PKM2 functions as a histidine kinase to phosphorylate PGAM1 and increase glycolysis shunts in cancer.

Phosphoglycerate mutase 1 (PGAM1) is a key node enzyme that diverts the metabolic reactions from glycolysis into its shunts to support macromolecule biosynthesis for rapid and sustainable cell proliferation. It is prevalent that PGAM1 activity is upregulated in various tumors; however, the underlying mechanism remains unclear. Here, we unveil that pyruvate kinase M2 (PKM2) moonlights as a histidine kinase in a phosphoenolpyruvate (PEP)-dependent manner to catalyze PGAM1 H11 phosphorylation, that is essential for PGAM1 activity. Moreover, monomeric and dimeric but not tetrameric PKM2 are efficient to phosphorylate and activate PGAM1. In response to epidermal growth factor signaling, Src-catalyzed PGAM1 Y119 phosphorylation is a prerequisite for PKM2 binding and the subsequent PGAM1 H11 phosphorylation, which constitutes a discrepancy between tumor and normal cells. A PGAM1-derived pY119-containing cell-permeable peptide or Y119 mutation disrupts the interaction of PGAM1 with PKM2 and PGAM1 H11 phosphorylation, dampening the glycolysis shunts and tumor growth. Together, these results identify a function of PKM2 as a histidine kinase, and illustrate the importance of enzyme crosstalk as a regulatory mode during metabolic reprogramming and tumorigenesis.

Rapid Continuous 3D Printing via Orthogonal Dual-Color Photoinitiation and Photoinhibition.

Stereolithographic additive manufacturing technology has developed from point-by-point scanning exposure to layer-by-layer masking curing and even volumetric printing. Rapid prototyping is one of the important goals pursued by researchers. A continuous three-dimensional (3D) printing system based on the dual-color photoinitiation and photoinhibition is proposed with the aim of further improving printing speed. The process of continuous 3D printing is realized through the anti-polymerization layer between the cured part and the window generated by the ultraviolet (UV) light sheet (355 nm), and dynamic masking with the blue light (470 nm). The volume of the anti-polymerization layer can be adjusted by the intensity ratio of the incident lights (IUV, 0/Iblue,0) and the size of UV laser spot to enhance the reflow filling rate of the liquid resin. For the orthogonal Gaussian anti-polymerization layer, an intensity ratio of 28.6 allows for an inhibition volume of 97.1% of the desired rectangular anti-polymerization zone with a height of 1 mm. The simulation analysis of continuous 3D printing process by flow-structure interaction reveals that the increase of the thickness of the anti-polymerization layer effectively improves the filling rate of the resin and the cross-sectional area of printing, and reduces the stress of the cured part. The experiments with two different 3D structures printing demonstrate that the filling rate and the stress have virtually no effect on the printing process at a large-scale thickness of the anti-polymerization layer, and the printing speed is capable of reaching 200 µm/s. Certainly, the printing volume and complexity can be further improved with the improvement of the system and the optimization of the resin.

M2-Type Macrophages and Cancer-Associated Fibroblasts Combine to Promote Colorectal Cancer Liver Metastases.

Purpose: This research explored the association between CD163-labeled M2-type macrophages and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) of 38 colorectal cancer (CRC) liver metastases. In addition, we investigated the correlation differences between M2-type macrophages and CAFs in the tumor microenvironments of 38 primary colorectal cancer patients with confirmed liver metastases and 946 colorectal cancer patients, as well as possible mechanisms of action between the two cells. Methods: The Immunohistochemistry (IHC) method was applied to detect the expression levels of M2-type macrophages and CAFs in the tissues of 984 cases of CRC and to analyze the correlation between M2-type macrophages and CAFs in colorectal cancer tissues. The IHC method was also applied to detect the expression levels of M2-type macrophages and CAFs in the liver metastases of 38 cases of CRC in the experimental group and to analyze the correlation between the two cells in liver metastases. Results: 1. M2-type macrophages and CAFs expression were significantly higher in 38 primary colorectal cancer patients compared to 946 controls, and the expression of M2-type macrophages was significantly positively correlated with CAFs. 2. In 984 CRC cases, M2-type macrophages and CAFs expression levels were significantly higher in the cancer tissues than in the paired paracancerous tissues. 3. The expression levels of M2-type macrophages and CAFs in primary colorectal cancer were significantly higher in the experimental group than in colorectal cancer tissues without distant metastasis. Conclusion: M2-type macrophages and CAFs are involved in the development of the colorectal cancer tumor microenvironment, and their interaction influences the initiation and progression of liver metastasis in colorectal cancer. It may provide new clinical ideas for early diagnosis of CRC liver metastases and searching for immune targets.

Multi-wavelength confocal displacement sensing using a highly dispersive flat-field concave grating.

A multi-wavelength confocal displacement sensor based on a flat-field concave grating (FFCG) was proposed and designed; the large dispersion and small volume of the FFCG make it an ideal candidate for replacing the complex dispersive lens group. The designed displacement sensor was calibrated by displacement meter, and the characteristics were measured. Consequently, for the proposed displacement sensor, the displacement range of 6.8 mm was measured with the R-square linearity evaluation coefficient of 0.998, and the sensitivity preceded 17.1 nm/mm. The resolution of the displacement sensor was characterized by 70 µm, as well as a full width at half maximum (FWHM) fluctuating around 1.63 nm, indicating high precision and accuracy in displacement measurement. Moreover, the stability and reliability of the sensor were verified within 20 min, with no significant wavelength shifts, and gentle power fluctuations of 557.73 counts at 520 nm and 563.67 counts at 545.05 nm, respectively.

Distance-based generation of a unicursal random path on a non-grid point set for optical polishing.

In modern ultra-precision polishing, sub-aperture technologies are prone to mid-spatial frequency errors due to identical patterns of a path. A random tool path on a regular point set is widely used to suppress mid-spatial frequency errors. In this study, two non-grid uniform point sets, the Fibonacci and the three-directional, were introduced into optical polishing. To solve the time-consuming problem caused by a large amount of distance calculation, a distance-based weighted random (DBWR) algorithm and a linear programming and connecting (LPC) algorithm were presented. The DBWR algorithm reduces the generation time by strengthening the weight of the neighboring points in a specific direction, while the LPC algorithm adjusts the order and distance of points artificially. Then a random stitching method was proposed for the large-scale point set applying to large-sized optical surfaces, which dramatically reduced the generation time. Finally, experiments validated that the algorithms for non-grid sets can be effectively used for optical surface figuring without introducing an apparent mid-spatial frequency.

Target-surface multiplexed quantitative dynamic phase microscopic imaging based on the transport-of-intensity equation.

Microscopic phase digital imaging based on the transport of intensity equation, known as TIE, is widely used in optical measurement and biomedical imaging since it can dispense with the dependence of traditional phase imaging systems on mechanical rotational scanning and interferometry devices. In this work, we provide a single exposure target-surface multiplexed phase reconstruction (SETMPR) structure based on TIE, which is remarkably easy to construct since it directly combines a conventional bright-field inverted microscope with a special image plane transmission structure that is capable of wavefront shaping and amplification. In practice, the SETMPR is able to achieve dynamic, non-interferometric, quantitative refractive index distribution of both static optical samples and dynamic biological samples in only one shot, meaning that the only limitation of measuring frequency is the frame rate. By comparing the measurement results of a microlens array and a grating with a standard instrument, the quantitative measurement capability and accuracy are demonstrated. Subsequently, both in situ static and long-term dynamic quantitative imaging of HT22 cells were performed, while automatic image segmentation was completed by introducing machine learning methods, which verified the application prospect of this work in dynamic observation of cellular in the biomedical field.

H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins.

The transmission and maintenance of genetic information in eukaryotic cells relies on the faithful duplication of the entire genome. In each round of division, excessive replication origins are licensed, with only a fraction activated to give rise to bi-directional replication forks in the context of chromatin. However, it remains elusive how eukaryotic replication origins are selectively activated. Here we demonstrate that O-GlcNAc transferase (OGT) enhances replication initiation by catalyzing H4S47 O-GlcNAcylation. Mutation of H4S47 impairs DBF4-dependent protein kinase (DDK) recruitment on chromatin, causing reduced phosphorylation of the replicative helicase mini-chromosome maintenance (MCM) complex and compromised DNA unwinding. Our short nascent-strand sequencing results further confirm the importance of H4S47 O-GlcNAcylation in origin activation. We propose that H4S47 O-GlcNAcylation directs origin activation through facilitating MCM phosphorylation, and this may shed light on the control of replication efficiency by chromatin environment.

Rotation-Assisted Separation Model of Constrained-Surface Stereolithography.

Among a variety of additive manufacturing technologies, constrained-surface image-projection-based stereolithography (SLA) technology has unique advantages in printing precision and commercial maturity. For the constrained-surface SLA process, separating the cured layer from the constrained surface is a crucial step that enables the fabrication of the current layer to accomplish. The separation process limits the accuracy of vertical printing and affects the reliability of fabricating. To reduce the separation force, current existing methods include coating nonsticky film, tilting the tank, sliding the tank, and vibrating the constrained glass. Compared with the above methods, the rotation-assisted separation method presented in this article has the advantages of simple structure and inexpensive equipment. The results of the simulation show that the pulling separation with rotating can reduce the separation force and shorten the separation time effectively. Besides, the timing of rotating is also crucial. A customized rotatable resin tank is used in the commercial liquid crystal display-based three-dimensional printer to reduce the separation force by breaking the vacuum environment between the cured layer and the fluorinated ethylene propylene film in advance. The analyzed results have demonstrated that this method can reduce the maximum separation force and the ultimate separation distance, and the reduction is related to the edge profile of the pattern.

CXC chemokine ligand-10 promotes the accumulation of monocyte-like myeloid-derived suppressor cells by activating p38 MAPK signaling under tumor conditions.

CXC chemokine ligand-10 (CXCL10) is a small (10 kDa) secretory protein in the CXC subfamily of cytokines. CXCL10 has been reported to play an important role in antitumor immunity as a chemotactic factor. Tumor development is always accompanied by the formation of an immunosuppressive tumor microenvironment, and the role of CXCL10 in tumor immunosuppression remains unclear. Here, we reported that CXCL10 expression was significantly upregulated in mice with melanoma, and tumor cells secreted large amounts of CXCL10. Myeloid-derived suppressor cells (MDSCs) are an important part of the immunosuppressive tumor microenvironment. Our results showed that CXCL10 promoted the proliferation of monocyte-like (mo)-MDSCs by activating the p38 MAPK signaling pathway through CXCR3, which led to the abnormal accumulation of mo-MDSCs under tumor conditions. This finding provides a new understanding of the mechanism by which a tumor-induced immunosuppressive microenvironment forms and suggests that CXCL10 could be a potential intervention target for slowing tumor progression.

EGF promotes PKM2 O-GlcNAcylation by stimulating O-GlcNAc transferase phosphorylation at Y976 and their subsequent association.

Epidermal growth factor (EGF) is one of the most well-characterized growth factors and plays a crucial role in cell proliferation and differentiation. Its receptor EGFR has been extensively explored as a therapeutic target against multiple types of cancers, such as lung cancer and glioblastoma. Recent studies have established a connection between deregulated EGF signaling and metabolic reprogramming, especially rewiring in aerobic glycolysis, which is also known as the Warburg effect and recognized as a hallmark in cancer. Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme controlling the final step of glycolysis and serves as a major regulator of the Warburg effect. We previously showed that PKM2 T405/S406 O-GlcNAcylation, a critical mark important for PKM2 detetramerization and activity, was markedly upregulated by EGF. However, the mechanism by which EGF regulates PKM2 O-GlcNAcylation still remains uncharacterized. Here, we demonstrated that EGF promoted O-GlcNAc transferase (OGT) binding to PKM2 by stimulating OGT Y976 phosphorylation. As a consequence, we found PKM2 O-GlcNAcylation and detetramerization were upregulated, leading to a significant decrease in PKM2 activity. Moreover, distinct from PKM2, we observed that the association of additional phosphotyrosine-binding proteins with OGT was also enhanced when Y976 was phosphorylated. These proteins included STAT1, STAT3, STAT5, PKCδ, and p85, which are reported to be O-GlcNAcylated. Together, we show EGF-dependent Y976 phosphorylation is critical for OGT-PKM2 interaction and propose that this posttranslational modification might be important for substrate selection by OGT.

Influence and suppression of periodic contour error of tool path and velocity deviations on surface error.

When using multi-axis machines with a pneumatic tool to polish large-aperture optical surfaces, the paths generated by the computer numerical control system deviate from the desired ones. This causes periodic contour errors and surface ripples. In addition, because of the different machine layouts, the tool end velocity also can change. We introduce a multi-axis machine and analyze the surface error and power spectral density (PSD) of three commonly used paths (raster, spiral, and random path) in terms of the contour error using different position interpolation methods. A cubic polynomial is introduced to smooth the axis motion, and a velocity compensation method is considered to diminish the velocity deviation from the machine layout. The results show that the circular interpolation method exhibits a balanced performance in terms of both the contour error and the PSD for various paths. In addition, the optimization can be performed before G-code generation without affecting the characteristics of the original optimization system.

ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation.

Inducible regulatory T (iTreg) cells play a central role in immune suppression. As iTreg cells are differentiated from activated T (Th0) cells, cell metabolism undergoes dramatic changes, including a shift from fatty acid synthesis (FAS) to fatty acid oxidation (FAO). Although the reprogramming in fatty acid metabolism is critical, the mechanism regulating this process during iTreg differentiation is still unclear. Here we have revealed that the enzymatic activity of ATP-citrate lyase (ACLY) declined significantly during iTreg differentiation upon transforming growth factor ß1 (TGFß1) stimulation. This reduction was due to CUL3-KLHL25-mediated ACLY ubiquitination and degradation. As a consequence, malonyl-CoA, a metabolic intermediate in FAS that is capable of inhibiting the rate-limiting enzyme in FAO, carnitine palmitoyltransferase 1 (CPT1), was decreased. Therefore, ACLY ubiquitination and degradation facilitate FAO and thereby iTreg differentiation. Together, we suggest TGFß1-CUL3-KLHL25-ACLY axis as an important means regulating iTreg differentiation and bring insights into the maintenance of immune homeostasis for the prevention of immune diseases.

Alginate-Based Amphiphilic Block Copolymers as a Drug Codelivery Platform.

Structured nanoassemblies are biomimetic structures that are enabling applications from nanomedicine to catalysis. One approach to achieve these spatially organized architectures is utilizing amphiphilic diblock copolymers with one or two macromolecular backbones that self-assemble in solution. To date, the impact of alternating backbone architectures on self-assembly and drug delivery is still an area of active research limited by the strategies used to synthesize these multiblock polymers. Here, we report self-assembling ABC-type alginate-based triblock copolymers with the backbones of three distinct biomaterials utilizing a facile conjugation approach. This "polymer mosaic" was synthesized by the covalent attachment of alginate with a PLA/PEG diblock copolymer. The combination of alginate, PEG, and PLA domains resulted in an amphiphilic copolymer that self-assembles into nanoparticles with a unique morphology of alginate domain compartmentalization. These particles serve as a versatile platform for co-encapsulation of hydrophilic and hydrophobic small molecules, their spatiotemporal release, and show potential as a drug delivery system for combination therapy.

Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation.

Inducible regulatory T (iTreg) cells play a crucial role in immune suppression and are important for the maintenance of immune homeostasis. Mounting evidence has demonstrated connections between iTreg differentiation and metabolic reprogramming, especially rewiring in fatty acid oxidation (FAO). Previous work showed that butyrate, a specific type of short-chain fatty acid (SCFA) readily produced from fiber-rich diets through microbial fermentation, was critical for the maintenance of intestinal homeostasis and capable of promoting iTreg generation by up-regulating histone acetylation for gene expression as an HDAC inhibitor. Here, we revealed that butyrate could also accelerate FAO to facilitate iTreg differentiation. Moreover, butyrate was converted, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote FAO and thereby iTreg differentiation. Mutation of CPT1A at Arg243, a reported amino acid required for MCoA association, impaired both MCoA and BCoA binding, indicating that Arg243 is probably the responsible site for MCoA and BCoA association. Furthermore, blocking BCoA formation by ACSS2 inhibitor compromised butyrate-mediated iTreg generation and mitigation of mouse colitis. Together, we unveil a previously unappreciated role for butyrate in iTreg differentiation and illustrate butyrate-BCoA-CPT1A axis for the regulation of immune homeostasis.

Hydrogel-Embedded Quantum Dot-Transcription Factor Sensors for Quantitative Progesterone Detection.

Immobilization of biosensors in or on a functional material is critical for subsequent device development and translation to wearable technology. Here, we present the development and assessment of an immobilized quantum dot-transcription factor-nucleic acid complex for progesterone detection as a first step toward such device integration. The sensor, composed of a polyhistidine-tagged transcription factor linked to a quantum dot and a fluorophore-modified cognate DNA, is embedded within a hydrogel as an immobilization matrix. The hydrogel is optically transparent, soft, and flexible as well as traps the quantum dot-transcription factor DNA assembly but allows free passage of the analyte, progesterone. Upon progesterone exposure, DNA dissociates from the quantum dot-transcription factor DNA assembly resulting in an attenuated ratiometric fluorescence output via Förster resonance energy transfer. The sensor performs in a dose-dependent manner with a limit of detection of 55 nM. Repeated analyte measurements are similarly successful. Our approach combines a systematically characterized hydrogel as an immobilization matrix and a transcription factor-DNA assembly as a recognition/transduction element, offering a promising framework for future biosensor devices.

Pyruvate Kinase M2 Promotes the Activation of Dendritic Cells by Enhancing IL-12p35 Expression.

Dendritic cells (DCs) play a central role in both innate and adaptive immunity. Emerging evidence has demonstrated metabolic reprogramming during DC activation. However, how DC activation is linked with metabolic reprogramming remains unclear. Here we show that pyruvate kinase M2 (PKM2), the rate-limiting enzyme in the last step of glycolysis, is critical for LPS-induced DC activation. Upon DC activation, JNK signaling stimulated p300 association with PKM2 for the acetylation of lysine 433, a classic posttranslational modification critical for PKM2 destabilization and nuclear re-localization. Subsequently, nuclear PKM2 partnered with c-Rel to enhance Il12p35 expression, which is important for Th1 cell differentiation. Meanwhile, decreased enzymatic activity of PKM2 due to detetramerization facilitated glycolysis and fatty acid synthesis, helping DCs meet their need for biomacromolecules. Together, we provide evidence for metabolic control of DC activation and offer insights into aberrant immune responses due to dysregulated Th1 functions.

Three-degree-of-freedom autocollimator based on a combined target reflector.

As an angle measuring instrument, the traditional autocollimator has the ability to measure the two-degree-of-freedom angles, namely, pitch and yaw, but fails to measure the roll angle. In this study, we propose a novel autocollimator that can simultaneously measure the three-degree-of-freedom (3-DOF) angles. As a key component, a combined target reflector (CTR) is meticulously designed to split the collimated laser beam into two beams. The 3-DOF angle measurement is achieved by sensing the displacements of the two beam spots reflected from the CTR. The measurement principle and simulation analysis are presented in detail. Experiments are conducted to assess the performance of the proposed autocollimator, and the results indicate that it has an accuracy of better than 0.74 arcsec over a range of ${ \pm 200}\,\,{\rm arcsec}$±200arcsec, and it can be used for 3-DOF angular motion error measurement of a precision displacement stage.

RNF8 promotes efficient DSB repair by inhibiting the pro-apoptotic activity of p53 through regulating the function of Tip60.

OBJECTIVES: RING finger protein 8 (RNF8) is an E3 ligase that plays an essential role in DSB repair. p53 is a well-established tumour suppressor and cellular gatekeeper of genome stability. This study aimed at investigating the functional correlations between RNF8 and p53 in DSB damage repair. MATERIALS AND METHODS: In this article, wild-type, knockout and shRNA-depleted HCT116 and U2OS cells were stressed, and the roles of RNF8 and p53 were examined. RT-PCR and Western blot were utilized to investigate the expression of related genes in damaged cells. Cell proliferation, apoptosis and neutral cell comet assays were applied to determine the effects of DSB damage on differently treated cells. DR-GFP, EJ5-GFP and LacI-LacO targeting systems, flow cytometry, mass spectrometry, IP, IF, GST pull-down assay were used to explore the molecular mechanism of RNF8 and p53 in DSB damage repair. RESULTS: We found that RNF8 knockdown increased cellular sensitivity to DSB damage and decreased cell proliferation, which was correlated with high expression of the p53 gene. RNF8 improved the efficiency of DSB repair by inhibiting the pro-apoptotic function of p53. We also found that RNF8 restrains cell apoptosis by inhibiting over-activation of ATM and subsequently reducing p53 acetylation at K120 through regulating Tip60. CONCLUSIONS: Taken together, these findings suggested that RNF8 promotes efficient DSB repair by inhibiting the pro-apoptotic activity of p53 through regulating the function of Tip60.

Interactive Manipulation of Nonconductive Microparticles in Scanning Electron Microscope by a Virtual Nano-hand Strategy.

Micro/nano-manipulation is the fabrication of particular constructs on devices at the micro/nano-scale. Precise manipulation of microparticles is one of the key technological difficulties in manufacturing micro/nano-scale components. Based on scanning electron microscopy and nanomanipulator, this paper adopts a direct push method to operate randomly distributed microparticles into ordered structures. A two-probe interaction strategy is proposed to enable microparticle movements in all directions efficiently and avoid scratching the substrate surface. To overcome the uncertainties in micromanipulation, a virtual nano-hand strategy was also implemented: long-range advance of each microparticle is realized by multiple single-step pushes, whose trajectory is theoretically analyzed. The pushes are well programmed to imitate effects of a more powerful and determined hand. Experimental results show that the theoretical single-step motion trajectory is in line with actual operation, and the proposed strategy can ensure precise operation of the microparticles in all directions and improve reliability and effectiveness of operation.

Design of a free-form diffuser for the entrance optic to correct the cosine error in the photometer.

The entrance optic is an important part of the photometer head. It can match the directional response of the photometer to the cosine function. In this paper, an entrance optic consisting of a free-form diffuser, a shadow ring, and an integrating cavity is introduced. An iterative optimization algorithm is presented to design a free-form diffuser that exhibits better cosine response characteristics. Diffusers of different materials and sizes are designed in a simulation experiment. After a finite number of iterations, in the absence of the shadow ring, the integral cosine error of the free-form diffuser is less than 1%. The directional cosine error is less than 3% for incidence angles between 0° and 70°. After adding a shadow ring to correct the directional response of the incident angle greater than 80°, for incident angles between 0° and 85°, the cosine errors are typically less than 3%, except that the cosine errors of very few large incident angles are close to 5%. The experimental results show the effectiveness and robustness of the proposed method. In addition, the influence of an important percentage constant σ on iterative optimization is studied. It is found that the larger the parameter σ, the fewer the number of iterations, and the directional cosine error may be slightly larger but still acceptable. The wide range of values of σ further embodies the versatility and flexibility of the proposed method.