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.

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.

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.

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.

Nephrogenous cyclic AMP levels in primary hyperparathyroidism.

The clinical utility of urinary cyclic adenosine-3',5'-monophosphate (cAMP) determinations has been limited by the overlap between hyperparathyroid and normal patients. We evaluated the potential of the parathyroid hormone (PTH)-dependent, nephrogenous cAMP in the diagnosis of hyperparathyroidism. Twenty-three patients with primary hyperparathyroidism and 19 control subjects had two-hour urine collections and blood sampling at midpoint. Nephrogenous cAMP level was calculated as total urinary cAMP excretion minus the amount filtered. The total urinary cAMP excretion (micromols per gram of creatinine) was higher in hyperparathyroid patients (6.8 +/- .5 SE), but overlapped with values obtained in controls (2.9 +/- .15). The level of nephrogenous cAMP (percent of total) was also higher in hyperparathyroid patients (72.5 +/- 1.8) than controls (26.3 +/- 4.1) and clearly separated the groups. Determination of nephrogenous cAMP levels may be useful in the diagnosis of hyperparathyroidism.

Effects of alpha-tocopherol acetate on the swimming endurance of trained swimmers.

Well-trained, competitive swimmers were divided into two groups. Group A was given 900 IU alpha-tocopherol acetate daily for 6 months while group B was given placebos. A swimming endurance test was given before the start of supplementation and after 1, 2, 5 and 6 months. No difference in swimmers' endurance was observed between the two groups during the 6-month period. There was also no difference in postexercise serum lactic acid levels. Younger, less well-trained, competitive swimmers were also divided into two groups. Group A received 900 IU alpha-tocopherol acetate daily while group B received placebos. Swimming times for these swimmers were erratic, reflecting a lack of training. alpha-Tocopherol did not appear to have any effect on their swimming endurance.

Synthesis and anti-herpes simplex activity of analogues of phosphonoacetic acid.

The synthesis of monoesters (P and C) of phosphonoacetic acid (PA) is given. The carboxyl esters were prepared by two methods: the reaction of chloroacetates with tris(trimethylsilyl) phosphite, followed by hydrolysis; and by the acid-catalyzed esterification of PA with the appropriate alcohol. P-Monoesters of PA were prepared either by the reaction of alkyl[bis(trimethylsilyl)] phosphite with benzyl chloroacetate followed by deprotection or by the reaction of dimethylphenyl phosphite with benzyl bromoacetate followed by hydrogenolysis. Three aryl- (alkyl-)phosphinic acid derivatives are reported. The above compounds were evaluated for anti-herpes activity against HSV-induced DNA polymerase and in animals infected with herpes dermatitis.

Low velocity vehicular injuries in children--"run-over" accidents.

Fourteen children, mostly in the toddler to preschool age group, were treated for injuries resultant from low velocity vehicular accidents in which the child was literally run over. Multiple system injury and visceral injury to brain, lungs, liver, and kidneys were noted. One death and several permanent disabilities resulted in this small group. Most of the accidents could have been prevented by closer supervision of the children. Devices such as childproof door handles, which are currently available, and audible backing signals, currently available on industrial vehicles, might further reduce the incidence of this type of accident.

Mesenteric cysts in children.

BACKGROUND: Mesenteric cysts are uncommon benign abdominal masses. Approximately one third of patients with these lesions are children. METHODS: We reviewed our clinical records for the past 14 years (corresponding to the period of time in which ultrasonography and computed tomography became reliable methods for imaging pediatric patients) and found 10 patients with mesenteric cysts. RESULTS: We were surprised to discover that abdominal pain was a presenting complaint in all but one patient. Five of the patients had the findings of an acute surgical abdomen and were thought to have appendicitis. Two patients operated on for appendicitis were transferred to our hospital with the diagnosis of an abdominal mass. In each case the mass was a mesenteric cyst. Cyst distribution included the small-bowel mesentery in seven patients, the transverse mesocolon in two patients, and the right mesocolon in one patient. Six cases required concomitant bowel resection for the cyst removal, and all were cystic lymphangiomas. The resected specimens were described as cystic lymphangiomas in eight of the 10 cases. CONCLUSIONS: Mesenteric cysts should be considered as an origin for abdominal pain in children, particularly after exclusion of more common diagnoses. We have found ultrasonographic imaging to be a reliable method for the diagnosis of appendicitis in children and advocate its use as an initial imaging study in patients with an acute surgical abdomen and presumed appendicitis. If appendicitis is indicated unlikely by ultrasonogram, the examination can be extended to the remainder of the abdomen, which can reveal mesenteric cysts or other pathologic conditions.

Ventilatory support and primary closure of gastroschisis.

Recent clinical reports suggest that the majority of neonates with gastroschisis are best managed with a silon pouch technique. Our results with primary closure and short-term ventilatory assistance show a survival rate equal to that of the silon pouch technique. In addition, multiple operations and the chances of complications related to the pouch are avoided. No complications relating to the ventilatory assistance were encountered. Better pediatric ventilators and improved pediatric respiratory management allow this technique to be used for gastroschisis now, whereas 10 years ago the complications ith neonatal ventilators may have outweighed the morbidity of the silon pouch.

The value of hepatobiliary scans in fasted patients receiving total parenteral nutrition.

Hepatobiliary scanning is considered to be a highly accurate method for the diagnosis of acute cholecystitis. False-positive scans (failure to visualize the gallbladder in the absence of cholecystitis) have been reported to occur in fasted patients receiving total parenteral nutrition (TPN). To determine the prevalence of false-positive scans in this patient population and identify factors that might be associated with scan outcome, hepatobiliary imaging was performed in fasted patients receiving TPN and without clinical evidence of acute cholecystitis. Gallbladder nonvisualization occurred in 18 of 50 (36%) patients. In the group whose gallbladders did not visualize, a significantly higher male to female ratio (15:3 versus 17:15; p = 0.016), alkaline phosphatase (346 +/- 84 IU/L versus 212 +/- 32 IU/L, p less than 0.04), total bilirubin (1.7 +/- 0.3 mg/dl versus 1.0 +/- 0.2 mg/dl, p less than 0.02), and lower serum albumin (2.4 +/- 0.01 gm/dl versus 2.8 +/- 0.2 gm/dl, p less than 0.02) levels were noted. In 18 patients, gallbladder ultrasonography was also performed, and the presence of sludge or a thickened gallbladder wall did not correlate with scan outcome. The prevalence of false-positive hepatobiliary scans in fasted patients receiving TPN is significant and does not always correlate with a syndrome of acute gallbladder inflammation. The results of such scans must therefore be interpreted with caution in these patients.

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.

Association between microorganism growth at the catheter insertion site and colonization of the catheter in patients receiving total parenteral nutrition.

Catheter-related sepsis is one of the major complications of total parenteral nutrition (TPN) therapy. The relationship between microbial colonization of the skin at the site of catheter insertion and colonization of the central venous catheter was investigated in 74 catheters used to administer TPN therapy in 53 patients. Semiquantitative culture specimens were obtained from the insertion site and intravascular and subcutaneous catheter segments at the time of catheter removal. Bacteria and/or fungi were recovered from 19 catheters and 19 insertion sites; of the 19 colonized catheters, 6 had sterile insertion sites. Organisms isolated from the remaining 13 catheters were isolated concurrently from the insertion site. Catheter-associated bacteremia or fungemia was observed in 10 of the 19 patients with colonized catheters. The association between colonization of catheters and the presence of more than 10(3) bacterial or fungal colony-forming units at the insertion site was significant (P less than 0.005). These results demonstrated that colonization of catheters by organisms present on the skin at the site of catheter insertion occurred twice as frequently as colonization by the hematogenous route. The results also suggested that colonization of catheters by organisms present at the insertion site occurred only after a threshold number of organisms was reached.

The effect of a new branched chain--enriched amino acid solution on postoperative catabolism.

Branched-chain amino acids (BCAAs) may regulate muscle amino acid flux. Metabolic studies of both experimental animals and humans utilizing comparatively large amounts of BCAAs infused with hypocaloric glucose have shown that catabolism and proteolysis can be blunted. These studies suggested that the nitrogen-sparing properties of amino acid solutions used in postoperative trauma or sepsis might be improved by increasing the amount of BCAAs. This hypothesis was tested on ten patients undergoing operations of moderate severity utilizing a peripheral amino acid mixture with a branched-chain:non-branched-chain ratio of 45:55% given in 5% dextrose. The patients received 1.7 gm of protein equivalent/kg of ideal body weight in 5% dextrose-crystalloid solution with a concentration of 3.5% amino acids for the first 5 postoperative days. Nitrogen balance, 3-methylhistidine excretion, blood chemistries, and plasma amino acid profile tests were done daily. The results showed that nitrogen equilibrium was maintained for 5 postoperative days without any untoward effects on patients, their surgical wounds, or hepatic function. Plasma amino acids showed no significant changes from baseline with the exception of elevations of the BCAAs. We conclude that this 45% BCAA-enriched solution may be safely administered to patients with postoperative traumatic injury and results in nitrogen equilibrium over a 5-day period.

Mediastinal masses in infants and children.

In a group of 93 pediatric patients with mediastinal masses, three quarters of them were diagnosed because they were symptomatic. Approximately one half of the masses were malignant, and of neurogenic or lymphomatous origin. Neurogenic tumors predominated before the age of 4 years, while lymphomas were most common beyond age 4. Two thirds of the malignancies were potentially curable by surgery, irradiation, and chemotherapy; 54% of the patients were salvaged. Enteric duplications, ganglioneuromas, bronchogenic cysts, hemangiolymphangiomas, thymic cyst, and teratomas were the most frequently occurring benign masses. Simple excision was uniformly effective in these lesions.

Controversial aspects of appendicitis management in children.

The areas of controversy in appendicitis management in children include antibiotic usage, drainage of the peritoneal cavity, and closure of contaminated incisions. Our results and bacteriologic data suggest that gentamicin sulfate and clindamycin phosphate should be routinely used in cases of suspected perforation, that only selective and limited use of drains is warranted, and that delayed primary closure should be used in all cases with perforation.

Postoperative enteral vs parenteral nutrition. A randomized controlled trial.

Twenty patients undergoing major upper-gastrointestinal-tract or pancreaticobiliary surgery were randomized to receive postoperative nutritional support by total parenteral nutrition (TPN) or elemental diet administered by needle-catheter jejunostomy (NCJ). Both routes of administration provided adequate nutritional support. No unexpected complications were encountered. The NCJ group compared favorably with the TPN group at the end of the seven-day trial. The NCJ group provided significant cost efficiency while maintaining adequate nutritional support.

Lipid-free total parenteral nutrition and macrophage function in rats.

Certain lipids are immunosuppressive when used for nutritional support, while other lipids and nutritional additives may enhance immunologic function. We hypothesized that total parenteral nutrition (TPN) may be immunosuppressive irrespective of lipids. Twenty-four rats underwent central vein catheterization and received either intravenous saline solution and oral chow or TPN alone. At 7 or 14 days, the animals were killed. Splenic and bone marrow macrophages were isolated and cultured in either M199 medium alone or were stimulated with Escherichia coli lipopolysaccharide. The supernatants were tested for prostaglandin E2 and C3. The splenic prostaglandin E2 levels were significantly higher in the TPN group following lipopolysaccharide stimulation at 7 days but not at 14 days. Administration of TPN to rats, even without lipids, may be immunosuppressive through the release of prostaglandin E2 from splenic macrophages following a septic challenge. This effect appears to be abolished after 14 days of TPN infusion.

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.