Nanomedicine carries the hope of precisely identifying and healing lesion sites in vivo. However, the reproducible synthesis of monodisperse protein nanoparticles smaller than 50 nm in diameter and up-scalable to industrial production still poses challenges to researchers. In this report, we describe nanoparticles, so called Absicles, based on an albumin matrix and prepared by a procedure developed by the authors. These Absicles are monodisperse with tunable diameters ranging from 15 nm to 70 nm respectively. They exhibit long term stability against decomposition and aggregation, exceeding many months. The synthesis of Absicles shows exceptionally high reproducibility concerning size, and is simple and cost-effective for up-scaling. Absicles, bearing appropriate targeting groups, bind with high specificity to colon carcinoma tissue ex vivo; they present an attractive platform for further development towards drug delivery applications.

Nanoparticles bearing specific targeting groups can, in principle, accumulate exclusively at lesion sites bearing target molecules, and release therapeutic agents there. However, practical application of targeted nanoparticles in the living organism presents challenges. In particular, intravasally applied nanoparticles encounter physical and physiological barriers located in blood vessel walls, blocking passage from the blood into tissue compartments. Whereas small molecules can pass out of the blood, nanoparticles are too large and need to utilize physiological carriers enabling passage across endothelial walls. The issues associated with crossing blood-tissue barriers have limited the usefulness of nanoparticles in clinical applications. However, nanoparticles do not encounter blood-tissue barriers if their targets are directly accessible from the blood. This review focuses on osteoporosis, a disabling and common disease for which therapeutic strategies are limited. The target sites for therapeutic agents in osteoporosis are located in bone resorption pits, and these are in immediate contact with the blood. There are specific targetable biomarkers within bone resorption pits. These present nanomedicine with the opportunity to treat a major disease by use of simple nanoparticles loaded with any of several available effective therapeutics that, at present, cannot be used due to their associated side effects.

Molecular imaging using magnetic resonance imaging (MRI) is expected to play a crucial future role in oncological diagnosis and in monitoring of therapeutic progress. Targeted nanoparticle contrast media (CM) with high relaxivities are required in order to obtain adequate signal-to-noise ratios as well as visualization of a desired pathologic area of the human body. The aims of this study were to synthesize and define certain physicochemical and enhancement properties of new doubly derivatized polylactic acid–bovine serum albumin (PLA-BSA) nanoparticles (NPs) modified by the covalent coupling of glutaraldehyde as a crosslinking agent. An additional functionalization with endothelial cells (ECs) targeting groups (tomato lectins; LEA) and signal-emitting moieties (DTPA-Gd) enables its use as a macromolecular, biodegradable contrast agent for MRI. The NPs were characterized by different spectroscopies, size exclusion chromatography, and scanning and transmission electron microscopy. In a human vein model, the dynamics of the nanoparticle interactions with the vein wall were examined in MRI, with correlative imaging in electron microscopy. In vitro studies were conducted to show endothelial binding and persistent enhancement at the apical EC surface. NPs with a diameter between 55 and 75 nm, able to carry simultaneous signal emitting, and targeting motifs on a single construct were successfully prepared. A high Gd payload and endothelial binding to blood vessel walls were observed. The binding affinity and specificity of LEA was preserved, and a strong enhancement at the endothelium was achieved. The stabilized core–shell structure of PLA-NP might allow for further encapsulation of lipophilic drugs or for attachment of other targeting molecules, such as antibodies.

This multidisciplinary study examined the pharmacokinetics of nanoparticles based on albumin-DTPA-gadolinium chelates, testing the hypothesis that these nanoparticles create a stronger vessel signal than conventional gadolinium-based contrast agents and exploring if they are safe for clinical use. Nanoparticles based on human serum albumin, bearing gadolinium and designed for use in magnetic resonance imaging, were used to generate magnet resonance images (MRI) of the vascular system in rats (“blood pool imaging”). At the low nanoparticle doses used for radionuclide imaging, nanoparticle-associated metals were cleared from the blood into the liver during the first 4 h after nanoparticle application. At the higher doses required for MRI, the liver became saturated and kidney and spleen acted as additional sinks for the metals, and accounted for most processing of the nanoparticles. The multiple components of the nanoparticles were cleared independently of one another. Albumin was detected in liver, spleen, and kidneys for up to 2 days after intravenous injection. Gadolinium was retained in the liver, kidneys, and spleen in significant concentrations for much longer. Gadolinium was present as significant fractions of initial dose for longer than 2 weeks after application, and gadolinium clearance was only complete after 6 weeks. Our analysis could not account quantitatively for the full dose of gadolinium that was applied, but numerous organs were found to contain gadolinium in the collagen of their connective tissues. Multiple lines of evidence indicated intracellular processing opening the DTPA chelates and leading to gadolinium long-term storage, in particular inside lysosomes. Turnover of the stored gadolinium was found to occur in soluble form in the kidneys, the liver, and the colon for up to 3 weeks after application. Gadolinium overload poses a significant hazard due to the high toxicity of free gadolinium ions. We discuss the relevance of our findings to gadolinium-deposition diseases.

We explore present-day trends and challenges in nanomedicine. Creativity in the laboratories continues: the published literature on novel nanoparticles is now vast. Nanoagents are discussed here which are composed entirely of strongly photoluminescent materials, tunable to desired optical properties and of inherently low toxicity. We focus on "quantum nanoparticles" prepared from allotropes of carbon. The principles behind strong, tunable photoluminescence are quantum mechanical: we present them in simple outline. The major industries racing to develop these materials can offer significant technical guidance to nanomedicine, which could help to custom-design strongly signalling nanoagents specifically for stated clinical applications. Since such agents are small, they can be targeted easily, making active targeting possible. We consider it timely now to study the interactions nanoparticles undergo with tissue components in living animals and to learn to understand and overcome the numerous barriers the organism interposes between the blood and targets in or on parenchymal cells. As the near infra-red spectrum opens up, detection of glowing nanoparticles several centimeters deep in a living human subject becomes calculable and we present a simple way to do this. Finally, we discuss the slow-fuse and resource-inefficient entry of nanoparticles into clinical application. A first possible reason is failure to target across the body's barriers, see above. Second, in the sparse translational landscape funding and support gaps yawn widely between academic research and subsequent development. We consider the agendas of the numerous "stakeholders" participating in this sad landscape and point to some faint glimmers of hope for the future.

In recent decades, targeted therapeutics have significantly improved therapy results in patients with malignant tumors of different origins. However, malignant diseases characterized by aggressiveness and increased capacity for metastatic spread still require basic researchers and clinicians to direct enormous efforts toward the development of novel therapeutic targets. Potential targets should be selected with the clinical endpoint in view; targeted therapeutics can be developed: for use in combination with currently existing therapeutic approaches in order to improve their efficacy; to overcome the treatment resistance of tumor cells and thus protect the patient from recurrence; to repress molecular mechanisms related to immune escape of cancer cells; and to combat the metastatic dissemination of carcinoma cells. Taking into account the specific clinical aim that should be achieved, different strategies and techniques can be proposed to identify the most promising candidate molecules for further development as therapeutic targets. Since cellular membranes contain a large number of druggable molecules, evaluation of the membrane protein profiles of carcinoma cells having different properties can provide a basis for further development of therapeutic targets. This review considers how cellular membranes obtained from different pre-clinical and clinical samples can be used in screening and to identify targets for cancer therapy.

The majority of tumor-related deaths are due to metastasis. Despite the clinical importance of understanding metastasis, we lack knowledge of the molecular mechanisms underlying tumor cell spreading and cell survival far from the primary tumor. Elucidating the molecular characteristics of highly metastatic carcinoma cells would help identify biomarkers or therapeutic targets relevant to predicting or combatting metastasis, and for this the phenotype of metastatic cells could be much more important than their genotype. Hence, proteomic approaches have wide potential utility. This review discusses possibilities of analyzing metastasis-specific protein patterns in a range of sample types, including in vitro and in vivo cancer models, and tissues and biological fluids from patients. Proteome approaches can identify proteins involved in regulating the metastatic capacities of tumors.

In this article, we review the recent updates in musculoskeletal ultrasound (MSK US). The progressive development in ultrasound scanners including both hard- and softwares has already been reflected in its ability to diagnose different MSK diseases efficiently. We discuss here the recent applications of conventional US techniques as peripheral nerve imaging and image-guided therapies. The role of color Doppler US and contrast-enhanced US is also discussed with its impact especially in soft tissue tumors and small joint arthropathies. Sonoelastography as a new and promising technique is discussed in detail in this review. It may provide better and more robust diagnosis for many MSK diseases and may replace some of the relatively old routine techniques applied nowadays. In the end, 3D imaging and image fusion are discussed with their impact in image guidance of therapy as well as their teaching applications.

Despite the fact that radiation therapy is a highly effective therapeutic approach, a small intratumoral cell subpopulation known as “cancer stem cells” (CSCs) is radiation-resistant and possesses specific molecular properties protecting it against radiation-induced damage. The exact mechanisms of this radioresistance are still not fully elucidated, but they relate to these cells’ enhanced DNA repair capacities and their low intracellular ROS concentrations, resulting from their up-regulation of ROS scavengers. The low ROS content is accompanied by disturbances in cell cycle regulation, so it can be assumed that either CSCs are quiescent or dormant themselves, or that this cell population consists of at least two cell subpopulations: the normally and the slowly proliferating cells (quiescent or dormant cells). Slowly dividing CSCs show concomitant dysregulation of the signaling molecules mediating both cell cycle progression and maintenance of cell stemness. Despite a massive accumulation of data concerning the mechanisms underlying DNA damage response in CSCs, it represents a challenge to researchers in the era of personalized medicine to elucidate the role of intracellular ROS and of signaling pathways associated with the radiation resistance of these cells; there is a clear need to understand the molecular mechanisms helping CSCs to survive radiation exposure.

DNA damaging agents (ionizing radiation and chemotherapeutics) are considered as most effective in cancer treatment. However, there is a subpopulation of carcinoma cells within the tumour demonstrating resistance to DNA damaging treatment approaches. It is suggested that limited tumour response to this kind of therapy can be associated with specific molecular properties of carcinoma stem cells (CSCs) representing the most refractory cell subpopulation. This review article presents novel data about molecular features of CSCs underlying DNA damage response and related intracellular signalling.

Radiation therapy plays an important role in the management of malignant tumors, however, the problem of radiation resistance resulting in tumor recurrences after treatment is still unsolved. The emergence of novel biomarkers to predict cancer cell insensitivity to ionizing radiation could help to improve therapy results in cancer patients receiving radiation therapy. The proteomic approach could be effectively used to identify proteins associated with cancer radiation resistance. It is generally believed that radiation resistance could be associated with cancer stem cell persistence within the tumor. Therefore, determination of the molecular characteristics of cancer stem cells could provide additional possibilities to discover novel biomarkers to predict radiation resistance in cancer patients. This review addresses proteome-based findings that could be used for further biomarker identification and preclinical and clinical validation.

Background: In order to improve therapy for HNSCC patients, novel methods to predict and combat local and/or distant tumour relapses are urgently needed. This study has been dedicated to the hypothesis that Rac1, a Rho GTPase, is implicated in HNSCC insensitivity to chemo-radiotherapy resulting in tumour recurrence development.Methods: Parental and radiation-resistant (IRR) HNSCC cells were used to support this hypothesis. All cells were investigated for their sensitivity to ionising radiation and cisplatin, Rac1 activity, its intracellular expression and subcellular localisation. Additionally, tumour tissues obtained from 60 HNSCC patients showing different therapy response were evaluated for intratumoral Rac1 expression.Results: Radiation-resistant IRR cells also revealed resistance to cisplatin accompanied by increased expression, activity and trend towards nuclear translocation of Rac1 protein. Chemical inhibition of Rac1 expression and activity resulted in significant improvement of HNSCC sensitivity to ionising radiation and cisplatin. Preclinical results were confirmed in clinical samples. Although Rac1 was poorly presented in normal mucosa, tumour tissues revealed increased Rac1 expression. The most pronounced Rac1 presence was observed in HNSCC patients with poor early or late responses to chemo-radiotherapy. Tissues taken at recurrence were characterised not only by enhanced Rac1 expression but also increased nuclear Rac1 content.Conclusions: Increased expression, activity and subcellular localisation of Rac1 could be associated with lower early response rate and higher risk of tumour recurrences in HNSCC patients and warrants further validation in larger independent studies. Inhibition of Rac1 activity can be useful in overcoming treatment resistance and could be proposed for HNSCC patients with primary or secondary chemo-radioresistance.

Purpose : New understanding of cancer stem cell (CSC) biology continues to emerge due to development of novel methods in genomics and proteomics. Analysis of nucleic acids (RNA, DNA) is widely used to elucidate molecular perturbations in malignant tumors and carcinoma cells, however genome data do not reflect the functional activities of encoded proteins. Therefore proteome-based methods could enhance knowledge about deregulation of pathways as a result of altered expression and activities of proteins in CSC.Methods and results: A sufficient number of CSC for proteomic analyses can be obtained in a variety of ways: Fluorescence (FACS) and magnetic (MACS) activated cell sorting, laser cell capture microdissection, and three-dimensional spheroid/organoid cell culture. These methods to enrich and isolate CSC can be performed either with or without staining using antibodies against currently known CSC-specific cell surface molecules, such as clusters of differentiation 44, 24, 133 (CD44, CD24, CD133), epithelial cell adhesion molecule (EpCAM), aldehydede-hydrogenase-1 (ALDH1), etc. The most important limitation on using antibody-based staining of CSC is that we still do not possess definitive CSC surface markers. This review article discusses methods that could be used to study protein profiling of CSC and to identify novel CSC-specific biomarkers and therapeutic targets.Conclusion : Despite an opinion that the proteomic approach is time-consuming, laborious and difficult, this method can be used effectively to clarify which pathways are involved in regulating various intratumoral processes, including activation of CSC. Based on this point of view, searching and identification of single molecules as biomarkers or therapeutic targets could become possible when CSC-associated pathways are well described and clearly understood due to detailed investigation of the protein patterns in pre-clinical models and clinical samples.

Metastatic progression of malignant tumors resistant to conventional therapeutic approaches is an ultimate challenge in clinical oncology. Despite the efforts of basic and clinical researchers, there is still no effective treatment schedule to prevent or combat metastatic spread of malignant tumors. This report presents recent findings that could help in the development of targeted therapeutics directed against the most aggressive and treatment-resistant carcinoma cells. It was demonstrated that HNSCC carcinoma cell lines with acquired treatment resistance possessed increased number of cells with carcinoma stem cell (CSC) properties. Furthermore, resistant cells were characterized by increased expression of Rac1, enhanced cell migration, and accelerated release of proangio- and vasculogenic factors (VEGF-A) and influence on endothelial cell (HMEC-1) migration. Inhibition of Rac1 signaling in the treatment-resistant carcinoma cells can interrupt metastatic process due to anoikis restoration and decrease of cell migration. It is also suggested that carcinoma cells with repressed survival capacities will be characterized by reduced release of proangiogenic factors, resulting in the decrease of endothelial cell migration. Therefore targeting of Rac1-related pathways may be considered as a promising therapeutic approach to prevent or combat metastatic lesions.

Next to nothing is known about nanoparticle and nanofiber trafficking at the feto-maternal interface in early human pregnancy. As the first trimester is thought to be crucial for the further placental and fetal development, it will be important to assess the possible risks of nanomaterial exposures during this period. There are some intriguing observations in nanotoxicology, however, indicating certain differences between classical toxicology and nanotoxicology. To understand nanomaterial-biokinetics and placental toxicity in early gestation, the special architecture, the hypoxic condition, the bilayer of villous trophoblast, the plugging of spiral arteries and the contribution of intrauterine glands to nutrition, as well as the delicate immunologic situation at the implantation site, will have to be considered. Unless nano-specific biokinetics are properly understood, it will be difficult to ensure identification of potential “nano-thalidomides” among all the newly engineered nanoparticles and fibers, based on the models available in reproductive toxicology.

Intermediate Filaments in Health and Disease'' were the focus of the scientific program of the 55th Symposium of the Society for Histochemistry in Prague, Czech Republic.Various aspects of intermediate filaments (IF) and associated proteins were presented and discussed with emphasis on imaging and sample preparation methods including both light and electron microscopy and specific labeling techniques.The Symposium was organized in cooperation with BMBS COST Action Nanomechanics of Intermediate Filaments Networks (NANONET) and took place in the Conference Center of the Institute of the Molecular Genetics, Academy of Science of the Czech Republic.Pavel Hoza ´k as the local organizer together with his dedicated team did put together a very exciting and representative program.

Each organ possesses its own particular barrier(s). We recently reviewed the bloodbreast barrier, a complex and tightly regulated barrier, as well as one of the extremely “high” barriers, the blood-brain barrier [1] (the other two high barriers are in the testis and the placenta). Tissues and organs require organ-characteristic internal fluid environments to enable and facilitate their specific functions. For example, each glandular secretion is different in composition from all the others, and all are different from the blood; for the purposes of this review, the blood can be considered a homogenous fluid [1, 2]. To sustain these different milieux, structural and physiological barriers located at blood-tissue interfaces regulate bidirectional material flow between the tissue aqueous compartment and the blood compartment. The blood side of the barrier is demarcated by specialized structures and functions regulating the transfer of materials and cells; the luminal side of the barrier is demarcated by another set of structures and functions at the apical side of the epithelial cells, which regulate access of the luminal contents to the blood. Two barrier systems thus work in a coordinated fashion to elaborate a single consolidated barrier. 1. The blood-tissue barriers are of considerable biological importance, defining the operating environments of the organs. They are dynamic, biologically active interfaces, at which bidirectional exchanges of materials and cells are actively and continuously regulated (Figure 1). At its barrier, a tissue can accumulate particular molecules by taking these up from the blood, yet simultaneously it prevents the entry of other molecules which are also present in the blood. The separation between aqueous compartments results from the presence of tight intercellular junctions (occluding junctions) between cells, either between the endothelial cells in the microvessel wall, or between the characteristic epithelial cells of the organ. Tight junctions are an essential component of all the blood-tissue barriers (except in the placenta, where the barrier bases on a syncytium). The > 20 different protein constituents of the tight junctions, for example the claudins [3, 4], occludin [5], the cytoplasmic plaque proteins ZO-1,2,3, cingulin, and others [6] vary widely amongst tight junctions in different tissues. This, together with active regulation of tight junction assembly by zonulin [7], explains the variable paracellular permeability found in diverse tissues [6, 8, 9]. The separated aqueous compartments are bridged by selective uptake and transfer of specific molecules, mediated by a wide range of specific transporters and ion channels. Furthermore, specific enzymes may also be present to degrade any molecules (such as peptides) capable of crossing the barrier in spite of the tight junctions (Figure 1). The Figure shows both sides of the intestinal barrier, the blood side described above, and the luminal side which will be mentioned next. 2. The epithelial cells maintain uptake and transport systems at their apical side, quite different from those at the basolateral side. If a brush border is present, as in most gut segments, this will contain enzymes regulating passage of materials, and in the gut these may be digestive enzymes. The epithelium may coat itself with a continuous layer of viscoelastic gel in the form of a chemically sophisticated mixture of interacting molecules, of which the mucins are the major part, and in this case the barrier regulates passage across a mucous epithelium. Both the blood side and the luminal side of mucosal barriers will next be described in more detail for the intestine.

Sperm are modified substantially in passing through both the male and the female reproductive tracts, only thereafter becoming functionally competent to fertilize eggs. Drosophila sperm become motile in the seminal vesicle; after ejaculation, they interact with seminal fluid proteins and undergo biochemical changes on their surface while they are stored in the female sperm storage organs. However, the molecular mechanisms underlying these maturation processes remain largely unknown. Here, we focused on Drosophila Neprilysin genes, which are the fly orthologs of the mouse Membrane metallo-endopeptidase-like 1 (Mmel1) gene. While Mmel1 knockout male mice have reduced fertility without abnormality in either testis morphology or sperm motility, there are inconsistent results regarding the association of any Neprilysin gene with male fertility in Drosophila. We examined the association of the Nep1-5 genes with male fertility by RNAi and found that Nep4 gene function is specifically required in germline cells. To investigate this in more detail, we induced mutations in the Nep4 gene by the CRISPR/Cas9 system and isolated two mutants, both of which were viable and female fertile, but male sterile. The mutant males had normal-looking testes and sperm; during copulation, sperm were transferred to females and stored in the seminal receptacle and paired spermathecae. However, following sperm transfer and storage, three defects were observed for Nep4 mutant sperm. First, sperm were quickly discarded by the females; second, the proportion of eggs fertilized was significantly lower for mutant sperm than for control sperm; and third, most eggs laid did not initiate development after sperm entry. Taking these observations together, we conclude that the Nep4 gene is essential for sperm function following sperm transfer to females.