A Cell Cycle-Regulated \u3cem\u3eToxoplasma\u3c/em\u3e Deubiquitinase, TgOTUD3A, Targets Polyubiquitins with Specific Lysine Linkages

The contribution of ubiquitin-mediated mechanisms in the regulation of the Toxoplasma gondii cell cycle has remained largely unexplored. Here, we describe the functional characterization of a T. gondii deubiquitinase (TGGT1_258780) of the ovarian-tumor domain-containing (OTU) family, which, based on its structural homology to the human OTUD3 clade, has been designated TgOTUD3A. The TgOTUD3A protein is expressed in a cell cycle-dependent manner mimicking its mRNA expression, indicating that it is regulated primarily at the transcriptional level. TgOTUD3A, which was found in the cytoplasm at low levels in G1 parasites, increased in abundance with the progression of the cell cycle and exhibited partial localization to the developing daughter scaffolds during cytokinesis. Recombinant TgOTUD3A but not a catalytic-site mutant TgOTUD3A (C229A) exhibited activity against poly- but not monoubiquitinated targets. This activity was selective for polyubiquitin chains with preference for specific lysine linkages (K48 \u3e K11 \u3e K63). All three of these polyubiquitin linkage modifications were found to be present in Toxoplasma, where they exhibited differential levels and localization patterns in a cell cycle-dependent manner. TgOTUD3A removed ubiquitin from the K48- but not the K63-linked ubiquitinated T. gondii proteins independently of the modified target protein, thereby exhibiting the characteristics of an exodeubiquitinase. In addition to cell cycle association, the demonstration of multiple ubiquitin linkages together with the selective deubiquitinase activity of TgOTUD3A reveals an unappreciated level of complexity in the T. gondii “ubiquitin code.

Novel Approaches Reveal that \u3cem\u3eToxoplasma gondii\u3c/em\u3e Bradyzoites within Tissue Cysts Are Dynamic and Replicating Entities \u3cem\u3eIn Vivo\u3c/em\u3e

Despite their critical role in chronic toxoplasmosis, the biology of Toxoplasma gondii bradyzoites is poorly understood. In an attempt to address this gap, we optimized approaches to purify tissue cysts and analyzed the replicative potential of bradyzoites within these cysts. In order to quantify individual bradyzoites within tissue cysts, we have developed imaging software, BradyCount 1.0, that allows the rapid establishment of bradyzoite burdens within imaged optical sections of purified tissue cysts. While in general larger tissue cysts contain more bradyzoites, their relative occupancy was typically lower than that of smaller cysts, resulting in a lower packing density. The packing density permits a direct measure of how bradyzoites develop within cysts, allowing for comparisons across progression of the chronic phase. In order to capture bradyzoite endodyogeny, we exploited the differential intensity of TgIMC3, an inner membrane complex protein that intensely labels newly formed/forming daughters within bradyzoites and decays over time in the absence of further division. To our surprise, we were able to capture not only sporadic and asynchronous division but also synchronous replication of all bradyzoites within mature tissue cysts. Furthermore, the time-dependent decay of TgIMC3 intensity was exploited to gain insights into the temporal patterns of bradyzoite replication in vivo. Despite the fact that bradyzoites are considered replicatively dormant, we find evidence for cyclical, episodic bradyzoite growth within tissue cysts in vivo. These findings directly challenge the prevailing notion of bradyzoites as dormant nonreplicative entities in chronic toxoplasmosis and have implications on our understanding of this enigmatic and clinically important life cycle stage. IMPORTANCE: The protozoan Toxoplasma gondii establishes a lifelong chronic infection mediated by the bradyzoite form of the parasite within tissue cysts. Technical challenges have limited even the most basic studies on bradyzoites and the tissue cysts in vivo. Bradyzoites, which are viewed as dormant, poorly replicating or nonreplicating entities, were found to be surprisingly active, exhibiting not only the capacity for growth but also previously unrecognized patterns of replication that point to their being considerably more dynamic than previously imagined. These newly revealed properties force us to reexamine the most basic questions regarding bradyzoite biology and the progression of the chronic phase of toxoplasmosis. By developing new tools and approaches to study the chronic phase at the level of bradyzoites, we expose new avenues to tackle both drug development and a better understanding of events that may lead to reactivated symptomatic disease

Reexamining Chronic \u3cem\u3eToxoplasma gondii\u3c/em\u3e Infection: Surprising Activity for a Dormant Parasite

Purpose of Review Despite over a third of the world’s population being chronically infected with Toxoplasma gondii, little is known about this largely asymptomatic phase of infection. This stage is mediated in vivo by bradyzoites within tissue cysts. The absence of overt symptoms has been attributed to the dormancy of bradyzoites. In this review, we reexamine the conventional view of chronic toxoplasmosis in light of emerging evidence challenging both the nature of dormancy and the consequences of infection in the CNS. Recent Findings New and emerging data reveal a previously unrecognized level of physiological and replicative capacity of bradyzoites within tissue cysts. These findings have emerged in the context of a reexamination of the chronic infection in the brain that correlates with changes in neuronal architecture, neurochemistry, and behavior that suggest that the chronic infection is not without consequence. Summary The emerging data driven by the development of new approaches to study the progression of chronic toxoplasma infection reveals significant physiological and replicative capacity for what has been viewed as a dormant state. The emergence of bradyzoite and tissue cyst biology from what was viewed as a physiological “black box” offers exciting new areas for investigation with direct implications on the approaches to drug development targeting this drug-refractory state. In addition, new insights from studies on the neurobiology on chronic infection reveal a complex and dynamic interplay between the parasite, brain microenvironment, and the immune response that results in the detente that promotes the life-long persistence of the parasite in the host

Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in \u3cem\u3eToxoplasma gondii\u3c/em\u3e

The Toxoplasma genome encodes the capacity for distinct architectures underlying cell cycle progression in a life cycle stage-dependent manner. Replication in intermediate hosts occurs by endodyogeny, whereas a hybrid of schizogony and endopolygeny occurs in the gut of the definitive feline host. Here, we characterize the consequence of the loss of a cell cycle-regulated ovarian tumor (OTU family) deubiquitinase, OTUD3A of Toxoplasma gondii (TgOTUD3A; TGGT1_258780), in T. gondii tachyzoites. Rather than the mutation being detrimental, mutant parasites exhibited a fitness advantage, outcompeting the wild type. This phenotype was due to roughly one-third of TgOTUD3A-knockout (TgOTUD3A-KO) tachyzoites exhibiting deviations from endodyogeny by employing replication strategies that produced 3, 4, or 5 viable progeny within a gravid mother instead of the usual 2. We established the mechanistic basis underlying these altered replication strategies to be a dysregulation of centrosome duplication, causing a transient loss of stoichiometry between the inner and outer cores that resulted in a failure to terminate S phase at the attainment of 2N ploidy and/or the decoupling of mitosis and cytokinesis. The resulting dysregulation manifested as deviations in the normal transitions from S phase to mitosis (S/M) (endopolygeny-like) or M phase to cytokinesis (M/C) (schizogony-like). Notably, these imbalances are corrected prior to cytokinesis, resulting in the generation of normal progeny. Our findings suggest that decisions regarding the utilization of specific cell cycle architectures are controlled by a ubiquitin-mediated mechanism that is dependent on the absolute threshold levels of an as-yet-unknown target(s). Analysis of the TgOTUD3A-KO mutant provides new insights into mechanisms underlying the plasticity of apicomplexan cell cycle architecture

In Situ Silver Nanowire Deposited Cross-Linked Carboxymethyl Cellulose: A Potential Transdermal Anticancer Drug Carrier

Recently, a novel biopolymeric nanocomposite hydrogel comprised of in situ formed silver nanowires (AgNWs) deposited chemically cross-linked carboxymethyl cellulose (CMC) has been developed, which demonstrates superior efficacy as anticancer drug-curcumin carrier. The cross-linked polymer has been prepared by grafting poly [2-(methacryloyloxy) ethyl trimethylammonium chloride] on CMC using diethylene glycol dimethacrylate cross-linker. The nanocomposite hydrogel has the capability to encapsulate both hydrophobic/hydrophilic transdermal drugs. With variation in reaction conditions/parameters, several composite materials have been synthesized and depending on lower swelling/higher cross-linking and greater gel strength, an optimized grade of nanocomposite hydrogel has been selected. The developed nanocomposite hydrogel is characterized with FTIR/NMR spectra, FESEM/XRD/TGA/AFM/XPS analyses, and UV–visible spectroscopy. Rheological study has been performed to enlighten the gel strength of the composite material. The synthesized nanocomposite hydrogel is biodegradable and nontoxic to mesenchymal stem cells (hMSCs). In vitro release of curcumin suggests that in situ incorporation of AgNWs on cross-linked CMC enhanced the penetration power of nanocomposite hydrogel and released the drug in sustained way (∼62% for curcumin released in 4 days). Ex vivo rat skin permeation study confirms that the drug from both the cross-linked and nanocomposite hydrogel was permeable through the rat skin in controlled fashion. Additionally the curcumin loaded composite hydrogel can efficiently kill the MG 63 cancer cells, which has been confirmed by apoptosis study and therefore, probably be a suitable carrier for curcumin delivery toward cancer cells

Stimulus-Responsive, Biodegradable, Biocompatible, Covalently Cross-Linked Hydrogel Based on Dextrin and Poly(<i>N</i>‑isopropylacrylamide) for in Vitro/in Vivo Controlled Drug Release

A novel stimulus-sensitive covalently cross-linked hydrogel derived from dextrin, <i>N</i>-isopropylacrylamide, and <i>N</i>,<i>N</i>′-methylene bis­(acrylamide) (c-Dxt/pNIPAm), has been synthesized via Michael type addition reaction for controlled drug release application. The chemical structure of c-Dxt/pNIPAm has been confirmed through Fourier transform infrared (FTIR) spectroscopy and ¹H and ¹³C NMR spectral analyses. The surface morphology of the hydrogel has been studied by field emission scanning electron microscopic (FE-SEM) and environmental scanning electron microscopic (E-SEM) analyses. The stimulus responsiveness of the hydrogel was studied through equilibrium swelling in various pH media at 25 and 37 °C. Rheological study was performed to measure the gel strength and gelation time. Noncytotoxicity of c-Dxt/pNIPAm hydrogel has been studied using human mesenchymal stem cells (hMSCs). The biodegradability of c-Dxt/pNIPAm was confirmed using hen egg lysozyme. The in vitro and in vivo release studies of ornidazole and ciprofloxacin imply that c-Dxt/pNIPAm delivers both drugs in a controlled way and would be an excellent alternative for a dual drug carrier. The FTIR, powder X-ray diffraction (XRD), and UV–vis–near infrared (NIR) spectra along with the computational study predict that the drugs remain in the matrix through physical interaction. A stability study signifies that the drugs (ornidazole ∼97% and ciprofloxacin ∼98%) are stable in the tablet formulations for up to 3 months

Ovary ecdysteroidogenic hormone functions independently of the insulin receptor in the yellow fever mosquito, Aedes aegypti

Most mosquito species must feed on the blood of a vertebrate host to produce eggs. In the yellow fever mosquito, Aedes aegypti, blood feeding triggers medial neurosecretory cells in the brain to release insulin-like peptides (ILPs) and ovary ecdysteroidogenic hormone (OEH). Theses hormones thereafter directly induce the ovaries to produce ecdysteroid hormone (ECD), which activates the synthesis of yolk proteins in the fat body for uptake by oocytes. ILP3 stimulates ECD production by binding to the mosquito insulin receptor (MIR). In contrast, little is known about the mode of action of OEH, which is a member of a neuropeptide family called neuroparsin. Here we report that OEH is the only neuroparsin family member present in the Ae. aegypti genome and that other mosquitoes also encode only one neuroparsin gene. Immunoblotting experiments suggested that the full-length form of the peptide, which we call long OEH (lOEH), is processed into short OEH (sOEH). The importance of processing, however, remained unclear because a recombinant form of lOEH (rlOEH) and synthetic sOEH exhibited very similar biological activity. A series of experiments indicated that neither rlOEH nor sOEH bound to ILP3 or the MIR. Signaling studies further showed that ILP3 activated the MIR but rlOEH did not, yet both neuropeptides activated Akt, which is a marker for insulin pathway signaling. Our results also indicated that activation of TOR signaling in the ovaries required co-stimulation by amino acids and either ILP3 or rlOEH. Overall, we conclude that OEH activates the insulin signaling pathway independently of the MIR, and that insulin and TOR signaling in the ovaries is coupled