Genomic, phenotypic, and clinical safety of Limosilactobacillus reuteri ATCC PTA 4659.

Evaluating the safety of probiotic microorganisms is an important part of the development of probiotic products. In this study, we have performed a systematic safety assessment of Limosilactobacillus reuteri American Type Culture Collection (ATCC) PTA 4659 based on genome analysis, antibiotic susceptibility testing, phenotypic characterization, and a human clinical safety study. Genome sequence analysis showed that the strain is free from virulence and antibiotic resistance genes. Connected to this, phenotypic characterization showed that the strain is susceptible to the main classes of antibiotics. Limosilactobacillus reuteri ATCC PTA 4659 was shown to produce histamine, which has previously been described as an anti-inflammatory mediator produced by certain L. reuteri strains. However, the amount of histamine, a biogenic amine, poses no safety concern of a potential product. The strain was investigated in a human clinical safety study and was shown to survive passage through the gastrointestinal tract, both when administered at high [1 × 1011 colony-forming units (CFU)/day] and low doses (1 × 109 CFU/day). The clinical safety evaluation showed that the doses administered are safe for human consumption. Furthermore, carbohydrate utilization, mucus adhesion, and tolerance to acid and bile were studied. It was shown that L. reuteri ATCC PTA 4659 has a very high adhesion to mucus and tolerance to both gastric pH and bile, all potentially important properties for a probiotic strain. Altogether, this study has demonstrated that Limosilactobacillus reuteri ATCC PTA 4659 is safe for human consumption and along with its phenotypic characteristics and previously described anti-inflammatory effects, makes it a promising strain for future probiotic development. NCT01033539.

Development of a machine learning framework for radiation biomarker discovery and absorbed dose prediction.

Background: Molecular radiation biomarkers are an emerging tool in radiation research with applications for cancer radiotherapy, radiation risk assessment, and even human space travel. However, biomarker screening in genome-wide expression datasets using conventional tools is time-consuming and underlies analyst (human) bias. Machine Learning (ML) methods can improve the sensitivity and specificity of biomarker identification, increase analytical speed, and avoid multicollinearity and human bias. Aim: To develop a resource-efficient ML framework for radiation biomarker discovery using gene expression data from irradiated normal tissues. Further, to identify biomarker panels predicting radiation dose with tissue specificity. Methods: A strategic search in the Gene Expression Omnibus database identified a transcriptomic dataset (GSE44762) for normal tissues radiation responses (murine kidney cortex and medulla) suited for biomarker discovery using an ML approach. The dataset was pre-processed in R and separated into train and test data subsets. High computational cost of Genetic Algorithm/k-Nearest Neighbor (GA/KNN) mandated optimization and 13 ML models were tested using the caret package in R. Biomarker performance was evaluated and visualized via Principal Component Analysis (PCA) and dose regression. The novelty of ML-identified biomarker panels was evaluated by literature search. Results: Caret-based feature selection and ML methods vastly improved processing time over the GA approach. The KNN method yielded overall best performance values on train and test data and was implemented into the framework. The top-ranking genes were Cdkn1a, Gria3, Mdm2 and Plk2 in cortex, and Brf2, Ccng1, Cdkn1a, Ddit4l, and Gria3 in medulla. These candidates successfully categorized dose groups and tissues in PCA. Regression analysis showed that correlation between predicted and true dose was high with R2 of 0.97 and 0.99 for cortex and medulla, respectively. Conclusion: The caret framework is a powerful tool for radiation biomarker discovery optimizing performance with resource-efficiency for broad implementation in the field. The KNN-based approach identified Brf2, Ddit4l, and Gria3 mRNA as novel candidates that have been uncharacterized as radiation biomarkers to date. The biomarker panel showed good performance in dose and tissue separation and dose regression. Further training with larger cohorts is warranted to improve accuracy, especially for lower doses.

Sevoflurane promotes premature differentiation of dopaminergic neurons in hiPSC-derived midbrain organoids.

Background: Conventional animal models used in corresponding basic studies are distinct from humans in terms of the brain's development trajectory, tissue cytoarchitecture and cell types, making it difficult to accurately evaluate the potential adverse effects of anesthetic treatments on human fetal brain development. This study investigated the effects of sevoflurane on the midbrain's development and cytopathology using human physiologically-relevant midbrain organoids. Methods: Monolayer human induced pluripotent stem cells (hiPSC)-derived human floor plate cells and three-dimensional hiPSC-derived midbrain organoids (hMBOs) were exposed to 2% (v/v) sevoflurane for 2 or 6 h, followed by expansion or differentiation culture. Then, immunofluorescence, real-time PCR, EdU assay, Tunnel assay, and transcriptome sequencing were performed to examine the effects of sevoflurane on the midbrain's development. Results: We found that 2% sevoflurane exposure inhibited hFPCs' proliferation (differentiation culture: 7.2% ± 0.3% VS. 13.3% ± 0.7%, p = 0.0043; expansion culture: 48% ± 2.2% VS. 35.2% ± 1.4%, p = 0.0002) and increased their apoptosis, but did not affect their differentiation into human dopaminergic neurons After 6 h, 2% sevoflurane exposure inhibited cell proliferation (62.8% ± 5.6% VS. 100% ± 5.5%, p = 0.0065) and enhanced the premature differentiation of hMBOs (246% ± 5.2% VS. 100% ± 28%, p = 0.0065). The RNA-seq results showed long-term exposure to sevoflurane up regulates some transcription factors in the differentiation of dopaminergic neurons, while short-term exposure to sevoflurane has a weak up-regulation effect on these transcription factors. Conclusion: This study revealed that long-term exposure to sevoflurane could promote the premature differentiation of hMBOs, while short-term exposure had negligible effects, suggesting that long-term exposure to sevoflurane in pregnant women may lead to fetals' midbrain development disorder.

Novel insights into the disease transcriptome of human diabetic glomeruli and tubulointerstitium.

BACKGROUND: Diabetic nephropathy (DN) is the most common cause of end-stage renal disease, affecting ∼30% of the rapidly growing diabetic population, and strongly associated with cardiovascular risk. Despite this, the molecular mechanisms of disease remain unknown. METHODS: RNA sequencing (RNAseq) was performed on paired, micro-dissected glomerular and tubulointerstitial tissue from patients diagnosed with DN [n = 19, 15 males, median (range) age: 61 (30-85) years, chronic kidney disease stages 1-4] and living kidney donors [n = 20, 12 males, median (range) age: 56 (30-70) years]. RESULTS: Principal component analysis showed a clear separation between glomeruli and tubulointerstitium transcriptomes. Differential expression analysis identified 1550 and 4530 differentially expressed genes, respectively (adjusted P < 0.01). Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses highlighted activation of inflammation and extracellular matrix (ECM) organization pathways in glomeruli, and immune and apoptosis pathways in tubulointerstitium of DN patients. Specific gene modules were associated with renal function in weighted gene co-expression network analysis. Increased messengerRNA (mRNA) expression of renal damage markers lipocalin 2 (LCN) and hepatitis A virus cellular receptor1 (HAVCR1) in the tubulointerstitial fraction was observed alongside higher urinary concentrations of the corresponding proteins neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in DN patients. CONCLUSIONS: Here we present the first RNAseq experiment performed on paired glomerular and tubulointerstitial samples from DN patients. We show that prominent disease-specific changes occur in both compartments, including relevant cellular processes such as reorganization of ECM and inflammation (glomeruli) as well as apoptosis (tubulointerstitium). The results emphasize the potential of utilizing high-throughput transcriptomics to decipher disease pathways and treatment targets in this high-risk patient population.

Identification of cell and disease specific microRNAs in glomerular pathologies.

MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression in physiological processes as well as in diseases. Currently miRs are already used to find novel mechanisms involved in diseases and in the future, they might serve as diagnostic markers. To identify miRs that play a role in glomerular diseases urinary miR-screenings are a frequently used tool. However, miRs that are detected in the urine might simply be filtered from the blood stream and could have been produced anywhere in the body, so they might be completely unrelated to the diseases. We performed a combined miR-screening in pooled urine samples from patients with different glomerular diseases as well as in cultured human podocytes, human mesangial cells, human glomerular endothelial cells and human tubular cells. The miR-screening in renal cells was done in untreated conditions and after stimulation with TGF-ß. A merge of the detected regulated miRs led us to identify disease-specific, cell type-specific and cell stress-induced miRs. Most miRs were down-regulated following the stimulation with TGF-ß in all cell types. Up-regulation of miRs after TGF-ß was cell type-specific for most miRs. Furthermore, urinary miRs from patients with different glomerular diseases could be assigned to the different renal cell types. Most miRs were specifically regulated in one disease. Only miR-155 was up-regulated in all disease urines compared to control and therefore seems to be rather unspecific. In conclusion, a combined urinary and cell miR-screening can improve the interpretation of screening results. These data are useful to identify novel miRs potentially involved in glomerular diseases.

Amplification of the Melanocortin-1 Receptor in Nephrotic Syndrome Identifies a Target for Podocyte Cytoskeleton Stabilization.

The melanocortin-1 receptor (MC1R) in podocytes has been suggested as the mediator of the ACTH renoprotective effect in patients with nephrotic syndrome with the mechanism of action beeing stabilization of the podocyte actin cytoskeleton. To understand how melanocortin receptors are regulated in nephrotic syndrome and how they are involved in restoration of filtration barrier function, melanocortin receptor expression was evaluated in patients and a rat model of nephrotic syndrome in combination with cell culture analysis. Phosphoproteomics was applied and identified MC1R pathways confirmed using biochemical analysis. We found that glomerular MC1R expression was increased in nephrotic syndrome, both in humans and in a rat model. A MC1R agonist protected podocytes from protamine sulfate induced stress fiber loss with the top ranked phoshoproteomic MC1R activated pathway beeing actin cytoskeleton signaling. Actin stabilization through the MC1R consisted of ERK1/2 dependent phosphorylation and inactivation of EGFR signaling with stabilization of synaptopodin and stressfibers in podocytes. These results further explain how patients with nephrotic syndrome show responsiveness to MC1R receptor activation by decreasing EGFR signaling and as a consequence restore filtration barrier function by stabilizing the podocyte actin cytoskeleton.

Transcriptomic and Proteomic Profiling Provides Insight into Mesangial Cell Function in IgA Nephropathy.

IgA nephropathy (IgAN), the most common GN worldwide, is characterized by circulating galactose-deficient IgA (gd-IgA) that forms immune complexes. The immune complexes are deposited in the glomerular mesangium, leading to inflammation and loss of renal function, but the complete pathophysiology of the disease is not understood. Using an integrated global transcriptomic and proteomic profiling approach, we investigated the role of the mesangium in the onset and progression of IgAN. Global gene expression was investigated by microarray analysis of the glomerular compartment of renal biopsy specimens from patients with IgAN (n=19) and controls (n=22). Using curated glomerular cell type-specific genes from the published literature, we found differential expression of a much higher percentage of mesangial cell-positive standard genes than podocyte-positive standard genes in IgAN. Principal coordinate analysis of expression data revealed clear separation of patient and control samples on the basis of mesangial but not podocyte cell-positive standard genes. Additionally, patient clinical parameters (serum creatinine values and eGFRs) significantly correlated with Z scores derived from the expression profile of mesangial cell-positive standard genes. Among patients grouped according to Oxford MEST score, patients with segmental glomerulosclerosis had a significantly higher mesangial cell-positive standard gene Z score than patients without segmental glomerulosclerosis. By investigating mesangial cell proteomics and glomerular transcriptomics, we identified 22 common pathways induced in mesangial cells by gd-IgA, most of which mediate inflammation. The genes, proteins, and corresponding pathways identified provide novel insights into the pathophysiologic mechanisms leading to IgAN.

Mesangial cells from patients with IgA nephropathy have increased susceptibility to galactose-deficient IgA1.

BACKGROUND: IgA nephropathy (IgAN) is the most common glomerulonephritis in the world, affecting close to a million people. Circulating galactose-deficient IgA (gd-IgA), present in patients with IgAN, form immune complex deposits in the glomerular mesangium causing local proliferation and matrix expansion. Intriguing though, individuals having gd-IgA deposits in the kidneys do not necessarily have signs of glomerular disease. Recurrence of IgAN only occurs in less than half of transplanted patients with IgAN, indicating that gd-IgA is not the only factor driving the disease. We hypothesize that, in addition to IgA complexes, patients with IgAN possess a subtype of mesangial cells highly susceptible to gd-IgA induced cell proliferation. METHODS: To test the hypothesis, we designed a technique to culture primary mesangial cells from renal biopsies obtained from IgAN patients and controls. The cell response to gd-IgA treatment was then measured both on gene and protein level and the proliferation rate of the cells in response to PDGF was investigated. RESULTS: When treated with gd-IgA, mesangial cells from patients with IgAN express and release more PDGF compared to controls. In addition, the mesangial cells from patients with IgAN were more responsive to treatment with PDGF resulting in an increased proliferation rate of the cells compared to control. Mesangial cells cultured from patients with IgAN expressed and released more IL-6 than controls and had a higher expression of matrix genes. Both mesangial cells derived from patients with IgAN and controls increased their expressed TGFß1 and CCL5 when treated with gd-IgA. CONCLUSION: We conclude that mesangial cells derived from IgAN patients have a mesangioproliferative phenotype with increased reactivity to IgA and that these cellular intrinsic properties may be important for the development of IgA nephropathy.