TYMS promotes genomic instability and tumor progression in Ink4a/Arf null background.

We previously showed that elevated TYMS exhibits oncogenic properties and promotes tumorigenesis after a long latency, suggesting cooperation with sequential somatic mutations. Here we report the cooperation of ectopic expression of human TYMS with loss of Ink4a/Arf, one of the most commonly mutated somatic events in human cancer. Using an hTS/Ink4a/Arf -/- genetically engineered mouse model we showed that deregulated TYMS expression in Ink4a/Arf null background accelerates tumorigenesis and metastasis. In addition, tumors from TYMS-expressing mice were associated with a phenotype of genomic instability including enhanced double strand DNA damage, aneuploidy and loss of G1/S checkpoint. Downregulation of TYMS in vitro decreased cell proliferation and sensitized tumor cells to antimetabolite chemotherapy. In addition, depletion of TYMS in vivo by TYMS shRNA reduced tumor incidence, delayed tumor progression and prolonged survival in hTS/Ink4a/Arf -/- mice. Our data shows that activation of TYMS in Ink4a/Arf null background enhances uncontrolled cell proliferation and tumor growth, supporting the development of new agents and strategies targeting TYMS to delay tumorigenesis and prolong survival.

First-in-class multifunctional TYMS nonclassical antifolate inhibitor with potent in vivo activity that prolongs survival.

Although thymidylate synthase (TYMS) inhibitors have served as components of chemotherapy regimens, the currently available inhibitors induce TYMS overexpression or alter folate transport/metabolism feedback pathways that tumor cells exploit for drug resistance, limiting overall benefit. Here we report a small molecule TYMS inhibitor that i) exhibited enhanced antitumor activity as compared with current fluoropyrimidines and antifolates without inducing TYMS overexpression, ii) is structurally distinct from classical antifolates, iii) extended survival in both pancreatic xenograft tumor models and an hTS/Ink4a/Arf null genetically engineered mouse tumor model, and iv) is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we verify the compound is a multifunctional nonclassical antifolate, and using a series of analogs, we identify structural features allowing direct TYMS inhibition while maintaining the ability to inhibit dihydrofolate reductase. Collectively, this work identifies nonclassical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile, highlighting the potential for enhanced cancer therapy.

Corrigendum: Targeting thymidylate synthase enhances the chemosensitivity of triple-negative breast cancer towards 5-FU-based combinatorial therapy.

[This corrects the article DOI: 10.3389/fonc.2021.656804.].

Intratunnel versus extratunnel fixation of hamstring autograft for anterior cruciate ligament reconstruction.

PURPOSE: To determine which is more effective: intratunnel or extratunnel anterior cruciate ligament soft-tissue graft fixation. A secondary purpose was to determine whether groups displayed differing relations between objective International Knee Documentation Committee (IKDC) grade and the timing of full weight bearing (FWB), jogging/running, and return to sports. The study hypotheses were that intratunnel fixation would display a greater percentage of normal or nearly normal objective IKDC grades and enable earlier FWB, return to jogging/running, and return to sports. METHODS: We performed a systematic review of prospective, Studies with Level I or II evidence published from 2000 to 2011 with at least 2 years' follow-up that used interference screw hamstring autograft fixation (intratunnel group) or button, staple, or post hamstring autograft fixation (extratunnel group) for primary anterior cruciate ligament reconstruction. We also compared IKDC grades; Tegner and Lysholm scores; instrumented anterior laxity and pivot-shift test findings; timing of FWB, jogging/running, and return to sports; and modified Coleman Methodology Scores. RESULTS: The groups showed comparable modified Coleman Methodology Scores, objective IKDC grades, Lysholm and Tegner scores, instrumented anterior laxity and pivot-shift test findings, and return-to-sports timing. The intratunnel group displayed earlier FWB and jogging/running; however, return-to-sports timing did not differ between groups. Early FWB and the percentage of patients with normal or nearly normal objective IKDC grades were directly related for the extratunnel group. CONCLUSIONS: Patients who received intratunnel fixation were released earlier to FWB and jogging/running, supporting the study hypotheses. Groups did not differ in return-to-sports timing or objective IKDC grades, not supporting the study hypotheses. Early FWB in the extratunnel group was related to a greater percentage of patients having normal or nearly normal objective IKDC grades. Return-to-sports timing and having a normal objective IKDC grade were related in both groups; however, this relation was stronger with intratunnel fixation. LEVEL OF EVIDENCE: Level II, systematic review of Level I and II studies.

Supplemental bio-tenodesis improves tibialis anterior allograft yield load in extremely low density tibiae.

INTRODUCTION: Improved soft tissue tendon graft mechanical properties have led to their increased use for anterior cruciate ligament (ACL) reconstruction. Because they do not have an osseous component; however, there are greater concerns regarding tibial graft slippage during early postoperative rehabilitation and activities of daily living, particularly in patients with poor bone mineral density (BMD), such as older patients, women, smokers, and patients undergoing revision ACL reconstruction surgery. METHODS: This in vitro biomechanical study attempted to determine the effectiveness of supplemental ACL graft fixation in low BMD tibiae. Eight paired knees (16 specimens) were harvested from female cadavers (mean age = 76, range = 60-88 years). Tibiae were assigned to either a combination bioabsorbable interference screw, bio-tenodesis screw group (Group 1, n = 8, apparent BMD = 0.44 ± 0.13 g/cm(2)) or a bioabsorbable interference screw group (Group 2, n = 8, apparent BMD = 0.44 ± 0.14 g/cm(2)). Double-strand (single loop) tibialis anterior tendon allografts were fixed in matched diameter tibial tunnels. Using a custom 6° of freedom jig, potted constructs were mounted on to a servo hydraulic device with the axial loading force aligned directly with the tibial tunnel. Constructs underwent progressive cyclic tensile loading from 10 to 150 N with a 25 N load increase every 20 cycles. This was followed by yield load to failure testing (20 mm/min). RESULTS: Groups did not display displacement differences during progressive cyclic loading. Group 1 (312.7 ± 67.5 N) displayed 25% greater yield load at failure than Group 2 (235.0 ± 47.6 N), P = 0.045. Both groups displayed fixation levels well below the previously reported minimal safe threshold estimate for early unrestricted weight bearing, accelerated rehabilitation and activities of daily living. CONCLUSION: Supplemental bio-tenodesis fixation may improve early tibial-soft tissue tendon graft fixation in patients that have poor tibial BMD, but study results suggest that both methods may require weightbearing, rehabilitation, and activity of daily living restrictions during the early postoperative period to prevent graft slippage.

Thymidylate synthase accelerates Men1-mediated pancreatic tumor progression and reduces survival.

Clinical studies of cancer patients have shown that overexpression or amplification of thymidylate synthase (TS) correlates with a worse clinical outcome. We previously showed that elevated TS exhibits properties of an oncogene and promotes pancreatic neuroendocrine tumors (PanNETs) with a long latency. To study the causal impact of elevated TS levels in PanNETs, we generated a mouse model with elevated human TS (hTS) and conditional inactivation of the Men1 gene in pancreatic islet cells (hTS/Men1-/-). We demonstrated that increased hTS expression was associated with earlier tumor onset and accelerated PanNET development in comparison with control Men1-/- and Men1+/ΔN3-8 mice. We also observed a decrease in overall survival of hTS/Men1+/- and hTS/Men1-/- mice as compared with control mice. We showed that elevated hTS in Men1-deleted tumor cells enhanced cell proliferation, deregulated cell cycle kinetics, and was associated with a higher frequency of somatic mutations, DNA damage, and genomic instability. In addition, we analyzed the survival of 88 patients with PanNETs and observed that high TS protein expression independently predicted worse clinical outcomes. In summary, elevated hTS directly participates in promoting PanNET tumorigenesis with reduced survival in Men1-mutant background. This work will refocus attention on new strategies to inhibit TS activity for PanNET treatment.

Targeting Thymidylate Synthase Enhances the Chemosensitivity of Triple-Negative Breast Cancer Towards 5-FU-Based Combinatorial Therapy.

BACKGROUND: The ongoing treatment modalities for breast cancer (BC) primarily rely on the expression status of ER, PR and HER-2 receptors in BC tissues. Our strategy of chemosensitization provides new insights to counter chemoresistance, a major obstacle that limits the benefits of chemotherapy of mammary cancers. METHODS: By utilizing a murine breast cancer model employing NSG mice bearing orthotopic triple-negative breast cancer (TNBC) xenografts, we have evaluated the ability of phytochemical curcumin in chemosensitizing BC to 5-Fluorouracil (5-FU) chemotherapy and the differential modulations of cellular events in response to this strategy, independent of their receptor status. RESULTS: A significant synergistic antitumor potential was observed in the murine model with a sub-optimal dose treatment of 5-FU plus curcumin, as evaluated by a reduction in the tumor-related parameters. We authenticated the pivotal role of thymidylate synthase (TS) in regulating the 5-FU-curcumin synergism using the TNBC pre-clinical model. Our study also confirmed the pharmacological safety of this chemotherapeutic plus phytoactive combination using acute and chronic toxicity studies in Swiss albino mice. Subsequently, the molecular docking analysis of curcumin binding to TS demonstrated the affinity of curcumin towards the cofactor-binding site of TS, rather than the substrate-binding site, where 5-FU binds. Our concomitant in vivo and in silico evidence substantiates the superior therapeutic index of this combination. CONCLUSION: This is the first-ever pre-clinical study portraying TS as the critical target of combinatorial therapy for mammary carcinomas and therefore we recommend its clinical validation, especially in TNBC patients, who currently have limited therapeutic options.

Biomechanical comparison of screw-in suture anchor-suture combinations used for Bankart repair.

INTRODUCTION: Bankart repair laxity may contribute to pathologic joint instability. This biomechanical study compared two screw-in suture anchor-suture combinations under tensile loads. METHODS: Twelve pairs of scapulae were implanted with either a 3 mm diameter, 14 mm long poly-L/D-lactide suture anchor with a suture eyelet (Group 1) or a 3.1 mm diameter, 11 mm long polylactide suture anchor with a molded eyelet (Group 2). Constructs were cyclically loaded between 25 and 50 N with a 25 N load increase every 25 cycles. RESULTS: Group 2 displayed greater displacement at failure, had more specimens with > or =2 mm displacement by the 50 N interval (P = 0.014), and had displaced more by 100 N (P < or = 0.046). Group 1 displayed a stronger load-displacement at failure relationship than Group 2 (r (2) = 0.67 vs. r (2) = 0.37). CONCLUSION: Construct differences may influence decisions regarding the required number of suture anchor-suture loops, the rehabilitation timetable, and the timing of return to unrestricted activities.

Tsc1 Regulates the Proliferation Capacity of Bone-Marrow Derived Mesenchymal Stem Cells.

TSC1 is a tumor suppressor that inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). TSC1 mutations are associated with Tuberous Sclerosis Complex (TSC), characterized by multiple benign tumors of mesenchymal and epithelial origin. TSC1 modulates self-renewal and differentiation in hematopoietic stem cells; however, its effects on mesenchymal stem cells (MSCs) are unknown. We investigated the impact of Tsc1 inactivation in murine bone marrow (BM)-MSCs, using tissue-specific, transgelin (Tagln)-mediated cre-recombination, targeting both BM-MSCs and smooth muscle cells. Tsc1 mutants were viable, but homozygous inactivation led to a dwarfed appearance with TSC-like pathologies in multiple organs and reduced survival. In young (28 day old) mice, Tsc1 deficiency-induced significant cell expansion of non-hematopoietic BM in vivo, and MSC colony-forming potential in vitro, that was normalized upon treatment with the mTOR inhibitor, everolimus. The hyperproliferative BM-MSC phenotype was lost in aged (1.5 yr) mice, and Tsc1 inactivation was also accompanied by elevated ROS and increased senescence. ShRNA-mediated knockdown of Tsc1 in BM-MSCs replicated the hyperproliferative BM-MSC phenotype and led to impaired adipogenic and myogenic differentiation. Our data show that Tsc1 is a negative regulator of BM-MSC proliferation and support a pivotal role for the Tsc1-mTOR axis in the maintenance of the mesenchymal progenitor pool.

Oxidative Stress and Antioxidant Status in High-Risk Prostate Cancer Subjects.

The oxidant/antioxidant balance has been implicated in the pathophysiology of prostate cancer. We investigated oxidative damage and antioxidant status in high-risk prostate cancer subjects. Reduced glutathione (GSH) levels were measured in erythrocytes, 8-hydroxydeoxyguanosine (8-OHdG) in leukocytes and plasma levels of catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-R), glutathione S-transferase (GST), superoxide dismutase (SOD), and lipid peroxide products were measured in high-risk and age-matched healthy subjects. Serum PSA levels were significantly higher (p < 0.0001) in high-risk subjects, whereas GST (p < 0.0001) and GSH (p < 0.002) were higher in healthy controls. Levels of 8-OHdG, an oxidized nucleoside of DNA, were significantly increased (p < 0.0001) in high-risk subjects. No marked difference in the levels of CAT (p = 0.237), GSH-Px (p = 0.74), GSH-R (p = 0.344), SOD (p = 0.109), and lipid peroxide products (p = 0129) were observed between two groups. Pearson's correlation between GST and PSA (r = -0.69 (p < 0.0001)), GST and 8-OHdG (r = -0.62 (p < 0.0004)), GSH and 8-OHdG (r= -0.39 (p = 0.038)), and CAT and GSH-Px (r= -0.33 (p = 0.04)) were found to be negatively correlated, whereas 8-OHdG and PSA were positively associated (r= 0.57 (p < 0.002). These results indicate a significant role of oxidative damage in prostate carcinogenesis, particularly during the early stages of development. In conclusion, our data support the importance of antioxidant defense as a valuable diagnostic and/or prognostic marker in prostate cancer.

Oncolytic virotherapy for small-cell lung cancer induces immune infiltration and prolongs survival.

Oncolytic virotherapy has been proposed as an ablative and immunostimulatory treatment strategy for solid tumors that are resistant to immunotherapy alone; however, there is a need to optimize host immune activation using preclinical immunocompetent models in previously untested common adult tumors. We studied a modified oncolytic myxoma virus (MYXV) that shows high efficiency for tumor-specific cytotoxicity in small-cell lung cancer (SCLC), a neuroendocrine carcinoma with high mortality and modest response rates to immune checkpoint inhibitors. Using an immunocompetent SCLC mouse model, we demonstrated the safety of intrapulmonary MYXV delivery with efficient tumor-specific viral replication and cytotoxicity associated with induction of immune cell infiltration. We observed increased SCLC survival following intrapulmonary MYXV that was enhanced by combined low-dose cisplatin. We also tested intratumoral MYXV delivery and observed immune cell infiltration associated with tumor necrosis and growth inhibition in syngeneic murine allograft tumors. Freshly collected primary human SCLC tumor cells were permissive to MYXV and intratumoral delivery into patient-derived xenografts resulted in extensive tumor necrosis. We confirmed MYXV cytotoxicity in classic and variant SCLC subtypes as well as cisplatin-resistant cells. Data from 26 SCLC human patients showed negligible immune cell infiltration, supporting testing MYXV as an ablative and immune-enhancing therapy.

NSC30049 inhibits Chk1 pathway in 5-FU-resistant CRC bulk and stem cell populations.

The 5-fluorouracil (5-FU) treatment induces DNA damage and stalling of DNA replication forks. These stalled replication forks then collapse to form one sided double-strand breaks, leading to apoptosis. However, colorectal cancer (CRC) stem cells rapidly repair the stalled/collapsed replication forks and overcome treatment effects. Recent evidence suggests a critical role of checkpoint kinase 1 (Chk1) in preventing the replicative stress. Therefore, Chk1 kinase has been a target for developing mono or combination therapeutic agents. In the present study, we have identified a novel orphan molecule NSC30049 (NSC49L) that is effective alone, and in combination potentiates 5-FU-mediated growth inhibition of CRC heterogeneous bulk and FOLFOX-resistant cell lines in culture with minimal effect on normal colonic epithelial cells. It also inhibits the sphere forming activity of CRC stem cells, and decreases the expression levels of mRNAs of CRC stem cell marker genes. Results showed that NSC49L induces 5-FU-mediated S-phase cell cycle arrest due to increased load of DNA damage and increased γ-H2AX staining as a mechanism of cytotoxicity. The pharmacokinetic analysis showed a higher bioavailability of this compound, however, with a short plasma half-life. The drug is highly tolerated by animals with no pathological aberrations. Furthermore, NSC49L showed very potent activity in a HDTX model of CRC stem cell tumors either alone or in combination with 5-FU. Thus, NSC49L as a single agent or combined with 5-FU can be developed as a therapeutic agent by targeting the Chk1 pathway in 5-FU-resistant CRC heterogeneous bulk and CRC stem cell populations.

Efficient Gene Delivery and Expression in Pancreas and Pancreatic Tumors by Capsid-Optimized AAV8 Vectors.

Despite efforts to use adeno-associated viral (AAV) vector-mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F; and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to KrasG12D+/-, KrasG12D+/-/Pten+/-, and wild-type mice. The transduction efficiency of these vectors expressing the mCherry reporter gene was evaluated 2 weeks post administration in pancreas or PDAC and correlated with viral genome copy numbers. Our comparative and quantitative analyses of the transduction profiles demonstrated that the Y-to-F double-mutant exhibited the highest mCherry expression in pancreatic tissues (range 45-70%) compared with WT-AAV8 (7%; p < 0.01). We also detected a 7-fold higher level of vector genome copy numbers in normal pancreas following transduction with the double-mutant AAV8 compared with WT-AAV8 (10,285 vs. 1,500 vector copies/µg DNA respectively, p < 0.05). In addition, we observed that intraperitoneal injection of the double-mutant AAV8 led to a 15-fold enhanced transduction efficiency as compared to WT-AAV8 in mouse PDAC, with a corresponding ∼14-fold increase in vector genome copy numbers (26,575 vs. 2,165 copies/µg DNA respectively, p < 0.05). These findings indicate that the Y447+Y733F-AAV8 leads to a significant enhancement of transduction efficiency in both normal and malignant pancreatic tissues, suggesting the potential use of this vector in targeting pancreatic diseases in general, and PDAC in particular.

A biomechanical comparison of the FasT-Fix meniscal repair suture system and the RapidLoc device in cadaver meniscus.

PURPOSE: This biomechanical study compared the fixation characteristics of horizontally or vertically implanted FasT-Fix devices (Smith & Nephew, Endoscopy Division, Andover, MA) consisting of two 5-mm PLLA suture T-bar anchors with a pretied self-sliding knot (No. 0 nonabsorbable, USP, braided polyester suture material) and the RapidLoc device (Mitek Surgical Products, Westwood, MA) consisting of a PLLA T-bar anchor or "backstop," a connecting suture (No. 2 nonbiodegradable Ethibond; Ethicon, Somerville, NJ), and a PLLA grommet, for repairing posterior third lesions in human menisci. TYPE OF STUDY: Controlled laboratory biomechanical study. METHODS: After repair of a vertical longitudinal meniscus lesion with either vertically or horizontally implanted FasT-Fix devices or RapidLoc devices, 3 groups of 6 specimens underwent cyclic loading (5 mm/minute, cycling between 5 and 50 N at 1 Hz for 500 cycles) before load to failure testing on a servo hydraulic device. One-way analysis of variance and Tukey HSD post hoc tests were used to evaluate group differences (P < .05). RESULTS: The vertical FasT-Fix device group (3.2 +/- 0.49 mm) had less displacement after cyclic testing than either the horizontal FasT-Fix (4.4 +/- 0.73 mm, P = .003) or the RapidLoc (4.6 +/- 0.22 mm, P = .002) device groups. The vertical FasT-Fix device group had greater stiffness during cyclic testing (14.4 +/- 2.1 N/mm) than the horizontal FasT-Fix (10.4 +/- 1.6 N/mm, P = .0001) or the RapidLoc (9.7 +/- 0.44 N/mm, P = .0001) device groups. During load to failure testing, the vertical FasT-Fix group (125.3 +/- 39 N) had 28% greater strength than the horizontal FasT-Fix device group (89.7 +/- 14 N, P = .02) and 30% greater strength than the RapidLoc device group (87.1 +/- 13 N, P = .028), whereas displacement and stiffness did not show statistically significant group differences. CONCLUSIONS: The vertical FasT-Fix group had superior biomechanical characteristics for meniscal fixation during cyclic and load to failure testing compared with horizontal FasT-Fix or RapidLoc devices. CLINICAL RELEVANCE: Although the RapidLoc devices provided fixation characteristics comparable to horizontally implanted FasT-Fix devices, vertically implanted FasT-Fix devices may provide superior all-inside fixation.

Biomechanical comparison of the bioabsorbable RetroScrew system, BioScrew XtraLok with stress equalization tensioner, and 35-mm Delta Screws for tibialis anterior graft-tibial tunnel fixation in porcine tibiae.

BACKGROUND: Achieving effective soft tissue graft-tibial tunnel fixation remains problematic. HYPOTHESIS: No differences would exist for tibialis anterior graft-tibial tunnel fixation when comparing the RetroScrew System (20-mm retrograde screw, 17-mm antegrade screw), the 35-mm tapered Delta Screw (manual tensioning), and the 35-mm BioScrew XtraLok (applied using an instrumented tensioner). STUDY DESIGN: Controlled laboratory study. METHODS: Porcine tibiae (apparent bone mineral density, 1.3 g/cm(2)) and human tendon allografts were divided into 3 matched groups of 6 specimens each before cyclic (500 cycles, 50-250 N, 1 Hz) and load-to-failure (20 mm/min) tests. RESULTS: The BioScrew XtraLok (210.9 +/- 54.9 N/mm) and the 35-mm Delta Screw (224.3 +/- 43.7 N/mm) displayed superior stiffness to the RetroScrew System (114.1 +/- 23.3 N/mm) (P = .0004) during cyclic testing. The BioScrew XtraLok (1.0 +/- 0.2 mm) and the Delta Screw (0.9 +/- 0.2 mm) also displayed less displacement during cyclic testing than the RetroScrew System (1.8 +/- 0.5 mm) (P = .001). During load-to-failure testing, the BioScrew XtraLok withstood greater loads (1436.3 +/- 331.3 N) (P = .001) and displayed greater stiffness (323.6 +/- 56.8 N/mm) (P = .002) than the 35-mm Delta Screw (load, 1042.2 +/- 214.4 N; stiffness, 257.2 +/- 22.2 N/mm) and the RetroScrew System (load, 778.7 +/- 177.5 N; stiffness, 204.4 +/- 52.9 N/mm). CONCLUSION: The BioScrew XtraLok with instrumented tensioning displayed superior fixation to the RetroScrew System and the 35-mm Delta Screw applied with manual tensioning. CLINICAL RELEVANCE: The BioScrew XtraLok may provide superior soft tissue graft-tibial tunnel fixation. Further in vitro studies using human tissue and in vivo clinical studies are needed.

Salvage rotator cuff repair using a biotenodesis screw.

Rotator cuff tear fixation can be difficult when humeral head bone stock is poor as a result of osteopenia or osteoporosis. This technical note describes biotenodesis screw use to achieve effective fixation after 2 previous attempts at implanting suture anchors failed.

Targeting Focal Adhesion Kinase and Resistance to mTOR Inhibition in Pancreatic Neuroendocrine Tumors.

BACKGROUND: Focal adhesion kinase (FAK) mediates survival of normal pancreatic islets through activation of AKT. Upon malignant transformation of islet cells into pancreatic neuroendocrine tumors (PanNETs), AKT is frequently overexpressed and mutations in the AKT/mTOR pathway are detected. Because mTOR inhibitors rarely induce PanNET tumor regression, partly because of feedback activation of AKT, novel combination strategies are needed to target FAK/AKT/mTOR signaling. METHODS: We characterized the activation of FAK in PanNETs using immunohistochemistry and Western blot analysis and tested the FAK inhibitor PF-04554878 in human PanNET cells in vitro and in vivo (at least three mice per group). In addition, we evaluated the effect of combined FAK and mTOR inhibition on PanNET viability and apoptosis. All statistical tests were two-sided. RESULTS: We found that FAK is overexpressed and hyperphosphorylated in human PanNETs and that PF-04554878 strongly inhibited FAK (Tyr397) autophosphorylation in a dose-dependent manner. We found that PF-04554878 inhibited cell proliferation and clonogenicity and induced apoptosis in PanNET cells. Moreover, oral administration of PF-04554878 statistically significantly reduced tumor growth in a patient-derived xenograft model of PanNET (P = .02) and in a human PanNET xenograft model of peritoneal carcinomatosis (P = .03). Importantly, PF-04554878 synergized with the mTOR inhibitor everolimus by preventing feedback AKT activation. CONCLUSIONS: We demonstrate for the first time that FAK is overexpressed in PanNETs and that inhibition of FAK activity induces apoptosis and inhibits PanNET proliferation. We found that the novel FAK inhibitor PF-04554878 synergizes with everolimus, a US Food and Drug Administration-approved agent for PanNETs. Our findings warrant the clinical investigation of combined FAK and mTOR inhibition in PanNETs.

Determination of organochlorine pesticides in agricultural soil with special reference to gamma-HCH degradation by Pseudomonas strains.

Soil samples were taken from different agricultural fields and analyzed for organochlorine pesticide residues by gas chromatography. The analysis indicated that the soil samples contained some common organochlorine pesticides DDT, DDD, DDE, HCH and Aldrin. gamma-HCH was detected as 47.35 ppb whereas the concentrations of alpha-HCH, beta-HCH, p('),p(')-DDE, o('),p(')-DDT were 38.81, 1.79, 7.10 and 13.30 ppb, respectively, in the same soil. Two Pseudomonas strains isolated from agricultural soil were found to possess gamma-hexachlorocyclohexane degrading ability when the isolates were grown in a mineral salt medium containing gamma-HCH as the sole source of carbon and a number of metabolites were produced and detected by the gas chromatography. These bacterial isolates were further tested for carbohydrate and amino acid utilization as well as for their susceptibility against 10 commonly used antibiotics namely amoxycillin, chloramphenicol, cloxacillin, doxycycline, methicillin, nalidixic acid, neomycin, nitrofurantoin, streptomycin and tetracycline. Both the isolates were also screened for plasmid DNA and found to harbour a single plasmid.

Second generation intramedullary nailing of subtrochanteric femur fractures: a biomechanical study of fracture site motion.

OBJECTIVES: To compare fracture site motion between different second-generation intramedullary nails used to fix subtrochanteric fractures of the proximal femur with and without femoral neck fractures. DESIGN: Nondestructive mechanical testing of four types of femoral intramedullary nails was undertaken to evaluate fracture site motion using a model that simulated single-leg and double-leg stance. METHODS: Three types of reconstruction nails (the Russell-Taylor Delta [Smith & Nephew, Memphis, TN], the Uniflex [Biomet, Warsaw, IN], Alta CFX [Howmedica-Osteonics, Rutherford, NJ]) and the Long Gamma nail (Howmedica-Osteonics, Rutherford, NJ), each measuring 11 x 380 mm, were inserted in fiberglass composite femurs. Four fracture patterns were studied (transverse subtrochanteric, subtrochanteric with posteromedial wedge comminution, subtrochanteric with one-centimeter gap, and a one-centimeter gap with a subcapital neck fracture). Single-and double-leg stance loading was simulated using a servohydraulic load frame (MTS, Eden Prairie, MN). Two-way analysis of variance and post hoc t tests were used to determine any statistically significant differences between groups. RESULTS: In single-leg stance there were significant differences in coronal plane rotation, shear, and axial translation across the subtrochanteric fracture site between the different nail types and the different fracture patterns (p < 0.001). In double-leg stance there were significant differences in coronal plane rotation and femoral head vertical motion between the different nail types and the different fracture patterns (p < 0.001), and there were significant differences in shear and axial translation between the different fracture patterns (p < 0.001) but not the different nail types (p > 0.05). CONCLUSIONS: For simple, well-reduced fractures the choice of implant is not critical. As fracture severity increased (comminution, gap, and combined neck fracture), the choice of implant, particularly with reference to proximal nail dimensions and implant materials, was a significant factor in reducing fracture site motion. Therefore, our laboratory data suggest that when subtrochanteric fractures are unstable (e.g., comminution, segmental bone loss) and early weight bearing is desirable, the choice of implant is critical and should be restricted to implants that allow minimal fracture site motion (Long Gamma and Russell-Taylor).

Endopearl augmentation of bioabsorbable interference screw fixation of a soft tissue tendon graft in a tibial tunnel.

In this technical note, we introduce a new technique for tibial side soft tissue graft fixation using a bioabsorbable interference screw augmented with an EndoPearl (Linvatec, Largo, FL) for ACL reconstruction. This technique will enable the knee surgeon to use shorter grafts while minimizing slippage without reducing fixation strength.