Understanding the Efficiency of Splenic Hemangiosarcoma Diagnosis Using Monte Carlo Simulations.

Hemangiosarcoma is a common neoplasm of the spleen in older dogs. However, diagnosis is complicated by necrosis and hemorrhage, which can mimic a number of benign processes. Currently, there is no consensus about the number of sections pathologists should examine to rule out hemangiosarcoma. To answer this question, we examined 413 histopathologic sections from 50 cases of canine hemangiosarcoma (mean: 8.1 sections per case; range, 5-14). Each section had the presence or absence of hemangiosarcoma determined by 2 board-certified anatomic pathologists. Then, 100 Monte Carlo simulations were performed, randomly selecting sections from each case 10 000 times and the results averaged. These simulations suggest that examination of 5 sections from a spleen with hemangiosarcoma yields a 95.02% chance of diagnosing hemangiosarcoma, while examination of 10 sections yields a 98.59% chance of diagnosis when hemangiosarcoma is in fact present. The data emphasize the need to submit the entire spleen for histopathologic examination in suspected cases of hemangiosarcoma and suggest that 5 sections obtained by a trained individual are likely sufficient for diagnosis.

C-H Arylation in the Formation of a Complex Pyrrolopyridine, the Commercial Synthesis of the Potent JAK2 Inhibitor, BMS-911543.

The development of an improved short and efficient commercial synthesis of the JAK2 inhibitor, a complex pyrrolopyridine, BMS-911543, is described. During the discovery and development of this synthesis, a Pd-catalyzed C-H functionalization was invented which enabled the rapid union of the key pyrrole and imidazole fragments. The synthesis of this complex, nitrogen-rich heterocycle was accomplished in only six steps (longest linear sequence) from readily available materials.

Symptomatic Vertebral Hemangioma of the Posterior Elements Sharing Blood Supply with a Radiculomedullary Artery.

Vertebral hemangiomas are common lesions usually restricted to the vertebral body. They are characterized by proliferation of endothelial cells and subsequent expansion of vascular spaces within the bone. These lesions are usually clinically silent and are discovered incidentally. Only rarely are vertebral hemangiomas symptomatic. Here, we present the case of a 68-year-old female with an aggressive hemangioma causing neurologic deficit. The lesion was localized within the posterior spinal elements, with no involvement of the vertebral body. Transarterial embolization was deemed unsafe due to the close proximity of a prominent radiculomedullary artery. The patient was treated with posterior decompression at T4-T6.

Strategy To Prepare 3-Bromo- and 3-Chloropyrazoles.

A general strategy to prepare substituted 3-bromo- and 3-chloropyrazoles is described. The three-step method involves condensation of crotonates or ß-chloro carboxylic acids with hydrazines, followed by halogenation and oxidation. Several condensation and oxidation protocols were developed to enable preparation of a wide variety of 3-halopyrazoles with good to excellent yields and regiocontrol.

Ni-Catalyzed C-H Functionalization in the Formation of a Complex Heterocycle: Synthesis of the Potent JAK2 Inhibitor BMS-911543.

BMS-911543 is a complex pyrrolopyridine investigated as a potential treatment for myeloproliferative disorders. The development of a short and efficient synthesis of this molecule is described. During the course of our studies, a Ni-mediated C-N bond formation was invented, which enabled the rapid construction of the highly substituted 2-aminopyridine core. The synthesis of this complex, nitrogen-rich heterocycle was accomplished in only eight steps starting from readily available materials.

Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas.

Carbonic anhydrase (CA) is one of nature's fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme's sensitivity to the harsh process conditions. Using directed evolution, the properties of a ß-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.

Synthesis of the 6-azaindole containing HIV-1 attachment inhibitor pro-drug, BMS-663068.

The development of a short and efficient synthesis of a complex 6-azaindole, BMS-663068, is described. Construction of the 6-azaindole core is quickly accomplished starting from a simple pyrrole, via a regioselective Friedel-Crafts acylation, Pictet-Spengler cyclization, and a radical-mediated aromatization. The synthesis leverages an unusual heterocyclic N-oxide α-bromination to functionalize a critical C-H bond, enabling a highly regioselective copper-mediated Ullmann-Goldberg-Buchwald coupling to install a challenging triazole substituent. This strategy resulted in an efficient 11 step linear synthesis of this complex clinical candidate.

(+)-Sorangicin A: evolution of a viable synthetic strategy.

An effective, asymmetric total synthesis of the antibiotic (+)-sorangicin A (1) has been achieved. Central to this venture was the development of first and second generation syntheses of the signature dioxabicyclo[3.2.1]octane core, the first featuring chemo- and stereoselective epoxide ring openings facilitated by a Co(2)(CO)(6)-alkyne complex, the second involving a KHMDS-promoted epoxide ring formation/opening cascade. Additional highlights include effective construction of the dihydro- and tetrahydropyran ring systems, respectively via a stereoselective conjugate addition/α-oxygenation protocol and a thioketalization/hydrostannane reduction sequence. Late-stage achievements entailed two Julia-Kociénski olefinations to unite three advanced fragments with high E-stereoselectivity, followed by a modified Stille protocol to introduce the Z,Z,E trienoate moiety, thereby completing the carbon skeleton. Mukaiyama macrolactonization, followed by carefully orchestrated Lewis and protic acid-promoted deprotections that suppressed isomerization and/or destruction of the sensitive (Z,Z,E)-trienoate linkage completed the first, and to date only, total synthesis of (+)-sorangicin A (1).

Total synthesis of (+)-sorangicin A.

The final synthetic challenges associated with (+)-sorangicin A have been overcome, thus leading to the first total synthesis of this complex macrolide antibiotic. Highlights of the highly convergent synthesis include two Julia-Kociénski olefinations to unite three advanced fragments with high E-stereoselectivity. Critical to the final-stage success was the use of a carefully defined Stille coupling and a Mukaiyama macrolactonization as well as Lewis and protic acid-promoted deprotections carefully designed to suppress E/Z isomerization and/or destruction of the delicate (Z,Z,E)-trienoate linkage.

Catalytic effectiveness, a measure of enzyme proficiency for industrial applications.

Recent attention has been paid to the inadequacy of using the ratio Vmax/KM as a measure of enzyme performance, particularly in the context of industrial biocatalysis. This can lead to misleading expectations of enzyme performance and can be troublesome when used to select among different variants for scale-up evaluation under process conditions. To address these issues, we derive the average velocity based on the time-integrated behavior of the enzyme over the course of the reaction. The resulting expression, deemed catalytic effectiveness, captures important features of the system that have heretofore been ignored (such as highly variable substrate and/or product concentrations and inhibition) and offers a rigorous way to compare enzymes for their capacity to carry out industrial transformations.

Rapid magnetic resonance imaging-guided reduction of craniovertebral junction deformities.

The authors demonstrate the utility of an MR imaging-compatible traction board for the rapid reduction of craniovertebral junction (CVJ) deformities. To choose the appropriate surgical management, patients with compressive CVJ deformities often undergo a trial of traction. Conventional traction trials require the treating surgeon to infer from plain radiographs the manner in which traction forces affect neural and ligamentous structures at the CVJ. To avoid overdistraction injury, low increments of weight are added in a gradual fashion, a process that typically requires 48-72 hours. The authors outline the use of an MR imaging-compatible traction board to determine reducibility safely and rapidly in 4 patients with compressive CVJ deformities. Four patients with advanced CVJ deformities underwent a trial of MR imaging-guided traction performed using an MR imaging-compatible spine board. Serial sagittal images were acquired at baseline and following each sequential addition of force. All patients tolerated traction without neurological worsening. The neural elements were seen to be adequately decompressed in all cases during a single MR imaging session. Patients subsequently underwent craniocervical stabilization and fusion. Postoperative imaging showed maintenance of the reduction without neural compression. An MR imaging-guided trial of traction can facilitate the rapid and safe determination of the reducibility of compressive lesions in patients with advanced CVJ deformities. Rapidly acquired sagittal MR images permit the surgeon to evaluate the effects of traction on the soft tissues at the CVJ, thereby expediting the traction trial and avoiding the risks of immobility in this often-fragile patient population.

Evolution of the Petasis-Ferrier union/rearrangement tactic: construction of architecturally complex natural products possessing the ubiquitous cis-2,6-substituted tetrahydropyran structural element.

The frequent low abundance of architecturally complex natural products possessing significant bioregulatory properties mandates the development of rapid, efficient, and stereocontrolled synthetic tactics, not only to provide access to the biologically rare target but also to enable elaboration of analogues for the development of new therapeutic agents with improved activities and/or pharmacokinetic properties. In this Account, the genesis and evolution of the Petasis-Ferrier union/rearrangement tactic, in the context of natural product total syntheses, is described. The reaction sequence comprises a powerful tactic for the construction of the 2,6- cis-substituted tetrahydropyran ring system, a ubiquitous structural element often found in complex natural products possessing significant bioactivities. The three-step sequence, developed in our laboratory, extends two independent methods introduced by Ferrier and Petasis and now comprises: condensation between a chiral, nonracemic beta-hydroxy acid and an aldehyde to furnish a dioxanone; carbonyl olefination; and Lewis-acid-induced rearrangement of the resultant enol acetal to generate the 2,6- cis-substituted tetrahydropyranone system in a highly stereocontrolled fashion. To demonstrate the envisioned versatility and robustness of the Petasis-Ferrier union/rearrangement tactic in complex molecule synthesis, we exploited the method as the cornerstone in our now successful total syntheses of (+)-phorboxazole A, (+)-zampanolide, (+)-dactylolide, (+)-spongistatins 1 and 2, (-)-kendomycin, (-)-clavosolide A, and most recently, (-)-okilactomycin. Although each target comprises a number of synthetic challenges, this Account focuses on the motivation, excitement, and frustrations associated with the evolution and implementation of the Petasis-Ferrier union/rearrangement tactic. For example, during our (+)-phorboxazole A endeavor, we recognized and exploited the inherent pseudo symmetry of the 2,6- cis-substituted tetrahydropyranone product to overcome the inherent chelation bias of an adjacent oxazolidine ring during the Lewis-acid-promoted rearrangement. In addition, we discovered that a more concentrated solution of Cp2TiMe2 (0.7 versus 0.5 M in THF) with the addition of ethyl pivalate dramatically improves the yield in the Petasis-Tebbe olefination. During the (+)-zampanolide and (+)-dactylolide programs, we observed that the addition of trifluoromethanesulfonic acid (TfOH), especially on a preparative scale, was crucial to the efficiency of the initial condensation/union reaction, while our efforts toward (-)-kendomycin led to the improved implementation of a modified Kurihara condensation of the beta-hydroxy acid and aldehyde involving i-PrOTMS and TMSOTf. Finally, the successful deployment of the Petasis-Ferrier tactic in our synthesis of (-)-clavosolide A validated the viability of this tactic with a system possessing the highly acid-labile cyclopropylcarbinyl moiety, while the challenges en route to (-)-okilactomycin demonstrated that a neighboring alkene functionality can participate in an intramolecular Prins cyclization during the TMSOTf-promoted union process, unless suitably protected.

Enzyme optimization: moving from blind evolution to statistical exploration of sequence-function space.

Directed evolution is a powerful tool for the creation of commercially useful enzymes, particularly those approaches that are based on in vitro recombination methods, such as DNA shuffling. Although these types of search algorithms are extraordinarily efficient compared with purely random methods, they do not explicitly represent or interrogate the genotype-phenotype relationship and are essentially blind in nature. Recently, however, researchers have begun to apply multivariate statistical techniques to model protein sequence-function relationships and guide the evolutionary process by rapidly identifying beneficial diversity for recombination. In conjunction with state-of-the-art library generation methods, the statistical approach to sequence optimization is now being used routinely to create enzymes efficiently for industrial applications.

Access to cerebrospinal fluid absorption sites by infusion into vascular channels of the skull diplö.

OBJECT: The purpose of this human cadaver study was to determine whether or not an intraosseous skull infusion would access the superior sagittal sinus (SSS) via intradural venous channels. The diploic space of the skull bone contains a sinusoidal vascular network that communicates with the underlying dura mater. Diploic veins in the parasagittal area connect with endothelium-lined intradural channels in the subjacent dura and ultimately with the dural venous sinuses. A significant proportion of cerebrospinal fluid (CSF) absorption is thought to occur via arachnoid granulations in the region of the SSS and especially along the parasagittal dura where arachnoid granulations are surrounded by intradural venous channels (lateral lacunae). The CSF is likely to be conducted from the subarachnoid space into the venous system via the fine intradural channels making up the lateral lacunae. METHODS: Infusion of vinyl acetate casting material into the diploic space of the human cadaveric skull resulted in complete filling of the lateral lacunae and SSS. Corrosion casting techniques and examination under magnification were used to characterize the anatomical connections between diploic spaces and dural venous sinuses. RESULTS: Corrosion casting, performed on five formalin-fixed cadavers, clearly showed the anatomical connections between the diploic infusion site and the venous sinuses in the underlying parasagittal dura where some of the CSF is thought to be absorbed. CONCLUSIONS: The diploic vascular channels of the human skull may represent an indirect pathway into the dural venous sinuses. Intraosseous skull infusion may represent another possible strategy for diversion of CSF into the vascular system in the treatment of hydrocephalus.

Intraosseous infusion into the skull: potential application for the management of hydrocephalus.

OBJECT: Hydrocephalus results from abnormal cerebrospinal fluid (CSF) volumes or flow patterns. The absorption of CSF is determined largely by pressures within veins and venous sinuses in the head and adjacent to the spine. Most surgical solutions for hydrocephalus involve diversion of excess CSF into alternative absorption sites, and most of these solutions are still suboptimal. The focus of this work has been to recreate more normal CSF absorption into the dural venous sinuses without having to directly access the superior sagittal sinus (SSS). METHODS: Intraosseous skull infusion for the purpose of accessing the SSS and the systemic venous system was tested by experimental skull infusions of tracer fluids into living large animals (14 adult pigs). Compared with control injections into an ear vein, infusions into the skull through specially designed infusion devices had similar systemic absorption characteristics. This suggested that intraosseous skull infusion in a living large animal was successful in gaining access to the SSS and systemic venous system. CONCLUSIONS: This study constitutes the first demonstration of the success of intraosseous skull infusion in gaining rapid access to the systemic venous system and it thus opens the possibility of using this strategy for diversion of CSF back into the intracranial venous system for the treatment of hydrocephalus.

Improving catalytic function by ProSAR-driven enzyme evolution.

We describe a directed evolution approach that should find broad application in generating enzymes that meet predefined process-design criteria. It augments recombination-based directed evolution by incorporating a strategy for statistical analysis of protein sequence activity relationships (ProSAR). This combination facilitates mutation-oriented enzyme optimization by permitting the capture of additional information contained in the sequence-activity data. The method thus enables identification of beneficial mutations even in variants with reduced function. We use this hybrid approach to evolve a bacterial halohydrin dehalogenase that improves the volumetric productivity of a cyanation process approximately 4,000-fold. This improvement was required to meet the practical design criteria for a commercially relevant biocatalytic process involved in the synthesis of a cholesterol-lowering drug, atorvastatin (Lipitor), and was obtained by variants that had at least 35 mutations.

A pilot study of magnetic resonance imaging-guided closed reduction of cervical spine fractures.

STUDY DESIGN: We report on a prospective selective case series of 17 patients with cervical fracture-dislocations treated with closed reduction under MRI guidance. OBJECTIVE: To demonstrate the safe and effective use of in-line axial traction in the reduction of cervical fracture-dislocations using MRI guidance. SUMMARY OF BACKGROUND DATA: Closed reduction of the cervical spine for acute fracture-dislocations has been a traditional technique used for restoring vertebral alignment and providing neural element decompression. The safety of this technique has been questioned, with concerns of disc migration and overdistraction causing neurologic worsening cited as reasons to choose operative reduction and decompression as a safer option in some circumstances. METHODS: Seventeen patients with fracture-dislocations of the subaxial cervical spine were given a trial of traction under MRI guidance between 1999 and 2003. The incidence of posteriorly herniated disc material was noted, and the diameter of the spinal canal at the injured level was recorded before and after traction. RESULTS: All patients tolerated traction without neurologic worsening. Pretraction disc disruption was found in 15 of 17 (88.2%) of patients, with posterior herniation in 4 of 17 (23.5\%). Traction caused a return of herniated disc material toward the disc space in all cases. Canal dimensions improved in 11 of 17 patients, with canal diameter increasing by a factor of 1.1 to 3.0, with a mean improvement of 1.73. The process of reduction was observed to be a gradual one, with progressive, significant improvement in canal dimensions occurring before anatomic realignment. As distracting force was increased, sequential MRIs showed that canal dimensions did not diminish at any time in any patient. CONCLUSIONS: MRI monitoring in closed cervical reduction is a useful research tool for this technique. Closed reduction appears to be safe as used in this preliminary study and is effective in achieving immediate spinal cord decompression.

A two-sample Bayesian t-test for microarray data.

BACKGROUND: Determining whether a gene is differentially expressed in two different samples remains an important statistical problem. Prior work in this area has featured the use of t-tests with pooled estimates of the sample variance based on similarly expressed genes. These methods do not display consistent behavior across the entire range of pooling and can be biased when the prior hyperparameters are specified heuristically. RESULTS: A two-sample Bayesian t-test is proposed for use in determining whether a gene is differentially expressed in two different samples. The test method is an extension of earlier work that made use of point estimates for the variance. The method proposed here explicitly calculates in analytic form the marginal distribution for the difference in the mean expression of two samples, obviating the need for point estimates of the variance without recourse to posterior simulation. The prior distribution involves a single hyperparameter that can be calculated in a statistically rigorous manner, making clear the connection between the prior degrees of freedom and prior variance. CONCLUSION: The test is easy to understand and implement and application to both real and simulated data shows that the method has equal or greater power compared to the previous method and demonstrates consistent Type I error rates. The test is generally applicable outside the microarray field to any situation where prior information about the variance is available and is not limited to cases where estimates of the variance are based on many similar observations.

(+)-Sorangicin A synthetic studies. Construction of the C(1-15) and C(16-29) subtargets.

[structure: see text] Effective stereocontrolled syntheses of subtargets (-)-2 and (-)-4, comprising respectively the C(16-29) and C(1-15) tetrahydropyran and dihydropyran moieties of the potent antibiotic (+)-sorangicin A (1), have been achieved. The cornerstone for the synthesis of (-)-2 involved an aldol tactic exploiting 1,4-induction, followed in turn by an acid-mediated cyclization/ketalization and hydrosilane reduction promoted by TMSOTf, while construction of (-)-4 entailed a stereoselective conjugate addition/alpha-oxygenation sequence.