Clinical predictive value of renalase in post-ERCP pancreatitis.

BACKGROUND AND AIMS: Plasma levels of renalase decrease in acute experimental pancreatitis. We aimed to determine if decreases in plasma renalase levels after ERCP predict the occurrence of post-ERCP pancreatitis (PEP). METHODS: In this prospective cohort study conducted at a tertiary hospital, plasma renalase was determined before ERCP (baseline) and at 30 and 60 minutes after ERCP. Native renalase levels, acidified renalase, and native-to-acidified renalase proportions were analyzed over time using a longitudinal regression model. RESULTS: Among 273 patients, 31 developed PEP. Only 1 PEP patient had a baseline native renalase >6.0 µg/mL, whereas 38 of 242 without PEP had a native renalase > 6.0 µg/mL, indicating a sensitivity of 97% (30/31) and specificity of 16% (38/242) in predicting PEP. Longitudinal models did not show differences over time between groups. CONCLUSIONS: Baseline native renalase levels are very sensitive for predicting PEP. Further studies are needed to determine the potential clinical role of renalase in predicting and preventing PEP.

Inhibition mechanism of crude lipopeptide from Bacillus subtilis against Aeromonas veronii growth, biofilm formation, and spoilage of channel catfish flesh.

Aeromonas veronii is associated with food spoilage and some human diseases, such as diarrhea, gastroenteritis, hemorrhagic septicemia or asymptomatic and even death. This research investigated the mechanism of the growth, biofilm formation, virulence, stress resistance, and spoilage potential of Bacillus subtilis lipopeptide against Aeromonas veronii. Lipopeptides suppressed the transmembrane transport of Aeromonas veronii by changing the cell membrane's permeability, the structure of membrane proteins, and Na+/K+-ATPase. Lipopeptide significantly reduced the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) by 86.03% and 56.12%, respectively, ultimately slowing Aeromonas veronii growth. Lipopeptides also restrained biofilm formation by inhibiting Aeromonas veronii motivation and extracellular polysaccharide secretion. Lipopeptides downregulated gene transcriptional levels related to the virulence and stress tolerance of Aeromonas veronii. Furthermore, lipopeptides treatment resulted in a considerable decrease in the extracellular protease activity of Aeromonas veronii, which restrained the decomposing of channel catfish flesh. This research provides new insights into lipopeptides for controlling Aeromonas veronii and improving food safety.

Plasma renalase levels are associated with the development of acute pancreatitis.

BACKGROUND/OBJECTIVES: Severe acute pancreatitis is associated with significant morbidity and mortality. Identifying factors that affect the risk of developing severe disease could influence management. Plasma levels of renalase, an anti-inflammatory secretory protein, dramatically decrease in a murine acute pancreatitis model. We assessed this response in hospitalized acute pancreatitis patients to determine if reduced plasma renalase levels occur in humans. METHODS: Plasma samples were prospectively and sequentially collected from patients hospitalized for acute pancreatitis. Two forms of plasma renalase, native (no acid) and acidified, were measured by ELISA and RNLS levels were compared between healthy controls and patients with mild and severe disease (defined as APACHE-II score ≥7) using nonparametric statistical analysis. RESULTS: Control (33) and acute pancreatitis (mild, 230 (76.7%) and severe, 70 (23.3%) patients were studied. Acidified RNLS levels were lower in pancreatitis patients: Control: 10.1 µg/ml, Mild 5.1 µg/ml, Severe 6.0 µg/ml; p < 0.001. Native RNLS levels were increased in AP: Control: 0.4 µg/ml, Mild 0.9 µg g/ml, Severe 1.2 µg/ml p < 0.001; those with severe AP trended to have higher native RNLS levels than those with mild disease (p = 0.056). In patients with severe AP, higher APACHE-II scores at 24 h after admission correlated with lower acid-sensitive RNLS levels on admission (r = -0.31, p = 0.023). CONCLUSION: Low plasma acidified RNLS levels, and increased native RNLS levels are associated with AP. Additional studies should assess the clinical correlation between plasma RNLS levels and AP severity and outcomes.

Competitive inhibition of a non-natural cofactor dependent formaldehyde dehydrogenase by imidazole.

OBJECTIVES: To better understand the unique inhibitory behavior of a non-natural cofactor preferred formaldehyde dehydrogenase (FalDH) mutant 9B2. RESULTS: We described our serendipitous observation that 9B2 was reversibly inhibited by residual imidazole introduced during protein preparation, while the wild-type enzyme was not sensitive to imidazole. Kinetic analysis showed that imidazole was a competitive inhibitor of formaldehyde with a Ki of 16 µM and an uncompetitive inhibitor of Nicotinamide Cytosine Dinucleotide for 9B2, indicating that formaldehyde and imidazole were combined in the same position. Molecular docking results of 9B2 showed that imidazole could favorably bind very close to the nicotinamide moiety of the cofactor, where formaldehyde was expected to reside for catalysis, which was in line with a competitive inhibition. CONCLUSION: The mutant 9B2 can be competitively inhibited by imidazole, suggesting that cautions should be taken to evaluate activities as protein mutants might attain unexpected sensitivity to a component in buffers for purification or activity assays.

Kidney-Targeted Renalase Agonist Prevents Cisplatin-Induced Chronic Kidney Disease by Inhibiting Regulated Necrosis and Inflammation.

BACKGROUND: Repeated administration of cisplatin causes CKD. In previous studies, we reported that the kidney-secreted survival protein renalase (RNLS) and an agonist peptide protected mice from cisplatin-induced AKI. METHODS: To investigate whether kidney-targeted delivery of RNLS might prevent cisplatin-induced CKD in a mouse model, we achieved specific delivery of a RNLS agonist peptide (RP81) to the renal proximal tubule by encapsulating the peptide in mesoscale nanoparticles (MNPs). We used genetic deletion of RNLS, single-cell RNA sequencing analysis, and Western blotting to determine efficacy and to explore underlying mechanisms. We also measured plasma RNLS in patients with advanced head and neck squamous cell carcinoma receiving their first dose of cisplatin chemotherapy. RESULTS: In mice with CKD induced by cisplatin, we observed an approximate 60% reduction of kidney RNLS; genetic deletion of RNLS was associated with significantly more severe cisplatin-induced CKD. In this severe model of cisplatin-induced CKD, systemic administration of MNP-encapsulated RP81 (RP81-MNP) significantly reduced CKD as assessed by plasma creatinine and histology. It also decreased inflammatory cytokines in plasma and inhibited regulated necrosis in kidney. Single-cell RNA sequencing analyses revealed that RP81-MNP preserved epithelial components of the nephron and the vasculature and suppressed inflammatory macrophages and myofibroblasts. In patients receiving their first dose of cisplatin chemotherapy, plasma RNLS levels trended lower at day 14 post-treatment. CONCLUSIONS: Kidney-targeted delivery of RNLS agonist RP81-MNP protects against cisplatin-induced CKD by decreasing cell death and improving the viability of the renal proximal tubule. These findings suggest that such an approach might mitigate the development of CKD in patients receiving cisplatin cancer chemotherapy.

Engineering Formaldehyde Dehydrogenase from Pseudomonas putida to Favor Nicotinamide Cytosine Dinucleotide.

The enzyme formaldehyde dehydrogenase (FalDH) from Pseudomonas putida is of particular interest for biotechnological applications as it catalyzes the oxidation of formaldehyde independent of glutathione. However, the consumption of a stoichiometric amount of nicotinamide adenine dinucleotide (NAD) can be challenging at the metabolic level as this may affect many other NAD-linked processes. A potential solution is to engineer FalDH to utilize non-natural cofactors. Here we devised FalDH variants to favor nicotinamide cytosine dinucleotide (NCD) by structure-guided modification of the binding pocket for the adenine moiety of NAD. Several mutants were obtained and the best one FalDH 9B2 had over 150-fold higher preference for NCD than NAD. Molecular docking analysis indicated that the cofactor binding pocket shrunk to better fit NCD, a smaller-sized cofactor. FalDH 9B2 together with other NCD-linked enzymes offer opportunities to assemble orthogonal pathways for biological conversion of C1 molecules.

The role of NAD and NAD precursors on longevity and lifespan modulation in the budding yeast, Saccharomyces cerevisiae.

Molecular causes of aging and longevity interventions have witnessed an upsurge in the last decade. The resurgent interests in the application of small molecules as potential geroprotectors and/or pharmacogenomics point to nicotinamide adenine dinucleotide (NAD) and its precursors, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide, and nicotinic acid as potentially intriguing molecules. Upon supplementation, these compounds have shown to ameliorate aging related conditions and possibly prevent death in model organisms. Besides being a molecule essential in all living cells, our understanding of the mechanism of NAD metabolism and its regulation remain incomplete owing to its omnipresent nature. Here we discuss recent advances and techniques in the study of chronological lifespan (CLS) and replicative lifespan (RLS) in the model unicellular organism Saccharomyces cerevisiae. We then follow with the mechanism and biology of NAD precursors and their roles in aging and longevity. Finally, we review potential biotechnological applications through engineering of microbial lifespan, and laid perspective on the promising candidature of alternative redox compounds for extending lifespan.

Comparative multi-omics analysis of hypoxic germination tolerance in weedy rice embryos and coleoptiles.

Hypoxic germination tolerance is an important trait for seedling establishment of direct-seeded rice. Our comparative metabolomics analysis revealed that weedy rice accumulated more sugar and amino acids than cultivated rice accumulated in the embryo and coleoptile tissues under hypoxic stress. At the transcriptional level, oxidative phosphorylation activity in weedy rice was higher than in cultivated rice that likely led to more efficient energy metabolism during hypoxic stress. Based on our comparative proteomics analysis, enriched proteins related to cell wall implied that the advantages in energy metabolism of weedy rice were ultimately reflected in the formation of tissue structures. In this study, we found that most of key hypoxic germination tolerance (HGT) genes shared the same genetic backgrounds with Oryza japonica, however, several of them genetically similar to other Oryza plant also play important roles. Our findings suggest weedy rice can serve as genetic resources for the improvement of direct-seeding rice.

Structural Insights into Malic Enzyme Variants Favoring an Unnatural Redox Cofactor.

The use of nicotinamide cytosine dinucleotide (NCD), a biocompatible nicotinamide adenosine dinucleotide (NAD) analogue, is of great scientific and biotechnological interest. Several redox enzymes have been devised to favor NCD, and have been successfully applied in creating NCD-dependent redox systems. However, molecular interactions between cofactor and protein have still to be disclosed in order to guide further engineering efforts. Here we report the structural analysis of an NCD-favoring malic enzyme (ME) variant derived from Escherichia coli. The X-ray crystal structure data revealed that the residues located at position 346 and 401 in ME acted as the "gatekeepers" of the adenine moiety binding cavity. When Arg346 was substituted with either acidic or aromatic residues, the corresponding mutants showed substantially reduced NCD preference. Inspired by these observations, we generated Lactobacillus helveticus derived d-lactate dehydrogenase variants at Ile177, the counterpart to Arg346 in ME, and found a similar trend in terms of cofactor preference changes. As many NAD-dependent oxidoreductases share key structural features, our results provide guidance for protein engineering to obtain more NCD-favoring variants.

Development of Novel Anti-influenza Thiazolides with Relatively Broad-Spectrum Antiviral Potentials.

Seasonal and pandemic influenza causes 650,000 deaths annually in the world. The emergence of drug resistance to specific anti-influenza virus drugs such as oseltamivir and baloxavir marboxil highlights the urgency of novel anti-influenza chemical entity discovery. In this study, we report a series of novel thiazolides derived from an FDA-approved drug, nitazoxanide, with antiviral activity against influenza and a broad range of viruses. The preferred candidates 4a and 4d showed significantly enhanced anti-influenza virus potentials, with 10-fold improvement compared to results with nitazoxanide, and were effective against a variety of influenza virus subtypes including oseltamivir-resistant strains. Notably, the combination using compounds 4a/4d and oseltamivir carboxylate or zanamivir displayed synergistic antiviral effects against oseltamivir-resistant strains. Mode-of-action analysis demonstrated that compounds 4a/4d acted at the late phase of the viral infection cycle through inhibiting viral RNA transcription and replication. Further experiments showed that treatment with compounds 4a/4d significantly inhibited influenza virus infection in human lung organoids, suggesting the druggability of the novel thiazolides. In-depth transcriptome analysis revealed a series of upregulated cellular genes that may contribute to the antiviral activities of 4a/4d. Together, the results of our study indicated the direction to optimize nitazoxanide as an anti-influenza drug and discovered two candidates with novel structures, compounds 4a/4d, that have relatively broad-spectrum antiviral potentials.

Engineering d-Lactate Dehydrogenase to Favor an Non-natural Cofactor Nicotinamide Cytosine Dinucleotide.

Synthetic nicotinamide adenine dinucleotide (NAD) analogues are of great scientific and biotechnological interest. One such analogue, nicotinamide cytosine dinucleotide (NCD), has been successfully applied to creating bioorthogonal redox systems. Yet, only a few redox enzymes have been devised to favor NCD. We have engineered Lactobacillus helveticus-derived NAD-dependent d-lactate dehydrogenase (LhDLDH) to favor NCD by semirational design. Sequence alignment and structural analysis revealed that amino acid residues I177 and N213 form a "gate" guarding the NAD adenine moiety binding cavity. Saturated mutagenesis libraries were constructed by using the mutant LhDLDH-V152R as the parental sequence. Mutants were obtained with good catalytic efficiency, and NCD preference increased by up to 940-fold. Experiments showed that Escherichia coli cells expressing mutants with higher NCD preference afforded much less d-lactate, thus suggesting the potential to construct NCD-mediated orthogonal metabolism.

Structure-Guided Design of Formate Dehydrogenase for Regeneration of a Non-Natural Redox Cofactor.

Formate dehydrogenase (FDH) has been widely used for the regeneration of the reduced nicotinamide adenine dinucleotide (NADH). To utilize nicotinamide cytosine dinucleotide (NCD) as a non-natural redox cofactor, it remains challenging as NCDH, the reduced form of NCD, has to be efficiently regenerated. Here we demonstrate successful engineering of FDH for NCDH regeneration. Guided by the structural information of FDH from Pseudomonas sp. 101 (pseFDH) and the NAD-pseFDH complex, semi-rational strategies were applied to design mutant libraries and screen for NCD-linked activity. The most active mutant reached a cofactor preference switch from NAD to NCD by 3700-fold. Homology modeling analysis showed that these mutants had reduced cofactor binding pockets and dedicated hydrophobic interactions for NCD. Efficient regeneration of NCDH was implemented by powering an NCD-dependent D-lactate dehydrogenase for stoichiometric and stereospecific reduction of pyruvate to D-lactate at the expense of formate.

Identification of Novel Influenza Polymerase PB2 Inhibitors Using a Cascade Docking Virtual Screening Approach.

To discover novel inhibitors that target the influenza polymerase basic protein 2 (PB2) cap-binding domain (CBD), commercial ChemBridge compound libraries containing 384,796 compounds were screened using a cascade docking of LibDock-LigandFit-GOLD, and 60 compounds were selected for testing with cytopathic effect (CPE) inhibition assays and surface plasmon resonance (SPR) assay. Ten compounds were identified to rescue cells from H1N1 virus-mediated death at non-cytotoxic concentrations with EC50 values ranging from 0.30 to 67.65 µM and could bind to the PB2 CBD of H1N1 with Kd values ranging from 0.21 to 6.77 µM. Among these, four compounds (11D4, 12C5, 21A5, and 21B1) showed inhibition of a broad spectrum of influenza virus strains, including oseltamivir-resistant ones, the PR/8-R292K mutant (H1N1, recombinant oseltamivir-resistant strain), the PR/8-I38T mutant (H1N1, recombinant baloxavir-resistant strain), and the influenza B/Lee/40 virus strain. These compounds have novel chemical scaffolds and relatively small molecular weights and are suitable for optimization as lead compounds. Based on sequence and structure comparisons of PB2 CBDs of various influenza virus subtypes, we propose that the Phe323/Gln325, Asn429/Ser431, and Arg355/Gly357 mutations, particularly the Arg355/Gly357 mutation, have a marked impact on the selectivities of PB2 CBD-targeted inhibitors of influenza A and influenza B.

Non-natural Cofactor and Formate-Driven Reductive Carboxylation of Pyruvate.

A non-natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non-natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD-dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO2 source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When 13 C-isotope-labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom-economic strategy for CO2 fixation where formate is used in lieu of CO2 and offers dedicated reducing power.

Regulated necrosis and failed repair in cisplatin-induced chronic kidney disease.

Cisplatin is an effective chemotherapeutic agent, but significant nephrotoxicity limits its clinical use. Despite extensive investigation of the acute cellular and molecular responses to cisplatin, the mechanisms of progression from acute to chronic kidney injury have not been explored. We used functional and morphological metrics to establish a time-point when the transition from acute and reversible kidney injury to chronic and irreparable kidney disease is clearly established. In mice administered 1 or 2 doses of intraperitoneal cisplatin separated by 2 weeks, kidney function returned toward baseline two weeks after the first dose, but failed to return to normal two weeks following a second dose. Multiphoton microscopy revealed increased glomerular epithelial and proximal tubular damage in kidneys exposed to two doses of cisplatin compared with those exposed to a single dose. In contrast, there was no evidence of fibrosis, macrophage invasion, or decrease in endothelial cell mass in chronically diseased kidneys. Pathway analysis of microarray data revealed regulated necrosis as a key determinant in the development of chronic kidney disease after cisplatin administration. Western blot analysis demonstrated activation of proteins involved in necroptosis and increased expression of kidney injury markers, cellular stress response regulators, and upstream activators of regulated necrosis, including Toll-like receptors 2 and 4. These data suggest that unresolved injury and sustained activation of regulated necrosis pathways, rather than fibrosis, promote the progression of cisplatin-induced acute kidney injury to chronic kidney disease.

Characterization of the substrate scope of an alcohol dehydrogenase commonly used as methanol dehydrogenase.

Many alcohol dehydrogenases (ADHs) catalyze oxidation of a broad scope of alcohols. When an NAD-dependent ADH oxidizes methanol, albeit at a poor rate, it may be treated as methanol dehydrogenase (MDH). One ADH from Geobacillus stearothermophilus DSM 2334 (GsADH) has been widely used as MDH, but its actual substrate scope remains less characterized. Here we purified recombinant GsADH from Escherichia coli and determined its crystal structure. We collected kinetics data of this enzyme towards a number of short chain alcohols, and found that isopropanol is by far the most favorable substrate. Moreover, molecular docking analysis suggested that substrate preference is mainly attributed to the conformer energy of the protein-substrate complex. Our data clarified the substrate scope of GsADH and provided structural insights, which may facilitate more efficient cofactor regeneration and rational metabolic engineering.

Extracellular renalase protects cells and organs by outside-in signalling.

Renalase was discovered as a protein synthesized by the kidney and secreted in blood where it circulates at a concentration of approximately 3-5 µg/ml. Initial reports suggested that it functioned as an NAD(P)H oxidase and could oxidize catecholamines. Administration of renalase lowers blood pressure and heart rate and also protects cells and organs against ischaemic and toxic injury. Although renalase's protective effect was initially ascribed to its oxidase properties, a paradigm shift in our understanding of the cellular actions of renalase is underway. We now understand that, independent of its enzymatic properties, renalase functions as a cytokine that provides protection to cells, tissues and organs by interacting with its receptor to activate protein kinase B, JAK/STAT, and the mitogen-activated protein kinase pathways. In addition, recent studies suggest that dysregulated renalase signalling may promote survival of several tumour cells due to its capacity to augment expression of growth-related genes. In this review, we focus on the cytoprotective actions of renalase and its capacity to sustain cancer cell growth and also the translational opportunities these findings represent for the development of novel therapeutic strategies for organ injury and cancer.

Effect of argon flow on promoting boron doping for in-situ grown silicon nitride thin films containing silicon quantum dots.

Boron-doped silicon nitride thin films (SiNx) containing silicon quantum dots (Si QD) were prepared in situ by plasma enhanced chemical vapor deposition. With the aim of optimizing the performance of thin films, the mixed gas including argon and hydrogen was applied as dilution. The effects of Ar flow on the structural, electrical and optical properties of B-doped SiNx thin films were systemically studied through various characterizations. By tuning the Ar flow, the properties, such as QD size, crystallinity and optical band gap, can be effectively controlled. The B-doping efficiency in thin films was proved to be promoted by introducing moderate Ar flow. The maximum values of dark conductivity (1.52 S cm-1) and carrier concentration (2.41 × 1019 cm-3) were obtained for the B-doped SiNx thin films at the Ar flow of 200 sccm. Furthermore, the mechanism on the promotion in B-doping was illustrated in detail in this paper.

Structural Transition of Cinnamate-Based Light-Responsive Ionic Liquids in Aqueous Solutions and Their Light-Tunable Rheological Properties.

Light-responsive wormlike micelles have important applications in fields such as microfluids, photoswichable fluids, and rheology control. However, single-component light-responsive wormlike micelles formed only from a single tail surfactant have not been reported in literature. In this work, self-assembly behavior of 1-alkyl-3-methylimidazolium trans-ortho-methoxycinnamate [Cnmim][OMCA] (n = 8, 10, 12, 14, 16) ionic liquids in aqueous solutions is studied by UV-vis spectroscopy, viscosity, rheology, conductivity, and cryo-TEM measurements. It is found for the first time that, among the single tail ionic liquid surfactants studied, [C16mim][OMCA] can form wormlike micelles in aqueous solutions without any additives and light irradiation. Then these wormlike micelles are able to transform into cylindrical micelles under UV light irradiation, resulting in significantly tunable rheological properties of the solutions. The photoisomerization of anion of [C16mim][OMCA] from trans- to cis-isomer as well as the relative hydrophilicity and structural feature of the cis-isomer are suggested to be responsible for such transition.

The novel tumor angiogenic factor, adrenomedullin-2, predicts survival in pancreatic adenocarcinoma.

BACKGROUND: Tumor angiogenesis has been demonstrated to have an important role in the development, progression, and metastasis of pancreas cancer. Adrenomedullin-2 (ADM2) is a calcitonin gene-related peptide that has recently been shown to be a novel tumor angiogenesis factor, acting via mitogen-activated protein kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/Akt, and vascular endothelial growth factor/vascular endothelial growth factor-2 signaling pathways. Through the use of tissue microarray (TMA) technology, we hypothesize that ADM2 is an important tumor angiogenesis factor in pancreatic cancer. METHODS: Multiple TMAs were created using tissue from pancreatic cancer patients resected between January 1996 and December 2006. Core tissue samples of formalin-fixed, paraffin-embedded blocks of pancreatic cancer tissue were collected through an institutional review board-approved protocol and linked to available clinicopathologic data. Two TMAs consisting of 112 and 60 patients with pancreatic adenocarcinoma were studied for ADM2 protein expression using a quantitative, automated immunofluorescent microscopy system, a technology that removes potential observer bias in TMA analysis. The results were analyzed using independent Student t-test, chi-square, log-rank regression, and Kaplan-Meier methods. RESULTS: One hundred sixteen patients were identified for complete analysis, and 56 patients had complete survival data. Median follow-up for survivors was 14.5 mo. Total cellular levels of ADM2 were found to be a predictor of survival in pancreatic cancer. Low ADM2 levels were associated with a higher 5-y survival compared with high ADM2 levels (18% versus 6%, P = 0.05). Median survival was also worse in high ADM2 expressers (18.7 versus 8.6 mo). In accordance with prior-published pancreatic cancer data, a worse histologic grade (P = 0.001), tumor (T) stage (P = 0.009), and overall disease stage (P = 0.004), all portended a worse survival. CONCLUSIONS: For the first time, we have demonstrated that high levels of ADM2 expression predict a poorer survival in patients with pancreatic adenocarcinoma. This suggests a possible role of ADM2 in pancreas cancer and as a novel biomarker that predicts poorer survival. Additional study of ADM2 in pancreatic cancer will help reveal its true angiogenic role in pancreas cancer and its potential role as a novel therapeutic target.