Dose Perturbations of Gold Fiducial Markers in the Prostate Cancer Intensity Modulated Proton Radiation Therapy (IMPT)

The objective of this study is to investigate the dose perturbations introduced by the implanted gold fiducial markers in the prostate cancer intensity modulated proton therapy (IMPT) and the impacts of different plan designs on the pertur-bations. Five proton plans: a single lateral field 3D-modulation (3D-mod) plan, 2 fields laterally opposing 3D-mod plan, 6-, 9-, and 18-field distal edge tracking (DET) plans were designed on the CT images of a prostate patient. The dose distributions were first generated for the plans free of fiducial markers with 78 Gy prescribed to 95% of the PTV. To derive the dose perturbations of the gold fiducial markers, three cylindrical shaped gold fiducial markers (3 mm long and 1 mm in diameter) were artificially inserted into the prostate, and the dose distributions were re-computed. Monte Carlo method was used for dose computation. It was found that the gold fiducial markers perturbed the dose distribu-tions, especially along the beam paths. The markers caused a shadowing effect reducing the doses in the areas beyond the markers. Overall, due to the presence of the fiducial markers, D99% of prostate were reduced by 2.96 Gy, 4.21 Gy, 0.16 Gy, 0.34 Gy, 0.15 Gy for the plans of single field 3D-mod, 2-field parallel opposed 3D-mod, 6-, 9-, and 18-field DET respectively. Our study showed these dose perturbation effects decreased with the increase of number of beam angles. Up to 6 beam angles may be required to reduce the dose perturbations from the gold fiducial markers to a clini- cally acceptable level in IMPT.

Security breach:peripheral nerves provide unrestricted access for toxin delivery into the central nervous system

We explore the hypothesis that a potential explanation for the initiation of motor neuron disease is an unappreciated vulnerability in central nervous system defense,the direct delivery of neurotoxins into motor neurons via peripheral nerve retrograde transport.This further suggests a mechanism for focal initiation of neuro-degenerative diseases in general,with subsequent spread by network degeneration as suggested by the Frost-Diamond hypothesis.We propose this vulnerability may be a byproduct of vertebrate evolution in a benign aquatic environment,where external surfaces were not exposed to concentrated neurotoxins.

Autophagy inhibition by chloroquine sensitizes HT-29 colorectal cancer cells to concurrent chemoradiation

AIM:To investigate whether the inhibition of autophagy by chloroquine(CQ)sensitizes rectal tumors to radiation therapy(RT)or concurrent chemoradiation(chemoRT).METHODS:In vitro,HCT-116 and HT-29 colorectal cancer(CRC)cell lines were treated as following:(1)PBS;(2)CQ;(3)5-fluorouracil(5-FU);(4)RT;(5)CQ and RT;(6)5-FU and RT;(7)CQ and 5-FU;and(8)5-FU and CQ and RT.Each group was then exposed to various doses of radiation(0-8 Gy)depending on the experiment.Cell viability and proliferative capacity were measured by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)and clonogenic assays.Clonogenic survivalcurves were constructed and compared across treatment groups.Autophagy status was determined by assessing the LC3-Ⅱto LC3-Ⅰratio on western blot analysis,autophagosome formation on electron microscopy and identification of a perinuclear punctate pattern with GFPlabeled LC3 on fluorescence microscopy.Cell cycle arrest and cell death were evaluated by FACS and AnnexinⅤanalysis.All experiments were performed in triplicate and statistical analysis was performed by the student’s t test to compare means between treatment groups.RESULTS:RT(2-8 Gy)induced autophagy in HCT-116and HT-29 CRC cell lines at 4 and 6 h post-radiation,respectively,as measured by increasing LC3-Ⅱto LC3-Ⅰratio on western blot.Additionally,electron microscopy demonstrated autophagy induction in HT-29 cells24 h following irradiation at a dose of 8 Gy.Drug treatment with 5-FU(25μmol/L)induced autophagy and the combination of 5-FU and RT demonstrated synergism in autophagy induction.CQ(10μmol/L)alone and in combination with RT effectively inhibited autophagy and sensitized both HCT-116 and HT-29 cells to treatment with radiation(8 Gy;P<0.001 and 0.00001,respectively).Significant decrease in clonogenic survival was seen only in the HT-29 cell line,when CQ was combined with RT at doses of 2 and 8 Gy(P<0.5 and P=0.05,respectively).There were no differences in cell cycle progression or Annexin V staining upon CQ addition to RT.CONCLUSION:Autophagy inhibition by CQ increases CRC cell sensitivity to concurrent treatment with 5-FU and RT in vitro,suggesting that addition of CQ to chemoRT improves CRC treatment response.

Suicide transport blockade of motor neuron survival generates a focal graded injury and functional deficit

We describe a pre-clinical spinal cord motor neuron injury model that is minimal invasive, reproducible, focal and easily applied to small rodents.Retrograde axonal transport of a pro-apoptotic phosphatidylinosotol 3'-kinase inhibitor, wortmannin, via the sciatic nerve results in loss of ipsilateral lumbar motor neurons proportional to the level of drug administered.Motor neuron loss was detected by choline acetyltransferase(ChAT) immunostaining and with a transgenic thy1-eGFP marker.The short half-life of wortmannin generates minimal wound spread, and wortmannin does not affect axon transport, as determined by co-injection of a pseudorabies virus tracer.Using quantitative transcript analysis, we found that ChAT transcripts significantly decreased at 14 days post-delivery of 1 μg wortmannin, relative to sham controls, and remained low after 90 days.Smaller effects were observed with 200 ng and 100 ng wortmannin.Wortmannin also generated a transient and significant increase in astrocyte Gfap transcripts after 14 days with a return to control levels at 90 days.Treated mice had hind limb spasticity and a forced motor function defect that was quantified using a water exit test.Controls rapidly exit a shallow water tray, and wortmannin treated animals were up to 12-fold slower, a phenotype that persisted for at least 3 months.Thus the focal delivery of wortmannin to motor neurons generates a reproducible and scalable injury that can facilitate quantitative studies on neural regeneration and repair.The efficacy of sciatic nerve suicide transport can also explain neurotoxin-mediated selective loss of motor neurons in diseases such as amyotrophic lateral sclerosis.All procedures were performed at Rutgers under established Institutional Animal Care and Use protocols(eIACUC_TR201800022, approved on March 20, 2018).

Pharmacological potential of ampelopsin in Rattan tea

Rattan tea,made from the leaves of Ampelopsis grossedentata,may potentially perform multiple pharmacological roles,including anti-bacterial,anti-cancer,antioxidant,hepatoprotective and anti-hypertension functions.These beneficial functions of Rattan tea are strongly associated with the bioactivity of ampelopsin,a major flavonoid compound in Rattan tea.In this review,we summarize current research related to the bioactivity and pharmacological mechanisms of ampelopsin,which will provide a better reference for its potential application in the prevention of chronic diseases.©2012 Production and hosting by Elsevier B.V.on behalf of Beijing Academy of Food Sciences.

Cell-free derivatives from mesenchymal stem cells are effective in wound therapy

AIM:To compare the efficacy of cell-free derivatives from Bone marrow derived human mesenchymal stem cells(hMSCs) in wound therapy.METHODS:hMSCs have been shown to play an important role in wound therapy.The present study sought to compare efficacy of hMSCs and cell-free derivatives of hMSCs,which may be clinically more relevant as they are easier to prepare,formulate and transport.hMSCs were isolated from human bone marrow and cultured.Multi lineage differentiation of hMSCs was performed to confirm their identity.The ability of hMSCs to migrate was evaluated using in vitro and in vivo migration assays.Cell lysates and conditioned medium concentrate was prepared from hMSCs(see Methods for details).Wounds were induced in mice and wound areas were measure before and after cell and cell-free derivative treatment.RNA and proteins were extracted from the skin and cytokine levels were measured.RESULTS:Co-culture of hMSCs with keratinocytes resulted in increased expression of CXCL-12(SDF1) and ENA78(CXCL-5) in the conditioned media indicating that the hMSCs can respond to signals from keratinocytes.Accelerated wound closure was observed when hMSCs were injected near the site of excisional wounds in athymic as well as NOD/SCID mice.Interestingly,cell-free lysates prepared from hMSCs were also effective in inducing accelerated wound closure and increased expression of SDF1 and CXCL-5 at the wound bed.Additionally,concentrated media from hMSCs as well as an emulsion containing lysates prepared from hMSCs was also found to be more effective in rapid re-epithelialization than fibroblasts or vehicle-alone control.Use of cell-free derivatives may help replace expensive wound care approaches including use of growth factors,epidermal/dermal substitutes,synthetic membranes,cytokines,and matrix components,and most importantly avoid transmission of pathogens from human and animal products.CONCLUSION:These results encourage development of derivatives of hMSCs for wound care and re-epithelialization applications.

Natural products for cancer prevention associated with Nrf2–ARE pathway

Cancer chemoprevention involves the application of natural or synthetic compounds to reduce the risk of cancer development.One of the most effective strategies for preventing human cancers might involve inducing phase II detoxifying enzymes and antioxidant enzymes via natural dietary compounds.The regulatory regions of these inducible genes encode the antioxidant response element(ARE).Nuclear factor-erythroid 2-related factor 2(Nrf2),as a transcription factor,plays a key role in the expression of ARE-mediated genes.Similarly,Nrf2 performs an essential function in the up-regulation of these genes in response to oxidative stress and treatment with dietary phytochemicals.In this article,we discuss the current state of knowledge regarding the Nrf2/ARE pathway as a potential molecular target for cancer chemoprevention and its molecular regulation mechanisms,and highlight Nrf2/ARE inducers derived from natural products,which may be used as chemopreventive agents for cancer patients.©2013 Beijing Academy of Food Sciences.Production and hosting by Elsevier B.V.All rights reserved.

Organ-specific enhancement of metastasis by spontaneous ploidy duplication and cell size enlargement

Aneuploidy 通常在乳癌被观察并且与差的预后被联系。aneuploidy 的一种经常的类型， hypertetraploidy，可以源于 hyperdiploid 房间的 ploidy 复制。然而，在乳癌前进的 ploidy 复制的病理学的后果没被描绘。这里，我们在场在 vitro 并且在 vivo 通过 ploidy 复制从 MDA-MB-231 乳癌房间线的器官特定的变形变体表明 hypertetraploid 房间的自发的外观的一个试验性的系统。hypertetraploid 子孙显示出增加的变形潜力到肺和大脑，然而并非到骨头，它可以被不同毛状的结构部分在这些器官解释与扩大尺寸授与微分住宿优点到肿瘤细胞。我们的结果建议在 ploidy 复制和变形潜力的改进之间的一个潜在的机械学的连接，在乳癌的以前的临床的研究被观察。

Pathological findings following radical prostatectomy in patients who are candidates for active surveillance： impact of varying PSA levels

Active surveillance is an acceptable treatment option in men with a low-risk prostate cancer. In the present study, we have retrospectively reviewed the outcomes of 509 men who fit the criteria for active surveillance but selected radical prostatectomy. Then, the impact of varying prostate-specific antigen （PSA） levels on the risk of upstaging and upgrading in these patients was assessed. Pathological characteristics of patients who fulfilled the inclusion criteria under three active surveillance criteria--those of the University of California-San Francisco, the National Cancer Institute and the European Association of Urology--were examined. The proportion of men who were deemed candidates for active surveillance but were subsequently upstaged or upgraded was determined. Of 509 patients, 186 （36.5%）, 132 （25.9%） and 88 （17.3%） men fulfilled the active surveillance criteria, respectively. Upgrading （Gleason scores 7-10） ranged from 32.8% to 38.6%, while upstaging （≥ pT3） ranged from 10.2% to 12.5%, depending on the three active surveillance criteria. After a median follow-up of 24 months, three patients developed a biochemical recurrence. When the impact of varying PSA levels was examined using a test for trend analysis in the context of PSA for each protocol, rates of upstaging were lower in men with PSA 〈4 ng m1-1. However, there was no impact of varying PSA levels on upgrading. In conclusion, commonly used active surveillance protocols carry the risks of upgrading and upstaging. More reliable and accurate markers are needed to better stratify the risks of men who are appropriate candidates for active surveillance.

PLGA-Polymer Encapsulating Tumor Antigen and CpG DNA Administered into the Tumor Microenvironment Elicits a Systemic Antigen-Specific IFN-γ Response and Enhances Survival

Critical to the generation of an effective therapeutic antitumor immune response is the elicitation of effective antigen presentation coupled with overcoming tumor-immune escape mechanisms. Towards this end, we aimed to understand the therapeutic effectiveness of a polymer based vaccine approach at enhancing the anti-tumor responses in a tumor-bearing mouse model. While we and others have previously demonstrated the effectiveness of PLGA based systems in delivering antigen etc., studies scarcely focus on understanding the immunological mechanisms of polymer based therapies in tumor bearing treatment models. Considering tumors modulate the immune system and consequently the efficacy of therapies, understanding treatment mechanisms in the presence of tumor will help lead to more efficacious treatment options. We demonstrate here that a poly(lactic-co-glycolic acid) (PLGA) based delivery system encapsulating tumor antigen (OVA) and the TLR9 agonist CpG motif DNA administered into the tumor microenvironment initiates an effective type 1 mediated (IFN-γ producing) anti-tumor response in a syngeneic murine model of T cell lymphoma (E.G7-OVA). Although E.G7-OVA tumors spontaneously generate antigen specific CTLs in draining lymph nodes (LN), tumors progress rapidly. Modulation of the tumor microenvironment via local PLGA based therapy led to the generation of a systemic antigen specific Th1 response, absent in the non-polymer delivery method, subsequently associated with reduced tumor growth and prolongation of survival. These studies provide further insight into the use of a PLGA-based therapeutic approach at modulating the tumor microenvironment and highlight the need for analyzing the treatment effects in a tumor bearing model.

第208位异亮氨酸对人细胞色素P4502A6尼古丁代谢活性的影响

目的探讨人体尼古丁主要代谢酶细胞色素P450(CYP)2A6及其同族成员CYP2A13肽链结构中,影响其尼古丁5′-羟化代谢活性的关键性氨基酸残基。方法使用前期制备的CYP2A6和CYP2A13系列氨基酸互换突变体:CYP2A6V117A,CYP2A6G164H,CYP2A6I208S,CYP2A6R372H和CYP2A6S465P以及CYP2A13A117V,CYP2A13H164G,CYP2A13S208I,CYP2A13H372R和CYP2A13P465S,比较其与相应野生蛋白酶的尼古丁5′-羟化催化反应的动力学参数。结果各突变体对2个CYP2A蛋白酶的尼古丁代谢活性影响不同。对于CYP2A6,I208S突变对酶活性的影响显著,导致表观反应常数Km及最大反应速度Vmax由野生型62.25μmol.L-1和6.53mol.min-1.mol-1变化为345μmol.L-1和2.19mol.min-1.mol-1,但该位点对CYP2A13酶活性无显著影响;对于CYP2A13,H372R突变对酶活性的影响最为显著,导致Km及Vmax由野生型的26.01μmol.L-1和24.51mol.min-1.mol-1变为148.7μmol.L-1和6.11mol.min-1.mol-1,此位点对CYP2A6无显著影响。其他位点突变对酶活性影响较小或不显著。结论CYP2A家族蛋白中,I208与H372分别是影响CYP2A6和CYP2A13对尼古丁代谢的关键残基。对于同家族蛋白酶而言,关键性氨基酸的作用并不总是一一对应。

Tumor suppressor p53:new functions of an old protein

p53 was discovered 30 years ago.Extensive studies have been done on p53 since then,which makes p53 one of the most extensively studied genes.p53 has long been recognized as a key tumor suppressor.Cell cycle arrest,apoptosis and senescence have been traditionally recognized as the main functions of p53 in tumor suppression.Recently,some novel functions of p53 have been identified,including the regulation of energy metabolism,antioxidant defense,and microRNA expression and maturation,which all contribute to the role of p53 in tumor suppression.Furthermore,the contribution of p53 to normal biologic processes(e.g.reproduction and aging)and some other aspects of diseases(e.g.neurodegenerative diseases)is only now being appreciated.Here we will review recent advances in the study of some new functions of p53.

Backdoor intrusion: retrotoxicity can explain targeted motor neuron death in amyotrophic lateral sclerosis

To the editor: Amyotrophic lateral sclerosis(ALS) is a fatal disease of unknown cause that selectively targets brain and spinal cord motor neurons(MNs).The lifetime risk is 1 in 2000, and most cases are sporadic although up to 10% of patients are predisposed by familial mutations in MN protection or repair genes(Bruijn et al., 2004).Risk factors include agrochemical exposure and trauma(Walters et al., 2019), although why they target MN is perplexing.Farmers are at a greater risk than non-farming rural residents(Kang et al, 2014), and ALS clusters occur in abrasion prone activities conducted on agrochemical treated fields such as baseball and soccer(Chio et al., 2005).

Critical Amino Acid Residues for Nicotine 5'-Hydroxylation in Human CYP2A Enzymes

Objective： We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-（3-pyridyl）-1-butanone（NNK） and aflatoxin BI（AFB1） carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods： A series of reciprocally substituted mutants of CYP2A6lle^300→ Phe, CYP2A6Gly^301aAla, CYP2A6Ser^369 → Gly, CYP2A13Phe^300→ Ile, CYP2A13Ala^301 → Gly and CYP2A13Gly^369 → Set were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5＇hydroxylatin by wild type and mutant CYP2A proteins was performed. Results：All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile^300→ Phe and CYP2A6Gly^301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe^300→Ile and CYP2A13Ala^301→Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by Vmax variations rather than Km changes. Substitution of #369 residue significantly affected both Km and Vmax values. CYP2A6Ser^369 → Gly increase the catalytic efficiency via a significant Km decrease versus Vmax enhancement, while the opposite effects were seen with CYP2A13Gly^369 → Ser. Conclusion：#300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5＇ -oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Set369 to Gly substitution indirectly affected nicotine binding by creating more space and conformational flexibility for the nearby residues, such as Leu^370 which is crucial for many hydroxylations.

Genomic instability and cancer:an introduction

Genomic instability refers to an increased tendency of alterations in the genome during the life cycle of cells.It is a major driving force for tumorigenesis.During a cell division,genomic instability is minimized by four major mechanisms:high-fidelity DNA replication in S-phase,precise chromosome segregation in mitosis,error free repair of sporadic DNA damage,and a coordinated cell cycle progression.This introduction summarizes the major molecular processes that contribute to these mechanisms in the context of prevention of genomic instability and tumorigenesis.