Feed-forward stimulation of CAMK2 by the oncogenic pseudokinase PEAK1 generates a therapeutically 'actionable' signalling axis in triple negative breast cancer.

The PEAK family of pseudokinases, comprising PEAK1-3, are signalling scaffolds that play oncogenic roles in several poor prognosis human cancers, including triple negative breast cancer (TNBC). However, therapeutic targeting of pseudokinases is challenging due to their lack of catalytic activity. To address this, we screened for PEAK1 effectors by affinity purification and mass spectrometry, identifying calcium/calmodulin-dependent protein kinase 2 (CAMK2)D and CAMK2G. PEAK1 promoted CAMK2D/G activation in TNBC cells via a novel feed-forward mechanism involving PEAK1/PLCγ1/Ca 2+ signalling and direct binding via a consensus CAMK2 interaction motif in the PEAK1 N-terminus. In turn, CAMK2 phosphorylated PEAK1 to enhance association with PEAK2, which is critical for PEAK1 oncogenic signalling. To achieve pharmacologic targeting of PEAK1/CAMK2, we repurposed RA306, a second generation CAMK2 inhibitor under pre-clinical development for treatment of cardiovascular disease. RA306 demonstrated on-target activity against CAMK2 in TNBC cells and inhibited PEAK1-enhanced migration and invasion in vitro . Moreover, RA306 significantly attenuated TNBC xenograft growth and blocked metastasis in a manner mirrored by CRISPR-mediated PEAK1 ablation. Overall, these studies establish PEAK1 as a critical cell signalling nexus, identify a novel mechanism for regulation of Ca 2+ signalling and its integration with tyrosine kinase signals, and identify CAMK2 as a therapeutically 'actionable' target downstream of PEAK1.

Microstructural Inhomogeneity in the Fusion Zone of Laser Welds.

This paper investigated evolutions of α-Al sub-grains' morphology and crystalline orientation in the fusion zone during laser welding of 2A12 aluminum alloys. Based on this, a new method for assessing the weldability of materials was proposed. In laser deep-penetration welding, in addition to the conventional columnar and equiaxed dendrites, there also exhibited a corrugated structure with several 'fine-coarse-fine' transformations. In such regions, an abnormal α-Al coarsening phenomenon was encountered, with a more dispersed crystalline orientation arrangement and a decreased maximum pole density value. Particularly, structural alterations appeared more frequently in the weld bottom than the top. The above results indicated that the laser-induced keyhole presented a continually fluctuating state. Under such a condition, the solid-liquid transformation exhibited an unstable solidification front, a fluctuant undercooling, and a variational solidification rate. Meanwhile, the welding quality of this material is in a critical state to generate pores. Therefore, the appearance and relevant number of corrugated regions can be considered as a new way for judging the weldability, which will help to narrow the processing window with better welding stability.

Structural mapping of PEAK pseudokinase interactions identifies 14-3-3 as a molecular switch for PEAK3 signaling.

PEAK pseudokinases regulate cell migration, invasion and proliferation by recruiting key signaling proteins to the cytoskeleton. Despite lacking catalytic activity, alteration in their expression level is associated with several aggressive cancers. Here, we elucidate the molecular details of key PEAK signaling interactions with the adapter proteins CrkII and Grb2 and the scaffold protein 14-3-3. Our findings rationalize why the dimerization of PEAK proteins has a crucial function in signal transduction and provide biophysical and structural data to unravel binding specificity within the PEAK interactome. We identify a conserved high affinity 14-3-3 motif on PEAK3 and demonstrate its role as a molecular switch to regulate CrkII binding and signaling via Grb2. Together, our studies provide a detailed structural snapshot of PEAK interaction networks and further elucidate how PEAK proteins, especially PEAK3, act as dynamic scaffolds that exploit adapter proteins to control signal transduction in cell growth/motility and cancer.

Ablation of porcine subcutaneous fat and porcine aorta tissues by a burst-mode nanosecond-pulsed laser at 355 nm.

High-energy laser pulses used in laser angioplasty are challenging the laser cost, delivery system damage, efficiency, and laser catheter operating time. 355 nm nanosecond-pulsed laser in burst mode has shown potentials in reducing the system complexity and selective ablation of tissues. In this paper, burst mode laser ablation of porcine subcutaneous fat and porcine aorta is investigated. A histopathological analysis demonstrates that porcine subcutaneous fat can be ablated at a rate of greater than 0.2 mm/s when the number of pulses per burst is 1500 (corresponding to a fluence of 0.12 mJ/mm2 per pulse and 180 mJ/mm2 per burst), and the temperature of tissue during lasing is lower than 45°C. The porcine aorta remains nearly unaffected at the same laser parameter, and the tissue temperature during lasing is lower than 35°C. It shows the feasibility of using a burst-mode laser for selective ablation of tissue.

The pseudokinase NRBP1 activates Rac1/Cdc42 via P-Rex1 to drive oncogenic signalling in triple-negative breast cancer.

We have determined that expression of the pseudokinase NRBP1 positively associates with poor prognosis in triple negative breast cancer (TNBC) and is required for efficient migration, invasion and proliferation of TNBC cells in culture as well as growth of TNBC orthotopic xenografts and experimental metastasis. Application of BioID/MS profiling identified P-Rex1, a known guanine nucleotide exchange factor for Rac1, as a NRBP1 binding partner. Importantly, NRBP1 overexpression enhanced levels of GTP-bound Rac1 and Cdc42 in a P-Rex1-dependent manner, while NRBP1 knockdown reduced their activation. In addition, NRBP1 associated with P-Rex1, Rac1 and Cdc42, suggesting a scaffolding function for this pseudokinase. NRBP1-mediated promotion of cell migration and invasion was P-Rex1-dependent, while constitutively-active Rac1 rescued the effect of NRBP1 knockdown on cell proliferation and invasion. Generation of reactive oxygen species via a NRBP1/P-Rex1 pathway was implicated in these oncogenic roles of NRBP1. Overall, these findings define a new function for NRBP1 and a novel oncogenic signalling pathway in TNBC that may be amenable to therapeutic intervention.

Identification of biological pathways and processes regulated by NEK5 in breast epithelial cells via an integrated proteomic approach.

Specific members of the Nima-Related Kinase (NEK) family have been linked to cancer development and progression, and a role for NEK5, one of the least studied members, in breast cancer has recently been proposed. However, while NEK5 is known to regulate centrosome separation and mitotic spindle assembly, NEK5 signalling mechanisms and function in this malignancy require further characterization. To this end, we established a model system featuring overexpression of NEK5 in the immortalized breast epithelial cell line MCF-10A. MCF-10A cells overexpressing NEK5 exhibited an increase in clonogenicity under monolayer conditions and enhanced acinar size and abnormal morphology in 3D Matrigel culture. Interestingly, they also exhibited a marked reduction in Src activation and downstream signalling. To interrogate NEK5 signalling and function in an unbiased manner, we applied a variety of MS-based proteomic approaches. Determination of the NEK5 interactome by Bio-ID identified a variety of protein classes including the kinesins KIF2C and KIF22, the mitochondrial proteins TFAM, TFB2M and MFN2, RhoH effectors and the negative regulator of Src, CSK. Characterization of proteins and phosphosites modulated upon NEK5 overexpression by global MS-based (phospho)proteomic profiling revealed impact on the cell cycle, DNA synthesis and repair, Rho GTPase signalling, the microtubule cytoskeleton and hemidesmosome assembly. Overall, the study indicates that NEK5 impacts diverse pathways and processes in breast epithelial cells, and likely plays a multifaceted role in breast cancer development and progression. Video Abstract.

Ultrafast inactivation of SARS-CoV-2 with 266 nm lasers.

Disinfection eliminates pathogenic microorganisms and ensures a biosafe environment for human beings. The rapid spread of COVID-19 is challenging traditional disinfection methods in terms of reducing harmful side effects and conducting faster processes. Spraying large-scale chemical disinfectants is harmful to individuals and the environment, while UV lamp and light-emitting diode (LED) disinfection still requires a long exposure time due to the low irradiance and highly divergent beam characteristics. Given that a laser maintains a high irradiance over a long distance, we studied the effectiveness of lasers as a new disinfection method, and the results show the capability for ultrafast inactivation of SARS-CoV-2 virus with a 266 nm laser. This work confirms UV lasers as a good candidate for disinfection.

Distinct PEAK3 interactors and outputs expand the signaling potential of the PEAK pseudokinase family.

The pseudokinase scaffolds PEAK1 and PEAK2 are implicated in cancer cell migration and metastasis. We characterized the regulation and role of the third family member PEAK3 in cell signaling. Similar to PEAK1 and PEAK2, PEAK3 formed both homotypic and heterotypic complexes. In addition, like PEAK1, it bound to the adaptors Grb2 and CrkII. However, unlike PEAK1 and PEAK2, homodimerized PEAK3 also interacted with the ARF GTPase-activating protein ASAP1, the E3 ubiquitin ligase Cbl, and the kinase PYK2. Dimerization and subsequent phosphorylation on Tyr24, likely by a Src family kinase, were required for the binding of PEAK3 to Grb2 and ASAP1. Interactions with Grb2, CrkII, ASAP1, Cbl, and PYK2 exhibited contrasting dynamics upon cell stimulation with epidermal growth factor (EGF), in part due to PEAK3 dephosphorylation mediated by the phosphatase PTPN12. Overexpressing PEAK3 in mesenchymal-like MDA-MB-231 breast cancer cells enhanced cell elongation in a manner dependent on PEAK3 dimerization, and manipulation of PEAK3 expression demonstrated a positive role for this scaffold in regulating cell migration. Overexpressing PEAK3 in PEAK1/2 double-knockout MCF-10A breast epithelial cells enhanced acinar growth, impaired basement membrane integrity, and promoted invasion in three-dimensional cultures, with the latter two effects dependent on the binding of PEAK3 to Grb2 and ASAP1. PEAK1 and PEAK2 quantitatively and temporally influenced PEAK3 function. These findings characterize PEAK3 as an integral, signal-diversifying member of the PEAK family with scaffolding roles that promote cell proliferation, migration, and invasion.

Deep Learning Based Monitoring of Spatter Behavior by the Acoustic Signal in Selective Laser Melting.

As one of the most promising metal additive manufacturing (AM) technologies, the selective laser melting (SLM) process has high expectations ofr its use in aerospace, medical, and other fields. However, various defects such as spatter, crack, and porosity seriously hinder the applications of the SLM process. In situ monitoring is a vital technique to detect the defects in advance, which is expected to reduce the defects. This work proposed a method that combined acoustic signals with a deep learning algorithm to monitor the spatter behaviors. The acoustic signals were recorded by a microphone and the spatter information was collected by a coaxial high-speed camera simultaneously. The signals were divided into two types according to the number and intensity of spatter during the SLM process with different combinations of processing parameters. Deep learning models, one-dimensional Convolutional Neural Network (1D-CNN), two-dimensional Convolutional Neural Network (2D-CNN), Recurrent Neural Network (RNN), Long Short Term Memory (LSTM), and Gated Recurrent Unit (GRU) were trained to establish the relationships between the acoustic signals and characteristics of spatter. After K-fold verification, the highest classification confidence of models is 85.08%. This work demonstrates that it is feasible to use acoustic signals in monitoring the spatter defect during the SLM process. It is possible to use cheap and simple microphones instead of expensive and complicated high-speed cameras for monitoring spatter behaviors.

Impact of the heat load on the laser performance of chirally-coupled-core fibers.

As the heat load within the central core of chirally-coupled-core (CCC) fibers will change the pre-designed refractive index profile through the thermo-optic effect, its impact on the laser performance of CCC fibers is investigated. Analysis and simulation results on two typical CCC fibers show that the effects of the heat load include the modal loss reduction and the transmission spectrum drift. The former comes from the thermal lensing effect in the central core, and the latter is caused by the change in the refractive index difference between the central core and the side core. Considering the non-uniform axial heat distribution in the actual laser operation, the overall laser performance of CCC fibers with different pump power is simulated. It is found that, because of the high pre-designed high-order mode loss, the single-mode operation of CCC fibers will be maintained but the slope efficiency may reduce dramatically if the fundamental mode loss is strongly dependent on the heat load.

Mode instabilities in Yb:YAG crystalline fiber amplifiers.

Mode instabilities (MI) threshold in the Yb:YAG crystalline fiber amplifier is simulated by a full numerical model. The propagation of signal fields is simulated by the finite-difference beam-propagation method combined with the rate equations, and the time-dependent heat equation is solved by the alternating-direction-implicit method. Considering the strong temperature-dependent laser performance of Yb:YAG, an iterative method is applied to reach the steady state of Yb:YAG, the crystalline fiber amplifier, before the simulation of MI behavior. The simulated MI thresholds in Yb:YAG crystalline fiber amplifiers are found to be at least 28 times of those in Yb-doped silica-glass fiber amplifiers, up to tens of kilowatts. Simulation results show that, in addition to the expected higher thermal conductivity and lower thermo-optic coefficient, strong gain saturation also plays an important role in the high MI threshold of the Yb:YAG crystalline fiber.

Evaluation of Cyclic Peptide Inhibitors of the Grb7 Breast Cancer Target: Small Change in Cargo Results in Large Change in Cellular Activity.

Grb7 is an adapter protein, overexpressed in HER2+ve breast and other cancers, and identified as a therapeutic target. Grb7 promotes both proliferative and migratory cellular pathways through interaction of its SH2 domain with upstream binding partners including HER2, SHC, and FAK. Here we present the evaluation of a series of monocyclic and bicyclic peptide inhibitors that have been developed to specifically and potently target the Grb7 SH2-domain. All peptides tested were found to inhibit signaling in both ERK and AKT pathways in SKBR-3 and MDA-MB-231 cell lines. Proliferation, migration, and invasion assays revealed, however, that the second-generation bicyclic peptides were not more bioactive than the first generation G7-18NATE peptide, despite their higher in vitro affinity for the target. This was found not to be due to steric hindrance by the cell-permeability tag, as ascertained by ITC, but to differences in the ability of the bicyclic peptides to interact with and penetrate cellular membranes, as determined using SPR and mass spectrometry. These studies reveal that just small differences to amino acid composition can greatly impact the effectiveness of peptide inhibitors to their intracellular target and demonstrate that G7-18NATE remains the most effective peptide inhibitor of Grb7 developed to date.

Proteomic Profiling of Human Prostate Cancer-associated Fibroblasts (CAF) Reveals LOXL2-dependent Regulation of the Tumor Microenvironment.

In prostate cancer, cancer-associated fibroblasts (CAF) exhibit contrasting biological properties to non-malignant prostate fibroblasts (NPF) and promote tumorigenesis. Resolving intercellular signaling pathways between CAF and prostate tumor epithelium may offer novel opportunities for research translation. To this end, the proteome and phosphoproteome of four pairs of patient-matched CAF and NPF were characterized to identify discriminating proteomic signatures. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a hyper reaction monitoring data-independent acquisition (HRM-DIA) workflow. Proteins that exhibited a significant increase in CAF versus NPF were enriched for the functional categories "cell adhesion" and the "extracellular matrix." The CAF phosphoproteome exhibited enhanced phosphorylation of proteins associated with the "spliceosome" and "actin binding." STRING analysis of the CAF proteome revealed a prominent interaction hub associated with collagen synthesis, modification, and signaling. It contained multiple collagens, including the fibrillar types COL1A1/2 and COL5A1; the receptor tyrosine kinase discoidin domain-containing receptor 2 (DDR2), a receptor for fibrillar collagens; and lysyl oxidase-like 2 (LOXL2), an enzyme that promotes collagen crosslinking. Increased activity and/or expression of LOXL2 and DDR2 in CAF were confirmed by enzymatic assays and Western blotting analyses. Pharmacological inhibition of CAF-derived LOXL2 perturbed extracellular matrix (ECM) organization and decreased CAF migration in a wound healing assay. Further, it significantly impaired the motility of co-cultured RWPE-2 prostate tumor epithelial cells. These results indicate that CAF-derived LOXL2 is an important mediator of intercellular communication within the prostate tumor microenvironment and is a potential therapeutic target.

Characterization of the Src-regulated kinome identifies SGK1 as a key mediator of Src-induced transformation.

Despite significant progress, our understanding of how specific oncogenes transform cells is still limited and likely underestimates the complexity of downstream signalling events. To address this gap, we use mass spectrometry-based chemical proteomics to characterize the global impact of an oncogene on the expressed kinome, and then functionally annotate the regulated kinases. As an example, we identify 63 protein kinases exhibiting altered expression and/or phosphorylation in Src-transformed mammary epithelial cells. An integrated siRNA screen identifies nine kinases, including SGK1, as being essential for Src-induced transformation. Accordingly, we find that Src positively regulates SGK1 expression in triple negative breast cancer cells, which exhibit a prominent signalling network governed by Src family kinases. Furthermore, combined inhibition of Src and SGK1 reduces colony formation and xenograft growth more effectively than either treatment alone. Therefore, this approach not only provides mechanistic insights into oncogenic transformation but also aids the design of improved therapeutic strategies.

All-in-fiber method of generating orbital angular momentum with helically symmetric fibers.

An all-in-fiber method of generating orbital angular momentum (OAM) is proposed. A simple device composed with a section of helically symmetric fiber and another section of regular fiber is designed to convert input light to optical vortices. Finite element method calculation of first- and second-order OAM generation based on the coordinates transformation technique is taken to show that the eigenmodes of the helically symmetric fiber structures carry orbital and spin angular momentum. Simulation using the self-developed beam propagation method algorithm is also performed to verify the orbital angular momentum generation and evaluate the performance of the OAM generator.

All-in-fiber method of generating orbital angular momentum with helically symmetric fibers: publisher's note.

This publisher's note amends a figure caption in Appl. Opt.57, 8182 (2018)APOPAI0003-693510.1364/AO.57.008182.

High dietary fat and sucrose results in an extensive and time-dependent deterioration in health of multiple physiological systems in mice.

Obesity is associated with metabolic dysfunction, including insulin resistance and hyperinsulinemia, and with disorders such as cardiovascular disease, osteoporosis, and neurodegeneration. Typically, these pathologies are examined in discrete model systems and with limited temporal resolution, and whether these disorders co-occur is therefore unclear. To address this question, here we examined multiple physiological systems in male C57BL/6J mice following prolonged exposure to a high-fat/high-sucrose diet (HFHSD). HFHSD-fed mice rapidly exhibited metabolic alterations, including obesity, hyperleptinemia, physical inactivity, glucose intolerance, peripheral insulin resistance, fasting hyperglycemia, ectopic lipid deposition, and bone deterioration. Prolonged exposure to HFHSD resulted in morbid obesity, ectopic triglyceride deposition in liver and muscle, extensive bone loss, sarcopenia, hyperinsulinemia, and impaired short-term memory. Although many of these defects are typically associated with aging, HFHSD did not alter telomere length in white blood cells, indicating that this diet did not generally promote all aspects of aging. Strikingly, glucose homeostasis was highly dynamic. Glucose intolerance was evident in HFHSD-fed mice after 1 week and was maintained for 24 weeks. Beyond 24 weeks, however, glucose tolerance improved in HFHSD-fed mice, and by 60 weeks, it was indistinguishable from that of chow-fed mice. This improvement coincided with adaptive ß-cell hyperplasia and hyperinsulinemia, without changes in insulin sensitivity in muscle or adipose tissue. Assessment of insulin secretion in isolated islets revealed that leptin, which inhibited insulin secretion in the chow-fed mice, potentiated glucose-stimulated insulin secretion in the HFHSD-fed mice after 60 weeks. Overall, the excessive calorie intake was accompanied by deteriorating function of numerous physiological systems.

Finite-difference beam-propagation method for anisotropic waveguides with torsional birefringence.

A new three-dimensional finite-difference (FD)-based beam propagation method (BPM) is proposed for simulating optical propagation in weakly guiding waveguides with torsional birefringence, which cannot be simulated by existing FD-BPM algorithms. We also demonstrate that this new BPM algorithm is capable of obtaining eigenmode solutions in a helically-symmetric z-dependent waveguide structure with torsional birefringence.

Structure of SgK223 pseudokinase reveals novel mechanisms of homotypic and heterotypic association.

The mammalian pseudokinase SgK223, and its structurally related homologue SgK269, are oncogenic scaffolds that nucleate the assembly of specific signalling complexes and regulate tyrosine kinase signalling. Both SgK223 and SgK269 form homo- and hetero-oligomers, a mechanism that underpins a diversity of signalling outputs. However, mechanistic insights into SgK223 and SgK269 homo- and heterotypic association are lacking. Here we present the crystal structure of SgK223 pseudokinase domain and its adjacent N- and C-terminal helices. The structure reveals how the N- and C-regulatory helices engage in a novel fold to mediate the assembly of a high-affinity dimer. In addition, we identified regulatory interfaces on the pseudokinase domain required for the self-assembly of large open-ended oligomers. This study highlights the diversity in how the kinase fold mediates non-catalytic functions and provides mechanistic insights into how the assembly of these two oncogenic scaffolds is achieved in order to regulate signalling output.

Discovery, Development, and Cellular Delivery of Potent and Selective Bicyclic Peptide Inhibitors of Grb7 Cancer Target.

Grb7 is a signaling protein with critical roles in tumor cell proliferation and migration and an established cancer therapeutic target. Here we explore chemical space to develop a new bicyclic peptide inhibitor, incorporating thioether and lactam linkers that binds with affinity (KD = 1.1 µM) and specificity to the Grb7-SH2 domain. Structural analysis of the Grb7-SH2/peptide complex revealed an unexpected binding orientation underlying the binding selectivity by this new scaffold. We further incorporated carboxymethylphenylalanine and carboxyphenylalanine phosphotyrosine mimetics and arrived at an optimized inhibitor that potently binds Grb7-SH2 (KD = 0.13 µM) under physiological conditions. X-ray crystal structures of these Grb7-SH2/peptide complexes reveal the structural basis for the most potent and specific inhibitors of Grb7 developed to date. Finally, we demonstrate that cell permeable versions of these peptides successfully block Grb7 mediated interactions in a breast cancer cell line, establishing the potential of these peptides in the development of novel therapeutics targeted to Grb7.