Following the cyclone, mean (± S.E.) hard coral cover ranged from just 2.1 (0.2) % to 5.3 (0.4) % on exposed reefs and no reproductively mature colonies of any species of Acropora remained. Although no fragments of Acropora were found at impacted exposed sites following the cyclone, small juvenile colonies of Acropora (<10 cm diameter) were present, suggesting that their small size and compact morphologies enabled them to survive the cyclone. By contrast, sheltered reefs appeared to be unaffected by the cyclone. Mean (± S.E.) hard coral cover ranged from 18.2 (2.4) % to 30.0 (1.0) % and a large proportion of colonies of Acropora were reproductively mature. Macroalgae accounted for 8 to 16% of benthic cover at exposed sites impacted by cyclone Yasi but were absent at sheltered sites. Mean (± S.E.) recruitment of acroporids to settlement tiles declined from 25.3 (4.8) recruits tile−1 in the pre-cyclone spawning event (2010) to 15.4 (2.2) recruits tile−1 in the first post-cyclone spawning event (2011). Yet, post-cyclone recruitment did not differ between exposed (15.2±2.1 S.E.) and sheltered sites (15.6±2.2 S.E.), despite the loss of reproductive colonies at the exposed sites, indicating larval input from external sources. Spatial variation in impacts, the survival of small colonies, and larval replenishment to impacted reefs suggest that populations of Acropora have the potential to recover from this severe disturbance, provided that the Palm Islands are not impacted by acute disturbances or suffer additional chronic stressors in the near future.Cyclone Yasi, one of the most severe tropical storms on record, crossed the central Great Barrier Reef (GBR) in February 2011, bringing wind speeds of up to 285 km hr Disturbance is part of the evolutionary history of coral reefs, however the increasing frequency and intensity of anthropogenic and climate-related disturbances, particularly coral bleaching and altered storm regimes Acropora, the most abundant and species-rich scleractinian genus in the Indo-Pacific Acropora larvae become competent to settle within a few days Acropora larvae can remain competent to settle for many months, highlighting their potential for long distance dispersal Acropora larvae is corroborated by their population genetic structures The potential for larval replenishment of coral populations following disturbance may vary among species that differ in mode of reproduction. Coral reproductive strategies fall into two broad categories, brooding corals that have internal fertilization and release mature planulae ready to settle close to parents, and broadcast-spawning corals that release gametes, typically in a timed single or bi-annual spawning event, for external fertilization at the ocean surface potentially leading to dispersal between reefs nd, 2011, severe tropical cyclone Yasi crossed the central GBR  approximately 5.5 hours before cyclone Yasi crossed the coast. The eye of cyclone Yasi passed just north of the Palm Islands, a group of inshore islands with fringing reefs in the central GBR , which can reduce coral populations catastrophically. On February 2tral GBR  as one otral GBR . Rapid eAcropora in the year before and after the cyclone.Tropical storms are among the most significant disturbances for coral reefs http://www.gbrmpa.gov.au/__data/assets/pdf_file/0014/28112/Map6-GDA94.pdf).The study was conducted on the exposed and sheltered fringing reefs of Orpheus and Pelorus Islands in the Palm Island group, which were in the direct path of very destructive tropical cyclone Yasi . The PalAcropora species, and recruitment to experimental substrata were collected at all six sites (details below). In 2010 we were not aware that cyclone Yasi would occur so surveys were only conducted at a subset of sites before the cyclone. Field data were collected before (2010) and after (2011) cyclone Yasi. In 2011, data for benthic cover, abundance and colony sizes for all Permits to conduct field research in the Great Barrier Reef Marine Park were obtained from the Great Barrier Reef Marine Park Authority (GBRMPA: permit G33638.1 issued to VL). All data collected in this study can be obtained by request from VL.Benthic cover following cyclone Yasi was quantified in November 2011 using visual census of 20 m line intercept transects. At each site , six replicate transects were sampled: three on the reef crest and three on the reef slope (10 m between transects within each habitat). On each transect, the benthic category lying underneath the tape was identified, and the intercept was measured to the nearest centimeter. The following benthic categories were recorded: live hard coral, soft coral, macroalgae, rock, and other . Hard corals were recorded at the genus level but, because this study focuses on the spawning acroporids, coral cover is presented as Acroporidae and other hard corals. Spatial variation in benthic cover was examined using hierarchical (nested) analysis of variance . Data were log (x +1) transformed to meet the assumptions of homogenous variances.Acropora assemblages, the abundance and colony sizes of all species of Acropora were estimated at exposed and sheltered sites. At each site, 20 m×2 m (40 m2) belt transects were surveyed on the reef crest and upper slope (six replicates per site). All colonies of Acropora were recorded and the maximum diameter of the colony (D1) and the diameter at right angles (D2) were measured for each colony (to the nearest cm) using a tape measure. The size (mean diameter) of each colony was calculated as (D1+ D2)/2. In order to quantify the potential for recovery at impacted sites via propagation from coral fragments, the abundance and sizes of all live fragments of Acropora spp. were also recorded at impacted sites (E1–E3). Spatial variation in density and size of colonies was examined using hierarchical (nested) analysis of variance . Data were log (x+1) transformed to meet the assumptions of homogenous variances.To quantify the impact of tropical cyclone Yasi on Acropora tenuis at exposed and sheltered sites were collected before (2010) and after (2011) cyclone Yasi. A. tenuis is a broadcast spawning species that reproduces in spring on the GBR and is representative of the majority of species of Acropora that participate in the annual synchronised spawning event A. tenuis present. Each colony of A. tenuis encountered was photographed with a 30 cm ruler in-situ, which was placed along the maximum diameter of the colony (D1). The ruler was used as calibration to estimate D1 and D2 to the nearest cm for colonies <30 cm and to the nearest 5 cm for colonies >30 cm. Colony size was calculated as (D1+ D2)/2. The camera and GPS clocks were synchronized and every A. tenuis photographed had a date and time stamp, which allowed the exact location of each colony to be mapped along the GPS path. Density was estimated by calculating the number of A. tenuis sampled along each 100 m of the GPS track, with the assumption that each meter swum sampled 4 m2 of reef area. Post-cyclone abundances and colony sizes for A. tenuis were estimated from the belt transect surveys used to census the abundance of the entire Acropora assemblage (described above). Spatial and temporal variation in the density of A. tenuis was examined using analysis of variance. Spatial (between exposed and sheltered sites) and temporal (pre- and post-cyclone) variation in mean colony size was tested using t-tests.Abundance and colony size data for Recruitment to experimental substrata was quantified for the spawning events in the years immediately before (2010) and after (2011) cyclone Yasi. In 2010 recruitment was quantified at two sites (E2 and S1) and in 2011 recruitment was quantified at all six sites . In each year, unglazed terracotta tiles (11×11×1 cm) were attached directly to the substratum in horizontal orientation th category labeled “unknown”. Data for brooding pocilloporids are part of a long-term recruitment study and will be reported in a separate publication. Spatial and temporal variation in recruitment was examined using hierarchical (nested) analysis of variance. Data were log (x +1) transformed to meet the assumption of homogenous variances where necessary.Following retrieval, tiles were systematically examined  using a dissecting photomicroscope. The availability of recruits to experimental substrata was quantified by counting all corallites , irrespective of whether or not they were alive at the time of sampling Wind conditions can have a major impact on surface currents and other hydrodynamic features that influence the retention and dispersal of coral larvae in the first few days after spawning 1, 30 = 71.6, p<0.001), where it ranged from 2.1 (0.2) to 5.3 (0.4) %, than at sheltered sites, where it ranged from 18.2 (2.4) to 30.0 (1.0) % . Acroporidae accounted for <0.1% of benthic cover and <1% of hard coral cover at exposed sites nine months after the cyclone. Hard corals that survived the cyclone at exposed sites were mostly encrusting or submassive non-acroporid spawning corals. Mean (± S.E.) soft coral cover was <1% at all three exposed sites, which was significantly lower than at sheltered sites , where it ranged from 8.4 (2.4) % to 17.0 (3.1) % . Macroalgae accounted for 8 to 16% of benthic habitats at exposed sites but were not recorded at any sheltered site (Mean (± S.E.) hard coral cover was significantly lower at exposed sites (F (1.0) %  but was  (3.1) % . There wred site . Rock cored site .Acropora spp. were highest at E3 (14.0±2.7 S.E. per 40 m2) and S1 (15.3±1.1 S.E. per 40 m2), where colonies were more than twice as abundant than at any of the other four sites  but no significant difference in overall density of colonies of Acropora spp. between exposures . However, the mean colony size of Acropora spp. at sheltered sites (15.2 cm ±1.2 S.E.) was almost four times larger than at exposed sites (4.3 cm ±0.2 S.E.), resulting in a highly significant difference between exposures . Specifically, all colonies of Acropora spp. at exposed sites were smaller than 10 cm  and large (adult) colonies (Acropora found at any of the exposed sites after cyclone Yasi.Mean densities of colonies of ur sites . As suchan 10 cm  and typicolonies . There wA. tenuis was at the exposed site (E2), with a mean density of 0.69±0.05 S.E. colonies per 40 m2, which was significantly greater than mean densities at S1 (0.41±0.08 S.E.) and S3 (0.36±0.04 S.E.) but not at S2 (0.55±0.13 S.E.) . Following the cyclone (2011), there were no colonies of A. tenuis found at any of the three exposed sites, while mean densities (per 40 m2) at the sheltered sites  remained unchanged from pre-cyclone levels .Before the cyclone (2010), the highest abundance of A. tenuis were larger than 10 cm at all sites surveyed  (A. tenuis at sheltered sites (21.3 cm ±1.6 S.E.) were not significantly different from mean sizes before the cyclone .Before the cyclone, more than 70% of colonies of ; S1–S3)  and meanin-situ following cyclone Yasi, whereas at exposed sites every tile deployed had been ripped out of the substratum by the cyclone and none were found.Although fieldwork conducted at exposed and sheltered sites prior to cyclone Yasi did not quantify benthic cover or habitat complexity, photos taken at exposed sites before  A–C and A total of 2874 identifiable coral recruits were sampled on the settlement tiles. In 2010, 958 recruits were sampled at two sites (E2 and S1): 606 recruits in T1 (37 tiles) and 352 recruits in T2 (39 tiles). In 2011, 1916 recruits were sampled across all six sites: 940 recruits in T1 (118 tiles) and 976 recruits in T2 (117 tiles). Acroporids accounted for 94% of identifiable recruits, followed by other spawners (4%) and pocilloporids (2%). In 2010, the maximum number of recruits on a tile was 45 (at E2 in T1) and six tiles had zero recruits, while in 2011, maximum number of recruits on a tile was 29 (at E3 in T1) and four tiles had zero recruits. The following results refer to Acroporidae recruits only.−1 (2010 T1 and T2 combined) did not differ between the exposed (E2: 25.7±4.9 S.E.) and sheltered (S1: 24.9±4.7 S.E.) sites sampled . Recruitment after the cyclone (2011 T1 and T2 combined: E2 − 9.4±1.5 S.E. and S1 − 17.9±2.5 S.E.) was significantly lower than recruitment before the cyclone .Before cyclone Yasi, the mean number of acroporid recruits tile1, 223 = 66.4, p<0.001, 4, 223 = 15.7, p<0.001) and this effect was primarily driven by the consistently higher recruitment at E3 than at either of the other two exposed sites, and the lower recruitment at S2 than at the other two sheltered sites (Following the cyclone (2011), mean recruitment of acroporids in the first two weeks post-spawning (T1) was significantly higher at exposed sites (10.1±1.2 S.E.) than at sheltered sites (5.0±0.8 S.E.) but this trend was reversed in T2 , resultied sites .−1 in the first week post-spawning and 7 to 20 km hr−1 in the second week  to 33.2 (±1.4) km hr−1.In 2010, daily averaged wind speeds recorded at Orpheus Island ranged from 10 to 16 km hrond week . Daily astronger , as werespawning . In 2011Acanthaster planci) on the GBR demonstrated that tropical cyclones account for large proportions of coral loss Acropora .Coral reefs around the world are being increasingly impacted by multiple stressors, which have reduced coral cover and degraded reef ecosystems Acropora cover to <0.1% of all benthic habitats at exposed sites of the Palm Islands, and the surviving colonies of Acropora were very small  and consisted mostly of encrusting bases with a few small branches. A. tenuis juveniles reach 1 cm in diameter at six months of age, and 3 to 5 cm by 1.5 to 2.5 years Acropora at exposed sites nine months after cyclone Yasi indicated that only immature colonies survived the cyclone. It will be several years before these juveniles reach reproductive maturity and contribute to the larval pool.Cyclones rarely reduce coral cover to zero. Damage is typically spatially heterogeneous, depending on the characteristics of the cyclone  and the reef habitats involved  Acropora is the most abundant and species-rich scleractinian genus on the GBR and on reefs throughout the Indo-Pacific Acropora assemblages is essential for the long-term persistence of coral reefs in this region. In the GBR (and the Indo-Pacific), recovery of local Acropora assemblages following disturbance relies heavily on replenishment by planktonic larvae Acropora species relies predominantly on regrowth of live fragments Acropora at exposed sites following cyclone Yasi, recruitment to settlement tiles did not drop to zero, indicating that impacted sites were receiving larval subsidies from external sources in the first spawning event after the cyclone. These findings are consistent with studies demonstrating that the relationship between acroporid abundance and larval supply operates at spatial scales larger than a single reef in the GBR A. millepora across inshore and mid-shelf reefs in the northern and far northern GBR The genus −1) was recorded at the Palm Islands nine months after the 1998 mass-bleaching event Acropora colonies at Orpheus Island) Coral fecundity can be reduced by stress Acropora larvae typically have a pre-competency period of two to three days, after which they begin to search for suitable substratum, and settle between four to seven days post-spawning −1), coral larvae formed slicks that were visible for up to 22 hours as they dispersed away from natal reefs, whereas under strong wind conditions (>25 km hr−1) coral larvae were quickly dispersed throughout the water column Acropora to settlement tiles has been documented for the GBR in the absence of an obvious disturbance A. tenuis at one exposed site in 2010  A. planci infestations A. planci infestations in the early 1990s were not seen in in the early 2000s on reefs impacted by both A. planci and coral bleaching in 1998 (or 2002) Acropora, suggesting that there had not been a strong recruitment pulse post-disturbance Time-series data documenting the recovery of reefs following outbreaks of Acropora assemblages in the Palm Islands suffered a major collapse between 1920 and 1955 Acropora populations in the Palm Islands Acropora from exposed Palm Island reefs. It is unlikely that regrowth of remnant live coral tissues will contribute substantially to recovery of coral communities impacted by cyclone Yasi, particularly as structural complexity has been shown to be important for rapid reef recovery Acropora at those sites, suggest that these impacted populations of Acropora have the potential to recover over time. However, if this relatively slow recovery process is again interrupted by disturbance  or hindered by other stressors , coral populations in the Palm Islands may fail to recover, with the consequence that reefs will undergo phase-shifts to less desirable algal-dominated states Recent paleoecological evidence suggests that


Zebrafish are becoming increasingly popular as an organism in which to model human disease and to study the effects of small molecules on complex physiological and pathological processes. Since larvae are no more than a few millimetres in length, and can live in volumes as small as 100 microliters, they are particularly amenable to high-throughput and high content compound screening in 96 well plate format. There is a growing literature providing evidence that many compounds show similar pharmacological effects in zebrafish as they do in mammals, and in particular humans. However, a major question regarding their utility for small molecule screening for neurological conditions is whether a molecule will reach its target site within the central nervous system. Studies have shown that Claudin-5 and ZO-1, tight-junction proteins which are essential for blood-brain barrier (BBB) integrity in mammals, can be detected in some cerebral vessels in zebrafish from 3 days post-fertilisation (d.p.f.) onwards and this timing coincides with the retention of dyes, immunoreactive tracers and fluorescent markers within some but not all cerebral vessels. Whilst these findings demonstrate that features of a BBB are first present at 3 d.p.f., it is not clear how quickly the zebrafish BBB matures or how closely the barrier resembles that of mammals. Here, we have combined anatomical analysis by transmission electron microscopy, functional investigation using fluorescent markers and compound uptake using liquid chromatography/tandem mass spectrometry to demonstrate that maturation of the zebrafish BBB occurs between 3 d.p.f. and 10 d.p.f. and that this barrier shares both structural and functional similarities with that of mammals.  In mammals, the blood-brain barrier (BBB) provides a complex obstacle to the penetration of drugs into the central nervous system (CNS). The first level of barrier is presented by the tight junctions between endothelial cells of the vasculature. These high resistance tight junctions, made up by proteins such as claudin-5 and ZO-1, render brain capillary endothelia tightly sealed, in contrast to “leaky” endothelial capillaries in the periphery. Thus, there is no paracellular movement of fluid and only minimal pinocytosis from capillaries into the CNS , and in Zebrafish are popular as a vertebrate model with which to perform compound screens ,4 and inExpression of claudin-5 and ZO-1 have been detected in cerebral microvessels of larval zebrafish from as early as 2 and 3 days post-fertilisation (d.p.f.) respectively ,9. FurthIn this study, we set out to investigate how size-dependent exclusion matures during larval development, to determine the degree of similarity in exclusion properties to those of mammalian BBB and to study the cellular components surrounding the vascular endothelium in the brain. We show that zebrafish do indeed gradually develop a sophisticated BBB with active transport systems. This allows for the design of rational strategies for screening compounds for neurological indications and interpretation of results, as well as potentially providing a system to predict whether a novel compound might penetrate the human brain.This work was licenced by the Home Office under the Animals (Scientific Procedures) Act (Home Office licence number PPL80/2074 for work performed at DanioLabs Ltd and PPL 80/2322 for work performed at the University of Cambridge). Protocols were approved by the local Animal Welfare Ethical Review Committee (AWERC); namely, DanioLabs AWERC and University of Cambridge AWERC. Appropriate steps were taken to ameliorate suffering in all work. Experiments were designed with the minimum number of animals necessary to produce meaningful results.y1 transgenic fish [2, 0.33 mM Mg2SO4, 10-5% Methylene Blue) and staged using standard criteria [Embryos were collected from matings of adults of wildtype  and Tg(fli1a:EGFP)nic fish  kept undnic fish . Embryoscriteria . All expin vivo were taken on a Zeiss AxioZoom V16 microscope with Apotome. Dye and saline injected larvae were anaesthetised at 4 or 6 hours after injection and fixed in 4% paraformaldehyde in PBS. Ten larvae per age group and injection treatment were embedded in Sakura Tissue-Tek OCT  and frozen parasagittal sections were cut at 10 µm thickness. Stocks of 2% Evans blue (961 Da) and 10% sodium fluorescein (376 Da) in 0.9% saline were prepared and stored at 4 °C. Larvae were anaesthetised by immersion in 0.2 mg/ml 3-amino benzoic acid (MS222) prior to being immobilised and positioned laterally in 3% methyl cellulose in embryo medium such that the heart was accessible. Larvae and juveniles from 2 days post-fertilisation (d.p.f.) to 30 d.p.f. were injected with 0.5 to 2 nl (depending on age) of dye or saline control into the pericardial region, using a standard zebrafish microinjection apparatus . Fish weSections were visualised without mounting medium by fluorescence microscopy on an Olympus BX51 microscope. Three midline sagittal sections from each larva were identified and images were captured using a ColorView camera (Olympus) and AnalySis software (Soft Imaging System). The fluorescence intensity within the brain was quantified for each midline section of each larva in each treatment group using colour threshold and area measurements in AnalySis. Mean values, standard deviations and Student’s t-tests were calculated using Excel software (Microsoft Office). Larvae were anaesthetised at 3, 5, 8 and 10 d.p.f. by immersion in 0.2 mg/ml MS222, fixed in 4% glutaraldehyde in cacodylate buffer containing 0.006% hydrogen peroxide for 3 hours and washed in cacodylate buffer before post-fixing in osmium tetroxide. Samples were bulk stained with uranyl acetate, dehydrated in ethanol and embedded in Spurr’s resin. Thin parasagittal sections (5 nm) were prepared with a Leica Ultracut UCT, stained with uranyl acetate and lead citrate and viewed in a Philips CM100 electron microscope at 80 KV.Protein sequences of mammalian and zebrafish ABCB gene family members were identified from ENSEMBL . BLAST searches were performed to identify whether un-annotated paralogues could be identified from the zebrafish genome. Sequence alignments were performed using ClustalW.Larvae were anaesthetised at 3, 5, 8 and 10 d.p.f. by immersion in 0.2 mg/ml MS222, fixed in 4% PFA and processed for wholemount antibody staining as previously described . Immunoh/tandem mass spectrometry (LC/MS/MS) analysis without causing toxicity. Maximum tolerated concentration (MTC) was determined using n=6 larvae per group. A non-toxic drug concentration of 15 µg/ml was selected for all subsequent compound exposure experiments except for scopolamine, which was used at 5 µg/ml. 60 larvae  were transferred into a single well of a 12 well plate (Corning) and the final volume of embryo medium per well was adjusted to 1.5 ml. Compounds of interest were prepared as 2 mg/ml stocks in dimethyl sulfoxide (DMSO). 11.25 µl of stock was added to each well, except for scopolamine (3.75 µl). Larvae were incubated in the drug for 1 hour at room temperature then anaesthetised on ice. For whole uptake analysis, anaesthetised larvae were transferred to 1.5 ml microfuge tubes and all excess liquid was removed. 100 µl ice cold PBS was added, samples were briefly centrifuged and all excess liquid was removed. For brain versus body uptake analysis, larvae were anaesthetised by chilling prior to decapitation using fine iridectomy scissors and No. 5 watchmaker forceps. Head and body tissue samples were collected in separate microfuge tubes on ice and excess liquid removed. 100 µl ice cold PBS was added and samples were briefly centrifuged and all excess liquid removed. The weight of the tissue sample was measured and the samples were frozen at -20 °C prior to extraction for LC/MS/MS. Initial drug exposure assays were performed in larval zebrafish to identify a sufficiently high concentration of drug that could be detected by liquid chromatographyFor studies investigating the effect of Abcb1/4/5 efflux on drug distribution, larvae were exposed to the ABCB1/4/5 efflux inhibitor verapamil at 50 µg/ml, a concentration previously demonstrated to be non-toxic, for 2 hours prior to exposure to the test compound. Test compound was added to the well containing 50 µg/ml verapamil and larvae were incubated for a further hour at room temperature prior to tissue collection as described. For subsequent LC/MS/MS analysis, tissue samples were thawed and after addition of an internal standard (clozapine) extracted into an organic solvent . The solvent extract was then evaporated and reconstituted in methanol/acetic acid, except for scopolamine exposed samples, which were reconstituted in acetonitrile/ammonia. Liquid chromatography for haloperidol, desloratadine and diphenhydramine (100 µl injections of extracted samples) was carried out using a Phenomenex Luna C18(2), 50 x 2 mm, 5 µm analytical column with a mobile phase gradient given in Rhodamine 123 (R123) was dissolved in 10% ethanol/0.9% saline to make a 0.5 mg/ml stock. Dye exclusion experiments on larvae at 3, 5, 8 and 10 d.p.f. were performed as described above using 0.2 nl R123. Some larvae were exposed to verapamil at 50 µg/ml for 2 hours prior to and during intrapericardial injection of R123 and for 3 hours after injection. At 3 hours after injection, larvae were anaesthetised and fixed in 4% PFA prior to processing for frozen sections. Quantification of fluorescence in the brain was performed as described above. y1 larvae at 3, 5, 7 and 10 d.p.f. were fixed in 4 % PFA, then processed for antibody staining as previously described [Tg(fli1a:EGFP)escribed . The GFADye exclusion experiments have long been used in rodent investigations into the integrity of the BBB ,18–21. Tabcb1  gene [abcb5  whereas in zebrafish, abcb1, abcb6 and tap2 are missing, abcb11 is duplicated (abcb11a and abcb11b) and, in addition, zebrafish possess abcb3l1. Phylogenetic analysis of the mammalian and zebrafish abcb family suggests that the zebrafish abcb5 (ENSDARG00000021787) and abcb4 genes (ENSDARG00000010936) are the closest homologues of mammalian ABCB1/4/5  gene . Howeverbcb1a by ) and ENSbcb1b by ). In manBCB1/4/5  , and since mammalian ABCB4 and ABCB5 are known to be expressed in the vascular endothelial cells of the CNS [y1 larvae at 10 d.p.f. revealed specific staining in the vascular endothelium of the CNS. A range of larval ages were then examined to determine the earliest time point with specific vascular staining. Staining was observed in the vascular endothelium of the CNS at 8 d.p.f. (abcb5 (ENSDARG00000021787) or abcb4 gene (ENSDARG00000010936) is the functional homologue of mammalian ABCB1 and is hereafter referred to as abcb1/4/5.Using an antibody against a highly conserved amino acid sequence found in all mammalian Pgp isoforms  and also8 d.p.f.  but not 8 d.p.f. . Based oDye exclusion experiments were performed with Rhodamine 123 (R123), a fluorescent substrate for ABCB1 , ABCB4  and ABCBA panel of 5 drugs known to either cross or not cross the BBB in mammals were selected to test in zebrafish larvae. Larvae of different ages were exposed to these compounds at non-toxic concentrations for 1 hour prior to collection of head and trunk tissue for analysis of drug concentration. A generic extraction method was developed and LC/MS/MS was performed to analyse the concentration of drug in head and trunk tissue in larvae of different ages. Drug/tissue concentrations were normalised against a standard and the relative amount of drug in head and trunk samples was compared to determine whether the drug distribution altered with larval age . The amoAll drugs were equally distributed in head and trunk samples at 5 d.p.f., indicating the absence of a BBB at this age. Furthermore, diphenhydramine and haloperidol, which do cross the BBB in mammals, showed an equal distribution in head and trunk samples at all ages examined, indicating that these drugs also cross the zebrafish BBB. In scopolamine treated larvae, the drug level was below the detection limits at 5 and 8 d.p.f. However, scopolamine was detected at equal concentrations in head and trunk samples in 10 d.p.f. larvae, suggesting that scopolamine also crossed the BBB, as in mammals. In contrast, scopolamine N-butyl bromide and desloratadine, two drugs that do not cross the mammalian BBB, showed lower concentrations in the head versus the trunk at 10 d.p.f. and from 8 d.p.f. respectively, suggesting that these compounds are also excluded from the brain in zebrafish larvae. Exclusion of desloratadine at 8 d.p.f. correlates with the earliest expression of Abcb1/4/5 in the vascular endothelium. Since it is known that this drug is actively exported by Pgp (ABCB1) in mammals, we investigated whether the distribution of this drug would be altered by co-incubation with the ABCB1, ABCB4 and ABCB5 inhibitor, verapamil. Co-incubation with verapamil resulted in equalisation of the distribution of desloratadine in head and trunk samples at 8 d.p.f and an accumulation in the head versus the body at 10 d.p.f. Therefore, the exclusion of desloratadine from 8 d.p.f is dependent on the function of Abcb1/4/5 in zebrafish. y1 larvae at 10 d.p.f. but not earlier , we undertook an anatomical timecourse analysis using transmission electron microscopy (TEM) to determine whether there was also anatomical evidence for a BBB and if so, to characterise the maturation of these features. At 3 d.p.f., no evidence of tight junctions in the vessels around the brain and spinal cord was observed . At 5 d. earlier .Several studies have demonstrated that dyes or enzymatically active compounds are retained within cerebral microvessels of the larval zebrafish brain from as early as 3 d.p.f. ,9–11, coActive transport systems play a critical role in the function of the BBB in mammals. The most significant transporter in mammals is ABCB1 protein (Pgp), although other ABCB family transporters and organic anion transporters are also expressed by brain endothelial cells , believed to be a full transporter (i.e. the single protein functions as an efflux pump) with expression restricted to the testis; ABCB5 beta (812 aa) encodes a half-transporter which is only active as a dimer and expression is restricted to pigment cells. Evolutionary analysis of ABCB5 has shown that the ancestral gene is a full transporter and that ABCB5, 1, 4 and 11 share a common ancestor which began duplicating early in the evolution of the chordate lineage. Zebrafish have homologues of ABCB4 and ABCB5, but not ABCB1. The expression of zebrafish Abcb1/4/5 in the vasculature suggests that these proteins may fulfil the role of mammalian Pgp. By immunohistochemical and dye exclusion experiments, we have shown here both anatomical and functional evidence for active transport of molecules across the BBB in zebrafish. Abcb1/4/5 is first detected in the vascular endothelium of the CNS at 8 d.p.f. Thus whilst non-Pgp/ABCB5 substrate small molecules (e.g. sodium fluorescein) can penetrate the BBB and remain in the brain until day 10, Pgp/ABCB1/4/5 substrates (e.g. R123 and desloratidine) are actively effluxed from day 8.The presence of the BBB has been one of the greatest obstacles in the development of drugs to treat neurological conditions, with less than 1% of small molecules penetrating this barrier. Various models have been developed to predict drug permeation through membranes. For example, measurements of drug partitioning in solvents is widely employed, although perfusion through solvents is not identical to diffusion across biological membranes and provides no information about active transport systems . Indeed,in vivo tool for the prediction of compound distribution. In accordance with other studies on compound uptake in larval zebrafish [in vivo, but not for accurate quantification. For example, analysis of compound uptake in head versus trunk at 5 d.p.f. and 10 d.p.f. could be used as a method for predicting BBB penetration. In addition, analysis of drug distribution at different larval ages (i.e. before and after the onset of Abcb1/4/5 expression) allows analysis of whether a particular drug is actively effluxed. Since the use of zebrafish screens to identify novel therapeutics is gaining popularity [Here, we have demonstrated that zebrafish larva may be used as an ebrafish ,33, we oebrafish . While tpularity ,36, thesin vivo model for the prediction of BBB permeability for novel compounds.In summary, the demonstration of a BBB in zebrafish larvae lends further weight to their importance as an experimental species for drug discovery, particularly for neurological indications. Furthermore, since the barrier is formed in larvae by 10 d.p.f., zebrafish may provide a rapid and accurate Figure S1Alignment of human, mouse and zebrafish ABCB4 and ABCB5 protein sequences.A) Alignment of human, mouse and zebrafish ABCB4 protein sequences. B) Alignment of human, mouse and zebrafish ABCB5 protein sequences. Peptide sequences were aligned using ClustalW. (DOCX)Click here for additional data file.Figure S2Expression of zebrafish ABCB1/4/5 homologue at 3 d.p.f.Maximum intensity projection of the cerebral vasculature of Tg(fli1a:EGFP)y1 transgenic embryos at 3 d.p.f. A) GFP labels the vasculature; B) Alexa 568 labelled antibody staining with ABCB1/4/5 antibody; C) overlay. ABCB1/4/5 antibody does not co-localise with the vascular endothelium at 3 d.p.f. although positive antibody staining is observed in the liver primordium (arrowhead). (TIF)Click here for additional data file.Figure S3Expression of GFAP around cerebral vessels.Maximum intensity projections of Tg(fli1a:EGFP)y1 transgenic larvae stained with GFAP antibody. A) At 7 d.p.f., GFAP staining is observed in the glia but does not co-localise with GFAP in the vasculature. B (low magnification) and C (high magnification) At 10 d.p.f., GFAP staining is observed in some cerebral vessels (arrowheads). Scale bar represents 50 µm. (TIF)Click here for additional data file.Table S1LC conditions for haloperidol, desloratadine and diphenhydramine.(DOCX)Click here for additional data file.Table S2LC conditions for scopolamine and scopolamine N-butyl bromide.(DOCX)Click here for additional data file.Table S3MS Scan Parameters under APCI conditions.(DOCX)Click here for additional data file.


Microbiological confirmation of a urinary tract infection (UTI) takes 24–48 h. In the meantime, patients are usually given empirical antibiotics, sometimes inappropriately. We assessed the feasibility of sequentially performing a Gram stain and MALDI-TOF MS mass spectrometry (MS) on urine samples to anticipate clinically useful information. In May-June 2012, we randomly selected 1000 urine samples from patients with suspected UTI. All were Gram stained and those yielding bacteria of a single morphotype were processed for MALDI-TOF MS. Our sequential algorithm was correlated with the standard semiquantitative urine culture result as follows: Match, the information provided was anticipative of culture result; Minor error, the information provided was partially anticipative of culture result; Major error, the information provided was incorrect, potentially leading to inappropriate changes in antimicrobial therapy. A positive culture was obtained in 242/1000 samples. The Gram stain revealed a single morphotype in 207 samples, which were subjected to MALDI-TOF MS. The diagnostic performance of the Gram stain was: sensitivity (Se) 81.3%, specificity (Sp) 93.2%, positive predictive value (PPV) 81.3%, negative predictive value (NPV) 93.2%, positive likelihood ratio (+LR) 11.91, negative likelihood ratio (−LR) 0.20 and accuracy 90.0% while that of MALDI-TOF MS was: Se 79.2%, Sp 73.5, +LR 2.99, −LR 0.28 and accuracy 78.3%. The use of both techniques provided information anticipative of the culture result in 82.7% of cases, information with minor errors in 13.4% and information with major errors in 3.9%. Results were available within 1 h. Our serial algorithm provided information that was consistent or showed minor errors for 96.1% of urine samples from patients with suspected UTI. The clinical impacts of this rapid UTI diagnosis strategy need to be assessed through indicators of adequacy of treatment such as a reduced time to appropriate empirical treatment or earlier withdrawal of unnecessary antibiotics. Urinary tract infections (UTI) are among the most common infections 5 colony forming units/ml Traditionally, a rapid diagnosis of UTI entailed a Gram stain on urine samples A new technology, Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS), has been recently introduced for the analysis of different biomolecules. This mass spectrometry procedure has been successfully used for the rapid identification of microorganisms already isolated by culture In this article, we assess the feasibility of systematically performing a Gram-stain followed by MALDI-TOF MS on urine samples and determine the capacity of this algorithm to anticipate clinically useful information. This algorithm predicted the presence or absence of bacteriuria and the causative pathogen.The study protocol received institutional review board approval by the “Comité Ético de Investigación Clínica Hospital General Universitario Gregorio Marañón” and the need for informed consent was waived.Our institution is a large teaching hospital with 1,550 beds that serves approximately 715,000 of Madrid’s inhabitants. It has an active surgery program including solid organ transplants.5 colony forming units (cfu) per milliliter of one or two different species ©, Siemens, Tarrytown, NY).During May-June 2012, we randomly selected 1000 urine samples from patients with UTI symptoms . All samples were cultured (2.5 µl) on Cystine Lactose-Electrolyte-Deficient (CLED) agar  and incubated in ambient air at 35°C for 18 h. Negative plates were incubated for a further 24 h. The cutoff for a positive culture was ≥10A Gram stain was performed on all uncentrifuged samples and those with bacteria of a single morphotype per 20 oil-immersion fields were processed for MALDI-TOF MS spectrometry.Agreement between the Gram stain and the culture result was defined as follows.Full agreement: the Gram stain result was consistent with the bacteria recovered in the culture. Partial agreement: only one of two microorganisms observed was recovered in culture, or viceversa, one of two microorganisms recovered in the culture was not observed. Disagreement: microorganisms observed were not recovered in culture and/or those not observed did appear on the culture plates.Sample preparation. Each 15 ml urine sample was centrifuged at 1,500 revolutions per minute (rpm) for 10 min to remove leukocytes. The supernatant was collected and aliquoted into 1.5 ml-microcentrifuge tubes and centrifuged at 13,000 rpm for 2 min. Pellets from all tubes were recovered and resuspended in 1 ml high-performance liquid chromatography (HPLC)-quality water . After another centrifugation at 13,000 for 1 min, the pellet was incubated in 0.2% Tween-80 (Sigma-Aldrich) for 5 min and centrifuged at 13,000 rpm for 1 min. The pellet was then washed to eliminate the detergent and centrifuged at 13,000 rpm for a further minute.After discarding the supernatant, the pellet was spotted onto a polished steel MALDI target plate using a 1 µl sterile loop and allowed to dry. Next, it was covered with 1 µl of formic acid (70% vol/vol) and left to dry. Then, 1 µl of matrix solution (α-cyano-4-hydroxy-cinnamic acid solution in 50% acetonitrile and 2.5% trifluoroacetic acid) was added prior to the acquisition of spectra in a mass spectrometer.Samples in which no microorganism identification was possible were further tested after a protein extraction step . All extracted and non-extracted samples were tested in duplicate to determine the reproducibility of the method. The final score for each sample was the average score of the two spots. The turnaround time was 30 min for the first method and 45 min for the second.MALDI-TOF MS. Measurements were acquired in a Microflex LT bench top mass spectrometer  using the default settings. According to the manufacturer, a score ≥2.0 indicated species identification, a score between 1.7 and 2.0 indicated genus identification and a score of <1.7 indicates no identification.true positive, the MALDI-TOF MS and culture result were consistent; MALDI-TOF MS identified only one microorganism when two different microorganisms were isolated in the culture; in patients with a recent UTI diagnosis, on antibiotic treatment and with a negative urine culture, MALDI-TOF MS identified a microorganism concordant with the prior positive urine culture; true negative, a negative culture in the absence of MALDI-TOF MS identification; false positive, MALDI-TOF MS identification differed from the microorganism(s) identified in the cultures; or false negative, a positive culture in the absence of MALDI-TOF MS identification.MALDI-TOF MS interpretation. The MALDI-TOF MS result was correlated with the culture result as follows: Our algorithm was correlated with the culture result, as follows:Match: the information that would be reported to the clinician according to the Gram stain ± MALDI-TOF MS results was anticipative of the culture result. We defined as a match any of the following situations:No microorganisms detected by Gram staining (and therefore no MALDI-TOF MS was performed) in samples returning a negative culture;Microorganisms detected by Gram staining and MALDI-TOF MS identification matched the microorganism(s) identified in samples;Mixed flora detected by Gram staining (and therefore no MALDI-TOF was performed) in samples returning a contaminated culture.Minor error: the information that would be reported to the clinician according to the Gram stain ± MALDI-TOF MS result was partially anticipative of the culture result. We defined as a minor error any of the following situations:No microorganisms detected by Gram staining (and therefore no MALDI-TOF was performed) in samples returning a contaminated culture;Mixed flora detected by Gram staining (and therefore no MALDI-TOF was performed) in samples returning a negative or positive culture;Microorganisms detected by Gram staining in the absence of MALDI-TOF MS identification in samples returning a negative or a matching positive culture.Major error: the information that would be reported to the clinician according to the Gram stain ± MALDI-TOF MS result was incorrect and would have potentially led to inappropriate changes in antimicrobial therapy. We defined as major error any of the following situations:No microorganisms detected by Gram staining (and therefore no MALDI-TOF was performed) in samples returning a positive culture.Microorganisms detected by Gram staining and MALDI-TOF MS identification did not match the culture result.Categorical variables are provided with their frequency distributions. Continuous variables are summarized as medians and inter-quartile ranges (IQR). For the Gram stain, mass spectrometry and sequential algorithm methods, sensitivity, specificity, positive and negative predictive values (PPV and NPV only for the Gram stain), and positive (+LR) and negative (−LR) likelihood ratios, with their 95% confidence intervals, were calculated. Accuracy was defined as the sum of true positive and true negative results. All statistical tests were performed using SPSS ver. 15.0.Over the study period, 1000 samples from 958 patients were randomly selected for inclusion in the study: 43.2% of these patients were adult men (n = 414), and 3.8% were under 18 years of age (n = 36). Median age was 60.2 years (IQR 41.2–76.3) with no differences between sexes. Distribution by sampling technique was: 91.6% midstream voided, 6.8% bladder catheterization, 1.1% obtained during surgery and 0.6% obtained from patients with a permanent urinary catheter. The origin of the samples was: inpatients, 42.1%; emergency department, 28.8%; outpatient clinics, 25.8%; primary care, 2.6%; unknown, 0.7%.Escherichia coli (58.3%), Enterococcus faecalis (8.3%) and Klebsiella pneumoniae (7.9%).Of the 1000 samples examined, 242 returned a positive culture. Out of 252 microorganisms isolated, 208 (82.5%) were Gram-negative, 36 (14.3%) were Gram-positive and 8 (3.2%) were yeasts. In only 10 samples (4.1%) were two different microorganisms isolated. The most frequently isolated microorganisms were All 1000 samples were Gram stained. Microorganisms were detected in 267 samples. Gram negative microorganisms were seen in 52.1% of the occasions, Gram positive microorganisms in 20.2%, yeasts in 5.2% and mixed flora in 22.5%. Out of 733 samples in which no microorganisms were detected by Gram staining, 93.2% (n = 683) returned negative cultures.Full agreement of the Gram stain with the culture result was recorded for 854 or 85.4% of the samples (95% CI 83.2–87.6); partial agreement for 46 or 4.6% (95% CI 3.3–5.9) and disagreement for 100 or 10.0% (95% CI 8.1–11.9). To define the diagnostic performance of the Gram stain, “partial agreements” were considered as true positive results. The diagnostic performance of the Gram stain was: Se 81.3% (95% CI 76.4–86.1), Sp 93.2% (95% CI 91.3–95.1), PPV 81.3% (95% CI 76.4–86.1), NPV 93.2% (95% CI 91.3–95.1), +LR 11.91 (95% CI 9.06–15.67), −LR 0.20 (95% CI 0.16–0.26) and accuracy 90.0% (95% CI 88.1–91.9).The 207 samples yielding a single morphotype in the Gram stain were subjected to MALDI-TOF MS. The performance of this test was as follows: correct identification, 130, and only one microorganism identified, 7 (true positives), for a total of 137 (66.2%); no identification in samples with a negative culture (true negative), 25 (12.1%); incorrect identification , 9 (4.3%); and no identification in samples returning a positive culture , 36 (17.4%).The protein extraction protocol was employed every time MALDI-TOF MS failed to offer an identification. That is, it was used on 84 out of 207 samples (40.6%). This procedure allowed to reach a final identification in 20/207 (9.7%) additional samples and gave 3 false positive results. Without extraction, MALDI-TOF’s diagnostic performance was: Se 68.0% (95% CI 60.8–75.2), Sp 81.3 (95% IC 66.2–96.3), +LR 3.63 (IC95% 1.75–7.51), and −LR 0.39 (95% CI 0.30–0.52). With extraction, MALDI-TOF’s diagnostic performance was: Se 79.2% (95% CI 72.9–85.5), Sp 73.5 (95% IC 57.2–89.8), +LR 2.99 (IC95% 1.70–5.27), and −LR 0.28 (95% CI 0.20–0.40). Thus, without extraction, sensitivity would have been lower.In our study, out of 1000 urine samples, 10 were found to have 2 different species in cultures; MALDI-TOF MS identified 1 of the 2 isolates in two samples, provided an unreliable identification in one sample and provided no identification in the remaining 7 samples.In False positive MALDI-TOF MS results did not match the Gram stain results. True negative MALDI-TOF MS identifications corresponded to false positive Gram stain results. False negative MALDI-TOF MS results did not correspond to any particular bacterial species.We did not find any differences in the performance of MALDI-TOF MS with the various types of organisms causing UTI. Nevertheless, this could be due to small sample size.The combined performance of the two techniques is shown in Of 207 samples in which a single morphotype was detected by Gram staining, MALDI-TOF MS provided an identification in 146 samples and provided no identification in 61 samples. When MALDI-TOF MS provided an identification, this was correct in 93.8% (137/146) of cases, whereas when no identification was given, this negative result was erroneous for 59.0% (36/61) of the samples.When we examined the spectra recorded in 31/36 positive samples for which no MALDI-TOF MS identification was achieved, three intense peaks were observed, corresponding to the human α-defensins 1, 2 and 3, at a mass to charge ratio of around 3440 Da. The presence of these molecules is known to suppress bacterial protein peaks and therefore prevent database matching In summary, when a urine sample in which a single morphotype was detected by Gram staining returned a positive MALDI-TOF MS result, the likelihood of this result being correct (as established by the subsequent culture result) was 93.8%. When MALDI-TOF MS provided no result but peaks corresponding to human α-defensins were observed in the MALDI-TOF MS spectrum, a positive culture was obtained the following day. However, when MALDI-TOF MS provided no result but no such peaks were detected, in 25/30 of the cases the subsequent cultures were negative.The time needed for these two rapid techniques was significantly shorter than that required for the more conventional culture methods. For all samples, results were available within one h. We have included in By performing a Gram stain followed by MALDI-TOF MS directly on urine samples we were able to predict the presence or absence of bacteriuria and the causative microorganism in patients with a suspicion of UTI reasonably well and within a working laboratory shift. According to our findings, physicians would receive therapeutically helpful information for 96.1% of samples in less than 1 h. Only in 0.4 out of 10 cases would inappropriate information be provided.Managing patients with a UTI entails a considerable amount of work for clinicians and microbiology laboratory personnel The usefulness of Gram staining uncentrifuged urine to identify significant bacteriuria was first demonstrated in 1968 5 cfu/ml should be reported, as these are 73.9 times more likely to correlate with a clinically significant UTI than lower counts Some authors propose that only counts ≥10The clinical impacts of rapidly reporting Gram stain information on urine samples submitted for culture have not been sufficiently addressed and only one previous study including 57 samples has examined this issue to date The general consensus is that MALDI-TOF MS is a fast, reliable and cost-effective technique that is easily implemented 5 cfu/ml and can therefore be identified by MALDI-TOF MS Some authors have warned of the need to improve the sensitivity of this technology, although they admitted that the majority of urine samples collected from patients with common urinary tract infections have bacterial counts >105 cfu/ml do, of course, exist. Present rapid screening tests  are not sensitive enough for samples with bacterial counts under this threshold UTI with counts under 102–3 cfu/ml.In patients with upper or complicated UTI, if there is no clinical improvement within 48 hours of starting antibiotic treatment, in the case of recurrent UTI, or if there are any other complicating factors, then no pre-screening should be done and these urine samples should all be cultured, giving value to counts ≥10Candida glabrata using our diagnostic algorithm only support an identification at the genus level, which is not more informative for the clinician than the Gram stain. The same applied to urine cultures positive for certain Gram positive microorganisms such as Streptococcus agalactiae and Streptococcus gallolyticus. Still, for the Bruker MALDI Biotyper system, lowering the species-secure score cutoff to >1.7 instead of the manufacturer’s recommendation of ≥2.0 is now deemed acceptable by some authors There is some concern regarding the performance of MALDI-TOF MS directly on clinical samples testing positive for specific microorganisms. It is well known that yeast identification at the species level achievable by MALDI-TOF MS in positive blood cultures, even after protein extraction, is poor Regarding mixed urine cultures, in a recent study by Wang et al., the MALDI-TOF MS detection of 2 microorganisms present in urine specimens was tested by spiking sterile saline aliquots with 2 different bacterial species at different dilutions. The authors observed that the two types of bacterium could be identified in a mixture only if they appeared at ratios of 1∶1 or 1∶2 a priori for high bacterial counts revealed an overall sensitivity of the MALDI-TOF MS method of 60.7% 5 cfu/ml.In a study by Köhling et al., 107 urine samples from patients with clinical suspicion of UTI that were not preselected In our study, after reviewing the medical records of the patients with human α-defensins, we could not find any common clinical feature among them with regard to type of patient, underlying disease, clinical presentation or outcome.The potential clinical impacts of being able to identify the causative pathogen of any infection is directly related to how much knowing the taxon would serve to direct empiric antibiotic therapy Enterobacteriaceae, Staphylococcus aureus).Our serial diagnostic algorithm Gram stain plus MALDI-TOF MS was able to provide in under an hour an etiologic diagnosis of UTI. This diagnosis allows for the adjustment of empiric antibiotic treatment, especially when the causative microorganism are not those most frequently associated with this type of infection  or when they have resistance mechanisms (i.e., AmpC betalactamase-producing As a limitation of our study, we should mention that being a single-center study, its results may only reflect local practice patterns. However, we included 1000 urine samples from all types of patients and did not preselect any subgroup with greater clinical suspicion of UTI. A disadvantage of the method proposed is that it requires a minimum sample volume of 15 ml of urine.Future studies should address the clinical impacts of our rapid diagnosis algorithm for UTI by examining its capacity to improve the adequacy of treatment in terms of reducing the time of empiric antibiotic treatment or allowing for the earlier withdrawal of unnecessary antibiotics.


Sus scrofa) genomic diversity within and between sites and compared them with human and other livestock. The current Porcine 60K single nucleotide polymorphism (SNP) panel has an average SNP distance in a range of 30 - 40 kb. Most of genetic variation was distributed within populations, and only a small proportion of them existed between populations. The average heterozygosity was lower in pig than in human and other livestock. Genetic inbreeding coefficient (ISF), population differentiation (STF), and Nei’s genetic distance between populations were much larger in pig than in human and other livestock. Higher average genetic distance existed between European and Asian populations than between European or between Asian populations. Asian breeds harboured much larger variability and higher average heterozygosity than European breeds. The samples of wild boar that have been analyzed displayed more extensive genetic variation than domestic breeds. The average linkage disequilibrium (LD) in improved pig breeds extended to 1 - 3 cM, much larger than that in human (~ 30 kb) and cattle (~ 100 kb), but smaller than that in sheep (~ 10 cM). European breeds showed greater LD that decayed more slowly than Asian breeds. We briefly discuss some processes for maintaining genomic diversity in pig, including migration, introgression, selection, and drift. We conclude that, due to the long time of domestication, the pig possesses lower heterozygosity, higher ISF, and larger LD compared with human and cattle. This implies that a smaller effective population size and less informative markers are needed in pig for genome wide association studies.We have reviewed the current pig ( Even so a large number of local breeds are maintained especially in China. It is commonly held that current European and Asian pig breeds are domesticated from different ancestors that might have different genome wide diversity and extent of linkage disequilibrium (LD). Worldwide, domestic pigs can be divided into two main clades: Asian- and European-types, which diverged from each other around 58,000 years ago. The European wild boars were domesticated at least in the 4th millennium BC and then rapidly spread throughout Europe , 2. AsiaInitial genetic studies in the pig mainly focused on investigating the genetic diversity of a breed at relatively small numbers of individual sites, which is essential for sustainable management of genetic resources for future utility. With the identification of the first useful DNA marker HAL1843) in the ryanodine receptor gene for marker assisted selection 3 in the , 6 or byThe development of high throughput genomic techniques provides us with an opportunity to assess genome wide diversity and its structure. Since the earlier draft sequence published for human 2001) and cattle (2009), an increasing number of studies have aimed at studying genomic diversity and LD in human and cattle -16. For  and cattet al. [The focus of this review is on pig, however, we will compare the pig with results from human as well as other livestock species. We begin by discussing pig genomic diversity within sites and then addressed the genomic diversity among sites described by LD. In each case, we briefly compare pig genomic diversity with human and other livestock. The mechanisms for maintaining genomic diversity in pig are briefly discussed, including the factors of migration, introgression, selection and drift. A comprehensive review on population genomic diversity from the perspective of evolutionary theories is detailed in this volume by Hu et al. . 2.2.1Pig genomic diversity within populations is quite variable. Observed heterozygosity ranged from 0.35 to 0.60, with an average ~ 0.5 across 17 autosomal chromosomes in 11 European pig breeds . These vA large number of SNPs are currently available for most species, such as human , cattle These SNPs were used to construct a panel using the Illumina platform, 64,232 SNPs were decoded and selected [2.2.1 [Since pig breeds from Asian and European populations were domesticated from different ancestors, they are expected to have different genetic diversities. One suggestion is that Asian wild boars had higher genetic diversity compared to European wild boars . The nuc1 , 25-26. Nei’s genetic distances between populations in Table 2 [Nei’s genetic distance was 0.531, with a range from 0.194 to 1.188. In European populations, the average genetic distance was 0.495, ranging from 0.163 to 1.122. Higher average genetic distance was observed between European and Asian clades (1.434), which was nearly three times larger than those within populations. The smallest value was even up to 1.119 between Duroc and Tibet pigs [et al. [The genetic distance among populations is very varied reflecting the differences in domestication model and history in the two regions. We have summarized the  Table 2 , 25, 26.STF from the literature [2. The average STF value (0.257) between Asian and European populations was much higher than those within populations. When compared within the two different regions, the overall genetic differentiation for Asian populations was ~ 0.227, ranging from 0.182 to 0.294, much higher than that for European (0.134). The STF value between domestic and wild boars for Asian (0.29) was also higher than that for European (0.194) [STF was between Asian and European breeds , and the smallest was observed within European breeds  [Most genetic variation occurred within populations rather than between breeds from Asia and Europe. A summary of terature , 25, 26 ropean 0.4 [19, 25 (0.194) . A recenrkshire) . All Asian and European pigs were closely related in terms of their maternal lineages, but they were different from each other. In wild boars, the average percentage sequence divergences calculated by mtDNA were 0.2370 within Chinese population, 0.3718 within European, and 0.5603 between Chinese and European. In domestic populations, the pairwise nucleotide sequence divergence was much lower (0.0056) between Chinese and European breeds . Asian d2.3.3. It shows that heterozygosity in pig was smaller than that in human and other livestock, but more flexible. Genetic variation within populations and genetic distance were much larger in pig than in human and other livestock. A comparison of pig genomic diversity with human and other livestock is summarized in Table STF) was lower in human than in pig . In human, STF was 0.05 - 0.13 for autosomal SNPs [Alu insertions polymorphism [et al. [STF).The average expected heterozygosity in human ranged from 0.7 to 0.9 , which wmal SNPs -43, ~ 0.morphism . Biswas  [et al.  indicate3). Recent studies [ISF) ranged from 0.03 to 0.32. The observed and expected heterozygosities were at ranges of 0.42 - 0.67and 0.61 - 0.73, respectively. Among different goat breeds, Nei’s genetic distance differed from 0.067 to 0.830. The genetic diversity between breeds (STF) ranged from 0.012 to 0.200. The overall proportion of genetic differentiation among breeds was around 0.14, such as 0.02 in Guadarrama goat [The genetic diversity was more extensive in goat than in pig . The observed and expected heterozygosities in cattle were at the ranges of 0.47 - 0.74 and 0.45 - 0.78, respectively. While the genetic diversities both within (ISF) and between breeds (STF) seem to be a slightly lower in cattle than in pig. Nei’s genetic distance was much larger in pig (0.163 - 1.794) than in cattle (0.015 - 0.382). Compared to pig, cattle genetic diversity has been more widely studied. Genetic diversity in Chinese breeds was higher than that in European breeds. This is similar to that found for the pig. In Chinese cattle breeds, the mean heterozygosity was at a range of 0.69 - 0.76, and Nei`s genetic distance ranged from 0.025 to 0.352 [ISF) and between (STF) populations were slightly lower. Nei’s distance was at a range of 0.084 - 0.382 [The average heterozygosity in cattle is slightly higher than that in pig  than for SSC15 (0.15 - 0.19) [D´ was higher on SSC7 than on SSC4, and LD decreased faster with the marker distance on SSC4 than on SSC7 [chromosomes 10 and 12 had the lowest average LD, chromosomes 1, 13, and 14 had the highest value [2r). When considering longer SNP pair distance (3 Mb), differences in LD between different chromosomes were even larger up to 0.2  [2r. The significant LD variation among different regions was mainly due to the strong artificial selection on economic traits.Within pig populations, a wide range of LD was observed on different chromosomes. With 29 and 5 microsatellite markers located on SSC15 and SSC2 in different lines, respectively, LD in terms of  - 0.19) . By inve on SSC7 . Using tst value . Using t2 value) . It indiD´ and 2r are widely used to evaluate inter-site associations in livestock. D´ strongly depends on allele frequency, and it decreases as the MAF increases. 2r is more influenced by linkage distance. By using ~ 4,500 autosomal SNPs in more than 6,000 pigs from six commercial lines [D´ was observed in case of MAF as 0.067. The 2r values indicated that the largest LD was in the most tightly linked group. As the linkage distance increased from 0.1 to 40 cM, the average 2 rreduced from 0.371 to 0.008. The observed average 2r began to rapidly decrease when the linkage distance approached to 3 cM. Amaral et al. [2r being 0.3 as a threshold. Marker density and sampling size are the main factors that affect the accuracy of LD evaluation, which further influences the design of whole genome wide association studies. Nsengimana et al. [D´ value was feasible in commercial pig populations for genome-wise association. Du et al. [et al. [al lines , the higl et al.  used higa et al.  suggesteu et al.  recommenu et al.  up to 40u et al.  with com [et al.  predicte3.2.2r = 0.3). However, for Chinese breeds with a similar sample size, the SNP spacing was 0.005 cM, and 500,000 SNPs per individual would be required [The genome wide inter-site associations in terms of LD differ among different populations. Modern breeding programs increased the extent of LD and caused significant differences between European and Chinese pig breeds. LD decayed more rapidly in Chinese breeds than in European breeds, indicating that the extent of LD was smaller in Chinese breeds than in European breeds. In European breeds, LD extended up to 2 cM, and large haploblocks could be up to 400 kb; whereas in Chinese breeds, the extent of LD was smaller (0.05 cM) and generally did not exceed 10 kb , 58. Chirequired . This larequired . 2r > 0.3 and 2 r> 0.2 were 49% and 57%, respectively; while the corresponding percentages for Yorkshire were 52% and 60%. At the average useful 2r > 0.2, the SNPs density in Landrace and Yorkshire extended to 1.0 and 1.5 Mb, respectively, which resulted in a smaller effective population size (eN) for Yorkshire (55) than for Landrace (80). Similar eN were also obtained in American Landrace (74) and Berkshire (77), but higher in Hampshire (109) and Duroc (113) [D´ was also highly significant in Yorkshire, Large White and Landrace populations. The lowest D´ was in Large White breeds, and the highest was in Duroc breeds [Contrary to Chinese breeds, European breeds showed significant differences in LD . By usinoc (113) . This, tc breeds . 3.3.4. It shows that, at the same 2 rthreshold, LD was significantly larger in pig than in human, but much smaller than in sheep. Compared with cattle, LD was much larger in pig at threshold of 2 r= 0.2 but similar to cattle at threshold of 2 r= 0.1. A comparison of LD among pig, human, and other livestock is summarized in Table 3.3.1.et al. [Due to a smaller effective population size and the stronger selection that has occurred in livestock, LD was significantly greater in pig (1 - 3 cM) than in human. The average LD in human was up to ~ 30 kb with a high variability, depending on the different populations and marker density used. Computer simulations and empirical data suggested that human LD extended only 3 - 5 kb for common disease SNPs, meaning that approximately 500,000 SNPs at least was needed for whole-genome studies , 61. Latet al.  analyzedeN in different populations (see above for examples in pigs). For the SNP-pair interval less than 100 kb across all of the genome, thee N estimate was ~3100 for western European, Japanese and Chinese, and ~7500 for the Yoruba population [eN and fewer informative markers required for genome wide studies, at least for the European pig populations.The LD information is very useful for estimating pulation . This re3.3.2.2r > 0.2 [et al. [2r was 0.14 at marker distance of 100 kb. It indicated that at least 50,000 SNP markers were required for whole genomic selection. McKay et al. [2r was 0.15 - 0.2 at a physical distance of 100 kb SNP loci apart, indicating that 30,000 - 50,000 markers were needed to conduct whole genome association studies. When microsatellite markers were used with four cattle breeds to estimate LD [2r value across the populations was 0.16 at a range of 0.11 - 0.22. For the distance greater than 50 cM, the average 2r declined to 0.07. Compared with cattle, pig LD was much stronger, especially for the useful 2r > 0.2.Generally, cattle have larger LD than human, but smaller LD than pig. LD in cattle was at a range of 40 - 100 kb at the threshold of r2 > 0.2 , 15, 67. [et al.  comparedy et al.  reportedimate LD , at a ma2r and 8.2 Mb for D´. It indicated that at least 75,000 SNPs and a sample size of 75 or 400 would be required for whole genome association study. Qanbari et al. [2r) was observed for SNPs less than 100 kb apart. For American Holstein cattle, 2r was much larger (0.59) at the same marker distance (100 kb) [2r > 0.8) in genomic regions with marker distance less than 50 kb. LD for SNP pair intervals 100 kb apart (2r = 0.14) was similar to that in Holstein-Friesian cattle. Compared with pig, LD among populations varied less in cattle. This is in agreement with the common notion that a larger proportion of genetic variation exists within than between populations in most organisms. Extensive LD in cattle varied a little among different populations. In Holstein-Friesian cattle, by analyzing 1,566,890 syntenic and 365,400 non-syntenic SNP pairs that cover all autosomes , the sigi et al.  used Ill(100 kb) . Kim & K(100 kb)  also rev3.3.3.D`) was observed for syntenic marker-pairs with the interval less than 60 cM. By using 490 markers in Soay sheep [D´ > 0.2 as for marker-pair distance < 10 cM. Meadows et al. [D´ value between non-syntenic marker pairs ranged from 0.266 (Poll Dorset) to 0.322 (Merino × Border Leicester). 2 rdecayed faster within Merino and Merino × Border Leicester. LD varied a lot among populations in sheep, similar to the results in pig.Compared with pig, LD in sheep seems much more extensive and flexible. High LD extending to 10 - 60 cM was observed in two domestic sheep breeds evaluated by microsatellite markers . Large fay sheep , 22% mars et al.  used 28 4.4.1.th centuries. When European wild boars were domesticated, they rapidly became the predominant lineage within European domestic swine [e N, subsequent migration can lead to a high level of polymorphism. While maternal introgression from European domestic pig has no or very little impact on Chinese breeds. That is why some European haplotypes were detected in some local breeds, but no European mtDNA haplotypes were detected [A significant differentiation in genetic diversity and LD exists between Asian and European pig populations. One reasonable explanation is that their domestications originated from different ancestors. However, they shared some important haplotypes with each other, which might come from extensive migration between populations and incomplete lineage sorting. Historic and demographic events revealed that Near Eastern pigs were definitely introduced into Europe during the 18 – 19ic swine . So the ic swine . There wic swine , 34, 35.4.2.Efficient artificial selection has a dramatic influence on livestock genome diversity and linkage disequilibrium. This effect depends on the direction, intensity, duration and consistency of selection over time. For a long time, in pigs selection focused on growth and fatness (representing demand for cheap lean meat). The availability of dense marker panels provides the opportunity to consider LD between regions and to search for signatures of selection as a means to identify causative mutations or other useful markers as has been done in several species. For example, two of the first QTL to be identified in the pig were on chromosomes 4 and 7. These include QTL for growth and fatness traits. However, despite significant efforts the genetic variation underlying these QTL has not been elucidated. The extent of LD for SSC7 was significantly larger compared to SSC4 . FurtherModern breeding practices and best linear unbiased prediction (BLUP) selection starting in the last century led to a rapid increase of genetic gain and large LD in pig breeds. This can cause inbreeding that increases LD but reduces genetic diversity. Taking Meishan for example, in order to increase the population size and reproductive performance, an inbred line of Meishan was developed in the late 1980s in a selection scheme. This may explain the lower genetic variability observed within Meishan  comparedSelection reduces genetic diversity on the next generation but enhances LD. Selection on one locus or multiple loci, will increase LD between the neighbouring loci and the selected locus. As a result of intensive artificial selection, livestock have a larger LD than human populations. In addition, selection can cause LD between unlinked loci that contribute to phenotypes undergoing selection . This co4.3.eN (bottleneck effects) and directional artificial selection. The reduction ine N can lead to lower genetic diversity. LD initially generated by genetic drift gradually reduces with time. The genetic drift effects are substantial in historic pig breeds. The development of European and Chinese pig breeds has been different and the study of these differences can provide additional insights on diversity and these processes. It appears that the differences described to date are the result of differences in the number of ancestral stocks as well as the differences in modern breeding systems. Selection is likely to have been stronger in Europe and N America than in Asia. European breeds had smaller effective population sizes, resulting in stronger LD [eN than Hampshire and Duroc breeds, producing different LDs for the same marker-intervals [Domestication in livestock essentially results in two processes: the decline in onger LD . Americantervals , 59. The5.Sus scrofa) and compared them with human and other livestock. It is concluded that genetic diversity is smaller in pig than in human and other livestock. A relatively larger genetic variation within populations (ISF) exists in pig compared with cattle and goat. A majority of genetic variation in pig was distributed within rather than between populations, similar to human and other livestock. A large genetic distance exists between Asian and European populations. Asian breeds harbour much larger variability than European breeds. For the different genetic diversity structures, the inter-site associations described by LD were much stronger in pig (1 - 3 cM) than in human (~ 30 kb) and cattle (~ 100 kb), but less than in sheep (~ 10 cM). LD varied largely among different populations in pig and sheep, but a little in cattle. This review confirms that, strong breeding and selection programs have occurred in pig for a relatively long time, which resulted in the relatively lower heterozygosity and higher ISF and larger LD, compared with human and cattle. This also implies that a smaller effective population size and less informative markers would be needed for pig whole genome association studies (at least for European populations). To some extent, this difference might arise from the joint effects of migration, selection, and drift during the process of pig domestication. In this review, we summarized genomic diversity within and between sites in pig (


Objective. To compare the clinical and radiographic outcomes in patients of different ages who underwent the Dynesys stabilization. Methods. This retrospective study included 72 patients (mean age 61.4 years) with one- or two-level lumbar spinal stenosis who underwent laminectomy and the Dynesys  dynamic stabilization system. Thirty-seven patients were younger than 65-year old while the other 35 were older. Mean followup was 46.7 months. Pre- and postoperative radiographic and clinical evaluations were analyzed. Results. The mean calibrated disc signal (CDS) at the index level was significantly improved from 60.2 ± 25.2 preoperatively to 66.9 ± 26.0 postoperatively (P > 0.001). Screw loosening occurred in 22.2% of patients and 5.1% of screws. The improvement in CDS at index level was seen to be significant in younger patients but not in older patients. Overall, the mean visual analogue scale (VAS) of back pain, VAS of leg pain, and the Oswestry disability index (ODI) scores improved significantly after operation. There were no significant differences in pre- and postoperative VAS and ODI and screw loosening rates between the younger and older patients. Conclusions. There is significant clinical improvement after laminectomy and dynamic stabilization for symptomatic lumbar spinal stenosis. Intervertebral disc rehydration was seen in younger patients. Instrumented spinal fusion is the treatment of choice for degenerative spondylosis with instability refractory to conservative treatment , 2. SpinIn the recent years, there is the emerging option of dynamic stabilization to spare spinal fusion and still yield satisfactory outcomes in the surgical management of lumbar spondylosis and back pain. Fischgrund and colleagues reported application of the Dynesys , a pedicle-based lumbar dynamic stabilization system, as an effective alternative to treat lumbar spondylosis in 1994 –16. TheoThis study aims to compare the clinical and radiographic outcomes in patients of different ages who underwent surgical decompression and Dynesys stabilization. This is the first study focusing on the discrepancies between the elderly and the younger patients. The pre- and postoperative magnetic resonance imaging (MRI) especially of each patient were compared to evaluate the condition of the discs at the index segments. All clinical data was prospectively collected and outcomes were measured by standardized parameters with more than two years of followup.From September 2007 to August 2009, the study enrolled 88 consecutive patients who underwent surgical decompression and dynamic stabilization with Dynesys  in the authors' service. The clinical and radiological data were collected prospectively and then analyzed retrospectively. Three experienced spine surgeons performed all operations in a very similar fashion by the same technique.The inclusion criteria were the patients who had lumbar DDD with persistent symptoms such as intermittent claudication, low back pain, buttock pain, leg pain, or any combination of the above. Each patient underwent preoperative MRI to confirm the diagnosis and had failed medical treatment for longer than 12 weeks. The exclusion criteria were degenerative scoliosis, prior lumbar surgery, disc diseases requiring discectomies, and spondylolisthesis worse than grade II. Patients with psychiatric disorders, cerebral vascular accidents, coexisting cervical or thoracic myelopathy, or neoplasms were also excluded. Among the 88 patients enrolled in this cohort, 72 completed the follow up for more than 24 months and were thus analyzed. These patients were divided into two groups according to the age at operation: young age (<65 years) and old age (≧65 years).Patients were placed in prone position under general anesthesia. Fluoroscopy was used routinely before sterilization to confirm the treated level and keep the lumbar spine in a neutral lordotic curve. Prophylactic antibiotics were infused thirty minutes before incision. After midline incision by subperiosteal dissection, the laminae and spinous process were exposed. The facet joints capsules were preserved intact. Standard total laminectomies and foraminotomies with resection of hypertrophic spur and ligamentum flavum were performed carefully to preserve the complex structure of the facet joints. The exiting and traversing nerve roots were probed through to confirm the adequate decompression without any bony fragment. Bilateral fascial incisions were made by subdermal dissection through the same wound. Along the Wiltse and Spencer intermuscular plane, the titanium alloy screws were placed transpedicularly without destruction of the facet joints . The modThe Oswestry disability index (ODI) scale was used for functional assessment. The 0–10 visual analogue scale (VAS) was used for back and leg pain evaluation. The patients themselves completed the ODI and VAS questionnaire preoperatively and at 1.5-, 3-, 6-, 12-, and 24-month followup regularly. Preoperative scores were compared to postoperative scores.Standard anterior/posterior and lateral radiographs, computed tomography (CT), and magnetic resonance imaging (MRI) studies were routinely performed in preoperative assessment. Radiographs were taken regularly at 1.5-, 3-, 6-, 12-, and 24-month followup.The presence of “halo zone sign” or “double halo sign” on anteroposterior radiographs was defined as screw loosening during followup . CT was Pre- and post-operative MRI images were compared focusing on the condition of the discs, including the indexed and adjacent segments. Due to the lack of an absolute unit of measuring the signal intensities on MRI, for example, the Hounsfield unit used in computed tomography to describe radiodensity, these signals of the same patient are difficult to compare. Ideally the pre- and post-operative MRI studies need to be taken by the very same MRI machine to unify the signal intensity references. We tried to allocate the same MRI machine to each patient; however, not every patient achieved the goal. To address this issue, the digital signal in central intervertebral disc of the levels T12-L1 in T2-weighted image (T2WI) was designated as a reference . The sigt-test, chi-square, and Mann-Whitney U test, where appropriate. All data analysis was processed with the statistical software, SPSS version 17.0 . Statistical significance was set at P value < 0.05.Clinical and radiographic assessments were compared and analyzed using Student's Of the 88 consecutive patients with lumbar spondylosis who underwent 1- or 2-level dynamic stabilization with the Dynesys system, 72 patients (81.8%), in whom 370 screws were placed, completed the clinical and radiological evaluations for at least 24 months postoperatively. There were 37 men (51.4%) and 35 women (48.6%) whose mean age was 61.4  ± 11.3 (31–82) years at the time of surgery .The mean followup duration was 46.7 ± 7.8 (33–58) months. In the 72 patients, 31 (43.1%) underwent a 1-level and 41 (56.9%) underwent a 2-level stabilization. The distributions of index levels were as follows: 3 (4.2%) at L3-4; 23 (31.9%) at L4-5; 5 (6.9%) at L5-S1; 1 (1.4%) at L2-3-4; 33 (45.8%) at L3-4-5; and 7 (9.7%) at L4-5-S1 .P = 0.006). The hypertensive patients in both groups had similar prevalence rates   patients diagnosed of type 2 (diabetes mellitus) DM and 9 (12.5%) patients of hypertension. There were more diabetic patients in older age group (34.3%) than in young age group (8.1%) in current study (= 0.656) .P < 0.001). There was significant improvement in both the young age group  and the old age group    . There w, resp.) .P < 0.001). There was significant improvement in both the young age group  and the old age group    . There w, resp.) .P < 0.001). The young age group specifically improved from 50.4  ±  21.3 preoperatively to 21.8 ± 22.0 postoperatively (P < 0.001) and the old group from 51.7  ± 17.8 preoperatively to 25.2 ± 20.8 postoperatively (P < 0.001)   . There w, resp.) .P = 0.900, P = 0.739, resp.) (There was screw loosening in 16 out of 72 patients (22.2% per patient) and 19 out of 370 screws (5.1% per screw). Specifically, there were 8 of 37 patients (21.6%) and 9 of 188 screws (4.8%) found loosened in the young age group. In contrast, there were 8 of 35 patients (22.9%) and 10 of 182 screws (5.5%) found loosened in the old age group. There was no statistically significant difference found between the two groups in the rates of screw loosening per patient or per screw  .P = 0.014). There was significant increase in the young age group  (P = 0.366). There were no significant differences in pre- and post-operative bridged level CDS between the young and old age groups   with statistical significance, from 60.2 ±25.2 preoperatively to 66.9 ±  26.0 postoperatively (= 0.013) . However, resp.) .n = 37) and the old age  groups demonstrated satisfactory improvement in clinical outcomes. The overall screw loosening rate was 22.2% per patient (16 in 72 patients) and 5.1% per screw (19 in 370 screws). The rate of screw loosening was slightly higher in the old age patients. Regarding disc signal, in the overall CDS of bridged discs, the change significantly improved from 60.2 ± 25.2 preoperatively to 66.9 ± 26.0 postoperatively (P = 0.014). However, the change in the young age group  appeared to be more obvious than in the old age group .The current study collected total 72 patients with lumbar spondylosis who underwent decompression and dynamic stabilization. A total 370 screws of Dynesys were placed during the operations. In a mean follow-up period of 46.7 months, the results of both the young age  of the ODI defined by Hägg et al., and the 1.2 unit improvement of VAS reported by Copay et al., overall there were 68.1% (49/72) of patients had MCID of VAS back pain, 84.7% (61/72) had MCID of VAS leg pain, and 81.9% (59/72) had MCID of ODI , 20. TheAccording to the population projections of the United Nations, the number of elderly patients (older than 65 years) in the world will increase from 8 to 14 percent and the percentage will increase far more to 25 percent in more developed nations between 2010 and 2040. Low back pain and lumbar spondylosis is a common problem in the elderly patients which may greatly affect their quality of life. Pain, numbness, claudication, and risk to fall are the frequent symptoms of the patients. The prevalence of lumbar spondylosis will increase as the population ages , 22. ConP < 0.001). They concluded that increasing age was a predicting factor for complication. However, the presence of complications had no association with final clinical outcomes [Regarding the influence of age, Deyo et al. reported that complications associated with procedures were primarily related to patients' age . The moroutcomes .Dynamic stabilization, Dynesys, is an alternative surgical treatment for lumbar spondylosis. It aims to change the mechanical loading of lumbar spinal segments. Several studies have proved the safety and efficacy of dynamic stabilization but there were only few series involving the Dynesys in the elderly patients , 28–30. P = 0.811). After decompression and Dynesys implant, they both had significant improvement. Regarding the improvement of VAS back pain, 64.9% (24/37) of patients in young age group had MCID and 71.4% (25/35) of patients in old age group had MCID. The treatment of back pain in elderly seems to be as effective as in young patients. The results of VAS leg pain were similar to back pain. Between the two groups, the preoperative VAS for leg pain was the same . After surgery, 78.4% (29/37) of patients in young age group had MCID and 91.4% (32/35) of patients in old age group had MCID. The elderly group seemed to have much better result than the young age group.However, there was no comparative study to discuss the outcomes between young and elderly patients. To date, there is a paucity of long-term data for the application of Dynesys in the elderly patients. The current retrospective study specifically aimed to compare the cohort of young to old age patients with lumbar spondylosis who underwent dynamic stabilization. Regarding the low back pain, the prevalence may be quite high and it may be often underestimated due to inconsistent study design and definition. Cho et al. reported the lifetime prevalence of low back pain was as high as 53.8% in the Asian population . In our P = 0.612). Thirty in 37 (81.1%) young patients and 29 in 35 (82.9%) old patients had MCID in ODI. Both groups had significant improvement after surgery.The preoperative ODI scores were similar between the two groups  at the treated levels. The data demonstrated progressive disc degeneration at bridged level after dynamic stabilization. The authors concluded that the degeneration may be due to natural disease progression [In concern of the bridged disc degeneration, Kumar et al. collected 32 patients with lumbar spondylosis who underwent Dynesys stabilization and completed 2-year follow-up MRI in 2008. Till now, there were only few in vivo reports to discuss disc change. By using Woodend scores to define the degeneration classification, they found that there was a significant increase in the scores from preoperative 1.95 to postoperative 2.52 , but no related symptom was recorded. No other complications (i.e. wound infection) or reoperations have happened to date in the current series.There was significant clinical improvement after laminectomy and dynamic stabilization with the Dynesys for symptomatic lumbar spinal stenosis in both the young and old age patients. The screw loosening rate was slightly higher in the old age patients. Disc degeneration may stop or reverse in the young age patients but not for the elderly patients. Further studies are needed to evaluate the regenerative effect of dynamic stabilization on the intervertebral discs.


Acute kidney injury (AKI) in cancer patients is a complication that causes substantial morbidity and mortality.A total of 1,500 cancer patients admitted to the medical intensive care unit (ICU) between November 2008 and February-March 2011 were evaluated for AKI, defined as an increase in serum creatinine (SCr) >0.3 mg/dl over the baseline value, according to the AKIN stage I definition. Univariate analysis was used to study ICU admission parameters associated with AKI occurrence during the ICU stay.P = 0.004), serum potassium , serum pH , base excess , serum phosphorus , baseline serum creatinine . At multivariate analysis, the following variables at ICU admission were associated with AKI: serum creatinine >1.0 mg/dl , pH <7.38  and need for vasopressors in the first 24 hours . Variables previously thought to be indicative of a poor prognosis  were not predictive of AKI.AKI incidence was 31%, with a mortality rate of 42%, compared with 20% for non-AKI patients. The mean age of all patients was 63.1 ± 11.3 years and 55% were male. Sepsis (44.8%) and respiratory failure (24.8%) were the major reasons for admission to the ICU. At univariate analysis, the following variables at ICU admission were significantly associated with AKI in cancer patients during the ICU stay: need for vasopressors (74.3% vs. 25.7%; AKI is frequent in critically ill cancer patients and has a great impact on mortality. AKI incidence can be better estimated by an evaluation of the acute organ dysfunction at ICU admission than by the characteristics of the underlying malignancy.


The goal of this study was to assess the amount of biofilm within the endotracheal tube (ETT) by CT scan and to evaluate in a bench test a device aimed at ETT biofilm removal. Biofilm grows within the ETT soon after intubation, resulting in increased resistance to airflow. Biofilm detachment from the ETT can result in bacterial colonization of the distal airways. The use of an ETT cleaning device could effectively reduce the patient's airflow resistance burden. The quantification and distribution of biofilm within the ETT might help to evaluate ETT cleaning device effectiveness.We collected ETTs of 11 critically ill patients after extubation. Micro-CT scan  was performed using a resolution of 35 μm. Axial sections of the 20 cm above the cuff were reconstructed, and the volume of secretions was assessed by a density criterion. Microbiological cultures of the ETT lavage fluid were then obtained. Patient's demographics and clinical data were recorded. In a different set of bench experiments, we injected 1 ml water-based polymer into new ETTs of different sizes. We measured resistance to airflow before and after using an ETT cleaning device . We also obtained resistance values of intact ETTs as controls.P = 0.032, R2 = 0.46). Bacterial growth was present in 9/11 (82%) ETT fluids cultures and Candida spp. showed an elevated prevalence . In the bench tests, the cleaning device reduced resistance to airflow (difference before and after cleaning 5.5 (95% CI = 8.9 to 1.6) cmH2O/l/second, P = 0.006). After cleaning, resistance resulted higher than intact ETTs, although with a clinically negligible difference (difference 0.3 (95% CI = 0.2 to 0.6 cmH2O/l/second), P = 0.032).The studied ETTs remained in place for a median of 7 days (IQR range 4 to 15). The amount of secretions assessed by CT scan was 0.293 ± 0.290 ml (range 0.032 to 0.777 ml). Secretion volumes were not related to patient severity at admission  or days of intubation; an inverse correlation with patient's age was present (Micro-CT scan is a feasible and promising technique to assess secretions volume in ETTs after extubation. The use of an ETT cleaning device decreases resistance to airflow in bench tests; the effectiveness of such a device in the clinical setting could be properly assessed by post-extubation CT scan.


It is believed that enteral nutrition (EN) support is the preferred route as compared to parenteral nutrition (PN). Critically ill patients on EN receive less than 60% of their metabolic requirements. To meet patients’ calorie goal addition of PN to EN was proposed. This study was conducted to determine whether supplemental PN have any difference with EN alone in regard to inflammatory indices.Twenty patients were randomized to either receive EN alone or EN+PN for 7 days. Pre albumin and inflammatory indices including interleukin IL-1, IL-6 and tumor necrosis factor-α (TNF-α) were measured on days of 0, 3,7. Also Sequential Organ Failure Assessment (SOFA) score and Therapeutic Intervention Scoring System-28 (TISS-28) score were calculated on days of 0, 3 and 7.IL-1, IL-6 and TNF-α did not show significant difference between two interventions. Pre-albumin was increased from baseline by 9% and 81% in EN and EN+PN groups respectively but it did not reach to statistical significance. SOFA score did not show significant difference. TISS score was higher in EN+PN group on days of 3 and 7.No difference was found between EN and EN+PN regimens in regard to inflammation, while severity of illness may not change with these regimens, nursing workload increases with implementation of supplemental PN. Providing nutrition to critically ill patients is being considered as a primary therapeutic strategy . It is bThis study was a randomized, controlled clinical trial carried out in a 10 bed ICU of Sina teaching hospital  approved by institute ethic board. Written consent forms obtained from patients’ relatives.Two groups of 10 patients were enrolled between November 2007 and May 2009. Patients eligible for inclusion were those of 18 years old or over, recent ICU admission (<24 hrs), having systemic inflammatory response syndrome (SIRS), and Acute Physiology and Chronic Health Evaluation II (APACHE II) score greater than 10 and expected not to feed via oral route for at least 5 days (Patients were fed via naso-gastric (NG) tube with Fresubin® Original  a solution with 1 kCal/ml energy. An average 70 kg patient received 50 ml of this solution initially every 3 hrs which increased with 50 ml increments to a maximum of 300 ml every 3 hrs with a rate of 100 ml/h. Prior to the next feeding, residual volume was checked and if it was greater than 300 ml feeding was delayed by 3 hrs and metoclopramide was administered.PN consisted of 500 ml of 10% amino acid solution , and 500 mL of 50% dextrose solution  infused over 24 hrs. Enteral support in this group was the same as EN group.The patient's demographic data, cause of admission, APACHE II score and serum albumin were recorded on admission (day 0). Sequential Organ Failure Assessment (SOFA) score and TISS-28 score were calculated on days of 0, 3 and 7 to evaluate severity of illness and to determine number of interventions, respectively , 11. LenP values less than 0.05 was considered significant.Statistical analysis was performed using SPSS- 11.5 software . Non-parametric data were analyzed using Mann-Whitney test. Normally distributed data were analyzed using repeated measures analysis of variances and student t-test. From 20 patients that were entered in the study , one patp=0.658; The patients serum pre-albumin was increased from baseline by 9% and 81% in EN and EN+PN groups respectively but it did not reach to statistical significance p=0.658; .Results showed that patients who received EN+PN received more energy than patients on EN alone , 13. EN+p>0.05; p>0.05). IL-1 concentrations on the day of 7 increased by 3% in EN group and decreased by 5.6% in EN+PN group from baseline (p>0.05).Levels of IL-6 did not change significantly from baseline up to day7 p>0.05; , but itsIn contrast to reports that TNF is significantly lower in EN than TPN patients  and malnIL-6 is an independent outcome predictor in the ICU  and in tIn agreement with results of other investigation , 19, 20 In a systematic review of five clinical trials comparing EN versus EN+PN, no significant differenc on mortality were reported and it was concluded that in well-nourished patients with intact GI tract, supplementing PN to EN has no clinical benefit . Route op=0.133), but on days of 3 and 7 two groups showed significant difference (p=0.03). TISS scores were higher in EN+PN group (35.40 vs.39.30 on day of 3 and 36.50 vs. 39.38 on day of 7).TISS score did not show significant difference on the day of 0 . The mean length of the patient stays in the ICU was 27.70 in EN and 25.70 days in EN+PN group (p=0.785).Mean length of hospitalization of the patients were 36.50 and 37.40 days in EN and EN+PN groups respectively (Similarly Huang et al have reported no difference in ICU and hospital length of stays . HoweverNo difference was found between EN and EN+PN regimens in regard to effects on inflammatory responses. Severity of illness may not change with these regimens. Nursing workload increases with implementation of supplemental PN. Until sufficient data from large randomized clinical trials is available using EN with parenteral supplementation is not recommended.