Dye Transfer Research Articles

Transfer of Dyes and Drugs into Cells Using EGFR-Targeted Nanosyringes

Selective targeting of drug loaded nanovectors to specific epitopes highly expressed on the surface of cancer cells is a goal for nanotechnologists. We have modified our previously described PEGylated-hydrophilic carbon clusters (PEG-HCCs) so that the epidermal growth factor receptor (EGFR) binding peptide, GE11, is attached using click chemistry at the end of each PEG. The resulting nanosyringe, PepEGFR-PEG-HCC, can be loaded with a wide range of hydrophobic drugs and dyes. We show that, both in vitro and in vivo, this payload can be delivered to cancer cells expressing EGFR. We can observe the activation of EGFR and track the normal physiological internalization and recycling/signaling pathways of this tyrosine kinase following binding of PepEGFR-PEG-HCC. We also demonstrate the competitive binding of the nanosyringe to EGFR with its normal activator, EGF, as well as observing the colocalization of the nanosyringe with clathrin, the coated pit integral protein. The internalization of the drug/dye loaded nanosyringe can be inhibited by using anti-EGFR antibodies, the drug erlotinib, or Pitstop-1, the clathrin coated pit formation specific inhibitor. To further demonstrate the specificity of the drug loaded nanovectors, we demonstrated that, in both flank and intracranial xenograft mouse models, dye delivery is highly specific to tumors and no other tissues. Finally, using nanosyringes loaded with esterase sensitive fluorescein diacetate, we demonstrated that the drug payloads can be in vivo delivered to the cytosol of cancer cells within the mouse brain.

Theoretical study of tribranched organic sensitizer for Efficient Dye-sensitized solar cells

ABSTRACTWe investigated the effect of heteroleptic dual electron acceptor as a photosensitizer for the dye-sensitized solar cells (DSSCs). Novel tribranched organic dyes were designed with heteroleptic dual electron acceptors [cyanoacrylic acid (R1), and indolinum carboxyl acid (R2)] on each side of a triazine (TRZ) based organic dye and triphenylamine and the additional phenothiazine as an antenna group for DSSCs. The density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations were used to estimate the photovoltaic properties of the dyes. The absorption spectrum of the dyes showed different forms because of the different energy levels of molecular orbital (MO) of each dye and intramolecular energy transfer. Considering overall properties, the asymmetric tribranched organic dye exhibited a high conversion efficiency performance for DSSCs. The findings of this work suggest that optimizing the branch of electron donors and acceptors in dye sensitizers based on asymmetric tribranched organic dye produces good photovoltaic properties for DSSCs.

Natural and synthetic polymers in fabric and home care applications

AbstractPolymers can be tailored to provide different benefits in Fabric & Home Care formulations depending on the monomers and modifications used, such as avoiding dye transfer inhibition in the wash, modifying the surface of tiles or increasing the viscosity and providing suspension properties to consumer products. Specifically, the rheology modification properties of synthetic and natural polymers are discussed in this chapter. The choice of a polymeric rheology modifier will depend on the formulation ingredients (charges, functional groups), the type and the amount of surfactants, the pH and the desired rheology modification. Natural polymeric rheology modifiers have been traditionally used in the food industry, being xanthan gum one of the most well-known ones. On the contrary, synthetic rheology modifiers are preferably used in paints & coats, textile printing and cleaning products.

Cyclic Nucleotides Differentially Regulate Cx43 Gap Junction Function in Uterine Artery Endothelial Cells From Pregnant Ewes.

Cell-cell communication is dependent on GJ (gap junction) proteins such as Cx43 (connexin 43). We previously demonstrated the importance of Cx43 function in establishing the enhanced pregnancy vasodilatory phenotype during pregnancy in uterine artery endothelial cells from pregnant (P-UAEC) ewes. Cx43 is regulated by elevating cAMP and PKA (protein kinase A)-dependent Cx43 S365 phosphorylation-associated trafficking and GJ open gating, which is opposed by PKC (protein kinase C)-dependent S368 phosphorylation-mediated GJ turnover and closed gating. However, the role of cyclic nucleotide-mediated signaling mechanisms that control Cx43 and GJ function in P-UAECs is unknown. We hypothesize that cAMP will mediate increases in S365 phosphorylation, thereby, enhancing GJ trafficking and open gating, while cGMP will stimulate S368, but not S365, phosphorylation to enhance GJ turnover and closed gating in P-UAECs. Treatment with 8-Bromo (8-Br)-cAMP signal significantly (P<0.05) increased nonphosphorylated S365 signal and total Cx43 phosphorylation, but not S368 phosphorylation, while 8-Br-cGMP significantly (P<0.05) increased Cx43 C-terminus-S365 signal, S368, and total Cx43 phosphorylation. Inhibition of PKA, but not PKG (protein kinase G), abrogated the 8-Br-cAMP-stimulated increase in nonphosphorylated S365 and total Cx43 phosphorylation and inhibited S368 below basal levels, whereas inhibition of PKG blocked (P<0.05) the 8-bromo-cGMP-stimulated rises in nonphosphorylated S365, total Cx43, and S368 phosphorylation levels in P-UAECs. Functional studies showed that 8-Br-cAMP increased dye transfer and sustained calcium bursts, while 8-Br-cGMP decreased both. Thus, in P-UAECs, only 8-Br-cAMP and not 8-Br-cGMP effectively enhances nonphosphorylated S365 and total Cx43 expression that correspondingly reduces S368 phosphorylation, allowing increased GJ communication. This provides new insights into the regulatory mechanisms behind Cx43 function and GJ communication.

Low interspecific pollen transfer between invasive aquatic Ludwigia grandiflora and native co-flowering plants

Showy invasive alien plants are often integrated in the diet of generalist pollinators and because of the lack of co-evolvement with the native plant community, a high amount of interspecific pollen transfer (IPT) can be expected. We investigated pollinator switching and magnitude plus distance of IPT between the alien aquatic Ludwigia grandiflora and the native Lythrum salicaria in both directions in uninvaded and invaded sites with a different relative abundance of L. grandiflora (% cover of the alien plant: no cover; low cover: <5%; high cover: 50–75%). A field experiment was conducted to include both pollinator interspecific movements and tracking of IPT, using fluorescent dye as a pollen analogue. Despite a substantial overlap in pollinators between L. grandiflora and the native L. salicaria, less than 10% of the observed flights were interspecific. Similar results were found in dye transfer patterns. The proportions of stigmas with conspecific dye were always higher than the proportions of stigmas with heterospecific dye for L grandiflora and L. salicaria. There were no differences in conspecific dye loads for L. salicaria between uninvaded and invaded sites. Conspecific pollen loss (native CPL) and heterospecific pollen deposition (alien HPD) were in general low and species-specific. The distance of HPD ranged respectively from 1.7 to 39 m and from 0.3 to 54.8 m in the low cover and high cover sites while CPL ranged respectively from 6.40 to 68.02 m and from 0.60 to 40.18 m in the low cover and high cover sites. We can conclude that, in this system, CPL and HPD will play a minor role in pollinator-mediated interaction. Furthermore, interspecific competition for pollinators will cover a larger distance than just neighboring individuals. Our results suggest the necessity to consider the combined effect of insect visitation, pollen deposition, relative alien abundance, distance and seed set when investigating pollinator-mediated interactions of invasive plants.

Progressive Atrial Conduction Defects Associated With Bone Malformation Caused by a Connexin-45 Mutation

BackgroundInherited cardiac conduction disease is a rare bradyarrhythmia associated with mutations in various genes that affect action potential propagation. It is often characterized by isolated conduction disturbance of the His-Purkinje system, but it is rarely described as a syndromic form. ObjectivesThe authors sought to identify the genetic defect in families with a novel bradyarrhythmia syndrome associated with bone malformation. MethodsThe authors genetically screened 15 European cases with genotype-negative de novo atrioventricular (AV) block and their parents by trio whole-exome sequencing, plus 31 Japanese cases with genotype-negative familial AV block or sick sinus syndrome by targeted exon sequencing of 457 susceptibility genes. Functional consequences of the mutation were evaluated using an in vitro cell expression system and in vivo knockout mice. ResultsThe authors identified a connexin-45 (Cx45) mutation (p.R75H) in 2 unrelated families (a de novo French case and a 3-generation Japanese family) who presented with progressive AV block, which resulted in atrial standstill without ventricular conduction abnormalities. Affected individuals shared a common extracardiac phenotype: a brachyfacial pattern, finger deformity, and dental dysplasia. Mutant Cx45 expressed in Neuro-2a cells showed normal hemichannel assembly and plaque formation. However, Lucifer yellow dye transfer and gap junction conductance between cell pairs were severely impaired, which suggested that mutant Cx45 impedes gap junction communication in a dominant-negative manner. Tamoxifen-induced, cardiac-specific Cx45 knockout mice showed sinus node dysfunction and atrial arrhythmia, recapitulating the intra-atrial disturbance. ConclusionsAltogether, the authors showed that Cx45 mutant p.R75H is responsible for a novel disease entity of progressive atrial conduction system defects associated with craniofacial and dentodigital malformation.

Ginsenoside Rg1 alleviates corticosterone-induced dysfunction of gap junctions in astrocytes

Ethnopharmacological relevanceGinsenoside Rg1 (Rg1), one of the major bioactive ingredients of Panax ginseng C. A. Mey, has neuroprotective effects in animal models of depression, but the mechanism underlying these effects is still largely unknown Aim of the studyGap junction intercellular communication (GJIC) dysfunction is a potentially novel pathogenic mechanism for depression. Thus, we investigated that whether antidepressant-like effects of Rg1 were related to GJIC. Materials and methodsPrimary rat prefrontal cortical and hippocampal astrocytes cultures were treated with 50μM CORT for 24h to induce gap junction damage. Rg1 (0.1, 1, or 10μM) or fluoxetine (1μM) was added 1h prior to CORT treatment. A scrape loading and dye transfer assay was performed to identify the functional capacity of gap junctions. Western blot was used to detect the expression and phosphorylation of connexin43 (Cx43), the major component of gap junctions. ResultsTreatment of primary astrocytes with CORT for 24h inhibited GJIC, decreased total Cx43 expression, and increased the phosphorylation of Cx43 at serine368 in a dose-dependent manner. Pre-treatment with 1μM and 10μM Rg1 significantly improved GJIC in CORT-treated astrocytes from the prefrontal cortex and hippocampus, respectively, and this was accompanied by upregulation of Cx43 expression and downregulation of Cx43 phosphorylation. ConclusionThese findings provide the first evidence indicating that Rg1 can alleviate CORT-induced gap junction dysfunction, which may have clinical significance in the treatment of depression.

Excited-State Intramolecular Proton Transfer (ESIPT) of Fluorescent Flavonoid Dyes: A Close Look by Low Temperature Fluorescence.

Flavonoids have emerged to be an important molecular frame for chemical sensors, due to their capability to give dual emission and their compatibility to biological cells. Two flavonoid compounds with Me2N- and Et2N-substituents were examined by acquiring their fluorescence at different temperatures, in order to evaluate the impact of intramolecular charge transfer (ICT) on ESIPT. By freezing the sample solution in liquid N2, the study detected the "locally excited" state at λem ≈ 460 nm, in which both ICT and ESIPT processes were not present. As temperature was warmed up (to about -90 °C), ICT process became gradually allowed and emission was shifted to λem ≈ 510 nm, which was attributed to the normal form N* of flavonoid. Emission from tautomeric form T* (λem ≈ 575 nm) could only be observed at a higher temperature, when ESIPT became allowed. With the aid of a model compound (without R2N-substituent) and computational study, the study led to improved understanding on the photophysical properties of flavonoid materials in general.

Photography and the Appropriation of Kodak Dye Transfer in Socialist China

Photography and the Appropriation of Kodak Dye Transfer in Socialist China

Quantitative analysis of cell proliferation by a dye dilution assay: Application to cell lines and cocultures.

Cell proliferation represents one of the most fundamental processes in biological systems, thus the quantitative analysis of cell proliferation is important in many biological applications such as drug screening, production of biologics, and assessment of cytotoxicity. Conventional proliferation assays mainly quantify cell number based on a calibration curve of a homogeneous cell population, and therefore are not applicable for the analysis of cocultured cells. Moreover, these assays measure cell proliferation indirectly, based on cellular metabolic activity or DNA content. To overcome these shortcomings, a dye dilution assay employing fluorescent cell tracking dyes that are retained within cells was applied and was diluted proportionally by subsequent cell divisions. Here, it was demonstrated that this assay could be implemented to quantitatively analyze the cell proliferation of different types of cell lines, and to concurrently analyze the proliferation of two types of cell lines in coculture by utilizing cell tracking dyes with different spectral characteristics. The mean division time estimated by the dye dilution assay is compared with the population doubling time obtained from conventional methods and values from literature. Additionally, dye transfer between cocultured cells was investigated and it was found that it is a characteristic of the cells rather than a characteristic of the dye. It was suggested that this method can be easily combined with other flow cytometric analyses of cellular properties, providing valuable information on cell status under diverse conditions. © 2017 International Society for Advancement of Cytometry.

Dual energy antenna for efficient dye-sensitized solar cells

ABSTRACTA novel 4-(dicyanomethylene)-2-methyl-6-[p-(dimethylamino) styryl]-4H-pyran (DCM)-based organic dye with a heteroleptic dual-electron donor was designed, and its electronic and optical properties were investigated theoretically for dye-sensitized solar cells (DSSCs). We designed three dyes with triphenylamine (T) and phenoxazine (P) moieties on each side of a DCM-based organic dye (D); namely, TPDPT, TPDTP, and PTDTP. In this study, heteroleptic TPDTP dye was compared with the homoleptic dyes (TPDPT and PTDTP). The absorption spectrum of the dyes showed different forms because of the different energy levels of the molecular orbital (MO) of each dye and intramolecular energy transfer. It was shown that the energy antenna group created an additional absorption band around 430 nm and caused a cascading two-electron process aiding the charge-separation process. Absorption Band 1 (around 640 nm) and Band 2 (around 520 nm) showed that P→D and T→D, respectively. It was expected that the TPDTD dye with the heteroleptic dual donor would perform competitively compared to the other dyes with homoleptic dual donors in terms of conversion efficiency in DSSCs

Inhibition of gap junction intercellular communication is involved in silica nanoparticles-induced H9c2 cardiomyocytes apoptosis via the mitochondrial pathway.

Gap junction intercellular communication (GJIC) between cardiomyocytes is essential for synchronous heart contraction and relies on connexin-containing channels. Connexin 43 (Cx43) is a major component involved in GJIC in heart tissue, and its abnormal expression is closely associated with various cardiac diseases. Silica nanoparticles (SNPs) are known to induce cardiovascular toxicity. However, the mechanisms through which GJIC plays a role in cardiomyocytes apoptosis induced by SNPs remain unknown. The aim of the present study is to determine whether SNPs-decreased GJIC promotes apoptosis in rat cardiomyocytes cell line (H9c2 cells) via the mitochondrial pathway using CCK-8 Kit, scrape-loading dye transfer technique, Annexin V/PI double-staining assays, and Western blot analysis. The results showed that SNPs elicited cytotoxicity in H9c2 cells in a time- and concentration-dependent manner. SNPs also reduced GJIC in H9c2 cells in a concentration-dependent manner through downregulation of Cx43 and upregulation of P-Cx43. Inhibition of gap junctions by gap junction blocker carbenoxolone disodium resulted in decreased survival and increased apoptosis, whereas enhancement of the gap junctions by retinoic acid led to enhanced survival but decreased apoptosis. Furthermore, SNPs-induced apoptosis through the disrupted functional gap junction was correlated with abnormal expressions of the proteins involved in the mitochondrial pathway-related apoptosis such as Bcl-2/Bax, cytochrome C, Caspase-9, and Caspase-3. Taken together, our results provide the first evidence that SNPs-decreased GJIC promotes apoptosis in cardiomyocytes via the mitochondrial pathway. In addition, downregulation of GJIC by SNPs in cardiomyocytes is mediated through downregulation of Cx43 and upregulation of P-Cx43. These results suggest that in rat cardiomyocytes cell line, GJIC plays a protective role in SNPs-induced apoptosis and that GJIC may be one of the targets for SNPs-induced biological effects.

Golgi-retained Cx32 mutants interfere with gene addition therapy for CMT1X

Numerous GJB1 gene mutations cause the X-linked form of Charcot-Marie-Tooth disease (CMT1X). GJB1 encodes connexin32 (Cx32), which forms trans-myelin gap junctions in Schwann cells. Most GJB1 mutations result in loss-of-function mechanisms, supporting the concept of gene replacement therapy. However, interactions between delivered wild type and endogenously expressed mutant Cx32 may potentially occur in the setting of gene replacement therapy. In order to screen for possible interactions of several representative CMT1X mutants with wild type Cx32 that may interfere with the functional gap junction formation, we established an in vitro screening method co-expressing in HeLa cells wild type Cx32 and one of eight different Cx32 mutants including A39P, A39V, T55I, R75W, M93V, L143P, N175D and R183S. Some of the Golgi-retained mutants hindered gap junction plaque assembly by Cx32 on the cell membrane, while co-immunoprecipitation analysis revealed a partial interaction of wild type protein with Golgi-retained mutants. Dye transfer studies confirmed that Golgi-retained R75W, M93V and N175D but not endoplasmic reticulum-retained T55I had a negative effect on wild type Cx32 function. Finally, in vivo intraneural delivery of the gene encoding the wild type Cx32 in mice bearing either the T55I or R75W mutation on Cx32 knockout background showed that virally delivered protein was correctly localized in mice expressing the endoplasmic reticulum-retained T55I whereas it did not traffic normally in mice expressing the Golgi-retained R75W. Thus, certain Golgi-retained Cx32 mutants may interfere with exogenously delivered Cx32. Screening for mutant-wild type Cx32 interactions should be considered prior to planning gene addition therapy for CMT1X.

Alterations in the skin of Labeo rohita exposed to an azo dye, Eriochrome black T: a histopathological and enzyme biochemical investigation.

Histopathological changes and alterations in the activity of certain metabolic and antioxidant enzymes were analyzed in the head skin of Labeo rohita, exposed to sublethal test concentrations of the azo dye, Eriochrome black T for 4days, using 24h renewal bioassay method. Hypertrophied epithelial cells, increased density of mucous goblet cells, and profuse mucous secretion at the surface were considered to protect the skin from toxic impact of the azo dye. Degenerative changes including vacuolization, shrinkage, decrease in dimension, and density of club cells with simultaneous release of their contents in the intercellular spaces were associated to plug them, preventing indiscriminate entry of foreign matter. On exposure of fish to the dye, significant decline in the activity of enzymes-alkaline phosphatase, acid phosphatase, carboxylesterase, succinate dehydrogenase, catalase, and peroxidase-was associated with the binding of dye to the enzymes. Gradual increase in the activity of lactate dehydrogenase was considered to reflect a shift from aerobic to anaerobic metabolism. On transfer of azo dye exposed fish to freshwater, skin gradually recovers and, by 8days, density and area of mucous goblet cells, club cells, and activity of the enzymes appear similar to that of controls. Alteration in histopathology and enzyme activity could be considered beneficial tool in monitoring environmental toxicity, valuable in the sustenance of fish populations.

SERINC Inhibits HIV-1 Env Induced Membrane Fusion and Slows Fusion Pore Enlargement

The SERINC family of proteins are integral membrane proteins that regulate the incorporation of serine into phospholipids, to create PS, and into sphingolipids. It has recently been shown that two members of the family, SERINC3 and SERINC5, inhibit HIV infectivity. Using a cell-cell fusion system to determine the extent to which inhibition of infectivity is due to reduced fusion, we found that the presence of SERINC3 or SERINC5 in either effector or target cells slows the kinetics and reduces the extent of fusion induced by HIV-1 Env. These two incorporators of serine greatly retard fusion pore enlargement, as determined by the rate of aqueous dye transfer once a pore forms. Nef is an auxiliary protein of HIV that is well-known to enhance HIV infectivity. The presence of SERINC5 and Nef in effector cells leads to the same extent of fusion induced by expression of Env alone, showing that Nef eliminates the reduction of fusion caused by SERINC. (R01 GM 101 539).

Abstract WP107: Igf-1-mediated Neuroprotection for Ischemic Stroke is Preceded by Its Actions at the Endothelial Cell Barrier

Background: Our studies show that Insulin like growth factor (IGF)-1 reduces MCAo-induced infarction in middle-aged female rats. We tested the hypothesis that IGF-1s neuroprotective effect is due to its action on the microvascular /endothelial barrier in the early acute phase of stroke. Methods: Middle-aged (12 mo) acyclic female rats were subject to intraluminal middle cerebral artery occlusion (MCAo) for 90 min followed by reperfusion and terminated 4h or 24h later. Animals received ICV infusion of artificial CSF, IGF-1 or IGF-1+JB-1. In separate experiments, we evaluated infarct volume, barrier assessment by uptake of Evan’s blue dye or histological evaluation of lectin stained microvessels. In parallel, cortical endothelial cells from middle-aged females were harvested and cultured ex vivo. Barrier properties were tested in cultures in transfer wells exposed to oxygen glucose deprivation (OGD) in the presence or absence of IGF-1 with FITC-labeled BSA in the media. Results: At 4h post MCAo, IGF-1 treatment significantly reduced brain uptake of Evan’s blue dye, while infarct volume was no different from controls. At 24h post MCAo, IGF-1 reduced infarct volume by 40% and decreased motor impairment (neurological score; p&lt;0.05). Lectin stained microvessels in the ischemic hemisphere displayed wider lumens (normal uncompressed lumen, p&lt;0.003) and more intense label (p&lt;0.01) in IGF-1 treated animals as compared to either vehicle or IGF-1+JB-1 groups. Ex vivo, IGF-1 reduced the loss of cell-cell contact due to OGD and reduced the dye transfer across the monolayer. Conclusions: Our findings indicate that IGF-1 acts in the early acute phase of ischemic stroke to reduce barrier permeability, which precedes its beneficial effect on infarct volume and behavior. Further, brain endothelial cells play a crucial role in IGF-1 mediated neuroprotection in middle-aged females.

Stable archaeal tetraether lipid liposomes for photodynamic application: transfer of carboxyfluorescein to cultured T84 tumor cells

Background: Archaeal membranes have phytanyl ether lipids instead of common fatty acid-glycerol esters in bacterial and eukaryotic cells. Sulfolobus and Thermoplasma species have unique membrane-spanning tetraether lipids (TEL), which form stable liposomes. Recently, we cultured Thermoplasma species from the Indonesian volcano Tangkuban Perahu and isolated TEL. The purpose of this in vitro study is to investigate the transfer of fluorescent dye from stable TEL liposomes to cultured colon carcinoma cells.Methods: TEL was extracted from cultured cells with chloroform-methanol (1:1), then it was fractionated and purified via diethylaminoethyl-cellulose-acetate columns and activated charcoal for the formation of stable liposomes. For the fluorescence exchange assay, TEL liposomes were loaded with water-soluble carboxyfluorescein (CF). Staining experiments were conducted with various cell cultures, and T84 colon carcinoma cells were chosen for the main experiments. Liposome stability was tested by light scattering and electron microscopic size determinations as well as by unspecific CF release at low pH (6.0–7.4) and increased temperature (4–50°C/70°C).Results: TEL liposomes exhibit high stability and extremely low proton permeability at low pH. CF staining of cultured T84 colon carcinoma cells appeares more intensive from TEL liposomes than from dipalmitoylphosphatidylcholine liposomes.Conclusion: The results of this in vitro study demonstrate CF staining of colon carcinoma cells and high stability of TEL liposomes at low pH, matching the condition in the gastro-intestinal (GI) route and in the urogentital (UG) tract. For this reason, in vivo studies on liposomal fluorescent photosensitizers for topical application of photodynamic cancer therapy in the GI and UG tracts should be carried out.

Gene therapy targeting oligodendrocytes provides therapeutic benefit in a leukodystrophy model.

Pelizaeus-Merzbacher-like disease or hypomyelinating leukodystrophy-2 is an autosomal recessively inherited leukodystrophy with childhood onset resulting from mutations in the gene encoding the gap junction protein connexin 47 (Cx47, encoded by GJC2). Cx47 is expressed specifically in oligodendrocytes and is crucial for gap junctional communication throughout the central nervous system. Previous studies confirmed that a cell autonomous loss-of-function mechanism underlies hypomyelinating leukodystrophy-2 and that transgenic oligodendrocyte-specific expression of another connexin, Cx32 (GJB1), can restore gap junctions in oligodendrocytes to achieve correction of the pathology in a disease model. To develop an oligodendrocyte-targeted gene therapy, we cloned the GJC2/Cx47 gene under the myelin basic protein promoter and used an adeno-associated viral vector (AAV.MBP.Cx47myc) to deliver the gene to postnatal Day 10 mice via a single intracerebral injection in the internal capsule area. Lasting Cx47 expression specifically in oligodendrocytes was detected in Cx47 single knockout and Cx32/Cx47 double knockout mice up to 12 weeks post-injection, including the corpus callosum and the internal capsule but also in more distant areas of the cerebrum and in the spinal cord. Application of this oligodendrocyte-targeted somatic gene therapy at postnatal Day 10 in groups of double knockout mice, a well characterized model of hypomyelinating leukodystrophy-2, resulted in significant improvement in motor performance and coordination at 1 month of age in treated compared to mock-treated mice, as well as prolonged survival. Furthermore, immunofluorescence and morphological analysis revealed improvement in demyelination, oligodendrocyte apoptosis, inflammation, and astrogliosis, all typical features of this leukodystrophy model in both brain and spinal cord. Functional dye transfer analysis confirmed the re-establishment of oligodendrocyte gap junctional connectivity in treated as opposed to untreated mice. These results provide a significant advance in the development of oligodendrocyte-cell specific gene therapy. Adeno-associated viral vectors can be used to target therapeutic expression of a myelin gene to oligodendrocytes. We show evidence for the first somatic gene therapy approach to treat hypomyelinating leukodystrophy-2 preclinically, providing a potential treatment for this and similar forms of leukodystrophies.

A small variation in average particle size of PLGA nanoparticles prepared by nanoprecipitation leads to considerable change in nanoparticles’ characteristics and efficacy of intracellular delivery

In this study, it was aimed to investigate characteristics and intracellular delivery of two different-sized PLGA nanoparticles in ouzo region by considering number of nanoparticles. To determine the effect of formulation parameters on average particle size, Dil labeled nanoparticles were prepared using a three-factor, two-level full factorial statistical experimental design. PLGA230 (230.8 ± 4.32 nm) and PLGA160 (157.9 ± 6.16 nm) nanoparticles were obtained by altering polymer amount based on experimental design results and characterized. Same number of PLGA230 and PLGA160 nanoparticles per cell were applied onto HEK293 cells; then, cytotoxicity, uptake kinetics and mechanism were evaluated by flow cytometry and fluorescent microscopy. Also same weight of PLGA230 and PLGA160 nanoparticles were applied and cellular uptake of these nanoparticles was evaluated. It was found that PLGA230 nanoparticles had higher encapsulation efficiency and slower dye release compared to PLGA160 nanoparticles. When they were applied at same counts per cell, PLGA230 nanoparticles displayed faster and higher intracellular dye transfer than PLGA160 nanoparticles. On the other hand, PLGA160 appeared to be a more effective vehicle than PLGA230 when applied at the same weight concentration. It was also shown that for both nanoparticles, HEK293 cells employed macropinocytic, caveolae- and clathrin-mediated endocytic pathways.

Virus Innexins induce alterations in insect cell and tissue function

Polydnaviruses are dsDNA viruses that induce immune and developmental alterations in their caterpillar hosts. Characterization of polydnavirus gene families and family members is necessary to understand mechanisms of pathology and evolution of these viruses, and may aid to elucidate the role of host homologues if present. For example, the polydnavirus vinnexin gene family encodes homologues of insect gap junction genes (innexins) that are expressed in host immune cells (hemocytes). While the roles of Innexin proteins and gap junctions in insect immunity are largely unclear, we previously demonstrated that Vinnexins form functional gap junctions and alter the junctional characteristics of a host Innexin when co-expressed in paired Xenopus oocytes. Here, we test the effect of ectopic vinnexin expression on host cell physiology using both a lepidopteran cell culture model and a dipteran whole organism model. Vinnexin expression in the cell culture system resulted in gene-specific alterations in cell morphology and a slight, but non-statistically significant, reduction in gap junction activity as measured by dye transfer, while ectopic expression of a lepidopteran innexin2 gene led to morphological alterations and increase in gap junction activity. Global ectopic expression in the model dipteran, Drosophila melanogaster, of one vinnexin (vinnexinG) or D. melanogaster innexin2 (Dm-inx2) resulted in embryonic lethality, while expression of the other vinnexin genes had no effect. Furthermore, ectopic expression of vinnexinG, but not other vinnexin genes or Dm-inx2, in D. melanogaster larval gut resulted in developmental arrest in the pupal stage. These data indicate the vinnexins likely have gene-specific roles in host manipulation. They also support the use of Drosophila in further analysis of the role of Vinnexins and other polydnavirus genes in modifying host physiological processes. Finally, our findings suggest the vinnexin genes may be useful to perturb and characterize the physiological functions of insect Innexins.