Energy-dependent Process Research Articles

EXTRACELLULAR AND INTRACELLULAR SYNTHESIS OF SILVER NANOPARTICLES

<p>ABSTRACT<br />Objective: The cellular synthesis of nanoparticle is a green process and alternative for a conventional process for the preparation of silver nanoparticles.<br />In our research, focus has been given to the development of an efficient and eco-friendly viable process for the synthesis of silver nanoparticles using<br />cancer and non-cancerous cells, a cell culture that was isolated. The results of this investigation are observed that silver nanoparticles could be<br />induced to synthesis intra- and extra-cellularly using mammalian cells such as cancerous and non-cancerous cells.<br />Methods: The silver nanoparticles are synthesized by the cancer and non-cancerous cells such as HeLa (Homo sapiens, human), SiHa, and human<br />embryonic kidney-293 cell lines. The silver nanoparticles were characterized by ultraviolet (UV)-visible spectroscopy, transmission electron<br />microscopy (TEM), and X-ray powder diffraction (XRD).<br />Results: The silver nanoparticles exhibited maximum absorbance at 415 nm in UV-visible spectroscopy. The XRD confirms the characteristic of the<br />crystal lattice of silver nanoparticles by observing three peaks: Peak at 38 is due to reflection from (111), peak at 44 is due to reflection from (200),<br />and peak at 65 is due to reflection from (220). TEM images showed the formation of stable silver nanoparticles in the cell lines.<br />Conclusion: The method of extraction of intracellular/extracellular synthesis of silver nanoparticles was inexpensive, simple, and effective in large<br />scale with no need to use of complex process equipment. The cancer cell considered as a biological factory at nanoscale dimension which continued to<br />grow after synthesis of silver nanoparticles. The silver reduction by these cancer cells has occurred through energy-dependent processes that lead to<br />the high output of this reaction. Hence, this new approach of using a mammalian cell for the successful synthesis of nanosized silvers could be easily<br />scaled up, which establishes its commercial viability and also useful in the drug delivery and drug targeting.<br />Keywords: Silver nanoparticles, Cancer cells, Biosynthesis and characteristics of silver nanoparticles.</p><p> </p>

Use of fluorescent nanoparticles to investigate nutrient acquisition by developing Eimeria maxima macrogametocytes.

The enteric disease coccidiosis, caused by the unicellular parasite Eimeria, is a major and reoccurring problem for the poultry industry. While the molecular machinery driving host cell invasion and oocyst wall formation has been well documented in Eimeria, relatively little is known about the host cell modifications which lead to acquisition of nutrients and parasite growth. In order to understand the mechanism(s) by which nutrients are acquired by developing intracellular gametocytes and oocysts, we have performed uptake experiments using polystyrene nanoparticles (NPs) of 40 nm and 100 nm in size, as model NPs typical of organic macromolecules. Cytochalasin D and nocodazole were used to inhibit, respectively, the polymerization of the actin and microtubules. The results indicated that NPs entered the parasite at all stages of macrogametocyte development and early oocyst maturation via an active energy dependent process. Interestingly, the smaller NPs were found throughout the parasite cytoplasm, while the larger NPs were mainly localised to the lumen of large type 1 wall forming body organelles. NP uptake was reduced after microfilament disruption and treatment with nocodazole. These observations suggest that E. maxima parasites utilize at least 2 or more uptake pathways to internalize exogenous material during the sexual stages of development.

Cellular compartmentation of energy metabolism: creatine kinase microcompartments and recruitment of B-type creatine kinase to specific subcellular sites.

There is an increasing body of evidence for local circuits of ATP generation and consumption that are largely independent of global cellular ATP levels. These are mostly based on the formation of multiprotein(-lipid) complexes and diffusion limitations existing in cells at different levels of organization, e.g., due to the viscosity of the cytosolic medium, macromolecular crowding, multiple and bulky intracellular structures, or controlled permeability across membranes. Enzymes generating ATP or GTP are found associated with ATPases and GTPases enabling the direct fueling of these energy-dependent processes, and thereby implying that it is the local and not the global concentration of high-energy metabolites that is functionally relevant. A paradigm for such microcompartmentation is creatine kinase (CK). Cytosolic and mitochondrial isoforms of CK constitute a well established energy buffering and shuttling system whose functions are very much based on local association of CK isoforms with ATP-providing and ATP-consuming processes. Here we review current knowledge on the subcellular localization and direct protein and lipid interactions of CK isoforms, in particular about cytosolic brain-type CK (BCK) much less is known compared to muscle-type CK (MCK). We further present novel data on BCK, based on three different experimental approaches: (1) co-purification experiments, suggesting association of BCK with membrane structures such as synaptic vesicles and mitochondria, involving hydrophobic and electrostatic interactions, respectively; (2) yeast-two-hybrid analysis using cytosolic split-protein assays and the identifying membrane proteins VAMP2, VAMP3 and JWA as putative BCK interaction partners; and (3) phosphorylation experiments, showing that the cellular energy sensor AMP-activated protein kinase (AMPK) is able to phosphorylate BCK at serine 6 to trigger BCK localization at the ER, in close vicinity of the highly energy-demanding Ca(2+) ATPase pump. Thus, membrane localization of BCK seems to be an important and regulated feature for the fueling of membrane-located, ATP-dependent processes, stressing again the importance of local rather than global ATP concentrations.

Spectrometric study on the interaction of indocyanine green with human serum albumin

The interaction of indocyanine green(ICG) with human serum albumin(HSA) was investigated via various spectrometric(UV-visible, fluorescence and circular dichroism) techniques. The experimental results indicate that the interaction of ICG with HSA depends on the values of R(R is defined as the molar ratio of HSA to ICG). The interaction of ICG with HSA can form two complexes with intrinsic binding constants(Ka) of 2.97×105(R≤2) and 2.63×104(R>2), respectively. The fluorescence and induced CD(ICD) spectra of ICG demonstrate that binding the first mole of HSA to ICG can form a chiral ICG-HSA complex with strong fluorescence emission, and the chirality and fluorescence of ICG-HSA complex can be significantly reduced by adding another mole of HSA to ICG. Furthermore, although both ICG and ICG-HSA complexes followed an energy-dependent endocytosis process to enter living cells, the cellular uptaken dynamic mechanism of ICG was significantly affected by the HSA conjugation.

A Comparative Study of Cellular Uptake and Subcellular Localization of Doxorubicin Loaded in Self-Assemblies of Amphiphilic Copolymers with Pendant Dendron by MDA-MB-231 Human Breast Cancer Cells.

Previously synthesized amphiphilic diblock copolymers with pendant dendron moieties have been investigated for their potential use as drug carriers to improve the delivery of an anticancer drug to human breast cancer cells. Diblock copolymer (P71 D3 )-based micelles effectively encapsulate the doxorubicin (DOX) with a high drug-loading capacity (≈95%, 104 DOX molecules per micelle), which is approximately double the amount of drug loaded into the diblock copolymer (P296 D1 ) vesicles. DOX released from the resultant P71 D3 /DOX micelles is approximately 1.3-fold more abundant, at a tumoral acidic pH of 5.5 compared with a pH of 7.4. The P71 D3 /DOX micelles also enhance drug potency in breast cancer MDA-MB-231 cells due to their higher intracellular uptake, by approximately twofold, compared with the vesicular nanocarrier, and free DOX. Micellar nanocarriers are taken up by lysosomes via energy-dependent processes, followed by the release of DOX into the cytoplasm and subsequent translocation into the nucleus, where it exert its cytotoxic effect.

The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme.

Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354–370 and that K354, R355, and K367 are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system.

Cellular Apoptosis Assay of Breast Cancer.

Apoptosis is an energy-dependent enzymatic cell suicide process. It almost always involves the activation of caspases. In this chapter, we systemically introduce methodologies to assay caspases dependent biochemical and morphological changes in vitro breast cancer cell lines and in vivo breast cancer tissues. In addition, mitochondrial involvement is crucial to distinguish two different apoptotic pathways. Methodology to assay dissipation of mitochondrial transmembrane potential, an early event of mitochondrial involvement, is also included. Of note, since apoptotic features may not appear to the same extent depending on the context of cell types and the death-inducing insults, a common practice is to use more than one method to assess apoptosis, qualitatively and quantitatively.

Aggregation Induced Emission Mediated Controlled Release by Using a Built-In Functionalized Nanocluster with Theranostic Features.

We report biological evaluation of a novel nanoparticle delivery system based on 1,1,2-triphenyl-2-(p-hydroxyphenyl)-ethene (TPE-OH, compound 1), which has tunable aggregation-induced emission (AIE) characteristics. Compound 1 exhibited no emission in DMSO. In aqueous media, compound 1 aggregated, and luminescence was observed. The novel membrane-cytoplasm-nucleus sequential delivery strategy could induce apoptosis in four different kinds of cancer cells (including three adherent cell lines and one suspension cell line). The nanoparticles remained in the cytoplasm with intense blue emissions, whereas doxorubicin was observed in the nucleus with striking red luminescence. The nanoassembly was internalized in cells through an energy-dependent process. Three sorts of chemical inhibitors were used to clarify the endocytosis mechanism based on the AIE type prodrug. Furthermore, we have developed the first AIE theranostic system where drug targeting and release have been applied in an animal model.

Enhanced Anti-Metastatic Activity of Etoposide Using Layered Double Hydroxide Nano Particles.

Cell migration and invasion are integral to lung cancer metastasis. In this study, we investigated the combination of traditional chemotherapy and a layered double hydroxide (LDH) carrier as a new strategy for the inhibition of migration and invasion. To investigate the characteristics and possible mechanisms of VP16-LDH [the Mg-Al/LDH containing etoposide (VP16)], we used several experimental techniques, such as transmission electron microscopy (TEM) and fluorescent microscopy. The TEM, X-ray diffraction (XRD) and zeta potential results indicated that VP16 binds well with LDH, with an average size of 70 nm, and the drug delivery system was confirmed to have the desired quality of slow release by the in vitro release test results. Fluorescent images showed that the cellular uptake of VP16-LDH was a caveolae-mediated and energy-dependent process. Moreover, A549 cells treated with VP16-LDH (5 μg/ml, 10 μg/ml) demonstrated significant inhibition of cell migration and invasion compared with the cells treated with free VP16 at the same concentration. The inhibition of AKT, mTOR and STAT3 phosphorylation and p-β-catenin up-regulation in VP16-LDH-treated cells revealed a possible molecular mechanism via the mTOR/AKT and STAT pathways, through which VP16-LDH had a stronger inhibitory effect on migration than the drug alone.

Multifunctional PLGA Nanobubbles as Theranostic Agents: Combining Doxorubicin and P-gp siRNA Co-Delivery Into Human Breast Cancer Cells and Ultrasound Cellular Imaging.

Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. One of the effective approaches to overcome MDR is to use nanoparticle-mediated the gene silence of chemotherapeutic export proteins by RNA interference to increase drug accumulation in drug resistant cancer cells. In this work, a new co-delivery system, DOX-PLGA/PEI/P-gp shRNA nanobubbles (NBs) around 327 nm, to overcome doxorubicin (DOX) resistance in MCF-7 human breast cancer was designed and developed. Positively charged polyethylenimine (PEI) were modified onto the surface of DOX-PLGA NBs through DCC/NHS crosslinking, and could efficiently condense P-gp shRNA into DOX-PLGA/PEI NBs at vector/shRNA weight ratios of 70:1 and above. An in vitro release profile demonstrated an efficient DOX release (more than 80%) from DOX-PLGA/PEI NBs at pH 4.4, suggesting a pH-responsive drug release for the multifunctionalized NBs. Cellular experimental results further showed that DOX-PLGA/PEI/P-gp shRNA NBs could facilitate cellular uptake of DOX into cells and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The IC50 of DOX-PLGA NBs against MCF-7/ADR cells was 2-fold lower than that of free DOX. The increased cellular uptake and nuclear accumulation of DOX delivered by DOX-PLGA/PEI/P-gp shRNA NBs in MCF-7/ADR cells was confirmed by fluorescence microscopy and fluorescence spectrophotometry, and might be owning to the down-regulation of P-gp and reduced the efflux of DOX. The cellular uptake mechanism of DOX-PLGA/PEI/P-gp shRNA NBs indicated that the macropinocytosis was one of the pathways for the uptake of NBs by MCF-7/ADR cells, which was also an energy-dependent process. Furthermore, the in vitro cellular ultrasound imaging suggested that the employment of the DOX-PLGA/PEI/P-gp shRNA NBs could efficiently enhance ultrasound imaging of cancer cells. These results demonstrated that the developed DOX-PLGA/PEI/P-gp shRNA NBs is a potential, safe and efficient theranotic agent for cancer therapy and diagnostics.

Abstract 18019: Activation of Hepatic Hif-2a Signaling Perturbs Cholesterol Homeostasis in Mice

Cardiovascular disease (CVD) is the leading cause of death in the United States. Therefore, identifying novel pathways involved in the pathogenesis of CVD becomes imperative. Inflammation and hypercholesterolemia are the two hallmarks of CVD. Patients with chronic intermittent hypoxia or obstructive sleep apnea are at high risk for CVD due to aberrant cholesterol levels. Evolutionarily, cholesterol synthesis is a highly oxygen and energy dependent process. Therefore, the molecular mechanism involved in the paradoxical increase in cholesterol during hypoxia remains unclear. Hypoxia signaling is mediated through a well-conserved hypoxia driven transcription factors, hypoxia-inducible factor (HIF)-1 and HIF-2. Mice with a liver-specific disruption of the Von Hippel Lindau protein ( Vhl LivKO ), a critical E3 ubiquitin ligase that degrades HIFs, exhibit increased levels of HIF-1 and HIF-2 protein and develop hepatic steatosis, systemic inflammation, hypercholesterolemia and have decreased survival. Moreover, we show that the transcription factor hypoxia-inducible factor (HIF)-2 is highly activated in the liver of rodent models of atherogenesis. Activation of HIF-2 in liver disrupts the circadian expression of cholesterol hydroxylases leading to hypercholesterolemia. Using a comprehensive metabolomic analysis, we identified an arginine metabolite, asymmetric dimethylarginine (ADMA) is highly induced in the serum and liver of C57BL/6 mice fed with high cholesterol diet. ADMA potently activates HIF-2 in human hepatoma HepG2 and colon-derived SW-480 cells. Our results demonstrate that metabolite induced HIF-2 signaling in liver perturbs cholesterol homeostasis leading to systemic inflammation and hypercholesterolemia.

Can thermogenic adipocytes protect from obesity?

The role of brown adipocytes and adipocytes of a new beige type in the energy metabolism of a healthy person and in the pathogenesis of obesity has extensively been discussed in recent years. The interest to these cells has been stimulated owing to the application of new noninvasive methods for studying the metabolic activity of tissues. Using these methods, the presence of thermogenically active adipocytes in adults and their reactivity to cold stimuli have been proved. These data, together with the results of animal experiments support the idea of thermogenic fat being a direct regulator of the energy balance of man. However, for several reasons there are some objections to this viewpoint. The main objection is that the total activity of the human thermogenic adipocytes is about 100 kJ/day, i.e., it is negligible. In addition, the burn of excessive nutrients is biologically inappropriate for an organism. Therefore, the idea that obesity is caused by the decreased activity of thermogenic adipocytes is erroneous. The statement that the causes of obesity are associated with the increased efficiency of energy-dependent processes seems more reasonable. The consequence is a reduction in energy expenditure to perform a unit of biological work. This results in excess of nutrients deposited in the form of fat.

Endocytosis‒Mediated Invasion and Pathogenicity of Streptococcus agalactiae in Rat Cardiomyocyte (H9C2)

Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae internalization, we chemically inhibited discrete parts of cellular uptake system in H9C2 cells using genistein, chlorpromazine, nocodazole and cytochalasin B. Chemical inhibition of microtubule and actin formation by nocodazole and cytochalasin B impaired S. agalactiae internalization into H9C2 cells. Consistently, reverse‒ transcription PCR (RT‒PCR) and quantitative real time‒PCR (RT-qPCR) analyses also detected higher levels of transcripts for cytoskeleton forming genes, Acta1 and Tubb5 in S. agalactiae‒infected H9C2 cells, suggesting the requirement of functional cytoskeleton in pathogenesis. Host survival assay demonstrated that S. agalactiae internalization induced cytotoxicity in H9C2 cells. S. agalactiae cells grown with benzyl penicillin reduced its ability to internalize and induce cytotoxicity in H9C2 cells, which could be attributed with the removal of surface lipoteichoic acid (LTA) from S. agalactiae. Further, the LTA extracted from S. agalactiae also exhibited dose‒dependent cytotoxicity in H9C2 cells. Taken together, our data suggest that S. agalactiae cells internalized H9C2 cells through energy‒dependent endocytic processes and the LTA of S. agalactiae play major role in host cell internalization and cytotoxicity induction.

Characteristics of oxygen transport through the surface of the isolated perfused rat liver

It is shown that the transport of oxygen through the surface of the isolated perfused rat liver is an energy-dependent process that requires the energy of ATP hydrolysis.

Emerging roles of apoptotic microtubules during the execution phase of apoptosis.

Apoptosis is a genetically programmed energy-dependent process of cell demise, characterized by specific morphological and biochemical events in which the activation of caspases has an essential role. During apoptosis the cytoskeleton participates actively in characteristic morphological rearrangements of the dying cell. This reorganisation has been assigned mainly to actinomyosin ring contraction, while microtubule and intermediate filaments are depolymerized at early stages of apoptosis. However, recent reports have showed that microtubules are reformed during the execution phase of apoptosis organizing an apoptotic microtubule network (AMN). AMN is organized behind plasma membrane, forming a cortical structure. Apoptotic microtubules repolymerization takes place in many cell types and under different apoptotic inducers. It has been hypothesized that AMN is critical for maintaining plasma membrane integrity and cell morphology during the execution phase of apoptosis. AMN disorganization leads apoptotic cells to secondary necrosis and the release of potential toxic molecules which can damage neighbor cells and promotes inflammation. Therefore, AMN formation during physiological apoptosis or in pathological apoptosis induced by anti-cancer treatments is essential for tissue homeostasis and the prevention of additional cell damage and inflammation.

Subcellular distribution and uptake mechanism of di-n-butyl phthalate in roots of pumpkin (Cucurbita moschata) seedlings.

Phthalate acid esters (PAEs) are of particular concern due to their potential environmental risk to human and nonhuman organisms. Although uptake of PAEs by plants has been reported by several researchers, information about the intracellular distribution and uptake mechanisms of PAEs is still lacking. In this study, a series of hydroponic experiments using intact pumpkin (Cucurbita moschata) seedlings was conducted to investigate how di-n-butyl phthalate (DnBP), one of the most frequently identified PAEs in the environment, enters and is distributed in roots. DnBP was transported into subcellular tissues rapidly in the initial uptake period (<12 h). More than 80% of DnBP was detected in the cell walls and organelles, which suggests that DnBP is primarily accumulated in these two fractions due to their high affinity to DnBP. The kinetics of DnBP uptake were fitted well with the Michaelis-Menten equation, suggesting that a carrier-mediated process was involved. The application of 2,4-dinitrophenol and sodium vanadate reduced the uptake of DnBP by 37 and 26%, respectively, while aquaporin inhibitors, silver and glycerol, had no effect on DnBP uptake. These data demonstrated that the uptake of DnBP included a carrier-mediated and energy-dependent process without the participation of aquaporins.

Features of Metabolic Processes in Children Born by Caesarean Section in the Early Adaptation Period: the Role of Cerebral Ischemia

Background : The postnatal adaptation comprises alteration of metabolic processes of a newborn, the disturbance of which can lead to dysfunction, and later — to the development of diseases. Objective : Our aim was to study the bond of cerebral ischemia in full-term children born by Caesarean section with the course of metabolic processes in the early neonatal period. Methods : A retrospective comparative study was conducted. Children born by Caesarean section, who had cerebral ischemia (n =120) and who didn't have it, (n = 150) were examined. Complex examination included an assessment of a number of factors: clinical, biochemical blood tests, acidbase balance, concentration of some hormones. Results : In children after Caesarean section with cerebral ischemia in the early period of adaptation we noted disturbances of the acid-base balance (BE 0.3 ± 1.9 mmol/l; р = 0.038), blood oxygenation (SaO 2 79.7 ± 2.8%; р = 0.001), frank lactataemia (4.3 ± 1.8 mmol/l; р = 0.002), signs of a catabolic orientation of metabolism (hypoproteinemia, increased protein metabolism products, frank primary loss of body weight). The considerable changes of activity of the blood enzymes, disturbance of glucose metabolism indicated the lack of energy-dependent processes. Due to this, the hyperbilirubinemia and a hormonal imbalance were more often formed. Conclusion : Cerebral ischemia in children born by Caesarean section is a reason for the development of a metabolic disadaptation. The detection of disturbances of metabolic processes in the early neonatal period is necessary for a timely start of the correctional therapy in order to prevent the development of diseases in more distant periods of a child's life.

Comparison of Hourly Solar Radiation from a Ground–Based Station, Remote Sensing and Weather Forecast Models at a Coastal Site of South Italy (Lamezia Terme)

The solar radiation is a critical input parameter when working with solar energy and radiation dependent surface processes. In this study, we present preliminary results from an inter-comparison between hourly values from a pyranometer, MSG-SEVIRI sensor and two meso-scale models, WRF and RAMS, in clear and cloudy sky conditions. Cloudy sky condition is the most important because the attenuation of solar radiation in the atmosphere is strongly dependent on the cloud variability. Bias and RMSE errors are evaluated at a coastal site in the Mediterranean area. These statistics show the tendency of both models to overestimate short-wave radiation.

In vivo spectroscopy and NMR metabolite fingerprinting approaches to connect the dynamics of photosynthetic and metabolic phenotypes in resurrection plant Haberlea rhodopensis during desiccation and recovery.

The resurrection plant Haberlea rhodopensis was used to study dynamics of drought response of photosynthetic machinery parallel with changes in primary metabolism. A relation between leaf water content and photosynthetic performance was established, enabling us to perform a non-destructive evaluation of the plant water status during stress. Spectroscopic analysis of photosynthesis indicated that, at variance with linear electron flow (LEF) involving photosystem (PS) I and II, cyclic electron flow around PSI remains active till almost full dry state at the expense of the LEF, due to the changed protein organization of photosynthetic apparatus. We suggest that, this activity could have a photoprotective role and prevent a complete drop in adenosine triphosphate (ATP), in the absence of LEF, to fuel specific energy-dependent processes necessary for the survival of the plant, during the late states of desiccation. The NMR fingerprint shows the significant metabolic changes in several pathways. Due to the declining of LEF accompanied by biosynthetic reactions during desiccation, a reduction of the ATP pool during drought was observed, which was fully and quickly recovered after plants rehydration. We found a decline of valine accompanied by lipid degradation during stress, likely to provide alternative carbon sources for sucrose accumulation at late stages of desiccation. This accumulation, as well as the increased levels of glycerophosphodiesters during drought stress could provide osmoprotection to the cells.

RGD functionalized polymeric nanoparticles targeting periodontitis epithelial cells for the enhanced treatment of periodontitis in dogs

Long term retention of antimicrobials with effective drug concentration in gingival crevicular fluid (GCF) is of vital importance for the treatment of chronic periodontitis. In this study, a novel epithelial cell-targeting nanoparticle drug delivery system by conjugating minocycline-loaded poly(ethylene glycol)–poly(lactic acid) (PEG–PLA) nanoparticles (NP-MIN) with RGD peptide were developed and administrated locally for targeting periodontitis epithelial cells and enhancing the treatment of periodontitis in dogs. Biodegradable NP-MIN was made with an emulsion/solvent evaporation technique. RGD peptide was conjugated to the surface of nanoparticles via Maleimide group reaction with hydrosulfide in RGD peptide (RGD-NP-MIN). Transmission electron microscopy examination and dynamic light scattering results revealed that RGD-NP-MIN had a sphere shape, with a mean diameter around 106nm. In vitro release of minocycline from RGD-NP-MIN showed that RGD modification did not change the remarkable sustained releasing characteristic of NP-MIN. To elucidate the interaction of RGD-NP and epithelial cells, RGD-NP binding, uptake and cellular internalization mechanisms by calu-3 cells were investigated. It was shown RGD modification significantly enhanced nanoparticles binding and uptake by Calu-3 cells, and RGD-NP uptake was an energy-dependent process through receptor-mediated endocytosis. Both clathrin-associated endocytosis and caveolae-dependent endocytosis pathway were involved in the RGD-NP uptake, and the intracellular transport of RGD-NP was related to lysosome and Golgi apparatus. Finally, in vivo pharmacokinetics of minocycline in the periodontal pockets and anti-periodontitis effects of RGD-NP-MIN on periodontitis-bearing dogs were evaluated. After local administration of RGD-NP-MIN, minocycline concentration in gingival crevicular fluid decreased slowly and maintained an effective drug concentration for a longer time than that of NP-MIN. Anti-periodontitis effects demonstrated that RGD-NP-MIN could significantly decrease symptoms of periodontitis, which was better than any other control group. These findings suggested that these epithelial cell-targeting nanoparticles offered a novel and effective local delivery system for the treatment of periodontitis.