Discovery and Optimization of Tambjamines as a Novel Class of Antileishmanial Agents.

Leishmaniasis is a neglected tropical disease that is estimated to afflict over 12 million people. Current drugs for leishmaniasis suffer from serious deficiencies, including toxicity, high cost, modest efficacy, primarily parenteral delivery, and emergence of widespread resistance. We have discovered and developed a natural product-inspired tambjamine chemotype, known to be effective against Plasmodium spp, as a novel class of antileishmanial agents. Herein, we report in vitro and in vivo antileishmanial activities, detailed structure-activity relationships, and metabolic/pharmacokinetic profiles of a large library of tambjamines. A number of tambjamines exhibited excellent potency against both Leishmania mexicana and Leishmania donovani parasites with good safety and metabolic profiles. Notably, tambjamine 110 offered excellent potency and provided partial protection to leishmania-infected mice at 40 and/or 60 mg/kg/10 days of oral treatment. This study presents the first account of antileishmanial activity in the tambjamine family and paves the way for the generation of new oral antileishmanial drugs.

Occurrence and Predictors of Postoperative Preauricular and Masticatory Muscle Pain Symptoms After Surgical Removal of Third Molars: A Single-Blind Randomized Controlled Trial Comparing Dental Assistants Supporting the Mandible and the Restful Jaw Device.

BACKGROUND: During third molar removal, the mandible is supported by a dental assistant (DA) to counter downward forces during surgery, and with sedation, to maintain airway patency. The Restful Jaw device (PEP Design; Saint Paul) provides this support instead of the DA. PURPOSE: This study compared the occurrence of postoperative preauricular and masticatory muscle pain symptoms (PMMPS) between the device and DAs providing mandibular support, using two outcome measures. Secondary aims identify predictors of outcome and providers' opinions of the device. STUDY DESIGN, SETTING, SAMPLE: In this multisite, single-blind, two-arm parallel randomized trial, participants without preoperative PMMPS had surgical removal of third molars, with sedation and bite blocks were randomly assigned to manual support or the device. EXPOSURE VARIABLE: The exposed group was randomly assigned to the device and the nonexposed group to manual support. MAIN OUTCOME VARIABLE(S): The primary outcome was patient-reported PMMPS. Two secondary outcomes were pain assessed with the temporomandibular disorder Pain Screener and providers' views on the device. Outcomes were assessed at 1-, 3-, and 6-month postsurgery. COVARIATES: The covariates are baseline demographics (eg, sex), clinical characteristics (eg, eruption status), and third molar surgeries. ANALYSES: For occurrence of pain, generalized estimating equations assessed differences between groups. Logistic regression analysis assessed predictors of pain at 1 month, per the Screener. The level for statistical significance was 5%. RESULTS: Enrollment was 86 and 83 participants in the device and DA groups, respectively. The average age was 20.8 years; the majority were female (65%) and Caucasian (66%). The retention rate was ≥95.9%. The groups did not differ significantly for occurrence of pain using the primary and secondary outcome measures at any follow-up (P ≥ .46). Fully impacted molars were associated with occurrence of pain (odds ratio = 3.44; 95% confidence interval 1.49-7.92; P = .004). CONCLUSION AND RELEVANCE: Occurrence of pain using the primary and secondary outcome measures did not differ significantly between groups at any follow-up and was associated with removal of fully impacted third molars. Four out of five surgeons reported wanting to use the device on a regular basis when performing this procedure in sedated patients.

Impact of Blast Overpressure on the Pharmacokinetics of Various Antibiotics in Sprague Dawley Rats.

INTRODUCTION: Combat injuries are complex and multimodal. Most injuries to the extremities occur because of explosive devices such as improvised explosive devices. Blast exposure dramatically increases the risk of infection in combat wounds, and there is limited available information on the best antibiotic treatments for these injuries. We previously demonstrated that mice exposed to blast displayed a delayed clearance of cefazolin from the plasma and liver; further semi-mechanistic modeling determined that cefazolin concentrations in the skin of these mice were reduced. Our objective was to investigate the effects of blast on the pharmacokinetics of antibiotics of different types used for the treatment of combat wounds in the rat model. MATERIALS AND METHODS: Male Sprague Dawley rats were exposed to blast overpressure followed by injection of a bolus of animal equivalent doses of an antibiotic (cefazolin, cefepime, ertapenem, or clindamycin) into the tail vein at 1-hour post-blast exposure. Blood was collected at predetermined time points via repeated sampling from the tail vein. Animals were also euthanized at predetermined time points, at which time liver, kidney, skin, and blood via cardiac puncture were collected. Antibiotic concentrations were determined by ultra-performance liquid chromatography-tandem mass spectrometry. RESULTS: Blast-exposed rats exhibited a similar rate of clearance compared to non-blasted rats in the blood, liver, kidney, and skin, which is inconsistent with the data regarding cefazolin in blast-exposed mice. CONCLUSIONS: Our results in rats do not recapitulate our previous observation of delayed cefazolin clearance in mice following the blast overpressure exposure. Although using rats permitted us to collect multiple blood samples from the same animals, rats may not be a suitable model for measuring the pharmacokinetics of antibiotics following blast. The interpretation of the results may be challenging because of variation in data among rat subjects in the same sample groups.

Systematic Review and Meta-Analysis of the Effect of Loop Diuretics on Antibiotic Pharmacokinetics.

Loop diuretics and antibiotics are commonly co-prescribed across many clinical care settings. Loop diuretics may alter antibiotic pharmacokinetics (PK) via several potential drug interactions. A systematic review of the literature was performed to investigate the impact of loop diuretics on antibiotic PK. The primary outcome metric was the ratio of means (ROM) of antibiotic PK parameters such as area under the curve (AUC) and volume of distribution (Vd) on and off loop diuretics. Twelve crossover studies were amenable for metanalysis. Coadministration of diuretics was associated with a mean 17% increase in plasma antibiotic AUC (ROM 1.17, 95% CI 1.09-1.25, I2 = 0%) and a mean decrease in antibiotic Vd by 11% (ROM 0.89, 95% CI 0.81-0.97, I2 = 0%). However, the half-life was not significantly different (ROM 1.06, 95% CI 0.99-1.13, I2 = 26%). The remaining 13 observational and population PK studies were heterogeneous in design and population, as well as prone to bias. No large trends were collectively observed in these studies. There is currently not enough evidence to support antibiotic dosing changes based on the presence or absence of loop diuretics alone. Further studies designed and powered to detect the effect of loop diuretics on antibiotic PK are warranted in applicable patient populations.

Beta-Lactam Probability of Target Attainment Success: Cefepime as a Case Study.

INTRODUCTION: Probability of target attainment (PTA) analysis using Monte Carlo simulations has become a mainstay of dose optimization. We highlight the technical and clinical factors that may affect PTA for beta-lactams. METHODS: We performed a mini review in adults to explore factors relating to cefepime PTA success and how researchers incorporate PTA into dosing decisions. In addition, we investigated, via simulations with a population pharmacokinetic (PK) model, factors that may affect cefepime PTA success. RESULTS: The mini review included 14 articles. PTA results were generally consistent, given the differences in patient populations. However, dosing recommendations were more varied and appeared to depend on the definition of pharmacodynamic (PD) target, definition of PTA success and specific clinical considerations. Only 3 of 14 articles performed formal toxicological analysis. Simulations demonstrated that the largest determinants of cefepime PTA were the choice of PD target, continuous vs. intermittent infusion and creatinine clearance. Assumptions for protein binding, steady state vs. first dose, and simulating different sampling schemes may impact PTA success under certain conditions. The choice of one or two compartments had a minimal effect on PTA. CONCLUSIONS: PTA results may be similar with different assumptions and techniques. However, dose recommendation may differ significantly based on the selection of PD target, definition of PTA success and considerations specific to a patient population. Demographics and the PK parameters used to simulate time-concentration profiles should be derived from patient data applicable to the purpose of the PTA. There should be strong clinical rationale for dose selection. When possible, safety and toxicity should be considered in addition to PTA success.

Pharmacokinetics of piperacillin and tazobactam in critically Ill patients treated with continuous kidney replacement therapy: A mini-review and population pharmacokinetic analysis.

WHAT IS KNOWN AND OBJECTIVE: Timely and appropriate dosing of antibiotics is essential for the treatment of bacterial sepsis. Critically ill patients treated with continuous kidney replacement therapy (CKRT) often have physiologic derangements that affect pharmacokinetics (PK) of antibiotics and dosing may be challenging. We sought to aggregate previously published piperacillin and tazobactam (pip-tazo) pharmacokinetic data in critically ill patients undergoing CKRT to better understand pharmacokinetics of pip-tazo in this population and better inform dosing. METHODS: The National Library of Medicine Database was searched for original research containing piperacillin or tazobactam clearance (CL) or volume of distribution (V) estimates in patients treated with CKRT. The search yielded 77 articles, of which 26 reported suitable estimates of CL or V. Of the 26 articles, 10 for piperacillin and 8 for tazobactam had complete information suitable for population pharmacokinetic modelling. Also included in the analysis was piperacillin and tazobactam PK data from 4 critically ill patients treated with CKRT in the Military Health System, 2 with burn and 2 without burn. RESULTS AND DISCUSSION: Median and range of literature reported PK parameters for piperacillin (CL 2.76 L/hr, 1.4-7.92 L/hr, V 31.2 L, 16.77-42.27 L) and tazobactam (CL 2.34 L/hr, 0.72-5.2 L/hr, V 36.6 L, 26.2-58.87 L) were highly consistent with population estimates (piperacillin CL 2.7 L/hr, 95%CI 1.99-3.41 L/hr, V 25.83 22.07-29.59 L, tazobactam CL 2.49 L/hr, 95%CI 1.55-3.44, V 30.62 95%CI 23.7-37.54). The proportion of patients meeting pre-defined pharmacodynamic (PD) targets (median 88.7, range 71%-100%) was high despite significant mortality (median 44%, range 35%-60%). High mortality was predicted by baseline severity of illness (median APACHE II score 23, range 21-33.25). Choice of lenient or strict PD targets (ie 100%fT >MIC or 100%fT >4XMIC) had the largest impact on probability of target attainment (PTA), whereas presence or intensity of CKRT had minimal impact on PTA. WHAT IS NEW AND CONCLUSION: Pip-tazo overexposure may be associated with increased mortality, although this is confounded by baseline severity of illness. Achieving adequate pip-tazo exposure is essential; however, risk of harm from overexposure should be considered when choosing a PD target and dose. If lenient PD targets are desired, doses of 2250-3375 mg every 6 h are reasonable for most patients receiving CKRT. However, if a strict PD target is desired, continuous infusion (at least 9000-13500 mg per day) may be required. However, some critically ill CKRT populations may need higher or lower doses and dosing strategies should be tailored to individuals based on all available clinical data including the specific critical care setting.

Factors Associated With the Mental Health and Satisfaction of Oral and Maxillofacial Surgery Residents in the United States: A Cross-Sectional Study and Analysis.

PURPOSE: Residents in training have reported high levels of stress and anxiety and have a greater risk of mental health problems compared with the general population. Mental health problems among residents have been correlated with decreased professional effectiveness, increased medical errors, emotional exhaustion, and depersonalization and could have significant negative effects on future practitioners. The purpose of the present study was to identify the factors that might be associated with the mental health and satisfaction of oral and maxillofacial surgery (OMS) residents and to determine the associations between these factors and OMS resident satisfaction as a surrogate of resident well-being. MATERIALS AND METHODS: We designed and implemented an online survey, which was e-mailed to all OMS residents in the 101 accredited training programs in the United States. The survey was designed to determine and assess the factors associated with OMS resident satisfaction. Differences between groups were tested using 1-way analysis of variance for continuous variables and χ2 tests for categorical variables. For each factor, we fit a logistic regression model to estimate the odds ratio of resident satisfaction for the factor, adjusting for gender, year in residency, and years of advanced training. RESULTS: Of the 1181 resident surveys sent out, 300 were completed (25.4% response rate). The satisfied OMS residents tended to be men, further along in their training program, and to have access to mental health resources. Dissatisfaction was associated with greater self-reported stress levels, working a greater number of hours per week, and believing one would be viewed differently for speaking to faculty about mental health. CONCLUSIONS: OMS resident satisfaction was associated with identifiable and potentially modifiable factors. These factors included workload characteristics, stress and coping ability, and mental health impact and resource availability. Evidence-based strategies for OMS resident well-being could lead to the development of best practice guidelines for promoting and optimizing resident mental health.

Investigation of an Opioid Prescribing Protocol After Third Molar Extraction Procedures.

PURPOSE: The United States is experiencing an epidemic of opioid overdoses and deaths. The relation between prescription opioids and opioid abuse is well documented. Oral and maxillofacial surgeons and other dentists are proportionately among the most prevalent prescribers of opioids. Practitioners are looking for evidence-based ways to decrease excess opioid prescriptions and adequately manage postoperative pain. The authors recently analyzed the impact of a mandated nonopioid prescribing protocol at their institution. Although broad guidelines have been useful for treating postoperative pain, there are no procedure-specific guidelines for managing pain after third molar extraction. The purpose of this study was to determine whether an opioid prescribing protocol was sufficient to decrease opioid prescribing after third molar extractions. MATERIALS AND METHODS: This retrospective study compared the use of opioids prescribed for patients undergoing third molar extraction before introducing and after implementing a postoperative opioid prescribing protocol. The inclusion criterion was third molar extraction performed at the Division of Oral and Maxillofacial Surgery at the University of Minnesota (Minneapolis, MN) during the fourth quarters of 2015 and 2017 with complete records. RESULTS: The number of opioid prescriptions decreased and the number of nonopioid analgesics prescribed increased for all procedure codes after implementation of the protocol. Higher Current Dental Terminology (CDT) codes were associated with increased opioid prescriptions, indicating increased surgical difficulty was a rationale for opioid prescriptions. The mean number of opioid tablets per prescription was 15.9 in 2015 and decreased to 11.5 in 2017. No statistical difference was observed for average tablets for various CDT codes. CONCLUSION: Data from this study suggest an acute postoperative pain opioid prescribing protocol leads to fewer opioid prescriptions after third molar extraction procedures, less variance in opioid prescribing among practitioners, a decreased number of opioid tablets prescribed per patient, and safe and effective management of acute postoperative pain.

Addressing the Opioid Epidemic: Impact of Opioid Prescribing Protocol at the University of Minnesota School of Dentistry.

Prescription opioid medications continue to be abused on an epidemic level and have been shown to be a "gateway" drug to heroin abuse. Individuals experimenting with opioids commonly fall in the 10- to 19-year age range in which dentists are the highest prescribers. To reduce the number of excess opioids, the Department of Oral and Maxillofacial Surgery, University of Minnesota, developed and implemented an evidence-based opioid prescribing policy. Data were collected via electronic health record for the previous year and compared with the year following the protocol implementation. The results showed a drastic decrease (>46%) in the number of prescriptions given over a 1-year period. All departments reported a decrease in opioid prescriptions and the average number of tablets per prescription. The concern of undertreating pain was not found to be significant, as there was no increase in after-hours calls, recall appointments, or documentable emergency room visits. The results support the efficacy of an opioid prescribing policy's ability to lower the frequency and number of opioids given to patients, while still adequately treating patients' pain. Continued evaluation and modifications of the protocol and close monitoring of prescriber habits will enhance patients' pain control while also limiting the number of opioids available for abuse.

Periodic slow slip triggers megathrust zone earthquakes in northeastern Japan.

Both aseismic and seismic slip accommodate relative motion across partially coupled plate-boundary faults. In northeastern Japan, aseismic slip occurs in the form of decelerating afterslip after large interplate earthquakes and as relatively steady slip on uncoupled areas of the subduction thrust. Here we report on a previously unrecognized quasi-periodic slow-slip behavior that is widespread in the megathrust zone. The repeat intervals of the slow slip range from 1 to 6 years and often coincide with or precede clusters of large [magnitude (M) ≥ 5] earthquakes, including the 2011 M 9 Tohoku-oki earthquake. These results suggest that inherently periodic slow-slip events result in periodic stress perturbations and modulate the occurrence time of larger earthquakes. The periodicity in the slow-slip rate has the potential to help refine time-dependent earthquake forecasts.

Promoter trapping method: transcription factor purification using human telomerase reverse transcriptase promoter.

BACKGROUND: Transcription factors bind to response elements on the promoter regions of genes to regulate transcriptional activity. One of the major problems with identifying transcription factors is their low abundance relative to other proteins in the cell. Developing a purification technique specific for transcription factors is crucial to the understanding of gene regulation. Promoter trapping is a method developed that uses the promoter regions as bait to trap proteins of interest and then purified using column chromatography. Here we utilize this technique to study the telomerase promoter, which has increased transcriptional activity in cancer cells. Gaining insight on how to control the enzyme at the promoter level may give new routes towards cancer treatments. RESULTS: Our findings show that the telomerase promoter (-170 - +91) and Promoter Trapping isolate a transcriptionally active and reproducible complex, when analyzed by liquid chromatography tandem mass spectrometry. We were also able to identify transcription factors, including AP-2 and SP1 known to bind this promoter, as well as show that these two proteins can bind to each other's response element. CONCLUSION: Here we focus on verifying the ability and versatility of Promoter Trapping coupled with additional well-characterized methods to identify already known factors responsible for telomerase transcriptional regulation.

Anti-CRLF2 Antibody-Armored Biodegradable Nanoparticles for Childhood B-ALL.

B-precursor acute lymphoblastic leukemia (B-ALL) lymphoblast (blast) internalization of anti-cytokine receptor-like factor 2 (CRLF2) antibody-armored biodegradable nanoparticles (AbBNPs) are investigated. First, AbBNPsaere synthesized by adsorbing anti-CRLF2 antibodies to poly(D,L-lactide- co -glycolide) (PLGA) nanoparticles of various sizes and antibody surface density (Ab/BNP) ratios. Second, AbBNPs are incubated with CRLF2-overexpressing (CRLF2+) or control blasts. Third, internalization of AbBNPs by blasts is evaluated by multicolor flow cytometry as a function of receptor expression, AbBNP size, and Ab/BNP ratio. Results from these experiments are con-firmed by electron microscopy, fluorescence microscopy, and Western blotting. The optimal size and Ab/BNP for internalization of AbBNPs by CRLF2+ blasts is 50 nm with 10 Ab/BNP and 100 nm with 25 Ab/BNP. These studies show that internalization of AbBNPs in childhood B-ALL blasts is AbBNP size-and Ab/BNP ratio-dependent. All AbBNP combinations are non-cytotoxic. It is also shown that CD47 is very slightly up-regulated by blasts exposed to AbBNPs. CD47 is "the marker of self" overexpressed by blasts to escape phagocytosis, or "cellular devouring", by beneficial macrophages. The results indicate that precise engineering of AbBNPs by size and Ab/BNP ratio may improve the internalization and selectivity of future biodegradable nanoparticles for the treatment of leukemia patients, including drug-resistant minority children and Down's syndrome patients with CRLF2+B-ALL.

Fault healing promotes high-frequency earthquakes in laboratory experiments and on natural faults.

Faults strengthen or heal with time in stationary contact, and this healing may be an essential ingredient for the generation of earthquakes. In the laboratory, healing is thought to be the result of thermally activated mechanisms that weld together micrometre-sized asperity contacts on the fault surface, but the relationship between laboratory measures of fault healing and the seismically observable properties of earthquakes is at present not well defined. Here we report on laboratory experiments and seismological observations that show how the spectral properties of earthquakes vary as a function of fault healing time. In the laboratory, we find that increased healing causes a disproportionately large amount of high-frequency seismic radiation to be produced during fault rupture. We observe a similar connection between earthquake spectra and recurrence time for repeating earthquake sequences on natural faults. Healing rates depend on pressure, temperature and mineralogy, so the connection between seismicity and healing may help to explain recent observations of large megathrust earthquakes which indicate that energetic, high-frequency seismic radiation originates from locations that are distinct from the geodetically inferred locations of large-amplitude fault slip.

Tremor-tide correlations and near-lithostatic pore pressure on the deep San Andreas fault.

Since its initial discovery nearly a decade ago, non-volcanic tremor has provided information about a region of the Earth that was previously thought incapable of generating seismic radiation. A thorough explanation of the geologic process responsible for tremor generation has, however, yet to be determined. Owing to their location at the plate interface, temporal correlation with geodetically measured slow-slip events and dominant shear wave energy, tremor observations in southwest Japan have been interpreted as a superposition of many low-frequency earthquakes that represent slip on a fault surface. Fluids may also be fundamental to the failure process in subduction zone environments, as teleseismic and tidal modulation of tremor in Cascadia and Japan and high Poisson ratios in both source regions are indicative of pressurized pore fluids. Here we identify a robust correlation between extremely small, tidally induced shear stress parallel to the San Andreas fault and non-volcanic tremor activity near Parkfield, California. We suggest that this tremor represents shear failure on a critically stressed fault in the presence of near-lithostatic pore pressure. There are a number of similarities between tremor in subduction zone environments, such as Cascadia and Japan, and tremor on the deep San Andreas transform, suggesting that the results presented here may also be applicable in other tectonic settings.

Remote triggering of fault-strength changes on the San Andreas fault at Parkfield.

Fault strength is a fundamental property of seismogenic zones, and its temporal changes can increase or decrease the likelihood of failure and the ultimate triggering of seismic events. Although changes in fault strength have been suggested to explain various phenomena, such as the remote triggering of seismicity, there has been no means of actually monitoring this important property in situ. Here we argue that approximately 20 years of observation (1987-2008) of the Parkfield area at the San Andreas fault have revealed a means of monitoring fault strength. We have identified two occasions where long-term changes in fault strength have been most probably induced remotely by large seismic events, namely the 2004 magnitude (M) 9.1 Sumatra-Andaman earthquake and the earlier 1992 M = 7.3 Landers earthquake. In both cases, the change possessed two manifestations: temporal variations in the properties of seismic scatterers-probably reflecting the stress-induced migration of fluids-and systematic temporal variations in the characteristics of repeating-earthquake sequences that are most consistent with changes in fault strength. In the case of the 1992 Landers earthquake, a period of reduced strength probably triggered the 1993 Parkfield aseismic transient as well as the accompanying cluster of four M > 4 earthquakes at Parkfield. The fault-strength changes produced by the distant 2004 Sumatra-Andaman earthquake are especially important, as they suggest that the very largest earthquakes may have a global influence on the strength of the Earth's fault systems. As such a perturbation would bring many fault zones closer to failure, it should lead to temporal clustering of global seismicity. This hypothesis seems to be supported by the unusually high number of M >or= 8 earthquakes occurring in the few years following the 2004 Sumatra-Andaman earthquake.

Nonvolcanic tremor evolution and the San Simeon and Parkfield, California, earthquakes.

Nonvolcanic tremors occur adjacent to locked faults and may be closely related to the generation of earthquakes. Monitoring of the San Andreas Fault in the Parkfield, California, region revealed that after two strong earthquakes, tremor activity increased in a nearly dormant tremor zone, increased and became periodic in a previously active zone, and has remained elevated and periodic for over 4 years. Static shear- and Coulomb-stress increases of 6 to 14 kilopascals from these two earthquakes are coincident with sudden increases in tremor rates. The persistent changes in tremor suggest that stress is now accumulating more rapidly beneath this part of the San Andreas Fault, which ruptured in the moment magnitude 7.8 Ft. Tejon earthquake of 1857.

Hepatocyte growth factor inhibits VEGF-forkhead-dependent gene expression in endothelial cells.

OBJECTIVE: Recently, we reported that the forkhead transcription factor, FKHR/FOXO1, is required for vascular endothelial growth factor (VEGF)-mediated upregulation of a number of genes in endothelial cells. Here, we tested the hypothesis that hepatocyte growth factor (HGF), a potent activator of PI3K-Akt in endothelial cells, is capable of depleting the nucleus of FKHR/FOXO1 and thus inhibiting VEGF induction of this class of genes. METHODS AND RESULTS: Incubation of human coronary artery endothelial cells with HGF induced prolonged PI3K/Akt-dependent phosphorylation and nuclear exclusion of FKHR/FOXO1. HGF-mediated inhibition of FKHR/FOXO1 activity resulted in secondary attenuation of VEGF-induced expression of FKHR/FOXO1-dependent genes including vascular cell adhesion molecule-1, manganese superoxide dismutase, endothelial specific molecule-1, CBP/p300 interacting transactivator with ED-rich tail-2, bone morphogenetic protein-2, matrix metalloproteinase (MMP)-10, and MGC5618. At a functional level, preincubation of HGF resulted in inhibition of VEGF-induced vascular cell adhesion molecule (VCAM)-1-mediated monocyte adhesion to endothelial cells. HGF-mediated inhibition of VEGF-inducible VCAM-1 expression and monocyte adhesion was reversed by overexpression of constitutively active phosphorylation-resistant triple mutant (TM)-FKHR. CONCLUSIONS: These findings suggest that physiological agonists of PI3K-Akt signaling pathway may modulate VEGF-FKHR/FOXO1-dependent gene expression in endothelial cells. The data underscore the importance of the "set point" of the endothelial cell when considering mechanisms of signal transduction.

Overexpression of Spry1 in chondrocytes causes attenuated FGFR ubiquitination and sustained ERK activation resulting in chondrodysplasia.

The FGF signaling pathway plays essential roles in endochondral ossification by regulating osteoblast proliferation and differentiation, chondrocyte proliferation, hypertrophy, and apoptosis. FGF signaling is controlled by the complementary action of both positive and negative regulators of the signal transduction pathway. The Spry proteins are crucial regulators of receptor tyrosine kinase-mediated MAPK signaling activity. Sprys are expressed in close proximity to FGF signaling centers and regulate FGFR-ERK-mediated organogenesis. During endochondral ossification, Spry genes are expressed in prehypertrophic and hypertrophic chondrocytes. Using a conditional transgenic approach in chondrocytes in vivo, the forced expression of Spry1 resulted in neonatal lethality with accompanying skeletal abnormalities resembling thanatophoric dysplasia II, including increased apoptosis and decreased chondrocyte proliferation in the presumptive reserve and proliferating zones. In vitro chondrocyte cultures recapitulated the inhibitory effect of Spry1 on chondrocyte proliferation. In addition, overexpression of Spry1 resulted in sustained ERK activation and increased expression of p21 and STAT1. Immunoprecipitation experiments revealed that Spry1 expression in chondrocyte cultures resulted in decreased FGFR2 ubiquitination and increased FGFR2 stability. These results suggest that constitutive expression of Spry1 in chondrocytes results in attenuated FGFR2 degradation, sustained ERK activation, and up-regulation of p21Cip and STAT1 causing dysregulated chondrocyte proliferation and terminal differentiation.

Notch2 signaling induces apoptosis and inhibits human MDA-MB-231 xenograft growth.

Notch functions as an oncogene or tumor inhibitor in various cancers, and decreases in Notch2 expression are associated with increasing grade of human breast cancer. We constitutively activated Notch signaling with intracellular domain (ICD) expression in the human adenocarcinoma line MDA-MB-231. Notch2 signaling increased apoptosis, whereas Notch4ICD (int3) significantly increased cell proliferation and growth. Cells with activated Notch2 or Notch4 were injected into nu/nu mice for analysis of in vivo tumor xenograft phenotype. Tumor growth was significantly altered depending on the receptor activated. Notch2ICD potently suppressed tumor take and growth, leading to a 60% decrease in tumors and significantly smaller, necrotic tumors. Despite this, Notch2ICD tumors were highly vascularized, although the vessels were smaller and comprised a more immature network compared with Notch4ICD tumors. Notch4ICD tumors were highly aggressive and well vascularized, indicating a role for Notch4 signaling in the promotion of the malignant phenotype in addition to its transforming ability. Although both NotchICD groups expressed angiogenic factors, Notch4ICD had selective vascular endothelial growth factor-D in both tumor and host stroma, suggesting a differential regulation of cytokines that may impact vascular recruitment and autocrine tumor signaling. Our results demonstrate that Notch2 signaling is a potent inhibitory signal in human breast cancer xenografts.