Cell-cycle-specific activators of the Mec1/ATR checkpoint kinase

Mec1 [ATR (ataxia telangiectasia mutated- and Rad3-related) in humans] is the principle kinase responsible for checkpoint activation in response to replication stress and DNA damage in Saccharomyces cerevisiae. The heterotrimeric checkpoint clamp, 9-1-1 (checkpoint clamp of Rad9, Rad1 and Hus1 in humans and Ddc1, Rad17 and Mec3 in S. cerevisiae; Ddc1-Mec3-Rad17) and the DNA replication initiation factor Dpb11 (human TopBP1) are the two known activators of Mec1. The 9-1-1 clamp functions in checkpoint activation in G1- and G2-phase, but its employment differs between these two phases of the cell cycle. The Ddc1 (human Rad9) subunit of the clamp directly activates Mec1 in G1-phase, an activity identified only in S. cerevisiae so far. However, in G2-phase, the 9-1-1 clamp activates the checkpoint by two mechanisms. One mechanism includes direct activation of Mec1 by the unstructured C-terminal tail of Ddc1. The second mech-anism involves the recruitment of Dpb11 by the phosphorylated C-terminal tail of Ddc1. The latter mechanism is highly conserved and also functions in response to replication stress in higher eukaryotes. In S. cerevisiae, however, both the 9-1-1 clamp and the Dpb11 are partially redundant for checkpoint activation in response to replication stress, suggesting the existence of additional activators of Mec1.     

Effects of accessory proteins on the bypass of a cis-syn thymine-thymine dimer by Saccharomyces cerevisiae DNA polymerase eta

Among several hypotheses to explain how translesion synthesis (TLS) by DNA polymerase eta (pol eta) suppresses ultraviolet light-induced mutagenesis in vivo despite the fact that pol eta copies DNA with low fidelity, here we test whether replication accessory proteins enhance the fidelity of TLS by pol eta. We first show that the single-stranded DNA binding protein RPA, the sliding clamp PCNA, and the clamp loader RFC slightly increase the processivity of yeast pol eta and its ability to recycle to new template primers. However, these increases are small, and they are similar when copying an undamaged template and a template containing a cis-syn TT dimer. Consequently, the accessory proteins do not strongly stimulate the already robust TT dimer bypass efficiency of pol eta. We then perform a comprehensive analysis of yeast pol eta fidelity. We show that it is much less accurate than other yeast DNA polymerases and that the accessory proteins have little effect on fidelity when copying undamaged templates or when bypassing a TT dimer. Thus, although accessory proteins clearly participate in pol eta functions in vivo, they do not appear to help suppress UV mutagenesis by improving pol eta bypass fidelity per se.     

Impact of mucosal inflammation on cervical human immunodeficiency virus (HIV-1)-specific CD8 T-cell responses in the female genital tract during chronic HIV infection

The female genital tract is the major route of heterosexual human immunodeficiency virus (HIV) acquisition and transmission. Here, we investigated whether HIV-specific CD8 T-cell-mediated immune responses could be detected in the genital mucosa of chronically HIV-infected women and whether these were associated with either local mucosal HIV shedding or local immune factors. We found that CD8⁺ T-cell gamma interferon responses to Gag were detectable at the cervix of HIV-infected women but that the magnitude of genital responses did not correlate with those similarly detected in blood. This indicates that ex vivo HIV responses in one compartment may not be predictive of those in the other. We found that increased genital tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) levels correlated significantly with levels of Gag-specific CD8⁺ T cells at the cervix. Women who were detectably shedding virus in the genital tract had significantly increased cervical levels of TNF-α, IL-1β, IL-6, and IL-8 compared to women who were not detectably shedding virus. We were, however, unable to detect any association between the magnitude of cervical HIV-specific responses and mucosal HIV shedding. Our results support the hypothesis that proinflammatory cytokines in the female genital tract may promote HIV replication and shedding. In addition, we further show that inflammatory cytokines are associated with increased levels of HIV-specific CD8 effector cells at the genital mucosa but that these were not able to control genital HIV shedding.     

Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

Background  

Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.     

The rate of false positive sequence matches of peptides profiled by MALDI MS and identified by MS/MS

In most MALDI peptide profiling cases, sequencing is required to identify peptides of interest, preferentially by using different mass spectrometry techniques. Using identical samples, we determined the number of false positive matches in sequence of peptide identification using different mass spectrometers. This paper demonstrates that the reliability of the identification phase greatly benefits from concerted MS-technologies and determines the influence of mass accuracy, signal-to-noise and statistical score on peptide identification.     

Division of labor at the eukaryotic replication fork

DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon) are both required for efficient replication of the nuclear genome, yet the division of labor between these enzymes has remained unclear for many years. Here we investigate the contribution of Pol delta to replication of the leading and lagging strand templates in Saccharomyces cerevisiae using a mutant Pol delta allele (pol3-L612M) whose error rate is higher for one mismatch (e.g., T x dGTP) than for its complement (A x dCTP). We find that strand-specific mutation rates strongly depend on the orientation of a reporter gene relative to an adjacent replication origin, in a manner implying that >90% of Pol delta replication is performed using the lagging strand template. When combined with recent evidence implicating Pol epsilon in leading strand replication, these data support a model of the replication fork wherein the leading and lagging strand templates are primarily copied by Pol epsilon and Pol delta, respectively.     

Ultrasound enhanced prehospital thrombolysis using microbubbles infusion in patients with acute ST elevation myocardial infarction: Rationale and design of the Sonolysis study

Background

The CONSORT Statement provides recommendations for reporting randomized controlled trials. We assessed the extent to which leading medical journals that publish reports of randomized trials incorporate the CONSORT recommendations into their journal and editorial processes.

Methods

This article reports on two observational studies. Study 1: We examined the online version of 'Instructions to Authors' for 165 high impact factor medical journals and extracted all text mentioning the CONSORT Statement or CONSORT extension papers. Any mention of the International Committee of Medical Journal Editors (ICMJE) or clinical trial registration were also sought and extracted. Study 2: We surveyed the editor-in-chief, or editorial office, for each of the 165 journals about their journal's endorsement of CONSORT recommendations and its incorporation into their editorial and peer-review processes.

Results

Study 1: Thirty-eight percent (62/165) of journals mentioned the CONSORT Statement in their online 'Instructions to Authors'; of these 37% (23/62) stated this was a requirement, 63% (39/62) were less clear in their recommendations. Very few journals mentioned the CONSORT extension papers. Journals that referred to CONSORT were more likely to refer to ICMJE guidelines (RR 2.16; 95% CI 1.51 to 3.08) and clinical trial registration (RR 3.67; 95% CI 2.36 to 5.71) than those journals which did not. Study 2: Thirty-nine percent (64/165) of journals responded to the on-line survey, the majority were journal editors. Eighty-eight percent (50/57) of journals recommended authors comply with the CONSORT Statement; 62% (35/56) said they would require this. Forty-one percent (22/53) reported incorporating CONSORT into their peer-review process and 47% (25/53) into their editorial process. Eighty-one percent (47/58) reported including CONSORT in their 'Instructions to Authors' although there was some inconsistency when cross checking information on the journal's website. Sixty-nine percent (31/45) of journals recommended authors comply with the CONSORT extension for cluster trials, 60% (27/45) for harms and 42% (19/45) for non-inferiority and equivalence trials. Few journals mentioned these extensions in their 'Instructions to Authors'.

Conclusion

Journals should be more explicit in their recommendations and expectations of authors regarding the CONSORT Statement and related CONSORT extensions papers.     

Survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in Wilted and Additive-Treated Grass Silage

Grass was field-dried to 3 different dry matter (DM) levels (200, 430 and 540 g/kg) and inoculated with 10⁶–10⁷ cfu/g of a Listeria monocytogenes strain sharing a phagovar occasionally involved in food-borne outbreaks of listeriosis. Formic acid (3 ml/kg) or lactic acid bacteria (8·10⁵/g) with cellulolytic enzymes were applied only to forages with low and intermediate DM levels. Forages were ensiled in laboratory silos (1700 ml) and were stored at 25°C for 30 or 90 days. After 90 days of storage, L. monocytogenes could not be detected in any silo, except one with the high dry matter grass without additive. After 30 days of storage, between 10² and 10⁶ cfu L. monocytogenes/g silage were isolated from the untreated silages. Increasing the DM content from 200 to 540 g/kg did not reduce listeria counts possibly because of the lower production of fermentation acids (higher pH). In silages treated with additives, counts of L. monocytogenes were always lower than in silages without additive. In wet silages (DM 200 g/kg) both additives were effective, but in the wilted silages (DM 430 g/kg) only the bacterial additive reduced listeria counts below detection level. Listeria counts were highly correlated to silage pH (r = 0.92), the concentration of lactic acid (r = -0.80) and the pooled amount of undissociated acids (r = -0.83).     

A New Black Aspergillus Species, A. vadensis, Is a Promising Host for Homologous and Heterologous Protein Production

A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted. The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A. vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.     

ATP utilization by yeast replication factor C. III. The ATP-binding domains of Rfc2, Rfc3, and Rfc4 are essential for DNA recognition and clamp loading.

The conserved lysine in the Walker A motif of the ATP-binding domain encoded by the yeast RFC1, RFC2, RFC3, and RFC4 genes was mutated to glutamic acid. Complexes of replication factor C with a N-terminal truncation (Delta2-273) of the Rfc1 subunit (RFC) containing a single mutant subunit were overproduced in Escherichia coli for biochemical analysis. All of the mutant RFC complexes were capable of interacting with PCNA. Complexes containing a rfc1-K359E mutation were similar to wild type in replication activity and ATPase activity; however, the mutant complex showed increased susceptibility to proteolysis. In contrast, complexes containing either a rfc2-K71E mutation or a rfc3-K59E mutation were severely impaired in ATPase and clamp loading activity. In addition to their defects in ATP hydrolysis, these complexes were defective for DNA binding. A mutant complex containing the rfc4-K55E mutation performed as well as a wild type complex in clamp loading, but only at very high ATP concentrations. Mutant RFC complexes containing rfc2-K71R or rfc3-K59R, carrying a conservative lysine --> arginine mutation, had much milder clamp loading defects that could be partially (rfc2-K71R) or completely (rfc3-K59R) suppressed at high ATP concentrations.