Researchers from Osaka University developed a new method for brain tissue analysis in high spatial resolution that requires no sample preparation.
Medical professionals all want to be able to quickly and correctly diagnose diseases. Their future ability to do so will depend on identifying what biochemicals are present in tissue sections, where the biomolecules are, and at what concentrations. For this purpose, mass spectrometry imaging–which can identify multiple biochemicals in a single experiment–will be useful. However, the stability of biomolecular sampling needs improvement to obtain the chemical distribution information with high spatial resolution.
In the recent study published in Analytical Chemistry, researchers from Osaka University used mass spectrometry to image the distribution of fat molecules in mouse brain tissue. They acquired data at a spatial resolution of 6.5 micrometers, enabling analysis on a cellular level.
The researchers used a very small capillary to gently extract lipid molecules from a tissue section, and a carefully designed setup for fine 3D directional control. Although biological tissue may often seem smooth to the naked eye, on an ultrasmall scale it’s rather rough. The ability to account for this ultrasmall-scale roughness is central to obtaining reproducible biochemical data at high spatial resolution.
“In our experiments, the probe’s vibration amplitude is constant even when the sample height changes,” says Yoichi Otsuka, first author. “We can also measure changes in sample height up to 20 micrometers, and it can be increased up to 180 micrometers.”
The researchers’ first experiments were to measure irregular distributions of molecules across an uneven surface: microwells filled with various concentrations of a dye. The measured concentrations correlated with the known concentrations, and the measured surface topography was close to the actual microwell diameter. Experiments with mouse brain sections yielded a multi-dimensional data of multiple molecules such as the distribution of certain hexosylceramides–lipids that are important in aging.
“Principle component analysis helped us integrate our wide-ranging data,” explains Takuya Matsumoto, senior author. “For example, we could assign the classes of lipids that are primarily present in the cortex and brainstem.”
Correlating such data with disease progression will require further study and perhaps additional development of the researchers’ biomolecule extraction setup. The researchers anticipate that their approach will be useful for quantitatively imaging the myriad neural networks in brain tissue. Ultimately, they hope to help medical practitioners reliably diagnose diseases such as brain cancer in a tissue section with the support of molecular information in high spatial resolution.
The article, “High-spatial-resolution multimodal imaging by tapping-mode scanning probe electrospray ionization with feedback control,” was published in Analytical Chemistry at DOI: https://doi.org/10.1021/acs.analchem.0c04144
Researchers at the University of Campinas conducted more than 120 experiments with dunes of up to 10 cm that interact for a few minutes, obtaining a model valid for dunes on the surface of Mars that are many miles long and take more than a thousand years.
Barchans are crescent-shaped sand dunes whose two horns face in the direction of the fluid flow. They appear in different environments, such as inside water pipes or on river beds, where they take the form of ten-centimeter ripples, and deserts, where they can exceed 100 meters, and the surface of Mars, where they can be a kilometer in length or more. If their size varies greatly, so does the time they take to form and interact. The orders of magnitude range from a minute for small barchans in water to a year for large desert formations and a millennium for the giants on Mars.
They are formed by the interaction between the flow of a fluid, such as gas or liquid, and granular matter, typically sand, under predominantly unidirectional flow conditions (read more at: agencia.fapesp.br/29178).
“What’s interesting is the similarity of their formation and interaction dynamics, regardless of size. As a result, we can study aquatic barchans in the laboratory to make predictions about the evolution of the dunes in Lençóis Maranhenses [a coastal ecosystem in the Northeast of Brazil] or to investigate the origins of the topography in the Hellespontus region on Mars,” said Erick Franklin, a researcher and professor at the University of Campinas’s School of Mechanical Engineering (FEM-UNICAMP) in the state of São Paulo, Brazil.
Working with his PhD student Willian Righi Assis, Franklin performed more than 120 experiments and identified five basic types of interaction between barchans.
A striking aspect of the topic is that as well as having a robust shape that appears in many different environments, barchans typically form corridors in which their sizes are approximately the same. Analysis of individual dunes suggests they should grow indefinitely, becoming steadily larger, but this is not the case. One explanation for their characteristic size in a given environment is that binary interactions, especially collisions, redistribute the mass of sand, and instead of growing continuously they subdivide into smaller dunes.
“This has been proposed in the past, but no one had extensively tested and mapped these interactions, as dune collisions take decades to happen in terrestrial deserts,” Franklin said. “Taking advantage of the fact that underwater barchans are small and move much faster, we conducted experiments in a hydrodynamic channel made of transparent material, with turbulent water flow forming and transporting pairs of barchans while a camera filmed the process. We identified for the first time the five basic types of binary interaction.”
In the experiments, the researchers varied independently each of the parameters involved in the problem, such as grain diameter, density and roundness, water flow velocity, and initial conditions. The images acquired were processed by computer using a numerical code written by the researchers. Based on the results, they proposed two maps that supplied a general classification of the possible interactions.
“Our experiments showed that when a binary collision occurs, the barchan that was originally downstream, i.e. in front, expelled a dune of an approximately equal mass to that of the barchan upstream, i.e. behind,” Franklin said. “The first impression was that the upstream barchan passed over the other barchan like a wave, but the use of colored grains helped us show this didn’t happen. Actually, the upstream barchan entered the downstream barchan, which became too large and released a mass more or less equal to the mass received.”
Interactions between the two barchans basically involved two mechanisms. One was the disturbance caused in the fluid, which bypassed the upstream barchan, accelerated and impacted the downstream barchan, which eroded. This is termed the “wake effect”. The other was the collision in which the colliding barchans’ grains merged.
“Our experimental data showed that these two mechanisms caused five types of barchan-barchan interaction,” Franklin said. “Bearing in mind that the velocity of a dune is inversely proportional to its size, the simplest two are what we call chasing and merging.”
Chasing occurs when the two barchans are roughly the same size and erosion due to the wake effect makes the downstream dune decrease in size. The two barchans then move at the same velocity and remain at a constant distance from each other. Merging happens when the upstream barchan is much smaller than the downstream barchan. Erosion caused by the wake does not substantially decrease the size of the upstream dune, so that the barchans collide and merge, forming a single dune.
The third type of interaction is exchange, which is more complicated. “Exchange happens when the upstream barchan is smaller than the downstream barchan, but not much smaller. Here, too, the upstream dune catches up with the downstream dune and they collide. As they do so, the smaller dune ascends and spreads over the larger one. During this process, however, the fluid flow, which is deflected by the new dune, strongly erodes the front of the dune, which ejects a new dune. Because it is smaller and emerges downstream, the new dune moves faster and a gap opens up between the two dunes,” Franklin said.
The last two types of interaction happen when fluid flow is very strong. “What we call ‘fragmentation-chasing’ is when the dunes are of different sizes. The wake effect on the downstream dune is so strong that it splits into two. Both the resulting dunes are smaller than the upstream dune. The result is three dunes with gaps widening between them. The last type is ‘fragmentation-exchange’, which is similar. The difference is that the upstream dune reaches the downstream dune before its division into two is complete,” Franklin said.
The five types are easy to understand in this video. In fact, the researchers were able to construct the typology thanks to the visual support afforded by the movies described in the article. “Our results, obtained for subaqueous barchans that were centimeters in length and developed in minutes, significantly advance the understanding of the dynamics and formation of this type of dune,” Franklin said. “Through laws of scale, they enable us to transpose the findings to other environments, where sizes are larger and timespans longer. Understanding the past of Mars or projecting its distant future, both of which are currently of interest to scientists, could be greatly facilitated by these findings.”
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Study shows that a tree frog endemic to a mountainous region of the Brazilian savanna is unable to disperse and find genetically closer mates when the terrain is rugged, potentially endangering survival of the species.
The savanna tree frog Bokermannohyla ibitiguara is about 4 cm long and is found only in gallery forest along streams in the Serra da Canastra mountain range in the state of Minas Gerais, Southeast Brazil. In this watery forest environment, it can grow, feed, mate, and lay eggs without needing to range very far throughout its life cycle, according to a study published in Diversity and Distributions.
According to the Brazilian and US researchers who conducted the study, topography rather than vegetation is the main factor leading to more or less dispersal of the species in the territory, and this information is even recorded in its DNA.
They analyzed genetic variation among groups of B. ibitiguara living inside and outside the Serra da Canastra National Park, a protected area in the region, discovering that the flatter the terrain, the more genetically diverse is the population.
In areas of highly variable elevation, individuals are genetically similar. In evolutionary terms, this can be harmful to the species, which becomes more susceptible to disease and climate change, for example.
“Genetic analysis and conservation studies typically take land cover into account, among other factors, but the Cerrado [Brazilian savanna] is topographically diverse, including montane regions with high plateaus [chapadões] separated by low areas. We set out to verify whether this variable terrain played a part in the genetic diversity of the species, and found that it did. The vegetation alone didn’t explain the genetic differences we identified between sites, or even within the same site. The topography did,” said Renato Christensen Nali, first author of the article and a professor at the Federal University of Juiz de Fora’s Institute of Biological Sciences (ICB-UFJF) in Minas Gerais, Brazil.
The study was one of the results of Nali’s doctoral research, conducted at São Paulo State University’s Bioscience Institute (IB-UNESP) in Rio Claro, Brazil, with a scholarship from FAPESP (São Paulo Research Foundation).
The research was part of the project “Reproductive ecology of anuran amphibians: an evolutionary perspective”, for which the principal investigator is Cynthia Peralta de Almeida Prado, a co-author of the article. She is a professor at UNESP’s School of Agrarian and Veterinary Sciences in Jaboticabal and teaches graduate students in zoology at IB-UNESP in Rio Claro.
The flatter the better
“The findings are very interesting because they bring to light a novel factor for conservation of the Cerrado, among other reasons. Ecological corridors and native forests are rightly considered important for conservation units, but more attention needs to be paid to the type of terrain. The topography should permit dispersal of the animals,” said Nali, who heads ICB-UFJF’s Amphibian Evolutionary Ecology Laboratory (Lecean).
To arrive at the results, the researchers analyzed 12 populations of B. ibitiguara, six inside Serra da Canastra National Park and six outside. Genetic diversity was much higher among the anurans living in the protected area than among those living outside the park. When the researchers correlated information on the degree of protection of the areas with the state of the vegetation, they found that these factors were less decisive for genetic diversity than the topography.
“The terrain is much more rugged outside the park, whereas inside it there’s a large, very even plateau where the anurans can disperse more, find mates in more distant areas, and increase their genetic diversity,” Nali said. “Outside the park, the rugged terrain and variable elevation appear to confine them to small areas.”
The influence of these factors was evidenced by genetic tests. The researchers used a technique known as macrosatellite marker analysis to examine specific regions of the genome and found higher allele diversity in the populations living in the park. Allele diversity is one of the determinants of genetic integrity and adaptive potential.
In addition, the populations living outside the park displayed a greater loss of heterozygosity. If this loss, which is associated with declining genetic variability, recurs across several generations, it can eventually threaten the population’s survival.
The study underscores the importance of topography as a factor to consider in conservation studies, as well as showing how the mere presence of a species in an area cannot ensure that it is not endangered.
“Molecular analysis enables us to find out if a population’s genetic status is favorable,” Nali said. “An area may have a large number of individuals, but DNA analysis may show that its genetic constitution is unfavorable, with few alleles and low heterozygosity. In practice, therefore, the population’s effective size is small.”
Although the study focused on only one species, he added, the findings can apply to others as well since the physical characteristics associated with dispersal are similar for other frogs and toads. More species need to be investigated to confirm the applicability of the findings.
The group noted that land cover nevertheless remains an important factor for conservation in the Cerrado, more than 50% of which has been converted into pasture or cropland, while less than 5% is protected by conservation units.
References: Nali, RC, Becker, CG, Zamudio, KR, Prado, CPA. Topography, more than land cover, explains genetic diversity in a Neotropical savanna tree frog. Divers Distrib. 2020; 26: 1798– 1812. https://doi.org/10.1111/ddi.13154