Automatic implementation of precise grid screens: the four-corners method

Hennessy, Daniel N.; Narayanan, Beena

doi:10.1107/s0907444909020320

Cited by 4 publications

(4 citation statements)

References 3 publications

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“…Consistent with this, several other studies have also suggested the predictive value of basal keratins on BRCA1 mutation 132 , 133 . Preclinical models of tumors with dysfunctional BRCA1 have been shown to be exclusively sensitive to cross-linking agents and inhibitors of the poly (ADP-ribose) polymerase 134 , indicating the efficient therapeutic treatment of tumors of this class.…”

Section: Hallmark 2: Activating Invasion and Metastasismentioning

confidence: 99%

Cancer Hallmarks, Biomarkers and Breast Cancer Molecular Subtypes

Dai¹,

Xiang²,

Li³

et al. 2016

J. Cancer

355

308

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Breast cancer is a complex disease encompassing multiple tumor entities, each characterized by distinct morphology, behavior and clinical implications. Besides estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, novel biomarkers have shown their prognostic and predictive values, complicating our understanding towards to the heterogeneity of such cancers. Ten cancer hallmarks have been proposed by Weinberg to characterize cancer and its carcinogenesis. By reviewing biomarkers and breast cancer molecular subtypes, we propose that the divergent outcome observed from patients stratified by hormone status are driven by different cancer hallmarks. 'Sustaining proliferative signaling' further differentiates cancers with positive hormone receptors. 'Activating invasion and metastasis' and 'evading immune destruction' drive the differentiation of triple negative breast cancers. 'Resisting cell death', 'genome instability and mutation' and 'deregulating cellular energetics' refine breast cancer classification with their predictive values. 'Evading growth suppressors', 'enabling replicative immortality', 'inducing angiogenesis' and 'tumor-promoting inflammation' have not been involved in breast cancer classification which need more focus in the future biomarker-related research. This review novels in its global view on breast cancer heterogeneity, which clarifies many confusions in this field and contributes to precision medicine.

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Section: Hallmark 2: Activating Invasion and Metastasismentioning

confidence: 99%

Cancer Hallmarks, Biomarkers and Breast Cancer Molecular Subtypes

Dai¹,

Xiang²,

Li³

et al. 2016

J. Cancer

355

308

View full text Add to dashboard Cite

show abstract

“…Crystallization plates for initial screening were prepared using the Mosquito micro volume crystallization robot at both 2:1 and 1:1 ratios of protein to precipitant with volumes of 0.1 µl:0.5 µl and 0.075 µl:0.075 µl. Optimization blocks were prepared using the Multiprobe II liquid handling robot producing 96-well grids varying precipitant concentrations using the four-corner approach to aid robotic setup (32). …”

Section: Methodsmentioning

confidence: 99%

Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

Cole

Ofer

Ryzhenkova

et al. 2013

Nucleic Acids Research

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Uracil-DNA glycosylase (UDG) compromises the replication strategies of diverse viruses from unrelated lineages. Virally encoded proteins therefore exist to limit, inhibit or target UDG activity for proteolysis. Viral proteins targeting UDG, such as the bacteriophage proteins ugi, and p56, and the HIV-1 protein Vpr, share no sequence similarity, and are not structurally homologous. Such diversity has hindered identification of known or expected UDG-inhibitory activities in other genomes. The structural basis for UDG inhibition by ugi is well characterized; yet, paradoxically, the structure of the unbound p56 protein is enigmatically unrevealing of its mechanism. To resolve this conundrum, we determined the structure of a p56 dimer bound to UDG. A helix from one of the subunits of p56 occupies the UDG DNA-binding cleft, whereas the dimer interface forms a hydrophobic box to trap a mechanistically important UDG residue. Surprisingly, these p56 inhibitory elements are unexpectedly analogous to features used by ugi despite profound architectural disparity. Contacts from B-DNA to UDG are mimicked by residues of the p56 helix, echoing the role of ugi’s inhibitory beta strand. Using mutagenesis, we propose that DNA mimicry by p56 is a targeting and specificity mechanism supporting tight inhibition via hydrophobic sequestration.

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“…The composition of the buffer systems may be altered during optimization experiments to change the pH. Obviously, all of these optimization experiments are very amenable to automation (Hennessy et al, 2009).…”

Section: Optimization Of Conditionsmentioning

confidence: 99%

The MORPHEUS protein crystallization screen

2009

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A 96-condition initial screen for protein crystallization, called MORPHEUS, has been developed at the MRC Laboratory of Molecular Biology, Cambridge, England (MRC-LMB). The concept integrates several innovative approaches, such as chemically compatible mixes of potential ligands, new buffer systems and precipitant mixes that also act as cryoprotectants. Instead of gathering a set of crystallization conditions that have already been successful, a selection of molecules frequently observed in the Protein Data Bank (PDB) to co-crystallize with proteins has been made. These have been put together in mixes of similar chemical behaviour and structure, and combined with buffers and precipitant mixes that were also derived from PDB searches, to build the screen de novo. Observations made at the MRC-LMB and many practical aspects were also taken into account when formulating the screen. The resulting screen is easy to use, comprehensive yet small, and has already yielded a list of crystallization hits using both known and novel samples. As an indicator of success, the screen has now become one of the standard screens used routinely at the MRC-LMB when searching initial crystallization conditions for biological macromolecules.

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Automatic implementation of precise grid screens: the four-corners method

Abstract: Example of four-corners grid mixing. Shown here are mixing proportions for the indicated grid, in a 24-well format for legibility. The proportions for the more common 96-well format are similar.short communications

Cited by 4 publications

References 3 publications

Cancer Hallmarks, Biomarkers and Breast Cancer Molecular Subtypes

Cancer Hallmarks, Biomarkers and Breast Cancer Molecular Subtypes

Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase

The MORPHEUS protein crystallization screen

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