(2'-Oxo)alkylresorcinols restore dehydration tolerance in a knockout line of PpORS, a bryophyte-specific type III polyketide synthase in Physcomitrium (Physcomitrella) patens.

MAIN CONCLUSION: PpORS-produced 2'-oxo-5-pentacosylresorcinol (2'-oxo-C25-RL) restored dehydration tolerance in ors-3, a knockout mutant of PpORS. Feeding experiments with [14C]-2'-oxo-C25-RL suggested the role of PpORS products in cuticular polymer that confer dehydration resistance. 2'-Oxoalkylresorcinol synthase from the moss Physcomitrium (Physcomitrella) patens (PpORS) is the earliest diverged member of plant type III polyketide synthases, and produces very-long-chain 2'-oxoalkylresorcinols in vitro. Targeted knockouts of PpORS (ors) exhibited an abnormal phenotype (increased susceptibility to dehydration), and a defective cuticle in ors was suggested (Li et al., Planta 247:527-541, 2018). In the present study, we investigated chemical rescue of the ors phenotype and also metabolic fates of the PpORS products in the moss. Using C24-CoA as substrate, 2'-oxo-5-pentacosylresorcinol (2'-oxo-C25-RL) and two minor pyrones were first enzymatically prepared as total in vitro products. When a knockout mutant (ors-3) and control strains were grown in the presence of the total in vitro products or purified 2'-oxo-C25-RL, the ability of ors-3 and the control to survive dehydration stress increased in a dose-dependent manner. Structurally analogous long-chain alkylresorcinols also rescued the ors phenotype, although less efficiently. When the moss was grown in the presence of 14C-radiolabeled 2'-oxo-C25-RL, 96% of the radioactivity was recovered only after acid hydrolysis. These findings led us to propose that 2'-oxoalkylresorcinols are the functional in planta products of PpORS and are incorporated into cuticular biopolymers that confer resistance to dehydration. In addition, the earliest diverging ORS clade in phylogenetic trees of plant type III PKSs exclusively comprises bryophyte enzymes that share similar active site substitutions with PpORS. Further studies on these bryophyte enzymes may shed light on their roles in early plant evolution and offer a novel strategy for improving dehydration tolerance in plants.

Wheat Consumption Aggravates Colitis in Mice via Amylase Trypsin Inhibitor-mediated Dysbiosis.

BACKGROUND & AIMS: Wheat has become the world's major staple and its consumption correlates with prevalence of noncommunicable disorders such as inflammatory bowel diseases. Amylase trypsin inhibitors (ATIs), a component of wheat, activate the intestine's innate immune response via toll-like receptor 4 (TLR4). We investigated the effects of wheat and ATIs on severity of colitis and fecal microbiota in mice. METHODS: C57BL/6 wild-type and Tlr4-/- mice were fed wheat- or ATI-containing diets or a wheat-free (control) diet and then given dextran sodium sulfate to induce colitis; we also studied Il10-/- mice, which develop spontaneous colitis. Changes in fecal bacteria were assessed by taxa-specific quantitative polymerase chain reaction and 16S ribosomal RNA metagenomic sequencing. Feces were collected from mice on wheat-containing, ATI-containing, control diets and transplanted to intestines of mice with and without colitis on control or on ATI-containing diets. Intestinal tissues were collected and analyzed by histology, immunohistochemistry, and flow cytometry. Bacteria with reported immunomodulatory effects were incubated with ATIs and analyzed in radial diffusion assays. RESULTS: The wheat- or ATI-containing diets equally increased inflammation in intestinal tissues of C57BL/6 mice with colitis, compared with mice on control diets. The ATI-containing diet promoted expansion of taxa associated with development of colitis comparable to the wheat-containing diet. ATIs inhibited proliferation of specific human commensal bacteria in radial diffusion assays. Transplantation of microbiota from feces of mice fed the wheat- or ATI-containing diets to intestines of mice on control diets increased the severity of colitis in these mice. The ATI-containing diet did not increase the severity of colitis in Tlr4-/- mice. CONCLUSIONS: Consumption of wheat or wheat ATIs increases intestinal inflammation in mice with colitis, via TLR4, and alters their fecal microbiota. Wheat-based, ATI-containing diets therefore activate TLR4 signaling and promote intestinal dysbiosis.

PpORS, an ancient type III polyketide synthase, is required for integrity of leaf cuticle and resistance to dehydration in the moss, Physcomitrella patens.

MAIN CONCLUSION: PpORS knockout mutants produced abnormal leaves with increased dye permeability and were more susceptible to dehydration, consistent with PpORS products being constituents of a cuticular structure in the moss. Type III polyketide synthases (PKSs) have co-evolved with terrestrial plants such that each taxon can generate a characteristic collection of polyketides, fine-tuned to its needs. 2'-Oxoalkylresorcinol synthase from Physcomitrella patens (PpORS) is basal to all plant type III PKSs in phylogenetic trees and may closely resemble their most recent common ancestor. To gain insight into the roles that ancestral plant type III PKSs might have played during early land plant evolution, we constructed and phenotypically characterized targeted knockouts of PpORS. Ors gametophores, unless submerged in water while they were developing, displayed various leaf malformations that included grossly misshapen leaves, missing or abnormal midribs, multicellular protuberances and localized necrosis. Ors leaves, particularly abnormal ones, showed increased permeability to the hydrophilic dye, toluidine blue. Ors gametophores lost water faster and were more susceptible to dehydration than those of the control strain. Our findings are consistent with ors leaves possessing a partially defective cuticle and implicate PpORS in synthesis of the intact cuticle. PpORS orthologs are present in a few moss species but have not been found in other plants. However, conceivably an ancestral ORS in early land plants may have contributed to their protection from dehydration.

Anti-enzymatic and DNA docking studies of montelukast: A multifaceted molecular scaffold with in vitro investigations, molecular expression analysis and molecular dynamics simulations.

Montelukast, an approved leukotriene receptor 1 (Cys-LT 1) antagonist with anti-inflammatory properties is used for the treatment of asthma and allergic rhinitis. In the present studies, montelukast was subjected to in vitro inhibitory assays followed by kinetic and in silico investigations. Montelukast demonstrated inhibitory activity against yeast α-glucosidase (IC50 44.31 ± 1.21 µM), jack bean urease (JB urease, IC50 8.72 ± 0.23 µM), human placental alkaline phosphatase (hPAP, IC50 17.53 ± 0.19 µM), bovine intestinal alkaline phosphatase (bIAP, IC50 15.18 ± 0.23 µM) and soybean 15-lipoxygenase (15-LOX, IC50 2.41 ± 0.13 µM). Kinetic studies against α-glucosidase and urease enzymes revealed its competitive mode of inhibition. Molecular expression analysis of montelukast in breast cancer cell line MCF-7 down-regulated AP by a factor of 0.27 (5 µM) compared with the 0.26 value for standard inhibitor levamisole (10 µM). Molecular docking estimated a binding affinity ranging -8.82 to -15.65 kcal/mol for the enzymes. Docking against the DNA dodecamer (ID: 1BNA) observed -9.13 kcal/mol via minor groove binding. MD simulations suggested stable binding between montelukast and the target proteins predicting strong inhibitory potential of the ligand. Montelukast features a chloroquinoline, phenyl ring, a cyclopropane group, a carboxylic group and a sulfur atom all of which collectively enhance its inhibitory potential against the said enzymes. These in vitro and computational investigations demonstrate that it is possible and suggested that the interactions of montelukast with more than one targets presented herein may be linked with the side effects presented by this drug and necessitate additional work. The results altogether suggest montelukast as an important structural scaffold possessing multitargeted features and warrant further investigations in repurposing beyond its traditional pharmacological use.

Antiurease Activity of Antibiotics: In Vitro, In Silico, Structure Activity Relationship, and MD Simulations of Cephalosporins and Fluoroquinolones.

Helicobacter pylori infection is widespread in 50% of the world's population and is associated with gastric ulcers and related disorders that ultimately culminate in gastric cancer. Levofloxacin-based, or clarithromycin-based, triple therapy is frequently used to inhibit the bacterial urease enzyme for the eradication of H. pylori. A comprehensive investigation based on the urease inhibitory profiles of antibiotics and their computational implications is lacking in the scientific literature. The present study was aimed specifically to determine the antiurease activities within the realms of cephalosporins and fluoroquinolones by in vitro methods supported with in silico investigations. The results demonstrate the jack bean urease inhibitory activity of cephalosporins, wherein cefadroxil, cefpodoxime, cefotaxime, and cefaclor displayed inhibitions (IC50 21.35 ± 0.64 to 62.86 ± 0.78 µM) compared with the standard thiourea (IC50 21.25 ± 0.15 µM). Among fluoroquinolones, levofloxacin, ofloxacin, and gemifloxacin (IC50 7.24 ± 0.29 to 16.53 ± 0.85 µM) unveiled remarkable inhibitory profiles. Levofloxacin and ofloxacin exhibited competitive inhibition against the said enzyme. Ciprofloxacin and moxifloxacin displayed weak urease inhibitions. During molecular docking studies, Asp362, Gly279, Arg338, Asn168, Asp223, Gln364, and Met366 were involved in hydrogen bonding in fluoroquinolones, and hydrogen bonding was established with Arg338, His248, Asn168 residues, and metal Ni601 and Ni602 of the enzyme. MD simulations and MMPBSA results demonstrated the existence of significant protein-ligand binding. Overall, these results warrant further investigations into the significance of these active molecules in relation to their inhibitory potential against the targeted urease enzyme.

Green innovation, globalization, financial development, and CO2 emissions: the role of governance as a moderator in South Asian countries.

The current study is designed to analyze the relationship between, environmental innovations, globalization, financial development, and CO2 emissions in the South Asian region over the period of 1996 to 2019. In this regard, the role of governance is also incorporated as a moderator along with Environmental Kuznets Curve (EKC) hypothesis. The sample size includes Bangladesh, India, Pakistan, Nepal, and Sri Lanka. The results of the robust least square show the validity of EKC in the sample countries. Environmental innovations show desirable results on CO2 emissions, while globalization, financial development, and governance are increasing environmental degradation. The role of governance as a moderator is only effective and favorable with environmental innovation. However, in the case of globalization and financial development, governance appeared to be ineffective in lessening the rate of emissions; rather, it contributes to emissions. It clearly shows the missing link in formulating coherent policy to achieve sustainability targets. Therefore, it is desirable to improve the role of governance with respect to environmental policies not only to handle directly environmental issues but also indirectly while promoting the process of globalization and financial development.

Unraveling the non-linear impact of financial development on environmental sustainability: insights from developing countries agreeing the accord.

This study delves into the intricate relationship between financial development and environmental sustainability by considering the role of the Paris Agreement in the context of developing countries. By employing advanced econometric techniques method of moment quantile regression (MMQR) and considering a period spanning from 1996 to 2021, this research unravels the non-linear impact of financial development on environmental degradation while considering population and GDP as control variables. The study reveals an inverted N-shaped relationship between financial development and environmental degradation, indicating that environmental degradation (ED) decreases as financial development increases. However, this is followed by a rise in ED before eventually witnessing a further decline. Additionally, the study highlights the positive correlation between GDP and population with ED across all quantiles, with a more pronounced impact observed in higher quantiles. Furthermore, the coefficient of the Paris Agreement demonstrates its effectiveness in decreasing environmental degradation, particularly at higher quantiles of ED. The findings of this study hold practical implications for policymakers, emphasizing the importance of designing and implementing coherent environmental and economic policies in developing countries. This study contributes to understanding the complex dynamics between financial development and environmental sustainability, offering valuable insights for fostering sustainable development pathways.

Fibroblast Activation Protein Activates Macrophages and Promotes Parenchymal Liver Inflammation and Fibrosis.

BACKGROUND & AIMS: Fibroblast activation protein (FAP) is expressed on activated fibroblast. Its role in fibrosis and desmoplasia is controversial, and data on pharmacological FAP inhibition are lacking. We aimed to better define the role of FAP in liver fibrosis in vivo and in vitro. METHODS: FAP expression was analyzed in mice and patients with fibrotic liver diseases of various etiologies. Fibrotic mice received a specific FAP inhibitor (FAPi) at 2 doses orally for 2 weeks during parenchymal fibrosis progression (6 weeks of carbon tetrachloride) and regression (2 weeks off carbon tetrachloride), and with biliary fibrosis (Mdr2-/-). Recombinant FAP was added to (co-)cultures of hepatic stellate cells (HSC), fibroblasts, and macrophages. Fibrosis- and inflammation-related parameters were determined biochemically, by quantitative immunohistochemistry, polymerase chain reaction, and transcriptomics. RESULTS: FAP+ fibroblasts/HSCs were α-smooth muscle actin (α-SMA)-negative and located at interfaces of fibrotic septa next to macrophages in murine and human livers. In parenchymal fibrosis, FAPi reduced collagen area, liver collagen content, α-SMA+ myofibroblasts, M2-type macrophages, serum alanine transaminase and aspartate aminotransferase, key fibrogenesis-related transcripts, and increased hepatocyte proliferation 10-fold. During regression, FAP was suppressed, and FAPi was ineffective. FAPi less potently inhibited biliary fibrosis. In vitro, FAP small interfering RNA reduced HSC α-SMA expression and collagen production, and FAPi suppressed their activation and proliferation. Compared with untreated macrophages, FAPi regulated macrophage profibrogenic activation and transcriptome, and their conditioned medium attenuated HSC activation, which was increased with addition of recombinant FAP. CONCLUSIONS: Pharmacological FAP inhibition attenuates inflammation-predominant liver fibrosis. FAP is expressed on subsets of activated fibroblasts/HSC and promotes both macrophage and HSC profibrogenic activity in liver fibrosis.

Dietary wheat amylase trypsin inhibitors promote features of murine non-alcoholic fatty liver disease.

We previously demonstrated that a common dietary protein component, wheat amylase trypsin inhibitors (ATI), stimulate intestinal macrophages and dendritic cells via toll like receptor 4. Activation of these intestinal myeloid cells elicits an inflammatory signal that is propagated to mesenteric lymph nodes, and that can facilitate extraintestinal inflammation. Mice were fed a well-defined high fat diet, with (HFD/ATI) or without (HFD) nutritionally irrelevant amounts of ATI. Mice on HFD/ATI developed only mild signs of intestinal inflammation and myeloid cell activation but displayed significantly higher serum triglycerides and transaminases compared to mice on HFD alone. Moreover, they showed increased visceral and liver fat, and a higher insulin resistance. ATI feeding promoted liver and adipose tissue inflammation, with M1-type macrophage polarization and infiltration, and enhanced liver fibrogenesis. Gluten, the major protein component of wheat, did not induce these pathologies. Therefore, wheat ATI ingestion in minute quantities comparable to human daily wheat consumption exacerbated features of the metabolic syndrome and non-alcoholic steatohepatitis, despite its irrelevant caloric value.

α-Mannosyl-Functionalized Cationic Nanohydrogel Particles for Targeted Gene Knockdown in Immunosuppressive Macrophages.

Immunosuppressive M2 macrophages govern the immunophathogenic micromilieu in many severe diseases including cancer or fibrosis, thus, their re-polarization through RNA interference is a promising concept to support combinatorial therapies. For targeted siRNA delivery, however, safe and stable carriers are required that manage cell specific transport to M2 macrophages. Here, siRNA-loaded cationic nanogels are reported with α-mannosyl decorated surfaces that target and modify M2 macrophages selectively. Via amphiphilic precursor block copolymers bearing one single α-mannosyl moiety at their chain end mannosylated cationic nanohydrogel particles (ManNP) were obtained of 20 nm diameter determined by dynamic light scattering and cryogenic electron transmission microscopy. α-Mannosyl surface modification is confirmed by agglutination with concanavalin A. SiRNA-loaded ManNP preferentially targets the overexpressed mannose receptor CD206 on M2 macrophages, as shown by in vitro cell uptake studies in M2 polarized primary macrophages. This specificity is confirmed, since ManNP uptake could be reduced by blocking of CD206 with mannan. Effective ManNP-guided siRNA delivery is confirmed by sequence-specific gene knockdown of CSF-1R in M2-type macrophages exclusively, while the expression levels in M1-polarized macrophages is not affected. In conclusion, α-mannosyl-functionalized ManNPs are promising universal siRNA carriers for targeted immunomodulatory treatment of immunosuppressive macrophages.

Enhanced protection of C57 BL/6 vs Balb/c mice to melanoma liver metastasis is mediated by NK cells.

The B16F10 murine melanoma cell line displays a low expression of MHC class I molecules favoring immune evasion and metastases in immunocompetent C57 BL/6 wild-type mice. Here, we generated metastases to the liver, an organ that is skewed towards immune tolerance, by intrasplenic injection of B16F10 cells in syngeneic C57 BL/6 compared to allogeneic Balb/c mice. Surprisingly, Balb/c mice, which usually display a pronounced M2 macrophage and Th2 T cell polarization, were ∼3 times more susceptible to metastasis than C57 BL/6 mice, despite a much higher M1 and Th1 T cell immune response. The anti-metastatic advantage of C57 BL/6 mice could be attributed to a more potent NK-cell mediated cytotoxicity against B16F10 cells. Our findings highlight the role of NK cells in innate anti-tumor immunity in the context of the liver - particularly against highly aggressive MHC I-deficient cancer cells. Moreover, the B16F10 model of melanoma liver metastasis is suited for developing novel therapies targeting innate NK cell related immunity in liver metastases and liver cancer.

Lessons from a Broad-based ACA Outreach Effort: Promises and Pitfalls.

The Marketplace Coverage Initiative (MCI) sought to expand awareness and ACA Marketplace enrollment in the greater Kansas City Area. The MCI was evaluated through interviews, surveys, and focus groups. Two main findings are particularly relevant for future Marketplace enrollment efforts. First, the link between contacting someone and actual enrollment is tenuous as follow-up is challenging. Outreach efforts that only track contacts, such as appointments and email addresses, lack information needed to assess enrollment. Linking outreach activities to enrollment outcomes leads us to a dramatically different conclusion about using big data and campaign-style tactics than evaluations of similar techniques such as that pioneered by Enroll America in 11 states. Second, there is a large chasm between the knowledge levels of the uninsured and the decisions they face on the Marketplace. Based on these findings, outreach efforts were redesigned for the 2014 open enrollment period to focus on smaller, community-driven projects.

SiRNA-mediated in vivo gene knockdown by acid-degradable cationic nanohydrogel particles.

Cationic nanohydrogel particles have become an attractive tool for systemic siRNA delivery, but improvement of their in vivo tolerance is desirable, especially to prevent potential long term side effects by tissue and cellular accumulation. Here, we designed novel ketal cross-linked cationic nanohydrogel particles that were assessed for reduced tissue accumulation and robust siRNA delivery in vitro and in vivo. An oligo-amine cross-linker equipped with a ketal moiety in its core was synthesized and applied to nanohydrogel cross-linking of self-assembled reactive ester block copolymers in DMSO. The resulting acid-sensitive cationic nanoparticles spontaneously disassembled over time in acidic milieu, as investigated by dynamic light scattering. Fluorescent correlation spectroscopy showed effective complexation with siRNA as well as its release upon particle degradation at endosomal pH. These properties resulted in an enhanced in vitro gene knockdown for the acid-degradable cationic nanoparticles compared to their non-degradable spermine analogues. In a murine liver fibrosis model enhanced carrier and payload accumulation in the fibrotic tissue facilitated sequence-specific gene knockdown and prevented fibrosis progression. Long-term monitoring of the carrier in the body showed an enhanced clearance for the acid-degradable carrier, even after multiple dosing. Therefore, these acid-degradable cationic nanohydrogel particles can be considered as promising siRNA carriers for in vivo purposes towards therapeutic applications.

In Vivo Gene-Silencing in Fibrotic Liver by siRNA-Loaded Cationic Nanohydrogel Particles.

Cationic nanohydrogel particles loaded with anti-Col1α1 siRNA suppress collagen synthesis and deposition in fibrotic mice: Systemically administered 40 nm sized nanogel particles accumulate in collagen-expressing cells in the liver. Their siRNA payload induces a sequence specific in vivo gene knockdown affording an efficient antifibrotic effect in mice with liver fibrosis.