Topological analysis of Hedgehog acyltransferase, a multipalmitoylated transmembrane protein.

Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo.

Effects of the Percutaneous Carbon Dioxide Therapy on Post-surgical and Post-traumatic Hematoma, Edema and Pain.

Introduction: Carbon dioxide therapy (CDT) has been used since the Roman Empire to improve the microcirculation and oxygenation of tissues altered. The classical indications are vasculopathies and ischemic diseases. Hypothesis: The CDT could be effective in favoring joint mobility recovery and pain reduction in patients with post-surgical or post-traumatic hematoma, edema, and pain. Study Design: Case report. Material and Methods: Eight patients were treated once a day for 5-10 days; a single session lasted 50 min. For post-surgical cases, the treatment began the day or the day after they were discharged from the hospital. Result: For all patients in this series, the CDT has brought clinical benefits in terms of decreasing pain and improving of joint mobility. Conclusion: The CDT is a safe and effective treatment to provide a greater amount of oxygen to the injured tissues. It favors the healing of post-surgical and post-traumatic hematoma and edema, promoting the mobility recovery of patients.

Partial Denervation for Neuroma Pain after Knee Anterior Tibial Tuberosity Exostectomy: A Case Report.

INTRODUCTION: Neuromas of the infrapatellar branch of the saphenous nerve (IBSN) can occur after direct trauma or surgical approaches to the anterior aspect of the knee. The purpose of this case report is to highlight the clinical presentation, diagnosis, and successful surgical management. CASE REPORT: A 20-year-old patient who had a history of recent anterior knee surgery at another institution presented with allodynia, dysesthesia, and a positive Tinel's test over the surgical scar. The pain was exacerbated by light touch, was progressively worse, and was limiting sports participation and activities of daily living. A clinical diagnosis of neuroma of the IBSN was made and this was successfully treated by microsurgical neurolysis, resection of the neuroma, and burying of the stump. This resulted in complete resolution of pain. CONCLUSION: Neuromas of the IBSN can cause significant symptoms affecting activities of daily living. Microsurgical neurolysis is a useful treatment modality associated with complete resolution of pain and high patient satisfaction.

Insulin-like growth factors and their binding proteins in the circulation of patients with echinococcosis, trichinellosis and toxoplasmosis.

BACKGROUND: Insulin-like growth factors (IGFs) are polypeptide hormones that play anabolic roles in cellular growth and metabolism. Their activity is regulated by binding proteins (IGFBPs) and degradation mechanisms. The liver is regarded as the main source of circulating forms and the levels change in various disease states. The aim of the study was to explore the effects of parasitic infections on the circulating IGFs and IGFBPs. METHODS: Peptide concentrations in sera of patients with echinococcosis, trichinellosis and toxoplasmosis were measured by radioimmunoassay, while IGFBP patterns were characterised by ligand-affinity blotting and gel chromatography. RESULTS: IGF-I levels were reduced in trichinellosis (p=0.016), with the increased relative amounts of IGFBP-1, -2 and -4. The significantly reduced levels of IGF-I (p<0.001), IGF-II (p=0.017) and IGFBP-3 and the increased presence of IGFBP-2 were found in patients with echinococcosis. CONCLUSIONS: In subjects with trichinellosis probably the combined effects of inadequate nutrition and the immunological response occurred. A possible explanation for the changes found in patients with echinococcosis is that the liver is a frequent target organ for Echinococcus granulosus. Since liver cysts were present in these patients, it can be expected that hepatic injury may affect liver metabolism leading to altered IGF/IGFBP profiles.

Attenuation of hedgehog acyltransferase-catalyzed sonic Hedgehog palmitoylation causes reduced signaling, proliferation and invasiveness of human carcinoma cells.

Overexpression of Hedgehog family proteins contributes to the aetiology of many cancers. To be highly active, Hedgehog proteins must be palmitoylated at their N-terminus by the MBOAT family multispanning membrane enzyme Hedgehog acyltransferase (Hhat). In a pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1 and transfected HEK293a cells Hhat localized to the endoplasmic reticulum. siRNA knockdown showed that Hhat is required for Sonic hedgehog (Shh) palmitoylation, for its assembly into high molecular weight extracellular complexes and for functional activity. Hhat knockdown inhibited Hh autocrine and juxtacrine signaling, and inhibited PDAC cell growth and invasiveness in vitro. In addition, Hhat knockdown in a HEK293a cell line constitutively expressing Shh and A549 human non-small cell lung cancer cells inhibited their ability to signal in a juxtacrine/paracrine fashion to the reporter cell lines C3H10T1/2 and Shh-Light2. Our data identify Hhat as a key player in Hh-dependent signaling and tumour cell transformed behaviour.

Bioorthogonal chemical tagging of protein cholesterylation in living cells.

We report the first chemical probe for bioorthogonal chemical tagging of post-translationally cholesterylated proteins with an azide in living cells. This enables rapid multiplexed fluorescence detection and affinity labelling of protein cholesterylation, as exemplified by Sonic hedgehog protein, opening up new approaches for the de novo identification of cholesterylated proteins.

Hand hygiene practices and adherence determinants in surgical wards across Europe and Israel: a multicenter observational study.

We examined hand hygiene practices in surgical wards in 9 countries in Europe and Israel through direct practice observation. There was marked interhospital variation in hand hygiene compliance (range, 14%-76%), as well as glove and alcohol-based handrub use. After multivariable analysis, surgical subspecialty, professional category, type of care activity, and workload were independently associated with compliance. Hand hygiene practices are influenced by numerous factors, and a tailored approach may be required to improve practices.

[Risk factors for the appearance of central venous catheters colonisation].

INTRODUCTION/AIM: Intravascular device placement (IVD) is a part of everyday medical practice, however, its application is associated with a high risk of onset of nosocomial infections (NI) and increased mortality and morbidity. Nosocomial blood infections (NBIs) account for 10% of all the registered NI. NBIs are more frequent in patients with a placed IVD and it present an important risk factor for the onset of NBI, i.e. catheter-associated NBIs (CANBIs). Pathogenesis of CANBIs is complex and conditioned by the presence of different characteristics related to a catheter, patient and a specific causative organism. The most common CRBSI causes include coagulase-negative staphylococcus, S. aureus, Enterobacter spp, Candida spp, Klebsiella spp, Pseudomonas spp. and Enterococcus spp. METHODS: All the patients hospitalized at the Intensive Care Department of the Clinic of Digestive Diseases over the period January 1, 2004-September 1, 2004 were retrospectively analyzed. The study included 107 patients in whom central venous catheter (CVC) was placed for more than 48 h. All the causes isolated from a CVC segment were recorded. Culture, isolation and identification of the causative organisms were performed using standard microbiological methods in the Bacteriological Laboratory within the Emergency Center, Clinical Center of Serbia. Catheter segment samples (tip of the CVC 3-5 cm long) were analyzed. Based on the insight into medical documentation, patients' examination and medical staff interview, catheter and patient-related characteristics were recorded. RESULTS: A total of 107 CVCs were analyzed, out of which 56 (52%) were sterile while 51 (48%) were colonized. The results of our study evidenced that total parenteral nutrition (TPN) (p < 0.05), number of catheterization days (p < 0.05), and central venous pressure measurement (p < 0.05) were significantly associated with CVC colonization. In this study, no statistically significant difference in catheter colonization was found with respect to sex, age, anatomical insertion site and CVC placement site. CONCLUSION: According to the results of our study, TPN, the number of catheterization days and measurement of central venous pressure play major roles in colonization of CVC. Understanding risk factors associated with CVC colonization and onset of CANBIs is a prerequisite for quality preventive work of health professionals.

[Nosocomial infections in the intensive care units].

BACKGROUND/AIM: The risk for nosocomial infections (NIs) is 5-10 times higher in patients hospitalized in the Intensive Care Units (ICUs) than in patients staying in other wards. The higher incidence rates of NIs in the ICUs may be explained by the fact that the patients in the ICUs have more severe underlying disease, and are exposed to the invasive diagnostic and therapeutical procedures. The unreasonable use of antibiotics leads to the selection of multiresistant agents, which have been increasingly recorded as the NIs causative agents. The aim of this study was to investigate the characteristics of NIs in the ICUs in the period January-June 2005. METHODS: The study of incidence was performed in accordance with the methodology of the Centers for Diseases and Prevention. Any infections in the patients hospitalized in the ICUs in the period from January to June 2005 were registered. The results both from medical documentation and from the direct contacts with the medical personnel were analyzed. The samples were tested using standard methods in the microbiological laboratory. RESULTS: The incidence rates of NIs patients ranged from 1.5 to 40.8, and the incidence rates of infections were 1.5 to 65.6 per 1 000 patient's days. Out of the total number of NIs, urinary infections accounted for 44.6%, blood infections for 37.6%, and surgical site infections for 16.9%. CONCLUSION: The most frequent cause of nosocomial urinary infections was Klebsiella, of nosocomial sepsis--coagulase--negative staphylococci, and of surgical site infections--Staphylococcus aureus.

Insulin-like growth factors in patients with liver cysts.

Insulin-like growth factors (IGFs) play an important role in cell growth and differentiation, and the liver is the main source of IGFs and IGF-binding proteins (IGFBPs) that appear in the circulation. The effect of liver cysts on the circulating IGF system was studied in this work. Serum concentrations of IGF-I and -II were measured by radioimmunoassay, IGFBP patterns were characterised by ligand-affinity and immunoblotting, and a lectin-binding assay was used to investigate the glyco component of IGFBP-3 complexes. IGF-I and -II concentrations in patients with cysts were significantly lower compared to those in healthy individuals (P<0.0001 and P<0.01, respectively), and the decrease was related to age but not sex. The overall mean concentrations of IGF-I and -II were not significantly different whether the cysts were caused by Echinococcus granulosus, cross-reactive pathologies, or some other factor. IGFBP profiles correlated with the amount of IGF present: patients with lower IGF-I concentrations expressed decreased IGFBP-3 and elevated IGFBP-2 levels. Increased IGFBP-3 proteolytic activity in the patients' blood was not detected by immunoblotting. In the lectin-binding assay, IGFBP-3 complexes in the circulation of patients demonstrated reactivity similar to that in healthy persons, suggesting that the overall structure of the saccharide moieties of the IGFBP-3 complexes was not significantly altered due to liver cyst formation.