Detecting Tumor Antigen-Specific T Cells via Interaction-Dependent Fucosyl-Biotinylation.

Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific T cells for studying their biology. Through this interaction-dependent labeling approach, intratumoral TSA-reactive CD4+, CD8+ T cells, and TSA-suppressive CD4+ T cells can be detected and separated from bystander T cells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct T cell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype, TSA-reactive CD8+ TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle, FucoID should have the potential of accelerating the pace of personalized cancer treatment.

4.4 kW Nd:YAG slab amplifier with a high repetition frequency and high beam quality.

Using the self-developed fused indium wetting technology and planar waveguide, the uniform heat dissipation of the slab crystal and uniform pumping of the pump light were achieved, respectively. Based on the master oscillator power amplification (MOPA) scheme, the power was then amplified when the seed light source passed through the Nd:YAG slab crystal three times. Additionally, the image transfer system that we added to the amplified optical path achieved high beam quality. Finally, we obtained a rectangular pulsed laser with an output average power of 4461 W, a repetition frequency of 20 kHz, a pulse width of 62 ns, an optical-to-optical conversion efficiency of 26.8%, and a beam quality of ß x=7.0 and ß y=7.7.

Polarization-multiplexed metasurface enabled tri-functional imaging.

Diffraction-limited focusing imaging, edge-enhanced imaging, and long depth of focus imaging offer crucial technical capabilities for applications such as biological microscopy and surface topography detection. To conveniently and quickly realize the microscopy imaging of different functions, the multifunctional integrated system of microscopy imaging has become an increasingly important research direction. However, conventional microscopes necessitate bulky optical components to switch between these functionalities, suffering from the system's complexity and unstability. Hence, solving the problem of integrating multiple functions within an optical system is a pressing need. In this work, we present an approach using a polarization-multiplexed tri-functional metasurface, capable of realizing the aforementioned imaging functions simply by changing the polarization state of the input and output light, enhancing the system structure's compactness and flexibility. This work offers a new avenue for multifunctional imaging, with potential applications in biomedicine and microscopy imaging.

Experimental demonstration of optical trapping and manipulation with multifunctional metasurface.

Chip-scale optical tweezers, which are usually implemented in a planar format without using bulky diffractive optical elements, are recognized as a promising candidate to be integrated with a lab-on-a-chip system. However, traditional chip-scale optical tweezers are often static and allow for only one type of manipulation functionality since the geometrical parameters of the tweezers are fixed. Herein, we introduce a new, to the best of our knowledge, class of on-chip optical tweezers for diverse types of manipulation of micro-particles. Utilizing both the propagation phase and Pancharatnam-Berry phase, we experimentally demonstrate the spin-dependent trapping, moving, and circling of micro-particles with the transfer of optical gradient force and orbital angular momentum to particles. We further show that the spin angular momentum of the output beam provides an additional degree of freedom to control the spinning rotation of particles. This new type of optical tweezers paves the way for multifunctional and dynamical trapping and manipulation of particles with a lab-on-a-chip system.

d-Idose, d-Iduronic Acid, and d-Idonic Acid from d-Glucose via Seven-Carbon Sugars.

A practical synthesis of the very rare sugar d-idose and the stable building blocks for d-idose, d-iduronic, and d-idonic acids from ido-heptonic acid requires only isopropylidene protection, Shing silica gel-supported periodate cleavage of the C6-C7 bond of the heptonic acid, and selective reduction of C1 and/or C6. d-Idose is the most unstable of all the aldohexoses and a stable precursor which be stored and then converted under very mild conditions into d-idose is easily prepared.

SuFEx Click Chemistry Enabled Late-Stage Drug Functionalization.

Sulfur(VI) Fluoride Exchange (SuFEx) is a new family of click chemistry transformations which relies on readily available materials to produce compounds bearing the SVI-F motif. The potential of SuFEx in drug discovery has just started to be explored. We report the first method of SuFEx chemistry for the conversion of phenolic compounds to their respective arylfluorosulfate derivatives in situ in 96-well plates. This method is compatible with automated synthesis and screening to quickly assess the biological activities of the in situ generated, crude products. Using this method, we perform late-stage functionalization of a panel of known anticancer drugs to generate the corresponding arylfluorosulfates. These in situ generated arylfluorosulfates are directly tested in a cancer-cell growth inhibition assay in parallel with their phenolic precursors. We discover three arylfluorosulfates that exhibit improved anticancer cell proliferation activities compared to their phenol precursors. Among these three compounds, the fluorosulfate derivative of Fulvestrant possesses significantly enhanced activity to down-regulate estrogen receptor (ER) expression in ER+ breast cancer cell line MCF-7 and the fluorosulfate derivative of Combretastatin A4-a general anticancer drug currently being evaluated under clinical trials-exhibits a 70-fold increase in potency in the drug resistant colon cancer cell line HT-29.

6-Deoxyhexoses from l-Rhamnose in the Search for Inducers of the Rhamnose Operon: Synergy of Chemistry and Biotechnology.

In the search for alternative non-metabolizable inducers in the l-rhamnose promoter system, the synthesis of fifteen 6-deoxyhexoses from l-rhamnose demonstrates the value of synergy between biotechnology and chemistry. The readily available 2,3-acetonide of rhamnonolactone allows inversion of configuration at C4 and/or C5 of rhamnose to give 6-deoxy-d-allose, 6-deoxy-d-gulose and 6-deoxy-l-talose. Highly crystalline 3,5-benzylidene rhamnonolactone gives easy access to l-quinovose (6-deoxy-l-glucose), l-olivose and rhamnose analogue with C2 azido, amino and acetamido substituents. Electrophilic fluorination of rhamnal gives a mixture of 2-deoxy-2-fluoro-l-rhamnose and 2-deoxy-2-fluoro-l-quinovose. Biotechnology provides access to 6-deoxy-l-altrose and 1-deoxy-l-fructose.

Synergistic therapeutic vascular cytoprotection against complement-mediated injury induced via a PKCα-, AMPK-, and CREB-dependent pathway.

Endothelial injury and dysfunction precede accelerated arterial disease in allograft vasculopathy and systemic autoimmune diseases and involve pathogenic Abs and complement. Recent reports suggest that switching to rapamycin from calcineurin antagonists reduces posttransplant vasculopathy and prolongs survival following cardiac transplantion. The majority of these patients also receive statin therapy. We examined potential mechanisms underlying this protective response in human endothelial cells and identified synergy between rapamycin and atorvastatin. Mechanistically, atorvastatin and rapamycin activated a protein kinase Cα, AMP-activated kinase, and CREB-dependent vasculoprotective pathway, which induced decay-accelerating factor (DAF) promoter activity via binding to the cAMP response element, mutation of which attenuated promoter activity. This response significantly increased endothelial cell surface DAF and enhanced protection against complement-mediated injury. Synergy with rapamycin was reproduced by simvastatin, whereas combining atorvastatin with cyclosporine or mycophenolate in place of rapamycin was ineffective. Importantly, synergy was reproduced in vivo, in which only atorvastatin and rapamycin therapy in combination was sufficient to induce DAF on murine aortic endothelium. We believe this pathway represents an important therapeutically inducible vasculoprotective mechanism for diseases mediated by pathogenic Abs and complement, including posttransplant vasculopathy and systemic lupus erythematosus. Although our study focuses on the vascular endothelium, the findings are likely to be broadly applicable, given the diverse cellular expression of DAF.

3-Fluoroazetidinecarboxylic Acids and trans,trans-3,4-Difluoroproline as Peptide Scaffolds: Inhibition of Pancreatic Cancer Cell Growth by a Fluoroazetidine Iminosugar.

Reverse aldol opening renders amides of 3-hydroxyazetidinecarboxylic acids (3-OH-Aze) unstable above pH 8. Aze, found in sugar beet, is mis-incorporated for proline in peptides in humans and is associated with multiple sclerosis and teratogenesis. Aze-containing peptides may be oxygenated by prolyl hydroxylases resulting in potential damage of the protein by a reverse aldol of the hydroxyazetidine; this, rather than changes in conformation, may account for the deleterious effects of Aze. This paper describes the synthesis of 3-fluoro-Aze amino acids as hydroxy-Aze analogues which are not susceptible to aldol cleavage. 4-(Azidomethyl)-3-fluoro-Aze and 3,4-difluoroproline are new peptide building blocks. trans,trans-2,4-Dihydroxy-3-fluoroazetidine, an iminosugar, inhibits the growth of pancreatic cancer cells to a similar degree as gemcitabine.

Nine of 16 stereoisomeric polyhydroxylated proline amides are potent ß-N-acetylhexosaminidase inhibitors.

All 16 stereoisomeric N-methyl 5-(hydroxymethyl)-3,4-dihydroxyproline amides have been synthesized from lactones accessible from the enantiomers of glucuronolactone. Nine stereoisomers, including all eight with a (3R)-hydroxyl configuration, are low to submicromolar inhibitors of ß-N-acetylhexosaminidases. A structural correlation between the proline amides is found with the ADMDP-acetamide analogues bearing an acetamidomethylpyrrolidine motif. The proline amides are generally more potent than their ADMDP-acetamide equivalents. ß-N-Acetylhexosaminidase inhibition by an azetidine ADMDP-acetamide analogue is compared to an azetidine carboxylic acid amide. None of the amides are good α-N-acetylgalactosaminidase inhibitors.

Short and sweet: (D)-glucose to (L)-glucose and (L)-glucuronic acid.

The scarcity and expense of access to L-sugars and other rare sugars have prevented the exploitation of their biological potential; for example D-psicose, only recently available, has been recognized as an important new food. Here we give the definitive and cheap synthesis of 99.4% pure L-glucose from D-glucose which requires purification of neither intermediates nor final product other than extraction into and removal of solvents; a simple crystallization will raise the purity to >99.8%.

Characterizing Foxp3+ and Foxp3- T cells in the homeostatic state and after allo-activation: resting CD4+Foxp3+ Tregs have molecular characteristics of activated T cells.

Due to the intracellular expression of Foxp3 it is impossible to purify viable Foxp3+ cells on the basis of Foxp3 staining. Consequently CD4+Foxp3+ regulatory T cells (Tregs) in mice have mostly been characterized using CD4+CD25+ T cells or GFP-Foxp3 reporter T cells. However, these two populations cannot faithfully represent Tregs as the expression of CD25 and Foxp3 does not completely overlap and GFP+Foxp3+ reporter T cells have been reported to be functionally altered. The aim of this study was to characterize normal Tregs without separating Foxp3+ and Foxp3- cells for the expression of the main functional molecules and proliferation behaviors by flow cytometry and to examine their gene expression characteristics through differential gene expression. Our data showed that the expressions of Foxp3, CD25, CTLA-4 (both intracellular and cell surface) and PD-1 was mostly confined to CD4+ T cells and the expression of Foxp3 did not completely overlap with the expression of CD25, CTLA-4 or PD-1. Despite higher levels of expression of the T cell inhibitory molecules CTLA-4 and PD-1, Tregs maintained higher levels of Ki-67 expression in the homeostatic state and had greater proliferation in vivo after allo-activation than Tconv. Differential gene expression analysis revealed that resting Tregs exhibited immune activation markers characteristic of activated Tconv. This is consistent with the flow data that the T cell activation markers CD25, CTLA-4, PD-1, and Ki-67 were much more strongly expressed by Tregs than Tconv in the homeostatic state.

Efficient characterizations of multiphoton states with an ultra-thin optical device.

Metasurface enables the generation and manipulation of multiphoton entanglement with flat optics, providing a more efficient platform for large-scale photonic quantum information processing. Here, we show that a single metasurface optical device would allow more efficient characterizations of multiphoton entangled states, such as shadow tomography, which generally requires fast and complicated control of optical setups to perform information-complete measurements, a demanding task using conventional optics. The compact and stable device here allows implementations of general positive operator valued measures with a reduced sample complexity and significantly alleviates the experimental complexity to implement shadow tomography. Integrating self-learning and calibration algorithms, we observe notable advantages in the reconstruction of multiphoton entanglement, including using fewer measurements, having higher accuracy, and being robust against experimental imperfections. Our work unveils the feasibility of metasurface as a favorable integrated optical device for efficient characterization of multiphoton entanglement, and sheds light on scalable photonic quantum technologies with ultra-thin optical devices.

Novel Approaches to Access Arylfluorosulfates and Sulfamoyl Fluorides Based on Sulfur (VI) Fluoride Exchange.

Sulfur (VI) fluoride exchange (SuFEx) is a new family of click chemistry reactions that relies on readily available sulfuryl fluoride (SO2 F2 ) and ethenesulfonyl fluoride to build diverse chemical structures bearing the SVI -F motif, such as fluorosulfate (-OSO2 F) and sulfonyl fluoride (-SO2 F). These motifs could be useful functional groups and connective linkers in organic synthesis. This unit describes two protocols for performing SuFEx. The first protocol describes an in situ method for rapid generation of arylfluorosulfates in 96-well plates for high-throughput screening. The second protocol outlines use of a shelf-stable fluorosulfuryl imidazolium salt for generating arylfluorosulfates and sulfamoyl fluorides. © 2019 by John Wiley & Sons, Inc.

A Single-Step Chemoenzymatic Reaction for the Construction of Antibody-Cell Conjugates.

Employing live cells as therapeutics is a direction of future drug discovery. An easy and robust method to modify the surfaces of cells directly to incorporate novel functionalities is highly desirable. However, genetic methods for cell-surface engineering are laborious and limited by low efficiency for primary cell modification. Here we report a chemoenzymatic approach that exploits a fucosyltransferase to transfer bio-macromolecules, such as an IgG antibody (MW∼ 150 KD), to the glycocalyx on the surfaces of live cells when the antibody is conjugated to the enzyme's natural donor substrate GDP-Fucose. Requiring no genetic modification, this method is fast and biocompatible with little interference to cells' endogenous functions. We applied this method to construct two antibody-cell conjugates (ACCs) using both cell lines and primary cells, and the modified cells exhibited specific tumor targeting and resistance to inhibitory signals produced by tumor cells, respectively. Remarkably, Herceptin-NK-92MI conjugates, a natural killer cell line modified with Herceptin, exhibit enhanced activities to induce the lysis of HER2+ cancer cells both ex vivo and in a human tumor xenograft model. Given the unprecedented substrate tolerance of the fucosyltransferase, this chemoenzymatic method offers a general approach to engineer cells as research tools and for therapeutic applications.

[Expression of Aurora Family Genes in Acute Leukemia and Its Clinical Significance].

OBJECTIVE: To explore the mRNA expression of Aurora-A,B,C(AUR-A,B,C) in acute leukemia(AL) and their correlations with the clinical indications. METHODS: The mRNA expression levels of AUR-A,B,C in 73 cases of newly diagnosed AL (untreated group), 20 cases of AL with remission (remission group) and 14 healthy volunteers as control (healthy group) were detected by QRT-PCR, and the difference of expression levels in difference groups, their correlations with clinical indicators and the correlation between the AUR-A,B,C mRNA expression levels themselves were analyzed. RESULTS: The mRNA expression levels of AUR-A,B,C in untreated group were all higher than those in healthy group and remission group(P<0.01), but there was not significant difference between healthy group and remission group(P>0.05); the mRNA expressions of AUR-A,B,C in acute lymphoblastic leukemia(ALL) group were all significantly higher than that in AML group(P<0.01). The mRNA expression of AUR-A,B,C in high risk group was higher than that in low risk group(P<0.05), but there was no difference in mRNA expression of AUR-A,B,C between high risk group and middle risk group as well as between middle risk group and low risk group(P>0.05). The mRNA expression of AUR-A, B, C in CD34, CD71 and CD56 negative group was not statistically different from that in CD34,CD71 and CD56 positive group(P>0.05). In 73 cases of newly diagnosed AL, the mRNA expression levels of AUR-A, B significantly were positively correlated with lactate dehydrogenase(LDH) level and risk stratification (r=0.279, P=0.017; r=0.314, P=0.007 and r=0.277, P=0.018; r=0.349, P=0.002), while the mRNA expression levels of AUR-A, B were not significantly correlated with age, WBC count, blast ratio in bone marrow at initial diagnosis and remission or no-remission after 1 cours of chemotherapy; the mRNA expression level of AUR-C was significantly positively correlated with WBC count (r=0.263, P=0.025), and LDH level (r=0.348, P=0.003) at initial diagnosis and risk stratificantion(r=0.376, P=0.001), and negatively correlated with age (r=-0.241, P=0.040), and was not significantly correlated with blast ratio in bone marrow at initial diagnosis and remission or noremission after 1 course of chemotherapy. There were significant positive correlations in the mRNA expression between AUR-A and B (r=0.444, P=0.000), AUR-B and C (r=0.763, P=0.000) as well as AUR-A and C (r=0.616, P=0.000). CONCLUSION: Aur-A, B, C mRNA were highly expressed in patients with newly diagnosed AL, moreover the mRNA expression levels of Aur-A,B,C were positively correlated with each other, the high expression of Aur-A, B, C are associated with leukemia types, risk stratification, WBC count and LDH level at initial diagnosis, so they all maybe used as the prognostic markers and potential therapeutic targets.

Triacetonide of Glucoheptonic Acid in the Scalable Syntheses of d-Gulose, 6-Deoxy-d-gulose, l-Glucose, 6-Deoxy-l-glucose, and Related Sugars.

Ease of separation of petrol-soluble acetonides derived from the triacetonide of methyl glucoheptonate allows scalable syntheses of rare sugars containing the l-gluco or d-gulo structural motif with any oxidation level at the C6 or C1 position of the hexose, usually without chromatography: meso-d-glycero-d-guloheptitol available in two steps is an ideal entry point for the study of the biotechnological production of heptoses.

Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the rhaBAD Promoter.

External control of gene expression is crucial in synthetic biology and biotechnology research and applications, and is commonly achieved using inducible promoter systems. The E. coli rhamnose-inducible rhaBAD promoter has properties superior to more commonly used inducible expression systems, but is marred by transient expression caused by degradation of the native inducer, l-rhamnose. To address this problem, 35 analogues of l-rhamnose were screened for induction of the rhaBAD promoter, but no strong inducers were identified. In the native configuration, an inducer must bind and activate two transcriptional activators, RhaR and RhaS. Therefore, the expression system was reconfigured to decouple the rhaBAD promoter from the native rhaSR regulatory cascade so that candidate inducers need only activate the terminal transcription factor RhaS. Rescreening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterized further to determine the strength, kinetics, and concentration-dependence of induction; whether the inducer was used as a carbon source by E. coli; and the modality (distribution) of induction among populations of cells. l-Mannose was found to be the most useful orthogonal inducer, providing an even greater range of induction than the native inducer l-rhamnose, and crucially, allowing sustained induction instead of transient induction. These findings address the key limitation of the rhaBAD expression system and suggest it may now be the most suitable system for many applications.

L-fucose from vitamin C with only acetonide protection.

Addition of human milk oligosaccharides (HMO) to baby foods may protect infants from disease. As many simple HMOs are fucosylated this is likely to increase the demand for L-fucose as a synthetic building block. Any chemical synthesis must be cheap to compete with a biotechnological process. Acetonide is the only protecting group we have used in this new synthesis of L-fucose from vitamin C in 27% overall yield (purification by recrystallization; no chromatography required in the entire sequence).