Harnessing ester bond chemistry for protein ligation.

The hydrolysis potential of ester bonds in covalently cross-linking proteins is captured in our novel protein ligation technology. This new type of "molecular superglue" based on the spontaneously-formed Thr-Gln ester bonds found in cell-surface adhesins, affords a unique mechanism to both rationally assemble and disassemble complex protein nanomaterials.

Total Chemical Synthesis of an Orf Virus Protein, ORFV002, an Inhibitor of the Master Gene Regulator NF-κB.

ORFV002 is a novel orf viral protein (117 Aa) that inhibits nuclear events through the regulation of the transcriptional activity of NF-κB, a master regulator of human gene expression (Diel et al., J Virol 2011, 85, 264-275). It is identified as the first nuclear inhibitor of NF-κB produced by orf virus (ORFV) and no homologues in other genera of the Chordopoxvirinae subfamily have been reported to date (Diel et al., J Virol 2011, 85, 264-275). Our molecular structure predictions suggest that ORFV002 may mimic part of IκB, an inhibitor and natural human partner of NF-κB. Recent advances in total chemical synthesis of proteins have provided solutions in overcoming challenges of current recombinant methods of protein isolation for structure elucidation. Aided by Boc solid phase peptide synthesis and native chemical ligation, ORFV002 was successfully synthesized in multimilligram amounts in good yield and high purity.

National audit of the sensitivity of double-contrast barium enema for colorectal carcinoma, using control charts For the Royal College of Radiologists Clinical Radiology Audit Sub-Committee.

AIM: To audit the sensitivity of double-contrast barium enema (DCBE) for colorectal carcinoma, as currently practised in UK departments of radiology. METHODS: As part of its programme of national audits, the Royal College of Radiologists Clinical Radiology Audit Sub-Committee undertook a retrospective audit of the sensitivity of DCBE for colorectal carcinoma during 2002. The following targets were set: demonstration of a lesion > or =95%; correct identification as a carcinoma > or =90%. RESULTS: Across the UK, 131 departments took part in the audit, involving 5454 examinations. The mean demonstration rate was 92.9% and the diagnosis rate was 85.9%, slightly below the targets set. The equivocal rate (lesion demonstrated, but not defined as malignant) was 6.9%, the perception failure rate was 2.8% and the technical failure rate was 4.4%. Control-chart methodology was used to analyze the data and to identify any departments whose performance was consistent with special-cause variation. CONCLUSION: When compared with the diagnosis rate (84.6%) and demonstration rate (92.7%) reported in the Wessex Audit 1995, [Thomas RD, Fairhurst JJ, Frost RA. Wessex regional audit: barium enema in colo-rectal carcinoma. Clin Radiol 1995;50:647-50.] a similar level of performance was observed in the NHS today, implying that the basic process for undertaking and reporting DCBE has remained relatively unchanged over the last few years. Improvement in the future will require fundamental changes to the process of reporting DCBE, in order to minimize the perception failure rate and accurately to describe lesions, so reducing the equivocal rate. Control-chart methodology has a useful role in identifying strategies to deliver continual improvement.

A national audit of radiotherapy in head and neck cancer.

AIMS: To undertake a national audit of radiotherapy practice in head and neck cancer to estimate compliance with published guidelines and national standards. METHODS: A two-part electronic data entry form was distributed to all U.K. radiotherapy centres in September 2000. The first part examined the centres' policies for managing interruptions, the second collected summaries of the management of 50 consecutive patients treated in each centre for head and neck cancer. The outcome measures were: frequency and causes of interruptions to therapy: policy and compliance with policy for managing interruptions; prolongation; and time between first visit to clinic and start of treatment. RESULTS: Fifty-five out of 56 centres returned data on a total of 2553 patients. Overall, 1467 (55%) patients had one or more treatment interruptions. Of patients whose treatment was interrupted, 56% still completed on time due to compensatory steps, but in 32% no attempted compensation was undertaken. Seven centres had no policy for dealing with treatment interruptions. Centres whose policies included treatment on bank (public) holidays achieved higher compliance and fewer prolonged cases than those whose policies did not. Average time from first visit to head and neck oncology clinic to starting radiotherapy was 40 days; six centres had an average wait of less than 28 days. CONCLUSIONS: This audit demonstrates wide variations in the quality of care between centres, failure to comply with guidelines for compensation for gaps and failure to meet national targets (for waiting times) that have serious implications both for patient outcomes and for the success of the National Cancer Plan.

Structures of m-iodo Hoechst-DNA complexes in crystals with reduced solvent content: implications for minor groove binder drug design.

The DNA photosensitisers m-iodo Hoechst and m-iodo, p-methoxy Hoechst have been co-crystallised with the oligonucleotide d(CGCGAATTCGCG)(2)and their crystal structures determined. The crystals were then subjected to slow dehydration, which reduced their solvent contents from 40 (normal) to 30 (partially dehydrated) and then 20% (fully dehydrated) and caused a reduction in cell volume from 68,000 to 60,000 then 51,000 A(3). The dehydration resulted in a dramatic enhancement of diffraction resolution from approximately 2.6 to beyond 1.5 A. Crystal structures have also been determined for the partially and fully dehydrated states. The fully dehydrated crystals consist of an infinite polymeric network, in which neighbouring dodecamer duplexes are crosslinked through phosphate oxygens via direct bonding to bridging magnesium cations. This unique three-dimensional structure for DNA is described in detail in the following companion paper. The present paper details evidence from the sequence of crystal structures that the DNA is able to breathe locally, allowing the ligand to leave the minor groove, re-orient in the surrounding solvent medium and then re-enter the groove in a different orientation and location. The rearrangement of the minor groove binding ligands during the dehydration process mimics the binding behaviour of these ligands in solution and in vivo. We also present details of the DNA-ligand interactions that are consistent with a hydrogen atom ion mechanism for photocleavage of DNA.

Intermolecular interactions and water structure in a condensed phase B-DNA crystal.

By controlled dehydration, the unit cells of dodecamer DNA-drug crystals have been shrunk from 68,000 (normal state) to 60,000 (partially dehydrated intermediate state) to 51,000 A(3) (fully dehydrated state), beyond which no further solvent loss occurs. The total solvent content in the normal crystals is approximately 40% by volume, reducing to approximately 20% in the fully dehydrated phase. The 25% reduction in cell volume induced a dramatic enhancement in the resolution of the X-ray diffraction data (from 2. 6 to beyond 1.5 A). We have determined the structures of the normal, partially dehydrated and fully dehydrated crystals. Details of the ligand binding have been presented in the preceding article. The present paper describes the unique features of the structure of the fully dehydrated phase. This structure was refined with 9,015 unique observed reflections to R = 14.9%, making it one of the most reliable models of B -form DNA available. The crystals exist as infinite polymeric networks, in which neighbouring dodecamer duplexes are crosslinked through phosphate oxygens via direct bonding to magnesium cations. The DNA is packed so tightly that there is essentially only a single layer of solvent between adjacent molecules. The details of the crystal packing, magnesium bridging, DNA hydration and DNA conformation are described and compared with other experimental evidence related to DNA condensation.

2-ethylthio-9-methyl-1,4-dihydroacridine-1,4-dione and 9-methyl-2-(4-tolylthio)-1,4-dihydroacridine-1,4-dione.

The title compounds, C16H13NO2S, (I), and C21H15NO2S, (II), have similar molecular structures that differ only in the side groups attached to the S atom. The crystal packing is dominated by pi-pi stacking interactions, involving acridinedione-acridinedione overlap in (I) and both acridinedione-acridinedione and acridinedione-tolyl overlap in (II).

Minor groove binding of a bis-quaternary ammonium compound: the crystal structure of SN 7167 bound to d(CGCGAATTCGCG)2.

The X-ray crystal structure of the complex between the synthetic antitumour and antiviral DNA binding ligand SN 7167 and the DNA oligonucleotide d(CGCGAATTCGCG)2 has been determined to an R factor of 18.3% at 2.6 A resolution. The ligand is located within the minor groove and covers almost 6 bp with the 1-methylpyridinium ring extending as far as the C9-G16 base pair and the 1-methylquinolinium ring lying between the G4-C21 and A5-T20 base pairs. The ligand interacts only weakly with the DNA, as evidenced by long range contacts and shallow penetration into the groove. This structure is compared with that of the complex between the parent compound SN 6999 and the alkylated DNA sequence d(CGC[e6G]AATTCGCG)2. There are significant differences between the two structures in the extent of DNA bending, ligand conformation and groove binding.

Designer DNA-binding drugs: the crystal structure of a meta-hydroxy analogue of Hoechst 33258 bound to d(CGCGAATTCGCG)2.

An analogue of the DNA binding compound Hoechst 33258, which has the para hydroxyl group altered to be at the meta position, together with the replacement of one benzimidazole group by pyridylimidazole, has been cocrystallized with the dodecanucleotide sequence d(CGCGAATTCGCG)2. The X-ray structure has been determined at 2.2 A resolution and refined to an R factor of 20.1%. The ligand binds in the minor groove at the sequence 5'-AATTC with the bulky piperazine group extending over the CxG base pair. This binding is stabilised by hydrogen bonding and numerous close van der Waals contacts to the surface of the groove walls. The meta-hydroxyl group was found in two distinct orientations, neither of which participates in direct hydrogen bonds to the exocyclic amino group of a guanine base. The conformation of the drug differs from that found previously in other X-ray structures of Hoechst 33258-DNA complexes. There is significant variation between the minor groove widths in the complexes of Hoechst 33258 and the meta-hydroxyl derivative as a result of these conformational differences. Reasons are discussed for the inability of this derivative to actively recognise guanine.

Non-surgical management of early breast cancer in the United Kingdom: work-load, referral patterns and staging. Clinical Audit Sub-committee of the Faculty of Clinical Oncology, Royal College of Radiologists, and the Joint Council for Clinical Oncology.

This is the first of five papers describing the results of a national survey of the non-surgical treatment of early breast cancer in the UK. The topics include: work-load, referral patterns, participation in specialist clinics, use of treatment policies, availability of outcome data, quality of pathology reports, and staging investigations.

Audit: the use of radiotherapy for NSCLC in the UK.

A questionnaire on the management of NSCLC was sent to all clinical oncologists in the UK. Responses were received from 121 individuals with at least one representative response from each of 54 British radiotherapy units. Results were then discussed at an open meeting attended by a cross section of clinical oncologists; a synopsis of responses to the questionnaire and discussion at this meeting is contained in this report. A majority of respondents estimated treatment of NSCLC to make up 10%-25% of their work-load. Radical and palliative treatments could be clearly distinguished, and aims of treatment, selection criteria and radiotherapy schedules were consistent with recommendations in the published literature. More than 90% of treatments were with palliative rather than radical intent. Radical treatment schedules could be divided according to dose (< 50 Gy, 50-55 Gy and >55 Gy), number of fractions (< 20, 20, > 20 fractions), overall time < 4/52, 4/52, > 4/52), dose per fraction (> 2.75 Gy, 2.1-2.75 Gy, < or = 2 Gy) and target volume (tumour alone, tumour and hilar nodes, or tumour, hilar and mediastinal nodes). Divided thus, radical techniques fell into three broad groups, each of the three techniques supported by a body of literature. Choice of schedule could be related to a heterogeneous referral pattern and unresolved controversies, identified as debate on the value of treating mediastinal lymphadenopathy with high dose radiation, the value of 'subradical doses' of radiation for microscopic disease, and the relative importance of volume treated, total dose, dose per fraction and overall treatment time in achieving an optimal therapeutic ratio.(ABSTRACT TRUNCATED AT 250 WORDS)