A panel of eGFP reporters for single base editing by APOBEC-Cas9 editosome complexes.

The prospect of introducing a single C-to-T change at a specific genomic location has become feasible with APOBEC-Cas9 editing technologies. We present a panel of eGFP reporters for quantification and optimization of single base editing by APOBEC-Cas9 editosomes. Reporter utility is demonstrated by comparing activities of seven human APOBEC3 enzymes and rat APOBEC1 (BE3). APOBEC3A and RNA binding-defective variants of APOBEC3B and APOBEC3H display the highest single base editing efficiencies. APOBEC3B catalytic domain complexes also elicit the lowest frequencies of adjacent off-target events. However, unbiased deep-sequencing of edited reporters shows that all editosomes have some degree of local off-target editing. Thus, further optimization is required to generate true single base editors and the eGFP reporters described here have the potential to facilitate this process.

Perspective: APOBEC mutagenesis in drug resistance and immune escape in HIV and cancer evolution.

The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) mutational signature has only recently been detected in a multitude of cancers through next-generation sequencing. In contrast, APOBEC has been a focus of virology research for over a decade. Many lessons learnt regarding APOBEC within virology are likely to be applicable to cancer. In this review, we explore the parallels between the role of APOBEC enzymes in HIV and cancer evolution. We discuss data supporting the role of APOBEC mutagenesis in creating HIV genome heterogeneity, drug resistance, and immune escape variants. We hypothesize similar functions of APOBEC will also hold true in cancer.

Stress concentration around an atelectatic region: a finite element model.

Lung parenchyma surrounding an atelectatic region is thought to be subjected to increased stress compared with the rest of the lung. Using 37 hexagonal cells made of linear springs, Mead et al. (1970) measured a stress concentration greater than 30% in the springs surrounding a stiffer central cell. We re-examine the problem using a 2D finite element model of 500 cells made of thin filaments with a non-linear stress-strain relationship. We study the consequences of increasing the central stiff region from one to nine contiguous cells in regular hexagonal honeycombs and random Voronoi honeycombs. The honeycomb structures were uniformly expanded with strains of 15%, 30%, 45% and 55% above their resting, non-deformed geometry. The curve of biaxial stress vs. fractional area change has a similar shape to that of the pressure-volume curve of the lung, showing an initial regime with relatively flat slope and a final regime with decreasing slope, tending toward an asymptote. Regular honeycombs had little variability in the maximum stress in radially oriented filaments adjacent to the central stiff region. In contrast, some filaments in random Voronoi honeycombs were subjected to stress concentration approximately 16 times the average stress concentration in the radially oriented filaments adjacent to the stiff region. These results may have implications in selecting the appropriate strategy for mechanical ventilation in ARDS and defining a "safe" level of alveolar pressure for ventilating atelectatic lungs.

Peripheral resistance: a link between global airflow obstruction and regional ventilation distribution.

Airflow obstruction and heterogeneities in airway constriction and ventilation distribution are well-described prominent features of asthma. However, the mechanistic link between these global and regional features has not been well defined. We speculate that peripheral airway resistance (R(p)) may provide such a link. Structural and functional parameters are estimated from PET and HRCT images of asthmatic (AS) and nonasthmatic (NA) subjects measured at baseline (BASE) and post-methacholine challenge (POST). Conductances of 35 anatomically defined proximal airways are estimated from airway geometry obtained from high-resolution computed tomography (HRCT) images. Compliances of sublobar regions subtended by 19 most distal airways are estimated from changes in regional gas volume between two lung volumes. Specific ventilations (sV) of these sublobar regions are evaluated from 13NN-washout PET scans. For each pathway connecting the trachea to sublobar region, values of R(p) required to explain the sV distribution and global airflow obstruction are computed. Results show that R(p) is highly heterogeneous within each subject, but has average values consistent with global values in the literature. The contribution of R(p) to total pathway resistance (R(T)) increased substantially for POST (P < 0.0001). The fraction R(p)/R(T) was higher in AS than NA at POST (P < 0.0001) but similar at BASE (range: 0.960-0.997, median: 0.990). For POST, R(p)/R(T) range was 0.979-0.999 (NA) and 0.981-0.995 (AS). This approach allows for estimations of peripheral airway resistance within anatomically defined sublobar regions in vivo human lungs and may be used to evaluate peripheral effects of therapy in a subject specific manner.

Functional effect of longitudinal heterogeneity in constricted airways before and after lung expansion.

Heterogeneity in narrowing among individual airways is an important contributor to airway hyperresponsiveness. This paper investigates the contribution of longitudinal heterogeneity (the variability along the airway in cross-sectional area and shape) to airway resistance (R(aw)). We analyzed chest high-resolution computed tomography scans of 8 asthmatic (AS) and 9 nonasthmatic (NA) subjects before and after methacholine (MCh) challenge, and after lung expansion to total lung capacity. In each subject, R(aw) was calculated for 35 defined central airways with >2 mm diameter. Ignoring the area variability and noncircular shape results in an underestimation of R(aw) (%U(total)) that was substantial in some airways (∼50%) but generally small (median <6%). The average contribution of the underestimation of R(aw) caused by longitudinal heterogeneity in the area (%U(area)) to %U(total) was 36%, while the rest was due to the noncircularity of the shape (%U(shape)). After MCh challenge, %U(area) increased in AS and NA (P < 0.05). A lung volume increase to TLC reduced %U(total) and %U(area) in both AS and NA (P < 0.0001, except for %U(total) in AS with P < 0.01). Only in NA, %U(shape) had a significant reduction after increasing lung volume to TLC (P < 0.005). %U(area) was highly correlated, but not identical to the mean-normalized longitudinal heterogeneity in the cross-sectional area [CV(2)(A)] and %U(shape) to the average eccentricity of the elliptical shape. This study demonstrates that R(aw) calculated assuming a cylindrical shape and derived from an average area along its length may, in some airways, substantially underestimate R(aw). The observed changes in underestimations of R(aw) with the increase in lung volume to total lung capacity may be consistent with, and contribute in part to, the differences in effects of deep inhalations in airway function between AS and NA subjects.

First Report of Fusarium Wilt of Canary Island Date Palm Caused by Fusarium oxysporum f. sp. canariensis in Louisiana.

Canary Island date palm (Phoenix canariensis Hort. Ex Chabaud) is a signature palm planted in New Orleans, LA. Currently, the city has approximately 1,000 mature Canary Island date palms. During the fall of 2009, 153 palms were inspected with 27 palms exhibiting typical symptoms of Fusarium wilt. Symptoms included one-sided death and a reddish brown streak on the rachis of affected fronds and death of the leaflets. Longitudinal sections of affected fronds showed vascular discoloration. Severely infected palms were completely dead. Small pieces of diseased tissue from five palms were surface sterilized with sodium hypochlorite (0.6%) for 2 to 3 min, then rinsed in sterile distilled water, blotted dry, and plated on potato dextrose agar (PDA). Fungal colonies on PDA produced a purple pigment, and both macro- and microconidia that are typical of Fusarium oxysporum were observed under a light microscope. A single-spore culture of isolate PDC-4701 was obtained. DNA from this isolate was extracted with a DNeasy Plant Mini kit (Qiagen Inc., Valencia, CA) and primers ef1 and ef2 were used to amplify and sequence the translation elongation factor 1-α gene (2). NCBI BLAST analysis of the 616-bp sequence resulted in 100% identity with F. oxysporum f. sp. canariensis isolates PLM-385B from Texas and PLM-511A from South Carolina (GenBank Accession Nos. HM 591538 and HM 591537, respectively). Isolate PDC-4701, grown on PDA for 2 weeks, was used to inoculate 10 9-month-old P. canariensis seedlings. An 18-gauge needle was used to inject 15 ml of a 107 conidia/ml suspension into the stem near the soil line. Each seedling was inoculated at two locations and covered with Parafilm at the inoculation sites. Ten control seedlings were injected with sterile distilled water in the same manner. Inoculated and control seedlings were maintained in a greenhouse at 28 ± 2°C. Leaves of all 10 inoculated seedlings started to wilt 3 months after inoculation. Internal vascular discoloration was observed and the pathogen was reisolated from the symptomatic seedlings. No symptoms developed on any of the 10 control seedlings. On the basis of morphology and DNA sequence data, this pathogen is identified as F. oxysporum f. sp. canariensis. Fusarium wilt of Canary Island date palm has been previously reported from California, Florida, Nevada, Texas, and South Carolina (1). To our knowledge, this is the first report of Fusarium wilt of Canary Island date palm caused by F. oxysporum f. sp. canariensis in Louisiana, extending its geographic range. The disease may adversely affect the tradition of planting Canary Island date palms in New Orleans. The sequence of isolate PDC-4701 has been submitted to the NCBI database (GenBank Accession No. JF826442) and a culture specimen has been deposited in the Fusarium Research Center culture collection (Accession No. O-2602) at the Pennsylvania State University, University Park, PA. References: (1) M. L. Elliott et al. Plant Dis. 95:356, 2011. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004.

Evidence for an extraterrestrial impact 12,900 years ago that contributed to the megafaunal extinctions and the Younger Dryas cooling.

A carbon-rich black layer, dating to approximately 12.9 ka, has been previously identified at approximately 50 Clovis-age sites across North America and appears contemporaneous with the abrupt onset of Younger Dryas (YD) cooling. The in situ bones of extinct Pleistocene megafauna, along with Clovis tool assemblages, occur below this black layer but not within or above it. Causes for the extinctions, YD cooling, and termination of Clovis culture have long been controversial. In this paper, we provide evidence for an extraterrestrial (ET) impact event at approximately equal 12.9 ka, which we hypothesize caused abrupt environmental changes that contributed to YD cooling, major ecological reorganization, broad-scale extinctions, and rapid human behavioral shifts at the end of the Clovis Period. Clovis-age sites in North American are overlain by a thin, discrete layer with varying peak abundances of (i) magnetic grains with iridium, (ii) magnetic microspherules, (iii) charcoal, (iv) soot, (v) carbon spherules, (vi) glass-like carbon containing nanodiamonds, and (vii) fullerenes with ET helium, all of which are evidence for an ET impact and associated biomass burning at approximately 12.9 ka. This layer also extends throughout at least 15 Carolina Bays, which are unique, elliptical depressions, oriented to the northwest across the Atlantic Coastal Plain. We propose that one or more large, low-density ET objects exploded over northern North America, partially destabilizing the Laurentide Ice Sheet and triggering YD cooling. The shock wave, thermal pulse, and event-related environmental effects (e.g., extensive biomass burning and food limitations) contributed to end-Pleistocene megafaunal extinctions and adaptive shifts among PaleoAmericans in North America.

Relationship between airway narrowing, patchy ventilation and lung mechanics in asthmatics.

Bronchoconstriction in asthma results in patchy ventilation forming ventilation defects (VDefs). Patchy ventilation is clinically important because it affects obstructive symptoms and impairs both gas exchange and the distribution of inhaled medications. The current study combined functional imaging, oscillatory mechanics and theoretical modelling to test whether the degrees of constriction of airways feeding those units outside VDefs were related to the extent of VDefs in bronchoconstricted asthmatic subjects. Positron emission tomography was used to quantify the regional distribution of ventilation and oscillatory mechanics were measured in asthmatic subjects before and after bronchoconstriction. For each subject, ventilation data was mapped into an anatomically based lung model that was used to evaluate whether airway constriction patterns, consistent with the imaging data, were capable of matching the measured changes in airflow obstruction. The degree and heterogeneity of constriction of the airways feeding alveolar units outside VDefs was similar among the subjects studied despite large inter-subject variability in airflow obstruction and the extent of the ventilation defects. Analysis of the data amongst the subjects showed an inverse relationship between the reduction in mean airway conductance, measured in the breathing frequency range during bronchoconstriction, and the fraction of lung involved in ventilation defects. The current data supports the concept that patchy ventilation is an expression of the integrated system and not just the sum of independent responses of individual airways.

Locally generated particulate pollution and respiratory symptoms in young children.

BACKGROUND: Particulate matter <10 mum (PM(10)) from fossil fuel combustion is associated with an increased prevalence of respiratory symptoms in children and adolescents. However, the effect of PM(10) on respiratory symptoms in young children is unclear. METHODS: The association between primary PM(10) (particles directly emitted from local sources) and the prevalence and incidence of respiratory symptoms was studied in a random sample cohort of 4400 Leicestershire children aged 1-5 years surveyed in 1998 and again in 2001. Annual exposure to primary PM(10) was calculated for the home address using the Airviro dispersion model and adjusted odds ratios (ORS) and 95% confidence intervals were calculated for each microg/m(3) increase. RESULTS: Exposure to primary PM(10) was associated with the prevalence of cough without a cold in both 1998 and 2001, with adjusted ORs of 1.21 (1.07 to 1.38) and 1.56 (1.32 to 1.84) respectively. For night time cough the ORs were 1.06 (0.94 to 1.19) and 1.25 (1.06 to 1.47), and for current wheeze 0.99 (0.88 to 1.12) and 1.28 (1.04 to 1.58), respectively. There was also an association between primary PM(10) and new onset symptoms. The ORs for incident symptoms were 1.62 (1.31 to 2.00) for cough without a cold and 1.42 (1.02 to 1.97) for wheeze. CONCLUSION: In young children there was a consistent association between locally generated primary PM(10) and the prevalence and incidence of cough without a cold and the incidence of wheeze which was independent of potential confounders.

Modeling kinetics of infused 13NN-saline in acute lung injury.

A mathematical model was developed to estimate right-to-left shunt (Fs) and the volume of distribution of 13NN in alveolar gas (VA) and shunt tissue (Vs). The data obtained from this model are complementary to, and obtained simultaneously with, pulmonary functional positron emission tomography (PET). The model describes 13NN kinetics in four compartments: central mixing volume, gas-exchanging lung, shunting compartment, and systemic recirculation. To validate the model, five normal prone (NP) and six surfactant-depleted sheep in the supine (LS) and prone (LP) positions were studied under general anesthesia. A central venous bolus of 13NN-labeled saline was injected at the onset of apnea as PET imaging and arterial 13NN sampling were initiated. The model fit the tracer kinetics well (mean r2 = 0.93). Monte Carlo simulations showed that parameters could be accurately identified in the presence of expected experimental noise. Fs derived from the model correlated well with shunt estimates derived from O2 blood concentrations and from PET images. Fs was higher for LS (54 +/- 18%) than for LP (5 +/- 4%) and NP (1 +/- 1%, P < 0.01). VA, as a fraction of PET-measured lung gas volume, was lower for LS (0.18 +/- 0.09) than for LP (0.96 +/- 0.28, P < 0.01), whereas Vs, as a fraction of PET-measured lung tissue volume, was higher for LS (0.46 +/- 0.26) than for LP (0.05 +/- 0.08, P < 0.01). The main conclusions are as follows: 1) the model accurately describes measured arterial 13NN kinetics and provides estimates of Fs, and 2) in this animal model of acute lung injury, the fraction of available gas volume participating in gas exchange is reduced in the supine position.

Epstein-Barr virus and the somatic hypermutation of immunoglobulin genes in Burkitt's lymphoma cells.

It has been suggested that Epstein-Barr virus (EBV) might suppress antibody maturation either by facilitating bypass of the germinal center reaction or by inhibiting hypermutation directly. However, by infecting the Burkitt's lymphoma (BL) cell line Ramos, which hypermutates constitutively and can be considered a transformed analogue of a germinal center B cell, with EBV as well as by transfecting it with selected EBV latency genes, we demonstrate that expression of EBV gene products does not lead to an inhibition of hypermutation. Moreover, we have identified two natural EBV-positive BL cell lines (ELI-BL and BL16) that hypermutate constitutively. Thus, contrary to expectations, EBV gene products do not appear to affect somatic hypermutation.

Appendiceal disease in women with endometriosis and right lower quadrant pain.

STUDY OBJECTIVE: To evaluate the frequency and range of appendiceal disease in women with endometriosis and right lower quadrant (RLQ) pain, and to estimate the value of preoperative gastrograffin enema (GGE) as a screen for the disease. DESIGN: Nonrandomized clinical trial (Canadian Task Force classification II-2). SETTING: University-affiliated hospital with a private practice setting. PATIENTS: A subpopulation of 65 women from a group of 337 patients undergoing laparoscopy for symptomatic endometriosis. INTERVENTION: Preoperative GGE was performed whenever possible in these women. At laparoscopy, the appendix was removed if it appeared abnormal or if the preoperative GGE was positive. MEASUREMENTS AND MAIN RESULTS: Of 65 women (19%) with symptomatic endometriosis and preoperative RLQ pain, 52 (80%) underwent appendectomy as part of surgery. Of these 52 excised appendixes, 39 (75%) had histologically confirmed pathology including appendicitis or periappendicitis, endometriosis, fibrous obliteration, lymphoid hyperplasia, and carcinoid tumor. Preoperative GGE had sensitivity of 74% and specificity of 83% for appendiceal disease. Its positive predictive value was 95% and negative predictive value was 42%. No complications from laparoscopic appendectomy occurred. CONCLUSION: Disease of the appendix is common in women with endometriosis and RLQ pain. Appendectomy is particularly likely if preoperative GGE is positive.

The SOS response regulates adaptive mutation.

Upon starvation some Escherichia coli cells undergo a transient, genome-wide hypermutation (called adaptive mutation) that is recombination-dependent and appears to be a response to a stressful environment. Adaptive mutation may reflect an inducible mechanism that generates genetic variability in times of stress. Previously, however, the regulatory components and signal transduction pathways controlling adaptive mutation were unknown. Here we show that adaptive mutation is regulated by the SOS response, a complex, graded response to DNA damage that includes induction of gene products blocking cell division and promoting mutation, recombination, and DNA repair. We find that SOS-induced levels of proteins other than RecA are needed for adaptive mutation. We report a requirement of RecF for efficient adaptive mutation and provide evidence that the role of RecF in mutation is to allow SOS induction. We also report the discovery of an SOS-controlled inhibitor of adaptive mutation, PsiB. These results indicate that adaptive mutation is a tightly regulated response, controlled both positively and negatively by the SOS system.

Use of recruitment maneuvers and high-positive end-expiratory pressure in a patient with acute respiratory distress syndrome.

OBJECTIVE: To present the use of a novel high-pressure recruitment maneuver followed by high levels of positive end-expiratory pressure in a patient with the acute respiratory distress syndrome (ARDS). DESIGN: Observations in one patient. SETTING: The medical intensive care unit at a tertiary care university teaching hospital. PATIENT: A 32-yr-old woman with severe ARDS secondary to streptococcal sepsis. INTERVENTIONS: The patient had severe gas exchange abnormalities because of acute lung injury and marked lung collapse. Attempts to optimize recruitment based on the inflation pressure-volume (PV) curve were not sufficient to avoid dependent lung collapse. We used a recruitment maneuver using 40 cm H2O of positive end-expiratory pressure (PEEP) and 20 cm H2O of pressure controlled ventilation above PEEP for 2 mins to successfully recruit the lung. The recruitment was maintained with 25 cm H2O of PEEP, which was much higher than the PEEP predicted by the lower inflection point (P(Flex)) of the PV curve. MEASUREMENTS AND MAIN RESULTS: Recruitment was assessed by improvements in oxygenation and by computed tomography of the chest. With the recruitment maneuvers, the patient had a dramatic improvement in gas exchange and we were able to demonstrate nearly complete recruitment of the lung by computed tomography. A PV curve was measured that demonstrated a P(Flex) of 16-18 cm H2O. CONCLUSION: Accumulating data suggest that the maximization and maintenance of lung recruitment may reduce lung parenchymal injury from positive pressure ventilation in ARDS. We demonstrate that in this case PEEP alone was not adequate to recruit the injured lung and that a high-pressure recruitment maneuver was required. After recruitment, high-level PEEP was needed to prevent derecruitment and this level of PEEP was not adequately predicted by the P(Flex) of the PV curve.

An objective analysis of the pressure-volume curve in the acute respiratory distress syndrome.

To assess the interobserver and intraobserver variability in the clinical evaluation of the quasi-static pressure-volume (P-V) curve, we analyzed 24 sets of inflation and deflation P-V curves obtained from patients with ARDS. We used a recently described sigmoidal equation to curve-fit the P-V data sets and objectively define the point of maximum compliance increase of the inflation limb (P(mci, i)) and the true inflection point of the deflation limb (P(inf,d)). These points were compared with graphic determinations of lower Pflex by seven clinicians. The graphic and curve-fitting methods were also compared for their ability to reproduce the same parameter value in data sets with reduced number of data points. The sigmoidal equation fit the P-V data with great accuracy (R(2) = 0.9992). The average of Pflex determinations was found to be correlated with P(mci,i) (R = 0.89) and P(inf,d) (R = 0.76). Individual determinations of Pflex were less correlated with the corresponding objective parameters (R = 0.67 and 0.62, respectively). Pflex + 2 cm H(2)O was a more accurate estimator of P(inf,d) (2 SD = +/-6.05 cm H(2)O) than Pflex was of P(mci,i) (2 SD = +/-8.02 cm H(2)O). There was significant interobserver variability in Pflex, with a maximum difference of 11 cm H(2)O for the same patient (SD = 1.9 cm H(2)O). Clinicians had difficulty reproducing Pflex in smaller data sets with differences as great as 17 cm H(2)O (SD = 2.8 cm H(2)O). In contrast, the curve-fitting method reproduced P(mci,i) with great accuracy in reduced data sets (maximum difference of 1.5 cm H(2)O and SD = 0.3 cm H(2)O). We conclude that Pflex rarely coincided with the point of maximum compliance increase defined by a sigmoid curve-fit with large differences in Pflex seen both among and within observers. Calculating objective parameters such as P(mci,i) or P(inf,d) from curve-fitted P-V data can minimize this large variability.

Mismatch repair is diminished during stationary-phase mutation.

This paper is an invited Response to a recent Commentary [P.L. Foster, Rev. Mut. Res. 436 (1999) 179-184] entitled "Are adaptive mutations due to a decline in mismatch repair? The evidence is lacking". The Commentary argues that no evidence exists supporting the idea that mismatch repair is limiting specifically during stationary-phase mutation. A primary concern of the author is to question the method that we used previously to measure growth-dependent mutation. In this method, mutation rates are calculated using counts of mutant colonies taken at times when those colonies arise, rather than at a predetermined, fixed time. Here we show further data that illustrate why this must be done to ensure accurate mutation measurements. Such accuracy was necessary for our published determination that mismatch repair proteins are not limiting during growth-dependent mutation, but become so during stationary-phase mutation. We review the evidence supporting the idea that stationary-phase reversion of a lac frameshift mutation occurs in an environment of decreased mismatch repair capacity. Those data are substantial. The data presented in the Commentary, in apparent contradiction to this idea, do not justify the conclusion presented there.

Somatic hypermutation and the three R's: repair, replication and recombination.

Somatic hypermutation introduces single base changes into the rearranged variable (V) regions of antigen activated B cells at a rate of approximately 1 mutation per kilobase per generation. This is nearly a million-fold higher than the typical mutation rate in a mammalian somatic cell. Rampant mutation at this level could have a devastating effect, but somatic hypermutation is accurately targeted and tightly regulated. Here, we provide an overview of immunoglobulin gene somatic hypermutation; discuss mechanisms of mutation in model organisms that may be relevant to the hypermutation mechanism; and review recent advances toward understanding the possible role(s) of DNA repair, replication, and recombination in this fascinating process.

Recombination-based mechanisms for somatic hypermutation.

We review some experiments designed to test recombination-based mechanisms for somatic hypermutation in mice, particularly mechanisms involving templated mutation or gene conversion. As recombination and repair functions are highly conserved among prokaryotes and eukaryotes, pathways of mutation in microorganisms may prove relevant to the mechanism of somatic hypermutation. Escherichia coli initiates a recombination-based pathway of mutation in response to environmental stimuli, and this "adaptive" pathway of mutation has striking similarities with somatic hypermutation, as does a process of mutagenic repair that occurs at double-strand breaks in Saccharomyces cerevisiae. We present a model for recombination-based hypermutation of the immunoglobulin loci which could result in either templated or non-templated mutation.

Transient and heritable mutators in adaptive evolution in the lab and in nature.

Major advances in understanding the molecular mechanism of recombination-dependent stationary-phase mutation in Escherichia coli occurred this past year. These advances are reviewed here, and we also present new evidence that the mutagenic state responsible is transient. We find that most stationary-phase mutants do not possess a heritable stationary-phase mutator phenotype, although a small proportion of heritable mutators was found previously. We outline similarities between this well-studied system and several recent examples of adaptive evolution associated with heritable mutator phenotype in a similarly small proportion of survivors of selection in nature and in the lab. We suggest the following: (1) Transient mutator states may also be a predominant source of adaptive mutations in these latter systems, the heritable mutators being a minority (Rosenberg 1997); (2) heritable mutators may sometimes be a product of, rather than the cause of, hypermutation that gives rise to adaptive mutations.