Jeckelmann1

Glow discharge apparatus for high quality and reproducible cryo-electron microscopy specimen preparation

Glow discharge apparatus for high quality and reproducible cryo-electron microscopy specimen preparation

Cryogenic electron microscopy (cryo-EM) has become the preferred method for determining the atomic-resolution structures of macromolecules 1. The preparation of cryo-EM specimens is a multi-step procedure, typically divided into four main stages: (i) glow-discharging the cryo-EM grids, (ii) applying the macromolecular sample to the grid, (iii) blotting off excess protein solution, and (iv) vitrifying the specimen in a cryogenic liquid. Specialized equipment is employed in steps (iii) and (iv) to meticulously control these processes. The importance of vitrification in cryo-EM grid preparation was highlighted when the Swiss researcher Jacques Dubochet received the Nobel Prize in Chemistry in 2017 for his pioneering work in this area (https://www.nobelprize.org/prizes/chemistry/2017/summary/).

Cryo-EM grids used for high-resolution protein structure determination are typically made of copper or gold with a holey carbon film attached on one side. Chemically, this surface is hydrophobic and would naturally repel hydrophilic protein samples. Therefore, cryo-EM grids must be rendered hydrophilic through a process called glow-discharge. Since the glow-discharging procedure significantly impacts protein absorption behavior on cryo-EM grids, precise control of this process is essential. With the funding acquired through the “UniBern Forschungsstiftungs” grant, we obtained a Dual-Chamber PELCO easiGlow™ glow-discharging device, which allows for controlled glow-discharging and ensures a more reliable and reproducible production of cryo-EM specimens (Figure 1A).

The PELCO easiGlow™ glow-discharging device was installed in the sample preparation room of the structural biological branch of the Microscopy Imaging Center of the University of Bern (MIC, https://www.mic.unibe.ch/). Since its installation, this device has been used to produce cryo-EM specimens that are analyzed on the MIC’s high-end electron microscope Titan KRIOS G4. The use of the PELCO easiGlow™ device has already proven invaluable to our research, enabling our group to recently publish the high-resolution structures of (i) the bacterial green-light-absorbing proton pump proteorhodopsin (GPR) 2 (Figure 1B) and (ii) the bacterial glucose transporter IIC(B) 3 (Figure 1C). Moreover, the PELCO easiGlow™ glow-discharging device holds significant value for all MIC users at the University of Bern.

Jean-Marc Jeckelmann, PhD
Institute of Biochemistry and Molecular Medicine

Links:

1             Guaita, M., Watters, S. C. & Loerch, S. Recent advances and current trends in cryo-electron microscopy. Curr. Opin. Struct. Biol. 77, 102484 (2022). https://doi.org/10.1016/j.sbi.2022.102484

2             Hirschi, S. et al. Structural insights into the mechanism and dynamics of proteorhodopsin biogenesis and retinal scavenging. Nature Commun. 15, 6950 (2024). https://doi.org/10.1038/s41467-024-50960-3

3             Roth, P. et al. Structure and mechanism of a phosphotransferase system glucose transporter. Nature Commun. 15, 7992 (2024). https://doi.org/10.1038/s41467-024-52100-3

 

Ausprey3

Microclimate Buffering within Alpine Landscapes

Microclimate Buffering within Alpine Landscapes: Linking Ecophysiology, Behavioral Plasticity & Microhabitat Selection of Alpine Avifauna in a Changing Climate

 Mountain ecosystems are among the regions most susceptible to temperature warming, and species adapted to alpine ecosystems are predicted to face local extinctions via physiological and/or behavioral intolerance to rapid increases in temperature. However, information is lacking regarding the ability of alpine birds to adapt both behaviorally and physiologically to rapidly warming temperatures.

Our research project leverages state-of-the-art advances in thermal imaging, GPS tracking, and animal biologging technologies to examine how alpine birds exhibit behavioral plasticity to temperature warming via selection of microhabitats that provide cool microclimates and buffering from high temperatures. We are focusing on two species predicted to respond differently to warming temperatures: the Alpine Rock Ptarmigan (cold adapted) and Rock Partridge (warm adapted). The ultimate goal of the project is to provide concrete recommendations for managing microhabitat structures within alpine landscapes that are predicted to provide microclimatic buffering and refugia from thermal warming.

Funding from the UniBern Forschungsstiftung allowed us to purchase a professional thermal imaging drone (DJI Mavic 3T) that maps ground surface temperatures at extremely fine resolutions relevant to the biology of our focal species (<10 cm). Funding also covered associated accessories that allowed efficient fieldwork in challenging alpine landscapes, including a professional base station that provided real-time kinetic (RTK) connectivity to the drone and centimeter-level spatial accuracy while mapping. During the summer of 2024, we successfully deployed the drone during the summer breeding season (June – August) within territories of our two target species. This work resulted in the creation of over 250 high resolution thermal maps of ground surface temperature that will be aligned with fine-scale movement data generated by GPS tags mounted on captured birds to understand how the two species are selecting for different thermal regimes. The drone work directly supported the MSc thesis of Luca Robbi, who conducted the fieldwork and mastered skills in drone piloting procedures, land cover mapping, and data management that will be of fundamental value for him when securing an internship and eventual full-time employment in the environmental sector. The drone will be integral to completing the project, which is planned to continue for two more years in 2025 – 2026.

Dr. Ian J. Ausprey
Institute of Ecology & Evolution
Division of Conservation Biology

 

Luca Robbi (MSc student) preparing the drone for a mapping mission at our field site near Becs de Bosson, Valais.
Luca Robbi (MSc student) preparing the base station used for real-time kinetic (RTK) positioning of the drone at our field site at Sanetsch Pass, Valais.
Flying the drone.
Raissig1

A hand-held porometer for high-throughput phenotyping of plant-atmosphere gas exchange in grasses

A hand-held porometer for high-throughput phenotyping of plant-atmosphere gas exchange in grasses

Land plants must balance water vapour loss through leaves with efficient carbon dioxide (CO2) uptake for photosynthesis. Specialised “breathing pores” on leaves called stomata can open and close to minimise water loss and maximise CO2 uptake. Thus, plants with efficient and fast stomatal pores are likely more resilient to the upcoming, climate-change-induced drought and heat periods.

The ”Stomatal Biology” group at the Institute for Plant Sciences is interested in how different stomatal morphologies affect gas exchange and how we can bioengineer stomatal form to prepare plants for the upcoming climatic challenges. We primarily work with grasses, which form morphologically innovative stomata with very rapid opening and closing dynamics. The rapid stomata of grasses contribute to the high water-use efficiency of grasses and their evolutionary success. Nowadays, grasses dominate many natural and agricultural ecosystems and our most important food crops like maize, rice and wheat are all grasses.

Gas exchange measurements are either very time-consuming and laborious or rather inaccurate. With the help of the UniBern Forschungsstiftung, we were able to acquire the hand-help porometer LI-600N, which allows for rapid and highly accurate measurements of steady-state gas exchange, while simultaneously assessing photosynthetic capacity. Therefore, the LI-600N will enable us to perform high-throughput screens of large populations of different grasses or grass genotypes. This will identify species that show a high photosynthetic efficiency, which is relevant for yield, yet low stomatal conductance, which is relevant for water-stress resilience.

Prof. Dr. Michael T. Raissig
Institute of Plant Sciences (IPS)

https://raissiglab.org/
https://www.ips.unibe.ch/

Raissig1

Figure 1: The measuring head of the porometer LI-600N for narrow leaves (left) and LI-600 for broad leaves (right).

Petelle2

Tracking chicks to understand individual movement phenotypes and their corresponding welfare outcomes

Tracking chicks to understand individual movement phenotypes and their corresponding welfare outcomes

Our research group focuses on the health and welfare of poultry and rabbits as it relates to their housing. To this end, one of the main themes in our research is individual variation in movement and space use within a commercial setting. Our past research shows that individuals are consistent in how they move throughout the aviary, and that individuals are distinctly different from one another. For example, some individuals are very active, moving throughout the different levels of the aviary rapidly throughout the day, while others tend to stay on one level for most of the day. However, we don’t know when distinct individual variation emerges or how it changes across the lifetime of the individual.

With the generous support of the UniBern Forschungsstiftung, we were able to purchase additional radio frequency identification (RFID) antennas to install in our rearing barn to gather positional data of chicks at one day of age. From this data, we will assess whether individuals differ in their space use right after hatch or whether differences develop slowly over the rearing period. We can then link these movement differences with overall health and welfare across their lives.

At present, we have already carried out a pilot study in our experimental barn (Figure 1) that demonstrates we are able to obtain data from chicks and that movement between chicks across the first weeks seem to be consistently different, however our validation is ongoing. We also recently installed antennas in two pens in our rearing barn and started tracking 600 chicks (300/pen) to determine movement differences in a commercial setting (Figure 2).

Drs. Michael Toscano and Matthew Petelle

ZTHZ – Center for Proper Housing: Poultry and Rabbits

VPHI – Veterinary Public Health Insitute

 

Figure 1: Fifteen chicks in one of our pilot pens. Each chick is outfitted with and RFID wing tag to monitor their location within the pen.

Figure 2: One day old chick with RFID tag in commercial rearing pen.  Antennas are under the brown chick paper. 

Mirra

Evaluation of the parasympathetic tone activity (PTA)™ index as biomarker of nociception in pigs

Evaluation of the parasympathetic tone activity (PTA)™ index as biomarker of nociception in pigs

One of the main aims of our group is to research on nociceptive processes, diagnostic strategies and treatment approaches in animals.

A major challenge for veterinarians is to correctly evaluate nociception during general anaesthesia. In this context, novel strategies should be investigated.

With the financial support of the UniBern Forschungsstiftung we were able to acquire the parasympathetic tone activity (PTA)™ monitor (Fig.1). Through the analysis of the activity of the parasympathetic nervous system (heart rate variability evaluation), it provides the anaesthesiologists with an index of nociception (PTA index), expressed on a scale between 0 and 100: values close to 100 indicate absence of nociception, while values below 50 suggest presence of nociception.

The present project aims at assessing the reliability of the PTA in pigs, species largely used for experimental and translational studies. Data were collected before, during, and after the application of various nociceptive stimuli. while the pigs were under propofol-based anaesthesia. Data collection has been completed, and analysis is ongoing.

The PTA index has the potential to serve as an additional tool for evaluating the nociceptive/anti-nociceptive status of animals under general anaesthesia.

Dr. med. vet. Alessandro Mirra
Anaesthesiology and Pain Therapy Section
Vetsuisse Faculty 

Figure 1: Parasympathetic tone activity (PTA)™ monitor

Mueller-U2

Reflection Microscope for characterization of collective mucociliary activity

Reflection Microscope for characterization of collective mucociliary activity

Primary ciliary dyskinesia (PCD) is a rare hereditary disease impairing the ciliary activity and resulting in a variety of respiratory symptoms, such as neonatal respiratory distress in term-born infants, chronic rhino-sinusitis and persistent wet cough from the day of birth, and recurrent respiratory tract infections often resulting in later bronchiectasis. Mutations in more than 50 genes are known to cause defects in the structure and function of cilia. Abnormalities in the ciliary structure lead to ciliary dyskinesia, which results in an impairment of the mucociliary transport. Reduced mucociliary transport then leads to less efficient removal of respiratory pathogens from the airways. Due to the high number of potentially involved genes, the diagnosis of PCD is very challenging. There is no single gold standard test and a combination of methods has to be performed. Therefore, we would like to use computational high-speed video reflection microscopy (CRM) to assess the collective mucociliary activity in air-liquid interface (ALI) cell cultures of nasal epithelial cells derived from patients with suspicion of PCD. This approach has the potential to become the first diagnostic approach allowing to diagnose PCD in a highly accurate as well as automated and cost-effective way.

With the support of the Berne University Research Foundation we bought a reflection microscope to record the surface of ALI-cultured, differentiated cells of patients suspicious for PCD. The addition of a climate chamber furthermore enabled temperature control during experiments. The videos can be analyzed using our Cilialyzer software and we will investigate which parameters of the collective ciliary activity could discriminate reliably and sensitively between healthy and PCD. Furthermore, the microscope is used to test the potential of various drugs (e.g. cystic fibrosis modulator drugs) to improve ciliary activity and mucociliary transport in vitro.

PD Dr. Loretta Müller

Department for BioMedical Research, Lung Precision Medicine (LPM)
Pädiatrische Pulmonologie, Inselspital, Kinderklinik

Figure 1: Reflection microscopy partially funded by the Berne University Research Foundation additionally equipped with a climate chamber to regulate the temperature (mostly kept to 37°C). 

Figure 2: Image example of a video taken with the reflection microscope of a healthy ALI cultured cell culture. 

Karousis22

Development of an in vitro translation-based screening system to identify mRNA translation inhibitors

Development of an in vitro translation-based screening system to identify mRNA translation inhibitors

The goal of our project is to develop a platform that allows the identification of small molecules that inhibit translation in human cells using cell-free lysates. The lysates are also used for studying the mode of action of proteins from coronaviruses (2,3).

With the funding that was acquired thanks to the UniBern Forschungsstiftungs grant we obtained a shaking incubator device that allows us to produce ample amounts of cells and lysates for our screening purposes, based on a previously published protocol (1).

 

Evangelos D. Karousis, PhD
Dept. of Chemistry, Biochemistry and Pharmaceutical Sciences

Leidel1

Using big data for the analysis of cellular translational control

Using big data for the analysis of cellular translational control

Protein synthesis is essential for any living organism. However, how the dynamics of mRNA translation affect the formation of functional proteins is poorly understood. To gain new insights into the relationship between translation dynamics and protein folding, we analyze mutants that show codon-specific translation defects.

We analyze these mutants in detail using a variety of omics techniques such as ribosome profiling or pulse-chase proteomics. As all these methods generate large amounts of data, it is essential to be able to store and handle such large datasets. With the funding we received from the UniBern Forschungsstiftung, we purchased an extension to our redundant NAS storage system and a new analysis server. We have already used it to characterize key enzymes in translation dynamics (Wu et al., bioRxiv 2024; Lin et al., Mol Cell 2024).

The next goal is to develop machine learning strategies to analyze and integrate these datasets. However, the extension of our NAS system, combined with the purchase of a new analysis server, has already led to exciting new discoveries and will continue to do so in the future.

Prof. Dr. Sebastian A. LEIDEL

Department of Chemistry, Biochemistry and Pharmaceutical Sciences

Links:

– Wu et al., BioRxiv 2024: DOI: 10.1101/2024.02.27.582385

www.biorxiv.org/content/10.1101/2024.02.27.582385v2

– Lin et al., Molecular Cell 2024: DOI: 10.1016/j.molcel.2024.06.013

https://doi.org/10.1016/j.molcel.2024.06.013

Towbin1

Robustness and Individuality in Organ Growth Control of C. elegans

Robustness and Individuality in Organ Growth Control of C. elegans

Correctly sized body parts are crucial for organismal function. For example, small discrepancies in limb length severely obstruct motility, and overgrowth of cardiac muscle is a prevalent cause of heart failure. The growth of different cells and organs must therefore be tightly coordinated to prevent that even small differences in growth rates amplify to large differences in size during development. How growth signals are propagated from cell to cell, and how organs integrate combinatorial signals from different tissues is a fundamental, yet poorly addressed question of high biomedical relevance. We address this question using quantitative live imaging experiments with the nematode worm C. elegans.

Specifically, we used strains expressing a green fluorescent protein in the pharynx of C. elegans and a red fluorescent protein in all cells of the body. Using a micro cultivation technique, we grew individual animals in small chambers that can be maintained on a fluorescent microscope over many days and the entire development of C. elegans. This technique allows us to precisely monitor the growth of the pharynx and the body size at high time resolution for individual animals and measure the heterogeneity in growth and size among individuals of an isogenic population. Our key finding is that there exists a molecular mechanism that coordinates the growth of the pharynx and the body, such that the relative proportions between the pharynx and the body length are very robust to even strong perturbations.

The “Nikon piezo stage” that we were able to acquire thanks to the UniBern Forschungsstiftungs’ grant, has become essential for our investigations. The piezo drive enables extremely rapid acquisition of three dimensional image stacks with minimal time delay between optical sections. This speed is crucial for our application, as the animals are not anesthetized inside the chambers and move rapidly. Thanks to the rapid acquisition, we can now effectively acquire large stacks of images and reconstruct 3-dimensional representations of the animals.

Prof. Dr. Benjamin Towbin
Institute of Cell Biology

C. elegans ...
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Fördersumme 2024

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