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ABSTRACT: A facile method for accurate detection of type 1 dengue virus (DV1) infection from complex biological mixtures, using type specific immunocapture coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. The biological mixtures were treated with magnetic particles coated with a monoclonal antibody directly against type 1 dengue virus. After immunocapture purification, the DV1 was eluted with 30% acetic acid, directly spotted with seed-layer method, and analyzed by MALDI-TOF MS for DV1 capsid protein. The detection limit of the assay was ∼10(5)pfu/mL by MALDI-TOF MS. The immunocapture could unambiguously differentiate the DV1 from other serotypes of the dengue viruses and Japanese encephalitis virus, and could be used as a specific probe to detect DV1 from complex biological mixtures.
Journal of chromatography. A 02/2013; · 4.19 Impact Factor
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ABSTRACT: G. hollisae thermostable direct hemolysin (Gh-TDH) is produced by most strains of G. hollisae. This toxin has been reported to be absorbed in the intestines in humans. Secondary liver injury might be caused by venous return of the toxin through the portal system. We aimed to firstly analyze the in vitro and in vivo hepatotoxicity of Gh-TDH.
Liver cells (primary human non-cancer cell and FL83B mouse cells) were treated and mice (BALB/c) were fed with this toxin to investigate its hepatotoxicity. Morphological examination and cytotoxicity assays using liver cells were also performed. Fluorescein isothiocyanate-conjugated toxin was used to analyze the localization of this protein in liver cells. Mice were subjected to liver function measurements and liver biopsies following toxin treatment and wild-type bacterial infection. PET (positron emission tomography)/CT (computed tomography) images were taken to assess liver metabolism during acute injury and recovery.
The effect of hepatotoxicity was dose and time dependent. Cellular localization showed that the toxin was initially located around the cellular margins and subsequently entered the nucleus. Liver function measurements and liver biopsies of the mice following treatment with toxin or infection with wild-type Grimontia hollisae showed elevated levels of transaminases and damage to the periportal area, respectively. The PET/CT images revealed that the reconstruction of the liver continued for at least one week after exposure to a single dose of the toxin or bacterial infection.
The hepatotoxicity of Gh-TDH was firstly demonstrated. The damage was located in the periportal area of the liver, and the liver became functionally insufficient.
PLoS ONE 01/2013; 8(2):e56226. · 4.09 Impact Factor
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ABSTRACT: A Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase mutant, ERG7(T384Y/Q450H/V454I), produced parkeol but not lanosterol as the sole end product. Parkeol undergoes downstream metabolism to generate compounds 9 and 10. In vitro incubation of parkeol produced a product profile similar to that of the in vivo experiment. In summary, parkeol undergoes a metabolic pathway similar to that of cycloartenol in yeast but distinct from that of lanosterol in yeast, suggesting that two different metabolic pathways of postoxidosqualene cyclization may exist in S. cerevisiae.
Organic Letters 10/2012; 14(20):5222-5. · 5.86 Impact Factor
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ABSTRACT: The Cys703 to Ile or His mutation within Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase ERG7 (ERG7(C703I/H)) generates an unusual truncated bicyclic rearranged intermediate, (8R,9R,10R)-polypoda-5,13E,17E,21-tetraen-3β-ol, related to iridal-skeleton triterpenoid. Numerous oxidosqualene-cyclized truncated intermediates, including tricyclic, unrearranged tetracyclic with 17α/β exocyclic hydrocarbon side chain, rearranged tetracyclic, and chair-chair-chair tricyclic intermediates (compounds 3-9), were also isolated from the ERG7(C703X) site-saturated mutations or the ERG7(F699T/C703I) double mutation, indicating the functional role of the Cys703 residue in stabilizing the bicyclic C-8 cation and the rearranged intermediate or interacting with Phe699, and opened a new avenue of engineering ERG7 for producing biological active agents.
Biochimie 06/2012; 94(11):2376-81. · 3.02 Impact Factor
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ABSTRACT: This study's aim was to determine the predictive factors of the duration of first-attack acute urticaria in children.
The sample included 1075 children admitted to the emergency department with first-attack acute urticaria. Variables comprising the clinical features and past histories of children with duration of disease of 3 days or less, 4 to 7 days, 8 to 14 days, and 15 days or more were compared to determine the predictors of duration of acute urticaria.
Age, various etiologies, clinical presentations, coexistent pyrexia or angioedema, and personal histories of allergic diseases were significant factors (all P < .05). Among allergic diseases, atopic dermatitis was the most significant predictor of duration of acute urticaria, and those with multiple allergic diseases had longer durations of urticaria (both P < .05). Oral plus injection forms of antihistamine or steroid were related to shorter duration of disease (P < .05).
Etiologies and personal allergy history may be the most important predictors of the duration of a first attack of acute urticaria.
The American journal of emergency medicine 10/2011; 29(8):883-9. · 1.54 Impact Factor
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ABSTRACT: The Chinese herb Radix astragalus (RA) has been widely used as a dietary supplement in Asia, and there are numerous reports on its bioactivities. However, there are no reports to date regarding the use of Aspergillus spp. in the culture medium of the RA plant for the production of phenolic antioxidants. In this study, utilizing the fungus Aspergillus to ferment the native RA has successfully resulted in a significant increase in the phenolic contents of RA, and the fermented RA also revealed much better antioxidant activity toward 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals, hydroxyl radical, superoxide radical and peroxyl radical than those of unfermented RA. Among these phenolics, a potent novel antioxidant was isolated and identified as 3,4-di(4'-hydroxyphenyl) isobutyric acid with a molecular weight of 272, by ESI-MS (electrospray ionization mass), ¹H NMR (nuclear magnetic resonance), ¹³C NMR, DEPT (distortionless enhancement by polarization transfer)-NMR, HMQC (heteronuclear multiple quantum coherence), and HMBC (heteronuclear multiple bond correlation) spectra. These data demonstrated that the solid-state bioprocessing strategy could be an innovative approach to enhance the antioxidant activity of RA.
Journal of Agricultural and Food Chemistry 06/2011; 59(12):6520-5. · 2.82 Impact Factor
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ABSTRACT: This paper reports the characteristics, outcomes and clinical features of children with poisoning treated at an emergency department (ED).
This retrospective study at an emergency department consisted of 140 children with poison exposure who were aged under 18 years. Their characteristics were analyzed in order to understand the differences between accidental and non-accidental poisoning. The poisonous materials were divided into two major categories (pharmaceuticals and non-pharmaceuticals) and their associations with patient outcomes were analyzed. Furthermore, the association was analyzed between the incidence of poison exposure and the season in which the poison exposure occurred.
The incidence of poison exposure was highest among adolescents and pre-school age children. Nonaccidental poisoning was more common in older girls and accidental poisoning was more common in younger boys (P<0.001). Neurological system agents were the most common cause of poisoning in the pharmaceutical group and cleansing products were the most common cause of poisoning in the non-pharmaceutical group. Neurological and gastrointestinal symptoms were the most common clinical presentations for the pharmaceutical and non-pharmaceutical groups, respectively. Furthermore, poisoning due to cleansing products and analgesics were associated with the longest duration of hospitalization. March was the highest risk month for pediatric poisoning (P=0.018).
Cleansing products and analgesics were associated with the longest duration of hospitalization and intentional poison was more common in girls.
World Journal of Pediatrics 05/2011; 7(2):143-9. · 1.22 Impact Factor
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ABSTRACT: The crystal structure of the microbial transglutaminase (MTGase) zymogen from Streptomyces mobaraense has been determined at 1.9-Å resolution using the molecular replacement method based on the crystal structure of the mature
MTGase. The overall structure of this zymogen is similar to that of the mature form, consisting of a single disk-like domain
with a deep active cleft at the edge of the molecule. A major portion of the prosequence (45 additional amino acid residues
at the N terminus of the mature transglutaminase) folds into an L-shaped structure, consisting of an extended N-terminal segment
linked with a one-turn short helix and a long α-helix. Two key residues in the short helix of the prosequence, Tyr-12 and
Tyr-16, are located on top of the catalytic triad (Cys-110, Asp-301, and His-320) to block access of the substrate acyl donors
and acceptors. Biochemical characterization of the mature MTGase, using N-α-benzyloxycarbonyl-l-glutaminylglycine as a substrate, revealed apparent Km and kcat/Km values of 52.66 mm and 40.42 mm−1 min−1, respectively. Inhibition studies using the partial prosequence SYAETYR and homologous sequence SQAETYR showed a noncompetitive
inhibition mechanism with IC50 values of 0.75 and 0.65 mm, respectively, but no cross-linking product formation. Nevertheless, the prosequence homologous oligopeptide SQAETQR, with
Tyr-12 and Tyr-16 each replaced with Gln, exhibited inhibitory activity with the formation of the SQAETQR-monodansylcadaverine
fluorophore cross-linking product (SQAETQR-C-DNS). MALDI-TOF tandem MS analysis of SQAETQR-C-DNS revealed molecular masses
corresponding to those of NSQAETQC-C-DNS and C-DNS-NQRC sequences, suggesting the incorporation of C-DNS onto the C-terminal Gln residue of the prosequence homologous oligopeptide.
These results support the putative functional roles of both Tyr residues in substrate binding and inhibition.
Journal of Biological Chemistry 03/2011; 286(9):7301-7307. · 4.77 Impact Factor
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ABSTRACT: We have developed a sensitive gold nanoparticle (AuNP)-based inductively coupled plasma mass spectrometry (ICP-MS) amplification and magnetic separation method for the detection of oligonucleotide sequences. The assay relies on (i) the sandwich-type binding of two designed probe sequences that specifically recognize the target oligonucleotide sequences, (ii) magnetic bead separation, and (iii) AuNP-based ICP-MS amplification detection. To enhance the analytical signal and minimize the background signal resulting from nonspecific binding, we performed a series of experiments to evaluate the effects of various parameters (the concentration of the capture probe; the time required for hybridization; the number of washings required to eliminate nonspecific binding) on the oligonucleotide detection. Under the optimized conditions, the detection limit was 80zmol (corresponding to 1.6fM of the target sequence in a sample volume of 50μL). Moreover, it employs a shorter hybridization step and ICP-MS, this procedure is relatively simple and rapid (ca. 1.5h). Based on the analytical results obtained using complementary and mismatched sequences, our method exhibits good performance in distinguishing complementary and random oligonucleotides. Compared with the "gold standard" methodology (plaque assay) for the quantification of dengue virus, our method has the capability to allow early detection of dengue virus in complicated and small-volume samples, with high specificity, good analytical sensitivity, and superior time-effectiveness.
Journal of chromatography. A 03/2011; 1218(14):1795-801. · 4.19 Impact Factor
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ABSTRACT: Site-saturated substitution in Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase at Ile705 position produced three chair-boat-chair (C-B-C) truncated tricyclic compounds, two 17α-exocyclic protosteryl intermediates, two protosteryl C-17 truncated rearranged intermediates and the normal biosynthetic product, lanosterol. These results indicated the importance of the Ile705 residue in affecting lanosterol's C/D ring stabilization including 6-6-5 tricyclic and protosteryl C-17 cations and 17α/β-exocyclic side chain stereochemistry.
Organic & Biomolecular Chemistry 02/2011; 9(4):1092-7. · 3.70 Impact Factor
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ABSTRACT: Vibrio hollisae, a halophilic species recently reclassified as Grimontia hollisae, is a causative agent of gastroenteritis and septicaemia. One important pathogenic Vibrio factor, thermostable direct haemolysin (TDH), has been purified and crystallized in two crystal forms using the vapour-diffusion method. The crystals belonged to an orthorhombic space group, with unit-cell parameters a = 104.8, b = 112.4, c = 61.3 Å and a = 122.9, b = 123.3, c = 89.8 Å. The crystals contained either four or eight molecules per asymmetric unit, with predicted solvent contents of 49.4 and 46.3% and Matthews coefficients (V(M)) of 2.4 and 2.3 Å(3) Da(-1), respectively. These crystals were suitable for structure determination, which would yield structural details related to the cytotoxicity and oligomeric structure of this pore-forming toxin.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 02/2011; 67(Pt 2):224-7. · 0.51 Impact Factor
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ABSTRACT: An artificial zinc porphyrin-myoglobin-based photo-chemical energy conversion system, consisting of ZnPP-Mb or ZnPE(1)-Mb as a photosensitizer, NADP(+) as an electron acceptor, and triethanolamine as an electron donor, has been constructed to mimic photosystem I. The photoirradiated product is able to reduce a single-electron acceptor protein cytochrome c, but cannot catalyze the two-electron reduction of acetaldehyde by alcohol dehydrogenase, thus demonstrating a single electron transfer mechanism. Furthermore, the artificial system can bifunctionally promote oxidoredox reactions, depending on the presence or absence of a sacrificial electron donor, thus suggesting its potential application in electrochemical regeneration steps involved in chemical transformation and/or energy conversion.
International journal of biological sciences 01/2011; 7(8):1203-13. · 2.70 Impact Factor
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Yu-Kuo Wang,
Sheng-Cih Huang,
Yi-Fang Wu,
Yu-Ching Chen,
Yen-Ling Lin,
Manoswini Nayak,
Yan Ren Lin,
Wen-Hung Chen,
Yi-Rong Chiu,
Thomas Tien-Hsiung Li,
Bo-Sou Yeh, Tung-Kung Wu
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ABSTRACT: Recombinant thermostable direct hemolysin from Grimontia hollisae (Gh-rTDH) exhibits paradoxical Arrhenius effect, where the hemolytic activity is inactivated by heating at 60 °C but is reactivated by additional heating above 80 °C. This study investigated individual or collective mutational effect of Tyr53, Thr59, and Ser63 positions of Gh-rTDH on hemolytic activity, Arrhenius effect, and biophysical properties. In contrast to the Gh-rTDH wild-type (Gh-rTDH(WT)) protein, a 2-fold decrease of hemolytic activity and alteration of Arrhenius effect could be detected from the Gh-rTDH(Y53H/T59I) and Gh-rTDH(T59I/S63T) double-mutants and the Gh-rTDH(Y53H/T59I/S63T) triple-mutant. Differential scanning calorimetry results showed that the Arrhenius effect-loss and -retaining mutants consistently exhibited higher and lower endothermic transition temperatures, respectively, than that of the Gh-rTDH(WT). Circular dichroism measurements of Gh-rTDH(WT) and Gh-rTDH(mut) showed a conspicuous change from a β-sheet to α-helix structure around the endothermic transition temperature. Consistent with the observation is the conformational change of the proteins from native globular form into fibrillar form, as determined by Congo red experiments and transmission electron microscopy.
International journal of biological sciences 01/2011; 7(3):333-46. · 2.70 Impact Factor
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ABSTRACT: Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including L-carnosine and L-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD(CAT). The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an "extra" domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD(CAT) exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD(CAT) for GABAergic therapies or neuroprotection.
Journal of Biological Chemistry 12/2010; 285(50):39500-10. · 4.77 Impact Factor
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ABSTRACT: Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase
H clan that catalyze a broad range of dipeptide and tripeptide substrates, including l-carnosine and l-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may
mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of
the PepD catalytic domain-alone truncated protein PepDCAT. The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two
zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices
to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding
residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains
an “extra” domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed
both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone
truncated PepDCAT exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepDCAT for GABAergic therapies or neuroprotection.
Journal of Biological Chemistry 12/2010; 285(50):39500-39510. · 4.77 Impact Factor
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ABSTRACT: The crystal structure of the microbial transglutaminase (MTGase) zymogen from Streptomyces mobaraense has been determined at 1.9-Å resolution using the molecular replacement method based on the crystal structure of the mature MTGase. The overall structure of this zymogen is similar to that of the mature form, consisting of a single disk-like domain with a deep active cleft at the edge of the molecule. A major portion of the prosequence (45 additional amino acid residues at the N terminus of the mature transglutaminase) folds into an L-shaped structure, consisting of an extended N-terminal segment linked with a one-turn short helix and a long α-helix. Two key residues in the short helix of the prosequence, Tyr-12 and Tyr-16, are located on top of the catalytic triad (Cys-110, Asp-301, and His-320) to block access of the substrate acyl donors and acceptors. Biochemical characterization of the mature MTGase, using N-α-benzyloxycarbonyl-L-glutaminylglycine as a substrate, revealed apparent K(m) and k(cat)/K(m) values of 52.66 mM and 40.42 mM(-1) min(-1), respectively. Inhibition studies using the partial prosequence SYAETYR and homologous sequence SQAETYR showed a noncompetitive inhibition mechanism with IC(50) values of 0.75 and 0.65 mM, respectively, but no cross-linking product formation. Nevertheless, the prosequence homologous oligopeptide SQAETQR, with Tyr-12 and Tyr-16 each replaced with Gln, exhibited inhibitory activity with the formation of the SQAETQR-monodansylcadaverine fluorophore cross-linking product (SQAETQR-C-DNS). MALDI-TOF tandem MS analysis of SQAETQR-C-DNS revealed molecular masses corresponding to those of (N)SQAETQ(C)-C-DNS and C-DNS-(N)QR(C) sequences, suggesting the incorporation of C-DNS onto the C-terminal Gln residue of the prosequence homologous oligopeptide. These results support the putative functional roles of both Tyr residues in substrate binding and inhibition.
Journal of Biological Chemistry 12/2010; 286(9):7301-7. · 4.77 Impact Factor
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ABSTRACT: The aim of this study was to determine the predictive factors of severity and duration of an initial episode of acute urticaria in children. This was a retrospective study of 1120 children of <18 yr who presented to the emergency department (ED) with an initial episode of acute urticaria during the period January 1, 2001, to December 31, 2007. These patients were followed in the ED or outpatient department (OPD) until their symptoms subsided. Variables comprising mild, moderate, and severe urticaria were compared to determine the predictors of severity. The relationships between duration of urticaria and clinical features, including physician-diagnosed causes and treatment modalities, were also analyzed. Significant predictive factors of severity of an initial episode of acute urticaria in children included age, physician-diagnosed causes of urticaria, clinical presentation, coexistent pyrexia or angioedema, and personal allergic history (all p < 0.001). The duration of urticaria was dependent on the physician-diagnosed causes and treatment. Inhalants and unknown causes were predictive of longer duration, while contact materials were associated with shorter duration of urticaria (p < 0.001). Combination treatment comprising an oral plus injectable form of antihistamine or corticosteroid significantly shortened the duration of urticaria compared to single treatment (p < 0.001), especially in children receiving short-term aggressive treatment in the pediatric observation unit (POU) of the ED.
Pediatric Allergy and Immunology 11/2010; 21(7):1043-51. · 2.46 Impact Factor
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ABSTRACT: The study aimed to determine the factors predictive of sustained return of spontaneous circulation (ROSC) in children with out-of-hospital cardiac arrest (OHCA) of noncardiac origin.
Eighty children were included in this retrospective study. The variables that lead to sustained ROSC and those that do not lead to sustained ROSC were analyzed. Survival analyses, including chance of achieving sustained ROSC and sum duration of ROSC, were conducted according to the duration of in-hospital cardiopulmonary resuscitation (CPR).
Etiologies of noncardiac OHCA differed significantly across different age groups (P < .001). Only 8.8% of children had initial arrest rhythms that were shockable. Predictors of sustained ROSC included the initial cardiac rhythm (P = .002), a shorter period between collapse and the first chest compression (P = .002), a shorter in-hospital CPR duration (P = .004), and prehospital CPR (P = .007). In children where ROSC was initially sustained, those with in-hospital CPR of more than 20 minutes, ROSC was sustained for less time (P < .001).
Few children with noncardiac OHCA present with shockable cardiac rhythms. Furthermore, long-term ROSC is difficult to maintain in children who receive in-hospital CPR for more than 20 minutes.
The American journal of emergency medicine 03/2010; 28(3):310-7. · 1.54 Impact Factor
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ABSTRACT: The Saccharomyces cerevisiae ERG7(Phe699) mutants produced one chair-chair-chair (C-C-C) and two chair-boat-chair (C-B-C) truncated tricyclic compounds, one tetracyclic 17alpha-exocyclic unrearranged intermediate, and two 17beta-exocyclic truncated rearranged intermediates. These results provided direct evidence for the importance of the residue in affecting mechanistic transitions between C-B-C and C-C-C substrate conformation and between the 17alpha- and 17beta-exocyclic side chain stereochemistry as well as in stabilizing the 6-6-5 tricyclic and the protosteryl C-17 cations.
Organic Letters 02/2010; 12(3):500-3. · 5.86 Impact Factor
