Synonymous edits in the Escherichia coli genome have substantial and condition-dependent effects on fitness.

CRISPR-Cas-based genome editing is widely used in bacteria at scales ranging from construction of individual mutants to massively parallel libraries. This procedure relies on guide RNA-directed cleavage of the genome followed by repair with a template that introduces a desired mutation along with synonymous "immunizing" mutations to prevent re-cleavage of the genome after editing. Because the immunizing mutations do not change the protein sequence, they are often assumed to be neutral. However, synonymous mutations can change mRNA structures in ways that alter levels of the encoded proteins. We have tested the assumption that immunizing mutations are neutral by constructing a library of over 50,000 edits that consist of only synonymous mutations in Escherichia coli. Thousands of edits had substantial effects on fitness during growth of E. coli on acetate, a poor carbon source that is toxic at high concentrations. The percentage of high-impact edits varied considerably between genes and at different positions within genes. We reconstructed clones with high-impact edits and found that 69% indeed had significant effects on growth in acetate. Interestingly, fewer edits affected fitness during growth in glucose, a preferred carbon source, suggesting that changes in protein expression caused by synonymous mutations may be most important when an organism encounters challenging conditions. Finally, we showed that synonymous edits can have widespread effects; a synonymous edit at the 5' end of ptsI altered expression of hundreds of genes. Our results suggest that the synonymous immunizing edits introduced during CRISPR-Cas-based genome editing should not be assumed to be innocuous.

Dynamic Fluorescence Sensing of Bromide Ions by Photochromic Bi(III)-Coordination Polymers Based on a Ligand Integrated by Naphthalene Diimides and Pyridinium in Solution and Films.

Bismuth(III)-based complexes have garnered increasing attention in fluorescence sensing due to their environmentally friendly and sustainable characteristics. A Bismuth(III) coordination polymer (CP),1-Cl based on a naphthalene diimides(NDI)-pyridinium is synthesized by an in situ reaction method. Notable for its sensitivity to visible light, 1-Cl shows excellent photochromic properties, and the integration of NDI and pyridinium in one ligand makes photogenerated radicals more stable. Structural analysis and theoretical calculations are employed to investigate the potential pathway of photoinduced electron transfer (ET) during the photochromic process. Notably, in aqueous solutions, 1-Cl displays an extraordinary fluorescence enhancement response to bromide ion (Br-), resulting in a distinct transition from yellow to orange in color. The potential mechanism of fluorescence sensing has been revealed through single-crystal X-ray diffraction analysis. This insight highlights a continuous substitution process where the Cl- ions are successively replaced by Br- ions. Consequently, a single-crystal-to-single-crystal transformation (SCSC) occurs, yielding the intermediate species, 1-Cl-Br, which ultimately transforms into the final product, 1-Br. Finally, the photochromic film is successfully prepared and applied to practical applications such as ink-free printing, information anti-counterfeiting, and the visual detection of Br- ions. This work combines photochromism with fluorescence sensing, broadening the research field and practical application of photochromic materials.

Three-Dimensional Viologen-Based Lanthanide-Organic Frameworks: Photochromism and Fluorescence Detection of Quinolone Antibiotics.

Quinolone antibiotic residues, norfloxacin (NORF) and ciprofloxacin (CIP), have attracted more attention due to their frequent detection in surface water and food field, which seriously threaten the health of animals and humans. Rapid and efficient detection of NORF and CIP is critical for environmental testing and ecosystems. Herein, two novel isostructural viologen-functionalized Ln(III) complexes [Ln2L0.5(IPA)3]n (Ln = Eu, 1; Tb, 2; L = N,N'-bis (2-carboxyethyl)-4,4'-bipyridridylium dichloride, H2IPA = isophthalic acid) with a three-dimensional structure have been synthesized solvothermally. Complexes 1 and 2 exhibited reversible photochromism under UV light. In addition, complex 1 exhibits excellent pH tolerance and can be seen as an efficient fluorescent probe for the detection of NORF and CIP with detection limits of 7.90 × 10-7 and 9.48 × 10-7 M, respectively. Furthermore, the good photoresponsive and outstanding fluorescent properties of 1 were further exploited in dual-function paper involving erasable inkless printing and detection of NORF and CIP. Our work reports a new strategy for recognizing NORF and CIP based on the luminescent color change of the viologen-based Ln-MOFs, providing a new direction for the development of multifunctional materials.

Conformation- and Coordination Mode-Dependent Stimuli-Responsive Salicylaldehyde Hydrazone Zn(II) Complexes.

Luminescent Zn(II) complexes that respond to external stimuli are of wide interest due to their potential applications. Schiff base with O,N,O-hydrazone shows excellent luminescence properties with multi-coordination sites for different coordination modes. In this work, three salicylaldehyde hydrazone Zn(II) complexes (1, 2a, 2b) were synthesized and their stimuli-responsive behaviors in different states were explored. Only complex 1 exhibits reversible and self-recoverable photochromic and photoluminescence properties in solution. This may be due to the configuration eversion and the excited-state intramolecular proton transfer (ESIPT) process. In the solid state, 2a has obvious mechanochromic luminescence property, which is caused by the destruction of intermolecular interactions and the transformation from crystalline state to amorphous state. 2a and 2b have delayed fluorescence properties due to effective halogen bond interactions in structures. 2a could undergo crystal-phase transformation into its polymorphous 2b by force/vapor stimulation. Interestingly, 2b shows photochromic property, which can be attributed to the electron transfer and generation of radicals induced by UV irradiation. Due to different conformations and coordination modes, the three Zn(II) complexes show different stimuli-responsive properties. This work presents the multi-stimuli-responsive behaviors of salicylaldehyde hydrazone Zn(II) complexes in different states and discusses the response mechanism in detail, which may provide new insights into the design of multi-stimuli-responsive materials.

Multistimuli-Responsive Materials Based on Zn(II)-Viologen Coordination Polymers and Their Applications in Inkless Print and Anticounterfeiting.

Recently, stimuli-responsive materials have attracted great attention, while most of them respond to single or two stimuli. Thus, it is essential to design multifunctional stimuli-responsive materials and develop their applications. The strategy that constructing high-dimensional coordination polymers facilitates the application scope of a viologen-based photochromic system is put forward and confirmed for the first time. Herein, a novel multistimuli-responsive viologen-based Zn-MOF with a two-dimensional framework has been successfully designed and synthesized. Complex 1 exhibits chromic behavior under a variety of external stimuli such as 365 nm UV, X-rays, heat, electricity, and ethylamine. More interestingly, the crystal state of complex 1 displays dual fluorescence and room-temperature phosphorescence (RTP) emission and emits a yellow afterglow when turning off the UV lamp. In addition, Eu(III)-functionalized hybrids, Eu3+@Zn-MOF, were prepared by coordinated postsynthetic modification based on viologen complexes for the first time. The sample of Eu3+@Zn-MOF inherits the photochromic characteristics of the viologen complexes and gives the distinctive fluorescence of the europium ions. Based on the multicolor switching of 1 and Eu3+@Zn-MOF, their possible practical utilization was successfully developed in the fields of inkless, erasable print media, electrochromic information tag printing, information encryption, and anticounterfeiting.

Solvent-Modulated Self-Assembly of Naphthalenediimide-Based Cd(II) Complexes and the Controllable Photochromism via Conformational Isomerization.

Rational regulation of the properties of photochromic materials is a challenging and meaningful work. In the present work, NDI-based complexes, namely, [Cd0.5(NDI)(HBDC)]·H2O (1) and a series of conformational isomers of {[Cd(NDI)0.5(BDC)]·MeCN}n (2), were synthesized by varying the solvent conditions (H2BDC = terephthalic acid, NDI = N,N'-bis(3-pyridylcarbonylhydrazine)-1,4,5,8-naphthalene diimide). Complex 1 exhibits a 0D mononuclear structure without photochromic behavior due to the bad conjugation of the naphthalene diimide moiety. The conformational isomers of complex 2 manifest a 3D network, showing ultra-fast photo-induced intermolecular electron transfer photochromic behavior under X-ray, UV, and visible light. However, they show different photochromic rates and coloring contrast upon photoirradiation, which originates from their difference in the distances of lone pair(COO)···π(NDI). This was realized via controlling the solvent ratio in the reaction system. In addition, compared to UV/X-ray light, 2 exhibits greater sensitivity to visible light and is an organic-inorganic hybrid material with photomodulated luminescence. Based on the excellent performance, complex 2 can be applied to filter paper, showing potential applications as an inkless printing medium and selective perception of ammonia and amine vapors in the solid state via different visual color changes.

De Winter electrocardiogram pattern due to type A aortic dissection: a case report.

BACKGROUND: De Winter electrocardiograph (ECG) pattern is an atypical presentation of acute myocardial infarction (AMI) due to severe stenosis of the left anterior descending (LAD). Complications of acute aortic dissection (AD) in the setting of acute myocardial infarction (AMI) with de Winter sign are relatively rare and physicians may easily miss the diagnosis of AD. We report a case of patient with acute chest pain and de Winter ECG pattern due to AD involving the left main coronary artery (LM), LAD and left circumflex artery (LCX). CASE PRESENTATION: A 57-year-old male patient was initially diagnosed with AMI and then the diagnosis of acute AD was supported by transthoracic echocardiograph (TTE). After two stents were implanted respectively into the proximal LM-LAD and LM-LCX, he recovered from cardiogenic shock. Two months later, the patient underwent the surgery of ascending aorta replacement. After the surgery, there was no obvious chest discomfort during follow-up. CONCLUSIONS: When an ECG shows a "de Winter pattern", we should also consider the possibility of AD which result in LAD occlusion. TTE is a useful tool in screening for AD. Further research is needed to prove that percutaneous coronary intervention (PCI) may be a useful treatment strategy in the case of AD leading to severe LAD occlusion and unstable hemodynamics when there's no condition to perform aortic replacement surgery immediately.

Evolutionary dynamics and structural consequences of de novo beneficial mutations and mutant lineages arising in a constant environment.

BACKGROUND: Microbial evolution experiments can be used to study the tempo and dynamics of evolutionary change in asexual populations, founded from single clones and growing into large populations with multiple clonal lineages. High-throughput sequencing can be used to catalog de novo mutations as potential targets of selection, determine in which lineages they arise, and track the fates of those lineages. Here, we describe a long-term experimental evolution study to identify targets of selection and to determine when, where, and how often those targets are hit. RESULTS: We experimentally evolved replicate Escherichia coli populations that originated from a mutator/nonsense suppressor ancestor under glucose limitation for between 300 and 500 generations. Whole-genome, whole-population sequencing enabled us to catalog 3346 de novo mutations that reached > 1% frequency. We sequenced the genomes of 96 clones from each population when allelic diversity was greatest in order to establish whether mutations were in the same or different lineages and to depict lineage dynamics. Operon-specific mutations that enhance glucose uptake were the first to rise to high frequency, followed by global regulatory mutations. Mutations related to energy conservation, membrane biogenesis, and mitigating the impact of nonsense mutations, both ancestral and derived, arose later. New alleles were confined to relatively few loci, with many instances of identical mutations arising independently in multiple lineages, among and within replicate populations. However, most never exceeded 10% in frequency and were at a lower frequency at the end of the experiment than at their maxima, indicating clonal interference. Many alleles mapped to key structures within the proteins that they mutated, providing insight into their functional consequences. CONCLUSIONS: Overall, we find that when mutational input is increased by an ancestral defect in DNA repair, the spectrum of high-frequency beneficial mutations in a simple, constant resource-limited environment is narrow, resulting in extreme parallelism where many adaptive mutations arise but few ever go to fixation.

Multistimulus Response of Two Tautomeric Zn(II) Complexes and Their White-Light Emission Based on Different Mechanisms.

A triphenylamine (TPA)-based 2H-quinazoline Zn(II) complex (Q-TPA-Zn) exhibiting dual fluorescence and phosphorescence emission in the solid state was designed and prepared. It possesses mechanochromic luminescence and thermochromic luminescence properties. In the solid state, the white afterglow luminescence could be observed at 77 K (CIExy: 0.27, 0.33) while cyan luminescence could be observed at 297 K. After thermolysis at 300 °C, Q-TPA-Zn could be transformed into Schiff base complex S-TPA-Zn with white fluorescence in the powder state (CIExy: 0.32, 0.38), in methanol (CIExy: 0.32, 0.39), and in dimethylformamide (CIExy: 0.26, 0.32) at room temperature. This arises from dual emission of normal* emission and tautomeric* emission induced by excited-state intramolecular proton transfer (ESIPT) from the benzimidazole NH group to the Schiff base N atom. Q-TPA-Zn could also be transformed into its isomeric form, S-TPA-Zn, through photochemical ring-opening reaction upon irradiation under 365 nm in the solution, exhibiting high-contrast photochromic luminescence. Interestingly, S-TPA-Zn could further be transformed into its zwitterionic isomer after continuous irradiation. The same ring-opening reaction could also take place for the orgainc compound Q-TPA via heating or 365 nm irradiation. The ring-opening reaction mechanism and ESIPT emission were interpreted via theoretical calculation.

A Multistimuli Responsive Crystalline Cd(II)-Viologen Coordination Polymer with Single-Crystal-Single-Crystal Transformation.

It is necessary to develop stable and fast multistimuli responsive materials due to the growing demand in our daily life. In this work, a new viologen-based Cd-complex (1) exhibits multiple thermochromic and photochromic behaviors through 10 states with 7 colors. For example, it responds to both Cu Kα/Mo Kα X-ray sources and UV dual light quickly with a color change from colorless to dark blue (1X) (Cu Kα/Mo Kα X-ray sources) and cyan (1-UV) (UV light), respectively. Interestingly, it exhibits a three-step coloration phenomenon when heated, which is unprecedented in viologen compounds. Crystal 1 undergoes a color change to pink, blue, and brown under 130, 180, and 240 °C, respectively. In addition, upon fumigation, both 1P and 1Q undergo a decoloration process to colorless (1K) and yellow (1T), respectively. Four more states (1P, 1K, 1T, and 1O) obtained via dehydration-hydration treatment are all photochromic. More importantly, via single-crystal-single-crystal transformation (SC-SC), the photochromic and thermochromic behaviors of 1 were investigated from the molecular level, which is also rather rare for thermochromic species. The detailed electron donor and the pathways for electron transfer were clearly given according to the results of crystal structure. The colorful states upon external stimuli may be attributed to the multiple pathways for electron transfer.

Multistimuli Responsive Solid-State Emission of a Zinc(II) Complex with Multicolour Switching.

The development of smart luminescent materials, especially those stimulus-responsive fluorescent materials that can switch between different colors repeatedly under external stimulation based on a single molecule, is of great significance but a challenge. In this work, a novel zinc(II)-Schiff base complex (ZnL2) was obtained and characterized. Upon exposure to the HCl and NH3 vapors, it displayed remarkable tricolor acidochromic behavior with high contrast and rapid response under the ambient light as well as UV light (365 nm). The XPS analyses of ZnL2 crystals before and after HCl/NH3 fuming show that the acidochromism originates principally from the adsorption of vapor and the gas-solid reaction equilibrium on the crystal surface. The reddish-brown color of the HCl-fumigated ZnL2 crystals could be attributed to the generation of HL at the surface of ZnL2, and red-shifted emission could be ascribed to the self-absorption effect. The single crystal X-ray diffraction data indicate that these processes cause slight changes in the molecular conformation and crystal packing. ZnL2 shows reversible mechanochromic luminescence behavior between yellow and orange emission during the grinding-fuming/heating cycles due to the modulation between amorphous and crystalline states. Moreover, ZnL2 was successfully made into test paper for the rapid detection of HCl/NH3 vapors and mechanical stimuli.

Fitness and Productivity Increase with Ecotypic Diversity among Escherichia coli Strains That Coevolved in a Simple, Constant Environment.

The productivity of a biological community often correlates with its diversity. In the microbial world this phenomenon can sometimes be explained by positive, density-dependent interactions such as cross-feeding and syntrophy. These metabolic interactions help account for the astonishing variety of microbial life and drive many of the biogeochemical cycles without which life as we know it could not exist. While it is difficult to recapitulate experimentally how these interactions evolved among multiple taxa, we can explore in the laboratory how they arise within one. These experiments provide insight into how different bacterial ecotypes evolve and from these, possibly new "species." We have previously shown that in a simple, constant environment a single clone of Escherichia coli can give rise to a consortium of genetically and phenotypically differentiated strains, in effect, a set of ecotypes, that coexist by cross-feeding. We marked these different ecotypes and their shared ancestor by integrating fluorescent protein into their genomes and then used flow cytometry to show that each evolved strain is more fit than the shared ancestor, that pairs of evolved strains are fitter still, and that the entire consortium is the fittest of all. We further demonstrate that the rank order of fitness values agrees with estimates of yield, indicating that an experimentally evolved consortium more efficiently converts primary and secondary resources to offspring than its ancestor or any member acting in isolation.IMPORTANCE Polymicrobial consortia occur in both environmental and clinical settings. In many cases, diversity and productivity correlate in these consortia, especially when sustained by positive, density-dependent interactions. However, the evolutionary history of such entities is typically obscure, making it difficult to establish the relative fitness of consortium partners and to use those data to illuminate the diversity-productivity relationship. Here, we dissect an Escherichia coli consortium that evolved under continuous glucose limitation in the laboratory from a single common ancestor. We show that a partnership consisting of cross-feeding ecotypes is better able to secure primary and secondary resources and to convert those resources to offspring than the ancestral clone. Such interactions may be a prelude to a special form of syntrophy and are likely determinants of microbial community structure in nature, including those having clinical significance such as chronic infections.

Long noncoding RNA HOTAIR silencing inhibits invasion and proliferation of human colon cancer LoVo cells via regulating IGF2BP2.

Colon cancer is one of the most life-threatening malignancies worldwide. Long noncoding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) is a cancer-associated biomarker involved in the metastasis and prognosis of several cancers. However, whether and how HOTAIR affects colon cancer progression is still unclear. Consequently, we used RNA interference to knock down HOTAIR to explore its effects on human colon cancer cells. The dual luciferase reporter gene assay was initially used for testify the regulating relationship between lncRNA HOTAIR and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). We determined the expressions of HOTAIR, IGF2BP2, E-cadherin, and vimentin. Meanwhile, cell growth, cycle and apoptosis, migration, and invasion were assayed. LoVo cells were transplanted into nude mice, and tumor formation and microvessel density were evaluated. LncRNA HOTAIR positively regulated IGF2BP2. Besides, the expressions of HOTAIR and E-cadherin and the apoptosis were increased, while the expressions of IGF2BP2 and vimentin, the growth, invasion and migration of LoVo cells, the average tumor weight, and microvessel density value were decreased. Of importance, overexpressed IGF2BP2 could reverse the above impacts. Taken together, the current study indicates that silencing of HOTAIR could inhibit the invasion, proliferation, and migration, and promote apoptosis of colon cancer LoVo cells through suppressing IGF2BP2 and the epithelial-mesenchymal transition.

Effects of Glut1 gene silencing on proliferation, differentiation, and apoptosis of colorectal cancer cells by targeting the TGF-ß/PI3K-AKT-mTOR signaling pathway.

This study aims to investigate the effects of glucose transport l (Glut1) gene on proliferation, differentiation, and apoptosis of colorectal cancer (CRC) cells by regulating the TGF-ß/PI3K-AKT-mTOR signaling pathway. Immunohistochemistry was conducted to detect the positive Glut1 expression. Normal human CRC epithelial cells (CCD-18Co) and CRC cell line HCT116 were grouped into the control, blank, negative control (NC), and shGlut1-1 groups. RT-qPCR and Western blotting were performed to detect the expressions of Glut1, TGF-ß1, PI3K, AKT, PTEN, mTOR, Bcl-2, and Bax. Protein expression of phosphorylated-PI3K (p-PI3K), p-S473-AKT, p-S389-S6K1, p-T70-4EBP1, Cleaved caspase-3 and Cleaved-PARP were detected. MTT assay, flow cytometry, and colony formation assay were performed in order to detect cell viability, cell cycle, and apoptosis, respectively. The positive expression rate of Glut1 in CRC tissues was 75% ± 8%, while in the adjacent normal tissues it was 0%. In comparison to adjacent normal tissues, CRC tissues had increased Glut1, TGF-ß1, PI3K, AKT, mTOR, and Bcl-2 expressions, and p-PI3K, p-S473-AKT, p-S389-S6K1, and p-T70-4EBP1 expressions; and decreased PTEN, Bax, Cleaved caspase-3, and Cleaved-PARP expressions. In comparison with the blank and NC groups, cells in the shGlut1-1 group showed decreased Glut1, TGF-ß1, PI3K, AKT, mTOR, and Bcl-2 expressions, and p-PI3K, p-S473-AKT, p-S389-S6K1, and p-T70-4EBP1 expressions; and increased PTEN, Bax, Cleaved caspase-3, and Cleaved-PARP expressions, along with more arrested cells in C0/C1 phase than in S phase and slower cell growth. These results suggested that silencing the Glut1 gene inhibited proliferation and promoted apoptosis of CRC cells by inactivating TGF-ß/PI3K-AKT-mTOR signaling pathway.

Deciphering the Origin, Evolution, and Physiological Function of the Subtelomeric Aryl-Alcohol Dehydrogenase Gene Family in the Yeast Saccharomyces cerevisiae.

Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). Yeast AAD genes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes, AAD4 and AAD14, encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeast AAD genes are undergoing pseudogenization. The 5' sequence of AAD15 has been deleted from the genome. Repair of an AAD3 missense mutation at the catalytically essential Tyr73 residue did not result in a functional enzyme. However, ancestral-state reconstruction by fusing Aad6 with Aad16 and by N-terminal repair of Aad10 restores NADPH-dependent aryl-alcohol dehydrogenase activities. Phylogenetic analysis indicates that AAD genes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeast AAD genes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeast AAD gene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role for AAD genes in lignin degradation that is degenerating as a result of yeast's domestication and use in brewing, baking, and other industrial applications.IMPORTANCE Functional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the first Saccharomyces cerevisiae genome sequence was published over 20 years ago, 22% of its estimated 6,603 open reading frames (ORFs) remain unverified. One outstanding example of this category of genes is the enigmatic seven-member AAD family. Here, we demonstrate that proteins encoded by two members of this family exhibit aliphatic and aryl-aldehyde reductase activity, and further that such activity can be recovered from pseudogenized AAD genes via ancestral-state reconstruction. The phylogeny of yeast AAD genes suggests that these proteins may have played an important ancestral role in detoxifying aromatic aldehydes in ligninolytic fungi. However, in yeast adapted to niches rich in sugars, AAD genes become subject to mutational erosion. Our findings shed new light on the selective pressures and molecular mechanisms by which genes undergo pseudogenization.

[Intrarectal local anesthesia versus periprostatic nerve block in transrectal prostate biopsy for patients with different prostate volumes: A prospective randomized controlled trial].

OBJECTIVE: To evaluate the analgesic effect of intrarectal local anesthesia (IRLA) versus that of periprostatic nerve block anesthesia (PPNB) in initial transrectal ultrasound-guided prostate biopsy (TRUS-PB) for patients with different prostate volumes (PV). METHODS: A total of 253 patients undergoing initial TRUS-PB in our hospital from January 2014 to November 2017 were divided into three PV groups (<50 ml, 50－100 ml, and >100 ml), each again randomized into three subgroups (control, IRLA, and PPNB) with the random number table method. The pain during the procedure was assessed based on the Visual Analogue Scale (VAS) scores and the blind method was used by the biopsy operator, VAS valuator and data analyst. RESULTS: Among the patients with PV <50 ml, the VAS scores in the blank control, IRLA, and PPNB subgroups were 4.39±0.87, 3.51±0.84 and 3.43±1.07, respectively, remarkably higher in the control than in the IRLA and PPNB groups (P<0.05), but with no statistically significant differences between the latter two (P>0.05). Among those with PV of 50－100 ml, the VAS scores in the three subgroups were 4.50±1.05, 4.38±1.13 and 3.38±1.44, respectively, markedly higher in the control and IRLA than in the PPNB group (P<0.05), but with no statistically significant differences between the former two groups (P>0.05). Among those with PV >100 ml, the VAS scores in the three subgroups were 5.19±1.05, 5.00±1.25 and 4.19±0.91, respectively, remarkably higher in the former two groups than in the latter (P<0.05), but with no statistically significant differences between the former two groups (P>0.05). CONCLUSIONS: Either IRLA or PPNB can be recommended for initial TRUS-PB in patients with PV <50 ml, PPNB for those with PV of 50－100 ml, and PPNB with other painkillers for those with PV >100 ml.

Neutrophil CD64 Index as a superior biomarker for early diagnosis of infection in febrile patients in the hematology department.

BACKGROUND: In the hematology department, the availability of biomarkers for early detection of infection is difficult to obtain. The present study aimed to compare the diagnostic values of neutrophil CD64 Index, procalcitonin (PCT), interleukin-6 (IL-6) and C-reactive protein (CRP) and to determine whether the combined analysis of these biomarkers offer stronger predictive power in the diagnosis for the infection of febrile patients. METHODS: Neutrophil CD64 Index, PCT, IL-6 and CRP levels were determined in 356 febrile patients in the hematology ward from May 2013 to May 2015. Sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values, receiver operating characteristic (ROC) areas under the curve (AUC), and logistic regression analysis were determined to evaluate the diagnostic values of these biomarkers. RESULTS: The levels of the four biomarkers were higher in the infection patients (p<0.001), and the PCT and IL-6 were higher in the patients with positive microbial blood culture (p<0.01). The neutrophil CD64 Index, PCT, IL-6, CRP had AUCs of 0.95, 0.83, 0.75 and 0.73, respectively. The best cut-off value of the neutrophil CD64 Index to detect infections was 5.06, with high specificity (87.5%) and sensitivity (88.4%). Furthermore, neutrophil CD64 Index, PCT and IL-6 offered the best combination of diagnosis with sensitivity of 93.9% and an AUC of 0.95. In addition, the neutrophil CD64 Index may have a special value to assist the physician to diagnose infection in the neutropenic patients with fever. CONCLUSIONS: The neutrophil CD64 Index is useful for early identification of infections in febrile patients in the hematology department. The combined analysis of the CD64 Index, PCT and IL-6 could further improve its sensitivity.

Expressions of Inhibitors of DNA Binding-1 and Matrix Metalloproteinase-9 in Colorectal Adenocarcinoma Tissues and Their Correlations with Microvessel Density.

Objective To explore the expressions of inhibitors of DNA binding-1 (Id-1) and matrix metalloproteinase-9 (MMP-9) in colorectal carcinoma tissues and its correlation with microvessel density (MVD). Methods The expressions of Id-1 and MMP-9 as well as CD34-labelled MVD in colorectal adenocarcinoma tissues (n=50) and normal adjacent tissues (n=50) were examined by immunohistochemistry. Results The positive expressions of Id-1 and MMP-9 were seen in 72.00% (36/50) and 78.00%(39/50) of colorectal adenocarcinoma tissues,which were significantly higher than those [24.00%(12/50) and 28.00% (14/50)] in normal adjacent tissues (P=0.000). The MVD value (17.22±2.08) in colorectal adenocarcinoma tissues was significantly higher than that (5.36±2.17) in normal adjacent tissues (P=0.000). The expressions of Id-1 and MMP-9 and MVD were significantly correlated with serosa invasion,TNM stage,carcinoembryonic antigen(+),lymph node metastasis,vascular invasion,and liver metastasis (all P<0.05) but not with the patient's age,gender,tumor size,and differentiation degree (all P>0.05). The MVD value with Id-1 and MMP-9 positive expression were significantly higher than those with Id-1 and MMP-9 negative expression (all P=0.000). The expression of Id-1 in colorectal adenocarcinoma tissues showed significantly positive correlation with that of MMP-9 (r=0.429,P=0.000). Cox multivariate analysis showed that Id-1 and MMP-9 expressions were independent prognostic factors for colorectal carcinoma. Conclusions The high expressions of Id-1 and MMP-9 have high correlations with the development and progression of colorectal adenocarcinoma and have positive correlation with MVD. Both of them may be involved in the microvascular generation and the invasion and hematogenous metastasis of colorectal carcinoma.

CRISPR-Cas9-assisted genome editing in E. coli elevates the frequency of unintended mutations.

Cas-assisted lambda Red recombineering techniques have rapidly become a mainstay of bacterial genome editing. Such techniques have been used to construct both individual mutants and massive libraries to assess the effects of genomic changes. We have found that a commonly used Cas9-assisted editing method results in unintended mutations elsewhere in the genome in 26% of edited clones. The unintended mutations are frequently found over 200 kb from the intended edit site and even over 10 kb from potential off-target sites. We attribute the high frequency of unintended mutations to error-prone polymerases expressed in response to dsDNA breaks introduced at the edit site. Most unintended mutations occur in regulatory or coding regions and thus may have phenotypic effects. Our findings highlight the risks associated with genome editing techniques involving dsDNA breaks in E. coli and likely other bacteria and emphasize the importance of sequencing the genomes of edited cells to ensure the absence of unintended mutations.

Saikosaponin D mitigate pilocarpine-induced astrocyte injury by regulating the NLRP3/caspase-1 signaling pathway.

Studies have shown that saikosaponin D (SSD) has favorable neurotherapeutic effects. Therefore, the objective of this study was to explore the efficacy and possible molecular mechanisms of SSD on pilocarpine (PP)-induced astrocyte injury. Primary astrocytes were isolated from juvenile rats and identified using immunofluorescence. The cells were treated with PP and/or SSD for 6 h and 12 h, respectively, followed by measurement of their viability through 3-(4,5-dimethylthiazol)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Next, quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of Glial fibrillary acidic protein (GFAP), C3, S100 calcium binding protein A10 (S100a10), pentraxin 3 (Ptx3), toll-like receptor 4 (TLR4), and RAG in astrocytes after different treatments. Enzyme-linked immunosorbent assay and biochemical tests were utilized to evaluate the level of inflammatory factors [interleukin (IL)-1ß, IL-6, and tumor necrosis factor alpha (TNF-α)] secreted by cells and the content of oxidative stress-related factors (malondialdehyde [MDA] and glutathione [GSH]) or enzyme activity (catalase [CAT] and glutathione peroxidase [GPX]) in cells. The JC-1 mitochondrial membrane potential (MMP) fluorescence probe was used to measure the MMP in astrocytes. Additionally, western blot was applied to test the expression of proteins related to the nod-like receptor protein 3 (NLRP3)/caspase-1 signaling pathway. PP treatment (1 mM) induced cell injury by significantly reducing the viability of astrocytes and expression of cellular markers. SSD treatment (4 µM) had no toxicity to astrocytes. Besides, SSD (4 µM) treatment could significantly up-regulate the cell viability and marker expression of PP-induced astrocytes. Furthermore, SSD could be employed to inhibit inflammation (reduce IL-1ß, IL-6, and TNF-α levels) and oxidative stress (decrease MDA level, elevate GSH level, the activity of CAT and GPX), and ameliorate mitochondrial dysfunction (upregulate JC-1 ratio) in PP-induced astrocytes. Moreover, further mechanism exploration revealed that SSD treatment significantly reduced the activity of the NLRP3/caspase-1 signaling pathway activated by PP induction. SSD increased cell viability, inhibited inflammation and oxidative stress response, and ameliorated mitochondrial dysfunction in PP-induced astrocyte injury model, thus playing a neuroprotective role. The mechanism of SSD may be related to the inhibition of the NLRP3/caspase-1 inflammasome.