Associations of Advanced Glycation End Products with Sleep Disorders in Chinese Adults.

BACKGROUND: Advanced glycation end products (AGEs), a group of food processing byproducts, have been implicated in the development of various diseases. However, the relationship between circulating AGEs and sleep disorders remains uncertain. METHODS: This cross-sectional study elucidated the association of plasma AGEs with sleep disorders among 1732 Chinese adults who participated in the initial visit (2019-2020) of the Tongji-Shenzhen Cohort (TJSZC). Sleep behavior was assessed using self-reported questionnaires and precise accelerometers. Plasma levels of AGEs, including Nε-(Carboxymethyl)lysine (CML), Nε-(Carboxyethyl)lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1), were quantified by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: In logistic regression, per IQR increment in individual AGEs was associated with an increased odds ratio of short sleep duration (CML: 1.11 [1.00, 1.23]; CEL: 1.16, [1.04, 1.30]), poor sleep quality (CML: 1.33 [1.10, 1.60]; CEL: 1.53, [1.17, 2.00]; MG-H1: 1.61 [1.25, 2.07]), excessive daytime sleepiness (CML: 1.33 [1.11, 1.60]; MG-H1: 1.39 [1.09, 1.77]), and insomnia (CML: 1.29 [1.05, 1.59]). Furthermore, in weighted quantile sum regression and Bayesian kernel machine regression analyses, elevated overall exposure levels of plasma AGEs were associated with an increased risk of sleep disorders, including short sleep duration, poor sleep quality, excessive daytime sleepiness, and insomnia, with CML being identified as the leading contributor. Insufficient vegetable intake and higher dietary fat intake was associated with an increase in plasma CEL. CONCLUSIONS: These findings support a significant association between plasma AGEs and sleep disorders, indicating that AGEs may adversely influence sleep health and reducing the intake of AGEs may facilitate preventing and ameliorating sleep disorders.

Peptidyl arginine deiminase-4 inhibitor ameliorates pulmonary fibrosis through positive regulation of developmental endothelial locus-1.

Recurring lung injury, chronic inflammation, aberrant tissue repair and impaired tissue remodelling contribute to the pathogenesis of pulmonary fibrosis (PF). Neutrophil extracellular traps (NETs) are released by activated neutrophils to trap, immobilise and kill invading pathogen and is facilitated by peptidyl arginine deiminase-4 (PAD-4). Dysregulated NETs release and abnormal PAD-4 activation plays a crucial role in activating pro-fibrotic events in PF. Developmental endothelial locus-1 (Del-1), expressed by the endothelial cells of lungs and brain acts as an endogenous inhibitor of inflammation and fibrosis. We have hypothesised that PAD-4 inhibitor exerts anti-inflammatory and anti-fibrotic effects in mice model of PF. We have also hypothesised by PAD-4 regulated the transcription of Del-1 through co-repression and its inhibition potentiates anti-fibrotic effects of Del-1. In our study, the PAD-4 inhibitor chloro-amidine (CLA) demonstrated anti-NETotic and anti-inflammatory effects in vitro in differentiated HL-60 cells. In a bleomycin-induced PF mice model, CLA administration in two doses (3 mg/kg, I.P and 10 mg/kg, I.P) improved lung function, normalized bronchoalveolar lavage fluid parameters, and attenuated fibrotic events, including markers of extracellular matrix and epithelial-mesenchymal transition. Histological analyses confirmed the restoration of lung architecture and collagen deposition with CLA treatment. ELISA, IHC, IF, RT-PCR, and immunoblot analysis supported the anti-NETotic effects of CLA. Furthermore, BLM-induced PF reduced Del-1 and p53 expression, which was normalized by CLA treatment. These findings suggest that inhibition of PAD-4 results in amelioration of PF in animal model and may involve modulation of Del-1 and p53 pathways, warranting further investigation.

Pre-diagnostic plasma advanced glycation end-products and soluble receptor for advanced glycation end-products and mortality in colorectal cancer patients.

Advanced glycation end-products (AGEs), formed endogenously or obtained exogenously from diet, may contribute to chronic inflammation, intracellular signaling alterations, and pathogenesis of several chronic diseases including colorectal cancer (CRC). However, the role of AGEs in CRC survival is less known. The associations of pre-diagnostic circulating AGEs and their soluble receptor (sRAGE) with CRC-specific and overall mortality were estimated using multivariable-adjusted Cox proportional hazards regression among 1369 CRC cases in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Concentrations of major plasma AGEs, Nε-[carboxy-methyl]lysine (CML), Nε-[carboxy-ethyl]lysine (CEL) and Nδ-[5-hydro-5-methyl-4-imidazolon-2-yl]-ornithine (MG-H1), were measured using ultra-performance liquid chromatography mass-spectrometry. sRAGE was assessed by enzyme-linked immunosorbent assay. Over a mean follow-up period of 96 months, 693 deaths occurred of which 541 were due to CRC. Individual and combined AGEs were not statistically significantly associated with CRC-specific or overall mortality. However, there was a possible interaction by sex for CEL (Pinteraction = .05). Participants with higher sRAGE had a higher risk of dying from CRC (HRQ5vs.Q1 = 1.67, 95% CI: 1.21-2.30, Ptrend = .02) or any cause (HRQ5vs.Q1 = 1.38, 95% CI: 1.05-1.83, Ptrend = .09). These associations tended to be stronger among cases with diabetes (Pinteraction = .03) and pre-diabetes (Pinteraction <.01) before CRC diagnosis. Pre-diagnostic AGEs were not associated with CRC-specific and overall mortality in individuals with CRC. However, a positive association was observed for sRAGE. Our findings may stimulate further research on the role of AGEs and sRAGE in survival among cancer patients with special emphasis on potential effect modifications by sex and diabetes.

Optimizing Systems for Robust Heterologous Production of Biosurfactants Rhamnolipid and Lyso-Ornithine Lipid in Pseudomonas putida KT2440.

Surfactants are amphiphilic molecules that are capable of mixing water and oil. Biosurfactants are eco-friendly, low-toxicity, and stable to a variety of environmental factors. Optimizing conditions for microorganisms to produce biosurfactants can lead to improved production suitable for scaling up. In this study, we compared heterologous expression levels of the luminescence system luxCDABE operon controlled by regulatable promoters araC-PBAD and its strong version araC-PBAD-SD in Escherichia coli K12, Pseudomonas aeruginosa PAO1, and P. putida KT2440. Real-time monitoring of luminescence levels in the three strains indicated that luxCDABE controlled by araC-PBAD-SD promoter with 0.2% arabinose supplementation in P. putida produced the highest level of luminescence. By using the araC-PBAD-SD promoter-controlled rhlAB expression in P. putida, we were able to produce mono-rhamnolipid at a level of 1.5 g L-1 when 0.02% arabinose was supplemented. With the same system to express olsB, lyso-ornithine lipid was produced at a level of 10 mg L-1 when 0.2% arabinose was supplemented. To our knowledge, this is the first report about optimizing conditions for lyso-ornithine lipid production at a level up to 10 mg L-1. Taken together, our results demonstrate that regulatable araC-PBAD-SD promoter in P. putida KT2440 is a useful system for heterologous production of biosurfactants.

Protein glycation products associate with progression of kidney disease and incident cardiovascular events in individuals with type 1 diabetes.

BACKGROUND: Despite improved glycemic treatment, the impact of glycation on pathological consequences may persist and contribute to adverse clinical outcomes in diabetes. In the present study we investigated the association between serum protein glycation products and progression of kidney disease as well as incident major adverse cardiovascular events (MACE) in type 1 diabetes. METHODS: Fructosamine, advanced glycation end products (AGEs), and methylglyoxal-modified hydro-imidazolone (MG-H1) were measured from baseline serum samples in the FinnDiane study (n = 575). Kidney disease progression was defined as steep eGFR decline (> 3 mL/min/1.73 m2/year) or progression of albuminuria (from lower to higher stage of albuminuria). MACE was defined as acute myocardial infarction, coronary revascularization, cerebrovascular event (stroke), and cardiovascular death. RESULTS: Fructosamine was independently associated with steep eGFR decline (OR 2.15 [95% CI 1.16-4.01], p = 0.016) in the fully adjusted model (age, sex, baseline eGFR). AGEs were associated with steep eGFR decline (OR 1.58 per 1 unit of SD [95% CI 1.07-2.32], p = 0.02), progression to end-stage kidney disease (ESKD) (HR 2.09 per 1 unit of SD [95% CI 1.43-3.05], p < 0.001), and pooled progression (to any stage of albuminuria) (HR 2.72 per 1 unit of SD [95% CI 2.04-3.62], p < 0.001). AGEs (HR 1.57 per 1 unit of SD [95% CI 1.23-2.00], p < 0.001) and MG-H1 (HR 4.99 [95% CI 0.98-25.55], p = 0.054) were associated with incident MACE. MG-H1 was also associated with pooled progression (HR 4.19 [95% CI 1.11-15.89], p = 0.035). Most AGEs and MG-H1 associations were no more significant after adjusting for baseline eGFR. CONCLUSIONS: Overall, these findings suggest that protein glycation products are an important risk factor for target organ damage in type 1 diabetes. The data provide further support to investigate a potential causal role of serum protein glycation in the progression of diabetes complications.

Glucoselysine, a unique advanced glycation end-product of the polyol pathway and its association with vascular complications in type 2 diabetes.

Glucoselysine (GL) is an unique advanced glycation end-product derived from fructose. The main source of fructose in vivo is the polyol pathway, and an increase in its activity leads to diabetic complications. Here, we aimed to demonstrate that GL can serve as an indicator of the polyol pathway activity. Additionally, we propose a novel approach for detecting GL in peripheral blood samples using liquid chromatography-tandem mass spectrometry and evaluate its clinical usefulness. We successfully circumvent interference from fructoselysine, which shares the same molecular weight as GL, by performing ultrafiltration and hydrolysis without reduction, successfully generating adequate peaks for quantification in serum. Furthermore, using immortalized aldose reductase KO mouse Schwann cells, we demonstrate that GL reflects the downstream activity of the polyol pathway and that GL produced intracellularly is released into the extracellular space. Clinical studies reveal that GL levels in patients with type 2 diabetes are significantly higher than those in healthy participants, while Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine (MG-H1) levels are significantly lower. Both GL and MG-H1 show higher values among patients with vascular complications; however, GL varies more markedly than MG-H1 as well as hemoglobin A1c, fasting plasma glucose, and estimated glomerular filtration rate. Furthermore, GL remains consistently stable under various existing drug treatments for type 2 diabetes, whereas MG-H1 is impacted. To the best of our knowledge, we provide important insights in predicting diabetic complications caused by enhanced polyol pathway activity via assessment of GL levels in peripheral blood samples from patients.

Levels of asymmetric dimethylarginine in plasma and aqueous humor: a key risk factor for the severity of fibrovascular proliferation in proliferative diabetic retinopathy.

Introduction: Proliferative diabetic retinopathy (PDR) is a common diabetes complication, significantly impacting vision and quality of life. Previous studies have suggested a potential link between arginine pathway metabolites and diabetic retinopathy (DR). Connective tissue growth factor (CTGF) plays a role in the occurrence and development of fibrovascular proliferation (FVP) in PDR patients. However, the relationship between arginine pathway metabolites and FVP in PDR remains undefined. This study aimed to explore the correlation between four arginine pathway metabolites (arginine, asymmetric dimethylarginine[ADMA], ornithine, and citrulline) and the severity of FVP in PDR patients. Methods: In this study, plasma and aqueous humor samples were respectively collected from 30 patients with age-related cataracts without diabetes mellitus (DM) and from 85 PDR patients. The PDR patients were categorized as mild-to-moderate or severe based on the severity of fundal FVP. The study used Kruskal-Wallis test to compare arginine, ADMA, ornithine, and citrulline levels across three groups. Binary logistic regression identified risk factors for severe PDR. Spearman correlation analysis assessed associations between plasma and aqueous humor metabolite levels, and between ADMA and CTGF levels in aqueous humor among PDR patients. Results: ADMA levels in the aqueous humor were significantly greater in patients with severe PDR than in those with mild-to-moderate PDR(P=0.0004). However, the plasma and aqueous humor levels of arginine, ornithine, and citrulline did not significantly differ between mild-to-moderate PDR patients and severe PDR patients (P>0.05). Binary logistic regression analysis indicated that the plasma (P=0.01) and aqueous humor (P=0.006) ADMA levels in PDR patients were risk factors for severe PDR. Furthermore, significant correlations were found between plasma and aqueous humor ADMA levels (r=0.263, P=0.015) and between aqueous humor ADMA and CTGF levels (r=0.837, P<0.001). Conclusion: Elevated ADMA levels in plasma and aqueous humor positively correlate with the severity of FVP in PDR, indicating ADMA as a risk factor for severe PDR.

Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation.

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications. KEY POINTS: • Ornithine lipid production in E. coli increases biomass yield under phosphate limitation. • Engineered strains show an enhanced production phenotype under low pH stress. • Transcriptome analysis and CCCP experiments revealed reduced proton leakage.

Radical S-Adenosyl-l-Methionine Enzyme PylB: A C-Centered Radical to Convert l-Lysine into (3R)-3-Methyl-d-Ornithine.

PylB is a radical S-adenosyl-l-methionine (SAM) enzyme predicted to convert l-lysine into (3R)-3-methyl-d-ornithine, a precursor in the biosynthesis of the 22nd proteogenic amino acid pyrrolysine. This protein highly resembles that of the radical SAM tyrosine and tryptophan lyases, which activate their substrate by abstracting a H atom from the amino-nitrogen position. Here, combining in vitro assays, analytical methods, electron paramagnetic resonance spectroscopy, and theoretical methods, we demonstrated that instead, PylB activates its substrate by abstracting a H atom from the Cγ position of l-lysine to afford the radical-based ß-scission. Strikingly, we also showed that PylB catalyzes the reverse reaction, converting (3R)-3-methyl-d-ornithine into l-lysine and using catalytic amounts of the 5'-deoxyadenosyl radical. Finally, we identified significant in vitro production of 5'-thioadenosine, an unexpected shunt product that we propose to result from the quenching of the 5'-deoxyadenosyl radical species by the nearby [Fe4S4] cluster.

The investigation of cytotoxic and apoptotic activity of Cl-amidine on the human U-87 MG glioma cell line.

BACKGROUND: Peptidyl (protein) arginine deiminases (PADs) provide the transformation of peptidyl arginine to peptidyl citrulline in the presence of calcium with posttranslational modification. The dysregulated PAD activity plays an important role on too many diseases including also the cancer. In this study, it has been aimed to determine the potential cytotoxic and apoptotic activity of chlorine-amidine (Cl-amidine) which is a PAD inhibitor and whose effectiveness has been shown in vitro and in vivo studies recently on human glioblastoma cell line Uppsala 87 malignant glioma (U-87 MG) forming an in vitro model for the glioblastoma multiforme (GBM) which is the most aggressive and has the highest mortality among the brain tumors. METHODS: In the study, the antiproliferative and apoptotic effects of Cl-amidine on GBM cancer model were investigated. The antiproliferative effects of Cl-amidine on U-87 MG cells were determined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate method at the 24th and 48th hours. The apoptotic effects were analyzed by Annexin V and Propidium iodide staining, caspase-3 activation, and mitochondrial membrane polarization (5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide) methods in the flow cytometry. RESULTS: It has been determined that Cl-amidine exhibits notable antiproliferative properties on U-87 MG cell line in a time and concentration-dependent manner, as determined through the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate assay. Assessment of apoptotic effects via Annexin V and Propidium iodide staining and 5,5', 6,6'-tetrachloro-1,1', 3,3' tetraethyl benzimidazolyl carbocyanine iodide methods has revealed significant efficacy, particularly following a 24-hour exposure period. It has been observed that Cl-amidine induces apoptosis in cells by enhancing mitochondrial depolarization, independently of caspase-3 activation. Furthermore, regarding its impact on healthy cells, it has been demonstrated that Cl-amidine shows lower cytotoxic effects when compared to carmustine, an important therapeutic agent for glioblastoma. CONCLUSION: The findings of this study have shown that Cl-amidine exhibits significant potential as an anticancer agent in the treatment of GBM. This conclusion is based on its noteworthy antiproliferative and apoptotic effects observed in U-87 MG cells, as well as its reduced cytotoxicity toward healthy cells in comparison to existing treatments. We propose that the antineoplastic properties of Cl-amidine should be further investigated through a broader spectrum of cancer cell types. Moreover, we believe that investigating the synergistic interactions of Cl-amidine with single or combination therapies holds promise for the discovery of novel anticancer agents.

Phenotypic and genotypic assessment of iron acquisition in diverse bovine-associated non-aureus staphylococcal strains.

Although the role of iron in bacterial infections has been well described for Staphylococcus (S.) aureus, iron acquisition in (bovine-associated) non-aureus staphylococci and mammaliicocci (NASM) remains insufficiently mapped. This study aimed at elucidating differences between four diverse bovine NASM field strains from two species, namely S. chromogenes and S. equorum, in regards to iron uptake (with ferritin and lactoferrin as an iron source) and siderophore production (staphyloferrin A and staphyloferrin B) by investigating the relationship between the genetic basis of iron acquisition through whole genome sequencing (WGS) with their observed phenotypic behavior. The four field strains were isolated in a previous study from composite cow milk (CCM) and bulk tank milk (BTM) in a Flemish dairy herd. Additionally, two well-studied S. chromogenes isolates originating from a persistent intramammary infection and from a teat apex were included for comparative purpose in all assays. Significant differences between species and strains were identified. In our phenotypical iron acquisition assay, while lactoferrin had no effect on growth recovery for all strains in iron deficient media, we found that ferritin served as an effective source for growth recovery in iron-deficient media for S. chromogenes CCM and BTM strains. This finding was further corroborated by analyzing potential ferritin iron acquisition genes using whole-genome sequencing data, which showed that all S. chromogenes strains contained hits for all three proposed ferritin reductive pathway genes. Furthermore, a qualitative assay indicated siderophore production by all strains, except for S. equorum. This lack of siderophore production in S. equorum was supported by a quantitative assay, which revealed significantly lower or negligible siderophore amounts compared to S. aureus and S. chromogenes. The WGS analysis showed that all tested strains, except for S. equorum, possessed complete staphyloferrin A (SA)-synthesis and export operons, which likely explains the phenotypic absence of siderophore production in S. equorum strains. While analyzing the staphyloferrin A and staphyloferrin B operon landscapes for all strains, we noticed some differences in the proteins responsible for iron acquisition between different species. However, within strains of the same species, the siderophore-related proteins remained conserved. Our findings contribute valuable insights into the genetic elements associated with bovine NASM pathogenesis.

pH-Dependent physicochemical properties of ornithine lipid in mono- and bilayers.

In certain bacteria, phosphatidylethanolamine lipids (PEL) get largely replaced by phosphate-free ornithine lipids (OL) under conditions of phosphate starvation. It has so far been unknown how much these two lipid types deviate in their physicochemical properties, and how strongly bacteria thus have to adapt in order to compensate for the difference. Here, we use differential scanning calorimetry, X-ray scattering, and X-ray fluorescence to investigate the properties of OL with saturated C14 alkyl chains in mono- and bilayers. OL is found to have a greater tendency than chain-analogous PEL to form ordered structures and, in contrast to PEL, even a molecular superlattice based on a hydrogen bonding network between the headgroups. This superlattice is virtually electrically uncharged and persists over a wide pH range. Our results indicate that OL and PEL behave very differently in ordered single-component membranes but may behave more similarly in fluid multicomponent membranes.

Pharmacokinetics/pharmacodynamics of L-ornithine phenylacetate in overt hepatic encephalopathy and the effect of plasma ammonia concentration reduction on clinical outcomes.

Hepatic encephalopathy (HE) is a serious neurocognitive complication of liver dysfunction, often associated with elevated plasma ammonia. Ornithine phenylacetate (OP), a potent ammonia scavenger, is being evaluated for the treatment of acute/overt HE. The pharmacokinetics and pharmacodynamics of OP in patients with HE were characterized in this phase IIb study (NCT01966419). Adult patients hospitalized with an overt HE episode, cirrhosis, and plasma ammonia above the upper limit of normal (ULN) who failed to improve after 48 hours' standard care were randomly assigned to continuous intravenous OP (10, 15, or 20 g/day, based on Child-Turcotte-Pugh score) or matching placebo for 5 days. Plasma levels of ornithine and phenylacetic acid (PAA) and plasma/urinary levels of phenylacetylglutamine (PAGN) (primary metabolite of PAA) were regularly assessed; plasma ammonia level was the primary pharmacodynamic variable. PAA demonstrated dose-dependent pharmacokinetics; ornithine and PAGN levels increased with dose. PAGN urinary excretion represented ~50%-60% of administered PAA across all doses. Mean reduction in plasma ammonia with OP at 3 hours postinfusion was significantly greater versus placebo (p = 0.014); and time to achieve plasma ammonia less than or equal to the ULN was significantly reduced (p = 0.028). Achievement of clinical response based on HE stage was associated with a greater reduction in mean plasma ammonia level (p = 0.009). OP effects on plasma ammonia were consistent with its proposed mechanism of action as a primary ammonia scavenger, with a significant association between reduced plasma ammonia and improvement in HE stage. OP should be further evaluated as a promising treatment for hyperammonemia in patients with overt HE.

Protein Modification with Ribose Generates Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine.

Advanced glycation end products (AGEs) are associated with diabetes and its complications. AGEs are formed by the non-enzymatic reactions of proteins and reducing sugars, such as glucose and ribose. Ribose is widely used in glycation research as it generates AGEs more rapidly than glucose. This study analyzed the AGE structures generated from ribose-modified protein by liquid chromatography-quadrupole time-of-flight mass spectrometry. Among these AGEs, Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine (MG-H1) was the most abundant in ribose-glycated bovine serum albumin (ribated-BSA) among others, such as Nε-(carboxymethyl) lysine, Nε-(carboxyethyl) lysine, and Nω-(carboxymethyl) arginine. Surprisingly, MG-H1 was produced by ribated-BSA in a time-dependent manner, whereas methylglyoxal levels (MG) were under the detectable level. In addition, Trapa bispinosa Roxb. hot water extract (TBE) possesses several anti-oxidative compounds, such as ellagic acid, and has been reported to inhibit the formation of MG-H1 in vivo. Thus, we evaluated the inhibitory effects of TBE on MG-H1 formation using ribose- or MG-modified proteins. TBE inhibited MG-H1 formation in gelatin incubated with ribose and ribated-BSA, but not in MG-modified gelatin. Furthermore, MG-H1 formation was inhibited by diethylenetriaminepentaacetic acid. These results demonstrated that ribose reacts with proteins to generate Amadori compounds and form MG-H1 via oxidation.

Inhibition of PAD4-mediated NET formation by cl-amidine prevents diabetes development in nonobese diabetic mice.

Many evidences indicated that neutrophil extracellular traps (NETs) play pathogenic roles in type 1 diabetes (T1D). Peptidylarginine deiminases 4 (PAD4) has been proved to be indispensable for generation of NETs. In the current study, we investigated whether oral administration of cl-amidine, an effective inhibitor of PAD4, protects non-obese diabetic (NOD) mice from T1D development. Female NOD mice were orally administrated with cl-amidine (5 µg/g body weight) from the age of 8 weeks up to 16 weeks. It showed that cl-amidine inhibit NET formation in vitro and in vivo. The onset of T1D was delayed nearly 8 weeks and the incidence of disease was significantly decreased in cl-amidine treated mice compared with the control group. Moreover, cl-amidine decreased the serum levels of anti-citrullinated peptide antibody (ACPA) and anti-neutrophil cytoplasmic antibodies (ANCA) in NOD mice. Also, it decreased generation of T1D autoantibodies such as glutamic acid decarboxylase antibody (GADA), tyrosine phosphatase-related islet antigen-2 antibody (IA2A) and zinc transporter 8 antibody (ZnT8A), which were strongly correlated with the reduced serum PAD4 and MPO-DNA levels. Furthermore, cl-amidine administration inhibited pancreatic inflammation and increased frequency of regulatory T cells in pancreatic lymph nodes (PLNs). In addition, cl-amidine improved gut barrier dysfunction and decreased the serum level of lipopolysaccharide (LPS), which was positively correlated with the NETs markers (PAD4 and MPO-DNA) and T1D autoantibody IA2A. In conclusion, our data showed that orally delivery of cl-amidine effectively prevent T1D development and suggested inhibition of PAD4-dependent NET formation as a potential way of clinical treatment in T1D.

Controlled Delivery of Pan-PAD-Inhibitor Cl-Amidine Using Poly(3-Hydroxybutyrate) Microspheres.

This study deals with the process of optimization and synthesis of Poly(3-hydroxybutyrate) microspheres with encapsulated Cl-amidine. Cl-amidine is an inhibitor of peptidylarginine deiminases (PADs), a group of calcium-dependent enzymes, which play critical roles in a number of pathologies, including autoimmune and neurodegenerative diseases, as well as cancer. While Cl-amidine application has been assessed in a number of in vitro and in vivo models; methods of controlled release delivery remain to be investigated. P(3HB) microspheres have proven to be an effective delivery system for several compounds applied in antimicrobial, wound healing, cancer, and cardiovascular and regenerative disease models. In the current study, P(3HB) microspheres with encapsulated Cl-amidine were produced in a size ranging from ~4-5 µm and characterized for surface morphology, porosity, hydrophobicity and protein adsorption, in comparison with empty P(3HB) microspheres. Cl-amidine encapsulation in P(3HB) microspheres was optimized, and these were found to be less hydrophobic, compared with the empty microspheres, and subsequently adsorbed a lower amount of protein on their surface. The release kinetics of Cl-amidine from the microspheres were assessed in vitro and expressed as a function of encapsulation efficiency. There was a burst release of ~50% Cl-amidine in the first 24 h and a zero order release from that point up to 16 days, at which time point ~93% of the drug had been released. As Cl-amidine has been associated with anti-cancer effects, the Cl-amidine encapsulated microspheres were assessed for the inhibition of vascular endothelial growth factor (VEGF) expression in the mammalian breast cancer cell line SK-BR-3, including in the presence of the anti-proliferative drug rapamycin. The cytotoxicity of the combinatorial effect of rapamycin with Cl-amidine encapsulated P(3HB) microspheres was found to be 3.5% more effective within a 24 h period. The cells treated with Cl-amidine encapsulated microspheres alone, were found to have 36.5% reduction in VEGF expression when compared with untreated SK-BR-3 cells. This indicates that controlled release of Cl-amidine from P(3HB) microspheres may be effective in anti-cancer treatment, including in synergy with chemotherapeutic agents. Using controlled drug-delivery of Cl-amidine encapsulated in Poly(3-hydroxybutyrate) microspheres may be a promising novel strategy for application in PAD-associated pathologies.

Metastatic Prostate Cancer Cells Secrete Methylglyoxal-Derived MG-H1 to Reprogram Human Osteoblasts into a Dedifferentiated, Malignant-like Phenotype: A Possible Novel Player in Prostate Cancer Bone Metastases.

Bone metastases from prostate cancer (PCa) result from a complex cross-talk between PCa cells and osteoblasts (OB). Thus, targeting this interplay has become an attractive strategy to interfere with PCa bone dissemination. The agents currently used in clinical trials have proved ineffective, boosting research to identify additional mechanisms that may be involved in this two-directional talk. Here, we investigated whether and how 5-hydro-5-methylimidazolone (MG-H1), a specific methylglyoxal (MG)-derived advanced glycation end product (AGE), was a novel player in the dialogue between PCa and OB to drive PCa bone metastases. Conditioned medium from osteotropic PC3 PCa cells, pre-treated or not with a specific MG scavenger, was administrated to human primary OB and cell morphology, mesenchymal trans-differentiation, pro-osteogenic determinants, PCa-specific molecules, and migration/invasion were studied by phase-contrast microscopy, real-time PCR, western blot and specific assays, respectively. We found that PC3 cells were able to release MG-H1 that, by binding to the receptor for AGEs (RAGE) on OB, reprogrammed them into a less-differentiate phenotype, endowed with some PCa-specific molecular features and malignant properties, in a mechanism involving reactive oxidative species (ROS) production and NF-kB pathway activation. These findings provide novel insights into the mechanisms of PCa osteoblastic metastases and foster in vivo research toward new therapeutic strategies interfering with PCa/OB cross-talk.

The Pbo Cluster from Pseudomonas syringae pv. Phaseolicola NPS3121 Is Thermoregulated and Required for Phaseolotoxin Biosynthesis.

The bean (Phaseolus vulgaris) pathogen Pseudomonas syringae pv. phaseolicola NPS3121 synthesizes phaseolotoxin in a thermoregulated way, with optimum production at 18 °C. Gene PSPPH_4550 was previously shown to be thermoregulated and required for phaseolotoxin biosynthesis. Here, we established that PSPPH_4550 is part of a cluster of 16 genes, the Pbo cluster, included in a genomic island with a limited distribution in P. syringae and unrelated to the possession of the phaseolotoxin biosynthesis cluster. We identified typical non-ribosomal peptide synthetase, and polyketide synthetase domains in several of the pbo deduced products. RT-PCR and the analysis of polar mutants showed that the Pbo cluster is organized in four transcriptional units, including one monocistronic and three polycistronic. Operons pboA and pboO are both essential for phaseolotoxin biosynthesis, while pboK and pboJ only influence the amount of toxin produced. The three polycistronic units were transcribed at high levels at 18 °C but not at 28 °C, whereas gene pboJ was constitutively expressed. Together, our data suggest that the Pbo cluster synthesizes secondary metabolite(s), which could participate in the regulation of phaseolotoxin biosynthesis.

Overt Hepatic Encephalopathy: Current Pharmacologic Treatments and Improving Clinical Outcomes.

Overt hepatic encephalopathy is a generally reversible neurologic complication of cirrhosis. Overt hepatic encephalopathy has been associated with poor hospitalization- and mortality-related outcomes, which is important given increasing hepatic encephalopathy-related hospitalizations over time. The aim of this narrative review is to provide an overview of hospital- and mortality-related outcomes in patients with overt hepatic encephalopathy and the pharmacologic therapies that may improve these outcomes. Guideline-recommended prophylaxis with lactulose (first-line therapy) or secondary prophylaxis with rifaximin plus lactulose decreases hospital admissions and mortality rates. Rifaximin or lactulose treatment was beneficial for reducing the hospitalization rate in patients with hepatic encephalopathy compared with no treatment. Further, retrospective studies have shown that rifaximin with or without lactulose was effective for decreasing the number of hepatic encephalopathy episodes, hepatic encephalopathy-related hospitalizations, and duration of hospitalization. Ornithine phenylacetate, an ammonia-reducing agent currently in development, is also being investigated in hospitalized patients with hepatic encephalopathy. Overall, data support that prophylaxis for the prevention of hepatic encephalopathy recurrence improves outcomes in patients with cirrhosis and a history of hepatic encephalopathy.

Protective Effects of Swertiamarin against Methylglyoxal-Induced Epithelial-Mesenchymal Transition by Improving Oxidative Stress in Rat Kidney Epithelial (NRK-52E) Cells.

Increased blood glucose in diabetic individuals results in the formation of advanced glycation end products (AGEs), causing various adverse effects on kidney cells, thereby leading to diabetic nephropathy (DN). In this study, the antiglycative potential of Swertiamarin (SM) isolated from the methanolic extract of E. littorale was explored. The effect of SM on protein glycation was studied by incubating bovine serum albumin with fructose at 60 °C in the presence and absence of different concentrations of swertiamarin for 24 h. For comparative analysis, metformin was also used at similar concentrations as SM. Further, to understand the role of SM in preventing DN, in vitro studies using NRK-52E cells were done by treating cells with methylglyoxal (MG) in the presence and absence of SM. SM showed better antiglycative potential as compared to metformin. In addition, SM could prevent the MG mediated pathogenesis in DN by reducing levels of argpyrimidine, oxidative stress and epithelial mesenchymal transition in kidney cells. SM also downregulated the expression of interleukin-6, tumor necrosis factor-α and interleukin-1ß. This study, for the first time, reports the antiglycative potential of SM and also provides novel insights into the molecular mechanisms by which SM prevents toxicity of MG on rat kidney cells.