A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking.

We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein's turnover time from such data.

Resistance to HSP90 inhibition involving loss of MCL1 addiction.

Inhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality.

Prevalence and correlates of exchanging sex for drugs or money among adolescents in the United States.

OBJECTIVE: This study examined the prevalence and correlates of exchanging sex for drugs or money among a nationally representative sample of 13,294 adolescents in the United States. METHODS: Data are from the National Longitudinal Study of Adolescent Health, waves I and II. The lifetime prevalence of exchanging sex was estimated and a cross sectional analysis of sociodemographic and behavioural correlates was conducted. Unadjusted odds ratios were obtained. RESULTS: 3.5% of adolescents had ever exchanged sex for drugs or money. Two thirds of these youths were boys. The odds of having exchanged sex were higher for youths who had used drugs, had run away from home, were depressed, and had engaged in various sexual risk behaviours. 15% of boys and 20% of girls who had exchanged sex reported they had ever been told they have HIV or another sexually transmitted infection (STI). CONCLUSIONS: Adolescents with a history of exchanging sex have engaged in other high risk behaviours and may experience poor health outcomes, including depression and HIV/STIs. These findings should help inform strategies to prevent this high risk sexual behaviour and its potential consequences.

An audit of the accuracy of fine needle aspiration using a liquid-based cytology system in the setting of a rapid access breast clinic.

We have assessed the effectiveness and accuracy of reporting fine needle aspirates of the breast (FNAB) using a liquid-based cytology (LBC) system (the Cytospin) method) in the pressure situation of a rapid access clinic (RAC). We have reviewed every case from the RAC from June 1997 to February 2001 inclusive. There were 1322 cases, which accounted for 26% of the total FNAB received in our department over the period. There were 323 cancers and 999 benign cases in the group. The inadequate/nondiagnostic rate (C1) was 18%. The absolute sensitivity, including C1 cases, was 73% with the complete sensitivity being 90%. The groups of 'atypical, probably benign' (C3) and 'suspicious, probably malignant' (C4) accounted for a total of 6.2%. There were 28 false negative cases and 1 false positive case (a borderline phyllodes tumour). Comparing our results with the standards recommended by the NHSBSP has shown that the diagnosis of FNAB using this LBC method is feasible, accurate and reliable even in the pressure situation of a RAC.

Borderline nuclear change; can a subgroup be identified which is suspicious of high-grade cervical intraepithelial neoplasia, i e CIN 2 or worse?

Borderline nuclear change; can a subgroup be identified which is suspicious of high-grade cervical intraepithelial neoplasia, i.e. CIN 2 or worse? Only 10% of first borderline smears are associated with a histological high-grade (HG) abnormality, i.e. CIN 2,3, invasive malignancy or glandular neoplasia on subsequent investigation. The advantages of highlighting this subgroup are obvious but is this possible? From 1996 and 1997, 242 borderline smears with histological follow-up were examined by two independent experienced observers (observer 1 and 2) without prior knowledge of further investigation results. For each smear a profile of nuclear details was produced, also noting the type of cell mainly affected by the process; then the observers were asked to assess the degree of worry of HG disease for each smear i.e. whether the smear fell into group 1 borderline changes indicative of low-grade (normal, inflammatory, CIN1/HPV) disease (BL/LG) or group 2 difficult borderline smear, HG disease (CIN 2,3, invasive neoplasia or glandular neoplasia) cannot be excluded (BL/HG). Observer 1 selected a group of BL/HG with a PPV for HG disease of 38%, with observer 2 having a PPV of 50%; this compared with the overall laboratory HG disease PPV for borderline smears of 14%. Both observers found the most useful criterion to be the increase in nuclear:cytoplasmic ratio. Our results show that it is possible to separate a small group of borderline smears which should be classified as 'borderline/high grade lesion difficult to exclude' (BL/HG). Both observers had some success in arriving at this classification although their method of selecting out this group was quite different.

Quantitative assessment of fetal ventricular function: establishing normal values of the myocardial performance index in the fetus.

OBJECTIVE: Assessment of ventricular function in the fetus has been limited for many reasons, including relative cardiac size and atypical orientation of fetal cardiac structures. A myocardial performance index (MPI) has been described in adult and pediatric populations as an echocardiographic measure of global (systolic and diastolic) ventricular performance. Because the MPI is a Doppler index, it is independent of ventricular geometry and can be applied to both left and right ventricular function. This study attempts to define the MPI in a group of normal fetuses and compare these data to other published studies of this index. STUDY DESIGN: The right ventricular (RV) and left ventricular (LV) MPI were measured in 125 normal fetuses (20--40 weeks gestation, mean age 28 weeks). These fetuses were divided into five gestational age groups for comparison. These data were compared to 152 normal children (age 3--18 years, mean age 9.3 years). RESULTS: In normal fetuses, the LV MPI was 0.36 +/- 0.06 and the RV MPI was 0.35 +/- 0.05. This was not statistically different from the group of normal children in whom the LV MPI was 0.35 +/- 0.03 and the RV MPI was 0.32 +/- 0.03. In addition, no significant change in the fetal MPI was seen with advancing gestational age. CONCLUSION: This study demonstrates that fetal ventricular function can be quantitatively measured with the MPI. The MPI provides an easily obtainable and reproducible measure of fetal ventricular performance that can be readily incorporated into all fetal echocardiographic examinations.

Long-term functional outcome of neurogenic thoracic outlet syndrome in surgically and conservatively treated patients.

PURPOSE: Neurogenic thoracic outlet syndrome (NTOS) in the absence of bony and electrodiagnostic abnormalities, often referred to as disputed NTOS, remains enigmatic. Optimal treatment, especially the role of surgery, is controversial. The long-term functional outcome of a cohort of patients undergoing independent medical examination for disputed NTOS with symptoms sufficiently severe to cause inability to work forms the basis for this report. METHODS: Patients with disputed NTOS and symptoms sufficiently severe to cause at least temporary inability to work seen for independent medical examinations from 1990-1998 formed the study group. None of the patients were treated by our group. Functional outcome was assessed with information from a standardized telephone interview or patient questionnaire. The patients' ability to return to work and an assessment of their current level of symptoms and symptom progression since the time of onset were determined. RESULTS: Seventy-nine patients were reevaluated at a mean follow-up of 4.2 years (range, 2-7.5 years) after our initial evaluation. Fifteen patients (19%) underwent first rib resection surgery performed by others, whereas 64 (81%) had no surgery. Patients undergoing surgery had missed more work time than those undergoing conservative management (27.6 +/- 6.0 months vs 14.9 +/- 2.6 months, P <.04). Return to work was achieved in nine patients who were operated on (60%) and in 50 patients who were not operated on (78%) (P = not significant [NS]). Among operated patients, current assessment of symptom severity was severe, moderate, mild, and asymptomatic in 7%, 47%, 40% and 7%, respectively. This distribution did not differ significantly from that observed in nonoperated patients (11%, 55%, 30%, 5%; P = NS). When asked about changes in symptomatic status since onset, 7% of the operated group had complete resolution, 27% had marked improvement, 40% had minimal improvement, 13% had no improvement, and 13% were worse. This did not differ significantly from the change in symptoms reported by the nonoperated group (2%, 30%, 22%, 31%, 16%; P = NS). CONCLUSION: Most patients with disputed NTOS in this nonrandomized series were able to return to work and demonstrated an improvement of symptoms with long-term follow-up. First rib resection did not improve functional outcome in this group.

Optimizing duplex follow-up in patients with an asymptomatic internal carotid artery stenosis of less than 60%.

OBJECTIVES: The Asymptomatic Carotid Atherosclerosis Study established benefit of carotid endarterectomy for 60% to 99% asymptomatic internal carotid artery (ICA) stenosis. Optimal follow-up intervals to detect progression from < 60% to 60%-99% ICA stenosis are unknown. In a previous study from our laboratory, we found that ICAs with < 60% stenosis and peak systolic velocities (PSVs) of 175 cm/s or more on initial duplex were at high risk for progression. Prospective evaluation of this hypothesis and determination of optimal duplex follow-up intervals for asymptomatic patients with < 60% ICA stenosis form the basis of this report. METHODS: All patients who underwent initial carotid duplex examination for any indication since January 1, 1995, with at least one patent, asymptomatic, previously nonoperated ICA with < 60% stenosis; with 6 months' or greater follow-up; and with one or more repeat duplex examinations were entered into the study. On the basis of the initial duplex examination, ICAs were classified into two groups: those with a PSV less than 175 cm/s and those with a PSV of 175 cm/s or more. Follow-up duplex examinations were performed at varying intervals to detect progression from < 60% to 60%-99% ICA stenosis with criteria previously reported (both PSV > or = 260 cm/s and end-diastolic velocity > or = 70 cm/s). RESULTS: A total of 407 patients (640 asymptomatic ICAs with < 60% stenosis) underwent serial duplex scans (mean follow-up, 22 months). Three ICAs (0.5%) became symptomatic and progressed to 60%-99% ICA stenosis at a mean of 21 months (all transient ischemic attacks), whereas four other ICAs occluded without stroke during follow-up. Progression to 60%-99% stenosis without symptoms was detected in 46 ICAs (7%) (mean, 18 months). Of the 633 patent asymptomatic arteries, 548 ICAs (87%) had initial PSVs less than 175 cm/s, and 85 ICAs (13%) had initial PSVs of 175 cm/s or more. Asymptomatic progression to 60%-99% ICA stenosis occurred in 22 (26%) of 85 ICAs with initial PSVs of 175 cm/s or more, whereas 24 (4%) of 548 ICAs with initial PSVs less than 175 cm/s progressed (P <.0001). The Kaplan-Meier method was used to determine freedom from progression at 6 months, 12 months, and 24 months, which was 95%, 83%, and 70% for ICAs with initial PSVs of 175 cm/s or more versus 100%, 99%, and 95%, respectively, for ICAs with initial PSVs less than 175 cm/s (P <.0001). CONCLUSIONS: Patients with < 60% ICA stenosis and PSVs of 175 cm/s or more on initial duplex examination are significantly more likely to progress asymptomatically to 60%-99% ICA stenosis, and progression is sufficiently frequent to warrant follow-up duplex studies at 6-month intervals. Patients with < 60% ICA stenosis and initial PSVs less than 175 cm/s may have follow-up duplex examinations safely deferred for 2 years.

Controlled non-heart-beating donor liver transplantation: a successful single center experience, with topic update.

BACKGROUND: The critical shortage of transplantable organs necessitates utilization of unconventional donors. We describe a successful experience of controlled non-heart-beating donor (NHBD) liver transplantation. METHODS: Controlled NHBDs had catastrophic head injury, prognosis for no meaningful recovery, decision to withdraw life support, and subsequent consent for donation. After stopping mechanical ventilation in the operating room, death determination by a nontransplant caregiver, and rapid aortic cannulation, liver and kidneys were recovered. RESULTS: Controlled NHBDs contributed 5% of hepatic allografts (8/164) from August 1996 through June 1999 (9% in 1998). Sixteen NHBDs afforded 8 livers and 24 kidneys. Liver donors (n=8) were 11-66 years old; half were >50 years old. Premortem alanine aminotransferase was 25-157 U/L. Arrest occurred 3-27 min after stopping ventilation. Perfusion started 3-5 min after incision, and <22 min after hypotension (mean arterial pressure: <50 mmHg). Patient and graft survivals are 100% at 18+/-12 months follow-up. There was no intraoperative complication, reperfusion syndrome, poor graft function, primary nonfunction, arterial thrombosis, biliary complication, or serious infection. Postoperative day 2 prothrombin time was 13+/-1 sec. Peak alanine aminotransferase was 980+/-601 U/L. Intensive care unit and posttransplant lengths of stay were 2+/-2 and 10+/-7 days, respectively. Soon after transplantation there was frequent temporary hyperbilirubinemia (five of eight recipients; bilirubin peak: 7-29 mg/dl, 2-3 weeks after transplantation) and rejection (4/8 recipients, <3 weeks after transplantation). CONCLUSIONS: NHBDs significantly and safely expanded our donor pool. NHBD surgeons must be capable of rapid procurement. Cautious liberalization of criteria for accepting livers from NHBDs with confounding risk factors is justified. Refined ethics guidelines would broaden approval of NHBDs.

Neuroblastomatous recurrence of ganglioglioma. Case report.

This case is believed to be the first reported recurrent intracranial ganglioglioma with purely neuroblastomatous malignant transformation. A complete macroscopic resection of a right frontal lobe tumor in an 18-year-old woman revealed differentiated ganglioglioma. Seven years later a large, well-demarcated recurrent tumor was again macroscopically totally resected in the same patient. Histological analysis showed malignant transformation in only the neuronal component of the original tumor. A review of the literature on recurrent gangliogliomas and their malignant transformation is included.

Patency and characteristics of lower extremity vein grafts requiring multiple revisions.

OBJECTIVES: Multiple (> 1) revisions of lower extremity vein grafts may be required to maintain patency. Characteristics of recurrent lower extremity vein graft lesions and the patency achieved after multiple revisions have not been emphasized in reports on infrainguinal vein graft stenosis. This study was performed to determine (1) the patency of multiply revised lower extremity vein grafts and (2) the timing, location, and angiographic and duplex features of the recurrent lesions. METHODS: Lower extremity vein grafts that were followed in a duplex surveillance protocol and required revisions from January 1990 through December 1998 were identified. All revisions were preceded by angiography. In multiply revised lower extremity vein grafts, the immediate preoperative angiogram and duplex examination findings, as well as the angiogram made before the previous revision and the duplex study done after the previous revision, were reviewed to characterize recurrent lesions at the time of previous and current graft revision. The patencies of grafts undergoing single and multiple revisions were compared. RESULTS: A total of 233 lower extremity vein graft revisions were performed; of these, 50 (21%) were repeat revisions. Of grafts requiring more than one revision, 98% were normal on duplex examination after the initial revision. Five-year assisted primary patency of multiply revised grafts (91%) was not different from that of grafts with a single revision (89%; P not significant). Of 60 lesions repaired in the 50 repeat revisions, 29 (48%) were at the previously revised site, and 31 (52%) were at new sites. The time between revisions was less if the same site was revised (11 +/- 2 months) than if a different site required revision (20 +/- 4 months; P <.05). Arteriographic evidence of a minor (< 50% diameter) lesion was present at the time of the initial revision in 23% of cases in which revision of a second site was subsequently required. CONCLUSION: In our experience, 21% of lower extremity vein grafts requiring initial revision ultimately require additional revisions. Multiply revised lower extremity vein grafts have excellent long-term patency. Lesions occur with equal frequency at the site of prior revision and new sites. Lesions prompting revision at new sites occur significantly later and are infrequently detected on prior imaging studies.

Revascularization of the superior mesenteric artery alone for treatment of intestinal ischemia.

OBJECTIVE: Complete revascularization is recommended by many authors for treatment of intestinal ischemia. The observation that postprandial intestinal hyperemia is limited to the superior mesenteric artery (SMA) has suggested to us that SMA revascularization alone should be adequate treatment. We preferentially manage intestinal ischemia with a single bypass graft to the SMA and herein update our results using this approach. METHODS: Patients were identified from a prospectively established vascular surgical registry. Each patient was assessed for acute versus chronic intestinal ischemia, preoperative angiographic findings, operation used, perioperative morbidity and mortality, late symptomatic relief, cause of death, and life table-determined survival and graft patency. Graft patency was determined by follow-up angiography or duplex scanning. RESULTS: Fifty bypass grafts to the SMA alone were performed in 49 patients (31 women, 18 men; mean age, 62 years) for treatment of intestinal ischemia. In all patients additional splanchnic arteries were available for bypass grafting. Operative indications were acute symptoms in 21 patients, 14 of whom had bowel infarction; chronic symptoms in 26 patients; and prophylaxis in conjunction with infrarenal aortic surgery in 3 patients. Thirty-two grafts originated from the aorta or an iliac artery, and 18 originated from an aortic graft. There were 40 prosthetic and 10 autogenous conduits. Perioperative mortality was 3% in patients with chronic symptoms and 12% overall. All survivors were symptomatically improved. Mean follow-up was 44 months. Nine-year assisted primary graft patency was 79%, and 5-year patient survival was 61%. Two late deaths occurred in patients with recurrent intestinal ischemia resulting from graft occlusions. CONCLUSIONS: Bypass grafting to the SMA alone appears to be both an effective and durable procedure for treatment of intestinal ischemia. Our results appear equal to those reported for "complete" revascularization for intestinal ischemia. When the SMA is a suitable recipient vessel, multiple bypass grafts to other splanchnic vessels are unnecessary in the treatment of intestinal ischemia.

Experience with neurocysticercosis in the UK: correct diagnosis and neurosurgical management of the small enhancing brain lesion.

Neurocysticercosis is a major cause of epilepsy and other neurological morbidity in endemic areas of the world but is exceptionally rare in the West. We have recently had experience of eight patients with this condition, seven presenting with epilepsy and single or multiple small, enhancing parenchymal lesions and one with hydrocephalus caused by a midbrain lesion. One lesion was stereotactically excised after it persisted, but in five other cases spontaneous cyst resolution was observed during expectant management with anticonvulsants. Two patients with multiple lesions were referred to us for further management but were free of active infection. Recent studies show that neurocysticercosis may often be diagnosed based upon the clinical, epidemiological and radiological features. Spontaneous cyst resolution is to be expected in this condition and suspected patients should be carefully observed and surgery avoided. We believe that this disease presents more commonly than has been appreciated in the UK and propose a protocol for management.

Is a single preoperative duplex scan sufficient for planning bilateral carotid endarterectomy?

PURPOSE: Duplex scanning is often the sole imaging study before carotid endarterectomy (CEA). Patients with bilateral severe internal carotid artery (ICA) stenosis may be considered for bilateral CEA. High-grade ICA stenosis, however, may artifactually elevate velocity measurements used to quantify stenosis in the contralateral ICA. It is unknown whether ipsilateral CEA will influence duplex determination of the presence of a contralateral 60% to 99% ICA stenosis. This study was performed to determine whether a single preoperative duplex scan is sufficient to plan bilateral CEA. METHODS: Preoperative and early postoperative carotid duplex scans in patients with bilateral ICA stenosis who underwent unilateral CEA were reviewed. Changes in duplex scans that determined stenosis in the ICA contralateral to the CEA were analyzed. Previously validated criteria used to determine 60% to 99% ICA stenosis were a peak systolic velocity (PSV) of 260 cm/sec or more combined with an end diastolic velocity (EDV) of 70 cm/sec or more. RESULTS: Over an 8-year period, 460 patients underwent CEA; 107 patients (23.3%) had an asymptomatic 50% to 99% contralateral ICA stenosis by standard criteria (PSV, >125 cm/sec) and an early postoperative duplex scan examination. Of these 107 patients, 38 patients (35.5%) had duplex scan criteria for 60% to 99% contralateral ICA stenosis. In these 38 patients, there was a mean postoperative PSV decrease of 47.7 cm/sec (10.1%) and a mean EDV decrease of 36.0 cm/sec (19.3%) in the ICA contralateral to the CEA. Eight of 38 (21.1%) preoperative contralateral 60% to 99% ICA lesions were reclassified as less than 60% on postoperative duplex scanning. Six of 69 (8.7%) preoperative lesions of less than 60% were reclassified as 60% to 99% on postoperative duplex scan. These six preoperative examinations were all close to the criteria for 60% to 99% stenosis (mean PSV, 232.5 cm/sec; mean EDV, 62.5 cm/sec). CONCLUSION: One-fifth of patients with apparent 60% to 99% contralateral ICA lesions before the operation have less than 60% stenosis when restudied with duplex scan after unilateral CEA. Lesions below but near the cutoff for 60% to 99% may be reclassified as 60% to 99% on the postoperative duplex scan. These findings mandate that when duplex scanning is used as the sole imaging modality before CEA, patients with severe bilateral carotid stenosis must have an additional carotid duplex examination before operation on the second side.

Hypothenar hammer syndrome: proposed etiology.

PURPOSE: Finger ischemia caused by embolic occlusion of digital arteries originating from the palmar ulnar artery in a person repetitively striking objects with the heel of the hand has been termed hypothenar hammer syndrome (HHS). Previous reports have attributed the arterial pathology to traumatic injury to normal vessels. A large experience leads us to hypothesize that HHS results from trauma to intrinsically abnormal arteries. METHODS: We reviewed the arteriography, histology, and clinical outcome of all patients treated for HHS in a university clinical research center study of hand ischemia, which prospectively enrolled more than 1300 subjects from 1971 to 1998. RESULTS: Twenty-one men had HHS. All had occupational (mechanic, carpenter, etc) or avocational (woodworker) exposure to repetitive palmar trauma. All patients underwent upper-extremity and hand arteriography, unilateral in eight patients (38%) and bilateral in 13 patients (62%). By means of arteriogram, multiple digital artery occlusions were shown in the symptomatic hand, with either segmental ulnar artery occlusion in the palm or characteristic "corkscrew" elongation, with alternating stenoses and ectasia. Similar changes in the contralateral asymptomatic (and less traumatized) hand were shown by means of 12 of 13 bilateral arteriograms (92%). Twenty-one operations, consisting of segmental ulnar artery excision in the palm and vein grafting, were performed on 19 patients. Histology was compatible with fibromuscular dysplasia with superimposed trauma. Patency of arterial repairs at 2 years was 84%. One patient (5%) required amputative debridement of necrotic finger tips. No other tissue loss occurred. There have been no recurrences of ischemia in patients with patent bypass grafts. CONCLUSION: To our knowledge, this is the largest reported group of HHS patients. The characteristic angiographic appearance, histologic findings, and striking incidence of bilateral abnormalities in patients with unilateral symptoms lead us to conclude that HHS occurs when persons with preexisting palmar ulnar artery fibrodysplasia experience repetitive palmar trauma. This revised theory for the etiology of HHS explains why HHS does not develop in most patients with repetitive palmar trauma.

Rapid growth in a cavernoma.

The availability of magnetic resonance imaging has greatly increased the detection of cavernous malformations of the central nervous system in both symptomatic and asymptomatic patients. These lesions may be responsible for previously unexplained neurological events or may even have been incorrectly diagnosed. We describe a patient presenting with focal neurological signs in whom an initial diagnosis of malignant glioma had been made. Following excision, the pathological diagnosis was cavernoma. This vascular lesion has continued to show rapid growth and aggressive behaviour despite multiple surgical resections. The indications for operative and non-operative intervention will be discussed.

Marjolin's ulcer presenting in the neck.

'Marjolin's ulcer' is a term that has come to be used for epidermal squamous cell carcinomas that have developed in areas of chronic inflammation. To our knowledge this is the first ever report of a Marjolin's ulcer developing following long-term irritation of the skin of the neck. The history of the eponym is traced and reveals that Marjolin probably never actually described this pathological process.