Investigating the symbiotic role of plasmid pMESCI01 in Mesorhizobium ciceri bv. biserrulae WSM1271 with Biserrula pelecinus

The establishment of a nitrogen-fixing symbiotic relationship between soil-dwelling bacteria (rhizobia) and legumes is a valuable source of bioavailable nitrogen in the biosphere. The genes required for the establishment and maintenance of this symbiosis may be encoded on plasmids or within the chromosome of the rhizobial microsymbiont as a Symbiosis Island (SI). Previous studies have found that Mesorhizobium ciceri bv. biserrulae WSM1271, which forms a nitrogen-fixing symbiosis with the pasture legume Biserrula pelecinus, transferred its tripartite SI to at least two presumably non-symbiotic Mesorhizobium spp., converting them into B. pelecinus-nodulating rhizobia. These newly evolved symbionts were not as effective in nitrogen fixation with B. pelecinus as WSM1271, even though they had acquired the entire SI from WSM1271. Importantly, WSM1271 harbours a plasmid (pMESCI01) that is absent from WSM2073 and WSM2075. This plasmid may carry additional determinants required for highly effective symbiotic interactions with B. pelecinus. Therefore, analysis of the potential symbiotic role and transmissibility of this plasmid may help explain the differences in nitrogen fixation of these three strains with B. pelecinus. Bioinformatic analysis of pMESCI01 revealed >50% of genes to have hypothetical functions only, with a diverse range of function predictions for the remainder of genes. Crucially, 12 putative nitrogen fixation genes were identified on pMESCI01 that could have a role in the symbiosis between WSM1271 and B. pelecinus. Homologs of known plasmid conjugal transfer type IV secretion systems (T4SS) or relaxases were not detected, but a potential origin of transfer (oriT) site was located, leaving open the question of whether pMESCI01 is self-transmissible. To determine whether pMESCI01 is self-transmissible, the plasmid was marked with an Ω-Sp/Sm cassette, encoding spectinomycin and streptomycin resistance. Possible transconjugants from a subsequent conjugation experiment between WSM1271-pMESCI01::Ω-Sp/Sm and WSM2073Nm could not be screened, owing to unexpected spectinomycin resistance of the WSM2073Nm recipient. However, WSM2073Nm was confirmed to be highly sensitive to streptomycin, so the conjugation experiment can be repeated in future to assess self-transmissibility of pMESCI01 with pMESCI01::Ω-Sp/Sm. To assess whether pMESCI01 was essential to nitrogen fixation in WSM1271 with B. pelecinus, the curing vector pSRKrepABC was constructed and plasmid-cured derivatives of WSM1271 were produced via a plasmid incompatibility approach. Phenotypic assessment of wild-type WSM1271 compared to the plasmid-cured derivatives inoculated onto B. pelecinus in a glasshouse trial revealed no difference in mean shoot dry weights or nodulation of plants across the treatments. This indicated that the plasmid-located genes are likely to not be essential to nitrogen fixation in WSM1271 with this host. Alignment of the sequence of pMESCI01 with accessory plasmids from other fully-sequenced M. ciceri strains revealed a high percentage of homology across the plasmids from these strains, which originate from geographically diverse locations. This suggests that these M. ciceri plasmids are widely dispersed and may be a common feature of this species. While the work in this thesis indicates that pMESCI01 is not essential in the symbiosis between WSM1271 and B. pelecinus, genes on the plasmid may still impart important regulatory or metabolic benefits to WSM1271. Future investigations into the possible function of the large number of genes (432 in total) on pMESCI01 may help to address the role of this plasmid in WSM1271 and, by extension, the role of accessory plasmids in other M. ciceri strains. Therefore, continued studies of Mesorhizobium spp. would be important in determining the evolution, transmissibility and role of these plasmids

The developmental implications of the regulatory relationship between cJun and OCT4 in murine embryonic stem cells

As cells transition from the point of fertilization through the process of embryonic development, many molecular changes occur that affect cell fate. At the blastocyst stage, the earliest distinction, two separate cell populations arise. The trophectoderm cells will generate all of the extraembryonic tissues while the inner cell mass will yield all of the embryonic tissues. These cells, which generate the organism, are termed pluripotent at this stage and found within the inner cell mass (ICM). A variety of genetic mechanisms that regulate this event have been characterized. Here, we examined the effect of cJun expression in regulating Oct4, a gene known to be a master regulator of pluripotency. Luciferase assays were performed to validate our bioinformatics analysis of the 3 kb Oct4 promoter, which identified a cJun binding site approximately 2500 bp upstream of the transcription start site. These experiments demonstrate transient expression of cJun off a reporter plasmid significantly increases luciferase activity of the Oct4-luciferase reporter construct, while expression of the transcriptionally inactive cJun mutant L40/42A represses it. Additionally, we corroborate previous reports that cJun can synergize with Beta-catenin, a member of the Wnt pathway, to significantly increase the promoter activity of Oct4. A role for regulation of endogenous Oct4 expression was evaluated by immunoblot analysis of murine embryonic stem cells transiently transfected with GFP cJun, which demonstrated increased OCT4A expression 36 hours post-transfection. To assess the developmental implications of this regulatory relationship, we evaluated the effect of cJun overexpression on early differentiation by transiently transfecting mESCs and initiating embryoid body formation. When evaluated for germ layer marker expression by immunoblot analysis, it was revealed that an increase in cJun correlated with an increase in GATA4 expression, suggesting cJun expression could lead to selection toward the endodermal germ layer. In light of this, we carried out directed differentiation into cardiomyocytes, using a hanging drop intermediate, and found that an increase in cJun led to a significant increase in cardiomyocyte formation and beating. Overall, this suggests a new role for cJun in the regulation of potency and the formation of the endoderm during those early cell fate choices

Personal README Files: User Manuals for Library Staff

Presentation given at the Designing for Digital Conference in Austin, Texas, on Monday, March 9, 2020.Teams at three libraries are using personal README files to improve communication. As README files tell you how to use software, personal README files tell you how best to interact with teammates. Presenters will share the hows, whys and benefits of incorporating personal README files into your team's practice.https://deepblue.lib.umich.edu/bitstream/2027.42/154114/1/Personal README Files- User Manuals for Library Staff.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154114/2/Personal README Files- User Manuals for Library Staff (with speaker notes).pdfDescription of Personal README Files- User Manuals for Library Staff.pdf : Presentation slidesDescription of Personal README Files- User Manuals for Library Staff (with speaker notes).pdf : Presentation slides with speaker note

Jargon-Free Librarianing: Speaking the Language of Our Patrons

Eliminating jargon from our reference interactions, information literacy classes, and online resources is an undertaking that requires cooperation and input from all library departments. Through collaboration with Reference & Research Services and Information Delivery Services, we examined ways our University Libraries currently presents itself both through user experience with our website, during chat and in-person reference interactions, and in information literacy instruction sessions. Our poster will identify core problems jargon-overload present. We will look at how these problems and inconsistencies impact user experience from a Resources Discovery perspective, and provide specific examples from our library. We will provide visuals that show how language is used in library instruction and reference interactions, both in person and virtually. By looking at examples of terminology used in resource discovery, face-to-face instruction and virtual interaction, we can identify areas where natural language can take precedence. Use of consistent natural language in information literacy instruction, reference interactions and resource discovery will provide patrons with the language that they need to develop information-seeking and analyzing skills that will benefit them long after they leave the University. Librarians are notorious for speaking in jargon, often to the detriment of our users. By examining the words we use to communicate with people from both the resource discovery and instruction perspectives, we are seeking ways to break down barriers between ourselves as librarians and our users by providing consistency in both face-to-face communication and virtual interactions with our resource discovery systems

Development of a multivariable risk model integrating urinary cell DNA methylation and cell-free RNA data for the detection of significant prostate cancer

Background: Prostate cancer exhibits severe clinical heterogeneity and there is a critical need for clinically implementable tools able to precisely and noninvasively identify patients that can either be safely removed from treatment pathways or those requiring further follow up. Our objectives were to develop a multivariable risk prediction model through the integration of clinical, urine-derived cell-free messenger RNA (cf-RNA) and urine cell DNA methylation data capable of noninvasively detecting significant prostate cancer in biopsy naïve patients. Methods: Post-digital rectal examination urine samples previously analyzed separately for both cellular methylation and cf-RNA expression within the Movember GAP1 urine biomarker cohort were selected for a fully integrated analysis (n = 207). A robust feature selection framework, based on bootstrap resampling and permutation, was utilized to find the optimal combination of clinical and urinary markers in a random forest model, deemed ExoMeth. Out-of-bag predictions from ExoMeth were used for diagnostic evaluation in men with a clinical suspicion of prostate cancer (PSA ≥ 4 ng/mL, adverse digital rectal examination, age, or lower urinary tract symptoms). Results: As ExoMeth risk score (range, 0-1) increased, the likelihood of high-grade disease being detected on biopsy was significantly greater (odds ratio = 2.04 per 0.1 ExoMeth increase, 95% confidence interval [CI]: 1.78-2.35). On an initial TRUS biopsy, ExoMeth accurately predicted the presence of Gleason score ≥3 + 4, area under the receiver-operator characteristic curve (AUC) = 0.89 (95% CI: 0.84-0.93) and was additionally capable of detecting any cancer on biopsy, AUC = 0.91 (95% CI: 0.87-0.95). Application of ExoMeth provided a net benefit over current standards of care and has the potential to reduce unnecessary biopsies by 66% when a risk threshold of 0.25 is accepted. Conclusion: Integration of urinary biomarkers across multiple assay methods has greater diagnostic ability than either method in isolation, providing superior predictive ability of biopsy outcomes. ExoMeth represents a more holistic view of urinary biomarkers and has the potential to result in substantial changes to how patients suspected of harboring prostate cancer are diagnosed

SEPATH: benchmarking the search for pathogens in human tissue whole genome sequence data leads to template pipelines

Background : Human tissue is increasingly being whole genome sequenced as we transition into an era of genomic medicine. With this arises the potential to detect sequences originating from microorganisms, including pathogens amid the plethora of human sequencing reads. In cancer research, the tumorigenic ability of pathogens is being recognized, for example Helicobacter pylori and human papillomavirus in the cases of gastric non-cardia and cervical carcinomas respectively. As of yet, no benchmark has been carried out on the performance of computational approaches for bacterial and viral detection within host-dominated sequence data. Results : We present the results of benchmarking over 70 distinct combinations of tools and parameters on 100 simulated cancer datasets spiked with realistic proportions of bacteria. mOTUs2 and Kraken are the highest performing individual tools achieving median genus level F1-scores of 0.90 and 0.91 respectively. mOTUs2 demonstrates a high performance in estimating bacterial proportions. Employing Kraken on unassembled sequencing reads produces a good but variable performance depending on post-classification filtering parameters. These approaches are investigated on a selection of cervical and gastric cancer whole genome sequences where Alphapapillomavirus and Helicobacter are detected in addition to a variety of other interesting genera. Conclusions : We provide the top performing pipelines from this benchmark in a unifying tool called SEPATH, which is amenable to high throughput sequencing studies across a range of high-performance computing clusters. SEPATH provides a benchmarked and convenient approach to detect pathogens in tissue sequence data helping to determine the relationship between metagenomics and disease

CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy.

Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals-including CD471, programmed cell death ligand 1 (PD-L1)2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)3. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy