Pharmacological expansion of type 2 alveolar epithelial cells promotes regenerative lower airway repair.

Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to lower airway repair. Agents that promote the selective expansion of these cells might stimulate regeneration of the compromised alveolar epithelium, an etiology-defining event in several pulmonary diseases. From a high-content imaging screen of the drug repurposing library ReFRAME, we identified that dipeptidyl peptidase 4 (DPP4) inhibitors, widely used type 2 diabetes medications, selectively expand AEC2s and are broadly efficacious in several mouse models of lung damage. Mechanism of action studies revealed that the protease DPP4, in addition to processing incretin hormones, degrades IGF-1 and IL-6, essential regulators of AEC2 expansion whose levels are increased in the luminal compartment of the lung in response to drug treatment. To selectively target DPP4 in the lung with sufficient drug exposure, we developed NZ-97, a locally delivered, lung persistent DPP4 inhibitor that broadly promotes efficacy in mouse lung damage models with minimal peripheral exposure and good tolerability. This work reveals DPP4 as a central regulator of AEC2 expansion and affords a promising therapeutic approach to broadly stimulate regenerative repair in pulmonary disease.

Discovery of CMX990: A Potent SARS-CoV-2 3CL Protease Inhibitor Bearing a Novel Warhead.

There remains a need to develop novel SARS-CoV-2 therapeutic options that improve upon existing therapies by an increased robustness of response, fewer safety liabilities, and global-ready accessibility. Functionally critical viral main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target due to its homology within the coronaviral family, and lack thereof toward human proteases. In this disclosure, we outline the advent of a novel SARS-CoV-2 3CLpro inhibitor, CMX990, bearing an unprecedented trifluoromethoxymethyl ketone warhead. Compared with the marketed drug nirmatrelvir (combination with ritonavir = Paxlovid), CMX990 has distinctly differentiated potency (∼5× more potent in primary cells) and human in vitro clearance (>4× better microsomal clearance and >10× better hepatocyte clearance), with good in vitro-to-in vivo correlation. Based on its compelling preclinical profile and projected once or twice a day dosing supporting unboosted oral therapy in humans, CMX990 advanced to a Phase 1 clinical trial as an oral drug candidate for SARS-CoV-2.

Response Surfaces Method and Artificial Intelligence Approaches for Modeling the Effects of Environmental Factors on Chlorophyll a in Isochrysis galbana.

This study reported the condition optimization for chlorophyll a (Chl a) from the microalga Isochrysis galbana. The key parameters affecting the Chl a content of I. galbana were determined by a single-factor optimization experiment. Then the individual and interaction of three factors, including salinity, pH and nitrogen concentration, was optimized by using the method of Box-Benhnken Design. The highest Chl a content (0.51 mg/L) was obtained under the optimum conditions of salinity 30‱ and nitrogen concentration of 72.1 mg/L at pH 8.0. The estimation models of Chl a content based on the response surfaces method (RSM) and three different artificial intelligence models of artificial neural network (ANN), support vector machine (SVM) and radial basis function neural network (RBFNN), were established, respectively. The fitting model was evaluated by using statistical analysis parameters. The high accuracy of prediction was achieved on the ANN, SVM and RBFNN models with correlation coefficients (R2) of 0.9113, 0.9127, and 0.9185, respectively. The performance of these artificial intelligence models depicted better prediction capability than the RSM model for anticipating all the responses. Further experimental results suggested that the proposed SVM and RBFNN model are efficient techniques for accurately fitting the Chl a content of I. galbana and will be helpful in validating future experimental work on the Chl a content by computational intelligence approach.

The Growth Inhibition of Polyethylene Nanoplastics on the Bait-Microalgae Isochrysis galbana Based on the Transcriptome Analysis.

The adverse effects of microplastics on microalgae species have been extensively studied, but their impact on the bait microalgae entering the food chain has not been well understood. This study investigated the cytological and physiological response of Isochrysis galbana to polyethylene microplastics (PE-MPs, 10 µm) and nanoplastics (PE-NPs, 50 nm). The results showed that PE-MPs had no significant impact on I. galbana, while PsE-NPs obviously inhibited cell growth, reduced chlorophyll content, and caused a decline in carotenoids and soluble protein. These changes in the quality of I. galbana could negatively affect its use as aquaculture feed. To understand the molecular response mechanism of I. galbana to PE-NPs, transcriptome sequencing was performed. The result revealed that the TCA cycle, purine metabolism, and some key amino acid syntheses were down-regulated by PE-NPs, while the Calvin cycle and fatty acid metabolism were up-regulated to tolerate PE-NP pressure. Microbial analysis showed that the bacterial community structure associated with I. galbana was significantly altered at the species level by PE-NPs. In conclusion, this study provides new insights into the physiological stress response caused by microplastic pollution based on transcriptome and bacterial community analysis. The findings highlight the need to mitigate the release of microplastics into the environment to prevent their harmful effects on aquatic ecosystems and will be helpful in understanding the impact of polyethylene nanoplastics on the bait microalgae.

Estimating the contribution of plant traits to light partitioning in simultaneous maize/soybean intercropping.

Spatial configuration and plant phenotypic plasticity contribute to increased light capture in relay intercropping, but there is little information on whether these factors also increase light capture in simultaneous intercropping. We developed and validated a three-dimensional functional-structural plant model to simulate light capture in maize and soybean sole crops and intercrop scenarios, using species traits observed in sole crops and intercrops. The intercrop maize phenotype had 2% greater light capture than the sole crop phenotype in a pure stand. The soybean intercrop phenotype had 5-10% lower light capture than the sole crop phenotype in a pure stand. The intercrop configuration increased the light capture of maize by 29% and reduced the light capture of soybean by 42%, compared with the light capture expected from sole crops. However, intercrop configuration only marginally affected total light capture by the intercrop system (+1%). Testing of individual soybean plant traits revealed that plasticity in leaf dimensions was the main reason for differences in light capture by soybean in simulated sole crops and intercrops. The results of this study illustrate a major shift of light capture from shorter species (soybean) to the taller component (maize) in a simultaneous strip intercrop. Plastic plant traits modulate this overall effect, but only marginally.

Estimation of maize plant height and leaf area index dynamics using an unmanned aerial vehicle with oblique and nadir photography.

BACKGROUND AND AIMS: High-throughput phenotyping is a limitation in plant genetics and breeding due to large-scale experiments in the field. Unmanned aerial vehicles (UAVs) can help to extract plant phenotypic traits rapidly and non-destructively with high efficiency. The general aim of this study is to estimate the dynamic plant height and leaf area index (LAI) by nadir and oblique photography with a UAV, and to compare the integrity of the established three-dimensional (3-D) canopy by these two methods. METHODS: Images were captured by a high-resolution digital RGB camera mounted on a UAV at five stages with nadir and oblique photography, and processed by Agisoft Metashape to generate point clouds, orthomosaic maps and digital surface models. Individual plots were segmented according to their positions in the experimental design layout. The plant height of each inbred line was calculated automatically by a reference ground method. The LAI was calculated by the 3-D voxel method. The reconstructed canopy was sliced into different layers to compare leaf area density obtained from oblique and nadir photography. KEY RESULTS: Good agreements were found for plant height between nadir photography, oblique photography and manual measurement during the whole growing season. The estimated LAI by oblique photography correlated better with measured LAI (slope = 0.87, R2 = 0.67), compared with that of nadir photography (slope = 0.74, R2 = 0.56). The total number of point clouds obtained by oblique photography was about 2.7-3.1 times than those by nadir photography. Leaf area density calculated by nadir photography was much less than that obtained by oblique photography, especially near the plant base. CONCLUSIONS: Plant height and LAI can be extracted automatically and efficiently by both photography methods. Oblique photography can provide intensive point clouds and relatively complete canopy information at low cost. The reconstructed 3-D profile of the plant canopy can be easily recognized by oblique photography.

An Internally Controlled Quantitative Target Occupancy Assay for Covalent Inhibitors.

Assessing target occupancy is critical for establishing proof-of-mechanism for novel inhibitors and to determine whether robust target inhibition can be achieved at tolerated doses. This is challenging in the clinic using conventional methods due to the need for untreated controls. We describe a new mass spectrometry approach to quantitatively assess target occupancy for covalent inhibitors that does not require untreated controls, and apply the method to the KRASG12C inhibitor ARS-1620.

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

Dual Shp2 and Pten Deficiencies Promote Non-alcoholic Steatohepatitis and Genesis of Liver Tumor-Initiating Cells.

The complexity of liver tumorigenesis is underscored by the recently observed anti-oncogenic effects of oncoproteins, although the mechanisms are unclear. Shp2/Ptpn11 is a proto-oncogene in hematopoietic cells and antagonizes the effect of tumor suppressor Pten in leukemogenesis. In contrast, we show here cooperative functions of Shp2 and Pten in suppressing hepatocarcinogenesis. Ablating both Shp2 and Pten in hepatocytes induced early-onset non-alcoholic steatohepatitis (NASH) and promoted genesis of liver tumor-initiating cells likely due to augmented cJun expression/activation and elevated ROS and inflammation in the hepatic microenvironment. Inhibiting cJun partially suppressed NASH-driven liver tumorigenesis without improving NASH. SHP2 and PTEN deficiencies were detected in liver cancer patients with poor prognosis. These data depict a mechanism of hepato-oncogenesis and suggest a potential therapeutic strategy.

Bridging cell surface receptor with nuclear receptors in control of bile acid homeostasis.

Bile acids (BAs) are traditionally considered as "physiological detergents" for emulsifying hydrophobic lipids and vitamins due to their amphipathic nature. But accumulating clinical and experimental evidence shows an association between disrupted BA homeostasis and various liver disease conditions including hepatitis infection, diabetes and cancer. Consequently, BA homeostasis regulation has become a field of heavy interest and investigation. After identification of the Farnesoid X Receptor (FXR) as an endogenous receptor for BAs, several nuclear receptors (SHP, HNF4α, and LRH-1) were also found to be important in regulation of BA homeostasis. Some post-translational modifications of these nuclear receptors have been demonstrated, but their physiological significance is still elusive. Gut secrets FGF15/19 that can activate hepatic FGFR4 and its downstream signaling cascade, leading to repressed hepatic BA biosynthesis. However, the link between the activated kinases and these nuclear receptors is not fully elucidated. Here, we review the recent literature on signal crosstalk in BA homeostasis.

Cytoplasmic tyrosine phosphatase Shp2 coordinates hepatic regulation of bile acid and FGF15/19 signaling to repress bile acid synthesis.

Bile acid (BA) biosynthesis is tightly controlled by intrahepatic negative feedback signaling elicited by BA binding to farnesoid X receptor (FXR) and also by enterohepatic communication involving ileal BA reabsorption and FGF15/19 secretion. However, how these pathways are coordinated is poorly understood. We show here that nonreceptor tyrosine phosphatase Shp2 is a critical player that couples and regulates the intrahepatic and enterohepatic signals for repression of BA synthesis. Ablating Shp2 in hepatocytes suppressed signal relay from FGFR4, receptor for FGF15/19, and attenuated BA activation of FXR signaling, resulting in elevation of systemic BA levels and chronic hepatobiliary disorders in mice. Acting immediately downstream of FGFR4, Shp2 associates with FRS2α and promotes the receptor activation and signal relay to several pathways. These results elucidate a molecular mechanism for the control of BA homeostasis by Shp2 through the orchestration of multiple signals in hepatocytes.

Nonreceptor tyrosine phosphatase Shp2 promotes adipogenesis through inhibition of p38 MAP kinase.

The molecular mechanism underlying adipogenesis and the physiological functions of adipose tissue are not fully understood. We describe here a unique mouse model of severe lipodystrophy. Ablation of Ptpn11/Shp2 in adipocytes, mediated by aP2-Cre, led to premature death, lack of white fat, low blood pressure, compensatory erythrocytosis, and hepatic steatosis in Shp2(fat-/-) mice. Fat transplantation partially rescued the lifespan and blood pressure in Shp2(fat-/-) mice, and administration of leptin also restored partially the blood pressure of mutant animals with endogenous leptin deficiency. Consistently, homozygous deletion of Shp2 inhibited adipocyte differentiation from embryonic stem (ES) cells. Biochemical analyses suggest a Shp2-TAO2-p38-p300-PPARγ pathway in adipogenesis, in which Shp2 suppresses p38 activation, leading to stabilization of p300 and enhanced PPARγ expression. Inhibition of p38 restored adipocyte differentiation from Shp2(-/-) ES cells, and p38 signaling is also suppressed in obese patients and obese animals. These results illustrate an essential role of adipose tissue in mammalian survival and physiology and also suggest a common signaling mechanism involved in adipogenesis and obesity development.

Dual faces of SH2-containing protein-tyrosine phosphatase Shp2/PTPN11 in tumorigenesis.

PTPN11, which encodes tyrosine phosphatase Shp2, is a critical gene mediating cellular responses to hormones and cytokines. Against original prediction as tumor suppressor for tyrosine phosphatases, PTPN11 was first identified as a proto-oncogene because activating mutations of this gene are associated with leukemogenesis. However, most recent experimental data suggest PTPN11/Shp2 acting as a tumor suppressor in hepatocarcinogenesis. This review focuses on the tumor-promoting or suppressing roles of the gene PTPN11/Shp2 in different cell types.

Shp2 controls female body weight and energy balance by integrating leptin and estrogen signals.

In mammals, leptin regulates food intake and energy balance mainly through the activation of LepRb in the hypothalamus, and estrogen has a leptin-like effect in the hypothalamic control of metabolism. However, it remains to be elucidated how estrogen signaling is intertwined with the leptin pathway. We show here that Shp2, a nonreceptor tyrosine phosphatase, acts to integrate leptin and estrogen signals. The expression of a dominant-active mutant (Shp2(D61A)) in forebrain neurons conferred female, but not male, transgenic mice resistance to high-fat diet (HFD)-induced obesity and liver steatosis, accompanied by improved insulin sensitivity and glucose homeostasis. Fed with either HFD or regular chow food, Shp2(D61A) female mice showed dramatically enhanced leptin sensitivity. Microinjection of Shp2(D61A)-expressing adeno-associated virus into mediobasal hypothalamus elicited a similar antiobese effect in female mice. Biochemical analyses showed a physical association of Shp2 with estrogen receptor alpha, which is necessary for the synergistic and persistent activation of Erk by leptin and estrogen. Together, these results elucidate a mechanism for the direct cross talk of leptin and estrogen signaling and offer one explanation for the propensity of postmenopausal women to develop obesity.

Ptpn11/Shp2 acts as a tumor suppressor in hepatocellular carcinogenesis.

The human gene Ptpn11, which encodes the tyrosine phosphatase Shp2, may act as a proto-oncogene because dominantly activating mutations have been detected in several types of leukemia. Herein we report a tumor-suppressor function of Shp2. Hepatocyte-specific deletion of Shp2 promotes inflammatory signaling through the Stat3 pathway and hepatic inflammation/necrosis, resulting in regenerative hyperplasia and development of tumors in aged mice. Furthermore, Shp2 ablation dramatically enhanced diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) development, which was abolished by concurrent deletion of Shp2 and Stat3 in hepatocytes. Decreased Shp2 expression was detected in a subfraction of human HCC specimens. Thus, in contrast to the leukemogenic effect of dominant-active mutants, Ptpn11/Shp2 has a tumor-suppressor function in liver.

Kit-Shp2-Kit signaling acts to maintain a functional hematopoietic stem and progenitor cell pool.

The stem cell factor (SCF)/Kit system has served as a classic model in deciphering molecular signaling events in the hematopoietic compartment, and Kit expression is a most critical marker for hematopoietic stem cells (HSCs) and progenitors. However, it remains to be elucidated how Kit expression is regulated in HSCs. Herein we report that a cytoplasmic tyrosine phosphatase Shp2, acting downstream of Kit and other RTKs, promotes Kit gene expression, constituting a Kit-Shp2-Kit signaling axis. Inducible ablation of PTPN11/Shp2 resulted in severe cytopenia in BM, spleen, and peripheral blood in mice. Shp2 removal suppressed the functional pool of HSCs/progenitors, and Shp2-deficient HSCs failed to reconstitute lethally irradiated recipients because of defects in homing, self-renewal, and survival. We show that Shp2 regulates coordinately multiple signals involving up-regulation of Kit expression via Gata2. Therefore, this study reveals a critical role of Shp2 in maintenance of a functional HSC/progenitor pool in adult mammals, at least in part through a kinase-phosphatase-kinase cascade.

Radiation Sensitivity and Tumor Susceptibility in ATM Phospho-Mutant ATF2 Mice.

The transcription factor ATF2 was previously shown to be an ATM substrate. Upon phosphorylation by ATM, ATF2 exhibits a transcription-independent function in the DNA damage response through localization to DNA repair foci and control of cell cycle arrest. To assess the physiological significance of this phosphorylation, we generated ATF2 mutant mice in which the ATM phosphoacceptor sites (S472/S480) were mutated (ATF2(KI)). ATF2(KI) mice are more sensitive to ionizing radiation (IR) than wild-type (ATF2 (WT)) mice: following IR, ATF2(KI) mice exhibited higher levels of apoptosis in the intestinal crypt cells and impaired hepatic steatosis. Molecular analysis identified impaired activation of the cell cycle regulatory protein p21(Cip/Waf1) in cells and tissues of IR-treated ATF2(KI) mice, which was p53 independent. Analysis of tumor development in p53(KO) crossed with ATF2(KI) mice indicated a marked decrease in amount of time required for tumor development. Further, when subjected to two-stage skin carcinogenesis process, ATF2(KI) mice developed skin tumors faster and with higher incidence, which also progressed to the more malignant carcinomas, compared with the control mice. Using 3 mouse models, we establish the importance of ATF2 phosphorylation by ATM in the acute cellular response to DNA damage and maintenance of genomic stability.

Retinoid signaling can repress blastula Wnt signaling and impair dorsal development in Xenopus embryo.

Vitamin A derivatives (retinoids) are actively involved during vertebrate embryogenesis. However, exogenous retinoids have also long been known as potent teratogens. The defects caused by retinoid treatment are complex. Here, we provided evidence that RAR-mediated retinoid signaling can repress Xenopus blastula Wnt signaling and impair dorsal development. Exogenous retinoic acid (RA) could antagonize the dorsalizing effects of lithium chloride-mediated Wnt activation in blastula embryos. The Wnt-responsive reporter gene transgenesis and luciferase assay showed that excess RA can repress the Wnt signaling in blastula embryos. In addition, the downstream target genes of the Wnt signaling that direct embryonic dorsal development, were also down-regulated in the RA-treated embryos. Mechanically, RA did not interfere with the stability of beta-catenin, but promoted its nuclear accumulation. The inverse agonist of retinoic acid receptors (RAR) rescued the Wnt signaling repression by RA and relieved the RA-induced nuclear accumulation of beta-catenin. Our results explain one of the reasons for the complicated teratogenic effects of retinoids and shed light on the endogenous way of interactions between two developmentally important signaling pathways.

Activin/nodal signaling modulates XPAPC expression during Xenopus gastrulation.

Gastrulation is the first obligatory morphogenesis during vertebrate development, by which the body plan is established. Nodal signaling is a key player in many developmental processes, including gastrulation. XPAPC has been found to exert its biological function through modifying the adhesion property of cells and interacting with other several important molecules in embryos. In this report, we show that nodal signaling is necessary and sufficient for XPAPC expression during Xenopus gastrulation. Furthermore, we isolated 4.8 kb upstream DNA sequence of Xenopus XPAPC, and proved that this 4.8-kb genomic contig is sufficient to recapitulate the expression pattern of XPAPC from gastrula to tail bud stage. Transgene and ChIP assays indicate that Activin/nodal signaling participates in regulation of XPAPC expression through a Smad binding element within the XPAPC promoter. Concomitant investigation suggests that the canonical Wnt pathway-activated XPAPC expression requires nodal signaling.

Xenopus Tbx6 mediates posterior patterning via activation of Wnt and FGF signalling.

In vertebrates, the patterning of anterior-posterior (AP) axis is a fundamental process during embryogenesis. Wnt and FGF signalling pathways play important roles in regulating the patterning of embryo AP axis. Mouse Tbx6 encodes a transcription factor that has been demonstrated to be involved in the specification of the posterior tissue in mouse embryonic body. Here, we prove that morpholino-induced knockdown of XTbx6 impairs posterior development, indicating the requirement of XTbx6 in this process. Meanwhile, gain of XTbx6 function is sufficient to induce ectopic posterior structures in Xenopus embryos. Furthermore, XTbx6 activates the expression of Xwnt8 and FGF8, which are two mediators of posterior development, suggesting a mechanism by which XTbx6 modulates posterior patterning via Wnt and FGF signalling pathway activation.