Toll-like receptor 9 antagonizes antibody affinity maturation.

Key events in T cell-dependent antibody responses, including affinity maturation, are dependent on the B cell's presentation of antigen to helper T cells at critical checkpoints in germinal-center formation in secondary lymphoid organs. Here we found that signaling via Toll-like receptor 9 (TLR9) blocked the ability of antigen-specific B cells to capture, process and present antigen and to activate antigen-specific helper T cells in vitro. In a mouse model in vivo and in a human clinical trial, the TLR9 agonist CpG enhanced the magnitude of the antibody response to a protein vaccine but failed to promote affinity maturation. Thus, TLR9 signaling might enhance antibody titers at the expense of the ability of B cells to engage in germinal-center events that are highly dependent on B cells' capture and presentation of antigen.

Identification and structural characterization of a mutant KRAS-G12V specific TCR restricted by HLA-A3.

Mutations in KRAS are some of the most common across multiple cancer types and are thus attractive targets for therapy. Recent studies demonstrated that mutant KRAS generates immunogenic neoantigens that are targetable by adoptive T-cell therapy in metastatic diseases. To expand mutant KRAS-specific immunotherapies, it is critical to identify additional HLA-I allotypes that can present KRAS neoantigens and their cognate T-cell receptors (TCR). Here, we identified a murine TCR specific to a KRAS-G12V neoantigen (7VVVGAVGVGK16) using a vaccination approach with transgenic mice expressing HLA-A*03:01 (HLA-A3). This TCR demonstrated exquisite specificity for mutant G12V and not WT KRAS peptides. To investigate the molecular basis for neoantigen recognition by this TCR, we determined its structure in complex with HLA-A3(G12V). G12V-TCR CDR3ß and CDR1ß formed a hydrophobic pocket to interact with p6 Val of the G12V but not the WT KRAS peptide. To improve the tumor sensitivity of this TCR, we designed rational substitutions to improve TCR:HLA-A3 contacts. Two substitutions exhibited modest improvements in TCR binding avidity to HLA-A3 (G12V) but did not sufficiently improve T-cell sensitivity for further clinical development. Our study provides mechanistic insight into how TCRs detect neoantigens and reveals the challenges in targeting KRAS-G12V mutations.

A method of genetic transformation and gene editing of succulents without tissue culture.

Succulents, valued for their drought tolerance and ornamental appeal, are important in the floriculture market. However, only a handful of succulent species can be genetically transformed, making it difficult to improve these plants through genetic engineering. In this study, we adapted the recently developed cut-dip-budding (CDB) gene delivery system to transform three previously recalcitrant succulent varieties - the dicotyledonous Kalanchoe blossfeldiana and Crassula arborescens and the monocotyledonous Sansevieria trifasciata. Capitalizing on the robust ability of cut leaves to regenerate shoots, these plants were successfully transformed by directly infecting cut leaf segments with the Agrobacterium rhizogenes strain K599. The transformation efficiencies were approximately 74%, 5% and 3.9%-7.8%, respectively, for K. blossfeldiana and C. arborescens and S. trifasciata. Using this modified CDB method to deliver the CRISPR/Cas9 construct, gene editing efficiency in K. blossfeldiana at the PDS locus was approximately 70%. Our findings suggest that succulents with shoot regeneration ability from cut leaves can be genetically transformed using the CDB method, thus opening up an avenue for genetic engineering of these plants.

High-affinity oligoclonal TCRs define effective adoptive T cell therapy targeting mutant KRAS-G12D.

Complete cancer regression occurs in a subset of patients following adoptive T cell therapy (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). However, the low success rate presents a great challenge to broader clinical application. To provide insight into TIL-based immunotherapy, we studied a successful case of ACT where regression was observed against tumors carrying the hotspot mutation G12D in the KRAS oncogene. Four T cell receptors (TCRs) made up the TIL infusion and recognized two KRAS-G12D neoantigens, a nonamer and a decamer, all restricted by human leukocyte antigen (HLA) C*08:02. Three of them (TCR9a, 9b, and 9c) were nonamer-specific, while one was decamer-specific (TCR10). We show that only mutant G12D but not the wild-type peptides stabilized HLA-C*08:02 due to the formation of a critical anchor salt bridge to HLA-C. Therapeutic TCRs exhibited high affinities, ranging from nanomolar to low micromolar. Intriguingly, TCR binding affinities to HLA-C inversely correlated with their persistence in vivo, suggesting the importance of antigenic affinity in the function of therapeutic T cells. Crystal structures of TCR-HLA-C complexes revealed that TCR9a to 9c recognized G12D nonamer with multiple conserved contacts through shared CDR2ß and CDR3α. This allowed CDR3ß variation to confer different affinities via a variable HLA-C contact, generating an oligoclonal response. TCR10 recognized an induced and distinct G12D decamer conformation. Thus, this successful case of ACT included oligoclonal TCRs of high affinity recognizing distinct conformations of neoantigens. Our study revealed the potential of a structural approach to inform clinical efforts in targeting KRAS-G12D tumors by immunotherapy and has general implications for T cell-based immunotherapies.

Canagliflozin Attenuates Lipotoxicity in Cardiomyocytes by Inhibiting Inflammation and Ferroptosis through Activating AMPK Pathway.

Diabetic cardiomyopathy (DCM) is a myocardial disease independent of other cardiovascular diseases, such as coronary heart disease, hypertension, etc. Lipotoxicity is closely related to DCM. In this study, we investigated the mechanism of lipid metabolism disturbance in DCM in HL-1 cells. Through bioinformatics and Western blotting analysis, we found that canagliflozin (CAN) significantly inhibited the expression of inflammatory factors cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Ferroptosis is mediated by lipid peroxidation. We demonstrated the presence of ferroptosis in cardiomyocytes by detecting intracellular Fe2+ content and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH), and mitochondrial membrane potential (MMP). CAN could significantly regulate the indicators of ferroptosis. By using specific inhibitors celecoxib (coxib), S-methylisothiourea sulfate (SMT), Ferrostatin-1 (Fer-1), and Compound C, we further found that CAN regulated inflammation and ferroptosis through AMP-activated protein (AMPK), and inflammation interacted with ferroptosis. Our study indicated that CAN attenuated lipotoxicity in cardiomyocytes by regulating inflammation and ferroptosis through activating the AMPK pathway. This study provides a new direction of myocardial lipotoxicity and some new information for the treatment of DCM.

All-flesh fruit in tomato is controlled by reduced expression dosage of AFF through a structural variant mutation in the promoter.

The formation of locule gel is an important process in tomato and is a typical characteristic of berry fruit. In this study, we examined a natural tomato mutant that produces all-flesh fruit (AFF) in which the locule tissue remains in a solid state during fruit development. We constructed different genetic populations to fine-map the causal gene for this trait and identified SlMBP3 as the locus conferring the locule gel formation, which we rename as AFF. We determined the causal mutation as a 416-bp deletion in the promoter region of AFF, which reduces its expression dosage. Generally, this sequence is highly conserved among Solanaceae, as well as within the tomato germplasm. Using BC6 near-isogenic lines, we determined that the reduced expression dosage of AFF did not affect the normal development of seeds, whilst producing unique, non-liquefied locule tissue that was distinct from that of normal tomatoes in terms of metabolic components. Combined analysis using mRNA-seq and metabolomics indicated the importance of AFF in locule tissue liquefaction. Our findings provide insights into fruit-type differentiation in Solanaceae crops and also present the basis for future applications of AFF in tomato breeding programs.

Structure of MHC-Independent TCRs and Their Recognition of Native Antigen CD155.

During normal T cell development in the thymus, αß TCRs signal immature thymocytes to differentiate into mature T cells by binding to peptide-MHC ligands together with CD4/CD8 coreceptors. Conversely, in MHC and CD4/CD8 coreceptor-deficient mice, the thymus generates mature T cells expressing MHC-independent TCRs that recognize native conformational epitopes rather than linear antigenic-peptides presented by MHC. To date, no structural information of MHC-independent TCRs is available, and their structural recognition of non-MHC ligand remains unknown. To our knowledge in this study, we determined the first structures of two murine MHC-independent TCRs (A11 and B12A) that bind with high nanomolar affinities to mouse adhesion receptor CD155. Solution binding demonstrated the Vαß-domain is responsible for MHC-independent B12A recognition of its ligand. Analysis of A11 and B12A sequences against various MHC-restricted and -independent TCR sequence repertoires showed that individual V-genes of A11 and B12A did not exhibit preference against MHC-restriction. Likewise, CDR3 alone did not discriminate against MHC binding, suggesting VDJ recombination together with Vα/Vß pairing determine their MHC-independent specificity for CD155. The structures of A11 and B12A TCR are nearly identical to those of MHC-restricted TCR, including the conformations of CDR1 and 2. Mutational analysis, together with negative-staining electron microscopy images, showed that the CDR regions of A11 and B12A recognized epitopes on D1 domain of CD155, a region also involved in CD155 binding to poliovirus and Tactile in human. Taken together, MHC-independent TCRs adopt canonical TCR structures to recognize native Ags, highlighting the importance of thymic selection in determining TCR ligand specificity.

High affinity binding of SARS-CoV-2 spike protein enhances ACE2 carboxypeptidase activity.

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) has emerged to a pandemic and caused global public health crisis. Human angiotensin-converting enzyme 2(ACE2) was identified as the entry receptor for SARS-CoV-2. As a carboxypeptidase, ACE2 cleaves many biological substrates besides angiotensin II to control vasodilatation and vascular permeability. Given the nanomolar high affinity between ACE2 and SARS-CoV-2 spike protein, we investigated how this interaction would affect the enzymatic activity of ACE2. Surprisingly, SARS-CoV-2 trimeric spike protein increased ACE2 proteolytic activity ∼3-10 fold against model peptide substrates, such as caspase-1 substrate and Bradykinin-analog. The enhancement in ACE2 enzymatic function was mediated by the binding of SARS-CoV-2 spike RBD domain. These results highlighted the potential for SARS-CoV-2 infection to enhance ACE2 activity, which may be relevant to the cardiovascular symptoms associated with COVID-19.

αß T cell receptors that do not undergo major histocompatibility complex-specific thymic selection possess antibody-like recognition specificities.

Major histocompatibility complex (MHC) restriction is the cardinal feature of T cell antigen recognition and is thought to be intrinsic to αß T cell receptor (TCR) structure because of germline-encoded residues that impose MHC specificity. Here, we analyzed αßTCRs from T cells that had not undergone MHC-specific thymic selection. Instead of recognizing peptide-MHC complexes, the two αßTCRs studied here resembled antibodies in recognizing glycosylation-dependent conformational epitopes on a native self-protein, CD155, and they did so with high affinity independently of MHC molecules. Ligand recognition was via the αßTCR combining site and involved the identical germline-encoded residues that have been thought to uniquely impose MHC specificity, demonstrating that these residues do not only promote MHC binding. This study demonstrates that, without MHC-specific thymic selection, αßTCRs can resemble antibodies in recognizing conformational epitopes on MHC-independent ligands.

Selection of Plasmodium falciparum cytochrome B mutants by putative PfNDH2 inhibitors.

Malaria control is threatened by a limited pipeline of effective pharmaceuticals against drug-resistant strains of Plasmodium falciparum Components of the mitochondrial electron transport chain (ETC) are attractive targets for drug development, owing to exploitable differences between the parasite and human ETC. Disruption of ETC function interferes with metabolic processes including de novo pyrimidine synthesis, essential for nucleic acid replication. We investigated the effects of ETC inhibitor selection on two distinct P. falciparum clones, Dd2 and 106/1. Compounds CK-2-68 and RYL-552, substituted quinolones reported to block P. falciparum NADH dehydrogenase 2 (PfNDH2; a type II NADH:quinone oxidoreductase), unexpectedly selected mutations at the quinol oxidation (Qo) pocket of P. falciparum cytochrome B (PfCytB). Selection experiments with atovaquone (ATQ) on 106/1 parasites yielded highly resistant PfCytB Y268S mutants seen in clinical infections that fail ATQ-proguanil treatment. In contrast, ATQ pressure on Dd2 yielded moderately resistant parasites carrying a PfCytB M133I or K272R mutation. Strikingly, all ATQ-selected mutants demonstrated little change or slight increase of sensitivity to CK-2-68 or RYL-552. Molecular docking studies demonstrated binding of all three ETC inhibitors to the Qo pocket of PfCytB, where Y268 forms strong van der Waals interactions with the hydroxynaphthoquinone ring of ATQ but not the quinolone ring of CK-2-68 or RYL-552. Our results suggest that combinations of suitable ETC inhibitors may be able to subvert or delay the development of P. falciparum drug resistance.

Pineapple Leaf Phenols Attenuate DSS-Induced Colitis in Mice and Inhibit Inflammatory Damage by Targeting the NF-κB Pathway.

Colitis is not fully curable, although currently, some treatment options are being adopted. In this study, we investigated the effects of pineapple leaf phenols (PLPs), natural phenol products from pineapple leaves, on DSS-induced colitis in mice. The results showed that PLPs dramatically decreased the inflammatory response by inhibiting NF-κB activation and the secretion of pro-inflammatory factors. Moreover, PLPs provided protection against DSS-induced acute colitis by maintaining epithelial integrity. Caffeic and P-coumaric acids had similar effects and could be the active components responsible for PLPs' effect on colitis. These results indicate that the oral administration of PLPs might be considered as a therapeutic strategy in the treatment of patients with colitis. However, further research on clinical applications and the exact effect of PLPs on colitis is required.

Helios+ and Helios- Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires.

The transcription factor Helios is expressed in a large subset of Foxp3+ Tregs. We previously proposed that Helios is a marker of thymic derived Treg (tTreg), while Helios- Treg were induced from Foxp3- T conventional (Tconv) cells in the periphery (pTreg). To compare the two Treg subpopulations, we generated Helios-GFP reporter mice and crossed them to Foxp3-RFP reporter mice. The Helios+ Treg population expressed a more activated phenotype, had a slightly higher suppressive capacity in vitro and expressed a more highly demethylated TSDR but were equivalent in their ability to suppress inflammatory bowel disease in vivo. However, Helios+ Treg more effectively inhibited the proliferation of activated, autoreactive splenocytes from scurfy mice. When Helios+ and Helios- Treg were transferred to lymphoreplete mice, both populations maintained comparable Foxp3 expression, but Foxp3 expression was less stable in Helios- Treg when transferred to lymphopenic mice. Gene expression profiling demonstrated a large number of differentially expressed genes and showed that Helios- Treg expressed certain genes normally expressed in CD4+ Foxp3- T cells. TCR repertoire analysis indicated very little overlap between Helios+ and Helios- Treg. Thus, Helios+ and Helios- Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires.

Development of an economic ultrafast liquid chromatography with tandem mass spectrometry method for trace analysis of multiclass mycotoxins in Polygonum multiflorum.

Rapid, economic, and highly effective determination of multiple mycotoxins in complex matrices has given huge challenges for the analytical method. In this study, an economic analytical strategy based on sensitive and rapid ultrafast liquid chromatography coupled to hybrid triple quadrupole/linear ion trap mass spectrometry technique was developed for the determination of seven mycotoxins of different chemical classes (aflatoxin B1 , B2 , G1 , and G2 , ochratoxin A, T-2 toxin, and HT-2 toxin) in Polygonum multiflorum. Target mycotoxins were completely extracted using a modified quick, easy, cheap effective, rugged, and safe method without additional clean-up steps. The types of extraction solvents and adsorbents for the extraction procedure were optimized to achieve high recoveries and reduce coextractives in the final extracts. Due to significant matrix effects for all analytes (≤68.9% and ≥110.0%), matrix-matched calibration curves were introduced for reliable quantification, exploring excellent linearity for the seven mycotoxins with coefficients of determination >0.9992. The method allowed high sensitivity with limit of detection in the range of 0.031-2.5 µg/kg and limit of quantitation in the range of 0.078-6.25 µg/kg, as well as satisfactory precision with relative standard deviations lower than 8%. Recovery rates were between 74.3 and 119.8% with relative standard deviations below 7.43%. The proposed method was successfully applied for 24 batches of P. multiflorum samples, and six samples were found to be positive with aflatoxin B1 , B2 , G1 , or ochratoxin A. The method with significant advantages, including minimum analytical time, low time and solvent consumption, and high sensitivity, would be a preferred candidate for economic analysis of multiclass mycotoxins in complex matrices.

Structural mechanism of high affinity FcγRI recognition of immunoglobulin G.

Antibody-based immunotherapies are becoming powerful means of modern medicine for treating cancers and autoimmune diseases. The increasing popularity of antibody-based treatment demands a better understanding of antibody functions and in particular, their interaction with Fc receptors as effectiveness of antibodies often depends on their ability to activate or avoid effector cell functions through Fc receptors. Until recently, our understanding of antibody recognition by Fc receptors is based on the structures of low affinity Fc receptor in complex with Fc. These structural studies provided significant insights to our understanding of how an IgG antibody generally docks on Fcγ receptor and the requirement of immune complex formation for effector cell activations. They are less informative, however, to the molecular forces underlying the vast different affinities between antibodies and their Fcγ receptors. Recently, the structure of the high affinity FcγRI in complex with IgG-Fc has been determined. This review will focus on the knowledge learned from the high affinity complex structural work and a potential receptor-glycan interaction as an important contribution to the receptor affinity.

[Study on famous prescription of prestigious traditional Chinese medicine master Lu Zhizheng based on QbD concept: optimization of extraction process and granule forming process of Meiyan Shuqi Prescription].

Based on the concept of quality by design( QbD),the design space of Meiyan Shuqi Prescription extraction process was established,and its granules forming process was optimized. The content of total flavonoids was the critical quality attribute( CQA).The amount of water,extraction time and frequency were used as the critical process parameters( CPPs). Box-Behnken experimental design was used to optimize the extraction process. The design space of Meiyan Shuqi Prescription extraction process was established and verified. The results of variance analysis showed that the P value of the quadratic regression model was less than 0. 01,which indicated that the model had a statistical significance,and the relationship between each factor and response value could be functionalized by the established model. Through further optimization of design space,the optimal extraction process of Meiyan Shuqi prescription was13-fold volume of water,extraction for 3 times and each extraction time of 1. 4 hours. Furthermore,the forming process optimized by single factor was as follows: the excipient was dextrin; the ratio of extract powder to excipient was 1 ∶1; the wetting agent was 70% ethanol; and the amount of wetting agent was 0. 3 m L·g-1. The Meiyan Shuqi Prescription extraction process and the forming process based on QbD concept provide reference for the study of other traditional Chinese medicine prescriptions.

Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding.

Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. The sole high-affinity IgG receptor, FcγRI plays a significant role in immunotherapy. To elucidate the molecular mechanism of its high-affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcγRI in complex with the Fc domain of human IgG1. FcγRI binds to the Fc in a similar mode as the low-affinity FcγRII and FcγRIII receptors. In addition to many conserved contacts, FcγRI forms additional hydrogen bonds and salt bridges with the lower hinge region of Fc. Unique to the high-affinity receptor-Fc complex, however, is the conformation of the receptor D2 domain FG loop, which enables a charged KHR motif to interact with proximal carbohydrate units of the Fc glycans. Both the length and the charge of the FcγRI FG loop are well conserved among mammalian species. Ala and Glu mutations of the FG loop KHR residues showed significant contributions of His-174 and Arg-175 to antibody binding, and the loss of the FG loop-glycan interaction resulted in an ∼ 20- to 30-fold decrease in FcγRI affinity to all three subclasses of IgGs. Furthermore, deglycosylation of IgG1 resulted in a 40-fold loss in FcγRI binding, demonstrating involvement of the receptor FG loop in glycan recognition. These results highlight a unique glycan recognition in FcγRI function and open potential therapeutic avenues based on antibody glycan engineering or small molecular glycan mimics to target FcγRI for certain autoimmune diseases.

[Research progress of pretreatment technology for mycotoxin detection in Chinese materia medica and complex matrices].

In the processes of planting, harvest, transport and storage, improper treatment of Chinese materia medica (CMM) and foodstuffs and agricultural products will result in fungal growth and mycotoxins contamination, which will not only directly affect the quality, safety and efficacy of these complex matrices, but also seriously threaten the consumers' health and lives. Therefore, the establishment of high-throughout analytical methods with high sensitivity for the determination of mycotoxins in CMM and foodstuffs and agricultural products at trace levels will provide reliable references for reducing the risk of mycotoxin exposure in humans. Due to the matrix complexity of CMM and foodstuffs and agricultural products, highly-effective pretreatment technologies are necessary for the establishment of such analytical techniques. In this review, the current extraction and purification methods commonly used for the detection of mycotoxins were summarized, the importance of pretreatment techniques for the precise quantification of mycotoxins in complex matrices such as Chinese herbal medicines was highlighted, as well as the development tendency about the pretreatment techniques for mycotoxins in complex matrices in the future was proposed.

Structural mechanism of serum amyloid A-mediated inflammatory amyloidosis.

Serum amyloid A (SAA) represents an evolutionarily conserved family of inflammatory acute-phase proteins. It is also a major constituent of secondary amyloidosis. To understand its function and structural transition to amyloid, we determined a structure of human SAA1.1 in two crystal forms, representing a prototypic member of the family. Native SAA1.1 exists as a hexamer, with subunits displaying a unique four-helix bundle fold stabilized by its long C-terminal tail. Structure-based mutational studies revealed two positive-charge clusters, near the center and apex of the hexamer, that are involved in SAA association with heparin. The binding of high-density lipoprotein involves only the apex region of SAA and can be inhibited by heparin. Peptide amyloid formation assays identified the N-terminal helices 1 and 3 as amyloidogenic peptides of SAA1.1. Both peptides are secluded in the hexameric structure of SAA1.1, suggesting that the native SAA is nonpathogenic. Furthermore, dissociation of the SAA hexamer appears insufficient to initiate amyloidogenic transition, and proteolytic cleavage or removal of the C-terminal tail of SAA resulted in formation of various-sized structural aggregates containing ∼5-nm regular repeating protofibril-like units. The combined structural and functional studies provide mechanistic insights into the pathogenic contribution of glycosaminoglycan in SAA1.1-mediated AA amyloid formation.

Structural Basis for Clonal Diversity of the Public T Cell Response to a Dominant Human Cytomegalovirus Epitope.

Cytomegalovirus (CMV) is a ubiquitous and persistent human pathogen that is kept in check by CD8(+) cytotoxic T lymphocytes. Individuals expressing the major histocompatibility complex (MHC) class I molecule HLA-A2 produce cytotoxic T lymphocytes bearing T cell receptors (TCRs) that recognize the immunodominant CMV epitope NLVPMVATV (NLV). The NLV-specific T cell repertoire is characterized by a high prevalence of TCRs that are frequently observed in multiple unrelated individuals. These public TCRs feature identical, or nearly identical, complementarity-determining region 3α (CDR3α) and/or CDR3ß sequences. The TCRs may express public CDR3α motifs alone, public CDR3ß motifs alone, or dual public CDR3αß motifs. In addition, the same public CDR3α motif may pair with different CDR3ß motifs (and the reverse), giving rise to highly diverse NLV-specific TCR repertoires. To investigate the structural underpinnings of this clonal diversity, we determined crystal structures of two public TCRs (C7 and C25) in complex with NLV·HLA-A2. These TCRs utilize completely different CDR3α and CDR3ß motifs that, in addition, can associate with multiple variable α and variable ß regions in NLV-specific T cell repertoires. The C7·NLV·HLA-A2 and C25·NLV·HLA-A2 complexes exhibit divergent TCR footprints on peptide-MHC such that C25 is more focused on the central portion of the NLV peptide than is C7. These structures combined with molecular modeling show how the public CDR3α motif of C25 may associate with different variable α regions and how the public CDR3α motif of C7 may pair with different CDR3ß motifs. This interchangeability of TCR V regions and CDR3 motifs permits multiple structural solutions to binding an identical peptide-MHC ligand and thereby the generation of a clonally diverse public T cell response to CMV.

Nonclassical Phosphomolybdates with Different Degrees of Reduction: Syntheses and Structural and Photo/Electrocatalytic Properties.

Four nonclassical phosphomolybdates, formulated as (H2pytty)8[{Mn(H2pytty)(H2O)3}{Sr⊂P6Mo6(V) Mo12(VI)O73}]2·16H2O (1), [{Mn(H3pytty)(H2O)3}2{Sr⊂P6Mo4(V)Mo14(VI)O73}]·18H2O (2), (H3pytp) (H2pytty)2[{Fe(H2O)4}{Sr⊂P6Mo3(V)Mo15(VI)O73}]·5H2O (3), and (H2pytty)2[{Cd(H2O)4}{Cd(H2O)3 (H3pytty)}{Sr⊂P6Mo5(V)Mo13(VI)O73}]·9H2O (4) (pytty = 3-(pyrazin-2-yl)-5-(1H-1,2,4-triazol-3-yl)-1,2,4-triazolyl; pytp = 4'-(4″-pyridyl)-2,4':6',4″-terpyridine) were hydrothermally synthesized and fully characterized. The penta- and hexa-reduced basket clusters represent the highest reduced level of basket-based polyoxometalate so far. In addition, transition metal complexes as bridge units were introduced to basket system for the first time to induce rare two-dimensional inorganic-organic hybrid layer. The results indicate that reduced degrees of the basket clusters of compounds 1-4 can be tuned by altering the molar ratio of organic ligand pytty and Na2MoO4. Compounds 1-4 exhibit bifunctional electrocatalytic behaviors for oxidation of dopamine and reduction of H2O2. Electrocatalytic mechanism, chronoamperometric experiments and electrocatalytic stability are studied in detail. In addition, the compounds show highly efficient catalytic ability for the degradation of typical dyes under UV irradiation.