20 years of crystal hits: progress and promise in ultrahigh-throughput crystallization screening.

Diffraction-based structural methods contribute a large fraction of the biomolecular structural models available, providing a critical understanding of macromolecular architecture. These methods require crystallization of the target molecule, which remains a primary bottleneck in crystal-based structure determination. The National High-Throughput Crystallization Center at Hauptman-Woodward Medical Research Institute has focused on overcoming obstacles to crystallization through a combination of robotics-enabled high-throughput screening and advanced imaging to increase the success of finding crystallization conditions. This paper will describe the lessons learned from over 20 years of operation of our high-throughput crystallization services. The current experimental pipelines, instrumentation, imaging capabilities and software for image viewing and crystal scoring are detailed. New developments in the field and opportunities for further improvements in biomolecular crystallization are reflected on.

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

Strong and recurring seasonality revealed within stream diatom assemblages.

Improving stream water quality in agricultural landscapes is an ecological priority and a legislative duty for many governments. Ecosystem health can be effectively characterised by organisms sensitive to water quality changes such as diatoms, single-celled algae that are a ubiquitous component of stream benthos. Diatoms respond within daily timescales to variables including light, temperature, nutrient availability and flow conditions that result from weather and land use characteristics. However, little consideration has been given to the ecological dynamics of diatoms through repeated seasonal cycles when assessing trajectories of stream function, even in catchments actively managed to reduce human pressures. Here, six years of monthly diatom samples from three independent streams, each receiving differing levels of diffuse agricultural pollution, reveal robust and repeated seasonal variation. Predicted seasonal changes in climate-related variables and anticipated ecological impacts must be fully captured in future ecological and water quality assessments, if the apparent resistance of stream ecosystems to pollution mitigation measures is to be better understood.

Major agricultural changes required to mitigate phosphorus losses under climate change.

Phosphorus losses from land to water will be impacted by climate change and land management for food production, with detrimental impacts on aquatic ecosystems. Here we use a unique combination of methods to evaluate the impact of projected climate change on future phosphorus transfers, and to assess what scale of agricultural change would be needed to mitigate these transfers. We combine novel high-frequency phosphorus flux data from three representative catchments across the UK, a new high-spatial resolution climate model, uncertainty estimates from an ensemble of future climate simulations, two phosphorus transfer models of contrasting complexity and a simplified representation of the potential intensification of agriculture based on expert elicitation from land managers. We show that the effect of climate change on average winter phosphorus loads (predicted increase up to 30% by 2050s) will be limited only by large-scale agricultural changes (e.g., 20-80% reduction in phosphorus inputs).The impact of climate change on phosphorus (P) loss from land to water is unclear. Here, the authors use P flux data, climate simulations and P transfer models to show that only large scale agricultural change will limit the effect of climate change on average winter P loads in three catchments across the UK.

Changing climate and nutrient transfers: Evidence from high temporal resolution concentration-flow dynamics in headwater catchments.

We hypothesise that climate change, together with intensive agricultural systems, will increase the transfer of pollutants from land to water and impact on stream health. This study builds, for the first time, an integrated assessment of nutrient transfers, bringing together a) high-frequency data from the outlets of two surface water-dominated, headwater (~10km(2)) agricultural catchments, b) event-by-event analysis of nutrient transfers, c) concentration duration curves for comparison with EU Water Framework Directive water quality targets, d) event analysis of location-specific, sub-daily rainfall projections (UKCP, 2009), and e) a linear model relating storm rainfall to phosphorus load. These components, in combination, bring innovation and new insight into the estimation of future phosphorus transfers, which was not available from individual components. The data demonstrated two features of particular concern for climate change impacts. Firstly, the bulk of the suspended sediment and total phosphorus (TP) load (greater than 90% and 80% respectively) was transferred during the highest discharge events. The linear model of rainfall-driven TP transfers estimated that, with the projected increase in winter rainfall (+8% to +17% in the catchments by 2050s), annual event loads might increase by around 9% on average, if agricultural practices remain unchanged. Secondly, events following dry periods of several weeks, particularly in summer, were responsible for high concentrations of phosphorus, but relatively low loads. The high concentrations, associated with low flow, could become more frequent or last longer in the future, with a corresponding increase in the length of time that threshold concentrations (e.g. for water quality status) are exceeded. The results suggest that in order to build resilience in stream health and help mitigate potential increases in diffuse agricultural water pollution due to climate change, land management practices should target controllable risk factors, such as soil nutrient status, soil condition and crop cover.

High frequency variability of environmental drivers determining benthic community dynamics in headwater streams.

Headwater streams are an important feature of the landscape, with their diversity in structure and associated ecological function providing a potential natural buffer against downstream nutrient export. Phytobenthic communities, dominated in many headwaters by diatoms, must respond to physical and chemical parameters that can vary in magnitude within hours, whereas the ecological regeneration times are much longer. How diatom communities develop in the fluctuating, dynamic environments characteristic of headwaters is poorly understood. Deployment of near-continuous monitoring technology in sub-catchments of the River Eden, NW England, provides the opportunity for measurement of temporal variability in stream discharge and nutrient resource supply to benthic communities, as represented by monthly diatom samples collected over two years. Our data suggest that the diatom communities and the derived Trophic Diatom Index, best reflect stream discharge conditions over the preceding 18-21 days and Total Phosphorus concentrations over a wider antecedent window of 7-21 days. This is one of the first quantitative assessments of long-term diatom community development in response to continuously-measured stream nutrient concentration and discharge fluctuations. The data reveal the sensitivity of these headwater communities to mean conditions prior to sampling, with flow as the dominant variable. With sufficient understanding of the role of antecedent conditions, these methods can be used to inform interpretation of monitoring data, including those collected under the European Water Framework Directive and related mitigation efforts.

Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase.

In all organisms, aminoacyl tRNA synthetases covalently attach amino acids to their cognate tRNAs. Many eukaryotic tRNA synthetases have acquired appended domains, whose origin, structure and function are poorly understood. The N-terminal appended domain (NTD) of glutaminyl-tRNA synthetase (GlnRS) is intriguing since GlnRS is primarily a eukaryotic enzyme, whereas in other kingdoms Gln-tRNA(Gln) is primarily synthesized by first forming Glu-tRNA(Gln), followed by conversion to Gln-tRNA(Gln) by a tRNA-dependent amidotransferase. We report a functional and structural analysis of the NTD of Saccharomyces cerevisiae GlnRS, Gln4. Yeast mutants lacking the NTD exhibit growth defects, and Gln4 lacking the NTD has reduced complementarity for tRNA(Gln) and glutamine. The 187-amino acid Gln4 NTD, crystallized and solved at 2.3 Å resolution, consists of two subdomains, each exhibiting an extraordinary structural resemblance to adjacent tRNA specificity-determining domains in the GatB subunit of the GatCAB amidotransferase, which forms Gln-tRNA(Gln). These subdomains are connected by an apparent hinge comprised of conserved residues. Mutation of these amino acids produces Gln4 variants with reduced affinity for tRNA(Gln), consistent with a hinge-closing mechanism proposed for GatB recognition of tRNA. Our results suggest a possible origin and function of the NTD that would link the phylogenetically diverse mechanisms of Gln-tRNA(Gln) synthesis.

Risk of uterine rupture in Australian women attempting vaginal birth after one prior caesarean section: a retrospective population-based cohort study.

OBJECTIVE: Higher risks of uterine rupture have been reported among women attempting vaginal birth after caesarean (VBAC) particularly following induction with prostaglandins, compared with women who do not labour. This study aimed to estimate these risks as well as that associated with oxytocin use. DESIGN: Population-based retrospective cohort study involving all women who had their first births by caesarean. In their second birth, risks of uterine rupture among women without labour and women who had labour augmented or induced were compared with women who gave birth after spontaneous labour. SETTING: Four Australian states in 1998-2000. POPULATION: Women on pregnancy outcome databases with a second birth after a prior caesarean for their first birth. METHODS: From 29, 008 women identified from the databases, those with uterine rupture were identified and validated using hospital case records. MAIN OUTCOME MEASURE: Uterine rupture. RESULTS: The risk of complete uterine rupture among women without labour was 0.01%. The risk in spontaneous labour without augmentation was 0.15%, considerably higher when there was augmentation with oxytocin (1.91%). The risk with induction of labour was 0.54% for oxytocin alone, 0.68% for prostaglandin alone, 0.63% without either and 0.88% when they were combined. Compared with spontaneous labour, risks were increased three- to five-fold for any induction, six-fold for prostaglandin combined with oxytocin and 14-fold for augmentation with oxytocin. CONCLUSIONS: Careful consideration should be given to the use of oxytocin for augmentation of labour or induction by any method for women with a previous caesarean in view of increased risks of uterine rupture.

Crystal cookery - using high-throughput technologies and the grocery store as a teaching tool.

Crystallography is a multidisciplinary field that links divergent areas of mathematics, science and engineering to provide knowledge of life on an atomic scale. Crystal growth, a key component of the field, is an ideal vehicle for education. Crystallization has been used with a 'grocery store chemistry' approach and linked to high-throughput remote-access screening technologies. This approach provides an educational opportunity that can effectively teach the scientific method, readily accommodate different levels of educational experience, and reach any student with access to a grocery store, a post office and the internet. This paper describes the formation of the program through the students who helped develop and prototype the procedures. A summary is presented of the analysis and preliminary results and a description given of how the program could be linked with other aspects of crystallography. This approach has the potential to bridge the gap between students in remote locations and with limited funding, and access to scientific resources, providing students with an international-level research experience.

PEGylation of cyanovirin-N, an entry inhibitor of HIV.

Cyanovirin-N (CV-N) is a potent inhibitor of human immunodeficiency virus and many other viruses. It has a high potential for use as a systemic compound to control viral load or in the development of microbicides to prevent primary viral infection. Due to its cyanobacterial origin it is likely to show the typical drawbacks associated with pharmaceutical use of foreign proteins such as short plasma half-life, proteolysis and immunogenicity. Several strategies were used to covalently bond poly(ethylene glycol) (PEGylate) to CV-N. Random PEGylation at lysine residues resulted in poor retention of antiviral activity. Many site-directed mutants were made to test site-specific PEGylation. One mutant, where glutamine 62 was replaced with cysteine (CV-N(Q62C)) and PEGylated with maleimide activated PEG, retained significant anti-HIV activity in vitro.

Lack of isohemagglutinin production following minor ABO incompatible unrelated HLA mismatched umbilical cord blood transplantation.

While immune hemolysis due to donor isohemagglutinin (IH) production often complicates minor ABO incompatible peripheral blood hematopoietic stem cell transplantation (PBSCT), it is not known if this occurs with umbilical cord blood transplantation (UCBT). We compared IH production and hemolysis following minor ABO allogeneic PBSCT and UCBT. We reviewed 24 ABO minor incompatible allogeneic PBSCTs and 14 ABO minor incompatible UCBTs. Patients were evaluated for donor-derived IH by reverse ABO grouping. Evaluation of hemolysis was based on clinical and laboratory findings of anemia associated with increased RBC transfusion need, concomitant with the appearance of donor-derived IH. Of the 24 ABO minor incompatible allogeneic PBSCTs, 15 produced donor-derived IH from 6 to 88 days following transplantation, with seven of 15 patients exhibiting clinically evident hemolysis. There was no significant difference in days to leukocyte engraftment or infused CD34 cells in patients with or without donor-derived IH. None of the 14 patients receiving ABO incompatible UCBTs showed evidence of donor-derived IH following transplantation with a median follow-up of 60 days. We conclude that donor IHs are not produced in patients undergoing minor ABO incompatible UCBTs suggesting fundamental immunologic differences between allogeneic PBSCT and UCBT.

Efficacy and acceptability of a new topical skin lotion of sodium cromoglicate (Altoderm) in atopic dermatitis in children aged 2-12 years: a double-blind, randomized, placebo-controlled trial.

BACKGROUND: Atopic dermatitis (AD) is a common inflammatory allergic disease of children. The primary anti-inflammatory therapy is topical steroids. An effective treatment without the topical and systemic adverse effects of corticosteroids would be useful. Topical formulations of sodium cromoglicate have been researched in the past, but without consistent results. We report a trial of a new aqueous skin lotion of sodium cromoglicate (Altoderm) in children with AD. OBJECTIVES: To compare the efficacy, safety and acceptability of Altoderm lotion with a placebo control in the treatment of AD in children. METHODS: A double-blind, controlled study in which children aged 2-12 years with AD were randomized to 12 weeks of treatment with a lotion containing 4% sodium cromoglicate (Altoderm) or the lotion base. To be included subjects had to have a SCORAD score of > or = 25 and < or = 60 at both of two clinic visits 14 days apart. Subjects continued using existing treatment which included emollients and topical steroids. The primary outcome was the change in the SCORAD score. The two groups were compared for the change in the SCORAD score from the second baseline visit to the visit after 12 weeks of treatment using an analysis of variance. Secondary outcome measures included parents' assessment of symptoms, usage of topical steroids recorded on daily diary cards, and final opinions of treatment by parent and clinician. Parents were asked about adverse effects at each clinic visit and the responses recorded. RESULTS: Fifty-eight children were randomized to Altoderm and 56 to placebo and all were included in the intention-to-treat analysis. The mean +/- SD SCORAD scores at baseline were 41.0 +/- 9.0 (Altoderm) and 40.4 +/- 8.73 (placebo). These scores were reduced after 12 weeks by 13.2 (36%) with Altoderm and by 7.6 (20%) with placebo. The difference of 5.6 (95% confidence interval 1.0-10.3) is statistically significant (P = 0.018). Diary card symptoms improved with both treatments but the improvement was greater in the Altoderm-treated patients. Topical steroid usage was reduced in both groups and was larger in the Altoderm-treated patients. The differences were statistically significant for the mean of all symptoms, the overall skin condition and use of topical steroids. Those for itching and sleep loss were not. Treatment-related adverse events were reported in 11 subjects (Altoderm seven, placebo four). Most of these referred to irritation, redness and burning at the site of application. There were four reports of erythema and pruritus (Altoderm three, placebo one), and three reports of application site burning (Altoderm two, placebo one). None was reported as severe or very severe. CONCLUSIONS: These results show a clinically useful benefit of this sodium cromoglicate lotion in children with moderately severe AD.

14-Week toxicity and cell proliferation of methyleugenol administered by gavage to F344 rats and B6C3F1 mice.

Methyleugenol, a food flavor and fragrance agent, was tested for toxicity in male and female F344/N rats and B6C3F1 mice. Groups of 10 males and 10 females per sex per species were administered 0, 10, 30, 100, 300 or 1000 mg methyleugenol/kg body weight in 0.5% aqueous methylcellulose by gavage, 5 days per week for 14 weeks. Additional groups of rats and mice of each sex were dosed similarly and used for hematology and clinical chemistry studies. Groups of 10 male and 10 female rats and mice received the vehicle by gavage on the same dosing schedule and served as vehicle controls. For serum gastrin, gastric pH and cell proliferation studies groups of 10 female rats were given 0, 37, 75 or 150 mg/kg, once daily 5 days per week for 30 or 90 days or 300 or 1000 mg/kg for 30 days; male mice were given 0, 9, 18.5, 37, 75, 150 or 300 mg/kg for 30 or 90 days. For the gastrin, pH and cell proliferation studies, groups of 10 female rats and 10 male mice were given the vehicle for 30 or 90 days and served as controls. Methyleugenol administration to rats induced erythrocyte microcytosis and thrombocytosis in male and female rats. It also caused an increase in serum alanine aminotransferase and sorbitol dehydrogenase activities and bile acid concentration, suggesting hepatocellular injury, cholestasis or altered hepatic function. Additionally, methyleugenol induced hypoproteinemia and hypoalbuminemia, evidenced by decreased total protein and albumin concentrations in both male and female rats, suggesting in inefficiency of dietary protein utilization due to methyleugenol-induced toxic effects on the liver and glandular stomach of rats and mice. The increase in gastrin and gastric pH of rats and mice given methyleugenol suggests that gastrin feedback was impaired and resulted in conditions not conducive to protein digestion. In rats, methyleugenol caused an increase in the incidences of hepatocyte cytologic alteration, cytomegaly, Kupffer cell pigmentation, mixed foci of cellular alteration and bile duct hyperplasia of the liver and atrophy and chronic inflammation of the mucosa of the glandular stomach. In mice, it caused an increase in the incidence of cytologic alteration, necrosis, bile duct hyperplasia and subacute inflammation of the liver and atrophy, degeneration, necrosis, edema, mitotic alteration, and cystic glands of the fundic region of the glandular stomach. The increased incidences of adrenal gland cortical hypertrophy and/or cytoplasmic alteration in the submandibular salivary glands, adrenal glands, testis and uterus of rats were considered secondary to the chemical-related effects observed in the liver and glandular stomach. Based on mortality, body weight gain, clinical chemistry and gross and microscopic evaluation of tissues of rats and mice, the no-observed-effect level (NOEL) of methyleugenol for both species was estimated at 10 mg/kg.

Yohimbine in neurally mediated syncope. Pathophysiological implications.

In this study, we evaluated if increased sympathetic stimulation is an essential requirement for the development of neurally mediated syncope (NMS) by manipulating overall sympathetic outflow in subjects susceptible to tilt-induced syncope. Eight previously characterized patients with recurrent NMS (five females and three males; 34+/-2 yr) were recruited from the Vanderbilt Syncope Unit and eight age-matched controls underwent initial administration of clonidine (CLO) or yohimbine (YHO). This was done, prospectively, to determine doses of these agents that would increase or decrease plasma norepinephrine levels by >/= 30%. On a different day, in all subjects we determined intraarterial blood pressure, EKG and muscle sympathetic nerve activity (MSNA) both supine and during upright tilt. After this, subjects randomly received either CLO or YHO, and 3 h later another tilt was performed. After 1 wk, a similar procedure with the other drug was performed. During the two basal tilts, all the control subjects completed the study, whereas all the NMS patients developed syncope. Reduction in sympathetic tone by CLO resulted in a decreased tolerance to tilt in three out of eight controls and in all the NMS patients. In contrast, YHO not only increased basal plasma NorEpi levels and MSNA, but also prevented syncope in seven out of eight patients. In a selected population of patients, increased sympathetic activity is not a prerequisite for the development of syncope. Yohimbine-induced enhancement of sympathetic tone in patients with NMS improves orthostatic tolerance and raises the possibility that this drug may be a useful agent in the treatment of NMS.

Chronic orthostatic intolerance: a disorder with discordant cardiac and vascular sympathetic control.

BACKGROUND: Chronic orthostatic intolerance (COI) is a debilitating autonomic condition in young adults. Its neurohumoral and hemodynamic profiles suggest possible alterations of postural sympathetic function and of baroreflex control of heart rate (HR). METHODS AND RESULTS: In 16 COI patients and 16 healthy volunteers, intra-arterial blood pressure (BP), ECG, central venous pressure (CVP), and muscle sympathetic nerve activity (MSNA) were recorded at rest and during 75 degrees tilt. Spectral analysis of RR interval and systolic arterial pressure (SAP) variabilities provided indices of sympathovagal modulation of the sinoatrial node (ratio of low-frequency to high-frequency components, LF/HF) and of sympathetic vasomotor control (LFSAP). Baroreflex mechanisms were assessed (1) by the slope of the regression line obtained from changes of RR interval and MSNA evoked by pharmacologically induced alterations in BP and (2) by the index alpha, obtained from cross-spectral analysis of RR and SAP variabilities. At rest, HR, MSNA, LF/HF, and LFSAP were higher in COI patients, whereas BP and CVP were similar in the two groups. During tilt, BP did not change and CVP fell by the same extent in the 2 groups; the increase of HR and LF/HF was more pronounced in COI patients. Conversely, the increase of MSNA was lower in COI than in control subjects. Baroreflex sensitivity was similar in COI and control subjects at rest; tilt reduced alpha similarly in both groups. CONCLUSIONS: COI is characterized by an overall enhancement of noradrenergic tone at rest and by a blunted postganglionic sympathetic response to standing, with a compensatory cardiac sympathetic overactivity. Baroreflex mechanisms maintain their functional responsiveness. These data suggest that in COI, the functional distribution of central sympathetic tone to the heart and vasculature is abnormal.

Sympathetic and baroreceptor reflex function in neurally mediated syncope evoked by tilt.

The pathophysiology of neurally mediated syncope is poorly understood. It has been widely assumed that excessive sympathetic activation in a setting of left ventricular hypovolemia stimulates ventricular afferents that trigger hypotension and bradycardia. We tested this hypothesis by determining if excessive sympathetic activation precedes development of neurally mediated syncope, and if this correlates with alterations in baroreflex function. We studied the changes in intraarterial blood pressure (BP), heart rate (HR), central venous pressure (CVP), muscle sympathetic nerve activity (MSNA), and plasma catecholamines evoked by upright tilt in recurrent neurally mediated syncope patients (SYN, 5+/-1 episodes/mo, n = 14), age- and sex-matched controls (CON, n = 23), and in healthy subjects who consistently experienced syncope during tilt (FS+, n = 20). Baroreflex responses were evaluated from changes in HR, BP, and MSNA that were obtained after infusions of phenylephrine and sodium nitroprusside. Compared to CON, patients with SYN had blunted increases in MSNA at low tilt levels, followed by a progressive decrease and ultimately complete disappearance of MSNA with syncope. SYN patients also had attenuation of norepinephrine increases and lower baroreflex slope sensitivity, both during tilt and after pharmacologic testing. FS+ subjects had the largest decrease in CVP with tilt and had significant increases in MSNA and heart rate baroreflex slopes. These data challenge the view that excessive generalized sympathetic activation is the precursor of the hemodynamic abnormality underlying recurrent neurally mediated syncope.

Modeling of arterial and cardiopulmonary baroreflex control of heart rate.

We evaluated R-R interval changes (delta R-R interval) in 13 subjects (27 +/- 6 yr; 7 men and 6 women) as a function of blood pressure changes at the carotid sinus and aortic arch and central venous pressure changes at the cardiopulmonary receptors. Neck chamber pressure and suction were used to change pressure at the carotid sinus while lower body negative pressure, phenylephrine infusion, and nitroprusside infusion were used to change pressure at the carotid sinus (delta CSP), aortic arch (delta AAP), and cardiopulmonary receptors (delta CPP). Random effects regression analysis showed a significant linear relationship for delta R-R interval (-1.75 + 1.64 delta CSP + 15.40 delta AAP + 29.02 delta CPP + error), and the correlation (r) between the observed and predicted delta R-R interval was 0.82 (P < 0.00001). Sixty-seven percent of the delta R-R interval variability observed in the study is explained by the model. delta AAP accounts for approximately 63%, delta CSP for 14%, and delta CPP for 23% of the explained delta R-R interval.