Parkin Overexpression Ameliorates PrP106–126-Induced Neurotoxicity via Enhanced Autophagy in N2a Cells

Transmissible spongiform encephalopathies (TSEs) are caused by the accumulation of the abnormal prion protein scrapie (PrP^Sc). Prion protein aggregation, misfolding, and cytotoxicity in the brain are the major causes of neuronal dysfunction and ultimate neurodegeneration in all TSEs. Parkin, an E3 ubiquitin ligase, has been studied extensively in all major protein misfolding aggregating diseases, especially Parkinson’s disease and Alzheimer’s disease, but the role of parkin in TSEs remains unknown. Here we investigated the role of parkin in a prion disease cell model in which neuroblastoma2a (N2a) cells were treated with prion peptide PrP106–126. We observed a gradual decrease in the soluble parkin level upon treatment with PrP106–126 in a time-dependent manner. Furthermore, endogenous parkin colocalized with FITC-tagged prion fragment106–126. Overexpression of parkin in N2a cells via transfection repressed apoptosis by enhancing autophagy. Parkin-overexpressing cells also showed reductions in apoptotic BAX translocation to the mitochondria and cytochrome c release to the cytosol, which ultimately inhibited activation of proapoptotic caspases. Taken together, our findings reveal a parkin-mediated cytoprotective mechanism against PrP106–126 toxicity, which is a novel potential therapeutic target for treating prion diseases.     

Growth of ectomycorrhizal mycelia and composition of soil microbial communities in oak forest soils along a nitrogen deposition gradient

Deciduous forests may respond differently from coniferous forests to the anthropogenic deposition of nitrogen (N). Since fungi, especially ectomycorrhizal (EM) fungi, are known to be negatively affected by N deposition, the effects of N deposition on the soil microbial community, total fungal biomass and mycelial growth of EM fungi were studied in oak-dominated deciduous forests along a nitrogen deposition gradient in southern Sweden. In-growth mesh bags were used to estimate the production of mycelia by EM fungi in 19 oak stands in the N deposition gradient, and the results were compared with nitrate leaching data obtained previously. Soil samples from 154 oak forest sites were analysed regarding the content of phospholipid fatty acids (PLFAs). Thirty PLFAs associated with microbes were analysed and the PLFA 18:2ω6,9 was used as an indicator to estimate the total fungal biomass. Higher N deposition (20 kg N ha⁻¹ y⁻¹ compared with 10 kg N ha⁻¹ y⁻¹) tended to reduce EM mycelial growth. The total soil fungal biomass was not affected by N deposition or soil pH, while the PLFA 16:1ω5, a biomarker for arbuscular mycorrhizal (AM) fungi, was negatively affected by N deposition, but also positively correlated to soil pH. Other PLFAs positively affected by soil pH were, e.g., i14:0, a15:0, 16:1ω9, a17:0 and 18:1ω7, while some were negatively affected by pH, such as i15:0, 16:1ω7t, 10Me17:0 and cy19:0. In addition, N deposition had an effect on the PLFAs 16:1ω7c and 16:1ω9 (negatively) and cy19:0 (positively). The production of EM mycelia is probably more sensitive to N deposition than total fungal biomass according to the fungal biomarker PLFA 18:2ω6,9. Low amounts of EM mycelia covaried with increased nitrate leaching, suggesting that EM mycelia possibly play an important role in forest soil N retention at increased N input.     

A synopsis of the genus <Emphasis Type="Italic">Poteranthera</Emphasis> (Melastomeae: Melastomataceae) with the description of a new,apparently pollinator deceiving species

A synopsis of the annual genus Poteranthera is presented here. Three species are recognized including the newly described Poteranthera windischii. Poteranthera is characterized by its annual life cycle, generally linear leaves that have conspicuous gland-tipped hairs on the lamina margin, 5-merous flowers with a constriction at the level of the torus, one cycle of stamens reduced to staminodia or absent altogether, three locular ovary, and reniform seeds with a foveolate testa. The flowers of the new species P. windischii are strongly heterandrous, where the usual set of stamens that bees harvest pollen from is reduced to staminodia and the stamen set that deposits pollen on the bee has large yellow ventral appendages that possibly function as pollen mimics and result in deceit pollination. This hypothesis is supported by experiments that have demonstrated innate preferences of female bees for yellow, UV absorbing colors in flowers. Species of Poteranthera are extremely rare, known from very few specimens and possibly highly endangered.     

A Novel Folate-Targeted Nanoliposomal System of Doxorubicin for Cancer Targeting

Targeted drug delivery systems for cancer improves anti-tumor efficacy and reduces systemic toxicity by restricting availability of cytotoxic drugs within tumors. Targeting moieties, such as natural ligands (folic acid, transferrin, and biotin) which are overexpressed on tumors, have been used to enhance liposome-encapsulated drug accumulation within tumors and resulted in better control. In this report, we explored the scope of targeting ligand folic acid, which is incorporated in liposome systems using folic acid-modified cholesterol (CPF), enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in increased cytotoxicity within tumors. Folate-tagged poloxamer-coated liposomes (FDL) were found to have significantly higher cellular uptake than conventional poloxamer-coated liposomes (DL), as confirmed by fluorometric analysis in B16F10 melanoma cells. Biodistribution study of the radiolabeled liposomal system indicated the significantly higher tumor uptake of FDL as compared to DL. Anti-tumor activity of FDL against murine B16F10 melanoma tumor-bearing mice revealed that FDL inhibited tumor growth more efficiently than the DL. Taken together, the results demonstrated the significant potential of the folate-conjugated nanoliposomal system for drug delivery to tumors.     

Lidocaine-loaded fish scale-nanocellulose biopolymer composite microneedles

Microneedle (MN) technology has emerged as an effective drug delivery system, and it has tremendous potential as a patient friendly substitute for conventional methods for transdermal drug delivery (TDD). In this paper, we report on the preparation of lidocaine-loaded biodegradable microneedles, which are manufactured from fish scale-derived collagen. Lidocaine, a common tissue numbing anaesthetic, is loaded in these microneedles with an aim of delivering the drug with controlled skin permeation. Evaluation of lidocaine permeation in porcine skin has been successfully performed using Franz diffusion cell (FDC) which has shown that the drug permeation rate increases from 2.5 to 7.5% w/w after 36 h and pseudo steady state profile is observed from 5.0 to 10.0% w/w lidocaine-loaded microneedle. Swelling experiments have suggested that the microneedles have negligible swellability which implies that the patch would stick to the tissue when inserted. The experiments on MN dissolution have depicted that the lidocaine loaded in the patch is lower than the theoretical loading, which is expected as there can be losses of the drug during initial process manufacture.     

Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition

Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.     

Growth enhancement of black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis>) by a newly isolated <Emphasis Type="Italic">Bacillus tequilensis</Emphasis> NII-0943

A Gram positive, rod-shaped potential strain was selected from the pool of bacterial isolates obtained from the Western Ghats forest (India) on the basis of zone of P-solubilization activity. Identification based on 16S rRNA gene sequence revealed that the strain is of Bacillus species, sharing highest sequence similarity to Bacillus tequilensis NRRL B-41771^T (99.5%). Strain NII-0943 was able to produce good amount of indole acetic acid (IAA) and was positive for siderophore production. In addition to IAA and siderophore attributes, strain NII-0943 also possessed the characteristics like Ca₃(PO₄)₂ solubilization and growth in nitrogen-free medium. Seed inoculation with the strain NII-0943 resulted in significantly higher root initiation in black pepper cuttings grown under pots. The contents of nitrogen and phosphorus in both soil and plant were also enhanced significantly in treatments inoculated with these bacterial inocula. Hence, based on this evidence it is proposed that strain NII-0943 could be deployed as a plant growth-promoting inoculant to attain the desired results of bacterization.     

Mycobacterium kansasii-induced death of murine macrophages involves endoplasmic reticulum stress responses mediated by reactive oxygen species generation or calpain activation

Although pathogenic mechanisms of tuberculosis have been extensively studied, little is known about the pathogenic mechanisms of Mycobacterium kansasii. In this work the influence of virulence and ER-stress mediated apoptosis of macrophages during two different strains of M. kansasii infection was investigated. We show that M. kansasii infection is associated with ER stress-mediated apoptosis in the murine macrophage cell line RAW 264.7. Infection of RAW 264.7 cells in vitro with apoptosis-inducing a clinical isolate of M. kansasii SM-1 (SM-1) resulted in strong induction of ER stress responses compared with M. kansasii type strain (ATCC 12478)-infected RAW 264.7 cells. Interestingly, inhibition of calpain prevented the induction of CHOP and Bip in ATCC 12478-infected RAW 264.7 cells but not in RAW 264.7 cells infected with SM-1. In contrast, reactive oxygen species (ROS) were significantly increased only in RAW 264.7 cells infected with SM-1. We propose that ROS generation is important for triggering ER stress-mediated apoptosis during SM-1 infection, whereas ATCC 12478-induced, ER stress-mediated apoptosis is associated with calpain activation. Our results demonstrate that the ER stress pathway plays important roles in the pathogenesis of M. kansasii infections, and that different strains of M. kansasii induce different patterns of ER stress-mediated apoptosis.     

Comparative Membrane Channel Size and Activity of Botulinum Neurotoxins A and E

In an effort to compare the molecular basis of differential toxic activity of botulinum neurotoxin A (BoNT/A) and BoNT/E, we have analyzed their membrane channel activity by measuring calcein release from liposomes. Both BoNT/A and /E showed a same level of membrane channel activity that was specifically blocked by IgG specific to the neurotoxins. With the use of fluorescein-labeled dextran, we determined that the size of the channel is at least 24.2 A which is appropriate for the translocation of a protein of 50 kDa (the light chain of BoNT). These findings would suggest that the difference in the toxicity level of the two BoNT serotypes might reflect differences in either endopeptidase activity or their binding to receptor(s).