Category Archives: DNA

'I don't think it's in her DNA': Man who co-authored Murdaugh book with Colleton Co. Clerk of Court discusses allegations  ABC NEWS 4

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'I don't think it's in her DNA': Man who co-authored Murdaugh book with Colleton Co. Clerk of Court discusses allegations - ABC NEWS 4

For many years, nobody in the Daly family knew what had become of their long lost relative who had simply upped and vanished one day. His mother, as well as his brothers and sisters, had all died without getting any answers.

Frances Daly (50), originally from Carrickmacross, Co Monaghan, but now living in Dublin, describes using her DNA to solve this family mystery of her fathers uncle, who was named John Daly, as one of her proudest moments.

He used to get remembered at various family events, recalls Daly. He would regularly get a decade of the rosary said for him. Everybody thought he just went off and made his fortune, but unfortunately that didnt happen.

It was only when Daly took an interest in tracing her family tree that she came up with answers.

I saw him in the Census, and then I found him two years later, dead in Australia at 23 from typhoid, she says. I found a guy coming in from London on a boat to Perth, and I think its him. His name is too common to be absolutely sure.

But if it is him, he got the whole way across Australia and then got bitten by something. He was in the sugar cane territory so there would have been regular outbreaks of diseases.

Daly managed to find his grave in Brisbane, and also found a small article in a newspaper marking his death. The big question left for Daly and her family was why this man had travelled all the way to the other side of the world without a word to anybody.

It was only when she sent her DNA to a genealogy company, which operates by building a database and cross-referencing peoples genetic codes, that she finally closed the circle and got what is at least plausible explanation for what happened.

He had two maternal aunts in New Zealand, and it seems he was going out to them, she says. Well never know for sure, but I didnt know about the New Zealand people until I did the DNA so that kind of closed a circle for me.

It brought great comfort to some of the older relatives to know he was buried properly and there was a priest and everything there with him. My father had a cousin who was a priest so to be able to tell him everything was done right was special.

Daly, whose family are spread over counties Louth, Monaghan and Kildare, also unearthed relatives from Melbourne, Montana, Canada, and New Zealand through the DNA testing.

I call them my new relatives, she says. Its great to be able to show them photographs of the homestead, or the graves weve some very old graves in the family and share stories. To be able to show them, this is your auntie Mary, or whatever the case may be.

Ive gone off and taken photographs of fields and old houses and farmyards and sent them away to people. You can see family resemblances and traits in photographs of people going back generations too.

A number of these people even made the long journeys back to Ireland to meet Daly and see these familial landmarks for themselves. Sometimes the biggest connection is to be able to go and put some flowers on a grave and acknowledge somebody, she says.

Part of the appeal of all this, Daly adds, is putting the pieces of the jigsaw together without having the picture on the box to help you along. For her, it isnt just about what happened, but why.

They were farmers from the country so they left for economic reasons a chance of a better life, she says. Back in the day, if there was a small farmer and the eldest son got the land and there were three other sons, the others had no choice but to go.

I have relatives who went out to mining towns in Montana. A lot of people from Carrickmacross went there for some reason. I dont know why because there isnt much of a mining tradition in Carrickmacross.

Claire Bradley, a professional genealogist specialising in Irish family history, says there are huge leaps that can be made in tracing family trees through the use of DNA, but stresses it must be used as a compliment to paper records.

In Ireland, we are really hampered by the fire in the Four Courts in 1922 when a lot of records were destroyed, largely Census records, but also wills and things like that, she says. I have gone from about 1,000 in my family tree when I started using DNA to about 4,000 now.

Claire Bradley, a professional genealogist specialising in Irish family history

All that being said, many people have raised concerns around the idea of handing their DNA over to private companies and what the implications of that could be down the road. Unsurprisingly, perhaps, Bradley dismisses much of this as scaremongering.

Youre agreeing to give it over, she says. You can have it removed from the database if you change your mind. I think there is a lot of scaremongering in it. I think people give away a lot more information on Facebook than they do with a DNA swab.

[Centuries of documents burned in the Four Courts in 1922. Now theyre being recreated]

[Irish DNA atlas maps genes of the people of Ireland]

Bradley says the DNA aspect of genealogy is more adept at widening family trees than it is at tracing backwards. This was the case for Gina Dooley (46) from Limerick who describes herself as a newcomer to all this.

About a month before the Covid-19 pandemic hit Irish shores, her grandmother died. During the grieving process, a lot of old photographs were taken out and pored over as the family took stock.

It was when she looked at a photograph of her great-grandmother that she was stopped in her tracks. I was struck by how I knew nothing about this woman, yet she looked so much like me and my mother, she says. So, she started digging, and later did the DNA test.

She didnt uncover any terrible secrets and describes her family history as a normal one regular peasant farmers but remarks that building a picture of the time they lived in and the town they came from makes them feel more real and me feel more grounded.

One particular story of a second cousin she discovered in Australia stands out. After they made contact, the man told her about an uncle of his with whom he had lived as a child. She then went to the Military Archives to find out more about him.

I dont know how to describe the feeling of sitting in the Military Archive holding pages that he wrote, she says. I never met this man he was my grandmothers uncle but it was like a piece of him was still alive.

I could share that with James in Australia, and he could relay to me things he remembered of this man, and how he used to go off on a rant about Ian Paisley. James was five years old, and he didnt know who Ian Paisley was, but that is all he remembers of this man.

Continued here:
Using DNA to solve a family mystery: 'It brought great comfort to ... - The Irish Times

Epigenetic factors regulate the genes youre born with and can cause them to malfunction, from the impact of stress, adversity and pollution to diet, exercise and whether or not you drink and/or smoke. Illustration / Getty Images

Halfway through a challenge to lower her cellular age before a significant birthday, Joanna Wane looks at how a biological clock based on data from the world-famous Dunedin Study can show whether youre speeding up or slowing down time

When Terrie Moffitt first came out from the United States to work on the Dunedin Study as a PhD graduate, the 1000-plus people whove been involved in the project since birth were in their early teens. Next year, when the latest round of exhaustive tests and interviews begins, theyll be turning 52.

Back then, in the mid-80s, a few of them were already causing minor havoc. Shoplifting at Woolworths. Converting cars. Sniffing glue in the Octagon. Drugs, alcohol, risky sex. Moffitts research over the following years into why some kids grow out of juvenile delinquency (or goofing around, as she calls it) and others grow up to be career criminals is still widely cited by criminologists worldwide.

Moffitt is now associate director of the Dunedin Study and the clear link she identified between serious adult offending and childhood trauma bears revisiting amid the current political rhetoric over getting tough on youth crime. Once they got out the other side [from their teens], we began to see those two groups diverge, she tells Canvas, from her home in North Carolina. Then you could look back and see that the ones who continued with crime into their 20s and 30s were the ones who had a lot of difficulties as very young children and grew up in very adverse homes.

The major contribution [of the research] was pointing out to governments and the justice system that the vast majority of teenagers who break the law are not going to develop into hardcore criminals, so you should really help them not get a prison record. Give them a chance to grow out of it and reform themselves and they will naturally.

What the Dunedin Study has shown over more than five decades now is that childhood exposure to poverty, trauma or victimisation leaves a lasting cellular imprint on the body, too. A tough start in life means youre likely to age faster and die earlier, you increase the risk of developing chronic age-related conditions such as cardiovascular disease, dementia, hypertension, type 2 diabetes and cancer.

Of course, the reverse is also true if youve been dealt a better hand and make the most of it, increasing your chances of an extended healthspan, defined as the number of quality years where youre generally healthy, active and free from disease.

The impact, for better or worse, of both the environment you live in and the way you behave in it holds more power over your life than you might think. Epigenetic factors regulate the genes youre born with and can cause them to malfunction, from the impact of stress, adversity and pollution to diet, exercise and whether or not you drink and/or smoke. This process of DNA methylation is the most significant influence on how well people age; some estimates put it as high as 70 to 80 per cent.

Moffitt, a professor of psychology and neuroscience at Duke University in North Carolina and a professor of social behaviour and development at Kings College, London, has remained closely involved with the Dunedin Study. Having followed the cohort from adolescence to midlife, shell be back in New Zealand next year for the latest phase of research, which will look at how well theyre preparing for old age.

Since the study members turned 26, data has been collected every five or six years on a series of key biomarkers widely used as an indicator of risk for disease, including cholesterol levels, blood pressure, gum recession, lung function, heart health and blood glucose (a potential red flag for pre-diabetes). Physical functions have been added: how long they can stand on one leg, how many times they can get up out of a chair in 30 seconds without using their hands. Brain scans, first done at age 45, will also be repeated next year.

The first epigenetic clock was invented a decade or so ago, using DNA methylation levels as a way to measure biological ageing. However, what makes the Dunedin Study so unique is its access to waves of progressive data on the same group of people. We figured out, hey, I bet we could do the same thing [as the other epigenetic clocks] but using our biomarkers, says Moffitt. So thats what we did. And we determined there were 173 of those methylation marks on top of genes that differentiated between those who were falling apart swiftly over the past 20 years, those who were holding steady and those who were staying young.

The DunedinPACE algorithm, developed in collaboration with Duke University and Columbia University in New York, analyses those epigenetic marks via a pin-prick blood test. Its now considered the most precise measure of how fast or slowly a person is ageing and the best predictor of future health outcomes. Unlike other epigenetic clocks that have been named after the scientists who invented them (Horvath, Hannum, Levine), DunedinPACE acknowledges the members of the study whove made it possible.

Likened to a speedometer, the test captures a specific moment in time and is a fluid measure that can show significant change in as little as eight weeks reflecting a lifestyle change, perhaps, or the efficacy of a new medication. The fastest rate of ageing recorded so far is 1.4, which means that a person is ageing biologically by almost five extra months for every chronological year. The slowest rate of ageing thats been measured is 0.6.

Moffitt hasnt been surprised to find such a huge variation between fast and slow agers. There are people [in the Dunedin Study] who are Olympic athletes and there are people who lead a really down-and-out life, in and out of prison, on and off addictive drugs. So I knew some were taking care of themselves and some were really not, she says. When study members came to the unit for assessment day, some would look so young while others really showed their age. Seeing that reflected in the [rate of ageing] numbers was quite amazing.

The Dunedin cohort is predominantly Pkeh but comparative studies internationally have applied the DunedinPACE algorithm successfully across ethnicities and among the elderly. Moffitt is also co-director of a longitudinal study following twins born into 1100 British families in the mid-90s and has analysed blood samples from them that show the same trajectories. Its miraculous, she says. Born 20,000 miles and 20 years apart from the Dunedin Study and yet the DunedinPACE can still tell you something about them.

Ironically, no equivalent trials have yet been done on Mori and Pasifika in New Zealand.

On my 59th birthday last November, I took a DNA Age test through Auckland-based biotechnology company SRW and sent it off to TruDiagnostics in the US for analysis. A whole swag of results came back, including my extrinsic epigenetic age (a shade under 44) and my DunedinPACE value at 0.71. A follow-up test, done six months later, shows my rate of ageing holding steady. The other big news was that the length of my telomeres had extended markedly.

Protective caps on the tips of chromosome DNA strands, telomeres have been likened to the aglets that protect shoelaces from fraying. Each time a cell divides, our telomeres shorten and they shorten faster under oxidative stress. Once they reach a critical length, the cell dies. Research in adults has shown that telomere length is a predictor of lifespan and is causally linked to age-related diseases. According to the latest test results, mine have actually extended from 7.01 to 7.24 kilobases, longer than 97.94 per cent of people my age. On this measurement, my predicted biological age is 30.90.

Before I celebrate too hard, though, the full reveal of how my baseline data has shifted over a period of 12 months will come when I do a final test, on the day of my 60th birthday.

Over the past year, Ive made some lifestyle tweaks in an attempt to shift the dial in the right direction more alcohol-free days, a bit more exercise (hampered by a knee and shoulder injury), a daily dose of the blackcurrant-based brain drink repa, a monthly visit to my osteopath Glyn Flutey, and a regime of SRW supplements, Cel1, Cel2 and Cel3, developed in collaboration with leading scientists around the world to support nine key cellular functions that decline with age.

One of the molecules in Cel1 is astragaloside, a compound found in a plant root that stimulates telomere repair. Ill never know for sure but it seems likely the supplements have contributed to my positive result. An observational trial by the company in New Zealand showed a biological age reduction of two and a half years in people who took the full suite of Cel supplements for six months. An independent 12-month trial SRW hopes will verify those findings is now underway in the US.

Apart from a regular yoga class, and a hypnotherapy session to help break a lifetime habit of grinding my teeth, I havent specifically targeted stress reduction yet as a way to work on lowering my biological age. I did a transcendental meditation course once, in my early 30s, and I know my brain needs some time to slow down, but fitting in two 20-minute sessions a day just never seemed realistic to me.

Rachel Grunwell, a former investigative journalist-turned-wellness coach, teaches mindfulness meditation through her company Inspired Health to a whole range of clients, from people like me to corporate directors and high-performance athletes. The mother of three boys and a former Herald columnist, she interviewed 30 global experts for her recent book Balance: Food, Health + Happiness and found plenty of science to support the idea that lowering stress levels can help slow down ageing.

We live on a planet thats speeding up; everything is getting faster and faster, she says. I feel anxious just thinking about the idea of having to fit 20 minutes of meditation into my life! But even taking a minute can be incredibly powerful to slow down your breathing, be in the moment and reset a stressed nervous system.

ONE-MINUTE MEDITATIONS

Chronic stress hammers the body physically, increasing your risk of everything from heart disease (particularly for women post-menopause) and high cholesterol to depression and cognitive problems. It can actually shrink your brain. Here are three mindful minute tools wellness coach Rachel Grunwell recommends to help de-stress your nervous system:

Box breathing meditation: Breathe in for four counts, pause for four counts, breathe out for four counts, pause for four counts. Repeat. This forces you to slow down your breathing and focusing on the count is a good distraction.

20-second hack: Practise belly breathing by imagining youre blowing up a balloon in your belly as you inhale. Then, as you breathe out, picture the balloon deflating. To check if youre doing it right, place one hand on your chest and the other on your stomach to see which one moves as you breathe.

Body scan meditation: You can do this sitting, lying down or with your legs up the wall. Taking deep belly breaths, scan yourself from the feet up, relaxing the different parts of your body on each exhalation. Especially good just before sleep.

10-second hack: Most of us carry tension in our shoulders. Grab a few seconds to take a deep breath, squeeze your shoulders up towards your ears as high as you can, hold for a beat or two, then release with a strong out-breath.

Senses meditation: When youre having a hot drink, at home or at a cafe, tune into all your senses to be present and aware in the moment. Savour the feeling of the warm cup in your hands, the patterns you see in the crema, the sounds you can hear around you, the aromas you can smell, the taste in your mouth.

10-minute hack: If youre in a virtual meeting where you need to listen but not participate, pop in your earbuds and go for a quick walk. You cant be entirely anxious when youre walking, says Grunwell. Part of your brain disengages. Or just be still and look for shapes in the clouds.

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How to live longer: DNA, wellbeing and ageing - New Zealand Herald

Summary

Terminators are some of the most formidable and deadly Sci-Fi villains in the history of the genre, but they didnt reach their peak until they merged with another science fiction monster, Aliens Xenomorph. The hybridization of the two creatures unlocked the Terminators' ultimate weapon, one that it could never have wielded without Xenomorph DNA.

Despite the fact that these respective franchises originally had nothing to do with each other upon their films initial releases, their crossover comic series Aliens vs Predator vs The Terminator asserts that Alien and The Terminator have been a part of the same continuity all along. Apparently, Alien takes place in the future after Skynets attack on Planet Earth and the AIs subsequent defeat to the Human Resistance led by John Connor. It seems Skynets reign was pretty short-lived, though remnants of the evil artificial intelligence remained scattered across the galaxy. As humanity rebuilt from the ashes of Skynets assault on the planet and became the space-faring race of humans introduced in 1979s Alien, a rogue squadron of surviving Terminators is plotting the return of Skynet. By the time of this crossover - which takes place after Alien Resurrection - those rogue Terminators finally figured out how they were going to pull it off.

In Aliens vs Predator vs The Terminator by Mark Schultz and Mel Rubi, the remaining Terminators are able to successfully create Terminator/Xenomorph hybrids. These creatures have the biological weapons inherent in every Xenomorph, along with the ferocity and hive-mind-like coordination of the perfect organism, but thats not all. This hybridization also gave the Terminators an entirely new ability, one that isnt derived from either the Xenomorph or the Terminators entirely, but is rather a unique result of their merging: the Absorption Matrix. This ability allows the hybrids to literally absorb their physical surroundings, which both makes themselves more durable in any given situation with the added benefit of absolutely demolishing their surroundings, which is an aspect to the ability that can be weaponized in and of itself given the right circumstances (like inside a spaceship or within a highly volatile laboratory, for instance).

This ability manifested through the sheer malleability of Xenomorph DNA, and the Terminators ability to manipulate it to suit their needs. It is well established in Alien canon that Xenomorphs adapt gradually to their surroundings with each generation, as their hosts allow them to evolve through genetic imprint, which naturally makes them better suited for their environment. The Terminators know this, but since they cant impart pieces of themselves with every new generation of Xenomorph in the more natural way (if one considers Facehugger impregnation natural), they opted to speed up that process through experimentation.

These experiments conducted by the Terminators to create the best versions of themselves for the resurgence of Skynet results in perhaps the most ferocious Terminator in the history of the franchise, one with an ultimate attack it could never have had without the DNA of Aliens Xenomorph.

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Terminator's Ultimate Weapon Was Only Unlocked by Alien DNA - Screen Rant

Evelyn M. Witkin, whose discovery of the process by which DNA repairs itself opened the door to significant advances in the treatment of cancer and genetic defects, died on Saturday in Plainsboro Township, N.J. She was 102.

Her son, Joseph, said her death, in a rehabilitation facility, resulted from complications of a fall.

In a career that began at the dawn of modern genetic research in the late 1940s, Dr. Witkin explored the ways in which radiation both damaged DNA and generated a repair mechanism, what she came to call the SOS response.

The repair mechanism produces an enzyme that in turn creates replacement parts for the damaged DNA. But its an imperfect process that can at times turn out slightly different versions, or mutations what scientists call mutagenesis.

Her insight into the SOS response, which Dr. Witkin developed with Miroslav Radman, then a scientist at the Free University of Brussels, shed new light on how solar radiation and chemicals in the environment affect humans genetic makeup.

She discovered the first coordinated response to stress in cells, Joann Sweasy, a geneticist at the University of Arizona who studied under Dr. Witkin, said in a phone interview. And thats so incredibly important for understanding evolution, and for understanding mutagenesis in terms of tumors.

Dr. Witkin was still a graduate student at Columbia when she spent the summer of 1944 working at the Cold Spring Harbor Laboratory, on the north shore of Long Island. Though she had no background in microbiology her research until then had been with fruit flies on her first day there she was assigned to generate mutations in cultures of the bacteria E. coli.

She placed several under a germicidal ultraviolet lamp. Almost all of them died. But four colonies survived.

At this point, I asked, Why did they survive? Maybe a mutation made them resistant, Dr. Witkin told The New York Times in 2016.

That single question set in motion nearly a half-century of research for Dr. Witkin, first at Cold Spring Harbor and then at the Downstate Medical Center at the State University of New York, in Brooklyn, and finally at Rutgers University, where she worked from 1971 until retiring in 1991.

She won the National Medal of Science some years later, in 2002, but the pinnacle of her career came in 2015, when she and another geneticist, Stephen J. Elledge, won the Albert Lasker Award for Basic Medical Research, the highest honor in the medical sciences after the Nobel Prize.

She had a remarkable ability to see into fundamental biological questions, Donna L. George, a geneticist at the University of Pennsylvania who studied under Dr. Witkin, said by phone. The central tenets of her ideas were validated, sometimes decades later, by the development of new experimental techniques and molecular probes.

Evelyn Ruth Maisel was born on March 9, 1921, in Manhattan. Her father, Joseph, was a pharmacist who died when Evelyn was 3. Her mother, Manya (Levin) Maisel, then married Jacob Bersin, another pharmacist, who moved the family to Forest Hills, Queens.

Evelyn attended New York public schools and studied zoology at New York University. During her senior year, she joined a group of students who were protesting the universitys policy of benching Black athletes whenever its sports teams played opponents from segregated schools.

They rallied around the case of a Black football player, Leonard Bates, who was to be left behind when the team traveled to the University of Missouri. They collected 4,000 names on a petition to let him play and organized 2,000 students to protest outside the central administration building.

No Missouri compromise! they chanted. Let Bates play!

Mr. Bates did not play against Missouri or, later, against other segregated teams. Other Black athletes faced similar discrimination. The protests continued through the school year, until the university put an end to them by suspending seven of the movements leaders, including Evelyn.

She had been planning to continue into graduate work at N.Y.U., but now, having also lost a graduate assistantship as punishment, she set her sights on Columbia. She graduated from N.Y.U. in the fall of 1941 and immediately went uptown to begin her doctorate.

My having gone to Columbia was the greatest blessing that ever happened to me professionally, she told the National Science and Technology Medals Foundation in 2016. She wasnt sure shed have been a National Medal of Science laureate, she said, if New York University hadnt decided that I was a bad girl in 1941.

She was already interested in genes, and especially in a theory espoused by the Russian scientist Trofim Lysenko that denied their existence and insisted that environment shaped evolution.

At Columbia, she worked with another Russian-born researcher, Theodosius Dobzhansky, considered a founder of evolutionary genetics. He not only disabused her of Dr. Lysenkos ideas; he also introduced her to a paper by Salvador Luria and Max Delbrck proving that bacteria had DNA.

Reporting on it for Dobzhanskys class, I jumped up and down with excitement, she told The Times. At the time, one of the big questions involved how genetic mutations occurred. Thanks to Luria and Delbrck, I now saw how we could use bacteria models to answer that.

She married Herman Witkin, a psychologist, in 1943. He died in 1979. Along with her son, Joseph, a doctor who is also a founding member of the rock n roll group Sha Na Na, she is survived by four grandchildren and three great-grandchildren. Another son, Andrew, died in 2010.

Dr. Witkin stayed at Cold Spring Harbor until 1955, when she moved to SUNY Downstate. She later joined the faculty at Douglass College in New Jersey, at the time an all-womens institution attached to Rutgers. In 1983 she became the director of the Waksman Institute of Microbiology, also at Rutgers, where she stayed until retiring.

In 2021, on her 100th birthday, the Waksman Institute renamed one of its premier research laboratories for her.

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Evelyn M. Witkin, Who Discovered How DNA Repairs Itself, Dies at ... - The New York Times

Arnold, F. H. Design by directed evolution. Acc. Chem. Res. 31, 125131 (1998).

Article CAS Google Scholar

Davis, A. M., Plowright, A. T. & Valeur, E. Directing evolution. The next revolution in drug discovery? Nat. Rev. Drug Discov. 16, 681698 (2017).

Article CAS PubMed Google Scholar

Packer, M. S. & Liu, D. R. Methods for the directed evolution of proteins. Nat. Rev. Genet. 16, 379394 (2015).

Article CAS PubMed Google Scholar

Arnold, F. H. Directed evolution. Bringing new chemistry to life. Angew. Chem. 57, 41434148 (2018).

Article CAS Google Scholar

Rix, G. & Liu, C. C. Systems for in vivo hypermutation. A quest for scale and depth in directed evolution. Curr. Opin. Chem. Biol. 64, 2026 (2021).

Article CAS PubMed PubMed Central Google Scholar

Morrison, M. S., Podracky, C. J. & Liu, D. R. The developing toolkit of continuous directed evolution. Nat. Chem. Biol. 16, 610619 (2020).

Article CAS PubMed Google Scholar

Meyer, A. J. & Ellington, A. D. Molecular evolution picks up the PACE. Nat. Biotechnol. 29, 502503 (2011).

Article CAS PubMed Google Scholar

Simon, A. J., dOelsnitz, S. & Ellington, A. D. Synthetic evolution. Nat. Biotechnol. 37, 730743 (2019).

Article CAS PubMed Google Scholar

Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420424 (2016).

Article CAS PubMed PubMed Central Google Scholar

Chen, H. et al. Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor. Nat. Biotechnol. 38, 165168 (2020).

Article CAS PubMed Google Scholar

Cravens, A., Jamil, O. K., Kong, D., Sockolosky, J. T. & Smolke, C. D. Polymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering. Nat. Commun. 12, 1579 (2021).

Article CAS PubMed PubMed Central Google Scholar

Hao, W. et al. Development of a base editor for protein evolution via in situ mutation in vivo. Nucleic Acids Res. 49, 95949605 (2021).

Article CAS PubMed PubMed Central Google Scholar

Schubert, M. G. et al. High-throughput functional variant screens via in vivo production of single-stranded DNA. Proc. Natl Acad. Sci. USA 118, e2018181118 (2021).

Article CAS PubMed PubMed Central Google Scholar

Jensen, E. D. et al. A synthetic RNA-mediated evolution system in yeast. Nucleic Acids Res. 49, e88 (2021).

Article CAS PubMed PubMed Central Google Scholar

Crook, N. et al. In vivo continuous evolution of genes and pathways in yeast. Nat. Commun. 7, 13051 (2016).

Article CAS PubMed PubMed Central Google Scholar

Halperin, S. O. et al. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature 560, 248252 (2018).

Article CAS PubMed Google Scholar

Yi, X., Khey, J., Kazlauskas, R. J. & Travisano, M. Plasmid hypermutation using a targeted artificial DNA replisome. Sci. Adv. 7, eabg8712 (2021).

Article CAS PubMed PubMed Central Google Scholar

Blum, T. R. et al. Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity. Science 371, 803810 (2021).

Article CAS PubMed PubMed Central Google Scholar

Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous directed evolution of biomolecules. Nature 472, 499503 (2011).

Article CAS PubMed PubMed Central Google Scholar

Ravikumar, A., Arzumanyan, G. A., Obadi, M. K., Javanpour, A. A. & Liu, C. C. Scalable, continuous evolution of genes at mutation rates above genomic error thresholds. Cell 175, 19461957 (2018).

Article CAS PubMed PubMed Central Google Scholar

Zhong, Z. & Liu, C. C. Probing pathways of adaptation with continuous evolution. Curr. Opin. Syst. Biol. 14, 1824 (2019).

Article PubMed PubMed Central Google Scholar

Ravikumar, A., Arrieta, A. & Liu, C. C. An orthogonal DNA replication system in yeast. Nat. Chem. Biol. 10, 175177 (2014).

Article CAS PubMed Google Scholar

Wellner, A. et al. Rapid generation of potent antibodies by autonomous hypermutation in yeast. Nat. Chem. Biol. 17, 10571064 (2021).

Javanpour, A. A. & Liu, C. C. Evolving small-molecule biosensors with improved performance and reprogrammed ligand preference using OrthoRep. ACS Synth. Biol. 10, 27052714 (2021).

Article CAS PubMed PubMed Central Google Scholar

Rix, G. et al. Scalable continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities. Nat. Commun. 11, 5644 (2020).

Article CAS PubMed PubMed Central Google Scholar

Muoz-Espn, D., Holguera, I., Ballesteros-Plaza, D., Carballido-Lpez, R. & Salas, M. Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid. Proc. Natl Acad. Sci. USA 107, 1654816553 (2010).

Article PubMed PubMed Central Google Scholar

van Nies, P. et al. Self-replication of DNA by its encoded proteins in liposome-based synthetic cells. Nat. Commun. 9, 1583 (2018).

Article PubMed PubMed Central Google Scholar

Gillis, A. & Mahillon, J. Phages preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis. Past, present and future. Viruses 6, 26232672 (2014).

Article PubMed PubMed Central Google Scholar

Meijer, W. J., Horcajadas, J. A. & Salas, M. Phi29 family of phages. Microbiol. Mol. Biol. Rev. 65, 261287 (2001).

Article CAS PubMed PubMed Central Google Scholar

Gillis, A. & Mahillon, J. Influence of lysogeny of Tectiviruses GIL01 and GIL16 on Bacillus thuringiensis growth, biofilm formation, and swarming motility. Appl. Environ. Microbiol. 80, 76207630 (2014).

Article PubMed PubMed Central Google Scholar

Biggel, M. et al. Whole genome sequencing reveals biopesticidal origin of Bacillus thuringiensis in foods. Front. Microbiol. 12, 775669 (2022).

Article PubMed PubMed Central Google Scholar

Verheust, C., Fornelos, N. & Mahillon, J. GIL16, a new gram-positive tectiviral phage related to the Bacillus thuringiensis GIL01 and the Bacillus cereus pBClin15 elements. J. Bacteriol. 187, 19661973 (2005).

Article CAS PubMed PubMed Central Google Scholar

Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018).

Article PubMed PubMed Central Google Scholar

Wannier, T. M. et al. Improved bacterial recombineering by parallelized protein discovery. Proc. Natl Acad. Sci. USA 117, 1368913698 (2020).

Article CAS PubMed PubMed Central Google Scholar

Wu, Y. et al. Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis. Nucleic Acids Res. 48, 9961009 (2020).

Article CAS PubMed Google Scholar

Soengas, M. S. et al. Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 35 exonuclease and strand-displacement activities. EMBO J. 11, 42274237 (1992).

Article CAS PubMed PubMed Central Google Scholar

Vega, M., de, Lazaro, J. M., Salas, M. & Blanco, L. Primer-terminus stabilization at the 35 exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases. EMBO J. 15, 11821192 (1996).

Article PubMed PubMed Central Google Scholar

Truniger, V., Lzaro, J. M., Salas, M. & Blanco, L. A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases. EMBO J. 15, 34303441 (1996).

Article CAS PubMed PubMed Central Google Scholar

Vega, M., de, Lzaro, J. M. & Salas, M. Phage 29 DNA polymerase residues involved in the proper stabilisation of the primer-terminus at the 35 exonuclease active site. J. Mol. Biol. 304, 19 (2000).

Article PubMed Google Scholar

Prez-Arnaiz, P., Lzaro, J. M., Salas, M. & de Vega, M. Functional importance of bacteriophage 29 DNA polymerase residue tyr148 in primer-terminus stabilisation at the 3'-5' exonuclease active site. J. Mol. Biol. 391, 797807 (2009).

Article PubMed Google Scholar

Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583589 (2021).

Article CAS PubMed PubMed Central Google Scholar

Liu, Y., Liu, L., Li, J., Du, G. & Chen, J. Synthetic biology toolbox and chassis development in Bacillus subtilis. Trends Biotechnol. 37, 548562 (2019).

Article CAS PubMed Google Scholar

Lu, Z. et al. CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in Bacillus subtilis. Nucleic Acids Res. 47, e40 (2019).

Article PubMed PubMed Central Google Scholar

Tian, R. et al. Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction. Nat. Commun. 11, 5078 (2020).

Article CAS PubMed PubMed Central Google Scholar

Cai et al. Cell-free chemoenzymatic starch synthesis from carbon dioxide. Science 373, 15231527 (2021).

Article CAS PubMed Google Scholar

Gao, B. et al. Constructing a methanol-dependent Bacillus subtilis by engineering the methanol metabolism. J. Biotechnol. 343, 128137 (2022).

Article CAS PubMed Google Scholar

Li, C., Zou, Y., Jiang, T., Zhang, J. & Yan, Y. Harnessing plasmid replication mechanism to enable dynamic control of gene copy in bacteria. Metab. Eng. 70, 6778 (2022).

Article CAS PubMed PubMed Central Google Scholar

Fornelos, N., Bamford, J. K. H. & Mahillon, J. Phage-borne factors and host LexA regulate the lytic switch in phage GIL01. J. Bacteriol. 193, 60086019 (2011).

Article CAS PubMed PubMed Central Google Scholar

Fornelos, N. et al. Lytic gene expression in the temperate bacteriophage GIL01 is activated by a phage-encoded LexA homologue. Nucleic Acids Res. 46, 94329443 (2018).

Article CAS PubMed PubMed Central Google Scholar

Fornelos, N. et al. Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage. Nucleic Acids Res. 43, 73157329 (2015).

Article CAS PubMed PubMed Central Google Scholar

del Solar, G., Giraldo, R., Ruiz-Echevarra, M. J., Espinosa, M. & Daz-Orejas, R. Replication and control of circular bacterial plasmids. Microbiol. Mol. Biol. Rev. 62, 434464 (1998).

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Engineered bacterial orthogonal DNA replication system for ... - Nature.com

July 14, 2023 If you have ever tried to build something as a kid, with a kid, or for a kid with the tiniest of Legos, you might have an idea of the challenge in creating at the small scale. Scientists at the University of Illinois Urbana Champaign face this type of challenge regularly in their study of how to create tiny motors inspired by DNA.

The added challenge for them, though, is the level of tiny at which they work the nanoscale. This is better understood by thinking about the thickness of one sheet of paper or the width of a single strand of human hair both equal to about 100,000 nanometers.

To study at this scale,ACCESSresources like the Expanse supercomputer at the San Diego Supercomputer Center at UC San Diego come in handy for researchers such as those led by Aleksei Aksimentiev, professor of biological physics at the University of Illinois Urbana Champaign and principal investigator for a recent study with findings titledDNA double helix, a tiny electromotor, published in Nature Nanotechnology.

A supercomputer such asExpanse enables researchers to peer deeper into elements of what they are studying by creating simulations that use a mathematical description, or model, of a real system embodied in a computer program. Aksimentiev explained that the first step in creating the Expanse simulations was to place DNA molecules in water, apply electric field flow and then observe the rotation or measure the torque generated.

Supercomputers like SDSCs Expanse are essential to the development of nanotechnology as they provide a window into the nanoscale world that is otherwise not accessible to experiments, Aksimentiev said.

For this study, a typical simulation took a little longer than a week to complete, which allowed the researchers to carefully create their simulations and measure the torque, enabling them to see the rotation. Once they carefully analyzed the results, they submitted their work to be published in the journal so that the broader research community had access to their findings.

People have thought about this for a while and gone back and forthcould it work or not, Aksimentiev said. We thought that maybe the water doesnt have enough traction to produce torque, but it did, and we were excited to see the DNA spin in the direction prescribed by its helicity despite significant fluctuations.

Aksimentiev said that he and the Illinois team plan to use lessons learned from this study to build systems at a nanoscale level that will be used as components in self-propelled systems or for nanoscale energy conversion.

You can read more about this story here (published April 28, 2023):Expanse Supercomputer Used for Tiny Torque Simulations of DNA Molecules.

Institution:SDSC (San Diego Supercomputer Center) University:University of Illinois at Urbana Champaign Funding Agency:NSF Allocation Number:MCA05S028

The science story featured here, allocated through August 31, 2022,was enabled through Extreme Science and Engineering Discovery Environment (XSEDE) and supported by National Science Foundation grant number #1548562. Projects allocated September 1, 2022 and beyondare enabled by the ACCESS program, which is supported by National Science Foundation grants #2138259, #2138286, #2138307, #2137603, and #2138296.

Source: ACCESS

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ACCESS and Expanse Supercomputer Aid in DNA-Based Motor ... - HPCwire

Description of the proposed method

Our proposed method, schematically depicted in Fig.1, takes advantage of using a modified primer (Modified Specific Primer, PSM) during the reverse transcription step of the protocol. Such a primer is specific for the RNA molecules to be quantified and its nucleotide sequence is designed to lack a perfect homology to the retro-transcribed template DNA. Generally, it is enough to add few mismatches with respect to the original sequence, preferably located in the close proximity to the 3'-OH terminal region. These modifications make the primer partially complementary to the target sequence but still able to hybridize at the temperatures of 3742C used during the reverse transcription step. Nevertheless, the PSM will dissociate from the partially homologous genomic DNA sequence during the PCR step, once the operating temperature reaches around 60C. The aim of using such conveniently modified specific primer is to achieve amplification specifically from cDNA template while successfully avoiding genomic DNA targets. The correct number of modifications to be applied, their effectiveness and proper discriminating temperatures should be experimentally tested for each and every transcript to be analyzed, by selecting those parameters that show negative and positive amplification tendencies towards DNA and cDNA targets, respectively. This optimization phase represents a preliminary step of our method that enables the setup of negative and positive controls and, advantageously, has to be carried out only once, since it always remains valid for a specific amplicon and can be applied to a varying number of replicates under different experimental conditions. Indeed, in current protocols the negative control (NC: RT, without reverse transcriptase) should ideally be prepared for each new sample to be tested, even though the target is the same, due to the random effectiveness of DNase I treatment. Using a PSM we are able to generate cDNA slightly different from its genomic DNA counterpart, due to the nucleotide mismatches present in the sequence.

Schematic illustration of the new method. (A) Basic model of nucleic acid metabolism from DNA to cDNA. Integration of modified specific primer into cDNA by means of reverse transcription makes it a permanent part of the sequence. (B) Amplification of target sequence by means of polymerase chain reaction. cDNA converted by modified specific primer is properly amplified at certain discriminating temperature, while genomic DNA targets are successfully avoided.

During the phase following reverse transcription (Fig.1B), the amplification of cDNA by PCR takes place using the same modified primer (PSM) from the previous step in addition to the unmodified specific primers (SP) starting from the opposite direction. Consequently, the resulting amplicon is a copy of the cDNA and not the DNA, due to the specifically selective annealing temperatures usually ranging from 55C to 62C. Therefore, with this procedure, there is no need to eliminate the co-purified DNA from the RNA sample since it is no longer a competing target and will not affect the final result of the assay. Indeed, in certain experimental conditions it could be useful and advantageous to have both DNA and RNA present together in the same sample if, for example, the results need to be normalized with respect to the gene copy number variation.

Our proposed new method can be utilized in various experimental investigations and for the purposes of this paper, it has been tested by analyzing three bacterial E. coli genes: ssb, sulA and recA (Figs.2, 3 and 4), and two satellite DNA transcripts: human alpha-satellite (ASAT) (Figs.5 and 6, and Suppl. Fig.2) and TCAST1 satellite from Tribolium castaneum (Suppl. Fig.1).

Transcription of ssb gene in exponentially growing E. coli cells harbouring ssb overexpression plasmid pID2 obtained by dPCR using current (A) and new method (B). Columns represent number of copies/l and the plotted error bar shows whether or not the events differ with 95% Poisson confidence interval.

Transcription of recA gene in exponentially growing E. coli cells obtained by dPCR using current and new method. Columns represent number of copies/l and the plotted error bar shows whether or not the events differ with 95% Poisson confidence interval.

Transcription of sulA gene in exponentially growing E. coli cells obtained by dPCR using current and new method. Columns represent number of copies/l and the plotted error bar shows whether or not the events differ with 95% Poisson confidence interval.

Delta Rn vs Cycle plot of alpha satellite DNA isolated from HeLa cells obtained by qPCR using current method (A) and new method (B).+RT and RT represent positive and negative controls, with and without reverse transcription, respectively.

Transcription level of alpha satellite DNA obtained by qPCR using current method (A) and new method (B). Columns show average of 2 different loaded samples in qPCR experiments performed in triplicate. N0 represents normalized average N0 value for alpha satellite. C represent alpha samples with reverse transcription and NC represents negative controls without reverse transcription and M is 100bp size marker.

Bacterial genes are a good experimental model to test our method because they do not contain introns in their coding region, removing the possibility of discriminating between transcripts and the DNA according to their different sizes. Hence, the technique could be applied to test the expression of all genes organized with a short or null intron (e.g. viral genes).

The bacterial strain used in this test was transformed with multicopy plasmid carrying a cloned ssb gene9, which could compete for amplification with ssb-cDNA during the transcripts quantification by PCR, unless additional DNase I treatments were implemented. The results indicated in Fig.2 show a large difference (more than 40-fold) in ssb transcription levels measured by our method, as compared to the currently used method. This really high level of amplified ssb sequence in the latter approach, when reverse transcription was not carried out, and the DNA was eliminated in both RNA isolation and RT steps (Fig.2A), is likely due to low efficiency of elimination of covalently closed circular plasmid DNA, meaning that it is false (i.e. it does not accurately represent the process of transcription) and is actually caused by DNA contamination.

This is likely a reason for all the observed cases of high levels of ssb sequence amplification using classical primers (Fig.2A). In contrast, ssb sequence was amplified by our new method only in those cases when reverse transcription was performed, i.e. when cDNA was created (Fig.2B). The level of ssb sequence amplification did not depend on DNA elimination (Fig.2B), thus confirming insensibility of our method to the presence of genomic (and plasmid) DNA. Next, we quantified expression of recA and sulA genes, which are present as single copies in the E. coli genome. In accord with the previous assay, no recA sequence amplification was observed using our method unless cDNA was created by reverse transcription (Fig.3). The level of recA sequence amplification was, again, independent from genomic DNA elimination from the sample (Fig.3). Conversely, the current method, which uses standard primers, showed a false positive signal even when reverse transcription step was skipped and the genomic DNA was (obviously incompletely) eliminated by DNase I treatment (Fig.3).

Finally, analysis of sulA gene expression using a modified primer was in accord with the previous assays since amplification of sulA sequence occurred only after reverse transcription, i.e. it was specific for cDNA (Fig.4). Accordingly, no effect was observed after genomic DNA elimination (Fig.4). In contrast, amplification of sulA sequence using standard primers was very different, and was not abolished even in situations where genomic DNA was eliminated and reverse transcription was not performed (Fig.4); theoretically, the RT/+DNase I sample should not contain any cDNA or genomic DNA.

The presented results clearly demonstrate that our method of using a modified primer during cDNA synthesis produces a cDNA-specific PCR signal, which is independent of genomic DNA, and therefore much more accurately quantifies gene expression when compared to the standard, commonly used method, which, unfortunately, does not produce real negative control since there is always possibility to have contaminating DNA in the sample.

Satellite DNA represents one of the best target candidates for demonstrating the effectiveness of our methodology since it is a highly repetitive non-coding genomic DNA, ever-present in large quantities in the sample and therefore difficult, if not impossible, to remove during RNA purification.

Alpha satellite DNA is the most abundant human satellite DNA of 171bp long, comprising up to 10% of the genome14. Figure5A, shows qPCR results obtained by following the current standard protocol (old method) which implies the elimination of DNA both during the RNA purification and reverse transcription phase. In spite of that, alpha satellite DNA continues to persist in the negative control samples ( RT). Furthermore, since it is not organized into exons and introns, satellite DNA cannot be discriminated from satellite cDNA based on its length; therefore, even a slightest trace of DNA contamination often produces false-positive results. The new method, however, successfully demonstrated the disappearance of the alpha satellite DNA contamination from the qPCR amplification results (Fig.5B, RT), as it can be clearly seen also by loading the amplicons on agarose gel (Suppl. Fig.2): ASAT amplicon of 126bp long is present only in+RT samples (C: controls) respect to RT samples (NC: negative control). The same results could be represented as in Fig.6A (current method) and Fig.6B (new method), where N0 value is the starting concentration of amplicon in the sample and columns show average of 2 different loaded samples in qPCR experiments performed in triplicate (see Materials & method section).

The highly abundant satellite DNA TCAST1 has previously been characterized as the major satellite that makes up to 30% of the beetle Tribolium castaneum genome, organizing the centromeric as well as pericentromeric regions of all 20 chromosomes10,13. Again, using the new method only cDNA was amplified (+RT samples) and almost nothing of genomic DNA contamination was detected in RT samples (Suppl. Fig.1). The results clearly show they are exactly the same as those obtained for human alpha satellite DNA.

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Reverse transcription-quantitative PCR (RT-qPCR) without the need ... - Nature.com

People with shorter telomeres, or chromosome caps, may be at a greater risk of developing idiopathic pulmonary fibrosis (IPF), a study reported.

This work also provided some interesting genetic evidence to prove that obesity and exposure to tobacco smoking as a fetus might also contribute to the development of the fibrotic [disease], the researchers wrote. Previous studies suggest a link between obesity and/or smoking and shorter telomeres, they added, noting the associations found here should be confirmed in future studies.

The study, Genetic association of telomere length, obesity and tobaccosmoking with idiopathic pulmonary fibrosis risk, was published in the journal BMC Public Health.

IPF, a form of pulmonary fibrosis (PF) that lacks a clear underlying cause, is characterized by progressive lung tissue scarring, or fibrosis, leading to shortness of breath and other disease symptoms. It also tends to occur in middle-age and older adults, and telomeres length can be affected by the aging process.

Telomeres are structures that cap the ends of chromosomes, helping to protect DNA from damage. Abnormally short telomeres have been tied to an higher risk of a variety of conditions, including PF.

An increasing number of observational studies focused on the potential effects of telomere-associated factors (i.e. tobacco smoking, alcohol drinking, and obesity) on the risk of IPF, and provided some interesting findings, the researchers wrote.

Scientists at institutes in Zhengzhou, China, aimed to validate these reported observations by using genome-wide association studies to explore the genetic effects of telomere length and telomere-associated factors on IPF risk. Genome-wide association studies are those that scan all genes, or the genome, looking for small genetic alterations, called single nucleotide polymorphisms (SNPs), that are found more often in people with a particular disease.

Data collected came from a U.K. biobank with 472,174 participants, with a focus on genetic variants potentially associated with telomere length and the linking of telomere length to certain conditions. IPF data were obtained from a database including 1,812 people in Europe with the disease and 338,784 without it as controls.

Longer telomere length was found to associate with a reduced risk of IPF, with risk being 52.5% lower for each standard deviation increase in telomere length. This risk was calculated after excluding SNPs linked to obesity and smoking. (Standard deviation or SD is a statistical measure of how much variation there is within a dataset, or how close or far apart data points spread from the mean.)

Next, they analyzed factors associated with telomere length, including obesity, tobacco smoking, and alcohol drinking. Obesity was determined by a persons body mass index (BMI) and body fat percentage (BFP), both measures of body fat.

Higher BMI and BFP values linked with an increased risk of developing IPF. That risk was 42.5% times higher per each SD increase in BMI, and 70.2% per each SD increase in BFP.

People whose mothers smoked during pregnancy also were at a higher risk of IPF a risk found to be 13.18 times higher per SD increase in the prevalence of maternal smoking.

These results were consistent with one important medical truism that many organs of the fetus (including the lungs) were at a developmental stage and were susceptible to damage from tobacco, the researchers wrote, and findings warrant further investigation.

No associations were seen between heavy smoking, smoking initiation, age of smoking initiation, smoking cessation, and the risk of developing IPF. Similarly, heavy drinking did not associate with a higher risk of the disease.

In conclusion, the present study confirmed the causal association of [telomere length] with the risk of IPF, the researchers wrote. It also demonstrated that factors that might influence the length of telomeres, such as obesity and exposure to tobacco smoking as a fetus might also contribute to the development of [IPF].

When exploring the effect of telomere length on one disease, it is important to consider that telomere length decreases progressively with age, so study groups should be of similar ages. A significant age difference between data sets in this study, and that was noted as an important limitation by its authors.

Therefore, these findings should be verified by future studies, the researchers noted.

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Shorter telomeres, protective caps for DNA, seen as potential... - Pulmonary Fibrosis News

L.L. Bean has just added a third shift at its factory in Brunswick, Maine, in an attempt to keep up with demand for its iconic boot.

Orders have quadrupled in the past few years as the boots have become more popular among a younger, more urban crowd.

The company says it saw the trend coming and tried to prepare, but orders outpaced projections. They expect to sell 450,000 pairs of boots in 2014.

People hoping to have the boots in time for Christmas are likely going to be disappointed. The bootsare back ordered through February and even March.

"I've been told it's a good problem to have but I"m disappointed that customers not getting what they want as quickly as they want," said Senior Manufacturing Manager Royce Haines.

Customers like, Mary Clifford, tried to order boots on line, but they were back ordered until January.

"I was very surprised this is what they are known for and at Christmas time you can't get them when you need them," said Clifford.

People who do have boots are trying to capitalize on the shortage and are selling them on Ebay at a much higher cost.

L.L. Bean says it has hired dozens of new boot makers, but it takes up to six months to train someone to make a boot.

The company has also spent a million dollars on new equipment to try and keep pace with demand.

Some customers are having luck at the retail stores. They have a separate inventory, and while sizes are limited, those stores have boots on the shelves.

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New DNA testing helps Maryland investigators arrest suspect 44 years after woman's killing - NBC4 Washington