Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML.

Therapy directed against oncogenic FLT3 has been shown to induce response in patients with acute myeloid leukemia (AML), but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant AML cell lines by sustained treatment with the FLT3 inhibitor sorafenib. Parental cell lines carry the FLT3-ITD (tandem duplication) mutation and are highly responsive to FLT3 inhibitors, whereas resistant cell lines display resistance to multiple FLT3 inhibitors. Sanger sequencing and protein mass-spectrometry did not identify any acquired mutations in FLT3 in the resistant cells. Moreover, sorafenib treatment effectively blocked FLT3 activation in resistant cells, whereas it was unable to block colony formation or cell survival, suggesting that the resistant cells are no longer FLT3 dependent. Gene expression analysis of sensitive and resistant cell lines, as well as of blasts from patients with sorafenib-resistant AML, suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype, which was further supported by next-generation sequencing and phospho-specific-antibody array analysis. Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in resistant cell lines. Gedatolisib significantly extended survival of mice in a sorafenib-resistant AML patient-derived xenograft model. Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD-dependent AML results in resistance to drugs targeting FLT3.

Architecture of the RNA polymerase II-Mediator core initiation complex.

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

Vaccination of sows reduces the prevalence of PCV-2 viraemia in their piglets under field conditions.

The objectives of this study were to further understand vertical transmission of porcine circovirus type 2 (PCV-2) and the effect of dam vaccination on PCV-2 viraemia in newborn piglets. Randomly selected sows from each of two breeding herds were designated as non-vaccinated or vaccinated groups. A commercial inactivated PCV-2 vaccine was administered at weaning and 18 days later to half of the sows on each farm. At parturition, colostrum was collected from 70 dams on each farm and postsuckle (Farm 1) or presuckle blood (Farm 2) was collected from five randomly selected piglets per litter. Colostrum samples had an anti-PCV-2 antibody prevalence of 98.5 per cent (135/137) with significantly (P = 0.0039) higher concentrations in vaccinated dams. Among piglets, 43.9 per cent (301/685) were seropositive for PCV-2 and 11.7 per cent (80/686) were PCV-2 DNA-positive. All the PCV-2 DNA-positive samples were further characterised and 28 were PCV-2a, 28 PCV-2b, and five mixed PCV-2a and PCV-2b infection. The prevalence of PCV-2 DNA in piglets was lower (0.7-22.8 per cent) compared with previous studies (44.8-90 per cent) indicating a change in PCV-2 ecology likely due to wide use of vaccination. Under the study conditions, dam vaccination reduced PCV-2 viraemia in the offspring with colostrum access.

Vaccination with inactivated or live-attenuated chimeric PCV1-2 results in decreased viremia in challenge-exposed pigs and may reduce transmission of PCV2.

The objectives were to determine transmissibility of PCV2 to naïve contact pigs 140 days after infection of resident pigs and the benefit of vaccination with live-attenuated or inactivated chimeric PCV2 vaccines on chronic PCV2 infection. Twelve 6-week old PCV2 naïve pigs were randomly divided into four groups of three pigs: negative controls, positive controls, and pigs vaccinated with either a live-attenuated or inactivated chimeric PCV1-2 vaccine. All animals were bled weekly and tested for anti-PCV2 antibodies and PCV2 and PCV1-2 DNA and all groups except negative controls were challenged at 10 weeks. Two pigs vaccinated with the live PCV2 vaccine were PCV1-2 viremic at a single observation point. Both vaccine regimens induced an anti-PCV2 antibody response which was detected sooner and reached a higher level with the commercial inactivated vaccine. Both vaccines significantly decreased the concentration and duration of PCV2 viremia compared to the positive controls. PCV2 DNA was detected in lymphoid tissues of 1/3 pigs in the live-attenuated vaccine group and 3/3 positive control pigs. Three, 2-week old, PCV2 naïve contact pigs were comingled with each group at 168 days post-vaccination or 140 days post-challenge. After seven days of co-housing, the resident pigs were removed and the contact pigs remained for six weeks. Evidence of chimeric PCV1-2 vaccine or PCV2 challenge virus transmission to naïve contact pigs was lacking in all groups. The results of this study suggest that 140-day closure of a small pig population in a controlled environment may result in stabilization and elimination of PCV2.

Studies on porcine circovirus type 2 vaccination of 5-day-old piglets.

Porcine circovirus type 2 (PCV2) vaccines have become widely used since they became available in 2006. It is not uncommon for producers to use PCV2 vaccines in pigs younger than what is approved by manufacturers. The objective of this study was to determine the efficacy of a chimeric and a subunit PCV2 vaccine administered at 5 or 21 days of age. Forty-eight PCV2-naïve piglets were randomly divided into six groups of eight pigs each. Vaccination was done at day 5 or day 21, followed by triple challenge with PCV2, porcine parvovirus (PPV), and porcine reproductive and respiratory syndrome virus (PRRSV) at day 49. Vaccinated pigs seroconverted to PCV2 approximately 14 days postvaccination and had a detectable neutralizing antibody response by 21 days postvaccination regardless of age at vaccination. At day 49, the pigs vaccinated with the chimeric vaccine had significantly higher levels of neutralizing antibodies than the pigs vaccinated with the subunit vaccine. After challenge, vaccinated pigs had significantly decreased levels of PCV2 viremia and a decreased prevalence and severity of microscopic lesions compared to the positive-control group, which had severe lymphoid lesions associated with abundant PCV2 antigen, compatible with PCV-associated disease. The results of this study indicate that, under the conditions of this study, vaccination of PCV2-naïve pigs at day 5 or day 21 resulted in development of a detectable humoral immune response and provided reduction or complete protection against PCV2 viremia and PCV2-associated lesions after triple challenge with PCV2, PPV, and PRRSV.

The impact of longissimus glycolytic potential and short-term feeding of magnesium sulfate heptahydrate prior to slaughter on carcass characteristics and pork quality.

The objective of this study was to compare the effects of longissimus glycolytic potential (GP) and of time of feeding of supplemental magnesium sulfate heptahydrate on carcass and pork quality traits. The study was carried out in a 2 x 2 x 4 factorial arrangement; the treatments were sex (castrate vs gilt), GP (Low [normal] vs High), and time of feeding of magnesium sulfate-fortified diets (0 [control] vs 2 vs 3 vs 5 d prior to slaughter). Glycolytic potential was determined on a biopsy sample of longissimus from the live animal prior to the start of the study. A total of 144 pigs were allotted to the feeding-time treatments on the basis of sex (castrate and gilt), weight, and GP. Pigs were placed in individual pens and had free access to water. Prior to the start of the study, pigs were given ad libitum access to a standard finisher diet. During the study, animals were fed at a fixed level of 2.75 kg of a standard finisher diet/day; the fortified diet contained 3.2 g/d of additional magnesium. At the end of the feeding period, animals were transported to a commercial packing facility and slaughtered within 15 min of arrival. Fresh meat quality was measured on the longissimus. There were no treatment interactions. Carcass traits were similar across time of feeding treatments. Backfat thickness at the last lumbar vertebra and 10th rib were lower (P < 0 .05) for High than for Low GP pigs. High GP pigs had lower ultimate pH (P < 0.001) and higher drip (P < 0.05) and purge loss (P < 0.01) than Low GP pigs. Drip loss was reduced (P < 0.05) for pigs fed the magnesium-fortified diet for 5 and 2 but not for 3 d compared to controls (8.98, 7.29, 7.89, and 7.41 for the 0-, 2-, 3-, and 5-d treatments, respectively, SEM 0.447). Purge loss was similar for all of the time of feeding treatments. Longissimus L* values were lower (P < 0.05) for the 2-d treatment than for the controls. Results from this study suggest an inconsistent effect of short-term feeding of magnesium sulfate on muscle color and drip loss in pigs with both Low (normal) and High GP.

The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability.

Loss of telomere function can induce cell cycle arrest and apoptosis. To investigate the processes that trigger cellular responses to telomere dysfunction, we crossed mTR-/- G6 mice that have short telomeres with mice heterozygous for telomerase (mTR+/-) that have long telomeres. The phenotype of the telomerase null offspring was similar to that of the late generation parent, although only half of the chromosomes were short. Strikingly, spectral karyotyping (SKY) analysis revealed that loss of telomere function occurred preferentially on chromosomes with critically short telomeres. Our data indicate that, while average telomere length is measured in most studies, it is not the average but rather the shortest telomeres that constitute telomere dysfunction and limit cellular survival in the absence of telomerase.

Effect of different protein sources on growth and carcass traits in growing-finishing pigs.

Crossbred gilts (n = 180) and barrows (n = 180) from the Louisiana State University (LSU) Agricultural Center and the University of Illinois (UI) were used to compare the effect of soybean meal in swine diets, relative to other protein sources, on growth performance and carcass traits of growing-finishing pigs. Four replications with five pigs each at each location were allotted to nine dietary treatments: soybean meal control (SBM), crystalline AA (corn-AA), extruded soybeans (ESB), canola meal (CAN), peanut meal (PNT), sunflower meal (SFLR), ground peas, meat and bone meal (MBM), and poultry by-product meal (PLTY). The diets were formulated to meet or exceed NRC nutrient requirements and to have equal Lys:ME according to dietary phase and sex. Corn was the grain source in all diets and the protein sources were the sole source of supplemental protein in all diets except when AA were added to meet the requirement. Pigs (three per pen at each location) were killed at an average final BW of 114 kg in the LSU or UI Meat Science Laboratories. Pigs fed SBM had greater (P < 0.05) ADG than pigs fed the corn-AA, CAN, SFLR, MBM, or PLTY and greater (P < 0.05) ADFI relative to pigs fed the corn-AA, ESB, MBM, or PLTY. Gain:feed was decreased (P < 0.05) in pigs fed corn-AA or SFLR but increased (P < 0.05) in pigs fed ESB compared with pigs fed the SBM diet. Loin muscle area was decreased (P < 0.05) in pigs fed the corn-AA or MBM diets compared with pigs fed the SBM diet. Tenth-rib backfat thickness was greater (P < 0.10) in pigs fed corn-AA, peas, or MBM than in those fed SBM. The NPPC percentage acceptable quality lean and kilograms of lean were decreased (P < 0.10) in pigs fed corn-AA, peas, or MBM compared with those fed SBM. Results from this experiment suggest that pigs fed SBM have equal or better growth performance and carcass traits than pigs fed other protein sources.

Telomere dysfunction triggers developmentally regulated germ cell apoptosis.

Telomere dysfunction results in fertility defects in a number of organisms. Although data from fission yeast and Caenorhabditis elegans suggests that telomere dysfunction manifests itself primarily as defects in proper meiotic chromosome segregation, it is unclear how mammalian telomere dysfunction results in germ cell death. To investigate the specific effects of telomere dysfunction on mammalian germ cell development, we examined the meiotic progression and germ cell apoptosis in late generation telomerase null mice. Our results indicate that chromosome asynapsis and missegregation are not the cause of infertility in mice with shortened telomeres. Rather, telomere dysfunction is recognized at the onset of meiosis, and cells with telomeric defects are removed from the germ cell precursor pool. This germ cell telomere surveillance may be an important mechanism to protect against the transmission of dysfunctional telomeres and chromosomal abnormalities.

The two RNA ligases of the Trypanosoma brucei RNA editing complex: cloning the essential band IV gene and identifying the band V gene.

Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase. A complex of seven different polypeptides purified from Trypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of approximately 57 kDa (band IV) and approximately 50 kDa (band V). From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex. It is indeed an RNA ligase, for when expressed in Escherichia coli, the protein autoadenylylates and catalyzes RNA joining. Overexpression studies revealed that T. brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels. The protein's mitochondrial targeting was confirmed by its location, size when expressed in T. brucei and E. coli, and N-terminal sequence. Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes. The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions. We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.

Wild-derived inbred mouse strains have short telomeres.

Telomere length and telomerase activity directly affect the replicative capacity of primary human cells. Some have suggested that telomere length influences organismal lifespan. We compared telomere length distributions in a number of inbred and outbred established mouse strains with those of strains recently derived from wild mice. Telomere length was considerably shorter in wild-derived strains than in the established strains. We found no correlation of telomere length with lifespan, even among closely related inbred mouse strains. Thus, while telomere length plays a role in cellular lifespan in cultured human cells, it is not a major factor in determining organismal lifespan.

G-strand overhangs on telomeres in telomerase-deficient mouse cells.

Telomeres of eukaryotic chromosomes contain 3' overhangs which are thought to be essential for the maintenance of proper chromosome end structure and function. We examined the requirement for telomerase activity for the generation of these G-strand overhangs in mammalian cells. Using non-denaturing in-gel hybridization to both tissue and cultured cells from mice deficient for the telomerase RNA component, we found that G-strand overhangs exist in the absence of telomerase activity. Quantitation of overhang signal intensity showed no significant reduction in telomerase-deficient cells relative to wild-type. These results support a telomerase-independent mechanism for generating G-strand overhangs.

Strain-dependent developmental relaxation of imprinting of an endogenous mouse gene, Kvlqt1.

Genomic imprinting is an epigenetic modification of the gamete or zygote leading to parental origin-specific differential expression of the two alleles of a gene in somatic cells of the offspring. We previously reported that the human KVLQT1 gene is imprinted and disrupted in patients with germline balanced chromosomal rearrangements and Beckwith-Wiedemann syndrome. In human, the gene is imprinted in most fetal tissues except the heart, and KVLQT1 is part of a 1-Mb cluster of imprinted genes on human chromosome 11p15. 5. We sought to determine whether the mouse Kvlqt1 gene is imprinted, by performing interspecific crosses of 129/SvEv mice with CAST/Ei (Mus musculus castaneus). We identified a transcribed polymorphism that distinguishes the two parental alleles in F1 offspring. Examination of embryonic, neonatal, and postnatal tissues revealed that Kvlqt1 is imprinted in mouse early embryos, in both female 129 x male CS and female CS x male 129 offspring, with preferential expression of the maternal allele, like the human homologue. Surprisingly, imprinting was developmentally relaxed, and the developmental stage and tissue specificity of relaxation of imprinting was strain-dependent. To our knowledge, this is the first example of an endogenous gene that shows strain-dependent developmental relaxation of imprinting.

A familial "balanced" 3;9 translocation with cryptic 8q insertion leading to deletion and duplication of 9p23 loci in siblings.

A child with phenotypic features of the 9p- syndrome, including metopic craniosynostosis, small ears, abdominal wall defect, and mental retardation, as well as hypopigmentation, was found to have a cytogenetically balanced 3;9 translocation, with breakpoints at 3p11 and 9p23, inherited from his phenotypically normal father. Molecular analysis showed heterozygous deletion of the TYRP (tyrosinase-related protein) locus, as well as loci D9S157, D9S274, D9S268, and D9S267, in the child but in neither parent. FISH analysis of the proband's father indicated that loci deleted in his son, including TYRP, were present on neither the der(3) nor the der(9) translocation products but had been inserted into the long arm of chromosome 8. Therefore, the apparent deletion of these loci in the proband was the result of meiotic segregation of the father's 3;9 translocation chromosomes together with his normal chromosome 8 (not bearing the insertion from 9p23). Neither the deletion of these 9p23 loci from the translocation chromosomes nor their insertion into 8q was detectable by standard chromosome banding techniques. The proband's sister exhibited speech delay, mild facial dysmorphism, and renal malformation, and her karyotype was 46,XX. Molecular analysis showed that she had inherited normal chromosomes 3 and 9, as well as the chromosome 8 with the insertion of 9p23 material, from her father.(ABSTRACT TRUNCATED AT 250 WORDS)