ALBA collaborates in the discovery of a new muscular disease: myoglobinopathy

An international collaboration led by IDIBELL identifies the first disease caused by a mutation in myoglobin.

At the MIRAS beamline of the ALBA Synchrotron they could demonstrate the presence of oxidized lipids in the damaged muscle cells.
Researchers of the Bellvitge Biomedical Research Institute (IDIBELL) led by Dr. Montse Olivé have described in Nature Communications a new muscular disease caused by a mutation in the myoglobin gene. The study has been possible thanks to a collaboration with a group of geneticists from the University of Western Australia (UWA), led by Prof. Nigel Laing, and researchers from the Karolinska Institute (Stockholm, Sweden).

Myoglobin, the protein that gives muscles their red colour, has as its main function the transportation and intracellular storage of oxygen, acting as an oxygen reservoir when there are low levels (hypoxia) or a total lack thereof (anoxia). It also acts as scavenger of free radicals and other reactive oxygen species, avoiding cell damage due to oxidative stress.

>Read more on the ALBA website

Image: Left, Typical μFTIR spectra and their second derivative of the muscle tissue where the lipid region has been highlighted in orange and the protein region in blue; the inset shows the lipid/protein ratio (calculated from the Infrared spectra) on an optical image of a tissue section with sarcoplasmic bodies. The color bar represents intensity of the ratio: blue and red mean low and high lipid content, respectively. The scale bar is 4 microns. Right,  Infrared second derivative spectrum of the amide region of one sarcoplasmic body (green) showing an increase of β-sheet structures indicating protein aggregation. Second derivative of the amide region corresponding to the tissue surrounding the sarcoplasmic bodies (black).

A step closer to early detection of multiple sclerosis

Synchrotron techniques identify the critical conditions that alter myelin structure

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease resulting in the destruction of myelin, a fatty substance that insulates nerves and increases the speed at which signals travel between nerve cells. MS affects more than 2.3 million people worldwide and has no cure. In work recently published in PNAS, a team of researchers from Tel Aviv University and the Technion-Israel Institute of Technology mapped, for the first time, the delicate and complicated force balance between the myelin sheath constituents, and their effect on the myelin structure. This new information will allow the identification of critical components involved in neurodegenerative diseases such as MS.

Structural insights into tiny bacterial harpoons

Bacteria produce complex nano-harpoons on their cell surface. One of their functions is to harpoon and inject toxins into cells that are close by. Producing such a complex weapon requires lots of different moving components that scientists are still trying to understand. Researchers from the University of Sheffield have been using some of Diamond’s crystallography beamlines to understand a particularly enigmatic piece of this tiny puzzle. The team led by David Rice and Mark Thomas worked on a protein component of the harpoon called TssA which they already knew was an integral piece of the machinery. However, unlike the other components of the harpoon, there are distinct variants of the TssA protein that contain radically different amino acid sequences at one end of the protein. The team showed that the structures of the variable region of two different TssA subunits were completely unrelated and they could assemble into distinctly different multisubunit complexes in terms of their size and geometry. This begged the question as to how different bacteria could use this protein with different structures to produce a harpoon with the same function across all species. They found that despite these differences, there was a very specific conserved region at the other end of the protein. They hypothesise that the conserved region is the part that does the work and helps the harpoon to function whereas the variable region acts as a scaffold. They used I02, I03 and I24 in their study and plan to do follow up work using X-ray crystallography and Cryo-EM such as those at the eBIC centre at Diamond. The research was published in Nature Communications.

>Read more on the Diamond Light Source website

Image: Macromolecular Crystallography (MX) at Diamond reveals the shape and arrangement of biological molecules at atomic resolution, knowledge of which provides a highly accurate insight into function. 

A timely solution for the photosynthetic oxygen evolving clock

XFEL Hub collaboration reveals the intermediates of the photosynthetic water oxidation clock

A large international collaborative effort aided by the XFEL Hub at Diamond Light Source has generated the most detailed time-resolved studies to date of a key protein involved in photosynthesis. The pioneering work, recently published in Nature, shows how photosystem II harnesses light energy to produce oxygen – insights that could direct a next generation of photovoltaic cells. 
>Read more on the Diamond Light Source website

Image: this figure is issued from a video you can watch here.

Know your ennemy

Light source identifies a key protein interaction during E. coli infection

Escherichia coli is a common source for contaminated water and food products, causing the condition known as gastroenteritis with symptoms that include diarrhea, vomiting, fever, loss of energy, and dehydration. In fact, for children or individuals with weakened immune systems, this bacterial infection in the gut can be life-threatening.

One of the microbes responsible for gastroenteritis, known formally as enteropathogenic E. coli (EPEC), causes infections by directing a pointed, needle-like projection into the human intestinal tract, releasing toxins that make people sick.

“Enteropathogenic E. coli can fire toxic proteins from inside the bacterium right into the cells of your gut lining,” says Dustin Little, a post-doctoral researcher in the Brian Coombes lab at McMaster University’s Department of Biochemistry and Biomedical Sciences.

>Read more on the Canadian Light Source website

Image: Dustin Little and Brian Coombes in the lab.
Credit: Dustin Little. 

Mycoplasma genitalium’s cell adhesion mechanism revealed

Mycoplasma genitalium is a sexually transmitted bacterium responsible for several genitourinary disorders.

An estimated 1% of the adult population is infected with this bacterium. Using XALOC beamline at the ALBA Synchrotron it has been defined the structure of the protein involved in the pathogen’s adhesion process. The discovery opens the door to defining new therapeutic strategies to fight this pathogen which is becoming more and more resistant to antibiotics.

Researchers from the Molecular Biology Institute of Barcelona (IBMB-CSIC) and the Institute of Biotechnology and Biomedicine (IBB-UAB) have discovered the mechanism by which the bacterium Mycoplasma genitalium (Mgen) adheres to human cells. This adhesion is essential for the onset of bacterial infection and subsequent disease development.
Mgen is an emerging pathogen responsible for several infectious genitourinary disorders. In men, it is the most common cause of urethritis (15-20%) while in women, it has been associated with cervicitis, pelvic inflammatory disease, premature birth and spontaneous abortions. So far, it was known that adherence to the genitourinary tract was possible thanks to proteins known as adhesins, which recognise specific cell surface receptors.
In this study, IBMB-CSIC researchers determined the three-dimensional structure of the Mgen’s P110 adhesins interacting with these cell receptors using X-rays diffraction and protein crystallography at the XALOC beamline. “We made a protein crystal of the P110 adhesin bound to these receptors and diffracted with the synchrotron’s X-rays to determine the exact position of the atoms within the protein, and we were able to decipher the three-dimensional structure”, explains IBMB researcher David Aparicio.

>Read more on the ALBA website

Image: Overall structure of P110. Two views, 90° apart from each other, of the extracellular region of P110 that is formed by a large N-domain, with a seven blade β-propeller (green), the crown (brown), and the C-domain (orange). In the right side panel the view is along the central axis of the β-propeller. The situation of the seven blades in the propeller is explicitly indicated showing that the two terminal blades I and VII are close to the C-terminal domain and opposite to the crown.

 

X-rays reveal L-shape of scaffolding protein

Structural biologists discover unexpected results at PETRA III at DESY in Germany.

An investigation at DESY’s X-ray light source PETRA III has revealed a surprising shape of an important scaffolding protein for biological cells. The scaffolding protein PDZK1 is comprised of four so-called PDZ domains, three linkers and a C-terminal tail. While bioinformatics tools had suggested that PDZK1’s PDZ domains and linkers would behave like beads on a string moving around in a highly flexible manner, the X-ray experiments showed that PDZK1 has a relatively defined L-shaped conformation with only moderate flexibility. The team led by Christian Löw from the Centre for Structural Systems Biology CSSB at DESY and Dmitri Svergun from the Hamburg branch of the European Molecular Biology Laboratory EMBL report their results in the journal Structure.

Similar to metal scaffolding which provides construction workers with access points to a building, scaffolding proteins mediate interactions between proteins situated on the membrane of the human cell. While the molecular structure of each of PDZK1’s four individual PDZ domains has been solved using X-ray crystallography and NMR spectroscopy, the overall arrangement of the domains in the protein as well as their interactions was not yet understood.

>Read more on the PETRA III at DESY website

Image: Artistic shape interpretation of the scaffolding protein PDZK1. (Credit: Manon Boschard)tistic shape interpretation of the scaffolding protein PDZK1.
Credit: Manon Boschard

Shining a new light on biological cells

Combined X-ray and fluorescence microscope reveals unseen molecular details

A research team from the University of Göttingen has commissioned at the X-ray source PETRA III at DESY a worldwide unique microscope combination to gain novel insights into biological cells. The team led by Tim Salditt and Sarah Köster describes the combined X-ray and optical fluorescence microscope in the journal Nature Communications. To test the performance of the device installed at DESY’s measuring station P10, the scientists investigated heart muscle cells with their new method.

Modern light microscopy provides with ever sharper images important new insights into the interior processes of biological cells, but highest resolution is obtained only for the fraction of biomolecules which emit fluorescence light. For this purpose, small fluorescent markers have to be first attached to the molecules of interest, for example proteins or DNA. The controlled switching of the fluorescent dye in the so-called STED (stimulated emission depletion) microscope then enables highest resolution down to a few billionth of a meter, according to principle of optical switching between on- and off-state introduced by Nobel prize winner Stefan Hell from Göttingen.

>Read more on the PETRA III at DESY website

Image: STED image (left) and X-ray imaging (right) of the same cardiac tissue cell from a rat. For STED, the network of actin filaments in the cell, which is important for the cell’s mechanical properties, have been labeled with a fluorescent dye. Contrast in the X-ray image, on the other hand, is directly related to the total electron density, with contributions of labeled and unlabeled molecules. By having both contrasts at hand, the structure of the cell can be imaged in a more complete manner, with the two imaging modalities “informing each other”.
Credit: University of Göttingen, M. Bernhardt et al.

Just like lego – studying flexible protein for drug delivery

Researchers from the Sapienza University of Rome and its spin-off company MoLiRom (Italy) are spending the weekend at the ESRF to study a protein that could potentially transport anticancer drugs.

Ferritin is a large spherical protein (20 times bigger than haemoglobin) that stores iron within its cavity in every organism. Just like a lego playset, Ferritin assembles and disassembles. It is also naturally targeted to cancer cells. These are the reasons why Ferritin is a great candidate as a drug-transport protein to fight cancer. An international team of scientists from “Sapienza” University of Rome and the SME MoLiRom (Italy) came to the ESRF to explore a special kind of ferritin that shows promising properties. “This is an archaebacterial ferritin that have transformed into a humanised ferritin to try to tackle cancer cells”, explains Matilde Trabuco, a scientist at the Italian SME MoLiRom.

The mechanism looks simple enough: “Ferritin has a natural attraction to cancer cells. If we encapsulate anti-cancer drugs inside it, it will act as a Trojan horse to go inside cells, then it will open up and deliver the drug”.

Ferritins have been widely used as scaffolds for drug-delivery and diagnostics due to their characteristic cage-like structure. Most ferritins are stable and disassemble only by a harsh pH jump that greatly limits the type of possible cargo. The humanised ferritin was engineered to combine assembly at milder conditions with specific targeting of human cancer cells.

 

>Read more on the European Synchrotron Website

 

Unprecedented 3D images of neurons in healthy and epileptic brains

Results open new perspectives for the study of neurodevelopment and neurodegenerative diseases.

A comprehensive understanding of the brain, its development, and eventual degeneration, depends on the assessment of neuronal number, spatial organization, and connectivity. However, the study of the brain architecture at the level of individual cells is still a major challenge in neuroscience.
In this context, Matheus de Castro Fonseca, from the Brazilian Biosciences National Laboratory (LNBio), and collaborators [1] used the facilities of the Brazilian Synchrotron Light Laboratory (LNLS) to obtain, for the first time, three-dimensional images in high resolution of part of the neuronal circuit, observed directly in the brain and with single cell resolution.

The researchers used the IMX X-Ray Microtomography beamline, in combination with the Golgi-Cox mercury-based impregnation protocol, which proved to be an efficient non-destructive tool for the study of the nervous system. The combination made it possible to observe the points of connectivity and the detailed morphology of a region of the brain, without the need for tissue slicing or clearing.
The mapping of neurons in healthy and unhealthy tissues should improve the research in neurodegenerative and neurodevelopmental diseases. As an example of this possibility, the work presents, for the first time in 3D, the neuronal death in an animal model of epilepsy.

The researchers are now working to extend the technique to animal models of Parkinson’s disease. The intention is to better understand the cellular mechanisms involved in the onset and progression of the disease. In the future, with the inauguration of the new Brazilian synchrotron light source, Sirius, the researchers believe that it will be possible to obtain images at the subcellular level, that is, images of the interior of the neurons.

>Read more on the Brazilian Synchrotron Light Laboratory website

Image: X-ray microtomography of the cerebral cortex showing the segmentation of individual neurons. Each color represents a single neuron or a group of neurons.

Infrared beams show cell types in a different light

Berkeley Lab scientists developing new system to identify cell differences.

By shining highly focused infrared light on living cells, scientists at the U.S. Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) hope to unmask individual cell identities, and to diagnose whether the cells are diseased or healthy.
They will use their technique to produce detailed, color-based maps of individual cells and collections of cells – in microscopic and eventually nanoscale detail – that will be analyzed using machine-learning techniques to automatically sort out cell characteristics.

Using microscopic color maps to unlock cell identity

Their focus is on developing a rapid way to easily identify cell types, and features within cells, to aid in biological and medical research by providing a way to probe living cells in their native environment without harming the cells or requiring obtrusive cell-labeling techniques.
“This is totally noninvasive,” said Cynthia McMurray, a biochemist and senior scientist in Berkeley Lab’s Molecular Biophysics and Integrated Bioimaging (MBIB) Division who is leading this new imaging effort with Michael Martin, a physicist and senior staff scientist at Berkeley Lab’s Advanced Light Source (ALS).
The ALS has dozens of beamlines that produce beams of intensely focused light, from infrared to X-rays, for a broad range of experiments.

>Read more on the Advanced Light Source website

Image: From left to right: Aris Polyzos, Edward Barnard, and Lila Lovergne, pictured here at Berkeley Lab’s Advanced Light Source, are part of a research team that is developing a cell-identification technique based on infrared imaging and machine learning.
Credit: Marilyn Chung/Berkeley Lab

Microfluidic mixing chips can reveal how biomolecules interact

Christopher Flynn, a fourth year student majoring in Physics and Mathematics at Fort Lewis College, and a SUnRiSE student at Cornell this summer, is contributing to the design of microfluidic mixing chips which could significantly enhance our understanding of proteins and living cells.

Microfluidic mixing chips are used by scientists to analyze biological molecules. They have small channels in which biological solutions, usually solutions of protein, are mixed. Biological small angle x-ray solution scattering (BioSAXS) is then used to study how these biomolecules change under different conditions, for example when they mix with hormones and drugs or when they interact with other biomolecules. These observations can help further our understanding of how cells function.

With the intention of opening a door to the inner workings of cells, Flynn and Gillilan are continuing the work of Gillilan’s former postdoctoral student, Jesse Hopkins, who started a project on microfluidic chips more than two years ago. Hopkins was working on fabricating chips that could be used to observe molecular interactions and structural changes on a millisecond scale.

While Hopkins successfully designed almost every aspect of the chip, he was unable to get the final x-ray transparent window fixed on the chip without it leaking. Flynn’s main task over the summer is to resolve this. He creates chips in the Cornell NanoScale Science and Technology Facility (CNF), using techniques including photolithography and lamination. The chips have different layers, the faulty transparent window being in one of the last. After the first few layers of the chips are made, Flynn uses them to investigate different possibilities for the window. He expects to test these windows by pumping liquids through the chips, and if they have been fit successfully, to compare any results to computer simulations that Hopkins had developed.

>Read more on the Cornell High Energy Synchrotron Source

Image: Richard Gillilan and Topher Flynn. The channels of the mixing chips are 30 microns wide, 500 microns deep.; a difficult feat but important feature of the chip. 

Open and shut: pain signals in nerve cells

Our daily function depends on signals traveling between nerve cells (neurons) along fine-tuned pathways. Central nervous system neurons contain acid-sensing ion channel 1a (ASIC1a), a protein important in sensing pain and forming memories of fear. An ion channel lodged in the cell membrane that provides a pathway for sodium ions to enter the cell, ASIC1a opens and closes in response to changes in extracellular proton concentrations. When protons accumulate outside the neuron, the channel opens, allowing sodium ions to flow into the cell, depolarizing the cell membrane and generating an electrical signal. The channel eventually becomes desensitized to protons and the gate closes. Scientists have visualized both the open and desensitized channel structures, but the third structure, which forms when the protons dissipate and the channel closes, remained elusive. Using protein crystallography at the ALS, researchers finally visualized the closed channel.

>Read more on the Advanced Light Source website

Animation: As the proton concentration increases or decreases, the gated channel ASIC1a toggles between open and closed positions, controlling the timing of signals traveling through the cell membrane of one neuron en route to the next.

An electrifying view on catalysis

The future of chemistry is ‘electrifying’: With increasing availability of cheap electrical energy from renewables, it will soon become possible to drive many chemical processes by electrical power. In this way, chemical products and fuels can be produced via sustainable routes, replacing current processes which are based on fossil fuels.

In most cases, such electrically driven reactions make use of so-called electrocatalysts, complex materials which are assembled from a large number of chemical componentAs. The electrocatalyst plays an essential role: It helps to run the chemical reaction while keeping the loss of energy minimal, thereby saving as much renewable energy as possible. In most cases, electrocatalysts are developed empirically and the chemical reactions at their interfaces are poorly understood. A better understanding of these processes is essential, however, for fast development of new electrocatalysts and for a directed improvement of their lifetime, one of the most important factors that currently limit their applicability.

>Read more on the Elettra website

Figure:  Introducing well-defined model electrocatalysts into the field of electrochemistry.

Google Maps for the cerebellum

A team of researchers from Göttingen has successfully applied a special variant of X-ray imaging to brain tissue. With the combination of high-resolution measurements at DESY’s X-ray light source PETRA III and data from a laboratory X-ray source, Tim Salditt’s group from the Institute of X-ray Physics at the Georg August University of Göttingen was able to visualize about 1.8 million nerve cells in the cerebellar cortex. The researchers describe the investigations with the so-called phase contrast tomography in the Proceedings of the National Academy of Sciences (PNAS).
The human cerebellum contains about 80 percent of all nerve cells in 10 percent of the brain volume – one cubic millimeter can therefore contain more than one million nerve cells. These process signals that mainly control learned and unconscious movement sequences. However, their exact positions and neighbourhood relationships are largely unknown. “Tomography in the so-called phase contrast mode and subsequent automated image processing enables the cells to be located and displayed in their exact position,” explains Mareike Töpperwien from the Institute of X-ray Physics at the University of Göttingen, lead author of the publication.

>Read more on the PETRA III at DESY website

Image: Result of the phase contrast X-ray tomography at DESY’s X-ray source PETRA III.
Credit: Töpperwien et al., Universität Göttingen

Probing tumour interiors

X-ray fluorescence mapping to measure tumour penetration by a novel anticancer agent.

A new anticancer agent developed by the University of Warwick has been studied using microfocus synchrotron X-ray fluorescence (SXRF) at I18 at Diamond Light Source. As described in The Journal of Inorganic Biochemistry, researchers saw that the drug penetrated ovarian cancer cell spheroids and the distribution of zinc and calcium was perturbed.  

Platinum-based chemotherapy agents are used to treat many cancer patients, but some can develop resistance to them. To address this issue, scientists from the University of Warwick sought to employ alternative precious metals. They developed an osmium-based agent, known as FY26, which exhibits high potency against a range of cancer cell lines. To unlock the potential of this novel agent and to test its efficacy and safety in clinical trials, the team need to fully understand its mechanism of action.

To explore how FY26 behaves in tumours, the team grew ovarian cancer spheroids and used SXRF at I18 to probe the depth of penetration of the drug. They noted that FY26 could enter the cores of the spheroids, which is critical for its activity and very encouraging for the future of the drug. SXRF also enabled them to probe other metals within the cells, which showed that the distribution of zinc and calcium was altered, providing new insights into the mechanism of FY26-induced cell death.

>Read more on the Diamond Light Source website

Figure: (extract) A) Structure of FY26and related complexes, [(ŋ6-p-cym)Os(Azpy-NMe2)X]+. B) Bright field images and SXRF elemental maps of Os, Ca and Zn in A2780 human ovarian carcinoma spheroid sections (500 nm thick) treated with 0.7 µM FY26(½ IC50) for 0 or 48 h. Raster scan: 2×2 µm2 step size, 1 s dwell time. Scale bar 100 µm. Calibration bar in ng mm-2. Yellow squares in bright field images indicate areas of the spheroid studied using SXRF. Red areas in SXRF elemental maps indicate the limits of the spheroids. C) Average Os content (in ng mm-2) as a function of distance from A2780 3D spheroid surface, after treatment for 16 h (green), 24 h (blue) or 48 h (red) with 0.7 µM FY26.