OBJECTIVE

To study the effects of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) in high-risk leukemia.

METHODS

From October 1995 to December 2000, 25 patients with high-risk leukemia (median age 34 years, range 5.5-52 years) were transplanted with peripheral blood stem cells from their HLA-identical sibling donors. Among them, 15 patients suffered from acute leukemia (ALL) (one ph+ ALL in CR1, seven in CR2 or greater, and seven in relapse, including two in relapse after the first all-BMT), four patients suffered from chronic myelocytic leukemia (CML) (in CP2, AP, BC, and relapse after BMT respecively), 6 patients suffered from myelodys plastic syndrome (MDS), including one case of refractory anemia with excess of blasts (REAB), one case of refractory anemia with excess of blasts in trransformation (REAB-T), and 4 cases of acute leukemia secondary to MDS. The graft versus host disease (GVHD) prophylaxis included administration of cyclosporine and methotrexate.

RESULTS

All patients were successfully engrafted. The median times (range) for their neutrophil returning to>> or = 0.5 x 10(9)/L and for platelet returning to>> or = 20 x 10(9)/L were 14 (10-18) days and 11 (7-45) days after transplantation resprctively. Grade II acute GVHD occurred in 12 patients with an incidence rate of 48%. Grade III GVHD was found in one patient (4%). No grade IV GVHD was seen. Among the 23 evaluable patients, 16 were diagnosed as chronic GVHD (70%). The actual transplant-related mortality was 16%. Leukemia relapse occurred in 6 patients, four of them received donor lymphocyte infusion (DLI) and achieved remission again. Nineteen patients were alive and disease-free with a median follow-up time of 304 (94-1,963) days. The two-year probability of overall survival, disease-free survival (DFS), and relapse rates were 64%, 58%, and 25% respectively.

CONCLUSIONS

Allo-PBSCT decreases the relapse rate, increases the disease-free survival rate for patients with high-risk leukemia. All-PBSCT may be a better choice for patients with high-risk hematological maligmancies.

Background: Aberrant DNA methylation has been firmly established as a factor contributing to the pathogenesis of colorectal cancer (CRC) via its capacity to silence tumour suppressor genes. However, the methylation status of multiple tumour suppressor genes and their roles in promoting CRC metastasis are not well characterised.

Methods: We explored the methylation and expression profiles of CPEB1 (the gene encoding cytoplasmic polyadenylation element-binding protein 1), a candidate CRC tumour suppressor gene, using The Cancer Genome Atlas (TCGA) database and validated these results in both CRC cell lines and cells from Han Chinese CRC patients (n = 104). The functional role of CPEB1 in CRC was examined in experiments performed in vitro and in vivo. A candidate transcription factor capable of regulating CPEB1 expression was predicted in silico and validated by luciferase reporter, DNA pull-down, and electrophoretic mobility shift assays.

Results: Hypermethylation and decreased expression of CPEB1 in CRC tumour tissues were revealed by TCGA database. We also identified a significant inverse correlation (Pearson's R = - 0.43, P < 0.001) between promoter methylation and CPEB1 expression. We validated these results in CRC samples and two CRC cell lines. We also demonstrated that up-regulation of CPEB1 resulted in significantly decreased tumour growth, migration, invasion, and tumorigenicity and promoted tumour cell apoptosis both in vitro and in vivo. We identified the transcription factors CCAAT enhancer-binding protein beta (CEBPB) and transcription factor CP2 (TFCP2) as critical regulators of CPEB1 expression. Hypermethylation of the CPEB1 promoter resulted in a simultaneous increase in the capacity for TFCP2 binding and a decreased likelihood of CEBPB binding, both of which led to diminished expression of CPEB1.

Conclusions: Our results identified a novel tumour-suppressive role of CPEB1 in CRC and found that hypermethylation of the CPEB1 promoter may lead to diminished expression due to decreased chromatin accessibility and transcription factor binding. Collectively, these results suggest a potential role for CPEB1 in the diagnosis and treatment of CRC.

Keywords: CEBPB; CPEB1; Chromatin accessibility; Colorectal cancer; Metastasis; Methylation; TFCP2.

The MoFe protein component of the nitrogenase enzyme complex is the substrate reducing site and contains two sets of symmetrically arrayed metallo centers called the P (Fe₈S₇) and the FeMoco (MoFe₇S₉-C-homocitrate) centers. The ATP-binding Fe protein is the specific reductant for the MoFe protein. Both symmetrical halves of the MoFe protein are thought to function independently during nitrogenase catalysis. Forming [AlF₄]^- transition-state complexes between the MoFe protein and the Fe protein of Azotobacter vinelandii ranging from 0 to 2 Fe protein/MoFe protein produced a series of complexes whose specific activity decreases with increase in bound Fe protein/MoFe protein ratio. Reduction of 2H⁺ to H₂ was inhibited in a linear manner with an x-intercept at 2.0 with increasing Fe protein binding, whereas acetylene reduction to ethylene decreased more rapidly with an x-intercept near 1.5. H⁺ reduction is a distinct process occurring independently at each half of the MoFe protein but acetylene reduction decreases more rapidly than H⁺ reduction with increasing Fe protein/MoFe protein ratio, suggesting that a response is transmitted between the two αβ halves of the MoFe protein for acetylene reduction as Fe protein is bound. A mechanistic model is derived to investigate this behavior. The model predicts that each site functions independently for 2H⁺ reduction to H₂. For acetylene reduction, the model predicts positive (synchronous) not negative cooperativity arising from acetylene binding to both sites before substrate reduction occurs. When this model is applied to inhibition by Cp2 and modified Av2 protein (L127∆) that form strong, non-dissociable complexes, positive cooperativity is absent and each site acts independently. The results suggest a new paradigm for the catalytic function of the MoFe protein during nitrogenase catalysis.

Keywords: Kinetic model; MoFe protein; Nitrogenase; Positive cooperativity; Synchronous kinetics; Transition-state complex.

Reaction of [Mo2 Cp2 (μ-κ1 :κ1 ,η6 -PMes*)(CO)2 ] with S or Se followed by protonation with [H(OEt2 )2 ](BAr'4 ) gave the cationic derivatives [Mo2 Cp2 {μ-κ2P,E :κ1P ,η5 -EP(C6 H3 tBu3 )}(CNR)(CO)2 ](BAr'4 ) (E=S; R=tBu, iPr, Ph, 4-C6 H4 OMe, Xyl; or E=Se; R=tBu; Ar'=3,5-C6 H3 (CF3 )2 ). Reaction of the latter with K[BHsBu3 ] yielded the aldimine complexes [Mo2 Cp2 {μ-κ2P,E :κ2P,N ,η4 -SP(C6 H3 tBu3 (CHNR))}(CO)2 ] and their aminocarbene isomers [Mo2 Cp2 {μ-κ2P,E :κ2P,C ,η4 -SP(C6 H3 tBu3 (NRCH))}(CO)2 ] (R ≠ Xyl), following C-C and C-N couplings, respectively. Monitoring of these reactions revealed that the initial H- attack takes place at a Cp ligand to give cyclopentadiene intermediates [Mo2 Cp{μ-κ2P,S :κ1P ,η5 -SP(C6 H3 tBu3 )}(η4 -C5 H6 )(CNR)(CO)2 ], which then undergo C-H oxidative addition to give the hydride isomers [Mo2 Cp2 {μ-κ2P,S :κ1P ,η3 -SP(C6 H3 tBu3 )}(H)(CNR)(CO)2 ]. In turn, the latter rearrange to give the aldimine and aminocarbene complexes. DFT calculations revealed that the hydride intermediates first undergo migratory insertion of the isocyanide ligand into the Mo-H bond to give unobservable formimidoyl intermediates, which then evolve either by nucleophilic attack of the N atom on the C6 ring (C-N coupling) or by migratory insertion of the formimidoyl ligand into the C6 ring (C-C coupling). Our data suggest that increasing the size of the substituent R at the isocyanide ligand destabilizes the aldimine isomer to a greater extent, thus favoring formation of the aminocarbene complex.

Herein we describe different C-C coupling reactions of permethyltitanocene and -zirconocene with disubstituted 1,3-butadiynes. The outcomes of these reactions vary depending on the metals and the diyne substituents. The reduction of [Cp2*MCl2] (Cp* = C5Me5; M = Ti, Zr) with Mg in the presence of disubstituted butadiynes RC triple bond C-C triple bond CR' is suitable for the synthesis of different C-C coupling products of the diyne and the permethylmetallocenes, and provides a new method for the generation of functionalized pentamethyl-cyclopentadienyl derivatives. For M = Zr and R = R' = tBu, the reaction gives, by a twofold activation of one pentamethylcyclopentadienyl ligand, the complex [Cp*Zr[-C(=C=CHtBu)-CHtBu-CH2-eta5-C5Me3-CH2-]] (3), containing a fulvene ligand that is coupled to the modified substrate (allenic subunit). When using the analogous permethyltitanocene fragment "Cp2*Ti", the reaction depends strongly on the substituents R and R'. The coupling product of the butadiyne with two methyl groups of one of the pentamethylcyclopentadienyl ring systems, [Cp*Ti[eta5-C5Me3-(CH2-CHR-eta2-C2-CHR'-CH2)]], is obtained with R = R' = tBu (4) and R = tBu, R' = SiMe3 (5). In these complexes one pentamethylcyclopentadienyl ligand is annellated to an eight-membered ring with a C-C triple bond, which is coordinated to the titanium center. A different activation of both pentamethylcyclopentadienyl ligands is observed for R = R' = Me, resulting in the complex [[eta5-C5Me4(CH2)-]Ti[-C(=CHMe)-C(=CHMe)-CH2-eta5-C5Me4]] (6), which displays a fulvene as well as a butadienyl-substituted pentamethylcyclopentadienyl ligand. The influence exerted by the size of the metal is illustrated in the reaction of [Cp2*ZrCl2] with MeC triple bond C-C triple bond CMe. Here the five-membered metallacyclocumulene complex [Cp2*Zr(eta4-1,2,3,4-MeC4Me)] (7) is obtained. The reaction paths found for R = R' = Me are identical to those formerly described for R = R' = Ph.

The reaction of the allene precursor Li2 (Me3 SiC3 SiMe3 ) with [Cp2 ZrCl2 ] (Cp=cyclopentadienyl) was examined. The selective formation of hitherto unknown linear, allene-bridged dizirconocene complexes [(Cp2 ZrCl)2 {-μ-(Me3 Si)C3 (SiMe3 )-}] and [(Cp2 Zr)2 {-μ-(Me3 Si)C3 (SiMe3 )-}2 ] was observed. Upon σ coordination of the allenediyl unit to {Cp2 Zr}, pyrophoric Li2 (Me3 SiC3 SiMe3 ) is tamed stepwise to yield a surprisingly robust 1,5-dizirconacyclooctatetra-2,3,6,7-ene with cumulated double bonds. This complex is unexpectedly inert against moisture, air, water and acetone. Surprisingly, it degrades under MS conditions to give the highly strained 1-zirconacyclobuta-2,3-diene. All compounds isolated have been fully characterised and the molecular structures are discussed. The stability and reactivity of these complexes are rationalised by DFT computations.

A series of two-dimensional (2D) coordination polymers (CPs), namely poly[[bis(μ-acetato)diaqua(μ₆-biphenyl-3,3',5,5'-tetracarboxylato)bis(N,N-dimethylacetamide)digadolinium(III)] N,N-dimethylacetamide monosolvate], {[Gd₂(C₁₆H₆O₈)(C₂H₃O₂)₂(C₄H₉NO)₂(H₂O)₂]·C₄H₉NO}_n (CP1), poly[[bis(μ-acetato)diaqua(μ₆-biphenyl-3,3',5,5'-tetracarboxylato)bis(N,N-dimethylacetamide)didysprosium(III)] N,N-dimethylacetamide monosolvate], {[Dy₂(C₁₆H₆O₈)(C₂H₃O₂)₂(C₄H₉NO)₂(H₂O)₂]·C₄H₉NO}_n (CP2), poly[bis(μ-acetato)diaqua(μ₆-biphenyl-3,3',5,5'-tetracarboxylato)bis(N,N-dimethylacetamide)dineodymium(III)], [Nd₂(C₁₆H₆O₈)(C₂H₃O₂)₂(C₄H₉NO)₂(H₂O)₂]_n (CP3), poly[bis(μ-acetato)diaqua(μ₆-biphenyl-3,3',5,5'-tetracarboxylato)bis(N,N-dimethylacetamide)disamarium(III)], [Sm₂(C₁₆H₆O₈)(C₂H₃O₂)₂(C₄H₉NO)₂(H₂O)₂]_n (CP4), has been synthesized from rigid biphenyl-3,3',5,5'-tetracarboxylic acid under solvothermal conditions. Their structures have been determined by single-crystal X-ray diffraction analyses, elemental analyses, IR spectra, powder X-ray diffraction and thermogravimetric analyses, and CP1-CP4 crystallize in the monoclinic space group P2₁/n. CP1-CP4 are isomorphous and feature similar 2D double layers, which are further extended via interlayer hydrogen-bonding interactions into a three-dimensional (3D) supramolecular structure. Hydrogen-bonding interactions between N,N-dimethylacetamide molecules and carboxylate O atoms strengthen the packing of the layers. The organic ligands interconnect with metal ions to generate 2D layered structures with a (4,4)-connected net having {4⁴.6²} topology. CP1 has been investigated for its magnetic properties and magnetic susceptibility measurements were carried out in the range 2.0-300 K. The results of the magnetic measurements show weak antiferromagnetic coupling between the Gd^III ions in CP1. Moreover, the strong luminescence of CP2 and CP4 can be selectively quenched by the Fe³⁺ ion and toxic solvents (e.g. acetone).

A zirconium/nickel-mediated one-pot synthesis of ketones is reported. In the presence of Zn or Mn, Cp2 ZrCl2 was found to dramatically accelerate the coupling and suppress side product formation via an I→SPy displacement at the same time. Unlike Zn/Pd- and Fe/Cu-mediated one-pot ketone syntheses, the new method is effective for nucleophiles bearing OR or equivalent functional groups at the α-position. A mechanism comprising a nickel catalytic cycle, a zirconium catalytic cycle, and Zr→Ni transmetalation is proposed, and Cp2 ZrCl2 and/or low-valent Zr species are suggested to play crucial dual roles.

The mechanisms of polymerization of epsilon-caprolactone (CL) initiated by either the rare-earth hydride [Cp2Eu(H)] or the borohydrides [Cp2Eu(BH4)] or [(N2NN')Eu(BH4)] were studied at the DFT level (Cp=eta5-C5H5; N2NN'=(2-C5H4N)CH2(CH2CH2NMe)2). For all compounds the reaction proceeds in two steps: a hydride transfer from the rare earth initiator to the carbonyl carbon of the lactone, followed by ring-opening of the monomer. In the last step a difference is observed between the hydride and borohydride complexes, because for the latter the ring-opening is induced by an additional B-H bond cleavage leading to a terminal--CH2OBH2 group. This corresponds to the reduction by BH3 of the carbonyl group of CL. Upon reaction of [Cp2Eu(H)] with CL, the alkoxy-aldehyde complex produced, [Cp2Eu(O(CH2)5C(O)H)], is the first-formed initiating species. In contrast, for the reaction of CL with the borohydride complexes [(Lx)Eu(BH4)] (Lx=Cp2 or N2NN'), an aliphatic alkoxide with a terminal--CH2OBH2 group, [(Lx)Eu(O(CH2)6OBH2)] is formed and subsequently propagates the polymerization. The present DFT investigations are fully compatible with previously reported mechanistic studies of experimental systems.

OBJECTIVE

To investigate whether the change of serum tumor markers profile after chemotherapy in epithelial ovarian carcinoma and evaluate the clinical significance.

METHODS

The levels of CA(125), CA(19-9) and CP2 before and after initial surgery, during primary chemotherapy and follow-up were serially measured and analyzed retrospectively in 28 cases of recurrent epithelial ovarian carcinoma patients and 20 cases of primary chemo-resistant ovarian carcinoma patients from Jan 1999 to July 2007. According to whether the change of serum tumor markers profile, all the patients were divided into two groups: marker changed-group and marker un-changed group. The average follow up period was 25 months.

RESULTS

(1) The changes of tumor marker profile were included the number and(or) types of markers, which included 13 cases (46%, 13/28) of the recurred cases and 9 cases (45%, 9/20) of the primary chemo-resistant cases. (2) For recurrent ovarian carcinoma changed tumor marker profile, the highest pathology type was serous histological type (77%, 10/13), while was mucinous histological type (4/9) for primary chemo-resistant patients. (3) For recurred patients, the median progression-free survival (PFS) and median overall survival (OS) in marker changed-group (22.2 and 60.0 months) were significantly longer than that in marker un-changed group (17.4, 46.0 months; P < 0.05). For primary chemo-resistant ovarian carcinoma patients, median OS in marker changed-group (15.9 months) was significantly shorter than that in marker un-changed group (25.0 months; P < 0.05).

CONCLUSIONS

The profile of serum tumor makers in epithelial ovarian carcinoma may be changed after chemotherapy, which should be concomitantly determinate different serum tumor markers for monitoring the response to chemotherapy and follow-up of patients.

Studying the therapeutic effects of focal vibration (FV) in neurorehabilitation is the focus of current research. However, it is still not fully understood how FV on upper limb muscles affects the sensorimotor cortex in healthy subjects. To explore this problem, this experiment was designed and conducted, in which FV was applied to the muscle belly of biceps brachii in the left arm. During the experiment, electroencephalography (EEG) was recorded in the following three phases: before FV, during FV, and two minutes after FV. During FV, a significant lower relative power at C3 and C4 electrodes and a significant higher connection strength between five channel pairs (Cz-FC1, Cz-C3, Cz-CP6, C4-FC6, and FC6-CP2) in the alpha band were observed compared to those before FV. After FV, the relative power at C4 in the beta band showed a significant increase compared to its value before FV. The changes of the relative power at C4 in the alpha band had a negative correlation with the relative power of the beta band during FV and with that after FV. The results showed that FV on upper limb muscles could activate the bilateral primary somatosensory cortex and strengthen functional connectivity of the ipsilateral central area (FC1, C3, and Cz) and contralateral central area (CP2, Cz, C4, FC6, and CP6). These results contribute to understanding the effect of FV over upper limb muscles on the brain cortical network.

The phosphide-bridged dimolybdenum complexes (H-DBU)[Mo2Cp2(mu-PR2)(CO)4] (R= Cy, Ph; DBU = 1,8-diazabicyclo[5.4.0.]undec-7-ene) react with p-benzoquinone to give the hemiquinone complexes [Mo(2)Cp2(OC6H4OH)(mu-PR2)(CO)4]. The latter experience facile homolytic cleavage of the corresponding Mo-O bonds and react readily at room temperature with HSPh or S2Ph2 to give the thiolate complexes [Mo2Cp2(mu-PCy2)(mu-SPh)(CO)4] or [Mo2Cp2(mu-PR2)(mu-SPh)(CO)2]. In contrast, PRH-bridged substrates experience overall insertion of quinone into the P-H bond to give the anionic compounds (H-DBU)[Mo(2)Cp2{mu-PR(OC6H4OH)}(CO)4], which upon acidification yield the corresponding neutral hydrides. The cyclohexyl anion experiences rapid nucleophilic displacement of the hemiquinone group by different anions ER- (ER = OH, OMe, OC4H5, OPh, SPh) to give novel anionic compounds (H-DBU)[Mo2Cp2{mu-PCy(ER)}(CO)4], which upon acidification yield the corresponding neutral hydrides. The structure of four of these hydride complexes [PPh(OC6H4OH), PCy(OH), PCy(OMe), and PCy(OPh) bridges] was determined by X-ray diffraction methods and confirmed the presence of cis and trans isomers in several of these complexes. In addition, it was found that the hydroxyphosphide anion [Mo2Cp2{mu-PCy(OH)}(CO)4]- displays in solution an unprecedented tautomeric equilibrium with its hydride-oxophosphinidene isomer [Mo2Cp2(mu-H){mu-PCy(O)}(CO)4]-.

The reaction of phenylmercuric chloride with an anionic dimanganese borylene (Li(+)[Cp2(CO)4Mn2B](-)) led to the formation of a trimetalloboride featuring the first reported bond between mercury and a non-cluster boron atom. Examination by (199)Hg NMR indicated (11)B-(199)Hg scalar coupling. Theoretical calculations indicated the nature of bonding to be σ-donation from a B-Mn π-orbital to Hg, in conjunction with weak Hgd→π* back-donation.

Caffeine is a common active adulterant found in illicit drugs of abuse, including coca paste (CP). CP is a smokable form of cocaine mainly consumed in South America, produced during the cocaine-extraction process. CP has high abuse liability and its chronic consumption induces severe sleep-wake alterations. However, the effect of CP on the sleep-wake cycle and the effect of the presence of caffeine as an adulterant remain unknown. We studied the effect of an acute intraperitoneal injection of 2.5 and 5 mg/kg of a representative CP sample adulterated with caffeine (CP1) on the rat sleep-wake cycle. Compared with saline, administration of CP1 induced an increase in wakefulness and a decrease in light (light sleep) and slow wave sleep that was larger than the effects produced by equivalent doses of cocaine. Compared with CP1, combined treatment with cocaine (5 mg/kg) and caffeine (2.5 mg/kg), a surrogate of CP1, elicited similar effects. In contrast, a nonadulterated CP sample (CP2) produced an effect that was not different from cocaine. Our data indicate that caffeine produces a significant potentiation of the wakefulness-promoting effect of cocaine, suggesting that caffeine should be explored as a causal agent of clinical symptoms observed in CP users.

In this study, two asymmetrical cyclopeptides (CP1 and CP2) were designed and synthesized. The self-assembly behaviors of the asymmetrical cyclopeptides at varying pHs were investigated in terms of transmission electron microscopy (TEM), circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy. It was found that the self-assembly of CP1 resulted in the formation of nanofibers with α-helix conformation, while CP2 self-assembled into well-ordered nanorods with anti-parallel β-sheet conformation. The strategy demonstrated here presents great potential for preparation of well-defined nanostructures via rationally designing the molecular structures of cyclopeptides.

Herein, we describe a reductive cross-coupling of alkynes and aryl iodides by using a novel catalytic system composed of a catalytic amount of palladium dichloride and a promoter precursor, hafnocene difluoride (Cp2 HfF2 , Cp=cyclopentadienyl anion), in the presence of a mild reducing reagent, a hydrosilane, leading to a one-pot preparation of trans-alkenes. In this process, a series of coupling reactions efficiently proceeds through the following three steps: (i) an initial formation of hafnocene hydride from hafnocene difluoride and the hydrosilane, (ii) a subsequent hydrohafnation toward alkynes, and (iii) a final transmetalation of the alkenyl hafnium species to a palladium complex. This reductive coupling could be chemoselectively applied to the preparation of trans-alkenes with various functional groups, such as an alkyl group, a halogen, an ester, a nitro group, a heterocycle, a boronic ester, and an internal alkyne.

Purpose: Cardiac power (CP) index is a product of mean arterial pressure (MAP) and cardiac output (CO). In aortic stenosis, however, MAP is not reflective of true left ventricular (LV) afterload. We evaluated the utility of a gradient-adjusted CP (GCP) index in predicting survival after transcatheter aortic valve replacement (TAVR), compared to CP alone.

Materials and methods: We included 975 patients who underwent TAVR with 1 year of follow-up. CP was calculated as (CO×MAP)/[451×body surface area (BSA)] (W/m²). GCP was calculated using augmented MAP by adding aortic valve mean gradient (AVMG) to systolic blood pressure (CP1), adding aortic valve maximal instantaneous gradient to systolic blood pressure (CP2), and adding AVMG to MAP (CP3). A multivariate Cox regression analysis was performed adjusting for baseline covariates. Receiver operator curves (ROC) for CP and GCP were calculated to predict survival after TAVR.

Results: The mortality rate at 1 year was 16%. The mean age and AVMG of the survivors were 81±9 years and 43±4 mm Hg versus 80±9 years and 42±13 mm Hg in the deceased group. The proportions of female patients were similar in both groups (p=0.7). Both CP and GCP were independently associated with survival at 1 year. The area under ROCs for CP, CP1, CP2, and CP3 were 0.67 [95% confidence interval (CI), 0.62-0.72], 0.65 (95% CI, 0.60-0.70), 0.66 (95% CI, 0.61-0.71), and 0.63 (95% CI 0.58-0.68), respectively.

Conclusion: GCP did not improve the accuracy of predicting survival post TAVR at 1 year, compared to CP alone.

Keywords: Aortic valve stenosis; hemodynamics; mortality; transcatheter aortic valve replacement.

With the pressure to reduce antibiotics use in poultry production, cost-effective alternative products need to be developed to enhance the bird's immunity. The present study evaluated the efficacy of cranberry fruit by-products to modulate immunity in broiler chickens. Broiler Cobb 500 chicks were fed a control basal diet, basal diet supplemented with bacitracin (BACI, 55 ppm), cranberry pomace at 1% and 2% (CP2), or cranberry pomace ethanolic extract at 150 and 300 ppm (COH300) for 30 d. Blood sera were analyzed at days 21 and 28 of age for Ig levels by ELISA. The innate and adaptive immune-related gene expression levels in the liver and bursa of Fabricius were investigated at 21 d of age by quantitative polymerase chain reaction arrays. At day 21, the highest IgY level was found in the blood serum of the CP2-fed birds. In the liver, 13 of the 22 differentially expressed genes were downregulated across all treatments compared with the control. Expression of genes belonging to innate immunity such as caspase 1 apoptosis-related cysteine peptidase, chemokine receptor 5, interferon gamma, myeloid differentiation primary response gene 88, and Toll-like receptor 3 were significantly downregulated mainly in BACI- and COH300-fed birds. In the bursa, 5 of 9 genes associated with the innate immunity were differentially expressed. The expression of anti-inflammatory IL-10 gene was upregulated in all treatment groups in bursa compared with the control. The expression of transferrin gene was significantly upregulated in livers of birds fed COH300 and in bursa of birds fed BACI, indicating feeding practices and organ-dependant modulation of this gene in broiler. Overall results of this study showed that cranberry product feed supplementation modulated the innate immune and suppressed proinflammatory cytokines in broilers, providing a platform for future investigations to develop berry products in poultry feeding.

Keywords: broiler; cranberry pomace; liver and bursa immunity; serum Ig.

The chemical investigation on endophytic fungus Annulohypoxylon cf. stygium in leaves of Anoectochilus roxburghii (Wall.) Lindl. Sixteen compounds were isolated and their structures were identified as palmarumycin CP2 ( 1 ), (-)-(3R)-mellein methyl ethe ( 2 ), kipukasins D ( 3 ), kipukasins E ( 4 ), secosterigmatocystin ( 5 ), notoamide D ( 6 ), notoamide C ( 7 ), (+)-versicolamide B ( 8 ), (-)-notoamide A ( 9 ), (-)-notoamide B ( 10 ), diorcinal ( 11 ), stephacidin A ( 12 ), sterigmatocystin ( 13 ), versiconol ( 14 ), dihydrosterigmatocystin ( 15 ), and averufanin ( 16 ) respectively by spectroscopic analysis and comparison with literature data. All the compounds were isolated from Annulohypoxylon genus for the first time. Compounds 1 and 13 showed selective cytotoxic activities against HepG2, Hela, MCF-7 and HT-29.

Keywords: Annulohypoxylon cf. stygium; Cytotoxic activity; Endophytic fungus.

T4 genotype Acanthamoeba are opportunistic pathogens that cause two types of infections, including vision-threatening Acanthamoeba keratitis (AK) and a fatal brain infection known as granulomatous amoebic encephalitis (GAE). Due to the existence of ineffective treatments against Acanthamoeba, it has become a potential threat to all contact lens users and immunocompromised patients. Metal nanoparticles have been proven to have various antimicrobial properties against bacteria, fungi, and parasites. Previously, different types of cobalt nanoparticles showed some promise as anti-acanthamoebic agents. In this study, the objectives were to synthesize and characterize the size, morphology, and crystalline structure of cobalt phosphate nanoparticles, as well as to determine the effects of different sizes of cobalt metal-based nanoparticles against A. castellanii. Cobalt phosphate octahydrate (CHP), Co₃(PO₄)₂•8H₂O, was synthesized by ultrasonication using a horn sonicator, then three different sizes of cobalt phosphates Co₃(PO₄)₂ were produced through calcination of Co₃(PO₄)₂•8H₂O at 200 °C, 400 °C and 600 °C (CP2, CP4, CP6). These three types of cobalt phosphate nanoparticles were characterized using a field emission scanning electron microscope (FESEM), energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction (XRD) analysis. Next, the synthesized nanoparticles were subjected to biological assays to investigate their amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects against A. castellanii, as well as cell cytotoxicity. The overall results showed that 1.30 ± 0.70 µm of CHP microflakes demonstrated the best anti-acanthemoebic effects at 100 µg/mL, followed by 612.50 ± 165.94 nm large CP6 nanograins. However, amongst the three tested cobalt phosphates, Co₃(PO₄)₂, the smaller nanoparticles had stronger antiamoebic effects against A. castellanii. During cell cytotoxicity analysis, CHP exhibited only 15% cytotoxicity against HeLa cells, whereas CP6 caused 46% (the highest) cell cytotoxicity at the highest concentration, respectively. Moreover, the composition and morphology of nanoparticles is suggested to be important in determining their anti-acathamoebic effects. However, the molecular mechanisms of cobalt phosphate nanoparticles are still unidentified. Nevertheless, the results suggested that cobalt phosphate nanoparticles hold potential for development of nanodrugs against Acanthamoeba.

Lettuce big-vein disease (BVD) is a serious virus disease of lettuce. Recent evidence has brought into question the role of Lettuce big-vein virus (LBVV) in the etiology of BVD, and suggested that Mirafiori lettuce virus (MiLV) and not LBVV is the causal agent of BVD (1,2). Lettuce plants (Lactuca sativa L.) with symptoms similar to those of BVD were observed during the winter of 2003 in open-field and hydroponic-grown lettuce plants located in the Chacabuco Province of central Chile. Symptomatic plants exhibited leaves with chlorotic vein banding that became ruffled and distorted. Symptoms were usually accompanied by reduced plant size and absence of head formation. Roots from symptomatic plants were analyzed by light microscopy-acid fuchsin staining. Zoosporangia and resting spores of Olpidium brassicae were identified on the basis of their morphology and structure. Additionally, soil transmission experiments were performed with 50 healthy lettuce seedlings replanted into contaminated soil collected from lettuce fields having symptomatic crops. After 3 weeks, one-half of the seedlings showed differing degrees of big-vein symptoms, and the presence of spores of O. brassicae was confirmed in the roots by light microscopy. Seedlings raised in sterilized soil showed no symptoms after the same period of time. On the basis of nucleotide sequences of LBVV and MiLV from the GenBank database, primers specific to the coat protein genes of each virus were designed as follows: MiLVV-CP1: 5'-CAAATCTGTCCACAATTCC-3'; MiLVV-CP2: 5'-TCTCACTTGAAAACCTTCC-3'; MiLVV-CP3: 5'-TTGCAACGTGATGAAACC-3'; MiLVV-CP4: 5'-AAAGAAGAGAAGCCTGTTCC-3'; LBVV-CP1: 5'-AAGCTTTCCGTACTGTCC-3'; LBVV-CP2: 5'-CCTTGATACAGTTTTTGACC-3'; LBVV-CP3: 5'-GTATGCTGATTTCTGTAGACC-3'; LBVV-CP4: 5'-TAGATGTTCTTCGCCACC-3'. The primers were used in reverse transcription-polymerase chain reaction (RT-PCR) assays with dsRNA as a template. Amplicons of the expected size were obtained in each of the five symptomatic plants analyzed, using each of the designated primer sets: MiLVV-CP1/MiLVV-CP2: 562 bp; MiLVV-CP3/MiLVV-CP4: 743 bp; LBVV-CP1/LBVV-CP2: 485 bp; and LBVV-CP3/ LBVV-CP4: 570 bp. No amplicons were obtained from healthy lettuce plants. The identity of both viruses was verified by cloning and sequencing of the amplicons. Nucleotide sequences were compared with those in the GenBank database. Sequences derived from the Chilean isolates resulted in identities of 87 to 97% for MiLV and 97 to 99% for LBVV. All samples analyzed were from the Chacabuco Province where 43% of the lettuce crops in Chile are grown. Thus, the impact that BVD may have on lettuce availability for local consumption may be significant. To our knowledge, this is the first report of Lettuce big-vein disease and Mirafiori lettuce virus infecting lettuce and the first report of BVD in Chile. References: (1) H. Lot et al. Phytopathology 92:288, 2002. (2) P. Roggero et al. Eur. J. Plant Pathol. 109:261, 2003.

Water-soluble polysaccharides were isolated from Colpomenia peregrina to determine their chemical characteristics and immunomodulatory properties. High extraction yields were obtained for CP1 (17.6%) and CP2 (5.2%) polysaccharides. Polysaccharides were mainly consisted of neutral sugars (67.01-73.79%), uronic acids (9.43-14.89%), proteins (3.44-14.89%) and small amounts of sulfates (4.87-4.91%). Polysaccharides were composed of fucose (20.62-24.56%), galactose (25.5-26.94%) and glucose (50.00-52.91%) residues. The average molecular weights of the CP1 and CP2 polysaccharides were 1890 × 103 g/mol and 639 × 103 g/mol, respectively. The polysaccharides exerted a relatively low cytotoxicity against HeLa cancer cells (< 40%). The CP1 and CP2 polysaccharides were nontoxic and induced RAW264.7 murine macrophage cells to release considerable amounts of nitric oxide (NO). Inflammatory cytokines including IL-1β, TNF-α, IL-6, IL-10 and IL-12 from were secreted from RAW264.7 cells induced with CP1 polysaccharides. As the most immunostimulating fraction, CP1 polysaccharides were homogeneous and formed of 1,3-linked galactose, 1,4-linked glucose and 1,3-linked fucose residues. Overall, these findings suggested that the polysaccharides isolated from C. peregrina can be utilized as potential natural immunostimulant in functional foods or pharmaceutical industries.

Mycobacterium tuberculosis (Mtb) is the pathogen responsible for tuberculosis, a leading cause of illness and death worldwide. Growing evidence suggests that the proinflammatory cytokine IL-32 plays a major role in host defences against pathogens such as Mtb. IL-32 exists in six alternatively spliced isoforms, but antituberculosis effects have been reported only for some of them. In this study, we examined the effect of all six IL-32 isoforms on Mtb replication in the murine macrophage cell line RAW 264.7. Compared with cells transfected with the other isoforms, IL-32ε-transfected cells exhibited the strongest antituberculosis effect and the highest rate of Mtb-induced apoptosis. Of note, this apoptosis pathway was independent of caspase-3 activation. Instead, N-Myc interactor (NMI), an inhibitor of Wnt signalling, was a key player in IL-32ε-mediated apoptosis by inhibiting Wnt/β-catenin signalling and thereby activating c-Myc-mediated apoptosis. Moreover, we identified two cis-acting elements that are binding sites for the transcriptional regulators paired box 6 (PAX6) and transcription factor CP2 (TFCP2) in the promoter of NMI and these elements proved essential for IL-32ε-induced upregulation of Nmi expression. Furthermore, IL-32ε-mediated activation of the mitogen-activated protein kinase p38 also contributed to NMI upregulation. In conclusion, our results demonstrate that Mtb infection-induced IL-32ε-mediated apoptosis in macrophages plays a key role in host defences against Mtb.