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The pandemic readiness toolbox needs to be extended, targeting different biomolecules, using orthogonal experimental set-ups. Here, we build on our Cov-MS effort using LC-MS, adding SISCAPA technology to enrich proteotypic peptides of the SARS-CoV-2 nucleocapsid (N) protein from trypsin-digested patient samples. The Cov

[Image: see text] A combination of focused library and virtual screening, hit expansion, and rational design has resulted in the development of a series of inhibitors of RET(V804M) kinase, the anticipated drug-resistant mutant of RET kinase. These agents do not inhibit the wild type (wt) isoforms of RET or KDR and therefore offer a potential adjunct to RET inhibitors currently undergoing clinical evaluation.

Objective: FLASH irradiation reportedly produces less normal tissue toxicity, while maintaining tumour response. To investigate oxygen’s role in the ‘FLASH effect’, we assessed DNA damage levels following irradiation at different oxygen tensions, doses and dose rates. Methods: Samples of whole blood were irradiated (20 Gy) at various oxygen tensions (0.25–21%) with 6 MeV electrons at dose rates of either 2 kGy/s (FLASH) or 0.1 Gy/s (CONV), and subsequently with various doses (0–40 Gy) and intermediate dose rates (0.3–1000 Gy/s). DNA damage of peripheral blood lymphocytes (PBL) were assessed by the alkaline comet assay. Results: Following 20 Gy irradiation, lower levels of DNA damage were induced for FLASH, the difference being significant at 0.25% (p < 0.05) and 0.5% O2 (p < 0.01). The differential in DNA damage at 0.5% O2 was found to increase with total dose and dose rate, becoming significant for doses ≥20 Gy and dose rates ≥30 Gy/s. Conclusion: This study shows, using the alkaline comet assay, that lower levels of DNA damage are induced following FLASH irradiation, an effect that is modulated by the oxygen tension, and increases with the total dose and dose rate of irradiation, indicating that an oxygen related mechanism, e.g. transient radiation-induced oxygen depletion, may contribute to the tissue sparing effect of FLASH irradiation. Advances in knowledge: This paper is first to directly show that FLASH-induced DNA damage is modulated by oxygen tension, total dose and dose rate, with FLASH inducing significantly lower levels of DNA damage for doses ≥20 Gy and dose rates ≥30 Gy/s, at 0.5% O2.

There are limits that emerge out of the interactive nature of interactive digital narrative that make authoring it challenging. These limits include exponential branching, where branches in the narrative increase the amount of content needed to be written progressively throughout the work; combinatorial explosion, where increasing combinations of possible game states makes writing additional contentcomplex; as well as programming scope problems that are seen in any digital project, wherein the range of features or game interactions that could be implemented is infinite but development time finite. These limits place on the authors of interactive digital narrative an authorial burden, increasing the amount of content needed to be written, states managed or features programmed. There are multiple strategies for tackling the burden, from reducing or reusing content, to decontextualising and generating content.

Acute stroke care requires rapid assessment and intervention. Replacing traditional sequential algorithms in stroke care with parallel processing using telestroke consultation could be useful in the management of acute stroke patients. The purpose of this study was to assess the feasibility of a nurse-driven acute stroke protocol using a parallel processing model.This is a prospective, nonrandomized, feasibility study of a quality improvement initiative. Stroke team members had a 1-month training phase, and then the protocol was implemented for 6 months and data were collected on a "run-sheet." The primary outcome of this study was to determine if a nurse-driven acute stroke protocol is feasible and assists in decreasing door to needle (intravenous tissue plasminogen activator [IV-tPA]) times.Of the 153 stroke patients seen during the protocol implementation phase, 57 were designated as "level 1" (symptom onset4.5 hours) strokes requiring acute stroke management. Among these strokes, 78% were nurse-driven, and 75% of the telestroke encounters were also nurse-driven. The average door to computerized tomography time was significantly reduced in nurse-driven codes (38.9 minutes versus 24.4 minutes; P .04).The use of a nurse-driven protocol is feasible and effective. When used in conjunction with a telestroke specialist, it may be of value in improving patient outcomes by decreasing the time for door to decision for IV-tPA.

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Introduction: Several serum based biomarkers for acute stroke have been identified, but the clinical values of these biomarkers have not been realized due to inadequate sensitivity and specificity in their measurements. 24 S -Hydroxycholesterol is a stable metabolite of cholesterol synthesized by neurons of the CNS, and released to the serum at a constant rate. Hypothesis: We hypothesized that serum 24 S -Hydroxycholesterol would be an ideal biomarker for acute stroke. Methods: Acute stroke patients presenting to ED were enrolled after informed consent. Serum samples were collected in ED and levels of 24 S -Hydroxycholesterol were determined by mass spectrometry. We enrolled 63 patients from October 2015 to October 2016. Of these 63 patients, 60 presented within 8 hours from onset of symptoms. Patients were categorized as no stroke, ischemic stroke, and intracerebral hemorrhage. We compared serum 24 S -Hydroxycholesterol levels in each group and the variation in 24 S -Hydroxycholesterol levels with regard to presenting NIHSS. Result: Out of the 63 patients enrolled, 22 did not have a stroke, 36 had ischemic stroke, and 5 had intracerebral hemorrhage. The mean 24 S -Hydroxycholesterol levels were 54 ng/ml, 58 ng/ml and 75 ng/ml in the no stroke group, ischemic stroke group and intracerebral hemorrhage group, respectively. No correlation was observed between NIHSS and 24 S -Hydroxycholesterol levels. Difference in 24 S -Hydroxycholesterol levels between no stroke and intracerebral hemorrhage patients was statistically significant, but was limited by the small sample size (P = 0.047). Conclusion: 24 S -Hydroxycholesterol may be a useful serum biomarker for intracerebral hemorrhage. Analyses in a larger population is needed to determine the practical importance of this biomarker.

DNA damage repair enzymes are promising targets in the development of new therapeutic agents for a wide range of cancers and potentially other diseases. The enzyme poly(ADP-ribose) glycohydrolase (PARG) plays a pivotal role in the regulation of DNA repair mechanisms; however, the lack of potent drug-like inhibitors for use in cellular and in vivo models has limited the investigation of its potential as a novel therapeutic target. Using the crystal structure of human PARG in complex with the weakly active and cytotoxic anthraquinone 8a, novel quinazolinedione sulfonamides PARG inhibitors have been identified by means of structure-based virtual screening and library design. 1-Oxetan-3-ylmethyl derivatives 33d and 35d were selected for preliminary investigations in vivo. X-ray crystal structures help rationalize the observed structure–activity relationships of these novel inhibitors.

The present report describes our efforts to convert an existing LXR agonist into an LXR antagonist using a structure-based approach. A series of benzenesulfonamides was synthesized based on structural modification of a known LXR agonist and was determined to be potent dual liver X receptor (LXR α/β) ligands. Herein we report the identification of compound 54 as the first reported LXR antagonist that is suitable for pharmacological in vivo evaluation in rodents.

Novel derivatives of the steroid DHEA 1, a known uncompetitive inhibitor of G6PD, were designed, synthesized, and tested for their ability to inhibit this dehydrogenase enzyme. Several compounds with approximately 10-fold improved potency in an enzyme assay were identified, and this improved activity translated to efficacy in a cellular assay. The SAR for steroid inhibition of G6PD has been substantially developed; the 3β-alcohol can be replaced with 3β-H-bond donors such as sulfamide, sulfonamide, urea, and carbamate. Improved potency was achieved by replacing the androstane nucleus with a pregnane nucleus, provided a ketone at C-20 is present. For pregnan-20-ones incorporation of a 21-hydroxyl group is often beneficial. The novel compounds generally have good physicochemical properties and satisfactory in vitro DMPK parameters. These derivatives may be useful for examining the role of G6PD inhibition in cells and will assist the future design of more potent steroid inhibitors with potential therapeutic utility.

The total synthesis of (±)-janoxepin, a novel antiplasmodial d-leucine derived oxepine-pyrimidinone-ketopiperazine isolated from the fungus Aspergillus janus, is described. The cornerstones of the synthetic route are pyrimidinone preparation, ring-closing metathesis, aldol introduction of the enamide, and dihydro-oxepine elaboration. This synthetic route proved very efficient for the formation of a number of janoxepin analogues, including dihydro-janoxepin and tetrahydro-janoxepin.

Objective: To observe the impact on stroke code time metrics after applying a “pit stop” model of bedside nursing for telestroke encounters. Background: Despite the recent push for target treatment times in acute stroke codes, no guidelines exist for optimizing practices specific to stroke care via telemedicine. Effective telestroke is dependent on efficient data gathering by remote staff, and lengthy metrics for real-world telestroke often preclude timely tPA treatment. By co-opting “pit stops” as inspiration, an optimized nursing workflow for telestroke can be created on the following principles: Identification of Shared Goals; Organized Urgency with the Removal of Gatekeepers; Multi-personnel, Non-Sequential Processes; Focus on Defined Staged Roles; and Empowered Engagement/Responsibility. Methods: The QCI-NASCAR protocol was implemented in Oct 2013, and data was collected prospectively on consecutive stroke code activations through Apr 2014 at St. Paul University Hospital (Dallas, TX), a telestroke spoke site. The nurse-driven protocol was reinforced by a paper checklist (i.e. “Driver Sheet”), which doubled as a data collection form. Timestamps were recorded in real time for: door time, MD at bedside, CT arrival, needle time, and/or code cancellation. The primary outcome was Door-to-CT (D2CT) times to reflect the portion of the stroke code most impacted by the nursing protocol. Results: Mean D2CT times were: all cases (n=152, 33.2 min), intervention-eligible cases (n=71, 27.0 min), and thrombolytic-eligible cases (n=57, 22.2 min). A trend for lower D2CT times and standard deviations was noted in comparing the first half of the data (n=76, 38.04 ± 58.1 min) to the second (n=77, 27.8 ± 19.1 min; p Conclusion: QCI-NASCAR demonstrates the feasibility of implementing a nursing-driven protocol for telestroke encounters. A larger, multi-institutional trial will demonstrate if such a protocol can significantly and reproducibly lower stroke code metrics to national guideline parameters.

α-Hydroxyketones undergo MnO2-mediated oxidation, followed by in situ trapping with 2-pyridylamidrazone, to give 3-pyridyl-5-substituted 1,2,4-triazines in a one-pot procedure, which avoids the need to isolate the reactive α-ketoaldehyde intermediates. By modifying this procedure to allow condensation prior to oxidation, the corresponding 6-substituted 1,2,4-triazines were obtained. The preparation of a novel unsymmetrical 2,2′-bipyridine using one of the pyridyl 1,2,4-triazines prepared herein is also described.

Summary Purpose: This is a phase II study to assess the role of induction chemotherapy in the management of stage IIIA non-small-cell lung cancer (NSCLC). We are now reporting the long-term follow-up of the Toronto phase II trial. Methods: Sixty five patients with mediastinoscopy proven stage IIIA NSCLC received two cycles of preoperative MVP or VLB/P followed by thoracotomy followed by two further courses of chemotherapy. Results: The overall response rate was 67.7% with three complete and 41 partial responders. Forty seven patients went on to thoracotomy with 35 complete resections. Pathologically 4.6% of patients had no tumour remaining. There were three postop deaths as well as five chemotherapy related deaths. Of the 35 patients completely resected 19 have recurred including eight in brain. The median survival for the entire 65 patients is 18.6 months with a 1 year survival of 66%, 5 year survival of 29% and a 10 year survival of 22%. Conclusions: The long-term survival of induction chemotherapy is maintained. The high incidence of brain recurrences warrants assessment of the role of prophylactic cranial radiation. The role of surgery for stage IIIA NSCLC following induction chemotherapy awaits further study.

A novel isonitrile derivative was synthesized and used in an Ugi four component coupling reaction to explore aryl group substitution effects on inhibition of the coagulation cascade serine protease factor Xa.

Abnormalities of hippocampal structure have been reported in schizophrenia subjects. However, such abnormalities have been difficult to discriminate from normal neuroanatomical variation. High dimensional brain mapping, which utilizes probabilistic deformations of a neuroanatomical template, was used to characterize disease-related patterns of changes in hippocampal volume, shape, and asymmetry.T(1)-weighted magnetic resonance scans were collected in 52 schizophrenia and 65 comparison subjects who were similar in age, gender, and parental socioeconomic status. The schizophrenia subjects were clinically stable at the time of assessment.Significant abnormalities of hippocampal shape and asymmetry (but not volume after total cerebral volume was included as a covariate) were found in the schizophrenia subjects. The pattern of shape abnormality suggested a neuroanatomical deformity of the head of the hippocampus, which contains neurons that project to the frontal cortex. The pattern of hippocampal asymmetry observed in the schizophrenia subjects suggested an exaggeration of the asymmetry pattern observed in the comparison subjects. No correlations were found between the magnitude of hippocampal shape and asymmetry abnormality and the severity of residual symptoms or duration of illness.Schizophrenia is associated with structural deformities of the hippocampus, which suggest a disturbance of the connections between the hippocampus and the frontal cortex. However, the magnitude of these deformities are not related to severity or duration of illness.

Activating gene fusions in the RET receptor tyrosine kinase have been found to drive 1-2% of lung adenocarcinomas and therefore offer an attractive target for targeted therapy. Whilst non-selective tyrosine kinase inhibitors with RET activity are efficacious in this setting, their use is generally limited by dose limiting toxicity associated with their more potent activity versus other targets, specifically KDR (VEGFR2) in the case of cabozantinib and vandetanib. Given this limitation, there is considerable interest in developing more selective inhibitors of RET kinase. Tyrosine kinase inhibitors are prone to early clinical failure due to mutations in the kinase ATPase binding domain, which render the kinase catalytically active but no longer sensitive to drug treatment. Such mutations often occur in the so-called “gatekeeper” region and in this specific case, resistance is predicted to arise from a Val-Met or Val-Leu mutation at residue 804. Through a combination of computational methods, structural biology and drug design, we have identified and further optimized a series of inhibitors of the V804M mutant RET kinase which show sub-micromolar cellular activity in cells driven by V804M RET. Moreover, these agents show excellent selectivity against the wtRET kinase and KDR. As such, these agents may offer valuable start-points for second-generation RET inhibitors for use in patents who relapse after treatment with first generation selective RET inhibitors. Citation Format: Allan M. Jordan, Rebecca Newton, Bohdan Waszkowycz, Richard Bayliss, Habiba Begum, Daniel Burschowsky, Aude Echalier, Samantha Hitchin, Colin Hutton, Shaun Johns, Stuart Jones, Li-Ying Lin, Mark Richards, Chitra Seewooruthun, Alex Stowell, Ian Waddell, Mandy Watson, Donald Ogilvie. Delivering selective and cell-active inhibitors of V804M mutant RET kinase through structure-guided drug discovery [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 3236. doi:10.1158/1538-7445.AM2017-3236

Factor Xa, a critical serine protease in the blood coagulation cascade, has become a target for inhibition as a strategy for the invention of novel anti-thrombotic agents. Here we describe the development of phenylglycine containing benzamidine carboxamides as novel, potent and selective inhibitors of factor Xa.

Pyridone derivatives, especially with 6-aryl substituents, have been shown to be useful P2-P3 peptidomimetic scaffolds for the design of potent inhibitors of ICE.

DNA repair is a critical process for the survival and normal proliferation of healthy cells. However, given the enhanced levels of cellular stress and genomic instability, these repair processes are even more critical to the survival of malignant cells, where rates of DNA damage are considerably increased. Given this, inhibitors of DNA damage repair have seen a resurgence of interest in recent years in an effort to exploit tumour cell vulnerabilities. One such example of this approach has resulted in the recent approval of the PARP inhibitor olaparib (Lynparza™) for women with advanced ovarian cancer associated with defective BRCA genes. Olaparib acts against the poly(ADP-ribose)polymerase (PARP) enzymes, more recently re-defined as the ARTD (Diphtheria toxin-like human ADP-ribosyltransferase) enzyme class. Whilst PARP is widely known to play critical and well-understood roles in DNA repair, poly(ADP ribose) glycohydrolase (PARG) is less well known but equally essential for effective DNA repair, degrading PAR chains and facilitating effective DNA repair. However, its inhibition may offer several key advantages over PARP inhibition. Most critically, whilst there are 18 isoforms, there exists only a single PARG protein, offering a specific point of therapeutic intervention. However, due to the open nature of the PARG binding cleft and the nature of the binding site, this protein has been considered to be difficult to inhibit with small, drug-like small molecules, particularly in the cellular context. This poster will describe our efforts to overcome these challenges against this challenging target and report our early successes achieved through innovative computational chemistry strategies. These efforts have delivered several credible, drug-like startpoints for further medicinal chemistry optimisation. Citation Format: Bohdan Waszkowycz, Dominic James, Steven Durant, Nicola Hamilton, Cliff Jones, Stuart Jones, Allan Jordan, Alan Lau, Alison MGonagle, Mark O’Connor, Kate Smith, Alex Stowell, Julie Tucker, Ian Waddell, Donald Ogilvie. Discovery of the first cell-active inhibitors of poly(ADP Ribose) glycohydrolase through high-throughput screening and computational approaches. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4352.

In recent years, many proteins involved in DNA repair, such as ATR, ATM and PARP, have received considerable attention as potential points of therapeutic intervention in cancer. Indeed, these efforts have recently delivered several agents into clinical evaluation or FDA regulatory approval. However, the DNA repair protein poly(ADP ribose) glycohydrolase (PARG), which plays an equally critical role in DNA single stand break repair, to successful drug discovery efforts. Through our innovative collaboration with AstraZeneca, we have discovered a novel PARG-binding pharmacophore and have employed this information to discover drug-like chemotypes, facilitating the development of potent and selective inhibitors. This poster will describe our emerging results in this area, where a novel benzimidazolone sulphonamide scaffold has been shown potently to inhibit PARG in both biochemical and cellular assays with potencies of 40 nM and 60 nM respectively. Moreover, these agents display pharmacology consistent with the anticipated mode of action, appropriate drug-like properties and are selective against PARP1 and the close glycohydrolase homologue ARH3. The medicinal chemistry optimisation of this scaffold will be described, alongside the recent biological results obtained. Ultimately, this work has helped deliver tool compounds which may help to elucidate the true pharmacology and roles of PARG in cancer and other disease settings. Citation Format: Allan Jordan, Ben Acton, Nicola Hamilton, James Hitchin, Colin Hutton, Dominic James, Cliff Jones, Stuart Jones, Alison McGonagle, Helen Small, Kate Smith, Alex Stowell, Julie Tucker, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. Benzimidazolone sulphonamides - potent, selective and drug-like inhibitors of poly(ADP Ribose) Glycohydrolase (PARG). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3715.

The macrodomain protein poly(ADP ribose) glycohydrolase (PARG) has been shown to be a critical component in the repair of single stand DNA breaks and counteracts the function of the ARTD family of poly(ADP ribose) polymerases, commonly known as the PARPs. As PARG exists as a single protein, it presents an attractive target for therapeutic intervention in cancer cells with enhanced dependence upon DNA repair. Inhibitors of this enzyme have proved difficult to discover and develop. Moreover, intact cell-active tool compounds which have the propensity to be used as robust chemical probes to understand PARG pharmacology, are absent from the literature. This poster will describe our work in this emerging area, optimising a series of drug-like quinazolinedione derivatives to deliver molecules with the correct physicochemical and biochemical properties to function as in vitro cell probe compounds. These unprecedented agents display potent on-target biochemical (5 nM) and cell (10 nM) activity with a significant window to acute 3-day cytotoxicity. Moreover, these agents are selective against PARP family members and the close glycohydrolase homologue ARH3. The medicinal chemistry optimisation of the scaffold will be described, alongside the outline pharmacology demonstrating on-target, selective inhibition of PARG in cells. Such tool compounds will be of value in revealing the detailed mechanisms of action of PARG in DNA repair and other PAR chain-mediated cellular processes, with the ultimate goal of delivering novel and clinically relevant therapeutic agents. Citation Format: Kate Smith, Ben Acton, Dominic James, Cliff Jones, Stuart Jones, Allan Jordan, Nicola Hamilton, Alison McGonagle, Daniel Mould, Helen Small, Alex Stowell, Julie Tucker, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. Optimisation of quinazolinedione sulphonamides as novel inhibitors of poly(ADP Ribose) glycohydrolase (PARG). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3714.

RET (REarranged during Transfection) is a receptor tyrosine kinase, which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activation of RET is a mechanism of oncogenesis in medullary thyroid carcinomas where both germline and sporadic activating somatic mutations are prevalent. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been opportunistically identified through selectivity profiling of compounds initially designed to target other tyrosine kinases. Vandetanib and cabozantinib, both multi-kinase inhibitors with RET activity, are approved for use in medullary thyroid carcinoma, but additional pharmacological activities, most notably inhibition of vascular endothelial growth factor - VEGFR2 (KDR), lead to dose-limiting toxicity. The recent identification of RET fusions present in ~1% of lung adenocarcinoma patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. In an earlier publication [Newton et al, 2016; 1] we reported the discovery of a series of 2-substituted phenol quinazolines as potent and selective RET kinase inhibitors. Here we describe the development of the robust screening cascade which allowed the identification and advancement of this chemical series. Furthermore we have profiled a panel of RET-active clinical compounds both to validate the cascade and to confirm that none display a RET-selective target profile.

The total synthesis of an analogue of FK506, in which the substituted cyclohexyl residue at C28 has been replaced by a phenyl group, is described. This synthesis demonstrates (i) the successful application of new methodology for the introduction of the masked tricarbonyl grouping (C8-C10), and (ii) new synthetic routes to the (C10-C19) and (C22-C26) regions.

The asymmetric synthesis of a fragment corresponding to the C 1 -C 8 region of the immunosuppressant dicodermolide is reported. A trihydroxylactone of defined absolute stereochemistry is also prepared. This compound is a potential reductive ozonolysis product of discodermolide and may aid the determination of the absolute stereochemistry of the natural product.

The 5-year survival rates for patients undergoing a potentially curative surgical resection for pre-operatively identified stage 3A N2 non-small cell lung cancer (NSCLC) vary from 2–13%. In an attempt to improve the curative potential of surgery, 39 patients with mediastinoscopy stage 3A unresectable N2 NSCLC received induction chemotherapy with 2 cycles of mitomycin-C, vindesine and cisplatin (MVP). Responding patients underwent thoracotomy for resection and 2 further courses of MVP. The overall response rate was 64% ( 25 39 ) with 3 complete and 22 partial responses. 22 patients were resected which included a radical mediastinal node dissection. 18 resections were complete and 4 were incomplete. Pathologically 3 patients (7.7%) had no tumor remaining. There were 2 post-op deaths secondary to a BP fistula. In addition to GI and neurologic side effects, toxicity of chemotherapy included mitomycin pulmonary toxicity in 2 patients and 4 septic deaths. 28 patients have died, 20 with recurrent or progressive disease. Of the 18 patients completely resected, 8 have recurred with a median time to recurrence of 20.6 months. Sites of recurrence include 2 loco-regional, 5 distant (2 in brain) and 1 both. Median survival of the entire 39 patients is 18.6 months with a 3-year survival of 26%. The median survival for those patients completely resected is 29.7 months with a 3-year survival of 40%. These findings suggest that MVP is an effective but toxic chemotherapeutic regimen for limited NSCLC, and although the median survival appears to be prolonged, the role of induction chemotherapy followed by surgery in stage IIIA N2 NSCLC requires a Phase III randomized trial comparing it to other treatment modalities.

An understanding of the mass transfer behavior of an impinging jet can be usefully applied to wet chemical processes such as water rinsing, photoresist development, and metal etching or plating. Theoretical and experimental methods were used to study the mass transfer characteristics of an axisymmetric impinging jet confined between two parallel plates. Such a configuration was used because of its potential applicability to the fabrication of printed wiring boards. The CFD (computational fluid dynamics) method was used to model fluid flow and mass transfer. An electrochemical probe based on the ferro-ferricyanide system was used to experimentally determine the mass transfer coefficients and to evaluate the applicability of the theoretical methods used. An etching method was used to characterize the mass transfer rates in a typical cupric chloride etching solution. A new observation of the effect of jet instability on the etching rate in the central impingement zone is discussed.

The total synthesis of an analogue of FK506, in which the substituted cyclohexyl residue at C28 has been replaced by a phenyl group, is described. This synthesis demonstrates (i) the successful application of new methodology for the introduction of the masked tricarbonyl grouping (C8-C10), and (ii) new synthetic routes to the (C10-C19) and (C22-C26) regions.

Mice immunized with an aromatic-dependent ( aro − ) S. dublin strain CS101 by either the intraperitoneal (i.p.) or oral route, were protected against oral challenge with a virulent S. dublin strain CS90, the degree of protection being the greatest when mice had received 3 immunizing doses at weekly intervals. Mice immunized with an aromatic-dependent ( aro − ) S. typhimurium strain CS332 by the i.p. or oral routes were protected against challenge with virulent S. dublin strain CS90 at 1 or 2 weeks but not at 3 or 4 weeks post-immunization. Mice immunized with 1 dose of aro − S. dublin strain CS101 by the i.p. route developed low levels of lipopolysaccharide (LPS) and flagellin-specific antibody but no delayed-type hypersensitivity (DTH) whereas those immunized with 2 or 3 doses developed significantly higher antibody titres and DTH. In contrast, mice immunized by the oral route developed neither significant antibody response nor DTH. The aro − S. dublin strain CS101 could not be detected beyond day 28 post-inoculation in visceral organs including liver, spleen, mesentery, small intestine, caecum or large intestine of mice inoculated by the i.p. route or in mice inoculated by the oral route with the exception of day 42 post-inoculation. Challenge of mice previously immunized with 3 doses of the aro − S. dublin strain CS101 by the i.p. or oral route with virulent S. dublin strain CS90 resulted in their rapid clearance from the above visceral organs. Calves immunized with the aro − S. dublin strain CS101 by either the intramuscular (i.m.) or oral routes were significantly protected against oral challenge with virulent S. dublin strain CS90. In contrast to the observations in mice, somatic (O) and flagellar (H) antibody titres of calves immunized by either route were negligible as were anti-LPS antibody titres. However, flagellin-specific antibody titres were higher in calves immunized by the i.m. than the oral route. These results indicate that the protection observed in immunized mice or calves against oral challenge with virulent S. dublin was unlikely to have been mediated by humoral salmonella-specific immune mechanism(s).

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases (PARPs). PARG depletion, using RNAi, results in several effects such as PAR chain persistence, progression of single- to double-strand DNA lesions and NAD+ depletion. Given these findings, inhibition of PARG with a small molecule agent offers a potential opportunity to interfere with DNA repair mechanisms and induce cell death in those cells with increased susceptibility to DNA damage, such as tumour cells. Previous efforts to develop small molecule inhibitors of PARG activity have generally been hampered by poor physicochemical properties, off-target pharmacology and a lack of cell permeability, leading some to suggest that PARG may be undruggable. In contrast, we have now developed a series of first-in-class PARG inhibitors which display drug-like properties and attractive pharmacokinetic parameters. These compounds have proved to be useful biological tool compounds. Moreover, displaying selective activity in both biochemical and, more importantly, cellular assays of PARG function, these derivatives have allowed an exploration of the phenotypes resulting from reversible, pharmacological PARG inhibition in both in vitro cell panels and in vivo models. Furthermore, our initial bioinformatic analysis suggests that deficiency of a known tumour suppressor confers sensitivity to PARG inhibition, suggesting patient populations that will potentially benefit from PARGi therapies. Citation Format: Bohdan Waszkowycz, Dominic James, Ben Acton, Emma Fairweather, Sam Fritzl, Niall Hamilton, Nicola Hamilton, Sarah Holt, James Hitchen, Colin Hutton, Stuart Jones, Allan Jordan, Alison McGonagle, Daniel Mould, Helen Small, Kate Smith, Alexandra Stowell, Ian D. Waddell, Donald Ogilvie. First-in-class inhibitors of the putatively undruggable DNA repair target Poly(ADP-ribose) glycohydrolase (PARG). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C39.

RET (REarranged during Transfection) is a receptor tyrosine kinase (TK), which plays pivotal roles in regulating cell survival, differentiation, proliferation, migration and chemotaxis. Activating mutations in RET (C634W and M918T) have been identified in both familial and sporadic forms of medullary thyroid carcinoma (MTC) and correlate with aggressive disease progression, validating RET as a classical oncogene. Furthermore the recent identification of RET fusions (CCDC6-RET and KIF5B-RET) present in ∼1% of lung adenocarcinoma (LAD) patients has renewed interest in the identification and development of more selective RET inhibitors lacking the toxicities associated with the current treatments. At present, there are no known specific RET inhibitors in clinical development, although many potent inhibitors of RET have been identified opportunistically through selectivity profiling of compounds initially designed to target other TKs. Such “secondary RET inhibitors” include the clinical agents Vandetanib and Cabozantinib, both approved for use in MTC, but additional pharmacological activities (most notably inhibition of KDR) lead to dose-limiting toxicity. Using a robust screening cascade developed in house, we have measured RET and KDR inhibitory activity in vitro and in relevant cell line models to assess compound potency and selectivity. Anti-proliferative activity and off-target toxicity of these agents have also been measured. Although these competitor compounds displayed reasonable RET potency in cellular assays and this translated into anti-proliferative effects in our MTC and LAD disease models, as expected none met our target candidate criteria, clearly highlighting the need for therapeutic agents with improved selectivity. Guided by structure-based drug design, we have identified and optimised a novel series of potent and selective inhibitors of the RET kinase domain. These agents met our stringent criteria for enzyme and cell selectivity and, whilst potent in a RET-driven cell line, display little overt toxicity in a matched non-RET driven cell line. Herein, we describe the chemical optimisation of these agents and, using structural information, rationalise their improved selectivity. Citation Format: Roger J. Butlin, Rebecca Newton, Mandy Watson, Gemma Hopkins, Ben Acton, Kate Bowler, Samantha Fritzl, Kristin Goldberg, Niall Hamilton, Sarah Holt, Stuart Jones, Allan Jordan, Nikki March, Daniel Mould, Helen Small, Alexandra Stowell, Ian Waddell, Bohdan Waszkowycz, Donald Ogilvie. The identification and structure-guided optimisation of potent and selective inhibitors of oncogenes in medullary thyroid carcinoma and lung adenocarcinoma. [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 778. doi:10.1158/1538-7445.AM2015-778

The fluid head is an exciting new technology developed at IBM Endicott. It is based on the fluid bearing principle, where small volumes of high speed fluid, hold and support and process a panel. This process is confined very close to the product and is able to deliver controlled amounts of fluids resulting in: less water usage; emission reduction; better chemical containment; reduced atomization; less hazardous waste; and less energy consumption. In this paper, comparisons of equipment utilizing the fluid head in rinsing, and drying devices are compared with conventional dip and spray processes. Parameters of ionic contamination, debris removal, electrical usage, as well as water usage and reduction in floor space are compared. >

RNA interference experiments in Caenorhabditis elegans suggest functional overlap in many ubiquitin-conjugating enzymes (UBCs). Phylogenetic analysis of C. elegans, Drosophila, and human genes implies that the numbers of UBCs increases with developmental complexity., Background The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs. Results The C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis of C. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species. The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis. Conclusions The number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.

The red alga Chondrus crispus (Irish moss) has been commercially harvested in Eastern Canada for almost 60 years. Its land-based tank aquaculture was initiated in the 1970s. In the 1990s, it became clear that production costs of these capital intensive systems were still too high for the carrageenan market but not for the production of edible seaweeds. Open-water aquaculture of cold-temperate species of carrageenophytes, and in particular of C. crispus, has rarely been attempted. This study re-examined the potential of the unique unattached and mostly vegetative population of C. crispus at Basin Head, in eastern Prince Edward Island (P.E.I.), and at 5 transplant sites in western P.E.I. Basin Head plants were successfully transplanted to other sites, providing similar or different environmental conditions, and yielding comparable, or even higher, productivity. During the peak growth periods (May to end of June and autumn), daily growth rates (DGRs) between 3 and 4% d−l were recorded at Basin Head and Freeland, with some plants exceeding 6% d−1. Over the whole study period (May to October), DGRs between 2 and 4% d−1 were lower than those reported for different species of Eucheuma and Kappaphycus alvarezii; they were, however, compensated for by extremely high carrageenan yields (between 58.1 and 71.0% DW) during the summer months when nutrients (phosphorus and nitrogen) levels in seawater and algal tissue were low. The DGRs could be increased by developing culture structures retaining fragmenting, but otherwise healthy, large distal clumps, lost with the present simple tying of plants on screens. Preliminary results demonstrated that transplantation and grow-out techniques are biologically successful, and that the Basin Head population of C. crispus has significant potential for open-water aquaculture in estuaries and basins of Atlantic Canada.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway (PPP) and catalyses the oxidation of glucose-6-phosphate to glucono-δ-lactone-6-phosphate with the concomitant production of NADPH. Alongside an important role in the production of biosynthetic precursors such as ribonucleotides, this pathway is a major cellular source of NADPH. As such, the pathway plays a critical role in both the biosynthesis of fatty acids & cholesterol biosynthesis (thus supporting cell division) and maintaining glutathione in its reduced state (GSH), the latter ameliorating cellular stress arising from reactive oxygen species (ROS). Given these key roles, modulation of the pathway has potential therapeutic application, and inhibition of G6PD is an attractive target, particularly in combination with standard of care radiotherapy or cytotoxic drugs which can increase ROS. We have developed a series of novel steroid G6PD inhibitors which, in our hands, modulate flux through the PPP and increase ROS-induced stress in cells. This disclosure will describe some of our results in this area. 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 1139. doi:1538-7445.AM2012-1139