Phase III randomized study of second line ADI-PEG 20 plus best supportive care versus placebo plus best supportive care in patients with advanced hepatocellular carcinoma.

Background: Arginine depletion is a putative target in hepatocellular carcinoma (HCC). HCC often lacks argininosuccinate synthetase, a citrulline to arginine-repleting enzyme. ADI-PEG 20 is a cloned arginine degrading enzyme-arginine deiminase-conjugated with polyethylene glycol. The goal of this study was to evaluate this agent as a potential novel therapeutic for HCC after first line systemic therapy. Methods and patients: Patients with histologically proven advanced HCC and Child-Pugh up to B7 with prior systemic therapy, were randomized 2 : 1 to ADI-PEG 20 18 mg/m2 versus placebo intramuscular injection weekly. The primary end point was overall survival (OS), with 93% power to detect a 4-5.6 months increase in median OS (one-sided α = 0.025). Secondary end points included progression-free survival, safety, and arginine correlatives. Results: A total of 635 patients were enrolled: median age 61, 82% male, 60% Asian, 52% hepatitis B, 26% hepatitis C, 76% stage IV, 91% Child-Pugh A, 70% progressed on sorafenib and 16% were intolerant. Median OS was 7.8 months for ADI-PEG 20 versus 7.4 for placebo (P = 0.88, HR = 1.02) and median progression-free survival 2.6 months versus 2.6 (P = 0.07, HR = 1.17). Grade 3 fatigue and decreased appetite occurred in <5% of patients. Two patients on ADI-PEG 20 had ≥grade 3 anaphylactic reaction. Death rate within 30 days of end of treatment was 15.2% on ADI-PEG 20 versus 10.4% on placebo, none related to therapy. Post hoc analyses of arginine assessment at 4, 8, 12 and 16 weeks, demonstrated a trend of improved OS for those with more prolonged arginine depletion. Conclusion: ADI-PEG 20 monotherapy did not demonstrate an OS benefit in second line setting for HCC. It was well tolerated. Strategies to enhance prolonged arginine depletion and synergize the effect of ADI-PEG 20 are underway. Clinical Trial number: www.clinicaltrials.gov (NCT 01287585).

RNA interference-aided knockdown of a putative saccharopine dehydrogenase leads to abnormal ecdysis in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

The brown planthopper Nilaparvata lugens is a serious phloem-feeding pest of rice in China. The current study focuses on a saccharopine dehydrogenase (SDH) that catalyzes the penultimate reaction in biosynthesis of the amino acid lysine (Lys), which plays a role in insect growth and carnitine production (as a substrate). The protein, provisionally designated as NlylsSDH [a SDH derived from yeast-like symbiont (YLS) in N. lugens], had a higher transcript level in abdomens, compared with heads, wings, legs and thoraces, which agrees with YLS distribution in N. lugens. Ingestion of Nlylssdh targeted double-stranded RNA (dsNlylssdh) for 5, 10 and 15 days decreased the mRNA abundance in the hoppers by 47, 70 and 31%, respectively, comparing with those ingesting normal or dsegfp diets. Nlylssdh knockdown slightly decreased the body weights, significantly delayed the development of females, and killed approximately 30% of the nymphs. Moreover, some surviving adults showed two apparent phenotypic defects: wing deformation and nymphal cuticles remained on tips of the legs and abdomens. The brachypterours/macropterours and sex ratios (female/male) of the adults on the dsRNA diet were lowered compared with the adults on diets without dsRNA. These results suggest that Nlylssdh encodes a functional SDH protein. The adverse effect of Nlylssdh knockdown on N. lugens implies the importance of Lys in hopper development. This study provides a proof of concept example that Nlylssdh could serve as a possible dsRNA-based pesticide for planthopper control.

The P450 enzyme Shade mediates the hydroxylation of ecdysone to 20-hydroxyecdysone in the Colorado potato beetle, Leptinotarsa decemlineata.

Ecdysone 20-monooxygenase (E20MO), a cytochrome P450 monooxygenase (CYP314A1), catalyses the conversion of ecdysone (E) to 20-hydroxyecdysone (20E). We report here the cloning and characterization of the Halloween gene Shade (Shd) encoding E20MO in the Colorado potato beetle, Leptinotarsa decemlineata. LdSHD has five conserved motifs typical of insect P450s, ie the Helix-C, Helix-I, Helix-K, PxxFxPE/DRF (PERF) and heme-binding motifs. LdShd was expressed in developing eggs, the first to fourth instars, wandering larvae, pupae and adults, with statistically significant fluctuations. Its mRNA was ubiquitously distributed in the head, thorax and abdomen. The recombinant LdSHD protein expressed in Spodoptera frugiperda 9 (Sf9) cells catalysed the conversion of E to 20E. Dietary introduction of double-stranded RNA (dsRNA) of LdShd into the second instar larvae successfully knocked down the LdShd expression level, decreased the mRNA level of the ecdysone receptor (LdEcR) gene, caused larval lethality, delayed development and affected pupation. Moreover, ingestion of LdShd-dsRNA by the fourth instars also down-regulated LdShd and LdEcR expression, reduced the 20E titre, and negatively influenced pupation. Introduction of 20E and a nonsteroidal ecdysteroid agonist halofenozide into the LdShd-dsRNA-ingested second instars, and of halofenozide into the LdShd-dsRNA-ingested fourth instars almost completely relieved the negative effects on larval performance. Thus, LdSHD functions to regulate metamorphotic processes by converting E to 20E in a coleopteran insect species Le. decemlineata.

Constructing the major biosynthesis pathways for amino acids in the brown planthopper, Nilaparvata lugens†Stål (Hemiptera: Delphacidae), based on the transcriptome data.

Nilaparvata lugens is a serious phloem-feeding pest of rice throughout Asia. Rice phloem sap can meet its nutrition requirement for sugars but not for some essential amino acids such as isoleucine, leucine, methionine, phenylalanine, tryptophan, lysine, arginine and histidine. N. lugens harbours yeast-like symbionts in mycetocytes formed by abdominal fat body cells. Removal of the symbionts results in negative physiological effects, suggesting that the symbionts play a pivotal role in the nitrogen metabolism. In the present paper, 521 mRNA expressed sequence tags (ESTs) encoding 126 enzymes that were involved in amino acid biosynthesis were identified based on a transcriptome data, reverse transcription (RT)-PCR and rapid amplification of cDNA ends. Similarity analysis, codon usage bias, along with tissue-biased expression and phylogenetic analysis of a subset of ESTs, suggest that 437 ESTs out of the 521 originate from symbionts, and the remaining 84 mRNA fragments come from N. lugens. Accordingly, the biosynthesis pathways for 20 amino acids were manually constructed. It is postulated that both N. lugens and its symbiont can independently assimilate ammonia and biosynthesize seven non-essential amino acids: glutamate; glutamine; aspartate; asparagine; alanine; serine; and glycine. N. lugens and symbiont enzymes may work collaboratively to catalyse the biosynthesis of proline, methionine, valine, leucine, isoleucine, phenylalanine and tyrosine. We infer from this that symbionts function in the biosynthesis of lysine, arginine, tryptophan, threonine, histidine and cysteine. Our data support the previously proposed hypothesis, i.e. the yeast-like symbionts compensate for, at least partially, the amino acid needs of N. lugens.

In vitro inhibitory effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri.

AIM: This study was conducted to evaluate the toxic effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri. METHODS AND RESULTS: Inhibitory effect of various concentrations of gossypol on the growth of E. ictaluri was determined. Bacterial recovery was performed by preincubation of bacteria in medium containing various concentrations of gossypol and subsequent activation of bacteria by inoculating on gossypol-free plates. Concentrations of racemic gossypol, (+)-gossypol and (-)-gossypol of 1.5 microg ml(-1) or higher significantly reduced the number of bacterial colonies compared with that of the control. The growth of E. ictaluri was completely inhibited on agar plates supplemented with 3 microg ml(-1), regardless of the forms of gossypol. The inhibitory effect of (+)-gossypol was higher than that of (-)-gossypol or gossypol-acetic acid. Recovery of E. ictaluri was <50% for all three forms of gossypol at concentrations of 5 microg ml(-1). Bacterial recovery remained relatively constant (6.5%) at gossypol concentrations from 10 to 100 microg ml(-1). Complete killing of E. ictaluri was not reached at gossypol levels up to 100 microg ml(-1). CONCLUSION: Gossypol-acetic acid, and (+)- and (-)-optical isomers have anti-bacterial effect against E. ictaluri. The results suggest the action is bacteriostatic rather than bactericidal. SIGNIFICANCE AND IMPACT OF THE STUDY: The therapeutic effect of gossypol against E. ictaluri may be useful in controlling enteric septicaemia of catfish.

Molecular weights and molecular-weight distributions from ultracentrifugation of nonideal solutions.

Ultracentrifugation, membrane osmometry and capillary viscometry experiments have been performed on two dextran samples, which have molecular-weight distributions (MWDs) similar to those of dextrans used as blood plasma extenders. The manufacturer reported values of Mn and MW, determined by end group analysis and by light scattering, respectively. Our values of Mn, determined by osmometry, and MW, calculated from ultracentrifugal and viscometry experiments, agreed quite well with the manufacturer's results. Good agreement was obtained with values of MW and BLS (the light scattering second virial coefficient) obtained from sedimentation equilibrium experiments at different speeds using sector or nonsector-shaped centerpieces. Several ways of obtaining MW, MZ and BLS from sedimentation equilibrium experiments are presented. We have also shown how to obtain the speed-dependent term of the sedimentation equilibrium second virial coefficient. Both BLS and the speed-dependent nonideal terms could be used to correct the sedimentation equilibrium data, so that ideal values of d in c/d(r2) or dc/d(r2) could be estimated and used to obtain the MWDs of the dextran samples. Both Donnelly's and Scholte's methods were used with the sedimentation equilibrium data. With both methods, unimodal MWDs were encountered, which gave good agreement with the manufacturer's MWDs, obtained by a combination of analytical gel chromatography and light scattering. Uncorrected sedimentation equilibrium data gave MWDs quite different from the manufacturer's results. The MWD calculated from the differential distribution of sedimentation coefficients also gave a unimodal MWD, but this MWD did not give a good agreement with the sedimentation equilibrium results or with the manufacturer's results.