Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing.

BACKGROUND: Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. RESULTS: In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. CONCLUSIONS: Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

Ir-Catalyzed Asymmetric Tandem Allylation/Iso-Pictet-Spengler Cyclization Reaction for the Enantioselective Construction of Tetrahydro-γ-carbolines.

Ir-catalyzed asymmetric tandem allylation/iso-Pictet-Spengler cyclization of arylidenaminomalonates with indolyl allylic methyl carbonates was successfully developed, which provided a direct and practical approach to access synthetically useful and biologically active tetrahydro-γ-carboline derivatives bearing multiple functional groups and stereogenic centers in good to high yields and excellent stereoselective control (44%-96% yields, >20:1 dr, 94% → 99% ee). A wide range of substrate generality, easily available substrates, and simple chiral catalytic system displayed great potential practicality of this efficient protocol.

A new entry to highly functionalized pyrroles via a cascade reaction of α-amino esters and alkynals.

Here, we developed an expedient access route to highly functionalized pyrroles from readily available α-amino acid ester hydrochlorides and alkynals via a cascade condensation/intramolecular cyclization followed by a unique C-N ester migration process. A variety of 1,2,3-trisubstituted pyrroles, which were difficult to acquire with the common methodologies, were successfully prepared in good yields under mild conditions.

Asymmetric synthesis of quaternary α-trifluoromethyl α-amino acids by Ir-catalyzed allylation followed by kinetic resolution.

Facile access to quaternary α-trifluoromethyl α-amino acids has been developed. This sequential reaction involves an Ir-catalyzed asymmetric allylation of α-trifluoromethyl aldimine esters followed by an unprecedented kinetic resolution.

Stereodivergent assembly of tetrahydro-γ-carbolines via synergistic catalytic asymmetric cascade reaction.

Enantiomerically enriched indole-containing heterocycles play a vital role in bioscience, medicine, and chemistry. As one of the most attractive subtypes of indole alkaloids, highly substituted tetrahydro-γ-carbolines are the basic structural unit in many natural products and pharmaceuticals. However, the syntheses of tetrahydro-γ-carbolines with high functionalities from readily available reagents are significant challenging. In particular, the stereodivergent syntheses of tetrahydro-γ-carbolines containing multi-stereogenic centers remain quite difficult. Herein, we report an expedient and stereodivergent assembly of tetrahydro-γ-carbolines with remarkably high levels of stereoselective control in an efficient cascade process from aldimine esters and indolyl allylic carbonates via a synergistic Cu/Ir catalyst system. Control experiments-guided optimization of synergistic catalysts and mechanistic investigations reveal that a stereodivergent allylation reaction and a subsequent highly stereoselective iso-Pictet-Spengler cyclization are the key elements to success.

Stereodivergent Synthesis of α,α-Disubstituted α-Amino Acids via Synergistic Cu/Ir Catalysis.

Cu/Ir dual catalysis has been developed for the stereodivergent α-allylation of aldimine esters. The method enables the preparation of a series of nonproteinogenic α-amino acids (α-AAs) bearing two contiguous stereogenic centers in high yield with excellent stereoselectivity. All four product stereoisomers could be obtained from the same set of starting materials via pairwise combination of two chiral catalysts. Notably, one-pot protocol could be successfully applied for the preparation of the bimetallic Cu/Ir complexes to simplify the manipulation of Cu/Ir dual catalysis. This method could be further utilized for the construction of the key intermediate of a bioactive pyrrolidine derivative and the concise synthesis of a plant growth regulator (2S,3S)-2-amino-3-cyclopropylbutanoic acid.

Synergistic Cu/Pd Catalysis for Enantioselective Allylic Alkylation of Aldimine Esters: Access to α,α-Disubstituted α-Amino Acids.

An unprecedented enantioselective allylic alkylation of readily available aldimine esters has been developed, and is catalyzed by a synergistic Cu/Pd catalyst system. This strategy provides facile access to nonproteinogenic α,α-disubstituted α-amino acids in high yield with excellent enantioselectivity. The more challenging double allylic alkylation of glycinate-derived imine esters was also realized. Furthermore, this methodology was applied for the construction of the key intermediate of PLG peptidomimetics.

Predicting liver metastasis of gastrointestinal tract cancer by diffusion-weighted imaging of apparent diffusion coefficient values.

AIM: To determine if efficacy of chemotherapy on liver metastasis of gastrointestinal tract cancer can be predicted by apparent diffusion coefficient (ADC) values of diffusion-weighted imaging (DWI). METHODS: In total, 86 patients with liver metastasis of gastrointestinal tract cancer (156 metastatic lesions) diagnosed in our hospital were included in this study. The maximum diameters of these tumors were compared with each other before treatment, 2 wk after treatment, and 12 wk after treatment. Selected patients were classified as the effective group and the ineffective group, depending on the maximum diameter of the tumor after 12 wk of treatment; and the ADC values at different treatment times between the two groups were compared. Spearman rank correlation was used to analyze the relationship between ADC value and tumor diameter. Receiver operating characteristic curve (ROC curve) was used to analyze the ADC values before treatment to predict the patient's sensitivity and specificity degree of efficacy to the chemotherapy. RESULTS: There was no difference in age between the two groups and in maximum tumor diameter before treatment and 2 wk after treatment. However, after 12 wk of treatment, maximum tumor diameter in the effective group was significantly lower than that in the ineffective group (P < 0.05). Before treatment, ADC values in the ineffective group were significantly higher than those in the effective group (P < 0.05). There was no difference in ADC values between the effective and ineffective groups after 2 and 12 wk of treatment. However, ADC values were significantly higher after 2 and 12 wk of treatment compared to before treatment in the effective group (P < 0.05). Spearman rank correlation analysis showed that ADC value before treatment and the reduced percentage of the maximum tumor diameter after 12 wk of treatment were negatively correlated, while the increase in the percentage of the ADC value 12 wk after treatment and the decrease in the percentage of the maximum tumor diameter were significantly positively correlated. The results of the ROC curve showed that ADC value with a chemotherapy ineffective threshold value of 1.14 × 10(-3) mm(2)/s before treatment had a sensitivity and specificity of 94.3% and 76.7%, respectively. CONCLUSION: DWI ADC values can be used to predict the response of patients with liver metastasis of gastrointestinal tract cancer to chemotherapy with high sensitivity and relatively high specificity.

An organocatalytic Michael-cyclization cascade of 4-oxa-α,ß-unsaturated carboxylic acids with aldehydes: facile synthesis of chiral γ-lactols and trisubstituted γ-lactones.

An organocatalytic Michael-cyclization cascade of aldehydes with 4-oxa-α,ß-unsaturated carboxylic acids has been developed, giving functionalized γ-lactols with high yields and enantioselectivities. The products could be easily transformed into complex trisubstituted γ-lactones and γ-lactams.

Ultrastructural uncoupling between T-tubules and sarcoplasmic reticulum in human heart failure.

AIMS: Chronic heart failure is a complex clinical syndrome with impaired myocardial contractility. In failing cardiomyocytes, decreased signalling efficiency between the L-type Ca(2+) channels (LCCs) in the plasma membrane (including transverse tubules, TTs) and the ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) underlies the defective excitation-contraction (E-C) coupling. It is therefore intriguing to know how the LCC-RyR signalling apparatus is remodelled in human heart failure. METHODS AND RESULTS: Stereological analysis of transmission electron microscopic images showed that the volume densities and the surface areas of TTs and junctional SRs were both decreased in heart failure specimens of dilated cardiomyopathy (DCM) and ischaemic cardiomyopathy (ICM). The TT-SR junctions were reduced by ~60%, with the remaining displaced from the Z-line areas. Moreover, the spatial span of individual TT-SR junctions was reduced by ~17% in both DCM and ICM tissues. In accordance with these remodelling, junctophilin-2 (JP2), a structural protein anchoring SRs to TTs, was down-regulated, and miR-24, a microRNA that suppresses JP2 expression, was up-regulated in both heart failure tissues. CONCLUSION: Human heart failure of distinct causes shared similar physical uncoupling between TTs and SRs, which appeared attributable to the reduced expression of JP2 and increased expression of miR-24. Therapeutic strategy against JP2 down-regulation would be expected to protect patients from cardiac E-C uncoupling.

Ultrastructural remodelling of Ca(2+) signalling apparatus in failing heart cells.

AIMS: The contraction of a heart cell is controlled by Ca(2+)-induced Ca(2+) release between L-type Ca(2+) channels (LCCs) in the cell membrane/T-tubules (TTs) and ryanodine receptors (RyRs) in the junctional sarcoplasmic reticulum (SR). During heart failure, LCC-RyR signalling becomes defective. The purpose of the present study was to reveal the ultrastructural mechanism underlying the defective LCC-RyR signalling and contractility. METHODS AND RESULTS: In rat models of heart failure produced by transverse aortic constriction surgery, stereological analysis of transmission electron microscopic images showed that the volume density and the surface area of junctional SRs and those of SR-coupled TTs were both decreased in failing heart cells. The TT-SR junctions were displaced or missing from the Z-line areas. Moreover, the spatial span of individual TT-SR junctions was markedly reduced in failing heart cells. Numerical simulation and junctophilin-2 knockdown experiments demonstrated that the decrease in junction size (and thereby the constitutive LCC and RyR numbers) led to a scattered delay of Ca(2+) release activation. CONCLUSIONS: The shrinking and eventual absence of TT-SR junctions are important mechanisms underlying the desynchronized and inhomogeneous Ca(2+) release and the decreased contractile strength in heart failure. Maintaining the nanoscopic integrity of TT-SR junctions thus represents a therapeutic strategy against heart failure and related cardiomyopathies.

Beta-adrenergic signaling accelerates and synchronizes cardiac ryanodine receptor response to a single L-type Ca2+ channel.

As the most prototypical G protein-coupled receptor, beta-adrenergic receptor (betaAR) regulates the pace and strength of heart beating by enhancing and synchronizing L-type channel (LCC) Ca(2+) influx, which in turn elicits greater sarcoplasmic reticulum (SR) Ca(2+) release flux via ryanodine receptors (RyRs). However, whether and how betaAR-protein kinase A (PKA) signaling directly modulates RyR function remains elusive and highly controversial. By using unique single-channel Ca(2+) imaging technology, we measured the response of a single RyR Ca(2+) release unit, in the form of a Ca(2+) spark, to its native trigger, the Ca(2+) sparklet from a single LCC. We found that acute application of the selective betaAR agonist isoproterenol (1 microM, < or = 20 min) increased triggered spark amplitude in an LCC unitary current-independent manner. The increased ratio of Ca(2+) release flux underlying a Ca(2+) spark to SR Ca(2+) content indicated that betaAR stimulation helps to recruit additional RyRs in synchrony. Quantification of sparklet-spark kinetics showed that betaAR stimulation synchronized the stochastic latency and increased the fidelity (i.e., chance of hit) of LCC-RyR intermolecular signaling. The RyR modulation was independent of the increased SR Ca(2+) content. The PKA antagonists Rp-8-CPT-cAMP (100 microM) and H89 (10 microM) both eliminated these effects, indicating that betaAR acutely modulates RyR activation via the PKA pathway. These results demonstrate unequivocally that RyR activation by a single LCC is accelerated and synchronized during betaAR stimulation. This molecular mechanism of sympathetic regulation will permit more fundamental studies of altered betaAR effects in cardiovascular diseases.

[Study on the effect of intervention about acquired immunodeficiency syndrome among men who have sex with men].

OBJECTIVE: To study the outcomes of AIDS intervention programs and to provide scientific evidence for developing pertinent strategy on intervention among men who have sex with men (MSM). METHODS: MSM were recruited through snowballing and investigated by face to face interview in 2006 and 2007 respectively. SPSS 12.0 was used to compare the change of cognition about AIDS, sexual behavior, prevalence rates of HIV and Syphilis before and after the intervention program among the population under study. RESULTS: The cognition about AIDS among MSM was obviously improved after the intervention with the rate increased from 74.3% to 82.4% (P = 0.01). The rate of last time condom use among MSM increased from 56.4% to 65.5% (P = 0.00). The rate of consistent condom use during six months among MSM increased from 31.8% to 41.9% (P = 0.00). The rates of both condom use during commercial sex with men and with women did not change much among MSM. The prevalence rates of HIV in 2006 and 2007 were 10.4% and 10.8% and of syphilis in 2006 and 2007 were 9.3% and 7.3% respectively which were not significantly different between before and after the intervention. CONCLUSION: The cognition about AIDS among MSM was improved obviously. However, the rate of consistent condom use was still low, reflecting the segregation phenomenon between their behavior and cognition. All our findings implied that it was crucial to carry out AIDS prevention and control programs.

Intermolecular failure of L-type Ca2+ channel and ryanodine receptor signaling in hypertrophy.

Pressure overload-induced hypertrophy is a key step leading to heart failure. The Ca(2+)-induced Ca(2+) release (CICR) process that governs cardiac contractility is defective in hypertrophy/heart failure, but the molecular mechanisms remain elusive. To examine the intermolecular aspects of CICR during hypertrophy, we utilized loose-patch confocal imaging to visualize the signaling between a single L-type Ca(2+) channel (LCC) and ryanodine receptors (RyRs) in aortic stenosis rat models of compensated (CHT) and decompensated (DHT) hypertrophy. We found that the LCC-RyR intermolecular coupling showed a 49% prolongation in coupling latency, a 47% decrease in chance of hit, and a 72% increase in chance of miss in DHT, demonstrating a state of "intermolecular failure." Unexpectedly, these modifications also occurred robustly in CHT due at least partially to decreased expression of junctophilin, indicating that intermolecular failure occurs prior to cellular manifestations. As a result, cell-wide Ca(2+) release, visualized as "Ca(2+) spikes," became desynchronized, which contrasted sharply with unaltered spike integrals and whole-cell Ca(2+) transients in CHT. These data suggested that, within a certain limit, termed the "stability margin," mild intermolecular failure does not damage the cellular integrity of excitation-contraction coupling. Only when the modification steps beyond the stability margin does global failure occur. The discovery of "hidden" intermolecular failure in CHT has important clinical implications.