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Being a father, you continue to seek out tools to help your children grow and learn. I stumbled across “The Histronauts: A Viking Adventure” one day and it immediately captured my attention. A children’s book aimed to teach the younger generation history in a relatable and fun way? Yes, please! After reading it myself and seeing my kids reaction to each page, I knew this book was a slam dunk. From the colorful artistic pages full of life, to the fun story of the Histronauts characters, to the well researched facts and activities to interact with, I could not recommend this book enough to any parent out there.
Frances Durkin is a doctoral researcher of medieval history from the University of Birmingham and author of The Histronauts book series. I was delighted to speak with Frances about the type of research she conducted in “A Viking Adventure”, her motivation in writing children’s books, the characters of the Histronauts, the art by illustrator Grace Cooke, and more.
Going Viking: For the people who don’t know, what is the Histronauts?
Frances Durkin: The Histronauts is a series of children’s books about a group of modern-day children (and their pet cat) who travel back in time to different periods of history. Our latest book is about the Vikings and in it the Histronauts meet a Norse girl whose father builds longships. Through her they find out about how people lived their lives: what they ate, where they lived, how they travelled and what the world was like through their eyes. Our readers share in their learning experience by following their story. The books are designed as graphic novels, they are full of detailed illustrations and we’ve packed them with as many facts and activities as we could fit inside.
I believe it is very important to implement reading and history at an early age. Stories woven like this can leave a lasting impact on how a child might remember periods of history. What motivated you to write a children’s book?
My own childhood. I wouldn’t be a historian now if I hadn’t loved it so much as a child. I was extremely lucky to grow up in a part of the world which was very connected to its history. I was never far from a castle or an old roman wall. But storytelling was absolutely at the heart of why I was so interested. Sherwood Forest was on my doorstep and the stories of Robin Hood were my first step towards ultimately becoming a medievalist. I loved finding out more about what the world around me would have been like in previous centuries. I’ve always wanted to focus on engaging a younger audience because I want to be able to inspire that same enthusiasm for history which was such a big part of my life growing up.
My kids really enjoyed the Histronauts. I believe it made the learning part more accessible to them because they connected with these characters. How did you come up with this group of characters?
I’m so pleased that they liked them! We really wanted them to be engaging and relatable. It was important to us that our readers see themselves as an extension of the Histronaut team. The four characters we have came about from the initial collaboration between me and illustrator Grace Cooke. We had a bigger group of characters to choose from at one point in the early stages of creating the books. In the end these were the characters that we loved the most. We knew their personalities and how they would respond to being in these extraordinary time-travelling situations. We’re both big sisters so we were really drawn to having a sibling relationship in the book as well as a little friendship group, so we have a brother and sister, and their best friend. And we love animals so it was completely natural to have a pet which travelled with them. I really wanted to name the cat Herodotus after the ancient Greek historian and a friend of mine brilliantly suggested that we shorten his name to Hero.
As they were concerned about the Histronauts, I was taken aback by how detailed you made this book between the artwork and all the information that was provided. I was enjoying it just as much as they were, maybe even more. What grabbed your attention to begin the research about the Vikings?
The Vikings were amazing! They were really high on the list of subjects that we wanted to cover when we first came up with the idea for the books. We make the decision about the topics with our UK publisher and they’re brilliant at listening to our reasons for choosing the periods of history that we want to explore. This is our third book and we knew that we wanted to travel back to a time which offered our story a wealth of colour and adventure. I grew up close enough to York that visits to the Jorvik Centre were a treat when I was younger. Jorvik allows you to travel through a reconstruction of the Viking city on the site where the archaeological excavations took place. It brilliantly builds something entirely real and relatable out of the evidence. The Saga Museum in Iceland does something similar and having these tangible reconstructions really sparked my imagination to step into the Viking world. There’s something so incredibly fascinating about the sense of adventure that the Vikings encapsulate. To be able to fill a boat with as much as you can, point it at the horizon, and just keep going fills me with awe.
There’s a tremendous amount of research that can be seen through this book on a variety of topics in the Viking Age. What type of sources did you look at to help write this book?
I started by reading as much as I could. I focused on books, journal articles and other academic research to build the text of the story and the boxes of information which are interspersed throughout the books. Library catalogues are my usual starting points and I read as many recent journal articles as I can get my hands on. Everything I present in the book is meticulously researched. That does sometimes mean that I have to make choices to omit something when the knowledge that we have isn’t concrete enough to present something as fact. For example I would have loved to write about female Viking warriors. The scholarly debate surrounding the Birka burial findings is fascinating, but it didn’t meet the criteria I set myself for including female warriors as a given fact. However, I did get to have our little Norse girl tell The Histronauts that there are stories of Viking warrior women. And I have to be very aware of my audience. One of my favourite pages in the book is an illustration of a boat burial and of course my research included Ahmad ibn Fadlan’s account from the tenth century. It’s an extraordinary version of the event but not at all suitable for children, even the ones who really love their gory stories.
When you started “A Viking Adventure” did you know a lot about this time, or did the research you conducted for this book really open your eyes about who the Vikings really were?
I certainly thought I knew lots about the period and the people before I started, but researching the book opened my eyes to a wealth of fascinating new information. As I’m sure you, and anyone who has engaged with a research project knows, one question inevitably leads to another. New things are being discovered all the time and that makes being a historian endlessly rewarding.
The art in “A Viking Adventure” is phenomenal. Grace Cooke really did a great job in portraying the life of the Vikings with the care of the details she drew. How much archaeological research went into recreating some of the illustrations like the longships?
She’s amazing! Her work opens up a whole world of visual communication which is so key to these books. The Histronauts books have two research stages; once I’ve researched and written the story and text, then Grace and I work together on investigating the details of the visual elements for her illustrations. We both drew on as much archaeological information as we could with everything from the largescale objects such as the longships, to the smaller more intimate objects such as the combs and jewellery. Museum exhibitions have always been the starting point for our research as they help us to build a visual idea of what we’re going to work on. When we first started this book, we took a trip up to York to see the Jorvik Centre together, we visited the Vale of York Hoard, the British Museum, and we explored museum catalogues online. We built up a visual scrapbook of our research that we could refer back to. Archaeology is absolutely key to this. To go back to the boat burial illustration I mentioned earlier, we looked very closely at the information about the unearthed boats such as those found at Gokstad, Oseberg and Scar. We used these to build up our image and to make sure it contained as much information as possible. We really wanted to be respectful of those burials and Grace has created an image which I find incredibly moving.
Besides the Vikings, you also have “An Egyptian Adventure” and “A Roman Adventure” books. What time period would you like to explore next with the Histronauts?
Oh, where do I start?! Our fourth book is about ancient Greece. I had a great time researching and writing it and I can’t wait to see the illustrations bring everything to life. My PhD research is based in medieval Europe so I would love to explore that period with The Histronauts. I think they’d have a great time in the Middle Ages and I have so many ideas – maybe too many – I might need three books for that one. But there are so many other, less familiar periods which I think would be incredible to explore. I’m also very greedy to see what the Grace Cooke versions of different periods of history look like.
You can find “The Histronauts: A Viking Adventure” on Amazon and Amazon UK, along with their other two books, “The Histronauts: An Egyptian Adventure” and “The Histronauts: A Roman Adventure.” For more information, go to https://www.thehistronauts.com/ and on Twitter at @HistoriaFrankie and @TheHistronauts.


Designed by Les George, the Kershaw XCOM was inspired by the classic WWII trench knife. It has been scaled down to facilitate functional everyday carry with a tactically inspired build. The XCOM features a thumb disk opener and an inset stainless steel liner lock. Item is outfitted with a stainless steel pocket clip for tip-up carry.
Check out some other awesome knives designed by Les George!
I bought this knife from a review I saw! I bought this to add to my collection! It is easy to open! I like the spear point blade! It feels comfortable in my hand! The handle is like the k-bar with the lines! You can open it left or right hand! Cool for a gift or add to your collection! Nice price!
Good knife,,solid,sharp an looks cool! Purchased 2,,1 for my grandsons 14th birthday and 1 for just cause I'm a good granpa.
I bought this knife after reading a magazine article on it. It is simply outstanding! Easy to carry, draw, and deploy. Good grips and a guard for your fingers. Been using it for months now with no issues.
You, know, there are some knives that you look at and just say to yourslef, "I've gotta have one of those." For me, the XCOM is one of those knives. That's saying something, coming from me. I generally am not a fan of Kershaw knives because I consider most of them a little too thick and/or heavy. Not so with this one! The knife is light in the hand and just feels comfortable. The handle fits the hand comfortably and the black finish and scales make it an outstanding looker. 8CR13MOV steel is tough and the knife does the job you bought it for. Sharp blade with the dagger look that appeals to me. This knife will make me take a second look the next time a Kershaw comes up. Nice work on the design. I'm sold. Well worth the bargain price.
Cool, smooth, awesome deployment, and smooth lock. Great deep clip too. Price is insane for such a fabulous blade. 5 stars if XCOM had been, well, bigger. 4 inch blade would have made this perfect.
Good: This is a good looking knife, well made, feels good in the hand. Not as good: This knife has a thumb disc, which makes it ambidextrous, good for lefties like me. Unfortunately a thumb disc is only good if the knife opens freely. That’s why I’ve always despised them. This knife is way too stiff for that. The stiffness of opening this blade turns it into a two handed knife, and the highly textured disc into a thumb grinder, instead of a thumb opener. Since this design is by definition a fighting knife, difficult deployment is a serious problem. A flipper would solve that, but as it is, it’s going to be a project to get it right.
Well made, sharp, and what you expect from Kershaw. Doesn't handle that well when retrieving from your pocket but it was designed to look like a trench knife so what else would you expect? Not an everyday carry for me but I like the look of it.


- Today Waninkoko
mentioned he agrees with Hermes
's recent views on the discussion of PS3 NPDRM SELFs, how their encryption differs, GeoHot and what PS3 Firmware 3.60 may bring to combat PlayStation 3 hackers.
Below is a rough translation from Elotrolado, to quote:
"Let's see... speaking of bans


text:
A Michelson interferometer uses light from a sodium lamp. Sodium atoms emit light having wavelengths and . The interferometer is initially set up with both arms of equal length , producing a bright spot at the center of the interference pattern. How far must mirror be moved so that one wavelength has produced one more new maximum than the other wavelength?
Length from one more new maximum than other wavelength, L
These lamps are mostly utilized for street and industrial lighting.
The lamp works by using vaporized sodium metal to create an electric arc.
To help begin the lamp or adjust its hue, other compounds and gases are utilized.
For the sake of clarity, we will refer to the distance travelled by rather than .
The gap between both the number of fringes produced by each wavelength will be determined by
We can find it by solving for the distance para metrically.
In our scenario, we will have the following numbers:
A 600 line/mm diffraction grating is in an empty aquarium tank. The index of refraction of the glass walls is . A helium-neon laser is outside the aquarium. The laser beam passes through the glass wall and illuminates the diffraction grating.
a. What is the first-order diffraction angle of the laser beam?
b. What is the first-order diffraction angle of the laser beam after the aquarium is filled with water ?
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domain: chemistry


text:
15 vestidos de novia de manga larga que te enamorarán
Instantanea Toma Primera
Panambi Novias Like um just cause they got my babies name Novia Marie so cute!
Bgo and me
How to Choose the Right Bride Gown?
From Lista with love
marta martí long-sleeve wedding dress with sheer lace cuff sleeves and v-neck back
Saja Wedding Dresses for Simple wedding dresses with delicate embellishment accents these elegantly understated bridal gowns from Saja. Long sleeve wedding dresses, and vintage wedding dresses handmade in NYC.

domain: design


Sensys ascends to the cloud
All of Sensys Networks wireless sensors are now being shipped with capability for use with Cloud Connect – the company’s new data hosting service. Traffic engineers can monitor an entire region’s intersections without necessity for servers or software. The technology is claimed to have completely eliminated uncertainty or guesswork over whether signal control detection equipment is working properly. Cloud Connect provides data in real-time, so there is no reason to have broken inductive loops at intersections.


Updated Tuesday, Jan. 26 at 1:30 p.m. PT.
The Senate filibuster allows a minority in the Senate to prevent the adoption of important legislation—even when the president and majorities in both the House and Senate are in favor.
Democracy requires two qualities—a government whose leaders are chosen by free and fair elections, and decisions that reflect the will of the majority.
Surprisingly, the U.S. does not always acknowledge the principle of majority vote. The unusual role of the Electoral College in presidential elections is but one example. On several occasions, the winner of the popular vote did not become president. In 2016, for example, Hillary Clinton received 48 percent of the popular vote, whereas Donald Trump received 46 percent. Yet the Electoral College vote—304 to 227—gave the presidency to Trump. Fortunately, the 2020 election did not suffer this same fate: Joe Biden defeated Trump in both the popular vote and the Electoral College (the views of a violent mob of insurrectionists notwithstanding).
The Senate Filibuster
Voting rules in Congress also reflect this unusual dynamic. This is most evident in the Senate. Under current rules, decisions requiring Senate approval are subject to debate. This includes the approval of pending bills as well as advice and consent for presidential appointments. But if debate does not end, no vote can be taken. The filibuster—a mechanism that allows a minority of senators to prevent debate from ending—precludes the Senate from taking action.
The filibuster is a powerful mechanism because it requires 60 senators (out of 100) to override it by voting to end debate, a process known as cloture. Recent changes in filibuster practice allow Senate votes on presidential appointments to executive branch positions and the federal judiciary to proceed based on a simple majority vote. However, the requirement that 60 senators must vote for cloture applies to most legislation (except for budget bills).
In other words, the Senate filibuster still allows a minority in the Senate to prevent the adoption of important legislation, even when the president and majorities in both the House and Senate are in favor. (As Ezra Klein writes in the New York Times: “President Biden’s agenda will live or die in the Senate. Odds are it will die, killed by the filibuster.”)
Senator Mitch McConnell, Kentucky Republican and new minority leader, is clearly aware of the sheer force of the filibuster, and he wielding what little influence he has in the Senate in an attempt to get Senate Majority Leader Chuck Schumer (D-N.Y.) to pledge not to get rid of it—an offer Schumer has vehemently opposed. Late Monday, McConnell signaled he’d drop this demand.
The filibuster is often described as a countermajoritarian mechanism that protects the Senate minority. It is also alleged to promote bipartisan collaboration. At times, the filibuster has fulfilled these important functions. But in most cases, the filibuster has been used as a partisan mechanism to undermine democratic governance.
It is no coincidence that the most notorious examples of the filibuster occurred in the midst of the civil rights era. In 1957, Strom Thurmond personally filibustered on the Senate floor for 24 hours to prevent debate on civil rights legislation. In 1967, Southern senators used the filibuster to prevent a vote on the Civil Rights Act for 74 days.
In recent years, the filibuster has been used with increasing frequency in the Senate. The consequences are significant. It has reduced the amount of legislation adopted by Congress. Both parties can point to the Senate filibuster to explain failed legislative efforts.
To be clear, the Senate’s voting rules are not required by the Constitution. They developed piecemeal in a process that can be traced to 1806. In fact, it was Aaron Burr who proposed changing the Senate rules that allowed a simple majority to end debate in the chamber. Burr’s proposal was not motivated by countermajoritarian concerns; it was a housekeeping measure designed to simplify Senate business. Because the filibuster is a Senate rule and not a constitutional mandate, it can be amended or rescinded by the Senate at any time—ironically by a simple majority.
Ending the Filibuster
With Democrats now in control of the White House and Congress, the Senate may soon consider whether to revise or eliminate the filibuster option for legislation.
For Democrats, there are many legislative proposals that would fail if the filibuster remains: Civil rights. Climate change. Immigration. Health care. Election reform. Even pandemic relief bills could founder in the face of the Senate filibuster.
Because the filibuster impedes the legislative process and counters democratic governance, it is time to reform it. But there is still an opportunity for the Senate to address legitimate goals of promoting bipartisan cooperation and addressing counter-majoritarian concerns.
For example, some commentators have suggested restructuring the filibuster process and placing the burden on those who want to continue debate. Instead of requiring 60 votes to override a filibuster and end debate, the Senate could require 40 votes to continue debate. Forcing senators to speak throughout their filibuster would also heighten the consequences of legislative obstruction by making it more difficult to sustain the filibuster.
Another procedural option would be to prevent the Senate from considering any other business while a filibuster is pending. This would heighten the consequences of the filibuster for both parties.
A different approach is to connect filibuster reform with other Senate procedures. One of the most consequential areas for Senate action involves providing advice and consent for judicial appointments, including appointments to the Supreme Court. While the filibuster has already been removed for such procedures, the Senate could still consider ways of addressing countermajoritarian concerns as well as the optics of voting on partisan lines for judicial appointments.
For example, the Senate could establish a bipartisan committee to consider revisions to the process used to review judicial appointments. And perhaps there are ways of defusing the significance of life tenure on the Court by revising the rules for senior status or developing staggered terms. With careful thought and cautious drafting, these changes would not even require a constitutional amendment.
One of the greatest failings of politicians who serve in the majority party is their refusal to consider how some rules will protect their interests when they are in the minority. This narrow time horizon promotes partisanship and deters cooperation. Yet bipartisan collaboration is a legitimate value.
As the Senate considers the future of the filibuster, it should establish procedures that uphold the principle of majority vote. But in crafting a solution, it should consider the shadow of the future. In the Senate, the Democratic party has at least two years of majority rule. In 2023, that may change. This is democracy at work.
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Rapid urbanisation has increased conflicting spatial claims for economic activities, housing, public space and infrastructure networks. These conflicts challenge the goal of urban vitality and quality of life. The cluster focuses on the following three subtopics:
- Research on community-linked urban developments explores how governmental decentralisation and devolution of traditional public responsibilities (e.g. housing, transport) gives rise to new forms of urban development with opportunities to experiment with democratic processes.
- Sustainable and inclusive mobility takes an individual and ethical perspective to understand how, where and why we move in our cities and urban regions. This perspective challenges the existing institutions that reinforce a technocratic and sectoral approach to planning transportation and land use.
- Urban expansion/sprawl provides methodological tools and empirical data with which to understand drivers of urban expansion and sprawl.

domain: sociology


text:
Creative collective collaborates with Comedy Central on its new animated series, ‘Pixelheads,’ with gameplay from Divekick, award-winning games from Electronic Arts, and the voices of comedians Steve Agee, H Michael Croner and Rich Fulcher, among others.
Twenty-five all-new math-infused episodes -- including a musical one-hour special – of Emmy-winning preschool hit set to roll out starting Spring 2016.
New sketch-based CG-animated comedy series lands on Disney XD in the U.S. and the popular PTDR cartoon slot on the Canal+ Kids channel Télétoon+ in France.
In an effort to encourage international co-production, representatives from FreemantleMedia Enterprises, Marvel Television, Cinema Management Group, Tricon Kids and PBS travel to the British capital in a three-day “familiarization” trip.
Genius Brands International expands its global distribution with new international broadcast, home entertainment and digital licensing partners for ‘Thomas Edison’s Secret Lab’ and ‘Stan Lee’s Mighty 7.’
Los Angeles-based animation studio 6PH leverages Toon Boom’s Harmony Software for the two-minute music video finale, “Rap Battle,” in ‘The SpongeBob Movie: Sponge Out of Water.’
26-episode series produced by Nickelodeon Animation Studios is created and executive produced by C.H. Greenblatt.
Animated feature to be produced by ‘The LEGO Movie’s Chris McKay and Roy Lee, with input from series creator Pendleton Ward.
Feature-length movie, ‘Sodor’s Legend of the Lost Treasure,’ features Oscar winner as the voice of new engine Ryan.
Deal with Cyber Group Studios to see both series introduced to U.S. audiences on Kabillion TV on Demand.
Preschool co-production with Nelvana wins big at the third International Emmy Kids Awards ceremony hosted in New York on February 20.
Preschool series from 9 Story Media Group and The Fred Rogers Company wins four awards for Best New Series, Best Animated Series, Best Voice Talent and Best Music.
Nickelodeon showcases new content slate and upcoming initiatives for 2015-2016 season at annual upfront presentation.
Feature selections include Studio Ghibli’s ‘When Marnie Was There’ and Aardman’s ‘Shaun the Sheep The Movie’; short film selections include Steven Woloshen’s ‘1000 Plateaus’ and Piotr Dumala’s ‘Hipopotamy.’
Andy Milonakis and James Adomian lead voice cast for new animated series created by Emmy Award-winning director Ryan Quincy.
Beginning in 2015, Netflix Will Premiere ‘Danger Mouse,’ ‘Inspector Gadget,’ ‘Some Assembly Required,’ ‘Bottersnikes & Gumbles’ and ‘SUPER 4.’
Mexico’s number-one Free-to-Air broadcaster to add reimagined series to its all-star preschool programming.
Fairytale-inspired animated series for preschoolers created by children's book author Jorge Aguirre is slated to debut in Fall 2015.
Kids TV & learning platform for preschoolers signs multi-territory content deal with DreamWorks Classics for seven new properties; confirms forthcoming Android launch.

domain: artistic


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The recovery of metagenome-assembled genomes (MAGs) from metagenomic data has recently become a common task for microbial studies. The strengths and limitations of the underlying bioinformatics algorithms are well appreciated by now based on performance tests with mock data sets of known composition. However, these mock data sets do not capture the complexity and diversity often observed within natural populations, since their construction typically relies on only a single genome of a given organism. Further, it remains unclear if MAGs can recover population-variable genes (those shared by >10% but <90% of the members of the population) as efficiently as core genes (those shared by >90% of the members). To address these issues, we compared the gene variabilities of pathogenic Escherichia coli isolates from eight diarrheal samples, for which the isolate was the causative agent, against their corresponding MAGs recovered from the companion metagenomic data set. Our analysis revealed that MAGs with completeness estimates near 95% captured only 77% of the population core genes and 50% of the variable genes, on average. Further, about 5% of the genes of these MAGs were conservatively identified as missing in the isolate and were of different (non-Enterobacteriaceae) taxonomic origin, suggesting errors at the genome-binning step, even though contamination estimates based on commonly used pipelines were only 1.5%. Therefore, the quality of MAGs may often be worse than estimated, and we offer examples of how to recognize and improve such MAGs to sufficient quality by (for instance) employing only contigs longer than 1,000 bp for binning.
IMPORTANCE Metagenome assembly and the recovery of metagenome-assembled genomes (MAGs) have recently become common tasks for microbiome studies across environmental and clinical settings. However, the extent to which MAGs can capture the genes of the population they represent remains speculative. Current approaches to evaluating MAG quality are limited to the recovery and copy number of universal housekeeping genes, which represent a small fraction of the total genome, leaving the majority of the genome essentially inaccessible. If MAG quality in reality is lower than these approaches would estimate, this could have dramatic consequences for all downstream analyses and interpretations. In this study, we evaluated this issue using an approach that employed comparisons of the gene contents of MAGs to the gene contents of isolate genomes derived from the same sample. Further, our samples originated from a diarrhea case-control study, and thus, our results are relevant for recovering the virulence factors of pathogens from metagenomic data sets.
The recovery of complete or nearly complete genomes from metagenomes, or genome binning, is still a challenging bioinformatics task influenced by several factors such as microbial community diversity, sequencing effort, sequence read length, intrapopulation sequence and gene diversity, and the assembly and binning algorithms used. Several studies have successfully recovered metagenome-assembled genomes (MAGs) from metagenomic data sets (1–4), while other studies have cited high intrapopulation strain heterogeneity as the cause of failure to recover MAGs (2, 5–7). High (target) population abundance relative to the rest of the microbial community has also been identified as one reason for poor MAG recovery (7, 8). The recovery of reliable and complete or nearly-complete MAGs provides key data for several downstream analyses and research objectives, including the quantification of intrapopulation diversity (9). Intrapopulation diversity has been attributed to various genetic and evolutionary mechanisms, such as horizontal gene transfer (HGT) (2), lack of recent genome-wide and gene-specific sweeps (1), and neutral mutations (6). Reliable detection of such genetic events within a population based on—for instance—(metagenomic) read recruitment plots of MAGs (10) can provide new quantitative insights into the dynamics of intrapopulation gene diversity and identify the ecological and genetic mechanisms responsible for the diversity patterns observed. Therefore, it is important to further understand the limitations and strengths of genome binning and MAGs in assessing intrapopulation diversity.
Several pipelines have recently been developed to deal with these issues and to recover population genomes from metagenomic data sets with relatively high intrapopulation heterogeneity, and these pipelines have even allowed the reconstruction of individual strains (or genotypes) from such data sets (11, 12). For example, ConStrains (11) used single-nucleotide polymorphism (SNP) patterns in a set of universal genes to infer levels of intrapopulation diversity. DESMAN (12), on the other hand, identified core genes within MAGs or groups of MAGs of the same species and subsequently calculated variant positions within these genes to identify different strains and their relative abundances. Both of these pipelines were tested with mock and real (field) samples; typically, they performed better with the former and with low-diversity data sets (11, 12). Thus, how reliably SNPs and genes can be assigned to specific reference strains (genomes) by using these and other tools, especially with actual experimental data sets, remains speculative.
Mock data sets of known composition, such as those developed as part of the Critical Assessment of Metagenome Interpretation (CAMI) challenge (13), are not ideal for assessing the effects of intrapopulation heterogeneity, because only a single (or, at best, only a few) strain per species has typically been included during the DNA sequencing step or used to simulate data in these mock data sets. Hence, how well the results based on mock data sets translate to real, more-diverse data sets often remains unclear. Nonetheless, the results of the CAMI challenge provide valuable insights into the strengths and limitations of different software for specific research questions and tasks. Most notable, for genome binning, is the fact that different algorithms showed variable performances depending on the complexity of the data set and the presence or absence of closely related strains and populations (13, 14); no binning algorithm performed the best for all types of samples. However, in most comparisons, MaxBin 2.0 performed reliably and often outperformed other binning algorithms.
Our recent study of natural populations described a method to statistically identify genes of MAGs that are absent (i.e., none or <10% of the members of the population carry the gene) or variable (i.e., carried by some members of the population but not all) based on the sequence coverage of these genes by metagenomic short reads that represent the natural population under study relative to the core genes (carried by all or almost all members of the population, i.e., >90% of total members) (2). That is, our study provided an approach to quantify gene-level diversity within a population based on read recruitment of a reference genome that represents the population. This study also determined the reference genome/MAG sequence coverage needed in order to reliably detect such variable genes (as opposed to missing them by chance due to low sequence coverage) at about 7× or more, which agreed with the results of the CAMI challenge for reliably recovering target genes in a metagenomic assembly. Here, we employed this method and these thresholds for detecting isolate-specific and population-variable genes based on metagenomic read coverage.
In this paper, we aimed to advance the metagenomic workflow for assessing intrapopulation gene-level diversity and to provide an independent assessment of MAG quality that does not rely on the common practice of detecting the presence or absence and copy number of (a rather small number of) universal protein-coding genes or clade-specific core genes (15, 16). To this end, we focused on all the genes that were recovered (or not) by pathogenic Escherichia coli MAGs based on whole-genome comparisons of MAGs against their corresponding isolate genomes recovered from the same diarrheal stool sample. We also assessed the number of population-variable and core genes that were identified based on a read recruitment plot of an isolate genome but were not captured by the corresponding MAG. Therefore, we assessed the quality of the MAG based on the true- and false-positive gene recovery rates by using the isolate genomes as a reference, in addition to the common tools available for this purpose. The metagenomic data sets used in the study were previously reported as part of our survey of diarrheal cases in Ecuador and our typing of the etiological agents (17). Here, we used a small subset of these data sets in which the E. coli isolate available represented the etiological agent of diarrhea and was abundant enough in the corresponding data set to not limit assembly and genome binning (>10× coverage; see Table S1 in the supplemental material). Hence, our study also represents a relevant case for public health, because it assesses the limitations in recovering the full gene content of the etiological agent of diarrheal disease using metagenomics and genome binning.
Identification and quality of MAGs and isolate genomes.
In total, eight isolate-MAG pairs were analyzed from eight distinct human fecal samples (Tables 1 and 2; Fig. 1). The estimated completeness of the genomes based on the Microbial Genomes Atlas (MiGA) workflow ranged from 91% to 95.5% (average, 94.5%) for the isolates and from 81.1% to 95.5% (average, 92%) for the MAGs (Table 1). Contamination estimates were 0.0% for all isolate genomes (as expected for pure isolate DNA sequencing) and ranged from 0.9% to 7.2% for the MAGs. The high level of estimated completeness of all isolates was also consistent with the high sequencing depth of their genomes obtained by the genome reads (average, 22×) (Tables 1 and 2; Fig. 2). MAGs were recovered from the same sample as the isolate (no coassembly was performed), using the large contigs of the assembly (longer than 500 bp) and MaxBin 2.0 with default settings (see Materials and Methods for details). Genome-aggregate average nucleotide identity (ANI) within all isolate-MAG pairs was 98.9% or above, with an average of 99.77%, indicating that the MAGs obtained belonged to the same population as the isolates and were members of E. coli, with the exceptions of samples Q196 (97.63% ANI) and Q51 (96.99% ANI) (Table 3), in which the MAGs apparently represented a strain(s) of the population distinct from the isolate.
TABLE 1 Properties of isolate genomes analyzed and metagenome-assembled genomesa
No. of genes
Isol, isolate genome; MAG, metagenome-assembled genome; ETEC, enterotoxigenic E. coli; DAEC, diffusely adhering E. coli. Completeness, contamination, and quality values are expressed as percentages.
TABLE 2 Sizes of metagenomes, genome isolates analyzed, and metagenome-assembled genomes, and numbers of contigsa
TABLE 3 Fractions of isolate variable and isolate-specific genes and of variable and strain-specific genes not recovered by MAGsa
ANI (isolate vs MAG) (%)
% of the following isolate genes/total isolate genes:
% of isolate genes shared with MAGs
% of isolate core genes missed by MAGs/core genes
% of isolate variable genes missed by MAGs/variable genes
aAbsolute numbers of genes are given in parentheses.
The number of isolate genes shared with the corresponding (paired) MAG recovered from the same sample ranged from 2,118 in Q51 to 4,371 in E230, representing, on average, 74% of the isolate genes, with the exception of Q51, in which they represented only 42% of the genes (Table 3). Similarly, MAG genes shared with the isolate represented, on average, 75% of total MAG genes, with the exception of Q51, where they represented only 24%. The variation in these values correlated well with the ANI between the isolate genome and its corresponding MAG; genome pairs showing relatively low ANI values also shared fewer genes. For instance, the isolate and MAG originating from Q51 showed 96.99% ANI. E230 was a different case in that a high number of genes (6,990) was recovered in the isolate genome compared to the average number of genes in E. coli genomes or the MAGs studied here (average, 4,902; standard deviation [SD], 154) (Tables 1 and 2), suggesting that there was a coculture of two strains, which would explain the low ratio of shared genes (ANI between the isolate and the MAG was 99.84%). Sample E158 was also an exception to the above-mentioned rule (high ANI; high percentage of shared genes) because the low ratio of shared genes was presumably due to low MAG completeness (<90%).
The rest of the MAG genes (nonshared) either had a distant match or did not match with isolate population genes (MAG specific) (Fig. 1) and could be considered the result of natural variability within the natural population present in the sample or could be due to assembly or binning errors as shown below. Given the ANI and genome content results, the Q51 isolate and its corresponding MAG clearly did not represent the same genotype (or strain) and thus were removed from further analysis. Although Q196 had a low ANI with its MAG (97.63%), similar to Q51, it was included in further analysis because the number of genes shared between the isolate and the MAG was not that low, representing 70.26% and 60.88% of the total isolate and MAG genes, respectively.
Frequencies of population core, variable, and isolate-specific genes.
Metagenomic reads were mapped onto the corresponding isolate genome from the same sample in order to reveal the core genes within the natural E. coli population in the sample and identify genes that were variable in the population, i.e., those that were carried by some but not all members of the population, as well as isolate-specific genes (those with no metagenomic reads mapping onto them) (Fig. 1; Fig. 3, panel 1.2; Fig. S1). Metagenomic reads were also mapped on the corresponding MAG sequence (Fig. 3, panel 1.3) in order to assess whether or not the MAG represented the metagenomic population as well as the isolate did (whether the average nucleotide identity of mapped reads [ANIr] to the MAG and the ANIr to the isolate sequence were similar). Indeed, average identity and coverage level values were similar between the MAG and the isolate, showing <10% difference in most cases (Table S1) except for the E230, Q51, and Q196 samples, which represented heterogeneous populations of pathogenic and commensal E. coli, as shown below. In all cases, including the samples with heterogeneous populations, the isolate genome was preferred over the MAG as the reference in the recruitment plot for the analysis of core variable and strain-specific genes because of the uncertainty about the extent to which the MAG may represent sequences, including contamination from other taxa.
Variable genes were defined as those with coverage significantly lower than the mean genome coverage (P < 0.01) but >10% of the mean coverage; isolate-specific genes were defined as those showing ≤10% of the mean genome coverage, including no coverage by reads (completely absent). To calculate the level of coverage that provided a probability (P value) of <0.01 (i.e., that the gene coverage was significantly lower than the mean genome coverage, with the null hypothesis being that the gene is present in the population and core), the resulting distribution of coverage values for all genes of a MAG or an isolate genome based on the recruitment plot with metagenome reads was fit to a log-normal distribution using the enveomics.R package, v1.4.1 (10). Core genes were defined as those that showed coverage similar to the average coverage of the whole genome (i.e., P ≥ 0.01).
Variable genes were low in frequency, in general, ranging from 0% to 2.24% of the total isolate genes (average, 0.997%), while isolate-specific genes ranged from 0.10% to 13.31%, with an average of 3.12% (Fig. 4; Table 3). Annotation of population-variable genes revealed that they were related to glycosyltransferases, membrane transporters, secretion proteins, transcriptional regulators, and type III effector proteins that could be related to increased virulence of the population and/or increased fitness in the gut (Table S2), implying that variable genes could provide different members of the population with different adaptive traits.
Samples B200, E230, and Q196 had the highest numbers of isolate-specific genes, which accounted for >1% of the total genes in the genome, in contrast to the other four isolates analyzed (Fig. 4; Table 3). Note that the isolate genome recovered from sample Q196 is likely to represent an E. coli population other than the abundant genotype present in the sample, as evidenced by a low ANI between the isolate genome and the MAG (97.6%). Annotation of these isolate-specific genes showed that >30% represented uncharacterized proteins, even reaching 49% in Q196, while 4.65% to 10.51% of isolate-specific genes were attributable to mobile elements, mainly phage proteins, transposases, and integrases (Table S3). The relative frequency of uncharacterized proteins among isolate-specific genes was significantly higher than that in the total isolate genome (P, 0.04 by the chi-square test). This was not the case for the mobile elements (P, 0.12 by the chi-square test), although ratios for mobile elements in samples B200 and E230 were much higher (>2-fold) among isolate-specific genes than in the whole genome. However, 20 to 38% of the isolate-specific genes identified were at the edges of contigs (<100 bp from the edge), in comparison to 5 to 40% for the whole genome, and this could be problematic for metagenomic read coverage estimation (fewer reads can typically be mapped at the ends of contigs with high stringency thresholds such as those used here). Therefore, we did not include isolate-specific genes from the isolates in the quality assessment of the MAGs (Fig. 1); in addition, such genes should not be expected to be captured by MAGs, since they are carried by only a few cells, or even a single cell, of the total population.
As also discussed previously with regard to the E230 isolate genome, an unexpectedly high number of genes (6,990) was observed compared to the average number of genes in E. coli isolate genomes and the MAGs studied here (average, 4,902, SD, 154) (Table 1). Taxonomic profiling of the isolate-specific genes in E230 showed that all these genes were assignable to E. coli, suggesting that there was a coculture of two strains that was sequenced, and this presumably accounted for the high numbers of isolate-specific genes not captured by the metagenome and core genes not detected by the MAG (Fig. 2 and 4). This was also supported by the high fragmentation of the E230 genome assembly (1,630 contigs; N50, 5,050 bp) (Table 2). Hence, the relatively lower performance of the MAG in the case of E230 (see below) was most likely due to a mixed coculture of the isolate genome rather than a low-quality MAG.
Population core and variable genes missed by MAGs (completeness).
We next examined if the population core and variable genes identified by the read recruitment analysis above using the isolate genome as the reference were captured by the corresponding MAGs from the same sample. Core genes not captured by the MAGs represented a fraction ranging from 11.31% (B45) to 34.7% (E158) (24.84%, on average) of the total core genes, while the fraction of variable genes missing in the MAGs was 13.63% to 69.56% (50.17%, on average) (Table 3; Fig. S2). As expected, MAGs missed all isolate-specific genes in these comparisons; these genes are too rare in-situ to be assembled, since they are specific to the isolate in question.
A fraction of missed core genes (ranging from 12% to 79%) and variable genes (from 19% to 39%) in our samples found matching MAG homologs in BLASTN whole-genome comparisons of isolates with MAGs. However, these matches showed ≤90% nucleotide identity or ≤90% alignment length, indicating that either these genes were indeed real population-divergent homologs—genes not shared with the isolate—or they resulted from assembly errors (Fig. S3). The remaining missed core (16% to 71%) and variable (64% to 71%) population genes that did not find any homolog among the MAG genes were assembled but binned in other Enterobacteriaceae MAGs from the same fecal sample (Fig. S3), revealing binning errors when closely related populations coexist in the same samples. Genes with relatively low sequence identity or alignment length and genes binned in other MAGs accounted for >95% of the total core genes and/or variable genes missed by the MAG. In two of the samples (E230, Q196), the percentage of genes that had no match with any MAG gene exceeded 30% (Fig. S3). Not surprisingly, these two samples were also among the samples with the highest numbers of isolate-specific genes (Table 3; Fig. 4) while isolate Q196 also had a low ANI value (<99%) with its corresponding MAG, suggesting the copresence of different genotypes.
Importantly, assessment of MAG completeness using either MiGA (based on the presence/absence and single/multiple copy of 111 essential universal genes) or CheckM (based on 1,173 marker genes conserved in the Enterobacteriaceae family) showed that all MAGs were of high completeness (86.5% to 91%) based on recently proposed standards (9), and the estimated completeness was slightly higher by CheckM than by MiGA (Table 1; Fig. 2). However, when genome completeness was estimated based on true-positive recovery rates for (i) core genes only (recovered population core genes/total population core genes, using the isolate as the reference in the read recruitment plot) and (ii) core and variable genes combined (recovered population core and variable genes/total core and variable genes), the rates ranged from 65.29% to 88.68% and 64.95 to 88.65%, respectively (Fig. 2). These rates were lower than the completeness assessments of MiGA and CheckM, by 16.9% and 20.4%, respectively, on average (Fig. 2).
Frequency of contamination from non-E. coli sequences in MAGs and underlying causes.
MAG genes that were not shared with the population after whole-genome comparison to the isolate genes with substantial metagenome coverage (core and variable population genes), ranged from 3.97% (B45) to 48.05% (E158) (Table 4). These genes were separated into two categories: (i) genes that had distant matches showing either >70% but ≤90% identity or alignment length, or both, with the isolate genes and (ii) genes with no matches with isolate genes at ≥70% identity and alignment length (Fig. 1), i.e., MAG-specific genes. The first category accounted, in total, for 25.8% to 45.02% of the nonshared MAG genes (Table 4), indicating true gene variability for cases of lower identities and potential assembly issues for cases of low alignment length values. The second category accounted for more than half of the nonshared MAG genes in all cases (Table 4). In the MAG-isolate pairs with low ANI values (Table 3), some of these MAG-specific genes could represent real population-variable genes not present in the isolate. However, we posit that this fraction, representing between 2.35% (B45) and 26.41% (E158) of the entire genome (average, 14.42%, excluding Q196), corresponds, for the most part, to MAG contamination: genome fragments erroneously included in the MAG.
TABLE 4 Fractions of MAG genes not shared with the population, MAG-specific genes, and non-Enterobacteriaceae genes among MAG genes and MAG-specific genes
% (absolute no.) of the following genes within the indicated group:
MAG genes not shared with isolate
Genes in short contigs (<1,000 bp)
Nonshared MAG genes
Total MAG genes
MAG-specific non-Enterobacteriaceae genes
A considerable fraction (20% to 45%; average, 30.6%, excluding Q196) of the MAG-specific genes were found in contigs shorter than 1,000 bp (Table 4) (reported toward the end of the MAG sequence file) and usually showed more-variable coverage than the rest of the genome (higher or lower [examples can be found in Fig. 3]), further corroborating the proposition that these genes represented binning errors. In agreement with this interpretation, best-match analysis of MAG-specific genes against available reference genomes showed that a substantial fraction, ranging from 11.87% (E158) to 46.63% (E230) of these MAG-specific genes or 2.42% to 10.03% of total MAG genes, did not match Enterobacteriaceae (Table 4); the remaining genes matched Enterobacteriaceae genomes. Some of the genes not matching Enterobacteriaceae were also parts of short contigs (7% to 36%) (Table 4). Importantly, both CheckM and MiGA failed to estimate MAG contamination by other bacterial families in most cases, i.e., their contamination estimates were 0.9% to 3.6%, because these tools are based on universal genes only, and the majority of the extraneous genes were not universal or core genes.
The highest numbers of MAG-specific genes were observed in the MAGs recovered from the E158, B200, Q196, and E184 metagenomes (>20% of total MAG genes). Overall, these MAGs had high completeness and low contamination (Table 1) but exhibited the highest numbers of non-Enterobacteriaceae genes, followed by the E230 MAG, which also exhibited 9% MAG-specific genes (Table 4).
Evaluation of the effect of contig length on MAG quality.
Our MAGs overall showed high completeness (>80%) and low contamination (<10%) according to MiGA (Table 1), and the N50 values (50% of the assembly is found in contigs longer than the N50 value) varied among the MAGs and were lower than the N50 values for the isolates in most cases (Table 2). The importance of contig number and length for retrieving accurate and complete MAGs has been discussed previously (18), and those studies suggested that fewer than 10 long contigs should ideally be used as candidates for complete MAGs (i.e., fully circularized genomes). In agreement with this suggestion, we found that the number of contigs making up the MAG sequence was positively correlated (R = 0.91) with the percentage of genes that were MAG specific in our data set (Fig. S4), implying that using longer contigs decreases false-positive binning errors. To more accurately quantify this parameter, we performed binning again, using different minimum lengths (1 kb, 2 kb, and 5 kb) for including contigs in the binning step. Our evaluation using MiGA showed that MAG completeness was not significantly decreased in this range of contig lengths except for the E158-MAG, while contamination decreased and quality increased in almost all cases (Table S4). A contig length of 1,000 bp offered the best compromise between lower contamination and minor decreases in quality and completeness, which was roughly consistent with what was reported previously based on different binning algorithms (19). Similarly, the number of nonshared MAG genes decreased in most cases, as expected (Table S4; Fig. 5), while isolate genes captured by the corresponding MAG decreased slightly and even increased in some cases, indicating that using longer contigs (lower fragmentation) also allowed for more-reliable binning (Table S4; Fig. 5).
Despite the common use of genome binning in recent molecular microbial ecology studies, investigations of how well MAGs represent the total population by direct comparison with isolate genomes from the same sample are lacking. Here, we compared population core and variable genes detected using genomes of E. coli isolates as the reference in read recruitment plots versus MAGs originating from the same sample. Taking into account that our samples were medium-to-high-complexity data sets (17), our results were consistent with those of the CAMI challenge in that we were able to recover MAGs with high completeness (>80%) and low contamination (<8%) from each metagenome using MaxBin 2.1.1 (13). Confoundingly, our study highlighted, in addition, consistent contamination of sequences from other bacterial families recovered by MAGs that often went unnoticed by the common strategies and tools used for this purpose.
Overall, MAGs missed 25% and 50% of the population core and variable genes, respectively, on average, which was substantially higher than the estimate of the quality-checking pipelines (Table 3; Fig. 4; see also Fig. S2 in the supplemental material). These findings were consistent with those reported recently based on a comparison of isolate genomes derived from a low-complexity (enrichment) culture of ∼20 bacterial species with the MAGs recovered from the metagenome of the culture (20), further highlighting the need for improvements in binning approaches and/or manual curation of the resulting MAGs (18). Nelson and colleagues reported, in addition, that isolate genes missed by MAGs were often found in short contigs of atypical tetranucleotide frequency (20), which was presumably specific to the approach used for binning (a tetranucleotide-based approach). The binner software employed in our study (MaxBin) is not (as) sensitive to tetranucleotide composition variation but is prone to splitting population genomes in multiple (independent) MAGs in some samples (13), which was also observed in our samples (E158, Q51) that yielded MAGs of low completeness. Therefore, different results than those reported here could be obtained depending on the binning strategy employed and the specific metagenomic data set(s). That said, our results should provide a reference guide for the binning of single data sets with no manual curation of the resulting MAGs. Further, the genes missed by MAGs often included virulence factors in both our study and the previous study (20), because these genes are commonly carried by short contigs (after assembly) of atypical coverage and/or tetranucleotide frequency. Therefore, applications of binning to clinical samples should examine contigs not binned as part of the (target) MAG for the presence of (known or suspected) virulence factors as well as applying the recommendations below for more-reliable MAGs.
The reverse comparison, i.e., population genes identified using the MAG as the reference that were not recovered by using the isolate genome, revealed even more MAG genes that were not shared with the isolate (Table 4). These genes are attributable, at least partly, to the fact that the isolates almost never represent all members of a natural population. However, our analysis indicated that the majority of these genes were classified in families other than Enterobacteriaceae (i.e., potential errors in binning), which would result in an overestimation of population gene variability. Our analysis showed that our MAGs have, on average, 4.4% (range, 2.3% to 6.7%, excluding Q196) of sequences not taxonomically classified into Enterobacteriaceae and attributed here to binning errors. This value is higher than the contamination levels estimated both by MiGA (0.9% to 2.7%; average, 1.5%, excluding Q196) and by CheckM (0.36% to 7.92%; average, 2.63%). Therefore, it appears that MAGs recovered using the most frequently used pipelines and thresholds commonly have a low but substantial frequency of sequences that belong to taxonomic groups other than the group in question and that this was not estimated by the best-performing tools for quality assessment, since these tools do not target the detection of extraneous sequences. Thus, further improvements in genome quality assessment as well as long-read sequencing technologies are needed for higher accuracy. For the time being, the recommendations below should help to identify such problematic cases and/or to obtain MAGs of sufficient quality.
Suggestions for reliable genome binning and MAGs.
Our analysis identified key parameters and metrics, such as the number of contigs and gene taxonomic classification, that could be explored for more-reliable genome binning or could help to identify problematic MAGs. The following suggestions are therefore presented; they typically result in high-quality MAGs, defined as representing >85% of core and variable population genes and showing <3% contamination (i.e., genes of different taxonomic origin). Notably, this quality level was achieved by default binning settings only for the MAGs of samples E124 and B45; six out of eight metagenomes produced MAGs of lower quality. Our suggestions include the following:
Identify high-quality MAGs based on MiGA or CheckM (>70% quality score) with >10× coverage in order to obtain homogenous gene coverage. Also, select MAGs with a low number of contigs (<500) and high N50 (>20,000 bp), since these parameters provide the best assemblies.
Generate metagenomic read recruitment plots of high-quality MAGs as suggested previously (7). These plots typically reveal uniform coverage across the MAG, with increased coverage where the rRNA and multicopy mobile genes are located. Manually inspect, and possibly remove, contigs of unusual coverage compared to the rest of the genome that do not carry rRNA or (known) multicopy genes. Tools such as Anvi’o could further facilitate the (manual) identification and removal of such contigs (21).
Keep in mind that sequences harboring contamination are more frequently found in short contigs and are characterized by more-variable coverage patterns than the average of the genome. Further, the sequence diversity within the population revealed by the recruitment plot (e.g., mapping patterns of reads showing >90% nucleotide identity to the MAG) often differs for chimeric contigs relative to the rest of the genome. To avoid such contigs, it is preferable to select longer contigs for binning (>1,000 bp).
Compare MAGs to their closest relatives (ANI, >93% to 95%) in terms of genome size and number of genes for expectation of the gene content from the MAG sequence. Note that taxonomy does not directly follow conservation of genome length (22). However, genome size deviations of more than 0.5 and 1 Mbp at the species and genus levels, respectively, could be indicative of problematic binning.
Obtain the taxonomic profiles of a majority of genes using tools such as MyTaxa, and examine their in situ coverage by metagenomic reads to detect chimeric MAGs, even in cases where the tools for quality assessment provide high-quality values.
Our study is based on a limited number of samples (pairs) analyzed and on a specific, limited level of complexity, that of the gut microbiome. Another limitation was the focus on E. coli isolates alone. Future studies should include metagenomes of varied complexities and isolates of different species in order to verify that our results apply to other prokaryotic taxa. Further, while we used only one binning tool in this study, our data set is appropriate for evaluating additional binning tools or strategies that combine the outputs of several tools. The suggestions provided here should facilitate the identification and removal or manual curation of “suspicious” MAGs not captured by the available quality-checking tools. Since MAGs are increasingly used in ecological and diversity/taxonomic studies (23, 24), it is possible that the coassembly of several metagenomes containing closely related populations could produce even more-erroneous MAGs (25). Thus, it is important that such efforts be combined with thorough inspection of the MAG sequences, especially for the handful of MAGs that are critical for the study of pathogens and other target organisms or are used to describe new Candidatus taxa (26).
MATERIALS AND METHODS
Study design, collection of samples, isolates, and sequencing.
This study was a part of the EcoZUR (E. coli en Zonas Urbanas y Rurales) project, a case-control study of diarrhea that was carried out over 18 months in northern coastal Ecuador (17, 27). One of the objectives of the EcoZUR project was the identification of diarrheal cases for which E. coli was the causative agent and the evaluation of signatures in the gut microbiome that might distinguish between infections caused by different E. coli pathotypes. For this purpose, samples from infected children with an E. coli pathotype, based on culture results and PCR-based identification of pathotype-specific genes carried by the isolates, and their age-matched controls were analyzed using whole shotgun metagenomic data, isolate genome sequencing, and epidemiological data (Fig. 1). Collection of samples, culturing of E. coli isolates, and metagenome and genome sequencing, as well as metagenome assembly, have been described previously (17). For the current study, a subset of EcoZUR samples was used (Tables 1 and 2), for which the isolated E. coli strain was presumably the causative disease agent based on a combination of data including metagenomic relative abundance and level of clonality (pathogenic E. coli populations tend to be more clonal than commensal E. coli), the presence of virulence factors, and epidemiological data (e.g., the presence of symptoms and the more-frequent association of the isolate with disease than with healthy samples) (17).
Population genome binning.
Contigs longer than 500 bp were binned into genomes using MaxBin v2.1.1 with default settings (28). Only contigs from the assembly of an individual sample were used for binning (i.e., no coassembly was performed). CheckM and the MiGA Web server (www.microbial-genomes.org) were used to estimate the completeness and contamination of each metagenome-assembled genome (MAG) based on the recovery of single-copy universal bacterial proteins (15, 16). Recruitment plots were constructed, as described previously (29), for isolates and MAG contigs and genes using the enveomics.R package, v1.4.1, from the enveomics collection (10).
Taxonomic assignments and functional annotation of predicted genes.
Genes were predicted in all MAGs or isolate genome sequences analyzed using Prodigal (v2.6.1; default parameters) (30). Taxonomic assignment based on phylogenetic reconstruction of the universal single-copy-number genes for each MAG was performed using CheckM and MiGA. Assessment of the taxonomic origins of individual genes and operons was based on MyTaxa (31) as implemented in MiGA. Genome-aggregate average nucleotide identity (ANI) was calculated between all MAGs assigned to Enterobacterales and the respective isolates using the ani.rb script from the enveomics collection. Functional annotation for each genome was performed using blastp searches (score, >60 bits; similarity, >40%) against UniProt 2017_11 (32).
Gene conservation in the natural (metagenomic) population.
Recruitment of metagenomic reads to the reference MAG or isolate sequences was used to identify areas of sequence discontinuity for the corresponding population as described previously (29). For all MAGs, 95% nucleotide identity, the most commonly used threshold for species demarcation (33), corresponding to an area of sequence discontinuity revealed by the read recruitment plots, was used as the threshold to identify reads representing the reference population and estimate the coverage of the population (i.e., relative abundance in the metagenome) (Table S1; Fig. 2). Reads with <95% identity were assumed to represent distinct (nontarget), co-occurring populations and were excluded from further analysis. Read mapping against the MAG or isolate genomes was performed with BLAT, and only one match (best match) per read was saved (34).
Eight MAG-isolate pairs from disease samples (Fig. 1; Tables 1 and 2) for which the isolate was concluded to be the causative agent and its population was abundant (>7× coverage) in the metagenome based on our previous study (17) were used in total. Metagenome read mapping was performed for each isolate to assess the level of gene conservation within the natural sequence-discrete population and to determine the relative abundance of each gene (Fig. 1). More specifically, metagenome reads were mapped onto isolate genomes in order to identify variable and core genes, and isolate genes missing (or less abundant) within the natural population (for exact definitions of these sets of genes, see Results). Variable and absent genes were calculated only for isolates with 7× or more average coverage across the genome in order to reduce the impact of spurious results. When population coverage is lower, genes may be missed due to sequence depth variation and not true absence, resulting in a false-positive signal (2). This level of coverage (>7×) and the associated P value (<0.01; see also below) ensured a low number (<1%) of potential false-positive results based on subsampling and simulation experiments performed previously (2). Gene coverage was calculated using the sequencing depth of the corresponding window in the recruitment plot using the BlastTab.seqdepth.pl script from the enveomics collection (10). All isolate genes were also searched against MAG genes using BLASTN searches (thresholds for a match: nucleotide identity, >90%; alignment length, >90%) to determine if the MAG included these genes (Fig. 1). For read mapping, the ANIr against the reference MAG or isolate genome sequence, using all mapped reads with identities above the selected nucleotide identity threshold (95%), was also calculated in order to estimate the level of intrapopulation sequence diversity (Table S1).
Funding for this study is provided by National Institute of Allergy and Infectious Diseases grants 1K01AI103544 (to K.L.) and R01AI137679 (to K.L. and K.T.K.) and by NSF award 1759831 (to K.T.K.). The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or NSF.
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text:
Mike Lindell, who is the CEO of MyPillow, is turning the tables on Nancy Pelosi.
Lindell has filed a lawsuit against Pelosi and other members of the Jan. 6 committee.
Left-wing Democrats and a few anti-Trump Republicans are continuing their witch hunt against President Donald Trump by looking to acquire Lindell’s personal phone records.
The committee issued a subpoena for the phone records.
Lindell fired back by suing the committee, Pelosi, and Verizon in order to stop the carrier from turning over the information.
He told CNBC, “I wasn’t there on January 6th and yes they did subpoena my phone records but we filed a complaint for declaratory and injunctive relief against the January 6th committee and Verizon to completely invalidate this corrupt subpoena.
The lawsuit, filed in the U.S. District Court for Minnesota, said Lindell’s actions challenging the results of the November 2020 election were “motivated, in part, by his strongly held religious beliefs.”
Top Democrats like Pelosi have continued to push a false narrative that the events of January 6, 2021 were an organized attempt by Trump to overturn the presidential election result.
The only problem is that an extensive FBI investigation found “no evidence that he Trump or people directly around him were involved in organizing the violence, according to the four current and former law enforcement officials.”
On the contrary, there is strong evidence that roughly 96% of cases were “one-off incidents” and only 4% could be tied to various militia-type groups. These groups were still not centrally coordinated.
The new report vindicates Trump as being falsely linked to allegedly inciting or organizing the events of Jan. 6th and wrongfully banned by social media platforms.
A former senior law enforcement official with knowledge of the investigation explained, “Ninety to ninety-five percent of these are one-off cases. Then you have five percent, maybe, of these militia groups that were more closely organized,” he added.
“But there was no grand scheme with Roger Stone and Alex Jones and all of these people to storm the Capitol and take hostages,” he concluded.
Watch the clip:
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It said enforcing the subpoena would violate his rights to “freedom of religion, speech, press, political expression, and to associate with others to advance their shared beliefs.”
The lawsuit also said members of the committee “acted without authority because they were not validly organized as a House committee” according to the rules of the House, The Hill reported.
In addition, it argued that the subpoena “exceeds the authority of the Select Committee” because it requests “records that are far beyond the scope of the Select Committee’s investigation.”
Lindell’s lawsuit is only the most recent legal action taken by Trump allies. Others have brought similar lawsuits against the committee and Pelosi.
At the end of December, Michael Flynn, the former national security adviser to Trump, sued the committee to block its subpoena for his phone records, CNN reported.
Others who have done so include former White House Chief of Staff Mark Meadows, radio host and commentator Alex Jones and attorney John Eastman.

domain: politics


Imagine Talents Kopenhagen Fur project day one
Dear Fur lovers
I want to share with you the first day of my experience with Kopenhagen Fur in Copenhagen fot the Imagine Talents project. What is it about?
Kopenhagen Fur has launched a new project for the future dedicated to the world of fashion and innovation.
Let me explains better.
The project is called IMAGINE TALENTS Kopenhagen fur (calling it a PROJECT is an understatement, really it is every student’s DREAM).
Kopenhagen Fur has brought in twenty one future designers coming from international in France, Korea, Japan, China, Denmark and the UK, and has given each of them these possibilities:
- create a fur garment
- present it during Copenhagen fashion week, with a show in a super modern location and 800 guests.
- exhibit the fur at the National Museum of Denmark, during an exhibition dedicated to fur from 28 January to 1 February.
On arrival, the designers were treated like royalty: They took part in a workshop on the fur sector, that gave them a complete vision of our sector, also permitting them to touch the raw materials with their own hands.
Later they visited the auction house, Kick
, a mink farm
and many others.
They could admire the town’s, but above all spend time with the Kopenhagen Fur and Kick team at imagine talents, meet the directors and have direct contact with them, which us always a real learning experience.
This, in short, is what they did and from here I want to tell you everything that happened day by day.
A great satisfaction at Imagine Talents
Kopenhagen Fur charged me not only with communicating Imagine Talents (I will share a post with interviewed and photos of their creations) but also with a part of Copenhagen Fashion Week.
Needless to say this project was not only satisfying but also formative because there is always something new to learn from them.
My Day at Imagine Talents Kopenhagen Fur
On my arrival at the hotel, the Phoenix situated right in the centre of Copenhagen, I got changed and had my make up done by a great make up artist, Saira Khan, then I went to Kick where I immediately fell in LOVE AT FIRST with a multicoloured mink fur vest with the writing POP.
I couldn’t resist the temptation of having some shots taken by Umberto Gorra
the photographer who followed me during this experience.
I had some meetings with Kick and Kopenhagen Fur, we planned the videos and the posts and in the afternoon after having met the twenty one designers we went to Kick’s Designer Centre as you can see in the photos.
This splendid day ended with a dinner together at the Tivoli Restaurant
where you can taste the best sushi in the Danish capital.
LADY FUR’S LOOK
In the photos I am wearing a POP look
Kick mink fur
Jeans Avedon Skynny Alex Jeans
Don’t miss my fashion stories on Grazia
Don’t miss the street style of the first day of the Copenhagen Fashion Week on elle.it
Look at the posts with Kopenhagen Fur


text:
Honduras' Caribbean Coast Restaurants
Crispy brick-oven pizzas served in five sizes...
This cheery little restaurant is a popular...
Nestled in the heart of Tela's Garífuna...
FODOR'S GO LIST
These are the top 25 trips we're looking to book right now -- and we hope you'll join us!More
View deals in Honduras' Caribbean Coast for vacation packages, hotels, airfare, and more from our partners!More

domain: travel


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DW1704 WIFI CARD DRIVER (dw1704_wifi_2072.zip)
Dell Inspiron 15 3531, Ervaringen & Discussie.
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text:
Fold the base shape into a double triangle.
- Bend the corners of the first triangle to the top of the “dead” corner.
- Fold the resulting triangles in half. Swipe by bending the left corner away from you. Turn over.
- Bend the right corner to the middle of the inflection line. Fold the bottom side in half.
- Bend, aligning the side with the inflection line. Turn the corner on the other side and repeat the steps in Figures 3-4.
- Flip through.
- Bend the upper part away from you. Bend the corners. Turn over.
- Draw the eyes, nose. Try on a figure.


Superstar Singer 2 2nd July 2022 Written Update, Dharmendra Special, Eliminations
Superstar Singer 2 2nd July 2022 Written Update: "Dharmendra" Special, Watch the full show online on Sony Liv This Week, Captains and Top 13 Contestants Selection Today, Elimination This Week
Sony TV's Superstar Singer 2 will have Dharmendra, Hema, and Esha Deol as guests on the show this weekend.
According to the latest promo for this weekend’s Superstar Singer 2 (2 July 2022), Mohammad Faiz will dedicate his performance to Hema Malini Deol.
Faiz will sing the song “Dream Girl” and win the hearts of the judges.
Another promo for this weekend’s Superstar Singer 2 (02-07-2022) shows the marvelous performances by Sayisha and Mani.
Dharmendra compliments the immense talent of the kids and conveys his regard for their future.
To get to know more, download Superstar Singer 2 all episodes, or to watch season 2022 full latest episode online, go to the SonyLiv app or sonyliv.com
Superstar Singer Season 2 2nd July 2022 Update, Top 13 Contestants, Latest Promo:
Superstar Singer 2 2nd July 2022 Written Update:
Today's Superstar Singer 2 2nd July 2022 written update starts with the host Aditya welcoming Darmendra Singh Deol to the show as a special guest.
The first performance is given by Pratyush Anand and Vishwaja Jadhav on "Chahe Rahe Paas, Chahe Rahe Door" and "Chal So Jaate Hai' which is highly appreciated by Dharam Ji.
The next performance is by Pranjal who sings a Bengali song and later "Tum Pukaar Lo" song and he later pleads for voting.
Rituraj comes next and sings "Aaj Mausam Bada Beimaan Hai" and sets the romantic mood.
The fourth performance is by Sayisha on "Abke Saajan Saawan Mein" which is highly praised by the judges and she gets a buzzer.
Next is the duet by Salman and Soyab on "Wo Mainu Pyaar Krdi Hai" which reminds Dharam Ji of his moments from the movie shooting.
Afterward, Mani shares village food with Dharam Ji and gives a performance on "Laad Ladawa" which makes everyone emotional.
Then comes Faiz with Arunita and sings " Mausam Hai Suhana Ki Jee Na Lge" and "Dreamgirl".
After some time, all the singers come together and sing in a chorus in their melodious voices.
Later, Chaitanya and Danish come and teases their beloved with their song "O Meri Mehbooba, Tuje Jaana Hai To Ja".
End of today's Superstar Singer 2 2nd July 2022 episode.


World Bulletin / News Desk
Director General of Turkey's Civil Aviation Department, Bilal Eksi on Wednesday said that Turkish planes did not use Syrian air space.
Speaking to the Anadolu Agency (AA), Eksi stated that the Syrian passenger plane, forced to land in Ankara's Esenboga International Airport earlier on Wednesday, had 37 people on board including crew members.
Reminded about warnings to Turkish planes not to use Syrian air space, Eksi said that Turkish planes were not presently using Syrian air space.
Güncelleme Tarihi: 11 Ekim 2012, 09:36


text:
Casino Keno Some Facts, Free Games
Bingo is a lottery game where the player notices a set of Eighty numbers which then must match the alternative by a accidental ball pull.
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Players may uniquely have one active bonus in their account at anybody time.
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Keno game was recognized as the White Pigeon Game, because the winning numbers were sent out by pigeons from more large subdivisions to the fewer towns.
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Bingo is an exciting game that looks much like a lottery ticket.
In the beginning a keno ticket contained 120 amounts in Chinese. Only in the center of the 19th century the game of keno game was brought in the United States by immigrants from People's Republic of China, and to be easier to play, the Chinese figures have been replaced by figures, and in exchange for 120 figures people commenced to play this game with Eighty numbers.
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Examine cards and outfit to be utilized in plays to assure that they are in fine level.
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domain: entertainment


text:
We are happy to share that we are tentatively planning a litter for Fall 2020.
The next litter after Fall 2020 will be some time in 2021. If you would like to be added to the waiting list, please submit a Puppy Application Form.
|During a practice|
|Playing Seaman during 1 of 3 of the performances|
|Waiting for his part|
|during one of the 3 performances|
|During the final performance|
|Pretending to pant like Axel!! hahahaha|
|My niece as Sacajawea and Axel as Seaman|
|Axel singing his silent part...|


text:
SEREMBAN, 11 May – Alumni Centre of UPM took the initiative to organize Tracing Alumni for Collaboration & Engagement (TRACE) programme at the Department of Agriculture and Department of Forestry, Negeri Sembilan.
The engagement sessions included activities such as sharing of the past and upcoming events, and promoting the importance of alumni to support the aspirations of UPM’s PG200. The sessions concluded with a dialogue session between the alumni and ACUPM where valuable feedback was gathered on the way forward to further strengthen the linkages with Alumni-Alma mater.
Director of ACUPM, Assoc. Prof. Dr. Mohd Bakri Haji Ishak thanked all the alumni who participated in the event and for making it a memorable day.
Date of Input: | Updated: | amir_peli
Office of Deputy Vice-Chancellor
(Research and Innovation)
Universiti Putra Malaysia
Off Lebuh Silikon
43400 UPM Serdang, Selangor

domain: news


text:
WHAT IS LOCALIZED VIBRATION TRAINING (LVT)
Localized Vibration Training is an evolution of Whole Body Vibration Training, whereby instead of applying a vibration to the whole body, the vibrations are targeted on individual muscles. In addition, these vibrations mimic attentional cues onto the muscle, improving the users proprioception and bodily awareness.
These devices create vibrations that elicit a physiological and mechanical response, which have been shown to drastically improve activation in the target muscle.
HOW DOES LVT INCREASE MUSCLE ACTIVATION
The vibrations exerted by LVT devices increase the activation of mechanoreceptors involved in proprioception , causing an increased neural activation of a muscle when initiating a movement [2,3], which results in increased muscle activations [4,5,6] and increased maximal voluntary contractions (MVC) [7,8,9].
LVT simultaneously causes the muscles opposing a given movement to experience a reduced neural activation, ‘turning them off’ for longer as a result of reduced excitability , further enhancing the positive effect of LVT on movement.
WHY IS MUSCLE ACTIVATION SO IMPORTANT
As a weightlifter, muscle activation is extremely important because it directly correlates to the effectiveness of your workout. With poor activation in the target muscle, you will engage accessory muscle groups and joints that you don't intend on working, which increases injury risk and decreases results where you want them.
Muscle activation also correlates to force output. The less muscle fibers that you activate, the less fibers that you have working to generate force and strength for you.
All in all, muscle activation is critical when trying to make progress in your fitness journey, so LVT could be great for you if you're trying to take your workout to the next level and get the results you desire.
5. Mosier, 2017

domain: literature


Poetins wapens uit Iran en Noord-Korea
October 31, 2023 - Russia has enlisted Iran and North Korea to provide weapons for its ongoing assault on Ukraine. President Vladimir Putin has even built a factory to construct thousands of Iranian-designed drones.
North Korea has been supplying Russia with munitions since September, Kyrylo Budanov, the head of Ukraine’s Main Intelligence Directorate, told state news agency Ukrinform.
“North Korea has been supplying Russia with 122mm and 152mm artillery rounds and rockets for Grad multiple rocket launchers for a month and a half,” Budanov said.
Engineers at Alabuga -- an industrial hub deep inside Russia -- have been secretly building a production line to manufacture thousands of Iranian-designed drones, weapons that Putin’s forces could use to bombard Ukrainian cities.
The Washington-based Institute for Science and International Security analyzed leaked documents from Russia’s Alabuga Special Economic Zone, supplied by the Washington Post.
The documents outlined the plan, code-named “Project Boat,” to build a variant of Iran’s Shahed-136, a self-detonating attack drone capable of travelling 2,500km.
Since January 2023, an estimated 200 Alabuga engineers have travelled to drone manufacturing centres in Iran to gain expertise.
Assembly of Iran-supplied Shahed-136s -- Russia calls the drone the Geran-2 -- began in February at Alabuga. Production of the Russian version will start fully in February 2024.