Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks

Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 and designed sgRNAs to build transcriptional logic gates and connect them to perform computation in living cells. We constructed a set of NOT gates by designing five synthetic Escherichia coli σ[subscript 70] promoters that are repressed by corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. These sgRNAs exhibit high on‐target repression (56‐ to 440‐fold) and negligible off‐target interactions (< 1.3‐fold). These gates were connected to build larger circuits, including the Boolean‐complete NOR gate and a 3‐gate circuit consisting of four layered sgRNAs. The synthetic circuits were connected to the native E. coli regulatory network by designing output sgRNAs to target an E. coli transcription factor (malT). This converts the output of a synthetic circuit to a switch in cellular phenotype (sugar utilization, chemotaxis, phage resistance).United States. Defense Advanced Research Projects Agency (CLIO N66001‐12‐C‐4016)National Institutes of Health (U.S.) (GM095765)National Institute of General Medical Sciences (U.S.) (Grant P50 GMO98792)Synthetic Biology Engineering Research Center (EEC0540879)United States. Defense Advanced Research Projects Agency (Ginkgo BioWorks. CLIO N66001‐12‐C‐4018)United States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant N00014‐13‐1‐0074)United States. Office of Naval Research. Multidisciplinary University Research Initiative (Boston University. Award 4500000552)United States. Air Force Office of Scientific Research (FA9550‐11‐C‐0028)American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship (32 CFR 168a

MMP-13 stimulates osteoclast differentiation and activation in tumour breast bone metastases

INTRODUCTION: The increased bone degradation in osteolytic metastases depends on stimulation of mature osteoclasts and on continuous differentiation of new pre-osteoclasts. Metalloproteinases (MMP)-13 is expressed in a broad range of primary malignant tumours and it is emerging as a novel biomarker. Recent data suggest a direct role of MMP-13 in dissolving bone matrix complementing the activity of MMP-9 and other enzymes. Tumour-microenvironment interactions alter gene expression in malignant breast tumour cells promoting osteolytic bone metastasis. Gene expression profiles revealed that MMP-13 was among the up-regulated genes in tumour-bone interface and its abrogation reduced bone erosion. The precise mechanism remained not fully understood. Our purpose was to further investigate the mechanistic role of MMP-13 in bone osteolytic lesions. METHODS: MDA-MB-231 breast cancer cells that express MMP-13 were used as a model for in vitro and in vivo experiments. Conditioned media from MDA-MB-231 cells were added to peripheral blood mononuclear cultures to monitor pre-osteoclast differentiation and activation. Bone erosion was evaluated after injection of MMP-13-silenced MDA-MB-231 cells into nude mice femurs. RESULTS: MMP-13 was co-expressed by human breast tumour bone metastases with its activator MT1-MMP. MMP-13 was up-regulated in breast cancer cells after in vitro stimulation with IL-8 and was responsible for increased bone resorption and osteoclastogenesis, both of which were reduced by MMP inhibitors. We hypothesized that MMP-13 might be directly involved in the loop promoting pre-osteoclast differentiation and activity. We obtained further evidence for a direct role of MMP-13 in bone metastasis by a silencing approach: conditioned media from MDA-MB-231 after MMP-13 abrogation or co-cultivation of silenced cells with pre-osteoclast were unable to increase pre-osteoclast differentiation and resorption activity. MMP-13 activated pre-MMP-9 and promoted the cleavage of galectin-3, a suppressor of osteoclastogenesis, thus contributing to pre-osteoclast differentiation. Accordingly, MMP-13 abrogation in tumour cells injected into the femurs of nude mice reduced the differentiation of TRAP positive cells in bone marrow and within the tumour mass as well as bone erosion. CONCLUSIONS: These results indicate that within the inflammatory bone microenvironment MMP-13 production was up-regulated in breast tumour cells leading to increased pre-osteoclast differentiation and their subsequent activation

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Rheumatoid Factor Positivity Is Associated with Increased Joint Destruction and Upregulation of Matrix Metalloproteinase 9

We evaluated changes in gene expression of mTOR, p21, caspase-3, ULK1, TNFα, matrix metalloproteinase (MMP)-9, and cathepsin K in the whole blood of rheumatoid arthritic (RA) patients treated with methotrexate (MTX) in relation to their rheumatoid factor status, clinical, immunological, and radiological parameters, and therapeutic response after a 24-month follow-up. The study group consisted of 35 control subjects and 33 RA patients without previous history of MTX treatment. Gene expression was measured using real-time RT-PCR. Decreased disease activity in patients at the end of the study was associated with significant downregulation of TNFα expression. Downregulation of mTOR was observed in seronegative patients, while no significant changes in the expression of p21, ULK1, or caspase-3 were noted in any RA patients at the end of the study. The increase in erosion numbers observed in the seropositive patients at the end of the follow-up was accompanied by upregulation of MMP-9 and cathepsin K, while seronegative patients demonstrated an absence of significant changes in MMP-9 and cathepsin K expression and no increase in the erosion score. Our results suggest that increased expression of MMP-9 and cathepsin K genes in the peripheral blood might indicate higher bone tissue destruction activity in RA patients treated with methotrexate. The clinical study registration number is 0120.0810610

The expression of interferon-stimulated genes (interferon “signature”) in patients with rheumatoid arthritis (Preliminary results)

Objective. To assess the expression of interferon-stimulated genes in patients with rheumatoid arthritis (RA).Material and methods. Twenty patients with RA were examined. All patients received methotrexate therapy at a stable dose (Me 15 [10-17.5] mg) for at least 4 weeks. To assess the Type I IFN gene signature (IFNGS) we selected five genes (IFI44L, MX1, IFIT1, RSAD2, EPSTI1). The expression of IFI44L and IFIT1 could not be determined, and further analysis took into account three genes - MX1, EPSTI1, RSAD2.Results. Baseline level of MX1 expression - 11.48 [5.45-19.38], EPSTI1 - 12.83 [5.62-19.64], RSAD2 - 5.16 [2.7310.4] and IFN score - 10.3 [5.18-17.12] in patients with rheumatoid arthritis was significantly higher compared with healthy donors - 1.26 [0.73-1.6], 1.06 [0.81-1.48], 0.93 [0.72-1.19], and 1.09 [0.92-1.42] respectively, p&lt;0.05. IFN signature was found in 15 (75%) patients, was absent - in 5 (15%) patients. A positive correlation was found between the IFNGS and the level of EPSTI1 expression with the duration of methotrexate therapy (r=0.46, p=0.03). Among patients who received methotrexate therapy for more than one year, there was a tendency to a higher level of EPSTI1 expression (10.74 [12.6-32.8]) and IFNGS (16.2 [8.9-38.3]) compared with patients taking methotrexate for less than a year (9.67 [5.4-14.2] and 7.9 [4.5-13.4]), (p=0.06).Conclusion. Preliminary results on the assessment of IFNGS indicate an increased expression of IFN-stimulated genes in patients with RA, which may be important for predicting the course of the disease and personalizing therapy