Pathogenic strains of Escherichia coli and Salmonella enterica modify the tricatecholic siderophore enterobactin (Ent) by glucosylation of three aryl carbon atoms, a process controlled by the iroA locus [Hantke, K., Nicholson, G., Rabsch,... more
Pathogenic strains of Escherichia coli and Salmonella enterica modify the tricatecholic siderophore enterobactin (Ent) by glucosylation of three aryl carbon atoms, a process controlled by the iroA locus [Hantke, K., Nicholson, G., Rabsch, W. & Winkelmann, G. (2003) Proc. Natl. Acad. Sci. USA 100, 3677-3682]. Here, we report the purification of the IroB protein and its characterization as the Ent C-glucosyltransferase. IroB transfers glucosyl groups from uridine-5'-diphosphoglucose to C5 of one, two, or three of the 2,3-dihydroxybenzoyl units of Ent to yield monoglucosyl-C-Ent (MGE), diglucosyl-C-Ent (DGE), and triglucosyl-C-Ent (TGE). DGE, also known as salmochelin S4, and macrolactone-opened derivatives have been isolated from the culture broths of S. enterica and uropathogenic E. coli [Bister, B., Bischoff, D., Nicholson, G. J., Valdebenito, M., Schneider, K., Winkelmann, G., Hantke, K. & Sussmuth, R. D. (2004) Biometals 17, 471-481], but MGE and TGE have not been reported previously. IroB has a k(cat) of approximately 10 min(-1) for the first C-glucosylation and is distributive, with sequential conversion and buildup of MGE and then DGE. The C5 to C1' regio-selectivity of the 2,3-dihydroxybenzoyl-glucose linkage at all three rings of TGE suggests a C5 carbanion, para to the C2 phenolate oxygen, as the carbon nucleophile in this novel enzymatic C-glucosylation.
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Research Interests:
To evaluate the therapeutic effect of intravitreal bevacizumab combined with intravitreal plasminogen and pneumatic retinopexy as treatment of subfoveal hemorrhages due to exudative age-related macular degeneration. Clinical... more
To evaluate the therapeutic effect of intravitreal bevacizumab combined with intravitreal plasminogen and pneumatic retinopexy as treatment of subfoveal hemorrhages due to exudative age-related macular degeneration. Clinical interventional case series study. Ten patients (10 eyes) with exudative age-related macular degeneration, presented with a subfoveal hemorrhage larger than 1 disc size and smaller than 5 disc sizes. They received an intravitreal injection of 50 μg of plasminogen and 0.3 mL of 100% sulfur hexafluoride gas combined with intravitreal 1.5 mg of bevacizumab, followed by 2 additional intravitreal injections of 1.5 mg of bevacizumab in an interval of 6 weeks. Mean visual acuity improved slightly, although not statistically significant (P = 0.24), from 1.56 ± 0.47 to 1.48 ± 0.60 logMAR at 1 month after the procedure and to 1.35 ± 0.54 logMAR at 3 months after baseline. Subfoveal hemorrhage recurred in none of the patients during the follow-up. In all patients, the subfoveal hemorrhage had at least partially been moved to the infrafoveal region. Pronounced degenerative subfoveal changes were the main reason for a lack of a marked increase in visual acuity after the procedure. For some patients with exudative age-related macular degeneration and a subfoveal hemorrhage larger than 1 disc size and smaller than 5 disc sizes, the combined intravitreal injection of bevacizumab, plasminogen, and gas followed by 2 additional intravitreal bevacizumab injections can lead to a stabilization or slight improvement in visual acuity, unless subfoveal degenerative changes are not too marked to prevent a gain in vision.
Inteins are naturally occurring protein elements that catalyze their own excision from within a larger protein together with the ligation of the flanking... more
Inteins are naturally occurring protein elements that catalyze their own excision from within a larger protein together with the ligation of the flanking "extein" sequences. Previously we reported the directed evolution of an intein-based molecular switch in which intein splicing in yeast cells was made dependent on the cell-permeable small molecule 4-hydroxytamoxifen (4-HT). Here we show that these evolved inteins are effective means of rendering protein function and biological signaling pathway activation dependent on 4-HT in mammalian cells. We have characterized the generality, speed, and dose dependence of ligand-induced protein splicing in murine NIH3T3 cells and in human HEK293 cells. Evolved inteins were used to control in mammalian cells the function of Gli1 and a truncated form of Gli3, two transcriptional mediators of the Hedgehog signaling pathway. Finally, we show that a complex biological process such as osteoblast differentiation can be made dependent on 4-HT using the evolved intein system. Our findings suggest that evolved small-molecule-dependent inteins may serve as a general means of achieving gene-specific, dose-dependent, post-translational, and small-molecule-induced control over protein activity in mammalian systems.
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Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via... more
Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large, multidomain enzymes that biosynthesize a number of pharmaceutically important natural products. The recognition of biosynthetic intermediates, displayed via covalent attachment to carrier proteins, by catalytic domains is critical for NRPS and polyketide synthase function. We report the use of combinatorial mutagenesis coupled with in vivo selection for the production of the Escherichia coli NRPS product enterobactin to map the surface of the aryl carrier protein (ArCP) domain of EntB that interacts with the downstream elongation module EntF. Two libraries spanning the predicted helix 2 and loop 2/helix 3 of EntB-ArCP were generated by shotgun alanine scanning and selected for their ability to support enterobactin production. From the surviving pools, we identified several hydrophobic residues (M249, F264, and A268) that were highly conserved. These residues cluster near the phosphopantetheinylated serine i...
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To evaluate the changes in aqueous vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) following intravitreal bevacizumab injections for choroidal neovascularization (CNV) secondary to age-related... more
To evaluate the changes in aqueous vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) following intravitreal bevacizumab injections for choroidal neovascularization (CNV) secondary to age-related macular degeneration or pathologic myopia. Aqueous samples were obtained at the time of injection from 51 eyes of 51 patients who underwent three monthly intravitreal bevacizumab (Avastin) injections at baseline, 1, and 2 months. Concentrations of VEGF and PEDF in the aqueous were evaluated using enzyme-linked immunosorbent assay and compared. For the 34 eyes with age-related macular degeneration CNV, the mean +/- standard deviation aqueous VEGF level reduced from 102.6 pg/mL +/- 90.6 pg/mL at baseline to 18.3 pg/mL +/- 22.5 pg/mL at 2 months (P < 0.001), whereas the mean PEDF level increased from 11.2 ng/mL +/- 10.4 ng/mL at baseline to 38.7 ng/mL +/- 47.9 ng/mL at 2 months (P = 0.001). For the 17 eyes with myopic CNV, the mean +/- standard deviation ...
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We tested the hypothesis that genetic variants in vasoactive and angiogenic factors regulating the retina vasculature contribute to the development of diabetic retinopathy (DR). A case-control study was performed to study the genetic... more
We tested the hypothesis that genetic variants in vasoactive and angiogenic factors regulating the retina vasculature contribute to the development of diabetic retinopathy (DR). A case-control study was performed to study the genetic association between DR and polymorphic variants of EDN1 (Lys198Asn), LTA (IVS1-80C>A, IVS1-206G>C, IVS1-252A>G), eNOS (Glu298Asp), and ITGA2 (BgI II) in a Chinese population with type 2 diabetes mellitus. A well defined population with type 2 diabetes, consisting of 127 controls and 216 DR patients, was recruited. A higher frequency of the Asn/Asn genotype of EDN1 was found in individuals with at least 10 years of diabetes and no retinopathy (controls) compared with DR patients with any duration of diabetes (DR: 2.3%; control: 11.0%; p=0.0002). The Asn allele was also more frequent in controls than DR patients (DR: 16.4%; control: 29.5%; p=0.007). Multiple logistic regression analysis showed that the Asn/Asn genotype was the factor most signifi...
Research Interests: Genetics, Polymorphism, China, Type 2 Diabetes, Molecular, and 20 moreHumans, Hypertension, Diabetic Retinopathy, Female, Male, Regression Analysis, Logistic Regression Analysis, Aged, Middle Aged, Optometry and Ophthalmology, Genotype, Lysine, Odds ratio, Endothelin-1, Genetic Association, Case Control Study, Type 2 Diabetes Mellitus, Asparagine, Age of Onset, and Case Control Studies
Probes that form covalent bonds with RNA molecules on the basis of their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with... more
Probes that form covalent bonds with RNA molecules on the basis of their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with unusually nucleophilic RNAs. We used these probes to identify reactive genome-encoded RNAs, resulting in the discovery of a 42-nt catalytic RNA from an archaebacterium that reacts with a 2,3-disubstituted epoxide at N7 of a specific guanosine. Detailed characterization of the catalytic RNA revealed the structural requirements for reactivity. We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest. This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate. We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind the ASH1 mRNA.
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Primary liver malignancies and liver metastases are affecting millions of individuals worldwide. Because of their late and advanced stage presentation, only 10% of patients can receive curative surgical treatment, including transplant or... more
Primary liver malignancies and liver metastases are affecting millions of individuals worldwide. Because of their late and advanced stage presentation, only 10% of patients can receive curative surgical treatment, including transplant or resection. Alternative treatments, such as systemic chemotherapy, ablative therapy, and chemoembolization, have been used with marginal survival benefits. Selective internal radiation therapy (SIRT), also known as radioembolization, is a compelling alternative treatment option for primary and metastatic liver malignancies with a growing body of evidence. In this article, an introduction to SIRT including background, techniques, clinical outcomes, and complications is reviewed.
Research Interests: Brachytherapy and Humans
To evaluate the efficacy of a safety enhanced photodynamic therapy (PDT) protocol with half-dose verteporfin for treating chronic central serous chorioretinopathy (CSC). Forty-eight eyes of 48 patients with symptomatic chronic CSC... more
To evaluate the efficacy of a safety enhanced photodynamic therapy (PDT) protocol with half-dose verteporfin for treating chronic central serous chorioretinopathy (CSC). Forty-eight eyes of 48 patients with symptomatic chronic CSC underwent indocyanine green angiography guided PDT with half dose (3 mg/m) verteporfin. Outcome measures included logMAR best-corrected visual acuity (BCVA), central retinal thickness, and angiographic changes during the 12-month study period. The mean CSC duration was 8.2 months (range, 3-40 months). At 12 months after PDT, the mean logMAR BCVA improved from 0.31 to 0.15 (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001). The mean improvement was 1.6 lines and 45 (95.8%) eyes had stable or improved vision. Eyes without pigment epithelial detachment (PED) had significantly greater visual improvement compared with eyes with PED (P = 0.031). Patients with CSC of 6 months or less or younger than 45 years were more likely to gain vision by two or more lines after treatment (P = 0.007 and P = 0.018, respectively). Forty (83.3%) eyes had complete resolution of serous detachment at 3 months, with 43 (89.6%) eyes at 12 months. The safety enhanced PDT protocol appeared to be beneficial for patients with chronic CSC. Further controlled study is warranted to evaluate the safety and efficacy of this treatment option.
Research Interests: Treatment Outcome, Optical coherence tomography, Porphyrins, Photodynamic Therapy, Visual acuity, and 14 moreProspective studies, Humans, Chronic Disease, Female, Male, Retina, Follow-up studies, Aged, Middle Aged, Optometry and Ophthalmology, Photochemotherapy, Adult, Retinal Diseases, and Prospective Study
To evaluate the efficacy of photodynamic therapy (PDT) with half-dose verteporfin for treating acute central serous chorioretinopathy (CSC). Prospective, double-masked, placebo-controlled, randomized clinical trial. Sixty-three eyes of 63... more
To evaluate the efficacy of photodynamic therapy (PDT) with half-dose verteporfin for treating acute central serous chorioretinopathy (CSC). Prospective, double-masked, placebo-controlled, randomized clinical trial. Sixty-three eyes of 63 patients with acute symptomatic CSC of 3 months&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; duration or less were recruited. Forty-three eyes were randomized to indocyanine green angiography (ICGA)-guided PDT with half-dose (3 mg/m(2)) verteporfin and 21 eyes were randomized to placebo. Patients in the verteporfin group received an infusion of half-dose verteporfin over 8 minutes, followed by ICGA-guided PDT 10 minutes from the start of infusion. Laser was applied for 83 seconds covering the choroidal abnormalities observed in ICGA, with a maximum laser spot size of 4500 mum. The primary outcome measure was the proportion of eyes with absence of subretinal fluid at the macula at 12 months. Secondary outcome measures included changes in mean logarithm of the minimum angle of resolution (logMAR) best-corrected visual acuity (BCVA), subjective symptoms, optical coherence tomography (OCT) results, central foveal thickness (CFT), and angiographic findings during the 12-month study period. Thirty-nine patients in the verteporfin group and 19 patients in the placebo group completed 12 months of follow-up. Thirty-seven (94.9%) eyes in the verteporfin group compared with 11 (57.9%) eyes in the placebo group showed absence of subretinal fluid at the macula at 12 months (P = 0.001). The mean logMAR BCVA at 12 months was significantly better in the verteporfin group compared with the placebo group: -0.05 and 0.05, respectively (P = 0.008). All 39 (100%) verteporfin-treated eyes had stable or improved vision, compared with 15 (78.9%) eyes in the placebo group (P = 0.009). The mean OCT CFT for the verteporfin group also was significantly lower compared with the placebo group at 12 months (P = 0.001). No ocular or systemic adverse event was encountered in the study. Photodynamic therapy with half-dose verteporfin is effective in treating acute symptomatic CSC, resulting in a higher proportion of patients with absence of exudative macular detachment and better visual acuity compared with placebo.
Research Interests: Ophthalmology, Treatment Outcome, Optical coherence tomography, Porphyrins, Photodynamic Therapy, and 15 moreVisual acuity, Prospective studies, Humans, Female, Male, Follow-up studies, Clinical Sciences, Middle Aged, Optometry and Ophthalmology, Photochemotherapy, Adult, Public health systems and services research, Retinal Diseases, Body Fluids, and Exudates and Transudates
Central serous chorioretinopathy (CSC) is a complicated disease with still unclear causes, pathogenesis and management strategy despite active research. CSC has been traditionally considered as a self-limiting disease where spontaneous... more
Central serous chorioretinopathy (CSC) is a complicated disease with still unclear causes, pathogenesis and management strategy despite active research. CSC has been traditionally considered as a self-limiting disease where spontaneous recovery occurs in 90% of the patients within a few months. This proclaimed &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;benign&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; nature of CSC, however, has been queried by increasing scientific evidence that permanent photoreceptors damage and neurosensory-cystoid degeneration of macula occur in the event of chronic CSC. CSC is probably not a benign disease. Treatments for CSC are still evolving. It is very difficult to define the proper timing for active treatment of CSC because it is not easy to define a universally accepted cut-off time point for active intervention. There is a recent suggestion that active CSC treatment should be considered if symptoms last longer than 3 months as atrophy of photoreceptors may occur as early as 4 months after initial presentation. The CSC patients may be stratified into two groups based on the initial presenting visual acuity and duration of symptom: the good visual prognosis group and the dubious visual prognosis group. The management may then be tailor-made based on the visual prognosis group. &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;Safety-enhanced&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; photodynamic therapy (PDT) using lower doses and reduced fluence is still the mainstay of treatment. Newer treatment modalities like intravitreal anti-VEGF therapy, micropulsed diode laser treatment, and the use of corticosteroid antagonists do warrant further investigation. Combination therapies involving two or more of the above modalities of treatments may have a role to play in this actively researched area.
The objective of the study was to determine the efficacy of contrast-enhanced ultrasound (CEUS) using ultrasound (US)-specific microbubbles in guiding radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC). A retrospective... more
The objective of the study was to determine the efficacy of contrast-enhanced ultrasound (CEUS) using ultrasound (US)-specific microbubbles in guiding radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC). A retrospective analysis of 50 patients with HCC treated with CEUS guided RFA using perflutren at our institution was performed. CEUS images were first compared to B-mode US images performed at the same RFA session to determine the ability of CEUS to increase the conspicuity of lesions. A qualitative score (1 = poor, 2 = fair, 3 = excellent) was used to grade the ability to visualize the lesions. The preprocedure CEUS images were then evaluated using the most recent prior contrast enhanced computed tomography (CT) or magnetic resonance imaging (MRI). The efficacy of the treatment was evaluated with short-term follow-up imaging (median 1 month) for presence of residual or recurrent disease. CEUS allows at least fair visualization (score ≥2) in 78% (reader 1) and 80% (reader 2) of the lesions not visualized by B-mode US, and 50% (reader 1) and 42% (reader 2) of the lesions poorly visualized by B-mode US. Lesion appearances on CEUS are largely concordant with those on CT or MRI: 88% for reader 1, 96% for reader 2. With CEUS-guided RFA, complete response was achieved in the vast majority of the lesions at short-term follow-up: 82% for reader 1, 94% for reader 2. CEUS increases the conspicuity and provides better characterization of hypervascular HCC that are either not seen or poorly seen on B-mode US, and CEUS provides real-time guidance of RFA with good short-term treatment responses.
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ABSTRACT The management of colorectal liver metastasis has undergone a significant change since the development of novel ablation and embolization. Drug-eluting microsphere platforms, designed to deliver targeted concentrations of... more
ABSTRACT The management of colorectal liver metastasis has undergone a significant change since the development of novel ablation and embolization. Drug-eluting microsphere platforms, designed to deliver targeted concentrations of systemic therapy directly into the tumor via its arterial vasculature, have garnered interest and gained in popularity in recent years. Based on in vitro and in vivo data, multiple factors contribute to locoregional exposure including carrier base, smaller particle size (larger surface area), chemotherapeutic and chemotherapeutic intensity. Based on the current published clinical data, therapy appears well tolerated but the questions remain as to the ideal technique, patient population and overall efficacy. The purpose of this article is to provide a perspective on the scientific basis, and clinical review of the current data supporting the use of this platform in the setting of metastatic colorectal carcinoma.
We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can in principle be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs... more
We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can in principle be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.
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Research Interests:
To evaluate the efficacy of intravitreal ranibizumab for the primary treatment of myopic choroidal neovascularization (CNV). Sixteen eyes of 16 consecutive patients who received 3 monthly injections of intravitreal ranibizumab for primary... more
To evaluate the efficacy of intravitreal ranibizumab for the primary treatment of myopic choroidal neovascularization (CNV). Sixteen eyes of 16 consecutive patients who received 3 monthly injections of intravitreal ranibizumab for primary treatment of myopic CNV were reviewed. Additional ranibizumab injections were performed in eyes with persistent or recurrent CNV after 3 months. The mean age of the patients was 60.8 years, and the spherical equivalent refractive error was -10.9 D. The mean logMAR best-corrected visual acuity at baseline was 0.58 (20/76). At 1 month and 12 months, the mean logMAR best-corrected visual acuity improved significantly to 0.39 (20/49) and 0.28 (20/37), respectively (P = 0.001 and P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001, respectively). The mean improvement at 12 months was 3.0 lines, and 12 (75.0%) eyes had improvement of 2 or more lines. Fifteen (93.8%) eyes had angiographic closure at 3 months and 1 (6.2%) required further treatment because of persistent leakage at 3 months. Two (12.5%) patients had recurrence of CNV and required retreatment between 3 months and 9 months. Optical coherence tomography showed significant reduction in the mean central foveal thickness after treatment (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001). None of the patients developed any ocular or systemic side effects associated with intravitreal ranibizumab. Intravitreal ranibizumab appeared to be effective for the primary treatment of myopic CNV, with a high proportion of patients sustaining visual gain after treatment.
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Research Interests: Genetics, Multidisciplinary, Macromolecular X-Ray Crystallography, Protein Stability, Escherichia coli, and 11 moreProtein structure, Enzyme, Protein Function, Molecular cloning, Functional Group, Amino Acid Profile, Boolean Satisfiability, aminoacyl-tRNA synthetase, Protein Structure Determination, Genetic Code, and Genetic Selection
Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced... more
Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced with a domain from a foreign NRPS to create a chimeric assembly line that produces a new variant of a natural NRP. However, such chimeric NRPS modules are often heavily impaired, impeding efforts to create novel NRP variants by swapping domains from different modules or organisms. Here we show that impaired chimeric NRPSs can be functionally restored by directed evolution. Using rounds of mutagenesis coupled with in vivo screens for NRP production, we rapidly isolated variants of two different chimeric NRPSs with approximately 10-fold improvements in enzyme activity and product yield, including one that produces new derivatives of the potent NRP/polyketide antibiotic andrimid. Because functional restoration in these examples required only modest library sizes (10(3) to 10(4) clones) and three or fewer rounds of screening, our approach may be widely applicable even for NRPSs from genetically challenging hosts.
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Research Interests:
Research Interests:
To evaluate the long-term efficacy of verteporfin photodynamic therapy (PDT) as the primary treatment for symptomatic circumscribed choroidal hemangioma (CCH). Prospective consecutive, 2-centered, noncomparative, interventional case... more
To evaluate the long-term efficacy of verteporfin photodynamic therapy (PDT) as the primary treatment for symptomatic circumscribed choroidal hemangioma (CCH). Prospective consecutive, 2-centered, noncomparative, interventional case series. Twenty-five subjects with symptomatic CCH. All patients had recent onset of visual symptoms and evidence of exudative macular changes on fluorescein angiography (FA) and optical coherence tomography (OCT). Verteporfin 6 mg/m(2) body surface area was administered intravenously over a 10-minute interval. Five minutes after infusion, a 689 nm laser was applied with a light dose of 50 J/cm(2) for the first 3 patients and a light dose of 100 J/cm(2) for all the other patients. Retreatments were performed in case of persistent exudation found on OCT. Evaluation of best-corrected visual acuity (BCVA) using Early Treatment of Diabetic Retinopathy Study (ETDRS) criteria, FA, indocyanine green angiography (ICGA), OCT, and ultrasound were performed before PDT and on follow-up examinations. All patients were followed for at least 5 years. Primary outcome measures were changes in BCVA and foveal center thickness (FCT) between baseline and month 60. Secondary measures were tumor thickness decrease, absence of leakage on FA, and adverse events. Twenty-two patients received 1 PDT session at 100 J/cm(2), and no recurrences were detected. Three eyes, treated with 50 J/cm(2), received a second PDT session at 100 J/cm(2) 1 month after the first session. After a follow-up of 60 months, BCVA improved an average of 18.5 ETDRS letters (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.001); BCVA improved by &amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; or =2 lines in 19 eyes (76%). The FCT decreased from a mean of 386.20 microm to 179.2 microm, and OCT showed the complete resolution of macular exudation in all cases. All tumors responded with a reduction in size. No treatment-related adverse events or complications were identified. The 5-year results of PDT in treating symptomatic CCH support treatment with a light dose of 100 J/cm(2) after slow intravenous infusion of verteporfin to stabilize or improve visual acuity and resolution of macular exudation.
Research Interests: Ophthalmology, Treatment Outcome, Optical coherence tomography, Porphyrins, Photodynamic Therapy, and 14 moreVisual acuity, Prospective studies, Humans, Female, Male, Follow-up studies, Clinical Sciences, Aged, Middle Aged, Optometry and Ophthalmology, Photochemotherapy, Adult, Public health systems and services research, and Antineoplastic Agents
Research Interests: Ophthalmology, Treatment Outcome, Optical coherence tomography, Visual acuity, Prospective studies, and 15 moreHumans, Diabetic Retinopathy, Female, Male, Retina, Clinical Sciences, Aged, Middle Aged, Optometry and Ophthalmology, Triamcinolone Acetonide, Public health systems and services research, Diabetic Macular Edema (DME), Glucocorticoids, Macular edema, and Injections
To evaluate the safety and efficacy of intravitreal bevacizumab in the treatment of choroidal neovascularization (CNV) secondary to pathologic myopia (PM). Prospective, consecutive, nonrandomized, interventional case series. Twenty-two... more
To evaluate the safety and efficacy of intravitreal bevacizumab in the treatment of choroidal neovascularization (CNV) secondary to pathologic myopia (PM). Prospective, consecutive, nonrandomized, interventional case series. Twenty-two eyes of 22 patients with CNV secondary to PM. Consecutive patients with subfoveal or juxtafoveal CNV secondary to PM were recruited prospectively to receive an initial course of 3 monthly intravitreal injections of bevacizumab. Three additional monthly injections were performed in eyes with persistent CNV leakage after 3 months. Patients were followed up for 6 months, and the best-corrected visual acuity (BCVA), changes in fluorescein angiography, and optical coherence tomography (OCT) results were assessed. Changes in BCVA, angiographic closure, and OCT central foveal thickness (CFT) at the 6-month follow-up. The mean+/-standard deviation (SD) spherical equivalent refractive error of the 22 eyes was -10.3+/-3.7 D (range, -6.0D to -18.0D). All patients completed follow-up at 6 months. Twenty (90.9%) eyes had angiographic closure after 3 monthly injections of intravitreal bevacizumab, and 2 (9.1%) eyes required further treatment up to 6 months. The mean+/-SD logarithm of the minimum angle of resolution (logMAR) BCVA at baseline was 0.60+/-0.18 (Snellen equivalent, 20/80). At 1 and 6 months, the mean+/-SD logMAR BCVA improved significantly to 0.43 (Snellen equivalent, 20/53; P = 0.003) and 0.35 (Snellen equivalent, 20/45; P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.001), respectively. The mean lines of improvements at 1 and 6 month compared with baseline were 1.7 and 2.6 lines, respectively. Fifteen (68.2%) eyes had an improvement of 2 or more lines at 6 months. The OCT results also showed significant reduction in CFT after treatment. No ocular or systemic complications were noted after intravitreal injections. The 6-month outcomes suggest intravitreal bevacizumab to be a promising treatment method for CNV secondary to PM, resulting in both visual and anatomic improvements. Treatment resulted in complete absence of angiographic leakage in 90.9% of eyes at 3 months. Further studies to evaluate the safety, efficacy, and optimal treatment regimen are justified.
Research Interests: Ophthalmology, Treatment Outcome, Optical coherence tomography, Visual acuity, Prospective studies, and 12 moreHumans, Female, Male, Monoclonal Antibodies, Clinical Sciences, Aged, Middle Aged, Optometry and Ophthalmology, Adult, Public health systems and services research, Pilot Projects, and Injections
To assess the efficacy of less than 3 cycles of intra-arterial chemotherapy (IAC) for retinoblastoma. Retrospective, nonrandomized, interventional case series. Eight patients. Intra-arterial chemotherapy. Tumor control and globe salvage.... more
To assess the efficacy of less than 3 cycles of intra-arterial chemotherapy (IAC) for retinoblastoma. Retrospective, nonrandomized, interventional case series. Eight patients. Intra-arterial chemotherapy. Tumor control and globe salvage. Eight patients received fewer than 3 cycles of IAC for retinoblastoma because there was complete tumor control with no residual viable tumor (n = 7) or poor response (n = 1) with little hope that further therapy would benefit the patient. In 3 cases, additional vascular compromise precluded further IAC. The treatment was primary in 6 cases and secondary after failure of other treatment in 2 cases. The 8 eyes were classified (International Classification of Retinoblastoma) as group C (n = 2), group D (n = 3), group E (n = 1), and secondary treatment (n = 2). At initial examination, the main tumor showed a mean basal diameter of 16 mm, a thickness of 8.6 mm, vitreous seeds (n = 2), subretinal seeds (n = 6), and iris neovascularization (n = 1). Three patients were treated with a single cycle of IAC, and 5 patients were treated with 2 cycles of IAC. After IAC, complete tumor response was found in 7 eyes (88%) and partial response was found in 1 eye (13%). Over a mean of 13 months follow-up, there was intraretinal tumor recurrence (n = 1), subretinal seed recurrence (n = 1), and no case of vitreous seed recurrence. Globe salvage was achieved in 2 of 2 group C eyes (100%), 3 of 3 group D eyes (100%), 0 of 1 group E eye (0%), and 1 of 2 secondary treatment eyes (50%). Globe salvage was achieved in 6 of 8 eyes (75%), and 2 of 8 eyes (25%) required enucleation. One or 2 cycles of IAC can be sufficient for selected eyes with group C or D retinoblastoma, with remarkable tumor control. The author(s) have no proprietary or commercial interest in any materials discussed in this article.
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Laser peripheral iridotomy is the standard treatment for acute angle-closure glaucoma. A patient with acute angle-closure glaucoma who developed central serous chorioretinopathy after uneventful laser iridotomies is described. Central... more
Laser peripheral iridotomy is the standard treatment for acute angle-closure glaucoma. A patient with acute angle-closure glaucoma who developed central serous chorioretinopathy after uneventful laser iridotomies is described. Central serous chorioretinopathy occurring after sequential argon-neodymium:YAG laser peripheral iridotomy is a novel complication in the English literature and is related to the stress induced by both the initial disease and the subsequent procedure, particularly in psychologically susceptible individuals.
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Research Interests:
The 5-HT3 receptor is a ligand-gated ion channel with significant structural similarity to the nicotinic acetylcholine receptor. Several regions that form the ligand binding site in the nicotinic acetylcholine receptor are partially... more
The 5-HT3 receptor is a ligand-gated ion channel with significant structural similarity to the nicotinic acetylcholine receptor. Several regions that form the ligand binding site in the nicotinic acetylcholine receptor are partially conserved in the 5-HT3 receptor, presumably reflecting the conserved signal transduction mechanism. Specific amino acid differences in these regions may account for their distinct ligand recognition properties. Using site-directed mutagenesis, we have replaced one of these residues, glutamate 106 (E106), with aspartate (D), asparagine (N), alanine (A) or glutamine (Q) and characterized the ligand-binding and electrophysiological properties of the mutant receptors after transient expression in HEK-293 cells. The affinity for the selective 5-HT3 receptor antagonist [3H]GR65630 was decreased 14-fold in the mutant E106D (Kd = 3.69 +/- 0.32 nM) when compared to wildtype (WT, E106) 5-HT3 receptor (0.27 +/- 0.03 nM), while the affinity for E106N was unchanged (0.42 +/- 0.07 nM, means +/- SEM, n = 3-10). Decreased affinities for both E106D and E106N were observed for the antagonists granisetron, ondansetron and renzapride and for the agonists 5-HT (130- and 30-fold) and 2-methyl-5-HT (250- and 20-fold), respectively. Both mutants still formed 5-HT-activatable ion channels, but the high Hill coefficient of the concentration effect curves in wildtype (2.0) was decreased to unity in both cases. The EC50 of 5-HT was increased seven-fold in E106N (8.7 microM) when compared to wildtype (1.2 microM), but unchanged in E106D, and the potency of the antagonist ondansetron for both mutants was decreased. E106A and E106Q expressed poorly preventing a detailed characterization. These data suggest that E106 contributes to the ligand-binding site of the 5-HT3 receptor and may form an ionic or hydrogen bond interaction with the primary ammonium group of 5-HT.
Research Interests: Psychology, Electrophysiology, Neuropharmacology, Patch-clamp and imaging techniques, Signal Transduction, and 18 moreSerotonin, Cell line, Glutamate, Humans, Kidney, Mice, Animals, Ligand Binding, Acetylcholine, Hydrogen Bond, Serotonin Receptors, Radioligand Assay, Amino Acid Profile, Structural Similarity Index, Site-directed Mutagenesis, Ligands, Neurosciences, and Glutamic Acid
Page 1. Correspondence Effects of chronic lead exposure on 1H MRS of hippocampus and frontal lobes in children To the Editor: The ideal of the scientific method is eloquently expressed in Bertolt Brecht's play “Galileo” when ...
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To characterize the contributions of individual amino acids to the structure or function of a protein, researchers have adopted directed evolution approaches, which use iterated cycles of mutagenesis and selection or screening to search... more
To characterize the contributions of individual amino acids to the structure or function of a protein, researchers have adopted directed evolution approaches, which use iterated cycles of mutagenesis and selection or screening to search vast areas of sequence space for sets of mutations that provide insights into the protein of interest.
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Using a system that accelerates the serendipitous discovery of new reactions by evaluating hundreds of DNA-encoded substrate combinations in a single experiment, we explored a broad range of reaction conditions for new bond-forming... more
Using a system that accelerates the serendipitous discovery of new reactions by evaluating hundreds of DNA-encoded substrate combinations in a single experiment, we explored a broad range of reaction conditions for new bond-forming reactions. We discovered reactivity that led to a biomolecule-compatible, Ru(II)-catalysed azide-reduction reaction induced by visible light. In contrast to current azide-reduction methods, this reaction is highly chemoselective and is compatible with alcohols, phenols, acids, alkenes, alkynes, aldehydes, alkyl halides, alkyl mesylates and disulfides. The remarkable functional group compatibility and mild conditions of the reaction enabled the azide reduction of nucleic acid and oligosaccharide substrates, with no detectable occurrence of side reactions. The reaction was also performed in the presence of a protein enzyme without the loss of enzymatic activity, in contrast to two commonly used azide-reduction methods. The visible-light dependence of this reaction provides a means of photouncaging functional groups, such as amines and carboxylates, on biological macromolecules without using ultraviolet irradiation.
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We developed a general method to detect cellular small molecule-RNA conjugates that does not rely on the reactivity of the small molecule. This technique revealed NAD-linked RNA in Escherichia coli and Streptomyces venezuelae. Subsequent... more
We developed a general method to detect cellular small molecule-RNA conjugates that does not rely on the reactivity of the small molecule. This technique revealed NAD-linked RNA in Escherichia coli and Streptomyces venezuelae. Subsequent characterization showed that NAD is a 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; modification of RNA, cannot be installed in vitro through aberrant transcriptional initiation, is only found among smaller cellular RNAs and is present at a surprisingly high abundance of approximately 3,000 copies per cell.
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Many bacteria, including numerous human pathogens, synthesize small molecules known as siderophores to scavenge iron. Enterobactin, a siderophore produced by enteric bacteria, is surprisingly ineffective as an iron-scavenging agent for... more
Many bacteria, including numerous human pathogens, synthesize small molecules known as siderophores to scavenge iron. Enterobactin, a siderophore produced by enteric bacteria, is surprisingly ineffective as an iron-scavenging agent for bacteria growing in animals because of its hydrophobicity and its sequestration by the mammalian protein siderocalin, a component of the innate immune system. However, pathogenic strains of Escherichia coli and Salmonella use enzymes encoded by the iroA gene cluster to tailor enterobactin by glycosylation and linearization. The resulting modified forms of enterobactin, known as salmochelins, can evade siderocalin and are less hydrophobic than enterobactin, restoring this siderophore&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s iron-scavenging ability in mammals.
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Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage... more
Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ∼15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;nickases&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;, recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least fourfold higher than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the versatility of this approach for highly specific genome-wide editing.
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Current approaches to reaction discovery focus on one particular transformation. Typically, researchers choose substrates based on their predicted ability to serve as precursors for the target structure, then evaluate reaction conditions... more
Current approaches to reaction discovery focus on one particular transformation. Typically, researchers choose substrates based on their predicted ability to serve as precursors for the target structure, then evaluate reaction conditions for their ability to effect product formation. This approach is ideal for addressing specific reactivity problems, but its focused nature might leave many areas of chemical reactivity unexplored. Here we report a reaction discovery approach that uses DNA-templated organic synthesis and in vitro selection to simultaneously evaluate many combinations of different substrates for bond-forming reactions in a single solution. Watson-Crick base pairing controls the effective molarities of substrates tethered to DNA strands; bond-forming substrate combinations are then revealed using in vitro selection for bond formation, PCR amplification and DNA microarray analysis. Using this approach, we discovered an efficient and mild carbon-carbon bond-forming reaction that generates an enone from an alkyne and alkene using an inorganic palladium catalyst. Although this approach is restricted to conditions and catalysts that are at least partially compatible with DNA, we expect that its versatility and efficiency will enable the discovery of additional reactions between a wide range of substrates.
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On the basis of the distance-dependence of DNA-templated reductive amination reactions and of recent findings of D. Lynn and co-workers, we developed DNA-templated polymerizations of synthetic peptide nucleic acid (PNA) aldehydes. The... more
On the basis of the distance-dependence of DNA-templated reductive amination reactions and of recent findings of D. Lynn and co-workers, we developed DNA-templated polymerizations of synthetic peptide nucleic acid (PNA) aldehydes. The coupling reactions proceed in a highly efficient and sequence-specific manner, even in the presence of mixtures of PNA aldehydes of different sequence. Synthetic peptide nucleic acid polymers containing as many as 40 PNA units (representing 10 consecutive coupling reactions) were formed efficiently. The ease of preparing PNAs containing tailor-made functional groups together with these findings raises the possibility of evolving synthetic sequence-defined polymers by iterated cycles of translation, selection, PCR amplification, and diversification previously available only to biological macromolecules.
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DNA-templated organic synthesis enables the translation, selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in vitro selection... more
DNA-templated organic synthesis enables the translation, selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in vitro selection of a pilot library of 65 macrocycles. In this work, we report several key developments that enable the DNA-templated synthesis of much larger (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;10,000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. Integrating these four developments, we executed the translation of 13,824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNA-templated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles.
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Carrier proteins are 80- to 100-residue way stations that are central to polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. Because the biosynthetic intermediates for catalytic operations are... more
Carrier proteins are 80- to 100-residue way stations that are central to polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. Because the biosynthetic intermediates for catalytic operations are presented on carrier proteins as covalently attached thioesters (via a 4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-phosphopantetheine prosthetic group), the specific protein-protein interactions between carrier proteins and other NRPS/PKS domains are critical for high-fidelity conversion to the final product. Here we show by combinatorial mutagenesis and selection that the aryl carrier protein of EntB (EntB-ArCP) contains localized protein interaction surfaces. Our strategy involved random mutagenesis of N-terminal regions of EntB-ArCP, then selection for clones that produce enterobactin by plating onto iron-deficient media. We identified several residues that were highly conserved from our selection, two of which (G242 and D244) constitute an interaction surface on EntB-ArCP for the phosphopantetheinyl transferases (PPTases) EntD and Sfp. This PPTase interface is distinct from a previously characterized interface on EntB-ArCP for the downstream elongation module, EntF. These results suggest that different protein components recognize different faces of EntB-ArCP in the enterobactin synthetase and that the majority of EntB-ArCP surface residues are not involved in these interactions. Therefore, designing noncognate carrier protein interactions in PKS and NRPS systems should be possible with very few mutations on a particular carrier protein.