Automation of macromolecular crystallography beamlines

The production of three-dimensional crystallographic structural information of macromolecules can now be thought of as a pipeline which is being streamlined at every stage from protein cloning, expression and purification, through crystallisation to data collection and structure solution. Synchrotron X-ray beamlines are a key section of this pipeline as it is at these that the X-ray diffraction data that ultimately leads to the elucidation of macromolecular structures are collected. The burgeoning number of macromolecular crystallography (MX) beamlines available worldwide may be enhanced significantly with the automation of both their operation and of the experiments carried out on them. This paper reviews the current situation and provides a glimpse of how a MX beamline may look in the not too distant future.     

Microsatellite DNA in Actinidia chinensis: isolation, characterisation, and homology in related species

 We have isolated and sequenced 263 microsatellite-containing clones from two small insert libraries of Actinidia chinensis enriched for (AC/GT) and (AG/CT) repeats, respectively. Primer pairs were designed for 203 microsatellite loci and successfully amplified from both plasmid and A. chinensis genomic DNA. In this paper we report the sequences of 40 primer pairs for which we have demonstrated Mendelian segregation in the progeny from controlled crosses. The polymorphism of ten microsatellites of each type was evaluated in four diploid and six tetraploid genotypes of A. chinensis. All microsatellites proved to be polymorphic, the number of alleles per locus detected in polyacrylamide sequencing gels ranging from 9 to 17. The high degree of polymorphism in Actinidia renders these markers useful either for mapping in A. chinensis or for fingerprinting cultivars of both domesticated kiwifruit species (A. chinensis and A. deliciosa). While most primer pairs produced single amplification products, about 20% generated banding patterns consistent with the amplification of two different loci. This supports the hypothesis that diploid species of Actinidia (2n=2x=58) are polyploid in origin with a basic chromosome number x=14/15 and that chromosome duplication may have occurred during the evolution of the genus. Finally, we have assayed the cross-species transportability of primer pairs designed from A. chinensis sequences and have found extensive cross-species amplification within the genus Actinidia; 75% of primer pairs gave successful amplification in the eight species assayed (A. arguta, A. rufa, A. polygama, A. chrysantha, A. callosa, A. hemsleyana, A. eriantha, and A. deliciosa), which are representative of the four sections into which the genus is currently split. Received: 14 February 1998 / Accepted: 26 May 1998     

Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.

Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved. Finally, placing E1A expression under the control of a strong promoter did not alter the requirement for E1B in the transformation of primary cells. One possibility is that the E1B 19K protein is required to overcome the cytotoxic effects of E1A protein expression and thereby enable primary cells to become transformed.     

Paternal inheritance of plastids in interspecific hybrids of the genus Actinidia revealed by PCR-amplification of chloroplast DNA fragments

RFLPs (restriction fragment length polymorphisms) of PCR (polymerase chain reaction) -amplified fragments were used to trace the pattern of plastid DNA inheritance in the genus Actinidia. A total of 51 progeny originating from interspecific crosses between three A. arguta cultivars and A. deliciosa, the kiwifruit, and 12 progeny originating from the cross between A. kolomikta and A. chinensis were analysed together with their parents. No reciprocal crosses could be tested since they all failed to set viable seeds. Attempts to rescue immature embryos failed in all cases as well. The A. arguta×A. deliciosa crosses were checked for the RFLP patterns of a sequence encoding part of the Rubisco large subunit (rbcL), using either AluI or MseI, and for a sequence encoding part of the photosystem II D1 protein (psbA), using HinfI. The A. kolomikta×A. chinensis cross was checked for the RFLP patterns of sequences encoding the spacers between trnT and the 5-trnL exon (a-b spacer DNA) and the trnL 3 exon and trnF (e-f spacer DNA), respectively. The first spacer revealed a natural polymorphism between the two parent species due to a large deletion occurring in A. kolomikta detectable without further restriction enzyme treatment. The e-f spacer DNA was digested with HinfI. The comparison of the RFLP patterns in the parents and their progeny showed a strictly paternal inheritance of chloroplast DNA in Actinidia, with no exception found in any of the crosses examined. As the reciprocal crosses were not available, we do not know whether paternal inheritance of plastids is restricted to the crosses we analysed or if this is the general rule for plastid inheritance in the genus Actinidia. Actinidia is dioecious and is the first purely outbreeding species for which a paternal plastid inheritance has so far been documented.     

Sex segregation ratio and gender expression in the genus Actinidia

The sex segregation ratio was checked in bi-parental families of Actinidia deliciosa (2n=6x=174) obtained by crossing four females (A12, Mo3, Br4, Hw1) with two males (T2, M1) and one fruiting male (M3h, subandroecious) according to a factorial mating design. The M3h fruiting male was also selfed. The sex ratio was checked in maternal families of A. kolomikta (2n=2x) and A. chinensis (2n=2x) as well as in A. deliciosa. Seedlings of both diploid species took 3–4 years to progress beyond juvenility, whereas a noticeable number of seedlings from biparental crosses of A. deliciosa involving A12 and Hw1 as seed parents were still non-flowering after seven growing seasons. Open-pollinated families of both diploid and hexaploid species as well as most families from biparental crosses showed a sex segregation ratio approaching 11. Subandroecious lines with different degrees of ovary and pistil development appeared in proportions of 0–4.2%, depending on the cross, but only 6 of the 2567 male vines checked were capable of setting fruit. No case of self-fertility or apomixis was detected among 1866 bagged female vines. Selfed M3h progenies gave only female and male phenotypes in a ratio of 1 female to 3 males. No off-type vines were found among these progenies. The same disomic sex segregation ratio seems to be operating at different ploidy levels in the genus Actinidia. Since selfed fruiting males produced both female and male individuals, the male sex appears to be the heterogametic one. Such evidence indicates that a monofactorial system based on one or more linked genes or on an X/Y chromosome set must be controlling sex expression. How a monofactorial sex-determining mechanism could operate in polyploids to give a 11 female: male ratio is discussed. Minor modifying gene(s) seem to be responsible for the feminization of males, and their expression appears enhanced by environmental conditions. Masculinizing gene(s) seem to be lacking in female genotypes.     

Lipid peroxidation contributes to immune reactions associated with alcoholic liver disease.

Increasing evidence indicates the involvement of immune reactions in the pathogenesis of alcoholic liver disease. We have investigated whether ethanol-induced oxidative stress might contribute to immune response in alcoholics. Antibodies against human serum albumin modified by reaction with malondialdehyde (MDA), 4-hydroxynonenal (HNE), 2-hexenal, acrolein, methylglyoxal, and oxidized arachidonic and linoleic acids were measured by ELISA in 78 patients with alcoholic cirrhosis and/or hepatitis, 50 patients with nonalcoholic cirrhosis, 23 heavy drinkers with fatty liver, and 80 controls. Titers of IgG-recognizing epitopes derived from MDA, HNE, and oxidized fatty acids were significantly higher in alcoholic as compared to nonalcoholic cirrhotics or healthy controls. No differences were instead observed in the titers of IgG-recognizing acrolein-, 2-hexenal-, and methylglyoxal-modified albumin. Alcoholics showing high IgG titers to one adduct tended to have high titers to all the others. However, competition experiments showed that the antigens recognized were structurally unrelated. Anti-MDA and anti-HNE antibodies were significantly higher in cirrhotics with more severe disease as well as in heavy drinkers with cirrhosis or extensive fibrosis than in those with fatty liver only. We conclude that antigens derived from lipid peroxidation contribute to the development of immune responses associated with alcoholic liver disease.     

Initial treatment choices to achieve sustained response in major depression: a systematic review and network meta‐analysis

Major depression is often a relapsing disorder. It is therefore important to start its treatment with therapies that maximize the chance of not only getting the patients well but also keeping them well. We examined the associations between initial treatments and sustained response by conducting a network meta‐analysis of randomized controlled trials (RCTs) in which adult patients with major depression were randomized to acute treatment with a psychotherapy (PSY), a protocolized antidepressant pharmacotherapy (PHA), their combination (COM), standard treatment in primary or secondary care (STD), or pill placebo, and were then followed up through a maintenance phase. By design, acute phase treatment could be continued into the maintenance phase, switched to another treatment or followed by discretionary treatment. We included 81 RCTs, with 13,722 participants. Sustained response was defined as responding to the acute treatment and subsequently having no depressive relapse through the maintenance phase (mean duration: 42.2±16.2 weeks, range 24‐104 weeks). We extracted the data reported at the time point closest to 12 months. COM resulted in more sustained response than PHA, both when these treatments were continued into the maintenance phase (OR=2.52, 95% CI: 1.66‐3.85) and when they were followed by discretionary treatment (OR=1.80, 95% CI: 1.21‐2.67). The same applied to COM in comparison with STD (OR=2.90, 95% CI: 1.68‐5.01 when COM was continued into the maintenance phase; OR=1.97, 95% CI: 1.51‐2.58 when COM was followed by discretionary treatment). PSY also kept the patients well more often than PHA, both when these treatments were continued into the maintenance phase (OR=1.53, 95% CI: 1.00‐2.35) and when they were followed by discretionary treatment (OR=1.66, 95% CI: 1.13‐2.44). The same applied to PSY compared with STD (OR=1.76, 95% CI: 0.97‐3.21 when PSY was continued into the maintenance phase; OR=1.83, 95% CI: 1.20‐2.78 when PSY was followed by discretionary treatment). Given the average sustained response rate of 29% on STD, the advantages of PSY or COM over PHA or STD translated into risk differences ranging from 12 to 16 percentage points. We conclude that PSY and COM have more enduring effects than PHA. Clinical guidelines on the initial treatment choice for depression may need to be updated accordingly.     

Selective publication of antidepressant trials and its influence on apparent efficacy: Updated comparisons and meta-analyses of newer versus older trials

BackgroundValid assessment of drug efficacy and safety requires an evidence base free of reporting bias. Using trial reports in Food and Drug Administration (FDA) drug approval packages as a gold standard, we previously found that the published literature inflated the apparent efficacy of antidepressant drugs. The objective of the current study was to determine whether this has improved with recently approved drugs.Methods and findingsUsing medical and statistical reviews in FDA drug approval packages, we identified 30 Phase II/III double-blind placebo-controlled acute monotherapy trials, involving 13,747 patients, of desvenlafaxine, vilazodone, levomilnacipran, and vortioxetine; we then identified corresponding published reports. We compared the data from this newer cohort of antidepressants (approved February 2008 to September 2013) with the previously published dataset on 74 trials of 12 older antidepressants (approved December 1987 to August 2002).Using logistic regression, we examined the effects of trial outcome and trial cohort (newer versus older) on transparent reporting (whether published and FDA conclusions agreed). Among newer antidepressants, transparent publication occurred more with positive (15/15 = 100%) than negative (7/15 = 47%) trials (OR 35.1, CI_95% 1.8 to 693). Controlling for trial outcome, transparent publication occurred more with newer than older trials (OR 6.6, CI_95% 1.6 to 26.4). Within negative trials, transparent reporting increased from 11% to 47%.We also conducted and contrasted FDA- and journal-based meta-analyses. For newer antidepressants, FDA-based effect size (ES_FDA) was 0.24 (CI_95% 0.18 to 0.30), while journal-based effect size (ES_Journals) was 0.29 (CI_95% 0.23 to 0.36). Thus, effect size inflation, presumably due to reporting bias, was 0.05, less than for older antidepressants (0.10).Limitations of this study include a small number of trials and drugs—belonging to a single class—and a focus on efficacy (versus safety).ConclusionsReporting bias persists but appears to have diminished for newer, compared to older, antidepressants. Continued efforts are needed to further improve transparency in the scientific literature.