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Background and Objectives: Intestinal ischaemia-reperfusion injury following resuscitated haemorrhagic shock (HS) leads to endothelial and microcirculatory dysfunction and intestinal barrier breakdown. Although vascular smooth muscle machinery remains intact, microvascular vasoconstriction occurs secondary to endothelial cell dysfunction, resulting in further ischaemia and organ injury. Resuscitation with fresh frozen plasma (FFP) improves blood flow, stabilizes the endothelial glycocalyx and alleviates organ injury. We postulate these improvements correlate with decreased tissue CO 2 concentrations, improved microvascular oxygenation and attenuation of intestinal microvascular endothelial dysfunction., Materials and Methods: Male Sprague-Dawley rats were randomly assigned to groups (n = 8/group): (1) sham, (2) HS (40% mean arterial blood pressure [MAP], 60 min) + crystalloid resuscitation (CR) (shed blood saline) and (3) HS + FFP (shed blood + FFP). MAP, heart rate (HR), ileal perfusion, pO 2 and pCO 2 were measured at intervals until 4 h post-resuscitation (post-RES). At 4 h post-RES, the ileum was rinsed in situ with Krebs solution. Topical acetylcholine and then nitroprusside were applied for 10 min each. Serum was obtained, and after euthanasia, tissues were harvested and snap-frozen in liquid N 2 and stored at -80°C., Results: FFP resuscitation resulted in sustained ileal perfusion as well as rapid sustained return to baseline microvascular pO 2 and pCO 2 values when compared to CR (p < 0.05). Endothelial function was preserved relative to sham in the FFP group but not in the CR group (p < 0.05)., Conclusion: FFP-based resuscitation improves intestinal perfusion immediately following resuscitation, which correlates with improved tissue oxygenation and decreased tissue CO 2 levels. CR resulted in significant damage to endothelial vasodilation response to acetylcholine, while FFP preserved this function., (© 2023 International Society of Blood Transfusion.)

The endoplasmic reticulum (ER)-resident protein fat storage-inducing transmembrane protein 2 (FIT2) catalyzes acyl-CoA cleavage in vitro and is required for ER homeostasis and normal lipid storage in cells. The gene encoding FIT2 is essential for the viability of mice and worms. Whether FIT2 acts as an acyl-CoA diphosphatase in vivo and how this activity affects the liver, where the protein was discovered, are unknown. Here, we report that hepatocyte-specific Fitm2 knockout (FIT2-LKO) mice fed a chow diet exhibited elevated acyl-CoA levels, ER stress, and signs of liver injury. These mice also had more triglycerides in their livers than control littermates due, in part, to impaired secretion of triglyceride-rich lipoproteins and reduced capacity for fatty acid oxidation. We found that challenging FIT2-LKO mice with a high-fat diet worsened hepatic ER stress and liver injury but unexpectedly reversed the steatosis phenotype, similar to what is observed in FIT2-deficient cells loaded with fatty acids. Our findings support the model that FIT2 acts as an acyl-CoA diphosphatase in vivo and is crucial for normal hepatocyte function and ER homeostasis in the murine liver., Competing Interests: Conflict of interest T. C. W. is an investigator of the Howard Hughes Medical Institute., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Intestinal injury from resuscitated hemorrhagic shock (HS) disrupts intestinal microvascular flow and causes enterocyte apoptosis, intestinal barrier breakdown, and injury to multiple organs. Fresh frozen plasma (FFP) resuscitation or directed peritoneal (DPR) resuscitation protect endothelial glycocalyx, improve intestinal blood flow, and alleviate intestinal injury. We postulated that FFP plus DPR might improve effective hepatic blood flow (EHBF) and prevent associated organ injury (liver, heart)., Study Design: Anesthetized Sprague-Dawley rats underwent HS (40% mean arterial pressure, 60 minutes) and were randomly assigned to groups (n = 8 per group): Sham; crystalloid resuscitation (CR; shed blood + 2 volumes CR); DPR (intraperitoneal 2.5% peritoneal dialysis fluid); FFP (shed blood + 1 vol IV FFP); FFP + DPR. EHBF was measured at postresuscitation timepoints. Organ injury was evaluated by serum ELISA (fatty acid-binding protein [FABP]-1 [liver], FABP-3 [heart], Troponin-I [heart], and Troponin-C [heart]) and hematoxylin and eosin. Differences were evaluated by 1-way ANOVA and 2-way repeated-measures ANOVA., Results: CR resuscitation alone did not sustain EHBF. FFP resuscitation restored EHBF after resuscitation (2 hours, 3 hours, and 4 hours). DPR resuscitation restored EHBF throughout the postresuscitation period but failed to restore serum FABP-1 VS other groups. Combination FFP + DPR rapidly and sustainably restored EHBF and decreased organ injury. CR and DPR alone had elevated organ injury (FABP-1 [hepatocyte], FABP-3 [cardiac], and Troponin-I/C), whereas FFP or FFP + DPR demonstrated reduced injury at 4 hours after resuscitation., Conclusion: HS decreased EHBF, hepatocyte injury, and cardiac injury as evidenced by serology. FFP resuscitation improved EHBF and decreased organ damage. Although DPR resuscitation resulted in sustained EHBF, this alone failed to decrease hepatocyte or cardiac injury. Combination therapy with DPR and FFP may be a novel method to improve intestinal and hepatic blood flow and decrease organ injury after HS/resuscitation., (Copyright © 2022 by the American College of Surgeons. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Background: A small number of patients treated with minimally invasive correction of pectus excavatum recur after bar removal. This risk appears to be greater in younger children who continue to grow following bar removal., Methods: We propose the use of wrist films to determine skeletal maturity and delay bar removal until it is completed. This is not possible in very young patients (less than 14 years of age) or necessary in patients older than 19., Results: In the 14-year to 18-year age group, we have used wrist films to determine skeletal maturity in 25 patients. Ten patients (age 14-18) demonstrated full maturation, and their bars were removed at 2 years. Five patients had films that demonstrated nearly closed growth plates, and those bars were removed 6 months later (2.5 years post-insertion). Ten patients had 2 sets of films taken, initially at 2 years post-operation demonstrating open growth plates. Films 12 months later showed skeletal maturation. Their bars were removed at 3 years post-operation. There were no recurrences with an average follow-up of 3 years., Discussion: Radiographic determination of skeletal maturity may be used as a guide to the timing of bar removal following the correction of pectus excavatum.

The endoplasmic reticulum is a cellular hub of lipid metabolism, coordinating lipid synthesis with continuous changes in metabolic flux. Maintaining ER lipid homeostasis despite these fluctuations is crucial to cell function and viability. Here, we identify a novel mechanism that is crucial for normal ER lipid metabolism and protects the ER from dysfunction. We identify the molecular function of the evolutionarily conserved ER protein FIT2 as a fatty acyl-coenzyme A (CoA) diphosphatase that hydrolyzes fatty acyl-CoA to yield acyl 4'-phosphopantetheine. This activity of FIT2, which is predicted to be active in the ER lumen, is required in yeast and mammalian cells for maintaining ER structure, protecting against ER stress, and enabling normal lipid storage in lipid droplets. Our findings thus solve the long-standing mystery of the molecular function of FIT2 and highlight the maintenance of optimal fatty acyl-CoA levels as key to ER homeostasis., (© 2020 Becuwe et al.)

Excessive levels of saturated fatty acids are toxic to cells, although the basis for this lipotoxicity remains incompletely understood. Here, we analyzed the transcriptome, lipidome, and genetic interactions of human leukemia cells exposed to palmitate. Palmitate treatment increased saturated glycerolipids, accompanied by a transcriptional stress response, including upregulation of the endoplasmic reticulum (ER) stress response. A comprehensive genome-wide short hairpin RNA (shRNA) screen identified >350 genes modulating lipotoxicity. Among previously unknown genetic modifiers of lipotoxicity, depletion of RNF213, a putative ubiquitin ligase mutated in Moyamoya vascular disease, protected cells from lipotoxicity. On a broader level, integration of our comprehensive datasets revealed that changes in di-saturated glycerolipids, but not other lipid classes, are central to lipotoxicity in this model. Consistent with this, inhibition of ER-localized glycerol-3-phosphate acyltransferase activity protected from all aspects of lipotoxicity. Identification of genes modulating the response to saturated fatty acids may reveal novel therapeutic strategies for treating metabolic diseases linked to lipotoxicity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Inflammation and endoplasmic reticulum (ER) stress are hallmarks of metabolic syndrome. While these metabolic derangements have been well-investigated in white adipose tissue, their existence and etiology in brown adipose tissue (BAT) are poorly understood. Here, we aimed to investigate ER homeostasis and the inflammatory status and of BAT lacking uncoupling protein-1 (UCP1), a protein required for BAT thermogenesis. H&E staining illustrated lipid accumulation and crown-like structures surrounding adipocytes in BAT of UCP1-/- mice housed at room temperature compared to control mice. Further, immunohistological evaluation of F4/80 and gene expression studies demonstrated BAT macrophage infiltration and robust elevation of pro-inflammatory markers in UCP1-/- BAT. ER stress was also present in BAT of UCP1-/- mice, as evidenced by elevated gene expression and post-translational modifications of unfolded protein response components. After four weeks of thermoneutral housing, UCP1-/- mice did not exhibit elevated BAT inflammation and ER stress gene expression compared to WT mice, but depot expansion persisted. Collectively, we demonstrate that the effects of UCP1 deficiency in BAT are not restricted to mitochondrial uncoupling. We conclude that brown adipose tissue of UCP1-/- mice exhibits pro-inflammatory immune cell infiltration and perturbations in ER homeostasis and that this phenotype is driven by cold exposure rather than lipid accumulation., Competing Interests: The authors have declared that no competing interests exist.

Traumatic spinal cord injury (SCI) is associated with a lifetime of disability stemming from loss of motor, sensory, and autonomic functions; these losses, along with increased comorbid sequelae, negatively impact health outcomes and quality of life. Early decompression surgery post-SCI can enhance patient outcomes, but does not directly facilitate neural repair and regeneration. Currently, there are no U.S. Food and Drug Administration-approved pharmacological therapies to augment motor function and functional recovery in individuals with traumatic SCI. After an SCI, the enzyme, Rho, is activated by growth-inhibitory factors and regulates events that culminate in collapse of the neuronal growth cone, failure of axonal regeneration, and, ultimately, failure of motor and functional recovery. Inhibition of Rho activation is a potential treatment for injuries such as traumatic SCI. VX-210, an investigational agent, inhibits Rho. When administered extradurally after decompression (corpectomy or laminectomy) and stabilization surgery in a phase 1/2a study, VX-210 was well tolerated. Here, we describe the design of the SPRING trial, a multicenter, phase 2b/3, randomized, double-blind, placebo-controlled clinical trial to evaluate the efficacy and safety of VX-210 (NCT02669849). A subset of patients with acute traumatic cervical SCI is currently being enrolled in the United States and Canada. Medical, neurological, and functional changes are evaluated at 6 weeks and at 3, 6, and 12 months after VX-210 administration. Efficacy will be assessed by the primary outcome measure, change in upper extremity motor score at 6 months post-treatment, and by secondary outcomes that include question-based and task-based evaluations of functional recovery.

Uncoupling protein-1 (UCP1) facilitates thermogenesis in brown and beige adipocytes and can promote energy expenditure by decreasing mitochondrial respiratory efficiency. Defects in UCP1 and brown adipose tissue thermogenesis subject animals to chronic cold stress and elicit compensatory responses to generate heat. How UCP1 regulates white adipose tissue (WAT) lipid biology and tissue crosstalk is not completely understood. Here, we probed the effect of UCP1 deficiency on FA metabolism in inguinal and epididymal WAT and investigated how these metabolic perturbations influence hepatic lipid homeostasis. We report that at standard housing temperature (21°C), loss of UCP1 induces inguinal WAT de novo lipogenesis through transcriptional activation of the lipogenic gene program and elevated GLUT4. Inguinal adipocyte hyperplasia and depot expansion accompany the increase in lipid synthesis. We also found that UCP1 deficiency elevates adipose stearoyl-CoA desaturase gene expression, and increased inguinal WAT lipolysis supports the transport of adipose-derived palmitoleate (16:1n7) to the liver and hepatic triglyceride accumulation. The observed WAT and liver phenotypes were resolved by housing animals at thermoneutral housing (30°C). These data illustrate depot-specific responses to impaired BAT thermogenesis and communication between WAT and liver in UCP1 -/- mice., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

An egg must be fertilized by a single sperm only. To prevent polyspermy, the zona pellucida, a structure that surrounds mammalian eggs, becomes impermeable upon fertilization, preventing the entry of further sperm. The structural changes in the zona upon fertilization are driven by the exocytosis of cortical granules. These translocate from the oocyte's centre to the plasma membrane during meiosis. However, very little is known about the mechanism of cortical granule translocation. Here we investigate cortical granule transport and dynamics in live mammalian oocytes by using Rab27a as a marker. We show that two separate mechanisms drive their transport: myosin Va-dependent movement along actin filaments, and an unexpected vesicle hitchhiking mechanism by which cortical granules bind to Rab11a vesicles powered by myosin Vb. Inhibiting cortical granule translocation severely impaired the block to sperm entry, suggesting that translocation defects could contribute to miscarriages that are caused by polyspermy.

A 36-year-old woman presented to the emergency department with a 3-day history of an occipital headache associated with transient visual impairment and short-term memory loss. MRI of the brain showed innumerable focal embolic infarcts of differing ages, for which a cause could not be determined. The patient was discharged and readmitted 7 weeks later with acute aphasia and a right-sided hemiplegia. CT of the abdomen revealed a right-sided ovarian mass and prominent retroperitoneal nodes, which cytology confirmed to be metastatic ovarian cancer., (2015 BMJ Publishing Group Ltd.)

In response to temperature, Blastomyces dermatitidis converts between yeast and mold forms. Knowledge of the mechanism(s) underlying this response to temperature remains limited. In B. dermatitidis, we identified a GATA transcription factor, SREB, important for the transition to mold. Null mutants (SREBΔ) fail to fully complete the conversion to mold and cannot properly regulate siderophore biosynthesis. To capture the transcriptional response regulated by SREB early in the phase transition (0-48 hours), gene expression microarrays were used to compare SREB∆ to an isogenic wild type isolate. Analysis of the time course microarray data demonstrated SREB functioned as a transcriptional regulator at 37°C and 22°C. Bioinformatic and biochemical analyses indicated SREB was involved in diverse biological processes including iron homeostasis, biosynthesis of triacylglycerol and ergosterol, and lipid droplet formation. Integration of microarray data, bioinformatics, and chromatin immunoprecipitation identified a subset of genes directly bound and regulated by SREB in vivo in yeast (37°C) and during the phase transition to mold (22°C). This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. Functional analysis suggested that lipid droplets were actively metabolized during the phase transition and lipid metabolism may contribute to filamentous growth at 22°C. Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX. Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition.

Hepatic steatosis is associated with detrimental metabolic phenotypes including enhanced risk for diabetes. Stearoyl-CoA desaturases (SCDs) catalyze the synthesis of MUFAs. In mice, genetic ablation of SCDs reduces hepatic de novo lipogenesis (DNL) and protects against diet-induced hepatic steatosis and adiposity. To understand the mechanism by which hepatic MUFA production influences adipose tissue stores, we created two liver-specific transgenic mouse models in the SCD1 knockout that express either human SCD5 or mouse SCD3, that synthesize oleate and palmitoleate, respectively. We demonstrate that hepatic de novo synthesized oleate, but not palmitoleate, stimulate hepatic lipid accumulation and adiposity, reversing the protective effect of the global SCD1 knockout under lipogenic conditions. Unexpectedly, the accumulation of hepatic lipid occurred without induction of the hepatic DNL program. Changes in hepatic lipid composition were reflected in plasma and in adipose tissue. Importantly, endogenously synthesized hepatic oleate was associated with suppressed DNL and fatty acid oxidation in white adipose tissue. Regression analysis revealed a strong correlation between adipose tissue lipid fuel utilization and hepatic and adipose tissue lipid storage. These data suggest an extrahepatic mechanism where endogenous hepatic oleate regulates lipid homeostasis in adipose tissues.

The development of novel pharmaceutical treatments for disorders of the cerebral vasculature is a serious unmet medical need. These vascular disorders are typified by a disruption in the delicate Rho signaling equilibrium within the blood vessel wall. In particular, Rho kinase overactivation in the smooth muscle and endothelial layers of the vessel wall results in cytoskeletal modifications that lead to reduced vascular integrity and abnormal vascular growth. Rho kinase is thus a promising target for the treatment of cerebral vascular disorders. Indeed, preclinical studies indicate that Rho kinase inhibition may reduce the formation/growth/rupture of both intracranial aneurysms and cerebral cavernous malformations.

Cervical spinal cord injury (SCI) results in partial to full paralysis of the upper and lower extremities. Traditional primary endpoints for acute SCI clinical trials are too broad to assess functional recovery in cervical subjects, raising the possibility of false positive outcomes in trials for cervical SCI. Endpoints focused on the recovery of hand and arm control (e.g., upper extremity motor score, motor level change) show the most potential for use as primary outcomes in upcoming trials of cervical SCI. As the field moves forward, the most reliable way to ensure meaningful clinical testing in cervical subjects may be the development of a composite primary endpoint that measures both neurological recovery and functional improvement.

Advances in screening and computational methods have enhanced recent efforts to discover/design small-molecule protein inhibitors. One attractive target for inhibition is the myosin family of motor proteins. Myosins function in a wide variety of cellular processes, from intracellular trafficking to cell motility, and are implicated in several human diseases (e.g., cancer, hypertrophic cardiomyopathy, deafness and many neurological disorders). Potent and selective myosin inhibitors are, therefore, not only a tool for understanding myosin function, but are also a resource for developing treatments for diseases involving myosin dysfunction or overactivity. This review will provide a brief overview of the characteristics and scientific/therapeutic applications of the presently identified small-molecule myosin inhibitors before discussing the future of myosin inhibitor and activator design.

Cellular signaling pathways underlie the transfer of information throughout the cell and to adjoining cells and so govern most critical cellular functions. Increasing evidence points to the molecular motor myosin 1c as a prominent player in many signaling cascades, from the integrin-dependent signaling involved in cell migration to the signaling events underlying insulin resistance. Myosin 1c functions on these pathways both via an important role in regulating lipid raft recycling and also via direct involvement in signaling cascades. This review provides an overview of the functional involvement of myosin 1c in cellular signaling and discusses the possible potential for myosin 1c as a target for drug-based treatments for human diseases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The actin-based molecular motor myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the myosin VI-binding partner Dab2 and is identical for full-length myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of myosin VI on clathrin-coated structures suggests that wild type myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of myosin VI.

Myosin-6 is an actin-based motor protein that moves its cargo towards the minus-end of actin filaments. Mutations in the gene encoding the myosin-6 heavy chain and changes in the cellular abundance of the protein have been linked to hypertrophic cardiomyopathy, neurodegenerative diseases, and cancer. Here, we present a detailed kinetic characterization of the human myosin-6 motor domain, describe the effect of 2,4,6-triiodophenol on the interaction of myosin-6 with F-actin and nucleotides, and show how addition of the drug reduces the number of myosin-6-dependent vesicle fusion events at the plasma membrane during constitutive secretion., (Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.)

In eukaryotes, the final steps in both the regulated and constitutive secretory pathways can be divided into four distinct stages: (i) the 'approach' of secretory vesicles/granules to the PM (plasma membrane), (ii) the 'docking' of these vesicles/granules at the membrane itself, (iii) the 'priming' of the secretory vesicles/granules for the fusion process, and, finally, (iv) the 'fusion' of vesicular/granular membranes with the PM to permit content release from the cell. Recent work indicates that non-muscle myosin II and the unconventional myosin motor proteins in classes 1c/1e, Va and VI are specifically involved in these final stages of secretion. In the present review, we examine the roles of these myosins in these stages of the secretory pathway and the implications of their roles for an enhanced understanding of secretion in general.

During constitutive secretion, proteins synthesized at the endoplasmic reticulum (ER) are transported to the Golgi complex for processing and then to the plasma membrane for incorporation or extracellular release. This study uses a unique live-cell constitutive secretion assay to establish roles for the molecular motor myosin VI and its binding partner optineurin in discrete stages of secretion. Small interfering RNA-based knockdown of myosin VI causes an ER-to-Golgi transport delay, suggesting an unexpected function for myosin VI in the early secretory pathway. Depletion of myosin VI or optineurin does not affect the number of vesicles leaving the trans-Golgi network (TGN), indicating that these proteins do not function in TGN vesicle formation. However, myosin VI and optineurin colocalize with secretory vesicles at the plasma membrane. Furthermore, live-cell total internal reflection fluorescence microscopy demonstrates that myosin VI or optineurin depletion reduces the total number of vesicle fusion events at the plasma membrane and increases both the proportion of incomplete fusion events and the number of docked vesicles in this region. These results suggest a novel role for myosin VI and optineurin in regulation of fusion pores formed between secretory vesicles and the plasma membrane during the final stages of secretion.

The inflexibility of double-stranded DNA with respect to bending and twisting is well established in vitro. Understanding apparent DNA physical properties in vivo is a greater challenge. Here, we exploit repression looping with components of the Escherichia coli lac operon to monitor DNA flexibility in living cells. We create a minimal system for testing the shortest possible DNA repression loops that contain an E. coli promoter, and compare the results to prior experiments. Our data reveal that loop-independent repression occurs for certain tight operator/promoter spacings. When only loop-dependent repression is considered, fits to a thermodynamic model show that DNA twisting limits looping in vivo, although the apparent DNA twist flexibility is 2- to 4-fold higher than in vitro. In contrast, length-dependent resistance to DNA bending is not observed in these experiments, even for the shortest loops constraining <0.4 persistence lengths of DNA. As observed previously for other looping configurations, loss of the nucleoid protein heat unstable (HU) markedly disables DNA looping in vivo. Length-independent DNA bending energy may reflect the activities of architectural proteins and the structure of the DNA topological domain. We suggest that the shortest loops are formed in apical loops rather than along the DNA plectonemic superhelix.

Purpose: Interventions to improve school ethos can reduce substance use but "upstream" causal pathways relating to implementation and school-level changes are uncertain. We use qualitative and quantitative data from a pilot trial to build hypotheses regarding these., Methods: The Healthy School Ethos intervention involved two schools being provided with facilitation, training, and funding to plan and implement actions (some mandatory and some locally determined) to improve school ethos over one year. The evaluation involved a pilot-trial with two intervention and two comparison schools; semi-structured interviews with facilitators, staff, and students; and baseline and follow-up surveys with students aged 11 to 12 years., Results: Student accounts linked participation in planning or delivering intervention activities with improved self-regard and relationships with staff and other students. Some activities such as re-writing school rules involved broad participation. Students in receipt of actions such as peer-mediation or motivational sessions reported benefits such as improved safety and relationships. Some student accounts linked improved self-regard and relationships with increased engagement and aspirations, and reduced substance use. At 9-month follow-up, students in intervention schools reported less hurting and teasing of others and feeling unsafe at school. Other outcomes suggested intervention benefits but were not significant., Conclusions: School-ethos interventions may reduce substance use through upstream pathways involving the aforementioned factors. Future phase-III trials should quantitatively model the extent to which these mediate intervention effects., (Copyright © 2010 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.)

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry-based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein-specific siRNA screen (38 SNAREs, 4 SNARE-like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post-Golgi SNAREs SNAP-29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP-29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP-29-depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19-interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP-23, SNAP-25, SNAP-29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post-Golgi-specific R-SNARE in this process.

Objective: To contrast the psychosocial profile of adolescents with risk factors for homelessness, identified using Chamberlain and MacKenzie's self-report scale, compared to the profiles of homeless adolescents., Methods: Multinomial logistic regression analyses were conducted contrasting profiles for (a) 137 homeless adolescents, (b) 766 secondary students reporting risk factors for homelessness, and (c) 4,844 students not reporting risks for homelessness., Results: Fourteen percent of a representative population of at-school adolescents, from Victoria, Australia, showed elevated risk of homelessness. These adolescents showed depressive symptoms at least equivalent to homeless adolescents (RR 6.0, 95% CI: 4.9, 7.3, and RR 3.5, 95% CI: 2.1, 5.8, respectively). In multivariate analyses, homeless and at risk adolescents reported equivalent levels of family conflict, early problem behaviour and low opportunities and rewards for family involvement. Compared to adolescents not at risk, at risk adolescents were more likely to be female and to show poorer social skills/assertiveness and depressive symptoms. Compared to at risk adolescents, homeless adolescents showed additional family, school, peer and individual risks, but lower depressive symptomatology., Conclusions: The findings highlight the potential we have to quickly and simply detect adolescents showing significant risk of homelessness. This sizable minority of adolescents report risks often equivalent to homeless adolescents. It is hoped that stakeholders working with young people will utilise this screening potential to identify and intervene effectively with this significant subpopulation of youth, and their families, while they are still at home and school.

Objective: To determine the extent to which comprehensive health screening of adolescents was undertaken in a tertiary inpatient setting., Design and Setting: Retrospective review of 100 consecutive medical records of 13-18-year-old adolescents admitted to The Royal Children's Hospital, Melbourne (first 20 consecutive admissions in 2001 to each of five units--general medicine, adolescent medicine, specialty medicine, general surgery, and specialty surgery)., Main Outcome Measures: Documentation of screening for biomedical (height, weight, pubertal staging, and hepatitis B vaccination) and psychosocial concerns (HEADSS framework categorised into four screening levels--none, incomplete, adequate, thorough). Risks identified and actions taken., Results: Weight was recorded for 98 patients, height for 17, pubertal staging for 12, and hepatitis B vaccination status for nine. Documentation of psychosocial screening was absent from 62 charts, inadequate in 29, thorough in three, and complete in seven charts. Adolescent medicine inpatients were more likely than patients in other units to have any screening of psychosocial risk recorded and more likely to be thoroughly screened (P < 0.005). Screening was more often documented for less sensitive issues (eg, home, tobacco) than higher risk behaviours (eg, illicit drug use) (P = 0.013). When screening identified risks, appropriate action was undertaken in most cases., Conclusions: This study highlights deficiencies in comprehensive health screening in adolescents admitted to a tertiary children's hospital. These results support the development of more consistent approaches to screening adolescent inpatients.

Clinical observations have shown that patients arriving in the PACU who had no experience using a pain scale had more difficulty rating their pain in the immediate postoperative period. The purpose of this pilot study was to determine if preoperative instruction on the use of a pain scale would improve the patient's ability to self-report pain in the Phase I PACU. The sample consisted of 50 English speaking, orthopedic patients between the ages of 19 and 75 years. A visual numerical rating scale (NRS) for pain was used to teach patients in the holding area. Twenty-six of these patients had previous experience with the NRS and 24 had no experience. Of those with experience, 21 (80%) could use the pain scale in the PACU. Of the 24 patients who were taught in the holding area, 20 (85%) could use the pain scale to rate their pain in the PACU. The results of this study suggest that if patients have previous experience with a pain scale, or if they are taught preoperatively, they can more effectively self-report pain postoperatively. Future studies should be conducted to involve a larger sample, a variety of surgeries, and non-English-speaking patients.

Research into adolescent health issues is hampered by absolute requirements for parental consent. Society's recognition of adolescents' autonomy and decision-making capacity has been embodied in the legal recognition of the mature minor's right to make decisions on matters affecting his or her life. Psychological research indicates that young people from 14 years have decision-making capacity. US and UK research ethics guidelines acknowledge the mature-minor principle, but Australian guidelines are out of step with international practice. An absolute requirement for parental consent in Australian research ethics guidelines is potentially unethical if it denies mature adolescents' autonomy and is a barrier to participation, study validity and improved health outcomes through research findings. There are grounds for considering a mature-minor clause in the National Health and Medical Research Council research ethics guidelines, particularly in the context of youth participation in minimal-risk research.

Background/purpose: Hospital mortality rate among children with hypoplastic left heart syndrome (HLHS) after cardiac repair is well documented, but comparable data after noncardiac, surgical procedures are unknown. The authors hypothesized an increasing number of noncardiac procedures were being performed on children with HLHS, less than 2 years of age, from 1988 to 1997, and that these procedures were associated with a substantial mortality rate., Methods: A retrospective review of hospital discharge data for 2,457 children less than 2 years of age with HLHS for 1988 through 1997 was performed. The authors examined the outcomes of HLHS children undergoing only noncardiac surgical procedures during their hospital stay. Differences in hospital mortality rates between 1988 through 1992 versus 1993 through 1997 were assessed using the Chi2 square statistic., Results: Nineteen percent of the 147 children with HLHS undergoing noncardiac, surgical procedures died (95% CI, 13% to 25%). Comparing the 2 study periods, there was no significant change in outcome among HLHS children undergoing noncardiac, surgical procedures (78% v. 83%; P >.1). There was no significant difference in the percentage of hospital discharges with noncardiac, surgical procedures performed per year., Conclusions: Although children with HLHS were not undergoing an increase in the number of noncardiac surgical procedures performed annually, even minor surgical procedures were associated with considerable mortality. Outcomes after noncardiac surgery in high-risk children with congenital heart disease warrant further investigation., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

A 73-year-old man with a history of postpneumonectomy empyema and a long-term chest tube since 1979 presented with fever, chills, leukocytosis, and purulent fluid from the left tube thoracostomy. CT scan and bronchoscopy demonstrated a right lower lobe pneumonia and a left mainstem dehiscence with direct communication to the left tube thoracostomy. He underwent primary closure of the bronchopleural fistula with latissimus dorsi muscle flap coverage after antibiotic therapy for right lower lobe pneumonia.

A 13-year-old Haitian girl presented with complex congenital heart disease consisting of transposition of the great arteries with total anomalous venous return. The patient exhibited clinical findings of long-standing congestive heart failure and cyanosis. She underwent complete repair with resolution of her symptoms.

We report the case of a 29-year-old man who suffered sub-arachnoid bleeding while stabilized on a biventricular assist device as a bridge to cardiac transplantation. We adjusted his anti-coagulation therapy to control the bleeding and to concurrently minimize thrombosis while on support. He underwent 2 craniotomy operations to evacuate sub-arachnoid hematomas, and he underwent a subsequent operation to debride and close the dura. Eighteen days later, he underwent successful orthotopic heart transplant and was discharged to home 3 weeks post-transplant.

Background: Early implantation of centrifugal devices in patients with postcardiotomy cardiogenic shock may provide a bridge to recovery and allow subsequent long-term survival., Methods: Since January 1989, 62 patients were supported with centrifugal pumps because of failure to wean from cardiopulmonary bypass. Indications were postcardiotomy cardiogenic shock (PCCS) (n = 60), bridge to cardiac retransplantation (n = 1), and right ventricular failure (n = 1). Patients' ages ranged from 23 to 78 years; 40 were men (65%), and 22 were women (35%). Twenty-two patients (35%) had a left ventricular assist device; 9 patients (15%) had a right ventricular assist device; and 31 patients (50%) had a biventricular assist device. Length of support ranged from 1 day to 19 days., Results: Forty-two patients (68%) were weaned successfully; 27 patients survived to discharge (44%). Complications included bleeding (n = 41, 66%), renal failure (n = 28, 45%), and respiratory failure (n = 26, 42%). Currently, 23 patients survived 10 or more years (n = 1), 6 to 10 years (n = 7), 1 to 5 years (n = 10), and less than 1 year (n = 5)., Conclusions: Centrifugal pumps are available, easy to use, and relatively inexpensive. Our experience justifies their continued use as a bridge to recovery for patients with postcardiotomy cardiogenic shock, despite the availability and increasing use of more expensive devices.

A 29-year-old gravid female presented at 22 weeks gestation with an acute Type I aortic dissection and coarctation of the aorta. She underwent emergent repair of her aortic dissection using cardiopulmonary bypass and hypothermia. At 25 weeks gestation, she underwent repair of her coarctation of the aorta. The patient had a cesarean delivery of a viable, normal male infant at 39 weeks gestation.

Allograft valves have been used in cardiac valve replacement for over 40 years. Early failures of allograft mitral valve replacement (AMVR) were attributed to inadequate allograft preservation or unsuccessful implantation techniques. Due to an improved understanding of mitral valve function gained from experience in mitral valve reconstruction, improved imaging techniques for preoperative assessment, and improved methods of allograft preservation, there has been a resurgence of interest in AMVR. The use of bioprosthetic valves in children and young adults is characterized by reduced durability and early valve failure. With the use of mechanical valves, there is a significant long-term risk of morbidity and mortality resulting from thrombosis or hemorrhagic complications from anticoagulation. Additionally, anticoagulation is often difficult to manage in children. The advantages of using allograft mitral valves include the avoidance of anticoagulation and the preservation of the subvalvar apparatus and its role in ventricular function. At our institution, successful early results with AMVR have been obtained in eight children and young adults with previous atrioventricular septal defects, previous placement of mitral valve prostheses, rheumatic valvular disease, and bacterial endocarditis. Our early results and those of others are encouraging for the continued use of AMVR in children and young adults. Long-term follow-up will be necessary to assess the durability and function of these allograft mitral valves. Copyright 1999 by W.B. Saunders Company

The Norwood stage I procedure is often used for the initial treatment of infants with hypoplastic left heart syndrome. This procedure creates a systemic arterial to pulmonary artery shunt to establish pulmonary blood flow. We describe a method to facilitate placement of this shunt by attaching a polytetrafluoroethylene shunt to a pulmonary artery homograft patch before performing the median sternotomy. This technique facilitates the performance of the proximal shunt anastomosis and expedites the procedure.

Background: Recent reports have demonstrated successful early outcomes using mitral valve homografts in adults. We report our early results after homograft mitral valve replacement in 4 children with previous atrioventricular septal defects, previous placement of a prosthetic valve, and rheumatic valvular disease., Methods: Between May 1996 and June 1997, 4 children (ages 5, 11, 13, and 15 years) underwent mitral valve replacement with cryopreserved mitral valve homografts at our institution. Preoperative echocardiography confirmed moderately severe to severe mitral regurgitation, stenosis, or both in all 4 patients., Results: Successful homograft valve replacement was achieved in all 4 patients. Based on symptoms, physical examinations, and echocardiographic follow-up, all four homograft mitral valves are functioning well with normal hemodynamics. None of these patients are receiving warfarin. Follow-up has been limited to 10 months., Conclusions: In children requiring mitral valve replacement, the use of mitral valve homografts offers advantages over prosthetic valves, such as the avoidance of complications associated with thrombosis and anticoagulation. Homograft mitral valve replacement is technically feasible in children with congenital and rheumatic heart disease and previous prosthetic valves.

Objective: To ascertain the effectiveness of a home vaccination service for children behind in their vaccination schedule., Design: Randomised controlled trial of nurse-administered vaccination at home. Children were allocated at random to the intervention or the control group before any contact with the parents was made., Setting: 10 council areas in north-west metropolitan Melbourne defined by 56 postcode zones. Six-week intervention period from November 1996., Participants: 405 children--all those in the study area (n = 2610) 90 days late (age 9 months) for their third diphtheria-tetanus-pertussis/poliomyelitis/Haemophilus influenzae type B (DTP/OPV/Hib) vaccination, or 120 days late (age 16 months) for their measles-mumps-rubella (MMR) vaccination, according to the Australia Childhood Immunisation Register., Main Outcome Measures: Number of children completing DTP/OPV/Hib or MMR during the intervention period, and number up to date before intervention., Results: Verification of vaccination status with the parents revealed that 123 (60%) of the children in the intervention group and 113 (56%) of those in the control group were up to date with their vaccinations, leaving a study population of 81 (intervention group) and 88 (control group). Vaccination was achieved in 46 (57%) intervention children and 24 (27%) control children (risk ratio [RR], 2.08; 95% CI, 1.4-3.1; P < 0.001). For DTP/OPV/Hib, 18/32 (56%) intervention children and 12/36 (33%) control children were vaccinated, (P = 0.06). For MMR, 28/49 (57%) and 12/52 (23%) children were vaccinated, respectively (P < 0.001). Home vaccinations were completed with 26 families (including five siblings). The average cost per child vaccinated as a result of the home program was $92.52., Conclusion: Home vaccination for children behind in their immunisation schedule is an effective, acceptable and relatively cheap method of completing recommended vaccinations. We recommend that a home vaccination program be widely implemented and made available, particularly for disadvantaged families.

Study Objectives: Studies estimating the cost of specific illnesses do not generally take into account the fact that health care costs would have been incurred in the absence of the disease of interest. The goal of this study was to develop a method of estimating age specific baseline health care costs. These costs were calculated for Australian men, and their magnitude was compared with the costs of caring for men with HIV infection., Design: Information about health service usage was obtained from the 1989-90 national health survey and linked with data on the costs of services to obtain average monthly costs for individual and total health services., Setting: The Australian community., Participants: Average total health service costs per man per month were $103 (Australian). Hospital admissions comprised approximately 40% of these costs and casualty/outpatient visits, consultations with a doctor, and prescribed medication comprised 10%, 13%, and 12%, respectively. Costs increased with age, from around $60 per month for men aged 20-39 years to $213 per month for men aged 60 and over., Conclusion: Baseline costs comprised around 18% of health care costs for men with asymptomatic HIV infection, but less than 1% of costs for men with AIDS. These estimates provide an essential baseline for determining the costs attributable to specific diseases.

Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease caused by a defective CD18 gene. The cell-surface glycoprotein encoded by this gene CD18 is normally expressed in cells of the hematopoietic system. An in vitro murine model of CD18 gene replacement therapy was developed to investigate the feasibility of an in vivo murine hematopoietic stem cell gene therapy model. Human CD18-transducing retroviruses were used to transfer a functional human CD18 gene into a variety of cells including (i) murine lymphoblasts (which express murine CD11a and murine CD18), (ii) murine fibroblasts (which have no endogenous murine CD11a/CD18 expression), and (iii) murine fibroblasts, which have been stably transfected with a human CD11a gene. In murine lymphoblasts, human CD18 was expressed on the cell surface as a heterodimer with murine CD11a. Cell-surface expression of human CD18 had no apparent effect on the level of endogenous murine CD11a/CD18 expression. Immunoprecipitation of cell-surface labeled proteins in murine lymphoblasts with a human CD18 specific antibody co-precipitated murine CD11a. Human CD18 can be detected by immunochemistry in the cytoplasm of fibroblasts infected with CD18 encoding retrovirus, but coexpression with CD11a is required for cell-surface expression of either subunit in fibroblasts. These studies suggest that human CD18 will form a heterodimer with murine CD11a and that human CD18 is not expressed on the cell surface of cells not expressing CD11. This provides the basis for the development of a murine hematopoietic stem cell gene replacement therapy model for the treatment of LAD.