[show abstract][hide abstract] ABSTRACT: While gene expression studies have proved extremely important in understanding cellular processes, it is becoming more apparent that there may be differences in individual cells that are missed by studying the population as a whole. We have developed a qRT-PCR protocol that allows us to assay multiple gene products in small samples, starting at 100 cells and going down to a single cell, and have used it to study radiation responses at the single-cell level. Since the accuracy of qRT-PCR depends greatly on the choice of "housekeeping" genes used for normalization, initial studies concentrated on determining the optimal panel of such genes. Using an endogenous control array, it was found that for IMR90 cells, common housekeeping genes tend to fall into one of two categories-those that are relatively stably expressed regardless of the number of cells in the sample, e.g., B2M, PPIA, and GAPDH, and those that are more variable (again regardless of the size of the population), e.g., YWHAZ, 18S, TBP, and HPRT1. Further, expression levels in commonly studied radiation-response genes, such as ATF3, CDKN1A, GADD45A, and MDM2, were assayed in 100, 10, and single-cell samples. It is here that the value of single-cell analyses becomes apparent. It was observed that the expression of some genes such as FGF2 and MDM2 was relatively constant over all irradiated cells, while that of others such as FAS was considerably more variable. It was clear that almost all cells respond to ionizing radiation but the individual responses were considerably varied. The analyses of single cells indicate that responses in individual cells are not uniform and suggest that responses observed in populations are not indicative of identical patterns in all cells. This in turn points to the value of single-cell analyses.
[show abstract][hide abstract] ABSTRACT: To perform high-throughput studies on the biological effects of ionizing radiation in vivo, we have implemented a microfluidic tool for microbeam irradiation of Caenorhabditis elegans. The device allows the immobilization of worms with minimal stress for a rapid and controlled microbeam irradiation of multiple samples in parallel. Adapted from an established design, our microfluidic clamp consists of 16 tapered channels with 10-μm-thin bottoms to ensure charged particle traversal. Worms are introduced into the microfluidic device through liquid flow between an inlet and an outlet, and the size of each microchannel guarantees that young adult worms are immobilized within minutes without the use of anesthesia. After site-specific irradiation with the microbeam, the worms can be released by reversing the flow direction in the clamp and collected for analysis of biological endpoints such as repair of radiation-induced DNA damage. For such studies, minimal sample manipulation and reduced use of drugs such as anesthetics that might interfere with normal physiological processes are preferable. By using our microfluidic device that allows simultaneous immobilization and imaging for irradiation of several whole living samples on a single clamp, here we show that 4.5-MeV proton microbeam irradiation induced DNA damage in wild-type C. elegans, as assessed by the formation of Rad51 foci that are essential for homologous repair of radiation-induced DNA damage.
[show abstract][hide abstract] ABSTRACT: The Radiological Research Accelerator Facility at Columbia University has recently added a UV microspot irradiator to a microbeam irradiation platform. This UV microspot irradiator applies multiphoton excitation at the focal point of an incident laser as the source for cell damage, and with this approach, a single cell within a 3D sample can be targeted and exposed to damaging UV. The UV microspot's ability to impart cellular damage within 3D is an advantage over all other microbeam techniques, which instead impart damage to numerous cells along microbeam tracks. This short communication is an overview, and a description of the UV microspot including the following applications and demonstrations of selective damage to live single cell targets: DNA damage foci formation, patterned irradiation, photoactivation, targeting of mitochondria, and targeting of individual cardiomyocytes in a live zebrafish embryo.
[show abstract][hide abstract] ABSTRACT: A novel technique for label-free imaging of live biological cells in aqueous medium that is insensitive to ambient vibrations is presented. This technique is a spin-off from previously developed immersion Mirau interferometry. Both approaches utilize a modified Mirau interferometric attachment for a microscope objective that can be used both in air and in immersion mode, when the device is submerged in cell medium and has its internal space filled with liquid. While immersion Mirau interferometry involves first capturing a series of images, the resulting images are potentially distorted by ambient vibrations. Overcoming these serial-acquisition challenges, simultaneous immersion Mirau interferometry incorporates polarizing elements into the optics to allow simultaneous acquisition of two interferograms. The system design and production are described and images produced with the developed techniques are presented.
The Review of scientific instruments 05/2013; 84(5):053701. · 1.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have developed a Quantitative Light Absorption Analysis (QLAA) method to rapidly estimate human lymphocyte concentrations isolated from small volumes of whole blood. Measurements of the light absorption analysis were calibrated for lymphocyte concentration levels using a hemocytometer. To validate the QLAA system, blood samples were collected from 17 healthy donors and lymphocyte absorption measurements were directly compared with the manual microscope counting. The results showed that lymphocyte measurements obtained using the QLAA system were comparable with the manually scored lymphocyte counts but with measurements taken in seconds.
[show abstract][hide abstract] ABSTRACT: This paper describes the fabrication and integration of light-induced dielectrophoresis for cellular manipulation in biological microbeams. An optoelectronic tweezers (OET) cellular manipulation platform was designed, fabricated, and tested at Columbia University's Radiological Research Accelerator Facility (RARAF). The platform involves a light induced dielectrophoretic surface and a microfluidic chamber with channels for easy input and output of cells. The electrical conductivity of the particle-laden medium was optimized to maximize the dielectrophoretic force. To experimentally validate the operation of the OET device, we demonstrate UV-microspot irradiation of cells containing green fluorescent protein (GFP) tagged DNA single-strand break repair protein, targeted in suspension. We demonstrate the optofluidic control of single cells and groups of cells before, during, and after irradiation. The integration of optofluidic cellular manipulation into a biological microbeam enhances the facility's ability to handle non-adherent cells such as lymphocytes. To the best of our knowledge, this is the first time that OET cell handling is successfully implemented in a biological microbeam.
The Review of scientific instruments 01/2013; 84(1):014301. · 1.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: It is unknown whether the observed increase in computed tomography pulmonary angiography (CTPA) utilization has resulted in increased detection of pulmonary emboli (PEs) with a less severe disease spectrum.
Trends in utilization, diagnostic yield, and disease severity were evaluated for 4,048 consecutive initial CTPAs performed in adult patients in the emergency department of a large urban academic medical center between 1/1/2004 and 10/31/2009. Transthoracic echocardiography (TTE) findings and peak serum troponin levels were evaluated to assess for the presence of PE-associated right ventricular (RV) abnormalities (dysfunction or dilatation) and myocardial injury, respectively. Statistical analyses were performed using multivariate logistic regression.
268 CTPAs (6.6%) were positive for acute PE, and 3,780 (93.4%) demonstrated either no PE or chronic PE. There was a significant increase in the likelihood of undergoing CTPA per year during the study period (odds ratio [OR] 1.05, 95% confidence interval [CI] 1.04-1.07, P<0.01). There was no significant change in the likelihood of having a CTPA diagnostic of an acute PE per year (OR 1.03, 95% CI 0.95-1.11, P = 0.49). The likelihood of diagnosing a less severe PE on CTPA with no associated RV abnormalities or myocardial injury increased per year during the study period (OR 1.39, 95% CI 1.10-1.75, P = 0.01).
CTPA utilization has risen with no corresponding change in diagnostic yield, resulting in an increase in PE detection. There is a concurrent rise in the likelihood of diagnosing a less clinically severe spectrum of PEs.
PLoS ONE 01/2013; 8(6):e65669. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 0.5% to 10% of clean surgeries result in surgical-site infections, and attempts to reduce this rate have had limited success. Germicidal UV lamps, with a broad wavelength spectrum from 200 to 400 nm are an effective bactericidal option against drug-resistant and drug-sensitive bacteria, but represent a health hazard to patient and staff. By contrast, because of its limited penetration, ∼200 nm far-UVC light is predicted to be effective in killing bacteria, but without the human health hazards to skin and eyes associated with conventional germicidal UV exposure.
The aim of this work was to test the biophysically-based hypothesis that ∼200 nm UV light is significantly cytotoxic to bacteria, but minimally cytotoxic or mutagenic to human cells either isolated or within tissues.
A Kr-Br excimer lamp was used, which produces 207-nm UV light, with a filter to remove higher-wavelength components. Comparisons were made with results from a conventional broad spectrum 254-nm UV germicidal lamp. First, cell inactivation vs. UV fluence data were generated for methicillin-resistant S. aureus (MRSA) bacteria and also for normal human fibroblasts. Second, yields of the main UV-associated pre-mutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) were measured, for both UV radiations incident on 3-D human skin tissue.
We found that 207-nm UV light kills MRSA efficiently but, unlike conventional germicidal UV lamps, produces little cell killing in human cells. In a 3-D human skin model, 207-nm UV light produced almost no pre-mutagenic UV-associated DNA lesions, in contrast to significant yields induced by a conventional germicidal UV lamp.
As predicted based on biophysical considerations, 207-nm light kills bacteria efficiently but does not appear to be significantly cytotoxic or mutagenic to human cells. Used appropriately, 207-nm light may have the potential for safely and inexpensively reducing surgical-site infection rates, including those of drug-resistant origin.
PLoS ONE 01/2013; 8(10):e76968. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: We fabricated ultrathin metal-semiconductor Schottky diodes for use as transmission particle detectors in the biological microbeam at Columbia University's Radiological Research Accelerator Facility (RARAF). The RARAF microbeam can deliver a precise dose of ionizing radiation in cell nuclei with sub-micron precision. To ensure an accurate delivery of charged particles, the facility currently uses a commercial charged-particle detector placed after the sample. We present here a transmission detector that will be placed between the particle accelerator and the biological specimen, allowing the irradiation of samples that would otherwise block radiation from reaching a detector behind the sample. Four detectors were fabricated with co-planar gold and aluminum electrodes thermally evaporated onto etched n-type crystalline silicon substrates, with device thicknesses ranging from 8.5 μm - 13.5 μm. We show coincident detections and pulse-height distributions of charged particles in both the transmission detector and the commercial detector above it. Detections are demonstrated at a range of operating conditions, including incoming particle type, count rate, and beam location on the detectors. The 13.5 μm detector is shown to work best to detect 2.7 MeV protons (H(+)), and the 8.5 μm detector is shown to work best to detect 5.4 MeV alpha particles ((4)He(++)). The development of a transmission detector enables a range of new experiments to take place at RARAF on radiation-stopping samples such as thick tissues, targets that need immersion microscopy, and integrated microfluidic devices for handling larger quantities of cells and small organisms.
Journal of Instrumentation 12/2012; 7. · 1.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: A completely automated, high-throughput biodosimetry workstation has been developed by the Center for Minimally Invasive Radiation Biodosimetry at Columbia University over the past few years. To process patients' blood samples safely and reliably presents a significant challenge in the development of this biodosimetry tool. In this paper, automated failure recognition methods of robotic manipulation of capillary tubes based on a torque/force sensor are described. The characteristic features of sampled raw signals are extracted through data preprocessing. The twelve-dimensional (12D) feature space is projected onto a two-dimensional (2D) feature plane by the optimized Principal Component Analysis (PCA) and Fisher Discrimination Analysis (FDA) feature extraction functions. For the three-class manipulation failure problem in the cell harvesting module, FDA yields better separability index than that of PCA and produces well separated classes. Three classification methods, Support Vector Machine (SVM), Fisher Linear Discrimination (FLD) and Quadratic Discrimination Analysis (QDA), are employed for real-time recognition. Considering the trade-off between error rate and computation cost, SVM achieves the best overall performance.
Expert Systems with Applications 08/2012; 39(10):9602-9611. · 1.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Protein oxidation can contribute to radiation-induced cell death by two mechanisms: (1) by reducing the fidelity of DNA repair, and (2) by decreasing cell viability directly. Previously, we explored the first mechanism by developing a mathematical model and applying it to data on Deinococcus radiodurans . Here we extend the model to both mechanisms, and analyze a recently published data set of protein carbonylation and cell survival in D. radiodurans and Escherichia coli exposed to gamma and ultraviolet radiation. Our results suggest that similar cell survival curves can be produced by very different mechanisms. For example, wild-type E. coli and DNA double-strand break (DSB) repair-deficient recA- D. radiodurans succumb to radiation doses of similar magnitude, but for different reasons: wild-type E. coli proteins are easily oxidized, causing cell death even at low levels of DNA damage, whereas proteins in recA- D. radiodurans are well protected from oxidation, but DSBs are not repaired correctly even when most proteins are intact. Radioresistant E. coli mutants survive higher radiation doses than the wild-type because of superior protection of cellular proteins from radiogenic oxidation. In contrast, wild-type D. radiodurans is much more radioresistant than the recA- mutant because of superior DSB repair, whereas protein protection in both strains is similar. With further development, the modeling approach presented here can also quantify the causes of radiation-induced cell death in other organisms. Enhanced understanding of these causes can stimulate research on novel radioprotection strategies.
Radiation Research 06/2012; 178(1):17-24. · 2.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: the risk of performing thoracic com-puted tomography (CT). The simplest way to reduce risk is not to perform thoracic CT in younger female patients, viz, those who are still menstruating. In male and older female patients, say (with apologies) those aged 50 years, the risk of breast cancer induction from thoracic CT is so low that the benefit greatly exceeds the cost. In considering radiation risk, therefore, it is essential to look separately at young versus old, at male versus female patients, and, in particular, at pediatric patients. These groups have widely differing risks and should not be mixed. For ex-ample, as Brenner et al have previously pointed out (3), the cancer induction risk of carrying out CT of the chest in a 15-year-old girl is high, whereas that in a 70-year-old man is very low. One hopes that the authors' contin-ued reinforcement of the differing risks at different ages will lead to their widespread use as a simple and logi-cal way of reducing radiation dose: spe-cifically to those with the highest risk. We thank Dr Milne for his interesting comments. In fact, the traditional view that radiation-induced cancer risks de-crease monotonically with increasing age at exposure (1) has been increasingly chal-lenged in recent years (2–4). This is pri-marily because one of the main path-ways to radiation-induced cancer is now thought to be promotion of preexisting premalignant cells by radiation to a malignant state, and our burden of such premalignant cells increases with age. Most radiation-induced cancers ap-pear in the "cancer-prone" ages (typically 50–80 years of age), independent of age at exposure (5), so the latency period be-tween radiation exposure and the poten-tial appearance of a cancer decreases markedly with increasing age at expo-sure. Taking these effects into account, more recent analyses of cancer incidence among atomic bomb survivors suggest that the lifetime risk of radiation-induced cancer is not so different, for example, for exposure at age 5 years versus expo-sure at age 55 years (3,6). Thus, it is not necessarily the case that "the older the better," as Dr Milne asserts. In summary, we agree that individual demographics such as age, and also po-tentially reduced life expectancy (the topic of the article  on which Dr Milne comments), should be consid-ered in assessing individual radiation risks. However, radiation-induced cancer risks probably vary less with age at ex-posure than had previously been thought, which is of particular impor-tance for those patients who are most likely to undergo CT—that is, patients aged 45–65 years (8). We reiterate that when CT is clini-cally justified, the comparatively small radiation risk will almost always be out-weighed by the potential clinical benefit (9). It is nevertheless prudent to mini-mize the potential risks from any radio-logic exposure, and the considerations briefly described herein suggest that it may not be wise to focus potential risk reduction strategies predominantly on pediatric patients, as Dr Milne sug-gests. Patients of all ages are important here, including older patients.
[show abstract][hide abstract] ABSTRACT: Since the 1960s, the Radiological Research Accelerator Facility (RARAF) has been providing researchers in biology, chemistry and physics with advanced irradiation techniques, using charged particles, photons and neutrons.We are currently developing a unique facility at RARAF, to simulate neutron spectra from an improvised nuclear device (IND), based on calculations of the neutron spectrum at 1.5 km from the epicenter of the Hiroshima atom bomb. This is significantly different from a standard fission spectrum, because the spectrum changes as the neutrons are transported through air, and is dominated by neutron energies between 0.05 and 8 MeV. This facility will be based on a mixed proton/deuteron beam impinging on a thick beryllium target.A second, novel facility under development is our new neutron microbeam. The neutron microbeam will, for the first time, provide a kinematically collimated neutron beam, 10-20 micron in diameter. This facility is based on a Proton Microbeam, impinging on a thin lithium target near the threshold of the (7)Li(p,n)(7)Be reaction. This novel neutron microbeam will enable studies of neutron damage to small targets, such as single cells, individual organs within small animals or microelectronic components.
Journal of Instrumentation 03/2012; 7(3). · 1.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: The risk of secondary lung malignancy (SLM) is a significant concern for women treated with whole-breast radiation therapy after breast-conserving surgery for early-stage breast cancer. In this study, a biologically based secondary malignancy model was used to quantify the risk of secondary lung malignancies (SLMs) associated with several common methods of delivering whole-breast radiation therapy (RT).
Both supine and prone computed tomography simulations of 15 women with early breast cancer were used to generate standard fractionated and hypofractionated whole-breast RT treatment plans for each patient. Dose-volume histograms (DVHs) of the ipsilateral breast and lung were calculated for each patient on each plan. A model of spontaneous and radiation-induced carcinogenesis was used to determine the relative risks of SLMs for the different treatment techniques.
A higher risk of SLMs was predicted for supine breast irradiation when compared with prone breast irradiation for both the standard fractionation and hypofractionation schedules (relative risk [RR] = 2.59, 95% confidence interval (CI) = 2.30-2.88, and RR = 2.68, 95% CI = 2.39-2.98, respectively). No difference in risk of SLMs was noted between standard fractionation and hypofractionation schedules in either the supine position (RR = 1.05, 95% CI = 0.97-1.14) or the prone position (RR = 1.01, 95% CI = 0.88-1.15).
Compared with supine whole-breast irradiation, prone breast irradiation is associated with a significantly lower predicted risk of secondary lung malignancy. In this modeling study, fractionation schedule did not have an impact on the risk of SLMs in women treated with whole-breast RT for early breast cancer.
International journal of radiation oncology, biology, physics 01/2012; 83(4):1101-6. · 4.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: superiority in prostate cancer detection including local recurrence and lymph node metastases over F-18 fluorodeoxyglucose (FDG) PET and a higher sensitivity and specificity compared with other imaging studies in prostate cancer. We report our experience. Materials/Methods: From January 2010 to December 2011, a total of 76 patients with prostate cancer were referred for evaluation. Of these, 32 patients underwent primary radiation therapy treatment, 23 patients were for adjuvant radiation therapy, and 10 patients received salvage radiation therapy as well as 11 patients were treated with palliative radiation therapy for painful bone metastases. High risk and patients with metastatic disease also received hormonal therapy. Patients with suspicious lymph nodes on CAT scan or MRI during staging work up underwent C11-acetate PET. The findings and changes to original treatment plan are reported. Results: A total of 19 patients had C11-acetate PET. Eleven patients (57%) had high risk prostate cancer. Nine patients (47%) underwent primary radiation therapy treatment. Seven patients (37%) had increased SUV values (Above 6). The involved areas on C11-acetate PET included external iliac lymph nodes in five patients as well as both, iliac external and inguinal lymph nodes in two patients; Modification of radiation therapy treatment volumes, dose or both was required in three , one and three patients respectively. The decision to add hormone therapy remained the same without any change. With a mean follow up of 18 months, all of these patients remained under biochemical control and C11-acetate PET showed decrease in SUV at the previously affected regions. There was no correlation between PSA level and lymph node involvement but most of the patients with positive C11-acetate PET were in high risk category. Conclusions: C11-acetate PET was useful in detecting lymph node metastases especially in high risk prostate cancer patients. The findings of C11-acetate PET resulted in the change of radiation therapy treatment field/ volume or dose in more than one third of the patients (37%), hopefully leading to better local regional control. SUV values may serve as a tool for monitoring radiation therapy response. Further studies including prospective clinical trials are needed. Purpose/Objectives: Radiation therapy for prostate cancer may be asso-ciated with an increased risk of secondary malignancies, including rectal and bladder cancer. Pelvic nodal irradiation (PNI) is often added as a strategy to improve outcomes in intermediate and high risk localized prostate cancer, and its use is currently being investigated by the RTOG. PNI can be delivered with a 3D conformal technique or IMRT to decrease toxicity. In this study, we model the secondary rectal and bladder malig-nancy risk associated with the addition of IMRT PNI in the treatment of prostate cancer. Materials/Methods: Ten high-risk prostate cancer patients who underwent definitive treatment with pelvic node and prostate/seminal vesicle irradi-ation using IMRT were retrospectively analyzed. For each patient, two plans were compared. Both plans treated the prostate and seminal vesicles (P+SV) to 8100 cGy, while one plan included PNI to 4500 cGy. Rectal and bladder wall dose-volume histograms were obtained. A previously
International Journal of Radiation OncologyBiologyPhysics 01/2012; · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: patients with completely resected stage IIIa (N2) NSCLC has not been reached the consensus. The CTV contouring guideline for PORT was developed in 2004. The rationale of the CTV was evaluated in this study. Materials/Methods: Basing on comprehensive evidence of regularity of mediastinal lymphatic drainage, the CTV contouring guideline for PORT in stage IIIa(N2) patients with completely resected was developed in our hospital. The postoperative CTV of the left lung cancer include #1R, #1L, #2R, #2L, #3, #4R, #4L, #5, #6, #7 and #10-11L station lymph nodes; the right lung cancer including #1R, #2R, #3, #4R, #7 and #10-11L station lymph nodes. (The lymph node grouping was according to the 1997 AJCC/UICC regional lymph node classification. Because of the change of 2009 AJCC lymph node grouping, the #1 station lymph nodes was accordingly removed from the irradiation field.) From 2005 to 2010, 50 patients with pathological stage IIIa(N2) NSCLC who underwent complete resection and received postoperative adjuvant radiation therapy in accordance with the CTV con-touring guideline at our hospital were included in this retrospective study. The patterns of first failure after PORT were evaluated in order to help determine whether or not the contouring protocol of PORT CTV is appropriate. Results: Of the 50 patients in this study, 54% were males and 46% were females. The median radiation dose was 50.4Gy at 1.8Gy per fraction. No patient received preoperative chemotherapy or radiation therapy. All patients underwent postoperative adjuvant chemotherapy in this study. The median follow-up time for the 26 living patients was 41.3 months (range, 20.3-80.6). The median survival time (MST) was 53.6 months. Up to the last follow-up, 47 patients had the reliable follow-up data. The first treatment failures were observed in 37 patients, including 7 (18.9%) with loco-regional recurrence alone, 29 (78.4%) with distant metastases, and 1 with both. Among the 7 patients with loco-regional recurrence alone, the site of the local recurrence was the supraclavicular lymph nodes in 4 patients, the mediastinal lymph nodes in 2 patients, multiple local recur-rence sites (both mediastinal LN and supraclavicular LN) in 1 patient. Out of the 3 patients presented with the mediastinal LN relapses, all patients had the in-field recurrence sites within the irradiated field of the post-operative radiation therapy. Conclusions: Basing on the available information, the CTV delineation protocol for completely resected stage IIIa(N2) patients is relatively adequate and appropriate. Purpose/Objective(s): Treatment of early-stage non-small cell lung cancer (NSCLC) with stereotactic body radiation therapy (SBRT) is associated with high rates of local control and long-term overall survival. SBRT is an increasingly preferred treatment option for operable tumors, leading to concerns about the risk of long-term toxicities such as radiation-induced secondary malignancies. There have been no previous studies estimating secondary malignancy risks associated with SBRT delivered with IMRT or with coplanar or non-coplanar VMAT. In this study, we seek to quantify mean lung doses, lung volumes receiving >20 Gy (V20), and predicted rates of secondary lung malignancies in patients treated with SBRT for stage I-II NSCLC and compare the risks associated with IMRT vs. coplanar VMAT vs. non-coplanar VMAT techniques. Materials/Methods: Thirty-six treatment plans for 12 stage I-II NSCLC patients treated with definitive-intent SBRT were retrospectively assessed.
International Journal of Radiation OncologyBiologyPhysics 01/2012; · 4.52 Impact Factor