ABSTRACT
Patients with Skraban-Deardorff syndrome (SKDEAS), a neurodevelopmental syndrome associated with a spectrum of developmental and intellectual delays and disabilities, harbor diverse mutations in WDR26, encoding a subunit of the multiprotein CTLH E3 ubiquitin ligase complex. Structural studies revealed that homodimers of WDR26 bridge two core-CTLH E3 complexes to generate giant, hollow oval-shaped supramolecular CTLH E3 assemblies. Additionally, WDR26 mediates CTLH E3 complex binding to subunit YPEL5 and functions as substrate receptor for the transcriptional repressor HBP1. Here, we mapped SKDEAS-associated mutations on a WDR26 structural model and tested their functionality in complementation studies using genetically engineered human cells lacking CTLH E3 supramolecular assemblies. Despite the diversity of mutations, 15 of 16 tested mutants impaired at least one CTLH E3 complex function contributing to complex assembly and interactions, thus providing first mechanistic insights into SKDEAS pathology.
Subject(s)
Adaptor Proteins, Signal Transducing , Intellectual Disability , Mutation , Ubiquitin-Protein Ligases , Humans , Adaptor Proteins, Signal Transducing/genetics , HEK293 Cells , Intellectual Disability/genetics , Intellectual Disability/metabolism , Models, Molecular , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/chemistryABSTRACT
Anti-TNF therapies are a core anti-inflammatory approach for chronic diseases such as rheumatoid arthritis and Crohn's Disease. Previously, we and others found that TNF blocks the emergence and function of alternative-activated or M2 macrophages involved in wound healing and tissue-reparative functions. Conceivably, anti-TNF drugs could mediate their protective effects in part by an altered balance of macrophage activity. To understand the mechanistic basis of how TNF regulates tissue-reparative macrophages, we used RNAseq, scRNAseq, ATACseq, time-resolved phospho-proteomics, gene-specific approaches, metabolic analysis, and signaling pathway deconvolution. We found that TNF controls tissue-reparative macrophage gene expression in a highly gene-specific way, dependent on JNK signaling via the type 1 TNF receptor on specific populations of alternative-activated macrophages. We further determined that JNK signaling has a profound and broad effect on activated macrophage gene expression. Our findings suggest that TNF's anti-M2 effects evolved to specifically modulate components of tissue and reparative M2 macrophages and TNF is therefore a context-specific modulator of M2 macrophages rather than a pan-M2 inhibitor.
Subject(s)
Macrophages , Transcription, Genetic , Tumor Necrosis Factor-alpha/metabolism , Animals , Cells, Cultured , Cytokines/metabolism , Female , Macrophage Activation/drug effects , Macrophages/drug effects , Macrophages/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Signal Transduction/drug effects , Signal Transduction/genetics , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , Tumor Necrosis Factor Inhibitors/pharmacologyABSTRACT
An injection seeded Nd:YAG laser oscillator has been set up and frequency stabilized following an rf-sideband scheme. This dual rod oscillator emits pulses with 23 ns duration and 20 mJ energy. The beam quality is almost diffraction limited (M(2)=1.2). The frequency stability was characterized with a heterodyne method to 1.0 MHz root mean square (rms). This oscillator will serve as the front end for a series of lidar devices for spectrally sensitive measurements.
ABSTRACT
In side pumped laser head geometries good extraction of energy has to be weighted against diffraction effects of the amplified beam. Beam clipping at the aperture of laser rods can be avoided by using an undoped cladding around the doped core. The wings of e.g. Gaussian beams can be accommodated in the cladding. Phase distortion by the refractive index step of the rod can be compensated by a phase conjugating mirror in double pass configuration. In our proof of principle experiment the brightness of the beam from core doped amplifier rods was shown to be doubled compared to a conventional rod of the same outer diameter.
ABSTRACT
The early status of strain development for the production of interleukin (IL)-6, IL-8, IL-10, and interferon (IFN) gamma is described. The general approach to generating such strains was to amplify gene sequences encoding the mature forms of the various cytokines by PCR from commercially available cDNA sources. The design of the amplificates allowed an in-frame fusion to an MFalpha1 leader segment contained in two basic expression vectors, pFPMT121-MFalpha1 and pTPSMT-MFalpha1. The two vectors differ in that one harbors the methanol-inducible FMD promoter and the other the constitutive TPS1 promoter as control elements for heterologous gene expression. The most advanced process development example is that of IFNalpha-2a. Here, the MOX promoter derived from another key gene of methanol metabolism is used for expression control. The successful development of a production process for Hansenula polymorpha-derived IFNalpha-2a is summarized. This was achieved by combining genetic engineering of suitable production strains with improved processing capabilities for the secreted cytokine, and by purification procedures from cultures grown in yeast extract-peptone-glycerol-based media.
