A Network Comprised of miR-15b and miR-29a Is Involved in Vascular Endothelial Growth Factor Pathway Regulation in Thymus Adipose Tissue from Elderly Ischemic Cardiomyopathy Subjects.

As the human thymus ages, it undergoes a transformation into adipose tissue known as TAT. Interestingly, in previous research, we observed elevated levels of vascular endothelial growth factor A (VEGFA) in TAT from patients with ischemic cardiomyopathy (IC), particularly in those over 70 years old. Moreover, in contrast to subcutaneous adipose tissue (SAT), TAT in elderly individuals exhibits enhanced angiogenic properties and the ability to stimulate tube formation. This makes TAT a promising candidate for angiogenic therapies and the regeneration of ischemic tissues following coronary surgery. MicroRNAs (miRNAs) have emerged as attractive therapeutic targets, especially those that regulate angiogenic processes. The study's purpose is to determine the miRNA network associated with both the VEGFA pathway regulation and the enrichment of age-linked angiogenesis in the TAT. RT-PCR was used to analyze angiogenic miRNAs and the expression levels of their predicted target genes in both TAT and SAT from elderly and middle-aged patients treated with coronary artery bypass graft surgery. miRTargetLink Human was used to search for miRNAs and their target genes. PANTHER was used to annotate the biological processes of the predicted targets. The expression of miR-15b-5p and miR-29a-3p was significantly upregulated in the TAT of elderly compared with middle-aged patients. Interestingly, VEGFA and other angiogenic targets were significantly upregulated in the TAT of elderly patients. Specifically: JAG1, PDGFC, VEGFA, FGF2, KDR, NOTCH2, FOS, PDGFRA, PDGFRB, and RHOB were upregulated, while PIK3CG and WNT7A were downregulated. Our results provide strong evidence of a miRNA/mRNA interaction network linked with age-associated TAT angiogenic enrichment in patients with IC.

ECM-Regulator timp Is Required for Stem Cell Niche Organization and Cyst Production in the Drosophila Ovary.

The extracellular matrix (ECM) is a pivotal component adult tissues and of many tissue-specific stem cell niches. It provides structural support and regulates niche signaling during tissue maintenance and regeneration. In many tissues, ECM remodeling depends on the regulation of MMP (matrix metalloproteinase) activity by inhibitory TIMP (tissue inhibitors of metalloproteinases) proteins. Here, we report that the only Drosophila timp gene is required for maintaining the normal organization and function of the germline stem cell niche in adult females. timp mutant ovaries show reduced levels of both Drosophila Collagen IV α chains. In addition, tissue stiffness and the cellular organization of the ovarian niche are affected in timp mutants. Finally, loss of timp impairs the ability of the germline stem cell niche to generate new cysts. Our results demonstrating a crucial role for timp in tissue organization and gamete production thus provide a link between the regulation of ECM metabolism and tissue homeostasis.

In Vitro Induction of Tendon-Specific Markers in Tendon Cells, Adipose- and Bone Marrow-Derived Stem Cells is Dependent on TGFß3, BMP-12 and Ascorbic Acid Stimulation.

Mesenchymal Stem Cells (MSCs) and tissue-specific progenitors have been proposed as useful tools for regenerative medicine approaches in bone, cartilage and tendon-related pathologies. The differentiation of cells towards the desired, target tissue-specific lineage has demonstrated advantages in the application of cell therapies and tissue engineering. Unlike osteogenic and chondrogenic differentiation, there is no consensus on the best tenogenic induction protocol. Many growth factors have been proposed for this purpose, including BMP-12, b-FGF, TGF-ß3, CTGF, IGF-1 and ascorbic acid (AA). In this study, different combinations of these growth factors have been tested in the context of a two-step differentiation protocol, in order to define their contribution to the induction and maintenance of tendon marker expression in adipose tissue and bone marrow derived MSCs and tendon cells (TCs), respectively. Our results demonstrate that TGF-ß3 is the main inducer of scleraxis, an early expressed tendon marker, while at the same time inhibiting tendon markers normally expressed later, such as decorin. In contrast, we find that decorin is induced by BMP-12, b-FGF and AA. Our results provide new insights into the effect of different factors on the tenogenic induction of MSCs and TCs, highlighting the importance of differential timing in TGF-ß3 stimulation.

Guideline of guidelines: follow-up after nephrectomy for renal cell carcinoma.

The purpose of this article was to review and compare the international guidelines and surveillance protocols for post-nephrectomy renal cell carcinoma (RCC). PubMed database searches were conducted, according to the PRISMA statement for reporting systematic reviews, to identify current international surveillance guidelines and surveillance protocols for surgically treated and clinically localized RCC. A total of 17 articles were reviewed. These included three articles on urological guidelines, three on oncological guidelines and 11 on proposed strategies. Guidelines and strategies varied significantly in relation to follow-up, specifically with regard to the frequency and timing of radiological imaging. Although there is currently no consensus within the literature regarding surveillance protocols, various guidelines and strategies have been developed using both patient and tumour characteristics.

Au@16-pH-16/miR-21 mimic nanosystem: An efficient treatment for obesity through browning and thermogenesis induction.

Despite the abundance of registered clinical trials worldwide, the availability of effective drugs for obesity treatment is limited due to their associated side effects. Thus, there is growing interest in therapies that stimulate energy expenditure in white adipose tissue. Recently, we demonstrated that the delivery of a miR-21 mimic using JetPEI effectively inhibits weight gain in an obese mouse model by promoting metabolism, browning, and thermogenesis, suggesting the potential of miR-21 mimic as a treatment for obesity. Despite these promising results, the implementation of more advanced delivery system techniques for miR-21 mimic would greatly enhance the advancement of safe and efficient treatment approaches for individuals with obesity in the future. Our objective is to explore whether a new delivery system based on gold nanoparticles and Gemini surfactants (Au@16-ph-16) can replicate the favorable effects of the miR-21 mimic on weight gain, browning, and thermogenesis. We found that dosages as low as 0.2 µg miR-21 mimic /animal significantly inhibited weight gain and induced browning and thermogenic parameters. This was evidenced by the upregulation of specific genes and proteins associated with these processes, as well as the biogenesis of beige adipocytes and mitochondria. Significant increases in miR-21 levels were observed in adipose tissue but not in other tissue types. Our data indicates that Au@16-ph-16 could serve as an effective delivery system for miRNA mimics, suggesting its potential suitability for the development of future clinical treatments against obesity.

A Noninvasive Method for Time-Lapse Imaging of Microbial Interactions and Colony Dynamics.

Complex interactions between microbial populations can greatly affect the overall properties of a microbial community, sometimes leading to cooperation and mutually beneficial coexistence, or competition and the death or displacement of organisms or subpopulations. Interactions between different biofilm populations are highly relevant in diverse scientific areas, from antimicrobial resistance to microbial ecology. The utilization of modern microscopic techniques has provided a new and interesting insight into how bacteria interact at the cellular level to form and maintain microbial biofilms. However, our ability to follow complex intraspecies and interspecies interactions in vivo at the microscopic level has remained somewhat limited. Here, we detailed BacLive, a novel noninvasive method for tracking bacterial growth and biofilm dynamics using high-resolution fluorescence microscopy and an associated ImageJ processing macro (https://github.com/BacLive) for easier data handling and image analysis. Finally, we provided examples of how BacLive can be used in the analysis of complex bacterial communities. IMPORTANCE Communication and interactions between single cells are continuously defining the structure and composition of microbial communities temporally and spatially. Methods routinely used to study these communities at the cellular level rely on sample manipulation which makes microscopic time-lapse experiments impossible. BacLive was conceived as a method for the noninvasive study of the formation and development of bacterial communities, such as biofilms, and the formation dynamics of specialized subpopulations in time-lapse experiments at a colony level. In addition, we developed a tool to simplify the processing and analysis of the data generated by this method.

Iron-Gold Nanoflowers: A Promising Tool for Multimodal Imaging and Hyperthermia Therapy.

The development of nanoplatforms prepared to perform both multimodal imaging and combined therapies in a single entity is a fast-growing field. These systems are able to improve diagnostic accuracy and therapy success. Multicomponent Nanoparticles (MCNPs), composed of iron oxide and gold, offer new opportunities for Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) diagnosis, as well as combined therapies based on Magnetic Hyperthermia (MH) and Photothermal Therapy (PT). In this work, we describe a new seed-assisted method for the synthesis of Au@Fe Nanoparticles (NPs) with a flower-like structure. For biomedical purposes, Au@Fe NPs were functionalized with a PEGylated ligand, leading to high colloidal stability. Moreover, the as-obtained Au@Fe-PEG NPs exhibited excellent features as both MRI and CT Contrast Agents (CAs), with high r2 relaxivity (60.5 mM-1⋅s-1) and X-ray attenuation properties (8.8 HU mM-1⋅HU). In addition, these nanoflowers presented considerable energy-to-heat conversion under both Alternating Magnetic Fields (AMFs) (∆T ≈ 2.5 °C) and Near-Infrared (NIR) light (∆T ≈ 17 °C). Finally, Au@Fe-PEG NPs exhibited very low cytotoxicity, confirming their potential for theranostics applications.

Enantioselective synthesis of 4-amino-3,4-dihydrocoumarins and their non-cyclic hydroxyester precursors: Biological evaluation for the treatment of glioblastoma multiforme.

The stereoselective addition of ethyl acetate enolate to the C═N bond of N-tert-butylsulfinylimines has been investigated in depth. A significant effect of the LHMDS amount and the N-sulfinylimine nature on the stereoselectivity of the process was observed. Conditions were found where sulfinylimines of differently substituted salicylaldehydes derivatives, ethyl acetate, and LHMDS afforded the corresponding addition products as a single diastereomer in good yields. The developed protocol was successfully applied to the first stereoselective synthesis of differently substituted 4-amino-3,4-dihydrocoumarin derivatives. Computational models confirmed the prominent role of the ortho aryl substituent in the stereoselectivity of the process. A significant and selective cytotoxic activity against Glioblastoma Multiforme (GBM) cancer line has been determined for the noncyclic hydroxy ester derivative.

Shell-adductor muscle attachment and Ca2+ transport in the bivalves Ostrea stentina and Anomia ephippium.

Among bivalve muscles, the adductors are particularly important for animal survival because they control valve closure. Most studies have addressed the type and morphology of this muscle in bivalves but few have focused on the mechanism that anchors it to the shell myostracum layer. Moreover, the possible calcium transport mechanism through the adductor muscle cells to the myostracum shell layer, which is necessary for bivalve biomineralisation, has never been addressed. Our results indicate that the muscle cell-shell attachment is mediated by the outer mantle epithelial cell layer, here termed tendon cells. These cells are modified at the muscle scar zone by the presence of actin cytoskeletal bundles, which anchor cells to the extracellular matrix via focal adhesion (or focal contact) junctions at the basal side and to extrapallial matrix at the apical side, both rich in collagen. From apical focal adhesions, bundles of collagen-rich fibres cross the extrapallial space and penetrate the myostracum shell layer. The latter constitutes one of the strongest anchoring structures among invertebrates. Numerous vesicles protrude from the tendon cells into the extrapallial space. TEM-EDX analysis reveals the presence of Ca2+ inside some of these vesicles both in tendon cells and in the extrapallial space. This suggests a potential mechanism for calcium transport from cells to the myostracum. STATEMENT OF SIGNIFICANCE: The interfaces between bivalve shells and muscular attachments are unique and of special interest as adhesive functional biomaterials, being one of the strongest invertebrate anchoring structures. We present an updated ultrastructural model of the adductor muscle-shell attachment. Muscle cells connect with the shell through epithelial `tendon cells`, which have a cytoskeleton of actin microfilaments that connect to the extracellular matrix via focal adhesions. Collagen-rich fibres arise from apical focal adhesions, cross the nanometric extrapallial space and penetrate the myostracum where they form an organic network. Calcium is present inside vesicles that are released into the extrapallial space. The lack of direct cellular control on secretion restricts the myostracal microstructure to prismatic aragonitic similar to its inorganic counterpart.

Stem cell niche organization in the Drosophila ovary requires the ECM component Perlecan.

Stem cells reside in specialized microenvironments or niches that balance stem cell proliferation and differentiation.1,2 The extracellular matrix (ECM) is an essential component of most niches, because it controls niche homeostasis, provides physical support, and conveys extracellular signals.3-11 Basement membranes (BMs) are thin ECM sheets that are constituted mainly by Laminins, Perlecan, Collagen IV, and Entactin/Nidogen and surround epithelia and other tissues.12 Perlecans are secreted proteoglycans that interact with ECM proteins, ligands, receptors, and growth factors such as FGF, PDGF, VEGF, Hedgehog, and Wingless.13-18 Thus, Perlecans have structural and signaling functions through the binding, storage, or sequestering of specific ligands. We have used the Drosophila ovary to assess the importance of Perlecan in the functioning of a stem cell niche. Ovarioles in the adult ovary are enveloped by an ECM sheath and possess a tapered structure at their anterior apex termed the germarium. The anterior tip of the germarium hosts the germline niche, where two to four germline stem cells (GSCs) reside together with a few somatic cells: terminal filament cells (TFCs), cap cells (CpCs), and escort cells (ECs).19 We report that niche architecture in the developing gonad requires trol, that niche cells secrete an isoform-specific Perlecan-rich interstitial matrix, and that DE-cadherin-dependent stem cell-niche adhesion necessitates trol. Hence, we provide evidence to support a structural role for Perlecan in germline niche establishment during larval stages and in the maintenance of a normal pool of stem cells in the adult niche.

Fe3O4-Au Core-Shell Nanoparticles as a Multimodal Platform for In Vivo Imaging and Focused Photothermal Therapy.

In this study, we report the synthesis of gold-coated iron oxide nanoparticles capped with polyvinylpyrrolidone (Fe@Au NPs). The as-synthesized nanoparticles (NPs) exhibited good stability in aqueous media and excellent features as contrast agents (CA) for both magnetic resonance imaging (MRI) and X-ray computed tomography (CT). Additionally, due to the presence of the local surface plasmon resonances of gold, the NPs showed exploitable "light-to-heat" conversion ability in the near-infrared (NIR) region, a key attribute for effective photothermal therapies (PTT). In vitro experiments revealed biocompatibility as well as excellent efficiency in killing glioblastoma cells via PTT. The in vivo nontoxicity of the NPs was demonstrated using zebrafish embryos as an intermediate step between cells and rodent models. To warrant that an effective therapeutic dose was achieved inside the tumor, both intratumoral and intravenous routes were screened in rodent models by MRI and CT. The pharmacokinetics and biodistribution confirmed the multimodal imaging CA capabilities of the Fe@AuNPs and revealed constraints of the intravenous route for tumor targeting, dictating intratumoral administration for therapeutic applications. Finally, Fe@Au NPs were successfully used for an in vivo proof of concept of imaging-guided focused PTT against glioblastoma multiforme in a mouse model.

Indolenine-Based Derivatives as Customizable Two-Photon Fluorescent Probes for pH Bioimaging in Living Cells.

Novel pH probes based on 2-(6-methoxynaphthalen-2-yl)-3,3-dimethyl-3H-indole have been synthesized and characterized. These compounds display excellent "off-on" fluorescence responses to acidic pH especially under two-photon (TP) excitation conditions as well as strong selectivity and sensitivity toward H+. These features are supported by fluorescence quantum yields over 35%, TP cross sections ∼60 GM, and good resistance to photodegradation under acidic conditions. The synthetic versatility of this model allows subcellular targets to be tuned through minor scaffold modifications without affecting its optical characteristics. The effectiveness of the probes' innate photophysical properties and the structural modifications for different pH-related applications are demonstrated in mouse embryonic fibroblast cells.

Dual functionality of the amyloid protein TasA in Bacillus physiology and fitness on the phylloplane.

Bacteria can form biofilms that consist of multicellular communities embedded in an extracellular matrix (ECM). In Bacillus subtilis, the main protein component of the ECM is the functional amyloid TasA. Here, we study further the roles played by TasA in B. subtilis physiology and biofilm formation on plant leaves and in vitro. We show that ΔtasA cells exhibit a range of cytological symptoms indicative of excessive cellular stress leading to increased cell death. TasA associates to the detergent-resistant fraction of the cell membrane, and the distribution of the flotillin-like protein FloT is altered in ΔtasA cells. We propose that, in addition to a structural function during ECM assembly and interactions with plants, TasA contributes to the stabilization of membrane dynamics as cells enter stationary phase.

The extracellular matrix protects Bacillus subtilis colonies from Pseudomonas invasion and modulates plant co-colonization.

Bacteria of the genera Pseudomonas and Bacillus can promote plant growth and protect plants from pathogens. However, the interactions between these plant-beneficial bacteria are understudied. Here, we explore the interaction between Bacillus subtilis 3610 and Pseudomonas chlororaphis PCL1606. We show that the extracellular matrix protects B. subtilis colonies from infiltration by P. chlororaphis. The absence of extracellular matrix results in increased fluidity and loss of structure of the B. subtilis colony. The P. chlororaphis type VI secretion system (T6SS) is activated upon contact with B. subtilis cells, and stimulates B. subtilis sporulation. Furthermore, we find that B. subtilis sporulation observed prior to direct contact with P. chlororaphis is mediated by histidine kinases KinA and KinB. Finally, we demonstrate the importance of the extracellular matrix and the T6SS in modulating the coexistence of the two species on melon plant leaves and seeds.

Housekeeping Gene Stability in Human Mesenchymal Stem and Tendon Cells Exposed to Tenogenic Factors.

The use of biochemical inducers of mesenchymal stem cell (MSC) differentiation into tenogenic lineage represents an investigated aspect of tendon disorder treatment. Bone morphogenetic protein 12 (BMP-12) is a widely studied factor, representing along with ascorbic acid (AA) and basic fibroblast growth factor (bFGF) one of the most promising stimulus in this context so far. Quantitative gene expression of specific tenogenic marker is commonly used to assess the efficacy of these supplements. Nevertheless, the reliability of these data is strongly associated with the choice of stable housekeeping genes. To date, no published studies have evaluated the stability of housekeeping genes in MSCs during tenogenic induction. Three candidate housekeeping genes (YWHAZ, RPL13A, and GAPDH) in human MSCs from bone marrow (BMSCs), adipose tissue (ASCs), and tendon cells (TCs) supplemented with BMP-12 or AA and bFGF in comparison with control untreated cells for 3 and 10 days were evaluated. GeNorm, NormFinder, and BestKeeper tools and the comparative ΔCt method were used to evaluate housekeeping gene stability and the overall ranking was determined by using by the RefFinder algorithm. In all culture conditions, YWHAZ was the most stable gene and RPL13A was the second choice. YWHAZ and RPL13A were the two most stable genes also for ASCs and BMSCs, regardless of the time point analyzed, and for TCs at 10 days of tenogenic induction. Only for TCs at 3 days of tenogenic induction were GAPDH and YWHAZ the best performers. In conclusion, our findings will be useful for the proper selection of housekeeping genes in studies involving MSCs cultured in the presence of tenogenic factors, to obtain accurate and high-quality data from quantitative gene expression analysis.

Intracellular accumulation and immunological properties of fluorescent gold nanoclusters in human dendritic cells.

We have studied the effect of highly fluorescent gold nanoclusters (Au NCs) (∅ < 3 nm) stabilized by different ligands on the intracellular accumulation and immune response of human derived-monocyte dendritic cells (DCs). Results indicate that the high uptake efficiency of Au NCs is strongly related to their small size and to the nature of the ligand, with zwitterionic ligands being more effective than PEGylated ones. Evidence from flow cytometry and microscopy demonstrate time and concentration-dependent Au NCs internalization by endocytic pathway(s) involving amorphous and laminar organelles, while maintaining their discrete size and photoluminescence properties. The uptake of zwitterionic ligand-stabilized Au NCs induced very low cytotoxicity and a strong immunosuppressive response (Th1/Treg pattern), associated with a DC maturation state. This behavior contrasts to the effect of bigger particles (∼12 nm size) which induced a cytotoxic response involving Natural Killer (CD56) cells. Overall, this study stresses the critical importance of particle size and ligand type on the immunostimulation of DCs and highlights the remarkable potential of this new class of nanomaterial as a novel vaccine platform.