Alexandrium catenella. Within the bacterial community, Gammaproteobacteria and Bacteroidetes were predominant during the initial bloom stage, while Alphaproteobacteria, Cyanobacteria, and Actinobacteria were the most abundant taxa present during bloom onset and termination. In the archaea biosphere, methanogenic members were present during the early bloom period while the majority of species identified in the late bloom stage were ammonia-oxidizing archaea and Halobacteriales. Dinoflagellates were the major eukaryotic group present during most stages of the bloom, whereas a mixed assemblage comprising diatoms, green-algae, rotifera, and other microzooplankton were present during bloom termination. Temperature and salinity were key environmental factors associated with changes in bacterial and archaeal community structure, respectively, whereas inorganic nitrogen and inorganic phosphate were associated with eukaryotic variation. The relative contribution of environmental parameters measured during the bloom to variability among samples was 35.3%. Interaction analysis showed that Maxillopoda, Spirotrichea, Dinoflagellata, and Halobacteria were keystone taxa within the positive-correlation network, while Halobacteria, Dictyochophyceae, Mamiellophyceae, and Gammaproteobacteria were the main contributors to the negative-correlation network. The positive and negative relationships were the primary drivers of mutualist and competitive interactions that impacted algal bloom fate, respectively. Functional predictions showed that blooms enhance microbial carbohydrate and energy metabolism, and alter the sulfur cycle. Our results suggest that microbial community structure is strongly linked to bloom progression, although specific drivers of community interactions and responses are not well understood. The importance of considering biotic interactions  when investigating the link between microbial ecological behavior and an algal bloom’s trajectory is also highlighted.Interactions between microorganisms and algae during bloom events significantly impacts their physiology, alters ambient chemistry, and shapes ecosystem diversity. The potential role these interactions have in bloom development and decline are also of particular interest given the ecosystem impacts of algal blooms. We hypothesized that microbial community structure and succession is linked to specific bloom stages, and reflects complex interactions among taxa comprising the phycosphere environment. This investigation used pyrosequencing and correlation approaches to assess patterns and associations among bacteria, archaea, and microeukaryotes during a spring bloom of the dinoflagellate  Alexandrium is a cosmopolitan dinoflagellate genus that proliferates annually worldwide, often forming dense blooms in near-shore waters. Blooms of certain toxin-producing species in these genus are responsible for widespread and significant impacts, including animal mortality events, and can contaminate shellfish with toxins that threaten human health  have been associated with blooms  over the complete bloom cycle, i.e., from bloom initiation through termination . In addiThis investigation used high throughput sequencing and association networking methods to analyze prokaryotic and eukaryotic microbial community dynamics during a natural dinoflagellate bloom to better characterize patterns of interaction and related ecological behavior during HAB events. We hypothesized that microbial community structure and succession is linked to specific bloom stages, thus driving complex interactions among taxa comprising the phycosphere environment. The specific aims of this study were to explore the population dynamics of microorganisms during an algal bloom, and to identify and characterize correlations among microbial taxa using network analysis. The ultimate goal of this study was to advance our current understanding of community structure and associations among microbial populations during algal blooms, and provide insights into the mechanisms underlying these interactions and dynamics.2 in area) located in Eastham, MA . The pond is connected to the Nauset Estuary by a shallow and narrow tidal channel approximately 1–3 m in depth, which permits tidal exchange with the wider marsh system . Salt Pond is a small kettle pond . Water samples were collected in triplicate (2 L each) using a Niskin bottle from depths of about 0.5–1.0 m. Samples were first pre-filtered using a 300-mesh sieve, and were then collected on isopore filters with a diameter of 47 mm and pore size of 0.22 μm . Filters were stored at -80°C until analysis. A 50 mL fraction of each sample was preserved in 1% glutaraldehyde for enumerating total bacterial abundance. In addition, 500 mL of each sample was preserved with 1% Lugol’s iodine for phytoplankton identification and enumeration. This latter sample was further concentrated via settling and siphoning to produce a 25 mL concentrate. Algae were observed and counted under an optical microscope (×100 magnification).Sampling was carried out in Salt Pond weekly from 19 March 2014 until 12 June 2014  were collected using an YSI Professional Pro Meter . The detection range of salinity and precision were 1–60 and 0.1, respectively. Ammonium (NH4+), nitrate (NO3-), and phosphate (PO43-) were measured according to methods outlined below. NH4+ was measured according to the indophenol blue method using a spectrophotometer  , and the purity was ≥99%.During each sampling event, physicochemical parameters of the seawater were recorded. 0, Hach) . NO3- anSeawater samples preserved in glutaraldehyde were stained with 4′-6′-diamidino-2-phenylindole  for 15 min  and filt260/OD280 to verify that the value was >1.8 (NanoDropTM 2000), and stored at -20°C until further use.DNA was extracted using a Fast DNA Spin Kit  according to the manufacturer’s instructions, and eluted in 50 μL TE buffer. DNA quality was estimated by the ratio of ODin silico to target nearly all archaea and bacteria (Prokaryote 16S rRNA genes (V4–V5 hypervariable region) were amplified as described by bacteria , and probacteria . An 8-bp2, 200 mM dNTPs, 0.8 μg/mL BSA, 2.5 U of GoTaq Flexi DNA Polymerase (Promega), and 10 ng of total DNA. PCR cycling conditions were as follows: 2 min at 94°C; 10 cycles 94°C for 30 s, 65°C for 30 s ; 18 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension of 72°C for 10 min.Amplification of eukaryotes was performed using primers 547F (5′-CCAGCASCYGCGGTAATTCC-3′) and 952R (5′-ACTTTCGTTCTTGATYRA-3′), which target the V4 region of the 18S RNA gene. Amplifications were carried out in triplicate 50 μL PCRs comprising 0.5 μM of each primer, 1× GoTaq Flexi Reaction Buffer , 2.5 mM MgClThe PCR products were pooled, cleaned, and concentrated using a QIAquick PCR Purification Kit (Qiagen). A single composite sample for pyrosequencing was prepared by combining approximately equimolar amounts of PCR products from each sample. Sequencing was carried out by Biolinker Biotech. Co. Ltd. .de novo method (2 pipeline (SRP114998).Raw sequences were processed and checked using Mothur and QIIMe software packages . Sequenco method . The rawo method . After lpipeline . The seqP < 0.05. These two filtering steps were applied to remove poorly represented OTUs and reduce network complexity, which assisted in determining the core microbial community in samples. An affiliation network was generated using the R package igraph, version 1.0.1. False positive correlations were filtered according to methods by P-value threshold. Unexpected links (a score below the thresholds) were identified, counted, and removed from the network. The false positives were discarded using the dirmult package in R. An affiliation network is defined as a network in which the members are affiliated with one another based on co-membership of a group or co-participation in some type of event. Direct connections among the different genera were extracted from the two-mode affiliation network using igraph’s bipartite.projection function. The nodes in the reconstructed network represented variable taxa, and the edges connecting the nodes represented correlations between genera. The topographic features of the network, including centrality and edge weights, were also analyzed using functions available in the igraph package. In addition, subsets of representative specialist genera from the samples were selected to help identify potential keystone taxa and their interactions within the network. Sub-networks were then generated from the meta-community networks using subgraph functions in the igraph package. Information on the target network was further organized in matrices and visualized in chord diagrams using the R package circlize, version 0.3.5.3Interaction networks and co-occurrence patterns were examined according to t-test in the STAMP program .The α- and β-diversity indices for each sample were calculated using Mothur . Statist program . The str program . A heatmatabases . The preFigure 1A). Nutrient concentrations at each time point are shown in Figure 1B. NH4++ NO3- and PO43- ranged from 1.52 to 4.53 μmol/L and 0.12 to 0.51 μmol/L, respectively. Highest and lowest concentrations of NH4+ and NO3- were detected during bloom onset and peak bloom stages, respectively (P < 0.01). A different trend was observed in PO43- concentrations in which PO43- was higher earlier in the bloom, and lower concentrations occurred during the peak- and decline bloom stages (P < 0.01). The approximate IN/IP ratio in surface waters ranged from 12.67 to 37.75.Five primary stages were documented over the course of the bloom: pre- [W1], onset [W2–W3], exponential growth [W4], peak [W5], and decline/termination stages [W6–W9]. Over the duration of the sampling period, temperature, salinity, and pH values ranged from 5.2 to 16.8°C, 29.8 to 33.1, and 8.06 to 8.36, respectively . The cellular morphology and size of A. catenella are shown in Supplementary Figure S1. Total abundance of bacteria ranged from 1.7 × 105 to 1.2 × 106 cells/mL, and the highest value appeared immediately after the bloom’s peak  (P < 0.01). Micrographs of DAPI-stained filter samples are shown in Supplementary Figure S2. Total bacterial abundance was correlated with the A. catenella bloom, and a hysteresis phenomenon was observed in the population dynamics of the two groups.Figure 2A). The Chao1 index calculated for the bacterial group remained relatively constant throughout the A. catenella bloom, and only increased at the starting point of bloom termination. The Shannon index was relatively constant throughout the bloom; Simpson indices exhibited a similar trend, but decreased slightly over the course of the bloom.Using a 97% similarity cut-off, a total of 4,859 bacterial OTUs, 517 archaea OTUs, and 3452 eukaryotes OTUs were obtained from the field samples. To investigate changes in biodiversity, stage variations in Chao1, Shannon, and Simpson indices were calculated for the bacterioplankton communities . The Shannon index values increased during the bloom, starting at the second time point , whereas Simpson index values decreased from this time point.In the archaeal biosphere, fluctuations were observed throughout the entire sampling period. The highest Chao1 diversity index value appeared during bloom onset, and the lowest value was observed at the bloom’s peak .Chao1 indices calculated for eukaryotic communities were significantly lower compared with those calculated for bacterial/archaeal communities, and were relatively stable throughout the bloom. Fluctuations were observed, however, in the Shannon and Simpson indices calculated for the different sampling periods. The most obvious change appeared at beginning of the bloom’s decline , in which the lowest Shannon and highest Simpson diversity indices were observed  > group II (W4 and W8) > group III (W5–W7 and W9). The biggest difference in community composition across samples was found for W3, which was collected during bloom development . Archaea samples clustered into three groups that largely corresponded to the time period sampled , in spite of some overlap. The biggest difference among sampling periods was found at the decline stage, which had a comparatively higher beta-diversity indices (W7 and W9) (group I); while the remaining samples exhibited similar community composition (group II) . High similarity in eukaryotic species composition was found in a grouping comprised of samples from the early and late bloom stages (W1–W3 and W6–W9), whereas some differences appeared in samples collected during exponential growth (W4–W5). In this group, the highest beta-diversity index value was found at the peak stage of the bloom .Patterns of microbial community beta diversity (among-sample differences in OTU composition) are shown in Supplementary Figure S3A. Bacteroidetes and Proteobacteria were the two major dominant phyla during all bloom stages. The mean proportion of Bacteroidetes during the bloom’s peak (65.3%) was higher than during pre-bloom (30.6–51.5%) and post-bloom stages (21.5–53.1%). Proteobacteria exhibited an inverse pattern compared with Bacteroidetes; specifically, the Gammaproteobacteria dominated during the pre-bloom stage, while Alphaproteobacteria were predominant during the peak and decline stages . Betaproteobacteria and Deltaproteobacteria appeared during the post-bloom stage, and the proportional abundance of these groups was 5.1 and 2.8%, respectively. As the bloom progressed, significant changes in the bacterial community were observed. The Cyanobacteria cluster significantly increased (30%) during the onset of bloom decline (W6) and gradually decreased thereafter. Similarly, the Actinobacteria group gradually increased over time, and the maximum proportional abundance was observed during bloom decline (about 4.2–10.7%). Low proportions of Spirochaetae, Candidate division WS3, and Acidobacteria occupied <1% at different bloom phases .Bacterial community structure (phylum level) during the bloom is shown in Flavobacteriales, Alteromonadales, Rhodobacterales, Oceanospirillales, Thiotrichales, Sphingobacteriales, Campylobacterales, Desulfobacterales, Clostridiales, and Cytophagales, which contributed >98% of the total quality reads . Among them, some taxa displayed obvious variations during algal bloom development. Flavobacteriales gradually increased during the bloom’s onset, reached maximum proportional abundance during the peak stage, and then declined along with the bloom; interestingly, abundance subsequently increased concurrent with the bloom’s termination. Similar to Flavobacteriales, Rhodobacterales increased significantly during the early bloom stages, reached highest proportional abundance when the bloom began to decline (W7), and decreased subsequently. Alteromonadales was the third most proportionally abundant order, and showed an overall decline as the bloom progressed. Thiotrichales and Oceanospirillales exhibited gradually increasing and decreasing patterns with bloom progression, respectively.The dominant bacterial clades  in samples were Thaumarchaeota) and II (Euryarchaeota) . Among the most abundant OTUs (genus level), >50% belonged to methanogenic species ; 11–21% belonged to ammonia-oxidizing archaea (AOA) ; 5–30% belonged to Halobacteriaceae; 3–10% belonged to Thermoplasmatales; and 2–9% belonged to other species . The Methanomicrobiales were dominant during the first sampling period, and their proportional abundance increased significantly (to 60%) from bloom onset to exponential growth stages. Halobacteriales and Thermoplasmatales were present during bloom decline. The proportional abundance of these taxa increased during the late bloom stage and comprised 35–40% of the total archaeal community .Compared with bacteria, relatively fewer archaea were detected. The ratio of archaea was about 6–10% of the total prokaryotic community. The sequences obtained in this study primarily fall within the Marine Groups I  significantly increased and reached peak abundance (>90%) at the height of the bloom . During the later bloom stages (W7–W9), dinoflagellates were gradually replaced by other algal taxa, including Chlorophyta (mainly Tetraselmis sp. and Micromonas sp.), Diatomea , and other low-abundance algae . In addition to the phytoplankton, zooplankton also exhibited distinct dynamics during the bloom. During the pre-bloom stage, Arthropoda, Apicomplexa, and some taxa  co-occurred. During the bloom’s peak stage, their abundances were significantly decreased and most of 18S OTUs were occupied by Alexandrium sp. When the algal bloom entered the termination stages, eukaryotic diversity gradually increased, and multiple organisms  co-occurred during this bloom phase .Operational taxonomic units profiles of eukaryotic community structure during algal blooms in Salt Pond are shown in Figure 7A, the bacterial samples were divided into four clusters according to the different bloom stages .To determine whether the apparent differences in microbial community composition among samples significantly correlated with the algal bloom stage, cluster analyses were carried out. As shown in Figure 7B). Samples from the pre-bloom stage (W1) were clustered together and separated from samples collected during bloom onset (W2–W4). Interestingly, a few samples (W8 and W9) exhibited some similarities with the samples collected during exponential growth and decline bloom stages (W5–W7).Differences in archaeal community composition among bloom stages were also depicted by assemblage analysis .Analysis of 18S OTU profiles also revealed obvious temporal structuring. W1 and W2 formed the first group (pre-bloom and initiation of bloom onset), W3–W5 formed the second group , and third group comprised the remaining samples (W6–W9), collected during the bloom decline stages. The distribution showed a distinct temporal progression, with overlap at some phases . In the archaea, NH4+ and PO43- were the controlling factors associated with population variation , whereas the strongest determinant of community structure in microeukaryotes was the IN/IP ratio . Among the physical parameters, temperature and salinity were related to the composition of bacteria and archaea, respectively. There were no obvious negative or positive correlations between population dynamics and pH value. Additional information about the relationships between genera and environmental parameters as determined by correlation analysis (Pearson correlation coefficient) is shown in Supplementary Figures S6.Correspondence canonical analysis (CCA) was used to determine whether correlations in microbial structure were associated with environmental parameters . Among the chemical factors, POFigure 9), indicating that they were significant factors in determining microbial composition. Independently, T, N, and SP were able to explain 13.9% (P = 0.01), 12.2% (P = 0.05), and 3.3% (P = 0.05) of the total variation observed, respectively. Interactions between T and N, T and SP, and N and SP explained 2.9, 1.8, and 1.2% of variation, respectively. About 64.7% of the microbial community variation in OTU data was not explained by these environmental parameters, indicating that other biotic and/or abiotic factors contribute to these processes.A VPA was carried out to partition the contributions of environmental parameters. Results of this analysis showed that temperature (T), nutrients (N), and “salinity + pH” (SP) explained 35.3% of microbial community variation in the OTU data  (P < 0.01). Active interplay was observed in the negative relationships, which appeared to the highest degree in the archaea. The Halobacteria was strongly negatively correlated (P < 0.01) with several organisms, including Dictyochophyceae, Mamiellophyceae, Gammaproteobacteria, Flavobacteria, and some zooplankton (such as Maxillopoda) .An interaction network based on community correlations is shown in A. catenella, a network was constructed based on significant OTUs . For simplicity, we selected the top 30 OTUs, which were analyzed at the genus level. Among the bacterial groups, three members  had strong positive correlations with A. catenella; five members  were significantly negatively correlated with A. catenella; and the remaining species (Marinobacterium and Pseudospirillum) exhibited weak correlations with the target algae. Within the archaea, three main members  were correlated with bloom-forming Alexandrium sp., whereas other archaea were not significantly associated with algal dynamics. In the microeukaryote biosphere, Thalassiosira and Woloszynskia directly influenced algal abundance, whereas Helicostomella displayed negative effects on algae concentration through grazing or parasitic infection . An interesting finding was that three species  were correlated with both bloom formation and collapse, and exhibited both positive and negative correlations with A. catenella.To further investigate the correlations between microbial species and the bloom-forming dinoflagellate Figure 12).Based on the detection of functional genes, differences in the functional potential of bacterial communities were found among the different bloom stages. The three groups representing major functions  showed higher activity in the onset/exponential growth stage [W2–W4] compared with the pre-bloom stage [W1]. Amino acid and carbohydrate metabolism were the main pathways present during the bloom’s peak, leading into decline [W5–W6]. Interestingly, when the bloom began to decline [W7–W9], these metabolic pathways were diminished, whereas sulfur, lipid metabolism, and environmental adaptation genes were enriched during this phase , Gammaproteobacteria was closely associated with A. catenella. Our results are similar to this observation and suggest that Gammaproteobacteria have a taxon-specific selective capability in the phycosphere niche. In addition, we observed that Gammaproteobacteria were still a dominant species during the later bloom stages . Gammaproteobacteria, and particularly members of the Alteromonas–Pseudomonas–Vibrio group, are frequently characterized as r-strategists  and Alphaproteobacteria (such as Roseobacter and SAR11). Flavobacteria are highly diverse, and are capable of degrading high-molecular-weight organics   is another active producer of S and N during blooms  . This gr pectin) . Indeed,drolysis . Prior f decline . In thisg blooms . In addintiation , and hasrum lima . During nditions , and thenditions . These rg blooms . Shifts  ability .Actinobacteria and Cyanobacteria . In the oceanic environment, Actinobacteria helps to decompose organic matter  for uptake by phytoplankton. Several researchers have reported that Actinobacteria are closely linked to the diatom/dinoflagellate blooms , which is in line with previous reports suggesting a role for Cyanobacteria in sinking particle flux   in response to nutrient-limiting conditions, which further stimulates heterotrophic bacterial activity . Indeed,e blooms , which cpetition . This ispetition . In addid algae) .Cytophaga–Flavobacterium–Bacteroides (CFB) and Rhodobacterales. In this work, the most marked fluctuations were found in heterotrophic taxa, including the CFB members, which may reflect their ability to quickly establish and grow under changing conditions  during the bloom. Previously, Rhodobacterales, which comprise large fractions of the total bacterioplankton. Given its antagonism toward other bacteria, this group may be an important regulator of HAB dynamics , and their communities during the bloom were less complex compared with bacterioplankton. Changes in archaeal community composition during blooms have been sporadically reported in blooms of diatoms (Scrippsiella trochoidea) . During the pre-bloom period, Methanomicrobiales and Methanocellales were the predominant genera. The Methanocellales are phylogenetically diverse, and may play a significant role in ecosystem functioning through their ability to process complex compounds into simpler molecules in both anaerobic and aerobic habitats  ; and moshabitats . This abMethanosarcinales and Methanobacteriales, co-occurred in the mid- and post-bloom stages, potentially as syntrophic partners inorganic biomass degradation , which indicated a lower level of α-diversity in whole bloom period. A significant shift in the phytoplankton community structure was also reflected in β-diversity measures . Pre-bloom phytoplankton communities were characterized by high diversity, which included both diatom and dinoflagellate taxa. Upon bloom initiation, diatom abundance declined markedly. This suggests that diatom growth may have been negatively influenced by the high abundance of Alexandrium sp. Allelopathic and auto-inhibition effects play important roles in competition between algae, and may have contributed to the dynamics we observed between these groups , with various green algae or diatoms  as minor contributors . Several days later, the number of algal cells peaked, and the eukaryotic community was comprised almost exclusively of Alexandrium sp. (90%). Subsequent to the bloom’s peak, multiple phytoplankton groups co-occurred, ranging from nutrient specialists to nutrient opportunists . During bloom decline, diatoms and green algae comprised nearly 50% of eukaryotic OTUs. Algal activities led to rapid exhaustion of nutrients, and in conjunction with other taxa , contributed to bloom termination. Other unmeasured hydrochemical characteristics and/or factors not examined in this study likely contributed as well. Algal mortality during this stage dramatically increased the substrate available to other microeukaryotic communities, and the most obvious shift in community composition occurred during bloom termination.During the early stages of the bloom, communities were dominated by Dinophyceae  different hosts have different associated microeukaryotic communities; (2) higher copy numbers of ciliate and diatom 18S rRNA gene sequences dwarfed haptophyte DNA; and (3) primer mismatches or difficulties amplifying GC-rich haptophyte DNA, which resulted in low haptophyte tag yield. Despite the relatively small OTU tag numbers in our dataset, haptophytes are known to be opportunistic, and utilize urea and phosphonate as alternative sources of N and P , which may indicate that these species have the ability to compete with algae for nutrients and thus contribute to Alexandrium bloom termination.Other non-abundant gellates , compara N and P . In thisApicomplexa), Arthropoda (Maxillopoda), ciliate (Ciliophora), and rotifera. Their abundance was enriched during the bloom termination and post-bloom stages. Most of these taxa are phytoplankton grazers, and are the primary drivers in top–down control that can play a critical role in bloom decline. Our results are similar to those reported by Parvilucifera sinerae) may be an important factor in the termination of Alexandrium minutum blooms in the natural environment. Regarding the observed increase in ciliates during the post-stage, one possible reason is the availability of easily digestible taxa such as green algae and diatoms, both of which were dominant at the end of the bloom . Fungi were another cornerstone taxa during the transition from a phytoplankton dominated community to non-phytoplankton-dominated community. In this work, Basidiomycota and Ascomycota were the primary members detected, and their abundance was increased during bloom termination. These results are similar to our previous observations  and structure . Recent work further indicates that viruses influence algal host physiology and behavior . These results indicate that temperature was the major abiotic force shaping microbial community structure in Salt Pond, which is consistent with past studies that documented a pronounced impact of temperature on plankton composition , which may exhibit sensitivity to osmotic pressure. Salinity was previously shown to be a principal driving force of archaeal community patterns at the global scale , presumably due to uptake and utilization by Alexandrium sp. . It partly supports the principle of “everything is everywhere, but the environment selects”  as explanatory variables. These factors independently explained 13.9, 12.2, and 3.3% of the total observed variations, respectively . Furthermore, the putative keystone nodes in the network were highly abundant , suggesting that these groups are important in maintaining network structures in the microbial community. Several examples of interplay (positive or negative) among these groups have been reported previously . Among the bacterial groups, positive correlations were observed during the bloom that suggested certain functional interdependencies between bacteria and algae. In this study, Alexandrium sp. was positively correlated with Caldithrix, Spirochaeta, and Sulfurovum. These keystone species are characterized as opportunistic and play multiple roles, including the degradation of organic matter  and Alexandrium sp. were also observed, reflecting the appearance of nutrient competitors or algicidal bacteria during the blooms. The former are more efficient than algae at acquiring P and N from organic compounds (A multiplicity of inter- and intra-specific associations (direct or indirect) within the network were further analyzed in order to illuminate correlations between the primary microbes and the bloom-forming algae c matter , metabolc matter . And essc matter . These pc matter . In contompounds , while tompounds .Alexandrium sp. dynamics. Methanolobus was the primary group present during bloom formation, whereas Nitrosopumilus and Nitrosoarchaeum were the key members during bloom decline and termination. As a member of the methanogenic archaea, Methanolobus exhibit positive effects as symbionts, due in part to their participation in the N cycle in the phycosphere environment  had a dual correlation with bloom growth and collapse, suggesting the ability to adapt the changing environmental conditions associated during the algal bloom, based on their documented genotypic and metabolic diversity . Using Geochip, Akashiwo sanguinea, which was consistent with our results. Energy metabolism was maintained at relatively high levels during the onset and exponential stages, which may have contributed to the Alexandrium bloom’s duration. Similar results were observed in A. sanguinea, and through promotion of nitrogen (ammonium) accumulation, organic acid utilization, and carbon assimilation  assemblages from the natural environment during a phytoplankton bloom event. The microbial response to algal blooms exhibited high heterogeneity and complex dynamics, which comprised the succession of distinct populations exhibiting distinct functional profiles. Further, the association networks showed that algal and microbial community structure observed during the bloom was shaped by environmental factors as well as biotic interactions. The results highlight the importance of microbial network interactions as a robust tool to help understand the fate of HABs. It should be noted that we could not investigate the metaproteomes or metabolomes of phycosphere microorganisms in the present study. Future research using integrated approaches, such as flow cytometry and “Omics” methods, will continue to improve our understanding of microbial community interactions during HAB events, and could be used to further explore causal relationships between microbial diversity and activity, and algal blooms.JZ and ZC performed the experiments. JZ and MR drafted the manuscript. TS and DK collected the samples and analyzed the data. JZ and MR prepared the figures and tables. DA, ZC, and DK completed critical comments and revision.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.


The recipient SVC was transected near the right atrium, its lower stump was sutured, and its upper stump was laterally mobilized, which exposed the right PA. A left atrial incision was made medially to the right superior pulmonary vein and extended in the interatrial groove. The donor heart was placed in the right side of the chest, and the 2 left atria were sutured in a position that allowed the donor left PA to easily reach the recipient right PA. The anterior surface of the recipient right PA was opened and a cobra hood posterior slit was made on the donor left PA, almost extending to the main PA, and a direct anastomosis was achieved without tension. This was followed by an end-to-end donor/recipient SVC anastomosis and then an end-to-side anastomosis of the donor IVC to the recipient IVC as low as possible, allowing the lower body venous return of the recipient to reach the donor right atrium. The donor ascending aorta, which is usually long, was anastomosed as distally as possible in the recipient ascending aorta to create space and access to the PA anastomosis in case of bleeding .The patient was easily weaned off cardiopulmonary bypass with good hemodynamic and functional recovery. The patient’s systolic PA pressure had fallen to half systemic arterial pressure by the time he left the intensive care unit.We believe that the method herein expounded has certain merits. The technique can prevent the theoretic complications of a PA prosthetic graft such as compression, infection, thrombosis, fibrosis, and difficulty in sternal closure. Moreover, achieving a bicaval anastomosis seems to have dual advantages. The first advantage on the SVC side is that the closure of the recipient cardiac end unloads the dysfunctional native heart and endomyocardial biopsy is facilitated because the SVC anastomosis leads the bioptome forceps easily to the donor right ventricle. The other positive point on the IVC side is the sharing of lower body blood and burden between the recipient and donor heart.


Renal allograft rupture (RAR) is a rare but lethal complication of renal transplantation. It potentially threatens graft and patient survival. RAR is frequently associated with acute rejection, but other causes like renal vein thrombosis and acute tubular necrosis have also been observed. Most commonly a graft nephrectomy is required, but graft repair can also be attempted in selected cases to salvage the graft. Herein, we describe a rare case of spontaneous renal allograft rupture in the early posttransplant period due to acute tubular necrosis. A 42-year-old male, living donor renal allograft recipient, experienced RAR on the sixth posttransplant day. Surgical exploration showed two lacerations of 10 cm and 5 cm length at the upper and mid pole of the kidney. Histologically, the graft demonstrated acute tubular injury; no features of humoral or cellular rejection were identified. The successful management of this complication resulted in the salvage of the patient and the graft. This case demonstrates that early diagnosis and prompt treatment of a life-threatening RAR can salvage the graft. Spontaneous renal allograft rupture is a rare yet potentially life-threatening complication of renal transplantation. It is associated with a high incidence of graft loss The consequences are fatal in 6% of the cases and graft loss is the outcome in another 53% Most of these cases are immunologically mediated and caused by acute rejection. Acute tubular necrosis is only rarely responsible for this complication , 4.With improvements in the immunosuppressive regimens, the incidence of this complication has decreased. Recognition and prompt management of allograft rupture are important because of its likely devastating outcomes. Usually, graft nephrectomy is necessary in these cases, but conservative surgical intervention has also been tried successfully.We report an unusual case of a renal allograft recipient who developed a spontaneous RAR. The rupture was secondary to a relatively uncommon cause, acute tubular necrosis. The graft was successfully salvaged by surgical repair.This case emphasises that the transplant team must be aware of this fatal complication and the current management strategies.A 42-year-old gentleman, suffering from End-Stage Renal Disease (ESRD) secondary to vesicoureteral reflux (VUR). VUR was detected at the age of four years. He developed progressive proteinuria 12 years later followed by slowly progressive decline of renal function culminating in ESRD and was started on hemodialysis in Jan 2010. He underwent a live spousal blood group compatible renal transplant on 19th Feb 2010. The graft was lost to chronic allograft nephropathy in 2015 and second live donor renal transplant was planned. Donor was 33-year-old male, altruistic with HLA mismatch 3/6, ABO compatible, and CDC cross-match negative. Optical internal urethrotomy was done one day prior to transplant as he had urethral stricture diagnosed by micturating cystourethrogram. Induction was done with antithymocyte globulin in the dose of 3 mg/kg and triple immunosuppression was given with tacrolimus, mycophenolate mofetil, and prednisolone. The second transplant was done on 20th April 2016. Donor nephrectomy was done laparoscopically, the warm ischaemia time being 10 minutes. There was no injury to the renal vein or artery during donor nephrectomy. After anastomosis of the renal vein to the external iliac vein, there was avulsion at the venous anastomotic site. There was no thrombosis or stenosis of the vein and no heparin was required intraoperatively.Venous avulsion was repaired with 6.0 Prolene immediately, after which the arterial and venous blood flows were normal. The recipient was hemodynamically stable in the intraoperative and immediate posttransplant period. Immediate postoperative graft function was poor.In the next six postoperative days, his urine output remained around 2–4 liters/day. His serum creatinine came down very slowly from 9.97 mg/dL to 8.37 mg/dL by the fifth posttransplant day. Drain fluid decreased from 190 ml/day to 80 ml/day by the fifth posttransplant day. Hemoglobin remained stable, and no significant perigraft collection was seen on sonography. In view of poor allograft function, a renal biopsy was planned on sixth postoperative day, but his drain volume suddenly increased on that day and a frank hemorrhagic collection of 1 liter was drained within half an hour. The patient complained of severe abdominal pain and the urine output dropped. The graft became swollen and tender.Aggressive resuscitation was started and the patient was immediately reexplored in the operation theatre. A hematoma with large amount of blood clots was evacuated. There were two ruptures in the transplant kidney, 10 cm and 5 cm, one at upper pole and one at midportion, respectively, along the convex border . There wAn allograft biopsy was done in the operating room.The patient was shifted to transplant unit and the patient was given two sessions of plasmapheresis followed by 5 gm of IVIG on the first and second posttransplant day, while awaiting the graft biopsy report.The donor specific antibodies were negative by flow cytometry. Histopathology showed normocellular and uniform glomeruli. There were no capillary wall thickening and no fibrin thrombi or necrosis. Tubules showed vacuolation and degeneration and necrosis of epithelial cells. There was severe acute tubular necrosis. There was no tubulitis. There were no interstitial infiltration, fibrosis, and endarteritis. C4d stain was negative in the peritubular capillaries and glomerular capillaries.In Figures His graft function improved significantly and he was discharged after 10 days of repair of the rupture with good urine output and a serum creatinine of 1.9 mg/dl .Renal allograft rupture is defined as a superficial or deep tear of the renal capsule as well as renal parenchyma .It was first reported in 1968 by Murray et al. [The prevalence of RAR ranges from 0.3% to 9.6% with a mean of 3.4% .It typically occurs within three weeks after transplantation .The most frequent cause of RAR is acute graft rejection. It is the main predisposing factor in 60–80% of the cases [Other factors contributing to RAR include ischaemic acute kidney injury, acute tubular necrosis, damaged hilar lymphatic channels, renal vein thrombosis, ureteral obstruction with subsequent hydronephrosis, renal biopsy, trauma, nephrostomy tubes, and development of renal cell cancer , 5, 9–11The pathogenetic mechanism of RAR is not fully understood. Cortical and capsular ischaemia resulting from interstitial oedema and cellular inflammatory cell infiltration, in the setting of acute rejection, is considered the major cause of RAR by exerting capsular tension, tearing, and rupture. This may occur even many years after transplantation .Interstitial oedema was found in 85% cases and major ischaemic damage in 15%. Graft biopsy is the etiological factor in 15% and hydronephrosis in 4%.Hemodialysis with heparin is the possible factor in 46% cases . ClinicaThe management of RAR is urgent exploratory operation to control the bleeding, perform nephrectomy if indicated, and evacuate the hematoma to reduce the possibility of secondary infection.Early reports of conservative management of RAR showed poor results with less than 30% success rates. The high rate of graft nephrectomies was due to the failure to control bleeding and inability to reverse rejection, postoperative multiorgan failure, and uncontrollable coagulopathy. Over the past two decades, improvements in the surgical techniques have significantly reduced the transplant related mortality and morbidity. Current literature demonstrate that the ruptured grafts can be salvaged with a success rate as high as 80%. Recurrent rupture occurs in only 5% patients .With salvage rates between 40 and 100% and variable long-term complications and performance of the repaired graft, it is worth trying to salvage spontaneously ruptured grafts. Only in those patients whose hemodynamic status cannot be stabilised by appropriate aggressive hemodynamic support, graft nephrectomy should be considered as a definite treatment , 9.RAR is still encountered in clinical practice. In spite of advances in immunosuppression, most of the cases are due to acute rejection. Acute tubular necrosis is an uncommon cause of RAR. The life-threatening nature of this complication mandates early recognition and treatment. Although transplant nephrectomy is a definite treatment, with advances in the surgical techniques, attempts can be made to salvage the graft when hemorrhage can be controlled and RAR does not compromise survival of the patient.


The first aim of this study was to analyze the relationships between liver stiffness measurement, hepatic venous pressure and liver fibrosis. The second aim was to demonstrate the utility of real-time shear wave elastography for evaluation of Budd-Chiari syndrome patients before and after balloon hepatic venous angioplasty.A total of 32 patients with Budd-Chiari syndrome slated for successful balloon angioplasty met the inclusion and exclusion criteria. Shear wave elastography was used to generate dynamic liver stiffness measurement 2 days before angioplasty and 2 days, 3 months, and 6 months after angioplasty. Hepatic venous pressures were measured during balloon angioplasty. Correlations among liver stiffness, hepatic venous pressure, and fibrosis were assessed.Mean liver stiffness was 35.17 ± 10.60 kPa, 20.15 ± 5.47 kPa, 15.36 ± 4.34 kPa and 15.68 ± 5.58 kPa at baseline and 2 days, 3 months, and 6 months after angioplasty, respectively. Liver stiffness measured at 2 days and 3 months after angioplasty was significantly decreased (P < 0.001); liver stiffness measured at 6 months after angioplasty was not significantly different from that measured at 3 months after angioplasty (P = 0.636). Analysis of liver stiffness measurement and hepatic venous pressure before balloon angioplasty yielded a coefficient of correlation r = 0.701 (P < 0.001). Before and 2d after angioplasty, liver stiffness measurement did not correlated with fibrosis , , respectively, while the LSM difference before and 2 days after angioplasty negatively correlated with stiffness severity . Liver stiffness measured at 2 days and 3 months after angioplasty was significantly decreased (P < 0.001), remaining stable at 3 months, though still in the cirrhotic range.The liver stiffness of Budd-Chiari syndrome patients, measured by shear wave elastography, decreased considerably after hepatic venous recanalization, and significantly correlated with hepatic venous pressure though not with degree of fibrosis. Shear wave elastography may be effective in monitoring short- and long-term treatment outcomes in Budd-Chiari syndrome. Elastography uses ultrasound to estimate the stiffness of a material when a mechanical stress is applied. Shear wave elastography (quantitative elastography), such as transient elastography (TE), real-time shear wave elastography (SWE), and point shear-wave elastography (pSWE), is a noninvasive methodology that has been used to monitor liver stiffness in patients with chronic viral hepatitis –3. TransSWE is the most recently developed technique . Like TEHowever, recent studies have found that LSM is not only affected by the fibrosis stage but also by other confounding factors, such as the levels of aminotransferases ,13, extr2O) can be measured during balloon angioplasty, and a decline in hepatic venous pressure is considered to be reliable sign of successful vessel dilatation [We collected Budd-Chiari syndrome (BCS) patients who have undergone balloon angioplasty. BCS is defined as any level of hepatic venous outflow obstruction from the junction of small vein to the inferior vena cava and the right atrium, regardless of the cause of obstruction . This lelatation .Therefore, the first aim of this study was to analyze the relationships among LSM, hepatic venous pressure and liver fibrosis. The second aim was to evaluate the usefulness of SWE for evaluation of patients with BCS before and after balloon angioplasty.This prospective single-institution study was performed in strict accordance with the ethical guidelines of the Declaration of Helsinki, and was approved by the Ethics Committee of Chinese People's Liberation Army General Hospital. All study participants provided written informed consent.Between October 2013 and May 2015, patients with primary BCS who were scheduled to undergo balloon angioplasty and liver biopsy in the Department of Interventional Radiology in Chinese People's Liberation Army General Hospital were prospectively considered for this study. Inclusion criteria were incomplete obstruction of a hepatic vein or retrohepatic segment of the IVC. These patients were candidates for balloon angioplasty with or without stenting. The exclusion criteria were: liver biopsy specimens with a length < 1.5 cm and/or including < 6 complete portal triads, hepatic space-occupying lesion measuring > 3cm, portal vein thrombosis, congestive heart failure, biliary tract obstruction, primary biliary cirrhosis, and other diseases that affect hepatic sinusoidal pressure.Ultrasound-guided percutaneous liver biopsy was performed using 16-gauge Magnum needles  in the right anterior lower lobe (S5) or right posterior lower lobe (S6) of the liver (according to the Couinaud system of liver segmentation) . The intThe specimens were fixed in formaldehyde and embedded in paraffin. The specimens were reviewed using the Metavir scoring system  by two pSWE was performed 2 days before angioplasty, and 2 days, 3 months, and 6 months after angioplasty. Two radiologists (H. W. and J. C.) performed the procedures independently. Both two radiologists had at least 7 years of experience in ultrasound examination of the liver and at least 2 months of experience in SWE. They were blinded to the baseline data and biopsy results of the patients.2, where E is Young’s modulus (kPa), ρ is tissue density (kg/m3); and C is shear wave velocity (m/s). The tissue density was assumed to be 1000 kg/m3 with the ultrasound system [An ultrasound system  was used for SWE with a convex probe . Liver stiffness was expressed as Young’s modulus according to the formula E = 3ρCd system .All patients fasted for at least 8h before SWE. The right arm was placed in maximum abduction to enlarge the space between the ribs. An intercostal space was located in the right anterior lower lobe (S5) or right posterior lower lobe of the liver (S6) (according to the Couinaud system of liver segmentation) and 1.5–3 cm below the liver capsule. A 4cm×3cm SWE box was used to define a 2 cm-diameter circular region of interest (ROI), known as the “Q-box”. The mean, maximal and minimal values of Young’s modulus within the Q- box were measured during a breath hold. Five measurements were performed for each patient with the mean calculated and used for analysis. The Q-box was placed away from the hepatic blood vessels and gall bladder. If the box was not filled completely with color, the measurement was considered a failed measurement, as recommended by the manufacturer.2O) was measured during and after angioplasty using a manometer.All patients underwent balloon angioplasty with or without stenting 2 days after satisfactory SWE using the following procedures: modified Seldinger technique was used for puncture of the right femoral vein or the right jugular vein, followed by angiography of the IVC and hepatic vein to locate the site and extent of obstruction. Then the occluded segment(s) were expanded with balloon catheters  to diameters up to 25mm. Hepatic venous pressure . Patient characteristics are reported as mean ± standard deviations. Shapiro–Wilk testing was conducted on all the data. Repeated measures of ANOVA were conducted for liver stiffness at each time point before angioplasty, and 2 days, 3 and 6 months following angioplasty. One-way ANOVA was used to analyze the differences in preprocedure LSMs and pressure values among the different METAVIR fibrosis stages. Least-significant-difference (LSD) post-hoc analysis was employed for pairwise comparison. The LSM of two cases that failed angioplasty before and after surgery was compared using a t-test. The correlation between SWE measurements and venous pressure was analyzed by Pearson correlation. SWE measurements and biopsy results were compared by Spearman correlation. The correlation between preprocedure and postprocedure LSM differences and fibrosis severity was analyzed using the Spearman correlation. P < 0.05 indicated a significant difference.Thirty-seven patients were eligible during the recruitment period . Five paThirty-two patients underwent successful balloon angioplasty with or without stenting. LSM after angioplasty decreased significantly , with liBalloon angioplasty failed in two patients as a result of long-segment stenosis, with LSM of 52.21 kPa, 37.14 kPa before angioplasty and 50.09 kPa, 38.67 kPa after angioplasty, respectively .Recurrence was detected on angiography in one patient at 6 months after angioplasty. The LSM had been approximately 40.12 kPa at 2d before angioplasty, 14.57 kPa at 2d after angioplasty, 15.14 kPa at 3 months after angioplasty and 33.32 kPa at 6 months after angioplasty. Since the patient was intolerant of warfarin anticoagulation, conservative treatment was administered instead of a second balloon angioplasty.2O, and that immediately after angioplasty was 16.91 ± 4.48 cm H2O (P < 0.001). The coefficient of correlation between LSM and hepatic venous pressure before angioplasty was r = 0.701, (P < 0.001) (Mean hepatic venous pressure before angioplasty was 27.18 ± 8.47 cm H< 0.001) .Before angioplasty, LSM did not correlated with the degree of fibrosis in the histologic specimens . Two days after surgery, LSM still did not correlated with the degree of fibrosis . There was no significant difference in hepatic venous pressures among the different fibrosis stages before angioplasty ; there was no significant difference in LSM among different fibrosis stages before angioplasty . After successful balloon angioplasty, there was no significant difference in hepatic venous pressure among the different fibrosis stages ; there was no significant difference in LSM among the different fibrosis stages . However, LSM difference before and 2 d after angioplasty was negatively correlated with fibrosis .In this study we assessed liver stiffness before and after balloon angioplasty in 32 patients with BCS using SWE. Preprocedure mean LSM was obviously increased above the cut-off value of 10.4 kPa , 30 abovBecause fibrosis does not change over a short period of time, the correlation between liver fibrosis revealed by biopsy and LSM before angioplasty and LSM at 2 d after surgery was investigated. We found LSM did not correlated with fibrosis ,  before and 2 d after angioplasty, respectively. Our findings do not reproduce previous results suggesting that LSM positively correlates with the degree of fibrosis , 29. TheNevertheless, the difference in elasticity 2 d before and 2 d after angioplasty was negatively correlated with the degree of fibrosis . Thus the more severe the fibrosis, the smaller the change in liver stiffness after recanalization will be. This is probably because severe fibrosis causes compression and obstruction of the intrahepatic vein and the portal vein still maintains a high pressure even after recanalization. Moreover, at 2 d after recanalization with balloon angioplasty, although the LSM after successful recanalization decreased significantly (20.15 ± 5.47 kPa), it remained far above the cut-off value for liver cirrhosis. One patient showed an obvious increase in liver stiffness during reexamination with SWE 6 months after angioplasty. This patient was diagnosed as having had a recurrence of thrombosis according to the ultrasound. Therefore, SWE may be a method for monitoring the recurrence of BCS patients after angioplasty. Color Doppler ultrasound is less satisfactory in detecting the recurrence of obstruction in BCS patients , while LThe evaluation of the effectiveness of BCS treatment is generally based on symptoms, which inevitably introduces subjective factors and errors . AlthougWe are aware of the limitations of our pilot study. First, different in obstruction sites were present among the recruited BCS patients. Despite these differences, liver stiffness was measured uniformly in the right lobe. It was uncertain whether the change in hepatic venous pressure would be uniform for different sites of obstruction. To control for potential differences, LSM was performed at the same site for all patients and IVC was the site of recanalization in most patients (26/32). Among the remaining six patients, four had obvious intrahepatic collateral circulation, so we believe that the site of obstruction was less likely to affect the experimental result. Second, the small sample size is also a limitation. And, there was only one recurrent case. The value of SWE in monitoring BCS recurrence remains to be investigated.In conclusion, liver stiffness in BCS patients, measured by SWE, decreased considerably after hepatic venous recanalization, and significantly correlated with hepatic venous pressure though not with degree of fibrosis. SWE may be effective in monitoring short- and long-term treatment outcomes of BCS.


There are errors in the Funding section. The correct funding information is as follows:https://fapes.es.gov.br); TGK, MOG, CFS—Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)  (http://www.cnpq.br); MOG—Fundação de Amparo à Pesquisa do Estado de Minas Gerais  (http://www.fapemig.br/pt-br); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (http://www.capes.gov.br); Financiadora de Estudos e Projetos (www.finep.gov.br); and Sistema Nacional de Laboratórios em Nanotecnologias (SisNANO)/Ministério da Ciência, Tecnologia e Informação (www.mcti.gov.br/sisnano).TGK—Fundação de Amparo à Pesquisa e Inovação do Espírito Santo  (The publisher apologizes for the error.


Death with graft function (DWGF) is an important cause of long-term loss of grafts and patients. In this study, we investigated clinical characteristics and causes of DWGF in kidney transplant recipients.n = 2137). We divided recipients into four groups: alive with graft function (AWGF), alive with graft loss (AWGL), DWGF, and death with graft loss (DWGL).We recruited kidney allograft recipients who underwent surgery during 1973–2016 at Seoul National University Hospital in Korea  lost graft function due to death. DWGF was responsible for 38.6% of a total of 228 deaths. Recipients with DWGF were older, more often diabetic, and experienced delayed graft function more often compared to patients with AWGF, AWGL, and DWGL. Additionally, they had fewer episodes of acute rejection than AWGF and AWGL patients. The majority of DWGF developed because of infection (40.9%), malignancy (28.4%), and cardiovascular disease (11.4%). Infection-related mortality was highest within the first year after transplantation. Death due to malignancy was lowest within the first year, but increased thereafter.In our center, DWGF was a significant cause of graft loss. Infection and malignancy were the leading causes of DWGF during the overall post-transplantation period. Therefore, close monitoring for infection and malignancy should be instituted to lessen the burden of graft loss. Death with graft function (DWGF) is the leading cause of long-term graft failure . DespiteIn previous studies, the main cause of DWGF was cardiovascular disease (CVD) –8. UnconDWGF in kidney recipients is common. Therefore, it is important to identify the causes of and risk factors for DWGF, because increasingly more KTs. The time period after KT may affect the cause of death in recipients, because the degree of immunosuppression and its cumulative effect may be different based on time. Therefore, studies are needed to identify the causes of DWGF according to the period after transplantation.Understanding the main cause of DWGF and the changes in possible causes may help control DWGF; the final goal is improving long-term graft survival. In this study, we investigated the causes of DWGF and evaluated those causes depending on the time period after transplantation.Our retrospective study cohort consisted of adult and pediatric patients who received a kidney allograft during 1973–2016 at Seoul National University Hospital in Korea. We excluded recipients without precise information regarding graft function or recipient survival. We divided the patients into four groups according to graft function and recipient survival: alive with graft loss (AWGL), alive with graft function (AWGF), DWGF, and death with graft loss (DWGL).Clinical information was extracted from electronic databases at our center. These data included both recipient and donor characteristics. Demographic factors such as age and sex, causes of end-stage renal disease, dialysis duration and modality, transplant number, transplantation era, and multi-organ transplants were gathered from recipients. In addition, donor characteristics, such as age, sex, underlying diabetes at the time of transplantation, and donor types, were obtained. Data regarding transplant-related characteristics, such as ABO mismatches, HLA mismatches, DGF development, and biopsy-proven acute rejection (BPAR) episodes, were also collected.Statistics Korea using unique identifiers that were extracted until December 2014.Graft losses were investigated from medical records and the national renal replacement therapy database maintained by the Korean Society of Nephrology . Data reDWGF was defined as death of a kidney allograft recipient who had preserved kidney function without the need for dialysis or re-transplantation. We excluded acute kidney injury (AKI)-related dialysis events from DWGF . Baselint test or Mann–Whitney test. Categorical variables were compared according to their proportions found using the Chi-square test.All analyses were performed with SPSS 21.0 . Parametric variables were provided as means and standard deviation (± SD). Non-parametric variables were provided as median and interquartile range. Continuous data were compared using the Student’s Cox logistic regression was used to identify risk factors related to DWGF compared with AWGF. Proportional hazards assumptions for Cox models were tested using log-minus-log plots. We chose several covariates found to be statistically significant by a univariate Cox hazard ratio analysis and retained them as potential predictors. A stepwise multivariate Cox regression analysis was performed to assess hazard ratios for DWGF after adjustment for recipient age, sex, pre-transplantation diabetes, dialysis duration, history of KT and multi-organ transplantation, donor age and type, and the number of HLA mismatches. To determine risk factors for DWGF compared with DWGL, we used logistic regression analysis.A total of 2137 patients received kidney transplants and were enrolled. Among them, 228 (10.2%) patients died and 455 (20.3%) patients had graft loss. DWGF occurred in 88 patients, accounting for 38.6% of patients who died and 19.3% of patients with graft loss. AWGF, AWGL, and DWGL patients comprised 78.7, 10.7 and 6.6% of total allograft recipients, respectively  years. More than 70% were men, who received their allograft from living donors. Pre-transplant diabetes cases were found in 23.9% of DWGF patients. Eighty-three percent of DWGF patients received hemodialysis before KT. Median dialysis duration was less than 1 year. Median time to death was 66.1 (11.3–148.9) months.Next, we compared the clinical characteristics of DWGF patients with other groups Table . CompareTable Distributions of DWGF causes changed according to time after transplantation Fig. . InfectiThe incidence of graft loss decreased during the past 30 years. However, DWGF accounted for 12.9% of all graft failure during era 1; recently, this gradually increased to 45% during era 3 , and DGF  compared to AWGF patients. Compared to DWGL, DWGF was more prevalent in older recipients .During the median follow-up (65.5 months), DWGF developed in recipients with older age [adjusted hazard ratio (HR), 1.064; 95% confidence interval (CI), 1.043–1.086; In this study, we demonstrated that infection was the most common cause in DWGF patients, followed by malignancy. Unexpectedly, cardiovascular death comprised only 10% of overall causes of death in our cohort. Infection was most prevalent during the early post-transplantation period. Although infection-related death decreased slightly over time, it was the main cause of death, even 10 years after transplantation. Malignancy was the most cause during the late post-transplantation period. We also found that recipients who were older, and had pre-transplantation diabetes and DGF should be closely monitored for infection and malignancy events, even though they have preserved renal function.DWGF developed in recipients with older age, pre-transplant DM, and DGF compared with the other three groups. These factors were independently associated with DWGF. Vulnerable immunity, ischemia reperfusion injury, and immunosuppressive therapy are regarded as risk factors for DWGF in recipients with older age, pre-transplant DM, and DGF , 17. FurOverall, infection was the leading cause of DWGF in this study, whereas CVD is the most common cause of DWGF in western countries. The most common infection after KT is pneumonia , 20. We Interestingly, despite medical developments, the proportion of infection increased more in era 3 than in eras 1 and 2 among the causes leading to the death of DWGF patients. In our study, median age increased from 40 years in era 1 to 60 years in era 3. The drug effects in older recipients are different from that in younger . BecauseWe know that kidney transplant recipients are at higher risk for development of malignancy than the general population . During In previous studies –8, cardiOur study has limitations inherent to its retrospective nature. Our study was a single-center study and involved patients who underwent transplantation over the course of several eras of different immunosuppression protocols spanning five decades. Despite these limitations, our study has strengths. First, we evaluated the causes of DWGF according to the time point after transplantation using a long observation period. Because the short-term outcome of transplantation has improved, graft survival is longer than ever. Therefore, there is more demand for evaluating time-dependent causes. Second, this study included high-risk kidney recipients, such as those with multiple transplants and second transplants, while the majority of other studies analyzed only low-risk patients.In conclusion, infection and malignancy were the main causes of DWGF during the overall post-transplantation period, whereas CVD was the most common cause of DWGF in western countries. Infection was the most common cause of DWGF within the first post-transplantation year, whereas malignancy was the main cause of late DWGF. Our study suggests that efforts to give attention to causes, according to the time after transplantation, will improve the long-term outcomes and that new guidelines are needed for Asian populations.


Transient starch provides carbon and energy for plant growth, and its synthesis is regulated by the joint action of a series of enzymes. Starch synthesis IV (SSIV) is one of the important starch synthase isoforms, but its impact on wheat starch synthesis has not yet been reported due to the lack of mutant lines.TaSSIVb-D, with a mutation density of 1/165 Kb. Among these, three missense mutations and one nonsense mutation were predicted to have severe impacts on protein function. In the mutants, TaSSIVb-D was significantly down-regulated without compensatory increases in the homoeologous genes TaSSIVb-A and TaSSIVb-B. Altered expression of TaSSIVb-D affected granule number per chloroplast; compared with wild type, the number of chloroplasts containing 0–2 granules was significantly increased, while the number containing 3–4 granules was decreased. Photosynthesis was affected accordingly; the maximum quantum yield and yield of PSII were significantly reduced in the nonsense mutant at the heading stage.Using the TILLING approach, we identified 54 mutations in the wheat gene TaSSIVb-D plays an important role in the formation of transient starch granules in wheat, which in turn impact the efficiency of photosynthesis. The mutagenized population created in this study allows the efficient identification of novel alleles of target genes and could be used as a resource for wheat functional genomics.These results indicate that The online version of this article (doi:10.1186/s12864-017-3724-4) contains supplementary material, which is available to authorized users. Starch granules in plants are classified into two types according to their physiological role. Some starch granules are stored for long periods in reserve tissues as storage starch. This type of starch is an important source of carbohydrates for human beings. Other types of starch are stored temporarily as transient starch to provide carbon and energy for plant growth. Transient starch is synthesized in chloroplasts during the day and consumed during the night. Storage starch granules are synthesized in the plastid stroma by joint action of ADP-glucose pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE), starch debranching enzymes (DBE) and their isoforms; however, the synthesis and regulation of transient granules hold a specific feature . Some syAGPase gene enhances the rate of starch biosynthesis [Arabidopsis genome; AtSSIV is involved in the initiation of starch granule formation in leaves [AtSSIV increases the levels of starch accumulation by 30–40% and results in a higher rate of growth [SSIV mutants have remarkably decreased numbers of granules and abnormal granules [AGPase is the first and rate-limiting enzyme in the biosynthesis of starch, it catalyzes the synthesis of ADP-glucose, which is the substrate of amylose starch, and over expression of the ynthesis –4. Amyloynthesis –8, whereynthesis , 10; andynthesis . Isoformynthesis , 13. Twoynthesis –17. Onlyn leaves , 19. Ovef growth ; SSIV mugranules , 19, 21.Arabidopsis. Two SSIV isoforms, SSIVa and SSIVb, are found in rice. SSIVa is mainly expressed in the endosperm and is responsible for starch accumulation in seeds, whereas SSIVb is mainly expressed in leaves at early development stages and is responsible for leaf granule biosynthesis [ZmSSIV is highly expressed in the embryo, endosperm and pericarp in maize. In wheat, only one isoform of SSIV has been identified until now. This gene is located on the long chromosome arm of homologous group I and shows high similarity with OsSSIVb, so this isoform is named TaSSIVb [TaSSIVb is preferentially expressed in leaves and is not regulated by the circadian clock [TaSSIVb is also highly expressed at the middle stages of seed development [TaSSIVb on wheat leaf and seed granule characteristics at different growth stages have not yet been reported due to the lack of mutant lines.The functions of SSIV isozymes in the leaves of cereal crops are not as clear as in ynthesis , 14, 22.ynthesis . ZmSSIV  TaSSIVb , 24. TaSelopment , 26; howTaSSII, TaSBEIIa, TaSBEIIb, have been identified using this platform [waxy homozygous mutations created through TILLING and a pre-existing deletion of a third waxy homoeolog displays a near-null phenotype [TaSBEII have been identified in both durum wheat and bread wheat. Double mutant lines combining a TaSBEIIa-A mutation with a TaSBEIIa-B mutation in durum wheat and triple mutant lines combining mutations in TaSBEIIa-A, TaSBEIIa-B with TaSBEIIa-D have a significant increase in both amylose and resistant starch content [TaSSIVb would be helpful in characterizing its role in transient starch granule synthesis in wheat leaves.TILLING  is a powerful approach for novel allele identification and has been used to screen mutagenized plant populations for novel alleles of target genes –34. Hundplatform , 36. A hhenotype . Novel a content –42. UsinTaSSIVb–D with mutations in functional regions in an Ethyl methanesulphonate (EMS) mutagenized population derived from an elite hexaploid wheat cultivar. Homozygous mutants were used to investigate the specific role of TaSSIVb-D in starch granule synthesis in leaves at different stages of growth. We found that TaSSIVb-D mutations decrease gene expression and the number of starch granules in leaves.In this paper, we used the TILLING approach to identify novel alleles of TaSSIVb-D were designed for allele mining  or C to T (35.19%) transition point mutations, and the other 2 are T insertions. Of these mutations, 26 are in the coding region  are predicted to have a severe impact on protein function. Based on the PSSM program, three missense mutations  are predicted to have a severe impact. As the nonsense mutant E054-13 results in the loss of both the starch catalytic domain and glycosyltranferase domain, it might have a severe impact on protein function. Because the E1137 mutation is located in the glycosyltranferase domain which has less similarity with other SS isoforms , and migTaSSIVb gene expression. In wild type, gene expression profiling revealed non-significant differences in expression between different growth stages  at the seedling stage .Specific primer sets for each sub-genome  and E1137 (84.70%), were significantly higher compared with wild type (70.30%)  and yield of PSII (Y(II)) of E054-13 were significantly lower than in wild type  and glycosyltranferase domain GT-1). GT-5 (amino acids 422–661) is more conserved than GT-1 (amino acids 706–886) between SS isoforms . Two mut. GT-5  also show significant phenotypic differences compared with the wild type [In many cases, homeologous genes from different sub-genomes of wheat have dosage effects on phenotypes. Only double or triple mutants of operties , 49, andoperties . Howeverild type , 50, so SSIV in Arabidopsis also has negative effects on photosynthesis efficiency [SSIV mutant in wheat shows the same effects; photosynthesis efficiency is significantly reduced. This is likely because SSIV mutants accumulate high concentrations of the substrate ADPglucose [It has been reported that the capacity for starch synthesis and the rate of photosynthesis are positively correlated . A starcficiency . Our SSIPglucose , which lPglucose .TaSSIVb-D. Gene expression, granule number and photosynthetic parameters are down-regulated or reduced in the missense mutant E1137 and the nonsense mutant E054-13, and the impact of E054-13 is much more severe than that of E1137. This demonstrates TaSSIVb-D plays an important role in the formation of transient starch granules in wheat, and mutation of TaSSIVb-D results in remarkable reduction in transitory starch granules in leavies. The mutagenized population generated in this study could be used as a resource for gene functional research.We identified novel alleles, including missense and nonsense mutations, of the starch synthesis gene, Triticum aestivum L.) cultivar Jing411 was used as the wild type (WT) to create the mutagenized population. Homozygous mutant lines grown in the field with normal management were used for RT-qPCR and phenotyping.Wheat (1 generation) were planted directly in the field and self-fertilized. The M2 was developed using the single-seed descent (SSD) method.Seeds from the wild type Jing411 were soaked in tap water for 10 h at 20°C, then incubated in 1.5% EMS in 0.1 M sodium phosphate buffer (pH7.0) for 4 h or 6 h at 20°C. This was followed by washing with running tap water for 4 h at room temperature. The mutagenized seeds . DNA concentration was measured with the NanoDrop2000 spectrophotometer  according to the manufacturer’s instructions, and the final concentration was adjusted to 50 ng/μl. Individual DNA samples were pooled 2-fold into 96-well plates for TILLING screening, which was done according to Till et al. [The mutagenized population consists of 3058 Ml et al. .TaSSIVb (DQ400416) was downloaded fromhttps://www.ncbi.nlm.nih.gov/nuccore/DQ400416, and the sequences of TaSSIVb-A, TaSSIVb-B and TaSSIVb-D were obtained through alignment with long arm of chromosome 1A, 1B and 1D (https://wheat-urgi.versailles.inra.fr/Seq-Repository). TaSSIVb-D was used as the target gene for mining and phenotyping novel alleles. Primer Premier 5.0  was used to design homoeolog-specific TILLING and qPCR primers , forward primers labeled with the fluorescent dye IRD700 and reverse primers labeled with the fluorescent dye IRD800  were mixed with the ratio of unlabeled forward: labeled forward: unlabeled reverse: labeled reverse = 4: 1: 1: 4. PCR amplification and polyacrylamide gel electrophoresis were carried out as previously described . Identifhttp://blocks.fhcrc.org/proweb/parsesnp/) and SIFT  programs were used to predict the severity of each mutation. Mutations with PSSM >10 or SIFT <0.05 are predicted to have a severe effect on protein function [The PARSESNP  according to the manufacturer’s instructions. RNA extraction and cDNA synthesis was done for three biological replicates per genotype with three technical replicates for each biological replicates, and their concentrations measured with the NanoDrop2000 spectrophotometer, the same volume and concentration of normalized template was used for the next step experiment. RT-qPCR was performed on a CFX96 system using the SsoFast EvaGreen Supermix kit (Bio-Rad), PCR was performed at 94°C for 3 min, followed by 40 cycles at 94°C for 30 s, annealing for 30 s, and 72°C for 10 s, and then a melt curve stage. Concentration of each primer was 0.3 μmol, amplification efficiency of the three genes ranged from 97% to 100%. The actin gene used as a control was the same as described by Gu et al. [Ct method according to the CFX96 manual, data were analyzed by one-way ANOVA using Microsoft Excel software.The effects of mutations on the expression of u et al. . The relet al. [At the seedling, elongation, and heading stages, leaves of mutants and WT at the same developmental stage were sampled with 3 replicates for transmission electron microscope analysis. Each sample was processed as described in Guo et al.  and photet al. .Fv/Fm and Y(II) were measured with a photosynthetically active radiation (PAR) of 281 μmol m-2s-1 according to the manufacturer’s instructions. The response of photosynthetic fluorescence parameters to light intensity changes was determined using rapid light curves. Imaging Win software  was used to collect and analyze fluorescence parameter data. Data were analyzed by one-way ANOVA with Microsoft Excel software.A pulse amplitude modulation fluorometer  was used to measure chlorophyll fluorescence parameters. At the seedling, elongation, and heading stages, individual plants at the same developmental stage were selected for measurements with three biological and three technical replicates. From 10:00-11:00 am on the same day, after dark adaption by Dark Leaf Clip for 30 min, the value of Additional file 1: Table S1.Table S2. Percentage of chloroplasts containing different numbers of starch granules. (DOCX 19 kb)Primers used for RT-qPCR. Additional file 2: Figure S1.TaSSIVb-D-specific primers; s4b-qA: TaSSIVb-A-specific primers; s4b-qB: TaSSIVb-B-specific primers. (DOCX 1759 kb)Validation of sub-genome-specific RT-qPCR primers using Chinese Spring nullisomic-tetrasomic lines. CS: Chinese Spring; N1DT1B, N1AT1B, N1AT1D, and N1BT1D: Chinese Spring nullisomic-tetrasomic lines; J411: wild type; s4b-qD:


The purpose of this study was to compare union rates for isolated subtalar arthrodesis with and without the use of bone grafts or bone graft substitutes. We retrospectively reviewed 135 subtalar fusions with a mean follow-up of 18 ± 14 months. The standard approach was used for all surgeries. Graft materials included b-tricalcium phosphate, demineralized bone matrix, iliac crest autograft and allograft, and allograft cancellous chips. Successful subtalar fusion was determined clinically and radiographically. There was an 88% (37/42) union rate without graft use and an 83% (78/93) union rate with bone graft use. Odds ratio of union for graft versus no graft was 0.703 . The average time to union in the graft group was 3 ± 0.73 months and 3 ± 0.86 in the non-graft group, with no statistically significant difference detected (p = 0.56). Level of Evidence IV, Case Series.Graft use did not improve union rates for subtalar arthrodesis.  Indications include primary degenerative arthritis, inflammatory arthritis, post-infectious arthritis, congenital hindfoot deformities, talocalcaneal coalitions, and posterior tibial tendon dysfunction. The main goals of STJ arthrodesis are pain relief, hindfoot realignment, and functional improvement.,Nonunion remains an important complication, with incidence and role of risk factors varying in the literature. Recent reports have highlighted a decrease in overall union rates from between 96% and 100% to 84%The purpose of this retrospective study was to compare the fusion rates  and the time to union of STJ arthrodesis with and without the use of concomitant bone grafting. We hypothesized that the use of bone grafts or bone graft substitutes would not improve union rates and time to union. We also evaluated the association of smoking and the occurrence of STJ nonunion.We reviewed the charts and radiographs of 133 patients who underwent 135 primary STJ arthrodesis between January 2010 and December 2013, after the approval of Research Ethics Committee of our Institution (IRB number: X160503004). There were 66 males and 67 females. Mean age was 48  years. Forty-one cases (feet) were smokers (26 in the graft group and 15 in the non-graft group) and 19 cases (feet) were diabetics (10 in the graft group and 9 in the non-graft group).  PatientsAll patients were evaluated clinically and radiographically  until union was achieved or the diagnosis of nonunion was established by CT. Clinically, fusion was defined by subtalar joint stability in the absence of symptoms. Radiographically, fusion was defined as obliteration of the joint space with the presence of crossing trabeculae. CT criteria for fusion was consolidation of at least 50% of the posterior facet of the subtalar joint. All suspected cases of delayed union or nonunion were evaluated by CT.p < 0.05.Patients were divided into one of two operative groups - graft group or non-graft group - for comparison of the primary outcome of interest (union rate) using Fisher’s exact test. Secondarily, Fisher’s exact test was used in comparing the union rates in smokers and nonsmokers. Logistic regression was also used to compare odds of union for graft versus non-graft and smoker versus nonsmoker. All statistical analyses were performed on SPSS 23.0 software  with significance level set at Patients were draped and prepped (including a thigh tourniquet) in a sterile fashion. Skin incision and joint exposure were performed as described,  until thOnce bony apposition was achieved with proper hindfoot alignment, K-wires were inserted from the calcaneal tuberosity into the talar dome across the posterior facet. Positioning was confirmed fluoroscopically. With satisfactory positioning confirmed, definitive fixation was achieved using either a single 7.3mm screw (16 feet) or two 6.5mm screws (119 feet) (the gold standard for STJ arthrodesis).  The screThe splint was removed at 2 weeks for a wound check and stitch removal. This was followed by 6 weeks of non-weight-bearing cast use, and then removable boot cast use until clinical and radiographic confirmation of healing/fusion. Assessment of healing/fusion was performed clinically and radiographically every 6 weeks. Patients were followed for a mean of 18 ± 14 months. Poor union was determined as the presence of persistent pain and tenderness as well as poor radiographic evidence of progressive healing (i.e. lack of trabeculae across the fusion site). Patients with residual symptoms by week 16 to 20 postoperatively were evaluated by CT  and werep = 0.61). Odds ratio of union for graft versus non-graft was 0.703 . The average time to union in the graft group was 3 ± 0.73 months and 3 ± 0.86 in the nongraft group with no statistically significant difference detected (p = 0.56). There was an overall union rate of 85% (115/135) and CT-confirmed nonunion rate of 15% (20/135).  The averp = 0.19). (p = 0.45). Excluding smokers from data analysis resulted in a union rate of 88% (83/94) (85% (115/135) when included).There were 41 (feet) smokers in the study population . The smoking population had a 78% (32/41) union rate compared to an 88% (83/94) union rate in nonsmokers. However, these union rates were not significantly different between smokers and nonsmokers (= 0.19).  The oddsAll of the 19 diabetic patients included in this study achieved union .  There weComplications included: 7 deep infections ; 1 wound dehiscence (resolved with wound care); 5 sural neuritis cases (2 required nerve block or neurectomy. Others self-resolved); 3 complex regional pain syndrome cases; 1 talar neck stress fracture (between 2 screws) (conservatively managed); 5 persistent pain cases requiring hardware removal; 2 subfibular impingements (1 required arthroscopic debridement. The other was managed non-operatively). The possible risk factors for nonunion cases are described in ,-n (ranging from 95 to 184 feet) usually report union rates (85%-90%) closer to what we find in our study (85%).,Despite the recent trends towards minimally invasive operative techniques and the increasing use of subtalar arthroscopic fusion,Compared to other bone graft types, autogenous bone grafts carry a lower risk of infection transmission and are more likely to incorporate at their new site since they have minimal to no immunogenicity.,In the past decade, there has been a revolutionary change in the array of bone grafting products availableThe emergence of new synthetic bone graft products has been of great interest to the orthopedic community during the last decade.Scranton recommends bone grafting to avoid nonunion12 conducted a prospective study comparing autogenous iliac crest bone graft to DBM in 55 patients undergoing hindfoot arthrodesis, finding no significant difference in healing between the two groups of bone graft patients. Similarly, there was no significant difference in the time to healing between the iliac crest bone graft fusions and the DBM fusions. In their patient series, DBM was used in 36 hindfoot fusions with union achieved in 35 feet (97.2%).With respect to our union rate findings for each graft type, it is difficult to make any conclusions due to scant sample sizes for each graft type except b-TCP. b-TCP, when prepared with bone marrow, provides an excellent osteoconductive structure, with osteogenic capabilities from the marrow.p < .01). Similarly, Ishikawa et al.Easley et al.Our study had several limitations. To begin with, it is a retrospective study based on reviewing patients’ clinical charts and radiographs, limiting information such as patient outcome scores. In the same vein, other issues associated with the lack of variable control in retrospective studies are also noted in this study. For example, we found that patients in the non-graft group were more likely to have single screw fixation when compared to patients in the graft group. Of note however, this finding reflected the inclination of a single surgeon for both single screw and non-grafted operative technique . Another weakness of this study is the fact that successful fusion was entirely based on clinical judgment supplemented by radiographic evidence of healing. CT scan was not obtained for every patient to confirm union. While this would be ideal, this would expose a large number of patients to unnecessary expense and radiation. Because these patients had no pain on weight bearing and their plain radiographs confirmed union, a CT scan was not thought necessary. The use of bone graft or bone graft substitutes in STJ arthrodesis did not result in higher fusion rates nor did they shorten the time to union when compared to STJ arthrodesis without graft use. In addition, smoking status did not negatively impact union outcome.