Acquired DNA damage repairs deficiency-driven immune evolution and involved immune factors of local versus distant metastases in non-small cell lung cancer.

The evolution of immune profile from primary tumors to distant and local metastases in non-small cell lung cancer (NSCLC), as well as the impact of the immune background of primary tumors on metastatic potential, remains unclear. To address this, we performed whole-exome sequencing and immunohistochemistry for 73 paired primary and metastatic tumor samples from 41 NSCLC patients, and analyzed the change of immune profile from primary tumors to metastases and involved genetic factors. We found that distant metastases tended to have a decreased CD8+ T cell level along with an increased chromosomal instability (CIN) compared with primary tumors, which was partially ascribed to acquired DNA damage repair (DDR) deficiency. Distant metastases were characterized by immunosuppression (low CD8+ T cell level) and immune evasion (high PD-L1 level) whereas local metastases (pleura) were immune-competent with high CD8+ T cell, low CD4+ T cell and low PD-L1 level. Primary tumors with high levels of CD4+ T cells were associated with distant metastases rather than local metastases. Analysis of TCGA data and a single-cell RNA-sequencing dataset revealed a decreasing trend of major immune cells, such as CD8+ T cells, and an increasing trend of CD4 T helper cells (Th2 and Th1) in primary tumors with metastases from local to distant sites. Our study indicates that there are differences in the immune evolution between distant and local metastases, and that acquired DDR deficiency contributes to the immunosuppression in distant metastases of NSCLC. Moreover, the immune background of primary tumors may affect their metastatic potential.

Correlation of exosomal microRNA clusters with bone metastasis in non-small cell lung cancer.

20-40% of lung cancer patients develop bone metastasis (BM) with significantly decreased overall survival. Currently, BM is mainly diagnosed by computerized tomography (CT) scan or magnetic resonance imaging (MRI) when symptom develops. Novel biomarkers with higher prediction value of BM are needed. Plasma-derived exosomal microRNAs had been isolated and sequenced of total 30 non-small cell lung cancer (NSCLC) patients including 16 with bone metastasis and 14 without bone metastasis. Hierarchical clustering based on the total miRNA profile can clearly separate cancer patients and healthy individuals (H), but not patients with (BM +) or without (BM-) BM. Weight Co-expression network of miRNAs (WGCNA) analyses identified three consensus clusters (A, B, C) of highly correlated miRNAs, among which cluster B (144 miRNAs) showed significantly differential expression in lung cancer patients, especially in BM + group. Pathway analysis of cluster B miRNAs revealed enrichment in metabolic pathways that may involve in preconditioning of the metastatic niche. Three differentially expressed miRNAs between BM + and BM- patients within cluster B were identified as miR-574-5p, a suppressor of Wnt/ß-catenin pathway, was down-regulated, while miR-328-3p and miR-423-3p, two activators of the same pathway, were up-regulated in BM + patients. Cluster A miRNAs (n = 49) also showed trend of upregulation in BM + patients. Interestingly, pathway analysis indicated that 43 of them are associated with chromosome14, which has been suggested to promote epithelial-mesenchymal transition (EMT) and bone metastasis.

One pot simultaneous preparation of both enantiomer of ß-amino alcohol and vicinal diol via cascade biocatalysis.

OBJECTIVES: To investigate the efficiency of a new cascade biocatalysis system for the conversion of R, S-ß-amino alcohols to enantiopure vicinal diol and ß-amino alcohol. RESULTS: An efficient cascade biocatalysis was achieved by combination of a transaminase, a carbonyl reductase and a cofactor regeneration system. An ee value of > 99% for 2-amino-2-phenylethanol and 1-phenyl-1, 2-ethanediol were simultaneously obtained with 50% conversion from R, S-2-amino-2-phenylethanol. The generality of the cascade biocatalysis was further demonstrated with the whole-cell approaches to convert 10-60 mM R, S-ß-amino alcohol to (R)- and (S)-diol and (R)- and (S)-ß-amino alcohol in 90-99% ee with 50-52% conversion. Preparative biotransformation was demonstrated at a 50 ml scale with mixed recombinant cells to give both (R)- and (S)-2-amino-2-phenylethanol and (R)- and (S)-1-phenyl-1, 2-ethanediol in > 99% ee and 40-42% isolated yield from racemic 2-amino-2-phenylethanol. CONCLUSIONS: This cascade biocatalysis system provides a new practical method for the simultaneous synthesis of optically pure vicinal diol and an ß-amino alcohol.

Cloning and functional expression of a chitinase cDNA from the apple leaf miner moth Lithocolletis ringoniella.

Insect chitinase plays essential roles in chitin catabolism involved in digestion and molting during insect development. In the current work, we cloned a chitinase cDNA, LrCht5, from the apple leaf miner moth Lithocolletis ringoniella and characterized its amino acid sequence and protein properties. The L. ringoniella chitinase cDNA was 2136 bp in length with an open reading frame of 1737 bp that encodes a polypeptide of 579 amino acid residues with a predicted molecular mass of 64.4 kDa and pI of 5.49. The catalytic domain has several phosphorylation and glycosylation sites. The recombinant LrCht5 was expressed in Escherichia coli and the Spodoptera frugiperda cell line Sf9, and the LrCht5 expressed in insect cells exhibited chitinolytic activity. LrCht5 was most stable at pH 6.0 and 45°C. This work has potential application in the development of novel and more specific synthetic chitinase inhibitors for use as bioinsecticides.

Effect of location of the His-tag on the production of soluble and functional Buthus martensii Karsch insect toxin.

The low yield and poor folding efficiency in vivo of soluble and active recombinant cysteine-rich proteins expressed in Escherichia coli are a major challenge for large-scale protein production and purification. Expression vectors containing Buthus martensii Karsch insect toxin (BmK IT) fused to the C terminus of the intein Ssp DnaB were constructed in an attempt to overcome this problem. Following purification and intein self-cleavage, the fusion protein His(6)-intein-IT produced insoluble BmK IT, while intein-IT-His(6) generated soluble and properly folded BmK IT. This result indicated that the positioning of the His(6) tag has a key role in the production of soluble and functional BmK IT.

Potential biochemical therapy of glioma cancer.

Glioma is a highly invasive, rapidly spreading form of brain cancer that is resistant to surgical and medical treatment. The recent progresses made in intracellular and ion channels of glioma cells provide a potential new approach for biochemical therapy of brain tumor. In this paper, we reviewed clinical data on chemotherapy by temozolomide and results from new studies on voltage-gated potassium channels, large-conductance Ca(2+)-activated K(+) channels, volume-activated chloride channels, glioma-specific chloride channel and their modulators. These new findings may represent future directions for brain tumor studies and treatment.

Therapeutic potential of chlorotoxin-like neurotoxin from the Chinese scorpion for human gliomas.

Chlorotoxin, one of the key toxins in scorpion Leiurus quinquestriatus venom, has been shown to bind specifically to glioma cell surface as a specific chloride channel blocker. In this study, a purified, recombinant chlorotoxin-like peptide from the scorpion Buthus martensii Karsch (named rBmK CTa) was characterized by in vivo and in vitro studies. The results from cell proliferation assay with human glioma (SHG-44) cells showed that rBmK CTa inhibits the growth of glioma cells in a dose-dependent manner, with an IC(50) value of approximately 0.28microM. Under the same conditions, the IC(50) value for normal astrocytes increased to 8microM. This clearly indicated that rBmK CTa had specific toxicity against glioma cells but not astrocytes. Results from whole-cell patch-clamp recording showed that chloride current in SHG-44 was inhibited by rBmK CTa in a voltage-dependent manner and percent inhibitions for the blocking action of rBmK CTa (0.07 and 0.14microM) on I(Cl) was 17.64+/-3.06% and 55.86+/-2.83%, respectively. Histological analysis of rBmK CTa treated mice showed that brain, leg muscle and cardiac muscle were the target organs of this toxin. These results suggest that rBmK CTa may have potential therapeutic application in clinical treatment of human glioma. It represents an approach for developing a novel therapeutic agent.