Studying age-associated pathologies through temperature controlled microscopy

Fluorescent labelled E. coli can be seen in the swollen pharynx of some dying C. elegans (P, left) but not others with an atrophied pharynx (p, right).

With improving healthcare humans are living longer than ever before, but with longer life comes ever more senescence-related pathologies. Understanding the role genes and environment play in the development of such pathologies in the complex system of our bodies is difficult.  The nematode Caenorhabditis elegans is a great model organism and has been used extensively to study the biology of ageing because just like more complex animals, C. elegans also develop senescent pathologies.

Researchers from the University College London and Washington University, MO studied age-related pathologies in C. elegans and their role in limiting lifespan. Understanding these processes in a simple organism may in turn help to further understand the origins of human age-related pathologies.

To investigate the causes of death, the group analysed the corpses of recently expired wildtype C. elegans and found two particular forms with different pharyngeal pathologies. One type, named P ("big P") death, occurred earlier than the other and had increases in the posterior bulb size of 20-120%. The other, named p ("small p") death, showed a shrinking of the posterior bulb by up to 70%.

The figures above show the age distribution and percentage survival of P and p deaths.

Dissections and RFP labelling of the E. coli food source established that the enlarged posterior bulb in P death individuals was due to E. coli infection in elderly C. elegans. The high pharyngeal pumping rates typical of young nematodes is thought to mechanically damage the cuticle, creating vulnerability to invasion. Their findings suggest there is a narrow time frame in which young nematodes are thus susceptible. Consistent with this, the group found that mutants with reduced pumping rate had fewer P deaths, and lived longer.  However, it was also found that worms dying with P and p death previously had similar pumping rates, so why did some worms but not others get an infected pharynx? 

The difference appeared to be due to the ability of some nematodes (p death type) to heal the cuticle thereby preventing invasion. 

When asked about their work and the role of the PE120 stage, Professor David Gems stated, “to help identify old age pathologies that limit life, we watched nematodes as they aged, measuring a range of pathologies, and then measured their lifespan. By measuring how well each pathology correlated with lifespan we could identify pathologies likely to cause death. But to do this required repeatedly putting immobilized nematodes under the microscope, and we needed to do this in a way that wasn’t so stressful that it shortened their lifespan. By using the PE120 Linkam stage to gently cool the worms, we were able to avoid using stressful anaesthetics. We were able to confirm that repeated viewing of nematodes using the PE120 Linkam stage in this way did not shorten their lifespan.”

The group used a novel approach to understand ageing by analysing and combining pathology and mortality profiles. Further work can now be conducted with a view to understanding how genes that affect lifespan differentially affect worms dying from different causes.

By Tabassum Mujtaba

Zhao, Y. et al. Two forms of death in ageing Caenorhabditis elegans. Nat. Commun. 8, 15458 doi: 10.1038/ncomms15458 (2017).

November's Paper of the Month

Solution printing is a novel technique which uses an ink solution, containing semi-conductor precursors or nanoparticles, and deposits these on substrates with desirable characteristics. This offers a cost-effective method of creating large area thin film optoelectronics whilst also offering precise control over the stoichiometry and adaptability of the material. Metal halide perovskites have superb optoelectronic properties and the last few years has seen their power conversion efficiency increase rapidly, largely through the optimisation of the crystal morphology. 

The requirement to control morphology has posed a problem for solution printing. The understanding of crystallisation in dynamic flow of perovskite inks is quite limited, thus imposing restrictions in achieving high-quality perovskite films by the solution-printing technique.

A group from the Georgia Institute of Technology and the University of Nebraska-Lincoln used a meniscus-assisted solution printing method to elucidate the crystallisation kinetics of perovskite inks and help create high efficiency perovskite solar cells. The thin-films were created with preferred crystal orientation with micrometre-scale grains. 

When discussing their work Professor Lin said, “through integrating the meniscus effect within the solution printing, we found that the solvent evaporation could be largely promoted by the meniscus effect instead of the thermal evaporation as in conventional doctor-blade coatings, thus leading to the low-temperature solution-based deposition of high-quality perovskite films with preferred crystal orientations. This low temperature feature circumvents thermal degradations and thermomechanical fatigues on perovskite and electrode materials, as well as decreases energy consumptions. Our technique paves the pathway for depositing perovskite thin films on flexible polymer substrates, and is anticipated to promote the future development and applications of perovskites in low-cost, large-area, and flexible optoelectronic devices.”

The group used an LTS350* to control the substrate temperature during the meniscus-assisted solution printing process, due to its capability of precisely controlling the temperature at ±0.1oC. Their investigation on the crystallisation kinetics of perovskite films revealed that a large temperature fluctuation would seriously impact the crystallisation kinetics of perovskite films during the meniscus-assisted printing process. The LTS350* was ideal for maintaining the substrate at a constant temperature and focusing on the exploration of the meniscus effect on the perovskite crystallisation process. 

Their study helped to uncover the crystallisation kinetics of perovskites during the printing process, providing rational guides to precisely control the crystallisation morphology of printed perovskite films. By improving the control over morphology, the group’s work helps to pave a route to large-area optoelectronic devices for commercial applications.

*The LTS350 has been superseded by the LTS420 offering a large temperature range and better temperature control to 0.01°C.

Lin et al., Meniscus-assisted solution printing of large-grained perovskite films for high-efficiency solar cells. Nat. Commun. 16045, doi:10.1038/ncomms16045 (2017).

October's Paper of the Month

Partly fossilized mycelium of fungal hyphae on a zeolite crystal from 740 m depth in fractured granite. Back-scattered Environmental Scanning Electron Microscopy image. Width of view 600 µm. Photo credit: Henrik Drake, Magnus Ivarsson.

Despite being considered vital for energy cycling of the earth; the deep biosphere is one of the least understood ecosystems. It is thought to have approximately 19% of the earth’s biomass yet samples are hard to come by, making their study difficult. Microorganisms from the deep biosphere that have been studied are generally prokaryotes, with microeukaryotes being largely ignored.

Recently samples were taken from a 740m deep drill core sample in Sweden after the site was investigated for its suitability for deep nuclear waste repositories. Findings have shown the presence of fossil and active fungi in these deep ecosystems, but little work has gone into understanding them.

Drake et al., studied the microorganisms in these deep crystalline fractured rock samples. Their aim was to gain a better understanding of the microbial processes in the continental crust. The knowledge of this vast realm is very scarce and tells us more about life forms and processes under extreme conditions which may also have important implications for nuclear waste storage. 

Their analyses found the microorganisms belonged to the Kingdom Fungi and were found to be anaerobic. The closest systems studied were that of anaerobic fungi in the rumina of ruminant animals. It was proposed that the fossilised fungi also shared a symbiotic relationship with bacteria in the deep biosphere. 

The group used a THMS600 to help indicate the approximate age of the fungi. Dr Drake said, “The THM600 was used to investigate fluid inclusions in calcite crystals that were spatially related to the fungi. The fluid inclusion signatures gave us information about past conditions (e.g. salinity) in the fracture void. Because no radiometric dating could be made of the fungi, the fluid inclusion signatures (when put in a paleohydrogeological context) serve as an important temporal indicator for when the fungi were active.” 

Their work highlighted an intimate relationship between the fungi and sulphate reducing bacteria, further drawing attention to the richness of the deep oligotrophic biosphere which is often neglected. These fungi were found to provide significant amounts of H2 to autotrophic microorganisms in the crystalline continental crust.

The group also looked at the biochemistry of these fungi and found they may pose a threat to repositories of toxic waste.  This is through either directly breaking down the barriers holding the waste, or by facilitating the bacterial community into doing so. 

Their work highlights the importance of studying these neglected geological microorganisms. With fossil fuels running out, nuclear energy may be the way forward. But to safely store away waste products, understanding their chemical and geological environment is of utmost importance as illustrated by Drake et al., As such it becomes vital to study ecosystems, such as the deep biosphere, in its entirety. 

By Tabassum Mujtaba

Drake et al., Anaerobic consortia of fungi and sulfate reducing bacteria in deep granite fractures. Nat. Comms 8, Article number: 55 doi:10.1038/s41467-017-00094-6 (2017)

September's Paper of the Month

Serpentinization is central to many theories of the origin of life. 

The hydrothermal alteration of mantle rocks, referred as serpentinization, occurs when the mantle is exposed to aqueous fluids circulating below 400°C, leading to the formation of serpentine, hydrogen and other minerals. It is a process heavily involved in mass exchange between the mantle and the surface and influences geochemical cycling and fluid-mobile elements. It occurs in various submarine environments including mid-ocean ridges and subduction zones and it affects the physical and chemical properties of the oceanic lithosphere.

 It is also pivotal to current theories on the origin of life. Serpentinization is likely to have provided the crucial chemical gradients required for life to being when the earth was simply rock, water and carbon dioxide. 

Despite being a process vital to our understanding of the origin of life and the Earth´s lithospheric mantle activity, the rates and the environmental factors affecting serpentinization are poorly understood. A collaborative effort from Virginia Tech, The Free University of Berlin, Woods Hole Oceanographic Institution and The University of Texas used synthetic fluid inclusions as micro-reactors in olivine crystals as a model to study the rate of serpentinization. This method allowed them to study mineral precipitation and water activity in real time and in situ.

When discussing their work, Dr. Lamadrid said “We trapped synthetic fluid inclusions (tiny droplets of fluid) with a seawater-like composition in gem quality olivine crystals and then we set the samples to serpentinization conditions (~280ºC). Within a few days, serpentine crystals begin to precipitate inside the synthetic fluid inclusions. Since the inclusions are isolated any changes inside the inclusion can be observed and we can model them as chemical micro-reactors. The serpentinization reaction consumes H2O, so the original salinity starts to increase as more H2O leaves the fluid to form new serpentine crystals. As such, we were able to monitor the amounts of H2O leaving the fluid by measuring changes of the salinity inside the inclusion. The salinity of the fluid inclusions were measured with high precision by measuring changes in the freezing point depression of the fluid inclusions with the Linkam THMSG600 stage.”

Their technique allowed them to study the mineralogy and chemistry of the reaction products. After carrying out experiments with different salinities and fluid compositions, they found the reaction to be highly sensitive to the salinity and chemistry of the fluid. This poses interesting concepts of where serpentinization may occur in the earth’s mantle as well on other planetary bodies. Their novel micro-reactor technique could also be applied to many other minerals, reaction products, and fluid compositions to study fluid-rock reactions in real time and in situ. 
 

By Tabassum Mujtaba

 Lamadrid, H. M. et al. Effect of water activity on rates of serpentinization of olivine. Nat. Commun. 8, 16107 doi: 10.1038/ncomms16107 (2017).