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We consider the initial value problem associated to the regularized Benjamin-Ono equation, rBO. Our aim is to establish local and global wellposedness results in weighted Sobolev spaces via contraction principle. We also prove a unique continuation property that implies that arbitrary polynomial type decay is not preserved yielding sharp results regarding well-posedness of the initial value problem in most weighted Sobolev spaces. [ABSTRACT FROM AUTHOR]

The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community., Competing Interests: Competing interests: N.L.K. is involved in entrepreneurial activities in top-down proteomics and consults for ThermoFisher Scientific. P.O.D. is the founder and principal of Eastwoods Consulting, providing business advisory services to life science companies. The remaining authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

BamA is the central component of the essential β-barrel assembly machine (BAM), a conserved multi-subunit complex that dynamically inserts and folds β-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., (© 2024. The Author(s).)

Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-β (Aβ) pathology (PS2APP) or combined Aβ and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Introduction: Hearing health is a public health concern that affects the quality of life and can be disturbed by noise exposure, generating auditory and extra-auditory symptoms. Objective. To identify the hearing health status in adults living in Bogotá and its association with environmental noise exposure and individual and otological factors., Objective: To identify the hearing health status in adults living in Bogotá and its association with environmental noise exposure and individual and otological factors., Materials and Methods: We conducted a cross-sectional study using a database with 10,311 records from 2014 to 2018, consigned in a structured survey of noise perception and hearing screening. We performed a descriptive, bivariate, and binary logistic regression analysis., Results: Of the included participants, 35.4% presented hearing impairment. In the perception component, 13.0 % reported not hearing well; 28.8 % had extra-auditory symptoms, 53.3 % informed otological antecedents and 69.0 % presented discomfort due to extramural noise. In the logistic regression, the variables with the highest association for hearing impairment were living in noisy areas (OR = 1.50) (95% CI: 1.34-1.69), being male (OR = 1.85) (95% CI: 1.64-2.09), increasing age (for each year of life, the risk of hearing impairment increased 6%), and having history of extra-auditory symptoms (OR = 1.86) (95% CI: 1.66-2.08)., Conclusions: Hearing impairment is multi-causal in the studied population. The factors that promote its prevalence are increasing age, being male, smoking, ototoxic medications, living in areas with high noise exposure, and extra-auditory symptoms.

Ferritin is a multivalent, self-assembling protein scaffold found in most human cell types, in addition to being present in invertebrates, higher plants, fungi, and bacteria, that offers an attractive alternative to polymer-based drug delivery systems (DDS). In this study, the utility of the ferritin cage as a DDS was demonstrated within the context of T cell agonism for tumor killing. Members of the tumor necrosis factor receptor superfamily (TNFRSF) are attractive targets for the development of anticancer therapeutics. These receptors are endogenously activated by trimeric ligands that occur in transmembrane or soluble forms, and oligomerization and cell-surface anchoring have been shown to be essential aspects of the targeted agonism of this receptor class. Here, we demonstrated that the ferritin cage could be easily tailored for multivalent display of anti-OX40 antibody fragments on its surface and determined that these arrays are capable of pathway activation through cell-surface clustering. Together, these results confirm the utility, versatility, and developability of ferritin as a DDS.

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics., (© 2024. The Author(s).)

Heterozygous mutations in the granulin ( GRN ) gene are a leading cause of frontotemporal lobar degeneration with TDP-43 aggregates (FTLD-TDP). Polymorphisms in TMEM106B have been associated with disease risk in GRN mutation carriers and protective TMEM106B variants associated with reduced levels of TMEM106B, suggesting that lowering TMEM106B might be therapeutic in the context of FTLD. Here, we tested the impact of full deletion and partial reduction of TMEM106B in mouse and iPSC-derived human cell models of GRN deficiency. TMEM106B deletion did not reverse transcriptomic or proteomic profiles in GRN-deficient microglia, with a few exceptions in immune signaling markers. Neither homozygous nor heterozygous Tmem106b deletion normalized disease-associated phenotypes in Grn -/- mice. Furthermore, Tmem106b reduction by antisense oligonucleotide (ASO) was poorly tolerated in Grn -/- mice. These data provide novel insight into TMEM106B and GRN function in microglia cells but do not support lowering TMEM106B levels as a viable therapeutic strategy for treating FTD- GRN ., Competing Interests: The authors have a published patent application WO 2019/154979 A1 related to this work., (© 2023 The Authors.)

During the course of biopharmaceutical production, heterologous protein expression in Chinese hamster ovary (CHO) cells imposes a high proteostatic burden that requires cellular adaptation. To mitigate such burden, cells utilize the unfolded protein response (UPR), which increases endoplasmic reticulum (ER) capacity to accommodate elevated rates of protein synthesis and folding. In this study, we show that during production the UPR regulates growth factor signaling to modulate growth and protein synthesis. Specifically, the protein kinase R-like ER kinase (PERK) branch of the UPR is responsible for transcriptional down-regulation of platelet-derived growth factor receptor alpha (PDGFRa) and attenuation of the IRE1-alpha (IRE1a) branch of the UPR. PERK knockout (KO) cell lines displayed reduced growth and viability due to higher rates of apoptosis despite having stabilized PDGFRa levels. Knocking out PERK in an apoptosis impaired (Bax/Bak double KO) antibody-expressing cell line prevented apoptotic cell death and revealed that apoptosis was likely triggered by increased ER stress and reactive oxygen species levels in the PERK KO hosts. Our findings suggest that attenuation of IRE1a and PDGFRa signaling by the PERK branch of the UPR reduces ER protein folding capacity and hence specific productivity of CHO cells in order to mitigate UPR and prevent apoptotic cell death. Last, Bax/Bak/PERK triple KO CHO cell lines displayed 2-3 folds higher specific productivity and titer (up to 8 g/L), suggesting that modulation of PERK signaling during production processes can greatly improve specific productivity in CHO cells., (© 2023 American Institute of Chemical Engineers.)

TGFβ signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFβ signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFβ and PD-L1 restrain intratumoral stem cell-like CD8 T cell (T SCL ) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFβ/PD-L1 blockade IFNγ hi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFβ therapy efficacy. Our data suggest that TGFβ works with PD-L1 to prevent T SCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state., (© 2023. Springer Nature Limited.)

Recent advances in native mass spectrometry (MS) and denatured intact protein MS have made these techniques essential for biotherapeutic characterization. As MS analysis has increased in throughput and scale, new data analysis workflows are needed to provide rapid quantitation from large datasets. Here, we describe the UniDec processing pipeline (UPP) for the analysis of batched biotherapeutic intact MS data. UPP is built into the UniDec software package, which provides fast processing, deconvolution, and peak detection. The user and programming interfaces for UPP read a spreadsheet that contains the data file names, deconvolution parameters, and quantitation settings. After iterating through the spreadsheet and analyzing each file, it returns a spreadsheet of results and HTML reports. We demonstrate the use of UPP to measure the correct pairing percentage on a set of bispecific antibody data and to measure drug-to-antibody ratios from antibody-drug conjugates. Moreover, because the software is free and open-source, users can easily build on this platform to create customized workflows and calculations. Thus, UPP provides a flexible workflow that can be deployed in diverse settings and for a wide range of biotherapeutic applications.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with significant mortality. Prognostic biomarkers to identify rapid progressors are urgently needed to improve patient management. Since the lysophosphatidic acid (LPA) pathway has been implicated in lung fibrosis in preclinical models and identified as a potential therapeutic target, we aimed to investigate if bioactive lipid LPA species could be prognostic biomarkers that predict IPF disease progression. LPAs and lipidomics were measured in baseline placebo plasma of a randomized IPF-controlled trial. The association of lipids with disease progression indices were assessed using statistical models. Compared to healthy, IPF patients had significantly higher levels of five LPAs (LPA16:0, 16:1, 18:1, 18:2, 20:4) and reduced levels of two triglycerides species (TAG48:4-FA12:0, -FA18:2) (false discovery rate < 0.05, fold change > 2). Patients with higher levels of LPAs had greater declines in diffusion capacity of carbon monoxide over 52 weeks (P < 0.01); additionally, LPA20:4-high (≥median) patients had earlier time to exacerbation compared to LPA20:4-low (

Idiopathic pulmonary fibrosis is a progressive fibrotic disease characterized by excessive deposition of (myo)fibroblast produced collagen fibrils in alveolar areas of the lung. Lysyl oxidases (LOXs) have been proposed to be the central enzymes that catalyze the cross-linking of collagen fibers. Here, we report that, while its expression is increased in fibrotic lungs, genetic ablation of LOXL2 only leads to a modest reduction of pathological collagen cross-linking but not fibrosis in the lung. On the other hand, loss of another LOX family member, LOXL4, markedly disrupts pathological collagen cross-linking and fibrosis in the lung. Furthermore, knockout of both Loxl2 and Loxl4 does not offer any additive antifibrotic effects when compared to Loxl4 deletion only, as LOXL4 deficiency decreases the expression of other LOX family members including Loxl2 . On the basis of these results, we propose that LOXL4 is the main LOX activity underlying pathological collagen cross-linking and lung fibrosis.

Immune monitoring in cancer immunotherapy involves screening CD8+ T-cell responses against neoantigens, the tumor-specific peptides presented by Major histocompatibility complex Class I (MHCI) on the cell surface. High-throughput immune monitoring requires methods to produce and characterize small quantities of thousands of MHCI-peptide complexes that may be tested for a patient's T-cell response. MHCI synthesis has been achieved using a photocleavable peptide that is exchanged by the neoantigen; however, assays that measure peptide exchange currently disassemble the complex prior to analysis─precluding direct molecular characterization. Here, we use native mass spectrometry (MS) to profile intact recombinant MHCI complexes and directly measure peptide exchange. Coupled with size-exclusion chromatography or capillary-zone electrophoresis, the assay identified all tested human leukocyte antigen (HLA)/peptide combinations in the nanomole to picomole range with minimal run time, reconciling the synthetic and analytical requirements of MHCI-peptide screening with the downstream T-cell assays. We further show that the assay can be "multiplexed" by measuring exchange of multiple peptides simultaneously and also enables calculation of Vc 50 , a measure of gas-phase stability. Additionally, MHCI complexes were fragmented by top-down sequencing, demonstrating that the intact complex, peptide sequence, and their binding affinity can be determined in a single analysis. This screening tool for MHCI-neoantigen complexes represents a step toward the application of state-of-the-art MS technology in translational settings. Not only is this assay already informing on the viability of immunotherapy in practice, the platform also holds promise to inspire novel MS readouts for increasingly complex biomolecules used in the diagnosis and treatment of disease.