The Use of Survival Dose-Rate Dependencies as Theoretical Discrimination Criteria for In-Silico Dynamic Radiobiological Models.

Introduction: Cell repair dynamics are crucial in optimizing anti-cancer therapies. Various assays (eg, comet assay and γ-H2AX) assess post-radiation repair kinetics, but interpreting such data is challenging and model-based data analyses are required. However, ambiguities in parameter calibration remain an unsolved challenge. To address this, we propose combining survival dose-rate effects with computer simulations to gain knowledge about repair kinetics. Methods: After a literature review, theoretical discriminators based on common fractionation/dose-rate-related effects were defined to discard unrealistic model dynamics. The Multi-Hit Repair (MHR) model was calibrated with canine osteosarcoma Abrams cell line data to study the discriminators' efficacy in scenarios with limited survival data. Additionally, survival dose-rate-dependent data from the human SiHa cervical cancer cell line were used to illustrate the survival behavior at diverse dose-rates and the capability of the MHR to model these data. Results: SiHa data confirmed the validity of the proposed discriminators. The discriminators filtered 99% of parameter sets, improving the calibration of Abrams cells data. Furthermore, results from both cell lines may hint universal aspects of cellular repair. Conclusions: Dose-rate theoretical discrimination criteria are an effective method to understand repair kinetics and improve radiobiological model calibration. Moreover, this methodology may be used to analyze diverse biological data using dynamic models in-silico.

BIOMERO: A scalable and extensible image analysis framework.

In the rapidly evolving field of bioimaging, the integration and orchestration of findable, accessible, interoperable, and reusable (FAIR) image analysis workflows remains a challenge. We introduce BIOMERO (bioimage analysis in OMERO), a bridge connecting OMERO, a renowned bioimaging data management platform; FAIR workflows; and high-performance computing (HPC) environments. BIOMERO facilitates seamless execution of FAIR workflows, particularly for large datasets from high-content or high-throughput screening. BIOMERO empowers researchers by eliminating the need for specialized knowledge, enabling scalable image processing directly from OMERO. BIOMERO notably supports the sharing and utilization of FAIR workflows between OMERO, Cytomine/BIAFLOWS, and other bioimaging communities. BIOMERO will promote the widespread adoption of FAIR workflows, emphasizing reusability, across the realm of bioimaging research. Its user-friendly interface will empower users, including those without technical expertise, to seamlessly apply these workflows to their datasets, democratizing the utilization of AI by the broader research community.

Exploiting a subtype-specific mitochondrial vulnerability for successful treatment of colorectal peritoneal metastases.

Peritoneal metastases (PMs) from colorectal cancer (CRC) respond poorly to treatment and are associated with unfavorable prognosis. For example, the addition of hyperthermic intraperitoneal chemotherapy (HIPEC) to cytoreductive surgery in resectable patients shows limited benefit, and novel treatments are urgently needed. The majority of CRC-PMs represent the CMS4 molecular subtype of CRC, and here we queried the vulnerabilities of this subtype in pharmacogenomic databases to identify novel therapies. This reveals the copper ionophore elesclomol (ES) as highly effective against CRC-PMs. ES exhibits rapid cytotoxicity against CMS4 cells by targeting mitochondria. We find that a markedly reduced mitochondrial content in CMS4 cells explains their vulnerability to ES. ES demonstrates efficacy in preclinical models of PMs, including CRC-PMs and ovarian cancer organoids, mouse models, and a HIPEC rat model of PMs. The above proposes ES as a promising candidate for the local treatment of CRC-PMs, with broader implications for other PM-prone cancers.

Super-resolution GSDIM microscopy unveils distinct nanoscale characteristics of DNA repair foci under diverse genotoxic stress.

DNA double-strand breaks initiate the DNA damage response (DDR), leading to the accumulation of repair proteins at break sites and the formation of the-so-called foci. Various microscopy methods, such as wide-field, confocal, electron, and super-resolution microscopy, have been used to study these structures. However, the impact of different DNA-damaging agents on their (nano)structure remains unclear. Utilising GSDIM super-resolution microscopy, here we investigated the distribution of fluorescently tagged DDR proteins (53BP1, RNF168, MDC1) and γH2AX in U2OS cells treated with γ-irradiation, etoposide, cisplatin, or hydroxyurea. Our results revealed that both foci structure and their nanoscale ultrastructure, including foci size, nanocluster characteristics, fluorophore density and localisation, can be significantly altered by different inducing agents, even ones with similar mechanisms. Furthermore, distinct behaviours of DDR proteins were observed under the same treatment. These findings have implications for cancer treatment strategies involving these agents and provide insights into the nanoscale organisation of the DDR.

Using picoliter droplet deposition to track clonal competition in adherent and organoid cancer cell cultures.

Clonal growth and competition underlie processes of key relevance in etiology, progression and therapy response across all cancers. Here, we demonstrate a novel experimental approach, based on multi-color, fluorescent tagging of cell nuclei, in combination with picoliter droplet deposition, to study the clonal dynamics in two- and three-dimensional cell cultures. The method allows for the simultaneous visualization and analysis of multiple clones in individual multi-clonal colonies, providing a powerful tool for studying clonal dynamics and identifying clonal populations with distinct characteristics. Results of our experiments validate the utility of the method in studying clonal dynamics in vitro, and reveal differences in key aspects of clonal behavior of different cancer cell lines in monoculture conditions, as well as in co-cultures with stromal fibroblasts.

High-Content and High-Throughput Clonogenic Survival Assay Using Fluorescence Barcoding.

The Clonogenic Survival Assay (CSA) is a fundamental tool employed to assess cell survival and proliferative potential in cancer research. Despite its importance, CSA faces limitations, primarily its time- and labor-intensive nature and its binary output. To overcome these limitations and enhance CSA's utility, several approaches have been developed, focusing on increasing the throughput. However, achieving both high-content and high-throughput analyses simultaneously has remained a challenge. In this paper, we introduce LeGO-CSA, an extension of the classical CSA that employs the imaging of cell nuclei barcoded with fluorescent lentiviral gene ontology markers, enabling both high-content and high-throughput analysis. To validate our approach, we contrasted it with results from a classical assay and conducted a proof-of-concept screen of small-molecule inhibitors targeting various pathways relevant to cancer treatment. Notably, our results indicate that the classical CSA may underestimate clonogenicity and unveil intriguing aspects of clonal cell growth. We demonstrate the potential of LeGO-CSA to offer a robust approach for assessing cell survival and proliferation with enhanced precision and throughput, with promising implications for accelerating drug discovery and contributing to a more comprehensive understanding of cellular behavior in cancer.

Perturbation of Copper Homeostasis Sensitizes Cancer Cells to Elevated Temperature.

Temporary elevation of tumor temperature, also known as hyperthermia, is a safe and well-tolerated treatment modality. The efficacy of hyperthermia can be improved by efficient thermosensitizers, and various candidate drugs, including inhibitors of the heat stress response, have been explored in vitro and in animal models, but clinically relevant thermosensitizers are lacking. Here, we employ unbiased in silico approaches to uncover new mechanisms and compounds that could be leveraged to increase the thermosensitivity of cancer cells. We then focus on elesclomol, a well-performing compound, which amplifies cell killing by hyperthermia by 5- to 20-fold in cell lines and outperforms clinically applied chemotherapy when combined with hyperthermia in vitro. Surprisingly, our findings suggest that the thermosensitizing effects of elesclomol are independent of its previously reported modes of action but depend on copper shuttling. Importantly, we show that, like elesclomol, multiple other copper shuttlers can thermosensitize, suggesting that disturbing copper homeostasis could be a general strategy for improving the efficacy of hyperthermia.

Evaluation of the Heat Shock Protein 90 Inhibitor Ganetespib as a Sensitizer to Hyperthermia-Based Cancer Treatments.

Hyperthermia is being used as a radio- and chemotherapy sensitizer for a growing range of tumor subtypes in the clinic. Its potential is limited, however, by the ability of cancer cells to activate a protective mechanism known as the heat stress response (HSR). The HSR is marked by the rapid overexpression of molecular chaperones, and recent advances in drug development make their inhibition an attractive option to improve the efficacy of hyperthermia-based therapies. Our previous in vitro work showed that a single, short co-treatment with a HSR (HSP90) inhibitor ganetespib prolongs and potentiates the effects of hyperthermia on DNA repair, enhances hyperthermic sensitization to radio- and chemotherapeutic agents, and reduces thermotolerance. In the current study, we first validated these results using an extended panel of cell lines and more robust methodology. Next, we examined the effects of hyperthermia and ganetespib on global proteome changes. Finally, we evaluated the potential of ganetespib to boost the efficacy of thermo-chemotherapy and thermo-radiotherapy in a xenograft murine model of cervix cancer. Our results revealed new insights into the effects of HSR inhibition on cellular responses to heat and show that ganetespib could be employed to increase the efficacy of hyperthermia when combined with radiation.

Metallated phthalocyanines and their hydrophilic derivatives for multi-targeted oncological photodynamic therapy.

BACKGROUND AND AIM: A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition. METHODS: Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts. RESULTS: The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis. CONCLUSIONS: AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization.

Super-Resolution Imaging of Intracellular Lipid Nanocarriers to Study Drug Delivery in Photodynamic Therapy.

Liposomal nanocarriers are intensively investigated as delivery vehicles for photoactivatable agents used in photodynamic therapy (PDT). The uptake, intracellular distribution, and processing of the nanocarriers are of paramount importance for the effectiveness of the therapy; visualization and analysis of these processes can, therefore, stimulate the development of improved PDT modalities. Here we describe a simple protocol, based on super-resolution imaging, that can be used for detailed quantification of concentration, distribution, and size of individual lipid nanocarriers in adherent mammalian cells.

The Effects of Heat Stress on the Transcriptome of Human Cancer Cells: A Meta-Analysis.

Hyperthermia is clinically applied cancer treatment in conjunction with radio- and/or chemotherapy, in which the tumor volume is exposed to supraphysiological temperatures. Since cells can effectively counteract the effects of hyperthermia by protective measures that are commonly known as the heat stress response, the identification of cellular processes that are essential for surviving hyperthermia could lead to novel treatment strategies that improve its therapeutic effects. Here, we apply a meta-analytic approach to 18 datasets that capture hyperthermia-induced transcriptome alterations in nine different human cancer cell lines. We find, in line with previous reports, that hyperthermia affects multiple processes, including protein folding, cell cycle, mitosis, and cell death, and additionally uncover expression changes of genes involved in KRAS signaling, inflammatory responses, TNF-a signaling and epithelial-to-mesenchymal transition (EMT). Interestingly, however, we also find a considerable inter-study variability, and an apparent absence of a 'universal' heat stress response signature, which is likely caused by the differences in experimental conditions. Our results suggest that gene expression alterations after heat stress are driven, to a large extent, by the experimental context, and call for a more extensive, controlled study that examines the effects of key experimental parameters on global gene expression patterns.

Zinc finger protein ZNF384 is an adaptor of Ku to DNA during classical non-homologous end-joining.

DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage as they can lead to mutations and chromosomal rearrangements, which underlie cancer development. Classical non-homologous end-joining (cNHEJ) is the dominant pathway for DSB repair in human cells, involving the DNA-binding proteins XRCC6 (Ku70) and XRCC5 (Ku80). Other DNA-binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair, but their role in cNHEJ remained elusive. Here we show that ZNF384, a member of the C2H2 family of ZnF proteins, binds DNA ends in vitro and is recruited to DSBs in vivo. ZNF384 recruitment requires the poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent expansion of damaged chromatin, followed by binding of its C2H2 motifs to the exposed DNA. Moreover, ZNF384 interacts with Ku70/Ku80 via its N-terminus, thereby promoting Ku70/Ku80 assembly and the accrual of downstream cNHEJ factors, including APLF and XRCC4/LIG4, for efficient repair at DSBs. Altogether, our data suggest that ZNF384 acts as a 'Ku-adaptor' that binds damaged DNA and Ku70/Ku80 to facilitate the build-up of a cNHEJ repairosome, highlighting a role for ZNF384 in DSB repair and genome maintenance.

Distinct pathways of homologous recombination controlled by the SWS1-SWSAP1-SPIDR complex.

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.

A simple microscopy setup for visualizing cellular responses to DNA damage at particle accelerator facilities.

Cellular responses to DNA double-strand breaks (DSBs) not only promote genomic integrity in healthy tissues, but also largely determine the efficacy of many DNA-damaging cancer treatments, including X-ray and particle therapies. A growing body of evidence suggests that activation of the mechanisms that detect, signal and repair DSBs may depend on the complexity of the initiating DNA lesions. Studies focusing on this, as well as on many other radiobiological questions, require reliable methods to induce DSBs of varying complexity, and to visualize the ensuing cellular responses. Accelerated particles of different energies and masses are exceptionally well suited for this task, due to the nature of their physical interactions with the intracellular environment, but visualizing cellular responses to particle-induced damage - especially in their early stages - at particle accelerator facilities, remains challenging. Here we describe a straightforward approach for real-time imaging of early response to particle-induced DNA damage. We rely on a transportable setup with an inverted fluorescence confocal microscope, tilted at a small angle relative to the particle beam, such that cells can be irradiated and imaged without any microscope or beamline modifications. Using this setup, we image and analyze the accumulation of fluorescently-tagged MDC1, RNF168 and 53BP1-key factors involved in DSB signalling-at DNA lesions induced by 254 MeV α-particles. Our results provide a demonstration of technical feasibility and reveal asynchronous initiation of accumulation of these proteins at different individual DSBs.

Apc-mutant cells act as supercompetitors in intestinal tumour initiation.

A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC, which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway1,2. It has previously been established that Apc-mutant cells have a competitive advantage over wild-type ISCs3. Consequently, Apc-mutant ISCs frequently outcompete all wild-type stem cells within a crypt, thereby reaching clonal fixation in the tissue and initiating cancer formation. However, whether the increased relative fitness of Apc-mutant ISCs involves only cell-intrinsic features or whether Apc mutants are actively involved in the elimination of their wild-type neighbours remains unresolved. Here we show that Apc-mutant ISCs function as bona fide supercompetitors by secreting WNT antagonists, thereby inducing differentiation of neighbouring wild-type ISCs. Lithium chloride prevented the expansion of Apc-mutant clones and the formation of adenomas by rendering wild-type ISCs insensitive to WNT antagonists through downstream activation of WNT by inhibition of GSK3ß. Our work suggests that boosting the fitness of healthy cells to limit the expansion of pre-malignant clones may be a powerful strategy to limit the formation of cancers in high-risk individuals.

Modulating the Heat Stress Response to Improve Hyperthermia-Based Anticancer Treatments.

Cancer treatments based on mild hyperthermia (39-43 °C, HT) are applied to a widening range of cancer types, but several factors limit their efficacy and slow down more widespread adoption. These factors include difficulties in adequate heat delivery, a short therapeutic window and the acquisition of thermotolerance by cancer cells. Here, we explore the biological effects of HT, the cellular responses to these effects and their clinically-relevant consequences. We then identify the heat stress response-the cellular defense mechanism that detects and counteracts the effects of heat-as one of the major forces limiting the efficacy of HT-based therapies and propose targeting this mechanism as a potentially universal strategy for improving their efficacy.

Attritional evaluation of lipophilic and hydrophilic metallated phthalocyanines for oncological photodynamic therapy.

BACKGROUND AND AIM: Oncological photodynamic therapy (PDT) relies on photosensitizers (PSs) to photo-oxidatively destroy tumor cells. Currently approved PSs yield satisfactory results in superficial and easy-to-access tumors but are less suited for solid cancers in internal organs such as the biliary system and the pancreas. For these malignancies, second-generation PSs such as metallated phthalocyanines are more appropriate. Presently it is not known which of the commonly employed metallated phtahlocyanines, namely aluminum phthalocyanine (AlPC) and zinc phthalocyanine (ZnPC) as well as their tetrasulfonated derivatives AlPCS4 and ZnPCS4, is most cytotoxic to tumor cells. This study therefore employed an attritional approach to ascertain the best metallated phthalocyanine for oncological PDT in a head-to-head comparative analysis and standardized experimental design. METHODS: ZnPC and AlPC were encapsulated in PEGylated liposomes. Analyses were performed in cultured A431 cells as a template for tumor cells with a dysfunctional P53 tumor suppressor gene and EGFR overexpression. First, dark toxicity was assessed as a function of PS concentration using the WST-1 and sulforhodamine B assay. Second, time-dependent uptake and intracellular distribution were determined by flow cytometry and confocal microscopy, respectively, using the intrinsic fluorescence of the PSs. Third, the LC50 values were established for each PS at 671 nm and a radiant exposure of 15 J/cm2 following 1-h PS exposure. Finally, the mode of cell death as a function of post-PDT time and cell cycle arrest at 24 h after PDT were analyzed. RESULTS: In the absence of illumination, AlPC and ZnPC were not toxic to cells up to a 1.5-µM PS concentration and exposure for up to 72 h. Dark toxicity was noted for AlPCS4 at 5 µM and ZnPCS4 at 2.5 µM. Uptake of all PSs was observed as early as 1 min after PS addition to cells and increased in amplitude during a 2-h incubation period. After 60 min, the entire non-nuclear space of the cell was photosensitized, with PS accumulation in multiple subcellular structures, especially in case of AlPC and AlPCS4. PDT of cells photosensitized with ZnPC, AlPC, and AlPCS4 yielded LC50 values of 0.13 µM, 0.04 µM, and 0.81 µM, respectively, 24 h post-PDT (based on sulforhodamine B assay). ZnPCS4 did not induce notable phototoxicity, which was echoed in the mode of cell death and cell cycle arrest data. At 4 h post-PDT, the mode of cell death comprised mainly apoptosis for ZnPC and AlPC, the extent of which was gradually exacerbated in AlPC-photosensitized cells during 8 h. ZnPC-treated cells seemed to recover at 8 h post-PDT compared to 4 h post-PDT, which had been observed before in another cell line. AlPCS4 induced considerable necrosis in addition to apoptosis, whereby most of the cell death had already manifested at 2 h after PDT. During the course of 8 h, necrotic cell death transitioned into mainly late apoptotic cell death. Cell death signaling coincided with a reduction in cells in the G0/G1 phase (ZnPC, AlPC, AlPCS4) and cell cycle arrest in the S-phase (ZnPC, AlPC, AlPCS4) and G2 phase (ZnPC and AlPC). Cell cycle arrest was most profound in cells that had been photosensitized with AlPC and subjected to PDT. CONCLUSIONS: Liposomal AlPC is the most potent PS for oncological PDT, whereas ZnPCS4 was photodynamically inert in A431 cells. AlPC did not induce dark toxicity at PS concentrations of up to 1.5 µM, i.e., > 37 times the LC50 value, which is favorable in terms of clinical phototoxicity issues. AlPC photosensitized multiple intracellular loci, which was associated with extensive, irreversible cell death signaling that is expected to benefit treatment efficacy and possibly immunological long-term tumor control, granted that sufficient AlPC will reach the tumor in vivo. Given the differential pharmacokinetics, intracellular distribution, and cell death dynamics, liposomal AlPC may be combined with AlPCS4 in a PS cocktail to further improve PDT efficacy.

In Vitro and In Vivo Studies on HPMA-Based Polymeric Micelles Loaded with Curcumin.

Curcumin-loaded polymeric micelles composed of poly(ethylene glycol)-b-poly(N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) were prepared to solubilize and improve the pharmacokinetics of curcumin. Curcumin-loaded micelles were prepared by a nanoprecipitation method using mPEG5kDa-b-p(HPMA-Bz) copolymers with varying molecular weight of the hydrophobic block (5.2, 10.0, and 17.1 kDa). At equal curcumin loading, micelles composed of mPEG5kDa-b-p(HPMA-Bz)17.1kDa showed better curcumin retention in both phosphate-buffered saline (PBS) and plasma at 37 °C than micelles based on block copolymers with smaller hydrophobic blocks. No change in micelle size was observed during 24 h incubation in plasma using asymmetrical flow field-flow fractionation (AF4), attesting to particle stability. However, 22-49% of the curcumin loading was released from the micelles during 24 h from formulations with the highest to the lowest molecular weight p(HPMA-Bz), respectively, in plasma. AF4 analysis further showed that the released curcumin was subsequently solubilized by albumin. In vitro analyses revealed that the curcumin-loaded mPEG5kDa-b-p(HPMA-Bz)17.1kDa micelles were internalized by different types of cancer cells, resulting in curcumin-induced cell death. Intravenously administered curcumin-loaded, Cy7-labeled mPEG5kDa-b-p(HPMA-Bz)17.1kDa micelles in mice at 50 mg curcumin/kg showed a long circulation half-life for the micelles (t1/2 = 42 h), in line with the AF4 results. In contrast, the circulation time of curcumin was considerably shorter than that of the micelles (t1/2α = 0.11, t1/2ß = 2.5 h) but ∼5 times longer than has been reported for free curcumin (t1/2α = 0.02 h). The faster clearance of curcumin in vivo compared to in vitro studies can be attributed to the interaction of curcumin with blood cells. Despite the excellent solubilizing effect of these micelles, no cytostatic effect was achieved in neuroblastoma-bearing mice, possibly because of the low sensitivity of the Neuro2A cells to curcumin.

The Challenge of Teaching Essential Immunology Laboratory Skills to Undergraduates in One Month-Experience of an Osteoimmunology Course on TLR Activation.

Acquiring immunology laboratory skills during undergraduate studies is often a prerequisite for admission to Masters' programs. Many broad liberal arts and sciences honors degree colleges struggle in teaching these essentials since only limited time is usually reserved for this. Here, we describe a new 1-month-course developed to train a small group of honors students in 6 techniques that are useful for immunology research. In essence, 15 students were divided into 3 groups of 5 students where each student became involved in current osteoimmunology research. Osteoimmunology is a relatively new branch of the immunology tree, where the effects of inflammation and the immune system on bone formation and bone degradation is studied. A broad, 3 weeks experiment on the chronic effects of molecules that specifically activate toll-like receptors TLR2 and TLR4 on bone formation or osteoclast differentiation was performed just before the start of the course. Control samples and samples treated with TLR2 (group A), TLR4 (group B), or TLR2+TLR4 (group C) agonists were harvested and analyzed using quantitative PCR, ELISA, biochemistry, microscopy of enzyme-histochemically stained osteoclasts, scanning electron microscopy, and confocal microscopy. Each technique was taught for 2 days by a specialized instructor, who was present at all laboratory activities. The primary research question for each group was: how does the experimental condition affect bone formation or osteoclast formation? The secondary research question specified per technique was: how does this technique answer part of the primary research question? Pedagogically, students were encouraged to collaborate within the group to analyze the obtained data. Secondly, at the end of the course, a representative of each group collaborated to summarize the TLR activation modalities of a technique of choice. Thirdly, each group wrote a report, where introduction and discussion were graded as a group; each technique part was graded individually. The summary of the results from the 3 treatment modalities was presented orally. The student evaluation of the course was high, students remarked that the course had a curriculum overarching function, since it created an awareness and appreciation for both the joy and the blood-sweat-and-tears aspects of pipetting, and writing research articles, making interpretation of those easier.

Ultra-soft X-ray system for imaging the early cellular responses to X-ray induced DNA damage.

The majority of the proteins involved in processing of DNA double-strand breaks (DSBs) accumulate at the damage sites. Real-time imaging and analysis of these processes, triggered by the so-called microirradiation using UV lasers or heavy particle beams, yielded valuable insights into the underlying DSB repair mechanisms. To study the temporal organization of DSB repair responses triggered by a more clinically-relevant DNA damaging agent, we developed a system coined X-ray multi-microbeam microscope (XM3), capable of simultaneous high dose-rate (micro)irradiation of large numbers of cells with ultra-soft X-rays and imaging of the ensuing cellular responses. Using this setup, we analyzed the changes in real-time kinetics of MRE11, MDC1, RNF8, RNF168 and 53BP1-proteins involved in the signaling axis of mammalian DSB repair-in response to X-ray and UV laser-induced DNA damage, in non-cancerous and cancer cells and in the presence or absence of a photosensitizer. Our results reveal, for the first time, the kinetics of DSB signaling triggered by X-ray microirradiation and establish XM3 as a powerful platform for real-time analysis of cellular DSB repair responses.