bluuebunny/crossref_metadata_2025 huggingface dataset:
Invasion by cultured human follicular thyroid cancer correlates with increasedβ1 integrins and production of proteases
Authors: Demeure Michael J. , Damsky Caroline H. , Elfman Fred , Goretzki Peter E. , Wong Mariwil G. , Clark Orlo H.
Published: August 2005

A recognized model of tumor invasion requires cells to adhere to epithelial basement membrane and extracellular matrix components triggering release of proteases thus allowing cancer cells to invade the substrate. This adhesion is mediated byβ1 integrins, a family of receptors to substrates such as collagen, laminin, and fibronectin. In order to study tumor invasion in follicular thyroid cancer (FTC), we used cell lines derived from a single patient's FTC primary tumor (FTC‐133), neck lymph node metastases (FTC‐236), and lung metastases (FTC‐238).In vitro invasion as determined by the ability of the tumor cells to penetrate Matrigel® was assessed by scanning electron microscopy. FTC‐133 did not invade, FTC‐236 was moderately invasive, and FTC‐238 was highly invasive. Immunoprecipation with a monoclonal antibody toβ1 integrin subunits and SDS‐PAGE showed increased synthesis and flow cytometry showed increased expression of this subunit in FTC‐236 and FTC‐238 compared to FTC‐133. Proteolytic activity was assessed by gelatin zymography. FTC‐238 cell extract and conditioned media exhibited a more complex array of proteases consistent with activated type I collagenase and stromelysin compared to the less invasive clones, however 72 and 92 kd gelatinases consistent with type IV collagenases were present in the conditioned media from all three lines. In conclusion,in vitro invasion parallelsin vivo metastasis by the source cells in the FTC‐133/236/238 cell‐lines. The ability to invade basement membrane preparation correlates with increased synthesis and expression ofβ1 integrins and activation of tumor proteases.
Primary hyperparathyroidism in Austria: Results of an 8‐year prospective study
Authors: Niederle Bruno , Stamm Ludwig , Längle Friedrich , Schubert Edith , Woloszczuk Wolfgang , Prager Rudolf
Published: August 2005

Serum calcium determinations in 45,217 hospitalized patients during an 8 year period revealed sustained hypercalcemia in 124 (0.3%) patients. Further clinical and biochemical investigations established primary hyperparathyroidism (PHP) in at least 40 (32%) patients, an incidence of PHP of 89.6±41.5 per 100,000 patients per year. The incidence of PHP in males and females was 31.5 and 124.2 per 100,000, respectively. The highest incidence was found in females >66 years of age (268.8 per 100,000). Nineteen (48%) patients showed classical symptoms of PHP, 14 (35%) patients were minimally symptomatic, and 7 (17%) patients were asymptomatic. The incidence and symptomatology of PHP in Austria is nearly the same as in other countries. Because of the multiple actions of parathyroid hormone in the body, PHP can cause debilitating symptoms but too often it remains undiagnosed clinically. Routine calcium determinations are very important and PHP must always be considered in the differential diagnosis, especially in minimally symptomatic patients.
Primary hyperparathyroidism in younger and older patients: Symptoms and outcome of surgery
Authors: Udén Per , Chan Allen , Duh Quan‐Yang , Siperstein Allan , Clark Orlo H.
Published: August 2005

Two‐hundred and fifty patients undergoing initial exploration for primary hyperparathyroidism were analyzed for differences in clinical presentation, biochemical status, pathology, and outcome of surgery. In patients <60 years of age (younger patients, n=119) the most common preoperative symptoms and signs were fatigue (40.3%), bone pain (33.6%), renal stones (31.0%), hypertension (27.7%), and psychiatric illness (27.7%). In patients ≥60 years of age (older patients, n=131) the most frequent symptoms and signs were hypertension (46.6%), fatigue (35.1%), bone pain (30.5%), muscle weakness (28.2%), and joint pain (22.9%). Renal stones were 2.6 times more common (p<0.001, chi2) in younger patients and hypertension 1.7 times more common (p<0.05, chi2) in older patients. There was no significant difference in the preoperative and postoperative laboratory values typically associated with primary hyperparathyroidism. Double adenomas were more common in older (9.2%) than in younger patients (2.5%,p<0.05, chi2). Surgical cure was obtained in 98.8% of patients, and after parathyroidectomy 83% of the younger and 82% of the older patients experienced substantial relief of pre‐operative symptoms. Specific questioning revealed most patients to be symptomatic and older patients appear to receive the same clinical and metabolic benefits from parathyroidectomy as younger patients.
The detection and management of dysplastic nevi and early melanoma
Authors: Rivers Jason K.
Published: August 2005

Worldwide, the incidence and mortality rates for melanoma are increasing at an alarming rate. Recently, the recognition of dysplastic nevi as markers and potential precursors for melanoma has identified one group of individuals with an increased risk to develop this disease. Close surveillance of these individuals should improve the ability of clinicians to detect melanoma at an early stage when surgical excision of the primary tumor is curative in the majority of patients.
Predicting survival and recurrence in localized melanoma: A multivariate approach
Authors: Soong Seng‐Jaw , Shaw Helen M. , Balch Charles M. , McCarthy William H. , Urist Marshall M. , Lee Jeannette Y.
Published: August 2005

Several clinical and pathologic factors appear to affect melanoma recurrence and survival. While much attention has been directed at identifying prognostic factors, few researchers have developed predictive models for survival and recurrence. Two major clinical questions are of interest in the management of melanoma: 1) what is the patient's chance of surviving for a given period, e.g., 5 or 10 years, after diagnosis of melanoma; and 2) after a patient has been disease free for a period of time, e.g., 5 years, what is his or her chance of melanoma recurrence or death in the following interval, e.g., 5 years or 10 years. In this paper, a generalized multivariate prognostic model to address both of these clinical questions is presented.

Tables of the estimated probabilities of melanoma recurrence and death for prognostic subgroups are shown to facilitate prediction of an individual patient's outcome. The model was based on a database of 4,568 patients with localized melanoma, one of the largest melanoma databases in the world with detailed clinical and pathologic information, and long‐term follow‐up. Tumor thickness at diagnosis was the single most important prognostic factor for all outcomes. Tumor ulceration, Clark's level, lesion location, and sex had an impact on overall survival from diagnosis for some of the subgroups defined by tumor thickness. Tumor thickness at diagnosis was strongly indicative of melanoma recurrence and death even after a disease free interval of 2, 5, or 10 years. Lesion location and ulceration were of prognostic importance after disease free intervals up to 5 years, but their impact on melanoma recurrence and death diminished after longer disease free intervals.

Prediction models for melanoma outcome at diagnosis and after a disease free period can provide useful information to clinicians in the management of melanoma patients. Utilization of the model will be valuable in identifying patients at high risk for melanoma recurrence and death.
Elective lymph node dissection for melanoma: Two perspectives
Authors: McCarthy W. H. , Shaw H. M. , Cascinelli N. , Santinami M. , Belli F.
Published: August 2005

Node dissection for melanoma remains the most important therapeutic decision for the optimal management of the patient with melanoma. In Australia and in some parts of the United States, the majority of patients with intermediate thickness melanoma (1.6–4.00 mm) will undergo elective lymph node dissection. In Europe, the majority of patients with similar lesions are treated with wide local excision only. The development of an agreed policy for node dissection for melanoma awaits the results of 2 current, prospective randomized trials confirming or disproving the efficacy of elective lymph node dissection. Non‐randomized studies showing a benefit or not showing a benefit are readily available but the only 2 prospective randomized studies published to date showing a benefit, have been criticized on methodologic grounds. This paper summarizes the evidence and opinions on both sides of this debate. The clinician charged with responsibility for this important decision must, as yet, make the decision to dissect lymph nodes on the basis of a perspective derived from the best available evidence.
Management of the regional lymph nodes in patients with cutaneous malignant melanoma
Authors: Cochran Alistair J. , Wen Duan‐Ren , Morton Donald L.
Published: August 2005

One of the most difficult decisions in the management of patients with melanoma remains whether or not to dissect electively the regional lymph nodes of patients with high risk primary tumors. There is abundant evidence that a proportion of such patients will eventually develop nodal metastases and that the probability of metastasis increases with increasing tumor thickness and depth of invasion. If elective node dissection is performed on all patients with high risk primaries, most patients (who have no tumor in the nodes) will be subjected to a potentially morbid operation from which they can achieve no benefit. On the other hand, a “wait and see” policy accepts that a minority of patients will go on to develop nodal metastases and that if definitive therapy is delayed until metastases are detected clinically their likelihood of survival is much reduced. We describe a new technique of dye‐directed selective lymphadenectomy that allows accurate and objective identification of individual patients who have subclinical metastases, the individuals whoa priori are most likely to benefit from lymphadenectomy. We also discuss new approaches to the assessment of prognosis after lymphadenectomy for node‐spread melanoma, a group of patients in whom the prognosis is not uniformly bad. We end with a consideration of quality assurance aspects of the pathological evaluation of lymphadenectomy specimens.
Isolated limb perfusion for melanoma: Effectiveness and toxicity of cisplatin compared with that of melphalan and other drugs
Authors: Thompson John F. , Gianoutsos Mark P.
Published: August 2005

In patients with advanced or recurrent melanoma confined to a limb, hyperthermic isolated limb perfusion (ILP) with melphalan produces complete remission in 35–40% of cases and partial remission in a further 35–40%. Mild or moderate limb toxicity is usual, but severe toxic reactions in the limb sometimes occur. After preliminary reports suggested that cisplatin administered by ILP was even more effective than melphalan yet less toxic, a study was undertaken to further assess the value of hyperthermic ILP with cisplatin in the management of limb melanoma. Ten patients were treated. The procedure failed to eliminate melanoma in the limb in 5 of the 6 who received therapeutic ILPs for recurrent disease, and recurrence developed in 2 of the 4 patients who received prophylactic ILPs. Toxicity in the perfused limbs was unacceptably high, with 2 of the 10 patients having severe reactions, one necessitating amputation. We conclude from the results of this study and from a review of literature that neither cisplatin nor any other drug or drug combination so far used for ILP in melanoma patients achieves results which are clearly superior to those achieved with melphalan. Studies are currently in progress investigating double perfusion protocols, new strategies with regional hyperthermia, and the administration by ILP of biological response modifiers such as tumor necrosis factor and interferon. However, for the present, hyperthermic ILP with melphalan remains the treatment most likely to be successful in eliminating or controlling advanced or recurrent melanoma in a limb.
Controversies concerning adjuvant regional isolated perfusion for stage I melanoma of the extremities
Authors: Koops Heimen Schraffordt , Kroon Bin B. R. , Oldhoff Jan , Hoekstra Harald J.
Published: August 2005

The use of isolated perfusion with melphalan in an adjuvant setting for stage I extremity melanoma continues to be controversial. In this article, 2 retrospective studies compare patients undergoing perfusion from Groningen, the Netherlands, with matched controls from the Sydney Melanoma Unit in Australia and from 5 hospitals in the Netherlands and Westphalia (a region of Germany bordering the Netherlands). All patients underwent wide local excision for a primary extremity melanoma of 1.5 mm or more in thickness. In the first study, women with a melanoma of the leg (excluding the foot) and treated with perfusion had a significantly higher survival than women treated with local excision alone. In the second study, a proportional hazards regression analysis for recurrent disease and survival identified no statistically significant difference in favor of adjuvant perfusion.

Two other studies from the literature evaluating the efficacy of perfusion are examined. Patients treated at the M.D. Anderson Cancer Center, Houston, with wide local excision and perfusion are compared retrospectively with a group of patients individually matched for prognostic factors from the University of Alabama in Birmingham, U.S.A. and the Sydney Melanoma Unit. A small subset of patients with tumors >2 mm thick benefited from perfusion. A prospective randomized study from the University Hospital, Cologne, West Germany found benefits for all perfused patients with tumors >1.5 mm but their patients did not appear to be individually matched for tumor thickness.

It is concluded after critical appraisal of all these studies that there is insufficient convincing evidence to date of an advantage of perfusion over wide local excision alone.
Hereditary melanoma and the search for the melanoma gene
Authors: Kefford Richard F.
Published: August 2005

The discovery and characterization of growth regulatory genes, in the form of oncogenes, and their counterparts, tumor suppressor (TS) or antioncogenes, has vastly expanded the basic understanding of tumorigenesis. Human solid tumors, such as colorectal cancer, for which the molecular genetics have been most clearly defined, display progressive evolution from cellular dysplasia to anaplasia and metastasis through the stepwise accumulation of genetic defects, involving the regulation and expression of both oncogenes and TS genes. The study of basic genetic abnormalities in melanoma and the identification of the most fundamental of these is critical both to the understanding of abnormal melanocyte proliferation and its potential pharmacologic or immunologic regulation, and also to the identification and screening of patients at high risk for the development of melanoma. The search for such genetic abnormalities has included an analysis of melanomas for defects in known characterized oncogenes and TS genes, and, more importantly, the use of families with hereditary melanoma (HM) and dysplastic nevi in an endeavor to find the melanoma gene. The importance of HM is fundamental, since in the case of other hereditary cancer syndromes for which the genetic basis has been identified, the same or similar genetic abnormalities underlie sporadic tumors of the same tissue type. Thus HM is likely to be the major signpost to the melanomagenic defect.
Active immunotherapy with viral lysates of micrometastases following surgical removal of high risk melanoma
Authors: Hersey Peter
Published: August 2005

Patients who develop lymph node metastases from melanoma are known to be at risk of developing further recurrences from melanoma following surgical removal of lymph node metastases. The purpose of the present study was to examine whether immunization over a 2 year period with a vaccine made from vaccinia viral lysates of an allogeneic melanoma cell (VMCL), following surgical removal of lymph node metastases, would help prevent the development of distant metastases and improve survival from the disease. Eighty patients treated with VMCL alone and followed for a minimum of 5.5 years had improved survival compared to a historical control group of 151 patients and a concurrent non‐randomized group of 55 patients. Similarly, the survival of 102 patients treated with VMCL + low dose cyclophosphamide for a minimum of 3.5 years was superior to that of the historical control group but not to that of the VMCL alone treated group. Improvement in survival was still evident when this was measured from the date of removal of the primary tumor. Analysis of subsets of patients showed that VMCL treatment appeared to benefit patients irrespective of the number of lymph nodes involved and whether surgery was carried out near to (synchronous metastases) or some time after removal of the primary delayed metastases). Analysis of the effect of treatment on duration to development of distant metastases suggested that there was a lower proportion of metastases during treatment with VMCL compared to the historical control groups. Treatment with VMCL also appeared to be associated with a lower incidence of cutaneous metastases but a higher proportion of visceral (including liver) metastases. Treatment was not associated with prolongation of survival after development of distant metastases. The results from this prolonged follow‐up provide further support for an apparent survival benefit from immunotherapy with VMCL and suggest that the duration to and site of distant metastases is altered by this treatment. A randomized control (Phase III) study based on these results is in progress.
Immunotherapy with monoclonal antibodies in metastatic melanoma
Authors: Steffens Thomas A. , Bajorin Dean F. , Houghton Alan N.
Published: August 2005

Therapy for metastatic melanoma has been disappointing to date. Treatment with chemotherapy only uncommonly results in complete responses and rarely results in long‐term survivors. The identification of human melanoma cell surface antigens has led to the development of an array of mouse monoclonal antibodies (MAb) for use in the diagnosis and therapy of patients with metastatic melanoma. Strategies utilizing MAbs based on immunologic approaches have been developed. Naked MAbs directed against glycoprotein surface antigens or conjugated to toxins or radionuclides have shown little biologic or clinical activity. However, phase I studies of MAb directed against glycolipid antigens have yielded objective tumor shrinkage with occasional complete responses. Severe toxicity has been seen infrequently. Possible anti‐tumor mechanisms include complement activation and antibody‐dependent cellular cytotoxicity utilizing natural killer cells or monocytes as effector cells. Strategies to enhance the anti‐tumor effects of MAb, including combinations with cytotoxic agents and cytokines, have been introduced with limited success thus far. The development of a human IgG anti‐mouse antibody has been seen in nearly all treated patients. A new generation of MAb engineered to overcome the immunogenicity of mouse MAb and to enhance immune effector function will soon enter clinical trials.
Therapy of disseminated melanoma by liposome‐activated macrophages
Authors: Fidler Isaiah J.
Published: August 2005

The uncontrolled growth of metastases is a major cause of death from melanoma. Metastases arise from the nonrandom spread of specialized malignant cells that preexist within a primary neoplasm. Macrophages are activated to become tumoricidal by interaction with phospholipid vesicles (liposomes) containing immunomodulators recognize and destroy neoplastic cellsin vitro andin vivo while leaving nonneoplastic cells unharmed. The mechanism(s) by which macrophages discriminate between tumorigenic and normal cells is independent of tumor cell characteristics such as immunogenicity, metastatic potential, and sensitivity to cytotoxic drugs. Moreover, macrophage destruction of tumor cells apparently is not associated with the development of tumor cell resistance.

Intravenously administered liposomes are cleared from the circulation by phagocytic cells. The administration of such liposomes has been shown to activate macrophagesin situ and to bring about eradication of cancer metastases in several experimental tumor systems and in dogs with spontaneous osteogenic sarcoma. Macrophage destruction of metastasesin vivo is significant, provided that the tumor burden at the start of treatment is minimal. For this reason, we have been investigating various methods to achieve maximal cytoreduction in metastases by modalities such as chemotherapy or radiotherapy prior to systemic activation of macrophages. Macrophage‐directed therapy has been studied in several human protocols, yielding important biological information about the safe use of liposome‐encapsulated macrophage activators in cancer patients.
Systemic chemotherapy for malignant melanoma
Authors: Coates Alan S.
Published: August 2005

Malignant melanoma is notoriously resistant to chemotherapy. Standard single agents include dacarbazine and the nitrosoureas, but most series treating patients with metastatic melanoma report response rates of about 20%. Occasional patients, however, achieve long term remission, amounting to cure, after such treatment. Adjuvant studies have failed to define any systemic therapy of value after surgery for primary or regional lymph node disease. Combinations of drugs have not been shown to be superior to single agents. Cisplatin and fotemustine are among the newer agents with activity. Isolated limb perfusion with melphalan and systemic therapy with high dose alkylating agents and marrow transplantation suggest that the resistance to conventional concentrations of alkylating agents is not absolute. One strategy to improve the efficacy of alkylating agents may therefore be the modulation of cellular glutathione or glutathione‐S‐transferase. Biological response modifiers remain of interest, though many of the protocols with the best response rates are impractical for routine use. Many patients with asymptomatic metastatic melanoma should be observed without treatment. Outside clinical trials, it is reasonable to use single agent dacarbazine or lomustine if chemotherapy is required.
Epidemiology of gastro‐esophageal reflux disease
Authors: Howard P. J. , Heading R. C.
Published: August 2005

Gastro‐esophageal reflux disease encompasses a spectrum of disorders in which gastric reflux leads to symptoms and/or damage to the esophageal mucosa. Although a common problem in clinical practice, our understanding of the pathophysiology of the condition has not been matched by our knowledge of its epidemiology and natural history. This review examines some of the difficulties inherent in epidemiological studies with particular emphasis on the nature and variety of reflux symptoms and their relationship to esophagitis, the natural history and complications of reflux disease, Barrett's esophagus, and the possible role of gastroesophageal reflux in lung disease, especially asthma.
Maintenance therapy: Is there still a place for antireflux surgery?
Authors: Armstrong David , Nicolet Marcel , Monnier Phillippe , Chapuis Germain , Savary Marcel , Blum André L.
Published: August 2005

Effective and safe maintenance medical therapy for uncomplicated reflux esophagitis is now feasible with omeprazole and it is likely that other H+K+ ATPase blockers, and possibly very high dose H2 receptor antagonist regimens, will also be acceptable. In addition, many patients with ulceration, strictures, and Barrett's esophagus will respond to conservative medical therapy and a proportion of patients with erosive esophagitis may remain in remission with cisapride or with low dose H2 receptor antagonists, if disease is less severe. Thus, there is now a medical “gold standard” against which surgical therapy for uncomplicated esophagitis must be judged and it is essential that all future studies be conducted with clearly defined criteria for the assessment of the symptoms and endoscopic signs of esophagitis and its complications.

As ever, the patient's wishes are paramount, but he or she must be allowed to select his or her therapy on the basis of a balanced and fully informed assessment of the long‐term and short‐term risks of all therapeutic modalities. The burdensome prospect of lifelong tablet ingestion and its potential dangers must be weighed against the alternative, in up to 30% of cases, that surgery may produce dysphagia, gas bloat, or dumping with no guarantee of a long‐term cure.
Does modern technology belong in gastro‐intestinal surgery? A step from subjective perception to objective information
Authors: Hill Lucius D. , Kraemer Stefan J. M.
Published: August 2005

The intra‐operative measurement of lower esophageal sphincter (LES) pressure can aid the surgeon in primary repair of the gastroesophageal junction. We evaluated 1,000 patients undergoing primary repairs; 540 patients underwent surgery before the introduction of LES pressure measurements and 460 patients after LES pressure measurement. The incidence of continued reflux was lower in the group having intraoperative LES pressure measurement (4.5% vs. 1.5%). The incidence of continued reflux in this group has been <1%. The measurement of intra‐operative LES pressure brings objective technology to our patients and improves their quality of life.
Endoscopic control of gastro‐esophageal reflux: Status report
Authors: Donahue Philip E. , Sugitani Atsushi , Carvalho Paulo
Published: August 2005

Gastro‐esophageal reflux disease is a common disorder which sometimes requires surgical treatment. Until recently, surgical treatment was the only alternative for severe complications of reflux. During the past 5 years endoscopic treatments for reflux have been reported from several medical centers. These new approaches may herald a new era for the treatment of reflux complications. However, at this time they are purely experimental approaches.

Since physiologic antireflux mechanisms involve the muscles of the proximal gastric cardia as well as those of the lower esophageal sphincter, it is not surprising that endoscopic treatments have been directed at these two areas. However, the first endoscopic attempts at reflux control were directed at the lower esophagus sphincter. The most recent efforts at endoscopic control have been aimed at the proximal stomach, based on the concept that scarification beneath the lower end of the esophagus reduces the amount or frequency of reflux. Neither of these latter approaches has been reported in human patients.

Since all endoscopic approaches are potentially dangerous, we recommend the adoption of a rigorous protocol for all human application. A possible protocol for this purpose is described herein; the authors will provide data analysis for any cohort of patients receiving endoscopic therapy, and will provide the results of all endoscopic treatments to any serious investigators.
What is the place of antrectomy with Roux‐en‐Y in the treatment of reflux disease? Experience with 83 total duodenal diversions
Authors: Fekete F. , Pateron D.
Published: August 2005

Antrectomy with Roux‐en‐Y gastrojejunostomy was performed in 83 patients with “complicated” forms of peptic esophagitis. The esophagitis was considered complicated either because of the severity of the lesions (stricture, brachyesophagus, or endobrachyesophagus) or because of postoperative conditions after one or more previous operations (Heller's myotomy, esophagogastric resections, or hiatal hernia repair).

A standard procedure was performed in 56 patients while technical adjustments were required for 27 patients who had previously undergone surgery. Two patients died from pulmonary embolism. Early postoperative complications occurred in 11% of patients. Healing of esophagitis was observed for all the patients treated with the standard procedures. Six partial regressions and 1 complete regression of Barrett's mucosa were observed. Digestive sequellae were minor and decreased with time. Assessment of pH and small bowel manometry showed that the reflux was controlled and no small bowel motility disturbance was observed when the standard technique was used including a small gastric resection. The main digestive sequellae, including lack of healing of esophagitis, dumping syndrome, and gastrojejunal anastomotic ulcer, occurred when a two‐thirds gastrectomy was performed in order to avoid vagotomy.
The quality of life following antireflux surgery
Authors: Pope Charles E.
Published: August 2005

To improve the quality of life after antireflux surgery, the patient should notice improvement or disappearance of the symptoms leading to the surgery and should not acquire any new symptoms resulting from antireflux surgery. Published accounts of antireflux surgery results vary widely. Part of this variation may be due to nonstandard methods of evaluating symptoms. A system of symptom evaluation is proposed which takes into account the intensity and frequency of symptoms. It is hoped that this system will allow surgeons to refine and improve their techniques and results.
Surgical management after failed antireflux operations
Authors: Skinner David B.
Published: August 2005

From 1973 to 1989, 117 (28%) patients underwent re‐operation for failed antireflux surgery from a total of 413 esophagogastric operations for gastro‐esophageal reflux disease. Seventy‐eight patients who underwent re‐operation before 1984 were reviewed in detail for classification and long‐term outcome. Forty re‐operations followed a failed Nissen fundoplication, while no other procedure was the most recent prior operation in more than 10 patients. Re‐operation rates were 3% following prior surgery in our clinic for reflux disease other than stricture and 9.6% if the prior operation was done for stricture. There was no difference in re‐operation rates for the Belsey Mark IV or Nissen fundoplication, the 2 most commonly used repairs. In each case, complete pre‐operative evaluations included symptom score, radiography, endoscopy, and esophageal function tests. Based on the results, the 78 patients were classified as pure sphincter mechanism failure to stop reflux (n=14), pure esophageal clearance failure (n=12), combined sphincter mechanism failure and clearance failure (n=29), alkaline reflux (n=9), or no reflux but another condition found (n=14). Patients having symptoms following a prior Nissen fundoplication or Angelchik prosthesis insertion were more likely to have esophageal clearance failure than those having other repairs. The classification proved to be a useful guide to the need for and types of re‐operation chosen. Among the 117 patients undergoing re‐operation, there were 2 (1.7%) deaths within 3 months of surgery and 25 (21%) complications. Long‐term results were directly related to the number of previous operations, with resection and intestinal interposition being the preferred procedure in patients having had 2 or more previous attempts at antireflux surgery.
Peak Seeker: An Algorithm for Rapid Determination of Solubility
Authors: Yuan Ding , Uvarova Victoria , Isbell John
Published: August 2005

In the quest for novel pharmaceuticals to treat important diseases, biologists often use in vitro assays to assess apparent activity. Thus, when testing novel synthetic compounds in search of a new class of drugs, it is important to know the solubility of the compound under the assay conditions used so that the activity data may be interpreted correctly. As the screening throughput increases, so do the demands on solubility determinations and the reliance on this data to aid in the selection of the more promising compounds for further studies. Since many compounds synthesized in drug discovery compounds contain UV chromophores, solubility determinations using UV plate readers are widely applicable to common druglike compounds. A more analytically rigorous approach calculates the solubility using a calibration curve rather than a ratio to a reference concentration. Rather than use the λmax of the analyte for quantification, an automated method is used for intelligent selection of the wavelength most appropriate for quantification. Using a BioMek FX and Peak Seeker, our visual basic application written in-house, 128 compounds may be assayed at a single pH within 5 h using MultiScreen Solubility plates.
Automated Forensic DNA Purification Optimized for FTA Card Punches and Identifiler STR-based PCR Analysis
Authors: Tack Lois C. , Thomas Michelle , Reich Karl
Published: August 2005

Forensic labs globally face the same problem—a growing need to process a greater number and wider variety of samples for DNA analysis. The same forensic lab can be tasked all at once with processing mixed casework samples from crime scenes, convicted offender samples for database entry, and tissue from tsunami victims for identification. Besides flexibility in the robotic system chosen for forensic automation, there is a need, for each sample type, to develop new methodology that is not only faster but also more reliable than past procedures. FTA is a chemical treatment of paper, unique to whatman Bioscience, and is used for the stabilization and storage of biological samples. Here, we describe optimization of the Whatman FTA Purification Kit protocol for use with the AmpFlSTR Identifiler PCR Amplification Kit. The conditions giving the best quality Identifiler results were used to automate a simple rapid method that processed forensic samples applied to FTA-treated paper punches in a 96-well plate in ∼30 min. We describe an automated DNA purification procedure using 1.25 mm bloodstained FTA punches that is easily scalable and can be used with both convicted offender and reference casework bloodstained and buccal-swiped FTA samples. It works equally well with the Profiler Plus PCR Amplification Kit. Fully automated drying and polymerase chain reaction (PCR) amplification setup of short tandem repeat (STR) loci can be added to expand the walk-away process.
Quantifying Bioactivity on a Large Scale: Quality Assurance and Analysis of Multiparametric Ultra-HTS Data
Authors: Heyse Stephan , Brodte Annette , Bruttger Oliver , Duerr Oliver , Freeman Tobe , Jung Tom , Lindemann Michael , Ottl Johannes , Rinn Bernd
Published: August 2005

There is a growing need to precisely quantify the selectivity of large compound sets in high throughput screening, directing investment in lead optimization towards compounds with a high chance of success. High-content, high-density screening technologies such as multiparametric ultra-HTS provide a basis for highly precise screening with unprecedented scope for delineating process artifacts from reliable signals. However, the full potential of these technologies can only be realized with suitable experimental design and sophisticated data analysis tools.

We present two advanced analysis workflows demonstrating how multiparametric readouts from a high throughput primary screen can improve decision quality in the hit identification process. The first involves discrete thresholding and the application of multiple selection criteria. The second uses machine learning algorithms and allows an unbiased consideration of all measured parameters.
Cellular Dielectric Spectroscopy: A Label-Free Technology for Drug Discovery
Authors: Leung Gordon , Tang H. Roger , McGuinness Ryan , Verdonk Edward , Michelotti Julia M. , Liu Vivian F.
Published: August 2005

Cellular dielectric spectroscopy (CDS) provides realtime, label-free, universal measurements, enabling comprehensive pharmacological evaluation of cell surface receptors in living cells. The sensitivity of the measurement allows monitoring of ligand-mediated activation of endogenous receptors, therefore generating physiologically relevant data. Activation of receptors results in CDS response profiles that are characteristic of main subsets of G-protein coupled receptors (GPCRs) within a cell line. This allows cluster analysis of response profiles that may be used in several important applications, which include identification of the G-protein coupling of orphan GPCRs and the cataloging of active endogenous receptors in cells. In this study, CDS technology is used in the pharmacological evaluation of multiple receptors in many cell types, including primary cells. Specifically, data is presented demonstrating hit confirmation, receptor selectivity analysis, ligand potency, and Schild analysis of receptor-selective antagonists. CDS results compare favorably to other cell-based assays, and the robustness and reproducibility of CDS assays are reflected by low assay coefficient of variation (CVs) and reliable Z'-scores of the data. Because CDS requires no stable or transiently transfected cells or special reagents, assay development and data acquisition is simple and fast. The ease of use, universality, and label-free nature of the CDS-based platform make it well suited to secondary screening applications in drug discovery.
A High-Throughput System for the Rapid Extraction of Plant Genomic DNA for Genome Mapping and Marker-Assisted Breeding Studies
Authors: Wang Danhui , Song Keming , Kreader Carol , Weber Scott , Van Dinther Jennifer , Valdes-Camin Rafael
Published: August 2005

Simple sequence repeats (SSR) or simple sequence length polymorphisms (SSLP) are PCR-based molecular markers that have been widely used in genomic mapping and marker-assisted selection. SSR markers have made it possible to establish a high-density genetic map and evaluate genes of interest via tight association between markers and phenotypes. Large-scale markerassisted breeding studies in plants have created the need for a high-throughput system for genomic DNA preparation and analysis from thousands of samples in a segregating population. However, the standard methods for purifying DNA from plant tissues can be laborious, time-intensive, and not readily amenable to automation. Thus, an automated system for the rapid extraction and subsequent amplification and analysis of plant genomic DNA has been developed to facilitate high-throughput genome mapping and marker-assisted breeding studies. This system utilizes Sigma's Extract-N-Amp Plant PCR kit, a novel system for the rapid extraction and subsequent amplification of genomic DNA from plant tissues, and the maize SSR primer set. This extraction system eliminates time-consuming steps such as organic extractions and mechanical disruption, releasing sufficient genomic DNA from plant tissues for direct use in SSR marker analysis.
Automation and Microfluidic Assays: In Vitro Models of the Mammalian Microcirculation
Authors: Spence Dana M.
Published: August 2005

The red blood cells (RBCs) from patients with such diseases as pulmonary hypertension (PH) and diabetes (Type I and II) have a unique physical property, namely, the deformability of their RBCs are different from those of healthy patients. For example, patients with PH and diabetes have RBCs whose deformability is less than that of healthy patients. Unfortunately for these patients, there have been very few reports relating these somewhat abnormal RBC deformabilities to a cause/effect of the aforementioned diseases. Our group believes there is a link between the physical properties of these RBCs, the chemical properties they induce, and the resultant physiological consequences of the induced chemical properties. However, characterization or verification of the physical, chemical, and physiological processes is difficult because current analytical tools do not enable all three processes to be monitored simultaneously. Here, the ability of microchip technology to monitor physical, chemical, and physiological events that may occur in each of the diseases will be discussed. These events, which are physically stimulated on-chip, are monitored both optically (chemiluminescence) and electrochemically (amperometry), and when taken collectively, become excellent in vitro models of important in vivo processes.
MicroRNAs acting in a double-negative feedback loop to control a neuronal cell fate decision
Authors: Johnston Robert J. , Chang Sarah , Etchberger John F. , Ortiz Christopher O. , Hobert Oliver
Published: August 2005

The elucidation of the architecture of gene regulatory networks that control cell-type specific gene expression programs represents a major challenge in developmental biology. We describe here a cell fate decision between two alternative neuronal fates and the architecture of a gene regulatory network that controls this cell fate decision. The two
Caenorhabditis elegans
taste receptor neurons “ASE left” (ASEL) and “ASE right” (ASER) share many bilaterally symmetric features, but each cell expresses a distinct set of chemoreceptors that endow the gustatory system with the capacity to sense and discriminate specific environmental inputs. We show that these left/right asymmetric fates develop from a precursor state in which both ASE neurons express equivalent features. This hybrid precursor state is unstable and transitions into the stable ASEL or ASER terminal end state. Although this transition is spatially stereotyped in wild-type animals, mutant analysis reveals that each cell has the potential to transition into either the ASEL or ASER stable end state. The stability and irreversibility of the terminal differentiated state is ensured by the interactions of two microRNAs (miRNAs) and their transcription factor targets in a double-negative feedback loop. Simple feedback loops are found as common motifs in many gene regulatory networks, but the loop described here is unusually complex and involves miRNAs. The interaction of miRNAs in double-negative feedback loops may not only be a means for miRNAs to regulate their own expression but may also represent a general paradigm for how terminal cell fates are selected and stabilized.
Rab11 GTPase-Regulated Membrane Trafficking Is Crucial for Tip-Focused Pollen Tube Growth in Tobacco
Authors: de Graaf Barend H.J. , Cheung Alice Y. , Andreyeva Tatyana , Levasseur Kathryn , Kieliszewski Marcia , Wu Hen-ming
Published: August 2005

Pollen tube growth is a polarized growth process whereby the tip-growing tubes elongate within the female reproductive tissues to deliver sperm cells to the ovules for fertilization. Efficient and regulated membrane trafficking activity incorporates membrane and deposits cell wall molecules at the tube apex and is believed to underlie rapid and focused growth at the pollen tube tip. Rab GTPases, key regulators of membrane trafficking, are candidates for important roles in regulating pollen tube growth. We show that a green fluorescent protein–tagged Nicotiana tabacum pollen-expressed Rab11b is localized predominantly to an inverted cone-shaped region in the pollen tube tip that is almost exclusively occupied by transport vesicles. Altering Rab11 activity by expressing either a constitutive active or a dominant negative variant of Rab11b in pollen resulted in reduced tube growth rate, meandering pollen tubes, and reduced male fertility. These mutant GTPases also inhibited targeting of exocytic and recycled vesicles to the pollen tube inverted cone region and compromised the delivery of secretory and cell wall proteins to the extracellular matrix. Properly regulated Rab11 GTPase activity is therefore essential for tip-focused membrane trafficking and growth at the pollen tube apex and is pivotal to reproductive success.
Monomethyl Histone H3 Lysine 4 as an Epigenetic Mark for Silenced Euchromatin in Chlamydomonas
Authors: van Dijk Karin , Marley Katherine E. , Jeong Byeong-ryool , Xu Jianping , Hesson Jennifer , Cerny Ronald L. , Waterborg Jakob H. , Cerutti Heriberto
Published: August 2005

Histone Lys methylation plays an important role in determining chromatin states and is mostly catalyzed by SET domain–containing proteins. The outcome, transcriptional repression or activation, depends on the methylated histone residue, the degree of methylation, and the chromatin context. Dimethylation or trimethylation of histone H3 Lys 4 (H3K4me2 or H3K4me3) has been correlated with transcriptionally competent/active genes. However, H3K4 methylation has also been implicated in gene silencing. This dualistic nature of the H3K4 methyl mark has thus far remained unresolved. In the green alga Chlamydomonas reinhardtii, Mut11p, related to a subunit of trithorax-like methyltransferase complexes, is required for transcriptional silencing. Here, we show that Mut11p interacts with conserved components of H3K4 methyltransferase machineries, and an affinity-purified Mut11p complex(es) methylates histones H3, H2A, and H4. Moreover, a Mut11 mutant showed global loss of monomethylated H3K4 (H3K4me1) and an increase in dimethylated H3K4. By chromatin immunoprecipitation analysis, this strain also displayed substantial reduction in H3K4me1 and enrichment in H3K4me2 associated with transcriptionally derepressed genes, transgenes, and retrotransposons. RNA interference–mediated suppression of Set1, encoding an H3K4 methyltransferase, induced similar phenotypes, but of lower magnitude, and no detectable increase in H3K4me2. Together, our results suggest functional differentiation between dimethyl H3K4 and monomethyl H3K4, with the latter operating as an epigenetic mark for repressed euchromatin.
Role of myofibrillogenesis regulator-1 in myocardial hypertrophy
Authors: Liu Xiuhua , Li Tianbo , Sun Sheng , Xu Feifei , Wang Yiguang
Published: August 2005

Myofibrillogenesis regulator-1 (MR-1) is a novel homologous gene, identified from a human skeletal muscle cDNA library, that interacts with contractile proteins and exists in human myocardial myofibrils. The present study investigated MR-1 protein expression in hypertrophied myocardium and MR-1 involvement in cardiac hypertrophy. Cardiac hypertrophy was induced by abdominal aortic stenosis (AAS) in Sprague-Dawley rats. Left ventricular (LV) hypertrophy was assessed by the ratio of LV wet weight to whole heart weight (LV/HW) or LV weight to body weight (LV/BW). Rat MR-1 (rMR-1) expression in the myocardium was detected by immunohistochemical and Western blotting analysis. Hypertrophy was induced by ANG II incubation in cultured neonatal rat cardiomyocytes. The effect of rMR-1 RNA interference on ANG II-induced hypertrophy was studied by transfection of cardiomyocytes with an RNA interference plasmid, pSi-1, which targets rMR-1. Hypertrophy in cardiomyocytes was assessed by [3H]Leu incorporation and myocyte size. rMR-1 protein expression in cardiomyocytes was detected by Western blotting. We found that AAS resulted in a significant increase in LV/HW and LV/BW: 89% and 86%, respectively ( P < 0.01). Immunohistochemistry and Western blot analysis demonstrated upregulated rMR-1 protein expression in hypertrophic myocardium. ANG II induced a 24% increase in [3H]Leu incorporation and a 65.8% increase in cell size compared with control cardiomyocytes ( P < 0.01), which was prevented by treatment with losartan, an angiotensin (AT1) receptor inhibitor, or transfection with pSi-1. rMR-1 expression increased in ANG II-induced hypertrophied cardiomyocytes, and pSi-1 transfection abolished the upregulation. These findings suggest that MR-1 is associated with cardiac hypertrophy in rats in vivo and in vitro.