The formation of ubiquitin rich condensates triggers recruitment of the ATG9A lipid transfer complex to initiate basal autophagy.

Autophagy is an essential cellular recycling process that maintains protein and organelle homeostasis. ATG9A vesicle recruitment is a critical early step in autophagy to initiate autophagosome biogenesis. The mechanisms of ATG9A vesicle recruitment are best understood in the context of starvation-induced non-selective autophagy, whereas less is known about the signals driving ATG9A vesicle recruitment to autophagy initiation sites in the absence of nutrient stress. Here we demonstrate that loss of ATG9A or the lipid transfer protein ATG2 leads to the accumulation of phosphorylated p62 aggregates in the context of basal autophagy. Furthermore, we show that p62 degradation requires the lipid scramblase activity of ATG9A. Lastly, we present evidence that poly-ubiquitin is an essential signal that recruits ATG9A and mediates autophagy foci assembly in nutrient replete cells. Together, our data support a ubiquitin-driven model of ATG9A recruitment and autophagosome formation during basal autophagy.

A combined metabonomic and proteomic approach identifies frontal cortex changes in a chronic phencyclidine rat model in relation to human schizophrenia brain pathology.

Current schizophrenia (SCZ) treatments fail to treat the broad range of manifestations associated with this devastating disorder. Thus, new translational models that reproduce the core pathological features are urgently needed to facilitate novel drug discovery efforts. Here, we report findings from the first comprehensive label-free liquid-mass spectrometry proteomic- and proton nuclear magnetic resonance-based metabonomic profiling of the rat frontal cortex after chronic phencyclidine (PCP) intervention, which induces SCZ-like symptoms. The findings were compared with results from a proteomic profiling of post-mortem prefrontal cortex from SCZ patients and with relevant findings in the literature. Through this approach, we identified proteomic alterations in glutamate-mediated Ca(2+) signaling (Ca(2+)/calmodulin-dependent protein kinase II, PPP3CA, and VISL1), mitochondrial function (GOT2 and PKLR), and cytoskeletal remodeling (ARP3). Metabonomic profiling revealed changes in the levels of glutamate, glutamine, glycine, pyruvate, and the Ca(2+) regulator taurine. Effects on similar pathways were also identified in the prefrontal cortex tissue from human SCZ subjects. The discovery of similar but not identical proteomic and metabonomic alterations in the chronic PCP rat model and human brain indicates that this model recapitulates only some of the molecular alterations of the disease. This knowledge may be helpful in understanding mechanisms underlying psychosis, which, in turn, can facilitate improved therapy and drug discovery for SCZ and other psychiatric diseases. Most importantly, these molecular findings suggest that the combined use of multiple models may be required for more effective translation to studies of human SCZ.

Evidence for disease and antipsychotic medication effects in post-mortem brain from schizophrenia patients.

Extensive research has been conducted on post-mortem brain tissue in schizophrenia (SCZ), particularly the dorsolateral prefrontal cortex (DLPFC). However, to what extent the reported changes are due to the disorder itself, and which are the cumulative effects of lifetime medication remains to be determined. In this study, we employed label-free liquid chromatography-mass spectrometry-based proteomic and proton nuclear magnetic resonance-based metabonomic profiling approaches to investigate DLPFC tissue from two cohorts of SCZ patients grouped according to their lifetime antipsychotic dose, together with tissue from bipolar disorder (BPD) subjects, and normal controls (n=10 per group). Both techniques showed profound changes in tissue from low-cumulative-medication SCZ subjects, but few changes in tissue from medium-cumulative-medication subjects. Protein expression changes were validated by Western blot and investigated further in a third group of subjects who were subjected to high-cumulative-medication over the course of their lifetime. Furthermore, key protein expression and metabolite level changes correlated significantly with lifetime antipsychotic dose. This suggests that the detected changes are present before antipsychotic therapy and, moreover, may be normalized with treatment. Overall, our analyses revealed novel protein and metabolite changes in low-cumulative-medication subjects associated with synaptogenesis, neuritic dynamics, presynaptic vesicle cycling, amino acid and glutamine metabolism, and energy buffering systems. Most of these markers were altered specifically in SCZ as determined by analysis of the same brain region from BPD patients.

Metabonomic characterization of the 3-nitropropionic acid rat model of Huntington's disease.

3-Nitropropionic acid (3-NP)-induced neurotoxicity can be used as a model for the genetic neurodegenerative disorder Huntington's disease (HD). A metabolic profiling strategy was adopted to explore the biochemical consequences of 3-NP administered to rats in specific brain regions. (1)H NMR spectroscopy was used to characterize the metabolite composition of several brain regions following 3-NP-intoxication. Dose-dependent increases in succinate levels were observed in all neuroanatomical regions, resulting from the 3-NP-induced inhibition of succinate dehydrogenase. Global decreases in taurine and GABA were observed in the majority of brain regions, whereas altered lipid profiles were observed only in the globus pallidus and dorsal striatum. Depleted phosphatidylcholine and elevated glycerol levels, which are indicative of apoptosis, were also observed in the frontal cortex of the 3-NP model. Many of the metabolic anomalies are consistent with those reported in HD. The 3-NP-induced model of HD provides a means of monitoring potential mechanisms of pathology and therapeutic response for drug interventions, which can be efficiently assessed using metabolic profiling strategies.

Metabonomic analysis identifies molecular changes associated with the pathophysiology and drug treatment of bipolar disorder.

Bipolar affective disorder is a severe and debilitating psychiatric condition characterized by the alternating mood states of mania and depression. Both the molecular pathophysiology of the disorder and the mechanism of action of the mainstays of its treatment remain largely unknown. Here, (1)H NMR spectroscopy-based metabonomic analysis was performed to identify molecular changes in post-mortem brain tissue (dorsolateral prefrontal cortex) of patients with a history of bipolar disorder. The observed changes were then compared to metabolic alterations identified in rat brain following chronic oral treatment with either lithium or valproate. This is the first study to use (1)H NMR spectroscopy to study post-mortem bipolar human brain tissue, and it is the first to compare changes in disease brain with changes induced in rat brain following mood stabilizer treatment. Several metabolites were found to be concordantly altered in both the animal and human tissues. Glutamate levels were increased in post-mortem bipolar brain, while the glutamate/glutamine ratio was decreased following valproate treatment, and gamma-aminobutyric acid levels were increased after lithium treatment, suggesting that the balance of excitatory/inhibitory neurotransmission is central to the disorder. Both creatine and myo-inositol were increased in the post-mortem brain but depleted with the medications. Lastly, the level of N-acetyl aspartate, a clinically important metabolic marker of neuronal viability, was found to be unchanged following chronic mood stabilizer treatment. These findings promise to provide new insight into the pathophysiology of bipolar disorder and may be used to direct research into novel therapeutic strategies.

Metabolic characterization of distinct neuroanatomical regions in rats by magic angle spinning 1H nuclear magnetic resonance spectroscopy.

High-resolution magic angle spinning (HRMAS) (1)H NMR spectroscopy has been applied to the biochemical characterization of specific brain regions in rats in order to establish baseline levels of tissue metabolite profiles with which to compare models of neuropathology or toxic lesion. Cores of tissue (20 mg) from the brain stem, cerebellum, frontal cortex, and hippocampus were obtained from histologically defined coronal slices of brain from 18 male Sprague-Dawley rats. HRMAS (1)H NMR spectra were acquired for each of the regions sampled and the degree of intersample variability, as assessed by principal components analysis and discriminant analysis by projection to latent structure was found to be low. Clear region-specific differences in the biochemical profiles were observed using both comparison of metabolite ratios and/or pattern recognition methods. Relatively low concentrations of GABA in the cerebellum, high concentrations of taurine and N-acetylaspartate in the cortex, and high levels of choline, glycerophosphocholine, and phosphocholine in the hippocampus predominantly influenced the classification of the different brain regions. Additionally, N-acetylaspartylglutamate was detected in the brain stem, but was largely absent from the other regions examined. Such analyses provide a baseline reference for further HRMAS NMR spectroscopic studies to monitor disease and pharmacological insults in specific regions of the brain.

The wandering spleen.

Wandering spleen is a rare condition that commonly presents with splenic infarction secondary to torsion. Splenectomy was previously advocated as the treatment of choice, though recent reports suggest that the organ may be distorted, and splenopexy performed. This paper documents how the latter procedure may be done using adherent omentum.

Management of coexisting coarctation of the aorta and oesophageal atresia.

The combination of neonatal coarctation of the aorta (CoA) and oesophageal atresia (oA) is rare and the optimum approach to surgical correction uncertain. Of 84 consecutive neonates with CoA over an 8-year period, 2 had coexisting oA. We consider that CoA repair by subclavian flap should be performed first to improve the environment for healing of the oesophageal anastomosis. A carefully co-ordinated approach between paediatric and cardiac surgical teams is recommended.

Diaphragmatic agenesis as a distinct clinical entity.

Anatomically, diaphragmatic agenesis (DA) is the most extreme form of congenital diaphragmatic defect, but clinically it has not been defined separately from Bochdalek's hernia (BH). Between 1986 and 1992, the authors treated 55 neonates who had diaphragmatic defects. Forty-eight of these cases presented within 24 hours of birth. Seventeen of these neonates (35.4%) were found to have DA; the other 31 (64.6%) had BH. There were no significant differences in maternal age, gestational age, gender ratio, birth weight, and incidence of coexisting congenital anomalies between the two groups. However, neonates with DA differed significantly from those with BH with respect to incidence of antenatal diagnosis (76.4% v 12.5%, P = .0004), mean Apgar scores at 1 (4.1 +/- 2.0 v 5.7 +/- 2.3; P = .034) and 5 (5.5 +/- 2.7 v 7.6 +/- 2.2; P = .016) minutes, mean duration of preoperative stabilization (2.8 +/- 2.0 v 2.1 +/- 1.9 days; P = .044), and postoperative respiratory support (27.7 +/- 13.6 v 9.3 +/- 8.0 days; P = .002). Complications occurred in all seven DA survivors (100%) and in only four (19.0%) of the 21 BH survivors (P = .0008). The long-term survival rate was significantly lower for neonates with DA (29.4% v 64.5%; P = .04). Diaphragmatic agenesis is a distinct clinical entity; its unique short- and long-term problems require careful management.

Obliteration of the distal bile duct in the development of congenital choledochal cyst.

The authors report the first case of antenatally diagnosed choledochal cyst having evidence of early fetal bile drainage and complete obliteration of the distal bile duct. This disputes the traditional hypothesis of reflux of pancreatic enzymes and supports the recent theory of primary obstruction as the etiologic cause of choledochal cyst. Coexisting congenital hypothyroidism and pulmonary stenosis had not been reported previously.

Diaphragmatic agenesis as a distinct clinical entity.

Anatomically, diaphragmatic agenesis (DA) represents the most extreme form of congenital diaphragmatic defects, but clinically it has not been defined separately from Bochalek's hernia (BH). Between 1986 and 1992, the authors treated 55 neonates who had diaphragmatic defects. Forty-eight of them presented within 24 hours of birth and comprised the study group. Seventeen neonates (35.4%) were found to have DA; the other 31 (64.6%) had BH. There were no differences in the maternal age, gestation course, gender ratio, birth weight, or incidence of co-existing congenital anomalies between the two groups. However, there were significant differences with respect to the incidence of antenatal diagnosis (76.4% for DA patients v 12.5% for BH patients; P = .0004), mean (+/- SD) Apgar scores at 1 (4.1 +/- 2.0 v 5.7 +/- 2.3; P = .034) and 5 minutes (5.5 +/- 2.7 v 7.6 +/- 2.2; P = .016), mean duration of preoperative stabilization (2.8 +/- 2.0 v 2.1 +/- 1.9 days; P = .044) and mean duration of postoperative respiratory support (27.7 +/- 13.6 v 9.3 +/- 8.0 days; P = .002). Complications occurred in all seven survivors of DA and in only four (19.0%) of 21 survivors of BH (P = .0008). The neonates with DA had a significantly poorer long-term survival rates (29.4% v 64.5%; P = .04). Diaphragmatic agenesis is a distinct clinical entity; its unique short-term and long-term problems require careful management.

Cytokine response of neonates to surgery.

Cytokines are early responders in the cascade of host mediators after injury. The cytokine response in neonates following surgery and its prognostic significance were studied prospectively. Twenty-one patients (oesophageal atresia [5], congenital diaphragmatic hernia [4], exomphalos [4], patent vitellointestinal duct [1], anorectal anomaly [2], choledochal cyst [1], renal cyst [1], ovarian cyst [1], myelomeningocoele [1], and pyloric stenosis [1]) operated on at a median age of 3 days (range, 1 to 24 days) and 12 age-matched controls were included in the study. Plasma samples were obtained once in the controls, and serially preoperatively and at 1, 3, 6, 12, 24, 36, and 48 hours postoperatively in the patients. The levels of the cytokines, interleukin-6 (IL-6), and interleukin-8 (IL-8) were measured using the enzyme-linked immunosorbent assay (ELISA) technique. The median plasma levels of IL-6 and IL-8 in normal controls were 2.4 pg/mL and 92.0 pg/mL, respectively. Of the 21 patients, four had postoperative complications (pulmonary consolidation [2], septicaemia [1], and oesophageal leak [1]) between days 4 and 6. All 17 uncomplicated cases had an increase in IL-6 and IL-8 in the early postoperative period, with the peak occurring within 12 hours after surgery. The mean (+/- SEM) peak levels of IL-6 and IL-8 in uncomplicated cases were of 92.6 +/- 15.8 pg/mL and 230.3 +/- 45.3 pg/mL, respectively. In the four complicated cases, there was a disproportionately higher increase in both IL-6 (peaks, 305.0, 125.0, 240.0, and 220.0 pg/mL) and IL-8 (peaks, 1500.0, 340.0, 245.0, 355.0 and pg/mL), which preceded the clinical onset of complications. The early postoperative increases in plasma IL-6 and IL-8 probably represent the stress response of neonates to surgery. Furthermore, the association of an exaggerated increase in postoperative levels of plasma IL-6 and IL-8 and postoperative complications may have prognostic significance.

Delayed primary wound closure using skin tapes for advanced appendicitis in children. A prospective, controlled study.

In a 3-year period, 63 consecutive patients with advanced perforated (n=53) and gangrenous (n=10) appendicitis were allocated to undergo either immediate wound closure or delayed primary wound closure after emergency appendectomy. The incidence of wound infection between delayed primary wound closure and immediate wound closure was similar (24.0% and 21.1%, respectively). The duration for complete healing of infected wounds was slightly shorter in the group undergoing delayed primary wound closure (mean +/- SD, 24.3 +/- 9.2 days) than in the group undergoing immediate wound closure (mean +/- SD, 32.6 +/- 16.5 days), but the difference was not significant. However, healing of noninfected wounds was significantly prolonged in the group undergoing delayed primary wound closure (mean +/- SD, 19.3 +/- 10.1 days) compared with the group undergoing immediate wound closure (mean +/- SD, 7.0 +/- 0 days). The latter had been shown to associate with more nonseptic wound complications and therefore required longer rehabilitation. Our study showed that delayed primary closure did not offer additional advantage over immediate closure in the treatment of wounds associated with advanced appendicitis in children.

Antibiotic prophylaxis in acute non-perforated appendicitis in children: single dose of metronidazole and gentamicin.

In a 3-year study, 103 children with acute non-perforated appendicitis who underwent appendicectomy were randomized for either a single preoperative dose of gentamicin and metronidazole or three doses of gentamicin and metronidazole given before and after the operation. The overall wound infection rate was 1.9%. There was no significant difference between wound infection rates of the single-dose group (2.1%) and the three-dose group (1.8%). The mean(s.d.) hospital stays of the single-dose and three-dose groups were similar: 6.6(2.2) days and 6.4(2.7) days. This study shows that a single preoperative dose of gentamicin and metronidazole is as effective as three doses of gentamicin and metronidazole in the control of post-appendicectomy wound sepsis.

Helicobacter pylori and associated duodenal ulcer.

Twenty three children with coexistent duodenal ulcer and Helicobacter pylori infection were treated with either two weeks of amoxycillin (25 mg/kg/day) in addition to six weeks of cimetidine, or cimetidine alone. Endoscopy with antral and duodenal biopsies for urease test, microaerophilic culture, and histological studies were performed at entry, six weeks, 12 weeks, and at six months. Children with persistent H pylori infection at six weeks were given a further two weeks' course of amoxycillin. H pylori persisted in all children not receiving amoxycillin treatment but cleared in six of the 13 children (46%) treated with amoxycillin. With failure of H pylori clearance at six months, only two out of six (33%) ulcers had healed and 50% of patients had experienced ulcer recurrence. In contrast, when H pylori remained cleared all ulcers healed and no ulcer recurred. Persistent H pylori infection was associated with persistent gastritis and duodenitis despite endoscopic evidence of ulcer healing. Detection and eradication of H pylori deserves particular attention in the routine management of duodenal ulceration in children.

Peptic ulcer in children.

From January 1983 to April 1989, 39 children were diagnosed as suffering from primary peptic ulcer. Of these ulcers, 30 were duodenal, seven prepyloric, and two patients had a combination of both. Among 36 patients with H2-blocker therapy, 33 had treatment completed with ulcer healing assessed by endoscopic examinations. Sixteen patients (49%) had ulcer healing within a single 6-week course of treatment, whereas 17 patients (51%) required multiple 6-week courses. The latter group had a mean basal acid output (BAO) of 0.13 +/- 0.06 mmol/kg/h and a mean maximal acid output (MAO) of 0.48 +/- 0.12 mmol/kg/h, which is significantly higher (P less than .05 and P less than .005, respectively) than the mean BAO (0.08 +/- 0.06 mmol/kg/h) and mean MAO (0.31 +/- 0.11 mmol/kg/h) in patients whose ulcers healed within 6 weeks of treatment. Subsequent follow-up with endoscopy over a mean period of 1.8 years (range, 3 months to 6 years) showed 10 patients with recurrent ulcers. Life-table analysis showed a recurrence rate of 35% within 1 year after ulcer healing. Five patients required surgery for ulcer complications--obstruction (2), perforation (2), and bleeding (1). In two of these patients initial H2-blocker therapy was unsuccessful.

Rapid endoscopy room diagnosis of Campylobacter pylori-associated gastritis in children.

A modified urease broth test was used in 109 gastric biopsy specimens of children that yielded a 96% sensitivity and a 100% specificity rate for Campylobacter pylori infection. The longest reaction time was less than or equal to 4 min. The correlation between the degree of C. pylori infection and the reaction time was found to be highly significant (gamma = 0.78, p less than 0.001). Patients were examined as outpatients, and immediate medications were prescribed for eradication of the bacteria on the basis of the urease broth test results. This is the first report on the use of this modified rapid urease test for the diagnosis of C. pylori infection in children.