Enhanced aldehyde dehydrogenase activity by regenerating NAD+ in Klebsiella pneumoniae and implications for the glycerol dissimilation pathways

In Klebsiella pneumoniae, 3-hydroxypropaldehyde is converted to 3-hydroxypropionic acid (3-HP) by aldehyde dehydrogenase (ALDH) with NAD⁺ as a cofactor. Although ALDH overexpression stimulates the formation of 3-HP, it ceases to accumulate when NAD⁺ is exhausted. Here we show that NAD⁺ regeneration, together with ALDH overexpression, facilitates 3-HP production and benefits cell growth. Three distinct NAD⁺-regenerating enzymes: NADH oxidase and NADH dehydrogenase from K. pneumoniae, and glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae, were individually expressed in K. pneumoniae. In vitro assay showed their higher activities than that of the control, indicating their capacities to regenerate NAD⁺. When they were respectively co-expressed with ALD4, an ALDH from S. cerevisiae, the activities of ALD4 were significantly elevated compared with that expressing ALD4 alone, suggesting that the regenerated NAD⁺ enhanced the activity of ALD4. More interestingly, the growth rates of all NAD⁺-regenerating strains were prolonged in comparison with the control, indicating that NAD⁺ regeneration stimulated cell proliferation. This study not only reveals the reliance of ALD4 activity on NAD⁺ availability but also provides a method for regulating the dha regulon.     

Targeting UHRF1-SAP30-MXD4 axis for leukemia initiating cell eradication in myeloid leukemia

Aberrant self-renewal of leukemia initiation cells (LICs) drives aggressive acute myeloid leukemia (AML). Here, we report that UHRF1, an epigenetic regulator that recruits DNMT1 to methylate DNA, is highly expressed in AML and predicts poor prognosis. UHRF1 is required for myeloid leukemogenesis by maintaining self-renewal of LICs. Mechanistically, UHRF1 directly interacts with Sin3A-associated protein 30 (SAP30) through two critical amino acids, G572 and F573 in its SRA domain, to repress gene expression. Depletion of UHRF1 or SAP30 derepresses an important target gene, MXD4, which encodes a MYC antagonist, and leads to suppression of leukemogenesis. Further knockdown of MXD4 can rescue the leukemogenesis by activating the MYC pathway. Lastly, we identified a UHRF1 inhibitor, UF146, and demonstrated its significant therapeutic efficacy in the myeloid leukemia PDX model. Taken together, our study reveals the mechanisms for altered epigenetic programs in AML and provides a promising targeted therapeutic strategy against AML.Subject terms: Leukaemia, Cell biology     

Synthetic robust perfect adaptation achieved by negative feedback coupling with linear weak positive feedback

Unlike their natural counterparts, synthetic genetic circuits are usually fragile in the face of environmental perturbations and genetic mutations. Several theoretical robust genetic circuits have been designed, but their performance under real-world conditions has not yet been carefully evaluated. Here, we designed and synthesized a new robust perfect adaptation circuit composed of two-node negative feedback coupling with linear positive feedback on the buffer node. As a key feature, the linear positive feedback was fine-tuned to evaluate its necessity. We found that the desired function was robustly achieved when genetic parameters were varied by systematically perturbing all interacting parts within the topology, and the necessity of the completeness of the topological structures was evaluated by destroying key circuit features. Furthermore, different environmental perturbances were imposed onto the circuit by changing growth rates, carbon metabolic strategies and even chassis cells, and the designed perfect adaptation function was still achieved under all conditions. The successful design of a robust perfect adaptation circuit indicated that the top-down design strategy is capable of predictably guiding bottom-up engineering for robust genetic circuits. This robust adaptation circuit could be integrated as a motif into more complex circuits to robustly implement more sophisticated and critical biological functions.     

Extensive up-regulation of gene expression in cancer: the normalised use of microarray data

Based on the assumption that only a few genes are differentially expressed in a disease and have balanced upward and downward expression level changes, researchers usually normalise microarray data by forcing all of the arrays to have the same probe intensity distributions to remove technical variations in the data. However, accumulated evidence suggests that gene expressions could be widely altered in cancer, so we need to evaluate the sensitivities of biological discoveries to violation of the normalisation assumption. Here, we show that the medians of the original probe intensities increase in most of the ten cancer types analyzed in this paper, indicating that genes may be widely up-regulated in many cancer types. Thus, at least for cancer study, normalising all arrays to have the same distribution of probe intensities regardless of the state (diseased vs. normal) tends to falsely produce many down-regulated differentially expressed (DE) genes while missing many truly up-regulated DE genes. We also show that the DE genes solely detected in the non-normalised data for cancers are highly reproducible across different datasets for the same cancers, indicating that effective biological signals naturally exist in the non-normalised data. Because the powers of current statistical analyses using the non-normalised data tend to be low, we suggest selecting DE genes in both normalised and non-normalised data and then filter out the false DE genes extracted from the normalised data that show opposite deregulation directions in the non-normalised data.     

Metabolic engineering of Corynebacterium glutamicum by synthetic small regulatory RNAs

Journal of Industrial Microbiology & Biotechnology - Corynebacterium glutamicum is an important platform strain that is wildly used in industrial production of amino acids and various other...     

Cell-free protein synthesis for proteomics.

The use of cell-free expression systems as an alternative to cell-based methods for protein production is greatly facilitating studies of protein functions. Recent improvements to cell-free systems, and the development of cell-free protein display and microarray technologies, have led to cell-free protein synthesis becoming a powerful tool for large-scale analysis of proteins. This paper reviews the most commonly used cell-free systems and their applications in proteomics.