Graphenylene Nanotubes.

A new type of carbon nanotube, based on the graphenylene motif, is investigated using density functional and tight-binding methods. Analogous to conventional graphene-based nanotubes, a two-dimensional graphenylene sheet can be "rolled" into a seamless cylinder in armchair, zigzag, or chiral orientations. The resulting nanotube can be described using the familiar (n,m) nomenclature and possesses 4-, 6-, and 12-membered rings, with three distinct bond lengths, indicating a nonuniform distribution of the electron density. The dodecagonal rings form pores, 3.3 Å in diameter in graphenylene, which become saddle-shaped paraboloids in smaller-diameter nanotubes. Density functional theory predicts zigzag nanotubes to be small-band gap semiconductors, with a generally decreasing band gap as the diameter increases. Interestingly, the calculations predict metallic characteristics for armchair nanotubes with small diameters (<2 nm), and small-band gap semiconducting characteristics for larger-diameter ones. Graphenylene nanotubes with indices mod(n-m,3) = 0 exhibit a band gap approximately equal to that of armchair graphenylene nanotubes with comparable diameter.

Design of aging intervention studies: the NIA interventions testing program.

The field of biogerontology has made great strides towards understanding the biological processes underlying aging, and the time is ripe to look towards applying this knowledge to the pursuit of aging interventions. Identification of safe, inexpensive, and non-invasive interventions that slow the aging process and promote healthy aging could have a significant impact on quality of life and health care expenditures for the aged. While there is a plethora of supplements and interventions on the market that purport to slow aging, the evidence to validate such claims is generally lacking. Here we describe the development of an aging interventions testing program funded by the National Institute on Aging (NIA) to test candidate interventions in a model system. The development of this program highlights the challenges of long-term intervention studies and provides approaches to cope with the stringent requirements of a multi-site testing program.

Optimizing detection of QTLs retarding aging: choice of statistical model and animal requirements.

Quantitative trait locus (QTL) analysis makes no assumptions about the identity of genes involved in regulating aging. Moreover, it may be used as the first step in identifying such genes and, thus QTL analysis may be instrumental in formulating new hypotheses about aging. Genetic experiments, however, require hundreds to thousands of animals and are very expensive in mammals. Statistical power to detect longevity genes could be improved by excluding accidental, unrelated to aging mortality. While many early deaths are probably accidental, excluding early mortality altogether eliminates the age-related component, too. We used computer simulations to assess the effect of excluding early age-related, mortality on the statistical power of several common tests, such as t-test, Mann-Whitney and chi(2). Surprisingly, even the age-related, Gompertz component of early mortality reduces the statistical power of the t- and Mann-Whitney tests. For example, in a backcross design, to detect a gene slowing down the rate of aging and increasing mouse life span by 10% (P=0.0001; power=0.8), a regular t-test will require 640 mice, all kept for the entire life span and genotyped. If life spans of only 25% of the longest-lived animals from each of the two groups, carrying a putative longevity allele and not carrying it, are compared, population size can be reduced by two-fold, to about 300, and genotyping by seven-fold, to 90. Confirming simulation results, the significance of the effect of caloric restriction on life span increased from P=3.4x10(-5) to 1.1x10(-7), when life spans of only 40% of the longest-lived mice from each of the two groups, ad libitum fed and calorie restricted, were compared. Finally, finding the optimal combination of statistical test, the number of phenotyped and the number of genotyped animals, which would minimize experimental costs was addressed.

Hematopoietic stem cell functional failure in interleukin-2-deficient mice.

The effects of interleukin-2 (IL-2) deficiency on hematopoiesis were tested by measuring cellular compositions in peripheral blood, spleen, thymus, and bone marrow of 3- to 5-month-old gene-targeted Il2 null (Il2(-/-)) mice using the Advia 120 Hematology system and fluorescence-activated cell staining (FACS). Il2(-/-) mice developed hematological failure and autoimmune responses, showing variable but significant degrees of anemia, lymphocytopenia, thrombocytopenia, splenomegaly, thymus involution, and weight loss. Surprisingly, Il2(-/-) mice had normal numbers of bone marrow cells (BMCs) with increased numbers of Lin(-)Kit(+)Sca1(+)CD34(-) and Lin(-)Kit(+)Sca1(+)CD34(+) cells that are normally associated with hematopoietic stem cells (HSCs) and progenitor cells. Day-12 colony-forming units-spleen cells were slightly reduced in Il2(-/-) mice. When Il2(-/-) and Il2(+/+) mice were compared for long-term HSC function in vivo in the competitive repopulation assay, BMCs from Il2(-/-) donors had 10- to 20-fold less HSC repopulating ability, which affected both myeloid and lymphoid cell lineages. Thus, HSCs from Il2(-/-) mice can proliferate normally but are functionally defective for reconstituting lethally irradiated recipients.

The effects of combined x-axis translations and y-axis rotations on projected lamina junction offset.

OBJECTIVES: To quantify the projection errors caused by the combining of selected x-axis translations with selected y-axis rotations as seen on plain film anteroposterior (AP) radiographs. STUDY DESIGN: A computer was used to model the projection of the lamina junction and points on the lateral body margins of a 2-dimensional vertebral model onto an AP radiograph. This simulation was done in the neutral position and in a number of combinations of x-axis translations and y-axis rotations. RESULTS: Some combinations of x-axis translation and y-axis rotation can increase or decrease the projected horizontal lamina offset as compared with what would be found with rotation alone. The projection of some combinations may lead the viewer to believe that rotation of the vertebral model is in the opposite direction of its true rotation. CONCLUSION: The projections of combined movements of x-axis translations and y-axis rotations can lead to confusion when the clinician attempts to discern quantity and direction of y-axis vertebral rotation from the AP radiograph.

Maximum life spans in mice are extended by wild strain alleles.

The genes that control basic aging mechanisms in mammals are unknown. By using two four-way crosses, each including a strain derived from wild, undomesticated stocks, we identified two quantitative trait loci that extend murine life spans by approximately 10%. In one cross, the longest-lived 18% of carriers of the D8Mit171 marker allele from the MOLD/Rk strain, Mus m. molossinus, outlived the longest lived 18% of noncarriers by 129 days (P = 5.4 x 10(-5)); in a second cross, carriers of the D10Mit267 allele from the CAST/Ei strain, Mus m. castaneus, outlived noncarriers by 125 days ( P = 1.6 x 10(-6)). In both crosses, P < 1.0 x 10(-4 )is considered significant. Because these life span increases required that all essential biological systems function longer than normal, these alleles most likely retarded basic aging mechanisms in multiple biological systems simultaneously.

Reliability of lateral bending and axial rotation with validity of a new method to determine axial rotation on anteroposterior cervical radiographs.

OBJECTIVE: To investigate the reliability of a new radiographic measurement of axial rotation and lateral bending on anterior-posterior cervical views by using a computer and sonic digitizer. DESIGN: A blind, repeated-measure design was used. Anteroposterior cervicothoracic radiographs were presented to each of 3 examiners in random order. Each film was digitized, and 1 week later the films were randomized for a second run. SETTING: Private, primary-care chiropractic clinic. MAIN OUTCOME MEASURES: The interclass and intraclass correlation coefficients (ICC) for intraexaminer and interexaminer reliability were calculated from measurements on radiographs for determining axial rotations (Ry) and lateral bending (Rz) of C3 to T3. RESULTS: When the new axial rotation method was applied to small rotations of a C3 plastic model, the average error was less than 1 degrees. For the calculations of axial rotation (Ry), the ICC values were in the good to excellent range. For axial rotation, the intraclass correlation coefficients were ICCs > or =0.78, and the interclass correlation coefficients were ICCs > or =0.67. For lateral flexions (Rz) of C3 to T3, all intraclass and interclass correlation coefficients were in the excellent range (ICCs > 0.87). CONCLUSIONS: Methods of calculating axial rotations in the spine have been reported for large angles (5 degrees to 30 degrees ) but not for smaller angles. A new method for determining axial rotations of the cervical segments on AP views, based on the chord across the arc displaced by the spinous-lamina junction, had reliability (ICC values) in the good to excellent range. Compared with measured rotations of a C3 model (-5 degrees to +5 degrees ), the new method had an average error of less than 1 degrees and approximately 11.5%. The reliability for the axial rotation measurements was in the good to excellent range, and the lateral bending measurements were all in the excellent range.

Reliability of centroid, Cobb, and Harrison posterior tangent methods: which to choose for analysis of thoracic kyphosis.

STUDY DESIGN: Thirty lateral thoracic radiographs were digitized twice by each of the three examiners. OBJECTIVES: To determine the reliability of the centroid, Cobb, and Harrison posterior tangent methods when applied to analysis of thoracic kyphosis. BACKGROUND DATA: Reliability studies on measurements of thoracic kyphosis are rare. METHODS: Blind, repeated-measures design was used. Thirty lateral thoracic radiographs were digitized twice by each of three examiners. To evaluate reliability of determining global and segmental alignment, vertebral bodies of T1-T12 were digitized. Centroids at the intersection of vertebral body diagonals and tangents to posterior vertebral bodies were constructed by computer. Also the computer constructed global and segmental centroid angles, Cobb angles (two-line method), and posterior tangent intersection angles from T1 to T12. Interclass and Intraclass correlation coefficients for these data were calculated and interpreted. RESULTS: From the points selected by examiners, all three methods have similar high ICC values for the global angles (> 0.94). For the segmental angles, the interobserver and intraobserver reliability is also very similar for all three methods, with ICCs in the good and excellent ranges (0.59-0.75 and 0.75-1.0, respectively). The mean absolute differences of observers' measurements are low, similar, and in the range of 0.9 degrees to 2.5 degrees. CONCLUSIONS: The centroid, two-line Cobb, and Harrison posterior tangent methods, when applied to measurements of kyphosis, are all reliable and have similar small error ranges. The centroid method does not give an accurate segmental analysis, uses more points and more time in clinical applications, and results in smaller angles of total kyphosis than the Cobb or posterior tangent methods. The posterior tangents are the slopes along the curve.

Radiographic analysis of lumbar lordosis: centroid, Cobb, TRALL, and Harrison posterior tangent methods.

STUDY DESIGN: Delayed, repeated measures, with three examiners each twice digitizing thirty lateral lumbar radiographs. OBJECTIVES: To determine the reliability and clinical utility of the centroid, Cobb, tangential radiologic assessment of lumbar lordosis (TRALL), and Harrison posterior tangent line-drawing methods for analysis of lumbar lordosis. BACKGROUND DATA: Cobb's method is commonly used for curvature analysis on lateral lumbar radiographs, whereas the centroid, TRALL, and Harrison posterior tangent methods are not widely used. METHODS: Thirty lateral lumbar radiographs were digitized twice by each of three examiners. To evaluate reliability of determining global and segmental alignment, all four vertebral body corners of T12-S1 and the superior margin of the femur head were digitized. Angles created were segmental and global centroid, (two-line) Cobb angles, and intersections of posterior tangents. A global TRALL angle was determined. Means, standard deviations, mean absolute differences, interclass and intraclass correlation coefficients (ICC), and confidence intervals were calculated. RESULTS: The interobserver and intraobserver reliabilities of measuring all segmental and global angles were in the high range (ICCs > 0.83). The mean absolute differences of observers' measurements were small (0.6 degrees -2.0 degrees ). Distal segmental (L4-S1) and global angles of lumbar curvature were dependent on the method of measurement. CONCLUSIONS: All four radiographic methods had high reliability and low mean absolute differences of observers' measurements. Because it lacks a segmental analysis, the TRALL method is not recommended. The centroid, Cobb, and Harrison posterior tangent methods provide global and segmental angles. However, the centroid segmental method requires three segments and is less useful for a stability analysis.

Slight head extension: does it change the sagittal cervical curve?

It is commonly believed that slight flexion/extension of the head will reverse the cervical lordosis. The goal of the present study was to determine whether slight head extension could result in a cervical kyphosis changing into a lordosis. Forty consecutive volunteer subjects with a cervical kyphosis and with flexion in their resting head position had a neutral lateral cervical radiograph followed immediately by a lateral cervical view taken in an extended head position to level the bite line. Subjects were patients at a spine clinic in Elko, Nevada. All radiographs were digitized. Global and segmental angles of the cervical curve were compared for any change in angle due to slight extension of the head. The average extension of the head required to level the bite line was 13.9 degrees. This head extension was not substantially correlated with any segmental or global angle of lordosis. Subjects were categorized into those requiring slight head extension (0 degree-13.9 degrees) and those requiring a significant head extension (> 13.9 degrees). In the slight head extension group, the average change in global angle between posterior tangents on C2 and C7 was 6.9 degrees, and 80% of this change occurred in C1-C4. In the significant head extension group, the average change in global angle between posterior tangents on C2 and C7 was 11.0 degrees, and the major portion of this change occurred in C1-C4. Out of 40 subjects, only one subject, who was in the significant head extension group and had only a minor segmental kyphosis, changed from kyphosis to lordosis. The results show that slight extension of the head does not change a reversed cervical curve into a cervical lordosis as measured on lateral cervical radiographs. Only small extension angle changes (mean sum = 4.8 degrees) in the upper cervical segments (C2-C4) occur in head extension of 14 degrees or less.

Comparison of axial and flexural stresses in lordosis and three buckled configurations of the cervical spine.

OBJECTIVE: To calculate and compare combined axial and flexural stresses in lordosis versus buckled configurations of the sagittal cervical curve. DESIGN: Digitized measurements from lateral cervical radiographs of four different shapes were used to calculate axial loads and bending moments on the vertebral bodies of C2-C7.Background. Osteoarthritis and spinal degeneration are factors in neck and back pain. Calculations of stress in clinically occurring configurations of the sagittal cervical spine are rare. METHODS: Center of gravity of the head (inferior-posterior sella turcica) and vertebral body margins were digitized on four different lateral cervical radiographs: lordosis, kyphosis, and two "S"-shapes. Polynomials (seventh degree) and stress concentrations on the concave and convex margins were derived for the shape of the sagittal cervical curvatures from C1 to T1. Moments of inertia were determined from digitizing and the use of an elliptical shell model of cross-section. Moment arms from a vertical line through the center of gravity of the head to the atlas and scaled neck extensor moment arms from the literature were used to compute the vertical component of extensor muscle effort. Segmental lever arms were calculated from a vertical line through C1 to each vertebra. RESULTS: In lordosis, anterior and posterior stresses in the vertebral body are nearly uniform and minimal. In kyphotic areas, combined stresses changed from tension to compression at the anterior vertebral margins and were very large (6-10 times as large in magnitude) compared to lordosis. In kyphotic areas at the posterior vertebral body, the combined stresses changed from compression (in lordosis) to tension. CONCLUSIONS: The stresses in kyphotic areas are very large and opposite in direction compared to a normal lordosis. This analysis provides the basis for the formation of osteophytes (Wolff's Law) on the anterior margins of vertebrae in kyphotic regions of the sagittal cervical curve. This indicates that any kyphosis is an undesirable configuration in the cervical spine. Relevance. Osteophytes and osteoarthritis are found at areas of altered stress and strain. Axial and flexural stresses at kyphotic areas in the sagittal cervical spine are abnormally high.

Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production.

Single-gene mutations that extend lifespan provide valuable tools for the exploration of the molecular basis for age-related changes in cell and tissue function and for the pathophysiology of age-dependent diseases. We show here that mice homozygous for loss-of-function mutations at the Pit1 (Snell dwarf) locus show a >40% increase in mean and maximal longevity on the relatively long-lived (C3H/HeJ x DW/J)F(1) background. Mutant dw(J)/dw animals show delays in age-dependent collagen cross-linking and in six age-sensitive indices of immune system status. These findings thus demonstrate that a single gene can control maximum lifespan and the timing of both cellular and extracellular senescence in a mammal. Pituitary transplantation into dwarf mice does not reverse the lifespan effect, suggesting that the effect is not due to lowered prolactin levels. In contrast, homozygosity for the Ghrhr(lit) mutation, which like the Pit1(dw) mutation lowers plasma growth hormone levels, does lead to a significant increase in longevity. Male Snell dwarf mice, unlike calorically restricted mice, become obese and exhibit proportionately high leptin levels in old age, showing that their exceptional longevity is not simply due to alterations in adiposity per se. Further studies of the Pit1(dw) mutant, and the closely related, long-lived Prop-1(df) (Ames dwarf) mutant, should provide new insights into the hormonal regulation of senescence, longevity, and late life disease.

Genetic variability in responses to caloric restriction in animals and in regulation of metabolism and obesity in humans.

Panel 5 focused on genetic factors that might mediate or moderate the effects of caloric restriction (CR) on longevity. Panel members stated that currently there is limited information directly addressing these issues. Therefore, they focused attention on what studies could be done. In addition, the panel believed that certain conceptual issues merited clarification and focused attention on this issue. Human studies and studies of nonhuman model organisms were discussed. The panel found at least three reasons why it would be valuable to find genes that influence the (putative) longevity-promoting effect of CR in humans. Such knowledge would offer: (a) the ability to predict individual responses to CR; (b) increased understanding of physiological mechanisms; and (c) the potential to develop mechanism-based interventions to promote longevity or healthy aging. In addition, the panel emphasized several macro-level recommendations regarding research strategies to avoid, research strategies to emphasize, and resources needing development.

Primitive hematopoietic stem cell function in vivo is uniquely high in the CXB-12 mouse strain.

Bone marrow cells (BMCs) from CXB-12/HiaJ (CXB-12) mice had 14 times the total long-term repopulating ability found in the best of 11 other CXB recombinant inbred (RI) lines. BMCs from each RI line donor were mixed with genetically marked standard competitor BMCs from the BALB/cByxC57BL/6 F1 (CByB6F1) hybrid, the mice used to produce the RI lines, and the mixtures repopulated lethally irradiated CByB6F1 recipients. Percentages of donor-type erythrocytes and lymphocytes measured the actual long-term repopulating functions of the donor RI lines relative to the standard competitor. CXB-12 BMCs repopulated better after 3 or 6 months than after 1 month, suggesting that the most primitive precursors were involved. Compared to CByB6F1 standard competitor cells, CXB-12 cells repopulated 3 to 12 times as well, with their advantage increasing when higher doses of cells were transplanted, probably because of hybrid resistance of the recipient against low doses. This was far better than expected, because F1 cells normally function 2 to 3 times as well as cells from an inbred strain. In competitive dilution, the advantage resulted from 2 factors: more precursor cells and more function per precursor. In the model that best fit the data, CXB-12 donors had 2.4 times the concentration of hematopoietic stem cells (HSCs) as the CByB6F1 standard, and each HSC repopulated 1.4 times as well. CXB-12 mice did not have elevated erythrocyte and lymphocyte numbers in blood and marrow and did not have unusually elevated concentrations of colony-forming unit spleen, cobblestone colonies, and long-term colony-initiating cells in marrow.

In vivo kinetics of murine hemopoietic stem cells.

We used stochastic modeling and computer simulation to study the replication, apoptosis, and differentiation of murine hemopoietic stem cells (HSCs) in vivo. This approach allows description of the behavior of an unobserved population (ie, HSCs) on the basis of the behavior of observed progeny cells (ie, granulocytes and lymphocytes). The results of previous limiting-dilution, competitive-repopulation studies in 44 mice were compared with the results of simulated transplantation studies to identify parameters that led to comparable outcomes. Using this approach, we estimated that murine HSCs replicate (on average) once every 2.5 weeks and that the frequency of murine HSCs is 8 per 10(5) nucleated marrow cells. If it is assumed that short-term repopulating cells are distinct from HSCs, that they contribute to hemopoiesis early after transplantation, and that they are independently regulated, a frequency of 4 HSCs per 10(5) nucleated marrow cells also allows simulations that best approximate the observed data. When stochastic modeling and computer simulation were applied to limiting-dilution, autologous-transplantation studies in cats heterozygous for glucose-6-phosphate-dehydrogenase, different estimates of HSC replication rate (1 per 8.3-10 weeks) and frequency (6 per 10(7) cells) were derived. Therefore, it appears that these parameters vary inversely with increased longevity, size, or both. An implication of these data is that human HSCs may be less frequent and replicate more slowly. These findings on cell kinetics have several implications.

Murine hematopoietic stem cell characterization and its regulation in BM transplantation.

Using 5-color fluorescence-activated cell sorting, we isolated a subset of murine pluripotent hematopoietic stem cells (PHSC) with the phenotype Lin(-) Sca(+) kit(+) CD38(+) CD34(-) that appears to fulfill the criteria for most primitive PHSC. In the presence of whole bone marrow (BM) competitor cells, these cells produced reconstitution in lethally irradiated primary, secondary, and tertiary murine transplant recipients over the long term. However, these cells alone could not produce reconstitution in lethally irradiated recipients. Rapid proliferation of these cells after BM transplantation required the assistance of another BM cell subset, which has the phenotype Lin(-) Sca(+) kit(+) CD38(-) CD34(+).

Cobb method or Harrison posterior tangent method: which to choose for lateral cervical radiographic analysis.

STUDY DESIGN: Thirty lateral cervical radiographs were digitized twice by three examiners to compare reliability of the Cobb and posterior tangent methods. OBJECTIVES: To determine the reliability of the Cobb and Harrison posterior tangent methods and to compare and contrast these two methods. SUMMARY OF BACKGROUND DATA: Cobb's method is commonly used on both anteroposterior and lateral radiographs, whereas the posterior tangent method is not widely used. METHODS: A blind, repeated-measures design was used. Thirty lateral cervical radiographs were digitized twice by each of three examiners. To evaluate reliability of determining global and segmental alignment, vertebral bodies of C1-T1 were digitized. Angles created were two global two-line Cobb angles (C1-C7 and C2-C7), segmental Cobb angles from C2 to C7, and posterior tangents drawn at each posterior vertebral body margin. Cobb's method and the posterior tangent method are compared and contrasted with these data. RESULTS: Of 34 intraclass and interclass correlation coefficients, 28 were in the high range (>0.7), and 6 were in the good range (0.6-0.7). The Cobb method at C1-C7 overestimated the cervical curvature (-54 degrees ) and, at C2-C7 it underestimated the cervical curve (-17 degrees ), whereas the posterior tangents were the slopes along the curve (-26 degrees from C2 to C7). The inferior vertebral endplates and posterior body margins did not meet at 90 degrees (C2: 105 degrees +/- 5.2 degrees, C3: 99.7 degrees +/- 5.2 degrees, C4: 99.9 degrees +/- 5.8 degrees, C5: 96.1 degrees +/- 4.5 degrees, C6: 97.0 degrees +/- 3.8 degrees, C7: 95.4 degrees +/- 4.1 degrees ), which caused the segmental Cobb angles to underestimate lordosis at C2-C3, C4-C5, and C6-C7. CONCLUSIONS: Although both methods are reliable with the majority of correlation coefficients in the high range (ICC > 0.7), from the literature, the posterior tangent method has a smaller standard error of measurement than four-line Cobb methods. Global Cobb angles compare only the ends of the cervical curve and cannot delineate what happens to the curve internally. Posterior tangents are the slopes along the curve and can provide an analysis of any buckled areas of the cervical curve. The posterior tangent method is part of an engineering analysis (first derivative) and more accurately depicts cervical curvature than the Cobb method.

Cervical coupling during lateral head translations creates an S-configuration.

OBJECTIVE: To determine cervical coupling during the posture of lateral head translation relative to a fixed thoracic cage. DESIGN: Digitized measurements from anteroposterior cervical radiographs of 20 volunteers were obtained in neutral, left, and right lateral translation posture of the head compared to a fixed thorax. BACKGROUND DATA: Clinically, lateral translation of the head is a common posture. Ranges of motion and spinal coupling have not been reported for this movement. METHODS: Vertebral body corners, mid-lateral articular pillars and the superior spinous-lamina junction of C3-T4 were digitized on 60 radiographs. Using the orthogonal axis of positive x-direction to the left, vertical as positive y and anterior as positive z, digitized points were used to measure projected segmental z-axis rotation, y-axis rotation, and segmental lateral translations of each vertebra. RESULTS: Subjects translated their heads laterally a mean of 51 mm. The major coupled motion was lateral bending (z-axis rotation), which changed direction at the C4-C5 disc space creating an S-shape. Upper cervical (C3-C4) lateral bending was contralateral to the main motion of head translation direction. Lower cervical and upper thoracic lateral bending were ipsilateral. Other segmental motions averaged less than 1 mm and 1 degrees. CONCLUSIONS: Lateral head translations (x-axis) compared to a fixed thoracic cage can be large with a mean of 51 mm to one side. The major spinal coupling was lateral bending which changed direction at C4-C5 resulting in an S-configuration. This might have application in side impacts. All other segmental movements were small, less than 1 mm and 1 degrees. RELEVANCE: The clinically common posture of lateral head translation results in an S-shaped cervical spine and may occur in side impact trauma. This posture has not been studied for cervical coupling patterns or range of motion (ROM).