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Silver nanoparticles (AgNPs) are particularly among the widely used nanomaterials in medicine, industry, and agriculture. The small size and large surface area of AgNPs and other nanomaterials result in their high reactivity in biological systems. To better understand the effects of AgNPs on plants at the molecular level, tomato (Lycopersicon esculentum L.) seedlings were exposed to 30 mg/L silver in the form of nanoparticle (AgNPs), ionic (AgNO3), or bulk (Ag0) in 50% Hoagland media for 7 days. The effects of silver on the expression of plant membrane transporters H+-ATPase, vacuolar type H+-ATPase (V-ATPase), and enzymes isocitrate dehydrogenase (IDH), and catalase in roots was assessed using RT-qPCR and immunofluorescence-confocal microscopy. We observed significantly higher expression of catalase in plants exposed to AgNPs (Fold of expression 1.1) and AgNO3 (Fold of expression 1.2) than the control group. The immunofluorescence imaging of the proteins confirmed the gene expression data; the expression of the enzyme catalase was upregulated 41, 216, and 770% higher than the control group in plants exposed to AgNPs, Ag0, and AgNO3, respectively. Exposure to AgnO3 resulted in the upregulation (fold of expression 1.2) of H+-ATPase and downregulation (fold of expression 0.7) of V-ATPase. A significant reduction in the expression of the redox-sensitive tricarboxylic cycle (TCA) enzyme mitochondrial IDH was observed in plants exposed to AgNPs (38%), AgNO3 (48%), or Ag0 (77%) compared to the control. This study shows that exposure to silver affects the expression of genes and protein involved in membrane transportation and oxidative response. The ionic form of silver had the most significant effect on the expression of genes and proteins compared to other forms of silver. The results from this study improve our understanding about the molecular effects of different forms of silver on important crop species. Novelty statement Silver nanoparticles released into the environment can be oxidized and be transformed into ionic form. Both the particulate and ionic forms of silver can be taken by plants and affect plants physiological and molecular responses. Despite the extensive research in this area, there is a scarce of information about the effects of silver nanoparticles on the expression of membrane transporters especially H+-ATPase involved in regulating cells' electrochemical charge, and the activity of enzymes involved in oxidative stress responses. This is a unique study that evaluates the expression of cellular proton transporters and enzymes of redox balance and energy metabolisms such as membrane transporters, H+-ATPase, and V-ATPases, and enzymes catalase and IDH. The results provide us valuable information about the impact of silver on plants at the molecular level by evaluating the expression of genes and proteins. The exposure of plants to silver as an environmental stressor affects the expression of genes and proteins involved in maintaining cell's electrochemical gradient (H+-ATPase, V-ATPase) and redox potential (IDH, catalase). [ABSTRACT FROM AUTHOR]

The assay presented here was designed to assess the immediate effects of ethanol (EtOH) exposure on intracellular signaling activated by BMPs (Bone Morphogenetic Proteins). Previous reports of the relationship between EtOH exposure and BMP-dependent signaling have primarily assessed the expression of individual BMPs, changes in BMP target genes or effects on the phosphorylation level of key downstream mediators after days or weeks of in vivo EtOH exposure. What happens to BMP-stimulated signaling immediately following exposure to EtOH remains largely unexplored. Here, the early events of BMP-evoked intracellular signaling were examined in an in vitro model of acute EtOH toxicity. The BMP/Ethanol Stimulation Assay involved first stimulating cultured cells with recombinant BMPs. BMP-evoked intracellular signaling was then allowed to develop for 30 minutes. Next, the cells were exposed to a range of EtOH concentrations for an additional 30 minutes. Finally, the cultures were processed for Western blot analysis or immunofluorescent labeling.This short-term assay:• Permits investigation of EtOH exposure during the initial signaling events downstream of BMP receptor activation• Enables assessment of how the presence of BMPs might protect against cellular injury caused by toxic EtOH levels

Barley (Hordeum vulgare L.) plants accumulate glycine betaine (GB), a major compatible solute, in response to salt stress. In barley, GB is produced by a two-step oxidation of choline in a cooperative way in the cytosol and peroxisomes. In this study, we investigated the localization of two GB biosynthetic enzymes, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), in the tissues of barley plants (cv. Haruna-nijyo) grown under normal and saline conditions. Three-week-old barley plants grown hydroponically were treated with a hydroponic culture solution containing 200 mM NaCl for 72 h. Salt treatment resulted in increased expression of CMO and BADH proteins mainly in the leaves of barley but not in the roots. The expression of CMO protein was increased by the presence of NaCl in younger leaves but decreased in older leaves. The tissue localization of CMO and BADH proteins was analyzed by immunofluorescent labeling method using their primary antibodies and a fluorescein-conjugated secondary antibody. CMO and BADH proteins were constitutively co-localized in mesophyll and bundle sheath cells under both normal and saline conditions. A possible physiological function of GB in the salt tolerance of barley plants is discussed.

Abstract: Thioglycolic-acid-stabilized CdTe quantum dots, synthesized directly in aqueous solution, are successfully conjugated with biotin and polyethylene glycol. Using these conjugates, we report the development of this kind of water-soluble quantum dot for immunofluorescent labeling of cancer cells. The results show that these conjugates have very low nonspecific binding and good stability against photobleaching, enabling them to be applied in many biological fields, such as cellular labeling, intracellular tracking, and other imaging applications. [Copyright &y& Elsevier]

Summary • Since mycorrhizal fungi constitute an important component of the soil–plant interface, their responses to changes in nutrient availability may mediate shifts in ecosystem function. We tested the hypothesis that initial soil nutrient availability may determine effects of nitrogen (N) and phosphorus (P) additions on the growth and community of arbuscular mycorrhizal (AM) fungi. • Extraradical hyphal lengths and degree of root colonization of AM fungi were measured in control and fertilized plots along a soil fertility gradient in Hawaii. Responses of individual AM genera were assessed through immunofluorescent labeling. • The AM biomass was increased by N and P additions in the N- and P-limited sites, respectively, and reduced by P fertilization in the fertile site only. The abundance of Scutellospora was lower under N than under P fertilization, whereas the incidence of Glomus was higher in the fertile site than the N-limited site. Gigaspora and Acaulospora did not vary among sites or treatments. • Our results indicate that a decrease in AM abundance following nutrient additions cannot be assumed to occur and the effects may differ among AM genera and ecosystems with varying soil nutrients. Limitation of N and P may be one possible explanation. [ABSTRACT FROM AUTHOR]

The assay presented here was designed to assess the immediate effects of ethanol (EtOH) exposure on intracellular signaling activated by BMPs (Bone Morphogenetic Proteins). Previous reports of the relationship between EtOH exposure and BMP-dependent signaling have primarily assessed the expression of individual BMPs, changes in BMP target genes or effects on the phosphorylation level of key downstream mediators after days or weeks of in vivo EtOH exposure. What happens to BMP-stimulated signaling immediately following exposure to EtOH remains largely unexplored. Here, the early events of BMP-evoked intracellular signaling were examined in an in vitro model of acute EtOH toxicity. The BMP/Ethanol Stimulation Assay involved first stimulating cultured cells with recombinant BMPs. BMP-evoked intracellular signaling was then allowed to develop for 30 minutes. Next, the cells were exposed to a range of EtOH concentrations for an additional 30 minutes. Finally, the cultures were processed for Western blot analysis or immunofluorescent labeling. This short-term assay: • Permits investigation of EtOH exposure during the initial signaling events downstream of BMP receptor activation • Enables assessment of how the presence of BMPs might protect against cellular injury caused by toxic EtOH levels., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Immunofluorescent staining (IF) uses antigen-antibody complexes tagged with fluorochromes to observe the expression of proteins within a tissue sample. Multiple groups have described optimized methods to visualize several proteins simultaneously within the same tissue section using immunofluorescence in both mouse and human FFPE tissues. Our group routinely uses an optimized protocol described here to examine nuclear receptor expression in experimental samples from conditional knockout in vivo studies.

Etheostoma is a genus of North American darter fish whose species have similar habitats and breeding seasons, yet hybridization is rare. Behavioral barriers have been demonstrated to play a key role in maintaining species boundaries. Further, conspecific (same species) sperm precedence has also been observed when the gametes of two different species come into contact. In this study, we investigated if physical characteristics of sperm could be a mechanism for the lower fertilization success of heterospecific (different species) males when eggs are simultaneously exposed to conspecific and heterospecific sperm. We chose to examine the sperm of two closely related species, E. zonale and E. barrenense. Using toluidine blue and immunofluorescent labeling methods, we compared head diameter and tail length of sperm cells between the two species. We found that head diameter was significantly larger for E. barrenense sperm compared to E. zonale . This difference in cell morphology may point to a physical mechanism underlying conspecific sperm precedence in Etheostoma . Our results are the first to describe a morphological difference in sperm between species in this genus and provide initial evidence for the role of sperm morphology in prezygotic reproductive isolation., Competing Interests: DISCLOSURE The authors certify that they have no conflict of interest with the subject matter of this manuscript.

Objectives To identify proteoglycans (PGs) and collagen fibrils (CF) within human dentin by means of a dual immunofluorescent labeling technique and to investigate the monomer infiltration of two etch-and-rinse adhesives to tosyl-phenylalanine chloromethyl-ketone (TPCK)-treated trypsin (TRY)-pretreated dentin. Methods Thirty-micrometer sections of middle coronal dentin were obtained and etched with 37% phosphoric acid gel for 15 s. After preconditioning with or without TRY digestion, the sections were subjected to dual immunofluorescent labeling and observed with a confocal laser scanning microscope (CLSM). Demineralized dentin matrixes treated with or without TRY were observed with field emission scanning electron microscope (FE-SEM). Two etch-and-rinse adhesives, Adper Single Bond 2 (SB) and Prime & Bond NT (PBNT), were applied to the dentin surfaces that were pretreated with or without TRY. The thickness of the hybrid layers was evaluated under confocal micro-Raman spectroscopy and analyzed with a two-way ANOVA. Results Green and red fluorescence was used to represent the PGs and the CF that were colocalized in the same section with different distributions. PGs were localized in the lumens of the dentin tubules and in peritubular dentin, while the type-I collagen fibrils were localized in intertubular dentin and peritubular dentin. After preconditioning with TRY digestion, the red fluorescence decreased or disappeared, the organic filaments in the lumens of the dentin tubules disappeared, the tubules were enlarged, and the hybrid layer thickness for adhesives bonded to the TRY-pretreated dentin surfaces were significantly increased ( p < 0.001 for both SB and PBNT). Significance The dual immunofluorescence labeling methodology can be used to study the human dentin matrix without decalcifying the entire dentin fragment. Proteoglycans were localized in the lumens of the dentin tubules and in peritubular dentin, which could depress the infiltration of the adhesive resin monomers. The use of TRY digestion increased the thickness of the hybrid layer created by the tested two-step etch-and-rinse adhesive. [ABSTRACT FROM AUTHOR]

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.

Eleven phosphodiesterase (PDE) families are known, each having several different isoforms and splice variants. Recent evidence indicates that expression of individual PDE family members is tissue-specific. Little is known concerning detailed PDE component expression in brain microvessels where the blood-brain-barrier and the local cerebral blood flow are thought to be regulated by PDEs. The present study attempted to identify PDE family members that are expressed in brain microvessels. Adult male F344 rats were sacrificed and blocks of the cerebral cortex and infratentorial areas were dissected. Microvessels were isolated using a filtration method, and total RNA was extracted. RNA quality and quantity were determined using an Agilent bioanalyzer. The isolated cortical and infratentorial microvessel total RNA amounts were 2720 ± 750 ng (n = 2) and 250 ± 40 ng (n = 2), respectively. Microarrays with 22 000 transcripts demonstrated that there were 16 PDE transcripts in the PDE superfamily, exhibiting quantifiable density in the microvessels. An additional immunofluorescent study verified that PDE4D (cAMP-specific) and PDE5A (cGMP-specific) were colocalized with RECA-1 (an endothelial marker) in the cerebral cortex using both F344 rats and Sprague-Dawley rats (n = 3-6/strain). In addition, PDE4D and PDE5A were found to be colocalized with alpha-smooth muscle actin which delineates cerebral arteries and arterioles as well as pericytes. In conclusion, a filtration method followed by microarray analyses allows PDE components to be identified in brain microvessels, and confirmed that PDE4D and PDE5A are the primary forms expressed in rat brain microvessels.