Phantom bursting is highly sensitive to noise and unlikely to account for slow bursting in beta-cells: considerations in favor of metabolically driven oscillations

Pancreatic beta-cells show bursting electrical activity with a wide range of burst periods ranging from a few seconds, often seen in isolated cells, over tens of seconds (medium bursting), usually observed in intact islets, to several minutes. The phantom burster model [Bertram, R., Previte, J., Sherman, A., Kinard, T.A., Satin, L.S., 2000. The phantom burster model for pancreatic beta-cells. Biophys. J. 79, 2880-2892] provided a framework, which covered this span, and gave an explanation of how to obtain medium bursting combining two processes operating on different time scales. However, single cells are subjected to stochastic fluctuations in plasma membrane currents, which are likely to disturb the bursting mechanism and transform medium bursters into spikers or very fast bursters. We present a polynomial, minimal, phantom burster model and show that noise modifies the plateau fraction and lowers the burst period dramatically in phantom bursters. It is therefore unlikely that slow bursting in single cells is driven by the slow phantom bursting mechanism, but could instead be driven by oscillations in glycolysis, which we show are stable to random ion channel fluctuations. Moreover, so-called compound bursting can be converted to apparent slow bursting by noise, which could explain why compound bursting and mixed Ca(2+) oscillations are seen mainly in intact islets.     

Simulations of action of DNA topoisomerases to investigate boundaries and shapes of spaces of knots

The configuration space available to randomly cyclized polymers is divided into subspaces accessible to individual knot types. A phantom chain utilized in numerical simulations of polymers can explore all subspaces, whereas a real closed chain forming a figure-of-eight knot, for example, is confined to a subspace corresponding to this knot type only. One can conceptually compare the assembly of configuration spaces of various knot types to a complex foam where individual cells delimit the configuration space available to a given knot type. Neighboring cells in the foam harbor knots that can be converted into each other by just one intersegmental passage. Such a segment-segment passage occurring at the level of knotted configurations corresponds to a passage through the interface between neighboring cells in the foamy knot space. Using a DNA topoisomerase-inspired simulation approach we characterize here the effective interface area between neighboring knot spaces as well as the surface-to-volume ratio of individual knot spaces. These results provide a reference system required for better understanding mechanisms of action of various DNA topoisomerases.     

Dose calculation validation of a convolution algorithm in a solid water phantom

PurposeThe dose calculated using a convolution algorithm should be validated in a simple homogeneous water-equivalent phantom before clinical use. The dose calculation accuracy within a solid water phantom was investigated.MethodsThe specific Gamma knife design requires a dose rate calibration within a spherical solid water phantom. The TMR10 algorithm, which approximates the phantom material as liquid water, correctly computes the absolute dose in water. The convolution algorithm, which considers electron density miscalculates the dose in water as the phantom Hounsfield units were converted into higher electron density when the original CT calibration curve was used. To address this issue, the electron density of liquid water was affected by modifying the CT calibration curve. The absolute dose calculated using the convolution algorithm was compared with that computed by the TMR10. The measured depth dose profiles were also compared to those computed by the convolution and TMR10 algorithms. A patient treatment was recalculated in the solid-water phantom and the delivery quality assurance was checked.ResultsThe convolution algorithm and the TMR10 calculate an absolute dose within 1% when using the modified CT calibration curve. The dose depth profile calculated using the convolution algorithms was superimposed on the TMR10 and measured dose profiles when the modified CT calibration curve was applied. The Gamma index was better than 93%.ConclusionsDose calculation algorithms, which consider electron density, require a CT calibration curve adapted to the phantom material to correctly compute the dose in water.     

Three roads to islet bursting: emergent oscillations in coupled phantom bursters

Glucose-induced membrane potential and Ca(2+) oscillations in isolated pancreatic beta-cells occur over a wide range of frequencies, from >6/min (fast) to <1/min (slow). However, cells within intact islets generally oscillate with periods of 10-60 s (medium). The phantom bursting concept addresses how beta-cells can generate such a wide range of frequencies. Here, we explore an updated phantom bursting model to determine how heterogeneity in a single parameter can explain both the broad frequency range observed in single cells and the rarity of medium oscillations. We then incorporate the single-cell model into an islet model with parameter heterogeneity. We show that strongly coupled islets must be composed of predominantly medium oscillating single cells or a mixture of fast and slow cells to robustly produce medium oscillations. Surprisingly, we find that this constraint does not hold for moderate coupling, and that robustly medium oscillating islets can arise from populations of single cells that are essentially all slow or all fast. Thus, with coupled phantom bursters, medium oscillating islets can be constructed out of cells that are either all fast, all slow, or a combination of the two.     

Development of a deformable phantom for experimental verification of 4D Monte Carlo simulations in a deforming anatomy

PurposeTo verify the accuracy of 4D Monte Carlo (MC) simulations, using the 4DdefDOSXYZnrc user code, in a deforming anatomy. We developed a tissue-equivalent and reproducible deformable lung phantom and evaluated 4D simulations of delivered dose to the phantom by comparing calculations against measurements.MethodsA novel deformable phantom consisting of flexible foam, emulating lung tissue, inside a Lucite external body was constructed. A removable plug, containing an elastic tumor that can hold film and other dosimeters, was inserted in the phantom. Point dose and position measurements were performed inside and outside the tumor using RADPOS 4D dosimetry system. The phantom was irradiated on an Elekta Infinity linac in both stationary and moving states. The dose delivery was simulated using delivery log files and the phantom motion recorded with RADPOS.ResultsReproducibility of the phantom motion was determined to be within 1 mm. The phantom motion presented realistic features like hysteresis. MC calculations and measurements agreed within 2% at the center of tumor. Outside the tumor agreements were better than 5% which were within the positional/dose reading uncertainties at the measurement points. More than 94% of dose points from MC simulations agreed within 2%/2 mm compared to film measurements.ConclusionThe deformable lung phantom presented realistic and reproducible motion characteristics and its use for verification of 4D dose calculations was demonstrated. Our 4DMC method is capable of accurate calculations of the realistic dose delivered to a moving and deforming anatomy during static and dynamic beam delivery techniques.     

The fingerprint of phantom mutations in mitochondrial DNA data

Phantom mutations are systematic artifacts generated in the course of the sequencing process itself. In sequenced mitochondrial DNA (mtDNA), they generate a hotspot pattern quite different from that of natural mutations in the cell. To identify the telltale patterns of a particular phantom mutation process, one first filters out the well-established frequent mutations (inferred from various data sets with additional coding region information). The filtered data are represented by their full (quasi-)median network, to visualize the character conflicts, which can be expressed numerically by the cube spectrum. Permutation tests are used to evaluate the overall phylogenetic content of the filtered data. Comparison with benchmark data sets helps to sort out suspicious data and to infer features and potential causes for the phantom mutation process. This approach, performed either in the lab or at the desk of a reviewer, will help to avoid errors that otherwise would go into print and could lead to erroneous evolutionary interpretations. The filtering procedure is illustrated with two mtDNA data sets that were severely affected by phantom mutations.     

Quality of stallion semen obtained by a new semen collection phantom (Equidame) versus a Missouri artificial vagina

A study was performed to test a new semen collection device (Equidame phantom) that fractionates the ejaculate by comparing the quality of semen obtained by the Equidame phantom with that obtained by a Missouri artificial vagina. Semen from 4 Finnhorse stallions was collected 4 times per stallion by both methods. Half of the ejaculate was frozen and the other half extended and loaded into 2 Equitainer transport containers (24- and 48-h samples). Motility parameters were determined by a Hamilton-Thorn motility analyzer after cooled storage for 24 and 48 h and again after freezing/thawing. Raw and chilled semen samples were cultured and the number of bacterial colonies counted after incubations of 24 and 48 h. After a 24-h incubation the number of colony-forming units (CFU) in raw semen was significantly higher (P<0.01) when collected by the Missouri artificial vagina than by the Equidame phantom. After cooled storage, 75% of the semen samples contained no bacteria after an incubation of 24 h, and 69% yielded no growth after 48 h. The sperm-rich fractions (Cup 2) collected by the Equidame phantom had lower mean volumes (22.1 +/- 2.3 mL [+/- SEM] versus 101.6 +/- 9.3 mL) and significantly higher mean sperm concentrations (218.0 +/- 25.8 x 10(6) vs 86.2 +/- 8.1 x 10(6) cells/mL; P<0.05) than the total ejaculates collected by the Missouri device. The total and progressive motility of chilled and frozen-thawed semen did not differ significantly between collection methods. The Equidame phantom yielded semen that was of a lower bacteriological colony counts, but had sperm motility similar to that of semen collected with the traditional method by the Missouri artificial vagina.     

Motor cortex representation of the upper-limb in individuals born without a hand

The body schema is an action-related representation of the body that arises from activity in a network of multiple brain areas. While it was initially thought that the body schema developed with experience, the existence of phantom limbs in individuals born without a limb (amelics) led to the suggestion that it was innate. The problem with this idea, however, is that the vast majority of amelics do not report the presence of a phantom limb. Transcranial magnetic stimulation (TMS) applied over the primary motor cortex (M1) of traumatic amputees can evoke movement sensations in the phantom, suggesting that traumatic amputation does not delete movement representations of the missing hand. Given this, we asked whether the absence of a phantom limb in the majority of amelics means that the motor cortex does not contain a cortical representation of the missing limb, or whether it is present but has been deactivated by the lack of sensorimotor experience. In four upper-limb amelic subjects we directly stimulated the arm/hand region of M1 to see 1) whether we could evoke phantom sensations, and 2) whether muscle representations in the two cortices were organised asymmetrically. TMS applied over the motor cortex contralateral to the missing limb evoked contractions in stump muscles but did not evoke phantom movement sensations. The location and extent of muscle maps varied between hemispheres but did not reveal any systematic asymmetries. In contrast, forearm muscle thresholds were always higher for the missing limb side. We suggest that phantom movement sensations reported by some upper limb amelics are mostly driven by vision and not by the persistence of motor commands to the missing limb within the sensorimotor cortex. We propose that prewired movement representations of a limb need the experience of movement to be expressed within the primary motor cortex.     

A continuously lit stimulus is perceived to be shorter than a flickering stimulus during a saccade

When subjects made a saccade across a single-flashed dot, a flickering dot or a continuous dot, they perceived a dot, an array (phantom array), or a line (phantom line), respectively. We asked subjects to localize both endpoints of the phantom array or line and calculated the perceived lengths. Based on the findings of Matsumiya and Uchikawa (2001), we predicted that the apparent length of the phantom line would be larger than that of the phantom array. In Experiment 1 of the current study, contrary to the prediction, the phantom line was found to be shorter than the phantom array. In Experiment 2, we investigated whether the function underlying the filled-unfilled space illusion (Lewis, 1912) instead of the function underlying the saccadic compression could explain the results. Subjects were asked to localize both endpoints of a line or an array while keeping their eyes fixated. Although the results of Experiment 2 showed that the perceived length of a line was shorter than that of an array, the function underlying the filled-unfilled illusion could not fully account for the results of Experiment 1. To explain the present results, we proposed a model for the localization process and discussed its validity.     

Development of a dynamic deformable thorax phantom for the quality management of deformable image registration

The purpose of this study was to develop a novel dynamic deformable thorax phantom for deformable image registration (DIR) quality assurance (QA) and to verify as a tool for commissioning and DIR QA.The phantom consists of a base phantom, an inner phantom, and a motor-derived piston. The base phantom is an acrylic cylinder phantom with a diameter of 180 mm. The inner phantom consists of deformable, 20 mm thick disk-shaped sponges. To evaluate the physical characteristics of the phantom, we evaluated its image quality and deformation. DIR accuracies were evaluated using the three types of commercially DIR software (MIM, RayStation, and Velocity AI) to test the feasibility of this phantom. We used different DIR parameters to test the impact of parameters on DIR accuracy in various phantom settings. To evaluate DIR accuracy, a target registration error (TRE) was calculated using the anatomical landmark points.The three locations (i.e., distal, middle, and proximal positions) had different displacement amounts. This result indicated that the inner phantom was not moved but deformed. In cases with different phantom settings and marker settings, the ranges of the average TRE were 0.63–15.60 mm (MIM). In cases with different DIR parameters settings, the ranges of the average TRE were as follows: 0.73–7.10 mm (MIM), 8.25–8.66 mm (RayStation), and 8.26–8.43 mm (Velocity). These results suggest that our phantom could evaluate the detailed DIR behaviors with TRE. Therefore, this is indicative of the potential usefulness of our phantom in DIR commissioning and QA.     

Space radiation absorbed dose distribution in a human phantom.

The radiation risk to astronauts has always been based on measurements using passive thermoluminescent dosimeters (TLDs). The skin dose is converted to dose equivalent using an average radiation quality factor based on model calculations. The radiological risk estimates, however, are based on organ and tissue doses. This paper describes results from the first space flight (STS-91, 51.65 degrees inclination and approximately 380 km altitude) of a fully instrumented Alderson Rando phantom torso (with head) to relate the skin dose to organ doses. Spatial distributions of absorbed dose in 34 1-inch-thick sections measured using TLDs are described. There is about a 30% change in dose as one moves from the front to the back of the phantom body. Small active dosimeters were developed specifically to provide time-resolved measurements of absorbed dose rates and quality factors at five organ locations (brain, thyroid, heart/lung, stomach and colon) inside the phantom. Using these dosimeters, it was possible to separate the trapped-proton and the galactic cosmic radiation components of the doses. A tissue-equivalent proportional counter (TEPC) and a charged-particle directional spectrometer (CPDS) were flown next to the phantom torso to provide data on the incident internal radiation environment. Accurate models of the shielding distributions at the site of the TEPC, the CPDS and a scalable Computerized Anatomical Male (CAM) model of the phantom torso were developed. These measurements provided a comprehensive data set to map the dose distribution inside a human phantom, and to assess the accuracy and validity of radiation transport models throughout the human body. The results show that for the conditions in the International Space Station (ISS) orbit during periods near the solar minimum, the ratio of the blood-forming organ dose rate to the skin absorbed dose rate is about 80%, and the ratio of the dose equivalents is almost one. The results show that the GCR model dose-rate predictions are 20% lower than the observations. Assuming that the trapped-belt models lead to a correct orbit-averaged energy spectrum, the measurements of dose rates inside the phantom cannot be fully understood. Passive measurements using 6Li- and 7Li-based detectors on the astronauts and inside the brain and thyroid of the phantom show the presence of a significant contribution due to thermal neutrons, an area requiring additional study.     

Laser‐induced plasmon‐mediated treatment of retinoblastoma in viscous vitreous phantom

Retinoblastoma (RB) is a rare form of cancer of the retina most prevalent in young children. We successfully show that laser‐induced cell disruption, mediated by gold plasmonic nanoparticle (NP), is a potential and efficient therapy to kill the cancerous cells. The proof of concept is demonstrated in vitro on cultured Y79 RB cancer cells with a nanosecond laser at 527 nm, for both attached cells at the bottom of a Petri dish and for floating, clustered cells in a viscous vitreous phantom comprised of hyaluronan. We report a cellular death of 82% after irradiation in classic culture medium and a cellular death of 98% in vitreous phantom, for similar number of NPs in each sample. It is found that the NPs efficiently penetrate the floating Y79 clusters cells in the vitreous phantom, leading to a cellular death of over 85% even within the centre of the aggregates. The proposed treatment technique is based on a similar nanosecond laser used to eliminate floaters in the vitreous, but with much lower (100‐1000 times) fluences of 20 J cm^?2.      

DNA polymerase activity in a repair-deficient human cell line

A human low-density-lipoprotein (LDL) receptor-deficient diploid fibroblast cell line (GM1915) was determined to be short patch competent (DNA polymerase-beta) and long patch deficient (DNA polymerase-alpha) for DNA excision repair. Analysis of DNA from GM1915 cells or from WI38 control cells, following treatment with a mutagen known to initiate long patch excision repair, showed that GM1915 cells exhibited decreased resynthesis of oligonucleotide segments excised during repair. When cells deficient in DNA polymerase-alpha activity were permeabilized to permit LDL entry, repair synthesis immediately increased. These data suggest that DNA polymerase-alpha is not activated by mutagen treatment in GM1915 cells and that introduction of LDL into the cells results in activation of the enzyme.     

Direct and bystander effects induced by scattered radiation generated during penetration of radiation inside a water-phantom

In this study, the dose distribution of photon (6 MV) and electron (22 MeV) radiation in a water-phantom was compared with the frequency of apoptotic and micronucleated cells of two human cell lines (BEAS-2B normal bronchial epithelial cells and A549 lung cancer epithelial cells). Formation of micronuclei and apoptotic-like bodies was evaluated by the cytokinesis-block micronucleus test. Measurements were performed for five different phantom depths (3-20 cm). Irradiated cells were placed in a water-phantom in three variants: directly on the axis in the beam, under shielding (only in photon radiation) and outside the beam field. The results reveal a discrepancy between the distribution of physical dose at different depths of the water-phantom and biological effects. This discrepancy is of special significance in case of cells irradiated at a greater depth or placed outside the field and under shield during the exposure to radiation. The frequency of cytogenetic damage was higher than the expected value based on the physical dose received at different depths. Cells placed outside the beam axis were exposed to scattered radiation at very low doses, so we tested if bystander effects could have had a role in the observed discrepancy between physical radiation dose and biological response. We explored this question by use of a medium-transfer technique in which medium (ICM-irradiation conditioned medium) from irradiated cells was transferred to non-irradiated (bystander) cells. The results indicate that when cells were incubated in ICM transferred from cells irradiated at bigger depths or from cells exposed outside the radiation field, the number of apoptotic and micronucleated cells was similar to that after direct irradiation. This suggests that these damages are caused by factors released by irradiated cells into the medium rather than being induced directly in DNA by X-rays. Evaluation of biological effects of scattered radiation appears useful for clinical practice.     

Slow variable dominance and phase resetting in phantom bursting

Bursting oscillations are common in neurons and endocrine cells. One type of bursting model with two slow variables has been called ‘phantom bursting’ since the burst period is a blend of the time constants of the slow variables. A phantom bursting model can produce bursting with a wide range of periods: fast (short period), medium, and slow (long period). We describe a measure, which we call the ‘dominance factor’, of the relative contributions of the two slow variables to the bursting produced by a simple phantom bursting model. Using this tool, we demonstrate how the control of different phases of the burst can be shifted from one slow variable to another by changing a model parameter. We then show that the dominance curves obtained as a parameter is varied can be useful in making predictions about the resetting properties of the model cells. Finally, we demonstrate two mechanisms by which phase-independent resetting of a burst can be achieved, as has been shown to occur in the electrical activity of pancreatic islets.     

Progressive changes in cutaneous trigger zones for sensation referred to a phantom hand: a case report and review with implications for cortical reorganization

The dominant model of cortical plasticity induced by peripheral denervation suggests that a physiologically-reorganized cortical area can acquire new perceptual meaning, including a change in the peripheral referral of sensation. An alternative view is that such an area may retain its former perceptual significance, even though it becomes responsive to new peripheral inputs. To examine evidence related to this issue, a clinical case is presented documenting the time course of changes in phantom limb sensation in a patient with accidental amputation of a hand. About 24 h after injury, a vivid phantom hand was present; tactile stimulation revealed cutaneous trigger zones on the arm, stimulation of which elicited sensation referred to specific fingers of the phantom. While the phantom hand percept remained fairly stable over time, the trigger zones expanded progressively in size during the next 1-8 weeks but had contracted and changed location considerably about one year later. At all times studied, the trigger zones were topographically related to specific fingers and other parts of the phantom hand. The implications of these and other recent clinical findings for cortical reorganization are discussed, and the following tentative conclusions are drawn. (1) A phantom percept is mediated by central neural networks which remain functionally intact after amputation. (2) Cutaneous trigger zones mapped in humans correspond to novel receptive fields of cortical neurons mapped in animals following peripheral denervation. (3) Cortical reorganization induced by denervation does not produce a major change in perceptual meaning or peripheral reference. In the present case, stimulation of new trigger zones (receptive fields) on the patient's arm presumably activated a reorganized cortical hand area but evoked sensation still referred to the (now missing) hand. Hence, physiological cortical remapping is not necessarily accompanied by functional respecification.     

Cytotoxic and genotoxic effect of inhibitor of vulcanisation N-cyclohexylthiophthalimide in a battery of in vitro assays

Mutagenicity of N-cyclohexylthiophthalimide (Duslin P) was tested first by the Ames test in the bacteria, Salmonella typhimurium. The negative results of the Ames test suggested that this compound does not induce mutations in the genome of S. typhimurium under the conditions used. To estimate the cytotoxicity of Duslin P to human cells, we measured cellular DNA and protein as well as cell proliferation, i.e., the mitotic index of treated and control cells. The genotoxic effects were assayed by two biochemical methods developed for detection of single-strand breaks of DNA in mammalian cells, i.e., by the alkaline single cell gel electrophoresis (comet assay) and by the DNA unwinding method, respectively. The DNA unwinding method showed that this compound did not induce DNA damage at concentrations < 7 micrograms/ml. Alkaline single cell gel electrophoresis revealed approximately double the level of DNA damage (in comparison to untreated control DNA) at a concentration of 2 micrograms/ml, which reduced proliferation to approximately 30%, and triple the level of DNA damage at higher concentrations (6 and 7 micrograms/ml), which inhibited completely both DNA synthesis and proteosynthesis. Cells with moderately damaged DNA were more common than cells with heavily damaged DNA. Parallel experiments with the strong mutagen and carcinogen MNNG showed that MNNG induced in cells a high level of DNA damage at concentrations which did not reduce the mitotic index or proteosynthesis, while DNA synthesis inhibited only partially. After treatment with MNNG, cells with heavily damaged DNA were more common than cells with moderately damaged DNA. Duslin P-treated VH10 cells were also tested cytogenetically, confirming that Duslin P induced neither chromosomal aberrations nor aneuploidy. We conclude that Duslin P has no mutagenic effect on bacteria, does not induce chromosomal aberrations and CREST positive or CREST negative micronuclei in human cells and induces only a small increase of DNA damage in human cells which is consistent with DNA fragmentation due to cell death.     

Evaluation of Non-Local Means Based Denoising Filters for Diffusion Kurtosis Imaging Using a New Phantom

Image denoising has a profound impact on the precision of estimated parameters in diffusion kurtosis imaging (DKI). This work first proposes an approach to constructing a DKI phantom that can be used to evaluate the performance of denoising algorithms in regard to their abilities of improving the reliability of DKI parameter estimation. The phantom was constructed from a real DKI dataset of a human brain, and the pipeline used to construct the phantom consists of diffusion-weighted (DW) image filtering, diffusion and kurtosis tensor regularization, and DW image reconstruction. The phantom preserves the image structure while minimizing image noise, and thus can be used as ground truth in the evaluation. Second, we used the phantom to evaluate three representative algorithms of non-local means (NLM). Results showed that one scheme of vector-based NLM, which uses DWI data with redundant information acquired at different b-values, produced the most reliable estimation of DKI parameters in terms of Mean Square Error (MSE), Bias and standard deviation (Std). The result of the comparison based on the phantom was consistent with those based on real datasets.     

Bystander effects induced by direct and scattered radiation generated during penetration of medium inside a water phantom

Background

The biological effects of ionizing radiation have long been thought to results from direct targeting of the nucleus leading to DNA damage. Over the years, a number of non-targeted or epigenetic effects of radiation exposure have been reported where genetic damage occurs in cells that are not directly irradiated but respond to signals transmitted from irradiated cells, a phenomenon termed the “bystander effects”.

Aim

We compared the direct and bystander responses of human A 549, BEAS-2-B and NHDF cell lines exposed to both photon (6 MV) and electron (22 MeV) radiation inside a water phantom. The cultures were directly irradiated or exposed to scattered radiation 4 cm outside the field. In parallel, non-irradiated cells (termed bystander cells) were incubated in ICM (irradiation conditioned medium) collected from another pool of irradiated cells (termed donor cells).

Materials and methods

In directly irradiated cells as well as ICM-treated cells, the frequency of micronuclei and condensation of chromatin characteristic for the apoptotic process were estimated using the cytokinesis-block micronucleus test.

Results

In all tested cell lines, radiation induced apoptosis and formation of micronuclei. A549 and BEAS-2B cells cultured in ICM showed increased levels of micronuclei and apoptosis, whereas normal human fibroblasts (NHDF line) were resistant to bystander response. In A549 and BEAS-2B cells placed outside the radiation field and exposed to scattered radiation the formation of micronuclei and induction of apoptosis were similar to that after ICM-treatment.

Conclusion

Results suggest that the genetic damage in cells exposed to scattered radiation is caused by factors released by irradiated cells into the medium rather than by DNA damage induced directly by X rays. It seems that bystander effects may have important clinical implications for health risk after low level radiation exposure of cells lying outside the radiation field during clinical treatment.     

State of hepatitis B viral DNA in a human hepatoma cell line.

PLC/PRF/5, a tissue culture cell line isolated from a human hepatocellular carcinoma and producing hepatitis B surface antigen, was studied for the presence of hepatitis B virus (HBV)-specific DNA and RNA. PLC/PRF/5 cell DNA accelerated the rate of reassociation of HBV [32P]DNA, and quantitative experiments indicated that the cells contained approximately four copies of viral DNA per haploid, mammalian cell DNA equivalent. PLC/PRF/5 DNA accelerated the rate of reassociation of all individual restriction endonucleases HincII and HaeIII fragments of HBV [32P]DNA, indicating that DNA from all regions of the viral genome is present in the cells. This suggests that these cells contain at least most, and possibly all, of the viral genome. Digestion of PLC/PRF/5 cell DNA with restriction endonuclease HindIII (an enzyme found not to cleave the DNA of any HBV isolate so far examined) yielded only three fragments, all larger than virion DNA, which contained HBV DNA base sequences, suggesting that HBV DNA is integrated in high-molecular-weight DNA at three different sites in these cells and that there is no viral DNA in an episomal form. PLC/PRF/5 cell [32P]RNA was found to hybridize with all restriction fragments of HBV DNA adequately tested, indicating that at least most, and possibly all, of the viral DNA in these cells is transcribed.