Hepatic nutrient and hormone signaling to mTORC1 instructs the postnatal metabolic zonation of the liver.

The metabolic functions of the liver are spatially organized in a phenomenon called zonation, linked to the differential exposure of portal and central hepatocytes to nutrient-rich blood. The mTORC1 signaling pathway controls cellular metabolism in response to nutrients and insulin fluctuations. Here we show that simultaneous genetic activation of nutrient and hormone signaling to mTORC1 in hepatocytes results in impaired establishment of postnatal metabolic and zonal identity of hepatocytes. Mutant hepatocytes fail to upregulate postnatally the expression of Frizzled receptors 1 and 8, and show reduced Wnt/ß-catenin activation. This defect, alongside diminished paracrine Wnt2 ligand expression by endothelial cells, underlies impaired postnatal maturation. Impaired zonation is recapitulated in a model of constant supply of nutrients by parenteral nutrition to piglets. Our work shows the role of hepatocyte sensing of fluctuations in nutrients and hormones for triggering a latent metabolic zonation program.

Carrageenan-Based Crowding and Confinement Combination Approach to Increase Collagen Deposition for In Vitro Tissue Development.

Connective tissue models grown from cell monolayers can be instrumental in a variety of biomedical fields such as drug screening, wound healing, and regenerative engineering. However, while connective tissues contain abundant fibrillar collagen, achieving a sufficient assembly and retention of fibrillar collagen in vitro is challenging. Unlike the dilute cell culture environment, the body's environment is characterized by a high density of soluble macromolecules (crowding) and macromolecular networks (confinement), which contribute to extracellular matrix (ECM) assembly in vivo. Consequently, macromolecular crowding (MMC) has been successfully used to enhance the processing of type I procollagen, leading to significant increases in fibrillar collagen assembly and accumulation during in vitro culture of a variety of cell types. In this study, we developed a combination approach using a carrageenan hydrogel, which released soluble macromolecules and served as a confinement barrier. We first evaluated the local carrageenan release and then confirmed the effectiveness of this combination approach on collagen accumulation by the human MG-63 bone cell line. Additionally, computational modeling of oxygen and glucose transport within the culture system showed no negative effects of the hydrogel and its releasates on cell viability.

Lower systemic inflammation is associated with gut firmicutes dominance and reduced liver injury in a novel ambulatory model of parenteral nutrition.

BACKGROUND: Total Parenteral Nutrition (TPN) provides lifesaving nutritional support to patients unable to maintain regular enteral nutrition (EN). Unfortunately, cholestasis is a significant side effect affecting 20-40% of paediatric patients. While the aetiology of TPN-associated injury remains ill-defined, an altered enterohepatic circulation in the absence of gut luminal nutrient content during TPN results in major gut microbial clonal shifts, resulting in metabolic endotoxemia and systemic inflammation driving liver injury and cholestasis. HYPOTHESIS: To interrogate the role of gut microbiota, using our novel ambulatory TPN piglet model, we hypothesized that clonal reduction of bacteria in Firmicutes phylum (predominant in EN) and an increase in pathogenic Gram-negative bacteria during TPN correlates with an increase in serum lipopolysaccharide and systemic inflammatory cytokines, driving liver injury. METHODS: Upon institutional approval, 16 animals were allocated to receive either TPN (n = 7) or EN only (n = 9). The TPN group was subdivided into a low systemic inflammation (TPN-LSI) and high systemic inflammation (TPN-HSI) based on the level of serum lipopolysaccharide. Culture-independent identification of faecal bacterial populations was determined by 16S rRNA. RESULTS: Piglets on TPN, in the TPN-HSI group, noted a loss of enterocyte protective Firmicutes bacteria and clonal proliferation of potent inflammatory and lipopolysaccharide containing pathogens: Fusobacterium, Bacteroidetes and Campylobacter compared to EN animals. Within the TPN group, the proportion of Firmicutes phylum correlated with lower portal lipopolysaccharide levels (r = -0.89). The TPN-LSI had a significantly lower level of serum bile acids compared to the TPN-HSI group (7.3 vs. 60.4 mg/dL; p = .018), increased day 14 weight (5.67 vs. 5.07 kg; p = .017) as well as a 13.7-fold decrease in serum conjugated bilirubin. CONCLUSION: We demonstrate a novel relationship between the gut microbiota and systemic inflammation in a TPN animal model. Pertinently, the degree of gut dysbiosis correlated with the severity of systemic inflammation. This study underscores the role of gut microbiota in driving liver injury mechanisms during TPN and supports a paradigm change in therapeutic targeting of the gut microbiota to mitigate TPN-related injury. KEY MESSAGESThis study identified a differential link between gut microbiota and inflammation-the higher the dysbiosis, the worse the systemic inflammatory markers.Higher levels of Firmicutes species correlated with reduced inflammation.

Beyond lipids: Novel mechanisms for parenteral nutrition-associated liver disease.

Parenteral nutrition (PN) is a therapy that delivers essential nutrients intravenously to patients who are unable to meet their nutrition requirements via standard enteral feeding. This methodology is often referred to as PN when accompanied by minimal or no enteral nutrition (EN). Although PN is lifesaving, significant complications can arise, such as intestinal failure-associated liver disease and gut-mucosal atrophy. The exact mechanism of injury remains ill defined. This review was designed to explore the available literature related to the drivers of injury mechanisms. The Farnesoid X receptor and fibroblast growth factor 19 signaling pathway seems to play an important role in gut-systemic signaling, and its alteration during PN provides insights into mechanistic links. Central line infections also play a key role in mediating PN-associated injury. Although lipid reduction strategies, as well as the use of multicomponent lipid emulsions and vitamin E, have shown promise, the cornerstone of preventing injury is the early establishment of EN.

Carbamazepine mitigates parenteral nutrition-associated liver disease in a novel ambulatory piglet model.

BACKGROUND: Parenteral nutrition (PN) remains a critical therapeutic option in patients who cannot tolerate enteral feeding. However, although lifesaving, PN is associated with significant side effects, including liver injury, the etiology of which is multifactorial. Carbamazepine (CBZ), an antiepileptic medication, is known to modulate hepatic fibrosis and hepatocellular injury in a variety of liver diseases. We hypothesized that CBZ could prevent PN-associated liver disease (PNALD), which we tested by using our novel ambulatory PN piglet model. METHODS: Piglets were fitted with jugular catheters and infusion pumps for PN and randomized to enteral nutrition (n = 7), PN (n = 6), or PN with parenteral CBZ (n = 6) for 2 weeks. Serum and liver tissue were analyzed via light microscopy, quantification of serum liver injury markers, Ki67 and cytokeratin-7 indexing, and real-time quantitative polymerase chain reaction. RESULTS: PN-fed piglets in our model developed manifestations of PNALD-particularly, increased serum bilirubin, gamma-glutamyltransferase, liver cholestasis, and Ki67 expression compared with that of EN-fed animals (P < 0.03). CBZ therapy in PN-fed animals led to a significant reduction in these markers of injury (P < 0.05). Investigation into the mechanism of these therapeutic effects revealed increased expression of sterol regulatory element-binding protein 1 (SREBP-1), peroxisome proliferator-activated receptor alpha (PPAR-α), and fatty acid binding protein (FABP) in PN-fed animals receiving CBZ (P < 0.03). Further investigation revealed increased LC3 expression and decreased lysosomal-associated membrane protein (LAMP1) expression with CBZ (P < 0.03). CONCLUSION: CBZ administration mitigates PNALD severity, suggesting a novel therapeutic strategy targeting PN-associated side effects, and may present a paradigm change to current treatment options.

Novel NMP split liver model recapitulates human IRI and demonstrates ferroptosis modulators as a new therapeutic strategy.

BACKGROUND: Almost 9%of deceased donor livers are discarded as marginal donor livers (MDL) due to concern of severe ischemia reperfusion injury (IRI). Emerging data supports ferroptosis (iron regulated hepatocellular death) as an IRI driver, however lack of robust preclinical model limits therapeutic testing. In this manuscript we describe the development of a novel rigorous internal control system utilizing normothermic perfusion of split livers to test ferroptosis regulators modulating IRI. METHODS: Upon institutional approval, split human MDLs were placed on our normothermic perfusion machine, Perfusion Regulated Organ Therapeutics with Enhanced Controlled Testing (PROTECT), pumping arterial and portal blood. Experiment 1 compared right (UR) and left (UL) lobes to validate PROTECT. Experiment 2 assessed ferroptosis regulator Deferoxamine in Deferoxamine Agent Treated (DMAT) vs. No Agent Internal Control (NAIC) lobes. Liver serology, histology, and ferroptosis genes were assessed. RESULTS: Successful MDL perfusion validated PROTECT with no ALT or AST difference between UR and UL (∆ALT UR: 235, ∆ALT UL: 212; ∆AST UR: 576, ∆AST UL: 389). Liver injury markers increased in NAIC vs. DMAT (∆ALT NAIC: 586, ∆ALT DMAT: -405; ∆AST NAIC: 617, ∆AST DMAT: -380). UR and UL had similar expression of ferroptosis regulators RPL8,HO-1 and HIFα. Significantly decreased intrahepatic iron (p = .038), HO-1 and HIFα in DMAT (HO-1 NAIC: 6.93, HO-1 DMAT: 2.74; HIFαNAIC: 8.67, HIFαDMAT: 2.60)and no hepatocellular necrosis or immunohistochemical staining (Ki67/Cytokeratin-7) differences were noted. CONCLUSION: PROTECT demonstrates the therapeutic utility of a novel normothermic perfusion split liver system for drug discovery and rapid translatability of therapeutics, driving a paradigm change in organ recovery and transplant medicine. Our study using human livers, provides preliminary proof of concept for the novel role of ferroptosis regulators in driving IRI.

Parenteral Nutrition and Cardiotoxicity.

Parenteral nutrition (PN) is a life-saving nutritional therapy for those situations when patients are unable to receive enteral nutrition. However, despite a multitude of benefits offered by PN, it is associated with a variety of side effects, most notably parenteral nutrition-associated liver disease (PNALD). Adverse effects of PN on other organ systems, such as brain and cardiovascular system, have been poorly studied. There have been several case reports, studies, and a recent animal study highlighting cardiotoxic effects of PN; however, much remains unclear about the underlying mechanisms causing cardiac damage. In this review, we propose a series of potential mechanisms behind PN-associated heart injury, and we provide an overview of therapeutic strategies and recent scientific advances.

Impaired Gut-Systemic Signaling Drives Total Parenteral Nutrition-Associated Injury.

BACKGROUND: Total parenteral nutrition (TPN) provides all nutritional needs intravenously. Although lifesaving, enthusiasm is significantly tempered due to side effects of liver and gut injury, as well as lack of mechanistic understanding into drivers of TPN injury. We hypothesized that the state of luminal nutritional deprivation with TPN drives alterations in gut-systemic signaling, contributing to injury, and tested this hypothesis using our ambulatory TPN model. METHODS: A total of 16 one-week-old piglets were allocated randomly to TPN (n = 8) or enteral nutrition (EN, n = 8) for 3 weeks. Liver, gut, and serum were analyzed. All tests were two-sided, with a significance level of 0.05. RESULTS: TPN resulted in significant hyperbilirubinemia and cholestatic liver injury, p = 0.034. Hepatic inflammation (cluster of differentiation 3 (CD3) immunohistochemistry) was higher with TPN (p = 0.021). No significant differences in alanine aminotransferase (ALT) or bile ductular proliferation were noted. TPN resulted in reduction of muscularis mucosa thickness and marked gut atrophy. Median and interquartile range for gut mass was 0.46 (0.30-0.58) g/cm in EN, and 0.19 (0.11-0.29) g/cm in TPN (p = 0.024). Key gut-systemic signaling regulators, liver farnesoid X receptor (FXR; p = 0.021), liver constitutive androstane receptor (CAR; p = 0.014), gut FXR (p = 0.028), G-coupled bile acid receptor (TGR5) (p = 0.003), epidermal growth factor (EGF; p = 0.016), organic anion transporter (OAT; p = 0.028), Mitogen-activated protein kinases-1 (MAPK1) (p = 0.037), and sodium uptake transporter sodium glucose-linked transporter (SGLT-1; p = 0.010) were significantly downregulated in TPN animals, whereas liver cholesterol 7 alpha-hydroxylase (CyP7A1) was substantially higher with TPN (p = 0.011). CONCLUSION: We report significant alterations in key hepatobiliary receptors driving gut-systemic signaling in a TPN piglet model. This presents a major advancement to our understanding of TPN-associated injury and suggests opportunities for strategic targeting of the gut-systemic axis, specifically, FXR, TGR5, and EGF in developing ameliorative strategies.

Concurrent Use of Calcium Chloride and Arginine Vasopressin Infusions in Pediatric Patients with Acute Cardiocirculatory Failure.

Acute heart failure (AHF) can cause low cardiac output and poor end-organ perfusion. Inotropic agents along with vasodilators can improve organ perfusion. Arginine vasopressin (AVP) and calcium chloride (CaCl) infusions are increasingly being used in low cardiac output states in pediatric AHF. We retrospectively reviewed 77 patients (0-18 years) with AHF admitted between January 2014 and May 2017 who received concurrent AVP and CaCl infusions. Surrogates of cardiac output and organ perfusion included hemodynamic vital signs, laboratory parameters, and urine output (UO). Organ dysfunction and vasopressor inotropic scores were also calculated. Median (IQR) age was 0.88 years (0, 3.75), and median weight was 6.62 kg (3.5, 13.7). Congenital heart disease was present in 70% (46/77) patients. Univentricular physiology was present in 25% (25/77) patients. None of the patients were in the immediate postoperative period. Median durations of AVP and CaCl were 2 days (1, 3) and 3 days (2, 6), respectively. Using Wilcoxon-signed rank test and Bonferroni correction, post hoc comparison showed that at 8 h post infusion, all systolic blood pressure (SBP) and diastolic blood pressure (DBP) results, and UO were greater than those 1 h prior to infusion. Median SBP increased from 79 mm Hg (71, 92) 1 h prior to 97 mm Hg (84, 107) 8 h post. Median DBP increased from 44 mm Hg (35, 52) 1 h prior to 54 mm Hg (44, 62) 8 h post. Heart rate showed a decrease between measurements 1 h prior to infusion and 8 h post, with median scores 146 (127, 162) and 136 (114, 150) beats per minute, respectively. Within first 8 h, median UO continuously increased from 6 mL/h. (0, 25) at 1 h post infusion to 20 mL/h. (2, 62) at 8 h post infusion. Median pediatric logarithmic organ dysfunction scores on days 4 through 7 post infusion were lower compared to day 1; median vasopressor inotropic scores on day 2 through 7 post infusion were lower compared to day 1. Serum lactate level, arterial pH, and base excess all showed favorable trend. Concurrent use of AVP and CaCl infusions may improve surrogates of cardiac output, and intensive care outcomes, and prevent organ dysfunction in children with AHF.

Role of the Gut⁻Liver Axis in Driving Parenteral Nutrition-Associated Injury.

For decades, parenteral nutrition (PN) has been a successful method for intravenous delivery of nutrition and remains an essential therapy for individuals with intolerance of enteral feedings or impaired gut function. Although the benefits of PN are evident, its use does not come without a significant risk of complications. For instance, parenteral nutrition-associated liver disease (PNALD)-a well-described cholestatic liver injury-and atrophic changes in the gut have both been described in patients receiving PN. Although several mechanisms for these changes have been postulated, data have revealed that the introduction of enteral nutrition may mitigate this injury. This observation has led to the hypothesis that gut-derived signals, originating in response to the presence of luminal contents, may contribute to a decrease in damage to the liver and gut. This review seeks to present the current knowledge regarding the modulation of what is known as the "gut⁻liver axis" and the gut-derived signals which play a role in PN-associated injury.

Validation Study of MaxSignal® Histamine Enzymatic Assay for the Detection of Histamine in Fish/Seafood.

Bioo Scientific Corp. has developed a rapid enzymatic quantitative assay for the determination of histamine in seafood. Fresh/frozen tuna, canned tuna, pouched tuna, and frozen mahi mahi samples were used for the validation study under the specific guidelines of the AOAC Research Institute Performance Tested MethodsSM program. Recoveries ranged from 82 to 107% at concentrations ranging from 6 to 72 ppm, with RSDr values between 0.8 and 6.5% (6-72 ppm). The linearity of the assay ranged from 0 to 108 ppm, with R2 values exceeding 0.99. The LOD was 0.9 ppm and the LOQ was 2.6 ppm for frozen tuna, which gave the lowest background level of contaminant. Cross-reactivity of the assay was tested against 14 other biogenic amines and was found to be minimal for all (<0.5%), except for agmatine (4.1%) and putrescine (0.9%). There was no observable interference from any tested biogenic amines. Product consistency was verified by validating lot-to-lot variations and variations within the same lot. Overall recoveries for all tested matrixes were within the acceptable range (80-120%). A 1-year claimed shelf life of the kit at 4°C was verified by accelerated stability study data collected on days 1, 15, and 32 at 25°C and by real-time stability testing at 1-month, 6-month, and 1-year at 4°C. No difference in histamine detection was observed in ruggedness testing, in which minor changes were introduced to the assay protocol. Good agreement was observed between AOAC Official MethodSM 977.13 and the MaxSignal® Histamine Enzymatic Assay method. Independent laboratory testing demonstrated that the MaxSignal method works with the same precision in the hands of minimally trained technicians as with the expert method developers. This study validates the performance of Bioo Scientific's rapid enzymatic method.

Novel FRET-based assay to detect reverse transcriptase activity using modified dUTP analogues.

We have developed a novel continuous assay to measure reverse transcriptase (RT) polymerase activity. The assay uses fluorescence energy transfer measurements to detect the incorporation of complementary pairs of fluorescently labeled deoxyuridine into cDNA product. The fluorescently labeled dUTP substrates were prepared using commercially available reagents with a simple coupling reaction. The fluorescent dye pairs have significant spectral overlap which allows FRET interaction between dyes incorporated into the cDNA. Using a polyA/oligo dT primer/template, the assay can readily detect DNA polymerase activity from any viral reverse transcriptase enzyme. The reaction proceeds linearly over time, and the rate is proportional to the enzyme concentration. We used the assay to compare the thermostability of a number of wild-type and mutant viral RT enzymes. Our results indicate that the wild-type AMV (avian myeloblastosis virus) enzyme is slightly more stable at 43 degrees C than the HIV-1 (human immunodeficiency virus) or MMLV (Moloney murine leukemia virus) enzymes. The thermostability of the RT enzyme was dramatically increased by the presence of primer/template with the enzyme. We also used the assay to study the effects of inhibitors on HIV-1 RT polymerase activity. This assay may be highly useful for the identification and characterization of potent RT inhibitors which could be candidates for development as therapeutic antiviral agents.

Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification.

The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Activation of PKR by short-interfering RNAs (siRNAs) and stimulation of the innate immune response has been suggested to explain certain off-target effects in some RNA interference experiments. Here we show that PKR's kinase activity is stimulated in vitro 3- to 5-fold by siRNA duplexes with 19 bp and 2 nt 3'-overhangs, whereas the maximum activation observed for poly(I)*poly(C) was 17-fold over background under the same conditions. Directed hydroxyl radical cleavage experiments indicated that siRNA duplexes have at least four different binding sites for PKR's dsRNA binding motifs (dsRBMs). The location of these binding sites suggested specific nucleotide positions in the siRNA sense strand that could be modified with a corresponding loss of PKR binding. Modification at these sites with N2-benzyl-2'-deoxyguanosine (BndG) blocked interaction with PKR's dsRBMs and inhibited activation of PKR by the siRNA. Importantly, modification of an siRNA duplex that greatly reduced PKR activation did not prevent the duplex from lowering mRNA levels of a targeted message by RNA interference in HeLa cells. Thus, these studies demonstrate that specific positions in an siRNA can be rationally modified to prevent interaction with components of cellular dsRNA-regulated pathways.

First-in-class pan caspase inhibitor developed for the treatment of liver disease.

A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular activity and in vivo efficacy. This structure-activity relationship study focused on the P4 oxamides and warhead moieties. Primarily on the basis of in vitro data, inhibitors were selected for study in a murine model of alpha-Fas-induced liver injury. IDN-6556 (1) was further profiled in additional in vivo models and pharmacokinetic studies. This first-in-class caspase inhibitor is now the subject of two Phase II clinical trials, evaluating its safety and efficacy for use in liver disease.

Structure-activity relationships within a series of caspase inhibitors. Part 2: Heterocyclic warheads.

Various heterocyclic hetero-methyl ketones of the 1-naphthyloxyacetyl-Val-Asp backbone have been prepared. A study of their structure-activity relationship (SAR) related to caspase-1, -3, -6, and -8 is reported. Their efficacy in a cellular model of cell death is also discussed. Potent broad-spectrum caspase inhibitors have been identified.