DNA methylation in necrotizing enterocolitis.

Epigenetic modifications, such as DNA methylation, are enzymatically regulated processes that directly impact gene expression patterns. In early life, they are central to developmental programming and have also been implicated in regulating inflammatory responses. Research into the role of epigenetics in neonatal health is limited, but there is a growing body of literature related to the role of DNA methylation patterns and diseases of prematurity, such as the intestinal disease necrotizing enterocolitis (NEC). NEC is a severe intestinal inflammatory disease, but the key factors that precede disease development remain to be determined. This knowledge gap has led to a failure to design effective targeted therapies and identify specific biomarkers of disease. Recent literature has identified altered DNA methylation patterns in the stool and intestinal tissue of neonates with NEC. These findings provide the foundation for a new avenue in NEC research. In this review, we will provide a general overview of DNA methylation and then specifically discuss the recent literature related to methylation patterns in neonates with NEC. We will also discuss how DNA methylation is used as a biomarker for other disease states and how, with further research, methylation patterns may serve as potential biomarkers for NEC.

The administration of a pre-digested fat-enriched formula prevents necrotising enterocolitis-induced lung injury in mice.

Necrotising enterocolitis (NEC) is a devastating gastrointestinal disease of prematurity that typically develops after the administration of infant formula, suggesting a link between nutritional components and disease development. One of the most significant complications that develops in patients with NEC is severe lung injury. We have previously shown that the administration of a nutritional formula that is enriched in pre-digested Triacylglyceride that do not require lipase action can significantly reduce the severity of NEC in a mouse model. We now hypothesise that this 'pre-digested fat (PDF) system' may reduce NEC-associated lung injury. In support of this hypothesis, we now show that rearing newborn mice on a nutritional formula based on the 'PDF system' promotes lung development, as evidenced by increased tight junctions and surfactant protein expression. Mice that were administered this 'PDF system' were significantly less vulnerable to the development of NEC-induced lung inflammation, and the administration of the 'PDF system' conferred lung protection. In seeking to define the mechanisms involved, the administration of the 'PDF system' significantly enhanced lung maturation and reduced the production of reactive oxygen species (ROS). These findings suggest that the PDF system protects the development of NEC-induced lung injury through effects on lung maturation and reduced ROS in the lung and also increases lung maturation in non-NEC mice.

The human milk oligosaccharides 2'-fucosyllactose and 6'-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling.

BACKGROUND: Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) can reduce NEC through inhibition of TLR4 signaling. METHODS: NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2'-FL, 6'-SL, or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, and human tissue explants and via in silico modeling. RESULTS: Supplementation of infant formula with either 2'-FL and/or 6'-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2'-FL and 6'-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa light-chain enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2'-FL and 6'-SL, but not lactose, to dock into the binding pocket of the TLR4-MD2 complex, explaining their ability to inhibit TLR4 signaling. CONCLUSIONS: 2'-FL and 6'-SL, but not lactose, prevent NEC in mice and piglet models and attenuate NEC inflammation in the human ileum, in part through TLR4 inhibition. IMPACT: Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants that occurs in the setting of bacterial colonization of the gut and administration of formula feeds and activation by the innate immune receptor toll-like receptor 4 (TLR4). Breast milk prevents NEC through unclear mechanisms. We now show that breast milk-enriched human milk oligosaccharides (HMOs) that are derived from lactose prevent NEC through inhibition of TLR4. The human milk oligosaccharides 2'-FL and 6'-SL, but not the backbone sugar lactose, prevent NEC in mice and piglets. 2'-FL and 6'-SL but not lactose inhibited TLR4 signaling in cultured enterocytes, in enteroids derived from mouse intestine, and in human intestinal explants obtained at the time of surgical resection for patients with NEC. In seeking the mechanisms involved, 2'-FL and 6'-SL but not lactose were found to directly bind to TLR4, explaining the inhibition and protection against NEC. These findings may impact clinical practice by suggesting that administration of HMOs could serve as a preventive strategy for premature infants at risk for NEC development.

A Dynamic Variation of Pulmonary ACE2 Is Required to Modulate Neutrophilic Inflammation in Response to Pseudomonas aeruginosa Lung Infection in Mice.

Angiotensin-converting enzyme 2 (ACE2) is a potent negative regulator capable of restraining overactivation of the renin-angiotensin system, which contributes to exuberant inflammation after bacterial infection. However, the mechanism through which ACE2 modulates this inflammatory response is not well understood. Accumulating evidence indicates that infectious insults perturb ACE2 activity, allowing for uncontrolled inflammation. In the current study, we demonstrate that pulmonary ACE2 levels are dynamically varied during bacterial lung infection, and the fluctuation is critical in determining the severity of bacterial pneumonia. Specifically, we found that a pre-existing and persistent deficiency of active ACE2 led to excessive neutrophil accumulation in mouse lungs subjected to bacterial infection, resulting in a hyperinflammatory response and lung damage. In contrast, pre-existing and persistent increased ACE2 activity reduces neutrophil infiltration and compromises host defense, leading to overwhelming bacterial infection. Further, we found that the interruption of pulmonary ACE2 restitution in the model of bacterial lung infection delays the recovery process from neutrophilic lung inflammation. We observed the beneficial effects of recombinant ACE2 when administered to bacterially infected mouse lungs following an initial inflammatory response. In seeking to elucidate the mechanisms involved, we discovered that ACE2 inhibits neutrophil infiltration and lung inflammation by limiting IL-17 signaling by reducing the activity of the STAT3 pathway. The results suggest that the alteration of active ACE2 is not only a consequence of bacterial lung infection but also a critical component of host defense through modulation of the innate immune response to bacterial lung infection by regulating neutrophil influx.

Clinical and Molecular Characteristics of Klebsiella pneumoniae Isolates Causing Bloodstream Infections in Japan: Occurrence of Hypervirulent Infections in Health Care.

Although hypervirulent Klebsiella pneumoniae (hvKp) has been associated with severe community-acquired infections that occur among relatively healthy individuals, information about hvKp infections in health care settings remains limited. Here, we systematically analyzed the clinical and molecular characteristics of K. pneumoniae isolates causing bloodstream infections in a cross-sectional study. Clinical characteristics of K. pneumoniae bloodstream infections from hospitals across Japan were analyzed by a review of the medical records. Whole-genome sequencing of the causative isolates was performed. Bacterial species were confirmed and hvKp were identified using whole-genome sequencing data. Clinical characteristics of hvKp infections were compared with those of non-hvKp infections by bivariate analyses. Of 140 cases of K. pneumoniae bloodstream infections, 26 cases (18.6%) were caused by various clones of hvKp defined by the carriage of cardinal virulence genes. Molecular identification revealed that 24 (17.1%) and 14 (10%) cases were caused by Klebsiella variicola and Klebsiella quasipneumoniae, respectively. Patients with hvKp infections had higher proportions of diabetes mellitus (risk ratio [RR], 1.75; 95% confidence interval [CI], 1.05 to 2.94), and their infections had significantly higher propensity to involve pneumonia (RR, 5.85; 95% CI, 1.39 to 24.6), liver abscess (RR, 5.85; 95% CI, 1.39 to 24.6), and disseminated infections (RR, 6.58; 95% CI, 1.16 to 37.4) than infections by other isolates. More than one-half of hvKp infections were health care associated or hospital acquired, and a probable event of health care-associated transmission of hvKp was documented. hvKp isolates, which are significantly associated with severe and disseminated infections, are frequently involved in health care-associated and hospital-acquired infections in Japan.

Attenuation of pulmonary ACE2 activity impairs inactivation of des-Arg9 bradykinin/BKB1R axis and facilitates LPS-induced neutrophil infiltration.

Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase with important functions in the renin-angiotensin system and plays a critical role in inflammatory lung diseases. ACE2 cleaves single-terminal residues from several bioactive peptides such as angiotensin II. However, few of its substrates in the respiratory tract have been identified, and the mechanism underlying the role of ACE2 in inflammatory lung disease has not been fully characterized. In an effort to identify biological targets of ACE2 in the lung, we tested its effects on des-Arg9 bradykinin (DABK) in airway epithelial cells on the basis of the hypothesis that DABK is a biological substrate of ACE2 in the lung and ACE2 plays an important role in the pathogenesis of acute lung inflammation partly through modulating DABK/bradykinin receptor B1 (BKB1R) axis signaling. We found that loss of ACE2 function in mouse lung in the setting of endotoxin inhalation led to activation of the DABK/BKB1R axis, release of proinflammatory chemokines such as C-X-C motif chemokine 5 (CXCL5), macrophage inflammatory protein-2 (MIP2), C-X-C motif chemokine 1 (KC), and TNF-α from airway epithelia, increased neutrophil infiltration, and exaggerated lung inflammation and injury. These results indicate that a reduction in pulmonary ACE2 activity contributes to the pathogenesis of lung inflammation, in part because of an impaired ability to inhibit DABK/BKB1R axis-mediated signaling, resulting in more prompt onset of neutrophil infiltration and more severe inflammation in the lung. Our study identifies a biological substrate of ACE2 within the airways, as well as a potential new therapeutic target for inflammatory diseases.

Fat composition in infant formula contributes to the severity of necrotising enterocolitis.

Necrotising enterocolitis (NEC) is a devastating disease that typically affects formula-fed premature infants, suggesting that dietary components may influence disease pathogenesis. TAG are the major fat components of infant formula, and their digestion requires pancreatic lipases, which may be naturally deficient in premature neonates. We hypothesise that NEC develops partly from the accumulation of incompletely digested long-chain TAG-containing unsaturated fatty acids within the intestinal epithelial cells, leading to oxidative stress and enterocyte damage. We further hypothesise that the administration of a formula that contains reduced TAG ('pre-digested fat') that do not require lipase action may reduce NEC severity. To test these hypotheses, we induced NEC in neonatal mice using three different fat formulations, namely 'standard fat', 'pre-digested fat' or 'very low fat', and determined that mice fed 'standard fat' developed severe NEC, which was significantly reduced in mice fed 'pre-digested fat' or 'very low fat'. The expression level of the critical fat-digesting enzyme carboxyl ester lipase was significantly lower in the newborn compared with older pups, leading to impaired fat digestion. The accumulation of mal-digested fat resulted in the significant accumulation of fat droplets within the intestinal epithelium of the distal ileum, resulting in the generation of reactive oxygen species and intestinal inflammation. Strikingly, these changes were prevented in pups fed 'pre-digested fat' or 'very low fat' formulas. These findings suggest that nutritional formula containing a pre-digested fat system may overcome the natural lipase deficiency of the premature gut, and serve as a novel approach to prevent NEC.

Pulmonary Epithelial TLR4 Activation Leads to Lung Injury in Neonatal Necrotizing Enterocolitis.

We seek to define the mechanisms leading to the development of lung disease in the setting of neonatal necrotizing enterocolitis (NEC), a life-threatening gastrointestinal disease of premature infants characterized by the sudden onset of intestinal necrosis. NEC development in mice requires activation of the LPS receptor TLR4 on the intestinal epithelium, through its effects on modulating epithelial injury and repair. Although NEC-associated lung injury is more severe than the lung injury that occurs in premature infants without NEC, the mechanisms leading to its development remain unknown. In this study, we now show that TLR4 expression in the lung gradually increases during postnatal development, and that mice and humans with NEC-associated lung inflammation express higher levels of pulmonary TLR4 than do age-matched controls. NEC in wild-type newborn mice resulted in significant pulmonary injury that was prevented by deletion of TLR4 from the pulmonary epithelium, indicating a role for pulmonary TLR4 in lung injury development. Mechanistically, intestinal epithelial TLR4 activation induced high-mobility group box 1 release from the intestine, which activated pulmonary epithelial TLR4, leading to the induction of the neutrophil recruiting CXCL5 and the influx of proinflammatory neutrophils to the lung. Strikingly, the aerosolized administration of a novel carbohydrate TLR4 inhibitor prevented CXCL5 upregulation and blocked NEC-induced lung injury in mice. These findings illustrate the critical role of pulmonary TLR4 in the development of NEC-associated lung injury, and they suggest that inhibition of this innate immune receptor in the neonatal lung may prevent this devastating complication of NEC.

The first case report of emphysematous pyelonephritis and bacteremia due to Oligella urethralis.

Oligella urethralis (O. urethralis) is an organism that rarely causes infections in humans. We report the case of a 90-year-old bedridden woman with progressive dementia who was placed in a long-term-care facility. She was admitted to our hospital due to fever and unconsciousness with pyuria. The abdominal computed tomography showed left pneumatosis and urinary stone. Fluoroquinolones-resistant O. urethralis, which was identified by the Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and the 16S rRNA gene sequencing, was isolated form the blood and urine cultures at admission. To the best of our knowledge, this is the first case of emphysematous pyelonephritis caused by O. urethralis.

Intestinal Epithelial TLR-4 Activation Is Required for the Development of Acute Lung Injury after Trauma/Hemorrhagic Shock via the Release of HMGB1 from the Gut.

The mechanisms that lead to the development of remote lung injury after trauma remain unknown, although a central role for the gut in the induction of lung injury has been postulated. We hypothesized that the development of remote lung injury after trauma/hemorrhagic shock requires activation of TLR4 in the intestinal epithelium, and we sought to determine the mechanisms involved. We show that trauma/hemorrhagic shock caused lung injury in wild-type mice, but not in mice that lack TLR4 in the intestinal epithelium, confirming the importance of intestinal TLR4 activation in the process. Activation of intestinal TLR4 after trauma led to increased endoplasmic reticulum (ER) stress, enterocyte apoptosis, and the release of circulating HMGB1, whereas inhibition of ER stress attenuated apoptosis, reduced circulating HMGB1, and decreased lung injury severity. Neutralization of circulating HMGB1 led to reduced severity of lung injury after trauma, and mice that lack HMGB1 in the intestinal epithelium were protected from the development of lung injury, confirming the importance of the intestine as the source of HMGB1, whose release of HMGB1 induced a rapid protein kinase C ζ-mediated internalization of surface tight junctions in the pulmonary epithelium. Strikingly, the use of a novel small-molecule TLR4 inhibitor reduced intestinal ER stress, decreased circulating HMGB1, and preserved lung architecture after trauma. Thus, intestinal epithelial TLR4 activation leads to HMGB1 release from the gut and the development of lung injury, whereas strategies that block upstream TLR4 signaling may offer pulmonary protective strategies after trauma.

The human milk oligosaccharide 2'-fucosyllactose attenuates the severity of experimental necrotising enterocolitis by enhancing mesenteric perfusion in the neonatal intestine.

Necrotising enterocolitis (NEC) is a common disease in premature infants characterised by intestinal ischaemia and necrosis. The only effective preventative strategy against NEC is the administration of breast milk, although the protective mechanisms remain unknown. We hypothesise that an abundant human milk oligosaccharide (HMO) in breast milk, 2'-fucosyllactose (2'FL), protects against NEC by enhancing intestinal mucosal blood flow, and we sought to determine the mechanisms underlying this protection. Administration of HMO-2'FL protected against NEC in neonatal wild-type mice, resulted in a decrease in pro-inflammatory markers and preserved the small intestinal mucosal architecture. These protective effects occurred via restoration of intestinal perfusion through up-regulation of the vasodilatory molecule endothelial nitric oxide synthase (eNOS), as administration of HMO-2'FL to eNOS-deficient mice or to mice that received eNOS inhibitors did not protect against NEC, and by 16S analysis HMO-2'FL affected the microbiota of the neonatal mouse gut, although these changes do not seem to be the primary mechanism of protection. Induction of eNOS by HMO-2'FL was also observed in cultured endothelial cells, providing a link between eNOS and HMO in the endothelium. These data demonstrate that HMO-2'FL protects against NEC in part through maintaining mesenteric perfusion via increased eNOS expression, and suggest that the 2'FL found in human milk may be mediating some of the protective benefits of breast milk in the clinical setting against NEC.

Toll-like receptor 4-mediated endoplasmic reticulum stress in intestinal crypts induces necrotizing enterocolitis.

The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4(ΔIEC-OVER) mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4(ΔIEC) mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.

Synthesis of anti-inflammatory α-and ß-linked acetamidopyranosides as inhibitors of toll-like receptor 4 (TLR4).

The low-molecular weight isopropyl 2-acetamido-α-glucoside 16 (C34) inhibits toll-like receptor 4 (TLR4) in enterocytes and macrophages in vitro, and reduces systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. We used a copper(II)-mediated solvolysis of anomeric oxazolines and an acid-mediated conversion of ß-glucosamine and ß-galactosamine pentaacetates to generate analogs of 16 at the anomeric carbon and at C-4 of the pyranose ring. These compounds were evaluated for their influence on TLR4-mediated inflammatory signaling in cultured enterocytes and monocytes. Their efficacy was confirmed using a NF-kB-luciferase reporter mouse, thus establishing the first structure-activity relationship (SAR) study in this series and identifying the more efficacious isopropyl 2-acetamido-α-galactoside 17.

Investigating the epidemiology and outbreaks of scabies in Japanese households, residential care facilities, and hospitals using claims data: the Longevity Improvement & Fair Evidence (LIFE) study.

Objectives: This study aimed to characterize the epidemiology of scabies and its outbreaks in Japanese households, residential care facilities (RCFs), and hospitals using claims data. Methods: This descriptive epidemiological study was conducted using claims data from eight municipalities in Japan. Scabies cases were identified using a combination of recorded diagnoses and administered medications. The study period was from April 2015 to March 2019. Outbreaks were defined as ≥2 cases of scabies occurring within a calendar month at a single household, RCF, or hospital. Results: We identified 857 scabies cases for analysis. The annual prevalence of scabies ranged from 40 to 67 per 100,000 beneficiaries. The annual attack rate of scabies was found to be highest in RCFs (21 per 1000 RCFs), followed by hospitals (11 per 1000 hospitals) and households (0.25 per 1000 households). The annual outbreak attack rate was also highest in RCFs (4.0 per 1000 RCFs), followed by hospitals (1.6 per 1000 hospitals) and household (0.027 per household). The patterns of outbreaks varied widely among the RCFs. Conclusions: The study showcases the potential of claims data for detecting infectious disease outbreaks, which could provide valuable insight for the future management and prevention of scabies. Infection control of scabies in RCFs is crucial in aging societies.

Sorafenib and HDAC inhibitors synergize with TRAIL to kill tumor cells.

The present studies were designed to compare and contrast the abilities of TRAIL (death receptor agonist) and obatoclax (BCL-2 family inhibitor) to enhance sorafenib + HDAC inhibitor toxicity in GI tumor cells. Sorafenib and HDAC inhibitor treatment required expression of CD95 to kill GI tumor cells in vitro and in vivo. In cells lacking CD95 expression, TRAIL treatment, and to a lesser extent obatoclax, enhanced the lethal effects of sorafenib + HDAC inhibitor exposure. In hepatoma cells expressing CD95 a similar data pattern emerged with respect to the actions of TRAIL. Downstream of the death receptor the ability of TRAIL to enhance cell killing correlated with reduced AKT, ERK1/2, p70 S6K, and mTOR activity and enhanced cleavage of pro-caspase 3 and reduced expression of MCL-1 and BCL-XL. Over-expression of BCL-XL or MCL-1 or expression of dominant negative pro-caspase 9 protected cells from drug toxicity. Expression of activated AKT, p70 S6K, mTOR, and to a lesser extent MEK1EE also protected cells that correlated with maintained c-FLIP-s expression, reduced BIM expression, and increased BAD phosphorylation. In vivo sorafenib + HDAC inhibitor toxicity against tumors was increased in a greater than additive fashion by TRAIL. Collectively, our data argue that TRAIL, rather than obatoclax, is the most efficacious agent at promoting sorafenib + HDAC inhibitor lethality.

Predictors of Poor Very Early Diuretic Response and Effectiveness of Early Tolvaptan in Symptomatic Acute Heart Failure.

BACKGROUND: Diuretic response (DR) in patients with symptomatic acute decompensated heart failure (ADHF) has an impact on prognosis. This study aimed to identify predictive factors influencing acute 6 h poor DR and to assess DR after early administration of tolvaptan (TLV). METHODS: This multicenter retrospective study included 1670 patients who were admitted for ADHF and received intravenous furosemide within 1 h of presentation in clinical scenario 1 or 2 defined based on initial systolic blood pressure ≥100 mmHg with severe symptoms (New York Heart Association class III or IV (n = 830). The score for the poor DR factors in the very acute phase was calculated in patients treated with furosemide-only diuretics (n = 439). The DR to TLV administration was also assessed in patients who received an additional dose of TLV within 6 h (n = 391). RESULTS: The time since discharge from the hospital for a previous heart failure < 3 months (odds ratio [OR] 2.78, 95% confidence interval [CI] 1.34-5.83; p = 0.006), loop diuretics at admission (OR 3.05, 95% CI 1.74-5.36; p < 0.0001), and estimated glomerular filtration rate (eGFR) < 45 mL/min/1.73 m2 (OR 2.99, 95% CI 1.58-5.74; p = 0.0007) were independent determinants of poor DR. The frequency of poor DR according to the risk stratification group was low risk (no risk factor), 18.9%; middle risk (one risk factor), 33.1%; and high risk (two to three risk factors), 58.0% (p < 0.0001). All risk groups demonstrated a significantly lower incidence of poor DR with early TLV administration: 10.7% in the early TLV group versus 18.9% in the loop diuretics group (p = 0.09) of the low-risk group; 18.4% versus 33.1% (p = 0.01) in the middle-risk group, and 20.2% versus 58.0% (p < 0.0001) in the high-risk group. CONCLUSION: Early administration of TLV in patients with predicted poor DR contributed to a significant diuretic effect and suppression of worsening renal function.

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field's understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Impact of the time-to-target rate of urine volume concept on the outcome of acute decompensated heart failure.

BACKGROUND: Early decongestion with diuretics could improve clinical outcomes. This study aimed to examine the impact of the time-to-target rate of urine volume (T2TUV) concept on the outcome of acute decompensated heart failure (ADHF). METHODS: This multicenter retrospective study included 1670 patients with ADHF who received diuretics within 24 h of admission. T2TUV was defined as the time from admission to the rate of urine volume of 100 ml/h. The primary outcomes were in-hospital death, mortality, and re-hospitalization for 1 year. RESULTS: A total of 789 patients met the inclusion criteria (T2TUV on day 1, n = 248; day 2-3, n = 172; no target rate UV, n = 369). In-hospital mortality in the day 1 group was significantly lower (2.7% vs. 5.9% vs. 11.1%; p < 0.001) than that of other groups. The mortality and re-hospitalization for 1 year in the day 1 group was significantly lower (event-free rate: 67.7% vs. 54.1% vs. 56.9%; log-lank p = 0.004) than that of other groups. In multivariate analysis, predictors of T2TUV at day 1 were age (odds ratio [OR]: 1.02, 95% confidence interval [CI]: 1.01-1.04, p = 0.007), previous hospitalized heart failure (OR: 1.47, 95% CI: [1.03-2.12], p = 0.03), N-terminal-pro B type natriuretic peptide per 1000 pg/ml (OR: 1.02, 95% CI: 1.01-1.04, p = 0.007), carperitide (OR: 0.69, 95% CI: 0.48-0.99, p = 0.05), and early administration of tolvaptan (OR: 0.6, 95% CI: 0.42-0.85, p = 0.004). CONCLUSIONS: T2TUV of less than day 1 was associated with lower in-hospital mortality and decreased mortality and re-hospitalization at 1 year.