Fusing the decarboxylase OleT JE and the reductase domain of P450BM3 creates a self-sufficient protein, OleT-BM3R, which is able to efficiently catalyze oxidative decarboxylation of carboxylic acids into linear α-olefins (LAOs) under mild aqueous conditions using O 2 as the oxidant and NADPH as the electron donor. The compatible electron transfer system installed in the fusion protein not only eliminates the need for auxiliary redox partners, but also results in boosted decarboxylation reactivity and broad substrate scope. Coupled with the phosphite dehydrogenasebased NADPH regeneration system, this enzymatic reaction proceeds with improved product titers of up to 2.51 g L −1 and volumetric productivities of up to 209.2 mg L −1 h −1 at low catalyst loadings (∼0.02 mol %). With its stability and scalability, this self-sufficient biocatalyst offers a nature-friendly approach to deliver LAOs.

The histidine kinase CheA plays a central role in signal integration, conversion, and amplification in the bacterial chemotaxis signal transduction pathway. The kinase activity is regulated in chemotaxis signaling complexes formed via the interactions among CheA's regulatory domain (P5), the coupling protein CheW, and transmembrane chemoreceptors. Despite recent advancements in the understanding of the architecture of the signaling complex, the molecular mechanism underlying this regulation remains elusive. An interdomain linker that connects the catalytic (P4) and regulatory domains of CheA may mediate regulatory signals from the P5-CheW-receptor interactions to the catalytic domain. To investigate whether this interdomain linker is capable of both activating and inhibiting CheA, we performed screens to search for P4-P5 linker mutations that result in different CheA autokinase activities. Several CheA variants were identified with kinase activities ranging from 30% to 670% of the activity of wild-type CheA. All of these CheA variants were defective in receptor-mediated kinase activation, indicating that the natural receptor-mediated signal transmission pathway was simultaneously affected by these mutations. The altered P4-P5 linkers were sufficient for making significant changes in the kinase activity even in the absence of the P5 domain. Therefore, the interdomain linker is an active module that has the ability to impose regulatory effects on the catalytic activity of the P4 domain. These results suggest that chemoreceptors may manipulate the conformation of the P4-P5 linker to achieve CheA regulation in the platform of the signaling complex. The molecular mechanism underlying kinase regulation in bacterial chemotaxis signaling complexes formed by the regulatory domain of the histidine kinase CheA, the coupling protein CheW, and chemoreceptors is still unknown. We isolated and characterized mutations in the interdomain linker that connects the catalytic and regulatory domains of CheA and found that the linker mutations resulted in different CheA autokinase activities in the absence and presence of the regulatory domain as well as a defect in receptor-mediated kinase activation. These results demonstrate that the interdomain linker is an active module that has the ability to impose regulatory effects on CheA activity. Chemoreceptors may manipulate the conformation of this interdomain linker to achieve CheA regulation in the platform of the signaling complex.

Background/Aim: Pancreatic cancer (PC) is currently the fourth leading cause of cancer-related mortality worldwide. Peripheral blood mononuclear cells (PBMCs) is a subpopulation of accessible and functional immune cells. Comparative analysis of the proteome of PBMCs can help us elucidate the mechanism of disease and find potential biomarkers for diagnosis. Materials and Methods: PBMCs were collected from healthy individuals, patients with benign diseases, and pancreatic cancer. iTRAQ-2DLC-MS/MS and SWATH methodologies were applied to make a comparative proteomics analysis of PBMCs. Results: A total of 3,357 proteins with a false discovery rate (FDR) <1% were identified, of which 114 proteins were found dysregulated in the PC group. An extensive SWATH library was constructed which showed a potential application for large scale clinical sample analysis. Conclusion: A PBMCs proteome with extensive protein representation was achieved, which will potentially allow the identification of novel biomarkers for PC. Pancreatic cancer (PC) is an aggressive malignancy, characterized by invasiveness, rapid progression and profound resistance to treatments (1-3). To date, there are no clinically validated screening methods for PC in the curative stage (4, 5). There is an urgent need for additional studies, applying modern technologies, on PC to increase our understanding of this disease, and improve the ability to diagnose PC in asymptomatic patients. In our previous work, surgically resected fresh PC tissues and adjacent non-tumor tissues were investigated and novel prognostic predictors of PC and PC-associated diabetes mellitus were discovered (6, 7). Given the asymptomatic nature of PC, a blood-based assay is preferred because it would be feasible and minimally invasive (8). Peripheral blood mononuclear cells (PBMCs), including monocytes, T-cells, B cells, and natural killer (NK) cells (9), play important roles in the function of the immune system and in monitoring immune-relevant events. They are readily accessible from routinely collected blood. Furthermore, it is known that the immune system is involved not only in the pathogenesis of autoimmune and infectious diseases, but also in cancer (10, 11), metabolic syndrome (12), atherosclerosis (13) and many other diseases (14). The PBMCs represent a biological sample which closely reflect the response of the body to pathogens and diseases like cancer. Therefore, they may allow for discovery of potentially pathology-relevant biomolecules of PC, such as DNA, RNA and proteins (15). Baine et al. reported an in-depth comparison of global gene expression profiles in PBMCs of PC patients and healthy controls, revealing that 383 genes were significantly different between PC and healthy controls (16). However, it is unknown whether these alterations have been transferred into the proteome of PBMCs in PC. Proteomic analysis provides large-scale determination of gene and cellular function at the protein level, so that it is quintessential to understanding health and disease of any organ ...

Asthma is a chronic airway inflammatory disease with obvious heterogeneity and complex pathophysiological manifestations. 1 At present, there are at least 300 million cases of asthma in the world, and the prevalence of asthma is increasing year by year. 2 Severe asthma refers to the severe state of asthma. Although severe asthma accounts for a small proportion of the total number of asthma, its direct medical costs account for 50% of the total cost of asthma treatment. 3 Severe asthma is difficult to control,

Deep mining the proteome of trace biological samples is critical for biomedical applications. However, it remains a challenge due to the loss of analytes caused by current sample preparation procedures. To address this, we recently developed a single-pot and miniaturized in-solution digestion (SMID) method for minute sample handling with three streamlined steps and completed within 3 h. The SMID approach outperformed the traditional workflow in substantially saving time, reducing sample loss, and exhibiting extensive applicability for 10−100 000 cell analysis. This user-friendly and high-sensitivity strategy enables ∼5300 proteins and 53 000 peptides to be confidently identified within 1 h of mass spectrometry (MS) time from a small amount of 1000 HeLa cells. In addition, we accurately and robustly detected proteomes in 10 mouse oocytes with excellent reproducibility. We further adopted SMID for the proteome analysis in cell migration under confinement, which induced cells to undergo a mesenchymal−amoeboid transition (MAT). During the MAT, a systematic quantitative proteome map of 1000 HeLa cells was constructed with seven expression profile clusters, which illustrated the application of SMID and provided a fundamental resource to investigate the mechanism of MAT.