Treatment of Chordomas and Chondrosarcomas With CyberKnife Robotic Hypofractionated Radiosurgery: A Single Institution Experience.

Introduction The aim of this study is to determine the efficacy and safety of CyberKnife® (Accuray, Inc., Sunnyvale, CA) hypofractionated radiosurgery (HfRS) in the treatment of chordomas and chondrosarcomas. Methods A total of 24 patients retrospectively identified with chordomas (19 patients) or chondrosarcomas (five patients) were treated between 2012 and 2019 with HfRS as monotherapy or an adjuvant, rescue, or combination therapy. Tumors were located in the skull base (75%) and vertebral spine (25%). Of these, 19 patients underwent previous partial resection and four patients received previous conventional external beam radiation therapy (EBRT) (60-74 Gy). Exclusive or rescue HfRS (20 patients) was administered in five fractions with a median dose of 37.5 Gy (30-40 Gy). Combined tomotherapy-EBRT treatment (median dose: 54 Gy) and HfRS (16.5-30 Gy in 3-12 fractions) were performed in four patients with bulky chordomas. Results The median follow-up from HfRS was 28 months. During clinical follow-up, no deaths were registered with overall survival (OS) of 100% and the actuarial local recurrence-free survival (LRFS) was 93% at one year, 85% at three years, and 68% at five years. Acute toxicity related to HfRS was present in a single patient. Conclusions It is seen that HfRS is effective and safe for chordomas and chondrosarcomas, with rates of LRFS comparable to other radiation modalities.

Malformaciones arteriovenosas del tronco cerebral tratadas con radiocirugía con acelerador lineal. Resultados a largo plazo / No disponible

Objetivo Las malformaciones arteriovenosas (MAV) del tronco cerebral conllevan un alto riesgo de hemorragia recurrente y de morbimortalidad. Las opciones de tratamiento son limitadas y principalmente asientan sobre la radiocirugía estereotáctica. Estudiamos los resultados de nuestra serie de MAV de tronco tratadas con acelerador lineal (LINAC) con seguimiento a largo plazo. Métodos Se analizan retrospectivamente los datos clínicos y radiológicos de 41 pacientes consecutivos con MAV de tronco tratadas mediante radiocirugía con acelerador lineal de 6MV entre 1992 y 2010. Comprendían 25 lesiones en mesencéfalo, 14 en protuberancia, una en bulbo y otra bulboprotuberancial. Se analizan los resultados del tratamiento en cuanto a (..) (AU)

[Linear accelerator radiosurgery for brainstem arteriovenous malformations. Long-term results]. / Malformaciones arteriovenosas del tronco cerebral tratadas con radiocirugía con acelerador lineal. Resultados a largo plazo.

OBJECTIVE: Arteriovenous malformations (AVM) in the brainstem carry a high risk of recurrent haemorrhage, mortality and morbidity. Treatment options are limited and mainly based on stereotactic radiosurgery. We studied the results of our series of brainstem AVM treated with linear accelerator (LINAC) and with a long-term follow-up. METHODS: We retrospectively analysed the clinical and radiological data of 41 consecutive patients with brainstem AVM treated by radiosurgery with a 6MV linear accelerator between 1992 and 2010. Twenty five lesions were located in the mesencephalon, 14 in the pons, one in the medulla oblongata and one was bulbopontine. We analysed the treatment results in relation to survival, rate of radiological obliteration, rebleeding, and treatment toxicity. RESULTS: The obliteration rate confirmed by angiography/MRA was 59.5% on 38 controlled patients. The mean follow-up period was 61 months (range: 6.7-178) and the margin dose was 14Gy in most treatments. Up to 39% of patients received more than one radiosurgery procedure to achieve closure of the malformation. No statistical correlation was found with the margin dose, presence of pretreatment haemorrhage, nidus diameter or score on the Pollock-Flickinger grading system. The annual haemorrhage rate after radiosurgery was 3.2%. Three patients died from rebleeding and actuarial survival rate was 88% at 5 and 10 years after treatment. Four patients suffered new transient neurological deficits due to toxicity, and only one presented a permanent deficit (2.6%). CONCLUSIONS: Nidus obliteration in brainstem AVM must be achieved according to three main criteria: maximum obliteration rate forced by the high rate of rebleeding, minimal morbidity given its critical location, and the greatest possible accuracy. Stereotactic radiosurgery with our moderate-dose protocol, which we believe achieved these three premises, may become an elective therapeutic modality for these patients.

A rhodanese-like protein is highly overrepresented in the mutant S. clavuligerus oppA2::aph: effect on holomycin and other secondary metabolites production.

A protein highly overrepresented in the proteome of Streptomyces clavuligerus oppA2::aph was characterized by MS/MS as a rhodanese-like enzyme. The rhlA gene, encoding this protein, was deleted from strains S. clavuligerus ATCC 27064 and S. clavuligerus oppA2::aph to characterized the RhlA enzyme activity, growth on different sulfur sources and antibiotic production by the mutants. Whereas total thiosulfate sulfurtransferase activity in cell extracts was not affected by the rhlA deletion, growth, cephamycin C and clavulanic acid production were impaired in the rhlA mutants. Holomycin production was drastically reduced (66-90%) in the rhlA mutants even when using S. clavuligerusΔrhlA pregrown cells, suggesting that this enzyme might be involved in the formation of the cysteine precursor for this sulfur-containing antibiotic. While growth on thiosulfate as the sole sulfur source was particularly low the volumetric and specific antibiotic production of the three antibiotics increased in all the strains in the presence of thiosulfate. This stimulatory effect of thiosulfate on antibiotic production was confirmed by addition of thiosulfate to pre-grown cells and appears to be a general effect of thiosulfate on oxidative stress as was also evident in the production of staurosporin by S. clavuligerus.

Different proteins bind to the butyrolactone receptor protein ARE sequence located upstream of the regulatory ccaR gene of Streptomyces clavuligerus.

Cell-free extracts from Streptomyces clavuligerus, purified by elution from heparin-agarose with an ARE-containing DNA fragment or by salt elution chromatography, bind to a 26 nt ARE sequence, for butyrolactone receptor proteins (ARE(ccaR)). This sequence is [corrected] located upstream of the ccaR gene, encoding [corrected] the activator protein CcaR required for clavulanic acid and cephamycin C biosynthesis. The binding is specific for the ARE sequence as shown by competition with a 34 nt unlabelled probe identical to the ARE sequence. A brp gene, encoding a butyrolactone receptor protein, was cloned from S. clavuligerus. Sixty-one nucleotides upstream of brp another ARE sequence (ARE(brp)) was found, suggesting that Brp autoregulates its expression. Pure recombinant rBrp protein binds specifically to the ARE sequences present upstream of ccaR and brp. A brp-deleted mutant, S. clavuligerus Deltabrp::neo1, produced 150-300% clavulanic acid and 120-220% cephamycin C as compared with the parental strain, suggesting that Brp exerts a repressor role in antibiotic biosynthesis. EMSA assays using affinity chromatography extracts from the deletion mutant S. clavuligerus Deltabrp::neo1 lacked a high-mobility band-shift due to Brp but still showed a [corrected] slow-mobility band-shift observed in the wild-type strain. These results indicate that two different proteins bind specifically to the ARE sequence and modulate clavulanic acid and cephamycin C [corrected] biosynthesis by its action on ccaR gene expression.

Two oligopeptide-permease-encoding genes in the clavulanic acid cluster of Streptomyces clavuligerus are essential for production of the beta-lactamase inhibitor.

orf7 (oppA1) and orf15 (oppA2) are located 8 kb apart in the clavulanic acid gene cluster of Streptomyces clavuligerus and encode proteins which are 48.0% identical. These proteins show sequence similarity to periplasmic oligopeptide-binding proteins. Mutant S. clavuligerus oppA1::acc, disrupted in oppA1, lacks clavulanic acid production. Clavulanic acid production is restored by transformation with plasmid pIJ699-oppA1, which carries oppA1, but not with the multicopy plasmid pIJ699-oppA2, which carries oppA2. The mutant S. clavuligerus oppA2::aph also lacks clavulanic acid production, shows a bald phenotype, and overproduces holomycin (5). Clavulanic acid production at low levels is restored in the oppA2-disrupted mutants by transformation with plasmid pIJ699-oppA2, but it is not complemented by the multicopy plasmid pIJ699-oppA1. Both genes encode oligopeptide permeases with different substrate specificities. The disrupted S. clavuligerus oppA2::aph is not able to grow on RPPGFSPFR (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; bradykinin), but both mutants grow on VAPG (Val-Ala-Pro-Gly) as the only nitrogen source, indicating differences in the peptide bound by the proteins encoded by both genes. The null S. clavuligerus oppA1::acc and S. clavuligerus oppA2::aph mutants are more resistant to the toxic tripeptide phosphinothricyl-alanyl-alanine (also named bialaphos) than the wild-type strain, suggesting that this peptide might be transported by these peptide-binding proteins.

Mutants of Streptomyces clavuligerus with disruptions in different genes for clavulanic acid biosynthesis produce large amounts of holomycin: possible cross-regulation of two unrelated secondary metabolic pathways.

A Streptomyces clavuligerus ccaR::aph strain, which has a disruption in the regulatory gene ccaR, does not produce cephamycin C or clavulanic acid, but does produce a bioactive compound that was identified as holomycin by high-performance liquid chromatography (HPLC) and infrared and mass spectrometry. S. clavuligerus strains with disruptions in different genes of the clavulanic acid pathway fall into three groups with respect to holomycin biosynthesis. (i) Mutants with mutations in the early steps of the pathway blocked in the gene ceaS (pyc) (encoding carboxyethylarginine synthase), bls (encoding a beta-lactam synthetase), or open reading frame 6 (ORF6; coding for an acetyltransferase of unknown function) are holomycin nonproducers. (ii) Mutants blocked in the regulatory gene ccaR or claR or blocked in the last gene of the pathway encoding clavulanic acid reductase (car) produce holomycin at higher levels than the wild-type strain. (iii) Mutants with disruption in cyp (coding for cytochrome P450), ORF12, and ORF15, genes that appear to be involved in the conversion of clavaminic acid into clavaldehyde or in secretion steps, produce up to 250-fold as much holomycin as the wild-type strain. An assay for holomycin synthetase was developed. This enzyme forms holomycin from holothin by using acetyl coenzyme A as an acetyl group donor. The holomycin synthase activities in the different clavulanic acid mutants correlate well with their production of holomycin.

The clavulanic acid biosynthetic cluster of Streptomyces clavuligerus: genetic organization of the region upstream of the car gene.

The genetic organization of the region upstream of the car gene of the clavulanic acid biosynthetic gene cluster of Streptomyces clavuligerus has been determined. Sequence analysis of a 12.1 kb region revealed the presence of 10 ORFs whose putative functions, according to database searches, are discussed. Three co-transcriptional units are proposed: ORF10-11, ORF12-13 and ORF15-16-17-18. Potential transcriptional terminators were identified downstream of ORF11 (fd) and ORF15. Targeted disruption of ORF10 (cyp) gave rise to transformants unable to produce clavulanic acid, but with a considerably higher production of cephamycin C. Transformants inactivated at ORF14 had a remarkably lower production of clavulanic acid and similar production of cephamycin C. Significant improvements of clavulanic acid production, associated with a drop in cephamycin C biosynthesis, were obtained with transformants of S. clavuligerus harbouring multiple copies of plasmids carrying different constructions from the ORF10-14 region. This information can be used to guide strain improvement programs, blending random mutagenesis and molecular cloning, to optimize the yield of clavulanic acid.