The Immune Contexture and Cancer Therapy Aspects of the TIM-3 Checkpoint Pathway

The advent of immune checkpoint therapy revolutionizes the oncology field by achieving durable anti-tumor efficacy and immense clinical progress. Among the immune checkpoints, T cell immunoglobulin and mucin domain 3 (TIM-3) has been reported to express on both innate and adaptive immune cells where it accounts for immune tolerance and tumor aggression. It shows promising effects that are applicable in several cancer types. This review provides a broad overview of the main TIM-3 relative signaling pathways and its immune regulatory role to shape cancer development positively. The importance of TIM-3 inhibitors in cancer control were also summarized according to the latest reports, discussing its clinical trial data that inform the rationale for TIM-3 blockade

Nonplanar On-shell Diagrams and Leading Singularities of Scattering Amplitudes

Bipartite on-shell diagrams are the latest tool in constructing scattering amplitudes. In this paper we prove that a Britto-Cachazo-Feng-Witten (BCFW)-decomposable on-shell diagram process a rational top-form if and only if the algebraic ideal comprised of the geometrical constraints is shifted linearly during successive BCFW integrations. With a proper geometric interpretation of the constraints in the Grassmannian manifold, the rational top-form integration contours can thus be obtained, and understood, in a straightforward way. All rational top-form integrands of arbitrary higher loops leading singularities can therefore be derived recursively, as long as the corresponding on-shell diagram is BCFW-decomposable.Comment: 13 pages with 12 figures; final version appeared in Eur.Phys.J. C77 (2017) no.2, 8

Improved production of docosahexaenoic acid in batch fermentation by newly-isolated strains of Schizochytrium sp. and Thraustochytriidae sp. through bioprocess optimization

Thraustochytrids, rich in docosahexaenoic acid (DHA, C22:6??3), represent a potential source of dietary fatty acids. Yet, the effect of culture conditions on growth and fatty acid composition vary widely among different thraustochytrid strains. Two different thraustochytrid strains, Schizochytrium sp. PKU#Mn4 and Thraustochytriidae sp. PKU#Mn16 were studied for their growth and DHA production characteristics under various culture conditions. Although they exhibited similar fatty acid profiles, PKU#Mn4 seemed a good candidate for industrial DHA fermentation while PKU#Mn16 displayed growth tolerance to a wide range of process conditions. Relative DHA content of 48.5% and 49.2% (relative to total fatty acids), respectively, were achieved on glycerol under their optimal flask culture conditions. Maximum DHA yield (Yp/x) of 21.0% and 18.9% and productivity of 27.6 mg/L-h and 31.9 mg/L-h were obtained, respectively, in 5-L bioreactor fermentation operated with optimal conditions and dual oxygen control strategy. A 3.4- and 2.8-fold improvement of DHA production (g/L), respectively, was achieved in this study. Overall, our study provides the potential of two thraustochytrid strains and their culture conditions for efficient production of DHA-rich oil

Transformation of \u3ci\u3eFusarium verticillioides\u3c/i\u3e with a polyketide gene cluster isolated from a fungal endophyte activates the biosynthesis of fusaric acid

A large number of bioactive natural products have been isolated from plant endophytic fungi. However, molecular mechanisms for the biosynthesis of these metabolites have lagged behind because genetic and biochemical studies are difficult to perform within many of the endophytes. In this work, we describe our attempt to express a putative mycoepoxydiene (MED) biosynthetic gene cluster in Fusarium verticillioides, which has a well-developed genetic system for the study fungal polyketide biosynthesis. MED was isolated from Phomopsis sp. A123, a fungal endophyte of the mangrove plant, Kandelia candel. It has several unusual structural features and interesting biological activities. Integration of this Phomopsis gene cluster into the F. verticillioides genome led to the biosynthesis of multiple metabolites. The most highly activated metabolite was isolated and its structure was shown by 1D- and 2D-NMR to be fusaric acid, which is a mycotoxin in Fusarium species and is implicated in fungal pathogenesis. Although fusaric acid was isolated more than 70 years ago, its biosynthetic mechanism remains unclear. These transformants produced 30–35 mg fusaric acid per 100 ml culture. The high level production of fusaric acid will greatly facilitate the genetic and biochemical study of its biosynthetic mechanism. Although we have not detected MED or its analogs from the heterologous host, this work represents the first attempt to express a fungal endophytic gene cluster in a Fusarium species

Iterative Assembly of Two Separate Polyketide Chains by the Same Single-module Bacterial Polyketide Synthase in the Biosynthesis of HSAF

HSAF (1) was isolated from the biocontrol agent Lysobacter enzymogenes (Figure 1).[1-4] This bacterial metabolite belongs to polycyclic tetramate macrolactams (PTM) that are emerging as a new class of natural products with distinct structural features. [5, 6] HSAF exhibits a potent antifungal activity and shows a novel mode of action.[1-4] The HSAF biosynthetic gene cluster contains only a single-module hybrid polyketide synthasenonribosomal peptide synthetase (PKS-NRPS), although the PTM scaffold is apparently derived from two separate hexaketide chains and an ornithine residue.[1-4] This suggests that the same PKS module would act not only iteratively, but also separately, in order to link the two hexaketide chains with the NRPS-activated ornithine to form the characteristic PTM scaffold. Recently, the Gulder group reported heterologous expression of the ikarugamycin (4) biosynthetic gene cluster in E. coli,[7] and the Zhang group reported the enzymatic mechanism for formation of the inner 5-memebered ring and demonstrated the polyketide origin of the ikarugamycin skeleton.[8] Ikarugamycin is a Streptomyces-derived PTM which has a 5,6,5-tricyclic system (Figure 1). Both the Gulder and Zhang groups showed that a three-gene cluster is sufficient for ikarugamycin biosynthesis. Despite the progress, this iterative polyketide biosynthetic mechanism had not been demonstrated using purified PKS and NRPS. In addition, HSAF has a 5,5,6-tricyclic system, and its gene cluster contains at least six genes.[3] Finally, unlike most PTM compounds, HSAF is produced by a Gramnegative bacterium, L. enzymogenes. Here, we report the heterologous production of HSAF analogs in Gram-positive Streptomyces hosts, in which the native PKS have been deleted. We also obtained evidence for the formation of the polyene tetramate intermediate in Streptomyces when only the single-module hybrid PKS-NRPS gene was expressed. Finally, we showed the in vitro production of the polyene tetramate using the individually purified PKS and NRPS. The results provide direct evidence for this iterative polyketide biosynthetic mechanism that is likely general for the PTM-type hybrid polyketide-peptides

Biosynthesis of HSAF, a Tetramic Acid-containing Macrolactam from \u3ci\u3eLysobacter enzymogenes\u3c/i\u3e

HSAF was isolated from Lysobacter enzymogenes, a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here, we described the HSAF biosynthetic gene cluster which contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS/ NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/ NRPS are six genes, encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activated L-ornithine and the fourdomain NRPS was able to catalyze the formation of a tetramic acid-containing product from acyl- S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms

Biosynthesis of HSAF, a Tetramic Acid-containing Macrolactam from \u3ci\u3eLysobacter enzymogenes\u3c/i\u3e

HSAF was isolated from Lysobacter enzymogenes, a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here, we described the HSAF biosynthetic gene cluster which contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS/ NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/ NRPS are six genes, encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activated L-ornithine and the fourdomain NRPS was able to catalyze the formation of a tetramic acid-containing product from acyl- S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms

Multi-ancestry genome-wide association meta-analysis of Parkinson?s disease

Although over 90 independent risk variants have been identified for Parkinson’s disease using genome-wide association studies, most studies have been performed in just one population at a time. Here we performed a large-scale multi-ancestry meta-analysis of Parkinson’s disease with 49,049 cases, 18,785 proxy cases and 2,458,063 controls including individuals of European, East Asian, Latin American and African ancestry. In a meta-analysis, we identified 78 independent genome-wide significant loci, including 12 potentially novel loci (MTF2, PIK3CA, ADD1, SYBU, IRS2, USP8, PIGL, FASN, MYLK2, USP25, EP300 and PPP6R2) and fine-mapped 6 putative causal variants at 6 known PD loci. By combining our results with publicly available eQTL data, we identified 25 putative risk genes in these novel loci whose expression is associated with PD risk. This work lays the groundwork for future efforts aimed at identifying PD loci in non-European populations

Studies on the biosynthesis of the antifungal polycyclic tetramate macrolactam HSAF and the AMPK-activating mycoepoxydiene

In search for antifungal compounds with novel chemistry and a new mode of action, we isolated a heat stable antifungal factor (HSAF) from Lysobacter enzymogenes C3. HSAF was determined to be dihydromaltophilin, a polycyclic tetramate macrolactam with novel antifungal mechanism. Our ultimate goal is to develop this type of compounds as new antifungal drug leads. As a key step toward this goal, we have investigated the mechanism for HSAF biosynthesis, its regulation and genetic engineering. Firstly, we constructed two NRPS adenylation domain swapping mutants (replacing ornithine adenylation domain with lysine adenylation domain) with expectation to generate the analogue of HSAF with one additional methylene group. The expected compound was not detected in the resulted strain, possibly due to the miscommunication between domains within the single-modular PKS-NRPS, the key enzyme for the construction of the HSAF skeleton. To explore the enzymes involving in the post-PKS-NRPS biosynthesis of HSAF, a zinc-dependent oxidoreductase (OX4) and a FAD-dependent oxidoreductase (OX3) were deleted. Both mutants abolished the production of HSAF. Then we isolated a cosmid that bears the complete HSAF biosynthetic gene cluster, and transferred the cosmid into Streptomyces for heterologous expression. Metabolite analysis indicates that HSAF and its analogues were produced in the transformed Streptomyces strains. This result demonstrates that a one-module PKS can synthesize two separate polyketide chains, an unprecedented mechanism for bacterial polyketide biosynthesis. In probing the regulatory mechanism of HSAF biosynthesis, we identified a cAMP receptor like protein (Clp) and demonstrated that a cis-acting element within its promoter region was essential for the regulation. In the fourth project, a cosmid bearing the complete gene cluster for mycoepoxydiene biosynthesis was transformed into Fusarium verticilloides for heterologous expression. Mycoepoxydiene was isolated from a fungal endophyte of a mangrove plant with unusual structure features and a strong AMPK-activating activity. The metabolites analysis of the Fusarium verticilloides mutants led to identification of fusaric acid, a mycotoxin with significance in food safety and agriculture

CD47 BLOCKING IMMUNOSTIMULATORY ANTIBODY CONJUGATES ELICIT ROBUST TUMOR-SPECIFIC IMMUNITY FROM PHAGOCYTIC CELLS

Cancer Immunotherapies targeting adaptive immune checkpoints have been substantially explored as they elicited durable clinical responses across multiple types of cancers. Asides from the success of anti-CTLA-4 and anti-PD-1/PD-L1 immune checkpoint inhibitors, more recent findings highlight the significance of checkpoints in the innate arm of immunity. As an innate immune checkpoint, CD47 has been shown to be highly expressed by various types of solid and hematologic tumors and to be correlated with aggressive disease and poor patient outcomes. CD47 delivers an inhibitory “don’t eat me” signal to macrophages through SIRP⍺, enabling the escape of cancer cells from macrophage-mediated phagocytosis. A growing number of studies have shown that interrupts the CD47-SIRP⍺ axis enhances the phagocytosis of tumor cells by macrophages and promotes the downstream activation of the adaptive immune system. However, in preclinical and early clinical stages, the blockade of CD47-SIRP⍺ still shows a relatively low response rate in the solid tumor. Solely blocking this phagocytosis checkpoint is not sufficient to engage macrophages, a pro-phagocytic cue as an “eat me” signal must also be present to activate the effector function of macrophages, which enhances the innate immune sensing and stimulates the essential immune surveillance process. Recent and ongoing clinical trials are demonstrating the therapeutic efficacy of CD47 blockade in combination with monoclonal antibodies, chemotherapy, or other checkpoint inhibitors for adult cancer. In this study, we propose an engineered CD47 antibody variant that exhibits strong immunostimulatory property while promoting tumor cell phagocytosis at the same time