Meta-omics analysis indicates the saliva microbiome and its proteins associated with the prognosis of oral cancer patients

Saliva is a biofluid that maintains the health of oral tissues and the homeostasis of oral microbiota. Studies have demonstrated that Oral squamous cell carcinoma (OSCC) patients have different salivary microbiota than healthy individuals. However, the relationship between these microbial differences and clinicopathological outcomes is still far from conclusive. Herein, we investigate the capability of using metagenomic and metaproteomic saliva profiles to distinguish between Control (C), OSCC without active lesion (L0), and OSCC with active lesion (L1) patients. The results show that there are significantly distinct taxonomies and functional changes in L1 patients compared to C and L0 patients, suggesting compositional modulation of the oral microbiome, as the relative abundances of Centipeda, Veillonella, and Gemella suggested by metagenomics are correlated with tumor size, clinical stage, and active lesion. Metagenomics results also demonstrated that poor overall patient survival is associated with a higher relative abundance of Stenophotromonas, Staphylococcus, Centipeda, Selenomonas, Alloscordovia, and Acitenobacter. Finally, compositional and functional differences in the saliva content by metaproteomics analysis can distinguish healthy individuals from OSCC patients. In summary, our study suggests that oral microbiota and their protein abundance have potential diagnosis and prognosis value for oral cancer patients. Further studies are necessary to understand the role of uniquely detected metaproteins in the microbiota of healthy and OSCC patients as well as the crosstalk between saliva host proteins and the oral microbiome present in OSCC.     

Surfactant disaturated-phosphatidylcholine kinetics in acute respiratory distress syndrome by stable isotopes and a two compartment model

Background

In patients with acute respiratory distress syndrome (ARDS), it is well known that only part of the lungs is aerated and surfactant function is impaired, but the extent of lung damage and changes in surfactant turnover remain unclear. The objective of the study was to evaluate surfactant disaturated-phosphatidylcholine turnover in patients with ARDS using stable isotopes.

Methods

We studied 12 patients with ARDS and 7 subjects with normal lungs. After the tracheal instillation of a trace dose of ¹³C-dipalmitoyl-phosphatidylcholine, we measured the ¹³C enrichment over time of palmitate residues of disaturated-phosphatidylcholine isolated from tracheal aspirates. Data were interpreted using a model with two compartments, alveoli and lung tissue, and kinetic parameters were derived assuming that, in controls, alveolar macrophages may degrade between 5 and 50% of disaturated-phosphatidylcholine, the rest being lost from tissue. In ARDS we assumed that 5–100% of disaturated-phosphatidylcholine is degraded in the alveolar space, due to release of hydrolytic enzymes. Some of the kinetic parameters were uniquely determined, while others were identified as lower and upper bounds.

Results

In ARDS, the alveolar pool of disaturated-phosphatidylcholine was significantly lower than in controls (0.16 ± 0.04 vs. 1.31 ± 0.40 mg/kg, p < 0.05). Fluxes between tissue and alveoli and de novo synthesis of disaturated-phosphatidylcholine were also significantly lower, while mean resident time in lung tissue was significantly higher in ARDS than in controls. Recycling was 16.2 ± 3.5 in ARDS and 31.9 ± 7.3 in controls (p = 0.08).

Conclusion

In ARDS the alveolar pool of surfactant is reduced and disaturated-phosphatidylcholine turnover is altered.     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

Thermal adaptation strategies of the extremophile bacterium Thermus filiformis based on multi-omics analysis

Extremophiles - Thermus filiformis is an aerobic thermophilic bacterium isolated from a hot spring in New Zealand. The experimental study of the mechanisms of thermal adaptation is important to...     

Surfactant disaturated phosphatidylcholine kinetics in infants with bronchopulmonary dysplasia measured with stable isotopes and a two-compartment model.

We previously found a shorter surfactant disaturated phosphatidylcholine palmitate (DSPC-PA) half-life in infants with bronchopulmonary dysplasia (BPD) by using a single stable isotope tracer and simple formulas based on a one-exponential fit of the final portion of the enrichment decay curve. The aim of this study was to apply noncompartmental and compartmental analysis on the entire enrichment decay curve of DSPC-PA and to compare the kinetic data with our previous results. We analyzed 10 preterm newborns with BPD (gestational age 26 +/- 0.6 wk, weight 777 +/- 199 g) and 6 controls (gestational age 26 +/- 1.4 wk, weight 787 +/- 259 g). All took part in our previous study. Endotracheal 13C-labeled dipalmitoyl phosphatidylcholine was administered, and the 13C-enrichment of surfactant DSPC-PA was measured from serial tracheal aspirates by gas chromatography-mass spectrometry. Noncompartmental and compartmental models were numerically identified from the tracer-to-tracee ratio and kinetic parameters related to the accessible (pool accessible to sampling, likely to be the lung alveolar pool) and to the nonaccessible pools (pools not accessible to samplings, likely to be the intracellular storage pool) were estimated in the two study groups. Comparison was performed by Mann-Whitney test. A two-compartment model provided the most reliable assessment of DSPC-PA kinetics. In BPD vs. controls, mean +/- SE residence time of DSPC-PA in the accessible was 17.5 +/- 2.6 vs. 32.2 +/- 6.4 h (P < 0.05), whereas it was 49.7 +/- 3.5 vs. 54.4 +/- 3.9 h (NS, not significant) in the nonaccessible pool; DSPC-PA recycling was 0.26 +/- 0.05 vs. 0.43 +/- 0.04% (NS), respectively. A two-compartment model of surfactant DSPC-PA kinetics allowed a thorough assessment of DSPC-PA kinetics, including masses, synthesis, and fluxes between pools. The most important findings of this study are that in BPD infants DSPC-PA loss from the alveolar pool was higher and recycling through the intracellular pool lower than in controls.     

Does Whole-Body Hypothermia in Neonates with Hypoxic–Ischemic Encephalopathy Affect Surfactant Disaturated-Phosphatidylcholine Kinetics?

BackgroundIt is unknown whether Whole-Body Hypothermia (WBH) affects pulmonary function. In vitro studies, at relatively low temperatures, suggest that hypothermia may induce significant changes to the surfactant composition. The effect of WBH on surfactant kinetics in newborn infants is unknown. We studied in vivo kinetics of disaturated-phosphatidylcholine (DSPC) in asphyxiated newborns during WBH and in normothermic controls (NTC) with no or mild asphyxia. Both groups presented no clinically apparent lung disease.MethodsTwenty-seven term or near term newborns requiring mechanical ventilation were studied (GA 38.6±2.2 wks). Fifteen during WBH and twelve NTC. All infants received an intra-tracheal dose of ¹³C labelled DSPC and tracheal aspirate were performed. DSPC amount, DSPC half-life (HL) and pool size (PS) were calculated.ResultsDSPC amount in tracheal aspirates was 0.42 [0.22–0.54] and 0.36 [0.10–0.58] mg/ml in WBH and NTC respectively (p = 0.578). DSPC HL was 24.9 [15.7–52.5] and 25.3 [15.8–59.3] h (p = 0.733) and DSPC PS was 53.2 [29.4–91.6] and 40.2 [29.8–64.6] mg/kg (p = 0.598) in WBH and NTC respectively.ConclusionsWBH does not alter DSPC HL and PS in newborn infants with no clinical apparent lung disease.     

Agrin and Perlecan Mediate Tumorigenic Processes in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.     

In Vivo Effect of Pneumonia on Surfactant Disaturated-Phosphatidylcholine Kinetics in Newborn Infants

Background

Bacterial pneumonia in newborns often leads to surfactant deficiency or dysfunction, as surfactant is inactivated or its production/turnover impaired. No data are available in vivo in humans on the mechanism of surfactant depletion in neonatal pneumonia. We studied the kinetics of surfactant''s major component, disaturated-phosphatidylcholine (DSPC), in neonatal pneumonia, and we compared our findings with those obtained from control newborn lungs.

Methods

We studied thirty-one term or near-term newborns (gestational age 39.7±1.7 weeks, birth weight 3185±529 g) requiring mechanical ventilation. Fifteen newborns had pneumonia, while 16 newborns were on mechanical ventilation but had no lung disease. Infants received an intratracheal dose of ¹³C labeled dipalmitoyl-phosphatidylcholine at the study start. We measured the amount and the isotopic enrichment of DSPC-palmitate from serial tracheal aspirates by gas chromatography and gas chromatography-mass spectrometry, respectively, and we calculated the DSPC half-life (HL) and pool size (PS) from the isotopic enrichment curves of surfactant DSPC-palmitate.

Results

The mean DSPC amount obtained from all tracheal aspirates did not differ between the two groups. DSPC HL was 12.7 (6.5–20.2) h and 25.6 (17.9–60.6) h in infants with pneumonia compared with control infants (p = 0.003). DSPC PS was 14.1 (6.6–30.9) mg/kg in infants with pneumonia and 34.1 (25.6–65.0) mg/kg in controls, p = 0.042. Myeloperoxidase (MPO) activity, as a marker of lung inflammation, was 1322 (531–2821) mU/ml of Epithelial Lining Fluid (ELF) and 371(174–1080) mU/ml ELF in infants with pneumonia and in controls, p = 0.047. In infants with pneumonia, DSPC PS and HL significantly and inversely correlated with mean Oxygenation Index (OI) during the study (DSPC PS vs. OI R = −0.710, p = 0.004 and HL vs. OI R = −0.525, p = 0.044, respectively).

Conclusions

We demonstrated for the first time in vivo in humans that DSPC HL and PS were markedly impaired in neonatal pneumonia and that they inversely correlated with the degree of respiratory failure.     

Metabolic precursors of surfactant disaturated-phosphatidylcholine in preterms with respiratory distress

Our objective was to study the metabolic precursors of surfactant disaturated-phosphatidylcholine (DSPC) in preterm infants with respiratory distress syndrome (RDS) on mechanical ventilation. We performed 46 DSPC kinetic studies in 23 preterms on fat-free parenteral nutrition and mechanical ventilation (birth weight = 1167 ± 451 g, gestational age = 28.5 ± 2.0 weeks). Eight infants received a simultaneous intravenous infusion of U¹³C-glucose and [16,16,16]²H-palmitate, eight infants received U¹³C-glucose and ²H₂O, and seven received U¹³C-palmitate and ²H₂O. Surfactant DSPC kinetics were calculated from the isotopic enrichments of DSPC-palmitate from sequential tracheal aspirates and its metabolic precursors in plasma or urine. DSPC fractional synthesis rate (FSR) was 17 ± 11, 21 ± 16, and 15 ± 6%/day from glucose, palmitate, and body water, respectively (P = 0.36). DSPC-FSR from U¹³C-glucose and ²H₂O were significantly correlated and yielded similar estimates (difference of –0.1 ± 3%) (P = 0.91). The difference in the 15 infants receiving palmitate versus ²H₂O or palmitate versus glucose was +6.0 ± 12%/day (P = 0.21). There was a significant correlation between DSPC-FSRs from plasma glucose and plasma FFA. The contribution of glucose versus palmitate to DSPC-FSR was 49 ± 20% versus 51 ± 20%, respectively. Plasma glucose and FFA showed similar contributions to DSPC-FSR in infants with RDS and fat-free parenteral nutrition. FSRs from ²H₂O or glucose were highly correlated.