SET-26, HCF-1, and HDA-1 are highly conserved chromatin factors with key roles in development and aging. Here we present mechanistic insights into how these factors regulate gene expression and ...modulate longevity in C. elegans. We show that SET-26 and HCF-1 cooperate to regulate a common set of genes, and both antagonize the histone deacetylase HDA-1 to limit longevity. HCF-1 localization at chromatin is largely dependent on functional SET-26, whereas SET-26 is only minorly affected by loss of HCF-1, suggesting that SET-26 could recruit HCF-1 to chromatin. HDA-1 opposes SET-26 and HCF-1 on the regulation of a subset of their common target genes and in longevity. Our findings suggest that SET-26, HCF-1, and HDA-1 comprise a mechanism to fine-tune gene expression and longevity and likely have important implications for the mechanistic understanding of how these factors function in diverse organisms, particularly in aging biology.
Misuse of prescription medications has led to higher mortality and morbidity in the U.S. This study investigates medical and nonmedical use of prescription medications (prescription pain relievers, ...stimulants, prescription tranquilizers, and sedatives) among adults aged ≥50 years.
Data were from the 2015–2017 National Survey of Drug Use and Health. For each prescription medication, medical and nonmedical use were mutually exclusive (prescription pain relievers, stimulants, tranquilizers, and sedatives). Nonmedical use (misuse) was defined as the use of any prescription medications in greater amounts, frequency, or duration than prescribed or the use of medications without a prescription of the individual's own. Descriptive analysis was performed to assess prescription medication use and misuse among adults aged ≥50 years (pooled N=26,322). Correlates of misuse were assessed within multivariable logistic regression analyses at p<0.05. All analyses were conducted in 2019.
In the past 12 months, 46.7% (51.7 million) of adults reported using any prescription medication, of which 7.7% (4.0 million) reported misuse. The proportion of past 12-month users reporting misuse was 5.4% for sedatives, 6.4% for prescription pain relievers, 6.5% for prescription tranquilizers, and 8.1% for stimulants. Overall, 16.2% (17.9 million) reported using 2 or more prescription medications within the past 12 months. Multiple prescription medication use was associated with female sex; White race; younger age; and use of nonprescription substances, including alcohol, tobacco, marijuana, and heroin.
It is important to monitor medication misuse behaviors among older adults who may be living with several chronic conditions requiring pain management. The strong associations with serious psychological distress underscore the need for increased access to mental health services among this key population.
Platinum-based cancer therapy is restricted by dose-limiting side effects and is associated with elevation of growth differentiation factor 15 (GDF-15). But whether this elevation contributes to such ...side effects has been unclear. Here, we explored the effects of GDF-15 blockade on platinum-based chemotherapy-induced emesis, anorexia, and weight loss in mice and/or nonhuman primate models. We found that circulating GDF-15 is higher in subjects with cancer receiving platinum-based chemotherapy and is positively associated with weight loss in colorectal cancer (NCT00609622). Further, chemotherapy agents associated with high clinical emetic score induce circulating GDF-15 and weight loss in mice. Platinum-based treatment-induced anorexia and weight loss are attenuated in GDF-15 knockout mice, while GDF-15 neutralization with the monoclonal antibody mAB1 improves survival. In nonhuman primates, mAB1 treatment attenuates anorexia and emesis. These results suggest that GDF-15 neutralization is a potential therapeutic approach to alleviate chemotherapy-induced side effects and improve the quality of life.
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•Plasma GDF-15 is higher in subjects with cancer receiving platinum-based chemotherapy•GDF-15 deletion attenuates anorexia and weight loss induced by platinum-based agents•GDF-15 neutralization lowered cisplatin-induced emesis/anorexia in nonhuman primates•GDF-15 neutralization + cisplatin treatment promotes survival in a mouse tumor model
In this work, circulating GDF-15 is higher in subjects with cancer receiving platinum-based chemotherapy. In mice and/or nonhuman primates, GDF-15 neutralization with a novel antibody (mAB1) alleviates emesis, anorexia, and weight loss induced by platinum-based chemotherapies, and mAB1 treatment improves survival in tumor-bearing mice when given in combination with cisplatin.
Assessment of the efficacy of a multi-agent chemotherapy protocol in which cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) are administered in canine lymphoma is generally performed ...by physical measurement of lymph node diameter. However, no consistent correlation has been made with prognostic indicators and the length or absence of clinical remission based on lymph node size. RNA disruption measured mid-therapy has been correlated with increased disease-free survival in recent studies of human cancer and was assessed in this study of canine lymphoma patients. Fine needle aspirate samples were taken before treatment and at weeks 3, 6, and 11 of CHOP therapy. RNA was isolated from these samples and assessed using an Agilent Bioanalyzer. RNA disruption assay (RDA) analysis was performed on the data from the resulting electropherograms.
An increased RNA disruption index (RDI) score was significantly associated with improved progression-free survival.
Predicting the risk of early relapse during chemotherapy could benefit veterinary patients by reducing ineffective treatment and could allow veterinary oncologists to switch earlier to a more effective drug regimen.
Phosphorylation of inhibitor of nuclear transcription factor κB (IκB) by IκB kinase (IKK) triggers the degradation of IκB and migration of cytoplasmic κB to the nucleus where it promotes the ...transcription of its target genes. Activation of IKK is achieved by phosphorylation of its main subunit, IKKβ, at the activation loop sites. Here, we report the 2.8 Å resolution crystal structure of human IKKβ (hIKKβ), which is partially phosphorylated and bound to the staurosporine analog K252a. The hIKKβ protomer adopts a trimodular structure that closely resembles that from Xenopus laevis (xIKKβ): an N-terminal kinase domain (KD), a central ubiquitin-like domain (ULD), and a C-terminal scaffold/dimerization domain (SDD). Although hIKKβ and xIKKβ utilize a similar dimerization mode, their overall geometries are distinct. In contrast to the structure resembling closed shears reported previously for xIKKβ, hIKKβ exists as an open asymmetric dimer in which the two KDs are further apart, with one in an active and the other in an inactive conformation. Dimer interactions are limited to the C-terminal six-helix bundle that acts as a hinge between the two subunits. The observed domain movements in the structures of IKKβ may represent trans-phosphorylation steps that accompany IKKβ activation.
Background: IκB kinase β is a key regulator in the NκB signaling pathway.
Results: Crystal structure of a human IKKβ asymmetric dimer shows one kinase active site phosphorylated and in the active conformation and the other unphosphorylated and inactive.
Conclusion: Depending on the phosphorylation state, IKKβ can adopt distinct dimeric geometry.
Significance: High resolution structure of hIKKβ provides structural basis for its activation and potential use of inhibitor design.
Implementation of in vitro assays that correlate with in vivo human pharmacokinetics (PK) would provide desirable preclinical tools for the early selection of therapeutic monoclonal antibody (mAb) ...candidates with minimal non-target-related PK risk. Use of these tools minimizes the likelihood that mAbs with unfavorable PK would be advanced into costly preclinical and clinical development. In total, 42 mAbs varying in isotype and soluble versus membrane targets were tested in in vitro and in vivo studies. MAb physicochemical properties were assessed by measuring non-specific interactions (DNA- and insulin-binding ELISA), self-association (affinity-capture self-interaction nanoparticle spectroscopy) and binding to matrix-immobilized human FcRn (surface plasmon resonance and column chromatography). The range of scores obtained from each in vitro assay trended well with in vivo clearance (CL) using both human FcRn transgenic (Tg32) mouse allometrically projected human CL and observed human CL, where mAbs with high in vitro scores resulted in rapid CL in vivo. Establishing a threshold value for mAb CL in human of 0.32 mL/hr/kg enabled refinement of thresholds for each in vitro assay parameter, and using a combinatorial triage approach enabled the successful differentiation of mAbs at high risk for rapid CL (unfavorable PK) from those with low risk (favorable PK), which allowed mAbs requiring further characterization to be identified. Correlating in vitro parameters with in vivo human CL resulted in a set of in vitro tools for use in early testing that would enable selection of mAbs with the greatest likelihood of success in the clinic, allowing costly late-stage failures related to an inadequate exposure profile, toxicity or lack of efficacy to be avoided.
Expression of vascular endothelial growth factor (VEGF) is tightly regulated to achieve normal angiogenesis. The objective was to examine regulation of VEGF by the activin-like kinase receptors ...(ALKs) ALK1 and ALK5. Transforming growth factor β1 (TGFβ1) and bone morphogenetic protein-9 (BMP-9) enhanced and suppressed VEGF expression, respectively, in aortic endothelial cells, as determined by real-time polymerase chain reaction, immunoblotting, cell proliferation, and tube formation. The use of small interfering RNA revealed that TGFβ1 stimulated VEGF expression by activating ALK5, TGFβ type II receptor, and SMAD2, whereas BMP-9 suppressed it by activating ALK1, BMP type II receptor, and SMAD1. ALK1 signaling occurred independently of ALK5 activity. Partial ALK1 deficiency in vitro and in vivo resulted in elevated VEGF expression. In vitro, increased BMP-9 levels normalized VEGF expression in cells with partial, but not severe, ALK1 deficiency. Time course experiments revealed that an increase in ALK1 expression induced by BMP-4, an angiogenic stimulus, preceded induction of ALK5 and VEGF in control cells. In ALK1-deficient cells, however, VEGF expression occurred earlier and was abnormally high, even though ALK5 was not induced. Our results suggest that ALK1 and ALK5 are both essential for correct regulation of VEGF, and that disruption of either pathway leads to disease.
For an antibody to be a successful therapeutic many competing factors require optimization, including binding affinity, biophysical characteristics, and immunogenicity risk. Additional constraints ...may arise from the need to formulate antibodies at high concentrations (>150 mg/ml) to enable subcutaneous dosing with reasonable volume (ideally <1.0 mL). Unfortunately, antibodies at high concentrations may exhibit high viscosities that place impractical constraints (such as multiple injections or large needle diameters) on delivery and impede efficient manufacturing. Here we describe the optimization of an anti-PDGF-BB antibody to reduce viscosity, enabling an increase in the formulated concentration from 80 mg/ml to greater than 160 mg/ml, while maintaining the binding affinity. We performed two rounds of structure guided rational design to optimize the surface electrostatic properties. Analysis of this set demonstrated that a net-positive charge change, and disruption of negative charge patches were associated with decreased viscosity, but the effect was greatly dependent on the local surface environment. Our work here provides a comprehensive study exploring a wide sampling of charge-changes in the Fv and CDR regions along with targeting multiple negative charge patches. In total, we generated viscosity measurements for 40 unique antibody variants with full sequence information which provides a significantly larger and more complete dataset than has previously been reported.
Structure and function of therapeutic antibodies can be modulated by a variety of post-translational modifications (PTM). Tyrosine (Tyr) sulfation is a type of negatively charged PTM that occurs ...during protein trafficking through the Golgi. In this study, we discovered that an anti-interleukin (IL)-4 human IgG1, produced by transiently transfected HEK293 cells, contained a fraction of unusual negatively charged species. Interestingly, the isolated acidic species exhibited a two-fold higher affinity to IL-4 and a nearly four-fold higher potency compared to the main species. Mass spectrometry (MS) showed the isolated acidic species possessed an +80-Dalton from the expected mass, suggesting an occurrence of Tyr sulfation. Consistent with this hypothesis, we show the ability to control the acidic species during transient expression with the addition of Tyr sulfation inhibitor sodium chlorate or, conversely, enriched the acidic species from 30% to 92% of the total antibody protein when the IL-4 IgG was co-transfected with tyrosylprotein sulfotransferase genes. Further MS and mutagenesis analysis identified a Tyr residue at the light chain complementarity-determining region-1 (CDRL-1), which was sulfated specifically. These results together have demonstrated for the first time that Tyr sulfation at CDRL-1 could modulate antibody binding affinity and potency to a human immune cytokine.
Next-generation site-specific cysteine-based antibody-drug-conjugates (ADCs) broaden therapeutic index by precise drug-antibody attachments. However, manufacturing such ADCs for clinical validation ...requires complex full reduction and reoxidation processes, impacting product quality. To overcome this technical challenge, we developed a novel antibody manufacturing process through cysteine (Cys) metabolic engineering in Chinese hamster ovary cells implementing a unique cysteine-capping technology. This development enabled a direct conjugation of drugs after chemoselective-reduction with mild reductant tris(3-sulfonatophenyl)phosphine. This innovative platform produces clinical ADC products with superior quality through a simplified manufacturing process. This technology also has the potential to integrate Cys-based site-specific conjugation with other site-specific conjugation methodologies to develop multi-drug ADCs and exploit multi-mechanisms of action for effective cancer treatments.
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