Ruthenium Phenoxo Complexes: An Isolobal Ligand to Cp with Improved Properties.

Catalytic π-arene activation is based on catalysts that allow for arene exchange. To date, cyclopentadiene (Cp)-derived catalysts are the most commonly used in π-arene activation despite their low arene exchange rates. Herein, we report the synthesis, analysis, and catalytic application of Ru(II) complexes supported by phenoxo ligands, which are isolobal alternatives to Cp. The phenoxo complexes exhibit arene exchange rates significantly faster than those of the corresponding Cp complexes. The rate can be further increased through the choice of appropriate counterions. The mechanism of the arene exchange process is elucidated by kinetic and computational analyses. We demonstrate the utility of the new catalysts through an SNAr reaction between fluorobenzene and alcohols, including secondary alcohols that could not be used previously in related reactions. Moreover, the catalytic thermal decarboxylation of phenylacetic acids is presented.

Screening of polysaccharides from the differently processed products of Angelica sinensis with the best liver protection effect on chicken and the intervention mechanism study based on tandem mass tag proteomics and multiple reaction monitoring approach.

The incidence of colibacillosis in poultry is on the rise, significantly affecting the chicken industry. Ceftiofur sodium (CS) is frequently employed to treat this disease, resulting in lipopolysaccharide (LPS) buildup. Processing plays a vital role in traditional Chinese veterinary medicine. The potential intervention in liver injury by polysaccharides from the differently processed products of Angelica sinensis (PDPPAS) induced by combined CS and LPS remains unclear. This study aims to investigate the protective effect of PDPPAS on chicken liver injury caused by CS combined with LPS buildup and further identify the polysaccharides with the highest hepatoprotective activity in chickens. Furthermore, the study elucidates polysaccharides' intervention mechanism using tandem mass tag (TMT) proteomics and multiple reaction monitoring (MRM) methods. A total of 190 1-day-old layer chickens were randomly assigned into 12 groups, of which 14 chickens were in the control group and 16 in other groups, for a 10-day trial. The screening results showed that charred A. sinensis polysaccharide (CASP) had the most effective and the best hepatoprotective effect at 48 h. TMT proteomics and MRM validation results demonstrated that the intervention mechanism of the CASP high-dose (CASPH) intervention group was closely related to the protein expressions of FCER2, TBXAS1, CD34, AGXT, GCAT, COX7A2L, and CYP2AC1. Conclusively, the intervention mechanism of CASPH had multitarget, multicenter regulatory features.

RNA methyltransferase BmMettl3 and BmMettl14 in silkworm (Bombyx mori) and the regulation of silkworm embryonic development.

N6-methyladenosine (m6A) is a ubiquitous reversible epigenetic RNA modification that plays an important role in regulating many biological processes, especially embryonic development. However, regulation of m6A methylation during silkworm embryonic development and diapause remains to be investigated. In this study, we analyzed the phylogeny of subunits of methyltransferases BmMettl3 and BmMettl14, and detected the expression patterns of BmMettl3 and BmMettl14 in different tissues and at different developmental stages in silkworm. To investigate the function of m6A on the development of silkworm embryo, we analyzed the m6A/A ratio in diapause and diapause termination eggs. The results showed that BmMettl3 and BmMettl14 were highly expressed in gonads and eggs. Moreover, the expression of BmMettl3 and BmMettl14 and the m6A/A ratio were significantly increased in diapause termination eggs compared with diapause eggs in the early stage of silkworm embryonic development. Furthermore, in BmN cell cycle experiments, the percentage of cells in the S phase increased when lacking BmMettl3 or BmMettl14. This work contributes to understanding the role of m6A methylation during insect embryogenesis and gametogenesis. It also provides a research orientation to further analyze the role of m6A methylation in diapause initiation and termination during insect embryonic development.

[Changes in Plasma Amyloid-ß Level and Their Relationship With White Matter Microstructure in Patients With Mild Cognitive Impairment].

Objective To investigate the changes in plasma amyloid-ß (Aß) level and their relationship with white matter microstructure in the patients with amnesic mild cognitive impairment(aMCI) and vascular mild cognitive impairment (vMCI).Methods A total of 36 aMCI patients,20 vMCI patients,and 34 sex and age matched healthy controls (HC) in the outpatient and inpatient departments of the First Affiliated Hospital of Anhui Medical University were enrolled in this study.Neuropsychological scales,including the Mini-Mental State Examination,the Montreal Cognitive Assessment,and the Activity of Daily Living Scale,were employed to assess the participants.Plasma samples of all the participants were collected for the measurement of Aß42 and Aß40 levels.All the participants underwent magnetic resonance scanning to obtain diffusion tensor imaging (DTI) data.The DTI indexes of 48 white matter regions of each individual were measured (based on the ICBM-DTI-81 white-matter labels atlas developed by Johns Hopkins University),including fractional anisotropy (FA) and mean diffusivity (MD).The cognitive function,plasma Aß42,Aß40,and Aß42/40 levels,and DTI index were compared among the three groups.The correlations between the plasma Aß42/40 levels and DTI index of aMCI and vMCI patients were analyzed.Results The Mini-Mental State Examination and the Montreal Cognitive Assessment scores of aMCI and vMCI groups were lower than those of the HC group (all P<0.001).There was no significant difference in the Activity of Daily Living Scale score among the three groups (P=0.654).The plasma Aß42 level showed no significant difference among the three groups (P=0.227).The plasma Aß40 level in the vMCI group was higher than that in the HC group (P=0.014),while it showed no significant difference between aMCI and HC groups (P=1.000).The plasma Aß42/40 levels in aMCI and vMCI groups showed no significant differences from that in the HC group (P=1.000,P=0.105),while the plasma Aß42/40 level was lower in the vMCI group than in the aMCI group (P=0.016).The FA value of the left anterior limb of internal capsule in the vMCI group was lower than those in HC and aMCI groups (all P=0.001).The MD values of the left superior corona radiata,left external capsule,left cingulum (cingulate gyrus),and left superior fronto-occipital fasciculus in the vMCI group were higher than those in HC (P=0.024,P=0.001,P=0.003,P<0.001) and aMCI (P=0.015,P=0.004,P=0.019,P=0.001) groups,while the MD values of the right posterior limb of internal capsule (P=0.005,P=0.001) and left cingulum (hippocampus) (P=0.017,P=0.031) in the aMCI and vMCI groups were higher than those in the HC group.In the aMCI group,plasma Aß42/40 level was positively correlated with FA of left posterior limb of internal capsule (r=0.403,P=0.015) and negatively correlated with MD of the right fonix (r=-0.395,P=0.017).In the vMCI group,plasma Aß42/40 level was positively correlated with FA of the right superior cerebellar peduncle and the right anterior limb of internal capsule (r=0.575,P=0.008;r=0.639,P=0.002),while it was negatively correlated with MD of the right superior cerebellar peduncle and the right anterior limb of internal capsule (r=-0.558,P=0.011;r=-0.626,P=0.003).Conclusions Plasma Aß levels vary differently in the patients with aMCI and vMCI.The white matter regions of impaired microstructural integrity differ in the patients with different dementia types in the early stage.The plasma Aß levels in the patients with aMCI and vMCI are associated with the structural integrity of white matter,and there is regional specificity between them.

Construction of a Structure-Switchable Toehold Dumbbell Probe for Sensitive and Label-Free Measurement of MicroRNA in Cancer Cells and Tissues.

MicroRNAs (miRNAs) play multiple crucial roles in post-transcriptional regulating gene expression, and the abnormal expression may induce various human diseases. Herein, we demonstrate the construction of a structure-switchable toehold dumbbell probe for sensitive and label-free measurement of microRNA in cancer cells and tissues on the basis of integrating exponential-rolling circle amplification (EXP-RCA) with linear-rolling circle amplification (LRCA). We designed a structure-switchable toehold dumbbell probe with annular and symmetric structure whose either side can hybridize with target miRNA to initiate EXP-RCA, greatly improving the detection sensitivity. Moreover, the dumbbell probe is designed with an appropriate standard free energy (G), and it cannot be activated by mismatched miRNAs, endowing this assay with good specificity. When target miRNA is present, it interacts with the dumbbell probe to activate EXP-RCA via toehold-mediated stand displacement, generating abundant triggers. The resulting triggers and target miRNA can function as primers to initiate LRCA, producing abundant long tandem repeats that can generate a distinct fluorescence signal using SYBR Gold as the indicator. This assay can be carried out homogeneously and isothermally without the requirement for either sophisticated modification/separation steps or any extra primers. It displays ultrahigh sensitivity with a limit of detection of 8.45 × 10-17 M and excellent specificity, and it can differentiate let-7a from its homologous analogues. Moreover, this method can accurately quantify let-7a expression at a single-cell level and can even distinguish the let-7a expression between non-small cell lung cancer patient tissues and healthy person tissues.

Construction of Bioluminescent Sensors for Label-Free, Template-Free, Separation-Free, and Sequence-Independent Detection of both Clustered and Isolated Damage in Genomic DNA.

DNA damage induced by endogenous/exogenous factors may cause various diseases, and the genomic DNA damage has become an important biomarker for clinical diagnosis and risk assessment, but it remains a great challenge to accurately quantify both clustered and isolated damage because of their random locations, large diversity, and low abundance. Herein, we demonstrate the development of bioluminescent sensors for label-free, template-free, separation-free, and sequence-independent detection of both clustered and isolated damage in genomic DNA based on the base-excision repair (BER) pathway and terminal transferase (TdT)-initiated template-free isothermal cyclic amplification. The damaged bases are cleaved by DNA glycosylase to generate a new 3'-OH terminus, and subsequently, TdT catalyzes the repeated incorporation of dTTPs into the 3'-OH terminus to produce poly-T structures which can hybridize with the signal probe to form a poly-T sequence/signal probe duplex. Under the lambda exonuclease hydrolysis, a large number of adenosine monophosphate (AMP) molecules are produced to generate a high bioluminescence signal through the cyclic interconversion of AMP-adenosine triphosphate (ATP)-AMP in the presence of luciferin and firefly luciferase. Moreover, the introduction of APE1-induced cyclic cleavage signal amplification can greatly improve the detection sensitivity. The proposed strategy can detect both clustered and isolated damage in genomic DNA with extremely high sensitivity and excellent specificity, and it can even distinguish 0.001% DNA damage in the mixture. Importantly, it can detect the cellular DNA damage with a detection limit of 0.011 ng and further extend to measure various DNA damage with the integration of appropriate DNA repair enzymes.

Construction of an Entropy-Driven Dumbbell-Type DNAzyme Assembly Circuit for Lighting Up Uracil-DNA Glycosylase in Living Cells.

Sensitive monitoring of intracellular uracil-DNA glycosylase (UDG) in living cells is essential to understanding the DNA repair pathways and discovery of anticancer drugs. Herein, we demonstrate the construction of an entropy-driven dumbbell-type DNAzyme assembly circuit for lighting up UDG in living cells via the integration of entropy-driven DNA catalysis (EDC) with the DNAzyme biocatalyst. Target UDG excises the damaged uracil base, causing the breakage of detection probe and the release of trigger. The released trigger can initiate the downstream EDC reaction to form two catalytically active DNAzyme units. The resultant dual Mg2+-DNAzyme units serve as the signal transducers to cyclically cleave the fluorophore/quenched-modified reporters, generating an enhanced fluorescence signal. In contrast to the single-layered EDC method with a linear amplification, the proposed doublet EDC-DNAzyme strategy exhibits high signal gain and achieves a detection limit of 8.71 × 10-6 U/mL. Notably, this assay can be performed in one-step manner at room temperature without the requirement of strict temperature control and complicated reaction procedures, and it can further screen the UDG inhibitors, measure kinetic parameters, and discriminate cancer cells from normal cells. Moreover, this strategy can monitor intracellular UDG activity with improved signal gain, and it may be exploited for sensing and imaging of other types of DNA modifying enzymes with the integration of the corresponding detection substrate, providing a facile and robust approach for biological research studies and clinical diagnosis.

MotifCNN-fold: protein fold recognition based on fold-specific features extracted by motif-based convolutional neural networks.

Protein fold recognition is one of the most critical tasks to explore the structures and functions of the proteins based on their primary sequence information. The existing protein fold recognition approaches rely on features reflecting the characteristics of protein folds. However, the feature extraction methods are still the bottleneck of the performance improvement of these methods. In this paper, we proposed two new feature extraction methods called MotifCNN and MotifDCNN to extract more discriminative fold-specific features based on structural motif kernels to construct the motif-based convolutional neural networks (CNNs). The pairwise sequence similarity scores calculated based on fold-specific features are then fed into support vector machines to construct the predictor for fold recognition, and a predictor called MotifCNN-fold has been proposed. Experimental results on the benchmark dataset showed that MotifCNN-fold obviously outperformed all the other competing methods. In particular, the fold-specific features extracted by MotifCNN and MotifDCNN are more discriminative than the fold-specific features extracted by other deep learning techniques, indicating that incorporating the structural motifs into the CNN is able to capture the characteristics of protein folds.

DeepSVM-fold: protein fold recognition by combining support vector machines and pairwise sequence similarity scores generated by deep learning networks.

Protein fold recognition is critical for studying the structures and functions of proteins. The existing protein fold recognition approaches failed to efficiently calculate the pairwise sequence similarity scores of the proteins in the same fold sharing low sequence similarities. Furthermore, the existing feature vectorization strategies are not able to measure the global relationships among proteins from different protein folds. In this article, we proposed a new computational predictor called DeepSVM-fold for protein fold recognition by introducing a new feature vector based on the pairwise sequence similarity scores calculated from the fold-specific features extracted by deep learning networks. The feature vectors are then fed into a support vector machine to construct the predictor. Experimental results on the benchmark dataset (LE) show that DeepSVM-fold obviously outperforms all the other competing methods.

Metabonomics study on the hepatoprotective effect mechanism of polysaccharides from different processed products of Angelica sinensis on layer chickens based on UPLC-Q/TOF-MS/MS, multivariate statistical analysis and conjoint analysis.

Chicken colibacillosis is one of the most severe diseases in the poultry industry. Ceftiofur sodium (CS) is often used to treat it in clinical practice and lipopolysaccharide (LPS) accumulates in the chicken's body. Previous experimental studies found that CS combined with LPS could induce liver injury in layer chickens, and polysaccharides from charred Angelica sinensis(CASP) had a better hepatoprotective effect than polysaccharides from unprocessed Angelica sinensis(UASP). However, the intervention mechanism was unclear. Thus, UPLC-Q/TOF-MS/MS-based metabonomics and transcriptomics were used in this study to clarify the hepatoprotective effect mechanism of CASP and UASP in layer chickens. Transcriptomics and enzyme-linked immunosorbent assay were used for biological verification of some critical mutual metabolic pathways screened with metabonomics. The comprehensive analysis results showed that in a layer chicken liver injury model built with LPS and CS, 12 critical metabolic pathways were disturbed, involving 10 important differential metabolites. The hepatoprotective effect mechanism of CASP is related to the arachidonic acid metabolism and mTOR signaling pathways, involving nine important differential metabolites. In contrast, the hepatoprotective effect mechanism of UASP is related to the arachidonic acid metabolism pathway, involving six important differential metabolites.

A new method providing complementary explanation of the blood-enriching function and mechanism of unprocessed Angelica sinensis and its four kinds of processed products based on tissue-integrated metabolomics and confirmatory analysis.

Angelica sinensis (AS) is a common Traditional Chinese Medicine used for tonifying blood in China. Unprocessed AS and its four kinds of processed products (ASs) are used to treat blood deficiency syndrome in the country. The different blood-tonifying mechanisms of ASs remain unclear. In this work, a novel method integrating metabolomics and hematological and biochemical parameters was established to provide a complementary explanation of blood supplementation mechanism of ASs. Our results revealed that different ASs exhibited various blood supplementation effect, and that AS parched with alcohol demonstrated the best blood supplementation effect. Eight metabolites from liver tissue and 12 metabolites from spleen tissue were considered to be potential biomarkers. These biomarkers were involved in four metabolic pathways. Correlation analysis results showed that l-aspartic acid and l-alanine (spleen tissue), linoleic acid, and l-cystathionine (liver tissue) exhibited a high positive or negative correlation with the aforesaid biochemical indicators. The blood-supplementation effect mechanism of ASs were related to four metabolic pathways. l-Aspartic acid and l-alanine (spleen tissue), linoleic acid, and l-cystathionine (liver tissue) were the four key metabolites associated with the blood supplementation effect of ASs. This study gives a complementary explanation of the blood supplementation effect and mechanism of action of ASs.

Carbonyl umpolung as an organometallic reagent surrogate.

Construction of new carbon-carbon bonds is the cornerstone of organic chemistry. Organometallic reagents are amongst the most robust and versatile nucleophiles for this purpose. Polarization of the metal-carbon bonds in these reagents facilitates their reactions with a vast array of electrophiles to achieve chemical diversification. The dependence on stoichiometric quantities of metals and often organic halides as feedstock precursors, which in turn produces copious amounts of metal halide waste, is the key limitation of the classical organometallic reactions. Inspired by the classical Wolff-Kishner reduction converting carbonyl groups in aldehydes or ketones into methylene derivatives, our group has recently developed strategies to couple various alcohols, aldehydes, and ketones with a broad range of both hard and soft carbon electrophiles in the presence of catalytic amounts of transition metals, via the hydrazone derivatives: i.e., as organometallic reagent surrogates. This Tutorial Review describes the chronological development of this concept in our research group, detailing its creation in the context of a deoxygenation reaction and evolution to a more general carbon-carbon bond-forming strategy. The latter is demonstrated by the employment of carbonyl-derived alkyl carbanions in various transition-metal catalyzed chemical transformations, including 1,2-carbonyl/imine addition, conjugate addition, carboxylation, olefination, cross-coupling, allylation, alkylation and hydroalkylation.

Development of a Single Quantum Dot-Mediated FRET Nanosensor for Sensitive Detection of Single-Nucleotide Polymorphism in Cancer Cells.

Single-nucleotide polymorphisms (SNPs) are important hallmarks of human diseases. Herein, we develop a single quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) nanosensor with the integration of multiple primer generation rolling circle amplification (MPG-RCA) for sensitive detection of SNPs in cancer cells. This assay involves only a linear padlock probe for MPG-RCA. The presence of a mutant target facilitates the circularization of linear padlock probes to initiate RCA, producing three short single-stranded DNAs (ssDNAs) with the assistance of nicking endonuclease. The resulting ssDNAs can function as primers to induce cyclic MPG-RCA, resulting in the exponential amplification and generation of large numbers of linker probes. The linker probes can subsequently hybridize with the Cy5-labeled reporter probes and the biotinylated capture probes to obtain the sandwich hybrids. The assembly of these sandwich hybrids on the 605 nm-emission quantum dot (605QD) generates the 605QD-oligonucleotide-Cy5 nanostructures, resulting in efficient FRET from the 605QD to Cy5. This nanosensor is free from both the complicated probe design and the exogenous primers and has distinct advantages of high amplification efficiency, zero background signal, good specificity, and high sensitivity. It can detect SNPs with a large dynamic range of 8 orders of magnitude and a detection limit of 5.41 × 10-20 M. Moreover, this nanosensor can accurately distinguish as low as 0.001% mutation level from the mixtures, which cannot be achieved by previously reported methods. Furthermore, it can discriminate cancer cells from normal cells and even quantify SNP at the single-cell level.

A Host-Guest Interaction-Based and Metal-Organic Gel-Based Biosensor with Aggregation-Induced Electrochemiluminescence Enhancement for Methyltransferase Assay.

Metal-organic gels (MOGs) are new soft materials with the characteristics of high colloidal stability, superb luminescence properties, and facile synthesis. Herein, we develop for the first time a host-guest interaction-based and MOG-based biosensor with aggregation-induced electrochemiluminescence (ECL) enhancement for M.SssI methyltransferase (M.SssI MTase) assay. This biosensor employs a MOG as the luminophor and potassium persulfate as the coreactant, and the formation of the Ag-MOG from the aggregation of silver nanoclusters can induce significant ECL enhancement. Two complementary single-stranded DNAs (ssDNAs, i.e., biotinylated DNA-1 and Fc-labeled DNA-2) that contain specific recognition sequence 5'-CCGG-3' can form a double-stranded DNA (dsDNA) probe. In the absence of M.SssI MTase, the dsDNA probe will be digested by restriction endonuclease HpaII, leading to the release of Fc from magnetic beads (MBs). The ß-CD can specifically recognize the released Fc through guest-host interaction, resulting in the quenching of an ECL signal. In contrast, the presence of M.SssI MTase enables the formation of fully methylated dsDNA, which cannot be cleaved by HpaII, making Fc remain on the MB surface and consequently generating an improved ECL signal. This biosensor can specifically detect M.SssI MTase with a linear range of 0.05-100 U mL-1 and a limit of detection of 3.5 × 10-3 U mL-1, and it enables accurate detection of M.SssI MTase in human serum. In addition, it can be used for inhibitor screening, with wide applications in drug discovery and disease diagnosis.

Label-Free and Template-Free Chemiluminescent Biosensor for Sensitive Detection of 5-Hydroxymethylcytosine in Genomic DNA.

5-Hydroxymethylcytosine (5hmC) is a modified base present at low levels in various mammalian cells, and it plays essential roles in gene expression, DNA demethylation, and genomic reprogramming. Herein, we develop a label-free and template-free chemiluminescent biosensor for sensitive detection of 5hmC in genomic DNAs based on 5hmC-specific glucosylation, periodate (IO4+) oxidation, biotinylation, and terminal deoxynucleotidyl transferase (TdT)-assisted isothermal amplification strategy, which we term hmC-GLIB-IAS. This hmC-GLIB-IAS exhibits distinct advantages of bisulfite-free, improved sensitivity, and genome-wide analysis of 5hmC at constant reaction temperature without the involvement of either specially labeled nucleic acid probes or specific templates for signal amplification. This method can sensitively detect 5hmC with a detection limit of 2.07 × 10-13 M, and it can detect 5hmC in the whole genome DNA with a detection limit of 3.92 × 10-5 ng/µL. Moreover, this method can distinguish 5hmC from 5-methylcytosine (5mC) and cytosine (C) and even discriminate 0.1% 5hmC in the mixture of 5hmC-DNA and 5mC-DNA. Importantly, this hmC-GLIB-IAS strategy enables genome-wide analysis without the involvement of either isotope-labeled substrates or specific antibodies, providing a powerful platform to detect 5hmC in real genomic DNA with high reproducibility and accuracy.

Construction of a Dye-Sensitized and Gold Plasmon-Enhanced Cathodic Photoelectrochemical Biosensor for Methyltransferase Activity Assay.

DNA methyltransferases may function as important biomarkers of cancers and genetic diseases. Herein, we develop a dye-sensitized and gold plasmon-enhanced cathodic photoelectrochemical (PEC) biosensor on the basis of p-type covalent organic polymers (COPs) for the signal-on measurement of M.SssI methyltransferase (M.SssI MTase). The cathodic PEC biosensor is constructed by the in situ growth of p-type COP films onto a glass coated with indium tin oxide and the subsequent assembly of biotin- and HS-labeled double-stranded DNA (dsDNA) probes onto the COP film via biotin-streptavidin interaction. The dsDNA probe contains the recognition sequence of M.SssI MTase. The COP thin films possess a porous ultrathin nanosheet structure with abundant active sites, facilitating the generation of a high photocurrent compared with the hydrothermally synthesized ones. The presence of DNA methyltransferases can prevent the digestion of restriction endonuclease HpaII, consequently inducing the introduction of gold nanoparticles (AuNPs) to the dsDNA probes via the S-Au bond and the intercalation of rhodamine B (RhB) into the DNA grooves to produce a high photocurrent due to the dye-photosensitized enhancement and AuNP-mediated surface plasmon resonance. However, in the absence of M.SssI MTase, HpaII digests the dsDNA probes, and neither AuNPs nor RhB can be introduced onto the electrode surface, leading to a low photocurrent. This cathodic PEC biosensor possesses high sensitivity and good selectivity, and it can screen the inhibitors and detect M.SssI MTase in serum as well.

Copper-Catalyzed Conjugate Addition of Carbonyls as Carbanion Equivalent via Hydrazones.

Copper-catalyzed conjugate addition is a classic method for forming new carbon-carbon bonds. However, copper has never showed catalytic activity for umpolung carbanions in hydrazone chemistry. Herein, we report a facile conjugate addition of hydrazone catalyzed by readily available copper complexes at room temperature. The employment of mesitylcopper(I) and electron-rich phosphine bidentate ligand is a key factor affecting reactivity. The reaction allows various aromatic hydrazones to react with diverse conjugated compounds to produce 1,4-adducts in yields of about 20 to 99%.

Medication appropriateness on an acute geriatric care unit: the impact of the removal of a clinical pharmacist.

BACKGROUND: evidence is largely available indicating benefits to adding a pharmacist on acute care wards. The benefits of maintaining pharmacotherapeutic consultant services on a geriatric ward remain unexplored. OBJECTIVES: to determine the impact of the removal of a clinical pharmacist from an acute geriatric ward on patients' Medication Appropriateness Index (MAI) scores, admission-related outcomes and drug burdens. METHODS: researchers consulted the archives for records of patients admitted to the geriatric care unit before and after the pharmacist's withdrawal. The primary outcome of differential MAI scores and secondary outcomes of rehospitalisations, emergency department visits, durations of hospitalisation and differential drug count were compared pre- and post-intervention. An interrupted time series analysis regression model was used for the primary outcome. RESULTS: a total of 305 patients admitted before (n = 208) and after (n = 97) the pharmacist's withdrawal were included in the study. The intervention had a significant impact on the primary outcome, increasing the relative differential MAI score (adjusted mean) by 9.3 points (95% confidence interval 3.9-14.6). As for the secondary outcomes, differences in admission-related outcomes were non-significant but the mean differential drug count significantly increased post-intervention from 0.02 to 1.36 (P < 0.001). CONCLUSION: the removal of the pharmacist led to an increase in inappropriate drug prescription. Careful consideration should be given to decisions regarding the removal of the pharmacist from acute geriatric care teams.

5-Hydroxymethylcytosine Glucosylation-Triggered Helicase-Dependent Amplification-Based Fluorescent Biosensor for Sensitive Detection of ß-Glucosyltransferase with Zero Background Signal.

ß-glucosyltransferase (ß-GT) catalyzes the glucosylation of 5-hydroxymethylcytosine (5-hmC) to enable the survival of bacteriophage and parasite in host cells, and it is a critical tool enzyme for 5-hmC assay. However, few methods are available for ß-glucosyltransferase assay, and they usually have the drawbacks of radioactive contamination, high background, laborious procedures, and unsatisfactory sensitivity. Herein, we develop a new fluorescent biosensor with zero background signal for sensitive detection of ß-GT activity based on 5-hmC glucosylation-triggered helicase-dependent amplification (HDA). The detection probe we designed may act as both a probe for ß-GT recognition and a template for HDA amplification. The ß-GT-catalyzed 5-hmC glucosylation can protect the detection probes from both the cleavage by MfeI restrictive enzyme and the digestion by exonucleases I and III. The remaining detection probes can subsequently act as the templates for exponential HDA amplification to generate numerous double-stranded DNA products, which can be easily detected by SYBR Green I in a label-free manner. The zero background can be achieved by efficient elimination of primer-dimer nonspecific amplification and complete digestion of nonglucosylated detection probes. This biosensor exhibits high sensitivity and good specificity, and it can be further used to analyze ß-GT kinetic parameters and screen the inhibitors, providing a powerful platform for deeper understanding of ß-GT biological functions and promoting ß-GT-related epigenetic studies. Furthermore, this biosensor can be extended to detect various DNA-modifying enzymes by simply replacing the recognition sequence and restriction enzyme.

Development of Oxidation Damage Base-Based Fluorescent Probe for Direct Detection of DNA Methylation.

DNA methylation has become a promising epigenetic biomarker for cancer diagnosis, prognosis, and therapy monitoring. Herein, we demonstrate for the first time the development of a new oxidation damage base (8-oxo-7,8-dihydroguanine (8-oxoG))-modified fluorescent probe for direct detection of DNA methylation. This fluorescent probe is labeled with carboxy-X-rhodamine (ROX) and black hole quencher 2 (BHQ2) at the 5' and 3' termini, respectively, with one 8-oxoG base modification in the middle position, and it can discriminate the methylated cytosine from the unmethylated cytosine. The presence of target methylated DNA may induce the recycle cleavage of fluorescent probes with the assistance of human 8-oxoG DNA glycosylase 1 (hOGG1) enzyme, resulting in an enhanced fluorescence signal. In comparison with the reported bisulfite treatment-based indirect approaches, this fluorescent probe can be used for direct detection of DNA methylation under isothermal conditions without the requirement of a stringent primer/template design, any thermal cycling, and ligation procedures, greatly simplifying the experimental processes. Moreover, this fluorescent probe exhibits good specificity and high sensitivity, and it can distinguish a 0.01% methylation level even in the presence of excess unmethylated DNA. Furthermore, this fluorescent probe can be used to detect DNA methylation in genomic DNA extracted from human colon cancer cells, holding great potential in epigenetic study and early clinical diagnosis.