Linear Assembly Research Articles

Nonlinear Biosynthetic Assembly of Alpiniamide by a Hybrid cis/trans-AT PKS-NRPS.

Alpiniamide A is a linear polyketide produced by Streptomyces endophytic bacteria. Despite its relatively simple chemical structure suggestive of a linear assembly line biosynthetic construction involving a hybrid polyketide synthase-nonribosomal peptide synthetase enzymatic protein machine, we report an unexpected nonlinear synthesis of this bacterial natural product. Using a combination of genomics, heterologous expression, mutagenesis, isotope-labeling, and chain terminator experiments, we propose that alpiniamide A is assembled in two halves and then ligated into the mature molecule. We show that each polyketide half is constructed using orthogonal biosynthetic strategies, employing either cis- or trans-acyl transferase mechanisms, thus prompting an alternative proposal for the operation of this PKS-NRPS.

A New Selective Assembly Model for Achieving Specified Tolerance in High Precision Assemblies

Manufacturing of high precision assemblies pose a great challenge to engineers. High precision assemblies are generally assembled using selective assembly when the assembly tolerance requirement is less than the sum of the part tolerances. Although extensive research has been done in selective assembly modelling for minimising surplus parts and tolerance variation, they do not suit given specifications. In this paper, a new selective assembly model using a genetic algorithm is proposed to provide a detailed method for assembling parts for achieving specified assembly tolerance with minimum surplus parts. This model provides the best combination of selective groups and the number of assemblies in each group and so the assembly process is simplified. Genetic Algorithm is used to find the best combinations and the number of assemblies in each combination to minimize surplus parts. This paper analyses the effectiveness of the model for given target assembly tolerance for a linear assembly and it can be extended to any type of product.

Base part centered assembly task precedence generation

The recognized potential of cobots for industrial applications has been motivating a growing body of literature. As further research uncovers new potential applications, cobots are permeating into more more complex industrial applications. The inherent safety of cobots enables them to work at multiple levels of autonomy in close proximity with human operators. While this opens a range of new possibilities for human-robot collaboration, it also requires a different approach to assembly sequence generation. Most assembly sequence generation methods lack the flexibility to consider assembly tasks concurrently performed by operator and cobot. By splitting the products into sub-assemblies, the focus of both robot and human can be divided over multiple sub-assemblies assembled simultaneously or centered into a single sub-assembly for mutual assistance. In this paper, an algorithm for the identification of assembly task precedence is presented. Unlike most previous methods, by identifying sub-assemblies, their assembly precedence and that of their comprising parts, parallel task execution is not hindered by rigid linear assembly sequences. The output of the proposed algorithm allows for the creation of collaborative assembly sequences embedding resource capability, collaborative workplace design, and safety considerations. The proposed algorithm uses liaison and collision matrices as input as well as base part and sub-assembly content.

SHARPIN regulates cell proliferation of cutaneous basal cell carcinoma via inactivation of the transcriptional factors GLI2 and c‑JUN.

SHANK-associated RH domain-interacting protein (SHARPIN) is a component of the linear ubiquitin chain assembly complex that can enhance the NF-κB and JNK signaling pathways, acting as a tumor-associated protein in a variety of cancer types. The present study investigated the role of SHARPIN in cutaneous basal cell carcinoma (BCC). Human BCC (n=26) and normal skin (n=5) tissues, and BCC (TE354.T) and normal skin (HaCaT) cell lines were used to evaluate SHARPIN expression level using immunohistochemistry and western blotting, respectively. A lentivirus carrying SHARPIN-targeting or negative control short hairpin RNA was infected into TE354.T cells, and the infected stable cells were assayed to analyze tumor cell proliferation, cell cycle, apoptosis, migration and invasion by Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine incorporation assays, flow cytometry and Transwell assays. Western blotting was performed to assess the protein expression levels of gene signaling in SHARPIN-silenced BCC cells. SHARPIN protein expression levels were downregulated or absent in BCC cancer nests and precancerous lesions compared with normal skin samples. In addition, SHARPIN expression levels were lower in TE354.T cells compared with HaCaT cells. SHARPIN shRNA enhanced tumor cell proliferation and the S phase of the cell cycle, whereas BCC cell apoptotic rates, and migratory and invasive abilities were not significantly altered. The expression levels of cyclin D1, cyclin-dependent kinase 4, phosphorylated-c-JUN and GLI family zinc finger 2 proteins were increased, whereas Patched 1 (PTCH1) and PTCH2 were decreased in the SHARPIN-shRNA-infected BCC cells. Therefore, the present results suggested that SHARPIN may act as a tumor suppressor during BCC development.

OTULIN Prevents Liver Inflammation and Hepatocellular Carcinoma by Inhibiting FADD- and RIPK1 Kinase-Mediated Hepatocyte Apoptosis

Inflammatory signaling pathways are tightly regulated to avoid chronic inflammation and the development of disease. OTULIN is a deubiquitinating enzyme that controls inflammation by cleaving linear ubiquitin chains generated by the linear ubiquitin chain assembly complex. Here, we show that ablation of OTULIN in liver parenchymal cells in mice causes severe liver disease which is characterized by liver inflammation, hepatocyte apoptosis, and compensatory hepatocyte proliferation, leading to steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). Genetic ablation of Fas-associated death domain (FADD) completely rescues and knockin expression of kinase inactive receptor-interacting protein kinase1 (RIPK1) significantly protects mice from developingliver disease, demonstrating that apoptosis of OTULIN-deficient hepatocytes triggers disease pathogenesis in this model. Finally, we demonstrate that type I interferons contribute to disease in hepatocyte-specific OTULIN-deficient mice. Our study reveals the critical importance of OTULIN in protecting hepatocytes from death, thereby preventing the development of chronic liver inflammation and HCC.

Linear ubiquitin assembly complex regulates lung epithelial-driven responses during influenza infection.

Influenza A virus (IAV) is among the most common causes of pneumonia-related death worldwide. Pulmonary epithelial cells are the primary target for viral infection and replication and respond by releasing inflammatory mediators that recruit immune cells to mount the host response. Severe lung injury and death during IAV infection result from an exuberant host inflammatory response. The linear ubiquitin assembly complex (LUBAC), composed of SHARPIN, HOIL-1L, and HOIP, is a critical regulator of NF-κB-dependent inflammation. Using mice with lung epithelial-specific deletions of HOIL-1L or HOIP in a model of IAV infection, we provided evidence that, while a reduction in the inflammatory response was beneficial, ablation of the LUBAC-dependent lung epithelial-driven response worsened lung injury and increased mortality. Moreover, we described a mechanism for the upregulation of HOIL-1L in infected and noninfected cells triggered by the activation of type I IFN receptor and mediated by IRF1, which was maladaptive and contributed to hyperinflammation. Thus, we propose that lung epithelial LUBAC acts as a molecular rheostat that could be selectively targeted to modulate the immune response in patients with severe IAV-induced pneumonia.

Linear Polyubiquitin Chain Modification of TDP-43-Positive Neuronal Cytoplasmic Inclusions in Amyotrophic Lateral Sclerosis.

Neuronal cytoplasmic inclusions (NCIs) containing TAR DNA-binding protein of 43 kDa (TDP-43) are pathological hallmarks of amyotrophic lateral sclerosis (ALS) and are known to be ubiquitinated. Eight linkage types of polyubiquitin chains have been reported, each type of chain exerting different intracellular actions. The linkage type of polyubiquitin chain involved in the formation of NCIs in sporadic ALS (sALS), however, has not yet been elucidated. We performed immunohistochemical study of the spinal cords of 12 patients with sALS and on those of 6 control subjects. Virtually all ubiquitinated NCIs were immunolabeled with lysine 48-linked polyubiquitin chain (K48-Ub). Although the majority of NCIs were triple-immunoreactive for K48-Ub, linear polyubiquitin chain (L-Ub), and lysine 63-linked polyubiquitin chain (K63-Ub), thin parts of K48-Ub-immunopositive NCIs were not labeled for K63-Ub or L-Ub. We also detected HOIP and SHARPIN, components of linear ubiquitin chain assembly complex, colocalizing with L-Ub on NCIs. Moreover, the immunosignal of optineurin, an autophagy receptor working with L-Ub, and that of activated NF-κB p65, were observed to be colocalizing with L-Ub on certain parts of NCIs. The L-Ub modification of TDP-43-positive NCIs may function as an inducer of autophagic clearance of NCIs, neuroinflammation, and neurodegeneration in sALS.

Post-translational Modification of OTULIN Regulates Ubiquitin Dynamics and Cell Death

Linear ubiquitination has emerged as an important post-translational modification that regulates NF-κB activation, inflammation, and cell death in both immune and non-immune compartments, including the skin. The deubiquitinase OTULIN specifically disassembles linear ubiquitin chains generated by the linear ubiquitin assembly complex (LUBAC) and is necessary to prevent embryonic lethality and autoinflammatory disease. Here, we dissect the direct role of OTULIN in cell death andfind that OTULIN limits apoptosis and necroptosis in keratinocytes. During apoptosis, OTULIN is cleaved by capase-3 at Asp-31 into a C-terminal fragment that restricts caspase activation and cell death. During necroptosis, OTULIN is hyper-phosphorylated at Tyr-56, which modulates RIPK1 ubiquitin dynamics and promotes cell death. OTULIN Tyr-56 phosphorylation is counteracted by the activity of dual-specificity phosphatase 14 (DUSP14), which we identify as an OTULIN phosphatase that limits necroptosis. Our data provide evidence of dynamic post-translational modifications of OTULIN and highlight their importance in cell death outcome.

Characterization and refinement of zein-based gels

Biopolymer-based gels have a significant impact on controlling rheological properties in a number of industrial processes. Many natural polymers such as animal and plant proteins are used as gelling agents. In particular, zein is a biocompatible protein that embraces almost 80% of the whole protein content of corn. The current study was designed to characterize various gels made up of zein, in order to provide useful formulations for various applications. Diffusing wave spectroscopy in association with dynamic rheology was used to obtain a full physico-chemical characterization of the zein dispersions. Zein gels were prepared using different amounts of protein (10%, 12.5%, 15% and 20% w/v) and their stability was evaluated by Turbiscan Lab®. The microrheological parameters e.g. SLB (solid-liquid balance), EI (elasticity index), MVI (macroscopic viscosity index) and the storage (G′) and loss (G″) moduli were evaluated as a function of strain, frequency, time, and temperature providing information on the linear viscoelastic region, structural assembly, and thermal characteristics. The formulations containing 15% and 20% w/v of zein formed stable dispersions especially at 37 °C. The high protein concentration showed storage moduli values significantly greater than those of viscous moduli, demonstrating that the elastic character became dominant. These findings were confirmed up to 50 °C. The viscosity of these samples decreased as the shear rate increased, thus demonstrating a typical pseudoplastic or shear thinning behavior. Our findings showed that dispersions containing 20% w/v of zein are promising low-cost formulations to be used as a gel in various fields of application.

Study of the SiPM double component recovery time

The recovery time of the Hamamatsu SiPM linear assembly was measured. The assembly contains eight SiPMs of 2.8mm active area diameter; pixel size is 15xl5mkm2. The assembly is used for Phase1 upgrade in hadron calorimeter of the CMS experiment. Recovering process is found to depend on the number of the pixels fired and varied from 7 ±1 ns to 23 ± 3 ns for 50 Ohm readout impedance.

Effect of Dipolar Interactions on the Assembly Process of Iron Oxide Nanoparticles Promoted by the CuAAC “Click” Chemistry Reaction

Nanoparticle assemblies are very attractive because they allow the fine-tuning of magnetic properties by taking advantage of collective behavior ruled by interparticle interactions. Nevertheless, great efforts to control the spatial arrangement of nanoparticles still have to be developed in order to integrate such nanomaterials in devices for applications in fields such as sensors and recording media. Herein, we report on the assembly of iron oxide magnetic nanoparticles promoted by copper-catalyzed alkyne–azide cycloaddition (CuAAC) “click” reaction. Azido-terminated nanoparticles were assembled onto alkyne-terminated gold substrates. The assembly mechanism was investigated by monitoring the density of nanoparticles as a function of their size and concentration. The kinetics of the assembly process is exponential and is favored by the increase of the nanoparticle size and of the concentration. We show that the assembly of nanoparticles is controlled by the random sequential adsorption (RSA) mechanism below a critical size. In contrast, large nanoparticles allow strong dipolar interactions which participate actively in the assembly process, thus they avoid the RSA pathway. These two different mechanism pathways have a significant influence on the spatial arrangement of nanoparticles. The RSA results in rather isolated small nanoparticles at the earliest reaction times which become gradually a discontinuous monolayer. In contrast, the dipolar interaction assisted RSA mechanism (DIA-RSA) favors linear or 2D small assemblies which quickly grow in tight-packed monolayers of nanoparticles. Finally, the magnetic collective properties of these assemblies are markedly favored by the increase of the nanoparticle size.

Diverse Nanoassemblies of Graphene Quantum Dots and Their Mineralogical Counterparts

AbstractComplex structures from nanoparticles are found in rocks, soils, and sea sediments but the mechanisms of their formation are poorly understood, which causes controversial conclusions about their genesis. Here we show that graphene quantum dots (GQDs) can assemble into complex structures driven by coordination interactions with metal ions commonly present in environment and serve a special role in Earth's history, such as Fe3+ and Al3+. GQDs self‐assemble into mesoscale chains, sheets, supraparticles, nanoshells, and nanostars. Specific assembly patterns are determined by the effective symmetry of the GQDs when forming the coordination assemblies with the metal ions. As such, maximization of the electronic delocalization of π‐orbitals of GQDs with Fe3+ leads to GQD‐Fe‐GQD units with D2 symmetry, dipolar bonding potential, and linear assemblies. Taking advantage of high electron microscopy contrast of carbonaceous nanostructures in respect to ceramic background, the mineralogical counterparts of GQD assemblies are found in mineraloid shungite. These findings provide insight into nanoparticle dynamics during the rock formation that can lead to mineralized structures of unexpectedly high complexity.

Diverse Nanoassemblies of Graphene Quantum Dots and Their Mineralogical Counterparts.

Complex structures from nanoparticles are found in rocks, soils, and sea sediments but the mechanisms of their formation are poorly understood, which causes controversial conclusions about their genesis. Here we show that graphene quantum dots (GQDs) can assemble into complex structures driven by coordination interactions with metal ions commonly present in environment and serve a special role in Earth's history, such as Fe3+ and Al3+ . GQDs self-assemble into mesoscale chains, sheets, supraparticles, nanoshells, and nanostars. Specific assembly patterns are determined by the effective symmetry of the GQDs when forming the coordination assemblies with the metal ions. As such, maximization of the electronic delocalization of π-orbitals of GQDs with Fe3+ leads to GQD-Fe-GQD units with D2 symmetry, dipolar bonding potential, and linear assemblies. Taking advantage of high electron microscopy contrast of carbonaceous nanostructures in respect to ceramic background, the mineralogical counterparts of GQD assemblies are found in mineraloid shungite. These findings provide insight into nanoparticle dynamics during the rock formation that can lead to mineralized structures of unexpectedly high complexity.

Coordinated regulation of scaffold opening and enzymatic activity during CARD11 signaling

The activation of key signaling pathways downstream of antigen receptor engagement is critically required for normal lymphocyte activation during the adaptive immune response. CARD11 is a multidomain signaling scaffold protein required for antigen receptor signaling to NF-κB, c-Jun N-terminal kinase, and mTOR. Germline mutations in the CARD11 gene result in at least four types of primary immunodeficiency, and somatic CARD11 gain-of-function mutations drive constitutive NF-κB activity in diffuse large B cell lymphoma and other lymphoid cancers. In response to antigen receptor triggering, CARD11 transitions from a closed, inactive state to an open, active scaffold that recruits multiple signaling partners into a complex to relay downstream signaling. However, how this signal-induced CARD11 conversion occurs remains poorly understood. Here we investigate the role of Inducible Element 1 (IE1), a short regulatory element in the CARD11 Inhibitory Domain, in the CARD11 signaling cycle. We find that IE1 controls the signal-dependent Opening Step that makes CARD11 accessible to the binding of cofactors, including Bcl10, MALT1, and the HOIP catalytic subunit of the linear ubiquitin chain assembly complex. Surprisingly, we find that IE1 is also required at an independent step for the maximal activation of HOIP and MALT1 enzymatic activity after cofactor recruitment to CARD11. This role of IE1 reveals that there is an Enzymatic Activation Step in the CARD11 signaling cycle that is distinct from the Cofactor Association Step. Our results indicate that CARD11 has evolved to actively coordinate scaffold opening and the induction of enzymatic activity among recruited cofactors during antigen receptor signaling.

Regulation of the endosomal SNX27-retromer by OTULIN

OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ-binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association, OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the VPS26A-retromer subunit and endosome-to-plasma membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of SNX27-retromer assembly and recycling to the cell surface.

Dichroic Plasmon Superstructures of Au Nanorods over Macroscopic Areas via Directed Self‐Assemblies of Diblock Copolymers

AbstractThe ability to organize metal nanoparticles over the macroscopic length scale has fundamental implications for advanced plasmonic applications. In particular, linear assemblies of anisotropic nanorods (NRs) may represent a dichroic superstructure, in which plasmonic extinction is inherently dependent on the direction of light polarization. As plasmonic properties are interconnected with optics, electronics, catalysis, and sensing, dichroism in plasmonic systems can stimulate interesting opportunities in many fields of chemistry. However, the preparation of dichroic structures with a single orientation at the centimeter scale remains a significant challenge. Herein, directed self‐assemblies of polystyrene‐block‐poly(methyl methacrylate) (PS‐b‐PMMA) diblock copolymers on lithographical patterns are applied to produce a linear pattern of Au NRs. Periodic nanogrooves from plasma‐etched PS‐b‐PMMA nanodomains facilitate the dense packing of Au NRs into end‐to‐end alignment, which entail strong plasmonic coupling among longitudinal plasmonic modes. Because the resulting NR assemblies have uniform orientation, their dichroic functions can be examined by conventional UV–vis spectroscopy without the aid of single‐particle measurements. Interestingly, both transverse and longitudinal localized surface plasmonic resonance peaks can be turned on and off under two polarized lights in orthogonal directions. Moreover, the resulting superstructures can be transferred onto various substrates, allowing the implantation of dichroic functions for numerous plasmonic applications.

Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP

The ubiquitylation of NF-κB essential modulator (NEMO) is part of the intracellular immune signalling pathway. Monoubiquitylated NEMO is required for exploring the mechanism of NEMO linear ubiquitylation by LUBAC (linear ubiquitin chain assembly complex), but is not accessible by biological techniques. Here we perform the chemical ubiquitylation of NEMO using a ligation auxiliary, which only requires a two-step synthesis, and is easily installed onto the lysine side-chain. Chemical ligation occurs directly on the lysine ε amine and remains efficient below pH 7. We show that ubiquitylated NEMO has similar affinity to linear di-ubiquitin chains as unmodified NEMO. The proximal ubiquitin of chemically synthesised NEMOCoZi-Ub is accepted as a substrate for linear extension by the (RING-Between-RING) RBR domain of HOIL-1-interacting protein (HOIP) alone. Our results indicate that NEMO linear ubiquitylation consists of two-steps, an initial priming event and a separate extension step requiring different LUBAC components.

Functional flow cytometry of monocytes for routine diagnosis of innate primary immunodeficiencies

Functional flow cytometry of monocytes for routine diagnosis of innate primary immunodeficiencies

Defect‐Driven Magnetization Configuration of Isolated Linear Assemblies of Iron Oxide Nanoparticles

AbstractThe magnetization state of 1D magnetic nanoparticle (NP) chains plays a key role in a wide range of applications ranging from diagnosis and therapy in medicine to actuators, sensors, and quantum recording media. The interplay between the exact particle orientation and the magnetic anisotropy is in turn crucial for controlling the overall magnetization state with high precision. Here, a 3D description of the magnetic structure of one‐NP‐wide chains is reported. Here, two complementary experimental techniques are combined, magnetic force microscopy (MFM) and electronic holography (EH) which are sensitive to out‐of‐plane and in‐plane magnetization components, respectively. The approach to micromagnetic simulations is extended, which provides results in good agreement with MFM and EH. The findings are at variance with the known results on unidirectional NP assemblies, and show that magnetization is rarely strictly collinear to the chain axis. The magnetic structure of one‐NP‐wide chains can be interpreted as head‐to‐head magnetic domain structures with off‐axis magnetization components, which is very sensitive to morphological defects in the chain structure such as minute size variation of NPs, tiny misalignment of NPs, and/or crystal orientation with respect to easy magnetization axis.

Non-proteolytic ubiquitination of OTULIN regulates NF-κB signaling pathway.

NF-κB signaling regulates diverse processes such as cell death, inflammation, immunity, and cancer. The activity of NF-κB is controlled by methionine 1-linked linear polyubiquitin, which is assembled by the linear ubiquitin chain assembly complex (LUBAC) and the ubiquitin-conjugating enzyme UBE2L3. Recent studies found that the deubiquitinase OTULIN breaks the linear ubiquitin chain, thus inhibiting NF-κB signaling. Despite the essential role of OTULIN in NF-κB signaling has been established, the regulatory mechanism for OTULIN is not well elucidated. To discover the potential regulators of OTULIN, we analyzed the OTULIN protein complex by proteomics and revealed several OTULIN-binding proteins, including LUBAC and tripartite motif-containing protein 32 (TRIM32). TRIM32 is known to activate NF-κB signaling, but the mechanism is not clear. Genetic complement experiments found that TRIM32 is upstream of OTULIN and TRIM32-mediated NF-κB activation is dependent on OTULIN. Mutagenesis of the E3 ligase domain showed that the E3 ligase activity is essential for TRIM32-mediated NF-κB activation. Further experiments found that TRIM32 conjugates polyubiquitin onto OTULIN and the polyubiquitin blocks the interaction between HOIP and OTULIN, thereby activating NF-κB signaling. Taken together, we report a novel regulatory mechanism by which TRIM32-mediated non-proteolytic ubiquitination of OTULIN impedes the access of OTULIN to the LUBAC and promotes NF-κB activation.