Proteolysis of MLL family proteins is essential for taspase1-orchestrated cell cycle progression

Taspase1 was identified as the threonine endopeptidase that cleaves mixed-lineage leukemia (MLL) for proper Hox gene expression in vitro. To investigate its functions in vivo, we generated Taspase1(-/-) mice. Taspase1 deficiency results in noncleavage (nc) of MLL and MLL2 and homeotic transformations. Remarkably, our in vivo studies uncover an unexpected role of Taspase1 in the cell cycle. Taspase1(-/-) animals are smaller in size. Taspase1(-/-) mouse embryonic fibroblasts (MEFs) exhibit impaired proliferation, and acute deletion of Taspase1 leads to a marked reduction of thymocytes. Taspase1 deficiency incurs down-regulation of Cyclin Es, As, and Bs and up-regulation of p16(Ink4a) . We show that MLL and MLL2 directly target E2Fs for Cyclin expression. The uncleaved precursor MLL displays a reduced histone H3 methyl transferase activity in vitro. Accordingly, chromatin immunoprecipitation assays demonstrate a markedly decreased histone H3 K4 trimethylation at Cyclin E1 and E2 genes in Taspase1(-/-) cells. Furthermore, MLL(nc/nc;2nc/nc) MEFs are also impaired in proliferation. Our data are consistent with a model in which precursor MLLs, activated by Taspase1, target to Cyclins through E2Fs to methylate histone H3 at K4, leading to activation. Lastly, Taspase1(-/-) cells are resistant to oncogenic transformation, and Taspase1 is overexpressed in many cancer cell lines. Thus, Taspase1 may serve as a target for cancer therapeutics.     

Identification of clinical isolates of indole-positive and indole-negative Klebsiella spp

Biochemical methods employed to classify bacterial species have limitations and may have contributed to the taxonomic complexity recently reported for the genus Klebsiella. The objective of the present study was to apply a simple biochemical test panel to classify a collection of human Klebsiella isolates. We found that with only three additional tests, it is possible to place most isolates in a defined species. Analysis of a 512-bp sequence of the rpoB gene was used as the reference. A total of 16 conventional and 4 supplementary tests were used to evaluate 122 recent isolates identified as Klebsiella from 120 patients, isolated at the clinical laboratory of a university hospital in Minas Gerais, Brazil. Of these, 102 (84%) isolates were identified as Klebsiella pneumoniae or Klebsiella variicola, 19 (15%) as Klebsiella oxytoca, and 1 (1%) as Raoultella planticola. Enterobacterial repetitive intergenic consensus-PCR typing revealed a diversity of genotypes. rpoB gene sequencing confirmed the phenotypic identification and detected five K. variicola isolates among the K. pneumoniae/K. variicola group. Three additional tests that include growth at 10 degrees C and histamine and d-melezitose assimilation should be considered essential tests for the typing of Klebsiella isolates.     

Three-Arm Noninferiority Trials with a Prespecified Margin for Inference of the Difference in the Proportions of Binary Endpoints

The design of a three-arm trial including the experimental treatment, an active reference treatment, and a placebo is recommended as a useful approach to the assessment of noninferiority of the experimental treatment. The inclusion of the placebo arm enables the assessment of assay sensitivity and internal validation, in addition to testing the noninferiority of the experimental treatment to the reference. Generally, the acceptable noninferiority margin Δ has been defined as the maximum clinically irrelevant difference between treatments in many two-arm noninferiority trials. However, many articles have considered a design in which the noninferiority margin Δ is relatively defined as a prespecified fraction f of the unknown effect size of the reference treatment. Therefore, these methods cannot be applied to cases where the margin is defined as a prespecified difference between treatments. In this article, we propose score-based statistical procedures for a three-arm noninferiority trial with a prespecified margin Δ for inference of the difference in the proportions of binary endpoints. In addition, we derive the approximate sample size and optimal allocation to minimize the total sample size and that of the placebo arm. A randomized controlled trial on major depressive disorder based on the difference in the proportions of remission is used to demonstrate our proposed method.     

Soluble lutein in combination with brilliant blue as a new dye for chromovitrectomy

Summary

A new dye for vitreoretinal surgery comprised of soluble lutein/zeaxanthin 1 % and brilliant blue 0.025 % is advantageous compared with other dyes currently used for chromovitrectomy, and showed no signs of toxicity at 1 month of follow-up.

Purpose

To evaluate the feasibility and safety of a dye [soluble lutein/zeaxanthin (LZ) 1 % and brilliant blue (BB) 0.025 %] for improving removal of vitreous, epiretinal membranes (ERM), and internal limiting membranes (ILM) in humans.

Methods

We prospectively evaluated 18 eyes treated surgically for a macular hole or ERM. Eighteen surgeons performed chromovitrectomy using the dye, and completed a questionnaire to evaluate the efficacy and safety of the dye. . Examinations included best-corrected visual acuity and intraocular pressure measurements and optical coherence tomography, fluorescein angiography, and autofluorescence performed at baseline and days 1, 7, and 30 postoperatively.

Results

The green dye was deposited on the posterior pole; vigorous dye flushing into the vitreous cavity was unnecessary. All surgeons reported that the ILM stained greenish-blue; 94.4 % reported ILM peeling adequate; the ERM stained poorly. No evidence of toxicity was observed.

Conclusion

The new dye deposited on the posterior pole due to its higher density. The ability to stain the ILM was similar to BB. The new dye has ability to stain the vitreous, hyaloid, and especially the ILM satisfactorily. The new dye may be useful during chromovitrectomy.     

Teamwork in the viscous oceanic microscale

Nutrient acquisition is crucial for oceanic microbes, and competitive solutions to solve this challenge have evolved among a range of unicellular protists. However, solitary solutions are not the only approach found in natural populations. A diverse array of oceanic protists form temporary or even long-lasting attachments to other protists and marine aggregates. Do these planktonic consortia provide benefits to their members? Here, we use empirical and modeling approaches to evaluate whether the relationship between a large centric diatom, Coscinodiscus wailesii, and a ciliate epibiont, Pseudovorticella coscinodisci, provides nutrient flux benefits to the host diatom. We find that fluid flows generated by ciliary beating can increase nutrient flux to a diatom cell surface four to 10 times that of a still cell without ciliate epibionts. This cosmopolitan species of diatom does not form consortia in all environments but frequently joins such consortia in nutrient-depleted waters. Our results demonstrate that symbiotic consortia provide a cooperative alternative of comparable or greater magnitude to sinking for enhancement of nutrient acquisition in challenging environments.

Global models of oxygen and carbon dioxide alterations depend upon transfer rates between small phytoplankton cells and surrounding surface waters of the world’s oceans (1–3). Although these cells are important on large scales, their individual interactions occur at microscopic dimensions that are dominated by viscosity. In this viscous environment, critical cellular processes, such as the exchange of nutrients, metabolites, and wastes, rely upon diffusion (4). While diffusion is an effective means of nutrient transport for the smallest microbes (5), it also creates a depleted region around the cell surface, referred to as the diffusion boundary layer (DBL), that limits nutrient consumption and cell growth (6, 7). The DBL for a cell at rest extends nine cell radii from the cell surface before the nutrient concentration reaches 90% of ambient levels (8), creating formidable disadvantages for nutrient acquisition by large cells requiring nutrient diffusion across large distances relative to their cell size (9). Phytoplankton have evolved mechanisms to mitigate the limitations of diffusive transfer rates by swimming or sinking (10) to generate relative motion between the cell and surrounding fluid. Diatoms—barrel-shaped, nonmotile eukaryotes—are considered to be one of the most ecologically important groups of phytoplankton (11–14) that absorb nutrients across their whole cell surface (15) but often increase sinking rates when experiencing nutrient limitation (16). Sinking thins the DBL surrounding the diatom cell and reduces the distance over which diffusion limits nutrient transport (8, 17). One potential disadvantage of this mechanism for DBL reduction is the high probability for a cell to sink out of sunlit regions, and sinking diatoms are major contributors to organic mass flux from surface to deep oceanic waters (1). A widespread but unevaluated alternative for such large cells involves teamwork with other smaller, motile cells that combine to form multicell consortia. Consortia are typically comprised of larger nonmotile host cells with smaller, surface-adhering motile cells termed epibionts. Although infrequently studied, epibionts are ubiquitous in the micrometer-scale world of planktonic organisms (18), and flagellated or ciliated epibionts often attach to larger objects (19) or marine snow (20). The selective forces favoring epibiont attachment remain in question. A range of fluid dynamic effects on prey encounter as well as biological factors such as reduced predator encounter risk or elevated prey availability surrounding host attachment sites have been proposed to explain the widespread nature of epibiont attachment (21, 22). However, the movement of motile epibiont cilia or flagella alters flows and, hence, creates an altered fluid dynamic environment surrounding the consortia. What are the consequences for the consortium host—does a larger, nonmotile cell benefit by membership in consortia? While advantages to the epibiont have been examined, the impacts on the host cells within a consortium have remained largely unconsidered. One reason for this is that physical associations between members of consortia are often temporary, and the short time scales of these relationships have hampered direct evaluation of the fluid mechanical interactions between consortium members.Here, we describe experimental work in combination with mathematical models that quantify the effect of the epibiont’s advective currents on nutrient availability to host cells within consortia formed by a large diatom, Coscinodiscus wailesii, and its peritrich ciliate epibiont, Pseudovorticella coscinodisci (Fig. 1A). Consortia composed of C. wailesii and P. coscinodisci are common along the Atlantic coast of South America (23, 24) and have provided a valuable opportunity for measuring fluid interactions characterizing a planktonic host–epibiont association.Open in a separate windowFig. 1.Diatom–ciliate association: (A) diatom C. wailesii and its epibiont P. coscinodisci. Scale bars represent 100 microns in length. (B) Flow around a single ciliate directed toward the diatom cell surface. (C) Velocity field for the same diatom–ciliate pair with a blue line indicating the transect line used for measurement of flow field velocities. The magenta segment represents the microcurrent component that directly intercepts the ciliary crown.     

Staphylococcus aureus elicits marked alterations in the airway proteome during early pneumonia

Pneumonia caused by Staphylococcus aureus is a growing concern in the health care community. We hypothesized that characterization of the early innate immune response to bacteria in the lungs would provide insight into the mechanisms used by the host to protect itself from infection. An adult mouse model of Staphylococcus aureus pneumonia was utilized to define the early events in the innate immune response and to assess the changes in the airway proteome during the first 6 h of pneumonia. S. aureus actively replicated in the lungs of mice inoculated intranasally under anesthesia to cause significant morbidity and mortality. By 6 h postinoculation, the release of proinflammatory cytokines caused effective recruitment of neutrophils to the airway. Neutrophil influx, loss of alveolar architecture, and consolidated pneumonia were observed histologically 6 h postinoculation. Bronchoalveolar lavage fluids from mice inoculated with phosphate-buffered saline (PBS) or S. aureus were depleted of overabundant proteins and subjected to strong cation exchange fractionation followed by liquid chromatography and tandem mass spectrometry to identify the proteins present in the airway. No significant changes in response to PBS inoculation or 30 min following S. aureus inoculation were observed. However, a dramatic increase in extracellular proteins was observed 6 h postinoculation with S. aureus, with the increase dominated by inflammatory and coagulation proteins. The data presented here provide a comprehensive evaluation of the rapid and vigorous innate immune response mounted in the host airway during the earliest stages of S. aureus pneumonia.