Kenneth A. Frankel

Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)

We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.

Stereotactic Heavy-Charged-Particle Bragg-Peak Radiation for Intracranial Arteriovenous Malformations

BACKGROUND Heavy-charged-particle radiation has several advantages over protons and photons for the treatment of intracranial lesions; it has an improved physical distribution of the dose deep in tissue, a small angle of lateral scattering, and a sharp distal falloff of the dose. METHODS We present detailed clinical and radiologic follow-up in 86 patients with symptomatic but surgically inaccessible cerebral arteriovenous malformations that were treated with stereotactic helium-ion Bragg-peak radiation. The doses ranged from 8.8 to 34.6 Gy delivered to volumes of tissue of 0.3 to 70 cm3. RESULTS Two years after radiation treatment, the rate of complete obliteration of the lesions, as detected angiographically, was 94 percent for lesions smaller than 4 cm3, 75 percent for those of 4 to 25 cm3, and 39 percent for those larger than 25 cm3. After three years, the rates of obliteration were 100, 95, and 70 percent, respectively. Major neurologic complications occurred in 10 patients (12 percent), of whom 8 had permanent deficits. All these complications occurred in the initial stage of the protocol, before the maximal dose of radiation was reduced to 19.2 Gy. In addition, hemorrhage occurred in 10 patients from residual malformations between 4 and 34 months after treatment. Seizures and headaches were less severe in 63 percent of the 35 and 68 percent of the 40 patients, respectively, who had them initially. CONCLUSIONS Given the natural history of these inaccessible lesions and the high risks of surgery, we conclude that heavy-charged-particle radiation is an effective therapy for symptomatic, surgically inaccessible intracranial arteriovenous malformations. The current procedure has two disadvantages: a prolonged latency period before complete obliteration of the vascular lesion and a small risk of serious neurologic complications.

Heavy-Charged-Particle Radiosurgery of the Pituitary Gland: Clinical Results of 840 Patients

Since 1954, 840 patients have been treated at Lawrence Berkeley Laboratory with stereotactic charged-particle radiosurgery of the pituitary gland. The initial 30 patients were treated with proton beams; the subsequent 810 patients were treated with helium ion beams. In the great majority of the 475 patients treated for pituitary tumors, marked and sustained biochemical and clinical improvement was observed. Variable degrees of hypopituitarism developed in about one-third of patients treated solely with radiosurgery. In the earlier years of the program, 365 patients underwent radiosurgery to treat selected systemic diseases by inducing hypopituitarism. Focal temporal lobe necrosis and cranial nerve injury occurred in about 1% of patients who were treated with doses less than 230 Gy.

Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source

The SIBYLS beamline of the Advanced Light Source at Lawrence Berkeley National Laboratory is a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline. Key features and capabilities are described along with implementation and performance.

Stereotactic heavy-charged-particle bragg peak radiosurgery for the treatment of intracranial arteriovenous malformations in childhood and adolescence

Forty patients aged 6 to 18 years have now been treated for inoperable intracranial arteriovenous malformations (AVMs) using stereotactic heavy-charged-particle Bragg peak radiosurgery at the Lawrence Berkeley Laboratory 184-inch Synchrocyclotron at the University of California, Berkeley. This paper describes the procedures for selection of patients, the treatment protocol, and the neurological and neuroradiological responses to stereotactic radiosurgery in this age group. The volumes of the treated AVMs ranged from 265 mm3 to 60,000 mm3. The results are favorable: thus far, 20 of 25 patients have experienced greater than or equal to 50% obliteration of their AVMs within 1 year after treatment, and 14 of 18 patients have experienced total obliteration of the AVM by 2 years after treatment. Two patients hemorrhaged from radiosurgically treated AVMs within 12 months after treatment, but none thereafter. Complications include vasogenic edema and arterial occlusion; three patients have had neurological worsening as definite or possible sequelae of treatment. The strengths and limitations of the method are discussed.

The minimum crystal size needed for a complete diffraction data set

A formula for absolute scattering power is derived to include spot fading arising from radiation damage and the crystal volume needed to collect diffraction data to a given resolution is calculated.

Comparison of different radiation types and irradiation geometries in stereotactic radiosurgery

Recent interest in stereotactic radiosurgery of intracranial lesions, and the development of stereotactic irradiation techniques has led to the need for a systematic and complete comparison of these methods. A method for conducting these comparisons is proposed and is applied to a set of currently-used stereotactic radiosurgical techniques. Three-dimensional treatment planning calculations are used to compare dose distributions for several different radiation types and irradiation geometries. Calculations were performed using charged particles (H, He, C, and Ne ions) and the irradiation geometry currently used at Lawrence Berkeley Laboratory. Photons in the Gamma Knife configuration and the Heidelberg Linac arc method are used. The 3-dimensional dose distributions were evaluated by means of dose-volume histograms and integral doses to the target volume and to normal brain. The effects of target volume, shape and location are studied. The charged particle dose distributions are more favorable than those of the photon methods. The differences between charged particles and photons increase with increasing target volume. The differences between different charged particle species are small, as are the effects of target shape and location.

Image correlation of MRI and CT in treatment planning for radiosurgery of intracranial vascular malformations

Magnetic resonance imaging (MRI) has been incorporated with stereotactic cerebral angiography and computed tomography (CT) in the treatment planning process of heavy ion radiosurgery of intracranial arteriovenous malformations (AVMs). Correlation of the images of the AVM and normal tissue on each of these neuroradiological imaging modalities is achieved by means of fiducial markers. The computerized transfer of angiographic information to the CT images regarding the size, shape, and location of the abnormal vasculature has been described in an earlier report. A separate computer program calculates a fit between individual fiducial markers on the CT and MR images that enables the transfer of contours between the two imaging modalities. The MR images aid in the determination of the 3-dimensional shape of the AVM, adding to the information derived from the two angiographic projections. Currently, MRI cannot replace cerebral angiography in delineating the entire arterial phase of the AVM. Magnetic resonance imaging is invaluable in the treatment planning of angiographically-occult AVMs, determining the location, size, and shape of the volume to be treated. Correlation of the CT and MRI images allows for the transfer of CT-calculated isodose contours to the MRI images to aid in the determination of optimal treatment plans.

The Structure of the CRISPR-Associated Protein Csa3 Provides Insight Into the Regulation of the CRISPR/Cas System

Adaptive immune systems have recently been recognized in prokaryotic organisms where, in response to viral infection, they incorporate short fragments of invader-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). In subsequent infections, the CRISPR loci are transcribed and processed into guide sequences for the neutralization of the invading RNA or DNA. The CRISPR-associated protein machinery (Cas) lies at the heart of this process, yet many of the molecular details of the CRISPR/Cas system remain to be elucidated. Here, we report the first structure of Csa3, a CRISPR-associated protein from Sulfolobus solfataricus (Sso1445), which reveals a dimeric two-domain protein. The N-terminal domain is a unique variation on the dinucleotide binding domain that orchestrates dimer formation. In addition, it utilizes two conserved sequence motifs [Thr-h-Gly-Phe-(Asn/Asp)-Glu-X(4)-Arg and Leu-X(2)-Gly-h-Arg] to construct a 2-fold symmetric pocket on the dimer axis. This pocket is likely to represent a regulatory ligand-binding site. The N-terminal domain is fused to a C-terminal MarR-like winged helix-turn-helix domain that is expected to be involved in DNA recognition. Overall, the unique domain architecture of Csa3 suggests a transcriptional regulator under allosteric control of the N-terminal domain. Alternatively, Csa3 may function in a larger complex, with the conserved cleft participating in protein-protein or protein-nucleic acid interactions. A similar N-terminal domain is also identified in Csx1, a second CRISPR-associated protein family of unknown function.

Heavy charged-particle Bragg peak radiosurgery for intracranial vascular disorders.

The program at Donner Pavilion has applied nuclear medicine research to the diagnosis and radiosurgical treatment of life-threatening intracranial vascular disorders that affect approximately one million Americans. Stereotactic heavy-ion Bragg peak radiosurgery, using narrow beams of heavy ions (helium), demonstrates superior biological and physical characteristics in brain over X and gamma rays and protons, viz., improved dose distribution in the Bragg peak, sharp lateral and distal borders, and less multiple scattering and range straggling for the same residual range in CNS tissue. Examination of CNS tissue response and alteration of cerebral blood-flow dynamics related to heavy-ion Bragg peak radiosurgery is being undertaken using three-dimensional treatment planning and quantitative imaging utilizing cerebral angiography, computerized tomography (CT), magnetic resonance imaging (MRI), cine-CT, xenon X-ray CT, and positron emission tomography (PET). Also under examination are the physical properties of narrow heavy-ion beams for improving methods of dose delivery and dose distribution and for establishing clinical RBE/LET and dose-response relationships for human CNS tissues. Based on the evaluation and treatment with stereotactically directed narrow beams of heavy ions of over 130 patients, with cerebral angiography and CT scanning, and with MRI and radioisotope scanning of selected patients, plus extensive clinical and neuroradiological follow-up, it appears that heavy-ion radiosurgery obliterates intracranial arteriovenous malformations or protects against rebleeding with reduced morbidity and mortality.