Homeostasis of cell wall integrity pathway phosphorylation is required for the growth and pathogenicity of Magnaporthe oryzae.

The cell wall provides a crucial barrier to stress imposed by the external environment. In the rice blast fungus Magnaporthe oryzae, this stress response is mediated by the cell wall integrity (CWI) pathway, consisting of a well-characterized protein phosphorylation cascade. However, other regulators that maintain CWI phosphorylation homeostasis, such as protein phosphatases (PPases), remain unclear. Here, we identified two PPases, MoPtc1 and MoPtc2, that function as negative regulators of the CWI pathway. MoPtc1 and MoPtc2 interact with MoMkk1, one of the key components of the CWI pathway, and are crucial for the vegetative growth, conidial formation, and virulence of M. oryzae. We also demonstrate that both MoPtc1 and MoPtc2 dephosphorylate MoMkk1 in vivo and in vitro, and that CWI stress leads to enhanced interaction between MoPtc1 and MoMkk1. CWI stress abolishes the interaction between MoPtc2 and MoMkk1, providing a means of deactivation for CWI signalling. Our studies reveal that CWI signalling in M. oryzae is a highly coordinated regulatory mechanism vital for stress response and pathogenicity.

Lentiviral globin gene therapy with reduced-intensity conditioning in adults with ß-thalassemia: a phase 1 trial.

ß-Thalassemias are inherited anemias that are caused by the absent or insufficient production of the ß chain of hemoglobin. Here we report 6-8-year follow-up of four adult patients with transfusion-dependent ß-thalassemia who were infused with autologous CD34+ cells transduced with the TNS9.3.55 lentiviral globin vector after reduced-intensity conditioning (RIC) in a phase 1 clinical trial ( NCT01639690) . Patients were monitored for insertional mutagenesis and the generation of a replication-competent lentivirus (safety and tolerability of the infusion product after RIC-primary endpoint) and engraftment of genetically modified autologous CD34+ cells, expression of the transduced ß-globin gene and post-transplant transfusion requirements (efficacy-secondary endpoint). No unexpected safety issues occurred during conditioning and cell product infusion. Hematopoietic gene marking was very stable but low, reducing transfusion requirements in two patients, albeit not achieving transfusion independence. Our findings suggest that non-myeloablative conditioning can achieve durable stem cell engraftment but underscore a minimum CD34+ cell transduction requirement for effective therapy. Moderate clonal expansions were associated with integrations near cancer-related genes, suggestive of non-erythroid activity of globin vectors in stem/progenitor cells. These correlative findings highlight the necessity of cautiously monitoring patients harboring globin vectors.

Enhanced topical corticosteroids delivery to the eye: A trade-off in strategy choice.

Topical corticosteroids are the primary treatment of ocular inflammation caused by surgery, injury, or other conditions. Drug pre-corneal residence time, drug water solubility, and drug corneal permeability coefficient are the major factors that determine the ocular drug bioavailability after topical administration. Although growing research successfully enhanced local delivery of corticosteroids utilizing various strategies, rational and dynamic approaches to strategy selection are still lacking. Within this review, an overview of the various strategies as well as their performance in retention, solubility, and permeability coefficient of corticosteroids are provided. On this basis, the tradeoff of strategy selection is discussed, which may shed light on the rational choice and application of ophthalmic delivery enhancement strategies.

Depletion of high-content CD14+ cells from apheresis products is critical for successful transduction and expansion of CAR T cells during large-scale cGMP manufacturing.

With the US Food and Drug Administration (FDA) approval of four CD19- and one BCMA-targeted chimeric antigen receptor (CAR) therapy for B cell malignancies, CAR T cell therapy has finally reached the status of a medicinal product. The successful manufacturing of autologous CAR T cell products is a key requirement for this promising treatment modality. By analyzing the composition of 214 apheresis products from 210 subjects across eight disease indications, we found that high CD14+ cell content poses a challenge for manufacturing CAR T cells, especially in patients with non-Hodgkin's lymphoma and multiple myeloma caused by the non-specific phagocytosis of the magnetic beads used to activate CD3+ T cells. We demonstrated that monocyte depletion via rapid plastic surface adhesion significantly reduces the CD14+ monocyte content in the apheresis products and simultaneously boosts the CD3+ content. We established a 40% CD14+ threshold for the stratification of apheresis products across nine clinical trials and demonstrated the effectiveness of this procedure by comparing manufacturing runs in two phase 1 clinical trials. Our study suggests that CD14+ content should be monitored in apheresis products, and that the manufacturing of CAR T cells should incorporate a step that lessens the CD14+ cell content in apheresis products containing more than 40% to maximize the production success.

Extracellular vesicles from human airway basal cells respond to cigarette smoke extract and affect vascular endothelial cells.

The human airway epithelium lining the bronchial tree contains basal cells that proliferate, differentiate, and communicate with other components of their microenvironment. One method that cells use for intercellular communication involves the secretion of exosomes and other extracellular vesicles (EVs). We isolated exosome-enriched EVs that were produced from an immortalized human airway basal cell line (BCi-NS1.1) and found that their secretion is increased by exposure to cigarette smoke extract, suggesting that this stress stimulates release of EVs which could affect signaling to other cells. We have previously shown that primary human airway basal cells secrete vascular endothelial growth factor A (VEGFA) which can activate MAPK signaling cascades in endothelial cells via VEGF receptor-2 (VEGFR2). Here, we show that exposure of endothelial cells to exosome-enriched airway basal cell EVs promotes the survival of these cells and that this effect also involves VEGFR2 activation and is, at least in part, mediated by VEGFA present in the EVs. These observations demonstrate that EVs are involved in the intercellular signaling between airway basal cells and the endothelium which we previously reported. The downstream signaling pathways involved may be distinct and specific to the EVs, however, as increased phosphorylation of Akt, STAT3, p44/42 MAPK, and p38 MAPK was not seen following exposure of endothelial cells to airway basal cell EVs.

A self-balancing circuit centered on MoOsm1 kinase governs adaptive responses to host-derived ROS in Magnaporthe oryzae.

The production of reactive oxygen species (ROS) is a ubiquitous defense response in plants. Adapted pathogens evolved mechanisms to counteract the deleterious effects of host-derived ROS and promote infection. How plant pathogens regulate this elaborate response against ROS burst remains unclear. Using the rice blast fungus Magnaporthe oryzae, we uncovered a self-balancing circuit controlling response to ROS in planta and virulence. During infection, ROS induces phosphorylation of the high osmolarity glycerol pathway kinase MoOsm1 and its nuclear translocation. There, MoOsm1 phosphorylates transcription factor MoAtf1 and dissociates MoAtf1-MoTup1 complex. This releases MoTup1-mediated transcriptional repression on oxidoreduction-pathway genes and activates the transcription of MoPtp1/2 protein phosphatases. In turn, MoPtp1/2 dephosphorylate MoOsm1, restoring the circuit to its initial state. Balanced interactions among proteins centered on MoOsm1 provide a means to counter host-derived ROS. Our findings thereby reveal new insights into how M. oryzae utilizes a phosphor-regulatory circuitry to face plant immunity during infection.

Enhancement of diallyl disulfide-induced apoptosis by inhibitors of MAPKs in human HepG2 hepatoma cells.

We examined the effects of diallyl disulfide (DADS), an oil-soluble organosulfur compound found in garlic, on human HepG2 hepatoma cells to better understand its effect on apoptosis and apoptosis-related genes. Our study has demonstrated that DADS affects cell proliferation activity and viability and elicits typical apoptotic morphologic changes (chromatic condensation and nuclear fragmentation) in human HepG2 hepatoma cells. Also, treatment with DADS induces a temporary increase in phosphorylated p38 MAPK (phospho-p38) and phosphorylated p42/44 MAPK (phospho-p42/p44) in a time- and concentration-dependent manner. Inhibition of activated/phosphorylated mitogen-activated protein kinase (MAPK) with phospho-p38 or phospho-p42/44 specific inhibitors, SB203580 or U0126, induces apoptosis without DADS treatment, indicating that at least the endogenous activated forms of p38 MAPK and p42/p44 MAPK markedly exert cytoprotective roles from cell apoptosis in the HepG2 hepatoma cells. Combined treatment with these inhibitors followed by DADS further enhances the DADS-induced apoptosis. Taken together, these results show that both DADS and the specific inhibitors of MAPKs could induce apoptosis in HepG2 hepatoma cells and that the MAPKs inhibitors further enhance the apoptotic effect in DADS-treated HepG2 hepatoma cells.

Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells.

Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are mechanistically distinct DNA repair pathways that contribute substantially to double-strand break (DSB) repair in mammalian cells. We have combined mutations in factors from both repair pathways, the HR protein Rad54 and the DNA-end-binding factor Ku80, which has a role in NHEJ. Rad54(-/-)Ku80(-/-) mice were severely compromised in their survival, such that fewer double mutants were born than expected, and only a small proportion of those born reached adulthood. However, double-mutant mice died at lower frequency from tumors than Ku80 single mutant mice, likely as a result of rapid demise at a young age from other causes. When challenged with an exogenous DNA damaging agent, ionizing radiation, double-mutant mice were exquisitely sensitive to low doses. Tissues and cells from double-mutant mice also showed indications of spontaneous DNA damage. Testes from some Rad54(-/-)Ku80(-/-) mice displayed enhanced apoptosis and reduced sperm production, and embryonic fibroblasts from Rad54(-/-)Ku80(-/-) animals accumulated foci of gamma-H2AX, a marker for DSBs. The substantially increased DNA damage response in the double mutants implies a cooperation of the two DSB repair pathways for survival and genomic integrity in the animal.

Adenovirus-mediated heat-activated antisense Ku70 expression radiosensitizes tumor cells in vitro and in vivo.

Ku70 is one component of a protein complex, Ku70 and Ku80, that functions as a heterodimer to bind DNA double-strand breaks and activates DNA-dependent protein kinase. Our previous study with Ku70-/- and Ku80-/- mice, and cell lines has shown that Ku70- and Ku80-deficiency compromises the ability of cells to repair DNA double-strand breaks, increases radiosensitivity of cells, and enhances radiation-induced apoptosis. In this study, we examined the feasibility of using adenovirus-mediated, heat-activated expression of antisense Ku70 RNA as a gene therapy paradigm to sensitize cells and tumors to ionizing radiation. First, we performed experiments to test the heat inducibility of heat shock protein (hsp) 70 promoter and the efficiency of adenovirus-mediated gene transfer in rodent and human cells. Replication-defective adenovirus vectors were used to introduce a recombinant DNA construct, containing the enhanced green fluorescent protein (EGFP) under the control of an inducible hsp70 promoter, into exponentially growing cells. At 24 h after infection, cells were exposed to heat treatment, and heat-induced EGFP expression at different times was determined by flow cytometry. Our data clearly show that heat shock at 42 degrees C, 43 degrees C, or 44 degrees C appears to be equally effective in activating the hsp70 promoter-driven EGFP expression (>300-fold) in various tumor cells. Second, we have generated adenovirus vectors containing antisense Ku70 under the control of an inducible hsp70 promoter. Exponentially growing cells were infected with the adenovirus vector, heat shocked 24 h later, and the radiosensitivity determined 12 h after heat shock. Our data show that heat shock induces antisense Ku70 RNA, reduces the endogenous Ku70 level, and significantly increases the radiosensitivity of the cells. Third, we have performed studies to test whether Ku70 protein level can be down-regulated in a solid mouse tumor (FSa-II), and whether this results in enhanced radiosensitivity in vivo, as assessed by in vivo/in vitro colony formation and by the tumor growth delay. Our data demonstrate that heat-shock-induced expression of antisense Ku70 RNA attenuates Ku70 protein expression in FSa-II tumors, and significantly sensitizes the FSa-II tumors to ionizing radiation. Taken together, our results suggest that adenovirus-mediated, heat-activated antisense Ku70 expression may provide a novel approach to radiosensitize human tumors.