Multisystem inflammatory syndrome in children: an Umbrella review.

We conducted an Umbrella review of eligible studies to evaluate what patient features have been investigated in the multisystem inflammatory syndrome in children (MIS-C) population, in order to guide future investigations. We comprehensively searched MEDLINE, EMBASE, and Cochrane Database of Systematic Reviews from December 1, 2019 to the May 6, 2022. The time period was limited to cover the coronavirus disease-2019 (COVID-19) pandemic period. The protocol was registered in the PROSPERO registry (CRD42022340228). Eligible studies included (1) a study population of pediatric patients ≤21 years of age diagnosed with MIS-C; (2) an original Systematic review or Mata-analysis; (3) published 2020 afterward; and (4) was published in English. A total of 41 studies met inclusion criteria and underwent qualitative analysis. 28 studies reported outcome data of MIS-C. 22 studies selected clinical features of MIS-C, and 6 studies chose demographic data as a main topic. The mortality rate for children with MIS-C was 1.9% (interquartile range (IQR) 0.48), the ICU admission rate was 72.6% (IQR 8.3), and the extracorporeal membrane oxygenation rate was 4.7% (IQR 2.0). A meta-analysis of eligible studies found that cerebral natriuretic peptide in children with MIS-C was higher than that in children with COVID-19, and that the use of intravenous immunoglobulin (IVIG) in combination with glucocorticoids to treat MIS-C compared to IVIG alone was associated with lower treatment failure. In the future, for patients with MIS-C, studies focused on safety of surgery requiring general anesthesia, risk factors, treatment, and long-term outcomes are warranted.

Argon inhalation attenuates systemic inflammation and rescues lung architecture during experimental neonatal sepsis.

PURPOSE: Neonatal sepsis is a systemic inflammatory infection common in premature infants and a leading cause of mortality. Argon is an emerging interest in the field of noble gas therapy. Neonates with severe sepsis are frequently mechanically ventilated creating an opportunity for inhalation therapy. We aimed to investigate argon inhalation as a novel experimental therapy in neonatal sepsis. METHODS: Sepsis was established in C57BL/6 neonatal mice by a lipopolysaccharide intraperitoneal injection on postnatal day 9. Septic pup mice were exposed to room air as well as non-septic controls. In the argon group, septic pup mice were exposed to argon (70% Ar, 30% O2) for 6 h in a temperature-controlled environment. RESULTS: At 6 h, survival was significantly enhanced when septic mice received argon compared to septic controls. Serum profiles of cytokine release were significantly attenuated as well as lung architecture restored. CONCLUSIONS: Our findings suggest that argon inhalation as a novel treatment for neonatal sepsis, reducing mortality and counteracting the acute systemic inflammatory response in the blood and preserving the architecture of the lung. This research can contribute to a paradigm shift in the treatment and outcome of neonates with sepsis.

Impact of Opioids on Cellular Metabolism: Implications for Metabolic Pathways Involved in Cancer.

Opioid utilization for pain management is prevalent among cancer patients. There is significant evidence describing the many effects of opioids on cancer development. Despite the pivotal role of metabolic reprogramming in facilitating cancer growth and metastasis, the specific impact of opioids on crucial oncogenic metabolic pathways remains inadequately investigated. This review provides an understanding of the current research on opioid-mediated changes to cellular metabolic pathways crucial for oncogenesis, including glycolysis, the tricarboxylic acid cycle, glutaminolysis, and oxidative phosphorylation (OXPHOS). The existing literature suggests that opioids affect energy production pathways via increasing intracellular glucose levels, increasing the production of lactic acid, and reducing ATP levels through impediment of OXPHOS. Opioids modulate pathways involved in redox balance which may allow cancer cells to overcome ROS-mediated apoptotic signaling. The majority of studies have been conducted in healthy tissue with a predominant focus on neuronal cells. To comprehensively understand the impact of opioids on metabolic pathways critical to cancer progression, research must extend beyond healthy tissue and encompass patient-derived cancer tissue, allowing for a better understanding in the context of the metabolic reprogramming already undergone by cancer cells. The current literature is limited by a lack of direct experimentation exploring opioid-induced changes to cancer metabolism as they relate to tumor growth and patient outcome.

Oligomeric assembly regulating mitochondrial HtrA2 function as examined by methyl-TROSY NMR.

Human High temperature requirement A2 (HtrA2) is a mitochondrial protease chaperone that plays an important role in cellular proteostasis and in regulating cell-signaling events, with aberrant HtrA2 function leading to neurodegeneration and parkinsonian phenotypes. Structural studies of the enzyme have established a trimeric architecture, comprising three identical protomers in which the active sites of each protease domain are sequestered to form a catalytically inactive complex. The mechanism by which enzyme function is regulated is not well understood. Using methyl transverse relaxation optimized spectroscopy (TROSY)-based solution NMR in concert with biochemical assays, a functional HtrA2 oligomerization/binding cycle has been established. In the absence of substrates, HtrA2 exchanges between a heretofore unobserved hexameric conformation and the canonical trimeric structure, with the hexamer showing much weaker affinity toward substrates. Both structures are substrate inaccessible, explaining their low basal activity in the absence of the binding of activator peptide. The binding of the activator peptide to each of the protomers of the trimer occurs with positive cooperativity and induces intrasubunit domain reorientations to expose the catalytic center, leading to increased proteolytic activity. Our data paint a picture of HtrA2 as a finely tuned, stress-protective enzyme whose activity can be modulated both by oligomerization and domain reorientation, with basal levels of catalysis kept low to avoid proteolysis of nontarget proteins.

Cancer proteome and metabolite changes linked to SHMT2.

Serine hydroxymethyltransferase 2 (SHMT2) converts serine plus tetrahydrofolate (THF) into glycine plus methylene-THF and is upregulated at the protein level in lung and other cancers. In order to better understand the role of SHMT2 in cancer a model system of HeLa cells engineered for inducible over-expression or knock-down of SHMT2 was characterized for cell proliferation and changes in metabolites and proteome as a function of SHMT2. Ectopic over-expression of SHMT2 increased cell proliferation in vitro and tumor growth in vivo. Knockdown of SHMT2 expression in vitro caused a state of glycine auxotrophy and accumulation of phosphoribosylaminoimidazolecarboxamide (AICAR), an intermediate of folate/1-carbon-pathway-dependent de novo purine nucleotide synthesis. Decreased glycine in the HeLa cell-based xenograft tumors with knocked down SHMT2 was potentiated by administration of the anti-hyperglycinemia agent benzoate. However, tumor growth was not affected by SHMT2 knockdown with or without benzoate treatment. Benzoate inhibited cell proliferation in vitro, but this was independent of SHMT2 modulation. The abundance of proteins of mitochondrial respiration complexes 1 and 3 was inversely correlated with SHMT2 levels. Proximity biotinylation in vivo (BioID) identified 48 mostly mitochondrial proteins associated with SHMT2 including the mitochondrial enzymes Acyl-CoA thioesterase (ACOT2) and glutamate dehydrogenase (GLUD1) along with more than 20 proteins from mitochondrial respiration complexes 1 and 3. These data provide insights into possible mechanisms through which elevated SHMT2 in cancers may be linked to changes in metabolism and mitochondrial function.

DYNC2H1 hypomorphic or retina-predominant variants cause nonsyndromic retinal degeneration.

PURPOSE: Determining the role of DYNC2H1 variants in nonsyndromic inherited retinal disease (IRD). METHODS: Genome and exome sequencing were performed for five unrelated cases of IRD with no identified variant. In vitro assays were developed to validate the variants identified (fibroblast assay, induced pluripotent stem cell [iPSC] derived retinal organoids, and a dynein motility assay). RESULTS: Four novel DYNC2H1 variants (V1, g.103327020_103327021dup; V2, g.103055779A>T; V3, g.103112272C>G; V4, g.103070104A>C) and one previously reported variant (V5, g.103339363T>G) were identified. In proband 1 (V1/V2), V1 was predicted to introduce a premature termination codon (PTC), whereas V2 disrupted the exon 41 splice donor site causing incomplete skipping of exon 41. V1 and V2 impaired dynein-2 motility in vitro and perturbed IFT88 distribution within cilia. V3, homozygous in probands 2-4, is predicted to cause a PTC in a retina-predominant transcript. Analysis of retinal organoids showed that this new transcript expression increased with organoid differentiation. V4, a novel missense variant, was in trans with V5, previously associated with Jeune asphyxiating thoracic dystrophy (JATD). CONCLUSION: The DYNC2H1 variants discussed herein were either hypomorphic or affecting a retina-predominant transcript and caused nonsyndromic IRD. Dynein variants, specifically DYNC2H1 variants are reported as a cause of non syndromic IRD.

A de novo mutation in PITX2 underlies a unique form of Axenfeld-Rieger syndrome with corneal neovascularization and extensive proliferative vitreoretinopathy.

BACKGROUND: Axenfeld-Rieger syndrome is characterized by a spectrum of anterior segment dysgenesis involving neural-crest-derived tissues, most commonly secondary to mutations in the transcription factor genes PITX2 and FOXC1. MATERIALS AND METHODS: Single retrospective case report. RESULTS: A full-term infant presented at 5 weeks of age with bilateral Peters anomaly and Axenfeld-Rieger syndrome, with development of atypical features of progressive corneal neovascularization and proliferative vitreoretinopathy. Despite surgical interventions, the patient progressed to bilateral phthisis bulbi by 22 months of age. Genetic testing revealed a novel de novo p.Leu212Valfs*39 mutation in PITX2, leading to loss of a C-terminal OAR domain that functions in transcriptional regulation. CONCLUSIONS: It is important to consider mutations in PITX2 in atypical cases of anterior segment dysgenesis that also present with abnormalities in the angiogenesis of the anterior and posterior segments.

Ultrasound-guided maxillary nerve block: an anatomical study using the suprazygomatic approach.

PURPOSE: Although a maxillary nerve (MN) block reportedly provides satisfactory analgesia for midface surgery and chronic maxillofacial pain syndromes, a safe and reliable MN block technique has not been reported. The goal of this anatomical study was to quantify the various angles and depth of the block needle, as well as to evaluate the impact of volume on the extent of injectate spread that might influence anesthetic coverage and block-related complications. METHODS: Following an ultrasound-guided suprazygomatic MN block with dye injection, a dissection was performed in the pterygopalatine fossa (PPF) of four lightly embalmed cadaveric specimens. Half of the specimens were injected with 5 mL of dye, and the other half with 1 mL of dye. The needle depth was measured from the ultrasound images and using rubber markers. Following injection, dissection was performed to map the area of dye spread. RESULTS: The median [interquartile range (IQR)] distance from the skin to the PPF was 37 [36-43] mm and 47 [40-50] mm by ultrasound and rubber marker methods, respectively. The median [IQR] needle orientation was 14 [11-32] degrees inferiorly and 15 [10-17] degrees posteriorly. The PPF was consistently dyed in the 5 mL group, but sporadically dyed in the 1 mL group. In the 5 mL group, spread outside of the PPF was seen. CONCLUSIONS: We showed that 5 mL of injectate far exceeds the capacity of the PPF, leading to drug spread outside of the PPF. Moreover, we found that 1 mL of injectate largely covered the nerve, suggesting a more efficacious and safer block procedure. This finding will need confirmation in future clinical studies.

Influence of Surgical Procedures and General Anesthesia on Child Development Before Primary School Entry Among Matched Sibling Pairs.

Importance: Substantial preclinical evidence suggests that the developing brain is susceptible to injury from anesthetic drugs. Findings from clinical studies of the neurotoxic effects of anesthesia are mixed, but these effects can be influenced by unmeasured confounding from biological and environmental risk and protective factors on child development. Objective: To examine the association between surgical procedures that require general anesthesia before primary school entry and child development in biological siblings. Design, Setting, and Participants: This retrospective sibling-matched cohort study included sibling pairs aged 5 to 6 years with the same birth mother who had Early Development Instrument (EDI) data completed. The EDI is a population-based measure of child development that assesses children's readiness to learn in 5 major domains (physical health and well-being, social knowledge and competence, emotional health and maturity, language and cognitive development, and communication skills and general knowledge). All eligible children in public and Catholic schools in Ontario, Canada, from 2004 through 2012 were included. Data were analyzed from December 13, 2017, through July 27, 2018. Exposures: Surgical procedures that require general anesthesia from the date of birth to EDI completion. Main Outcomes and Measures: Early developmental vulnerability, defined as any major domain of the EDI in the lowest 10th percentile of the Ontario population. Results: Of the 187 226 eligible children for whom the EDI was completed, a total of 10 897 sibling pairs (21 794 children; 53.8% female; mean [SD] age, 5.7 [0.3] years) were subsequently identified, including 2346 with only 1 child exposed to surgery. No significant differences were found between exposed and unexposed children in early developmental vulnerability (697 of 3080 [22.6%] vs 3739 of 18 714 [20.0%]; adjusted odds ratio [aOR], 1.03; 95% CI, 0.98-1.14; P = .58) or for each of the 5 major EDI domains (aOR for language and cognitive development, 0.96 [95% CI, 0.80-1.14]; aOR for physical health and well-being, 1.09 [95% CI, 0.96-1.24]; aOR for social knowledge and competence, 0.98 [95% CI, 0.84-1.14]; aOR for emotional health and maturity, 0.98 [95% CI, 0.84-1.14]; and aOR for communication skills and general knowledge, 0.90 [95% CI, 0.77-1.05]), after adjusting for confounding factors (age at EDI completion, sex, mother's age at birth, and eldest sibling status). Conclusions and Relevance: In this provincial cohort study, children who had surgical procedures that require general anesthesia before primary school entry were not found to be at increased risk of adverse child development outcomes compared with their biological siblings who did not have surgery. These findings further support that anesthesia exposure in early childhood is not associated with detectable adverse child development outcomes.

An autoantibody identifies arrhythmogenic right ventricular cardiomyopathy and participates in its pathogenesis.

Aims: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by right ventricular myocardial replacement and life-threatening ventricular arrhythmias. Desmosomal gene mutations are sometimes identified, but clinical and genetic diagnosis remains challenging. Desmosomal skin disorders can be caused by desmosomal gene mutations or autoantibodies. We sought to determine if anti-desmosome antibodies are present in subjects with ARVC. Methods and results: We evaluated ARVC subjects and controls for antibodies to cardiac desmosomal cadherin proteins. Desmoglein-2 (DSG2), desmocollin-2, and N-cadherin proteins on western blots were exposed to sera, in primary and validation cohorts of subjects and controls, as well as the naturally occurring Boxer dog model of ARVC. We identified anti-DSG2 antibodies in 12/12 and 25/25 definite ARVC cohorts and 7/8 borderline subjects. Antibody was absent in 11/12, faint in 1/12, and absent in 20/20 of two control cohorts. Anti-DSG2 antibodies were present in 10/10 Boxer dogs with ARVC, and absent in 18/18 without. In humans, the level of anti-DSG2 antibodies correlated with the burden of premature ventricular contractions (r = 0.70), and antibodies caused gap junction dysfunction, a common feature of ARVC, in vitro. Anti-DSG2 antibodies were present in ARVC subjects regardless of whether an underlying mutation was identified, or which mutation was present. A disease-specific DSG2 epitope was identified. Conclusion: Anti-DSG2 antibodies are a sensitive and specific biomarker for ARVC. The development of autoimmunity as a result of target-related mutations is unique. Anti-DSG2 antibodies likely explain the cardiac inflammation that is frequently identified in ARVC and may represent a new therapeutic target.

Evaluating the effects of general anesthesia on sleep in children undergoing elective surgery: an observational case-control study.

Study Objectives: Previous research has suggested that general anesthetics can disturb postoperative sleep patterns by affecting the sleep-wake cycle. The objective was to identify the effects of general anesthetics on sleep quality and related behavioral changes in children. Methods: This was a prospective, observational case-control study with children, aged 18 months to 8 years, undergoing general anesthesia for elective surgery. Participants wore an actigraph for 7 days on three occasions: prior to surgery, the immediate postoperative period, and 3 months after surgery. Data regarding behavior patterns were collected using behavioral assessments at baseline, the first postoperative week, and 3 months following surgery. Results: Thirty-one participants (mean age 4.8 ± 2.0 years, 81% male) underwent urologic or otolaryngologic surgery. The median (interquartile range) anesthetic duration was 132.0 (80.0-184.0) min. No significant differences were found in sleep efficiency, total sleep time, wake time after sleep onset, or sleep onset latency between baseline, 7 day postoperative period, and the 3 month follow-up. No significant differences were found in sleep-related behavioral metrics including internalizing and externalizing behaviors, and executive functioning. Data were compared with a control group of 18 participants (mean age 5.3 ± 1.8 years, 61% male). No significant differences were found in sleep patterns and related behavioral metrics between both groups. Conclusions: In this study, general anesthesia did not result in disturbed sleep or associated negative behavioral changes in otherwise healthy children undergoing elective surgeries of low complexity. Physicians can advise parents that a child's surgery and associated general anesthetic exposure may not result in significant changes in postoperative sleep patterns.

Inhalational Anesthetics Induce Neuronal Protein Aggregation and Affect ER Trafficking.

Anesthetic agents have been implicated in the causation of neurological and cognitive deficits after surgery, the exacerbation of chronic neurodegenerative disease, and were recently reported to promote the onset of the neurologic respiratory disease Congenital Central Hypoventilation Syndrome (CCHS), related to misfolding of the transcription factor Phox2B. To study how anesthetic agents could affect neuronal function through alterations to protein folding, we created neuronal cell models emulating the graded disease severity of CCHS. We found that the gas anesthetic isoflurane and the opiate morphine potentiated aggregation and mislocalization of Phox2B variants, similar to that seen in CCHS, and observed transcript and protein level changes consistent with activation of the endoplasmic reticulum (ER) unfolded protein response. Attenuation of ER stress pathways did not result in a correction of Phox2B misfolding, indicating a primary effect of isoflurane on protein structure. We also observed that isoflurane hindered the folding and activity of proteins that rely heavily on ER function, like the CFTR channel. Our results show how anesthetic drugs can alter protein folding and induce ER stress, indicating a mechanism by which these agents may affect neuronal function after surgery.

Anesthetic Practices for Laser Rehabilitation of Pediatric Hypertrophic Burn Scars.

The use of ablative fractional carbon dioxide laser therapy and pulsed dye laser therapy has led to significant improvements in the rehabilitation of hypertrophic burn scars. However, laser procedures are associated with appreciable pain among pediatric patients. Clinical consensus suggests using general anesthesia for pediatric laser procedures; however, guidelines for perioperative care are lacking. The objective of this quality improvement study is to determine whether a difference exists in postoperative pain outcomes in pediatric patients who receive intraoperative opioid regimens compared with patients who receive opioid-sparing regimens for laser therapy of hypertrophic burn scars. A retrospective review of patients who received laser therapy at a pediatric burn center from April 2014 to May 2015 was performed. Overall, 88 of the 92 procedures reviewed were included. A statistically significant difference was not found between the likelihood of postoperative pain when intraoperative opioid regimens (n = 63) were given compared with opioid-sparing regimens (n = 25) X (1, n = 88) = 2.870, P = .0902. There was also no difference between short-acting (n = 48), long-acting (n = 9), or combination (n = 6) intraoperative opioids compared with opioid-sparing regimens (n = 25) in the likelihood of postoperative pain. Despite the small sample size, the low number of postoperative pain cases is encouraging. Ultimately, these data provide a foundation for developing anesthetic guidelines for pediatric laser procedures. Specifically, clinicians should consider the potential to deliver adequate perioperative care via an opioid-sparing regimen ± adjuvant.

A Population-based Study Evaluating the Association between Surgery in Early Life and Child Development at Primary School Entry.

BACKGROUND: It is unclear whether exposure to surgery in early life has long-term adverse effects on child development. The authors aimed to investigate whether surgery in early childhood is associated with adverse effects on child development measured at primary school entry. METHODS: The authors conducted a population-based cohort study in Ontario, Canada, by linking provincial health administrative databases to children's developmental outcomes measured by the Early Development Instrument (EDI). From a cohort of 188,557 children, 28,366 children who underwent surgery before EDI completion (age 5 to 6 yr) were matched to 55,910 unexposed children. The primary outcome was early developmental vulnerability, defined as any domain of the EDI in the lowest tenth percentile of the population. Subgroup analyses were performed based on age at first surgery (less than 2 and greater than or equal to 2 yr) and frequency of surgery. RESULTS: Early developmental vulnerability was increased in the exposed group (7,259/28,366; 25.6%) compared with the unexposed group (13,957/55,910; 25.0%), adjusted odds ratio, 1.05; 95% CI, 1.01 to 1.08. Children aged greater than or equal to 2 yr at the time of first surgery had increased odds of early developmental vulnerability compared with unexposed children (odds ratio, 1.05; 95% CI, 1.01 to 1.10), but children aged less than 2 yr at the time of first exposure were not at increased risk (odds ratio, 1.04; 95% CI, 0.98 to 1.10). There was no increase in odds of early developmental vulnerability with increasing frequency of exposure. CONCLUSIONS: Children who undergo surgery before primary school age are at increased risk of early developmental vulnerability, but the magnitude of the difference between exposed and unexposed children is small.

OTX2 mutations cause autosomal dominant pattern dystrophy of the retinal pigment epithelium.

PURPOSE: To identify the genetic cause of autosomal-dominant pattern dystrophy (PD) of the retinal pigment epithelium (RPE) in two families. METHODS AND RESULTS: Two families with autosomal-dominant PD were identified. Eight members of family 1 (five affected) were subjected to whole-genome SNP genotyping; multipoint genome-wide linkage analysis identified 7 regions of potential linkage, and genotyping four additional individuals from family 1 resulted in a maximum logarithm of odds score of 2.09 observed across four chromosomal regions. Exome sequencing of two affected family 1 members identified 15 shared non-synonymous rare coding sequence variants within the linked regions; candidate genes were prioritised and further analysed. Sanger sequencing confirmed a novel heterozygous missense variant (E79K) in orthodenticle homeobox 2 (OTX2) that segregated with the disease phenotype. Family 2 with PD (two affected) harboured the same missense variant in OTX2. A shared haplotype of 19.68 cM encompassing OTX2 was identified between affected individuals in the two families. Within the two families, all except one affected demonstrated distinct 'patterns' at the macula. In vivo structural retinal imaging showed discrete areas of RPE-photoreceptor separation at the macula in all cases. Electroretinogram testing showed generalised photoreceptor degeneration in three cases. Mild developmental anomalies were observed, including optic nerve head dysplasia (four cases), microcornea (one case) and Rathke's cleft cyst (one case); pituitary hormone levels were normal. CONCLUSIONS: This is the first report implicating OTX2 to underlie PD. The retinal disease resembles conditional mice models that show slow photoreceptor degeneration secondary to loss of Otx2 function in the adult RPE.

Integrin-linked kinase mediates force transduction in cardiomyocytes by modulating SERCA2a/PLN function.

Human dilated cardiomyopathy (DCM) manifests as a profound reduction in biventricular cardiac function that typically progresses to death or cardiac transplantation. There is no effective mechanism-based therapy currently available for DCM, in part because the transduction of mechanical load into dynamic changes in cardiac contractility (termed mechanotransduction) remains an incompletely understood process during both normal cardiac function and in disease states. Here we show that the mechanoreceptor protein integrin-linked kinase (ILK) mediates cardiomyocyte force transduction through regulation of the key calcium regulatory protein sarcoplasmic/endoplasmic reticulum Ca(2+)ATPase isoform 2a (SERCA-2a) and phosphorylation of phospholamban (PLN) in the human heart. A non-oncogenic ILK mutation with a synthetic point mutation in the pleckstrin homology-like domain (ILK(R211A)) is shown to enhance global cardiac function through SERCA-2a/PLN. Thus, ILK serves to link mechanoreception to the dynamic modulation of cardiac contractility through a previously undiscovered interaction with the functional SERCA-2a/PLN module that can be exploited to rescue impaired mechanotransduction in DCM.

Losartan ameliorates "upstream" pulmonary vein vasculopathy in a piglet model of pulmonary vein stenosis.

OBJECTIVES: Pulmonary vein stenosis (PVS) is a relentless disease with a poor prognosis. Although surgical repair can effectively treat "downstream" (near left atrial junction) PVS, residual "upstream" (deep in lung parenchyma) PVS commonly dictates long-term survival. Our initial studies revealed an association between PVS and transforming growth factor-ß signaling, which led us to investigate the effect of losartan on upstream pulmonary vein vasculopathy in a piglet model of PVS. METHODS: Neonatal Yorkshire piglets underwent sham surgical banding (sham, n = 6), staged bilateral pulmonary vein banding of all pulmonary veins except the right middle pulmonary vein (banded, n = 6), and staged pulmonary vein banding with losartan treatment (losartan, 1 mg/kg/d, n = 7). After 7 weeks, the hemodynamic data were obtained and the piglets killed. RESULTS: Pulmonary vein banding (compared with sham) was associated with continuous turbulent flow in banded pulmonary veins, pulmonary hypertension (pulmonary artery/systemic blood pressure ratio 0.51 ± 0.06 vs 0.23 ± 0.02, P < .001), and diffuse pulmonary vein intimal hyperplasia in the upstream pulmonary veins (P < .001). Losartan administration decreased the pulmonary artery/systemic blood pressure ratios compared with those in the banded piglets (0.36 ± 0.08 vs 0.51 ± 0.06, P = .007) but it remained greater than those in the sham group (P = .001). Losartan was also associated with diminished pulmonary vein intimal hyperplasia compared with that in the banded piglets (P < .001) but still remained more than that in the sham group (P = .035). Pulmonary vein banding reduced vascular endothelial-cadherin expression, indicative of diminished endothelial integrity, which was restored with losartan. CONCLUSIONS: Losartan treatment improved PVS-associated pulmonary hypertension and intimal hyperplasia and might be a beneficial prophylactic therapy for patients at high risk of developing PVS after pulmonary vein surgery.

Pulmonary vein stenosis and the pathophysiology of "upstream" pulmonary veins.

BACKGROUND: Surgical and catheter-based interventions on pulmonary veins are associated with pulmonary vein stenosis (PVS), which can progress diffusely through the "upstream" pulmonary veins. The mechanism has been rarely studied. We used a porcine model of PVS to assess disease progression with emphasis on the potential role of endothelial-mesenchymal transition (EndMT). METHODS: Neonatal piglets underwent bilateral pulmonary vein banding (banded, n = 6) or sham operations (sham, n = 6). Additional piglets underwent identical banding and stent implantation in a single-banded pulmonary vein 3 weeks postbanding (stented, n = 6). At 7 weeks postbanding, hemodynamics and upstream PV pathology were assessed. RESULTS: Banded piglets developed pulmonary hypertension. The upstream pulmonary veins exhibited intimal thickening associated with features of EndMT, including increased transforming growth factor (TGF)-ß1 and Smad expression, loss of endothelial and gain of mesenchymal marker expression, and coexpression of endothelial and mesenchymal markers in banded pulmonary vein intimal cells. These immunopathologic changes and a prominent myofibroblast phenotype in the remodeled pulmonary veins were consistently identified in specimens from patients with PVS, in vitro TGF-ß1-stimulated cells isolated from piglet and human pulmonary veins, and human umbilical vein endothelial cells. After stent implantation, decompression of a pulmonary vein was associated with reappearance of endothelial marker expression, suggesting the potential for plasticity in the observed pathologic changes, followed by rapid in-stent restenosis. CONCLUSIONS: Neonatal pulmonary vein banding in piglets recapitulates critical aspects of clinical PVS and highlights a pathologic profile consistent with EndMT, supporting the rationale for evaluating therapeutic strategies designed to exploit reversibility of upstream pulmonary vein pathology.

Crystal structures of protein phosphatase-1 bound to motuporin and dihydromicrocystin-LA: elucidation of the mechanism of enzyme inhibition by cyanobacterial toxins.

The microcystins and nodularins are tumour promoting hepatotoxins that are responsible for global adverse human health effects and wildlife fatalities in countries where drinking water supplies contain cyanobacteria. The toxins function by inhibiting broad specificity Ser/Thr protein phosphatases in the host cells, thereby disrupting signal transduction pathways. A previous crystal structure of a microcystin bound to the catalytic subunit of protein phosphatase-1 (PP-1c) showed distinct changes in the active site region when compared with protein phosphatase-1 structures bound to other toxins. We have elucidated the crystal structures of the cyanotoxins, motuporin (nodularin-V) and dihydromicrocystin-LA bound to human protein phosphatase-1c (gamma isoform). The atomic structures of these complexes reveal the structural basis for inhibition of protein phosphatases by these toxins. Comparisons of the structures of the cyanobacterial toxin:phosphatase complexes explain the biochemical mechanism by which microcystins but not nodularins permanently modify their protein phosphatase targets by covalent addition to an active site cysteine residue.

The crystal structure of 1-D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) from Mycobacterium tuberculosis reveals a zinc hydrolase with a lactate dehydrogenase fold.

Mycothiol (1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside, MSH or AcCys-GlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn2+ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a C-terminal domain consisting of two beta-sheets and two alpha-helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD+ co-factor of lactate dehydrogenase. The Zn2+ is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn2+ providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol S-conjugate amidase based on the structure of MshB.