6,064 research outputs found

    Advancing Intact Protein Quantitation with Updated Deconvolution Routines

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    Analysis of intact proteins by mass spectrometry enables direct quantitation of the specific proteoforms present in a sample and is an increasingly important tool for biopharmaceutical and academic research. Interpreting and quantifying intact protein species from mass spectra typically involves many challenges including mass deconvolution and peak processing as well as determining optimal spectral averaging parameters and matching masses to theoretical proteoforms. Each of these steps can present informatic hurdles, as parameters often need to be tailored specifically to the data sets. To reduce intact mass deconvolution data analysis burdens, we built upon the widely used ÔÇťsliding windowÔÇŁ mass deconvolution technique with several additional concepts. First, we found that how spectra are averaged and the overlap in spectral windows can be tuned to favor either sensitivity or speed. A multiple window averaging approach was found to be the most effective way to increase mass detection and yielded a >2-fold increase in the number of masses detected. We also developed a targeted feature-finding routine that boosted sensitivity by >2-fold, decreased coefficient of variation across replicates by 50%, and increased the quality of mass elution profiles through 3-fold more detected time points. Lastly, we furthered existing approaches for annotating detected masses with potential proteoforms through spectral fitting for possible proteoform family modifications and network viewing. These proteoform annotation approaches ultimately produced a more accurate way of finding related, but previously unknown proteoforms from intact mass-only data. Together, these quantitation workflow improvements advance the information obtainable from intact protein mass spectrometry analyses

    The promiscuous development of an unconventional Qa1b-restricted T cell population

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    MHC-E restricted CD8 T cells show promise in vaccine settings, but their development and specificity remain poorly understood. Here we focus on a CD8 T cell population reactive to a self-peptide (FL9) bound to mouse MHC-E (Qa-1b) that is presented in response to loss of the MHC I processing enzyme ERAAP, termed QFL T cells. We find that mature QFL thymocytes are predominantly CD8╬▒╬▓+CD4-, show signs of agonist selection, and give rise to both CD8╬▒╬▒ and CD8╬▒╬▓ intraepithelial lymphocytes (IEL), as well as memory phenotype CD8╬▒╬▓ T cells. QFL T cells require the MHC I subunit ╬▓-2 microglobulin (╬▓2m), but do not require Qa1b or classical MHC I for positive selection. However, QFL thymocytes do require Qa1b for agonist selection and full functionality. Our data highlight the relaxed requirements for positive selection of an MHC-E restricted T cell population and suggest a CD8╬▒╬▓+CD4- pathway for development of CD8╬▒╬▒ IELs

    Probable Domain Generalization via Quantile Risk Minimization

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    Domain generalization (DG) seeks predictors which perform well on unseen test distributions by leveraging data drawn from multiple related training distributions or domains. To achieve this, DG is commonly formulated as an average- or worst-case problem over the set of possible domains. However, predictors that perform well on average lack robustness while predictors that perform well in the worst case tend to be overly-conservative. To address this, we propose a new probabilistic framework for DG where the goal is to learn predictors that perform well with high probability. Our key idea is that distribution shifts seen during training should inform us of probable shifts at test time, which we realize by explicitly relating training and test domains as draws from the same underlying meta-distribution. To achieve probable DG, we propose a new optimization problem called Quantile Risk Minimization (QRM). By minimizing the ╬▒\alpha-quantile of predictor's risk distribution over domains, QRM seeks predictors that perform well with probability ╬▒\alpha. To solve QRM in practice, we propose the Empirical QRM (EQRM) algorithm and provide: (i) a generalization bound for EQRM; and (ii) the conditions under which EQRM recovers the causal predictor as ╬▒Ôćĺ1\alpha \to 1. In our experiments, we introduce a more holistic quantile-focused evaluation protocol for DG and demonstrate that EQRM outperforms state-of-the-art baselines on datasets from WILDS and DomainBed.Comment: NeurIPS 2022 camera-ready (+ minor corrections

    Application of Chiral Transfer Reagents to Improve Stereoselectivity and Yields in the Synthesis of the Antituberculosis Drug Bedaquiline

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    Bedaquiline (BDQ) is an important drug for treating multidrug-resistant tuberculosis (MDR-TB), a worldwide disease that causes more than 1.6 million deaths yearly. The current synthetic strategy adopted by the manufacturers to assemble this molecule relies on a nucleophilic addition reaction of a quinoline fragment to a ketone, but it suffers from low conversion and no stereoselectivity, which subsequently increases the cost of manufacturing BDQ. The Medicines for All Institute (M4ALL) has developed a new reaction methodology to this process that not only allows high conversion of starting materials but also results in good diastereo- and enantioselectivity toward the desired BDQ stereoisomer. A variety of chiral lithium amides derived from amino acids were studied, and it was found that lithium (R)-2-(methoxymethyl)pyrrolidide, obtained from d-proline, results in high assay yield of the desired syn-diastereomer pair (82%) and with considerable stereocontrol (d.r. = 13.6:1, e.r. = 3.6:1, 56% ee), providing BDQ in up to a 64% assay yield before purification steps toward the final API. This represents a considerable improvement in the BDQ yield compared to previously reported conditions and could be critical to further lowering the cost of this life-saving drug

    SP7 gene silencing dampens bone marrow stromal cell hypertrophy, but it also dampens chondrogenesis

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    For bone marrow stromal cells (BMSC) to be useful in cartilage repair their propensity for hypertrophic differentiation must be overcome. A single day of TGF-╬▓1 stimulation activates intrinsic signaling cascades in BMSCs which subsequently drives both chondrogenic and hypertrophic differentiation. TGF-╬▓1 stimulation upregulates SP7, a transcription factor known to contribute to hypertrophic differentiation, and SP7 remains upregulated even if TGF-╬▓1 is subsequently withdrawn from the chondrogenic induction medium. Herein, we stably transduced BMSCs to express an shRNA designed to silence SP7, and assess the capacity of SP7 silencing to mitigate hypertrophy. SP7 silencing dampened both hypertrophic and chondrogenic differentiation processes, resulting in diminished microtissue size, impaired glycosaminoglycan production and reduced chondrogenic and hypertrophic gene expression. Thus, while hypertrophic features were dampened by SP7 silencing, chondrogenic differentation was also compromised. We further investigated the role of SP7 in monolayer osteogenic and adipogenic cultures, finding that SP7 silencing dampened characteristic mineralization and lipid vacuole formation, respectively. Overall, SP7 silencing affects the trilineage differentiation of BMSCs, but is insufficient to decouple BMSC hypertrophy from chondrogenesis. These data highlight the challenge of promoting BMSC chondrogenesis whilst simultaneously reducing hypertrophy in cartilage tissue engineering strategies.</p

    Application of Chiral Transfer Reagents to Improve Stereoselectivity and Yields in the Synthesis of the Anti-Tuberculosis Drug Bedaquiline

    No full text
    Bedaquiline (BDQ) is an important drug for treating multidrug-resistant tuberculosis (MDR-TB), a worldwide disease that causes more than 1.6 million deaths yearly. The current synthetic strategy adopted by the manufacturers to assemble this molecule relies on a nucleophilic addition reaction of two complex starting materials, but suffers from low conversion and no stereoselectivity, which subsequently increases the cost of manufacturing BDQ. M4ALL has developed a new approach to this process that not only allows high conversion of starting materials, but also results in good diastereo- and enantioselectivity towards the desired BDQ stereoisomer. A variety of chiral lithium amides derived from amino acids were studied, and it was found that lithium (R)-2-(methoxymethyl)pyrrolidide, obtained from D-proline, results in high assay yield of the syn-diastereomer pair (82 %) and with considerable stereocontrol (d.r. = 13.6:1, e.r. = 3.6:1, 56 % ee) providing bedaquiline in up to 64 % assay yield before purification steps towards the final API. This represents a considerable improvement in the BDQ yield compared to previously reported conditions and could be critical to further lowering the cost of this life-saving drug

    dealii/dealii: deal.II version 9.4.2

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    All download files are mirrored at https://dealii.43-1.org/downloads/ This is a minor update to 9.4.1 with the following changes: a compilation issue with step-70 has been resolved CMake: prefer -pthread for posix thread support a type mismatch for suitesparse has been fixed that lead to compilation failures on certain platforms a number of Microsoft Visual Code compatibility fixes concerning extern declaration

    Murine cytomegalovirus downregulates ERAAP and induces an unconventional T cell response to self

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    Summary: The endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) plays a crucial role in shaping the peptide-major histocompatibility complex (MHC) class I repertoire and maintaining immune surveillance. While murine cytomegalovirus (MCMV) has multiple strategies for manipulating the antigen processing pathway to evade immune responses, the host has also developed ways to counter viral immune evasion. In this study, we find that MCMV modulates ERAAP and induces an interferon γ (IFN-γ)-producing CD8+ T cell effector response that targets uninfected ERAAP-deficient cells. We observe that ERAAP downregulation during infection leads to the presentation of the self-peptide FL9 on non-classical Qa-1b, thereby eliciting Qa-1b-restricted QFL T cells to proliferate in the liver and spleen of infected mice. QFL T cells upregulate effector markers upon MCMV infection and are sufficient to reduce viral load after transfer to immunodeficient mice. Our study highlights the consequences of ERAAP dysfunction during viral infection and provides potential targets for anti-viral therapies

    Technology and Mental Health Counseling

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    The use of technology in the field of mental health has been an increasing interest of study and utilization in recent years, especially during and after the Covid 19 pandemic in which social distancing practices were encouraged to limit the spread of the virus. During this time, the use of technology for health-related appointments became vital as many offices shut down and/ or had limited in-person access. Furthermore, technology has already been integrated and researched significantly in other areas of the healthcare fields such as psychiatry and nursing. However, there is a lack of understanding of the role of technology in the counseling field, understanding its use, defining what it is, and exploring its outcomes. This paper will highlight the development of technology in counseling and specifically focus on the available types of technology such as virtual reality and augmented reality
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