Halide inhibition of the copper-catalysed azide-alkyne cycloaddition.

Chloride, bromide and iodide are inhibitors of the copper-catalysed azide-alkyne cycloaddition, with iodide exhibiting the most detrimental effects on rates and yields. A study of this inhibition is presented, along with experimental protocols to accommodate the presence of halides in this widely used reaction.

Radiographically measured total knee wear is constant and predicts failure.

Knee wear is commonly measured with the most recent radiograph based on the assumption that wear progresses at a constant rate. Changing patient activity or in vivo polyethylene deterioration are examples of factors that could cause wear rates to change over time. Using six or more radiographs on each of 251 knees over a mean 10-year follow-up, we determined the pattern of polyethylene wear. 92% of knees had linear wear with a mean wear rate of 0.09 ± 0.12 mm/yr. Ten knees (4%) had late accelerated wear. Knees with accelerated wear and those with a wear rate of 0.15 mm/yr or greater had lower survivorship rates. We conclude that wear is linear with rare exceptions and that higher wear is correlated with failure.

High-throughput differential scanning fluorimetry (DSF) and cellular thermal shift assays (CETSA): Shifting from manual to automated screening.

Biophysical affinity screening is increasingly being adopted as a high-throughput hit finding technique in drug discovery. Automation is highly beneficial to high-throughput screening (HTS) since a large number of compounds need to be reproducibly tested against a biological target. Herein, we describe how we have automated two biophysical affinity screening methods that rely on a thermal shift in protein melting temperature upon small molecule binding: differential scanning fluorimetry (DSF) and the cellular thermal shift assay (CETSA).

Fragment-Based Screening Identifies New Quinazolinone-Based Inositol Hexakisphosphate Kinase (IP6K) Inhibitors.

A high-throughput fragment-based screen has been employed to discover a series of quinazolinone inositol hexakisphosphate kinase (IP6K) inhibitors. IP6Ks have been studied for their role in glucose homeostasis, metabolic disease, fatty liver disease, chronic kidney disease, blood coagulation, neurological development, and psychiatric disease. IP6Ks phosphorylate inositol hexakisphosphate (IP6) to form pyrophosphate 5-diphospho-1,2,3,4,6-pentakisphosphate (IP7). Molecular docking studies and investigation of structure-activity relationships around the quinazolinone core resulted in compounds with submicromolar potency and interesting selectivity for IP6K1 versus the closely related IP6K2 and IP6K3 isoforms.

Advancing automation in high-throughput screening: Modular unguarded systems enable adaptable drug discovery.

Challenged by ageing infrastructure and increasingly demanding screening cascades, AstraZeneca High Throughput Screening department has developed advanced automation systems that can support both current needs and future strategies in drug discovery. Through collaboration with HighRes Biosolutions and other third-party vendors, highly versatile automated modular platforms have been designed. Safety features such as collaborative robots allow enhanced system accessibility, and adaptive scheduling software has improved protocol design and system recovery. These innovations have led to significant improvements in system flexibility while maintaining screening productivity.

Use of an alarm pheromone against ants for gaining access to aphid/scale prey by the red velvet mite Balaustium sp. (Erythraeidae) in a honeydew-rich environment.

This study shows that honeydew prompts arrestment and reduced activity, but not attraction, by the mite Balaustium sp. nr. putmani. When presented with short-range, two-choice bioassays, mites ceased their characteristic rapid crawling activity when they encountered honeydew-treated surfaces, resulting in them clustering around the honeydew. Approximately 80% of mites were retained by honeydew, with responses being independent of both mite life-history stage and source of honeydew (coccid scale insect or aphid). No obvious crawling movements or redirection of running path were made to the honeydew by the mites, implying the lack of any kind of attractant. Response of mites to single-sugar presentations of the main honeydew components--glucose, sucrose, fructose and trehalose--(0.001-0.1 mmol l(-1)) were inconsistent and failed to reproduce the arrestment/clustering associated with raw honeydew, suggesting that none of these sugars is an active arrestant ingredient. Formation of feeding clusters on honeydew does not contribute to enhancing water conservation by suppressing net transpiration (water loss) rates of individual mites as group size increases, indicating that the clustering is an artifact of arrestment. We hypothesize that release of neryl formate by the mites reduces negative interactions with the local ant species commonly associated with honeydew. We hypothesize that honeydew serves as: (1) a cue that facilitates discovery of scale/aphid prey; (2) a retainer on plants where these prey are present, signaling abundance and quality; and (3) an alternative and supplemental food source like that noted for other plant-inhabiting predatory mites. Neryl formate serves as an alarm pheromone and foul-tasting allomonal defense secretion that prevents predation of mites by ants that co-exist with aphid/scale insects in these honeydew-rich habitats.

Development of a High-Throughput Cytometric Screen to Identify Anti- Wolbachia Compounds: The Power of Public-Private Partnership.

The Anti- Wolbachia (A·WOL) consortium at the Liverpool School of Tropical Medicine (LSTM) has partnered with the Global High-Throughput Screening (HTS) Centre at AstraZeneca to create the first anthelmintic HTS for neglected tropical diseases (NTDs). The A·WOL consortium aims to identify novel macrofilaricidal drugs targeting the essential bacterial symbiont ( Wolbachia) of the filarial nematodes causing onchocerciasis and lymphatic filariasis. Working in collaboration, we have validated a robust high-throughput assay capable of identifying compounds that selectively kill Wolbachia over the host insect cell. We describe the development and validation process of this complex, phenotypic high-throughput assay and provide an overview of the primary outputs from screening the AstraZeneca library of 1.3 million compounds.

Industrial scale high-throughput screening delivers multiple fast acting macrofilaricides.

Nematodes causing lymphatic filariasis and onchocerciasis rely on their bacterial endosymbiont, Wolbachia, for survival and fecundity, making Wolbachia a promising therapeutic target. Here we perform a high-throughput screen of AstraZeneca's 1.3 million in-house compound library and identify 5 novel chemotypes with faster in vitro kill rates (<2 days) than existing anti-Wolbachia drugs that cure onchocerciasis and lymphatic filariasis. This industrial scale anthelmintic neglected tropical disease (NTD) screening campaign is the result of a partnership between the Anti-Wolbachia consortium (A∙WOL) and AstraZeneca. The campaign was informed throughout by rational prioritisation and triage of compounds using cheminformatics to balance chemical diversity and drug like properties reducing the chance of attrition from the outset. Ongoing development of these multiple chemotypes, all with superior time-kill kinetics than registered antibiotics with anti-Wolbachia activity, has the potential to improve upon the current therapeutic options and deliver improved, safer and more selective macrofilaricidal drugs.

Posterior cruciate-retaining total knee arthroplasty for valgus osteoarthritis.

UNLABELLED: The valgus, osteoarthritic knee is challenging technically and it is unknown whether and how technical and implant variables influence outcomes. We therefore determined the influence of surgical technique of soft tissue balancing and patient and implant factors from 100 unselected cruciate-retaining TKAs for valgus osteoarthritis in patients younger than 75 years of age. From 1987 to 1990, lateral soft tissue balancing was done with an outside-in progression in which the lateral collateral ligament and popliteus were typically released from the femur. From 1991 to 1994, an inside-out technique was use in which the lateral collateral ligament and/or popliteus were typically preserved. The minimum followup was 0.1 year (mean, 8.2 years; range, 0.1-18.2 years). Fourteen of 16 revisions were for wear and/or instability. Popliteus release, lateral collateral ligament release, or greater polyethylene shelf age increased the risk of revision. At 10 postoperative years, survival (end point, revision) was 89% (100 knees), 94% when the shelf age was less than 1 year (n = 73 knees), 97% when the popliteus or lateral collateral ligament was not released (n = 57 knees), and 100% when both conditions were met (n = 39 knees). Cruciate-retaining implants can be successfully used in knees with any degree of valgus osteoarthritis and survival is improved when the surgeon preserves at least one of the structures providing lateral stability in flexion and uses polyethylene with a short shelf life. LEVEL OF EVIDENCE: Level III, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

Factors associated with the loss of thickness of polyethylene tibial bearings after knee arthroplasty.

BACKGROUND: Wear of the polyethylene tibial bearing is a leading cause of failure of knee replacements done prior to the current decade. The objective of this study was to determine how patient-related factors, implant-related factors, and limb or tibial component alignment influenced the amount of thickness loss in polyethylene tibial bearings that were retrieved at the time of revision surgery or after the death of the patient. METHODS: We retrieved polyethylene tibial bearings from eighty-one unicondylar and eighty-nine total knee replacements that had been performed because of osteoarthritis with varus deformity from 1984 to 1998. All of the polyethylene bearings had been sterilized with gamma radiation in air. Polyethylene loss was quantified as the change in the minimum bearing thickness per years in vivo (the mean time in vivo [and standard deviation] was 8 +/- 4 years). Multiple linear regression was used to assess whether polyethylene loss was associated with age, weight, gender, varus angle of the tibial component, postoperative hip-knee-ankle angle, initial thickness of the polyethylene, shelf age of the polyethylene, and either the type of polyethylene (for total knee replacements, which were of one posterior cruciate ligament-retaining design) or the manufacturer (for unicondylar knee replacements), and to determine the magnitude by which polyethylene loss would change if any of the significant risk factors were changed. RESULTS: The mean loss (and standard deviation) of polyethylene thickness in the medial compartment of total knee replacements (0.33 +/- 0.28 mm/yr) and that in medial unicompartmental knee replacements (0.49 +/- 0.40 mm/yr) were significantly (p < 0.05) associated with the same three variables: patient age, postoperative hip-knee-ankle angle, and shelf age of the polyethylene. A total knee bearing with a one-year increase in shelf age, a unicondylar knee bearing with a six-month increase in shelf age, a patient who was ten years younger at the time of operation, or a limb that was aligned in 5 degrees more varus (less valgus) had similar effects on the loss of polyethylene thickness in the medial compartment; the coefficients of the linear regression equations indicated that any one of these changes would increase polyethylene loss by 0.11 to 0.14 mm/yr. CONCLUSIONS: The wear-related loss of thickness in gamma-irradiated-in-air polyethylene bearings from unicondylar and total knee replacements implanted in osteoarthritic knees with varus deformity is influenced mainly by the shelf age of the polyethylene, the age of the patient, and the postoperative angulation of the knee in the coronal plane. CLINICAL RELEVANCE: Although polyethylene bearings have not been sterilized with gamma radiation in air since the end of the last decade, many arthroplasty patients have polyethylene bearings that were sterilized with use of this method. An understanding of the findings of this study may be of value as these patients return for follow-up care. Whether the study findings have relevance to bearings sterilized with other methods is unclear and will remain so for many years. LEVEL OF EVIDENCE: Prognostic Level II.

Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Recurrent MLK4 Loss-of-Function Mutations Suppress JNK Signaling to Promote Colon Tumorigenesis.

MLK4 is a member of the mixed-lineage family of kinases that regulate the JNK, p38, and ERK kinase signaling pathways. MLK4 mutations have been identified in various human cancers, including frequently in colorectal cancer, where their function and pathobiological importance have been uncertain. In this study, we assessed the functional consequences of MLK4 mutations in colon tumorigenesis. Biochemical data indicated that a majority of MLK4 mutations are loss-of-function (LOF) mutations that can exert dominant-negative effects. In seeking to understand the abrogated activity of these mutants, we elucidated a new MLK4 catalytic domain structure. To determine whether MLK4 is required to maintain tumorigenic phenotypes, we reconstituted its signaling axis in colon cancer cells harboring MLK4-inactivating mutations. We found that restoring MLK4 activity reduced cell viability, proliferation, and colony formation in vitro and delayed tumor growth in vivo. Mechanistic investigations established that restoring the function of MLK4 selectively induced the JNK pathway and its downstream targets, cJUN, ATF3, and the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. Our work indicates that MLK4 is a novel tumor-suppressing kinase harboring frequent LOF mutations that lead to diminished signaling in the JNK pathway and enhanced proliferation in colon cancer.

Osteolysis after total knee arthroplasty: influence of tibial baseplate surface finish and sterilization of polyethylene insert. Findings at five to ten years postoperatively.

BACKGROUND: Debris displaced from the articular and backside surfaces of the polyethylene inserts of modular tibial components is considered a chief cause of osteolysis at the sites of total knee arthroplasties. One design of total knee replacement featured changes, over time, in the proximal surface roughness of the tibial baseplate and the method of sterilization of the polyethylene insert. We hypothesized that polishing the baseplate surface and sterilizing the insert with means other than gamma radiation in air had reduced the prevalence of osteolysis. METHODS: Three hundred and sixty-five posterior cruciate ligament-retaining Anatomic Modular Knee primary total knee arthroplasties were performed in 300 patients from 1987 to 1998. Anteroposterior and lateral radiographs of the knees were made within a five to ten-year postoperative interval. Two arthroplasty specialists independently examined the radiographs for evidence of osteolysis (defined as any nonlinear region of cancellous bone loss with delineable margins). RESULTS: Osteolysis was identified in 34% (eighty-two) of 242 knees treated with an insert that had been gamma-irradiated in air and affixed to a rough baseplate surface, and it was identified in 9% (nine) of ninety-eight knees treated with an insert that had been gamma-irradiated in an inert gas, or had not been irradiated, and joined to a polished surface. Osteolysis was associated with six factors, including one related to the patient (male gender), one related to the tibial baseplate (the proximal surface finish), three related to the polyethylene insert (the material from which it was machined, the sterilization method, and the shelf age), and one related to the technique (hyperextension of the femoral component relative to the tibial component). CONCLUSIONS: In this design of a total knee prosthesis, polishing the tibial baseplate counterface and implementing a more contemporary sterilization practice (as opposed to gamma radiation in air) noticeably diminished but did not eliminate osteolysis.

Shelf age of the polyethylene tibial component and outcome of unicondylar knee arthroplasty.

BACKGROUND: A recent report linked shelf-aging of unicondylar polyethylene tibial components with accelerated fatigue wear and rapid failure. We retrospectively reviewed our experiences with another unicondylar knee system to investigate the relationship between the shelf age of the polyethylene components and clinical outcome. METHODS: One hundred Single Compartment Replacement unicompartmental knee arthroplasties were performed with cement between 1990 and 1996. The median shelf age for the polyethylene inserts was 1.7 years. At the time of the review, four knees had been lost to follow-up, sixteen were in patients who had died, and nineteen had been revised. Sixty-one knee replacements remained in situ at a mean and standard deviation of 8 +/- 2 years postoperatively. RESULTS: With revision as the end point, Kaplan-Meier survivorship analysis demonstrated a six-year rate of survival of 96% when the shelf age of the insert was less than the median shelf age but only 71% when the median shelf age was exceeded. Fatigue wear was identified on all retrieved components except for two that had shelf ages under one year and had been revised within three years. At four to six years of clinical follow-up, the group of knees in which the median shelf age had been exceeded also had poorer Knee Society knee scores and function scores. CONCLUSIONS: Aging on the shelf accelerated fatigue failure of polyethylene inserts sterilized with gamma irradiation in air and compromised our intermediate-term clinical outcomes with this unicondylar knee replacement system. Attention to the relationship between the shelf age and the clinical performance of the polyethylene component continues to be warranted with unicondylar knee arthroplasty implants.

Tibial interface wear in retrieved total knee components and correlations with modular insert motion.

BACKGROUND: Wear occurring at the interface between the polyethylene insert and metal baseplate of a modular tibial component has become an increasingly common finding at the time of revision total knee arthroplasty. Although this so-called backside wear on retrieved polyethylene inserts has been evaluated in prior studies, wear on retrieved metal baseplates has not been described, to our knowledge. The purposes of the present study were to characterize backside wear on retrieved polyethylene inserts and on the mating surfaces of their corresponding baseplates and to investigate if there is a relationship between backside wear and relative motion of the modular elements. METHODS: Twenty-nine retrieved modular tibial components of twelve fixed-bearing designs were analyzed in vitro with regard to backside wear and relative motion between the polyethylene insert and the metal baseplate. We graded the backside of each polyethylene insert and the mating surface of the metal baseplate for wear with use of a scoring system that consisted of three modes of wear and three levels of severity of wear. Relative motion between the insert and the baseplate was measured in the transverse plane with use of a mechanical testing machine. These measurements were used to compute the insert motion index, which served to quantify unrestricted motion of the insert with respect to the baseplate. RESULTS: The mean insert motion index for the tibial components was 416 micro m (range, 104 micro m to 760 micro m). On a wear-grading scale ranging from 0 to 54 (with 0 indicating no wear), the mean backside wear score was 30 (range, 12 to 48) for the inserts and 28 (range, 7 to 51) for the baseplates. Insert motion was positively correlated with backside polyethylene wear (p = 0.003) and baseplate wear (p < 0.001). Baseplate wear was strongly correlated with backside polyethylene wear (p < 0.001). CONCLUSIONS: Backside wear was correlated with the relative motion between the polyethylene insert and the metal baseplate. New locking mechanism designs directed toward better methods of securing the polyethylene insert to the tibial tray are needed to minimize the generation of particulate wear debris at the modular interface.

Evaluation of contemporary software methods used to quantify polyethylene wear after total hip arthroplasty.

BACKGROUND: Radiographic measurements of the wear of total hip arthroplasty implants are indirect measurements based on radiographic determinations of the location of the femoral head relative to the acetabular component. Using the simplest case of zero wear, we assessed the reproducibility and accuracy of two software applications designed to quantify wear from clinical radiographs. METHODS: After a cobalt-chromium head was glued into the polyethylene liner of a titanium shell, one cross-table lateral radiograph and three anteroposterior pelvic radiographs were made for twelve permutations of acetabular component angulation. The three anteroposterior radiographs differed only with regard to the cephalocaudal positioning of the prosthesis relative to the x-ray tube. To assess method reproducibility, each anteroposterior radiograph was assumed to be both the initial and the latest follow-up radiograph of a wear analysis. To assess method accuracy, each anteroposterior radiograph was paired in a wear analysis with each of the two anteroposterior radiographs made when the component was in the same angulation but at a different cephalocaudal position relative to the tube (one radiograph was the initial follow-up radiograph while the second was the latest follow-up radiograph). The analyses of reproducibility and accuracy were performed both with and without inclusion of the lateral radiograph made with the component in the same angulation. RESULTS: Both methods fared well in the reproducibility analyses, with mean linear and volumetric wear values of 0.00 to 0.07 mm and 0 to 24 mm(3), respectively. In the accuracy analyses, the mean linear and volumetric wear values derived with the two methods were 0.26 to 0.40 mm and 78 to 126 mm(3), respectively. CONCLUSIONS: Whereas the results of the reproducibility analyses showed that the methods were consistent in determining the relative positions of the head and shell from a given anteroposterior radiograph or pair of anteroposterior and lateral radiographs, the non-zero wear results obtained in the accuracy analyses proved that these positional determinations were often inaccurate. Thus, while contemporary software methods may yield reproducible results, their accuracy is limited by their inability to correctly determine the position of the head relative to the acetabular component.

Minisatellite DNA mutation rate in dandelions increases with leaf-tissue concentrations of Cr, Fe, Mn, and Ni.

We have examined whether mutation rates at minisatellite DNA loci in dandelions (Taraxacum officinale Weber, sensu lato: Asteraceae) increase with increasing exposure to metal pollution. From 16 sites (Colorado to Pennsylvania, USA) covering a range of airborne particulate-matter exposures, soil metal concentrations, and leaf-tissue metal concentrations, we grew an average of 7.9 offspring from each of 10 parent plants, and we analyzed the parent-offspring transmission of 82,715 minisatellite DNA markers to 1,258 offspring for rates of mutation. The mean number of markers examined per individual (using six minisatellite probes) was 65.8. Detection of mutations is facilitated by agamospermous reproduction (clonal seed production) in dandelions. Across sites, the average single-event, parent-offspring marker transmission mutation rate was 0.0067, ranging from 0.002 to 0.015 (a 7.5-fold difference). No significant correlation was detected between site single-event mutation rates and either airborne particulate-matter or soil concentrations for any of the metals. However, across sites, mutation rates were significantly (p < 0.05) and positively correlated to increasing leaf-tissue concentrations of Cr, Fe, Mn, and Ni (Cd, Cu, Pb, and Zn exhibited no correlation). Multiple-regression analyses suggest that a model including three metals--in order of importance: Cr (p = 0.002), Fe (p = 0.02), and Ni (p = 0.005); overall, p = 0.001--may improve the ability to predict mutation rate relative to leaf metal concentrations in dandelions. Mutations at minisatellite DNA loci in sexually apomictic organisms may thus provide convenient biomarkers by which to assess the mutagen stressor risk in environments.

Temporal germ cell development strategy during continuous spermatogenesis within the montane lizard, Sceloporus bicanthalis (Squamata; Phrynosomatidae).

Sceloporus bicanthalis is a viviparous lizard that lives at higher elevations in Mexico. Adult male S. bicanthalis were collected (n = 36) from the Nevado de Toluca, Mexico (elevation is 4200 m) during August to December, 2007 and January to July, 2008. Testes were extracted, fixed in Trumps, and dehydrated in a graded series of ethanol. Tissues were embedded, sectioned (2 µm), stained, and examined via a light microscope to determine the spermatogenic developmental strategy of S. bicanthalis. In all months examined, the testes were spermiogenically active; based on this, plus the presence of sperm in the lumina of seminiferous tubules, we inferred that S. bicanthalis had year-round or continuous spermatogenesis, unlike most reptiles that occupy a temperate or montane habitat. It was recently reported that seasonally breeding reptiles had a temporal germ cell development strategy similar to amphibians, where germ cells progress through spermatogenesis as a single population, which leads to a single spermiation event. This was much different than spatial development within the testis of other derived amniotes. We hypothesized that germ cell development was temporal in S. bicanthalis. Therefore, we wanted to determine whether reptiles that practice continuous spermatogenesis have a mammalian-like spatial germ cell development, which is different than the typical temperate reptile exhibiting a temporal development. In the present study, S. bicanthalis had a temporal development strategy, despite its continuous spermatogenic cycle, making them similar to tropical anoles.

Histological analysis of spermatogenesis and the germ cell development strategy within the testis of the male Western Cottonmouth Snake, Agkistrodon piscivorus leucostoma.

Cottonmouth (Agkistrodon piscivorus leucostoma) testes were examined histologically to determine the germ cell development strategy employed during spermatogenesis. Testicular tissues from Cottonmouths were collected monthly from swamps around Hammond, Louisiana. Pieces of testis were fixed in Trump's fixative, dehydrated in ethanol, embedded in Spurr's plastic, sectioned with an ultramicrotome, and stained with toluidine blue and basic fuchsin. Spermatogenesis within Cottonmouths occurs in two independent events within a single calendar year. The testes are active during the months of March-June and August-October with spermiation most heavily observed during April-May and October. To our knowledge, this is the first study that describes bimodal spermatogenesis occurring in the same year within the subfamily Crotalinae. During spermatogenesis, no consistent spatial relationships are observed between germ cell generations. Typically, either certain cell types were missing (spermatocytes) or the layering of 3-5 spermatids and/or spermatocytes within the same cross-section of seminiferous tubule prevented consistent spatial stages from occurring. This temporal pattern of sperm development is different from the spatial development found within birds and mammals, being more reminiscent of that seen in amphibians, and has now been documented within every major clade of reptile (Chelonia, Serpentes, Sauria, Crocodylia). This primitive-like sperm development, within a testis structurally similar to mammals and birds, may represent an intermediate testicular model within the basally positioned (phylogenetically) reptiles that may be evolutionarily significant.