Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-κB and HSP27/p38/AKT Pathways and by Inhibiting ABCB1

Despite advances in cancer detection and prevention, a diagnosis of metastatic disease remains a death sentence due to the fact that many cancers are either resistant to chemotherapy (conventional or targeted) or develop resistance during treatment, and residual chemoresistant cells are highly metastatic. Metastatic cancer cells resist the effects of chemotherapeutic agents by upregulating drug transporters, which efflux the drugs, and by activating proliferation and survival signaling pathways. Previously, we found that c-Abl and Arg non-receptor tyrosine kinases are activated in breast cancer, melanoma, and glioblastoma cells, and promote cancer progression. In this report, we demonstrate that the c-Abl/Arg inhibitor, imatinib (imatinib mesylate, STI571, Gleevec), reverses intrinsic and acquired resistance to the anthracycline, doxorubicin, by inducing G2/M arrest and promoting apoptosis in cancer cells expressing highly active c-Abl and Arg. Significantly, imatinib prevents intrinsic resistance by promoting doxorubicin-mediated NF-κB/p65 nuclear localization and repression of NF-κB targets in a STAT3-dependent manner, and by preventing activation of a novel STAT3/HSP27/p38/Akt survival pathway. In contrast, imatinib prevents acquired resistance by inhibiting upregulation of the ABC drug transporter, ABCB1, directly inhibiting ABCB1 function, and abrogating survival signaling. Thus, imatinib inhibits multiple novel chemoresistance pathways, which indicates that it may be effective in reversing intrinsic and acquired resistance in cancers containing highly active c-Abl and Arg, a critical step in effectively treating metastatic disease. Furthermore, since imatinib converts a master survival regulator, NF-κB, from a pro-survival into a pro-apoptotic factor, our data suggest that NF-κB inhibitors may be ineffective in sensitizing tumors containing activated c-Abl/Arg to anthracyclines, and instead might antagonize anthracycline-induced apoptosis.     

Heterogeneity in the replicating population of cultured human epidermal keratinocytes

We studied the replication of keratinocytes in stratified squamous epithelia. Other studies have revealed functional and morphological heterogeneity in the replicating population of such cells. To examine possible kinetic heterogeneity, we determined the cell-cycle lengths of replicating cells in cultures of human epidermal keratinocytes. A double-label assay was developed, which measures the time between two successive cycles of DNA synthesis. The first cycle of DNA synthesis was marked by pulse labeling cultures for a brief period with 14C-thymidine (dThd), and the second cycle was detected by labeling at a later time with bromodeoxyuridine (BrdUrd). The time taken for the 14C-labeled DNA to become doubly labeled with BrdUrd was shown to correspond to the length of the cell cycle. In subconfluent cultures in which the cell number increased at an exponential rate, the average cell-cycle time was 21.5 h. In confluent cultures in which desquamation was balanced by cell renewal, the average cell cycle was 31.5 h. However, in confluent cultures, three populations of replicating cells were evident, these having cycle times of 22, 33, and 40 h. In subconfluent cultures, there was no clear evidence for cell-cycle heterogeneity of the replicating cells, although the most rapidly cycling cells in these cultures had a cycle time (16 h) considerably less than the most rapidly cycling cells in the confluent cultures (21 h). It is possible that the rapidly cycling cells seen in the subconfluent cultures were stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Forage quality's influence on mule deer fawns

In many mule deer (Odocoileus hemionus) populations, recruitment of fawns drives population dynamics. The quality of food available to females and their fawns in summer and autumn may play an important role in fawn recruitment. We examined direct links between digestible energy (DE) content of food and the DE intake of females on the nutrient concentration of milk and between the nursing behavior, DE intake, growth, and survival in captive mule deer fawns. We offered females and their fawns diets that simulated the natural decline in DE content of forage from mid-summer to late autumn in many western landscapes. Fawns fed a higher DE diet weighed 14% more at the onset of winter, had fewer unsuccessful nursing attempts, consumed milk with more protein and energy, and had higher survival than fawns fed a low DE diet. Differences between fawn performances among treatments were greatest when diet quality began decreasing earlier in the summer. Because our results indicate that summer and autumn nutrition is likely to influence fawn recruitment, wildlife biologists should include metrics for summer precipitation and late autumn fawn mass in population models, and land managers should focus on methods for improving the nutritional carrying capacity of summer and early autumn habitats. © 2011 The Wildlife Society.     

Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed‐batch media

Perfusion medium was successfully developed based on our fed‐batch platform basal and feed media. A systematic development approach was undertaken by first optimizing the ratios of fed‐batch basal and feed media followed by targeted removal of unnecessary and redundant components. With this reduction in components, the medium could then be further concentrated by 2× to increase medium depth. The medium osmolality was also optimized where we found ～360 mOsm/kg was desirable resulting in a residual culture osmolality of ～300 mOsm/kg for our cell lines. Further building on this, the amino acids Q, E, N, and D were rebalanced to reduce lactate and ammonium levels, and increase the cell‐specific productivity without compromising on cell viability while leaving viable cell density largely unaffected. Further modifications were also made by increasing certain important vitamin and lipid concentrations, while eliminating other unnecessary vitamins. Overall, an effective perfusion medium was developed with all components remaining in the formulation understood to be important and their concentrations increased to improve medium depth. The critical cell‐specific perfusion rate using this medium was then established for a cell line of interest to be 0.075 nL/cell‐day yielding 1.2 g/L‐day at steady state. This perfusion process was then successfully scaled up to a 100 L single‐use bioreactor with an ATF6 demonstrating similar performance as a 2 L bioreactor with an ATF2. Large volume handling challenges in our fed‐batch facility were overcome by developing a liquid medium version of the powder medium product contained in custom totes for plug‐and‐play use with the bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:891–901, 2017     

Quinolines as a novel structural class of potent and selective PDE4 inhibitors. Optimisation for inhaled administration

Crystallography driven optimisation of a lead derived from similarity searching of the GSK compound collection resulted in the discovery of quinoline-3-carboxamides as highly potent and selective inhibitors of phosphodiesterase 4B. This series has been optimized to GSK256066, a potent PDE4B inhibitor which also inhibits LPS induced production of TNF-α from isolated human peripheral blood mononuclear cells with a pIC₅₀ of 11.1. GSK256066 also has a suitable profile for inhaled dosing.     

Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria

Background

Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness.

Methods

Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits.

Results

Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions.

Conclusions

These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic ‘supermales’ could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings.     

Effect of Soy Protein Subunit Composition on the Rheological Properties of Soymilk during Acidification

The effect of soy protein subunit composition on the acid-induced aggregation of soymilk was investigated by preparing soymilk from different soybean lines lacking specific glycinin and β-conglycinin subunits. Acid gelation was induced by glucono-δ-lactone (GDL) and analysis was done using diffusing wave spectroscopy and rheology. Aggregation occurred near pH 5.8 and the increase in radius corresponded to an increase in the elastic modulus measured by small deformation rheology. Diffusing wave spectroscopy was also employed to follow acid gelation, and data indicated that particle interactions start to occur at a higher pH than the pH of onset of gelation (corresponding to the start of the rapid increase in elastic modulus). The protein subunit composition significantly affected the development of structure during acidification. The onset of aggregation occurred at a higher pH for soymilk samples containing group IIb (the acidic subunit A₃) of glycinin, than for samples prepared from Harovinton (a commercial variety containing all subunits) or from genotypes null in glycinin. The gels made from lines containing group I (A₁, A₂) and group IIb (A₃) of glycinin resulted in stiffer acid gels compared to the lines containing only β-conglycinin. These results confirmed that the ratio of glycinin/β-conglycinin has a significant effect on gel structure, with an increase in glycinin causing an increase in gel stiffness. The type of glycinin subunits also affected the aggregation behavior of soymilk.     

Density, dispersal, and feeding impact of western flower thrips (Thysanoptera: Thripidae) on flowering chrysanthemum at different spatial scales

Abstract.  1. This study evaluated the effect of dispersal on the density and feeding impact of a phytophagous insect in relation to the spatial distribution of its host plants.
2. The interaction between density, dispersal, and feeding impact of western flower thrips on flowering chrysanthemum was quantified at three spatial scales, with infested and uninfested plants either isolated in 0.25 m² individual cages, or enclosed together in 2.25 m² communal cages or 75 m² greenhouses.
3. In individual cages, the rate of dispersal from chrysanthemum plants to blue sticky traps increased with the density of thrips for females but not males. Uninfested plants consistently had fewer thrips when they were individually caged rather than enclosed with plants infested with adults, indicating that dispersal mediates inter-plant distribution of thrips.
4. The feeding impact of thrips on inflorescences was evaluated using the absorbance of ethanol extracts at wavelengths characteristic of yellow carotenoid pigments associated with chrysanthemum inflorescences (415, 445, and 472 nm). Increasing absorbance of extracts with increasing density of thrips per inflorescence suggests that feeding by thrips results in ruptured cells leaching carotenoid pigments.
5. In communal cages, the distribution of thrips was uniform for infested and uninfested plants, whereas the density and feeding impact of thrips in greenhouses were higher for infested than uninfested plants. These results suggest that short-range dispersal by adults homogenises the density and feeding impact of thrips among host plants only on a small spatial scale.