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Cancer cells migrating in confined microenvironments exhibit plasticity of migration modes. Confinement of contractile cells in a nonadhesive environment drives “leader bleb–based migration” (LBBM), morphologically characterized by a long bleb that points in the direction of movement separated from a cell body by a contractile neck. Although cells undergoing LBBM have been visualized within tumors, the organization of organelles and actin regulatory proteins mediating LBBM is unknown. We analyzed the localization of fluorescent organelle-specific markers and actin-associated proteins in human melanoma and osteosarcoma cells undergoing LBBM. We found that organelles from the endolysosomal, secretory, and metabolic systems as well as the vimentin and microtubule cytoskeletons localized primarily in the cell body, with some endoplasmic reticulum, microtubules, and mitochondria extending into the leader bleb. Overexpression of fluorescently tagged actin regulatory proteins showed that actin assembly factors localized toward the leader bleb tip, contractility regulators and cross-linkers in the cell body cortex and neck, and cross-linkers additionally throughout the leader bleb. Quantitative analysis showed that excess filamin-A and fascin-1 increased migration speed and persistence, while their depletion by small interfering RNA indicates a requirement in promoting cortical tension and pressure to drive LBBM. This indicates a critical role of specific actin crosslinkers in LBBM.

Plant-based diets, both vegan and vegetarian, which emphasize grains, vegetables, fruits, legumes, nuts, and seeds are increasingly popular for health as well as financial, ethical, and religious reasons. The medical literature clearly demonstrates that whole food plant-based diets can be both nutritionally sufficient and medically beneficial. However, any person on an intentionally restrictive, but poorly-designed diet may predispose themselves to clinically-relevant nutritional deficiencies. For persons on a poorly-designed plant-based diet, deficiencies are possible in both macronutrients (protein, essential fatty acids) and micronutrients (vitamin B12, iron, calcium, zinc, and vitamin D). Practitioner evaluation of symptomatic patients on a plant-based diet requires special consideration of seven key nutrient concerns for plant-based diets. This article translates these concerns into seven practical questions that all practitioners can introduce into their patient assessments and clinical reasoning. Ideally, persons on plant-based diets should be able to answer these seven questions. Each serves as a heuristic prompt for both clinician and patient attentiveness to a complete diet. As such, these seven questions support increased patient nutrition knowledge and practitioner capacity to counsel, refer, and appropriately focus clinical resources.

This paper examines the pursuit of legitimacy by the self-proclaimed “republics” in Ukraine. While these “republics” are illegal, questions of their legitimacy are commonly discussed almost...

Heterochromatin protein 1α (HP1α) regulates chromatin specification and plasticity during cell fate decision. Different structural determinants account for HP1α localization and function during cell division cycle. Our earlier study showed that centromeric localization of HP1α depends on the epigenetic mark H3K9me3 in interphase, while its centromeric location in mitosis relies on uncharacterized PXVXL-containing factors. Here, we identified a PXVXL-containing protein, ligand-dependent nuclear receptor-interacting factor 1 (LRIF1), which recruits HP1α to the centromere of mitotic chromosomes and its interaction with HP1α is essential for accurate chromosome segregation during mitosis. LRIF1 interacts directly with HP1α chromoshadow domain via an evolutionarily conserved PXVXL motif within its C-terminus. Importantly, the LRIF1-HP1α interaction is critical for Aurora B activity in the inner centromere. Mutation of PXVXL motif of LRIF1 leads to defects in HP1α centromere targeting and aberrant chromosome segregation. These findings reveal a previously unrecognized direct link between LRIF1 and HP1α in centromere plasticity control and illustrate the critical role of LRIF1-HP1α interaction in orchestrating accurate cell division.

This paper investigates outcomes of building students’ intuitive understanding of a limit as a function's predicted value by examining introductory calculus students’ conceptions of limit both befo...

Bradykinin has been implicated as a mediator of the acute pathophysiological and inflammatory consequences of respiratory tract infections and in exacerbations of chronic diseases such as asthma. Bradykinin may also be a trigger for the coughing associated with these and other conditions. We have thus set out to evaluate the pharmacology of bradykinin-evoked coughing in guinea pigs. When inhaled, bradykinin induced paroxysmal coughing that was abolished by the bradykinin B2 receptor antagonist HOE 140. These cough responses rapidly desensitized, consistent with reports of B2 receptor desensitization. Bradykinin-evoked cough was potentiated by inhibition of both neutral endopeptidase and angiotensin-converting enzyme (with thiorphan and captopril, respectively), but was largely unaffected by muscarinic or thromboxane receptor blockade (atropine and ICI 192605), cyclooxygenase, or nitric oxide synthase inhibition (meclofenamic acid and N(G)-nitro-L-arginine). Calcium influx studies in bronchopulmonary vagal afferent neurons dissociated from vagal sensory ganglia indicated that the tachykinin-containing C-fibers arising from the jugular ganglia mediate bradykinin-evoked coughing. Also implicating the jugular C-fibers was the observation that simultaneous blockade of neurokinin2 (NK2; SR48968) and NK3 (SR142801 or SB223412) receptors nearly abolished the bradykinin-evoked cough responses. The data suggest that bradykinin induces coughing in guinea pigs by activating B2 receptors on bronchopulmonary C-fibers. We speculate that therapeutics targeting the actions of bradykinin may prove useful in the treatment of cough.

Cell migration is orchestrated by dynamic interaction of microtubules with the plasma membrane cortex. However, the regulatory mechanisms underlying the cortical actin cytoskeleton and microtubule dynamics are less characterized. Our earlier study showed that small GTPase-activating proteins, IQGAPs, regulate polarized secretion in epithelial cells (1). Here, we show that IQGAP1 links dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein, SKAP. Biochemical characterizations revealed that IQGAP1 and SKAP form a cognate complex and that their binding interfaces map to the WWIQ motif and the C-terminal of SKAP, respectively. The WWIQ peptide disrupts the biochemical interaction between IQGAP1 and SKAP in vitro, and perturbation of the IQGAP1-SKAP interaction in vivo using a membrane-permeable TAT-WWIQ peptide results in inhibition of directional cell migration elicited by EGF. Mechanistically, the N-terminal of SKAP binds to EB1, and its C terminus binds to IQGAP1 in migrating cells. Thus, we reason that a novel IQGAP1 complex orchestrates directional cell migration via coupling dynamic microtubule plus-ends to the cell cortex.

Heterochromatin protein 1α (HP1α) is involved in regulation of chromatin plasticity, DNA damage repair, and centromere dynamics. HP1α detects histone dimethylation and trimethylation of Lys-9 via its chromodomain. HP1α localizes to heterochromatin in interphase cells but is liberated from chromosomal arms at the onset of mitosis. However, the structural determinants required for HP1α localization in interphase and the regulation of HP1α dynamics have remained elusive. Here we show that centromeric localization of HP1α depends on histone H3 Lys-9 trimethyltransferase SUV39H1 activity in interphase but not in mitotic cells. Surprisingly, HP1α liberates from chromosome arms in early mitosis. To test the role of this dissociation, we engineered an HP1α construct that persistently localizes to chromosome arms. Interestingly, persistent localization of HP1α to chromosome arms perturbs accurate kinetochore-microtubule attachment due to an aberrant distribution of chromosome passenger complex and Sgo1 from centromeres to chromosome arms that prevents resolution of sister chromatids. Further analyses showed that Mis14 and perhaps other PXVXL-containing proteins are involved in directing localization of HP1α to the centromere in mitosis. Taken together, our data suggest a model in which spatiotemporal dynamics of HP1α localization to centromere is governed by two distinct structural determinants. These findings reveal a previously unrecognized but essential link between HP1α-interacting molecular dynamics and chromosome plasticity in promoting accurate cell division.

To investigate the potential role of mtDNA alterations during the onset of colorectal cancer, the occurrence of mtDNA variants in colorectal adenomatous (Tubular, Tubulovillous, and Villous) polyps, were studied. High resolution endonucleases and PCR-based sequence were applied to examine mtDNA variants in the ND and ATPase genes of 64 primary tissues of colorectal adeno-polyps and their matched normal controls. Forty-two variants were observed and 57% (24/42) were not previously reported in the MITODAT reference. Fifty-eight percent of these variants were germline and homoplasmic transitions. The distribution of observed mtDNA variants includes: 31% (13/42) tubular, 52% (22/42) tubulovillous, 45% (19/42) villous, and 45% (19/42) cancer (including FAP and JVP). Notably, an unreported germline variant in the ATPase 8 gene at nucleotide position (np) G8573A was observed in tubulovillous adenomas tissues. The results suggest that some specific mtDNA variants may serve as a potential biomarker for colorectal adenomatous polyps.

Cell migration is orchestrated by dynamic interactions of microtubules with the plasma membrane cortex. How these interactions facilitate these dynamic processes is still being actively investigated. TIP150 is a newly characterized microtubule plus end tracking protein essential for mitosis and entosis (Ward, T., Wang, M., Liu, X., Wang, Z., Xia, P., Chu, Y., Wang, X., Liu, L., Jiang, K., Yu, H., Yan, M., Wang, J., Hill, D. L., Huang, Y., Zhu, T., and Yao, X. (2013) Regulation of a dynamic interaction between two microtubule-binding proteins, EB1 and TIP150, by the mitotic p300/CBP-associated factor (PCAF) orchestrates kinetochore microtubule plasticity and chromosome stability during mitosis. J. Biol. Chem. 288, 15771–15785; Xia, P., Zhou, J., Song, X., Wu, B., Liu, X., Li, D., Zhang, S., Wang, Z., Yu, H., Ward, T., Zhang, J., Li, Y., Wang, X., Chen, Y., Guo, Z., and Yao, X. (2014) Aurora A orchestrates entosis by regulating a dynamic MCAK-TIP150 interaction. J. Mol. Cell Biol. 6, 240–254). Here we show that TIP150 links dynamic microtubules to steer cell migration by interacting with cortactin. Mechanistically, TIP150 binds to cortactin via its C-terminal tail. Interestingly, the C-terminal TIP150 proline-rich region (CT150) binds to the Src homology 3 domain of cortactin specifically, and such an interaction is negatively regulated by EGF-elicited tyrosine phosphorylation of cortactin. Importantly, suppression of TIP150 or overexpression of phospho-mimicking cortactin inhibits polarized cell migration. In addition, CT150 disrupts the biochemical interaction between TIP150 and cortactin in vitro, and perturbation of the TIP150-cortactin interaction in vivo using a membrane-permeable TAT-CT150 peptide results in an inhibition of directional cell migration. We reason that a dynamic TIP150-cortactin interaction orchestrates directional cell migration via coupling dynamic microtubule plus ends to the cortical cytoskeleton.

Evidence suggests that cognitive training interventions can improve older adults' cognitive performance. Successful training programs are adaptable and train multiple cognitive domains to target individual strengths and weaknesses. Computerized training programs are useful because they allow older adults to easily access training. This pilot study used an online attention training program, ATTENTION WORKOUT™, to enhance three aspects of attention– coordination, allocation, and selective focus –in community-dwelling older adults randomized to either an abbreviated (n=13) or an extended (n=17) practice training program over a 6-week period. Participants in the extended practice group significantly improved on selective focus reading distraction tasks with unrelated words (U=39.5; Z=-2.34; p=.02) and blanks (U=26.5; Z=-3.05; p=.002) as well as a matching attributes task (U=49.5; Z=-2.33; p=.02). The extended practice group significantly improved on three tasks of coordinating attention – radio-tuning (U=30; Z=-2.73; p=.01), circuit-breaker resetting (U=46; Z=-2.24; p=.03), and the combination of the two tasks (U=15; Z=-3.51; p

This note explains why the difference‐in‐differences methodology, as applied by Deborah Haas‐Wilson and Christopher Garmon in their paper “Hospital Mergers and Competitive Effects: Two Retrospective Analyses” contained in this issue, does not produce credible results when applied to an assessment of the merger of Evanston and Highland Park Hospital, north of Chicago. Because of data deficiencies, an inability to control adequately for variation in factors that might affect the rate of price changes, and questionable assumptions about the pre‐merger equilibrium, the econometric results are inconsistent with other market factors that indicated that the merging hospitals were not close competitors. These market conditions are instead consistent with the view of the largest payer, who opined that the transaction had no effect on its ability to obtain competitive rates.

To varying degrees, parents who adopt children from China attempt to bring Chinese people, language, and culture into daily life as a way to help their adopted children feel proud about their ethnic heritage. Do attempts to provide bi-cultural socialization really influence ethnic identity? And, if so, what are the costs and benefits? In this article we draw on a longitudinal study of families with children adopted from China to describe their pre-teen and teenage daughters' orientations toward Chinese identity. The results indicate that Chinese ethnicity is an important component of adolescent identity for many adoptees, and that the odds of its being important are directly related to bi-cultural socialization influences within and outside of the family. Chinese ethnic identity does not appear to compromise the importance of American identity or closeness with family members who are not of Chinese descent. However, it does have the potential to magnify feelings of loss of birth parents and distre...

The main objective of this research was to test the hypothesis that bioavailable protein and, more specifically, the sulfur-containing amino acids within the protein, can be degraded by proteolytic enzymes to produce odor-causing compounds--mainly volatile sulfur compounds (VSCs)--during biosolids storage. To achieve these objectives, samples of digester effluent and cake solids were collected at 11 different wastewater treatment plants in North America, and the samples were analyzed for protein and amino acid content and general protein-degrading enzyme activity. At the same time, cake samples were stored using headspace bottles, the concentration of VSCs were measured using gas chromatography, and olfactometry measurements were made by a trained odor panel. The results showed that the bound cake protein content and methionine content was well-correlated with VSC production and the detection threshold measured by the odor panel.

Eleven dewatered sludge cakes collected from anaerobic digesters at different treatment plants were evaluated for the amount, type, and pattern of odorous gas production. All but one of the sludge cakes were from mesophilic anaerobic digesters. One was from a thermophilic digester. The pattern and quantities of sulfur gases were found to be unique for each of the samples with regard to the products produced, magnitude, and subsequent decline. The main odor-causing chemicals were volatile sulfur compounds, which included hydrogen sulfide, methanethiol, and dimethyl sulfide. Volatile sulfur compound production peaked in 3 to 8 days and then declined. The decline was a result of conversion of organic sulfur compounds to sulfide. In one side-by-side test, a high-solids centrifuge cake generated more odorous compounds than the low-solids centrifuge cake. The data show that anaerobic digestion does not eliminate the odor potential of anaerobically digested dewatered cakes. Water Environ. Res., 78, 821 (2006).

The development of attitudes towards one's own as well as other ethnic groups are childhood precursors of ethnic identity. The current paper examines the formation of ethnic attitudes among 266 school-age children who were born in China and adopted by Americans and, at the time of the study, were attending 254 different elementary schools across the country. We hypothesized that a disposition to associate socially desirable traits with being Chinese would be fostered by a school environment that was itself racially and ethnically diverse. In order to test this hypothesis, we linked attitudinal data from a photo preference task with archival data quantifying the number and distribution of students of different races and ethnicities in each child's school and other relevant data elements from a parents' questionnaire. The results do not support the assumption that diversity at school encourages children adopted from China to associate socially desirable traits with being Chinese. To the contrary, c...

Background: The aim of this study was to compare the reproducibility and sensitivity of four commonly used methods for QT interval assessment when applied to ECG data obtained after infusion of ibutilide. Methods: Four methods were compared: (1) 12-lead simultaneous ECG (12-SIM), (2) lead II ECG (LEAD II), both measured on a digitizing board, (3) 3-LEAD ECG using a manual tangential method, and (4) a computer-based, proprietary algorithm, 12SL™ ECG Analysis software (AUT). QT intervals were measured in 10 healthy volunteers at multiple time points during 24 hours at baseline and after single intravenous doses of ibutilide 0.25 and 0.5 mg. Changes in QT interval from baseline were calculated and compared across ECG methods, using Bland–Altman plots. Variability was studied using a mixed linear model. Results: Baseline QT values differed between methods (range 376–395 ms), mainly based on the number of leads incorporated into the measurement, with LEAD II and 3-LEAD providing the shortest intervals. The 3-LEAD generated the largest QT change from baseline, whereas LEAD II and 12-SIM generated essentially identical result within narrow limits of agreement (0.4 ms mean difference, 95% confidence interval ± 20.5 ms). Variability with AUT (standard deviation 15.8 ms for within-subject values) was clearly larger than with 3-LEAD, LEAD II, and 12-SIM (9.6, 10.0, and 11.3 ms). Conclusion: This study demonstrated significant differences among four commonly used methods for QT interval measurement after pharmacological prolongation of cardiac repolarization. Observed large differences in variability of measurements will have a substantial impact on the sample size required to detect QT prolongation in the range that is currently advised in regulatory guidance.

VB4-845 is a Targeted Protein Therapeutic (TPT) comprised of a scFv fragment specific for the Epithelial Cell Adhesion Molecule (EpCAM) genetically fused to a truncated form of Pseudomonas exotoxin A (ETA), via a flexible linker. Due to the inherent immunogenicity of the ETA moiety, VB4-845 is only used for the treatment of loco-regionally accessible tumors and is currently in a Phase III clinical trial for the treatment of high grade, non-muscle invasive bladder cancer. In a Phase I study in late stage squamous cell carcinoma of the head and neck, direct injection of VB4-845 led to shrinkage of the principal injected tumor, as well as non-targeted tumors in some patients, suggesting the activation of a T cell-mediated anti-tumor response through cross-priming. Only cells undergoing immunogenic cell death, or ICD, are capable of stimulating cross-priming. ICD is characterized by distinct “eat-me” signals such as the release of ATP and HMGB1, and cell surface translocation of calreticulin, an endoplasmic reticulum chaperone protein. To evaluate whether VB4-845-mediated cell killing results in ICD events, treated tumor cell lines were assessed for these distinct signaling molecules. In vitro studies using flow cytometry and ELISA showed both translocation of calreticulin and release of ATP and HMGB1 by VB4-845-treated tumor cells. Immune T cell activation via an ICD pathway suggests a complimentary outcome when combined with checkpoint inhibitors. A study was performed in PDX tumor-bearing NOG mice reconstituted with a human immune system to assess the combination of intratumoral injection of VB4-845 with an anti-PD1 antibody, Nivolumab, given i.p. VB4-845 showed growth suppression of the injected tumor whereas the growth delay of the contralateral, non-injected tumor was more pronounced with the addition of Nivolumab. In summary, the data presented suggests that VB4-845 mediates tumor cell killing by an ICD pathway and that the resulting cross-priming effect can enhance the anti-tumoral activity of immune checkpoint inhibitors. Citation Format: Rachelle Lee Dillon, Shilpa Chooniedass, Arjune Premsukh, Glen MacDonald, Jeannick Cizeau, Gregory A. Adams. VB4-845 tumor cell killing in a combination study with the anti-PD-1, Nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 614. doi:10.1158/1538-7445.AM2017-614

ArfGAP With Coiled-Coil, Ankyrin Repeat And PH Domains 4 (ACAP4) is an ADP-ribosylation factor 6 (ARF6) GTPase-activating protein essential for EGF-elicited cell migration. However, how ACAP4 regulates membrane dynamics and curvature in response to EGF stimulation is unknown. Here, we show that phosphorylation of the N-terminal region of ACAP4, named the Bin, Amphiphysin, and RSV161/167 (BAR) domain, at Tyr34 is necessary for EGF-elicited membrane remodeling. Domain structure analysis demonstrates that the BAR domain regulates membrane curvature. EGF stimulation of cells causes phosphorylation of ACAP4 at Tyr34, which subsequently promotes ACAP4 homodimer curvature. The phospho-mimicking mutant of ACAP4 demonstrates lipid-binding activity and tubulation in vitro, and ARF6 enrichment at the membrane is associated with ruffles of EGF-stimulated cells. Expression of the phospho-mimicking ACAP4 mutant promotes ARF6-dependent cell migration. Thus, the results present a previously undefined mechanism by which EGF-elicited phosphorylation of the BAR domain controls ACAP4 molecular plasticity and plasma membrane dynamics during cell migration.

To evaluate the safety of tubeless percutaneous nephrolithotomy in patients undergoing supracostal percutaneous renal access.Between October 1999 and October 2010, 302 patients underwent percutaneous nephrolithotomy via a supracostal access tract. Two hundred forty-eight (82.1%) patients had a nephrostomy tube placed at the end of the case and 54 (17.9%) did not. The medical records of both cohorts were compared regarding patient demographics (age, sex, body mass index, preoperative creatinine level), operative characteristics (estimated blood loss, length of stay, treatment efficacy), and complication rates (overall, thoracic, hemorrhage necessitating transfusion).Patient demographics did not differ between the tubeless and nephrostomy tube groups. Estimated blood loss was significantly less in the tubeless patients (67 mL vs 123 mL; P=0.019). The tubeless group had a shorter mean length of stay than the nephrostomy tube group (2.5 vs 3.4 days, P0.01). Treatment success was comparable between the two groups (tubeless 81.5% vs nephrostomy tube 77.8%; P=0.553). Overall complication (P=0.765) and blood transfusion (P=0.064) rates were equivalent. Chest complications were higher in the tubeless group (22.2%) compared with the nephrostomy tube patients (10.9%) (P=0.024). Nevertheless, chest complications necessitating intervention were not different (P=0.152).Tubeless supracostal percutaneous nephrolithotomy was associated with less intraoperative blood loss and a shorter hospital stay. Although the tubeless group experienced more chest complications overall, the need for intervention was no different among the two cohorts. Tubeless supracostal percutaneous nephrolithotomy appears safe.

After the death of Jesus, followers of the new Christian religious movement struggled to define both their mission and identity as a distinct faith community. As the members of this fledgling church grappled with the significance of their Jewish heritage and growing ambition to spread Christianity to Gentiles, circumcision became the divisive issue within the early church. As we will see, this controversy was the driving force in forming Christianity as a distinctly Gentile religion. This allowed Gentile Christians to escape the cultural devastation of the Jewish Diaspora and positioned the religion to become the dominant faith throughout Western Europe. But the debate over circumcision did not end there. Centuries of the greatest leaders, scholars, and artists in Western history continued to ponder the practice of circumcision and its role in Christian spirituality.

ARF6 GTPase is an important regulator of membrane trafficking and actin-based cytoskeleton dynamics active at the leading edge of migrating cells. The integrin family heterodimeric transmembrane proteins serve as major receptors for extracellular matrix proteins, which play essential roles in cell adhesion and migration. Our recent proteomic analyses of ARF6 effectors have identified a novel ARF6 GTPase-activating protein, ACAP4, essential for EGF-induced cell migration. However, molecular mechanisms underlying ACAP4-mediated cell migration have remained elusive. Here, we show that ACAP4 regulates integrin β1 dynamics during EGF-stimulated cell migration by interaction with Grb2. Our biochemical study shows that EGF stimulation induces phosphorylation of tyrosine 733, which enables ACAP4 to bind Grb2. This interaction of ACAP4 with Grb2 regulates integrin β1 recycling to the plasma membrane. Importantly, knockdown of ACAP4 by siRNA or overexpression of ACAP4 decreased recycling of integrin β1 to the plasma membrane and reduced integrin-mediated cell migration. Taken together, these results suggest a novel function for ACAP4 in the regulation of cell migration through controlling integrin β1 dynamics.

Colorectal tumors mostly arise from sporadic adenomatous polyps. Polyps are defined as a mass of cells that protrudes into the lumen of the colon. Adenomatous polyps are benign neoplasms that, by definition display some characteristics of dysplasia. It has been shown that polyps were benign tumors which may undergo malignant transformation. Adenomatous polyps have been classified into three histologic types; tubular, tubulovillous, and villous with increasing malignant potential. The ability to differentially diagnose these colorectal adenomatous polyps is important for therapeutic intervention. To date, little efforts have been directed to identifying genetic changes involved in adenomatous polyps. This study was designed to examine the relevance of mitochondrial genome alterations in the three adenomatous polyps. Using high resolution restriction endonucleases and PCR-based sequencing, fifty-seven primary fresh frozen tissues of adenomatous polyps (37 tumors and 20 matched surrounding normal tissues) obtained from the southern regional Cooperative Human Tissue Network (CHTN) and Grady Memorial Hospital at Atlanta were screened with three mtDNA regional primer pairs that spanned 5.9 kbp. Results from our data analyses revealed the presence of forty-four variants in some of these mitochondrial genes that the primers spanned; COX I, II, III, ATP 6, 8, CYT b, ND 5, 6 and tRNAs. Based on the MITODAT database as a sequence reference, 25 of the 44 (57%) variants observed were unreported. Notably, a heteroplasmic variant C8515G/T in the MT-ATP 8 gene and a germline variant 8327delA in the tRNAlys was observed in all the tissue samples of the three adenomatous polyps in comparison to the referenced database sequence. A germline variant G9055A in the MT-ATP 6 gene had a frequency of 100% (17/17) in tubular and 57% (13/23) in villous adenomas; no corresponding variant was in tubulovillous adenomas. Furthermore, A9006G variant at MT-ATP 6 gene was observed at frequency of 57% (13/23) in villous adenomas only. Interestingly, variants A9006G and G9055A were absent in the villous tissue samples that were clinicopathological designated as "polyvillous adenomas". Our current data provide a basis for continued investigation of certain mtDNA variants as predictors of the three adenomatous polyps in a larger number of clinicopathological specimens.

The safety, tolerability, and pharmacokinetics of PNU-96391, an orally active weak dopamine D2 receptor antagonist with modulatory properties of central dopaminergic function, was characterized. Fifty-three healthy normal volunteers were enrolled in this randomized, double-blinded, placebo-controlled, single-dose study. Subjects were assigned to single oral doses of placebo and 1, 3, 10, 30, 100, 150, and 200 mg PNU-96391. Safety and tolerability were assessed using telemetry, Holter monitoring, surface ECG, vital signs, safety laboratories, and adverse event reports. Pharmacokinetic parameters were determined by model-independent techniques. Adverse events were infrequent, of mild to moderate intensity, and in the dose range of 1 to 150 mg. Dose escalation was stopped at 200 mg because of severe nausea, dizziness, lightheadedness, and tachycardia. Besides the increase in heart rate, no other drug-related effects on vital signs were observed. Safety laboratory measurements were not significantly changed. Evidence of drug activity was demonstrated by a dose-dependent elevation in serum prolactin. PNU-96391 was rapidly absorbed, with maximum concentrations achieved between 0.5 and 4 hours in all subjects. The half-life of the drug was short (2 to 6 h). The main metabolite, PNU-100014, was rapidly formed, with a t(max) ranging from 1 to 6 hours. Peak levels of the metabolite are approximately half of the parent drug, and the half-life is slightly longer (4 to 10 h). Increases in dose resulted in linear increases in exposure for both PNU-96391 and PNU-100014. Hence, PNU-96391 was well tolerated at doses ranging from 1 to 150 mg.

Despite advances in the surgical and chemotherapeutic management of ovarian cancer over the last decade, survival rates remain low, largely due to late diagnosis. Up to now very few candidate biomarkers have been investigated for early detection of ovarian cancer. The implication of ROS in female reproductive system and ovarian cancer are evidenced. Enzymatic antioxidants neutralize excessive ROS in cells. Under special condition, the cellular antioxidants are depleted. The ultimate aim of this study is to identify a biomarker for early detection of ovarian cancer. In this study 155 snap-freeze primary serous epithelial ovarian tissues including, normal, normal surrounding, cystadenoma, borderline and carcinoma with age range of 65±12,(55% Caucasian 45% Africa American), were analyzed. The objectives are: (a) to evaluate the protein expression profiles using two dimension gel electrophoresis (2-D SDS-PAGE) and LC/MS technology, (b) to identify and evaluate the level of oxidative modified protein, using derivatisation of protein carbonyl group with 2,4-dinitrophenylhyrdozine(DNPH) assay and western blot technique. Our results showed that the index of carbonyl protein /total protein increased by onset and progression of the serous ovarian malignancy. The elevation of oxidized protein among African American was 40 % higher than Caucasian in advanced stages of diseases. SDS-PAGE profiles showed a distinct protein pattern among cancerous and non-cancerous tissues. LC/MS analysis for elucidation of molecular changes of selected proteins bands is in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4508. doi:1538-7445.AM2012-4508

PURPOSE: Colorectal cancer is the third most common cause of cancer-related deaths in both men and women in the western hemisphere. The prognosis is based on the stage of tumor at the time of diagnosis, with surgery being the most effective treatment; beginning at distinct adenomatous stages- tubular, tubulovillous, and villous, to carcinoma. The best evaluation of colorectal tumor progression, especially the early adenomas, continues to confound clinicians and the presence of colorectal adenomas dysplasia subtype, increases one's risk of developing colorectal cancer. The overall objective of this study was to evaluate mtDNA variants in three stages of colorectal adenomatous polyps; Tubular (T), Tubulovillous, (TV), Villous (V) adenomas, and Cancer (C). DESIGN METHODS: PCR-based sequencing and High resolution endonucleases were used to detect mtDNA variants in 1.8 kb of ATP 6 and 8, 2.2 kb of Complex 1 ND5 and 2.6 Complex 1 MT-ND4 of Cytochrome B genes from 64 clinicopathological colorectal adenomatous polyps; T, TV, V, Cancer, and their paracancerous/normal surrounding tissue (NST) that were obtained from Cooperative Human Tissues Network and Grady Memorial Hospital, Atlanta, Georgia. HYPOTHESIS: Our hypothesis is that alterations in the mitochondrial genome of early colorectal tumors compared to non-cancerous, normal surrounding, tissue will allow us to identify a profile of polymorphisms worthy of follow-up investigation as candidate for early colorectal cancer morphological and stage-specific markers. RESULTS: Thirty-nine of mtDNA variants were detected with primers spanning 1.8 kb of ATP 6 and 8 regions, of which 25 (64%) were previously unreported based on the MITODAT reference database. Twenty four of 39 (62%) mtDNA variants were somatic mutations. Notably, three germ-line variants; G8407C, G8680C, and G8681 were observed in tubular adenomas only and four somatic variants; 8714delC, 8766delC, 8668delC and 8670delA were observed in villous adenomas. With primers spanning 2.2 kb of Complex 1 ND5 and 2.6 Complex 1 MT-ND4 of Cytochrome B genes, 104 mtDNA variants were detected of which 6 (5%) were previously reported based on the MITODAT reference database. Ninety-nine of 104 (95%) mtDNA variants were unreported mutations. Two germ-line variants; G11914A, T11944C were observed in tubulovillous adenomas; 10 somatic variants were observed in tubular adenomas; 42 somatic variants were observed in villous; and all other mutations were an insertion or deletion of base pairs in all polyp types and/or cancer. CONCLUSION: Our current data provide a basis for continued investigation of certain mtDNA variants as potential genetic biomarkers for colorectal adenomatous polyps in a larger number of clinicopathological tissue specimens, and other regions of the mt genome. Research supported by NIH-NCI Grant # CA150317-01A2 and Grant # CA150039-01 awarded to Felix O. Aikhionbare at Morehouse School of Medicine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3806. doi:10.1158/1538-7445.AM2011-3806

Survival rates of ovarian cancer patients are low, largely due to late diagnosis. Early detection plays a major role towards improving outcomes in ovarian cancer, however few candidate biomarkers have shown efficacy for the detection of preclinical disease. Human mitochondrial gene mutations have increasingly been associated with various cancers. The mitochondrial genome is highly susceptible to oxidative damage and may accumulate damage over a long period of time. Consequently, variations in (mtDNA) may potentially play a role as a modifying risk factor in the development of age-related diseases such as ovarian cancer. The goal of this study is to examine correlations between mitochondrial gene alterations and three subtypes of ovarian tumor progression. Mitochondrial DNA fragments were obtained from 120 epithelial ovarian cancer tissue samples (40 serous; 40 endometrioid and 40 mucinous ovarian tumors respectively). The entire mitochondrial genome was PCR amplified using 9 overlapping primers sets and analyzed using both high resolution restriction digest and PCR-based sequencing techniques. Additionally, the levels of carbonyl proteins in cytosolic fraction of tissues were assessed by standard colorimetric technique, using 2-4-dinitrophenylhydrazine (DNPH) to derivatize protein, and measure the absorbance of the DNP-hydrazones at 370 nm. In this study we revealed the presence thirty-nine polymorphisms of which 28 were unreported. The observed mutations with notably frequencies (41-93%) among these samples were at np C7028T, C7256T, G7520A, T8548G, T8588C, A8860G, C9488G, C9500T, T9540C, C9857T, and T9951C. Furthermore, six unreported point mutations with frequencies of 14-41 % were observed at np G7520A, T8548G, C9488G, C9500T, C9857 and T9951C. A combined mutation of G7520A and C7256T was frequent at 45% in endometrioid stage III only. Interestingly, variants C7020T (56%) and at np A8860G (92%) were evenly distributed in all three studied ovarian tumor subtype and stages. Variants T9540C and C7520T showed significant higher frequency in African American samples compare to Caucasian samples and these may be used for further investigation for cancer disparity study. A number of the observed sequence variants were germ-lines with variants found in these mitochondrial genes/regions; D-Loop, 12S rRNA-tRNAphe, tRNAval, COX I, tRNAser, tRNAasp, COX II, tRNAlys, ATPase 6, ATPase 8, COX III, ND2, and ND3 genes among the three subtypes of ovarian cancer. Our results showed the elevation of the ratio of protein carbonyl/total protein in all three types of serous polyp as compare to their surrounding tissue. The level of the ratio of protein carbonyls/total protein decreased as a result of serous ovarian tumor progression. Our study suggests that certain mitochondrial DNA sequence variants and protein carbonyl (CO) groups, oxidative stress may play a potential role in etiological differences that may exist between the pathogenicity of subtypes and stages of benign and invasive epithelial ovarian tumors. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):A115.

B41 Mullerian inhibiting substance (MIS) is a member of the transforming growth factor β family of growth and differentiation factors known primarily for its role in regulating in-utero sexual differentiation of males. In the female breast, MIS inhibits elongation and branching of mammary ducts. In vitro and in animal models, MIS inhibits growth of mammary tumors. Thus, we hypothesized that MIS may be a novel biomarker of decreased breast cancer risk. The objective of this study was to prospectively determine if MIS levels in serum are related to breast cancer risk. A case-control study was performed nested in the Columbia, Missouri Serum Bank. Each of 129 cases with prediagnostic serum collected before menopause were matched to 2 controls on age, date, menstrual cycle day, and time of day of blood collection. MIS was measured in serum by ELISA. Data analysis was performed using conditional logistic regression. Contrary to expectations, results of preliminary analysis indicate that premenopausal women with elevated serum MIS levels are at an increased risk of breast cancer. Median MIS concentrations in cases and controls were .3 ng/ml and .1 ng/ml, respectively (p=.002). Women in the highest MIS quartile had a RR = 6.6 (95% CI = 2.5 - 17.4; trend-p Citation Information: Cancer Prev Res 2008;1(7 Suppl):B41.

This report covers the application of Artificial Intelligence Techniques to the problem of creating automated tools to assist officers; it also examines the use of an emerging AI field, neural computing, as it might be applied to this problem domain. Artificial intelligence, Expert systems, Automated aids to testing.