Airway secretory cell fate conversion via YAP‐mTORC1‐dependent essential amino acid metabolism

Tissue homeostasis requires lineage fidelity of stem cells. Dysregulation of cell fate specification and differentiation leads to various diseases, yet the cellular and molecular mechanisms governing these processes remain elusive. We demonstrate that YAP/TAZ activation reprograms airway secretory cells, which subsequently lose their cellular identity and acquire squamous alveolar type 1 (AT1) fate in the lung. This cell fate conversion is mediated via distinctive transitional cell states of damage‐associated transient progenitors (DATPs), recently shown to emerge during injury repair in mouse and human lungs. We further describe a YAP/TAZ signaling cascade to be integral for the fate conversion of secretory cells into AT1 fate, by modulating mTORC1/ATF4‐mediated amino acid metabolism in vivo. Importantly, we observed aberrant activation of the YAP/TAZ‐mTORC1‐ATF4 axis in the altered airway epithelium of bronchiolitis obliterans syndrome, including substantial emergence of DATPs and AT1 cells with severe pulmonary fibrosis. Genetic and pharmacologic inhibition of mTORC1 activity suppresses lineage alteration and subepithelial fibrosis driven by YAP/TAZ activation, proposing a potential therapeutic target for human fibrotic lung diseases.     

The structure of AcrIE4-F7 reveals a common strategy for dual CRISPR inhibition by targeting PAM recognition sites

Bacteria and archaea use the CRISPR-Cas system to fend off invasions of bacteriophages and foreign plasmids. In response, bacteriophages encode anti-CRISPR (Acr) proteins that potently inhibit host Cas proteins to suppress CRISPR-mediated immunity. AcrIE4-F7, which was isolated from Pseudomonas citronellolis, is a fused form of AcrIE4 and AcrIF7 that inhibits both type I-E and type I-F CRISPR-Cas systems. Here, we determined the structure of AcrIE4-F7 and identified its Cas target proteins. The N-terminal AcrIE4 domain adopts a novel α-helical fold that targets the PAM interaction site of the type I-E Cas8e subunit. The C-terminal AcrIF7 domain exhibits an αβ fold like native AcrIF7, which disables target DNA recognition by the PAM interaction site in the type I-F Cas8f subunit. The two Acr domains are connected by a flexible linker that allows prompt docking onto their cognate Cas8 targets. Conserved negative charges in each Acr domain are required for interaction with their Cas8 targets. Our results illustrate a common mechanism by which AcrIE4-F7 inhibits divergent CRISPR-Cas types.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

Use of electrophoretic enzyme patterns for streptomycete systematics

Abstract The relative electrophoretic mobilities of various enzymes from 24 different streptomycetes were determined on polyacrylamide gels in order to examine the relatedness of species and strains of the genus Streptomyces . Of 11 different enzymes tested in this study, hexokinase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase showed a limited number of constant and reproducible polymorphic enzyme patterns, by comparing which the inter-specific relationships could be examined. In contrast, glucose dehydrogenase, alcohol dehydrogenase, 3-hydroxybutyrate dehydrogenase, phosphoglucose isomerase, peroxidase and esterase exhibited either weak non-reproducible or highly heterogeneous band patterns which were suitable for dissecting the strains within a species and a cluster group.     

Carbamylation of hemoglobin in vivo with chronic sublethal dietary cyanide: implications for hemoglobin S

Carbamylation of the hemoglobin in sickle cell anemia has been demonstrated to improve the status of this hemoglobinopathy. Using an animal model of West African human patterns of chronic sublethal dietary cyanide ingestion, 12 miniature swine consuming either 0, 0.4, 0.7, or 1.2 mg of cyanide/kg body weight/day were studied for 24 weeks to determine if this dietary regime could produce irreversible carbamylated hemoglobin. Throughout the study, the hematological status of all animals remained similar; however, the levels of carbamylated hemoglobin as measured by nanomoles of valine hydantoin varied proportionally to dietary sublethal cyanide intakes, indicating that these natural dietary levels could effect an important and presumably permanent modification of the hemoglobin's beta chain. Serum thiocyanate levels were also positively correlated with cyanide ingestion loads (r = 0.83, P less than 0.01). The implications of these findings in swine are important for the millions of humans with hemoglobin S who regularly consume similar levels of dietary cyanide and for our assessments of the biochemical and medical status of hemoglobin S under natural conditions.     

Nucleotide sequence of the gene coding for a 130-kDa mosquitocidal protein of Bacillus thuringiensis israelensis

The nucleotide sequence of pVB131 containing the gene coding for a 130-kDa Bacillus thuringiensis israelensis (B.t.isr) mosquitocidal protein was determined. The pVB131 plasmid was constructed by Sekar and Carlton [Gene 33 (1985) 151-158]. Our sequencing revealed only one open reading frame large enough to code for a protein of 130 kDa. The translation start site was determined by sequencing the protein isolated from B.t.isr. The amino acid sequence of the protein was deduced from the nucleotide sequence, and its Mr was determined as 128,505. Immunological and biochemical analyses of B.t.isr mosquitocidal proteins indicated that the 130-kDa protein coded by pVB131 was indeed expressed in B.t.isr. Comparing the peptide sequence of the 130-kDa B.t.isr toxin with the sequences of other B.t. toxins having activities specific to lepidopteran species showed that several domains were highly homologous. This suggests that they are evolutionarily related to each other, and in the evolutionary process the sequences in the homologous domains that are important to the insecticidal activity have been conserved.     

Further characterization of a novel lectin derived from silkworm faeces; specific binding to immunoglobulins,and activation of immunocytes

In previous studies a novel lectin-like glycoprotein was isolated from silkworm faeces and shown to recognize sugar chains, especially mannose on the cell surface as an epitope, and to cause aggregation of various types of cells in suspension. However, this substance caused detachment and aggregation of only some types of plated cells, such as QM-RSV cells, which are quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV) at 35.5 °C, a permissive temperature for RSV. As described here, during studies on the mechanism of cell detachment and aggregation of QM-RSV cells by this new lectin, some novel biological activities of the lectin were recognized. This lectin was found to bind to immunoglobulins (Ig) specifically at specific amino acid sequences, not via recognition of their molecular conformation. It also recognized the neural cell adhesion molecule (NCAM), which is one of the members of the Ig-superfamily that have Ig-like domains. Furthermore, it had a strong mitogenic effect on lymphocytes, and also caused about 3-fold of phagocytosis by macrophages within 24 hr after its addition.     

Immune Checkpoint Blockade Augments Changes Within Oncolytic Virus-induced Cancer MHC-I Peptidome,Creating Novel Antitumor CD8 T Cell Reactivities

The combination cancer immunotherapies with oncolytic virus (OV) and immune checkpoint blockade (ICB) reinstate otherwise dysfunctional antitumor CD8 T cell responses. One major mechanism that aids such reinstatement of antitumor CD8 T cells involves the availability of new class I major histocompatibility complex (MHC-I)-bound tumor epitopes following therapeutic intervention. Thus, therapy-induced changes within the MHC-I peptidome hold the key to understanding the clinical implications for therapy-reinstated CD8 T cell responses. Here, using mass spectrometry–based immuno-affinity methods and tumor-bearing animals treated with OV and ICB (alone or in combination), we captured the therapy-induced alterations within the tumor MHC-I peptidome, which were then tested for their CD8 T cell response-stimulating activity. We found that the oncolytic reovirus monotherapy drives up- as well as downexpression of tumor MHC-I peptides in a cancer type and oncolysis susceptibility dependent manner. Interestingly, the combination of reovirus + ICB results in higher numbers of differentially expressed MHC-I-associated peptides (DEMHCPs) relative to either monotherapies. Most importantly, OV+ICB-driven DEMHCPs contain biologically active epitopes that stimulate interferon-gamma responses in cognate CD8 T cells, which may mediate clinically desired antitumor attack and cancer immunoediting. These findings highlight that the therapy-induced changes to the MHC-I peptidome contribute toward the reinstated antitumor CD8 T cell attack established following OV + ICB combination cancer immunotherapy.