting that infected animals without signs of disease are able to shed virus and may be infectious, which is consistent with reports of asymptomatic spread in human cases of COVID-19. These results suggest that differences in exposure dose may be a factor influencing disease presentation in humans, and reinforce the importance of public health measures that limit exposure dose, such as social distancing, masking, and increased ventilation. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, and, ultimately, mitigation strategies. Additionally, these data will be useful to inform dose selection in future studies examining the efficacy of therapeutics and vaccines against inhalational COVID-19, and as a baseline in healthy, young adult animals for assessment of the importance of other factors, such as age, comorbidities, and viral variant, on the infectious dose and disease presentation.

Concussion susceptibility is mediated by spreading depolarization-induced neurovascular dysfunction.

The mechanisms underlying the complications of mild traumatic brain injury, including post-concussion syndrome, post-impact catastrophic death, and delayed neurodegeneration remain poorly understood. This limited pathophysiological understanding has hindered the development of diagnostic and prognostic biomarkers and has prevented the advancement of treatments for the sequelae of mild traumatic brain injury. We aimed to characterize the early electrophysiological and neurovascular alterations following repetitive mild traumatic brain injury and sought to identify new targets for the diagnosis and treatment of individuals at risk of severe post-impact complications. We combined behavioural, electrophysiological, molecular, and neuroimaging techniques in a rodent model of repetitive mild traumatic brain injury. In humans, we used dynamic contrast-enhanced MRI to quantify blood-brain barrier dysfunction after exposure to sport-related concussive mild traumatic brain injury. Rats could clearly be classified based on their susceptibility to neurological complications, including life-threatening outcomes, following repetitive injury. Susceptible animals showed greater neurological complications and had higher levels of blood-brain barrier dysfunction, transforming growth factor ß (TGFß) signalling, and neuroinflammation compared to resilient animals. Cortical spreading depolarizations were the most common electrophysiological events immediately following mild traumatic brain injury and were associated with longer recovery from impact. Triggering cortical spreading depolarizations in mild traumatic brain injured rats (but not in controls) induced blood-brain barrier dysfunction. Treatment with a selective TGFß receptor inhibitor prevented blood-brain barrier opening and reduced injury complications. Consistent with the rodent model, blood-brain barrier dysfunction was found in a subset of human athletes following concussive mild traumatic brain injury. We provide evidence that cortical spreading depolarization, blood-brain barrier dysfunction, and pro-inflammatory TGFß signalling are associated with severe, potentially life-threatening outcomes following repetitive mild traumatic brain injury. Diagnostic-coupled targeting of TGFß signalling may be a novel strategy in treating mild traumatic brain injury.

3'-O-ß-Glucosyl-4',5'-didehydro-5'-deoxyadenosine Is a Natural Product of the Nucleocidin Producers Streptomyces virens and Streptomyces calvus.

3'-O-ß-Glucosyl-4',5'-didehydro-5'-deoxyadenosine 13 is identified as a natural product of Streptomyces calvus and Streptomyces virens. It is also generated in vitro by direct ß-glucosylation of 4',5'-didehydro-5'-deoxyadenosine 12 with the enzyme NucGT. The intact incorporation of oxygen-18 and deuterium isotopes from (±)[1-18O,1-2H2]-glycerol 14 into C-5' of nucleocidin 1 and its related metabolites precludes 3'-O-ß-glucosyl-4',5'-didehydro-5'-deoxyadenosine 13 as a biosynthetic precursor to nucleocidin 1.

Hepatitis C virus in people with serious mental illness: an analysis of the care cascade at a tertiary health service with a pilot 'identify and treat' strategy.

BACKGROUND: People with serious mental illness (SMI) are underserved from a hepatitis C virus (HCV) screening and treatment perspective. AIMS: To examine the HCV care cascade in people with SMI and to pilot a supported HCV treatment integration programme. METHODS: HCV prevalence was retrospectively analysed from 4492 consecutive individuals admitted to a tertiary hospital mental health service between January 2017 and December 2018. Subcohort analysis of screening patterns and predictors of seropositive infection was performed. Referral pathways and community care integration were analysed for HCV-positive individuals, and a prospective community-based 'identify and treat' HCV programme was assessed. RESULTS: Screening for HCV had been performed in 18.6% (835/4492) of the cohort. Seroprevalence was 4.6% (207/4492). HCV seropositivity was associated with age >40 years (odds ratio (OR) = 9.30; confidence interval (CI) 3.69-23.45; P < 0.01), injecting drug use (OR = 24.26; CI 8.99-65.43; P < 0.01) and previous incarceration (OR = 12.26; CI 4.51-33.31; P < 0.01). In a cohort of treatment-eligible individuals, 43.3% (90/208) had neither been referred to specialist services or general practitioners for HCV management. Amongst those referred to specialist services, 64.7% (57/88) did not attend scheduled follow up, and 48.3% (15/31) of attendees were lost to follow up. Through an intensified community access programme, 10 people were successfully treated for HCV, although 22 could not be engaged. CONCLUSION: People with SMI are underserved by traditional models of HCV healthcare. Intensified community-based support can partially bolster the treatment cascade, although investment in innovative screening and management strategies are required to achieve healthcare parity.

The use of recovery animals in nonclinical safety assessment studies with monoclonal antibodies: further 3Rs opportunities remain.

Assessment of reversibility from nonclinical toxicity findings in animals with potential adverse clinical impact is required during pharmaceutical development, but there is flexibility around how and when this is performed and if recovery animals are necessary. For monoclonal antibodies (mAbs) and in accordance with ICH S6(R1) if inclusion of recovery animals is warranted, this need only occur in one study. Data on study designs for first-in-human (FIH)-enabling and later-development toxicity studies were shared from a recent collaboration between the NC3Rs, EPAA, Netherlands Medicines Evaluation Board (MEB) and 14 pharmaceutical companies. This enabled a review of practices on recovery animal use during mAb development and identification of opportunities to reduce research animal use. Recovery animals were included in 68% of FIH-enabling and 69% of later-development studies, often in multiple studies in the same program. Recovery groups were commonly in control plus one test article-dosed group or in all dose groups (45% of studies, each design). Based on the shared data review and conclusions, limiting inclusion of recovery to a single nonclinical toxicology study and species, study design optimisation and use of existing knowledge instead of additional recovery groups provide opportunities to further reduce animal use within mAb development programs.

Re-evaluating the need for chronic toxicity studies with therapeutic monoclonal antibodies, using a weight of evidence approach.

To support registration of monoclonal antibodies (mAbs) for chronic indications, 6-month toxicity studies have historically been conducted. Experience with mAb development has shown a relatively benign and well-understood safety profile for this class, with most toxicity findings anticipated based on pharmacology. We evaluated whether a 6-month toxicity study is necessary to assess the long-term safety of mAbs. Data on First-in-Human (FIH)-enabling and chronic toxicity studies were shared for 142 mAbs submitted by 11 companies. Opportunities to further optimize study designs to reduce animal usage were identified. For 71% of mAbs, no toxicities or no new toxicities were noted in chronic studies compared to FIH-enabling study findings. New toxicities of potential concern for human safety or that changed trial design were identified in 13.5% of cases, with 7% being considered critical and 2% leading to program termination. An iterative, weight-of-evidence model which considers factors that influence the overall risk for a mAb to cause toxicity was developed. This model enables an evidence-based justification, suggesting when 3-month toxicity studies are likely sufficient to support late-stage clinical development and registration for some mAbs.

Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates.

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.