Intestinal Alkaline Phosphatase Research Articles

Alternative Therapy in the Prevention of Experimental and Clinical Inflammatory Bowel Disease. Impact of Regular Physical Activity, Intestinal Alkaline Phosphatase and Herbal Products.

Inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease, are multifactorial, chronic, disabling, and progressive diseases characterised by cyclical nature, alternating between active and quiescent states. While the aetiology of IBD is not fully understood, this complex of diseases involve a combination of factors including the genetic predisposition and changes in microbiome as well as environmental risk factors such as high-fat and low-fibre diets, reduced physical activity, air pollution and exposure to various toxins and drugs such as antibiotics. The prevalence of both IBD and obesity is increasing in parallel, undoubtedly proving the existing interactions between these risk factors common to both disorders to unravel poorly recognized cell signaling and molecular alterations leading to human IBD. Therefore, there is still a significant and unmet need for supportive and adjunctive therapy for IBD patients directed against the negative consequences of visceral obesity and bacterial dysbiosis. Among the alternative therapies, a moderate-intensity exercise can benefit the health and well-being of IBD patients and improve both the healing of human IBD and experimental animal colitis. Intestinal alkaline phosphatase (IAP) plays an essential role in the maintenance of intestinal homeostasis intestinal and the mechanism of mucosal defence. The administration of exogenous IAP could be recommended as a therapeutic strategy for the cure of diseases resulting from the intestinal barrier dysfunction such as IBD. Curcumin, a natural anti-inflammatory agent, which is capable of stimulating the synthesis of endogenous IAP, represents another alternative approach in the treatment of IBD. This review was designed to discuss potential "nonpharmacological" alternative and supplementary therapeutic approaches taking into account epidemiological and pathophysiological links between obesity and IBD, including changes in the functional parameters of the intestinal mucosa and alterations in the intestinal microbiome.

Dephosphorylation of DNA Fragments with Alkaline Phosphatase.

The removal of 5' phosphates from nucleic acids is used to enhance subsequent labeling with [γ-32P]-ATP, reduce the circularization of plasmid vectors in ligation reactions, and render DNA susceptible or resistant to other enzymes that act on nucleic acids (e.g., λ exonuclease). Essentially, any nucleotide phosphatase (e.g., bacterial alkaline phosphatase, calf intestinal alkaline phosphatase [CIP], placental alkaline phosphatase, shrimp alkaline phosphatase [SAP], or several acid phosphatases such as sweet potato and prostate acid phosphatase) will catalyze the removal of 5' phosphates from nucleic acid templates. In fact, these enzymes prefer small substrates such as p-nitrophenyl phosphate (PNPP) and the exposed 5' phosphates of nucleic acids to bulky globular protein substrates.

Alkaline Phosphatase.

Several types of alkaline phosphatases (or alkaline phosphomonoesterase) are commonly used in molecular cloning, including bacterial alkaline phosphatase (BAP) and calf intestinal alkaline phosphatase (CIP, CIAP, or CAP). Similar enzymes isolated from more esoteric cold-blooded organisms (e.g., SAP from shrimp) have become available in recent years and have the advantage of being easier to inactivate than BAP or CIP at the end of dephosphorylation reactions. The uses and properties of these enzymes are introduced here.

Maternal diet during pregnancy and intestinal markers are associated with early gut microbiota

Diet has an important role in host-microbiome interplay, which may result in intestinal permeability changes and physiopathological effects at a systemic level. Despite the importance of maternal microbiota as the main contributor to the initial microbial seeding, little is known about the effects of maternal diet during pregnancy on maternal-neonatal microbiota. This study aimed at ascertainingthe possible associations between maternal dietary intake during pregnancy and neonatal microbiota at birth and to evaluate the relationship with maternal intestinal markers. In a nested cross-sectional study in the longitudinal MAMI cohort, maternal-neonatal microbiota profiling at birth (n = 73) was assessed by 16S rRNA gene sequencing. Maternal intestinal markers as zonulin, intestinal alkaline phosphatase (IAP) activity and faecal calprotectin were measured in faeces. Furthermore, maternal-neonatal clinical and anthropometric data, as well as maternal nutrient intake during pregnancy obtained by FFQ questionnaires, were collected. Maternal diet is associated with both maternal and neonatal microbiota at the time of birth, in a delivery mode-dependent manner. The existing link between maternal diet, intestinal makers and neonatal gut microbiota would be mainly influenced by the intake of saturated (SFA) and monounsaturated fatty acids (MUFA). Members of Firmicutes in the neonatal microbiota were positively associated with maternal fat intake, especially SFA and MUFA, and negatively correlated to fibre, proteins from vegetable sources and vitamins. Maternal diet during pregnancy, mainly fat intake (SFA and MUFA), was related to intestinal markers, thus likely shifting the microbial transmission to the neonate and priming the neonatal microbial profile with potential health outcomes. NCT03552939.

Zeta potential changing nanoemulsions based on phosphate moiety cleavage of a PEGylated surfactant

The aim of this study was to develop and evaluate a zeta potential changing nanoemulsion (NE) containing polyoxyethylene (9) nonylphenol monophosphate ester (PNPP) as emulsifier.2% (v/v) of PNPP and 0.18% (5 μM) of cetyltrimethylammonium bromide (CTAB) were incorporated into the lipophilic NE preconcentrate (PEG-40 castor oil, glyceryl tricaprylate/tricaprate, propylene glycol dicaprylocaprate, propylene glycol monolaurate, highly purified diethylene glycol monoethyl ether; 20/20/10/30/20, v/v). After diluting the lipophilic preconcentrate, resulting NE was analyzed regarding droplet size, polydispersity index (PDI), storage stability and zeta potential change after incubation with intestinal alkaline phosphatase (IAP). Phosphate release due to cleavage by IAP was quantified by malachite green assay and toxicity as well as cellular uptake behavior on Caco-2 cells was determined. The NE containing PNPP and CTAB displayed a droplet size of 113 nm and a PDI of 0.20. It was stable over 4 h. A zeta potential change from −33.7 to +8.2 mV was observed due to cleavage of phosphate groups by isolated IAP. PNPP could be identified as non-toxic up to concentrations of 0.01% (v/v). Furthermore, an enhanced cellular uptake of the NE changing its zeta potential on Caco-2 cells was determined. Therefore, PNPP seems to be a promising tool for the development of zeta potential changing NE.

Synthesis, characterization, in vitro tissue-nonspecific alkaline phosphatase (TNAP) and intestinal alkaline phosphatase (IAP) inhibition studies and computational evaluation of novel thiazole derivatives

Alkaline phosphatases (APs) are a class of homodimeric enzymes which physiologically possess the dephosphorylation ability. APs catalyzes the hydrolysis of monoesters into phosphoric acid which in turn catalyze a transphosphorylation reaction. Thiazoles are nitrogen and sulfur containing aromatic heterocycles considered as effective APs inhibitors. In this context, the current research paper presents the successful synthesis, spectroscopic characterization and in vitro alkaline phosphatase inhibitory potential of new thiazole derivatives. The structure activity relationship and molecular docking studies were performed to find out the binding modes of the screened compounds with the target site of tissue non-specific alkaline phosphatase (h-TNAP) as well as intestinal alkaline phosphatase (h-IAP). Compound 5e was found to be potent inhibitor of h-TNAP with IC50 value of 0.17 ± 0.01 µM. Additionally, compounds 5a and 5i were found to be highly selective toward h-TNAP with IC50 values of 0.25 ± 0.01 µM and 0.21 ± 0.02 µM, respectively. In case of h-IAP compound 5f was the most potent inhibitor with IC50 value of 1.33 ± 0.10 µM. The most active compounds were resort to molecular docking studies on h-TNAP and h-IAP to explore the possible binding interactions of enzyme-ligand complexes. Molecular dynamic simulations were carried out to investigate the overall stability of protein in apo and holo state.

Assessment of Efficacy and Safety of Clindamycin Against Methicillin- Resistant Staphylococcus aureus (MRSA) Infected Subcutaneous Abscess Model

Background: Methicillin-resistant Staphylococcus aureus (MRSA) causes infection in the wound leading to life-threatening bacteriemia. It causes recalcitrant infections because of being resistant to various antibiotics. The recent studies reported clindamycin to be effective against MRSA rather than inducible clindamycin resistance and decrease the incidence of new infections after clindamycin treatment. The study focused on assessing the efficacy and safety of clindamycin against MRSA infected rats. Methods: The rats become neutropenic by intraperitoneal administration of cyclophosphamide at a dose of 150 mg/kg and 100 mg/kg for 4 days and 5th day respectively. The neutropenic rats were infected with MRSA by subcutaneous administration of 106 CFU/ml of MRSA. The 3 groups of rats such as Normal, MRSA infected, MRSA infected rats that were administered clindamycin orally at a dose of 90 mg/kg/ thrice daily for 14 days were used in the study. The abscess size, weight, and bacterial load were measured at the end of the study. The blood and liver samples were collected for biochemical analysis and histopathological evaluation. Results: The MRSA was confirmed by Polymerase Chain Reaction (PCR) method. The clindamycin minimum inhibitory concentration was 0.125 - 0.5 μg/ml. The MRSA showed negative D test for clindamycin indicating the absence of inducible clindamycin resistance. The decreased abscess size, weight, bacterial count, Intestinal Alkaline Phosphatase (IAP), weight loss, alteration in hematological parameters, mild changes in cholesterol, ALT and liver histology, no significant (P > 0.05) change in triglycerides, AST, ALP, bilirubin, lactate, urea, and creatinine were seen in clindamycin treated MRSA infected rats. The MRSA infected clindamycin treated rats showed mild irritation and diarrhea. Conclusion: Our study concludes that the clindamycin showed better anti- MRSA activity and tolerable adverse effects such as anemia, weight loss, and mild irritation after oral treatment, but the intestinal dysbiosis is a severe adverse effect and causes diarrhea.

Dietary Supplementation with Omega-6 LC-PUFA-Rich Microalgae Regulates Mucosal Immune Response and Promotes Microbial Diversity in the Zebrafish Gut.

The effect of dietary omega-6 long-chain polyunsaturated fatty acid (LC-PUFA) on host microbiome and gut associated immune function in fish is unexplored. The effect of dietary supplementation with the omega-6 LC-PUFA-rich microalga Lobosphaera incisa wild type (WT) and its delta-5 desaturase mutant (MUT), rich in arachidonic-acid and dihomo-gamma-linolenic acid (DGLA), respectively, on intestinal gene expression and microbial diversity was analyzed in zebrafish. For 1 month, fish were fed diets supplemented with broken biomass at 7.5% and 15% (w/w) of the two L. incisa strains and a control nonsupplemented commercial diet. Dietary supplementation resulted in elevated expression of genes related to arachidonic acid metabolism-cyclooxygenase 2 (cox-2), lipoxygenase 1(lox-1), anti-inflammatory cytokine-interleukin 10 (il-10), immune defense-lysozyme (lys), intestinal alkaline phosphatase (iap), complement (c3b), and antioxidants-catalase (cat), glutathione peroxidase (gpx). Microbiome analysis of the gut showed higher diversity indices for microbial communities in fish that were fed the supplemented diets compared to controls. Different treatment groups shared 237 operational taxonomic units (OTUs) that corresponded to the core microbiome, and unique OTUs were evident in different dietary groups. Overall, the zebrafish gut microbiome was dominated by the phylum Fusobacteria and Proteobacteria (averaging 38.4% and 34.6%, respectively), followed by Bacteroidetes (12.9%), Tenericutes, Planctomycetes, and Actinobacteria (at 3.1–1.3%). Significant interaction between some of the immune-related genes and microbial community was demonstrated.

Bisthioureas of pimelic acid and 4-methylsalicylic acid derivatives as selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP) and intestinal alkaline phosphatase (IAP): Synthesis and molecular docking studies

Alkaline phosphatases (ALPs) are membrane bound metalloenzymes, distributed all over the body. Recent studies have revealed that by targeting ALPs can lead towards the treatment of many deadliest diseases including cardiac, cancerous and brain diseases. Thioureas and their derivatives are of considerable significance and are privileged scaffolds in medicinal chemistry. They show a wide range of pharmacological activities such as antibacterial, antiparasitic, anti-inflammatory and antioxidants etc. On the other hand, salicylic acid and its derivatives are known for its broad spectrum of activities. The work presented comprises of synthesis of N-acyl-N'-aryl substituted bisthioureas of pimelic acid (1–7) and 3,5-dimethyl pyrazole (11), 1-aroyl-3-aryl thiourea (12) and 1,3,4-oxadiazole (13) derivatives of 4-methyl salicylic acid. Structures of all the synthesized compounds were characterized by FT-IR and 1H NMR spectroscopic analysis. Synthesized compounds were evaluated for their alkaline phosphatases inhibition potential and exhibited high potency as well as selectivity towards h-TNAP and h-IAP. Compound 7 and 12 which were the bisthiourea derivative of pimmelic acid and thiourea derivative of 4-methyl salicylic acid, respectively, showed excellent selectivity against h-TNAP and h-IAP, respectively.

Maternal treatment with oral intestinal alkaline phosphatase mitigates high fat diet-induced cognitive disorders in offspring mice

Intestinal alkaline phosphatase (IAP) is an endogenous enzyme that promotes gastrointestinal homeostasis by detoxifying inflammatory mediators, tightening the gut barrier and promoting a healthy microbiome. Oral IAP administration was efficacious in ameliorating diabetes in a high fat diet (HFD)-induced murine model. In humans, maternal obesity and diabetes during pregnancy have been associated with an increased risk of autism spectrum disorders (ASD). In mice, HFD-induced maternal obesity leads to offspring with cognitive deficiency. Here we investigated whether IAP administration to obese dams could ameliorate autism-like disorders in mice. Using a HFD murine model, we recapitulated that maternal obesity leads to male offspring with social deficits as shown by the three chamber test and reciprocal social interaction analyses. Notably, oral delivery of IAP to dams improved those deficiencies. In addition, a jumping behavior was noted in pups from obese dams, which was rescued by maternal IAP treatment. Our findings suggest that maternal treatment with IAP can relieve some ASD-like symptoms in offspring mice.

Altered intestinal microbiota induced by chronic hypoxia drives the effects on lipid metabolism and the immune response of oriental river prawn Macrobrachium nipponense

Feed management and seasonal changes can cause long-term episodes of hypoxia in prawn ponds during aquaculture. Changes to prawn morphology and their intestinal microbiota in response to chronic hypoxia are poorly understood. In the present study, we exposed the oriental river prawn Macrobrachium nipponense to normoxic, moderately hypoxic, and hypoxic conditions for four weeks, and then studied their fatty acid composition, metabolic and immune enzyme activities, and intestinal microbiota. Hypoxia clearly affected the intestinal microbiota and mucosal morphology. Compared with the normoxia and moderate hypoxia groups, the prawns in the hypoxia group had a significantly higher levels of intestinal pathogenic bacteria and a clearly different microbial community structure. Functional prediction revealed that the prawns' adaptation to hypoxia might involve microbiota-induced modulation of lipid metabolic pathways. Furthermore, significant decreases in the levels of intestinal short-chain fatty acids and the levels of hepatic highly unsaturated fatty acids (e.g., eicosapentaenoic acid and eicosapentenoic acid) were observed on prawns in response to hypoxia (P < .05). Hepatic metabolic enzymes activities and haemolymph metabolites were also markedly affected by chronic hypoxia (e.g., fatty acid synthetase, acetyl-CoA carboxylase, lipo-protein lipase, carnitine palmitoyltransferase I, and the free fatty acid content). Hypoxia also significantly reduced intestinal immune enzyme activities in the prawns (e.g., phenoloxidase, alkaline phosphatase, and acid phosphatase) (P < .05). During hypoxia, the prawns' intestinal microbial communities correlated significantly with the intestinal alkaline phosphatase activity (P < .05). The results suggested that chronic hypoxia negatively affected M. nipponense intestinal health by altering the composition of the microbiota, inhibiting the immune response, and decreasing the content of short-chain fatty acids in the intestines.

Syncytiotrophoblast extracellular microvesicle profiles in maternal circulation for noninvasive diagnosis of preeclampsia

Preeclampsia is the most common placental pathology in pregnant females, with increased morbidity and mortality incurred on the mother and the fetus. There is a need for improved biomarkers for diagnosis and monitoring of this condition. Placental syncytiotrophoblasts at the maternal-fetal interface release nanoparticles, including extracellular microvesicles, into the maternal blood during pregnancy. Syncytiotrophoblast extracellular microvesicles (STEVs) are being studied for their diagnostic potential and for their potential physiologic role in preeclampsia. We hypothesized that STEV profiles in maternal circulation would be altered under conditions of preeclampsia compared to normal pregnancy. Extracellular vesicles (EVs) released by BeWo cells in vitro showed high expression of syncytin-1, but no plac1 expression, demonstrating that trophoblast cell EVs express syncytin-1 on their surface. Placental alkaline phosphatase also showed high expression on BeWo EVs, but due to concern for cross reactivity to highly prevalent isoforms of intestinal and bone alkaline phosphatase, we utilized syncytin-1 as a marker for STEVs. In vivo, syncytin-1 protein expression was confirmed in maternal plasma EVs from Control and Preeclampsia subjects by Western blot, and overall, lower expression was noted in samples from patients with preeclampsia (n = 8). By nanoparticle analysis, EV profiles from Control and Preeclampsia groups showed similar total plasma EV quantities (p = 0.313) and size distribution (p = 0.415), but STEV quantitative signal, marked by syncytin-1 specific EVs, was significantly decreased in the Preeclampsia group (p = 2.8 × 10−11). Receiver operating characteristic curve demonstrated that STEV signal threshold cut-off of <0.316 was 95.2% sensitive and 95.6% specific for diagnosis of preeclampsia in this cohort (area under curve = 0.975 ± 0.020). In conclusion, we report that the syncytin-1 expressing EV profiles in maternal plasma might serve as a placental tissue specific biomarker for preeclampsia.

Sulfonylhydrazones: Design, synthesis and investigation of ectonucleotidase (ALP & e5'NT) inhibition activities.

Medicinal importance of the sulfonylhydrazones is well-evident owing to their binding ability with zinc containing metalloenzymes. In the present study, we have synthesized different series of sulfonylhydrazones by using facile synthetic methods in good to excellent yield. All the successfully prepared sulfonylhydrazones were screened for ectonucleotidase (ALP & e5'NT) inhibitory activity. Among the chromen-2-one scaffold based sulfonylhydrazones, the compounds 7 was found to be most potent inhibitor for h-TNAP (human tissue non-specific alkaline phosphatase) and h-IAP (human intestinal alkaline phosphatase) with IC50 values of 1.02±0.13 and 0.32±0.0 3µM respectively, compared with levamisole (IC50=25.2±1.90µM for h-TNAP) and l-phenylalanine (IC50=100±3.00µM for h-IAP) as standards. Further, the chromen-2-one based molecule 5a showed excellent activity against h-ecto 5'-NT (human ecto-5'-nucleotidase) with IC50 value of 0.29±0.004µM compared to standard, sulfamic acid (IC50=42.1±7.8µM). However, among the series of phenyl ring based sulfonylhydrazones, compound 9d was found to be most potent against h-TNAP and h-IAP with IC50 values of 0.85±0.08 and 0.52±0.03µM, respectively. Moreover, in silico studies were also carried to demonstrate their putative binding with the target enzymes. The potent compounds 5a, 7, and 9d against different ectonucleotidases (h-ecto 5'-NT, h-TNAP, h-IAP) could potentially serve as lead for the development of new therapeutic agents.

Supplementation with Brush Border Enzyme Alkaline Phosphatase Slows Aging.

Diminished integrity of the intestinal epithelial barrier with advanced age is believed to contribute to aging-associated dysfunction and pathologies in animals. In mammals, diminished gut integrity contributes to inflammaging, the increase in inflammatory processes observed in old age. Recent work suggests that expression of intestinal alkaline phosphatase (IAP) plays a key role in maintaining gut integrity. IAP expression decreases with increasing age in mice and humans. Absence of IAP leads to liver inflammation and shortened life-spans in mice lacking the IAP gene. In normal mice, exogenous supplemental IAP reverses age-induced barrier dysfunction, improves aging-associated metabolic dysfunction, prevents microbiome dysbiosis (imbalance), and extends life-span. Consistent with IAP playing a conserved role in maintaining gut integrity, increased dietary IAP increases aging-diminished physical performance in flies. IAP helps maintain gut integrity in part by supporting the expression of tight junction proteins that maintain the intestinal epithelial barrier and by inactivating bacterial pro-inflammatory factors such as lipopolysaccharides (LPS) by dephosphorylation. Recombinant IAP is in late clinical trials for sepsis-associated acute kidney injury, suggesting it may soon become available as a therapeutic. Taken together, these reports support the idea that directly increasing IAP levels by supplemental recombinant IAP or by indirectly increasing IAP levels using dietary means to induce endogenous IAP may slow the development of aging-associated pathologies.

Glycosylation in a Common Mechanism of Colitis and Sepsis

Glycosylation is a post‐translation modification common to proteins produced in the secretory pathway. In the bloodstream and at cell surfaces, glycan structures operate in multiple processes including the determination of protein half‐life, abundance, and function. Disruption of the normal mammalian N‐glycan repertoire can occur in part from acquired environmental and metabolic factors, and are responsible for the pathogenesis of multiple common diseases including autoimmunity, diabetes, colitis, and sepsis. Microbial infections are common throughout mammalian lifespan and can be life‐threatening as in the case of sepsis. Bacterial pathogens have also been considered as a possible environmental triggers precipitating chronic diseases including the human inflammatory bowel diseases (IBDs). For example, seasonal bacterial infections have been associated with increases in clinical diagnoses of IBD. To test the possibility that recurrent self‐resolving bacterial infections may trigger chronic disease, we developed a mouse model of recurrent human food poisoning using low‐titer non‐lethal gastric infections of Gram‐negative Salmonella enterica Typhimurium (ST). The ST pathogen produces a greater human disease burden than any other foodborne bacterial pathogen in the United States and is endemic throughout the world. Although the mouse rapidly cleared multiple recurrent low titer ST infections, colitis developed after several months, escalating in severity and failing to resolve after the cessation of infection. ST progressively disabled a mechanism of host protection by inducing intestinal neuraminidase activity with Neu3 gene induction among duodenal enterocytes. This accelerated the rate of molecular aging of intestinal alkaline phosphatase (IAP) measured by extent of de‐sialylation and led to its rapid endocytic clearance, thereby diminishing IAP levels in the small intestine and colon. Pathogenesis required host Tlr4 function through neuraminidase induction which was deleterious to the host as IAP deficiency increased the abundance of the LPS‐phosphate endotoxin in the colon. Oral administration of IAP or the marketed anti‐viral neuraminidase inhibitor Zanamivir were therapeutic reducing both LPS‐phosphate and inflammatory tissue damage. Humans with IBD have been reported with low IAP levels, while IAP‐null humans develop IBD. In both mouse and human colitis studies, IAP augmentation is remarkably therapeutic. This pathogenic mechanism emanating from altered protein glycosylation may be a trigger of one or more forms of IBD among the human population. In related studies where the ST pathogen gains access to the bloodstream in sepsis, we discovered a similar mechanism operating among Gram‐negative pathogens including ST and E. coli (EC). Both pathogens operated through Tlr4 to increase host neuraminidases Neu1 and Neu3 in the bloodstream, and thereby diminished alkaline phosphatase activities via endocytic clearance by the Ashwell‐Morell receptor of hepatocytes. These findings reveal a similar pathogenic process operating among ST and EC pathogens to subvert host protection by diminishing the expression of anti‐inflammatory enzymes. These findings further support published data and rationale for clinical trials augmenting alkaline phosphatase activity in sepsis and IBD patients.Support or Funding InformationNIH DK048247; HL131474; HL125352; AI141394

Intestinal alkaline phosphatase is a diagnostic biomarker for necrotizing enterocolitis in preterm infants

Necrotizing enterocolitis (NEC) in preterm infants is an often fatal, gastrointestinal emergency. There is a need for a robust NEC biomarker that can be adopted in pathology labs and that is not confounded by sepsis. Use of such a NC biomarker could improve bedside management, lower morbidity and mortality, and permit patient selection in clinical trials for therapeutic candidates. We evaluated if aberrant intestinal alkaline phosphatase (iAP) biochemistry in infant stool is a molecular biomarker for necrotizing enterocolitis and not correlated to sepsis in the absence of NEC. There were 136 premature infants enrolled in three hospitals in this observational and prospective study. Median birthweight (IQR) was 1050 g (790‐350 g) and median gestational age was 28.4 weeks (26‐0.9 wks). Twenty‐five infants were diagnosed with severe NEC, 19 were suspected of having NEC, and 91 were non‐NEC infants. Twenty‐six patients were diagnosed with late‐onset sepsis and 14 had other non‐GI infections. Infant stool samples were collected between 24–40+ weeks postconceptual age (PCA). Enrolled infants were subject to abdominal radiography at physician and hospital site discretion. Enzyme activity and relative abundance of iAP were measured using fluorometric detection and immunoassays, respectively. Post‐measurement, biochemical data were evaluated against clinical entries from infants’ hospital stay. Fecal iAP content was 99±19, 123±41, and 5±3 (median±SE) for severe, suspected, and non‐NEC samples, respectively. Enzyme activity was 183±108, 355±180, and 613±218 kU/g (median±SE) for severe, suspected, and non‐NEC samples, respectively. ROC area under the curve (AUC) values were 0.96, 0.97, 0.54, and 0.58 for relative iAP content measurements at time of severe NEC, suspected NEC, sepsis, and other non‐GI infections; AUC values for iAP activity were 0.60, 0.76, 0.52 and 0.57, respectively. High amounts of iAP protein in stool and low iAP enzyme activity are reliable and specific biomarkers for NEC: iAP biochemistry uniquely has clinical utility in distinguishing NEC from sepsis.Support or Funding InformationThis work was supported by the National Institutes of Health (R41HD095779 to RB and SK; R01GM097350 to SK; K08DK101608, R03DK111473 and R01DK118568 to MG), National Science Foundation (IIP‐1713220 and IIP‐1547932 to RB and SK); March of Dimes (5‐FY17‐79 to MG), Louisiana Board of Regents (LEQSF‐RD‐D‐07 to SK), LSU LIFT2 (HSCNO‐2017‐LIFT‐006 to SK), Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital (MG), Department of Pediatrics at Washington University School of Medicine, St. Louis (MG), LSU School of Medicine (BB, SK, and MH), and LSU Health Foundation (MH, ZG, and SK).

Development and In Vitro Evaluation of Stearic Acid Phosphotyrosine Amide as New Excipient for Zeta Potential Changing Self-Emulsifying Drug Delivery Systems

PurposeDevelopment of zeta potential changing SEDDS containing newly synthesized derivative stearic acid phosphotyrosine amide.MethodsStearoyl chloride was conjugated with phosphotyrosine, which is substrate for the brush border enzyme intestinal alkaline phosphate. The synthesized derivative was implemented in different SEDDS formulations and the zeta potential changing properties and the concluding mucus diffusion abilities were evaluated.ResultsStearic acid phosphotyrosine amide was successfully synthesized and incorporated into SEDDS. A SEDDS formulation containing the new derivative showed a zeta potential of −14 mV before, and + 2 mV after enzymatic cleavage by intestinal alkaline phosphatase. Experiments on a Caco-2 monolayer demonstrated that the phosphate cannot only be cleaved by isolated enzyme, but also by enzyme, which was expressed by cells. The mucus diffusion abilities of the untreated, negatively charged SEDDS were significantly higher compared to the enzymatically cleaved, positively charged SEDDS.ConclusionThe developed stearic acid phosphotyrosine represents a promising excipient for zeta potential changing SEDDS.Graphical

Intestinal lncRNA H19 and miRNA‐675 expression Influenced by Metal Transporter ZIP14

Zinc has been shown to influence intestinal barrier function. To function properly, zinc must be placed at specific sites of action in cells. Previously, we demonstrated that the zinc transporter Zip14 (Slc39a14) is highly expressed in the small intestine and is localized to the basolateral membrane of enterocytes. Ablation of Zip14 in mice produces a phenotype that includes increased intestinal permeability and metabolic endotoxemia. To investigate the mechanism involved, we performed genome‐wide examination of gene expression in the small intestine from WT and Zip14 KO mice using Illumina RNA sequencing. This RNAseq analysis revealed that among 1079 long non‐coding (lnc) RNAs profiled, 36 annotated lncRNAs were identified as overexpressed in intestines of Zip14 KO mice. H19 RNA was the most highly overexpressed lncRNA. Read densities of other selected lncRNAs demonstrated specificity in the differential expression of H19 in the intestine of the KO mice. Upon confirmation using qPCR, we found that H19 was significantly up‐regulated in intestines of the Zip14 KO mice. Up‐regulation of miR‐675‐5p, a microRNA derived from H19, was also demonstrated. Intestinal epithelial cells (IEC) isolated by a chelation method revealed high expression of intestinal alkaline phosphatase and Zip14 mRNAs, indicative of substantial enterocyte‐enrichment. Expression of H19, but not miR‐675‐5p, was localized to the isolated IECs. Markedly enhanced expression of H19 was detected in IECs from the Zip14 KO mice compared to WT mice. The increased H19 expression in Zip14 KO mice was limited to IECs and skeletal muscle of the six tissues examined. IECs were also isolated from intestine‐specific Zip14 KO (I‐KO) mice. The up‐regulation of H19 was observed in IECs from the I‐KO mice compared to those from flox/flox controls, but miR‐675‐5p expression was not different between cells from the two genotypes. These data suggest that intestinal H19 is produced by IECs and is up‐regulated with Zip14 ablation. H19 overexpression suggests it is involved in a molecular defect in intestinal function of Zip14, perhaps including intestinal barrier dysfunction.Support or Funding InformationNIH Grant R01 DK 094244

Aberrant expression of placental-like alkaline phosphatase in chronic myeloid leukemia cells in vitro and its modulation by vitamin E.

To analyze the expression patterns of various AP forms in cells originated from CML patients in blast crisis and to modify their expression by vitamin E. RNA extracted from leukemic cells was converted to cDNA and real-time reverse transcription polymerase chain reaction was performed using SYBR Green protocol with primers to tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase and CCAAT-enhancer-binding proteins alpha (C/EBPα). To analyze the modulation of expression of APs and C/EBPα, CML cells were incubated with 100µM vitamin E. We have observed the aberrant expression of mRNA intestinal alkaline phosphatase in CML cells that upon sequencing demonstrated the significant alignment with PLAP sequence while no gene homology with tissue placental alkaline phosphatase (PAP) was revealed. Vitamin E decreases mRNA PLAP expression and increases mRNA TNAP expression. Moreover, along with down-regulation of aberrant PLAP and up-regulation of TNAP, vitamin E increases C/EBPα mRNA expression. The loss of TNAP in CML may contribute to pathogenesis of this disease. PLAP may be considered as a putative target in differentiation therapies in myeloid neoplasms. Our findings suggest the potential role of vitamin E as the inducer of differentiation potential of leukemic cells in CML.

Intestinal alkaline phosphatase targets the gut barrier to prevent aging.

Gut barrier dysfunction and gut-derived chronic inflammation play crucial roles in human aging. The gut brush border enzyme intestinal alkaline phosphatase (IAP) functions to inhibit inflammatory mediators and also appears to be an important positive regulator of gut barrier function and microbial homeostasis. We hypothesized that this enzyme could play a critical role in regulating the aging process. We tested the role of several IAP functions for prevention of age-dependent alterations in intestinal homeostasis by employing different loss-of-function and supplementation approaches. In mice, there is an age-related increase in gut permeability that is accompanied by increases in gut-derived portal venous and systemic inflammation. All these phenotypes were significantly more pronounced in IAP-deficient animals. Oral IAP supplementation significantly decreased age-related gut permeability and gut-derived systemic inflammation, resulted in less frailty, and extended lifespan. Furthermore, IAP supplementation was associated with preserving the homeostasis of gut microbiota during aging. These effects of IAP were also evident in a second model system, Drosophilae melanogaster. IAP appears to preserve intestinal homeostasis in aging by targeting crucial intestinal alterations, including gut barrier dysfunction, dysbiosis, and endotoxemia. Oral IAP supplementation may represent a novel therapy to counteract the chronic inflammatory state leading to frailty and age-related diseases in humans.