iGATE analysis improves the interpretability of single-cell immune landscape of influenza infection.

Influenza poses a persistent health burden worldwide. To design equitable vaccines effective across all demographics, it is essential to better understand how host factors such as genetic background and aging affect the single-cell immune landscape of influenza infection. Cytometry by time-of-flight (CyTOF) represents a promising technique in this pursuit, but interpreting its large, high-dimensional data remains difficult. We've developed a new analytical approach iGATE (in-silico gating annotating training elucidating) based on probabilistic support vector machine classification. By rapidly and accurately "gating" tens of millions of cells in silico into user-defined types, iGATE enabled us to track 25 canonical immune cell types in mouse lung over the course of influenza infection. Applying iGATE to study effects of host genetic background, we show that the lower survival of C57BL/6 mice compared to BALB/c is associated with a more rapid accumulation of inflammatory cell types and decreased IL-10 expression. Further, we demonstrate that the most prominent effect of aging is a defective T-cell response, reducing survival of aged mice. Finally, iGATE reveals that the 25 canonical immune cell types exhibit differential influenza infection susceptibility and replication permissiveness in vivo, but neither property varies with host genotype or aging. Software is available at https://github.com/UmichWenLab/iGATE.

Antigen-Clustered Nanovaccine Achieves Long-Term Tumor Remission by Promoting B/CD 4 T Cell Crosstalk.

Current cancer vaccines using T cell epitopes activate antitumor T cell immunity through dendritic cell/macrophage-mediated antigen presentation, but they lack the ability to promote B/CD4 T cell crosstalk, limiting their anticancer efficacy. We developed antigen-clustered nanovaccine (ACNVax) to achieve long-term tumor remission by promoting B/CD4 T cell crosstalk. The topographic features of ACNVax were achieved using an iron nanoparticle core attached with an optimal number of gold nanoparticles, where the clusters of HER2 B/CD4 T cell epitopes were conjugated on the gold surface with an optimal intercluster distance of 5-10 nm. ACNVax effectively trafficked to lymph nodes and cross-linked with BCR, which are essential for stimulating B cell antigen presentation-mediated B/CD4 T cell crosstalk in vitro and in vivo. ACNVax, combined with anti-PD-1, achieved long-term tumor remission (>200 days) with 80% complete response in mice with HER2+ breast cancer. ACNVax not only remodeled the tumor immune microenvironment but also induced a long-term immune memory, as evidenced by complete rejection of tumor rechallenge and a high level of antigen-specific memory B, CD4, and CD8 cells in mice (>200 days). This study provides a cancer vaccine design strategy, using B/CD4 T cell epitopes in an antigen clustered topography, to achieve long-term durable anticancer efficacy through promoting B/CD4 T cell crosstalk.

Smoking-induced subgingival dysbiosis precedes clinical signs of periodontal disease.

Smoking accelerates periodontal disease and alters the subgingival microbiome. However, the relationship between smoking-associated subgingival dysbiosis and progression of periodontal disease is not well understood. Here, we sampled 233 subgingival sites longitudinally from 8 smokers and 9 non-smokers over 6-12 months, analyzing 804 subgingival plaque samples using 16 rRNA sequencing. At equal probing depths, the microbial richness and diversity of the subgingival microbiome was higher in smokers compared to non-smokers, but these differences decreased as probing depths increased. The overall subgingival microbiome of smokers differed significantly from non-smokers at equal probing depths, which was characterized by colonization of novel minority microbes and a shift in abundant members of the microbiome to resemble periodontally diseased communities enriched with pathogenic bacteria. Temporal analysis showed that microbiome in shallow sites were less stable than deeper sites, but temporal stability of the microbiome was not significantly affected by smoking status or scaling and root planing. We identified 7 taxa-Olsenella sp., Streptococcus cristatus, Streptococcus pneumoniae, Streptococcus parasanguinis, Prevotella sp., Alloprevotella sp., and a Bacteroidales sp. that were significantly associated with progression of periodontal disease. Taken together, these results suggest that subgingival dysbiosis in smokers precedes clinical signs of periodontal disease, and support the hypothesis that smoking accelerates subgingival dysbiosis to facilitate periodontal disease progression.

The Genotypic Variability among Short-Season Soybean Cultivars for Nitrogen Fixation under Drought Stress.

Soybean fixes atmospheric nitrogen through the symbiotic rhizobia bacteria that inhabit root nodules. Drought stress negatively affect symbiotic nitrogen fixation (SNF) in soybean. The main objective of this study was to identify allelic variations associated with SNF in short-season Canadian soybean varieties under drought stress. A diversity panel of 103 early-maturity Canadian soybean varieties was evaluated under greenhouse conditions to determine SNF-related traits under drought stress. Drought was imposed after three weeks of plant growth, where plants were maintained at 30% field capacity (FC) (drought) and 80% FC (well-watered) until seed maturity. Under drought stress, soybean plants had lower seed yield, yield components, seed nitrogen content, % nitrogen derived from the atmosphere (%Ndfa), and total seed nitrogen fixed compared to those under well-watered conditions. Significant genotypic variability among soybean varieties was found for yield, yield parameters, and nitrogen fixation traits. A genome-wide association study (GWAS) was conducted using 2.16 M single nucleotide single nucleotide polymorphisms (SNPs) for different yield and nitrogen fixation related parameters for 30% FC and their relative performance (30% FC/80% FC). In total, five quantitative trait locus (QTL) regions, including candidate genes, were detected as significantly associated with %Ndfa under drought stress and relative performance. These genes can potentially aid in future breeding efforts to develop drought-resistant soybean varieties.

Virus-inspired strategies for cancer therapy.

The intentional use of viruses for cancer therapy dates back over a century. As viruses are inherently immunogenic and naturally optimized delivery vehicles, repurposing viruses for drug delivery, tumor antigen presentation, or selective replication in cancer cells represents a simple and elegant approach to cancer treatment. While early virotherapy was fraught with harsh side effects and low response rates, virus-based therapies have recently seen a resurgence due to newfound abilities to engineer and tune oncolytic viruses, virus-like particles, and virus-mimicking nanoparticles for improved safety and efficacy. However, despite their great potential, very few virus-based therapies have made it through clinical trials. In this review, we present an overview of virus-inspired approaches for cancer therapy, discuss engineering strategies to enhance their mechanisms of action, and highlight their application for overcoming the challenges of traditional cancer therapies.

Stability of healthy subgingival microbiome across space and time.

The subgingival microbiome is one of the most stable microbial ecosystems in the human body. Alterations in the subgingival microbiome have been associated with periodontal disease, but their variations over time and between different subgingival sites in periodontally healthy individuals have not been well described. We performed extensive, longitudinal sampling of the subgingival microbiome from five periodontally healthy individuals to define baseline spatial and temporal variations. A total of 251 subgingival samples from 5 subjects were collected over 6-12 months and deep sequenced. The overall microbial diversity and composition differed significantly between individuals. Within each individual, we observed considerable differences in microbiome composition between different subgingival sites. However, for a given site, the microbiome was remarkably stable over time, and this stability was associated with increased microbial diversity but was inversely correlated with the enrichment of putative periodontal pathogens. In contrast to microbiome composition, the predicted functional metagenome was similar across space and time, suggesting that periodontal health is associated with shared gene functions encoded by different microbiome consortia that are individualized. To our knowledge, this is one of the most detailed longitudinal analysis of the healthy subgingival microbiome to date that examined the longitudinal variability of different subgingival sites within individuals. These results suggest that a single measurement of the healthy subgingival microbiome at a given site can provide long term information of the microbial composition and functional potential, but sampling of each site is necessary to define the composition and community structure at individual subgingival sites.

Robot-Assisted Laparoscopic Removal of a Large Primary Retroperitoneal Mature Cystic Teratoma in an Adult.

Mature teratomas are unique and generally benign neoplasms. They are derived from embryonic tissues and typically located within the gonadal region. Primary retroperitoneal teratomas are uncommon in adults and often challenging to treat, given their location and size. Here, we offer a rare case of a large primary retroperitoneal mature cystic teratoma, detected on abdominal ultrasound during the work-up of abdominal bloating and nausea and treated with robot-assisted laparoscopic excision in a 58-year-old male. In this report, we sought to describe the evaluation, treatment, and follow-up of this condition, as well as review the associated literature.

Primary prostatic squamous cell carcinoma.

Primary prostate squamous cell malignancies are rare and very aggressive. A 65-year-old man presented to our institution with clinical and radiographic findings concerning for advanced prostatic malignancy. Surgical pathology of the tumor revealed poorly differentiated carcinoma with squamous differentiation, and histopathological markers were positive for markers of squamous differentiation negative for all urothelial and prostatic markers.

The Intermucosal Connection between the Mouth and Gut in Commensal Pathobiont-Driven Colitis.

The precise mechanism by which oral infection contributes to the pathogenesis of extra-oral diseases remains unclear. Here, we report that periodontal inflammation exacerbates gut inflammation in vivo. Periodontitis leads to expansion of oral pathobionts, including Klebsiella and Enterobacter species, in the oral cavity. Amassed oral pathobionts are ingested and translocate to the gut, where they activate the inflammasome in colonic mononuclear phagocytes, triggering inflammation. In parallel, periodontitis results in generation of oral pathobiont-reactive Th17 cells in the oral cavity. Oral pathobiont-reactive Th17 cells are imprinted with gut tropism and migrate to the inflamed gut. When in the gut, Th17 cells of oral origin can be activated by translocated oral pathobionts and cause development of colitis, but they are not activated by gut-resident microbes. Thus, oral inflammation, such as periodontitis, exacerbates gut inflammation by supplying the gut with both colitogenic pathobionts and pathogenic T cells.

Effects of Nitrogen Application on Nitrogen Fixation in Common Bean Production.

The nitrogen fixing ability of common bean (Phaseolus vulgaris L.) in association with rhizobia is often characterized as poor compared to other legumes, and nitrogen fertilizers are commonly used in bean production to achieve high yields, which in general inhibits nitrogen fixation. In addition, plants cannot take up all the nitrogen applied to the soil as a fertilizer leading to runoff and groundwater contamination. The overall objective of this work is to reduce use of nitrogen fertilizer in common bean production. This would be a major advance in profitability for the common bean industry in Canada and would significantly improve the ecological footprint of the crop. In the current work, 22 bean genotypes [including recombinant inbred lines (RILs) from the Mist × Sanilac population and a non-nodulating mutant (R99)] were screened for their capacity to fix atmospheric nitrogen under four nitrogen regimes. The genotypes were evaluated in replicated field trials on N-poor soils over three years for the percent nitrogen derived from atmosphere (%Ndfa), yield, and a number of yield-related traits. Bean genotypes differed for all analyzed traits, and the level of nitrogen significantly affected most of the traits, including %Ndfa and yield in all three years. In contrast, application of rhizobia significantly affected only few traits, and the effect was inconsistent among the years. Nitrogen application reduced symbiotic nitrogen fixation (SNF) to various degrees in different bean genotypes. This variation suggests that SNF in common bean can be improved through breeding and selection for the ability of bean genotypes to fix nitrogen in the presence of reduced fertilizer levels. Moreover, genotypes like RIL_38, RIL_119, and RIL_131, being both high yielding and good nitrogen fixers, have potential for simultaneous improvement of both traits. However, breeding advancement might be slow due to an inconsistent correlation between these traits.

Yeast Intracellular Staining (yICS): Enabling High-Throughput, Quantitative Detection of Intracellular Proteins via Flow Cytometry for Pathway Engineering.

The complexities of pathway engineering necessitate screening libraries to discover phenotypes of interest. However, this approach is challenging when desirable phenotypes cannot be directly linked to growth advantages or fluorescence. In these cases, the ability to rapidly quantify intracellular proteins in the pathway of interest is critical to expedite the clonal selection process. While Saccharomyces cerevisiae remains a common host for pathway engineering, current approaches for intracellular protein detection in yeast either have low throughput, can interfere with protein function, or lack the ability to detect multiple proteins simultaneously. To fill this need, we developed yeast intracellular staining (yICS) that enables fluorescent antibodies to access intracellular compartments of yeast cells while maintaining their cellular integrity for analysis by flow cytometry. Using the housekeeping proteins ß actin and glyceraldehyde 3-phophate dehydrogenase (GAPDH) as targets for yICS, we demonstrated for the first time successful antibody-based flow cytometric detection of yeast intracellular proteins with no modification. Further, yICS characterization of a recombinant d-xylose assimilation pathway showed 3-plexed, quantitative detection of the xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) enzymes each fused with a small (6-10 amino acids) tag, revealing distinct enzyme expression profiles between plasmid-based and genome-integrated expression approaches. As a result of its high-throughput and quantitative capability, yICS enabled rapid screening of a library created from CRISPR-mediated XDH integration into the yeast δ site, identifying rare (1%) clones that led to an 8.4-fold increase in XDH activity. These results demonstrate the utility of yICS for greatly accelerating pathway engineering efforts, as well as any application where the high-throughput and quantitative detection of intracellular proteins is desired.

Enhancing the Yield and Quality of Influenza Virus-like Particles (VLPs) Produced in Insect Cells by Inhibiting Cytopathic Effects of Matrix Protein M2.

To provide broader protection and eliminate the need for annual update of influenza vaccines, biomolecular engineering of influenza virus-like particles (VLPs) to display more conserved influenza proteins such as the matrix protein M2 has been explored. However, achieving high surface density of full-length M2 in influenza VLPs has been left unrealized. In this study, we show that the ion channel activity of M2 induces significant cytopathic effects in Spodoptera frugiperda (Sf9) insect cells when expressed using M2-encoding baculovirus. These effects include altered Sf9 cell morphology and reduced baculovirus replication, resulting in impaired influenza protein expression and thus VLP production. On the basis of the function of M2, we hypothesized that blocking its ion channel activity could potentially relieve these cytopathic effects, and thus restore influenza protein expression to improve VLP production. The use of the M2 inhibitor amantadine indeed improves Sf9 cellular expression not only of M2 (∼3-fold), but also of hemagglutinin (HA) (∼7-fold) and of matrix protein M1 (∼3-fold) when coexpressed to produce influenza VLPs. This increased cellular expression of all three influenza proteins further leads to ∼2-fold greater VLP yield. More importantly, the quality of the resulting influenza VLPs is significantly improved, as demonstrated by the ∼2-fold, ∼50-fold, and ∼2-fold increase in the antigen density to approximately 53 HA, 48 M1, and 156 M2 per influenza VLP, respectively. Taken together, this study represents a novel approach to enable the efficient incorporation of full-length M2 while enhancing both the yield and quality of influenza VLPs produced by Sf9 cells.

Agronomic Performance and Nitrogen Fixation of Heirloom and Conventional Dry Bean Varieties Under Low-Nitrogen Field Conditions.

Common beans (Phaseolus vulgaris) form a relationship with nitrogen-fixing rhizobia and through a process termed symbiotic nitrogen fixation (SNF) which provides them with a source of nitrogen. However, beans are considered poor nitrogen fixers, and modern production practices involve routine use of N fertilizer, which leads to the down-regulation of SNF. High-yielding, conventionally bred bean varieties are developed using conventional production practices and selection criteria, typically not including SNF efficiency, and may have lost this trait over decades of modern breeding. In contrast, heirloom bean genotypes were developed before the advent of modern production practices and may represent an underutilized pool of genetics which could be used to improve SNF. This study compared the SNF capacity under low-N field conditions, of collections of heirloom varieties with and conventionally bred dry bean varieties. The heirloom-conventional panel (HCP) consisted of 42 genotypes from various online seed retailers or from the University of Guelph Bean Breeding program seedbank. The HCP was genotyped using a single nucleotide polymorphism (SNP) array to investigate genetic relatedness within the panel. Field trials were conducted at three locations in ON, Canada from 2014 to 2015 and various agronomic and seed composition traits were measured, including capacity for nitrogen fixation (using the natural abundance method to measure seed N isotope ratios). Significant variation for SNF was found in the panel. However, on average, heirloom genotypes did not fix significantly more nitrogen than conventionally bred varieties. However, five heirloom genotypes fixed >60% of their nitrogen from the atmosphere. Yield (kg ha-1) was not significantly different between heirloom and conventional genotypes, suggesting that incorporating heirloom genotypes into a modern breeding program would not negatively impact yield. Nitrogen fixation was significantly higher among Middle American genotypes than among Andean genotypes, confirming previous findings. The best nitrogen fixing line was Coco Sophie, a European heirloom white bean whose genetic makeup is admixed between the Andean and Middle American genepools. Heirloom genotypes represent a useful source of genetics to improve SNF in modern bean breeding.

The female-biased factor VGLL3 drives cutaneous and systemic autoimmunity.

Autoimmune disease is 4 times more common in women than men. This bias is largely unexplained. Female skin is "autoimmunity prone," showing upregulation of many proinflammatory genes, even in healthy women. We previously identified VGLL3 as a putative transcription cofactor enriched in female skin. Here, we demonstrate that skin-directed overexpression of murine VGLL3 causes a severe lupus-like rash and systemic autoimmune disease that involves B cell expansion, autoantibody production, immune complex deposition, and end-organ damage. Excess epidermal VGLL3 drives a proinflammatory gene expression program that overlaps with both female skin and cutaneous lupus. This includes increased B cell-activating factor (BAFF), the only current biologic target in systemic lupus erythematosus (SLE); IFN-κ, a key inflammatory mediator in cutaneous lupus; and CXCL13, a biomarker of early-onset SLE and renal involvement. Our results demonstrate that skin-targeted overexpression of the female-biased factor VGLL3 is sufficient to drive cutaneous and systemic autoimmune disease that is strikingly similar to SLE. This work strongly implicates VGLL3 as a pivotal orchestrator of sex-biased autoimmunity.

Engineering Virus-like Particles for Antigen and Drug Delivery.

Virus-like particles (VLPs) are nanoscale biological structures consisting of viral proteins assembled in a morphology that mimic the native virion but do not contain the viral genetic material. The possibility of chemically and genetically modifying the proteins contained within VLPs makes them an attractive system for numerous applications. As viruses are potent immune activators as well as natural delivery vehicles of genetic materials to their host cells, VLPs are especially well suited for antigen and drug delivery applications. Despite the great potential, very few VLP designs have made it through clinical trials. In this review, we will discuss the challenges of developing VLPs for antigen and drug delivery, strategies being explored to address these challenges, and the genetic and chemical approaches available for VLP engineering.

The Coming Age of Insect Cells for Manufacturing and Development of Protein Therapeutics.

Protein therapeutics is a rapidly growing segment of the pharmaceutical market. Currently, the majority of protein therapeutics are manufactured in mammalian cells for their ability to generate safe and efficacious human-like glycoproteins. The high cost of using mammalian cells for manufacturing has motivated a constant search for alternative host platforms. Insect cells have begun to emerge as a promising candidate, largely due to the development of the baculovirus expression vector system. While there are continuing efforts to improve insect-baculovirus expression for producing protein therapeutics, key limitations including cell lysis and the lack of homogeneous humanized glycosylation still remain. The field has started to see a movement toward virus-less gene expression approaches, notably the use of clustered regularly interspaced short palindromic repeats to address these shortcomings. This review highlights recent technological advances that are realizing the transformative potential of insect cells for the manufacturing and development of protein therapeutics.

Follow-up Visits and Changes in Pain Scores Reported by Oncology Outpatients After Initial Presentation With Severe Pain.

BACKGROUND: In addition to tumour treatment, the management of symptoms such as pain is an important component of cancer care. Pain management is a complex field and prior studies have highlighted many different clinical care responses to a cancer patient presenting with severe pain. We explored follow-up and how pain screening scores changed over time, among a cohort of cancer outpatients, and how follow-up was scheduled after the initial visit. METHODS: The care provided to 96 patients seen at the London Regional Cancer Program was reviewed for the 12-week period following presentation with severe pain >7/10. Follow-up ESAS (Edmonton Symptom Assessment System) scores, visits, and compliance were documented. RESULTS: Follow-up ESAS data was available for 41/96 patients. Mean ESAS pain decreased from 8.4/10 to 3.6/10 among those patients with follow-up; however, for 55/96 patients, no follow-up ESAS score was available (deceased n=3, no follow-up visit n=41, no pain score reported, n=11). CONCLUSIONS: Despite a very high proportion of documented active pain management plans in the case of cancer patients presenting with severe pain, very little follow-up directed specifically at pain management was performed. Cancer treatment appears to be the primary determinant of oncology follow-up timing at our centre.

Co-composting of Beef Cattle Feedlot Manure with Construction and Demolition Waste.

With increased availability of dried distillers' grains with solubles (DDGS) as cattle feed and the need to recycle organic wastes, this research investigated the feasibility of co-composting DDGS cattle feedlot manure with construction and demolition (C&D) waste. Manure was collected from cattle fed a typical western Canadian finishing diet (CK) of 860 g rolled barley ( L.) grain, 100 g barley silage, and 40 g vitamin and mineral supplement kg dry matter (DM) and from cattle fed the same diet but (DG manure) with 300 g kg DM barley grain being replaced by DDGS. The CK and DG manures were co-composted with and without C&D waste in 13 m bins. Compost materials were turned on Days 14, 37, and 64, and terminated on Day 99. Adding C&D waste led to higher compost temperatures (0.4 to 16.3°C, average 7.2°C) than manure alone. Final composts had similar total C, total N, C/N ratios, and water-extractable K, Mg, and NO content across all treatments. However, adding C&D waste increased δC, δN, water-extractable SO, and Ca contents and decreased pH, total P (TP), water-extractable C, N, and P and most volatile fatty acids (VFA). The higher C&D compost temperatures should reduce pathogens while reduced VFA content should reduce odors. When using the final compost product, the increased SO and reduced TP and available N and P content in C&D waste compost should be taken into consideration. Increased S content in C&D compost may be beneficial for some crops grown on S-deficient soils.

Sorption of atmospheric ammonia by soil and perennial grass downwind from two large cattle feedlots.

Livestock manure in feedlots releases ammonia (NH3), which can be sorbed by nearby soil and plants. Ammonia sorption by soil and its effects on soil and perennial grass N contents downwind from two large cattle feedlots in Alberta, Canada were investigated from June to October 2002. Atmospheric NH3 sorption was measured weekly by exposing air-dried soil at sampling points downwind along 1700-m transects. The amount of NH3 sorbed by soil was 2.60 to 3.16 kg N ha(-1) wk(-1) near the source, declining to about 0.25 kg N ha(-1) wk(-1) 1700 m downwind, reflecting diminishing atmospheric NH3 concentrations. Ammonia sorption at a control site away from NH3 sources was much lower: 0.085 kg N ha(-1) wk(-1). Based on these rates, about 19% of emitted NH3 is sorbed by soil within 1700 m downwind of feedlots. Field soil and grass samples from the transect lines were analyzed for total N (TN) and KCl-extractable N content (soil only). Nitrate N content in field soil followed a trend similar to that of atmospheric NH3 sorption. Soil TN contents, because of high background levels, showed no clear pattern. The TN content of grass, downwind of the newer feedlot, followed a pattern similar to that of NH3 sorption; downwind of the older feedlot, grass TN was correlated to soil TN. Our results suggest that atmospheric NH3 from livestock operations can contribute N to local soil and vegetation, and may need to be considered when determining fertilizer rates and assessing environmental impact.