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PURPOSE Chordoma is a locally aggressive tumor that most commonly affects the base of the skull/clivus, cervical, and sacral spine. Conventional radiotherapy (RT), cannot be safely increased further to improve disease control due to the risk of toxicity to the surrounding critical structures. Tumor-targeted hyperthermia (HT) combined with Proton Beam Radiation Therapy (PBRT) is known to act as a potent radiosensitizer in cancer control. In this study, we investigated whether PBRT efficacy for chordoma can be enhanced in combination with HT as a radiosensitizer. MATERIAL AND METHODS Human chordoma cell lines, U-CH2 and Mug-chor1 were treated in vitro with HT followed by PBRT with variable doses. The colony-forming assay was performed, and dose-response was characterized by linear-quadratic model fits. HSP-70 and Brachyury (TBXT) biomarkers for chordoma aggression levels were quantified by western blot analysis. Gene microarray analysis was performed by U133 Arrays. Pathway Analysis was also performed using IPA bioinformatic software. RESULTS Our findings in both U-CH2 and Mug-Chor1 cell lines demonstrate that hyperthermia followed by PBRT has an enhanced cell killing effect when compared with PBRT-alone (p

Pancreatic cancer (PC) is the fourth-most-deadly cancer in the United States with a 5-year survival rate of only 8%. The majority of patients with locally advanced pancreatic cancer undergo chemotherapy and/or radiation therapy (RT). However, current treatments are inadequate and novel strategies are desperately required. 3-Bromopyruvate (3-BP) is a promising anticancer drug against pancreatic cancer. It exerts potent anticancer effects by inhibiting hexokinase II enzyme (HK2) of the glycolytic pathway in cancer cells while not affecting the normal cells. 3-BP killed 95% of Panc-2 cells at 15 μM concentration and severely inhibited ATP production by disrupting the interaction between HK2 and mitochondrial Voltage Dependent Anion Channel-1 (VDAC1) protein. Electron microscopy data revealed that 3-BP severely damaged mitochondrial membrane in cancer cells. We further examined therapeutic effect of 3-BP in syngeneic mouse pancreatic cancer model by treating animals with 10, 15 and 20 mg/kg dose. 3-BP at 1520 mg/kg dose level significantly reduced tumor growth by approximately 75-80% in C57BL/6 female mice. Immunohistochemistry data showed complete inhibition of hexokinase II (HK2) and TGFβ, in animals treated with 3-BP drug. We also observed enhanced expression of active caspase-3 in tumor tissues exhibited apoptotic death. Flow Cytometry analysis showed significant inhibition in MDSC (CD11b) population in treated tumor which may have allowed infiltration of CD8+ T cells and inhibited tumor growth. Notably, metabolomic data also revealed severe inhibition in glycolysis, NADP, ATP and lactic acid production in cancer cells treated with 40 μM 3-BP. Importantly, we also observed inhibition in lactic acid production responsible for tumor aggression. These results provide new evidence that 3-BP severely inhibit glucose metabolism in cancer cells by blocking hexokinase II, and disrupting mitochondria by suppressing BCL2L1 in pancreatic cancer.

Purpose/Objective(s): Pancreatic ductal adenocarcinoma (PDAC) is characterized by an unusually high degree of hypoxia in the tumor microenvironment, which activates hypoxic signaling pathways orchestrated by hypoxia-inducible factor 1α (HIF1α). It has been shown previously that HIF1α significantly increases PDAC radioresistance and growth, though its mechanism of action is unknown. Here, we test the hypothesis that HIF1α confers these traits in PDAC through involvement in DNA damage repair and KRAS signaling in vitro. Materials/Methods: We performed studies with the KPC cell line (KrasLSL-G12D/+;TP53LSL-R172H/+). We used CRISPR-Cas9, a genome editing tool, to generate a HIF1α knockout (KO) cell line. Hypoxic microenvironments were induced using 100uM of the oxygen scavenger CoCl2. Radioresistance and double-strand DNA damage was evaluated and compared between wild-type (WT) and KO cells after treatment with increasing doses of ionizing radiation using neutral comet assays. Tumor signaling pathways were mapped and analyzed using western blot and co-immunoprecipitation. The KRASG12D inhibitor, BI-2852, was obtained from Boehringer-Ingelheim. Cancer cells were evaluated for growth and migration using cell proliferation assays and scratch wound assays following treatment with 50 uM BI-2852. Bioinformatic analysis was conducted using TCGA (The Cancer Genome Atlas) data; high and low expression were defined as samples with mRNA expression z-scores ≥ 1 and ≤ -1, respectively, Results: HIF1α KO cells demonstrated significantly increased radioresistance compared to WT cells following 8 and 10 Gy of radiation in hypoxic conditions (t-test, P Conclusion: Overall, this study demonstrates that HIF1α promotes DNA damage repair in PDAC and reveals a HIF1α/KRAS/p53 signaling axis. Future studies will test anti-HIF1α and anti-KRAS combination therapies in PDAC mouse models. Citation Format: Kevin J. Tu, Sanjit K. Roy, Amit Sawant, Hem D. Shukla. HIF1α is involved in radiation-induced DNA damage repair and anti-apoptotic pathways in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4810.

Hyperthermia treatment of tumors (43-47°C) is used to sensitize breast cancer tumors to radiotherapy. Thermoradiotherapy significantly increases complete response rate and provides a treatment modality for more aggressive forms of the disease for which treatment options are generally more limited. However, the cellular mechanism underlying hyperthermia-induced radiosensitivity remains unclear. To better understand this phenomenon, we analyzed microarray data from four cell lines following treatment at 45°C for 30 minutes (Gene Expression Omnibus GSE48398). Three of the cell lines, MDA231 (37ºC, n=6; 45ºC, n=3), MDA486 (37ºC, n=6; 45ºC, n=3), and MCF7 (37ºC, n=6; 45ºC, n=6), were malignant breast cancer cell lines. The fourth cell line, MCF10A (37ºC, n=3; 45ºC, n=3), was from non-malignant mammary epithelial tissue. We identified 164 DEGs (adjP < 0.05, Fold change > 2) shared between the four cell lines and 182 DEGS shared by the three cancerous cell lines. Genes involved in radiosensitivity regulation as identified by dbCRSR, a database of radioresistance genes, were significantly dysregulated (Chi-squared test, p = 0.04). Gene ontology analysis using Cytoscape (v3.8.2) found 53 significantly enriched GO terms from an analysis off the DEGs (Benjamini-Hochberg adjP < 0.05). In particular, DEGs were enriched in processes involving G2 cell cycle arrest, apoptosis, and DNA damage checkpoints (adjP Citation Format: Kevin Jili Tu, Hong Zhang, Dario Rodrigues, Jason K. Molitoris, Amit Sawant, Hem D. Shukla. Gene expression profile analysis of hyperthermia-induced radiosensitivity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1104.

Pancreatic cancer (PC) is the deadly cancer in both men and women, with a one-year relative survival rate of 20%, and a five-year relative survival rate of only 8%. Pancreatic cancer is characterized by dense fibrotic stroma that constitutes up to 90% of the total tumor volume and contains extracellular matrix (ECM), immune cells, vasculature, and cancer-associated fibroblasts which are hallmark of immunosuppressive tumor microenvironment. In present investigation we have tested anti-cancer effect of 3-BromoPyruvate (3-BP), an inhibitor of aerobic glycolysis employing Panc-2 cancer cells, and syngeneic pancreatic cancer mouse model. Panc-2 cancer cells treated with 40 uM of 3-BP showed significant inhibition in cell proliferation and ATP production. Cells treated with 60 uM dose level, showed drastic phenotypic changes (membrane blebbing) within 2 hours of treatment. The qRT PCR data showed 3BP inhibited hexokinase II (HK2) expression at 40uM, suggesting 3-BP regulate HK2 expression at transcriptional level. We also observed that 3-BP in combination with low doses of irradiation was more effective in killing cancer cells than 3-BP alone in both 2D and 3D culture model. Treatment of C57BL/6 subcutaneous pancreatic tumor model with 10 and 20 mg/kg of 3BP also showed significant reduction of tumor growth. The Transmission Electron Microscopy of tumor tissues treated with 3-BP revealed severely damaged mitochondrial structure which might have severely affected ATP generation in cancer cell. Altogether, the results in this study demonstrated that 3-BP could be a promising anti-tumor drug against pancreatic cancer and its efficacy could be improved in combination with radiation therapy. Citation Format: Sanjit Roy, Tijana Dukic, Binny Bhandary, Jason Molitoris, Young H Ko, Hem D Shukla. 3-Bromopyruvate reduces pancreatic tumor growth by damaging mitochondria and inhibiting glucose metabolism in both cell culture and mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1094.

Prostate cancer (PCa) treatment with radiation therapy (RT) has an excellent cure rate. However, Radiation-induced Erectile Dysfunction (RiED) is a common and irreversible toxicity impacting quality of life, and there is no FDA approved specific drug for RiED. We previously showed that prostate RT increased RhoA/ROCK signaling in the cavernous nerve (CN) and penile tissues, which may lead to RiED in rats. In this study, we investigated whether RhoA/ROCK pathway inhibition by a specific inhibitor called Hydroxyfasudil (HF) can improve RiED in our well-established rat model.Male Sprague-Dawley rats were randomized to the following groups: sham-RT, HF-only, RT-only, and RT + HF. Rats were either exposed to a single dose of 25 Gy prostate-confined RT or a sham procedure. 10 mg/kg HF or normal saline was injected intraperitoneally. Erectile function was evaluated by intracavernosal pressure (ICP) and mean arterial pressure (MAP) measurements at week 14 post-RT. Cavernous nerve (CN) injury was evaluated by transmission electron microscopy (TEM), and penile tissue fibrosis by Masson trichrome staining (MT).We have found that the HF treatment prior to RT showed significant (p 0.001) improvement in ICP/MAP ratio, area under the curve, and maximum ICP value, compared to RT-alone rats. Furthermore, RT + HF treated rats exhibited increased CN myelination and decreased axonal atrophy, comparted to RT-only. HF treatment showed significantly decreased penile tissue fibrosis (p 0.05) compared to RT-alone treated rats.Our results provide the first preclinical evidence that targeting RhoA/ROCK pathway by HF may provide a novel therapeutic option for the treatment of RiED.

3-Bromopyruvate (3 BP) is a promising anticancer drug against pancreatic cancer. It exerts potent anticancer effects by inhibiting hexokinase II enzyme of glycolysis pathway and ATP generation in cancer cells. In present investigation we have examined the effect of 3-BromoPyruvate employing panc-2 cancer cells, and syngeneic pancreatic cancer mouse model. The clonogenic survival assay demonstrated dose dependent inhibition of cell growth, and we observed 95% cell death at 15 uM conc. Further, ATP production was severely inhibited in cancer cells treated with same concentration of 3-BP. We also observed that 3-BP in combination with low doses of irradiation was more effective in killing cancer cells than 3-BP alone. Electron microscopy revealed that 3-BromoPyruvate severely damaged mitochondrial integrity which might have severely affected ATP generation in cancer cells. We further tested 3-BromoPyruvate (2.5-10.0 mg/kg) in syngeneic panc-2 tumor mouse model and observed that 3-BP at10.0 mg/kg dose significantly reduced tumor growth (volume) about 35-40%. Immunohistochemistry (IHC) data showed that 3-BP inhibits hexokinase II (HK2) and TGFbeta1 and enhanced active caspase-3 expression in tumor tissues as compared to untreated control. The disruption of the interaction between VDAC1 and HK2 due to 3BP treatment was confirmed by CO-IP. Interestingly, 3 BP at higher dose induces caspase-3 activation in tumor derived organoid without showing any effect on normal pancreatic organoid. Altogether, the results in this study demonstrated that 3 BP could be a promising anti-tumor drug against pancreatic cancer and its efficacy could be improved in combination with radiation therapy. Citation Format: Sanjit K. Roy, Tijana Dukic, Binny Bhandary, Aneesh Acharya, Kevin J. Tu, Young H. Ko, Hem D. Shukla. 3-Bromopyruvate in combination with radiation inhibits pancreatic cancer growth by dismantling mitochondria and ATP generation in a preclinical mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5243.

Background: Pancreatic cancer (PC) is the fourth-most-deadly cancer in the United States with a 5-year survival rate of only 8%. Unfortunately, only 10&ndash, 20% of PC patients are candidates for surgery, with the vast majority of patients with locally-advanced disease undergoing chemotherapy and/or radiation therapy (RT). Current treatments are clearly inadequate and novel strategies are crucially required. We investigated a novel tripartite treatment (combination of tumor targeted hyperthermia (HT), radiation therapy (RT), and immunotherapy (IT)) to alter immunosuppressive PC-tumor microenvironment (TME). (2). Methods: In a syngeneic PC murine tumor model, HT was delivered before tumor-targeted RT, by a small animal radiation research platform (SARRP) followed by intraperitoneal injections of cytotoxic T-cell agonist antibody against OX40 (also known as CD134 or Tumor necrosis factor receptor superfamily member 4, TNFRSF4) that can promote T-effector cell activation and inhibit T-regulatory (T-reg) function. (3). Results: Tripartite treatment demonstrated significant inhibition of tumor growth (p &lt, 0.01) up to 45 days post-treatment with an increased survival rate compared to any monotherapy. Flow cytometric analysis showed a significant increase (p &lt, 0.01) in cytotoxic CD8 and CD4+ T-cells in the TME of the tripartite treatment groups. There was no tripartite-treatment-related toxicity observed in mice. (4). Conclusions: Tripartite treatment could be a novel therapeutic option for PC patients.

Simple Summary Chordoma is a rare, slow-growing cancer of the spinal cord. Photon radiation therapy and surgery are the standard of care for chordoma. Proton radiation therapy has become an increasingly common treatment in comparison to photon radiation therapy due to the ability to reduce off-target radiation dose. However, there is still an increased risk of toxicity to the surrounding critical structures that lead to poor treatment outcomes. Moreover, the biologic effectiveness of protons to sterilize chordoma cells remains uncertain and likely varies according to the proton energy spectrum throughout the proton field. We aim to investigate the tumoricidal properties of proton radiation therapy at the middle and end of the proton radiation field and elucidate variations in the relative biological effectiveness for chordoma cells. Our study helps quantify the therapeutic value of treating chordoma near the end of the proton field, where linear energy transfer is relatively high. Abstract Background: Chordoma is a cancer of spinal cord, skull base, and sacral area. Currently, the standard of care to treat chordoma is resection followed by radiation therapy. Since, chordoma is present in the spinal cord and these are very sensitive structures and often complete removal by surgery is not possible. As a result, chordoma has a high chance of recurrence and developing resistance to radiation therapy. In addition, treatment of chordoma by conventional radiation therapy can also damage normal tissues surrounding chordoma. Thus, current therapeutic options to treat chordoma are insufficient and novel therapies are desperately needed to treat locally advanced and metastatic chordoma. (2) Methods: In the present investigation, human chordoma cell lines of sacral origin MUG-Chor1 and U-CH2 were cultured and irradiated with Proton Beam Radiation using the clinical superconducting cyclotron and pencil-beam (active) scanning at Middle and End of the Spread-Out Bragg Peak (SOBP). Proton radiation was given at the following doses: Mug-Chor1 at 0, 1, 2, 4, and 8 Gy and U-CH2 at 0, 4, 8, 12, and 16 Gy. These doses were selected based on a pilot study in our lab and attempted to produce approximate survival fractions in the range of 1, 0.9, 0.5, 0.1, and 0.01, respectively, chosen for linear quadratic model fitting of the dose response. (3) Results: In this study, we investigated relative biological effectiveness (RBE) of proton radiation at the end of Spread Out Bragg Peak assuming that the reference radiation is a proton radiation in the middle of the SOBP. We observed differences in the survival of both Human chordoma cell lines, U-CH2 and MUG-Chor1. The data showed that there was a significantly higher cell death at the end of the Bragg peak as compared to middle of the Bragg peak. Based on the linear quadratic (LQ) fit for cell survival we calculated the RBE between M-SOBP and E-SOBP at 95% CI level and it was observed that RBE was higher than 1 at E-SOBP and caused significantly higher cell killing. Proton field at E-SOBP caused complex DNA damage in comparison to M-EOBP and the genes such as DNA topoisomerase 1, GTSE1, RAD51B were downregulated in E-SOBP treated cells. Thus, we conclude that there seems to be substantial variation in RBE (1.3–1.7) at the E-SOBP compared with the M-SOBP.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most lethal cancer in the United States with an estimated 60,430 new cases and 48,220 deaths in 2021. PDACs are characterized by extensive desmoplastic stroma and severe hypovascularity, resulting in an intra-tumoral hypoxic microenvironment. In response to hypoxic stress, cancer cells set off many adaptive responses including metabolism regulation, cell survival, and inflammation through the stabilization and activation of the hypoxia-inducible factor (HIF) family of transcription factors. In this study, we used the gene-editing tool CRISPR-cas9 to knock out HIF1A and investigate whether HIF1A affects the radioresistant and invasive characteristics of PDACs in a KPC cell line model. To first observe how HIF1A affects PDAC radioresistance, we performed a clonogenic survival assay with increasing doses of radiation on both wild-type and HIF1A knockout (KO) cells treated and untreated with CoCl2-induced hypoxia conditions (100 uM and 200 uM CoCl2). Our data showed that under hypoxia, KO cells exhibited significant cell death when treated with 6, 8, and 10 Gy of radiation as compared to wild-type KPC cells, emphasizing the role of HIF1A in radiation resistance. In addition, to understand the role of HIF1A in regulating the invasive behavior of PDACs, we performed a cell proliferation assay on wild-type and KO KPC cells. HIF1A KO cells treated with CoCl2 exhibited significantly reduced proliferation compared to wild-type cells also treated with CoCl2 (p0.05). Because western blot demonstrated increased HIF1A expression in wild-type cells following CoCl2 treatment, our results provided evidence for the role of HIF1A activation in promoting PDAC invasiveness. Through western blot, we also confirmed the association between HIF1A expression with p53 degradation in PDAC. We used COREMINE, a literature mining tool, to map a direct interaction between HIF1A with KRAS in PDACs (p=0.000016). We propose HIF1A as a switch to activate KRAS and degrade p53 under hypoxic conditions in PDAC proliferation. Thus, modulating the HIF1A switch may be an important mechanism to reduce the tumor-promoting microenvironment and inhibit cancer growth. Citation Format: Kevin J. Tu, Sanjit K. Roy, Binny Bhandary, Amit Sawant, Hem D. Shukla. Loss of HIF1A decreases resistance to radiation and invasiveness in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-126.

Radiation therapy plays a major role in the treatment of lung cancer patients. However, cancer cells develop resistance to radiation. Tumor radioresistance is a complex multifactorial mechanism which may be dependent on DNA damage and repair, hypoxic conditions inside tumor microenvironment, and the clonal selection of radioresistant cells from the heterogeneous tumor site, and it is a major cause of treatment failure in non–small cell lung cancer (NSCLC). In the present investigation caveolin-1 (CAV-1) has been observed to be highly expressed in radiation resistant A549 lung cancer cells. CRISPR-Cas9 knockout of CAV-1 reverted the cells to a radio sensitive phenotype. In addition, CAV-1 overexpression in parental A549 cells, led to radiation resistance. Further, gene expression analysis of A549 parental, radiation resistant, and caveolin-1 overexpressed cells, exhibited overexpression of DNA repair genes RAD51B, RAD18, SOX2 cancer stem cell marker, MMPs, mucins and cytoskeleton proteins in resistant and caveolin-1 over expressed A549 cells, as compared to parental A549 cells. Bioinformatic analysis shows upregulation of BRCA1, Nuclear Excision DNA repair, TGFB and JAK/STAT signaling pathways in radioresistant and caveolin-1 overexpressed cells, which may functionally mediate radiation resistance. Immunohistochemistry data demonstrated heterogeneous expression of CAV-1 gene in human lung cancer tissues, which was analogous to its enhanced expression in human lung cancer cell line model and mouse orthotopic xenograft lung cancer model. Also, TCGA PanCancer clinical studies have demonstrated amplification, deletions and missense mutation in CAV-1 gene in lung cancer patients, and that CAV-1 alteration has been linked to poor prognosis, and poor survival in lung cancer patients. Interestingly, we have also optimized ELISA assay to measure caveolin-1 protein in the blood of A549 radiation resistant human xenograft preclinical mouse model and discovered higher level of caveolin-1 (950 pg/ml) in tumor bearing animals treated with radiation, as compared to xenograft with radiosensitive lung cancer cells (450 pg/ml). Thus, we conclude that caveolin-1 is involved in radio-resistance and contributes to tumor aggression, and it has potential to be used as prognostic biomarker for radiation treatment response, and tumor progression for precision medicine in lung cancer patients.

Caveolin-1 is a membrane protein highly expressed in many tumors and plays an important role in tumor progression and metastasis. This review describes the structure of the Caveolin-1 protein and its pre-clinical and clinical significance, demonstrating that Caveolin-1 is a novel biomarker for radioresistance which has the promising potential to improve the clinical outcome of cancer patients undergoing radiation treatment.Targeted radiation therapy has shown immense benefits for cancer treatment. However, one of the major challenges for effective clinical outcome of radiation therapy for cancer patients is the development of radioresistance during radiation treatment. As a consequence, radiation therapy becomes a less effective modality for successful clinical outcome. Furthermore, a radioresistant tumor has the ability to repair its genome, and therefore becomes more aggressive and metastasizes. The plausible mechanisms for tumor radioresistance include the rapid DNA repair, somatic mutations in tumor oncogenes, aberrant activation of kinase pathways, and changes in the tumor microenvironment including tumor hypoxia, tumor vasculature, and cancer stem cells. Caveolin-1 is significantly upregulated in certain cancer cells and aberrantly mediates downstream signaling mechanisms. Notably, numerous recent research reports have shown the role of Caveolin-1 in tumor radioresistance and poor treatment outcome. Thus, Caveolin-1 could be a novel prognostic biomarker to monitor tumor radioresistance in cancer patients undergoing radiation therapy.Caveolin-1 has the promising potential to become a novel prognostic biomarker to monitor tumor radioresistance and radiation response specifically in the prostate, pancreas, and lung cancer.

Cancer is the leading cause of mortality among men and women worldwide. Despite the availability of numerous diagnostic techniques for various cancers, the overall survival rate remains low and the majority of patients die due to late diagnosis and advanced stage of the disease. Diagnosing and treating cancer at its early stages ideally during the precancerous phase could significantly increase survival rate with the possibility of cure and prolong survival. Cancer is a genetic disease and it is illicitly activated by the acquisition of somatic DNA lesions and aberrations in genome structure and defects in maintenance and repair. These somatic DNA mutations known as driver mutations seem to be the prime cause in initiating tumorigenesis. The advances in genomic technologies have immensely facilitated the understanding of cancer progression and metastasis, and the discovery of novel biomarkers. However, changes in somatic mutational landscape of the oncogenome are translated into aberrantly regulated oncoproteome which drives the cancer initiation. Thus, combination of proteomic and genomic technologies is urgently required to discover biomarkers for early diagnosis. The recent advances in human genome based detection of cancer using advanced genomic technologies like NextGen Sequencing, digital PCR, cfDNA technology have shown promise; for example oncogenic somatic mutation variants, transcriptomic analysis, copy number variant, and methylation data from the Cancer Genome Atlas. Similarly, oncoproteomics has the potential to revolutionize clinical management of the disease, including cancer diagnosis and screening based on new proteomic database which embodies somatic variants and post translational modifications, thus devising proteomic technologies as a complement to histopathology. Further, the use of multiple proteomic and genomic biomarkers rather than a single gene or protein could greatly improve diagnostic accuracy and enhance the predictive power for treatment outcome and may enable adequate monitoring of the response to treatment and could be an important option for personalized medicine. The proteogenomic approach has the promise to identify new biomarkers for radiation therapy (RT) which could reliably predict the tumor radiation resistance and which could also accurately predict normal tissue toxicity, and at the same time radiotherapy effectiveness. In this review we have summarize the recent advances in proteogenomic approaches to develop more sensitive diagnostic and prognostic biomarkers which could be translated into improved clinical care and management of the disease.

Pancreatic cancer (PC) has the highest mortality rate amongst all other cancers in both men and women, with a one-year relative survival rate of 20%, and a five-year relative survival rate of 8% for all stages of PC combined. The Whipple procedure, or pancreaticoduodenectomy, can increase survival for patients with resectable PC, however, less than 20% of patients are candidates for surgery at time of presentation. Most of the patients are diagnosed with advanced PC, often with regional and distant metastasis. In these advanced cases, chemotherapy and radiation have shown limited tumor control, and PC continues to be refractory to treatment and results in a poor survival outcome. In recent years, there has been intensive research on checkpoint inhibitor immunotherapy for PC, however, PC is characterized with dense stromal tissue and a tumor microenvironment (TME) that is highly immunosuppressive, which makes immunotherapy less effective. Interestingly, when immunotherapy is combined with radiation therapy (RT) and loco-regional hyperthermia (HT), it has demonstrated enhanced tumor responses. HT improves tumor killing via a variety of mechanisms, targeting both the tumor and the TME. Targeted HT raises the temperature of the tumor and surrounding tissues to 42–43 °C and makes the tumor more immunoresponsive. HT can also modulate the immune system of the TME by inducing and synthesizing heat shock proteins (HSP), which also activate an anti-tumor response. It is well known that HT can enhance RT-induced DNA damage in cancer cells and simultaneously help to oxygenate hypoxic regions. Thus, it is envisaged that combined HT and RT might have immunomodulatory effects in the PC-TME, making PC more responsive to immunotherapies. Moreover, the combined tripartite approach of immunotherapy, RT, and HT could reduce the overall toxicity associated with each individual therapy, while concomitantly enhancing the immunotherapeutic effect of overall individual therapies to treat local and metastatic PC. Thus, the use of a tripartite combinatorial approach could be promising and more efficacious than monotherapy or dual therapy to treat and increase the survival of the PC patients.

Prostate cancer is one of the most prevalent cancers and the second leading cause of cancer-related deaths in men in the United States. A large number of patients undergo radiation therapy (RT) as a standard care of treatment; however, RT causes erectile dysfunction (radiation-induced erectile dysfunction; RiED) because of late side effects after RT that significantly affects quality of life of prostate cancer patients. Within 5 years of RT, approximately 50% of patients could develop RiED. Based on the past and current research findings and number of publications from our group, the precise mechanism of RiED is under exploration in detail. Recent investigations have shown prostate RT induces significant morphologic arterial damage with aberrant alterations in internal pudendal arterial tone. Prostatic RT also reduces motor function in the cavernous nerve which may attribute to axonal degeneration may contributing to RiED. Furthermore, the advances in radiogenomics such as radiation induced somatic mutation identification, copy number variation and genome-wide association studies has significantly facilitated identification of biomarkers that could be used to monitoring radiation-induced late toxicity and damage to the nerves; thus, genomic- and proteomic-based biomarkers could greatly improve treatment and minimize arterial tissue and nerve damage. Further, advanced technologies such as proton beam therapy that precisely target tumor and significantly reduce off-target damage to vital organs and healthy tissues. In this review, we summarize recent advances in RiED research and novel treatment modalities for RiED. We also discuss the possible molecular mechanism involved in the development of RiED in prostate cancer patients. Further, we discuss various readily available methods as well as novel strategies such as stem cell therapies, shockwave therapy, nerve grafting with tissue engineering, and nutritional supplementations might be used to mitigate or cure sexual dysfunction following radiation treatment.

Botulinum neurotoxins (BoNTs) are highly potent poisons produced by seven serotypes of Clostridium botulinum. The mechanism of neurotoxin action is a multistep process which leads to the cleavage of one of three different SNARE proteins essential for synaptic vesicle fusion and transmission of the nerve signals to muscles: synaptobrevin, syntaxin, or SNAP-25. In order to understand the precise mechanism of neurotoxin in a host, the domain structure of the neurotoxin was analyzed among different serotypes of C. botulinum. The results indicate that neurotoxins type A, C, D, E and F contain a coiled-coil domain while types B and type G neurotoxin do not. Interestingly, phylogenetic analysis based on neurotoxin sequences has further confirmed that serotypes B and G are closely related. These results suggest that neurotoxin has multi-domain structure, and coiled-coil domain plays an important role in oligomerisation of the neurotoxin. Domain analysis may help to identify effective antibodies to treat Botulinum toxin intoxication.

The accelerated growth of proteomics data presents both opportunities and challenges. Large-scale proteomic profiling of biological samples such as cells, organelles or biological fluids has led to discovery of numerous key and novel proteins involved in many biological/disease processes including cancers, as well as to the identification of novel disease biomarkers and potential therapeutic targets. While proteomic data analysis has been greatly assisted by the many bioinformatics tools developed in recent years, a careful analysis of the major steps and flow of data in a typical highthroughput analysis reveals a few gaps that still need to be filled to fully realize the value of the data. To facilitate functional and pathway discovery for large-scale proteomic data, we have developed an integrated proteomic expression analysis system, iProXpress, which facilitates protein identification using a comprehensive sequence library and functional interpretation using integrated data. With its modular design, iProXpress complements and can be integrated with other software in a proteomic data analysis pipeline. This novel approach to complex biological questions involves the interrogation of multiple data sources, thereby facilitating hypothesis generation and knowledge discovery from the genomic-scale studies and fostering disease diagnosis and drug development.

Cold environments are common throughout the Galaxy. We are conducting a series of experiments designed to probe the low-temperature limits for growth in selected methanogenic and halophilic Archaea. This paper presents initial results for two mesophiles, a methanogen, Methanosarcina acetivorans, and a halophile, Halobacterium sp. NRC-1, and for two Antarctic cold-adapted Archaea, a methanogen, Methanococcoides burtonii, and a halophile, Halorubrum lacusprofundi. Neither mesophile is active at temperatures below 5 °C, but both cold-adapted microorganisms show significant growth at sub-zero temperatures (−2 °C and −1 °C, respectively), extending previous low-temperature limits for both species by 4–5 °C. At low temperatures, both H. lacusprofundi and M. burtonii form multicellular aggregates, which appear to be embedded in extracellular polymeric substances. This is the first detection of this phenomenon in Antarctic species of Archaea at cold temperatures. The low-temperature limits for both psychrophilic species fall within the temperature range experienced on present-day Mars and could permit survival and growth, particularly in sub-surface environments. We also discuss the results of our experiments in the context of known exoplanet systems, several of which include planets that intersect the Habitable Zone. In most cases, those planets follow orbits with significant eccentricity, leading to substantial temperature excursions. However, a handful of the known gas giant exoplanets could potentially harbour habitable terrestrial moons.

The genome of Halobacterium sp. strain NRC-1 contains a large gene cluster, gvpMLKJIHGFEDACNO , that is both necessary and sufficient for the production of buoyant gas-filled vesicles. Due to the resistance of gas vesicles to solubilization, only the major gas vesicle protein GvpA and a single minor protein, GvpC, were previously detected. Here, we used immunoblotting analysis to probe for the presence of gas vesicle proteins corresponding to five additional gvp gene products. Polyclonal antisera were raised in rabbits against LacZ-GvpF, -GvpJ, and -GvpM fusion proteins and against synthetic 15-amino-acid peptides from GvpG and -L. Immunoblotting analysis was performed on cell lysates of wild-type Halobacterium sp. strain NRC-1, gas vesicle-deficient mutants, and purified gas vesicles, after purification of LacZ fusion antibodies on protein A and β-galactosidase affinity columns. Our results show the presence of five new gas vesicle proteins (GvpF, GvpG, GvpJ, GvpL, and GvpM), bringing the total number of proteins identified in the organelles to seven. Two of the new gas vesicle proteins are similar to GvpA (GvpJ and GvpM), and two proteins contain predicted coiled-coil domains (GvpF and GvpL). GvpL exhibited a multiplet ladder on sodium dodecyl sulfate-polyacrylamide gels indicative of oligomerization and self-assembly. We discuss the possible functions of the newly discovered gas vesicle proteins in biogenesis of these unique prokaryotic flotation organelles.

The light-induced proton efflux and active carbon uptake are inhibited by mercury and cadmium ions in Anabaena flos-aquae. The inhibitory effects of these heavy metal ions are reversed by 40 mM concentration of sodium. Here we report that light-induced proton efflux is sodium-dependent which leads to a characteristic enhancement in the rate of photosynthetic oxygen generation and carbon fixation. A low concentration (10 μM) of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) significantly inhibited the rate of oxygen generation while 10 μM carbonyl cyanide-m-chlorophenylhydrazone (CCCP) completely blocked the oxygen generation activity in the organism. The chlorophyll-a fluorescence yield indicates that little fluorescence quenching occurred in the absence of sodium ion. Increasing the extracellular sodium ion accelerated both the initial rate and the extent of fluorescence quenching. These results support the assumption that metal-induced inhibition of the photosynthetic machinery may be mediated by the movement of protons.

Interferon response factor 3 (IRF-3) is a transcription factor that plays an essential role in controlling the synthesis of interferon-β (IFN-β) and is a protein consisting of two well-defined domains, the N-terminal DNA-binding and the C-terminal dimerization domains, connected by a 75-residue linker, supposedly unfolded. However, it was not clear whether in intact IRF-3 this linker segment of the chain, which carries the nuclear export signal and includes a region of high helical propensity, remains unfolded. This has been investigated using nuclear magnetic resonance by ligating the (15)N-labeled linker to the unlabeled N-terminal and C-terminal domains. It was found that, while the linker alone is indeed in a completely unfolded state, when ligated to the C-terminal domain it shows some ordering, and this ordering becomes much more pronounced when the linker is also ligated to the N-terminal domain. Thus, in intact IRF-3, the linker represents a folded structural domain; i.e., IRF-3 is a three-domain globular protein. Light scattering studies of wild-type IRF-3 showed that these three domains are tightly packed, and therefore, the dimer of IRF-3, which is formed upon phosphorylation of its C-terminal domains following virus invasion, must be a rather rigid and compact construction. One would then expect that binding of such a dimer to its tandem recognition sites PRDIII and PRDI, which are located on opposing faces of the IFN-β enhancer DNA, should result in deformation of the DNA. Analysis of the characteristics of binding of the monomeric and dimeric IRF-3 to the enhancer DNA indeed showed that formation of this complex requires considerable work for deformation of its components, most likely bending of the DNA. Such bending was confirmed by atomic force microscopy of dimeric IRF-3 bound to the PRDII-PRDI tandem recognition sites placed at the middle of a 300 bp DNA probe. Bending of DNA by IRF-3 must be significant in the assembly and function of the IFN-β enhancer.

Heat shock proteins (HSP) are a family of ubiquitous and phylogenically highly conserved proteins which play an essential role as molecular chaperones in protein folding and transport. Heat Shock Protein 90 (Hsp90) is not mandatory for the biogenesis of most proteins, rather it participate in structural maturation and conformational regulation of a number of signaling molecules and transcription factors. Hsp90 has been shown to play an important role in antigen presentation, activation of lymphocytes, macrophages, maturation of dendritic cells, and in the enhanceosome mediated induction of inflammation. Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease with complex immunological and clinical manifestations. Dysregulated expression of Type I interferonα, activation of B cells and production of autoantibodies are hallmarks of SLE. The enhanced levels of Hsp90 were detected in the serum of SLE patients. The elevated level of Hsp90 in SLE has also been correlated with increased levels of IL-6 and presence of autoantibodies to Hsp90. This suggests that Hsp90 may contribute to the inflammation and disease progression and that targeting of Hsp 90 expression may be a potential treatment of SLE. The pharmacologic inhibition of Hsp90 was successfully applied in mouse models of autoimmune encephalomyelitis and SLE—like autoimmune diseases. Thus targeting Hsp90 may be an effective treatment for SLE, especially if combined with other targeted therapeutic approaches.

Membrane proteomic analysis has been proven to be a promising tool for identifying new and specific biomarkers that can be used for prognosis and monitoring of various cancers. Membrane proteins are of great interest particularly those with functional domains exposed to the extracellular environment. Integral membrane proteins represent about one-third of the proteins encoded by the human genome and assume a variety of key biological functions, such as cell-to-cell communication, receptor-mediated signal transduction, selective transport, and pharmacological actions. More than two-thirds of membrane proteins are drug targets, highlighting their immensely important pharmaceutical significance. Most plasma membrane proteins and proteins from other cellular membranes have several PTMs; for example, glycosylation, phosphorylation, and nitrosylation, and moreover, PTMs of proteins are known to play a key role in tumor biology. These modifications often cause change in stoichiometry and microheterogeneity in a protein molecule, which is apparent during electrophoretic separation. Furthermore, the analysis of glyco- and phosphoproteome of cell membrane presents a number of challenges mainly due to their low abundance, their large dynamic range, and the inherent hydrophobicity of membrane proteins. Under pathological conditions, PTMs, such as phosphorylation and glycosylation are frequently altered and have been recognized as a potential source for disease biomarkers. Thus, their accurate differential expression analysis, along with differential PTM analysis is of paramount importance. Here we summarize the current status of membrane-based biomarkers in various cancers, and future perspective of membrane biomarker research.

Background Our earlier studies have shown that red blood cell (RBC) morphology in Alzheimer's disease (AD) subjects was altered (> 15% of the RBCs were elongated as compared to 5.9% in normal controls (p < 0.0001)). These results suggested alterations in the RBC membrane architecture in AD subjects, possibly due to RBC-β-amyloid interactions and/or changes in the expression of membrane proteins. We hypothesized that the observed changes could be due to changes in the level of the protein components of the cytoskeleton and those linked to the RBC membrane. To examine this, we performed a proteomic analysis of RBC membrane proteins of AD subjects, and their age-matched controls using one pool of samples from each group, following their separation by SDS-PAGE, in-gel Tryptic digestion, LC-MS-MS of peptides generated, and a label-free approach of semi-quantitative analysis of their relative MS spectral intensities. Results The data suggest, (1) RBC shape/morphology changes in AD subjects are possibly attributed primarily to the changes (elevation or decrease) in the level of a series of membrane/cytoskeleton proteins involved in regulating the stability and elasticity of the RBC membrane, and (2) changes (elevation or decrease) in the level of a second series of proteins in the RBC membrane proteome reflect similar changes reported earlier by various investigators in AD or animal model of AD. Of particular interest, elevation of oxidative stress response proteins such as heat shock 90 kDa protein 1 alpha in AD subjects has been confirmed by western blot analysis in the RBC membrane proteome. Conclusions The results suggest that this study provides a potential link between the alterations in RBC membrane proteome in AD subjects and AD pathology.

The accurate delivery of external beam radiation therapy is often facilitated through the implantation of radio-opaque fiducial markers (seeds). Before the delivery of each treatment fraction, seed positions can be determined via volumetric imaging. By registering these seed locations with the corresponding locations in the previously acquired treatment planning CT, it is possible to adjust the patient position or the treatment plan so that seed displacement is accommodated. We present an automatic algorithm that identifies seeds in both planning and pretreatment images and subsequently determines the geometric transformation between the two sets. The algorithm is applied to the imaging series of 10 prostate cancer patients. Each series is comprised of a single multislice planning CT and several megavoltage conebeam CT images obtained immediately prior to a subsequent treatment session. Seed locations were determined for 164 images to within 1 mm with an accuracy of 98 plusmn 6.3%.

Properties of attachment of Mycobacterium marinum to hydrophobic surfaces and subsequent␣biofilm formation were investigated. Binding of M. marinum to polypropylene occured under aerobic and anaerobic/microaerophilic conditions. However, aerobic conditions were necessary for biofilms to persist. Highly non-polar organic solvents were found to efficiently remove attached bacteria from the polypropylene surface, indicating strong hydrophobic interactions between the M. marinum cell wall and the surface. Increased capsular material, occurring during stationary phase, correlated with a decrease in attachment of cells to polypropylene. A protein of approximately 40 kDa appears to be present in increased amounts during the stationary phase. The protein has been identified by LC MS/MS analysis as alanine dehydrogenase.

Publisher Summary This chapter describes a selection of experimental methods used to characterize heat shock proteins, starting with the identification of the heat shock regulons. It is suggested that their small genome size, coupled with the necessity to carry out ongoing protein salvage processes in the cell during normal growth, has resulted in minimal but highly efficient protein folding machinery in hyperthermophiles. Some proteins, such as AAA+ATPase of unknown function, are part of the heat shock response. In addition, a homolog of the Nascent Associated Complex has recently been described in the Archaea, although it is not clear how it relates to protein folding. Chaperone functions can be difficult to study in mesophiles, due to the multiplicity of chaperone encoding genes and parallel functions that they carry out. Heat shock regulation and chaperone functions in hyperthermophiles provide fresh insights into basic protein-folding mechanisms. The study of heat shock proteins involves decision-making as to which proteins are regulated by heat stress, the mechanisms of heat shock regulation, and the assignment of functions to the heat shock proteins by biophysical and biochemical approaches. The determination of chaperone functions is a very significant challenge, involving recombinant expression and characterization of individual HSPs, and elucidation of the functional roles of HSPs and their cooperative interactions with target proteins.

Clostridium botulinum, a Gram-positive, anaerobic spore-forming bacteria, is distinguished by its significant clinical applications as well as its potential to be used as bioterror agent. Growing cells secrete botulinum neurotoxin (BoNT), the most poisonous of all known poisons. While BoNT is the causative agent of deadly neuroparalytic botulism, it also serves as a remarkably effective treatment for involuntary muscle disorders such as blepharospasm, strabismus, hemifacial spasm, certain types of spasticity in children, and other ailments. BoNT is also used in cosmetology for the treatment of glabellar lines, and is well-known as the active component of the anti-aging medications Botox and Dysport. In addition, recent reports show that botulinum neurotoxin can be used as a tool for pharmaceutical drug delivery. However, BoNT remains the deadliest of all toxins, and is viewed by biodefense researchers as a possible agent of bioterrorism (BT). Among seven serotypes, C. botulinum type A is responsible for the highest mortality rate in botulism, and thus has the greatest potential to act as biological weapon. Genome sequencing of C. botulinum type A Hall strain (ATCC 3502) is now complete, and has shown the genome size to be 3.89 Mb with a G+C content of approximately 28.2%. The bacterium harbors a 16.3 kb plasmid with a 26.8% G+C content--slightly lower than that of the chromosome. Most of the virulence factors in C. botulinum are chromosomally encoded; bioinformatic analysis of the genome sequence has shown that the plasmid does not harbor toxin genes or genes for related virulence factors. Interestingly, the plasmid does harbor genes essential to replication, including dnaE, which encodes the alpha subunit of DNA polymerase III which has close similarity with its counterpart in C. perfringens strain 13. The plasmid also contains similar genes to those that encode the ABC-type multidrug transport ATPase, and permease. The presence of ABC-type multidrug transport ATPase, and permease suggests putative involvement of efflux pumps in bacteriocin production, modification, and export in C. botulinum. The C. botulinum plasmid additionally harbors genes for LambdaBa04 prophage and site-specific recombinase that are similar to those found in the Ames strain of Bacillus anthracis; these genes and their products may play a role in genomic rearrangement. Completion of genome sequencing for C. botulinum will provide an opportunity to design genomic and proteomic-based systems for detecting different serotypes of C. botulinum strains in the environment. The completed sequence may also facilitate identification of potential virulence factors and drug targets, as well as help characterize neurotoxin-complexing proteins, their polycistronic expression, and phylogenetic relationships between different serotypes.

Purpose: We present a novel image acquisition and reconstruction scheme for dramatically increasing the field‐of‐view (FOV) in cone beam computed tomography(CBCT) using a standard centered detector. The method enables CBCT datasets to be employed as a basis for treatment planning, since the entire extent of even very large patients can be imaged in a quantitative sense. Applications include planning for emergency cases based on CBCTimages, and at Third World facilities that lack multislice‐CT systems using megavoltage‐CBCT. Methods and Materials: The projection acquisition scheme proceeds as follows: 1) The patient couch is offset (in height and lateral position) from its intended position ( x 0) at the start of treatment by a distance related to the amount of additional FOV required. 2) Projections are acquired over a limited arc. 3) The couch is offset from x 0 by the same distance as in (1), but in the opposite direction 4) Projections are acquired over a second arc. Projections are corrected for scatter, beam‐hardening and non‐uniform beam profile, and then simultaneously reconstructed using an iterative algorithm. Evaluation: We image a pelvic anthropomorphic phantom using a Siemens ONCOR linac equipped with an amorphous‐silicon detector. Dose calculation is then performed on the resulting extended FOV megavoltage‐CBCT and a multislice‐CT of the same phantom. Results: The full cross section of the phantom is reconstructed with only minor artifacts. The dose study shows almost identical dose distributions for the reference and EFOV dataset: the central percentage depth‐dose profiles for a 10×10 cm2 field exhibits a maximum deviation of 0.8%, illustrating the insignificance of the artifacts from a dosimetric perspective. Conclusion: The method increases the FOV of an ONCOR linac by 67% to 45 cm. This exceeds the capabilities of current extended FOV implementations that are based on offset‐detector systems. The reconstructed volumes appear suitable for planning purposes.

We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodium-proton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.

Purpose: A novel rotational IMRT (rIMRT) technique using burst delivery (continuous gantry rotation with beam off during MLC repositioning) is investigated. We evaluate the plan quality and delivery efficiency and accuracy of this dynamic technique with a conventional flat 6MV photon beam. Methods: Burst‐delivery rIMRT was implemented in a planning system (Panther, Prowess) and delivered with a 160‐MLC linac (Artiste, Siemens). Ten rIMRT plans were generated for five anonymized patient cases encompassing head and neck, brain, prostate, and prone breast. All plans were analyzed retrospectively and not used for treatment. Among the varied plan parameters were the number of optimization points, number of arcs, gantry speed, and gantry angle range (alpha) over which the beam is turned on at each optimization point. Combined rotational/step‐and‐shoot rIMRT plans were also created by superimposing multiple‐segment static fields at several optimization points. The rIMRT trial plans were compared with each other and with plans generated using helical tomotherapy. Plans were delivered and verified using a diode array, ionization chambers, and thermoluminescent dosimeters. Results: Burst‐mode rIMRT can achieve plan quality comparable to helical tomotherapy, while the former may lead to slightly better OAR sparing for certain cases and the latter generally achieves slightly lower hot spots. Few instances were found in which increasing the number of optimization points above 36, or superimposing step‐and‐shoot IMRT segments, led to statistically significant improvements in OAR sparing. Using an additional rIMRT partial arc yielded substantial OAR dose improvements for the brain case. Measured doses from the rIMRT plan delivery were within 4% of the plan calculation in low dose gradient regions. Delivery time range was 228–375 seconds for single‐arc rIMRT 200‐cGy prescription with a 300 MU/min dose rate. Conclusions: Rotational IMRT with burst delivery, whether combined with static fields or not, yields clinically acceptable and deliverable treatment plans.

Purpose: To demonstrate the ability to deliver unflattened (high dose rate), modulated arc plans using the Siemens Artiste linear accelerator using prototype linac firmware developed by Siemens and prototype TPS software developed by Prowess. Method and Materials: Prototype Prowess treatment planning software was used to develop a set of high dose rate (1,000 mu/min) modulated arc plans for four treatment sites ‐ head/neck, lung, prostate and breast. A step‐and‐shoot plan (SS) was also created for delivery efficiency comparison to arc‐based plans. The same planning criteria were used for all plans to ensure their ‘equivalence’. With the linear accelerator converted to the experimental state, all plans were delivered to a solid water phantom. Dose was measured using an ion chamber for point dose measurements and film for evaluation of relative dose distribution with a gamma criteria of 3%/3mm. Delivery times were collected to assess delivery efficiency. Results: Plans developed using all arc modalities were successfully delivered for all cases. The maximum difference between measured and calculated doses was 3.08% for ion chamber measurements and the minimum 3%/3mm gamma agreement was 87% of pixels. All arc treatments were delivered in less time than the corresponding SS plans, with the lungSBRT plans being delivered in 5 minutes for a 20 Gy prescription. Conclusion: Unflattened, modulated arc delivery is possible using prototype firmware on a Siemens Artiste machine and the Prowess treatment planning system accurately predicted doses for such deliveries. Modulated arc delivery in combination with unflat (high dose rate i.e. 1,000+ mu/min) mode may allow for very efficient delivery of highly conformai isodose distributions. Conflict of Interest: This work is funded by a grant from Siemens Oncology.

Purpose: Cone beam therapy (CBT) has been proposed as a form of rotational IMRT that can potentially offer fast delivery with equal or superior plan quality. This work aims to investigate CBT planning methods to generate high‐quality plans. Methods and Materials: The idea of CBT is to spread apertures for a beam to neighboring beam angles enabling non‐overlapping apertures to be delivered with gantry rotation. A prototype (Prowess Inc) with this algorithm implemented was used for this study. A full arc consisting of 72 equally‐spaced initial coplanar beams, optionally combined with a partial arc and non‐zero couch angle selected to avoid direct irradiation of organs‐at‐risk (OAR), was used to generate single‐ or two‐arc CBT plans. Plans for TomoTherapy, and conventional IMRT were also generated using the same CT and contours. For CBT and IMRT plans, a linac equipped with 160 MLC (Artiste, Siemens) with a possible dose rate of 1000 MU/min was considered. Plans for 10 representative brain and head and neck cancer patients were generated. Results: Two‐arc CBT offers equal or better plan quality than single‐arc. On average, the uniformity index for PTV was improved by 1.4% and the EUD for organs at risk (OAR) reduced by 25% when comparing two‐ and single‐arc CBT plans. Compared with TomoTherapy, the two‐arc CBT can lead to reduced EUD for OAR (12.9% on average) but with reduced dose uniformity in target (5.6% on average). The average delivery times for single‐ and two‐arc CBT are estimated to be 2 and 5 minutes, respectively, shorter than TomoTherapy (8 minutes). Conclusion: Two‐arc CBT can generate equal or better plans than single‐arc CBT and conventional IMRT. Compared with TomoTherapy plans, the two‐arc CBT plans offer improved OAR sparing but with decreased dose uniformity in targets. The delivery times of CBT were estimated to be 2–5 minutes.

Purpose: The purpose of this work was to investigate the technical feasibility of using the widely deployed Megavoltage Cone Beam CT (MVCBCT) for 3D IGRT applications with mMLC based treatment delivery. Method and Materials: In most clinical applications of MVCBCT IGRT, the largest possible field of view (FOV) is sought for evaluating adjacent organs‐at‐risk. However, an add‐on mMLC with maximum field size of 10cm×12cm is preferred for precise small‐field hypofractionated treatments. mMLC based treatments utilizing 2.5mm leaf widths are usually performed for SRS, SRT and SBRT applications, where the higher accuracy of 3D IGRT localization is also warranted. Current clinical systems do not allow restricting the field size of CBCT acquisition in the X direction. Performing imaging with the mMLC mounted and the field size opened to ∼27cm results in major truncation artifacts rendering the images unusable, as well as the irradiation of the mMLC electronics during image acquisition, which is undesirable. The current study was conducted with MVision™ and Moduleaf™ mMLC (Siemens Medical Systems), using anthropomorphic head and neck, and chest phantoms. A custom cerrobend aperture block was fabricated to shield the mMLC from radiation damage during imaging with the X Jaw open. The clinically used reconstruction algorithm was set to only reconstruct a smaller field of view corresponding to the clinically useful restricted treatmentfield size, although the data acquisition field size was set to the required 27.4 cm in the X direction. Results: With the changes described above, we were able to accomplish high quality images with the mMLC. The image quality was better than images obtained for the full field acquisition in the clinical mode for the same phantoms. Conclusion: We believe this to be the first study demonstrating high quality 3D IGRT through a small mMLC collimator designed for SRT.

Purpose: Large patient anatomies and limited imaging field‐of‐view (FOV) lead to truncation of CT projections. Truncation introduces serious artifacts into reconstructed images, including central cupping and bright external rings. FOV may be increased using laterally offset detectors, but this requires advanced imaging hardware and full angular scanning. When linacs equipped with mini‐MLCs are used for megavoltage cone beam CTimaging, truncation is inevitable. We propose a novel method to complete truncated projections based on the observation that the thickness of the patient at all angles may be estimated along the projection rays by calculating water‐equivalent thicknesses (WET). These values are not at all affected by truncation and constitute valuable auxiliary information. Methods: We parameterize a pair of points along each ray that intersects the unknown object boundary. These points are separated by the measured WET value (obtained from projections that have been corrected for scatter and beam‐hardening). We assume, for all large body parts, that the patient outline may be roughly approximated as an ellipse. We use a deterministic optimization algorithm to simultaneously estimate the point positions and ellipse parameters by minimizing the distance between point sets and the ellipse boundary. The ellipse obtained as solution is used to complete the truncated projections before reconstruction. We apply the algorithm to a severely truncated CT dataset of a typical abdomen. Results: The RMS error between non‐truncated and truncated reconstructions is 176%. The correction algorithm reduces this error to 1.0%. Conclusion: Even thought the algorithm assumes an elliptical patient cross‐section, truly impressive increases in quantitative image quality are observed. The presence of pelvic bone in the image does not appreciably bias the ellipse position even though it produces incorrect thickness estimates for some rays. The algorithm incurs low computational burden and is suitable for on‐line clinical work flows. Supported by Siemens.

Purpose:CTimaging of patients for radiotherapytreatment planning frequently includes anatomy that extends beyond the 50cm nominal field‐of‐view (nFOV): 28.35% of the 575 CT scans we acquired from July–December 2006. The purpose of this study was to evaluate 1) the degradation of Hounsfield units (HU) in the extended field of view (eFOV), and 2) the dosimetric impact of ignoring or correcting this degradation within the treatment planning system (TPS). Method and Materials:CTimages were acquired at maximum FOV (82cm) on a diamond‐shaped 30×30×13cm solid‐water phantom, challenging the reconstruction algorithm's ability to model the truncated projection data adequately. A unique dataset was acquired for 19 phantom locations (1cm intervals outside of the nFOV) and each imported into a TPS. Each phantom was contoured using a threshold of −200HU. A single treatment beam with isocenter placed at the center of the phantom was planned to deliver 100cGy to isocenter for each CT data set; dose map comparisons with and without homogenous correction for density (HU>−700 =1gm/cc) were performed relative to the control phantom within the nFOV. Results: Significant variation of HU was observed as a function of phantom displacement outside the nFOV (range +513 to −873HU); most dramatically ∼5cm beyond the nFOV border. If uncorrected, these changes in HU produced significant dose errors. Plans were compared to control plans and dose difference maps were generated; uncorrected images 3cm outside the nFOV demonstrated >5% difference, where overriding HU values above −700HU maintained

BackgroundHalobacteriumsp. NRC-1 is an extremely halophilic archaeon and has adapted to optimal growth under conditions of extremely high salinity. Its proteome is highly acidic with a median pI of 4.9, a unique characteristic which helps the organism to adapt high saline environment. In the natural growth environment,HalobacteriumNRC-1 encounters a number of stressful conditions including high temperature and intense solar radiation, oxidative and cold stress. Heat shock proteins and chaperones play indispensable roles in an organism's survival under many stress conditions. The aim of this study was to develop an improved method of 2-D gel electrophoresis with enhanced resolution of the acidic proteome, and to identify proteins with diverse cellular functions using in-gel digestion and LC-MS/MS and MALDI-TOF approach.ResultsA modified 2-D gel electrophoretic procedure, employing IPG strips in the range of pH 3–6, enabled improved separation of acidic proteins relative to previous techniques. Combining experimental data from 2-D gel electrophoresis with available genomic information, allowed the identification of at least 30 cellular proteins involved in many cellular functions: stress response and protein folding (CctB, PpiA, DpsA, and MsrA), DNA replication and repair (DNA polymerase A α subunit, Orc4/CDC6, and UvrC), transcriptional regulation (Trh5 and ElfA), translation (ribosomal proteins Rps27ae and Rphs6 of the 30 S ribosomal subunit; Rpl31eand Rpl18e of the 50 S ribosomal subunit), transport (YufN), chemotaxis (CheC2), and housekeeping (ThiC, ThiD, FumC, ImD2, GapB, TpiA, and PurE). In addition, four gene products with undetermined function were also identified: Vng1807H, Vng0683C, Vng1300H, and Vng6254. To study the heat shock response ofHalobacteriumNRC-1, growth conditions for heat shock were determined and the proteomic profiles under normal (42°C), and heat shock (49°C) conditions, were compared. Using a differential proteomic approach in combination with available genomic information, bioinformatic analysis revealed five putative heat shock proteins that were upregulated in cells subjected to heat stress at 49°C, namely DnaJ, GrpE, sHsp-1, Hsp-5 and sHsp-2.ConclusionThe modified 2-D gel electrophoresis markedly enhanced the resolution of the extremely acidic proteome ofHalobacteriumNRC-1. Constitutive expression of stress proteins and chaperones help the organism to adapt and survive under extreme salinity and other stress conditions. The upregulated expression pattern of putative chaperones DnaJ, GrpE, sHsp-1, Hsp-5 and sHsp-2 under elevated temperature clearly suggests thatHalobacteriumNRC-1 has a sophisticated defense mechanism to survive in extreme environments.