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Evolving CRISPR
A team of Harvard Medical School researchers at Massachusetts General Hospital has found a way to expand the use and precision of powerful gene-editing tools called CRISPR-Cas9 RNA-guided nucleases.
In their report in Nature, the investigators describe evolved versions of the DNA-cutting Cas9 enzyme that can recognize a different range of nucleic acid sequences than is now possible with the naturally occurring form of Cas9 scientists have been using.
"In our paper we show that sites in human and zebrafish genes that could not previously be modified by wild-type Cas9 can now be targeted with the new variants we have engineered," said Benjamin Kleinstiver, HMS research fellow in pathology at Mass General and lead author of the Nature paper. "This will allow researchers to target an expanded range of sites in a variety of genomes, which will be useful for applications requiring highly precise targeting of DNA sequences."
CRISPR-Cas9 nucleases consist of the Cas9 bacterial enzyme and a short, 20-nucleotide RNA molecule that matches the target DNA sequence. In addition to the RNA/DNA match, the Cas9 enzyme needs to recognize a specific nucleotide sequence called a protospacer adjacent motif (PAM) adjacent to the target DNA.
The most commonly used form of Cas9, derived from the bacteria Streptococcus pyogenes and known as SpCas9, recognizes PAM sequences in which any nucleotide is followed by two guanine DNA bases. This limits the DNA sequences that can be targeted using SpCas9 to only those that include two sequential guanines.
To get around this limitation, the team set up an engineering system that allowed them to rapidly evolve the ability of SpCas9 to recognize different PAM sequences. From a collection of randomly mutated SpCas9 variants, they identified combinations of mutations that enabled SpCas9 to recognize new PAM sequences.
These evolved variants essentially double the range of sites that can now be targeted for gene editing using SpCas9. They also identified an SpCas9 variant that was less likely to induce the off-target gene mutations sometimes produced by CRISPR-Cas9 nucleases, a problem originally described in a 2013 study led by J. Keith Joung, HMS professor of pathology at Mass General and senior author of the current study.
"This additional evolved variant with increased specificity should be immediately useful to all researchers who currently use wild-type SpCas9 and should reduce the frequencies of unwanted off-target mutations," Joung said. "Perhaps more important, our findings provide the first demonstration that the activities of SpCas9 can be altered by directed protein evolution."
The scientists showed in their paper that the forms of Cas9 found in two other bacteria—Staphylococcus aureus and Streptococcus thermophiles—can also function in their bacterial evolution system, suggesting that their functions can be modified as well, Joung said.
"This work just scratches the surface of the range of PAMs that can be targeted by Cas9," Joung said. "We believe that other useful properties of the enzyme may be modified by a similar approach, allowing potential customization of many important features."
The study was supported by National Institutes of Health Director's Pioneer Award DP1 GM105378, NIH grants R01 GM107427 and R01 GM088040, a Jim and Ann Orr Research Scholar Award, and a National Sciences and Engineering Research Council of Canada Postdoctoral Fellowship. The MGH has filed a patent application on the use of the SpCas9 variants described in this paper.
Adapted from a Mass General news release.
FISHing for Insight
Geneticists just got a new pair of glasses.
By improving an imaging technology called FISH, they've made it possible to view genetic material in more detail than ever before.
One modification achieves "super resolution" imaging. The other can distinguish maternal from paternal chromosomes.
What the researchers are starting to see promises to help them better understand how DNA gets packaged into chromosomes, how that structure relates to health and disease, what the biological significance may be when genetic material is inherited from one parent versus the other—and more.
"Geneticists have looked at chromosomes for over a century, but they've longed for higher resolution and an easier and more affordable strategy for looking at any part of any genome, not just the most accessible regions," said Ting Wu, professor of genetics at Harvard Medical School and senior author of the study, published in Nature Communications. "With this new technology, we've enabled significant steps forward in all those regards."
"Scientists have a lot of models where we draw in cartoon form what we think is happening," said the study's first author, Brian Beliveau, who conducted the work as a graduate student in the Wu lab. "When a region of the genome is silenced, we draw it as compacted. When a gene is expressed, we draw it as more open or active. But we don't actually know yet what any of these look like."
"Now we have a tool to start looking at this systematically, which is very exciting," said Beliveau, currently a research fellow in the lab of HMS associate professor of systems biology Peng Yin at the Wyss Institute for Biologically Inspired Engineering.
Teach a person to FISH…
FISH, short for fluorescence in situ hybridization, is a decades-old visualization technique that locates and illuminates specific genes in the nuclei of cells.
Scientists use FISH to find out things like how many chromosomes a person has or where a particular gene is located on a chromosome.
But conventional FISH has its limits, and scientists want to look closer.
For instance, it hasn't been able to show the details of how chromosomes are folded, whether there are different ways of folding or how two genome segments interact—all of which would help elucidate how our bodies work.
Researchers have had some success getting high-resolution FISH images of small segments of the genome. But the cost to look at such questions genome-wide has been "beyond anybody's purse," said Wu. "The flexibility to target a specific region on the genome has been very difficult also."
In 2012, Beliveau devised an enhancement to FISH called Oligopaint that made this possible at low cost.
Traditional FISH works by attaching a fluorescent tag to a short, single strand of DNA called a probe, which has a sequence complementary to the segment of DNA a researcher wants to study. When released into a cell, the probe binds to the desired sequence in the nucleus and lights it up so it can be seen under a microscope.
A limiting factor to scaling up FISH has been that "it's a ton of work to try to find those sequences in nature to make the necessary DNA probes and isolate them in a way that would be compatible with the tool," said Beliveau.
He solved the problem by developing computer software that lets scientists design the probes they need and then build them with synthetic components—hundreds or thousands at a time.
Super resolution
In the new paper, Beliveau taught Oligopaint two new tricks.
First, he paired it up with two other technologies—STORM from the lab of Xiaowei Zhuang at Harvard University, and DNA-PAINT from Peng Yin's lab—to zoom in on chromosomes at "super resolution."
The DNA double helix is about 2 nanometers wide. When it gets wound around a bundle called a nucleosome, one of the simplest building blocks of chromosomes, it grows to about 10 nanometers. Since conventional microscopy can resolve images to only about 200 to 300 nanometers, it's been impossible to see such infinitesimal structures.
The new Oligopaint combinations can bring chromosomes into focus down to about 20 nanometers. That's possible because each DNA probe is able to bind to a second probe; the resulting fluorescent output blinks, enabling researchers to distinguish the fluorescent tags one at a time and achieve a finer resolution.
"This is an unprecedented level of detail," said Wu.
"Right now when you do FISH on structures below a certain size, you get a spot," said Beliveau. "With these technologies, we're seeing interesting shapes and structures, loops and protrusions, start to drop out of these things."
Wu, Beliveau and their colleagues have already begun to find "intriguing organizational themes" in the chromatin of mammalian and fruit fly cells. But they consider the current paper mostly a proof of concept.
"The goal is to communicate these techniques to the research community as quickly as possible so everyone can start making discoveries," said Beliveau.
They hope researchers generate "a huge bolus of images" that will reveal more about what our genetic structure looks like, Wu added.
"Of course, naturally, if we get down to 20 nanometers then people will want 10 nanometers, and then 5," she said wryly.
Distinguishing mom from dad
We normally inherit our chromosomes pairs: one from our mother and one from our father. The second trick Beliveau taught Oligopaint was to tell them apart.
"This was not thought possible but, to our surprise, our technology managed to break this barrier too," said Wu.
Beliveau modified his Oligopaint probes so they could detect the presence or absence of the many single nucleotide variants, or SNPs, that distinguish maternal from paternal chromosomes.
As long as researchers know which SNPs to look for and have hundreds or thousands of them to target—just a few isn't enough—Oligopaint can now light up a chromosome to identify "mom" or "dad."
Wu, for one, is excited to use this new ability to study things like how one of two X chromosomes gets inactivated in female mammals. It's also become clear to scientists that maternal and paternal genes don't behave the same way, and that this might have effects on human health and disease. Wu is particularly interested in Down syndrome, in which children inherit three instead of two copies of chromosome 21.
"I'd love to look at a cell and ask, are two copies from the mother? From the father? How do they behave relative to each other?" she said.
This study was supported by the National Institutes of Health (R01GM61936, R01GM090278, 1R01EB018659, 5DP1GM106412, F32CA157188, 1DP2OD007292, 5R21HD072481, 1DP2OD004641), National Science Foundation (CCF1054898, CCF1162459), Office of Naval Research (N000141110914, N000141010827, N000141310593), Harvard Medical School, Wyss Institute for Biologically Inspired Engineering, Centre National de la Recherche Scientifique, Howard Hughes Medical Institute, Damon Runyon Cancer Research Foundation, Fulbright Visiting Scholar Program and Alexander von Humboldt-Foundation.
Beyond Average
Imagine someone hands you a smoothie and asks you to identify everything that went into it.
You might be able to discern a hint of strawberry or the tang of yogurt. But overall it tastes like a blend of indiscernible ingredients.
Now imagine that the smoothie is made of 20,000 ground-up cells from, say, the brain.
You could run tests to determine what molecules are in the sample, which is what scientists do now. That would certainly give you useful information, but it wouldn't tell you which cells those molecules originally came from. It would provide only an average cell profile for the whole smoothie.
And when it comes to the tissues in our bodies, averages are almost always misleading. Just as you know there isn't an "average" food called strawbanaspinach-orangegurt, scientists know there isn't just one cell type in the brain.
"If you take a hunk of tissue and grind it up and analyze the RNA, you have no idea if it represents what every cell in that population is doing or what no cell in the population is doing," said Marc Kirschner, the John Franklin Enders University Professor of Systems Biology and chair of the Department of Systems Biology at Harvard Medical School. "Imagine if you had a population of men and women. If you assume everyone is an average of men and women, you [probably] wouldn't represent a single person in that population."
The trouble is, it's expensive, time-consuming and tricky to characterize tissues one cell, or cell type, at a time.
Kirschner and Steven McCarroll, assistant professor of genetics at HMS, reported this week in separate papers that their labs have developed high-throughput techniques to quickly, easily and inexpensively give every cell in a sample a unique genetic barcode before it goes into the blender.
As a result, scientists can analyze complex tissues by profiling each individual cell—no averaging required.
"Different cells in a tissue use the same genome in amazingly diverse ways: to engineer specialized cell shapes, accomplish diverse feats of physiology, and mount distinct functional responses to the same stimulus. These techniques will finally let science understand how biological systems operate at that single-cell level," said McCarroll, who is also director of genetics for the Stanley Center for Psychiatric Research at the Broad Institute of Harvard and MIT. "We are so excited about the work ahead."
To make their tools, both teams collaborated with David Weitz, the Mallinckrodt Professor of Physics and Applied Physics at Harvard's School of Engineering and Applied Sciences and a pioneer in the field of microfluidics.
The teams expect that their techniques, published concurrently in the journal Cell, will equip biologists to discover and classify cell types in the body in much greater depth, map cell diversity in complex tissues such as the brain, better understand stem cell differentiation and gain more insights into the genetics of disease.
Harvard's Office of Technology Development has been working closely with the researchers to develop patent applications for various aspects of the technology, all with an eye toward commercialization.
'Two roads diverged in a yellow wood'
Evan Macosko and Allon Klein met in a microfluidics class a few years ago. Then they went their separate ways.
Unbeknownst to each other, they decided to develop methods to answer the same question: How could they obtain gene expression profiles for thousands of individual cells to better understand the complexity of gene expression within a tissue?
Gene expression—the pattern of gene activity in a particular cell—underlies every process in biology, from cognition in the brain to development in the egg. Scientists have known for 50 years that gene expression varies from cell to cell like a fingerprint, making skin cells different from liver cells and making some liver cells different from others. But they haven't been able to measure it efficiently at the single-cell level in samples with many cell types.
Macosko, HMS instructor in psychiatry at Massachusetts General Hospital and a Stanley Neuroscience Fellow in the McCarroll lab, came up with a technique he called Drop-seq. Klein, assistant professor of systems biology at HMS, devised a method he called indexing droplets for sequencing, or inDrops.
Last fall, they learned about each other's work through the scientific conference circuit.
"It was kind of like meeting your doppelgänger," said Macosko. "He had been thinking about the same things I had for two years. Human beings have different ways of solving problems, and it was really cool to see how he did it."
How they work
The teams each developed ways of using tiny beads to deliver vast numbers of different DNA barcodes into hundreds of thousands of nanometer-sized water droplets simultaneously.
Thanks to Weitz's expertise, both methods were able to use microfluidic devices to co-encapsulate cells in these droplets along with the beads. The droplets get created in a tiny assembly line, streaming along a channel the width of a human hair.
The bead barcodes get attached to the genes in each cell, so that scientists can sequence the genes all in one batch and still trace each gene back to the cell it came from.
Macosko and Klein make their beads in different ways. The droplets get broken up at different steps in the process. Other aspects of the chemistry diverge. But the result is the same.
After running a single batch of cells through Drop-seq or inDrops, scientists "can see which genes are expressed in the entire sample—and can sort by each individual cell," said Klein.
They can then use computer software to uncover patterns in the mix, including which cells have similar gene expression profiles. That provides a way to classify what cell types were in the original tissue—and to possibly discover new ones.
Current methods allow researchers to generate 96 single-cell expression profiles in a day for several thousand dollars. Drop-seq, by comparison, enables 10,000 profiles a day for 6.5 cents each.
"If you're a biologist with an interesting question in mind, this approach could shine a light on the problem without bankrupting you," said Macosko. "It finally makes gene expression profiling on a cell-by-cell level tractable and accessible. I think it's something biologists in a lot of fields will want to use."
Rather than competing with each other, the teams believe that having two options available in Drop-seq and inDrops will benefit the scientific community.
"Each method has unique elements that makes it better for different applications. Biologists will be able to choose which one is most appropriate for them," said Macosko.
Different goals
McCarroll, Macosko and their colleagues are excited to explore the brain with Drop-seq.
With luck, that will include discovering new cell types, constructing a global architecture of those cell types in the brain and understanding brain development and function as they relate to disease.
Among the questions they want to pursue are: What are all the cell types that make the brain work? How do these cell types vary in their functions and responses to stimuli? What cell populations are missing or malfunctioning in schizophrenia, autism and other disorders of the brain?
Classifying cell types may not sound exciting, said Joshua Sanes, the Jeff C. Tarr Professor of Molecular and Cellular Biology and the Paul J. Finnegan Family Director of the Center for Brain Science at Harvard University and a co-author of the Drop-seq paper, but it lays the foundation for mapping neuronal circuits and one day being able to probe the mystery of how the "wetware" of the brain gives rise to thoughts, emotions and behaviors.
In the shorter term, Sanes looks forward to completing a catalog of cell types in the mouse retina. Drop-seq has already revealed several new ones.
Kirschner, Klein and their colleagues, meanwhile, are keenly interested in other areas, including stem cell development.
"Does a population of cells that we initially think is uniform actually have some substructure?" Klein wants to know; he's trying to find out by studying immune cells and different kinds of adult stem cells. "What is the nature of an early developing stem cell? What endows those cells with a pluripotent state? Is gene expression more plastic or does it have a well-defined state that's different from a more mature cell? How is its fate determined?"
Using inDrops, Klein and team have confirmed prior findings that suggest even embryonic stem cells are not uniform. They found previously undiscovered cell types in the population they studied, as well as cells in intermediate stages that they suspect are converting from one type to another.
Although both teams are excited by the massive amounts of data they and other researchers will obtain from Drop-seq and inDrops, they realize the sheer volume of information poses a problem as well.
"We have thousands of cells expressing tens of thousands of genes. We can't look in 20,000 directions to pick out interesting features," said Klein.
Machine learning is able to do some of that, and the teams have already employed new statistical techniques. Still, Kirschner has called on mathematicians and computer scientists to develop new ideas about how to analyze and extract useful information about our biology from the mountains of data that are on the horizon.
Financial disclosures and funding information
Allon Klein, Linas Mazutis, Ilke Akartuna, David Weitz and Mark Kirschner have submitted patent applications (US62/065,348, US62/066,188, US62/072,944) for the work described.
A patent application has also been filed for the work described by Macosko et al.
The Kirschner lab's study was supported by the National Institutes of Health (SCAP Grant R21DK098818), a Career Award at the Scientific Interface from the Burroughs-Wellcome Fund, and a Marie Curie International Outgoing Fellowship (300121).
The McCarroll lab's work was supported by the Stanley Center for Psychiatric Research, the Simons Foundation, the National Institutes of Health (P50HG006193, U01MH105960, R25MH094612, F32HD075541), the Klarman Cell Observatory, a Stewart Trust Fellows Award and the Howard Hughes Medical Institute.
Microfluidic device fabrication was performed at the Harvard Center for Nanoscale Systems, a member of the National Nanotechnology Infrastructure Network, with support from the National Science Foundation and the Harvard Materials Research Science and Engineering Center.
Splitting Hair Cells
Tiny hair cells in the inner ear play an outsized role.
For balance, five separate patches of hair cells sense movement and tell the brain where the head is in space while translating the pull of gravity.
For hearing, a five cell-wide ribbon of 16,000 hair cells spirals inside the cochlea, the snail-shaped structure where hair cells vibrate in response to sound waves. Every cycle of sound waves sends microscopic cilia on the tips of these cells back and forth, riding a trampoline of cells suspended between two fluid-filled spaces.
The movement opens pores in the cells, allowing electrical current to flow inside. This conversion of mechanical to electrical signals sends nerve impulses to the brain, which then "hears" the sound.
David Corey, the Bertarelli Professor of Translational Medical Science at Harvard Medical School and a Howard Hughes Medical Institute investigator, has spent his scientific life studying this mechanosensory apparatus, asking which proteins are involved in converting sounds into nerve impulses. So far, only about one-third of those proteins are known. Déborah Scheffer, HMS research associate in the Corey lab, is interested in what makes hair cells different from the cells that surround them in the inner ear.
Their most recent work, published in The Journal of Neuroscience, has revealed that many genes implicated in hereditary deafness are much more active in hair cells than in surrounding cells. This suggests that other genes that produce proteins only in hair cells might also cause inherited deafness. About 1 in 1,000 children are born deaf, and mutations in as many as 300 different genes might cause deafness.
Their findings may also have implications for age-related hearing loss, which affects about half of adults aged 75 or older. Sometimes impairment occurs much earlier, after exposure to harmful amounts of noise.
"This work gives us a parts list that the hair cell uses to assemble different components, helping us figure out the molecular mechanism of sensing sound," Corey said. "It also tells us which genes drive the unique development of a hair cell, raising the hope that this information can be used to create new hair cells to restore hearing in cases of age- or noise-related hearing loss."
To understand the hair cells of the inner ear, Corey and Scheffer collaborated with colleagues Jun Shen, HMS instructor in pathology at Brigham and Women's Hospital, and Zheng-Yi Chen, associate professor of otolaryngology at Massachusetts Eye and Ear, to find out which genes hair cells use that neighboring cells do not. What makes one cell different from another is the choice and the timing of the genes expressed by a cell.
Working in mice engineered to make green fluorescent hair cells, Scheffer devised a way to separately purify hair cells and surrounding cells at different points over about two weeks of mouse inner-ear development. At each point and for each sample, she sequenced the RNA used to make proteins for all 20,000 genes in the mouse genome.
"Now we have a panel of all the genes that are involved in hair cell development," Scheffer said.
Scheffer and Corey deposited their data in a publicly available database that Shen established three years ago. The Shared Harvard Inner Ear Laboratory Database, or SHIELD, holds gene expression data integrated with comprehensive annotation, including potential locations for deafness genes. Scientists from around the world access the data more than 400 times a day.
Some scientists interested in the molecular biology of hearing and deafness can use SHIELD to identify new deafness genes, which may lead to specific gene therapies. Others want to know what makes a hair cell a hair cell, so they can find a way to make surrounding cells in the inner ear turn into hair cells. These cells do not normally divide, so once they are lost, the only hope is to somehow induce them to divide or to turn neighboring cells into hair cells.
Next, Scheffer will explore microRNA expression in hair cells to see which genes they regulate. That will yield a genetic network of gene expression, messenger RNA and protein production.
"I want to know all the genes that interact with each other and what transcription factors are involved at each step," she said.
Corey said their work is only the foundation.
"Someday this work will go to the clinic, but first you have to know the parts list."
This research was supported by National Institutes of Health grants R01-DC000304, R01-DC002281, R03-DC013866 and R01-DC006908; the Frederick and Ines Yeatts Hair Cell Regeneration Grant; P30 DC05209 to the Eaton-Peabody Laboratory of Massachusetts Eye and Ear and a Hearing Health Foundation Emerging Research Grant.
Evolutionary Relic
Pseudogenes, a subclass of long noncoding RNA (lncRNA) that developed from the human genome's 20,000 protein-coding genes but has lost the ability to produce proteins, have long been considered nothing more than genomic "junk."
Yet the retention of these 20,000 mysterious remnants during evolution suggests that they may in fact possess biological functions and contribute to the development of disease.
Now, a team led by investigators at Harvard Medical School and the Cancer Center at Beth Israel Deaconess Medical Center has provided some of the first evidence that one of these noncoding "evolutionary relics" actually has a role in causing cancer.
In a new study published in the journal Cell on April 2, the scientists report that, independent of any other mutations, abnormal amounts of the BRAF pseudogene led to the development of an aggressive lymphoma-like disease in a mouse model, a discovery suggesting that pseudogenes may play a primary role in a variety of diseases.
The new discovery also suggests that with the addition of this vast "dark matter" the functional genome could be tremendously larger than previously thought—three or four times its current known size.
"Our mouse model of the BRAF pseudogene developed cancer as rapidly and aggressively as it would if you were to express the protein-coding BRAF oncogene," explained senior author Pier Paolo Pandolfi, the HMS George C. Reisman Professor of Medicine and co-founder of the Institute for RNA Medicine in the Cancer Center at Beth Israel Deaconess.
"It's remarkable that this very aggressive phenotype, resembling human diffuse large B-cell lymphoma, was driven by a piece of so-called 'junk RNA,'" he said.
"In the past, we have found noncoding RNA to be overexpressed, or misexpressed, but because no one knew what to do with this information, it was swept under the carpet. Now we can see that it plays a vital role. We have to study this material, we have to sequence it and we have to take advantage of the tremendous opportunity that it offers for cancer therapy," Pandolfi said.
Competing endogenous RNAs
The new discovery hinges on the concept of competing endogenous RNAs (ceRNA), a functional capability for pseudogenes first described by Pandolfi almost five years ago when his laboratory discovered that pseudogenes and other noncoding RNAs could act as decoys to divert and sequester tiny pieces of RNA, known as microRNAs, away from their protein-coding counterparts to regulate gene expression.
In this new paper, the authors wanted to determine whether this same ceRNA "cross talk" took place in a living organism and whether it would result in similar consequences.
"We conducted a proof-of-principle experiment using the BRAF pseudogene," explained first author Florian Karreth, who conducted this work as a postdoctoral fellow in the Pandolfi laboratory.
"We investigated whether this pseudogene exerts critical functions in the context of a whole organism and whether its disruption contributes to the development of disease," Karreth said.
The investigators focused on the BRAF pseudogene because of its potential ability to regulate the levels of the BRAF protein, a well-known proto-oncogene linked to numerous types of cancer.
In addition, said Karreth, the BRAF pseudogene is known to exist in both humans and mice.
The investigators began by testing the BRAF pseudogene in tissue culture. Their findings demonstrated that when overexpressed, the pseudogene did indeed operate as a microRNA decoy that increased the amounts of the BRAF protein.
This, in turn, stimulated the MAP-kinase signaling cascade, a pathway through which the BRAF protein controls cell proliferation, differentiation and survival and which is commonly found to be hyperactive in cancer.
Aggressive lymphoma-like cancer
When the team went on to create a mouse model in which the BRAF pseudogene was overexpressed, they found that the mice developed an aggressive lymphoma-like cancer.
Similar to their findings in their cell culture experiments, the investigators found that the mice overexpressing the BRAF pseudogene displayed higher levels of the BRAF protein and hyperactivation of the MAP kinase pathway, which suggests that this axis is indeed critical to cancer development.
They confirmed this by inhibiting the MAP kinase pathway with a drug that dramatically reduced the ability of cancer cells to infiltrate the liver in transplantation experiments.
The Pandolfi team further validated the microRNA decoy function of the BRAF pseudogene by creating two additional transgenic mice, one overexpressing the front half of the BRAF pseudogene and the other overexpressing the back half.
Both of these mouse models developed the same lymphoma phenotype as the mice overexpressing the full-length pseudogene, a result that the authors described as "absolutely astonishing."
The investigators also found that the BRAF pseudogene is overexpressed in human B-cell lymphomas and that the genomic region containing the BRAF pseudogene is commonly amplified in a variety of human cancers. Moreover, the authors said, silencing the BRAF pseudogene in human cancer cell lines that expressed higher levels led to reduced cell proliferation, a finding that highlights the importance of the pseudogene in these cancers and suggests that a therapy that reduces BRAF pseudogene levels may be beneficial in cancer patients.
This work was supported, in part, by the National Institutes of Health (CA170158-01), the Department of Defense Prostate Cancer Research Program, the American Cancer Society, the German National Academy of Sciences Leopoldina, the Italian Association for Cancer Research, the International Association for Cancer Research, Cancer Research UK and the Wellcome Trust.
Adapted from a Beth Israel Deaconess news release.
Personal Genetics and the Law
The need for a better understanding of personal genetics has never been more urgent. That was the message an expert panel of speakers relayed in a Congressional briefing on the intersection of personal genetics and law enforcement.
"There is no time to lose," said Lauren Tomaselli, director of curriculum and training for the Personal Genetics Education Project (pgEd) at Harvard Medical School, citing a recent appeal to the Supreme Court on a ruling that allows a person's DNA to be collected and tested without their knowledge or permission. The case was declined by the court.
The pgEd leaders organized the March 19 briefing on Capitol Hill in cooperation with the offices of U.S. Rep. Louise Slaughter, D-N.Y., and Sen. Elizabeth Warren, D-Mass.
The mission of pgED is to educate young people through school programs and to accelerate public awareness of genetics issues by advising the entertainment industry. It also seeks to engage lawmakers—the "eyes and ears of the nation"— in discussions.
pgEd takes no position on policy, preferring to educate from a neutral position so that its audience can make better-informed decisions.
At the briefing, Duana Fullwiley, associate professor of anthropology at Stanford University, said in some cases genetic technologies that are being utilized by the U.S. criminal justice system are leapfrogging not just public understanding but also peer-reviewed scientific evaluation.
One case in point: DNA phenotyping, a tool that generates the image of a human face based on genetic samples that have been taken from a crime scene.
Police in Columbia, South Carolina, recently relied on such an image provided by Parabon NanoLabs as they searched for suspects in a double murder.
The science behind this service, called Snapshot, has not been analyzed by people outside the company, Fullwiley said.
She criticized the database on which Snapshot is based for two reasons, saying it skews toward an over-representation of African-Americans and its results offer a false sense of precision.
Focusing on a single type of suspect can implicate a whole group, she said, citing the generic image of a young man with dark hair, eyes and skin.
"When, as a society, we are already dealing with racial bias in policing and civil rights, we have to be very careful about rolling out technologies that can potentially have racial impacts that are disparate for different groups," she said.
David Kaye, associate dean for research at Penn State Law, said DNA screening in criminal investigations is often racially based because it relies on witness accounts.
He asked, "If you use the information at your disposal, is it truly discriminatory?"
Courts have also allowed involuntary collection of genetic samples, even through subterfuge, he said.
For example, he said detectives duped a suspect into replying to a letter that offered money via a class-action suit. DNA that was recovered from the paper form was returned to a fabricated law office created by the detectives. In another instance, he said a case was built against a serial killer based on DNA retrieved from his daughter's Pap smear.
The Microbe Question
Claire Fraser, director of the Institute for Genome Sciences at the University of Maryland School of Medicine, explained how microbial DNA might one day be used for forensic purposes. Her past work identified genetic mutations in anthrax spores in the deadly 2001 anthrax mailing. That laid the foundation for the new field of microbial forensics.
"Mother Nature is the best bioterrorist," she said, using SARS, Ebola and West Nile virus as examples.
The microbes we carry with us, collectively known as our microbiomes, could potentially be used as identifiers, she said, but added that that day is far in the future.
Henry Greely, director of the Center for Law and the Biosciences at Stanford Law School, said he worries about the ethnic disproportion in the database of 11 million records now held by federal and state law enforcement.
"There is a much higher chance for a black American than a white American" to be implicated by a family member's DNA sample, he said. "That's troubling."
While it would be politically difficult, Greely said, he would prefer to see a system in which all Americans would have their samples included in a federal database, making it more representative of the nation. He did concede that privacy could be a problem. If privacy were breached, he said, public trust in law enforcement and in genetics would suffer.
Genetic Privacy Rights
Slaughter is a longtime champion of genetic privacy, having sponsored a bill that in 2000 became the Genetic Information Nondiscrimination Act, also known as GINA. She was introduced at the briefing as "the only microbiologist in Congress."
"GINA was all about privacy," she said, recalling the battle for its passage. "We wanted to make sure that the social policy kept up with science, but science fiction intervened. Everybody thought we were talking about cloning."
Protecting genetic information in the workplace and for insurance purposes is still an urgent issue, Slaughter said.
"Your genetics belongs to you and the information is yours," she said to applause from the audience, which included congressional staffers as well as people from the U.S. Department of Justice, the FBI, the National Institutes of Health, the American Society of Human Genetics and the American Association for the Advancement of Science.
In the discussion that followed the speakers' presentations, Ting Wu, HMS professor of genetics and a founder of pgEd, asked if somehow racial discrimination could be minimized.
"Obviously it's a problem," she said. "We can think of Ferguson and see where that goes."
Wu, who founded pgEd in 2006, said she feels a deep responsibility to educate people about genetics. She has said it's not a choice but a necessity.
In an interview after the briefing, George Church, the Robert Winthrop Professor of Genetics at HMS, raised the issue of "DNA exceptionalism," in which genetic tools are seen as different from other modalities, and not just in jurisprudence.
In medicine, for example, gene therapy is viewed as an extraordinary category of treatment.
The pace of public understanding and scientific advancement are not moving in step, he said.
"We have a long way to go, but that's because genetics is a moving target," Church said.
Samantha Schilit, a pgEd affiliate and a graduate student in genetics, said she hopes to pursue personal genetics as a genetic counselor. She attended the briefing as a guest of pgEd after winning a contest in her department to add the most pins to Map-Ed, an online quiz on key concepts and topics in genetics.
"What shocked me is how truly new these topics are," she said, citing the DNA phenotyping news from South Carolina in February.
Schilit said she is uneasy about the possibility that information gathered by a direct-to-consumer company, for example, could find its way into a forensic investigation, a possibility that was raised by Greely.
"These issues are ethically complicated," she said. "This field is moving so fast."
The briefing was the third of five planned by pgEd. The first briefing highlighted the science of genomics, personalized medicine and genetic engineering as well as ways to reach out to the public. The second briefing focused on two topics: the role of genetics research in the unfolding Ebola outbreak in West Africa and the issues addressed by GINA. The third briefing on law enforcement grew out of topics touched on in the first two.
pgEd is supported by the HMS Department of Genetics and private funding from Sigma-Aldrich, Autodesk, Genentech, IDT (targeted specifically for GETed conferences and Map-Ed), and an anonymous donor.
NSAIDs and Cancer Risk
An analysis of genetic and lifestyle data from 10 large epidemiologic studies has confirmed that regular use of aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs) appears to reduce the risk of colorectal cancer in most individuals.
The study, published in JAMA, also found that a few individuals with rare genetic variants do not share this benefit. Additional questions need to be answered before preventive treatment with these medications can be recommended for anyone, the study authors cautioned.
"Previous studies, including randomized trials, demonstrated that NSAIDS, particularly aspirin, protect against the development of colorectal cancer, but it remains unclear whether an individual's genetic makeup might influence that benefit," said Andrew Chan, HMS associate professor of medicine at Massachusetts General Hospital and co-senior author of the JAMA report. "Since these drugs are known to have serious side effects—especially gastrointestinal bleeding—determining whether certain subsets of the population might not benefit is important for our ability to tailor recommendations for individual patients."
The research team analyzed data from the Colon Cancer Family Registry and from nine studies included in the Genetics and Epidemiology of Colorectal Cancer Consortium, which includes the Nurses' Health Study, the Health Professionals Follow-up Study and the Women's Health Initiative. They compared genetic data for 8,624 individuals who developed colorectal cancer with genetic data for 8,553 individuals who did not, matched for factors such as age and gender.
The comprehensive information on lifestyle and general health data provided by participants in the studies again confirmed that regular use of aspirin or NSAIDs was associated with a 30 percent reduction in colorectal cancer risk for most individuals. However, that preventive benefit did not apply to everyone. The study found no risk reduction in participants with relatively uncommon variants in genes on chromosome 12 and chromosome 15.
"Determining whether an individual should adopt this preventive strategy is complicated, and currently the decision needs to balance one's personal risk for cancer against concerns about internal bleeding and other side effects," Chan said. "This study suggests that adding information about one's genetic profile might help in making that decision. However, it is premature to recommend genetic screening to guide clinical care, since our findings need to be validated in other populations. An equally important question that also needs to be investigated is whether there are genetic influences on the likelihood that someone might be harmed by treatment with aspirin and NSAIDs."
Support for this study includes several grants from the National Cancer Institute and the National Institute of Diabetes and Digestive and Kidney Diseases.
Adapted from a Mass General news release.
Rett Syndrome Revelation
Scientists have known for 15 years that mutations in a single gene lead to Rett syndrome, a severe neurological disorder that affects girls around their first birthdays. In the years since the MECP2 gene was pinpointed, researchers have struggled to understand how it functions in the brain in Rett syndrome.
Now the enigma of Rett syndrome and perhaps other disorders on the autism spectrum could be one step closer to being solved.
A Harvard Medical School team has discovered that when MECP2 is mutated in Rett syndrome, the brain loses its ability to regulate genes that are unusually long. Their finding suggests new ways to consider reversing the intellectual and physical debilitation this disruption causes with a drug that could potentially target this error. The team, led by Michael Greenberg, reported its findings in Nature.
"The longer the gene, the more disrupted it becomes when you lose MECP2," said Greenberg, the Nathan Marsh Pusey Professor of Neurobiology at HMS. "Rett syndrome may be a defect in this process of fine-tuning the expression of long genes."
Scientists, including Greenberg, have figured out over the last 10 years that MECP2 plays a role in sculpting the connections between neurons in the developing brain. These synapses are refined by exposure to sensory experiences, just the sort of stimulation a one-year-old would encounter as she learns to walk and talk.
MECP2 is present in all cells in the body, but when the brain is forming and maturing its synapses in response to sensory input, MECP2 levels in the brain are almost 10 times as high as in other parts of the body. The new study connects MECP2 mutations to long genes, which may be more prone to errors simply because their length leaves more room for mistakes.
Speed Bump
"Normally, MECP2 may act like a speed bump, fine-tuning long genes by slowing down the machinery that transcribes long genes," said Harrison Gabel, a postdoctoral fellow in the Greenberg lab and co-first author of the Nature paper. In transcription, the information in a strand of DNA is copied onto a new molecule of messenger RNA, which is then turned into a protein. "Without MECP2, the machinery may be moving too fast, making too much mRNA from these genes, resulting in problems for the neurons."
Finding this effect of MECP2 on long genes was no small feat. In a typical search for the mechanism behind a genetic mutation, mice are engineered to lack the normal gene so that its absence reveals how it functions. However, work in many different labs has shown that knocking out MECP2 had only subtle effects when analyzed across the genome. The changes in gene expression were inconsistent, small and, using Gabel's word, "fuzzy."
Gabel took another approach, querying massive genomic databases such as ENCODE to ask a simple question: What do genes that are affected by mutated MECP2 have in common?
Answer: They are long. Most of them are at least five times longer than the average gene, with many of them more than 50 times longer than the average. It is important to note that the genes identified across dozens of data sets were very long, giving the researchers a common finding where previous conclusions from these data sets had lacked a common theme.
Harrison and co-first author Benyam Kinde, an MD-PhD student in the Greenberg lab, found the long-gene misregulation in multiple mouse models of Rett syndrome and confirmed it in the brain tissue of deceased Rett patients.
For MECP2 to function normally as a speed bump, it binds to a form of methylated DNA found in long genes in the brain. Methyl groups are chemical modifiers of gene activity, and in other parts of the body MECP2 binds methylated CG sites on genes. The methylation pattern that appears to be important for MECP2 in regulating long genes is known as methylated CA, and there appears to be a special mechanism operating as synapses are forming.
"It seems that evolution has used MECP2 and methylated CA to put in place this speed bump so that the expression of long genes is restrained in the brain," Greenberg said. "As far as Rett syndrome, the thought is now that this subtle but widespread overexpression of long genes might be contributing to the disorder."
Corrective Strategy
The scientists can't be sure of what these overexpressed long genes do, but many of them appear to be very important to the function of the brain. This suggests that if they could correct the defect in long-gene expression, they might be able to reverse at least some of the symptoms of Rett syndrome. As a first attempt at a corrective strategy, the researchers selected a cancer drug called topotecan because it blocks an enzyme known to be important for long-gene transcription.
In a lab dish, they added topotecan to neurons lacking MECP2. The drug reversed the long-gene misregulation, suggesting that restoring normal long-gene expression might be a way to correct neurological dysfunction in Rett syndrome and in other autism spectrum disorders with long genes, such as fragile X syndrome. Topotecan, a chemotherapeutic agent, is too toxic, Greenberg said, but derivatives of topotecan might be a worthwhile avenue to pursue.
"We think this issue of long-gene misregulation may be more generally occurring in other disorders of human cognition," Greenberg said. "The potential is pretty significant because one now has a common regulatory mechanism to target with drugs."
This work was supported by grants from the Rett Syndrome Research Trust and the National Institutes of Health (1RO1NS048276 and T32GM007753), the Damon Runyon Cancer Research Foundation (DRG-2048-10), the William Randolf Hearst fund and the Howard Hughes Medical Institute.
Variety Show
Researchers have begun to appreciate the importance of copy number variation when considering the connections between DNA and disease.
Most people have two copies of most genes. But some have only one copy, or three, or none. There have been hints that copy number variation (CNV) might range much more widely than zero to three, but such extremes have been hard to analyze in gene sequencing data.
"For all the excitement about copy number variation in human genetics, most earlier research has been limited to the simplest form of CNV, in which you have either a missing segment or an extra copy of it," said Steven McCarroll, assistant professor of genetics at Harvard Medical School and director of genetics for the Stanley Center for Psychiatric Research at the Broad Institute of MIT and Harvard.
"Here we came up with a way to analyze extreme forms of CNV," he said. "Now we can start to use this exuberant form of genetic variation to help illuminate the genetic basis of disease."
McCarroll and colleagues reported their insights about extreme CNV in Nature Genetics on Jan. 26. Their discoveries were made possible by new computational techniques that first author Bob Handsaker developed to analyze whole-genome sequence data from thousands of genomes at once.
"Before, we had no good way to study genes that have a really high copy number, above four," said Handsaker, a research scientist in the McCarroll lab. "Now we can find places where people's gene copy number ranges from zero to 15. It's the first time we've been able to measure this kind of variation with such precision."
"We've found that in hundreds of genes, there's a wide variation in copy numbers. Now that we can measure these variations accurately, we can ask whether there are health repercussions," said Handsaker.
The results also enrich the understanding of human genome evolution, said McCarroll.
Once they had developed a way to study extreme CNV, Handsaker, McCarroll and their team made four primary discoveries.
First: About 88 percent of gene copy number variation among humans arises from extreme copy number variants rather than simple copy number variants.
"These extreme copy number variants are a small fraction of all CNVs, but they have broader effects on genes than we anticipated," said McCarroll.
Second: The more copies of a gene a person has, the more that gene is expressed.
"You might think this was obvious," said Handsaker, "but in some organisms, such as plants, when you have more copies, most of them are turned off. It turns out that in humans, they're all turned on in almost all cases."
Third: With simple CNV, most people have two copies, while a few outliers have one or three or none. McCarroll's team found that with extreme CNV, most people don't have two copies but instead have CNVs scattered across a wide range.
"For a lot of these CNVs with these especially exuberant differences, two randomly chosen people are actually more likely to have different numbers of copies than the same number," said Handsaker.
Fourth: Sequences with more copies are more likely to mutate further, expanding in copy number quickly and dramatically.
The team found what they call "runaway duplication haplotypes," in which some versions of a chromosome have acquired as many as 10 copies of a gene over the past thousand or so generations, while other versions of the same chromosome continue to have just one copy.
"The fast, dramatic expansion in copy number of specific genes appears to have been evolutionarily recent and geographically localized," said McCarroll.
One gene involved in resistance to trypanosomes—parasites that cause human illnesses including sleeping sickness and Chagas disease—evolved to have a high copy number on a subset of the chromosomes in West African populations. Another gene, related to a gene that contributes to asthma resistance, evolved to have a high copy number in Europe.
"These variations show really unusual patterns in some parts of the world," said McCarroll. "But it's too soon to know whether they're doing something important."
The team is now offering to the research community "the first data resource on extreme forms of CNV and how they actually vary across a large number of people" as well as a software toolkit to analyze extreme CNV in huge sequencing data sets, McCarroll said.
"Until recently, whole-genome sequencing was quite expensive. Today, that's changing quickly," McCarroll added. "This work gives us a sense of the kinds of things it's going to be possible to see in whole-genome sequences that it wasn't possible to see before."
Coauthor Jennifer R. Berman is an employee of Bio-Rad Inc.
This research was supported by National Human Genome Research Institute grant R01 HG006855. Additional funding from NHGRI (U01 HG006510) is supporting follow-on work to develop production-ready software that can be used by any research laboratory.
No Escape
The creation of genetically modified and entirely synthetic organisms continues to generate excitement as well as worry.
Such organisms are already churning out insulin and other drug ingredients, helping produce biofuels, teaching scientists about human disease and improving fishing and agriculture. While the risks can be exaggerated to frightening effect, modified organisms do have the potential to upset natural ecosystems if they were to escape.
Physical containment isn't enough. Lab dishes and industrial vats can break; workers can go home with inadvertently contaminated clothes. And some organisms are meant for use in open environments, such as mosquitoes that can't spread malaria.
So attention turns to biocontainment: building in biological safeguards to prevent modified organisms from surviving where they're not meant to. To do so, geneticists and synthetic biologists find themselves taking a cue from safety engineers.
"If you make a chemical that's potentially explosive, you put stabilizers in it. If you build a car, you put in seat belts and airbags," said George Church, Robert Winthrop Professor of Genetics at Harvard Medical School and core faculty member at the Wyss Institute.
And if you've created the world's first genomically recoded organism, a strain of Escherichia coli with a radically changed genome, as Church's group announced in 2013, you make its life dependent on something only you can supply.
Church and colleagues report Jan. 21 in Nature that they further modified their 2013 E. coli to incorporate a synthetic amino acid in many places throughout their genomes. Without this amino acid, the bacteria can't perform the vital job of translating their RNA into properly folded proteins.
The E. coli can't make this unnatural amino acid themselves or find it anywhere in the wild; they have to eat it in specially cooked-up lab cultures.
A separate team reports in Nature that it was able to engineer the same strain of E. coli to become dependent on a synthetic amino acid using different methods. That group was led by a longtime collaborator of Church's, Farren Isaacs of Yale University.
The two studies are the first to use synthetic nutrient dependency as a biocontainment strategy, and suggest that it might be useful for making genetically modified organisms safer in an open environment.
In addition, "We now have the first example of genome-scale engineering rather than gene editing or genome copying," said Church. "This is the most radically altered genome to date in terms of genome function. We have not only a new code, but also a new amino acid, and the organism is totally dependent on it."
Church's team, led by first authors Dan Mandell and Marc Lajoie, HMS research fellows in genetics, also made the E. coli resistant to two viruses, with plans to expand that list.
The modifications offer theoretically safer E. coli strains that could be used in biotechnology applications with less fear that they will be contaminated by viruses, which can be financially disastrous, or cause ecological trouble if they spill. (E. coli is one of the main organisms used in industry.)
Hooked on amino acids
Scientists have been exploring two main biocontainment methods, but each has weaknesses. Church was determined to fix them.
One method involves turning normally self-sufficient organisms like E. coli into auxotrophs, which can't make certain nutrients they need for growth. Humans are auxotrophs, which is why we need to include vitamins and other "essential" nutrients in our diets.
Altering the genetics of E. coli so they can't make a naturally occurring nutrient doesn't always work, said Church, because some of them manage to scavenge the nutrient from their surroundings. He lowered that risk by making the E. coli dependent on a nutrient not found in nature.
Another pitfall of making auxotrophs is that some E. coli could evolve a way to synthesize the nutrient they need. Or they could acquire the ability while exchanging bits of DNA with other E. coli in a process called horizontal gene transfer.
Church believes his team protected against those possibilities because it had to make 49 genetic changes to the E. coli to make them dependent on the artificial nutrient. The chance one of the bacteria could randomly undo all of those changes without also acquiring a harmful mutation, he said, is incredibly slim.
Church's solution also took care of concerns he had with another biocontainment technique, in which genetic "kill switches" make bacteria vulnerable to a toxin so spills can be quickly neutralized. "All you have to do to kill a kill switch is turn it off," which can be done in any number of ways, Church said. Routing around the dependency on the artificial amino acid is much harder.
Church determined that another key to making a successful "synthetic auxotroph" was to ensure that the E. coli's lives depended on the artificial amino acid. Otherwise, escaped E. coli could keep rolling along even if they couldn't make or scavenge it. So his group targeted proteins that drive the essential functions of the cell.
"If you put it off on the periphery, like on the paint job of your car, the car will still run," he explained. "You have to embed the dependency smack in the middle of the engine, like the crank shaft, so it now has a particular part you can only get from, say, one manufacturer in Europe."
Building a safer bacterium
The need to choose a process essential to E. coli survival and a nutrient not found in nature "limited us to a small number of genes," Church said. His team used computational tools to design proteins that might cause the desired "irreversible, inescapable dependency." They took the best candidates, synthesized them and tested them in actual E. coli.
They ended up with three successful redesigned essential proteins and two dependent E. coli strains. "Using three proteins together is more powerful than using them separately," Church said. He envisions future E. coli modified to require even more synthetic amino acids to make escape virtually impossible.
As it was, the escape rate—the number of E. coli able to survive without being fed the synthetic amino acid—was "so low we couldn't detect it," Church said.
The group grew a total of 1 trillion E. coli cells from various experiments, and after two weeks none had escaped. "That's 10,000 times better than the National Institutes of Health's recommendation for escape rate for genetically modified organisms," said Church.
The weaknesses in Church's methods remain to be seen. For now, he is satisfied with the results his group has obtained by pushing the limits of available testing.
"As part of our dedication to safety engineering in biology, we're trying to get better at creating physically contained test systems to develop something that eventually will be so biologically contained that we won't need physical containment anymore," said Church.
In the meantime, he said, "we can use the physical containment to debug it and make sure it actually works."
This work was funded by the U.S. Department of Energy (grant DE-FG02-02ER63445).


text:
La Terrasse
Crib Museum
The nativity-scene spectacle at the Crib Museum is a short walk off Manger Sq.
Old Bethlehem Museum
This museum is located in a typical Palestinian home of the 19th century. See native costumes, peruse the collection of...
Church of the Nativity
Though many have argued over whether X (or, in this case, a star) really does mark the spot, the Church of the Nativity nevertheless...
The walls of this tiny locally run bar just outside the Old City are emblazoned with flags from across the world. It serves a range of...
Abu Shanab
Succulent lamb chops and traditionally butchered shish kebab are said to turn vegetarians around, but there's plenty of meatless mezze....
La Terrasse information
Beauty and politics merge in the panoramic view from La Terrasse of Bethlehem and the Jewish settlement of Har Homa. Find a stepped-up Mediterranean-Mexican menu, comfy-chic ambience, a variety of wines and an international clientele. Alfredo comes with mushrooms, unless you decline. The creamy, crunchy almond soup is divine.

subset: commoncrawl


text:
All quotes tagged Understanding
(5376 quotes found)
"We want our conscious focus to entrain our subconscious and unconscious awareness. That is mastery in a nutshell."
"Foolish people can make wise decisions and wise people can make foolish decisions we are human and sinners but we have different mindsets"
"To understand people, is an impossible thing for a man or women to accomplish, but to understand a person is to not only know there greatness of success but there flaws in life. That we not only celebrate our success but we celebrate our flaws in life that we have overcome, to respect each others courage that we have shown in ourselves."
"thanks universe for all I have,success,special persons,what I have seen ,felt and loved,learning nothing is a mistake and goodness melt any badness"
"He wanted to be there with us, but everybody understands the situation and he wishes us the best. He still calls this team part of his family."


text:
September Is Apple Harvesting Time For The Fruit And The Stock 11 commentsJul 26, 2013 3:09 PM | about stocks: AAPL
Apple Inc. (NASDAQ:AAPL) shares have had a bounce since they reported a lower than expected 20 % drop in year over year net income for the June quarter and Tim Cook advised they had something "big" up their sleeve to be announced later. Apple bulls have been vocal about the terrific ecosystem; the high quality products; the massive cash position enhanced by an equally massive stock buy back; and, of course, the dividend. These are all good things.
IDC reports smartphone markets grew 59% in units in the June quarter to a worldwide volume of 238 million and Apple market share slumped to 13%.
LG Electronics, Lenovo and ZTE all expanded market share at enormous rates as did the "Other" category which makes up 42% of the market and (necessarily) includes BlackBerry, Nokia and a handful of others.
The implications for Apple are dire. Apple owned the smart phone market not that long ago and it is following a BlackBerry like trend of shedding share at an enormous rate and keeping an "its not important" demeanour just as many Apple bulls commenting on my recent SA article maintained that Apple's share of the so-called "high end" of the market is what is important.
Smartphones is not the only arena where Apple market share is taking a drubbing. Despite creating the Tablet market, Apple sales of iPads dropped substantially last quarter as the market flattened and consumers opted for more lower priced Android tablets. The total market reported dropped 14% in the June quarter while Apple sales dropped a whopping 25% from its first quarter volume shipping 14.6 million units.
Margins are falling as well and year over year Apple saw sales declines in key markets like China, Europe and Asia Pacific. This is not a trend favourable to Apple investors.
At this point, seven out of eight smart phone users choose a product not made by Apple. Over half of tablet users choose a product not made by Apple. It is starting to sound a lot like the PC market where more than 7 of 8 computer users choose a PC not made by Apple.
New products with new features may be Apple's salvation but they better come quickly. The flattening of markets for tablets suggests this trend has run its course and possibly consumers have all the tablets they want or need and may even upgrade their old laptop now that Intel has introduced processors with longer battery lives. The long battery life of the tablet in my view was one of the major contributors to its success despite its limitations as a computing device.
Ultrabooks are now thinner, lighter and have battery lives (thanks to Haswell and Silvermont) that compare favourably to tablets and offer a full range of computing power that is more compelling than a trip to YouTube or Facebook with a stop at Instagram and a few tweets on Twitter. I am seeing a lot more people at the coffee shop sporting a new laptop than a new iPad.
Fortunately for Apple, many of those new laptops are MacBooks which remain a compelling choice for many and with crisper display and Haswell processors are likely to remain a favourite for many users. iPads, not so much.
In my view, despite its recent torrid pace of growth, the smartphone market is rapidly maturing. With over 1 billion smartphones sold each year it is a refresh cycle rather than initial demand that will determine future growth, and Android assemblers are taking full advantage by offering a relentless array of new phones with new features. A once every year or so refresh by Apple of its iPhone is just not going to cut it in my view.
What does it mean for Apple? Without new products that capture the imagination of consumers - a real possibility given its history, but by no means a certainty - Apple will do well to hold on to the share it has today. If that is the case, you can expect it to become a less profitable and less interesting investment.
The September quarter is likely to be reasonably profitable for Apple if it meets its guidance but is not going to make anyone rich. In my view, it may well be the last decent quarter Apple puts up for a while. That suggests that Apple investors take advantage of the Apple harvesting season not only to fill their baskets with Royal Gala or Granny Smith's to make preserves but also to preserve the value of their investment portfolio by cashing out or hedging with options.
With its current strength, I believe Apple is a better short than long today.
Disclosure: I have no positions in any stocks mentioned, but may initiate a short position in AAPL over the next 72 hours. I wrote this article myself, and it expresses my own opinions. I am not receiving compensation for it (other than from Seeking Alpha). I have no business relationship with any company whose stock is mentioned in this article.Stocks: AAPLBack To Michael Blair's Instablog HomePage »
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Well thought out and brilliant!
Biz Break: Apple continues to cede ground in smartphone battle
Investors have dumped its stock for months now. Techies are demanding it come up with the Next Big iThing. Yet for all the bashing it's taken of late, Apple and its products apparently remain close to Americans' hearts.
Hewlett-Packard (HP) Computers
Dell Computers
Sony Computers
Dear Michael:
I mentioned that non-consumer class Cn citizens were enjoying Apple products.
Apple's Secret Weapon: the iPhone 4
Three-Year-Old Smartphone Propels Company's Sales in Price-Conscious Markets
Guo Bing, a vendor at a computer mall in Beijing's business district, said about 30% of his total sales are still iPhone 4 and 4S smartphones, compared with iPhone 5s, which make up about 50%.
According to most rumors, this will be replaced soon with an iPhone lite:
- same screen size across all product lines (which could later open the road for a new, larger screen size)
- get more lower-end customers since they don't perceive the product as "old"
$aapl - I got this one right http://seekingalpha.com/p/2wiz6 Apr 26, 2016
BlackBerry Ekes Out A Profit Despite Sales Tanking $BBRY http://seekingalpha.com/a/1ncez Dec 20, 2014
Enerplus - Good Management Should Provide Good Rewards To Investors $ERF http://seekingalpha.com/a/1naqp Dec 19, 2014
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text:
Increase SEO and traffic to my website
This project received 42 bids from talented freelancers with an average bid price of $3425 HKD.Get free quotes for a project like this
Skills Required
Project Budget$2000 - $6000 HKD
Total Bids42
Project Description
I just launched a consulting company providing services to European companies dealing with China on one hand and to Chinese company dealing with Europe on the other hand. For now, only the website targeting a Western audience is online : http://oshengconsulting.com.
I am looking to boost the SEO ranking of my website.
The project will include :
- Existing html code optimisation for SEO (website designed using wordpress)
- SEO/Internet Marketing comprehensive program
If you are interested in this project, please provide me with a clear proposition of a SEO/Internet Marketing program you would like to suggest, and the kind of results I should expect of it.
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- Forbes
- The New York Times
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text:
Please engage mind before opening mouth!
That pretty much sums it up Don.
Don't bother Don, the Liberal mind set is to talk without thinking. It is easier for them to go on. It is part of the "ignorance is bliss" lifestyle.
That is funny & i could not resist! Coming from Inbred DON- how would you know anything about mind! You have none.
I do like Donny boy. He never posts anything logical. It's either a personal attack or a fact dump.
it's just a robot lib-tard mouth


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Note: Never assume you are or know who the most important person in a room is. Ever.
Note 2: Never assume the small slice of someone's life you see is ALL there is to their story. Most successful folks play cards privately.
Note 3: Don't feel obligated to justify your decisions or success to anyone. If they need it, they don't matter.
To avoid drama: Live right, be worthy, make a difference.
The good stuff: Cultivate kindness and truth, stop making excuses and LIVE.
Rant over. Happy Independence Day!


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In legal documents obtained by PEOPLE, Soules alleges the dating service used his likeness in advertisements for Farmers Only, which caters to rural singles. While the advertisements did not use Soules' name, the lawsuit alleges it advertised "a bachelor farmer in search of love" who fell for a city girl, a relationship that ended in failure.
The lawsuit claims that Farmers Only capitalized on Soules' time on The Bachelor, which saw the farmer from Iowa propose to Whitney Bischoff, a fertility nurse from Chicago. Their engagement ended in May.
RELATED VIDEO: Who Is The Bachelor's Latest 'It' Girl?
According to the lawsuit, FarmersOnly.com made Soules "identifiable from the combination of a picture with a blacked-out person" as well as a reference to how his relationship with his ex-fianceé from the show failed because he "picked a city girl." The lawsuit claims the ad said that the man would have fared better had he gone to FarmersOnly.com and met a country girl.
Soules is seeking compensation for the alleged use of his image as well as legal fees.


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member for 7 years, 3 months, 1 day
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There is are now virtual cemeteries setup for all
Lycoming Co. Veterans
or click on the 'Lycoming Co Veterans' link under my friends.
stop by and check out and pay a visit to the veterans interned Lycoming County.
More than happy to make corrections and add relationship links to other memorials. Please use the "Edit" tab to submit corrections, additions or transfer request.
Please remember that cemetery listings are primary a historical record, which can be a great help to genealogical research. It is not important who manages the information as much as having it available to the public for research. The listings and/or memorials are not up for grabs based on lineage. Therefore I do not transfers memorials based these. I will continue to transfer memorials within Find A Grave guidelines and those that have been sponsored.
If you are considering asking for a transfer; read the Find A Grave guidelines.
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Lycoming Co Vet...
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Darlene Russell
right cemetery?
Are you sure she is buried in Salem Cem. The ground cover looks like Ellenton Cemetery in Lycoming Co., PA
Added by
Darlene Russell
on Sep 21, 2016 1:30 PM
Dawn Dickens
I sent a correction for DOB thru system and posted as Oct...I typed wrong info. George Gorrie Meek was born April 3 1847 . His half brother John Leonard, both died on same day. Do I send a correction or can you catch this ?
Added by
Dawn Dickens
on Sep 15, 2016 8:47 AM
Dawn Dickens
correction 46097747 edit
my deepest regard to incorrect info sent a few minutes ago. That's Leonard Meek, not John Meek and the screen keeps popping around due to these ads, difficult to type anything. I found the edit key and starting to send corrections, additions.. Lets hold off on a transfer,for now. Lets see where this goes.
Added by
Dawn Dickens
on Sep 14, 2016 10:49 AM
Dawn Dickens
correction 46097747 edit
my deepest regard to incorrect info sent a few minutes ago. That's Leonard Meek, not John Meek and the screen keeps popping around due to these ads, difficult to type anything. I found the edit key and starting to send corrections, additions.. Lets hold off on a transfer,for now. Lets see where this goes.
Added by
Dawn Dickens
on Sep 14, 2016 10:43 AM
Dawn Dickens
correction mem# 46097746
Steve, please correct this obvious one: John Meek. Posted picture clearly states: Death occurred on Dec 13 1855 age 72 years. Please correct dob: to 1783 and DOD: Dec 13 1855. By the way he was born in England and middle name is not Meek, only his last name (listed as John Meek Meek. He is the Senior to a long line. Q: I have a slew of family that needs to be connected as John Meek is my 4X great grandfather. Do you want me to write a BIO or may I request a transfer? Would be my desire to see this all flow for future generations to access. Again...I don't clearly understand what form I need to fill out...or easiest way? thanks for patience: firstname.lastname@example.com
Added by
Dawn Dickens
on Sep 14, 2016 9:21 AM
Ron Singley
Coea Pepperman
Cora Peppererman was not a Longenburger she was a Singley. She was the daughter of Levi Singley and was my grandfathers sister. She was married to Loyed Berminger before she was married to Elmer Pepperman and had a son John Freas Berminger who died at age 5 and a daughter Bessie Berminger. She was my fathers Ant and I new her well. I have a large wall picture Of Cora,Bessie and little John taken just before he died. That hung in her living room.
Ron Singley
Added by
Ron Singley
on Sep 06, 2016 7:26 AM
L. Moore
W. H. Mott
Your Witt Mott should be William H. Mott. Thank you for this correction.
Added by
L. Moore
on Aug 19, 2016 3:03 AM
Fred Winner
Warner W Winner
Please correct the MOTHERS NAME in your note... It should read ELIZABETH CHESLER as evidenced by Quaker burial records I posted ... She is buried in Corning, NY... Awaiting updates on many memorials recendtly posted.
Added by
Fred Winner
on Aug 15, 2016 3:49 PM
Fred Winner
Warner W Winner
Added by
Fred Winner
on Aug 15, 2016 3:47 PM
Bill Aurand
Find A Grave Memorial # 42097005
Hello: I believe that the memorial that you have under the name of --ra Hall is the following person:
Nora Virginia Hall
b. 07 Jun 1903 Hughesville, Lycoming, Pennsylvania
d. Sep 1977 Williamsport, Lycoming, Pennsylvania
She was married to Bernard George Herlt, Find A Grave Memorial # 42097002
Thought you would add the information. Thank You, Bill Aurand, Simi Valley, CA
Added by
Bill Aurand
on Aug 11, 2016 3:39 PM
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27 Reviews
My son's favorite. Great source of protein, good healthy snack anytime.
I too have been eating this since I was little, I put raisins or dried cranberries on top. A great snack for young and old. My DS puts chocolate chips on it, weird with the celery but it works for him.
This takes me back to when I was younger! I still crave this snack sometimes.
My aunt used to make me these when I went over to her house. She also makes celery with cream cheese. Simple and good. Very kid friendly. When I make them, I use less PB.
This is such a great way to get some veggies and protein into my children and grandchildren; this is an appetizer/snack that everybody enjoys. I got lots of good ideas for additons to these that I will use to change it up a bit. Thanks Bone Man!
A favorite snack at our house. We all love this recipe. Hubby has to have Reese's peanut butter or it just doesn't taste right LOL.
This is one of my favorite snacks...I sometimes use cream cheese or velveeta cheese instead. The kids add raisins on theirs and call it ants on a log'
The best way to eat celery--haha! I've made this dozens of times-easy, quick and good. And peanut butter is a great protein fix.
I tried this on a vacation to Canada, and when I saw it, I thought that the idea of celery and peanut butter was strange at first, but when I tried it, delicious! It is a healthy snack with an unique and pleasing flavor. Thank you Bone Man for bringing back good memories.
I'm allergic to peanuts so I made this with Cashew butter and it was quick, easy, and really enjoyable.


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The 5 members of popular Korean girl group 4minute have decided to part ways, thus disbanding after 7 years. The news came out at the beginning of the week. 109 more words
Tags » Gayoon
New trending GIF tagged kpop crazy blackandwhite 4minute...
New trending GIF tagged kpop, crazy, blackandwhite, 4minute, hyuna, gayoon, sohyun, jiyoung, jihun, 4minutecrazy via http://ift.tt/1ZR3l2b
4MINUTE Hate Outfit Inspiration
Since 4MINUTE's Crazy era, they have gathered a lot of fans nailing a fierce concept and for their latest comeback for HATE, they also got the hype for their unique music and choreography. 355 more words
4Minute - Hate (싫어)
네 눈빛이 말해주고 있어
Liar liar liar 더 날 속이지마
이젠 더 이상 알고 싶지 않아
I don't wanna know know know
더는 No no no 다 끝났어 758 more words
Name: 4Minute
Debut: June 18, 2009 (Hot Issue)
Company: Cube Entertainment
Fandom Name: 4NIA (Meaning: 4Minute + Mania)
Social Media: 1,273 more words
4Minute Release Individual Teasers For Future Masterpiece "Hate"
Hyunah and the Hyunaette's 4Minute's next comeback, entitled "Hate," is set to drop on February 1st and the group recently released individual teasers for the group's upcoming assuredly immortal single. 159 more words
4Minute's "Hit Your Heart" review
Artist: 4Minute
Album: "Hit Your Heart"
Label: Cube Entertainment
Year: 2010
Let's be honest here. 4Minute has a lot of hits. They have a lot of songs that are full of bass and could cause you to turn the volumes up. 212 more words


text: Happy Birthday to Me -- And My Mom and Grandma By Scott Gordon UP NEXT XHappy Birthday to Me -- And My Mom and GrandmaLinkEmbedEmailCopyCloseLink to this videohttp://www.nbcdfw.com/news/weird/Happy_Birthday_to_Me_--_And_My_Mom_and_Grandma_Dallas-Fort_Worth.htmlCopyClose Embed this video Replay More videos (1 of 9)«»Three generations of a Cleburne family share the same birthday. The odds of that? 1 in 133,225.Published at 12:30 PM GMT on Apr 2, 2009 NEWSLETTERS Receive the latest local updates in your inboxPrivacy policy | More Newsletters


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The Voyage of the 'Fox' in the Arctic SeasIn 1845, Sir John Franklin set off from England to locate and chart the elusive Northwest Passage. He and his crew of 129 men never returned.Over the following decade, forty expeditions were launched in an effort to establish the fate of the missing men. But it wasn't until 1854 that traces of their demise were discovered along the western shore of King William Island.However, without proof,
Polar Imperative: A History of Arctic Sovereignty in North America
A History of Arctic Sovereignty in North AmericaBased on Shelagh Grant 's groundbreaking archival research and drawing on her reputation as a leading historian in the field, Polar Imperative is a compelling overview of the historical claims of sovereignty over this continent's polar regions. This engaging, timely history examines: the unfolding implications of major climate changes the impact of resource exploitation on the indigenous peoples
$11.99In the early 1950s, an eleven-year-old boy in Colombo boards a ship bound for England. At mealtimes he is seated at the "cat's table"—as far from the Captain's Table as can be—with a ragtag group of "insignificant" adults and two other boys, Cassius and Ramadhin. As the ship makes its way across the Indian Ocean, through the Suez Canal, into the Mediterranean, the boys tumble from one adventure to
Shackleton's Incredible VoyageBound for Antarctica, where polar explorer Ernest Shackleton planned to cross on foot the last uncharted continent, the Endurance set sail from England in August 1914. In January 1915, after battling its way for six weeks through a thousand miles of pack ice and only a day's sail short of its destination, the Endurance became locked in an island of ice. For ten months the ice-moored Endurance
The Girl with the Dragon TattooThe Girl in the Spider's Web, the new book in the Millennium Series, is available now!Murder mystery, family saga, love story, and financial intrigue combine into one satisfyingly complex and entertainingly atmospheric novel.Harriet Vanger, a scion of one of Sweden's wealthiest families disappeared over forty years ago. All these years later, her aged uncle continues to seek the truth. He hires
$9.99Man Booker Prize Finalist 2011Shortlisted for the Scotiabank Giller PrizeShortlisted for the Governor General's Literary Award for FictionBerlin, 1939. The Hot Time Swingers, a popular jazz band, has been forbidden to play by the Nazis. Their young trumpet-player Hieronymus Falk, declared a musical genius by none other than Louis Armstrong, is arrested in a Paris café. He is never heard from again
$6.99From the author of the number one Canadian bestseller The Birth House comes the story of a young girl abandoned to the streets of post-Civil War New York City. "I am Moth, a girl from the lowest part of Chrystie Street, born to a slum-house mystic and the man who broke her heart." Set on the streets of Lower Manhattan in 1871, The Virgin Cure is the story of Moth, a girl abandoned by her
$6.99"A gorgeous, wise, riveting work of, among other things, cowboy noir…Honestly, I can't recall ever being this fond of a pair of psychopaths." —David Wroblewski, New York Times bestselling author of The Story of Edgar Sawtelle "A masterful, hilarious picaresque that keeps company with the best of Charles Portis and Mark Twain…a relentlessly absorbing feat of novelistic art."—Wells Tower, author of
$9.99The circus arrives without warning. No announcements precede it. It is simply there, when yesterday it was not. Within the black-and-white striped canvas tents is an utterly unique experience full of breathtaking amazements. It is called Le Cirque des Rêves, and it is only open at night. But behind the scenes, a fierce competition is underway—a duel between two young magicians, Celia and Marco,
A Novel of Catherine the GreatFrom award-winning author Eva Stachniak comes this passionate novel that tells the epic story of Catherine the Great's improbable rise to power—as seen through the ever-watchful eyes of an all-but-invisible servant close to the throne. Her name is Barbara—in Russian, Varvara. Nimble-witted and attentive, she's allowed into the employ of the Empress Elizabeth, amid the glitter and cruelty of the
$11.99Winner of the 2011 Man Booker PrizeBy an acclaimed writer at the height of his powers, The Sense of an Ending extends a streak of extraordinary books that began with the best-selling Arthur & George and continued with Nothing to Be Frightened Of and, most recently, Pulse. This intense new novel follows a middle-aged man as he contends with a past he has never much thought about—until his closest
$11.99A rare meeting of literary genius: P. D. James, long among the most admired mystery writers of our time, draws the characters of Jane Austen's beloved novel Pride and Prejudice into a tale of murder and emotional mayhem. It is 1803, six years since Elizabeth and Darcy embarked on their life together at Pemberley, Darcy's magnificent estate. Their peaceful, orderly world seems almost unassailable


text: URMC / Medicine Welcome to the Department of Medicine The Department of Medicine is the largest department within the University of Rochester School of Medicine and Dentistry. It is made up of nearly 300 faculty members who are involved in patient care, research and education. Again and again, the Department of Medicine has distinguished itself as both a national and world-leader. Take a moment to read about some of our achievements that are helping to move all of medicine forward. News & Events Sep 28, 2016URMC Researchers Discover Rare Flu-Thwarting Mutation Sep 20, 2016UR Medicine Cardiac Surgeons Perform 200th Heart TransplantSep 09, 2016Wilmot Cancer Institute to celebrate cancer survivorship at fourth annual Wilmot Warrior Walk


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400 US personnel and equipment would continue to flow into Turkey over next several days by US military airlift.
ANKARA -- US personnel and equipment began arriving at Incirlik Air Base to support NATO's Patriot battery deployment to Turkey, said the US Embassy in Ankara, the Turkish capital.
In a press release, the embassy said approximately 400 US personnel and equipment would continue to flow into Turkey over the next several days by US military airlift.
"The troops will man two Patriot batteries supporting NATO's mission there. Additional equipment will be moved by sea, arriving later this month," the embassy said.
The deployment of six Patriot batteries, including two each from Germany and the Netherlands, is in response to Turkey's request to NATO against possible missile threat from Syria.
"The forces will augment Turkey's air defense capabilities and contribute to the de-escalation of the crisis along the Alliance's border," the embassy said.
"Turkey is an important NATO ally and we welcome the opportunity to support the Turkish government's request in accordance with the NATO standing defense plan," the embassy quoted Navy Vice Adm. Charles Martoglio, US European Command deputy commander, as saying.
The duration of the deployment will be determined by the contributing nations in coordination with Turkey and NATO, the embassy said.
US Personnel, Patriot Missile Equipment Begin Arriving in Turkey
January 5, 2013
Source: Anadolu Agency
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This would truly be an historic, revolutionary and visionary enterprise empowering consumers as personal generators saving the planet one radioactive rod at a time.
Blind Faith•June 15, 2016 • Leave a Comment
Addiction in Indian Country•June 1, 2016 • 1 Comment
Meth epidemic overwhelms Rosebud Indian Reservation.
Evils of Soy•May 28, 2016 • Leave a Comment
Deforestation, murder, tyranny, toxic pollution, birth defects and cancer are just some of the impacts of soy monoculture.
Clean Not Green•May 19, 2016 • Leave a Comment
Clean Not Green
Generating alternative energy, using solar cells, requires the fossil-fueled extraction of minerals and the fossil-fueled manufacture of solar panels, that are made using rare earth minerals, and that produce highly toxic materials.
Alternative energy or 'clean energy' from solar cells not only cannot technologically displace fossil fuel in meeting current electrical demand; it does not even reduce CO2.
To make matters worse, the toxic greenhouse gas emissions from the manufacture and disposal of solar cells are 10,000-25,000 times more potent than the CO2 emissions generated from burning fossil fuel.
–Ozzie Zehner, author of Green Illusions
The Spectacle of Breaking Free•May 16, 2016 • Leave a Comment
Breaking Free from fossil fuels. The latest hoax from 350.
Michael Ratner 1943-2016•May 12, 2016 • Leave a Comment
Michael Ratner, human rights lawyer, 1943-2016.


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Steer your military vehicle towards swarms of offending soldiers! Drive up ramps to flip your buggy or tank at their defenses. Your mission is to detonate the machine and kill every enemy on the battlefield. Unlock more powerful weapons to become unstoppable!
Demolition Drive is one of our selected Racing Games. Also very popular on this website right now are Vehicles 2, Crushing Cars, Park My Car 2 and Desert Rage 3D.
Report game as broken
Cancel report of broken game


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Thank you!
Great! DotWav, if you can´t open it, use onlineconverter (Type in google) to convert it ;)
Thank you for the sound!
Wow!Thanks for share!!
This was one I was looking for. Thank you.
thanks, i m using it in a game
nice recording but the transitions are a little choppy. try to blur or blend them next time. nice job!
nice clean recording
the download is funky
OORAH Marines Semper FI!!
Thank you!
Thank you, needed something like this :)
Just amazing!!!!!!
Great job!
I love this! Fantastic job.
Thank you!!
thank you good sir :)
perfect for my project- THX
Thank you!
Great Sound. Thank you!
download not working
Thank you for this!
Thank you so much!
Really good sound, like it
Just what I need
so dope! i'd like to use this as a sound for the Batmobile in Batman Beyond: The Series (live action)! ^_^
Used here:
Nice! Thanks!!!
I download the sound but I get nothing when I edit it in sony vegas. Help please.
uhm.... what was that?! xD
Surprisingly used for an SF play - very useful xx
brilliant work!