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.

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.

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.

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.

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.

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.

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.

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.

BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation.

During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1-binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 recruitment, but not MCM2-7 loading, is impaired in BRPF3-depleted cells, identifying a BRPF3-dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3-HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby replication origins.

Abrogation of Lupus Nephritis in Somatic Hypermutation-Deficient MRL/lpr Mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease posing threats to multiple organs in the human body. As a typical manifestation of SLE, lupus nephritis is characterized by a series of pathological changes in glomerulus as well as accumulation of pathogenic autoreactive IgG with complement in the kidney that dramatically disrupts renal functions. Activation-induced deaminase (AID), which governs both somatic hypermutation (SHM) and class-switch recombination (CSR), has been shown to be essential for the regulation of SLE. However, the relative contributions of SHM and CSR to SLE pathology have not been determined. Based on the available AIDG23S mice, we successfully established an AIDG23S MRL/lpr mouse model, in which SHM is specifically abolished, although CSR is largely unaffected. We found that the abrogation of SHM effectively alleviated SLE-associated histopathological alterations, such as expansion of the mesangial matrix and thickening of the basement membrane of Bowman's capsule as well as infiltration of inflammatory cells. Compared with SLE mice, AIDG23S MRL/lpr mice exhibited decreased proteinuria, blood urea nitrogen, and creatinine, indicating that the loss of SHM contributed to the recovery of renal functions. As a consequence, the life span of those SHM-deficient MRL/lpr mice was extended. Together, we provide direct evidence pinpointing a vital role of SHM in the control of SLE development.

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.

O-GlcNAcylation destabilizes the active tetrameric PKM2 to promote the Warburg effect.

The Warburg effect, characterized by increased glucose uptake and lactate production, is a well-known universal across cancer cells and other proliferating cells. PKM2, a splice isoform of the pyruvate kinase (PK) specifically expressed in these cells, serves as a major regulator of this metabolic reprogramming with an adjustable activity subjected to numerous allosteric effectors and posttranslational modifications. Here, we have identified a posttranslational modification on PKM2, O-GlcNAcylation, which specifically targets Thr405 and Ser406, residues of the region encoded by the alternatively spliced exon 10 in cancer cells. We show that PKM2 O-GlcNAcylation is up-regulated in various types of human tumor cells and patient tumor tissues. The modification destabilized the active tetrameric PKM2, reduced PK activity, and led to nuclear translocation of PKM2. We also observed that the modification was associated with an increased glucose consumption and lactate production and enhanced level of lipid and DNA synthesis, indicating that O-GlcNAcylation promotes the Warburg effect. In vivo experiments showed that blocking PKM2 O-GlcNAcylation attenuated tumor growth. Thus, we demonstrate that O-GlcNAcylation is a regulatory mechanism for PKM2 in cancer cells and serves as a bridge between PKM2 and metabolic reprogramming typical of the Warburg effect.

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.

Lewis-antigen-containing ICAM-2/3 on Jurkat leukemia cells interact with DC-SIGN to regulate DC functions.

Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is an important C-type lectin and plays a critical role in the recognition of pathogens and self-antigens. It has recently been shown that DC-SIGN directly interacts with acute T lymphoblastic leukemia cells. However, the mechanism regulating DC-SIGN-dependent DC association as well as related functions is still elusive. Here we showed that DC-SIGN preferentially bound to a set of malignant T lymphocytes, including Jurkat, CCRF-HSB2 and CCRF-CEM. ICAM-2/3 on Jurkat cells appeared to be the responsible ligands and the block of ICAM-2/3 dramatically impaired DC-SIGN association. We also found that ICAM-2/3 bear a considerable amount of Lewis X, Lewis Y and Lewis A residues, which are important for DC-SIGN recognition. Furthermore, transcriptome analysis revealed an upregulation of fucosyltransferase 4 (FUT4) in Jurkat cells and downregulating FUT4 limited DC-SIGN binding, indicating a previously unappreciated role of FUT4 in the control of Lewis antigens on malignant T lymphocytes. In addition, the presence of Jurkat cells impaired DC maturation and the block of DC-SIGN improved Jurkat cell-mediated effects on DC function and T cell differentiation. Together, we provide evidence that DC-SIGN orients DC association with acute T lymphoblastic leukemia cells and orchestrates DC functions.

Regulation of Tumor Suppressor Gene CDKN2A and Encoded p16-INK4a Protein by Covalent Modifications.

CDKN2A is one of the most studied tumor suppressor genes. It encodes the p16-INK4a protein that plays a critical role in the cell cycle progression, differentiation, senescence, and apoptosis. Mutations in CDKN2A or dysregulation of its functional activity are frequently associated with various types of human cancer. As a cyclin-dependent kinase inhibitor, p16-INK4a forms a complex with cyclin-dependent kinases 4/6 (CDK4/6) thereby competing with cyclin D. It is believed that the helix-turn-helix structures in the content of tandem ankyrin repeats in p16-INK4a are required for the protein interaction with CDK4. Until recently, the mechanisms considered to be involved in the regulation of p16-INK4a functions and cancer development have been mutations in DNA, homozygous or heterozygous gene loss, and methylation of CDKN2A promoter region. In this review, we discuss recent findings on the regulation of p16-INK4a by covalent modifications at both transcriptional and post-translational levels.

Removal of millimeter-scale rolled edges using bevel-cut-like tool influence function in magnetorheological jet polishing.

Subaperture polishing techniques usually produce rolled edges due to edge effect. The rolled edges, especially those in millimeter scale on small components, are difficult to eliminate using conventional polishing methods. Magnetorheological jet polishing (MJP) offers the possibility of the removal of these structures, owing to its small tool influence function (TIF) size. Hence, we investigate the removal characters of inclined MJP jetting models by means of computational fluid dynamics (CFD) simulations and polishing experiments. A discrete phase model (DPM) is introduced in the simulation to get the influence of abrasive particle concentration on the removal mechanism. Therefore, a more accurate model for MJP removal mechanisms is built. With several critical problems solved, a small bevel-cut-like TIF (B-TIF), which has fine acentric and unimodal characteristics, is obtained through inclined jetting. The B-TIF proves to have little edge effect and is applied in surface polishing of thin rolled edges. Finally, the RMS of the experimental section profile converges from 10.5 nm to 1.4 nm, and the rolled edges are successfully suppressed. Consequently, it is validated that the B-TIF has remarkable ability in the removal of millimeter-scale rolled edges.

Disruption and restoration of nucleolar FC and DFC during S phase in HeLa cells.

In the higher eukaryotic nucleolus, fibrillar centers (FCs), the dense fibrillar components (DFCs), and the granular components (GCs) are functional domains structurally relatively well-defined by electron microscopy (EM). However, ultrastructural alterations in FC, DFC, and GC during the cell cycle and their associated cellular functions are still largely unclear. Based on synchronized HeLa cells, we followed the structural dynamics of nucleolus during cell cycle by EM. We found that nucleolus structure shifted from tripartite to bipartite organization and FC/DFCs were reorganized in S phase with three distinct stages: (1) In early-S phase, FC/DFC structures were disassembled. (2) In mid-S phase, a transition from FC/DFC disruption to restoration occurred. As FC/DFC structures were completely disassembled, nucleoli became structurally homogenous. (3) In late-S phase, the number of small FC/DFCs increased and subsequently large FC/DFCs were constructed. Our data demonstrated that nucleolar FC/DFCs in interphase are presented in two different forms or states due to disassembly and reassembly. FC/DFCs in G1 are nucleolar structures constructed concomitantly with the establishment of nucleoli derived from the nucleolar organizer region (NOR). FC/DFCs in G2 are nucleolar components reconstituted after the global reassembly in mid-S phase. Dynamic nucleolus structures revealed in this study may serve as ultrastructural characteristics to reflect distinct stages of the cell cycle. By providing evidence for the temporal and spatial regulation of nucleolus, our findings contribute to the coupling of nucleolus structures to cell cycle dependent functions.

Improvement of high-power laser performance for super-smooth optical surfaces using electrorheological finishing technology.

Laser-induced damage threshold (LIDT) is a key parameter for optical components heavily influenced by the surface roughness in high-power laser uses. Present polishing technologies often bring about directional micro waviness to the optical surfaces due to path effect. Roughness features of a K9 glass surface were studied in this paper. A new evaluating restriction for power spectral density specification was established, and the off-specification frequency contents were found out. Then the electromagnetic simulation of light field modulation was carried out, and the field enhancement factor reached 12.04, verifying the impact of these contents on the laser damage performance of optical components. To restrain the modulation effect by the textures, electrorheological finishing (ERF) technology was proposed, and the processing was undertaken on the K9 surface. Roughness data converged to minimal Ra 1.00 nm, and the angular spectrum decreased in expected ranges. ERF proved to be effective in eliminating the directional textures and restraining the light intensity modulation of the textures. As a result, the LIDTs of optical components can be improved by ERF processing.

Method to measure the position offset of multiple light spots in a distributed aperture laser angle measurement system.

In this paper, an accurate measurement method of multiple spots' position offsets on a four-quadrant detector is proposed for a distributed aperture laser angle measurement system (DALAMS). The theoretical model is put forward, as well as the corresponding calculation method. This method includes two steps. First, as the initial estimation, integral approximation is applied to fit the distributed spots' offset function; second, the Boltzmann function is employed to compensate for the estimation error to improve detection accuracy. The simulation results attest to the correctness and effectiveness of the proposed method, and tolerance synthesis analysis of DALAMS is conducted to determine the maximum uncertainties of manufacturing and installation. The maximum angle error is less than 0.08° in the prototype distributed measurement system, which shows the stability and robustness for prospective applications.