A novel framework to assess haematology and oncology registration trials: The THEOREMM project.

BACKGROUND: Methodological limitations affect a significant number of oncology and haematology trials, raising concerns about the applicability of their results. For example, a suboptimal control arm or limited access to best care upon progression may skew the trial results toward a benefit in the experimental arm. Beyond the fact that such limitations do not prevent drugs reaching the market, other assessment tools, such as those developed by professional societies-ESMO-MCBS and ASCO Value Framework-do not integrate these important shortcomings. METHODS: We propose creating a novel framework with the scope of assessing registration cancer clinical trials in haematology and oncology (randomized or single arm)-that is trials leading to a marketing authorization. The main steps of the methods are (1) assembling a scientific board; (2) defining the scope, goal and methods through pre-specified, pre-registered and protocolized methodology; (3) preregistration of the protocol; (4) conducting a scoping review of limitations and biases affecting oncology trials and assessing existing scores or methods; (5) developing a list of features to be included and assessed within the framework; (6) assessing each feature through a questionnaire sent to highly cited haematologists and oncologists involved in clinical trials; and (7) finalizing the first version of framework. RESULTS: Not applicable. CONCLUSIONS: Our proposal emerged in response to the lack of consideration for key limitations in current trial assessments. The goal is to create a framework specifically designed to assess single trials leading to marketing authorization in the field of oncology and haematogy.

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.

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.

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 ].

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.

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.

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.

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.

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.

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.

Dynamical remodeling of the transcriptome during short-term anaerobiosis in Saccharomyces cerevisiae: differential response and role of Msn2 and/or Msn4 and other factors in galactose and glucose media.

In contrast to previous steady-state analyses of the O(2)-responsive transcriptome, here we examined the dynamics of the response to short-term anaerobiosis (2 generations) in both catabolite-repressed (glucose) and derepressed (galactose) cells, assessed the specific role that Msn2 and Msn4 play in mediating the response, and identified gene networks using a novel clustering approach. Upon shifting cells to anaerobic conditions in galactose medium, there was an acute ( approximately 10 min) yet transient (<45 min) induction of Msn2- and/or Msn4-regulated genes associated with the remodeling of reserve energy and catabolic pathways during the switch from mixed respiro-fermentative to strictly fermentative growth. Concomitantly, MCB- and SCB-regulated networks associated with the G(1)/S transition of the cell cycle were transiently down-regulated along with rRNA processing genes containing PAC and RRPE motifs. Remarkably, none of these gene networks were differentially expressed when cells were shifted in glucose, suggesting that a metabolically derived signal arising from the abrupt cessation of respiration, rather than O(2) deprivation per se, elicits this "stress response." By approximately 0.2 generation of anaerobiosis in both media, more chronic, heme-dependent effects were observed, including the down-regulation of Hap1-regulated networks, derepression of Rox1-regulated networks, and activation of Upc2-regulated ones. Changes in these networks result in the functional remodeling of the cell wall, sterol and sphingolipid metabolism, and dissimilatory pathways required for long-term anaerobiosis. Overall, this study reveals that the acute withdrawal of oxygen can invoke a metabolic state-dependent "stress response" but that acclimatization to oxygen deprivation is a relatively slow process involving complex changes primarily in heme-regulated gene networks.

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.

Characterization of MUC1 glycoprotein on prostate cancer for selection of targeting molecules.

MUC1 glycoprotein that is overexpressed in aberrant forms in epithelial cancers has been used for diagnosis, staging and therapy. As normal prostate and prostate cancer tissues express MUC1, it represents a potential target, but MUC1 epitopes specific to prostate cancer have not been well characterized. In order to assess MUC1 epitopes in prostate cancer, and their correlation with Gleason grades, binding of 7 well-characterized anti-MUC1 monoclonal antibodies (MAbs) (BrE-3, SM3, BC2, EMA, B27.29, HMFG-1 and NCL MUC1 core), were studied on a prostate tissue microarray. This microarray contained 197 prostate tissue cores representing: i) normal/benign prostate; ii) prostatic intraepithelial neoplasia and Gleason grades 1 and 2; and iii) Gleason grades 3-5. These MAbs bind the MUC1 extracellular domain, but have variable sensitivity to MUC1 glycosylation. To further characterize the effect of glycosylation on their binding, MAb reactivities with unglycosylated MUC1 core peptide and breast and prostate cancer cell lysates were compared. These studies demonstrated strong binding of BrE-3, BC2 and EMA to the peptide core and recognition by BrE-3, SM3, BC2 and EMA of hypoglycosylated MUC1. The results for the microarray indicated that higher Gleason grades were associated with markedly increased cellular staining by MAbs that preferentially recognize less glycosylated MUC1 (BrE-3, p<0.001; SM3, p<0.004; EMA, p=0.009; and BC2, p<0.001). Staining by MAbs that bind preferentially to hyperglycosylated MUC1 (B27.29, p=0.33; HMFG-1, p=0.89; and NCL MUC1 core, p=0.96) did not correlate with Gleason grade. These results demonstrated that hypoglycosylated MUC1 expression increased with Gleason grade, thus supporting the targeting of hypoglycosylated MUC1 epitopes in prostate cancer for more specific imaging and therapy applications.

Enhancement of the therapeutic index: from nonmyeloablative and myeloablative toward pretargeted radioimmunotherapy for metastatic prostate cancer.

PURPOSE: New strategies that target selected molecular characteristics and result in an effective therapeutic index are needed for metastatic, hormone-refractory prostate cancer. EXPERIMENTAL DESIGN: A series of preclinical and clinical studies were designed to increase the therapeutic index of targeted radiation therapy for prostate cancer. (111)In/90Y-monoclonal antibody (mAb), m170, which targets aberrant sugars on abnormal MUC1, was evaluated in androgen-independent prostate cancer patients to determine the maximum tolerated dose and efficacy of nonmyeloablative radioimmunotherapy and myeloablative combined modality radioimmunotherapy with paclitaxel. To enhance the tumor to liver therapeutic index, a cathepsin degradable mAb linkage ((111)In/90Y-peptide-m170) was used in the myeloablative combined modality radioimmunotherapy protocol. For tumor to marrow therapeutic index improvement in future studies, anti-MUC1 scFvs modules were developed for pretargeted radioimmunotherapy. Anti-MUC1 and anti-DOTA scFvs were conjugated to polyethylene glycol scaffolds tested on DU145 prostate cancer cells and prostate tissue arrays, along with mAbs against MUC1 epitopes. RESULTS: The nonmyeloablative maximum tolerated dose of 90Y-m170 was 0.74 GBq/m2 for patients with not more than 10% axial skeleton involvement. Metastatic prostate cancer was targeted in all 17 patients; mean radiation dose was 10.5 Gy/GBq and pain response occurred in 7 of 13 patients reporting pain. Myeloablative combined modality radioimmunotherapy with 0.4 GBq/m2 of 90Y-peptide-m170 and paclitaxel showed therapeutic effects in 4 of 6 patients and 30% less radiation to the liver per unit of activity. Neutropenia was dose limiting without marrow support and patient eligibility was a major limitation to dose escalation. Hypoglycosylated MUC1 epitopes were shown to be abundant in prostate cancer and to increase with disease grade. Anti-MUC1 scFvs binding to prostate cancer tissue and live cells were developed into di-scFv binding modules. CONCLUSIONS: The therapeutic index enhancement for prostate radioimmunotherapy was achieved in clinical studies by the addition of cathepsin cleavable linkers to 90Y-conjugated mAbs and the use of paclitaxel. However, the need for marrow support in myeloablative combined modality radioimmunotherapy restricted eligible patients. Therefore, modular pretargeted radioimmunotherapy, aiming at improving the tumor to marrow therapeutic index, is being developed.

Cilengitide targeting of alpha(v)beta(3) integrin receptor synergizes with radioimmunotherapy to increase efficacy and apoptosis in breast cancer xenografts.

Although metastatic breast cancer is responsive to radioimmunotherapy (RIT), a systemic targeted radiation modality, complete and permanent remissions are not typical with single-modality treatment. Antiangiogenic agents, which target normal, proliferating endothelial cells, have the potential to provide relatively nontoxic continuous inhibition of tumor growth by blocking new blood vessel growth and may synergize with RIT to increase efficacy. This study was designed to determine whether, and how, the cyclic Arg-Gly-Asp peptide Cilengitide (EMD 121974), which targets the alpha(v)beta(3) integrin receptor expressed on neovasculature, could increase systemic RIT efficacy of therapy in a human breast cancer tumor model having mutant p53 and expressing bcl-2. HBT 3477 breast cancer tumor response in nude mice was compared between groups of untreated mice (n = 24), Cilengitide-treated mice (n = 18), RIT (200-260 mu Ci (90)Y-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-peptide ChL6; n = 46), and combined modality RIT (CMRIT) using RIT and six doses of Cilengitide (250 microg/dose; n = 41). Tumor size, survival, body weight, and blood counts were monitored for efficacy and toxicity of therapy. To clarify the mechanism of synergistic effect, tumors were evaluated at selected time points through 6 days for apoptosis, proliferation, and microvessel density. Cilengitide alone did not alter tumor growth when compared with untreated mice, but CMRIT with Cilengitide increased efficacy of treatment, with the cure rate for mice that received 260 mu Ci RIT increasing from 15 to 53% (P = 0.011). Lower-dose RIT (200 mu Ci) combined with Cilengitide resulted in less increase in cures (36 compared with 25% for RIT alone; P = 0.514). Combined analysis for high- and low-dose groups demonstrated increased efficacy of CMRIT (P = 0.020). Analysis of tumors from CMRIT mice indicated significantly increased apoptosis of tumor and endothelial cells 5 days after RIT compared with tumors from mice given RIT alone. Proliferation was decreased in CMRIT tumors compared with RIT tumors at 6 days (ANOVA, P < 0.05). Microvessel density in tumors from RIT and CMRIT mice was not different. No increased toxicity attributable to Cilengitide was observed based upon pooled blood sample and no statistical increase in mortality. In conclusion, CMRIT, combining Cilengitide and RIT, significantly increased the efficacy of therapy and increased apoptosis compared with single-modality therapy with either agent, in an aggressive, well-studied breast cancer model. The enhanced therapeutic synergy is of particular note, having been achieved without additional toxicity.

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.

Effect of molecular size of pegylated peptide on the pharmacokinetics and tumor targeting in lymphoma-bearing mice.

PURPOSE: Rapid blood and body clearances have hampered effective tumor targeting by small molecules. We used branched poly(ethylene glycol) (pegylated) polymers (M(r) 40,000, M(r) 70,000, M(r) 100,000, and M(r) 150,000) conjugated to tumor-specific and control peptides to assess the effect of both molecular weight and tumor specificity on pharmacokinetics and biodistribution. EXPERIMENTAL DESIGN: Pegylated specific lymphoma-binding peptide and control peptide (containing stereoisomers of proline and aspartate) were synthesized, radiolabeled with (111)In, fractionated by size, and injected into Raji lymphoma-bearing athymic mice (4-6 mice/group). Pharmacokinetics were followed for 2 days to evaluate effects of specificity and molecular size on blood clearance, body clearance, and biodistribution. RESULTS: As molecular size increased, blood and body clearances decreased (P < 0.001). The effect of molecular size on blood clearance was not altered by ligand binding specificity (P = 0.21), with t(1/2) ranging from 5.4 h (M(r) 40,000) to 17.7 h (M(r) 150,000). However, ligand specificity did alter body clearance, with pegylated control peptides clearing the body more slowly than pegylated specific peptides [P = 0.03; range, 19.1-91.3 h (specific peptides) versus 23.6-115.7 h (control peptides)]. At 24 h, there was more uptake of specific versus control pegylated peptides in tumor, liver, and marrow, but there was less uptake in kidneys, with a more pronounced difference for the higher molecular weight peptides (P < 0.01). CONCLUSIONS: These results demonstrate that the pharmacokinetics and biodistribution of peptides and resultant uptake in tumor and normal tissues can be altered by both molecular size and ligand specificity, with molecular size affecting pharmacokinetics and organ uptake in a predictable manner.

Selection and characterization of anti-MUC-1 scFvs intended for targeted therapy.

PURPOSE: The selection and characterization of anti-MUC-1 single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer molecules designed for optimal blood clearance and tumor penetration. The mucin MUC-1 was chosen as an antigen because it is abundantly expressed on epithelial cancers in an aberrantly glycosylated form, making it structurally and antigenically distinct from MUC-1 expressed on normal cells. EXPERIMENTAL DESIGN: A previously constructed anti-MUC-1 phage display library from hyperimmunized mice, with 5 x 10(5) calculated variants, was screened for the selection of anti-MUC-1 scFvs. Selection criteria were high binding to a MUC-1 peptide containing 4 tandem repeats of 20 amino acids and to MUC-1-positive MCF-7 (human breast cancer) cell lysates in ELISA. RESULTS: Six anti-MUC-1 scFv clones were selected and characterized. Nucleotide sequencing showed that four of them were full length scFv genes (variable heavy chain + variable light chain), whereas the remaining two contained either a variable heavy chain or a variable light chain alone. Their binding affinities (K(a)) range between 8 x 10(7) and 10(9) M(-1). Immunohistopathology demonstrated reactivity with breast cancer cells (MCF-7 and BT20) and human breast biopsy tissue. Molecular modeling revealed high structural similarity of the anti-MUC-1 scFvs with the X-ray-determined structure of the anti-CEA scFv (MFE-23). CONCLUSIONS: In vitro antigen binding was demonstrated for the selected anti-MUC-1 scFvs. The binding affinities of these scFvs are in a promising range for efficient in vivo antigen binding. These anti-MUC-1 scFvs will be evaluated as antigen-binding modules in new multifunctional agents for the detection and therapy of cancer.