Efficiency based updating of evidence based surgical guidelines - Experiences from a multidisciplinary guideline upon Minimally Invasive Surgery.

Background: Updating evidence-based clinical practice guidelines is an onerous process and there is a call for more efficient determination of key questions that need updating. Especially for surgical techniques it is unclear if new evidence will result in substantial changes after wide implementation and if continuous updating is always necessary. Objectives: This study analyses the impact of updating a surgical guideline and proposes suggestions for optimising this process. Materials and methods: The Dutch Minimally Invasive Surgery guideline was developed in 2011 and updated in 2021. For both versions a multidisciplinary guideline working group (GDG) was created, that determined key questions. Changes in conclusions and recommendations were analysed by the GDG and statements for expected change of recommendations in the future were made. Results: 15 key questions were formed, of which 12 were updates of the previous guideline. For only 27% of the updated key questions, the conclusions changed. In ten years, the body grew only marginally for most key questions and quality of the evidence did not improve substantially for almost all key questions. However, in this first update of the MIC guideline, many recommendations did change due to a more robust interpretation of the conclusions by the GDG. Based on analysis of this updating process, the GDG expects that only four out of 15 recommendations may change in the future. Conclusion: We propose an additional step at the end of guideline development and updating, where the necessity for updating in the future is determined for each key question by the GDG, using their valuable knowledge gained from developing or updating the guideline. For surgical guidelines, the authors suggest updating key issues if it includes a relatively newly introduced surgical- or adapted technique or a new patient group. Low quality or small body of evidence should not be a reason in itself for updating, as this mostly does not lead to new evidence-based conclusions. This new step is expected to result in a more efficient prioritising of key questions that need updating. What's new?: By adding one additional step at the end of the updating process, the future updating process could become more efficient.

Further insights into the treatment of perineal hernia based on a the experience of a single tertiary centre.

AIM: There is little evidence concerning the optimal surgical technique for the repair of perineal hernia. This study aimed to report on the evolution of a technique for repair of perineal hernia by analysing the experience in a tertiary referral centre. METHOD: This was a retrospective review of consecutive patients who underwent perineal hernia repair after abdominoperineal excision in a tertiary referral centre. The main study end-points were rate of recurrent perineal hernia, perineal wound complications and related re-intervention. RESULTS: Thirty-four patients were included: in 18 patients a biological mesh was used followed by 16 patients who underwent synthetic mesh repair. Postoperative perineal wound infection occurred in two patients (11%) after biological mesh repair compared with four (25%) after synthetic mesh repair (P = 0.387). None of the meshes were explanted. Recurrent perineal hernia following biological mesh was found in 7 of 18 patients (39%) after a median of 33 months. The recurrence rate with a synthetic mesh was 5 of 16 patients (31%) after a median of 17 months (P = 0.642). Re-repair was performed in four (22%) and two patients (13%), respectively (P = 0.660). Eight patients required a transposition flap reconstruction to close the perineum over the mesh, and no recurrent hernias were observed in this subgroup (P = 0.030). No mesh-related small bowel complications occurred. CONCLUSION: Recurrence rates after perineal hernia repair following abdominoperineal excision were high, and did not seem to be related to the type of mesh. If a transposition flap was added to the mesh repair no recurrences were observed, but this finding needs confirmation in larger studies.

Comparison of complications and shocks in paediatric and young transvenous and subcutaneous implantable cardioverter-defibrillator patients.

BACKGROUND: Young implantable cardioverter-defibrillator (ICD) patients are prone to complications and inappropriate shocks (IAS). The subcutaneous ICD (S-ICD) may avoid lead-related complications. This study aims to describe the incidence and nature of device-related complications in young transvenous ICD (TV-ICD) and S­ICD patients. METHODS: Single-chamber TV-ICD and S­ICD patients up to and including the age of 25 years implanted between 2002 and 2015 were retrospectively analysed. Complications were defined as device-related complications requiring surgical intervention. IAS were defined as shocks for anything other than ventricular tachycardia or ventricular fibrillation. Follow-up data were collected 5 years post-implantation. Kaplan-Meier estimates for complications at 5­year follow-up were calculated with a corresponding 95% confidence interval. RESULTS: Eighty-one patients (46 TV-ICD, 35 S-ICD) were included (median age 19.0 (IQR 16.0-23.0) and 16.5 (IQR 13.0-20.2) years respectively). Median follow-up was 60 and 40 months respectively. All-cause complication rate was 34% in the TV-ICD group and 25% in the S­ICD group (p = 0.64). TV-ICD patients had more lead complications: 23% (10-36%) versus 0% (p = 0.02). The rate of infections did not differ between TV-ICD and S­ICD: 2% (0-6%) versus 10% (0-21%) (p = 0.15). No systemic infections occurred in the S­ICD patients. The rates of IAS were similar, TV-ICD 22% (9-35%) versus S­ICD 14% (0-30%) (p = 0.40), as were those for appropriate shocks: 25% (11-39%) versus 27% (6-48%) (p = 0.92). CONCLUSION: The rates of all-cause complications in this cohort were equal, though the nature of the complications differed. S­ICD patients did not suffer lead failures or systemic infections. An era effect is present between the two groups.

An Overview of Clinical Outcomes in Transvenous and Subcutaneous ICD Patients.

PURPOSE OF REVIEW: Clear guidelines on when to select a subcutaneous ICD (S-ICD) over a transvenous ICD (TV-ICD) are lacking. This review will provide an overview of the most recent clinical data on S-ICD and TV-ICD therapy by pooling comparison studies in order to aid clinical decision making. RECENT FINDINGS: Pooling of observational-matched studies demonstrated an incidence rate ratio (IRR) for device-related complication of 0.90 (95% CI 0.58-1.42) and IRR for lead-related complications of 0.15 (95% CI 0.06-0.39) in favor of S-ICD. The IRR for device infections was 2.00 (95% CI 0.95-4.22) in favor of TV-ICD. Both appropriate shocks (IRR 0.67 (95% CI 0.42-1.06)) and inappropriate shocks (IRR 1.17 (95% CI 0.77-1.79)) did not differ significantly between both groups. With randomized data underway, the observational data demonstrate that the S-ICD is associated with reduced lead complications, but this has not yet resulted in a significant reduction in total number of complications compared to TV-ICDs. New technologies are expected to make the S-ICD a more attractive alternative.

Accumulation of advanced glycation end products is associated with macrovascular events and glycaemic control with microvascular complications in Type 2 diabetes mellitus.

AIM: The United Kingdom Prospective Diabetes Study (UKPDS) study showed that glycaemic control (HbA1c ) can predict vascular complications in Type 2 diabetes mellitus. The Diabetes Control and Complications Trial (DCCT) study showed that accumulation of advanced glycation end products (AGEs) from skin biopsies predicts vascular complications in Type 1 diabetes. Previously, we showed that tissue AGEs can be measured non-invasively using skin autofluorescence (SAF). The aim of this study was to compare the predictive value of HbA1c and SAF for new macrovascular events and microvascular complications in people with Type 2 diabetes. METHODS: A prospective cohort study of 563 participants, median age 64 years [interquartile range (IQR) 57-72], diabetes duration of 13 years, from five Dutch hospitals was performed. RESULTS: After a median follow-up of 5.1 (IQR 4.3-5.9) years, 79 (15%) participants had died and 49 (9%) were lost to follow-up. Some 133 (26%) developed a microvascular complication and 189 (37%) a macrovascular event. Tertiles of HbA1c were significantly associated with development of microvascular complications (log rank P = 0.022), but not with macrovascular events. Tertiles of SAF were significantly associated with macrovascular events (log rank P = 0.003). Cox regression analysis showed SAF was associated with macrovascular events: crude hazard ratio (HR) 1.53 (P < 0.001) per unit increase, HR 1.28 (P = 0.03) after correction for UKPDS score. HbA1c was predictive for microvascular complications: crude HR 1.20 (P = 0.004), HR 1.20 (P = 0.004) after correction for UKPDS score. CONCLUSION: This study shows that tissue accumulation of AGEs, assessed by SAF, is associated with development of macrovascular events in people with Type 2 diabetes, whereas HbA1c is associated with the development of microvascular complications.

Combined leadless pacemaker and subcutaneous implantable defibrillator therapy: feasibility, safety, and performance.

AIMS: The subcutaneous implantable cardioverter-defibrillator (S-ICD) and leadless pacemaker (LP) are evolving technologies that do not require intracardiac leads. However, interactions between these two devices are unexplored. We investigated the feasibility, safety, and performance of combined LP and S-ICD therapy, considering (i) simultaneous device-programmer communication, (ii) S-ICD rhythm discrimination during LP communication and pacing, and (iii) post-shock LP performance. METHODS AND RESULTS: The study consists of two parts. Animal experiments: Two sheep were implanted with both an S-ICD and LP (Nanostim, SJM), and the objectives above were tested. Human experience: Follow-up of one S-ICD patient with bilateral subclavian occlusion who received an LP and two LP (all Nanostim, SJM) patients (without S-ICD) who received electrical cardioversion (ECV) are presented. Animal experiments : Simultaneous device-programmer communication was successful, but LP-programmer communication telemetry was temporarily lost (2 ± 2 s) during ventricular fibrillation (VF) induction and 4/54 shocks. Leadless pacemaker communication and pacing did not interfere with S-ICD rhythm discrimination. Additionally, all VF episodes (n = 12/12), including during simultaneous LP pacing, were detected and treated by the S-ICD. Post-shock LP performance was unaltered, and no post-shock device resets or dislodgements were observed (24 S-ICD and 30 external shocks). Human experience : The S-ICD/LP patient showed adequate S-ICD sensing during intrinsic rhythm, nominal, and high-output LP pacing. Two LP patients (without S-ICD) received ECV during follow-up. No impact on performance or LP dislodgements were observed. CONCLUSION: Combined LP and S-ICD therapy appears feasible in all animal experiments (n = 2) and in one human subject. No interference in sensing and pacing during intrinsic and paced rhythm was noted in both animal and human subjects. However, induced arrhythmia testing was not performed in the patient. Defibrillation therapy did not seem to affect LP function. More data on safety and performance are needed.

Unexplained False Negative Results in Noninvasive Prenatal Testing: Two Cases Involving Trisomies 13 and 18.

Noninvasive prenatal testing (NIPT) validation studies show high sensitivity and specificity for detection of trisomies 13, 18, and 21. False negative cases have rarely been reported. We describe a false negative case of trisomy 13 and another of trisomy 18 in which NIPT was commercially marketed directly to the clinician. Both cases came to our attention because a fetal anatomy scan at 20 weeks of gestation revealed multiple anomalies. Karyotyping of cultured amniocytes showed nonmosaic trisomies 13 and 18, respectively. Cytogenetic investigation of cytotrophoblast cells from multiple placental biopsies showed a low proportion of nontrisomic cells in each case, but this was considered too small for explaining the false negative NIPT result. The discordant results also could not be explained by early gestational age, elevated maternal weight, a vanishing twin, or suboptimal storage or transport of samples. The root cause of the discrepancies could, therefore, not be identified. The couples involved experienced difficulties in accepting the unexpected and late-adverse outcome of their pregnancy. We recommend that all parties involved in caring for couples who choose NIPT should collaborate to clarify false negative results in order to unravel possible biological causes and to improve the process of patient care from initial counseling to communication of the result.

Impact of rapid genetic counselling and testing on the decision to undergo immediate or delayed prophylactic mastectomy in newly diagnosed breast cancer patients: findings from a randomised controlled trial.

BACKGROUND: Female breast cancer patients with a BRCA1/2 mutation have an increased risk of contralateral breast cancer. We investigated the effect of rapid genetic counselling and testing (RGCT) on choice of surgery. METHODS: Newly diagnosed breast cancer patients with at least a 10% risk of a BRCA1/2 mutation were randomised to an intervention group (offer of RGCT) or a control group (usual care; ratio 2 : 1). Primary study outcomes were uptake of direct bilateral mastectomy (BLM) and delayed contralateral prophylactic mastectomy (CPM). RESULTS: Between 2008 and 2010, we recruited 265 women. On the basis of intention-to-treat analyses, no significant group differences were observed in percentage of patients opting for a direct BLM (14.6% for the RGCT group vs 9.2% for the control group; odds ratio (OR) 2.31; confidence interval (CI) 0.92-5.81; P=0.08) or for a delayed CPM (4.5% for the RGCT group vs 5.7% for the control group; OR 0.89; CI 0.27-2.90; P=0.84). Per-protocol analysis indicated that patients who received DNA test results before surgery (59 out of 178 women in the RGCT group) opted for direct BLM significantly more often than patients who received usual care (22% vs 9.2%; OR 3.09, CI 1.15-8.31, P=0.03). INTERPRETATION: Although the large majority of patients in the intervention group underwent rapid genetic counselling, only a minority received DNA test results before surgery. This may explain why offering RGCT yielded only marginally significant differences in uptake of BLM. As patients who received DNA test results before surgery were more likely to undergo BLM, we hypothesise that when DNA test results are made routinely available pre-surgery, they will have a more significant role in surgical treatment decisions.

Skin autofluorescence and risk of micro- and macrovascular complications in patients with Type 2 diabetes mellitus-a multi-centre study.

AIMS: Skin autofluorescence is a non-invasive marker of advanced glycation end product accumulation. In a previous study, skin autofluorescence correlated with and predicted micro- and macrovascular complications in Type 2 diabetes in a primary care setting. The present cross-sectional study aims to confirm the association between skin autofluorescence and diabetic complications in patients with Type 2 diabetes in a multi-centre secondary care setting. METHODS: We analysed 563 subjects with Type 2 diabetes mellitus from five Dutch hospitals. RESULTS: Median age was 64 years, median duration of diabetes 13 years and median HbA(1c) 58 mmol/mol (7.5%). Sixty-one per cent of patients had microvascular complications (38% nephropathy, 36% retinopathy, 35% neuropathy) and 42% had macrovascular complications. Median UK Prospective Diabetes Study 10-year risk for coronary events was 19%. Median skin autofluorescence was elevated compared with age-matched healthy control subjects: 2.77 (interquartile range 2.39-3.28) vs. 2.46 (2.08-2.84) arbitrary units. Skin autofluorescence was particularly increased in patients with complications: no complications, median 2.56 (2.26-2.90); microvascular complications, 2.79 (2.38-3.29); macrovascular complications, 2.85 (2.41-3.41); both micro- and macrovascular complications, 2.96 (2.56-3.60) arbitrary units, P < 0.001. Logistic regression analysis showed that age, duration of diabetes, renal function, gender, atrial fibrillation and skin autofluorescence were independently associated with macrovascular complications. Multiple regression analysis identified age, smoking, renal function, macrovascular complications and the number of microvascular complications as the determinants of skin autofluorescence. CONCLUSIONS: This study confirms that skin autofluorescence is increased in patients with Type 2 diabetes in a secondary care setting. Skin autofluorescence was associated with macrovascular complications in patients with diabetes and this association was independent of classical risk factors.

[DNA-based diagnosis of hereditary tumour predisposition]. / DNA-diagnostiek naar erfelijke aanleg voor tumoren.

Of all forms of cancer, approximately 5% are caused by factors leading to a strong genetic predisposition. DNA diagnosis is currently used in families with hereditary tumour syndromes, such as familial adenomatous polyposis, hereditary non-polyposis colorectal carcinoma (Lynch syndrome), and hereditary breast and ovarian cancer. Those persons who have not inherited the predisposition no longer have to undergo regular examinations. DNA diagnosis for a hereditary predisposition is currently also performed in patients with cancer at a relatively young age, even if the family history is unclear or negative. Consideration of the patient in the context of his or her family is important for both medico-technical and psychosocial reasons. This is true of both diagnostic and presymptomatic DNA diagnosis. For these reasons, the clinical application of the DNA diagnosis of hereditary tumours has become an integral part of the work of the multidisciplinary cancer family clinics of the university medical centres and the cancer centres. Guidelines for the management of hereditary tumours have recently been issued, with criteria for referral to the specialised outpatient clinics.

Hemoglobin and hematocrit values after saline infusion and tourniquet.

This study attempted to contribute to standardization of blood testing in sport, and to investigate the effect of artificial dilution with saline. In 10 healthy, physically active males and 3 healthy physically active females hemoglobin (Hb), hematocrit (Ht), and % reticulocytes (%retics) were measured at different time points to look for possible fluctuations during day time, while the subjects had regular coffee breaks and lunch. In 7 of the subjects in a separate experiment 500 ml of saline were infused around 8 am and Hb, Ht, and %retics were measured before and every hour thereafter until 7 hours after infusion. In addition Ht was measured on a hematological analyzer as well as with a centrifuge. In a separate experiment the effect of tourniquet duration on Hb and Ht was studied in 9 of the subjects. The results show that Hb, Ht, and %retics are stable from 8 am to 4 pm, but that infusion of 500 ml of saline induces an acute decrease in Hb and Ht within one hour (Hb decreased from 15.2+/-0.9 g/dl to 14.5+/-1.0 g/dl, and Ht from 45.6+/-2.8 % to 44.0+/-2.5 %). The decline in Hb and Ht was maintained during the 7-hour observation period. Ht values of the same samples measured with a hematological analyzer and a centrifuge were not different. Application of the tourniquet did significantly affect Hb and Ht values only from two minutes, and thereafter Hb and Ht remained stable during the rest of the 5-minute tourniquet. With blood testing in sport these results have to be taken into consideration.

Experimental evaluation of vitellogenin as a predictive biomarker for reproductive disruption.

Vitellogenin (VTG) synthesis in male oviparous vertebrates is used as an indicator of environmental estrogen exposure, but the relationship between elevated VTG levels and the effects of environmental estrogens on reproductive success are poorly understood. To examine whether altered VTG expression predicts reproductive impairment, we exposed medaka (Oryzias latipes) for 2 or 8 weeks posthatch to 0, 0.5, 1.0, 2.5, and 7.5 ppb of the environmental estrogen o,p'-DDT. Fish were sampled 2, 4, and 8 weeks after hatch to examine VTG expression and gonad development. After exposure, fish were transferred to clean water, grown to sexual maturity, and placed in mating pairs. We collected eggs for 7 days and scored them for fecundity (number of eggs), fertility (percent fertilized), and hatching success (percent hatched). DDT had no effect on VTG expression after a 2-week exposure, whereas all doses induced VTG after 8 weeks. At both exposure durations, the highest doses of DDT caused a female-skewed sex ratio in adults. Gonadal feminization appeared to be progressive: some ovotestes were observed after 2- or 4-week exposure to the two highest doses, but the proportion of ovaries increased after 8 weeks. Both 2- and 8-week exposures significantly reduced fertility and hatching success at all doses, with lower doses having a greater effect after longer exposure. Fertility and hatching success were more sensitive to estrogenic disruption than were gonad differentiation and vitellogenin expression. We suggest that VTG expression may be interpreted as a warning of reproductive consequences, but absence of expression cannot be interpreted as absence of consequences.

Peroxisomal membrane proteins are properly targeted to peroxisomes in the absence of COPI- and COPII-mediated vesicular transport.

The classic model for peroxisome biogenesis states that new peroxisomes arise by the fission of pre-existing ones and that peroxisomal matrix and membrane proteins are recruited directly from the cytosol. Recent studies challenge this model and suggest that some peroxisomal membrane proteins might traffic via the endoplasmic reticulum to peroxisomes. We have studied the trafficking in human fibroblasts of three peroxisomal membrane proteins, Pex2p, Pex3p and Pex16p, all of which have been suggested to transit the endoplasmic reticulum before arriving in peroxisomes. Here, we show that targeting of these peroxisomal membrane proteins is not affected by inhibitors of COPI and COPII that block vesicle transport in the early secretory pathway. Moreover, we have obtained no evidence for the presence of these peroxisomal membrane proteins in compartments other than peroxisomes and demonstrate that COPI and COPII inhibitors do not affect peroxisome morphology or integrity. Together, these data fail to provide any evidence for a role of the endoplasmic reticulum in peroxisome biogenesis.

Long-term pyrene exposure of grass shrimp, Palaemonetes pugio, affects molting and reproduction of exposed males and offspring of exposed females.

The objective of this study was to investigate the impact of long-term pyrene exposure on molting and reproduction in the model estuarine invertebrate, the grass shrimp (Palaemonetes pugio). Grass shrimp were exposed to measured concentrations of 5.1, 15.0, and 63. 4 ppb (microg/L) pyrene for 6 weeks, during which time we determined molting and survivorship. At the end of the exposure, we immediately sacrificed some of the shrimp for biomarker (CYP1A and vitellin) analyses. The remaining shrimp were used to analyze fecundity and embryo survivorship during an additional 6 weeks after termination of pyrene exposure. Male shrimp at the highest pyrene dose (63 ppb) experienced a significant delay in molting and in time until reproduction, and showed elevated ethoxycoumarin o-deethylase (ECOD) activity immediately after the 6-week exposure period. In contrast, 63 ppb pyrene did not affect these parameters in female shrimp. Females produced the same number of eggs per body weight, with high egg viability (98-100%) at all exposure levels, but with decreased survival for the offspring of the 63-ppb pyrene-exposed females. In addition, vitellin levels were elevated only in females at 63 ppb pyrene after the 6-week exposure. We hypothesize that the elevated vitellin binds pyrene and keeps it biologically unavailable to adult females, resulting in maternal transfer of pyrene to the embryos. This would account for the lack of effect of pyrene exposure on ECOD activity, molting, and reproduction in the adult females, and for reduced survival of their offspring.

Pex8p, an intraperoxisomal peroxin of Saccharomyces cerevisiae required for protein transport into peroxisomes binds the PTS1 receptor pex5p.

We report the characterization of ScPex8p, which is essential for peroxisomal biogenesis in Saccharomyces cerevisiae. Cells lacking Pex8p are characterized by the presence of peroxisomal membrane ghosts and mislocalization of peroxisomal matrix proteins of the PTS1 and PTS2 variety to the cytosol. Pex8p is tightly associated with the lumenal face of the peroxisomal membrane. Consistent with its intraperoxisomal localization, Pex8p contains a peroxisomal targeting signal 1, and it interacts with the PTS1 receptor Pex5p. However, the Pex5p/Pex8p association is also observed upon deletion of the PTS1 of Pex8p, suggesting that Pex8p contains a second binding site for Pex5p. The pex8Delta mutant phenotype and the observed PTS1-independent interaction with the PTS1 receptor suggest that Pex8p is involved in protein import into the peroxisomal matrix. In pex8Delta cells, the PTS1 and PTS2 receptor still associate with membrane bound components of the protein import machinery, supporting the assumption that the Pex8p function in protein translocation follows the docking event.

Cloning and sequencing of cDNAs encoding for a novel copper-specific metallothionein and two cadmium-inducible metallothioneins from the blue crab Callinectes sapidus.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins found in micro-organisms, plants and all invertebrate and vertebrate animals. Unicellular eukaryotes such as yeast have a copper-MT whose synthesis is induced by a copper-activated transcription factor. Most higher organisms have two major cadmium/zinc MT isoforms, whose synthesis is controlled by a zinc-activated transcription factor. The blue crab, Callinectes sapidus, has two cadmium-inducible isoforms, CdMT-I and CdMT-II, and a third isoform, CuMT-II, which is induced by copper, but not by cadmium. The cDNA sequence of the copper-specific MT, along with those of the two CdMTs, was determined utilizing 3' and 5' rapid amplification of cDNA ends (RACE). CuMT-II cDNA encodes a 63 amino acid protein containing 21 cysteine residues. CdMT-I and CdMT-II cDNA encode a 58 and 57 amino acid protein, respectively, each with 18 cysteines. Molecular phylogeny analysis shows that the CdMT isoforms cluster with other crustacean CdMTs, whereas the copper-specific MT is more closely related to mollusk MTs. CuMT-II shows considerable homology to a copper-specific, non-cadmium inducible, MT from the snail, Helix pomatia. The presence of copper-specific MTs in mollusks and crustaceans, both of which are dependent on hemocyanin for oxygen transport, suggests that CuMT-II is involved in copper homeostasis associated with the synthesis and degradation of hemocyanin.

Validation of an ultrasound scanner for determing urinary volumes in surgical patients and volunteers.

OBJECTIVE: As bladder distension related to anaesthesia puts patients at risk for permanent dysfunction, peri-operative determination of bladder volume is of great importance. The aim of this study is to validate an ultrasonic imaging device for determing bladder urine volume. METHOD: To evaluate a broad volume range, ultrasonically scanned volumes were compared to true urinary volumes both in surgical patients and in volunteers. After institutional approval and informed consent 60 healthy volunteers were asked not to void for as long as possible. After ultrasound measurements (BladderScan BVI 2500, Diagnostic Ultrasound, Redmond WA, U.S.A.) they voided and true urinary volumes were measured. Fifty surgical patients scheduled for procedures requiring urinary catheterisation were studied. Pre- and post-induction of anaesthesia ultrasound measurements were recorded, followed by urinary catheterisation and measurement of true urinary volume. Urine volumes were compared using Student t-tests and Wilcoxon Rank Tests (p < 0.05). For validation linear regression was used together with Bland-Altman analyses. RESULTS: Ultrasonic scanning underestimated the true urine volume by about 7% over the whole volume range (17 ml to 970 ml). Underestimation was larger in females than in males (p < 0.02). R2 values for correlation of measured and scanned urinary volumes ranged between 0.92 and 0.95. Bland and Altman analyses showed a bias of 31 ml in volunteers and of 19 ml in patients and a precision of 110 ml and 80 ml, respectively. CONCLUSIONS: The ultrasonic imaging device can be used peri-operatively to establish bladder volume, taking into account the 7% underestimation of the bladder volume.

Biochemical defense mechanisms against copper-induced oxidative damage in the blue crab, Callinectes sapidus.

The blue crab (Callinectes sapidus) has a very dynamic copper metabolism associated with the biosynthesis and degradation of its respiratory pigment hemocyanin. In this study we report on the cellular defense mechanisms used by the crab to protect itself from copper toxicity. Short-term copper-exposure studies, conducted by incubating hepatopancreas tissue explants in copper-containing medium, show that copper taken up by the cells during the first 60 min combines with low-molecular-weight copper complex(es), which include Cu(I)-glutathione. Thereafter, copper binds to newly synthesized metallothionein (MT), with a concomitant decrease in Cu(I)-glutathione. Copper does not displace zinc from the endogenous ZnMT pool. Long-term exposure by means of copper-rich diets results in the synthesis of two MT isoforms in the hepatopancreas: CuMT-I and CuMT-II (D. Schlenk and M. Brouwer, 1991, Aquat. Toxicol. 20, 25-34). Transfer of copper from Cu(I)-glutathione to apoMT-I and apoMT-II can be accomplished in vitro. Cu(I) binding by the two isoforms is very different. Cu(I) binds to apoMT-I in a strictly cooperative manner. No partially filled Cu(I)-thiolate clusters appear to be present. In contrast, the Cu(I)-thiolate clusters in MT-II are formed only after more than four Cu(I) ions are bound. Long-term copper exposure leads to increased activity of two antioxidant enzymes: glutathione peroxidase and manganese superoxide dismutase (SOD). No CuZnSOD is found. Activities of catalase and glutathione reductase and the intracellular levels of glutathione are unaffected by copper. The defense mechanisms are not entirely sufficient for preventing copper-induced oxidative damage. Levels of oxidized lipids are significantly higher in copper-exposed crabs, but oxidized protein levels are nearly the same.

The paradigm that all oxygen-respiring eukaryotes have cytosolic CuZn-superoxide dismutase and that Mn-superoxide dismutase is localized to the mitochondria does not apply to a large group of marine arthropods.

The enzyme superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide radical, is present in the cytosol and mitochondria of all oxygen-respiring eukaryotes. The cytosolic form contains copper and zinc (CuZnSOD), whereas the mitochondrial form contains manganese (MnSOD). The latter protein is synthesized in the cytosol as a MnSOD precursor, containing an N-terminal mitochondrial-targeting sequence. CuZnSOD is sensitive toward cyanide (CN) and hydrogen peroxide (H2O2), but MnSOD is not. Assays for SOD activity in cytosol from the hepatopancreas of the blue crab, Callinectes sapidus, showed the presence of a CN/H2O2-insensitive form of SOD. No CN/H2O2-sensitive CuZnSOD was found. This unexpected phenomenon was shown to occur in all decapod crustacea (crabs, lobsters, shrimp) examined. The cytosolic and mitochondrial SODs of C. sapidus were purified by means of ion-exchange, size-exclusion, and reverse-phase HPLC. The cytosolic SOD is a homodimeric protein, which exists in a monomer-dimer equilibrium (24 kDa left and right arrow 48 kDa). The protein contains approximately 1 Mn per subunit. No copper or zinc is present. Amino acid sequence analysis identified the novel cytosolic SOD as a MnSOD precursor with an abnormal mitochondrial-targeting sequence. The mitochondrial SOD of C. sapidus is similar to the MnSOD found in other eukaryotes. N-Terminal amino sequences of mitochondrial and cytosolic blue crab MnSOD differ in several positions. The MnSODs are thus encoded for by two different genes. The paradigm that all eukaryotes contain intracellular CuZnSOD and that MnSOD occurs exclusively in the mitochondria appears not to apply to a large group of marine arthropods.

The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins.

We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pex13p, that is essential for protein import. A point mutation in the COOH-terminal Src homology 3 (SH3) domain of Pex13p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pex13p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pex13p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein.