Effects, uptake, and translocation of aluminum oxide nanoparticles in lettuce: A comparison study to phytotoxic aluminum ions.

The widespread use of aluminum oxide nanoparticles (Al2O3 NPs) unavoidably causes the release of NPs into the environment, potentially having unforeseen consequences for biological processes. Due to the well-known issue of Al phytoxicity, plant interactions with Al2O3 NPs are cause for concern, but these interactions remain poorly understood. This study investigated the effects of Al2O3 NPs on lettuce (Lactuca sativa L.) to elucidate the similarities and differences in plant growth responses when compared to those of Al ions. Seed germination, root length, biomass production, and uptake of Al and nutrients were measured from hydroponically-grown lettuce with varying concentrations of Al2O3 NPs (0, 0.4, 1, and 2 mg/mL) or AlCl3 (0, 0.04, 0.4, and 1 mg/mL). The Al2O3 NPs treatments had a positive influence on root elongation, whereas AlCl3 significantly reduced emerging root lengths. While 0.4 mg/mL Al2O3 NPs promoted biomass, 1 and 2 mg/mL showed a 10.4% and 17.9% decrease in biomass, respectively, when compared to the control. Similarly, 0.4 and 1 mg/mL AlCl3 reduced biomass to 22.3% and 9.96%, respectively. Both treatments increased Al uptake by roots linearly; however, translocation of Al2O3 NPs into shoots was limited, whereas translocation of AlCl3 increased with increasing treatment concentration. Further, Al2O3 NPs adsorbed on the roots serve as adsorbents for macronutrients, promoting their absorption and uptake in plants, but not micronutrients. Calcium uptake was the most inhibited by AlCl3. A new in vivo imaging technique, with elemental analysis, confirmed that Al2O3 NPs were assimilated as particles, not ions, suggesting that the observed phytotoxicity is not due to Al ions being released from the NPs. Thus, it is concluded that Al2O3 NPs pose less phytoxicity than AlCl3, primarily due to NPs role on stimulated root growth, significant adsorption/aggregation on roots, limited lateral translocation to shoots, and increased uptake of macronutrients.

PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.

In the event of a radiologic catastrophe, endothelial cell and neutrophil dysfunction play important roles in tissue injury. Clinically available therapeutics for radiation-induced vascular injury are largely supportive. PKCδ was identified as a critical regulator of the inflammatory response, and its inhibition was shown to protect critical organs during sepsis. We used a novel biomimetic microfluidic assay (bMFA) to interrogate the role of PKCδ in radiation-induced neutrophil-endothelial cell interaction and endothelial cell function. HUVECs formed a complete lumen in bMFA and were treated with 0.5, 2, or 5 Gy ionizing radiation (IR). At 24 h post-IR, the cells were treated with a PKCδ inhibitor for an additional 24 h. Under physiologic shear flow, the role of PKCδ on endothelium function and neutrophil adherence/migration was determined. PKCδ inhibition dramatically attenuated IR-induced endothelium permeability increase and significantly decreased neutrophil migration across IR-treated endothelial cells. Moreover, neutrophil adhesion to irradiated endothelial cells was significantly decreased after PKCδ inhibition in a flow-dependent manner. PKCδ inhibition downregulated IR-induced P-selectin, intercellular adhesion molecule 1, and VCAM-1 but not E-selectin overexpression. PKCδ is an important regulator of neutrophil-endothelial cell interaction post-IR, and its inhibition can serve as a potential radiation medical countermeasure.-Soroush, F., Tang, Y., Zaidi, H. M., Sheffield, J. B., Kilpatrick, L. E., Kiani, M. F. PKCδ inhibition as a novel medical countermeasure for radiation-induced vascular damage.

A Biomimetic Microfluidic Tumor Microenvironment Platform Mimicking the EPR Effect for Rapid Screening of Drug Delivery Systems.

Real-time monitoring of tumor drug delivery in vivo is a daunting challenge due to the heterogeneity and complexity of the tumor microenvironment. In this study, we developed a biomimetic microfluidic tumor microenvironment (bMTM) comprising co-culture of tumor and endothelial cells in a 3D environment. The platform consists of a vascular compartment featuring a network of vessels cultured with endothelial cells forming a complete lumen under shear flow in communication with 3D solid tumors cultured in a tumor compartment. Endothelial cell permeability to both small dye molecules and large liposomal drug carriers were quantified using fluorescence microscopy. Endothelial cell intercellular junction formation was characterized by immunostaining. Endothelial cell permeability significantly increased in the presence of either tumor cell conditioned media (TCM) or tumor cells. The magnitude of this increase in permeability was significantly higher in the presence of metastatic breast tumor cells as compared to non-metastatic ones. Immunostaining revealed impaired endothelial cell-cell junctions in the presence of either metastatic TCM or metastatic tumor cells. Our findings indicate that the bMTM platform mimics the tumor microenvironment including the EPR effect. This platform has a significant potential in applications such as cell-cell/cell-drug carrier interaction studies and rapid screening of cancer drug therapeutics/carriers.

A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip.

Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier (BBB). To establish such a model, we have developed a novel blood-brain barrier on a chip (B3C) that comprises a tissue compartment and vascular channels placed side-by-side mimicking the three-dimensional morphology, size and flow characteristics of microvessels in vivo. Rat brain endothelial cells (RBEC) isolated from neonatal rats were seeded in the vascular channels of B3C and maintained under shear flow conditions, while neonatal rat astrocytes were cultured under static conditions in the tissue compartment of the B3C. RBEC formed continuous endothelial lining with a central lumen along the length of the vascular channels of B3C and exhibited tight junction formation, as measured by the expression of zonula occludens-1 (ZO-1). ZO-1 expression significantly increased with shear flow in the vascular channels and with the presence of astrocyte conditioned medium (ACM) or astrocytes cultured in the tissue compartment. Consistent with in vivo BBB, B3C allowed endfeet-like astrocyte-endothelial cell interactions through a porous interface that separates the tissue compartment containing cultured astrocytes from the cultured RBEC in the vascular channels. The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0 ± 0.9 x 10-6 cm/s to 2.9 ± 1.0 x 10-6 cm/s or 1.1±0.4 x 10-6 cm/s, respectively). Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC. Moreover, B3C exhibits significantly improved barrier characteristics compared to the transwell model and B3C permeability was not significantly different from the in vivo BBB permeability in neonatal rats. In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics.

Gram's Stain Does Not Cross the Bacterial Cytoplasmic Membrane.

For well over a century, Hans Christian Gram's famous staining protocol has been the standard go-to diagnostic for characterizing unknown bacteria. Despite continuous and ubiquitous use, we now demonstrate that the current understanding of the molecular mechanism for this differential stain is largely incorrect. Using the fully complementary time-resolved methods: second-harmonic light-scattering and bright-field transmission microscopy, we present a real-time and membrane specific quantitative characterization of the bacterial uptake of crystal-violet (CV), the dye used in Gram's protocol. Our observations contradict the currently accepted mechanism which depicts that, for both Gram-negative and Gram-positive bacteria, CV readily traverses the peptidoglycan mesh (PM) and cytoplasmic membrane (CM) before equilibrating within the cytosol. We find that not only is CV unable to traverse the CM but, on the time-scale of the Gram-stain procedure, CV is kinetically trapped within the PM. Our results indicate that CV, rather than dyes which rapidly traverse the PM, is uniquely suited as the Gram stain.

A spiroligomer α-helix mimic that binds HDM2, penetrates human cells and stabilizes HDM2 in cell culture.

We demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers. Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM. The spiroligomer 1 penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner increases the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.

Combining visual information from the two eyes: the relationship between isthmotectal cells that project to ipsilateral and to contralateral optic tectum using fluorescent retrograde labels in the frog, Rana pipiens.

The frog nucleus isthmi (homolog of the mammalian parabigeminal nucleus) is a visually responsive tegmental structure that is reciprocally connected with the ipsilateral optic tectum; cells in nucleus isthmi also project to the contralateral optic tectum. We investigated the location of the isthmotectal cells that project ipsilaterally and contralaterally using three retrograde fluorescent label solutions: Alexa Fluor 488 10,000 mw dextran conjugate; Rhodamine B isothiocyanate; and Nuclear Yellow. Dye solutions were pressure-injected into separate sites in the superficial optic tectum. Following a 6-day survival, brains were fixed, sectioned, and then photographed. Injection of the different labels at separate, discrete locations in the optic tectum result in retrograde filling of singly labeled clusters of cells in both the ipsilateral and contralateral nucleus isthmi. Generally, ipsilaterally projecting cells are dorsal to the contralaterally projecting cells, but there is a slight overlap between the two sets of cells. Nonetheless, when different retrograde labels are injected into opposite tecta, there is no indication that individual cells project to both tecta. The set of cells that project to the ipsilateral tectum and the set of cells that project to the contralateral tectum form a visuotopic map in a roughly vertical, transverse slab. Our results suggest that nucleus isthmi can be separated into two regions with cells in the dorsolateral portion projecting primarily to the ipsilateral optic tectum and cells in the ventrolateral nucleus isthmi projecting primarily to the contralateral optic tectum.

Cdk9 phosphorylates p53 on serine 392 independently of CKII.

The tumor suppressor p53 is an important cellular protein, which controls cell cycle progression. Phosphorylation is one of the mechanisms by which p53 is regulated. Here we report the interaction of p53 with another key regulator, cdk9, which together with cyclin T1 forms the positive transcription elongation complex, p-TEFb. This complex cooperates with the HIV-1 Tat protein to cause the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II and this facilitates the elongation of HIV-1 transcription. We demonstrate that cdk9 phosphorylates p53 on serine 392 through their direct physical interaction. Results from protein-protein interaction assays revealed that cdk9 interacts with the C-terminal domain (aa 361-393) of p53, while p53 interacts with the N-terminal domain of cdk9. Transfection and protein binding assays (EMSA and ChIP) demonstrated the ability of p53 to bind and activate the cdk9 promoter. Interestingly, cdk9 phosphorylates serine 392 of p53, which could be also phosphorylated by casein kinase II. Kinase assays demonstrated that cdk9 phosphorylates p53 independently of CKII. These studies demonstrate the existence of a feedback-loop between p53 and cdk9, pinpointing a novel mechanism by which p53 regulates the basal transcriptional machinery.