Crystal Growth of Aspirin Using a Temperature-Controlled Microfluidic Device

Identifying the most appropriate polymorph of active pharmaceutical ingredients is one of the important steps in drug development, since their bioactivities are largely dependent on their solid forms. However, the sample preparation for the characterization of crystal forms is time-consuming and requires large quantities of sample. Here, we introduce a microfluidic device-based method to prepare a sub-millimeter-sized single aspirin crystal from a small quantity of material. For the crystal preparation, a device equipped with a solution flow system and temperature controller was placed under the microscope. To use the device, concentration–temperature phase diagrams were generated, and regions where dominant nucleation or crystal growth with specific directions were clearly determined. By observing time-dependent changes of crystal number and size with solution temperature, a pathway to grow a single crystal of aspirin was determined and applied to prepare a sub-millimeter-sized crystal from 250 μg of aspirin. The obtained crystal was sufficiently large for single-crystal X-ray diffraction analysis, which usually requires 10 mg to 1 g of material per crystallization experiment. Thus, this method can be adapted as an efficient approach to uncovering the crystallization process to obtain required crystal forms with minimal sample consumption

Vertebrate Ssu72 Regulates and Coordinates 3′-End Formation of RNAs Transcribed by RNA Polymerase II

<div><p>In eukaryotes, the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is composed of tandem repeats of the heptapeptide YSPTSPS, which is subjected to reversible phosphorylation at Ser2, Ser5, and Ser7 during the transcription cycle. Dynamic changes in CTD phosphorylation patterns, established by the activities of multiple kinases and phosphatases, are responsible for stage-specific recruitment of various factors involved in RNA processing, histone modification, and transcription elongation/termination. Yeast Ssu72, a CTD phosphatase specific for Ser5 and Ser7, functions in 3′-end processing of pre-mRNAs and in transcription termination of small non-coding RNAs such as snoRNAs and snRNAs. Vertebrate Ssu72 exhibits Ser5- and Ser7-specific CTD phosphatase activity <i>in vitro</i>, but its roles in gene expression and CTD dephosphorylation <i>in vivo</i> remain to be elucidated. To investigate the functions of vertebrate Ssu72 in gene expression, we established chicken DT40 B-cell lines in which Ssu72 expression was conditionally inactivated. Ssu72 depletion in DT40 cells caused defects in 3′-end formation of U2 and U4 snRNAs and <i>GAPDH</i> mRNA. Surprisingly, however, Ssu72 inactivation increased the efficiency of 3′-end formation of non-polyadenylated replication-dependent histone mRNA. Chromatin immunoprecipitation analyses revealed that Ssu72 depletion caused a significant increase in both Ser5 and Ser7 phosphorylation of the Pol II CTD on all genes in which 3′-end formation was affected. These results suggest that vertebrate Ssu72 plays positive roles in 3′-end formation of snRNAs and polyadenylated mRNAs, but negative roles in 3′-end formation of histone mRNAs, through dephosphorylation of both Ser5 and Ser7 of the CTD.</p></div

Ssu72 is required for 3′-end formation of <i>GAPDH</i> mRNA and U2 and U4 snRNAs.

<p>(A) Diagrams of the <i>GAPDH</i> (upper) and <i>U snRNA</i> genes (lower), shown as open arrows, with the cleavage sites depicted as arrowheads and RNA processing elements indicated in the open boxes. The black arrows represent the gene-specific primers used for reverse transcription of precursor transcripts. Dotted lines indicate the RT-qPCR amplicons used to quantitate the precursor transcripts. (B–D) RT-qPCR analysis of the relative expression levels of precursor (pre) and total transcripts of <i>GAPDH</i> (B), U2 snRNA (C) or U4 snRNA (D) in DT40 P3 (−/−) cells treated with Dox for the indicated number of days. Relative expression levels were normalized the corresponding levels on day 0. Error bars represent standard deviations of at least two independent experiments.</p

Establishment of conditional Ssu72-knockout DT40 cell lines.

<p>(A) Schematic representations of the chicken Ssu72 genomic fragment, knockout constructs, and configuration of the targeted alleles. Exons are shown as black boxes (E1–5), and the location of the 5′ probe used for Southern blotting is shown as a grey box. The double-headed arrows above the genes indicate length (in nucleotides). The <i>XbaI</i> and <i>EcoRI</i> restriction sites are indicated by vertical lines labeled X and R, respectively. (B) Southern blot analysis of wild-type (WT), heterozygous mutant (B15), homozygous mutant (P1, P2, P3), and unanticipated rearranged mutant (P10) clones. Genomic DNA obtained from each clone was digested with <i>Xba</i>I and <i>Eco</i>RI, and then hybridized with the 5′ probe shown in panel A. (C) RT-PCR analysis of the wild-type and mutant clones using primer pairs specific for the indicated gene. (D) Immunoblotting analysis of DT40 P3 (−/−) whole-cell extracts treated with Dox for the indicated times, using the indicated antibodies. Western blotting of β-actin was used as to confirm equal protein loading.</p

Ssu72 dephosphorylates Ser5P and Ser7P <i>in vitro</i>.

<p>(A) Fifty ng phosphorylated GST-CTD was incubated with 3.4 µg GST (lane 1), 0.2 µg SCP1 (lane 2), 0.2 µg, 1 µg, or 5 µg wild-type human Ssu72 (lanes 3–5), or 5 µg mutant (C12S) Ssu72 (lane 6), followed by Immunoblotting using the phospho CTD–specific antibodies 3E10 (Ser2P), 3E8 (Ser5P), and 4E12 (Ser7P) and an antibody against the CTD (8WG16). (B) Purified human Pol II was incubated with wild-type (lane 1) or mutant (C12S) Ssu72 (lane 2) and analyzed by Western blotting using phospho CTD–specific antibodies 3E10 (Ser2P), 3E8 (Ser5P), 4E12 (Ser7P), and 6D7 (Thr4P).</p

Ssu72 is essential for cell proliferation and viability in DT40 cells.

<p>(A, B) Growth curve of DT40 wild-type (WT) and conditional mutant [P1 (−/−)] DT40 cells. WT (A) or P1 (−/−) (B) cells were seeded in triplicate in 12-well plates (5×10³ cells/well) in media with or without doxycycline (Dox), and split every 2 days. Concentrations of live cells were determined by Giemsa staining and counted at the indicated time points. Cell density is shown on a logarithmic scale. (C) WT cells (left) or P1 (−/−) cells (right) treated with Dox for 6 days (lower) or untreated (upper) were subjected to FACS analysis after staining with propidium iodide. Gates M1, M2, M3, and M4, delimited by the vertical bars above the FACS traces, indicate the cell-cycle distributions of the sub-G1, G0/G1, S and G2 populations, respectively.</p

Ssu72 depletion results in a modest increase in CTD phosphorylation.

<p>(A) Whole-cell extracts from DT40 P3 (−/−) cells treated with Dox for the indicated number of days were separated by SDS-PAGE, and then analyzed by Immunoblotting with antibodies against FLAG-Ssu72 (anti-FLAG M2), the largest subunit (Rpb1) of Pol II (N20), Ser2P (3E10), Ser5P (3E8), Ser7P (4E12), and β-actin. Western blotting of β-actin was used to confirm equal protein loading. The positions corresponding to the hyper-phosphorylated form of Rpb1 (IIo) and hypo-phosphorylated form of Rpb1 (IIa) are indicated by arrows. (B) Fold change of phosphorylation levels of each serine (Ser2/5/7P) relative to the corresponding levels on day 0. The Western blot signals were quantified by using a luminescent image analyzer LAS-4000 mini (Fujifilm). The each value was normalized by total Pol II signal (Rpb1). Error bars represent standard deviations of two independent experiments.</p

Ssu72 depletion causes hyperphosphorylation of the CTD on Pol II–transcribed genes.

<p>(A–C) ChIP analysis of the downstream region of the <i>GAPDH</i> gene (A), U2 and U4 snRNA genes (B), and histone H3 and H4 genes (C). (Upper) Diagrams of these genes with the coding regions, polyadenylation signals (pA), 3′ box signals, and stem-loop sequences (SL) shown as open boxes. The numbers above these genes represent distance (in nucleotides) from the end of the coding region (for <i>GAPDH</i>) or the transcription start site (for snRNA and histone genes). Dotted lines indicate the ChIP amplicons. (Lower) ChIP analysis of region 1 or 2 on the indicated genes, using antibodies against Pol II (N20), Ser2P (3E10), Ser5P (3E8), or Ser7P (4E12), in DT40 P3 (−/−) cells treated with Dox for 4 days (grey bars) or untreated (black bars). The y axe represents the fold change relative to the corresponding levels on day 0. The values for Ser2P, Ser5P or Ser7P signals were normalized by the total Pol II signal. Error bars represent standard deviations of two independent experiments.</p

Ssu72 inhibits 3′-end formation of replication-dependent histone mRNAs.

<p>(A) Diagram of the histone gene, shown as an open arrow, with the cleavage site depicted an arrowhead and the stem-loop sequence (SL) indicated by the open box. The black arrow represents the gene-specific primer used for reverse transcription of precursor transcripts. Dotted lines indicate the RT-qPCR amplicons used to quantitate the precursor transcripts. (B-E) RT-qPCR analysis of the relative expression levels of precursor (pre) and total transcripts of histone H3 (B, D) or H4 (C, E) in DT40 P3 (−/−) cells treated with Dox for the indicated number of days. Gene-specific primers (B, C) or an oligo-dT primer (D, E) was used for reverse transcription to detect the expression levels of precursor transcripts. Relative expression levels were normalized the corresponding levels on day 0. Error bars represent standard deviations of at least two independent expriments.</p