Conditional activation of Pik3ca(H1047R) in a knock-in mouse model promotes mammary tumorigenesis and emergence of mutations.

Oncogenic mutations in PIK3CA, which encodes the phosphoinositide-3-kinase (PI3K) catalytic subunit p110α, occur in ∼25% of human breast cancers. In this study, we report the development of a knock-in mouse model for breast cancer where the endogenous Pik3ca allele was modified to allow tissue-specific conditional expression of a frequently found Pik3ca(H1047R) (Pik3ca(e20H1047R)) mutant allele. We found that activation of the latent Pik3ca(H1047R) allele resulted in breast tumors with multiple histological types. Whole-exome analysis of the Pik3ca(H1047R)-driven mammary tumors identified multiple mutations, including Trp53 mutations that appeared spontaneously during the development of adenocarinoma and spindle cell tumors. Further, we used this model to test the efficacy of GDC-0941, a PI3K inhibitor, in clinical development, and showed that the tumors respond to PI3K inhibition.

Evaluation of genetic potential of the polyvoltine silkworm (Bombyx mori L.) germplasm and identification of parents for breeding programme.

In the present study, polyvoltine germplasm stock of Andhra Pradesh State Sericulture Research and Development, Institute (APSSRDI) was evaluated for its performance based on quantitative and qualitative traits. Twenty-one oval and 10 peanut cocoon shaped lines were reared in different seasons of the year. Since the polyvoltines are non-diapausing, six generations were reared and evaluated for various economically important traits based on evaluation index and sub-ordinate function statistical methods. Ten top ranked lines obtained by using both the methods were identified as potential parental strains. Among oval lines, APM14, APM11, APM18, APMW9, and APM19, and among peanut lines APMD5, APMD1, APMD3, APMD9 and APMD8 were selected as base material. The identified high yielding lines will be used in various breeding programmes as initial parents for the synthesis of superior polyvoltine breeds/hybrids.

Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma.

Caspases are cysteine proteases essential to apoptosis. We have identified two families of caspase-like proteins, Paracaspases (found in metazoans and Dictyostelium) and metacaspases (found in plants, fungi, and protozoa). Metazoan paracaspase prodomains contain a death domain and immunoglobulin domains. Several plant metacaspase prodomains contain zinc finger motifs resembling those in the plant hypersensitive response/cell death protein Isd-1. The human paracaspase prodomain binds Bcl10, a protein involved in the t(1;14)(p22;q32) translocation of mucosa-associated lymphoid tissue (MALT) lymphoma. Another MALT lymphoma translocation, t(11;18)(q21;q21), fuses the IAP-2 gene to the MLT1/MALT1 locus, which encodes the human paracaspase. We find that this fusion activates NF-kappaB and that the caspase domain is required for this function, since mutation of the conserved catalytic cysteine attenuates NF-kappaB activation.

Output feedback control of nonlinear systems using RBF neural networks.

An adaptive output feedback control scheme for the output tracking of a class of continuous-time nonlinear plants is presented. An RBF neural network is used to adaptively compensate for the plant nonlinearities. The network weights are adapted using a Lyapunov-based design. The method uses parameter projection, control saturation, and a high-gain observer to achieve semi-global uniform ultimate boundedness. The effectiveness of the proposed method is demonstrated through simulations. The simulations also show that by using adaptive control in conjunction with robust control, it is possible to tolerate larger approximation errors resulting from the use of lower order networks.

Baculovirus-based genetic screen for antiapoptotic genes identifies a novel IAP.

The prototype baculovirus, Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) expresses p35, a potent anti cell-death gene that promotes the propagation of the virus by blocking host cell apoptosis. Infection of insect Sf-21 cells with AcMNPV lacking p35 induces apoptosis. We have used this pro-apoptotic property of the p35 null virus to screen for genes encoding inhibitors of apoptosis that rescue cells infected with the p35 defective virus. We report here the identification of Tn-IAP1, a novel member of the IAP family of cell death inhibitors. Tn-IAP1 blocks cell death induced by p35 null AcMNPV, actinomycin D, and Drosophila cell-death inducers HID and GRIM. Given the conserved nature of the cell death pathway, this genetic screen can be used for rapid identification of novel inhibitors of apoptosis from diverse sources.

Mutational analysis of Caenorhabditis elegans CED-4.

Much of our knowledge concerning the genetics that regulate cell death has come from the studies of cell death during the development of the nematode Caenorhabditis elegans. Of the 14 genes identified as components of nematode cell death pathways, two genes, ced-3 and ced-4, are required to promote cell death and a third, ced-9, blocks cell death. Recent studies show CED-4 to be an activator of CED-3 and CED-9 to be an inhibitor of CED-4. Two published sequence alignments suggest that CED-4 contains a death effector domain (DED), a protein sequence motif present in other death signaling proteins like Fadd and Flice; one study suggests a DED sequence similarity near the N-terminus while the other found sequence similarity near the C-terminus of CED-4. Using mutational analysis we have tested the functional significance of the conserved residues found within the putative DEDs of CED-4. Mutations in two conserved residues within the putative N-terminal DED of CED-4 affected its function, while mutations in the conserved residues within the putative C-terminal DED had no effect on CED-4 function. Our results do not support the presence of a DED in the C-terminus of CED-4 and suggest a potential role for the N-terminus in CED-4 function, possibly as a DED or as a CARD (caspase recruitment domain). We also found that CED-9 associated with all the CED-4 mutants and inhibited the activity of all the active-CED-4 mutants.

Caenorhabditis elegans CED-4 stimulates CED-3 processing and CED-3-induced apoptosis.

BACKGROUND: Programmed cell death or apoptosis is a key feature of normal development, tissue homeostasis and disease progression in metazoans. Genetic studies in the nematode C. elegans have identified three key genes involved in apoptosis, ced-3, ced-4 and ced-9. Expression of ced-3 and ced-4 is required for the induction of cell death, whereas expression of ced-9 is necessary to inhibit cell death. The precise mechanism by which these genes influence the life or death decision of a cell is not known. In this study, we have expressed the genes in an insect cell line to explore their role in the apoptotic pathway. RESULTS: Co-expression of ced-4 with ced-3 in insect cells stimulated both the induction and the level of CED-3-mediated apoptosis. Stimulation of CED-3-dependent apoptosis by CED-4 was accompanied by accelerated processing of CED-3, which was dependent on the presence of a wild-type CED-3 prodomain and a conserved lysine residue within a putative ATP/GTP-binding motif of CED-4. Co-expression of ced-9 with ced-4 and ced-3 inhibited the ability of CED-4 to stimulate CED-3 processing and CED-3-dependent apoptosis. Although a temperature-sensitive CED-9 mutant was unable to block CED-4 activity and failed to associate with CED-4, a deletion mutant of CED-9 lacking the carboxy-terminal hydrophobic domain could associate with CED-4 and block CED-4 activity. CONCLUSIONS: Our results establish a role for CED-4 in the processing of CED-3 and the stimulation of CED-3-induced apoptosis. Furthermore, we show that CED-9 achieves its anti-apoptotic effect by associating with CED-4 and blocking the ability of CED-4 to process CED-3.

Characterization of reaper- and FADD-induced apoptosis in a lepidopteran cell line.

Expression of the reaper gene (rpr) correlates with the initiation of apoptosis in Drosophila melanogaster. Transient expression of rpr in the lepidopteran SF-21 cell line induced apoptosis displaying nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Inhibitors of ICE-family proteases p35 and crmA, as well as members of the iap class of genes, Op-iap and D-iap2, but not bcl-2 family members, blocked rpr-induced apoptosis. Mutational analysis of rpr provided no support for the proposed sequence similarity of Reaper and death domain proteins. Mutations in the N-terminal region of Reaper, which displays sequence similarity to Hid and Grim, other Drosophila gene products correlated with the initiation of apoptosis, suggested that these residues might be functionally important. The mammalian cDNA encoding FADD (Fas-associating protein with a death domain) also induced cell death in SF-21 cells, but death progressed more slowly and with features which distinguished it from rpr-induced apoptosis. Several bcl-2 family members delayed or blocked FADD-induced cellular death. Thus, apoptosis initiated by Reaper progressed by a faster path which appeared to differ from that of FADD-induced apoptosis.

Baculovirus inhibitors of apoptosis (IAPs) block activation of Sf-caspase-1.

We have investigated the ability of Sf-caspase-1 and two mammalian caspases, caspase-1 and caspase-3, to induce apoptosis in Spodoptera frugiperda Sf-21 insect cells. While the transient expression of the pro-Sf-caspase-1 did not induce apoptosis, expression of the pro-domain deleted form, p31, or coexpression of the two subunits of mature Sf-caspase-1, p19 and p12, induced apoptosis in Sf-21 cells. The behavior of Sf-caspase-1 resembled that of the closely related mammalian caspase, caspase-3, and contrasted with that of the mammalian caspase-1, the pro-form of which was active in inducing apoptosis in Sf-21 cells. The baculovirus caspase inhibitor P35 blocked apoptosis induced by active forms of all three caspases. In contrast, members of the baculovirus inhibitor of apoptosis (IAP) family failed to block active caspase-induced apoptosis. However, during viral infection, expression of OpIAP or CpIAP blocked the activation of pro-Sf-caspase-1 and the associated induction of apoptosis. Thus, the mechanism by which baculovirus IAPs inhibit apoptosis is distinct from the mechanism by which P35 blocks apoptosis and involves inhibition of the activation of pro-caspases like Sf-caspase-1.

Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35.

The baculovirus antiapoptotic protein p35 inhibited the proteolytic activity of human interleukin-1 beta converting enzyme (ICE) and three of its homologs in enzymatic assays. Coexpression of p35 prevented the autoproteolytic activation of ICE from its precursor form and blocked ICE-induced apoptosis. Inhibition of enzymatic activity correlated with the cleavage of p35 and the formation of a stable ICE-p35 complex. The ability of p35 to block apoptosis in different pathways and in distantly related organisms suggests a central and conserved role for ICE-like proteases in the induction of apoptosis.

Complications of extracorporeal life support systems using heparin-bound surfaces. The risk of intracardiac clot formation.

Extracorporeal life support with heparin-coated extracorporeal membrane oxygenation circuits are being used with increased frequency in patients who have cardiogenic shock. We report our experience in 30 patients with cardiogenic shock, looking specifically at the complications associated with this form of life support. Thirty patients with a mean age of 46.5 +/- 16.6 years received extracorporeal life support for a mean of 62.8 +/- 41.1 hours (range 0.5 to 159 hours). Twenty-three patients had postcardiotomy cardiogenic shock, five had acute myocardial infarction, and one each had acute cardiac deterioration after a balloon coronary angioplasty and another after pulmonary artery balloon angioplasty. Peripheral (femoral vein to femoral artery) cannulation was used in 24 patients. Limb ischemia developed in 21 patients (70%), renal failure in 17 patients (57%), oxygenator failure requiring change in 13 patients (43%), bleeding requiring reexploration in 12 (40%), and infection in 9 patients (30%). Transesophageal echocardiography revealed intracardiac thrombus formation in 6 patients (20%) and clot was visualized grossly in the pump head in 2 patients (6%) necessitating pump-head change. Nine patients (30%) were discharged home. We conclude that the use of heparin-coated extracorporeal life support without systemic heparinization, especially after protamine has been used to reverse systemic heparinization in patients having postcardiotomy cardiogenic shock, may be dangerous. Extracorporeal life support has introduced new complications unique to itself specifically limb ischemia, oxygenator failure, and pump-head thrombus.

Recognition of chest radiograph orientation for picture archiving and communications systems display using neural networks.

A neural network classification scheme was developed that enables a picture archiving and communications system workstation to determine the correct orientation of posteroanterior or anteroposterior chest images. This technique permits thoracic images to be displayed conventionally when called up on the workstation, and therefore reduces the need for reorientation of the image by the observer. Feature data were extracted from 1,000 digitized chest radiographs and used to train a two-layer neural network designed to classify the image into one of the eight possible orientations for a posteroanterior chest image. Once trained, the neural network identified the correct image orientation in 888 of 1,000 images that had not previously been seen by the neural network. Of the 112 images that were incorrectly classified, 106 were mirror images of the correct orientation, whereas only 6 actually had the caudal-cranial axis aligned incorrectly. The causes for misalignment are discussed.