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The fungal communities provide the nutrients and drive the cycles of elements in nature, and the rare fungal taxa are proved to be crucial for these communities in many environments. However, the ecological functions of rare taxa for the fungal communities in mangrove ecosystems are poorly assessed until now. This work aims to reveal the importance of rare taxa for the assembly of fungal communities in mangrove sediments by using the amplicon sequencing analysis of different spatiotemporal samples collected from Sanya mangroves, China. The results showed that Ascomycota and Basidiomycota were the dominant phyla in the conditionally rare taxa (CRT). The fungal communities possessed outstanding stability against the spatiotemporal variation and most collected environmental factors. The CRT possessed narrower niches and were more affected by the environmental variables than the abundant taxa. The current work demonstrated that the CRT had significantly higher relative abundances, degrees (the number of adjacent edges), clustering coefficients, and closeness centralities in the top 8 modules of the co-occurrence network (p 0.05), indicating the important role of the CRT for the interaction of fungal communities in mangrove sediments. These findings indicate the importance of the CRT for the fungal community structures in mangrove sediments, and would deepen our understanding of dynamic functions of mangrove fungi, thereby facilitating the management, utilization, and protection of mangrove ecosystems. KEY POINTS: • Fungal communities in mangrove sediments are stable against environment variations. • The conditionally rare taxa (CRT) possessed narrower niches than the abundant fungal taxa. • The CRT are central for the co-occurrence network and interaction of fungal communities.

Mangrove is among the most carbon-rich biomes on earth, and viruses are believed to play a significant role in modulating local and global carbon cycling. However, few viruses have been isolated from mangrove sediments to date. Here, we report the isolation of a novel Bacillus phage (named phage vB_BviS-A10Y) from mangrove sediments. Phage vB_BviS-A10Y has a hexameric head with a diameter of ~ 79.22 nm and a tail with a length of ~ 548.56 nm, which are typical features of siphophages. vB_BviS-A10Y initiated host lysis at 3.5 h postinfection with a burst size of 25 plaque-forming units (PFU)/cell. The genome of phage vB_BviS-A10Y is 162,435 bp long with 225 predicted genes, and the GC content is 34.03%. A comparison of the whole genome sequence of phage vB_BviS-A10Y with those of other phages from the NCBI viral genome database showed that phage vB_BviS-A10Y has the highest similarity (73.7% identity with 33% coverage) to Bacillus phage PBC2. Interestingly, abundant auxiliary metabolic genes (AMGs) were identified in the vB_BviS-A10Y genome. The presence of a β-1,3-glucosyltransferase gene in the phage genome supported our previous hypothesis that mangrove viruses may manipulate carbon cycling directly through their encoded carbohydrate-active enzyme (CAZyme) genes. Therefore, our study will contribute to a better understanding of the diversity and potential roles of viruses in mangrove ecosystems.

The objective of this study was to remove heavy metals from the metallurgical industry sludge by bioleaching alone and bioleaching combined with persulfate (PDS). The results showed that the removal of Cu, Zn, Pb and Mn reached to 70%, 83.8%, 25.2% and 76.9% by bioleaching alone after 18 d, respectively. The experiment of bioleaching combined with PDS was carried out in which the optimal additive dosage of K2S2O8, 8 g/L, was added to bioleaching after 6 d. After 1 h, the removal of 4 heavy metals reached 75.1, 84.3, 36.7 and 81.6%, respectively. Compared with bioleaching alone, although the increase in removal efficiency was not obvious, the treatment cycle was distinctly shortened from 18 d to 6 d + 1 h. Scanning electron microscopy (SEM) results showed that the surface morphology of the sludge was changed significantly by the combined treatment. The content of heavy metals was significantly reduced after bioleaching combined with PDS by energy dispersive X-ray spectroscopy (EDX). The treated sludge mainly existed in a stable form, and the bioavailability was reduced with European Community Bureau of Reference (BCR) morphology analysis. Therefore, this study proved that the combination of bioleaching and PDS was an efficient method to remove heavy metals from metallurgical industry sludge.

A novel gene aga3027 from the genome of Flammeovirga sp. OC4, isolated from the deep sea, was screened and expressed in Escherichia coli BL21. This gene encoded the genetic information of a potential agarase that consists of 851 amino acids and belongs to 16 β-agarase family of glycoside hydrolase. Purified recombinant Aga3027 demonstrated the maximum activity of agarase at 40 °C and pH 9.0, displaying excellent thermostability and pH-stability. The agarase retained more than 80% of its maximum activity after incubation at 30–40 °C for 48 h, or after incubation at pH 6.0–9.0 for 60 min, which indicated that this agarase was suitable for industrial applications. Silica gel chromatography was used to purify the hydrolysates of agar treated by agarase from the recombinant Aga3027. The hydrolysates were identified as neoagarotetraose and neoagarohexaose by thin layer chromatography and further confirmed by ion chromatography.

An agarase gene (agaM1) was cloned, expressed and characterized by using Escherichia coli as host strain, revealing the outstanding properties of recombinant AgaM1 (rAgaM1) in agarose degradation and neoagaro-oligosaccharides (NAs) production in our previous work. In current study, agaM1 was extracellularly expressed in Bacillus subtilis, and we aim to assess the ability of the supernatant of recombinant B. subtilis fermentation broth containing rAgaM1 to degrade agarose without protein purification, which would save the cost of purification and avoid the activity loss during purification. The pH and temperature optima for the supernatant were 7.0 and 50 °C, respectively. The supernatant containing rAgaM1 has outstanding stability against 40 °C and 50 °C. Besides, we detailedly studied the possible influence factors of rAgaM1 expression in the supernatant, including pH, temperature, isopropyl β-D-thiogalactoside (IPTG) concentration, initial optical density at a wavelength of 600 nm (OD600 ), and induction time, and the optimum conditions for rAgaM1 expression by B. subtilis were confirmed. Moreover, the supernatant was able to produce NAs by using the Gracilaria lemaneiformis, whose cells were broken by autoclaving, as substrate, and a total of 1.41 µmol ml-1 of NA, including neoagarotetraose and neoagarohexaose, was produced after degradation for 48 h. This ability could save the cost of substrates in NA production, although the method requires a further study. Our results reveal that the NAs with great potential in food and pharmaceutical industries could be inexpensive to make by the supernatant containing rAgaM1 of B. subtilis fermentation broth in the foreseeable future.

A novel Gram-staining-negative, catalase- and oxidase-positive, facultatively anaerobic and rod-shaped motile bacterial strain, designated as ZWAL4003T, was isolated from mangrove sediments of the Zini Mangrove Forest, Zhangzhou City, PR China. Phylogenetic analysis based on its 16S rRNA gene sequence indicated that ZWAL4003T was grouped into a separated branch with Vibrio plantisponsor MSSRF60T (97.38% nucleotide sequence identity) and Vibrio diazotrophicus NBRC 103148T (97.27%). The major cellular fatty acids were C14 : 0 (12.6%), C16 : 0 (17.6%), and summed feature 3 (C16 : 1ω6c /C16 : 1 ω7c, 45.6%). Its genome had a length of 4650556 bp with 42.8% DNA G+C content, and contained genes involved in the biosynthesis of bacteriocin, β-lactone, resorcinol, N-acyl amino acid, and arylpolyene. The in silico DNA–DNA hybridization and average nucleotide identity values for whole-genome sequence comparisons between ZWAL4003T and V. plantisponsor LMG 24470T were clearly below the thresholds used for the delineation of a novel species. The morphological and chemotaxonomic characteristics and the genotypic data of ZWAL4003T indicated that it represented a novel species of the genus Vibrio . Its proposed name is Vibrio ziniensis sp. nov., and the type strain is ZWAL4003T (=KCTC 72971T=MCCC 1A17474T).

In this study, cattail was researched as a natural cellulose source to extract cellulose. Dewaxing, alkali and bleaching treatments were carried out for the cattail fibers (CFs). The FTIR, SEM and XRD results indicated that hemicellulose and lignin were successfully removed from the CFs, and the content of cattail cellulose increased from 41.66 ± 1.11% to 89.72 ± 1.07%. Subsequently, cellulose aerogel was prepared by the extracted cattail cellulose. The Zeolitic imidazolate framework-8 (ZIF-8) was uniformly loaded onto the surface of cellulose aerogel by the in situ growth, and ZIF-8 Cattail Cellulose Aerogel (ZCCA) was finally prepared. The SEM, FTIR, XRD and TGA results further confirmed the successful preparation of ZCCA. Additionally, the results of the adsorption experiment showed that ZCCA had excellent adsorption performance for enrofloxacin, and the maximum adsorption capacity of enrofloxacin reached 172.09 mg·g−1 while showing good reusability. The adsorption process followed the pseudo-second-order kinetic model and the Langmuir isotherm model. Thermodynamic studies showed that the adsorption of enrofloxacin was a spontaneous endothermic reaction and that the adsorption mechanism involves the interaction of hydrogen bonds, electrostatic and π-π stacking.

Seaweed oligosaccharides possess great bioactivities. However, different microbial strains are required to degrade multiple polysaccharides due to their limited biodegradability, thereby increasing the cost and complexity of production. Shewanella sp. WPAGA9 was isolated from deep-sea sediments in this study. According to the genomic and biochemical analyses, the extracellular fermentation broth of WPAGA9 had versatile degradation abilities for three typical seaweed polysaccharides including agar, carrageenan, and alginate. The maximum enzyme activities of the extracellular fermentation broth of WPAGA9 were 71.63, 76.4, and 735.13 U/ml for the degradation of agar, alginate, and carrageenan, respectively. Moreover, multiple seaweed oligosaccharides can be produced by the extracellular fermentation broth of WPAGA9 under similar optimum conditions. Therefore, WPAGA9 can simultaneously degrade three types of seaweed polysaccharides under similar conditions, thereby greatly reducing the production cost of seaweed oligosaccharides. This finding indicates that Shewanella sp. WPAGA9 is an ideal biochemical tool for producing multiple active seaweed oligosaccharides at low costs and is also an important participant in the carbon cycle process of the deep-sea environment.

This study aimed to observe the molecular mechanism underlying the effect of tumor necrosis factor–inducible protein 6 (TSG-6) on the bone morphogenetic protein-4 (BMP-4)/drosophila mothers against decapentaplegic protein(Smad) signaling pathway and mineralization of dental pulp stem cells (DPSCs) in inflammatory environment. Normal and TSG-6 gene–modified DPSCs were cultured in a mineralization-inducing fluid containing 0 or 50 ng/mL TNF-α separately. The real-time polymerase chain reaction was used to measure the expression of TSG-6 and odonto/osteogenic differentiation makers at the mRNA level. Western blot analysis and cellular immunofluorescence were used to observe the odonto/osteogenic differentiation of DPSCs and the variation of BMP-4/Smad signaling pathway at the protein level. Moreover, normal and modified DPSCs combined with hydrogel were used for subcutaneous implantation in nude mice. The levels of odonto/osteogenic markers and BMP-4/Smad-related proteins were lower in Ad-TSG-6 DPSCs than in normal DPSCs after mineralization induction, and were higher in TSG-6-RNAi DPSCs than in normal DPSCs after culturing with mineralization-inducing fluid containing 50 ng/mL TNF-α. The subcutaneous transplantation of normal and modified DPSCs combined with hydrogel in nude mice demonstrated that normal DPSCs were formed in the tissue containing collagen. The tissue formed by Ad-TSG-6 DPSCs was highly variable, and the cells were very dense. We can know that TNF-α regulates the expression of TSG-6, thereby inhibiting the BMP-4/Smad signaling pathway and the odonto/osteogenic differentiation ability of DPSCs.

Dexmedetomidine is widely used for sedating patients in operation rooms or intensive care units. Its protective functions against oxidative stress, inflammation reaction, and apoptosis have been widely reported. In present study, we explored the effects of dexmedetomidine on monocyte-endothelial adherence. We built lipopolysaccharide- (LPS-) induced monocyte-endothelial adherence models with U937 monocytes and human umbilical vein endothelial cells (HUVECs) and observed the effects of dexmedetomidine on U937-HUVEC adhesion. Specific siRNA was designed to knock-down Connexin43 (Cx43) expression in U937 monocytes. Gö6976, GSK2795039, and NAC were used to inhibit PKC-α, NOX2, and ROS, respectively. Then, we detected whether dexmedetomidine could downregulate Cx43 expression and its downstream PKC-α/NOX2/ROS signaling pathway activation and ultimately result in the decrease of U937-HUVEC adhesion. The results showed that dexmedetomidine, at its clinically relevant concentrations (0.1 nM and 1 nM), could inhibit adhesion of molecule expression (VLA-4 and LFA-1) and U937-HUVEC adhesion. Simultaneously, it also attenuated Cx43 expression in U937 monocytes. With the downregulation of Cx43 expression, the activity of PKC-α and its related NOX2/ROS signaling pathway were reduced. Inhibiting PKC-α/NOX2/ROS signaling pathway with Gö6976, GSK2795039, and NAC, respectively, VLA-4, LFA-1 expression, and U937-HUVEC adhesion were all decreased. In summary, we concluded that dexmedetomidine, at its clinically relevant concentrations (0.1 nM and 1 nM), decreased Cx43 expression in U937 monocytes and PKC-α associated with carboxyl-terminal domain of Cx43 protein. With the downregulation of PKC-α, the NOX2/ROS signaling pathway was inhibited, resulting in the decrease of VLA-4 and LFA-1 expression. Ultimately, U937-HUVEC adhesion was reduced.

The Paris Agreement calls for countries to pursue efforts to limit global-mean temperature rise to 2 °C compared to the pre-industrial level. To achieve this, it is essential to accelerate the low-carbon transition of energy system. China is the largest carbon emitter and plays a decisive role in mitigating global climate change. The transition pathways for China to contribute to meeting the global 2 °C target, however, have not been extensively explored. Here we develop a bottom-up national energy technology model (C3IAM/NET), a linear optimization model, to reveal the energy consumption, carbon emissions and technology pathway at the national and sectoral levels in line with the 2 °C climate target. Results show that China's carbon emissions need to peak at the year 2023 and reduce to 3.56 GtCO2 by mid-century. During the 2020–2050 planning horizon, the remaining carbon budget is estimated to be controlled within 234 GtCO2, with a cumulative emission reduction of 165.3 GtCO2, of which the power sector bearing the largest share of responsibility, followed by the industry, transportation and building sectors. We project that China's primary energy consumption needs to peak before 2040 and the proportion of non-fossil energy in energy structure needs to reach 76% by 2050, and about 88.4% of electricity production comes from renewables and nuclear energy.

As the most abundant biological entities, viruses are major players in marine ecosystems. However, our knowledge about virus-host interactions and viral ecology in the deep sea remains very limited. In this study, a novel bacteriophage (designated as phage BVE2) infecting Bacillus cereus group bacteria, was isolated from deep-sea sediments. Phage BVE2 caused host lysis within 1.5 h after infection. However, the presence of two integrase-encoding genes in the BVE2 genome suggested that BVE2 may also follow a temperate strategy. The genome of phage BVE2 is approximately 20 kb in length and is predicted to encode 28 proteins. Genomic and phylogenetic analysis suggested that BVE2 is a highly mosaic phage that has inherited genetic features from Wbeta-like viruses, B. cereus prophages, and its host, suggesting that frequent horizontal gene transfer events occurred during its evolution. This study will help to reveal the evolutionary history of Wbeta-like viruses and improve our understanding of viral diversity and virus–host interactions in the deep sea.

Nitrogen pollution in aquaculture needs the efficient and cost-effective in-situ technology. This study aims to apply Marichromatium gracile YL28 to in-situ bioremediation and test its ability to maintain the nitrogen balance in aquaculture. In laboratory aquaculture system, approximately 99.96% of nitrite (1 mg/L) was removed within 7 d through denitrification coupled with assimilatory nitrate reduction. Ammonium (3.5 mg/L) of 95.6% was directly assimilated by YL28 within 7 d. Moreover, in zero exchange water from shrimp (Penaeus vannamei) aquaculture field trials (20 days), YL28 significantly reduced the ammonium accumulation (0.6 mg/L) and 99.3% of nitrite (1.25 mg/L). Toxicological studies with the Institute of Cancer Research (ICR) mice and Oryzias melastigma indicated that M. gracile YL28 can be safely applied in aquatic ecosystems. All results demonstrate that strain YL28 has high promise for future applications of removing inorganic nitrogen and maintaining the nitrogen balance from in-situ aquaculture.

A motile, Gram-stain-negative, non-pigmented bacterial strain, designated MGL06T, was isolated from seawater of the South China Sea on selection medium containing 0.1 % (w/v) malachite green. Strain MGL06T showed highest 16S rRNA gene sequence similarity to Rhizobium vignae CCBAU 05176T (97.2 %), and shared 93.2–96.9 % with the type strains of other recognized Rhizobium species. Phylogenetic analyses based on 16S rRNA and housekeeping gene sequences showed that strain MGL06T belonged to the genus Rhizobium. Mean levels of DNA–DNA relatedness between strain MGL06T and R. vignae CCBAU 05176T, Rhizobium huautlense S02T and Rhizobium alkalisoli CCBAU 01393T were 20 ± 3, 18 ± 2 and 14 ± 3 %, respectively, indicating that strain MGL06T was distinct from them genetically. Strain MGL06T did not form nodules on three different legumes, and the nodD and nifH genes were also not detected by PCR or based on the draft genome sequence. Strain MGL06T contained Q-10 as the predominant ubiquinone. The major fatty acid was C18 : 1ω7c/C18 : 1ω6c with minor amounts of C19 : 0 cyclo ω8c, C16 : 0 and C18 : 1ω7c 11-methyl. Polar lipids of strain MGL06T included unknown glycolipids, phosphatidylcholine, aminolipid, phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, an unknown polar lipid and aminophospholipid. Based on its phenotypic and genotypic data, strain MGL06T represents a novel species of the genus Rhizobium, for which the name Rhizobium marinum sp. nov. is proposed. The type strain is MGL06T ( = MCCC 1A00836T = JCM 30155T).

AgaM1, a β-agarase belonging to glycoside hydrolases family 16 (GH16), was cloned from the environmental DNA of mangrove sediments. The gene agaM1 is 2136 bp in length and encodes a protein of 712 amino acids. The properties of recombinant AgaM1 (rAgaM1) were studied using prokaryotic expression. The optimum temperature and pH were 50 °C and 7.0, respectively, and rAgaM1 exhibited a high adaptability to wide ranges of temperature and pH. A relatively high activity was retained at from 30 to 60 °C and from pH 6.0 to 9.0. Thermal stability was showed more than 70% relative activity after pre-incubation at 40 °C for 60 h. Outstanding pH stability were observed for rAgaM1 from pH 5.0 to 10.0 after pre-incubation for 60 h. Thin-layer chromatography revealed neoagarotetraose (NA4) and neoagarohexaose (NA6) were the end-products of rAgaM1-degraded agarose. Besides, rAgaM1 were found with a Km of 1.82 mg ml-1 and a Vm of 357.14 U mg-1 for agarose. The Km was smaller than those of most agarases reported previously. This discrepancy revealed the high affinity of rAgaM1 to agarose. Overall, the results indicated the potential of rAgaM1 in future industrial application.

A thermostable α-amylase (designated as Amy16) has been previously identified in Flammeovirga pacifica isolated from deep-sea sediments. The DNA sequence of Amy16 exhibited no significant similarity with those of any known protein, including the glycoside hydrolases. Amino acid sequence analysis revealed that Amy16 belonged to GH13 family and possessed a conserved DXEXD motif, which was essential for its hydrolysis activities. The recombinant Amy16 purified with Ni(+) affinity column after its heterologous expression in Escherichia coli cells was most active at 50 °C and retained more than 81% of its initial activity after incubation at 60 °C for 20 min. The optimal pH for Amy16 was determined to be 6.5, and a good tolerance to alkaline environment was observed. Low concentration of Mg(2+), Sr(2+), Na(+) and K(+) slightly increased the activity of Amy16. Results of thin layer chromatography experiments revealed that Amy16 was able to hydrolyse starch into maltose in a time-dependent manner, suggesting that Amy16 is a liquid-type endoenzyme with starch hydrolysis activities. Therefore, our study presented thermostable and alkali-stable Amy16, which may be suitable for use as an additive in detergents.

Nine novel Gram-stain-positive bacteria were investigated by a polyphasic taxonomic approach. Based on the analysis of 16S rRNA gene sequences, these strains belonged to the Bacillus cereus group, sharing over 97 % similarity with the known species of this group, and less than 95 % similarity with other species of the genus Bacillus . Multilocus sequence typing analysis showed that they formed nine robust and well-separated branches from the known species. The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the nine strains were, respectively, below the 70 and 96 % threshold values for species definition, and between each strain and the known type strains of this group were also below the two threshold values. On the basis of the phenotypic and phylogenetic data, along with low dDDH and ANI values among these strains, these bacteria are assigned to the following nine novel species of the B. cereus group: Bacillus paranthracis sp. nov., type strain Mn5T (=MCCC 1A00395T=KCTC 33714T=LMG 28873T); Bacillus pacificus sp. nov., type strain EB422T (=MCCC 1A06182T=KCTC 33858T); Bacillus tropicus sp. nov., type strain N24T (=MCCC 1A01406T=KCTC 33711T=LMG 28874T); Bacillus albus sp. nov., type strain N35-10-2T (=MCCC 1A02146T=KCTC 33710T=LMG 28875T); Bacillus mobilis sp. nov., type strain 0711P9-1T (=MCCC 1A05942T=KCTC 33717T=LMG 28877T); Bacillus luti sp. nov., type strain TD41T (=MCCC 1A00359T=KCTC 33716T=LMG 28872T); Bacillus proteolyticus sp. nov., type strain TD42T (=MCCC 1A00365T=KCTC 33715T=LMG 28870T); Bacillus nitratireducens sp. nov., type strain 4049T (=MCCC 1A00732T=KCTC 33713T=LMG 28871T); and Bacillus paramycoides sp. nov., type strain NH24A2T (=MCCC 1A04098T=KCTC 33709T=LMG 28876T).

Since the 1970s, several dsRNA viruses, including Radish yellow edge virus, Raphanus sativus virus 1, Raphanus sativus virus 2, and Raphanus sativus virus 3, have been identified and reported as infecting radish. In the present study, in conjunction with a survey of seed-borne viruses in cultivated Brassica and Raphanus using the dsRNA diagnostic method, we discovered 3 novel cryptoviruses that infect Brassica and Raphanus: Raphanus sativus partitivirus 1, which infects radish (Raphanus sativus); Sinapis alba cryptic virus 1, which infects Sinapis alba; and Brassica rapa cryptic virus 1 (BrCV1), which infects Brassica rapa. The genomic organization of these cryptoviruses was analyzed and characterized. BrCV1 might represent the first plant partitivirus found in Gammapartitivirus. Additionally, the evolutionary relationships among all of the partitiviruses reported in Raphanus and Brassica were analyzed.

Aim Although lymph nodes are preferred resources for isolation of lymphocytes for crossmatches, the nodes may not be available at the time of crossmatching. An unusual case led us to revisit 39 deceased donor’s crossmatch results to see if enough B cell events were acquired in flow crossmatching. Methods Cells were isolated using Density Gradient of Ficoll-Hypaque from pronase-treated PBL, and/or from lymph nodes. Flow crossmatches (FCXM) were performed on a BD FACSCalibur. In FCXM, anti-CD3 was used to mark T-cells, anti-CD19 was used to mark B-cells and goat anti-human IgG was used as a secondary antibody for detection of donor specific antibody. Results A 38 years old Caucasian female suffered a severe injury from a car accident was evaluated as the potential deceased donor for three kidney recipients. Three tubes of PBL were received for initial FCXM. The crossmatch failed due to lower B cell events ( Conclusions Our findings suggest the PBLs, as the resources of cells for crossmatch, most of the times (94%) could result in successful crossmatches.

A 12 y/o female from Saudi Arabia with chronic granulomatous disease was evaluated at Duke for HSCT. Patient and sibling typing were performed by SBT method (Protrans, Germany) which separates alleles by group-specific PCR before sequencing. The patient was identical to her sibling at HLA-A, B, DRB1, DRB3/4/5 and DQB1 loci. However, the analysis software could not assign alleles at HLA-C locus for both the patient and sibling due to the presence of double peaks in exon 3 electropherogram. For both the patient and the sibling, exon 2 and exon 4 sequences showed no polymorphism. Sequence with forward primer of exon 3 was good 1–224 nt in exon 3 but became double peaks after 224. Reverse primer sequence of exon 3, however, had double peaks 1–224 nt then good after 224. Analyzing the results by ignoring the double peak sequences showed a perfect match to C*06:02 in the database. This indicated there were C*06:02 and another variant of C*06:02 with a G deletion at 224 of exon 3. This corresponds to nt 568 in cDNA which changes codon 166 from GAG to.AG and is predicted to be a null allele due to shift of open reading frame. In order to investigate whether the new variant was inherited from the family, we also obtained the samples from the parents. It turned out the C*06 new null allele was carried by the father. The sequence was submitted to GenBank (accession number MH031344). The patient’s sibling was identified to be the 10/10 matched donor and HSCT was performed in 2018 successfully.

Psychrotolerant Pseudomonas stutzeri strain 7193 capable of producing an extracellular α-amylase was isolated from deep sea sediments of Prydz Bay, Antarctic. The 59678-Da protein (AmyP) was encoded by 1665-bp gene (amyP). The deduced amino acid sequence was identified with four regions, which are conserved in amylolytic enzymes and form a catalytic domain, and was predicted to be maltotetraose forming extracellular amylase by using the I-TASSER online server. Purification of AmyP amylases from both the recombinant of Escherichia coli Top 10 F′ and strain 7193 was conducted. Biochemical characterization revealed that the optimal amylase activity was observed at pH 9.0 and temperature 40°C. The enzymes were unstable at temperatures above 30°C, and only retain half of their highest activity after incubation at 60°C for 5 min. Thin-layer chromatography analysis of the products of the amylolytic reaction showed the presence of maltotetraose, maltotriose, maltose and glucose in the starch hydrolysate.

The modular polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) have been found to be involved in natural product synthesis in many microorganisms. Study on their diversities in natural environment may provide important ecological insights, in addition to opportunities for antibacterial drugs development. In this study, the PKS and NRPS gene diversities in two coast sediments near China Zhongshan Station were studied. The phylogenetic analysis of amino acid (AA) sequences indicated that the identified ketosynthase (KS) domains were clustered with those from diverse bacterial groups, including Proteobacteria, Firmicutes, Planctomycetes, Cyanobacteria, Actinobacteria, and some uncultured symbiotic bacteria. One new branch belonging to hybrid PKS/NRPS enzyme complexes and five independent clades were found on the phylogenetic tree. The obtained adenylation (A) domains were mainly clustered within the Cyanobacteria and Proteobacteria group. Most of the identified KS and A domains showed below 80 and 60% identities at the AA level to their closest matches in GenBank, respectively. The diversities of both KS and A domains in natural environmental sample were different from those in sewage-contaminated sample. These results revealed the great diversity and novelty of both PKS and NRPS genes in Antarctic sediment.

Seventeen psychrotrophic bacteria with cold-adaptive amylolytic, lipolytic or proteolytic activity were isolated from deep sea sediment of Prydz Bay, Antarctic. They were affiliated with γ-Proteobacteria (12 strains) and gram-positive bacteria (5 strains) as determined by 16S rDNA sequencing. The amylase-producing strains belonged to genus Pseudomonas, Rhodococcus, and Nocardiopsis. Two Pseudomonas strains, 7193 and 7197, which showed highest amylolytic activity were chosen for further study. The optimal temperatures for their growth and amylase-producing were between 15 and 20°C. Both of the purified amylases showed highest activity at 40°C and pH 9.0, and retained 50% activity at 5°C. The SDS-PAGE and zymogram activity staining showed that the molecular mass of strain 7193 and 7197 amylases were about 60 and 50 kDa respectively. The Pseudomonas sp. 7193 amylase hydrolyzed soluble starch into glucose, maltose, maltotriose, and maltotetraose, indicating that it had both activities of α-amylase and glucoamylase. The product hydrolyzed by Pseudomonas sp. 7197 amylase was meltotetraose.

The Pacific nodule province covered about 4.5 million km(2) in the east tropical Pacific with an abundance of polymetallic nodules at the seafloor. In view of the environmental protection and resource preservation, the survey of biodiversity was important during the reconnaissance and exploitation in this area. As one of the important component of the deep sea ecosystem, the microbial community in the Pacific nodule province was still largely unknown. The chitinolytic bacteria diversity in deep-sea sediment of a station within the Pacific nodule province was examined by molecular technology. A total of 18 chitinase genes were detected by a set of degenerate PCR primer specific for chiA gene fragment of family 18 chitinase. Most of them belonged to the Serratia-like chitinase. Eight genes had different amino acid sequences in the conserved motif, encompassing the catalytic site among the ChiA protein of family 18 glycosyl hydrolases, and clustered in an independent clade on the phylygenetic tree.

A novel sulfatase gene, ary423 (1,536 bp ORF), encoding a protein of 511 amino acids with a calculated molecular mass of 56 kDa, was identified from Flammeovirga pacifica, which was isolated from deep-sea sediments of west Pacific Ocean. Amino acid sequence analysis revealed that Ary423 possessed a conserved C-X-A-X-R motif, which was recognized as the sulfatase signature. Phylogenetic analysis suggested that Ary423 belonged to arylsulfatases. After heterologous expression in Escherichia coli cells, the recombinant Ary423 was purified with a Ni(+) affinity column, and was shown to be highly active at a broad range of temperatures from 30° to 70°C, with maximum activity at 40°C. Furthermore, recombinant Ary423 retained more than 70% and 40% of its maximum activity after 12 h of incubation at 50°C and 60°C, respectively, exhibiting good thermostability at high temperatures. The optimal pH for Ary423 was determined to be 8.0 and the activity of Ary423 could be slightly enhanced by Mg(2+). The recombinant enzyme could hydrolyze sulfate ester bonds in pnitrophenyl sulfate (NPS) and Asparagus crude polysaccharides with a specific activity of 64.8 U/mg and 25.4 U/mg, respectively. These favorable properties could make Ary423 attractive for application in the desulfating process of agar production.

A taxonomic study was carried out on strain YQH10T, which was isolated from mangrove sediment collected from Zhangzhou, China during the screening of acetaldehyde-degrading bacteria. Cells of strain YQH10T were Gram-stain-negative rods and pale brown-pigmented. Growth was observed at salinities from 0 to 11 % and at temperatures from 4 to 42 °C. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YQH10T is affiliated to the genus Shewanella, showing the highest similarity with Shewanella haliotis DW01T (95.7 %) and other species of the genus Shewanella (91.4–95.6 %). The principal fatty acids were iso-C15 : 0 and C17 : 1ω8c. The major respiratory quinone was Q-8. The polar lipids comprised phosphatidylethanolamine and phosphatidylglycerol. The genomic DNA had a G+C content of 48.3 mol%. Strain YQH10T can completely degrade 0.02 % (w/v) acetaldehyde on 2216E at 28 °C within 48 h. Based on these phenotypic and genotypic data, strain YQH10T represents a novel species of the genus Shewanella, for which the name Shewanella mangrovi sp. nov. is proposed. The type strain is YQH10T ( = MCCC 1A00830T = JCM 30121T).

Aim The linkage between DPB1 and DPA1 was extensively studied in 5,944 European American stem cell donors (Hollenbach et al., 2012). Thirty-three DPB1 alleles and associated DPA1 were reported. In 2014, Voorter et al. reported the special association of DPB1∗105:01 with DPA1∗03:01 in contrast to DPB1∗04:02-DPA1∗01:03 association although both DPB1∗105:01 and DPB1∗04:02 belong to the same G group. Here we retrospectively reviewed 1938 renal recipients with DP typing and report new DPB1/DPA1 associations frequently seen in our cohort. Methods HLA-DPB1 and DPA1 typing was performed by rSSO (One Lambda, Thermo-Fisher). Haplotypes were inferred by Clark’s method published in 1990. Results We identified 11 DPB1 alleles whose association with DPA1 has never been reported previously (Table 1). Five DPB1 alleles, DPB1∗124:01, 126:01, 131:01, 350:01 and 352:01, are not in the 2013 CWD list. Of note, DPB1∗131:01 was detected at high frequency (2.9%) in our cohort. The association of these five DPB1s with DPA1 was the same as the most frequent allele in the same G group, e.g. DPB1∗126:01 and DPB1∗350:01 are both associated with DPA1∗01:03 the same as DPB1∗04:01. Six DPB1 CWD alleles observed in our cohort were not reported in Hollenbach’s study. In 2015, Aaron et al. reported significant variation of DPB1 frequency in four major ethnic groups. In their study, DPB1∗29:01, 39:01, 40:01, and 85:01 were much more frequent in African American than other three ethnic groups. High percentage of African American in our renal recipients (44%) may explain the detection of these alleles in our cohort. Interestingly DPB1∗106:01 was associated with DPA1∗02:01, unlike PB1∗19:01 which is in the same G group however is associated with DPA1∗02:02. Conclusions We reported 11 new DPB1/DPA1 haplotypes frequently seen in DP low resolution typing. Five non-CWD DPB1 alleles were identified frequently. The enrichment of some alleles in our cohort could be attributed to high incidence of African American in our patients. Download : Download high-res image (190KB) Download : Download full-size image

Aim Our aim is to evaluate the performance of a computerized virtual crossmatch within the Duke University Medical Center HLA laboratory. Our HLA lab has used virtual crossmatch (vXM) to select deceased donors for heart and lung transplantations for more than ten years, which has allowed us to accept donors from outside of our Organ Procurement Organization territory. The virtual crossmatches were done by manually checking the recipient’s HLA antibody profiles within our lab database against the potential donor’s HLA typing to identify if the recipient has any donor-specific antibodies (DSA) in the most recent serum tested and in historical sera. To ensure no DSA was missed, the vXM was manually done by multiple transplant professionals at different stages for each sensitized recipient when a donor offer was available. Laboratory informatics vendor HLA Data Systems, developers of the mTilda Lab Management System, has developed a stand-alone software program which performs a computerized vXM (cVXM) under the product name VxMatch. The VxMatch program directly connects to any HLA lab database to query required data for analysis. Methods We performed 56 cVXMs by using the new program for patients who had previously received manual vXMs (mVXM) over the past few months. 35 of the 56 patients have calculated panel reactive antibodies (cPRA) = >80%. Results We found a 100% concordance for identification of DSA between cVXM and mVXM. It took less than 5 min to complete the cVXM while the conventional mVXM took 30 min on average. For each identified unacceptable antigen, the cVXM program provides detailed information, including antibody specificities, mean fluorescence intensity (MFI), serum date and a graph showing the MFI changes of DSA over time. The mTilda cVXM can operate in two different modes: a technologist’s level and supervisory level. The supervisory module allows us to review each individual bead carrying potential unacceptable HLA antigen at alpha chain, beta chain, or allele level. This cVXM can also be used to identify the sera (for example the serum with peak DSA) for final XM and the ability to track the change of DSA post-transplant. Conclusion Our evaluation suggests that this new computerized vXM program is a user-friendly, reliable and powerful tool for HLA testing and management.

One clone (ACPGA001) exhibiting penicillin G acylase (PGA) activity was screened from a metagenomic library by using a medium containing penicillin G. A novel PGA gene from the inserted fragment of ACPGA001 was obtained by sequencing. The amino acid sequence of ACPGA001 PGA exhibited

An extracellular protease was purified from a deep-sea psychrophilic bacterium strain DY-A which was identified as a Pseudomonas species. The optimal growth and protease-producing temperatures of the strain were all 10 degrees C, and the protease was secreted only at temperatures under 20 degrees C. The enzyme was most active at 40 degrees C and at pH 10.0. It was inhibited by phenylmethyl sulfonylfluoride and diisopropyl fluorophosphate, indicating that it is a serine protease. Chelators such as EDTA, EGTA, 1,10-phenanthroline and 2,2'-bipyridyl produced a decrease of activity. The enzyme was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and resistant to thiol-containing reducing agents such as dithiotreitol. The enzyme was active towards N-succinyl-Ala-Ala-Pro-Phe- p-nitroanilide and N-succinyl-Ala-Ala-Pro-Leu- p-nitroanilide. The native molecular mass of the enzyme determined by native PAGE and SDS-PAGE was 25 kDa.

[Departement_IRSTEA]Territoires [TR1_IRSTEA]SEDYVIN; International audience; The rhizosphere is a key zone for pollutant removal in treatment wetlands; therefore, studies on microbial activity may provide helpful information for a better understanding of purification processes. We studied microbial activity in a vertical-downflow constructed wetland system treating waste activated sludge with high organic matter concentrations, under Mediterranean climate. The aims of the work were to study the influence of (i) the presence of plants, (ii) the plant species (Phragmites australis Cav., Typha latifolia L., Iris pseudacorus L.), and (iii) the plant growth stage (plant senescence and plant fast growing stage) on total respiration rate and phosphatase activity in the substrate (intented here as the solid support on which the plants grow). The presence of plants had a positive influence on microbial activity, since substrate respiration and both acid and alkaline phosphatase activity were always higher in planted than in unplanted mesocosms. Among the three tested species, Phragmites was the one that most stimulated both substrate respiration rate and phosphatase activity, followed by Typha and Iris. These differences of microbial activity between mesocosms were corresponding to differences of removal efficiency. Substrate respiration and phosphatase activity were of similar magnitude at the two growth stages, while the stimulating effect of plants seemed to have been delayed and microbial activity showed higher fluctuations at plant fast growing stage than at plant senescence.

Strain WPAGA1T was isolated from marine sediment of the west Pacific Ocean. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the genus Flammeovirga . Strain WPAGA1T exhibited highest 16S rRNA gene sequence similarity with Flammeovirga yaeyamensis NBRC 100898T (98.1 %) and lower sequence similarity with Flammeovirga arenaria IFO 15982T (94.6 %) and other members of the genus Flammeovirga (T was distinct from F. yaeyamensis NBRC 100898T and F. arenaria NBRC 15982T (43±4 % and 32±2 % relatedness values, respectively). Strain WPAGA1T could be distinguished from all known members of the genus Flammeovirga by a number of phenotypic features. However, the dominant fatty acids of strain WPAGA1T (iso-C15 : 0, C16 : 0 and C20 : 4ω6,9,12,15c), the major polyamine (cadaverine) and the G+C content of the chromosomal DNA (32.9 mol%) were consistent with those of members of the genus Flammeovirga . Based on phenotypic and chemotaxonomic features and 16S rRNA gene sequences, strain WPAGA1T can be assigned to the genus Flammeovirga as a representative of a novel species, for which the name Flammeovirga pacifica sp. nov. is proposed; the type strain is WPAGA1T ( = CCTCC AB 2010364T = LMG 26175T = DSM 24597T = MCCC 1A06425T).

International audience; The aim of this study was to investigate the potential role of three macrophyte species (Iris pseudacorus, Typha latifolia and Phragmites australis) for detoxication of xenobiotics, and to study their variations with seasons or concentrations of sewage sludge from the food industry. For this purpose, some aspects of the green liver concept were explored through peroxidase measurements in three compartments in roots: intracellular, cell wall and extracellular. In addition, phenol concentrations were also measured in order to assess heavy metal detoxication potential. Enzyme activities and phenol concentrations were overall lower in winter according to the phenological stages and some sludge effects occurred. Results show that P. australis roots exuded and contained more peroxidase in all seasons: 17 U/g (1373 U/g protein), 0.8 U/g (613 U/g protein) and 4.8 U/g (1329 U/g protein) in intracellular compartments, cell wall and exudates, respectively. In contrast, the highest phenol concentration was found in I. pseudacorus roots: 143.3 mg eq. gallic acid/g. Hence, in constructed wetlands, P. australis is suitable for organic waste water treatment, while I. pseudacorus should be used in the case of waters highly charged with heavy metals.

A metagenomic cosmid library was constructed, in which the insert DNA was derived from the coastal sediment near Antarctic China Zhongshan Station. One clone (ACPRO001) expressing protease activity was isolated from the library using milk agar plates. Sequencing of the clone revealed a novel protease gene. The amino acid sequence comparison and phylogenetic analysis indicated that it could be classified as a subtilisin-like serine protease, though the highly conserved residue Asp was replaced by Ala. The ACPRO001 protease gene was expressed in pET-His and purified for characterization. The optimal temperature and pH for the activity of the ACPRO001 protease were 60°C and pH 9.0, respectively. The enzyme retained about 73% of residual activity after 2 h incubation at 50°C in the presence of Ca(2+). The presence of Ca(2+) increased the thermostability of ACPRO001 protease obviously. The enzymatic activity was inhibited by 1 mM phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease.

A vertical-flow wetland system was tested for treatment of liquid sludge with high organic concentrations using an organic substrate (peat/crushed pine bark, 1/1) as growing medium. Mesocosms (1 m 3 ) were planted with either Phragmites australis Cav . , Typha latifolia L., or Iris pseudacorus L. The aim of the work was to determine the feasibility of using an organic substrate in treatment wetlands, through the study of its temporal patterns and of its impact on the water output quality. Results confirmed that the organic substrate can be used in such wetlands treating highly organic sludge, without clogging phenomena for the experimental period. The organic substrate released soluble organic matter but few mineral elements. Over the experimental period, substrate TOC concentration did not change while N concentration increased. Plants showed positive impact on substrate temporal patterns and also on the outflow water quality. Overall, Phragmites seemed to be more beneficial than Typha and Iris .

Aim An MDS patient has become a candidate for allogeneic hematopoietic stem cell transplantation (HSCT). Samples from patient and five donors were received for HLA typing. HLA-C results were ambiguous in the presence of HLA-C*02:06. We investigated the reason for the variation observed. Methods HLA-Class I high resolution typing was performed by SBT using the group specific strategy (Protrans®S3 HLA-C), allowing most of the alleles to be amplified individually and sequenced as hemizygous. The S3 HLA-C kit sequences the complete exons 2 and 3 of all HLA-C alleles and exon 4 of some. The kit has a locus specific reaction including “all alleles” that we always sequence, since it provides useful data to resolve several relevant ambiguities within exon 4; with the exception of the alleles not having exon 4 data available. This case was unique because some family members were assigned as HLA-C*02:06, 07: AAGAJ (07:01/07:06/07:18/07:343), and others, including the patient, were HLA-C*02:06, 07: ADCTY (07:01/07:06/07:18/07:166/07:343). Upon data review, it was noticed that the multiple NMDP codes referring to the same allele were confusing the clinicians needing to make decisions based on the HLA typing results. We re-sequenced one SBT HLA-C reaction from patient and potential donors to obtain hemizygous data of HLA-C*02:06 exon 4. Results Lack of exon 4 data of HLA-C*02:06 has caused nucleotide position 623 to be ambiguous since only the HLA-C*07:01+ alleles had exon 4 data available. Position 623 was heterozygous on “all alleles” sequencing data of exon 4. We submitted the re-sequenced exon 4 data to IMGT/HLA for review. The submission, HWS 10025170, was confirmed by the WHO Nomenclature Committee for Factors of the HLA System on 02/28/2015. Upon having HLA-C*02:06 exon 4 sequencing data confirmed, we edited the HLA typing reports, the clinicians acknowledged our effort to reduce ambiguities, facilitating their donor selection process; and we made a contribution to the HLA community making exon 4 data of HLA-C*02:06 available at the IMGT/HLA. Conclusions Ambiguities, such as this one, caused by incomplete data of HLA alleles may be prevented when all HLA alleles have complete sequences available, until then, we will keep generating the best resolution results possible using wisely the methods and tools we have available.

The celX gene encoding an extracellular cold-active cellulase was isolated from a psychrotrophic bacterium, which was isolated from deep-sea sediment and identified as a Pseudoalteromonas species. It encoded a protein consisting of 492 amino acids with a calculated molecular mass of 52.7 kDa. The CelX consisted of an N-terminal catalytic domain belonging to glycoside hydrolase family 5 and a C-terminal cellulose-binding domain belonging to carbohydrate-binding module family 5. The long linker sequence connecting both domains was composed of 105 residues. The optimal temperature for cellulase activity of CelX was 40 degrees C. The enzyme was most active at pH 6-7 and showed better resistance to alkaline condition. The zymogram activity analysis indicated that the CelX consisted of single enzyme component. The cellobiose was main hydrolysate of CelX.

The genomes of the three temperate bacteriophages contained in the chromosome of Staphylococcus aureus 8325 have been extracted from the sequence database and analyzed. phi 11, phi 12 and phi 13 are members of the same lytic group but different serogroups and consequently co-habitate the same host cell. Their genomes are approximately 42 kb to 45 kb and contain about 90 ORFs of at least 50 codons. Of these, about 50 have similarities to known genes or to genes of other staphylococcal phages. Each of the phages clusters within a homology group that share large regions of sequence identity while intergroup homology is comparatively low. The arrangement of genes on the chromosomes of the three phages is similar and consistent with current modular theory of phage gene organization. The replicated genomes appear to be packaged by different mechanisms. Phage phi 11 and phi 12 have been found to contain sequences consistent with pac-site phages while phi 13 has sequences consistent with cos-site phages. The attBsite for phi 11 is located in an intergenic region of the S. aureus chromosome while phi 12 and phi 13 integrate into specific genes. The phi 12 att-site is within an unknown gene, but the phi 13 att-site is within the beta-toxin gene. In contrast to the other two phages, phi 13 also introduces the staphylokinase gene (sak) and a second gene related to expression of fib.

Aim Ever increasing novel HLA allele discoveries raise in questions about the clinical relevance of these new alleles and their implications of further working on the donor searching process without compromising the timing of patient’s treatment. Our purpose is to bring attention to the novel allele discovery and provide helpful information to clinicians to make timely and right decisions on hematopoietic stem cell transplantation. Method HLA class I and class II high resolution (HR) typing for recipients and donors were performed by Sequence Based Typing (SBT) methodology. When a novel allele was detected, we examined its clinical significance in three locations: (1) peptide binding grooves; (2) T cell Receptor contacting area; (3) HLA Epitope-specific positions. Results Seven novel alleles and two confirmatory alleles (highlighted) have been identified in our routine HLA HR typing by SBT (see table). The position and the substitution of each novel allele was identified and listed on the table. Pocket Residue, TCR accessibility to HLA antigen and Epitope specificity were also described in the table. Conclusions/Discussions Taken together, our analysis suggest thats: (1) a mutation of a silent point mutation without change in amino acid would be considered as clinically irrelevant (C ∗ 08:02:08); (2) a mutation located outside of α1 and α2 domains of HLA class I or outside of β 1 of HLA class II and not HLA Epitope-specific should be considered as clinical insignificant (C ∗ 07:308). (3) a mutation located inside of α1 or α2 domains of HLA class I or inside of β 1 of HLA class II could be considered having less clinical relevance if it does not falls into one of the following three criteria: (a) accommodating any peptide-binding pockets, (b) being TCR accessible or (c) being HLA Epitope specific ( ∗ 44:173, C ∗ 03:183, C ∗ 04:158 and C ∗ 16:39:02); however, it would be considered having potential clinical relevance if it falls into one of those three criteria (DQB1 ∗ 03:45, DQB1 ∗ 03:48 and DQB1 ∗ 06:49).

Aim Genomic alterations and somatic mutations have been widely identified in hematologic malignancies. Recent studies have described mutations in different HLA genes of these patients. These mutations are identified when high resolution typing is needed for patients and potential donors. An African American patient treated for myeloma, received an autologous stem cell transplant in 2010. During 2011 he was diagnosed with a secondary plasma cell leukemia, becoming a candidate for an allogeneic stem cell transplant. Methods HR HLA typing was performed by Sequence Based Typing (SBT) methodology and a single base mismatch on one of his HLA-DQB1 allele was identified in exon 2, codon 13, leading to an amino acid substitution (Glycine to Valine). Three siblings, considered potential donors, had HLA typing performed and the same HLA-DQB1 ∗ 06:XX was identified in two of them. To investigate the HLA-DQB1 novel allele segregation and its origin within the family, their mother was typed. Results One of the HLA-DQB1 alleles identified did not match with DQB1 alleles from HLA database 3.6.0, used for the SBT data analysis. Their mother HLA typing identified HLA-DQB1∗06:02:01 and DQB1 ∗ 06:XX. The alleles DQB1 ∗ 06:XX and DQB1 ∗ 06:02:01 differ by 1 nucleotide mismatch in exon 2, which caused us to question what genetic alterations that may have occurred in her DQB1 genes. Conclusions Our findings demonstrate that the hematological malignancy is not always the cause for HLA genes alterations, which reinforces the value of family studies to explain gene segregation. Beyond the implications of obtaining an accurate HLA typing and donor selection, these genetic alterations on HLA genes may allow some mechanisms of immune surveillance to fail when associated with other complications caused by hematological malignancies affecting bone marrow transplant candidates. The allele in question was also identified by another HLA laboratory, and is included in the most recent IMGT HLA alleles update, it was named HLA-DQB1 ∗ 06:49.

Aim Increased novel allele discovery has a huge contribution to HLA diversity, which could, however, complicate donor search. Currently, no clear guidelines of unrelated hematopoietic stem cell (HSC) donor search are available when novel alleles are involved. Novel alleles raise questions of the clinical significance of the substitution and its effect on unrelated donor search. In this study we present an approach to evaluate a novel allele for donor search. Methods HLA high resolution typing was performed by SBT. We examined the clinical significance of a novel DQB1 ∗ 06:XX allele for the difference between it and the common DQB1 ∗ 06:02, the nature of the changed amino acid, and the location of the changed amino acid on three dimensional structure (α helix or β sheet). Results DQB1 high resolution typing revealed a novel allele identical to DQB1 ∗ 06:02 with except for a mismatch at position 25 within exon 2. The Nucleotide changed from ‘G’ to ‘T’ which caused amino acid changing from Glycine to Valine at codon 13(β13) in the amino-terminal region. The mutation is located on the floor of groove accommodating anchor position pocket 4 (P4). The side group of Glycine in DQB1 ∗ 06:02 changed from –H to –C3H7 for Valine in novel DQB1 ∗ 06:XX. The replaced side group –C3H7 occupies more space and possesses more hydrogen bonds. Although the shape and affinity of the peptide binding pocket might be altered due to changing of the side chain, both Valine and Glycine still belong to hydrophobic amino acid group. High resolution HLA typing of his mother and three siblings were also performed. The novel allele was inherited from the mother. Conclusions Our findings suggest that the amino acid change of the novel allele DQB1 ∗ 06:XX might not be clinically significant based on its location and characteristics when compared to the common DQB1 ∗ 06:02. The method we used to evaluate a novel allele could be a practical approach for the unrelated donor search for a potential HSC recipient with a novel allele of any loci.

Aim Single antigen bead (SAB) assay allows in-depth characterization of HLA antibody specificities. In addition to detection of antibodies against natural HLA antigens, the SAB may also detect antibodies against denatured HLA antigens. Recently, we observed antibody reactivity to all DR antigens (pan-DR) in serum samples of two transplant candidates using the Luminex SAB assay. This study was to characterize the pan-DR antibodies detected by Luminex SAB on two serum samples. Methods Serum samples received from two transplant candidates were analyzed in the study. Initial antibody screening and specificity tests were performed using Flow PRA and Luminex SAB. Additional tests using Flow SAB, Flow phenotype bead assay and Flow T and B cell crossmatch were performed to confirm the presence of HLA antibodies. Results Flow PRA was clearly negative for both samples, while Luminex SAB surprisingly revealed antibody reactivity to all DR antigens. No antibodies to DQ and DP were detected. However, no HLA antibodies were detected by Flow phenotype bead assay. In the Flow SAB assay, patient #1 showed reactivity with majority of the DR antigen beads, while patient #2 showed no any binding reactivity. The crossmatch with a donor cell carrying DR4, DR15, DR51, and DR53 was completely negative for both serum samples. No specific antibody removals were observed on serum samples adsorbed using the donor cells in a Luminex SAB assay. Conclusions Flow PRA and Flow phenotype beads are coated with natural HLA antigens extracted from cell lines, while SABs are coated with recombinant HLA antigens. The antibody binding detected by SAB, but not by FlowPRA and Flow phenotype, may indicate the presence of antibodies against denatured antigens. Our results suggest that the pan-DR antibodies detected in these patients were possibly antibodies against denatured HLA antigens, more likely to a shared epitope uncovered on denatured DR antigens. Antibodies to denatured antigens would not result in crossmatch reactivity.