The effectiveness and specificity of the ablation were evaluated upon days 1–10 after CB-SAP microinjection into the brain ventricular system. The vital status, survival, and common physiological parameters of the model animals were also assessed during the experimental period. The results demonstrated that CB-SAP damaged only the CSF-contacting nucleus, but not other functional structures, in the brain. The complete ablation occurred by day 7 after CB-SAP microinjection. A model animal that had no CSF-contacting nucleus was established after survival beyond that time point. No obvious effects were observed in the vital status of the model animals, and their survival was ensured. The common physiological parameters of model animals were stable. The present study provides a method to establish a CSF-contacting nucleus “knockout” model animal, which is similar to a gene knockout model animal for studying this particular nucleus  The brain-cerebrospinal fluid barrier is like a mysterious veil that blocks information transmission, substance exchange and functional modulation between the brain parenchyma and the cerebrospinal fluid (CSF). During several decades, the CSF-contacting structures were found near the walls of the cerebral ventricles and the central canal of the spinal cord. Vigh et al. regard the CSF-contacting neuron (CSF-CN) as a peculiar cell type within the central nervous system and hypothesized that it performs a pivotal role in non-synaptic signal transmission in the brain  and CB-horseradish peroxidase (CB-HRP) specifically label CSF-CNs in the parenchyma  specifically labels the CSF-contacting nucleus  were acquired from the Experimental Animal Center of Xuzhou Medical University. All experiments were approved and performed in accordance with the Committee for Ethical Use of Laboratory Animals, Xuzhou Medical University. Rats were anesthetized with pentobarbital sodium , and their heads were fixed on a stereotaxic instrument . CB-SAP  was dissolved in PBS , and 3 μl (500 ng) CB-SAP was microinjected into the lateral ventricle according to the stereotaxic coordinates provided by Paxinos and Watson . The injRats were divided into three groups . Group A was perfused at approximately 24 h after CB-SAP microinjection into the lateral ventricle, which corresponds to the length of time required for the CSF to finish circulating in the brain ventricular system. The brain and the spinal cord were sectioned coronally at 40 μm thickness  and underwent SAP immunofluorescence reaction . The sections containing the CSF-contacting nucleus underwent CB/SAP immunofluorescent double staining .Group B was the ablation group that was perfused at 5 days after CB-SAP microinjection into the lateral ventricle to investigate the influence of the degeneration agent CB-SAP on the CSF-contacting nucleus and other structures in the brain that directly contact the CSF. These other structures include tanycytes, ependymal cells, and other ultrastructures within the ventricle wall as well as the DR, which is the nearest structure adjacent to the CSF-contacting nucleus in the parenchyma. A segment of the CSF-contacting nucleus was cut into slices and underwent a CB immunofluorescent reaction . The tanycytes and ependymal cells were labeled with vimentin via immunofluorescent staining . The DR, which is adjacent to the CSF-contacting nucleus, was cut into slices and stained for its specific marker, 5-HT, using immunofluorescence . Group C was the control group and only received CB-HRP microinjection into the ventricle to label the CSF-contacting nucleus. The morphologies of the CSF-contacting nucleus, tanycytes, ependymal cells, ventricle wall surface, and DR in group C was observed at the same manner as for group B.The immunofluorescent results were captured by confocal laser microscopy . The ultrastructural changes of the ventricle wall surface in groups B and C were observed and captured with a scanning electron microscope .n = 6). Rats were perfused at the indicated time points. Brain segments slightly longer than the CSF-contacting nucleus were acquired on a stereotaxic instrument (Bregma: −7.40 to −9.80 mm). The segments were cut coronally on a cryostat  at 40 μm thickness. All sections underwent CB immunofluorescent staining . Sections were serially mounted. Morphological changes within the CB-positive neurons were observed by confocal laser microscopy . The remnant morphology of the CSF-contacting nucleus at the different times after ablation  was reconstructed spatially with Imaris software, version 8.4.1 . The number of neurons in the CSF-contacting nucleus was counted using Image-Pro Plus 7.0 software.The rats were divided into the control group (0 d) and the ablation group. The latter group was further subdivided into ablation groups at 1 d, 3 d, 5 d, 7 d, and 10 d .The rats' vital status and survival were recorded for 10 days after CB-SAP microinjection into the lateral ventricle. Baseline scores of the rats' physiological parameters  were measured before the CSF-contacting nucleus ablation. Then, the rats received CB-SAP microinjections into the lateral ventricle. After surgery, the rats were housed with a 12 h light/12 h dark cycle at room temperature (23 ± 1°C) with t-test. The number of neurons in the CSF-contacting nucleus at different time points after CB-SAP microinjection into the ventricle was analyzed by the Kruskal-Wallis test followed by the Mann-Whitney U-test. Any difference with P < 0.05 was considered significant.SPSS 13.0 software was used for data analysis. The data are presented as the mean ± standard deviation (SD). The morphological changes of the CSF-contacting nucleus, tanycytes, ependymal cells, and DR in the ablation and control groups were compared with Student's The CB-SAP distribution was evaluated at nearly 24 h after CB-SAP microinjection into the lateral ventricle. The SAP fluorescent product (green) was confined to the ventricular system. The outlines of the lateral ventricle (LV), the third ventricle (3V), the aqueduct (Aq), the fourth ventricle (4V), the central canal of the spinal cord (CC), and the pia mater of the brain presented clearly. The nearby brain parenchyma had no immunofluorescent labeling Figure .After CB-SAP was microinjected into the ventricle, it was transported in retrograde from the CSF to the CSF-contacting nucleus. Immunofluorescent labeling for neurons containing CB (CSF-contacting nucleus labeling agent), SAP (damaging agent), and CB/SAP (double stained) was found in the same neuron in the CSF-contacting nucleus. Other structures in the brain parenchyma displayed no immunofluorescent labeling Figure .The morphology of corresponding structures was assessed at 5 days after CB-SAP microinjection into the lateral ventricle.t(4) = 31.72, P < 0.001] after ablation  = 0.5658, P = 0.60 for tanycytes; t(4) = 0.1208, P = 0.91 for ependymal cells] after ablation  = 0.2938, P = 0.78] , the nearest non-CSF-contacting structure adjacent to the CSF-contacting nucleus, was labeled via immunofluorescence for serotonin (5-HT) and was not changed significantly after ablation  Figure .The ultrastructures within the ventricle wall that directly contact the CSF were evaluated by scanning electron microscopy after CSF-contacting nucleus ablation. These structures were not changed significantly after ablation when compared with the control group Figure .The neurons in the CSF-contacting nucleus present with CB-positive red fluorescent products. In the control group (0 d), the neurons were intact and regular. The neural processes were clear and dense. On the first day after ablation, the neurons shrank and were irregularly shaped, and the processes became sparse. On the third to fifth days after ablation, neural somas and processes were damaged into fragments. On the seventh day after ablation, only a few fragmented positive structures were detected. On the tenth day after ablation, no positive structures existed in the CSF-contacting nucleus Figure .2 = 34.241, P < 0.001), and the neuron number reduced significantly compared with that at prior time points , there were 1,439 ± 78 (defined as 100%) neurons in the CSF-contacting nucleus. On the first, third and fifth days after ablation, the numbers of neurons were 1,252 ± 72 (approximately 87%), 651 ± 24 (approximately 50%), and 268 ± 15 (approximately 26%), respectively. On the seventh and tenth days after ablation, no intact neurons existed (0%). The number of neurons in the CSF-contacting nucleus decreased significantly after ablation . Therefore, the model animal was considered stable and reliable.Model animals are indispensable tools in scientific studies. They provide a method for studying certain biological functions. The CSF-contacting nucleus is a special nucleus within the brain that has been recognized in recent years. Its anatomical structure is unique among the already known functional structures in the brain. Its neural somas are located in the parenchyma and communicate with other structures, such as non-CSF-contacting neurons, glia cells, and blood vessels in the brain. The processes have sensing, secreting and releasing functions and stretch directly into the CSF. Uncovering its functions will certainly help interpret the information transmission between the brain and body fluid with respect to the existence of the barriers in the brain  in the CSF-contacting nucleus under inflammatory pain   microinjection into the lateral ventricle according to the stereotaxic coordinates provided by Paxinos and Watson . The injS-YS conducted the studies. L-CZ and S-YS designed the study and prepared the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Setaria viridis is a C4 grass used as a model for bioenergy feedstocks. The elongating internodes in developing S. viridis stems grow from an intercalary meristem at the base, and progress acropetally toward fully expanded cells that store sugar. During stem development and maturation, water flow is a driver of cell expansion and sugar delivery. As aquaporin proteins are implicated in regulating water flow, we analyzed elongating and mature internode transcriptomes to identify putative aquaporin encoding genes that had particularly high transcript levels during the distinct stages of internode cell expansion and maturation. We observed that SvPIP2;1 was highly expressed in internode regions undergoing cell expansion, and SvNIP2;2 was highly expressed in mature sugar accumulating regions. Gene co-expression analysis revealed SvNIP2;2 expression was highly correlated with the expression of five putative sugar transporters expressed in the S. viridis internode. To explore the function of the proteins encoded by SvPIP2;1 and SvNIP2;2, we expressed them in Xenopus laevis oocytes and tested their permeability to water. SvPIP2;1 and SvNIP2;2 functioned as water channels in X. laevis oocytes and their permeability was gated by pH. Our results indicate that SvPIP2;1 may function as a water channel in developing stems undergoing cell expansion and SvNIP2;2 is a candidate for retrieving water and possibly a yet to be determined solute from mature internodes. Future research will investigate whether changing the function of these proteins influences stem growth and sugar yield in S. viridis. Saccharum officinarum), sorghum (Sorghum bicolor), switchgrass (Panicum virgatum), and miscanthus (Miscanthus X giganteum) provide the majority of soluble sugars and lignocellulosic biomass used for food and biofuel production worldwide  and rice (Oryza sativa) genomes 30–70 aquaporin homologs have been identified, respectively . Protein sequences used to generate the phylogenetic tree were obtained for S. viridis and Z. mays from Phytozome 11.0.5  .Expression data on identified e tissue . Protein HMMscan 1.S. viridis  were grown in 2 L pots, two plants per pot, in a soil mixture that contained one part coarse river sand, one part perlite, and one part coir peat. The temperatures in the glasshouse, located at the University of Newcastle  were 28°C during the day (16 h) and 20°C during the night (8 h). The photoperiod was artificially extended from 5 to 8 am and from 3 to 9 pm by illumination with 400 W metal halide lamps suspended ∼40 cm above the plant canopy. Water levels in pots were maintained with an automatic irrigation system that delivered water to each pot for 2 min once a day. Osmocote® exact slow release fertilizer  was applied at 20 g per pot, 2 weeks post-germination. Additional fertilization was applied using Wuxal® liquid foliar nutrient and Wuxal® calcium foliar nutrient  alternately each week.Seeds of ® Reagent  as per manufacturer’s instruction. Genomic DNA was removed using an Ambion TURBO DNase Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. cDNA was synthesized from 230 ng of isolated RNA from the cell expansion, transitional, and maturing developmental zones as described in Harvesting of plant material from a developing internode followed ® Green Master Mix 2x . A two-step cycling program was used following the manufacturer’s instructions. The green channel was used for data acquisition. Gene expression of the candidate genes was measured as relative to the housekeeper S. viridis PP2A . The PP2A gene was selected as a housekeeper gene because it is established as a robust reference gene in many plant species  and GoTaq species  and it w species . The forS. viridis elongating internode transcriptome (S. viridis sugar transporters from the Sucrose Transporter (SUT), Sugar Will Eventually be Exported Transporter (SWEET), and Tonoplast Monosaccharide Transporter (TMT) families were identified by homology to rice SUT, SWEET, and TMT genes  and cis-acting elements of Arabidopsis and rice SUT genes reported by Raw FPKM values of putative aquaporins and sugar transporters were extracted from the criptome . PutativMetscape . A gene Metscape . The 1.5database 3 and cisSvPIP2;1 and SvNIP2;2, from S. viridis transcriptome data  and purified using the MinElute PCR Purification Kit (QIAGEN). Complimentary RNA (cRNA) for SvPIP2;1 and SvNIP2;2 was transcribed using the Ambion mMessage mMachine Kit .Extracted consensus coding sequences for ome data , were syXenopus laevis oocytes were injected with 46 ng of SvPIP2;1 or SvNIP2;2 cRNA in 46 μL of water, or 46 μL of water alone as a control. Injected oocytes were incubated for 72 h in Ca-Ringer’s solution. Prior to undertaking permeability assays oocytes were transferred into ND96 solution pH 7.4  and allowed to acclimate for 30 min. Oocytes were then individually transferred into a 1:5 dilution of ND96 solution (42 osmol/L), pH 7.4, and swelling was measured for 1 min for SvPIP2;1 injected oocytes and 2 min for SvNIP2;2 injected oocytes. Oocytes were viewed under a dissecting microscope  at 2× magnification. The changes in volume were captured with a Vicam color camera  at 2× magnification and recorded with IC Capture 2.0 software  as AVI format video files. Images were acquired every 2.5 s for 2 min measurements and every 2 s for 1 min measurements. The osmotic permeability (fP) was calculated for water injected and cRNA injected oocytes from the initial rate of change in relative volume (dVrel/Idt) determined from the cross sectional area images captured assuming the oocytes were spherical:Vi and Ai are the initial volume and area of the oocyte, respectively, Vw is the partial molar volume of water and ΔCo is the change in external osmolality. The osmolality of each solution was determined using a Fiske® 210 Micro-Sample freezing point osmometer . pH inhibition of oocyte osmotic permeability was determined as above where oocytes where bathed in 1:5 diluted ND96 solution with the addition of 50 mM Na-Acetate, pH 5.6. Topological prediction models of SvPIP2;1 and SvNIP2;2 were generated in TMHMM4 . S. viridis proteins separated as expected into the major aquaporin subfamilies referred to as PIPs, TIPs, NIPs, and SIPs. Within S. viridis 41 full length aquaporins were identified: 12 PIPs, 14 TIPs, 12 NIPs, and three SIPs. One predicted aquaporin identified in the genome, transcript Sevir.6G061300.1, has very high similarity to SvNIP5;3 (Sevir.6G06000.1) but may be a pseudogene as it has two large deletions in the transcript relative to SvNIP5;3. Sevir.6G061300.1 only encodes for two out of the typical six transmembrane domains characteristic of aquaporins, and no transcripts have been detected in any of the S. viridis RNA-seq libraries available through the Joint Genome Institute (JGI) Plant Gene Atlas Project . We observed that SvPIP1;2 transcripts were abundant in all regions; and SvTIP1;1 transcripts were also abundant, particularly in cell expansion regions. SvPIP2;1, SvPIP1;1, SvTIP2;2, and SvTIP2;1 transcripts were detected in all regions with the highest transcript levels in cell expansion and transitional regions. Transcripts for SvTIP4;4, SvNIP3;1, and SvPIP1;5 were highest in the meristem relative to other regions; whereas SvTIP4;2, SvNIP2;2, and SvTIP1;2 transcripts were at their highest in transitional or mature regions. Low transcript levels were observed for SvSIP1;2, SvNIP1;1, and SvPIP2;4 in all regions, with maximum transcripts for SvNIP1;1 and SvPIP2;4 detected in the transitional region, and very low transcript levels were detected for SvPIP2;6, SvSIP1;1, and SvNIP2;1.We compared the relative transcript levels of putative SvPIP1;2 . Previous research has indicated that the related ZmPIP1;2 interacts with PIP2 subgroup proteins targeting PIP2s to plasma membrane, and a number of PIP1 aquaporins are not associated with osmotic water permeability when expressed alone in oocytes . The protein sequences of SvPIP2;1 and SvNIP2;2 were analyzed by the HMMER tool HMMscan which identified these candidates as belonging to the aquaporin (Major Intrinsic Protein) protein family.Overall the highest aquaporin transcript levels detected across the internode developmental zones were those of  oocytes . Our intSvPIP2;1 and SvNIP2;2 in the S. viridis internode regions by RT-qPCR. Stem samples were harvested from S. viridis plants grown under glasshouse conditions with the light period artificially supplemented by use of metal halide lamps to replicate as closely as possible the conditions used by Figure 3). SvPIP2;1 transcript levels were high in the cell expansion region and decreased toward the maturation region and SvNIP2;2 transcript levels were highest in mature stem tissues.To confirm our RNA-seq expression profile observations, we measured the transcript levels of S. viridis sugar transporters were identified from the internode transcriptome . This analysis revealed that for a number of aquaporins and sugar transport related genes there was a high correlation in expression: SvPIP2;1 expression correlated with the expression of SvPIP2;3, SvTIP2;1, and SvNIP1;1 (0.8–0.9); and the correlation coefficients for co-expression of SvPIP2;1 with SvPIP2;5, SvTIP4;1, SvTIP1;2, and SWEET1a were in the range of 0.8–0.9. Most notable was the high correlation (0.95–1.0) of expression of SvNIP2;2 with sugar transport related genes SvSUT5, SvSUT1, SvSWEET4a and with SvTIP4;2 and SvPIP2;6. The correlation between expression of SvNIP2;2 and SvSWEET13b and SvSWEET16 was also high (0.9–0.95). The cis-acting regulatory elements of the promoter regions of the aquaporin candidates SvNIP2;2 and SvPIP2;1, and the putative sugar transporter genes SvSUT1, SvSUT5, and SvSWEET4a were analyzed  was measured . The osmotic permeability (fP) of cRNA injected oocytes was calculated and compared to the osmotic permeability of water injected oocytes. Water injected oocytes had a fP of 0.60 ± 0.08 × 10-2 mm s-1. Relative to water injected control oocytes SvPIP2;1 and SvNIP2;2 cRNA injected oocytes had significantly higher fP of 14.13 ± 1.66 × 10-2 mm s-1 and 3.22 ± 0.28 × 10-2 mm s-1, respectively (p < 0.05).To explore whether the proteins encoded by Figure 5B). Reduced osmotic permeability of the cRNA injected oocyte membrane was observed in response to the low pH treatment. A reduction in Pf was observed for SvPIP2;1 and SvNIP2;2 cRNA injected oocytes bathed in an external hypo-osmotic solution at pH 5.6 relative to the pH 7.4 solution indicating that SvPIP2;1 and SvNIP2;2 have pH gating mechanisms . Water injected oocytes in the pH 5.6 Na-Acetate solution had Pf of 0.84 ± 0.13 × 10-2 mm s-1. SvNIP2;2 and SvPIP2;1 cRNA injected oocytes in the pH 5.6 solution had significantly lower fP of 2.46 ± 0.32 × 10-2 mm s-1 and 0.97 ± 0.13 × 10-2 mm s-1, respectively, compared to those in pH 7.4 solution (p < 0.05). SvPIP2;1 and SvNIP2;2 associated osmotic permeability and pH gating observations indicate that these proteins can function as water channels. The mechanism of pH gating for other plant aquaporins is the protonation of a Histidine residue in the Loop D structure; topological modeling of SvPIP2;1 and SvNIP2;2 predicted that the Loop D of SvPIP2;1 contains a Histidine residue while SvNIP2;2 Loop D does not contain a His residue . We note that Setaria aquaporins in an order consecutive with where they are found in the genome. For ease of comparing related aquaporins in C4 grasses of interest, we named the Setaria aquaporins based on their homology to previously named maize aquaporins   , of courd plants . Of thospression .The molecular and physiological mechanisms that determine stem cell number and cell size in turn determine the capacity of the stem as a sink . ExampleS. viridis internode, SvPIP2;1 was highly expressed in regions undergoing cell expansion . Positive correlations have been reported for the relationship between PIP mRNA and protein expression profiles of PIP isoforms in the expanding regions of embryos, roots, hypocotyls, leaves, and reproductive organs indicating that gene expression is a key mechanisms to regulate PIP function  PIP gene expression resulted in delayed lateral root emergence. Previously AtPIP2;2 anti-sense mutants were reported to have lower (25–30%) hydraulic conductivity of root cortex cells than control plants .The roles of a number of PIP proteins in hydraulic conductivity in plant roots and leaves have been reported but PIP function in stems is largely unexplored. The regulation of the hydraulic properties of expanding root tissues by PIP expression was analyzed by l plants . PIP2 fal plants . The roll plants . Expressl plants . Proteinto water . Here, wArabidopsis ovules a switch between apoplasmic and symplasmic pathways of delivering sucrose to storage sites has been reported . Our analysis of gene co-expression in stem tissues revealed high correlation between the expression of SvNIP2;2 and five putative S. viridis sugar transporter genes . Co-expression can indicate that genes are controlled by the same transcriptional regulatory program, may be functionally related, or be members of the same pathway or protein complex  NIP2;2 proteins are permeable to silicic acid and silicon, respectively . Gating of water channel activity has been reported for PIPs, including SvPIP2;1 , and for the TIP2;1 isoform found in grapevine (SvNIP2;2 water permeability was gated by pH (rapevine . The mecrapevine . Howeverrapevine , hence fSvPIP2;1 in expanding S. viridis stem regions and high transcript levels of SvNIP2;2 in mature stems inspired us to test the function of the proteins encoded by these genes. We found that SvPIP2;1 and SvNIP2;2 can function as pH gated water channels. We hypothesize that in stem tissues SvPIP2;1 is involved in cell growth and that SvNIP2;2 may facilitate water movement and potentially the flow of other solutes into the apoplasm to sustain solute transportation by bulk-flow, and possibly ‘recycle’ water used for solute delivery back to the xylem. It is expected that SvPIP2;1 could have additional roles, as other PIP water channels have been shown to also be permeable to CO2, hydrogen peroxide, urea, sodium and arsenic (S. viridis will require testing of the permeability of these proteins to a range of other solutes and modification of their function in planta.Our observations of high transcript levels of  arsenic . SvNIP2; arsenic . TranspoCG conceived and designed the work. SM, HO, LC, and JP acquired the data. SM, ST, CB, and CG analyzed and interpreted the data. SM and CB drafted and revised the work. All authors commented on the manuscript. SM, ST, RF, CB, and CG revised the work critically for intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pelophylax water frogs. We characterized the genetic makeup of Italian hybridogenetic hybrids (P. kl. hispanicus and esculentus) and identified a new endemic lineage of Eastern-Mediterranean origin as one parental ancestor of P. kl. hispanicus. This taxon is nowadays extinct in the wild but its germline subsists through its hybridogenetic descendant, which can thus be considered as a “semi living fossil”. Such rare situation calls for realistic efforts of de-extinction through selective breeding without genetic engineering, and fuels the topical controversy of reviving long extinct species. “Ghost” species hidden by taxa of hybrid origin may be more frequent than suspected in vertebrate groups that experienced a strong history of hybridization and semi-sexual reproduction.Hybridogenesis is a special mode of hybrid reproduction where one parental genome is eliminated and the other is transmitted clonally. We propose that this mechanism can perpetuate the genome of extinct species, based on new genetic data from  So-called “de-extinction” programs, like the “mammophant” (mammoth × elephant in vitro hybrid) or the Lazarus project (cloning of the extinct platypus frog from frozen specimens) are taking this fantasy to the experimental level, following recent advances in cloning biotechnology7. Here we present a new opportunity: the preservation of the genome of an extinct species through hybridogenesis, a special reproductive mode of hybrids. In this mode, the parental genomes do not recombine in F1s: one is eliminated before meiosis, and the other is solely transmitted in a clonal manner8  hybridize with marsh frogs (P. ridibundus s. l.) to form hybridogenetic hybrid species, aka kleptons, (P. kl. esculentus s. l.), in which the P. lessonae s. l. genome is excluded from the germline8. This group thus makes a fascinating model system to study hybridogenesis, where it can have far-reaching consequences: invasive marsh frogs are currently replacing pool frogs across Western Europe10; human-mediated introductions have led to the formation of novel hybridogenetic systems from naturally allopatric taxa, involving new klepton species11. As such, and while they benefit from a specific status, kleptons usually have a poor conservation value as they act as genetic parasites. In this report, we propose that hybridogenesis has perpetuated the lineage of an extinct clade of water frogs in Italy, and that it could be regarded as a potential mean to resurrect such extinct species.In a context of the worldwide mass extinction of biodiversity, the hopes for rediscovering or reviving extinct wildlife have become a popular scientific fantasyP. lessonae with P. kl. esculentus north of the Apennine Mountains and its sister species P. bergeri with P. kl. hispanicus in the Apennines and in Sicily12  together with microsatellite data, we characterized these ancestral marsh frog germlines in a comprehensive phylogeographic framework including most Pelophylax taxa known from the Western Palearctic. We made three striking discoveries.The Apennine Peninsula  is widely inhabited by pool frogs and their associated kleptons: P. kl. hispanicus as a new monophyletic lineage that we refer to as Pelophylax n. t. 1 . Potential signs of ancient admixture also arise from the microsatellite data: P. n. t. 1 is distinguished from the North-Italian marsh frog germline (see below), but few individuals have intermediate clustering , a Pleistocene period characterized by extreme cold and desiccation. Nowadays, P. n. t. 1 appears to be extinct as such, and its genome only persists in the hybridogenetic hybrid P. kl. hispanicus , before secondary contact and introgression by P. lessonae. Alternatively, it could be of hybrid origin resulting from leaky hybridogenesis between pool and marsh frogs, which would explain the intermediate phylogenetic position  also argue for an independent evolution of P. n. t. 1 . However, intense efforts of multiple controlled crossing experiments may allow to obtain few viable offspring, i.e. those where different deleterious mutations were fixed in their parental germlines. Although audacious, these might be rewarding given the flexibility of amphibian development; e.g. eggs can sometimes still develop under artificial haploid parthenogenesis (notably in Pelophylax25). An alternative approach would be to cross P. kl. hispanicus with its closest living relatives, e.g. the Anatolian lineage P. bedriagae c. f. 2. Subsequent backcrossing of the resulting F1 P. n. t. 1 × P. c. f. bedriagae hybrids with P. kl. hispanicus might allow to dilute the P. c. f. bedriagae genome while simultaneously purging deleterious mutations accumulated on the P. n. t. 1 germline. Selective breeding is a common de-extinction practice, e.g. envisaged to resurrect the aurochs26 and applied to recreate the quagga (an extinct subspecies of zebra), although the resulting individuals could not be genetically identical27. The implementation of such approaches would contribute to the ethical debate of reviving long extinct species. De-extinction is questionable when it pursues a technological achievement28. While this would not be the case here, the responsibility of mankind in restoring a biodiversity that “naturally” vanished prior to the sixth mass extinction still remains a topic of extreme controversy28. More generally, the de-extinction possibilities offered by scientific advances should not defuse the dramatic measures required to protect wildlife on the verge of extinction.The persistence of the genetic legacy of an extinct taxon provides exciting insights. By carrying this lineage, 30). Unknown lineages underlie the genetic makeup of some polyploid plants31 and fishes32. Such taxa may be more frequent than imagined in vertebrates, especially in amphibians and fishes where these reproductive modes have been well-documented33.This study calls for the tracking of “ghost” vertebrate lineages that remained hidden and protected in other species of hybrid origins, through hybridogenesis or other mechanisms with potentially similar consequences , representing 23 localities Table . Eleven 16, individuals from the study area were genotyped at microsatellite loci with diagnostic alleles to distinguish between pool (here P. bergeri /lessonae) from marsh frogs (P. ridibundus s. l.) frogs, as well as their kleptons (here P. kl. esculentus/hispanicus)34. This also confirmed the expected absence of marsh frogs (P. ridibundus s. l.).Given the uncertainty to identify water frogs by morphology16). Furthermore, we were able to parse the genotypes of 8 markers in 26 kleptons and reconstructed their marsh frog haplotypes, as previously done in Western Switzerland10. Both datasets (pool frogs/kleptons and phased marsh frog haplotypes) were analyzed by Principal Component Analyses (PCA) on individual genotypes, using the ade4 and adegenet packages in R. The differences between pool frogs and their associated kleptons stem from their marsh frog germlines. The genetic structure of kleptons should thus mirror the genetic structure of pool frogs if their marsh frog germlines only co-evolved in the kleptons after hybridogenesis was initiated, but not necessarily if the divergence predate hybridogenesis.To this purpose and to infer population structure, we analyzed nine polymorphic microsatellites in 72 individuals of pool and edible frogs from the study area  and Cellular myelocytomatosis  in respectively 45 (34 pool frogs and 11 kleptons) and 60 (49 pool frogs and 11 kleptons) individuals from the study area. These two markers were chosen as they have been widely used for phylogeography of water frogs and were consistently shown to discriminate between currently recognized taxa35. PCR conditions and primers are available in Table P. bergeri/lessonae alleles and a set of primers specific to the alleles of other taxa . However, kleptons with a P. n. t. 2 allele, as well as heterozygous pool frogs had to be cloned. Cloning was performed with the TOPO-cloning kit (Invitrogen), and a minimum of eight clones per individual was sequenced.We amplified and sequenced intronic portions of the nuclear genes axa Fig. . This al18 , with the Asian P. nigromaculatus and the early-diverged European P. perezi as outgroups. Phylogenetic Bayesian inferences were performed with MrBayes37, with 10 million iterations and 2 chains. Evolutionary models were chosen according to JModelTest38 . We also computed haplotype networks using TCS39 to visualize haplotype variation, including indels. In addition to marker-specific trees, we built a phylogeny based on concatenated sequences representative of the variation of each species for the two markers.We complemented our new data with published sequences available for these two genes. For SAI-1, this included the haplotypes found across the ranges of all speciesP. cretensis at the end of the Messinian salinity crisis (~5.3-5.5 Mya)41. To this purpose, we estimated the mutation rates of our nuclear markers beforehand from the evolution of the mitochondrial genomes published for ten species of European and Asian water frogs, which provides a well-resolved phylogeny42. Analyses were conducted in BEAST using a Yule-process prior, an uncorrelated lognormal relaxed clock43 and different partitions for each sequence set. This calibration involved a normally distributed prior at 5.4 Mya (95% HDP 4.8-5.9) for the split of P. cretensis. Then, we applied the molecular clock on the nuclear dataset using the estimated mutations rates with normally-distributed priors covering their 95% HDP (SAI-1: 0.0056 (0.0027-0.0112); CMI-2: 0.0113 (0.0026-0.0286)). We ran two independent chains of 20 million iterations each (with the first 2 million excluded as burnin) and used the Tracer module to check for convergence and effective sample size of parameters. We built a time-calibrated phylogeny from the BEAST runs with the module TreeAnnotator and the R package phyloch44.We performed molecular dating by calibrating the phylogeny to the known split of Supplementary Information


N-methyl-D-aspartate receptor, the α-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid receptor, the γ-amino-butyric acid B-receptor, the leucine-rich, glioma-inactivated 1, the contactin-associated protein-like 2, and the dipeptidyl-peptidase-like protein-6), all previously associated with psychopathology, in CSF samples from 29 unmedicated suicide attempters. Five of these subjects had high CSF/serum albumin ratio, indicative of increased blood-brain-barrier permeability. We were not able to detect any of these autoantibodies in the CSF samples. These pilot data do not support a role for autoimmune encephalopathies in suicidal behaviour, although the presence of lower levels of these autoantibodies cannot be ruled out in these patients.Previous findings suggest a link between neuroinflammatory processes and suicidality. Despite several lines of evidence supporting this link, including increased pro-inflammatory markers in blood-, cerebrospinal fluid (CSF)- and in post-mortem brain samples from suicidal individuals, the underlying mechanisms remain poorly understood. In this pilot study, we explored the possibility that autoimmune encephalopathies might be found among suicide attempters. We analysed the presence of six different autoantibodies ( Several lines of evidence suggest that neuroinflammatory processes are involved in the pathophysiology of suicidal behavior . ImmunomToxoplasma gondii [N-methyl-D-aspartate receptor (NMDAR) IgG in CSF [The underlying pathobiology behind neuroinflammation in suicidality is not fully understood. Suggested causal mechanisms include latent infections such as a gondii –14, as wa gondii , 16. Impa gondii , multipla gondii , and cela gondii . Severala gondii , 21. A fa gondii , 23, alta gondii , 25. Fura gondii . MoreoveG in CSF .The etiologic mechanisms behind the generation of autoantibodies in individuals with psychiatric manifestations are not fully understood, but may involve a compromised blood-brain-barrier (BBB) .In the present study we set out to detect autoantibodies in CSF samples from suicide attempters, a group often characterized by depressive symptoms, emotional instability, and even psychotic symptoms. The main purpose was to explore and develop preliminary data on the possibility that autoimmune processes may be involved in suicidal behavior. To this end, we analyzed six different autoantibodies in CSF samples from 29 suicide attempters, randomly selected from a larger cohort, employing an assay used for clinical diagnosis of autoimmune encephalitis. Within this larger cohort of suicide attempters, CSF/serum albumin ratio, an indicator of BBB integrity, has been quantified and this data have been previously published , 30.All parts of this study were approved by the Lund University Medical Ethics Commitee. All subjects gave verbal informed consent to participate in the study. Written informed consent was not required per the ethical approval. Consent was registered in the patients’ chart. Subjects who, in the investigator's judgment, lacked the ability to make an informed decision regarding study participation were not included in the study.Between 1987 and 2001, 29 subjects were enrolled in the study on admission to Lund University after a suicide attempt, as defined by Beck et al. . SuicideSix different autoantibodies previously implicated in autoimmune encephalitis  were anaAntibody detection was done using the Autoimmune Encephalitis 6 Biochip mosaics  by trained laboratory personnel at a specialized diagnostic laboratory affiliated with Lund University Hospital . Each biochip contains transfected cells expressing a specific antigen . Biochip mosaics were incubated each with 30 μl of un-diluted CSF for 30 minutes at room temperature, washed with PBS-Tween, and immersed in PBS-Tween for 5 minutes. Bound antibodies were stained with fluorescein isothiocyanate–labeled goat anti-human IgG, (Euroimmun) for 30 minutes at room temperature. Slides were washed again with a flush of PBS-Tween and then immersed in PBS-Tween for 5 minutes. Drops of PBS-buffered glycerol were placed onto a cover glass, and the biochip slides were embedded in this mounting medium simultaneously and examined by fluorescence microscopy. Positive and negative controls were included with every test procedure. Autoantibodies against the corresponding antigen react specifically with the corresponding transfected cells and results are given as positive or negative. NMDA receptor autoantibodies react specifically with the corresponding transfected cells and induce a fine granular cytoplasmic fluorescence, while the cell nuclei are only slightly stained. Antibodies against AMPA receptors, CASPR2, LGI1, and GABAB1/B2 receptors react specifically with the cytoplasm of the corresponding transfected cells inducing a cytoplasmic fluorescence, with some fluorescence of the cell membrane, while the cell nuclei are only slightly stained. Antibodies against DPPX react with the transfected cells of the test substrate. They produce a spread, smooth to fine-speckled cytoplasmic fluorescence, partly with fluorescence of the cell membrane. The cell nuclei are only slightly stained.Samples with no autoantibodies or negative control cells results in no fluorescent staining. The test results were interpreted manually by extensively trained personnel by reading the fluorescence of fixated cells in a microscopy and results are reported as positive or negative. There are no predetermined cut-off values, since a negative result will show no fluorescence.Albumin was determined in serum and CSF as previously described . CSF/serDemographic and clinical characteristics of the study participants are summarized in Most of the subjects (n = 21) had made a non-violent suicide attempt, e.g. intoxication, while the remainder (n = 8) had made a violent suicide attempt, e.g. carbon monoxide poisoning or jumping in front of a train . Ten of None of the following autoantibodies were detected in any of the CSF samples: NMDAR, AMPAR, GABAbR, LGI1, Caspr2, DPPX.Five subjects had increased CSF/serum albumin ratio, in relation to age-adjusted reference values previously described . For indIn this pilot study we investigated, the presence of autoimmune antibodies in CSF samples from a group of recent suicide attempters. Given that neuroinflammation has been implicated in the pathophysiology of suicidality , this isVarious psychiatric manifestations have been documented in subjects with anti-NMDAR encephalitis and other autoimmune encephalitis. These include psychotic symptoms, cognitive impairment, depression, irritability, and personality disturbances , 38. FewIn those cases autoantibodies are detected in individuals with psychiatric and neurologic manifestations, their origin is not fully understood. Several potential mechanisms have been proposed including paraneoplastic processes, past influenza infections and genetic susceptibility , 39. MorThe present study is among the first to investigate autoantibodies in CSF samples from psychiatric subjects. An important strength of the study is the inclusion of a well-characterized sample of unmedicated recent suicide attempters. The present study, however, also comes with several caveats. Firstly, the sample size was relatively small, thus any conclusions must be considered preliminary and the results should serve primarily as pilot data for testing in future larger scale studies. Even though the test panel that was used included six different autoantibodies, thus increasing the possibility of detection, the number of subjects tested might have been insufficient. Secondly, the assays used in our study were originally designed as a diagnostic tool for targeted populations of neurology patients where autoimmune processes in the brain are suspected. Thus, we cannot rule out that a more sensitive experimental assay would have been able to detect more subtle levels of autoantibodies. Future studies might consider using experimental assays where lower concentrations of autoantibodies might be detected. Finally, this is a cross-sectional study and we cannot completely rule the possibility that autoantibodies, if present, might have been detectable immediately after the suicide attempt but not after the wash-out period at the time of the lumbar puncture. Longitudinal studies of anti-NMDA receptor encephalitis, however, have shown that although titres may decrease over time, antibodies are often detected in consecutive samples .In conclusion, we were not able to detect any autoantibodies in CSF samples from recent suicide attempters. This is one of the first studies of its kind, and future larger scale studies using sensitive assays are warranted before any firm conclusions may be drawn.


GJB2/Cx26) and beta 6 (GJB6/Cx30). Our knowledge of their expression in humans is insufficient due to the limited availability of tissue. Here, we sought to establish the molecular arrangement of GJs in the epithelial network of the human cochlea using surgically obtained samples.Mutations in the GJB2 gene, which encodes the Connexin26 (Cx26) protein, are the most common cause of childhood hearing loss in American and European populations. The cochlea contains a gap junction (GJ) network in the sensory epithelium and two connective tissue networks in the lateral wall and spiral limbus. The syncytia contain the GJ proteins beta 2 .Cx30 plaques (<5 μm) dominated, while Cx26 plaques were subtle and appeared as ‘mini-junctions’ (2–300 nm). 3-D volume rendering of Z-stacks and orthogonal projections from single optical sections suggested that the GJs are homomeric/homotypic and consist of assemblies of identical GJs composed of either Cx26 or Cx30. Occasionally, the two protein types were co-expressed, suggesting functional cooperation.Establishing the molecular composition and distribution of the GJ networks in the human cochlea may increase our understanding of the pathophysiology of Cx-related hearing loss. This information may also assist in developing future strategies to treat genetic hearing loss. GJ plaques are composed of closely gathered hemi-channels composed of connexin (Cx) proteins arranged as hexameric rings or connexons with a central pore. These plaques are thought to play multiple roles in fluid homeostasis, metabolic supply, nerve excitation, and intercellular Ca kD, ions+ concentration important for hair cell transduction. K+ recycling from hair cells is assumed to be mediated by GJs, which allow the passage of K+ to the spiral ligament, where it is taken up by fibrocytes  and Cx30 (GJB6) cause non-syndromic inherited deafness  study on human cochlear material suggested recently that Cx26 and Cx30 proteins are not co-expressed in the lateral wall of the human cochlea but, rather, form closely associated GJ plaques . As variThe use of human materials was approved by the local ethics committee , and patient consent was obtained. The use of animal cochlear material was also approved by the local ethics committee . The study adhered to the guidelines of the Helsinki Declaration.Two archival specimens collected during surgery and two specimens collected after perilymphatic perfusion were analyzed in Uppsala ,36 and IFive cochleae from five adult patients . Its selectivity for human Cx30 was confirmed by western blotting. The anti-Cx26 monoclonal antibody was derived from mice and has a high specificity . The antibody against laminin β2 was a rat monoclonal antibody  specific for the laminin β2 chain. This antibody was used to demarcate the basal lamina at the bottom border of the epithelium in the organ of Corti (OC). It recognizes and is specific for the laminin β2 chain. The anti-laminin antibody showed no cross-reaction with other basement membrane components, such as type IV collagen. Both a polyclonal antibody  and a monoclonal tubulin antibody  against neuron-specific class III beta-tubulin (Tuj-1) were used. The combinations, characteristics, and sources of antibodies used in this study are summarized in X–Y) resolution of approximately 100 nm and an axial (Z) resolution in the approximately 300 nm range .The stained sections were visualized with an inverted fluorescence microscope (Nikon TE2000) equipped with a spot digital camera with three filters . Image-processing software  including image merging and fluorescence intensity analyzer features was installed on a computer system connected to the microscope. For laser confocal microscopy, we used the same microscope equipped with a three-channel laser emission system. The optical scanning and image-processing tasks, including the reconstruction of Z-stack images into projections and 3-D images, were performed using Nikon EZ-C1 (ver. 3.80) software. Structured illumination microscopy (SR-SIM) was performed with a Zeiss Elyra S.1 SIM system using a 63x/1.4 oil Plan-Apochromat lens (Zeiss), a sCMOS camera (PCO Edge), and ZEN 2012 software (Zeiss). Multicolor SR-SIM imaging was achieved with the following laser and filter setup: first channel, 405-nm laser excitation and BP 420-480 + LP 750 filter; second channel, 488-nm laser excitation and BP 495-550 + LP750 filter; and third channel, 561-nm laser excitation and BP 570-620 + LP 750 filter. To maximize image quality, five grid rotations and five phases were used for each image plane and channel. The grid size was automatically adjusted by the ZEN software for each excitation wavelength. SR-SIM images were processed with ZEN software using automatic settings and theoretical point spread function (PSF) calculation. A 3-D reconstruction was performed from the SR-SIM dataset using Imaris 8.2 software . The microscope was capable of achieving a lateral . Isolated scans, 3-D rendering, and maximum-intensity projections (MIPs) were analyzed and compared. Cx26 was weakly expressed. Numerous Cx30-positive plaques were found between all supporting cells. The shapes of the Cx30 plaques varied , and their size was up to five microns in diameter. In contrast, the Cx26 plaques were small (0.1–0.5 μm) and dot-like . No labeSupplementary Figure 2, available online). Annular patterns of Cx30 staining were frequent. These rings had a diameter of 100–1000 nm, with a central subdomain composed of Cx26 . Nerve endings beneath the inner hair cells (IHCs) were generally swollen and did not show any staining.Cx26 staining was mostly independent of Cx30. Although some areas showed superimposed Cx26 and Cx30 staining, orthogonal views of single optical sections showed separate color signals  and associated with Cx26 plaques , inset. Supplementary Figure 3, available online). In some cases, folded IPhCs were squeezed between an IHC and an inner pillar cell (IPC). The presence of GJs could not be verified with certainty between the IPC and IPhC , available online). No GJs were observed between sensory and supporting cells. The opposing lateral cell membranes of the Claudius cells (CCs) often had an undulating outline decorated with GJs . Annular GJs were not observed with TEM. The lateral cell membranes between HCs and Boettcher cells showed occasional sub-plasma membrane sacs suggestive of rough ER (not shown). The basal plasma membrane of the supporting cells, facing the basilar membrane (BM), often displayed focal densities . On confocal microscopy and SR-SIM, these sites frequently demonstrated Cx30 positivity, suggesting that they represented hemi-junctions  surrounding the IHCs displayed large numbers of GJs , which can differ between heteromeric and homomeric GJs . The close relationship between Cx26 and Cx30 homomeric/homotypic GJ plaques suggests a yet-undefined functional link and cooperation.There was surprisingly little expression of Cx26 in the sensory epithelium compared to Cx30. However, a fairly large amount of diffuse intracellular Cx26 staining was observed in DCs, particularly near the base of OHCs. This finding suggests that the epithelial GJ network consists mostly of Cx30 in human adults. MIP results indicated possible co-expression, but 3-D rendering revealed that Cx26 and Cx30 were expressed separately in different plaques. These findings indicate that GJs consist mainly of homomeric/homotypic Cx pairs . A simileric GJs ,29. AccoThe widespread distribution of Cx30-positive GJ plaques suggests that they are crucial for human hearing. The GJ syncytium connecting most supporting cells may supply the avascular sensory epithelium with nutrients as well as remove metabolic waste products from the highly active hair cells. The outer pillar columns also showed lateral intercellular Cx30 strands. Such coupling may be important to synchronize the motion of these cells, allowing them to act in concert for cochlear amplification . GJ stra+ ions from the sensory epithelium back to the connective tissue GJ networks in the lateral wall  deletion was believed to be due to defective Cx26 expression, and Cx30 was considered dispensable for cochlear function . Accordi0 deletio+ transport by supporting cells may lead to a degeneration of the OC (+ recycling may not be a deafness mechanism for GJ deficiency-induced hearing loss in these animals (Considering the important role played by Cx26 in human deafness, there is a discrepancy between embryonic and adult human tissue that may be explained by a maturation process. The development of the cochlear GJ system precedes the functional maturation of the rat inner ear that takes place between the second and third postnatal weeks . Kamiya f the OC . These cf the OC . Therefof the OC ,52 and lf the OC ,54. Mousf the OC ,55. GJB2f the OC . Deafnesf the OC . The aut animals .Remarkably, Cx26 subdomains appeared inside annular Cx30 plaques . Cx protIn the present study, TEM showed membrane densities, and SR-SIM demonstrated Cx30 plaques at the basal surface of the supporting cells facing the BM, which may represent hemi-channels. Hemi-channels facing the extra-cellular tissue have been described in the cochlea . Hemi-chIn summary, the present results seem to support the notion that GJs in the human cochlear sensory epithelium are homomeric/homotypic and that plaques are mostly populated by assemblies of identical GJs that express either Cx26 or Cx30. The possibility of mixed plaques cannot be excluded as the diameter of each GJ is below the maximal resolution of SR-SIM. Establishing the molecular composition of the GJ networks in the human cochlea is essential for understanding the pathophysiology of Cx-related hearing loss and may also assist in developing future strategies to treat genetic hearing loss ,63.Click here for additional data file.


Posttransplant Lymphoproliferative Disorder (PTLD) is one of the most common malignancies complicating solid organ transplantation. In contrast, PTLD accounts for a minority of secondary cancers following allogeneic hematopoietic cell transplantation (HCT). Here we report on a 61-year-old woman who received an ABO-mismatched, HLA-matched unrelated donor hematopoietic cell transplantation from a presumably healthy donor for a diagnosis of acute myeloid leukemia (AML). Eighteen months following her transplant, she developed a monoclonal gammopathy. Bone marrow studies revealed 10% plasma cells, but the patient lacked clinical defining features of multiple myeloma (MM); thus a diagnosis of smoldering multiple myeloma (SMM) was established. Cytogenetic and molecular studies of the bone marrow confirmed the plasma cells were donor-derived. The donor lacks a diagnosis of monoclonal gammopathy of undetermined significance, SMM, or MM. Infrequently, acute leukemia and lymphoma can develop de novo in engrafted cells of donor origin. Donor cell leukemia/lymphoma (DCL) was first described in 1971 . In manyAn otherwise healthy 61-year-old Caucasian woman presented in 2010 with anemia and thrombocytopenia after one month of recurrent upper respiratory infections. White blood cell (WBC) count at the time of presentation was 8.7 with 48% circulating blasts, hemoglobin was 9.6, and platelets were 39,000. Bone marrow biopsy was consistent with a diagnosis of acute myeloid leukemia (AML) with translocation of chromosomes 11 and 19 involving the MLL locus . She received two rounds of induction therapy with idarubicin and cytarabine with complete response. She then received one cycle of High-Dose Cytarabine (HiDAC) consolidation. She also received 1 cycle of decitabine as there were significant delays in identifying a suitable stem cell donor.She underwent a matched unrelated donor (MUD) hematopoietic cell transplantation (HCT) with fludarabine, busulfan, and thymoglobulin conditioning seven months following the initial diagnosis from a presumably healthy male donor. Graft versus Host Disease (GvHD) prophylaxis regimen consisted of methotrexate, tacrolimus, and mycophenolate mofetil. Day +30 post-HCT bone marrow showed complete remission with complete donor cell engraftment. Her immediate posttransplant course was complicated by mild acute gastrointestinal GvHD and human polyomavirus (BK) cystitis. Post-HCT bone marrow biopsy on days +100 and +180 again showed complete remission with complete donor cell engraftment. In the absence of any evidence of ongoing GvHD, at 18 months after HCT all immunosuppressive agents were discontinued after a prolonged taper schedule.a = 0.1, b = 2.4). Serum protein immunofixation classified the protein as IgG lambda monoclonal protein. Additional laboratory work-up was significant for increased lambda free light chain (FLC) , normal kappa FLC , low kappa/lambda FLC ratio , and a raised beta-2-microglobulin . Bone marrow biopsy revealed a normocellular bone marrow with trilineage hematopoiesis with no excess blasts but was significant for infiltrating plasma cells. An immunohistochemical stain for CD138 was performed to better quantify the plasma cells in a tissue context and showed singly dispersed and clustered plasma cells comprising approximately 10% of the marrow cellularity. Flow cytometric analysis showed approximate 1% of total events to be lambda light chain restricted plasma cells that did not express CD56. In situ hybridization for EBV (EBER) on the bone marrow biopsy was performed and was negative. Patient's hemoglobin, calcium level, and kidney function were within normal limits. A bone survey was performed and did not identify any lytic or blastic lesions. To further characterize the origin of plasma cells, CD138+ cells were isolated using a Miltenyi autoMACS device (Miltenyi Biotech) and then short tandem repeat (STR) polymorphic DNA markers were amplified by polymerase chain reaction (PCR), labeled with fluorescent markers, and used to distinguish patient and donor cells as different on a Capillary Electrophoresis Fluorescence Detection instrument. The results were consistent with complete donor cell engraftment (<5% recipient cells present).Five hundred and sixty days following her transplant, the patient was found to have abnormally high IgG levels during routine follow-up . Serum protein electrophoresis (SPEP) showed two abnormal restricted peaks in gamma region  that could be attributed to the underlying plasma cell disorder or other myeloma defining events, a diagnosis of donor-derived smoldering multiple myeloma (SMM) was established. The National Marrow Donor Program (NMDP) was notified. The NMDP has confirmed that the donor does not have a diagnosis of SMM or MM. It is unclear if the donor has ever had a bone marrow biopsy or SPEP to confirm or if he simply lacks the diagnosis.We continued to observe our patient regularly for changes in her myeloma markers. At the time of preparation of this manuscript, her myeloma markers have been stable for over four years. With regard to AML, she is almost 6 years out of MUD-HCT and has been in remission ever since.To date very few cases of donor origin PTLD-plasma cell neoplasms have been reported in literature. Three cases have been described with solid organs in kidney and heart-lung transplantation , 5. PeriKim et al. reported a 40-year-old woman with a diagnosis of refractory anemia with ring sideroblast,who underwent an allogeneic HCT from a 32-year-old healthy male donor. Almost four months after HCT, she developed progressive pancytopenia, biclonal gammopathy, and IGH gene rearrangement. A bone marrow biopsy revealed 12% plasma cells with an immunoglobulin heavy-chain gene rearrangement. Cytogenetics and chimerism studies confirmed a diagnosis of donor cell origin PTLD-MM . Upon adThere have been two reported cases of PTLD-MM with possible transfer of the malignant clone from donor to recipient. Kumar et al. reported on a 42-year-old man transplanted for chronic phase chronic myeloid leukemia (CP-CML). The donor was diagnosed with MM 27 months following the stem cell donation; the recipient developed IgA myeloma 40 months after HCT. Serum electrophoresis and bone marrow investigations established a diagnosis of IgA kappa MM in both donor and recipient . Her broPTLD is a life-threatening complication after HCT or solid organ transplantation. The majority of PTLD is of B-cell origin and associated with Epstein-Barr virus (EBV). During the past decade, progress has been made in better understanding both the pathogenesis of PTLD and early detection strategies . SeveralDespite elaborate efforts in identifying the risk factors associated with the development of PTLD, the pathogenesis of donor cell origin PTLD after HCT has not been thoroughly understood. Various components in the marrow tightly regulate hematopoietic cell fate. These players include osteoblasts, their mesenchymal precursors, CXCL12 rich reticular cells, and adipocytes. In addition to these cells, hormones such as estrogen, PTH, and catecholamines also contribute to the activation of the microenvironment . The intGiven the lack of a diagnosis of MGUS/SMM/MM in the donor described in our case, we hypothesized that the tumor microenvironment played a critical role in the development of donor-derived MM in our patient.Here we report on a patient who received an ABO-mismatched, MUD-HCT for a diagnosis of AML. Eighteen months after HCT, the patient developed donor origin SMM. To the best of our research to date, the donor lacks a diagnosis of MGUS/SMM/MM. This is the second case report of donor-derived MM following HCT in a patient whose donor lacks the diagnosis.


Oreochromis niloticus  with emphasis on growth performance, digestive enzyme activities as well as serum biochemical parameters. Also, lysozyme activity (LYZ), respiratory burst (RB), superoxide dismutase (SOD), catalase (CAT) and resistance to Streptococcus iniae were investigated. Fish were stocked in 15 rectangular fiber glass tanks (150× 60× 40 cm) at 40 fish per tank with water maintained at 210 litres. Fish were fed five isonitrogenous (33% crude protein) and isolipidic (10% lipid) diets with PO included at 0% (0% PO), 25% (25% PO), 50% (50%PO), 75% (75% PO) and 100% (100% PO) for 8 weeks. The findings demonstrated that growth, and feed utilization was not compromised when PO was used in place of FO either partially or totally. Except for protease activity which was not significantly altered, lipase and amylase activities were significantly altered when FO was replaced with PO. There were no significant differences among treatments for CAT, SOD and LYZ. Serum malondialdehyde (MDA) in fish fed 100% PO was significantly lower than all other groups whiles total antioxidant capacity (TAC) of fish fed 0% PO was significantly higher than all other groups. Fish fed 0% PO, 25% PO and 50% PO had glutathione reductase (GR) significantly higher than fish fed 75% PO and 100% PO. RB in fish fed 0% PO were significantly lower than fish fed 75% PO and 100% PO. Also, fish fed 0% PO had significantly lower total protein (TP) compared with groups fed 50% PO and 75% PO. Fish fed diets with PO had similar resistance ability to Streptococcus iniae as those fed diets with FO. However, the liver function was likely to be compromised due to the increase in aspartate amino transferase (AST) and alkaline phosphatas (ALP) along increasing PO inclusion levels. AST, total protein, triacylglycerol (TAG) and low-density lipoprotein cholesterol (LDL-C) were significantly higher (p<0.05) in groups fed higher levels of PO. This study therefore concludes that feeding tilapia fingerlings with diets containing PO affects antioxidant and innate immune parameters negatively due to the reduction in LYS, TAC, GR, MDA, CAT, SOD and GSHpx.This study was conducted to investigate the effects of replacing dietary fish oil (FO) with palm oil (PO) in juvenile Nile tilapia,  Dietary lipid cannot be replaced by other nutrients because it plays important biological functions, and serve as a major source of essential fatty acid and provide energy . Fish oiVegetable oils (VO) are potential and suitable candidates to replace fish oils in aquaculture feeds  because Lysozyme is a lytic protein that is important in the non-specific defense system. Serum lysozyme activity are relevant since it provides a defense line in preventing proliferation and colonization of pathogenic microbes as a consequence ensuring in the diminution of disease . Serum pSuperoxide dismutase (SOD) and catalase (CAT) are some essential antioxidant enzymes in fish, they are responsible for the breakdown and activation of the main metabolically produced endogenous radicals, hydrogen peroxide and superoxide , thus, tS. iniae in tilapia. This study will give a fair idea concerning how replacing FO with PO could impact the health of O. niloticus. The objective of the present study is therefore to determine the effects of replacing FO with PO on growth performance, antioxidant capacity, immune response, serum metabolites, digestive enzyme activities and response to S. iniae in juvenile Nile tilapia.To our knowledge, limited information is available on the effects of replacing FO with PO on antioxidant capacity, immune response, digestive enzyme activities and resistance to Five diets; isonitrogenous (33% crude protein) and isolipidic (10% crude lipid) were formulated for this study with ingredients supplied by Nonghao Feed Company . The differences in diets are with respect to fish oil and palm composition . Fish oiThe present study was performed in strict accordance with the Standard Operation Procedures (SOPs) of the Guide for the Use of Experimental Animals of Shanghai Ocean University. All animal care and use procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Ocean University. Fish were anesthetized with excess tricaine methane sulfonate (MS-222 at 200mg/L) before sampling in order to reduce suffering and stress.Mixed sex Nile tilapia fingerlings were obtained from Tilapia Germplasm Station of Shanghai Ocean University, China. Fish were sent to Shanghai Ocean University’s aquarium facility where they were fed twice a day to apparent satiation on commercial diets (30% crude protein) purchased from Shanghai Jin Yuan Trade. Twenty fours before trial, fish were starved, distributed into their respective tanks (fiber glass tanks of 150× 60× 40 cm) at a stocking density of 40 fish per tank. Each group was then fed their corresponding diet for eight week, two times a day at 08:00 and 16:00 to apparent satiation. Following standard methods [Feed was withheld from fish 24 hours before sampling. Fifteen fish were then sampled from each treatment (five from each tank), euthanized with an overdose of tricaine methane sulfonate (MS-222 at 200mg/L) as previously described by . Weight (1)Total weight gain (WG)FW and IW represent final weight and initial weight, respectively.(2)Feed intake (FI) is the total feed consumed (g) during the 56 days trial.(3)Feed conversion ratio (FCR)(4)Survival (S)TF and TFT represent total number of fish stocked and total number of fish at end of trial, respectively.(5)Condition factor (C)BW and TL represent body weight (g) and length (cm) respectively.Analysis of fatty acids in diets, muscle and liver were performed as previously described in . Feed asDigestive tract of five fish per tank that were previously bled were excised and divided into three portions: anterior, middle and posterior. The posterior intestine was distinguished from the mid intestine by increased diameter, darker mucosa, and annular rings. The remaining part was divided into two identical parts to obtain the anterior and mid portions. The samples were then frozen in liquid nitrogen and stored at -80 until measurement of enzyme activity. All enzyme activities were measured as previously described by .Fifteen fish were sampled from each treatment (five from a tank), anesthetized with excess amount of tricaine methane sulfonate  needle. Sampled blood was then transferred into a 1.5-mL micro centrifuge tubes, centrifuged at 3500 rpm for 15 min. The supernatant were then transferred to new tubes and stored at −20°C for the subsequent analyses. Contents of alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphate (ALP) high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), total cholesterol (TC) and triacylglycerol (TAG), were analyzed using a biochemical analyzer  as previously described by . The kitSerum total protein content was analyzed using a biochemical analyzer  as described above. Turbidimetric assay method according to  with somCommercial kit from Nanjing Jiancheng Biotech Co.  was purchased for the analysis of SOD activity Analysis was performed as described by the manufacturer. The amount of superoxide dismutase needed to inhibit the reduction of nitro blue tetrazolium by 50% defined one unit of SOD activity and was expressed as Aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) activities were measured as previously described by  whiles C-1 per min defined one unit of GSH-Px whiles a unit of GR activity was defined as the amount of enzyme depleting 1 mmol NADPH in 1 min [Commercial kits were purchased from Nanjing Jiancheng Bioengineering Institute  for analysis of Glutathione peroxidase (GSH-Px), glutathione reductase (GR), and total antioxidant capacity (TAC) activities in serum using a spectrophotometer  at 412, 340, and 520 nm, respectively. 0.01 increment of absorbance of the reaction system caused by serum per milliliter reacting at 37 C for 1 min defined one unit of TAC. The amount of enzyme that reduces the concentration of GSH in the reaction system at 1mmol lin 1 min . Using 1in 1 min .Streptococcus iniae (S. iniae—ARS-98-60) was obtained from National Pathogen Collection Center for Aquatic Animals (Shanghai Ocean University). The bacterium was cultured following procedures described previously by [S. iniae . The results showed that the 96 h LD50 was 105 CFU per fish.Frozen stock-culture of ously by . In orde6 cfu/mL of S. iniae (10 × 105 cfu/fish) intra-peritoneal (IP) using tuberculin syringe. The fish were fed their respective diets for 14 days whiles they were monitored and mortality recorded twice daily.Ten fish from each tank (30 per treatment) were randomly sampled after the 56 days feeding trial for the bacterial challenge. All fish were kept in a 57-L aquaria containing 50 L water. Water temperature was maintained at 28–29 C. Fish were injected with a dose of 0.1mL of 1 × 10P<0.05. Normality of data and homogeneity of variance were tested using Shapriro-Wilk normality test and Bartlett's test respectively. Graph Pad Prism (V.5.03) was used to perform all analysis data presented as mean ± standard error of the mean (SEM).Data was analyzed using both univariate and multivariate GLM and no significant interactions were observed so we only report one-way ANOVA. Tukey’s multiple tests were used to compare means of all treatments. Significant differences was tested at P<0.05). However there was no difference among groups for condition factor (K) among the different groups (P>0.05). There was no difference among groups with respect to feed intake and ranged between 76.27 and 77.19.Survival, growth performance, feed utilization and morphometric index are shown in O.niloticus has been reported and discussed in our previous study [Effects of replacing fish oil with palm oil on fatty acid composition of us study . A summaP>0.05). However, the highest activity in AI, MI and PI were recorded in fish fed 50% PO.Replacing dietary FO with PO resulted in different response with respect to the analyzed digestive enzyme activities . In thisP<0.05). On the contrary, there were no differences among treatment groups for total cholesterol (TC), Glutamic-pyruvic transaminase (GPT) and Glutamic- oxalacetic transaminase (GOT). HDL-C and HDL-C/LDL-C ratio significantly decreased with increasing dietary PO inclusion whiles ALP, AST, and LDL-C and TAG significantly increased with increasing dietary PO inclusion. Generally, GOT and GPT increased with increasing PO inclusion levels but were not significant.Serum biochemical indices analyzed are summarized in -1), whiles LYZ and RB ranged between 159.7 to 184.8 (Uml-1) and 12.10 to 16.18 (540nm) respectively.The results for non-specific immune response such as serum lysozyme (LYZ), total protein and respiratory burst (RB) are shown in The effects of dietary palm oil on antioxidant capacity are shown in P>0.05) in mortality among groups post bacteria (S. iniae) challenge  [Generally, the results of this study showed that PO inclusion had no negative impact on growth performance in  tilapia , juvenilambicus)  and commambicus)  when dieambicus) . This isambicus) . There wambicus) , indicatambicus) . The aboO. niloticus with higher levels of palm in place of fish oil could be compromised. This is evident from the fact that compared to the control, groups fed 75% PO and 100% PO recorded significantly low amounts of EPA and DHA. This is in agreement to previous studies by [O.niloticus with PO in place could negatively affect the nutritional status of the final product. This is because groups fed 75% PO and 100% PO recorded the least n-3: n-6 ratio and is in agreement to previous study [O. niloticus with coconut oil reduces n-3: n-6 ratios.The effects of different dietary palm oil inclusion on fatty acid composition and lipid levels (liver and muscle) , a studyudies by  when fisus study  which reO. niloticus with varying levels of PO in place of FO increased ALP activity with fish fed 100% PO recording the highest value (O. niloticus) with PO could affect the liver negatively although this study had no data on liver ALP. Fish fed 0% PO diet might have recorded the least ALP activity value due to a decrease in trans-phosphorylation activity [O. niloticus were fed diet containing 100% PO as the sole lipid source and was significantly higher than those fed diets with 0% PO. This is an indication that feeding O. niloticus on PO diets could influence the liver negatively even though [It has been reported that an increase in liver ALP as well as increase in plasma ALP is an indicator of damage to the parenchyma in the liver . In thisst value . This inactivity . The hign though  documentn though . This stn though . Althougn though . The resn though . Also, dn though  and indin though .O. niloticus did not deplete disease resistance. This is in agreement to previous studies, which reported that serum lysozyme activity was not affected by FO substitution with VOs in Eurasian perch [Although the defence capability of the mucosal surface was reduced by the inclusion of PO due to the reduction in lysozyme activity, the lysozyme activity not being significantly altered by PO inclusion suggested that replacing FO with PO in diets of an perch , Nile tian perch  and Giltan perch . On the an perch . These dan perch , 44. ResO. niloticus when fed diets with different PO. This is in agreement to the study of [There was a decrease in the antioxidant activities of GSHpx in this study as dietary PO levels increased. This signifies that feeding tilapia with PO decreases peroxidative damage through removal of excessive ROS. This study recorded a decline in SOD and CAT activities in the serum of study of  in whichstudy of . There wstudy of  in whichstudy of .Seriola lalandi) did not affect lipase activity. Lipase activity in AI in fish fed diets containing 25%, 50%, 75% and 100% PO were higher compared to the values in MI and PI. This phenomenon is considered artificial and might have been caused by a possible drag of secreted mucus to that part of the digestive tract [O. niloticus, which indicates that O. niloticus can digest different palm oil levels, and utilized them as an energy source. In this study, fish fed 50% PO had the highest protease enzyme activity in AI, MI and DI. There existed a correlation between enzyme activity and growth rate implying digestive enzyme play significant role in the growth of Nile tilapia.Replacement of fish oil with palm oil affected the amylase activity in AI, MI as well as PI . The leave tract . The stuO. niloticus. This was in agreement to previous study where dietary lipid levels had no effects on the resistance of Nile tilapia to S. iniae infection [Ictalurus punctatus [S. iniae is important because S. iniae is known to cause serious mortalities of more than 40%. The above results shows that replacing FO with PO, although wouldn’t improve resistance wouldn’t either compromise the ability of Nile tilapia to fight S. iniae.Replacement of FO with PO did not significantly influence on the resistance ability of nfection . Contrarunctatus . These dunctatus . The difunctatus , 49. TheO. niloticus diets with higher levels of PO (75% PO and 100% PO) is reduced due to the decrease in EPA and ARA with corresponding increase in n-3:n-6 ratios. There was an increase in AST and ALP which suggested that supplementing fish diet with PO could lead to liver damage. Compared to FO, PO improved innate immune parameters  of juvenile Nile tilapia. With the exception of GST which increased significantly with increasing dietary PO, replacing FO with PO significantly reduced GR, TAC and MDA. Finally, there was no difference in resistance ability of juvenile tilapia when challenged with S. iniae.In conclusion, this study demonstrated that FO could be replaced with PO without distinct compromise in growth performance as well as feed utilization in Nile tilapia. However, nutritional and health benefits of feeding


To assess the incidence of anatomical anomalies in patients with retractile testis.We studied prospectively 20 patients (28 testes) with truly retractile testis and compared them with 25 human fetuses (50 testes) with testis in scrotal position. We analyzed the relations among the testis, epididymis and patency of the processus vaginalis (PV). To analyze the relations between the testis and epididymis, we used a previous classification according to epididymis attachment to the testis and the presence of epididymis atresia. To analyze the structure of the PV, we considered two situations: obliteration of the PV and patency of the PV. We used the Chi-square test for contingency analysis of the populations under study (p <0.05).The fetuses ranged in age from 26 to 35 weeks post-conception (WPC) and the 20 patients with retractile testis ranged in ages from 1 to 12 years (average of 5.8). Of the 50 fetal testes, we observed complete patency of the PV in 2 cases (4%) and epididymal anomalies (EAs) in 1 testis (2%). Of the 28 retractile testes, we observed patency of the PV in 6 cases (21.4%) and EA in 4 (14.28%). When we compared the incidence of EAs and PV patency we observed a significantly higher prevalence of these anomalies in retractile testes (p=0.0116).Retractile testis is not a normal variant with a significant risk of patent processus vaginalis and epididymal anomalies. A retractile testis is defined as a supra-scrotal testis that can be manipulated easily into the scrotum and remain there without traction until the cremasteric reflex is induced . Recent Structural and ultrastructural studies have demonstrated morphological alterations in retractile testicle cases , 6, and Anomalies of the tunica vaginalis and the epididymis are associated with testicular torsion  and are The objective of the present study was to assess the incidence of anatomical anomalies in patients with retractile testes.This study was approved and was carried out in accordance with the ethical standards of the hospital's institutional committee on human experimentation.We studied 62 patients prospectively with truly retractile testis during the period from January 2010 through January 2015. The retractile testis in this sample was defined based on physical examination findings. We included only patients with testis that can be brought down into the scrotum without tension and, after gentle massaging of the cord stay there upon release for a while.We submitted to surgery 20 (32.25%) of the 62 patients. In eight cases of operated patients the retractile testis was bilateral. The surgery was performed because of parent anxiety and/or the impossibility for the periodic follow-up. We compared the anatomical findings of 28 retractile testes with 25 human fetuses (50 testes) with the testes in the scrotal position.During the surgery, after the induction of anesthesia all patients had the testis in scrotal position and we used the trans-scrotal approach with a little midline scrotal incision with dissection of the cremaster muscle and fixation of the testis in dartos tunica in all cases.The 25 fetuses were macroscopically well preserved. Their gestational age was determined in WPC, according to the foot-length criterion, which is currently considered the most acceptable parameter to calculate gestational age –12. The After measurement, the fetuses were carefully dissected with the aid of a stereoscopic lens with 16/25X magnification. The abdomen and pelvis were opened to identify and expose the urogenital organs and inguinal canal and to show the testicular position. We observed patency of the processus vaginalis and the relationship between the testis and epididymis in fetuses and the patients.To analyze the relations between the testis and epididymis in surgical patients and fetuses, we used a previous classification , 14: TypWe used the Chi-square test for contingency analysis of the populations under study (p <0.05), calculated by the Graph Pad Prism software.The patients ranged in ages from 1 to 12 years old (average of 5.8). Of the 28 retractile testes, we observed patency of the PV in 6 cases (21.4%) and EAs in 4 cases (14.28%). Of the 6 cases of PV patency, 4 (66.6%) were on the right side and 2 (33.3%) on the left side. Of the 4 cases of epididymal anomalies, 2 (50%) were on the right side and 2 (50%) on the left side. The majority of epididymal anomalies (3 - 75%) were tail disjunction .Retractile testis has traditionally been considered a variant of normal testis because it usually descends into the scrotum during adolescence . In geneAlthough the question is controversial, some authors have reported histological changes and spermiogram abnormalities in follow-up of adult patients with retractile testis , 17. PreIn an interesting study with 3433 boys the authors observed that the prevalence of undescended testis in 6-year, 9-year and 13-year olds had a variation from 1.2% to 2.2% and after the age of 5 years, only acquired UDT was observed .Studies applying ultrasound confirm that retractile testes show reduced volume in relation to normal testes . One recAgarwal , in an iIn our sample, in which surgery was performed on 28 retractile testes, we found processus vaginalis patency in 21.4% of the cases. These findings confirm that the chance of patients with retractile testis presenting patent processus vaginalis is not negligible. In the control group composed of fetuses in which the testes had completed their migration, patency was only observed in 4% of the cases, a much lower rate than in the patients with retractile testis.Cryptorchidism can be associated with various anatomical anomalies, but epididymal anomalies and patency of the processus vaginalis are among the most frequent. Epididymal anomalies are associated with cryptorchidism in over one-third of these cases , 24. AnoPatients with disjunction anomalies  can present a longer distance between the testis and epididymis, the region called the mesorchium , 13, 14.The rate of epididymal anomalies in patients with retractile testis is not well defined in the literature. In our sample, we observed that 14% of the patients with retractile testis submitted to orchiopexy presented epididymal anomalies. In three cases we observed tail disjunction, an anomaly where the mesorchium is elongated, and in one case there was total disjunction between the testis and epididymis, a situation associated with infertility and also increased size of the mesorchium.This article presents the first description in the literature of the presence of epididymal anomalies associated with retractile testes. Despite the small sample, these findings can be significant. Future studies with larger samples will be necessary to confirm this association between epididymal anomalies and retractile testes, to provide further evidence that retractile testis is not a normal variant and does need treatment.The main limitation of this work is the small sample of patients with retractile testis, but because of the controversy over treatment, surveys with large samples of patients having this condition who underwent surgery are not common in the literature. Another limitation is the control group. The ideal control group would be boys without inguinal-scrotal anomalies having the same average age as the group with such anomalies. However, ethical considerations regarding use of living subjects and the extreme rarity of cadavers to study requires the use of human fetuses with testicles located in the scrotum as the control group.Retractile testis is not a normal variant with a significant risk of patent processus vaginalis and epididymal anomalies.


Chest pain is one of the most common presenting symptoms in patients attending the emergency department; yet, less than one in five patients receive a final diagnosis of acute myocardial infarction.et alet al recommend serial cardiac troponin testing in all patients, although alternative approaches do exist.In their expert consensus document, Tan The latest European Society of Cardiology guidelines allow rule-out of acute coronary syndrome in patients with at least 6 hours of symptoms who are <99th centile on presentation to hospital.6There are now a number of validated studies demonstrating a single high-sensitivity cardiac troponin I or T test on presentation may reliably exclude the diagnosis of myocardial infarction with high negative predictive value. Such approaches use a cardiac troponin decision threshold far below the 99th centile, often at or close to the limit of detection, exploiting the robust performance of high-sensitivity assays. For example, a high-sensitivity cardiac troponin I concentration <5 ng/L gave a negative predictive value of 99.6% in 6304 consecutive patients presenting with suspected acute coronary syndrome.et al recommend serial testing on presentation and at 3 hours in line with other international guidelines.et al is the recognition of the importance of changes in cardiac troponin concentration within the normal reference range. For example, consider a male patient with a high-sensitivity cardiac troponin I concentration of 10 ng/L at presentation and a concentration of 25 ng/L at 3 hours. Despite a >50% change in concentration, they would be ruled out by the 3-hour diagnostic pathway of the European Society of Cardiology if they had no ongoing chest pain and had a low GRACE score (<140). In the pathway proposed by Tan et al, this patient would be considered at high risk of myocardial infarction and would be recommended for further testing. This will reduce the likelihood of missed index diagnoses. While Tan et al recommend a relative change in cardiac troponin concentration, absolute changes in cardiac troponin concentration may also be informative at lower concentrations. An absolute change of >3 ng/L at 3 hours in those patients with troponin concentrations ≤99th centile at presentation identifies patients with increasing cardiac troponin concentrations who would benefit from further testing at 6 hours.6Tan et al, laboratory turnaround times in some centres may prevent a clinician having a presentation troponin result within 1 hour, which they suggest may preclude use of this approach. One could argue that a serial sample may still be obtained at 1 hour although a delay in clinical decision-making would be necessary until both results were available. Current debate focuses on whether the thresholds for change in cardiac troponin I concentration as proposed in the 1-hour pathway of the European Society of Cardiology (≥2 ng/L) may be reliably distinguished from analytical variability, since as many as 50% of patients without acute coronary syndrome can have a change of 2 ng/L on serial testing.The latest iteration of the European Society of Cardiology guidelines includes an alternative testing strategy using a presentation and 1-hour rule-out pathway with thresholds of cardiac troponin below the 99th centile.with myocardial infarction is equally important as this may facilitate earlier treatment and transfer to an appropriate care setting. The Asia-Pacific consortium proposes a rule-in threshold for myocardial infarction of 10 times the upper limit of normal (260 ng/L for the Abbott ARCHITECTSTAT high-sensitivity troponin I assay). This threshold was recommended by the Asia-Pacific consensus group, as the current European Society of Cardiology threshold of 52 ng/L only has a positive predictive value of 84%.While one of the major advantages of using a high-sensitivity assay is the earlier rule-out of myocardial infarction, accurate identification of patients STAT high-sensitive cardiac troponin I assay, where concentrations in men are two-fold higher than those in women. Use of gender-specific thresholds identify women at higher risk of future myocardial infarction or cardiac death, and there is the potential for outcomes to be improved.et al highlight this difference in 99th centile concentration between men and women is consistent across different populations, despite the influence of age, renal function, ethnicity and specimen type.3The authors recommend clinicians adopt gender-specific thresholds when using the Abbott ARCHITECTet al acknowledge there may be a role for contemporary point-of-care testing. It is imperative that patients with suspected acute coronary syndrome are not ‘ruled out’ on the basis of a normal or undetectable point-of-care result until repeat testing has been performed at 6 hours or the patient has undergone formal laboratory testing. We firmly agree with the authors' recommendation for an ongoing programme of education for those clinicians who are requesting and interpreting cardiac troponin tests. This is of greatest importance for new or rotating clinical staff who may be unfamiliar with the cardiac troponin assay in use at their new institution, as cardiac troponin decision thresholds and concentrations are not interchangeable between different assay types.In settings where access to laboratory facilities is limited, Tan et al should be commended for compiling a large volume of evidence into a clear and concise series of recommendations for the clinician who may be seeking to introduce high-sensitivity cardiac troponin testing into clinical practice. In this constantly evolving field, it is important that all clinicians remain abreast of the latest developments, with further changes in the recommended strategy for rule-out of acute coronary syndrome likely in the fourth iteration of the universal definition of myocardial infarction in 2018.In summary, Tan