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This article is a Commentary on Wang et al., 216: 205–215.

A number of genes that confer resistance to coffee leaf rust (SH1–SH9) have been identified within the genus Coffea, but despite many years of research on this pathosystem, the complementary avirulence genes of Hemileia vastatrix have not been reported. After identification of H. vastatrix effector candidate genes (HvECs) expressed at different stages of its lifecycle, we established an assay to characterize HvEC proteins by delivering them into coffee cells via the type‐three secretion system (T3SS) of Pseudomonas syringae pv. Garcae (Psgc). Employing a calmodulin‐dependent adenylate cyclase assay, we demonstrate thatPsgc recognizes a heterologous P. syringae T3SS secretion signal which enables us to translocate HvECs into the cytoplasm of coffee cells. Using this Psgc‐adapted effector detector vector (EDV) system, we found that HvEC‐016 suppresses the growth of Psgcon coffee genotypes with the SH1 resistance gene. Suppression of bacterial blight symptoms in SH1 plants was associated with reduced bacterial multiplication. By contrast, HvEC‐016 enhanced bacterial multiplication in SH1‐lacking plants. Our findings suggest that HvEC‐016 may be recognized by the plant immune system in a SH1‐dependent manner. Thus, our experimental approach is an effective tool for the characterization of effector/avirulence proteins of this important pathogen.

Many pathogens use Type III and Type IV protein secretion systems to secrete virulence factors from the bacterial cytosol into host cells. These systems operate through a one-step mechanism. The secreted substrates (protein or nucleo-protein complexes in the case of Type IV conjugative systems) are guided to the base of the secretion channel, where they are directly delivered into the host cell in an ATP-dependent unfolded state. Despite the numerous disparities between these secretion systems, here we have focused on the structural and functional similarities between both systems. In particular, on the structural similarity shared by one of the main ATPases (EscN and VirD4 in Type III and Type IV secretion systems, respectively). Interestingly, these ATPases also exhibit a structural resemblance to F 1 -ATPases, which suggests a common mechanism for substrate secretion. The correlation between structure and function of essential components in both systems can provide significant insights into the molecular mechanisms involved. This approach is of great interest in the pursuit of identifying inhibitors that can effectively target these systems.

Summary Type III secretion enables bacteria to intoxicate eukaryotic cells with anti‐host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted α‐helix motif at the C‐terminus. Mutants YopDI262P and YopDK267P poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host‐membrane insertion of the YopD–YopB translocon. Mutants YopDA263P and YopDA270P had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.

This chapter contains sections titled: Introduction Role of Mammalian Mb in O 2 Homeostasis What's Missing in the Mb Knockout Mouse Evolutionary Origins of Mb and the Nitrogen Cycle Human Hb: Evolved Sensor of pO 2 and Redox Broad Reactivity and Influence of NO: Lessons from the Microcosm Hb Some Fish Demonstrate a Fundamental “Need” for Hb‐dependent NO Cycling, as in Humans Reactions of NO with Hb that Preserve NO Bioactivity Mammalian RBC/Hb–NO Interactions A Mutant Mouse Challenges the SNO‐Hb Hypothesis, but does not Overthrow it Signaling by Hb‐derived SNO: A Metabolically Responsive, Regulated Pathway Signaling by Hb‐derived SNO: Pathway Complexity Revealed by Multiple Defects in Disease States Therapeutic Implications of the Hb–NO Signaling System HBOCs, NO, and SNO Other Gaseous Hb Ligands of Potential Therapeutic Significance NO‐related Enzymatic Activities of Hb: Reconciling Nitrite Reductase and SNO Synthase Functions Measuring Biologically Relevant Hb–NO Adducts Conclusion Acknowledgments References

This chapter contains sections titled: Introduction Cancer Transkingdom RNAi (tkRNAi) Conclusion References

Background Tumor growth is closely linked to the activities of various cells in the tumor microenvironment (TME), particularly immune cells. During tumor progression, circulating monocytes and macrophages are recruited, altering the TME and accelerating growth. These macrophages adjust their functions in response to signals from tumor and stromal cells. Tumor-associated macrophages (TAMs), similar to M2 macrophages, are key regulators in the TME. Methods We review the origins, characteristics, and functions of TAMs within the TME. This analysis includes the mechanisms through which TAMs facilitate immune evasion and promote tumor metastasis. Additionally, we explore potential therapeutic strategies that target TAMs. Results TAMs are instrumental in mediating tumor immune evasion and malignant behaviors. They release cytokines that inhibit effector immune cells and attract additional immunosuppressive cells to the TME. TAMs primarily target effector T cells, inducing exhaustion directly, influencing activity indirectly through cellular interactions, or suppressing through immune checkpoints. Additionally, TAMs are directly involved in tumor proliferation, angiogenesis, invasion, and metastasis. Summary Developing innovative tumor-targeted therapies and immunotherapeutic strategies is currently a promising focus in oncology. Given the pivotal role of TAMs in immune evasion, several therapeutic approaches have been devised to target them. These include leveraging epigenetics, metabolic reprogramming, and cellular engineering to repolarize TAMs, inhibiting their recruitment and activity, and using TAMs as drug delivery vehicles. Although some of these strategies remain distant from clinical application, we believe that future therapies targeting TAMs will offer significant benefits to cancer patients.

Immunometabolic intervention has been applied to treat cancer via inhibition of certain enzymes associated with intratumoral metabolism. However, small-molecule inhibitors and genetic modification often suffer from insufficiency and off-target side effects. Proteolysis targeting chimeras (PROTACs) provide an alternative way to modulate protein homeostasis for cancer therapy; however, the always-on bioactivity of existing PROTACs potentially leads to uncontrollable protein degradation at non-target sites, limiting their in vivo therapeutic efficacy. We herein report a semiconducting polymer nano-PROTAC (SPN pro ) with phototherapeutic and activatable protein degradation abilities for photo-immunometabolic cancer therapy. SPN pro can remotely generate singlet oxygen ( 1 O 2 ) under NIR photoirradiation to eradicate tumor cells and induce immunogenic cell death (ICD) to enhance tumor immunogenicity. Moreover, the PROTAC function of SPN pro is specifically activated by a cancer biomarker (cathepsin B) to trigger targeted proteolysis of immunosuppressive indoleamine 2,3-dioxygenase (IDO) in the tumor of living mice. The persistent IDO degradation blocks tryptophan (Trp)-catabolism program and promotes the activation of effector T cells. Such a SPNpro-mediated in-situ immunometabolic intervention synergizes immunogenic phototherapy to boost the antitumor T-cell immunity, effectively inhibiting tumor growth and metastasis. Thus, this study provides a polymer platform to advance PROTAC in cancer therapy. Proteolysis targeting chimeras (PROTACs) is an effective alternative to modulate protein homeostasis but can lead to uncontrollable protein degradation and off-target side effects. Here, the authors developed semiconducting polymer nano-PROTACs with phototherapeutic and activatable protein degradation abilities for photo-immunometabolic cancer therapy.

Immunotherapy has emerged as a major therapeutic modality in oncology. Currently, however, the majority of patients with cancer do not derive benefit from these treatments. Vascular abnormalities are a hallmark of most solid tumours and facilitate immune evasion. These abnormalities stem from elevated levels of proangiogenic factors, such as VEGF and angiopoietin 2 (ANG2); judicious use of drugs targeting these molecules can improve therapeutic responsiveness, partially owing to normalization of the abnormal tumour vasculature that can, in turn, increase the infiltration of immune effector cells into tumours and convert the intrinsically immunosuppressive tumour microenvironment (TME) to an immunosupportive one. Immunotherapy relies on the accumulation and activity of immune effector cells within the TME, and immune responses and vascular normalization seem to be reciprocally regulated. Thus, combining antiangiogenic therapies and immunotherapies might increase the effectiveness of immunotherapy and diminish the risk of immune-related adverse effects. In this Perspective, we outline the roles of VEGF and ANG2 in tumour immune evasion and progression, and discuss the evidence indicating that antiangiogenic agents can normalize the TME. We also suggest ways that antiangiogenic agents can be combined with immune-checkpoint inhibitors to potentially improve patient outcomes, and highlight avenues of future research.

The development of innovative targeted therapeutic approaches are expected to surpass the efficacy of current forms of treatments and cause less damage to healthy cells surrounding the tumor site. Since the first development of targeting agents from hybridoma's, monoclonal antibodies (mAbs) have been employed to inhibit tumor growth and proliferation directly or to deliver effector molecules to tumor cells. However, the full potential of such a delivery strategy is hampered by the size of mAbs, which will obstruct the targeted delivery system to access the tumor tissue. By serendipity, a new kind of functional homodimeric antibody format was discovered in camelidae, known as heavy-chain antibodies (HCAbs). The cloning of the variable domain of HCAbs produces an attractive minimal-sized alternative for mAbs, referred to as VHH or nanobodies (Nbs). Apart from their dimensions in the single digit nanometer range, the unique characteristics of Nbs combine a high stability and solubility, low immunogenicity and excellent affinity and specificity against all possible targets including tumor markers. This stimulated the development of tumor-targeted therapeutic strategies. Some autonomous Nbs have been shown to act as antagonistic drugs, but more importantly, the targeting capacity of Nbs has been exploited to create drug delivery systems. Obviously, Nb-based targeted cancer therapy is mainly focused toward extracellular tumor markers, since the membrane barrier prevents antibodies to reach the most promising intracellular tumor markers. Potential strategies, such as lentiviral vectors and bacterial type 3 secretion system, are proposed to deliver target-specific Nbs into tumor cells and to block tumor markers intracellularly. Simultaneously, Nbs have also been employed for molecular imaging to diagnose diseased tissues and to monitor the treatment effects. Here, we review the state of the art and focus on recent developments with Nbs as targeting moieties for drug delivery systems in cancer therapy and cancer imaging.