TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis.

OBJECTIVES: Tuberculosis (TB) remains one of the public health problems worldwide. Rapid, sensitive and cost-effective diagnosis of Mycobacterium tuberculosis (M.tb) is critical for TB control. METHODS: We developed a novel M.tb DNA detection platform (nominated as TB-QUICK) which combined loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12b detection. TB-QUICK was performed on pulmonary or plasma samples collected from 138 pulmonary TB (PTB) patients, 21 non-TB patients and 61 close contacts to TB patients. Acid-fast bacillus (AFB) smear, M.tb culture and GeneXpert MTB/RIF (Xpert) assays were routinely conducted in parallel. RESULTS: By targeting M.tb IS6110, TB-QUICK platform could detect as low as 1.3 copy/µL M.tb DNA within 2 h. In pulmonary TB samples, TB-QUICK exhibited improved overall sensitivity of 86.8% over M.tb culture (66.7%) and Xpert (70.4%), with the specificity of 95.2%. More significantly, TB-QUICK exhibited a superior sensitivity in AFB-negative samples (80.5%) compared to Xpert (57.1%) and M.tb culture (46.2%). In the detection of plasma M.tb DNA by TB-QUICK, 41.2% sensitivity for AFB-positive and 31.7% for AFB-negative patients were achieved. CONCLUSION: In conclusion, TB-QUICK exhibits rapidity and sensitivity for M.tb DNA detection with the superiority in smear-negative paucibacillary TB patients. The clinical application of TB-QUICK in TB diagnosis needs to be further validated in larger cohort.

The analysis of the impact of the Belt and Road initiative on the green development of participating countries.

China has emphasized the importance of implementing the concept of green development into the process of the Belt and Road initiative. Therefore, in the process of promoting the initiative, it is necessary to clarify its impact on the green development of the participating countries. Based on such consideration, this paper establishes Green Development Capability (GDC) index to measure the green development level, and uses the Spatial Durbin Model for empirical testing basing on relevant data on different cooperative patterns between China and participating countries. The results show: (i) Economic Development Cooperation, Environmental Governance Cooperation and Sustainable Cooperation are conducive to enhancing the GDC, while the cooperation of Resource Utilization based on fossil energy trading has adversely affected on GDC. (ii) The current cooperation approaches have spillover effects, but not yet broken the Spatial Club imbalance. (iii) Economic Development Cooperation, Environmental Governance Cooperation and Sustainable Cooperation contribute to the promotion of developing countries' GDC, but over-reliance on mineral exploitation has caused these countries to fall into Resource Curses and hinder them from playing Backward Advantage.

Interleukin-22 secreted by ectopic endometrial stromal cells and natural killer cells promotes the recruitment of macrophages through promoting CCL2 secretion.

PROBLEM: During endometriosis, there is an increase in the number of dysfunctional macrophages; however, the mechanisms underlying macrophage recruitment are not well understood. The aim of the present study was to determine the role of natural killer (NK) cell-mediated secretion of chemokine (C-C motif) ligand 2 (CCL2) from endometrial stromal cells (ESCs) in the recruitment of macrophages. METHOD OF STUDY: Normal ESCs (nESC) and ectopic ESCs (eESCs) were separately co-cultured with NK cells for a macrophage chemotaxis assay, and the number of chemotactic macrophages was counted. The expression of interleukin-22 (IL-22) and IL-22 receptors was detected by ELISA and flow cytometry, respectively. eESCs were treated with 0.01, 0.1, and 1 ng/mL recombinant human IL-22 (rhIL-22) to determine the most effective concentration for stimulating CCL2 production. Following treatment with 1 ng/mL rhIL-22, secretion of CCL2 was detected from both the eESC monoculture and the eESC/NK co-culture. RESULTS: Compared with the eESC monoculture, the eESC/NK co-culture recruited a significantly higher number of chemotactic macrophages. There was also an increase in the levels of IL-22 and CCL2 secreted when eESCs were co-cultured compared with the monoculture. Treatment with rhIL-22 resulted in an increase in the levels of CCL2 secreted by eESCs, and the IL-22-induced CCL2 secretion was reversed by the IL-22 antagonist, αIL-22. Increased expression of IL-22 resulted in an increase in the number of chemotactic macrophages, but was reversed by αIL-22 and CCL2 antagonist (αCCL2). CONCLUSION: Interleukin-22 and CCL2 secretion by eESCs stimulated by NK cells contributes to the induction of macrophage recruitment and is thus implicated in the development of endometriosis.

Erratum: Author Correction: CRISPR-Cas12a-assisted nucleic acid detection.

[This corrects the article DOI: 10.1038/s41421-018-0028-z.].

CXCL16/CXCR6 interaction promotes endometrial decidualization via the PI3K/AKT pathway.

Decidualization renders the endometrium transiently receptive to an implanting blastocyst although the underlying mechanisms remain incompletely understood. The aim of this study was to determine the role of chemokine CXCL16 and its receptor CXCR6 in the decidualization during pregnancy. Here, the expression of CXCL16 was investigated in endometrial tissues, decidua and placenta in this study. Compared with endometrial tissue, protein expression of CXCL16 was significantly higher in tissues from the fertile control samples, especially in villus. Meanwhile, the primary trophoblast cells and decidual stromal cells (DSCs) secreted more CXCL16 and expressed higher CXCR6 compared to endometrial stromal cells (ESCs) in vitro. Stimulation with the inducer of decidualization (8-bromoadenosine 3',5'-cyclic with medroxyprogesterone acetate, 8-Br-cAMP plus MPA) significantly upregulated the expression of CXCL16 and CXCR6 in ESCs in vitro. After treatment with exogenous recombinant human CXCL16 (rhCXCL16) or trophoblast-secreted CXLC16, decidualised ESCs showed a significant decidual response, mainly characterised by increased prolactin (PRL) secretion. Simultaneously, PI3K/PDK1/AKT/Cyclin D1 pathway in decidualised ESCs were activated by rhCXCL16, and AKT inhibitor GS 690693 abolished the PRL secretion of ESCs that was triggered by rhCXCL16. Finally, the impaired CXCL16/CXCR6 expression could be observed at the maternal-foetal interface from patients who have experienced spontaneous abortion. This study suggests that the CXCL16/CXCR6 axis contributes to the progression of ESC decidualization by activating PI3K/PDK1/AKT/Cyclin D1 pathway. It unveils a new paradigm at the maternal-foetal interface in which CXCL16 is an initiator for the molecular crosstalk that enhances decidualization of ESCs.

CADS: CRISPR/Cas12a-Assisted DNA Steganography for Securing the Storage and Transfer of DNA-Encoded Information.

Because DNA has the merit of high capacity and complexity, DNA steganography, which conceals DNA-encoded messages, is very promising in information storage. The classical DNA steganography method hides DNA with a "secret message" in a mount of junk DNA, and the message can be extracted by polymerase chain reaction (PCR) using specific primers (key), followed by DNA sequencing and sequence decoding. As leakage of the primer information may result in message insecurity, new methods are needed to better secure the DNA information. Here, we develop a pre-key by either mixing specific primers (real key) with nonspecific primers (fake key) or linking a real key with 3'-end redundant sequences. Then, the single-stranded DNA (ssDNA) trans cleavage activity of CRISPR/Cas12a is employed to cut a fake key or remove the 3'-end redundant sequences, generating a real key for further information extraction. Therefore, with the Cas12a-assisted DNA steganography method, both storage and transfer of DNA-encoding data can be better protected.

Protocols for C-Brick DNA Standard Assembly Using Cpf1.

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites - the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) - flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short "GGATCC" scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

An efficient system for deletion of large DNA fragments in Escherichia coli via introduction of both Cas9 and the non-homologous end joining system from Mycobacterium smegmatis.

Accompanied with the internal non-homologous end joining (NHEJ) system, Cas9 can be used to easily inactivate a gene or delete a fragment through introduction of DNA double-stranded breaks (DSBs) in eukaryotic cells. While in most prokaryotes (e.g. Escherichia coli), due to the lack of NHEJ, homologous recombination (HR) is required for repair of DSBs, which is less convenient. Here, a markerless system was developed for rapid gene inactivation or fragment deletion in E. coli via introduction of both Cas9 and a bacterial NHEJ system. Three bacterial NHEJ systems, i.e. Mycobacterium smegmatis (Msm), Mycobacterium tuberculosis (Mtb) and Bacillus subtilis (Bs), were tested in E. coli, and the MsmNHEJ system showed the best efficiency. With the employment of Cas9 and MsmNHEJ, we efficiently mutated lacZ gene, deleted glnALG operon and two large DNA fragments (67 kb and 123 kb) in E. coli, respectively. Moreover, the system was further designed to allow for continuous inactivation of genes or deletion of DNA fragments in E. coli. We envision this system can be extended to other bacteria, especially those with low HR efficiency.

The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro.

As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in DNA assembly. Here, the Cpf1 cleavage sites were precisely characterized, where the cleavage site on the target strand was around the 22nd base relative to the protospacer adjacent motif site, but the cleavage on the non-target strand was affected by the spacer length. When the spacer length was 20 nt or longer, Cpf1 mainly cleaved around the 14th and the 18th bases on the non-target strand; otherwise, with a shorter spacer (i.e. 17-19 nt), Cpf1 mainly cleaved after the 14th base, generating 8-nt sticky ends. With this finding, Cpf1 with a 17-nt spacer crRNA were employed for in vitro substitution of the actII-orf4 promoter in the actinorhodin biosynthetic cluster with a constitutively expressing promoter. The engineered cluster yielded more actinorhodin and produced actinorhodin from an earlier phase. Moreover, Taq DNA ligase was further employed to increase both the ligation efficiency and the ligation accuracy of the method. We expect this CCTL (Cpf1-assisted Cutting and Taq DNA ligase-mediated Ligation) method can be widely used in in vitro editing of large DNA constructs.

C-Brick: A New Standard for Assembly of Biological Parts Using Cpf1.

So far, several DNA assembly standards have been developed, enabling scientists to conveniently share and modify characterized DNA parts. However, a majority of the restriction endonucleases used in these standards bear short recognition sites (e.g., 6 bps in BioBrick standard), which are widely distributed and need to be removed before further construction, causing much inconvenience. Although homing endonucleases, which recognize long DNA sequences, can be used for DNA assembly (e.g., iBrick standard), long scars will be left between parts, limiting their application. Here, we introduce a new DNA assembly standard, namely C-Brick, which employs the newly identified class 2 type V CRISPR-Cas systems protein Cpf1 endonuclease. C-Brick integrates both advantages of long recognition sites and short scars. With C-Brick standard, three chromoprotein cassettes were assembled and further expressed in Escherichia coli, producing colorful pigments. Moreover, C-Brick standard is also partially compatible with the BglBrick and BioBrick standards.

[A Concept Design of Flat-Field Spectrograph for Wide Wavelength Range].

The radiation spectrum from the plasmas contains a large amount of information of plasmas. Thus, one of the most effective methods to detecting the plasma parameters is measure the plasma radiation spectrum. Until now, since the restriction of the Toshiba mechanically ruled aberration-corrected concave gratings, the measurable wavelength range of the incidence flat-field grazing spectrometer in the soft X-ray range are only from 5 to 40 nm. In order to extend the wavelength rang of grazing incidence flat-field spectrometer, first, a grazing incidence concave reflection grating ray-trace code is written using optical path equation. Second, under the same conditions with reference 6, we compare our numerical results with Harada's results. The results show that our results agree very well with the results of Harada. The results of comparison show that our ray-trace code is believable. Finally, the variety of the flat-field curves are detailedly investigated using the ray-trace code with the different grazing incidence conditions. The results show that the measurable wavelength range of the incidence flat-field grazing spectrometer are extended to 5~80 nm from the soft X-ray wavelength range of 5~40 nm. This result theoretically demonstrates the possibility of expanded the traditional band flat-field grazing incidence spectrometer from soft X-ray band to the extreme ultraviolet (XUV), and also bring a new design ideas for improving the use of grazing incidence flat field concave grating.

Phenolic glycosides from Ficus tikoua and their cytotoxic activities.

Four new phenolic glycosides, named 2-ethylene-3,5,6-trimethyl-4-phenol-1-O-ß-d-xylopyranosyl-(1→6)-ß-d-glucopyranoside (1), 3-methoxy-4-O-ß-d-apiofuranosyl-(1→2)-ß-d-glucopyranosylpropiophenone (2), 3-hydroxy-1-(4-O-ß-d-glucopyranosyl-3-methoxyphenyl)propan-1-one (3) and 4-hydroxy-3,5-bis(3'-methyl-2-butenyl)benzoic acid-O-ß-d-glucopyranoside (4), were isolated from the ethanol extract of Ficus tikoua, together with six known compounds: 3,4,5-trimethoxyphenol-1-O-ß-d-apiofuranosyl-(1→6)-ß-d-glucopyranoside (5), 3,4,5-trimethoxyphenol-1-O-ß-d-glucopyranoside (6), 3-methoxy-4-O-ß-d-apiofuranosyl-(1→6)-ß-d-glucopyranosylpropiophenone (7), baihuaqianhuoside (8), 3,5-dimethoxy-4-hydroxybenzoic acid-O-ß-d-glucopyranoside (9) and 2-methoxy-4-allylphenyl-1-O-ß-d-apiofuranosyl-(1→6)-ß-d-glucopyranoside (10). The structures of the four new compounds were elucidated by chemical methods and MS and IR, as well as 1D and 2D NMR analyses. The cytotoxicities of the 10 compounds against HeLa, K562, HL60 and HepG2 cell lines were assessed.

Loss-of-function variation in the DPP6 gene is associated with autosomal dominant microcephaly and mental retardation.

The molecular basis of autosomal dominant microcephaly, a disorder associated with small head circumferences that results in variable mental retardation, is largely unknown. In the present study, we conducted a variation analysis of the DPP6 gene in patients with autosomal dominant microcephaly and variable mental retardation. The copy number variation analysis of DPP6 was performed on DNA samples from 22 patients with microcephaly using high-resolution, array-based genomic hybridization, and sequence analysis was performed to screen mutations in another 50 microcephalic patients. Two de novo deletions and one missense mutation in familial microcephalic patients were identified. The transfection of plasmids encoding green fluorescent protein-pLLU2G-shDPP6 fusion proteins in mouse brains revealed that the decreased expression of the DPP6 gene slightly reduced the weight of the mouse brains and resulted in mouse learning disabilities compared with their wild-type littermates. Our data indicate that the loss-of-function variations in DPP6 are associated with autosomal dominant microcephaly and mental retardation. DPP6 appears to play a major role in the regulation of proliferation and migration of neurons in neurogenesis, most likely by participating in neuronal electrical excitability, synaptic integration, and plasticity.

Enhancing isoprenoid production through systematically assembling and modulating efflux pumps in Escherichia coli.

Enhancement of the cellular exportation of heterologous compounds is an important aspect to improve the product yield in microbial cell factory. Efflux pumps can expel various intra- or extra-cellular substances out of microbial hosts and increase the cellular tolerance. Thus in this study, by using the hydrophobic sesquiterpene (amorphadiene) and diterpene (kaurene) as two model compounds, we attempted to improve isoprenoid production through systematically engineering the efflux pumps in Escherichia coli BL21(DE3). The pleiotropic resistant pumps, AcrAB-TolC, MdtEF-TolC from E. coli and heterologous MexAB-OprM pump from Pseudomonas aeruginosa, were overexpressed, assembled, and finely modulated. We found that overexpression of AcrB and TolC components can effectively enhance the specific yield of amorphadiene and kaurene, e.g., 31 and 37 % improvement for amorphadiene compared with control, respectively. The heterologous MexB component can enhance kaurene production with 70 % improvement which is more effective than TolC and AcrB. The results suggest that the three components of tripartite efflux pumps play varied effect to enhance isoprenoid production. Considering the highly organized structure of efflux pumps and importance of components interaction, various component combinations were constructed and the copy number of key components AcrB and TolC was finely modulated as well. The results exhibit that the combination TolC and TolC and AcrB improved the specific yield of amorphadiene with 118 %, and AcrA and TolC and AcrB improved that of kaurene with 104 %. This study indicates that assembling and finely modulating efflux pumps is an effective strategy to improve the production of heterologous compounds in E. coli.

Analysis of methylprednisolone-induced inhibition on the proliferation of neural progenitor cells in vitro by gene expression profiling.

Recently, it has been proved that methylprednisolone has inhibition effect on the proliferation of endogenous neural progenitor cells (NPCs) after spinal cord injury (SCI). Similar effect has also been found on NPCs cultured in vitro. However, the mechanism remains to be fully delineated. The purpose of this study is to investigate the potential molecular mechanism of this effect in NPCs cultured in vitro by gene expression profiling. Fetal mouse brain-derived NPCs were divided into 2 groups: NPCs incubated with methylprednisolone as a model of the methylprednisolone treatment after SCI, and without methylprednisolone as the control group. After the cell quantitative analysis and CCK-8 assay, the microarray analysis was carried out. Genes differentially expressed between NPCs treated with and without methylprednisolone were extracted. It was observed that the expression of 143 genes, including many members of distinct families, such as hypoxia inducible factors and neurotransmitter receptors, were significantly changed in response to the methylprednisolone treatment. Our results provide global molecular insights into the mechanisms of methylprednisolone-induced proliferation inhibition effect and suggest that EdnrB may play an important role in this effect.