Toxicogenomic module associations with pathogenesis: a network-based approach to understanding drug toxicity.

Despite investment in toxicogenomics, nonclinical safety studies are still used to predict clinical liabilities for new drug candidates. Network-based approaches for genomic analysis help overcome challenges with whole-genome transcriptional profiling using limited numbers of treatments for phenotypes of interest. Herein, we apply co-expression network analysis to safety assessment using rat liver gene expression data to define 415 modules, exhibiting unique transcriptional control, organized in a visual representation of the transcriptome (the 'TXG-MAP'). Accounting for the overall transcriptional activity resulting from treatment, we explain mechanisms of toxicity and predict distinct toxicity phenotypes using module associations. We demonstrate that early network responses complement traditional histology-based assessment in predicting outcomes for longer studies and identify a novel mechanism of hepatotoxicity involving endoplasmic reticulum stress and Nrf2 activation. Module-based molecular subtypes of cholestatic injury derived using rat translate to human. Moreover, compared to gene-level analysis alone, combining module and gene-level analysis performed in sequence identifies significantly more phenotype-gene associations, including established and novel biomarkers of liver injury.

Overview of worldwide diversity of Diaphorina citri Kuwayama mitochondrial cytochrome oxidase 1 haplotypes: two Old World lineages and a New World invasion.

Relationships among worldwide collections of Diaphorina citri (Asian citrus psyllid) were analyzed using mitochondrial cytochrome oxidase I (mtCOI) haplotypes from novel primers. Sequences were produced from PCR amplicons of an 821bp portion of the mtCOI gene using D. citri specific primers, derived from an existing EST library. An alignment was constructed using 612bps of this fragment and consisted of 212 individuals from 52 collections representing 15 countries. There were a total of eight polymorphic sites that separated the sequences into eight different haplotypes (Dcit-1 through Dcit-8). Phylogenetic network analysis using the statistical parsimony software, TCS, suggests two major haplotype groups with preliminary geographic bias between southwestern Asia (SWA) and southeastern Asia (SEA). The recent (within the last 15 to 25 years) invasion into the New World originated from only the SWA group in the northern hemisphere (USA and Mexico) and from both the SEA and SWA groups in the southern hemisphere (Brazil). In only one case, Reunion Island, did haplotypes from both the SEA and SWA group appear in the same location. In Brazil, both groups were present, but in separate locations. The Dcit-1 SWA haplotype was the most frequently encountered, including ~50% of the countries sampled and 87% of the total sequences obtained from India, Pakistan and Saudi Arabia. The second most frequently encountered haplotype, Dcit-2, the basis of the SEA group, represented ~50% of the countries and contained most of the sequences from Southeast Asia and China. Interestingly, only the Caribbean collections (Puerto Rico and Guadeloupe) represented a unique haplotype not found in other countries, indicating no relationship between the USA (Florida) and Caribbean introductions. There is no evidence for cryptic speciation for D. citri based on the COI region included in this study.

Comparison of laboratory colonies and field populations of Tamarixia radiata, an ectoparasitoid of the Asian citrus psyllid, using internal transcribed spacer and cytochrome oxidase subunit I DNA sequences.

The genetic diversity of Tamarixia radiata Waterston (Hymenoptera: Eulophidae) laboratory colonies derived from collections in China, northern Vietnam, Pakistan, and a mixed colony from Taiwan and southern Vietnam was evaluated using the internal transcribed spacer (ITS) region 1, ITS-2, and the 5' end of the cytochrome oxidase subunit I gene. The strains share the same ITS sequence, consistent with the morphological hypothesis that the collections represent a single species. The COI marker was variable and could distinguish the northern Vietnam and Pakistan colonies from each other and from the other colonies. Comparison of COI sequences from field-collected populations of Puerto Rico, Guadeloupe, and Texas indicates that Florida is not a likely source of the introduction into Puerto Rico but is a likely source of the introduction into Texas.

Proinflammatory consequences of transgenic fas ligand expression in the heart.

Expression of Fas ligand (FasL) renders certain tissues immune privileged, but its expression in other tissues can result in severe neutrophil infiltration and tissue destruction. The consequences of enforced FasL expression in striated muscle is particularly controversial. To create a stable reproducible pattern of cardiomyocyte-specific FasL expression, transgenic (Tg) mice were generated that express murine FasL specifically in the heart, where it is not normally expressed. Tg animals are healthy and indistinguishable from nontransgenic littermates. FasL expression in the heart does result in mild leukocyte infiltration, but despite coexpression of Fas and FasL in Tg hearts, neither myocardial tissue apoptosis nor necrosis accompanies the leukocyte infiltration. Instead of tissue destruction, FasL Tg hearts develop mild interstitial fibrosis, functional changes, and cardiac hypertrophy, with corresponding molecular changes in gene expression. Induced expression of the cytokines TNF-alpha, IL-1beta, IL-6, and TGF-beta accompanies these proinflammatory changes. The histologic, functional, and molecular proinflammatory consequences of cardiac FasL expression are transgene-dose dependent. Thus, coexpression of Fas and FasL in the heart results in leukocyte infiltration and hypertrophy, but without the severe tissue destruction observed in other examples of FasL-directed proinflammation. The data suggest that the FasL expression level and other tissue-specific microenvironmental factors can modulate the proinflammatory consequences of FasL.

Cardiac compartment-specific overexpression of a modified retinoic acid receptor produces dilated cardiomyopathy and congestive heart failure in transgenic mice.

Retinoids play a critical role in cardiac morphogenesis. To examine the effects of excessive retinoid signaling on myocardial development, transgenic mice that overexpress a constitutively active retinoic acid receptor (RAR) controlled by either the alpha- or beta-myosin heavy chain (MyHC) promoter were generated. Animals carrying the alpha-MyHC-RAR transgene expressed RARs in embryonic atria and in adult atria and ventricles, but developed no signs of either malformations or disease. In contrast, beta-MyHC-RAR animals, where expression was activated in fetal ventricles, developed a dilated cardiomyopathy that varied in severity with transgene copy number. Characteristic postmortem lesions included biventricular chamber dilation and left atrial thrombosis; the incidence and severity of these lesions increased with increasing copy number. Transcript analyses showed that molecular markers of hypertrophy, alpha-skeletal actin, atrial natriuretic factor and beta-MyHC, were upregulated. Cardiac performance of transgenic hearts was evaluated using the isolated perfused working heart model as well as in vivo, by transthoracic M-mode echocardiography. Both analyses showed moderate to severe impairment of left ventricular function and reduced cardiac contractility. Thus, expression of a constitutively active RAR in developing atria and/ or in postnatal ventricles is relatively benign, while ventricular expression during gestation can lead to significant cardiac dysfunction.

Protein interaction networks at the host-microbe interface in Diaphorina citri, the insect vector of the citrus greening pathogen.

The Asian citrus psyllid (Diaphorina citri) is the insect vector responsible for the worldwide spread of 'Candidatus Liberibacter asiaticus' (CLas), the bacterial pathogen associated with citrus greening disease. Developmental changes in the insect vector impact pathogen transmission, such that D. citri transmission of CLas is more efficient when bacteria are acquired by nymphs when compared with adults. We hypothesize that expression changes in the D. citri immune system and commensal microbiota occur during development and regulate vector competency. In support of this hypothesis, more proteins, with greater fold changes, were differentially expressed in response to CLas in adults when compared with nymphs, including insect proteins involved in bacterial adhesion and immunity. Compared with nymphs, adult insects had a higher titre of CLas and the bacterial endosymbionts Wolbachia, Profftella and Carsonella. All Wolbachia and Profftella proteins differentially expressed between nymphs and adults are upregulated in adults, while most differentially expressed Carsonella proteins are upregulated in nymphs. Discovery of protein interaction networks has broad applicability to the study of host-microbe relationships. Using protein interaction reporter technology, a D. citri haemocyanin protein highly upregulated in response to CLas was found to physically interact with the CLas coenzyme A (CoA) biosynthesis enzyme phosphopantothenoylcysteine synthetase/decarboxylase. CLas pantothenate kinase, which catalyses the rate-limiting step of CoA biosynthesis, was found to interact with a D. citri myosin protein. Two Carsonella enzymes involved in histidine and tryptophan biosynthesis were found to physically interact with D. citri proteins. These co-evolved protein interaction networks at the host-microbe interface are highly specific targets for controlling the insect vector responsible for the spread of citrus greening.

Reduced oviposition by Diaprepes abbreviatus (Coleoptera: Curculionidae) and growth enhancement of citrus by Surround particle film.

Regularly applied sprays of a particle film, Surround WP, greatly enhanced the growth of citrus trees on a poorly drained Winder soil at Fort Pierce, FL. After 3 yr of applications every 3 or 4 wk, Surround-treated trees had at least 5 times the mass, 6 times the canopy volume, and approximately 4 times the cross-sectional area of the tree stems at the graft union compared with untreated trees. The larger Surround-treated trees attracted a higher number of adult weevil Diaprepes abbreviatus (L.) and to a lesser extent citrus root weevil, Pachnaeus litus (Germar), per tree, but there was an equivalent number of egg masses per tree compared with the control trees. The number of egg masses per female weevil oviposited on Surround-treated trees was significantly less than either the control trees or trees treated biannually with an entomopathogenic nematode, BioVector. The number of larvae per tree recovered from the roots of excavated trees was greater from trees treated with Surround once every 3 wk compared with control trees. The data suggest that Surround particle film greatly enhanced the growth of citrus trees grown in a poorly drained soil. The reduction in oviposition by D. abbreviatus was insufficient to significantly reduce the number of root weevil larvae per tree feeding on the roots. However, the more vigorous trees resulting from Surround applications may be more resistant or tolerant to root weevil feeding.

Field comparison of chemical attractants and traps for Caribbean fruit fly (Diptera: Tephritidae) in Florida citrus.

Field studies in citrus were conducted to compare the following as attractants for the Caribbean fruit fly, Anastrepha suspensa (Loew): torula yeast-borax; propylene glycol (10%); a two-component lure consisting of ammonium acetate and putrescine; a two-component lure consisting of ammonium bicarbonate and putrescine; and a three-component lure consisting of ammonium bicarbonate, methylamine hydrochloride, and putrescine. Various combinations of these attractants in glass McPhail, plastic McPhail-type (Multi-Lure), and sticky panel traps were investigated in two replicated studies. In one study on wild flies, the most effective and least complex trap-lure combination tested was the Multi-Lure with propylene glycol baited with ammonium acetate and putrescine. This trap-lure combination captured significantly more female and male flies than the standard glass McPhail baited with torula yeast-borax in water. All of the trap-lure combinations were female biased, with an overall average of 80.8% (SEM 1.4) flies captured being female. A second study on laboratory-reared, irradiated flies indicated no significant differences among these trap-lure combinations with respect to number of flies recaptured, although rankings based on mean number of flies recovered per trap per day supported results of the first study. The percentage of flies recaptured that were female (83.0%, SEM 0.9) was statistically the same as in the first study. Weekly percentage recovery of flies during the second study was low, possibly due to our fly release strategy. Future release/recovery studies with laboratory-reared flies would benefit from some basic research on release strategies by using different trap densities and on relating recapture rates of laboratory-reared flies (nonsterile and sterile) to capture rates of wild flies.

Effect of the bisphosphonate risedronate on bone metastases in a rat mammary adenocarcinoma model system.

Risedronate (NE-58095) is a third-generation bisphosphonate with very potent antiresorptive activity but few toxic effects. The purpose of this work was to evaluate the effect of risedronate treatment on bone metastases produced in a rat breast cancer model. Berlin Druckrey IV rats inoculated with ENU1564 mammary adenocarcinoma cells were treated daily with risedronate or a saline placebo. Survival times, dictated by extraskeletal metastases (lung, heart, and brain), were not affected by risedronate treatment. Risedronate-treated animals had skeletal changes associated with decreased remodeling of bones undergoing endochondral ossification, most prominently affecting the appendicular skeleton. Despite the skeletal alterations induced by the treatment, the distribution of bone metastases throughout the surveyed skeletal sites was similar for treated and untreated animals. Bone metastases were enumerated in histologic sections of distal femur, spine, and skull. Tumor size was estimated from area measurements obtained from histologic lesions in distal femoral metaphyses and vertebral bodies. A greater number of treated rats had no bone metastases in any of the examined sections (30 versus 16.1% of untreated rats). Multiple bone metastases were observed less frequently in treated rats (33.3 versus 71% of untreated rats). Treated rats had fewer observed bone metastases in each examined site than untreated rats (p < or = 0.025). Mean tumor areas in femora and vertebrae were smaller in treated rats (p < or = 0.05), due to the less frequent presence of very large lesions. In untreated animals, osteoclasts appeared to be active at the tumor/bone interface and osseous structures were often completely replaced by expanding tumors. In contrast, metastases in treated animals caused less disruption of skeletal histoarchitecture. The apparent lack of osteoclastic activity and retention of bone within lesions suggested a decreased contribution of osteoclasts to the bone resorptive process. An in vivo immunohistochemical cell proliferation assay failed to reveal differences in the percentage of dividing tumor cells in bone metastatic sites in treated versus untreated animals. The results demonstrate significant effects of risedronate treatment on the incidence and size of observed skeletal metastases in this model.

Characterization of brain and bone-metastasizing clones selected from an ethylnitrosourea-induced rat mammary carcinoma.

An animal model for breast cancer, brain and bone metastasis was developed using ENU1564, a cell line established from a metastatic mammary adenocarcinoma induced by N-ethyl-N-nitrosourea in a female Berlin-Druckrey IV rat. The original tumor isolate (designated FP1) spontaneously metastasizes to regional lymph nodes and lung following orthotopic inoculation into mammary fat pad (mfp) and metastasizes widely following left cardiac ventricle (LV) inoculation. From FP1, two sublines were selected from brain metastases (designated Br7-C5) or from slowly growing colonies in vitro (FP2-A11), then cloned and compared in assays of spontaneous and experimental metastasis. After inoculation of 10(5) cells into mfp, Br7-C5 formed large tumors at the inoculation site (9.4 +/- 3.3 g) and spontaneously metastasized to lung and lymph node by 55 days post-inoculation (dpi). In contrast, FP2-A11 produced much smaller tumors within mfp (0.6 +/- 0.3 g) and failed to metastasize by 55 dpi. Rats inoculated via the LV with 10(4) Br7-C5 cells developed signs of weight loss, head tilt, and dyspnea by 24 +/- 1.4 dpi with consistent colonization of brain, bone, lung, heart, kidney, and stomach. Rats inoculated similarly with FP2-A11 showed no signs until 53 +/- 12.3 dpi, when all developed rear limb paresis. There was significant colonization of only brain and bone, with only minor lung involvement. These ENU1564 sublines thus differ in their apparent rates of tumor growth and lesion development in vivo, their capacity to metastasize from orthotopic implantation sites, and in the spectrum of tissues colonized in experimental metastasis assays. Both clones provide reproducible models of breast cancer metastasis in syngeneic hosts, particularly to brain and bone.

Beneficial metabolic effects of methylprednisolone sodium succinate in acute myocardial ischemia.

The metabolic and hemodynamic effects of methylprednisolone sodium succinate (40 mg/kg body weight) after acute myocardial ischemia were determined in 24 heparinized mongrel dogs. Myocardial ischemia was produced by ligation of the left anterior descending coronary artery. Catheters in the coronary sinus and the vein draining the left anterior descending coronary arterial area were used to collect blood samples from nonischemic and ischemic myocardium. Lactate, pyruvate, glucose, free fatty acids and oxygen were measured in arterial and venous blood from ischemic and nonischemic areas before and 3, 30 and 60 minutes after myocardial ischemia in animals with (Group II) and without (Group I) steroid treatment. In both Groups I and II glucose, lactate, free fatty acids, oxygen and coronary blood flow in nonischemic areas were not significantly changed, whereas glucose uptake in ischemic areas was significantly increased with myocardial ischemia and remained elevated. In Group I lactate uptake in ischemic areas became negative after coronary arterial ligation and remained so; in Group II, it increased after 30 (70%) and 60 (111%) minutes. Free fatty acid uptake in ischemic areas was reduced after myocardial ischemia in Group I, but in Group II it increased after 30 (224%) and 60 minutes (173%), and there was a concomitant increase in oxygen uptake. Pyruvate uptake in nonischemic areas decreased after 60 minutes in Group I, whereas it was reduced after 30 (68%) and 60 minutes (513%) in Group II. The changes were similar in ischemic myocardium. There were no significant changes in hemodynamic indexes. Coronary blood flow in ischemic areas decreased in Group I after myocardial ischemia and further after 30 and 60 minutes, but in Group II it increased after 30 (82%) and 60 minutes (53%). The data indicate that administration of methylprednisolone results in improved collateral blood flow into the infarcted area and a significantly improved metabolic response of ischemic myocardium. The glucocorticoid may also have a direct benefical effect on carbohydrate metabolism and cause the increased pyruvate neccesary to maintain the generation of energy-producing substrates. The results also suggest that methylprednisolone increases cell survival time and results in greater salvage of ischemic myocardium.

Severe left main coronary arterial stenosis with right coronary arterial occlusion: results of bypass graft surgery.

A detailed study was made of preoperative, operative and postoperative data from 69 patients with severe (70 percent or greater) luminal narrowing of the left main coronary artery and occlusion of the right coronary artery who underwent bypass surgery from December 1970 through December 1978. Preoperatively, 40.6 percent of patients were in functional class III and 55.1 percent in class IV. Ninety-six percent of those tested had a positive electrocardiographic treadmill test. Coronary bypass grafting was accomplished using standard techniques in all patients. An average of 2.7 grafts/patient were placed. The hospital mortality rate was 4.3 percent, and an additional 4.3 percent died before the end of 1 year. A history of congestive heart failure was a significant predictor (p less than 0.05) of postoperative mortality. An intraaortic balloon pump was not inserted in 64 patients, and our experience suggests that it was a necessary preoperative adjunct. A postoperative treadmill test was negative in 92 percent of those patients tested. Of those surviving 1 year postoperatively, 89 percent were in functional class I and 8 percent in class II. This study demonstrates a surgical mortality rate comparable with that of patients with left main coronary stenosis alone and a significantly better survival rate than that of similar patients treated medically.

Basic-level individuals.

In this experiment, 4-year-old children interpreted a novel count noun taught ostensively for an unfamiliar object as referring to a basic-level kind (such as PERSON or DOG) rather than to a kind that individuates its members by type of situation (such as PASSENGER or PUPPY). Because ostension does not distinguish a basic-level kind from a situation-restricted kind, this finding suggests that children assume, as a default, reference to a basic-level kind rather than to a situation-restricted kind when they hear a count noun applied to an unfamiliar object in an ostensive definition. The experiment shows that the assumption guides children's interpretation only if the object is unfamiliar, and if no specific information is available calling for a situation-restricted kind interpretation. The proposed word-learning assumption is critical to an understanding of the psychologically privileged manner of individuation, and therefore, of quantification (e.g., counting) involving objects.

Cardiac surgery under age two years. A review.

To assess the current status and risks of both open and closed cardiac procedures for congenital heart disease in patients under the age of 2 years, we reviewed all cardiac catheterizations and cardiac operations done from January, 1974, through December, 1977, at The Children's Orthopedic Hospital and Medical Center in Seattle, Washington. In this interval 370 patients under 2 years of age were catheterized. Eighty open procedures were performed in patients under 2 years of age, with seven hospital deaths. One hundred twenty-four closed heart procedures were performed on children under the age of 2 years, with eight deaths, for a hospital mortality rate of 6.5 percent. This review of consecutive cases over a 4 year period suggests that the judicious application of palliation or open repair using current techniques can lead to an overall mortality rate of between 6 and 7 percent for both open and closed heart procedures in children under 2 years of age. Since all deaths except one in the open-heart group occurred in patients with the most complex multiple defects, it seems reasonable to suggest that improved intraoperative and postoperative techniques have lowered the time for repair of straightforward congenital heart defects to under 2 years of age.

Tetralogy of Fallot: selective staged management.

One hundred twenty-four patients with tetralogy of Fallot have undergone either primary total repair (61), shunt and later repair (30), or an initial shunt (33). The mean ratio of pulmonary anulus to descending thoracic aorta increased from 0.80 +/- 0.25 before the shunt to 1.22 +/- 0.26 before the repair (p less than 0.0001). The mean ratio in the primary repair group was 1.23 +/- 0.25. A transannular patch was necessary in only six of 91 patients (6.6%). Postrepair right ventricular/left ventricular pressure ratio averaged 0.50 +/- 0.11 in the shunt plus repair group and 0.43 +/- 0.12 in the primary repair group. Only four patients had a right ventricular/left ventricular pressure ratio less than 0.65. A significant inverse linear relationship existed between this ratio and the pulmonary anulus size measured at operation and normalized for the patient's height (p less than 0.01). Postoperative complications occurred in 21% of patients after a shunt and 20% of patients after open heart repair. The early mortality was 0.8% (1/124). An initial shunt in patients with a small pulmonary anulus can result in an increased anulus size and better hemodynamic result with frequent avoidance of a transannular patch. Staged repair may result in improved overall mortality rates.

Solid-phase synthesis of cleavable N-arylmaleimides: applications in 1,3-dipolar cycloaddition and in thiol scavenging.

[reaction: see text]. An efficient solid-phase synthesis of polymer-supported N-arylmaleimides has been developed. Various N-arylmaleimidobenzoic acids (MBA) were elaborated onto Rink and SASRIN resins by reaction of the aniline precursors with maleic anhydride followed by facile cyclative dehydration of the resulting maleimic acids. Applications of these acid-cleavable MBA resins in the solid-phase synthesis of highly decorated pyrrolidines and as thiol scavengers are presented.

Direct mono-N-methylation of solid-supported amino acids: a useful application of the Matteson rearrangement of alpha-aminoalkylboronic esters.

[reaction: see text] A novel solid-phase method for the mono-N-methylation of resin-supported amino acids was developed on the basis of Matteson's 1,2-carbon-to-nitrogen migration of boron in alpha-aminoalkylboronic esters. Amino acids supported on either Wang resin or the highly acid-sensitive SASRIN resin can be methylated by reaction with pinacol chloromethylboronic ester, followed by rearrangement of the resulting aminomethylboronate and subsequent cleavage of the boronate group. This direct method requires only a simple and expedient oxidative resin wash to repair overalkylated sites.

Solid phase syntheses of polyamine toxins HO-416b and PhTX-433. Use of an efficient polyamide reduction strategy that facilitates access to branched analogues.

[structure--se text] Polyamine toxins HO-416b (1) and PhTX-433 (2) isolated from the venom of insects are important lead compounds in neuropharmacology. Their total synthesis has been achieved on a trityl derivatized resin in good yield and purity using a mild borane reduction protocol to access the polyamine chains from polyamide precursors. The synthesis of PhTX isomer 3 demonstrates the potential of this strategy for the generation of branched analogues.

Geometric comparison of aortic bifurcation prostheses to the normal and atherosclerotic human aorta, and clinical trial of a vertical seamed bifurcation prosthesis.

The 12 bifurcation prostheses generally available in the United States were compared. One was woven Teflon, two woven Dacron, and nine knitted Dacron. Ten were "seamless" in construction, and two were "seamed," but close examination revealed that all prostheses actually were seamed at the bifurication point. The grafts varied considerably in construction, porosity, and geometry. To compare the geometries, each prosthesis was preclotted in plasma, inflated to 120 mm. Hg, and quick frozen in alcohol and dry ice. Transverse and frontal bisections of the frozen specimens were photographed with a millimeter scale for sizing. For comparison, eight human aortofemoral bifurcations were distended and frozen similarly. The prostheses had far wider bifurcation shelves than did the normal aortas. The five normal human aortofemoral bifurcations had an average ratio of limb: body diameter of 0.61; the three atherosclerotic specimens averaged 0.49. The prostheses had an average limb:body diameter ratio of 0.48. or the "seamless" grafts, 0.48 for the circular transverse limb-seamed grafts, and 0.58 for the vertical-seamed grafts. Vertical-seamed aortic bifurcation grafts implanted in 102 patients between 1970 and 1976 (average follow-up, 29.5 months) have developed only a single limb closure, at 53 months.

Indications for surgical sterilization.

PIP: Indications, categorized as medical, obstetric, or parity for 842 female sterilizations between 1957 and 1968 are analyzed graphically by year. Overall medical and obstetric indications are listed. Medical indications fell from 100% to about 10%, and parity rose from 0 to about 80%. Average parity fell from 8.3 to 5.5. Types of operative procedures, supplemental procedures, complication rate in the University of Missouri Hospital, and contribution of parity to maternal death rates in Missouri are detailed. There were 2 failures and no mortalities.^ieng