Benefits of automated crystallization plate tracking, imaging, and analysis

We describe the design of a database and software for managing and organizing protein crystallization data. We also outline the considerations behind the design of a fast web interface linking protein production data, crystallization images, and automated image analysis. The database and associated interfaces underpin the Oxford Protein Production Facility (OPPF) crystallization laboratory, collecting, in a routine and automatic manner, up to 100,000 images per day. Over 17 million separate images are currently held in this database. We discuss the substantial scientific benefits automated tracking, imaging, and analysis of crystallizations offers to the structural biologist: analysis of the time course of the trial and easy analysis of trials with related crystallization conditions. Features of this system address requirements common to many crystallographic laboratories that are currently setting up (semi-)automated crystallization imaging systems.     

Antibody humanization by framework shuffling

We report here the humanization of a mouse monoclonal antibody (mAb B233) using a new technique which we call framework shuffling. mAb B233 was raised against the human receptor tyrosine kinase EphA2 which is selectively up-regulated in many cancer cell lines and as such constitutes an attractive target for cancer therapy. The six CDRs of B233 were fused in-frame to pools of corresponding individual human frameworks. These human frameworks encompassed all known heavy and light (kappa) chain human germline genes. The resulting Fab combinatorial libraries were then screened for binding to the antigen. A two-step selection process, in which the light and heavy chains of the parental mAb were successively humanized, resulted in the identification of several humanized variants that retained binding to EphA2. More precisely, after conversion to human IgG1, the dissociation constants of three select fully humanized variants ranged from 3 to 48 nM. This brings the best framework-shuffled, humanized binder within 5-fold of the avidity of parental mAb B233. Importantly, these humanized IgGs also possessed biochemical activities similar to those of parental mAb B233 as judged by induction of EphA2 phosphorylation. Thus, without requiring any rational design or structural information, this new humanization approach allows to rapidly identify various human framework combinations able to support the structural feature(s) of the CDRs which are essential for binding and functional activity.     

Taxol-stabilized microtubules can position the cytokinetic furrow in mammalian cells

How microtubules act to position the plane of cell division during cytokinesis is a topic of much debate. Recently, we showed that a subpopulation of stable microtubules extends past chromosomes and interacts with the cell cortex at the site of furrowing, suggesting that these stabilized microtubules may stimulate contractility. To test the hypothesis that stable microtubules can position furrows, we used taxol to rapidly suppress microtubule dynamics during various stages of mitosis in PtK1 cells. Cells with stabilized prometaphase or metaphase microtubule arrays were able to initiate furrowing when induced into anaphase by inhibition of the spindle checkpoint. In these cells, few microtubules contacted the cortex. Furrows formed later than usual, were often aberrant, and did not progress to completion. Images showed that furrowing correlated with the presence of one or a few stable spindle microtubule plus ends at the cortex. Actin, myosin II, and anillin were all concentrated in these furrows, demonstrating that components of the contractile ring can be localized by stable microtubules. Inner centromere protein (INCENP) was not found in these ingressions, confirming that INCENP is dispensable for furrow positioning. Taxol-stabilization of the numerous microtubule-cortex interactions after anaphase onset delayed furrow initiation but did not perturb furrow positioning. We conclude that taxol-stabilized microtubules can act to position the furrow and that loss of microtubule dynamics delays the timing of furrow onset and prevents completion. We discuss our findings relative to models for cleavage stimulation.     

Physical fitness of an industrial fire department vs. a municipal fire department

Both industrial and municipal firefighters need to maintain high levels of physical fitness and minimize cardiovascular risk factors. The nature of firefighter responsibilities in industrial and municipal settings may vary, affecting the ability to sustain high levels of physical fitness. We compared the working conditions, physical fitness, and exercise training practices of an industrial fire department (n = 17) to those of a nearby municipal fire department (n = 55). After informed consent, aerobic capacity, muscular strength, muscular endurance, body composition, flexibility, blood lipid concentrations, and blood pressure levels were measured. Exercise training practices and related factors were assessed using a questionnaire. Despite programmatic differences, these departments demonstrated similar, relatively high degrees of physical fitness and similar blood lipid concentrations, blood pressure levels, and cardiac risk factors. It is recommended that fire departments involve appropriately trained staff, schedule on-duty times for exercise, offer well-equipped exercise facilities, and follow National Strength and Conditioning Association (NSCA) and American College of Sports Medicine (ACSM) guidelines for exercise conditioning in order to maintain a high degree of physical fitness.     

Assembly of an APC-Cdh1-substrate complex is stimulated by engagement of a destruction box

The anaphase-promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators. Cdh1p is an APC coactivator that directly binds APC substrates. A genetic screen in budding yeast identified residues within Cdh1p critical for its function. Cdh1p proteins containing mutations within the "C box" or the "IR" motif could bind substrate, but not the APC, whereas mutants that only bound the APC were not identified, suggesting an ordered assembly of the ternary APC-Cdh1p-substrate complex. Supporting this hypothesis, we found that substrate binding to wild-type Cdh1p enhanced its association with the APC in yeast cells. We used peptide competition assays to demonstrate that Cdh1p interacts directly with the D box and the KEN box, two motifs within APC substrates known to be required for APC-mediated degradation. Moreover, an intact D box domain within a substrate was required to stimulate the association between the Cdh1p-substrate complex and the APC.     

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.     

Autoregressive modeling of analytical sensor data can yield classifiers in the predictor coefficient parameter space

SUMMARY: The analysis of chromatographic data resulting from complex chemical mixtures is challenging. Components may co-elute, causing their signals to overlap. An algorithm that will increase the signal-to-noise ratio so compounds present in low abundance can be better distinguished from noise is useful in this type of analysis. The autoregressive (AR) filter offers the advantage of smoothing chromatograms to increase this ratio, while also offering data compression and increased resolution. Furthermore, this filter can be useful for classification, as the roots of the predictor coefficient vectors represent features present in the data and can therefore be used for pattern recognition. In this paper, we present a novel method for applying AR filtering to chromatogram data. We show that the AR filter outperforms the Savitzky-Golay filter for smoothing noise while retaining important information within chromatograms, and also that AR correlation coefficients have the potential to be used to classify chromatogram data into groups. CONTACT: cdavis@draper.com.     

Drosophila Myt1 is a Cdk1 inhibitory kinase that regulates multiple aspects of cell cycle behavior during gametogenesis

The metazoan Wee1-like kinases Wee1 and Myt1 regulate the essential mitotic regulator Cdk1 by inhibitory phosphorylation. This regulatory mechanism, which prevents Cdk1 from triggering premature mitotic events, is also induced during the DNA damage response and used to coordinate cell proliferation with crucial developmental events. Despite the previously demonstrated role for Myt1 regulation of Cdk1 during meiosis, relatively little is known of how Myt1 functions at other developmental stages. To address this issue, we have undertaken a functional analysis of Drosophila Myt1 that has revealed novel developmental roles for this conserved cell cycle regulator during gametogenesis. Notably, more proliferating cells were observed in myt1 mutant testes and ovaries than controls. This can partly be attributed to ectopic division of germline-associated somatic cells in myt1 mutants, suggesting that Myt1 serves a role in regulating exit from the cell cycle. Moreover, mitotic index measurements suggested that germline stem cells proliferate more rapidly, in myt1 mutant females. In addition, male myt1 germline cells occasionally undergo an extra mitotic division, resulting in meiotic cysts with twice the normal numbers of cells. Based on these observations, we propose that Myt1 serves unique Cdk1 regulatory functions required for efficient coupling of cell differentiation with cell cycle progression.