Clinical governance and chronic pain: towards a practical solution.

There have been many studies into the effectiveness of single interventions in pain, however, little is known of performance or outcome of pain clinics where treatment often consists of multiple, complex interventions. Many pain clinicians currently experience considerable difficulty in fulfilling the requirements of clinical governance and completing a personal portfolio. There is a clear and urgent need for a viable method of monitoring performance. This study describes a well-developed computer-based system - Pain Audit Collection System (PACS). PACS has been designed by pain clinicians through consensus and its success in uptake suggests that it is a viable method for outcome evaluation. An analysis is provided of outcome data in typical pain clinics. Further work is needed to investigate the utility of this data.

G-overhang dynamics at Tetrahymena telomeres.

To learn more about the structure of the DNA terminus at Tetrahymena thermophila telomeres, we have devised a ligation-mediated primer extension protocol to accurately measure the length of the G-strand overhang. We show that overhang length and the identity of the 3'-terminal nucleotide are tightly regulated. The majority of overhangs terminate in the sequence 5'-TTGGGGT and >80% are either 14-15 or 20-21 nucleotides in length. No significant changes in overhang length were detected as cells traversed the cell cycle. However, changes in length distribution were observed when cells exited the cell cycle, indicating an altered balance between DNA synthesis and degradation or end protection. We also provide evidence that rDNA molecules have overhangs on both telomeres. Full-length rDNA could be cloned by a strategy that depends on overhangs being present at both ends. Moreover, analysis of leading strand telomeres revealed that a significant fraction have overhangs > or =5 nucleotides. Our results indicate that generation of the terminal telomeric DNA structure is highly regulated and requires several distinct DNA-processing events.

How two causes are different from one: the use of (un)conditional information in Simpson's paradox.

In a causally complex world, two (or more) factors may simultaneously be potential causes of an effect. To evaluate the causal efficacy of a factor, the alternative factors must be controlled for (or conditionalized on). Subjects judged the causal strength of two potential causes of an effect that covaried with each other, thereby setting up a Simpson's paradox--a situation in which causal judgments should vary widely depending on whether or not they are conditionalized on the alternative potential cause. In Experiments 1 (table format) and 2 (trial-by-trial format), the subjects did conditionalize their judgments for one causal factor on a known alternative cause. The subjects also demonstrated that they knew what information was needed to properly make causal judgments when two potential causes are available. In Experiment 3 (trial-by-trial), those subjects who were not told about the causal mechanism by which the alternative cause operated were less likely to conditionalize on it. However, the more a subject recognized the covariation between the alternative cause and the effect, the more the subject conditionalized on it. Such behavior may arise from the interaction between bottom-up and top-down processing.

Comparison of the caudal and lumbar approaches to the epidural space.

OBJECTIVES: To investigate the accuracy of placement of epidural injections using the lumbar and caudal approaches. To identify which factors, if any, predicted successful placement. METHODS: 200 consecutive patients referred to a pain clinic for an epidural injection of steroid were randomly allocated to one of two groups. Group L had a lumbar approach to the epidural space and group C a caudal approach to the epidural space. Both groups then had epidurography performed using Omnipaque and an image intensifier to determine the position of the needle. RESULTS: Body mass index (BMI), grade of operator, and route of injection were predictors of a successful placement. 93% of lumbar and 64% of caudal epidural injections were correctly placed (p< 0.001). 97% of lumbar and 85% of caudal epidural injections clinically thought to be correctly placed were confirmed radiographically. For epidural injections where the clinical impression was "maybe", 91% of lumbar injections, but only 45% of caudal injections were correctly placed. Obesity was associated with a reduced chance of successful placement (odds ratio (OR) 0.34 (95% confidence interval (CI) 0.17 to 0.72) BMI >30 v BMI <30). A more senior grade of operator was associated with a reduced chance of successful placement (OR 0.16 (95% CI 0.03 to 0.89) consultant v other). However, small numbers may have accounted for the latter result. CONCLUSIONS: The weight of the patient and intended approach need to be considered when deciding the method used to enter the epidural space. In the non-obese patient, lumbar epidural injections can be accurately placed without x ray screening, but caudal epidural injections, to be placed accurately, require x ray screening no matter what the weight of the patient.

p16INK4A and p19ARF act in overlapping pathways in cellular immortalization.

The INK4A locus encodes two independent but overlapping genes, p16INK4A and p19ARF, and is frequently inactivated in human cancers. The unusual structure of this locus has lead to ambiguity regarding the biological role of each gene. Here we express, in primary mouse embryonic fibroblasts (MEFs), antisense RNA constructs directed specifically towards either p16INK4A or p19 ARF. Such constructs induce extended lifespan in primary MEFs; this lifespan extension is reversed upon subsequent elimination of the p16INK4A or p19ARF antisense constructs. In immortal derivatives of cell lines expressing antisense p16INK4A or p19ARF RNA, growth arrest induced by recovery of p16INK4A expression is bypassed by compromising the function of the retinoblastoma protein (Rb), whereas growth arrest induced by re-expression of p19ARF is overcome only by simultaneous inactivation of both the Rb and the p53 pathways. Thus, the physically overlapping p16INK4A and p19ARF genes act in partly overlapping pathways.

Representations of motion and direction.

In 6 experiments, incidental memory was tested for direction of motion in an old-new recognition paradigm. Ability to recognize previously shown directions depended greatly on motion type. Memory for translation and expansion-contraction direction was highly veridical, whereas memory for rotation direction was conspicuously absent. Similar results were obtained in conditions in which motions were illustrated with pictures. Results suggest that explicit representations of direction in long-term memory are not so much related to motion per se as to the consequences of motion, the displacements of objects. Memory for all motions following circular pathways was found to be corrupted by a generic bias to regard the clockwise direction as familiar. Assessment of memory in these cases required disentangling familiarity bias for the clockwise direction from explicit recognition of direction.

Telomeres and telomerase: broad effects on cell growth.

During the past year, major advances have been made in understanding the link between telomerase expression and cell immortality. Studies of yeast telomeres have revealed an unexpected role for the non-homologous end-joining machinery in telomere maintenance and have provided the first definitive evidence that telomeres play a critical role in meiosis. Identification of new telomere proteins has led to a better understanding of vertebrate telomere structure and function.

Differential expression of linker histone variants in Euplotes crassus.

Two genes have been cloned from the ciliate Euplotes crassus that encode proteins with sequence similarity to the linker histones from a variety of organisms. One gene, H1-1, is present on a 1.3-kb macronuclear DNA molecule and encodes a 16.2- kDa protein. The second gene, H1-2, is present on a 0.7-kb DNA molecule and encodes an 18.8-kDa protein. Both H1-1 and H1-2 are expressed in vegetative cells, but the two genes exhibit very different patterns of expression during macronuclear development. H1-1 transcripts accumulate during conjugation and during the final rounds of DNA amplification. H1-2 transcripts accumulate after the onset of polytene chromosome formation and remain high throughout the remainder of macronuclear development. H1-1 is the major perchloric-acid-soluble protein from macronuclei. The pattern of gene expression and the macronuclear location of the H1-1 protein indicate that H1-1 is the predominant linker histone in vegetative macronuclei.

Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.

Association and clonal distribution of trisomy 12 and 13q14 deletions in chronic lymphocytic leukaemia.

The coexistence of trisomy 12 and deletions of chromosome 13 (13q12-q32) has rarely been observed in chronic lymphocytic leukaemia (CLL). Fluorescence in situ hybridization (FISH) performed on 600 consecutive CLL patients revealed the association of trisomy 12 and 13q14 deletion, of at least one of the three markers analysed (RB1, D13S319 and D13S25), in 55 cases (9% of 600 and 46% of 120 trisomy 12 cases). Trisomy 12 and isolated RB1 deletion were seen in 14/120 cases, trisomy 12 and D13S319/D13S25 deletion with diploid RB1 in 19/118, and trisomy 12 and deletion encompassing the three 13q markers studied in 22/118 cases. The heterogenous distribution of trisomy 12 and 13q deletions within the neoplastic B cells suggests that they are secondary rather than primary events in CLL leukaemogenesis.

Fetal origins of the TEL-AML1 fusion gene in identical twins with leukemia.

The TEL (ETV6)-AML1 (CBFA2) gene fusion is the most common reciprocal chromosomal rearrangement in childhood cancer occurring in approximately 25% of the most predominant subtype of leukemia- common acute lymphoblastic leukemia. The TEL-AML1 genomic sequence has been characterized in a pair of monozygotic twins diagnosed at ages 3 years, 6 months and 4 years, 10 months with common acute lymphoblastic leukemia. The twin leukemic DNA shared the same unique (or clonotypic) but nonconstitutive TEL-AML1 fusion sequence. The most plausible explanation for this finding is a single cell origin of the TEL-AML fusion in one fetus in utero, probably as a leukemia-initiating mutation, followed by intraplacental metastasis of clonal progeny to the other twin. Clonal identity is further supported by the finding that the leukemic cells in the two twins shared an identical rearranged IGH allele. These data have implications for the etiology and natural history of childhood leukemia.

Molecular cytogenetic analysis in splenic lymphoma with villous lymphocytes: frequent allelic imbalance of the RB1 gene but not the D13S25 locus on chromosome 13q14.

Structural abnormalities of chromosome 13q are one of the most frequent genetic aberrations in human tumors. 13q rearrangements are, however, infrequent in splenic lymphoma with villous lymphocytes (SLVL) by karyotype analysis. We have investigated the incidence of 13q14 deletions in a series of 74 SLVL cases by interphase fluorescence in situ hybridization using unique sequence probes for the RB1 and the D13S25 loci, which are frequently deleted in chronic lymphocytic leukemia. Chromosome 12 was also evaluated by fluorescence in situ hybridization using a pericentromeric DNA probe. 13q14 deletion was detected in 37 of 74 (50%) tumors. Thirty-five cases (47%) exhibited monoallelic loss of RB1, and 9 (12%) showed hemizygous D13S25 deletion. Seven cases displayed coexistence of RB1 and D13S25 deletion. Trisomy 12 was detected in 2 of 74 (3%) tumors. G-banding analysis in 40 tumors showed no interstitial deletion of 13q14 in any case. In contrast with the molecular findings observed in chronic lymphocytic leukemia, our results indicate that trisomy 12 is an uncommon chromosomal aberration in SLVLs, and microdeletion of 13q14 at the RB1 locus but not D13S25 is a frequent and specific genetic event in this disease, suggesting that allelic loss of the RB1 gene may play a role in the pathogenesis of SLVL.

Methylation of the multi tumor suppressor gene-2 (MTS2, CDKN1, p15INK4B) in childhood acute lymphoblastic leukemia.

The multi tumor suppressor genes MTS1 (CDKN2 p16INK4A) and MTS2 (CDKN1, p15INK4B) located at 9p21-22 are inactivated in some human cancers via several mechanisms including deletion and hypermethylation. We have investigated the deletion and methylation status of MTS1 and MTS2 in childhood acute lymphoblastic leukemia (ALL) of both T-cell (17 cases) and B-cell phenotypes (29 cases), and p16INK4A and p15INK4B mRNA expression in 36 of these cases. Biallelic or monoallelic loss of both MTS1 and MTS2 was observed in 12 cases of B-ALL and nine cases of T-ALL. Two cases of T-ALL showed deletion of MTS1 but not MTS2. The 5' CpG region of MTS2 was hypermethylated in 12 cases of precursor B-ALL and eight cases of T-ALL but no hypermethylation was found in the 5' CpG region of MTS1. All cases with homozygous deletion of MTS1 or MTS2 had no or low levels of mRNA expression and similar low levels of expression were found in cases in which MTS2 was present but fully methylated. Thus hypermethylation of MTS2, in contrast to MTS1, is frequent in childhood ALL. Furthermore our data show that although inactivation of MTS1 by deletion is common, inactivation of MTS2 by a combination of deletion and hypermethylation is more frequent in both B-ALL (20/29, 69%) and T-ALL (17/17, 100%). This suggests that both MTS1 and MTS2 are important targets of the 9p21-22 deletion.

Coordinate regulation of G- and C strand length during new telomere synthesis.

We have used the ciliate Euplotes to study the role of DNA polymerase in telomeric C strand synthesis. Euplotes provides a unique opportunity to study C strand synthesis without the complication of simultaneous DNA replication because millions of new telomeres are made at a stage in the life cycle when no general DNA replication takes place. Previously we showed that the C-strands of newly synthesized telomeres have a precisely controlled length while the G-strands are more heterogeneous. This finding suggested that, although synthesis of the G-strand (by telomerase) is the first step in telomere addition, a major regulatory step occurs during subsequent C strand synthesis. We have now examined whether G- and C strand synthesis might be regulated coordinately rather than by two independent mechanisms. We accomplished this by determining what happens to G- and C strand length if C strand synthesis is partially inhibited by aphidicolin. Aphidicolin treatment caused a general lengthening of the G-strands and a large increase in C strand heterogeneity. This concomitant change in both the G- and C strand length indicates that synthesis of the two strands is coordinated. Since aphidicolin is a very specific inhibitor of DNA pol alpha and pol delta, our results suggest that this coordinate length regulation is mediated by DNA polymerase.

Interphase cytogenetics in chronic lymphocytic leukemia.

The incidence of trisomy 12 and 13q12-q14 abnormalities in patients with chronic lymphocytic leukemia (CLL) was determined by conventional cytogenetics and interphase fluorescence in situ hybridization (FISH). In the analysis of 580 consecutive patients, trisomy 12 was detected by conventional cytogenetics in 39 cases (9%) and 117 cases (20%) by FISH. Trisomy 12 was shown to be associated with advanced clinical stage, atypical morphology, and higher proliferative activity. Combined immunophenotyping and FISH showed that trisomy 12 was present only in a proportion of the clonal B-cells. These data suggest that trisomy 12 is a secondary event associated with features of disease progression. Sequential FISH showed clonal progression of the trisomic clone over time. Three hundred patients also were investigated for 13q deletions using FISH analysis of the RB1 locus (13q14). Monoallelic RB1 deletion was seen in 104 (34%) of cases. One case had a homozygous deletion in 90% of the cells. Dual-color FISH detected the presence of trisomy 12 and RB1 in 17 (5%) cases. DNA probes for 13q12.3 (BRCA2) and 13q14 (RB1 and DBM locus) were used in 35 cases. Twenty-eight (80%) cases showed deletion of a 1Mb 13q12.3 encompassing the BRCA2 locus, whereas 22/35 (63%) were deleted at 13q14. Our data suggest that abnormalities of 13q are more frequent than trisomy 12 in CLL and provide evidence for the presence of a new candidate gene at 13q12.3 that may be involved in the pathogenesis of CLL.

MLL self fusion mediated by Alu repeat homologous recombination and prognosis of AML-M4/M5 subtypes.

Fifty-six patients with de novo acute myeloid leukemia M4/M5 subtypes were studied for rearrangements of the mixed lineage leukemia gene, MLL (also called HRX, Htrx-1, or ALL-1). Ten patients (18%) showed rearrangements of the MLL gene, 9 in a major breakpoint cluster region within a centromeric 8.3-kb BamHI fragment, whereas rearrangement in one patient was the result of a direct tandem duplication of exons 2-6 of MLL. Analysis of sequences at the duplication junction revealed that the points of MLL fusion within introns 6 and 1 both lie within Alu elements. This suggests the involvement of Alu repeat mediated homologous recombination in MLL self fusion. For the 10 rearranged samples, cytogenetics analysis revealed a normal karyotype in 3, and 3 had abnormalities other than 11q23. Survival analysis of patients revealed no difference between those with rearrangement of MLL and those showing the germ-line configuration.

DNA-Binding properties of the replication telomere protein.

The replication Telomere Protein, rTP, is a nuclear protein from the ciliate Euplotes crassus that appears to be a novel telomere replication factor. rTP shares extensive amino acid sequence identity with the two proteins that bind and protect the macronuclear telomeres from the ciliates Oxytricha and Euplotes. Since the most extended regions of conservation fall within the DNA-binding domains of the telomere-binding proteins, when rTP was first identified it was predicted to be another structural telomere-binding protein. However, subsequent research demonstrated that rTP transcripts accumulate only during DNA replication and the rTP protein localizes to the sites of DNA replication within Euplotes macronuclei. We have now expressed rTP in a heterologous expression system and have examined the DNA-binding properties of the recombinant protein. We show that rTP binds specifically to the G-strand of Euplotes telomeric DNA and hence has some of the same DNA-binding characteristics as the Euplotes and Oxytricha telomere-binding proteins. However, other aspects of rTP binding are unique. In particular, the protein exhibits a very high off-rate and can bind double-stranded DNA as well as internal tracts of telomeric sequence. We conclude that rTP and the telomere-binding proteins are members of a class of proteins that have a conserved DNA-binding motif tailored to bind the G-strand of telomeric DNA. However, the unique DNA-binding characteristics of rTP indicate that the protein has evolved to fulfil a specialized role during telomere replication.