Networks of marine protected areas (MPAs) are being adopted globally to protect ecosystems and supplement fisheries management. The state of California recently implemented a coast-wide network of MPAs, a statewide seafloor mapping program, and ecological characterizations of species and ecosystems targeted for protection by the network. The main goals of this study were to use these data to evaluate how well seafloor features, as proxies for habitats, are represented and replicated across an MPA network and how well ecological surveys representatively sampled fish habitats inside MPAs and adjacent reference sites. Seafloor data were classified into broad substrate categories (rock and sediment) and finer scale geomorphic classifications standard to marine classification schemes using surface analyses  done on the digital elevation model derived from multibeam bathymetry data. These classifications were then used to evaluate the representation and replication of seafloor structure within the MPAs and across the ecological surveys. Both the broad substrate categories and the finer scale geomorphic features were proportionately represented for many of the classes with deviations of 1-6% and 0-7%, respectively. Within MPAs, however, representation of seafloor features differed markedly from original estimates, with differences ranging up to 28%. Seafloor structure in the biological monitoring design had mismatches between sampling in the MPAs and their corresponding reference sites and some seafloor structure classes were missed entirely. The geomorphic variables derived from multibeam bathymetry data for these analyses are known determinants of the distribution and abundance of marine species and for coastal marine biodiversity. Thus, analyses like those performed in this study can be a valuable initial method of evaluating and predicting the conservation value of MPAs across a regional network. Human impacts on the oceans continue to increase , 2, 3, 4One major consideration when designing a network of MPAs for the purpose of conserving biodiversity and ecosystems is the representation of habitat and the ability to capture the diversity and heterogeneity of habitat features that support biodiversity , 16, 26.in situ and remotely sensed, has emerged as a much needed tool to determine the level of representation of the different habitat types inside and outside MPAs [in situ observations such as SCUBA or remotely operated vehicles (ROVs) are widely utilized but are limited in their depth ranges and the ability to efficiently sample large areas [Seabed mapping, both ide MPAs , 29, 30.ge areas . With thge areas , 29, 34.Products produced from remotely-sensed data have become fundamental to many applications of coastal marine science . Maps geThe state waters of California offer a unique opportunity to develop methods to assess the representation of habitat or habitat surrogates inside and outside of MPAs across a regional network and the replication of those habitats. Not only has California adopted the Marine Life Protection Act (MLPA), which produced a statewide network of MPAs that extends along the 1200 km of coastline from the Oregon border to the Mexico border and out to the three nautical mile limit of state waters , they alThe Central Coast Region of California is a particularly valuable example for evaluating the representation of habitats across the network of MPAs created by the MLPA design process. The Central Coast Region was the first section of the statewide network to be designed and preceded the high resolution seafloor mapping conducted by the CSMP. During the design of the Central Coast Region, seafloor information was incomplete and in the absence of comprehensive seafloor mapping, a variety of proxies were used including aerial images of the surface canopy of kelp forests as proxy for nearshore rock, and rockfish “fishing areas” based on recreational and commercial fishing landings as a proxy for offshore rocky reefs. Analysis of seafloor maps eventually produced in the central coast provides an opportunity to evaluate how well those proxies accurately represented the distribution and types of seafloor features, both across the region and within the MPAs created in that region.http://www.dfg.ca.gov/marine/mpa/centralcoast_rsg.asp). Stakeholders designed the network of MPAs based on ecological guidelines  provided them by a science advisory team, but were also allowed to consider socio-economic consequences of their design. As such, in the Central Coast and other regions of the statewide network, the design of individual MPAs and the network deviated from that prescribed by the science guidelines. Although habitats were identified for representation  capture the habitat types designated for protection by the MLPA design process?How well are MLPA habitats replicated throughout the region (i.e. is there sufficient habitat within MPAs to contribute to population connectivity)?How well are classes of geomorphological features based on finer scale topographic metrics, which were not considered when the MPAs were designed, represented across the MPA network?Does the kelp forest monitoring program representatively sample the diversity of seafloor structure inside the MPAs?How well does seafloor structure within reference sites outside MPAs match those sampled within the MPAs?The null hypotheses are that relative amounts of habitat within the MPAs are representative of  the region, habitats are adequately replicated with sufficient habitat area based on the guidelines in the MLPA, and that the kelp forest monitoring program has adequately captured all available habitats in their monitoring design. Therefore, we test for deviation from these hypotheses.The purpose of this study is to use multibeam bathymetry data acquired by the CSMP and the baseline monitoring data acquired by PISCO to evaluate the distributions of habitat defined in the MLPA process in the Central Coast MPA network and the representation of reef structure in the kelp forest monitoring design. To evaluate the placement of MPAs within the Central Coast network, we classified the seafloor data from the CSMP into potential habitat variables of known importance to many species  and used this information on seafloor structure to answer the following questions for the region:http://www.dfg.ca.gov/marine/mpa/ccmpas_list.asp). This region extends from Pigeon Point in the north (37°10′57“ N 122°23′38” W) to Point Conception in the south (34°26′55” N 120°28′14” W) and consists of a network of 29 MPAs with differing levels of protection . Each of the cells in these DEMs were classified into the MLPA habitat categories by first assigning “sediment” or “rock” substratum types based on thresholds of ‘ruggedness’ and then breaking them up by specified depth ranges  rocky reef and sandy bottom habitat, and the area (km2) of deeper (30–100m depth) rocky reef and sandy bottom. Replication guidelines were only available for the 0–30m and 30–100m habitats so only four MLPA habitats within those depth zones were assessed for replication. The linear distance measurements for the kelp forest habitat were derived from maps of the maximum coverage of kelp using LANDSAT data [Deviations from proportional representation of habitat types in MPAs is particularly problematic if there is insufficient representation of a habitat within MPAs to achieve replication . If individual MPAs are found to contain an inadequate area of habitat, they may not contribute to the network . GuideliSAT data , 57. LinSAT data . Thus, wSAT data .As specified in the MLPA, substrate (i.e. rock or sediment) was deemed an important habitat factor affecting the distribution of species. However, habitat structure, including habitat complexity and heterogeneity, has also been shown to affect variation in fish population size and assemblage structure , 60, 61.Once we classified rocky reef into distinct geomorphic classes, we tabulated the area of each of those classes for both the regions and for MPAs within the region to determine representation of those geomorphic classes across MPA networks. We then compared the proportionality of each of these fine-scale geomorphic classes across the region to the proportions within the MPAs to see how well these classes represented their availability throughout the region. Again, we used a 20% difference threshold to determine if there were significant differences between the percentages of each of the habitat classes found throughout the region compared to the percentages observed in the MPAs . We rejeBecause species assemblages vary across habitat types, it is important to design MPA monitoring programs to incorporate representative habitat inside and outside of MPAs so that any differences are not confounded by the sampling of different proportions of habitat types. We assessed habitat representation in the kelp forest monitoring dataset, which is only one of the studies used for baseline characterization of the Central Coast MLPA region. The kelp forest monitoring program (PISCO) relies on divers and, therefore, samples habitats in depths shallower than 20 meters. This analysis provides an example of how seafloor data can be used to help stratify monitoring locations across variations in rocky reef.http://www.piscoweb.org/research/science-by-discipline/ecosystem-monitoring/kelp-forest-monitoring/subtidal-sampling-protoco>. We then placed a 10m buffer around each transect to incorporate the spatial uncertainty in the exact location of that transect, with the assumption that each transect was conducted within that 10m buffer. In addition to the PISCO transects, we created 3,250 transects with the same dimensions as the diver transects that we placed randomly throughout the region in rocky reefs at comparable depths of the kelp forest surveys. These transects allowed us to characterize variation in rocky reef across the region and within MPAs to determine the availability of fine-scale features, which could then be used to assess how well the kelp forest monitoring surveys representatively sampled these features.To determine the habitat representation in the kelp forest monitoring design, we used GPS waypoints along with the initial diver recorded compass heading for the kelp forest survey transects to estimate the location where the transects were conducted in ArcGIS. To do this, we created a 100m polyline from the waypoint utilizing the initial heading of the diver and adjusted to the bathymetric contours, as specified in the PISCO sampling protocols <Once we created the buffered survey and random transects, we quantified the structure of the rocky reef within each transect using the same fine-scale geomorphic categories that were used for the geomorphic classification. After quantifying the reef structure within each of those transects, we used a cluster analysis within the statistical software package Primer to designate separate classes. Once clustered, we searched the dendrogram for the appropriate merging distance to categorize transects into a number of reef structure classes. Although the MLPA did not take into consideration this scale of variation across rocky reef, these reef structure classes were defined by certain combined characteristics of the rocky reef that were previously shown to be important to the distributions of fish , 36, 49.Across the Central Coast Region, abundance of the four MLPA-designated habitat categories in the 0–100m depth range (based on substrate classification derived from the CSMP data) were similar to the MLPA-predicted habitat from the best available data during the MLPA design process . None ofAlthough the best data available when setting up the MPAs provided good region-wide estimates of percent cover of habitat in shallow depth zones, the CSMP data revealed that, at the scale of individual MPAs, there were some very large differences in the MLPA-predicted percentage of habitat and the CSMP-derived habitat estimates across all depth zones. The percentage deviation between the MLPA-predicted and CSMP-derived substrate coverage of rock and sediment ranged from 0.2% to 332.8% across the Central Coast MPAs with an average deviation of 39.1% . The PieThe CSMP-derived classifications of the MLPA habitat were also used to determine the number of replicates of four of the habitat classes contained within the Central Coast network of MPAs. Three of the four habitat classes are adequately replicated within the MPAs as specified in the MLPA guidelines . Based oThe relative abundance of geomorphic classes based on fine-scale seafloor structure metrics within the MPA network were close approximations of the regional availability of these features for the majority of habitat categories . The devThe cluster analysis of the habitat within the surveyed and our randomly generated transects produced six distinct reef structure classes that were defined by distinctive combinations of geomorphological characteristics. Each covered a certain percentage of the region: low complexity (8%), low to moderate complexity (5%), moderate complexity (26%), moderate to high complexity (11%), high complexity (9%), high complexity on slope (42%). These reef structure classes were significantly clustered at close distances. To reduce the number of unique classes to a number of categories more amenable to the sample size of kelp monitoring transects, a Bray-Curtis dissimilarity distance of 1000 was used. This cutoff value allowed for the incorporation of slightly outlying clusters to be merged into larger clusters without forcing dissimilar clusters to become combined.Using these reef structure classes, we analyzed the percentage of each class found throughout the Central Coast MPAs and compared those percentages to the percentage of baseline monitoring transects that fell in those classes to determine how well the surveyed transects sampled the rocky reefs representatively within the MPAs. In most MPAs, the monitoring sites did not sample the reef structure classes proportionately . In someUsing the same reef structure classes from the cluster analysis that were used to evaluate the representation of MPA habitat in the PISCO monitoring design, we compared representation between the MPA sites and their corresponding reference sites. Habitat classes were sampled disproportionately between MPAs and reference sites in all thirteen MPAs . For exaResults of this study provide an assessment of how well habitat categories targeted for protection by California’s MLPA are represented and replicated across the network of MPAs along the central coast of California. During the designation of these MPAs, there were limited data on the availability (i.e. amount and distribution) of habitats across the region. Since the completion of the California State Mapping Program (CSMP), an unprecedented dataset is now available that allows for the detailed delineation of the habitat categories used in the design of the network, as well as fine-scale geomorphic features. This assessment of habitat representation is a first step in evaluating how well the currently designated network of MPAs representatively include the habitat categories used to achieve representation of ecosystems and their associated biodiversity in the Central Coast Region of the statewide network.At the spatial scale of the Central Coast network of MPAs, our assessment of how well the current network of MPAs representatively (i.e. proportionately) captures the habitat types designated for protection by the MLPA design process indicates mixed results depending on depth. Therefore, we rejected the hypothesis of representation of some habitat categories and not others. For the shallower MLPA depth classes (0–30m and 30–100m), our results indicate that the distribution and abundance of the designated MLPA habitat categories were successfully represented in the network of MPAs in proportion to their availability in the region. On the other hand, at depths below 100m there were significant discrepancies (> 20%) between the MLPA-predicted and the CSMP-derived estimates of both rocky reef and sediment habitat representation. It is likely that proxies of habitat type such as the availability of relatively well-mapped kelp forests and local fishermen knowledge provided a good basis for the distribution of habitat categories (i.e. rocky versus soft bottom by large depth zones) in the shallow nearshore, but less so in deeper waters. One reason for the bigger discrepancies between MLPA-predicted and CSMP-derived habitat coverage in deeper depth zones could be the coarse scale data used in the MLPA-predicted habitat maps to define the rock and sediment habitats in the deeper depth zones . These cThe analysis to determine how well regional geomorphic classes based on finer scale topographic metrics, which were not considered when the MPAs were designed, showed that there was good representation within the Central Coast network of MPAs for the more abundant classes. On the other hand, the fine-scale geomorphic classes that were rarer throughout the region were not sufficiently represented. It is somewhat surprising that the finer-scale categories of reef structure were so well represented in the shallow depths, given the lack of knowledge of these features from traditional sources. One implication of this result is that the finer scale geomorphic classes are sufficiently ubiquitous such that simply capturing hard bottom was sufficient to ensure representation of these higher resolution seafloor structures as well. In addition, variation in the abundance of the fine-scale categories appears to occur at large geographic scales. Therefore, by distributing these large MPAs across the broad geographic variation in coarse-scale habitat categories (i.e. rock and sand at each depth zone) these finer scale geomorphic classes were representatively included in the network.2) required of a replicate of this habitat relative to the typical size of MPAs across the network despite the abundance of this habitat in the region.When the substrate cover in the MLPA-predicted habitat maps was compared to the substrate coverage in the CSMP-derived habitat maps at the scale of individual MPAs there are much larger discrepancies in the representation of habitat, such as the under-representation of rock in the MLPA-predicted substrate maps of the Piedras Blancas MPA . The undWe considered only finer scale features and geomorphic variation across rocky reefs, not the soft bottom. However, more studies have also identified ecologically important variation in sediment habitats. For example, rippled scour depressions (RSDs) are features that contain coarser grained sediment and are depressed relative to the surrounding sediment . Davis eBecause of the inability of traditional, vessel-based seafloor mapping to collect data in the shallow (0–5m) surf zone in the nearshore environment along the coast, the use of seafloor data to estimate the distribution of habitats may be an under-representation of the rocky reef in the 0–30m depth class. Seafloor mapping is usually terminated at shallower depths due to navigational hazards such as the presence of emergent rocks, thick kelp canopy, or unsafe wave environment. Most of these impediments to surveying are usually indicators of subsurface rock. Therefore, the benthic maps for the state waters often end slightly offshore from the coastline in many areas where there is most likely rocky habitat. The development of new sampling methods and platforms that allow mapping of these shallow nearshore habitats, or analytical tools that allow for accurate extrapolation of adjacent habitat into these zones, is critical because of the abundance and diversity of species in these habitats and the ecosystem functions and services they produce . UnfortuDensities of most marine species are typically correlated with seafloor features . TherefoUsing metrics derived from seafloor maps to evaluate MPA design has fundamental limitations. This analysis focused on a small subset of variables (geomorphology and depth) that contribute to the characterization of habitat. However, habitat is ultimately defined by the collection of organisms associated with it and seafloor structure is only one set of variables that can influence the distribution and abundance of species across the region. Oceanographic variables, such as currents, water temperature and nutrients that characterize areas of coastal upwelling, riverine input, or differing levels of swell exposure can greatly influence the composition of species that constitute ecosystems. Similarly, biogenic habitat structure such as kelps and corals also contribute to variation in species assemblages that define habitats , 51. UltOur application of spatially explicit geomorphic metrics derived from seafloor maps proved to be a valuable approach in evaluating the design (habitat representation and replication) of the MPA network along the central coast of California. We found that how well habitat representation and replication was achieved differed by spatial scale and depth. Habitats were best represented and replicated at shallower depths at the scale of the entire network, and were not as well achieved at the scale of individual MPAs and at deeper depths. We also found that the spatial design of ecological monitoring programs (kelp forest surveys) designed to evaluate ecological responses to MPA establishment would have benefited greatly by the existence of high resolution seafloor maps when those surveys were designed. As marine conservation continues to move in the direction of ecosystem-based management and the designation of marine protected areas (MPAs), there is a need for methods that facilitate the design of MPA networks and to evaluate the likelihood of their design in meeting their conservation and management goals . BecauseS1 File(DOCX)Click here for additional data file.


A seven-year-old blue-necked male ostrich was found dead after a few days of illness. The animal was living in an open yard of 25 square meters along with three other females. They were given concentrate-rich ration with free access to green leaves and water. Autopsy revealed cardiac enlargement due to left ventricular hypertrophy and right ventricular dilatation. The left aterioventricular valves were irregularly thickened and contracted. The lungs were engorged with blood and the liver had nutmeg appearance. The small intestine showed segmental sub-serosal petechial hemorrhages. Histological examination revealed myxomatous degeneration of the left aterioventricular valves, pulmonary congestion and edema, congestion of periacinar hepatic zone and fatty degeneration of outer zones, renal glomerulosclerosis and arteriosclerosis. The affected parts of the small intestine showed villous atrophy with lacteal distention. The venules in the affected intestinal segment were severely dilated while the arterioles had narrow lumen and irregular wall thickening with hyaline deposition. The current article reports an endocardiosis in ostrich and discusses other vascular disorders. Endocardiosis is a well-known cardiac disease, which affects certain breeds of dogs. It is a myxomatous degeneration of the valves, which mainly involves the left aterio-ventricular valves leading to malformation and mitral insufficiency.Other cardiac valves are also affected, but with less severity and frequency. The affected valve is shortened and thickened, opaque white with smooth surface and no signs of inflammation. Microscopically, the most prominent features of endocardiosis are thickening of the valvular spongiosa and degeneration of the fibrosa.et al.,1997; Jubb et al., 2008; Kittleson, 2011; Fox, 2012). The cause of endocardiosis is not known, but is likely a genetically-influenced degeneration of connective tissue.The thickening of the former is due to proliferation of loose fibroblastic tissue rich with proteoglycans, hyaluronic acid and chondroitin sulfate , horses , pigs , Gambian rats , rhesus monkeys  and in aviary birds . This condition bears some resemblance to a condition in human referred to as mitral valve prolapse . The current paper describes and discusses a condition in a captive ostrich, which corresponds well with endocardiosis in other animals.Though infrequent, valvular abnormalities suggestive of endocardiosis have also been reported in cats , dehydrated in rising concentrations of Ethyl alcohol, cleared in Xylol and embedded in paraffin wax. Sections of 5-6 microns were stained with Hematoxylin and Eosin (H&E). Alcian blue was used to stain mucopoly-saccharides in the affected valves and Crystal violet for detection of amyloidosis in the arterioles of the intestine and myocardium.The main necropsy findings included cardiac enlargement due to left ventricular hypertrophy and right ventricular dilatation .The left ventricle had thick muscular wall, prominent papillary muscles and reduced lumen. The mitral valve leaflets were unevenly thickened, fleshy and shrunk .The wall of the right ventricle was thin and flabby. The left atrium was moderately dilated, but jet lesions were not seen. The lungs were severely congested and oozed blood from cut surfaces .The trachea contained bloody froth. The liver was enlarged and showed typical nutmeg appearance .The kidneys were also swollen and congested. The small intestine contained little food and showed sub-serosal petechial and ecchymotic hemorrhages on a segment measuring about 25 cm . Marked On microscopy, the valves showed extensive myxomatous degeneration formed of loosely arranged stellate fibroblast and mucopolysaccharide-rich ground substance  and 7.The pulmonary vessels and capillaries were severely congested with accumulation of transudate in air capillaries . The hepA considerable number of renal glomeruli showed variable extents of hypercellularity, sclerosis and capsular adhesion. The renal arterioles in many instances had onion like thickening of their walls and narrow lumen .Septic mural thrombi were observed in many medium-sized vessels, while homogenous micro-thrombi were noticed in many capillaries . The affSome arterioles of the cardiac muscle have also shown such changes .Amyloidosis was ruled out by the negative reaction of Crystal violet.Necropsy and microscopic examination have illustrated that the most possible cause of death, regardless of other recorded pathological abnormalities, is pulmonary failure. Those abnormalities, however, have certainly participated in deteriorating the general body condition and enhanced death.The pronounced pulmonary congestion and edema which led to fatal flooding of air capillaries were obviously secondary to cardiac disorder and are probably augmented by the natural sporadic attenuation of the blood - air barrier in ostrich lungs .et al., 2008).Left ventricle hypertrophy and associated pulmonary congestion encountered in this bird can well be explained on the basis of incompetence in the mitral valve. In mitral insufficiency, the back pressure in the left atrium is transmitted to the pulmonary veins and by raising pulmonary arterial pressure causes effects on the right side of the heart including ventricular dilatation  and in other animals . Some authors  have reported the occurrence of such condition in different birds with no special reference to ostrich.Mitral insufficiency can be a sequel to different valvular abnormalities including endocardiosis. In this case, the gross and microscopic changes detected in the mitral valves were concomitant with those stated for endocardiosis in dogs  who stated that it is sporadic and of unknown pathogenesis in animals. In this bird, however, we think that it has interfered effectively with normal blood flow and led to ischemic villous cicatrisation and atrophy.The nutmeg appearance of the liver is characteristic of venous congestion, which is a usual sequel to right ventricular failure. The abnormal thickening confined to the arterial vascular walls in the affected intestinal loop is comparable to hyaline arteriosclerosis described by Jones Ischemic changes were not obvious in the cardiac muscle probably because of milder arteriolar involvement. In the kidney, arterial wall thickening was devoid of hyalinization and resembled hyperplastic arteriosclerosis, which was also described by the former authors.They stated that hyperplastic arteriosclerosis is less encountered in animals and is associated with chronic renal diseases and other disorders associated with hypertension.et al., 2008). The septic thrombi noticed also in some renal venules might be due to extension from bacterial pyelitis noticed in some sections.Blood flow in those arterioles is certainly diminished due to narrowing in their lumen and this may explain the sclerotic changes in the nearby glomeruli. The existence of micro-thrombi in the renal capillaries is thought to be associated with renal ischemia (Jubb The vascular changes noticed in the heart, kidney and intestine can be secondary to the back pressures in the vascular system created by the endocardiosis and mitral insufficiency. Available literatures concerning endocardiosis in other animals have not mentioned such changes. The current investigation documents for endocardiosis in ostrich.


Ocular defects result from tumor, congenital anomaly and external injury not only lead to serious impairment of function and esthetics but also make the patient psychologically disabled. Prosthetic rehabilitation attempts to restore these disfgurements may improve esthetic, level of function, general psychologic improvement and quality of life. This clinical report details an attempt to rehabilitate a pediatric patient who has undergone orbital enucleation resulting from retinoblastoma with the aid of custom ocular prosthesis using commercially available prefabricated eye shell.How to cite this article: Janya S, Gubrellay P, Purwar A, Khanna S. Prosthetic Rehabilitation of Ocular Defect resulting from Pediatric Retinoblastoma. Int J Clin Pediatr Dent 2014; 7(3):209-212. Midline defects refer to the complete or partial involvement of the nose, and/or upper lip.3 A lateral defect may include complete or partial content of the cheek and-or orbit.4 The challenge of restoring such defect has always perplexed head and neck surgeon, plastic surgeon and maxillofacial prosthodontist.3Ocular malignancies have the potential for producing gross disfgurement and dysfunction, among them retino-blastoma is the most common and aggressive primary ocular malignancy of infancy and childhood.This clinical report describes the use of prefabricated eye shell to develop a customized ocular prosthesis for rehabilitation of postenucleation of right eye in a pediatric patient.An 8-year-old female patient referred to the Department of Maxillofacial Prosthodontics, MS Ramaiah Dental College and Hospital, Bengaluru, India, in August 2012, from Minto Eye Hospital, Bengaluru, for prosthetic rehabilitation of right ocular defect. History of present illness discovered that she was diagnosed with retinoblastoma, after which enucleation of right eye was conducted from same hospital in 2010. On extraoral examination, the margins and base of clinical defect were free from signs of infammation and completely healed. In this line of treatment plan, patient's guardians were explained about the fabrication of ocular prosthesis, and an informed, consent was obtained. The patient was seated in the physiological rest position, determined by the operator to make an impression of ocular defect . The subject was draped with sterile hospital towel. Patient's eyebrow was lubricated the patient's eyebrow by light application of petroleum jelly for the easy removal of the impression material after it sets. Impression of affected area was made with the help of a disposable syringe cover and custom tray made of autopolymerizing resin tray . Extension of this resin tray was checked in patient's eye socket to prevent any irritation of soft tissue. Needle end cover was attached to resin tray and used for impression. This custom made resin tray was used as a suitable carrier for impression material  to make fine and detailed functional impression of the tissue bed of eye socket. Automatic mixing gun system was used to inject the material into defect and simultaneously remaining impression material was loaded on to the impression surface of resin tray and passively placed in defect. Tray adhesive  was applied on the intaglio surface of resin tray for the retention of impression material. After the material was set, impression was removed gently . Flaskin The position of the iris was determined with the help of landmarks by making the patient look in a straight line. A vertical midline was marked passing through the forehead crease, glabella, tip of the nose and chin. The distance from the right eye medial canthus to the midline and left eye medial canthus to the midline was measured and calibrated. The fnished pattern was then fasked in a small two piece fask. Flasking was done taking care that the iris button should be secured to one counter of the fask and remaining part in the other portion of the fask. To achieve this acrylic stalk was attached to iris button before fasking. This acrylic stalk will ensure exact positioning of iris button. After dewaxing, packing was done with the selected heat-cure tooth color acrylic resin whose shade was matching with the sclera of contralateral eye . Long cy Patient and her guardians were instructed about hygiene practices . The need for regular recall appointments was emphasized to check the retention and comfort of prosthesis during observation period of 1 year .1Retinoblastoma is a most common, aggressive and invasive ocular malignancy of infancy and childhood. Survival and the chance of saving vision depend on severity of disease at presentation. Despite good understanding of its aetiology, mortality from retinoblastoma is about 70% in countries of low and middle income, where most affected children live.In the present case due to deep invasion of cancer, surgical excision of eye ball (enucleation) was conducted. Patient was kept under observation period for the complete healing of eye socket and referred to the department of maxillofacial prosthodontics for ocular rehabilitation. Occupational history of patient revealed that patient was a school-going child and hence it was an utmost social need of the patient to be esthetically acceptable by the society as early in life as possible. Hence, a custom ocular prosthesis using commercially available prefabricated eye shell was planned in order to enhance the confdence and quality of life of the patient.56 and custom made ocular prosthesis.9 Stock or prefabricated eye prosthesis has certain disadvantages of improper adaptation, compromised esthetic and limited eye movements where as custom made prosthesis advantages include improved adaptation to underlying tissues, increased mobility of the prosthesis, improved facial contours, and enhanced esthetics gained from control over the size of the iris and pupil, and color of the iris and sclera.9 It also involves technical sensitive procedures in various steps of fabrication which are quite difficult and based purely on artistic maneuverability of the maxillo-facial prosthodontist. Nevertheless, a custom prosthesis is more expensive than a stock prosthesis, and several steps are required for its fabrication. In our case, we have used prefabricated iris shell or iris button matched with patient contralateral eye and artificial custom made sclera was developed by heat-cure tooth color acrylic resin making it inexpensive and less time consuming. This combined approach for the development of ocular prosthesis surmounts the problem of prefabricated prosthesis and include the advantages of custom ocular prosthesis.Ocular prosthesis is broadly classified as stock,10 Various ocular impression techniques described so far have been based upon the materials available and the dexterity of the operator, making fabrication of an extraoral prosthesis more art than science.11 But each has its own integral advantages and disadvantages. Most can be placed into one of several broad categories: direct impression/external impression, impression with a stock ocular tray or modified stock ocular tray, impression using a stock ocular prosthesis, ocular prosthesis modif-cation and the wax scleral blank technique.12 In this case, impression with custom ocular tray was implemented. Miller suggested that a custom ocular tray is necessary in certain situations. For example, the anophthalmic socket could be highly irregular or stock trays may not be available.13The scientific aspect requires an accurate impression for the development of an accurately fitting extraoral prosthesis.14 Although osseointegrated implant may provide the most reliable prosthesis retention; additional surgeries, expenses, inadequate bone and prior radiation to the area may contraindicate this type of treatment.15 Inpresent case, retention was primarily achieved through anatomic tissue undercut.Today, numerous methods of retention for extraoral prostheses have been described in the literature; they include tissue undercuts, magnets and osseointegrated implants.16 Concept of QoL has emerged as an organizing scheme around which to describe and evaluate the experience of cancer patient in clinical research. In the described case, after one year of follow-up patient explained that retention and overall appearance was fair and satisfactory, wearing and cleaning of prosthesis was comfortable with no tissue discomfort was reported. This inferred that patient had overall improvement in QoL during observation period.A subjective measurement of patient was evaluated in this case to assess the improvement in quality of life (QoL). Chang et al proposed a standard questionnaire to evaluate the patient's satisfaction with facial prosthesis.Multidisciplinary approach is required to treat maxillo-facial rehabilitation cases. The use of custom-made ocular prosthesis has provided better option to the vast majority where implant is not a firsthand choice in developing countries due to its economic factor and other associated contraindications. In the described case, rehabilitation of pediatric patient is challenging as it require long-term follow-up till development of the enucleated socket is complete at the age of 12 years. In this long-observation period, childhood patient needs psychological support to recover confdence and self esteem in today's cosmetically challenging world.


Segmentation of the left ventricle is very important to quantitatively analyze global and regional cardiac function from magnetic resonance. The aim of this study is to develop a novel algorithm for segmenting left ventricle on short-axis cardiac magnetic resonance images (MRI) to improve the performance of computer-aided diagnosis (CAD) systems. In this research, an automatic segmentation method for left ventricle is proposed on the basis of local binary fitting (LBF) model and dynamic programming techniques. The validation experiments are performed on a pool of data sets of 45 cases. For both endo- and epi-cardial contours of our results, percentage of good contours is about 93.5%, the average perpendicular distance are about 2 mm. The overlapping dice metric is about 0.91. The regression and determination coefficient between the experts and our proposed method on the LV mass is 1.038 and 0.9033, respectively; they are 1.076 and 0.9386 for ejection fraction (EF). The proposed segmentation method shows the better performance and has great potential in improving the accuracy of computer-aided diagnosis systems in cardiovascular diseases. Cardiovascular disease is an important health concern due to its largest cause of death on human beings Currently, manual segmentation of LV by an expert reader is the standard clinical practice. However, this work is a long and tedious task, with inter- and intra-observer variability. Therefore, it is attractive to develop algorithms that are accurate and require as little user interaction as possible for clinical applications MR cardiac images present a number of features that make them difficult to segment. First, it is much difficult to segment LV with left ventricular outflow tract (LVOT), since edge information is severely missing for such myocardium. Second, they may present blurred edges due to the partial volume effect (PVE) and can be affected by motion artifacts, low contrast between the myocardium and surrounding tissues complicates the segmentation further. Third, the myocardium/background contrast is lower than the myocardium/blood pool contrast. It makes the inferior and lateral sectors of the epicardial boundary very ambiguous. Finally, there is variability in the shape of the endo- and epi-cardial contours across slices and phases which make the myocardium detection much complex.In recent years, many algorithms have been introduced to for (semi-) automated LV segmentation. They can be classified into two generic categories: (i) Deformable models, and (ii) Image-based techniques. A complete review of recent literature describing cardiac segmentation techniques is given in Deformable models include snakes Image-based techniques include thresholding In this study, an image-based segmentation schema which combines deformable model and image-based techniques is proposed to improve the segmentation performance. In our schema, a slice by slice segmentation of the LV image starts from mid-slice to the top end slice and the bottom end slice separately. For a slice image, LBF model http://sourceforge.net/projects/cardiac-mr/files/). There are 45 cases containing 12 heart failure with ischemia (HF-I) cases, 12 heart failure without ischemia (HF-NI) cases, 12 hypertrophy (HYP) cases and 9 normal (N) cases. The image data were obtained during 10-15 second breath-holds with a temporal resolution of 20 cardiac phases over the heart cycle. 6 to 12 SAX images were obtained from the atrioventricular ring to the apex  blood pool, lung and stomach. Different tissue has different image intensity and edge information. Hence, the intensity distribution and edge information are good characters in active contour model for image segmentation. Let Where The energy mask see . The binFor most slices, the Otsu threshold value is suitable for locating blood pool approximately, but in some extreme cases, the blood pool is missing using Otsu thresholding method. To determine automatically the fitness of a threshold, the ROI image is segmented with different threshold values, the overlap area of detected blood pool and the contour mask by LBF model is measured. The optimal threshold corresponding to the maximal overlap value is the one we are looking for. Using above criteria, we can search a better threshold and obtain an ideal segmented blood pool see .In the process of endocardial contour detection, we can find LVOT in the basal slice. In other slices, LVOT is non-existing. These two different types of slices are processed by different methods.The basal slice with LVOT is identified by the following steps. First of all, choose the middle slice image as the start image, and process each image sequentially in the basal direction. The convex hull of the blood pool is calculated. Calculate the length of the major axis L of the ellipse that has the same normalized second central moments as the binary blood pool obtained by LBF model. Then the ratio of the current major axis L to preceding L from the previous slice image is computed. If the ratio is larger than a predefined threshold , basal slice with LVOT is identified For a slice image with LVOT, we use the above LBF model to convert the gray ROI image into binary image and obtain a binary image see . The dilFor the points For the ROI image, the center point of obtained endocardial contour is used as the circle center, a circle is drawn with predetermined radius. The ROI image in the circle shape is transformed into a rectangular image by polar transform with the circle center point as the pole. The derived rectangular image is used to calculate the epicardial contour.The Extracting of epicardial boundary is under polar system and limited to certain regions. Region restricted dynamic programming from our previous method In our previous method, region restricted dynamic programming is employed in the polar coordinate; the searching scope of the edge points for epicardial is started from the endocardial contour. Bright regions such as the right ventricle, pericardial fat and abdominal fat are excluded in the search scope for epicardial edge points; a binary mask is constructed from the context information of these bright regions. The binary mask from our previous method is improved in our schema, that is to say, the derived epicardial contour region from the preceding slice image is dilated and added to the binary mask of our previous method to construct the last binary mask which can be adopted for the following dynamic programming (DP).In this study, we obtain an edge map of the LV image by using the non-maxima gradient suppression technique The LV image is denoted as Let N to 1 in the columns, the complete coordinates The cost function of the point in column The endo- and epi-cardial contours are transformed back to the original coordinates. Then, the 1D FFT and Bezier curve fitting are used to smooth the endo- and epi-cardial contours respectively. The 1D FFT method used in this paper is defined as follows:We adopt the evaluation software published by the MICCAI Clinical Image Segmentation Grand Challenge Workshop to analyze our results Several measures are employed in our experiments; these are percentage of good contours, average perpendicular distances, overlapping dice metric, left ventricle ejection fraction (EF) and mass (LVM) by including papillary muscles and trabeculations in the ventricular cavity.The ground truth of LV is manually delineated by the clinical experts from the data from the same workshop. Contours generated by automatic algorithms is considered as good if the average distance from ground truth is less than 5 mm. Percentage of good contours is calculated by the number of good contours over the number of all contours of ground truth. Perpendicular distance and overlapping dice metric are calculated only in the good contours from automatic algorithms. Average perpendicular distance evaluation calculates the distance from the automatically segmented contour to the corresponding one manually drawn by an expert, averaged over all contour points. Overlapping dice metric calculates the contour areas overlapping proportion between the automatic segmentation results and the ground truth.Clinical studies have employed different quantification methods for calculation of left ventricle volume, left ventricle mass and ejection fractions are critical parameters for cardiac diagnosis Where in the end-diastole phase, In this article, 45 cases have been tested in the experiment. The statistics of automatic segmentation results compared to the ground truth are listed in We use regression and Bland-Altman analysis to evaluate our results of 45 cases. To test the performance, we compare our present approach with Huang's method We compare percentage of good contours of 15 cases from the second dataset among Lu's We reveal some segmentation outputs from 9 studies with LVOT by our method in To demonstrate the advantages of our proposed method, we compare the derived endocardial contours from 2 studies  obtained from two methods in Furthermore, we display segmentation outputs for both endo- and epi-cardial contours from 6 studies obtained by two approaches in In this article, an automatic segmentation schema is developed for left ventricle on short-axis cardiac in multislice MRI. A series of image processing techniques are adopted in the schema, such as active contour model, optimal thresholding, non-maxima gradient suppression, and region constrained dynamic programming. The experiments show that the performance of our method is very promising.In LV segmentation, a very important step is to locate the LV blood pool which is often solved by region based technique, such Gaussian-mixture model and Otsu thresholding. The performance of Gaussian-mixture based segmentation algorithms clearly depends on how well the histogram of an image approximates a Gaussian mixture and the accuracy of the estimates of the model parameters. If the histograms of real LV images do not approximate a Gaussian mixture, it may produce wrong segmentation results. The Otsu method This study designs a simple but effective measure to solve the above problem. At first, local binary fitting model which combines edge and region information is used to locate blood pool of the LV image. Then the optimal threshold is obtained for LV segmentation. For the slice image with LVOT, the endocardial contour is extracted using context information from the previous slice image.For epicardial boundary extraction, we improve dynamic programming with some techniques, such as non-maxima gradient suppression, region constrained technique and context information constrain from the epicardial contour of the preceding slice image. Experimental results for the epicardial contour show that the above techniques are very effective.In our automatic LV segmentation in short-axis cardiac MRI, a slice-by-slice segmentation of LV image starts from mid-slice and extends to two-end slices, and the current slice center and the relevant masks from the obtained contours is used for next slice. The use of prior knowledge about spatial relationships between slice images makes our method accurate and robust.Segmentation performance of our approach and other methods are compared by using the measures of percentage of good contours, average perpendicular distance, and overlapping dice metric. Clinic parameters (EF and LVM) are studied using regression and Bland-Altman analysis. The results indicate that our proposed method has more accurate and robust performance.Although our schema is robust enough to cope with most studies, some extreme cases are still a challenge: 1) overlap between the intensity distributions within the cardiac regions in some slices; 2) the small blood pool at the apex is difficult to detect correctly. How to further improve accuracy and time efficiency of LV segmentation will be studied in the future.


Breathlessness is a prevalent and distressing symptom in intensive care unit patients. Patients' perception of breathing during a spontaneous breathing trial (SBT) might be correlated to extubation success. There is little evidence of the ability of health care workers to assess the patients' experiences of breathing.To assess mechanically ventilated patients' experiences of breathlessness during SBT.A prospective observational multicenter study of 100 mechanically ventilated patients. We assessed the agreement between nurses, physicians and patients' scores of breathlessness, perception of feeling secure and improvement of respiratory function at the end of a SBT. We also determined the association between breathlessness and demographic factors or respiratory observations. Self-reported breathlessness, feeling secure and improvement of respiratory function was reported at the end of a SBT by 11-point Numerical Rating Scales.Sixty-two patients (62%) reported moderate or severe breathlessness. The median intensity of breathlessness reported by the patients was 5 compared to 2 by nurses and physicians (p < 0.001). Patients felt less secure and reported less improvement of respiratory function compared to nurses and physicians ratings. About half of the nurses and physicians underestimated breathlessness (difference score ≤ -2) compared to the patients´ self-reports. Underestimation of breathlessness was not associated with professional competencies, whether the nurse or physician was involved in previous patient care or number of years working in an intensive care unit. Breathlessness was not related to objective assessments of respiratory function.Patients reported higher breathlessness after SBT compared to nurses and physicians. The data suggests that patients' self-report of breathlessness should if possible be included in the evaluation of a SBT.The study was supported by a grant from the Norwegian Nurses Organisation. The funder had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data.


Direct measurement of detailed kinematics of individual anatomical structures in the foot during human locomotion is crucial for understanding morphofunctional roles of the human foot structure that mechanically interacts with the ground in a favourable manner to maintain stable gait. In the present study, we constructed a dynamic gait simulator to load and mobilize the cadaver foot and directly measured the three-dimensional kinematics of foot bones using a biplane X-ray fluoroscopy.A robotic gait simulator was developed to load and mobilize the cadaver foot in a manner similar to the way human foot actually moves and interacts with the ground during walking. The simulator has three legs, fore, middle and hind legs, with the cadaver foot fixed to the middle leg Figure . After tA biplanar dynamic X-ray fluoroscopic system was developed with Shimadzu Corporation, Kyoto, Japan. The system consists of two sets of x-ray sources and flat panels with a resolution of 2688 x 2208 pixels. We recorded biplanar X-ray videos of foot movement using the system using the dynamic cadaver model at 15 fps. For direct measurement of 3D kinematics of the foot bones, we developed an automatic method to register bone surface models with the two fluoroscopic images Figure . SpecifiUsing the biplanar X-ray fluoroscopic images and the proposed reconstruction methodology based on CT, we reconstructed 3D movements of the calcaneus, talus, navicular and cuboid when a human cadaveric foot walked on a flat surface using the simulator. The surface models of the four bones were successfully matched with the corresponding fluoroscopic images and the joint movements were quantified and visualized. The present methodology must be undergone further evaluation, but the proposed framework may serve as an effective tool for understanding the morphofunctional roles of the human foot structure during walking.


International Brazilian Journal of Urology, in the Difference of Oppinion, there is a highly controversy subject: The managing of bulbar stenosis Dr. Santucci, from Detroit, favors the buccal urethoplasty against the complications of substitution uretroplaty and the bad results of internal urethtrotomy; on the other hand, Drs. Siegel and Morey justify why the excision and primary anastomotic urethroplasty is considered the gold standard in Dallas. A third vision came from Drs. Cavalcanti and FIeder, from Rio de Janeiro, which suggest that in selected cases  the internal urethrotomy followed by office dilatations may be an attractive alternative. On the page 623, a Review article brings actual information regarding the the plead meshes devices avialble for vaginal prolapses surgeries. This is fundamental, since at the same time the use of the mesh became popular, the posts about their complications and malpractice questions are so common.In this issue of There are reports and concerns about the risks of high intraocular and intracranial pressures during robotic assisted radical prostatectomy (RALP), due the long time of Trendelemburg position and pneumoperitoneum during the surgery. The Connecticut's group (page 661) analyzed retrospectively the post RALP outcomes of 40 patients with pre existing retinal or central nervous system comorbidities. There were no more Retinal or SNC complications in comparison with the remaining 1828 patients.The use of use of pre-procedural computed tomography angiography, can helps to know better in advance the renal vascular anathomy, reducing the surgical bleeding in miny percutaneous surgery (page 690). Despite the use of External Shockwave litothripsy has decreased for the ureteral stones management, it is yet a valid alternative. Yazici et al. reported the independent prognostic factors for stone fragmentation in proximal and distal ureteral stones, in a cohort of 204 patients. (page 677). Higher body mass index (BMI) patients had lower stone fragmentation in distal calculi. Higher BMI patients were also investigate in this journal number about the their rates for surgical complications after nephrectomy (page 697) and about the effectiveness of transobturator mid urethral versus mini slings in this population(page 714), reflecting the general interest in the repercussion of progressive obesity in the world.The modern lifestyle and environmental conditions may be among the reasons of the reduction of semen quality in Brazilian patients, according Borges Jr. et al. (page 717). At page 773, the protective effects of dexamethasone and vitamin E in the testicular parenchyma oxidative damage of a varicocele induced rat model are shown.Pediatric Urology: A group from Bahia reported the acute and the late urodynamics effects in children with idiopathic overactive bladder underwent parasacral transcutaneous electrical.


Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media (middle ear infections) in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In view of the huge global burden of disease caused by M. catarrhalis, the development of vaccines to prevent these infections and better approaches to treatment have become priorities. In previous work, we used a genome mining approach that identified three substrate binding proteins (SBPs) of ATP-binding cassette (ABC) transporters as promising candidate vaccine antigens. In the present study, we performed a comprehensive assessment of 19 SBPs of 15 ABC transporter systems in the M. catarrhalis genome by engineering knockout mutants and studying their role in assays that assess mechanisms of infection. The capacity of M. catarrhalis to survive and grow in the nutrient-limited and hostile environment of the human respiratory tract, including intracellular growth, account in part for its virulence. The results show that ABC transporters that mediate uptake of peptides, amino acids, cations and anions play important roles in pathogenesis by enabling M. catarrhalis to 1) grow in nutrient-limited conditions, 2) invade and survive in human respiratory epithelial cells and 3) persist in the lungs in a murine pulmonary clearance model. The knockout mutants of SBPs and ABC transporters showed different patterns of activity in the assay systems, supporting the conclusion that different SBPs and ABC transporters function at different stages in the pathogenesis of infection. These results indicate that ABC transporters are nutritional virulence factors, functioning to enable the survival of M catarrhalis in the diverse microenvironments of the respiratory tract. Based on the role of ABC transporters as virulence factors of M. catarrhalis, these molecules represent potential drug targets to eradicate the organism from the human respiratory tract. Moraxella catarrhalis is an exclusively human pathogen that is a common cause of otitis media (middle ear infections) in children and respiratory tract infections in adults with chronic obstructive pulmonary disease , mod  showed reduce invasion. Of the 9 mutants in which genes that encode SBPs were knocked out, 4  showed reduced invasion. These observations have important implications in how M. catarrhalis survives and persists in the human respiratory tract and are considered in the Discussion.M. catarrhalis wild type strain O35E and the mutant being evaluated (109 cfu of each), the concentration of each strain was determined 3 hours after challenge. We tested the gene cluster knockout mutants when they were available for an ABC transporter. For those which we were unable to knock out the entire gene cluster, we tested the SBP knockout of that transporter. oppA [peptides], met [methionine], and bcaa [branched chain amino acids]; 2 transporters of ions znu [zinc] and pst [phosphate]; and one transporter of an unknown substrate (unk). We conclude that these ABC transporters play a role in persistence of M. catarrhalis in the murine respiratory tract.We assessed the role of ABC transporters in persistence in the respiratory tract using the mouse pulmonary clearance model. Following aerosol challenge of groups of 10 mice with equal concentrations of the M. catarrhalis. Guided by the mechanisms of pathogenesis used by M. catarrhalis, the present study focused on three broad virulence properties: 1) growth and survival in nutrient-limited and hostile conditions such as those of the human respiratory tract, 2) invasion of human respiratory epithelial cells and 3) persistence in the respiratory tract in a murine pulmonary clearance model.In this manuscript we report the first comprehensive assessment of ABC transporters of the human respiratory tract pathogen, M. catarrhalis infection is colonization of the human nasopharynx, which is common in infancy and childhood [M. catarrhalis migrates from the nasopharynx to the middle ear where it multiplies and causes inflammation [M. catarrhalis has mechanisms to survive in multiple environmental niches in the human respiratory tract. It must survive at an air liquid interface in a nutritionally limited environment that varies over the course of colonization and infection. In the present study, we show that six ABC transporters play a role in growth in nutritionally limited conditions (The first step in the pathogenesis of hildhood , 35–38. ammation . In the nditions .M. catarrhalis colonizes the human respiratory tract, it adheres to mucin which coats the epithelial cell layer, adheres to epithelial cells, and invades cells, including epithelial cells and lymphoid cells [M. catarrhalis in the respiratory tract, indicating that the capacity of M. catarrhalis to invade and survive inside cells of the human respiratory tract is a virulence factor. We assessed ABC transporters by studying wild type and mutant strains for their capacity to invade A549 cells. The results showed that 9 ABC transporters contribute to invasion of human respiratory epithelial cells (When id cells , 39–41. al cells .We used the mouse pulmonary clearance model as a quantitative, in vivo model to assess wild type and ABC transporter mutant strains for their capacity to persist in the respiratory tract . Six ABCM catarrhalis in the diverse microenvironments of the respiratory tract.M. catarrhalis. While the assays used in this study test functions that are important in pathogenesis, none of the assays completely reproduce the conditions exactly as they exist in the human respiratory tract. As an exclusively human pathogen, no good animal model systems that parallel human disease by M. catarrhalis have yet been developed. A widely used model to assess persistence of M. catarrhalis in the respiratory tract is the mouse pulmonary clearance model. The model has the limitation that M. catarrhalis does not colonize the mouse for extended periods; however, this in vivo model is reproducible, is used by multiple research groups, and has been used extensively to assess putative vaccine antigens [A limitation of the present study is the drawbacks of the model systems to study the pathogenesis of antigens –47.lol knockout mutants likely had structural impairments because the genes encode lipoprotein outer membrane localization pathway machinery, creating slow growth and reduced viability in several assays.Another limitation of the present study is that we may have missed some ABC transporters because we relied on annotation of the genome to identify the genes to study. In addition, it was not possible to study each mutant in all three model systems because viability of selected mutants under the conditions of the assays limited the use of some models for some mutants. For example the M. catarrhalis ABC transporters likely function with more than one transporter and some SBPs function exclusively with their associated transporter. We did not perform the detailed growth studies that are required to draw such conclusions.In addition, SBPs of some of the Finally, because experiments in the present study did not include complementation of each mutation, we cannot exclude the possibility of downstream effects of the deleted genes. Guided by the results of the present study, more detailed studies of selected ABC transporters should include complementation of mutations.M. catarrhalis in the respiratory tract make that molecule a potential valuable drug or vaccine target. Further study of ABC transporters identified in the present study represent fertile areas for drug development.In view of the importance of ABC transporters in multiple cellular functions of bacteria, these molecules have potential therapeutic value as potential drug targets. In addition to uptake of nutrients, ABC transporters participate in bacterial chemotaxis, transduction signals from ligand binding to chemosensory apparatus and binding of transcription factors , 48, 49.


Essential genes are thought to be critical for the survival of the organisms under certain circumstances, and the natural selection acting on essential genes is expected to be stricter than on nonessential ones. Up to now, essential genes have been identified in approximately thirty bacterial organisms by experimental methods. In this paper, we performed a comprehensive comparison between the essential and nonessential genes in the genomes of 23 bacterial species based on the Ka/Ks ratio, and found that essential genes are more evolutionarily conserved than nonessential genes in most of the bacteria examined. Furthermore, we also analyzed the conservation by functional clusters with the clusters of orthologous groups (COGs), and found that the essential genes in the functional categories of G (Carbohydrate transport and metabolism), H (Coenzyme transport and metabolism), I (Transcription), J , K (Lipid transport and metabolism) and L  tend to be more evolutionarily conserved than the corresponding nonessential genes in bacteria. The results suggest that the essential genes in these subcategories are subject to stronger selective pressure than the nonessential genes, and therefore, provide more insights of the evolutionary conservation for the essential and nonessential genes in complex biological processes. They play significant roles in many critical cellular processes, and hence are also considered the foundation of cellular lifeet al. performed a genome-wide evolutionary analysis in three bacterial species, including Escherichia coli, Helicobacter pylori, and Neisseria meningitides, and found that the essential genes are evolutionarily conserved than the nonessential geneset al. also analyzed 16 different biological features on the evolutionary rate, and found that function essentiality is one of main contributors to the protein evolutionary rate variationIt is well known that the rates of evolution have significant variations among protein-coding genes. If a protein plays a significant role in the cellular life, it should be under rigorous functional or structural constraints in response to the strong purifying (negative) selective pressureWe constructed a database of essential genes named DEG, which has collected and organized the records of both essential genes and essential non-coding elements by genome-wide gene essentiality screens, including bacteria, archaea and eukaryotes316−5 based on BLASTP searches, and the orthologous protein with the highest score by BLASTP was selected for the further analysis2022http://tubic.tju.edu.cn/deg/. During the Ka/Ks estimation, the majority of protein sequences in some homologous strains are precisely the same with the protein sequences in the original organism, so that the ratios of Ka and Ks of these homologous sequences could not be determined based on the Nei–Gojobori method. As a result, we only selected the strains that keep enough diversity in most protein-coding sequences.The Ka/Ks ratio is the ratio of the number of non-synonymous substitutions per non-synonymous site (Ka) to the number of synonymous substitutions per synonymous site (Ks), which could be used as an indicator of selective pressure acting on the protein-coding geneIn order to estimate the difference of evolutionary conversation between the essential and nonessential genes in each organism, we performed a bootstrap analysis by half-sampling with replacement from the original gene set over 1000 replicates. The average values for these resampled sets including Ka, Ks and Ka/Ks were then calculated. It should be noted that the genes without valid Ka or Ks value were excluded from the analysis.http://www.ncbi.nlm.nih.gov/COG/). It should be noted that owing to the absence of COG annotations, the genes of Bacteroides fragilis 638R were excluded in the COG analysis. The significance of difference for the Ka/Ks values between the essential gene and nonessential genes in each COG scope by organism was performed by Mann-Whitney U test. And the P-value less than 0.01 was considered statistically significant.The Clusters of Orthologous Groups of proteins (COGs) database is a useful tool for the functional annotation, and provides a consistent classification of bacterial and eukaryotic species based on orthologous groupsAll the pipelines including BLASTP, ClustalW2, Pal2Nal and KaKs_calculator were executed by a custom python script. Biopython module was used to parse the GenBank and aln format files−5, the most pairs of proteins we found are orthologous. Then the average Ka, Ks and Ka/Ks ratios of the essential and nonessential gene were calculated in each organism, and the levels of significance for the difference between the essential and nonessential genes were determined using the Mann-Whitney U test . The lower three ratios of the essential genes, in particular, suggest that the essential genes are more conserved during the evolution, and consistent with the fact that the negative selection against amino acid replacements acting on the essential genes are more strict than on the nonessential genes.In order to estimate the difference of evolutionary conservation between the essential and nonessential genes, we constructed a data set containing the essential and nonessential genes, which were determined in genome-wide screen. The gene set consists of more than 70,000 genes from 23 genomes of bacterial organisms. y U test . Based oIn order to rule out the effect of the extreme values upon our results, the average values for Ka/Ks were evaluated by bootstrap analysis of 1000 replicates through half-sample resampling. Mycobacterium tuberculosis H37Rv and Vibrio cholerae N1696. The highly conserved nonessential genes, throughout distantly related bacteria, have been found in other organism, which are termed persistent nonessential (PNE) genesM. tuberculosis H37Rv and V. cholerae N1696 .


Studies with genetically different strains of mice showed different responses for diabetes, glucose levels and insulin levels. Several experimental models have shown that wound healing and angiogenesis are phenotypes dependent on the collection of genes present in an organism. However, we found no study that investigated the influence of genetic heterogeneity in the process of inner healing in diabetic animals.Evaluate the influence of genetic background in the components of repair process (angiogenesis and inflammation) induced by synthetic matrix in mice with type I diabetes .Angiogenesis and inflammation were assessed at 10 days after implantation in polyether-polyurethane sponge discs implanted subcutaneously in female Swiss, C57, and Balb/c control and diabetic mice induced by streptozotocin .The strains responded distinctly to the diabetogenic treatment as assessed by fasting glucose levels . Hemoglobin content, (µg/mg) in implants of Swiss diabetic animals decreased by 59% compared with the control group. The diabetogenic treatment did not alter this parameter in the other two strains. In all strains the number of vessels was decreased in implants of diabetic animals compared with their control groups . In contrast, VEGF levels (pg/mg) were increased in implants of Swiss and C57/BL diabetic mice The inflammatory parameters  were markedly influenced by the genetic background. In implants of Swiss and Balb/c diabetic animals, MPO increased, but NAG activity was lower in implants of Swiss diabetic mice. Furthermore, the levels of nitric oxide were also reduced in implants of the all diabetic mouse strains. The inflammatory cytokines  also showed distinct profiles after the diabetogenic treatment.The genetic background influenced the systemic and local response to the diabetogenic treatment in the strains of mice evaluated. Swiss mice were the most affected strain analyzed whereas the least was the Balb/c. These important strainrelated differences to diabetes must be considered in the design and analysis of studies internal healing hyperglicemic environment.


The respiratory tract, once believed to be sterile, harbors diverse bacterial communities. The role of microorganisms within health and disease is slowly being unraveled. Evidence points to the neonatal period as a critical time for establishing stable bacterial communities and influencing immune responses important for long-term respiratory health. This review summarizes the evidence of early airway and lung bacterial colonization and the role the microbiome has on respiratory health in the short and long term. The challenges of neonatal respiratory microbiome studies and future research directions are also discussed. Respiratory insufficiency is the major issue limiting viability at the extremes of prematurity. Chronic lung disease of prematurity (CLD), also known as bronchopulmonary dysplasia, is the most common long-term complication of prematurity  with redThe microbiome is defined as the whole habitat of microorganisms (or of their genes) and the surrounding environment, with the term microbiota more specifically referring to the community of microorganisms living within a particular environment . The divThe human host has evolved in symbiosis with microorganisms, which colonize multiple body sites. The interactions between host and the microbiota are only now being understood with microbiome science demonstrating that bacterial communities living within a human host have an influence over a diverse range of diseases . Within The traditional notion of the lungs of healthy individuals being free from bacterial colonization is now defunct , 17. TheIn addition to the ethical and practical challenges that complicate neonatal research, respiratory microbiome work has to overcome challenges of difficult sample collection, contamination, and the low bacterial load in the lungs.Bacteria are able to colonize the full length of the airways, from the nostril to the alveoli. The changes in anatomy and environmental exposure along this length lead to the creation of many varied niches for bacterial colonization. Sampling of the proximal airways at any age is straight forward using nasal swabs or nasopharyngeal aspirates. The inaccessibility of the lungs poses significant challenges for researchers. Sampling from the lungs and distal airways in adults has involved using sputum or endoscopic bronchoalveolar lavage , 21, 22.The particular difficulties of sampling the lung microbiome in neonates results in published studies concentrating on the upper airway or on ventilated infants , 28, 29.Culture-independent techniques utilize sequencing technologies to identify organisms by sequencing target genes. The most commonly used gene for bacterial species identification is the 16S ribosomal RNA gene. The difference in nucleotide sequence within the nine hypervariable regions of the gene are used to identify the bacterial species present within a sample using next generation sequencing . All bacMany respiratory samples, particularly those from neonates, generate a low biomass  with lowDespite the challenges and limitations of studying the airway and lung microbiome, a significant body of work is available relevant to adults, with several comprehensive reviews published , 41. Thein utero environment is traditionally considered to be physiologically sterile. This assumption has been challenged with the finding of bacteria in the placenta  with Proteobacteria making up around 10% of the lung microbiome , 20, 52.t common . The preStaphylococcus, in 19 samples, 17 of these being coagulase negative staphylococci. Ureaplasma species were dominant in nine samples from six subjects. Other species that predominated in a single sample, all between 14 and 21 days of life were Pseudomonas aeruginosa, Enterococcus faecalis, and Escherichia coli  in 31 of 32 tracheal aspirate samples taken in the first month of life. The most common dominant genus was hia coli . A similylococci . A subse of life . Taken tThe constituents of the infant gut microbiota have been shown to have been strongly influenced by delivery mode, feeding choices, and the perinatal use of antibiotics . The useEvidence from the airways is less substantial, but the nasopharynx of term-born infants share the same initial colonizing organisms as various skin sites and the mouth. All sites demonstrated a large influence of the delivery method . InfantsMechanically ventilated preterm infants exposed to chorioamnionitis appear to have decreased species diversity in their tracheal aspirates although the trend did not reach statistical significance compared to unexposed infants . This maAlloiococcus and Corynebacterium, with an increased proportion of potential pathogens, including Haemophilus, Streptococcus, and Moraxella  are first present in the stool . This suAccurate determination of the source of the microbiota would require deep sequencing of the genome of organisms from multiple sites including maternal skin, breast milk, birth canal, and from the skin of midwifery or neonatal staff caring for the infant, as well as range of environmental sites – a major undertaking.Evidence suggests that the airway microbiome is affected by multiple environmental factors. Interpreting the studies is difficult given the different sampling techniques and anatomical sites tested; however, evidence is consistent with studies of bowel colonization showing an effect of delivery mode, antibiotics, and feeding methods.Dysbiosis is defined as an imbalance in the microbes present in a particular niche that contributes or predisposes to disease , 16. AduUreaplasma species repeatedly implicated  were at significantly higher risk of pneumonia and bronchiolitis by the age of 3 years  asymptomatic carriage of streptococcus species significantly increases the risk of chronic wheeze at 5 years of age . In a sita group .The combined evidence suggests early colonization of the gut and the respiratory tract may have a role in determining future respiratory health. The factors previously discussed affecting the early colonization patterns may therefore have a lasting impact beyond the neonatal period. Further work to understand the mechanisms of microbiological programing may assist in developing novel treatments or reveal possible ways to influence the microbiota to promote long term health.via immunological modulation of mucosal immune responses and the plethora of small metabolite molecules produced by bacteria  may also be a mechanism for gut microbes influencing respiratory health . SCFA acvia this mechanism. DCs are critical to antigen presentation. Probiotics lead to DC secretion of the regulatory cytokines IL-10 and IL-12 leading to a shift in the T-helper cell population toward Th1 dominance (The common mucosal response theory suggests that antigens presented from one mucosal surface can affect lymphoid cell migration to other mucosal sites, influencing immune responses at the distal site . The immominance . The impThere may also be a lung–gut axis with the reverse processes in action. Exposing the lungs of mice to lipopolysaccharide results in changes to the bowel flora . The impThe gut–lung axis is an example of the potential influence of the gut microbiome over many body systems –93. A moFigure Cytomegalovirus and Neisseria gonorrhoeae used routinely (Overcoming technical challenges in obtaining samples from the lower airways and lungs of preterm infants can potentially lead to the development of accurate biomarkers for accurately identifying lung microbial colonization. Certainly, molecular methods provide improved sensitivity to traditional culture-based techniques, but modern methods will need to separate infection from colonization, viable from non-viable and commensals from pathogenic bacteria. In clinical work, the transition from culture-based pathogen identification to molecular-based techniques is already underway with PCR tests for, among others, outinely . These toutinely . It is lLactobacillus and/or Bifidobacterium species (Clostridium difficile infection (Ureaplasma infection in pre-terms has been suggested as a way to reduce rates of CLD (Probiotics and prebiotics are attempts to manipulate the microbiome of the gut to promote a healthy microbiome. This approach has received widespread support in the neonatal community and a favorable Cochrane review showing efficacy of probiotics in preventing NEC in preterm infants treated with  species . Alternanfection . Managinnfection , and liks of CLD , 99. TheRising antibiotics resistance and further understanding of host–microbe interactions may lead to a change in emphasis from eradication of pathogens by antibiotics to out competing pathogenic organisms by restoring the healthy, premorbid microbiome. Dysbiosis in the lungs may perhaps be amenable to treatment with nebulized probiotics. Early mouse work has demonstrated nasal administration of probiotic bacteria can protect against respiratory infection . FurtherStudying the gut–lung axis may provide significant insights into cultivating the optimal respiratory microbiome. Dietary changes are known to affect the gut microbiome . Breast Early microbial programing demonstrates the importance of a long-term view of all areas of neonatal care. Future studies focusing only on the neonatal period will not detect the effects of microbiological programing.As the understanding of the role of the microbiome increases from observational studies, it is likely that interventional studies will seek to manipulate the respiratory microbiome using established and novel techniques. Detailed work to grasp the complex symbiosis of host and colonizers is needed to minimize the risks of these treatments.The proponents of microbiome research suggest understanding the microbiome could lead to highly individualized care based on a patients colonization patterns, preventing acute infections, and long-term disease risks by microbiome manipulation , 103. ThDG performed the literature search and wrote the manuscript. SK reviewed the manuscript and authorized the final version.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.


Atypical infratemporal fossa infections are rare and potentially fatal.A case of an aspergillosis localized in the infratemporal fossa and another case of tuberculosis of the infratemporal fossa originating from the maxillary sinus, is described. The first patient was immunocompromised and showed symptoms of facial numbness; whereas the other was an immunocompetent man who complained of trigeminal neuralgia type pain. It was difficult to differentiate between infection and tumour despite the utilization of computed tomography scans and magnetic resonance imaging.These cases illustrate the need for a high index of suspicion; in addition to endoscopic confirmation and histopathology to establish precise diagnosis and early intervention. Atypical infratemporal fossa (ITF) infections are extremely uncommon and can be potentially fatal. They have been known to arise from odontogenic infections, dental extractions, maxillary sinus wall fractures, and maxillary sinusitis -5. Two uCase 1: A 37–year-old immunocompro- mised post renal transplant man presented himself to the Otolaryngology Department with a four-month history of left facial numbness and left upper molar pain that persisted despite dental extraction. There were no other otorhinolaryngological symptoms. Past medical history revealed that the patient was on an immuno- suppressive regime and suffered from hypertension. Clinical examination and flexible nasoendoscopy findings were unremarkable. Computed tomography (CT) scan and magnetic resonance imaging (MRI) reported an irregular soft tissue infiltrating the temporalis and lateral pterygoid muscle . There wThe pre-operative diagnosis was a left maxillary sinus tumour. The patient underwent a left endoscopic excision of the tumour via maxillotomy. Peri-operatively, it was noted that the lesion had a light yellowish colour and was solely confined to the ITF with no involvement of the maxillary sinus. Histopathological examination reported granulation tissue with inflammatory cells and fungal hyphae. Apergillus fumigatus was confirmed during fungal culture of the specimen. The final diagnosis was localised aspergillosis of the ITF.Post-operative recovery was uneventful and the patient was commenced on a six-week course of voriconazole. As there was minimal improvement in the patient’s condition and slight reduction in the size of the mass, he underwent further debulking via an extended endoscopic medial maxillectomy. He was followed up for a year with no signs of recurrence.Case 2: A 56-year-old immunocompetent man presented himself to the Otolaryngology Department with a three-month history of left facial pain and swelling, limited mouth opening, and left upper molar pain, which persisted despite dental extraction. There were no other otorhinolaryngological symptoms. Past medical history was unremarkable with no apparent immune deficiencies.Clinical examination revealed marked trismus with interincisal opening of 4 mm, making intra-oral examination difficult with an indurated swelling over his left maxillary region. Flexible nasoendoscopy showed medialisation of the left medial wall of the maxilla. CT scan and MRI reported a soft tissue mass in the maxillary sinus with involvement of the left pterygoid muscles and left masseter muscle . There wPost-operative recovery was uneventful. The patient was started on an antitubercular regime and was followed up for a year with no recurrence.The ITF is defined as a space bound anteriorly by the posterior surface of the maxilla, posteriorly by the mastoid and tympanic portions of the temporal bone, superiorly by the greater wing of the sphenoid and the squamous portion of the temporal bone, medially by the pterygoid process of the sphenoid, laterally by the zygomatic arch and ascending ramus of the mandible, and inferiorly by the posterior belly of the digastric muscle and the angle of the mandible. Its contents are the temporalis muscle, medial and lateral pterygoid muscles, pterygoid venous plexus, the maxillary artery and its branches, the mandibular nerve and the chorda tympani.The presence of ITF infections is largely determined by which part of the anatomy is affected. Facial swelling can manifest as a result of an abscess occupying the ITF and fullness of this area is usually apparent on clinical examination. Trismus also occurs as a result of extension to the pterygoid muscles. Additionally, there may be trigeminal neuralgia type pain, hypothesia or paraesthesia that is indicative of the mandibular nerve involvement. Interestingly, both these patients presented different symptoms, with one complaining of facial numbness whereas the other had facial swelling and trigeminal neuralgia type pain. Hence, even if the clinical signs and symptoms are rational from an anatomic basis, the diagnosis of ITF infections remains a challenging task as they can present themselves in a variety of ways.Imaging studies are fundamental for diagnosis and surgical planning. CT can distinctly distinguish between cellulitis and abscess formation whereas MRI is better for assessing soft-tissue. Nevertheless, it is often difficult to differentiate between infection and tumour, particularly in these cases where radiological and clinical features are nonspecific. Chronic infections may mimic neoplasm by showing signs of trismus and swelling without fever; as opposed to acute infections where there is usually pain and fever. In fact, our initial impression was a maxillary sinus tumour. As such, endoscopic confirmation with histopathology and culture are necessary for a definitive diagnosis. Achieving the correct diagnosis is crucial so that effective therapy can be tailored according to the pathology. In the first case of aspergillosis, surgical treatment was necessary when medical therapy alone failed. It is concluded that endoscopic debridement combined with systemic antifungals is imperative to improve survival, especially in immunocompromised patients. The second patient with TB improved significantly once he was commenced on an antitubercular therapy. To the best of our knowledge, the first case of aspergillosis solely localized in the ITF is reported. Goyal et al reported four patients presenting invasive fungal sinusitis with infratemporal fossa extension . The rouOur second patient presented an exceedingly rare case of TB in the ITF with the primary involvement of the maxillary sinus. Very few cases, documented in literature, show the maxillary sinus as the primary site of infection -10. The The venous drainage of the ITF is done through the pterygoid plexus, which anastomoses with the cavernous sinus, ophthalmic veins, and pharyngeal venous plexus. As a result, infections involving the ITF can spread haematogenously into the cavernous sinus and orbit leading to ominous consequences . InfectiThese cases illustrate the need for a high index of suspicion to achieve a precise diagnosis so that the treatment can be tailored according to the pathology. We believe that prompt diagnosis of the disease based on radiological and pathological findings and early intervention are crucial in establishing a positive outcome.


Applications of these techniques illuminate the developmental mechanisms underlying reptilian corticogenesis, which provides significant insight into the evolutionary steps of different types of cortex and the origin of the mammalian neocortex.The mammalian neocortex is a remarkable structure that is characterized by tangential surface expansion and six-layered lamination. However, how the mammalian neocortex emerged during evolution remains elusive. Because all modern reptiles have a homolog of the neocortex at the dorsal pallium, developmental analyses of the reptilian cortex are valuable to explore the origin of the neocortex. However, reptilian cortical development and the underlying molecular mechanisms remain unclear, mainly due to technical difficulties with sample collection and embryonic manipulation. Here, we introduce a method of embryonic manipulations for the Madagascar ground gecko and Chinese softshell turtle. We established  The mammalian cerebral cortex is a remarkable brain structure that is responsible for intricate social behaviors and intelligence. The cerebral cortex is characterized by tangential expansion of its surface area, which is particularly enhanced in the primate and human neocortex, and a six-layered laminar structure composed of multiple types of excitatory and inhibitory neurons , which develops in the dorsal part of the telencephalon in all vertebrate species  and the Chinese softshell turtle (Pelodiscus sinensis). Surgical techniques on developing reptilian embryos enable us to utilize various experimental approaches. We established the introduction of exogenous genes into the reptilian cortex by in ovo electroporation. Furthermore, we developed an ex ovo culture system for gecko and turtle embryos, which remarkably increased accessibility to the embryos and improved the efficiency of gene introduction. Successful manipulation techniques of non-mammalian embryos are valuable for studies of the evolutionary developmental biology of the cerebral cortex.Here, we describe a method of embryonic manipulation techniques for two reptilian species: the Madagascar ground gecko (P. pictus) is a ground crawling gecko that commonly lives on Madagascar Island. Gecko, lizard, snakes and Tuatara (Sphenodon) are included in the group of lepidosaurs  is a freshwater-living turtle that is widely distributed in eastern and southeastern Asia ; after these stages, the eyes and jaws increase rapidly in size, which make it difficult to access and electroporate to brains.After oviposition, the laid eggs should be isolated from the mother animal to avoid accidental crushing of the eggs. In our laboratory, the eggs are immediately transferred to a plastic container filled with dried vermiculite Figure . To mainin ovo electroporation, the egg is placed on a depression slide  with moistened papers  with a non-toxic dye (0.01% fast green). Typically, we prepare 2.5–5 μg/μL of plasmid solution for the electroporation. Holding the head of the embryo with a fine forceps, the DNA solution is injected into the left or right side of the lateral ventricle with a fine glass capillary  that is connected to a mouth-pipette  or mini-injector . Subsequently, needle-type electrodes  is inserted into the extra-embryonic space. Because DNA is negatively charged, a positive electrode was positioned at the target region , and a negative electrode was placed at the opposite side of the head .n Figure . FertiliThe position of an embryo within the egg can be monitored by illuminating the egg with a fiber light. To open the turtle eggs, a small hole is made in the shell by drilling the top of the shell with a micro drill  under the dissecting microscope Figures . After oex ovo culture system for the middle stages of reptilian embryos  provides higher expression of transgenes in amniotic brains, particularly in the neural stem/progenitor cells .To perform immunohistochemical analysis, embryos are fixed with standard fixative  for overnight at 4°C and immersed in 20% sucrose for cryoprotection. The samples were embedded in OCT compound , and 14 μm of cryosections are made with a cryostat . Several commercial antibodies are potentially applicable for immunohistochemistry in gecko and turtle embryos Table , 2012, aThe expression of exogenous genes can be monitored soon after electroporation. We collected gecko and turtle embryos at several time points after the electroporation, and examined the expression of fluorescent reporters under a fluorescent microscope. At 2–4 days after electroporation, intense GFP expression was detected in the dorsal part of the gecko and turtle telencephalon Figures . Even atAt 2 days after electroporation, GFP expression was exclusively detected in mitotic neural stem/progenitor cells that were localized at the ventricular zone of the developing gecko cortex. These neural stem/progenitor cells have a radial fiber similar to the mammalian radial glial cells, but the fibers extend in a curved manner at the neuronal layer as in the case of avian cortical radial fibers  multipolar neurons in the reptilian cortex do not exhibit mitotic activity and (2) multipolar-to-bipolar transition of migratory modes is not detected during the reptilian corticogenesis. These data support the idea that both amplification of IPCs  lineage tracing of neural stem/progenitor cells, (2) quantification of reporter activities for signaling molecules, and (3) gain- and loss-of-function analyses of specific genes in the developing reptilian cortex , a related species to the gecko, have been published .Recently, whole genome sequences of Chinese softshell turtle and sew turtle have been performed, which have confirmed that turtles must be positioned phylogenetically in archosaur groups in amniotes . Because reptilian eyes and jaws rapidly increase in size during embryogenesis, positioning the electrodes to target dorsal cortex is technically difficult at later embryonic stages. Application of an ex ovo culture system for gecko and turtle embryos is also limited for 3–4 days, most likely due to the lack of some essential nutrients and/or sufficient oxygen supply. Further improvements of gene delivery tools and/or culture conditions are required to manipulate embryos at any developmental stage.Currently, we have only successfully performed in vivo delivery of CRISPR/Cas vectors induces direct somatic recombination in target tissues, which enables the site-specific mutation of endogenous genes (Xue et al., Electroporation with a transposon-mediated genomic integration system provides permanent lineage tracing in mammalian and non-mammalian vertebrates (Garcia-Moreno et al., 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.