Experimental evidence for the thermophilicity of ancestral life.

Theoretical studies have focused on the environmental temperature of the universal common ancestor of life with conflicting conclusions. Here we provide experimental support for the existence of a thermophilic universal common ancestor. We present the thermal stabilities and catalytic efficiencies of nucleoside diphosphate kinases (NDK), designed using the information contained in predictive phylogenetic trees, that seem to represent the last common ancestors of Archaea and of Bacteria. These enzymes display extreme thermal stabilities, suggesting thermophilic ancestries for Archaea and Bacteria. The results are robust to the uncertainties associated with the sequence predictions and to the tree topologies used to infer the ancestral sequences. Moreover, mutagenesis experiments suggest that the universal ancestor also possessed a very thermostable NDK. Because, as we show, the stability of an NDK is directly related to the environmental temperature of its host organism, our results indicate that the last common ancestor of extant life was a thermophile that flourished at a very high temperature.

Crystal structure and site-directed mutagenesis analyses of haloalkane dehalogenase LinB from Sphingobium sp. strain MI1205.

The enzymes LinB(UT) and LinB(MI) (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of ß-hexachlorocyclohexane (ß-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB(MI) and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB(MI) were categorized into three groups based on the efficiency of the first-step (from ß-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB(MI) and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinB(MI). The dynamics simulation analyses of wild-type LinB(MI) and LinB(UT) revealed that the entrance of the substrate access tunnel of LinB(UT) was more flexible than that of LinB(MI), which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.

The flavoenzyme azobenzene reductase AzoR from Escherichia coli binds roseoflavin mononucleotide (RoFMN) with high affinity and is less active in its RoFMN form.

The Gram-positive bacterium Streptomyces davawensis is the only organism known to produce the antibiotic roseoflavin. Roseoflavin is a structural riboflavin analogue and is converted to the flavin mononucleotide (FMN) analogue roseoflavin mononucleotide (RoFMN) by flavokinase. FMN-dependent homodimeric azobenzene reductase (AzoR) (EC 1.7.1.6) from Escherichia coli was analyzed as a model enzyme. In vivo and in vitro experiments revealed that RoFMN binds to the AzoR apoenzyme with an even higher affinity compared to that of the "natural" cofactor FMN. Structural analysis (at a resolution of 1.07 Å) revealed that RoFMN binding did not affect the overall topology of the enzyme and also did not interfere with dimerization of AzoR. The AzoR-RoFMN holoenzyme complex was found to be less active (30% of AzoR-FMN activity) in a standard assay. We provide evidence that the different physicochemical properties of RoFMN are responsible for its reduced cofactor activity.

Crystallization and preliminary X-ray analysis of the haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58.

Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 Å resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 Å. The crystal contained two molecules in the asymmetric unit.

Expression, purification, crystallization and preliminary X-ray analysis of 4-hydroxy-3-methyl-2-keto-pentanoate aldolase (asHPAL) from Arthrobacter simplex strain AKU 626.

4-Hydroxy-3-methyl-2-keto-pentanoate aldolase (asHPAL), an enzyme used in the synthesis of (2S,3R,4S)-4-hydroxyisoleucine, was crystallized in the absence and the presence of 2-ketobutyrate as one of its substrates by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. Crystals of asHPAL grown without and with 2-ketobutyrate diffracted to 1.60 and 1.55 Šresolution and belonged to space group C2, with unit-cell parameters a = 116.8, b = 88.2, c = 85.3 Å, ß = 122.3° and a = 116.2, b = 88.1, c = 85.0 Å, ß = 122.3°, respectively.

Glaucoma Diagnostic Performance of Retinal Blood Flow Measurement With Doppler Optical Coherence Tomography.

Purpose: The purpose of this study was to evaluate the diagnostic performance of retinal blood flow (RBF) measured with the Doppler optical coherence tomography (OCT) segmental scanning method to distinguish between healthy and glaucoma eyes. Methods: Fifty-eight patients with normal tension glaucoma (NTG) who had a single-hemifield visual field defect and 44 age-matched healthy subjects were enrolled. Retinal nerve fiber layer thickness (RNFLT) was measured with swept-source OCT. Superior and inferior temporal arteries (TAs) and temporal veins (TVs) RBF were measured with Doppler OCT. The area under the curve (AUC) of the receiver operating characteristic (ROC) was used to compare the diagnostic performances in the damaged and normal hemispheres. Results: Multivariate regression analysis showed TA RBF and TV RBF were significantly reduced in the damaged and normal hemispheres. The ROC analysis showed that the AUC for quadrant RNFLT, TA RBF, and TV RBF were 0.973, 0.909, and 0.872 in the damaged hemisphere, respectively. The AUC values in the normal hemisphere were 0.783, 0.744, and 0.697, respectively. The combination of quadrant RNFLT and TA/TV RBF had a greater AUC than quadrant RNFLT alone in both damaged (AUC = 0.987) and normal (AUC = 0.825) hemispheres. Conclusions: In NTG eyes with single-hemifield damage, the RBF was found to be significantly reduced in the damaged and normal hemispheres independent from structural changes. The combination of RNFLT and RBF could improve diagnostic performances for glaucoma. Translational Relevance: Combining morphological and blood flow measurements with Doppler OCT may be useful in glaucoma diagnosis.

Structure of flap endonuclease 1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus.

Flap endonuclease 1 (FEN1) is a key enzyme in DNA repair and DNA replication. It is a structure-specific nuclease that removes 5'-overhanging flaps and the RNA/DNA primer during maturation of the Okazaki fragment. Homologues of FEN1 exist in a wide range of bacteria, archaea and eukaryotes. In order to further understand the structural basis of the DNA recognition, binding and cleavage mechanism of FEN1, the structure of FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus (DaFEN1) was determined at 2.00 Šresolution. The overall fold of DaFEN1 was similar to those of other archaeal FEN1 proteins; however, the helical clamp and the flexible loop exhibited a putative substrate-binding pocket with a unique conformation.

Macular Microvasculature and Associated Retinal Layer Thickness in Pediatric Amblyopia: Magnification-Corrected Analyses.

Purpose: The purpose of this study was to characterize macular microvasculature and structural retinal layers using magnification-corrected optical coherence tomography angiography (OCTA) images in children with amblyopia. Methods: This prospective cross-sectional study included 22 children with unilateral amblyopia (4-11 years of age) receiving spectral-domain OCTA. Vessel densities in foveal and parafoveal regions of the superficial capillary plexus (SCP) and deep capillary plexus (DCP) were measured in amblyopic and fellow eyes using a customized image analysis program correcting the scale of retinal image with axial length. Iowa Reference Algorithms (Iowa Institute for Biomedical Imaging) were used to measure mean thickness values of 10 intra-retinal layers rescaled for image size correction. Results: Foveal and parafoveal vessel densities in amblyopic eyes were lower than that of the fellow eyes in the SCP (fovea: P = 0.006 and parafovea: P = 0.003) and the DCP (P = 0.024 and P = 0.025, respectively). Amblyopic eyes had significantly smaller foveal avascular zone (FAZ) area than fellow eyes (P < 0.001). There were significant differences in retinal layer thickness between paired eyes, particularly in the inner retina in both foveal and parafoveal regions; retinal nerve fiber layer (RNFL) (P = 0.024 and P = 0.095, respectively), ganglion cell layer (P < 0.001 and P = 0.008), inner plexiform layer (IPL; P = 0.12 and P = 0.037), inner nuclear layer (P = 0.005 and P = 0.005), and outer plexiform layer (OPL; P = 0.02 and P = 0.057), except in the foveal IPL, the parafoveal RNFL, and OPL. Conclusions: Unilateral amblyopic eyes demonstrate reduced macular vessel density and thicker inner retinal layers compared with fellow eyes even after correcting for image magnification. Changes in macular microvasculature and structural layers may offer valuable insights in the development of amblyopia.

Retinal Blood Velocity Waveform Characteristics With Aging and Arterial Stiffening in Hypertensive and Normotensive Subjects.

Purpose: We aimed to explore the velocity waveform characteristics of the retinal artery associated with age and the cardio-ankle vascular index (CAVI) as a conventional arterial stiffness marker by applying the Doppler optical coherence tomography (DOCT) flowmeter. Methods: In this cross-sectional study, DOCT flowmeter imaging was performed in 66 participants aged 21 to 83 years (17 men, 49 women) with no history of eye diseases and no systemic diseases, except for hypertension. Retinal blood velocity waveform was analyzed where several parameters in time (upstroke time, T1, T2, T3, and T4) and area under the waveform (area elevation, area declination, A1, A2, A3, and A4) were extracted. Systolic blood pressure-adjusted Pearson's coefficients were calculated to determine the correlations of each parameter with age or CAVI. Results: Corrected upstroke time (UTc) was the waveform parameter most positively correlated with age (r = 0.497, P < 0.001). Area declination was the waveform parameter most negatively correlated with age (r = -0.682, P < 0.001) and CAVI (r = -0.601, P < 0.001). Conclusions: We extracted the waveform parameters associated with the risks of arterial stiffening. The velocity waveform analysis of the retinal artery with DOCT flowmeter potentially could become a new method for arterial stiffness identification. Translational Relevance: DOCT flowmeter could evaluate arterial stiffening in a different way from the conventional method of measuring arterial stiffening using pressure waveform. Because the DOCT flowmeter can easily, quickly, and noninvasively provide a retinal blood velocity waveform, this system could be useful as a routine medical examination for arterial stiffening.

Effect of Background Brightness on Preferred Retinal Loci in Patients With Macular Disease.

Purpose: To evaluate the effect of background brightness on the preferred retinal locus (PRL) in patients with macular disease. Methods: The study included 27 eyes (27 patients) with macular disease. Microperimetry (MP) was performed to evaluate the PRL and retinal sensitivity (RS) at 10 cd/m2. A prototypical device was used to evaluate the PRL at 650 cd/m2. Patients were divided into two groups: central fixation (CF) and eccentric fixation (EF). Results: The PRLs under different brightness levels differed significantly (P < 0.001) in 15 of 27 eyes (two of 13 eyes in the CF group and 13 of 14 eyes in the EF group). The best-corrected visual acuities (BCVAs) in eyes with different PRLs were significantly worse (P = 0.019) than in eyes with one PRL, although the foveal RS did not differ significantly. In patients with BCVAs over 0.1, the PRLs differed in four of 13 eyes in the CF group and in three of four eyes in the EF group (P > 0.05); in patients with BCVAs of 0.1 or lower, the PRLs differed in one of four eyes and 10 of 10 eyes, respectively (P = 0.011). Conclusions: In patients with macular disease, PRLs can change depending on the surrounding brightness. It may be beneficial to evaluate PRLs under brighter background conditions (e.g., in ambient light) when performing visual rehabilitation for these patients. Translational Relevance: This study provides important information for visual rehabilitation of patients with macular disease.

Effect of illumination on reading performance in Japanese patients with age-related macular degeneration.

PURPOSE: Several studies of Western patients with age-related macular degeneration (AMD) have investigated reading performance that improved at high levels of illumination; however, the relation between reading performance and macular function has not been evaluated in detail in Japan. The goals of this study were to evaluate the effect of different levels of illumination on reading performance in Japanese patients with AMD and determine the factors, such as macular function, that affect these results. STUDY DESIGN: Cross-sectional study. METHODS: We prospectively included 39 patients with bilateral AMD or maculopathy. We evaluated reading performance; reading acuity (RA), critical print size (CPS), and maximal reading speed (MRS) using charts with Japanese sentences based on the MNREAD-J in 500-7500 lx. Patients were classified into two groups based on the presence of a central scotoma (CS) or no CS (NCS) diagnosed by microperimetry. RESULTS: The RA improved significantly in 500-7500 lx in both groups (NCS, p = 0.001; CS, p = 0.046). The RA improvement differed significantly (2000 lx, p = 0.021; 5,000 lx; p = 0.021; 7500 lx, p = 0.047) between 500 lx and other illumination levels only in the NCS group and then plateaued over 2000 lx. The CPS and MRS did not improve significantly at any illumination level. CONCLUSIONS: These results suggest that the difference in macular function was related to improvement in the RA with increased illumination in Japanese patients with AMD.

Association Between Alterations of the Choriocapillaris Microcirculation and Visual Function and Cone Photoreceptors in Patients With Diabetes.

Purpose: The purpose of this study was to investigate the association between the choriocapillaris microcirculation and the visual function and cone photoreceptor structure in patients with diabetes. Methods: Thirteen control subjects and 26 patients with type 2 diabetes were recruited. The patients with diabetes were divided into three groups based on the grade of diabetic retinopathy (DR). The retinal sensitivity (RS) was evaluated using microperimetry. Cone photoreceptor mosaics were imaged using an adaptive optics retinal camera, and the cone heterogeneity packing index (HPi) was calculated. Optical coherence tomography angiography (OCTA) images of the choriocapillaris were obtained using spectral-domain OCTA, and the area of flow deficit (FD) was evaluated. All parameters were evaluated in the foveal and parafoveal areas. Results: The study included four patients with diabetes without retinopathy, 12 patients with nonproliferative diabetic retinopathy (NPDR), and 10 patients with proliferative diabetic retinopathy (PDR). The foveal and parafoveal FDs were correlated significantly (fovea, r = -0.58; P = 0.046 and r = -0.82; P = 0.003; parafovea, r = -0.59; P = 0.044 and r = -0.72; and P = 0.019, respectively) with the RS in patients with NPDR and PDR, but not in control and no diabetic retinopathy (NDR) groups. There were no differences in the foveal HPi among the groups. Conclusions: Impaired choriocapillaris microcirculation is associated with impaired visual function but not cone photoreceptor integrity in eyes with DR.

Circumpapillary collateral vessel development in iatrogenic central retinal artery occlusion observed using OCT angiography.

PURPOSE: It has been reported that peripapillary loops develop after central retinal artery occlusion (CRAO). Although cilioretinal anastomoses have been shown by fluorescein angiography (FA) and indocyanine green angiography, these examinations cannot confirm the structural continuity between the retinal arteries and the posterior ciliary arteries. In the current report, we followed a patient with iatrogenic CRAO in which circumpapillary collaterals formed and assessed the connection between these two vascular systems using optical coherence tomography angiography (OCTA). OBSERVATION: Iatrogenic CRAO developed in the left eye of a 30-year-old woman during preoperative embolization of a convexity meningioma. FA and OCTA showed complete impairment of the retinal circulation. Two weeks after this event, OCTA images showed flow in the retinal vessels and the beginning of collateral vessel development on the optic disc margin. Six months later, OCTA images showed that these circumpapillary collaterals connected branches of the retinal arteries with the vessels supplying the optic disc, which originate from the posterior ciliary arteries. CONCLUSION AND IMPORTANCE: OCTA aided our understanding of the three-dimensional configuration of the circumpapillary collaterals that developed after iatrogenic CRAO, which included anastomosis of the retinal and posterior ciliary artery systems. OCTA is useful for noninvasively monitoring the status of retinal circulatory dynamics after iatrogenic CRAO.

A Proposed Classification of Intraretinal Microvascular Abnormalities in Diabetic Retinopathy Following Panretinal Photocoagulation.

Purpose: To investigate the characteristics of intraretinal microvascular abnormalities (IRMAs) before and after panretinal photocoagulation (PRP) for diabetic retinopathy (DR) by using optical coherence tomography angiography (OCTA). Methods: Forty-six eyes of 29 patients with DR were included (26 eyes with severe nonproliferative diabetic retinopathy [SNPDR] and 20 eyes with proliferative diabetic retinopathy [PDR]). En face OCTA images of IRMAs in a 6 × 6-mm area were acquired by using Cirrus 5000 with AngioPlex. The morphological changes in IRMAs were evaluated before and after PRP. The changes in the IRMAs were divided into five subtypes: unchanged; tuft regression; reperfusion; mixed (combined tuft regression/reperfusion); and worsening (new appearance of tuft). Results: Unchanged IRMAs were identified in 15 SNPDR eyes and 2 PDR eyes; all neovascularization (NV) had regressed after PRP. Tufts were more frequently observed in the PDR eyes (15/20, 75%) than in the SNPDR eyes (8/26, 31%) (P = 0.003), and two tufts tended to exceed the inner limiting membrane, which showed progression to NV before PRP. The reperfusion phenomenon was observed in 7/26 SNPDR eyes and 4/20 PDR eyes, including the mixed type, and showed two vascular patterns: abnormal (dilated, tortuous, and twisted) and normal vessels. The worsening type was observed in 1/26 SNPDR eye and 2/20 PDR eyes. Conclusions: OCTA enabled classification of IRMA into more detailed types. The unchanged and reperfusion types suggested that IRMAs had aspects of remodeling. However, IRMAs with tufts were observed in 75% of the PDR eyes, and the tufts had aspects of NV.

Crystallization and preliminary X-ray analysis of flap endonuclease 1 (FEN1) from Desulfurococcus amylolyticus.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes 5'-overhanging flaps in DNA repair and removes the RNA/DNA primer during maturation of the Okazaki fragment in lagging-strand DNA replication. FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with monoammonium dihydrogen phosphate as the precipitant at pH 8.3. X-ray diffraction data were collected to 2.00 A resolution. The space group of the crystal was determined as the primitive hexagonal space group P321, with unit-cell parameters a = b = 103.76, c = 84.58 A. The crystal contained one molecule in the asymmetric unit.

A Case of Progressive Diabetic Retinopathy Related to Pregnancy Followed on Optical Coherence Tomography Angiography.

A 27-year-old woman with type 2 diabetes mellitus had mild diabetic retinopathy (DR) during the early gestation period. Optical coherence tomography angiography (OCTA) showed microaneurysms and small capillary nonperfusion with little change until before delivery. The patient later developed pregnancy-induced hypertension, which continued after delivery, and the DR worsened markedly. OCTA showed onset and recovery of paracentral acute middle maculopathy. Macular edema (ME) also developed, and OCTA showed irregular dilation in the radial peripapillary capillaries. After starting antihypertensive therapy, the capillary dilation and ME decreased. OCTA enables close follow-up of DR related to pregnancy during the perinatal period. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:393-397.].

Effect of ripasudil on diabetic macular edema.

The current study aimed to address whether ripasudil, a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor developed to treat glaucoma and ocular hypertension (OH), improves diabetic macular edema (DME) since it is known that ROCK upregulates vascular endothelial growth factor. We retrospectively investigated the foveal thickness (FT) measured by spectral-domain optical coherence tomography, visual acuity (VA), and intraocular pressure (IOP) in 12 eyes with DME that received ripasudil treatment for primary open-angle glaucoma or OH and compared them with 14 eyes that received no treatment. One month after ripasudil therapy, the mean FT decreased significantly from 439 ± 72 µm to 395 ± 62 µm (P = 0.003); this change was significantly different from that in the controls, in which the mean FT increased by 1 ± 39 µm (P = 0.01). Ripasudil also caused a significant decrease in IOP from 17.3 ± 5.2 mmHg to 14.6 ± 4.0 mmHg (P = 0.02); this change was significantly greater than that in the controls, in which IOP changed by 0.0 ± 1.6 mmHg (P < 0.008). There was no significant difference in the VA changes between groups. Our results suggested that ripasudil may have positive effects on both IOP and DME.

EN-FACE OPTICAL COHERENCE TOMOGRAPHY IMAGING IN A CASE OF CHOROIDAL RUPTURE.

PURPOSE: To report a case of choroidal rupture resulting due to blunt trauma using en-face optical coherence tomography (OCT) imaging. METHODS: Case report. RESULTS: A 22-year-old man sustained a blunt trauma to his right eye. At the initial examination, four lesions with a subretinal hemorrhage were observed during a fundus examination of the macular area including the fovea, but the findings under the lesions were unclear. The authors observed the extent of the minute findings noninvasively by en-face OCT imaging from the initial examination. Six months after the trauma, the fine crescent-shaped lines seen by angiography and OCT B-scan images were consistent with findings observed on the en-face OCT images. CONCLUSION: The en-face OCT imaging would be useful to examine the pathological changes in a choroidal rupture noninvasively during frequent follow-up examinations in a case of choroidal rupture.

Radial Peripapillary Capillary Network Visualized Using Wide-Field Montage Optical Coherence Tomography Angiography.

PURPOSE: We quantitatively analyzed the features of a radial peripapillary capillary (RPC) network visualized using wide-field montage optical coherence tomography (OCT) angiography in healthy human eyes. METHODS: Twenty eyes of 20 healthy subjects were recruited. En face 3 × 3-mm OCT angiograms of multiple locations in the posterior pole were acquired using the RTVue XR Avanti, and wide-field montage images of the RPC were created. To evaluate the RPC density, the montage images were binarized and skeletonized. The correlation between the RPC density and the retinal nerve fiber layer (RNFL) thickness measured by an OCT circle scan was investigated. RESULTS: The RPC at the temporal retina was detected as far as 7.6 ± 0.7 mm from the edge of the optic disc but not around the perifoveal area within 0.9 ± 0.1 mm of the fovea. Capillary-free zones beside the first branches of the arterioles were significantly (P < 0.0001) narrower than those beside the second ones. The RPC densities at 0.5, 2.5, and 5 mm from the optic disc edge were 13.6 ± 0.8, 11.9 ± 0.9, and 10.4 ± 0.9 mm-1. The RPC density also was correlated significantly (r = 0.64, P < 0.0001) with the RNFL thickness, with the greatest density in the inferotemporal region. CONCLUSIONS: Montage OCT angiograms can visualize expansion of the RPC network. The RPC is present in the superficial peripapillary retina in proportion to the RNFL thickness, supporting the idea that the RPC may be the vascular network primarily responsible for RNFL nourishment.

En face swept-source optical coherence tomographic analysis of X-linked juvenile retinoschisis.

PURPOSE: To clarify the area of retinoschisis by X-linked juvenile retinoschisis (XLRS) using swept-source optical coherence tomography (SS-OCT) en face images. OBSERVATIONS: We report two cases of XLRS in the same family. The patients presented with bilateral blurred vision. The posterior segment examination showed a spoked-wheel pattern in the macula. SS-OCT cross-sectional images revealed widespread retinal splitting at the level of the inner nuclear layer bilaterally. We diagnosed XLRS. To evaluate the area of retinoschisis, we obtained en face SS-OCT images, which clearly visualized the area of retinoschisis seen as a sunflower-like structure in the macula. CONCLUSION AND IMPORTANCE: We report the findings on en face SS-OCT images from patients with XLRS. The en face images using SS-OCT showed the precise area of retinoschisis compared with the SS-OCT thickness map and are useful for managing patients with XLRS.