Delivering drugs using nanocrystals

Monash University researchers have used advanced techniques at ANSTO to investigate the production of new, elongated polymer nanocapsules with a high payload of drug nanocrystals to potentially increase drug targetability, and also decrease dosage frequency and side effects.

This method had not been investigated previously and represents a pioneering method of investigation in the field of colloidal science applications for drug delivery.

Nanoparticles have been used to increase the delivery efficiency of cancer therapy because of their biocompatibility, versatility and the easiness of functionalisation.

The team engineered novel elongated polymer nanocapsules, which are unlike the more well-known spherical nanocapsules.

The elongated polymer nanocapsules were made with elongated liposomes or surfactant vesicles and used drug nanocrystals as a template. 

The results provided strong evidence that the elongated structure could be retained, and also confirmed that the loading method to form rod-like drug nanocrystals inside liposomes was a practical solution.

The combination of the high drug payload, in the form of encapsulated nanocrystals, and the non-spherical feature of liposomes represented a more efficient delivery system.

Spherical hollow nanocapsules have been studied extensively, but the formation of elongated nanocapsules containing active pharmaceuticals as therapeutic agents has been previously largely unsuccessful. 

Read more on the ANSTO website

Image: Elongated nanocapsules can be prepared by polymerisation at the surface of elongated liposome templates with drug nanocrystals

Molecular IgG3 structure paves the way for new applications of antibodies

A combination of scattering and analytical techniques has provided the first atomic-level structural model for the IgG3 antibody

In humans, Immunoglobulin (IgG) is the most common type of antibody found in blood circulation. IgG molecules are created by plasma B cells, and there are four subclasses. Of the four, IgG3 is the least understood. It has a uniquely long hinge region separating its Fab antigen-binding and Fc receptor-binding regions. The presence of this elongated hinge makes it challenging to perform structural studies, for example, with X-ray crystallography. Due to this lack of structural information, IgG3 is the only subclass not currently exploited for therapeutic uses. In work recently published in the Journal of Biological Chemistry, researchers from University College London and the University of Birmingham have used a combination of imaging and analytical methods to provide the first experimentally determined molecular structural model for a full-length IgG3 antibody. This new information should enable the use of IgG3 to develop new therapies and antibody tests. 

Getting a good look at IgG3

A high-resolution structure for part of the IgG3 molecule, the globular IgG3-Fc fragment, is available. And previous studies of the whole molecule using Small Angle X-ray Scattering (SAXS) and analytical ultracentrifugation (AUC) showed that IgG3 is elongated compared to IgG1, IgG2 and IgG4. SAXS also showed that IgG3 has a more extended central hinge than IgG1 and IgG2 that links its three globular regions together.  

Read more on the Diamond website

Image: The IgG3 structural model is formed from two globular Fab regions, a long hinge in the centre, and one Fc region, as shown from the scattering modelling fits. The structure is reminiscent of a giraffe with an extended and semi-rigid neck.

Credit:
Dr Valentina Spiteri, UCL.

Insights into coronavirus proteins using SAXS

A collaboration led by researchers from the European Molecular Biology Laboratory (EMBL) used small angle X-ray scattering (SAXS) at the European XFEL and obtained interesting data on samples containing coronavirus spike proteins including proteins of the isolated receptor biding domain. The results can, for example, help investigate how antibodies bind to the virus. This gives researchers a new tool that may improve understanding of our bodies’ immune response to coronavirus and help to develop medical strategies to overcome COVID-19

SAXS is a powerful technique as it allows researchers to gain insights into protein shape and function at the micro- and nanoscales. The technique has proven to be extremely useful in investigating macromolecular structures such as proteins, especially because it removes the need to crystallize these samples. This means researchers can study the sample in its native form under physiological conditions under which biological reactions occur.

Read more on the European XFEL website

Image: Seen here, the instrument SPB/SFX, where the SAXS experiment was carried out. Using this instrument researchers can study the three-dimensional structures of biological objects. Examples are biological molecules including crystals of macromolecules and macromolecular complexes as well as viruses, organelles, and cells.

Credit: European XFEL / Jan Hosan

Researchers discover foam “Fizzics”

Chemical engineers at the University of Illinois Chicago and UCLA used the U.S. Department of Energy’s Advanced Photon Source (APS) in answering longstanding questions about the underlying processes that determine the life cycle of liquid foams. The breakthrough in understanding how liquid foams dissipate could help improve the commercial production and application of foams in a broad range of industries and could lead to improved products. Findings of the research were featured in the Proceedings of the National Academy of Sciences of the United States of America.

Foams are a familiar phenomenon in everyday lives — mixing soaps and detergents into water when doing dishes, blowing bubbles out of soapy water toys, sipping the foam off a cup of lattes or milk shake. Liquid foams can occur in a variety of natural and artificial settings. While some foams are produced naturally, as in bodies of water creating large ocean blooms on the beaches, others arise in industrial processes. In oil recovery and fermentation, for example, foams are a byproduct.

Whenever soapy water is agitated, foams are formed. They are mostly gas pockets separated by thin liquid films that often contain tiny molecular aggregates called micelles. Oily dirt, for example, is washed away by hiding in the water-phobic cores of micelles. In addition, fat digestion in our bodies relies on the role of micelles formed by bile salts.

Over time, foams dissipate as liquid within the thin films is squeezed out. Soap and detergent molecules that are by very nature amphiphilic (hydrophilic and hydrophobic) aggregate within water to form spherical micelles, with their outward-facing heads being hydrophilic and water-phobic tails forming the core.

Read more on the ANL website

Image: Micellar foam films show grayscale intensity variations that correspond to rich nanoscopic topography mapped using IDIOM protocols.

Credit: Chrystian Ochoa and Vivek Sharma/UIC

Quantifying oriented myelin in mouse and human brain

Myelin “insulates” our neurons enabling fast signal transduction in our brain; myelin levels, integrity, and neuron orientations are important determinants of brain development and disease. However, myelin imaging methods used in clinics or research are non-specific or destructive.

Using small-angle X-ray scattering tensor tomography (SAXS-TT), we exploited myelin’s ~17nm periodicity to non-invasively derive 3D myelin and neuron orientation maps in macroscopic tissue volumes (Figure). We demonstrated the method on a mouse brain (a-d), a mouse spinal cord, a human visual cortex and two human white matter specimens. We validated the readouts with 2D and 3D histology, and correlated the results with MRI contrasts.

read more on the PSI website

Image: Figure. a) SAXS-TT setup. b) SAXS projection of the mouse brain, with myelin signal intensity and 2D fiber orientation color-encoded. c-d) Tomographic reconstruction results in quantitative 3D myelin maps (c) and a tensor representing neuron orientations in each voxel (d). e-f) Distinct myelin periodicities in the central and peripheral nervous system (CNS/PNS) enable multiplexed imaging (e) and reconstruction (f) of CNS and PNS structures. g) Control and dysmyelinated mouse brain signals, showcasing SAXS-TT’s sensitivity in quantifying minute myelin signals (see colorbar), and myelin integrity.

How X-rays could make reliable, rapid COVID-19 tests a reality

Vaccines are turning the tide in the pandemic, but the risk of infection is still present in some situations. If you want to visit a friend, get on a plane, or go see a movie, there is no highly accurate, instant test that can tell you right then and there whether or not you have a SARS-CoV-2 infection. But new research from Lawrence Berkeley National Laboratory (Berkeley Lab) could help get reliable instant tests on the market.

A study led by Michal Hammel and Curtis D. Hodge suggests that a highly sensitive lateral flow assay – the same type of device used in home pregnancy tests – could be developed using pairs of rigid antibodies that bind to the SARS-CoV-2 nucleocapsid protein. Such a test would only require a small drop of mucus or saliva, could give results in 15 minutes, and could detect a COVID-19 infection one day before the onset of symptoms. Their work was published in the journal mABs.

The current gold standard tests for COVID-19 use a form of polymerase chain reaction (PCR) to identify the presence of SARS-CoV-2 nucleic acid (RNA) rather than a viral protein. They are quite accurate, with false negative rates ranging less then 5%  (depending primarily on the sampling site, sample type, and stage of infection). However, PCR tests must be sent away for analysis at an accredited lab.

Read more on the Berkeley Lab website

Image: Molecular models constructed from the X-ray data show different antibodies bound to the SARS-CoV-2 nucleocapsid protein (pink). The scientists determined that the linear arrangement (right) has higher detection sensitivity than the sandwich arrangement (left).

Credit: Berkeley Lab

Virus recognition skills

A virus recognizes the starting point on the DNA to be packaged inside its protein shell

A bacteriophage – a virus that attacks bacteria – assembles into an infectious species using a powerful nanomachine to stuff its DNA into a protein shell. In several types of phage, this genome packaging motor is composed of several copies of large and small terminase subunits (TerL and TerS, respectively) that attach to a portal into the protein procapsid. 

Figure 1. Envelope of NV1 TerS from SAXS data, overlaid with modeled structure with open HTHs. Circle highlights one HTH motif.

The Cingolani group (Thomas Jefferson U) has now determined the structure of TerS from the Pseudomonas phage PaP3. Phage DNA to be packaged contains multiple copies of the genome, but just one copy is needed to fill a procapsid. Terminases attempt to package this one copy by various methods; in PaP3 a termination signal is provided by the interaction of a specific sequence in the DNA (the cos sequence) with TerS.

A crystal structure of PaP3 TerS reveals a nonameric ring of mixed alpha/beta composition, sitting atop a 9-stranded beta-barrel. Projecting out from the ring are spokes tipped with helix-turn-helix (HTH) DNA-binding domains. In the crystal, with no DNA present, the HTH domains are packed tightly against the inner parts of the nonamer (a “closed” form). Crystals of TerS from the related NV1 phage were also studied; their quality was not as good but the same conformation was found.  BioSAXS coupled to size-exclusion chromatography, at CHESS, was then used to examine the PaP3 TerS structure, and that of the related NV1 protein, in solution. Both turned out to be ~25% larger than predicted from the crystal structure. The molecular envelope determined from SAXS data for NV1 clearly showed protuberances on the outside of the nonameric ring that did not match the crystal structure. However, by rotating the HTH domain of each monomer about an obvious hinge region, an “open” model could be built that fit the SAXS envelope well (Figure 1). 

Read more on the CHESS website

Image: Figure 1. Envelope of NV1 TerS from SAXS data, overlaid with modeled structure with open HTHs. Circle highlights one HTH motif.

Unravelling the molecular structure, self-assembly, and properties of a cephalopod protein variant

Cephalopods, such as the loliginid in Figure 1A, are known for their remarkable ability to rapidly change the color and appearance of their skin. These capabilities are enabled in part by unique structural proteins called reflectins, which play essential roles in optical behavior of cephalopod skin cells. Moreover, reflectins have demonstrated exciting potential as functional materials within the context of biophotonic and bioelectronic systems. Given reflectins’ demonstrated significance from both fundamental biology and applications perspectives, some research effort has been devoted to resolving their three-dimensional (3D) structures. However, the peculiar sequence composition of reflectins has made them extremely sensitive to subtle changes in environmental conditions and prone to aggregation, thus significantly complicating the study of their structure-function relationships and precluding their definitive molecular-level structural characterization. In this work, we have elucidated the structure of a reflectin variant at the molecular level, demonstrated a robust methodology for controlling its assembly and optical properties.


We began our studies by rationally selecting a prototypical reflectin variant (RfA1TV) by using a bioinformatics-guided approach (Figure 1B). Next, we not only produced the variant in high yield and purity but also optimized conditions for maintaining this protein in a monomeric state (Figure 1C). We then probed the protein with small angle X-ray scattering (SAXS) using the Austrian SAXS beamline at the Elettra Synchrotron Laboratory in Trieste, Italy. For this purpose, a well-dispersed solution of RfA1TV was prepared in a low-pH buffer and transferred into a glass capillary, which was positioned in the path of an incident X-ray beam. The X-rays scattered by the solution-borne RfA1TV molecules formed a 2-D pattern on a Pilatus3 1M detector (Figure 1D). Subsequently, radial averaging and image calibration of the two-dimensional data furnished corresponding one-dimensional curves, which were further processed, analyzed, and correlated with other experiments to obtain insight into the protein’s geometry (Figure 1E).

Read more on the Elettra website

Image: (A) A camera image of a Doryteuthis pealeii squid. (B) An illustration of the selection of the prototypical truncated reflectin variant (RfA1TV) from full-length Doryteuthis pealeii reflectin A1. (C) A digital camera image of a solution of primarily monomeric RfA1TV (Upper) and a corresponding cartoon of RfA1TV monomers (Lower Inset). (D) An illustration of the SAXS analysis of the reflectin variant, wherein incident X-rays are scattered by the solution-borne proteins to furnish a corresponding scattering pattern. (E)The 3D structure of RfA1TV (random coils – gray, helices – orange, β-strands – purple). 

Credit: This figure has been adapted from M. J. Umerani*, P. Pratakshya* et al.Proc. Natl. Acad. Sci. U.S.A 117, 32891-32901 (2020).

Blood disorder mechanism discovered

G6PD deficiency affects about 400M people worldwide and can pose serious health risks. Uncovering the causes of the most severe cases could finally lead to treatments.

With a name like glucose-6-phosphate dehydrogenase deficiency, one would think it is a rare and obscure medical condition, but that’s far from the truth. Roughly 400 million people worldwide live with potential of blood disorders due to the enzyme deficiency. While some people are asymptomatic, others suffer from jaundice, ruptured red blood cells and, in the worst cases, kidney failure. 

Now, a team led by researchers at the Department of Energy’s SLAC National Accelerator Laboratory has uncovered the elusive mechanism behind the most severe cases of the disease: a broken chain of amino acids that warps the shape of the condition’s namesake protein, G6PD. The team, led by SLAC Professor Soichi Wakatsuki, report their findings January 18th in Proceedings of the National Academy of Sciences

Read more on the SLAC website

Image: The G6PD enzyme plays a crucial role in red blood cells, removing molecules such as hydrogen peroxide from the body. In some cases, mutations can bend the molecule awkwardly, interfering with G6PD’s function. In the worst cases, the mutations lead red blood cells to rupture.

Credit: Mio Wakatsuki, from protein images by Naoki Horikoshi/SLAC National Accelerator Laboratory

How a very “sociable” protein can hold clues about Alzheimer’s origin

The origin of the most prevalent form of Alzheimer’s disease, which accounts for 95% of cases, is still not clear despite decades of scientific studies. “Before understanding the pathology, we need to understand the biology”, explains Montse Soler López, scientist leading research on Alzheimer’s disease at the ESRF. “The only thing we are sure about is that the most common form of Alzheimer’s is linked with ageing”, she asserts.

So researchers have been focusing on parts of the body that degrade dramatically with age. Neurons, for example, are long-lived cells, meaning that they don’t renew themselves like other cells do. Neurons lodge mitochondria, which are so-called the “powerhouse of cell” because of their active role generating energy in the body. With time, mitochondria suffer oxidative stress and this leads to their malfunction. It has been recently discovered that people with Alzheimer’s may have an accumulation of amyloids inside mitochondria (previously it was thought amyloids were only outside the neurons). Montse Soler López is trying to find whether there is a link between mitochondrial dysfunction, presence of amyloids and early disease symptoms. “We believe that malfunctioning of the mitochondria can take place 20 years before the person shows symptoms of the disease”.

Read more on the ESRF website

Newly discovered photosynthesis enzyme yields evolutionary clues

Rubisco is one of the oldest carbon-fixing enzymes on the planet, taking CO2 from the atmosphere and fixing it into sugar for plants and other photosynthetic organisms. Form I (“form one”) rubisco goes back nearly 2.4 billion years and is a key focus of scientists studying the evolution of life as well as those seeking to develop bio-based fuels and renewable-energy technologies. A newly discovered form of rubisco—dubbed form I′ (“one prime”)—is thought to represent a missing link in the evolution of photosynthetic organisms, potentially providing clues as to how this enzyme changed the planet.

To learn how form I′ rubisco compares to other rubisco enzymes, researchers performed x-ray crystallography at Advanced Light Source (ALS) Beamline 8.2.2. Then, to capture how the enzyme’s structure changes during different states of activity, they applied small-angle x-ray scattering (SAXS) using Beamline 12.3.1 (SIBYLS). This combination of approaches enables scientists to construct unprecedented models of complex molecules as they appear in nature.

Read more on the ALS website

Image: A ribbon diagram (left) and molecular surface representation (right) of carbon-fixing form I′ rubisco, showing eight molecular subunits without the small subunits found in other forms of rubisco. An x-ray diffraction pattern of the enzyme, also generated by the research team, is in the background.

Credit: Henrique Pereira/Berkeley Lab

“foot-2-foot” interaction sheds light on bacterial conjugation

Bacteria possess mechanisms to establish communication between cells. This is especially important in bacterial conjugation, a process that allows bacteria to share genetic material. This is often used by bacteria to transfer antibiotic resistance genes and other virulence factors to neighbor cells, increasing the antibiotic resistance spread.

Now, a research team of ALBA scientists report the structural mechanism by which two proteins, Rap and Rco, act together to regulate conjugation. Rco is a repressor of conjugation, whereas Rap binds Rco and prevents Rco-mediated conjugation repression, thus resulting in an activation of the conjugation mechanism. The main results of the study show that Rap contains a binding pocket were a short peptide can bind, producing structural changes in Rap that forces its tetramerization, releasing Rco for blocking conjugation. Tetramerization occurs through an interaction that scientists named “foot-2-foot”, which differs significantly from the model proposed for other proteins of the Rap family.

Read more on the ALBA website

Image: RappLS20 tetramerization, side view of the peptide-bound tetramer. The red arrows indicate the loops connecting helices H4 and H5. (C) Zoom of the area around the N-terminus of helix H4, showing the insertion of this helix into the opposite monomer. The homotetramerization caused by the foot-2-foot interactions of the NTDs of RappLS20 provides an explanation for the activation of the RcopLS20 partner. In the absence of the peptide, the NTDs are positioned such that they allow the interaction with RcopLS20. However, upon binding the signaling peptide, the NTDs shift outwards, facilitating the formation of the homotetramer, leading to a change of the interaction surface of the NTDs that is no longer available for interactions with RcopLS20

Measuring interfaces in 3D printing

3D printing (3DP) leads to many defects and interfaces within printed parts. Failure during performance in the road-to-road and layer-by-layer processed parts appears at these interfaces and defects. Understanding the root cause of these limitations is key. 

Only by mapping the sample via µ-beam SAX was it possible to determine the source of a peculiar defect and interface morphology. To the surprise of the scientists the alignment of nanoparticles is not uniform and not random within roads and layers of an epoxy carbon fiber reinforced composite and explains some of the achieved mechanical properties and microscopy results.

Read more on the Cornell High Energy Synchrotron Source (CHESS) website

Image: 3D printing degree of orientation

Credit: CHESS

Bone breakages and hip fracture risk is linked to nanoscale bone inflexibility

Experiments carried out at Diamond using high energy intense beams of X-rays examined bone flexibility at the nanoscale. This allowed scientists to assess how collagen and minerals within bone flex and then break apart under load – in the nanostructure of hip bone samples.  

The report’s findings suggest that doctors should look not only at bone density, but also bone flexibility, when deciding how to prevent bone breakages. 

New research undertaken at Diamond’s Small Angle X-ray Scattering beamline (I22) has highlighted a gap in preventative treatment in patients prone to bone fractures.  The study, published in Scientific Reports and led by Imperial College London, found that flexibility as well as density in the bone nanostructure is an important factor in assessing how likely someone is to suffer fractures. 

Read more on the Diamond website

Image: Nanostructure: Collagen and mineral strain under load. Image: Shaocheng Ma, Imperial College London.

Significant progress on ultraflexible solar cells

Research from Monash University, the University of Tokyo and RIKEN, partly undertaken at the Australian Synchrotron, has produced an ultra-flexible ultra-thin organic solar cell that delivered a world-leading performance under significant stretching and strain.

The development paves the way forward for a new class of stretchable and bendable solar cells in wearable devices, such as fitness and health trackers, and smart watches with complex curved surfaces.

The advance, which was published in Joule, was made possible by designing an ultra-thin material based on a blend of polymer, fullerene and non-fullerene molecules with the desired mechanical properties and power efficiency, according to Dr Wenchao Huang, a Research Fellow at Monash University and the article’s first author.
The thickness of the solar cell film is only three micrometres, which is ten times smaller than the width of a human hair.

Dr Huang, who completed his PhD in the lab of Prof Chris McNeill at Monash on flexible organic solar cells, received the Australian Synchrotron’s Stephen Wilkins Medal in 2016 for his exceptional doctoral thesis that made use of the synchrotron-based research capabilities at the facility.

>Read more no the Autralian Lightsource at ANSTO website

Image: Schematic of ultraflexible solar cell

Assembly lines for designer bioactive compounds

Researchers successfully bioengineered changes to a molecular “assembly line” for bioactive compounds, based in part on insights gained from small-angle x-ray scattering at the Advanced Light Source (ALS).

The ability to re-engineer these assembly lines could improve their performance and facilitate the synthesis of new medically useful compounds.

Microbes are known to possess molecular “assembly lines” that produce an important class of compounds, many of which have uses as antibiotics, antifungals, and immunosuppressants. The compounds are peptides—chains of amino acids like RNA, but shorter and produced, not by ribosomes, but by cellular machines known as nonribosomal peptide synthetases (NRPSs).

>Read more on the Advanced Light Source website

Image: Top: Comparison of experimental SAXS scattering data (black) with theoretical curves (green) obtained using an ensemble optimization method (EOM) shows excellent agreement. Bottom: LgrA structural models corresponding to the EOM analyses show large differences in conformation, similar to the differences observed using crystallography.