miR-200 deficiency promotes lung cancer metastasis by activating Notch signaling in cancer-associated fibroblasts.

Lung adenocarcinoma, the most prevalent lung cancer subtype, is characterized by its high propensity to metastasize. Despite the importance of metastasis in lung cancer mortality, its underlying cellular and molecular mechanisms remain largely elusive. Here, we identified miR-200 miRNAs as potent suppressors for lung adenocarcinoma metastasis. miR-200 expression is specifically repressed in mouse metastatic lung adenocarcinomas, and miR-200 decrease strongly correlates with poor patient survival. Consistently, deletion of mir-200c/141 in the KrasLSL-G12D/+ ; Trp53flox/flox lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. miR-200 deficiency in lung cancer cells promotes the proliferation and activation of adjacent cancer-associated fibroblasts (CAFs), which in turn elevates the metastatic potential of cancer cells. miR-200 regulates the functional interaction between cancer cells and CAFs, at least in part, by targeting Notch ligand Jagged1 and Jagged2 in cancer cells and inducing Notch activation in adjacent CAFs. Hence, the interaction between cancer cells and CAFs constitutes an essential mechanism to promote metastatic potential.

RNA sequencing by direct tagmentation of RNA/DNA hybrids.

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.

Design of UAV Downwash Airflow Field Detection System Based on Strain Effect Principle.

Accurate measurement of the downwash flow field of plant protection unmanned aerial vehicles (UAVs) is essential for analyzing the spatial distribution of droplets. To realize on-line rapid detection of the downwash flow field of a multi-rotor UAV, a flexible polypropylene detection device based on the principle of full bridge strain effect was proposed. Its performance principle was based on the physical deformation caused by wind pressure. The Fluid Flow and Static Structural modules of ANSYS 16.0 finite element software were used to simulate one-way fluid-solid coupling interaction. The surface of the resistive strain gauge embedded in the flexible detecting structure responded well to wind speed variation, hence it was suitable for downwash airflow wind field detection. By solving the strain force on the surface of the flexible detection structure, the length and layout of the grating wire of the strain gauge on the surface of the flexible detection structure were optimized. Meanwhile at 4 m·s-1 wind speed, the output voltage at varied bridge flexible acquisition systems in the acquisition card was measured. Results indicated coefficient of variation of 3.67%, 1.63% and 1.5%, respectively, which proved the good data acquisition consistency of the system. Through calibration test, the regression equation for the relationship between output voltage and wind speed for three unique sensor signal measuring circuits was established. The determination coefficients R2 for single bridge, half bridge and full bridge circuits were 0.9885, 0.9866 and 0.9959, respectively. In conclusion, by applying the multi-rotor plant protection UAV test platform, the results indicated the maximum relative error of the wind speed at each sampling point of the system at 1.0 m altitude was below 5.61%. Simulated and measured value had an RMSE maximum error of 0.1246 m·s-1. Moreover, downwash airflow detection not only has high accuracy but also has high sensitivity. Thus, there is convenience and practicability in the plant protection offered by this approach. The rapid measurement of UAV wind field and the established two-dimensional wind field model can provide a basis for precise application of agricultural aviation.

Microdissection of the Ah01 chromosome in upland cotton and microcloning of resistance gene anologs from the single chromosome.

BACKGROUND: Chromosome microdissection is one of the most important techniques in molecular cytogenetic research. Cotton (Gossypium Linnaeus, 1753) is the main natural fiber crop in the world. The resistance gene analog (RGA) cloning after its single chromosome microdissection can greatly promote cotton genome research and breeding. RESULTS: Using the linker adaptor PCR (LA-PCR) with the primers of rice disease-resistance homologues, three nucleotide sequences PS016 (KU051681), PS054 (KU051682), and PS157 (KU051680) were obtained from the chromosome Ah01 of upland cotton (cv. TM-1). The Blast results showed that the three sequences are the nucleotide binding site-leucine rich repeat (NBS-LRR) type RGAs. Clustering results indicated that they are homologous to these published RGAs. Thus, the three RGAs can definitely be confirmed as NBS-LRR class of RGAs in upland cotton. CONCLUSIONS: Using single chromosome microdissection technique, DNA libraries containing cotton RGAs were obtained. This technique can promote cotton gene cloning, marker development and even the improvement of cotton genome research and breeding.

Postoperative prophylaxis of venous thromboembolism (VTE) in patients undergoing high ligation and stripping of the great saphenous vein (GSV).

To analyze the necessity of venous thromboembolism (VTE) prophylaxis for patients undergoing high ligation and stripping of the great saphenous vein (GSV) and to estimate the efficacy and safety of different anticoagulant protocols in a single-center randomized controlled trial with large sample size. A total of 2196 patients undergoing high ligation and stripping of the GSV were randomized to one of the following postoperative VTE prophylaxis protocols: group A, no VTE prophylaxis (n=542); group B, subcutaneous low-dose unfractionated heparin (LDUH) hypodermic injection, 125 U/kg per day in three divided doses (n=531); group C, low-molecular-weight heparin (LMWH) 6000 IU once a day (n=573); and group D, LMWH 4000 IU twice daily (n=550). Groups were compared for the incidence of VTE and major hemorrhage within 1 month following surgery. Varicose vein severity was classified by CEAP (Clinical, Etiologic, Anatomic, Pathophysiologic elements) score. The clinical characteristics of the patients were equally matched between groups. Postoperative deep vein thrombosis (DVT) and pulmonary embolism (PE) were significantly higher in group A (DVT 5.17%, PE 1.48%) compared to groups B (0.56%, 0%), C (0.35%, 0%) and D (0.36%, 0%) (p<0.01). The incidence of VTE did not differ between the three active chemoprophylaxis arms. Hemorrhagic complications were low for each group but higher in group B (0.75%) compared to the other groups (group A 0.18%; group C 0.17%; group D 0.18%, p<0.01). Hemorrhagic complications did not differ amongst groups A, C and D. In conclusion, postoperative VTE chemoprophylaxis following high ligation and GSV stripping effectively reduces the venous thrombosis complications of this procedure. Of the three active strategies tested, no difference in efficacy was noted; however, thrice daily LDUH did increase bleeding complications.

Effect of Fiber Content on Mechanical Properties of Fiber-Reinforced CGF All-Solid-Waste Binder-Solidified Soil.

In order to realize the resource utilization of solid waste and improve the tensile strength and toughness of soil, CCR-GGBS-FA all-solid-waste binder (CGF) composed of general industrial solid waste calcium carbide residue (CCR), ground granulated blast furnace slag (GGBS) and fly ash (FA) was used instead of cement and combined with polypropylene fiber to strengthen the silty soil taken from Dongying City, China. An unconfined compressive strength test (UCS test) and a uniaxial tensile test (UT test) were carried out on 10 groups of samples with five different fiber contents to uncover the effect of fiber content on tensile and compressive properties, and the reinforcement mechanism was studied using a scanning electron microscopy (SEM) test. The test results show that the unconfined compressive strength, the uniaxial tensile strength, the deformation modulus, the tensile modulus, the fracture energy and the residual strength of fiber-reinforced CGF-solidified soil are significantly improved compared with nonfiber-solidified soil. The compressive strength and the tensile strength of polypropylene-fiber-reinforced CGF-solidified soil reach the maximum value when the fiber content is 0.25%, as the unconfined compressive strength and the tensile strength are 3985.7 kPa and 905.9 kPa, respectively, which are 116.60% and 186.16% higher than those of nonfiber-solidified soil, respectively. The macro-micro tests identify that the hydration products generated by CGF improve the compactness through gelling and filling in solidified soil, and the fiber enhances the resistance to deformation by bridging and forming a three-dimensional network structure. The addition of fiber effectively improves the toughness and stiffness of solidified soil and makes the failure mode of CGF-solidified soil transition from typical brittle failure to plastic failure. The research results can provide a theoretical basis for the application of fiber-reinforced CGF-solidified soil in practical engineering.

Carvacrol affects breast cancer cells through TRPM7 mediated cell cycle regulation.

As the most prevalent cancer for females, breast cancer is also the second most popular cancer type overall. More efforts are needed to research new drugs and combination therapies for this disease. A naturally derived transient receptor potential melastatin-like 7 channel (TRPM7) inhibitor, carvacrol, was found to have anti-cancer potentials. We hypothesized that carvacrol affects breast cancer cells through TRPM7 mediated cell cycle regulation. Cell viability and apoptosis of breast cancer cell lines BT-483, BT-474, MCF-7, MDA-MB-231, and MDA-MB-453 were determined using the CCK-8 assay and ELISA respectively. TRPM7 in MDA-MB-231, MCF-7 was knocked down. Functional TRPM7 in MDA-MB-231, MCF-7, and HEK293 cells were tested with western blotting, patch-clamp, and fura-2 quench assay. The cell cycle and the regulatory proteins were determined by flow cytometry and western blotting. Results showed that carvacrol inhibited the viability of breast cancer cells with different potency. At 200 µM, MDA-MB-231 was the most sensitive, and MCF-7 was the least sensitive. At >200 µM, the apoptosis was dramatically induced. Carvacrol inhibited TRPM7 functions in MDA-MB-231, MCF-7, and HEK293. Carvacrol at 200 µM increased cells in the G1/G0 phase and decreased cells in the S and G2/M phase by regulating some cyclin proteins in MDA-MB-231. These effects were blocked by the knockdown of TRPM7. This study demonstrated that carvacrol suppresses breast cancer cells by cell cycle regulation and the TRPM7 pathway is one of the pharmacological mechanisms.

Tumor necrosis factor ligand-receptor superfamily and arthritis.

The current studies of apoptosis in rheumatoid arthritis (RA) suggest that the TNF ligand-receptor superfamily (TNFRsF) molecules, downstream pathways (activation of proapoptosis or anti-apoptosis pathway), cell types (lymphocytes and synovial fibroblast), and the mechanism that triggers apoptosis (tolerance induction-related, downmodulation of inflammation-related, or DNA damage-related) all exhibit a capability to determine the induction or prevention of RA. This series of defects at different levels and in different cells have been shown to lead to T cell and synovial hyperproliferation, defective apoptosis, excessive apoptosis, or bone erosion. In this chapter, we summarize the available knowledge of the regulation of TNFRsF and their likely pathogenic roles in RA to help identify candidate target cells and target molecules for delivery of gene constructs to modulate apoptosis to prevent the development of RA in both humans and mice.

Multi-label spacecraft electrical signal classification method based on DBN and random forest.

In spacecraft electrical signal characteristic data, there exists a large amount of data with high-dimensional features, a high computational complexity degree, and a low rate of identification problems, which causes great difficulty in fault diagnosis of spacecraft electronic load systems. This paper proposes a feature extraction method that is based on deep belief networks (DBN) and a classification method that is based on the random forest (RF) algorithm; The proposed algorithm mainly employs a multi-layer neural network to reduce the dimension of the original data, and then, classification is applied. Firstly, we use the method of wavelet denoising, which was used to pre-process the data. Secondly, the deep belief network is used to reduce the feature dimension and improve the rate of classification for the electrical characteristics data. Finally, we used the random forest algorithm to classify the data and comparing it with other algorithms. The experimental results show that compared with other algorithms, the proposed method shows excellent performance in terms of accuracy, computational efficiency, and stability in addressing spacecraft electrical signal data.

Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don't allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%.

Publisher Correction: Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.

In the version of this Article originally published, owing to a technical error, the Life Sciences Reporting Summary was not included; this summary is now available.

Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) efficiently generate all embryonic cell lineages but rarely generate extraembryonic cell types. We found that microRNA miR-34a deficiency expands the developmental potential of mouse pluripotent stem cells, yielding both embryonic and extraembryonic lineages and strongly inducing MuERV-L (MERVL) endogenous retroviruses, similar to what is seen with features of totipotent two-cell blastomeres. miR-34a restricts the acquisition of expanded cell fate potential in pluripotent stem cells, and it represses MERVL expression through transcriptional regulation, at least in part by targeting the transcription factor Gata2. Our studies reveal a complex molecular network that defines and restricts pluripotent developmental potential in cultured ESCs and iPSCs.

A Spacecraft Electrical Characteristics Multi-Label Classification Method Based on Off-Line FCM Clustering and On-Line WPSVM.

This paper proposes a novel multi-label classification method for resolving the spacecraft electrical characteristics problems which involve many unlabeled test data processing, high-dimensional features, long computing time and identification of slow rate. Firstly, both the fuzzy c-means (FCM) offline clustering and the principal component feature extraction algorithms are applied for the feature selection process. Secondly, the approximate weighted proximal support vector machine (WPSVM) online classification algorithms is used to reduce the feature dimension and further improve the rate of recognition for electrical characteristics spacecraft. Finally, the data capture contribution method by using thresholds is proposed to guarantee the validity and consistency of the data selection. The experimental results indicate that the method proposed can obtain better data features of the spacecraft electrical characteristics, improve the accuracy of identification and shorten the computing time effectively.

TaqMan® OpenArray® high-throughput transcriptional analysis of human embryonic and induced pluripotent stem cells.

It is widely accepted that somatic cells can be reprogrammed by a set of transcription factors to become embryonic stem cell-like: These reprogrammed cells, induced pluripotent stem cells (iPSCs), are nearly identical to embryonic stem cells (ESCs), because both have the capacity to self-renew and to form all cellular lineages of the body. Transcriptional differences between ESCs, iPSCs, and fibroblasts can be analyzed by quantitative PCR (qPCR) using TaqMan(®) Gene Expression assays, a widely used tool for rapid analysis of different cell types. In this chapter, we describe the OpenArray(®) platform which generates qPCR data from high-throughput instrumentation. We examined the gene signature profiles of ESCs, fibroblasts, and iPSCs with a TaqMan(®) OpenArray(®) Human Stem Cell Panel containing 631 TaqMan(®) Gene Expression assays that represent pathways involved in self-renewal, pluripotency, lineage patterning, transcriptional networks, stem cell differentiation, and development.

Multiple roles for CCR2 during fracture healing.

Bone injury induces an inflammatory response that involves neutrophils, macrophages and other inflammatory cells. The recruitment of inflammatory cells to sites of injury occurs in response to specific signaling pathways. The CC chemokine receptor type 2 (CCR2) is crucial for recruiting macrophages, as well as regulating osteoclast function. In this study, we examined fracture healing in Ccr2-/- mice. We first demonstrated that the expression of Ccr2 transcripts and the filtration of macrophages into fracture calluses were most robust during the early phases of fracture healing. We then determined that the number of macrophages at the fracture site was significantly lower in Ccr2-/- mice compared with wild-type controls at 3 days after injury. As a result, impaired vascularization, decreased formation of callus, and delayed maturation of cartilage were observed at 7 days after injury in mutant mice. At day 14, Ccr2-/- mice had less bone in their calluses. At day 21, Ccr2-/- mice had larger calluses and more bone compared with wild-type mice, suggesting a delayed remodeling. In addition, we examined the effect of Ccr2 mutation on osteoclasts. We found that a lack of Ccr2 did not affect the number of osteoclasts within fracture calluses at 21 days after injury. However, Ccr2-/- osteoclasts exhibited a decreased ability to resorb bone compared with wild-type cells, which could contribute to the delayed remodeling of fracture calluses observed in Ccr2-/- mice. Collectively, these results indicate that a deficiency of Ccr2 reduces the infiltration of macrophages and impairs the function of osteoclasts, leading to delayed fracture healing.

Osteoclasts - the innate immune cells of the bone.

Osteopenia and periarticular bony erosion are consequences of chronic inflammatory autoimmune disease due to an imbalance of osteoclast activity relative to new bone formation. Osteoclasts, which are specialized as the only bone resorbing cell type, are differentiated from hematopoietic myeloid precursor cells. Inflammatory signals mediated by multiple types of immune cells and cytokines have significant influence over osteoclast differentiation and function through direct effects on osteoclast precursors and indirect effects via osteoblasts and other cells in the bony microenvironment including synovial cells, stromal cells, osteocytes and chondrocytes. Recent studies have demonstrated that osteoclasts themselves express a number of immune receptors and are regulated similarly to macrophages and dendritic cells, closely related cells in the innate immune system. Though we are only beginning to understand the roles of innate immune receptors in osteoclasts, some of these receptors have been shown to be critical regulators of differentiation and function of osteoclasts. Osteoclasts likely function as the innate immune cells of the bone, thus are highly regulated to appropriately respond to stress and inflammatory changes in their microenvironment.

Bone microenvironment specific roles of ITAM adapter signaling during bone remodeling induced by acute estrogen-deficiency.

Immunoreceptor tyrosine-based activation motif (ITAM) signaling mediated by DAP12 or Fcepsilon receptor Igamma chain (FcRgamma) have been shown to be critical for osteoclast differentiation and maturation under normal physiological conditions. Their function in pathological conditions is unknown. We studied the role of ITAM signaling during rapid bone remodeling induced by acute estrogen-deficiency in wild-type (WT), DAP12-deficient (DAP12-/-), FcRgamma-deficient (FcRgamma-/-) and double-deficient (DAP12-/-FcRgamma-/-) mice. Six weeks after ovariectomy (OVX), DAP12-/-FcRgamma-/- mice showed resistance to lumbar vertebral body (LVB) trabecular bone loss, while WT, DAP12-/- and FcRgamma-/- mice had significant LVB bone loss. In contrast, all ITAM adapter-deficient mice responded to OVX with bone loss in both femur and tibia of approximately 40%, relative to basal bone volumes. Only WT mice developed significant cortical bone loss after OVX. In vitro studies showed microenvironmental changes induced by OVX are indispensable for enhanced osteoclast formation and function. Cytokine changes, including TGFbeta and TNFalpha, were able to induce osteoclastogenesis independent of RANKL in BMMs from WT but not DAP12-/- and DAP12-/-FcRgamma-/- mice. FSH stimulated RANKL-induced osteoclast differentiation from BMMs in WT, but not DAP12-/- and DAP12-/-FcRgamma-/- mice. Our study demonstrates that although ITAM adapter signaling is critical for normal bone remodeling, estrogen-deficiency induces an ITAM adapter-independent bypass mechanism allowing for enhanced osteoclastogenesis and activation in specific bony microenvironments.

Overexpression of activation-induced cytidine deaminase in B cells is associated with production of highly pathogenic autoantibodies.

Defective receptor editing or defective B cell checkpoints have been associated with increased frequency of multireactive autoantibodies in autoimmune disease. However, Ig somatic hypermutation and/or class switch recombination may be mechanisms enabling the development of pathogenic multireactive autoantibodies. In this study, we report that, in the BXD2 mouse model of autoimmune disease, elevated expression of activation-induced cytidine deaminase (AID) in recirculating follicular CD86(+) subsets of B cells and increased germinal center B cell activity are associated with the production of pathogenic multireactive autoantibodies. CD4 T cells from BXD2 mice that expressed increased levels of CD28 and an increased proliferative response to anti-CD3 and anti-CD28 stimulation are required for this process. Inhibition of the CD28-CD86 interaction in BXD2 mice with AdCTLA4-Ig resulted in normalization of AID in the B cells and suppression of IgG autoantibodies. This treatment also prevented the development of germinal center autoantibody-producing B cells, suggesting that an optimal microenvironment enabling AID function is important for the formation of pathogenic autoantibodies. Taken together, our data indicate that AID expression in B cells is a promising therapeutic target for the treatment of autoimmune diseases and that suppression of this gene may be a molecular target of CTLA4-Ig therapy.

Synovial fibroblasts promote osteoclast formation by RANKL in a novel model of spontaneous erosive arthritis.

OBJECTIVE: Erosion of cartilage and bone is a hallmark of rheumatoid arthritis (RA). This study was undertaken to explore the roles of hyperproliferating synovial fibroblasts and macrophages in abnormal osteoclast formation, using the recently described BXD2 mouse model of RA. METHODS: Cell distribution in the joints was analyzed by immunohistochemistry, using tartrate-resistant acid phosphatase (TRAP) staining to identify osteoclasts. To identify the defective cells in BXD2 mice, mouse synovial fibroblasts (MSFs) were cultured with bone marrow-derived macrophages. Osteoclast formation was assayed by TRAP staining and bone resorption pit assay, and the cytokine profiles of the MSFs and macrophages were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: In BXD2 mice, TRAP-positive osteoclasts were found at sites of active bone erosion, in close proximity to hyperproliferating synovial fibroblasts. On coculture, MSFs from BXD2 mice, but not C57BL/6 mice, produced high levels of RANKL messenger RNA, induced macrophages to form osteoclasts, and actively eroded bone slices, through a mechanism(s) that could be blocked by pretreatment with osteoprotegerin. Although macrophages from BXD2 mice expressed higher basal levels of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-6 than those from C57BL/6 mice, abnormal osteoclast formation was not due to enhanced sensitivity of the BXD2 mouse macrophages to RANKL. TNFalpha, produced by both BXD2 MSFs and BXD2 mouse macrophages, had a strong stimulatory effect on RANKL expression. CONCLUSION: BXD2 MSFs produce RANKL and induce the development of osteoclasts from macrophages. The enhanced production of RANKL is possibly due to autocrine stimulation, together with paracrine stimulation by factors produced by macrophages.

Jab1 antagonizes TGF-beta signaling by inducing Smad4 degradation.

Tumor suppressor Smad4 is the common signaling effector in the transforming growth factor beta (TGF-beta) superfamily. Phosphorylated regulatory Smads (R-Smads) interact with Smad4, and the complex translocates into the nucleus to regulate gene transcription. Proper TGF-beta signaling requires precise control of Smad functions. Smurfs have been shown to mediate the degradation of R-Smads but not the common-partner Smad4. We report a novel mechanism of Smad4 degradation. Jab1 interacts directly with Smad4 and induces its ubiquitylation for degradation. Jab1 was initially identified as a co-activator of c-Jun, and it also induces degradation of cell cycle inhibitor p27 and tumor suppressor p53. Ectopic expression of Jab1 decreased endogenous Smad4 steady-state levels. The 26S proteasome inhibitors lactacystin and MG132 reduced the degradation rate of Smad4 protein. Examination of the effects of JAB1-induced Smad4 degradation indicates that Jab1 inhibited TGF-beta-induced gene transcription. Our data suggest that Jab1 antagonizes TGF-beta function by inducing degradation of Smad4 through a distinct degradation pathway.