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Mammalian cell culture is a fundamental tool used to study living cells. Presently, the standard protocol for performing cell culture involves the use of commercial media that contain an excess of nutrients to prevent depletion during experiments. While this reduces the likelihood of cell starvation, it creates non-physiological culture conditions that have been shown to ‘re-wire’ cellular metabolism and broadly influence cell physiology. Recently, researchers have developed physiological media like Plasmax, formulated to approximate the nutrient composition of human blood plasma. Although this represents an improvement in cell culture practice, such physiologic media may be vulnerable to nutrient depletion over the course of an experiment  extending over several days. In this study we directly addressed this concern by measuring the rates of glucose and amino acid depletion from Plasmax by several cancer cell lines (PC3, LNCaP, MCF-7 and SH-SY5Y) over 48 hours. In all cell lines, depletion of glucose from Plasmax was rapid such that, by 48h, cells were hypoglycemic (less than 2mM glucose). Most amino acids were similarly rapidly depleted to sub-physiological levels by 48h. In contrast, glucose and most amino acids remained within the physiological range at 24h. Using RNA sequencing, we show that this nutrient depletion is associated with enrichment of cellular starvation responses, amino acid biosynthesis, apoptotic signalling, and endoplasmic reticulum stress response. Alterations in mitochondrial respiration using Seahorse extracellular flux analysis were also observed. Taken together, these results exemplify the metabolic considerations for Plasmax use, highlighting that cell culture in Plasmax requires daily media exchange.

SEB Conference 2022

Rapid nutrient depletion by cancer cells cultured in the physiologic medium Plasmax  

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2022 will find over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years.
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2022 found over 1500 experimental biologists from across the globe converging on the third-largest French city near the Mediterranean coast and the fastest-growing city in the country over the past 25 years - Montpellier, France.

Ocean acidification and warming caused by increased anthropogenic carbon dioxide emissions poses a tremendous challenge to marine calcifiers such as intertidal bivalves. The approach to studying acclimation potential or adaptation to climate change are often conducted under artificial conditions where food is provided ad libitum and do not consider tidal emersion. These limitations present problems for extrapolating results to natural ecosystems. For instance, food provisioning and regular emersion can enhance or limit acclimation capacities of marine organisms. Here we investigate the physiological response of young Pacific oyster Crassostrea gigas exposed for 3 months to ambient and future (+3°C, -0.3 pH unit) climate scenarios under two food regimes (ad libitum vs restricted) and two tidal levels (subtidal vs intertidal, emersion twice daily) under laboratory conditions. Oysters were sampled for biometric and physiological rate measurements (respiration and food intake), biochemistry (energetic reserves and enzyme assay) and histology to evaluate their reproductive status. At the end of the experiment, oysters were further challenged with a viral disease to investigate the physiological cost of acclimation to these conditions and their immune response. Preliminary analyses suggest that food level was the most influential factor, followed by climate scenario and tidal level. We indeed found that growth, food intake, reproduction and disease resistance of oysters were negatively affected by food restriction. Future conditions enhanced oyster physiological rates and sexual maturation, particularly at high food levels. Finally, tidal level had no major effect on these variables, suggesting physiological compensatory mechanisms during emersion. Our study suggests that the oyster is robust to future climatic conditions, even when feeding was restricted. A detailed analysis of the results will be presented at the conference.

Physiological and immune response of oysters to climate change as a function of diet and tidal regime

The genus Primula L. (Primulaceae) represents approximately 430 species, distributed all over the Northern Hemisphere in temperate to alpine regions. It is an important floristic element of the Himalayan flora and is considered an ecological indicator of the high-altitude vegetation. Many Primula species show two morphs (pin and thrum) based on style length. The stigma of pin plants is positioned at the entrance of the flower, exhibiting a long style, while in thrum flowers, the style is much shorter, leaving the stigma around halfway up the floral tube. Among these species, Primula denticulata exhibits a large extent of occurrence in a broad altitudinal range (2096-3700 m). A study was undertaken to understand the frequency, morphological and phenological variations between two morphs of the species along an altitudinal gradient in the Western Himalayas. We recorded the numbers of pin and thrum flower morphs at three different elevations (2337m, 3000m, and 3700m) during 2021 and 2022. Our results indicate variation in morph frequency at middle elevation i.e., 3000m, while at lower and higher elevations, both the morphs have an equal frequency. A delay was observed in the onset of phenological events with increasing elevation. We observed more numbers of floral visitors at lower and middle elevations than at higher elevations. The stigmatic surface of pin and thrum morphs were found to be different under Scanning Electron Microscope (SEM). Along with these morphological and phenological observations, genetic diversity analysis of P. denticulata is underway with 290 samples collected from different populations along the altitudinal gradient and 100 primer pairs designed from Simple Sequence Repeats (SSRs). As a result of this study, the correlation of morphological and phenological studies with genetic variation existing along the altitudinal gradient can be observed. The study will show the impact of changing environmental conditions on the diversity of the plant along the altitudinal gradient.
Keywords: Primula denticulata, Morph frequency, Altitudinal gradient, Morphological variations, Phenological variations, Changing environmental conditions








Effect of morphological, phenological variations and morph frequencies on population genetic diversity along an altitudinal gradient in heterostylous Primula denticulata

Microsphere super-resolution microscopy has recently received a lot of attention due to its simplicity and its capability to resolve features beyond the diffraction limit for living systems, in real time, without being invasive. Current systems which are able to image small biological structures are destructive, invasive (they use fluorescent dyes) or they provide reconstructed images (i.e. not a real time imaging of living organisms). The authors designed and built a bio-microscope tailored around the SMAL® (Super-Resolution Microsphere Amplifying Lens) objective lens which overcomes these drawbacks. The novelty of the setup is the ability to study cell’s structures and viruses which previously were not possible to be imaged using conventional white light microscopy. In this work, along various plant cells, label-free tobacco mosaic virus was employed for imaging in this work due to its stability, reliability, and its dimensions beyond the diffraction limit. The results were compared with images obtained using an X100/1.40 NA oil immersion objective lens fitted onto a white light microscope, in the same location. Microsphere assisted microscopy is a novel type of optical microscopy with great potential for high resolution imaging of in vivo biological structures and pathogens.

Optical super-resolution imaging of tissues and tobacco mosaic virus (TMV) using microsphere technology

Improving the performance of the CO2-fixing enzyme Rubisco is among targets for increasing crop yields. Here, Earth system models (ESM) of canopy C3 and C4 photosynthesis were combined with species-specific Rubisco parameters to quantify the consequences of bioengineering foreign Rubiscos into C3 and C4 crops under field conditions. The sunlit/shaded model for canopy photosynthesis was used together with species-specific Rubisco kinetics parameters including maximum rate (Kcat), Michaelis-Menten constant for CO2 (Kc21%O2), specificity for CO2 to O2 (Sc/o), and associated heat activation (Ha) values. Canopy scale consequences of replacing native Rubiscos in wheat, maize and sugar beet with foreign enzymes from 27 species were modelled using data from Ameriflux and Fluxnet databases. Variation among the included Rubisco kinetics differentially affected modelled carbon uptake rates, and Rubiscos from several species of C4 grasses showed the greatest potential of over 50% carbon uptake improvement in wheat, and over 25% improvement in sugar beet and maize. 

Rubisco substitutions predicted to enhance crop performance through carbon uptake modelling

To cope with a sustained drop in temperature plants need to adjust their photosynthesis to better suit the prevailing conditions. Fully developed leaves with fixed morphology alter their protein content by upregulating limiting factors. This dynamic response is called dynamic acclimation and requires days to be completed. For acclimation to be completed energy and elements need to be invested in de novo protein synthesis. A limitation to soil nitrogen availability can hinder this response and affect plant survival after cold exposure. To test this hypothesis, we grew Arabidopsis thaliana plants in three different N soil availabilities and exposed them to cold for 7 days. Plants were grown in sand and fertilized with nutrient solution containing 2.5mM, 5mM and 15mM total N at 20oC day/ 18 oC night. After 8 weeks when the first fully developed leaves immerged plants were transferred at 5 oC for 7 days before acclimation was assessed. Our results indicate that N availability plays a pivot role in plant response to cold stress. Only plants grown at the highest N availability were able to upregulate their maximum photosynthetic capacity while plants grown at the lowest N availability were unable to cope with the temperature shift and as a result had lower photosynthetic capacity after cold exposure. Plants grown in 5mM total N were able to maintain the same photosynthetic capacity after cold exposure. 

Plants cold ressponse and how is affected by nitrogen availability

So called ‘drop-in’ Biofuels produced from plants are becoming increasingly important for the aviation and shipping industries, which urgently need to move away from fossil fuels in order to limit their impact on the climate. Algae farms provide a number of benefits over traditional biofuel crops, such as sugar cane, by alleviating competition for arable land. When exposed to high light, the green alga Chlamydomonas reinhardtii produces triacylglycerols (TAGs) which can be converted to drop-in biodiesel. Excess light results in the over-reduction of the photosynthetic electron transport chain, and it has been suggested that the production of TAGs is in response to this redox imbalance. A deeper understanding, therefore, of how Chlamydomonas cells address redox imbalance will help to inform future work aiming to maximise TAG production in these conditions. Our work has highlighted a potential role for the phenomenon known as light enhanced respiration (LER) in responding to redox stress induced by high light. We show that presence of acetate in the growth media may allow cells to better acclimate to high light by increasing the capacity of the cells for LER. Autotrophic cells (cells grown without acetate) appear to be more susceptible to photodamage than mixotrophic (grown with acetate) when grown in high light. Our results display the complexity of the high light response and provide preliminary evidence for the potential of the mitochondria as a tool for addressing redox imbalance. 

A role for mitochondria during high light stress in Chlamydomonas reinhardtii

Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus anti-gravitropic offset (AGO) mechanisms. Here we report a new root angle regulatory gene termed ENHANCED GRAVITROPISM1 (EGT1) that encodes a putative AGO component. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes a F-box and Tubby domain containing protein which is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wildtype. Transcript profiling revealed Hvegt1 roots deregulate cell wall-loosening enzymes and ROS cofactors. In situ analysis identified that HvEGT1 is highly expressed in inner root tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. Atomic Force Microscopy measurements revealed that elongating cell walls in Hvegt1 mutant roots are significantly less stiff than wildtype. Hence EGT1 appears to control root angle by regulating cell wall stiffness in elongating root tissues, counteracting the gravitropic machinery’s known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing a novel anti-gravitropic mechanism.

Root angle in cereals is controlled by a novel anti-gravitropic mechanism 

Programmed cell death (PCD) is a genetically managed biological process that results in the targeted sacrifice of unwanted or diseased cells. Plants use PCD for various purposes such as development, defence against pathogens, response to abiotic stress caused by UV radiation or heat shock. Several proteinases are involved in the PCD process; among them, cathepsin B (CathB) which plays an important role in pathogen defence, plant development, germination, senescence, microspore embryogenesis and abiotic stress response. A greater understanding of the molecular mechanisms underlying the processes downstream of CathB is the next important step for new discoveries in cell biology and applications in biotechnology. To achieve this goal, it is important to discover and test substrates cleaved by plant CathB. Up to 13 substrates for CathB were identified in animal cells, and we found orthologous genes in Arabidopsis thaliana for eight of them. Among these, we are testing some candidate substrates for CathB. To experimentally validate the cleavage site, we used leaf agro-infiltration to co-expressed substrate GFP-fusion and CathB in tobacco cells and carried out cleavage assays. in vivo and in vitro results will be presented. 
















Characterising the proteolytic network of plant cathepsin B

Polyploids are of common occurrence among the plants. Several studies have been conducted to understand the impact of polyploidy on the evolution of plant genome. Natural polyploids do occur in the wild, but it is difficult to decipher which changes are due to polyploidy as the plants accumulate variation over the history of their existence. It is important to study the impact of polyploidy away from natural variation. Therefore, we generated synthetic neoautopolyploids in two species of Vigna L. using colchicine-based cotton swab method. The polyploids were screened via flowcytometry and confirmed polyploids were taken to the field to study biological parameters. The two species were found to be behaving differently based on various parameters like photosynthetic efficiency, cell density, stomatal count, cell size and nucleus size. We will be studying inheritance pattern of these traits in subsequent generations. Our study provides a proof that polyploidy does not always have a positive scaling effect on plants at various biological parameters and the changes are species specific. We will be studying genomic changes in future and will correlate them with morphological changes observed.  

Keywords: Polyploids, WGD, Autopolyploid, Flowcytometry, morphometery

Whole Genome Duplication Show Species-Specific Morphological Changes In Neoautopolyploids

Rising temperatures pose a significant threat to the survival of ectothermic animals. Thermal physiological traits, including upper thermal limits, are proven useful traits to assess the vulnerability of ectotherms to changing temperatures. For instance, one may use upper thermal limits to estimate current and future thermal safety margins (i.e., the proximity of upper thermal limits to experienced temperatures), use this trait in coercion with other physiological traits in species distribution models, or investigate the plasticity, heritability and evolvability of these traits for buffering the impacts of changing temperatures. While datasets on thermal tolerance limits have been previously compiled, they often report single estimates for a given species, do not present measures of data dispersion, and are biased towards certain parts of the globe. In this project, we systematically searched the literature in seven languages and produced a dataset of over 3000 heat tolerance estimates for 616 amphibian species. I will first present how the dataset we compiled can be used to address various questions in ecophysiology, and its limitations. Indeed, despite its scope, this dataset also suffered from geographical biases and captured less than 10% of described amphibian species. Here, I will demonstrate how we can fill the missing parts of this puzzle using powerful imputation procedures and estimate the heat tolerance of most described amphibian species. I will then demonstrate how we used this comprehensive database to estimate the vulnerability of the worlds’ amphibians to current and projected temperatures. 

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Physiological and immune response of oysters to climate change as a function of diet and tidal regime

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