DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis. Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.

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Cnu, a Novel oriC-Binding Protein of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.20.6998-7008.2005 ◽

2005 ◽

Vol 187 (20) ◽

pp. 6998-7008 ◽

Cited By ~ 15

Author(s):

Myung Suk Kim ◽

Sung-Hun Bae ◽

Sang Hoon Yun ◽

Hee Jung Lee ◽

Sang Chun Ji ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid Identity ◽

Replication Initiation ◽

In Vitro Assays ◽

Wild Type ◽

Genetic Method ◽

Dna Replication Initiation ◽

Pair Sequence

ABSTRACT We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

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Neural Stimulation in vitro and in vivo by Photoacoustic Nanotransducers

10.1101/2020.05.20.106898 ◽

2020 ◽

Author(s):

Yimin Huang ◽

Ying Jiang ◽

Xuyi Luo ◽

Jiayingzi Wu ◽

Haonan Zong ◽

...

Keyword(s):

Acoustic Waves ◽

Near Infrared ◽

Neurological Diseases ◽

Laser Pulses ◽

Neural Stimulation ◽

Nanosecond Laser ◽

Genetic Method ◽

Precise Control

AbstractNeuromodulation is an invaluable approach for study of neural circuits and clinical treatment of neurological diseases. Here, we report semiconducting polymer nanoparticles based photoacoustic nanotransducers (PANs) for neural stimulation. Our PANs strongly absorb light in the near-infrared second window and generate localized acoustic waves. PANs can also be surface-modified to selectively bind onto neurons. PAN-mediated activation of primary neurons in vitro is achieved with ten 3-nanosecond laser pulses at 1030 nm over a 3 millisecond duration. In vivo neural modulation of mouse brain activities and motor activities is demonstrated by PANs directly injected into brain cortex. With millisecond-scale temporal resolution, sub-millimeter spatial resolution and negligible heat deposition, PAN stimulation is a new non-genetic method for precise control of neuronal activities, opening potentials in non-invasive brain modulation.

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isoTarget: a genetic method for analyzing the functional diversity of splicing isoforms in vivo

10.1101/2020.04.30.070847 ◽

2020 ◽

Author(s):

Hao Liu ◽

Sarah Pizzano ◽

Ruonan Li ◽

Wenquan Zhao ◽

Macy W. Veling ◽

...

Keyword(s):

Alternative Splicing ◽

Biochemical Properties ◽

Protein Isoforms ◽

Genetic Method ◽

Axon Development ◽

Splicing Isoforms ◽

Multi Level ◽

The Difference ◽

Endogenous Proteins

SUMMARYProtein isoforms generated by alternative splicing contribute to proteome diversity. Due to the lack of effective techniques, isoform-specific functions, expression, localization, and signaling mechanisms of endogenous proteins in vivo are unknown for most genes. Here we report a genetic method, termed isoTarget, for blocking the expression of a targeted isoform without affecting the other isoforms and for conditional tagging the targeted isoform for multi-level analyses in select cells. Applying isoTarget to two mutually exclusive isoforms of Drosophila Dscam, Dscam[TM1] and [TM2], we found that endogenous Dscam[TM1] is localized in dendrites while Dscam[TM2] is in both dendrites and axons. We demonstrate that the difference in subcellular localization between Dscam[TM1] and [TM2], rather than any difference in biochemical properties, leads to the two isoforms’ differential contributions to dendrite and axon development. Moreover, with isoTarget, we discovered that the subcellular enrichment of functional partners results in a DLK/Wallenda-Dscam[TM2]-Dock signaling cascade specifically in axons. isoTarget is an effective technique for studying how alternative splicing enhances proteome complexity.

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A Chemical-genetics and Nanoparticle Enabled Approach for in vivo Protein Kinase Analysis

10.1101/2020.05.13.094573 ◽

2020 ◽

Author(s):

Fengqian Chen ◽

Qi Liu ◽

Terrell Hilliard ◽

Tingzeng Wang ◽

Hongjun Liang ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinases ◽

Intracellular Delivery ◽

Binding Pocket ◽

Live Cells ◽

Genetic Method ◽

Chemical Genetic ◽

Methionine Residue ◽

Two Factors

AbstractThe human kinome contains >500 protein kinases, and regulates up to 30% of the proteome. Kinase study is currently hindered by a lack of in vivo analysis approaches due to two factors: our inability to distinguish the kinase reaction of interest from those of other kinases in live cells and the cell impermeability of the ATP analogs. Herein, we tackled this issue by combining the widely used chemical genetic method developed by Dr. Kevan Shokat and colleagues with nanoparticle-mediated intracellular delivery of the ATP analog. The critical AKT1 protein kinase, which has been successfully studied with the method, was used as our initial prototype. Briefly, enlargement of the ATP binding pocket, by mutating the gate-keeper Methionine residue to a Glycine, prompted the mutant AKT1 to preferentially use the bulky ATP analog N6-Benzyl-ATP-γ-S (A*TPγS) and, thus, differentiating AKT1-catalyzed and other phosphorylation events. The lipid/calcium/phosphate (LCP) nanoparticle was used for efficient intracellular delivery of A*TPγS, overcoming the cell impermeability issue. The mutant, but not wild-type, AKT1 used the delivered A*TPγS for autophosphorylation and phosphorylating its substrates in live cells. Thus, an in vivo protein kinase analysis method has been developed. The strategy should be widely applicable to other protein kinases.

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OZONE THERAPY: MECHANISMS OF ACTION, RECENT LITERATURE AND NEWLY DISCOVERED BIOCHEMICAL PATHWAYS

Journal of Ozone Therapy ◽

10.7203/jo3t.2.2.2018.11124 ◽

2018 ◽

Vol 2 (2) ◽

Author(s):

Lamberto Re

Keyword(s):

Mitochondrial Respiratory Chain ◽

Complementary Therapy ◽

Pharmacological Action ◽

Cellular Level ◽

Point Of View ◽

Ozone Therapy ◽

Xenobiotic Stress ◽

Biochemical Pathways ◽

Endogenous Antioxidant

Ozone therapy is widely used in many countries since many years. Recently, the increasing widespread of this complementary therapy has been accomplished by an increased number of basic and clinic papers published on international journals. This lecture will deal on the first approach by a pharmacological point of view in the aim to characterize the mechanisms activated at sub-cellular level by ozone when used for medical and beauty application at low graded doses. The first theory was based upon the fact that the exposure to low, non-toxic, ozone concentrations could increase the efficacy of the endogenous antioxidant system by increasing the production or the activity of some enzymes exerting a key role in the mitochondrial respiratory chain. Many of the basic mechanisms of the ozone action are now well outlined. In addition, the modulation of interleukins productions and of some biochemical pathways related to inflammation and pain, indicates the rationale of its use in many pathological conditions related to pain, inflammation and age disorders. We also discuss on the mode of action of ozone that, with an hormetic mechanism, appear to be more similar to xenobiotic stress model than a pure pharmacological action. Indeed, our recent work in vivo on patients treated with major autohemo therapy (MAH) demonstrated the increase of Nrf2 level (P

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