The resting cyst of dinoflagellate Scrippsiella acuminata host bacterial microbiomes with more diverse trophic strategies under conditions typically observed in marine sediments.

Variation in the condition of marine sediments provides selective preservation milieus, which act as a key determinant for the abundance and distribution of dinoflagellate resting cysts in natural sediments. Microbial degradation is an understudied biological factor of potential importance in the processes. However, gaps remain in our knowledge about the fundamental information of the bacterial consortia associated with dinoflagellate resting cysts both in laboratory cultures and in the field. Here we used Scrippsiella acuminata as a representative of cyst-producing dinoflagellates to delineate the diversity and composition of bacterial microbiomes co-existing with the laboratory-cultured resting cysts, and to explore possible impacts of low temperature, darkness, and anoxia (the mock conditions commonly observed in marine sediments) on the associated bacterial consortia. Bacterial microbiome with high diversity were revealed associated with S. acuminata at resting stage. The mock conditions could significantly shift bacterial community structure and exert notably inhibitory effects on growth-promoting bacteria. Resting cysts under conditions typically observed in marine sediments fostered bacterial microbiomes with more diverse trophic strategies, characteristic of prominently enriched anaerobic chemotrophic bacteria generating energy via respiration with several different terminal electron acceptors, which yielded more acidic milieu unfavorable for the preservation of calcareous resting cysts. Our findings suggest that there is complex and dynamic interaction between dinoflagellates resting cysts and the associated bacterial consortia in natural sediments. This intrinsic interaction may influence the maintenance and/or accumulation of dinoflagellate resting cysts with potential of germination and initiation blooms in the field.

Evidence for the production of asexual resting cysts in a free-living species of Symbiodiniaceae (Dinophyceae).

Coral reef ecosystems are the most productive and biodiverse marine ecosystems, with their productivity levels highly dependent on the symbiotic dinoflagellates belonging to the family Symbiodiniaceae. As a unique life history strategy, resting cyst production is of great significance in the ecology of many dinoflagellate species, those HABs-causing species in particular, however, there has been no confirmative evidence for the resting cyst production in any species of the family Symbiodiniaceae. Based on morphological and life history observations of cultures in the laboratory and morpho-molecular detections of cysts from the marine sediments via fluorescence in situ hybridization (FISH), cyst photography, and subsequent singe-cyst PCR sequencing, here we provide evidences for the asexual production of resting cysts by Effrenium voratum, the free-living, red tide-forming, and the type species of the genus Effrenium in Symbiodiniaceae. The evidences from the marine sediments were obtained through a sequential detections: Firstly, E. voratum amplicon sequence variants (ASVs) were detected in the cyst assemblages that were concentrated with the sodium polytungstate (SPT) method from the sediments collected from different regions of China Seas by high-throughput next generation sequencing (NGS); Secondly, the presence of E. voratum in the sediments was detected by PCR using the species-specific primers for the DNA directly extracted from sediment; Thirdly, E. voratum cysts were confirmed by a combined approach of FISH using the species-specific probes, light microscopic (LM) photography of the FISH-positive cysts, and a subsequent single-cyst PCR sequencing for the FISH-positive and photographed cysts. The evidences from the laboratory-reared clonal cultures of E. voratum include that: 1) numerous cysts formed in the two clonal cultures and exhibited a spherical shape, a smooth surface, absence of ornaments, and a large red accumulation body; 2) cysts could maintain morphologically intact for a storage of two weeks to six months at 4 °C in darkness and of which 76-92 % successfully germinated through an internal development processes within a time period of 3-21 days after being transferred back to the normal culturing conditions; 3) two or four germlings were released from each cyst through the cryptopylic archeopyle in all cysts with continuous observations of germination processes; and 4) while neither sexual mating of gametes nor planozygote (cells with two longitudinal flagella) were observed, the haploidy of cysts was proven with flow cytometric measurements and direct LM measurements of fluorescence from cells stained with either propidium iodide (PI) or DAPI, which together suggest that the cysts were formed asexually. All evidences led to a conclusion that E. voratum is capable of producing asexual resting cysts, although its sexuality cannot be completely excluded, which guarantees a more intensive investigation. This work fills a gap in the knowledge about the life cycle, particularly the potential of resting cyst formation, of the species in Symbiodiniaceae, a group of dinoflagellates having unique life forms and vital significance in the ecology of coral reefs, and may provide novel insights into understanding the recovery mechanisms of coral reefs destructed by the global climate change and suggest various forms of resting cysts in the cyst assemblages of dinoflagellates observed in the field sediments, including HABs-causing species.

[Genetic study of a rare Chinese pedigree with a recombination occurring between the HLA-A/C loci in both parents].

OBJECTIVE: To analyze a Chinese pedigree with a recombination occurring between the HLA-A/C loci in both parents. METHODS: A patient who was planning to undergo hematopoietic stem cell transplantation due to "aplastic anemia" in February 2022 was selected as the study subject. Peripheral blood samples were collected from the patient, his parents and brother. HLA-A/C/B/DRB1/DQB1 high-resolution typing was carried out by using sequence-based typing and sequence-specific oligonucleotides. The recombination was identified by pedigree analysis. The HLA haplotype of each individual was identified by genealogical analysis. The parentage possibility was determined by short tandem repeat analysis. HLA-A/C/B/DRB1/DRB345/DQA1/DQB1/DPA1/DPB1 were determined with next-generation high-throughput sequence-based typing. The recombination sites were analyzed by family study. RESULTS: The high parentage possibilities of the family was confirmed by short tandem repeat analysis. Recombination was found between the HLA-A*24:02 A*33:03/C*14:03 in the paternally transmitted haplotype, whilst HLA-A*01:01 A*03:01/C*08:02 was found in the maternally transmitted haplotype, which had resulted in two novel HLA haplotypes in the proband. CONCLUSION: A rare case with simultaneous recombination of the paternal and maternal HLA-A/C loci has been discovered, which may facilitate further study of the mechanisms of the HLA recombination.

Prolonged joint cavity retention of tranexamic acid achieved by a solid-in-oil-in-gel system: A preliminary study.

Tranexamic acid (TXA) is an anti-fibrinolysis agent widely used in postoperative blood loss management. As a highly water-soluble drug, TXA is suffering from rapid clearance from the action site, therefore, large amount of drug is required when administered either by intravenously or topically. In this study, a TXA preparation with prolonged action site residence was designed using the nano-micro strategy. TXA nanoparticles were dispersed in oil by emulsification followed by lyophilization to give a solid-in-oil suspension, which was used as the oil phase for the preparation of TXA-loaded solid-in-oil-in-water (TXA@S/O/W) system. The particle size of TXA in oil was 207.4 ± 13.50 nm, and the particle size of TXA@S/O/W was 40.5 µm. The emulsion-in-gel system (TXA@S/O/G) was prepared by dispersing TXA@S/O/W in water solution of PLGA-b-PEG-b-PLGA (PPP). And its gelling temperature was determined to be 26.6 ℃ by a rheometer. Sustained drug release was achieved by TXA@S/O/G with 72.85 ± 7.52 % of TXA released at 120 h. Formulation retention at the joint cavity was studied by live imaging, and the fluorescent signals dropped gradually during one week. Drug escape from the injection site via drainage and absorption was investigated by a self-made device and plasma TXA concentration determination, respectively. TXA@S/O/G showed the least drug drainage during test, while more than 70 % of drug was drained in TXA@S/O/W group and TXA solution group. Besides, low yet steady plasma TXA concentration (less than 400 ng/mL) was found after injecting TXA@S/O/G into rat knees at a dosage of 2.5 mg/kg, which was much lower than those of TXA dissolved in PPP gel or TXA solution. In conclusion, sustained drug release as well as prolonged action site retention were simultaneously achieved by the designed TXA@S/O/G system. More importantly, due to the steady plasma concentration, this strategy could be further applied to other highly water-soluble drugs with needs on sustained plasma exposure.

Temporal Dynamics and Influential Factors of Taste and Odor Compounds in the Eastern Drinking Water Source of Chaohu Lake, China: A Comparative Analysis of Global Freshwaters.

The escalating proliferation of cyanobacteria poses significant taste and odor (T/O) challenges, impacting freshwater ecosystems, public health, and water treatment costs. We examined monthly variations in four T/O compounds from September 2011 to August 2012 in Chaohu Lake's eastern drinking water source (DECL). More importantly, we compared the reported T/O occurrence and the related factors in freshwater bodies worldwide. The assessment of T/O issues indicated a severe and widespread problem, with many cases surpassing odor threshold values. Remarkably, China reported the highest frequency and severity of odor-related problems. A temporal analysis revealed variations in odor occurrences within the same water body across different years, emphasizing the need to consider high values in all seasons for water safety. Globally, T/O issues were widespread, demanding attention to variations within the same water body and across different layers. Algae were crucial contributors to odor compounds, necessitating targeted interventions due to diverse odorant sources and properties. A correlation analysis alone lacked definitive answers, emphasizing the essential role of further validation, such as algae isolation. Nutrients are likely to have influenced the T/O, as GSM and MIB correlated positively with nitrate and ammonia nitrogen in DECL, resulting in proposed control recommendations. This study offers recommendations for freshwater ecosystem management and serves as a foundation for future research and management strategies to address T/O challenges.

Transoceanic ships as a source of alien dinoflagellate invasions of inland freshwater ecosystems.

Ships' ballast water and sediments have long been linked to the global transport and expansion of invasive species and thus have become a hot research topic and administrative challenge in the past decades. The relevant concerns, however, have been mainly about the ocean-to-ocean invasion and sampling practices have been almost exclusively conducted onboard. We examined and compared the dinoflagellate cysts assemblages in 49 sediment samples collected from ballast tanks of international and domestic routes ships, washing basins associated with a ship-repair yard, Jiangyin Port (PS), and the nearby area of Yangtze River (YR) during 2017-2018. A total of 43 dinoflagellates were fully identified to species level by metabarcoding, single-cyst PCR-based sequencing, cyst germination and phylogenetic analyses, including 12 species never reported from waters of China, 14 HABs-causing, 9 toxic, and 10 not strictly marine species. Our metabarcoding and single-cyst sequencing also detected many OTUs and cysts of dinoflagellates that could not be fully identified, indicating ballast tank sediments being a risky repository of currently unrecognizable invasive species. Particularly important, 10 brackish and fresh water species of dinoflagellate cysts (such as Tyrannodinium edax) were detected from the transoceanic ships, indicating these species may function as alien species potentially invading the inland rivers and adjacent lakes if these ships conduct deballast and other practices in fresh waterbodies. Significantly higher numbers of reads and OTUs of dinoflagellates in the ballast tanks and washing basins than that in PS and YR indicate a risk of releasing cysts by ships and the associated ship-repair yards to the surrounding waters. Phylogenetic analyses revealed high intra-species genetic diversity for multiple cyst species from different ballast tanks. Our work provides novel insights into the risk of bio-invasion to fresh waters conveyed in ship's ballast tank sediments and washing basins of shipyards.

Correlation between human leukocyte antigen ligands and killer cell immunoglobulin-like receptors in aplastic anemia patients from Shaanxi Han.

Regulating natural killer (NK) cell responses in hematological malignancies largely depend on molecular interactions between killer cell immunoglobulin-like receptors (KIR) and human leukocyte antigen (HLA) class I ligands. The goal of the current study was to examine the key functions of KIR genes, gene combinations of KIR-HLA, and KIR genotypes in genetic predisposition to aplastic anemia (AA). Herein, the genotyping of 16 KIR genes and HLA-A, -B, and -C ligands were performed in 72 AA patients and 150 healthy controls using PCR evaluations with sequence-specific primers using standard assays. According to the obtained results, AA patients had an increased incidence of activating KIR and KIR2DS4 (P = 0.465 × 10-4, Pc = 0.837 × 10-3, OR = 20.81, 95% CI = 2.786-155.5) compared to controls. KIR/HLA class I ligand profile KIR2DS4/C1 (P = 0.350 × 10-4, Pc = 0.630 × 10-3, OR = 8.944, 95% CI = 2.667-29.993) was significantly elevated in AA patients compared to healthy controls. Genotype AA1 (P = 0.003, OR = 2.351, 95% CI = 1.325-4.172) were increased, and AA195 (P = 0.006, OR = 0.060, 95% CI = 0.004-1.023) was decreased among AA cases compared to controls. Our findings indicated that KIR2DS4 may play a role in the pathogenesis of AA. This study revealed the contribution of KIR genes in the etiology of AA cases.

Improved salt-tolerance of transgenic soybean by stable over-expression of AhBADH gene from Atriplex hortensis.

KEY MESSAGE: The salt-tolerance of transgenic soybean cleared for environmental release was improved by stable over-expression of AhBADH gene from Atriplex hortensis, which was demonstrated through molecular analysis and field experiments. An effective strategy for increasing the productivity of major crops under salt stress conditions is the development of transgenics that harbor genes responsible for salinity tolerance. Betaine aldehyde dehydrogenase (BADH) is a key enzyme involved in the biosynthesis of the osmoprotectant, glycine betaine (GB), and osmotic balance in plants, and several plants transformed with BADH gene have shown significant improvements in salt tolerance. However, very few field-tested transgenic cultivars have been reported, as most of the transgenic studies are limited to laboratory or green house experiments. In this study, we demonstrated through field experiments that AhBADH from Atriplex hortensis confers salt tolerance when transformed into soybean (Glycine max L.). AhBADH was successfully introduced into soybean by Agrobacterium mediated transformation. A total of 256 transgenic plants were obtained, out of which 47 lines showed significant enhancement of salt tolerance compared to non-transgenic control plants. Molecular analyses of the transgenic line TL2 and TL7 with the highest salt tolerance exhibited stable inheritance and expression of AhBADH in progenies with a single copy insertion. TL1, TL2 and TL7 exhibited stable enhanced salt tolerance and improved agronomic traits when subjected to 300mM NaCl treatment. Currently, the transgenic line TL2 and TL7 with stable enhanced salt tolerance, which have been cleared for environmental release, are under biosafety assessment. TL 2 and TL7 stably expressing AhBADH could then be applied in commercial breeding experiments to genetically improve salt tolerance in soybean.

Deficiency of nitrogen but not phosphorus triggers the life cycle transition of the dinoflagellate Scrippsiella acuminata from vegetative growth to resting cyst formation.

Nitrogen (N) and phosphorus (P) are essential elements for algal growth. When N and P are deficient, dinoflagellates will take a series of measures to achieve population continuation including formation of resting cysts, an important ecological strategy of dinoflagellates that plays a key role in the initiation and termination of harmful algal blooms (HABs). How the deficiency of N and P affects algal growth and cyst formation has been investigated in some dinoflagellate species, but how it affects the life cycle transition in dinoflagellates has been poorly understood. In this study, we further explored the effect of N and P deficiency on the algal growth and resting cyst production in the cosmopolitan HABs-causing species Scrippsiella acuminata via refining the N and P concentration gradients. Further, we tracked the expression patterns of one CyclinB and one CDK1 genes of S. acuminata at different growth stages under three deficiency concentrations (1/1000 dilutions of N, P, and both N and P). The results suggest that N deficiency always triggered the cyst formation but P deficiency mainly inhibited the vegetative growth instead of inducing cyst formation. We also observed the highest cyst production when S. acuminata was cultured in the f/2-Si medium that was a one-thousandth dilution of N and P (N∼ 0.882 µM; P∼ 0.0362 µM). Our results for the expressions of CyclinB and CDK1 were well consistent with the results of algal growth and cyst formation at different deficiencies of N and P in terms of that higher expressions of these two genes were corresponding to higher rates of vegetative cell growth, while their expressions in resting cysts maintained to be moderate but significantly lower than that in fast-growing vegetative cells. Although we are still not sure whether the changing expressions of the two genes did regulate the transition of life cycle (i.e. cyst formation), or happened as parallels to the expressions of other truly regulating genes, our observations are surely inspirational for further investigations on the genetic regulation of life cycle transition in dinoflagellates. Our work will provide clues to probe the physiological and molecular mechanisms underlying the nutrient deficiency-induced alternation between life cycle stages in dinoflagellates.

Measuring viability of dinoflagellate cysts and diatoms with stains to test the efficiency of facsimile treatments possibly applicable to ships' ballast water and sediment.

Expansion of harmful algal bloom (HAB) species through ships' ballast water and sediment has been an increasing concern. Determining whether a microalgal cell, particularly for the toxic and HAB-forming species, is "viable" or "dead" is fundamental to understanding the effectiveness of the many ballast-water treatments that have been considered. To this end, we screened a variety of stains to assess the viability of dinoflagellate (Gymnodinium catenatum, GC) cysts and diatom (Corethron hystrix) vegetative cells to test the efficiency of ballast water treatments. Results showed that the stains fluorescing red or green are not sound candidates for viability measurements due to the interference of chlorophyll-induced red fluorescence or cytosolic green autofluorescence, while the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is limited by its toxicity, pseudo-positive judgment and the consequent confusion between cysts and vegetative cells. We further demonstrated that the stain Neutral Red (NR) is a sound candidate as the "vital stain" and can be easily applied for functionally defining the viability of both dinoflagellate cysts and diatoms. Another stain, the Evans Blue (EB), could be used as a "mortal stain" for the vegetative diatom cells but not a sensitive indicator of viability for GC cysts. The NR staining for GC cysts generally needs a higher dosage (0.005%) and longer staining time (24 h) than that were used for staining zooplankton, diatoms, and vegetative cells of dinoflagellates. In all cases, EB staining defined a "percentage of viable cells" significantly higher than that defined by NR. We conclude that the viability of a population is highly dependent on the species of stains used thus must be referred as a method-defined indicator.

Geographic distribution and historical presence of the resting cysts of Karenia mikimotoi in the seas of China.

The toxic dinoflagellate Karenia mikimotoi frequently forms harmful algal blooms (HABs) and thus causes massive kills of fish and shellfish in worldwide coastal waters, which has led to intensive investigations on multiple facets of the species. Following our recent discovery of K. mikimotoi forming resting cyst, a very possible mechanism for the inoculation of blooms and geographic expansion for this and many other HABs-causing species, here we report our detection of K. mikimotoi resting cysts in 125 surface sediment samples collected from the coastal waters (covering a latitude range from 18.29°N to 39.85°N) and 3 sediment cores (accumulated in 70‒100 years) collected from the East China Sea where are adjacent to the frequent blooming areas of K. mikimotoi. Via applications of quantitative real-time PCR (LSU rDNA-targeted), species-specific fluorescence in situ hybridization (FISH), and nested-PCR-and-sequencing to both types of the sediment samples that were pretreated with sodium polytungstate solution (SPT), we demonstrated that 1) K. mikimotoi cysts are widely present in surface sediments of the China seas (Bohai Sea (BS), Yellow Sea (YS), East China Sea (ECS), and South China Sea (SCS)), 2) the abundance of cysts is generally low (0 to 33 cysts in 32 g wet sediment), with that in the ECS and the SCS being higher than that in the YS and the BS, and the highest abundance was observed in sites of the ECS (e.g., Ningde, Fujian province) where the blooms of the species occurred frequently, as quantified by both methods, and 3) the cysts of K. mikimotoi have been present in the sediments of the ECS since 1970s, a short time prior to the first recorded bloom of K. mikimotoi in the SCS at 1980s. Our results not only demonstrated the wide geographic distribution of resting cyst of K. mikimotoi along the coast of China, but also proved a 50 years preservation of the cysts in the sediments of coastal area prone to forming frequent blooms. We consider our results have provided critical insights into the mechanisms of frequent bloom outbreaks and global distribution of K. mikimotoi in general, and particularly into the historical origin of K. mikimotoi in China. Further investigations are suggested to focus on on-site surveys for the cyst production and germination rates.

Dependence of genome size and copy number of rRNA gene on cell volume in dinoflagellates.

Dinoflagellates are an ecologically important group of protists in aquatic environment and have evolved many unusual and enigmatic genomic features such as immense genome sizes, high repeated genes, and a large portion of hydroxymethyluracil in DNA. Although previous studies have observed positive correlations between the large subunit (LSU) rRNA gene copy number and genome size of a variety of eukaryotic organisms (e.g. higher plants and animals), or between cell volume and LSU rRNA gene copy number, and/or between genome size and cell size, which suggests a possible co-evolution among these three features in different lineages of life, it remains an open question regarding the relationships among these three parameters in dinoflagellates. For the first time, we estimated the copy numbers of the LSU rRNA gene, the genome sizes, and cell volumes within a broad range of dinoflagellates (covering 15 species of 11 genera) using single-cell qPCR-based assay (determining LSU rRNA gene copy number), FlowCAM (cell volume measurement), and ultraviolet spectrophotometry (genome size estimation). The measured copy number of LSU rRNA gene ranged from 398 ± 184 (Prorocentrum minimum) to 152,078 ± 33,555 copies•cell-1 (Alexandrium pacificum), while the genome size and the cell volume ranged from 5.6 ± 0.2 (Karlodinium veneficum) to 853 ± 19.9 pg•cell-1 (Pseliodinium pirum), and from 1,070 ± 225 (Kar. veneficum) to 168,474 ± 124,180 µm3 (Ps. pirum), respectively. Together with the three parameters measured in literature, there are significant positive linear correlations between LSU rRNA gene copy numbers and genome sizes, cell volumes and LSU rRNA gene copy numbers, and between genome sizes and cell volumes via comparisons of multi-model regression analyses, suggesting a dependence of genome size and rRNA gene copy number on the cell volumes of dinoflagellates. Validation of the measurement methods was conducted via comparisons between reported data in the literature and that predicted using the linear equations we obtained, and between genome size measured by flow cytometry (FCM) and ultraviolet spectrophotometry (Nanodrop). These results provide insightful understandings of dinoflagellate evolution in terms of the relationships among genomes, gene copy number, and cell volume, and of rRNA gene-based studies in intra-populational and intra-individual genetic diversity, taxonomy, and diversity assessment in the environment of dinoflagellates. The results also provide a dataset useful for reads calibration in environmental metabarcoding studies of dinoflagellates and selection of candidate species for whole genome sequencing.

Exploration of resting cysts (stages) and their relevance for possibly HABs-causing species in China.

The studies on the species diversity, distribution, environmental implications, and molecular basis of resting cysts (stages) of dinoflagellates and a few species of other groups conducted in China during the last three decades are reviewed. The major achievements are summarized as the following five aspects: 1) The continual efforts in detecting the species diversity of resting cysts (spores) in dinoflagellates and other classes using either morphological or molecular approaches, or both, in the four seas of China, which led to identifications of 106 species of dinoflagellate resting cysts and 4 species of resting stages from other groups of microalgae, with a total of 64 species of dinoflagellate cysts and the resting stage of the brown tide-causing Aureococcus anophagefferens being unequivocally identified via molecular approaches from the sediments of Chinese coastal waters; 2) The well-known toxic and HABs-causing dinoflagellates Karenia mikimotoi, Karlodinium veneficum, Akashiwo sanguinea and the pelagophyte A. anophagefferens were proven to be resting cyst (stage) producers via laboratory studies on their life cycles and field detections of resting cysts (resting stage cells). And, via germination experiment and subsequent characterization of vegetative cells, numerous dinoflagellate species that had never been described or found to form cysts were discovered and characterized; 3) The distributions of the resting cysts of Alexandrium catenella, A. pacificum, Gymnodinium catenatum, K. mikimotoi, K. veneficum and Azadinium poporum and the resting stage cells of A. anophagefferens were morphologically and molecularly mapped in all four seas of China, with A. anophagefferens proven to have been present in the Bohai Sea for at least 1,500 years; 4) Obtaining important insights into the 'indicator' values of the dinoflagellate cyst assemblages in sediment cores for tracking eutrophication, environmental pollution and other anthropological influences in coastal waters; 5) Studies on the cyst-pertinent processes and genetic basis (transcriptomics together with physiological and chemical measurements) of resting cyst dormancy not only revealed the regulating patterns of some environmental factors in cyst formation and germination, but also identified many characteristically active or inactive metabolic pathways, differentially expressed genes, and the possibly vital regulating function of the phytohormone abscisic acid and a group of molecular chaperones in resting cysts. We also identified seven issues and three themes that should be addressed and explored by Chinese scientists working in the area in the future.

[Characterization of a rare HLA-C*08:84 allele and analysis of its 3-D molecular structure].

OBJECTIVE: To verify a rare allele of human leukocyte antigen (HLA) and analyze its inheritance and 3D molecular structure. METHODS: PCR-sequence-based typing, PCR-single strand oligonucleotide polymorphism and single allele-specific sequencing were carried out to characterize the rare HLA-C allele and its transmission in the family. Its protein structure was modeled by using SWISS-MODEL, Phyre2 and FATCAT software. RESULTS: Analysis indicated that the rare allele (HLA-C*08:84) has transmitted from the proband's mother and has differed from HLA-C*08:01 by a single base (g.512G>C), resulting in substitution of an amino acid (p.Trp147Ser). Modeling of the 3D structure of the encoded protein indicated that the amino acid residue variation is located at the alpha 2 helix, which participates the formation of pocket F. Modeling of the structures of C*08:84, C*08:01, C*08:02, C*08:03 and C*08:22 has suggested significant variation in the peptide binding regions of the backbone, with root mean square errors being 1.70 nm, 1.79 nm, 0.71 nm and 1.70 nm, respectively. CONCLUSION: A rare HLA-C*08:84 allele has been identified, and its clinical significance has been analyzed.

Potent allelopathy and non-PSTs, non-spirolides toxicity of the dinoflagellate Alexandrium leei to phytoplankton, finfish and zooplankton observed from laboratory bioassays.

The dinoflagellate genus Alexandrium has been well known for causing paralytic shellfish poisoning (PSP) worldwide. Several non-PSP toxin-producing species, however, have shown to exhibit fish-killing toxicity. Here, we report the allelopathic activity of Alexandrium leei from Malaysia to other algal species, and its toxicity to finfish and zooplankton, via laboratory bioassays. Thirteen microalgal species that co-cultured with Al. leei revealed large variability in the allelopathic effects of Al. leei on the test algae, with the growth inhibition rates ranging from 0 to 100%. The negative allelopathic effects of Al. leei on microalgae included loss of flagella and thus the motility, damages of chain structure, deformation in cell morphology, and eventually cell lysis. The finfish experienced 100% mortality within 24 h exposed to the live culture (2000-6710 cells·mL-1), while the rotifer and brine shrimp exhibited 96-100% and 90-100% mortalities within 48 h when exposed to 500-6000 cells·mL-1 of Al. leei. The mortality of the test animals depended on the Al. leei cell density exposed, leading to a linear relationship between mortality and cell density for the finfish, and a logarithmic relationship for the two zooplankters. When exposed to the treatments using Al. leei whole live culture, cell-free culture medium, extract of algal cells in the f/2-Si medium, extract of methanol, and the re-suspended freeze-and-thaw algal cells, the test organisms (Ak. sanguinea and rotifers) all died at the cell density of 8100 cells·mL-1 within 24 h. Toxin analyses by HILIC-ESI-TOF/MS and LC-ESI-MS/MS demonstrated that Al. leei did not produce PSP-toxins and 13-desmethyl spirolide C. Overall, our findings demonstrated potent allelopathy and toxicity of Al. leei, which do not only pose threats to the aquaculture industry, fisheries, and marine ecosystems but may also play a part role in the population dynamics and bloom formation of this species.

Pseudocochlodinium profundisulcus Resting Cysts Detected in the Ballast Tank Sediment of Ships Arriving in the Ports of China and North America and the Implications in the Species' Geographic Distribution and Possible Invasion.

Over the past several decades, much attention has been focused on the dispersal of aquatic nonindigenous species via ballast tanks of shipping vessels worldwide. The recently reclassified dinoflagellate Pseudocochlodinium profundisulcus (previously identified as Cochlodinium sp., Cochlodinium geminatum, or Polykrikos geminatus) was not reported in China until 2006. However, algal blooming events caused by this organism have been reported almost every year since then in the Pearl River Estuary and its adjacent areas in China. Whether P. profundisulcus is an indigenous or an invasive species has thus become an ecological question of great scientific and practical significance. In this study, we collected the sediments from ballast tanks of ships arriving in the ports of China and North America and characterized dinoflagellate resting cysts via a combined approach. We germinated two dark brownish cysts from the tank of an international ship (Vessel A) arriving at the Jiangyin Port (China) into vegetative cells and identified them as P. profundisulcus by light and scanning electron microscopy and phylogenetic analyses for partial LSU rDNA sequences. We also identified P. profundisulcus cyst from the ballast tank sediment of a ship (Vessel B) arriving in the port of North America via single-cyst PCR and cloning sequencing, which indicated that this species could be transported as resting cyst via ship. Since phylogenetic analyses based on partial LSU rDNA sequences could not differentiate all sequences among our cysts from those deposited in the NCBI database into sub-groups, all populations from China, Australia, Japan, and the original sources from which the cysts in the two vessels arrived in China and North America were carried over appeared to share a very recent common ancestor, and the species may have experienced a worldwide expansion recently. These results indicate that P. profundisulcus cysts may have been extensively transferred to many regions of the world via ships' ballast tank sediments. While our work provides an exemplary case for both the feasibility and complexity (in tracking the source) of the bio-invasion risk via the transport of live resting cysts by ship's ballast tanks, it also points out an orientation for future investigation.

Transcriptional Responses of the Heat Shock Protein 20 (Hsp20) and 40 (Hsp40) Genes to Temperature Stress and Alteration of Life Cycle Stages in the Harmful Alga Scrippsiella trochoidea (Dinophyceae).

The small heat shock protein (sHsp) and Hsp40 are Hsp members that have not been intensively investigated but are functionally important in most organisms. In this study, the potential roles of a Hsp20 (StHsp20) and a Hsp40 (StHsp40) in dinoflagellates during adaptation to temperature fluctuation and alteration of different life stages were explored using the representative harmful algal blooms (HABs)-causative dinoflagellate species, Scrippsiella trochoidea. We isolated the full-length cDNAs of the two genes via rapid amplification of cDNA ends (RACE) and tracked their differential transcriptions via real-time qPCR. The results revealed StHsp20 and StHsp40 exhibited mRNA accumulation patterns that were highly similar in response to heat stress but completely different toward cold stress, which implies that the mechanisms underlying thermal and cold acclimation in dinoflagellates are regulated by different sets of genes. The StHsp20 was probably related to the heat tolerance of the species, and StHsp40 was closely involved in the adaptation to both higher and lower temperature fluctuations. Furthermore, significantly higher mRNA abundance of StHsp40 was detected in newly formed resting cysts, which might be a response to intrinsic stress stemmed from encystment. This finding also implied StHsp40 might be engaged in resting cyst formation of S. trochoidea. Our findings enriched the knowledge about possible cross-talk of different Hsp members in dinoflagellates and provided clues to further explore the molecular underpinnings underlying resting cyst production and broad temperature tolerance of this group of HABs contributors.

Contact micropredation may play a more important role than exotoxicity does in the lethal effects of Karlodinium australe blooms: Evidence from laboratory bioassays.

Multiple dinoflagellate species from the genus Karlodinium have been well known to form massive and toxic blooms that consequently cause fish kills in many coastal waters around the world. Karlodinium australe is a mixotrophic and potentially ichthyotoxic species associated with fish kills. Here, we investigated phagotrophy of K. australe (isolate KaJb05) established from a bloom event in the West Johor Strait, Malaysia, using several prey species (phytoplankton, zooplankton, and larval fish). The results showed that K. australe ingested relatively small prey cells of co-occurring microalgae by direct engulfment, while it fed on larger prey cells of microalgae by tube feeding. The results of animal exposure bioassays using rotifer (Brachionus plicatilis), brine shrimp (Artemia salina), and larval fish (Oryzias melastigma) demonstrated that phagotrophy (in terms of the trophic mode of the dinoflagellate), or micropredation (in terms of the mechanism of lethal effects on prey), played a more important role than the toxicity did in causing the lethal effects of K. australe on these aquatic animals under low cell densities of K. australe, while the mortalities of animals observed in the exposure to cell lysates of K. australe were solely caused by the toxicity. A comparison of the lethal effects between K. australe and K. veneficum revealed that the lethal effect of K. australe on rotifers was much stronger than that of K. veneficum at all cell densities applied in the experiments and the more "aggressive" micropredation of K. australe is suggested to explain the difference in lethal effect between K. austale and K. veneficum. Our results may explain why K. australe exhibited fish killings during moderate blooms at cell densities < 2.34 × 106 cells L-1, whereas K. veneficum was observed to cause massive fish kills only if the cell density was above 107 cells L-1. We believe these findings provide new insights into the ecological consequences of phagotrophy exhibited in some mixotrophic and harmful algae such as species of Karlodinium and of HAB events in general.

Association of HLA class II (-DRB1,-DQB1,-DPB1) alleles and haplotypes on susceptibility to aplastic anemia in northern Chinese Han.

The Human Leukocyte Antigen (HLA) genes, playing key roles in mediating the immune response, especially HLA class II alleles were suggested to play a role in the activation of autoreactive T-cells in aplastic anemia (AA). Previous studies in different ethnic groups have indicated that some of HLA-A,-B,-DRB1 alleles had a protective or susceptive association with the prevalence, pathogenesis and development of AA. HLA class II genes, especially HLA-DQB1 and -DPB1 alleles or haplotypes at high-resolution level associated with AA have not been fully identified in northern Chinese Han populations. The aim of this study was to identify association of the variations in HLA class II region with AA in northern Chinese Han population. A recent case-control study, including 96 AA patients and 824 healthy controls was performed. The high-resolution HLA genotyping was conducted by PCR-SBT, -SSO and NGS-ION S5TM platform. Based on genotypic data of the three loci, haplotype estimation was carried out. HLA-DRB1*15:01 (Pc = 2.87 × 10-3; OR = 2.11, 95% CI = 1.45-3.07) and HLA-DQB1*06:02 (Pc = 1.86 × 10-2; OR = 2.01, 95% CI = 1.32-3.06) were the risk and predisposition alleles to AA in northern Chinese Han after considering multiple testing. Moreover, the HLA-DRB1*15:01-DQB1*06:02 (Pc = 4.90 × 10-3; OR = 2.09, 95% CI = 1.37-3.19) and HLA-DRB1*14:05-DQB1*05:03 (Pc = 2.65 × 10-2; OR = 2.82, 95%CI = 1.45-5.50) haplotypes had direct strong relevance to AA and were the susceptible haplotypes. HLA-DPB1 alleles and 23 polymorphic amino acid residues spanning exon 2 ~ 4 of DPß1 molecules have showed no statistically significant associations between AA and controls. The present findings establish a novel link between inherited HLA-DRB1,-DQB1,-DPB1 risk alleles and haplotypes in northern Chinese Han with AA, and open new avenues for development of targeted therapies to prevent or redirect immunopathology in AA.

A Novel Eyes Topical Drug Delivery System: CsA-LNC for the Treatment of DED.

PURPOSE: The objective of the present work was to prepare safe and effective Ciclosporin A Lipid nanocapsule (CsA-LNC) eye-drops for the treatment of DED. METHODS: The phase-inversion method was used to prepared different sizes CsA-LNC. CsA biodistribution in ocular after topical administration in rabbits was analyzed by a validated UPLC-MS/MS method. The efficacy of CsA-LNCs (25 nm, 50 nm, 85 nm) was evaluated using the tear breakup time, fluorescein staining, tear production, inflammatory cytokines and histopathology tests. The safety of CsA-LNCs was study by the score of ocular irritation and histological examination study. RESULTS: CsA-LNCs(20-100 nm) were successfully prepared, An in vivo PK study showed significant improvement of the bioavailability (4.20-fold (25 nm), 2.15-fold (50 nm) and 2.33-fold (85 nm)) in bulbar conjunctiva, and great permeability was observed in the cornea for CsA-LNCs compared with CsA emulsion. An in vivo PD study showed that CsA-LNCs have great efficacy for DED, and the effect was improved over CsA emulsion. CsA-LNCs were safe and not cause significant irritation to the eyes surface of rabbits. CONCLUSION: This work has demonstrated CsA-LNCs, in particular small sizes CsA-LNC, are safe and effective with promising potential to treat DED. Grapical abstract.