Histochemistry for Molecular Imaging in Nanomedicine.

All the nanotechnological devices designed for medical purposes have to deal with the common requirement of facing the complexity of a living organism. Therefore, the development of these nanoconstructs must involve the study of their structural and functional interactions and the effects on cells, tissues, and organs, to ensure both effectiveness and safety. To this aim, imaging techniques proved to be extremely valuable not only to visualize the nanoparticles in the biological environment but also to detect the morphological and molecular modifications they have induced. In particular, histochemistry is a long-established science able to provide molecular information on cell and tissue components in situ, bringing together the potential of biomolecular analysis and imaging. The present review article aims at offering an overview of the various histochemical techniques used to explore the impact of novel nanoproducts as therapeutic, reconstructive and diagnostic tools on biological systems. It is evident that histochemistry has been playing a leading role in nanomedical research, being largely applied to single cells, tissue slices and even living animals.

A single protective polymorphism in the prion protein blocks cross-species prion replication in cultured cells.

The bank vole (BV) prion protein (PrP) can function as a universal acceptor of prions. However, the molecular details of BVPrP's promiscuity for replicating a diverse range of prion strains remain obscure. To develop a cultured cell paradigm capable of interrogating the unique properties of BVPrP, we generated monoclonal lines of CAD5 cells lacking endogenous PrP but stably expressing either hamster (Ha), mouse (Mo), or BVPrP (M109 or I109 polymorphic variants) and then challenged them with various strains of mouse or hamster prions. Cells expressing BVPrP were susceptible to both mouse and hamster prions, whereas cells expressing MoPrP or HaPrP could only be infected with species-matched prions. Propagation of mouse and hamster prions in cells expressing BVPrP resulted in strain adaptation in several instances, as evidenced by alterations in conformational stability, glycosylation, susceptibility to anti-prion small molecules, and the inability of BVPrP-adapted mouse prion strains to infect cells expressing MoPrP. Interestingly, cells expressing BVPrP containing the G127V prion gene variant, identified in individuals resistant to kuru, were unable to become infected with prions. Moreover, the G127V polymorphic variant impeded the spontaneous aggregation of recombinant BVPrP. These results demonstrate that BVPrP can facilitate cross-species prion replication in cultured cells and that a single amino acid change can override the prion-permissive nature of BVPrP. This cellular paradigm will be useful for dissecting the molecular features of BVPrP that allow it to function as a universal prion acceptor.

Impact of antigen retrieval protocols on the immunohistochemical detection of epigenetic DNA modifications.

This study compares three different pretreatment protocols for the immunohistochemical detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. The human biological samples analyzed included formalin-fixed and paraffin-embedded (FFPE) normal squamous epithelium, ethanol-fixed cultured cells, and metaphase chromosomes. The antigen retrieval methods included low pH Citrate and high pH Tris-ethylenediaminetetraacetic acid (EDTA) protocols, as well as a method using Pepsin pretreatment combined with HCl for DNA denaturation. A gradual increase in the detection levels of 5-mC and 5-hmC was observed when going from Citrate via Tris/EDTA to Pepsin/HCl retrieval. While the Citrate retrieval protocol was the least efficient for the detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled visualization of differences in intra- and internuclear distribution patterns in tissue and cell culture samples by single- and double-fluorescence detection. Quantification of (hydroxy)methylation levels in FFPE material demonstrated a significant heterogeneity and differences in 5-mC and 5-hmC levels within and between nuclei in the different compartments of normal squamous epithelium. It was concluded that immunohistochemical detection of 5-mC and 5-hmC enables the correlation of these DNA modifications with histomorphological features in heterogeneous tissues, but this is influenced by different pretreatment protocols that must be carefully chosen to allow an appropriate interpretation of these epigenetic switches.

Irinotecan/scFv co-loaded liposomes coaction on tumor cells and CAFs for enhanced colorectal cancer therapy.

BACKGROUND: Cancer-associated fibroblasts (CAFs), as an important component of stroma, not only supply the "soils" to promote tumor invasion and metastasis, but also form a physical barrier to hinder the penetration of therapeutic agents. Based on this, the combinational strategy that action on both tumor cells and CAFs simultaneously would be a promising approach for improving the antitumor effect. RESULTS: In this study, the novel multifunctional liposomes (IRI-RGD/R9-sLip) were designed, which integrated the advantages including IRI and scFv co-loading, different targets, RGD mediated active targeting, R9 promoting cell efficient permeation and lysosomal escape. As expected, IRI-RGD/R9-sLip showed enhanced cytotoxicity in different cell models, effectively increased the accumulation in tumor sites, as well as exhibited deep permeation ability both in vitro and in vivo. Notably, IRI-RGD/R9-sLip not only exhibited superior in vivo anti-tumor effect in both CAFs-free and CAFs-abundant bearing mice models, but also presented excellent anti-metastasis efficiency in lung metastasis model. CONCLUSION: In a word, the novel combinational strategy by coaction on both "seeds" and "soils" of the tumor provides a new approach for cancer therapy, and the prepared liposomes could efficiently improve the antitumor effect with promising clinical application prospects.

Optogenetic Potentials of Diverse Animal Opsins: Parapinopsin, Peropsin, LWS Bistable Opsin.

Animal opsin-based pigments are light-activated G-protein-coupled receptors (GPCRs), which drive signal transduction cascades via G-proteins. Thousands of animal opsins have been identified, and molecular phylogenetic and biochemical analyses have revealed the unexpected diversity in selectivity of G-protein activation and photochemical property. Here we discuss the optogenetic potentials of diverse animal opsins, particularly recently well-characterized three non-canonical opsins, parapinopsin, peropsin, and LWS bistable opsin. Unlike canonical opsins such as vertebrate visual opsins that have been conventionally used for optogenetic applications, these opsins are bistable; opsin-based pigments do not release the chromophore retinal after light absorption, and the stable photoproducts revert to their original dark states upon subsequent light absorption. Parapinopsins have a "complete photoregeneration ability," which allows a clear color-dependent regulation of signal transductions. On the other hand, peropsins serve as a "dark-active and light-inactivated" GPCR to regulate signal transductions in the opposite way compared with usual opsins. In addition, an LWS bistable opsin from a butterfly was revealed to be the longest wavelength-sensitive animal opsin with its absorption maximum at ~570 nm. The property-dependent optical regulations of signal transductions were demonstrated in mammalian cultured cells, showing potentials of new optogenetic tools.

Manmade Electromagnetic Fields and Oxidative Stress-Biological Effects and Consequences for Health.

Concomitant with the ever-expanding use of electrical appliances and mobile communication systems, public and occupational exposure to electromagnetic fields (EMF) in the extremely-low-frequency and radiofrequency range has become a widely debated environmental risk factor for health. Radiofrequency (RF) EMF and extremely-low-frequency (ELF) MF have been classified as possibly carcinogenic to humans (Group 2B) by the International Agency for Research on Cancer (IARC). The production of reactive oxygen species (ROS), potentially leading to cellular or systemic oxidative stress, was frequently found to be influenced by EMF exposure in animals and cells. In this review, we summarize key experimental findings on oxidative stress related to EMF exposure from animal and cell studies of the last decade. The observations are discussed in the context of molecular mechanisms and functionalities relevant to health such as neurological function, genome stability, immune response, and reproduction. Most animal and many cell studies showed increased oxidative stress caused by RF-EMF and ELF-MF. In order to estimate the risk for human health by manmade exposure, experimental studies in humans and epidemiological studies need to be considered as well.

Cellular models for discovering prion disease therapeutics: Progress and challenges.

Prions, which cause fatal neurodegenerative disorders such as Creutzfeldt-Jakob disease, are misfolded and infectious protein aggregates. Currently, there are no treatments available to halt or even delay the progression of prion disease in the brain. The infectious nature of prions has resulted in animal paradigms that accurately recapitulate all aspects of prion disease, and these have proven to be instrumental for testing the efficacy of candidate therapeutics. Nonetheless, infection of cultured cells with prions provides a much more powerful system for identifying molecules capable of interfering with prion propagation. Certain lines of cultured cells can be chronically infected with various types of mouse prions, and these models have been used to unearth candidate anti-prion drugs that are at least partially efficacious when administered to prion-infected rodents. However, these studies have also revealed that not all types of prions are equal, and that drugs active against mouse prions are not necessarily effective against prions from other species. Despite some recent progress, the number of cellular models available for studying non-mouse prions remains limited. In particular, human prions have proven to be particularly challenging to propagate in cultured cells, which has severely hindered the discovery of drugs for Creutzfeldt-Jakob disease. In this review, we summarize the cellular models that are presently available for discovering and testing drugs capable of blocking the propagation of prions and highlight challenges that remain on the path towards developing therapies for prion disease.

IGF-binding proteins 3 and 4 are regulators of sprouting angiogenesis.

PURPOSE: We have previously identified insulin-like growth factor 2 (IGF2) and insulin-like growth factor 1 receptor (IGF1R) as essential proteins for tip cell maintenance and sprouting angiogenesis. In this study, we aim to identify other IGF family members involved in endothelial sprouting angiogenesis. METHODS: Effects on sprouting were analyzed in human umbilical vein endothelial cells (HUVECs) using the spheroid-based sprouting model, and were quantified as mean number of sprouts per spheroid and average sprout length. RNA silencing technology was used to knockdown gene expression. Recombinant forms of the ligands (IGF1 and IGF2, insulin) and the IGF-binding proteins (IGFBP) 3 and 4 were used to induce excess effects. Effects on the tip cell phenotype were analyzed by measuring the fraction of CD34+ tip cells using flow cytometry and immunohistochemistry in a 3D angiogenesis model. Experiments were performed in the presence and absence of serum. RESULTS: Knockdown of IGF2 inhibited sprouting in HUVECs, in particular when cultured in the absence of serum, suggesting that components in serum influence the signaling of IGF2 in angiogenesis in vitro. We then determined the effects of IGFBP3 and IGFBP4, which are both present in serum, on IGF2-IGF1R signaling in sprouting angiogenesis in the absence of serum: knockdown of IGFBP3 significantly reduced sprouting angiogenesis, whereas knockdown of IGFBP4 resulted in increased sprouting angiogenesis in both flow cytometry analysis and immunohistochemical analysis of the 3D angiogenesis model. Other IGF family members except INSR did not affect IGF2-IGF1R signaling. CONCLUSIONS: Serum components and IGF binding proteins regulate IGF2 effects on sprouting angiogenesis. Whereas IGFBP3 acts as co-factor for IGF2-IGF1R binding, IGFBP4 inhibits IGF2 signaling.

Renal proximal tubular epithelial cells: review of isolation, characterization, and culturing techniques.

The kidney is a complex organ, comprised primarily of glomerular, tubular, mesangial, and endothelial cells, and podocytes. The fact that renal cells are terminally differentiated at 34 weeks of gestation is the main obstacle in regeneration and treatment of acute kidney injury or chronic kidney disease. Furthermore, the number of chronic kidney disease patients is ever increasing and with it the medical community should aim to improve existing and develop new methods of renal replacement therapy. On the other hand, as polypharmacy is on the rise, thought should be given into developing new ways of testing drug safety. A possible way to tackle these issues is with isolation and culture of renal cells. Several protocols are currently described to isolate the desired cells, of which the most isolated are the proximal tubular epithelial cells. They play a major role in water homeostasis, acid-base control, reabsorption of compounds, and secretion of xenobiotics and endogenous metabolites. When exposed to ischemic, toxic, septic, or obstructive conditions their death results in what we clinically perceive as acute kidney injury. Additionally, due to renal cells' limited regenerative potential, the profibrotic environment inevitably leads to chronic kidney disease. In this review we will focus on human proximal tubular epithelial cells. We will cover human kidney culture models, cell sources, isolation, culture, immortalization, and characterization subdivided into morphological, phenotypical, and functional characterization.

ß-blockers Reverse Agonist-Induced ß2-AR Downregulation Regardless of Their Signaling Profile.

Altered ß-adrenergic receptor (ß-AR) density has been reported in cells, animals, and humans receiving ß-blocker treatment. In some cases, ß-AR density is upregulated, but in others, it is unaffected or even reduced. Collectively, these results would imply that changes in ß-AR density and ß-blockade are not related. However, it has still not been clarified whether the effects of ß-blockers on receptor density are related to their ability to activate different ß-AR signaling pathways. To this aim, five clinically relevant ß-blockers endowed with inverse, partial or biased agonism at the ß2-AR were evaluated for their effects on ß2-AR density in both human embryonic kidney 293 (HEK293) cells expressing exogenous FLAG-tagged human ß2-ARs and human lymphocytes expressing endogenous ß2-ARs. Cell surface ß2-AR density was measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Treatment with propranolol, carvedilol, pindolol, sotalol, or timolol did not induce any significant change in surface ß2-AR density in both HEK293 cells and human lymphocytes. On the contrary, treatment with the ß-AR agonist isoproterenol reduced the number of cell surface ß2-ARs in the tested cell types without affecting ß2-AR-mRNA levels. Isoproterenol-induced effects on receptor density were completely antagonized by ß-blocker treatment. In conclusion, the agonistic activity of ß-blockers does not exert an important effect on short-term regulation of ß2-AR density.

Impact of processing on the nutritional and functional value of mandarin juice.

BACKGROUND: Although phenolic compounds have a role in the health benefits of fruit juice consumption, little is known about the effect of processing on their bioaccessibility. The release of phenolic compounds from the food matrix during digestion is an important pre-requisite for their effectiveness within the human body, and so it is fundamental to identify technological treatments able to preserve not only the concentration of phytochemicals, but also their bioaccessibility. In the present study, we investigated the impact of high-pressure homogenization (HPH), alone and in the presence of 100 g kg-1 trehalose or Lactobacillus salivarius, on the bioaccessibility of flavonoids in mandarin juice. In addition, digested mandarin juices were supplemented to liver cultured cells in basal and stressed conditions to evaluate their protective effect in a biological system. RESULTS: HPH reduced the concentration of total phenolics and main flavonoids but increased their bioaccessibility after in vitro digestion (P < 0.001). In the basal condition, supplementation with all digested juices significantly reduced intracellular reactive oxygen species (ROS) concentration (P < 0.001). Thiobarbituric acid reactive substances concentration in the medium was also reduced by supplementation with HPH-treated juices. Although pre-treatment with juices did not completely counteract the applied oxidative stress, it preserved cell viability, and cells pre-treated with juices submitted to HPH in the presence of probiotics showed the lowest ROS concentration. CONCLUSION: The present study represents an important step ahead in the evaluation of the impact of processing on the nutritional and functional value of food, which cannot simply be assessed based on chemical composition. © 2020 Society of Chemical Industry.

Metabolic Adjustment of Arabidopsis Root Suspension Cells During Adaptation to Salt Stress and Mitotic Stress Memory.

Sessile plants reprogram their metabolic and developmental processes during adaptation to prolonged environmental stresses. To understand the molecular mechanisms underlying adaptation of plant cells to saline stress, we established callus suspension cell cultures from Arabidopsis roots adapted to high salt for an extended period of time. Adapted cells exhibit enhanced salt tolerance compared with control cells. Moreover, acquired salt tolerance is maintained even after the stress is relieved, indicating the existence of a memory of acquired salt tolerance during mitotic cell divisions, known as mitotic stress memory. Metabolite profiling using 1H-nuclear magnetic resonance (NMR) spectroscopy revealed metabolic discrimination between control, salt-adapted and stress-memory cells. Compared with control cells, salt-adapted cells accumulated higher levels of sugars, amino acids and intermediary metabolites in the shikimate pathway, such as coniferin. Moreover, adapted cells acquired thicker cell walls with higher lignin contents, suggesting the importance of adjustments of physical properties during adaptation to elevated saline conditions. When stress-memory cells were reverted to normal growth conditions, the levels of metabolites again readjusted. Whereas most of the metabolic changes reverted to levels intermediate between salt-adapted and control cells, the amounts of sugars, alanine, γ-aminobutyric acid and acetate further increased in stress-memory cells, supporting a view of their roles in mitotic stress memory. Our results provide insights into the metabolic adjustment of plant root cells during adaptation to saline conditions as well as pointing to the function of mitotic memory in acquired salt tolerance.

Effect of diflufenican on total carotenoid and phytoene production in carrot suspension-cultured cells.

MAIN CONCLUSION: Diflufenican increased 493-fold the level of phytoene. Diflufenican-induced inhibition of phytoene desaturase gene expression in carrot cells resulted in an increased production of phytoene. This work analyzes the effect of diflufenican, an inhibitor of phytoene desaturase, on the gene expression profiles of the biosynthetic pathway of carotenoids related with the production of these compounds in carrot cell cultures. The results showed that the presence of 10 µM diflufenican in the culture medium increased phytoene levels, which was 493-fold higher than in control cells after 7 days of treatment but did not alter cell growth in carrot cell cultures. The maximal production of phytoene was reached with 10 µM diflufenican after 7 days of incubation in the presence of light and with 30 g/L sucrose in the culture medium. Moreover, diflufenican decreased the expression of phytoene synthase and phytoene desaturase genes at all the times studied. This diflufenican-induced inhibition of phytoene desaturase gene expression in carrot cell cultures resulted in an increased production of phytoene. Our results provide new insights into the action of diflufenican in carrot cell cultures, which could represent an alternative more sustainable and environmentally friendly system to produce phytoene than those currently used.

Enhanced transfection efficiency by using a novel semi-attachment method in cell line and primary cells.

DNA transfection in cells is a key technique in biological studies. Cationic liposomes can form nanoparticle complexes with DNA and are widely used for gene delivery in mammalian cells. However, the major drawback of cationic liposomes is their low transfection efficiency in hard-to-transfect cells, such as primary cultured cells. In this study, we established a novel semi-attachment transfection method that showed remarkably improved transfection efficiency compared with traditional forward transfection method.

IGF2 and IGF1R identified as novel tip cell genes in primary microvascular endothelial cell monolayers.

Tip cells, the leading cells of angiogenic sprouts, were identified in cultures of human umbilical vein endothelial cells (HUVECs) by using CD34 as a marker. Here, we show that tip cells are also present in primary human microvascular endothelial cells (hMVECs), a more relevant endothelial cell type for angiogenesis. By means of flow cytometry, immunocytochemistry, and qPCR, it is shown that endothelial cell cultures contain a dynamic population of CD34+ cells with many hallmarks of tip cells, including filopodia-like extensions, elevated mRNA levels of known tip cell genes, and responsiveness to stimulation with VEGF and inhibition by DLL4. Furthermore, we demonstrate that our in vitro tip cell model can be exploited to investigate cellular and molecular mechanisms in tip cells and to discover novel targets for anti-angiogenesis therapy in patients. Small interfering RNA (siRNA) was used to knockdown gene expression of the known tip cell genes angiopoietin 2 (ANGPT2) and tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), which resulted in similar effects on tip cells and sprouting as compared to inhibition of tip cells in vivo. Finally, we identified two novel tip cell-specific genes in CD34+ tip cells in vitro: insulin-like growth factor 2 (IGF2) and IGF-1-receptor (IGF1R). Knockdown of these genes resulted in a significant decrease in the fraction of tip cells and in the extent of sprouting in vitro and in vivo. In conclusion, this study shows that by using our in vitro tip cell model, two novel essential tip cells genes are identified.

Intracellular water preexchange lifetime in neurons and astrocytes.

PURPOSE: To determine the intracellular water preexchange lifetime, τi , the "average residence time" of water, in the intracellular milieu of neurons and astrocytes. The preexchange lifetime is important for modeling a variety of MR data sets, including relaxation, diffusion-sensitive, and dynamic contrast-enhanced data sets. METHODS: Herein, τi in neurons and astrocytes is determined in a microbead-adherent, cultured cell system. In concert with thin-slice selection, rapid flow of extracellular media suppresses extracellular signal, allowing determination of the transcytolemmal-exchange-dominated, intracellular T1 . With this knowledge, and that of the intracellular T1 in the absence of exchange, τi can be derived. RESULTS: Under normal culture conditions, τi for neurons is 0.75 ± 0.05 s versus 0.57 ± 0.03 s for astrocytes. Both neuronal and astrocytic τi s decrease within 30 min after the onset of oxygen-glucose deprivation, with the astrocytic τi showing a substantially greater decrease than the neuronal τi . CONCLUSIONS: Given an approximate intra- to extracellular volume ratio of 4:1 in the brain, these data imply that, under normal physiological conditions, an MR experimental characteristic time of less than 0.012 s is required for a nonexchanging, two-compartment (intra- and extracellular) model to be valid for MR studies. This characteristic time shortens significantly (i.e., 0.004 s) under injury conditions. Magn Reson Med 79:1616-1627, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Effect of terbinafine on the biosynthetic pathway of isoprenoid compounds in carrot suspension cultured cells.

KEY MESSAGE: Terbinafine induced a significant increase of squalene production. Terbinafine increased the expression levels of squalene synthase. Cyclodextrins did not work as elicitors due to the gene expression levels obtained. Plant sterols are essential components of membrane lipids, which contributing to their fluidity and permeability. Besides their cholesterol-lowering properties, they also have anti-inflammatory, antidiabetic and anticancer activities. Squalene, which is phytosterol precursor, is widely used in medicine, foods and cosmetics due to its anti-tumor, antioxidant and anti-aging activities. Nowadays, vegetable oils constitute the main sources of phytosterols and squalene, but their isolation and purification involve complex extraction protocols and high costs. In this work, Daucus carota cell cultures were used to evaluate the effect of cyclodextrins and terbinafine on the production and accumulation of squalene and phytosterols as well as the expression levels of squalene synthase and cycloartenol synthase genes. D. carota cell cultures were able to produce high levels of extracellular being phytosterols in the presence of cyclodextrins (12 mg/L), these compounds able to increase both the secretion and accumulation of phytosterols in the culture medium. Moreover, terbinafine induced a significant increase in intracellular squalene production, as seen after 168 h of treatment (497.0 ± 23.5 µg g dry weight-1) while its extracellular production only increased in the presence of cyclodextrins.The analysis of sqs and cas gene expression revealed that cyclodextrins did not induce genes encoding enzymes involved in the phytosterol biosynthetic pathway since the expression levels of sqs and cas genes in cyclodextrin-treated cells were lower than in control cells. The results, therefore, suggest that cyclodextrins were only able to release phytosterols from the cells to the extracellular medium, thus contributing to their acumulation. To sum up, D. carota cell cultures treated with cyclodextrins or terbinafine were able to produce high levels of phytosterols and squalene, respectively, and, therefore, these suspension-cultured cells of carrot constitute an alternative biotechnological system, which is at the same time more sustainable, economic and ecological for the production of these bioactive compounds.

Co-ordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat-binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

Functional characterization of the transcriptional regulatory elements of three highly expressed constitutive genes in the jewel wasp, Nasonia vitripennis.

The jewel wasp, Nasonia vitripennis Ashmead (Hymenoptera: Pteromalidae), is an easily reared parasitoid that is providing an ever increasingly malleable model for examining the biology and genetics of Hymenoptera. Utilizing genomic and transcriptome resources, 5' upstream transcriptional regulatory sequences (TREs) from three highly expressed genes were identified and cloned. Criteria for TRE selection included the presence of an adjacent gene 5' of the translation initiation site. One gene was methylated whereas the other two were nonmethylated. Each TRE, heat-shock protein 70 (hsp70), activator of 90 kDa hsp ATPase protein 1 (hsp90A), and lipid storage droplet surface-binding protein 1 (lsdp) was linked with enhanced green fluorescent protein (EGFP) coding sequence and cloned into both pDP9e somatic and piggyBac germline transformation vectors. EGFP expression patterns under control of each TRE were compared with patterns of DsRed fluorescence produced from the transformation vector cassette. Functional activity of each TRE was observed in cultured Spodoptera frugiperda 9 (Sf9) cells and Drosophila melanogaster as well as in N. vitripennis embryos demonstrating that all three sequences had functional transcriptional regulatory activity in three different insect orders. Identification and functional characterization of these three TREs will provide critical and necessary resources for further genetic analyses of N. vitripennis, Hymenoptera and other insects.

An advanced mouse model for human skin wound healing.

Here, we report a model for studying wound repair based on skin regenerated from human tissue culture-expanded cells. The reconstituted skin (hRSK) responds to injury similar to that of intact human skin, and its constituent cells contribute to the healing process. As we have demonstrated that hRSK composed of GFP-labelled cells also heals "normally," we believe this model will be useful in analysing the wound repair process using genetically modified human cells.