A Novel Integrated Clinical-Biochemical-Radiological and Sonographic Classification of Necrotizing Enterocolitis.

OBJECTIVE: To evaluate the sensitivity and specificity of clinical, laboratory, and radiological markers and the neonatologist-performed intestinal ultrasound (NP-IUS) for treatment interventions in preterm neonates who developed necrotizing enterocolitis (NEC). STUDY DESIGN: This was a case-control study of preterm neonates < 35 weeks with a diagnostic workup for NEC. The diagnostic workup included NP-IUS performed by trained neonatologists using a standard protocol, abdominal roentgenogram (AXR), and laboratory investigations. Intestinal ultrasound (IUS) performed by two neonatologists was standardized to detect 11 injury markers. AXRs were read independently by experienced pediatric radiologists. The investigators who retrospectively interpreted the IUS were blinded to the clinical and treatment outcomes. RESULTS: A total of 111 neonates were assessed. Fifty-four did not require intervention and formed the control group. Twenty cases were treated medically, 21 cases were treated with late surgery for stricture or adhesions, and 16 were treated with early surgery. The integrated model of cumulative severity of ultrasound markers, respiratory and hemodynamic instability, abdominal wall cellulitis, and C- reactive protein > 16 mg/L had an area under the curve (AUC) of 0.89 (95% confidence interval [CI]: 0.83-0.94%, p < 0.0001) for diagnosing NEC requiring surgical intervention. We also investigated the utility of Bell's classification to diagnose either the need for surgery or death, and it had an AUC of 0.74 (95% CI: 0.65-0.83%, p < 0.0001). CONCLUSION: In this cohort, a combination of specific IUS markers and clinical signs of instability, abdominal wall cellulitis, plus laboratory markers were diagnostic of NEC requiring interventions. KEY POINTS: · The diagnosis of necrotizing enterocolitis requires a combination of markers.. · The combination of specific ultrasound markers, clinical signs, and laboratory markers were diagnostic of NEC requiring intervention.. · The intestinal ultrasound performed by a trained neonatologist was the most sensitive diagnostic marker of NEC requiring surgical intervention..

Hemispherectomy for hemimegalencephaly in a 6.5-week-old infant with tuberous sclerosis complex.

The aim of this report is to present a unique case of hemimegalencephaly and concomitant tuberous sclerosis complex (TSC1 mutation) with severe neonatal-onset epilepsy, which successfully underwent an anatomical hemispherectomy at 6.5 weeks of age for refractory seizures. Genetic testing confirmed a rare pathogenic, sporadic, heterozygous c.2041 + 1G > A gene mutation in intron 16 of the TSC1 gene, diagnostic for tuberous sclerosis. Post-operatively, the infant remained seizure free for at least 1 year. Following recurrence of her seizures, she has continued on multiple anti-seizure medications and everolimus therapy. We review the pathological and molecular features of this condition and highlight the ethics of intervention and steps taken toward safe neurosurgical intervention in this very young infant.

The isolation, purification and complete characterization of the diterpene forskolin from nutritional supplements.

Forskolin (1) is a diterpene found in the Coleus forskohlii plant that has been examined for its medical properties resulting from adenylyl cyclase activation. This article describes a straightforward purification method of 1 from commercially available weight loss capsules. In addition, there has been some ambiguity with respect to the use of the name 'forskolin' to describe 1 and related diterpenes, which this report serves to eliminate. Herein we detail the complete spectroscopic characterization of purified 1 as well as its single crystal X-ray structure.

The perfusion index histograms predict patent ductus arteriosus requiring treatment in preterm infants.

The impact of patent ductus arteriosus (PDA) on vital sign trends represented as histograms, and perfusion index in particular, is unknown. This study aimed to split continuously obtained PI and other vital signs before, during, and after medical treatment of PDA, into histogram bins, and determine the utility of PI and other vital sign histograms in the early prediction of hemodynamically significant PDA (hsPDA). In 34 infants at a mean gestational age of 26 ± 2.1 weeks, we prospectively collected vital signs for three different periods, 24 h before starting treatment of PDA, during PDA treatment, and 24 h after completion of the course of treatment, and confirmed PDA closure by echo. Histograms with three comparable periods were obtained from preterm infants who did not require treatment for PDA and analyzed for comparison. The duration of time spent in each histogram bin was determined for each time epoch. Episodes of low PI < 0.4 and high PI > 2 were significantly longer in duration in infants with PDA before treatment compared to those in infants with PDA during and after treatment. The arterial oxygen saturation (SpO2) < 80% was also longer in duration in infants with PDA before compared to that in infants with PDA during and after treatment. Low PI < 0.4 correlated with most echocardiography indices of hsPDA.Conclusion: We conclude that a patent ductus arteriosus requiring treatment in preterm infants ≤ 29 weeks GA was associated with significant fluctuations between a low PI < 0.4 alternating with a high PI > 2, reflecting the dynamic nature of hsPDA shunt volume. PI variability may be an early marker of hsPDA. What is Known: • The perfusion index is a continuous underutilized parameter provided by pulse oximetry to assess the peripheral perfusion. • The perfusion index helps predict conditions with hemodynamic instability. What is New: • The perfusion index assessed as daily histogram trends can predict patent ductus arteriosus requiring treatment.

Effects of Post-translational Modifications on Membrane Localization and Signaling of Prostanoid GPCR-G Protein Complexes and the Role of Hypoxia.

G protein-coupled receptors (GPCRs) play a pivotal role in the adaptive responses to cellular stresses such as hypoxia. In addition to influencing cellular gene expression profiles, hypoxic microenvironments can perturb membrane protein localization, altering GPCR effector scaffolding and altering downstream signaling. Studies using proteomics approaches have revealed significant regulation of GPCR and G proteins by their state of post-translational modification. The aim of this review is to examine the effects of post-translational modifications on membrane localization and signaling of GPCR-G protein complexes, with an emphasis on vascular prostanoid receptors, and to highlight what is known about the effect of cellular hypoxia on these mechanisms. Understanding post-translational modifications of protein targets will help to define GPCR targets in treatment of disease, and to inform research into mechanisms of hypoxic cellular responses.

Study of adenylyl cyclase-GαS interactions and identification of novel AC ligands.

Adenylyl cyclases (ACs) are membrane bound enzymes that catalyze the production of cAMP from ATP in response to the activation by G-protein Gαs. Different isoforms of ACs are ubiquitously expressed in different tissues involved in regulatory mechanisms in response to specific stimulants. There are 9 AC isoforms present in humans, with AC5 and AC6 proposed to play a vital role in cardiac functions. The activity of AC6 is sensitive to nitric oxide, such that nitrosylation of the protein might regulate its function. However, the information on structural determinants of nitrosylation in ACs and how they interact with Gαs is limited. Here we used homology modeling to build a molecular model of human AC6 bound to Gαs. Based on this 3D model, we predict the nitrosylation amenable cysteines, and identify potential novel ligands of AC6 using virtual ligand screening. Our model suggests Cys1004 in AC6 (subunit C2) and Cys174 in Gαs present at the AC-Gαs interface as the possible residues that might undergo reversible nitrosylation. Docking analysis predicted novel ligands of AC6 that include forskolin-based compounds and its derivatives. Further work involving site-directed mutagenesis of the predicted residues will allow manipulation of AC activity using novel ligands, and crucial insights on the role of nitrosylation of these proteins in pathophysiological conditions.

Hypoxic challenge of hyperoxic pulmonary artery myocytes increases oxidative stress due to impaired mitochondrial superoxide dismutase activity.

Infants with lung disease may be exposed to high O2 concentrations, and may have transient hypoxic episodes due to worsening lung pathophysiology, aggravating pulmonary arterial (PA) oxidative stress. NADPH oxidase (NOX) converts O2 to superoxide. Mitochondrial antioxidants such as superoxide dismutase (SOD) are sensitive to O2 tension. We previously reported decreased SOD2 activity in hypoxic PA myocytes due to nitration. In this study, we examined whether a transient hypoxic episode exposes antioxidant defense defects in hyperoxic PA myocytes. PA myocytes of term newborn piglets were cultured in hyperoxia (60% O2) or normoxia (21% O2) for 72 h; cells from both groups were challenged with transient hypoxia (10% O2) for 2 h. We measured NOX activity, SOD activities (fractionated by centrifugation and concavalin A- Sepharose chromatography), total ROS and superoxide generation, 8-isoprostane, and calcium responses to thromboxane mimetic. NOX activity increased in hyperoxic myocytes. Hyperoxia increased SOD1 activity but decreased SOD2 activity. Total ROS were reduced in hyperoxia, and hyperoxia + hypoxia groups. While hyperoxia alone did not alter superoxide content, superoxide increased after a hypoxic challenge of both normoxic and hyperoxic myocytes. Increased 8-isoprostane was seen only in hyperoxic myocytes challenged by transient hypoxia. We conclude that hyperoxic PA myocytes can limit total ROS despite increased NOX activity, but with inhibited SOD2 activity. Transient hypoxia increases superoxide formation; in the face of impaired SOD2, despite induction of SOD1, this oxidative stress causes increased 8-isoprostane generation. This may contribute to the mechanism of pulmonary arterial reactivity in infants with severe lung disease.

Analysis of the expression of human bitter taste receptors in extraoral tissues.

The 25 bitter taste receptors (T2Rs) in humans perform a chemosensory function. However, very little is known about the level of expression of these receptors in different tissues. In this study, using nCounter gene expression we analyzed the expression patterns of human TAS2R transcripts in cystic fibrosis bronchial epithelial (CuFi-1), normal bronchial epithelial (NuLi-1), airway smooth muscle (ASM), pulmonary artery smooth muscle (PASM), mammary epithelial, and breast cancer cells. Our results suggest a specific pattern of TAS2R expression with TAS2R3, 4, 5, 10, 13, 19, and 50 transcripts expressed at moderate levels and TAS2R14 and TAS2R20 (or TASR49) at high levels in the various tissues analyzed. This pattern of expression is mostly independent of tissue origin and the pathological state, except in cancer cells. To elucidate the expression at the protein level, we pursued flow cytometry analysis of select T2Rs from CuFi-1 and NuLi-1 cells. The expression levels observed at the gene level by nCounter analysis correlate with the protein levels for the T2Rs analyzed. Next, to assess the functionality of the expressed T2Rs in these cells, we pursued functional assays measuring intracellular calcium mobilization after stimulation with the bitter compound quinine. Using PLC inhibitor, U-73122, we show that the calcium mobilized in these cells predominantly takes place through the Quinine-T2R-Gαßγ-PLC pathway. This report will accelerate studies aimed at analyzing the pathophysiological function of T2Rs in different extraoral tissues.

Antihypertensive and neuroprotective actions of pyridoxine and its derivatives.

Vitamin B6 plays a crucial role in the nervous system as the amino acid decarboxylases involved in the synthesis of all putative neurotransmitters requires the coenzyme pyridoxal phosphate. Vitamin B6 in its various forms has antioxidant properties. Pyridoxal phosphate has a role in regulating cellular calcium transport through both the voltage-mediated and ATP-mediated purinergic mechanisms of cellular calcium influx and, hence, has a role in the control of hypertension. Pharmacological doses of vitamin B6 appear to decrease the high blood pressure associated with both genetic and nongenetic models of hypertension. Vitamin B6 has a crucial role in the normal function of the central and peripheral nervous systems. It also protects against ischemia and glutamate-induced neurotoxicity.

A role for actin polymerization in persistent pulmonary hypertension of the newborn.

Persistent pulmonary hypertension of the newborn (PPHN) is defined as the failure of normal pulmonary vascular relaxation at birth. Hypoxia is known to impede postnatal disassembly of the actin cytoskeleton in pulmonary arterial myocytes, resulting in elevation of smooth muscle α-actin and γ-actin content in elastic and resistance pulmonary arteries in PPHN compared with age-matched controls. This review examines the original histological characterization of PPHN with attention to cytoskeletal structural remodeling and actin isoform abundance, reviews the existing evidence for understanding the biophysical and biochemical forces at play during neonatal circulatory transition, and specifically addresses the role of the cortical actin architecture, primarily identified as γ-actin, in the transduction of mechanical force in the hypoxic PPHN pulmonary circuit.

Palmitoylation of Gαq determines its association with the thromboxane receptor in hypoxic pulmonary hypertension.

Pulmonary arterial vasoconstriction is a hallmark of persistent pulmonary hypertension of the newborn (PPHN). We reported increased calcium responses to thromboxane and selectively increased thromboxane prostanoid (TP) association with Gαq in hypoxic pulmonary artery. Palmitoylation of Gαq is important for efficient receptor-Gαq-phospholipase-C interactions. TPα receptor is not itself amenable to palmitoylation. We studied the role of Gαq palmitoylation in constriction of hypoxic pulmonary artery using pharmacological palmitoylation inhibition, the effects of hypoxia on palmitoylation, and the effects of site-specific cysteine substitution mutations of Gαq on Gαq membrane targeting, TPα association, and calcium dose-response curve to a TP agonist. PPHN pulmonary artery and HEK293T cells expressing TPα were exposed to irreversible palmitoylation inhibitor 2-bromopalmitate before challenge with TP agonist U46619. Palmitate uptake was studied in hypoxic and normoxic myocytes. Wild-type Gαq and Gαq cysteine-to-alanine mutants C9A, C10A, and C9A/C10A were transiently coexpressed in HEK293T cells stably expressing TPα. We examined membrane localization of Gαq, TP receptor-Gαq association by coimmunoprecipitation, and Ca(2+) responses to U46619 in hypoxic and normoxic cells. Gαq palmitoylation is essential for the Ca(2+) response to TPα stimulation. Inhibition of palmitoylation reduces contractile force to thromboxane in PPHN but not in control pulmonary artery. Hypoxia increases palmitoylation of Gαq; the hypoxic. but not the normoxic, response to thromboxane is palmitoylation sensitive. Palmitoylation of one N-terminal cysteine is required for physical association of Gαq with the TPα receptor. Palmitoylation of both cysteines is required for Gαq membrane localization and Ca(2+) mobilization. Depalmitoylation of any one Gαq cysteine reduces the hypoxic response to thromboxane challenge to equal that of normoxic cells.

Thromboxane-induced actin polymerization in hypoxic neonatal pulmonary arterial myocytes involves Cdc42 signaling.

In hypoxic pulmonary arterial (PA) myocytes, challenge with thromboxane mimetic U46619 induces marked actin polymerization and contraction, phenotypic features of persistent pulmonary hypertension of the newborn (PPHN). Rho GTPases regulate the actin cytoskeleton. We previously reported that U46619-induced actin polymerization in hypoxic PA myocytes occurs independently of the RhoA pathway and hypothesized involvement of the Cdc42 pathway. PA myocytes grown in normoxia or hypoxia for 72 h were stimulated with U46619, then analyzed for Rac/Cdc42 activation by affinity precipitation, phosphatidylinositide-3-kinase (PI3K) activity by phospho-Akt, phospho-p21-activated kinase (PAK) by immunoblot, and association of Cdc42 with neuronal Wiskott Aldrich Syndrome protein (N-WASp) by immunoprecipitation. The effect of Rac or PAK inhibition on filamentous actin was quantified by laser-scanning cytometry and by cytoskeletal fractionation; effects of actin-modifying agents were measured by isometric myography. Basal Cdc42 activity increased in hypoxia, whereas Rac activity decreased. U46619 challenge increased Cdc42 and Rac activity in hypoxic cells, independently of PI3K. Hypoxia increased phospho-PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia but increased after U46619 exposure. Hypoxia doubled filamentous-to-globular ratios of α- and γ-actin isoforms. Jasplakinolide stabilized γ-filaments, increasing force; cytochalasin D depolymerized all actin isoforms, decreasing force. Rac and PAK inhibition decreased filamentous actin in tissues although without decrease in force. Rho inhibition decreased myosin phosphorylation and force. Hypoxia induces actin polymerization in PA myocytes, particularly increasing filamentous α- and γ-actin, contributing to U46619-induced contraction. Hypoxic PA myocytes challenged with a thromboxane mimetic polymerize actin via the Cdc42 pathway, reflecting increased Cdc42 association with N-WASp. Mechanisms regulating thromboxane-mediated actin polymerization are potential targets for future PPHN pharmacotherapy.

Adenylyl cyclase isoforms 5 and 6 in the cardiovascular system: complex regulation and divergent roles.

Adenylyl cyclases (ACs) are crucial effector enzymes that transduce divergent signals from upstream receptor pathways and are responsible for catalyzing the conversion of ATP to cAMP. The ten AC isoforms are categorized into four main groups; the class III or calcium-inhibited family of ACs comprises AC5 and AC6. These enzymes are very closely related in structure and have a paucity of selective activators or inhibitors, making it difficult to distinguish them experimentally. AC5 and AC6 are highly expressed in the heart and vasculature, as well as the spinal cord and brain; AC6 is also abundant in the lungs, kidney, and liver. However, while AC5 and AC6 have similar expression patterns with some redundant functions, they have distinct physiological roles due to differing regulation and cAMP signaling compartmentation. AC5 is critical in cardiac and vascular function; AC6 is a key effector of vasodilatory pathways in vascular myocytes and is enriched in fetal/neonatal tissues. Expression of both AC5 and AC6 decreases in heart failure; however, AC5 disruption is cardio-protective, while overexpression of AC6 rescues cardiac function in cardiac injury. This is a comprehensive review of the complex regulation of AC5 and AC6 in the cardiovascular system, highlighting overexpression and knockout studies as well as transgenic models illuminating each enzyme and focusing on post-translational modifications that regulate their cellular localization and biological functions. We also describe pharmacological challenges in the design of isoform-selective activators or inhibitors for AC5 and AC6, which may be relevant to developing new therapeutic approaches for several cardiovascular diseases.

Assessment of Autoregulation of the Cerebral Circulation during Acute Lung Injury in a Neonatal Porcine Model.

In neonates with acute lung injury (ALI), targeting lower oxygenation saturations is suggested to limit oxygen toxicity while maintaining vital organ function. Although thresholds for cerebral autoregulation are studied for the management of premature infants, the impact of hypoxia on hemodynamics, tissue oxygen consumption and extraction is not well understood in term infants with ALI. We examined hemodynamics, cerebral autoregulation and fractional oxygen extraction, as measured by near-infrared spectroscopy (NIRS) and blood gases, in a neonatal porcine oleic acid injury model of moderate ALI. We hypothesized that in ALI animals, cerebral oxygen extraction would be increased to a greater degree than kidney or gut oxygen extraction as indicative of the brain's adaptive efforts to increase cerebral oxygen extraction at the expense of splanchnic end organs. Fifteen anesthetized, ventilated 5-day-old neonatal piglets were divided into moderate lung injury by treatment with oleic acid or control (sham injection). The degree of lung injury was quantified at baseline and after establishment of ALI by blood gases, ventilation parameters and calculated oxygenation deficit, hemodynamic indices by echocardiography and lung injury score by ultrasound. PaCO2 was maintained constant during ventilation. Cerebral, renal and gut oxygenation was determined by NIRS during stepwise decreases in inspired oxygen from 50% to 21%, correlated with PaO2 and PvO2; changes in fractional oxygen extraction (ΔFOE) were calculated from NIRS and from regional blood gas samples. The proportion of cerebral autoregulation impairment attributable to blood pressure, and to hypoxemia, was calculated from autoregulation nomograms. ALI manifested as hypoxemia with increasing intrapulmonary shunt fraction, decreased lung compliance and increased resistance, and marked increase in lung ultrasound score. Brain, gut and renal NIRS, obtained from probes placed over the anterior skull, central abdomen and flank, respectively, correlated with concurrent SVC (brain) or IVC (gut, renal) PvO2 and SvO2. Cerebral autoregulation was impaired after ALI as a function of blood pressure at all FiO2 steps, but predominantly by hypoxemia at FiO2 < 40%. Cerebral ΔFOE was higher in ALI animals at all FiO2 steps. We conclude that in an animal model of neonatal ALI, cerebrovascular blood flow regulation is primarily dependent on oxygenation. There is not a defined oxygenation threshold below which cerebral autoregulation is impaired in ALI. Cerebral oxygen extraction is enhanced in ALI, reflecting compensation for exhausted cerebral autoregulation due to the degree of hypoxemia and/or hypotension, thereby protecting against tissue hypoxia.

Diagnosis and management of congenital diaphragmatic hernia: a 2023 update from the Canadian Congenital Diaphragmatic Hernia Collaborative.

OBJECTIVE: The Canadian Congenital Diaphragmatic Hernia (CDH) Collaborative sought to make its existing clinical practice guideline, published in 2018, into a 'living document'. DESIGN AND MAIN OUTCOME MEASURES: Critical appraisal of CDH literature adhering to Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology. Evidence accumulated between 1 January 2017 and 30 August 2022 was analysed to inform changes to existing or the development of new CDH care recommendations. Strength of consensus was also determined using a modified Delphi process among national experts in the field. RESULTS: Of the 3868 articles retrieved in our search that covered the 15 areas of CDH care, 459 underwent full-text review. Ultimately, 103 articles were used to inform 20 changes to existing recommendations, which included aspects related to prenatal diagnosis, echocardiographic evaluation, pulmonary hypertension management, surgical readiness criteria, the type of surgical repair and long-term health surveillance. Fifteen new CDH care recommendations were also created using this evidence, with most related to the management of pain and the provision of analgesia and neuromuscular blockade for patients with CDH. CONCLUSIONS: The 2023 Canadian CDH Collaborative's clinical practice guideline update provides a management framework for infants and children with CDH based on the best available evidence and expert consensus.

Phenotype modulation of airway smooth muscle in asthma.

The biological responses of airway smooth muscle (ASM) are diverse, in part due to ASM phenotype plasticity. ASM phenotype plasticity refers to the ability of ASM cells to change the degree of a variety of functions, including contractility, proliferation, migration and secretion of inflammatory mediators. This plasticity occurs due to intrinsic or acquired abnormalities in ASM cells, and these abnormalities or predisposition of the ASM cell may alter the ASM response and in some cases recapitulate disease hallmarks of asthma. These phenotypic changes are ultimately determined by multiple stimuli and occur due to alterations in the intricate balance or reversible state that maintains ASM cells in either a contractile or synthetic state, through processes termed maturation or modulation, respectively. To elucidate the role of ASM phenotype in disease states, numerous in vitro studies have suggested a phenotypic switch in ASM primary cell cultures as an explanation for the plethora of responses mediated by ASM cells. Moreover, there is overwhelming evidence suggesting that the immunomodulatory response of ASM is due to the acquisition of a synthetic phenotype; however, whether this degree of plasticity is present in vivo as opposed to cell culture-based models remains speculative. Nonetheless, this review will give an overall scope of ASM phenotypic markers, triggers of ASM phenotype modulation and novel therapeutic approaches to control ASM phenotype plasticity.

Functional phenotype of airway myocytes from asthmatic airways.

In asthma, the airway smooth muscle (ASM) cell plays a central role in disease pathogenesis through cellular changes which may impact on its microenvironment and alter ASM response and function. The answer to the long debated question of what makes a 'healthy' ASM cell become 'asthmatic' still remains speculative. What is known of an 'asthmatic' ASM cell, is its ability to contribute to the hallmarks of asthma such as bronchoconstriction (contractile phenotype), inflammation (synthetic phenotype) and ASM hyperplasia (proliferative phenotype). The phenotype of healthy or diseased ASM cells or tissue for the most part is determined by expression of key phenotypic markers. ASM is commonly accepted to have different phenotypes: the contractile (differentiated) state versus the synthetic (dedifferentiated) state (with the capacity to synthesize mediators, proliferate and migrate). There is now accumulating evidence that the synthetic functions of ASM in culture derived from asthmatic and non-asthmatic donors differ. Some of these differences include an altered profile and increased production of extracellular matrix proteins, pro-inflammatory mediators and adhesion receptors, collectively suggesting that ASM cells from asthmatic subjects have the capacity to alter their environment, actively participate in repair processes and functionally respond to changes in their microenvironment.