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Background: Intracranial occlusion site, contrast permeability, and clot burden are thrombus characteristics that influence alteplase-associated reperfusion. In this study, we assessed the reperfusion efficacy of tenecteplase and alteplase in subgroups based on these characteristics in a pooled analysis of the EXTEND-IA TNK trial (Tenecteplase Versus Alteplase Before Endovascular Therapy for Ischemic Stroke). Methods: Patients with large vessel occlusion were randomized to treatment with tenecteplase (0.25 or 0.4 mg/kg) or alteplase before thrombectomy in hospitals across Australia and New Zealand (2015–2019). The primary outcome, early reperfusion, was defined as the absence of retrievable thrombus or >50% reperfusion on first-pass angiogram. We compared the effect of tenecteplase versus alteplase overall, and in subgroups, based on the following measured with computed tomography angiography: intracranial occlusion site, contrast permeability (measured via residual flow grades), and clot burden (measured via clot burden scores). We adjusted for covariates using mixed effects logistic regression models. Results: Tenecteplase was associated with higher odds of early reperfusion (75/369 [20%] versus alteplase: 9/96 [9%], adjusted odds ratio [aOR], 2.18 [95% CI, 1.03–4.63]). The difference between thrombolytics was notable in occlusions with low clot burden (tenecteplase: 66/261 [25%] versus alteplase: 5/67 [7%], aOR, 3.93 [95% CI, 1.50–10.33]) when compared to high clot burden lesions (tenecteplase: 9/108 [8%] versus alteplase: 4/29 [14%], aOR, 0.58 [95% CI, 0.16–2.06]; P interaction =0.01). We did not observe an association between contrast permeability and tenecteplase treatment effect (permeability present: aOR, 2.83 [95% CI, 1.00–8.05] versus absent: aOR, 1.98 [95% CI, 0.65–6.03]; P interaction =0.62). Tenecteplase treatment effect was superior with distal M1 or M2 occlusions (53/176 [30%] versus alteplase: 4/42 [10%], aOR, 3.73 [95% CI, 1.25–11.11]), but both thrombolytics had limited efficacy with internal carotid artery occlusions (tenecteplase 1/73 [1%] versus alteplase 1/19 [5%], aOR, 0.22 [95% CI, 0.01–3.83]; P interaction =0.16). Conclusions: Tenecteplase demonstrates superior early reperfusion versus alteplase in lesions with low clot burden. Reperfusion efficacy remains limited in internal carotid artery occlusions and lesions with high clot burden. Further innovation in thrombolytic therapies are required.

The paper described the design and fabrication of a robotically-milled brass chandelier using a bespoke vertical axial revolving material holder as a robotic fixture. While the technique described is for a chandelier design, it has potential architectural applications, as demonstrated by architects such as Barkow Leibinger. The significance of this research lies in the increased flexibility of the technique performed using a robotic arm compared to the current industrial method using tubematic laser cutter. In addition, the paper outlined the design of the robotic fixture and the computational workflow to create an integrated design-to-fabrication workflow. The research highlighted robotic systems as a potential design environment through reflection on Material Engagement Theory (MET) framework. Critically, the workflow constructed design feedback as robotic agencies that provide affordances through the fabrication setup. Such affordances contribute to the designing process and refine craftsmanship by creating transactional relationships between tools and material as a digital repertoire. This emerging design environment extends robotic research into design practice.

Background: Middle meningeal artery (MMA) embolization has recently emerged as a potential treatment for chronic subdural hematoma (cSDH). Numerous retrospective studies have suggested that it can potentially reduce the risk of hematoma recurrence following surgical evacuation. We have conducted a randomized controlled trial to investigate the effectiveness of postoperative MMA embolization in reducing recurrence rate, residual hematoma thickness as well as improving functional outcome. Methods: Patients aged 18 or above were recruited. Following evacuation through burr hole or craniotomy, patients were randomly allocated to undergo either MMA embolization or standard care (monitoring). The primary outcome was symptomatic recurrence requiring redo evacuation. Secondary outcomes include residual hematoma thickness and modified Rankin Scale (mRS) at 6 weeks and 3 months. Results: Thirty-six patients (41 cSDHs) were recruited between April 2021 and September 2022. Seventeen patients (19 cSDHs) were allocated to the embolization group and 19 patients (22 cSDHs) were in the control group. No symptomatic recurrence was observed in the treatment group while 3 control patients (15.8%) underwent repeat surgery for symptomatic recurrence, however, it was not statistically significant (P = 0.234). Furthermore, there was no significant difference in residual hematoma thickness at 6 weeks or 3 months between the two groups. All patients in the embolization group had a good functional outcome (mRS 0–1) at 3 months, which was significantly higher than the 53% observed in the control group. No complications related to MMA embolization were reported. Conclusion: Further study with larger sample size is required to evaluate the efficacy of MMA embolization.

This paper discusses the design, fabrication and operational workflow of a novel hot-wire cutter used as an end effector for a robotic arm. Typically, hot wire cutters used a linear cutting element which results in ruled surfaces geometry. While several researchers have examined the use of hot wire cutter with cooperative robotic arms to create non-ruled surface geometry, this research explores the use of an actuated hot wire cutter manoeuver by a single robotic arm to produce similar form. The paper outlines the machine making process and its workflow resulting in a 1:1 scale prototype. The paper concludes by examining how the novel tool can be applied to an urban stage design. The research set up a fabrication procedure that has the potential to be deployed as an on-site fabrication methodology.

Importance Intravenous thrombolysis with tenecteplase improves reperfusion prior to endovascular thrombectomy for ischemic stroke compared with alteplase. Objective To determine whether 0.40 mg/kg of tenecteplase safely improves reperfusion before endovascular thrombectomy vs 0.25 mg/kg of tenecteplase in patients with large vessel occlusion ischemic stroke. Design, setting, and participants Randomized clinical trial at 27 hospitals in Australia and 1 in New Zealand using open-label treatment and blinded assessment of radiological and clinical outcomes. Patients were enrolled from December 2017 to July 2019 with follow-up until October 2019. Adult patients (N = 300) with ischemic stroke due to occlusion of the intracranial internal carotid, \basilar, or middle cerebral artery were included less than 4.5 hours after symptom onset using standard intravenous thrombolysis eligibility criteria. Interventions Open-label tenecteplase at 0.40 mg/kg (maximum, 40 mg; n = 150) or 0.25 mg/kg (maximum, 25 mg; n = 150) given as a bolus before endovascular thrombectomy. Main outcomes and measures The primary outcome was reperfusion of greater than 50% of the involved ischemic territory prior to thrombectomy, assessed by consensus of 2 blinded neuroradiologists. Prespecified secondary outcomes were level of disability at day 90 (modified Rankin Scale [mRS] score; range, 0-6); mRS score of 0 to 1 (freedom from disability) or no change from baseline at 90 days; mRS score of 0 to 2 (functional independence) or no change from baseline at 90 days; substantial neurological improvement at 3 days; symptomatic intracranial hemorrhage within 36 hours; and all-cause death. Results All 300 patients who were randomized (mean age, 72.7 years; 141 [47%] women) completed the trial. The number of participants with greater than 50% reperfusion of the previously occluded vascular territory was 29 of 150 (19.3%) in the 0.40 mg/kg group vs 29 of 150 (19.3%) in the 0.25 mg/kg group (unadjusted risk difference, 0.0% [95% CI, -8.9% to -8.9%]; adjusted risk ratio, 1.03 [95% CI, 0.66-1.61]; P = .89). Among the 6 secondary outcomes, there were no significant differences in any of the 4 functional outcomes between the 0.40 mg/kg and 0.25 mg/kg groups nor in all-cause deaths (26 [17%] vs 22 [15%]; unadjusted risk difference, 2.7% [95% CI, -5.6% to 11.0%]) or symptomatic intracranial hemorrhage (7 [4.7%] vs 2 [1.3%]; unadjusted risk difference, 3.3% [95% CI, -0.5% to 7.2%]). Conclusions and relevance Among patients with large vessel occlusion ischemic stroke, a dose of 0.40 mg/kg, compared with 0.25 mg/kg, of tenecteplase did not significantly improve cerebral reperfusion prior to endovascular thrombectomy. The findings suggest that the 0.40-mg/kg dose of tenecteplase does not confer an advantage over the 0.25-mg/kg dose in patients with large vessel occlusion ischemic stroke in whom endovascular thrombectomy is planned. Trial registration ClinicalTrials.gov Identifier: NCT03340493.

Background: Mechanical thrombectomy (MT) became standard care in 2015 after positive trials in patients presenting with acute ischemic stroke and large vessel occlusion (LVO) 0-6h and in 2018 for selected patients up to 24h from symptom onset. Objective: To evaluate whether patients receiving MT at our center would have comparable outcomes in patients presenting to our comprehensive stroke center (direct) vs transfer patients (drip-and-ship) Methods: This is a retrospective observational study utilising prospectively collected stroke database for patients receiving MT for LVO in anterior and posterior circulation in South Brisbane network of 7 hospitals (6 drip-and-ship centers and 1 MT-capable center), Australia which serves 1.6 million. Day 90 modified Rankin scale (mRS) was used to assess functional outcomes via outpatient follow up at direct or referral center. The association of drip and ship versus mothership treatment with day 90 mRS was tested in ordinal logistic regression adjusted for age, baseline NIHSS and IV thrombolysis. Results: Of 191 patients who underwent Mechanical Thrombectomy from 2015 to June 2018 at our center, 22 patients were excluded from analysis as either their baseline mRS was >1 (13) or follow up data was missing (9). The mean age was 64.4 years. Median (inter-quartile range, IQR) NIHSS was 16 (9-21) on admission and 7 (2-18) on day 1. Thrombolysis in Cerebral Infarction (TICI) ≥2b was achieved in 88.9%. At 90 days, 50.9% achieved excellent functional outcome (mRS 0-1), 61.4% achieved good functional outcome (mRS 0-2) and 69% achieved favorable outcome (mRS 0-3). Median mRS was 1 (IQR 0-5) in 96 patients presenting directly to the endovascular center and 1 (IQR 1-4) in 73 drip-and-ship patients (common odds ratio 1.07 (95%CI 0.62-1.83), p=0.82) Conclusion: Our 7-center network experience confirms real world reproducibility of trial results, interestingly with no difference in functional outcomes for direct vs drip-and-ship patients.

Casting is one of the most widely used construction techniques. Complex geometries produced via computational design processes are not readily achievable through traditional rigid formwork and are subject to increased material waste. More suitable casting techniques are required to represent digital design output efficiently. This article presents a variable fabric formwork developed to work in conjunction with a 6-axis robotic arm for casting doubly curved panels based on hyperbolic paraboloid geometry. The variable formwork is designed to be extendable in length and width so that it can produce a wide range of outcomes within a single formwork. The interface established in the workflow allows the physical formwork and digital design to influence each other. The article concludes by discussing a verification method used to confirm the accuracy of the outcome. This variable fabric form-work reduces construction waste and is a more sustainable method for casting complex geometries. Highlights Robotic arm used to manipulate a bespoke mould for fabric formwork casting. Constraints of mould design used to inform robotic arm of its fabrication limits. Workflow allows the physical formwork and digital design to influence each other. Verification of result reveals future area of research.

Background and hypothesis Intravenous thrombolysis with alteplase remains standard care prior to thrombectomy for eligible patients within 4.5 h of ischemic stroke onset. However, alteplase only succeeds in reperfusing large vessel arterial occlusion prior to thrombectomy in a minority of patients. We hypothesized that tenecteplase is non-inferior to alteplase in achieving reperfusion at initial angiogram, when administered within 4.5 h of ischemic stroke onset, in patients planned to undergo endovascular therapy. Study design EXTEND-IA TNK is an investigator-initiated, phase II, multicenter, prospective, randomized, open-label, blinded-endpoint non-inferiority study. Eligibility requires a diagnosis of ischemic stroke within 4.5 h of stroke onset, pre-stroke modified Rankin Scale≤3 (no upper age limit), large vessel occlusion (internal carotid, basilar, or middle cerebral artery) on multimodal computed tomography and absence of contraindications to intravenous thrombolysis. Patients are randomized to either IV alteplase (0.9 mg/kg, max 90 mg) or tenecteplase (0.25 mg/kg, max 25 mg) prior to thrombectomy. Study outcomes The primary outcome measure is reperfusion on the initial catheter angiogram, assessed as modified treatment in cerebral infarction 2 b/3 or the absence of retrievable thrombus. Secondary outcomes include modified Rankin Scale at day 90 and favorable clinical response (reduction in National Institutes of Health Stroke Scale by ≥8 points or reaching 0–1) at day 3. Safety outcomes are death and symptomatic intracerebral hemorrhage. Trial registration ClinicalTrials.gov NCT02388061

This paper presents a novel advance digital fabrication method to produce doubly curved concrete panel with no immediate waste as a facade system. Using a bespoke CNC adjustable mould frame system coupled with robotic trimming techniques, the research examines the streamlining of data within a cohesive fabrication workflow. The paper concludes by reviewing an integrated workflow that points towards a multifaceted system of design, engineering and advanced manufacturing that propel research to design application.

A healthy 51-year-old female presented with a spontaneous direct carotid-cavernous sinus fistula associated with a persistent primitive trigeminal artery. She had no history of connective tissue or cerebrovascular disorders or significant head trauma. This is a rare lesion with only 18 previously reported cases. It had similar clinical presentation and imaging appearance to a high-flow direct carotid-cavernous fistula and was uncovered after successful trans-venous coil embolisation of the fistula. It therefore needs to be considered in cases of direct carotid-cavernous fistula without history of trauma. Knowledge of types of persistent primitive trigeminal artery is also important for their critical treatment implications.

Intravenous infusion of alteplase is used for thrombolysis before endovascular thrombectomy for ischemic stroke. Tenecteplase, which is more fibrin-specific and has longer activity than alteplase, is given as a bolus and may increase the incidence of vascular reperfusion.We randomly assigned patients with ischemic stroke who had occlusion of the internal carotid, basilar, or middle cerebral artery and who were eligible to undergo thrombectomy to receive tenecteplase (at a dose of 0.25 mg per kilogram of body weight; maximum dose, 25 mg) or alteplase (at a dose of 0.9 mg per kilogram; maximum dose, 90 mg) within 4.5 hours after symptom onset. The primary outcome was reperfusion of greater than 50% of the involved ischemic territory or an absence of retrievable thrombus at the time of the initial angiographic assessment. Noninferiority of tenecteplase was tested, followed by superiority. Secondary outcomes included the modified Rankin scale score (on a scale from 0 [no neurologic deficit] to 6 [death]) at 90 days. Safety outcomes were death and symptomatic intracerebral hemorrhage.Of 202 patients enrolled, 101 were assigned to receive tenecteplase and 101 to receive alteplase. The primary outcome occurred in 22% of the patients treated with tenecteplase versus 10% of those treated with alteplase (incidence difference, 12 percentage points; 95% confidence interval [CI], 2 to 21; incidence ratio, 2.2; 95% CI, 1.1 to 4.4; P=0.002 for noninferiority; P=0.03 for superiority). Tenecteplase resulted in a better 90-day functional outcome than alteplase (median modified Rankin scale score, 2 vs. 3; common odds ratio, 1.7; 95% CI, 1.0 to 2.8; P=0.04). Symptomatic intracerebral hemorrhage occurred in 1% of the patients in each group.Tenecteplase before thrombectomy was associated with a higher incidence of reperfusion and better functional outcome than alteplase among patients with ischemic stroke treated within 4.5 hours after symptom onset. (Funded by the National Health and Medical Research Council of Australia and others; EXTEND-IA TNK ClinicalTrials.gov number, NCT02388061 .).

Partial or even complete cancer regression can be achieved in some patients with current cancer treatments. However, such initial responses are almost always followed by relapse, with the recurrent cancer being resistant to further treatments. The discovery of therapeutic approaches that counteract relapse is, therefore, essential for advancing cancer medicine. Cancer cells are extremely heterogeneous, even in each individual patient, in terms of their malignant potential, drug sensitivity, and their potential to metastasize and cause relapse. Indeed, hypermalignant cancer cells, termed cancer stem cells or stemness-high cancer cells, that are highly tumorigenic and metastatic have been isolated from cancer patients with a variety of tumor types. Moreover, such stemness-high cancer cells are resistant to conventional chemotherapy and radiation. Here we show that BBI608, a small molecule identified by its ability to inhibit gene transcription driven by Stat3 and cancer stemness properties, can inhibit stemness gene expression and block spherogenesis of or kill stemness-high cancer cells isolated from a variety of cancer types. Moreover, cancer relapse and metastasis were effectively blocked by BBI608 in mice. These data demonstrate targeting cancer stemness as a novel approach to develop the next generation of cancer therapeutics to suppress cancer relapse and metastasis.

In patients with primary aldosteronism, adrenal vein sampling (AVS) is considered the only reliable technique to distinguish between unilateral and bilateral autonomous production of aldosterone, but agreement is lacking on the best criteria indicating successful cannulation and lateralization. The objective of this study was to assess the impact of differing criteria for the successful cannulation and lateralization on the reproducibility of subtype diagnosis. Sixty-two patients with confirmed primary aldosteronism underwent AVS on 2 separate occasions, because the first was unsatisfactory. We compared the different diagnoses of primary aldosteronism subtype reached using AVS data assessed by permissive (type 1), intermediate (type 2), and strict (type 3) criteria. Although 91.1% of all of the (both first and second) AVSs were “successful” by type 1 criteria (50.8% by type 2 and 33.9% by type 3), in only 35.3% of patients was the diagnosis concordant between the first and second AVS. Type 1 criteria also led to a higher rate of diagnosis of unilateral primary aldosteronism (67.3% of successful procedures) than type 2 (36.5%) or type 3 (26.2%). There was considerable disparity in the diagnosis reached using the 3 different criteria, with concordance in only 32.2%. Using either type 1 or 2 criteria, the minimal adrenal/peripheral vein cortisol ratio necessary to obtain the same diagnosis in the first and second AVS procedures was ≥2.75. In conclusion, permissive criteria for successful cannulation and lateralization on AVS achieve poor diagnostic reproducibility and should be avoided.

Activation of DNA damage checkpoint pathways, including Chk2, serves as an anticancer barrier in precancerous lesions. In an effort to identify small-molecule activators of Chk2, the authors developed a quantitative cell-based assay using a high-content analysis (HCA) platform. Induction of phosphorylated Chk2 was evaluated using several different parameters, including fold induction, Kolmogorov-Smirnov score, and percentage of positively stained cells. These measurements were highly correlated and provided an accurate method for compound ranking/binning, structure-activity relationship studies, and lead identification. Screening for Chk2 activators was undertaken with a target-focused library and a diversified library from ArQule chemical space. Several compounds exhibited submicromolar EC( 50) values for phosphorylated Chk2 induction. These compounds were further analyzed for Chk2-dependent cytotoxicity, as assessed through a high-content cell death assay in combination with siRNA silencing of Chk2 expression. Several compounds were identified and showed specific inhibition or lethality in a target-dependent manner. Therefore, identification of DNA damage checkpoint pathway activators by HCA is an attractive approach for discovering the next generation of targeted cancer therapeutics.

SummaryUbiquitin chains serve as a recognition motif for the proteasome, a multisubunit protease, which degrades its substrates into polypeptides while releasing ubiquitin for reuse. Yeast proteasomes contain two deubiquitinating enzymes, Ubp6 and Rpn11. Rpn11 promotes protein breakdown through its degradation-coupled activity. In contrast, we show here that Ubp6 has the capacity to delay the degradation of ubiquitinated proteins by the proteasome. However, delay of degradation by Ubp6 does not require its catalytic activity, indicating that Ubp6 has both deubiquitinating activity and proteasome-inhibitory activity. Delay of degradation by Ubp6 appears to provide a time window allowing gradual deubiquitination of the substrate by Ubp6. Rpn11 catalyzes en bloc chain removal, and Ubp6 interferes with degradation at or upstream of this step, so that degradation delay by Ubp6 is accompanied by a switch in the mode of ubiquitin chain processing. We propose that Ubp6 regulates both the nature and magnitude of proteasome activity.

The need for good aircraft handling qualities has been apparent since the days of the Wright Flyer. In the past decade, there has been a perception that this need has lessened as advanced concepts have evolved, in parallel with acquisition reform. The former has led those who are unfamiliar with the field of handling qualities to conclude that quantitative requirements are not necessary, as the latter has resulted in the elimination of the military specifications for handling qualities. This paper reviews the evolution of handling qualities and their specifications. It presents some ongoing challenges in the field to illustrate that handling qualities are still a critical issue for future aircraft.

Cycloheximide acts at the large subunit of the ribosome to inhibit translation. Here we report that ubiquitin levels are critical for the survival of Saccharomyces cerevisiae cells in the presence of cycloheximide: ubiquitin overexpression confers resistance to cycloheximide, while a reduced ubiquitin level confers sensitivity. Consistent with these findings, ubiquitin is unstable in yeast (t(1/2) = 2 h) and is rapidly depleted upon cycloheximide treatment. Cycloheximide does not noticeably enhance ubiquitin turnover, but serves principally to block ubiquitin synthesis. Cycloheximide also induces UBI4, the polyubiquitin gene. The cycloheximide-resistant phenotype of ubiquitin overexpressors is also characteristic of partial-loss-of-function proteasome mutants. Ubiquitin is stabilized in these mutants, which may account for their cycloheximide resistance. Previous studies have reported that ubiquitin is destabilized in the absence of Ubp6, a proteasome-associated deubiquitinating enzyme, and that ubp6 mutants are hypersensitive to cycloheximide. Consistent with the model that cycloheximide-treated cells are ubiquitin deficient, the cycloheximide sensitivity of ubp6 mutants can be rescued either by ubiquitin overexpression or by mutations in proteasome subunit genes. These results also show that ubiquitin wasting in ubp6 mutants is proteasome mediated. Ubiquitin overexpression rescued cells from additional translational inhibitors such as anisomycin and hygromycin B, suggesting that ubiquitin depletion may constitute a widespread mechanism for the toxicity of translational inhibitors.

We have identified proteins that are abundant in affinity-purified proteasomes, but absent from proteasomes as previously defined because elevated salt concentrations dissociate them during purification. The major components are a deubiquitinating enzyme (Ubp6), a ubiquitin-ligase (Hul5), and an uncharacterized protein (Ecm29). Ecm29 tethers the proteasome core particle to the regulatory particle. Proteasome binding activates Ubp6 300-fold and is mediated by the ubiquitin-like domain of Ubp6, which is required for function in vivo. Ubp6 recognizes the proteasome base and its subunit Rpn1, suggesting that proteasome binding positions Ubp6 proximally to the substrate translocation channel. ubp6Delta mutants exhibit accelerated turnover of ubiquitin, indicating that deubiquitination events catalyzed by Ubp6 prevent translocation of ubiquitin into the proteolytic core particle.

The yeast protein Rad23 belongs to a diverse family of proteins that contain an amino-terminal ubiquitin-like (UBL) domain. This domain mediates the binding of Rad23 to proteasomes, which in turn promotes DNA repair and modulates protein degradation, possibly by delivering ubiquitinylated cargo to proteasomes. Here we show that Rad23 binds proteasomes by directly interacting with the base subcomplex of the regulatory particle of the proteasome. A component of the base, Rpn1, specifically recognizes the UBL domain of Rad23 through its leucine-rich-repeat-like (LRR-like) domain. A second UBL protein, Dsk2, competes with Rad23 for proteasome binding, which suggests that the LRR-like domain of Rpn1 may participate in the recognition of several ligands of the proteasome. We propose that the LRR domain of Rpn1 may be positioned in the base to allow the cargo proteins carried by Rad23 to be presented to the proteasomal ATPases for unfolding. We also report that, contrary to expectation, the base subunit Rpn10 does not mediate the binding of UBL proteins to the proteasome in yeast, although it can apparently contribute to the binding of ubiquitin chains by intact proteasomes.

Substrates enter the proteasome core particle (CP) through a channel that opens upon association with the regulatory particle (RP). Using yeast mutants, we show that channel opening is mediated by the ATPase domain of Rpt2, one of six ATPases in the RP. To test whether degradation products exit through this channel, we analyzed their size distribution. Their median length from an open-channel CP mutant was 40% greater than that from the wild-type. Thus, channel opening may enhance the yield of peptides long enough to function in antigen presentation. These experiments demonstrate that gating of the RP channel controls both substrate entry and product release, and is specifically regulated by an ATPase in the base of the RP.

The standards of what is considered moral or immoral behaviour in politics are heavily influenced by the cultural context and social traditions. Not only does each democracy have its own idea of right and proper conduct for politicians, it also has a view of how relations between elected representatives and officials should be managed. These points are clearly illustrated by looking at recent moves towards reform in the political life of Japan. Despite ‘Westernization’ over many decades, Japanese traditions of gift giving are embedded in political life and political and industrial groupings have highly developed mechanisms of influencing the administration.

Cancer cells are extremely heterogeneous, even in each individual patient, in terms of their malignant potential, drug-senstivity, and their potential to metastasize and cause relapse. Subpopulations of cancer cells with extremely high tumorigenic potential have been isolated from cancer patients with a variety of tumor types and found to have high stemness properties termed cancer stem cells. These stemness-high cancer cells are extremely tumorigenic and are resistant to conventional therapeutics due to activation of pro-survival and anti-apoptotic pathways, overexpression of drug efflux pumps, and increased DNA repair capacity. Moreover, chemotherapy and radiation have been found to induce stemness genes in cancer cells, converting stemness-low cancer cells to stemness-high cancer cells. Such highly tumorigenic and drug-resistant stemness-high cancer stem cells are, therefore, likely to be “left-over” following chemotherapy or radiotherapy and ultimately responsible for relapse. We hypothesized that cancer stemness inhibition is sufficient to suppress metastasis and relapse. Stemness, initially defined by the expression of stem cells genes, is a property shared by embryonic stem cells and adult stem cells. It has been demonstrated that the gene expression profiles of cancer stem cells more closely resemble embryonic stem cells than adult stem cells, suggesting the feasibility to identify molecular targets that are required for cancer stemness, but not (or less so) by normal adult stem cells. Through gene-silencing approaches, we have identified Stat3 as critically important for maintaining cancer stemness, yet largely dispensable for adult stem cells. Here we show that BBI608, a small molecule identified by its ability to inhibit gene-transcription driven by Stat3 and cancer cell stemness properties, displays anticancer properties that are highly different from chemotherapeutics agents. Stemness-high cancer cells enriched by multiple techniques are resistance to chemotherapeutics, yet highly sensitive to the stemness inhibitor BBI608. Blockade of spherogenesis and reduction of stemness gene expression by BBI608 were observed in stemness-high cancer cells isolated from a variety of cancer types. While treatment of xenografted tumor models with chemotherapeutics enriched stemness-high cancer cells, BBI608 induced significant depletion of stemness-high populations in vivo. Moreover, the inhibition of stemness by BBI608 is sufficient to suppress cancer relapse and metastasis in xenografted human cancers in mice. These data demonstrate targeting cancer stemness as an effective way to suppress cancer relapse and metastasis. Citation Format: Youzhi Li, Harry A. Rogoff, Sarah Keates, Yuan Gao, Sylaja Murikipudi, Keith Mikule, David Leggett, Wei Li, Arthur Pardee, Chiang J. Li. Inhibition of stemness by BBI608 is sufficient to suppress cancer relapse and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-253. doi:10.1158/1538-7445.AM2015-LB-253

The proteasome is a highly complex, ATP-dependent protease, consisting of over 30 subunits, and dedicated mainly to the degradation of ubiquitin-protein conjugates. Proteasomes are evolutionarily conserved in the eukaryotic kingdom, and those of yeast are well suited to serve as a general model. We describe techniques for the purification of proteasomes from budding yeast in milligram amounts via conventional and affinity-based strategies. While both approaches yield highly purified material, the affinity method is faster and easier. In addition, the affinity method is more suitable for identifying proteasome-associated proteins. We also describe methods for purifying the major subassemblies of the proteasome, such as the CP, the RP, the lid, and the base. A variety of activity assays and native gel procedures are available to evaluate purified proteasomes functionally. When coupled with the genetic methods available in yeast, these biochemical procedures allow for detailed functional analysis of this unique protein complex.

The evaluation of reactive chemical hazards at the pilot and manufacturing scale, using laboratory testing, is increasingly used and has been well documented. However, reactive chemical hazard evaluation at the R&D scale presents special challenges. The typical hazard testing program requires a significant amount of sample, often takes time (>3 days) to complete, and is can be quite costly. On the other hand, the synthesis of new molecules in the R&D environment often produces only a few grams, occurs quickly (

2546 Background: BBI608, an orally-administered first-in-class cancer stemness inhibitor, blocks CSC self-renewal and induces cell death in CSC as well as non-stem cancer cells by inhibiting Stat3,...

2530 Background: BBI608 is an oral first-in-class cancer stemness inhibitor which inhibits the Stat3, β-catenin and Nanog pathways. Preclinically, potent, broad-spectrum anti-tumor and anti-metasta...

A quaternion-based attitude control system is developed for the X-33 in the ascent flight phase. A non-linear control law commands body-axis rotation rates that align the angular velocity vector with an Euler-axis defining the axis of rotation that takes the body-axis system into a desired-axis system. The magnitude of the commanded body rates are deter- mined by the magnitude of the rotation error. The commanded body rates form the input to a dynamic inversion-based adaptive/reconfigurable control law. The indirect adaptive control portion uses on-line system identification to estimate the current control effectiveness matrix to update a control allocation module. The control allocation runs in a null-space injection mode that excites and decorrelates the ef- fectors without degrading the vehicle response in or- der to enable on-line system identification. A direct adaptive control scheme uses the output of a neural network to compensate for dynamic inversion error. The overall system is designed to provide fault and damage tolerance for the X-33 on ascent. Prelimi- nary results are shown to demonstrate the feasibility of the approach.

The Caenorhabditis elegans ubiquitin-conjugating enzyme UBC-1 is distinct from other RAD6 homologues in possessing a C-terminal tail 40 amino acid residues long [Leggett, Jones and Candido (1995) DNA Cell Biol. 14, 883-891]. Such extensions from the core catalytic domain have been found in a subset of known conjugating enzymes, where they have been shown to have diverse roles including target recognition, membrane attachment and sporulation. In the present study we used mutagenesis in vitro to examine the role of the tail in specific aspects of UBC-1 structure and activity. Cross-linking experiments with purified recombinant UBC-1 reveal that it forms dimers and probably tetramers. The acidic tail of UBC-1 has an important role in this interaction because deletions of the tail significantly decrease, but do not abolish, this self-association. Ubiquitin conjugation assays show that, in addition to accepting a thiol-bound ubiquitin at its active site, UBC-1 is stably mono-ubiquitinated. Deletion analysis and site-directed mutagenesis localize the site of ubiquitination to Lys-162 in the tail. These findings demonstrate that the C-terminal tail of UBC-1 is important both for its quaternary structure and post-translational modification in vitro.

2542 Background: Cancer Stem Cells (CSC) are considered to be fundamentally responsible for malignant growth, relapse, metastasis, and resistance to conventional therapies. BBI608 is an orally-administered first-in-class cancer stemness inhibitor which blocks CSC self-renewal and induces cell death in CSC as well as non-stem cancer cells by inhibition of the Stat3, Nanog and b-catenin pathways, and has shown potent anti-tumor and anti-metastatic activities pre-clinically. Methods: A phase 1 dose escalation studyin adult patients with advanced cancer who had failed standard therapies was conducted to determine the safety, tolerability, recommended phase 2 dose (RP2D), pharmacokinetics and preliminary anti-tumor activity of BBI608. A modified Simon accelerated titration scheme was used for dose escalation, with a cycle consisting of twice-daily oral administration of BBI608 for 4 weeks. Cycles were repeated every 4 weeks (28 days) until progression of disease, unacceptable toxicity, or other discontinuation criteria were met. Results: Fourteen cohorts (N=41) were dosed from 20 mg to 2000 mg/day with adverse events being generally mild; the most common being grade 1-2 diarrhea, nausea, anorexia and fatigue. Four grade 3 events included diarrhea (n=3) and fatigue (n=1). MTD was not reached and further dose escalation was limited by pill burden. By the 400 mg/day dose level the plasma concentration of BBI608 was sustained for over 8 hours at a concentration above 1.5 uM (several fold above the IC50). 17/26 patients evaluable for tumor response achieved SD, for a DCR of 65%. Prolonged TTP was observed in 12/26 evaluable patients (46%), including patients with colorectal (CRC), head and neck, gastric, ovarian, melanoma, and breast cancer. In the subset of patients with CRC (N=18), SD was seen in 8/12 evaluable (67%). A median PFS of 14 weeks and median OS of 47 weeks were observed in evaluable CRC patients. Conclusions: Dose escalation of BBI608, a first-in-class cancer stem cell pathway inhibitor, has been achieved without dose limiting toxicity. BBI608 has shown an excellent safety profile, favorable pharmacokinetics, and encouraging signs of clinical activity particularly in CRC Clinical trial information: NCT01775423.

Introduction A 1985 USAF Test Pilot School project, HAVE PIO, performed a flight evaluation of the longitudinal pilotinduced oscillation (PIO) tendencies of several representative aircraft configurations. The evaluations have been duplicated on ground-based simulators using the same precision offset landing task. Comparisons are presented based on Cooper-Harper ratings, PIO ratings, time history data and pilot comments. Trends are established and an evaluation of the concepts proposed to make ground-based simulators a more effective tool for detecting PIO are presented.

Background: Cancer stem cells (CSC) have self-renewal capability and can differentiate into heterogeneous cancer cells. CSC have been isolated from a variety of human cancers and are considered to be fundamentally responsible for malignant growth, relapse, metastasis, and resistance to conventional therapies [J Clin Oncol. 2008 Jun 10;26(17)]. Therefore, targeting CSC may hold significant clinical promise for treating cancer. BBI608 is the first-in-class cancer cell stemness inhibitor discovered from Boston Biomedical’s cancer stem cell pathway inhibitor program (BBI6000). This novel orally-administered cancer cell stemness inhibitor blocks CSC self-renewal and induces cell death in CSC as well as non-stem cancer cells. In preclinical models, BBI608 showed potent and broad-spectrum anti-tumor and anti-metastatic activities in vitro and in vivo. Methods: A phase 1 dose escalation study in adult patients with advanced cancer who had failed standard therapies was initiated to determine the safety, tolerability, recommended phase 2 dose (RP2D), pharmacokinetics and preliminary anti-tumor activity of BBI608. A cycle consists of twice-daily oral administration of BBI608 for 4 weeks. Cycles were repeated every 4 weeks (28 days) until progression of disease, unacceptable toxicity, or another discontinuation criterion was met. A modified Simon accelerated titration scheme was used for dose escalation. Results: As of March 26th, 2010, eighteen patients have been enrolled with safety data available for 15 patients. Thus far, seven cohorts have been dosed from 20 mg to 600 mg/day and the dose escalation has been well tolerated. Adverse events have generally been mild with the most common being grade 1-2 diarrhea and nausea. Two grade 3 events included fatigue and diarrhea. To date, neither MTD nor RP2D has been reached. Thus far, BBI608 has exhibited favorable pharmacokinetics. At the 400 mg/day dose level the plasma concentration of BBI608 was sustained for over 8 hours at a concentration above 1.5 μM (several folds above the IC50). Of the patients enrolled to date, 9 were evaluable for tumor response; 6 (6/9 evaluable patients) have achieved stable disease for at least 8 weeks. Prolonged stable disease was observed in two patients with colon cancer (54+ and 20+ weeks) and one with head and neck cancer (17 weeks). Complete regression of a metastatic lesion to the kidney was also observed in one colon cancer patient. No new metastatic lesions have been observed in any patients except one patient with gastric adenocarcinoma on 40mg/day of BBI608. Conclusions: Initial dose escalation of BBI608, a first-in-class cancer stem cell pathway inhibitor, has been achieved without dose limiting toxicity. Thus far, BBI608 has shown an excellent safety profile, favorable pharmacokinetics, and preliminary signs of clinical activity. Enrollment and dose escalation are continuing and updated results will be presented. Citation Format: Adrian Langleben, Jeffrey G. Supko, Sebastien Hotte, Patricia Lorusso, Wilson H. Miller, Annie Hurtubise, David S. Leggett, Wei Li, Laura Borodyanski, Chiang J. Li. A phase 1 dose escalation study of BBI608, a first-in-class cancer stem cell pathway inhibitor in patients with advanced malignancies [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-171.

The STOL and Maneuver Technology Demonstrator (S/MTD) program was defined to develop technologies that would enable a supersonic fighter to land on a 1500 x 50 ft usable strip of runway. A short ground roll is achieved using thrust reversing plus control laws to minimize touchdown dispersion. The final /h4TD configuration includes a speed hold system (i.e. augmented airspeed stability) and pitch rate command from the stick. Pilot ratings in a piloted simulation were Level 1 with the speed hold and Level 2 without it. An additional piloted simulation experiment was conducted to investigate the trade-off of pitch axis bandwidth and speed stability. The 03jectives of this paper are to, first, document the rationale and SIMTD approach to precision landing flying qualities and second, to discuss more general design criteria developed from the piloted simulation.