[Establishment of a Patient-Derived T-Cell Acute Lymphoblastic Leukemia Xenograft Model in Novel Immunodeficient NCG Mice].

OBJECTIVE: The leukemia cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into NCG mice to establish a stable human T-ALL leukemia animal model. METHODS: Leukemia cells from bone marrow of newly diagnosed T-ALL patients were isolated, and the leukemia cells were inoculated into NCG mice via tail vein. The proportion of hCD45 positive cells in peripheral blood of the mice was detected regularly by flow cytometry, and the infiltration of leukemia cells in bone marrow, liver, spleen and other organs of the mice was detected by pathology and immunohistochemistry. After the first generation mice model was successfully established, the spleen cells from the first generation mice were inoculated into the second generation mice, and after the second generation mice model was successfully established, the spleen cells from the second generation mice were further inoculated into the third generation mice, and the growth of leukemia cells in peripheral blood of the mice in each group was monitored by regular flow cytometry to evaluate the stability of this T-ALL leukemia animal model. RESULTS: On the 10th day after inoculation, hCD45+ leukemia cells could be successfully detected in the peripheral blood of the first generation mice, and the proportion of these cells was gradually increased. On average, the mice appeared listless 6 or 7 weeks after inoculation, and a large number of T lymphocyte leukemia cells were found in the peripheral blood and bone marrow smear of the mice. The spleen of the mice was obviously enlarged, and immunohistochemical examination showed that hCD3+ leukemia cells infiltrated into bone marrow, liver and spleen extensively. The second and third generation mice could stably develop leukemia, and the average survival time was 4-5 weeks. CONCLUSION: Inoculating leukemia cells from bone marrow of patients with T-ALL into NCG mice via tail vein can successfully construct a patient-derived tumor xenografts (PDTX) model.

Parallel subgenome structure and divergent expression evolution of allo-tetraploid common carp and goldfish.

How two subgenomes in allo-tetraploids adapt to coexistence and coordinate through structure and expression evolution requires extensive studies. In the present study, we report an improved genome assembly of allo-tetraploid common carp, an updated genome annotation of allo-tetraploid goldfish and the chromosome-scale assemblies of a progenitor-like diploid Puntius tetrazona and an outgroup diploid Paracanthobrama guichenoti. Parallel subgenome structure evolution in the allo-tetraploids was featured with equivalent chromosome components, higher protein identities, similar transposon divergence and contents, homoeologous exchanges, better synteny level, strong sequence compensation and symmetric purifying selection. Furthermore, we observed subgenome expression divergence processes in the allo-tetraploids, including inter-/intrasubgenome trans-splicing events, expression dominance, decreased expression levels, dosage compensation, stronger expression correlation, dynamic functionalization and balancing of differential expression. The potential disorders introduced by different progenitors in the allo-tetraploids were hypothesized to be alleviated by increasing structural homogeneity and performing versatile expression processes. Resequencing three common carp strains revealed two major ecotypes and uncovered candidate genes relevant to growth and survival rate.

Optimum dredging time for inhibition and prevention of algae-induced black blooms in Lake Taihu, China.

Dredging, which is the removal of polluted surface sediments from a water body, is an effective means of preventing the formation of algae-induced black blooms. However, an inappropriate dredging time may contribute to rather than inhibit the formation of black blooms. To determine the optimum dredging time, four treatments were simulated with sediment samples collected from Lake Taihu: dredging in January 2014 (DW), April 2014 (DA), July 2014 (DS), and no dredging (UD). Results showed that typical characteristics associated with black blooms, such as high levels of nutrients (NH4 (+)-N and PO4 (3-)-P), Fe(2+), ∑S(2-) ([HS(-)] + [S(2-)]), and volatile organic sulfur compounds (VOSCs), including dimethyl sulfide (DMS), dimethyl disulfide (DMDS), and dimethyl trisulfide (DMTS), were more effectively suppressed in the water column by DW treatment than by UD treatment and the other two dredging treatments. The highest concentrations of NH4 (+)-N and PO4 (3-)-P in the UD water column were 4.09 and 4.03 times, respectively, those in the DW water column. DMS levels in the UD and DS water columns were significantly higher (p < 0.05) than those in the DW water column, but DMDS and DMTS levels were not significantly different between the treatments. After several months of dredging, surface sediments of the DW and DA treatments were well oxidized, and concentrations of Fe(2+) and ∑S(2-) were lower than those in UD and DS treatments because of material circulation between sediments and the water column. Water content, which is important for the transport of matter to the overlying water, was lower in the dredged sediments than in the undredged sediments. These factors can suppress the release of Fe(2+) and ∑S(2-) into the water column, thereby inhibiting the formation of black blooms. Black coloration occurred in the UD water column on the seventh day, 2 days later, and earlier, respectively, than the DW and DS water columns and almost on the same day as in the DA water column. This phenomenon is mainly attributed to the oxidation of the new sediment surface in the DW and DA water columns, suppressing the release of sulfur into the water column, because of a long incubation period. Thus, dredging in the winter can prevent the formation of black blooms, while dredging in summer may contribute to them.

[Environmental Effect of Substrate Amelioration on Lake: Effects on Phragmites communis Growth and Photosynthetic Fluorescence Characteristics].

Growth of rooted aquatic macrophytes was affected by the nature and composition of lake bottom sediments. Obviously, it has been recognized as an important ecological restoration measure by improving lake substrate and then reestablishing and restoring aquatic macrophytes in order to get rid of the environmental problem of lake. This study simulated five covering thickness to give an insight into the influence of substrate amelioration on Phragmites communis growth and photosynthetic fluorescence characteristics. The results showed that the total biomass, plant height, leaf length and leaf width of Phragmites communis under capping 5 cm were much more significant than those of capping 18 cm (P < 0.01), at the 120 d, the underground: shoot biomass ratio and fine root: underground biomass ratio were also much higher than those of other treatments (P < 0.05), which indicated that capping 18 cm treatment would significantly inhibit the growth of Phragmites communis , but the growth of control group Phragmites communis was slightly constrained by eutrophicated sediment. In addition, as the capping thickness growing, the underground: shoot biomass ratio of the plant would be reduced dramatically, in order to acquire much more nutrients from sediment for plant growing, the underground biomass of Phragmites communis would be preferentially developed, especially, the biomass of fine root. However, Photosystem II (PS II) photochemical efficiency (Fv/Fm), quantum yield (Yield), photochemical quenching (qP), non-photochemical quenching (qN) of Phragmites communis under different treatments had no significant differences (P > 0.05), furthermore, with much greater capping thickness, the photosynthesis structure of PS II would be much easier destroyed, and PS II would be protected by increasing heat dissipating and reducing leaf photosynthetic area and leaf light-captured pigment contents. In terms of the influence of sediment amelioration by soil exchange on the growth and photosynthetic fluorescence characteristics of Phragmites communis, plant growth could be effectively promoted under capping 2 cm and capping 5 cm by increasing the Eh value and nutrient content, whereas plant under capping 18 cm would be much easier adaptive to low-light stress in winter season, of which capping 2 cm treatment was conducive to enhance the initial slope of RLCs (α), maximum electron transport rate (ETRmax) and minimum saturating irradiance (Ek). With regard to the harness of environmental problem of lake, the eutrophication status of lake will be mitigated by using multi-ecological measures to control the internal nutrients content once the external loading was first effectively controlled.

[Simulation research on the release of internal nutrients affected by different dredging methods in lake].

A simulated experiment was carried out to study release features of internal source under different sediment dredging methods and the difference between two lake areas in Lake Taihu was also studied. The contaminated sediments were sampled from two sites in Meiliang Bay which were the Inner Bay (A) and the Outer Bay(B). Release rates of phosphorus after ideal dredging and suction dredging are about 20% and 72% of the control and the phosphorus release rate in Inner Bay(A) is about 80% of Outer Bay(B). Release rates of ammonia after ideal dredging and suction dredging are about 40% and 83% of the scallop dredging, but dredging process may even promote the release of ammonia in a short time, the ammonia release rate in Inner Bay(A) is about 150% higher than that in Outer Bay(B). Under the microcosm experiment condition, the ideal dredging method and the suction dredging method may have a better control of internal source in contrast with the scallop dredging. Altogether, sediment dredging may be a useful approach to decrease the release of internal source in the selected sites when the external nutrients are effectively controlled. Consider all kinds of dredging projects, the suction dredging should be the ideal option for sediment dredging in Lake Taihu.

[Sequences analysis of jlFABP2 and the correlation between polymorphisms and body weight gain in Cyprinus carpio var. jian].

Two replicate intestine fatty acid binding protein genes (jlFABP2a and jlFABP2b) were cloned from Cyprinus carpio var. jian using PCR. Both ORFs were 399 bp in length sharing 92.2% similarity with each other, and 88.0% and 90.5% with their counterpart in zebrafish, respectively. The gene structure of jlFABP2s was same as other FABPs, which contained four exons and three introns. Sequences and lengths of introns between 2a and 2b. were obviously different Phylogenetic tree displayed that two jlFABP2s corresponded to one zebrafish FABP2 which matches the fact that the chromosome number of common carp was twice of zebrafish. Real time-PCR showed that jlFABP2 genes mainly expressed in intestine and the expression level was very significantly higher than other tissues such as brain, liver, muscle, kidney, and gonad (P<0.01). The expression level of jlFABP2a was significantly (male, P<0.05) or very significantly (females, P<0.01) higher than 2b in intestine; and 2b was expressed slightly higher than 2a in other tissues. It seemed that 2a expressed specifically in intestine, while 2b expressed ubiquitously. Twelve and four SNP loci were found at jlFABP2a and 2b introns through comparison sequences from 8 individuals, respectively. Genotypes of I1-A15G, I1-A99G, I2-C487T, and I3-A27T on jlFABP2a were detected using PCR-RFLP in selection population of C. carpio var. jian. The SNP genotypes and individual weight gain correlation indicated that four SNPs were significantly (P<0.05 or P<0.01) associated with adult weight gain. Diplotype analysis displayed that individuals with genotype AGGGCCXX or AGGGXXAT grew faster than other individuals by 15%. The individuals with these two genotypes only occupied 9% in total selection populations, indicating the presence of large selection space. The 4 SNPs detected in this experiment can be used in C. carpio var. Jian growth selection breeding plan.

Two Δ6-desaturase-like genes in common carp (Cyprinus carpio var. Jian): structure characterization, mRNA expression, temperature and nutritional regulation.

Δ6-Desaturase is the rate-limiting enzyme involved in highly unsaturated fatty acid (HUFA) biosynthesis. There is very little information on the evolution and functional characterization of Δ6Fad-a and Δ6Fad-b in common carp (Cyprinus carpio var. Jian). In the present study, the genomic sequences and structures of two putative Δ6-desaturase-like genes in common carp genome were obtained. We investigated the mRNA expression patterns of Δ6Fad-a and Δ6Fad-b in tissue, hatching carp embryos, larvae by temperature shock and juveniles under nutritional regulation. Our results showed that the two Δ6Fad genes had identical coding exon structures, being comprised of 12 coding exons, and with introns of distinct size and sequence composition. They were not allelic variants of a single gene. Both Δ6Fad genes were highly expressed in liver, intestine (pyloric caeca) and brain. The Δ6Fad-a and Δ6Fad-b mRNAs showed an increase in expression from newly hatched to 25 days after hatching. The expression levels of Δ6Fad-a were obviously regulated by temperature, whereas Δ6Fad-b was not affected by temperature. The regulation of Δ6Fad-a and Δ6Fad-b in response to dietary fatty acid composition was determined in liver, brain and intestine (pyloric caeca) of common carp fed with diets: diet1with fish oil (FO) rich in n-3 HUFA, diet2 with corn oil (CO, 18:2n-6) and diet3 with linseed oil (LO, 18:3n-3). The differential expression of Δ6Fad-a and Δ6Fad-b genes in liver, brain and intestine in common carps was fed with different oil sources, respectively. Further work is in progress to determine the mechanism of differential expression of the Δ6Fad-a and Δ6Fad-b genes in different tissues and the roles of transcription factors in regulating HUFA synthesis.

[Influence of dredging on sediment resuspension and phosphorus transfer in lake: a simulation study].

A simulated experiment was conducted to investigate the impacts of sediment dredging on sediment resuspension and phosphorus transfer in the summer and winter seasons under the common wind-wave disturbance, and the contaminated sediment used in this study was from Meiliang Bay, Taihu lake. The result showed that 20 cm dredging could effectively inhibit the sediment resuspension in study area, dredging in winter has a better effect than that in summer, and the higher values of the total suspended solid (TSS) in undredged and dredged water column during the process of wind wave disturbance were 7.0 and 2.2, 24.3 and 6.4 times higher than the initial value in summer and winter simulation respectively. The paired-samples t-test result demonstrated that total phosphorus (TP) and phosphate (PO4(3-)-P) loading positively correlated to TSS content in dredged (P<0.01) and undredged water column (P<0.05), which proved that internal phosphorus fulminating release induced by wind-wave disturbance would significantly increase the TP and PO4(3-)-P loading in the water column. The effect of dredging conducted in summer on the TP and PO4(3)-P loading in the water column was negative, but not for winter dredging (P<0.01). The pore water dissolved reactive phosphorus (DRP) profile at water-sediment interface in summer simulation was also investigated by diffusive gradients in thin films (DGT) technique. Diffusion layer of the DRP profile in undredged sediment was wider than that in dredged sediment. However, the DRP diffusion potential in dredged sediment was greater than that in undredged sediment, showing that dredging can effectively reduce the risk of the DRP potential release in dredged pore water, but also would induce the DRP fulminating release in the short time under hydrodynamic action. Generally, dredging was usually deployed during the summer and the autumn. Considering Taihu Lake is a large, shallow, eutrophic lake and the contaminant distribution is spatially heterogeneous, it is vital to determine the optimal time, depth and scope of dredging.

[Microsatellite markers for parentage identification in Jian Carp (Cyprinus carpio var. Jian)].

Using 16 microsatellite loci we estimated the parentage of 647 progeny in 10 Jian Carp full-sib families. Cervus 3.0 analysis showed that mean PIC value of 16 microsatellites, mean number of allele, and mean expected heterozygosity were 0.7025, 6.63, and 0.7405, respectively. The combined probability of exclusion was 0.99922456 when both parents were unknown and the combined probability of exclusion was 0.99999557 when only one of the parental genotype was known, with the confidence level of 95%. Further simulations based on allele frequencies suggested that to achieve the requirements of paternity test usually took 8 to 12 microsatellite loci when both parents were unknown and 5 to 8 microsatellite loci when one parent was known. Out of 647 progenies, 94.6% were assigned to their parental pairs without the information of both parents in parentage analysis, which were lower than the theoretical assignment rates predicted by the Cervus simulations. This could be explained by the relationship between the candidate parents or existence of null and by typing errors. The identification of 9 families was useful for linkage analysis of Jian Carp and QTL location, also for marker assisted selection for economical traits.

Identification of housekeeping genes suitable for gene expression analysis in Jian carp (Cyprinus carpio var. jian).

Jian carp (Cyprinus carpio var. jian) is an important economic fish species cultured in China. In this report, we performed a systematic analysis to identify an appropriate housekeeping (HK) gene for the study of gene expression in Jian carp. For this purpose, partial DNA sequences of four potential candidate genes (elongation factor 1 alpha (EF-1α), glyceraldehyde-3-phosphate (GAPDH), beta-actin (ACTB), and 18S ribosomal RNA (18S rRNA) were isolated, and their expression levels were studied using RNA extracted from nine tissues (forebrain, hypothalamus, liver, fore-intestine, hind-intestine, ovary, muscle, heart, kidney) in juvenile and adult Jian carp. Gene expression levels were quantified by quantitative real time RT-PCR (qRT-PCR), and expression stability was evaluated by comparing the coefficients of variation (CV) of the Ct values. The results showed that EF-1α was the most suitable HK gene in all tissues of juvenile and adult Jian carp. However, at distinct juvenile and adult developmental stages, there was not a single optimal gene for normalization of expression levels in all tissues. EF-1α was the most stable gene only in forebrain, hypothalamus, liver, heart, and kidney. These results provide data that can be expected to aid gene expression analysis in Jian carp research, but underline the importance of identifying the optimal HK gene for each new experimental paradigm.

Influence of dietary fatty acids on muscle fatty acid composition and expression levels of Δ6 desaturase-like and Elovl5-like elongase in common carp (Cyprinus carpio var. Jian).

The effects of dietary fatty acids on muscle fatty acid composition and liver expression levels of Δ6 desaturase-like and Elovl5-like elongase were investigated in common carp (Cyprinus carpio var. Jian). Two Δ6 desaturase-like cDNAs (Fad6-a and Fad6-b) and two Elovl5-like elongase (Elovl5-a and Elovl5-b) cDNAs were cloned. Juvenile carp were fed three semi-purified diets (D1-3) for 6 weeks with different lipid sources: D1, fish oil with high highly unsaturated fatty acids (HUFAs); D2, corn oil with high linoleic acid (LA), but no HUFAs; and D3, linseed oil with high α-linolenic acid (LNA), but no HUFAs. Comparing muscle fatty acids among fish fed D1-3, the content of LA and arachidonic acid (AA) in common carp fed D2 and the content of LNA, EPA and DHA in common carp fed D3 were higher than initial levels (P<0.05), respectively. The liver transcript levels of Fad6-a and Elovl5-a in fish fed D2 and D3 were higher than initial levels (P<0.05), but Fad6-b and Elovl5-b levels were seldom affected by the diets. The dietary fatty acids affect the muscle fatty acid composition and the liver Fad6-a and Elovl5-a gene expression levels in common carp, and further studies should be undertaken.

Cloning, structure, and expression pattern of the P-450 aromatase gene in rice field eel (Monopterus albus).

We report the cloning, tissue expression, and structural analysis of the aromatase gene in the rice field eel (Monopterus albus). The ovary-derived cDNA (1,802 bp) has a 49 bp 5'-untranslated region (UTR), a 202 bp 3'-UTR, and a 1,551 bp open-reading frame, which encodes a protein of 517 amino acid residues with a predicted molecular weight of 58.2 kDa. The amino acid sequence alignment suggests that the rice field eel ovarian P-450 aromatase shares 63-80% identity with that of other fish species, reduced to 59-60% with brain-derived aromatases of other fishes and to 50% with human placenta aromatases. Between the 5' and 3' untranslated terminal regions, the rice field eel CYP19 gene contained seven introns at the same sites as in medaka and human but lacked an intron between the I-helix and the aromatase-specific conserved region. All introns conformed to the GT/AG rule. Sequence analysis of the 1,065 bp upstream of the translation start site revealed that the transcription initiation site was 51 bp upstream from the translation start site. This region had one estrogen receptor recognition half site (nt -62), five copies of an SRY/iSRY binding motif, a C/EBP (CCAAT enhancer binding protein) binding site (nt -751), chicken ovalbumin upstream promoter-transcription factor (nt -986) and GATA-2 (nt -186, -249) recognition sequences, but no binding sequence for steroidogenic factor-1 and the cAMP response element binding protein activating transcription factor family. In females, levels of relative expression were, in descending order, hypothalamus, pituitary, forebrain, ovary, and liver. In males, P450arom was detected only in the pituitary and the liver, with half the expression found in females. In fry, the P450arom expression level increased during development and was significantly higher in the brain than in the gonad.