Astrocytic connexin 43 deletion ameliorates SNI-induced neuropathic pain by reducing microglia activation.

Neuropathic pain (NP) is a chronic disease caused by damage to the peripheral or central nervous system. Connexin 43 (Cx43), the primary connexin expressed by astrocytes, has been reported to be significantly increased in NP. However, the roles and mechanisms of Cx43 in the development and maintenance of NP remain largely unknown, while microglia activation has been commonly regarded as a key factor of NP. In the present study, we found that Cx43 deletion significantly ameliorated spared nerve injury (SNI)-induced NP and suppressed SNI induced c-Fos expression in the spinal cord. Notably, Cx43 deletion led to much less SNI-induced microglia activation in the spinal cord. These results suggest that astrocyte Cx43 may play a significant role in regulating microglial activation and NP.

Inducing Global Expression of Actinobacterial Biosynthetic Gene Clusters.

Bacteria produce an impressive array of bioactive specialized metabolites, with Streptomyces (and the actinobacteria more generally) being unusually diverse and prolific producers. However, the biosynthetic potential of these organisms has yet to be fully explored, as many of the biosynthetic gene clusters that direct the synthesis of these natural products are transcriptionally silent under laboratory growth conditions. Here, we describe strategies that can be employed to broadly stimulate the expression of biosynthetic gene clusters in Streptomyces and their relatives, follow the transcription of these genes, and assess the antimicrobial activity of the resulting molecules.

Interplay between Nucleoid-Associated Proteins and Transcription Factors in Controlling Specialized Metabolism in Streptomyces.

Lsr2 is a small nucleoid-associated protein found throughout the actinobacteria. Lsr2 functions similarly to the well-studied H-NS, in that it preferentially binds AT-rich sequences and represses gene expression. In Streptomyces venezuelae, Lsr2 represses the expression of many specialized metabolic clusters, including the chloramphenicol antibiotic biosynthetic gene cluster, and deleting lsr2 leads to significant upregulation of chloramphenicol cluster expression. We show here that Lsr2 likely exerts its repressive effects on the chloramphenicol cluster by polymerizing along the chromosome and by bridging sites within and adjacent to the chloramphenicol cluster. CmlR is a known activator of the chloramphenicol cluster, but expression of its associated gene is not upregulated in an lsr2 mutant strain. We demonstrate that CmlR is essential for chloramphenicol production, and further reveal that CmlR functions to "countersilence" Lsr2's repressive effects by recruiting RNA polymerase and enhancing transcription, with RNA polymerase effectively clearing bound Lsr2 from the chloramphenicol cluster DNA. Our results provide insight into the interplay between opposing regulatory proteins that govern antibiotic production in S. venezuelae, which could be exploited to maximize the production of bioactive natural products in other systems. IMPORTANCE Specialized metabolic clusters in Streptomyces are the source of many clinically prescribed antibiotics. However, many clusters are not expressed in the laboratory due to repression by the nucleoid-associated protein Lsr2. Understanding how Lsr2 represses cluster expression, and how repression can be alleviated, is key to accessing the metabolic potential of these bacteria. Using the chloramphenicol biosynthetic cluster from Streptomyces venezuelae as a model, we explored the mechanistic basis underlying Lsr2-mediated repression, and activation by the pathway-specific regulator CmlR. Lsr2 polymerized along the chromosome and bridged binding sites located within and outside the cluster, promoting repression. Conversely, CmlR was essential for chloramphenicol production and further functioned to countersilence Lsr2 repression by recruiting RNA polymerase and promoting transcription, ultimately removing Lsr2 polymers from the chromosome. Manipulating the activity of both regulators led to a >130× increase in chloramphenicol levels, suggesting that combinatorial regulatory strategies can be powerful tools for maximizing natural product yields.

MicroRNA-191 regulates differentiation and migration of mesenchymal stem cells and their paracrine effect on angiogenesis.

MicroRNAs (miRNAs) are critical regulators in organ development. Among them, miR-191 is known to be regulated in early embryogenesis and dysregulated in cancer. This role in undifferentiated tissues suggests a possible part of miR-191 also in bone marrow derived mesenchymal stem cells (BMSCs) physiology. Here, we report that miR-191 decreased MMP expression and migration of BMSCs. Conditioned media of miR-191 overexpressing BMSCs block VEGF expression, and inhibit angiogenesis of HUVECs. Under osteogenic culture conditions, inhibition of miR-191 significantly induces bone formation. Moreover, our studies showed miR-191 might influence chondrogenesis of BMSCs by directly targeting CCAAT Enhancer Binding Protein Beta (CEBPB). Taken together, here we demonstrate the role of miR-191 in differentiation, migration and paracrine function of BMSCs.

[Questionnaire survey and analysis of implementability of clinical practice guidelines for acupuncture-moxibustion].

The current usage and the existing problems in the implementability of clinical practice guidelines for acupuncture-moxibustion were investigated by questionnaire survey, aiming to provide reference for the development or update of clinical practice guidelines for acupuncture-moxibustion in the future. The results showed most of the acupuncture-moxibustion clinicians did not have a deep understanding of the guidelines, but they had a strong will of uniform standards and related guidelines. Although the published clinical practice guidelines for acupuncture-moxibustion achieved some success, they still had not got rid of the shackles of the previous textbook. The main existing problems in the guidelines included insufficient promotion, poor credibility, no evaluation criteria for curative effect, and lack of consideration for patients' will, etc. As the guidelines for acupuncture-moxibustion were based on the latest evidence of current clinical research, it reflected the low quality of current clinical research on acupuncture-moxibustion and lacking of evidence-based concept among acupuncture-moxibustion clinicians. The implementability of clinical practice guidelines is a key step in evidence-based translational medicine, while the research on the implementability of acupuncture-moxibustion guidelines is still blank. More attention should be paid to this field in the future.

Streptomyces IHF uses multiple interfaces to bind DNA.

BACKGROUND: Nucleoid associated proteins (NAPs) are essential for chromosome condensation in bacterial cells. Despite being a diverse group, NAPs share two common traits: they are small, oligomeric proteins and their oligomeric state is critical for DNA condensation. Streptomyces coelicolor IHF (sIHF) is an actinobacterial-specific nucleoid-associated protein that despite its name, shares neither sequence nor structural homology with the well-characterized Escherichia coli IHF. Like E. coli IHF, sIHF is needed for efficient nucleoid condensation, morphological development and antibiotic production in S. coelicolor. METHODS: Using a combination of crystallography, small-angle X-ray scattering, electron microscopy and structure-guided functional assays, we characterized how sIHF binds and remodels DNA. RESULTS: The structure of sIHF bound to DNA revealed two DNA-binding elements on opposite surfaces of the helix bundle. Using structure-guided functional assays, we identified an additional surface that drives DNA binding in solution. Binding by each element is necessary for both normal development and antibiotic production in vivo, while in vitro, they act collectively to restrain negative supercoils. CONCLUSIONS: The cleft defined by the N-terminal and the helix bundle of sIHF drives DNA binding, but the two additional surfaces identified on the crystal structure are necessary to stabilize binding, remodel DNA and maintain wild-type levels of antibiotic production. We propose a model describing how the multiple DNA-binding elements enable oligomerization-independent nucleoid condensation. GENERAL SIGNIFICANCE: This work provides a new dimension to the mechanistic repertoire ascribed to bacterial NAPs and highlights the power of combining structural biology techniques to study sequence unspecific protein-DNA interactions.

Silencing cryptic specialized metabolism in Streptomyces by the nucleoid-associated protein Lsr2.

Lsr2 is a nucleoid-associated protein conserved throughout the actinobacteria, including the antibiotic-producing Streptomyces. Streptomyces species encode paralogous Lsr2 proteins (Lsr2 and Lsr2-like, or LsrL), and we show here that of the two, Lsr2 has greater functional significance. We found that Lsr2 binds AT-rich sequences throughout the chromosome, and broadly represses gene expression. Strikingly, specialized metabolic clusters were over-represented amongst its targets, and the cryptic nature of many of these clusters appears to stem from Lsr2-mediated repression. Manipulating Lsr2 activity in model species and uncharacterized isolates resulted in the production of new metabolites not seen in wild type strains. Our results suggest that the transcriptional silencing of biosynthetic clusters by Lsr2 may protect Streptomyces from the inappropriate expression of specialized metabolites, and provide global control over Streptomyces' arsenal of signaling and antagonistic compounds.

Unlocking the trove of metabolic treasures: activating silent biosynthetic gene clusters in bacteria and fungi.

Bacteria and fungi are prolific producers of secondary metabolites, yet they house a multitude of silent biosynthetic gene clusters that are poorly expressed and whose products are unknown or 'cryptic'. Stimulating the expression of these clusters and accessing their associated molecules is a major priority, as they are expected to have a veritable cornucopia of bioactivites. Here, we highlight three strategies that have been the focus of recent developments. Co-culture and elicitor screening, genetic regulator investigation and exploitation, and pathway refactoring and heterologous cluster expression, are collectively being employed to activate the expression of cryptic biosynthetic gene clusters (BGCs), and stimulate the production of novel metabolites having diverse activities.

Synthesis and Evaluation of a Chitosan Oligosaccharide-Streptomycin Conjugate against Pseudomonas aeruginosa Biofilms.

Microbial biofilms are considerably more resistant to antibiotics than planktonic cells. It has been reported that chitosan coupling with the aminoglycoside antibiotic streptomycin dramatically disrupted biofilms of several Gram-positive bacteria. This finding suggested the application of the covalent conjugate of antimicrobial natural polysaccharides and antibiotics on anti-infection therapy. However, the underlying molecular mechanism of the chitosan-streptomycin conjugate (CS-Strep) remains unclear and the poor water-solubility of the conjugate might restrict its applications for anti-infection therapy. In this study, we conjugated streptomycin with water-soluble chitosan oligosaccharides (COS). Unlike CS-Strep, the COS-streptomycin conjugate (COS-Strep) barely affected biofilms of tested Gram-positive bacteria. However, COS-Strep efficiently eradicated established biofilms of the Gram-negative pathogen Pseudomonas aeruginosa. This activity of COS-Strep was influenced by the degree of polymerization of chitosan oligosaccharide. The increased susceptibility of P. aeruginosa biofilms to antibiotics after conjugating might be related to the following: Suppression of the activation of MexX-MexY drug efflux pump system induced by streptomycin treatment; and down-regulation of the biosynthesis of biofilm exopolysaccharides. Thus, this work indicated that covalently linking antibiotics to chitosan oligosaccharides was a possible approach for the development of antimicrobial drugs against biofilm-related infections.

New Kid on the Block: LmbU Expands the Repertoire of Specialized Metabolic Regulators in Streptomyces.

Streptomyces has an extensive natural product repertoire, including most of the naturally derived antibiotics. Understanding the control of natural product biosynthesis is central to antibiotic discovery and production optimization. Here, Hou et al. (J. Bacteriol. 200:00447-17, 2018, https://doi.org/10.1128/JB.00447-17) report the identification and characterization of a novel regulator-LmbU-that functions primarily as an activator of lincomycin production in Streptomyces lincolnensis Importantly, members of this new regulator family are associated with natural product biosynthetic clusters throughout the streptomycetes and their actinomycete relatives.

A selective and label-free strategy for rapid screening of telomere-binding Ligands via fluorescence regulation of DNA/silver nanocluster.

Herein, the conformational switch of G-rich oligonucleotide (GDNA) demonstrated the obvious functional switch of GDNA which was found to significantly affect the fluorescence of the in-situ synthesized DNA/silver nanocluster (DNA-AgNC) in homogeneous solution. We envisioned that the allosteric interaction between GDNA and DNA-AgNC would be possible to be used for screening telomere-binding ligands. A unimolecular probe (12C5TG) is ingeniously designed consisting of three contiguous DNA elements: G-rich telomeric DNA (GDNA) as molecular recognition sequence, T-rich DNA as linker and C-rich DNA as template of DNA-AgNC. The quantum yield and stability of 12C5TG-AgNC is greatly improved because the nearby deoxyguanosines tended to protect DNA/AgNC against oxidation. However, in the presence of ligands, the formation of G-quadruplex obviously quenched the fluorescence of DNA-AgNC. By taking full advantage of intramolecular allosteric effect, telomere-binding ligands were selectively and label-free screened by using deoxyguanines and G-quadruplex as natural fluorescence enhancer and quencher of DNA-AgNC respectively. Therefore, the functional switching of G-rich structure offers a cost-effective, facile and reliable way to screen drugs, which holds a great potential in bioanalysis as well.

Sensitive detection of intracellular RNA of human telomerase by using graphene oxide as a carrier to deliver the assembly element of hybridization chain reaction.

Since the level of human telomerase RNA (hTR) in tumor cells is higher than that in normal somatic cells, the quantitative assay of hTR is of significant importance in tumor diagnosis. Herein, graphene oxide (GO) was simultaneously exploited as a fluorescence quencher and a carrier of nucleic acid to successfully deliver two hairpin DNA probes of hybridization chain reaction (HCR) into the cancer cell for detecting telomerase RNA based on DNA nanoassembly of HCR. The sticky end of HCR probes could tightly absorb on the surface of GO, resulting in fluorescence quenching of the dye which was tagged at the sticky end of two hairpin probes. When faced with hTR, the fluorescence of DNA probes is subsequently recovered because hTR could trigger HCR to autonomous assembly of a DNA polymer which released from the GO and led to fluorescence recovery. Taking advantage of nucleic acid nanoassembly of HCR, this intracellular HCR strategy creates enormous signal amplification, and enables ultra-sensitive fluorescence imaging of hTR expression. By monitoring fluorescence change, human telomerase RNA could be specifically studied and this method can also be used for detecting single-base mutation. The GO-aided HCR strategy allowed us to sensitively detect hTR in a living cell, which holds great potential for analyzing other low-abundance biomolecules in living cells via HCR.

A PCR-free fluorescence strategy for detecting telomerase activity via double amplification strategy.

As a universal tumor biomarker, research on the activity and inhibition of telomerase is of great importance for cancer diagnosis and therapy. Although the telomeric repeat amplification protocol (TRAP) has served as a powerful assay for detecting telomerase activity, its application has been significantly limited by amplification related errors and time-consuming procedure. To address the limitations of PCR-based protocol, a dual amplification fluorescence assay was developed for PCR-free detecting telomerase activity. Briefly, we designed an arch-structure DNA probe to specifically control strand displacement reaction and subsequent enzyme-aided amplification. Telomerase substrate (TS) primer was extended by telomerase to form long elongation products which contain several TTAGGG repeat units. So, one elongation product can release more than one trigger DNA (t-DNA) via strand displacement reaction to realize first amplification. Subsequently, t-DNA specifically opened molecular beacon (MB) to restore the fluorescence of MB. Meanwhile, t-DNA was recycled by the aid of nicking endonuclease to continuously open more and more MBs, leading to a second amplification. Owing to the double amplification strategy, the proposed method allowed the measurement of telomerase activity in crude cell extracts equivalent to 5 HeLa cells and 10 CCRF-CEM cells without PCR amplification. Besides, the influence of telomere-binding ligands on the telomerase activity demonstrated that the proposed method holds the potential to evaluate the inhibition efficiency of telomerase inhibitors.

A label-free cyclic assembly of G-quadruplex nanowires for cascade amplification detection of T4 polynucleotide kinase activity and inhibition.

Several fluorescence methods have been developed for sensitive detection of PNK activity based on signal amplification techniques, but they need fluorescently labeled DNA probes and superabundant assistant enzymes. We have addressed these limitations and report here a label-free and enzyme-free amplification strategy for sensitively and specifically studying PNK activity and inhibition via hybridization chain reaction (HCR). First, the phosphorylation of hairpin DNA H1 by T4 PNK makes it be specifically digested by lambda exonuclease (λ exo) from 5' to 3' direction to generate a single-stranded initiator which can successively open hairpins H2 and H3 to trigger an autonomous assembly of long DNA nanowires. Meanwhile, an intermolecular G-quadruplex is formed between H2 and H3, thereby providing fluorescence enhancement of N-methyl mesoporphyrin IX (NMM) which is a highly quadruplex-selective fluorophore. So, the PNK activity can be facilely and sensitively detected by using NMM as a signal probe which provides a low background signal to improve the overall sensitivity, resulting in the detection limit of 3.37 × 10(-4) U mL(-1). More importantly, its successful application for detecting PNK activity in a complex biological matrix and studying the inhibition effects of PNK inhibitors demonstrated that it provides a promising platform for screening PNK inhibitors as well as detecting PNK activity. Therefore, it is a highly sensitive, specific, reliable and cost-effective strategy which shows great potential for biological process research, drug discovery, and clinical diagnostics.

Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research.

[Effectiveness of preoperative autologous plateletpheresis combined with intraoperative autotransfusion on the blood coagulation in orthopaedic patients].

OBJECTIVE: To investigate the effectiveness of preoperative plateletpheresis combined with intraoperative autotransfusion on the blood coagulation of orthopaedic patients. METHODS: Sixty patients (ASA I-II) undergoing selective orthopaedic surgery were randomized into three groups (n = 20), that is, preoperative plateletpheresis combined with intraoperative autotransfusion for group I, intraoperative autotransfusion for group II, and group III without any managements of blood conservation. Coagulation parameters (prothrombin time, partial thromboplastin time, fibrinogen), hemoglobin and hematocrit values, platelet counts and aggregability were evaluated before the anaesthesia, 10 minutes after plateletpheresis, 10 minutes before the infusion of platelet rich plasma or autologous blood, 10 minutes after infusion, 24 and 48 hours postoperation. Intra- and postoperation blood loss and homologous blood transfusion requirements were also recorded. RESULTS: Among three groups, there were no differences in intraoperative blood loss, perioperative haemoglobin level (Hb and Hct). As compared with group I, significant lower level of platelet counts and aggregability were observed in group II and III at the time of 24 and 48 hours after operation (P < 0.05), while postoperation blood loss and homologous blood-transfusion requirements increased at the same period (P < 0.01). CONCLUSIONS: Preoperative plateletpheresis combined with intraoperative autotransfusion can ameliorate the blood coagulation in orthopaedic patients, and it is an effective way to decrease blood loss and homologous blood-transfusions requirements.