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Objectives: Esthesioneuroblastoma is an uncommon cancer of the nasal cavity. We describe the outcomes for 26 patients treated with curative intent with photon radiotherapy (RT) at the University of Florida., Methods: Between May 1972 and June 2007, 26 patients received RT for previously untreated esthesioneuroblastoma of the nasal cavity. Sixteen patients were males and 10 were females with a median age of 55 years (range, 3 to 82 y). The modified Kadish stage distribution was: B, 7 patients; C, 17 patients; and D, 2 patients. Treatment modalities included the following: definitive RT, 5 patients; preoperative RT, 2 patients; and postoperative RT after resection, 19 patients. Elective neck irradiation (ENI) was performed in 17 (71%) of 24 N0 patients., Results: Rates of local control, cause-specific survival, and absolute overall survival at 5 years were 79%, 72%, and 69%, respectively. Overall survival among patients treated with definitive RT was 20% at 5 years, compared with 81% among those who underwent surgery and adjuvant RT (P=0.01). One (6%) of 17 patients who received ENI developed a recurrence in the neck and was successfully salvaged. Ultimate neck control was 100% at 5 years for patients who received ENI versus 69% among those not receiving ENI (P=0.0173)., Conclusions: Resection combined with adjuvant RT is more effective than surgery or RT alone in the treatment of esthesioneuroblastoma. ENI reduces the risk of regional relapse in patients with Kadish stage B and C cancers.

Objectives: To evaluate the efficacy of postoperative radiotherapy for oral cavity squamous cell carcinoma (OCSCC) by comparing outcomes of high-risk subgroups., Study Design: Retrospective review., Methods: Outcome study of 139 patients with OCSCC treated with gross total resection and postoperative radiotherapy ± chemotherapy and at least one high-risk pathologic finding: positive margin (52%), close (0.1-5 mm) margin (27%), or extracapsular nodal extension (ECE; 45%)., Results: Median follow-up was 2.3 years. Local-regional control (LRC), freedom from distant metastases, cause-specific survival, and overall survival (OS) rates at 5 years were 64%, 85%, 51%, and 36%, respectively. Five-year LRC for negative (>5 mm), close (0.1-5 mm), and positive (carcinoma in situ or tumor at ink) margins were 73%, 83%, and 63%, respectively (P = not significant). Five-year neck control was 100% for node-negative patients, 88% for node-positive patients with no ECE, and 86% for node-positive patients with ECE (P = not significant). The combination of close/positive margin and ECE resulted in worse 5-year LRC (37% vs. 70%, P < 0.001), progression-free survival (26% vs. 60%, P < 0.001), and OS (13 vs. 43%, P < 0.001) compared with a single high-risk indication., Conclusions: Local-regional control was the predominant mode of treatment failure. Outcome in our series was not statistically different based on margin status or nodal ECE. This finding is indirect evidence of the efficacy of adjuvant radiotherapy in this setting., (© 2014 The American Laryngological, Rhinological and Otological Society, Inc.)

The expression, localization and activity of the serum- and glucocorticoid-induced protein kinase, Sgk-1, are regulated by multiple hormonal and environmental cues including cellular stress. Biochemical fractionation and indirect immunofluorescence demonstrated that sorbitol induced hyperosmotic stress stimulated expression and triggered the localization of endogenous Sgk-1 into the mitochondria of NMuMG mammary epithelial cells. The immunofluorescence pattern of endogenous Sgk-1 was similar to that of a green fluorescent linked fusion protein linked to the N-terminal Sgk-1 fragment that encodes the mitochondrial targeting signal. In the presence or absence of cellular stress, exogenously expressed wild type Sgk-1 efficiently compartmentalized into the mitochondria demonstrating the mitochondrial import machinery per se is not stressed regulated. Co-immunoprecipitation and GST-pull down assays identified the IF-1 mitochondrial matrix inhibitor of the F1F0-ATPase as a new Sgk-1 binding partner, which represents the first observed mitochondrial target of Sgk-1. The Sgk-1/IF-1 interaction requires the 122-176 amino acid region within the catalytic domain of Sgk-1 and is pH dependent, occurring at neutral pH but not at slightly acidic pH, which suggests that this interaction is dependent on mitochondrial integrity. An in vitro protein kinase assay showed that the F1F0-ATPase can be directly phosphorylated by Sgk-1. Taken together, our results suggest that stress-induced Sgk-1 localizes to the mitochondria, which permits access to physiologically appropriate mitochondrial interacting proteins and substrates, such as IF-1 and the F1F0-ATPase, as part of the cellular stressed induced program., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Intrathyroidal parathyroid carcinoma is an exceedingly rare cause of primary hyperparathyroidism. A 51-year-old African American female presented with goiter, hyperparathyroidism, and symptomatic hypercalcemia. Sestamibi scan revealed diffuse activity within an enlarged thyroid gland with uptake in the right thyroid lobe suggestive of hyperfunctioning parathyroid tissue. The patient underwent thyroidectomy and parathyroidectomy. At exploration, a 2.0 cm nodule in the usual location of the right inferior parathyroid was sent for intraoperative frozen consultation, which revealed only ectopic thyroid tissue. No parathyroid glands were identified grossly on the external aspect of the thyroid. Interestingly, postoperative parathyroid hormone levels normalized after removal of the thyroid gland. Examination of the thyroidectomy specimen revealed a 1.4 cm parathyroid nodule located within the parenchyma of the right superior thyroid, with capsular and vascular invasion and local infiltration into surrounding thyroid tissue. We present only the eighth reported case of intrathyroidal parathyroid carcinoma and review the literature.

Purpose: To analyze the results of definitive radiation therapy (RT) for squamous cell carcinoma of the pharyngeal wall., Methods and Materials: Between 1964 and 2009, 170 patients were treated with definitive RT; all living patients had a 1.7-year minimum follow-up., Results: The 5-year rates of local control and ultimate local control were the following: T1, 93% and 93%; T2, 84% and 91%; T3, 60% and 62%; and T4, 44% and 44%. Multivariate analysis revealed stage I-II tumors, female gender, and altered fractionation were associated with improved local-regional control. The 5-year cause-specific and overall survival rates were the following: I, 88% and 50%; II, 89% and 57%; III, 49% and 31%; IV, 35% and 21%; and overall, 50% and 31%, respectively. Fatal complications occurred in 9 patients (5%)., Conclusions: Local-regional control and survival are related to extent of disease and treatment technique. Although outcomes have improved in recent years, the morbidity of treatment is significant and a substantial proportion of patients die due to cancer., (Copyright © 2012 American Society for Radiation Oncology. Published by Elsevier Inc. All rights reserved.)

A unique case of IgG4(+) sclerosing disease was diagnosed in the sphenoid sinus, a previously unreported location, and was treated in a novel manner. This study describes the clinical presentation and management of IgG4 sclerosing disease in the paranasal sinuses. A retrospective case review and review of the medical literature were performed. A 38-year-old woman with a 2-year history of constant frontal headaches presented to our clinic. Imaging showed bony destruction of the sphenoid sinus and sellar floor. The patient underwent a right-sided sphenoidotomy with debridement and biopsy. Pathological evaluation showed a dense plasmacytic infiltrate with >150 IgG4(+) cells/high-power field. She was subsequently started on a nasal corticosteroid with improved patency of the sphenoid antrostomy. We report an unusual case of a middle-aged woman who presented with IgG4-sclerosing disease (IGSD) isolated to the sphenoid sinus. Although our knowledge concerning treatment in extrapancreatic organs is lacking, there is evidence that glucocorticoid treatment improves nasal sinus opacification on CT findings (Sato Y, Ohshima K, Ichimura K, et al., Ocular adnexal IgG4-related disease has uniform clinicopathology, Pathol Int 58:465-470, 2008). This case study and literature review adds to the growing literature describing IGSD in the head and neck and more specifically isolated to the sphenoid sinus with preliminary data concerning local control with topical steroids.

Objective: The objective of the study was to discuss the optimal management and treatment outcomes for patients with head and neck osteosarcomas., Study Design: Review article., Methods: Review of the pertinent literature., Results: Osteosarcomas account for approximately 1% or less of all head and neck cancers. The vast majority occur in the mandible and maxilla. The median age is in the fourth decade, with a wide range. They are more likely to recur locally after treatment and distant metastases are observed less often than with the more common osteosarcomas arising in the long bones. The optimal treatment is complete resection. The role of adjuvant chemotherapy is ill-defined. The vast majority of recurrences are observed within 5 years. The 5-year disease-specific and overall survival rates are approximately 60% to 70%., Conclusions: Osteosarcoma of the head and neck is a rare entity that occurs primarily in the mandible and maxilla. The optimal treatment is surgery. Adjuvant radiotherapy should be considered for those with close or positive margins. The role of adjuvant chemotherapy is ill-defined. The likelihood of cure is approximately 60% to 70%., (Copyright © 2011 Elsevier Inc. All rights reserved.)

B cell lymphoma survival requires tonic or ligand-independent signals through activation of Syk by the B cell receptor. The Epstein-Barr virus (EBV) protein latent membrane 2a (LMP2a), a mimic of the B cell receptor, provides constitutive survival signals for latently infected cells through Syk activation; however, the precise downstream mechanisms coordinating this survival response in EBV+ B cell lymphomas remain to be elucidated. Herein, we assess the mechanism of Syk survival signaling in EBV+ B cell lymphomas from post-transplant lymphoproliferative disorder (PTLD) to discover virally controlled therapeutic targets involved in lymphomagenesis and tumor progression. Using small molecule inhibition and siRNA strategies, we show that Syk inhibition reduces proliferation and induces apoptosis of PTLD-derived EBV+ B cell lines. Syk inhibition also reduces autocrine IL-10 production. Although Syk inhibition attenuates signaling through both the PI3K/Akt and Erk pathways, only PI3K/Akt inhibition causes apoptosis of PTLD-derived cell lines. Loss of the endogenous caspase inhibitor XIAP is observed after Syk or PI3K/Akt inhibition. The loss of XIAP and apoptosis that results from Syk or PI3K/Akt inhibition is reversed by inhibition of the mitochondrial protease HtrA2. Thus, Syk drives EBV+ B cell lymphoma survival through PI3K/Akt activation, which prevents the HtrA2-dependent loss of XIAP. Syk, Akt, and XIAP antagonists may present potential new therapeutic strategies for PTLD through targeting of EBV-driven survival signals.

Background: The purpose of this study was to describe the natural history and optimal treatment for head and neck paragangliomas (PGs)., Methods: Our methods were the review of the pertinent literature., Results: PGs are rare tumors seen most commonly in the head and neck. Approximately 90% are sporadic; the remainder are familial and related to mutations of the succinate dehydrogenase (SDH) gene complex. Most PGs are benign and slow growing; 6% to 19% are malignant, as evidenced by the development of metastases. PGs may be treated by complete resection or moderate-dose radiotherapy with a ≥90% likelihood of cure. The optimal radiotherapy dose is approximately 45 Gy/25 fractions/5 weeks. The treatment modality selected depends on the risk of complications. Due to their rarity, the optimal treatment for malignant PGs is unclear., Conclusion: PGs may be treated by either complete resection or radiotherapy with a high likelihood of success. Treatment depends on the location and extent of the PG and the morbidity associated with treatment., (Copyright © 2010 Wiley Periodicals, Inc.)

Objectives: Maxillary sinus squamous cell carcinoma is commonly diagnosed at an advanced stage and treated using radiotherapy, with or without surgical resection., Methods: Fifty-four patients with maxillary sinus squamous cell carcinoma were treated from 1969 to 2006, using radiotherapy, with or without surgical resection. Fifty-two (96%) patients had American Joint Committee on Cancer stages III to IV disease, and 45 (83%) patients had N0 neck disease., Results: Five-year local control (LC) rates by T-stage were 63% for T2/T3; and 43% for T4. Five-year LC rates for patients treated with radiotherapy preoperatively, postoperatively, and definitively were 61%, 65%, and 37%, respectively. Initially, overall 5-year LC, neck control, and local-regional control were 49%, 82%, and 45%, respectively. The ultimate 5-year LC, neck control, and local regional control after salvage of failures were 51%, 87%, and 50%, respectively. The overall 5-year cause-specific survival was 41%. Thirty-three percent of patients had a severe complication., Conclusions: Radiotherapy, with or without surgical resection, remains an effective tool in treating patients with this disease. LC, cause-specific survival, and complication rates need significant improvement. Treatment details and recommendations are discussed herein.

We have identified a new role for the matrix enzyme lysyl oxidase-like-2 (LOXL2) in the creation and maintenance of the pathologic microenvironment of cancer and fibrotic disease. Our analysis of biopsies from human tumors and fibrotic lung and liver tissues revealed an increase in LOXL2 in disease-associated stroma and limited expression in healthy tissues. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023) was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, desmoplasia and endothelial cells, decreased production of growth factors and cytokines and decreased transforming growth factor-beta (TGF-beta) pathway signaling. AB0023 outperformed the small-molecule lysyl oxidase inhibitor beta-aminoproprionitrile. The efficacy and safety of LOXL2-specific AB0023 represents a new therapeutic approach with broad applicability in oncologic and fibrotic diseases.

In this report, we assessed the steady-state enzymatic activity of lysyl oxidase-like 2 (LOXL2) against the substrates 1,5-diaminopentane (DAP), spermine, and fibrillar type I collagen. We find that both DAP and spermine are capable of activating LOXL2 to the same extent and have similar Michaelis constants (K(m) approximately 1 mm) and catalytic rates (k(cat) approximately 0.02 s(-1)). We also show that LOXL2 is capable of being inhibited by a known suicide inhibitor of lysyl oxidase (LOX), beta-aminopropionitrile, which we find is a potent inhibitor of LOXL2 activity. The modality of inhibition of beta-aminopropionitrile was also examined and found to be competitive with respect to the substrates DAP and spermine. In addition, we identified an antibody inhibitor (AB0023) of LOXL2 enzymatic function and have found that the inhibition occurs in a non-competitive manner with respect to both spermine and DAP. The binding epitope of AB0023 was mapped to the scavenger receptor cysteine-rich domain four of human LOXL2. AB0023 binds to a region remote from the catalytic domain making AB0023 an allosteric inhibitor of LOXL2. This affords AB0023 several advantages, because it is specific for LOXL2 and inhibits the enzymatic function of LOXL2 in a non-competitive manner thereby allowing inhibition of LOXL2 regardless of substrate concentration. These results suggest that antibody allosteric modulators of enzymatic function represent a novel drug development strategy and, in the context of LOXL2, suggest that inhibitors such as these might be useful therapeutics in oncology, fibrosis, and inflammation.

Background: The aim of this review was to discuss the role of altered fractionation and adjuvant chemotherapy for patients treated with definitive radiotherapy (RT) for head and neck squamous cell carcinoma (HNSCC)., Methods: This review explores the pertinent literature and discusses the optimal management of previously untreated patients with stage III-stage IVA and/or -B HNSCCs., Results: Depending on the schedule, altered fractionation improves locoregional control and survival. Both hyperfractionation and concomitant boost RT improve locoregional control and are associated with improved overall survival (OS). Adjuvant chemotherapy improves OS; the greatest impact is observed after concomitant versus induction or maintenance chemotherapy. Monochemotherapy appears to be equivalent to polychemotherapy. Drugs associated with the greatest survival benefit include fluorouracil and cisplatin. Intraarterial chemotherapy offers no advantage over intravenous chemotherapy. Concomitant cetuximab and RT results in improved outcomes similar to those observed after concomitant cisplatin-based chemotherapy and RT., Conclusions: Altered fractionation and/or concomitant chemotherapy result in improved outcomes compared with conventionally fractionated definitive RT alone for stage III-stage IV HNSCC. The optimal combination of RT fractionation and chemotherapy remains unclear., ((c) 2009 Wiley Periodicals, Inc.)

Objectives/hypothesis: To define the role of elective neck dissection during salvage surgery for locally recurrent head and neck squamous cell carcinoma (SCCA) initially treated with elective nodal irradiation (ENI)., Study Design: Retrospective chart review., Methods: We reviewed the medical records of patients treated with ENI at our institution from 1965 to 2006 for T1-4 N0 M0 SCCA of the oropharynx, hypopharynx, or larynx who developed an isolated local recurrence and remained N0. Fifty-seven patients were salvaged, 40 with neck dissection and 17 with neck observation. We then compared toxicity and actuarial outcomes between the two groups. Results were compared to the pertinent literature in a pooled analysis., Results: Four of 46 (9%) heminecks were found to have occult metastases in dissected specimens. The 5-year local-regional control rate was 75% for all patients. Neck dissection resulted in poorer outcomes compared with observation. In the dissected group, the 5-year local control, regional control, cause-specific survival, and overall survival rates were 71%, 87%, 60%, and 45%, respectively, compared to 82%, 94%, 92%, and 56%, respectively, for the observed group. Toxicity was more likely with dissection. In the pooled analysis totaling 230 patients, the overall pathologic positive rate of neck-dissection specimens was 9.6%; the compiled data showed no improvement in outcomes when salvage included neck dissection., Conclusions: Routine elective neck dissection should not be included during salvage surgery for locally recurrent head and neck SCCA if initial radiotherapy includes ENI. The risk of occult neck disease is low, outcomes do not improve, and the likelihood of toxicity increases. Laryngoscope, 2010.

Educational Objectives: Describe the clinical presentation and management of tracheoceles., Study Design: Retrospective case review and review of the medical literature., Results: A 56 year old female patient presented with progressive dysphonia and history of benign thyroid nodules. Flexible laryngoscopy revealed a severely paretic right true vocal cord. Computed tomography revealed a right sided air filled sac in the tracheoesophageal groove suspicious for causing compression of the right recurrent laryngeal nerve. The patient underwent an elective right hemithyroidectomy and resection of the air filled sac. Post operative pathology of the air filled sax was consistent with a tracheocele. A laryngoscopy performed 6 months postoperatively demonstrated recovery of right cord function., Conclusions: Tracheoceles are an uncommon entity first described in 1846 by Rokitansky (1) with only a paucity of case reports in the literature describing the surgical management and treatment of this disease. Herein, we report a patient who presented with dysphonia from a tracheocele in the right tracheoesophageal groove who subsequently underwent surgical resection ultimately improving vocal cord function.

Objective: Squamous cell carcinoma (SCCA) of the external auditory canal (EAC) is often treated with a combination of surgery and radiotherapy (RT) to optimize the chance of achieving locoregional control. This retrospective review describes a 27-year experience of treating these tumors at the University of Florida., Methods: Thirty patients with histologically confirmed SCCA of the EAC received external-beam radiation (RT) alone or combined with surgical resection between 1976 and 2003. Seven patients were treated with RT alone, 22 with postoperative RT, and 1with preoperative RT. Patients were grouped according to nodal status (N0/N1) and the Stell staging system for tumors of the EAC and middle ear. Early stage was defined as T1/T2 (n = 12) and advanced stage as T3 (n = 18). Median follow-up was 2 years (range, 0.1-19.4 years) with no patients lost to follow-up., Results: The 5-year actuarial probabilities for local control, locoregional control, and cause-specific survival for patients with early stage (T1/T2) versus advanced-stage (T3) tumors were 74% and 55% (P = 0.27), 63% and 38% (P = 0.16), and 70% and 41% (P = 0.04), respectively. The regional control rate was 83% (P = 0.6). There were 12 local recurrences and 4 neck recurrences as the first site of failure. One failure was successfully salvaged with surgery. Five of 23 (21%) patients undergoing surgery had significant complications (grade 3 or 4), whereas 2 of 30 (7%) patients receiving RT experienced grade 3 complications., Conclusion: Patients with early stage disease achieved better local control, locoregional control, and cause-specific survival than those with advanced tumors. Less than half of the patients (13 of 30; 43%) were cured without significant complications, suggesting a suboptimal therapeutic ratio, using current treatment methods.

Objectives/hypothesis: To determine the outcomes after radiotherapy (RT) alone or combined with surgery at the University of Florida for patients with carcinomas of the nasal cavity and paranasal sinuses., Methods: Between November 1964 and June 2005, 109 patients were treated with curative intent. Patients with maxillary sinus carcinomas were excluded. Fifty-six patients were treated with definitive RT, and 53 patients received surgery and preoperative (eight patients) or postoperative (45 patients) RT. Median follow-up was 4.3 years (range, 0.2-35.9 years). Median follow-up on living patients was 9.4 years (range, 2.0-35.9 years)., Results: The 5-year local control rates were: T1-T3, 82%; T4, 50%; and overall, 63%. Local control at 5 years was 43% after definitive RT versus 84% after surgery and adjuvant RT (P < .0001). Multivariate analysis of local control revealed that both overall stage and treatment group (definitive RT versus surgery and adjuvant RT) significantly impacted this endpoint. Cause-specific survival rates were: stages I to III, 81%; stage IV, 54%; and overall, 62%. Multivariate analysis revealed that T-stage, N-stage, and treatment group significantly influenced this endpoint. Thirty-one (20%) of 109 patients sustained severe complications; 17 of 56 patients (16%) after definitive RT and 14 of 53 patients (25%) after surgery and adjuvant RT., Conclusions: The probability of local control and cause-specific survival is better after surgery and RT compared with definitive RT. There is a modest increase in the risk of complications after surgery and RT. Thus, the preferred treatment is surgery combined with preoperative or postoperative RT.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a proven oncogene that is essential for transformation of human B cells by the virus. LMP1 induces constitutive activation of several signal transduction pathways involving nuclear factor kappaB, phosphatidylinositol 3-kinase/Akt, and the mitogen-activated protein kinases (MAPK) p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (Erk). Sequencing of LMP1 isolated from a panel of EBV+ B cell lymphomas identified three different variants of LMP1, each distinct from the B95.8 prototype isoform. All tumor variants of LMP1 as well as the B95.8 LMP1 isoform were able to induce rapid p38 phosphorylation as well as Akt and JNK activation. Additionally all variants showed similar ability to activate nuclear factor kappaB. In contrast, only tumor-derived LMP1 variants induced prolonged Erk activation and c-Fos expression. Sequence analysis revealed only two amino acids, 212 and 366, shared by the tumor variants but distinct from B95.8. Point mutation of either amino acids 212 (glycine to serine) or 366 (serine to threonine) from the B95.8 isoform to the tumor variant version of LMP1 was sufficient for gain of function characterized by sustained activation of Erk and subsequent c-Fos induction and binding to the AP1 site. Our results indicate that the enhanced ability of tumor-derived LMP1 to induce and stabilize the c-Fos oncogene can be localized to two amino acids in the C terminus of LMP1.

Background: Posttransplant lymphoproliferative disease (PTLD) is a serious complication of solid organ and bone marrow transplantation and is closely associated with Epstein-Barr virus (EBV) infection. We have previously shown that rapamycin (RAPA) directly inhibits the in vitro and in vivo proliferation of EBV-infected B lymphoblastoid cell lines (SLCL), derived from patients with PTLD, by arresting cells in the G1 phase of the cell cycle. The aim of this study is to elucidate the mechanism by which RAPA causes cell cycle arrest in EBV+ B cells., Methods: SLCL were cultured without or with RAPA (10 ng/ml) and G1-associated cell cycle proteins were analyzed by immunoblot and densitometric analysis. CDK complexes were immunoprecipitated and incubated with retinoblastoma protein (Rb) substrate. Kinase activity of the complex was determined by Western blot with anti-phospho-Rb antibodies., Results: We show that RAPA decreased both Cyclin D2 and Cyclin D3 protein levels. Furthermore, RAPA decreased the protein levels of cyclin dependent kinase 4 (CDK4) and increased the expression of the CDK inhibitor p27. In contrast, expression of the CDK inhibitor p21 was markedly inhibited by RAPA in the SLCL. Finally, in vitro kinase assays revealed that downstream hyperphosphorylation of Rb by CDK complexes was also decreased by RAPA., Conclusion: The results presented here elucidate key targets of RAPA-induced cell cycle arrest, provide insight into the growth pathways of EBV+ B-cell lymphomas, and demonstrate the potential for RAPA as a therapeutic option in the treatment of PTLD and other EBV+ lymphomas.

We describe here a microarray-based method for multiplexed, antigen-specific assessment of immunoglobulin (Ig) subclasses. We used 1152-feature arrays composed of 140 antigens or antigen fragments to detect isotype-specific mAb, to quantitatively monitor changes in isotype mAb concentration, and to profile antigen-specific antibody isotype production in a murine model of autoimmunity. This platform can be easily adapted to a variety of applications, and has the potential to elucidate mechanisms that govern development and evolution of antibody responses in in vivo and in vitro systems.

Functional analysis of a genome requires accurate gene structure information and a complete gene inventory. A dual experimental strategy was used to verify and correct the initial genome sequence annotation of the reference plant Arabidopsis. Sequencing full-length cDNAs and hybridizations using RNA populations from various tissues to a set of high-density oligonucleotide arrays spanning the entire genome allowed the accurate annotation of thousands of gene structures. We identified 5817 novel transcription units, including a substantial amount of antisense gene transcription, and 40 genes within the genetically defined centromeres. This approach resulted in completion of approximately 30% of the Arabidopsis ORFeome as a resource for global functional experimentation of the plant proteome.

The genome of the flowering plant Arabidopsis thaliana has five chromosomes. Here we report the sequence of the largest, chromosome 1, in two contigs of around 14.2 and 14.6 megabases. The contigs extend from the telomeres to the centromeric borders, regions rich in transposons, retrotransposons and repetitive elements such as the 180-base-pair repeat. The chromosome represents 25% of the genome and contains about 6,850 open reading frames, 236 transfer RNAs (tRNAs) and 12 small nuclear RNAs. There are two clusters of tRNA genes at different places on the chromosome. One consists of 27 tRNA(Pro) genes and the other contains 27 tandem repeats of tRNA(Tyr)-tRNA(Tyr)-tRNA(Ser) genes. Chromosome 1 contains about 300 gene families with clustered duplications. There are also many repeat elements, representing 8% of the sequence.