Francisella tularensis by host macrophages involves CR3/TLR2 crosstalk. Although experimental data provide evidence that Lyn kinase and PI3K are essential components of the CR3 pathway that influences TLR2 activity, additional responsible upstream signaling components remain unknown. In this paper we construct a mathematical model of CR3 and TLR2 signaling in response to F. tularensis. After demonstrating that the model is consistent with experimental results we perform numerical simulations to evaluate the contributions that Akt and Ras-GAP make to ERK inhibition. The model confirms that phagocytosis-associated changes in the composition of the cell membrane can inhibit ERK activity and predicts that Akt and Ras-GAP synergize to inhibit ERK.Complement Receptor 3 (CR3) and Toll-like Receptor 2 (TLR2) are pattern recognition receptors expressed on the surface of human macrophages. Although these receptors are essential components for recognition by the innate immune system, pathogen coordinated crosstalk between them can suppress the production of protective cytokines and promote infection. Recognition of the virulent Schu S4 strain of the intracellular pathogen  F. tularensis. The model is used to test the hypothesis that phagocytic signaling downstream from CR3 is responsible for an early inhibition of ERK activity, which is seen subsequent to contact with the complement C3-opsonized Schu S4 strain of F. tularensis. In addition, the model predicts that Akt and Ras-GAP synergize to inhibit ERK. To the best of our knowledge this is the first mathematical model to investigate crosstalk between these pathways within the context of infection. By providing a comprehensive picture of the initial host-pathogen interaction, and pathogen-induced crosstalk between cell surface receptors in particular, this model is important in the context of microbial immunopathogenesis.In the current work we construct a highly contextual model of membrane-proximal crosstalk between the ERK and PI3K cascades that is initiated through contact with  Receptor-mediated engagement followed by phagocytosis by professional phagocytes is the first critical step in microbial clearance or, in the case of intracellular pathogens, entry to a safe niche. The molecular mechanisms underlying phagocytosis are complex, usually involving more than one receptor and rapidly culminating in the combinatorial generation of a variety of biochemical signals along with rearrangement of the actin cytoskeleton to engulf the microbe Complement Receptor 3 , the major Francisella tularensis is an extremely virulent intracellular pathogen of macrophages and potential bioweapon. Indeed, the bacteria may be aerosolized and inhalation of as few as ten bacteria can result in the fatal disease pneumonic tularemia F. tularensis is rapidly phagocytosed by alveolar macrophages while suppressing their cytokine production. One mechanism the bacterium uses to accomplish this feat is to selectively engage only a few choice receptors. Although multiple types of receptors can mediate phagocytosis of Francisella, appreciable phagocytosis of the most virulent strains requires CR3 engagement by complement C3-opsonized bacteria F. tularensis as an intracellular pathogen Francisella comes almost exclusively from its stimulation of TLR2 F. tularensis. The model, which serves as a formal hypothesis, is shown to be consistent with the experimental results of S. Dai et al (unpublished data). Its implications are explored via numerical simulations.In what follows we construct a model of immediate membrane proximal signaling in response to F. tularensis depends heavily on the presence of complement. Dai et al found that complement opsonization substantially decreases cytokine production in response to F. tularensis, and identified key players in this immuno suppressive pathway (unpublished data). Their results are summarized as follows: ERK activation in response to F. tularensis is suppressed by complement-mediated signaling through CR3. Furthermore, ERK inhibition is rapid, being evident just 5 minutes post infection. In addition to suppressing ERK activation, CR3 ligation induces the rapid activation of Lyn kinase, which functions to inhibit cytokine production in response to F. tularensis. Finally, TLR2 and CR3 signaling intersect at the PI3K/Akt pathway, and the two receptors cooperate to support a complement dependent enhancement of Akt activity in response to F. tularensis.The response of macrophages to F. tularensis infection. In this model TLR2-induced ERK activation occurs through a previously characterized MyD88 independent pathway in which Rac and Ras associate with the cytoplasmic domain of TLR2 and undergo rapid activation in response to bacterial stimuli The observations of Dai et al were supplemented with existing literature to construct a model of the very earliest signaling events that occur in response to Francisella tularensis. Because the network is complex, we resort to mathematical modeling in order to deduce function from structure, that is, in order to check that the model is in fact consistent with experimental observations. The model equations, based on We next present the results of numerical simulations on the mathematical model. In order to test the model's consistency we compare the results of simulations in the presence and absence of complement.Francisella will elicit a slight increase in 3 phosphoinositides and a substantial increase in active Ras, Rac and Raf. As Raf is a proxy for ERK, we see that in the absence of complement the model is in agreement with the experimental results of S. Dai et al (unpublished data). In particular, TLR2 signaling stimulates both the ERK and PI3K pathways.Francisella. A comparison of F. tularensis induced Raf stimulation is markedly inhibited in the presence of complement.Having confirmed that, as parameterized, the mathematical model is consistent with complement-mediated ERK inhibition we performed an uncertainty and sensitivity analysis in order to assess how uncertainty in the model's parameters impacts its consistency with experimental data. In particular, as some of the model's parameters are uncertain, we wished to know if complement-mediated ERK inhibition is robust to variations in the model's parameters, i.e. is the model consistent with experimental result over a wide range of parameter values. We ran 10,000 numerical simulations in which the model's parameters were varied according to a Latin hypercube sampling scheme. The sensitivity of the model's output (as measured by the concentration of active Raf at 5 minutes post infection) to uncertainty in the parameters was then quantified through a partial rank correlation coefficient F.tularensis was compared to that when the parameters were set to their baseline values. A ten percent reduction in In addition to showing that the model's consistency is robust to parameter variations, the uncertainty and sensitivity analysis can be used to identify molecules and parameters that are important for complement-mediated ERK inhibition. Although many of the parameters show a small but significant correlation with the concentration of Raf at 5 minutes post infection, the strongest negative correlations are associated with the parameters for GAP-mediated Ras deactivation, namely Francisella undergo PI3K-independent phagocytosis Crosstalk between the complement and TLR systems is an essential determinant of the early immune response to pathogens Francisella infection, we also defined crosstalk between the ERK and PI3K signaling cascades within this context. Although several computational analyses of differential ERK signaling and ERK/PI3K crosstalk have already been performed In defining crosstalk between CR3 and TLR2 within the context of Francisella is different from CR3-mediated inhibition of TLR2 signaling initiated by Porphyromonas gingivalis, wherein IL-12 production is inhibited by ERK P. gingivalis binds to CR3 via its natural fimbriae, while Francisella cannot efficiently bind CR3 unless opsonized with C3bi. Or, it could also be that P. gingivalis engages other receptors that augment or interfere with CR3/TLR2 crosstalk. However, complement receptor-mediated PI3K activation has been observed to inhibit TLR-induced IL-12 production in response to Hepatitis C virus According to our model, CR3-mediated inhibition of TLR2 signaling initiated by F. tularesis. Recruitment, the process through which diverse proteins translocate to a common location, is a central aspect of receptor signaling. Indeed, receptor-mediated recruitment concentrates activators, enzymes and substrates to specific locations on the inner leaflet of the cell membrane. In order to model recruitment we allow local concentrations of molecules at the immunological synapse to far exceed membrane and cellular concentrations. For example, receptors may be recruited to the immunological synapse through their ability to bind bacterial ligands. In this case, the maximal concentration of receptors at the synapse is bounded by In this section we briefly describe the mechanisms through which the molecules of our model are regulated. Each verbal description is followed by a mathematical one. When coupled together, these mathematical descriptions form a model of membrane proximal receptor signaling in response to TLRs are pattern recognition receptors (PRRs) that detect and respond to a broad range of pathogen products including bacterial lipoproteins In According to CR3 has an active conformation that binds to and mediates the phagocytosis of C3bi-opsonized particles and an inactive conformation that does not Cai et al Ross et al Src family kinases, of which Lyn is a member, are rapidly activated in response to integrin ligation Francisella, the precise rates of Lyn activation and deactivation within this context are not known. Several in vitro studies, however, enable us to estimate Although we observe rapid activation of Lyn in response to The activity of Akt is regulated by multiple kinases, phosphatases and lipids. In resting cells Akt is sequestered in the cytoplasm. Upon stimulation Akt translocates to the membrane where it achieves full activation through phosphorylation at Ser and Thr Rac and Ras are examples of GTPases which are proteins that cycle between an inactive GDP bound form and an active GTP bound form. GTPase activity is tightly regulated by guanosine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in some cases guanosine dissociation inhibitors (GDIs) FrancisellaRac is a GTPase of the Rho family that is activated by both CR3 and TLR2 regulates a variety of cellular processes including cytoskeleton rearrangements and cytokine production Our model of Rac activity assumes that the concentrations of active Vav and GAP depend on the concentration of Lyn and PI(345)P as follows:As PI(45)P has been shown to inhibit the GEF activity of Vav, we consider fully active Vav to be that which is both bound to PI(354)P and phosphorylated by Lyn. The fraction of Vav bound to PI(345)P is determined by We were unable to determine the rates of Vav phosphorylation and dephosphorylation. As a result the fraction of phosphorylated Vav is determined by the unknown parameter Ras is a GTPase that mediates ERK activation by recruiting Raf to the plasma membrane Combing these two processes, i.e. the exchange of GDP for GTP and the hydrolysis of GTP to GDP, we obtain the following equation for Ras.Phosphatidylinositol, or PtdIns, is a membrane lipid that mediates signal transduction between cell surface receptors and the cytosol. Its inositol head group contains several free hydroxyls which can be phosphorylated to generate a variety of distinct derivatives termed phosphoinositides In vivo the inositol head group of PtdIns can be phosphorylated at positions 3, 4, and 5 Experiments conducted in vivo suggest that phosphoinositide production proceeds according to the following diagram The concentration of PI(45)P, PI(345)P and PI(34)P in resting neutrophils In its inactive state Raf is sequestered by 14-3-3 binding proteins, the association of which is supported by phosphorylation at Ser 259 We should note that Raf is also subject to ERK-mediated negative feedback. Indeed ERK was shown to inhibit Raf kinase activity by phosphorylating Raf at several sites


Water-soluble dendritic iridium catalysts were synthesized by attaching a reactive metal complex to DAB-Am dendrimers via an adapted asymmetric bipyridine ligand. These dendritic catalysts were applied in the aqueous reductive amination of valine while contained in a dialysis bag. Comparable conversions were observed as for the noncompartmentalized counterparts, albeit with somewhat longer reaction times. These results clearly show that the encapsulated catalyst system is suitable to successfully drive a complex reaction mixture with various equilibrium reactions to completion. Cascade catalysis, a bioinspired strategy to conduct multiple consecutive catalytic steps in one pot, is attracting the attention of an increasing number of chemists –9. AdvanDendrimers can also be used as scaffolds for creating macromolecular catalysts , which g3, which has been previously successfully applied in aqueous reductive aminations . Fi. Fi8 was16 and 17 was examined in the aqueous reductive amination of valine (18, 20 and iminium ion 21 are compensated by the iridium-catalyzed reduction to form the benzylated amino acid 19. Iridium complexes 3, 16 and 17 were capable of reducing the intermediate imine in the presence of HCO2K, which acted as the hydride source. The reaction was monitored by taking aliquots of the reaction mixture and by HPLC analysis. An excess of benzaldehyde was used, because it was also partially reduced to benzyl alcohol in a side reaction. The optimal pH appeared to be 5, comparable with a similar catalyst published by Fukuzumi [The reactivity of macromolecular catalysts line 18, . This reFukuzumi –30.3, formation of the N-alkylated amino acid 19 proceeded considerably faster than with the dendrimer-supported reductive aminations (In the presence of the nondendritic catalyst inations . However16 and 17 in a dialysis bag, a different setup was used based on commercially available materials. This consisted of a cup which could be closed and contained a dialysis membrane with a molecular-weight cut-off value of 2,000. We assumed that all reaction components except the catalyst would be able to pass through the membrane, turning the dialysis tube into a compartment suitable for catalysis. To verify this hypothesis, catalyst 16 was injected in the dialysis device and the reductive amination of valine was performed under otherwise identical conditions (In order to study the same reaction with the dendritic catalysts nditions . HPLC annditions , demonst16 still showed catalytic activity (After the appropriate reaction time, the dialysis tube was removed from the reaction mixture, dialyzed in buffer to remove all the other reaction contents and added to a fresh reaction mixture. In the second run, the recovered G3 dendrimer activity , albeit 17 (16, however, the G4 catalyst 17 was equally active in a second run, clearly demonstrating the viability of containment of the catalyst in the dialysis bag. This result also suggests that the immobilized catalyst should be applicable in cascade reactions with reagents that are normally incompatible with the iridium complex. Since our process was successfully conducted in water, combinations with enzymes should also be possible.Similar experiments were conducted with the G4 dendrimer 17 . The figDendritic catalysts were successfully synthesized by connecting Ir-bipyridine complexes to third and fourth generation DAB-Am dendrimers. The dendritic catalysts showed good activity in the multistep reductive amination of a free amino acid in water, albeit that prolonged reaction times as compared to the free catalyst were required. Due to the size of the macromolecular catalysts, it was possible to employ them in a dialysis device for conducting reductive aminations of free amino acids in water. The G4 catalyst showed the same reaction rate in a second run, which validates the concept of maintaining the macromolecular catalyst in the compartment. The work described here could be more widely applicable for compartmentalization of catalytic systems in aqueous media, where the iridium system may be used either in combination with other catalysts, or with enzymes. These topics are currently under investigation in our group.File 1Experimental details and spectroscopic data.File 2Spectra of compounds.


Sarcophaga crassipalpis, and ask whether there is an ontogeny of aggression that coincides with reproductive maturity. After establishing that reproductive maturity occurs by day 3 of age (post-eclosion), we examined the behavior of socially isolated males from different age cohorts  upon introduction, in a test arena, with another male of the same age. The results show a pronounced development of aggression with age. The change from relative indifference to heightened aggression involves a profound increase in the frequency of high-intensity aggressive behaviors between days 1 and 3. Also noteworthy is an abrupt increase in the number of statistically significant transitions involving these full-contact agonistic behaviors on day 2. This elevated activity is trimmed back somewhat by day 3 and appears to maintain a stable plateau thereafter. No convincing evidence was found for escalation of aggression nor the establishment of a dominance relationship over the duration of the encounters. Despite the fact that aggressive interactions are brief, lasting only a few seconds, a major reorganization in the relative proportions of four major non-aggressive behaviors  accompanies the switch from low to high aggression. A series of control experiments, with single flies in the test arenas, indicates that these changes occur in the absence of the performance of aggressive behaviors. This parallel ontogeny of aggressive and non-aggressive behaviors has implications for understanding how the entire behavioral repertoire may be organized and reorganized to accommodate the needs of the organism.Aggression, costly in both time and energy, is often expressed by male animals in defense of valuable resources such as food or potential mates. Here we present a new insect model system for the study of aggression, the male flesh fly  Aggression is widespread throughout the animal kingdom In recent years, a number of invertebrate model systems have been developed in an attempt to understand the neurophysiological and genetic determinants of agonistic behaviors Drosophila melanogaster. In nature, territorial behavior and aggression are observed in various Hawaiian Drosophila species that exhibit lek behavior in which males defend territories established on leaves, fern fronds, or tree limbs D. melanogaster, male territoriality is seen under laboratory conditions fruitless gene plays a major role in determining sex-specific patterns of aggression Drosophila appears to possess a great deal of complexity, involving extensive pleiotropy and epistasis Among the more thoroughly investigated invertebrate model systems is the fruit fly Drosophila melanogaster, male stalk-eyed flies defend valuable resources such as mates or food Teleopsis dalmanni, males appear to use a sequential assessment strategy in which pairs of male flies perform ritualized, escalating agonistic behaviors that may lead to direct physical contact but do not result in injury Similar to Sarcophaga crassipalpis). Male flesh flies appear to establish territories in nature that are different from those observed in D. melanogaster or stalk-eyed flies. Rather than defending a well-defined resource such as food or a female fly at a particular location, males defend a space in the environment. This space presumably serves as a sentinel position, from which to detect females that may travel through the surroundings. For example, males position themselves at equidistant intervals along fence rails and roof tops and will fly from these perches to pursue passing females . Analogous to what is found in nature, male flesh flies, placed in rectangular enclosures in the laboratory, show significantly lower spatial tolerance of same-sex conspecifics than do females. When placed in groups of four in circular arenas, males and females show a significant difference in the degree of clustering (measured by nearest neighbor statistics): males show a tendency towards a uniform distribution whereas females tend slightly towards a clustered distribution S. crassipalpis as a comparative model system for the study of aggression.An interesting contrast to the behavior of fruit flies and stalk-eyed flies is that of the flesh fly  of behavior? Does the transition to the aggressive condition require a radical reorganization of behavioral patterns or just slight modifications? How much of an impact will the change in condition have on the performance of behaviors that are not involved in aggression? The answers to these questions are relevant to understanding the rules that govern the neural control of behavior and may provide insights into the relationship between consistency and plasticity Detailed examination of the ontogeny of aggressive behavior in flesh flies enables some insights into the organization of behavior that have not yet been pursued in other invertebrate model systems. For example, what is the process by which the organism accomplishes the ontogenetic transition from being relatively non-aggressive to aggressive? Is the transition gradual or sudden? Do aggressive behaviors exist from an early age onward or do they appear S. crassipalpis) were maintained under nondiapause conditions  in a long-term (19 years) colony at East Tennessee State University, derived from another long-term colony established about 1975 at The Ohio State University in the laboratory of Dr. David Denlinger. All of the paired male experiments in this study were conducted within an aluminum shed built in the laboratory and maintained at 24±2°C as previously described Flesh flies  into clear glass jars (4 liter) provisioned with sugar cubes and water. The number of mating pairs in each jar was recorded once each hour throughout the photophase of the 15∶9 hour light:dark cycle through day 5. The experiment was performed at 24±2°C under low density  and high density  conditions. The proportion of flies mating under each density condition was determined by dividing the number of mating pairs by the total number of potential mating pairs, corrected for mortalities, at each observation time.The age at which 2). Provided ad libitum were sucrose (in the form of sugar cubes) and water (available from miniature microfuge tubes plugged with cotton). The isolation chambers were housed under a 12∶12 hour light:dark cycle at 24°C±2°C and visually separated from one another by black cardboard partitions. The isolation ensured that male flies were socially naïve at the time at which they subsequently were tested.Male flies were collected at emergence by chilling briefly until immobilized and placed in isolation chambers . To facilitate (1) the construction of raster plots showing the simultaneous behavior of both members of the opponent pairs, (2) determining of the durations of the bouts of agonistic behaviors, and (3) discerning the levels of agonistic behaviors through successive 10-min intervals within the encounters, we re-coded the video recordings using the Observer XT version 11.5  using a single observer (J.D.S.).Encounters between same-age male flies were performed in a relatively simple observation arena to ascertain if the performance of aggressive behaviors is age-related. Randomly chosen, socially naïve, same-age flies were removed from their isolation chambers and individually marked on the dorsal thorax with a dot of colored enamel. Pairs of 1-, 2-, 3-, 4-, and 6-day old flies were released into a circular arena (15-cm diameter petri dish), one fly on each side of a black partition separating each half of the arena . The areTo provide a control for the paired male experiments, individual males  were monitored in the arena. For this portion of the study, males were handled exactly as described above for the paired male experiments and their behaviors were video recorded in the same manner. However, the Observer XT version 11.5 software was used to compile and analyze the data. Also, the first and last 5 min of the 60-min recordings were not used, thus leaving 50 min of activity in each session for analyses. There were 10 males observed for each age cohort. All of the behavioral scoring of the single male data was done by a single researcher (J.D.S.).standing, walking, grooming, and upside-down) plus stilt, bobbing, and jump. All grooming movements were performed from a standing posture but were considered a separate behavior from standing. Interactive/non-aggressive behaviors were those in which the fly apparently was aware of the other fly in the arena but did not physically engage its adversary; the fly moved toward  or away from  its opponent. Avoid and retreat represented two different velocities of movement: avoid was relatively slow, involving locomotion via leg movements only whereas retreat was much faster, using propulsion from wing movements  and high-intensity aggressive behaviors  involved physical contact between the two flies. The difference between low-intensity and high-intensity aggression was characterized by limited body contact between individuals in the former and full-body contact in the latter. Because of the limited occurrence of each of the six different low-intensity aggressive behaviors, for purposes of analyses, they were combined and treated as a single behavior – low-intensity aggression. Also, because immobilized in one fly was the consequence of its opponent performing a hold behavior, the two behaviors always exhibited the same frequency in our analyses.Based on extensive video analyses, an ethogram  was creaovements . Low-int−1 individual−1 was calculated for each of eleven behaviors . In all cases involving the same types of comparisons, the same statistical analyses were performed for both the paired male and single male experiments.To determine if behavioral aggression varied with age in the paired male experiments, the mean number of occurrences houriors see  for eachstanding, walking, grooming, and upside-down, all from the non-interactive category) was determined for the entire age group and compared to the total observation time of the group within the arena. The resulting proportions were arcsine transformed and then evaluated across age cohorts using Tukey-type multiple comparisons To determine if the four most common behaviors change with age, the amount of observation time (in minutes) occupied by the four behaviors  in the following ascending order: <1%, 1–5%, 5–10%, 10–15%, and >15%. Significant behavioral transitions were represented by arrows connecting the symbols; the degree of departure from randomness was depicted by using three different sizes of arrows  according to the following ranges of Freeman-Tukey values, in ascending order: 1–5 , 5–10 , and >10 .To determine the age of onset of mating behavior, equal numbers of newly emerged (day 0) male and female flesh flies were placed under high or low density conditions and then monitored for the presence of mating pairs every hour throughout the daylight hours through day 5 of age. The proportion mating  for both density conditions was essentially zero on days 0 and 1, appeared at relatively low levels on day 2, and was maintained at relatively high levels from day 3 through day 5 . We concwalking, standing, grooming, and upside-down occupied the vast majority of time during the one-hour encounters in the arena. Individual bouts of these four behaviors were several seconds to several tens of seconds in duration. All other behaviors observed in the arenas were very short in duration, from fractions of a second to several seconds. Transitions from one behavior to another were instantaneous. In the 1-min example from the day 1 age cohort , then resumed walking. It transitioned again from walking to approach and then from approach to low-intensity aggression. Neither approach nor low-intensity aggression from fly 1 yielded a change in behavior from fly 2. The flies in the day 3 example , holding (performed in this case by fly 2), a longer bout of wrestling (approximately 3 seconds), followed by upside-down in fly 1 and walking in fly 2. Further representing the short-duration characteristics of interactive behaviors in male flesh flies were the interactions shown in the example from the day 6 age cohort (approached fly 2 which responded by retreating (lasting less than 1 second). Later in the record (beginning at about 27 seconds), fly 1 performed two lunges (each less than 0.5 second in duration), one immediately before and the other immediately after a hold. Just before the second lunge, fly 2 broke away from being immobilized and initiated its retreat (lasting less than one second). The second lunge from fly 1 was successful in targeting fly 2 during its retreat and the two opponents immediately engaged in two short bouts of wrestling, interrupted briefly by both flies being upside-down. Please see lunge, lunge followed by hold, and hold followed by wrestle.A raster plot  showing e cohort , there w example  displayee cohort . At abouAfter determining the age of sexual maturity as well as the fact that male flesh flies do indeed fight each other, a primary objective was to determine if the expression of agonistic behaviors develops in concert with sexual maturation.wrestling  all showed a significant, progressive increase in occurrence as the flies aged from day 1 to day 6  under the null hypothesis of equal probability of performance of the behavior by both members of the pair. A similar absence of domination by one fly over another was observed for high-intensity aggressive behaviors, represented by the combination of hold and lunge observed in pairs exhibiting at least two occurrences of these behaviors. Two other high-intensity aggressive behaviors were not included in this measure: immobilized, because it is the result of being held by the opponent fly, and wrestling, because both flies in the pair participate in the behavior. The distribution (lunge and hold) performed by individual 1 for all of the eligible pairs possessed a mean of 0.49±0.05 (s.e.m.). Although significantly different from a bimodal distribution , 60% of the eligible pairs showed ratios in the range from 0.25 through 0.75.No convincing evidence was found for a stable dominance relationship between opponents. Using the interactive, non-aggressive behavior unimodal  with a mribution  of the pS. crassipalpis were any indications of escalation of agonistic behavior during the one-hour encounters. Excluding the first and last 5 min of the one-hour bouts, the number of occurrences of high intensity aggression (hold plus lunge) per pair was calculated for five consecutive 10-min intervals for each of the age cohorts .Also absent in  cohorts . No signwalking, standing, grooming, and upside-down), taken together, occupied a consistently large percentage of the time observed for all age cohorts: 97.6%, 98.1%, 98.1%, 98.0%, and 96.4% for days 1, 2, 3, 4, and 6, respectively. However, the proportions of time occupied by each of the individual behaviors exhibited significant changes with age. Walking  and between behavior 2 and age  thus revealing age-related changes in behavioral frequencies. Furthermore, the third order interaction among behavior 1, behavior 2, and age also was significant , indicating that the transition probabilities were not the same among the age cohorts.To examine the temporal organization of behavior in greater detail, a behavioral transition matrix was constructed for each age-matched cohort . We obseP<0.05). There were 38, 58, 55, 54, and 53 significant behavioral transitions for age cohorts 1, 2, 3, 4, and 6 days, respectively.Within each age-cohort transition matrix, Freeman-Tukey deviates Kinematic diagrams  were conlunging (from approach and low intensity aggression) in the day 1 cohort but there are five significant transitions to lunging in the day 3 cohort . Similarly, there is only one significant transition to the high intensity aggressive behavior wrestling in the day 1 cohort (from upside-down) compared to five in the day 3 cohort . Many of the transitions involving high intensity aggression in the day 3 cohort are from one high intensity aggressive behavior to another. For example, the transitions holding–wrestling and lunging–wrestling are absent in day 1 flies but present in the day 3 cohort. Interestingly, there are a limited number of other behaviors that lead to one or more of the high intensity aggressive behaviors. These are low intensity aggression, approach, and upside-down in the day 1 cohort. The same three pathways to high intensity aggression are present in the day 3 cohort, with the additions of turn toward, avoid, and retreat.Many salient aspects of behavioral organization and reorganization can be seen by comparing the kinematic diagrams for the day 1 and day 3 age cohorts . Most obbobbing and jump. Bobbing contributes no significant behavioral transitions in the day 1 cohort but is paired with grooming, standing, and stilt in the day 3 flies. Jump is paired only with walking in day 1 flies but this transition is lost in the day 3 cohort and is replaced by five different associations.Not all changes in behavioral transitions between the day 1 and day 3 cohorts involve aggressive behaviors . Two notwalking–approach, approach–avoid, and grooming–standing transitions, all of which involve behaviors occurring at high frequency and transitions exhibiting a high degree of departure from randomness  standing–grooming, present on days 1 and 2 but absent thereafter, (2) grooming–stilt, absent on day 1 but present thereafter, (3) stilt–standing, absent on days 1 and 6 but present on days 2, 3, and 4, (4) walking–upside-down, absent on days 1, 2, and 3 but present on days 4 and 6, and (5) upside-down–walking which appeared only on day 6. There were no significant transitions involving the behavior jump in any of the age cohorts.The rather limited behavioral repertoire performed by single flies nevertheless showed some significant transitions common to all age cohorts and other transitions that changed with age . The walS. crassipalpis as a model system for exploring fundamental questions concerning how the nervous system may organize a variety of behaviors, including aggressive acts, and how these patterns may change with age. After establishing a high-resolution ethogram, we investigated the ontogeny of aggressive behavior, an aspect of aggression that has received limited attention. For this study, individual flies were kept isolated from one another beginning shortly after eclosion until the age at which they were tested in a minimal arena, thereby excluding most potential external influences on behavior  except that of light:dark cycles and chronological age. In contrast with other model systems, our observations were not limited to agonistic actions but encompassed non-aggressive behaviors as well, providing insights into how aggressive behaviors are integrated with other behavioral programs.Many gaps exist in our understanding about the neural control of behavior in animals S. crassipalpis begins as early as day 2 of age but most flies exhibit mating behavior by day 3  showed a significant, progressive increase in frequency with age . In another study, adult D. melanogaster males given a brief exposure to females failed to show courtship gene expression changes relative to naïve adult males The mechanisms underlying the age-related increases in aggressive behaviors in the flesh fly are unknown. There may, however, be some insights from gene expression patterns in S. crassipalpis apparently is based upon major increases in the frequency of behaviors involving full-body contact (high-intensity aggressive behaviors) whereas those behaviors characterized as low-intensity aggression , however, low-intensity aggression declined to less than half of its previous level and occurred less frequently than either of the individual high-intensity behaviors lunge and wrestle. These results suggest that low-intensity aggression does not play a major role in agonistic interactions in S. crassipalpis. Further underscoring this point is the finding that several statistically significant transitions involving other behaviors serve as pathways to or from the high-intensity aggressive behaviors  S. crassipalpis behavior limits our ability to interpret our findings with respect to natural conditions. However, the lack of escalation as well as the absence of dominant individuals during the encounters in the arena are contrary to findings in D. melanogasterS. crassipalpis. This scenario would be consistent with the finding that dominance relationships and fighting escalation did not occur during the encounters but difficult to reconcile with the apparent ferocity of the fights  and the significant increases in levels of interactive behaviors with age  while two others (turn toward and approach) showed no significant variation with age  and decreases  in the amount of time occupied by these non-interactive behaviors occur in the absence of an opponent. Therefore, the reapportioning of the non-aggressive behaviors relative to one another with age does not require the performance of aggressive behaviors. These findings suggest interesting hypotheses concerning the organization of behavior. For example, aggressive and non-aggressive behaviors may be connected functionally to one another by common control circuits such that the levels of standing, walking, grooming, and upside-down are related to thresholds for the release of aggressive behaviors. Alternatively, the aggressive and non-aggressive behaviors may have no functional connections or common modulatory controls but their age-related changes simply may be programmed to occur simultaneously. This aspect of behavioral organization has received little study in any model system and deserves further examination. One possible consequence of these findings is that experimental manipulation of aggression levels  may have collateral effects on very disparate behavioral programs.One of the basic questions concerning the ontogeny of aggression in the present study was how changes in aggressive behaviors might affect the performance of non-aggressive behaviors. In the case of interactive, non-aggressive behaviors, two changed significantly with age  perhaps reflects an age-related up-regulation of an internal program to ensure that sensory organs are clear of obstructions in preparation for agonistic interactions and mating. Exhibiting an even greater change was the amount of time spent in the behavior upside-down in the paired male experiments, increasing from 1% on day 1 to about 4% on day 3 and nearly 20% on day 6. This extraordinary escalation is not understood but possibly may be the result of a general, heightened state of arousal associated with the increase in aggressive tendencies or an increase in failed attempts to escape the confines of the arena. The finding that upside-down also increases with age in isolated males , there are pronounced increases in both the frequency of occurrence of high-intensity aggressive acts  or after the age of sexual maturity , low-intensity aggression exhibited statistically significant transitions only to two high-intensity aggressive behaviors at relatively low frequencies  in which the flies progress through low-intensity aggressive behaviors in order to assess the strength of the opponent and avoid high-intensity agonistic interactions. Instead, after sexual maturity, S. crassipalpis males readily engage in high-intensity aggressive behaviors. In fact, flesh flies transition from one high-intensity behavior to another at relatively high frequencies  for each day of age thereafter. In the single male experiments, bobbing was nonexistent, suggesting that this behavior may play some role in the interactions between flies. Stilt, in contrast, showed a significant decline in frequency with age in the paired male experiments (stilt–standing). The age-related pattern for stilt was different in the single male experiments, occurring at low levels on day 1 and exhibiting a significant peak in frequency on day 3  were noted in studies of male-male agonistic interactions in D. melanogasterT. dalmanniD. melanogasterwing threat, classified as a low-intensity aggressive act. Also absent from our observations were two low-intensity aggressive behaviors performed by T. dalmanniflex, involving flexion and flicking of the forelegs in the direction of the opponent, and line up eye stalks, in which the two opponents confront each other face-to-face with their eye stalks aligned in parallel.The utility of three behaviors performed by male flesh flies in our study,  4 and 6 . There weriments , althougon day 3 . Occurreeriments . In accocal leap  but may S. crassipalpis provides an intriguing comparative model system for the study of aggression because its natural lifestyle, including mating behavior, is very different from that observed in fruit flies and stalk-eyed flies. Our findings of fundamental differences in the organization of aggressive behaviors between the flesh fly and these other model systems are consistent with these lifestyle differences. Although our investigation of aggression in S. crassipalpis males shares some of the same analytical approaches used in the other model systems , it also adds two perspectives not previously explored in detail: the influences of age on the expression of aggressive behavior and the potential interactions of aggressive behaviors with other behaviors not performed during agonistic encounters. Extrapolating from our results, we maintain that such an approach will lead to a deeper understanding of the mechanisms controlling aggression.The flesh fly File S1Behavioral transition matrices for the day 1, 2, 4, and 6 age cohorts in the paired male experiments.(DOC)Click here for additional data file.Video S1Exemplar of the behavior lunge. Fly at the top lunges at its opponent.(AVI)Click here for additional data file.Video S2Exemplars of the behaviors lunge and hold. Fly to the right lunges and then holds its opponent; behaviors are shown at actual speed and ¼ speed.(MPG)Click here for additional data file.Video S3Exemplars of the behaviors hold and wrestle. Fly to the left approaches and then holds its opponent. This is followed quickly by wrestling, in which both flies are grasping and striking with their forelegs.(AVI)Click here for additional data file.Video S4Exemplars of the behaviors approach and avoid. Fly to the right approaches its opponent (standing). Then both flies avoid each other.(AVI)Click here for additional data file.Video S5Exemplars of the behaviors approach and retreat. Fly to the left approaches its opponent. Opponent then retreats to opposite side of the arena.(AVI)Click here for additional data file.


Homo neanderthalensis could use speech or complex language remains controversial. Similarity in overall shape does not necessarily demonstrate that the Kebara 2 hyoid was used in the same way as that of Homo sapiens. The mechanical performance of whole bones is partly controlled by internal trabecular geometries, regulated by bone-remodelling in response to the forces applied. Here we show that the Neanderthal and modern human hyoids also present very similar internal architectures and micro-biomechanical behaviours. Our study incorporates detailed analysis of histology, meticulous reconstruction of musculature, and computational biomechanical analysis with models incorporating internal micro-geometry. Because internal architecture reflects the loadings to which a bone is routinely subjected, our findings are consistent with a capacity for speech in the Neanderthals.The description of a Neanderthal hyoid from Kebara Cave (Israel) in 1989 fuelled scientific debate on the evolution of speech and complex language. Gross anatomy of the Kebara 2 hyoid differs little from that of modern humans. However, whether  Homo heidelbergensis from Sierra de Atapuerca (Spain) dated at ∼530 ka Australopithecus afarensis from Dikika  The Kebara 2 Neanderthal dates from approximately 60 ka and is part of a near-complete adult male skeleton unearthed in 1983 Pan troglodytes, which includes a cup-shaped extension or bulla , is very different to that of modern humans  number of points in the continuum. This process of modeling an object by dividing it into a system of smaller elements of known geometry (finite elements), interconnected at points common to two or more elements (nodes), is called discretisation. FEA is now increasingly used in biology It is important to note that the modelling approach used in the present study is entirely comparative, as in previous broadly similar studies Only two studies to date have analysed micro-biomechanical models that capture the trabecular network geometry of whole bones Examination of internal microscopic anatomy reveals that the medial sagittal microCT section from the Kebara 2 hyoid body  shows a Visual plots of von Mises (VM) stress distributions are given in H. neanderthalensis specimen (6.3 MPa) falls just outside the range determined for the H. sapiens sample (6.4 MPa to 10.8 MPa), it is in fact both qualitatively and quantitatively much closer to SAT37 than this modern human hyoid is to the remaining two  are functionally ‘shorter’ and the posterior group functionally ‘longer’ in speech than in feeding Modern-human-like gross anatomy in the hyoid body of a fossil specimen is not, in itself, clear demonstration that the individual was capable of speech sensu Duchin 1990) Pan troglodytes and/or Pan paniscus, are needed before any firmer conclusions could be drawn.We conclude that the presence of modern-human-like histological features and micro-biomechanical behavior in the Kebara 2 hyoid indicates that this bone not only resembled that of a modern human, but that it was used in very similar ways. This is because the internal microarchitecture is a response to the vectors and magnitudes of the forces to which it is routinely subjected. These findings are consistent with the suggestion that the Kebara 2 Neanderthal practiced speech (H. heidelbergensisH. heidelbergensis (cranium 5 from Sima de los Huesos) and H. neanderthalensis (La Ferrassie 1) and comparisons with modern humans, it has been inferred that not only H. neanderthalensis, but perhaps this common ancestor of both Neanderthals and modern humans may have been capable of speech H. heidelbergensis could help to resolve this question.Previous studies have shown that anatomical features of the outer and middle ear associated with the perception of speech were also present in Given that our results add support for the proposition that the Kebara 2 Neanderthal engaged in speech, the question may then become was he capable of the critical thought and syntactical ability necessary for complex language? Conclusive resolution of this question is not possible with the data and analytical tools currently available. However, speculation on this issue might be considered in light of the mounting body of evidence that continues to expand the known repertoire of sophisticated subsistence strategies and symbolism practiced by Neanderthals The Kebara 2 Neanderthal hyoid is stored in the Department of Anatomy and Anthropology of the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. The modern human samples  are stored in the University Museum of the University “G. d’Annunizio”, Chieti, Italy. These are all thought to be male, as is Kebara 2, with estimated ages of 20–40, 40–60 and 35–40 years respectively.Microfocus X-ray microCT was performed at the TomoLab station, Elettra Synchrotron Light Laboratory, Trieste Italy. Volumes of the whole hyoid samples were reconstructed from tomographic projections acquired through sample rotations over 360 degrees . Volume analysis was carried out both on the reconstructed 2D slices and on the rendered volumes. Volume renderings were obtained using VGStudio MAX 2.0©.‘Porous’ three dimensional Finite Element Models capturing the cortical bone and trabecular networks were created in MIMICS 13.4 for the Kebara hyoid and those of the three modern humans  and 5. PFEMs for the Kebara 2 hyoid and those of SAT37, OPIT37, and SAT41 comprised 1,482,720, 1,512,145, 1,526,070 and 1,496,338 4-noded tetrahedral ‘brick’ elements respectively. Specific material properties for the human hyoid are unknown. In this study, elements were designated as isotropic and assigned a Young’s modulus of 13 GPa and Poisson’s ratio of 0.3 following previously published methods Forces and vectors were calculated for individual muscle fibers based on detailed 3D reconstruction . The steAll Finite Element Analyses were performed in Strand7 (2.4) using a Dell Precision T1500 . Finite Element Models were scaled


Natural extracts play an important role in traditional medicines for the treatment of diabetes mellitus and are also an essential resource for new drug discovery. Dipeptidyl peptidase IV (DPP-IV) inhibitors are potential candidates for the treatment of type 2 diabetes mellitus, and the effectiveness of certain antidiabetic extracts of natural origin could be, at least partially, explained by the inhibition of DPP-IV.genus as plants with known antidiabetic properties. Moreover, none of the 18 molecules that we predicted as DPP-IV inhibitors exhibits chemical similarity with a group of 2,342 known DPP-IV inhibitors.Using an initial set of 29,779 natural products that are annotated with their natural source and an experimentally validated virtual screening procedure previously developed in our lab  genus). Moreover, none of the 18 molecules exhibits chemical similarity with a large group of known DPP-IV inhibitors.Our study identified 18 potential DPP-IV inhibitors in 18 different plant extracts (12 of these plants have known antidiabetic properties, whereas, for the remaining 6, antidiabetic activity has been reported for other plant species from the same  Notably, when these 446 molecules were merged with 2,342 known DPP-IV inhibitors, and the resulting set was classified into 50 clusters according to chemical similarity, there were 12 clusters that contained only natural products for which no DPP-IV inhibitory activity has been previously reported. Nine molecules from 7 of these 12 clusters were then selected for in vitro activity testing, and 7 out of the 9 molecules were shown to inhibit DPP-IV  The DPP-IV binding site is highly druggable in the sense that tight, specific binding to the enzyme can be achieved with small molecules with drug-like physicochemical properties The goal of the present work was to identify natural extracts with known antidiabetic activity that contain at least one molecule that we predict to be a DPP-IV inhibitor through a slightly modified version of the VS workflow described above We used a slightly modified version of a VS workflow that was previously developed and experimentally validated i.e., one positive/donor and one hydrophobic/aromatic ring) and 5 optional sties . Both mandatory sites interact with crucial molecular anchors for DPP-IV inhibition  aims to smooth differences in chemical structures and translate them into criteria important for their intermolecular interactions with the ligand-binding site. This filter has been reported to be a valuable VS tool for the discovery of novel scaffolds ET_pb ≥0.468 and (b) ST ≥0.237. Interestingly, the fact that DPP-IV inhibitors (a) have a significant positive electrostatic potential in the region that interacts with the Glu205/Glu206 dyad  dyad see .http://www.pubmed.org) revealed that the extracts of 12 out of these 95 natural sources have been reported to exhibit antidiabetic activity (see http://www.cas.org/products/sfacad) revealed that 6 out of these 12 natural compounds correspond  to molecules with known antidiabetic properties  and was identified as a DPP-IV inhibitor by our VS procedure. Extracts from Erythrina variegata, Erythrina senegalensis, Erythrina addisoniae, Erythrina abyssinica, and Erythrina mildbraedii are reported to enhance antidiabetic activity Erythrina mildbraedii and prenylflavonoids isolated from the Erythrina senegalensis roots have been described as inhibitors of two other proteins frequently targeted in T2DM treatment . Therefore, although it is possible that most of these activities are exerted by the phenolic compounds present in the Tecoma stans aqueous extract, bioguided studies are necessary to confirm this hypothesis Tecostamine, which is found in Scoparia dulcis, has been reported to improve hypoglycemia Scoparia dulcis has been described as a folk-medicinal plant and has been traditionally used as a remedy for diabetes mellitus in India and for hypertension in Taiwan Scoparia dulcis, an antidiabetic compound named amellin was also isolated and characterized by Nath Epinephrine , which is found in Ephedra species . However, Ephedra distachya and Ephedra alata are the only species reported to have antidiabetic properties Ephedra species may be new sources of antidiabetic extracts.From the same family as epinephrine, two additional compounds were predicted by our VS to be potential DPP-IV inhibitors. These compounds are (+)-pseudoephedrine (CAS number 90-82-4) and (−)-ephedrine (CAS number 299-42-3), and both have also been reported to have hypoglycemic activity Rauwolfia species. Rauwolfia vomitoria has been investigated for the content of alkaloids, especially those alkaloids with hypotensive and anti-inflammatory properties Rauwolfia serpentina is also used as antidiabetic extract Rauwolfia canescensRauwolfia degeneriRauwolfia densifloraRauwolfia heterophyllaRauwolfia indecora, Rauwolfia obscuraRauwolfia tetraphyllaThe molecules ajmaline and isosandwichine, which are enantiomers with the same CAS number, 509-37-5, are found in several Vinca major and several Rauwolfia species  is isolated from Vitis vinifera. An antihyperglycemic effect in streptozotocin-induced diabetic rats and insulinomimetic activity in insulin-sensitive cell lines have been described for grape seed procyanidin extracts (GSPE) One interesting hit predicted to be a DPP-IV inhibitor is an epicatechin derivate that is found in Galega orientalisHaloxylon salicornicumPennisetum typhoideumThe remaining 3 molecules predicted to be DPP-IV inhibitors through our VS workflow and that are found in extracts with described antidiabetic properties are hydroxysmirnovine from genus may share a high number of components, we also determined if any of our VS hits that were isolated from plants with no described antidiabetic activity belong to the same genus as species with known antidiabetic properties. We identified 6 molecules isolated from 6 different plants, Aconitum japonicum, Ervatamia officinalis, Solanum nudum, Solanum sodomaeum, Stephania cepharantha and Tabernaemontana eglandulosa  were not structurally related to any known molecule that inhibits DPP-IV and (b) have never been reported to have antidiabetic activity. In the present work, we applied a slightly modified version of this VS workflow to an in-house database of 29,779 NPs annotated with their corresponding natural source(s). From this initial set of NPs, our VS procedure identified as potential DPP-IV inhibitors 84 hit molecules that have been isolated from 95 different natural sources. Interestingly, after an exhaustive bibliographic search, our results demonstrate that we are able to predict (a) 12 DPP-IV inhibitors that are present in 12 plant extracts with known antidiabetic activity and (b) 6 DPP-IV inhibitors that are present in 6 different plants species with no described antidiabetic activity but that share the same genus as plants with known antidiabetic properties . Moreover, none of these 18 hits exhibits chemical similarity with 2,342 known DPP-IV inhibitors, and, therefore, it is expected that a significant number of these hits could be lead-hopping candidates for the development of new DPP-IV inhibitors. At this point, it is also interesting to note that the analysis of the chemical structures of these 18 NP hits revealed that the majority of them are alkaloids containing basic nitrogen atoms . Moreover, our results provide a new hypothesis about the mechanisms by which, at least partially, these 12 extracts exert their antidiabetic effects .In a previous study Lastly, we predicted that there are 77 other sources with no described antidiabetic activity that contain at least one VS hit. Consequently, our work opens the door to the discovery of new antidiabetic extracts of natural origin that could be of use, for example, in the design of functional foods aimed at preventing/treating T2DM. Therefore, the characterization of such extracts merits further attention, and such work is currently underway.e.g., plants, and fungi) with appropriate ADME/Tox properties and no chiral ambiguities. An important characteristic of this database is that each molecule is annotated with (a) the natural sources from which it has been obtained and (b) the bibliographic references that describe how to extract the relevant molecule from each natural source. The 3D structures of the molecules in this NP database were processed with LigPrep v2.3  using the following parameters: (a) the force field used was OPLS 2005; (b) all possible ionization states at pH 7.0±2.0 were generated with Ionizer; (c) the desalt option was activated; (d) tautomers were generated for all ionization states at pH 7.0±2.0; (e) chiralities were determined from the 3D structure; and (f) one low-energy ring conformation per ligand was generated. Conformations and sites for the resulting ligand structures were determined during the generation of the corresponding Phase Generate Phase Database graphic front-end. Default parameter values were used during this conformer generation process, with the exception of the maximum number of conformers per structure, which was increased from 100  to 200. The conformer sites were generated with definitions made by adding to the default built-in Phase definitions the ability to consider aromatic rings as hydrophobic groups.The database of NPs that was screened by the VS workflow contains 29,779 NPs from different origins  were slightly different, as outlined below.The VS workflow used in this work is the same as that described previously i.e., IC50≤10 nM) that bind reversibly to the enzyme; (2) using their corresponding DPP-IV coordinates to guide the superposition of the remaining PDB files ; (3) using the resulting coordinates to derive the corresponding energetic structure-based pharmacophores; and (4) building the common structure-based pharmacophore for reversible DPP-IV inhibition by prioritizing energetically favorable features over energetically weaker ones. The resulting pharmacophore consists of two compulsory sites (one positive/donor and one hydrophobic/aromatic ring) and five optional sites  and was completed with receptor-based excluded volumes that schematically represent the location of the DPP-IV residues that form the binding pocket in the PDB file 3C45.The VS protocol used a structure-based pharmacophore that was built by (1) selecting from the PDB those reliable complexes of human DPP-IV and potent inhibitors of non-peptide nature (http://www.schrodinger.com) and allows the reorientation of the conformers to determine if they match the pharmacophore. Only those ligands with at least one conformer that matches the two compulsory sites of the common pharmacophore and at least one of the optional sites  survive this VS step. These ligands were docked onto the binding pocket of the PDB file 3C45 with eHiTS v2009  survived this filter. Finally, in the last step of the VS protocol, the poses that were hits in this second pharmacophore screen were submitted to a shape and electrostatic potential comparison with the DPP-IV inhibitor present in the 3C45 PDB file to rescore the hits. This comparison was completed with EON v2.0.1  and used the Electrostatic Tanimoto combo (ET_combo) score as the similarity criterion. The ET_combo score is the sum of two calculations: (a) the Shape Tanimoto (ST) score, which is a quantitative measure of three-dimensional overlap , and (b) the Poisson-Boltzmann Electrostatic Tanimoto (ET_pb) score, which compares the electrostatic potential of two small molecules and ranges from 1  to a negative value that results from the overlap of positive and negative charges. In this work, we determined the EON threshold scores by comparing the inhibitor in 3C45 with the inhibitors found in a group of 24 PDB complexes of DPP-IV and reversible drugs that inhibit this protein . This comparison of the experimental poses of DPP-IV inhibitors yielded threshold values for DPP-IV inhibition that were slightly lower than the ones used in the original VS workflow ET_pb ≥0.468 and ST ≥0.237 instead of ET_pb ≥0.623 and ST ≥0.244), probably because the original threshold values were obtained exclusively using potent DPP-IV inhibitors ; in this study, this condition was relaxed slightly. After rescoring with EON, only those NPs with at least one conformation with ET_pb ≥0.468 and ST ≥0.237 relative to 3C45’s inhibitor were considered to be DPP-IV inhibitor candidates.The first step of the VS workflow uses the common structure-based pharmacophore to screen the conformer database with Phase v3.1 . MOLPRINT2D fingerprints The molecules that survived the electrostatics/shape similarity filter were merged with 2,342 known DPP-IV inhibitors obtained from the BindingDB database Table S1Natural extracts with reported antidiabetic activity that contain molecules that were predicted to be DPP-IV inhibitors by our VS protocol. The first column shows the 2D structure of each molecule and, when available, the corresponding common name and/or CAS number. The second column shows the number of the cluster in which the corresponding molecule was classified when its structure was compared with those of a group of 2,342 known DPP-IV inhibitors. The third column shows the scientific name of one of the sources in which the antidiabetic activity has been reported . Bibliographic references for each molecule are divided into three columns in which (a) the first column presents papers that describe the purification of the molecule from the corresponding extract; (b) the second column lists papers that describe the antidiabetic activity of the corresponding extract; and (c) the third column lists papers, when available, that describe the antidiabetic activity of the corresponding molecule or one that is very similar to it.(DOC)Click here for additional data file.Table S2Natural extracts with no described antidiabetic activity (but from the same genus as plants with extracts with described antidiabetic activity) that contain molecules that are predicted to be DPP-IV inhibitors by our VS protocol. The first column shows the 2D structure of each molecule and, when available, the corresponding common name or CAS number. The second column shows the number of the cluster in which the corresponding molecule was classified when its structure was compared with those of a group of 2,342 known DPP-IV inhibitors. The third column lists the source from which the VS hits have been purified . The fourth column lists the papers that describe the purification of the each molecule from the corresponding extract. The fifth column shows which are the extracts from the same genus where the antidiabetic activity has been described. Finally, the last column lists papers that describe the antidiabetic activity of the corresponding extract.(DOC)Click here for additional data file.


The olfactory neuroepithelium lines the upper nasal cavity and is in direct contact with the external environment and the olfactory bulbs. The ability to self-renew throughout life and the reproducible recovery after injury, make it a model tissue to study mechanisms underlying neurogenesis. In this study, X-rays were used to disrupt proliferating olfactory stem cell populations and to assess their role in the cellular and morphological changes involved in olfactory neurogenic processes.in vivo.We have analysed the histological and functional effects of a sub-lethal dose of X-rays on the adult mouse olfactory neuroepithelium at 2 hours, 24 hours, 1 week, 2 weeks and 5 weeks. We have shown an immediate cessation of proliferating olfactory stem cells as shown by BrdU, Ki67 and pH3 expression. At 24 hours there was an increase in the neural transcription factors Mash1 and Pax6 expression, and a disruption of the basal lamina and increase in glandular cell marker expression at 1 week post-irradiation. Coincident with these changes was an impairment of the olfactory function We have shown significant changes in basal cell proliferation as well as morphological changes in the olfactory neuroepithelium following X-ray irradiation. There is involvement of the basal lamina as well as a clear role for glandular and sustentacular cells. The olfactory neuroepithelium (ON) is a specialized epithelium responsible for the perception of odours and undergoes neurogenesis throughout life in order to cope with damage suffered by being in direct contact with the external environment. It is also an easily accessible source of stem cells for cell therapy, so that the ON has become a useful model of neurogenesis ,2.The ON is a simple pseudostratified epithelium composed, in a basal to apical organization, of horizontal basal cells, globose basal cells, olfactory sensory neurons (OSN), Bowman's gland duct cells, microvillar cells and sustentacular cells. Underlining the ON, the lamina propria is composed of connective tissue, OSN axon bundles and respective olfactory ensheathing glia, blood vessels and Bowman's glands ,4. Many 2[Different models of ON degeneration have been used, including the exposure to the solvents zinc sulphate ,13, acet2 and meth2,20. All 2,22.In this study, we analysed the effects on neuronal and non-neuronal cells within the ON following X-ray irradiation of proliferating olfactory stem cells. Neural stem cells are more vulnerable than other cell types to ionizing radiation. It has been shown that ionizing radiation induces apoptosis of proliferating stem cells in the dentate gyrus of the adult rat hippocampus . X-ray iWe hypothesize, based upon previous work published by our group, that glandular cells, apically-situated sustentacular cells as well as suspected basal cells will be affected by X-ray irradiation and cause both morphological and behavioural disruption of the mouse ON. This study contributes to the understanding of cellular events that occur within several olfactory cell populations after an induced selective damage to the proliferating olfactory stem cell populations.Anaesthetized adult mice were irradiated with a sub-lethal dose of X-rays on the mouse ON. The olfactory bulbs (OB) were protected from irradiation by use of a lead shield designed to protect the whole body except the nose snout Figure . ExperimHaematoxylin and eosin (H&E) staining was performed on both ON  and OB and sections analyzed at roughly the same coronal level for each group. For the ON, 5 areas  were defined for morphological analysis and for posterior detailed immunohistochemical analysis Figure . The groWe analysed the morphology of the ON, OB as well as the nerve fibres connecting them. We studied these structures following staining of βIIITubulin. We showed that in the controls and at all time points following irradiation (only 24 hour and 5 week post-irradiation in figure) βIIITubulin was present in the cell soma of OSNs, processes and axon bundles in the ON Figure ,D,F. FurWe quantified the number of OSNs positively stained for βIIITubulin (immature and some mature OSNs) , carnosiFor analysis of cell proliferation, three different markers were analyzed: detection of BrdU incorporation during the S phase of the cell cycle Figure ,D,E; Ki6We found an increase in Ki67-positive cells in the basal layer of the ON at 2 hours, which was significantly reduced below normal levels after 24 hours Figure . Figure In order to more clearly dissect cell proliferation within the ON, we have analyzed also pH3, a specific marker for mitosis. Results are in agreement with BrdU, in that we have found mitosis at the level of the basal lamina, showing a reduction at 2 hours and 24 hours, with values returning almost to normal after 1 week Figure . PositivThe overall analysis of proliferation markers shows a significant reduction in olfactory progenitor cell proliferation following X-ray irradiation, which is almost abolished after 24 hours, and returning to normal levels after 5 weeks.Apoptotic cells were quantified by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), which measured fragmented DNA by incorporating fluorescein-12-dUTP at 3'-OH DNA ends, using the terminal deoxynucleotidyl transferase enzyme. Apoptotic cells were quantified separately at the level of the basal, intermediate and sustentacular cell layers and we saw a significantly higher number of apoptotic cells within the basal and intermediate cell layers of the ON Figure ,C. This We noticed a significant disruption of the basal lamina starting 1 week post-irradiation, as seen by keratin-positive horizontal basal cells that line the basal lamina Figure ,C, compaCD15 labels duct cells of the Bowman's gland in the ON and acinus cells in the lamina propria Figure -H. ImporMoreover we saw many CD15-positive cells in between the lamina propria and the ON where they do not normally appear Figure ,G. Not aWe observed a statistically significant increase in both Mash1 Figure  and Pax6To assess the olfactory function following X-ray irradiation of the mouse nose, two olfactory tests were performed: the Buried Food Pellet and the Habituation/Dishabituation test at 24 hours, 1 week, 2 weeks and 5 weeks . The Buried Food Pellet measures the mouse's ability to find a buried piece of food using olfactory cues. The Habituation/Dishabituation test assesses the mouse’s ability to discriminate between different odours. A decrement in the amount of time the mouse spends with its nose to the odour is inferred to signal its ability to habituate to a repeatedly exposed odour. The increase in time exploring a new odour is interpreted to reflect dishabituation. Irradiated mice had no detectable change in behaviour with respect to controls either during their time in the home cage or during behavioural testing.Results for the Buried Food Pellet test Figure  show a sFor the Habituation/Dishabituation test, we measured two different odours: strawberry and coconut Figure . For strFollowing X-ray irradiation, we observed an overall decrease in cell proliferation at 24 hours, a complete abolition of mitosis at 2 and 24 hours and a decrease in apoptosis at 2 hours. These data seem to indicate that the ON, and especially the active cycling zone of the ON, the basal lamina, has suffered a cell cycle arrest immediately after and as a consequence of ionizing radiation. X-ray irradiation has been shown before to induce permanent cell cycle arrest and the exhibition of senescence-like phenotypes in normal human cells  and to iContrary to other models of ON degeneration, which result in high morphological alterations and overall degeneration of the ON, our model seems to target specifically the proliferating cell populations, with minor morphological changes. We have found a disruption of the basal lamina at 1 and 2 weeks post-irradiation, but the overall gross morphology of the ON remained unaltered.As expected, for all groups, the changes in proliferation and apoptosis mostly occurred in the basal lamina, indicating that this area is indeed actively cycling. This area is historically considered the locus for olfactory stem cells in the ON and the only zone for cell proliferation. Our results have also shown that apical sustentacular cells undergo proliferation and apoptosis. Following X-ray irradiation, there are greater numbers of cells in the apical layer of the ON expressing Ki67 compared to BrdU. It has been shown before that nestin, an intermediate filament protein expressed in progenitor cells of the neural lineage, in the ON is mainly expressed by endfeet and inferior processes of sustentacular cells . It has CD15-positive olfactory glandular cells migrate from the lamina propria into the basal layer of the ON corresponding to a severe disruption in the basal lamina at 1 and 2 weeks post-X-ray irradiation. A previous study showed that degeneration of the ON during normal aging spatially co-localizes with lesions in the Bowman's gland in the mouse olfactory mucosa . We haveMash1 is a transcription factor involved in neurogenic cell fate -52, and X-ray irradiation has undoubtedly resulted in impairment in the olfactory function, measured by both the Buried Food Pellet test and the Habituation/Dishabituation test. It is evident at 24 hours post-irradiation that there is olfactory impairment. As olfactory bulbs were protected by a lead shield during irradiation, we do not think the olfactory impairment is due to a deficit in olfactory bulb function, OSN signaling via axons or odorant detection by olfactory neuron cilia. The examination of βIIITubulin expression in the cell soma, nerve fibres and cilia of OSNs in the ON and the nerve fibres, glomerular and mitral cells of the bulbs showed no change following irradiation. Previous studies have shown that βIIITubulin forms microtubules that form neurites  and thatThis study has demonstrated that X-ray irradiation of the ON induces a reversible cell cycle arrest in the ON as well as a series of transcriptional alterations and a disruption of olfactory function which are mostly recovered 5 weeks post-irradiation. Coincident with changes to the basal lamina and basally situated olfactory stem cell populations, the sustentacular and glandular cells of the ON were shown for the first time to respond to irradiation by undergoing anatomical and spatial alterations. This study extends the understanding of ON plasticity and chemosensory function to include the role of olfactory supporting cells. Moreover, it might impact on the understanding of the effects on proliferating cells of ionizing radiation, which is commonly used in computed tomography scans, cancer radiotherapy and nuclear medicine.th edition, 2004). Animals were kept in a 12-hour light/dark cycle, with food and water ad libitum and handled for 1 week before experimentation in order to diminish their stress or anxiety.All experiments were carried out using wild-type male C57BL/6 J mice at 8 weeks of age and have been approved by the Garvan Institute of Medical Research/St Vincent’s Hospital Animal Ethics Committee and conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes  was diluted to 10 mg/ml in 0.9% saline solution and an intraperitoneal injection was administered at a dose of 50 mg/Kg body weight, 2 hours before transcardiac perfusion.Mice were anaesthetized with an intraperitoneal injection of 100 mg/Kg Ketamine plus 20 mg/Kg Xylazine and perfused with heparinized saline (10 IU/ml) followed by 4% paraformaldehyde . Olfactory tissue was dissected together with the olfactory bulbs and post-fixed in 4% PFA overnight. The ON was decalcified for 7 days in 15% EDTA/4% PFA and processed for paraffin embedding. Coronal serial sections of 6 μm were collected via a microtome (Leica). Tissue sections were left at 60°C for 1 hour for melting of the paraffin, dewaxed in Histoclear solution  and rehydrated through a graded series of alcohol.H&E staining was performed for each animal, at intervals of 180 μm for the entire length of the mouse nose , in order to determine the same coronal level for all animals analyzed. Sections were stained with hematoxylin for 45 seconds and washed in tap water and then stained with eosin for 30 seconds and washed in tap water. Tissue was dehydrated in a graded series of alcohols and slides mounted with Eukitt (Sigma).2O2 in PBS for 15 min. Sections were incubated with the respective biotinylated goat anti-rabbit, horse anti-mouse or rabbit anti-goat secondary antibodies  for 30 minutes at room temperature. Avidin-Horseradish Peroxidase complex was prepared using the Vector ABC Elite kit (Vector Laboratories) according to the manufacturer’s recommendations. Immunoreactivity was visualised using DAB as the chromogen and slides mounted using Aquamount .For immunohistochemical analysis against olfactory marker protein (OMP) and carnosine, antigen retrieval was performed by immersing slides in TE 1X pH 9.0 at high temperature. Non-specific staining was blocked in 10% goat and rabbit serum (Sigma) plus 1% bovine serum albumin for 1 h. Sections of ON and OB from control and irradiated mice were incubated with the primary antibody anti-βIIITubulin. The ON further examined using anti-OMP, anti-carnosine and , according to manufacturer's instructions. Briefly, tissue was hydrated and permeabilized by Proteinase K (20 μg/ml) digestion for 8 minutes at room temperature. No further PFA fixation was performed. Tissue was incubated for 1 hour at 37°C with Terminal Deoxynucleotidyl Transferase (recombinant) and fluorescein-12-dUTP and reaction stopped by incubating slides in 2x SSC for 15 minutes. Cell nuclei counterstaining was performed by use of DAPI .The TUNEL assay was performed by use of the DeadEndFor all immunofluorescence analysis, sections were examined using an upright fluorescence microscope  and pictures were acquired using a 40X and 100X oil objectives and an AxioCam digital camera (Carl Zeiss MicroImaging GmbH). For all histochemical analysis, sections were examined using an upright microscope  and pictures acquired using a 2.5X and 40X dry objectives and an AxioCam digital camera (Carl Zeiss MicroImaging GmbH). No image manipulation has been performed of any kind, apart from rotation and cropping.For each animal and marker, 5 different areas of the ON in the coronal section of the nose were quantified by counting the number of positively stained cells, corresponding to the middle of the nasal septum, one more superior and one more inferior area of both the left and right turbinates Figure . For allOlfactory tests have been performed as previously described , with miThis test measures the mouse’s ability to find a buried food pellet using olfactory cues. Mice (n=5 per group) were food deprived overnight with free access to water, between 5 pm the day prior to the test and the beginning of the test at 8 am the following day. To avoid problems associated with neophobia, mice were habituated to eat the new food by placing pieces in the home cage overnight for 3 days before the test. On the day of the test, a sultana was placed underneath the bedding material within the home cage. The floor of the cage was then completely covered with new bedding material to a depth of approximately 2.5 cm. The latency to find the sultana and start eating was recorded using a stopwatch. A maximum cut-off time of 15 minutes was used and mice reaching the cut-off were included in the analysis as if they had found the pellet after 15 minutes.Odorants were prepared by diluting strawberry and coconut essences (Queen Fine Foods Pty Ltd) 1:100 in distilled water. Mice were placed individually into a clean cage. Cotton buds were dipped into distilled water or odorant and are then placed through the wire cage lid and fixed with the tip at a height of 5 cm above the cage floor. Each stimulus was presented 3 times consecutively in the following order: water 3x, strawberry 3x, coconut 3x, for 3 minutes each and with the mouse returning to his home cage for 1 minute between each stimulus presentation. The cumulative time spent sniffing each stimulus was measured. The behaviour sniffing is defined as orienting the nose towards the stimulus within 2 cm of the cotton tip or in contact with the tip. Chewing the cotton tip or contacting it with an open mouth is not counted as sniffing. The percentage of time spent sniffing the first strawberry presentation with respect to the third water was calculated. The percentage of the time spent sniffing the first coconut presentation with respect to the third strawberry was calculated.All results were expressed as MEAN ± SEM plotted on graph and statistical analysis was performed using GraphPad Prism software (version 5.0), with statistical significance set at p ≤ 0.05. For both olfactory tests, a one-way ANOVA was performed, followed by Dunnet’s post hoc test, with respect to control group. For all immunohistochemistry analysis, a one-way ANOVA was performed, followed by Tukey's post hoc test, for comparison between all groups.BrdU: 5-bromo-2’deoxyuridine; GFAP: glial fibrillary associated protein; OB: olfactory bulb; OMP: olfactory marker protein; ON: olfactory neuroepithelium; OSN: olfactory sensory neuron; PBS: phosphate buffered saline; PFA: paraformaldehyde; TUNEL: terminal deoxynucleotidyl transferase mediated UTP nick end labeling.The authors declare that they have no competing interests.CC designed the study, carried out the immunofluorescence and animal studies, performed the statistical analysis and wrote the manuscript. YH carried out the immunohistochemistry studies. JS participated in the design of the study. KD conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.High magnification photomicrographs of βIIITubulin labeling of the olfactory bulbs in control and irradiated mice.Click here for file


Animal models have become a popular platform for the investigation of the molecular and systemic mechanisms of pathological cardiovascular physiology. Chronic pacing studies with implantable pacemakers in large animals have led to useful models of heart failure and atrial fibrillation. Unfortunately, molecular and genetic studies in these large animal models are often prohibitively expensive or not available. Conversely, the mouse is an excellent species for studying molecular mechanisms of cardiovascular disease through genetic engineering. However, the large size of available pacemakers does not lend itself to chronic pacing in mice. Here, we present the design for a novel, fully implantable wireless-powered pacemaker for mice capable of long-term (>30 days) pacing. This design is compared to a traditional battery-powered pacemaker to demonstrate critical advantages achieved through wireless inductive power transfer and control. Battery-powered and wireless-powered pacemakers were fabricated from standard electronic components in our laboratory. Mice (n = 24) were implanted with endocardial, battery-powered devices (n = 14) and epicardial, wireless-powered devices (n = 10). Wireless-powered devices were associated with reduced implant mortality and more reliable device function compared to battery-powered devices. Eight of 14 (57.1%) mice implanted with battery-powered pacemakers died following device implantation compared to 1 of 10 (10%) mice implanted with wireless-powered pacemakers. Moreover, device function was achieved for 30 days with the wireless-powered device compared to 6 days with the battery-powered device. The wireless-powered pacemaker system presented herein will allow electrophysiology studies in numerous genetically engineered mouse models as well as rapid pacing-induced heart failure and atrial arrhythmia in mice. The use of animal models of human disease processes plays an essential role in bio-medical research. Models of cardiovascular disease offer unique challenges since these diseases are multifactorial, often with contributing environmental causes that take years to fully manifest. One versatile maneuver is altering heart rate with the implantation of a cardiac pacemaker, a small electronic device connected to the heart with wires or leads. In animal models, the induction of rapid heart rates over a 4–6 week period results in cardiac remodeling and dysfunction, providing a model system of high impact cardiac diseases including congestive heart failure Although pacemakers were originally and primarily designed as a treatment for slow heart rate (bradyarrhythmias), recent developments have begun to highlight an array of more complex and subtle effects of ectopic pacing on cardiac electrical function. One seemingly simple example of pacemaker induced remodeling is the observation that after a period of ectopic pacing, a persistent alteration in T-wave axis (observed on the body surface ECG) occurs, which can linger for weeks or months One challenging aspect of studying pacemaker-induced phenomenon is the choice of model system. Traditionally, larger animal models, primarily canine, have been used to allow easier implantation of the pacemaker. On the other hand, the mouse, with its long history as a versatile and affordable genetic and molecular model, offers a much wider range of tools to investigate electrical remodeling in models of heart failure on the gene/protein levels We designed our printed circuit board (PCB) layout and pacemaker circuit using free ExpressPCB CAD software (expresspcb.com). Our PCB . Adult mice (n = 14) were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally and then restrained in a supine position on a heated magnetic stainless steel surgical board under a low power binocular dissecting microscope. In our original attempts to surgically implant a battery-powered pacemaker, we used an external jugular approach. For this procedure, mice were prepared for surgery with aseptic and anesthetic techniques. The mice were intubated with a 20-gauge 1″ smooth needle through the oral cavity as verified by direct visualization of the intubation device through the exposed trachea. Mice were then ventilated at a tidal volume of 200–500 µl and a rate of 140 strokes/minute. After mice reached an anesthetic level for surgery, the neck skin was cut to expose the jugular vein. The vein was isolated and secured with suture. A sterile battery-powered pacemaker was then inserted into a subcutaneous pocket formed by blunt dissection along the animal’s flank and back. The pacing electrode was then fed through a sterile trocar under the skin and into the site of the jugular isolation. A small incision was made in the jugular vessel and the pacemaker lead was inserted through the vessel and into the right ventricle of the heart. After confirmation of pacing capture and good positioning of the extra wire length, the pacemaker lead was secured into the jugular by tying down the suture and placing a small drop of sterile tissue glue on the vessel at the site of lead entrance. Both the neck and back skin incisions were closed and mice were allowed to recover on a warmer until being returned to their cage.th intercostal space. The chest was then opened by a lateral cut along the left side of the sternum between the ribs. The chest wall was retracted to better expose the apex of the left ventricle. The sterile pacemaker electrode was then sewn with 8–0 suture to the apical myocardial wall through either the anodal or cathodal eyelet. It was also ensured that both electrodes were touching the surface of the ventricular wall. The chest wall was then closed around the electrode with a purse string. The surgical incision was closed in two layers with an interrupted suture pattern. Mice were then allowed to recover with oxygen on a heating pad until extubation. Mice were monitored closely for 3 days following surgery.For the wireless-powered pacemaker, aseptic and anesthetic techniques were followed as previously described in adult mice (n = 10). Once a surgical plane of anesthesia was achieved in the mouse, a small incision was made in the mid-thorax region followed by blunt dissection to expose the larynx and trachea. A short midline skin incision was made with reflection of the skin directly above the base of the sternum. A subcutaneous skin pocket was made below the sternum and right above the abdomen by blunt dissection for placement of the flat pacemaker device. The pacemaker was then positioned under the skin in this pocket to meet the exact length required for the electrodes to enter the chest at the 4Following implantation and recovery, mice were monitored daily. In cases of erosion of the device through the skin, mice were euthanized at the time the erosion was identified. In all other cases of death, no symptoms were evident prior to death. At the time of sacrifice, mice were anesthetized with Avertin (0.5 mg/g) followed by removal of the heart for inspection and identification of complications.All statistical analysis was performed in Prism . An F-test was performed on the slope of the regression line of the mean pacing pulse width thresholds to determine significance compared to a slope of 0.We began by creating a battery-powered design similar to Bilchick et al. in order to better understand design criteria for an implantable mouse pacemaker Figure 1Similar to human pacemakers, this battery-powered mouse pacemaker operates as a constant voltage pulse generator. Methods section. Following initial implantation, device functionality was tested via an electrocardiogram (ECG) and an echocardiogram to verify electrical and mechanical cardiac capture. Mice were monitored daily for survival and device functionality via an ECG. All ECG measurements were performed under inhaled isofluorane anesthesia (1–2%). Eight of 14 (57.1%) mice died within 1–2 days of implantation due to various causes . Of the survivors, 2 of 6 (33.3%) survived 4 days, 1 of 6 (16.7%) survived 6 days, 1 of 6 (16.7%) survived 8 days, and 2 of 6 (33.3%) survived 11 days. With respect to device functionality, pacing was achieved in 9 of 14 (64.3%) mice. Chronic pacing was achieved in two of the 4 (50%) mice that survived >4 days for a maximum of 5 days and 6 days. Battery-powered devices were implanted in mice (n = 14) as specified in the , top) . An external function generator  is used to generate a pulsed input . As a result of the large voltage from the transmitter, we were able to design a passive receiver without additional amplifiers. Due to the high post-implant mortality with the battery-powered pacemaker, we attempted to refine our pacemaker design. We hypothesized that implant intolerance was primarily due to device size as well as mechanical complications resulting from placement of the endocardial lead. One of the larger components of the battery-powered design, by mass and volume, is the battery. Presently, lithium ion batteries with smaller size and sufficient charge capacity do not exist. As a result, we began exploring wireless energy transfer as a means for device power without the use of a battery. Inductive energy transfer has been used extensively for both power and data communication in implantable devices for various biological applications ed input Fig. 3b 1). Next, the device is pressed into the bead of Silastic (2) and an additional layer is added to the top (3). A final layer of areaseal film is pressed on the top of the device to join the upper and lower layers of Silastic (4). The use of gas-permeable areaseal film was found to be essential to allow curing of Silastic. The device is cured at room temperature for 3 days and then trimmed of excess. Methods section.The receiver coil and pacing circuit are assembled on a flexible polyimide PCB  to reduce device thickness and attached to a platinum bipolar pacing electrode. Methods section. Device functionality was verified in all ten (100%) mice at implant via ECG. Echocardiographic measurements were not acquired due to interference concerns between the transmitter and the echo machine. Following a five-day recovery period, ventricular pacing was conducted under isofluorane anesthesia (1–2%) at 5 day intervals for 30 days. Due to the fact that a cage system incorporating the transmitter coils remains under development, the wireless device was not continuously powered (in contrast to the battery device described above) but was only powered with a transmitter coil during testing at 5 day intervals to verify stability of device function. At each follow-up, pacing pulse-width threshold at 600–630 BPM was tested. On post-operative day 30, ability to pace above intrinsic physiologic rates (>750 BPM) was tested.Wireless-powered devices were implanted in mice (n = 10) as specified in the Nine of 10 (90%) mice survived device implantation with the wireless-powered device. Of the survivors, 6 of 9 (66.7%) mice survived 20 days . Necropsies were performed but no cause of death was identified. Twenty days post implant, 5 of 6 devices functioned properly. One device exhibited unreliable capture at 600 BPM; therefore pacing with reliable cardiac capture was successfully achieved in 4 of 6 (66.7%) mice for 20 days. In addition, three devices continued to exhibit stable function for up to 30 days. This study describes the design and implementation of a miniature, implantable wireless pacemaker for mice with off-the-shelf electronic components. This wireless design described herein has considerable advantages over a traditional battery-powered pacemaker. A list of contrasting device features is provided in First and foremost, our wireless-powered pacemaker is 82% smaller by mass and 77% smaller by volume compared to our battery-powered design. Compared to the battery-powered device described by Bilchick et al. Second, we demonstrate successful pacing for 30 days with our wireless device, a significant improvement compared to the one week of reliable pacing reported in previous studies in vivo.External control of our wireless device increases device flexibility and functionality. Pulse width and pacing rate can be adjusted on demand and are not limited to values preprogrammed onto a microchip. Additionally, programmed stimulation protocols  can be created on an external function generator to perform sophisticated electrophysiological testing Finally, cost and ease of implant were carefully considered during the development of a fully implantable pacemaker for mice. In our original battery-powered design, we used a 1 Fr. murine pacing catheter developed by NuMed, Inc. as our pacing electrode. While the placement of this electrode in the RV endocardium more closely resembles pacing in canines, consistent placement of the pacing catheter in the RV required a skilled mouse surgeon and occasional use of echocardiography and, even so, increased peri-operative mortality due to RV perforation. Additionally, the NuMed catheter added significant cost to the battery-powered design . By decreasing device size and pursuing an epicardial pacing strategy, we were able to greatly reduce the complexity of the implant procedure. Moreover, by replacing the NuMed catheter with standard platinum-iridium wire, a complete build for a wireless-powered pacemaker can be assembled for approximately $30.Here we demonstrate a successful implementation of a fully implantable mouse pacemaker based on wireless power transfer and control capable of 30 days of in vivo pacing. For the purpose of device testing, all experiments in this study were performed on anesthetized animals with a hand-held transmitter. In order to chronically pace freely roaming mice, we integrated a transmitter coil into the mouse housing. This system is designed to generate voltage fields in excess of the 3.9 V cap on the receiver throughout the cage to eliminate communication issues between the transmitter and receiver. This will ensure that mice will always be paced independent of the location they occupy in the cage.The development of a practical pacemaker for long-term studies in the mouse will permit a range of future studies. First, the development of the mouse as a model of cardiac memory will allow the well-developed molecular and genetic tools available in this animal to be brought to bear on the mechanism(s) underlying pacemaker-induced cardiac electrical remodeling. Second, long-term pacing at supra-physiological rates can result in heart failure and atrial fibrillation, both common pathologies amenable to further genetic studies in the mouse. While the current design is sufficient for physiologic testing, future development will focus on potential improvement to the pacing electrode and the creation of an electrical sensing circuit. During some experiments, subtle chest-wall captured was observed during pacing. Further modifications of the lead design with novel printing strategies on flexible substrates and improvements to the surgical method should mitigate this issue. Additionally, our receiver circuit is a dedicated pacing circuit without sensing capabilities. Stimulation and sensing with radiofrequency induction has been demonstrated previously for neural applications File S1Source code used in the first generation of implantable pacemaker.(DOCX)Click here for additional data file.