Light availability and rhizobium variation interactively mediate the outcomes of legume-rhizobium symbiosis.

PREMISE: Nutrients, light, water, and temperature are key factors limiting the growth of individual plants in nature. Mutualistic interactions between plants and microbes often mediate resource limitation for both partners. In the mutualism between legumes and rhizobia, plants provide rhizobia with carbon in exchange for fixed nitrogen. Because partner quality in mutualisms is genotype-dependent, within-species genetic variation is expected to alter the responses of mutualists to changes in the resource environment. Here we ask whether partner quality variation in rhizobia mediates the response of host plants to changing light availability, and conversely, whether light alters the expression of partner quality variation. METHODS: We inoculated clover hosts with 11 strains of Rhizobium leguminosarum that differed in partner quality, grew plants under either ambient or low light conditions in the greenhouse, and measured plant growth, nodule traits, and foliar nutrient composition. RESULTS: Light availability and rhizobium inoculum interactively determined plant growth, and variation in rhizobium partner quality was more apparent in ambient light. CONCLUSIONS: Our results suggest that variation in the costs and benefits of rhizobium symbionts mediate host responses to light availability and that rhizobium strain variation might more important in higher-light environments. Our work adds to a growing appreciation for the role of microbial intraspecific and interspecific diversity in mediating extended phenotypes in their hosts and suggests an important role for light availability in the ecology and evolution of legume-rhizobium symbiosis.

Characterization of the anti-CD22 targeted therapy, moxetumomab pasudotox, for B-cell precursor acute lymphoblastic leukemia.

Moxetumomab pasudotox is a second-generation recombinant immunotoxin against CD22 on B-cell lineages. Antileukemic activity has been demonstrated in children with chemotherapy-refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL), with variable responses. Here, we report in vitro and in vivo evaluation of moxetumomab pasudotox treatment of human cell lines and patient-derived cells as a preliminary study to understand characteristics of sensitivity to treatment. Binding, internalization, and apoptosis were evaluated using fluorescently tagged moxetumomab pasudotox. Studies in NOD-scid IL2Rgnull mice showed a modest survival benefit in mice engrafted with 697 cells but not in NALM6 or the two patient-derived xenograft models.

Intrapopulation genomics in a model mutualist: Population structure and candidate symbiosis genes under selection in Medicago truncatula.

Bottom-up evolutionary approaches, including geographically explicit population genomic analyses, have the power to reveal the mechanistic basis of adaptation. Here, we conduct a population genomic analysis in the model legume, Medicago truncatula, to characterize population genetic structure and identify symbiosis-related genes showing evidence of spatially variable selection. Using RAD-seq, we generated over 26,000 SNPs from 191 accessions from within three regions of the native range in Europe. Results from STRUCTURE analysis identify five distinct genetic clusters with divisions that separate east and west regions in the Mediterranean basin. Much of the genetic variation is maintained within sampling sites, and there is evidence for isolation by distance. Extensive linkage disequilibrium was identified, particularly within populations. We conducted genetic outlier analysis with FST -based genome scans and a Bayesian modeling approach (PCAdapt). There were 70 core outlier loci shared between these distinct methods with one clear candidate symbiosis related gene, DMI1. This work sets that stage for functional experiments to determine the important phenotypes that selection has acted upon and complementary efforts in rhizobium populations.

Decoupled genomic elements and the evolution of partner quality in nitrogen-fixing rhizobia.

Understanding how mutualisms evolve in response to a changing environment will be critical for predicting the long-term impacts of global changes, such as increased N (nitrogen) deposition. Bacterial mutualists in particular might evolve quickly, thanks to short generation times and the potential for independent evolution of plasmids through recombination and/or HGT (horizontal gene transfer). In a previous work using the legume/rhizobia mutualism, we demonstrated that long-term nitrogen fertilization caused the evolution of less-mutualistic rhizobia. Here, we use our 63 previously isolated rhizobium strains in comparative phylogenetic and quantitative genetic analyses to determine the degree to which variation in partner quality is attributable to phylogenetic relationships among strains versus recent genetic changes in response to N fertilization. We find evidence of distinct evolutionary relationships between chromosomal and pSym genes, and broad similarity between pSym genes. We also find that nifD has a unique evolutionary history that explains much of the variation in partner quality, and suggest MoFe subunit interaction sites in the evolution of less-mutualistic rhizobia. These results provide insight into the mechanisms behind the evolutionary response of rhizobia to long-term N fertilization, and we discuss the implications of our results for the evolution of the mutualism.

Trichomonas vaginalis: Clinical relevance, pathogenicity and diagnosis.

Trichomonas vaginalis is the etiological agent of trichomoniasis, the most prevalent non-viral sexually transmitted disease worldwide. Trichomoniasis is a widespread, global health concern and occurring at an increasing rate. Infections of the female genital tract can cause a range of symptoms, including vaginitis and cervicitis, while infections in males are generally asymptomatic. The relatively mild symptoms, and lack of evidence for any serious sequelae, have historically led to this disease being under diagnosed, and under researched. However, growing evidence that T. vaginalis infection is associated with other disease states with high morbidity in both men and women has increased the efforts to diagnose and treat patients harboring this parasite. The pathology of trichomoniasis results from damage to the host epithelia, caused by a variety of processes during infection and recent work has highlighted the complex interactions between the parasite and host, commensal microbiome and accompanying symbionts. The commercial release of a number of nucleic acid amplification tests (NAATs) has added to the available diagnostic options. Immunoassay based Point of Care testing is currently available, and a recent initial evaluation of a NAAT Point of Care system has given promising results, which would enable testing and treatment in a single visit.

Loop-mediated isothermal amplification (LAMP) for the rapid detection of Mycoplasma genitalium.

Mycoplasma genitalium is a sexually transmissible, pathogenic bacterium and a significant cause of nongonococcal urethritis in both men and women. Due to the difficulty of the culture of M. genitalium from clinical samples, the laboratory diagnosis of M. genitalium infection is almost exclusively carried out using nucleic acid amplification tests. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology, utilising a set of 4 primers specific to 6 distinct regions of the target DNA sequence, in order to amplify target DNA in a highly specific and rapid manner. A LAMP assay was designed to the pdhD gene of M. genitalium, and the limit of detection of the assay was determined as 10 fg of M. genitalium genomic DNA, equating to ~16 copies of the M. genitalium genome, which was equally sensitive as a gold standard 16S rRNA polymerase chain reaction assay.

Inoculation with an enhanced N2 -fixing Bradyrhizobium japonicum strain (USDA110) does not alter soybean (Glycine max†Merr.) response to elevated [CO2 ].

This study tested the hypothesis that inoculation of soybean (Glycine max Merr.) with a Bradyrhizobium japonicum strain (USDA110) with greater N2 fixation rates would enhance soybean response to elevated [CO2 ]. In field experiments at the Soybean Free Air CO2 Enrichment facility, inoculation of soybean with USDA110 increased nodule occupancy from 5% in native soil to 54% in elevated [CO2 ] and 34% at ambient [CO2 ]. Despite this success, inoculation with USDA110 did not result in greater photosynthesis, growth or seed yield at ambient or elevated [CO2 ] in the field, presumably due to competition from native rhizobia. In a growth chamber experiment designed to study the effects of inoculation in the absence of competition, inoculation with USDA110 in sterilized soil resulted in nodule occupation of >90%, significantly greater (15) N2 fixation, photosynthetic capacity, leaf N and total plant biomass compared with plants grown with native soil bacteria. However, there was no interaction of rhizobium fertilization with elevated [CO2 ]; inoculation with USDA110 was equally beneficial at ambient and elevated [CO2 ]. These results suggest that selected rhizobia could potentially stimulate soybean yield in soils with little or no history of prior soybean production, but that better quality rhizobia do not enhance soybean responses to elevated [CO2 ].

Phase I dose-escalation study of MEDI-573, a bispecific, antiligand monoclonal antibody against IGFI and IGFII, in patients with advanced solid tumors.

PURPOSE: This phase I, multicenter, open-label, single-arm, dose-escalation, and dose-expansion study evaluated the safety, tolerability, and antitumor activity of MEDI-573 in adults with advanced solid tumors refractory to standard therapy or for which no standard therapy exists. EXPERIMENTAL DESIGN: Patients received MEDI-573 in 1 of 5 cohorts (0.5, 1.5, 5, 10, or 15 mg/kg) dosed weekly or 1 of 2 cohorts (30 or 45 mg/kg) dosed every 3 weeks. Primary end points included the MEDI-573 safety profile, maximum tolerated dose (MTD), and optimal biologic dose (OBD). Secondary end points included MEDI-573 pharmacokinetics (PK), pharmacodynamics, immunogenicity, and antitumor activity. RESULTS: In total, 43 patients (20 with urothelial cancer) received MEDI-573. No dose-limiting toxicities were identified, and only 1 patient experienced hyperglycemia related to treatment. Elevations in levels of insulin and/or growth hormone were not observed. Adverse events observed in >10% of patients included fatigue, anorexia, nausea, diarrhea, and anemia. PK evaluation demonstrated that levels of MEDI-573 increased with dose at all dose levels tested. At doses >5 mg/kg, circulating levels of insulin-like growth factor (IGF)-I and IGFII were fully suppressed. Of 39 patients evaluable for response, none experienced partial or complete response and 13 had stable disease as best response. CONCLUSIONS: The MTD of MEDI-573 was not reached. The OBD was 5 mg/kg weekly or 30 or 45 mg/kg every 3 weeks. MEDI-573 showed preliminary antitumor activity in a heavily pretreated population and had a favorable tolerability profile, with no notable perturbations in metabolic homeostasis.

Loop-mediated isothermal amplification test for detection of Neisseria gonorrhoeae in urine samples and tolerance of the assay to the presence of urea.

A loop-mediated isothermal amplification (LAMP) assay for open reading frame 1 (ORF1) of the glutamine synthetase gene of Neisseria gonorrhoeae was able to tolerate urea concentrations of ≤ 1.8 M, compared with a PCR assay that was functional at concentrations of <100 mM. The LAMP assay was as sensitive as the PCR assay while being faster and simpler to perform.

Circulating tumor cells: application as a biomarker for molecular characterization and predictor of survival in an all-comer solid tumor phase I clinical study.

PURPOSE: Clinical development of cancer drugs has a low success rate. Prognostic and predictive biomarkers using minimally invasive approaches hold promise for increasing the probability of success by enabling disease characterization, patient selection and early detection of drug treatment effect. Enumeration and molecular characterization of circulating tumor cells (CTC) may address some of these needs, and thus were evaluated for utility in a Phase I solid tumor clinical study. EXPERIMENTAL DESIGN: Blood samples for CTC analysis were obtained from 24 cancer patients in a multi-center all-comer Phase I study of MEDI-575, a novel anti-PDGFRα antibody. Samples were taken at screening and analyzed for enumeration of CTC using the CellSearch(®) platform and for molecular characterization using a novel quantitative RT-PCR assay. RESULTS: Fifty-nine percent of the patients showed at least 1 CTC per 7.5 ml of blood at baseline. Progression-free survival (PFS) and overall survival (OS) of patients with 0 CTCs at baseline were longer than PFS and Os for patients with 1-3 and >3 CTCs (8.8 versus 1.4 and 1.3 months PFS, P = 0.02; 9.0 vs 7.4 and 3.5 months OS, P = 0.20, respectively). Patients with 0 CTC showed a greater percentage of stable disease than the other 2 groups with 1-3 and >3 CTCs (57% vs 29% and 0%). The multimarker qRT-PCR method detected CTC in 40% of the patients, and 80% of these patients were positive for pre-selected drug target genes. CONCLUSION: CTC enumeration of patients in an all-comer study is feasible and may allow for patient stratification for PFS and Os to evaluate the clinical response of investigational agents. Gene expression profiling of isolated CTC may provide a means for molecular characterization of selected tumor targets.

Coevolutionary genetic variation in the legume-rhizobium transcriptome.

Coevolutionary change requires reciprocal selection between interacting species, where the partner genotypes that are favoured in one species depend on the genetic composition of the interacting species. Coevolutionary genetic variation is manifested as genotype × genotype (G × G) interactions for fitness in interspecific interactions. Although quantitative genetic approaches have revealed abundant evidence for G × G interactions in symbioses, the molecular basis of this variation remains unclear. Here we study the molecular basis of G × G interactions in a model legume-rhizobium mutualism using gene expression microarrays. We find that, like quantitative traits such as fitness, variation in the symbiotic transcriptome may be partitioned into additive and interactive genetic components. Our results suggest that plant genetic variation had the largest influence on nodule gene expression and that plant genotype and the plant genotype × rhizobium genotype interaction determine global shifts in rhizobium gene expression that in turn feedback to influence plant fitness benefits. Moreover, the transcriptomic variation we uncover implicates regulatory changes in both species as drivers of symbiotic gene expression variation. Our study is the first to partition genetic variation in a symbiotic transcriptome and illuminates potential molecular routes of coevolutionary change.

The hematopoietic-specific beta1-tubulin is naturally resistant to 2-methoxyestradiol and protects patients from drug-induced myelosuppression.

Taxanes and other microtubule-targeting drugs (MTDs) represent one of the most effective classes of cancer chemotherapeutics. However, ultimately their utility is limited due to drug-induced myelosuppression. Here we identify 2-Methoxyestradiol (2ME2) as the first MTD able to specifically target tumor cells while sparing the bone marrow from dose-limiting, life-threatening toxicities. Following drug selection with 2ME2, epithelial cancer cells acquired a tubulin mutation at Vbeta236I that impaired the 2ME2-tubulin interaction and rendered cells resistant to 2ME2. We further show that the hematopoietic-specific Hbeta1 tubulin isotype naturally encodes Ibeta236 and is insensitive to 2ME2. Systemic administration of 2ME2 in C57BL6 mice revealed that there was no effect on bone marrow microtubules, in contrast to the taxane or Vinca alkaloid induced toxicities. Similar results were obtained upon drug treatment of human bone marrow and CD34-positive stem/progenitor cells. Herein, we describe the first isotype-targeted chemotherapeutic, setting a new paradigm for the entire class of MTDs, and providing a model that could allow the design of new tubulin inhibitors devoid of myelosuppression. The ability to design a drug with minimal side-effects would significantly augment the chances of clinical success by allowing the use of a truly therapeutic dose rather than the maximally tolerated.

Audit of an inpatient liaison psychiatry consultation service.

PURPOSE: The purpose of this paper is to examine an audit that was performed of all patients referred to a liaison psychiatry inpatient consultation service which sought to establish a baseline for demographics, type of referral, and management of referrals, with a view to introducing improved evidence-based treatments. It also aims to examine timeliness of response to referrals benchmarked against published standards. DESIGN/METHODOLOGY/APPROACH: All inpatient referrals to a liaison psychiatry service were recorded over a six-month period, including demographics, diagnosis, management and timeliness of response to referrals. The data were retrospectively analysed and compared against international standards. FINDINGS: A total of 172 referrals were received in the six months. Commonest referral reasons included assessments regarding depressive disorders (23.8 per cent), delirium/other cognitive disorders (19.2 per cent), alcohol-related disorders (18.6 per cent), anxiety disorders (14.5 per cent), and risk management (12.2 per cent). Evidence-based practices were not utilised effectively for a number of different types of presentations. A total of 40.1 per cent of referrals were seen on the same day, 75.4 per cent by the end of the next day, and 93.4 per cent by the end of the following day. PRACTICAL IMPLICATIONS: Use of a hospital protocol for management of delirium may improve outcomes for these patients. Evidence-based techniques, such as brief intervention therapies, may be beneficial for referrals involving alcohol dependence. Referrals were seen reasonably quickly, but there is room for improvement when compared with published standards. ORIGINALITY/VALUE: This paper provides valuable information for those involved in management of liaison psychiatry consultation services, providing ideas for development and implementation of evidence based practices.

Biogeography of the Sulfolobus islandicus pan-genome.

Variation in gene content has been hypothesized to be the primary mode of adaptive evolution in microorganisms; however, very little is known about the spatial and temporal distribution of variable genes. Through population-scale comparative genomics of 7 Sulfolobus islandicus genomes from 3 locations, we demonstrate the biogeographical structure of the pan-genome of this species, with no evidence of gene flow between geographically isolated populations. The evolutionary independence of each population allowed us to assess genome dynamics over very recent evolutionary time, beginning approximately 910,000 years ago. On this time scale, genome variation largely consists of recent strain-specific integration of mobile elements. Localized sectors of parallel gene loss are identified; however, the balance between the gain and loss of genetic material suggests that S. islandicus genomes acquire material slowly over time, primarily from closely related Sulfolobus species. Examination of the genome dynamics through population genomics in S. islandicus exposes the process of allopatric speciation in thermophilic Archaea and brings us closer to a generalized framework for understanding microbial genome evolution in a spatial context.

Comparison of the transcriptomic "stress response" evoked by antimycin A and oxygen deprivation in Saccharomyces cerevisiae.

BACKGROUND: Acute changes in environmental parameters (e.g., O2, pH, UV, osmolarity, nutrients, etc.) evoke a common transcriptomic response in yeast referred to as the "environmental stress response" (ESR) or "common environmental response" (CER). Why such a diverse array of insults should elicit a common transcriptional response remains enigmatic. Previous functional analyses of the networks involved have found that, in addition to up-regulating those for mitigating the specific stressor, the majority appear to be involved in balancing energetic supply and demand and modulating progression through the cell cycle. Here we compared functional and regulatory aspects of the stress responses elicited by the acute inhibition of respiration with antimycin A and oxygen deprivation under catabolite non-repressed (galactose) conditions. RESULTS: Gene network analyses of the transcriptomic responses revealed both treatments result in the transient (10 - 60 min) down-regulation of MBF- and SBF-regulated networks involved in the G1/S transition of the cell cycle as well as Fhl1 and PAC/RRPE-associated networks involved in energetically costly programs of ribosomal biogenesis and protein synthesis. Simultaneously, Msn2/4 networks involved in hexose import/dissimilation, reserve energy regulation, and autophagy were transiently up-regulated. Interestingly, when cells were treated with antimycin A well before experiencing anaerobiosis these networks subsequently failed to respond to oxygen deprivation. These results suggest the transient stress response is elicited by the acute inhibition of respiration and, we postulate, changes in cellular energetics and/or the instantaneous growth rate, not oxygen deprivation per se. After a considerable delay (> or = 1 generation) under anoxia, predictable changes in heme-regulated gene networks (e.g., Hap1, Hap2/3/4/5, Mot3, Rox1 and Upc2) were observed both in the presence and absence of antimycin A. CONCLUSION: This study not only differentiates between the gene networks that respond to respiratory inhibition and those that respond to oxygen deprivation but suggests the function of the ESR or CER is to balance energetic supply/demand and coordinate growth with the cell cycle, whether in response to perturbations that disrupt catabolic pathways or those that require rapidly up-regulating energetically costly programs for combating specific stressors.

Significant antitumor activity in vivo following treatment with the microtubule agent ENMD-1198.

Clinical studies using the microtubule-targeting agent 2-methoxyestradiol (2ME2; Panzem) in cancer patients show that treatment is associated with clinical benefit, including prolonged stable disease, complete and partial responses, and an excellent safety profile. Studies have shown that 2ME2 is metabolized by conjugation at positions 3 and 17 and oxidation at position 17. To define structure-activity relationships for these positions of 2ME2 and to generate metabolically stable analogues with improved anti-tubulin properties, a series of analogues was generated and three lead analogues were selected, ENMD-1198, ENMD-1200, and ENMD-1237. These molecules showed improved metabolic stability with >65% remaining after 2-h incubation with hepatocytes. Pharmacokinetic studies showed that oral administration of the compounds resulted in increased plasma levels compared with 2ME2. All three analogues bind the colchicine binding site of tubulin, induce G(2)-M cell cycle arrest and apoptosis, and reduce hypoxia-inducible factor-1alpha levels. ENMD-1198 and ENMD-1200 showed improved in vitro antiproliferative activities. Significant reductions in tumor volumes compared with vehicle-treated mice were observed in an orthotopic breast carcinoma (MDA-MB-231) xenograft model following daily oral treatment with all compounds (ANOVA, P < 0.05). Significantly improved median survival time was observed with ENMD-1198 and ENMD-1237 (200 mg/kg/d) in a Lewis lung carcinoma metastatic model (P < 0.05). In both tumor models, the high-dose group of ENMD-1198 showed antitumor activity equivalent to that of cyclophosphamide. ENMD-1198 was selected as the lead molecule in this analogue series and is currently in a phase I clinical trial in patients with refractory solid tumors.

Top-down proteomics on a chromatographic time scale using linear ion trap fourier transform hybrid mass spectrometers.

Proteomics has grown significantly with the aid of new technologies that consistently are becoming more streamlined. While processing of proteins from a whole cell lysate is typically done in a bottom-up fashion utilizing MS/MS of peptides from enzymatically digested proteins, top-down proteomics is becoming a viable alternative that until recently has been limited largely to offline analysis by tandem mass spectrometry. Here we describe a method for high-resolution tandem mass spectrometery of intact proteins on a chromatographic time scale. In a single liquid chromatography-tandem mass spectrometry (LC-MS/MS) run, we have identified 22 yeast proteins with molecular weights from 14 to 35 kDa. Using anion exchange chromatography to fractionate a whole cell lysate before online LC-MS/MS, we have detected 231 metabolically labeled (14N/15N) protein pairs from Saccharomyces cerevisiae. Thirty-nine additional proteins were identified and characterized from LC-MS/MS of selected anion exchange fractions. Automated localization of multiple acetylations on Histone H4 was also accomplished on an LC time scale from a complex protein mixture. To our knowledge, this is the first demonstration of top-down proteomics (i.e., many identifications) on linear ion trap Fourier transform (LTQ FT) systems using high-resolution MS/MS data obtained on a chromatographic time scale.

Selective high-affinity ligand antibody mimics for cancer diagnosis and therapy: initial application to lymphoma/leukemia.

PURPOSE: More than two decades of research and clinical trials have shown radioimmunotherapy to be a promising approach for treating various forms of cancer. Lym-1 antibody, which binds selectively to HLA-DR10 on malignant B-cell lymphocytes, has proved to be effective in delivering radionuclides to non-Hodgkin's lymphoma and leukemia. Using a new approach to create small synthetic molecules that mimic the targeting properties of the Lym-1 antibody, a prototype, selective high-affinity ligand (SHAL), has been developed to bind to a unique region located within the Lym-1 epitope on HLA-DR10. EXPERIMENTAL DESIGN: Computer docking methods were used to predict two sets of small molecules that bind to neighboring cavities on the beta subunit of HLA-DR10 surrounding a critical amino acid in the epitope, and the ligands were confirmed to bind to the protein by nuclear magnetic resonance spectroscopy. Pairs of these molecules were then chemically linked together to produce a series of bidentate and bisbidentate SHALs. RESULTS: These SHALs bind with nanomolar to picomolar K(d)'s only to cell lines expressing HLA-DR10. Analyses of biopsy sections obtained from patients also confirmed that SHAL bound to both small and large cell non-Hodgkin's lymphomas mimicking the selectivity of Lym-1. CONCLUSIONS: These results show that synthetic molecules less than 1/50th the mass of an antibody can be designed to exhibit strong binding to subtle structural features on cell surface proteins similar to those recognized by antibodies. This approach offers great potential for developing small molecule therapeutics that target other types of cancer and disease.

Metabolic-state-dependent remodeling of the transcriptome in response to anoxia and subsequent reoxygenation in Saccharomyces cerevisiae.

We conducted a comprehensive genomic analysis of the temporal response of yeast to anaerobiosis (six generations) and subsequent aerobic recovery ( approximately 2 generations) to reveal metabolic-state (galactose versus glucose)-dependent differences in gene network activity and function. Analysis of variance showed that far fewer genes responded (raw P value of or=2 generations) with the activation of Upc2- and Mot3-regulated networks involved in sterol and cell wall homeostasis. The response to reoxygenation was rapid (<5 min) and similar in both media, dominated by Yap1 networks involved in oxidative stress/redox regulation and the concomitant activation of heme-regulated ones. Our analyses revealed extensive networks of genes subject to combinatorial regulation by both heme-dependent (e.g., Hap1, Hap2/3/4/5, Rox1, Mot3, and Upc2) and heme-independent (e.g., Yap1, Skn7, and Puf3) factors under these conditions. We also uncover novel functions for several cis-regulatory sites and trans-acting factors and define functional regulons involved in the physiological acclimatization to changes in oxygen availability.