Psoralen mapping reveals a bacterial genome supercoiling landscape dominated by transcription.

DNA supercoiling is a key regulator of all DNA metabolic processes including replication, transcription, and recombination, yet a reliable genomic assay for supercoiling is lacking. Here, we present a robust and flexible method (Psora-seq) to measure whole-genome supercoiling at high resolution. Using this tool in Escherichia coli, we observe a supercoiling landscape that is well correlated to transcription. Supercoiling twin-domains generated by RNA polymerase complexes span 25 kb in each direction - an order of magnitude farther than previous measurements in any organism. Thus, ribosomal and many other highly expressed genes strongly affect the topology of about 40 neighboring genes each, creating highly integrated gene circuits. Genomic patterns of supercoiling revealed by Psora-seq could be aptly predicted from modeling based on gene expression levels alone, indicating that transcription is the major determinant of chromosome supercoiling. Large-scale supercoiling patterns were highly symmetrical between left and right chromosome arms (replichores), indicating that DNA replication also strongly influences supercoiling. Skew in the axis of symmetry from the natural ori-ter axis supports previous indications that the rightward replication fork is delayed several minutes after initiation. Implications of supercoiling on DNA replication and chromosome domain structure are discussed.

The predictive and prognostic significance of liquid biopsy in advanced epidermal growth factor receptor-mutated non-small cell lung cancer: A prospective study.

OBJECTIVE: To determine the predictive and prognostic roles of three blood-based biomarkers: circulating tumour DNA (ctDNA), circulating tumour cells (CTC) and carcinoembryonic antigen (CEA), in patients with advanced epidermal growth factor receptor-mutated (EGFR+) lung cancer. MATERIALS AND METHODS: We recruited 28 patients with 103 serial blood samples. We performed mutational analyses for EGFR mutations using droplet digital PCR (ddPCR) on ctDNA. We evaluated the accuracy of EGFR mutation detection in ctDNA compared with tissue biopsy. We also quantified CTCs, ctDNA and CEA in serially collected blood samples, and evaluated the baseline and changes in these blood-based biomarkers with clinical outcomes. RESULTS: EGFR mutation detection in plasma was highly concordant as compared with tissue biopsy. Detectable baseline ctDNA was associated with higher disease burden (p < 0.01). Early disappearance of ctDNA at 4 weeks was associated with radiological response at 12 weeks of treatment (p = 0.01) and improved progression free survival (PFS) (HR 5.47, 95%CI 1.32-22.72, p = 0.02) and overall survival (OS) (HR 5.46, 95%CI 1.28-23.22, p = 0.02). A decrease in CTC count at 4 weeks was associated with improved PFS (HR 3.81, 95%CI 1.13-12.79, p = 0.03) but not OS. 85% of patients with radiological progression had a ctDNA rise compared with 22% of patients with stable disease (p=0.01). ctDNA rise was seen on average 170 days prior to radiological progression. There is a significant association between the rise of CEA level with radiological progression (p=0.001). CONCLUSION: Early change in ctDNA, CTC and CEA levels may be long-term predictors of treatment benefit and failure prior to availability of radiological response data.

Apoptosis of alcohol-exposed human placental cytotrophoblast cells is downstream of intracellular calcium signaling.

BACKGROUND: Apoptosis is induced by ethanol (EtOH) in human placental trophoblast cells, possibly disrupting placentation and contributing to intrauterine growth restriction in fetal alcohol spectrum disorder (FASD). EtOH induces programmed cell death in several embryonic tissues by raising intracellular Ca(2+) . Therefore, the role of Ca(2+) signaling in EtOH-induced apoptosis was examined using human first trimester cytotrophoblast cell lines, examining the hypothesis that apoptosis is dependent on intracellular Ca(2+) signaling. METHODS: Using HTR-8/SVneo and SW.71 cytotrophoblast cell lines, real-time intracellular Ca(2+) concentration was monitored by fluo-4 epifluorescence microscopy and apoptosis was assessed by flow cytometry of cells fluorescently labeled for DNA fragmentation (TUNEL) and annexin V binding. RESULTS: Intracellular Ca(2+) concentrations increased synchronously in all cells within 10 seconds of exposure to 50 mM EtOH, but not at lower EtOH concentrations (10 to 25 mM) incapable of inducing apoptosis. Trophoblast cells treated with inhibitors of Ca(2+) signaling (BAPTA-AM, U73122, xestospongin D, BAPTA, SKF-96365) produced no intracellular Ca(2+) transients after exposure to 50 mM EtOH and were protected from cell death induced by EtOH. CONCLUSIONS: EtOH-induced apoptosis in human cytotrophoblast cells, identified by DNA fragmentation and externalized phosphatidylserine, was dependent upon Ca(2+) signaling. Both intracellular Ca(2+) mobilization and extracellular Ca(2+) influx were required, as well as phosphatidylinositol signaling. Inhibition by SKF-96365 suggests that the capacitative Ca(2+) entry mechanism that utilizes TRPC channels was activated by EtOH. Apoptosis occurs downstream of Ca(2+) signaling in trophoblasts and may contribute to placental insufficiency and poor fetal growth associated with FASD.

Time-frequency analysis of chewing activity in the natural environment.

UNLABELLED: The aim of this observational study was to investigate the features of the chewing activity and the variability of the human chewing pace, as assessed in the natural environment. It was hypothesized that the chewing pace is relatively constant within individuals across different days but is variable across individuals. Electromyographic surface activity was recorded unilaterally from the masseter in 21 participants for 3 hours over 3 recording days, in the natural environment, by means of portable recorders. The time-frequency properties of chewing activity were assessed with a previously validated algorithm. Repeated-measurements ANOVA was used for statistical analysis. Chewing activity mainly occurred in the range of 0.94 Hz (5(th) percentile) and 2.17 Hz (95(th) percentile). Mean and median chewing frequencies were 1.57 Hz and 1.58 Hz, respectively (95% confidence intervals: 1.45-1.68 Hz). The mean duration of chewing episodes was 13.0 sec, the 5(th) and 95(th) percentiles being 2.7 sec and 34.9 sec, respectively. Variability of the mean chewing frequency between individuals was much greater than that within individuals (F = 29.8; p < 0.001). The individual chewing paces were stable across different days (intraclass correlation coefficient = 0.88; 95% confidence intervals = 0.79-0.94). Our findings provide evidence that each individual, in the natural environment, chews with a consistent pace across different days. ABBREVIATIONS: ANOVA, analysis of variance; CPG, central pattern generator; EMG, electromyography; ICC, Intra-class Correlation coefficient; SD, standard deviation.

Mitotic separation of daughter cells in the human lymphoma B cell line Daudi involves L-selectin engagement and shedding.

BACKGROUND AND OBJECTIVE: A novel role for shedding of the surface molecule L-selectin has been proposed as an adjunctive phenomenon during cell detachment from marrow stroma or vessel endothelium. We wished to examine whether variations in expression of L-selectin on a lymphoma B cell line were linked to shedding. DESIGN AND METHODS: Mapping of L-selectin expression on the surface of Daudi lymphoma cells was performed by flow cytometry, fluorescence microscopy, and electron microscopy. Levels of shed L-selectin were evaluated by Western blotting of culture supernatants. Evaluation of cell cycle and proliferative activity was performed by flow cytometry. RESULTS: Large Daudi cells in S+G(2)/M phases were L-selectin positive, whereas small Daudi cells in G(0)/G(1) phase were L-selectin negative. During mitosis, L-selectin was distributed along the cleavage furrow, and gradually lost. Electron microscopy revealed that separating Daudi cells were negative for L-selectin on the entire surface, except minute aggregates of L-selectin within the cleavage furrow. Addition of agents known to interfere with the ligand-binding portion of L-selectin (sulfatides, MoAbs: Lam1.3 and TQ1) results in loss of L-selectin. Removal of L-selectin by digestion with chymotrypsin inhibits Daudi proliferation. The MoAb FMC46 did not interfere with proliferation. Proliferating Daudi cells produced large quantities of shed L-selectin. Inhibition of Daudi proliferation resulted in levels of shed L-selectin below the limit of detection. INTERPRETATION AND CONCLUSIONS: L-selectin is re-distributed on the cell surface of Daudi cells during the last phase of mitosis, in which plasma membrane invagination occurs between newly formed daughter cells. Shedding of L-selectin is involved in the cytokinesis of Daudi cells.

Apoptosis induced in human colorectal carcinoma by anti-Fas antibody.

BACKGROUND: Apoptosis or programmed cell death has been shown to play an important role in the progression from polyps to carcinomas. Fas/APO-1 is a cell surface protein that can induce apoptosis in a variety of cell types upon specific binding. In this study seven human colorectal carcinoma (HCRC) cell lines of varying differentiation were analyzed for cell surface Fas expression. Fas-mediated apoptosis, and correlation of apoptosis with bcl-2 expression. METHODS AND RESULTS: Using flow cytometry, all seven lines expressed varying amounts of cell surface Fas antigen. Exposure to anti-Fas antibody induced cell death in all the cell lines, albeit to varying degrees. The rate of apoptosis was quantitated using flow cytometry with propidium iodide staining of nuclear DNA. The poorly differentiated cell lines had a significantly decreased (p < 0.05) anti-Fas sensitivity as compared with the well-differentiated lines. Measurement of bcl-2 expression by flow cytometry showed an inverse correlation with anti-Fas sensitivity. CONCLUSIONS: This study confirms that HCRC cell lines express Fas antigen and, more importantly, provides the first evidence that exposure to anti-Fas antibody can induce apoptosis. Fas-mediated apoptosis in HCRC cell lines may be regulated by bcl-2 and may correlate with the degree of differentiation.

The L-selectin antibody FMC46 mediates rapid, transient increase in intracellular calcium in human peripheral blood mononuclear cells and Daudi lymphoma cells.

We report the induction of intracellular calcium mobilization [Ca2+]i in normal peripheral blood mononuclear cells (PBMC) and Daudi cells following binding with the L-selectin monoclonal antibody FMC46. The [Ca2+]i signal was mediated directly by binding of FMC46 without cross-linking antibodies. Increased [Ca2+]i was not induced by other L-selectin antibodies tested (TQ1, Leu8, Lam1.3). The increase in [Ca2+]i was rapid and was blocked completely by BAPTA, an agent which chelates intracellular calcium. The increase in [Ca2+]i was observed in calcium-containing as well as calcium free medium, suggesting that FMC46 caused release of Ca2+ from intracellular stores. In both PBMC and Daudi cells, previous signaling via L-selectin still allowed signaling through cross-linking of surface antigen receptor. These data provide evidence for direct alteration of the state of lymphocytes after ligation of a specific L-selectin epitope. L-selectin-mediated signaling does not desensitize signaling through the antigen-receptor and could therefore play a role in preactivating lymphocytes during endothelial transmigration into lymphoid tissues.

Coronary artery bypass grafts: new universal markers.

This report describes a new system to facilitate the identification of coronary artery bypass grafts during repeat coronary angiography. Geometric markers (square, triangle, circle, hexagon) are placed over the aortic end of the graft, with each geometric shape designating the target vessel of the graft, e.g. square, right coronary; triangle, anterior descending. This system significantly simplifies cannulation of the postoperative patient and also allows immediate recognition of specific vessels bypassed by mere perusal of the chest film.

Measurement of soluble pyrophosphate in plasma and synovial fluid of patients with various rheumatic diseases.

Soluble pyrophosphate was measured in the plasma and synovial fluid of various groups of patients and in the plasma of two control groups. The two control groups consisted of 13 healthy subjects and 19 patients suffering from benign lumbar back pain. The other group of patients had rheumatoid arthritis (RA) (14 plasma and 19 synovial fluid examinations), osteoarthrosis (OA) (19 plasma and 26 synovial fluids) and articular chondrocalcinosis (ACC) (27 plasma and 43 synovial fluids). The level of soluble pyrophosphate in the plasma was 3.5 mumol/l in healthy subjects, 4.0 mumol/l in patients with lumbar back pain, 4.1 mumol/l in individuals having OA and 3.5 mumol/l in the group suffering from RA as well as for those with ACC. The differences between these values are not significant statistically. In the synovial fluid the values were 4.6 mumol/l for the group with RA, 12.7 mumol/l for those with OA and 34.2 mumol/l in the group having ACC. If a normal distribution of these values is assumed and the average values and standard deviations recalculated for each group after elimination of cases more than 3 standard deviations above the mean, then we obtain 9.8 mumol/l for the group with OA and 23.8 mumol/l for those with ACC. The difference between the group with RA and that with OA is highly significant (p greater than 0.0001). Even more significant is the difference between the group with RA and ACC (p less than 0.0005). The difference between the OA and the ACC is also highly significant (p less than 0.001). On the basis of these observations various mechanisms leading to the pyrophosphage crystal deposition disease are discussed.

Myocardial revascularization in high-risk coronary patients.

It is recognized that postoperative mortality, infarction and the need for inotropic support are increased following myocardial revascularization in highrisk patients. Operations were carried out in 57 such patients in whom one or more of the following factors were present: ventricular dysfunction-ejection fraction less than 0.4 (17), unstable (8) or preinfarction angina (29), evolving infarction (8), recent infarction (less than two weeks before) (5) and refractory ventricular tachyarrhythmia (4). Combined risk factors were present in nine patients. The following principles were utilized to minimize ischemic injury: (1) avoidance of prebypass hypertension and hypotension, (2) avoidance of extreme hemodilution, (3) avoidance of ventricular fibrillation, (4) maintenance of beating empty heart, when possible, (5) the limiting of ischemic periods to less than 12 minutes (hypothermia 32 degrees C) and (6) repaying myocardial oxygen debt with total (vented) bypass, when necessary. The following results were obtained: inotropic support was required in five patients (9 percent), "new" postoperative infarction occurred in five patients (9 percent) and one patient died (2 percent). These results are comparable to those reported in good-risk patients, and indicate that optimal myocardial protection will allow safe revascularization in a high-risk patient.

Coronary revascularization in "high" versus "low-risk" patients: The role of myocardial protection.

Postoperative mortality, infarction, and need for inotropic support are reportedly increased following myocardial revascularization in "high-risk" patients. We believe these complications result from inadequate protection of the compromised myocardium and should not occur with greater frequency in "high-risk" than "Low-risk" patients if the heart is optimally protected during the entire course of the operative procedure. Results following revascularization in 50 consecutive "low-risk" and 50 consecutive "high-risk" patients were analyzed. One or more of the followin factors were present in the "high-risk" group: ventricular dysfunction--ejection fraction less than 0.4, preinfarction angina, evolving infarction, recent infarction (less than 2 weeks), and refractory ventricular tachyarrhythmia. The following principles were used in all patients to minimize ischemic injury: 1) avoidance of pre-bypass hypo- or hypertension, 2) limitation of ischemic arrest to less than 12 minutes, 3) avoidance of ventricular fibrillation, and 4) prolongation of total bypass as necessary to repay the myocardial oxygen debt. Postoperative inotropic support was required in 10% of "high" and 10% of "low-risk" patients, new postoperative infarction developed in 10% of "high" vs. 10% "low-risk" patients; death occurred in 2% of "high" vs. 4% "low-risk" patients. These results are comparable and indicate that optimum myocardial protection allows safe revascularization in the "high-risk" patient.

105 mm photofluorography: an adjunct to the study of coronary arterial and vascular disease.

Obtaining high resolution static films through an image intensifier has been made possible by recent developments in radiographic equipment. Serial photofluorography 105 mm spot filming has been used successfully to complement cine coronary arteriography in approximately 700 patients. Many patients with coronary heart disease have diffuse peripheral atheromatous changes which are also suitable to study by serial 105 mm photofluorography. Aortic arch, abdominal aortography, and peripheral studies are easily and quickly performed in conjunction with coronary arteriography. Satisfactory results still depend upon skillful application by the angiographer of this imaging mode and proper processing of the film. Advantages of photofluorographic filming over conventional large film techniques include lower radiation exposure, reduction of costs, and easier film handling and storage. Most important is the facilitation of the performance of angiographic studies since constant television monitoring of the catheter and the contrast injections is possible. Disadvantages include restricted area of coverage and slight decrease in resolution. Our experience indicates that photofluorography may be relied upon in the study of vascular disease eliminating the need for large film changers in a laboratory used primarily for cardiac diagnosis.

The need for family planning information activities: a conceptual model.

PIP: This paper presents a general model demonstrating the need for informational and educational methodology within family planning programs. There are several steps in designing a family planning program's goals and objectives in quantitative and qualitative terms. In addition, it is necessary to determine what conditions must obtain in order to achieve the goals of the program. Within thes conceptual framework the implementation of a program must be seen as the creation of the conditions necessary for success; the need for informational and educational components in family planning is a must. Much of this responsibility falls on the educational community and the health educato r, in particular. Assuming that there are resources to make contraceptive technology available to the target population, the 1st step is to make this availability known. Next it is important that those in the target group who respond are educated in the proper use and inherent risks of the contraceptive method they choose. (The great problem the program administrators and health educators face is the development of a comprehensive program to reach those who are not really conscious of the relevance of family planning). The goals of the educational component have to be defined 1st, however. Thus, it is apparent from this study that information and educational programs are essential for the success of family planning programs.^ieng