KPC-luciferase-expressing cells elicit an anti-tumor immune response in a mouse model of pancreatic cancer.

Mouse models for the study of pancreatic ductal adenocarcinoma (PDAC) are well-established and representative of many key features observed in human PDAC. To monitor tumor growth, cancer cells that are implanted in mice are often transfected with reporter genes, such as firefly luciferase (Luc), enabling in vivo optical imaging over time. Since Luc can induce an immune response, we aimed to evaluate whether the expression of Luc could affect the growth of KPC tumors in mice by inducing immunogenicity. Although both cell lines, KPC and Luc transduced KPC (KPC-Luc), had the same proliferation rate, KPC-Luc tumors had significantly smaller sizes or were absent 13 days after orthotopic cell implantation, compared to KPC tumors. This coincided with the loss of bioluminescence signal over the tumor region. Immunophenotyping of blood and spleen from KPC-Luc tumor-bearing mice showed a decreased number of macrophages and CD4+ T cells, and an increased accumulation of natural killer (NK) cells in comparison to KPC tumor mice. Higher infiltration of CD8+ T cells was found in KPC-Luc tumors than in their controls. Moreover, the immune response against Luc peptide was stronger in splenocytes from mice implanted with KPC-Luc cells compared to those isolated from KPC wild-type mice, indicating increased immunogenicity elicited by the presence of Luc in the PDAC tumor cells. These results must be considered when evaluating the efficacy of anti-cancer therapies including immunotherapies in immunocompetent PDAC or other cancer mouse models that use Luc as a reporter for bioluminescence imaging.

OrganoIDNet: a deep learning tool for identification of therapeutic effects in PDAC organoid-PBMC co-cultures from time-resolved imaging data.

PURPOSE: Pancreatic Ductal Adenocarcinoma (PDAC) remains a challenging disease due to its complex biology and aggressive behavior with an urgent need for efficient therapeutic strategies. To assess therapy response, pre-clinical PDAC organoid-based models in combination with accurate real-time monitoring are required. METHODS: We established stable live-imaging organoid/peripheral blood mononuclear cells (PBMCs) co-cultures and introduced OrganoIDNet, a deep-learning-based algorithm, capable of analyzing bright-field images of murine and human patient-derived PDAC organoids acquired with live-cell imaging. We investigated the response to the chemotherapy gemcitabine in PDAC organoids and the PD-L1 inhibitor Atezolizumab, cultured with or without HLA-matched PBMCs over time. Results obtained with OrganoIDNet were validated with the endpoint proliferation assay CellTiter-Glo. RESULTS: Live cell imaging in combination with OrganoIDNet accurately detected size-specific drug responses of organoids to gemcitabine over time, showing that large organoids were more prone to cytotoxic effects. This approach also allowed distinguishing between healthy and unhealthy status and measuring eccentricity as organoids' reaction to therapy. Furthermore, imaging of a new organoids/PBMCs sandwich-based co-culture enabled longitudinal analysis of organoid responses to Atezolizumab, showing an increased potency of PBMCs tumor-killing in an organoid-individual manner when Atezolizumab was added. CONCLUSION: Optimized PDAC organoid imaging analyzed by OrganoIDNet represents a platform capable of accurately detecting organoid responses to standard PDAC chemotherapy over time. Moreover, organoid/immune cell co-cultures allow monitoring of organoid responses to immunotherapy, offering dynamic insights into treatment behavior within a co-culture setting with PBMCs. This setup holds promise for real-time assessment of immunotherapeutic effects in individual patient-derived PDAC organoids.

Effect of tissue fixation on the optical properties of structural components assessed by non-linear microscopy imaging.

Fixation methods such as formalin are commonly used for the preservation of tissue with the aim of keeping their structure as close as possible to the native condition. However, fixatives chemically interact with tissue molecules, such as collagen in the extracellular matrix (ECM) or myosin, and may thus modify their structure. Taking advantage of the second- and third-harmonic generation (SHG and THG) emission capabilities of such components, we used nonlinear two-photon microscopy (NL2PM) to evaluate the effect that preservation methods, such as chemical fixatives, have on the nonlinear capabilities of protein components within mouse tissues. Our results show that depending on the preservation technique used, the nonlinear capabilities of collagen, lipid droplets and myosin microarchitecture are strongly affected. Parameters of collagen fibers, such as density and branch points, especially in collagen-sparse regions, e.g., in kidneys, were found to be altered upon formalin fixation. Moreover, cryo-freezing drastically reduced SHG signals from myosin. Our findings provide valuable information to select the best tissue fixation method for visualization and quantification of structural proteins, such as collagen and myosin by advanced NL2PM imaging techniques. This may advance the interpretation of the role these proteins play in disease.

Longitudinal x-ray based lung function measurement for monitoring Nintedanib treatment response in a mouse model of lung fibrosis.

Lung fibrosis (LF) is a chronic progressive, incurable, and debilitating condition of the lung, which is associated with different lung disease. Treatment options are still sparse. Nintedanib, an oral tyrosine kinase inhibitor, significantly slows the LF progression. However, there is a strong need of further research and the development of novel therapies. In this study, we used a correlative set-up that combines X-ray based lung function (XLF) with microCT and whole body plethysmography (WBP) for a comprehensive functional and structural evaluation of lung fibrosis (LF) as well as for monitoring response to orally administered Nintedanib in the mouse model of bleomycin induced LF. The decline in lung function as early as one week after intratracheal bleomycin instillation was reliably detected by XLF, revealing the lowest decay rate in the LF mice compared to healthy ones. Simultaneously performed microCT and WBP measurements corroborated XLF findings by exhibiting reduced lung volume [Formula: see text] and tidal volume [Formula: see text]. In LF mice XLF also revealed profound improvement in lung function one week after Nintedanib treatment. This positive response to Nintedanib therapy was further substantiated by microCT and WBP measurements which also showed significantly improved [Formula: see text] and [Formula: see text] in the Nintedanib treated mice. By comparing the XLF data to structural features assessing the extent of fibrosis obtained by ex-vivo high-resolution synchrotron radiation-based imaging and classical histology we demonstrate that: (1) a simple low dose x-ray measurement like XLF is sensitive enough to pick up treatment response, (2) Nintedanib treatment successfully improved lung function in a bleomycin induced LF mouse model and (3) differences between the fully restored lung function and the partially reduced fibrotic burden compared to healthy and untreated mice. The presented analysis pipeline underlines the importance of a combined functional and anatomical readout to reliably measure treatment response and could easily be adapted to other preclinical lung disease models.

Content aware image restoration improves spatiotemporal resolution in luminescence imaging.

Luminescent reporters are due to their intrinsically high signal-to-noise ratio a powerful labelling tool for microscopy and macroscopic in vivo imaging in biomedical research. However, luminescence signal detection requires longer exposure times than fluorescence imaging and is consequently less suited for applications requiring high temporal resolution or throughput. Here we demonstrate that content aware image restoration can drastically reduce the exposure time requirements in luminescence imaging, thus overcoming one of the major limitations of the technique.

An Alternative Technique for Monitoring the Live Interaction of Monocytes and Tumor Cells with Nanoparticles in the Mouse Lung.

Nanomaterials are increasingly used for the diagnosis and treatment of cancer, including lung cancer. For the clinical translation of nano-based theranostics, it is vital to detect and monitor their accumulation in the tumor, as well as their interaction with tumor, immune cells, and the tumor microenvironment (TME). While high resolution microscopy of fixed tumor specimens can provide some of this information from individual thin slices, it cannot capture cellular events over time and lacks 3D information of the tumor tissue. On the other hand, in vivo optical procedures either fall short of providing the necessary cellular resolution, as in the case of epifluorescence optical imaging, or are very demanding, as for instance intravital lung microscopy. We describe an alternative approach to investigate nanoparticle-cell interactions in entire mouse lung lobes, by longitudinal live cell confocal microscopy at nanometer resolution. By filling the lung ex vivo with 1% agarose, we were able to stabilize the lung lobes and visualize the interaction of fluorescent cells and nanoparticles for at least 4 hours post mortem. This high resolution ex vivo live cell imaging approach is an easy 4D tool for assessing several dynamic processes in tumor tissue, such as the traffic of cells, shedding of extracellular vesicles (EVs), and the accumulation of nanoparticles in tumor tissue. Graphic abstract: Schematic of the workflow for live cell imaging in the mouse lung.

Elastic transformation of histological slices allows precise co-registration with microCT data sets for a refined virtual histology approach.

Although X-ray based 3D virtual histology is an emerging tool for the analysis of biological tissue, it falls short in terms of specificity when compared to conventional histology. Thus, the aim was to establish a novel approach that combines 3D information provided by microCT with high specificity that only (immuno-)histochemistry can offer. For this purpose, we developed a software frontend, which utilises an elastic transformation technique to accurately co-register various histological and immunohistochemical stainings with free propagation phase contrast synchrotron radiation microCT. We demonstrate that the precision of the overlay of both imaging modalities is significantly improved by performing our elastic registration workflow, as evidenced by calculation of the displacement index. To illustrate the need for an elastic co-registration approach we examined specimens from a mouse model of breast cancer with injected metal-based nanoparticles. Using the elastic transformation pipeline, we were able to co-localise the nanoparticles to specifically stained cells or tissue structures into their three-dimensional anatomical context. Additionally, we performed a semi-automated tissue structure and cell classification. This workflow provides new insights on histopathological analysis by combining CT specific three-dimensional information with cell/tissue specific information provided by classical histology.

Ex vivo Live Cell Imaging of Nanoparticle-Cell Interactions in the Mouse Lung.

A successful clinical translation of novel nanoparticle-based cancer therapeutics requires a thorough preclinical investigation of their interaction with immune, tumor and endothelial cells as well as components of the tumor-microenvironment. Although high-resolution microscopy images of fixed tumor tissue specimens can provide valuable information in this regard, they are only static snapshots of a momentary event. Here we describe a superior alternative fluorescence microscopy approach to assess the feasibility of investigating nanoparticle-cell interactions in the mouse lung live and over time at nanometer resolution. We applied fluorescent lung tumor cells and Barium-based fluorescently labeled nanoparticles to nude mice or to CD68-EGFP transgenic mice for visualization of the monocyte-macrophage lineage. Shortly before imaging, fluorescently labeled lectin was intravenously injected for staining of the blood vessels. The lung was filled ex vivo with 1% agarose and individual lung lobes were imaged over time using a confocal microscope with Airyscan technology. Time series demonstrate that live cell imaging of lung lobes can be performed for at least 4 h post mortem. Time-lapse movies illustrate the dynamics of the nanoparticles within the pulmonary circulation and their uptake by immune cells. Moreover, the exchange of nanoparticle material between cancer cells was observed over time. Fluorescent monocytes in lungs of CD68-EGFP transgenic mice could be visualized within blood vessels in the process of interaction with tumor cells and nanoparticles. This high resolution ex vivo live cell imaging approach provides an excellent 4D tool to obtain valuable information on the behavior of tumor and immune cells at first encounter with nanoparticles and may contribute to the understanding of how nanoparticles interact with cells supporting the development of therapeutic strategies based on nanoparticulate drug delivery systems.

X-ray based virtual histology allows guided sectioning of heavy ion stained murine lungs for histological analysis.

Examination of histological or immunohistochemically stained 2D sections of embedded tissue is one of the most frequently used tools in biomedical research and clinical routine. Since to date, targeted sectioning of specific regions of interest (ROI) in the sample is not possible, we aimed at developing a guided sectioning approach based on x-ray 3D virtual histology for heavy ion stained murine lung samples. For this purpose, we increased the contrast to noise ratio of a standard benchtop microCT by 5-10-fold using free-propagation phase contrast imaging and thus substantially improved image quality. We then show that microCT 3D datasets deliver more precise anatomical information and quantification of the sample than traditional histological sections, which display deformations of the tissue. To quantify these deformations caused by sectioning we developed the "Displacement Index (DI)", which combines block-matching with the calculation of the local mutual information. We show that the DI substantially decreases when a femtosecond laser microtome is used for sections as opposed to a traditional microtome. In conclusion, our microCT based virtual histology approach can be used as a supplement and a guidance tool for traditional histology, providing 3D measurement capabilities and offering the ability to perform sectioning directly at an ROI.

Therapeutic Fluorescent Hybrid Nanoparticles for Traceable Delivery of Glucocorticoids to Inflammatory Sites.

Treatment of inflammatory disorders with glucocorticoids (GCs) is often accompanied by severe adverse effects. Application of GCs via nanoparticles (NPs), especially those using simple formulations, could possibly improve their delivery to sites of inflammation and therefore their efficacy, minimising the required dose and thus reducing side effects. Here, we present the evaluation of NPs composed of GC betamethasone phosphate (BMP) and the fluorescent dye DY-647 (BMP-IOH-NPs) for improved treatment of inflammation with simultaneous in vivo monitoring of NP delivery. Methods: BMP-IOH-NP uptake by MH-S macrophages was analysed by fluorescence and electron microscopy. Lipopolysaccharide (LPS)-stimulated cells were treated for 48 h with BMP-IOH-NPs (1×10-5-1×10-9 M), BMP or dexamethasone (Dexa). Drug efficacy was assessed by measurement of interleukin 6. Mice with Zymosan-A-induced paw inflammation were intraperitoneally treated with BMP-IOH-NPs (10 mg/kg) and mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI) were treated intranasally with BMP-IOH-NPs, BMP or Dexa (each 2.5 mg/kg). Efficacy was assessed in vivo by paw volume measurements with µCT and ex vivo by measurement of paw weight for Zymosan-A-treated mice, or in the AAI model by in vivo x-ray-based lung function assessment and by cell counts in the bronchoalveolar lavage (BAL) fluid and histology. Delivery of BMP-IOH-NPs to the lungs of AAI mice was monitored by in vivo optical imaging and by fluorescence microscopy. Results: Uptake of BMP-IOH-NPs by MH-S cells was observed during the first 10 min of incubation, with the NP load increasing over time. The anti-inflammatory effect of BMP-IOH-NPs in vitro was dose dependent and higher than that of Dexa or free BMP, confirming efficient release of the drug. In vivo, Zymosan-A-induced paw inflammation was significantly reduced in mice treated with BMP-IOH-NPs. AAI mice that received BMP-IOH-NPs or Dexa but not BMP revealed significantly decreased eosinophil numbers in BALs and reduced immune cell infiltration in lungs. Correspondingly, lung function parameters, which were strongly affected in non-treated AAI mice, were unaffected in AAI mice treated with BMP-IOH-NPs and resembled those of healthy animals. Accumulation of BMP-IOH-NPs within the lungs of AAI mice was detectable by optical imaging for at least 4 h in vivo, where they were preferentially taken up by peribronchial and alveolar M2 macrophages. Conclusion: Our results show that BMP-IOH-NPs can effectively be applied in therapy of inflammatory diseases with at least equal efficacy as the gold standard Dexa, while their delivery can be simultaneously tracked in vivo by fluorescence imaging. BMP-IOH-NPs thus have the potential to reach clinical applications.

X-ray-based lung function measurement reveals persistent loss of lung tissue elasticity in mice recovered from allergic airway inflammation.

Chronic asthma patients experience difficulties even years after the inciting allergen. Although studies in small animal asthma models have enormously advanced progress in uncovering the mechanisms of inception and development of the disease, little is known about the processes involved in the persistence of asthma symptoms in the absence of allergen exposure. Long-term asthma mouse models have so far been scarce or not been able to reproduce the findings in patients. Here we used a common ovalbumin-induced acute allergic airway inflammation mouse model to study lung function and remodeling after a 4-mo recovery period. We show by X-ray-based lung function measurements that the recovered mice continue to show impaired lung function by displaying significant air trapping compared with controls. High-resolution synchrotron phase-contrast computed tomography of structural alterations and diaphragm motion analysis suggest that these changes in pulmonary function are the result of a pronounced loss in lung elasticity. Histology of lung sections confirmed that this is most likely caused by a decrease in elastic fibers, indicating that remodeling can develop or persist independent of acute inflammation and is closely related to a loss in lung function. Our findings demonstrate that this X-ray-based imaging platform has the potential to comprehensively and noninvasively unravel long-term effects in preclinical mouse models of allergic airway inflammation and thus benefits our understanding of chronic asthma.

Transcriptome-wide targets of alternative splicing by RBM4 and possible role in cancer.

This study determined transcriptome-wide targets of the splicing factor RBM4 using Affymetrix GeneChip(®) Human Exon 1.0 ST Arrays and HeLa cells treated with RBM4-specific siRNA. This revealed 238 transcripts that were targeted for alternative splicing. Cross-linking and immunoprecipitation experiments identified 945 RBM4 targets in mouse HEK293 cells, 39% of which were ascribed to "alternative splicing" by in silico pathway analysis. Mouse embryonic stem cells transfected with Rbm4 siRNA hairpins exhibited reduced colony numbers and size consistent with involvement of RBM4 in cell proliferation. RBM4 cDNA probing of a cancer cDNA array involving 18 different tumor types from 13 different tissues and matching normal tissue found overexpression of RBM4 mRNA (p<0.01) in cervical, breast, lung, colon, ovarian and rectal cancers. Many RBM4 targets we identified have been implicated in these cancers. In conclusion, our findings reveal transcriptome-wide targets of RBM4 and point to potential cancer-related targets and mechanisms that may involve RBM4.

Tracking of Inhaled Near-Infrared Fluorescent Nanoparticles in Lungs of SKH-1 Mice with Allergic Airway Inflammation.

Molecular imaging of inflammatory lung diseases, such as asthma, has been limited to date. The recruitment of innate immune cells to the airways is central to the inflammation process. This study exploits these cells for imaging purposes within the lung, using inhaled polystyrene nanoparticles loaded with the near-infrared fluorescence dye Itrybe (Itrybe-NPs). By means of in vivo and ex vivo fluorescence reflectance imaging of an ovalbumin-based allergic airway inflammation (AAI) model in hairless SKH-1 mice, we show that subsequent to intranasal application of Itrybe-NPs, AAI lungs display fluorescence intensities significantly higher than those in lungs of control mice for at least 24 h. Ex vivo immunofluorescence analysis of lung tissue demonstrates the uptake of Itrybe-NPs predominantly by CD68(+)CD11c(+)ECF-L(+)MHCII(low) cells, identifying them as alveolar M2 macrophages in the peribronchial and alveolar areas. The in vivo results were validated by confocal microscopy, overlapping tile analysis, and flow cytometry, showing an amount of Itrybe-NP-containing macrophages in lungs of AAI mice significantly larger than that in controls. A small percentage of NP-containing cells were identified as dendritic cells. Flow cytometry of tracheobronchial lymph nodes showed that Itrybe-NPs were negligible in lung draining lymph nodes 24 h after inhalation. This imaging approach may advance preclinical monitoring of AAI in vivo over time and aid the investigation of the role that macrophages play during lung inflammation. Furthermore, it allows for tracking of inhaled nanoparticles and can hence be utilized for studies of the fate of potential new nanotherapeutics.

Non-invasive optical imaging of eosinophilia during the course of an experimental allergic airways disease model and in response to therapy.

BACKGROUND: Molecular imaging of lung diseases, including asthma, is limited and either invasive or non-specific. Central to the inflammatory process in asthma is the recruitment of eosinophils to the airways, which release proteases and proinflammatory factors and contribute to airway remodeling. The aim of this study was to establish a new approach to non-invasively assess lung eosinophilia during the course of experimental asthma by combining non-invasive near-infrared fluorescence (NIRF) imaging with the specific detection of Siglec-F, a lectin found predominantly on eosinophils. METHODOLOGY/PRINCIPAL FINDINGS: An ovalbumin (OVA)-based model was used to induce asthma-like experimental allergic airway disease (EAAD) in BALB/c mice. By means of a NIRF imager, we demonstrate that 48 h-72 h after intravenous (i.v.) application of a NIRF-labeled anti-Siglec-F antibody, mice with EAAD exhibited up to 2 times higher fluorescence intensities compared to lungs of control mice. Furthermore, average lung intensities of dexamethasone-treated as well as beta-escin-treated mice were 1.8 and 2 times lower than those of untreated, EAAD mice, respectively and correlated with the reduction of cell infiltration in the lung. Average fluorescence intensities measured in explanted lungs confirmed the in vivo findings of significantly higher values in inflamed lungs as compared to controls. Fluorescence microscopy of lung cryosections localized the i.v. applied NIRF-labeled anti-Siglec-F antibody predominantly to eosinophils in the peribronchial areas of EAAD lungs as opposed to control lungs. CONCLUSION/SIGNIFICANCE: We show that monitoring the occurrence of eosinophils, a prominent feature of allergic asthma, by means of a NIRF-labeled antibody directed against Siglec-F is a novel and powerful non-invasive optical imaging approach to assess EAAD and therapeutic response in mice over time.

ZRANB2 localizes to supraspliceosomes and influences the alternative splicing of multiple genes in the transcriptome.

Alternative splicing is a major source of protein diversity in humans. The human splicing factor zinc finger, Ran-binding domain containing protein 2 (ZRANB2) is a splicing protein whose specific endogenous targets are unknown. Its upregulation in grade III ovarian serous papillary carcinoma could suggest a role in some cancers. To determine whether ZRANB2 is part of the supraspliceosome, nuclear supernatants from human embryonic kidney 293 cells were prepared and then fractioned on a glycerol gradient, followed by Western blotting. The same was done after treatment with a tyrosine kinase to induce phosphorylation. This showed for the first time that ZRANB2 is part of the supraspliceosome, and that phosphorylation affects its subcellular location. Studies were then performed to understand the splicing targets of ZRANB2 at the whole-transcriptome level. HeLa cells were transfected with a vector containing ZRANB2 or with a vector-only control. RNA was extracted, converted to cDNA and hybridized to Affymetrix GeneChip(®) Human Exon 1.0 ST Arrays. At the FDR ≤1.3 significance level we found that ZRANB2 influenced the alternative splicing of primary transcripts of CENTB1, WDR78, C10orf18, CABP4, SMARCC2, SPATA13, OR4C6, ZNF263, CAPN10, SALL1, ST18 and ZP2. Several of these have been implicated in tumor development. In conclusion ZRANB2 is part of the supraspliceosome and causes differential splicing of numerous primary transcripts, some of which might have a role in cancer.

Resveratrol, by modulating RNA processing factor levels, can influence the alternative splicing of pre-mRNAs.

Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 µM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 µg/ml and 50 µg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.

The molecular basis of longevity, and clinical implications.

The determinants of length of life are multifactorial and involve complex processes, most of which are not as yet understood completely. Tremendous advances have, however, been made in recent times in understanding some of the key molecular mechanisms that influence ageing and lifespan. Herein we highlight many of the more important findings and their potential clinical implications. Most of the intracellular factors involved in the ageing process, such as members of the sirtuin family, as well as insulin and insulin-like growth factor-I and their genes, are part of interconnected pathways. The manipulation of these and other genes in animal models can increase or decrease lifespan. Transcriptional and post-transcriptional regulatory mechanisms, some of which involve microRNAs, as well as modifications to chromatin and histones, can influence longevity. A decline in the function of stem cells might also be responsible for some aspects of mammalian ageing. Calorie restriction, polyphenols such as resveratrol, rapamycin, spermidine and angiotensin I converting enzyme inhibitor, are able to increase lifespan by modulation of branches of the longevity pathways. Molecular genetic studies of long-lived subjects have identified several potential candidate genes, but genetic research on ageing is in its infancy. Large genome-wide association studies should provide insights. Although new biomarkers for ageing and health, such as ones that might reveal telomere dysfunction, have been described, advances in the genetics and molecular biology of longevity will require interdisciplinary approaches if the much-hoped for success in alleviating the diseases of ageing, and an extension of both lifespan and healthspan is to be achieved.

Resveratrol: cellular actions of a potent natural chemical that confers a diversity of health benefits.

Resveratrol is a polyphenolic flavonoid with potent antioxidant activity. It is found in a diversity of plants, notably berry fruit, and is attracting increased attention due to its health benefits, especially in common age-related diseases such as cancer, type 2 diabetes, cardiovascular disease, and neurological conditions. Resveratrol has positive effects on metabolism and can increase the lifespan of various organisms. Its effects arise from its capacity to interact with multiple molecular targets involved in diverse intracellular pathways. Most well known is the ability of resveratrol to activate sirtuins, a class of NAD(+)-dependent deacetylases that affect multiple transcription factors and other protein targets. More potent sirtuin activators have now been discovered by large-scale screening programs. Resveratrol and the new compounds are the subject of clinical trials to determine their consumer safety and suitability for the prevention and treatment of most common diseases of aging.

RBM4: a multifunctional RNA-binding protein.

RBM4, also known as Lark, was described initially as having a role in circadian rhythm control in Drosophila. In the last 5 years data have emerged from studies of mammalian cells. It is now clear that RBM4 is an RNA-binding protein involved in diverse cellular processes that include alternative splicing of pre-mRNA, translation, and RNA silencing. Its structure, similar to other RNA-binding proteins, contains two RNA recognition motifs and a CCHC-type zinc finger. Here we review current information about the function of RBM4 and its localization within the cell. We then speculate about its possible relationship to disease.

Hormesis as a pro-healthy aging intervention in human beings?

Hormesis is a phenomenon in which adaptive responses to low doses of otherwise harmful factors (also called mild stressors) make cells and organisms more robust. Aging is a complex and poorly understood process. This review explores the positive effects of hormesis on aging in animal models and human cell cultures, and discusses whether it might apply to humans. As an example, repeated mild heat stress confers anti-aging benefits to normal human cells in culture. Calorie restriction and xenohormetic compounds such as resveratrol, in large part via activation of sirtuins, decrease risk of common age-related conditions, such as cancer, cardiovascular disease, type 2 diabetes, and neurological diseases, so lengthening lifespan. Mild stressors and xenohormetic dietary components have diverse molecular targets and affect many pathways. Despite experimental advances in aging research, findings in humans are still quite limited. Moderate-intensity exercise, weight management and healthy diet ameliorate diseases of aging to increase lifespan and this could involve hormesis.