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Age-related T cell dysfunction can lead to failure of immune tolerance mechanisms, resulting in aberrant T cell-driven cytokine and cytotoxic responses that ultimately cause tissue damage. In this Review, we discuss the role of T cells in the onset and progression of age-associated conditions, focusing on cardiovascular disorders, metabolic dysfunction, neuroinflammation and defective tissue repair and regeneration. We present different mechanisms by which T cells contribute to inflammageing and might act as modulators of age-associated diseases, including through enhanced pro-inflammatory and cytotoxic activity, defective clearance of senescent cells or regulation of the gut microbiota. Finally, we propose that 'resetting' immune system tolerance or targeting pathogenic T cells could open up new therapeutic opportunities to boost resilience to age-related diseases.

Simple Summary Photodynamic therapy (PDT) is used for the treatment of in situ cutaneous squamous cell carcinoma (cSCC), the second most common form of skin cancer, as well as for its precancerous form, actinic keratosis. However, relapses after the treatment can occur. Transforming growth factor β1 (TGFβ1) produced by cancer-associated fibroblasts (CAFs) in the tumor microenvironment has been pointed as a key player in the development of cSCC resistance to other therapies, such as chemotherapy. Here, we demonstrate that TGFβ1 produced by CAFs isolated from patients with cSCC can drive resistance to PDT in SCC cells. This finding opens up novel possibilities for strategy optimization in the field of cSCC resistance to PDT and highlights CAF-derived TGFβ1 as a potential target to improve the efficacy of PDT. Abstract As an important component of tumor microenvironment, cancer-associated fibroblasts (CAFs) have lately gained prominence owing to their crucial role in the resistance to therapies. Photodynamic therapy (PDT) stands out as a successful therapeutic strategy to treat cutaneous squamous cell carcinoma. In this study, we demonstrate that the transforming growth factor β1 (TGFβ1) cytokine secreted by CAFs isolated from patients with SCC can drive resistance to PDT in epithelial SCC cells. To this end, CAFs obtained from patients with in situ cSCC were firstly characterized based on the expression levels of paramount markers as well as the levels of TGFβ1 secreted to the extracellular environment. On a step forward, two established human cSCC cell lines (A431 and SCC13) were pre-treated with conditioned medium obtained from the selected CAF cultures. The CAF-derived conditioned medium effectively induced resistance to PDT in A431 cells through a reduction in the cell proliferation rate. This resistance effect was recapitulated by treating with recombinant TGFβ1 and abolished by using the SB525334 TGFβ1 receptor inhibitor, providing robust evidence of the role of TGFβ1 secreted by CAFs in the development of resistance to PDT in this cell line. Conversely, higher levels of recombinant TGFβ1 were needed to reduce cell proliferation in SCC13 cells, and no induction of resistance to PDT was observed in this cell line in response to CAF-derived conditioned medium. Interestingly, we probed that the comparatively higher intrinsic resistance to PDT of SCC13 cells was mediated by the elevated levels of TGFβ1 secreted by this cell line. Our results point at this feature as a promising biomarker to predict both the suitability of PDT and the chances to optimize the treatment by targeting CAF-derived TGFβ1 in the road to a more personalized treatment of particular cSCC tumors.

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology. pre-print 245 KB

Traditional methods of localizing and quantifying the presence of pathogenic microorganisms in living experimental animal models of infections have mostly relied on sacrificing the animals, dissociating the tissue and counting the number of colony forming units. However, the discovery of several varieties of the light producing enzyme, luciferase, and the genetic engineering of bacteria, fungi, parasites and mice to make them emit light, either after administration of the luciferase substrate, or in the case of the bacterial lux operon without any exogenous substrate, has provided a new alternative. Dedicated bioluminescence imaging (BLI) cameras can record the light emitted from living animals in real time allowing non-invasive, longitudinal monitoring of the anatomical location and growth of infectious microorganisms as measured by strength of the BLI signal. BLI technology has been used to follow bacterial infections in traumatic skin wounds and burns, osteomyelitis, infections in intestines, Mycobacterial infections, otitis media, lung infections, biofilm and endodontic infections and meningitis. Fungi that have been engineered to be bioluminescent have been used to study infections caused by yeasts (Candida) and by filamentous fungi. Parasitic infections caused by malaria, Leishmania, trypanosomes and toxoplasma have all been monitored by BLI. Viruses such as vaccinia, herpes simplex, hepatitis B and C and influenza, have been studied using BLI. This rapidly growing technology is expected to continue to provide much useful information, while drastically reducing the numbers of animals needed in experimental studies.

Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are cell-derived membranous structures that were originally catalogued as a way of releasing cellular waste products. Since the discovery of their function in intercellular communication as carriers of proteins, lipids, and DNA and RNA molecules, numerous therapeutic approaches have focused on the use of EVs, in part because of their minimized risk compared to cell-based therapies. The skin is the organ with the largest surface in the body. Besides the importance of its body barrier function, much attention has been paid to the skin in regenerative medicine because of its cosmetic aspect, which is closely related to disorders affecting pigmentation and the presence or absence of hair follicles. The use of exosomes in therapeutic approaches for cutaneous wound healing has been reported and is briefly reviewed here. However, less attention has been paid to emerging interest in the potential capacity of EVs as modulators of hair follicle dynamics. Hair follicles are skin appendices that mainly comprise an epidermal and a mesenchymal component, with the former including a major reservoir of epithelial stem cells but also melanocytes and other cell types. Hair follicles continuously cycle, undergoing consecutive phases of resting, growing, and regression. Many biomolecules carried by EVs have been involved in the control of the hair follicle cycle and stem cell function. Thus, investigating the role of either naturally produced or therapeutically delivered EVs as signaling vehicles potentially involved in skin homeostasis and hair cycling may be an important step in the attempt to design future strategies towards the efficient treatment of several skin disorders., Fondo de Investigación Sanitaria, Instituto de Salud Carlos III (CP 14/00219), Fondo Europeo de Desarrollo Regional (FEDER), H2020-EU.1.1.—European Research Council (ERC-2016-StG 715322-EndoMitTalk), and Instituto de Salud Carlos III (FIS16/188). E.C. was supported by the Atracción de Talento Investigador grant 2017-T2/BMD-5766 (Comunidad de Madrid and Universidad Autónoma de Madrid). G.S.-H. was funded by an FPI grant (Universidad Autónoma de Madrid). M.M. was supported by the Miguel Servet program. info:eu-repo/grantAgreement/EC/H2020/715322

Licensee MDPI, Basel, Switzerland. Photodynamic therapy (PDT) constitutes a cancer treatment modality based on the administration of a photosensitizer, which accumulates in tumor cells. The subsequent irradiation of the tumoral area triggers the formation of reactive oxygen species responsible for cancer cell death. One of the compounds approved in clinical practice is methyl-aminolevulinate (MAL), a protoporphyrin IX (PpIX) precursor intermediate of heme synthesis. We have identified the mitotic catastrophe (MC) process after MAL-PDT in HeLa human carcinoma cells. The fluorescence microscopy revealed that PpIX was located mainly at plasma membrane and lysosomes of HeLa cells, although some fluorescence was also detected at endoplasmic reticulum and Golgi apparatus. Cell blockage at metaphase-anaphase transition was observed 24 h after PDT by phase contrast microscopy and flow cytometry. Mitotic apparatus components evaluation by immunofluorescence and Western blot indicated: multipolar spindles and disorganized chromosomes in the equatorial plate accompanied with dispersion of centromeres and alterations in aurora kinase proteins. The mitotic blockage induced by MAL-PDT resembled that induced by two compounds used in chemotherapy, taxol and nocodazole, both targeting microtubules. The alterations in tumoral cells provided evidence of MC induced by MAL-PDT, resolving mainly by apoptosis, directly or through the formation of multinucleate cells, This research was funded by Spanish grants from Instituto de Salud Carlos III MINECO and Feder Funds (FIS PI15/00974 and PI18/00708)

Photodynamic Therapy (PDT) with methyl-aminolevulinate acid (MAL-PDT) is being used for the treatment of Basal cell carcinoma (BCC), but recurrences have been reported. In this work, we have evaluated resistance mechanisms to MAL-PDT developed by three BCC cell lines (ASZ, BSZ and CSZ), derived from mice on a ptch+/− background and with or without p53 expression, subjected to 10 cycles of PDT (10thG). The resistant populations showed mesenchymal-like structure and diminished proliferative capacity and size compared to the parental (P) cells. The resistance was dependent on the production of the endogenous photosensitiser protoporphyrin IX in the CSZ cell line and on its cellular localisation in ASZ and BSZ cells. Moreover, resistant cells expressing the p53 gene presented lower proliferation rate and increased expression levels of N-cadherin and Gsk3β (a component of the Wnt/β-catenin pathway) than P cells. In contrast, 10thG cells lacking the p53 gene showed lower levels of expression of Gsk3β in the cytoplasm and of E-cadherin and β-catenin in the membrane. In addition, resistant cells presented higher tumorigenic ability in immunosuppressed mice. Altogether, these results shed light on resistance mechanisms of BCC to PDT and may help to improve the use of this therapeutic approach.

The hair follicle is a biological oscillator that alternates growth, regression, and rest phases driven by the sequential activation of the proliferation/differentiation programs of resident stem cell populations. The activation of hair follicle stem cell niches and subsequent entry into the growing phase is mainly regulated by Wnt/β-catenin signalling, while regression and resting phases are mainly regulated by Tgf-β/Bmp/Smad activity. A major question still unresolved is the nature of the molecular switch that dictates the coordinated transition between both signalling pathways. Here we have focused on the role of Endoglin (Eng), a key co-receptor for members of the Tgf-β/Bmp family of growth factors. Using an Eng haploinsufficient mouse model, we report that Eng is required to maintain a correct follicle cycling pattern and for an adequate stimulation of hair follicle stem cell niches. We further report that β-catenin binds to the Eng promoter depending on Bmp signalling. Moreover, we show that β-catenin interacts with Smad4 in a Bmp/Eng-dependent context and both proteins act synergistically to activate Eng promoter transcription. These observations point to the existence of a growth/rest switching mechanism in the hair follicle that is based on an Eng-dependent feedback cross-talk between Wnt/β-catenin and Bmp/Smad signals., This work was supported by grants from Ministerio de Economía y Competitividad of Spain (RTC-2014-2626-1 to J.E., SAF2013-46183-R to M.Q., and SAF2013-43421-R to C.B.), Comunidad de Madrid (S2010/BMD-2359, SkinModel to J.E. and M.Q.), Instituto de Salud Carlos III (PI15/01458 to J.E.), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to C.B.) financed jointly by the European Regional Development Funds (FEDER). E.C. and M.I.C. were supported by Spanish MECD-FPU and UAM-FPI fellowships, respectively.

The hair follicle is a biological oscillator that sequentially alternates phases of growth, regression and rest. The cyclic activity of this oscillator is driven by a stem cell population regulated by nurse cells and located in the hair follicle bulge region. As a whole, this morpho‐functional structure constitutes a suitable model to study the dynamics of the stem cell niches that actually maintain homeostatic and regeneration processes during development and in adult tissues. In the hair follicle, the activation of the stem cell niche and subsequent entry into the growing phase is mainly regulated by Wnt/β‐catenin signalling, while regression and resting phases are mainly regulated by Tgf‐β/Bmp/Smad activity. A major question still unresolved is the nature of the molecular switch that dictates the transition between both signalling pathways during the hair follicle growth cycle. Here we have focused on the role of Endoglin (Eng), a key co‐receptor for members of the Tgf‐β/Bmp family of growth factors. Using a haploinsufficient mouse model we have demonstrated that Eng is required for the stimulation of the hair follicle stem cell niche and to maintain a correct follicle cycling pattern. We further report that β‐catenin binds to the Eng promoter depending on Bmp signalling. Moreover, β‐catenin interacts with Smad4 in a Bmp/Eng dependent context and both proteins act synergistically to activate Eng promoter transcription. These observations point to the existence of a growth/rest switching mechanism in the hair follicle that is based on an Eng‐dependent feedback crosstalk between Wnt/β‐ catenin and Bmp/Smad signals.

et al., The tremendous development of nanotechnology is bringing us closer to the dream of clinical application of nanoparticles in photothermal therapies of tumors. This requires the use of specific nanoparticles that must be highly biocompatible, efficient light-to-heat converters and fluorescent markers. Temperature reading by the heating nanoparticles during therapy appears of paramount importance to keep at a minimum the collateral damage that could arise from undesirable excessive heating. In this work, this thermally controlled therapy is possible by using Nd3+ ion-doped LaF3 nanocrystals. Because of the particular optical features of Nd3+ ions at high doping concentrations, these nanoparticles are capable of in vivo photothermal heating, fluorescent tumor localization and intratumoral thermal sensing. The successful photothermal therapy experiments here presented highlight the importance of controlling therapy parameters based on intratumoral temperature measurements instead of on the traditionally used skin temperature measurements. In fact, signifi cant differences between intratumoral and skin temperatures do exist and could lead to the appearance of excessive collateral damage. These results open a new avenue for the real application of nano-particle-based photothermal therapy at clinical level., The work was supported by the Ministerio de Innovación y Ciencia of Spain under projects MAT2010–16161 and MAT2013–47395-C4–1-R. P.H. thanks the Spanish Ministerio de Economia y Competitividad (MINECO) for a Juan de la Cierva grant. The authors also thank the Brazilian agencies FAPEAL-Fundação de Amparo a Pesquisa do Estado de Alagoas (Project PRONEX 2009–09–006), FINEP (Financiadora de Estudos e Projetos), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) through Grant INCT NANO(BIO)SIMES, and CAPES (Coordenadoria de Aperfeiçoamento de Pessoal de Ensino Superior). D.J. (Pesquisador Visitante Especial (PVE)-CAPES) thanks the CAPES by means of the Project PVE number A077/2013. B. del Rosal thanks Universidad Autónoma de Madrid for an FPI grant.

There is a growing interest in the physiological roles of reactive oxygen species (ROS) as essential components of molecular mechanisms regulating key cellular processes, including proliferation, differentiation and apoptosis. This interest has fostered the development of new molecular tools to localize and quantify ROS production in cultured cells and in whole living organisms. An equally important but often neglected aspect in the study of ROS biology is the development of accurate procedures to introduce a ROS source in the biological system under study. At present, this experimental requirement is solved in most cases by an external and systemic administration of ROS, usually hydrogen peroxide. We have previously shown that a photodynamic treatment based on the endogenous photosensitizer protoporphyrin IX and further irradiation of the target with adequate light source can be used to transiently switch on an in situ ROS production in human cultured keratinocytes and in mouse skin in vivo. Using this approach we reported that qualitatively low levels of ROS can activate cell proliferation in cultured cells and promote a transient and reversible hyperproliferative response in the skin, particularly, in the hair follicle stem cell niche, promoting physiological responses like acceleration of hair growth and supporting the notion that a local and transient ROS production can regulate stem cell function and tissue homeostasis in a whole organism. Our principal aim here is to provide a detailed description of this experimental methodology as a useful tool to investigate physiological roles for ROS in vivo in different experimental systems., This work has been supported by grants from Ministerio de Economía y Competitividad of Spain (RTC-2014-2626-1 to JE), Comunidad de Madrid (S2010/BMD-2359, SkinModel to JE and AJ), and Instituto de Salud Carlos III (PI15/01458 to JE and PI15/00974 to AJ, Plan Estatal de I+D+i 2013-2016) financed jointly by the European Regional Development Funds (FEDER). EC was supported by Spanish MECD-FPU and Alfonso Martín Escudero Foundation fellowships. MIC was supported by an UAM-FPI fellowship. AB-C is supported by the Marie Curie AIAS-COFUND program (609033).

Nowadays, one of the most exciting applications of nanotechnology in biomedicine is the development of localized, noninvasive therapies for diverse diseases, such as cancer. Among them, nanoparticle-based photothermal therapy (PTT), which destroys malignant cells by delivering heat upon optical excitation of nanoprobes injected into a living specimen, is emerging with great potential. Two main milestones that must be reached for PTT to become a viable clinical treatment are deep penetration of the triggering optical excitation and real-time accurate temperature monitoring of the ongoing therapy, which constitutes a critical factor to minimize collateral damage. In this work, a yet unexplored capability of near-infrared emitting semiconductor nanocrystals (quantum dots, QDs) is demonstrated. Temperature self-monitored ­QD-based PTT is presented for the first time using PbS/CdS/ZnS QDs emitting in the second biological window. These QDs are capable of acting, simultaneously, as photothermal agents (heaters) and high-resolution fluorescent thermal sensors, making it possible to achieve full control over the intratumoral temperature increment during PTT. The differences observed between intratumoral and surface temperatures in this comprehensive investigation, through different irradiation conditions, highlight the need for real-time control of the intratumoral temperature that allows for a dynamic adjustment of the treatment conditions in order to maximize the efficacy of the therapy., This project was supported by the Spanish Ministerio de Economía y Competitividad under project and MAT2013-47395-C4-1-R. B.d.R. thanks Universidad Autónoma de Madrid for an FPI grant. F.R. acknowledges scholarship support from the Fonds de recherche du Québec—Nature et technologies (FRQNT) under the Programme de Bourses d’Excellence (Merit Scholarship Program for Foreign Students). A.B. is thankful to the Canadian Institutes of Health Research and the Breast Cancer Society of Canada (CIHR-BCSC), for postdoctoral funding granted to him through an Eileen Iwanicki Fellowship in Breast Cancer Imaging. F.V. is grateful to the Natural Sciences and Engineering Research Council (NSERC) of Canada, FRQNT, and the Canada Foundation for Innovation (CFI) for supporting his research program. F.V. also gratefully acknowledges the Fondation Sibylla Hesse. D.M. is grateful for the financial support from NSERC, FRQNT, CFI, and the Centre Québécois sur les Matériaux Fonctionnels (CQMF), Canada

et al., Nanoparticle-mediated photothermal therapy (NP-PTT) constitutes a flexible, highly selective, cost effective, and accurate tool for cancer treatment alone or in combination with other therapies such as radiotherapy or chemotherapy. The future application of NP-PTT in real life mainly depends on the design and synthesis of novel multifunctional nanoparticles that could overcome the current limitations of NP-PTT such as limited penetration depth and absence of therapy control. In this work, ultrasmall (≈2.4 nm) NdVO stoichiometric (100% constituent Nd ions) nanoparticles are reported, which are capable of in vivo sub-tissue localized heating under 808 nm optical excitation while providing, simultaneously, the possibility of high penetration near-infrared fluorescence imaging. Ultrasmall stoichiometric NdVO nanoparticles have evidenced a superior light-to-heat conversion efficiency. This is explained in terms of their large absorption cross-section at 808 nm (consequence of the particular spectroscopic properties of neodymium ions in NdVO and of the high neodymium content) as well as to their ultrasmall size that leads to large nonradiative decay rates. Results included in this work introduce ultrasmall, NdVO stoichiometric nanoparticles to the scientific community as multifunctional photothermal agents that could be considered as an alternative to traditional systems such as metallic, organic, or carbon-based nanoparticles., This project was supported by the Spanish Ministerio de Economía y Competitividad under project and MAT2013-47395-C4-1-R. The authors acknowledge support from Instituto de Salud Carlos III (FIS, PI15/00974) and Comunidad Autónoma de Madrid (CAM, Skin-Model, S2010/BMD-2359). B.d.R. thanks Universidad Autónoma de Madrid for an FPI grant. D.J. and M.D.D. acknowledge fi nancial support of the Ministry of Education, Science, and Technological development of the Republic of Serbia (grant number 45020). G.D. acknowledges the fi nancial support of the Slovenian Research Agency (ARRS) through program no. P2-0148 and project J2-6754. The authors thank COST action CM1403.

Photodynamic therapy using methyl 5-aminolevulinate (MAL) as a precursor of the photosensitizing agent protoporphyrin IX is widely used in clinical practice for the treatment of different pathologies, including cancer. In this therapeutic modality, MAL treatment promotes the forced accumulation of the endogenous photoactive compound protoporphyrin IX in target malignant cells. Subsequent irradiation of treated tissues with an appropriate visible light source induces the production of reactive oxygen species (ROS) that, once accumulated above a critical level, promote cell death. Here we demonstrate that a photodynamic treatment with low MAL concentrations can be used to promote a moderate production of endogenous ROS, which efficiently stimulates cell growth in human immortalized keratinocytes (HaCaT). We also show that this proliferative response requires Src kinase activity and is associated to a transient induction of cyclin D1 expression. Taken together, these results demonstrate for the first time that a combination of light and a photoactive compound can be used to modulate cell cycle progression through Src kinase activation and that a moderate intracellular increase of photogenerated ROS efficiently stimulates cell proliferation. © 2011 Elsevier GmbH., This work was supported by Grants SAF2008-00609 (Ministerio de Ciencia e Innovación, Spain) to J. Espada and FIS PS09/01099 (Ministerio de Sanidad; Spain) to A. Juarranz. A. Blázquez-Castro and E. Carrasco are recipients of FPU fellowships (Ministerio de Ciencia e Innovación, Spain). J. Espada is a hired investigator under the Programa Ramón y Cajal (Ministerio de Ciencia e Innovación, Spain).

Regenerative properties of skin decrease with age, and thus, the search for substances that minimize cutaneous ageing has increased in the last few years. The secretion of the mollusc Cryptomphalus Aspersa (SCA) is a natural product that bears regenerative properties when applied topically. The purpose of this work is to study the in vitro effects of SCA on cell proliferation and migration, as well as on cell-cell (E-cadherin and β-catenin) and cell-substrate (vinculin and β1-integrin) adhesion proteins expression, using a human keratinocyte cell line (HaCaT cells) and primary dermal fibroblasts (HF). We tested the effects of SCA on cell proliferation using a colorimetric assay. In addition, SCA-induced changes on cell migration were studied by wound-healing assays. Besides, Western blot and immunofluorescence microscopy were carried out to test the expression of different cell adhesion proteins. We found that SCA promotes proliferation and migration of HaCaT cells in a time- and dose-dependent manner. Moreover, treatment with SCA increases the migratory behaviour and the expression of adhesion molecules in both HaCaT and HF. Finally, SCA also improves cell survival and promotes phosphorylation of FAK and nuclear localization of β-catenin. These results shed light on the molecular mechanisms underlying the regenerative properties of SCA, based on its promoting effect on skin cell migration, proliferation and survival. Moreover, these results support future clinical uses of SCA in the regeneration of wounded tissues.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License.-- et al., Mammalian DNA methyltransferase 1 (DNMT1) is essential for maintaining DNA methylation patterns after cell division. Disruption of DNMT1 catalytic activity results in whole genome cytosine demethylation of CpG dinucleotides, promoting severe dysfunctions in somatic cells and during embryonic development. While these observations indicate that DNMT1-dependent DNA methylation is required for proper cell function, the possibility that DNMT1 has a role independent of its catalytic activity is a matter of controversy. Here, we provide evidence that DNMT1 can support cell functions that do not require the C-terminal catalytic domain. We report that PCNA and DMAP1 domains in the N-terminal region of DNMT1 are sufficient to modulate E-cadherin expression in the absence of noticeable changes in DNA methylation patterns in the gene promoters involved. Changes in E-cadherin expression are directly associated with regulation of ß-catenin-dependent transcription. Present evidence suggests that the DNMT1 acts on E-cadherin expression through its direct interaction with the E-cadherin transcriptional repressor SNAIL1., MEC (SAF2008-00609 to J.E.); MEC (SAF2007-63051 and Consolider CSD2007-00017); EU (MRTN-CT-2004-005428 to A.C.); MEC (SAF2004-07729, Consolider CSD2006-49); EU (FP7-CANCERDIP-2007-200620 to M.E.); MEC (SAF2010-19152 to J.R.). M.E. is an ICREA Research Professor. J.E. is a Ramon y Cajal Program researcher (MICIIN). Funding for open access charge: Consolider CSD2006-49 (Grant of the Spanish Science Governmental Department).

Over the past decade, near-infrared (NIR)-emitting nanoparticles have increasingly been investigated in biomedical research for use as fluorescent imaging probes. Here, high-quality water-dispersible core/shell/shell PbS/CdS/ZnS quantum dots (hereafter QDs) as NIR imaging probes fabricated through a rapid, cost-effective microwave-assisted cation exchange procedure are reported. These QDs have proven to be water dispersible, stable, and are expected to be nontoxic, resulting from the growth of an outer ZnS shell and the simultaneous surface functionalization with mercaptopropionic acid ligands. Care is taken to design the emission wavelength of the QDs probe lying within the second biological window (1000-1350 nm), which leads to higher penetration depths because of the low extinction coefficient of biological tissues in this spectral range. Furthermore, their intense fluorescence emission enables to follow the real-time evolution of QD biodistribution among different organs of living mice, after low-dose intravenous administration. In this paper, QD platform has proven to be capable (ex vivo and in vitro) of high-resolution thermal sensing in the physiological temperature range. The investigation, together with the lack of noticeable toxicity from these PbS/CdS/ZnS QDs after preliminary studies, paves the way for their use as outstanding multifunctional probes both for in vitro and in vivo applications in biomedicine. Low-dose in vivo near-infrared (NIR) fluorescence imaging is achieved by using carefully designed PbS/CdS/ZnS quantum dots (QDs), intensely emitting within the second biological window (1000-1350 nm). Moreover, preliminary studies both in vitro and in vivo have proven the lack of noticeable toxicity of these QDs. As an additional advantage, this NIR-fluorescence imaging platform has demonstrated useful multifunctionality, thus being capable, both ex vivo and in vitro, of high-resolution thermal sensing in the physiological temperature range., A.B., F.R., and E.C. contributed equally to this work. A.B. thanks the Fonds de recherche du Québec-Nature et Technologies (FRQNT) and the Canadian Institutes of Health Research–Breast Cancer Society of Canada (CIHR-BCSC) for postdoctoral funding through the Programme de Bourses d’Excellence and an Eileen Iwanicki Fellowship in Breast Cancer Imaging, respectively. F.R. greatly appreciates financial support from the Merit Scholarship Program for Foreign Students from the Ministére de L’Education, du Loisir et du Sport du Québec (MELS). F.V. and D.M. are grateful to the Natural Sciences and Engineering Research Council of Canada (NSERC) and FRQNT for funding. F.V. wholeheartedly thanks the Foundation Sibylla Hesse for supporting his research. D.M. also thanks the Quebec Center for Functional Materials for support. This work was also supported by Spanish Ministerio de Economia y Competitividad under projects MAT2010–16161 and MAT2013–47395-C4–1-R.

Novel approaches for high contrast, deep tissue, in vivo fluorescence biomedical imaging are based on infrared-emitting nanoparticles working in the so-called second biological window (1,000–1,400 nm). This allows for the acquisition of high resolution, deep tissue images due to the partial transparency of tissues in this particular spectral range. In addition, the optical excitation with low energy (infrared) photons also leads to a drastic reduction in the contribution of autofluorescence to the in vivo image. Nevertheless, as is demonstrated here, working solely in this biological window does not ensure a complete removal of autofluorescence as the specimen’s diet shows a remarkable infrared fluorescence that extends up to 1,100 nm. In this work, we show how the 1,340 nm emission band of Nd3+ ions embedded in SrF2 nanoparticles can be used to produce autofluorescence free, high contrast in vivo fluorescence images. It is also demonstrated that the complete removal of the food-related infrared autofluorescence is imperative for the development of reliable biodistribution studies.

The role of reactive oxygen species (ROS) in the regulation of hair follicle (HF) cycle and skin homeostasis is poorly characterized. ROS have been traditionally linked to human disease and aging, but recent findings suggest that they can also have beneficial physiological functions in vivo in mammals. To test this hypothesis, we transiently switched on in situ ROS production in mouse skin. This process activated cell proliferation in the tissue and, interestingly, in the bulge region of the HF, a major reservoir of epidermal stem cells, promoting hair growth, as well as stimulating tissue repair after severe burn injury. We further show that these effects were associated with a transient Src kinase phosphorylation at Tyr416 and with a strong transcriptional activation of the prolactin family 2 subfamily c of growth factors. Our results point to potentially relevant modes of skin homeostasis regulation and demonstrate that a local and transient ROS production can regulate stem cell and tissue function in the whole organism., JE was supported by Spanish MINECO grants SAF11-23493 and RTC-2014-2626-1 and Comunidad Autónoma de Madrid grant SkinModel CAM S10/BMD-2359. DV and MRH were supported by US NIH grant R01AI050875. AJ was supported by Spanish MINECO grant FIS PI12/01253 and CAM S10/BMD-2359. JCS was supported by Spanish MCINN grant CTQ2010-20870-C03-03. EC and MIC were supported by Spanish MECD-FPU and UAM-FPI fellowships, respectively.

SynopsisBackground The search of substances that minimize cutaneous ageing has increased in the last few years. Previous studies have described the regenerative properties of the secretion of the mollusc Cryptomphalus aspersa (C. aspersa) when applied topically. Objective We evaluate the in vitro effects of a new product derived from the eggs of C. aspersa, IFC-CAF, on cell proliferation, migration, distribution of cytoskeletal proteins, production of extracellular components as well as its ability to prevent cutaneous ageing because of intrinsic or extrinsic factors (exposure to UVB) by determination of ageing markers. Methods We have used the human keratinocyte cell line (HaCaT cells), primary dermal fibroblasts (HDF) and senescent dermal fibroblasts (SHDF). The effects of the compound on cell proliferation and on the cell cycle were determined by the MTT colorimetric assay, estimation of total protein and/or trypan blue test and by flow cytometry, respectively. We also studied cell migration using the wound-healing migration assay, whereas ELISA assays, Western Blot and immunofluorescence microscopy were carried out to test the expression of proteins related to cytoskeleton, extracellular matrix and with ageing. Results We have found that IFC-CAF does not promote proliferation but induces migration of HaCaT, HDF and SHDF in a time- and dose-dependent manner; a better organization of cytoskeletal proteins (F-actin and vimentin) and promotes the production of extracellular components (fibronectin, collagen 1 and MMPs) and the adhesion to cell-substrate vinculin protein. IFC-CAF also prevents cutaneous ageing. The treatment decreases the expression of the ageing-related markers b-Gal, p53 and p16INK4 in SDDF cells, and improves cell survival after UVB irradiation and nuclear repair in HaCaT cells. Conclusion IFC-CAF has regenerative properties and protects against ageing factors being, therefore, a potential therapeutic agent for treating or preventing skin ageing.

DNA labeling in vivo using nucleoside analogues is a current experimental approach to determine cell proliferation rates in cell cultures and tissues. It has also been successfully used to localize adult stem cell niches through the identification of nucleoside label-retaining cells (LRC) in long-term experiments. A major hindrance of this methodology relies on the selection of adequate procedures to quantify the nucleoside analogue content from image data files. Here we propose a simple procedure using Fiji image processing software to accurately calculate nucleoside analogue retaining chromatin/total chromatin (LRC/DAPI) signal ratios in the well-known mouse hair follicle stem cell niche., This work was supported by grants of the Spanish Ministerio de Economía y Competitividad (SAF 11-23493) and the Comunidad Autónoma de Madrid (SkinModel, CAM S10/BMD-2359) to J.E.; E.C and M.I.C. are supported by Ph.D. fellowship grants of the Spanish Ministerio de Educación and Universidad Autónoma de Madrid, respectively.

PMCID: PMC3970436.-- et al., The benzophenothiazinium dye EtNBS has previously been tested as a photosensitizer to mediate photodynamic therapy (PDT). It has been employed to kill cancer cells and microbial cells in vitro and to treat tumors and infections in vivo. We synthesized a panel of derivatives substituted at the 1-position of the benzene ring with electron donating or electron withdrawing groups (amino, acetamido and nitro) and tested their production of reactive oxygen species (ROS) and light-mediated killing of two species of Gram-positive and two species of Gram-negative bacteria. All three compounds showed lower fluorescence, lower yield of ROS and less microbial killing than parent EtNBS, while the order of activity (nitro > amino > acetamido) showed that an electron withdrawing substituent was better than electron donating. To test the hypothesis that 1-substitution distorts the planar structure of the conjugated rings we compared two compounds substituted with N-ethylpropylsulfonamido either at the 1-position or at the 4-position. The 4-isomer was significantly more photoactive than the 1-isomer. We also prepared an EtNBS derivative with a guanidinium group attached to the 5-amino group. This compound had high activity against Gram-negative bacteria due to the extra positive charge. Cellular uptake of the compounds by the four bacterial species was also measured and broadly correlated with activity. These results provided three separate pieces of structure-activity relationship data for antimicrobial photosensitizers based on the EtNBS backbone., This work was supported by US NIH grant R01AI050875 to MRH and US NIH Director's New Innovator Award, grant number DP2OD007096 to CLE.

DNA labeling in vivo using nucleoside analogues is a current experimental approach to determine cell proliferation rates in cell cultures and tissues. It has also been successfully used to localize adult stem cell niches through the identification of nucleoside label-retaining cells (LRC) in long-term experiments. A major hindrance of this methodology relies on the selection of adequate procedures to quantify the nucleoside analogue content from image data files. Here we propose a simple procedure using Fiji image processing software to accurately calculate nucleoside analogue retaining chromatin/total chromatin (LRC/DAPI) signal ratios in the well-known mouse hair follicle stem cell niche.

In mammals, methylation of cytosine C-5 position is a major heritable epigenetic mark on the DNA molecule. Maintenance of proper DNA methylation patterns is a key process during embryo development and in the maintenance of adult tissue homeostasis. The use of experimental procedures based on the chemical modification of cytosine by sodium bisulfite and the development of antibodies recognizing 5mC have essentially contributed to our knowledge on DNA methylation dynamics in normal and disease states. Here we describe standard procedures for bisulfite sequencing, methylation-specific PCR, and 5mC immunodetection using mouse skin and the hair follicle stem cell niche as model tissues.

In mammals, methylation of cytosine C-5 position is a major heritable epigenetic mark on the DNA molecule. Maintenance of proper DNA methylation patterns is a key process during embryo development and in the maintenance of adult tissue homeostasis. The use of experimental procedures based on the chemical modification of cytosine by sodium bisulfite and the development of antibodies recognizing 5mC have essentially contributed to our knowledge on DNA methylation dynamics in normal and disease states. Here we describe standard procedures for bisulfite sequencing, methylation-specific PCR, and 5mC immunodetection using mouse skin and the hair follicle stem cell niche as model tissues., This work was supported by grants of the Spanish Ministerio de Economía y Competitividad (SAF 11-23493) and the Comunidad Autónoma de Madrid (SkinModel, CAM S10/BMD-2359) to J.E.; E.C. and M.I.C. are supported by PhD fellowship grants of the Spanish Ministerio de Educación and Universidad Autónoma de Madrid, respectively.

Achieving bright, reliable, robust, and stable probes for in vivo imaging is becoming extremely urgent for the cancer imaging research community. To date very few works have reported on elucidating in the varied and chemically complex biological milieu. The authors report detailed investigations of the synthesis of near-infrared, water dispersive, strongly luminescent, and highly stable PbS/CdS/ZnS core/shell/shell quantum dots (QDs). These QDs are extremely stable, they could keep their initial morphology, dispersion status, and photoluminescence (PL) in phosphate buffered saline buffer for as long as 14 months. The QDs also show excellent photostability and could keep ≈80% of their initial PL intensity after 1 h continuous, strong UV illumination. More interestingly, they show negligible toxicity to cultured cells even at high QDs concentration. Given these outstanding properties, the QDs are explored for in vivo, tumor imaging in mice. With one order of magnitude lower QD concentration (0.04 mg mL–1), significantly weaker laser intensity (0.04 W cm–2 vs ≈1 W cm–2), and considerably shorter signal integration time (≤1 ms vs hundreds of ms) as compared to the best reported rare earth doped nanoparticles, the QDs show high emission intensity even at injection depth of ≈2.5 mm.

Photodynamic therapy (PDT) is a treatment modality for different forms of cancer based on the combination of light, molecular oxygen, and a photosensitizer (PS) compound. When activated by light, the PS generates reactive oxygen species leading to tumor destruction. Phthalocyanines are compounds that have already shown to be efficient PSs for PDT. Several examples of carbohydrate substituted phthalocyanines have been reported, assuming that the presence of carbohydrate moieties could improve their tumor selectivity. This work describes the photoeffects of symmetric and asymmetric phthalocyanines with D-galactose (so-called GPh1, GPh2, and GPh3) on HeLa carcinoma cells and their involvement in cell death. Photophysical properties and in vitro photodynamic activities for the compounds considered revealed that the asymmetric glycophthalocyanine GPh3 is very efficient and selective, producing higher photocytotoxicity on cancer cells than in nonmalignat HaCaT. The cell toxiticy after PDT treatment was dependent upon light exposure level and GPh3 concentration. GPh3 causes cell cycle arrest at the metaphase stage leading to multiple spindle poles, mitotic catastrophe, followed by apoptosis in cancer cells. These effects were partially negated by the pancaspase inhibitor Z-VAD-FMK. Together, these results indicate that GPh3 is an excellent candidate drug for PDT, able to induce selective tumor cell death.

Photodynamic therapy (PDT) employing methyl δ-aminolevulinic acid (Me-ALA), as a precursor of the photosensitizer protoporphyrin IX (PpIX), is used for the treatment of non melanoma cutaneous cancer (NMCC). However, one of the problems of PDT is the apparition of resistant cell populations. The aim of this study was to isolate and characterize squamous carcinoma cells SCC-13 resistant to PDT with Me-ALA. The SCC-13 parental population was submitted to successive cycles of Me-ALA-PDT and 10 resistant populations were finally obtained. In parental and resistant cells there were analyzed the cell morphology (toluidine blue), the intracellular PpIX content (flow cytometry) and its localization (fluorescence microscopy), the capacity of closing wounds (scratch wound assay), the expression of cell-cell adhesion proteins (E-cadherin and β-catenin), cell-substrate adhesion proteins (β1-integrin, vinculin and phospho-FAK), cytoskeleton proteins (α-tubulin and F-actin) and the inhibitor of apoptosis protein survivin, in the activated form as phospho-survivin (indirect immunofluorescence and Western blot). The results obtained indicate that resistant cells showed a more fibroblastic morphology, few differences in intracellular content of the photosensitizer, higher capacity of closing wounds, higher number of stress fibers, more expression of cell-substrate adhesion proteins and higher expression of phospho-survivin than parental cells. These distinctive features of the resistant cells can provide decisive information to enhance the efficacy of Me-ALA applications in clinic dermatology.

et al., Quantum dot based-thermometry, in combination with double beam confocal microscopy and infrared thermal imaging, has been used to investigate the heating efficiency of multi-walled carbon nanotubes (MWCNTs) under optical excitation within the first (808 nm) and second (1090 nm) biological windows as well as in the spectral region separating them (980 nm). It has been found that for the three excitation wavelengths the heating efficiency of MWCNTs (10 nm in diameter and 1.5 μm in length) is close to 50%. Despite this >flat> heating efficiency, we have found that the excitation wavelength is, indeed, critical during in vivo experiments due to the spectral dependence of both tissue absorption and scattering coefficients. It has been concluded that efficiency and selectivity of in vivo photothermal treatments based on MWCNTs are simultaneously optimized when laser irradiation lies within the first or second biological window. © 2013 The Royal Society of Chemistry., This work was supported by the Universidad Autónoma de Madrid and Comunidad Autónoma de Madrid (Project S2009/MAT-1756), by the Spanish Ministerio de Educacion y Ciencia (MAT2010-16161 and MAT2010-21270-C04-02) and by Fundación Dr Manuel Morales. Laura Martínez Maestro thanks FPI fellowship from the Spanish Ministerio de Economía y Competitividad, E.C. thanks FPU fellowship from the Spanish Ministerio de Educación and Blanca del Rosal thanks FPI fellowship from Universidad Autónoma de Madrid.