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The WAC-downWAC domain in the yeast ISW2 nucleosome remodeling complex forms a structural module essential for ISW2 function but not cell viability.
No full textBACKGROUND
ATP-dependent nucleosome remodeling complexes of the imitation switch (ISWI) family slide and space nucleosomes. The ISWI ATPase subunit forms obligate complexes with accessory subunits whose mechanistic roles remain poorly understood. In baker's yeast, the Isw2 ATPase subunit associates with Itc1, the orthologue of human ACF1/BAZ1A. Prior data suggested that the genomic deletion of the 374 N-terminal amino acids from Itc1 (hereafter called itc1) leads to a gain-of-toxic-function phenotype with severe growth defects in the BY4741 genetic background, possibly due to defective nucleosome spacing activity of the mutant complex.
RESULTS
Here we show that the deletion encompasses a novel motif termed downWAC that forms a conserved structural module with the N-terminal WAC domain. The module is predicted to interact with DNA. However, it does not form a stable interaction interface with the remainder of the complex. Instead, it is connected through a long disordered polypeptide linker to the remainder of the complex. Curiously, the itc1 allele does not lead to measurable growth defects in haploid BY4741 and diploid BY4743 strains. It also does not alter genome-wide nucleosome organization in wild-type cells. To rule out that potentially redundant remodeling factors obscure itc1-associated phenotypes, we repeated experiments in cells devoid of ISW1 and CHD1 remodelers with the same results. Only at known target genes of the ISW2 complex was the nucleosome organization perturbed in itc1 cells.
CONCLUSIONS
We conclude that the deletion of Itc1 N-terminus is indistinguishable from the full deletion of either ITC1 or ISW2. As such, itc1 should be considered a null allele of ISW2. We propose a model, in which the WAC-downWAC module, along with a flexible protein linker, helps ISW2 in searching for its target genes and positioning + 1-nucleosomes
In vivo biomechanical correlates of gait and stair climbing are predictors of ex vivo cartilage quality in gonarthrotic patients
No full textObjective: Previous evidence highlights the important role of knee joint malalignment and excessive joint moments for the development to knee osteoarthritis. To date most studies used X-ray imaging techniques to draw conclusions about cartilage quality. The present study aimed to systematically investigate the interrelationship between three-dimensional knee kinematics during walking and stair climbing and ex-vivo electromechanical cartilage quality in patients with end-stage knee osteoarthritis.
Methods: The prospective cohort study was conducted in 119 patients with end-stage knee OA. Patients scheduled for total knee arthroplasty surgery underwent radiographic assessment and biomechanical analysis in gait and stair climbing assessing in vivo knee joint angles and moments during movement dynamics prior to surgery. After surgery, ex vivo electromechanical properties of the tibial cartilage as an indicator or cartilage damage were evaluated using Arthro-BST. Associations between medial and lateral tibia cartilage quality and biomechanical parameters and radiographic, knee joint malalignment or joint width space were assessed using linear regression modelling.
Results: Peak knee adduction angle was found to be a significant predictor of medial cartilage quality during walking (R2 = 22%, p < 0.001) and stair climbing (R2 = 13%, p < 0.001). During walking, knee flexion moment was found to be a strong predictor of medial (R2 = 9%, p = 0.002) and lateral cartilage quality (R2 = 12%, p < 0.001). Knee adduction angle as determined on the frontal X-ray was found to be a significant predictor of medial cartilage quality (R2 = 12%, p = 0.003). Knee adduction (R2 = 19%, p < 0.001) and abduction angles (R2 = 44%, p < 0.01) during x-ray was significantly associated with dynamic assessments during 3D kinematics. Medial joint space width significantly predicted medial tibial cartilage quality (R2 = 16%, p < 0.001).
Conclusion: This study provides a biological rationale for the use of biomechanical analysis as a potential candidate to supplement diagnostic options for OA. The results showed that biomechanical parameters such as varus malalignment during walking and stair climbing are significantly associated with tibial cartilage deterioration and contribute to a variance explanation of 13-22%. Given the predictive value of these non-invasive examinations, kinematic analysis could be included in the test battery for OA patients and help to detect cartilage damage in addition to other recognized markers and imaging techniques.
Keywords: biomechanics, ArthroBST, cartilage quality, varus, valgus
Translational potential
This prospective cohort study translates in vivo biomechanical values of gait and stair climbing as well as joint width and static malalignment of images to cartilage quality of the medial and lateral tibia plateau as determined ex vivo by ArthroBSTTM
Cross-industry demonstration of the validity of the mixed matrix method for the assessment of cross-species exposure coverage of human circulating drug metabolites.
No full textThe mixed matrix method (MmM) is a widely used approach by the pharmaceutical industry for early assessment of whether exposures to major human circulating metabolites, of traditional small-molecule drugs, are adequately covered by the species used for toxicology assessment, which is a key requirement of the safety testing of drug metabolites (metabolites in safety testing guidelines). However, questions remain regarding its accuracy and utility in replacing conventional bioanalytical approaches. Furthermore, the available literature on the topic is not fully consistent in terms of how the assay should be conducted. As a result, encouraged by health authority advice on this topic, a cross-industry group under the European Federation of Pharmaceutical Industries and Associations was formed to: (1) further investigate the MmM accuracy, including a robust statistical analysis covering a diverse chemical space of commercially available drugs and drug candidates as well as their metabolites; (2) propose recommendations for best practice including a decision tree that the industry should consider when using the MmM; and (3) discuss whether the MmM could be used to support metabolites in safety testing assessment and could potentially be included into new drug application submissions without the need for additional measurements using the conventional bioanalytical approach. The outcome of this European Federation of Pharmaceutical Industries and Associations assessment shows that MmM measured exposure ratios of 1.9 and 1.4 are statistically sufficient to demonstrate adequate exposure coverage of human metabolites above 50% or between 10% and 50% of drug-related exposure, respectively, by toxicology species. The aim is to encourage both industry and regulatory agencies to consider MmM as an acceptable approach to compare major human circulating metabolite exposures across species. SIGNIFICANCE STATEMENT: The outcome of our mixed matrix method assessment showed that measured exposure ratios of 1.9 and 1.4 are adequate to demonstrate coverage of human metabolites above or below 50% drug-related exposure by toxicology species. Recommendations for best practice and a decision tree for conducting metabolites in safety testing evaluations are proposed. Our investigations show that mixed matrix method data are sufficiently robust for the intended purpose and that the assay provides an opportunity to streamline drug development and reduce the need for resource-intensive bioanalysis and certain animal studies
Looking Inside the SPPS Reactor through a Refractometer: Online Quantification of the Resin Loading
No full textThe methodology of solid phase peptide synthesis (SPPS) is the main architect of the positive boom that peptides are having in drug discovery. SPPS has many advantages, such as ease of scaling-up, high yields, and short production times. However, it is like a black box as the intermediates are not isolated, and the results are not known until the end of the entire process.
A key point of SPPS is to know the initial loading of the resin. Considering a higher loading compared to the real one will result in the use of a greater excess of reagents with a positive impact on the purity of the final product, but negative in economic terms. On the other hand, in case of lower loading compared to real one, the formation of deletion peptides can take place.
The most frequent method to calculate the loading is incorporating a Fmoc derivative, then removing the Fmoc group with piperidine followed by the UV spectrophotometric determination of the dibenzofulvene-piperidine adduct. This operation requires stopping the synthetic process and consumes time.
Herein, the quantitative use of the refractometry index is proposed to perform the on-line determination of the resin loading. This allows real-time knowledge of the reaction process at all times and to be able to stop the reaction if the loading achieved is the desired one or to add more coupling reagents to improve the loading
Best Practices and Recommendations for Non-Liquid Matrices Bioanalysis
No full textThe analysis of Non-liquid Matrices (NLMs) can provide key information on many aspects in drug discovery and development. These include but are not limited to drug uptake and distribution, engagement and modulation, and target exposure. A thorough understanding of these aspects is fundamental to the progression of drug development. In many cases, such an understanding can only be achieved through quantitative analysis of NLMs. Such dependence can lead to bottlenecks in the drug development process—as the practices and regulations that govern bioanalysis of conventional liquid matrices typically cannot be directly applied to NLMs. This paper strives to fill this crucial gap. To this end, subject matter experts from across the industry, through the auspices of the AAPS Bioanalytical Community, have combined their collective best practices for NLM bioanalysis in this paper. Certainly, this endeavor came with challenges, the most prominent of which also serves as the impetus for this project, the lack of literature on NLM bioanalysis dealing with different types of NLM, analysis rigor, and best practices to draw from. This paper aims to serve as a comprehensive set of best practices drawn from the experiences of leading scientists across the industry—for NLM bioanalysis in drug development
High Performance Countercurrent Membrane Purification for HCP depletion in biologics
No full textSubmission of Bachelor-Thesis (CDA between Novartis and University FHNW Muttenz in place)
As research and development of biologics manufacturing advances, high performance membrane
purification (HPCMP) for host cell protein (HCP) depletion in biologics becomes an attractive
alternative to conventional protein A and therapeutic protein capture methods, as it provides
a non-specific, cost effective and easily scalable process for capture. In this study, Repligen
Cytiva and Solventum membranes with various molecular weight cut-off as well as the addition
of L-arginine as an excipient were tested with model proteins such as immunoglobulin G,
myoglobin (Mb), lysozyme (Lyz), and albumin to test their reduction performance. Repligen
membranes show a lower mass transfer coefficient kc,Repligen = 3.710-07 m
s compared to
kc,Solventum = 1 * 10-06 m
s Solventum membranes for Mb / Lyz, which makes Repligen and Cytiva
membranes not suitable for HPCMP, although they show potential if further investigated.
HPCMP performed with 50 kDa therapeutic protein intermediate (TP50) and 100 kDa therapeutic
protein intermediate (TP100) resolved with a reduction factor rTP50 = 17 and rTP100 = 2.25
while maintaining a yield YTP50 = 69.61 % and YTP100 = 84.17 %, although HPCMP with TP50
proves to have a higher yield loss and inconsistent results, overall it reached a higher HCP depletion
than HPCMP with TP100 due to the greater difference in HCP concentration
Copper(II) Coordination Polymers Driven by 3,4-Pyridinedicarboxylic Acid: Synthesis, Scale Up, Crystal Structures, and Catalytic Behavior in Allylic Oxidation of a-Pinene
No full texta-Pinene is one of the most abundant and low-cost terpenes that can be used as a
renewable feedstock for generating added-value oxidation products via allylic oxidation.
Aiming at the development of novel catalytic systems for the functionalization of
terpenes, in this work two new isostructural 2D coordination polymers (CPs), {[Cu2(µ-
pdc)(µ3-pdc)(H2mdea)(H2O)2].2H2O}n (Cu-mdea) and {[Cu2(µ-pdc)(µ3-
pdc)(H3tipa)(H2O)2].4H2O}n (Cu-tipa) {H2pdc = 3,4-pyridinedicarboxylic acid, H2mdea
= methyldiethanolamine and H3tipa = triisopropanolamine} were synthesized via selfassembly
method under green conditions. These compounds were fully characterized, and their crystal structures were established. The topological analysis depicted the formation of uninodal 4-connected nets with the sql topology, which are driven by the 5-coordinated copper(II) units and pdc2- linkers. Through an optimized scale-up process from milligram to gram scale, a larger quantity of pure crystalline materials (~8.0 g) was obtained with higher yields (~70%) compared to the small-scale reaction (~60%). The obtained CPs were applied as efficient and recyclable heterogeneous catalysts for the mild oxidation of a-pinene with tert-butyl hydroperoxide. The effects of various reactions parameters, temperature-dependent and mechanistic studies, and catalyst stability were investigated in detail, leading to up to 93% of a-pinene conversions with good yields of 4-tert-butylperoxy-2-pinene (42%) and verbenone (25%) as main oxidation products. This study opens up the use of copper(II) coordination polymers as promising heterogeneous catalysts for the allylic oxidation of a-pinene into value-added products
Host cell protein-mediated adjuvanticity and immunogenicity risks of biotherapeutics.
No full textHost cell proteins (HCPs) are process-related impurities of biotherapeutic production that might affect product quality and/or patient safety. In a few cases, adverse events were attributed to HCPs present in the administered biotherapeutic. HCP-associated immune risks include adjuvanticity and immunogenicity with potential cross-reactivity. Based on the published data, some HCPs can act as adjuvants increasing the immunogenicity of the biotherapeutic as a bystander effect. HCPs may also induce immunogenicity against themselves, resulting in anti-HCP T cell responses and anti-HCP antibody formation. Depending on sequence similarities, these anti-HCP immune responses might theoretically be cross-reactive to the biotherapeutic or human endogenous proteins. In this review, we examine HCP-associated immune-related risks reported from non-clinical and clinical studies. We also discuss the potential and limitations of in vitro and in silico methods to evaluate the adjuvanticity and immunogenicity potential of HCPs. A risk-based assessment of the safety impact of HCPs may include the identity of the HCP and similarity to the biotherapeutic and human proteins, as well as product, treatment-, and patient-related factors
Integrating environmental, economic, and social pillars for a holistic decision-making approach in optimal portfolio and trial decisions: results of an international multi-stakeholder survey
No full textThe pharmaceutical industry is increasingly shifting to decentralized clinical trials (DCTs) conducted at the patient's home, sometimes including trial material home delivery. The traditional clinical trial (CT) is conducted at the investigational site. Research suggests that trials in centralized and decentralized settings have a large carbon footprint, with DCTS potentially providing patient-centric solutions. However, leaders must determine how to integrate environmental, economic, and social sustainability pillars into their portfolios and subsequent downstream trial-level decisions.
An online survey was designed and deployed via ETH’s SurveySelect software to capture international multi-stakeholder perceptions of priorities and tradeoffs when deciding between a DCT and a traditional CT for each pillar. The survey closed on 31st January 2023. A total of 447 participants responded. The findings revealed that the overall cohort prioritized greenhouse gas emissions (22.4%) for environmental impact, trial probability of success (15%) for economic considerations, and patient convenience (23.3%) for social criteria. Overall, the DCT setting was perceived as more sustainable in all pillars. Participants reported tradeoffs centered on patient engagement and bringing new medicines to the market.
The results from this survey provide initial insights into international multi-stakeholder perceptions of the priorities and tradeoffs when choosing between a traditional CT and DCT. The synthesized perceptions inform three key recommendations: the need (1) for simulation studies to guide holistic decision-making across all pillars as empirical data accumulates, (2) to protect the environment, and (3) to protect the supply chain. As empirical data accumulates, these recommendations provide directionality for further research