The Effect of Oxygen and Micronutrient Composition of Cell Growth Media on Cancer Cell Bioenergetics and Mitochondrial Networks.

Cancer cell culture is routinely performed under superphysiologic O2 levels and in media such as Dulbecco's Modified Eagle Medium (DMEM) with nutrient composition dissimilar to mammalian extracellular fluid. Recently developed cell culture media (e.g., Plasmax, Human Plasma-Like Medium (HPLM)), which are modeled on the metabolite composition of human blood plasma, have been shown to shift key cellular activities in several cancer cell lines. Similar effects have been reported with respect to O2 levels in cell culture. Given these observations, we investigated how media composition and O2 levels affect cellular energy metabolism and mitochondria network structure in MCF7, SaOS2, LNCaP, and Huh7 cells. Cells were cultured in physiologic (5%) or standard (18%) O2 levels, and in physiologic (Plasmax) or standard cell culture media (DMEM). We show that both O2 levels and media composition significantly affect mitochondrial abundance and network structure, concomitantly with changes in cellular bioenergetics. Extracellular acidification rate (ECAR), a proxy for glycolytic activity, was generally higher in cells cultured in DMEM while oxygen consumption rates (OCR) were lower. This effect of media on energy metabolism is an important consideration for the study of cancer drugs that target aspects of energy metabolism, including lactate dehydrogenase activity.

Media composition and O2 levels determine effects of 17ß-estradiol and selective estrogen receptor modulators on mitochondrial bioenergetics and cellular reactive oxygen species.

Estradiol (E2) and selective estrogen receptor modulators (SERMs) have broad-ranging cellular effects that include mitochondrial respiration and reactive oxygen species (ROS) metabolism. Many of these effects have been studied using cell culture models. Recent advances have revealed the extent to which cellular metabolism is affected by the culture environment. Cell culture media with metabolite composition similar to blood plasma [e.g., Plasmax, Human Plasma-Like Medium (HPLM)] alter cellular behaviors including responses to drugs. Similar effects have been observed with respect to O2 levels in cell culture. Given these observations, we investigated whether the effects of E2 and SERMs are also influenced by media composition and O2 level during cell culture experiments. We analyzed mitochondrial network characteristics, cellular oxidative metabolism, and H2O2 production in C2C12 myoblasts growing in physiological (5%) or standard cell culture (18%)O2 and in physiological (Plasmax) or standard cell culture [Dulbecco's modified Eagle's medium (DMEM)] media. Although E2 significantly lowered H2O2 production from cells growing in 18% O2/DMEM (standard cell culture), it had no effect on cells growing in Plasmax. Moreover, culture conditions significantly altered the effects of E2 and SERMs on mitochondrial abundance and network characteristics. These results indicate that the effects of E2 and SERMs on various aspects of cell physiology strongly depends on growth conditions, which in turn emphasizes the need to consider this carefully in cell culture experiments.

Bioabsorbable metal zinc differentially affects mitochondria in vascular endothelial and smooth muscle cells.

Zinc is an essential trace element having various structural, catalytic and regulatory interactions with an estimated 3000 proteins. Zinc has drawn recent attention for its use, both as pure metal and alloyed, in arterial stents due to its biodegradability, biocompatibility, and low corrosion rates. Previous studies have demonstrated that zinc metal implants prevent the development of neointimal hyperplasia, which is a common cause of restenosis following coronary intervention. This suppression appears to be smooth muscle cell-specific, as reendothelization of the neointima is not inhibited. To better understand the basis of zinc's differential effects on rat aortic smooth muscle (RASMC) versus endothelial (RAENDO) cells, we conducted a transcriptomic analysis of both cell types following one-week continuous treatment with 5 µM or 50 µM zinc. This analysis indicated that genes whose protein products regulate mitochondrial functions, including oxidative phosphorylation and fusion/fission, are differentially affected by zinc in the two cell types. To better understand this, we performed Seahorse metabolic flux assays and quantitative imaging of mitochondrial networks in both cell types. Zinc treatment differently affected energy metabolism and mitochondrial structure/function in the two cell types. For example, both basal and maximal oxygen consumption rates were increased by zinc in RASMC but not in RAENDO. Zinc treatment increased apparent mitochondrial fusion in RASMC cells but increased mitochondrial fission in RAENDO cells. These results provide some insight into the mechanisms by which zinc treatment differently affects the two cell types and this information is important for understanding the role of zinc treatment in vascular cells and improving its use in biodegradable metal implants.

Divergent mechanisms for regulating growth and development after imaginal disc damage in the tobacco hornworm, Manduca sexta.

Holometabolous insects have been able to radiate to vast ecological niches as adults through the evolution of adult-specific structures such as wings, antennae and eyes. These structures arise from imaginal discs that show regenerative capacity when damaged. During imaginal disc regeneration, development has been shown to be delayed in the fruit fly Drosophila melanogaster, but how conserved the delay-inducing mechanisms are across holometabolous insects has not been assessed. The goal of this research was to develop the hornworm Manduca sexta as an alternative model organism to study such damage-induced mechanisms, with the advantage of a larger hemolymph volume enabling access to the hormonal responses to imaginal disc damage. Upon whole-body X-ray exposure, we noted that the imaginal discs were selectively damaged, as assessed by TUNEL and Acridine Orange stains. Moreover, development was delayed, predominantly at the pupal-to-adult transition, with a concomitant delay in the prepupal ecdysteroid peak. The delays to eclosion were dose dependent, with some ability for repair of damaged tissues. We noted a shift in critical weight, as assessed by the point at which starvation no longer impacted developmental timing, without a change in growth rate, which was uncoupled from juvenile hormone clearance in the body. The developmental profile was different from that of D. melanogaster, which suggests species differences may exist in the mechanisms delaying development.

Collectively Improving Our Teaching: Attempting Biology Department-wide Professional Development in Scientific Teaching.

Many efforts to improve science teaching in higher education focus on a few faculty members at an institution at a time, with limited published evidence on attempts to engage faculty across entire departments. We created a long-term, department-wide collaborative professional development program, Biology Faculty Explorations in Scientific Teaching (Biology FEST). Across 3 years of Biology FEST, 89% of the department's faculty completed a weeklong scientific teaching institute, and 83% of eligible instructors participated in additional semester-long follow-up programs. A semester after institute completion, the majority of Biology FEST alumni reported adding active learning to their courses. These instructor self-reports were corroborated by audio analysis of classroom noise and surveys of students in biology courses on the frequency of active-learning techniques used in classes taught by Biology FEST alumni and nonalumni. Three years after Biology FEST launched, faculty participants overwhelmingly reported that their teaching was positively affected. Unexpectedly, most respondents also believed that they had improved relationships with departmental colleagues and felt a greater sense of belonging to the department. Overall, our results indicate that biology department-wide collaborative efforts to develop scientific teaching skills can indeed attract large numbers of faculty, spark widespread change in teaching practices, and improve departmental relations.

Central neural alterations predominate in an insect model of nociceptive sensitization.

Many organisms respond to noxious stimuli with defensive maneuvers. This is noted in the hornworm, Manduca sexta, as a defensive strike response. After tissue damage, organisms typically display sensitized responses to both noxious or normally innocuous stimuli. To further understand this phenomenon, we used novel in situ and in vitro preparations based on paired extracellular nerve recordings and videography to identify central and peripheral nerves responsible for nociception and sensitization of the defensive behavior in M. sexta. In addition, we used the in vivo defensive strike response threshold assayed with von Frey filaments to examine the roles that N-methyl-D-aspartate receptor (NMDAR) and hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels play in this nociceptive sensitization using the inhibitors MK-801 and AP5 (NMDAR), and ivabradine and ZD7288 (HCN). Using our new preparations, we found that afferent activity evoked by noxious pinch in these preparations was conveyed to central ganglia by axons in the anterior- and lateral-dorsal nerve branches, and that sensitization induced by tissue damage was mediated centrally. Furthermore, sensitization was blocked by all inhibitors tested except the inactive isomer L-AP5, and reversed by ivabradine both in vivo and in vitro. Our findings suggest that M. sexta's sensitization occurs through central signal amplification. Due to the relatively natural sensitization method and conserved molecular actions, we suggest that M. sexta may be a valuable model for studying the electrophysiological properties of nociceptive sensitization and potentially related conditions such as allodynia and hyperalgesia in a comparative setting that offers unique experimental advantages. J. Comp. Neurol. 525:1176-1191, 2017. © 2016 Wiley Periodicals, Inc.

Use of von Frey filaments to assess nociceptive sensitization in the hornworm, Manduca sexta.

BACKGROUND: The hornworm Manduca sexta exhibits a defensive strike to noxious assaults, a response that is robust and is easily observed by experimenters. Von Frey filaments and methods typical for studying nociception in other animals were used to assess the strike response in M. sexta. NEW METHODS: A series of von Frey filaments was applied to the body wall in ascending order and the data generated were used to determine the strike threshold by (i) the up-and-down method, (ii) the first response method, and (iii) the simplified up-and-down order method (SUDO). The effect of a noxious pinch on strike threshold was assessed. COMPARISON WITH EXISTING METHODS: To our knowledge none of these methods has been used on M. sexta previously, making the use of the up-and-down and SUDO methods the first in an invertebrate. The use of the first response method has been used in other invertebrates, and the method appears equally suited to M. sexta. RESULTS: All three methods were successful in monitoring the threshold sensitivity to touch, which was lowered (sensitized) by tissue damage induced with a pinch. Sensitization lasted 19h. CONCLUSIONS: All three methods of assessing nociception were successfully applied to quantify the defensive strike response in M. sexta, although the SUDO method required empirical assessment of which filament to start the test sequence with. The results revealed both short- and long-term sensitization. These methods should prove to be useful for quantifying sensitization in M. sexta.

Ecdysis behaviors and circadian rhythm of ecdysis in the stick insect, Carausius morosus.

Successful ecdysis in insects depends on proper timing and sequential activation of an elaborate series of motor programs driven by a relatively conserved network of neuropeptides. The behaviors must be activated at the appropriate times to ensure successful loosening and shedding of the old cuticle, and can be influenced by environmental cues in the form of immediate sensory feedback and by circadian rhythms. We assessed the behaviors, components of the neural network and the circadian basis of ecdysis in the stick insect, Carausius morosus. C. morosus showed many of the characteristic pre-ecdysis and ecdysis behaviors previously described in crickets and locusts. Ecdysis was described in three phases, namely the (i) preparatory or pre-ecdysis phase, (ii) the ecdysial phase, and (iii) the post-ecdysis or exuvial phase. The frequencies of push-ups and sways during the preparatory phase were quantified as well as durations of all the phases. The regulation of ecdysis appeared to act via elevation of cGMP, as described in many other insects, although eclosion hormone-like immunoreactivity was not noted using a lepidopteran antiserum. Finally, C. morosus showed a circadian rhythm to the onset of ecdysis, with ecdysis occurring just prior to or at lights on. Ecdysis could be induced precociously with mechanical stimulation.

Male chemosignals inhibit the neural responses of male mice to female chemosignals.

Reproductive function in mice is regulated by reproductively-stimulating and reproductively-inhibiting primer pheromones released by conspecifics. When experienced simultaneously, their responses to reproductively-inhibiting chemosignals take precedence over their responses to reproductively-stimulating chemosignals. For example, while female urine induces luteinizing hormone (LH) release in males, this response is blocked when male urine is presented in conjunction with female urine. In the present study, we examined the neural correlates of these responses to male and female urine. Sexually experienced, male CF1 mice were exposed to water, female urine, or a mixture of male and female urine. The resulting patterns of Fos-immunoreactivity (Fos-ir) were then compared between groups. Female urine induced significantly more Fos-ir within the main and accessory olfactory systems (MOS and AOS, respectively) than did water, male urine or mixed urine. Notably, within the main and accessory olfactory bulbs, male urine attenuated the responses of mitral cells, but not granule cells, to female urine. Overall, the results indicate that exposure to male urine inhibited the responses of cells within the MOS and AOS to female urine. The specific pattern of Fos-ir in the olfactory bulbs suggests that this may be due to an inhibition in the responses of mitral cells to female urine.

Steroid hormone modulation of olfactory processing in the context of socio-sexual behaviors in rodents and humans.

Primer pheromones and other chemosensory cues are important factors governing social interactions and reproductive physiology in many species of mammals. Responses to these chemosignals can vary substantially within and between individuals. This variability can stem, at least in part, from the modulating effects steroid and non-steroid hormones exert on olfactory processing. Such modulation frequently augments or facilitates the effects that prevailing social and environmental conditions have on the reproductive axis. The mechanisms underlying the hormonal regulation of responses to chemosensory cues are diverse. They are in part behavioral, achieved through the modulation of chemoinvestigative behaviors, and in part a product of the modulation of the intrinsic responsiveness of the main and accessory olfactory systems to conspecific, as well as other classes, of chemosignals. The behavioral and non-behavioral effects complement one another to ensure that mating and other reproductive processes are confined to reproductively favorable conditions.