The effect of metformin in EML4-ALK+ lung cancer alone and in combination with crizotinib in cell and rodent models.

Cell based studies have suggested that the diabetes drug metformin may combine with the anaplastic lymphoma kinase receptor (ALK) inhibitor crizotinib to increase ALK positive lung cancer cell killing and overcome crizotinib resistance. We therefore tested metformin alone and in combination with crizotinib in vivo, by employing a xenograft mouse model of ALK positive lung cancer. We found that 14 days of daily oral metformin (100 mg/kg) alone had a moderate but statistically significant effect on tumour growth suppression, but in combination with crizotinib, produced no greater tumour suppression than crizotinib (25 mg/kg) alone. We also reassessed the effect of metformin on EML4-ALK positive lung cancer (H3122) cell viability. Although metformin alone did have a moderate effect on cell viability (30% suppression) this was only at a clinically irrelevant concentration (5 mM) and there was no additive effect with cytotoxic concentrations of crizotinib. Moreover, metformin did not overcome crizotinib resistance in our resistant cells. Nevertheless, we were able to show that metformin induces a G1-cell cycle arrest and apoptosis alone and in combination with crizotinib. Also, consistent with earlier work, the addition of insulin-like growth factor-1 (IGF-1) to EML4-ALK positive cancer cells reduced cell killing by crizotinib. We therefore hypothesised that the effect of metformin in vivo was not due to direct cytotoxicity on cancer cells, but by modulation of IGF-1 expression. We therefore measured levels of IGF-1 in plasma taken from mice treated with metformin, but found no difference between the drug treatment and control groups. We further hypothesised that the effect of metformin could be due to modulation of thrombospondin 1 (TSP-1), which metformin has been proposed to regulatein vivo, but again we found no difference between the experimental groups. Finally, we investigated the potential for liver and kidney toxicity, as well as CYP3A based interactions, from the combination of metformin with crizotinib.

Inhibition of Mitogen-Activated Protein Kinase Kinase Alone and in Combination with Anaplastic Lymphoma Kinase (ALK) Inhibition Suppresses Tumor Growth in a Mouse Model of ALK-Positive Lung Cancer.

Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer most commonly arises through EML4 (Echinoderm Microtuble Like 4)-ALK chromosomal fusion. We have previously demonstrated that combination of the ALK inhibitor crizotinib with the MEK inhibitor selumetinib was highly effective at reducing cell viability of ALK-positive non-small-cell lung cancer (H3122) cells. In this study, we further investigated the efficacy of crizotinib and selumetinib combination therapy in an in vivo xenograft model of ALK-positive lung cancer. Crizotinib decreased tumor volume by 52% compared with control, and the drug combination reduced tumor growth compared with crizotinib. In addition, MEK inhibition alone reduced tumor growth by 59% compared with control. Crizotinib and selumetinib alone and in combination were nontoxic at the dose of 25 mg/kg, with values for ALT (<80 U/l) and creatinine (<2 mg/dl) within the normal range. Our results support the combined use of crizotinib with selumetinib in ALK-positive lung cancer but raise the possibility that a sufficient dose of an MEK inhibitor alone may be as effective as adding an MEK inhibitor to an ALK inhibitor. SIGNIFICANCE STATEMENT: This study contains in vivo evidence supporting the use of combination MEK inhibitors in ALK+ lung cancer research, both singularly and in combination with ALK inhibitors. Contrary to previously published reports, our results suggest that it is possible to gain much of the benefit from combination treatment with an MEK inhibitor alone, at a tolerable dose.

The discovery of a galaxy-wide superwind from a young massive galaxy at redshift z approximately 3.

High-velocity galactic outflows, driven by intense bursts of star formation and black hole accretion, are processes invoked by current theories of galaxy formation to terminate star formation in the most massive galaxies and to deposit heavy elements in the intergalactic medium. From existing observational evidence (for high-redshift galaxies) it is unclear whether such outflows are localized to regions of intense star formation just a few kiloparsecs in extent, or whether they instead have a significant impact on the entire galaxy and its surroundings. Here we present two-dimensional spectroscopy of a star-forming galaxy at redshift z = 3.09 (seen 11.5 gigayears ago, when the Universe was 20 per cent of its current age): its spatially extended Lyalpha line emission appears to be absorbed by H i in a foreground screen covering the entire galaxy, with a lateral extent of at least 100 kpc and remarkable velocity coherence. This screen was ejected from the galaxy during a starburst several 10(8) years earlier and has subsequently swept up gas from the surrounding intergalactic medium and cooled. This demonstrates the galaxy-wide impact of high-redshift superwinds.

Structural and functional characteristics of a dominant-negative isoform of porcine MHC class II transactivator.

The MHC class II transactivator, CIITA, is critical for MHC class II gene expression in all species studied to date. We isolated an interferon (IFN)-gamma-inducible isoform of porcine CIITA (pCIITA') encoding a protein of 566 amino acids (aa) with significant homology to human CIITA (hCIITA). Analysis indicated that pCIITA' lacks the entire GTP-binding domain that is important for nuclear translocation and activation of target genes by hCIITA. In pCIITA' this region is replaced by a 14-aa motif with homology to several signalling peptide sequences. Expression of pCIITA' in porcine (ST-IOWA) and human (HeLa) cell lines resulted in suppression of IFN-gamma-stimulated MHC class II gene expression, at the protein and mRNA levels. We also identified two IFN-gamma-inducible variants of hCIITA, hCIITAlo and hCIITA' from Hela cells, both exhibiting dominant-negative suppression of MHC class II gene expression. Interestingly, hCIITA' encodes a predicted protein of 546 aa with a strikingly similar organization to pCIITA' including the 14-aa GTP-binding domain-replacement motif in which 10 out of 14 amino acids are identical to the pig sequence. Expression of hCIITA' and hCIITAlo sequences in Hela cells suppressed IFN-gamma-induced MHC class II gene expression. hCIITAlo, a predicted 303-aa protein with deleted GTP-binding and carboxy-terminal domain, displayed a more subtle suppression of IFN-gamma-induced MHC class II expression. These in vitro data indicate that there may be a role in vivo for isoforms of CIITA that can suppress full-length CIITA-mediated MHC class II gene expression. Both humans and now, potentially, pigs are candidate donors for organ and tissue allografts and xenografts, respectively. Regulation of MHC class II gene expression by manipulation of CIITA isoform expression in humans and pigs may provide a useful strategy for attenuation of T-cell-mediated cellular rejection.

Player sensitivity to changes in string tension in a tennis racket.

Forty-one advanced recreational tennis players were tested to determine their ability to detect differences in string tension in a tennis racket. Subjects were given pairs of rackets that varied in tension by up to 98 N (10 kg) and were asked whether they noticed a difference in tension and if so, which racket was strung at a higher tension. Only 11 (27%) of those tested could correctly identify a tension difference of 5 kg (11 lb) or less. Fifteen (37%) could not pick a difference of 10 kg (22 lb). To examine the importance of sound as a means of discrimination, an additional test was undertaken where participants wore earplugs. Of the 26 subjects undertaking this additional test, only 6 (23%) were successful. It was concluded that advanced recreational tennis players demonstrated limited ability to correctly identify differences in string tension and that impact sound was an important factor for those participants who were successful at various levels of discrimination.

Mapping full-length porcine endogenous retroviruses in a large white pig.

Xenotransplantation may bridge the widening gap between the shortage of donor organs and the increasing number of patients waiting for transplantation. However, a major safety issue is the potential cross-species transmission of porcine endogenous retroviruses (PERV). This problem could be resolved if it is possible to produce pigs that do not contain replication-competent copies of this virus. In order to determine the feasibility of this, we have determined the number of potentially replication-competent full-length PERV proviruses and obtained data on their integration sites within the porcine genome. We have screened genomic DNA libraries from a Large White pig for potentially intact proviruses. We identified six unique PERV B proviruses that were apparently intact in all three genes, while the majority of isolated proviruses were defective in one or more genes. No intact PERV A proviruses were found in this pig, despite the identification of multiple defective A proviruses. Genotyping of 30 unrelated pigs for these unique proviruses showed a heterogeneous distribution. Two proviruses were uncommon, present in 7 of 30 and 3 of 30 pigs, while three were each present in 24 of 30 pigs, and one was present in 30 of 30 animals examined. Our data indicate that few PERV proviruses in Large White pigs are capable of productive infection and suggest that many could be removed by selective breeding. Further studies are required to determine if all potentially functional proviruses could be removed by breeding or whether gene knockout techniques will be required to remove the residuum.

Insights into the structural organization of the I1 inner arm dynein from a domain analysis of the 1beta dynein heavy chain.

To identify domains in the dynein heavy chain (Dhc) required for the assembly of an inner arm dynein, we characterized a new motility mutant (ida2-6) obtained by insertional mutagenesis. ida2-6 axonemes lack the polypeptides associated with the I1 inner arm complex. Recovery of genomic DNA flanking the mutation indicates that the defects are caused by plasmid insertion into the Dhc10 transcription unit, which encodes the 1beta Dhc of the I1 complex. Transformation with Dhc10 constructs encoding <20% of the Dhc can partially rescue the motility defects by reassembly of an I1 complex containing an N-terminal 1beta Dhc fragment and a full-length 1alpha Dhc. Electron microscopic analysis reveals the location of the missing 1beta Dhc motor domain within the axoneme structure. These observations, together with recent studies on the 1alpha Dhc, identify a Dhc domain required for complex assembly and further demonstrate that the intermediate and light chains are associated with the stem regions of the Dhcs in a distinct structural location. The positioning of these subunits within the I1 structure has significant implications for the pathways that target the assembly of the I1 complex into the axoneme and modify the activity of the I1 dynein during flagellar motility.

Gone with the wind: the origin of S0 galaxies in clusters

We present three-dimensional, high-resolution hydrodynamical simulations of the interaction between the hot ionized intracluster medium and the cold interstellar medium of spiral galaxies. Ram pressure and turbulent/viscous stripping remove 100% of the atomic hydrogen content of luminous galaxies like the Milky Way within 100 million years. These mechanisms naturally account for the morphology of S0 galaxies and the rapid truncation of star formation implied by spectroscopic observations, as well as a host of observational data on the neutral hydrogen (HI) morphology of galaxies in clusters.

Cholangiography during laparoscopic cholecystectomy--cumulative sum analysis of an institutional learning curve.

The ability to perform intraoperative cholangiography during laparoscopic cholecystectomy is an essential skill for the laparoscopic biliary surgeon. The volume of experience required to be able to consistently obtain a cholangiogram during laparoscopic cholecystectomy has not been determined. Cumulative sum analysis is a statistical technique which generates a graphical display that identifies periods of performance that fall below a predetermined standard for a given task. The cumulative sum (S(n)) for a series of observations is defined as: S(n)= summation operatorX(I) - X(o), where X(I) = 0 for a success, X(I) = 1 for a failure, and X(o) is the acceptable failure rate for the process under study. This function is plotted against the number of observations to create a curve. When the curve has a positive slope, the acceptable failure rate is being exceeded. When it reaches a plateau, the observed failure rate is equal to the acceptable failure rate. When the curve has a negative slope, the observed failure rate is lower than the acceptable failure rate. We performed a cumulative sum analysis of the first 97 intraoperative cholangiograms attempted during lap-aroscopic cholecystectomy at our institution. The results demonstrated that 46 cases were required to reach a level of proficiency where a cholangiogram could be obtained in 95% of attempts. Success rates of 85% and 90% were achieved at 16 and 25 cases, respectively. This form of analysis is a useful tool for estimating the number of attempts required to achieve a desired success rate when learning new procedures.

Cytoplasmic dynein heavy chain 1b is required for flagellar assembly in Chlamydomonas.

A second cytoplasmic dynein heavy chain (cDhc) has recently been identified in several organisms, and its expression pattern is consistent with a possible role in axoneme assembly. We have used a genetic approach to ask whether cDhc1b is involved in flagellar assembly in Chlamydomonas. Using a modified PCR protocol, we recovered two cDhc sequences distinct from the axonemal Dhc sequences identified previously. cDhc1a is closely related to the major cytoplasmic Dhc, whereas cDhc1b is closely related to the minor cDhc isoform identified in sea urchins, Caenorhabditis elegans, and Tetrahymena. The Chlamydomonas cDhc1b transcript is a low-abundance mRNA whose expression is enhanced by deflagellation. To determine its role in flagellar assembly, we screened a collection of stumpy flagellar (stf) mutants generated by insertional mutagenesis and identified two strains in which portions of the cDhc1b gene have been deleted. The two mutants assemble short flagellar stumps (<1-2 micrometer) filled with aberrant microtubules, raft-like particles, and other amorphous material. The results indicate that cDhc1b is involved in the transport of components required for flagellar assembly in Chlamydomonas.

Patterns of morbidity and resource consumption associated with laparoscopic cholecystectomy in a VA medical center.

BACKGROUND: The generally low incidence of morbidity and reduced rate of health care resource consumption commonly associated with laparoscopic cholecystectomy (LC) have been established from studies of patient populations which are distinct from that served by the Department of Veterans Affairs (VA) health care system. We sought to assess the outcomes of this procedure when performed on VA beneficiaries. MATERIALS AND METHODS: Demographic and perioperative data for all patients undergoing attempted LC in our facility were recorded in a prospective database beginning 1 January 1993. The information in this registry was analyzed to determine the demographics of the treated population, the spectrum of biliary tract disease encountered, and patterns of morbidity and resource consumption. RESULTS: LC was attempted in 141 cases. Median patient age was 62 years. The indication for surgery was either acute cholecystitis or biliary pancreatitis in 63 cases (45%). Thirteen patients (9%) developed major complications. These patients were significantly older (mean age 68 vs 59 years) than patients whose course was uncomplicated. Twenty-seven cases (19%) required conversion to an open procedure, most commonly for acute cholecystitis. Progressive cholecystitis was associated with a conversion rate of 64%. Both conversion and the development of a major complication produced significant increases in length of stay. CONCLUSIONS: The population undergoing attempted LC in the VA system is characterized by relatively advanced age and high incidences of comorbid illness and complicated biliary tract disease. These attributes increase the frequency of major morbidity and of conversion to open cholecystectomy, which in turn increase resource consumption. Comparisons between the outcomes of attempted LC in VA centers and "benchmark" results obtained in other settings should be controlled for these factors.

Implementation of a clinical pathway decreases length of stay and hospital charges for patients undergoing total colectomy and ileal pouch/anal anastomosis.

BACKGROUND: Clinical pathways are increasingly being used by hospitals to improve efficiency in the care of certain patient populations; however, little prospective data are available to support their use. This study examined whether using a clinical pathway for patients undergoing ileal pouch/anal anastomosis, a complex procedure in which we had extensive practical experience, affected hospital charges or length of stay (LOS). METHODS: A clinical pathway was developed to serve patients undergoing elective total colectomy and ileal pouch/anal anastomosis. All operations were performed by two attending physicians (J.E.F., M.S.N.). Before implementation, 10 pilot patients were prospectively monitored to ensure that hospital charges were accurately generated. In addition, charge audits were performed by an outside agency to verify the accuracy of the hospital bills. The pathway was then implemented, and 14 patients were prospectively analyzed. RESULTS: In all patients the principal diagnosis was ulcerative colitis, with the exception of three patients with familial polyposis. Mean external audit charges were within 2% of the hospital bills; therefore the hospital bills were used in all calculations. The mean LOS decreased from 10.3 days to 7.5 days (p = 0.046) for patients on the pathway versus pilot patients. Mean hospital charges also decreased significantly, from $21,650 to $17,958 per patient (p = 0.005). CONCLUSIONS: Implementation of a clinical pathway, even for an operation in which the surgeon has much experience, is an effective method for reducing LOS and charges for patients. This is likely the result of interdisciplinary cooperation, elimination of unnecessary interventions, and streamlined involvement of ancillary services. These results support the development of clinical pathways for procedures that involve routine preoperative and postoperative care. In addition, the benefits of clinical pathways should increase proportionally with increasing case volume for a particular procedure.

Peri-implantitis.

The anatomy of the periodontal tissues of a tooth differs from that of a dental implant. The largest differences are in the lack of a periodontal membrane between the bone and a dental implant and its lack of an organized connective tissue attachment at the collar. These anatomical differences may influence the inflammatory response of each to bacterial plaque. Similarity has been demonstrated between the bacterial plaque on implants and teeth, but the significance of the difference between plaques at healthy and diseased sites on implants has not been determined. The consequence of periodontitis is loss of fibrous tissue attachment to the tooth and loss of supporting bone. A similar loss of supporting bone adjacent to dental implants has also been observed. Regenerative surgical techniques have been developed to reform the fibrous tissue attachment to the tooth and replace the lost supporting bone and these techniques have not been applied to dental implants. Healing and regeneration after periodontitis is now better understood, but regeneration around implants remains controversial. The evidence for the existence of a distinct entity, 'peri-implantitis', and its treatment are discussed.

Determining light dose for photodynamic therapy of atherosclerotic lesions in the Yucatan miniswine.

PURPOSE: To determine the light dose required for photodynamic therapy of atherosclerotic lesions in the miniswine. METHODS: Aortic atherosclerosis was created in seven Yucatan miniswine by a combination of balloon endothelial injury and 2% cholesterol and 15% lard for 7 weeks. Six animals received the photosensitizer Photofrin 2.5 mg/kg, while an additional swine received no drug. After 24 hours, the abdominal aorta was exposed and the aorta opened longitudinally in each animal. Three 1-cm spots were illuminated with energy densities of 60, 120, and 240 J/cm2 from an argon-pumped dye laser tuned to 630 nm with a laser output of 1 W. Four weeks later, the animals were killed, abdominal aortae removed, and intimal thickness determined by morphometry. RESULTS: The percentage intimal thickness (mean +/- SD) was 36.7 +/- 27.1, 9.1 +/- 5.0, and 6.4 +/- 8.1 for the three energy densities, respectively. Although both 120 and 240 J/cm2 energy densities produced significant (p < 0.05) reduction in atheroma, considerable damage to the underlying media was also observed in the 240 J/cm2 group. CONCLUSIONS: A Photofrin dose of 2.5 mg/kg and 120 J/cm2 light are necessary for adequate ablation of atheroma while avoiding extensive medial damage.

Early enteral administration of a formula (Impact) supplemented with arginine, nucleotides, and fish oil in intensive care unit patients: results of a multicenter, prospective, randomized, clinical trial.

OBJECTIVE: To determine if early enteral feeding, in an intensive care unit (ICU) patient population, using a formula supplemented with arginine, dietary nucleotides, and fish oil (Impact), results in a shorter hospital stay and a reduced frequency of infectious complications, when compared with feeding a common use enteral formula (Osmolite.HN). DESIGN: A prospective, randomized, double-blind, multicenter trial. SETTING: ICUs in eight different hospitals. PATIENTS: Of 326 patients enrolled in the study, 296 patients were eligible for analysis. They were admitted to the ICU after an event such as trauma, surgery, or sepsis, and met a risk assessment screen (Acute Physiology and Chronic Health Evaluation II [APACHE II] score of > or = 10, or a Therapeutic Intervention Scoring System score of > or = 20) and study eligibility requirements. Patients were stratified by age (< 60 or > or = 60 yrs of age) and disease (septic or systemic inflammatory response syndrome). INTERVENTIONS: Patients were enrolled and full-strength tube feedings were initiated within 48 hrs of the study entry event. Enteral feedings were advanced to a target volume of 60 mL/hr by 96 hrs of the event. One hundred sixty-eight patients were randomized to receive the experimental formula, and 158 patients were randomized to receive the common use control formula. MEASUREMENTS AND MAIN RESULTS: Both groups tolerated early enteral feeding well, and the frequency of tube feeding-related complications was low. There were no significant differences in nitrogen balance between groups on study days 4 and 7. Patients receiving the experimental formula had a significant (p = .0001) increase in plasma arginine and ornithine concentrations by study day 7. Plasma fatty acid profiles demonstrated higher concentrations of linoleic acid (p < .01) in the patients receiving the common use formula and higher concentrations of eicosapentaenoic and docosahexaenoic acid (p < .01) in the patients receiving the experimental formula. The mortality rate was not different between the groups and was significantly (p < .001) lower than predicted by the admission severity scores in both feeding groups. In patients who received at least 821 mL/day of the experimental formula, the hospital median length of stay was reduced by 8 days (p < .05). In patients stratified as septic, the median length of hospital stay was reduced by 10 days (p < .05), along with a major reduction in the frequency of acquired infections (p < .01) in the patients who received the experimental formula. In the septic subgroup fed at least 821 mL/day, the median length of stay was reduced by 11.5 days, along with a major reduction in acquired infections (both p < .05) in the patients who received the experimental formula. CONCLUSIONS: Early enteral feeding of the experimental formula was safe and well tolerated in ICU patients. In patients who received the experimental formula, particularly if they were septic on admission to the study, a substantial reduction in hospital length of stay was observed, along with a significant reduction in the frequency of acquired infections.