Correction of ATM mutations in iPS cells from two ataxia-telangiectasia patients restores DNA damage and oxidative stress responses.

Patients with ataxia-telangiectasia (A-T) lack a functional ATM kinase protein and exhibit defective repair of DNA double-stranded breaks and response to oxidative stress. We show that CRISPR/Cas9-assisted gene correction combined with piggyBac (PB) transposon-mediated excision of the selection cassette enables seamless restoration of functional ATM alleles in induced pluripotent stem cells from an A-T patient carrying compound heterozygous exonic missense/frameshift mutations, and from a patient with a homozygous splicing acceptor mutation of an internal coding exon. We show that the correction of one allele restores expression of ~ 50% of full-length ATM protein and ameliorates DNA damage-induced activation (auto-phosphorylation) of ATM and phosphorylation of its downstream targets, KAP-1 and H2AX. Restoration of ATM function also normalizes radiosensitivity, mitochondrial ROS production and oxidative-stress-induced apoptosis levels in A-T iPSC lines, demonstrating that restoration of a single ATM allele is sufficient to rescue key ATM functions. Our data further show that despite the absence of a functional ATM kinase, homology-directed repair and seamless correction of a pathogenic ATM mutation is possible. The isogenic pairs of A-T and gene-corrected iPSCs described here constitute valuable tools for elucidating the role of ATM in ageing and A-T pathogenesis.

Measurement of the real part of the Clausius-Mossotti factor of dielectrophoresis for Brownian particles.

A method is proposed for measuring the real part of the Clausius-Mossotti factor ( Kr ) of dielectrophoresis for Brownian particles based on a solution of the Smoluchowski equation using a designed polydimethysilloxane microchannel with planar hyperbolic electrodes on its glass substrate. An approximate two-dimensional spring-like dielectrophoretic force is generated in the device, and the data necessarily measured is the time evolution of the in-plane particle displacement undergoing confined Brownian motion. Validity of the measurement was checked against the zeta potentials in the literature based on the classical theory of surface conductance using polystyrene particles of size of one micron. As the dielectrophoretic force depends on Kr , which is usually unknown for bio-particles and some engineered particles, and is seldom measured; this study is important from the academic point of view and could be helpful for the manipulation and characterization of sub-micron particles using dielectrophoresis. Extension of the method to the measurement of permanent dipole moment and total polarizability of particle was developed theoretically and discussed by incorporating an optical tweezer into the present device.

Combination of ablative fractional laser and daylight-mediated photodynamic therapy for actinic keratosis in organ transplant recipients - a randomized controlled trial.

BACKGROUND: Topical photodynamic therapy (PDT) for actinic keratoses (AK) is hampered by pain during illumination and inferior efficacy in organ-transplant recipients (OTR). OBJECTIVES: We assessed ablative fractional laser (AFL)-assisted daylight photodynamic therapy (PDT) (AFL-dPDT) compared with daylight PDT (dPDT), conventional PDT (cPDT) and AFL alone (AFL) in field treatment of AK in OTR. METHODS: In each patient, four areas in the same region were randomized to one treatment with AFL-dPDT, dPDT, cPDT and AFL. AFL was delivered with a 2940-nm AFL at 2·3 mJ per pulse, 1·15 W, two stacks, 50-µs pulse-duration, 2·4% density. In dPDT and AFL-dPDT, methyl aminolaevulinate (MAL) was applied for 2·5 h without occlusion during daylight exposure. For cPDT, MAL was occluded for 3 h followed by red-light (630 nm) irradiation at 37 J cm(-2). The primary end-point was complete response (CR) 3 months post-treatment. RESULTS: Sixteen patients with 542 AK (grades I-III) in field-cancerized skin of the scalp, chest and extremities were treated during August and September 2012. After 3 months, CR (AK I-III) rates were 74% after AFL-dPDT, 46% after dPDT, 50% after cPDT and 5% after AFL (P < 0·001). CR rates in AFL-dPDT, dPDT and cPDT were also significantly different (P = 0·004). Median maximal pain scores differed significantly during AFL-dPDT (0), dPDT (0), AFL (0) and cPDT (5) (P < 0·001). Erythema and crusting were more intense following AFL-dPDT than dPDT and cPDT, but only transient hypopigmentation was observed. CONCLUSIONS: AFL-dPDT is a novel PDT modality that enhances CR with excellent tolerability compared with dPDT and cPDT in difficult-to-treat AK in OTR.

Monitoring atmospheric 85Kr by atom counting.

Radioactive 85Kr is a major gaseous fission product emitted into the air by the nuclear fuel reprocessing industry. Measuring atmospheric 85Kr has applications in environmental monitoring, atmospheric transport model validation and dating of environmental water samples, including groundwater, sea water and glacier ice. We present an ultra-sensitive method for fast analysis of atmospheric 85Kr at 10-5 parts per trillion level. This method is based on laser cooling and trapping and is capable of counting individual 85Kr atoms. Measurements at the 3% precision level can be made on krypton extracted from 1L STP of air with a turnaround time of 1.5 h. Moreover, we have realized a system for continuous air sampling over days to weeks. Based on this atom-counting technology and a portable air sample integrator we have realized atmospheric 85Kr baseline monitoring in Hefei, China, for over 20 months. The technological advances presented in this work lay the ground for a global atmospheric 85Kr monitoring network.

Measurement of the Imaginary Part of the Clausius-Mossotti Factor of Particle/Cell via Dual Frequency Electrorotation.

A simple and inexpensive method using planar electrodes was proposed for the measurement of the imaginary part of the Clausius-Mossotti factor, K i , of particle/cell for electrorotation (ER) and travelling wave dielectrophoresis (twDEP). It is based on the balance between the dielectrophoretic and viscous torques on a particle undergoing ER subject to dual frequency operation in an ER chamber. A four-phase ac voltage signal with a given frequency is applied for generating ER for measurement, and another two-phase signal is applied at a selected frequency for generating a negative dielectrophoretic force for confining the particle motion, instead of using laser tweezer or three-dimensional electrodes in the literature. Both frequencies can be applied to the same electrodes in a four-electrode ER system and to alternative different electrodes in an eight-electrode ER system, and both systems are capable for providing accurate measurement. The measurements were validated by comparing with the theoretical result using sephadex particles in KCl solution, and with the existing experimental results for various human cancer cells in medium with conductivity from 0.01-1.2 S/m, using ER with optical tweezer and dual frequency twDEP. Contrast between the ER and the twDEP methods (the current two available methods) was discussed and commented. The present method could provide measurement for wider frequency range and more accurate result near K i = 0, in comparison with the results using the twDEP method. However, the twDEP method could perform much more rapid measurement. Detailed forces and torque were calculated inside the ER chamber for understanding the physics and assessing the characteristics of the dual frequency ER method. This study is of academic interest as the torque in ER and the force in twDEP can be calculated only when K i is known. It also finds biomedical applications as the K i -spectra can be served as physical phenotypes for different cells, and can be applied for deriving dielectric properties of cells.

Hemostatic Enhancement via Chitosan Is Independent of Classical Clotting Pathways-A Quantitative Study.

Hemostasis is a process causing bleeding to stop, and it is known from the literature that hemostasis can be enhanced using chitosan on wound gauzes. We proposed here a continuous flow-through device, with the test blood flowing through the gauze sample at a constant flow rate and the pressure drop across the gauze measured, for assessing the hemostatic performance of the gauze. Experiments were performed using the device with both whole blood and washed blood (with clotting factors and platelets removed from the whole blood), and their results agree with each other within 10% discrepancy, indicating quantitatively that hemostatic enhancement via chitosan is essentially independent of classical clotting pathways, which was demonstrated qualitatively through animal tests in the literature. The proposed device and method can be applied for evaluating quantitatively the hemostatic performance of various gauzes in a flowing blood environment (in comparison with static tests) with less test blood (20-60% less, in comparison with that of a flow-through device driven by a constant pressure gradient), and are thus, helpful for designing better wound gauzes. In particular, it is effective to enhance the hemostatic performance further (additional 30%) through acidification (changing the amino group to the ammonium group) of the gauze for chitosan-based wound gauzes.

A travelling wave dielectrophoretic pump for blood delivery.

The travelling wave dielectrophoretic pump studied here is essentially a rectangular straight micro-channel with an electrode array on part of its wall, and operated under an ac voltage with phase shift at neighbouring electrodes. The travelling wave dielectrophoretic force drives the cells, which drag the plasma, and after some sophisticated interaction between conventional dielectrophoresis, travelling wave dielectrophoresis and fluid mechanics, the whole blood is delivered. The pump was fabricated using MEMS techniques and studied in details for different parameters. It is found that the pumping velocity is maximized at an intermediate frequency around 20-30 MHz (varies with phase shift), and at an intermediate channel height at about 40 microm. The quasi-static average cell velocity can reach 15 microm s(-1) for a pump with 1 mm length and 16 electrodes (total array length 465 microm) operated at 5 V and 20 MHz with 90 degrees phase shift.

A Continuous Flow-through Microfluidic Device for Electrical Lysis of Cells.

In contrast to the delicate 3D electrodes in the literature, a simple flow-through device is proposed here for continuous and massive lysis of cells using electricity. The device is essentially a rectangular microchannel with a planar electrode array built on its bottom wall, actuated by alternating current (AC) voltages between neighboring electrodes, and can be incorporated easily into other biomedical systems. Human whole blood diluted 10 times with phosphate-buffered saline (about 6 108 cells per mL) was pumped through the device, and the cells were completely lysed within 7 s after the application of a 20 V peak-to-peak voltage at 1 MHz, up to 400 µL/hr. Electric field and Maxwell stress were calculated for assessing electrical lysis. Only the lower half-channel was exposed to an electric field exceeding the irreversible threshold value of cell electroporation (Eth2), suggesting that a cross flow, proposed here primarily as the electro-thermally induced flow, was responsible for bringing the cells in the upper half-channel downward to the lower half-channel. The Maxwell shear stress associated with Eth2 was one order of magnitude less than the threshold mechanical stresses for lysis, implying that an applied moderate mechanical stress could aid electrical lysis.

alpha-fetoprotein levels in maternal serum during pregnancy and maternal breast cancer incidence.

BACKGROUND: A full-term pregnancy is associated with a reduced risk of breast cancer, but the underlying biologic mechanism has not been elucidated. During pregnancy, maternal serum levels of alpha-fetoprotein, an estradiol-binding protein, rise sharply. In culture, alpha-fetoprotein inhibits the growth of estrogen-sensitive cells, including estrogen-sensitive breast cancer cells. Thus, we investigated whether a high level of alpha-fetoprotein in maternal serum during pregnancy is associated with a reduced risk of breast cancer. METHODS: From a population-based cohort of 42057 pregnant women in Denmark, enrolled in an alpha-fetoprotein-screening program from 1978 through 1996, we obtained a complete reproductive history, vital status, and a possible diagnosis of breast cancer (in 117 women) to the end of follow-up on September 1, 1998. RESULTS: During pregnancy, women with an alpha-fetoprotein level greater than or equal to the median value had a 41% lower risk of breast cancer than women with an alpha-fetoprotein level below the median value (relative risk [RR] = 0.59; 95% confidence interval [CI] = 0.41-0. 85). RRs for breast cancer by mother's age at childbirth were as follows: 29 years or younger, RR = 0.21 (95% CI = 0.08-0.56); 30-34 years, RR = 0.61 (95% CI = 0.32-1.14); 35-37 years, RR = 0.96 (95% CI = 0.49-1.89); and 38 years or older, RR = 0.71 (95% CI = 0.29-1. 75) (P for trend =.02). Further analyses suggested that high levels of alpha-fetoprotein were associated with a reduced incidence of aggressive disease. The most striking finding was that women with high levels of serum alpha-fetoprotein, compared with women with low levels of serum alpha-fetoprotein, showed a particularly reduced incidence of large tumors (>2 cm; RR = 0.24 [95% CI = 0.11-0.50]). CONCLUSION: A high level of alpha-fetoprotein in maternal serum during any pregnancy is associated with a low overall incidence of breast cancer and, in particular, with a low incidence of advanced breast cancer at diagnosis. This association appears particularly strong for a pregnancy occurring at a young age.

Second-trimester maternal serum alpha-fetoprotein and risk of adverse pregnancy outcome(1).

OBJECTIVE: To determine the risk of adverse pregnancy outcome by maternal serum alpha-fetoprotein (MSAFP) level. METHODS: We followed 77,149 pregnant women and their infants from MSAFP screening in the 15th to 20th week of gestation until 1 year after birth. Information on pregnancy outcome was obtained from national registries. The relative risks (RRs) and 95% confidence intervals (CIs) for adverse pregnancy outcome were estimated according to the level of MSAFP, with adjustment for confounders. RESULTS: A total of 638 pregnancies resulted in spontaneous abortion, 289 in stillbirth, and 437 in infant death. Compared with women with MSAFP levels at 0.75-1.24 multiples of the median (MoM), those with MSAFP levels greater than or equal to 2.5 MoM had an increased risk of spontaneous abortion (RR 12.5; 95% CI 9.7, 16.1), preterm birth (RR 4.8; 95% CI 4.1, 5.5), small for gestational age (RR 2.8; 95% CI 2.4, 3.2), low birth weight (RR 5.8; 95% CI 5.0, 6.6), and infant death (RR 1.9; 95% CI 1.2, 2.8). Women with MSAFP levels below 0.25 MoM had an increased risk of spontaneous abortion (RR 15.1; 95% CI 9.3, 24.8), preterm birth (RR 2.2; 95% CI 1.3, 3.8), and stillbirth (RR 4.0; 95% CI 1.0, 16.0); those with levels less than 0.5 MoM had an increased risk of infant death (RR 1.9; 95% CI 1.2, 3.0). The increased risk of infant death remained after the subtraction of recognized conditions associated with extreme MSAFP values. CONCLUSION: Pregnant women with extreme MSAFP values in the second trimester have an increased risk of fetal and infant deaths. Obstet Gynecol 2001;97:277-82.

Review of the theory of generalised dielectrophoresis.

Generalised dielectrophoresis (gDEP), including conventional dielectrophoresis (cDEP), electrorotation (ER) and travelling wave dielectrophoresis (twDEP), is an effective tool for particle (cell) manipulation and characterisation, even down to the level of nano-sized objects such as DNA, proteins and viruses. All the disciplines of gDEP are originated from the interaction of an applied electric field with its polarisation effect on the particle and can be studied systematically in a unified approach under electrostatics. In this review, the authors discuss both the quasi-static and transient theory of gDEP in an unbounded medium for both spherical and ellipsoidal particles. Then the quasi-static theory of wall effect is discussed on gDEP for a spherical particle. The wall effect is minor for ER, twDEP and cDEP parallel to wall(s), but could be significant for cDEP normal to wall(s). Force and torque expressions in terms of electric potential and its derivatives are provided and suggested for a robust calculation of the twDEP force and DEP torque. Discussions are provided for the application of the theory to nano-sized particles. The authors also illustrate some features of the Clausius-Mossotti factor using erythrocyte as an example, including both the crossover (DEP) and peak frequencies (ER) at low and high-frequency limits.

Refinement of the theory for extracting cell dielectric properties from dielectrophoresis and electrorotation experiments.

A modified theory is proposed for extracting cell dielectric properties from the peak frequency measurement of electrorotation (ER) and the crossover frequency measurement of dielectrophoresis (DEP). Current theory in the literature is based on the low frequency (DC) approximations for the equivalent cell permittivity and conductivity, which are valid when the measurements are performed in a medium with conductivity less than 1 mS/m. The present theory extracts the cell properties through optimizing an expression for the medium conductivity in terms of the peak ER, or DEP crossover, frequency according to its definition using full expressions of equivalent cell permittivity and conductivity. Various levels of approximation of the theory are proposed and discussed through a scaling analysis. The present theory can extract both membrane and interior properties from the low and the high peak ER, or DEP crossover, frequencies for any medium conductivity provided the peak ER, or DEP crossover, frequency exists. It can be reduced to the linear theory for the low peak ER and DEP crossover frequencies in the literature when the medium conductivity is less than 10 mS/m. However, we can determine the membrane capacitance and conductance via the slope and intercept, respectively, of the straight line fitting of the ER peak and DEP frequency against medium conductivity data according to the linear theory only when the intercept dominates the experimental uncertainty, which occurs when the medium conductivity is less than 1 mS/m in practice.

Occupational non-melanoma skin cancer.

Non-melanoma skin cancer is historically known to be associated with certain professions. Reporting is mandatory in Denmark when occupational exposure is suspected. In a retrospective register-based study of all cases of suspected occupational non-melanoma skin cancer reported to the Directorate of National Labour Inspection and the National Board of Industrial Injuries in Denmark in the period January 1, 1984 to December 31, 1994, we assessed the extent to which occupational exposures today are of importance in the occurrence of non-melanoma skin cancer. A total of 74 individuals (11 women and 63 men) aged 32-82 years (median 58 years) had been reported. Of these, 15 cases (20%) were approved as being occupational, 37 (50%) were rejected and 22 (30%) were either shelved or could not be further clarified. Most commonly approved were exposures such as asphalt, tar, and the like, and ionizing radiation, and localization on the arms or multiple tumours. Unexpected occupational exposure could not be identified but continued reporting is recommended in order to follow this in the future.

Neonatal level of thyroid-stimulating hormone and acute childhood leukemia.

One of the more consistent findings in leukemia research is the association between birth weight and childhood leukemia. Because thyroid hormones are critically involved in growth and differentiation, we speculated that hormone levels could be of significance to the development of leukemia in early life. Specifically, we hypothesized that high levels of thyroid-stimulating hormone (TSH) would be associated with a low risk of leukemia. Accordingly, high TSH (low free thyroid hormone) early after birth most likely reflects low function of the thyroid accompanied by low rate of cell turnover and so lower risk of faulty cell divisions leading to cancer. In a matched case-control study nested from all singleton children born in Denmark between 1986 and 1998, we compared levels of TSH (as measured in a neonatal screening program for congenital hypothyroidism) in 188 cases of acute lymphoblastic leukemia (ALL) and 28 of acute myeloid leukemia (AML) with levels in 1,450 and 216 matched controls, respectively. Data were analyzed using conditional logistic regression and odds ratios (OR) were adjusted for birth weight. As hypothesized, we found a decreased risk of ALL and AML associated with high TSH (OR(ALL) = 0.7 [0.5-1.0]; OR(AML) = 0.3 [0.1-1.0]). However, both conditions were also associated with low levels of TSH (OR(ALL) = 0.4 [0.2-0.7]; OR(AML) = 0.3 [0.1-1.4]). In conclusion, extreme TSH levels a few days after birth appears to be associated with a decreased risk of acute childhood leukemia.