Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains.

Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 µM at the promastigote stage and 32 and 195 µM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.

The global diversity of Haemonchus contortus is shaped by human intervention and climate.

Haemonchus contortus is a haematophagous parasitic nematode of veterinary interest. We have performed a survey of its genome-wide diversity using single-worm whole genome sequencing of 223 individuals sampled from 19 isolates spanning five continents. We find an African origin for the species, together with evidence for parasites spreading during the transatlantic slave trade and colonisation of Australia. Strong selective sweeps surrounding the ß-tubulin locus, a target of benzimidazole anthelmintic drug, are identified in independent populations. These sweeps are further supported by signals of diversifying selection enriched in genes involved in response to drugs and other anthelmintic-associated biological functions. We also identify some candidate genes that may play a role in ivermectin resistance. Finally, genetic signatures of climate-driven adaptation are described, revealing a gene acting as an epigenetic regulator and components of the dauer pathway. These results begin to define genetic adaptation to climate in a parasitic nematode.

Application of Bayesian statistics to estimate nitrous oxide emission factors of three nitrogen fertilisers on UK grasslands.

Trapezoidal integration by linear interpolation of data points is by far the most commonly used method of cumulative flux calculations of nitrous oxide (N2O) in studies that use flux chambers; however, this method is incapable of providing accurate uncertainty estimates. A Bayesian approach was used to calculate N2O emission factors (EFs) and their associated uncertainties from flux chamber measurements made after the application of nitrogen fertilisers, in the form of ammonium nitrate (AN), urea (Ur) and urea treated with Agrotain® urease inhibitor (UI) at four grassland sites in the UK. The comparison between the cumulative fluxes estimated using the Bayesian and linear interpolation methods were broadly similar (R2 = 0.79); however, the Bayesian method was capable of providing realistic uncertainties when a limited number of data points is available. The study reports mean EF values (and 95% confidence intervals) of 0.60 ±â€¯0.63, 0.29 ±â€¯0.22 and 0.26 ±â€¯0.17% of applied N emitted as N2O for the AN, Ur and UI treatments, respectively. There was no significant difference between N2O emissions from the Ur and UI treatments. In the case of the automatic chamber data collected at one site in this study, the data did not fit the log-normal model, implying that more complex models may be needed, particularly for measurement data with high temporal resolution.

Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression.

Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania-a protozoan parasite that kills more than 30,000 people each year-is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this.

Haemonchus contortus: Genome Structure, Organization and Comparative Genomics.

One of the first genome sequencing projects for a parasitic nematode was that for Haemonchus contortus. The open access data from the Wellcome Trust Sanger Institute provided a valuable early resource for the research community, particularly for the identification of specific genes and genetic markers. Later, a second sequencing project was initiated by the University of Melbourne, and the two draft genome sequences for H. contortus were published back-to-back in 2013. There is a pressing need for long-range genomic information for genetic mapping, population genetics and functional genomic studies, so we are continuing to improve the Wellcome Trust Sanger Institute assembly to provide a finished reference genome for H. contortus. This review describes this process, compares the H. contortus genome assemblies with draft genomes from other members of the strongylid group and discusses future directions for parasite genomics using the H. contortus model.

In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization.

In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb-S; and antimony resistant Sb-R). MIL-R was easily induced in both strains using the promastigote-stage, but a significant increase in MIL-R in the intracellular amastigote compared to the corresponding wild-type did not occur until promastigotes had adapted to 12.2 µM MIL. A variety of common and strain-specific genetic changes were discovered in MIL-adapted parasites, including deletions at the LdMT transporter gene, single-base mutations and changes in somy. The most obvious lipid changes in MIL-R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL-R parasites, with more genetic changes occurring in Sb-R compared with Sb-S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb-R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully.

Distinct cellular origin and genetic requirement of Hedgehog-Gli in postnatal rhabdomyosarcoma genesis.

Dysregulation of the Hedgehog (Hh)-Gli signaling pathway is implicated in a variety of human cancers, including basal cell carcinoma (BCC), medulloblastoma (MB) and embryonal rhabdhomyosarcoma (eRMS), three principle tumors associated with human Gorlin syndrome. However, the cells of origin of these tumors, including eRMS, remain poorly understood. In this study, we explore the cell populations that give rise to Hh-related tumors by specifically activating Smoothened (Smo) in both Hh-producing and -responsive cell lineages in postnatal mice. Interestingly, we find that unlike BCC and MB, eRMS originates from the stem/progenitor populations that do not normally receive active Hh signaling. Furthermore, we find that the myogenic lineage in postnatal mice is largely Hh quiescent and that Pax7-expressing muscle satellite cells are not able to give rise to eRMS upon Smo or Gli1/2 overactivation in vivo, suggesting that Hh-induced skeletal muscle eRMS arises from Hh/Gli quiescent non-myogenic cells. In addition, using the Gli1 null allele and a Gli3 repressor allele, we reveal a specific genetic requirement for Gli proteins in Hh-induced eRMS formation and provide molecular evidence for the involvement of Sox4/11 in eRMS cell survival and differentiation.

Parallel signatures of sequence evolution among hearing genes in echolocating mammals: an emerging model of genetic convergence.

Recent findings of sequence convergence in the Prestin gene among some bats and cetaceans suggest that parallel adaptations for high-frequency hearing have taken place during the evolution of echolocation. To determine if this gene is an exception, or instead similar processes have occurred in other hearing genes, we have examined Tmc1 and Pjvk, both of which are associated with non-syndromic hearing loss in mammals. These genes were amplified and sequenced from a number of mammalian species, including echolocating and non-echolocating bats and whales, and were analysed together with published sequences. Sections of both genes showed phylogenetic signals that conflicted with accepted species relationships, with coding regions uniting laryngeal echolocating bats in a monophyletic clade. Bayesian estimates of posterior probabilities of convergent and divergent substitutions provided more direct evidence of sequence convergence between the two groups of laryngeal echolocating bats as well as between echolocating bats and dolphins. We found strong evidence of positive selection acting on some echolocating bat species and echolocating cetaceans, contrasting with purifying selection on non-echolocating bats. Signatures of sequence convergence and molecular adaptation in two additional hearing genes suggest that the acquisition of high-frequency hearing has involved multiple loci.

Individualised assessment of response to clopidogrel in patients presenting with acute coronary syndromes: a role for short thrombelastography?

INTRODUCTION: There is considerable interindividual variation in response to the antiplatelet agent clopidogrel. Hyporesponse predicts negative outcomes in patients presenting with a variety of ischemic cardiac conditions and following intracoronary stent placement. Many tests of clopidogrel activity are time consuming and complex. Short thromboelastography (s-TEG) allows rapid measurement of platelet clopidogrel response. AIMS: We initiated this study to investigate the utility of s-TEG in assessing the response to clopidogrel in patients presenting with acute coronary syndromes (ACS) and to compare these results with established clopidogrel monitoring techniques. METHODS: Patients admitted with unstable angina (UA) or Non ST elevation myocardial infarction (NSTEMI) undergoing coronary angiography were recruited. After routine loading with clopidogrel, all patients were tested with s-TEG and Accumetrics Verify-Now rapid platelet function analyzer (VN-RPFA). We used the modified TEG technique of measuring area under the curve at 15 min (AUC15), which allows a rapid estimation of antiplatelet response. Vasodilator-stimulated phosphoprotein phosphorylation (VASP) was also tested in a subgroup of patients. Clinical follow-up was obtained at 1 year. s-TEG results were correlated with VN-RPFA and VASP findings. RESULTS: A total of 49 patients (33 male, mean age 63) were recruited and tested with s-TEG and VN-RPFA and a total of 39 patients were also assessed with VASP. s-TEG readings correlated well with VN-RPFA (r(2)= 0.54, P < 0.0001) and VASP (r(2)= 0.26, P= 0.001). CONCLUSION: s-TEG provides timely results which compare to current tests of clopidogrel activity. This technique can also be used to measure a variety of other clotting parameters and as such could develop into a valuable near patient test for the interventional cardiologist.

Non-skeletal scintigraphy of the horse: indications and validity.

Gamma scintigraphy is an established imaging modality in the horse and is principally utilised to investigate skeletal disease using bone-seeking radiopharmaceuticals. However, depending on the radiopharmaceutical and imaging sequence, scintigraphy can be used to investigate disease in virtually any organ system. In this article the indications and validity of scintigraphy as a clinical diagnostic tool to investigate non-skeletal disease in the horse are reviewed. These indications include: investigation of dental disease, identification of inflammatory foci, vascular lesions, muscle and tendon pathology, assessment of physiological function of the lungs, gastrointestinal tract and other organs. Some of the techniques described for use in the horse have not been validated fully and as a result non-skeletal scintigraphy requires further investigation and validation using well-designed prospective studies. Such information can be used by clinicians to make informed decisions regarding the clinical and economic issues associated with a specific scintigraphic technique and may help when interpreting the results.

Strain patterns during tensile, compressive, and shear fatigue of human cortical bone and implications for bone biomechanics.

It is a common theme in basic bone biomechanics and in biomechanical applications that much of the behavior can be determined and is dictated by the level of strain, whether this pertains to bone physiology, bone remodeling, osseoinduction, osseointegration, or the development of damage. The development of damage, demonstrated by stiffness loss measurements, has already been reported in detail in the literature. However, the systematic study of the development of "plastic" (residual) strains, which are associated with the inelastic mechanical behavior of bone tissue, has generally been overlooked. The present study compares the rates at which the elastic (e(a)) and plastic components (e(p)) of strain developed during tensile, compressive, and shear fatigue in human cortical bone of six individuals aged between 53 and 79 years. The overall hypothesis of this investigation is that there is a common underlying factor in the damage-related behavior of bone, which may allow us to link together the various aspects of the damage related behavior of bone. The rate of development of plastic strain (Deltae(p)/DeltaN) and the rate of growth in elastic strain amplitude (Deltae(a)/DeltaN) are described as a function of the stress (sigma), and/or stress normalized by the modulus of elasticity (sigma/E). The implications of our findings are discussed with respect to simple models/mechanisms, which may underlie the observed behavior.

The importance of the elastic and plastic components of strain in tensile and compressive fatigue of human cortical bone in relation to orthopaedic biomechanics.

The longevity, success, or failure of an orthopaedic implant is dependent on its osseointegration especially within the initial six months of the initial surgery. The development of strains plays a crucial role in both bone modelling and remodelling. For remodelling, in particular, strains of substantial values are required to activate the osteoblastic and osteoclastic activity for the osseointegration of the implant. Bone, however, is subject to "damage" when strain levels exceed a certain threshold level. Damage is manifested in the form of microcracks; it is linked to increased elastic strain amplitudes and is accompanied by the development of "plastic" (irrecoverable, residual) strains. Such strains increase the likelihood for the implant to subside or loosen. The present study examines the rates (per cycle) by which these two components of strain (elastic and "plastic") develop during fatigue cycling in two loading modes, tension and compression. The results of this study show that these strain rates depend on the applied stress in both loading modes. It also shows that elastic and plastic strain rates can be linked to each other through simple power law relationships so that one can calculate or predict the latter from the former and vice versa. We anticipate that such basic bone biomechanics data would be of great benefit to both clinicians and bioengineers working in the field of FEA modelling applications and orthopaedic implant surgery.

Effect of fluid forcing on vestibular hair bundles.

A dynamic 3-D hair bundle model including inertia and viscous fluid drag effects based on the finite element method is presented. Six structural components are used to construct the hair bundle--kinocilium, stereocilia, upper lateral links, shaft links, tip links, and kinocilial links. Fluid drag is distributed on the surface of cilia columns. Bundle mechanics are analyzed under two distinct loading conditions: (1) drag caused by the shear flow of the surrounding endolymph fluid (fluid-forced), (2) a single force applied to the tip of the kinocilium (point-forced). A striolar and a medial extrastriolar vestibular hair cell from the utricle of a turtle are simulated. The striolar cell bundle shows a clear difference in tip link tension profile between fluid-forced and point-forced cases. When the striolar cell is fluid forced, it shows more evenly distributed tip link tensions and is far more sensitive, responding like an on/off switch. The extrastriolar cell does not show noticeable differences between the forcing types. For both forcing conditions, the extrastriolar cell responds serially--the nearest tip links to the kinocilium get tensed first, then the tension propagates to the farther tip links.

Mood and cognitive performance effects of "energy" drink constituents: caffeine, glucose and carbonation.

Three studies using healthy volunteers (n = 271) investigated the effects of caffeine, carbohydrates and carbonation in functional "energy" drinks (EDs) with the aim of determining their benefit in every-day life. The results showed caffeine to be the main ED constituent responsible for the effects found, with possible minor, relatively weak effects of carbohydrates. EDs were found to improve and/or maintain mood and performance during fatiguing and cognitively demanding tasks relative to placebo. In terms of absolute values, EDs maintained levels of arousal compared to a deterioration in arousal where placebo was consumed. These effects were found in caffeine-deprived participants, and so may be largely due to "withdrawal reversal". There were only minor differences in the effects of water vs. "sensory-matched" placebo, supporting previous findings indicating that the type of placebo does not alter the conclusions drawn about the effects of the full ED. Finally, carbonation had various effects on mood, some of which were present immediately following consumption, others were consistent with slower absorption of caffeine (and possibly carbohydrates) from carbonated drinks.

Idiopathic constriction of the fetal ductus arteriosus.

Constriction of the ductus arteriosus in the fetus is well studied, but mostly secondary to maternal exposure to indomethacin or other non-steroidal anti-inflammatory medications, or structural cardiac lesions. We present a case of a fetus presenting with right ventricular hypertrophy secondary to an S-shaped ductus arteriosus with distal constriction diagnosed by pulsed Doppler imaging. A review of the recent English literature of similar cases is presented.

Direct coronary stenting compared with stenting after predilatation is feasible, safe, and more cost-effective in selected patients: evidence to date indicating similar late outcomes.

OBJECTIVES: To review the currently available data from studies assessing feasibility, safety, clinical outcome and cost-effectiveness of direct stenting. BACKGROUND: With technical advances of stent designs and their delivery systems a new strategy has become increasingly popular: direct stent implantation without prior balloon dilatation. METHODS: The Medline database was searched from January 1996 to March 2001 for clinical trials investigating direct stenting using the index terms direct stenting, coronary intervention, percutaneous transluminal coronary angioplasty (PTCA), PCI, angioplasty and ischemic heart disease. Studies were chosen based on the number of patients involved and endpoints mentioned. Data not yet published but presented at recent international meetings were also included. A comparison between direct stenting and stenting with predilatation was performed using for the latter results of the randomized trials supplemented with Benestent II data. RESULTS: At least 26 studies have investigated direct stenting, showing high primary and final success rates with few complications. Direct stenting provides a way to reduce costs, shorten procedural and fluoroscopy times and lower material consumption. Immediate and long-term clinical outcomes appear to be similar to stenting with predilatation. Preliminary results of large randomized trials with angiographic follow-up indicate that restenosis rates are similar to those of conventional stenting strategies. CONCLUSIONS: Direct stenting compared with stenting with predilatation is feasible, safe, faster and more cost-effective. The evidence to date shows similar late outcomes.