ABSTRACT
The combination of X-ray free-electron lasers (XFELs) with serial femtosecond crystallography represents cutting-edge technology in structural biology, allowing the study of enzyme reactions and dynamics in real time through the generation of `molecular movies'. This technology combines short and precise high-energy X-ray exposure to a stream of protein microcrystals. Here, the XFEL structure of carbonic anhydrase II, a ubiquitous enzyme responsible for the interconversion of CO2 and bicarbonate, is reported, and is compared with previously reported NMR and synchrotron X-ray and neutron single-crystal structures.
Subject(s)
Carbonic Anhydrase II , Carbonic Anhydrase II/chemistry , Crystallography, X-Ray , Proteins/chemistry , Synchrotrons , X-Rays , HumansABSTRACT
BACKGROUND: Anaplastic thyroid cancer (ATC) is a rare but aggressive type of thyroid carcinoma. BRAF V600E-mutation, which is found in 10%-50% of ATCs, is associated with poor prognosis. A recent clinical trial reported a substantial clinical benefit of concomitant treatment of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) for treating BRAF V600E-mutant ATC. However, reports on patients with ATC treated with this regimen following surgery are lacking. CASE SUMMARY: We report the case of a 63-year-old female patient diagnosed with BRAF V600E-mutant ATC. Following three surgeries-total thyroidectomy, total laryngectomy, and neck dissection-she was diagnosed with lung metastasis during follow-up. The metastatic ATC was successfully treated with dabrafenib and trametinib. The patient achieved a complete response at the 32-mo follow-up. CONCLUSION: Adjuvant chemotherapy with dabrafenib plus trametinib is efficacious for treatment and prevention of recurrent ATC with BRAF mutation following surgery.
ABSTRACT
Thus far, attempts to develop drugs that target corticotropin-releasing hormone receptor 1 (CRF1R), a drug target in stress-related therapy, have been unsuccessful. Studies have focused on using high-resolution G protein-coupled receptor (GPCR) structures to develop drugs. X-ray free-electron lasers (XFELs), which prevent radiation damage and provide access to high-resolution compositions, have helped accelerate GPCR structural studies. We elucidated the crystal structure of CRF1R complexed with a BMK-I-152 antagonist at 2.75 Å using fixed-target serial femtosecond crystallography. The results revealed that two unique hydrogen bonds are present in the hydrogen bond network, the stalk region forms an alpha helix and the hydrophobic network contains an antagonist binding site. We then developed two antagonists-BMK-C203 and BMK-C205-and determined the CRF1R/BMK-C203 and CRF1R/BMK-C205 complex structures at 2.6 and 2.2 Å, respectively. BMK-C205 exerted significant antidepressant effects in mice and, thus, may be utilized to effectively identify structure-based drugs against CRF1R.
Subject(s)
Corticotropin-Releasing Hormone , Electrons , Mice , Animals , Binding Sites , Drug Discovery , Lasers , Crystallography, X-RayABSTRACT
BACKGROUND: Non-dipping or reverse dipping patterns are known to be associated with adverse cardiovascular prognosis among the general population and clinical cohort. Few large sized studies have explored factors including sleep duration and sleep quality related to nighttime blood pressure (BP) and nocturnal dipping patterns. METHODS: Among 5,360 patients enrolled in Korean multicenter nationwide prospective Registry of ambulatory BP monitoring (KORABP), 981 subjects with complete data on sleep duration, sleep quality assessed using a 4-point Likert scale, and clinical variables were included in the analysis. Phenotypes of nighttime BP pattern were categorized as extreme dipper, dipper, non-dipper, and reverse dipper. Hypertension was defined as a 24-h ambulatory BPs were 130/80 mmHg or higher. RESULTS: Among 981 subjects, 221 were normotensive, 359 were untreated hypertensive, and 401 were treated hypertensive. Age of the participants were 53.87 ± 14.02 years and 47.1% were female. In overall patients, sleep duration was 431.99 ± 107.61 min, and one to four points of sleep quality were observed in 15.5%, 30.0%, 30.4%, and 24.2%, respectively. Of the 760 hypertensive patients, extreme dipper, dipper, non-dipper, and reverse dipper were observed in 58 (7.63%), 277 (36.45%), 325 (42.76%), and 100 (13.16%), respectively. In multiple linear regression analysis, sleep duration (ß = 0.0105, p < 0.001) and sleep quality (ß = -0.8093, p < 0.001) were associated with nighttime systolic BP and sleep quality was associated with extent of nighttime systolic BP dipping (ß = 0.7622, p < 0.001) in hypertensive patients. In addition, sleep quality showed positive association with dipper pattern (odds ratio [OR] = 1.16, 95% confidence interval [CI] = 1.03-1.30) and showed negative association with reverse dipper pattern (OR = 0.73, 95% CI = 0.62-0.86) in multiple logistic regression analyses. CONCLUSION: When adjusted covariates, less sleep duration and poor sleep quality were positively associated with nighttime systolic BP. Additionally, sleep quality was the independent associated factor for dipper and reverse dipper phenotypes. The study also found that male sex, low estimated glomerular filtration rate, high ambulatory BP, low office BP, and poor sleep quality were associated with blunted nighttime SBP dipping.
ABSTRACT
A Gram-stain-negative, non-motile by gliding and moderately halophilic rod-shaped bacterium HN-2-9-2T was isolated from seawater in Tongyeong, Republic of Korea. The strain grew at concentrations of 0.5â7â% (w/v) NaCl, at pH 5.5â8.5 and in a temperature range of 18â45 °C. HN-2-9-2T shared the highest 16S rRNA gene sequence percentage with Salinimicrobium xinjiangense BH206T (98.2â%). The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridisation (dDDH) values between HN-2-9-2T and the S. xinjiangense BH206T were 76.0â%, 81.9â% and 19.7â%, respectively. The genome comprised 3â509â958 bp with a DNA G+C content of 43.0%. HN-2-9-2T contained MK-6 as the sole menaquinone. The predominant fatty acids were iso-C15â:â0, anteiso-C15 : 0, iso-C17â:â0 3-OH, iso-C16â:â0, iso-C15â:â1G and summed feature 9, comprising iso-C17â:â1ω6c/C16â:â1 10-methyl. The polar lipids contained phosphatidylethanolamine, one unidentified phospholipid, two unidentified aminolipids, an unidentified glycolipid and six unidentified lipids. The polyphasic taxonomic properties indicate that the strain represents a novel species within the genus Salinimicrobium, for which the name Salinimicrobium tongyeongense sp. nov. is proposed. The type strain is HN-2-9-2T (=KCTC 82934T=NBRC 115920T).
Subject(s)
Fatty Acids , Seawater , Fatty Acids/chemistry , RNA, Ribosomal, 16S/genetics , Base Composition , Phylogeny , Bacterial Typing Techniques , DNA, Bacterial/genetics , Sequence Analysis, DNA , Seawater/microbiology , Vitamin K 2/chemistryABSTRACT
The taxonomic position of strain EF45031T, isolated from the Neungam Carbonate hot spring, was examined using the polyphasic taxonomic approach. Strain EF45031T shared the highest percentage of 16S rRNA gene sequence with Brachybacterium nesterenkovii CIP 104813 T (97.7%). The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values between strain EF45031T and the type strains B. nesterenkovii CIP 104813 T and B. phenoliresistens Phenol-AT were 77.0%, 69.15%, 21.9% and 75.73%, 68.81%, 20.5%, respectively. Phylogenomic analysis using an up-to-date bacterial core gene (UBCG) set revealed that strain EF45031T belonged to the genus Brachybacterium. Growth occurred between 25 and 50 â at pH 6.0-9.0 and could tolerate salinity up to 5% (w/v). Strain had anteiso-C15:0 and anteiso-C17:0 as major fatty acids. Menaquinone-7 (MK-7) was the predominant respiratory menaquinone. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, three aminolipids, and two unidentified glycolipids. The cell-wall peptidoglycan contained meso-diaminopimelic acid as a diagnostic diamino acid. The genome comprised 2,663,796 bp, with a G + C content of 70.9%. Stress-responsive periplasmic chaperone/protease coding genes were identified in the genome of EF45031T and were not detected in other Brachybacterium species. The polyphasic taxonomic properties indicate that the strain represents a novel species within the genus Brachybacterium, for which the name Brachybacterium sillae sp. nov. is proposed. The type strain is EF45031T (= KCTC 49702 T = NBRC 115869 T).
Subject(s)
Actinomycetales , Hot Springs , Phospholipids/chemistry , RNA, Ribosomal, 16S/genetics , Fatty Acids/chemistry , Phylogeny , Vitamin K 2/chemistry , DNA , DNA, Bacterial/genetics , Sequence Analysis, DNA , Bacterial Typing TechniquesABSTRACT
Microbial fermentation provides a valorization strategy, through biotransformation, to convert plant-derived raw materials into health-promoting agents. In this study, we have investigated the antioxidative activity of Abelmoschus manihot fermented with various Bacillaceae strains from specific environments and demonstrated the anti-inflammatory effects of Bacillus licheniformis CP6 fermented A. manihot extract (FAME) in lipopolysaccharide (LPS)-stimulated Raw264.7 macrophages. Of 1500 bacteria isolated from various specific environments, 47 extracellular protease- and amylase-producing strains with qualified presumption safety status, belonging to the family Bacillaceae, were selected for A. manihot fermentation. Among them, strain CP6, a halophilic bacterium isolated from Tongyeong seawater in Korea and identified as B. licheniformis, showed the highest antioxidant activity. In particular, FAME exerted anti-inflammatory effects on LPS-stimulated Raw264.7 macrophages. Consequently, FAME had a potent inhibitory effect on nitric oxide (NO) production in LPS-stimulated macrophages, without cytotoxicity. Moreover, FAME downregulated LPS-induced pro-inflammatory mediator and enzyme levels in LPS-induced Raw264.7 cells, including IL-1ß, IL-6, TNF-α, iNOS, and COX-2, compared to levels when cells were incubated in A. manihot extract (IAME). Further detailed characterization indicated that FAME suppresses inflammation by blocking NF-κB via IKK phosphorylation inhibition and IκB-α degradation and by downregulating NO production, and inflammatory mediators also decreased NF-κB translocation. Furthermore, FAME inhibited LPS-stimulated activation of MAPKs, including ERK1/2, JNK, and p38, compared to that with either IAME. Therefore, we suggest that FAME could be used for inflammation-related disorders.
Subject(s)
Abelmoschus , Bacillus licheniformis , NF-kappa B/metabolism , Signal Transduction , Bacillus licheniformis/metabolism , Lipopolysaccharides/pharmacology , Fermentation , Anti-Inflammatory Agents/pharmacology , Inflammation , Plant Extracts/pharmacology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/metabolismABSTRACT
Thermophiles that produce extracellular hydrolases are of great importance due to their applications in various industries. Thermophilic enzymes are of interest for industrial applications due to their compatibility with industrial processes, and the availability of the organisms is essential to develop their full potential. In this study, a culture-dependent approach was used to identify thermophilic bacteria from five hot springs in Republic of Korea. Characterization, taxonomic identification, and extracellular hydrolase (amylase, lipase, and protease) activity of 29 thermophilic bacterial isolates from the Neungam carbonate, Mungang sulfur, Deokgu, Baegam, and Dongnae hot springs were investigated. Identification based on the full-length 16S rRNA gene sequence revealed that strains belonged to the phylum Bacillota and were classified as Aeribacillus, Bacillus, Caldibacillus, Geobacillus, and Thermoactinomyces genera. It was found that 22 isolates could produce at least one extracellular enzyme. Geobacillus, representing 41.4% of the isolates, was the most abundant. The highest amount of proteolytic and lipolytic enzymes was secreted by strains of the genus Geobacillus, whereas Caldibacillus species produced the highest amount of amylolytic enzyme. The Geobacillus species producing hydrolytic extracellular enzymes appeared to be the most promising.
ABSTRACT
The Gram-positive, nonmotile, rod-shaped bacterium EF45044T was isolated from a hot spring in Chungju, South Korea. The strain was able to grow at concentrations of 0â5% (w/v) NaCl, at pH 6.0â10.0 and in the temperature range of 18â50 °C. Strain EF45044T showed the highest 16S rRNA gene sequence similarity (98.2%) with Microbacterium ketosireducens DSM 12510T, and the digital DNAâDNA hybridization (dDDH), average amino acid identity (AAI), and average nucleotide identity (ANI) values were all lower than the accepted species threshold. Strain EF45044T contained MKâ12 and MKâ13 as the predominant respiratory quinones and anteisoâC17:0, anteisoâC15:0, and isoâC16:0 as the major fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, and glycolipid were detected as the major polar lipids. The cell-wall peptidoglycan contained ornithine. The DNA G + C content was 71.4 mol%. Based on the polyphasic data, strain EF45044T (= KCTC 49703T) presents a novel species of the genus Microbacterium, for which the name Microbacterium neungamense sp. nov. is proposed.
Subject(s)
Fatty Acids , Microbacterium , RNA, Ribosomal, 16S/genetics , Microbacterium/genetics , Phylogeny , Bacterial Typing Techniques , DNA, Bacterial/genetics , Sequence Analysis, DNA , Fatty Acids/analysis , Nucleic Acid Hybridization , Phospholipids/chemistryABSTRACT
Staphylococcus aureus is a notorious and globally distributed pathogenic bacterium. New strategies to develop novel antibiotics based on intrinsic bacterial toxin-antitoxin (TA) systems have been recently reported. Because TA systems are present only in bacteria and not in humans, these distinctive systems are attractive targets for developing antibiotics with new modes of action. S. aureus PemIK is a type II TA system, comprising the toxin protein PemK and the labile antitoxin protein PemI. Here, we determined the crystal structures of both PemK and the PemIK complex, in which PemK is neutralized by PemI. Our biochemical approaches, including fluorescence quenching and polarization assays, identified Glu20, Arg25, Thr48, Thr49, and Arg84 of PemK as being important for RNase function. Our study indicates that the active site and RNA-binding residues of PemK are covered by PemI, leading to unique conformational changes in PemK accompanied by repositioning of the loop between ß1 and ß2. These changes can interfere with RNA binding by PemK. Overall, PemK adopts particular open and closed forms for precise neutralization by PemI. This structural and functional information on PemIK will contribute to the discovery and development of novel antibiotics in the form of peptides or small molecules inhibiting direct binding between PemI and PemK.
Subject(s)
Antitoxins , Staphylococcus aureus , Anti-Bacterial Agents/metabolism , Antitoxins/genetics , Antitoxins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , RNA/metabolism , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolismABSTRACT
PURPOSE: To evaluate the effectiveness of preoperative lacrimal endoscopic evaluation (LEE) of lacrimal duct system (LDS). DESIGN: Retrospective comparative case series METHODS: From March 2016 to February 2020, the charts of patients chosen to undergo endoscopic dacryocystorhinostomy (EDCR) or silicone tube intubation (STI) were reviewed retrospectively. Group 1 included patients that underwent EDCR, and group 2 included patients that underwent STI. Preoperative LEE was performed for all patients. In group 1, we compared the functional success rate for patients who had been converted to STI with the patients who had undergone EDCR. In group 2, we compared the functional success rate of STI with those who had had STI without LEE. RESULTS: In group 1, 19 (54.3%) eyes were converted to STI following LEE, and the functional success rate was 84.2%, which is not significantly different from that of the EDCR group following LEE (p = 0.608). The functional success rate of EDCR without LEE was not different from that of STI following LEE (p = 1.000). In group 2, five eyes (26.3%) were converted to EDCR following LEE. The group undergoing STI following LEE showed a significantly higher functional success rate (95.7%) than the group without LEE (66.6%, p = 0.023). CONCLUSION: Preoperative LEE enables direct visualization of the LDS and helps to obtain more accurate diagnosis. This allows for the best surgical option based on LEE findings, which can contribute to better results. Therefore, LEE would be expected to change the paradigm of the classical management of LDS.
Subject(s)
Dacryocystorhinostomy , Lacrimal Apparatus , Lacrimal Duct Obstruction , Nasolacrimal Duct , Endoscopy , Eyelids , Humans , Lacrimal Apparatus/diagnostic imaging , Lacrimal Apparatus/surgery , Lacrimal Duct Obstruction/diagnosis , Lacrimal Duct Obstruction/therapy , Nasolacrimal Duct/diagnostic imaging , Nasolacrimal Duct/surgery , Retrospective Studies , Treatment OutcomeABSTRACT
Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at the center that are involved in electron transfer, but shows very different efficiency along the two branches. We have determined the structure of cyanobacterial PS I at room temperature (RT) using femtosecond X-ray pulses from an X-ray free electron laser (XFEL) that shows a clear expansion of the entire protein complex in the direction of the membrane plane, when compared to previous cryogenic structures. This trend was observed by complementary datasets taken at multiple XFEL beamlines. In the RT structure of PS I, we also observe conformational differences between the two branches in the reaction center around the secondary electron acceptors A1A and A1B. The π-stacked Phe residues are rotated with a more parallel orientation in the A-branch and an almost perpendicular confirmation in the B-branch, and the symmetry breaking PsaB-Trp673 is tilted and further away from A1A. These changes increase the asymmetry between the branches and may provide insights into the preferential directionality of electron transfer.
Subject(s)
Photosystem I Protein Complex/chemistry , Vitamin K 1/chemistry , Crystallography, X-Ray , Photosynthesis , Protein Structure, Tertiary , Temperature , ThermosynechococcusABSTRACT
BACKGROUND: This study aimed to determine the morphological changes in Asian lower eyelid epiblepharon patients after surgery. METHODS: The medical records of 59 patients who underwent lower eyelid epiblepharon repair were reviewed retrospectively. Eighty-nine patients who underwent strabismus surgery were set as the control group. The photographs for each group were analyzed based on the following factors: inferior half area (IHA) of the eye, eyelash angular direction (EAD), angle between the eyelashes and the cornea, marginal reflex distance 1 (MRD1) and marginal reflex distance 2 (MRD2). RESULTS: After surgery, the medial EAD changed from 92.45° ± 20.21° (mean ± SD) to 79.43° ± 23.31°, while the central and lateral EADs were unchanged. IHA increased from 36.33 ± 9.78 mm3 to 43.06 ± 10.57 mm3, and MRD1 increased from 1.92 ± 0.99 mm to 2.50 ± 0.93 mm, whereas MRD2 did not change. The mean angle between the eyelashes and the cornea increased from 39.64° to 72.19° immediately postoperatively, but had reduced to 58.75° 3 months later, followed by no further significant change at the 6-month and 9-month postoperative follow-ups. CONCLUSIONS: There is morphological changes of the eyelid after lower eyelid epiblepharon surgery, with increases in the IHA and MRD1. In addition, contact between the eyelashes and the cornea occurred mainly in the medial portion of the eyelid the position, which everted and stabilized over 3 months. Thus, follow-up observations are required for at least 3 months to properly evaluate the surgical outcome.
Subject(s)
Eyelashes , Eyelid Diseases , Asian People , Child , Cornea , Eyelid Diseases/surgery , Humans , Oculomotor Muscles , Retrospective StudiesABSTRACT
Excavating the molecular details of many diverse enzymes from metagenomes remains challenging in agriculture, food, health, and environmental fields. We present a versatile method that accelerates metabolic enzyme discovery for highly selective gene capture in metagenomes using next-generation sequencing. Culture-independent enzyme mining of environmental DNA is based on a set of short identifying degenerate sequences specific for a wide range of enzyme superfamilies, followed by multiplexed DNA barcode sequencing. A strategy of 'focused identification of next-generation sequencing-based definitive enzyme research' enabled us to generate targeted enzyme datasets from metagenomes, resulting in minimal hands-on obtention of high-throughput biological diversity and potential function profiles, without being time-consuming. This method also provided a targeted inventory of predicted proteins and molecular features of metabolic activities from several metagenomic samples. We suggest that the efficiency and sensitivity of this method will accelerate the decryption of microbial diversity and the signature of proteins and their metabolism from environmental samples.
Subject(s)
DNA Barcoding, Taxonomic , Enzymes/genetics , High-Throughput Nucleotide Sequencing , MetagenomicsABSTRACT
The metallo-ß-lactamase fold is the most abundant metal-binding domain found in two major kingdoms: bacteria and archaea. Despite the rapid growth in genomic information, most of these enzymes, which may play critical roles in cellular metabolism, remain uncharacterized in terms of structure and function. In this study, X-ray crystal structures of SAV1707, a hypothetical metalloenzyme from Staphylococcus aureus, and its complex with cAMP are reported at high resolutions of 2.05 and 1.55â Å, respectively, with a detailed atomic description. Through a functional study, it was verified that SAV1707 has Ni2+-dependent phosphodiesterase activity and Mn2+-dependent endonuclease activity, revealing a different metal selectivity depending on the reaction. In addition, the crystal structure of cAMP-bound SAV1707 shows a unique snapshot of cAMP that reveals the binding mode of the intermediate, and a key residue Phe511 that forms π-π interactions with cAMP was verified as contributing to substrate recognition by functional studies of its mutant. Overall, these findings characterized the relationship between the structure and function of SAV1707 and may provide further understanding of metalloenzymes possessing the metallo-ß-lactamase fold.
Subject(s)
Endonucleases/chemistry , Phosphoric Diester Hydrolases/chemistry , Staphylococcus aureus/enzymology , beta-Lactamases/chemistry , Manganese/metabolism , Nickel/metabolism , Protein ConformationABSTRACT
Anti-CRISPR (Acr) proteins are phage-borne inhibitors of the CRISPR-Cas immune system in archaea and bacteria. AcrIIC2 from prophages of Neisseria meningitidis disables the nuclease activity of type II-C Cas9, such that dimeric AcrIIC2 associates with the bridge helix (BH) region of Cas9 to compete with guide RNA loading. AcrIIC2 in solution readily assembles into oligomers of variable lengths, but the oligomeric states are not clearly understood. In this study, we investigated the dynamic assembly of AcrIIC2 oligomers, and identified key interactions underlying the self-association. We report that AcrIIC2 dimers associate into heterogeneous high-order oligomers with the equilibrium dissociation constant KD â¼8 µM. Oligomerization is driven by electrostatic interactions between charged residues, and rational mutagenesis produces a stable AcrIIC2 dimer with intact Cas9 binding. Remarkably, the BH peptide of Cas9 is unstructured in solution, and undergoes a coil-to-helix transition upon AcrIIC2 binding, revealing a unique folding-upon-binding mechanism for Acr recognition.
Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems , Viral Proteins/metabolism , Bacteriophages/metabolism , Gene Editing , Gene Expression Regulation, Bacterial , Mutagenesis , Neisseria/virology , Neisseria meningitidis/genetics , Neisseria meningitidis/metabolism , RNA, Guide, Kinetoplastida/geneticsABSTRACT
The purpose of this study was to determine and compare the effects between injecting botulinum toxin A (BTX-A) transconjunctivally into the palpebral lobe and transcutaneously into the orbital lobe of the lacrimal gland in patients with epiphora due to lacrimal outflow obstruction. This randomized controlled study included 53 eyes of 31 patients with unilateral or bilateral epiphora. Patients were randomly allocated to receive an injection of BTX-A (3 units) either transconjunctivally (n = 15, 25 eyes) or transcutaneously (n = 16, 28 eyes). For objective assessments, the tear meniscus height and Schirmer's I test with topical anesthesia were measured at baseline and after 2, 6, 12, and 24 weeks of follow-up. Subjective evaluations were performed using the Munk score. After BTX-A injection, patients in both groups experienced significant objective and subjective reductions in tearing at all follow-up times compared to pre-injection (success rate 86.8%), and the effect lasted for a mean duration of 5.63 months. The two delivery routes showed similar clinical effectiveness for a single injected dose of BTX-A. In conclusion, injecting BTX-A via either a transconjunctival or transcutaneous route helps to reduce normal tear production and results in significant improvements in the symptoms in patients with epiphora.
Subject(s)
Acetylcholine Release Inhibitors/administration & dosage , Botulinum Toxins, Type A/administration & dosage , Lacrimal Apparatus/drug effects , Lacrimal Duct Obstruction/drug therapy , Tears/metabolism , Acetylcholine Release Inhibitors/adverse effects , Aged , Aged, 80 and over , Botulinum Toxins, Type A/adverse effects , Female , Humans , Injections, Intradermal , Injections, Intraocular , Lacrimal Apparatus/metabolism , Lacrimal Apparatus/physiopathology , Lacrimal Duct Obstruction/diagnosis , Lacrimal Duct Obstruction/metabolism , Lacrimal Duct Obstruction/physiopathology , Male , Prospective Studies , Recovery of Function , Seoul , Single-Blind Method , Time Factors , Treatment OutcomeABSTRACT
NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an essential enzyme in human cholesterol synthesis and a regulator of epidermal growth factor receptor (EGFR) trafficking pathways, has attracted interest as a therapeutic target due to its crucial relevance to cholesterol-related diseases and carcinomas. However, the development of pharmacological agents for targeting NSDHL has been hindered by the absence of the atomic details of NSDHL. In this study, we reported two X-ray crystal structures of human NSDHL, which revealed a detailed description of the coenzyme-binding site and the unique conformational change upon the binding of a coenzyme. A structure-based virtual screening and biochemical evaluation were performed and identified a novel inhibitor for NSDHL harboring suppressive activity towards EGFR. In EGFR-driven human cancer cells, treatment with the potent NSDHL inhibitor enhanced the antitumor effect of an EGFR kinase inhibitor. Overall, these findings could serve as good platforms for the development of therapeutic agents against NSDHL-related diseases.
Subject(s)
3-Hydroxysteroid Dehydrogenases/metabolism , Enzyme Inhibitors/metabolism , 3-Hydroxysteroid Dehydrogenases/antagonists & inhibitors , 3-Hydroxysteroid Dehydrogenases/chemistry , 3-Hydroxysteroid Dehydrogenases/genetics , Binding Sites , Cell Line, Tumor , Cell Survival/drug effects , Cholesterol/chemistry , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Erlotinib Hydrochloride/chemistry , Erlotinib Hydrochloride/metabolism , Erlotinib Hydrochloride/pharmacology , Humans , Kinetics , Molecular Docking Simulation , Mutagenesis, Site-Directed , NAD/chemistry , NAD/metabolism , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Signal TransductionABSTRACT
PURPOSE: To compare the variations in ocular torsion measurements made using different fundus photographic methods. METHODS: We enrolled subjects with three conditions: (1) patients with intermittent exotropia (IXT) (n = 44), (2) patients with unilateral superior oblique palsy (SOP) (n = 10), and (3) normal subjects as controls (n = 85). Ocular torsion was measured by disc-center-fovea angle (DFA) using three different imaging modalities: (1) conventional fundus photography (CFP) with a 45° field of view (FV), (2) wide-field fundus photography (WFP) with a 200° FV, and (3) optical coherence tomography (OCT) with a 55° FV. RESULTS: In the IXT group, the DFAs in the right and left eyes were 5.70±3.35° and 6.37±3.36°, respectively, for CFP, 8.39±5.24° and 8.61±3.67° for WFP, and 5.73±3.61° for 6.16±3.50° for OCT. In the SOP group, the DFAs in paretic and nonparetic eyes were 12.19±1.69° and 6.71±1.09°, respectively, for CFP, 14.29±2.36° and 8.23±3.31° for WFP, and 12.12±1.73° and 6.91±1.12° for OCT. In the control group, the DFAs in the right and left eyes were 5.39±2.65° and 5.71±3.16°, respectively, for CFP, 8.77±5.56° and 8.90±6.24° for WFP, and 5.27±2.67° and 5.72±3.20° for OCT. There was no difference between the results from CFP and OCT among the three groups. However, the torsional angle was larger when measured using WFP than the other two photographic methods (CFP and OCT) in all three groups (all p<0.05). CONCLUSION: The ocular torsion measurement varies with the fundus photographic method used to measure it. Clinicians should be careful to avoid overestimating ocular extorsion when it is evaluated using WFP.
Subject(s)
Eye Diseases/diagnosis , Eye Movements , Fundus Oculi , Tomography, Optical Coherence/methods , Torsion Abnormality/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Child , Child, Preschool , Diagnostic Techniques, Ophthalmological , Eye Diseases/surgery , Female , Follow-Up Studies , Humans , Male , Middle Aged , Ophthalmologic Surgical Procedures , Prognosis , Retrospective Studies , Young AdultABSTRACT
Accurate knowledge of extraocular muscle (EOM) tension is important for the diagnosis of and surgical planning for strabismus, such as choosing which eye to operate or determining the amount of muscle displacement. Previous evaluations of passive EOM tension have relied extensively on the experience and skill of ophthalmic surgeons, who generally perform such evaluations by gripping the eyeball and then pushing and pulling it. This methodology, named the forced duction test, has the significant limitation that the tension is felt subjectively via the forceps, with the results therefore not being quantifiable. Previous quantitative analyses have utilized several different types of equipment with implanted force transducers or have involved connecting the muscle tendon to a strain gauge. However, the associated equipment setups and recording systems are highly complex and rarely used outside research settings. This situation prompted the present study to develop a novel compact, quantifiable and clinically applicable device for measuring the passive tension in human EOMs for use in clinical practice. The device employs locking forceps and a tilting sensor to rule out effects of the gripping force and to compensate for changes in the force due to tilting, which improves the measurement accuracy. The performance of the device was investigated in 60 eyes of 30 consecutive anaesthetized patients immediately prior to ophthalmic surgery. The results showed that the measured EOM tension in each rectus muscles agreed with previous findings: 48.3 ± 14.5 g (0.82 ± 0.28 g/deg, mean ± SD) for the lateral rectus, 45.6 ± 13.2 g (0.82 ± 0.23 g/deg) for the medial rectus, 48.6 ± 14.7 g (0.71 ± 0.21 g/deg) for the inferior rectus and 53.4 ± 13.7 g (0.77 ± 0.25 g/deg) for the superior rectus.