Cytotoxicity profile of pristine graphene on brain microvascular endothelial cells.

Graphene-based nanomaterials hold the potential to be used in a wide variety of applications, including biomedical devices. Pristine graphene (PG) is an un-functionalized, defect-free type of graphene that could be used as a material for neural interfacing. However, the neurotoxic effects of PG, particularly to the blood-brain barrier (BBB), have not been fully studied. The BBB separates the brain tissue from the circulating substances in the blood and is essential to maintain the brain homeostasis. The principal components of the BBB are brain microvascular endothelial cells (BMVECs), which maintain a protectively low permeability due to the expression of tight junction proteins. Here we analyzed the effects of PG on BMVECs in an in vitro model of the BBB. BMVECs were treated with PG at 0, 10, 50 and 100 µg/mL for 24 hours and viability and functional analyses of BBB integrity were performed. PG increased lactate dehydrogenase release at 50 and 100 µg/mL, suggesting the induction of necrosis. Surprisingly, 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium (XTT) conversion was increased at 10 and 50 µg/mL. In contrast, XTT conversion was decreased at 100 µg/mL, suggesting the induction of cell death. In addition, 100 µg/mL PG increased DNA fragmentation, suggesting induction of apoptosis. At the same time, 50 and 100 µg/mL of PG increased the endothelial permeability, which corresponded with a decrease in the expression of the tight junction protein occludin at 100 µg/mL. In conclusion, these results suggest that PG negatively affects the viability and function of the BBB endothelial cells in vitro.

Raman spectroscopy using plasmonic and carbon-based nanoparticles for cancer detection, diagnosis, and treatment guidance.Part 1: Diagnosis.

Optical techniques, including Raman, photothermal and photoacoustic microscopy and spectroscopy, have been intensively explored for the sensitive and accurate detection of various diseases. Rapid advances in lasers, photodetectors, and nanotechnology have led to the development of Raman spectroscopy, particularly surface-enhanced Raman scattering (SERS), as a promising imaging modality that can help diagnose many diseases. This review focuses on the major recent advances in Raman spectroscopy and SERS-enhancing contrast nanoagents, as well as their potential to transition from a proof-of-concept approach to a cancer detection tool in vitro and in vivo.

Autophagy function and its relationship to pathology, clinical applications, drug metabolism and toxicity.

Autophagy is a cellular process that facilitates nutrient turnover and removal of expended macromolecules and organelles to maintain homeostasis. The recycling of cytosolic macromolecules and damaged organelles by autophagosomes occurs through the lysosomal degradation pathway. Autophagy can also be upregulated as a prosurvival pathway in response to stress stimuli such as starvation, hypoxia or cell damage. Over the last two decades, there has been a surge in research revealing the basic molecular mechanisms of autophagy in mammalian cells. A corollary of an advanced understanding of autophagy has been a concurrent expansion of research into understanding autophagic function and dysfunction in pathology. Recent studies have revealed a pivotal role for autophagy in drug toxicity, and for utilizing autophagic components as diagnostic markers and therapeutic targets in treating disease and cancer. In this review, advances in understanding the molecular basis of mammalian autophagy, methods used to induce and evaluate autophagy, and the diverse interactions between autophagy and drug toxicity, disease progression and carcinogenesis are discussed. Copyright © 2016 John Wiley & Sons, Ltd.

The role of surface chemistry in the cytotoxicity profile of graphene.

Graphene and its derivative, because of their unique physical, electrical and chemical properties, are an important class of nanomaterials being proposed as foundational materials in nanomedicine as well as for a variety of industrial applications. A major limitation for graphene, when used in biomedical applications, is its poor solubility due to its rather hydrophobic nature. Therefore, chemical functionalities are commonly introduced to alter both its surface chemistry and biochemical activity. Here, we show that surface chemistry plays a major role in the toxicological profile of the graphene structures. To demonstrate this, we chemically increased the oxidation level of the pristine graphene and compared the corresponding toxicological effects along with those for the graphene oxide. X-ray photoelectron spectroscopy revealed that pristine graphene had the lowest amount of surface oxygen, while graphene oxide had the highest at 2.5% and 31%, respectively. Low and high oxygen functionalized graphene samples were found to have 6.6% and 24% surface oxygen, respectively. Our results showed a dose-dependent trend in the cytotoxicity profile, where pristine graphene was the most cytotoxic, with decreasing toxicity observed with increasing oxygen content. Increased surface oxygen also played a role in nanomaterial dispersion in water or cell culture medium over longer periods. It is likely that higher dispersity might result in graphene entering into cells as individual flakes ~1 nm thick rather than as more cytotoxic aggregates. In conclusion, changes in graphene's surface chemistry resulted in altered solubility and toxicity, suggesting that a generalized toxicity profile would be rather misleading. Copyright © 2016 John Wiley & Sons, Ltd.

Distinct sets of Rab6 effectors contribute to ZW10--and COG-dependent Golgi homeostasis.

The organization of the Golgi apparatus is determined in part by the interaction of Rab proteins and their diverse array of effectors. Here, we used multiple approaches to identify and characterize a small subset of effectors that mimicked the effects of Rab6 on Golgi ribbon organization. In a visual-based, candidate protein screen, we found that the individual depletion of any of three Rab6 effectors, myosin IIA (MyoIIA), Kif20A and Bicaudal D (BicD), was sufficient to suppress Golgi ribbon fragmentation/dispersal coupled to retrograde tether proteins in a manner paralleling Rab6. MyoIIA and Kif20A depletions were pathway selective and suppressed ZW10-dependent Golgi ribbon fragmentation/dispersal only whereas BicD depletion, like Rab6, suppressed both ZW10- and COG-dependent Golgi ribbon fragmentation. The MyoIIA effects could be produced in short-term assays by the reversible myosin inhibitor, blebbistatin. At the electron microscope level, the effects of BicD-depletion mimicked many of those of Rab6-depletion: longer and more continuous Golgi cisternae and a pronounced accumulation of coated vesicles. Functionally, BicD-depleted cells were inhibited in transport of newly synthesized VSV-G protein to the cell surface. In summary, our results indicate small, partially overlapping subsets of Rab6 effectors are differentially important to two tether-dependent pathways essential to Golgi organization and function.

Interdependence of DNA mismatch repair proteins MLH1 and MSH2 in apoptosis in human colorectal carcinoma cell lines.

The mammalian DNA mismatch repair (MMR) system consists of a number of proteins that play important roles in repair of base pair mismatch mutations and in maintenance of genomic integrity. A defect in this system can cause genetic instability, which can lead to carcinogenesis. For instance, a germline mutation in one of the mismatch repair proteins, especially MLH1 or MSH2, is responsible for hereditary non-polyposis colorectal cancer. These MMR proteins also play an important role in the induction of apoptosis. Accordingly, altered expression of or a defect in MLH1 or MSH2 may confer resistance to anti-cancer drugs used in chemotherapy. We hypothesized that the ability of these two MMR proteins to regulate apoptosis are interdependent. Moreover, a defect in either one may confer resistance to chemotherapy by an inability to trigger apoptosis. To this end, we studied three cell lines-SW480, LoVo, and HTC116. These cell lines were selected based on their differential expression of MLH1 and MSH2 proteins. SW480 expresses both MLH1 and MSH2; LoVo expresses only MLH1 but not MSH2; HCT116 expresses only MSH2 but not MLH1 protein. MTT assays, a measure of cytotoxicity, showed that there were different cytotoxic effects of an anti-cancer drug, etoposide, on these cell lines, effects that were correlated with the MMR status of the cells. Cells that are deficient in MLH1 protein (HCT116 cells) were resistant to the drug. Cells that express both MLH1 and MSH2 proteins (SW480 cells) showed caspase-3 cleavage, an indicator of apoptosis. Cells that lack MLH1 (HCT116 cells) did not show any caspase-3 cleavage. Expression of full-length MLH1 protein was decreased in MMR proficient (SW480) cells during apoptosis; it remained unchanged in cells that lack MSH2 (LoVo cells). The expression of MSH2 protein remained unchanged during apoptosis both in MMR proficient (SW480) and deficient (HCT116) cells. Studies on translocation of MLH1 protein from nucleus to cytosolic fraction, an indicator of apoptosis, showed that MLH1 translocation only occurred in MMR proficient (SW480) cells upon induction of apoptosis further suggested a MSH2 dependent role of MLH1 in apoptosis. These data suggest a role of MLH1 in mediation of apoptosis in a MSH2-dependent manner. Taken together, our data supported an interdependence of mismatch repair proteins, particularly MLH1 and MSH2, in the mediation of apoptosis in human colorectal carcinoma cell lines.

Biological control potential of entomopathogenic fungal strains against peach Fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae).

The peachfruit fly, Bactrocera zonata (Saunders) is a polyphagous pest in nature, belonging to order, Diptera and their respective family is Tephritidae. It mostly feeds on different crops, vegetables and fruits. Different traditional chemical insecticides have been used to control this notorious pest. Excessive consumption of pesticides has become a major threat to the fresh fruits trade since many importing countries refused to accept the shipments due to public health and environmental concerns. There is a growing trend to control these pests using the most effective biological control methods and other preventive measures have been adopted for reducing their attacks. Fungal agents have been used as biological agents to manage the attack of different insects pest through biological means. The present study was conducted to assess the virulence of three entomopathogenic fungi, Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii, against Bactrocera zonata stages under different laboratory conditions. The results showed that B. bassiana and M. anisopliae were more effective in pathogenicity and potentially kill at all stages of B. zonata as compared to V. lecanii. The highest mortality rate for the third larval instar and the pupal stage were recorded after exposure to the 1 × 1010 conidia/ml concentrations, B. bassiana, with 68.67% and 89.67%, respectively. Adult B. zonata flies were the most susceptible to all entomopathogenic fungi. However, M. anisopliae was more virulent against B. zonata adult flies than B. bassiana and V. lecanii at 1 × 1010 conidial concentration. Therefore, the entomopathogenic fungi B. bassiana and M. anisopliae can be used as an cost effective bio-insecticide in the integrated pest management programs to control B. zonata. This study will be helpful to overcome this pest through biological control means.

Epistatic Analysis of the Contribution of Rabs and Kifs to CATCHR Family Dependent Golgi Organization.

Multisubunit members of the CATCHR family: COG and NRZ complexes, mediate intra-Golgi and Golgi to ER vesicle tethering, respectively. We systematically addressed the genetic and functional interrelationships between Rabs, Kifs, and the retrograde CATCHR family proteins: COG3 and ZW10, which are necessary to maintain the organization of the Golgi complex. We scored the ability of siRNAs targeting 19 Golgi-associated Rab proteins and all 44 human Kifs, microtubule-dependent motor proteins, to suppress CATCHR-dependent Golgi fragmentation in an epistatic fluorescent microscopy-based assay. We found that co-depletion of Rab6A, Rab6A', Rab27A, Rab39A and two minus-end Kifs, namely KIFC3 and KIF25, suppressed both COG3- and ZW10-depletion-induced Golgi fragmentation. ZW10-dependent Golgi fragmentation was suppressed selectively by a separate set of Rabs: Rab11A, Rab33B and the little characterized Rab29. 10 Kifs were identified as hits in ZW10-depletion-induced Golgi fragmentation, and, in contrast to the double suppressive Kifs, these were predominantly plus-end motors. No Rabs or Kifs selectively suppressed COG3-depletion-induced Golgi fragmentation. Protein-protein interaction network analysis indicated putative direct and indirect links between suppressive Rabs and tether function. Validation of the suppressive hits by EM confirmed a restored organization of the Golgi cisternal stack. Based on these outcomes, we propose a three-way competitive model of Golgi organization in which Rabs, Kifs and tethers modulate sequentially the balance between Golgi-derived vesicle formation, consumption, and off-Golgi transport.

Quantification of cellular associated graphene and induced surface receptor responses.

The use of graphene for biomedical and other applications involving humans is growing and shows practical promise. However, quantifying the graphitic nanomaterials that interact with cells and assessing any corresponding cellular response is extremely challenging. Here, we report an effective approach to quantify graphene interacting with single cells that utilizes combined multimodal-Raman and photoacoustic spectroscopy. This approach correlates the spectroscopic signature of graphene with the measurement of its mass using a quartz crystal microbalance resonator. Using this technique, we demonstrate single cell noninvasive quantification and multidimensional mapping of graphene with a detection limit of as low as 200 femtograms. Our investigation also revealed previously unseen graphene-induced changes in surface receptor expression in dendritic cells of the immune system. This tool integrates high-sensitivity real-time detection and monitoring of nanoscale materials inside single cells with the measurement of induced simultaneous biological cell responses, providing a powerful method to study the impact of nanomaterials on living systems and as a result, the toxicology of nanoscale materials.

Modifying Dendritic Cell Activation with Plasmonic Nano Vectors.

Dendritic cells (DCs) can acquire, process, and present antigens to T-cells to induce an immune response. For this reason, targeting cancer antigens to DCs in order to cause an immune response against cancer is an emerging area of nanomedicine that has the potential to redefine the way certain cancers are treated. The use of plasmonically active silver-coated gold nanorods (henceforth referred to as plasmonic nano vectors (PNVs)) as potential carriers for DC tumor vaccines has not been presented before. Effective carriers must be able to be phagocytized by DCs, present low toxicity, and induce the maturation of DCs-an early indication of an immune response. When we treated DCs with the PNVs, we found that the cell viability of DCs was unaffected, up to 200 µg/ml. Additionally, the PNVs associated with the DCs as they were phagocytized and they were found to reside within intracellular compartments such as endosomes. More importantly, the PNVs were able to induce expression of surface markers indicative of DC activation and maturation, i.e. CD40, CD86, and MHC class II. These results provide the first evidence that PNVs are promising carriers for DC-based vaccines and warrant further investigating for clinical use.

Identification of Rab41/6d Effectors Provides an Explanation for the Differential Effects of Rab41/6d and Rab6a/a' on Golgi Organization.

Unexpectedly, members of the Rab VI subfamily exhibit considerable variation in their effects on Golgi organization and trafficking. By fluorescence microscopy, neither depletion nor overexpression of the GDP-locked form of Rab6a/a', the first trans Golgi-associated Rab protein discovered, affects Golgi ribbon organization while, on the other hand, both Rab41/6d depletion and overexpression of GDP-locked form cause Golgi fragmentation into a cluster of punctate elements, suggesting that Rab41/6d has an active role in maintenance of Golgi ribbon organization. To establish a molecular basis for these differences, we screened for Rab41/6d interacting proteins by yeast two-hybrid assay. 155 non-repetitive hits were isolated and sequenced, and after searching in NCBI database, 102 different proteins and protein fragments were identified. None of these hits overlapped with any published Rab6a/a' effector. Eight putative Rab41 interactors involved in membrane trafficking were found. Significantly, these exhibited a preferential interaction with GTP- vs. GDP-locked Rab41/6d. Of the 8 hits, the dynactin 6, syntaxin 8, and Kif18A plasmids were the only ones expressing the full-length protein. Hence, these 3 proteins were selected for further study. We found that depletion of dynactin 6 or syntaxin 8, but not Kif18A, resulted in a fragmented Golgi apparatus that displayed a Rab41/6d knockdown phenotype, i.e., the Golgi apparatus was disrupted into a cluster of punctate Golgi elements. Co-immunoprecipation experiments verified that the interaction of dynactin 6 and syntaxin 8 with GTP-locked Rab41/6d was stronger than that with wild type Rab41/6d and least with the GDP-locked form. In contrast, co-immunoprecipitation interaction with Rab6a was greatest with the GDP-locked Rab6a, suggestive of a non-physiological interaction. In conclusion, we suggest that dynactin 6, a subunit of dynactin complex, the minus-end-directed, dynein motor, provides a sufficient molecular basis to explain the active role of Rab41/6d in maintaining Golgi ribbon organization while syntaxin 8 contributes more indirectly to Golgi positioning.

Metabolic Engineering and Comparative Performance Studies of Synechocystis sp. PCC 6803 Strains for Effective Utilization of Xylose.

Wood sugars such as xylose can be used as an inexpensive carbon source for biotechnological applications. The model cyanobacterium Synechocystis sp. PCC 6803 lacks the ability to catabolize wood sugars as an energy source. Here, we generated four Synechocystis strains that heterologously expressed XylAB enzymes, which mediate xylose catabolism, either in combination with or without one of three xylose transporters, namely XylE, GalP, or Glf. Except for glf, which is derived from the bacterium Zymomonas mobilis ZM4, the heterologous genes were sourced from Escherichia coli K-12. All of the recombinant strains were able to utilize xylose in the absence of catabolite repression. When xylose was the lone source of organic carbon, strains possessing the XylE and Glf transporters were most efficient in terms of dry biomass production and xylose consumption and the strain lacking a heterologous transporter was the least efficient. However, in the presence of a xylose-glucose mixed sugar source, the strains exhibited similar levels of growth and xylose consumption. This study demonstrates that various bacterial xylose transporters can boost xylose catabolism in transgenic Synechocystis strains, and paves the way for the sustainable production of bio-compounds and green fuels from lignocellulosic biomass.

RpaA regulates the accumulation of monomeric photosystem I and PsbA under high light conditions in Synechocystis sp. PCC 6803.

The response regulator RpaA was examined by targeted mutagenesis under high light conditions in Synechocystis sp. PCC 6803. A significant reduction in chlorophyll fluorescence from photosystem I at 77 K was observed in the RpaA mutant cells under high light conditions. Interestingly, the chlorophyll fluorescence emission from the photosystem I trimers at 77 K are similar to that of the wild type, while the chlorophyll fluorescence from the photosystem I monomers was at a much lower level in the mutant than in the wild type under high light conditions. The RpaA inactivation resulted in a dramatic reduction in the monomeric photosystem I and the D1 protein but not the CP47 content. However, there is no significant difference in the transcript levels of psaA or psbA or other genes examined, most of which are involved in photosynthesis, pigment biosynthesis, or stress responses. Under high light conditions, the growth of the mutant was affected, and both the chlorophyll content and the whole-chain oxygen evolution capability of the mutant were found to be significantly lower than those of the wild type, respectively. We propose that RpaA regulates the accumulation of the monomeric photosystem I and the D1 protein under high light conditions. This is the first report demonstrating that inactivation of a stress response regulator has specifically reduced the monomeric photosystem I. It suggests that PS I monomers and PS I trimers can be regulated independently for acclimation of cells to high light stress.

The high light-inducible polypeptides stabilize trimeric photosystem I complex under high light conditions in Synechocystis PCC 6803.

The high light-inducible polypeptides (HLIPs) are critical for survival under high light (HL) conditions in Synechocystis PCC 6803. In this article, we determined the localization of all four HLIPs in thylakoid protein complexes and examined effects of hli gene deletion on the photosynthetic protein complexes. The HliA and HliB proteins were found to be associated with trimeric photosystem I (PSI) complexes and the Slr1128 protein, whereas HliC was associated with PsaL and TMP14. The HliD was associated with partially dissociated PSI complexes. The PSI activities of the hli mutants were 3- to 4-fold lower than that of the wild type. The hli single mutants lost more than 30% of the PSI trimers after they were incubated in intermediate HL for 12 h. The reduction of PSI trimers were further augmented in these cells by the increase of light intensity. The quadruple hli deletion mutant contained less than one-half of PSI trimers following 12-h incubation in intermediate HL. It lost essentially all of the PSI trimers upon exposure to HL for 12 h. Furthermore, a mutant lacking both PSI trimers and Slr1128 showed growth defects similar to that of the quadruple hli deletion mutant under different light conditions. These results suggest that the HLIPs stabilize PSI trimers, interact with Slr1128, and protect cells under HL conditions.