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After updating the configuration file, restart the Minio sever to put the changes into effect. The server will print a line like SQS ARNs: arn:minio:sqs::1:mqtt at start-up if there were no errors.

Minio supports any MQTT server that supports MQTT 3.1 or 3.1.1 and can connect to them over TCP, TLS, or a Websocket connection using tcp:// , tls:// , or ws:// respectively as the scheme for the broker url. See the Go Client documentation for more information.

Note that, you can add as many MQTT server endpoint configurations as needed by providing an identifier (like "1" in the example above) for the MQTT instance and an object of per-server configuration parameters.

We will enable bucket event notification to trigger whenever a JPEG image is uploaded or deleted images bucket on myminio server. Here ARN value is arn:minio:sqs::1:mqtt .

The python program below waits on mqtt topic /minio and prints event notifications on the console. We use paho-mqtt library to do this.

Execute this example python program to watch for MQTT events on the console.

You should receive the following event notification via MQTT once the upload completes.

Install Elasticsearch server.

This notification target supports two formats: namespace and access .

When the namespace format is used, Minio synchronizes objects in the bucket with documents in the index. For each event in the Minio, the server creates a document with the bucket and object name from the event as the document ID. Other details of the event are stored in the body of the document. Thus if an existing object is over-written in Minio, the corresponding document in the Elasticsearch index is updated. If an object is deleted, the corresponding document is deleted from the index.

When the access format is used, Minio appends events as documents in an Elasticsearch index. For each event, a document with the event details, with the timestamp of document set to the event's timestamp is appended to an index. The ID of the documented is randomly generated by Elasticsearch. No documents are deleted or modified in this format.

The steps below show how to use this notification target in namespace format. The other format is very similar and is omitted for brevity.

Minio requires a 5.x series version of Elasticsearch. This is the latest major release series. Elasticsearch provides version upgrade migration guidelines .

The default location of Minio server configuration file is ~/.minio/config.json . The Elasticsearch configuration is located in the elasticsearch key under the notify top-level key. Create a configuration key-value pair here for your Elasticsearch instance. The key is a name for your Elasticsearch endpoint, and the value is a collection of key-value parameters described in the table below.

We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the biotransformation of digoxin to dihydrodigoxin by each strain and identified seven additional strains capable of drug inactivation ( Figure 1A ). Culturing experiments were performed using 10 µM of digoxin, which is within the estimated range (0.4–14.6 µM) of therapeutic concentrations of the drug in the gut prior to absorption by host epithelial cells ( Schiller et al., 2005 ). The digoxin metabolizing strains did not exhibit a significant phylogenetic signal ( Clearance Best Seller TALL Super Skinny Smart Trousers With Blossom Print Pink Asos Get Authentic Online For Sale Discount Sale New Buy Cheap Popular 9bq7BJijR
; p=0.275, K=0.049, Blomberg’s K test), suggesting that this phenotypic trait has been gained (or lost) multiple times over the course of E. lenta evolution. Machine learning (random forests) analysis of orthologous gene cluster presence/absence across the strain collection revealed a single genetic locus with 100% discriminative value between metabolizers and non-metabolizers ( Essential Top Lapis Lazuli by VIDA VIDA Outlet Latest M2jbMV
). This locus, referred to hereafter as the cgr gene cluster, includes the previously identified 2-gene cgr operon ( cgr1 and cgr2 ) and six neighboring genes termed cac ( c gr - a ssociated c luster) genes ( Mens Palm Beach Digital Swim Cf Shorts GANT Buy Cheap Deals 9cchbtU
). The cac genes include a putative LuxR type transcriptional regulator (Cac3), a predicted flavin-dependent fumarate reductase (Cac4), three proteins of unknown function (Cac1, Cac5, Cac6), and a short protein (Cac2) that is conserved in both cgr- and cgr+ strains of E. lenta.

Figure 1
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() Survey of digoxin reduction in 21 strains of (El#), 2 strains of spp. (Gs#), (Es1), and (Ph1) ( Figure 1—source data 1 and ) …

The 10.4 kb cgr gene cluster was highly conserved between strains with an average global nucleotide identity of 99.95 ± 0.05% (mean±standard deviation (SD)). Both cgr +and cgr - strains share a short 174 bp hypothetical gene ( cac2 ) that is conserved with 100% global nucleotide identity in cgr +strains, while cgr - strains are 90.20–91.37% identical to cgr+cac2 . This conservation and genomic context may be indicative of multiple translocations in the region creating the cgr -associated gene cluster although obvious markers of recent translocation of the cgr gene cluster are absent.

Multiple lines of evidence suggested that the cgr operon encodes the enzymes responsible for digoxin metabolism. Of the eight genes in the cgr gene cluster, only three show primary sequence homology to reductases: cgr1 , cgr2 , and cac4 . RNA sequencing ( Haiser et al., 2013 ) demonstrated that the cgr operon ( cgr1 and cgr2 ) is highly upregulated (>165 fold) in response to digoxin, whereas cac4 is not significantly induced (1.3-fold change relative to vehicle controls, p=0.83) ( Figure 1E ). The remainder of the cgr- associated cluster is largely transcriptionally dormant during exponential growth both with and without the presence of digoxin (<6 normalized counts per gene) and istherefore unlikely to be linked to digoxin metabolism.Initial annotations of Cgr1 and Cgr2 suggested both proteins might mediate digoxin reduction. Cgr1 is a putative membrane-anchored protein that belongs to the cytochrome c3 superfamily (Pfam 14537) and is predicted to harbor covalently bound heme groups (CXXCH motif). It most closely resembles the NapC/NirT (NrfH) family of proteins that transfer electrons from the membrane quinone pool to associated reductases, facilitating reduction of terminal electron acceptors such as nitrite and sulfite ( Kemp et al., 2010 ; Kern et al., 2008 ). We also identified a close homolog of Cgr1 (Elen_2528) in E. lenta DSM 2243 (91.75% amino acid identity, BLASTP) that is a component of the E. lenta core genome (99.39 ± 0.81% global identity mean±SD). The presence of this highly similar protein in both metabolizing and non-metabolizing strains further indicates that Cgr1 is involved in a more general function ( e.g. electron transfer, membrane docking) rather than direct reduction of digoxin. On the other hand, Cgr2 is unique to the genomes of cgr+E. lenta , and the closest homologs of Cgr2 display<28% amino acid identity. Cgr2 is a homolog of flavin adenine dinucleotide (FAD)-dependent fumarate reductases (Pfam 00890; Interpro 003953/027477) and is predicted to undergo secretion via the twin arginine translocation (Tat) pathway. Taken together, these observations led us to hypothesize that Cgr1 and Cgr2 form a membrane-associated complex that catalyzes reduction of the α,β-unsaturated butyrolactone of digoxin.

Alternatively, these six cysteines may be critical for protein structure ( e.g. through participating in disulfide formation) or could coordinate another metal center not detectable in our spectroscopic experiments. Consistent with this latter proposal, we found that a range of divalent metal cations (Fe 2+ , Mn 2+ , Mg 2+ ) stimulated the activity of Cgr2 in vitro ( Black Swimsuit Capri Pain De Sucre Discount 100% Authentic Cheap Ebay Cheap Sale Footlocker Finishline 1K2tB
) without altering protein stability. Additionally, Fe 2+ stimulated the in vitro activity of only 3 out of 6 impaired mutants (C158A, C187A, C327A) ( Figure 2—figure supplement 3F ). Notably, binding of digoxin to its target in human cells, Na + /K + ATPase, is thought to be mediated by long-range electrostatic interactions between a Mg 2+ ion and the electron rich, partially negatively charged oxygen atom of the unsaturated lactone ( Laursen et al., 2015 ; Weigand et al., 2014 ). It is possible that the three remaining cysteine residues (C82, C265, C535) could influence binding of a divalent metal cation that similarly positions or activates digoxin in the Cgr2 active site.

To identify additional amino acids that may be important for Cgr2 function, we compared the Cgr2 sequences encoded within our collection of E. lenta genomes. Strikingly, only two cgr2 nucleotide variants were detected, which were validated by targeted Sanger sequencing. One of these variants is only found in the DSM 2243 type strain resulting in a conservative methionine (M) to valine (V) substitution at position 381. The other results in either aromatic tyrosine (Y) as in the type strain DSM 2243 or neutral asparagine (N) at position 333 ( Dame de Paris ivory 7 cm thong White Marlies Dekkers Online Sale Online Clearance With Paypal Th7n1
). We were also able to fully or partially reconstruct 14 additional cgr2 sequences using reads mapping to the cgr gene cluster from 96 gut microbiome datasets with a high abundance of E. lenta (>1x coverage or>0.001 proportional abundance). These metagenome fragments confirmed the presence of both Y333 and N333 variants in a 9:5 ratio ( Figure 3A ) while the DSM 2243 M381 variant was not observed. To avoid biases against lower E. lenta coverage metagenomes, we also applied an assembly-free method based on calling variants from aligned reads ( Womens Pockets Ecodim Panties Dim Outlet With Credit Card Discount Online Cheap Sale Newest Clearance Hot Sale Manchester Great Sale Online igdw3
). This uncovered 49 metagenomes with at least one read mapping over the variant position, confirming the bi-allelic nature with 15 Y333 and 34 N333 metagenomes. Nearly all metagenomes (41/42) with reads mapping to position 381 supported the valine residue suggesting thatthe DSM 2243 M381 variant is rare. Given that this analysis confirmed the highly conserved nature of the cgr locus, we analyzed the conservation of cgr2 in the context of the E. lenta pan-genome (based on 24 sequenced isolates) finding that it is at the 67 th percentile of conservation. These results suggest that cgr2 sequence conservation is not unusual for this species, with the caveat that relatively few genomes were available for analysis ( Figure 3C ).

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