Using a 3-d model system to screen for drugs effective on solid tumors

There is a large medical need for the development of effective anticancer agents with minimal side effects. The present thesis represents an attempt to identify potent drugs for treatment of solid tumors. We used a strategy where 3-D multicellular tumor spheroids (cancer cells grown in three dimensional culture) were utilized as in vitro models for solid tumors. Drug libraries were screened using spheroids as targets and using apoptosis induction and loss of cell viability as endpoints. The hanging drop method for production of spheroids was modified to allow spheroid generation in the 96-well format. Initial studies showed that the screening of multicellular spheroids resulted in the identification of different hit compounds compared to screening of monolayer cultures. Interestingly, we found that spheroid screening enriched for hydrophobic compounds (XlogP >4), a finding of considerable interest for chemical library design and lead optimization in the field of anticancer drug development. An approach based on the analysis of drug-induced gene profiles was used to unravel the mechanism of action of hits identified in the screen. The proposed mechanisms of action were subsequently confirmed by specific in vitro assays. The generation of a caspase-cleaved product of cytokeratin 18 was used to determine apoptosis of carcinoma cells in spheroids. The same method could be used as a plasma biomarker to evaluate whether candidate compounds induced apoptosis in xenograft tumor models. The antibodies used in the assay recognize human but not mouse cytokeratin 18 – an advantage when xenograft models are used since tumor apoptosis can be specifically measured in blood samples. The screening work resulted in the identification of a novel topoisomerase inhibitor (thaspine), a novel iron chelator (CB21) and a number of microtubuli inhibitors. - Thaspine (an alkaloid from Croton lechleri) was identified in both monolayer and spheroid screening experiments. Thaspine was found to inhibit both topoisomerase I and II. Interestingly, thaspine was effective on cell lines overexpressing drug efflux transporters and showed in vivo activity. - CB21 was of particular interest, since it was toxic to the hypoxic quiescent cell population in spheroid cores which are known to be resistant to many chemotherapeutical drugs. The compound was not toxic to quiescent immortalized cells. CB21 was shown to be a very potent iron chelator. The compound induced marked induction of autophagy both in the outer and inner layers of spheroids. Interestingly, CB21 increased glucose uptake and reduced cellular oxygen consumption. The cytotoxicity of the compound was found to be increased during low glucose conditions, known to occur in the cores of spheroids. The compound showed a significant inhibitory effect in tumor xengrafts. - A number of novel microtubuli inhibitors were identified in the spheroid screen. This result was unexpected since such compounds are expected to be preferentially active on dividing cells. We conclude that drug screening using multicellular spheroids is a promising approach for anticancer drug discovery. A number of novel compounds were identified by screening, and some may be possible to develop for clinical use

Simulation of the effect of microstructure on electromigration induced failure of interconnects

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering, 1997.Includes bibliographical references (leaves 85-87).by Walid R. Fayad.M.S

Microstructure evolution and interconnect realiability

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering, 2000.Includes bibliographical references (leaves 207-213).In the context of predicting the effects of geometry, microstructure, and processing conditions on electromigration (EM) induced interconnect failure, normal grain growth in thin films was studied, analytic models were built for the grain structure statistics in 2D and 3D interconnects, and simulation programs were developed for generation of process and complex-geometry-sensitive interconnect structures. The models were validated through simulations and experiments and were integrated into tools for circuit-design level interconnect reliability predictions. The universal scaling behavior of 2D normal grain growth was demonstrated and characterized using a simulation of 2D grain growth (GGSim). We showed that the constant rate of change of the average grain area is equal to the grain boundary mobility constant pt. We also found that the steady state grain size distribution obtained using our simulation technique, as well as those reported in experiments on simple model systems and those reported for very different simulation techniques, are all very well fit by a Weibull distribution function with the dimensionless parameter p = 5/2, and are better fit by this function than the log normal, Gamma or Rayleigh functions. The 2D simulation was used to simulate the development of film structures with drag induced lognormal grain size distributions from which interconnect strips were etched and then annealed, in order to analyze the statistics of as-patterned, as well as post-pattern annealed, interconnect grain structures. These statistics were characterized as a function of the ratios of the line-widths to the initial-grain-sizes. Among the important findings is that polygranular cluster and bamboo segment length distributions for as-patterned lines are best fit by Weibull distribution functions. Analytic formulae describing grain structure statistics were reported, for usage in EM simulations and reliability predictions. A differential model predicting the evolution of the polygranular cluster length distribution during post-patterning annealing was developed. It was shown that the rate of bamboo-segment nucleation per unit time and unit of untransformed length is proportional to [mu]/w 3 , and is negligible in the growth-dominated steady-state. The cluster shrinkage velocity was demonstrated to reach a constant steady-state value proportional to [mu]/w (assuming constant and uniform [mu]). This was shown to lead to a time-invariant, steady-state exponential cluster length distribution with an average cluster length proportional to the strip width, and a cluster length fraction decaying exponentially with U=[mu]/w2 . The distribution of grain lengths in the resulting final bamboo grain structure is well fit by a log normal distribution, with a median grain length scaling with the line width, and a line-width-independent normalized deviation in the grain length. This result was used to show, using an EM simulation, that grain-orientation-dependent variations in surface diffusivities constitute a likely cause for the variabilities in lifetimes observed experimentally. The 2D simulation GGSim was also substantially modified to simulate the patterning of interconnect features of general shapes from polygranular thin film structures, as well as to simulate further grain structure evolution due to post-patterning annealing in these complex shapes. A grain structure extraction tool, PolySeg, was developed to allow extraction of the atomic transport details in the case of complex interconnect trees for EM reliability predictions using EM simulations. To assess the 3D effects on grain structure evolution, and therefore on interconnect reliability, a soap froth experiment was used to study 3D normal grain growth in long rectangular prisms. The kinetics were found to scale with the normalized time [mu]/w 2 (with w being the largest of the two prism cross-sectional dimensions). It was found that the normalized duration of the conversion from 3D (non-columnar) to 2D (columnar) structures and the normalized duration of the initial phase during which the structure was polygranular became longer as w/h approached 1. The same results obtained in the 2D case for the scaling behaviors of the bamboo nucleation rate and the polygranular cluster shrinkage rate were demonstrated. Based on a 2D approach, a prism-geometry-sensitive analytic model was developed for the transformation to fully-bamboo structures. These results were compared with preliminary results obtained using a 3D grain growth simulation and qualitative agreement was demonstrated. We have successfully captured with simple analytic models as well as elaborate simulations the physics of microstructure evolution in complex patterned thin-film structures. In particular, we have developed an array of models and simulations that can be used to investigate the impact of geometry and process history on microstructure evolution, and ultimately on EM-induced failure statistics.by Walid R. Fayad.Ph.D

Development of spiro-3-indolin-2-one containing compounds of antiproliferative and anti-SARS-CoV-2 properties

Abstract: A series of 1″-(alkylsulfonyl)-dispiro[indoline-3,2′-pyrrolidine-3′,3″-piperidine]-2,4″-diones 6a‒o has been synthesized through regioselective multi-component azomethine dipolar cycloaddition reaction of 1-(alkylsulfonyl)-3,5-bis(ylidene)-piperidin-4-ones 3a‒h. X-ray diffraction studies (6b‒d, h) confirmed the structures. The majority of the synthesized analogs reveal promising antiproliferation properties against a variety of human cancer cell lines (MCF7, HCT116, A431 and PaCa2) with good selectivity index towards normal cell (RPE1). Some of the synthesized agents exhibit potent inhibitory properties against the tested cell lines with higher efficacies than the standard references (sunitinib and 5-fluorouracil). Compound 6m is the most potent. Multi-targeted inhibitory properties against EGFR and VEGFR-2 have been observed for the synthesized agents. Flow cytometry supports the antiproliferation properties and shows the tested agents as apoptosis and necrosis forming. Vero cell viral infection model demonstrates the anti-SARS-CoV-2 properties of the synthesized agents. Compound 6f is the most promising (about 3.3 and 4.8 times the potency of the standard references, chloroquine and hydroxychloroquine). QSAR models explain and support the observed biological properties

Identification of a Novel Topoisomerase Inhibitor Effective in Cells Overexpressing Drug Efflux Transporters

BACKGROUND:Natural product structures have high chemical diversity and are attractive as lead structures for discovery of new drugs. One of the disease areas where natural products are most frequently used as therapeutics is oncology. METHOD AND FINDINGS:A library of natural products (NCI Natural Product set) was screened for compounds that induce apoptosis of HCT116 colon carcinoma cells using an assay that measures an endogenous caspase-cleavage product. One of the apoptosis-inducing compounds identified in the screen was thaspine (taspine), an alkaloid from the South American tree Croton lechleri. The cortex of this tree is used for medicinal purposes by tribes in the Amazonas basin. Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells. Analysis of the gene expression signature of thaspine-treated cells suggested that thaspine is a topoisomerase inhibitor. Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme. Interestingly, in contrast to the topoisomerase II inhibitors doxorubicin, etoposide and mitoxantrone, thaspine was cytotoxic to cell lines overexpressing the PgP or MRP drug efflux transporters. We finally show that thaspine induces wide-spread apoptosis in colon carcinoma multicellular spheroids and that apoptosis is induced in two xenograft mouse models in vivo. CONCLUSIONS:The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Technical Analysis - A comparative study between a Moving average and a buy-and-hold strategy

Background and research question: There are shared opinions about whether it is possible to forecast the direction of prices through studying previous of market data. According to the efficient market hypothesis, using technical analysis is not an efficient method to predict asset prices, implying that stock market prices are unpredictable. In the light of this and also because there is relatively little research on technical analysis, we find this research question relevant for stock market participants and other that find this interesting. Is it possible to accomplish positive return on the Swedish stock market by using the technical analysis and moving average method in particular as a trading strategy? - Purpose: The purpose of this study is to determine the efficiency of using the moving average as a trading strategy when forecasting the direction of asset prices of companies listed on Nasdaq Stockholm to exceed the buy-and-hold strategy. - Conclusion: The price development on OMXS30 suffer from serial correlation and are therefore not possible to predict with help of technical analysis

Screening of natural products for therapeutic activity against solid tumors

258-264Most of the currently used cancer therapeutics are natural products. These agents were generally discovered based on their toxicity to tumour cells using various bioassays. Although the exact mechanisms of action of the most commonly used cancer therapeutics such as anthracyclins, podophyllotoxins and camptothecin are incompletely understood, it is becoming increasingly clear that these agents often show complex modes of action at the cellular level, interacting with numerous targets. Such complex modes of action may be the very reason for clinical efficacy. For discovering new cytotoxic anticancer drugs sophisticated screening methods were used. The principles of such screening projects conducted, using collections of purified natural products or extracts from plants have been described. By performing simple but robust pre-screening tests such as the brine shrimp assay, bioactive extracts can be identified. Extracts (65) prepared from a collection of Egyptian plants were identified that showed cytotoxity on HepG2 cells. Interestingly, 22 (33%) of these raw extracts, induced > 2-fold induction of caspase-cleavage activity in a colon carcinoma cell line, consistent with induction of apoptosis. Only a fraction of the diversity of the biosphere has been tested for biological activity and novel cancer therapeutics remains to be discovered

A Naturally Derived Carrier for Photodynamic Treatment of Squamous Cell Carcinoma: In Vitro and In Vivo Models

Photodynamic therapy (PDT) is a non-invasive treatment strategy that includes the combination of three components—a photosensitizer, a light source, and tissue oxygen. PDT can be used for the treatment of skin diseases such as squamous cell carcinoma. The photosensitizer used in this study is the naturally derived chlorophyll derivative chlorin e6 (Ce6), which was encapsulated in ultradeformable ethosomes. Singlet oxygen production by Ce6 upon laser light irradiation was not significantly affected by encapsulation into ethosomes. PDT of squamous cell carcinoma cells treated with Ce6 ethosomes triggered increased mitochondrial superoxide levels and increased caspase 3/7 activity, resulting in concentration- and light-dose-dependent cytotoxicity. Ce6 ethosomes showed good penetration into 3D squamous cell carcinoma spheroids, which upon laser light irradiation exhibited reduced size, proliferation, and viability. The PDT effect of Ce6 ethosomes was specific and showed higher cytotoxicity against squamous cell carcinoma spheroids compared to normal skin fibroblast spheroids. In addition, PDT treatment of squamous cell carcinoma xenografts grown on chorioallantoic membranes of chick eggs (CAM) exhibited reduced expression of Ki-67 proliferation marker and increased terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining, indicating reduced proliferation and activation of apoptosis, respectively. The results demonstrate that Ce6-loaded ethosomes represent a convenient formulation for photodynamic treatment of squamous cell carcinoma

High-sensitivity C-reactive protein in chronic low back pain with vertebral end-plate Modic signal changes.

International audienceOBJECTIVE: To assess high-sensitivity C-reactive protein (hsCRP) level as a measure of low-grade inflammation in relation to Modic vertebral end-plate marrow signal change on magnetic resonance imaging (MRI) in patients with chronic low back pain. METHODS: All patients hospitalized for chronic low back pain in our institution were prospectively enrolled in this pilot study. Serum hsCRP concentration was measured by immunoturbidimetric assay. MR images were evaluated independently by a panel of 2 spine specialists and a radiologist. Recording of clinical parameters, MRI evaluation, and hsCRP level of each patient was blinded. RESULTS: Three groups of 12 consecutive patients (Modic 0, Modic I, and Modic II signal changes on MRI) were prospectively selected. Serum hsCRP level was significantly different in the 3 groups (P = 0.002) and especially high in the Modic I group (P = 0.002 compared with Modic 0 and II groups): mean +/- SD 1.33 +/- 0.77 mg/liter in the Modic 0 group, 4.64 +/- 3.09 mg/liter in the Modic I group, and 1.75 +/- 1.30 mg/liter in the Modic II group. The only difference in clinical parameters among the 3 groups (P = 0.001) was that the worst painful moment during the previous 24 hours occurred during the late night and morning for all Modic I patients (P = 0.001 compared with Modic 0 and P = 0.002 compared with Modic II). CONCLUSION: Low-grade inflammation indicated by high serum hsCRP level in patients with chronic low back pain could point to Modic I signal changes. This result could help physicians predict the patients with Modic I signals to more precisely prescribe the correct imaging procedure and local antiinflammatory treatment in such patients