Guy Garty - Academia.edu (original) (raw)

Papers by Guy Garty

Neuro-Oncology

Diffuse Midline Glioma (DMG), H3K27M altered, confers a dismal survival of 9-15 months and has a ... more Diffuse Midline Glioma (DMG), H3K27M altered, confers a dismal survival of 9-15 months and has a non-inflammatory tumor immune microenvironment (TIME). Radiation therapy (RT) is the mainstay treatment for DMG and has been shown in other cancers to recruit an immune component. However, the effect of RT on the DMG TIME has not been explored. In a syngeneic murine model of pontine DMG (PDGFB+, H3.3K27M, p53−/−), mice were treated with single fraction 15Gy RT or sham control, four mice per group. We performed single cell sequencing after CD45 isolation to evaluate the TIME 4 days post RT and compare to untreated tumor (sham control). Unsupervised clustering of 14,848 CD45+ cells revealed 16 immune cell subsets, most abundantly microglia at 75% of cells, with four subtypes representing a spectrum of homeostatic to activated. Microglia from RT are more concentrated in the activated subtypes with an upregulation of interferon response (i.e. Isg15, Ifit3) compared to untreated tumor with an...

Figure S2. Comparison of fold-change of Crip2, Chst3, Add3, Eif3f, Rpl26, Rpl27, Rps17, Rps19, an... more Figure S2. Comparison of fold-change of Crip2, Chst3, Add3, Eif3f, Rpl26, Rpl27, Rps17, Rps19, and c-Myc by qPCR and DNA microarray. (PDF 61 kb)

Table S1. Protein ubiquitination processes identified by PANTHER analysis. Benjamini-corrected p ... more Table S1. Protein ubiquitination processes identified by PANTHER analysis. Benjamini-corrected p values are shown. (PDF 30 kb)

Excel file with complete output of IPA canonical pathways. The "EIF2 Signaling" pathway... more Excel file with complete output of IPA canonical pathways. The "EIF2 Signaling" pathway and the related "mTOR Signaling", "Regulation of eIF4 and p70S6K Signaling", and "p70S6K Signaling" pathways are highlighted in red. The most statistically significant canonical pathways (Benjamini-corrected p value

Excel file with 5 tabs containing a summary of differentially expressed genes by radiation type. ... more Excel file with 5 tabs containing a summary of differentially expressed genes by radiation type. (XLSX 2429 kb)

Journal of Instrumentation, 2022

Standard dosimetry protocols exist for highly penetrating photon and particle beams used in the c... more Standard dosimetry protocols exist for highly penetrating photon and particle beams used in the clinic and in research. However, these protocols cannot be directly applied to shallow penetration MeV-range ion beams. The Radiological Research Accelerator Facility has been using such beams for almost 50 years to irradiate cell monolayers, using self-developed dosimetry, based on tissue equivalent ionization chambers. To better align with the internationally accepted standards, we describe implementation of a commercial, NIST-traceable, air-filled ionization chamber for measurement of absorbed dose to water from low energy ions, using radiation quality correction factors calculated using TRS-398 recommendations. The reported dose does not depend on the ionization density in the range of 10–150 keV/μm.

ABSTRACTPurposeIt has been suggested that heavy-ion radiation therapy may contribute to the contr... more ABSTRACTPurposeIt has been suggested that heavy-ion radiation therapy may contribute to the control of distal metastases. These distant responses may include immune cell activation. Immunostimulation resulting from radiation-induced immunogenic cell death (ICD) of cancer cells, leads to the recruitment of anti-tumor T cells. Specific markers of ICD include translocation of calreticulin (CRT) and extracellular release of high mobility group box 1 protein (HMGB1), and ATP. However, the LET dependence of these effects remains unknown.Materials and MethodsExpression of the molecular indicators described above were tested in a panel of human cancer cell lines, that included pancreatic cancer (Panc1 and Paca2), glioblastoma (U87 and LN18) and melanoma (HTB129 and SK-Mel5). Cells were irradiated with 5 Gy of particles spanning a range of LETs, from 10 KeV/μm to 150 KeV/μm and assayed for relocalization of calreticulin and release of HMGB1 and ATP were assayed 24 hours later.ResultsIn the p...

Journal of Proteome Research, 2021

An important component of ionizing radiation (IR) exposure after a radiological incident may incl... more An important component of ionizing radiation (IR) exposure after a radiological incident may include low-dose rate (LDR) exposures either externally or internally, such as from 137 Cs deposition. In this study, a novel irradiation system, VAriable Dose-rate External 137 Cs irradiatoR (VADER), was used to expose male and female mice to a variable LDR irradiation over a 30-day time span to simulate fallout type exposures in addition to biofluid collection from a reference dose rate (0.8 Gy/min). Radiation markers were identified by untargeted metabolomics and Random Forests. Mice exposed to LDR exposures were successfully identified from control groups based on their urine and serum metabolite profiles. In addition to metabolites commonly perturbed after IR exposure, we identified and validated a novel metabolite (hexosamine-valineisoleucine-OH) that increased up to 150-fold after LDR and 80-fold after conventional exposures in urine. A multiplex panel consisting of hexosamine-valine-isoleucine-OH with other urinary metabolites (N6,N6,N6-trimethyllysine, carnitine, 1-methylnicotinamide, α-ketoglutaric acid) achieved robust classification performance using receiver operating characteristic curve analysis irrespective of dose rate or sex. These results show that in terms of biodosimetry, dysregulated energy metabolism is associated with IR exposure for both LDR and conventional IR exposures. These mass spectrometry data have been deposited to the NIH data repository via Metabolomics Workbench with study ID ST001790,

Scientific Reports, 2021

We implemented machine learning in the radiation biodosimetry field to quantitatively reconstruct... more We implemented machine learning in the radiation biodosimetry field to quantitatively reconstruct neutron doses in mixed neutron + photon exposures, which are expected in improvised nuclear device detonations. Such individualized reconstructions are crucial for triage and treatment because neutrons are more biologically damaging than photons. We used a high-throughput micronucleus assay with automated scanning/imaging on lymphocytes from human blood ex-vivo irradiated with 44 different combinations of 0–4 Gy neutrons and 0–15 Gy photons (542 blood samples), which include reanalysis of past experiments. We developed several metrics that describe micronuclei/cell probability distributions in binucleated cells, and used them as predictors in random forest (RF) and XGboost machine learning analyses to reconstruct the neutron dose in each sample. The probability of “overfitting” was minimized by training both algorithms with repeated cross-validation on a randomly-selected subset of the ...

Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment, 2018

A horizontal multipurpose microbeam system with a single electrostatic quadruplet focusing lens h... more A horizontal multipurpose microbeam system with a single electrostatic quadruplet focusing lens has been developed at the Columbia University Radiological Research Accelerator Facility (RARAF). It is coupled with the RARAF 5.5 MV Singleton accelerator (High Voltage Engineering Europa, the Netherlands) and provides micrometer-size beam for single cell irradiation experiments. It is also used as the primary beam for a neutron microbeam and microPIXE (particle induced x-ray emission) experiment because of its high particle fluence. The optimization of this microbeam has been investigated with ray tracing simulations and the beam spot size has been verified by different measurements.

Journal of Instrumentation, 2006

We present two methods of independently mapping the dimensions of the sensitive volume in an ion-... more We present two methods of independently mapping the dimensions of the sensitive volume in an ion-counting nanodosimeter. The first method is based on a calculational approach simulating the extraction of ions from the sensitive volume, and the second method on probing the sensitive volume with 250 MeV protons. Sensitive-volume maps obtained with both methods are compared and systematic errors inherent in both methods are quantified.

In the search for biological markers after a large-scale exposure of the human population to radi... more In the search for biological markers after a large-scale exposure of the human population to radiation, gene expression is a sensitive endpoint easily translatable to in-field high throughput applications. Primarily, the ex-vivo irradiated healthy human blood model has been used to generate available gene expression datasets. This model has limitations i.e., lack of signaling from other irradiated tissues and deterioration of blood cells cultures over time. In vivo models are needed; therefore, we present our novel approach to define a gene signature in mouse blood cells that quantitatively correlates with radiation dose (at 1 Gy/min). Starting with available microarray datasets, we selected 30 radiation-responsive genes and performed cross-validation/training-testing data splits to downselect 16 radiation-responsive genes. We then tested these genes in an independent cohort of irradiated adult C57BL/6 mice (50:50 both sexes) and measured mRNA by quantitative RT-PCR in whole blood a...

BackgroundRadiation exposure due to the detonation of an improvised nuclear device remains a majo... more BackgroundRadiation exposure due to the detonation of an improvised nuclear device remains a major security concern. Radiation from such a device involves a combination of photons and neutrons. Although photons will make the greater contribution to the total dose, neutrons will certainly have an impact on the severity of the exposure as they have high relative biological effectiveness.ResultsWe investigated the gene expression signatures in the blood of mice exposed to 3 Gy x-rays, 0.75 Gy of neutrons, or to mixed field photon/neutron with the neutron fraction contributing 5, 15%, or 25% of a total 3 Gy radiation dose. Gene ontology and pathway analysis revealed that genes involved in protein ubiquitination pathways were significantly overrepresented in all radiation doses and qualities. On the other hand, eukaryotic initiation factor 2 (EIF2) signaling pathway was identified as one of the top 10 ranked canonical pathways in neutron, but not pure x-ray, exposures. In addition, the r...

Journal for ImmunoTherapy of Cancer

BackgroundDiffuse intrinsic pontine gliomas (DIPG’s) are immunologically inert tumors with a medi... more BackgroundDiffuse intrinsic pontine gliomas (DIPG’s) are immunologically inert tumors with a median survival of 9–15 months. Radiation therapy (RT) is the mainstay treatment for DIPG but is associated with immunodepletion of the tumor microenvironment (TME) at high dose ranges. FLASH, or ultra-fast dose rate RT, represents a novel ablative technique that may spare TME immune responses while decreasing tumor burden. Here, we present single-cell immune profiling of DIPG tumors treated with FLASH, conventional dose rate RT (CONV) or no RT (SHAM).MethodsMurine H3.3K27M mutant DIPG cells were stereotactically injected and tumor induction confirmed by magnetic resonance imaging (MRI) 15 days later. DIPG-bearing mice were randomly assigned to one of three treatment groups (n=4/group), FLASH, CONV or SHAM. A fourth group with no tumor (NML) was included as a negative biological control. A modified linear accelerator was used to deliver 15 Gy of electron RT to the brainstem at dose rates of ...

Radiation Research

An automated platform for cytogenetic biodosimetry, the "Rapid Automated Biodosimetry Tool I... more An automated platform for cytogenetic biodosimetry, the "Rapid Automated Biodosimetry Tool II (RABiT-II)," adapts the dicentric chromosome assay (DCA) for high-throughput mass-screening of the population after a large-scale radiological event. To validate this test, the U.S. Federal Drug Administration (FDA) recommends demonstrating that the high-throughput biodosimetric assay in question correctly reports the dose in an in vivo model. Here we describe the use of rhesus macaques (Macaca mulatta) to augment human studies and validate the accuracy of the high-throughput version of the DCA. To perform analysis, we developed the 17/22-mer peptide nucleic acid (PNA) probes that bind to the rhesus macaque's centromeres. To our knowledge, these are the first custom PNA probes with high specificity that can be used for chromosome analysis in M. mulatta. The accuracy of fully-automated chromosome analysis was improved by optimizing a low-temperature telomere PNA FISH staining in multiwell plates and adding the telomere detection feature to our custom chromosome detection software, FluorQuantDic V4. The dicentric frequencies estimated from in vitro irradiated rhesus macaque samples were compared to human blood samples of individuals subjected to the same ex vivo irradiation conditions. The results of the RABiT-II DCA analysis suggest that, in the lymphocyte system, the dose responses to gamma radiation in the rhesus macaques were similar to those in humans, with small but statistically significant differences between these two model systems.

The cytokinesis-block micronucleus (CBMN) assay is considered as the most suitable biodosimetry m... more The cytokinesis-block micronucleus (CBMN) assay is considered as the most suitable biodosimetry method for automation. Previously, we automated this assay on a commercial robotic biotech high-throughput system (RABiT-II) adopting both a traditional and an accelerated micronucleus protocol, both using centrifugation steps for lymphocyte harvesting and washing, after whole blood culturing. Here we describe further development of our accelerated CBMN assay protocol for using on High Throughput/High Content Screening (HTS/HCS) robotic systems without a centrifuge. This opens the way for implementation of the CBMN assay on a wider range of commercial automated HTS/HCS systems and thus increases the potential capacity of dose estimates following a mass-casualty radiological event.

In recent years we have automated the CBMN assay using microvolumes of blood, processed in multiw... more In recent years we have automated the CBMN assay using microvolumes of blood, processed in multiwell plates. We have seen that at doses above 6 Gy the detected yield of micronuclei actually declines with dose, likely because of mitotic delay, preventing cells from forming micronuclei and also, when using one color imaging, resulting in many false binucleated cells, consisting of two randomly-adjacent nuclei. By using the inverse mitotic index (the ratio of mononuclear to binuclear cells) to adjust the micronucleus yield we were able to obtain a monotonic increasing dose response curve at doses of up to at least 10 Gy from the same samples which generated dose-response curve with a peak near 6 Gy, when scored using the traditional micronucleus yield.