The bZIP transcription factor BIP1 of the rice blast fungus is essential for infection and regulates a specific set of appressorium genes.

The rice blast fungus Magnaporthe oryzae differentiates specialized cells called appressoria that are required for fungal penetration into host leaves. In this study, we identified the novel basic leucine zipper (bZIP) transcription factor BIP1 (B-ZIP Involved in Pathogenesis-1) that is essential for pathogenicity. BIP1 is required for the infection of plant leaves, even if they are wounded, but not for appressorium-mediated penetration of artificial cellophane membranes. This phenotype suggests that BIP1 is not implicated in the differentiation of the penetration peg but is necessary for the initial establishment of the fungus within plant cells. BIP1 expression was restricted to the appressorium by both transcriptional and post-transcriptional control. Genome-wide transcriptome analysis showed that 40 genes were down regulated in a BIP1 deletion mutant. Most of these genes were specifically expressed in the appressorium. They encode proteins with pathogenesis-related functions such as enzymes involved in secondary metabolism including those encoded by the ACE1 gene cluster, small secreted proteins such as SLP2, BAS2, BAS3, and AVR-Pi9 effectors, as well as plant cuticle and cell wall degrading enzymes. Interestingly, this BIP1 network is different from other known infection-related regulatory networks, highlighting the complexity of gene expression control during plant-fungal interactions. Promoters of BIP1-regulated genes shared a GCN4/bZIP-binding DNA motif (TGACTC) binding in vitro to BIP1. Mutation of this motif in the promoter of MGG_08381.7 from the ACE1 gene cluster abolished its appressorium-specific expression, showing that BIP1 behaves as a transcriptional activator. In summary, our findings demonstrate that BIP1 is critical for the expression of early invasion-related genes in appressoria. These genes are likely needed for biotrophic invasion of the first infected host cell, but not for the penetration process itself. Through these mechanisms, the blast fungus strategically anticipates the host plant environment and responses during appressorium-mediated penetration.

Circadian activation of the mitogen-activated protein kinase MAK-1 facilitates rhythms in clock-controlled genes in Neurospora crassa.

The circadian clock regulates the expression of many genes involved in a wide range of biological functions through output pathways such as mitogen-activated protein kinase (MAPK) pathways. We demonstrate here that the clock regulates the phosphorylation, and thus activation, of the MAPKs MAK-1 and MAK-2 in the filamentous fungus Neurospora crassa. In this study, we identified genetic targets of the MAK-1 pathway, which is homologous to the cell wall integrity pathway in Saccharomyces cerevisiae and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mammals. When MAK-1 was deleted from Neurospora cells, vegetative growth was reduced and the transcript levels for over 500 genes were affected, with significant enrichment for genes involved in protein synthesis, biogenesis of cellular components, metabolism, energy production, and transcription. Additionally, of the ~500 genes affected by the disruption of MAK-1, more than 25% were previously identified as putative clock-controlled genes. We show that MAK-1 is necessary for robust rhythms of two morning-specific genes, i.e., ccg-1 and the mitochondrial phosphate carrier protein gene NCU07465. Additionally, we show clock regulation of a predicted chitin synthase gene, NCU04352, whose rhythmic accumulation is also dependent upon MAK-1. Together, these data establish a role for the MAK-1 pathway as an output pathway of the circadian clock and suggest a link between rhythmic MAK-1 activity and circadian control of cellular growth.

A cis regulatory element in the TAPNAC promoter directs tapetal gene expression.

The tapetum is a single cell layer surrounding the anther locule and its major function is to provide nutrients for pollen development. The ablation of tapetal cells interferes with pollen production and results in plant male sterility. In spite of the importance of this tissue in the quality and production of pollen grains, studies on promoter gene regulation of tapetal expressed genes are very few and there are no reports on specific cis regulatory sequences that control tapetal gene expression. We have identified a NAC gene, TAPNAC (At1g61110), specifically expressed in the Arabidopsis tapetum via transcriptional profiling. The TAPNAC promoter was studied in detail to identify cis regulatory sequences that confer tapetal specific expression. For this purpose, TAPNAC promoter elements were fused to the ß-glucuronidase (GUS) reporter gene, and spatial and temporal GUS expression was monitored. The results showed that TAPNAC promoter-driven GUS expression emulates the expression of TAPNAC mRNA in anthers. A conserved TCGTGT motif was identified in the TAPNAC promoter and other tapetal expressed promoters. The TCGTGT motif enhances GUS expression in anthers of transgenic plants but only in the context of the TAPNAC promoter proximal region.

Transcription factors in light and circadian clock signaling networks revealed by genomewide mapping of direct targets for neurospora white collar complex.

Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.

Gene regulation logic in retinal ganglion cell development: Isl1 defines a critical branch distinct from but overlapping with Pou4f2.

Understanding gene regulatory networks (GRNs) that control neuronal differentiation will provide systems-level perspectives on neurogenesis. We have previously constructed a model for a GRN in retinal ganglion cell (RGC) differentiation in which four hierarchical tiers of transcription factors ultimately control the expression of downstream terminal genes. Math5 occupies a central node in the hierarchy because it is essential for the formation of RGCs and the expression of the immediate downstream factor Pou4f2. Based on its expression, we also proposed that Isl1, a LIM-homeodomain factor, functions in parallel with Pou4f2 and downstream of Math5 in the RGC GRN. To determine whether this was the case, a conditional Isl1 allele was generated and deleted specifically in the developing retina. Although RGCs formed in Isl1-deleted retinas, most underwent apoptosis, and few remained at later stages. By microarray analysis, we identified a distinct set of genes whose expression depended on Isl1. These genes are all downstream of Math5, and some of them, but not all, also depend on Pou4f2. Additionally, Isl1 was required for the sustained expression of Pou4f2, suggesting that Isl1 positively regulates Pou4f2 after Math5 levels are diminished. The results demonstrate an essential role for Isl1 in RGC development and reveal two distinct but intersecting branches of the RGC GRN downstream of Math5, one directed by Pou4f2 and the other by Isl1. They also reveal that identical RGC expression patterns are achieved by different combinations of divergent inputs from upstream transcription factors.

Broadening the impact of plant science through innovative, integrative, and inclusive outreach.

Population growth and climate change will impact food security and potentially exacerbate the environmental toll that agriculture has taken on our planet. These existential concerns demand that a passionate, interdisciplinary, and diverse community of plant science professionals is trained during the 21st century. Furthermore, societal trends that question the importance of science and expert knowledge highlight the need to better communicate the value of rigorous fundamental scientific exploration. Engaging students and the general public in the wonder of plants, and science in general, requires renewed efforts that take advantage of advances in technology and new models of funding and knowledge dissemination. In November 2018, funded by the National Science Foundation through the Arabidopsis Research and Training for the 21st century (ART 21) research coordination network, a symposium and workshop were held that included a diverse panel of students, scientists, educators, and administrators from across the US. The purpose of the workshop was to re-envision how outreach programs are funded, evaluated, acknowledged, and shared within the plant science community. One key objective was to generate a roadmap for future efforts. We hope that this document will serve as such, by providing a comprehensive resource for students and young faculty interested in developing effective outreach programs. We also anticipate that this document will guide the formation of community partnerships to scale up currently successful outreach programs, and lead to the design of future programs that effectively engage with a more diverse student body and citizenry.

Sugarcane DIRIGENT and O-methyltransferase promoters confer stem-regulated gene expression in diverse monocots.

Transcription profiling analysis identified Saccharum hybrid DIRIGENT (SHDIR16) and Omicron-Methyltransferase (SHOMT), putative defense and fiber biosynthesis-related genes that are highly expressed in the stem of sugarcane, a major sucrose accumulator and biomass producer. Promoters (Pro) of these genes were isolated and fused to the beta-glucuronidase (GUS) reporter gene. Transient and stable transgene expression analyses showed that both Pro( DIR16 ):GUS and Pro( OMT ):GUS retain the expression characteristics of their respective endogenous genes in sugarcane and function in orthologous monocot species, including rice, maize and sorghum. Furthermore, both promoters conferred stem-regulated expression, which was further enhanced in the stem and induced in the leaf and root by salicylic acid, jasmonic acid and methyl jasmonate, key regulators of biotic and abiotic stresses. Pro( DIR16 ) and Pro( OMT ) will enable functional gene analysis in monocots, and will facilitate engineering monocots for improved carbon metabolism, enhanced stress tolerance and bioenergy production.

Isolating promoters of multigene family members from the polyploid sugarcane genome by PCR-based walking in BAC DNA.

The availability of a wider range of promoters for regulated expression in valuable transgenic crops would benefit functional genomics studies and current biotechnology programs aimed at improved productivity. Polymerase chain reaction (PCR)-based genome walking techniques are commonly used to isolate promoters or 5' flanking genomic regions adjacent to known cDNA sequences in genomes that are not yet completely sequenced. However, these techniques are problematic when applied directly to DNA isolated from crops with highly complex and large genomes. An adaptor ligation-mediated PCR-based BAC genome walking method is described here for the efficient isolation of promoters of multigene family members, such as the putative defense and fiber biosynthesis DIRIGENT genes that are abundant in the stem of sugarcane, a species with a highly polyploid genome. The advantage of this method is the efficient and specific amplification of the target promoter using BAC genomic DNA as template for the adaptor ligation-mediated PCR walking.

Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.

Advancement in our understanding of the biology of adult stem cells and their therapeutic potential relies heavily on meaningful functional assays that can identify and measure stem cell activity in vivo and in vitro. In the mammalian nervous system, neural stem cells (NSCs) are often studied using a culture system referred to as the neurosphere assay. We previously challenged a central tenet of this assay, that all neurospheres are derived from a NSC, and provided evidence that it overestimates NSC frequency, rendering it inappropriate for quantitation of NSC frequency in relation to NSC regulation. Here we report the development and validation of the neural colony-forming cell assay (NCFCA), which discriminates stem from progenitor cells on the basis of their proliferative potential. We anticipate that the NCFCA will provide additional clarity in discerning the regulation of NSCs, thereby facilitating further advances in the promising application of NSCs for therapeutic use.

Circadian genomics of the chick pineal gland in vitro.

BACKGROUND: Chick pinealocytes exhibit all the characteristics of a complete circadian system, comprising photoreceptive inputs, molecular clockworks and an easily measured rhythmic output, melatonin biosynthesis. These properties make the in vitro pineal a particularly useful model for exploring circadian control of gene transcription in a pacemaker tissue, as well as regulation of the transcriptome by primary inputs to the clock (both photic and noradrenergic). RESULTS: We used microarray analysis to investigate the expression of approximately 8000 genes within cultured pinealocytes subjected to both LD and DD. We report that a reduced subset of genes was rhythmically expressed in vitro compared to those previously published in vivo, and that gene expression rhythms were lower in amplitude, although the functional distribution of the rhythmic transcriptome was largely similar. We also investigated the effects of 6-hour pulses of light or of norepinephrine on gene expression in free-running cultures during both subjective day and night. As expected, both light and norepinephrine inhibited melatonin production; however, the two treatments differentially enhanced or suppressed specific sets of genes in a fashion that was dependent upon time of day. CONCLUSION: Our combined approach of utilizing a temporal, photic and pharmacological microarray experiment allowed us to identify novel genes linking clock input to clock function within the pineal. We identified approximately 30 rhythmic, light-responsive, NE-insensitive genes with no previously known clock function, which may play a role in circadian regulation of the pineal. These are candidates for future functional genomics experiments to elucidate their potential role in circadian physiology. Further, we hypothesize that the pineal circadian transcriptome is reduced but functionally conserved in vitro, and supports an endogenous role for the pineal in regulating local rhythms in metabolism, immune function, and other conserved pathways.

Prospective trial of gross-total resection with Gliadel wafers followed by early postoperative Gamma Knife radiosurgery and conformal fractionated radiotherapy as the initial treatment for patients with radiographically suspected, newly diagnosed glioblastoma multiforme.

OBJECT: The purpose of this study was to determine whether increased local control and improved survival can be achieved in patients with glioblastoma multiformes (GBMs) who undergo aggressive resection, Gliadel wafer implantation, Gamma Knife radiosurgery (GKS), and fractionated radiotherapy (RT) as the initial treatment. METHODS: Thirty patients with radiographically suspected GBMs were screened for enrollment in a Phase I/II prospective clinical trial. Twenty-seven patients were eligible and underwent gross-total resection and Gliadel wafer implantation. Gamma Knife radiosurgery (12 Gy at 50%) was administered to the resection cavity within 2 weeks of surgery. Patients then received standard fractionated RT (total dose 60 Gy over 6 weeks). Temozolomide was prescribed for patients at the time of recurrence. Surveillance MR imaging, neurological examination, and quality-of-life evaluations were performed at 2-month intervals. To estimate the potential effects on the DNA repair mechanism, tumor tissue was analyzed with methylation-specific polymerase chain reaction analysis and immunohistochemical assays for MGMT gene promoter methylation and protein expression. RESULTS: The median survival for all patients was 50 weeks and the 2-year survival rate was 22%. When stratified into standard and high-risk patient groups, the median survivals were 76 and 33 weeks, respectively. Two patients remain alive at the time of this report with no clinical or radiographic evidence of disease at > 189 and 239 weeks posttreatment and excellent performance status. Local tumor control was achieved in 53% of patients, and local failure occurred in 47%. No acute early toxicity was noted; however, delayed symptomatic radionecrosis occurred in 47% of patients, which required repeated operations 9-24 months after the initial treatment. Delayed hydrocephalus requiring ventriculoperitoneal shunt placement occurred in 47% of patients. There was a significant difference in survival between patients whose tumors contained the methylated and unmethylated MGMT promoter, 103 versus 45 weeks, respectively (p = 0.0009, log-rank test). CONCLUSIONS: The combination of aggressive resection, Gliadel wafer implantation, and GKS in addition to standard fractionated RT in selected patients resulted in increased local control and increased survival compared with a historical control group treated with surgery and involved-field RT alone. Delayed focal radionecrosis was increased to 47% in this series and was managed with steroids and repeated resection. Aggressive local tumor control with these multimodal therapies should be approached judiciously for a select group of high performance patients and the probability of developing symptomatic radionecrosis requiring surgery should be anticipated and fully disclosed to patients who undergo this treatment.

Circadian profiling of the transcriptome in NIH/3T3 fibroblasts: comparison with rhythmic gene expression in SCN2.2 cells and the rat SCN.

To screen for output signals that may distinguish the pacemaker in the mammalian suprachiasmatic nucleus (SCN) from peripheral-type oscillators in which the canonical clockworks are similarly regulated in a circadian manner, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared relative to SCN2.2 cells in vitro and the rat SCN. Similar to the circadian profiling of the SCN2.2 and rat SCN transcriptomes, NIH/3T3 fibroblasts exhibited circadian fluctuations in the expression of the core clock genes, Per2, Cry1, and Bmal1, and 323 functionally diverse transcripts, many of which regulate cellular communication. Overlap in rhythmic transcripts among NIH/3T3 fibroblasts, SCN2.2 cells, and the rat SCN was limited to these clock genes and four other genes that mediate fatty acid and lipid metabolism or function as nuclear factors. Compared with NIH/3T3 cells, circadian gene expression in SCN oscillators was more prevalent among genes mediating glucose metabolism and neurotransmission. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase (iNos) in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of a NOS inhibitor on metabolic rhythms in cocultures containing SCN2.2 cells and untreated NIH/3T3 cells suggest that the gaseous neurotransmitter nitric oxide may play a key role in SCN pacemaker function. This comparative analysis of circadian gene expression in SCN and non-SCN cells may have important implications in the selective analysis of circadian signals involved in the coupling of SCN oscillators and regulation of rhythmicity in downstream cells.

Spinal cord gliomas: A multi-institutional retrospective analysis.

PURPOSE: To determine the impact of postoperative radiation therapy (POXRT) on outcome in spinal cord gliomas. PATIENTS AND METHODS: Data from 242 patients were collected retrospectively from six institutions using a standardized data sheet. Pathology specimens, when available, were centrally reviewed. RESULTS: A total of 183 patients were analyzed: 82 received surgery alone as initial treatment, whereas 101 had surgery and POXRT. Demographic, diagnostic, and treatment factors were analyzed for impact on progression-free (PFS) and overall survival (OS). PFS in ependymoma patients was 74%, 60%, and 35% at 5, 10, 15 years, respectively, and was significantly influenced by treatment type, race, age, tumor grade, and type of surgery on univariate analysis, with age being the only significant factor on multivariate analysis (MVA) (p = 0.01). OS of ependymoma patients was 91%, 84%, and 75% at 5, 10, and 15 years, respectively, and was significantly influenced by both complete resection (p = 0.04) and age (p = 0.03) on MVA. In astrocytomas, PFS was 42%, 29%, and 15% at 5, 10, and 15 years, and was significantly influenced by POXRT in low- and intermediate-grade tumors on MVA (p = 0.02). OS at 5, 10, and 15 years was 59%, 53%, and 32%, respectively, and was significantly influenced by grade on MVA (p < 0.01). CONCLUSION: Postoperative radiation therapy reduced disease progression in low- and moderate-grade astrocytomas. In ependymomas, complete resection significantly influenced OS.

Circadian profiling of the transcriptome in immortalized rat SCN cells.

Endogenous oscillations in gene expression are a prevalent feature of the circadian clock in the mammalian suprachiasmatic nucleus (SCN) and similar timekeeping systems in other organisms. To determine whether immortalized cells derived from the rat SCN (SCN2.2) retain these intrinsic rhythm-generating properties, oscillatory behavior of the SCN2.2 transcriptome was analyzed and compared with that found in the rat SCN in vivo using rat U34A Affymetrix GeneChips. In SCN2.2 cells, 116 unique genes and 46 ESTs or genes of unknown function exhibited circadian fluctuations with a 1.5-fold or greater difference in their mRNA abundance for two cycles. Many (35%) of these rhythmically regulated genes in SCN2.2 cells also exhibited circadian profiles of mRNA expression in the rat SCN in vivo. Functional analyses and cartography indicate that a diverse set of cellular pathways are strategically regulated by the circadian clock in SCN2.2 cells and that the largest categories of rhythmic genes are those involved in cellular and systems-level communication or in metabolic processes like cellular respiration, fatty acid recycling, and steroid synthesis. Because many of the same genes or nodes within these functional categories were rhythmically expressed in both SCN2.2 cells and the rat SCN, the circadian regulation of these pathways may be important in modulating input to or output from the SCN clock mechanism. In summary, global expression and circadian regulation of the SCN2.2 transcriptome retain many SCN-like properties, suggesting that genes displaying rhythmic profiles in both experimental models may be integral to their function as both circadian oscillators and pacemakers.

Is gross-total resection sufficient treatment for posterior fossa ependymomas?

OBJECT: The goals of this study were to analyze outcomes in patients with posterior fossa ependymomas, determine whether gross-total resection (GTR) alone is appropriate treatment, and evaluate the role of radiation therapy. METHODS: All patients with newly diagnosed intracranial ependymomas treated at Barrow Neurological Institute between 1983 and 2002 were identified. Those with supratentorial primary lesions, subependymomas, or neuraxis dissemination were excluded. Forty-five patients met the criteria for the study. Gross-total resection was accomplished in 32 patients (71%) and subtotal resection (STR) in 13 (29%). Radiation therapy was given to 25 patients: 13 following GTR and 12 after STR. The radiation fields were craniospinal followed by a posterior fossa boost in six patients and posterior fossa or local only in the remaining patients. With a median follow-up period of 66 months, the median duration of local control was 73.5 months with GTR alone, but has not yet been reached for patients with both GTR and radiotherapy (p = 0.020). The median duration of local control following STR and radiotherapy was 79.6 months. The 10-year actuarial local control rate was 100% for patients who underwent GTR and radiotherapy, 50% for those who underwent GTR alone, and 36% for those who underwent both STR and radiotherapy, representing significant differences between the GTR-plus-radiotherapy and GTR-alone cohorts (p = 0.018), and between the GTR-plus-radiotherapy and the STR-plus-radiotherapy group (p = 0.003). There was no significant difference in the 10-year actuarial local control rate between the GTR-alone and STR-plus-radiotherapy cohorts (p = 0.370). The 10-year overall survival was numerically superior in patients who underwent both GTR and radiotherapy: 83% compared with 67% in those who underwent GTR alone and 43% in those who underwent both STR and radiotherapy. These differences did not achieve statistical significance. Univariate analyses revealed that radiotherapy, tumor grade, and extent of resection were significant predictors of local control. CONCLUSIONS: Gross-total resection should be the intent of surgery when it can be accomplished with an acceptable degree of morbidity. Even after GTR has been confirmed with postoperative imaging, however, adjuvant radiotherapy significantly improves local control. The authors currently recommend the use of postoperative radiotherapy, regardless of whether the resection is gross total or subtotal.

Transcriptional profiling of the chick pineal gland, a photoreceptive circadian oscillator and pacemaker.

The avian pineal gland contains both circadian oscillators and photoreceptors to produce rhythms in biosynthesis of the hormone melatonin in vivo and in vitro. The molecular mechanisms for melatonin biosynthesis are largely understood, but the mechanisms driving the rhythm itself or the photoreceptive processes that entrain the rhythm are unknown. We have produced cDNA microarrays of pineal gland transcripts under light-dark and constant darkness conditions. Rhythmic transcripts were classified according to function, representing diverse functional groups, including phototransduction pathways, transcription/translation factors, ion channel proteins, cell signaling molecules, and immune function genes. These were also organized relative to time of day mRNA abundance in light-dark and constant darkness. The transcriptional profile of the chick pineal gland reveals a more complex form of gene regulation than one might expect from a gland whose sole apparent function is the rhythmic biosynthesis of melatonin. The mRNAs encoding melatonin biosynthesis are rhythmic as are many orthologs of mammalian "clock genes." However, the oscillation of phototransductive, immune, stress response, hormone binding, and other important processes in the transcriptome of the pineal gland, raises new questions regarding the role of the pineal gland in circadian rhythm generation, organization, and avian physiology.

Gene discovery and gene expression in the rice blast fungus, Magnaporthe grisea: analysis of expressed sequence tags.

Over 28,000 expressed sequence tags (ESTs) were produced from cDNA libraries representing a variety of growth conditions and cell types. Several Magnaporthe grisea strains were used to produce the libraries, including a nonpathogenic strain bearing a mutation in the PMK1 mitogen-activated protein kinase. Approximately 23,000 of the ESTs could be clustered into 3,050 contigs, leaving 5,127 singleton sequences. The estimate of 8,177 unique sequences indicates that over half of the genes of the fungus are represented in the ESTs. Analysis of EST frequency reveals growth and cell type-specific patterns of gene expression. This analysis establishes criteria for identification of fungal genes involved in pathogenesis. A large fraction of the genes represented by ESTs have no known function or described homologs. Manual annotation of the most abundant cDNAs with no known homologs allowed us to identify a family of metallothionein proteins present in M. grisea, Neurospora crassa, and Fusarium graminearum. In addition, multiply represented ESTs permitted the identification of alternatively spliced mRNA species. Alternative splicing was rare, and in most cases, the alternate mRNA forms were unspliced, although alternative 5' splice sites were also observed.

Surgery and permanent 125I seed paraspinal brachytherapy for malignant tumors with spinal cord compression.

PURPOSE: To evaluate the functional outcome, predictors of response, and toxicity from spinal surgery and 125I brachytherapy in patients with malignant tumors resulting in spinal cord compression. METHODS AND MATERIALS: Between July 1985 and September 2001, after surgical resection, 30 patients underwent 31 intraoperative paraspinal brachytherapy procedures at Barrow Neurological Institute. Twenty-four (with 25 procedures) had follow-up at our clinic and form the basis for this report. Surgical procedures were based on the location of the impinging lesion: corpectomy or spondylectomy in 13 cases and laminectomy in 12. Permanent 125I seeds in absorbable suture were placed with open exposure after resection. RESULTS: Spinal cord compression was cervical in 4 (16%), thoracic in 14 (56%), and lumbar in 7 (28%) of the 25 cases. One patient underwent two separate procedures at different spinal sites. Of the 25 brachytherapy sites, 22 also received external beam radiotherapy (EBRT): 5, EBRT with a planned brachytherapy boost; 4, brachytherapy and prompt EBRT after recovery; and 13, brachytherapy as salvage for local failure after prior EBRT. Three had no EBRT: 1 had lymphoma treated with chemotherapy, 1 had remote previous EBRT for a childhood tumor, and 1 refused EBRT. The mean follow-up was 19.8 months. The 2- and 3-year actuarial local control rate was 87.4% and 72.9%, respectively. Four sites (16.0%) experienced local failure. The mean time to recurrence for these 4 patients was 20.3 months. Three of the four had failed prior EBRT, with surgery and brachytherapy used for salvage. The 2- and 3-year actuarial overall survival rates were 24.0% and 16.0%, mean 19.2 months. An ambulatory function score was assigned pre- and postoperatively: I, normal ambulation; II, abnormal not requiring assistance; III, abnormal requiring assistance; and IV, unable to ambulate. All patients with score I, 91% of those with score II, 67% of those with score III, and 67% of those with score IV were ambulatory after the procedure; 84% had either normal or improved ambulation postoperatively. Morbidity was restricted to four postoperative events: one cerebrospinal fluid leak, two wound infections treated in situ without removal of seeds or instrumentation, and one pulmonary embolus. No myelopathies or other neurologic sequelae were encountered. CONCLUSION: This is the largest series in the literature exploring surgery and 125I brachytherapy in the treatment of malignant spinal cord compression. We found this to be well tolerated and to result in durable local control and ambulatory function. Our results suggest a benefit to aggressive local therapy in selected patients with spinal cord compression.