Distinct mutational signatures characterize concurrent loss of polymerase proofreading and mismatch repair.

Fidelity of DNA replication is maintained using polymerase proofreading and the mismatch repair pathway. Tumors with loss of function of either mechanism have elevated mutation rates with characteristic mutational signatures. Here we report that tumors with concurrent loss of both polymerase proofreading and mismatch repair function have mutational patterns that are not a simple sum of the signatures of the individual alterations, but correspond to distinct, previously unexplained signatures: COSMIC database signatures 14 and 20. We then demonstrate that in all five cases in which the chronological order of events could be determined, polymerase epsilon proofreading alterations precede the defect in mismatch repair. Overall, we illustrate that multiple distinct mutational signatures can result from different combinations of a smaller number of mutational processes (of either damage or repair), which can influence the interpretation and discovery of mutational signatures.

Whole-genome sequencing reveals activation-induced cytidine deaminase signatures during indolent chronic lymphocytic leukaemia evolution.

Patients with chromosome 13q deletion or normal cytogenetics represent the majority of chronic lymphocytic leukaemia (CLL) cases, yet have relatively few driver mutations. To better understand their genomic landscape, here we perform whole-genome sequencing on a cohort of patients enriched with these cytogenetic characteristics. Mutations in known CLL drivers are seen in only 33% of this cohort, and associated with normal cytogenetics and unmutated IGHV. The most commonly mutated gene in our cohort, IGLL5, shows a mutational pattern suggestive of activation-induced cytidine deaminase (AID) activity. Unsupervised analysis of mutational signatures demonstrates the activities of canonical AID (c-AID), leading to clustered mutations near active transcriptional start sites; non-canonical AID (nc-AID), leading to genome-wide non-clustered mutations, and an ageing signature responsible for most mutations. Using mutation clonality to infer time of onset, we find that while ageing and c-AID activities are ongoing, nc-AID-associated mutations likely occur earlier in tumour evolution.

The calcium-sensing receptor regulates calcium absorption in MDCK cells by inhibition of PMCA.

Calcium transport across a monolayer of Madin-Darby canine kidney (MDCK) cells was measured in response to stimulation of the basal surface with calcium-sensing receptor (CaR) agonists. Stimulation of the CaR resulted in a time- and concentration-dependent inhibition of calcium transport but did not change transepithelial voltage or resistance. Inhibition of transport was not altered by pretreatment of cells with pertussis toxin but was blocked by the phospholipase C (PLC) inhibitor U-73122. To determine a potential mechanism by which the CaR could inhibit calcium transport, we measured activity of the plasma membrane calcium ATPase (PMCA). Stimulation of the CaR on the basal surface resulted in an inhibition of the PMCA in a concentration- and PLC-dependent manner. Thus stimulation of the CaR inhibits both calcium transport and PMCA activity through a PLC-dependent pathway. These studies provide the first direct evidence that calcium can inhibit its own transcellular absorption in a model of the distal tubule. In addition, they provide a potential mechanism for the CaR to inhibit calcium transport, inhibition of PMCA.

Patient-oriented outcomes from low back surgery: a community-based study.

STUDY DESIGN: This study used a prospective cohort design. OBJECTIVE: To examine factors associated with favorable self-reported patient outcomes 1 year after elective surgery for degenerative back problems. SUMMARY OF BACKGROUND DATA: Many previous studies addressing the results of low back surgery have been conducted in academic institutions or by single surgeons. As part of a quality improvement effort, surgeons in private practice led a community-based outcomes management project in Washington State. METHODS: Patients ages 18 and older with the following diagnoses were eligible for the study: degenerative changes, herniated disc, instability, and spinal stenosis. Nine orthopedists and neurosurgeons enrolled a total of 281 patients. Participants were asked to complete baseline and 1-year follow-up surveys. Data concerning diagnoses, clinical signs, and operative procedures were provided by the surgeons. The researchers examined sociodemographic characteristics, self-reported symptoms before surgery, preoperative clinical signs, diagnoses, and operative procedures associated with three primary outcomes: better functioning, improved quality of life, and overall treatment satisfaction. RESULTS: Follow-up surveys were completed by 236 (84%) of the enrolled patients. Approximately two thirds of the study participants reported much better functioning (65%), a great quality of life improvement (64%), and a very positive perspective about their treatment outcome (68%). The following variables were associated with worse patient outcomes: older age, previous low back surgery, workers' compensation coverage, and consultation with an attorney before surgery. Patients undergoing a fusion procedure were more likely to report good outcomes. CONCLUSIONS: The authors' experience indicates that community-based outcomes data collection efforts are feasible and can be incorporated into usual clinical practice. The study results indicate that compensation payments and litigation are two important predictors of poor outcomes after low back surgery in community practice. Because of small numbers, varied diagnoses, and possible selection bias, the findings with respect to fusion should be interpreted cautiously.

Limitations in the neuroprotective potential of gene therapy with Bcl-2.

Considerable attention has focused on the therapeutic transfer of genes with viral vectors into neurons for the purpose of protecting against neurological insults. A number of papers have reported that overexpression of the anti-apoptotic protein Bcl-2 can protect neurons both in vitro and in vivo against a variety of necrotic insults. An emerging literature suggests that the availability of energy tends to modulate a neuron towards dying apoptotically, rather than necrotically, in the aftermath of an insult. This suggests that an anti-apoptotic protein such as Bcl-2 should be minimally protective, at best, against purely energetic insults. In support of this idea, we report that overexpression of Bcl-2 with a herpes simplex viral vector fails to protect hippocampal neurons, either in vitro or in vivo, against the electron transport uncoupler 3-acetylpyridine (3AP). As a positive control, the same vector significantly protected against the excitotoxin kainic acid. This finding supports the view that neurotoxicity induced by 3AP is likely to have only minimal apoptotic facets. On a broader level, it suggests some limitations in the neuroprotective potential of gene therapy with Bcl-2.

Inflammatory responses and their impact on beta-galactosidase transgene expression following adenovirus vector delivery to the primate caudate nucleus.

An E1, E3 deleted adenovirus vector, serotype 5, carrying the marker gene LacZ was bilaterally microinfused into the caudate nuclei of 10 St Kitts green monkeys. The location and number of cells expressing transgene and host immunologic response were evaluated at 1 week (n = 2) and 1 month (n = 8) following vector infusion. A large number of cells expressed beta-galactosidase in some monkeys, exceeding 600000 in one monkey, but no expression was seen in three of 10. All monkeys had positive adenoviral antibody titers before vector infusion, indicating the possibility of previous exposure to some adenovirus, but only one showed a significant increase in titer afterwards. Inflammatory cell markers revealed an inverse correlation between transgene expression and the extent of inflammatory response. Dexamethasone administered immediately before and for 8 days following vector delivery, however, had no effect on transgene expression. The demonstration of significant inflammatory responses in the brain of some individual primates, including demyelination, indicates the need for new generations of adenovirus vectors, or the successful suppression of inflammatory responses, before this vector is suitable for non-cytotoxic clinical applications in the CNS.

Herpes simplex viral vectors expressing Bcl-2 are neuroprotective when delivered after a stroke.

Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system for the purpose of decreasing necrotic neuronal injury. To that end, we have previously shown that a herpes simplex virus (HSV) vector expressing Bcl-2 could protect neurons from ischemia. In that study, vector was delivered before the ischemia. However, for such gene therapy to be of clinical use, vectors must be protective even if delivered after the onset of the insult. In the present study, we show that an HSV vector expressing Bcl-2 protects striatal neurons when delivered after focal ischemia. Rats were exposed to middle cerebral artery occlusion for 1 hour, followed by reperfusion, and damage was assessed 48 hours later. Delivery of the Bcl-2 vector 30 minutes after reperfusion (i.e., 1.5 hours after ischemia onset) prevented any significant loss of virally-targeted neurons in the striatum. In contrast, in rats microinfused with a vector only expressing a reporter gene, a highly significant loss of neurons occurred. By 4 hours into the reperfusion period (5 hours after ischemia onset), delivery of the Bcl-2 vector was no longer protective. These data show the efficacy of postinsult gene therapy strategies for the brain, underline the finite length of this temporal therapeutic window, and support the growing evidence attesting to the neuroprotective potential of Bcl-2.

Overexpression of Bcl-2 with herpes simplex virus vectors protects CNS neurons against neurological insults in vitro and in vivo.

Previous studies have demonstrated that overexpression of the proto-oncogene bcl-2 can protect neuron and neuron-like cell lines from growth factor deprivation, calcium ionophores, glutamate excitotoxicity, hypoglycemia, free radicals, and lipid peroxidation. To determine whether Bcl-2 exhibits a similar protective effect in CNS neurons, we generated defective herpes simplex virus (HSV) vectors capable of overexpressing Bcl-2 in primary cultures and in the intact brain. Infection of hippocampal cultures with Bcl-2 vectors enhanced neuron survivorship after exposure to adriamycin, a potent oxygen radical generator. Furthermore, dichlorofluorescein measurements indicated that there was a significant reduction in the accumulation of oxygen radicals associated with this insult. Bcl-2 vectors also enhanced survival in cultured neurons after exposure to glutamate and hypoglycemia. Most significantly, the in vivo delivery of the vector protected neurons against adriamycin toxicity in the dorsal horn of the dentate gyrus and focal ischemia in the striatum.

Glucocorticoids accelerate ATP loss following metabolic insults in cultured hippocampal neurons.

Glucocorticoids (GCs), adrenal steroids released during stress, can damage the hippocampus outright and increase hippocampal vulnerability to metabolic insults. Changes in ATP levels were measured in response to aglycemia and to cyanide in cultured hippocampal neurons that had been exposed to high- and low-GC conditions. GCs did not depress baseline ATP levels but did accelerate the rate of the decline in ATP concentrations observed during the metabolic insults. These results support the hypothesis that GCs increase neuronal vulnerability by disrupting cellular metabolism and agree with similar findings in hippocampal astrocytes.

List size, screening methods, and other characteristics of practices in relation to preventive care.

Twenty eight practices carried out a review of patient records for information about preventive procedures on two occasions in 1980 and 1982. We have now undertaken a survey of certain characteristics of the practices in an attempt to demonstrate features associated with effective preventive care. Significant favourable factors are a small list size, the setting up of a formal screening programme for cervical cytology and measuring blood pressure, and few registered patients in social classes IV and V. More successful practices also tend to be training practices, have principals with higher qualifications, and have developed good records organization. Opportunistic screening for cervical cytology and measuring blood pressure was not shown to be more effective than no policy of screening at all.

Impact of audit on preventive measures.

The results of two reviews of practice records for information about various preventive measures are reported. The reviews were conducted two and a half years apart in the same 29 practices, on roughly 6500 records on each occasion. The first review was carried out during a postgraduate education course involving audit. Two main analyses were undertaken: one concerned with the entire record sample and the other with the individual practice results. Important and substantial improvements were shown in both analyses, which were spread throughout the practices and were not confined to practices at the lower end of the range simply "catching up." The change between the reviews in the mean recorded rates for cervical cytology was 56% to 64%; for rubella immunity 28% to 40%; for polio immunity in adults 15% to 21%; for completed primary immunisation 68% to 78%; for recorded blood pressure 53% to 61%; and for smoking information 22% to 30%. Improvement has been steady but the rate of improvement, especially for recording blood pressure and rubella immunity, has increased since the postgraduate education course.

An evaluation of recorded information about preventive measures in 38 practices.

This paper reports the results of a review of practice records for information about various preventive measures; 8,500 records from 38 practices were studied. In collecting the data a Practice Activity Analysis data sheet was used.The results are presented in a way which makes it possible for others undertaking these analyses to compare their results with those reported here. They point to a number of conclusions, in particular:1. Much less cervical cytology has been done for women in their 50s than for those in their 30s and 40s; in particular 45 per cent of women in their 50s have never had a smear.2. Only 35 per cent of girls aged 15 to 19 were known to be immune to rubella.3. Only 14 per cent of adults aged 20 to 40 were known to be immune to polio.4. Of men in their 40s, 47 per cent had no record of their blood pressure having been taken during the previous 10 years.5. Information about smoking habits was available in 23 per cent of records.