Multiple Omics Uncovers Host–Gut Microbial Mutualism During Prebiotic Fructooligosaccharide Supplementation

Fructooligosaccharide (FOS), a prebiotic well known for its health-promoting properties, can improve the human gut ecosystem most likely through changes in its microbial composition. However, the detailed mechanism(s) of action of FOS in the modulation of the gut ecosystem remain(s) obscure. Traditional methods of profiling microbes and metabolites could barely show any significant features due to the existence of large interindividual differences, but our novel microbe–metabolite correlation approach, combined with faecal immunoglobulin A (IgA) measurements, has revealed that the induction of mucosal IgA by FOS supplementation correlated with the presence of specific bacteria. Furthermore, the metabolic dynamics of butyrate, l-phenylalanine, l-lysine and tyramine were positively correlated with that of these bacteria and IgA production, whereas p-cresol was negatively correlated. Taken together, our focused intraindividual analysis with omics approaches is a powerful strategy for uncovering the gut molecular network and could provide a new vista for understanding the human gut ecosystem.     

Combination of an agonistic anti-CD40 monoclonal antibody and the COX-2 inhibitor celecoxib induces anti-glioma effects by promotion of type-1 immunity in myeloid cells and T-cells

Malignant gliomas are heavily infiltrated by immature myeloid cells that mediate immunosuppression. Agonistic CD40 monoclonal antibody (mAb) has been shown to activate myeloid cells and promote antitumor immunity. Our previous study has also demonstrated blockade of cyclooxygenase-2 (COX-2) reduces immunosuppressive myeloid cells, thereby suppressing glioma development in mice. We therefore hypothesized that a combinatory strategy to modulate myeloid cells via two distinct pathways, i.e., CD40/CD40L stimulation and COX-2 blockade, would enhance anti-glioma immunity. We used three different mouse glioma models to evaluate therapeutic effects and underlying mechanisms of a combination regimen with an agonist CD40 mAb and the COX-2 inhibitor celecoxib. Treatment of glioma-bearing mice with the combination therapy significantly prolonged survival compared with either anti-CD40 mAb or celecoxib alone. The combination regimen promoted maturation of CD11b⁺ cells in both spleen and brain, and enhanced Cxcl10 while suppressing Arg1 in CD11b⁺Gr-1⁺ cells in the brain. Anti-glioma activity of the combination regimen was T-cell dependent because depletion of CD4⁺ and CD8⁺ cells in vivo abrogated the anti-glioma effects. Furthermore, the combination therapy significantly increased the frequency of CD8⁺ T-cells, enhanced IFN-γ-production and reduced CD4⁺CD25⁺Foxp3⁺ T regulatory cells in the brain, and induced tumor-antigen-specific T-cell responses in lymph nodes. Our findings suggest that the combination therapy of anti-CD40 mAb with celecoxib enhances anti-glioma activities via promotion of type-1 immunity both in myeloid cells and T-cells.     

Correlation between Plasma DNA and Tumor Status in an Animal Model

Overcoming metastasis is one of the most important issues with lung cancer. Since metastasis arises through complex steps, a suitable animal model is indispensable for investigation of metastasis. To establish an animal model reflecting human metastatic lung cancers, we used NOD/SCID/Jak3^null (NOJ) mice, which exhibit deficiencies in NK cell activity, macrophage and dendritic cell function, and complement activation, as well as T and B cell deficiencies. After screening twenty human lung cancer cell lines through expression patterns of E-cadherin and vimentin according to epithelial mesenchymal transition features, an H1975 cell line carrying EGFR mutations, L858R and T790M, was selected for investigation. Inoculation of the cells into the dorsal flanks caused systemic metastases after one month in lymph nodes, liver, lung, and peritoneum, suggesting that metastases occurred both lymphogenically and hematogenously. We confirmed the existence of H1975 cells in metastatic lesions by detection of T790M and L858R using the mutation-biased PCR and quenching probe (MBP-QP) system previously established in our laboratory. In addition, tumor-derived plasma DNA could be detected using the MBP-QP method. The amount of tumor-derived DNA was associated with tumor volume, whereas an unrelated large amount of tumor-derived DNA was circulating in the presence of metastasis. We present a novel animal model with systemic metastasis with human lung cancer cells. The amount of tumor derived DNA would be related with tumor volume and tumor progression such as metastasis.     

Circulating AIM as an Indicator of Liver Damage and Hepatocellular Carcinoma in Humans

Background

Hepatocellular carcinoma (HCC), the fifth most common cancer type and the third highest cause of cancer death worldwide, develops in different types of liver injuries, and is mostly associated with cirrhosis. However, non-alcoholic fatty liver disease often causes HCC with less fibrosis, and the number of patients with this disease is rapidly increasing. The high mortality rate and the pathological complexity of liver diseases and HCC require blood biomarkers that accurately reflect the state of liver damage and presence of HCC.

Methods and Findings

Here we demonstrate that a circulating protein, apoptosis inhibitor of macrophage (AIM) may meet this requirement. A large-scale analysis of healthy individuals across a wide age range revealed a mean blood AIM of 4.99±1.8 µg/ml in men and 6.06±2.1 µg/ml in women. AIM levels were significantly augmented in the younger generation (20s–40s), particularly in women. Interestingly, AIM levels were markedly higher in patients with advanced liver damage, regardless of disease type, and correlated significantly with multiple parameters representing liver function. In mice, AIM levels increased in response to carbon tetrachloride, confirming that the high AIM observed in humans is the result of liver damage. In addition, carbon tetrachloride caused comparable states of liver damage in AIM-deficient and wild-type mice, indicating no influence of AIM levels on liver injury progression. Intriguingly, certain combinations of AIM indexes normalized to liver marker score significantly distinguished HCC patients from non-HCC patients and thus could be applicable for HCC diagnosis.

Conclusion

AIM potently reveals both liver damage and HCC. Thus, our results may provide the basis for novel diagnostic strategies for this widespread and fatal disease.     

Stress-induced Δ1-pyrroline-5-carboxylate synthetase (P5CS) gene confers tolerance to salt stress in transgenic sugarcane

High salinity interferes in sugarcane growth and development, affecting not only crop yield but also reducing sucrose concentration in culms. Sugarcane plants submitted to salt stress can accumulate compatible solutes, such as proline, which may counteract the effects of salt accumulation in the vacuole and scavenge reactive oxygen species. The objective of this study was to evaluate the response to salt stress of sugarcane plants transformed with the Vigna aconitifolia P5CS gene, which encodes ?1-pyrroline-5-carboxylate synthetase, under the control of a stress-induced promoter AIPC (ABA-inducible promoter complex). For this, 4-month-old clonally multiplied sugarcane plants from two transformation events were irrigated every 2 days with 1/10 Hoagland’s solution supplemented with 100, 150 and 200 NaCl, progressively, during 28 days. Transgenic lines showed increased transgene expression in 3.75-fold when compared with the control plants after 9 days of irrigation with saline water, which can explain the higher proline concentration found in these plants. At the end of the experiment (day 28), the transgenic lines accumulated up to 25 % higher amounts of proline when compared with non-transformed control plants. Stress response in transgenic plants was also accompanied by a reduction of malondialdehyde (MDA) derived from cellular lipid peroxidation in leaves, lower Na⁺ accumulation in leaves and maintenance of photochemical efficiency of PSII. Thus, proline contributed to the protection of the photosynthetic apparatus and the prevention of oxidative damage in transgenic sugarcane under salt stress.     

Immature male gibbons produce female-specific songs

Gibbons are apes that are well known to produce characteristic species-specific loud calls, referred to as “songs.” Of particular interest is the sex specificity of the “great calls” heard in gibbon songs. However, little is known about the development of such calls. While great calls are given by female gibbons of various ages, they have never been recorded from males. Here, we report two observations of immature male gibbons from two different species, wild Hylobates agilis and captive H. lar, which spontaneously sang female-specific great calls. Based on the video clips, we conclude that immature males also have the potential to produce great calls. Our observations led us to propose a new hypothesis for the development of sexual differentiation in the songs of gibbons, and its implications for the general issue of sex-specific behavior in primates.     

The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I

Using rat dopaminergic and human neuroblastoma cell lines transduced with the NDI1 gene encoding the internal NADH dehydrogenase (Ndi1) from Saccharomyces cerevisiae, we investigated reactive oxygen species (ROS) generation caused by complex I inhibition. Incubation of non-transduced cells with rotenone elicited oxidative damage to mitochondrial DNA as well as lipid peroxidation. In contrast, oxidative stress was significantly decreased when the cells were transduced with NDI1. Furthermore, mitochondria from the NDI1-transduced cells showed a suppressed rate of ROS formation by the complex I inhibitors. We conclude that the Ndi1 enzyme is able to suppress ROS overproduction from defective complex I.     

Protein dolichylation in Plasmodium falciparum

We performed reverse-phase thin-layer chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of polyisoprenoids released by sulfonium-salt cleavage with methyl iodide from Plasmodium falciparum proteins labeled with [3H]FPP or [3H]GGPP and showed that a dolichol of 11 isoprene units is bound to 21-28-kDa protein clusters from trophozoite and schizont stages. The dolichol structure was confirmed by electrospray-ionization mass spectrometry analysis. Treatment with protein synthesis inhibitors and RP-HPLC analysis of the proteolytic digestion products from parasite proteins labeled with [35S]cysteine and [3H]FPP showed that the attachment of dolichol to protein is a post-translational event and probably occurs via a covalent bond to cysteine residues.     

Convenient structural analysis of glycosphingolipids using MALDI-QIT-TOF mass spectrometry with increased laser power and cooling gas flow

Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was applied to the structural characterization of neutral glycosphingolipids. Lithium adduct ions of glycosphingolipids were analyzed using MALDI-QIT-TOF MS under strong conditions of increased laser power and cooling gas flow. The relative intensities of fragment ions were increased under the strong conditions, and the resulting spectra revealed the presence of oligosaccharide ions fragmented from the glycosphingolipids. Consequently, the oligosaccharide sequences of the glycosphingolipids were readily obtained. To obtain more detailed structural information, MS/MS (MS2) and MS/MS/MS (MS3) analyses were performed with selection of the lactosylceramide and ceramide ions, respectively. The resulting data were sufficient to determine the structures of both the oligosaccharide and the ceramide moiety of each glycosphingolipid. The fragmentation patterns of MS2 and MS3 for Forssman glycolipid under the strong conditions were comparable to those of MS3 and MS4 obtained under standard conditions, respectively. Thus, MALDI-QIT-TOF MS with increased laser power and cooling gas flow is a convenient method for glycosphingolipid analysis.     

Possibility of transkingdom gene therapy for complex I diseases

Defects of complex I are involved in many human mitochondrial diseases, and therefore we have proposed to use the NDI1 gene encoding a single subunit NADH dehydrogenase of Saccharomyces cerevisiae for repair of respiratory activity. The yeast NDI1 gene was successfully introduced into mammalian cell lines. The expressed NDI1 protein was correctly targeted to the matrix side of the inner mitochondrial membranes, was fully functional and restored the NADH oxidase activity to the complex I-deficient cells. The NDI1-transduced cells were more resistant to complex I inhibitors and diminished production of reactive oxygen species induced by rotenone. It was further shown that the NDI1 protein can be functionally expressed in tissues such as skeletal muscles and the brain of rodents, which scarcely induced an inflammatory response. The use of NDI1 as a potential molecular therapy for complex I-deficient diseases is briefly discussed, including the proposed animal model.