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Background: Targeting the immune checkpoint pathway has demonstrated antitumor cytotoxicity in treatment-refractory head and neck squamous cell carcinoma (HNSC). To understand the molecular mechanisms underpinning its antitumor response, we characterized the immune landscape of HNSC by their tumor and stromal compartments to identify novel immune molecular subgroups. Patients and methods:A training cohort of 522 HNSC samples from the Cancer Genome Atlas profiled by RNA sequencing was analyzed. We separated gene expression patterns from tumor, stromal, and immune cell gene using a non-negative matrix factorization algorithm. We correlated the expression patterns with a set of immune-related gene signatures, potential immune biomarkers, and clinicopathological features. Six independent datasets containing 838 HNSC samples were used for validation.Results: Approximately 40% of HNSCs in the cohort (211/522) were identified to show enriched inflammatory response, enhanced cytolytic activity, and active interferon-c signaling (all, P < 0.001). We named this new molecular class of tumors the Immune Class. Then we found it contained two distinct microenvironment-based subtypes, characterized by markers of active or exhausted immune response. The Exhausted Immune Class was characterized by enrichment of activated stroma and antiinflammatory M2 macrophage signatures, WNT/transforming growth factor-b signaling pathway activation and poor survival (all, P < 0.05). An enriched proinflammatory M1 macrophage signature, enhanced cytolytic activity, abundant tumor-infiltrating lymphocytes, high human papillomavirus (HPV) infection, and favorable prognosis were associated with Active Immune Class (all, P < 0.05). The robustness of these immune molecular subgroups was verified in the validation cohorts, and Active Immune Class showed potential response to programmed cell death-1 blockade (P ¼ 0.01).

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We performed a comprehensive immuno-genomic analysis of tumor microenvironment immune types (TMITs), which is classified into four groups based on PD-L1+CD8A or PD-L1+cytolytic activity (CYT) expression, across a broad spectrum of solid tumors in order to help identify patients who will benefit from anti- PD-1/PD-L1 therapy. The mRNA sequencing data from The Cancer Genome Atlas (TCGA) of 14 solid cancer types representing 6,685 tumor samples was analyzed. TMIT was classified only for those tumor types that both PD-L1 and CD8A/CYT could prefict mutation and/or neoantigen number. The mutational and neoepitope features of the tumor were compared according to the four TMITs. We found that PD-L1/CD8A/CYT subgroups could not distinguish different mutation and neoantigen numbers in certain tumor types such as glioblastoma multiforme, prostate adenocarcinoma, and head and neck and lung squamous cell carcinoma. For the remaining tumor types, compared with TIMT II (low PD-L1 and CD8A/CYT), TIMT I (high PD-L1 and CD8A/CYT) had a significantly higher number of mutations or neoantigens in bladder urothelial carcinoma, breast and cervical cancer, colorectal, stomach and lung adenocarcinoma, and melanoma. In contrast, TMIT I of kidney clear cell, liver hepatocellular, and thyroid carcinoma were negatively correlated with mutation burden or neoantigen numbers. Our findings show that the TMIT stratification proposed could serve as a favorable approach for tailoring optimal immunotherapeutic strategies in certain tumor types. Going forward, it will be important to test the clinical practicability of TMIT based on quantification of immune infiltrates using mRNA-seq to predict clinical response to these and other immunotherapeutic strategies in more different tumors.

These authors contributed equally to this work. SUMMARYStarch is the most widespread form of energy storage in the plant kingdom. Although many enzymes and related factors have been identified for starch biosynthesis, unknown players remain to be identified, given that it is a complicated and sophisticated process. The endosperm of rice (Oryza sativa) has been used for the study of starch synthesis. Here, we report the cloning and characterization of the FLOURY ENDO-SPERM6 (FLO6) gene in rice. In the flo6 mutant, the starch content is decreased and the normal physicochemical features of starch are changed. Significantly, flo6 mutant endosperm cells show obvious defects in compound granule formation. Map-based cloning showed that FLO6 encodes a protein of unknown function. It harbors an N-terminal transit peptide that ensures its correct localization and functions in the plastid, and a C-terminal carbohydrate-binding module 48 (CBM48) domain that binds to starch. Furthermore, FLO6 can interact with isoamylase1 (ISA1) both in vitro and in vivo, whereas ISA1 does not bind to starch directly. We thus propose that FLO6 may act as a starch-binding protein involved in starch synthesis and compound granule formation through a direct interaction with ISA1 in developing rice seeds. Our data provide a novel insight into the role of proteins with the CBM48 domain in plant species.

The efficient delivery of biopharmaceutical drugs such as proteins and peptides into the cytosol of target cells poses substantial challenges owing to their large size and susceptibility to degradation. Current protein delivery vehicles have limitations such as the need for protein modification, insufficient delivery of large-size proteins or small peptides, and loss of protein function after the delivery. Here, we adopted a rational approach to design a polymer with robust efficacy for intracellular protein and peptide delivery. The polymer is composed of a dendrimer scaffold, a hydrophobic membrane-disruptive region, and a multivalent protein binding surface. It allows efficient protein/peptide binding, endocytosis, and endosomal disruption and is capable of efficiently delivering various biomacromolecules including bovine serum albumin, R-phycoerythrin, p53, saporin, β-galactosidase, and peptides into the cytosol of living cells. Transduction of apoptotic proteins and peptides successfully induces apoptosis in cancer cells, suggesting that the activities of proteins and peptides are maintained during the delivery. This technology represents an efficient and useful tool for intracellular protein and peptide delivery and has broad applicability for basic research and clinical applications.

Cytosolic protein delivery is a prerequisite for the development of protein therapeutics that act on intracellular targets. Proteins are generally membrane‐impermeable and thus need a carrier such as a polymer to facilitate their internalization. However, the efficient binding of proteins with different isoelectric points to polymeric carriers is challenging. In this study, we designed a coordinative dendrimer to solve this problem. The dendrimers modified with dipicolylamine/zinc(II) complex were capable of binding proteins through a combination of ionic and coordination interactions. The best polymer efficiently delivered 30 cargo proteins and peptides into the cytosol, while maintaining their bioactivity after intracellular release. The removal or replacement of zinc ions in the polymer with other transition‐metal ions lead to significantly decreased efficiency in cytosolic protein delivery. This study provides a new strategy to develop robust and efficient polymers for cytosolic protein delivery.

Biomedical applications of fluoropolymers in gene delivery, protein delivery, drug delivery, 19F MRI, PDT, anti-fouling, anti-bacterial, cell culture, and tissue engineering.