Targeted glycan degradation potentiates cellular immunotherapy for solid tumors.

Immune cell-based cancer therapies, such as chimeric antigen receptor T (CAR-T)-cell immunotherapy, have demonstrated impressive potency against hematological tumors. However, the efficacy of CAR-T cells against solid tumors remains limited. Herein, we designed tumor-targeting molecule-sialidase conjugates that potently and selectively stripped different sialoglycans from a variety of cancer cells. Desialylation enhanced induced pluripotent stem cell-derived chimeric antigen receptor-macrophage (CAR-iMac) infiltration and activation. Furthermore, the combination of cancer cell desialylation and CAR-iMac adoptive cellular therapy exerted a dramatic therapeutic effect on solid tumors and significantly prolonged the survival of tumor-bearing mice; these effects were mainly dependent on blockade of the checkpoint composed of sialic acid-binding immunoglobulin-like lectin (Siglec)-5 and Siglec-10 on the macrophages, and knockout of the glycoimmune checkpoint receptors could construct a CAR-iMac cell with stronger anticancer activity. This strategy that reverts the immune escape state ("cold tumor") to a sensitive recognition state ("hot tumor") has great significance for enhancing the effect of cellular immunotherapy on solid tumors. Therefore, desialylation combined with CAR-iMac cellular immunotherapy is a promising approach to enhance treatment with cellular immunotherapy and expand the valid indications among solid tumors, which provides inspiration for the development of cellular immunotherapies with glycoimmune checkpoint inhibition for the treatment of human cancer.

An Approach for Highly Enantioselective Synthesis of meta-Disubstituted [n]Paracyclophanes.

Atroposelective synthesis of meta-disubstituted [n]paracyclophanes is a difficult task in organic chemistry. We describe a facile approach for the synthesis of meta-disubstituted [n]paracyclophanes using Pd-catalyzed enantioselective C-H olefination and sequential reductive cleavage. A wide range of [n]paracyclophanes was obtained with excellent enantioselectivity. Thermodynamic analysis revealed that the rotational barrier of meta-disubstituted [n]paracyclophanes was lower than that of para-disubstituted [n]paracyclophanes. The synthesized planar-chiral [14]paracyclophane showed a bright fluorescence emission and impressive circularly polarized luminescence activity.

Tea polyphenol-engineered hybrid cellular nanovesicles for cancer immunotherapy and androgen deprivation therapy.

Androgen deprivation therapy (ADT) is a crucial and effective strategy for prostate cancer, while systemic administration may cause profound side effects on normal tissues. More importantly, the ADT can easily lead to resistance by involving the activation of NF-κB signaling pathway and high infiltration of M2 macrophages in tumor microenvironment (TME). Herein, we developed a biomimetic nanotherapeutic platform by deriving cell membrane nanovesicles from cancer cells and probiotics to yield the hybrid cellular nanovesicles (hNVs), loading flutamide (Flu) into the resulting hNVs, and finally modifying the hNVs@Flu with Epigallocatechin-3-gallate (EGCG). In this nanotherapeutic platform, the hNVs significantly improved the accumulation of hNVs@Flu-EGCG in tumor sites and reprogramed immunosuppressive M2 macrophages into antitumorigenic M1 macrophages, the Flu acted on androgen receptors and inhibited tumor proliferation, and the EGCG promoted apoptosis of prostate cancer cells by inhibiting the NF-κB pathway, thus synergistically stimulating the antitumor immunity and reducing the side effects and resistance of ADT. In a prostate cancer mouse model, the hNVs@Flu-EGCG significantly extended the lifespan of mice with tumors and led to an 81.78% reduction in tumor growth compared with the untreated group. Overall, the hNVs@Flu-EGCG are safe, modifiable, and effective, thus offering a promising platform for effective therapeutics of prostate cancer.

Engineered Cellular Vesicles Displaying Glycosylated Nanobodies for Cancer Immunotherapy.

Immune checkpoint blockade targeting the CD47/SIRPα axis represents an alluring avenue for cancer immunotherapy. However, the compromised efficacy and safety concerns in vivo of conventional anti-CD47 antibodies impede their wide clinical applications. Here we introduced a single type of high-mannose glycans into the nanobody against CD47 (HM-nCD47) and subsequently displayed HM-nCD47 on cellular vesicles (CVs) for enhanced cancer immunotherapy. In this platform, the CVs significantly improved the circulation time of HM-nCD47-CVs, the nCD47 enabled the blockade of the CD47/SIRPα axis, and the HM enhanced recognition of mannose-binding lectin, all synergistically activating the macrophage-mediated antitumor immunity. In both subcutaneous and metastatic murine tumor models, the HM-nCD47-CVs possessed significantly extended half-lives and increased accumulation at the tumor site, resulting in a remarkable macrophage-dependent inhibition of tumor growth, a transcriptomic remodeling of the immune response, and an increase in survival time. By integrating the chemical biology toolbox with cell membrane nanotechnology, the HM-nCD47-CVs represent a new immunotherapeutic platform for cancer and other diseases.

Dynamic Kinetic Stereoselective Glycosylation via RhII and Chiral Phosphoric Acid-Cocatalyzed Carbenoid Insertion to the Anomeric OH Bond for the Synthesis of Glycoconjugates.

Chemical synthesis of glycoconjugates is essential for studying the biological functions of carbohydrates. We herein report an efficient approach for the stereoselective synthesis of challenging α-linked glycoconjugates via a RhII /chiral phosphoric acid (CPA)-cocatalyzed dynamic kinetic anomeric O-alkylation of sugar-derived lactols via carbenoid insertion to the anomeric OH bond. Notably, we observed excellent anomeric selectivity, excellent diastereoselectivity, broad substrate scope, and high efficiency for this glycosylation reaction by exploring various parameters of the cocatalytic system. DFT calculations suggested that the anomeric selectivity was mainly determined by steric interactions between the C2-carbon of the carbohydrate and the phenyl group of the metal carbenoid, while π/π interactions with the C2-OBn substituent on the carbohydrate substrate play a significant role for diastereoselectivity at the newly generated stereogenic center.

General Strategy for the Synthesis of Rare Sugars via Ru(II)-Catalyzed and Boron-Mediated Selective Epimerization of 1,2-trans-Diols to 1,2-cis-Diols.

Human glycans are primarily composed of nine common sugar building blocks. On the other hand, several hundred monosaccharides have been discovered in bacteria and most of them are not readily available. The ability to access these rare sugars and the corresponding glycoconjugates can facilitate the studies of various fundamentally important biological processes in bacteria, including interactions between microbiota and the human host. Many rare sugars also exist in a variety of natural products and pharmaceutical reagents with significant biological activities. Although several methods have been developed for the synthesis of rare monosaccharides, most of them involve lengthy steps. Herein, we report an efficient and general strategy that can provide access to rare sugars from commercially available common monosaccharides via a one-step Ru(II)-catalyzed and boron-mediated selective epimerization of 1,2-trans-diols to 1,2-cis-diols. The formation of boronate esters drives the equilibrium toward 1,2-cis-diol products, which can be immediately used for further selective functionalization and glycosylation. The utility of this strategy was demonstrated by the efficient construction of glycoside skeletons in natural products or bioactive compounds.

Virus Dynamics and Decay in Evaporating Human Saliva Droplets on Fomites.

The transmission of most respiratory pathogens, including SARS-CoV-2, occurs via virus-containing respiratory droplets, and thus, factors that affect virus viability in droplet residues on surfaces are of critical medical and public health importance. Relative humidity (RH) is known to play a role in virus survival, with a U-shaped relationship between RH and virus viability. The mechanisms affecting virus viability in droplet residues, however, are unclear. This study examines the structure and evaporation dynamics of virus-containing saliva droplets on fomites and their impact on virus viability using four model viruses: vesicular stomatitis virus, herpes simplex virus 1, Newcastle disease virus, and coronavirus HCoV-OC43. The results support the hypothesis that the direct contact of antiviral proteins and virions within the "coffee ring" region of the droplet residue gives rise to the observed U-shaped relationship between virus viability and RH. Viruses survive much better at low and high RH, and their viability is substantially reduced at intermediate RH. A phenomenological theory explaining this phenomenon and a quantitative model analyzing and correlating the experimentally measured virus survivability are developed on the basis of the observations. The mechanisms by which RH affects virus viability are explored. At intermediate RH, antiviral proteins have optimal influence on virions because of their largest contact time and overlap area, which leads to the lowest level of virus activity.

The nuclear matrix protein HNRNPU maintains 3D genome architecture globally in mouse hepatocytes.

Eukaryotic chromosomes are folded into higher-order conformations to coordinate genome functions. In addition to long-range chromatin loops, recent chromosome conformation capture (3C)-based studies have indicated higher levels of chromatin structures including compartments and topologically associating domains (TADs), which may serve as units of genome organization and functions. However, the molecular machinery underlying these hierarchically three-dimensional (3D) chromatin architectures remains poorly understood. Via high-throughput assays, including in situ Hi-C, DamID, ChIP-seq, and RNA-seq, we investigated roles of the Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU), a nuclear matrix (NM)-associated protein, in 3D genome organization. Upon the depletion of HNRNPU in mouse hepatocytes, the coverage of lamina-associated domains (LADs) in the genome increases from 53.1% to 68.6%, and a global condensation of chromatin was observed. Furthermore, disruption of HNRNPU leads to compartment switching on 7.5% of the genome, decreases TAD boundary strengths at borders between A (active) and B (inactive) compartments, and reduces chromatin loop intensities. Long-range chromatin interactions between and within compartments or TADs are also significantly remodeled upon HNRNPU depletion. Intriguingly, HNRNPU mainly associates with active chromatin, and 80% of HNRNPU peaks coincide with the binding of CTCF or RAD21. Collectively, we demonstrated that HNRNPU functions as a major factor maintaining 3D chromatin architecture, suggesting important roles of NM-associated proteins in genome organization.

Site-Selective and Stereoselective O-Alkylation of Glycosides by Rh(II)-Catalyzed Carbenoid Insertion.

Carbohydrates are synthetically challenging molecules with vital biological roles in all living systems. Selective synthesis and functionalization of carbohydrates provide tremendous opportunities to improve our understanding on the biological functions of this fundamentally important class of molecules. However, selective functionalization of seemingly identical hydroxyl groups in carbohydrates remains a long-standing challenge in chemical synthesis. We herein describe a practical and predictable method for the site-selective and stereoselective alkylation of carbohydrate hydroxyl groups via Rh(II)-catalyzed insertion of metal carbenoid intermediates. This represents one of the mildest alkylation methods for the systematic modification of carbohydrates. Density functional theory (DFT) calculations suggest that the site selectivity is determined in the Rh(II)-carbenoid insertion step, which prefers insertion into hydroxyl groups with an adjacent axial substituent. The subsequent intramolecular enolate protonation determines the unexpected high stereoselectivity. The most prevalent trans-1,2-diols in various pyranoses can be systematically and predictably differentiated based on the model derived from DFT calculations. We also demonstrated that the selective O-alkylation method could significantly improve the efficiency and stereoselectivity of glycosylation reactions. The alkyl groups introduced to carbohydrates by OH insertion reaction can serve as functional groups, protecting groups, and directing groups.

C-terminal site-specific PEGylated Exendin-4 analog: A long-acting glucagon like Peptide-1 receptor agonist, on glycemic control and beta cell function in diabetic db/db mice.

PEG modification is a common clinical strategy for prolonging the half-life of therapeutic proteins or polypeptides. In a previous work, we have successfully synthesized PEG-modified Exendin-4 (PE) by conjugating a 20 kDa PEG to the C-terminal of Exendin-4. Then, we introduced an integrative characterization for PE to evaluate its hypoglycemic activity and pharmacokinetic properties. The normoglycemic efficacies and therapeutic activity of PE were investigated in db/db mice. The hypoglycemic time after single administration of PE on db/db mice was prolonged from 8.4 h to 54.9 h. In multiple treatment with PE, the fasting blood glucose in various PE dosages (50, 150, and 250 nmol/kg) were remarkably reduced, and the glycosylated hemoglobin level was decreased to 2.0%. When the in vivo single- and multiple-dose pharmacokinetics of PE were examined in Sprague-Dawley rats, the half-life was prolonged to 31.7 h, and no accumulation effect was observed. Overall, this study provided a novel promising therapeutic approach to improving glucose-controlling ability and extending half-life without accumulation in vivo for long-acting treatment of type-2 diabetes.

Enantioselective Intermolecular Enamide-Aldehyde Cross-Coupling Catalyzed by Chiral N-Heterocyclic Carbenes.

The unprecedented N-heterocyclic carbene (NHC)-catalyzed intermolecular cross-coupling of enamides and aldehydes is described. Upon exposure of enamides to aldehydes in the presence of a NHC catalyst, catalytic C-C bond formation occurs, providing highly enantioselective N-protected amines, bearing a quaternary carbon center, in good yields and with high enantioselectivities.

The roles of parafibromin expression in ovarian epithelial carcinomas: a marker for differentiation and prognosis and a target for gene therapy.

Parafibromin is a protein encoded by hyperparathyroidism 2 (HRPT2) and its downregulated expression is involved in the pathogenesis of parathyroid, breast, gastric, colorectal, lung, head and neck cancers. We aimed to investigate the roles of parafibromin expression in tumorigenesis, progression, or prognostic evaluation of ovarian cancers. HRPT2-expressing plasmid was transfected into ovarian cancer cells with the phenotypes and related molecules examined. The messenger RNA (mRNA) and protein expression of parafibromin were also examined in ovarian normal tissue, benign and borderline tumors and cancers by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, or immunohistochemistry respectively. It was found that parafibromin overexpression caused a lower growth, migration and invasion, higher sensitivity to cisplatin and apoptosis than the mock and control (P < 0.05). The transfectants showed the hypoexpression of phosphoinositide 3-kinase (PI3K), Akt, p70 ribosomal protein S6 kinase (p70s6k), Wnt5a, B cell lymphoma-extra large (Bcl-xL), survivin, vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9) than the mock and control at both mRNA and protein levels (P < 0.05). According to real-time PCR, parafibromin mRNA level was lower in ovarian benign tumors and cancers than normal ovary (P < 0.05), while parafibromin was strongly expressed in metastatic cancers in omentum than primary cancers by Western blot. Immunohistochemically, parafibromin expression was stronger in primary cancers than that in ovarian normal tissue (P < 0.05) but weaker than the metastatic cancers (P < 0.05) with a positive correlation with dedifferentiation, ki-67 expression and the lower cumulative survival rate (P < 0.05). These findings indicate that parafibromin downregulation might promote the pathogenesis, dedifferentiation and metastasis of ovarian cancers possibly by suppressing aggressive phenotypes, such as proliferation, cell cycle, apoptosis, migration and invasion.

Ruthenium-Catalyzed Formal Dehydrative [4 + 2] Cycloaddition of Enamides and Alkynes for the Synthesis of Highly Substituted Pyridines: Reaction Development and Mechanistic Study.

Reported herein is a ruthenium-catalyzed formal dehydrative [4 + 2] cycloaddition of enamides and alkynes, representing a mild and economic protocol for the construction of highly substituted pyridines. Notably, the features of broad substrate scope, high efficiency, good functional group tolerance, and excellent regioselectivities were observed for this reaction. Density functional theory (DFT) calculations and experiments have been carried out to understand the mechanism and regiochemistry. DFT calculations suggested that this formal dehydrative [4 + 2] reaction starts with a concerted metalation deprotonation of the enamide by the acetate group in the Ru catalyst, which generates a six-membered ruthenacycle intermediate. Then alkyne inserts into the Ru-C bond of the six-membered ruthenacycle, giving rise to an eight-membered ruthenacycle intermediate. The carbonyl group (which comes originally from the enamide substrate and is coordinated to the Ru center in the eight-membered ruthenacycle intermediate) then inserts into the Ru-C bond to give an intermediate, which produces the final pyridine product through further dehydration. Alkyne insertion step is a regio-determining step and prefers to have the aryl groups of the used alkynes stay away from the catalyst in order to avoid repulsion of aryl group with the enamide moiety in the six-membered ruthenacycle and to keep the conjugation between the aryl group and the triple C-C bond of the alkynes. Consequently, the aryl groups of the used alkynes are in the ß-position of the final pyridines, and the present reaction has high regioselectivity.

Synthesis of Highly Substituted Furans via Intermolecular Enynone-Aldehyde Cross-Coupling/Cyclization Catalyzed by N-Heterocyclic Carbenes.

A new strategy for facile access to multifunctionalized furans via N-heterocyclic carbene-catalyzed cross-coupling/cyclization of ynenones with aldehydes has been explored. This protocol features readily obtainable starting materials, mild and metal-free conditions, broad substrate scope, good functional group tolerance, excellent yields, and easy scale-up. Synthetic utility of the protocol has been further corroborated through functionalization of complex substrates and postmodifications of the product.

Genetically engineered cellular nanoparticles loaded with curcuminoids for cancer immunotherapy.

Background: Inducing immunogenic cell death (ICD) is a promising strategy to enhance immune responses for immune checkpoint blockade (ICB) therapy, but the lack of a simple and effective platform to integrate ICD and ICB therapy limits their clinical application. Methods: Here, we developed programmed cell death protein 1 (PD1)-overexpressing genetically engineered nanovesicles (NVs)-coated curcumin (Cur)-loaded poly (lactic-co-poly-polyglycolic acid) nanoparticles (PD1@Cur-PLGA) to integrate ICD and ICB therapy for enhancing tumor immunotherapy. Results: Genetically engineered NVs greatly enhanced the tumor targeting of nanoparticles, and the PD1 on NVs dramatically blocked the PD1/PDL1 signaling pathway and stimulated antitumor immune responses. Meanwhile, the delivered Cur successfully induced tumor cell apoptosis and activated ICD by inhibiting NF-κB phosphorylation and Bcl-2 protein expression and activating caspase and Bax apoptotic signaling. By synergizing the ICD effect of Cur and the PD1/PDL1 axis blocking function of genetically engineered NVs, the PD1@Cur-PLGA enhanced the intratumoral infiltration rate of mature dendritic cells and CD8+ T cells in tumor tissues, resulting in significantly inhibiting tumor growth in breast and prostate tumor-bearing mouse models. Conclusion: This synergistic ICD and ICB therapy based on genetically engineered NVs provides a low-cost, safe, and effective strategy to enhance cancer immunotherapy.

Rational Design of Untranslated Regions to Enhance Gene Expression.

How to improve gene expression by optimizing mRNA structures is a crucial question for various medical and biotechnological applications. Previous efforts focus largely on investigation of the 5' UTR hairpin structures. In this study, we present a rational strategy that enhances mRNA stability and translation by engineering both the 5' and 3' UTR sequences. We have successfully demonstrated this strategy using green fluorescent protein (GFP) as a model in Escherichia coli and across different expression vectors. We further validated it with luciferase and Plasmodium falciparum lactate dehydrogenase (PfLDH). To elucidate the underlying mechanism, we have quantitatively analyzed both protein, mRNA levels and half-life time. We have identified several key aspects of UTRs that significantly influence mRNA stability and protein expression in our system: (1) The optimal length of the single-stranded spacer between the stabilizer hairpin and ribosome binding site (RBS) in the 5' UTR is 25-30 nucleotide (nt) long. An optimal 32% GC content in the spacer yielded the highest levels of GFP protein production. (2) The insertion of a homodimerdizable, G-quadruplex structure containing RNA aptamer, "Corn", in the 3' UTR markedly increased the protein expression. Our findings indicated that the carefully engineered 5' UTRs and 3' UTRs significantly boosted gene expression. Specifically, the inclusion of 5 × Corn in the 3' UTR appeared to facilitate the local aggregation of mRNA, leading to the formation of mRNA condensates. Aside from shedding light on the regulation of mRNA stability and expression, this study is expected to substantially increase biological protein production.

Chemoimmunological Cascade Cancer Therapy Using Fluorine Assembly Nanomedicine.

Classical chemotherapeutic drugs may cause immunogenic cell death (ICD), followed by activating CD8+ T cells to promote cell-mediated antitumor immune responses. However, CD8+ T cells become exhausted due to tumor antigens' continuous stimulation, creating a major obstacle to effectively suppressing tumor growth and metastasis. Here, we develop an approach of chemo-gene combinational nanomedicine to bridge and reprogram chemotherapy and immunotherapy. The dually loaded nanomedicine induces ICD in tumor cells through doxorubicin and reverses the antitumor effects of exhausted CD8+ T cells through the small interfering RNA. The synergistic chemo-gene and fluorine assembly nanomedicine enriched in reactive oxygen species and acid-sensitive bonds results in enhanced cancer immunotherapy to inhibit tumor growth and the lung metastasis of breast cancer in a mouse model of breast cancer and melanoma. This study provides an efficient strategy and insights into chemoimmunological cascade therapy for combating malignant metastatic tumors.

Soil enzyme activities, soil physical properties, photosynthetic physical characteristics and water use of winter wheat after long-term straw mulch and organic fertilizer application.

Introduction: Inappropriate residue and nutrient management leads to soil degradation and the decline of soil quality and water storage capacity. Methods: An ongoing field experiment has been conducted since 2011 to investigate the effects of straw mulching (SM), and straw mulching combined with organic fertilizer (SM+O), on winter wheat yield, including a control treatment (CK, no straw). We studied the effects of these treatments on soil microbial biomass nitrogen and carbon, soil enzyme activity in 2019, photosynthetic parameters, evapotranspiration (ET), water use efficiency (WUE), and yields over five consecutive years (2015-2019). We also analyzed the soil organic carbon, soil structure, field capacity, and saturated hydraulic conductivity in 2015 and 2019. Results: Results indicate that compared with CK, SM and SM+O treatments increased the proportion of >0.25mm aggregates, soil organic carbon, field capacity, and saturated hydraulic conductivity, but decreased the soil bulk density. In addition, the SM and SM+O treatments also increased soil microbial biomass nitrogen and carbon, the activity of soil enzymes, and decreased the carbon-nitrogen ratio of microbial biomass. Therefore, SM and SM+O treatments both increased the leaf water use efficiency (LWUE) and photosynthetic rate (Pn), and improved the yields and water use efficiency (WUE) of winter wheat. The combination SM (4.5 t/ha)+O (0.75 t/ha) was more effective than SM alone, and both treatments were superior to the control. Conclusion: Based on the results of this study, SM+O is recommended as the most effective cultivation practice.

Acid-base transformative HADLA micelles alleviate colitis by restoring adaptive immunity and gut microbiome.

Inflammatory bowel disease (IBD) is a worldwide public health issue with an increasing number of patients annually. However, there is no curative drug for IBD, and the present medication for IBD generally focuses on suppressing hyperactive immune responses, which can only delay disease progression but inevitably induce off-target side effects, including infections and cancers. Herein, late-model orally administered nanotherapeutic micelles (HADLA) were developed based on a conjugate of hyaluronic acid (HA) and dehydrolithocholic acid (DLA), which was simple to achieve and obtained satisfactory therapeutic efficacy in a murine colitis model with a full safety profile. HADLA is capable of targeting inflammatory colon tissues, restoring intestinal barrier function and reducing intestinal epithelial cell death. Moreover, it modulates the adaptive immune system by inhibiting the activation of pathogenic T helper 17 (Th17) cells, and it exhibits more remarkable effects in preventing colitis than DLA alone. Finally, HADLA exhibits a remarkable ability to modulate dysregulated gut microbiomes by increasing beneficial probiotics and decreasing pathogenic bacteria, such as Turicibacter. Compared with the current systemic or subcutaneous administration of biologics, this study opens new avenues in the oral delivery of immune-modulating nanomedicine and introduces DLA as a new medication for IBD treatment.

Copper-catalyzed C-H oxidation/cross-coupling of α-amino carbonyl compounds.

Keeping options open: the new and mild title reaction involving indoles selectively furnishes 1 and 2 with the aid of tert-butyl hydroperoxide (TBHP). The method represents the first example of a copper-catalyzed α arylation of α-amino carbonyl substrates leading to α-aryl α-imino and α-aryl α-oxo carbonyl compounds using a C-H oxidation strategy.