Chromatin three-dimensional interactions mediate genetic effects on gene expression.

Studying the genetic basis of gene expression and chromatin organization is key to characterizing the effect of genetic variability on the function and structure of the human genome. Here we unravel how genetic variation perturbs gene regulation using a dataset combining activity of regulatory elements, gene expression, and genetic variants across 317 individuals and two cell types. We show that variability in regulatory activity is structured at the intra- and interchromosomal levels within 12,583 cis-regulatory domains and 30 trans-regulatory hubs that highly reflect the local (that is, topologically associating domains) and global (that is, open and closed chromatin compartments) nuclear chromatin organization. These structures delimit cell type-specific regulatory networks that control gene expression and coexpression and mediate the genetic effects of cis- and trans-acting regulatory variants on genes.

High-level recombination specific to polyomavirus genomes targeted to the integration-transformation pathway.

An unusually high incidence of interviral recombination was found in the process of integration of the polyomavirus genome concomitant with neoplastic transformation of nonpermissive cells. Transformants were isolated after mixed infections of Fischer rat cells with two mutants lacking restriction endonuclease sites and were analyzed for the presence of unselected integrated recombinant restriction fragments. A large fraction of the transformants isolated (38% of the 64 transformed cell lines studied) contained recombinant viral genomes that had undergone recombination in a 1.3-, 1.7-, or 3.6-kilobase-pair interval. More than 90% of these recombinant transformants showed evidence of crossovers in multiple intervals. To our knowledge, the recombination frequencies observed in these experiments represent the highest frequencies of homologous recombination reported for a mitotic mammalian system that does not involve transfection. In contrast to the elevated level of recombination in the integrated viral genomes, no evidence of recombination was obtained among the replicated unintegrated pool of viral genomes isolated from the same population of infected cells from which the recombinant transformants were derived. Either of two hypotheses can provide an explanation for the segregated recombination: either recombination occurs at elevated levels in a small, recombination-prone fraction of the population destined to become transformed, or recombination occurs only among those viral genomes which are engaged in the process of integration and thus interact with a recombinogenic host machinery (for example, the host scaffold). We favor the latter hypothesis.

Viral DNA synthesis in nonpermissive rat F-111 cells and its role in neoplastic transformation by polyomavirus.

We have investigated the occurrence and role of polyomavirus DNA synthesis in neoplastic transformation by this virus. We show that after infection of Fischer rat F-111 cells at 37 degrees C, there is two- to threefold increase in the level of viral DNA as compared with the input signal, with a peak observed between 5 and 7 days postinfection. Viral DNA synthesis is about 10 times higher at 33 degrees C and increases up to 15 days postinfection. Most of the viral DNA produced is supercoiled (form I DNA). On the basis of in situ hybridization, it appears that viral replication is restricted to a small fraction of the population. At the lower temperature, more cells are permissive for viral DNA synthesis and the level of synthesis per permissive cell is higher. The DNA synthesis observed is large T-antigen dependent, and the increase in viral DNA synthesis at 33 degrees C is paralleled by an increase in the expression of this viral protein. When large T antigen is inactivated, the half-life of de novo-synthesized viral DNA is less than 12 h, suggesting that large T antigen may be responsible for the stability of the viral genomes as well as their synthesis. Surprisingly, at early times postinfection (0 to 48 h), when the essential function of large T antigen in transformation is expressed (as demonstrated in shift-up experiments with tsa mutants), the level of large T antigen is below the detection level and is at least 10-fold lower than the levels observed in permissive infections at the start of viral DNA synthesis. The difference in viral DNA at 37 and 33 degrees C allowed us to study its effect on transformation. Although an increase in transformation frequency is observed in wild-type A2 infections carried at 33 degrees C (frequencies two to three times higher than at 37 degrees C), this increase appears to be unrelated to the increase in viral DNA synthesis. Furthermore, the overall level of viral DNA and large T antigen in F-111 cells may not affect the integration of the viral genome, since the patterns of integration in cells transformed by wild-type A2 at 33 and 37 degrees C appear similar. The results are compatible with a role for large T antigen in integration-transformation which is not simply to amplify the viral genome to enhance the probability of its integration.

Incidence of cancer among patients with rheumatoid arthritis.

BACKGROUND: To evaluate hypotheses about the relationship between immune alterations and cancer, several investigators have determined cancer incidence in groups of patients with rheumatoid arthritis (RA), a chronic autoimmune disease. The primary finding has been an increased risk of hematopoietic cancers. PURPOSE: In this study, we have attempted to refine estimates of the association between RA and subsequent development of specific cancers. METHODS: We investigated site-specific cancer risk associated with RA in a population-based cohort study of 11683 Swedish men and women with a hospital (inpatient) diagnosis of RA. These case patients were identified from 1965 to 1983 and had follow-up through 1984 by computer linkage of the Swedish Hospital Inpatient Register to the National Swedish Cancer Registry (840 case patients with cancer) and the Swedish Registry of Causes of Death. Cancer risk was estimated by standardized incidence ratios (SIRs) for specific cancers. RESULTS: For men and women overall, there were decreased risks for cancers of the colon (SIR = 0.63; 95% confidence interval [CI] = 0.5-0.9), rectum (SIR = 0.72; 95% CI = 0.5-1.1), and stomach (SIR = 0.63; 95% CI = 0.5-0.9) and an increased risk for lymphomas (SIR = 1.98; 95% CI = 1.5-2.6). CONCLUSIONS: The reduced risk for colorectal cancer in patients with RA is consistent with previous studies of RA patients and with reports which state that use of nonsteroidal anti-inflammatory drugs may protect against the development of large bowel cancers. The excess of lymphomas also confirms a number of earlier investigations of RA patients.

Morphology and immunohistochemistry of spontaneous chronic corneal epithelial defects (SCCED) in dogs.

PURPOSE: To determine the morphologic features of the epithelium and extracellular matrix in spontaneous chronic corneal epithelial defects (SCCED) in dogs. METHODS: Forty-eight superficial keratectomy specimens were obtained after confirmation of the presence of a superficial corneal erosion for longer than 3 weeks with no discernible underlying cause. Histologic samples were examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. Immunolocalization of laminin, collagen IV, fibronectin, and collagen VII was performed. RESULTS: Epithelial cells adjacent to the defect were poorly attached to the underlying extracellular matrix. A prominent superficial stromal hyaline acellular zone composed of collagen fibrils in the area of the erosion was present in most specimens. Samples exhibited a varying degree of fibroplasia, vascularization, and leukocytic infiltrate. Laminin, collagen IV, and collagen VII were usually either not present or were present only in discontinuous segments on the surface of the erosion. Fibronectin usually coated the surface of the erosion, either as a continuous sheet or in discontinuous segments. Transmission electron microscopy of 15 samples revealed that the basement membrane was either absent in the area of the erosion or was present only in discontinuous segments. Scanning electron microscopy of eight of nine samples confirmed the absence of continuous basement membrane. Epithelial and extracellular matrix components in the peripheral cornea appeared normal. CONCLUSIONS: Most canine patients with spontaneous chronic corneal epithelial defects do not have a normal basement membrane structure in the region of the epithelial defect and have other abnormalities in the subjacent extracellular matrix that may reflect a part of the underlying pathophysiology of chronic and recurrent erosions.

Breast cancer mortality after diagnostic radiography: findings from the U.S. Scoliosis Cohort Study.

STUDY DESIGN: A retrospective cohort study was conducted in 5573 female patients with scoliosis who were referred for treatment at 14 orthopedic medical centers in the United States. Patients were less than 20 years of age at diagnosis which occurred between 1912 and 1965. OBJECTIVES: To evaluate patterns in breast cancer mortality among women with scoliosis, with special emphasis on risk associated with diagnostic radiograph exposures. SUMMARY OF BACKGROUND DATA: A pilot study of 1030 women with scoliosis revealed a nearly twofold statistically significant increased risk for incident breast cancer. Although based on only 11 cases, findings were consistent with radiation as a causative factor. METHODS: Medical records were reviewed for information on personal characteristics and scoliosis history. Diagnostic radiograph exposures were tabulated based on review of radiographs, radiology reports in the medical records, radiograph jackets, and radiology log books. Radiation doses were estimated for individual examinations. The mortality rate of the cohort through January 1, 1997, was determined by using state and national vital statistics records and was compared with that of women in the general U. S. population. RESULTS: Nearly 138,000 radiographic examinations were recorded. The average number of examinations per patient was 24.7 (range, 0-618); mean estimated cumulative radiation dose to the breast was 10.8 cGy (range, 0-170). After excluding patients with missing information, 5466 patients were included in breast cancer mortality analyses. Their mean age at diagnosis was 10.6 years and average length of follow-up was 40.1 years. There were 77 breast cancer deaths observed compared with the 45.6 deaths expected on the basis of U.S. mortality rates (standardized mortality ratio [SMR] = 1.69; 95% confidence interval [CI] = 1.3-2.1). Risk increased significantly with increasing number of radiograph exposures and with cumulative radiation dose. The unadjusted excess relative risk per Gy was 5.4 (95% CI = 1.2-14.1); when analyses were restricted to patients who had undergone at least one radiographic examination, the risk estimate was 2.7 (95% CI = -0. 2-9.3). CONCLUSIONS: These data suggest that exposure to multiple diagnostic radiographic examinations during childhood and adolescence may increase the risk of breast cancer among women with scoliosis; however, potential confounding between radiation dose and severity of disease and thus with reproductive history may explain some of the increased risk observed.

Use of the mental health on-call system on a university campus.

Over the course of 1 calendar year, clinicians at a university mental health service collected data on every clinical case in which students presented after normal business hours or on weekends for urgent mental health consultation. During the year, 50 such incidents were recorded, which translated to a rate of 6.6 on-call events per year per 1,000 students. Students were primarily self-referred or referred by the student health center or residence life staff. Suicidal concerns, panic/anxiety, and depressive states were the three most common presenting complaints. Average clinician time per case was 1 1/2 hours, with sexual assault cases taking the most time per case, followed by substance abuse and suicidality. Follow-up outpatient counseling was employed in 76% of the cases. The results highlight the importance of on-call mental health services on college campuses.

Effect of topical phenylephrine on the equine pupil.

The mydriatic effect of 10% phenylephrine was evaluated in 9 horses. Base-line pupillary size in mesopic conditions and during light stimulation was ascertained before application of pharmacologic agents. In study 1, 10% phenylephrine was applied to each eye (n = 5 horses). After 15 minutes, the pupillary size was determined in both lighting conditions. Phenylephrine was again applied to each eye, and after an additional 15 minutes, the pupillary size was determined. In study 2, 1% tropicamide was applied to each eye (n = 4 horses), and after 30 minutes, the pupillary size was determined in both lighting conditions. Tropicamide and phenylephrine were applied to each eye immediately after determination of pupillary size, and after an additional 30 minutes, the pupillary size was determined. There was no significant change (P greater than 0.9) in pupillary size when phenylephrine was used alone or when phenylephrine was used in an eye that had been dilated with tropicamide.

Ocular angiosarcoma in four horses.

In 4 horses with ocular angiosarcomas, the common characteristics were that all horses were aged, and the neoplasms developed initially on conjunctival surfaces, grew slowly, and metastasized despite excision and radiation treatment. Histopathologically, the neoplasms were of 2 types: a capillary pattern of scant stroma and endothelial-lined vascular channels, and a solid pattern composed of sheets of pleomorphic cells with hyperchromatic nuclei and poorly defined cytoplasmic borders. Definitive diagnosis was made by use of factor VIII:RAg, which stained the tissues intensely.

Bilateral eyelid swelling attributable to lymphosarcoma in a horse.

Bilateral swelling of upper and lower eyelids was caused by lymphocytic infiltration in an 8-year-old Thoroughbred mare. The condition worsened with pregnancy and became associated with subcutaneous dissemination of lymphosarcoma at distant sites.

Squamous cell carcinoma causing blindness and ophthalmoplegia in a cat.

Ophthalmoplegia and blindness caused by squamous cell carcinoma were reported in a cat. Unilateral functional deficits of cranial nerves II, III, IV, and VI and of the sympathetic nerve supply caused blindness, complete ophthalmoplegia, and Horner syndrome. Radiography and computed tomography revealed a proliferating bony lesion associated with the right tympanic bulla, right temporal bone, right side of the mandible, and left frontal bone. A focal area of bony destruction involved the right sphenoid bone. The cat was euthanatized and necropsied. Squamous cell carcinoma was identified invading the bones and rostral part of the right side of the skull. The tumor had extended through the sphenoid bones into the region of the cavernous sinus and had surrounded the cranial nerves passing through this region. It also had invested connective tissue surrounding the optic nerves and had invaded the right globe through the lamina cribrosa. This represents an unusual distribution for ocular cranial squamous cell carcinoma in a cat.

Eyelid malformation in four cockatiels.

Four unrelated cockatiels had various degrees of maldevelopment of the eyelids. Only 1 eye of the 8 was normal. The condition had features compatible with ankyloblepharon or cryptophthalmos. Attempts were made to surgically establish an artificial palpebral fissure. Despite extensive surgical intervention and postoperative topical administration of corticosteroids, the skin over the orbit reverted to the preoperative state in all birds. Anecdotal information along with our findings suggest that this condition, although uncommon, is widespread in the cockatiel population in the United States.

Genetic characterization of CHO production host DG44 and derivative recombinant cell lines.

The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the cytogenetic level. Here, the first detailed karyotype analysis of DG44 and several recombinant derivative cell lines is described. In contrast to the 22 chromosomes in diploid Chinese hamster cells, DG44 has 20 chromosomes, only seven of which are normal. In addition, four Z group chromosomes, seven derivative chromosomes, and 2 marker chromosomes were identified. For all but one of the 16 DG44-derived recombinant cell lines analyzed, a single integration site was detected by fluorescence in situ hybridization regardless of the gene delivery method (calcium phosphate-DNA coprecipitation or microinjection), the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 51). Chromosomal aberrations, observed in more than half of the cell lines studied, were mostly unbalanced with examples of aneuploidy, deletions, and complex rearrangements. The results demonstrate that chromosomal aberrations are frequently associated with the establishment of recombinant CHO DG44 cell lines. Noteworthy, there was no direct correlation between the stability of the genome and the stability of recombinant protein expression.

Use of air-cleaning devices to create airborne particle-free spaces intended to alleviate allergic rhinitis and asthma during sleep.

UNLABELLED: Comprehensive quantitative experiments were performed to assess the capabilities of several air-cleaning devices to create a particle-free microenvironment as a therapy for sleeping persons affected by allergic rhinitis and asthma. Six devices were evaluated, of which five were portable and intended to provide general air cleaning for bedroom-sized spaces. The sixth was intended for installation in front of the headboard of a bed and was designed to provide clean air focused in a space occupied by a sleeping person. The air-cleaning methods of the selected devices included high-efficiency particulate air (HEPA) filtering and electrostatic precipitation. Particle concentration measurements for six particle-size ranges and sound intensity measurements were made during 8-h, sleep-simulating periods. The effects of four parameters were studied: (i) device location, (ii) controlled air motion in the laboratory, (iii) airflow rate setting of the air-cleaning device, and (iv) controlled disturbances. To ensure a totally objective study, a special laboratory facility was constructed which enabled complete control of the experimental conditions. The measured concentration histories provided comprehensive evidence of the relative capabilities of the various devices for the specific air-cleaning function. It was found that the device designed to focus the cleaned air in the sleeping space fulfilled its goal and, in that regard, was clearly superior to all of the other air-cleaning devices. PRACTICAL IMPLICATIONS: There is evidence that allergic reactions are triggered by the presence of airborne particles and that these reactions can be mitigated by particle removal. This strategy can be implemented by the use of air-cleaning devices which are capable of creating particle-free zones at locations where human activity occurs. In particular, the creation of a particle-free zone which encompasses the breathing space of a sleeping person holds promise of mitigating sleep-disturbing allergic reactions.

Identification of a coat protein binding site on southern bean mosaic virus RNA.

The cowpea strain of Southern bean mosaic virus (SBMV-C), a T = 3 icosahedral RNA virus, was dissociated to yield a ribonucleoprotein complex (RNPC) composed of the viral RNA and coat protein subunits. To determine if the coat protein subunits were bound to a specific site on the viral RNA, the RNPC was treated with ribonuclease, and the remaining coat protein--RNA complexes were recovered by filter binding. A single species of RNA was isolated by this procedure and further characterized by sequencing. The RNA was mapped to nucleotides 1410-1436 of the SBMV-C genome. This region of the viral RNA was predicted to fold into a hairpin with a 4-base loop and a duplex stem of 24 nucleotides. The stability and specificity of the coat protein-RNA complex isolated from dissociated virus suggest a possible role for this interaction in the selective encapsidation of the viral RNA.