MRE11-RAD50-NBS1 Complex Is Sufficient to Promote Transcription by RNA Polymerase II at Double-Strand Breaks by Melting DNA Ends.

The MRE11-RAD50-NBS1 (MRN) complex supports the synthesis of damage-induced long non-coding RNA (dilncRNA) by RNA polymerase II (RNAPII) from DNA double-strand breaks (DSBs) by an unknown mechanism. Here, we show that recombinant human MRN and native RNAPII are sufficient to reconstitute a minimal functional transcriptional apparatus at DSBs. MRN recruits and stabilizes RNAPII at DSBs. Unexpectedly, transcription is promoted independently from MRN nuclease activities. Rather, transcription depends on the ability of MRN to melt DNA ends, as shown by the use of MRN mutants and specific allosteric inhibitors. Single-molecule FRET assays with wild-type and mutant MRN show a tight correlation between the ability to melt DNA ends and to promote transcription. The addition of RPA enhances MRN-mediated transcription, and unpaired DNA ends allow MRN-independent transcription by RNAPII. These results support a model in which MRN generates single-strand DNA ends that favor the initiation of transcription by RNAPII.

From self-management to a systemized process: the implementation of lean management in a Chinese hospital's pharmacy intravenous admixture services center.

PURPOSE: The purpose of this paper is to explore the importance of lean leadership in an implementation in a Chinese hospital, considering a particular focus on the attitudes of nursing professionals while identifying specific cultural or institutional factors in China that might affect the implementation. DESIGN/METHODOLOGY/APPROACH: The authors use Harrison et al.'s (2016) framework to explore the outcomes of a nine-month action research project whereby the authors observed the process and outcomes of implementing lean in a pharmacy intravenous admixture service of a Chinese hospital. FINDINGS: The implementation of lean had positive results, which improved the efficiency of the operation, reduced the work start time and the amount of staff, and improved clinical satisfaction. In the process of implementation, nursing professionals showed a positive attitude toward the implementation and showed no obvious resistance under the positive influence of the head nurse. The combination of Chinese cultural characteristics, nursing culture and strong leadership enabled lean success. ORIGINALITY/VALUE: The unit moved from self-management to a systemized process of using lean concepts and methods, it is an important change for hospital managers.

CDK7 Inhibition Potentiates Genome Instability Triggering Anti-tumor Immunity in Small Cell Lung Cancer.

Cyclin-dependent kinase 7 (CDK7) is a central regulator of the cell cycle and gene transcription. However, little is known about its impact on genomic instability and cancer immunity. Using a selective CDK7 inhibitor, YKL-5-124, we demonstrated that CDK7 inhibition predominately disrupts cell-cycle progression and induces DNA replication stress and genome instability in small cell lung cancer (SCLC) while simultaneously triggering immune-response signaling. These tumor-intrinsic events provoke a robust immune surveillance program elicited by T cells, which is further enhanced by the addition of immune-checkpoint blockade. Combining YKL-5-124 with anti-PD-1 offers significant survival benefit in multiple highly aggressive murine models of SCLC, providing a rationale for new combination regimens consisting of CDK7 inhibitors and immunotherapies.

Productive Oilseed Rape Strips Supplement Seminatural Field-Margins in Promoting Ground-Dwelling Predatory Invertebrates in Agricultural Landscapes.

Intensively managed flowering crops like canola (Brassicales: Brassicaceae) (oilseed rape, OSR) provide significant short-term nectar resources for pollen consumers. They may also play important roles as annual "service strips" in temporarily promoting predatory invertebrates. We set out to test this assumption by comparing overall and functional group-specific species richness, activity density, and assemblage composition of carabids (Coleoptera: Carabidae) and spiders (Araneae), in three types of service strips-OSR, woody, and grassy strips established in direct vicinity to cropland. OSR strips were found to harbor the highest carabid species richness and activity density of small carabids. The activity density of carabids overall and of omnivorous species, the species richness and activity density of spiders across size classes and feeding strategies were all significantly reduced in woody strips. The percentage of seminatural habitat in the wider landscape was positively linked to the activity density of spiders overall, ground hunting and large spiders, whereas in carabids, positive effects were limited to large species occurring in grassy strips. Habitat type was the main predictor of both carabid and spider assemblage composition. Our results indicate that carabid and spider activity density across functional groups responded more strongly to changes in the landscape composition than the diversity of individual taxonomic groups. For agricultural landscape management, the establishment of habitat mosaics that include regular OSR could promote abundant, species-rich predatory invertebrates particularly in early spring. In contrast, structurally homogenous woody strips represent limited value in promoting the investigated biological pest control agents.

Fluorescence correlation spectroscopy for multiple-site equilibrium binding: a case of doxorubicin-DNA interaction.

Quantitative description of the interaction between doxorubicin (DOX), a broadly used anticancer drug, and DNA is the key to understand the action mechanism and side effects of its clinical use. However, the reported equilibrium constants of DOX-DNA interaction obtained using a range of different analytical methods vary even by several orders of magnitude. Herein, we propose a novel application of a single-molecule technique - fluorescence correlation spectroscopy (FCS) - to probe the interaction between DOX and two types of DNA (pUC19 and calf thymus DNA), taking advantage of intrinsic self-fluorescence of DOX. We provide an analytical formula for autocorrelation analysis to determine the equilibrium constant of DOX-DNA complex-formation, where binding of multiple DOX molecules to a DNA chain is included in the reaction-diffusion model. Our FCS-based method not only quantitatively revealed the values of equilibrium constant, but also implied that the stability of DOX-DNA complex is related to the types of base pair rather than the length or structure of the DNA. This work opens a promising pathway toward quantitative determination of molecular interactions in complex systems such as living cells or organisms at single-molecule level.

Effects of Plant Diversity, Vegetation Composition, and Habitat Type on Different Functional Trait Groups of Wild Bees in Rural Beijing.

The loss of flower-rich habitats and agricultural intensification have resulted in significant losses of wild bee diversity from agricultural landscapes that is increasingly threatening the pollination of zoochorous agricultural crops and agricultural sustainability. However, the links of different wild bee functional trait groups with habitat types and plant resources in agricultural landscapes remain poorly understood, thus impeding the formulation of effective policies for bee conservation. We therefore analyzed how bees representing different functional groups responded to variations in habitat type, vegetation composition and plant diversity. Natural shrubland sustained the highest diversity in bees overall, in large-sized bees, solitary bees and belowground-nesting bees, while each habitat harbored unique species. In half of the functional bee groups, species were negatively linked to tree coverage and herb coverage, respectively, while plant diversity was positively related to all functional groups except large-sized bees and aboveground-nesting bees. Overall bee abundance was positively related to abundance of plants in the Sympetalae, and negatively related to abundance of plants in the Archichlamydeae. Different bee functional groups showed distinct preferences for different plant communities. In order to conserve the diversity of wild bees across functional groups to optimize associated pollination services, a diverse habitat mosaic, and particularly plant species in Sympetalae need to be promoted in agricultural landscapes. Future studies should aim to enhance our understanding of plant-pollinator associations and specific food requirement of different wild bee species for their effective conservation.

Nanoscopic Approach to Quantification of Equilibrium and Rate Constants of Complex Formation at Single-Molecule Level.

Equilibrium and rate constants are key descriptors of complex-formation processes in a variety of chemical and biological reactions. However, these parameters are difficult to quantify, especially in the locally confined, heterogeneous, and dynamically changing living matter. Herein, we address this challenge by combining stimulated emission depletion (STED) nanoscopy with fluorescence correlation spectroscopy (FCS). STED reduces the length-scale of observation to tens of nanometres (2D)/attoliters (3D) and the time-scale to microseconds, with direct, gradual control. This allows one to distinguish diffusional and binding processes of complex-formation, even at reaction rates higher by an order of magnitude than in confocal FCS. We provide analytical autocorrelation formulas for probes undergoing diffusion-reaction processes under STED condition. We support the theoretical analysis of experimental STED-FCS data on a model system of dye-micelle, where we retrieve the equilibrium and rates constants. Our work paves a promising way toward quantitative characterization of molecular interactions in vivo.

Analytical form of the autocorrelation function for the fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) can provide information about diffusion coefficients and rate constants of chemical reactions in small systems of interacting molecules. However, the interpretation of FCS experiments depends crucially on the model of the autocorrelation function for the fluorescence intensity fluctuations. In this theoretical work, we consider a system of fluorescent molecules that diffuse and interact with massive particles, e.g. surfactant micelles. Using the general formalism of FCS, we derive a new analytical approximation of the autocorrelation function for systems in which both diffusion and a binary reaction occur. This approximation provides a smooth interpolation between the limit of fast reaction (much faster than diffusion), and the opposite limit of slow reaction. Our studies of noncovalent interactions of micelles with dyes by FCS provided an experimental case to which the approximate autocorrelation function was successfully applied [X. Zhang, A. Poniewierski, A. Jelinska, A. Zagozdzon, A. Wisniewska, S. Hou and R. Holyst, Soft Matter, 2016, 12, 8186-8194].

Determination of equilibrium and rate constants for complex formation by fluorescence correlation spectroscopy supplemented by dynamic light scattering and Taylor dispersion analysis.

The equilibrium and rate constants of molecular complex formation are of great interest both in the field of chemistry and biology. Here, we use fluorescence correlation spectroscopy (FCS), supplemented by dynamic light scattering (DLS) and Taylor dispersion analysis (TDA), to study the complex formation in model systems of dye-micelle interactions. In our case, dyes rhodamine 110 and ATTO-488 interact with three differently charged surfactant micelles: octaethylene glycol monododecyl ether C12E8 (neutral), cetyltrimethylammonium chloride CTAC (positive) and sodium dodecyl sulfate SDS (negative). To determine the rate constants for the dye-micelle complex formation we fit the experimental data obtained by FCS with a new form of the autocorrelation function, derived in the accompanying paper. Our results show that the association rate constants for the model systems are roughly two orders of magnitude smaller than those in the case of the diffusion-controlled limit. Because the complex stability is determined by the dissociation rate constant, a two-step reaction mechanism, including the diffusion-controlled and reaction-controlled rates, is used to explain the dye-micelle interaction. In the limit of fast reaction, we apply FCS to determine the equilibrium constant from the effective diffusion coefficient of the fluorescent components. Depending on the value of the equilibrium constant, we distinguish three types of interaction in the studied systems: weak, intermediate and strong. The values of the equilibrium constant obtained from the FCS and TDA experiments are very close to each other, which supports the theoretical model used to interpret the FCS data.

The Hinge Region Strengthens the Nonspecific Interaction between Lac-Repressor and DNA: A Computer Simulation Study.

LacI is commonly used as a model to study the protein-DNA interaction and gene regulation. The headpiece of the lac-repressor (LacI) protein is an ideal system for investigation of nonspecific binding of the whole LacI protein to DNA. The hinge region of the headpiece has been known to play a key role in the specific binding of LacI to DNA, whereas its role in nonspecific binding process has not been elucidated. Here, we report the results of explicit solvent molecular dynamics simulation and continuum electrostatic calculations suggesting that the hinge region strengthens the nonspecific interaction, accounting for up to 50% of the micro-dissociation free energy of LacI from DNA. Consequently, the rate of microscopic dissociation of LacI from DNA is reduced by 2~3 orders of magnitude in the absence of the hinge region. We find the hinge region makes an important contribution to the electrostatic energy, the salt dependence of electrostatic energy, and the number of salt ions excluded from binding of the LacI-DNA complex.

How can macromolecular crowding inhibit biological reactions? The enhanced formation of DNA nanoparticles.

In contrast to the already known effect that macromolecular crowding usually promotes biological reactions, solutions of PEG 6k at high concentrations stop the cleavage of DNA by HindIII enzyme, due to the formation of DNA nanoparticles. We characterized the DNA nanoparticles and probed the prerequisites for their formation using multiple techniques such as fluorescence correlation spectroscopy, dynamic light scattering, fluorescence analytical ultracentrifugation etc. In >25% PEG 6k solution, macromolecular crowding promotes the formation of DNA nanoparticles with dimensions of several hundreds of nanometers. The formation of DNA nanoparticles is a fast and reversible process. Both plasmid DNA (2686 bp) and double-stranded/single-stranded DNA fragment (66 bp/nt) can form nanoparticles. We attribute the enhanced nanoparticle formation to the depletion effect of macromolecular crowding. This study presents our idea to enhance the formation of DNA nanoparticles by macromolecular crowding, providing the first step towards a final solution to efficient gene therapy.

Tracking structural transitions of bovine serum albumin in surfactant solutions by fluorescence correlation spectroscopy and fluorescence lifetime analysis.

The structural dynamics of proteins is crucial to their biological functions. A precise and convenient method to determine the structural changes of a protein is still urgently needed. Herein, we employ fluorescence correlation spectroscopy (FCS) to track the structural transition of bovine serum albumin (BSA) in low concentrated cationic (cetyltrimethylammonium chloride, CTAC), anionic (sodium dodecyl sulfate, SDS), and nonionic (pentaethylene glycol monododecyl ether, C12E5 and octaethylene glycol monododecyl ether, C12E8) surfactant solutions. BSA is labelled with the fluorescence dye called ATTO-488 (ATTO-BSA) to obtain steady fluorescence signals for measurements. We find that the diffusion coefficient of BSA decreases abruptly with the surfactant concentration in ionic surfactant solutions at concentrations below the critical micelle concentration (CMC), while it is constant in nonionic surfactant solutions. According to the Stokes-Sutherland-Einstein equation, the hydrodynamic radius of BSA in ionic surfactant solutions amounts to ∼6.5 nm, which is 1.7 times larger than in pure water or in nonionic surfactant solutions (3.9 nm). The interaction between BSA and ionic surfactant monomers is believed to cause the structural transition of BSA. We confirm this proposal by observing a sudden shift of the fluorescence lifetime of ATTO-BSA, from 2.3 ns to ∼3.0 ns, in ionic surfactant solutions at the concentration below CMC. No change in the fluorescence lifetime is detected in nonionic surfactant solutions. Moreover, by using FCS we are also able to identify whether the structural change of protein results from its self-aggregation or unfolding.

[Spatial distribution pattern of carabid assemblage in agricultural landscape of Miyun County, Beijing].

By the method of pitfall trap sampling, an investigation was conducted on the carabid assemblage in four typical habitats (maize field, peanut field, orchard, and semi-natural woodland) in the agricultural landscape in Xitiange Village of Miyun County, Beijing. Among the four habitats, orchard had the highest alpha-diversity of carabid assemblage, followed by woodland and maize field, and peanut field had the lowest one. The species composition of the assemblage in woodland, peanut field, and orchard had evident difference, but was similar to that in maize field to some extent. The number of the individuals of predatory and omnivorous carabid sub-assemblages was larger in orchard, and the predatory sub-assemblage presented more distinct difference in its species turnover rate than the omnivore sub-assemblage among the habitats. This study showed that low-intensively managed orchard habitat could sustain higher alpha-diversity of carabid assemblage than semi-natural woodland habitat, and the landscape with diversified habitats could benefit the beta-diversity of carabid assemblage and its predatory sub-assemblage. To attach importance to the conservation of diversified habitats and low-intensively managed farmland habitat would have significance for the conservation of carabid assemblage diversity and its pest control function in agricultural landscape.

Fabrication of biodegradable micelles with reduction-triggered release of 6-mercaptopurine profile based on disulfide-linked graft copolymer conjugate.

This research is aimed to develop a biodegradable micelle delivery system with sheddable poly (ethylene glycol) shell to achieve the reduction-triggered intracellular sustained release of 6-mercaptopurine (6-MP) and decreased toxicity. Firstly, the amino-disulfide linked poly (ethylene glycol) monomethyl ether (mPEG-SS-NH(2)) was synthesized by the amidation reaction between cystamine and active ester of mPEG and p-nitrophenyl chloroformate (p-NPC) (mPEG-NPC). And then, the five-member rings in poly (l-succinimide) (PSI) were successively opened by mPEG-SS-NH(2) and 2-(pyridyldithio)-ethylamine (PDA) to produce the graft copolymer of mPEG-SS-NH-graft-PAsp-PDA. To avoid the drug initial burst, 6-MP was covalently conjugated with mPEG-SS-NH-graft-PAsp-PDA by thoil-disulfide exchange reaction to give the resultant product mPEG-SS-NH-graft-PAsp-MP. The product was found to form spherical micelles in aqueous media because of its amphiphilic nature with average particle size of 160 nm measured by dynamic light scattering (DLS). It was found that the mPEG-SS-NH-graft-PAsp-MP micelles, though stable in phosphate buffer solution (PBS), were prone to aggregation in the presence of dithiothreitol (DTT). The in vitro drug release studies revealed the release of 6-MP were distinct from the conventional micelles whose drugs loaded by physical encapsulation. Sustained release profile of 6-MP over 85 h was found in the presence of DTT (40 mM) simulating the intracellular condition while minimal drug release was observed within 24h at the level of DTT corresponding to extracellular environment. Remarkably, the cell viability results showed there was essential decrease of cytotoxicity to HL-60 cell line compared to free 6-MP.

Anti-tumor efficacy of polymer-platinum(II) complex micelles fabricated from folate conjugated PEG-graft-α,ß-poly [(N-amino acidyl)-aspartamide] and cis-dichlorodiammine platinum(II) in tumor-bearing mice.

To develop a tumor-targeting nano-sized delivery system of cis-dichlorodiammine platinum(II) (CDDP), polymer-metal complex micelles were fabricated from folate-conjugated PEG-graft-α,ß-poly [(N-amino acidyl)-aspartamide] (FA-PEG-g-PAAsp) and CDDP. The formation of polymer-metal complex micelles was confirmed by the measurements of critical aggregation concentration (CAC) and particle size, and the morphological observation. It was found that all the micelles showed spherical shapes with clear core-shell structures in narrow size distributions. The typical particle size measured by dynamic laser scattering (DLS) was ca. 105 nm, suggesting their passive targeting to tumor tissue and endocytosis potential. FA-PEG-g-PAAsp-CDDP micelles showed sustained drug release profiles over 40 h, and their accumulative drug release was ranked in the order of FA-PEG-g-PAsp-Ami-CDDPFA-PEG-g-PAAsp-CDDP>mPEG-g-PAAsp-CDDP, the severe toxicity of CDDP in vivo limited its use as ideal anti-tumor drug. Furthermore, FA-PEG-g-PAAsp-CDDP and mPEG-g-PAAsp-CDDP showed rather low toxicity against mice, just similar to that of PBS. It indicated the great potential utilization of the FA-PEG-g-PAAsp-CDDP micelles as the tumor-targeted drug carriers of CDDP with improved anti-tumor efficacy.