Transcriptome and 16S rRNA analysis revealed the response of largemouth bass (Micropterus salmoides) to Rhabdovirus infection.

To better understand the response of largemouth bass (Micropterus salmoides) to Micropterus salmoides rhabdovirus (MSRV) infection, we investigated the intestinal bacterial flora and transcriptome profile of fish at 72 hours post-infection (hpi). Total of 1574 differentially expressed genes (DEGs) were identified in largemouth bass spleen following MSRV infection, including 573 upregulated and 1001 downregulated genes. KEGG and GO enrichment analysis revealed that upregulated genes were enriched in certain antiviral related signaling pathway, including NOD-like receptor (NLR), RIG-I like receptors (RLR) and regulation of the interferon (IFN)-γ-mediated signaling pathway, whereas some immune-related DEGs enriched in focal adhesion (FA) and ECM-receptor interaction(ECM-RI) were downregulated, as well as genes associated with metabolic processes, such as peroxisome proliferator-activated receptors (PPAR), adipocytokine signaling pathway, Glycerolipid and Retinol metabolism. Furthermore, the principal component analysis (PCA) and phylogenetic analysis revealed that MSRV infection significantly affected the microbiota of largemouth bass intestine; the LEfSe analysis showed that relative abundances of Streptococcus were significantly increased, while the content of Akkermansia, Enterococcus and Lactobacillus were remarkably decreased in the fish intestine following MSRV infection. Additionally, a high correlation was determined between the expressions of interferon-related upregulated genes and the relative abundance of Streptococcus by redundancy analysis (RDA). These results collectively illustrated that intestinal microbiota composition might be associated with the immune-related gene expression in largemouth bass in response to MSRV infection.

Leukemic progenitor cells enable immunosuppression and post-chemotherapy relapse via IL-36-inflammatory monocyte axis.

Chemotherapy can effectively reduce the leukemic burden and restore immune cell production in most acute myeloid leukemia (AML) cases. Nevertheless, endogenous immunosurveillance usually fails to recover after chemotherapy, permitting relapse. The underlying mechanisms of this therapeutic failure have remained poorly understood. Here, we show that abnormal IL-36 production activated by NF-κB is an essential feature of mouse and human leukemic progenitor cells (LPs). Mechanistically, IL-36 directly activates inflammatory monocytes (IMs) in bone marrow, which then precludes clearance of leukemia mediated by CD8+ T cells and facilitates LP growth. While sparing IMs, common chemotherapeutic agents stimulate IL-36 production from residual LPs via caspase-1 activation, thereby enabling the persistence of this immunosuppressive IL-36­IM axis after chemotherapy. Furthermore, IM depletion by trabectedin, with chemotherapy and PD-1 blockade, can synergistically restrict AML progression and relapse. Collectively, these results suggest inhibition of the IL-36­IM axis as a potential strategy for improving AML treatment.

Screen of anti-migraine active compounds from Duijinsan by spectrum-effect relationship analysis and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Duijinsan (DJS) is a famous Chinese medicine prescription composed of Radix scutellariae (RS) and Rhei Radix (RRR), which has been mainly used for treating migraine. AIM OF THE STUDY: This study aimed to uncover the anti-migraine active compounds from DJS and preliminary predicted the pharmacological mechanism by evaluating the spectrum-effect relationship between high-performance liquid chromatography (HPLC) fingerprints and anti-migraine effects of Duijinsan (DJS) extract combined with molecular docking. MATERIALS AND METHODS: HPLC and LC-MS were applied for chemical analyses of DJS extracts in different proportions. Inhibition of DJS extracts on trigeminal nerve cell releasing calcitonin gene related peptide (CGRP) experiment was performed. The active compounds were screened by spectrum-effect relationship analysis and confirmed by molecular docking and the activities of major predicted compounds were validated in vitro. RESULTS: Twenty-six common peaks were assigned and identified from the fingerprints of different proportions DJS extracts. In vitro experimental results showed that DJS extracts inhibited inflammation and release of CGRP from trigeminal nerve cells. Five predicted active compounds, Chrysin 6-C-arabinoside 8-C-glucoside, Chrysin 6-C-glucoside 8-C-arabinoside, baicalin, Chrysin-7-O-Beta-D-glucoronide and Oroxylin A 7-O-glucuronide were sorted out according to spectrum-effect relationship analysis and molecular docking comprehensively. In vitro validation experiments showed that all the predicted compounds inhibited the CGRP releasing and the activation of TRPV1 channel. Baicalin, chrysin-7-O-ß-D-glucuronide and Oroxylin A-7-glucoronide significantly inhibited the activation of TRPV1 channel. CONCLUSION: Chrysin 6-C-arabinoside 8-C-glucoside, Chrysin 6-C-glucoside 8-C-arabinoside, baicalin, Chrysin-7-O-Beta-D-glucoronide and Oroxylin A 7-O-glucuronide which can inhibit the CGRP releasing and the activation of TRPV1 channel were screened as the anti-migraine active compounds by spectrum-effect relationship analysis and molecular docking.

Effect of biological additives on Japanese eel (Anguilla japonica) growth performance, digestive enzymes activity and immunology.

Japanese eel (Anguilla japonica) has become a commercially important fish species all over the world. High-density aquaculture has led to congestion and contributed to bacterial infection outbreaks that have caused high mortality. Therefore a 56-days feeding trial was conducted to determine the effects of dietary Bacillus amyloliquefaciens (GB-9) and Yarrowia lipolytica lipase2 (YLL2) on growth performance, digestive enzymes activity, innate immunity and resistance to pathogens of A. japonica. Fish growth performance was significantly affected by dietary YLL2 supplementation but not by GB-9. Fish fed diets with YLL2 at 2.0 g/kg diet in combination of high and low levels of GB-9 (5.0 g/kg and 2.0 g/kg) produced the highest growth. For digestive enzyme, lipase and trypsin activities was promoted by dietary containing YLL2, while amylase activities was increased by dietary containing YLL2, GB-9 single or combination. For innate immunity, the mucus lysozyme activity, leukocytes phagocytosis activity and reactive oxygen species level of skin, peroxidase and lysozyme activity of serum were enhanced in fish fed with GB-9 compared to those in control group (p < 0.05). The highest resistance to Vibrio anguillarum and Aeromonas hydrophila was determined in fish fed with 5.0 g kg-1 GB-9 + 2.0 g/kg YLL2. This study demonstrated that GB-9 and YLL2 enhanced non-specific immune defense system of A. japonica, providing them with higher resistance to pathogens. The present results suggested that the combination of these supplements could be considered as potential biological additives for aquaculture farmed fish.

Effects of Bacillus amyloliquefaciens and Yarrowia lipolytica lipase 2 on immunology and growth performance of Hybrid sturgeon.

A 12-weeks feeding trial was performed to investigate the possible effects of supplementation of Hybrid sturgeon diet with Bacillus amyloliquefaciens (GB-9) and Yarrowia lipolytica lipase2 (YLL2) single or combined on immune response and growth performance of Hybrid sturgeon (Acipenser schrenkii ♂and Acipenser baeri ♀). For this aim, Hybrid sturgeons were fed with four experimental diets namely: Diet 1 (0-control), Diet 2 (5.0 g/kg GB-9), Diet 3 (4.0 g/kg YLL2), and Diet 4 (5.0 g/kg GB-9 + 4.0 g/kg YLL2), respectively. After fed with varied diets, growth performance, mucosal immune response, leukocytes immune response and serum immunological response were measured. The results indicated that supplementations of GB-9 + YLL2 resulted in a significant increase in final weight, Docosahexaenoic acid (DHA) and Eicosapentenoic acid (EPA) concentration, compared with that of control (p < 0.05). For innate immunity, the results showed that skin mucus lysozyme activity, leukocytes phagocytosis activity and reactive oxygen species level, and serum alternative complement pathway activity, peroxidase and lysozyme activity were significantly higher in supplemented groups compared to the control (p < 0.05). The highest values were recorded in fish fed both YLL2 and GB-9 with respect to the individual application. The present results suggested that the combination of these supplementation could be considered as potential feed-additives for aquaculture farmed fish.

Pseudooceanicola lipolyticus sp. nov., a marine alphaproteobacterium, reclassification of Oceanicola flagellatus as Pseudooceanicola flagellatus comb. nov. and emended description of the genus Pseudooceanicola.

A Gram-stain-negative, rod-shaped bacterium, designated 157T, was isolated from seawater collected from the Philippine Sea. Cells of strain 157T grew in medium containing 0.5-10.0 % NaCl (w/v, optimum 3 %), at pH 6.0-8.5 (optimum 7.0) and at 15-40 °C (optimum 30 °C). Tweens 20, 40 and 80 as well as urea were hydrolysed. The 16S rRNA gene sequence of strain 157T had a high sequence similarity with respect to Pseudooceanicola marinus AZO-CT (97.2 %), and exhibited less than 97.0 % sequence similarity to other type strains of the species with validly published names. Phylogenetic analyses revealed that strain 157T fell within a cluster comprising the Pseudooceanicola species and formed a coherent clade with P. marinus AZO-CT and Pseudooceanicola antarcticus Ar-45T. Strain 157T exhibited average nucleotide identity values of 74.5 and 74.9 % to P. marinus LMG 23705T and P. antarcticus Ar-45T, respectively. In silico DNA-DNA hybridization analysis revealed that strain 157T shared 20.2 % DNA relatedness with P. marinus LMG 23705T and 20.6 % with P. antarcticus Ar-45T, respectively. The sole isoprenoid quinone was ubiquinone 10. The major fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C19 : 0 cyclo ω8c, C16 : 0 2-OH and C16 : 0. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminolipid and one unidentified glycolipid. The DNA G+C content was 64.6 mol%. According to the phylogenetic, chemotaxonomic and phenotypic data, it represents a novel species of the genus Pseudooceanicola, for which the name Pseudooceanicolalipolyticus is proposed. The type strain is 157T (=KCTC 52654T=MCCC 1K03317T). In addition, the description of the genus Pseudooceanicola is emended and Oceanicola flagellatus is reclassified as Pseudooceanicola flagellatus comb. nov., with the type strain DY470T (=CGMCC 1.12664T=LMG 27871T) proposed.

Hyphococcus flavus gen. nov., sp. nov., a novel alphaproteobacterium isolated from deep seawater.

A Gram-staining-negative, aerobic, non-spore-forming, coccoid to rod shaped bacteria with prosthecate and flagellum, designated as HSF6T, was isolated from deep seawater samples collected from the South China Sea at depth of 2.5 km and subjected to a polyphasic taxonomic investigation. Colonies of strain HSF6T were 1-2 mm in diameter, smooth, circular, convex and yellow. Strain HSF6T was found to grow at 15-37 °C (optimum, 25-35 °C), pH 5.0-9.5 (optimum, pH 7.0-7.5) and with 0-8 % (w/v) NaCl (optimum, 2 %). Chemotaxonomic analysis showed the predominant respiratory quinone of strains HSF6T were ubiquinone-10, and the major fatty acids were C18 : 1ω7c, C16 : 0 and 11-methyl C18 : 1ω7c. The polar lipids were monoglycosyldiglyceride (MGDG), sulfo-quinovosyl diacylglycerol (SQDG), three unknown glycolipids (GL1-3) and five unknown lipids (L1-5). The DNA G+C content of strain HSF6T was determined to be 51.0 mol% with HPLC. The comparison of 16S rRNA gene sequence similarities show that strain HSF6T was related most closely to genus Parvularcula with similarity ranging from 91.0 to 91.8 %. The phylogenetic trees, using the 16S rRNA gene sequence, reconstructed with neighbour-joining, maximum-parsimony and maximum-likelihood methods showed that strain HSF6T constituted a separated branch in the family 'Parvularculaceae'. Differential phenotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain HSF6T is clearly distinct from validly published genera. On the basis of these features, we propose strain HSF6T (=MCCC 1K03223T=KCTC 52486T) represents a novel species of a novel genus with the name Hyphococcus flavus gen. nov., sp. nov.

Multi-glycoside of Tripterygium wilfordii Hook. f. attenuates glomerulosclerosis in a rat model of diabetic nephropathy by exerting anti-microinflammatory effects without affecting hyperglycemia.

Multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) has been proven to be clinically effective in relieving microinflammation in patients with early diabetic nephropathy (DN). However, the therapeutic mechanisms involved in vivo remain unclear. In the process of early DN, microinflammation and activation of p38 mitogen­activated protein kinase (MAPK) and canonical nuclear factor (NF)-κB signaling pathways are the important mechanisms by which hyperglycemia contributes to glomerulosclerosis (GS). Therefore, this study aimed to examine the ameliorative effects of GTW on GS, and then to clarify its anti­microinflammatory mechanisms by inhibiting p38 MAPK and NF-κB signaling activities in the kidney. All rats were divided into 4 groups: the sham group, the sham + GTW group, the vehicle group and the GTW group. The suitable dose of GTW and vehicle were daily administered for 8 weeks after the induction of DN by unilateral nephrectomy combined with intraperitoneal injections of streptozotocin (STZ). The general status of the rats, biochemical parameters, renal histological changes and macrophages in glomeruli, as well as expression of the key proteins in the p38 MAPK and canonical NF-κB signaling pathways and inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and transforming growth factor (TGF)-ß1 in the kidney were examined, respectively. The results revealed that, GTW improved the general cond-ition and biochemical parameters of the rats, but did not lower blood glucose; GTW attenuated GS and suppressed glomerular microinflammation including the infiltration of ED1+ cells in glomeruli and the protein overexpression of TNF-α, IL-1ß and TGF-ß1 in the kidney; GTW inhibited the protein overexpression of key signaling molecules of p38 MAPK and canonical NF-κB pathways in the kidney including phosphorylated p38 MAPK, phosphorylated inhibitor protein IκB and NF-κB (p65). On the whole, we expounded that GTW, as a natural regulator in vivo, alleviates GS without affecting hyperglycemia, by exerting anti-microinflammatory effects, including reducing macrophage infiltration in glomeruli, suppressing TNF-α, IL-1ß and TGF-ß1 overexpression in the kidney and inhibiting p38 MAPK and NF-κB signaling activities.

Hyphobacterium vulgare gen. nov., sp. nov., a novel alphaproteobacterium isolated from seawater.

A Gram-stain-negative, aerobic, non-spore-forming, non-motile, oval to rod-shaped, prosthecate bacterium, designated strain WM6T, was isolated from a seawater sample collected from the South China Sea at a depth of 150 m and subjected to a polyphasic taxonomic investigation. Cells of strain WM6T were approximately 0.5-0.6 µm in width and 0.8-1.2 µm in length, and colonies were smooth, circular, convex and whitish yellow. Strain WM6T was found to grow at 10-45 °C (optimum, 30 °C), at pH 6.5-9.0 (optimum, pH 7.5-8.5) and with 1-6 % (w/v) NaCl (optimum, 1-2 %). Chemotaxonomic analysis showed the predominant respiratory quinone and the major fatty acid of strains WM6T were ubiquinone-10 and C18 : 1ω7c, respectively. The polar lipids of strain WM6T were phosphatidylglycerol, glucuronopyranosyldiglyceride, monoglycosyldiglyceride, sulfo-quinovosyl diacylglycerol, seven unknown glycolipids and two unknown lipids. The DNA G+C content of strain WM6T was determined to be 59.8 mol% by HPLC. 16S rRNA gene sequence similarities showed that strain WM6T was related most closely to the genus Maricaulis with a similarity range from 92.3 to 93.8 %. Phylogenetic trees reconstructed with the neighbour-joining and maximum-likelihood methods using mega and maximum-likelihood methods using arb showed that strain WM6T constituted a separated branch in the family Hyphomonadaceae. Differential phenotypic properties, together with phylogenetic distinctiveness, demonstrated that strain WM6T is clearly distinct from any validly published genus. On the basis of these features, strain WM6T represents a novel species of a new genus with the name Hyphobacterium vulgare gen. nov., sp. nov. The type strain of Hyphobacterium vulgare is WM6T (=MCCC 1K03222T=KCTC 52487T).

[Absorption and metabolism of icariin in different osteoporosis rat models].

To compare the intestinal absorption and metabolism of icariin in different osteoporosis rat models. Ovariectomy and intragastric administration of cyclophosphamide were used to establish two kinds of rat osteoporosis models. Then the rat intestinal perfusion was conducted, and HPLC was used to measure and calculate the permeability coefficients of icariin in different intestines and production amount of metabolites. Western blot was used to detect LPH enzyme expression in two models. Experimental results showed that both ovariectomy and intragastric administration of cyclophosphamide 4.5 mg•kg⁻¹ could reduce rat bone density and successfully construct the rat osteoporosis models. The apparent permeability coefficient Papp of 20 µmol icariin in duodenum, jejunum, ileum, colon was 5.695, 5.224, 1.492, 0.520 respectively in sham operation group; 3.876, 3.608, 0.863, and 0.291 in ovariectomized group; 4.945, 3.601, 1.990, 1.042 in normal saline group; 3.301, 2.108, 1.209, 1.233 in cyclophosphamide-induced osteoporosis model group. In addition, the protein expression levels of LPH enzyme in two model groups were lower than those in normal group. The absorption and metabolism of icariin in two kinds of osteoporosis models was lower than that in sham operation group and normal saline group; the reduction of expression level of LPH enzymes in rat intestine of different osteoporosis models was one of the reasons for leading to the reduced intestinal absorption and metabolism of icariin.

[The Hydrothermal Synthesis, Structure and Spectroscopy Study on (H2dap)6H[V12B16O54(OH)4] · 12H2O (dap = 1, 2-diaminopropane)].

A polyoxovanadium borate (H2 dap)6H[V12B16O54(OH)4] · 12H2O (dap=1,2-diaminopropane) with novel structure was hydrothermally synthesized and characterized by the single crystal X-ray diffraction. It crystallizes in triclinic system with space group Piand unit cell parameters a=19.027(4), b=16.142(3) Å, c=26.679(5) Å, α=90°, ß=101.06(3)°, γ=90°, V=8042(3) Å3, Z=4, Dc=1.962 g · cm(-3), µ=1.456 mm(-1), F(000)=4776, the final R1=0.0626, wR2=0.1927, S=1.003,for 7635 observed reflections with I>2σ(I). It is showed that the compound 1 is composed of V12B16 clusters unit and dap which is as a counter ion, and a two-dimensional layered structure is obtained by the effect of hydrogen bonding between the cluster units and dap, and between the layers via strong hydrogen bonds to form a three-dimensional supramolecular structure. The compound 1 were also characterized by IR, two-dimensional infrared (2D IR) correlation spectroscopy with magnetic and thermal perturbation, UV/Vis DRS spectra. The relationship between the structure and spectroscopy properties was discussed. The IR spectrum showed that the antisymmetric stretching vibration absorption peak νas (V-Oµ) and symmetric stretching vibration absorption peaks νs (V-Oµ) appeared at 775 and 683 cm(-1) respectively, whereas the vibration absorption peak in ν(B-O) of BO3 and the vibration absorption peak in ν(B-O) of BO4 appeared at 1350 and 1050 cm(-1) respectively. The response of the stretching vibrations of B-O and V-O was detected in the 2D IR correlation spectra with magentic perturbation. In addition, the response of the stretching vibrations of B-OH, B-O and V-O-V was detected in the 2D IR correlation spectra with thermal perturbation.

Valproic Acid Ameliorates Graft-versus-Host Disease by Downregulating Th1 and Th17 Cells.

Graft-versus-host disease (GVHD) is the major complication after allogeneic bone marrow transplantation. Valproic acid (VPA) was described as a histone deacetylase inhibitor that had anti-inflammatory effects and reduced the production of proinflammatory cytokines in experimental autoimmune disease models. Using well-characterized mouse models of MHC-mismatched transplantation, we studied the effects of VPA on GVHD severity and graft-versus-leukemia (GVL) activity. Administration of VPA significantly attenuated the clinical severity of GVHD, the histopathology of GVHD-involved organs, and the overall mortality from GVHD. VPA downregulated Th1 and Th17 cell responses and cytokine production in vitro and in vivo, whereas its effect on GVHD was regulatory T cell independent. The effect of VPA was related to its ability to directly reduce the activity of Akt, an important regulator of T cell immune responses. Importantly, when mice received lethal doses of host-type acute leukemia cells, administration of VPA did not impair GVL activity and resulted in significantly improved leukemia-free survival. These findings reveal a unique role for VPA as a histone deacetylase inhibitor in reducing the donor CD4(+) T cells that contribute to GVHD, which may provide a strategy to reduce GVHD while preserving the GVL effect.

Huangkui capsule attenuates renal fibrosis in diabetic nephropathy rats through regulating oxidative stress and p38MAPK/Akt pathways, compared to α-lipoic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM), Abelmoschus manihot (L.) medic (AM) is a natural medicinal plant used for the treatment of inflammatory diseases. Recently, Huangkui capsule (HKC), a Chinese patent medicine extracted from AM, has been widely applied to the clinical therapy of renal fibrosis in patients with early diabetic nephropathy (DN). However, the therapeutic mechanisms involved in vivo remain ambiguous. The goal of this study is to expound the mechanism in vivo of HKC in order to deepen the understanding of its clinical effects, by using the approaches of contrasting the dose-effects of HKC on oxidative stress (OS) in the kidney compared to α-lipoic acid (LA), and then demonstrating whether and how anti-oxidative properties of HKC or LA might be beneficial for the treatment of renal fibrosis in vivo. MATERIALS AND METHODS: Thirty-three rats were divided into 5 groups, a Sham group, a Vehicle group, a L-HKC group, a H-HKC group and a LA group. The different doses of HKC, LA and distilled water were daily administrated for 8 weeks after the induction of DN by the unilateral nephrectomy combined with streptozotocin (STZ) intraperitoneal injections. Rat's general status, biochemical parameters, renal histological changes and OS indicators, as well as the key protein expressions in p38 mitogen-activated protein kinase (p38MAPK)/serine-threonine kinase (Akt) signaling pathways and downstream cytokines including transforming growth factor (TGF)-ß1 and tumor necrosis factor (TNF)-α were examined, respectively. RESULTS: HKC and LA ameliorated body weight, kidney weight, urinary albumin and renal function including blood urea nitrogen and serum uric acid, attenuated renal fibrosis including the cell numbers and extracellular matrix rate in glomerulus, and controlled OS indicators including malondialdehyde, total superoxide dismutase, 8-hydroxy-2'-deoxyguanosine and nicotinamide adenine dinucleotide phosphate oxidase 4, but did not lower blood glucose in DN model rats. Among them, the anti-renal fibrosis effect of H-HKC was better than that of LA. In addition, HKC simultaneously down-regulated the protein expressions of phosphorylated p38MAPK, phosphorylated Akt (p-Akt), TGF-ß1 and TNF-α in the kidney of DN model rats, unlike HKC, LA only down-regulated p-Akt and TNF-α protein expressions. CONCLUSION: We have demonstrated that HKC, similar to LA, is renoprotective via attenuating OS and renal fibrosis in the DN rat model. The potential mechanisms by which HKC and LA exert their therapeutic effects in vivo are respectively through down-regulating the activation of p38MAPK and/or Akt pathways as well as the expressions of TGF-ß1 and/or TNF-α in the kidney. Our findings thus provide the useful information about a clinical combination of HKC and LA in early DN patients.

The regulation of normal and leukemic hematopoietic stem cells by niches.

The origin and propagation of normal and leukemic hematopoietic cells critically depend on their interplays with the hematopoietic microenvironment (or so-called niche), which represent important biological models for understanding organogenesis and tumorigenesis. Nevertheless, the anatomic and functional characterizations of the niche cells for normal hematopoietic stem cells (HSCs) have proved a formidable task. It is uncertain whether the combinational effects of a few sets of molecular niche elements, behind the long-sought cellular architectures with preferred anatomic locations, actually meets the functional definition of HSC niche. Moreover, even much less is known about the niche components for numerous types of leukemia-stem cells (LSCs) that originate via discrete cellular and molecular transforming mechanisms. However, one interesting scenario is emerging, i.e., the leukemia cells can positively remodel the hematopoietic microenvironment favorable for their competition over the normal hematopoiesis that co-exists within the same eco-system. This property probably represents a previously unappreciated essential trait of a functional LSC. Obviously, the further exploration into how the hematopoietic microenvironment interplay with normal or malignant hematopoiesis will shed light onto the designing of novel types of niche-targeting therapies for leukemia.