Many studies have investigated endogenous loss of proteins and amino acids (AAs) at the ileal level in growing pigs. However, only a few studies have researched this subject in piglets. Knowledge regarding AA ileal digestibility in piglets would be helpful during the formulation of diets for weaning piglets, rather than just using coefficients obtained in growing pigs. Therefore, in this study, we sought to estimate endogenous protein and AA ileal losses in piglets. Furthermore, apparent and true ileal digestibility (AID and TID) of protein and AAs from casein were measured.The average flow of protein was 20.8 g/kg of dry matter intake (DMI). Basal protein loss, as estimated by regression, was 16.9 g/kg DMI. Glutamic acid, arginine, and aspartic acid  were the AAs for which greater losses were seen. The AID of protein and AAs increased as the protein level in the diet increased. A higher increment in AID was observed between diets with 80 and160 g CP/kg of feed; this finding was mainly attributable to increases in glycine and arginine . The TID of protein was 97.8, and the TID of AAs varied from 93.9 for histidine to 100.2 for phenylalanine.The basal endogenous protein loss in piglets was 16.9 g/kg DMI. Endogenous protein was rich in glutamic acid, aspartic acid, and arginine, which represented 32.7% of endogenous protein loss in weaning piglets. The TID of casein was high and varied from 93.0 for histidine to 100.2 for phenylalanine. Many studies have investigated endogenous loss of proteins and amino acids (AAs) at the ileal level in growing pigs -3. HowevTherefore, the objectives of this study were: 1) measure endogenous ileal loss of protein and AAs in weaned piglets, avoiding changes in the AA profile of endogenous protein through prolonged intake of a low protein diet; 2) measure the apparent and true ileal digestibility  of protein and AAs derived from casein.The experiment was performed at the experimental farm of CENID-Physiology. The care of animals used in this study was in accordance with the guidelines issued by the Mexican Official Standard for the Production, Protection and Use of Lab Animals  and the Eighteen piglets , weaned at 18 ± 1 d of age and weighing 6 ± 1 kg, were used. The piglets were placed in individual metabolism cages provided with a self-feeder and a low pressure drinking nipple, in a temperature controlled room (26 ± 3°C). In order to modify the enzymatic profile of suckling animals as little as possible; piglets were fed from 18 to 20 d of age with a mixture of dry whole milk (80%) and maize starch (20%) 3 times/d, at 0800, 1300 and 1800 h, at a concentration of 30 g/kg body weight (BW). When the piglets were 21 d old, a T cannula was fit at the terminal ileum as previously described . After sFrom the ninth d after surgery, piglets received 1 of 3 experimental treatments Table  15]. Al. Al15]. Experimental diets were supplied starting on d 9 after surgery, according to schedules described previously. The experimental period lasted for 7 d, that is 5 d for adaptation and 2 d for collection of ileal digesta. Feed intake was fixed at 26 g/kg BW during the experimental period to maintain feed intake at a constant level in relation to BW for all animals. The piglets always had free access to water.Ileal digesta were collected in plastic bags (5 cm × 3 cm) containing 2 mL of a 0.2 mol/L solution of HCl to minimize further bacterial activity. Bags were attached to the barrel of the cannula with a rubber band. Ileal digesta were collected continuously over the course of 12 h when the piglets were 36 and 37 d of age (d 16 and 17 after surgery). When ileal digesta filled the bags, they were transferred to a container and frozen at -20°C until lyophilization.Ileal digesta were lyiophilized and ground in a laboratory mill by using a 0.5-mm mesh . Experimental diets and ileal digesta were analyzed for dry matter (DM) and CP, according to AOAC methods 934.01 and 976.05  respectiEndogenous protein and AA losses in the ileal digesta were estimated using the following equation :ILAA is the ileal loss of CP or AA expressed in g/kg DMI, AF is the nutrient concentration in ileal digesta (g/kg DM), ID is the marker concentration in the diet (g/kg DM), and IF is the marker concentration in the ileal digesta (g/kg DM).Where The AIDs of DM, protein and AAs were estimated using the following equation :AID is the apparent ileal digestibility of a nutrient in the diet, ID is the marker concentration in the diet (g/kg DM), AF is the nutrient concentration in the ileal digesta (g/kg DM), AD is the nutrient concentration in diet (g/kg DM), and IF is the marker concentration in the ileal digesta (g/kg DM). The calculi excluded crystalline amino acids  because they were considered completely digested at the end of the ileum [Where he ileum ,22.Basal protein and AA ileal losses and the TIDs of protein and AAs from casein were estimated by linear regression :y is the protein or amino acid amount disappearing from the digestive tract in terms of g/kg DMI, a is the protein or amino acid basal ileal loss in terms of g/kg DMI, b is the protein or amino acid disappearing in the digestive tract, , and x is the intake in g/kg DMI. Regression was performed using the REG procedure of the SAS software [R2, because almost the total amount of nutrient ingested disappeared at the ileal level. The intercept was negative because an endogenous loss was, by definition, an amount found at zero intake, and consequently, at zero intake there was no dietary nutrient absorption, only excretion. Nevertheless, the absolute value of the intercept using excreted (non-digestible) or absorbed (digestible) nutrient was the same and could be used as reported previously [Where software . PerformHomogeneity of variance for all data was tested by Levene’s test using the hovtest of the SAS software . ProteinYij is the dependent variable; μ is the general mean; Ti is the treatmenti 1, 2, 3; and eij is the experimental error, calculated using the GLM procedure of the SAS software [α-value of 0.05 was used to assess significance and orthogonal polynomial contrast were performed to find a linear or quadratic response [Where software . The pigresponse .P < 0.05, Table P < 0.05), threonine (P < 0.05), aspartic acid (P < 0.05), glutamic acid (P < 0.05), serine (P < 0.01) and histidine (P < 0.001). Protein flow was similar between digesta of piglets fed 160 or 240 g CP/kg of feed (P > 0.05).Protein flow was higher in the digesta of piglets fed 160 g CP/kg of feed than in the digesta of piglets fed 80 g CP/kg of feed . These amounts represented 17.6%, 8.7%, 7.9% and 7.0% of total AAs. AAs with lower flow included methionine, histidine and tyrosine ; these flow represented 1.6%, 3.0%, and 3.1% of total AA flow, respectively.P < 0.001) and had determination coefficients of at least 0.98.The basal endogenous protein ileal loss estimated by regression was 16.9 g/kg DMI Table . GlutamiP < 0.01) in their AIDs as protein level increased in the diet.The AIDs of protein, arginine, leucine, isoleucine, valine, phenylalanine, methionine, aspartic acid, serine, glycine, alanine and tyrosine, increased as casein levels increased Table . The AIDThe AAs with the greatest differences in their AIDs between piglets consuming 80 and 160 g CP/kg were glycine (46.1%), arginine (18%), valine (16.3%) and alanine (14.4%).The difference in the AID of protein between piglets consuming 80 and 240 g CP/kg feed was 9.2%, which was much lower differences in the AIDs of glycine (66.2%), arginine (35%) and alanine (28.2%) between these groups. The lowest difference in AID between these groups was for Lysine (2.9%).The TID of protein was 97.8, and the average TID for AA was 97.6. The minimum TID value was obtained for histidine (93.0) and the maximum TID value was obtained for phenylalanine (100.2). The TID values for lysine and threonine were 99.4 and 96.0, respectively  and for AA were similar to those reported by Mariscal-Landín and Reis de Souza , as obseThe lack of variation in the AIDs of lysine, histidine, threonine, cystine and proline between treatments (160 and 240 g CP/kg of feed) could be due to the high levels of these AAs in protein. Jørgensen  fed growThe basal endogenous protein loss in piglets was 16.9 g/kg DMI; Endogenous protein was rich in glutamic acid, aspartic acid and arginine, which represented 32.7% of endogenous protein loss in weaning piglets. The TID of casein was high and varied from 93.0 for histidine to 100.2 for phenylalanine.AA: Amino acids; AID: Apparent ileal digestibility; TID: True ileal digestibility; BW: Body weight; DM: Dry matter; CP: Crude protein.The authors declare that they have no competing interests.All authors contributed equally to this research. All authors read and approved the final manuscript.


The catalytic activity of palladium (Pd) nanostructures highly relies on their size and morphology, especially enclosed with high-index facets, which provide more active sites so as to enhance their catalytic performance comparing with their low-index facet counterparts. Herein, Pd concave nanocubes enclosed with {730} facets by a one-pot scalable liquid method, with various high-index facets are synthesized via tuning reduction kinetics. Due to their high-index facets, the Pd concave nanocubes exhibit much higher electrocatalytic activity and stability for methanol oxidation than the Pd nanocubes enclosed by {100} facets and commercial Pd/C. Furthermore, we scale up synthesis of Pd concave nanocubes by expanding the volume of all species to fifty times with high-yield production. It achieves the insights into high-volume production and controllable morphologies by manipulating the reaction temperature and the concentration of CTAB and AA. Generally, elevating the reaction temperature is beneficial to the formation of thermodynamic favored production—Pd nanocubes. While, the high-index facets PdCNs could be obtained by increasing concentration of AA but reducing the reaction temperature. Hence, the evolution of controllable Pd morphologies from nanocubes structures to various concave nanocubes is conducted by a series of optimization routine. Furthermore, we scale up synthesis of Pd concave nanocubes by approaching the volume of all species to fifty times as shown in scheme S1 and commercial Pd/C (9.651 m2/g), respectively , a Pt plate (1 × 1 cm2) counter electrode, and a saturated calomel electrode (SCE) at an electrochemical station (CHI660E). Representatively, 0.6 mg of Pd nano-catalyst and 150 μL of Nafion solution (0.1 wt%) were dispersed in 300 μL of water-ethanol solution with volume ratio of 3:1 followed by ultra-sonication for 0.5 h to form a homogeneous ink. Then 15 μL of the dispersion  was loaded onto the GCE . Prior to test, the solution of 1.0 M KOH and 1.0 M CH3OH was purged with pure Ar gas for 1 h. Methanol oxidation measurements were conducted in a solution containing 1.0 M KOH and 1.0 M CH3OH using GCE at a sweep rate of 50 mV/s. The stability test was performed at a sweep rate of 0.5 V/s in a 1.0 M KOH and 1.0 M CH3OH solution for 1500 cycles.The electrochemical activities of Pd nanocatalysts were performed at room temperature using a three-electrode system consisting a glassy carbon electrode , high resolution bright-field TEM, and selected area electron diffraction (SAED) measurements were carried out with the field emission FEI-F20, operated at 200 kV.X.X., G.G. and L.C. conceived and designed the study, prepared all figures, and they all wrote the manuscript. X.X. performed the experiments mainly, and Z.P., T.W. and X.M. helped to do the measurements. L.C. is the corresponding author. All authors reviewed the manuscript.Supporting informaion


The resulting spatiotemporal contrast sensitivity (CS) functions revealed that the OKR of zebrafish is tuned to spatial frequency and speed but not to temporal frequencies. Thereby, optimal test parameters for CS measurements were identified. At photopic light levels, a spatial frequency of 0.116 ± 0.01 c/d (mean ± SD) and a grating speed of 8.42 ± 2.15 degrees/second (d/s) was ideal; at scotopic light levels, these values were 0.110 ± 0.02 c/d and 5.45 ± 1.31 d/s, respectively. This study allows to better characterize zebrafish mutants with altered vision and to distinguish between defects of rod and cone photoreceptors as measurements were performed under different light conditions.Sensitivity to spatial and temporal patterns is a fundamental aspect of vision. Herein, we investigated this sensitivity in adult zebrafish for a wide range of spatial (0.014 to 0.511 cycles/degree [c/d]) and temporal frequencies (0.025 to 6 cycles/s) to better understand their visual system. Measurements were performed at photopic (1.8 log cd m Contrast sensitivity (CS) reflects the lowest contrast at which an image can be discriminated from its background  is a popular model in visual system research, in part due to its relatively large eyes and cone-rich retina  of the AB (Oregon) strain aged 9–12 months were used. The fish were raised under standard conditions  and −6.3 log cd m−2. The light levels were attenuated with cylindrical neutral density filters  that encircled the examination chamber of the zebrafish from the bottom to the top of the optomotor device. These measurements were performed at a constant temporal frequency of 1.5 cycles/second (c/s) for different spatial frequencies ranging from 0.014 to 0.511 cycles/degree (c/d). To further characterize CSFs at scotopic and photopic light levels, CS was evaluated at spatial frequencies ranging from 0.014 to 0.511 cycles/degree (c/d)—namely 0.014, 0.031, 0.064, 0.128, 0.236, 0.383, and 0.511 c/d—and at speeds of rotation ranging from 0.4 to 24 degrees/second (d/s)—namely, 0.4, 0.75, 1.5, 3, 6, 12, and 24 d/s. These parameters corresponded to temporal frequencies ranging from 0.025 to 6 c/s—namely, 0.025, 0.05, 0.1, 0.2, 0.4, 0.75, 1.5, 3, and 6 c/s. These measurements were performed at luminance levels of 1.8 log cd m−2 and −4.5 log cd m−2. The same six zebrafish were used throughout the study and for each set of parameters.CSFs were measured in adult zebrafish using a commercially available optomotor device originally designed for mice  but modified for measuring zebrafish, as previously described by our group  , temporal frequency , and grating speed . To further improve the fitting of our results and to account for the fact that CSFs do not necessarily level at a CS of 1 in all setups, an additional parameter (b) was added:where b, k, sof, tof, pos) for each individual curve automatically .Curve fitting for Figures The maxima of the fitted curves correspond to the best CS. The corresponding value of the abscissa directly represents ideal settings to measure visual functions  and Figure −2, n = 6 animals for all conditions).CSFs were measured over a wide range of speed, spatial and temporal frequencies at photopic and scotopic light levels. The results are summarized in Figure The maxima of the CSFs are at a very similar spatial frequency Figures , 3A. ThiIn Figure The sensitivity to spatial and temporal patterns is a fundamental aspect of the visual system Wandell, . Here, w−2 are not possible. However, studies in larval zebrafish suggest a stable CS above a luminance of 0.47 log cd m−2  given that temporal frequency is the result of spatial frequency multiplied by its speed. In mice, the tuning preference changes from speed to temporal frequency after the transition from cone to rod vision (Umino et al., The obtained CSFs in this study allow for the identification of ideal test parameters for visual acuity and CS tests. Specifically, a spatial frequency of 0.116 c/d and a grating speed of 8.42 d/s are optimal for photopic light levels, whereas a spatial frequency of 0.110 c/d and a grating speed of 5.45 d/s are optimal for scotopic light levels. Measurements at low light levels enable the differentiation of defects in rod and cone photoreceptors. In conclusion, this study increases our understanding of basic aspects of the zebrafish visual system, in part by describing the tuning of zebrafish vision. Furthermore, these results lay the basis for optimal testing of adult zebrafish visual acuity and CS and may allow for improved characterization and identification of mutants with altered vision.Conceived and designed the experiments: MT NH CT VE AJ. Performed the experiments NH. Analyzed the data CT MT NH. Wrote the paper MT NH CT. All authors contributed to and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cercopithifilaria (Spirurida: Onchocercidae) have been described in dogs in association with Rhipicephalus sanguineus group ticks, which act as their biological vectors. This study represents the first investigation on Cercopithifilaria spp. in dogs from Portugal.Filarioids belonging to the genus Cercopithifilaria, namely C. bainae (9.8%), C. grassii (3.9%) and Cercopithifilaria sp. II sensu Otranto et al., 2013 (13.7%). Results were confirmed by molecular amplification of partial cytochrome c oxidase subunit I and 12S rRNA genes and sequence analysis. Co-infections with more than one Cercopithifilaria species were detected in 3.9% of the animals.Dogs (n = 102) from the Algarve region  were sampled by skin snip collection and tissues were left to soak overnight in saline solution. Sediments were observed under a light microscope and the detected microfilariae identified according to their morphology. Twenty-four dogs (23.5%) were found infected with at least one species of Cercopithifilaria spp. in dogs from Portugal. The estimated level of infection with C. bainae, C. grassii and Cercopithifilaria sp. II suggests that these filarioids are prevalent in the canine population of southern Portugal.This is the first report of  Dirofilaria immitis, also known as the canine heartworm, and Dirofilaria repens are the agents of cardiopulmonary and subcutaneous dirofilarioses, respectively, which represent diseases of veterinary importance and zoonotic potential [Acanthocheilonema dracunculoides and Acanthocheilonema reconditum, have microfilariae that circulate in the bloodstream. However, dogs may also be infected with other less well-known filarial species, such as Onchocerca lupi and Cercopithifilaria spp., whose microfilariae are found in dermal tissues [Several species of vector-borne filarial nematodes (Spirurida: Onchocercidae) have been found parasitizing dogs in Europe. In particular, otential . These t tissues ,3.Cercopithifilaria, originally described as a subgenus of Dipetalonema by Eberhard [Cercopithifilaria have been described in dogs from Brazil and Europe [Cercopithifilaria grassii, Cercopithifilaria bainae and Cercopithifilaria sp. II sensu Otranto et al., 2013 have been morphologically and molecularly differentiated in canine populations in Europe [c oxidase subunit I (cox1) and 12S rRNA genes [The genus Eberhard , currentEberhard -7. AdultEberhard . Filariod Europe ,8. Up unn Europe -11. MicrNA genes ,10.C. bainae overlaps with that of Rhipicephalus sanguineus group ticks [C. bainae has been found in dogs and ticks from Italy, Spain and Greece [The field distribution of up ticks , which aup ticks , as expeup ticks ,14. C. bd Greece ,15, in od Greece  and alsod Greece ; M.S. LaD. immitis, A. dracunculoides, A. reconditum and O. lupi, have previously been identified in Portugal [Cercopithifilaria spp. is available from dogs. In order to fill this gap of knowledge, in November 2012, 102 dogs  housed in two shelters located in center-eastern areas of the Algarve region  were sampled. The animals were estimated to be 1-3 years (n = 19), 4-8 years (n = 78), 9-11 years (n = 4) or 12-15 years old (n = 1). An informed consent for inclusion of dogs in this survey was obtained from the owner before sampling. The study design and experimental procedures were approved by the ethical commission of the University of Évora as complying with the Portuguese legislation for the protection of animals (Law no. 92/1995). Skin snips of around 0.01 cm3 (0.2 × 0.2 × 0.2 cm) were collected from the forehead region using a disposable scalpel and soaked in saline solution (pH 7.4) for 12 h in an incubator at 30°C. Dermal microfilariae were searched for in 20 μl sediment placed on a glass slide, using a light microscope (magnifications of ×40 and ×100), and identified according to their morphological features, as previously described [Infections with canine filarioids, such as Portugal -19, but cox1 and 12S rRNA gene fragments , with primers and procedures as described elsewhere [Following morphological analysis of microfilariae, sediments scoring positive were stored in 70% ethanol and species identification was subsequently assessed by PCR amplification and DNA sequencing ,10. Micrhttp://blast.ncbi.nlm.nih.gov/Blast.cgi).Amplicons were purified using Ultrafree-DA columns  and sequenced directly with the Taq DyeDeoxyTerminator Cycle Sequencing Kit  in an automated sequencer . The sequences were determined from both strands, using the same primers individually as for PCR, and the electropherograms verified by eye. The sequences were aligned using ClustalW program and compared among them and with those available in GenBank dataset by Basic Local Alignment Search Tool , but no differences were found in pairwise comparison between C. bainae and C. grassii or C. bainae and Cercopithifilaria sp. II. The prevalence of single infections (19.6%) was significantly higher (p < 0.001) than that of mixed infections (3.9%)  and male dogs (21.6%) or between age groups, i.e. 1-3 years (31.6%), 4-8 years (21.8%), 9-11 years (25.0%) and 12-15 years (0.0%).By microscopy, microfilariae of d Figure , with anfections 9.6% was C. bainae (n = 10) had an average length ± standard deviation (SD) of 181.1 ± 7.5 μm (range: 170.1-196.4 μm). The average length ± SD of C. grassii microfilariae (n = 6) was 630.8 ± 13.8 μm (range: 610.1-643.5 μm). Microfilariae of Cercopithifilaria sp. II (n = 13) had an average length ± SD of 277.0 ± 15.5 μm (range: 260.9-307.4 μm).Microfilariae of C. bainae (JF461457 and JF461461), C. grassii (JQ837810 and JQ837812) and Cercopithifilaria sp. II (JQ837809 and JQ837811) available in GenBank database .The BLAST analysis revealed 99% homology of the amplified sequences with the closest accession numbers of Cercopithifilaria. Infections with C. bainae and Cercopithifilaria sp. II were detected in dogs from the Italian regions of Basilicata [C. bainae and C. grassii were detected in Sicily [C. bainae, C. grassi and Cercopithifilaria sp. II populations were found in dogs from central Spain [Cercopithifilaria spp. has been found to range from 9.4% in Sardinia [C. bainae in northeastern Greece was 4.3% [Morphological and molecular data concurred in indicating that the dermal microfilariae detected in dogs from southern Portugal belong to three distinct known species of silicata  and Sardsilicata , whereasn Sicily . Sympatral Spain . The 23.al Spain . In ItalSardinia  to 13.3%Sardinia . Still bCercopithifilaria spp. did not present physical abnormalities that could be associated to the presence of microfilariae in the skin (data not shown). However, based on clinical and histological findings, a pruritic, diffuse and erythematous dermatitis without any other apparent cause was previously described in two dogs infected with microfilariae of C. bainae[Cercopithifilaria sp. II [Dogs found infected with C. bainae or Cercoa sp. II .Cercopithifilaria spp. should be considered among those parasites that can cause dermal microfilariae infection in dogs in Portugal. In the meantime, further studies should be carried out in order to elucidate the pathogenic relevance of these filarioids for dogs and other potential hosts.In conclusion, the present study highlights that The authors declare that they have no competing interests.HCEC designed and supervised the study, collected samples, analyzed data, assisted in drafting and reviewed the manuscript; LC collected samples, analyzed data and wrote the manuscript; AG performed morphometric assessments and reviewed the manuscript; MSL performed DNA extraction and molecular analyses and reviewed the manuscript; FDT analyzed data and reviewed the manuscript; DO planned and supervised the study, analyzed data and reviewed the manuscript. All authors read and approved the final manuscript.


To assess and compare choroidal thickness changes related to aging, we determined whether changes are due to thinning of the choriocapillaris plus Sattler's (CS) layer and/or the large vessel layer in healthy eyes using swept-source optical coherence tomography (SS-OCT) at a wavelength of 1,050-nm.We studied 115 normal eyes of 115 healthy volunteers, all with refractive errors of less than -6 diopters. All 115 eyes underwent analysis of choroidal thickness at the fovea, the CS layer and the large choroidal vessel layer. In 68 of the 115 eyes, choroidal thickness was determined at five sites  using SS-OCT with an Early Treatment of Diabetic Retinopathy grid scan.Total choroidal thicknesses at each of the five sites were related to subject age (P<0.0001). The choroid was thinnest at the nasal site, followed by the temporal, inferior, superior and finally the subfoveal site itself. The total choroidal thickness at the nasal site was significantly less than those at the other four sites (p<0.05). The CS layer showed thinning which correlated with age (P<0.0001). The thickness of the choroidal large vessel layer also decreased with age (p = 0.02). Subfoveal choroidal thickness was calculated as follows: 443.89–2.98×age (μm) (P<0.0001).Subfoveal choroidal thickness decreases by 2.98 μm each year. Total choroidal thickness diminishes with age. The CS and large vessel layers of the choroid at the subfovea showed significant decreases, though only the former correlated strongly with age. The choroid contains a large number of blood vessels, pigments, and immune system components. The choroid absorbs light, supplies oxygen, and provides nourishment to protect the retina. However, the choroid is also the site of many pathological processes such as inflammation, ischemia and neovascularization. Optical coherence tomography (OCT), which became available in the late 1990s, allows non-invasive observations of the ocular fundus. Most recent ophthalmological studies have used OCT as a routine medical examination along with standard visual acuity testing. Time-domain OCT with an 820nm wavelength and 400 A-scans/sec represents a major advancement in the quality of OCT imaging as it allows better visualization of the retina. Spectral-domain (SD) OCT, providing in excess of 40,000 A-scans/sec, allows even more precise assessment of retinal structure, comparable to that of a histopathological specimen. Even SD-OCT is not, however, sufficient for detailed observations of the choroid.Spaide et al.  reportedBranchini et al.  reportedNeither of these prior reports examined, or even took into consideration, the age-change correlation with choroidal thickness. Therefore, using SS-OCT, we investigated choroidal thickness (choriocapillaris plus Sattler's (CS) layer and/or large vessel layer) in relation to age. We manually measured layer thicknesses, to obtain clear images and to derive formulas that would allow us to calculate thickness based on OCT data.We assessed choroidal thinning using SS-OCT manually to assure clear measurements, in relation to age decades, in order to determine whether the CS and/or the large vessel layer contributes to the widely recognized decrease in the thickness of the choroidal layer with advancing age. We also endeavored to clarify the etiology of choroidal thinning.Between September 2013 and August 2014, healthy patients were recruited for this study. All subjects were at least 21 years of age and had normal eyes. None had visual symptoms or a history of ocular disease. The only inclusion criterion was refractive error less than -6 diopters (D). This criterion was applied to eliminate the potential effects of axial length and high myopia [The institutional review board of Nihon University Hospital, Tokyo, Japan, deemed approval to be unnecessary for this study . For this study, we recruited volunteers, who visited our hospital to undergo periodic eye examinations, obtaining verbal consent without a signature in all cases. Furthermore, all participants had the right to opt-out of the study. If the volunteers agreed to participate in this study, they did not need to sign a consent form. If a volunteer decided to drop out of the study, we requested that they sign a consent form to opt-out of the study.We chose 115 eyes  for analysis of the thickness of each subfoveal layer (the CS layer and the large vessel layer) as the primary endpoint. Out of these 115 eyes, we chose 68  eyes for subgroup analysis of choroidal thickness in five areas .We examined only one eye, mainly the right eye, in each subject to eliminate individual variation. However, if the right met any of our exclusion criteria, we studied the left eye instead.It is necessary to measure all 512  and 64  lines clearly when measuring mean choroidal thicknesses of the five aforementioned areas. If even one slice is missing, due to blinking, the mean choroidal thickness value will not be accurate. Therefore, five of the 68 eyes were excluded from the analysis of choroidal thickness due to blinking during the examination. There are no effects of blinking on choroidal thickness at the fovea.We performed horizontal and axial scans and three-dimensional analysis of a 6 by 6 mm area at the center of the fovea using SS-OCT . The three-dimensional images were obtained with SS-OCT using raster scans of 512  and 64  lines. The A-scan value per dataset  was 0.8 seconds. Choroidal thickness and the chorioscleral border were measured manually with a built-in caliper (Y.W.), according to Rhaman et al . In eachChoroidal thickness was measured at the fovea and at each of the other four sites  3mm from the fovea for each age decade employing the Early Treatment Diabetic Retinopathy Study (ETDRS) grid . The mea2, 6,255 measured points) was calculated for each of the four areas. The foveal area was taken to be that surrounded by the innermost circle  and its average thickness was calculated.We defined “site” as a pin-point portion of the retina and used the term “area” for the broader portion. We defined the superior area as the total of the outer and inner superior areas as well as those of the inferior, nasal, and temporal areas . The aveChan-Ling et al. reported that the undifferentiated human’s capillary plexus forms first and that the layers of larger vessels form thereafter , and calThe following items were investigated; subfoveal choroidal thickness and the choroidal thicknesses at the aforementioned four sites 3mm from the fovea for each age decade and each eye, as well as the mean choroidal thickness at each of these five areas. The thicknesses of the subfoveal CS layer and the large vessel layer were also determined .The relationships between age and choroidal thicknesses were evaluated using regression models and regression coefficients. For the comparisons among choroidal thicknesses adjusted for age, a mixed model was applied, and we evaluated the differences of choroidal thickness and the distance between regression lines. The statistical power was 0.493, which was calculated based on a previous report, employing a distance of ‘23 μm’ for our 68 subjects (significance level of 0.05) . We defiThe subfoveal choroidal thickness and those at the four sites 3mm from the fovea are shown in Mean choroidal thickness values (±SD) for each age decade were; 20s: 336.33±52.38 μm, 30s: 377.67±117.86 μm, 40s: 345.44±57.78 μm, 50s: 286.88±83.01 μm, 60s: 208.50±59.43 μm, 70s: 230.62±69.57 μm, and 80s: 215.00±68.69 μm.These data confirm a significant linear correlation between subfoveal choroidal thickness and age (p<0.0001). The regression formula is: macular choroidal thickness (μm) = 443.89–2.98×age (p<0.0001). Mean subfoveal choroidal thickness decreased by 2.98 μm per year.Mean choroidal thickness values (±SD) at the site superior to the fovea for each age decade were; 20s: 300.22±50.79 μm, 30s: 308.17±68.86 μm, 40s: 327.69±63.84 μm, 50s: 259.87±76.08 μm, 60s: 211.50±89.20 μm, 70s: 208.64±76.25 μm, and 80s: 191.17±51.88 μm.There was a linear relationship between superior site choroidal thickness and age. The regression formula is: superior choroidal thickness (μm) = 396.36–2.52×age (p<0.0001). Mean choroidal thickness at the superior site decreased by 2.52 μm per year.Mean choroidal thickness values (±SD) at the site inferior to the fovea for each age decade were; 20s: 289.50±62.61 μm, 30s: 286.25±73.83 μm, 40s: 310.94±78.98 μm, 50s: 237.60±78.12 μm, 60s: 185.93±80.23 μm, 70s: 185.36±59.77 μm, and 80s: 133.33±41.25 μm.There was a linear relationship between choroidal thickness at the inferior site and age. The regression formula is: inferior choroidal thickness (μm) = 396.66–2.99×age (p<0.0001). Mean choroidal thickness at the inferior site decreased by 2.99 μm per year.Mean choroidal thickness values (±SD) at the nasal site for each age decade were; 20s: 181.67±35.17 μm, 30s: 178.50±53.52 μm, 40s: 183.19±48.81 μm, 50s: 145.00±46.33 μm, 60s: 110.64±50.38 μm, 70s: 101.82±42.08 μm, and 80s: 117.17±46.45 μm.There was a linear relationship between choroidal thickness at the nasal site and age. The regression formula is: nasal choroidal thickness (μm) = 236.01–1.70×age (p<0.0001). Mean choroidal thickness at the nasal site decreased by 1.70 μm per year.Mean choroidal thickness values (±SD) at the temporal site for each age decade were; 20s: 312.17±50.80 μm, 30s: 268.25±73.82 μm, 40s: 259.13±55.64 μm, 50s: 209.93±54.79 μm, 60s: 191.79±79.52 μm, 70s: 184.45±50.71 μm, and 80s: 153.00±45.14 μm.There was a linear relationship between choroidal thickness at the temporal site and age. The regression formula is: temporal choroidal thickness (μm) = 372.95–2.73×age (p<0.0001). Mean choroidal thickness at the temporal site decreased by 2.73 μm per year.The choroid was thinnest at the nasal site, followed in order by the temporal, inferior, superior and subfoveal sites. There was no significant difference in choroidal thickness between the temporal and inferior sites (p = 0.19) for any of the age groups .Mean choroidal thickness values (±SD) for each age decade were; 20s: 296.44±43.56 μm, 30s: 295.17±111.87 μm, 40s: 306.50±69.36 μm, 50s: 234.55±85.77 μm, 60s: 187.64±57.97 μm, 70s: 206.73±65.38 μm, and 80s: 204.80±57.10 μm.There was a linear relationship between mean choroidal thickness in the foveal area and age. The regression formula is: mean foveal choroidal thickness (μm) = 366.27–2.24×age (p<0.0001). Mean choroidal thickness in the foveal area decreased by 2.24 μm per year.Mean choroidal thickness (±SD) values for each age decade were; 20s: 300.44±35.67 μm, 30s: 277.42±89.60 μm, 40s: 300.75±48.45 μm, 50s: 246.18±75.39 μm, 60s: 179.91±55.65 μm, 70s: 208.91±70.85 μm, and 80s: 213.20±57.84 μm.There was a linear relationship between mean choroidal thickness in the superior area and age. The regression formula is: mean superior choroidal thickness (μm) = 364.66–2.22×age (p<0.0001). Mean choroidal thickness in the superior area decreased by 2.22 μm per year.Mean choroidal thickness values (±SD) for each age decade were; 20s: 286.11±58.33 μm, 30s: 289.58±104.49 μm, 40s: 302.35±79.85 μm, 50s: 236.82±73.33 μm, 60s: 189.59±59.53 μm, 70s: 194.73±59.70 μm, and 80s: 168.90±46.24 μm.There was a linear relationship between mean choroidal thickness in the inferior area and age. The regression formula is: mean inferior choroidal thickness (μm) = 373.99–2.51×age (p<0.0001). Mean choroidal thickness in the inferior area decreased by 2.51 μm per year.Mean average choroidal thickness values (±SD) for each age decade were; 20s: 254.33±37.89 μm, 30s: 256.25±95.87 μm, 40s: 259.25±61.84 μm, 50s: 218.59±64.90 μm, 60s: 154.23±56.46 μm, 70s: 168.64±56.91 μm, and 80s: 167.50±53.18 μm.There was a linear relationship between mean choroidal thickness in the nasal area and age. The regression formula is: mean nasal choroidal thickness (μm) = 325.52–2.14×age (p<0.0001). Mean choroidal thickness in the nasal area decreased by 2.14 μm per year.Mean choroidal thickness values (±SD) for each age decade were; 20s: 309.11±36.70 μm, 30s: 288.50±101.75 μm, 40s: 278.75±52.78 μm, 50s: 217.23±74.28 μm, 60s: 184.95±53.66 μm, 70s: 205.14±50.72 μm, and 80s: 182.90±41.87 μm.There was a linear relationship between mean choroidal thickness in the temporal area and age. The regression formula is: mean temporal choroidal thickness (μm) = 369.69–2.47×age (p<0.0001). Mean choroidal thickness in the temporal area decreased by 2.47 μm per year.The choroid was significantly thinner in the nasal area than in the other four areas (p<0.05), while there were no significant differences among the other four areas (p>0.05) .Mean CS layer thickness values (±SD) for each age decade were; 20s: 110.78±40.80 μm, 30s: 89.89±25.16 μm, 40s: 73.11±28.25 μm, 50s: 54.56±23.03 μm, 60s: 50.57±29.06 μm, 70s: 55.42±38.37 μm, and 80s: 39.89±32.47 μm.2 = 0.29).There was a linear relationship between CS layer thickness and age . The regMean subfoveal choroidal thickness values (±SD) of the large vessel layer for each age decade were; 20s: 214.56±74.54 μm, 30s: 239.5±88.86 μm, 40s: 225.59±47.75 μm, 50s: 183.40±55.08 μm, 60s: 185.86±58.76 μm, 70s: 189.26±49.82 μm, and 80s: 200.00±48.53 μm.2 = 0.056). A significant age-dependent difference in choroidal thickness was observed for both the CS layer (p<0.0001) and the large vessel layer (p<0.0209) (There was a linear relationship between large vessel layer thickness and age . The reg<0.0209)  and 5.The statistical power was 0.493, which was calculated based on a previous report, employing a distance of ‘23 μm’ for our 68 subjects (significance level of 0.05).2 = 0.1) [Chen et al reported axial length to be the main predictor of foveal choroidal thickness but they did not examine refractive error . High my2 = 0.1) .Therefore, high myopia -6D or greater was excluded in our present study to eliminate the effect of axial length and high myopia. Since lymphatics are also highly related to axial length changes in myopia, we need to consider this factor in future studies .The subfoveal choroidal thickness regression formula yielded differences ranging from 2.98 μm  to 2.24 μm .There are many reports describing choroidal thickness in each of the aforementioned areas, i.e. the fovea itself, and the sites superior, inferior, nasal, and temporal to the fovea –10.The choroidal thicknesses of both sites and areas decreased with age. The choroid was thinnest at the nasal site, followed by the temporal, inferior, superior, and foveal sites. The choroid was significantly thinner at the nasal site than at the other four sites. Choroidal thickness averages for each site were derived from a broad area extending from the outer portion of the choroid to the fovea itself, but the choroidal thicknesses of each site were obtained from only a single measurement. Therefore, a lack of measurement data might have produced differences.There are no reports, to our knowledge, presenting a formula relating the thicknesses of distinct parts of the choroid with age. The correlations between mean choroidal thicknesses and age are presented as scatter diagrams with minor deflection, with p values for degree of skewing and the intercept being <0.0001. We thus consider our data to be reliable.Esmaeelpour et al reported automatic measurements of the thicknesses of Sattler's layer and Haller's layer at the choroid using 3-dimensional OCT with a 1060-nm wavelength . We manuWe endeavored to assess choroidal thickness related to age decades and to determine which layer(s) might be involved in the observed choroidal thinning. Therefore, we conducted manual segmentation, employing a built-in caliper, and analyzed the thickness of both the CS layer and the choroidal large vessel layer. The thickness of the subfoveal CS layer decreased with age and the correlation was statistically significant (p<0.0001). The thickness of the large vessel layer also decreased with age (p = 0.0204), indicating a trend for the thickness of this layer to decrease with aging. The p values indicated, however, that the decrease in the CS layer was more than 200 times that in the large choroidal vessel layer. Therefore, we speculate that the aging change in total choroidal thickness at the fovea is mainly attributable to a decrease in CS layer thickness. The resolution quality of SS-OCT is not sufficient to allow the choriocapillaris layer to be distinguished from Sattler’s layer. In addition, the choriocapillaris is too thin to be evaluated by itself.In this study, we were also interested in vascular endothelial growth factor (VEGF)-A, which is secreted by the RPE and exerts a trophic influence on the choriocapillaris. The RPE secretes VEGF toward its basal side, where VEGF receptors are located on the adjacent choriocapillaris endothelium, suggesting VEGF to play a role in the maintenance of choriocapillaris fenestration in the normal eye ,22. An iIt has been suggested that in hypoxic states and with aging, this paracrine relationship is disturbed by a thickened Bruch’s membrane, and that this age-associated change may block the passage of VEGF-A from the RPE to the choriocapillaris ,26,27. IOur results reflect the choroidal layer changing with age, possibly in relation to the functions of VEGF-A and lipid accumulation. The CS layer is closer to the RPE layer than to the choroidal large vessel layer, i.e., the CS layer would presumably be more affected by the aging changes described in prior reports. However, the CS layer consists of Sattler’s layer and the choriocapillaris layer. Whether one or both of these layers contributes to diminished choroidal thickness with aging remains as yet unknown.We speculate that not only VEGF but also systemic water may exert effects on the thicknesses of ocular layers. The total amount of water in the body decreases with age, possibly contributing to the observed decreases in choroidal thickness with aging. The water drinking test revealed that as the amount of water in the body increases, choroidal thickness also increases . AlthougWe found that subfoveal choroidal thickness decreases by 2.98 μm per year, on average. Choroidal thickness decreased at all measurement sites with age. Thicknesses of the subfoveal CS layer and the large vessel layer decreased significantly, and that of the CS layer correlated especially strongly with age.


AbstractParagrantiawaguensis Hôzawa is reported from coastal reefs of the island of Okinawa. This rare species was previously known only from Central Japan, Mie Prefecture. It has peculiar apopylar tetractine spicules, so far unique among Calcarea. We present in situ images of the species and a full description including SEM images of skeletal structure and spicule complement. The status of Paragrantia as a separate genus of the family Grantiidae distinct from Grantia Fleming is confirmed on the basis of a morphological and molecular comparison with the European type species of Grantia, Grantiacompressa (Fabricius). ParagrantiawaguensisPageBreakThe species was first collected in Okinawa in 2006 and mentioned briefly in a paper reporting its chemistry  of a rare and curious calcareous sponge species from Okinawa, hemistry . SubsequParagrantiaGrantiidaePorifera, Calcarea, Calcaronea, Leucosolenida) on the basis of a curious structure in the exhalant openings of its type species, Paragrantiawaguensis. The genus and its type species were not seriously evaluated since they were described in 1940: the genus was assigned to the synonymy of the genus Scypha Gray (1821) in Burton’s literature review of the Calcarea (1963: 448) and the type species was considered a junior synonym of the boreal widespread species Scyphacompressa  (= Grantiacompressa), meriting only the comment that its general appearance resembled that of the well-known Grantiacompressa. Burton (l.c.) simply summarized Hôzawa’s description as a ‘named form’ under the synonyms of Scyphacompressa, in the process omitting to provide information on its diactines (see below). Calcarea were severely criticized at the time, and his synonymy assignments were generally considered unacceptable.Calcarea presented in the framework of the Systema Porifera project , was not available to us), and pose the question of the validity of the genus Paragrantia. For that purpose, we compared our specimens with a representative sample of the type species of the genus Grantia, Grantiacompressa  and performed a molecular sequence analysis of the two species and some of its assumed relatives.Despite Hôzawa’s excellent description and generally good illustrations of RMNH collection. For our comparison with Grantiacompressa, we used a specimen from Roscoff, W coast of France, incorporated in the ZMA collection, now housed in Naturalis Biodiversity Center.Specimens were collected using SCUBA by J. Tanaka (University of the Ryukyus) in 2006, and by BWH, as a guest of JDR, in 2014, subsequently identified by NJDV and RWMVS, and registered in the PageBreakSpecimens were sectioned by hand: cross sections perpendicular to the surface and tangential sections of the outer surface , the inner surface , and the oscular fringe. The sections were air dried, mounted on stubs, and sputter coated for examination under SEM. Spicules were dissociated using household bleach, washed five times in distilled water, and subsequently plated on glass slides for light-microscopic measurements and on SEM stubs for examination and micrographing under SEM.average-largest of 25 spicules of each distinct type.Measurements of the spicules are given as smallest-Paragrantiawaguensis with Grantiacompressa, a 28S rDNA sequence (430 bp) of Paragrantiawaguenis (sample RMNH Por. 9317) was provided by the Naturalis Barcode Laboratory. DNA was extracted using the NucleoMag 96 Tissue kit by Macherey-Nagel on a Thermo Scientific Kingfisher Flex magnetic bead extraction robot with a final elution volume of 150 µl. The forward and reverse of the C2-D2 region of the nuclear ribosomal 28S was amplified (Forward primer 5’GAAAAGAACTTTGRARAGAGAGT 3’ and Reverse primer 5’TCCGTGTTTCAAGACGGG 3’). Template was diluted ten times before amplification and added with 18.8 µl of ultrapure MQ water, 2.5 µl PCR buffer, 0.5 µl dNTP (containing 2.5 mM) and 0.25 µl Taq (5 units per µl) to a total reaction volume of 25 µl. PCR cycling consisted of an initial denaturation step at 94 °C for 3 mins, followed by 40 cycles each consisting of 15 secs at 95 °C, 30 secs at 50 °C, 40 secs at 72 °C, and a final extension of 5 mins at 72 °C. Bidirectional sequencing was performed at BaseClear (http://www.baseclear.com/). Sequences were edited manually with Sequencher 4.10.1 (Gene Codes Corporation).To verify the conclusions from the morphological comparison of http://blast.ncbi.nlm.nih.gov/Blast.cgi), and downloaded a representative set of partial 28S sequences of calcaronean species showing up in the BLAST result. The set of sequences included a sequence of Grantiacompressa provided by Leucosolenida-sequences submitted by various research groups belonging to Grantiidae, Sycettidae, Jenkinidae, Amphoriscidae and Lelapiidae. We added as outgroup sequence the calcinean Pericharaxheteroraphis . The combined dataset of 17 sequences (see Table http://www.megasoftware.net/megamac.php). For the phylogeny reconstruction we chose the Maximum Likelihood statistical method with a Bootstrap method set at 100 replicates. As Substitution Model we chose the Tamura-Nei model and – based on model testing algorithm in MEGA – we used GTR+G as Evolutionary Model. Further parameters were used in their default settings.With the obtained sequence we performed a BLAST analysis provided by the NCBI website . Hôzawa, 1940: 40, pl. V figs 8–11, text-fig. 4; Grantiawaguensis ; RMNH Por. 9317 , Japan, South Kuroshio ecoregion, Okinawa, Manza, approximately 26.5°N, 127.8°E, vertical rocky wall, 25–30 m, coll. B.W. Hoeksema, 10 August 2014; Naturalis Biodiversity Center, reg.nr. RMNH Por. 3901 , Japan, South Kuroshio PageBreakecoregion, Okinawa, Onna village, approximately 26.5°N, 127.8°E, coral reef slope, 20–55 m, coll. J. Tanaka, 6 May 2006.Naturalis Biodiversity Center, reg. nr. TUMC 110908, Japan, Central Kuroshio ecoregion, Mie Prefecture, Wagu, approximately 34.25°N, 136.8°E, coll. S. Hôzawa, July 1933.Syntype, 8 specimens (not seen) Tôhoku University Museum, reg.nr. Algae or detritus may stick to the rim. Inside the cup, most specimens are likewise smooth and white, but one of the specimens is mottled greenish due to encrusting algae growing on its inner surface . There is no evidence of branching choanocyte chambers. Subcortical lacunae are present, regularly distributed and apparently serving as inhalant reservoirs.Skeleton of the walls.  Figs , 4c1, bu703.2–990 × 12–21.6–29 µm.Diactines Fig.  from thePageBreak284.8–528 × 9–10.6–15 µm, paired actines 81–167.6–273 × 8–10.3–12 µm, apical actines 16–39.3–76 × 5–7.2–10 µm.Tetractines Fig.  from the223.5–279 × 6–8.8–10 µm, paired actines 63–84.8–105 × 7–9.2–10 µm.Triactines Fig.  from theNo data were provided by Hôzawa, but the Okinawa specimens were from a steep reef slope at 20–55 m depth, growing among coralline and turf algae, and encrusting sponges.Warm-temperate (Mie Prefecture) and subtropical (Okinawa) regions of Japan.The habit of Hozawa’s specimens is described from eight preserved individuals. A photo is given of two specimens , that thThe general structure of the skeleton and the overall diversity and sizes of the spicules likewise match closely  limits the generic relationships of waguensis to Grantia Fleming (1828), SycandraTeichonopsis Dendy & Row (1913). All three share the general structure of the skeleton with the present species. The latter two genera are monotypic and are distinguished on unique features, the presence of a special atrial network of tissue strands supported by small diactines (Sycandra), or an elaborate shape (Teichonopsis). Sycandrautriculus  has a similar structure and spiculation as our specimens (and many Grantia species), but the peculiar atrial network forms a unique distinction. Teichonopsislabyrinthica , vaguely resembles preserved specimens of the present species, but the spiculation differs clearly by the lack of any tetractines and the presence of brushes of small oxeas on the cortical and atrial surfaces. Grantia itself, in contrast, has approximately 40 accepted species , Grantiacompressa, the well known Purse Sponge of intertidal rocky coasts of Northern Europe. Remarkably, neither the Systema Porifera, chapter on Leucosolenida . The accepted status of many of those species is uncertain, as there has been no recent revision of the genus and the names were taken more or less uncritically from Grantiakempfi, was made by Grantia s.l. there appears to be a wide variety of shapes and skeletal features, possibly divisible into distinct types, which may eventually lead to the distinction of subgenera or genera, leaving the genus name Grantia restricted to those species that share the properties of the above described Grantiacompressa. Candidate species for such a restricted Grantia appear to be Grantiacupula , Grantiaextusarticulata , GrantiafistulataGrantiafoliacea Breitfuss (1898), GrantiastylataGrantiatenuisGrantiaaculeataGrantiatransgrediensGrantiauchidai Hôzawa & Tanita (1941).To date the World Database  lists 41Grantia’s), or those lacking tetractines, etc.Other, not further specified groups of species may be distinguished e.g. on the possession of long protruding diactines . We obtained from an analysis of 17 aligned Calcaronea sequences  raphanus and Leucandraaspera.A further differentiation between the two type species of the genera see e.g. , so it iGrantia along the lines sketched above has been made – preferably guided by independent molecular markers of the studied taxa – we propose to PageBreakmaintain Paragrantia as a separate genus, so far monotypic with Paragrantiawaguensis as the only species. Its status is comparable to other such genera (Sycandra and Teichonopsis), recognizable by unique features, as in this case the unique butterfly-shaped apopylar tetractines, for which we introduce the term ‘aliactines’  = wing).Until a revision of We propose here the following definition Grantiidae with cortical skeleton of triactines, articulate tubar skeleton composed of aligned triactines, and an oscular fringe with giant diactines and sagittal tri-and tetractines. Atrial skeleton composed of subatrial triactines, and atrial triactines and/or tetractines. Choanocyte chambers connect with the atrial lumen through apopylar chambers lined with modified specialized tetractine spicules .Syconoid


Toxocara canis, or cats, Toxocara cati. Toxocara is responsible for the presentation of various syndromes in humans depending on the affected organs.Toxocariasis is a zoonotic disease that poses a threat to public health worldwide. This disease primarily affects children and is caused by the presence in the digestive tract of a common roundworm of dogs, T. canis antibodies was investigated in children aged 3–16 years from semirural populations in the municipalities of Amecameca and Chalco in México. An ELISA was used to determine the presence of anti-T. canis antibodies in blood samples.In this study, the prevalence of anti-T. canis antibodies (12.02 %). Of these, 6.50 % were from males and 5.4 % were from females. Risk factors were investigated and it was found that living near a cattle operation had a statistically significant association with (Chi2 = 5.51 and p = 0.01) and was a risk factor for  seropositivity to T. canis. Keeping dogs with short hair (Chi2 = 3.24 and p = 0.07) showed a tendency toward seropositivity for T. canis, as did the habit of sleeping with pets (Chi2 = 3.46 and p = 0.06).Of the 183 sera obtained for this study, 22 were positive for anti-T. canis was confirmed in children in the Amecameca and Chalco regions of México and the risk factors were identified. These findings provide important insight into the prevalence and spread of this zoonotic parasite.Seropositivity to  Dogs are associated with more than 60 zoonotic diseases worldwide, mainly by parasitic organisms, posing a serious health threat to humans ,2. Many Toxocara canis and Toxocara cati [One of the most common infections worldwide caused by parasites is toxocariasis, a zoonotic disease  caused bara cati . The eggara cati  into theara cati ,8.Some risk factors associated with this parasite include: gender, age, socioeconomic status, close contact with domestic animals , ingestiT. canis in recreational areas, and in soil samples and the feces of stray dogs. Rates of 24 % and 67.5 % were obtained [T. canis; for example, 66 % prevalence in Spain and 67 % prevalence in Argentina [In México, there have been studies to investigate the prevalence of obtained ,12. The rgentina .T. canis antibodies and risk factors for T. canis were investigated in a semirural population in the State of México.Furthermore, seroprevalence studies in children reported rates of 22.22 % in México  comparedA total of 183 blood samples were collected from children in the municipalities of Amecameca and Chalco, México .A 3-ml sample of blood was obtained from each participant through phlebotomy and these samples were deposited in tubes without anticoagulant and incubated at room temperature for 40 min. Then, samples were subjected to centrifugation at 4000 × T. canis antibodies using a commercial ELISA kit, with a sensitivity of 87.5 % and specificity of 93.3 % (DIAGMEX-Toxocara® SA De CV. México). The optical density of the samples was determined using a Microplate Modulus® multiplate spectrophotometer  at a wavelength of 460 nm [Serological tests were performed on the blood samples to measure anti-f 460 nm . The optp < 0.05 was considered significant. The relative risk of T. canis antibodies was calculated from the odds ratio, with a significance of p < 0.05. Statistical analysis was performed using JMP® 8.0 software .To compare the rates of positivity between groups, Fisher’s exact test  was applT. canis antibodies were detected in 22 (12.02 %). No statistically significant difference was found between the prevalence of anti-T. canis antibodies in males and females (Table p = 0.64).Of the 183 sera tested, anti-2 = 3.24 and p = 0.07) showed a tendency toward seropositivity for T. canis, as did the habit of sleeping with pets (Chi2 = 3.46 and p = 0.06). By contrast, practicing preventive medicine and coexistence did not show a significant association (Table T. canis seropositivity (Chi2 = 5.51 and p = 0.01) and was also a risk factor for T. canis seropositivity  , and a higher percentage of seropositive males (53.1 %) than females, which they attributed to differences in the games played by children and their resulting close contact with the environment [p = 0.69) and age (p = 0.99) were not associated with parasitoses. These findings were in agreement with the present study in which no significant association was reported between gender and the presence of T. canis antibodies (p = 0.64). A study by Getaz and colleagues in 2007 found no significant association between seropositivity to T. canis and asthma, but did identify an association with nocturnal wheezing [In Mexicali, México, a previous study analyzing the seroprevalence of ironment , a studywheezing .T. canis, and the risk of transmission increases with the degree of environmental pollution. Three research teams [Toxocara spp. from the land in parks, playgrounds, gardens and beaches in urban and suburban areas is considered a critical factor for toxocariasis, as well as contact with dogs. The results of the current study showed that the potential risk factors of keeping dogs  and sleeping with dogs  showed a tendency toward seropositivity for T. canis but this was not a significant association. Another study of seroprevalence in children in México [2 = 0.80, p = 0.37). A study in Argentina, however, found that contact with animals was an important risk factor for T. canis exposure in children  [2 = 5.51, p = 0.01) with the presence of anti-T. canis antibodies in children, potentially linked with the presence of stray dogs on such premises.Hygiene habits, housing area and customs are factors that primarily influence the variation of seroprevalence of ch teams –21 condun México  found th= 0.001) . These dT. canis in children in the municipalities of Amecameca and Chalco, in the State of México. We observed a higher percentage of seropositive males than females, and attributed this to their increased activity in recreational areas. Living near livestock farms was identified as a risk factor for exposure to T. canis, as assessed by the presence of anti-T. canis antibodies.The results of this investigation revealed a seroprevalence of 12.02 % for


Pteromylaeus bovinus, Myliobatis aquila) and pelagic rays (Pteroplatytrygon violacea) from the northern Adriatic Sea (Gulf of Trieste) in relation to their size (age) was investigated. High arsenic concentrations were found in both groups with tendency of more efficient arsenic accumulation in benthic species, particularly in muscle . This was attributed to species differences in arsenic access, uptake and retention. In liver most arsenic was present in a form of arsenobetaine, dimethylarsinic acid and arsenoipids, whereas in muscle mainly arsenobetaine was found. The good correlations between total arsenic/arsenobetaine and size reflect the importance of accumulation of arsenobetaine with age. Arsenobetaine is an analogue of glycine betaine, a known osmoregulator in marine animals and both are very abundant in mussels, representing an important source of food for benthic species P. bovinus and M. aquila.The difference in arsenic concentration and speciation between benthic ( One of main environmental concerns in this region is pollution by toxic metals . The Iso arsenic , somewhanic [−1) . Concentt (d.w.) ,9, confiw.) [−1, ). Arseni−1 d.w. and for zooplankton, a single concentration of 15.1 µg·g−1 d.w. was found [−1 d.w., [−1 wet weight [−1 (average 4.48 mg·kg−1) [−1 total arsenic on a wet weight basis, arsenic speciation data showed the existence of a variety of arsenic compounds, from inorganic arsenic to various arsenosugars, depending on the plant species [et al. [Marine organisms have the ability to accumulate and transform inorganic arsenic into less toxic organic arsenic species like arsenosugars, methylarsonic acid (MA), dimethylarsinic acid (DMA), trimethylarsine oxide (TMAO), arsenobetaine (AB), arsenocholine (AsC) and several less common other arsenic compounds including sulfur analogues  and arseas found  and conf−1 d.w., ). For sot weight  and from species . A small species . The maj species ,17,18, a species ,20. The  species ,22,23 su species , and lit species . It is k species . Accordi [et al.  arsenic  [et al. , biomagnPrevious research has pointed out that phosphate should be in limited supply in the northern Adriatic ,28. Due Pteromylaeus bovinus and Myliobatis aquila) and pelagic rays (Pteroplatytrygon violacea) in relation to their size (age) and feeding habits. Three species from our previous study [The aim of this study was to investigate arsenic concentrations and speciation in liver and muscle of benthic  and from 32.4 to 362 µg·g−1 d.w. in muscle (113 ± 84 µg·g−1). P. bovinus) contain much higher concentrations of arsenic in muscle compared to pelagic ones (P. violacea), while liver arsenic does not differ as much between benthic and pelagic adult specimens. Regardless of size, the ratio of muscle to liver arsenic concentration is 5.75 ± 1.43 for P. bovinus and almost three times lower 2.02 ± 0.83, with one outlier at 8.15 (sample 038 PV), for P. violacea. But even with the outlier included the difference was statistically significant (p = 0.003) by the nonparametric Wilcoxon Mann-Whitney test on comparing the medians (including the outlier) of both species.Total arsenic concentrations, together with basic data on the ray samples, are shown in R2 values) for weight; R2 of 0.854 (n = 14) was found for muscle and 0.642 (n = 15) for liver  of the specimens investigated (43.7–60.0 cm DW).Correlation analysis of the age-related parameters disk width (DW) or disk length (DL), total length (TL) or weight (W) with total arsenic concentration yielded the best correlations . In liver the major arsenic compound was AB representing on average 59% of the total extractable arsenic, followed by lipidic  fraction accounting for 32% of the extractable arsenic. DMA represented on average about 9% of arsenic, while inorganic AsIII and AsV could only be detected in some samples and in most of cases was <0.1 µg·g−1.The sum of all arsenic species found was 62% ± 13% of the total arsenic for liver samples  and 81% R2 = 0.848) but poorer for liver (R2 = 0.403). For P. bovinus alone the correlations are even better (p < 0.05) are given between biometric variables and arsenic concentrations for all samples regardless the species using the Pearson’s coefficient of correlation.When the AB concentration in liver and in muscle of all three ray species was plotted against the weight of the animals investigated, a high correlation was found for muscle  from the Upper Gulf of California and De Gieter et al. [Raja clavata) from the English and Bristol Channel, both belonging to benthic species. Essumang [Manta birostris) from Ghana. The data from all three studies are included in M. aquila and P. bovinus) are comparable to the data of De Gieter et al. [R. clavata, although total arsenic concentrations in muscle samples of P. bovinus from our study reach very high values  [M. birostris have to be attributed to their diet,  consisting of plankton, known to contain less AB than higher marine organisms (see Introduction). The diet of the three species in our study is based on the analysis of food items in stomachs of the three studied species. All remains of living organisms found in the stomach were isolated, identified to the lowest possible taxonomical category, weighed and counted. The diet of Dasyatis violacea was already published [Dasyatis violacea) preyed mostly on fishes, living in the water column (predominantly anchovies) containing on average 0.43 µg·g−1 d.w. of As [Myliobatis aquila and Pteromlyaeus bovinus prey almost exclusively on benthic macroinvertebrates especially bivalves and gastropods, which contain between 2.4–33.0 µg·g−1 d.w. of As [In the literature there are very few data on arsenic concentrations in rays and none, to the best of our knowledge, for these particular species. Gutiérrez-Mejía et al.  studied r et al.  investigEssumang  reportedr et al.  for R. ch values . It coul  , the difublished , whereasw. of As  and to aw. of As ,37.et al. [The lower extractability of arsenic from liver resulting in underestimation of arsenolipids might be a logical consequence of the extraction procedure  but might also indicate the presence of entirely unknown non-extractable arsenic compound(s). According to Zeng et al.  we cannoet al. . This iset al. ,41. ThisSqualus acanthias) contains mainly AB  from the south Adriatic and almost no inorganic arsenic (0.5%–3.5%). The matrices of similar NRCC Certified Reference Materials DORM 1 and 2 consisting of Dogfish muscle contain mainly AB, some DMA and traces of AsC, TETRA and an unknown compound [No arsenic speciation data could be found in the literature for the same animals, but data for the liver samples of comparable marine predators show similar picture. For example, National Research Council Canada (NRCC) Certified Reference Material DOLT 3, Dogfish Liver  were higher than those in liver. Selenium was more uniformly distributed in levels up to 5 µg·g−1 d.w. and statistically correlated with liver levels of the total arsenic [While some authors could not find any correlation between the size of tuna and the arsenic concentration , other d arsenic .Mytilus galloprovincialis, filter-feeder consuming plankton and detritus from uncontaminated northern Adriatic Sea containing 14.7–29.9 mg·kg−1 As d.w. [M. galloprovincialis mentioned above was from 7.44 to 15.7 mg·kg−1 d.w. [Mytilus edulis) [−1 d.w.) while AB levels remained low (3.19–7.2 mg·kg−1 d.w.) thus we speculate that this might be related to the difference in in local food chain, uptake and retention of AB. Thus, AB is supposed to be absorbed in large amounts from diet, especially for benthic predators, and retained in muscle due to its similarity with the natural osmolyte (and methyl donor) glycine betaine, which is an important osmolite in cartilaginous fish [et al. [Since arsenate is taken up along the phosphate pathway it might be expected that its uptake would be higher in phosphate deficient environments like the northern Adriatic ,28, resu As d.w. , also po As d.w. . AB is s As d.w. ,52 and cg−1 d.w. ; It is i edulis) . In theious fish . Gailer  [et al.  reported [et al. . This is [et al.  while ot [et al. . High co [et al. . Larsen  [et al.  found th [et al.  while ot [et al. .−1), the high total arsenic in several invertebrates (2.4–33.0 mg·kg−1 d.w.) [M. galloprovincialis, 14.7–29.9 mg·kg−1 d.w.) [−1 d.w. of AB) [P. bovinus and M. aquila seem to be logically connected and explain the high AB levels in the fish investigated. Collected data clearly point to AB biomagnification throughout the bentic food chain.The AB concentration in muscle exhibits high correlations with the biometric parameters of the animals studied, indicating bioaccumulation of arsenobetaine in rays with age. In general it is believed that total arsenic does not accumulate along the food chain, as is well documented in the literature ,24. The −1 d.w.)  and in b−1 d.w.)  with abo. of AB) , which aet al. [For the determination of total arsenic concentration the most common methods include microwave digestion followed by ICP-MS measurement. In arsenic speciation extraction is followed by either HPLC separation or capillary electrophoresis, followed by element specific arsenic detection with either ICP-MS, MS-MS or HGAFS. Overview of analytical methods in use for arsenic speciation is given in Chen et al. . In our Pteromylaeus bovinus), 6 common eagle rays (Myliobatis aquila), both bottom dwelling benthic species from family Myliobatidae, and 8 pelagic stingrays (Pteroplatytrygon violacea) representing pelagic species from family Dasyatidae, were captured in the Gulf of Trieste, northern Adriatic Sea, during September and October 2005 as a part of a wider survey of these animals. The same specimens were previously analyzed for mercury and its chemical species [P. bovinus is a large ray found around the coasts of Europe and Africa. Mature males have a disk width (DW) larger than 95 cm, while adult females are even bigger (DW ≥ 120 cm). They are often 150 cm and sometimes up to 250 cm in total length (from snout to the end of the tail) and weigh up to 100 kg. The bull ray is generally a benthic dweller, but can also swim in surface waters and is known to live up to 14 years [M. aquila (Linnaeus 1758), the common eagle ray is distributed throughout the eastern Atlantic, including the North Sea, as well as the Mediterranean. In the Mediterranean Sea, M. aquila reaches a maximum size of 150 cm DW and 260 cm total length (TL) [P. violacea generally reaches around 60 cm DW. It almost exclusively inhabits the open ocean, is typically found in surface waters down to a depth of 100 m, but also in the Gulf of Trieste where the maximal depth does not exceed 25 m. Since it is a pelagic dwelling predator, it is an active hunter preying on squids and pelagic bony fishes [15 bull rays  until freeze-drying , −40 °C, 0.020 mbar). d.w. was determined and dry samples were homogenized in a planetary mill  using agate capsules and balls. The ground samples were tightly closed and kept in a refrigerator until analysis.All chemicals used were of at least p.a. quality and Milli-Q water (18.2 MΩcm) was used for preparation of all solutions. Arsenite (AsIII), arsenate (AsV), methylarsonic acid (MA), dimethylarsinic acid (DMA), arsenobetaine (AB), arsenocholine (AsC), trimethylarsine oxide (TMAO) and tetramethyl arsonium ion (TETRA) were used as standards and were prepared as described previously .2S2O8 in 3% NaOH), hydride generation and atomic fluorescence spectrometry determination as described earlier [Samples in duplicate portions were accurately weighed (0.15–0.25 g) into 50 mL polypropylene conical vials  and shaken overnight at 40 °C (muscle) or 50/40 °C (liver) with 20 mL of a mixture of methanol and water (9 + 1). After centrifuging  the extracts were decanted and the extraction repeated twice more. All three extracts were combined and evaporated on a rotary evaporator at 45 °C. Muscle residues were taken up in 5.00 mL of Milli-Q water, while liver residues were first defatted with diethyl ether (20–30 mL) and only then dissolved in 5.00 mL of water; the final aqueous extracts were then filtered  and kept frozen until speciation analysis. The diethyl ether fraction was transferred to an irradiation vial, evaporated to dryness and sealed. Total arsenic in extracts was determined using FI-UV-HGAFS method comprising of flow injection, on-line UV decomposition  step  was applied to convert non-volatile arsenic compounds into the corresponding volatile hydrides, which were detected in an Excalibur atomic fluorescence spectrometer . For the separation of cationic species  a Zorbax 300-SCX cation exchange column was used with 7.5 mM pyridine, pH 2.33 (adjusted with HCl) as mobile phase. In the case of cationic compounds the column eluent was subjected on-line to UV decomposition as described earlier [The chromatographic separation was performed under two different conditions. Anionic arsenic compounds  were separated on a Hamilton PRP-X100 anion exchange column using 15 mM KH earlier . Cationi3 and 1 mL of 30% H2O2, both suprapur quality, were added and the samples subjected to closed vessel microwave digestion  at max. power of 1500 W: ramp to 130 °C 10 min, ramp to 200 °C 10 min, hold 20 min, cooling 20 min. The same procedure was applied for blank samples and Standard Reference Materials. Digested solutions were diluted to 20 mL. Measurements of aliquots of digests were made by an Octapole Reaction System (ORS) Inductively Coupled Plasma Mass Spectrometer  equipped with an ASX-510 Autosampler . The instrumental conditions were set as follows: Babington nebulizer , Scott-type spray chamber , spray chamber temperature 5 °C, plasma gas flow rate 15 L·min−1, carrier gas flow rate 0.8 L·min−1, make-up gas flow rate 0.1 L·min−1, sample solution uptake flow rate 1 mL·min−1, RF power 1500 W, reaction cell gases helium or hydrogen 4 mL·min−1. Tuning of the instrument was done daily.About 0.2 g of muscle or liver sample was weighed into a quartz tube. 4 mL of 65% HNO1/2 = 26 h) was measured on an HP Ge detector for 1–3 min using the 559 keV γ-ray peak. Detection limits were in a range of ng·g−1 d.w.Dried diethyl ether fractions, containing lipidic fraction of arsenic, were irradiated in the Institute’s Triga MARK II nuclear reactor together with standard solutions, cooled for 3–4 days and 76As -HGAFS method the DORM 3 was analyzed together with samples, and the results  agreed well with the literature data for this SRM [−1, for AsV and DMA 1 and for AB 25 ng·g−1 of extract corresponding to 0.01, 0.03 and 0.62 µg·g−1 g d.w., respectively, all expressed as arsenic; quantification was possible at 3 times higher concentrations.Along with samples Standard Reference Materials were analysed with the same procedures. Total arsenic concentration determined with ICP-MS in DORM 3 (Dogfish Muscle) was 6.8 ± 0.5  and in DOLT 4 (Dogfish Liver) 9.7 ± 0.6 , all in µg·gthis SRM . DetectiHigh arsenic concentrations in liver and especially in muscle of all three ray species studied were found.Pteromylaeus bovinus, Myliobatis aquila) in comparison with the pelagic one (Pteroplatytrygon violacea).Higher arsenic levels were observed in muscle of both benthic species  our finding of high AB levels point on favorable retention of glycine betaine and coincidentally also AB for the purpose of osmoregulation; this hypothesis needs further investigation. In fact we are missing basic biological  data for particular ray species.


Microbacterium sp. strain HM58-2, which produces hydrazidase, an enzyme hydrolyzing acylhydrazides. The estimated genome size is 3.9 Mb. Genome sequence information of this strain will help to identify an assimilating mechanism of nonnatural compounds in this strain and to develop ecological applications.We report the draft genome sequence of  Microbacterium sp. strain HM58-2 has been isolated from soil to utilize an acylhydrazide, 4-hydroxybenzoic acid 1-phenyletylidene hydrazide (HBPH). This strain produces intracellular hydrazidase, which is a member of the amidase family and is considered to be a key enzyme for hydrazide catabolism , and 3 µg DNA were fragmented to 500 bp using the Covaris S2-A system . A library for sequencing was prepared with a NEBNext DNA sample prep master mix set for Illumina  and NEXTflex PCR-free bar codes . Raw reads were trimmed and de novo assembled using CLC Genomics Workbench version 6.0.5 . Parameters for the trimming were as follows: ambiguous trim, yes; quality trim, yes; quality limit, 0.05; search on reversed sequence, no; save discard sequence, no; remove 5′ terminal nucleotides, no; discard short reads, no; remove 3′ terminal nucleotides, no; trim adapter list, new trim adapter list; discard long reads, no; and same broken pairs, no. Parameters for the de novo assembly were as follows: masking mode, no masking; mismatch cost, 3; insertion cost, 3; deletion cost, 3; length cost, 3; length fraction, 0.5; similarity fraction, 0.8; global alignment, no; and auto-detect paired distances, yes.Genomic DNA of Microbacterium sp. strain HM58-2 was assembled into 39 contigs, with an accumulated length of 3,792,788 bp  and an average GC content of 70.1%. The longest contig was composed of 1.84 Mb, and only the five longest contigs made up a length of 3.46 Mb. The genome was annotated by the Microbial Genome Annotation Pipeline (MiGAP) version 2.19 (M. testaceum StLB037. Morohoshi et al. reported the complete genome sequence of M. testaceum StLB037 comprising 3.98 Mb with 3,532 CDSs and an average GC content of 70.28% (The draft genome of Since the expression of the hydrazidase gene is under catabolite repression, where it is induced by substrates, such as HBPH, as sole carbon source and repressed by the existence of glucose . TherefoBDCY01000001 to BDCY01000039.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession numbers


Journal of Abnormal Psychology, 2014, Vol. 123, No. 3, pp. 566–577. http://dx.doi.org/10.1037/a0037228) was missing the acknowledgement of the funding source in the author note. The work in this article was supported by the Economic and Social Research Council: ERSC (grant number ES/J00751X/1). The online version of this article has been corrected.The article “The Effect of Disgust and Fear Modeling on Children’s Disgust and Fear for Animals” by Chris Askew, Kübra Çakır, Liine Põldsam, and Gemma Reynolds (


Capsular outcomes of anterior/posterior capsulorhexis opening (ACO/PCO) are essential for performing a secondary in-the-bag intraocular lens implantation. To compare the capsular outcomes with different primary capsulorhexis sizes, Thirty-eight eligible patients (45 eyes) were randomly assigned to three groups by anterior capsulorhexis diameter . The areas of ACO/PCO and posterior capsule opening opacity (PCOO) as primary outcomes, while, the incidence of visual axis opacity (VAO) as secondary outcome were measured at follow-up visits. Among the thirty eyes included in the final analysis, the mean area of the ACO decreased significantly, whereas the PCO enlarged with time. Group A had the highest anterior capsule constriction and percentage reduction, which increased with time. There were significant differences in the percentage reductions at 6 months and 1 year compared to 1 month in Group A and B. Group C had the highest posterior capsule enlargement. The percentage of PCOO to PCO area and the incidence of VAO was highest in Group A and lowest in Group C. Thus, Capsulorhexis diameter of 4.0–5.0 mm may yield better capsular outcomes, considering moderate contraction of ACO, moderate enlargement of PCO, and lower percentage of PCOO and VAO. Pediatric cataract is one of the common cause of childhood blindness and are responsible for approximately 10–20% of blindness in children worldwide245It is well known that the pediatric eye is more reactive after cataract surgery891213In total, 8 patients dropped out after inclusion. Three patients (4 eyes) who were unable to complete the designed follow-up, 4 patients (6 eyes) whose retroillumination images could not be analyzed due to the small size of their pupils or iris synechiae and 1 patient (1 eye) with a high intraocular pressure after surgery were excluded from the analysis. Additionally, 2 eyes in Group A, 1 eye in Group B and 1 eye in Group C received Nd: YAG laser capsulotomy due to serious VAO during the follow-up . Therefo2 at baseline and postoperative 1 month, 6 months, and 1 year, respectively. The ACOs of Group B were 15.22 ± 1.18, 13.43 ± 0.97 (p = 0.007), 12.79 ± 1.00 (p = 0.003), and 12.41 ± 1.09 (p = 0.002) mm2, respectively; those of Group C were 22.92 ± 1.45, 20.86 ± 0.87 (p = 0.028), 20.49 ± 1.04 (p = 0.014), and 19.88 ± 0.81 (p = 0.020) mm2, respectively. For all three groups, the mean area of the ACO decreased significantly postoperatively  , 9.08 ± 0.53 (p = 0.005), and 9.00 ± 0.85 (p = 0.001) mms above) .2 at baseline and postoperative 1 month, 6 months, and 1 year, respectively. The PCOs of Group B were 7.26 ± 1.21, 7.75 ± 1.35 (p = 0.078), 8.29 ± 1.21 (p = 0.045), and 8.53 ± 1.25 (p = 0.033) mm2, respectively, and those of Group C were 10.37 ± 2.91, 11.29 ± 2.49 (p = 0.086), 12.07 ± 2.86 (p = 0.001), and 12.46 ± 2.95 (p = 0.005) mm2, respectively. For all three groups, the mean area of the PCO increased postoperatively , 8.00 ± 0.66 (p = 0.185), and 8.22 ± 0.74 (p = 0.101) mmratively .The percentage decreases of the ACO in Group A at 1 and 6 months and 1 year after surgery were 16.97 ± 1.93, 25.26 ± 2.19, and 26.04 ± 2.85%, respectively; in Group B, the respective values were 12.92 ± 1.73, 15.95 ± 0.60, and 17.74 ± 1.09%; and in Group C, the respective values were 8.88 ± 2.17, 10.55 ± 1.66, and 13.18 ± 2.60%. Group A had the highest capsule constriction, and there was a significant difference among the three groups at the various time points . The percentage reduction in the ACO area increased with time, and the differences between 6 months, 1 year and 1 month were significant in Group A  and Group B . The differences among the various visits in Group C were not significant  .The percentage increases in the PCO in Group A at 1 and 6 months and 1 year after surgery were 1.12 ± 6.63, 5.93 ± 5.13, and 8.80 ± 4.98, respectively; the respective values in Group B were 6.67 ± 3.35, 14.52 ± 5.95, and 17.84 ± 6.24; and the respective values in Group C were 11.94 ± 5.44, 17.59 ± 6.26, and 21.45 ± 6.73. Group C had the highest posterior capsule enlargement compared with the other two groups, although the differences did not reach statistical significance . Furthermore, there was no significant difference among the various postoperative visits in any of the groups .Overall, Group A had the highest capsule constriction and percentage reduction, which increased with time. There was a significant difference in the percentage reduction between 6 months, 1 year and 1 month in Group A and Group B (*p < 0.05). Group C had the highest posterior capsule enlargement, although the difference did not reach statistical significance.The percentage of PCOO was highest in Group A compared with Groups B and C . There was a significant difference between 6 months and 1 year after surgery in Groups A, B and C  investigated the capsular outcomes of pediatric cataract surgery performed with different anterior capsulorhexis sizes. We found that a diameter of 4.0–5.0 mm yielded optimal capsular outcomes, based on its relatively moderate contraction of ACO, moderate enlargement of PCO, and lower percentage of PCOO and VAO. To the best of our knowledge, this is the first RCT to investigate the relationship between the primary capsulorhexis size and capsular outcomes after pediatric cataract surgery.Currently, primary anterior and posterior capsulectomy and anterior vitrectomy are the main surgical interventions for pediatric cataracts, particularly among young children48et al.et al.et al.Several devices have been reported to control the diameter and shape of capsulorhexis, such as capsulotomy diameter marks, ring-shaped calipers, or the data-injection system171819et al.As opposed to the ACO, for the initial PCO, a 3.0-mm diameter capsulorhexis was well accepted and had a tendency to widen. Michael The results and interpretation of the current study must be understood within the context of its strengths and limitations. The strengths of the study include its randomized controlled design, the performance of the surgeries by a single experienced pediatric cataract surgeon using radiofrequency diathermy with a self-made needle calibration that ensured accuracy of the three controlled groups at baseline, and the investigation’s adherence to a unified and strict postoperative regimen and follow-up protocol within the same study group (CCPMOH). The weaknesses of the study must also be acknowledged. The sample size of the study groups was relatively small, and the results were most likely slightly biased by the exclusion of patients with serious iris synechiae and patients whose pupils could not dilate after surgery. This study only evaluated aphakic rather than pseudophakic eyes, and our findings could not be applied to pseudophakic eyes. However, the aim of this paper is to investigate the relationship between primary capsulorhexis size and capsular outcome of aphakic patients and eventually provide the theoretical basis to the successful in-the-bag IOL implantation at the second surgery. We employed a self-made calibrated needle in the chamber to measure the size of the capsule openings during surgery, but these measurements were made externally and postoperatively. Because of corneal magnification, immediate postoperative readings would be larger than intraoperative readings, yielding another potential bias. Despite these limitations, this study remains one of the first to investigate the relationship between the primary capsulorhexis size and capsular outcomes after pediatric cataract surgery.There are important potential implications for our present findings. Our research has demonstrated that an ideal capsulorhexis size for pediatric cataracts not only would decrease the rates of capsular complications, including capsule contraction and VAO, but also would increase the probability of successful in-the-bag IOL implantation at the second surgery. We are currently following the pediatric patients from our study to assess their rates of accurate in-the-bag IOL implantation.https://www.clinicaltrials.gov, registration number: NCT02158325, First received date: June 4, 2014). The authors confirm that all ongoing and related trials for this study are registered. Patients with glaucoma, ocular trauma, corneal disorders, persistent hyperplastic primary vitreous, rubella, Lowe syndrome, capsular fibrosis, or surgical complications, as well as those whose pupils could not dilate normally postoperation or who could not complete the follow-up, were excluded. Three patients were excluded, and thirty-eight patients (45 eyes) were included in this study  between March 2011 and November 2012 as registered members of the Childhood Cataract Program of the Chinese Ministry of Health (CCPMOH)is study .Preoperatively, all patients were randomly divided into 3 groups  using a random number chart and underwent cataract surgery without IOL implantation. The patients in Group A received a small anterior capsulorhexis that was 3.0 mm to 3.9 mm in diameter, Group B received a medium capsulorhexis that was 4.0 mm to 5.0 mm in diameter, and group C received a large capsulorhexis that was 5.1 to 6.0 mm in diameter.All patients underwent cataract surgery performed by a single experienced pediatric cataract surgeon (YZL) using a technique that has been previously describedPostoperatively, Tobradex eye drops  were used 6 times per day, and Tobradex eye ointment  was applied once per night for 2 weeks. From 2 weeks to 1 month postoperatively, the eye drops were used 4 times per day. For the second postoperative month, the patient switched to pranoprofen eye drops 4 times per day . This follow-up protocol was also used in our previous study. All patients were examined at 1 week, 1 month, 3 months, 6 months, 1 year, 18 months, and two years after surgery. Patients were followed for two years or until the development of severe VAO requiring Nd: YAG laser capsulotomy, whichever came first. The examinations consisted of visual acuity, IOP measurement by a Tonopen tonometer , fundoscopy, an assessment by high-resolution digital retroillumination imaging (detailed protocol presented below).All of the postoperative pediatric patients underwent pupil dilation with compound tropicamide eye drops  3–5 times (once every 10 minutes) until the pupil size was larger than 6 mm. Then, the children underwent slit-lamp-adapted anterior-segmental photography  for each operated eye, including one digital coaxial retroillumination photograph (DCRP), one diffuse light photo and one slit-light photo across the central visual axis. For uncooperative children, a set of assistance procedures for slit-lamp-adapted photography was developed to instruct the parents to help prepare the child’s head position and to help the child open his or her eyes as described in our previous studies11The postoperative capsular DCRPs were divided and defined as follows: area of the ACO, area of the PCO and area of the posterior capsule opening opacity (PCOO) . The ACOThe primary outcomes were the areas of the ACO and PCO at different postoperative follow-up visits. The secondary outcomes were the percent reduction or increase in the ACO, PCO and PCOO at different postoperative follow-up visits and the total incidence of VAO. We also assessed surgical complications, such as capsular tears, inflammation, corneal edema, glaucoma, iris synechiae and postoperative macular edema. The surgery was video recorded, and pictures of the completed ACO and PCO were taken intraoperatively. Both the intraoperative and postoperative pictures were standardized and documented on an analysis computer using our ECO software, a custom image analysis algorithm developed by our team using MATLAB . The contours of the ACO, PCO and PCOO were traced by hand using the ECO image processing software. Next, the capsule opening area, capsulorhexis diameter and opacity area were measured by computer. Two researchers independently used the software to quantify the values of the ACO, PCO and PCOO, and the results are the mean values determined by the two researchers. The mean values of the ACO, PCO and PCOO of each capsular type were calculated. The mean areas of the PCOO were analyzed at 6 months and 1 year. The percentage of PCOO was calculated as follows: PCOO area × 100/PCO area at the same time. If the patient underwent the Nd: YAG laser capsulotomy or a secondary surgical intervention due to serious VAO, the percentage of PCOO was recorded as 100%.The SPSS software package  was used for statistical analysis. The sample size calculation was based on power analysis. Power analysis adopts a hypothesis-testing method to determine the sample size according to several parameters, which include the pre-specified significance level, desired power level and expected effect size. Assuming a two-tailed alpha of 0.05, a probability of 0.2 for beta error (80% power) and an effect size of 0.28 after calculating with respect to the same primary outcome measure (area of the ACO) from the result of our preliminary research, 10 participants per group were required. A paired T test was used to compare the areas of the ACO and PCO between the postoperative visits and baseline. ANOVA was used for changes in the ACO and PCO areas among the three groups at different postoperative visits. Due to the non-normal distribution, Kruskal-Wallis nonparametric tests were used to compare the area percentage of PCOO among the three groups, and the Wilcoxon signed rank test was used to compare the difference between the postoperative visits. All statistical tests were two-tailed, with α = 0.05. A p value of <0.05 was considered statistically significant.How to cite this article: Lin, H. et al. Capsular Outcomes Differ with Capsulorhexis Sizes after Pediatric Cataract Surgery: A Randomized Controlled Trial. Sci. Rep.5, 16227; doi: 10.1038/srep16227 (2015).