Free and Immobilized Microbial Culture–Mediated Crude Oil Degradation and Microbial Diversity Changes Through Taxonomic and Functional Markers in a Sandy Loam Soil

Crude oil contamination of soil and water resources is a widespread issue. The present study evaluated the degradation of aliphatic hydrocarbons (C11–C36) in crude oil by 17 bacteria isolated from a crude oil–contaminated soil. The results suggested that Pseudomonas sp. and Bacillus amyloliquefaciens were the best hydrocarbon-degrading bacteria in the presence of surfactant Tween-80 (0.1% w/v). Based on the present investigation and a previous study, Pseudomonas sp. + B. amyloliquefaciens and fungus Aspergillus sydowii were identified as best oil degraders and were immobilized in alginate–bentonite beads, guargum–nanobenonite water dispersible granules (WDGs), and carboxy methyl cellulose (CMC)–bentonite composite. Sandy loam soil was fortified with 1, 2, and 5% crude oil, and total petroleum hydrocarbon (TPH) degradation efficiency of free cultures and bio-formulations was evaluated in sandy loam soils. Compared to a half-life (t1/2) of 69.7 days in the control soil (1% oil), free cultures of Pseudomonas sp. + B. amyloliquefaciens and A. sydowii degraded TPH with t1/2 of 10.8 and 19.4 days, respectively. Increasing the oil content slowed down degradation, and the t1/2 in the control and soils inoculated with Pseudomonas sp. + B. amyloliquefaciens and A. sydowii was 72.9, 14.7, and 22.2 days (2%) and 87.0, 23.4, and 30.8 days (5%), respectively. Supplementing soil with ammonium sulfate (1%) enhanced TPH degradation by Pseudomonas sp. + B. amyloliquefaciens (t1/2–10 days) and A. sydowii (t1/2–12.7 days). All three bio-formulations were effective in degrading TPH (1%), and the t1/2 was 10.7–11.9 days (Pseudomonas sp. + B. amyloliquefaciens and 14–20.2 days (A. sydowii) and were at par with free cultures. Microbial diversity analysis based on taxonomic markers and functional markers suggested that the bioaugmentation process helped keep soil in the active stage and restored the original microbial population to some extent. The present study concluded that bio-formulations of crude oil–degrading microbes can be exploited for its degradation in the contaminated environment.

Determination of salt tolerance levels and genetic relationships of Vicia sativa cultivars using gene targeted functional markers

The objectives of the present study were to determine salt tolerance levels of 12 different common vetch (Vicia sativa L.) cultivars at germination stage in the presence of 250 mM NaCl and to reveal genetic relationships based on gene targeted functional markers (GTFMs) associated with salt tolerance. The results revealed the presence of a significant genetic variation among the cultivars although s alt stress significantly reduced all germination parameters tested. The cultivar Ozveren was the most salt tolerant with 20.1% reduction in final germination percentage compared to control seeds while cultivars Alınoglu, Ayaz and Bakir did not germinate. The maximum delays in germination rate (G50 = 3.78 days) and synchrony (G10-90 = 3.45 days) were obtained from the cultivars Urkmez and Ozveren, respectively. The GTFMs provided a total of 53.1% polymorphism. The primers of MtSOS2 gene gave the highest numbers of alleles per primer pair while the highest polymorphism rate (77.8%) was obtained from the MtP5CS gene. The first three components of principal component analysis explained 57.63% of total variation. This study concluded that the cultivars determined to be salt tolerant and sensitive at germination stage distributed into three main clades determined by UPGMA analysis while the GTFMs associated with salt tolerance successfully determined the genetic relationships of common vetch cultivars.

Development of a Porcine Slaughterhouse Kidney Perfusion Model

Machine perfusion techniques are becoming standard care in the clinical donation and transplantation setting. However, more research is needed to understand the mechanisms of the protective effects of machine perfusion. For preservation related experiments, porcine kidneys are acceptable alternatives to human kidneys, because of their size and similar physiology. In this experiment, the use of slaughterhouse kidneys was evaluated with normothermic kidney perfusion (NKP), thereby avoiding the use of laboratory animals. Porcine kidneys were derived from two local abattoirs. To induce different degrees of injury, different warm ischemic times and preservation techniques were used. After preservation, kidneys were reperfused for 4 h with two different NKP solutions to test renal function and damage. The effect of the preservation technique or a short warm ischemic time was clearly seen in functional markers, such as creatinine clearance and fractional sodium excretion levels, as well as in the generic damage marker lactate dehydrogenase (LDH). Porcine slaughterhouse kidneys are a useful alternative to laboratory animals for transplantation- and preservation-related research questions. To maintain kidney function during NKP, a short warm ischemic time or hypothermic machine perfusion during the preservation phase are mandatory.

Green-Synthesized Magnesium Hydroxide Nanoparticles Induced Osteoblastic Differentiation in Bone Co-Cultured Cells

In this work, magnesium hydroxide NPs were synthesized using water (Mg(OH)2 NPs) or a rose hip (RH) extract (Mg(OH)2RH NPs) and tested for the bone cells’ effects in co-cultured osteoblastic and osteoclastic cells, using a Transwell® insert system, allowing reciprocal cell paracrine interactions. Behavior of each cell population was characterized for typical phenotype markers, at days 1 and 6. Cell cultures treated with osteogenic/osteoclastogenic inducers were used as positive control of cell differentiation. The NPs presented a round shape morphology with an average diameter ~90 nm (Mg(OH)2 NPs) and below 10 nm (Mg(OH)2RH NPs. Both NPs induced osteoblastic and osteoclastic behavior similarly to that observed in induced osteoblastic and osteoclastic cultures (positive controls). Differences between the two types of particles were evident at the gene expression level. Compared to Mg(OH)2 NPs, the green-synthesized NPs greatly increased the expression of osteoblastic genes coding for the early markers ALP and collagen type 1 and the later transcription factor osterix, while decreasing the expression of osteoclastogenic genes, namely the essential transcription factor NFATC1, TRAP and the genes coding for the functional markers CA2 and CTSK. Overall, a positive added effect could be hypothesized for Mg(OH)2RH NPs with potential usefulness to promote bone formation in regenerative applications.

USE OF RADIOMIC MARKERS IN PREOPERATIVE DETECTION OF EXTRA-AXIAL TUMOR GRADE

Backgraund: Extra-axial tumors are one of the tumor groups which are difficult for primary differential diagnostics. Detection and standardization of radiomic markers is one of the main problems of our time. Aim: To detect radiomic markers for preoperative assessment of extra-axial tumor grade. Materials and methods: Retrospective analysis of MRI (1.5T) data of 156 patients with extra-axial tumors. The patients were divided into 2 groups: Group 1(n=106) with perifocal changes, Group 2 (n=50) extra-axial tumor without perifocal changes. Diffusion and perfusion sequences were included in the scanning protocol. The areas of interest were (1) the lesion and (2) the area of perifocal changes. Measurements were made from the lesion and the area of perifocal changes on ACD and DSE maps, DCE was analyzed. Results: The maximum lesion size in Group 1 was 2.2cm (1.4; 4.3), in Group 2 - 1.2cm (0.9; 3.5). In Group 1 diffusion restriction from the lesion was detected in 42 patients (39.6%), in Group 2 in 7 patients (14%). The maximum size of perifocal changes in Group 1 was 2.85cm (1.5; 4.7). Diffusion restriction was detected in 52 cases (49.1%). In patients of Group 2 with verified meningioma multivariate linear regression analysis showed that the maximum size of the lesion demonstrated a 3.3-time increase of rCBF from the area of perifocal changes (coef. 3.3 CI 1.27; 5.28) p = 0.003, however, it demonstrated a 4-time decrease of rCBF (coef. 4 CI -7.46;-0.71) p = 0.02. Conclusions: Perfusion and diffusion method combined with anatomical sequences show potential and can be used as radiomic markers for diagnostic assessment and treatment of extra-axial tumors. There is further potential in detecting radiomic functional markers from the area of perifocal changes.

Hypoxia-Induced miR-210 Overexpression Promotes the Differentiation of Human-Induced Pluripotent Stem Cells to Hepatocyte-Like Cells on Random Nanofiber Poly-L-Lactic Acid/Poly (ε-Caprolactone) Scaffolds

An alternative treatment to liver transplantation includes the use of differentiated stem cells. Hypoxia has been shown to endow human-induced pluripotent stem cells (hiPSCs) with enhanced hepatic differentiation. We have investigated a new strategy for hepatocyte differentiation from hiPSCs using a three-step differentiation protocol with lentiviral overexpression of hypoxia-microRNA-210 of cells grown on a hybrid scaffold. We analyzed the transduction of the miR-210 lentiviral and definitive endoderm and pluripotency gene markers, including SRY-box 17 (SOX17), forkhead box A2 (FOXA2), and octamer-binding transcription factor 4 (OCT-4) by Real-Time PCR and fluorescent microscope. The scanning electron microscopy (SEM) examined the 3D cell morphological changes. Immunocytochemistry staining was used together with assays for aspartate aminotransferase, alanine aminotransferase, and urea secretion to analyze hepatocyte biomarkers and functional markers consisting of α-fetoprotein (AFP), low-density lipoprotein (LDL) uptake, fat accumulation, and glycogen. The flow cytometry analyzed the generation of reactive oxygen species (ROS). Compared to cells transfected with the blank lentiviral vectors as a control, overexpressing miR-210 was at higher levels in hiPSCs. The expression of endodermal genes and glycogen synthesis significantly increased in the differentiated lentiviral miR-210 cells without any differences regarding lipid storage level. Additionally, cells containing miR-210 showed a greater expression of ALB, LDL, AST, ALT, urea, and insignificant lower AFP and ROS levels after 18 days. However, SEM showed no significant differences between cells under the differentiation process and controls. In conclusion, the differentiation of hiPSCs to hepatocyte-like cells under hypoxia miR-210 may be a suitable method for cell therapy and regenerative medicine.

Developing functional markers for vitamin E biosynthesis in oil palm

Vitamin E is essential for human health and plays positive roles in anti-oxidation. Previously, we detected large variation in vitamin E content among 161 oil palm accessions. In this study, twenty oil palm accessions with distinct variation in vitamin E contents (171.30 to 1 258.50 ppm) were selected for genetic variation analysis and developing functional markers associated with vitamin E contents. Thirty-seven homologous genes in oil palm belonging to vitamin E biosynthesis pathway were identified via BLASTP analysis, the lengths of which ranged from 426 to 25 717 bp (average 7 089 bp). Multiplex PCR sequencing for the 37 genes found 1 703 SNPs and 85 indels among the 20 oil palm accessions, with 226 SNPs locating in the coding regions. Clustering analysis for these polymorphic loci showed that the 20 oil palm accessions could be divided into five groups. Among these groups, group I included eight oil palm accessions whose vitamin E content (mean value: 893.50 ppm) was far higher than other groups (mean value 256.29 to 532.94 ppm). Correlation analysis between the markers and vitamin E traits showed that 134 SNP and 7 indel markers were significantly (p < 0.05) related with total vitamin E content. Among these functional markers, the indel EgTMT-1-24 was highly correlated with variation in vitamin E content, especially tocotrienol content. Our study identified a number of candidate function associated markers and provided clues for further research into molecular breeding for high vitamin E content oil palm.

Selection of the Salt Tolerance Gene GmSALT3 During Six Decades of Soybean Breeding in China

Salt tolerance is an important trait that affects the growth and yield of plants growing in saline environments. The salt tolerance gene GmSALT3 was cloned from the Chinese soybean cultivar Tiefeng 8, and its variation evaluated in Chinese wild soybeans and landraces. However, the potential role of GmSALT3 in cultivation, and its genetic variation throughout the history of Chinese soybean breeding, remains unknown. Here we identified five haplotypes of GmSALT3 in 279 Chinese soybean landraces using a whole genome resequencing dataset. Additionally, we developed five PCR-based functional markers: three indels and two cleaved amplified polymorphic sequences (CAPS) markers. A total of 706 Chinese soybean cultivars (released 1956–2012), and 536 modern Chinese breeding lines, were genotyped with these markers. The Chinese landraces exhibited relatively high frequencies of the haplotypes H1, H4, and H5. H1 was the predominant haplotype in both the northern region (NR) and Huanghuai region (HHR), and H5 and H4 were the major haplotypes present within the southern region (SR). In the 706 cultivars, H1, H2, and H5 were the common haplotypes, while H3 and H4 were poorly represented. Historically, H1 gradually decreased in frequency in the NR but increased in the HHR; while the salt-sensitive haplotype, H2, increased in frequency in the NR during six decades of soybean breeding. In the 536 modern breeding lines, H2 has become the most common haplotype in the NR, while H1 has remained the highest frequency haplotype in the HHR, and H5 and H1 were highest in the SR. Frequency changes resulting in geographically favored haplotypes indicates that strong selection has occurred over six decades of soybean breeding. Our molecular markers could precisely identify salt tolerant (98.9%) and sensitive (100%) accessions and could accurately trace the salt tolerance gene in soybean pedigrees. Our study, therefore, not only identified effective molecular markers for use in soybean, but also demonstrated how these markers can distinguish GmSALT3 alleles in targeted breeding strategies for specific ecoregions.

825 High-dimensional image cytometry reveals spatially organized tumor-immune microenvironment in hepatocellular carcinoma

BackgroundStructured and spatial-nuanced interactions between components in tumor microenvironment (TME) regulates the efficacy of anti-tumor regimens. Insights into this orchestrated behavior in therapeutic responders and non-responders will facilitate immunotherapies. High-multiplex imaging and spatial statistics enable deep profiling of TMEs by simultaneous arraying cell phenotypes and locations. In this study, we quantified the landscape of TMEs from neoadjuvant cabozantinib and nivolumab administered locally advanced hepatocellular carcinoma (HCC) biospecimen.Methods14 patients with HCC were treated with the combination of cabozantinib and nivolumab through the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center. Among them, 12 patients (5 responders + 7 non-responders) underwent successful margin negative resection and are subjects to tissue microarray (TMAs) construction containing 37 representative tumor region cores. Using the TMAs, we performed imaging mass cytometry (IMC) with a panel of 27-cell lineage and functional markers (figure 1). All multiplexed images were then segmented to generate a single-cell dataset that enables (1) tumor-immune compartment analysis and (2) cell community analysis based on graph-embedding technology. Results from these hierarchies are merged to response-associated biological process patterns.ResultsImage processing on 37 multiplexed images discriminated 59,453 cells and then clustered into 17 cell types. Multi-level spatial quantification revealed distinct TME arrangements across cores from responders (R) and non-responders (NR): compartment analysis showed that at immune-tumor boundaries from NR, PD-L1 level on tumor cells is significantly higher than remote regions; however, Granzyme B level is lower (figure 2B). We also identified the proximity of CD8+ T cells to a subset of macrophages – Arginase 1hi CD163- macrophages (hazard macrophage) and CD4+ T cells, is a prognostic biomarker to neoadjuvant therapy (figure 3A and 3B). In-depth cell community analysis extracted cell-cell interaction networks based on spatial proximity. Next, hierarchical clustering grouped all networks with similar components (cell types) into 8 community categories (CC). Using graph-embedding and correlation test, we observed that in NR, macrophage-enriched CC (MCC) and lymphocytes-enriched CC (LCC) are strongly communicating with tumor CC; whereas in R, such communications were weakened by the engagement between MCC and LCC (figure 3C).ConclusionsIn conclusion, we employed an unbiased, quantitative spatial analysis to determine how tumor and immune components interact in responding and nonresponding HCC tumors. Based on our results, four immune-regulating factors are derived and summarized as a communication landscape (figure 4). The proposed framework represents a novel application of multiplexed imaging in translational medicine and has potential in initialization and validation of computational immuno-oncology models.AcknowledgementsThe authors acknowledge financial support from Bristol-Myers Squibb, Exelixis, the National Cancer Institute Specialized Program of Research Excellence (SPORE) in Gastrointestinal Cancers (P50 CA062924), the Passano Foundation, the National Institutes of Health (Grant No. U01CA212007 and R01CA138264) and Emerson Collective Cancer Research Fund (640183).Ethics ApprovalThe studies involving human participants were reviewed and approved by Institutional Review Board of the Johns Hopkins Medical Institutions.ConsentWritten informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.Abstract 825 Figure 1A panel of 27 markers was used to stain the hepatocellular carcinoma tumor region cores and processed using IMC. The marker names and descriptions are includedAbstract 825 Figure 2(A) Color overlays of lineage proteins covering Pan-Keratin and CD45 (rainbow) and functional markers covering PD-L1 and Granzyme B (white) in whole tissue core and subregions. (B) and (C) Protein expression analytical strategy. For compartmentalized cores, functional marker expressions on target cells were examined adjacent and remote to tumor-immune border and truncated to treatment response criteria for comparisonsAbstract 825 Figure 3(A) Diagram of CD8+ T cell RiskScore. Denote each CD8+ T cell to its nearest hazard macrophages as d1 and to its nearest CD4+ T cell as d2, thus the RiskScore is formally computed by taking the proportion of d2 to the combined distance of d1 and d2. (B) RiskScore on per-cell basis for responders and non-responders. (C) Cell community communication maps in tumor microenvironment associated with responders and non-respondersAbstract 825 Figure 4The synergistic anti-tumor immunity of macrophages and lymphocytes favors cabozantinib and nivolumab; immune function regulators (i.e., GranB and PD-L1) were upregulated throughout the immune compartment in non-responders; close proximity to hazard macrophages and distance away from CD4+ T cells associate with poorer effector function of CD8+ T cells

763 Intratumoral delivery of high potency STING agonists modulates the immunosuppressive myeloid compartment and induces curative responses in checkpoint-refractory glioblastoma models

BackgroundGlioblastoma is an aggressive primary brain malignancy that is characterized by a highly suppressive tumor microenvironment, including myeloid-derived suppressor cells, tumor-associated macrophages, and brain-resident microglia, but lacking significant T cell infiltration.1 2 This phenotype is reflected in the recently developed QKi-/- Pten-/- P53-/- (QPP) tumor model,3 which we show is resistant to PD1 or CTLA-4 blockade, but sensitive to agonists of the innate immune sensor Stimulator of Interferon Genes (STING). We have previously shown that agonists of the innate dsDNA-sensing cGAS-STING pathway are capable of proinflammatory repolarization in in vitro models of suppressive myeloid cells, although their function in the context of the Glioblastoma myeloid compartment in vivo remains poorly understood.4MethodsWe utilized the synthetic cyclic di-nucleotide STING agonists IACS-8803 (8803) and ML-RR-S2-CDA (MLRR) to assess survival and tumor immune infiltrate functional reprogramming in two orthotopic transplantable human and murine Glioblastoma tumor models, U87 and the recently developed QPP8 (Qki-/- Pten-/- P53-/-). Using in vitro models of M2-polarized microglia, we investigated the ability of natural (2'3'-cGAMP) and synthetic (MLRR and 8803) STING agonists to reverse immunosuppressive microglial polarization.ResultsWe found that intratumoral delivery of STING agonists significantly prolonged survival in the murine QPP8 orthotopic Glioblastoma tumor model, in contrast to checkpoint blockade which had no benefit on survival. In huNOG-EXL mice engrafted with human hematopoietic stem cells implanted with orthotopic U87 Glioblastoma, intratumoral delivery of STING agonists significantly prolonged survival and reduced expression of CD163 and CD206 on human tumor-infiltrating myeloid populations. Preliminary data suggests that in vitro suppressively-polarized microglia reduce expression of M2 functional markers, and increase expression of iNOS, PD-L1, CD80, and CD86 in a STING agonist potency-dependent manner.ConclusionsWe found that STING agonists can induce curative responses in checkpoint-refractory murine Glioblastoma models and mediate significant extension of survival in a humanized mouse U87 xenograft setting. This prolonged survival is associated with a decrease in immunosuppressive M2 functional markers in human tumor infiltrating myeloid populations. Additionally, M2-polarized microglia demonstrated a reduction in M2 functional markers and upregulation of proinflammatory M1 markers following treatment with STING agonists. Together these results indicate that delivery of STING agonists can induce proinflammatory repolarization of the Glioblastoma myeloid stroma, including both infiltrating myeloid populations and brain-resident microglia, to drive prolonged survival in refractory models of Glioblastoma.ReferencesGabrusiewicz K, Rodriguez B, Wei J, et al. Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype. JCI Insight 2016;1(2).Quail DF, Joyce JA. The microenvironmental landscape of brain tumors. Cancer Cell 2017;31(3):326–41.Shingu T, Ho AL, Yuan L, et al. Qki deficiency maintains stemness of glioma stem cells in suboptimal environment by downregulating endolysosomal degradation. Nat Genet 2017;49(1):75–86.Ager C, Boda A, Rajapakshe K, et al. (2021) “High potency STING agonists engage unique myeloid pathways to reverse pancreatic cancer immune privilege. JITC (in press)Ethics ApprovalAll experiments were conducted according to protocols approved by the University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee.