A proteolytic nanobiocatalyst with built-in disulphide reducing properties

We report a method to equip proteolytic nanobiocatalysts with intrinsic disulphide bond reducing properties. After immobilisation onto silica particles, selected protease enzymes are partially shielded in a nanometre-thick mercaptosilica layer acting not only as a protective system but also as a substrate reducing agent. The biocatalysts produced efficiently perform simultaneous disulphide bond reduction and protein digestion. Besides a significant simplification of the proteolysis process, this strategy allows for a drastic increase of the enzyme stability.

Proteolytic nanobiocatalysts are equipped with intrinsic disulphide bond reducing properties.     

Management of chronic neuropathic pain with methadone: a review of 13 cases

The synthetic opioid methadone has generated much interest in recent years among clinicians involved in the management of intractable chronic cancer pain. Its use as an analgesic is starting to extend to the treatment of noncancer pain, particularly neuropathic pain. Unfortunately, the evidence for its use in the management of neuropathic pain is limited to a few case studies. We examined retrospectively during a 12-month study period the clinical response of all 13 patients at our pain clinic who were prescribed methadone in an attempt to control neuropathic pain resistant to conventional analgesics. A questionnaire was also administered to the 9 patients who continued to take methadone at 12 months posttreatment. A total of 4 patients (31%) discontinued it by the end of the 12-month study period. Patients discontinued methadone due to the absence of pain relief and due to various intractable, undesirable side effects. Somnolence was the most common adverse effect reported, followed by nausea, constipation, and vomiting. All patients took coanalgesics (eg, amitriptyline, gabapentin) or other analgesics (eg, morphine, nonsteroidal anti-inflammatory drugs) during methadone treatment to control pain. The 9 patients who continued to take methadone at 12 months reported experiencing on average 43% pain relief (range 0-80%), 47% improvement in quality of life (range 0-100%), and 30% improvement in quality of sleep (range 0-60%). Methadone was effective at relieving pain and ameliorating quality of life and sleep in 62% of patients. These findings suggest that methadone can offer an acceptable success rate for the treatment of neuropathic pain. Prospective randomized, placebo-controlled studies are now needed to examine more rigorously the benefits of methadone for this type of pain.     

Ultimate journey of the terminally ill: Ways and pathways of hope

ObjectiveTo better understand the role of hope among terminally ill cancer patients.DesignQualitative analysis.SettingA tertiary specialized cancer centre in Canada.ParticipantsCancer patients in palliative care with an estimated remaining life expectancy of 12 months or less (N = 12) and their loved ones (N = 12) and treating physicians (N = 12).MethodsEach patient underwent up to 3 interviews and identified a loved one who participated in 1 interview. Treating physicians were also interviewed. All interviews were fully transcribed and analyzed by at least 2 investigators. Interviews were collected until saturation occurred.ConclusionApproaches aimed at sustaining hope need to reflect that patients’ reactions might fluctuate between despair and a form of acceptance that leads to a certain serenity. Clinicians need to maintain some degree of hope while remaining as realistic as possible. The findings also raise questions about how hope influences patients’ perceptions and acceptance of their treatments.     

Collagen‐mediated hemostasis

Collagens mediate essential hemostasis by maintaining the integrity and stability of the vascular wall. Imbalanced turnover of collagens by uncontrolled formation and/or degradation may result in pathologic conditions such as fibrosis. Thickening of the vessel wall because of accumulation of collagens may lead to arterial occlusion or thrombosis. Thinning of the wall because of collagen degradation or deficiency may lead to rupture of the vessel wall or aneurysm. Preventing excessive hemorrhage or thrombosis relies on collagen‐mediated actions. Von Willebrand factor, integrins and glycoprotein VI, as well as clotting factors, can bind collagen to restore normal hemostasis after trauma. This review outlines the essential roles of collagens in mediating hemostasis, with a focus on collagens types I, III, IV, VI, XV, and XVIII.     

Unconjugated bile salts shuttle through hepatocyte peroxisomes for taurine conjugation

Bile acid-CoA:amino acid N-acyltransferase (BAAT) conjugates bile salts to glycine or taurine, which is the final step in bile salt biosynthesis. In addition, BAAT is required for reconjugation of bile salts in the enterohepatic circulation. Recently, we showed that BAAT is a peroxisomal protein, implying shuttling of bile salts through peroxisomes for reconjugation. However, the subcellular location of BAAT remains a topic of debate. The aim of this study was to obtain direct proof for reconjugation of bile salts in peroxisomes. Primary rat hepatocytes were incubated with deuterium-labeled cholic acid (D(4)CA). Over time, media and cells were collected and the levels of D(4)CA, D(4)-tauro-CA (D(4)TCA), and D(4)-glyco-CA (D(4)GCA) were quantified by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Subcellular accumulation of D(4)-labeled bile salts was analyzed by digitonin permeabilization assays and subcellular fractionation experiments. Within 24 hours, cultured rat hepatocytes efficiently (>90%) converted and secreted 100 μM D(4)CA to D(4)TCA and D(4)GCA. The relative amounts of D(4)TCA and D(4)GCA produced were dependent on the presence of glycine or taurine in the medium. Treatment of D(4)CA-exposed hepatocytes with 30-150 μg/mL digitonin led to the complete release of D(4)CA, D(4)GCA, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cytosolic marker). Full release of D(4)TCA, catalase, and BAAT was only observed at 500 μg/mL digitonin, indicating the presence of D(4)TCA in membrane-enclosed organelles. D(4)TCA was detected in fractions of purified peroxisomes, which did not contain D(4)CA and D(4)GCA. Conclusion: We established a novel assay to study conjugation and intra- and transcellular transport of bile salts. Using this assay, we show that cholic acid shuttles through peroxisomes for taurine-conjugation.     

Trends in Nontherapeutic Laparotomy Rates in Patients Undergoing Surgical Therapy for Hepatic Colorectal Metastases

Surgery is the treatment of choice in selected patients with hepatic colorectal metastases. Despite improvements in preoperative imaging, patients can undergo unnecessary nontherapeutic laparotomy. The aim of this study was to examine trends in nontherapeutic laparotomy rates in patients undergoing planned surgical therapy for hepatic colorectal metastases. Data from 530 operations (461 patients) undergoing potentially curative surgical therapy for colorectal liver metastases between 1994 and 2005 were analyzed. The incidence of nontherapeutic laparotomy was determined and factors associated with nontherapeutic laparotomy were identified. Overall, 49 nontherapeutic laparotomies were performed (9.2%). Higher nontherapeutic laparotomy rates were seen in patients with multiple metastases and tumor size >5 cm (both P < 0.05). Preoperative positron emission tomography (PET) imaging was associated with lower risk of nontherapeutic laparotomy [5.6% versus 12.4%, P = 0.009, odds ratio (OR) = 0.42]. At laparotomy, extrahepatic findings were the reason for nontherapeutic laparotomy in 44.9% of cases. The nontherapeutic laparotomy rate significantly decreased over time (14.9% for 1994–1997 versus 9.6% for 1998–2001 versus 4.7% for 2002–2005; P = 0.003). While patients in each time period were similar with regard to tumor specific factors, utilization of PET imaging (P < 0.001) as well as resection plus ablation (P = 0.004) increased over time. We conclude that prevalence of nontherapeutic laparotomy for patients undergoing surgical exploration for hepatic colorectal metastases has decreased significantly in recent years to less than 5%. The reasons for this trend are probably multifactorial and may include improved preoperative assessment, such as PET imaging, as well as salvage surgical options.     

Peptidoglycan increases firm adhesion of monocytes under flow conditions and primes monocyte chemotaxis

The Toll-like receptor (TLR) 2/nucleotide-binding oligomerization domain ligand peptidoglycan (PG) has been shown to be present in macrophage-rich regions within atherosclerotic lesions, and stimulation of TLR2 promotes atherosclerotic plaque and intima formation in in vivo mouse models. We determined the effect of a PG preparation and Pam(3)Cys-SK(4), a synthetic TLR2 activator, on (1) adhesion molecule expression by flow cytometry; (2) monocyte adhesion under flow conditions, and (3) monocyte migration. The total adhesion (rolling and firm adhesion) of the PG-preparation-stimulated monocytes to L cells, constitutively expressing ICAM-1 (intercellular adhesion molecule-1) and E-selectin, was decreased. This was most likely due to the L-selectin shedding, since monocyte incubation with a blocking L-selectin antibody resulted in a comparable number of adherent monocytes as PG-stimulated cells. The PG preparation induced an increased percentage of firmly adherent, polarized cells and a beta(2)-integrin-dependent binding to ICAM-1-coated beads. Interestingly, the PG preparation induced a priming of the monocytes for increased migration towards the chemoattractant C5a which was TLR2 and beta(2)-integrin dependent. Pam(3)Cys-SK(4) gave comparable results to the PG preparation in all assays tested. This study demonstrates that PG activation of monocytes results in an increase in adhesive and migratory capacities of these cells. This might be a mechanism by which PG promotes atherosclerotic disease in vivo.     

Specificity of the anti-glycolytic activity of 3-bromopyruvate confirmed by FDG uptake in a rat model of breast cancer

Summary   Purpose: To evaluate the anti-glycolytic effects of 3-BrPA on rats bearing RMT mammary tumors, by determining FDG uptake after intravenous administration of the therapeutic dose. Materials and Methods: Sixteen rats bearing RMT tumors were treated either with 15 mM 3-BrPA in 2.5 ml of PBS or with 2.5 ml of PBS. After treatment, all rats received FDG and were sacrificed 1 h later. Results: 3-BrPA treatment significantly decreased FDG uptake in tumors by 77% (p = 0.002). FDG uptake did not significantly decrease in normal tissues after treatment. Conclusion: Our study showed that 3-BrPA exhibits a strong anti-glycolytic effect on RMT cells implanted in rats. Buijs and Vossen contributed equally to this work.     

Validity of the Actical accelerometer step-count function

PURPOSE: To assess the validity of the new Actical accelerometer step count function. METHODS: Actical step counts were compared according to two criterion standards. 1) Eight Acticals were assessed using a mechanical shaker table under six different testing conditions. 2) Thirty-eight volunteers (aged 9-59 yr) wore eight Acticals and eight Actigraphs during treadmill walking (50 and 83 m.min(-1)) and running (133 m.min(-1)) for 6 min at each speed. Steps were counted during the second and fourth minutes of each speed by a trained observer. RESULTS: The correlation between Actical step counts and the mechanical shaker step counts was excellent (r = 1.0). Compared with visually counted steps, both the Actical and Actigraph step counts were significantly different at 50 m.min(-1); however, no significant differences were evident at 83 and 133 m.min(-1). The criterion-related validity correlations (r) for the Actical and Actigraph, respectively, were 0.73 and 0.52 at the slow walk condition and 0.99 and 0.99 at the normal walk and run conditions. CONCLUSION: The new step count function of the Actical accelerometer provides valid estimates of step counts at 83 and 133 m.min(-1) on a range of healthy participants.