Susana Gonzalez

A once-rising star in stem cell biology who recently lost both her job and a sizable grant has had a fourth paper retracted.

The notice issued by Nature for a 2006 letter cites duplicated images, and a lack of raw data to verify the findings. First author Susana Gonzalez who wasdismissed from her position at the National Center for Cardiovascular Research (CNIC)in Spain last February over allegations of misconduct couldnt be reached by the journal.

Heres the full text of the retraction notice:

In this Letter, some PCR input panels contain duplicated bands (Figs 1b and 2a; and Supplementary Figs 4 and 6a). In Fig. 2e, theARFpromoter panel is a duplicate of the RD panel in Supplementary Fig. 8c. The raw data were not available to verify the data. Given these issues, the authors wish to retract the Letter. The authors deeply regret these errors and apologize to the community. Peter Klatt, Sonia Delgado, Esther Conde, Fernando Lopez-Rios, Montserrat Sanchez-Cespedes, Juan Mendez, Francisco Antequera and Manuel Serrano agree with the Retraction of the Letter. Susana Gonzalez could not be reached.

Oncogenic activity of Cdc6 through repression of the INK4/ARF locus has been cited 133 times, according to Clarivate Analytics Web of Science. The letter has been discussed on PubPeer.

Following misconduct allegations, Gonzalez was fired from the CNIC (a decision which she appealed), one of her grants (totaling nearly 2 million Euros)was suspended. Earlier this year, she received two retractions inNature Communications, which also citedfigure duplications and missing raw data. Cell Cycle has also retracted a 2012 paper she co-authored.

Jose F. de Celis, head of theCentre for Molecular Biology Severo Ochoa (CBMSO), where Gonzalez was working in March (but on sick leave), told us:

Susana is not longer at CBMSO, she requested a transfer and we though it was the best option.

Hat tip:Khalid El Bairi

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Nature retracts paper by stem cell scientist appealing her dismissal - Retraction Watch (blog)

Nir Hacohen, an immunologist and geneticist at the Broad Institute of the Massachusetts Institute of Technology and Harvard University, knew that biology had a problem. He wanted to understand the human immune responses role in cancer and other diseases. But to do that, he first had to address a more fundamental issue: The definition of the immune cell types themselves seemed insufficient, incomplete and outdated.

For over a century, distinctions between types of cells relied on how they appeared under a microscope: their shapes, sizes, locations and their uptake of staining dyes. Recent decades, however, witnessed a shift to molecular methods that use fluorescently labeled antibodies to target protein markers on the cells surface. Although this approach allowed researchers to isolate more cell types, it was not enough, according to Hacohen. Until 2009, biologists could analyze cells only in bulk, averaging signals from multitudes of them to get a picture of what was going on in a tissue. When sequencing RNA from individual cells finally became possible, the initial analyses were what Hacohen called biased and shallow because the few markers used to classify the cells were too insensitive to nuances of differences among them. Does this really capture the complexity of the cell? Hacohen said.

In a study published in Science this past April, he and his team showed that, as expected, much of this complexity had been obscured. Analyzing patterns of gene expression in individual human immune system cells, the researchers refined the definitions of the types known as dendritic cells and monocytes and identified a novel type that had been overlooked. Moreover, they discovered that a cell population thought to comprise one subtype was actually a mixture of two, which perform different functions.

Hacohens work represents one component of a much larger project. Last October, an international community of researchers led by Aviv Regev of the Broad Institute and Sarah Teichmann of the Wellcome Trust Sanger Institute launched the Human Cell Atlas to apply this kind of modeling to the entire body. It aims to catalog not just cell types which are predicted to extend far beyond the 200 types most often cited in textbooks but also the hallmarks of cell types under different conditions and in individuals with different genetic and epigenetic variations. That knowledge is important because it would provide a more comprehensive overview of the dynamic complexity of life. Immune cell subtypes might shift in someone who has an infection or an allergy or an autoimmune disease, for example; or they may vary for different population groups.

This is not comparable to the Human Genome Project, Hacohen said. That was a fairly well-prescribed problem. Here the problem is much more difficult and in a sense encompasses a lot of biology.

The Human Cell Atlas is only one of several projects in molecular and cellular biology looking to synthesize enormous quantities of data to gain deeper insights into just how diverse the cells in our bodies really are, and how complex life is. In 2003, researchers at the KTH Royal Institute of Technology, in Sweden, launched the Human Protein Atlas, which aims to catalog comprehensively the expression, location and spatial distribution of proteins within individual cells. Only within the past few years were members of the project able to start classifying, annotating and analyzing the millions of images they had captured of subcellular structures in different cell types. To reach that point, they first had to spend a decade standardizing, optimizing and scaling up their procedures, which involved using targeted antibodies to stain proteins and then looking for those markers inside healthy and cancerous tissue cells with high-resolution microscopy.

In January 2015, the team charted protein expression across more than 30 human tissues. This past May, they published the second part of their undertaking in Science. Turning their attention to the single-cell level, they mapped more than 12,000 proteins to 30 subcellular structures, in turn defining the proteomes the complete sets of expressed proteins of more than a dozen major organelles. The researchers identified which proteins were found where, explored variations in protein expression from cell to cell and analyzed how cells segregate chemical reactions within themselves.

One of the papers most salient findings, according to its principal investigator, Emma Lundberg, was that as many as half of our proteins can be found in multiple compartments of a cell. Everything that proteins do is specific within the context of their environment, Lundberg said. If one protein is present in the nucleus but also in the plasma membrane, it might have different functions in those compartments.

Take HER2, a receptor protein often overexpressed in certain breast cancers. When found in tumor cell membranes, HER2 correlates with a better prognosis than when it is in the cytoplasm or nucleus. There are more and more and more studies of single proteins showing that this is actually a common phenomenon, Lundberg said. But its the scale of it, she added, that is most exciting.

As much as 50 percent of the proteins that her group observed were expressed in more than one part of a cell. If that figure indicates how big multi-functionality could be, Lundberg said, it makes the cell much more complex and the functionality of the proteome greater.

This heterogeneity offers deeper insights into the fundamentals of protein function, but it may also explain why, for instance, certain drugs result in unwanted side effects.

Another group of scientists, who hope to publish their work in the fall, have been mapping the distribution of proteins in the cell types of the testis home to the greatest number of uniquely expressed protein-coding genes. In doing so, they are reclassifying the cell subtypes that occur during spermatogenesis. Many things are happening in these cells before they become mature, said Cecilia Lindskog Bergstrm of Uppsala University in Sweden, who is collaborating on the research. Proteins that are expressed in a certain sub-stage of sperm development will tell more about the function of these proteins.

This dynamic way of defining cell type is what Hacohen sought to establish further in his study of blood cells. In the findings it reported in May, the Human Protein Atlas began to demonstrate why these refinements may be necessary. The team observed that approximately 15 percent of the proteins exhibited single-cell variation: In a tissue that looked superficially uniform, some cells might differ from their neighbors in the amount or spatial distribution of the proteins they expressed, when one would expect them to be the same. The single-cell RNA sequencing approach of the Human Cell Atlas will allow researchers to create cell profiles based on molecules other than proteins.

In the past, we typically looked at a tissue or an organ in the way youd look at a smoothie, said Bart Deplancke, a biological systems engineer at the cole Polytechnique Fdrale de Lausanne in Switzerland. Based on its overall color and taste, one might assume that a smoothie consists of strawberries and bananas. But that way of looking at it may miss key ingredients and makes it seem as if all parts of the smoothie are identical. With modern techniques, Deplancke said, they can do the tissue-analysis equivalent of looking at a smoothie and saying, I see these different pieces of fruit. And they can see how that full diversity of cell types makes a functional organ. Similarly, they can learn how the full spectrum of cells involved in cancers and other diseases relates to prognosis and recovery.

Deplancke is one of three researchers who have begun organizing the Fly Cell Atlas, which seeks to characterize all the cell types in Drosophila fruit flies. The Allen Institute in Seattle is working toward a similar understanding of the mouse brain. Both hope to apply their findings to explain human behavior and disease, just as the Human Cell Atlas does. Ultimately, integrating the vast datasets generated by these different atlases may prove the greatest challenge of all but, the researchers hope, it will also be the most rewarding, combining structural, genomic and epigenetic approaches under the umbrella of a new kind of cartographic exploration.

Read the rest here:
Cell Atlases Reveal Biology's Frontiers - Quanta Magazine

A series of fluorescence microscopy images detail the blinking behavior of the teams nanoparticle buckyswitches. Credit: Nano Letters 17 (6) pp. 38963901

Visualizing biological cells under a microscope was just made clearer, thanks to research conducted by graduate student Yifei Jiang and principal investigator Jason McNeill of Clemson University's department of chemistry.

With the help of Rhonda Powell and Terri Bruce of Clemson's Light Imaging Facility, the team was able to develop a nanoparticle "switch" that fluoresces to sharpen the resolution of microscopic images that depict small cellular structures. As recently published in Nano Letters, this switch improves upon an imaging method that won the 2014 Nobel Prize in Chemistry.

Because cellular structures emit light at wavelengths smaller than 400-700 nanometers on the electromagnetic spectrum, they often appear blurred through a light microscope. This constraint is referred to as the diffraction limit, and it occurs because of the wave properties of light. As light passes around structures within biological cells, it diffracts, or bends, to a point that light microscopes cannot clearly resolve. The 2014 prize-winning imaging method - single molecule localization microscopy - was invented to surpass this limitation.

"Single molecule localization microscopy is based on molecular 'photoswitches' - fluorescent molecules that you can turn on and off, like a light switch, to beat the diffraction limit," McNeill said. "With this imaging method, the sample is imaged one fluorescent molecule at a time and a computer is used to construct an image that is much sharper than what you could get with a regular light microscope."

The catch, however, is that the fluorescence provided by photoswitches is dim at best, with only a slight improvement in image resolution. Single molecule localization microscopy also requires specialized equipment that can be expensive to obtain.

Cue the "buckyswitch" - the Clemson researchers' enhanced version of a photoswitch. This new type of nanoparticle retains the photoswitch's on-off capability, but is 10 times brighter and easier to use. It also allows microscopes to capture images up to the terapixel level. (That's the equivalent of one trillion pixels, or one million megapixels.)

"These nanoparticles are the first photoswitches to achieve precision down to approximately 1 nanometer, which greatly improves the resolution of super-resolution imaging," Jiang said. "Also, our method only requires one excitation light source, where conventional super-resolution techniques require two lasers; thus, we have simplified the microscope setup."

Jiang assembled the buckyswitch out of a fluorescent, semiconducting conjugated polymer complexed with a chemical derivative of buckminsterfullerene: a soccer-ball-shaped form of carbon.

"The hard part of making a fluorescent nanoparticle that you can turn on and off is that there are lots of areas emitting fluorescence at once," McNeill said. "In the case of fluorescent conjugated polymer, there are dozens or hundreds of chain segments. You can try to make a lot of little switches for each segment, but it's hard to get them all to switch off at the same time. You can't get them synchronized."

By adding the derivative of buckminsterfullerene, called PCBM, to the making of buckyswitches, a "master switch" is formed that regulates the atomic charge of the polymer's segments, thus synchronizing fluorescence. PCBM is able to seize electrons from the polymer segment, giving the segment an overall positive charge. This positive charge reduces the fluorescence of nearby segments, which has a domino effect that turns off fluorescence in the entire nanoparticle.

Bruce - whose background traverses the topics of chemical engineering, applied biology, cell biology, and experience in teaching and industry - likens this imaging method to the view of a suspension bridge at night.

"The wires of the bridge are often illuminated, and when you are standing far away from the bridge, the lights look like one continuous 'rope' of light, instead of individual bulbs. However, if you can make the bulbs blink - such that only every other bulb is 'on' at any time - your eyes can discern the individual bulbs from far away," Bruce said. "The basis for super-resolution microscopy lies in the ability to make fluorescent labels 'blink' just like the lights on the bridge. The work that Dr. McNeill's lab is doing is vital for the advancement of this technology because it focuses on making those individual blinks much brighter, so that our current photon detectors can actually see the blinks. If we can see the blinks with a camera or other photon detector, we can map where the blink occurs, and create an image where we can discern two points of light that are within 10-20 nanometers of one another."

Once the buckyswitch was synthesized, Jiang tested it in E. coli, but not before developing a unique growth media for the bacteria. Typically, E. coli is grown in media that is autofluorescent, meaning that it naturally emits light. Without the proper media, the buckyswitch's fluorescence would be obscured by background light, something that Powell underlined.

"A study like the one Yifei conducted required very little background fluorescence, so I researched media components that would be less likely to be autofluorescent and prepared a 'recipe' for a non-conventional, less autofluorescent nutrient media for bacteria culture," said Powell, who studied both biological sciences and microbiology at Clemson before becoming the research lab manager of the Clemson Light Imaging Facility. Powell and Bruce also worked to provide Jiang with the E. coli for the study.

After all of the necessary components were squared away, Jiang attached the nanoparticle buckyswitches to the surface of E. coli. As hoped, the buckyswitches emitted small flashes of light, which allowed the researchers to determine their precise positions. They then pieced together each flash of light to reconstruct the shape of the E. coli, yielding a super-resolution image.

"We hope this breakthrough will eventually be able to help researchers tackle difficult problems in biology, leading to breakthroughs in the understanding and treatment of disease," the Clemson team said.

The team designed the buckyswitches to work with standard fluorescent microscopes and free software that's available online, making the technology inexpensive and accessible for labs worldwide.

Their publication, titled "Improved Superresolution Imaging Using Telegraph Noise inOrganic Semiconductor Nanoparticles," is featured in the June 14 issue of Nano Letters.

Explore further: Background suppression for super-resolution light microscopy

More information: Yifei Jiang et al, Improved Superresolution Imaging Using Telegraph Noise in Organic Semiconductor Nanoparticles, Nano Letters (2017). DOI: 10.1021/acs.nanolett.7b01440

Journal reference: Nano Letters

Provided by: Clemson University

Excerpt from:
Researchers illuminate the field of microscopy with nanoparticle 'buckyswitch' - Phys.Org