The increased accessibility of soft-tissue data through diffusible iodine-based contrast-enhanced computed tomography (diceCT) enables comparative biologists to increase the taxonomic breadth of their studies with museum specimens. However, it is still unclear how soft-tissue measurements from preserved specimens reflect values from freshly collected specimens and whether diceCT preparation may affect these measurements. Here, we document and evaluate the accuracy of diceCT in museum specimens based on the soft-tissue reconstructions of brains and eyes of five bats. Based on proxies, both brains and eyes were roughly 60% of the estimated original sizes when first imaged. However, these structures did not further shrink significantly over a 4-week staining interval, and 1 week in 2.5% iodine-based solution yielded sufficient contrast for differentiating among soft-tissues. Compared to six “fresh” bat specimens imaged shortly after field collection (not fixed in ethanol), the museum specimens had significantly lower relative volumes of the eyes and brains. Variation in field preparation techniques and conditions, and long-term storage in ethanol may be the primary causes of shrinkage in museum specimens rather than diceCT staining methodology. Identifying reliable tissue-specific correction factors to adjust for the shrinkage now documented in museum specimens requires future work with larger samples.

The distribution of lignin, 8-5′ and 8-8′ linked lignin substructure, and noncellulosic polysaccharides in hemp (Cannabis sativa L.) phloem fibers were explored based on histochemical and immunological methods. Ultraviolet absorption and potassium permanganate staining were observed mainly in the compound middle lamella (CML) and S1 layers, and rarely in the G-layer of phloem fibers, suggesting that lignin concentration is high at the CML and S1 layers, and very low at the G-layer of hemp fibers. Acriflavine staining, uniform KM1 labeling (8-5′ linked lignin substructure), and no KM2 labeling (8-8′ linked structure) were observed in the G-layer, suggesting that there is a small amount of lignin-like compound with 8-5′ linked structure in the G-layer. In addition, some fiber cells showed a multilayered structure. Uniform arabinogalactan protein (AGP) labeling was observed on the S1 layers and G-layers using JIM14, but little appeared in the CML of hemp fibers, indicating that these layers of the phloem fibers contain AGP. Immunogold labeling of xylan (LM11) and glucomannan (LM21) showed that xylan and glucomannan were mainly present in the S1 layers and the G-layers, respectively. In some phloem fibers, LM21 immunofluorescence labeling showed multilayered structure, suggesting the heterogeneous distribution of glucomannan.

The intracellular concentration of calcium ion ([Ca2+]i) is a critical regulator of cell signaling and contractility of vascular smooth muscle cells (VSMCs). In this study, we employed an atomic force microscopy (AFM) nanoindentation-based approach to investigate the role of [Ca2+]i in regulating the cortical elasticity of rat cremaster VSMCs and the ability of rat VSMCs to adhere to fibronectin (Fn) matrix. Elevation of [Ca2+]i by ionomycin treatment increased rat VSMC stiffness and cell adhesion to Fn-biofunctionalized AFM probes, whereas attenuation of [Ca2+]i by 1,2-Bis (2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) treatment decreased the mechanical and matrix adhesive properties of VSMCs. Furthermore, we found that ionomycin/BAPTA-AM treatments altered expression of α5 integrin subunits and α smooth muscle actin in rat VSMCs. These data suggest that [Ca2+]i regulates VSMC elasticity and adhesion to the extracellular matrix by a potential mechanism involving changing dynamics of the integrin–actin cytoskeleton axis.

Emerging evidence from various studies indicates that plasmid DNA (pDNA) is internalized by cells through an endocytosis-like process when it is used for electrotransfection. To provide morphological evidence of the process, we investigated ultrastructures in cells that were associated with the electrotransfected pDNA, using immunoelectron microscopy. The results demonstrate that four endocytic pathways are involved in the uptake of the pDNA, including caveolae- and clathrin-mediated endocytosis, macropinocytosis, and the clathrin-independent carrier/glycosylphosphatidylinositol-anchored protein-enriched early endosomal compartment (CLIC/GEEC) pathway. Among them, macropinocytosis is the most common pathway utilized by cells having various pDNA uptake capacities, and the CLIC/GEEC pathway is observed primarily in human umbilical vein endothelial cells. Quantitatively, the endocytic pathways are more active in easy-to-transfect cells than in hard-to-transfect ones. Taken together, our data provide ultrastructural evidence showing that endocytosis plays an important role in cellular uptake and intracellular transport of electrotransfected pDNA.

A scanning electron microscopy (SEM) investigation of pine (Pinus sylvestris) and oak (Quercus sp.) wood samples exposed to various types of natural degradation is presented with the aim of discussing the correct identification of multiple degradation signs in waterlogged wood. This is part of an experiment performed at the archeological site of Biskupin (Poland) to evaluate the dynamics of short-term wood degradation during reburial and the suitability of excavated wood as substrate for the fungal attack. The final aim is to support and inform the in situ conservation strategy currently applied to archeological woods. To replicate the burial conditions, wood samples were put into lake water and peat. The samples were removed from the burial environments after 4, 6, 8, and 10 years, and then exposed to laboratory-controlled attack by a brown rot fungus Coniophora puteana and a white rot fungus Coriolus versicolor. SEM images were acquired for all samples before and after the fungal attack. The results showed a slight degradation occurred in the burial environments (soft rot and bacteria). In addition, both typical and previously neglected features of fungal attack were observed, highlighting that the extent of the fungal decay varies according to the previous degree of wood degradation. Some comparisons are provided with archeological wood samples from the Biskupin site.

Polistes paper wasps can be used to monitor trace metal contaminants, but the effects of pollution on the health of these insects are still unknown. We evaluated, in a south-eastern area of Spain, whether workers of Polistes dominula collected at urban and rural sites differ in health of midgut tissue and in fluctuating asymmetry, an estimate of developmental noise. We found that wasps collected at the urban sites had abundant lead (Pb)-containing spherites, which were less visible in wasps from the rural sites. Evident ultrastructural alterations in the epithelium of the midgut of the wasps collected at the urban sites included broken and disorganized microvilli, a high amount and density of heterochromatin in the nucleus of epithelial cells, cytoplasmic vacuolization and mitochondrial disruptions. Altogether, these findings suggest a negative effect on the transmembrane transport and a less efficient transcription. On the contrary, a healthy epithelium was observed in wasps from the rural sites. These differences may be preliminarily linked with levels of lead pollution, given that wasps from urban sites had double the Pb concentrations of wasps from rural sites. Level of fluctuating asymmetry was unrelated to wasp origin, thus suggesting no link between developmental noise and Pb-driven pollution.

Venous thrombosis is associated with combined oral contraceptive (COC) use. We investigated the impact of two ethinyl estradiol (EE) and drospirenone (DRSP) containing COCs (3 mg DRSP/20 µg EE and 3 µg DRSP/30 µg EE) on the viscoelasticity of whole blood clots along with the biophysical and biochemical characteristics of erythrocytes. Thromboelastography (TEG) analysis showed a tendency toward a hypercoagulable state in the COCs groups that was more pronounced with higher EE concentrations. Light microscopy and scanning electron microscopy (SEM) showed rouleaux formation of erythrocytes and alterations to the erythrocyte shape for both COC groups, which was attributed to membrane damage. SEM analysis showed spontaneous activation of fibrin and platelets in the COC groups, along with interactions between erythrocytes and platelets and/or fibrin. Confocal microscopy confirmed compromised membrane integrity in the COC groups compared to controls. Global thrombosis test analysis showed increased platelet activation and low thrombolysis in both COC groups when compared to controls. In conclusion, DRSP/EE formulations impact erythrocytes’ biophysical and biochemical properties to cause a shift in hemostasis to a prothrombotic state. Although these effects are mostly subclinical the long-term effects and risks involved with the use of these hormones should be considered carefully for each individual.

The production of Amarone wine is governed by a disciplinary guideline to preserve its typical features; however, postharvest infections by the fungus Botrytis cinerea (B. cinerea) not only represent a phytosanitary problem but also cause a significant loss of product. In this study, we tested a treatment with mild ozoniztion on grapes for Amarone wine production during withering in the fruttaio (the environment imposed by the disciplinary guideline) and evaluated the impact on berry features by a multimodal imaging approach. The results indicate that short and repeated treatments with low O3 concentrations speed up the naturally occurring berry withering, probably inducing a reorganization of the epicuticular wax layer, and inhibit the development of B. cinerea, blocking the fungus in an intermediate vegetative stage. This pilot study will pave the way to long-term research on Amarone wine obtained from O3-treated grapes.

The present study planned to assess the medicinally important plants in the lower Margalla Hills of Islamabad, Pakistan using scanning electron microscopy of their pollens. Both quantitative and qualitative characters of the pollens were analyzed. The shapes of the pollen were prolate, spheroidal, elliptical, and oblate. Exine sculpturing showed rugulate, scabrate, psilate gemmate, and reticulate ornamentation. The people residing in the rural areas are thought to depend, primarily, on medicinal plant resources for many health care needs. A sample of 140 respondents was selected and interviewed by using a stratified random sampling technique. Results showed that the local communities of lower Margalla Hills are highly dependent on plants to treat various ailments, for example, respiratory (28%), digestive (46%) nervous, circulatory and dermatological problems (6% each), and reproductive problems (8%). The continuous dependence of the local community on ethnomedicinally important plants has led to serious degradation of the natural forest of lower Margalla Hills. Priority-based conservation and sustainable use of these forest resources are necessary, and use of alternative resources will also help in the forest conservation.

This work explores the combination of µ-Raman spectroscopy and scanning electron microscopy with X-ray energy dispersive spectrometry (SEM-EDS) for the study of the glazes in 15th–16th century Hispano-Moresque architectural tiles. These are high lead glazes that can be tin-opacified or transparent, and present five colors: tin-white, cobalt-blue, copper-green, iron-amber, and manganese-brown. They are generally homogenous and mineral inclusions are mostly concentrated in the glaze-ceramic interface. Through SEM-EDS, these inclusions were observed and chemically analyzed, whereas µ-Raman allowed their identification on a molecular level. K-feldspars, wollastonite and diopside were the most common compounds, as well as cassiterite agglomerates that render the glaze opaque. Malayaite was identified in green glazes, and andradite and magnesioferrite in amber glazes. Co–Ni–ferrites were identified in blue glazes, as well as Ni–Fe–olivines. Manganese-brown is the color where most compounds were identified: bustamite, jacobsite, hausmannite, braunite, and kentrolite. Through the µ-Raman analysis of different areas in large inclusions previously observed by SEM, it was possible to identify intermediate phases that illustrate the reaction process that occurs between the color-conferring compounds and the surrounding lead glaze. Furthermore, the obtained results allowed inference of the raw materials and firing temperatures used on the manufacture of these tiles.