Escaping from CRISPR-Cas-mediated knockout: the facts, mechanisms, and applications.

Clustered regularly interspaced short palindromic repeats and associated Cas protein (CRISPR-Cas), a powerful genome editing tool, has revolutionized gene function investigation and exhibits huge potential for clinical applications. CRISPR-Cas-mediated gene knockout has already become a routine method in research laboratories. However, in the last few years, accumulating evidences have demonstrated that genes knocked out by CRISPR-Cas may not be truly silenced. Functional residual proteins could be generated in such knockout organisms to compensate the putative loss of function, termed herein knockout escaping. In line with this, several CRISPR-Cas-mediated knockout screenings have discovered much less abnormal phenotypes than expected. How does knockout escaping happen and how often does it happen have not been systematically reviewed yet. Without knowing this, knockout results could easily be misinterpreted. In this review, we summarize these evidences and propose two main mechanisms allowing knockout escaping. To avoid the confusion caused by knockout escaping, several strategies are discussed as well as their advantages and disadvantages. On the other hand, knockout escaping also provides convenient tools for studying essential genes and treating monogenic disorders such as Duchenne muscular dystrophy, which are discussed in the end.

Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling.

Accurate mitosis is coordinated by the spindle assembly checkpoint (SAC) through the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C). As an essential regulator, Cdc20 promotes mitotic exit through activating APC/C and monitors kinetochore-microtubule attachment through activating SAC. Cdc20 requires multiple interactions with APC/C and MCC subunits to elicit these functions. Functionally assessing these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNA interference (RNAi). Here we generated Cdc20 RNAi-sensitive cell lines which display a penetrant metaphase arrest by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron, was found critical for SAC by promoting MCC formation and its interaction with APC/C. These data reveal additional regulation within the SAC and establish a novel method to interrogate Cdc20.

Zombies Never Die: The Double Life Bub1 Lives in Mitosis.

When eukaryotic cells enter mitosis, dispersed chromosomes move to the cell center along microtubules to form a metaphase plate which facilitates the accurate chromosome segregation. Meanwhile, kinetochores not stably attached by microtubules activate the spindle assembly checkpoint and generate a wait signal to delay the initiation of anaphase. These events are highly coordinated. Disruption of the coordination will cause severe problems like chromosome gain or loss. Bub1, a conserved serine/threonine kinase, plays important roles in mitosis. After extensive studies in the last three decades, the role of Bub1 on checkpoint has achieved a comprehensive understanding; its role on chromosome alignment also starts to emerge. In this review, we summarize the latest development of Bub1 on supporting the two mitotic events. The essentiality of Bub1 in higher eukaryotic cells is also discussed. At the end, some undissolved questions are raised for future study.

Analysis and pharmacokinetic study of polyphyllin H in beagle dog plasma after oral administration of Rhizoma Paridis extracts by LC-MS/MS.

A highly sensitive, rapid assay method has been developed and validated for the analysis of polyphyllin H in beagle dog plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves extraction of polyphyllin H and ginsenoside Re (IS) from beagle dog plasma. Chromatographic separation was carried out on an Agilent Zorbax XDB-C18 (100 × 2.1 mm, 1.8µm) column by isocratic elution with acetonitrile and water (50:50, v/v) at a flow rate of 0.25 mL/min with a total run time of 2.5 min. The MS/MS ion transitions monitored were m/z 869.60 → 869.60 for polyphyllin H and m/z 969.60 → 969.60 for IS. [corrected] Linear responses were obtained for polyphyllin H ranging from 1 to 50 ng/mL. The intra-and inter-day precisions (RSDs) <1.77 and 3.39% and the extraction recovery ranged from 91.89 to 93.33% with RSD <2.68%. Stability studies showed that polyphyllin H was stable in the preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration-time profiles of polyphyllin H.

Specificity profiling of protein phosphatases toward phosphoseryl and phosphothreonyl peptides.

A combinatorial library method was developed to systematically profile the substrate specificity of protein phosphatases toward phosphoseryl (pS) and phosphothreonyl (pT) peptides. Application of this method and a previously reported phosphotyrosyl (pY) library screening technique to dual-specificity phosphatase (DUSP) VH1 of vaccinia virus revealed that VH1 is highly active toward both pS/pT and pY peptides. VH1 exhibits different and more stringent sequence specificity toward pS/pT than pY substrates. Unlike previously characterized protein tyrosine phosphatases (PTPs), the activity and specificity of VH1 are primarily determined by the amino acid residues C-terminal to the pS, pT, or pY residue. In contrast, the mammalian VH1-related (VHR) DUSP has intrinsically low catalytic activity toward pS and pT substrates, suggesting that its primary physiological function is to dephosphorylate pY residues in substrate proteins. This method is applicable to other DUSPs and protein-serine/threonine phosphatases, and the substrate specificity data will be useful for identifying the physiological substrates of these enzymes.

Development and validation of a highly sensitive LC-ESI-MS/MS method for the determination of hyperoside in beagle dog plasma: application to a pharmacokinetic study.

A highly sensitive, rapid assay method has been developed and validated for the analysis of hyperoside in beagle dog plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves extraction of hyperoside and ginsenoside Re (IS) from beagle dog plasma. Chromatographic separation was carried out on an Agilent Zorbax XDB-C18 (100 × 2.1 mm, 1.8 µm) column by isocratic elution with acetonitrile and water (50:50, v/v) at a flow rate of 0.25 mL/min with a total run time of 2.0 min. The MS/MS ion transitions monitored were 464.4 → 463.4 for hyperoside and 947.12 → 969.60 for IS. Linear responses were obtained for hyperoside ranging from 10 to 5000 ng/mL. The intra-and inter-day precisions (RSDs) were <5.38 and 3.39% and the extraction recovery ranged from 94.39 to 100.78% with an RSD <3.82%. Stability studies showed that hyperoside was stable in preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration-time profiles of hyperoside.

Simultaneous determination and pharmacokinetic study of polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII in beagle dog plasma after oral administration of Rhizoma Paridis extracts by LC-MS-MS.

For the first time, a rapid and specific LC-MS-MS method has been developed for the analysis of polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII in beagle dog plasma. The method was applied to study the pharmacokinetics of Rhizoma Paridis extracts containing polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII. The analysis was carried out on an Agilent Zorbax XDB-C(18) reversed-phase column (100 × 2.1 mm, 1.8 µm) by isocratic elution with acetonitrile and water (50:50, v/v). The flow rate was 0.25 mL/min. All analytes including internal standards were monitored by selected reaction monitoring with an electrospray ionization source. Linear responses were obtained for polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII ranging from 10 to 5000 ng/mL. The intra-and inter-day precisions (RSDs) were less than 6.66 and 9.15%. The extraction recovery ranged from 95.53 to 104.21% with RSD less than 8.69%. Stability studies showed that polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII were stable in preparation and analytical process. The validated method was successfully used to determine the concentration-time profiles of polyphyllin I, polyphyllin II, polyphyllin VI and polyphyllin VII.

Optimization of 5-hydroxytryptamines as dual function inhibitors targeting phospholipase A2 and leukotriene A4 hydrolase.

Dual function inhibitors targeting phospholipase A(2) (PLA(2)) and leukotriene A(4) hydrolase (LTA(4)H) may balance the arachidonic acid (AA) metabolic network and be used as new anti-inflammatory drugs. In previous study, we discovered multi-target drugs towards the AA metabolic network, among which a dual-target inhibitor (JMC08-4) for human nonpancreatic secretory phospholipase A(2) (hnps-PLA(2)) and human leukotriene A(4) hydrolase (LTA(4)H-h) was found. Based on the structure of compound JMC08-4, new dual-target inhibitors were designed assisted by molecular docking. In this report, a series of 5-hydroxytryptamine compounds were synthesized; and most of these title compounds showed more potent inhibitory activity than compound JMC08-4 in the in vitro bioassay against these two enzymes. The best one inhibited hnps-PLA(2) and LTA(4)H-h with IC(50) values of 9.2 ± 0.5 µM and 2.4 ± 1.4 µM, respectively.

Anxiolytic-like effects of compound zhi zhu xiang in rats.

The purpose of this study was to determine whether compound zhi zhu xiang (CZZX) exerts anxiolytic-like effects in rats. The animals were orally administered CZZX (0.75, 1.5, and 3 g/kg daily) for 10 days and tested in the elevated plus maze (EPM), Vogel conflict test (VCT), and open field. Repeated treatment with CZZX (3 g/kg/day, p.o.) significantly increased the percentage of both entries into and time spent on the open arms of the EPM compared with saline controls. In the VCT, repeated treatment with CZZX (1.5 and 3 g/kg/day, p.o.) significantly increased the number of punished licks. The drug did not change the total entries into the open arms of the EPM or interfere with water consumption or nociceptive threshold, discarding potential confounding factors in the two tests. In the open field, locomotion was not reduced, discarding the possible sedative effect of CZZX. In the binding assay, the binding of [(3)H] Ro 15-1788 (flumazenil) to the benzodiazepine binding site in washed crude synaptosomal membranes from rat cerebral cortex was affected by CZZX. These data indicate an anxiolytic-like profile of action for CZZX without sedative side effects, and this activity may be mediated by benzodiazepine binding site modulation at γ-aminobutyric acid-A receptors.

[Fluorescence spectra and protonation of ofloxacin in strong acidic solutions].

Fluorescence and UV-Vis spectra of ofloxacin (OFL) in sulfuric acid were studied. In the present paper, a new protonation state of OFL was observed. In hydrochloric acid, OFL produced bright green fluorescence upon excitation by UV radiation. The maximal emission wavelength of OFL is about 505 nm. However, OFL produces violet fluorescence when dissolved in concentrated sulfuric acid. The maximal emission wavelength changes into 400 nm. Further analysis demonstrated that the above changes arise from the variation of protonation states of OFL molecule. In dilute sulfuric acid, OFL accepted one proton, resulting in a protonation state that is similar to the OFL molecule dissolved HCl solution. The corresponding fluorescence band occurs at 505 nm. In concentrated sulfuric acid solution, OFL might accept additional protons. As a result, the size of the conjugated system is reduced and the fluorescence band exhibits a blue shift. In sulfuric acid of moderate concentrations, two bands at 505 and 400 nm respectively were found in the fluorescence emission spectra, indicating that OFL in two different protonation states coexists in the solution. In addition, both excitation band in excitation spectra and absorption bands in UV-Vis spectra exhibit red-shifted with the decrease in the concentration of sulfuric acid. Based on the above result, OFL can be used as a spectral probe to reflect the variation of H+ in strong acid environment.