In our previous work, including analysis of more than 10,000 sera from control patients and patients with a variety of liver diseases, we have demonstrated that with the use of recombinant autoantigens, antimitochondrial autoantibodies (AMA) are only found in PBC and that a positive AMA is virtually pathognomonic of either PBC or future development of PBC. Although the mechanisms leading to the generation of AMA are enigmatic, we have postulated that xenobiotic-induced and/or oxidative modification of mitochondrial autoantigens is a critical step leading to loss of tolerance. This thesis suggests that a severe liver oxidant injury would lead to AMA production. We analyzed 217 serum samples from 69 patients with acute liver failure (ALF) collected up to 24 months post-ALF, compared with controls, for titer and reactivity with the E2 subunits of pyruvate dehydrogenase (PDC-E2), branched chain 2-oxo-acid dehydrogenase (BCOADC-E2) and 2-oxo-glutarate dehydrogenase (OGDC-E2). AMA were detected in 28/69 (40.6%) ALF patients with reactivity found against all of the major mitochondrial autoantigens. In addition, and as further controls, sera were also analyzed for autoantibodies to gp210, Sp100, centromere, chromatin, soluble liver antigen (SLA), tissue transglutaminase (tTG) and deaminated gliadin peptides (DGP) where the most frequently detected non-mitochondrial autoantibody was against tTG (57.1% of ALF patients). In conclusion, the strikingly high frequency of AMA in ALF supports the thesis that oxidative stress-induced liver damage may lead to AMA induction. The rapid disappearance of AMA in these patients provides further support for the contention that PBC pathogenesis requires additional factors including genetic susceptibility.
Tolerance; Oxidative injury; Self-reactivity; Epitopes
Recombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids. We have previously adapted this technology to generate, directly from fosmid-based genomic clones, fusion gene reporter constructs designed to investigate gene expression patterns in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection cassette (RT-cassette) is first inserted and then, under negative selection, seamlessly replaced with the desired sequence. We report here on the generation and application of a resource comprising two sets of constructs designed to facilitate this particular recombineering approach.
Two complementary sets of constructs were generated. The first contains different fluorescent protein reporter coding sequences and derivatives while the second set of constructs, based in the copy-number inducible vector pCC1Fos, provide a resource designed to simplify RT-cassette-based recombineering. These latter constructs are used in pairs the first member of which provides a template for PCR-amplification of an RT-cassette while the second provides, as an excised restriction fragment, the desired fluorescent protein reporter sequence. As the RT-cassette is flanked by approximately 200 bp from the ends of the reporter sequence the subsequent negative selection replacement step is highly efficient. Furthermore, use of a restriction fragment minimizes artefacts negating the need for final clone sequencing. Utilizing this resource we generated single-, double- and triple-tagged fosmid-based reporters to investigate expression patterns of three C. elegans genes located on a single genomic clone.
We describe the generation and application of a resource designed to facilitate counter-selection recombineering of fosmid-based C. elegans genomic clones. By choosing the appropriate pair of ‘insertion’ and ‘replacement’ constructs recombineered products, devoid of artefacts, are generated at high efficiency. Gene expression patterns for three genes located on the same genomic clone were investigated via a set of fosmid-based reporter constructs generated with the modified protocol.
C. elegans; Recombineering; Fosmid; Fluorescent protein; Deoxyribose-phosphate aldolase; Peroxiredoxin; Metallocarboxypeptidase
As an intracellular protozoan parasite, Toxoplasma gondii is likely to exploit proteases for host cell invasion, acquisition of nutrients, avoidance of host protective responses, escape from the parasitophorous vacuole, differentiation, and other activities. T. gondii serine protease inhibitor 1 (TgPI1) is the most abundantly expressed protease inhibitor in parasite tachyzoites. We show here that alternative splicing produces two TgPI1 isoforms, both of which are secreted via dense granules into the parasitophorous vacuole shortly after invasion, become progressively more abundant over the course of the infectious cycle, and can be detected in the infected host cell cytoplasm. To investigate TgPI1 function, the endogenous genomic locus was disrupted in the RH strain background. ΔTgPI1 parasites replicate normally as tachyzoites but exhibit increased bradyzoite gene transcription and labeling of vacuoles with Dolichos biflorus lectin under conditions promoting in vitro differentiation. The differentiation phenotype can be partially complemented by either TgPI1 isoform. Mice infected with the ΔTgPI1 mutant display ∼3-fold-increased parasite burden in the spleen and liver, and this in vivo phenotype is also complemented by either TgPI1 isoform. These results demonstrate that TgPI1 influences both parasite virulence and bradyzoite differentiation, presumably by inhibiting parasite and/or host serine proteases.
Greater use of computerized decision support (DS) systems could address continuing safety and quality problems in healthcare, but the healthcare field has struggled to implement DS technology. This study surveys DS experience across multiple non-healthcare disciplines for new insights that are generalizable to healthcare provider decisions. In particular, it sought design principles and lessons learned from the other disciplines that could inform efforts to accelerate the adoption of clinical decision support (CDS).
Our systematic review drew broadly from non-healthcare databases in the basic sciences, social sciences, humanities, engineering, business, and defense: PsychINFO, BusinessSource Premier, Social Sciences Abstracts, Web of Science, and Defense Technical Information Center. Because our interest was in DS that could apply to clinical decisions, we selected articles that (1) provided a review, overview, discussion of lessons learned, or an evaluation of design or implementation aspects of DS within a non-healthcare discipline and (2) involved an element of human judgment at the individual level, as opposed to decisions that can be fully automated or that are made at the organizational level.
Clinical decisions share some similarities with decisions made by military commanders, business managers, and other leaders: they involve assessing new situations and choosing courses of action with major consequences, under time pressure, and with incomplete information. We identified seven high-level DS system design features from the non-healthcare literature that could be applied to CDS: providing broad, system-level perspectives; customizing interfaces to specific users and roles; making the DS reasoning transparent; presenting data effectively; generating multiple scenarios covering disparate outcomes (e.g., effective; effective with side effects; ineffective); allowing for contingent adaptations; and facilitating collaboration. The article provides examples of each feature. The DS literature also emphasizes the importance of organizational culture and training in implementation success. The literature contrasts “rational-analytic” vs. “naturalistic-intuitive” decision-making styles, but the best approach is often a balanced approach that combines both styles. It is also important for DS systems to enable exploration of multiple assumptions, and incorporation of new information in response to changing circumstances.
Complex, high-level decision-making has common features across disciplines as seemingly disparate as defense, business, and healthcare. National efforts to advance the health information technology agenda through broader CDS adoption could benefit by applying the DS principles identified in this review.
Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii (the causative agents of malaria and toxoplasmosis, respectively), are responsible for considerable morbidity and mortality worldwide. These pathogenic protozoa replicate within an intracellular vacuole inside of infected host cells, from which they must escape to initiate a new lytic cycle. By integrating cell biological, pharmacological, and genetic approaches, we provide evidence that both Plasmodium and Toxoplasma hijack host cell calpain proteases to facilitate parasite egress. Immunodepletion or inhibition of calpain-1 in hypotonically lysed and resealed erythrocytes prevented the escape of P. falciparum parasites, which was restored by adding purified calpain-1. Similarly, efficient egress of T. gondii from mammalian fibroblasts was blocked by either small interfering RNA–mediated suppression or genetic deletion of calpain activity and could be restored by genetic complementation.
Recent biochemical studies have identified a cell surface receptor for thyroid and steroid hormones that bind near the arginine-glycine-aspartate (RGD) recognition site on the heterodimeric αvβ3 integrin. To further characterize the intermolecular interactions for a series of hormone analogues, combined quantum mechanical and molecular mechanical (QM/MM) methods were used to calculate their interaction energies. All calculations were performed in the presence of either calcium (Ca2+) or magnesium (Mg2+) ions. These data reveal that 3,5′-triiodothyronine (T3) and 3,5,3′,5′-tetraiodothyroacetic acid (T4ac) bound in two different modes, occupying two alternate sites, one of which is along the Arg side chain of the RGD cyclic peptide site. These orientations differ from those of the other ligands whose alternate binding modes placed the ligands deeper within the RGD binding pocket. These observations are consistent with biological data that indicate the presence of two discrete binding sites that control distinct downstream signal transduction pathways for T3.
Toxoplasma gondii is a prevalent obligate intracellular parasite which chronically infects more than a third of the world's population. Key to parasite prevalence is its ability to form chronic and nonimmunogenic bradyzoite cysts, which typically form in the brain and muscle cells of infected mammals, including humans. While acute clinical infection typically involves neurological and/or ocular damage, chronic infection has been more recently linked to behavioral changes. Establishment and maintenance of chronic infection involves a balance between the host immunity and parasite evasion of the immune response. Here, we outline the known cellular interplay between Toxoplasma gondii and cells of the central nervous system and review the reported effects of Toxoplasma gondii on behavior and neurological disease. Finally, we review new technologies which will allow us to more fully understand host-pathogen interactions.
Toxoplasmosis is characterized by fast lytic replication cycles leading to severe tissue lesions. Successful host cell invasion is essential for pathogenesis. The division cycle of Toxoplasma gondii is characterized by an unusual cell cycle progression and a distinct internal budding mechanism. To identify essential genes involved in the lytic cycle we devised an insertional gene trapping strategy using the Tet-transactivator system. In essence, a random, active promoter is displaced with a tetracycline regulatable promoter, which if in an essential gene, will result in a conditionally lethal phenotype upon tetracycline addition. We isolated eight mutants with growth defects, two of which displayed modest invasion defects, one of which had an additional cell cycle defect. The trapped loci were identified using expression microarrays, exploiting the tetracycline dependent expression of the trapped genes. In mutant 3.3H6 we identified TCP-1, a component of the chaperonin protein folding machinery under the control of the Tet promoter. However, this gene was not critical for growth of mutant 3.3H6. Subsequently, we identified a suppressor gene encoding a protein with a hypothetical function by guided cosmid complementation. In mutant 4.3B13, we identified TAF250, an RNA polymerase II complex component, as the trapped, essential gene. Furthermore, by mapping the plasmid insertion boundaries we identified multiple genomic rearrangements, which hint at a potential replication dependent DNA repair mechanism. Furthermore, these rearrangements provide an explanation for inconsistent locus rescue results observed by molecular biological approaches. Taken together, we have added an approach to identify and study essential genes in Toxoplasma.
Toxoplasma gondii; invasion; cell cycle; TAF250; chaperonin; genetic screen
Curation of information from bioscience literature into biological knowledge databases is a crucial way of capturing experimental information in a computable form. During the biocuration process, a critical first step is to identify from all published literature the papers that contain results for a specific data type the curator is interested in annotating. This step normally requires curators to manually examine many papers to ascertain which few contain information of interest and thus, is usually time consuming. We developed an automatic method for identifying papers containing these curation data types among a large pool of published scientific papers based on the machine learning method Support Vector Machine (SVM). This classification system is completely automatic and can be readily applied to diverse experimental data types. It has been in use in production for automatic categorization of 10 different experimental datatypes in the biocuration process at WormBase for the past two years and it is in the process of being adopted in the biocuration process at FlyBase and the Saccharomyces Genome Database (SGD). We anticipate that this method can be readily adopted by various databases in the biocuration community and thereby greatly reducing time spent on an otherwise laborious and demanding task. We also developed a simple, readily automated procedure to utilize training papers of similar data types from different bodies of literature such as C. elegans and D. melanogaster to identify papers with any of these data types for a single database. This approach has great significance because for some data types, especially those of low occurrence, a single corpus often does not have enough training papers to achieve satisfactory performance.
We successfully tested the method on ten data types from WormBase, fifteen data types from FlyBase and three data types from Mouse Genomics Informatics (MGI). It is being used in the curation work flow at WormBase for automatic association of newly published papers with ten data types including RNAi, antibody, phenotype, gene regulation, mutant allele sequence, gene expression, gene product interaction, overexpression phenotype, gene interaction, and gene structure correction.
Our methods are applicable to a variety of data types with training set containing several hundreds to a few thousand documents. It is completely automatic and, thus can be readily incorporated to different workflow at different literature-based databases. We believe that the work presented here can contribute greatly to the tremendous task of automating the important yet labor-intensive biocuration effort.
WormBase (www.wormbase.org) has been serving the scientific community for over 11 years as the central repository for genomic and genetic information for the soil nematode Caenorhabditis elegans. The resource has evolved from its beginnings as a database housing the genomic sequence and genetic and physical maps of a single species, and now represents the breadth and diversity of nematode research, currently serving genome sequence and annotation for around 20 nematodes. In this article, we focus on WormBase’s role of genome sequence annotation, describing how we annotate and integrate data from a growing collection of nematode species and strains. We also review our approaches to sequence curation, and discuss the impact on annotation quality of large functional genomics projects such as modENCODE.
Caenorhabditis elegans; annotation; community resource; genome; model organism database; nematode; parasitic nematode; sequence curation
A cell surface receptor for thyroid hormone that activates extracellular regulated kinase (ERK) 1/2 has been identified on integrin αvβ3. We have examined the actions of thyroid hormone initiated at the integrin on human NCI-H522 non-small cell lung carcinoma and NCI-H510A small cell lung cancer cells. At a physiologic total hormone concentration (10−7 M), T4 significantly increased proliferating cell nuclear antigen (PCNA) abundance in these cell lines, as did 3, 5, 3′-triiodo-L-thyronine (T3) at a supraphysiologic concentration. Neutralizing antibody to integrin αvβ3 and an integrin-binding Arg-Gly-Asp (RGD) peptide blocked thyroid hormone-induced PCNA expression. Tetraiodothyroacetic acid (tetrac) lacks thyroid hormone function but inhibits binding of T4 and T3 to the integrin receptor; tetrac eliminated thyroid hormone-induced lung cancer cell proliferation and ERK1/2 activation. In these estrogen receptor-α (ERα)-positive lung cancer cells, thyroid hormone (T4>T3) caused phosphorylation of ERα; the specific ERα antagonist ICI 182,780 blocked T4-induced, but not T3-induced ERK1/2 activation, as well as ERα phosphorylation, proliferating-cell nuclear antigen (PCNA) expression and hormone-dependent thymidine uptake by tumor cells. Thus, in ERα-positive human lung cancer cells, the proliferative action of thyroid hormone initiated at the plasma membrane is at least in part mediated by ERα. In summary, thyroid hormone may be one of several endogenous factors capable of supporting proliferation of lung cancer cells. Activity as an inhibitor of lung cancer cell proliferation induced at the integrin receptor makes tetrac a novel anti-proliferative agent.
Since its release in 2000, WormBase (http://www.wormbase.org) has grown from a small resource focusing on a single species and serving a dedicated research community, to one now spanning 15 species essential to the broader biomedical and agricultural research fields. To enhance the rate of curation, we have automated the identification of key data in the scientific literature and use similar methodology for data extraction. To ease access to the data, we are collaborating with journals to link entities in research publications to their report pages at WormBase. To facilitate discovery, we have added new views of the data, integrated large-scale datasets and expanded descriptions of models for human disease. Finally, we have introduced a dramatic overhaul of the WormBase website for public beta testing. Designed to balance complexity and usability, the new site is species-agnostic, highly customizable, and interactive. Casual users and developers alike will be able to leverage the public RESTful application programming interface (API) to generate custom data mining solutions and extensions to the site. We report on the growth of our database and on our work in keeping pace with the growing demand for data, efforts to anticipate the requirements of users and new collaborations with the larger science community.
Apicomplexan parasites release factors via specialized secretory organelles (rhoptries, micronemes) that are thought to control host cell responses. In order to explore parasite-mediated modulation of host cell signaling pathways, we exploited a phylogenomic approach to characterize the Toxoplasma gondii kinome, defining a 44 member family of coccidian-specific secreted kinases, some of which have been previously implicated in virulence. Comparative genomic analysis suggests that ‘ROPK’ genes are under positive selection, and expression profiling demonstrates that most are differentially expressed between strains and/or during differentiation. Integrating diverse genomic-scale analyses points to ROP38 as likely to be particularly important in parasite biology. Upregulating expression of this previously uncharacterized gene in transgenic parasites dramatically suppresses transcriptional responses in the infected cell. Specifically, parasite ROP38 down-regulates host genes associated with MAPK signaling and the control of apoptosis and proliferation. These results highlight the value of integrative genomic approaches in prioritizing candidates for functional validation.
Tai Chi Chuan (TCC) is an integrative medicine mind-body practice with a physical activity component that has positive effects on aerobic capacity, muscular strength, and quality of life among cancer survivors, similar to the effects elicited by other modes of moderate intensity exercise. Inflammatory cytokines, and insulin and insulin-related signaling molecules may contribute to weight gain and affect cancer recurrence rates and survival; exercise can curb cancer- and treatment-related weight gain, increase survival, and reduce levels of insulin and inflammatory cytokines. Despite knowing the beneficial effects of conventional exercise interventions on these mediators, little is known about the physiologic effects of TCC, a mind-body practice with a physical activity component, on these pathways in breast cancer survivors.
We assessed the effects of a 12-week, moderately intense, TCC intervention (n=9) compared to a non-physical activity control (n=10) consisting of psychosocial support therapy (PST) on levels of insulin, IGF-1, IGFBP-1, IGFBP-3 and cytokines IL-6, IL-2, and IFN-γ in breast cancer survivors.
Levels of insulin are significantly different in TCC and PST groups; levels remained stable in the TCC group, but increased in the PST control group (p=0.099). Bivariate analysis revealed novel and significant correlations (all r >0.45, all p≤0.05) of both decreased fat mass and increased fat-free mass with increased IL-6 and decreased IL-2 levels.
This pilot study shows that TCC may be associated with maintenance of insulin levels and changes in cytokine levels that may be important for maintenance of lean body mass in breast cancer survivors.
Tai Chi Chuan; survivorship; weight gain; insulin; cytokines; biomarkers
The E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2) is the immunodominant autoantigen of primary biliary cirrhosis. Whereas lipoylation of PDC-E2 is essential for enzymatic activity and predominates under normal conditions, other biochemical systems exist that also target the lysine residue, including acylation of fatty acids or xenobiotics and ubiquitinylation. More importantly, the immunogenicity can be affected by derivatization of the lysine residue, as the recognition of lipoylated PDC-E2 by patient autoantibodies is enhanced compared with octanoylated PDC-E2. Furthermore, our laboratory has shown that various xenobiotic modifications of a peptide representing the immunodominant region of PDC-E2 are immunoreactive against patient sera. The only purported regulatory system that prevents the accumulation of potentially autoreactive PDC-E2 is glutathionylation, in which the lysine-lipoic acid moiety is further modified with glutathione during apoptosis. Interestingly, this system is found in several cell lines, including HeLa, Jurkat, and Caco-2 cells, but not in cholangiocytes and salivary gland epithelial cells, both of which are targets for destruction in primary biliary cirrhosis. Hence, the failure of this or other regulatory system(s) may overwhelm the immune system with immunogenic PDC-E2 that can initiate the breakdown of tolerance in a genetically susceptible individual. In this review the authors survey the data available on the biochemical life of PDC-E2, with particular emphasis on the lysine residue and its known interactions with machinery involved in various posttranslational modifications.
Angiotensin II (Ang II) signaling occurs via two major receptors which activate non-receptor tyrosin kinases that then interact with protein tyrosin-phosphatases (PTPs) to regulate cell function. SHP-2 is one such important PTP that also functions as an adaptor to promote downstream signaling pathway. Its role in Ang II signaling remains to be clarified.
Using cultured normal human fibroblasts, immunoprecipitation and western blots, we show for the first time that SHP-2 and PLCβ1 are present as a preformed complex. Complex PLCβ1 is tyr-phosphorylated basally and Ang II increased SHP-2-PLCβ1 complexes and caused complex associated PLCβ1 tyr-phosphorylation to decline while complex associated SHP-2's tyr-phosphorylation increased and did so via the Ang II type 1 receptors as shown by Ang II type 1 receptor blocker losartan's effects. Moreover, Ang II induced both increased complex phosphatase activity and decreased complex associated PLCβ1 tyr-phosphorylation, the latter response required regulator of G protein signaling (RGS)-2.
Ang II signals are shown for the first time to involve a preformed SHP-2-PLCβ1 complex. Changes in the complex's PLCβ1 tyr-phosphorylation and SHP-2's tyr-phosphorylation as well as SHP-2-PLCβ1 complex formation are the result of Ang II type 1 receptor activation with changes in complex associated PLCβ1 tyr-phosphorylation requiring RGS-2. These findings might significantly expand the number and complexity of Ang II signaling pathways. Further studies are needed to delineate the role/s of this complex in the Ang II signaling system.
Angiotensin II signaling; SHP-2; PLCβ1; SHP-2-PLCβ1 complex
The present study compared the effects of two different music interventions on changes in emotional states before and during running, and also explored effects of music interventions upon performance outcome. Volunteer participants (n = 65) who regularly listened to music when running registered online to participate in a three-stage study. Participants attempted to attain a personally important running goal to establish baseline performance. Thereafter, participants were randomly assigned to either a self-selected music group or an Audiofuel music group. Audiofuel produce pieces of music designed to assist synchronous running. The self-selected music group followed guidelines for selecting motivating playlists. In both experimental groups, participants used the Brunel Music Rating Inventory-2 (BMRI-2) to facilitate selection of motivational music. Participants again completed the BMRI-2 post- intervention to assess the motivational qualities of Audiofuel music or the music they selected for use during the study. Results revealed no significant differences between self-selected music and Audiofuel music on all variables analyzed. Participants in both music groups reported increased pleasant emotions and decreased unpleasant emotions following intervention. Significant performance improvements were demonstrated post-intervention with participants reporting a belief that emotional states related to performance. Further analysis indicated that enhanced performance was significantly greater among participants reporting music to be motivational as indicated by high scores on the BMRI-2. Findings suggest that both individual athletes and practitioners should consider using the BMRI-2 when selecting music for running.
Listening to music with a high motivational quotient as indicated by scores on the BMRI-2 was associated with enhanced running performance and meta-emotional beliefs that emotions experienced during running helped performance.
Beliefs on the effectiveness of music intended to alter emotions were associated with high scores on the BMRI-2.
Runners seeking to use music as an emotion regulating strategy should consider using the BMRI-2 as an effective means by which to identify potentially motivating tracks.
Psychological skills; affect; mood; endurance; performance; meta-emotional beliefs
The time required to initiate clinical trials, from declaration of the investigator’s intent to opening of the study for participant accrual, is cited as often being so long that clinical research is seriously impeded. Efforts to improve operational efficiency of trial initiation are confounded by the work flow complexity and the variations encountered with different types of trials and institutional environments. A computer Protocol Lifecycle Tracking (PLT) tool would enable study initiation staff to manage the process, and the various clinical research stakeholders to monitor the progress of a study’s initiation, as well as obtain data on the work flow to identify those activities that are in need of operational efficiency improvement. The objective of our work was to develop use cases and system requirements for a PLT tool. The result of our study is a use case document that can serve as the specifications for developing a PLT application.
Unmodified or as a poly[lactide-co-glycolide] nanoparticle, tetraiodothyroacetic acid (tetrac) acts at the integrin αvβ3 receptor on human cancer cells to inhibit tumor cell proliferation and xenograft growth. To study in vitro the pharmacodynamics of tetrac formulations in the absence of and in conjunction with other chemotherapeutic agents, we developed a perfusion bellows cell culture system. Cells were grown on polymer flakes and exposed to various concentrations of tetrac, nano-tetrac, resveratrol, cetuximab, or a combination for up to 18 days. Cells were harvested and counted every one or two days. Both NONMEM VI and the exact Monte Carlo parametric expectation maximization algorithm in S-ADAPT were utilized for mathematical modeling. Unmodified tetrac inhibited the proliferation of cancer cells and did so with differing potency in different cell lines. The developed mechanism-based model included two effects of tetrac on different parts of the cell cycle which could be distinguished. For human breast cancer cells, modeling suggested a higher sensitivity (lower IC50) to the effect on success rate of replication than the effect on rate of growth, whereas the capacity (Imax) was larger for the effect on growth rate. Nanoparticulate tetrac (nano-tetrac), which does not enter into cells, had a higher potency and a larger anti-proliferative effect than unmodified tetrac. Fluorescence-activated cell sorting analysis of harvested cells revealed tetrac and nano-tetrac induced concentration-dependent apoptosis that was correlated with expression of pro-apoptotic proteins, such as p53, p21, PIG3 and BAD for nano-tetrac, while unmodified tetrac showed a different profile. Approximately additive anti-proliferative effects were found for the combinations of tetrac and resveratrol, tetrac and cetuximab (Erbitux), and nano-tetrac and cetuximab. Our in vitro perfusion cancer cell system together with mathematical modeling successfully described the anti-proliferative effects over time of tetrac and nano-tetrac and may be useful for dose-finding and studying the pharmacodynamics of other chemotherapeutic agents or their combinations.
Clinical treatment protocols for specific solid cancers have favorable response rates of 20%–25%. Cancer cells frequently become resistant to treatment. Therefore, novel anti-cancer drugs and combination regimens need to be developed. Conducting enough clinical trials to evaluate combinations of anti-cancer agents in several regimens to optimize treatment is not feasible. We showed that tetrac inhibits the growth of various cancer cell lines. Our newly developed in vitro system allowed studying the effects of tetrac over time in various human cancer cell lines. Our mathematical model could distinguish two effects of tetrac and may be used to predict effects of other than the studied dosage regimens. Human breast cancer cells were more sensitive to the effect on success of replication than the effect on growth rate, whereas the maximum possible effect was larger for the latter effect. Nanoparticulate tetrac, which does not enter into cells, had a larger effect than unmodified tetrac. The combinations of tetrac and resveratrol, tetrac and cetuximab (Erbitux), and nano-tetrac and cetuximab showed approximately additive effects. Our in vitro perfusion system together with mathematical modeling may be useful for dose-finding, translation from in vitro to animal and human studies, and studying effects of other chemotherapeutic agents or their combinations.
The pathophysiology of brain damage that is common to ischemia–reperfusion injury and brain trauma include disodered neuronal and glial cell energetics, intracellular acidosis, calcium toxicity, extracellular excitotoxic glutamate accumulation, and dysfunction of the cytoskeleton and endoplasmic reticulum. The principal thyroid hormones, 3,5,3′-triiodo-l-thyronine (T3) and l-thyroxine (T4), have non-genomic and genomic actions that are relevant to repair of certain features of the pathophysiology of brain damage. The hormone can non-genomically repair intracellular H+ accumulation by stimulation of the Na+/H+ exchanger and can support desirably low [Ca2+]i.c. by activation of plasma membrane Ca2+–ATPase. Thyroid hormone non-genomically stimulates astrocyte glutamate uptake, an action that protects both glial cells and neurons. The hormone supports the integrity of the microfilament cytoskeleton by its effect on actin. Several proteins linked to thyroid hormone action are also neuroprotective. For example, the hormone stimulates expression of the seladin-1 gene whose gene product is anti-apoptotic and is potentially protective in the setting of neurodegeneration. Transthyretin (TTR) is a serum transport protein for T4 that is important to blood–brain barrier transfer of the hormone and TTR also has been found to be neuroprotective in the setting of ischemia. Finally, the interesting thyronamine derivatives of T4 have been shown to protect against ischemic brain damage through their ability to induce hypothermia in the intact organism. Thus, thyroid hormone or hormone derivatives have experimental promise as neuroprotective agents.
thyroid hormone; thyronamines; sodium-proton exchanger; calcium ATPase; seladin-1; transthyretin; ischemia–reperfusion injury
Malaria caused by Plasmodium falciparum is a catastrophic disease worldwide (880,000 deaths yearly). Vaccine development has proved difficult and resistance has emerged for most antimalarials. In order to discover new antimalarial chemotypes, we have employed a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library, many of which exhibited potent in vitro activity against drug resistant strains, and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in multiple organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Overall, our findings provide the scientific community with new starting points for malaria drug discovery.
The simple nematode, Caenorhabditis elegans, possesses the most extensive known gene family of nicotinic acetylcholine receptor (nAChR)-like subunits. Whilst all show greatest similarity with nAChR subunits of both invertebrates and vertebrates, phylogenetic analysis suggests that just over half of these (32) may represent other members of the cys-loop ligand-gated ion channel superfamily. We have introduced a novel nomenclature system for these ‘Orphan’ subunits, designating them as lgc genes (ligand-gated ion channels of the cys-loop superfamily), which can also be applied in future to unnamed and uncharacterised members of the cys-loop ligand-gated ion channel superfamily. We present here the resulting updated version of the C. elegans nAChR gene family and related ligand-gated ion channel (lgc) genes.
Caenorhabditis elegans; gene family; ion channel; nematode; nicotinic acetylcholine receptor
The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) having independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-dependent PGM (dPGM) requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM) does not. The PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms.
To examine the distribution of PGM NISE throughout the Bacteria, and gain insight into the evolutionary processes that shape their phyletic profiles, we searched bacterial genome sequences for the presence of dPGM and iPGM. Both forms exhibited patchy distributions throughout the bacterial domain. Species within the same genus, or even strains of the same species, frequently differ in their PGM repertoire. The distribution is further complicated by the common occurrence of dPGM paralogs, while iPGM paralogs are rare. Larger genomes are more likely to accommodate PGM paralogs or both NISE forms. Lateral gene transfers have shaped the PGM profiles with intradomain and interdomain transfers apparent. Archaeal-type iPGM was identified in many bacteria, often as the sole PGM. To address the function of PGM NISE in an organism encoding both forms, we analyzed recombinant enzymes from E. coli. Both NISE were active mutases, but the specific activity of dPGM greatly exceeded that of iPGM, which showed highest activity in the presence of manganese. We created PGM null mutants in E. coli and discovered the ΔdPGM mutant grew slowly due to a delay in exiting stationary phase. Overexpression of dPGM or iPGM overcame this defect.
Our biochemical and genetic analyses in E. coli firmly establish dPGM and iPGM as NISE. Metabolic redundancy is indicated since only larger genomes encode both forms. Non-orthologous gene displacement can fully account for the non-uniform PGM distribution we report across the bacterial domain.
Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators.
Taking advantage of available genome sequences and annotation for Toxoplasma gondii (a pathogenic parasite responsible for illness in immunocompromised individuals) and Plasmodium falciparum (a related parasite responsible for severe human malaria), we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP)-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis) and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional T. gondii isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at pilot scale to inform future array designs.
In addition to providing an initial global view of the T. gondii transcriptome across major lineages and permitting detailed resolution of recombination points in a historical sexual cross, the multifunctional nature of this array also allowed opportunities to exploit probes for purposes beyond their intended use, enhancing analyses. This array is in widespread use by the T. gondii research community, and several aspects of the design strategy are likely to be useful for other pathogens.