Pharmaceutical targeting of OTUB2 sensitizes tumors to cytotoxic T cells via degradation of PD-L1.

PD-1 is a co-inhibitory receptor expressed by CD8+ T cells which limits their cytotoxicity. PD-L1 expression on cancer cells contributes to immune evasion by cancers, thus, understanding the mechanisms that regulate PD-L1 protein levels in cancers is important. Here we identify tumor-cell-expressed otubain-2 (OTUB2) as a negative regulator of antitumor immunity, acting through the PD-1/PD-L1 axis in various human cancers. Mechanistically, OTUB2 directly interacts with PD-L1 to disrupt the ubiquitination and degradation of PD-L1 in the endoplasmic reticulum. Genetic deletion of OTUB2 markedly decreases the expression of PD-L1 proteins on the tumor cell surface, resulting in increased tumor cell sensitivity to CD8+ T-cell-mediated cytotoxicity. To underscore relevance in human patients, we observe a significant correlation between OTUB2 expression and PD-L1 abundance in human non-small cell lung cancer. An inhibitor of OTUB2, interfering with its deubiquitinase activity without disrupting the OTUB2-PD-L1 interaction, successfully reduces PD-L1 expression in tumor cells and suppressed tumor growth. Together, these results reveal the roles of OTUB2 in PD-L1 regulation and tumor evasion and lays down the proof of principle for OTUB2 targeting as therapeutic strategy for cancer treatment.

Gastrointestinal microbiome, resistance genes, and risk assessment of heavy metals in wild giant pandas.

The gastrointestinal microbiome (GM) of giant panda (GP) plays an important role in food utilization and health and is also an essential reservoir of resistance genes. Currently, little knowledge is available on the GM, acid resistance genes (AcRGs), antibiotic resistance genes (ARGs), metal resistance genes (MRGs), and mobile genetic elements (MGEs) in wild GPs. We sampled the gastrointestinal tract of a dead GP and explored the composition and function of GM and resistance genes through cryo-scanning electron microscopy, metagenomic sequencing, and genome-resolved metagenomics. The concentration of metals in the gastrointestinal lumen, feces, bamboo, and soil was measured by inductively coupled plasma mass spectrometry. Results showed that the composition of the microbiota varied in different gastrointestinal regions. Fecal microbiota was highly associated with small intestinal and colonic microbes. The lignocellulosic cross-linked structure of bamboo was destroyed in the stomach initially and destroying degree increased from stomach to anus. Reconstruction of metagenome-assembled-genomes confirmed that core GM, e.g., Streptococcus, Clostridium, Lactococcus, Leuconostoc, and Enterococcus, carried genes encoding the lignocellulose degradation enzyme. There were no significant differences of resistance genes between gastrointestinal and fecal samples, except MGEs. Multidrug and multi-metal resistance genes were predominant in all samples, while the transposase gene tnpA was the major type of MGE. Significant correlations were observed among the abundance of GM, resistance genes, and MGEs. Gastrointestinal and fecal mercury and chromium were the main metals influencing GM and resistance genes. The content of gastrointestinal and fecal metals was significantly associated with the presence of the same metals in bamboo, which could pose a threat to the health of wild GPs. This study characterized the gastrointestinal microbiome of wild GPs, providing new evidence for the role of the gastrointestinal microbiome in degrading lignocellulose from bamboo and highlighting the urgent need to monitor metal levels in soil and bamboo.

Diet, habitat environment and lifestyle conversion affect the gut microbiomes of giant pandas.

Gut microbiota (GM) are important for the health of giant pandas (GPs), in addition to the utilization of bamboo in their diets. However, it is not fully understood how diet, habitat environment and lifestyle contribute to the composition of GM in GP. Consequently, we evaluated how dietary changes, habitat environment conversions and lifestyle shifts influence the GM of GPs using high-throughput sequencing and genome-resolved metagenomics. The GM of GPs were more similar when their hosts exhibited the same diet. High fiber diets significantly increased the diversity and decreased the richness of gut bacterial communities alone or interacted with the age factor (p < 0.05). The abundances of Streptococcus, Pseudomonas, Enterococcus, Lactococcus, Acinetobacter, and Clostridium significantly increased during diet conversion process (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA > 4). Reconstruction of 60 metagenome-assembled-genomes (MAGs) indicated that these bacteria were likely responsible for bamboo digestion via gene complements involved in cellulose, hemicellulose, and lignin degradation. While habitat environment may play a more important role in shaping the GM of GP, lifestyle can also greatly affect bacterial communities. The GM structure in reintroduced GPs notably converged to that of wild pandas. Importantly, the main bacterial genera of wild GPs could aid in lignin degradation, while those of reintroduced GPs were related to cellulose and hemicellulose digestion. Streptococcus, Pseudomonas, Enterococcus, Lactococcus, Acinetobacter, and Clostridium may contribute to lignocellulose digestion in GP. The results revealed that diet conversion, habitat environment and lifestyle could remarkably influence the GM of GP. In addition, results suggested that increasing the ability of lignin degradation with GM may aid to change the GM of reintroduced pandas to resemble those of wild pandas.

Assessment of fluid responsiveness by inferior vena cava diameter variation in post-pneumonectomy patients.

AIM: First, the inferior vena cava dilatation index (DIVC) was measured by ultrasound, and then the reliability of DIVC as an indicator to predict volume responsiveness in patients undergoing mechanical ventilation after pneumonectomy was evaluated. METHODS: Pulse indicator continuous cardiac output (Picco) as gold standard was performed to sedated mechanically ventilated post-pneumonectomy patients in intensive care unit of Nanjing Thoracic Hospital from August 2014 to December 2016. Meanwhile, ultrasound measurement to inferior vena cava (IVC) diameter at the end inspiration (Dmax ) and the end of expiration (Dmin ) was performed. DIVC = (Dmax  - Dmin )/Dmin . Above values were recorded at baseline and then after fluid resuscitation challenge (7 mL/kg hydroxyethyl starch). An increase in cardiac index of more than 15% was used as the standard for fluid responsiveness. Patients were divided into responsive group and non-responsive group. A receiver operating characteristic (ROC) curve was then used to determine the sensitivity and specificity of DIVC in predicting fluid responsiveness after pneumonectomy. RESULTS: Eighteen patients were enrolled. 10 patients were divided into responsive group and eight in non-responsive group. DIVC in responsive group was significantly higher than in non-responsive group (P < 0.01). By setting DIVC ≥ 15% as a measure of fluid responsiveness, sensitivity was 81.8% and specificity was 85.7%. CONCLUSION: DIVC is a reliable indicator of capacity responsiveness in mechanically ventilated post-pneumonectomy patients.

Endotoxin removing method based on lipopolysaccharide binding protein and polyhydroxyalkanoate binding protein PhaP.

Polyhydroxyalkanoates (PHAs) granule associated protein PhaP has a strong affinity to PHA and other hydrophobic polymers. Human lipopolysaccharide binding protein (hLBP) is a natural endotoxin receptor in plasma. In this study, genes encoding hLBP fused with PhaP were expressed in Pichia pastoris GS115 for production of the fusion protein. The purified rhLBP-PhaP fusion protein was immobilized on particles of polyhydroxybutyrate (PHB), which is a member of microbial polyhydroxyalkanoates (PHA). The rhLBP-PhaP-coated PHB particles were added to endotoxin containing water and protein solutions to study their endotoxin removal and protein recovery efficiencies. The influences of ionic strengths and pH on endotoxin removal and protein recovery in different protein solutions were also studied using acidic proteins including bovine serum albumin (BSA), ovalbumin, and basic protein α-chymotrypsinogen as model proteins. The results showed that rhLBP-PhaP particles could remove endotoxin with an efficiency of over 90%. All endotoxin removal and protein recovery efficiencies were only slightly affected by ionic strengths but were drastically affected by pH changes. Our results demonstrated that rhLBP-PhaP particles with their high efficiency, ease of preparation, and nontoxicity will be a suitable system for endotoxin removal in the protein purification industry.

A novel self-cleaving phasin tag for purification of recombinant proteins based on hydrophobic polyhydroxyalkanoate nanoparticles.

A novel protein purification method was developed using microbial polyhydroxyalkanoates (PHA) granule-associated protein phasin, a pH-inducible self-cleaving intein and PHA nanoparticles. Genes for the target proteins to be produced and purified were fused to genes of intein and phasin, the genes were jointly over-expressed in vivo, such as in E. coli cells in this study. The fused proteins containing target protein, intein and phasin produced by the recombinant E. coli were released together with all other E. coli proteins via a bacterial lysis process. They were then adsorbed in vitro to the surfaces of the hydrophobic polymer nanoparticles incubated with the cell lysates. The nanoparticles attached with the fused proteins were concentrated via centrifugation. Then, the reasonably purified target protein was released by self-cleavage of intein and separated with nanoparticles by a simple centrifugation process. Using this system, enhanced green fluorescent protein (EGFP), maltose binding protein (MBP) and beta-galactosidase were successfully purified in their active forms with reasonable yields, respectively, demonstrating the effectiveness and reliability of this purification system. This method allows the production and purification of high value added proteins in a continuous way with low cost.