Acute central nervous system graft-versus-host-disease after liver transplantation.

Acute Central Nervous System (CNS) Graft Versus Host Disease (GvHD) is a rare form of GvHD, only described in case reports. Knowledge about this condition is extrapolated from chronic CNS GvHD cases occurring mostly after hematopoietic stem cell transplantation. GvHD following solid organ transplantation is an unexpected complication. GvHD after liver transplantation has a poor prognosis, and the optimal management is not yet known. Here we describe the case of a 63-year-old man who underwent deceased donor liver transplantation and subsequently developed skin rash, colitis and pancytopenia followed by refractory status epilepticus. Following the identification of lymphocytes of donor origin in the cerebrospinal fluid of the patient, he was diagnosed with acute CNS GvHD. He was treated with an intensive immunosuppressive regimen, but care was withdrawn due to lack of improvement and worsening neurologic prognosis. It is the second known case of acute CNS GvHD following liver transplantation. Clinicians should be aware of this possible, although rare, complication of liver transplantation, especially when there is refractory status epilepticus of unknown origin.

The Use of UAS to Release the Egg Parasitoid Trichogramma spp. (Hymenoptera: Trichogrammatidae) Against an Agricultural and a Forest Pest in Canada.

The egg parasitoid Trichogramma spp. (Hymenoptera: Trichogrammatidae) is a widely used biocontrol agent against lepidopteran pests. Historically, Trichogramma were deployed either by plane or by using cardboard cards on which parasitized eggs are glued and manually installed at sites. Plane deployment is costly and card installation is time consuming, but the use of Trichogramma has been shown to be efficient against several pests. In 2016 and 2017, a research project investigated the potential use of unmanned aerial system for distributing Trichogramma as biocontrol agents against two major pests: an agricultural pest of maize, the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), and a forest pest, the eastern spruce budworm, Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae). Exposure duration of parasitized eggs to field conditions (temperature, predation, etc.) in maize fields influenced the Trichogramma's emergence rate, suggesting that timing of parasitoid releases with their emergence is essential. Although parasitism of naturally occurring eggs in maize fields could not be compared due to the low density of the European corn borer, parasitism of sentinel eggs by Trichogramma was more prominent in plots with unmanned aircraft systems (UAS)-releases compared to control plots. For spruce budworm, treatment with Trichogramma increased egg parasitism and there was no difference between the deployment by UAS and by Trichocards. We discuss these results in the context of pest biology and management. We also discuss the advantages and shortcomings of both methods and offer insights into where future work might go to further leverage the use of UAS in managing these important pests.

Narcolepsy and Hypothalamic Region Tumors: Presentation and Evolution.

OBJECTIVE: Because most cases of brain tumor-associated narcolepsy have been published in the form of case reports or small series, the clinical presentation and evolution have not been well described. We sought to better define the epidemiology, etiology, and outcome of brain tumor-related narcolepsy. METHODS: We conducted an extensive review of the literature to identify cases of narcolepsy associated with brain tumors. Only cases of brain tumors involving the hypothalamic region including the suprasellar, sellar, and thalamus were included in this study. RESULTS: We report a child with possible narcolepsy in a child with a brain tumor. Through our literature review, we identified 25 additional cases of narcolepsy associated with brain tumors affecting the hypothalamic area. Most symptomatic narcolepsy cases were reported in children (70%). Half of the patients (13 of 25, 52%) developed narcolepsy after surgery, whereas 11 patients (44%) were symptomatic at the time of the tumor diagnosis. Ten patients had narcolepsy without cataplexy. Most cases were associated with craniopharyngioma (38%), adenoma (24%), and glioma (14%). Three, including our patient, experienced a complete resolution of symptoms. All patients underwent biopsy and were treated with adjuvant therapy. For patients with persistent symptoms, most (60%) improved following medical management of narcolepsy. CONCLUSION: This study represents the largest compilation of patients with this association. Our study allows us to better understand the etiology and outcome of patients with narcolepsy-related brain tumors.

Do natural enemies explain fluctuations in low-density spruce budworm populations?

Understanding the causal pathways through which forest insect outbreaks are triggered is important for resource managers. However, detailed population dynamics studies are hard to conduct in low-density, pre-outbreak populations because the insects are difficult to sample in sufficient numbers. Using laboratory-raised larvae installed in the field across a 1,000 km east-west gradient in Québec (Canada) over an 11-yr period, we examined if parasitism and predation were likely to explain fluctuations in low-density spruce budworm (Choristoneura fumiferana; SBW) populations. Parasitism rates by the two main larval parasitoid species, Elachertus cacoeciae and Tranosema rostrale, peaked during different years. This suggests that temporal fluctuations in overall parasitism were partly buffered by compensatory dynamics among parasitoid species. Still, spatial covariance analyses indicate that the residual interannual variation in parasitism rates was substantial and correlated over large distances (up to 700 km). On the other hand, interannual variation in predation rates was not spatially correlated. Piecewise structural equation models indicate that temporal variation in parasitism and predation does not influence temporal variation in wild SBW abundance. Spatially, however, SBWs installed in warmer locations tended to show higher parasitism rates, and these higher rates correlated with lower wild SBW population levels. Overall, the results indicate that large-scale drops in parasitism occur and could potentially contribute to SBW population increases. However, during the period covered by this study, other factors such as direct effects of weather on SBW larval development or indirect effects through host tree physiology or phenology were more likely to explain large-scale variation in wild SBW populations.

Impact of rearing temperature on encapsulation and the accumulation of transcripts putatively involved in capsule formation in a parasitized lepidopteran host.

Encapsulation and melanisation are innate immune reactions of insects against foreign intruders such as parasitoids. In an earlier study, we observed that immature life stages of the endoparasitoid Tranosema rostrale (Hymenoptera: Ichneumonidae) parasitizing Choristoneura fumiferana (Lepidoptera: Tortricidae) larvae experienced higher mortality due to encapsulation and melanisation when reared at high (30 °C) than at lower (10 °C, 20 °C) temperatures. Downregulation of T. rostrale polydnavirus genes in parasitized hosts and upregulation of two genes involved in the spruce budworm's melanisation process were identified as likely contributors to parasitoid mortality at high temperature. However, levels of transcripts of genes involved in the spruce budworm's cellular encapsulation process were not measured inasmuch as candidate genes, in the spruce budworm, had not yet been identified. In addition, our assessment of temperature-dependent encapsulation and melanisation of foreign objects in spruce budworm larvae was only partial. To fill these knowledge gaps, we injected Sephadex™ beads into unparasitized spruce budworm larvae and assessed their encapsulation/melanisation after the insects had been held at three different temperatures (10, 20, and 30 °C), and we identified spruce budworm genes putatively involved in the encapsulation process and quantified their transcripts at the same three temperatures, using a qPCR approach. As expected, both encapsulation and melanisation of Sephadex™ beads increased as a function of temperature. At the molecular level, three of the five genes examined (Integrin ß1, Hopscotch, Stat92E) clearly displayed temperature-dependent upregulation. The results of this study further support the hypothesis that a temperature-dependent increase in the encapsulation response of C. fumiferana against T. rostrale is due to the combined effects of reduced expression of polydnavirus genes and enhanced expression of host immune genes.

The sterile insect technique for the management of the spotted wing drosophila, Drosophila suzukii: Establishing the optimum irradiation dose.

The spotted wing drosophila Drosophila suzukii Matsumura (Diptera: Drosophilidae), a pest of berries stone fruits, invaded North America and Europe in 2008. Current control methods rely mainly on insecticides. The sterile insect technique (SIT) has potential as an additional control tactic for the integrated management of D. suzukii. As a step towards the development of the SIT, this study aimed at finding the optimum irradiation dose to sterilize D. suzukii under controlled laboratory conditions. Four-day-old D. suzukii pupae were irradiated 12 to 24 hours prior to adult emergence in a 60Co Gamma Cell 220 and in a 137Cs Gamma Cell 3000 with doses of 30, 50, 70, 80, 90, 100 or 120 Gy. Emergence rate (88.1%), percent of deformed flies (4.0%) and survival curves were not affected by the tested irradiation doses. However, some reproductive parameters of the flies were affected by irradiation. Females irradiated with a dose of 50 Gy or more had almost no fecundity. When non-irradiated females were mated with irradiated males, egg hatch decreased exponentially with irradiation dose from 82.6% for the untreated control males to 4.0% for males irradiated with 120 Gy. Mortality of F1 individuals from the irradiated treatment also occurred during larval and pupal stages, with an egg to adult survival of 0.2%. However, descendants produced by the irradiated generation were fertile. These results are an encouraging first experimental step towards the development of the SIT for the management of D. suzukii populations.

Measuring alterations in oscillatory brain networks in schizophrenia with resting-state MEG: State-of-the-art and methodological challenges.

OBJECTIVE: Neuroimaging studies provide evidence of disturbed resting-state brain networks in Schizophrenia (SZ). However, untangling the neuronal mechanisms that subserve these baseline alterations requires measurement of their electrophysiological underpinnings. This systematic review specifically investigates the contributions of resting-state Magnetoencephalography (MEG) in elucidating abnormal neural organization in SZ patients. METHOD: A systematic literature review of resting-state MEG studies in SZ was conducted. This literature is discussed in relation to findings from resting-state fMRI and EEG, as well as to task-based MEG research in SZ population. Importantly, methodological limitations are considered and recommendations to overcome current limitations are proposed. RESULTS: Resting-state MEG literature in SZ points towards altered local and long-range oscillatory network dynamics in various frequency bands. Critical methodological challenges with respect to experiment design, and data collection and analysis need to be taken into consideration. CONCLUSION: Spontaneous MEG data show that local and global neural organization is altered in SZ patients. MEG is a highly promising tool to fill in knowledge gaps about the neurophysiology of SZ. However, to reach its fullest potential, basic methodological challenges need to be overcome. SIGNIFICANCE: MEG-based resting-state power and connectivity findings could be great assets to clinical and translational research in psychiatry, and SZ in particular.

Alterations of Intrinsic Brain Connectivity Patterns in Depression and Bipolar Disorders: A Critical Assessment of Magnetoencephalography-Based Evidence.

Despite being the object of a thriving field of clinical research, the investigation of intrinsic brain network alterations in psychiatric illnesses is still in its early days. Because the pathological alterations are predominantly probed using functional magnetic resonance imaging (fMRI), many questions about the electrophysiological bases of resting-state alterations in psychiatric disorders, particularly among mood disorder patients, remain unanswered. Alongside important research using electroencephalography (EEG), the specific recent contributions and future promise of magnetoencephalography (MEG) in this field are not fully recognized and valued. Here, we provide a critical review of recent findings from MEG resting-state connectivity within major depressive disorder (MDD) and bipolar disorder (BD). The clinical MEG resting-state results are compared with those previously reported with fMRI and EEG. Taken together, MEG appears to be a promising but still critically underexploited technique to unravel the neurophysiological mechanisms that mediate abnormal (both hyper- and hypo-) connectivity patterns involved in MDD and BD. In particular, a major strength of MEG is its ability to provide source-space estimations of neuromagnetic long-range rhythmic synchronization at various frequencies (i.e., oscillatory coupling). The reviewed literature highlights the relevance of probing local and interregional rhythmic synchronization to explore the pathophysiological underpinnings of each disorder. However, before we can fully take advantage of MEG connectivity analyses in psychiatry, several limitations inherent to MEG connectivity analyses need to be understood and taken into account. Thus, we also discuss current methodological challenges and outline paths for future research. MEG resting-state studies provide an important window onto perturbed spontaneous oscillatory brain networks and hence supply an important complement to fMRI-based resting-state measurements in psychiatric populations.

High temperature induces downregulation of polydnavirus gene transcription in lepidopteran host and enhances accumulation of host immunity gene transcripts.

Endoparasitoids face the challenge of overcoming the immune reaction of their hosts, which typically consists of encapsulation and melanisation of parasitoid eggs or larvae. Some endoparasitic wasps such as the solitary Tranosema rostrale (Hymenoptera: Ichneumonidae) that lay their eggs in larvae of the spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae), have evolved a symbiotic relationship with a polydnavirus (PDV), which in turn helps them suppress the host's immune response. We observed an increase in mortality of immature T. rostrale with increasing temperature, and we tested two hypotheses about the mechanisms involved: high temperatures (1) hamper the expression of T. rostrale PDV genes and (2) enhance the expression of spruce budworm immunity-related genes. Dissections of parasitized spruce budworm larvae reared at 30°C revealed that most parasitoid eggs or larvae had died as a result of encapsulation and melanisation by the host. A qPCR analysis of T. rostrale PDV (TrIV) gene expression showed that the transcription of several TrIV genes in host larvae was downregulated at high temperature. On the other hand, encapsulation, but not melanisation, of foreign bodies in spruce budworm larvae was enhanced at high temperatures, as shown by the injection of Sephadex™ beads into larvae. However, at the molecular level, the transcription of genes related to spruce budworm's melanisation process (prophenoloxidase 1 and 2) was upregulated. Our results support the hypothesis that a temperature-dependent increase of encapsulation response is due to the combined effects of reduced expression of TrIV genes and enhanced expression of host immune genes.

Developmental and reproductive responses of the spruce budworm (Lepidoptera: Tortricidae) parasitoid Tranosema rostrale (Hymenoptera: Ichneumonidae) to temperature.

The temperature-dependent development and survival of immatures, as well as adult longevity and potential fecundity of the endoparasitoid Tranosema rostrale (Hymenoptera: Ichneumonidae) parasitizing spruce budworm Choristoneura fumiferana (Lepidoptera: Tortricidae) larvae was investigated under laboratory conditions at several constant temperatures ranging from 5 to 30°C. Maximum likelihood modeling approaches were used to estimate thermal responses in development, survival, and longevity. A model describing the effect of temperature on potential fecundity of the parasitoid was also developed taking oogenesis and oosorption into account. In-host and pupal development rates of the parasitoid increased with temperature up to 25°C, and decreased thereafter. Immature survival was highest below 20°C, and rapidly decreased at higher temperatures. Adult longevity decreased exponentially with increasing temperature for both males and females. Highest potential fecundity was reached at 10°C. Considering survival and potential fecundity, the parasitoid seems best adapted to cool temperatures below 20°C. Simulations of the life-history traits under variable temperature regimes indicate that temperature fluctuations decrease survival and increase realised fecundity compared to constant temperatures. The temperature-dependent fecundity model developed can be applied to other non-host-feeding synovigenic parasitoids. The equations and parameter estimates provided in this paper can be used to build comprehensive models predicting the seasonal phenology of this parasitoid and spruce budworm parasitism under changing climatic conditions.

Seasonal Parasitism and Host Instar Preference by the Spruce Budworm (Lepidoptera: Tortricidae) Larval Parasitoid Tranosema rostrale (Hymenoptera: Ichneumonidae).

The seasonal pattern of parasitism by a parasitoid can be influenced by many factors, such as interspecific competition and host instar preference. We conducted field and laboratory experiments to describe the seasonal pattern of parasitism of spruce budworm Choristoneura fumiferana (Clemens) larvae by Tranosema rostrale (Brischke), and to investigate whether this pattern can be explained by interaction with other parasitoid species, or by host instar preference. Larval survival, developmental time, sex ratio, and adult size of T. rostrale developing in different host instars were also measured to further assess the potential importance of host instar on parasitoid life history. Parasitism by T. rostrale increased over the season, reaching the highest rate during the fourth-instar larva, and then decreased again until the sixth-instar. At the same time, parasitism by another parasitoid, Elachertus cacoeciae (Howard), increased over the season, and multiparasitism with T. rostrale suggests potential competition between these two parasitoids. Tranosema rostrale showed no host instar preference when third- to sixth-instar larvae were exposed simultaneously in a manipulative field experiment. The proportion of females emerging from spruce budworm larvae increased over the season; however, no difference in sex ratio was observed in the manipulative field experiment. Only male pupal development time and adult size were marginally increased in fifth-instar spruce budworm larvae. We conclude that T. rostrale's seasonal phenology or competition with E. cacoeciae, but not host instar preference, were possibly responsible for the observed seasonal pattern of parasitism.

The male mate search: an optimal foraging issue?

Male insects must find and mate females to have some descendants; male fitness therefore depends on the number of females they inseminate. Males are for this reason expected to optimize the behaviors related to mate location, orientation and copulation. Although optimization of the reproductive behavior of males has long been neglected in the literature, recent studies suggest a renewed interest for this idea. Here we discuss the parallel between male mate-finding and mating strategies in insects and optimal foraging theory (OFT), a class of models which formalize the behavior of organisms seeking and exploiting resources, generally food. We highlight the different facets of male mating systems allowing such a parallel, and claim for a unifying approach of foraging behavior. Finally, we discuss novel research perspective emerging from the application of OFT to male reproductive behavior.

Sperm-depleted males influence the reproductive behaviour of conspecifics.

In many insect species, sperm-depleted males (SDMs, i.e. males that have exhausted their sperm after a given number of matings) remain sexually active and continue to mate females. Here, we investigated the behavioural modifications that occur in both sexes of the parasitoid Asobara tabida Nees (Hymenoptera: Braconidae), after matings by fertile males and sperm-depleted males. We show that (i) virgin females, mated females and females mated to a SDM exhibited different behaviours and that (ii) males responded differently to females depending on whether the females had previously mated with an SDM or not. Our findings demonstrate that SDM influenced the reproductive behaviour of both males and females, especially with regard to male responsiveness and female attractiveness. These findings are discussed in the context of adaptive behaviour and fitness maximization in both males and females.

When parasitoid males make decisions: information used when foraging for females.

Optimal foraging models predict how an organism allocates its time and energy while foraging for aggregated resources. These models have been successfully applied to organisms such as predators looking for prey, female parasitoids looking for hosts, or herbivorous searching for food. In this study, information use and patch time allocation were investigated using male parasitoids looking for mates. The influence of the former presence of females in absence of mates and the occurrence of mating and other reproductive behaviours on the patch leaving tendency was investigated for the larval parasitoid Asobara tabida. Although males do not modify their patch residence time based on the number of females that visited the patch, they do show an increase in the patch residence time after mating a virgin female and performing courtship behaviour such as opening their wings. These results are in concordance with an incremental mechanism, as it has been described for females of the same species while foraging for hosts. The similarities between males and females of the same species, and the conditions under which such a patch-leaving decision rule is fitted are discussed. This is the first study describing an incremental effect of mating on patch residence time in males, thus suggesting that similar information use are probably driving different organisms foraging for resource, regardless of its nature.

Size-induced reproductive constraints in an egg parasitoid.

The size of adult parasitoids is influenced by the quantity and quality of resources available during immature development. Gregarious development of endoparasitoids results in scramble competition where the resources are shared among individuals developing into the same host. Individuals that developed gregariously are therefore smaller and that reduced size generally translates into lower fitness due to reductions in several life history traits including longevity, mobility and traits linked to reproduction. We measured the reproductive constraints induced by size in Anaphes listronoti (Hymenoptera: Mymaridae), a facultative gregarious egg parasitoid of Curculionidae. Size decreased with number of immatures developing in a host egg for both male and female A. listronoti. This reduction in size induced a reproductive cost as both males and females produced fewer gametes when developing gregariously. Contrarily to other egg parasitoids, A. listronoti males are not prospermatogenic and produced some sperm during their adult life. Their spermatogeny index is estimated at 0.6-0.7 that places this species as moderately synspermatogenic. Female A. listronoti have an ovigeny index of 0.70 and are therefore moderately synovigenic. Large females that developed singly received a full sperm complement only when mated by a large male that also developed singly. When mating with a small triplet male, a large female received less than half her sperm complement. Large males were able to mate three females before the number of sperm transferred started to decrease.

Mosquito feeding affects larval behaviour and development in a moth.

Organisms are attacked by different natural enemies present in their habitat. While enemies such as parasitoids and predators will kill their hosts/preys when they successfully attack them, enemies such as micropredators will not entirely consume their prey. However, they can still have important consequences on the performance and ecology of the prey, such as reduced growth, increased emigration, disease transmission. In this paper, we investigated the impact of a terrestrial micropredator, the yellow fever mosquito Aedes aegypti, on its unusual invertebrate host, the Egyptian cotton leaf worm, Spodoptera littoralis. Larvae developing in presence of mosquitoes showed a slower development and reached a smaller pupal weight when compared to a control without mosquitoes, apparently because of a reduced feeding time for larvae. In addition, larvae tended to leave the plant in presence of mosquitoes.These results suggest that mosquitoes act as micropredators and affects lepidopteran larvae behaviour and development. Ecological impacts such as higher risks of food depletion and longer exposure to natural enemies are likely to be costly consequences. The importance of this phenomenon in nature - the possible function as last resort when vertebrates are unavailable - and the evolutionary aspects are discussed.

Phenotypic plasticity in the reproductive traits of a parasitoid.

Organisms show phenotypic plasticity--the capacity for a given genotype to express different phenotypes--in response to changes in the environment. Among the several factors that can cause phenotypic plasticity, nutritional constraints during development can affect the size of organisms and, consequently, affect most life-history traits, including reproductive traits. As their larvae are restricted by the amount of food contained in their host, parasitoids are a good model to study phenotypic plasticity related to size. The phenotypic plasticity of reproductive traits was investigated in the egg parasitoid Trichogramma euproctidis (Hymenoptera: Trichogrammatidae) by using host species of different sizes. Adult size, sperm storage organs (seminal vesicles and spermatheca), number of sperm stored and gamete size (sperm and oocyte) are all influenced by the host species; larger individuals have larger organs which contain more sperm, and both sperm and oocytes are correlated with adult size. However, while females become larger than males and mature larger oocytes in larger hosts, increase in sperm length stops after a given threshold.

Peripheral modulation of olfaction by physiological state in the Egyptian leaf worm Spodoptera littoralis (Lepidoptera: Noctuidae).

Insects show behavioural plasticity based on their physiological state. Deprivation from a resource will normally make them more responsive to it or to perform behaviour increasing the probability of encountering such a resource. Modulation of the olfactory system has been shown mainly in the central nervous system, but also in the periphery. In this study, antennal sensitivity of females of the Egyptian cotton leaf worm Spodoptera littoralis to different plant and sex pheromone odours was measured using electroantennography (EAG). Different mating status, age, and feeding status were used to investigate peripheral changes in olfactory responses. Virgin females were found to be more sensitive to plant odours and sex pheromone compared to mated females. Age also had an impact on antennal sensitivity, 2 and 4 days old females being more sensitive than just-emerged ones. Such an increase in sensitivity could be explained by maturation of olfactory receptor neurons after emergence or by an increase in motivation as the available expected life time remaining decreases. Finally, feeding status did not modify the antennal sensitivity of females. A peripheral modulation could thus be demonstrated in the olfactory system of female Spodoptera littoralis at certain life stages.

Protein kinase CK2 and cell polarity.

There is increasing evidence that protein kinase CK2 is involved, among a wide variety of cellular processes, in the maintenance of mammalian cell morphology and cell polarity. Here, we show that in epithelial cells, a fraction of CK2 is associated to the plasma membrane and that this localization is controlled by cell-matrix interactions. In addition, inhibition of CK2 activity in mammary epithelial cells (MCF10A), using either the specific CK2 inhibitor TBB or siRNA-mediated CK2beta knockdown, induced differential phenotypes revealing an important role of this enzyme in epithelial cell morphology.

Highlighting protein kinase CK2 movement in living cells.

Protein kinase CK2 has traditionally been described as a stable heterotetrameric complex (alpha2beta2) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol 23: 975-987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio-temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell.