ACR Appropriateness Criteria® Tinnitus.

Tinnitus is the perception of sound in the absence of an external source. It is a common symptom that can be related to hearing loss and other benign causes. However, tinnitus may be disabling and can be the only symptom in a patient with a central nervous system process disorder. History and physical examination are crucial first steps to determine the need for imaging. CT and MRI are useful in the setting of pulsatile tinnitus to evaluate for an underlying vascular anomaly or abnormality. If there is concomitant asymmetric hearing loss, neurologic deficit, or head trauma, imaging should be guided by those respective ACR Appropriateness Criteria® documents, rather than the presence of tinnitus. Imaging is not usually appropriate in the evaluation of subjective, nonpulsatile tinnitus that does not localize to one ear. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment.

Multisession Radiosurgery for Hearing Preservation.

Clinically relevant dose-tolerance limits with reliable estimates of risk in 1-5 fractions for cochlea are still unknown. Timmerman׳s limits from the October 2008 issue of Seminars in Radiation Oncology have served as the basis for clinical practice, augmented by updated constraints in TG-101 and QUANTEC, but the corresponding estimates of risk have not yet been well-reported. A total of 37 acoustic neuroma CyberKnife cases from Medstar Georgetown University Hospital treated in 3 or 5 fractions were combined with single-fraction Gamma Knife data from the 69 cases in Timmer 2009 to form an aggregate dataset of 106 cochlea cases treated in 1-5 fractions. Probit dose-response modeling was performed in the DVH Evaluator software to estimate normal tissue complication probability. QUANTEC recommends keeping single-fraction maximum dose to the cochlea less than 14Gy to maintain less than 25% risk of serviceable hearing loss, and our 17.9% risk estimate for 14Gy in 1 fraction is within their predicted range. In 5 fractions, our estimate of the Timmerman 27.5Gy maximum cochlea dose limit was 17.4%. For cases in which lower risk is required, the Timmerman 12Gy in 1 fraction and the TG-101 limit of 25Gy in 5 fractions had an estimated risk level of 11.8% and 13.8%, respectively. High-risk and low-risk dose tolerance with risk estimates in 1-5 fractions are all presented.

Mapping gene clusters within arrayed metagenomic libraries to expand the structural diversity of biomedically relevant natural products.

Complex microbial ecosystems contain large reservoirs of unexplored biosynthetic diversity. Here we provide an experimental framework and data analysis tool to facilitate the targeted discovery of natural-product biosynthetic gene clusters from the environment. Multiplex sequencing of barcoded PCR amplicons is followed by sequence similarity directed data parsing to identify sequences bearing close resemblance to biosynthetically or biomedically interesting gene clusters. Amplicons are then mapped onto arrayed metagenomic libraries to guide the recovery of targeted gene clusters. When applied to adenylation- and ketosynthase-domain amplicons derived from saturating soil DNA libraries, our analysis pipeline led to the recovery of biosynthetic clusters predicted to encode for previously uncharacterized glycopeptide- and lipopeptide-like antibiotics; thiocoraline-, azinomycin-, and bleomycin-like antitumor agents; and a rapamycin-like immunosuppressant. The utility of the approach is demonstrated by using recovered eDNA sequences to generate glycopeptide derivatives. The experiments described here constitute a systematic interrogation of a soil metagenome for gene clusters capable of encoding naturally occurring derivatives of biomedically relevant natural products. Our results show that previously undetected biosynthetic gene clusters with potential biomedical relevance are very common in the environment. This general process should permit the routine screening of environmental samples for gene clusters capable of encoding the systematic expansion of the structural diversity seen in biomedically relevant families of natural products.

Frameless fractionated stereotactic radiosurgery for vestibular schwannomas: a single-institution experience.

OBJECTIVE: To examine tumor control, hearing preservation, and complication rates after frameless fractionated stereotactic radiosurgery (SRS) in patients with vestibular schwannomas (VS). METHODS: Thirty-seven patients treated with fractionated SRS from 2002 to 2011 were retrospectively analyzed. Ninety-five percent were treated with 25 Gy in five fractions, targeting a median tumor volume of 1.03 cc (range 0.14-7.60). RESULTS: With a median follow-up of 4.25 years (range, 15 months-9 years), no tumors required an additional treatment resulting in 100% tumor control rate. Radiographic control rate was 91% in 32 patients at a median follow-up of 3 years. Of the 14 patients with serviceable hearing and with audiograms, the hearing preservation rate was 78% at a median follow-up of 18 months. Twenty-six patients with serviceable hearing pretreatment, were evaluated by a phone survey with a hearing preservation rate of 73% at a 5 year median follow-up. There were two cases that developed both new increased trigeminal parasthesias and facial spasms but there were no cases of facial weakness. Patient had 96% of good to excellent satisfaction rate with the treatment at a median follow-up of 5 years. CONCLUSION: Frameless fractionated SRS treatment of VS results in good rate of tumor control. Hearing preservation rate and rates of cranial nerve toxicity are comparable to what is reported in the literature. Patients choose this modality because of its non-invasive nature and are generally very satisfied with their long term outcome.

Natural product biosynthetic gene diversity in geographically distinct soil microbiomes.

The number of bacterial species estimated to exist on Earth has increased dramatically in recent years. This newly recognized species diversity has raised the possibility that bacterial natural product biosynthetic diversity has also been significantly underestimated by previous culture-based studies. Here, we compare 454-pyrosequenced nonribosomal peptide adenylation domain, type I polyketide ketosynthase domain, and type II polyketide ketosynthase alpha gene fragments amplified from cosmid libraries constructed using DNA isolated from three different arid soils. While 16S rRNA gene sequence analysis indicates these cloned metagenomes contain DNA from similar distributions of major bacterial phyla, we found that they contain almost completely distinct collections of secondary metabolite biosynthetic gene sequences. When grouped at 85% identity, only 1.5% of the adenylation domain, 1.2% of the ketosynthase, and 9.3% of the ketosynthase alpha sequence clusters contained sequences from all three metagenomes. Although there is unlikely to be a simple correlation between biosynthetic gene sequence diversity and the diversity of metabolites encoded by the gene clusters in which these genes reside, our analysis further suggests that sequences in one soil metagenome are so distantly related to sequences in another metagenome that they are, in many cases, likely to arise from functionally distinct gene clusters. The marked differences observed among collections of biosynthetic genes found in even ecologically similar environments suggest that prokaryotic natural product biosynthesis diversity is, like bacterial species diversity, potentially much larger than appreciated from culture-based studies.

Cloning large natural product gene clusters from the environment: piecing environmental DNA gene clusters back together with TAR.

A single gram of soil can contain thousands of unique bacterial species, of which only a small fraction is regularly cultured in the laboratory. Although the fermentation of cultured microorganisms has provided access to numerous bioactive secondary metabolites, with these same methods it is not possible to characterize the natural products encoded by the uncultured majority. The heterologous expression of biosynthetic gene clusters cloned from DNA extracted directly from environmental samples (eDNA) has the potential to provide access to the chemical diversity encoded in the genomes of uncultured bacteria. One of the challenges facing this approach has been that many natural product biosynthetic gene clusters are too large to be readily captured on a single fragment of cloned eDNA. The reassembly of large eDNA-derived natural product gene clusters from collections of smaller overlapping clones represents one potential solution to this problem. Unfortunately, traditional methods for the assembly of large DNA sequences from multiple overlapping clones can be technically challenging. Here we present a general experimental framework that permits the recovery of large natural product biosynthetic gene clusters on overlapping soil-derived eDNA cosmid clones and the reassembly of these large gene clusters using transformation-associated recombination (TAR) in Saccharomyces cerevisiae. The development of practical methods for the rapid assembly of biosynthetic gene clusters from collections of overlapping eDNA clones is an important step toward being able to functionally study larger natural product gene clusters from uncultured bacteria.

Expanding small-molecule functional metagenomics through parallel screening of broad-host-range cosmid environmental DNA libraries in diverse proteobacteria.

The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.

Probing cell-division phenotype space and Polo-like kinase function using small molecules.

Cell-permeable small molecules that inhibit their targets on fast timescales are powerful probes of cell-division mechanisms. Such inhibitors have been identified using phenotype-based screens with chemical libraries. However, the characteristics of compound libraries needed to effectively span cell-division phenotype space, to find probes that target different mechanisms, are not known. Here we show that a small collection of 100 diaminopyrimidines (DAPs) yields a range of cell-division phenotypes, including changes in spindle geometry, chromosome positioning and mitotic index. Monopolar mitotic spindles are induced by four inhibitors, including one that targets Polo-like kinases (Plks), evolutionarily conserved serine/threonine kinases. Using chemical inhibitors and high-resolution live-cell microscopy, we found that Plk activity is needed for the assembly and maintenance of bipolar mitotic spindles. Plk inhibition destabilizes kinetochore microtubules while stabilizing other spindle microtubules, leading to monopolar spindles. Further testing of compounds based on 'privileged scaffolds', such as the DAP scaffold, could lead to new cell-division probes and antimitotic agents.

Allosteric inhibition of kinesin-5 modulates its processive directional motility.

Small-molecule inhibitors of kinesin-5 (refs. 1-3), a protein essential for eukaryotic cell division, represent alternatives to antimitotic agents that target tubulin. While tubulin is needed for multiple intracellular processes, the known functions of kinesin-5 are limited to dividing cells, making it likely that kinesin-5 inhibitors would have fewer side effects than do tubulin-targeting drugs. Kinesin-5 inhibitors, such as monastrol, act through poorly understood allosteric mechanisms, not competing with ATP binding. Moreover, the microscopic mechanism of full-length kinesin-5 motility is not known. Here we characterize the motile properties and allosteric inhibition of Eg5, a vertebrate kinesin-5, using a GFP fusion protein in single-molecule fluorescence assays. We find that Eg5 is a processive kinesin whose motility includes, in addition to ATP-dependent directional motion, a diffusive component not requiring ATP hydrolysis. Monastrol suppresses the directional processive motility of microtubule-bound Eg5. These data on Eg5's allosteric inhibition will impact these inhibitors' use as probes and development as chemotherapeutic agents.

The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks.

During cell division, mitotic spindles are assembled by microtubule-based motor proteins. The bipolar organization of spindles is essential for proper segregation of chromosomes, and requires plus-end-directed homotetrameric motor proteins of the widely conserved kinesin-5 (BimC) family. Hypotheses for bipolar spindle formation include the 'push-pull mitotic muscle' model, in which kinesin-5 and opposing motor proteins act between overlapping microtubules. However, the precise roles of kinesin-5 during this process are unknown. Here we show that the vertebrate kinesin-5 Eg5 drives the sliding of microtubules depending on their relative orientation. We found in controlled in vitro assays that Eg5 has the remarkable capability of simultaneously moving at approximately 20 nm s(-1) towards the plus-ends of each of the two microtubules it crosslinks. For anti-parallel microtubules, this results in relative sliding at approximately 40 nm s(-1), comparable to spindle pole separation rates in vivo. Furthermore, we found that Eg5 can tether microtubule plus-ends, suggesting an additional microtubule-binding mode for Eg5. Our results demonstrate how members of the kinesin-5 family are likely to function in mitosis, pushing apart interpolar microtubules as well as recruiting microtubules into bundles that are subsequently polarized by relative sliding.