Additional file 1 of Sarcoma of the uterine cervix: experience of a single center

Supplementary Material

DataSheet1_Comprehensive screening strategy coupled with structure-guided engineering of l-threonine aldolase from Pseudomonas putida for enhanced catalytic efficiency towards l-threo-4-methylsulfonylphenylserine.docx

l-Threonine aldolases (TAs) can catalyze aldol condensation reactions to form β-hydroxy-α-amino acids, but afford unsatisfactory conversion and poor stereoselectivity at the Cβ position. In this study, a directed evolution coupling high-throughput screening method was developed to screen more efficient l-TA mutants based on their aldol condensation activity. A mutant library with over 4000 l-TA mutants from Pseudomonas putida were obtained by random mutagenesis. About 10% of mutants retained activity toward 4-methylsulfonylbenzaldehyde, with five site mutations (A9L, Y13K, H133N, E147D, and Y312E) showing higher activity. Iterative combinatorial mutant A9V/Y13K/Y312R catalyzed l-threo-4-methylsulfonylphenylserine with a 72% conversion and 86% diastereoselectivity, representing 2.3-fold and 5.1-fold improvements relative to the wild-type. Molecular dynamics simulations illustrated that additional hydrogen bonds, water bridge force, hydrophobic interactions, and π-cation interactions were present in the A9V/Y13K/Y312R mutant compared with the wild-type to reshape the substrate-binding pocket, resulting in a higher conversion and Cβ stereoselectivity. This study provides a useful strategy for engineering TAs to resolve the low Cβ stereoselectivity problem and contributes to the industrial application of TAs.</p

Xbp1 Directs Global Repression of Budding Yeast Transcription during the Transition to Quiescence and Is Important for the Longevity and Reversibility of the Quiescent State

<div><p>Pure populations of quiescent yeast can be obtained from stationary phase cultures that have ceased proliferation after exhausting glucose and other carbon sources from their environment. They are uniformly arrested in the G1 phase of the cell cycle, and display very high thermo-tolerance and longevity. We find that G1 arrest is initiated before all the glucose has been scavenged from the media. Maintaining G1 arrest requires transcriptional repression of the G1 cyclin, <i>CLN3</i>, by Xbp1. Xbp1 is induced as glucose is depleted and it is among the most abundant transcripts in quiescent cells. Xbp1 binds and represses <i>CLN3</i> transcription and in the absence of Xbp1, or with extra copies of <i>CLN3</i>, cells undergo ectopic divisions and produce very small cells. The Rad53-mediated replication stress checkpoint reinforces the arrest and becomes essential when Cln3 is overproduced. The <i>XBP1</i> transcript also undergoes metabolic oscillations under glucose limitation and we identified many additional transcripts that oscillate out of phase with <i>XBP1</i> and have Xbp1 binding sites in their promoters. Further global analysis revealed that Xbp1 represses 15% of all yeast genes as they enter the quiescent state and over 500 of these transcripts contain Xbp1 binding sites in their promoters. Xbp1-repressed transcripts are highly enriched for genes involved in the regulation of cell growth, cell division and metabolism. Failure to repress some or all of these targets leads <i>xbp1</i> cells to enter a permanent arrest or senescence with a shortened lifespan.</p></div

Genes with Xbp1 binding sites in their promoters are repressed after the diauxic shift.

<p>(A) Consensus Xbp1 binding site generated from the 520 Xbp1 repressed genes identified in this study. (B) Dot plot of Next-Generation RNA sequencing data for all polyadenylated transcripts from wild type (X-axis) and <i>xbp1</i> (Y-axis) cells grown to log phase (8 hours), after the DS (14 hours), after 18 or 24 hours of growth into stationary phase, and from purified Q cells. 520 Xbp1-repressed genes with Xbp1 binding sites in their promoters are highlighted in red. All other transcripts are plotted in grey.</p

G1 arrest is initiated before the diauxic shift.

<p>(A) Culture density based on optical density at 600 nm wavelength (OD600), (B) cell number increase, (C) percentage of cells in G1 are plotted for wild type cells grown in YEPD medium from log to stationary phase (stationary phase.) Values are the average of four growth curves and error bars are included. Cell number after the DS (14 hr) and at the end of cell division are indicated. (D) DNA fluorescence shown as scatter plots and histograms of log phase wild type cells, cells at the DS, and one hour later.</p

Xbp1 and Rad53 are required to stop cell division and maintain repression of <i>CLN3</i> transcription in stationary phase.

<p>(A) Transcript levels of <i>CLN3</i> and <i>ACT1</i> as cells grow for 8 to 48 hours into stationary phase (SP) were measured and reported as a ratio of <i>CLN3/ACT1</i>, (B) Cell volume distribution in log phase cultures (left) of <i>xbp1</i> (red) and wild type (black) cells and cultures that have ceased dividing after 50 hours of growth (right), relevant genotypes are indicated (C) Cell number, and (D and E) percent of cells in G1 as cells grow from log phase to SP. (F) Cell viability based on FungaLight dye exclusion in log phase cells (day 1) and after seven days of further growth into SP. (G) Upper panel, flow cytometry assays of ROS accumulation in strains indicated after five days (OD₆₀₀ = 24) of growth into SP. Percent of ROS positive cells within the M2 gate are shown in upper right of each panel. Below are micrographs and quantification of TUNEL positive cells after five days of growth into SP. Relevant genotypes indicated (BY6500 WT, BY5654 <i>5XCLN3</i>, BY6602 <i>xbp1</i>, BY6873 <i>cln3</i>, BY7131 <i>cln3xbp1</i>, BY7146 <i>xbp1 rad53-21</i>, BY6848 <i>rad53-21</i>, BY6697 <i>rad53-21 5XCLN3, BY7406 rad9 5XCLN3</i>.).</p

Fifteen percent of yeast transcripts are derepressed three-fold or more in <i>xbp1</i> cells during post-diauxic growth.

<p>(Left panel) derepressed genes with, or (right panel) without Xbp1 binding sites are shown after, 8, 14, 24, or 48 hours of growth and from purified Q cells (Q). RNA Next Generation sequence data are displayed as a ratio of BY6602 <i>xbp1</i>/BY6500 wild type.</p

Xbp1 is important for maintaining a reversible quiescent state.

<p>(A) Percent budding as a function of time as purified Q cells are returned to fresh YEPD media and re-enter the cell cycle. (B) Long term viability and (C) colony formation of purified Q cells over 8 weeks of incubation in water. (D) Samples were taken from BY6500 wild type (WT) and <i>xbp1</i> Q cells and 150 minutes after those Q cells were re-fed. Differential image contrast (DIC) and calcofluor-stained bud scars show the budded and unbudded populations. Relevant genotypes indicated (BY6500 wild type, BY6602 <i>xbp1</i>, BY6873 <i>cln3</i>, BY7131 <i>cln3xbp1</i>.).</p

Xbp1 binds and represses <i>CLN3</i> transcription in post-diauxic cells.

<p>(A) ChIP was performed on the promoters indicated in log phase cells and after 24 hours of growth. (B) <i>XBP1</i> and (C) <i>CLN3</i> mRNA levels in wild type and <i>xbp1</i> cells grown from log phase to stationary phase quantified from Next-Generation RNA sequencing data. (D) <i>xbp1</i> and (E) <i>5XCLN3</i> cells harvested for FACS analysis as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003854#pgen-1003854-g001" target="_blank">Figure 1</a> after 14 and 20 hours of growth (BY6500 WT, BY5654 <i>5XCLN3</i>, BY6602 <i>xbp1</i>.).</p

Ultrasensitive Fluorescent Probes Reveal an Adverse Action of Dipeptide Peptidase IV and Fibroblast Activation Protein during Proliferation of Cancer Cells

Dipeptide peptidase IV (DPPIV) and fibroblast activation protein (FAP) are isoenzymes. Evidence shows that DPPIV is related to antitumor immunity, and FAP may be a drug target in cancer therapy, making it seem that the two enzymes might have a synergistic role during the proliferation of cancer cells. Surprisingly, herein, we find an adverse action of DPPIV and FAP in the proliferation process by analyzing their changes with two tailor-made ultrasensitive fluorescent probes. First, the up-regulation of DPPIV and down-regulation of FAP in cancer cells under the stimulation of genistein are detected. Then, we find that MGC803 cells with a higher FAP but lower DPPIV level than SGC7901 cells exhibit a faster proliferation rate. Importantly, inhibiting the DPPIV expression with siRNA increases the proliferation rate of MGC803 cells, whereas the FAP inhibition decreases the rate. These findings suggest that the two enzymes play an adverse role during the proliferation of cancer cells, which provides us a new viewpoint for cancer studies