Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships [published erratum appears in Mol Biol Evol 1991 May;8(3):398]

The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down- weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird- crocodilian clade. Separate analyses of four other genes (alpha- crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.      

Rilonacept in the treatment of acute gouty arthritis: a randomized,controlled clinical trial using indomethacin as the active comparator

Introduction

In phase-3 clinical trials, the interleukin (IL-1) blocker, rilonacept (IL-1 Trap), demonstrated efficacy for gout flare prevention during initiation of urate-lowering therapy. This trial evaluated rilonacept added to a standard-of-care, indomethacin, for treatment of acute gout flares.

Methods

Adults, aged 18-70 years, with gout presenting within 48 hours of flare onset and having at least moderate pain as well as swelling and tenderness in the index joint were randomized to subcutaneous (SC) rilonacept 320 mg at baseline plus oral indomethacin 50 mg TID for 3 days followed by 25 mg TID for up to 9 days (n = 74); SC placebo at baseline plus oral indomethacin as above (n = 76); or SC rilonacept 320 mg at baseline plus oral placebo (n = 75). The primary efficacy endpoint was change in pain in the index joint (patient-reported using a Likert scale (0 = none; 4 = extreme)) from baseline to the average of values at 24, 48 and 72 hours (composite time point) for rilonacept plus indomethacin versus indomethacin alone. Comparison of rilonacept monotherapy with indomethacin monotherapy was dependent on demonstration of significance for the primary endpoint. Safety evaluation included clinical laboratory and adverse event (AE) assessments.

Results

Patient characteristics were comparable among the groups; the population was predominantly male (94.1%), white (75.7%), with mean ± SD age of 50.3 ± 10.6 years. All treatment groups reported within-group pain reductions from baseline (P < 0.0001). Although primary endpoint pain reduction was greater with rilonacept plus indomethacin (-1.55 ± 0.92) relative to indomethacin alone (-1.40 ± 0.96), the difference was not statistically significant (P = 0.33), so formal comparison between monotherapy groups was not performed. Pain reduction over the 72-hour period with rilonacept alone (-0.69 ± 0.97) was less than that in the other groups, but pain reduction was similar among groups at 72 hours. Treatment with rilonacept was well-tolerated with no reported serious AEs related to rilonacept. Across all groups, the most frequent AEs were headache and dizziness.

Conclusions

Although generally well-tolerated, rilonacept in combination with indomethacin and rilonacept alone did not provide additional pain relief over 72 hours relative to indomethacin alone in patients with acute gout flare.

Trial registration

ClinicalTrials.gov registration number {"type":"clinical-trial","attrs":{"text":"NCT00855920","term_id":"NCT00855920"}}NCT00855920.     

COPI–TRAPPII activates Rab18 and regulates its lipid droplet association

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII‐specific subunits by various methods including siRNA depletion and CRISPR–Cas9‐mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII‐deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD. We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.     

Correlation between delta-endotoxin and proteolytic activities produced by Bacillus thuringiensis var. kurstaki growing in an economic production medium

Abstract

Bacillus thuringiensis is a Gram positive bacterium that produces an insecticidal crystalline protein making it one of the most important biocontrol agents for pest management. Bioinsecticides based on B. thuringiensis were produced by fermentation processes in liquid media. Cultural conditions controlling proteolytic activities in different culture media were investigated to study the possible correlations between B. thuringiensis production of proteases and delta-endotoxins in a low-cost complex medium. Aeration appeared to play an important role in delta-endotoxin production. The correlation between proteolytic activity and aeration does not seem to be reliable. A negative correlation (correlation coefficient =? 0.774) was established between protease activity and delta-endotoxin production. In order to prove this correlation, protease hypo-producing and overproducing mutants were isolated through random mutagenesis of two wild strains, BUPM13 and BUPM5, by using nitrous acid. Interestingly, delta-endotoxin production of BUPM13-1, BUPM13-2 and BUPM13-3 was markedly improved when compared to the wild strain BUPM 13, reaching 2.1-fold, 3.69-fold and 8.13-fold, respectively. Maximal protease activity (540-2468 UI) obtained by BUPM5-1 and BUPM5-2 was 2.34-fold and 10.7-fold, respectively, more than that obtained by the wild strain BUPM5 with a drastic decrease of their delta-endotoxin production. Study of delta-endotoxin production by the selected mutants confirmed that insecticidal crystal protein stability in the culture strongly depends on the level of endogenous protease activity. This was also confirmed by bioassays measuring the LC₅₀ using larvae of Ephestia kuehniella. Determining protease activity in fermentation culture could be useful in indirectly predicting the potency of B. thuringiensis strains with high insecticidal activities. This would allow low-cost selection of overproducing wild isolates or mutants in the screening programmes for the reduction of production cost, which is important from a practical point of view.     

Evidence for independent recruitment of zeta-crystallin/quinone reductase (CRYZ) as a crystallin in camelids and hystricomorph rodents

Zeta-crystallin/quinone reductase (CRYZ) is an NADPH oxidoreductase expressed at very high levels in the lenses of two groups of mammals: camelids and some hystricomorph rodents. It is also expressed at very low levels in all other species tested. Comparative analysis of the mechanisms mediating the high expression of this enzyme/crystallin in the lens of the Ilama (Lama guanacoe) and the guinea pig (Cavia porcellus) provided evidence for independent recruitment of this enzyme as a lens crystallin in both species and allowed us to elucidate for the first time the mechanism of lens recruitment of an enzyme- crystallin. The data presented here show that in both species such recruitment most likely occurred through the generation of new lens promoters from nonfunctional intron sequences by the accumulation of point mutations and/or small deletions and insertions. These results further support the idea that recruitment of CRYZ resulted from an adaptive process in which the high expression of CRYZ in the lens provides some selective advantage rather than from a purely neutral evolutionary process.      

Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer

The HER-2/neu transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target for breast cancer. Accurate determination of HER-2/neu status is a prerequisite for selecting breast tumors for HER-2/neu immunotherapy or for taxan based chemotherapy. Unfortunately, there is no consensus concerning how this determination should be reached. We compared assessment of HER-2/neu status using Multiplex ligation-dependent probe amplification (MLPA) and immunohistochemistry (IHC). The patient group comprised 60 Indonesian breast cancers patients. IHC was performed on paraffin sections using the CB11 antibody from Novocastra. Results were scored according to the Hercept test. For MLPA, DNA was extracted from frozen samples, PCR amplified with a probe set containing three hemi-primer sets for the HER-2 locus and another nine control probes spread over chromosome 17 and other chromosomes, and analyzed on a gene scanner. A ratio above two for at least two HER-2 locus probes compared to the control probes was regarded as amplification. IHC for HER-2/neu was negative in 36 cases, and 24 cases (40%) showed expression. Seven, eight and nine of the latter cases were 1+, 2+ and 3+ positive, respectively. Forty-seven cases showed no amplification by MLPA, and 13 cases (22%) were amplified. Comparison of IHC and MPLA showed that none of the 36 IHC-negative or seven IHC 1+ cases was amplified. Five of the eight (63%) 2+ cases were amplified, and eight of nine (89%) of the IHC 3+ tumors showed gene amplification by MLPA assay. For HER-2/neu, there is a good correlation between gene amplification detected by MLPA and overexpression by IHC in invasive breast cancer. It appears that MLPA can detect the HER-2 amplified cases in the IHC 2+ class. Because MLPA is quick and inexpensive, it is an attractive method for detecting HER-2/neu amplification in daily laboratory practice.     

Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.      

Improvement of Bacillus thuringiensis bioinsecticide production by sporeless and sporulating strains using response surface methodology

Statistical experimental designs, involving a Plackett-Burman design followed by a rotatable central composite design were used to optimize the culture medium constituents for Bacillus thuringiensis bioinsecticide production. This was carried out by using firstly an asporogenic strain and extrapolated to some sporeless and sporulating strains. Initial screening of production parameters was performed and the variables with statistically significant effects on delta-endotoxin production were identified: glucose, glycerol, yeast extract and MnSO(4). These variables were selected for further optimization by response surface methodology. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 22.5 g/l of glucose, 4.8g/l of glycerol, 5.8 g/l of yeast extract and 0.008 g/l of MnSO(4). Under these conditions, delta-endotoxin production was 2,130 and 2,260 mg/l into 250 and 1,000 ml flask respectively, which represent more than 38% improvement in toxin production over the basal medium (1,636 mg/l). Such medium composition was shown to be suitable for overproducing delta-endotoxins by sporeless and sporulating strains.     

MEDIUM OPTIMIZATION OF ANTIFUNGAL ACTIVITY PRODUCTION BY Bacillus amyloliquefaciens USING STATISTICAL EXPERIMENTAL DESIGN

In order to overproduce biofungicides agents by Bacillus amyloliquefaciens BLB371, a suitable culture medium was optimized using response surface methodology. Plackett–Burman design and central composite design were employed for experimental design and analysis of the results. Peptone, sucrose, and yeast extract were found to significantly influence antifungal activity production and their optimal concentrations were, respectively, 20 g/L, 25 g/L, and 4.5 g/L. The corresponding biofungicide production was 250 AU/mL, corresponding to 56% improvement in antifungal components production over a previously used medium (160 AU/mL). Moreover, our results indicated that a deficiency of the minerals CuSO₄, FeCl₃ · 6H₂O, Na₂MoO₄, KI, ZnSO₄ · 7H₂O, H₃BO₃, and C₆H₈O₇ in the optimized culture medium was not crucial for biofungicides production by Bacillus amyloliquefaciens BLB371, which is interesting from a practical point of view, particularly for low-cost production and use of the biofungicide for the control of agricultural fungal pests.