αCT1 peptide sensitizes glioma cells to temozolomide in a glioblastoma organoid platform

Glioblastoma (GBM) is the most common form of brain cancer. Even with aggressive treatment, tumor recurrence is almost universal and patient prognosis is poor because many GBM cell subpopulations, especially the mesenchymal and glioma stem cell populations, are resistant to temozolomide (TMZ), the most commonly used chemotherapeutic in GBM. For this reason, there is an urgent need for the development of new therapies that can more effectively treat GBM. Several recent studies have indicated that high expression of connexin 43 (Cx43) in GBM is associated with poor patient outcomes. It has been hypothesized that inhibition of the Cx43 hemichannels could prevent TMZ efflux and sensitize otherwise resistance cells to the treatment. In this study, we use a three-dimensional organoid model of GBM to demonstrate that combinatorial treatment with TMZ and αCT1, a Cx43 mimetic peptide, significantly improves treatment efficacy in certain populations of GBM. Confocal imaging was used to visualize changes in Cx43 expression in response to combinatorial treatment. These results indicate that Cx43 inhibition should be pursued further as an improved treatment for GBM.     

Callosal transfer of impulses originating from superficial and deep nerves of the cat forelimb.

1. Experiments were performed in 18 chloralose-anaesthetized, curarized cats in order to study the callosal transfer of somatic information originated in exteroceptive and proprioceptive receptors. Several cutaneous and deep nerves of the forelimb were prepared and stimulated with graded intensities, so as to activate selectively afferent fibres pertaining to the different groups of Lloyd's classification. Simultaneous records were taken (and averaged on-line by means of a multichannel analyzer) from the distal end of a cut dorsal rootlet (C7-C8), from the cerebral cortex (SI, SII or area 3a, according to the experiment) and from the somesthetic callosal region (SCR). 2. The low-threshold afferent fibres (Group II) of cutaneous origin were found to have a wide projection to the SCR, with the maximal density in its middle portion. Some of the fastest corticocallosal impulses are relayed monosynaptically at cortical level. Plots of the amplitude of cortical and callosal responses as a function of stimulus strength showed that both central responses have the same threshold and exhibit a parallel, sharply-rising amplitude increase, thus suggesting that the cortico-callosal re-transmission system for afferent impulses of cutaneous origin is very powerful in nature. Impulses elicited in afferent fibres of higher threshold (Group III) do not enhance the cortical and callosal positive waves provoked by Group II afferent volleys. 3. Afferent fibres of deep origin were also found to send a wide projection to the SCR, although less substantial than that of cutaneous fibres. Stimulation of the deep radial nerve elicited mass responses in the whole SCR, provided the strength of stimuli was high enough to engage the Group II fibres. Only in the central portion of the SCR were small potentials recorded in response to pure Group I volleys of DRN. Experiments performed with selective stimulation of pure muscular branches of forelimb deep nerves as well as of articular and mixed (muscular and articular) branches gave evidence making it possible to ascertain the origin of deep afferent fibres projecting to the SCR. Stimulation of the forelimb muscular branches with strength provoking full activation of Group I afferent and additional engagement of those of Group II, did not provoke mass responses in the whole extent of the SCR. In order to obtain callosal potentials upon stimulation of pure muscular nerves, it was necessary to increase the stimulus strength at or above the threshold for Group III fibres. On the contrary, the same callosal foci unresponsive to Group I and II muscular afferent volleys exhibited clear-cut responses to stimulation of the lowest-threshold Group I and/or Group II afferents of articular and mixed nerves. From the results it might be inferred that only proprioceptive information originating from articular receptors and from extrafusal muscular afferents has access to the callosal interhemispheric transfer.     

Prolonged lifespan with enhanced exploratory behavior in mice overexpressing the oxidized nucleoside triphosphatase hMTH1

The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1‐Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1‐Tg mice express high levels of the hMTH1 hydrolase that degrades 8‐oxodGTP and 8‐oxoGTP and excludes 8‐oxoguanine from both DNA and RNA. Compared to wild‐type animals, hMTH1‐overexpressing mice have significantly lower steady‐state levels of 8‐oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age‐dependent accumulation of DNA 8‐oxoguanine that occurs in wild‐type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1‐Tg animals live significantly longer than their wild‐type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1‐Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1‐Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1‐Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids.     

Adenosine 2A Receptor Antagonist Prevented and Reversed Liver Fibrosis in a Mouse Model of Ethanol-Exacerbated Liver Fibrosis

The effect of moderate alcohol consumption on liver fibrosis is not well understood, but evidence suggests that adenosine may play a role in mediating the effects of moderate ethanol on tissue injury. Ethanol increases the concentration of adenosine in the liver. Adenosine 2A receptor (A2AR) activation is known to enhance hepatic stellate cell (HSC) activation and A2AR deficient mice are protected from fibrosis in mice. Making use of a novel mouse model of moderate ethanol consumption in which female C57BL/6J mice were allowed continued access to 2% (vol/vol) ethanol (11% calories) or pair-fed control diets for 2 days, 2 weeks or 5 weeks and superimposed with exposure to CCl₄, we tested the hypothesis that moderate ethanol consumption increases fibrosis in response to carbon tetrachloride (CCl₄) and that treatment of mice with an A2AR antagonist prevents and/or reverses this ethanol-induced increase in liver fibrosis. Neither the expression or activity of CYP2E1, required for bio-activation of CCl₄, nor AST and ALT activity in the plasma were affected by ethanol, indicating that moderate ethanol did not increase the direct hepatotoxicity of CCl₄. However, ethanol feeding enhanced HSC activation and exacerbated liver fibrosis upon exposure to CCl_4. This was associated with an increased sinusoidal angiogenic response in the liver. Treatment with A2AR antagonist both prevented and reversed the ability of ethanol to exacerbate liver fibrosis.

Conclusion

Moderate ethanol consumption exacerbates hepatic fibrosis upon exposure to CCl₄. A2AR antagonism may be a potential pharmaceutical intervention to decrease hepatic fibrosis in response to ethanol.     

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.     

Evaluating outcomes of restoration ecology projects on limited budgets: assessment of variation in sampling intensity and sampling frequency for four habitat types

While best practices for evaluating restoration ecology projects are emerging rapidly, budget constraints often limit postrestoration monitoring, which emphasizes the need for practical and efficient monitoring strategies. We examined the postrestoration outcome for an ENGO (Nature Conservancy of Canada) project, to assess retroactively how variation in intensity and frequency of sampling would have affected estimates of plant species composition, diversity, and richness over time. The project restored four habitat types (mesic forest, oak woodland, wet meadow, and sand barren) using sculptured seeding of tallgrass prairie and woody species. Species‐level plant cover was monitored annually for 10 years in 168 2 × 2–m quadrats. We performed randomization tests to examine estimates of species diversity and richness as a function of the number of quadrats sampled, and assessed the necessity of annual sampling for describing changes in species composition and successional trajectories. The randomization tests revealed that sampling 10–17 quadrats, depending on habitat type, was sufficient to obtain estimates of species diversity that were at least 95% of values obtained from the whole dataset. Species richness as a function of number of quadrats sampled did not plateau, which suggests that rather than increasing the number of sampling quadrats, richness could be estimated more efficiently using nonquadrat based sampling techniques. Nonmetric multidimensional scaling analysis revealed that plant species composition largely stabilized by 3–5 years postrestoration depending on habitat type. By that time, native, seeded species dominated the restoration, and the benefits of annual sampling for tracking changes in species composition diminished.     

HIV-1 Nef assembles a Src family kinase-ZAP-70/Syk-PI3K cascade to downregulate cell-surface MHC-I

HIV-1 Nef, which is required for the efficient onset of AIDS, enhances viral replication and infectivity by exerting multiple effects on infected cells. Nef downregulates cell-surface MHC-I molecules by an uncharacterized PI3K pathway requiring the actions of two Nef motifs-EEEE(65) and PXXP(75). We report that the Nef EEEE(65) targeting motif enables Nef PXXP(75) to bind and activate a trans-Golgi network-localized Src family tyrosine kinase (SFK). The Nef/SFK complex then recruits and phosphorylates the tyrosine kinase ZAP-70, which binds class I PI3K to trigger MHC-I downregulation in primary CD4+ T cells. In promonocytic cells, Nef/SFK recruits the ZAP-70 homolog Syk to downregulate MHC-I, implicating this PI3K pathway in multiple HIV-1 reservoirs. Isoform-specific PI3K inhibitors repress MHC-I downregulation, identifying them as potential therapeutic agents to combat HIV-1. The discovery of this Nef-SFK-ZAP-70/Syk-PI3K signaling pathway explains the hierarchal role of the Nef motifs in effecting immunoevasion.     

Local FK506 dose-dependent study using a novel three-dimensional organotypic assay

Local administration of FK506, an FDA approved immunosuppressant with neuroregenerative properties, is a promising technique to achieve improved peripheral nerve regeneration while preventing the side effects associated with the systemic administration of this drug. Although considerable research has been devoted to the development of clinically suitable systems for local delivery of FK506 to the site of nerve injury and repair, the optimal dose of FK506 for enhancement of axon regeneration in the peripheral nerve has not yet been established. To this end, we devised a three-dimensional (3D) organotypic assay capable of mimicking the peripheral nerve. This assay consisted of a neonatal rat dorsal root ganglion (DRG) extending its neurites into the native peripheral nerve scaffold provided by an acellular nerve allograft (ANA). A novel 3D compartmented cell culture system was adapted from the 3D organotypic assay to achieve local delivery of FK506 just to the growing neurites in vitro and establish the required local dose of FK506 for peripheral nerve regeneration. A bimodal dose response was observed by culturing the entire DRG–ANA construct with media containing different concentrations of FK506. Low drug concentration of 1 pg/ml and high drug concentration of 100 ng/ml lead to the longest neurite extension in vitro. Furthermore, regardless of the FK506 concentration, concentrating the drug to the growing neurites resulted in significant increase in both neurite extension and neurite density, an effect that was not observed with the FK506 delivery to both neurites and neural cell bodies within DRG. The findings in this study provide valuable insight into the optimal local dose of FK506 for peripheral nerve regeneration. Furthermore, for the first time, this study suggests the potential interaction of FK506 with axons at the level of the growth cone.     

Biotechnological applications of bioluminescence and chemiluminescence

Recent progress in molecular biology has made available several biotechnological tools that take advantage of the high detectability and rapidity of bioluminescence and chemiluminescence spectroscopy. These developments provide inroads to in vitro and in vivo continuous monitoring of biological processes (e.g. gene expression, protein-protein interaction and disease progression), with clinical, diagnostic and drug discovery applications. Furthermore, combining luminescent enzymes or photoproteins with biospecific recognition elements at the genetic level has led to the development of ultrasensitive and selective bioanalytical tools, such as recombinant whole-cell biosensors, immunoassays and nucleic acid hybridization assays. The high detectability of the luminescence analytical signal makes it appropriate for miniaturized bioanalytical devices (e.g. microarrays, microfluidic devices and high-density-well microtiter plates) for the high-throughput screening of genes and proteins in small sample volumes.