Hepatotoxicity in Patients with Hepatocellular Carcinoma on Treatment with Immune Checkpoint Inhibitors

Risk factors for hepatic immune-related adverse events (HIRAEs) in patients with advanced/unresectable hepatocellular carcinoma (HCC) treated with immune checkpoint inhibitors (ICIs) are unclear. We investigated: (i) clinical and morpho-pathological predictors of HIRAEs in 27 pretreatment tumor specimens, including surrogate biomarkers of the HCC immune class (based on intratumoral tertiary lymphoid structures, and glutamine synthase, CD3, and CD79 expression); and (ii) the relationship between HIRAE onset and subsequent treatment outcomes. Fifty-eight patients were included—20 (34%) received ICIs alone, and 38 (66%) received ICIs plus targeted agents as first- or further-line treatment. After a median time of 0.9 months (range, 0.4–2.7), nine patients (15.5%) developed grade ≥ 3 hepatitis, which was significantly associated with higher baseline ALT levels (p = 0.037), and an infectious HCC etiology (p = 0.023). ICIs were safely resumed in six out of nine patients. Time to treatment failure (TTF) was not significantly different in patients developing grade ≥ 3 hepatitis vs. lower grades (3.25 vs. 3.91 months, respectively; p = 0.81). Biomarker surrogates for the HCC immune class were not detected in patients developing grade ≥ 3 hepatitis. Grade ≥ 3 hepatitis has a benign course that does not preclude safe ICI reintroduction, without any detrimental effect on TTF.

Enhancing glutamine production by optimising the GS-GOGAT pathway in Corynebacterium glutamicum and NH4+-limited fermentation

The GS-GOGAT pathway is a key metabolic pathway of glutamate and glutamine. Optimising this pathway, leading to metabolic flux to glutamine, can increase glutamine production and reduce the production of the by-product glutamate. The NH-limited fermentation process limits the concentration of NH to increase the activity of GS and further increase the yield of glutamine. The GS-GOGAT pathway was optimised by knocking out the GOGAT genes NCgl0181 and NCgl0182 and the glutaminase genes NCgl2395 and NCgl2500 and by integrating a copy of the GS gene glnAbsu from Bacillus subtilis and a copy of the glutamine synthase gene glnAlcb from Lactobacillus acidophilus into the genomic NCgl0182 and NCgl2500 sites. Furthermore, the pXT01 plasmid with the strong promoter tuf was used to overexpress glnAbsu and glnAlcb. To obtain an optimal NH-limited fermentation process, the effects of starting feeding with (NH)SO at different times of fermentation and three (NH)SO feeding strategies on glutamine fermentation were studied, and a NH-limited fermentation process that was the most suitable for glutamine fermentation was determined. After optimising the GS-GOGAT pathway, Corynebacterium glutamicum G-6 was subjected to the NH-limited fermentation process to greatly increase the production of glutamine. The yield of glutamine reached 98.7 g/L, which was 104.8% higher than that in the original strain GM34; the content of glutamate reached 4.5 g/L, which then decreased by 85.2%; the GS activity increased significantly, and the sugar-acid conversion rate reached 41.2%.

Enhancing glutamine production by optimising the GS-GOGAT pathway in Corynebacterium glutamicum and NH4+-limited fermentation

The GS-GOGAT pathway is a key metabolic pathway of glutamate and glutamine. Optimising this pathway, leading to metabolic flux to glutamine, can increase glutamine production and reduce the production of the by-product glutamate. The NH-limited fermentation process limits the concentration of NH to increase the activity of GS and further increase the yield of glutamine. The GS-GOGAT pathway was optimised by knocking out the GOGAT genes NCgl0181 and NCgl0182 and the glutaminase genes NCgl2395 and NCgl2500 and by integrating a copy of the GS gene glnAbsu from Bacillus subtilis and a copy of the glutamine synthase gene glnAlcb from Lactobacillus acidophilus into the genomic NCgl0182 and NCgl2500 sites. Furthermore, the pXT01 plasmid with the strong promoter tuf was used to overexpress glnAbsu and glnAlcb. To obtain an optimal NH-limited fermentation process, the effects of starting feeding with (NH)SO at different times of fermentation and three (NH)SO feeding strategies on glutamine fermentation were studied, and a NH-limited fermentation process that was the most suitable for glutamine fermentation was determined. After optimising the GS-GOGAT pathway, Corynebacterium glutamicum G-6 was subjected to the NH-limited fermentation process to greatly increase the production of glutamine. The yield of glutamine reached 98.7 g/L, which was 104.8% higher than that in the original strain GM34; the content of glutamate reached 4.5 g/L, which then decreased by 85.2%; the GS activity increased significantly, and the sugar-acid conversion rate reached 41.2%.

DIFFERENT NITRATE AND AMMONIUM LEVELS MEDIA ON CHANGES OF NITROGEN ASSIMILATION ENZYMES IN RICE

Nitrogen (N) is an important nutrient for the growth and development of rice plants, required in large quantity and often limiting factor of rice yields. The research was to understand the different sources and levels of nitrogen in rice plant on the activity of N assimilation enzymes, including nitrate reductase (NR), glutamine synthase (GS) content, glutamate synthase (Gogat) content, content, ammonium (NH4+) and nitrate (NO3-) content on the leaves. Paddy (Ciherang variety) was grown in sand media containing Hoagland solution with different sources (ammonium and nitrate) and levels (0.4, 0.8, 1.6, 3.2, 6.4, and 12.8 mM) of nitrogen. Nitrogen assimilation was observed from leaves at one month of age. The NR activity increased on both Nitrogen sources, it was a higher activity in media contained nitrate. Also, the activity of GS showed higher in media contains nitrate, but its activity was decreased after application 1.6 mM of nitrate and 3.2 mM of ammonium. Western blot analysis of GS1 and GS2 showed that the band pattern of protein was similar to these enzyme activities. Nitrate content in leaves gradually increased in both sources of nitrogen and higher than 3.2 mM ammonium application caused an increase in ammonium content in leaves, but the nitrate content decreased. This research resulted that the available source of N for rice was in nitrate form, easily by the rice plants during the growth stage.

Kinetic properties of glutamate metabolism in the nematode parasite Haemonchus contortus (L3)

The key steps in cell metabolism of all organisms are the synthesis of both glutamate and glutamine because they denote the only means of incorporating inorganic nitrogen into carbon backbones. In this study, an assay for the activity of two key enzymes in nitrogen metabolisms such as glutamate dehydrogenase (GDH) and glutamine synthase (GOGAT) was conducted using homogenates of L3 larvae of Haemonchus contortus. GDH was assayed both in the direction of glutamate utilisation and glutamate formation. GOGAT activity was monitored in the direction of glutamine utilisation. The present result showed that H.contortus had a high Km for ammonia (27.22mM) and glutamine (15.04 mM). The high Km for ammonia suggests a very low affinity for ammonia, meaning that in the reversible amination of 2-oxoglutarate to glutamate, the predominant direction is likely to be glutamate deamination and not the incorporation of ammonia. The activity of GOGAT was also demonstrated but with a high Km, which indicates a low binding affinity of glutamine to the enzyme. Nevertheless, the presence of the two key enzymes of nitrogen metabolism, i.e. GDH and GOGAT, may provide a potential target for anthelmintic action.

Phylogenetic Analysis, Vegetative Compatibility, Virulence, and Fungal Filtrates of Leaf Curl Pathogen Colletotrichum fioriniae From Celery

Leaf curl of celery, caused by Colletotrichum acutatum sensu lato, has been reported in the U.S. A multi-locus phylogenetic analysis with three genes was conducted with a collection of isolates from celery (23) and non-celery (29) hosts to evaluate their taxonomic position within C. acutatum sensu lato. The three DNA regions used for phylogenetic analysis included the introns of glutamine synthase (GS) and glyceraldehyde-3-phosphate dehydrogenase (GPDH), and the partial sequence of the histone3 (his3) gene. Moreover, celery and non-celery isolates were evaluated for vegetative compatibility and pathogenicity on celery. Culture filtrates from celery and non-celery isolates were also evaluated for their ability to reproduce leaf curl symptoms. A total of 23 celery isolates were evaluated based on phylogenetic analysis, which showed that all celery isolates were closely related and belonged to the newly described species C. fioriniae. The celery isolates were grouped into six vegetative compatibility groups indicating the population was not clonal. Twenty two of 23 isolates of C. fioriniae from celery and other hosts (26 of 29) caused leaf curl symptoms. Isolates of C. acutatum, C. nymphaeae, and C. godetiae were pathogenic but did not cause leaf curl symptoms. Isolates of C. lupini, C. johnstonii, and C. gloeosporioides were not pathogenic on celery. In addition, cell-free fungal culture filtrates caused leaf curl symptoms on celery indicating that certain isolates produce a metabolite that can cause leaf curl symptoms on celery, possibly indole acetic acid (IAA).

Deletion of P2X7 Receptor Decreases Basal Glutathione Level by Changing Glutamate-Glutamine Cycle and Neutral Amino Acid Transporters

Glutathione (GSH) is an endogenous tripeptide antioxidant that consists of glutamate-cysteine-glycine. GSH content is limited by the availability of glutamate and cysteine. Furthermore, glutamine is involved in the regulation of GSH synthesis via the glutamate–glutamine cycle. P2X7 receptor (P2X7R) is one of the cation-permeable ATP ligand-gated ion channels, which is involved in neuronal excitability, neuroinflammation and astroglial functions. In addition, P2X7R activation decreases glutamate uptake and glutamine synthase (GS) expression/activity. In the present study, we found that P2X7R deletion decreased the basal GSH level without altering GSH synthetic enzyme expressions in the mouse hippocampus. P2X7R deletion also increased expressions of GS and ASCT2 (a glutamine:cysteine exchanger), but diminished the efficacy of N-acetylcysteine (NAC, a GSH precursor) in the GSH level. SIN-1 (500 μM, a generator nitric oxide, superoxide and peroxynitrite), which facilitates the cystine–cysteine shuttle mediated by xCT (a glutamate/cystein:cystine/NAC antiporter), did not affect basal GSH concentration in WT and P2X7R knockout (KO) mice. However, SIN-1 effectively reduced the efficacy of NAC in GSH synthesis in WT mice, but not in P2X7R KO mice. Therefore, our findings indicate that P2X7R may be involved in the maintenance of basal GSH levels by regulating the glutamate–glutamine cycle and neutral amino acid transports under physiological conditions, which may be the defense mechanism against oxidative stress during P2X7R activation.

Detection of microbial DNA in the blood of colorectal cancer patients for the diagnosis of microbial translocation.

171 Background: Dysbiosis has been associated with diseases and is of a major public health importance. Can lead to the passage of viable bacteria, their products or their fragments from the intestinal lumen through the mesenteric lymph nodes and other sites, known as bacterial translocation. The aim of the study was to determine whether microbial translocation occurs in stage II/III-IV colorectal cancer (CRC) patients and evaluate the usefulness of blood PCR for diagnosis of such translocation. Also to correlate the presence of Toll-Like Receptor and Vitamin D Receptor polymorphisms with the detection of microbial DNA fragments in the blood of CRC patients. Methods: Peripheral blood was obtained from 397 CRC patients (adjuvant n = 202 and metastatic n = 195) and 32 healthy individuals. DNA from all subjects was analyzed using PCR for amplification of genomic DNA encoding 16S rRNA, β-galactosidase gene of E. coli, Glutamine synthase gene of B. fragilis and 5.8S rRNA found in C. albicans. Results: Significantly higher rates of 16S rRNA, β-galactosidase, Glutamine synthase and 5.8S rRNA detection was observed in the pool of CRC patients than the controls ( p< 0.001). All microbial DNA fragments detected were also significantly associated with the metastatic disease ( p< 0.001) leading to shorter survival rates ( p< 0.001). Moreover, individuals with the homozygous mutant alleles of either TLR or VDR gene polymorphisms had significantly higher detection rates of microbial DNA fragments. Conclusions: The detection of microbial DNA fragments in patients with CRC highlights the role of these microbes in cancer development, progression and therefore in patients’ survival.

Effects of Brassinosteroids on Photosynthetic Performance and Nitrogen Metabolism in Pepper Seedlings under Chilling Stress

To investigate the effects of brassinosteroids on plant growth and nitrogen metabolism in pepper seedlings under chilling stress, pepper seedlings with three true leaves were foliar pretreated with 0.1 μM exogenous 24-epibrassinolide (EBR) before carrying out chilling stress for 7 days. The results showed that perapplication of EBR mitigated the chill-induced decrease in plant growth via maintenance of a high net photosynthetic rate (Anet), maximum quantum efficiency (Fv/Fm), and photochemical quenching coefficient (qP). Exogenous EBR markedly increased the levels of partial free amino acids (proline, arginine, aspartic acid, and glycine) and promoted nitrogen metabolism through increasing the activities of nitrate reductase (NR), glutamine synthase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in the leaves of pepper seedlings under chilling stress. The effect of exogenous EBR on the content of reactive oxygen species was also investigated. Pretreatment with EBR reduced the accumulation of hydrogen peroxide (H2O2) and superoxide anion (O2−·), and concomitantly alleviated membrane lipid peroxidation of pepper leaves under chilling stress. These results suggest that foliar pretreatment of EBR has a positive effect on improving the chilling tolerance of pepper seedlings via maintaining a high photosynthetic capability and enhancing the nitrogen metabolism.