Flexible nasal bronchoscopy vs. Airtraq® videolaryngoscopy for awake tracheal intubation: a randomised controlled non-inferiority study.

Videolaryngoscopy is a suitable alternative to flexible bronchoscopy to facilitate awake tracheal intubation. The relative effectiveness of these techniques in clinical practice is unknown. We compared flexible nasal bronchoscopy with Airtraq® videolaryngoscopy in patients with an anticipated difficult airway scheduled for awake tracheal intubation. Patients were allocated randomly to flexible nasal bronchoscopy or videolaryngoscopy. All procedures were performed with upper airway regional anaesthesia blockade and a target-controlled intravenous infusion of remifentanil. The success rate with the allocated technique was the primary outcome. A non-inferiority analysis with a predefined limit of 8% was planned. Seventy-eight patients were recruited, allocated randomly and analysed. The rate of successful intubation was 97% and 82% in the flexible bronchoscopy and videolaryngoscopy groups, respectively, p = 0.032. The median (IQR [range]) time to tracheal intubation was shorter with the Airtraq, 163 (105-332 [40-1004]) vs. 217 (180-364 [120-780]) s, p = 0.030. There were no significant differences for complications found between the groups. The median visual analogue scale for ease of intubation was 8 (7-9 [0-10]) for Airtraq vs. 8 (7-9 [0-10]) for flexible bronchoscopy, p = 0.710. The median visual analogue scale for patient comfort for Airtraq was 8 (6-9 [2-10]) vs. 8 (7-9 [3-10]) for flexible bronchoscopy, p = 0.370. The Airtraq videolaryngoscope is not non-inferior to flexible bronchoscopy for awake tracheal intubation in a clinical setting when awake tracheal intubation is indicted. It may be a suitable alternative when judged on a case-by-case basis.

A National Referral Service for Paediatric Brachytherapy: An Evolving Practice and Outcomes Over 13 Years.

AIMS: Most children requiring radiotherapy receive external beam treatment and few have tumours suitable for brachytherapy. No paediatric radiotherapy centre will treat enough patients from its own normal catchment population for expertise in brachytherapy to be developed and sustained. Following discussion and agreement in the national paediatric radiotherapy group, a service for paediatric brachytherapy in the UK has been developed. We report the process that has evolved over more than 10 years, with survival and functional outcome results. MATERIALS AND METHODS: Since 2009, potential patients have been referred to the central paediatric oncology multidisciplinary team meeting, where imaging, pathology and treatment options are discussed. Since 2013, the National Soft Tissue Sarcoma Advisory Panel has also reviewed most patients, with the principal aim of advising on the most suitable primary tumour management for complex patients. Clinical assessment and examination under anaesthetic with biopsies may be undertaken to confirm the appropriateness of brachytherapy, either alone or following conservative surgery. Fractionated high dose rate brachytherapy was delivered to a computed tomography planned volume after implantation of catheters under ultrasound imaging guidance. Since 2019, follow-up has been in a dedicated multidisciplinary clinic. RESULTS: From 2009 to 2021 inclusive, 35 patients (16 female, 19 male, aged 8 months to 17 years 6 months) have been treated. Histology was soft-tissue sarcoma in 33 patients and carcinoma in two. The treated site was pelvic in 31 patients and head and neck in four. With a median follow-up of 5 years, the local control and overall survival rates are 100%. Complications have been few, and functional outcome is good. CONCLUSION: Brachytherapy is effective for selected paediatric patients, resulting in excellent tumour control and good functional results. It is feasible to deliver paediatric brachytherapy at a single centre within a national referral service.

Artificial intelligence for detection of microsatellite instability in colorectal cancer-a multicentric analysis of a pre-screening tool for clinical application.

BACKGROUND: Microsatellite instability (MSI)/mismatch repair deficiency (dMMR) is a key genetic feature which should be tested in every patient with colorectal cancer (CRC) according to medical guidelines. Artificial intelligence (AI) methods can detect MSI/dMMR directly in routine pathology slides, but the test performance has not been systematically investigated with predefined test thresholds. METHOD: We trained and validated AI-based MSI/dMMR detectors and evaluated predefined performance metrics using nine patient cohorts of 8343 patients across different countries and ethnicities. RESULTS: Classifiers achieved clinical-grade performance, yielding an area under the receiver operating curve (AUROC) of up to 0.96 without using any manual annotations. Subsequently, we show that the AI system can be applied as a rule-out test: by using cohort-specific thresholds, on average 52.73% of tumors in each surgical cohort [total number of MSI/dMMR = 1020, microsatellite stable (MSS)/ proficient mismatch repair (pMMR) = 7323 patients] could be identified as MSS/pMMR with a fixed sensitivity at 95%. In an additional cohort of N = 1530 (MSI/dMMR = 211, MSS/pMMR = 1319) endoscopy biopsy samples, the system achieved an AUROC of 0.89, and the cohort-specific threshold ruled out 44.12% of tumors with a fixed sensitivity at 95%. As a more robust alternative to cohort-specific thresholds, we showed that with a fixed threshold of 0.25 for all the cohorts, we can rule-out 25.51% in surgical specimens and 6.10% in biopsies. INTERPRETATION: When applied in a clinical setting, this means that the AI system can rule out MSI/dMMR in a quarter (with global thresholds) or half of all CRC patients (with local fine-tuning), thereby reducing cost and turnaround time for molecular profiling.

Conditional risk of diverticulitis after non-operative management.

BACKGROUND: The objective of this study was to describe conditional recurrence-free survival (RFS) of patients after an index episode of diverticulitis managed without surgery, and to estimate the difference in conditional RFS for diverticulitis according to specific risk factors. METHODS: This was a multicentre retrospective cohort study including all patients managed without surgery for acute sigmoid diverticulitis at two university-affiliated hospitals in Montreal, Quebec, Canada. Conditional RFS for diverticulitis was estimated over 10 years of follow-up. A Cox proportional hazards model was performed at the index episode and again 2 years later. RESULTS: In total, 991 patients were included for analysis. The 1, 2- and 3-year actuarial diverticulitis RFS rates were 81·1, 71·5 and 67·5 per cent respectively. Compared with the 1-year actuarial RFS rate of 81·1 per cent, the 1-year conditional RFS increased with each additional year survived recurrence-free, reaching 96·0 per cent after surviving the first 4 years recurrence-free. A similar phenomenon was observed for 2-year diverticulitis conditional RFS. Lower age (hazard ratio (HR) 0·98, 95 per cent c.i. 0·98 to 0·99), Charlson Co-morbidity Index score of 2 or above (HR 1·78, 1·32 to 2·39) and immunosuppression (HR 1·85, 1·38 to 2·48) were independently associated with recurrence of diverticulitis from the index episode. At 2 years from the index episode, immunosuppression was no longer associated with diverticulitis recurrence (HR 1·02, 0·50 to 2·09). CONCLUSION: The conditional RFS of patients with diverticulitis improved with each year that was survived recurrence-free. Although several factors at index presentation may be associated with early recurrence, the conditional probability of recurrence according to many of these risk factors converged with time.

ANTECEDENTES: El objetivo de este estudio fue describir la supervivencia condicional libre de recidiva de diverticulitis (diverticulitis recurrence-free survival, Div-RFS) en pacientes tras un episodio de diverticulitis tratado de forma conservadora, y calcular la diferencia en la Div-RFS condicional de acuerdo con factores de riesgo específicos. MÉTODOS: Estudio de cohorte retrospectivo multicéntrico que incluyó a todos los pacientes tratados de forma no quirúrgica por diverticulitis sigmoidea aguda en dos hospitales afiliados a la universidad en Montreal, Quebec, Canadá. La supervivencia condicional libre de recidiva de la diverticulitis se calculó durante 10 años de seguimiento. Se realizó un análisis mediante un modelo de riesgos proporcionales de Cox en el episodio índice y nuevamente 2 años después. RESULTADOS: En total, se incluyeron 991 pacientes en el análisis. La Div-RFS actuarial a 1, 2 y 3 años fue del 81,1%, 71,5% y 67,5%, respectivamente. En comparación con la Div-RFS actuarial a 1 año del 81,1%, la Div-RFS condicional a 1 año aumentó con cada año adicional sobrevivido sin recidiva, alcanzando el 96,0% después de sobrevivir los primeros 4 años sin recidiva. Se observó un fenómeno similar para Div-RFS condicional a los 2 años. Una menor edad (cociente de riesgos instantáneos, hazard ratio, HR: 0,98; i.c. del 95%: 0,98 a 0,99), la puntuación de comorbilidad de Charlson ≥ 2 (HR: 1,78; i.c. del 95%: 1,32 a 2,39) y la inmunosupresión (HR: 1,85; i.c. del 95%: 1,38 a 2,48) se asociaron de forma independiente con la recidiva de la diverticulitis desde el episodio índice. En la regresión de Cox a los 2 años del episodio índice, la inmunosupresión ya no se asoció con recidiva de diverticulitis (HR: 1,02; i.c. del 95% 0,50-2,09). CONCLUSIÓN: La Div-RFS condicional mejoró con cada año de supervivencia sin recidiva. Si bien varios factores en la presentación del episodio índice pueden estar asociados con una recidiva precoz, la probabilidad condicional de recidiva en relación con muchos de estos factores de riesgo coincidió con el tiempo.

Clinical relevance of gene expression in localized and metastatic prostate cancer exemplified by FABP5.

PURPOSE: Fatty acid-binding protein 5 (FABP5), a transport protein for lipophilic molecules, has been proposed as protein marker in prostate cancer (PCa). The role of FABP5 gene expression is merely unknown. METHODS: In two cohorts of PCa patients who underwent radical prostatectomy (n = 40 and n = 57) and one cohort of patients treated with palliative transurethral resection of the prostate (pTUR-P; n = 50) FABP5 mRNA expression was analyzed with qRT-PCR. Expression was correlated with clinical parameters. BPH tissue samples served as control. To independently validate findings on FABP5 expression, three microarray and sequencing datasets were reanalyzed (MSKCC 2010 n = 216; TCGA 2015 n = 333; mCRPC, Nature Medicine 2016 n = 114). FABP5 expression was correlated with ERG-fusion status, TCGA subtypes, cancer driver mutations and the expression of druggable downstream pathway components. RESULTS: FABP5 was overexpressed in PCa compared to BPH in the cohorts analyzed by qRT-PCR (radical prostatectomy p = 0.003, p = 0.010; pTUR-P p = 0.002). FABP5 expression was independent of T stage, Gleason Score, nodal status and PSA level. FABP5 overexpression was associated with the absence of TMPRSS2:ERG fusion (p < 0.001 in TCGA and MSKCC). Correlation with TCGA subtypes revealed FABP5 overexpression to be associated with SPOP and FOXA1 mutations. FABP5 was positively correlated with potential drug targets located downstream of FABP5 in the PPAR-signaling pathway. CONCLUSION: FABP5 overexpression is frequent in PCa, but seems to be restricted to TMPRESS2:ERG fusion-negative tumors and is associated with SPOP and FOXA1 mutations. FABP5 overexpression appears to be indicative for increased activity in PPAR signaling, which is potentially druggable.

Infantile and adult mortality in precarious conditions.

INTRODUCTION: Maxillofacial surgery plays an important role in humanitarian surgery because this specialty includes common procedures such as facial reconstruction after burns, facial clefts or Noma's sequelae, which do not require complex infrastructures and thus can be carried out in sub-optimal conditions. Nevertheless, there is a mortality rate during those missions and few studies evaluating their rate and circumstances have been published. The aim of our study was threefold: to study the deaths reported during humanitarian missions, to analyze the circumstances of those deaths, and to determine the risk factors in order to propose possible adaptations in operative indications and therapeutic attitudes so that those mortalities may be avoided in the future. MATERIAL AND METHOD: We evaluated the early deaths (between day 0 and day 8) observed among 3,494 patients operated on during humanitarian missions conducted between 2007 and 2018 in Africa and Asia by the charity organization "Les Enfants du Noma" (EDN). Information (patient, type of surgery, circumstances of death) was collected from the medical files and by questioning the medical teams who took part in the missions. RESULTS: Five deaths were recorded from 2007 to 2018, representing a mortality rate of 0.14%. The deceased were between 1 and 20 years of age and the deaths occurred between the day of the surgical procedure and the third post-operative day. The causes of death were as follows: two cases of respiratory failure due to mucous plugs (one in the endotracheal tube and one in the tracheotomy canule), one cardio-respiratory failure, one bilateral tension pneumothorax and one peritonitis from several duodenal perforations. DISCUSSION: In order to minimize the risks of lethal complications, it is important to identify as early as possible certain high-risk situations, to ensure the adequacy of the means available, and to establish an indication for surgery by a concerted discussion among the surgeons and anesthetists involved. One of the general limitations of humanitarian missions is the lack of resuscitation facilities and the lack of availability of further exams. Rites, beliefs, superstitions, gri-gri and ancestral practices often hold a prominent place in these cultures. The absence of well-kept medical records (including personal and family histories), language barriers, large volumes of patients to manage, the inexperience of first-time teams, the lack of knowledge of the terrain and local health conditions, and an inability to empathize with patients, limit the quality of preoperative assessment and sometimes lead to poor decisions.

Prediction model and web-based risk calculator for postoperative ileus after loop ileostomy closure.

BACKGROUND: Postoperative ileus (POI) is a significant complication after loop ileostomy closure given both its frequency and impact on the patient. The purpose of this study was to develop and externally validate a prediction model for POI after loop ileostomy closure. METHODS: The model was developed and validated according to the TRIPOD checklist for prediction model development and validation. The development cohort included consecutive patients who underwent loop ileostomy closure in two teaching hospitals in Montreal, Canada. Candidate variables considered for inclusion in the model were chosen a priori based on subject knowledge. The final prediction model, which modelled the 30-day cumulative incidence of POI using logistic regression, was selected using the highest area under the receiver operating characteristic curve (AUC) criterion. Model calibration was assessed using the Hosmer-Lemeshow goodness-of-fit test. The model was then validated externally in an independent cohort of similar patients from the University of British Columbia. RESULTS: The development cohort included 531 patients, in whom the incidence of POI was 16·8 per cent. The final model included five variables: age, ASA fitness grade, underlying pathology/treatment, interval between ileostomy creation and closure, and duration of surgery for ileostomy closure (AUC 0·68, 95 per cent c.i. 0·61 to 0·74). The model demonstrated good calibration (P = 0·142). The validation cohort consisted of 216 patients, and the incidence of POI was 15·7 per cent. On external validation, the model maintained good discrimination (AUC 0·72, 0·63 to 0·81) and calibration (P = 0·538). CONCLUSION: A prediction model was developed for POI after loop ileostomy closure and included five variables. The model maintained good performance on external validation.

ANTECEDENTES: El íleo postoperatorio (postoperative ileus, POI) es una complicación importante tras el cierre de la ileostomía en asa, dada su frecuencia e impacto en el paciente. El propósito de este estudio fue desarrollar y validar externamente un modelo de predicción para el POI después del cierre de la ileostomía en asa. MÉTODOS: El modelo fue desarrollado y validado de acuerdo con la lista de verificación TRIPOD para el desarrollo y validación de un modelo de predicción. La cohorte de desarrollo incluyó pacientes consecutivos en los que se realizó el cierre de la ileostomía en asa en dos hospitales universitarios en Montreal, Canadá. Las variables candidatas consideradas para su inclusión en el modelo se seleccionaron a priori en función del conocimiento del problema. El modelo de predicción final, que modeló la incidencia acumulada a 30 días de POI mediante regresión logística, se seleccionó según el criterio del área más alta bajo la curva operativa del receptor (area under the receiver operating curve, AUC). La calibración del modelo se evaluó utilizando la prueba de bondad de ajuste de Hosmer-Lemeshow. El modelo fue posteriormente validado externamente en una cohorte independiente de pacientes similares de la Universidad de British Columbia. RESULTADOS: La cohorte de desarrollo incluyó a 531 pacientes, y la incidencia de POI fue de 16,7%. El modelo final incluyó cinco variables: edad, clasificación ASA (American Society of Anaesthesiologists), patología inicial y tratamiento, tiempo entre las dos intervenciones quirúrgicas y tiempo operatorio del cierre de ileostomía (AUC = 0,68; i.c. del 95%: 0,61 a 0,74). El modelo demostró buena calibración (P = 0,142). La cohorte de validación consistió en 216 pacientes, y la incidencia de POI fue de 15,7%. En la validación externa, el modelo mantuvo una buena discriminación (AUC = 0,72; i.c. del 95%: 0,63 a 0,81) y calibración (P = 0,538). CONCLUSIÓN: Se ha desarrollado un modelo de predicción de POI después del cierre de la ileostomía en asa que incluía cinco variables. El modelo mantuvo un buen funcionamiento en la validación externa.

Assessing the readability, quality and accuracy of online health information for patients with low anterior resection syndrome following surgery for rectal cancer.

AIM: Management of low anterior resection syndrome (LARS) requires a high degree of patient engagement. This process may be facilitated by online health-related information and education. The aim of this study was to systematically review current online health information on LARS. METHOD: An online search of Google, Yahoo and Bing was performed using the search terms 'low anterior/anterior resection syndrome' and 'bowel function/movements after rectal cancer surgery'. Websites were assessed for readability (eight standardized tests), suitability (using the Suitability Assessment of Materials instrument), quality (the DISCERN instrument), accuracy and content (using a LARS-specific content checklist). Websites were categorized as academic, governmental, nonprofit or private. RESULTS: Of 117 unique websites, 25 met the inclusion criteria. The median readability level was 10.4 (9.2-11.7) and 11 (44.0%) websites were highly suitable. Using the DISCERN instrument, seven (28.0%) websites had clear aims, two (8.0%) divulged the sources used and four (16.0%) had high overall quality. Only eight (32.0%) websites defined LARS and ten (40.0%) listed all five major symptoms associated with the LARS score. There was variation in the number of websites that discussed dietary modifications (80.0%), self-help strategies (72.0%), medication (68.0%), pelvic floor rehabilitation (60.0%) and neuromodulation (8.0%). The median accuracy of websites was 93.8% (88.2-96.7%). Governmental websites scored highest for overall suitability (P = 0.0079) and quality (P < 0.001). CONCLUSIONS: Current online information on LARS is suboptimal. Websites are highly variable, important content is often lacking and material is too complex for patients.

Study protocol evaluating the use of bowel stimulation before loop ileostomy closure to reduce postoperative ileus: a multicenter randomized controlled trial.

AIM: Postoperative ileus is the most commonly observed morbidity following ileostomy closure. Studies have demonstrated that the defunctionalized bowel of a loop ileostomy undergoes a series of functional and structural changes, such as atrophy of the intestinal villi and muscular layers, which may contribute to ileus. A single-centre study in Spain demonstrated that preoperative bowel stimulation via the distal limb of the loop ileostomy decreased postoperative ileus, length of stay and time to gastrointestinal function. METHOD: A multicentre randomized controlled trial involving patients from Canadian institutions was designed to evaluate the effect of preoperative bowel stimulation before ileostomy closure on postoperative ileus. Stimulation will include canalizing the distal limb of the ileostomy loop with an 18Fr Foley catheter and infusing it with a solution of 500 ml of normal saline mixed with 30 g of a thickening agent (Nestle© Thicken-Up© ). This will be performed 10 times over the 3 weeks before ileostomy closure in an outpatient clinic setting by a trained Enterostomal Therapy nurse. Surgeons and the treating surgical team will be blinded to their patient's group allocation. Data regarding patient demographics, and operative and postoperative variables, will be collected prospectively. Primary outcome will be postoperative ileus, defined as an intolerance to oral food in the absence of clinical or radiological signs of obstruction, that either requires nasogastric tube insertion or is associated with two of the following on or after post-operative day 3: nausea/vomiting; abdominal distension; and the absence of flatus. Secondary outcomes will include length of stay, time to tolerating a regular diet, time to first passage of flatus or stool and overall morbidity. A cost analysis will be performed to compare the costs of conventional care with conventional care plus preoperative stimulation. DISCUSSION: This manuscript discusses the potential benefits of preoperative bowel stimulation in improving postoperative outcomes and outlines our protocol for the first multicenter study to evaluate preoperative bowel stimulation before ileostomy closure. The results of this study could have considerable implications for the care of patients undergoing ileostomy closure.

Wnt signaling in cancer.

Wnt signaling is one of the key cascades regulating development and stemness, and has also been tightly associated with cancer. The role of Wnt signaling in carcinogenesis has most prominently been described for colorectal cancer, but aberrant Wnt signaling is observed in many more cancer entities. Here, we review current insights into novel components of Wnt pathways and describe their impact on cancer development. Furthermore, we highlight expanding functions of Wnt signaling for both solid and liquid tumors. We also describe current findings how Wnt signaling affects maintenance of cancer stem cells, metastasis and immune control. Finally, we provide an overview of current strategies to antagonize Wnt signaling in cancer and challenges that are associated with such approaches.

CT-based adaptive high-dose-rate endorectal brachytherapy in the preoperative treatment of locally advanced rectal cancer: Technical and practical aspects.

PURPOSE: During the last decade due to the availability of a CT scan in the brachytherapy suite, high-dose-rate endorectal brachytherapy (HDREBT) has evolved as a CT-based daily adaptive treatment. An update of the technical and practical aspects of HDREBT is provided. METHODS AND MATERIALS: Description of technical and practical aspects of HDREBT focused on the preoperative treatment of locally advanced rectal cancer. During preoperative HDREBT, 26 Gy is delivered in four daily applications of 6.5 Gy prescribed to the 100% isodose, covering the clinical target volume. Daily CT scans are obtained and used for plan optimization, leaving patient positioning unchanged between CT scan and treatment delivery. RESULTS: All steps of HDREBT treatment procedure are discussed in detail: flexible proctosigmoidoscopy and clipping; patient setup; applicator placement; target delineation; treatment planning and delivery; and patient care. Afterward, treatment results are reviewed. CONCLUSIONS: CT-based adaptive preoperative HDREBT is a practical and feasible therapy for locally advanced rectal cancer, offering excellent local control with a favorable toxicity profile.

Biliary leakage from gallbladder bed after cholecystectomy: Luschka duct or hepaticocholecystic duct?

Anatomic variations in the biliary tract are common and can cause difficulties when a cholecystectomy is performed. One of the most common ones are hepaticocholecystic ducts and Luschka ducts, connecting the gallbladder or its bed to the bile ducts but distinction between these two types of ducts can be difficult. We do discuss here the differences between these anatomical variations, their origin and their clinical implications. These aberrant ducts may go unnoticed and may require further complementary procedures in case of postoperative biliary leakage. In addition to a careful surgical procedure and an examination of the cystic bed in the end of the intervention, an intraoperative cholangiography should be performed as often as possible.

Role of prophylactic ureteric stents in colorectal surgery.

Ureteric injury is a feared complication in colorectal surgery, with a reported incidence of 0.2%-7.6%. Prophylactic ureteric catheter placement has the advantage of facilitating intraoperative ureter identification and assisting in immediate injury recognition and repair. However, its use has been controversial because of fear of ureteric damage during catheter insertion and postoperative urinary complications such as obstructive oliguria and urinary tract infection. Although the exact indications for prophylactic catheter placement are not clearly defined, it is generally used for reoperative cases, large tumors, previous radiation therapy, diverticulitis, fistulas, Crohn's disease and obesity. Herein, we review the incidence and risk factors for ureteric injury, the role of prophylactic ureteric stents and the complications and costs associated with its use in both open and laparoscopic colorectal surgery.

Gene knockdown studies revealed CCDC50 as a candidate gene in mantle cell lymphoma and chronic lymphocytic leukemia.

The two B-cell non-Hodgkin's lymphoma entities, chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), show recurrent chromosomal gains of 3q25-q29, 12q13-q14 and 18q21-q22. The pathomechanisms affected by these aberrations are not understood. The aim of this study was to identify genes, located within these gained regions, which control cell death and cell survival of MCL and CLL cancer cells. Blood samples collected from 18 patients with CLL and 6 patients with MCL, as well as 6 cell lines representing both malignancies were analyzed by gene expression profiling. By a comparison of genomic DNA and gene expression, 72 candidate genes were identified. We performed a limited RNA interference screening with these candidates to identify genes affecting cell survival. CCDC50 (coiled coil domain containing protein 50), SERPINI2 and SMARCC2 mediated a reduction of cell viability in primary CLL cells as well as in cell lines. Gene knockdown and a nuclear factor kappa B (NFkappaB) reporter gene assay revealed that CCDC50 is required for survival in MCL and CLL cells and controls NFkappaB signaling.

An integrated gene annotation and transcriptional profiling approach towards the full gene content of the Drosophila genome.

BACKGROUND: While the genome sequences for a variety of organisms are now available, the precise number of the genes encoded is still a matter of debate. For the human genome several stringent annotation approaches have resulted in the same number of potential genes, but a careful comparison revealed only limited overlap. This indicates that only the combination of different computational prediction methods and experimental evaluation of such in silico data will provide more complete genome annotations. In order to get a more complete gene content of the Drosophila melanogaster genome, we based our new D. melanogaster whole-transcriptome microarray, the Heidelberg FlyArray, on the combination of the Berkeley Drosophila Genome Project (BDGP) annotation and a novel ab initio gene prediction of lower stringency using the Fgenesh software. RESULTS: Here we provide evidence for the transcription of approximately 2,600 additional genes predicted by Fgenesh. Validation of the developmental profiling data by RT-PCR and in situ hybridization indicates a lower limit of 2,000 novel annotations, thus substantially raising the number of genes that make a fly. CONCLUSIONS: The successful design and application of this novel Drosophila microarray on the basis of our integrated in silico/wet biology approach confirms our expectation that in silico approaches alone will always tend to be incomplete. The identification of at least 2,000 novel genes highlights the importance of gathering experimental evidence to discover all genes within a genome. Moreover, as such an approach is independent of homology criteria, it will allow the discovery of novel genes unrelated to known protein families or those that have not been strictly conserved between species.

HCF-1 amino- and carboxy-terminal subunit association through two separate sets of interaction modules: involvement of fibronectin type 3 repeats.

When herpes simplex virus infects permissive cells, the viral regulatory protein VP16 forms a specific complex with HCF-1, a preexisting nuclear protein involved in cell proliferation. The majority of HCF-1 in the cell is a complex of associated amino (HCF-1(N))- and carboxy (HCF-1(C))-terminal subunits that result from an unusual proteolytic processing of a large precursor polypeptide. Here, we have characterized the structure and function of sequences required for HCF-1(N) and HCF-1(C) subunit association. HCF-1 contains two matched pairs of self-association sequences called SAS1 and SAS2. One of these matched association sequences, SAS1, consists of a short 43-amino-acid region of the HCF-1(N) subunit, which associates with a carboxy-terminal region of the HCF-1(C) subunit that is composed of a tandem pair of fibronectin type 3 repeats, a structural motif known to promote protein-protein interactions. Unexpectedly, the related protein HCF-2, which is not proteolyzed, also contains a functional SAS1 association element, suggesting that this element does not function solely to maintain HCF-1(N) and HCF-1(C) subunit association. HCF-1(N) subunits do not possess a nuclear localization signal. We show that, owing to a carboxy-terminal HCF-1 nuclear localization signal, HCF-1(C) subunits can recruit HCF-1(N) subunits to the nucleus.

Signaling specificity by Frizzled receptors in Drosophila.

Wnt-Frizzled (Fz) signaling pathways play recurring important roles during the development and homeostasis of vertebrates and invertebrates. Fz receptors can signal through beta-catenin-dependent and -independent pathways. In Drosophila, Fz and Fz2 are redundant receptors for Wg. In addition, Fz conveys signals through a distinct pathway to organize planar polarization of epithelial structures. We demonstrate that the cytoplasmic sequences of Fz2 and Fz preferentially activate the beta-catenin and planar polarity cascade, respectively. Both receptors activate either pathway, but with different efficiencies. Intrinsic differences in signaling efficiency in closely related receptors might be a general mechanism for generating signaling specificity in vivo.

The molecular basis of cystinuria: an update.

Cystinuria is a hereditary disorder of cystine and dibasic amino acid transport across the luminal membrane of renal proximal tubule and small intestine. In 1992, a cDNA (rBAT) was isolated from kidney which induced high-affinity, sodium-independent uptake of cystine and dibasic amino acids when expressed in Xenopus oocytes. The rBAT gene was mapped to a region of chromosome 2p known to contain a cystinuria locus, and rBAT expression was demonstrated in the straight (S3) portion of renal proximal tubule and small intestine. Over 30 distinct rBAT mutations have been described in patients who inherit two fully recessive (type I) cystinuria genes. Recently, the second cystinuria gene (SLC7A9) on chromosome 19q was identified; SLC7A9 mutations were shown to cause the incompletely recessive form of cystinuria (types II and III). Patients who inherit two mutant SLC7A9 genes have recurrent nephrolithiasis comparable to those with two rBAT mutations. In some cystinuria families, patients inherit a fully recessive allele from one parent and an incompletely recessive allele from the other parent; patients with this 'mixed type' of cystinuria have somewhat milder disease. It is not yet clear whether this form of cystinuria involves rBAT as well as SLC7A9 mutations. Current evidence suggests that the transmembrane channel mediating uptake of cystine and dibasic amino acids at the luminal surface is encoded by SLC7A9; the smaller rBAT protein forms a heterodimeric complex with this channel and is critical for its targetting to the luminal membrane.