Ultrastructural Examination of the Corneal Interface after Predescemetic Deep Anterior Lamellar Keratoplasty (DALK) - A Case Report with Light and Transmission Electron Microscopy.

PURPOSE: To examine corneal buttons with light and transmission electron microscopy (TEM) to visualize the interface area and highlight the ultrastructural corneal changes after deep anterior lamellar keratoplasty (DALK). METHODS: Two patients underwent excimer laser-assisted penetrating repeat keratoplasty after predescemetic DALK. The corneal buttons were examined by light microscopy and TEM. RESULTS: The light microscopic examination of the corneal buttons revealed fragments of a second Descemet's membrane in the central and midperipheral areas (Case 1). In both cases, visualization of the interface area was not possible by light microscopy. The donor and host stroma were tightly attached without dehiscence. TEM identified the interface area by irregularities in the collagen distribution between the donor and host stroma. The thickness of the remaining recipient corneal stroma measured approximately 30 µm (Case 1) and 100 µm (Case 2), respectively. In the host stroma, TEM revealed the absence or degeneration of keratocytes, accumulation of amorphous material between the collagen lamellae, and vacuolar inclusions dispersed in the stroma, forming a band-like zone anterior to Descemet's membrane. CONCLUSION: The interface area after DALK has been mainly investigated by in vivo confocal microscopy. Light microscopy and TEM findings indicate remodeling processes after DALK that are associated with increased keratocyte degeneration and structural alterations of the extracellular matrix in the host stroma. The choice of surgical method may influence the postoperative morphological and functional outcome since these findings were primarily apparent in the remaining host stroma. Therefore, complete exposure of Descemet's membrane is an important prognostic factor for the postoperative visual outcome.

Compound Danshen Dripping Pills pretreatment protects the heart from ischemia/reperfusion injury by enhancing autophagic flux

Abstract Compound Danshen Dripping Pills (CDDPs) have been used in clinical treatment to protect the heart from ischemia/reperfusion (IR) injury for many years. However, the underlying mechanism implicated in the protective effects remains to be explored. Here, we determined the effects of CDDPs in Sprague-Dawley rats with the IR model. Cardiac function in vivo was assessed by echocardiography. Transmission electron microscopy, histological and immunohistochemical techniques, Western blotting and recombinant adeno-associated virus 9 transfection were used to illustrate the effects of CDDPs on IR and autophagy. Our results showed that pretreatment with CDDPs decreased the level of serum myocardial enzymes and infarct size in rats after IR. Apoptosis evaluation showed that CDDPs significantly ameliorated the cardiac apoptosis level after IR. Meanwhile, CDDPs pretreatment increased myocardial autophagic flux, with upregulation of LC3B, downregulation of p62, and increased autophagosomes and autolysosomes. Moreover, the autophagic flux inhibitor chloroquine could increase IR injury, while CDDPs could partially reverse the effects. Furthermore, our results showed that the activation of AMPK/mTOR was involved in the cardioprotective effect exerted by CDDPs. Herein, we suggest that CDDPs partially protect the heart from IR injury by enhancing autophagic flux through the activation of AMPK/mTOR.

SARS-CoV-2: Ultrastructural Characterization of Morphogenesis in an In Vitro System.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted public health and the world economy and fueled a worldwide race to approve therapeutic and prophylactic agents, but so far there are no specific antiviral drugs. Understanding the biology of the virus is the first step in structuring strategies to combat it, and in this context several studies have been conducted with the aim of understanding the replication mechanism of SARS-CoV-2 in vitro systems. In this work, studies using transmission and scanning electron microscopy and 3D electron microscopy modeling were performed with the goal of characterizing the morphogenesis of SARS-CoV-2 in Vero-E6 cells. Several ultrastructural changes were observed-such as syncytia formation, cytoplasmic membrane projections, lipid droplets accumulation, proliferation of double-membrane vesicles derived from the rough endoplasmic reticulum, and alteration of mitochondria. The entry of the virus into cells occurred through endocytosis. Viral particles were observed attached to the cell membrane and in various cellular compartments, and extrusion of viral progeny took place by exocytosis. These findings allow us to infer that Vero-E6 cells are highly susceptible to SARS-CoV-2 infection as described in the literature and their replication cycle is similar to that described with SARS-CoV and MERS-CoV in vitro models.

The effect of charge-balanced transcutaneous electrical nerve stimulation on rodent facial nerve regeneration.

This study aimed to investigate the effect of charge-balanced transcutaneous electrical nerve stimulation (cb-TENS) in accelerating recovery of the facial function and nerve regeneration after facial nerve (FN) section in a rat model. The main trunk of the left FN was divided and immediately sutured just distal to the stylomastoid foramen in 66 Sprague-Dawley rats. The control group had no electrical stimulus. The other two groups received cb-TENS at 20 Hz (20 Hz group) or 40 Hz (40 Hz group). Cb-TENS was administered daily for seven days and then twice a week for three weeks thereafter. To assess the recovery of facial function, whisker movement was monitored for four weeks. Histopathological evaluation of nerve regeneration was performed using transmission electron microscopy (TEM) and confocal microscopy with immunofluorescence (IF) staining. In addition, the levels of various molecular biological markers that affect nerve regeneration were analyzed. Whisker movement in the cb-TENS groups showed faster and better recovery than the control group. The 40 Hz group showed significantly better movement at the first week after injury (p < 0.0125). In histopathological analyses using TEM, nerve axons and Schwann cells, which were destroyed immediately after the injury, recovered in all groups over time. However, the regeneration of the myelin sheath was remarkably rapid and thicker in the 20 Hz and 40 Hz groups than in the control group. Image analysis using IF staining showed that the expression levels of S100B and NF200 increased over time in all groups. Specifically, the expression of NF200 in the 20 Hz and 40 Hz groups increased markedly compared to the control group. The real-time polymerase chain reaction was performed on ten representative neurotrophic factors, and the levels of IL-1ß and IL-6 were significantly higher in the 20 and 40 Hz groups than in the control group (p < 0.015). Cb-TENS facilitated and accelerated FN recovery in the rat model, as it significantly reduced the recovery time for the whisker movement. The histopathological study and analysis of neurotrophic factors supported the role of cb-TENS in the enhanced regeneration of the FN.

A Disulfide-Stabilized Aß that Forms Dimers but Does Not Form Fibrils.

Aß dimers are a basic building block of many larger Aß oligomers and are among the most neurotoxic and pathologically relevant species in Alzheimer's disease. Homogeneous Aß dimers are difficult to prepare, characterize, and study because Aß forms heterogeneous mixtures of oligomers that vary in size and can rapidly aggregate into more stable fibrils. This paper introduces AßC18C33 as a disulfide-stabilized analogue of Aß42 that forms stable homogeneous dimers in lipid environments but does not aggregate to form insoluble fibrils. The AßC18C33 peptide is readily expressed in Escherichia coli and purified by reverse-phase HPLC to give ca. 8 mg of pure peptide per liter of bacterial culture. SDS-PAGE establishes that AßC18C33 forms homogeneous dimers in the membrane-like environment of SDS and that conformational stabilization of the peptide with a disulfide bond prevents the formation of heterogeneous mixtures of oligomers. Mass spectrometric (MS) studies in the presence of dodecyl maltoside (DDM) further confirm the formation of stable noncovalent dimers. Circular dichroism (CD) spectroscopy establishes that AßC18C33 adopts a ß-sheet conformation in detergent solutions and supports a model in which the intramolecular disulfide bond induces ß-hairpin folding and dimer formation in lipid environments. Thioflavin T (ThT) fluorescence assays and transmission electron microscopy (TEM) studies indicate that AßC18C33 does not undergo fibril formation in aqueous buffer solutions and demonstrate that the intramolecular disulfide bond prevents fibril formation. The recently published NMR structure of an Aß42 tetramer (PDB: 6RHY) provides a working model for the AßC18C33 dimer, in which two ß-hairpins assemble through hydrogen bonding to form a four-stranded antiparallel ß-sheet. It is anticipated that AßC18C33 will serve as a stable, nonfibrilizing, and noncovalent Aß dimer model for amyloid and Alzheimer's disease research.

The selective detection of Fe3+ ions using citrate-capped gold nanoparticles.

Citrate is a ubiquitous biological molecule that functions as Fe3+ chelators in some bacteria and the blood plasma of humans. Inspired by the strong affinity between citrate and Fe3+, a colorimetric Fe3+ probe based on citrate-capped AuNPs without any additional modification was designed. Citrate-capped AuNPs with a diameter of 22 nm were applied to detect Fe3+ without other reagents' assistance. This easily-prepared and low-cost colorimetric sensor exhibited good selectivity towards Fe3+ among common metal ions, a good linear relationship in the range of 0.1-0.8 µM of Fe3+ and quick response time of 10 min.

SPIRE-a software tool for bicontinuous phase recognition: application for plastid cubic membranes.

Bicontinuous membranes in cell organelles epitomize nature's ability to create complex functional nanostructures. Like their synthetic counterparts, these membranes are characterized by continuous membrane sheets draped onto topologically complex saddle-shaped surfaces with a periodic network-like structure. Their structure sizes, (around 50-500 nm), and fluid nature make transmission electron microscopy (TEM) the analysis method of choice to decipher their nanostructural features. Here we present a tool, Surface Projection Image Recognition Environment (SPIRE), to identify bicontinuous structures from TEM sections through interactive identification by comparison to mathematical "nodal surface" models. The prolamellar body (PLB) of plant etioplasts is a bicontinuous membrane structure with a key physiological role in chloroplast biogenesis. However, the determination of its spatial structural features has been held back by the lack of tools enabling the identification and quantitative analysis of symmetric membrane conformations. Using our SPIRE tool, we achieved a robust identification of the bicontinuous diamond surface as the dominant PLB geometry in angiosperm etioplasts in contrast to earlier long-standing assertions in the literature. Our data also provide insights into membrane storage capacities of PLBs with different volume proportions and hint at the limited role of a plastid ribosome localization directly inside the PLB grid for its proper functioning. This represents an important step in understanding their as yet elusive structure-function relationship.

Manual DALK in Keratoconus: An Ex Vivo Light and Transmission Electron Microscopy Analysis 2 Years After Surgery.

PURPOSE: The aim of this study was to evaluate the microscopic structure of a human cornea 2 years after manual deep anterior lamellar keratoplasty (DALK) for keratoconus with a recipient residual stromal bed thickness of 100 µm, using light and transmission electron microscopy. METHODS: A human cornea treated with manual DALK for keratoconus 2 years before was removed during penetrating keratoplasty because of stromal opacity of unknown origin, involving about half of the sample. The transparent half of the specimen was processed for light and transmission electron microscopy. RESULTS: Light microscopy examination performed with different staining techniques (hematoxylin and eosin, Picrosirius red, and Masson trichrome) revealed a homogeneous stroma. No interface was detected. Electron microscopy confirmed these findings. CONCLUSIONS: This study confirmed the available clinical and confocal studies that show progressive stromal remodeling after manual DALK. Two years after surgery, no posterior stromal interface was detected.

The perinuclear region concentrates disordered proteins with predicted phase separation distributed in a 3D network of cytoskeletal filaments and organelles.

Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.

Desenvolvimento e caracterização de lipossomas peguilados para veiculação de L-asparaginase / Development and characterization of peg-grafted liposomes for lasparaginase encapsulation

A Leucemia Linfoide Aguda (LLA) é um câncer de maior incidência em crianças, e tem a Lasparaginase (ASNase) como fármaco amplamente utilizado no tratamento dos afetados. A ASNase catalisa a hidrólise do aminoácido L-asparagina (Asn), presente na corrente sanguínea, a ausência do aminoácido no meio extracelular leva à morte células leucêmicas, que necessitam deste aminoácido para as funções celulares. Fatores envolvendo a eficiência do tratamento com ASNase como reações adversas e curta meia-vida, principalmente devido ao reconhecimento pelo sistema imune e degradação por proteases, limitam a sua eficácia. A encapsulação da enzima em lipossomas pode conferir proteção à degradação, melhorar seu perfil farmacocinético e diminuir os efeitos adversos, de forma a melhorar o tratamento da LLA sendo este o objetivo desse trabalho. Lipossomas de DOPC (1,2-dioleoil-sn-glicero-3-fosfocolina) e DMPC (1,2-dimiristoil-snglicero-3-fosfocolina) foram desenvolvidos empregando-se o método de hidratação do filme lipídico e diferentes protocolos de preparo contendo ou não diferentes concentrações de 18:0 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polietilenogicol)-2000] (DSPE-PEG). Os lipossomas produzidos foram utilizados para encapsular a ASNase e os sistemas contendo ou não ASNase encapsulada foram caracterizados por espalhamento de luz dinâmico (DLS), potencial zeta, microscopia eletrônica de transmissão (MET) e criomicroscopia de transmissão. Adicionalmente, foram avaliados a taxa de encapsulação e o perfil de permeabilidade das vesículas à L-asparagina. As análises de DLS mostraram que as nanoestruturas formadas empregando-se agitação magnética a partir de sistemas contendo 10% e 20% de DSPE-PEG possuem diâmetro hidrodinâmico menor (~ 25 nm a 60 nm) que os mesmos sistemas sem o fosfolipídio peguilado (~190 nm a 222 nm), demonstrando a relação entre a diminuição do tamanho e o aumento da quantidade de fosfolipídio peguilado e possível formação de estruturas micelares ou bicelares. O emprego de agitação em vórtex para hidratação do filme lipídico, adição do antioxidante -tocoferol e redução da concentração de DSPE-PEG (5% e 10%) levou à formação de sistemas com diâmetro hidrodinâmico maior, sendo esse protocolo e concentrações de PEG definidos como padrão. As análises de MET comprovaram a formação de lipossomas com diâmetro hidrodinâmico semelhante ao observado por DLS; com a utilização da criomicroscopia foi possível observar os lipossomas sem deformações. Os lipossomas de DMPC/DSPE-PEG 10% apresentaram maior permeabilidade à L-asparagina ao longo do tempo e, portanto, poderiam funcionar como nanoreatores, depletando o aminoácido da circulação. Estudos in vitro com células tumorais devem ser realizados e em seguida estudos in vivo, para confirmar este potencial

L-asparaginase (ASNase) is a first-choice drug, combined with other drugs, in therapeutic schemes to treat Acute Lymphoblastic Leukemia (ALL) in children and adolescents. ASNase catalyzes the hydrolysis of L-asparagine (Asn) in the bloodstream; since ALL cells cannot synthesize this amino acid, protein synthesis is impaired leading to leukemic cells death by apoptosis. In spite of its therapeutic importance, treatment with ASNase is associated to side effects, mainly hypersensitivity and immunogenicity. Another drawback refers to degradation by plasma proteases that altogether with immunogenicity shortens the enzyme half-life. Encapsulation of ASNase in liposomes, vesicular nanostructures formed by the self-aggregation of phospholipids, is an attractive alternative that possibly will protect the enzyme from plasma proteases, resulting on better pharmacokinetics profile. In this work, we prepared by thin film hydration liposomal formulations of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC) containing or not different concentrations of 18:0 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and encapsulated ASNase by electroporation. The systems containing or not ASNase were analyzed by Dynamic Light Scattering, zeta potential and Electron Microscopy. The encapsulation efficiency and vesicles permeability were also evaluated. According to the DLS analysis, the nanostructures formed by film hydration under magnetic stirring employing 10% or 20% DSPE-PEG presented smaller hydrodynamic diameter (~ 25 nm to 60 nm) than the same systems without the pegylated phospholipid (~ 190 nm to 222 nm), demonstrating the relation between size and the amount of pegylated phospholipid that results in formation of micellar or bicellar structures. The protocol was stabilize by hydration of the lipid film under vortex agitation, addition of the antioxidant - tocopherol and reduction of the concentration of DSPE-PEG (5% and 10%), what altogether led to the formation of nanostructures of higher hydrodynamic diameter and monodisperse systems. TEM analyzes confirmed the formation of liposomes with hydrodynamic diameter similar to that observed by DLS; with the use of cryomicroscopy it was possible to observe the liposomes without deformations. Liposomes of DMPC/DSPE-PEG 10% showed permeability to L-asparagine over time and, therefore, could function as nanoreactors, depleting the circulating amino acid

Therapeutic effects of benzoylaconitine and paeoniflorin in rats with collagen-induced arthritis

Abstract To explore the effects and mechanisms of benzoylaconitine and paeoniflorin on collagen-induced arthritis (CIA) rats. Weight, paw swelling, arthritis index and joint pathologic changes were examined in each group after CIA induction. PGE2, IL-1ß, IL-6, IL-10, TNF-α, VEGF, MMP-3, IgG and anti-CII Ab were assessed by ELISA; STAT1 and STAT3 expressions were analyzed immunohistochemically, and the ultrastructure of synovial cells was observed by transmission electron microscopy. Therapeutic effects were determined in CIA rats via injecting benzoylaconitine and paeoniflorin, which could alleviate the degree of swelling and arthritis index (AI) and pathological lesions of the sacroiliac gland; decrease the levels of PGE2, IL-1ß, TNF-α, VEGF and IgG in serum; reduce STAT1 and STAT3 expression in the membrane tissue; and inhibit the secretion and proliferation of synovial cells. These results showed that benzoylaconitine and paeoniflorin could significantly palliate the arthritic symptoms of CIA rats, and better therapeutic effects could be achieved if the two components were used in combination

Polystyrene-b-poly (acrylic acid) nanovesicles coated by modified chitosans for encapsulation of minoxidil

Abstract In this work, polystyrene-b-poly (acrylic acid) (PS-b-PAA) nanovesicles were coated by modified chitosans aiming at studying its physicochemical parameters. The chitosan (CS) was chemically modified to add hydrophilic and/or hydrophobic groups, obtaining three modified chitosans. The PS-b-PAA nanovesicles were obtained by organic (1,4-dioxane) cosolvent method in water, resulting in nanovesicles with less than 150 nm of diameter (polydispersibility index - PDI at 90° = 0.106), measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and negative zeta potential (-37.5 ± 3.2 mV), allowing the coating of its surface with oppositely charged polysaccharides, such as the CS and the modified chitosans. The coating process was made by mixing the colloidal suspensions with the CS and the modified chitosans at specific ENT#091;CS-xENT#093;/ENT#091;PS-b-PAAENT#093; ratios (0.001 to 1.0 wt %) and measuring the change in size and surface charge by DLS and zeta potential. Upon reaching maximum adsorption, the zeta potential parameter was positively stabilized (+26.7 ± 4.1 mV) with a hydrodynamic diameter slightly longer (< 200 nm of diameter). The encapsulation efficiency (EE) of minoxidil, quantified by capillary electrophoresis, was 50.7%, confirming their potential as drug delivery carriers and the coating process showed the possibility of controlling the surface charge nature of these nanovesicles

Transmission Electron Microscopy as a Powerful Tool to Investigate the Interaction of Nanoparticles with Subcellular Structures.

Nanomedical research necessarily involves the study of the interactions between nanoparticulates and the biological environment. Transmission electron microscopy has proven to be a powerful tool in providing information about nanoparticle uptake, biodistribution and relationships with cell and tissue components, thanks to its high resolution. This article aims to overview the transmission electron microscopy techniques used to explore the impact of nanoconstructs on biological systems, highlighting the functional value of ultrastructural morphology, histochemistry and microanalysis as well as their fundamental contribution to the advancement of nanomedicine.

Green synthesis of bimetallic ZnO-CuO nanoparticles and their cytotoxicity properties.

In this study, a simple and green strategy was reported to prepare bimetallic nanoparticles (NPs) by the combination of zinc oxide (ZnO) and copper oxide (CuO) using Sambucus nigra L. extract. The physicochemical properties of these NPs such as crystal structure, size, and morphology were studied by X-ray diffraction (XRD), field emission gun scanning electron microscopy (FEG-SEM), and transmission electron microscopy (TEM). The results suggested that these NPs contained polygonal ZnO NPs with hexagonal phase and spherical CuO NPs with monoclinic phase. The anticancer activity of the prepared bimetallic NPs was evaluated against lung and human melanoma cell lines based on MTT assay. As a result, the bimetallic ZnO/CuO NPs exhibited high toxicity on melanoma cancer cells while their toxicity on lung cancer cells was low.

The Role of the pH in the Impregnation of Spherical Mesoporous Silica Particles with L-Arginine Aqueous Solutions.

In the context of the development of carriers for amino acids delivery, Spherical Mesoporous Silica Particles (SMSP), characterized by particles size ranging from 0.15 µm to 0.80 µm and average pore diameter of 2.4 nm, were synthesised and loaded with L-arginine (ARG), a basic amino acid involved in several physiological processes. The loading was performed using water as a solvent through the wet impregnation method (with a final arginine content of 9.1% w/w). The material was characterized before and after impregnation by means of X-Ray Diffraction (XRD), nitrogen sorption analysis, Field Emission Scanning Electron Microscopy (FESEM) and Fourier Transform Infrared (FT-IR) spectroscopy. SMSP are shown to suffer degradation upon impregnation, which dramatically affects their porosity. To elucidate the role of the pH of the ARG impregnating solution (originally set at pH ≈ 11) on SMSP degradation, the loading was performed under different pH conditions (5 and 9) keeping constant the ARG concentration. The impregnation performed with acidic solution did not modify the carrier. All samples displayed ARG in amorphous form: zwitterionic species were present in SMSP impregnated at basic pH whereas positive protonated species in that impregnated at acidic pH.

A Potential New Role for Zinc in Age-Related Macular Degeneration through Regulation of Endothelial Fenestration.

Age-related macular degeneration (AMD) is a common blinding disease in the western world that is linked to the loss of fenestration in the choriocapillaris that sustains the retinal pigment epithelium and photoreceptors in the back of the eye. Changes in ocular and systemic zinc concentrations have been associated with AMD; therefore, we hypothesized that these changes might be directly involved in fenestrae formation. To test this hypothesis, an endothelial cell (bEND.5) model for fenestrae formation was treated with different concentrations of zinc sulfate (ZnSO4) solution for up to 20 h. Fenestrae were visualized by staining for Plasmalemmal Vesicle Associated Protein-1 (PV-1), the protein that forms the diaphragms of the fenestrated endothelium. Size and distribution were monitored by transmission electron microscopy (TEM). We found that zinc induced the redistribution of PV-1 into areas called sieve plates containing ~70-nm uniform size and typical morphology fenestrae. As AMD is associated with reduced zinc concentrations in the serum and in ocular tissues, and dietary zinc supplementation is recommended to slow disease progression, we propose here that the elevation of zinc concentration may restore choriocapillaris fenestration resulting in improved nutrient flow and clearance of waste material in the retina.

Ultra-Fine Control of Silica Shell Thickness on Silver Nanoparticle-Assembled Structures.

To study the distance-dependent electromagnetic field effects related to the enhancement and quenching mechanism of surface-enhanced Raman scattering (SERS) or fluorescence, it is essential to precisely control the distance from the surface of the metal nanoparticle (NP) to the target molecule by using a dielectric layer (e.g., SiO2, TiO2, and Al2O3). However, precisely controlling the thickness of this dielectric layer is challenging. Herein, we present a facile approach to control the thickness of the silica shell on silver nanoparticle-assembled silica nanocomposites, SiO2@Ag NPs, by controlling the number of reacting SiO2@Ag NPs and the silica precursor. Uniform silica shells with thicknesses in the range 5-40 nm were successfully fabricated. The proposed method for creating a homogeneous, precise, and fine silica coating on nanocomposites can potentially contribute to a comprehensive understanding of the distance-dependent electromagnetic field effects and optical properties of metal NPs.

Scleroderma-like Impairment in the Network of Telocytes/CD34+ Stromal Cells in the Experimental Mouse Model of Bleomycin-Induced Dermal Fibrosis.

Considerable evidence accumulated over the past decade supports that telocytes (TCs)/CD34+ stromal cells represent an exclusive type of interstitial cells identifiable by transmission electron microscopy (TEM) or immunohistochemistry in various organs of the human body, including the skin. By means of their characteristic cellular extensions (telopodes), dermal TCs are arranged in networks intermingled with a multitude of neighboring cells and, hence, they are thought to contribute to skin homeostasis through both intercellular contacts and releasing extracellular vesicles. In this context, fibrotic skin lesions from patients with systemic sclerosis (SSc, scleroderma) appear to be characterized by a disruption of the dermal network of TCs, which has been ascribed to either cell degenerative processes or possible transformation into profibrotic myofibroblasts. In the present study, we utilized the well-established mouse model of bleomycin-induced scleroderma to gain further insights into the TC alterations found in cutaneous fibrosis. CD34 immunofluorescence revealed a severe impairment in the dermal network of TCs/CD34+ stromal cells in bleomycin-treated mice. CD31/CD34 double immunofluorescence confirmed that CD31-/CD34+ TC counts were greatly reduced in the skin of bleomycin-treated mice compared with control mice. Ultrastructural signs of TC injury were detected in the skin of bleomycin-treated mice by TEM. The analyses of skin samples from mice treated with bleomycin for different times by either TEM or double immunostaining and immunoblotting for the CD34/α-SMA antigens collectively suggested that, although a few TCs may transition to α-SMA+ myofibroblasts in the early disease stage, most of these cells rather undergo degeneration, and then are lost. Taken together, our data demonstrate that TC changes in the skin of bleomycin-treated mice mimic very closely those observed in human SSc skin, which makes this experimental model a suitable tool to (i) unravel the pathological mechanisms underlying TC damage and (ii) clarify the possible contribution of the TC loss to the development/progression of dermal fibrosis. In perspective, these findings may have important implications in the field of skin regenerative medicine.

Thermoresponsive polymer assemblies via variable temperature liquid-phase transmission electron microscopy and small angle X-ray scattering.

Herein, phase transitions of a class of thermally-responsive polymers, namely a homopolymer, diblock, and triblock copolymer, were studied to gain mechanistic insight into nanoscale assembly dynamics via variable temperature liquid-cell transmission electron microscopy (VT-LCTEM) correlated with variable temperature small angle X-ray scattering (VT-SAXS). We study thermoresponsive poly(diethylene glycol methyl ether methacrylate) (PDEGMA)-based block copolymers and mitigate sample damage by screening electron flux and solvent conditions during LCTEM and by evaluating polymer survival via post-mortem matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). Our multimodal approach, utilizing VT-LCTEM with MS validation and VT-SAXS, is generalizable across polymeric systems and can be used to directly image solvated nanoscale structures and thermally-induced transitions. Our strategy of correlating VT-SAXS with VT-LCTEM provided direct insight into transient nanoscale intermediates formed during the thermally-triggered morphological transformation of a PDEGMA-based triblock. Notably, we observed the temperature-triggered formation and slow relaxation of core-shell particles with complex microphase separation in the core by both VT-SAXS and VT-LCTEM.