A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens

Identification of seedling resistance in wild oat relatives against oat crown rust

Faculty advisor: Melania FigueroaThis research was supported by the Undergraduate Research Opportunities Program (UROP)

EVALITA Evaluation of NLP and Speech Tools for Italian - December 17th, 2020

Welcome to EVALITA 2020! EVALITA is the evaluation campaign of Natural Language Processing and Speech Tools for Italian. EVALITA is an initiative of the Italian Association for Computational Linguistics (AILC, http://www.ai-lc.it) and it is endorsed by the Italian Association for Artificial Intelligence (AIxIA, http://www.aixia.it) and the Italian Association for Speech Sciences (AISV, http://www.aisv.it)

The role of costimulatory molecules during protective and pathogenic immune responses following infection with Toxoplasma gondii

Toxoplasma gondii is an intracellular protozoan parasite that is the etiologic agent of toxoplasmosis. Infection with T. gondii elicits a strong Th1 immune response characterized by the ability of NK and T cells to produce IFN-γ, the major mediator of resistance to this infection. However, in the absence of counter-regulatory mechanisms, in particular IL-10, this T cell mediated immune response results in infection-induced immunopathology. Given the importance of T cells in protective and pathogenic immune responses to this infection, it is important that we understand the events that regulate their responses. Costimulation is important in the development of T cell mediated immune responses and the B7/CD28, CD40/CD40L, and B7RP-1/ICOS interactions are pivotal in the regulation of these responses. The aim of this study is to understand the roles of costimulatory molecules in the regulation of protective and pathogenic T cell responses during toxoplasmosis. Results from these studies identified an important role for the B7/CD28 and CD40/CD40L pathways in the development of infection-induced immunopathology in IL-10 −/− mice following infection with T. gondii. While these studies expand our understanding of the roles of costimulatory molecules in pathogenic T cell responses during toxoplasmosis, additional studies identified a CD28 independent mechanism that regulates protective T cell responses during primary infection with T. gondii. However, CD28 has an important role in the generation/maintenance of optimal effector/memory CD4+ T cells required for protective T cell responses during the secondary immune response against T. gondii. Additionally, these studies reveal the importance of IL-2 in resistance to toxoplasmosis. The identification of ICOS, which can also provide costimulation to activated T cells, led to the hypothesis that this molecule may be involved in the CD28-independent mechanism of resistance to infection. Indeed, in vitro and vivo studies reveal that stimulation through ICOS largely accounts for the CD28-independent component of T cell mediated resistance to T. gondii and that ICOS has an essential role in resistance to this pathogen in the absence of CD28. Together, these studies expand our understanding of the roles of costimulatory molecules in the activation and maintenance of protective and pathogenic T cell responses during toxoplasmosis

Characterization of host and non-host interactions of the bacterial wilt pathogen, Erwinia tracheiphila

Bacterial wilt, caused by insect-transmitted Erwinia tracheiphila, is one of the most devastating diseases of cucurbit crops in the United States. Yield losses on susceptible cucumber ( Cucumis sativus), melon (Cucumis melo), and squash (Cucurbita pepo) cultivars reach up to 80%. Despite its economic importance, the pathogen has received inadequate research attention due to the prevalence and success of insecticidal sprays to control the vectors—a conventional disease management strategy—in addition to the pathogen’s difficulty being isolated and manipulated in the laboratory. As a consequence, the pathogenic mechanisms and basic biology of the bacterium are poorly understood. To answer basic questions and investigate more about the pathogen, I aimed to characterize how E. tracheiphila behaves in both compatible and incompatible systems. The first part (Chapter 2) confirms a previous finding that different strains of the pathogen possess specific compatibility with different cucurbit hosts. I developed modified inoculation and disease assessment methods and validated host-specificity of the strains. Cucumber, melon, and squash plants were inoculated with selected strains HCa1-5N, UnisCu1-1N ( Cucumis strains) and MISpSq-N (Cucurbita strain). The results suggest that Cucumis strains are more virulent in cucumber and melon but less virulent in squash. Similarly, the Cucurbita strain is more virulent in squash than in cucumber and melon. The second part (Chapter 3) discusses the incompatible, non-host interaction of the pathogen with tobacco (Nicotina tabacum). The induction of hypersensitive response (HR) as an immunity reaction in non-host plants is one of the classic characteristics of Gram-negative phytobacteria. I surveyed 21 E. tracheiphila strains for HR induction, characterized the plant physiological changes during the response, compared the sequences of the HR-inducing protein Harpin (HrpN), and conducted a HrpN-based phylogenetic analysis among erwiniae. The results indicate that E. tracheiphila induces authentic HR in tobacco through its HrpN protein, similar to other known HR-inducing Erwinia species. These findings provide basic information about the pathogen that can be exploited to develop more resistant cucurbit cultivars. For instance, the host-specific mechanism can be used to create host-pathogen incompatibility in susceptible hosts and lessen the ability of the pathogen to cause disease

Toward Intrinsically Flame-Retardant, Bioenabled Nitrogen Aromatic Nylon 6,6 Comonomers

Muconic acid and its hydrogenation product trans-3-hexenedioic acid (t3HDA) are promising diacid monomers for inserting property enhancements as functionalities into nylon 6,6. A present challenge is related to low reaction yields and the high sophistication of syntheses. A green, atom-economical functionalization reaction that minimizes processing steps is desired. In this work base catalyzed isomerization was used to produce trans-2-hexenedioic acid (t2HDA) under mild conditions, and graft nitrogen based aromatic thiols to t2HDA via Michael addition in gamma-Valerolactone (GVL). The grafted monomers were co-polymerized with adipic acid and hexamethylenediamine (HMDA), and the flame-retardant properties of the synthesized polyamides screened to identify promising nitrogen-containing aromatics to afford improvements in end use performance without compromising on the polyamide’s properties.This is a manuscript of an article published as Hadel, Joseph, Sohaima Noreen, Marco Nazareno Dell’anna, Dhananjay Dileep, Brent H. Shanks, Jean-Philippe Tessonnier, and Eric W. Cochran. "Toward Intrinsically Flame-Retardant, Bioenabled Nitrogen Aromatic Nylon 6, 6 Comonomers." In Sustainable Green Chemistry in Polymer Research. Volume 2. Sustainable Polymers and Applications, pp. 163-176. American Chemical Society, 2023. doi:10.1021/bk-2023-1451.ch008. Posted with Permission. Copyright © 2023American Chemical Societ

Hydropersulfides (RSSH) Outperform Post-Conditioning and Other Reactive Sulfur Species in Limiting Ischemia–Reperfusion Injury in the Isolated Mouse Heart

Hydrogen sulfide (H2S) exhibits protective effects in cardiovascular disease such as myocardial ischemia/reperfusion (I/R) injury, cardiac hypertrophy, and atherosclerosis. Despite these findings, its mechanism of action remains elusive. Recent studies suggest that H2S can modulate protein activity through redox-based post-translational modifications of protein cysteine residues forming hydropersulfides (RSSH). Furthermore, emerging evidence indicates that reactive sulfur species, including RSSH and polysulfides, exhibit cardioprotective action. However, it is not clear yet whether there are any pharmacological differences in the use of H2S vs. RSSH and/or polysulfides. This study aims to examine the differing cardioprotective effects of distinct reactive sulfur species (RSS) such as H2S, RSSH, and dialkyl trisulfides (RSSSR) compared with canonical ischemic post-conditioning in the context of a Langendorff ex-vivo myocardial I/R injury model. For the first time, a side-by-side study has revealed that exogenous RSSH donation is a superior approach to maintain post-ischemic function and limit infarct size when compared with other RSS and mechanical post-conditioning. Our results also suggest that RSSH preserves mitochondrial respiration in H9c2 cardiomyocytes exposed to hypoxia-reoxygenation via inhibition of oxidative phosphorylation while preserving cell viability

Increased virulence of Puccinia coronata f. sp.avenae populations through allele frequency changes at multiple putative Avr loci.

Pathogen populations are expected to evolve virulence traits in response to resistance deployed in agricultural settings. However, few temporal datasets have been available to characterize this process at the population level. Here, we examined two temporally separated populations of Puccinia coronata f. sp. avenae (Pca), which causes crown rust disease in oat (Avena sativa) sampled from 1990 to 2015. We show that a substantial increase in virulence occurred from 1990 to 2015 and this was associated with a genetic differentiation between populations detected by genome-wide sequencing. We found strong evidence for genetic recombination in these populations, showing the importance of the alternate host in generating genotypic variation through sexual reproduction. However, asexual expansion of some clonal lineages was also observed within years. Genome-wide association analysis identified seven Avr loci associated with virulence towards fifteen Pc resistance genes in oat and suggests that some groups of Pc genes recognize the same pathogen effectors. The temporal shift in virulence patterns in the Pca populations between 1990 and 2015 is associated with changes in allele frequency in these genomic regions. Nucleotide diversity patterns at a single Avr locus corresponding to Pc38, Pc39, Pc55, Pc63, Pc70, and Pc71 showed evidence of a selective sweep associated with the shift to virulence towards these resistance genes in all 2015 collected isolates

Age at diagnosis is a determinant factor of renal cell carcinoma-specific survival in patients treated with nephrectomy.

International audienceOBJECTIVE: Based on combined data for 4880 patients, 2 previous studies reported that advanced age is a predictor of increased renal cell carcinoma-specific mortality (RCC-SM). We explored the effect of age in cubic spline analyses to identify the age groups with the most elevated risk for renal cell carcinoma (RCC). METHODS: Our study included 3595 patients from 14 European centres who had partial or radical nephrectomies. We used the Kaplan-Meier method to compile life tables, and we performed Cox regression analyses to assess RCC-SM. Covariates included age at diagnosis, sex, TNM (tumour, node, metastasis) stage, tumour size, Fuhrman grade, symptom classification and histological subtype. RESULTS: Age ranged from 10 to 89 (mean 63, median 67) years. The median duration of follow-up was 2.9 years. The median survival for the cohort was 13.4 years. Stage distribution was as follows: 1915 patients (53.3%) had stage I disease, 388 (10.8%) had stage II, 895 (24.9%) had stage III and 397 (11.0%) had stage IV disease. In multivariate analyses, we coded age at diagnosis as a cubic spline, and it achieved independent predictor status (p < 0.001). The risk of RCC-SM was lowest among patients younger than 50 years. We observed an increase in RCC-SM until the age of 50, at which point the level of risk reached a plateau. We observed a second increase among patients aged 75-89 years. We found similar patterns when we stratified patients according to the 2002 American Joint Committee on Cancer (AJCC) stages. CONCLUSION: The effect of age shows prognostic significance and indicates that follow-up and possibly secondary treatments might need to be adjusted according to the age of the patient

Age at diagnosis is a determinant factor of renal cell carcinoma–specific survival in patients treated with nephrectomy

Item does not contain fulltextOBJECTIVE: Based on combined data for 4880 patients, 2 previous studies reported that advanced age is a predictor of increased renal cell carcinoma-specific mortality (RCC-SM). We explored the effect of age in cubic spline analyses to identify the age groups with the most elevated risk for renal cell carcinoma (RCC). METHODS: Our study included 3595 patients from 14 European centres who had partial or radical nephrectomies. We used the Kaplan-Meier method to compile life tables, and we performed Cox regression analyses to assess RCC-SM. Covariates included age at diagnosis, sex, TNM (tumour, node, metastasis) stage, tumour size, Fuhrman grade, symptom classification and histological subtype. RESULTS: Age ranged from 10 to 89 (mean 63, median 67) years. The median duration of follow-up was 2.9 years. The median survival for the cohort was 13.4 years. Stage distribution was as follows: 1915 patients (53.3%) had stage I disease, 388 (10.8%) had stage II, 895 (24.9%) had stage III and 397 (11.0%) had stage IV disease. In multivariate analyses, we coded age at diagnosis as a cubic spline, and it achieved independent predictor status (p < 0.001). The risk of RCC-SM was lowest among patients younger than 50 years. We observed an increase in RCC-SM until the age of 50, at which point the level of risk reached a plateau. We observed a second increase among patients aged 75-89 years. We found similar patterns when we stratified patients according to the 2002 American Joint Committee on Cancer (AJCC) stages. CONCLUSION: The effect of age shows prognostic significance and indicates that follow-up and possibly secondary treatments might need to be adjusted according to the age of the patient