NDC1 promotes hepatocellular carcinoma tumorigenesis by targeting BCAP31 to activate PI3K/AKT signaling.

Hepatocellular carcinoma (HCC) is among the world's worst malignancies. Nuclear division cycle 1 (NDC1) is an essential membrane-integral nucleoporin, found in this study to be significantly increased in primary HCC. A multivariate analysis revealed that higher NDC1 expression was linked to worse outcome in HCC patients. Mouse xenograft tumors overexpressing NDC1 grew rapidly, and HCC cells overexpressing NDC1 showed enhanced proliferation, invasion, and migration in vitro. In contrast, knocking down NDC1 had the opposite effects in vitro. Furthermore, co-immunoprecipitation and liquid chromatograph mass spectrometer analyses revealed that NDC1 activated PI3K/AKT signaling by interacting with BCAP31. In summary, NDC1 and BCAP31 cooperate to promote the PI3K/AKT pathway, which is essential for HCC carcinogenesis. This suggests that NDC1 is predictive of prognosis in HCC.

Alphaherpesvirus upregulates NDRG1 expression to facilitate the nuclear import of viral UL31 and UL34 proteins.

Proteins UL31 and UL34 encoded by alphaherpesvirus are critical for viral primary envelopment and nuclear egress. We report here that pseudorabies virus (PRV), a useful model for research on herpesvirus pathogenesis, uses N-myc downstream regulated 1 (NDRG1) to assist the nuclear import of UL31 and UL34. PRV promoted NDRG1 expression through DNA damage-induced P53 activation, which was beneficial to viral proliferation. PRV induced the nuclear translocation of NDRG1, and its deficiency resulted in the cytosolic retention of UL31 and UL34. Therefore, NDRG1 assisted the nuclear import of UL31 and UL34. Furthermore, in the absence of the nuclear localization signal (NLS), UL31 could still translocate to the nucleus, and NDRG1 lacked an NLS, thus suggesting the existence of other mediators for the nuclear import of UL31 and UL34. We demonstrated that heat shock cognate protein 70 (HSC70) was the key factor in this process. UL31 and UL34 interacted with the N-terminal domain of NDRG1 and the C-terminal domain of NDRG1 bound to HSC70. Replenishment of HSC70ΔNLS in HSC70-knockdown cells, or interference in importin α expression, abolished the nuclear translocation of UL31, UL34, and NDRG1. These results indicated that NDRG1 employs HSC70 to facilitate viral proliferation in the nuclear import of PRV UL31 and UL34.

Acute transcriptomic changes in murine RAW 264.7 cells following pseudorabies virus infection.

Next-generation sequencing enables the evaluation of gene expression changes resulting from virus-host interactions at the RNA level. Pseudorabies virus (PRV) causes substantial economic loss in the swine industry. Recent research has revealed that PRV can be transmitted to and infect humans as well. To identify physiopathological and pathological responses post-PRV infection, we characterized transcriptomic changes in the murine RAW 264.7 cell line over the course of 36 h. In total, 156, 153, and 190 differentially expressed genes were identified at 2 h, 12 h, and 36 h, respectively. Seven differentially expressed genes (Trim27, Ccdc117, Mrps12, Ccl4, Cerkl, Ubald1, and Hmga1-rs1) were present across all treatment groups. Our findings expand our knowledge of gene regulation and immune response following PRV infection. These differentially expressed genes can subsequently improve our understanding of PRV pathogenesis.

Endoscopic submucosal dissection treatment of metachronous early gastric adenocarcinoma in a case with diffuse large B-cell lymphoma.

Cases of primary gastric lymphoma complicated with gastric adenocarcinoma are rare in clinical practice. We report a case of metachronous early gastric adenocarcinoma diagnosed eight years after the onset of gastric diffuse large B-cell lymphoma (DLBCL) and treated with endoscopic submucosal dissection (ESD).

Loncastuximab tesirine in relapsed or refractory diffuse large B-cell lymphoma (LOTIS-2): a multicentre, open-label, single-arm, phase 2 trial.

BACKGROUND: Patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) who do not respond to or who have progressive disease after salvage therapies have a poor prognosis. Loncastuximab tesirine is a CD19-directed antibody-drug conjugate with encouraging phase 1 single-agent antitumour activity and acceptable safety in non-Hodgkin lymphoma. We aimed to evaluate the antitumour activity and safety of loncastuximab tesirine in patients with relapsed or refractory DLBCL. METHODS: We did a multicentre (28 hospital sites in the USA, UK, Italy, and Switzerland), open-label, single-arm, phase 2 trial (LOTIS-2) in patients aged 18 years or older with relapsed or refractory DLBCL after two or more multiagent systemic treatments, who had measurable disease and Eastern Cooperative Oncology Group performance status 0-2. Eligible patients received loncastuximab tesirine intravenously on day 1 of each 21-day cycle, at 150 µg/kg for two cycles, then 75 µg/kg thereafter, for up to 1 year or until disease relapse or progression, unacceptable toxicity, death, major protocol deviation, pregnancy, or patient, investigator, or sponsor decision. The primary endpoint was overall response rate assessed by central review. Primary antitumour activity and safety analyses were done in the as-treated population (patients who received at least one dose of loncastuximab tesirine), when all responding patients had at least 6 months of follow-up after initial documented response. Enrolment is complete. This trial is registered with ClinicalTrials.gov, NCT03589469. FINDINGS: Between Aug 1, 2018, and Sept 24, 2019, 184 patients were assessed for eligibility and 145 (79%) were enrolled and received at least one dose of loncastuximab tesirine, including patients with high-risk characteristics for poor prognosis, such as double-hit, triple-hit, transformed, or primary refractory DLBCL. 70 of 145 patients had complete or partial response (overall response rate 48·3% [95% CI 39·9-56·7]); 35 had complete response and 35 had partial response. The most common grade 3 or higher treatment-emergent adverse events were neutropenia (37 [26%] of 145 patients), thrombocytopenia (26 [18%]), and increased gamma-glutamyltransferase (24 [17%]). Serious adverse events were reported in 57 (39%) of 145 patients. Treatment-emergent adverse events with a fatal outcome occurred in eight (6%) of 145 patients; none were considered related to loncastuximab tesirine. INTERPRETATION: Loncastuximab tesirine has substantial single-agent antitumour activity and produces durable responses with an acceptable safety profile, potentially offering a new therapeutic option for heavily pretreated patients with relapsed or refractory DLBCL. FUNDING: ADC Therapeutics.

Identification of Potential Dipeptidyl Peptidase (DPP)-IV Inhibitors among Moringa oleifera Phytochemicals by Virtual Screening, Molecular Docking Analysis, ADME/T-Based Prediction, and In Vitro Analyses.

Moringa oleifera Lam. (MO) is called the "Miracle Tree" because of its extensive pharmacological activity. In addition to being an important food, it has also been used for a long time in traditional medicine in Asia for the treatment of chronic diseases such as diabetes and obesity. In this study, by constructing a library of MO phytochemical structures and using Discovery Studio software, compounds were subjected to virtual screening and molecular docking experiments related to their inhibition of dipeptidyl peptidase (DPP-IV), an important target for the treatment of type 2 diabetes. After the four-step screening process, involving screening for drug-like compounds, predicting the absorption, distribution, metabolism, excretion, and toxicity (ADME/T) of pharmacokinetic properties, LibDock heatmap matching analysis, and CDOCKER molecular docking analysis, three MO components that were candidate DPP-IV inhibitors were identified and their docking modes were analyzed. In vitro activity verification showed that all three MO components had certain DPP-IV inhibitory activities, of which O-Ethyl-4-[(α-l-rhamnosyloxy)-benzyl] carbamate (compound 1) had the highest activity (half-maximal inhibitory concentration [IC50] = 798 nM). This study provides a reference for exploring the molecular mechanisms underlying the anti-diabetic activity of MO. The obtained DPP-IV inhibitors could be used for structural optimization and in-depth in vivo evaluation.

Two new naphthalene glycosides from the seeds of Cassia obtusifolia.

A phytochemical study on the seeds of Cassia obtusifolia was carried out, which finally led to obtain two naphthalenes (1 and 2), two naphthopyrans (3 and 4) and twelve anthraquinones (5-16). The structures of all compounds were established mainly by NMR and MS experiments as well as the necessary chemical evidence. Among them, 1 and 2 (obtusinaphthalensides A and B) were identified to be new naphthalene glycosides.

Ruxolitinib versus standard therapy for the treatment of polycythemia vera.

BACKGROUND: Ruxolitinib, a Janus kinase (JAK) 1 and 2 inhibitor, was shown to have a clinical benefit in patients with polycythemia vera in a phase 2 study. We conducted a phase 3 open-label study to evaluate the efficacy and safety of ruxolitinib versus standard therapy in patients with polycythemia vera who had an inadequate response to or had unacceptable side effects from hydroxyurea. METHODS: We randomly assigned phlebotomy-dependent patients with splenomegaly, in a 1:1 ratio, to receive ruxolitinib (110 patients) or standard therapy (112 patients). The primary end point was both hematocrit control through week 32 and at least a 35% reduction in spleen volume at week 32, as assessed by means of imaging. RESULTS: The primary end point was achieved in 21% of the patients in the ruxolitinib group versus 1% of those in the standard-therapy group (P<0.001). Hematocrit control was achieved in 60% of patients receiving ruxolitinib and 20% of those receiving standard therapy; 38% and 1% of patients in the two groups, respectively, had at least a 35% reduction in spleen volume. A complete hematologic remission was achieved in 24% of patients in the ruxolitinib group and 9% of those in the standard-therapy group (P=0.003); 49% versus 5% had at least a 50% reduction in the total symptom score at week 32. In the ruxolitinib group, grade 3 or 4 anemia occurred in 2% of patients, and grade 3 or 4 thrombocytopenia occurred in 5%; the corresponding percentages in the standard-therapy group were 0% and 4%. Herpes zoster infection was reported in 6% of patients in the ruxolitinib group and 0% of those in the standard-therapy group (grade 1 or 2 in all cases). Thromboembolic events occurred in one patient receiving ruxolitinib and in six patients receiving standard therapy. CONCLUSIONS: In patients who had an inadequate response to or had unacceptable side effects from hydroxyurea, ruxolitinib was superior to standard therapy in controlling the hematocrit, reducing the spleen volume, and improving symptoms associated with polycythemia vera. (Funded by Incyte and others; RESPONSE ClinicalTrials.gov number, NCT01243944.).

S100 calcium-binding protein A12 as a diagnostic index for subclinical mastitis in cows.

Diagnosis of subclinical mastitis is very important in management of the dairy industry and improvement of dairy cow productivity. S100A12, that is found in related tissues of mammals, is considered as an index for diagnosing inflammatory reaction. To evaluate whether S100A12 is involved in subclinical mastitis, milk somatic cell mRNA from 276 dairy cows was used to detect the transcriptional level of S100A12 by real-time quantitative polymerase chain reaction. A predictive analysis for mastitis was performed, and the correlation between S100A12 and other subclinical mastitis indicators was also assessed. The transcriptional levels of S100A12 in the milk of cows with mastitis were significantly higher than those in the milk of healthy cows (p < 0.05). The correlation analysis showed that S100A12 was positively associated with the somatic cell count and the sodium and chloride concentrations of milk. In contrast, a negative correlation was found between S100A12 and the potassium concentration and pH of milk. However, no significant correlation was detected between S100A12 and the other parameters, such as protein, lactose, ash, fat, density, Ca2+ and SNF. These results suggested that the S100A12 level in milk may serve as a diagnostic tool for subclinical mastitis in cows without obvious clinical signs.

Polyoxypregnane Glycosides from the Roots of Marsdenia tenacissima and Their Anti-HIV Activities.

A continuous phytochemical study on the roots of Marsdenia tenacissima led to the isolation and identification of 13 new polyoxypregnane glycosides named marstenacissides B10-B17 (1, 2, 4, 7, 8, 11, 12, and 14) and marstenacissides A8-A12 (3, 9, 10, 13, and 15) in addition to two known polyoxypregnane glycosides marsdenosides M and L (5 and 6). Their structures were established by spectroscopic techniques and by comparison with the reported data in the literature. Moreover, the anti-HIV activities of these isolates and the previous isolated marstenacissides A1-A7 and B1-B9 were assessed, some of which exhibited slight or negligible effects against HIV-1.

The Pharmacological Effects of Lutein and Zeaxanthin on Visual Disorders and Cognition Diseases.

Lutein (L) and zeaxanthin (Z) are dietary carotenoids derived from dark green leafy vegetables, orange and yellow fruits that form the macular pigment of the human eyes. It was hypothesized that they protect against visual disorders and cognition diseases, such as age-related macular degeneration (AMD), age-related cataract (ARC), cognition diseases, ischemic/hypoxia induced retinopathy, light damage of the retina, retinitis pigmentosa, retinal detachment, uveitis and diabetic retinopathy. The mechanism by which they are involved in the prevention of eye diseases may be due their physical blue light filtration properties and local antioxidant activity. In addition to their protective roles against light-induced oxidative damage, there are increasing evidences that L and Z may also improve normal ocular function by enhancing contrast sensitivity and by reducing glare disability. Surveys about L and Z supplementation have indicated that moderate intakes of L and Z are associated with decreased AMD risk and less visual impairment. Furthermore, this review discusses the appropriate consumption quantities, the consumption safety of L, side effects and future research directions.

NF-κB is involved in the LPS-mediated proliferation and apoptosis of MAC-T epithelial cells as part of the subacute ruminal acidosis response in cows.

OBJECTIVES: To determine the effect of NF-κB on cell proliferation and apoptosis, we investigate the expression of inflammation and apoptosis-related factors in the bovine mammary epithelial cell line, MAC-T. RESULTS: MAC-T cells were cultured in vitro and MTT and LDH assays used to determine the effects of lipopolysaccharide (LPS) on proliferation and cytotoxicity respectively. RT-PCR and western blotting were used to evaluate the effect of LPS and NF-κB inhibition [pyrrolidine dithiocarbamate (PDTC) treatment] on the expression of inflammation and apoptosis-related factors. LPS significantly inhibited MAC-T cell proliferation in a dose- and time-dependent manner. Furthermore, LPS promoted apoptosis while the NF-кB inhibitor PDTC attenuated this effect. After LPS treatment, the NF-кB signaling pathway was activated, and the expression of inflammation and apoptosis-related factors increased. When PDTC blocked NF-кB signaling, the expression of inflammation and apoptosis-related factors were decreased in MAC-T cells. CONCLUSIONS: LPS activates the TLR4/NF-κB signaling pathway, inhibits proliferation and promotes apoptosis in MAC-T cells. NF-кB inhibition attenuates MAC-T cell apoptosis and TLR4/NF-κB signaling pathway. NF-кB inhibitor alleviating MAC-T cell apoptosis is presumably modulated by NF-кB.

[Research on optimal modeling strategy for licorice extraction process based on near-infrared spectroscopy technology].

The manufacture of traditional Chinese medicine (TCM) products is always accompanied by processing complex raw materials and real-time monitoring of the manufacturing process. In this study, we investigated different modeling strategies for the extraction process of licorice. Near-infrared spectra associate with the extraction time was used to detemine the states of the extraction processes. Three modeling approaches, i.e., principal component analysis (PCA), partial least squares regression (PLSR) and parallel factor analysis-PLSR (PARAFAC-PLSR), were adopted for the prediction of the real-time status of the process. The overall results indicated that PCA, PLSR and PARAFAC-PLSR can effectively detect the errors in the extraction procedure and predict the process trajectories, which has important significance for the monitoring and controlling of the extraction processes.

Heparin inhibits the inflammatory response induced by LPS and HMGB1 by blocking the binding of HMGB1 to the surface of macrophages.

High mobility group box 1 protein (HMGB1), a nuclear non-histone DNA-binding protein, is secreted extracellularly during inflammation and is a late mediator of inflammatory responses. The pro-inflammatory activity of recombinant HMGB1 proteins is dependent upon the formation of complexes with other mediators, such as lipopolysaccharide (LPS). This study investigated the influence of heparin on LPS+HMGB1-mediated inflammatory responses in cultured macrophages and a murine sepsis model. HMGB1 promoted the phosphorylation of p38 and ERK1/2. HMGB1 enhanced the induction of the pro-inflammatory cytokine, TNF-α, by LPS in macrophages. Heparin blocked the binding of HMGB1 to the surface of macrophages, and suppressed the phosphorylation of p38 and ERK1/2, but not JNK; TNF-α secretion was also decreased. However, heparin alone did not affect LPS-induced production of TNF-α. Heparin reduced lethality in mice exposed to LPS+HMGB1. To conclude, heparin inhibited LPS-induced HMGB1-amplified inflammatory responses by blocking HMGB1 binding to macrophage surfaces. Heparin could be used therapeutically as an effective inhibitor of HMGB1-associated inflammation.

SRC2-1 is required in PcINF1-induced pepper immunity by acting as an interacting partner of PcINF1.

Elicitins are elicitors that can trigger hypersensitive cell death in most Nicotiana spp., but their underlying molecular mechanism is not well understood. The gene Phytophthora capsici INF1 (PcINF1) coding for an elicitin from P. capsici was characterized in this study. Transient overexpression of PcINF1 triggered cell death in pepper (Capsicum annuum L.) and was accompanied by upregulation of the hypersensitive response marker, Hypersensitive Induced Reaction gene 1 (HIR1), and the pathogenesis-related genes SAR82, DEF1, BPR1, and PO2. A putative PcINF1-interacting protein, SRC2-1, was isolated from a pepper cDNA library by yeast two-hybrid screening and was observed to target the plasma membrane. The interaction between PcINF1 and SRC2-1 was confirmed by bimolecular fluorescence complementation and co-immunoprecipitation. Simultaneous transient overexpression of SRC2-1 and PcINF1 in pepper plants triggered intensive cell death, whereas silencing of SRC2-1 by virus-induced gene silencing blocked the cell death induction of PcINF1 and increased the susceptibility of pepper plants to P. capsici infection. Additionally, membrane targeting of the PcINF1-SRC2-1 complex was required for cell death induction. The C2 domain of SRC2-1 was crucial for SRC2-1 plasma membrane targeting and the PcINF1-SRC2-1 interaction. These results suggest that SRC2-1 interacts with PcINF1 and is required in PcINF1-induced pepper immunity.

A CD44 specific peptide developed by phage display for targeting gastric cancer.

OBJECTIVE: To develop a peptide probe that could be used for gastric cancer detection via binding to CD44 protein with specificity and affinity. RESULTS: A 12-mer phage peptide library was screened against immobilized CD44 protein. Bound phage counts using ELISA were performed to identify phage clones carrying the most highly selective peptide, which termed RP-1. Immunofluorescence and flow cytometry analysis indicated that the consensus peptide RP-1 could bind to CD44-positive gastric cancer cells with mean fluorescence intensities significantly higher than that of CD44-negative cells. CD44 knockdown led to decreased binding activity of RP-1 to the same cell line. Tissue array technique was used to identify the relationship (r = 0.556) between peptide binding and CD44 detection on gastric cancer tissues. Further, the hyaluronan-binding domain of CD44 was docked with RP-1 using computer modeling/docking approaches, revealing a RP-1/CD44 interaction with geometrical and energy match (-8.6 kcal/mol). CONCLUSIONS: The RP-1 peptide we screened exhibits affinity and specificity to CD44 on cells and has the potential to be used as a candidate probe for gastric cancer cell targeting.

Analysis of the Constituents in "Zhu She Yong Xue Shuan Tong" by Ultra High Performance Liquid Chromatography with Quadrupole Time-of-Flight Mass Spectrometry Combined with Preparative High Performance Liquid Chromatography.

"Zhu She Yong Xue Shuan Tong" lyophilized powder (ZSYXST), consists of a series of saponins extracted from Panax notoginseng, which has been widely used in China for the treatment of strokes. In this study, an ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) combined with preparative high performance liquid chromatography (PHPLC) method was developed to rapidly identify both major and minor saponins in ZSYXST. Some high content components were removed through PHPLC in order to increase the sensitivity of the trace saponins. Then, specific characteristic fragment ions in both positive and negative mode were utilized to determine the types of aglycone, saccharide, as well as the saccharide chain linkages. As a result, 94 saponins, including 20 pairs of isomers and ten new compounds, which could represent higher than 98% components in ZSYXST, were identified or tentatively identified in commercial ZSYXST samples.

[Comparative Study on Photosynthetic Characteristics and Medicinal Ingredients in Different Months of Inula nervosa].

OBJECTIVE: To compare the photosynthetic characteristics and medicinal ingredients in different months in order to provide a theoretical basis for cultivation and harvest of Inula nervosa. METHODS: The photosynthetic characteristics was measured by using LI-6400 and morphological characteristics were compared in different months, and the contents of total flavonoids and total phenols were determined by UV spectrophotometry. RESULTS: Net photosynthetic rate of Inula nervosa was the highest in June, which showed a single peak curve, and the average of daily change reached to 8.50 µmol/(m2 x s). Light response curve data showed the ability of using the strongest light was in June. Chlorophyll fluorescence parameters values also displayed that, openness of reflect center and photochemical efficiency of leaves' photosystem II were the highest, which also had the fastest rate of electron transfer in June. Morphological indicators showed that the single leaf area and leaf area of Inula nervosa were significantly higher in June than those in other months. The content of total phenols were much higher than that of total flavonoids in Inula nervosa. And the medicinal ingredient content of the underground part was higher than that in the aerial part. CONCLUSION: The best harvest time of underground part of Inula nervosa should be after autumn, when the weight and active ingredients are accumulated to a considerable level.

[Comparative Analysis of Content of Four Alkaloids in Fritillaria hupehensis and Fritillaria ebeiensis var. purpurea].

OBJECTIVE: To establish an assay method for simultaneous determination of peimine, peiminine, peimissine and hupehenine and to make a comparative analysis of the content of four alkaloids in Fritillaria hupehensis and Fritillaria ebeiensis var. purpurea for the first time. METHODS: A Unitary C18 column(250 mm x 4.6 mm, 5 µm) was chosen with acetonitrile-water (containing 0.05% diethylamine) as mobile phase in a gradient program. The column temperature was 35 degrees C and the flow-rate was 1.0 mL/min. RESULTS: There was high content of peiminine and the content of peimissine was inferior to peiminine in Fritillaria hupehensis. Relatively speaking, peimine and hupehenine were much lower than the other two ingredients. Fritillaria ebeiensis var. purpurea also contained high levels of peiminine, the minimum content of peimine and equivalent content of peimissine comparing with Fritillaria hupehensis. In addition, it didn't contain hupehenine in Fritillaria ebeiensis var. purpurea. CONCLUSION: This method is simple and fast, and it has good separation, reproducibility and reliable results. Also, it can be used as basis for the quality evaluation of Fritillaria hupehensis and Fritillaria ebeiensis var. purpurea.

A phase 2 study of ruxolitinib, an oral JAK1 and JAK2 Inhibitor, in patients with advanced polycythemia vera who are refractory or intolerant to hydroxyurea.

BACKGROUND: Polycythemia vera (PV) is a myeloproliferative neoplasm associated with somatic gain-of-function mutations of Janus kinase-2 (JAK2). Therapeutic options are limited in patients with advanced disease. Ruxolitinib, an oral JAK1/JAK2 inhibitor, is active in preclinical models of PV. The long-term efficacy and safety of ruxolitinib in patients with advanced PV who are refractory or intolerant to hydroxyurea were studied in a phase 2 trial. METHODS: Response was assessed using modified European LeukemiaNet criteria, which included a reduction in hematocrit to <45% without phlebotomy, resolution of palpable splenomegaly, normalization of white blood cell and platelet counts, and reduction in PV-associated symptoms. RESULTS: Thirty-four patients received ruxolitinib for a median of 152 weeks (range, 31 weeks-177 weeks) or 35.0 months (range, 7.1 months-40.7 months). Hematocrit <45% without phlebotomy was achieved in 97% of patients by week 24.Only 1 patient required a phlebotomy after week 4. Among patients with palpable splenomegaly at baseline, 44% and 63%, respectively, achieved nonpalpable spleen measurements at weeks 24 and 144. Clinically meaningful improvements in pruritus, night sweats, and bone pain were observed within 4 weeks of the initiation of therapy and maintained with continued treatment. Ruxolitinib treatment also reduced elevated levels of inflammatory cytokines and granulocyte activation. Thrombocytopenia and anemia were the most common adverse events.Thrombocytopenia of grade 3 or anemia of grade 3 (according to National Cancer Institute Common Terminology Criteria for Adverse Events,version 3.0) occurred in 3 patients each (9%) (1 patient had both) and were managed with dose modification. CONCLUSIONS: Ruxolitinib was generally well tolerated and provided rapid and durable clinical benefits in patients with advanced PV who were refractory or intolerant to hydroxyurea.