Czechia

Acute monocytic leukaemia (AML-M5) is a bone marrow disease that affects young children. Astonishingly, the discovery of disease-specific induced pluripotent stem cells (iPSCs) capable of recapitulating human pathogenesis allows us to model this disease in vitro. We had previously generated AML-M5-specific-iPSC (AML-M5-iPSCs) from THP-1 cells obtained from a patient[1]. These AML-M5-iPSCs were then induced with growth supplements to enter haematopoietic differentiation to generate monocytic cells. We noticed that reprogramming transgenes Oct3/4, Sox2 and c-Myc were unintentionally integrated into the genome of AML-M5-iPSC during reprogramming. Out of scientific interest, the effects of transgene integration on drug responses were investigated. Firstly, the monocytic cells were characterised to determine their morphological and phagocytotic properties, and compared with their parental cell line THP-1 across 5-, 10-, 15-, 20- and 25-day post-differentiation. Subsequently, cytotoxic effect of Doxorubicin at 0.5, 1, 2 and 4μM on both cells were investigated with CCK-SK viability assay, and apoptotic effect investigated with cell cycle analysis. 
Our results revealed that in terms of size and morphology, both THP-1 and differentiated monocytic cells remained comparable up to 25 days post-differentiation. Within the same duration, their phagocytotic rates also exhibited no significant differences (p&gt;0.05) when investigated with carboxylate-modified red fluorescent latex beads. However, after 24h Doxorubicin treatment, the IC50 value determined for THP-1 cells was 0.59µM, but the IC50 value for differentiated monocytic cells could not be determined; suggesting that they were significantly (p&lt;0.05) less responsive to Doxorubicin. Upon similar treatment conditions, 92.5±3.9% of THP-1 cells entered late apoptosis stage. However, for differentiated monocytic cells, only 0.3±0.2% entered late apoptosis stage, 37.2±1.5% entered necrosis stage and 62.5±1.6% remained in viable stage. Our results showed that transgenes integration was causing monocytic cells to be resistant to Doxorubicin despite the fact the they should only affect haematopoietic differentiation. In addition, the apoptotic effect of Doxorubicin had also been switched to necrotic effect. More interesting, transgene integration did not seem to alter the morphology and phagocytosis of monocytic cells. We postulate that transgenes Oct3/4, Sox2 and c-Myc interfered with the intercalation of Doxorubicin into the DNA, subsequently altering downstream pathways thereby disrupting cell death via apoptosis. In conclusion, further investigations are warranted to determine the mechanism of reprogramming transgene-induced drug resistance in monocytic cells derived from AML-M5-iPSC.


SEB Conference 2024

Integration of reprogramming transgenes into AML-M5-iPSC caused differentiated monocytic cells to be resistant to Doxorubicin

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SEB’s Annual Conference is celebrated for creating the perfect composition of research, new discoveries and collaborative connections. Be creative and innovative by exchanging ideas and knowledge with masters of biology from all over the world.

Acute monocytic leukaemia (AML-M5) is a bone marrow disease that affects young children. Astonishingly, the discovery of disease-specific induced pluripotent stem cells (iPSCs) capable of recapitulating human pathogenesis allows us to model this disease in vitro. We had previously generated AML-M5-specific-iPSC (AML-M5-iPSCs) from THP-1 cells obtained from a patient[1]. These AML-M5-iPSCs were then induced with growth supplements to enter haematopoietic differentiation to generate monocytic cells. We noticed that reprogramming transgenes Oct3/4, Sox2 and c-Myc were unintentionally integrated into the genome of AML-M5-iPSC during reprogramming. Out of scientific interest, the effects of transgene integration on drug responses were investigated. Firstly, the monocytic cells were characterised to determine their morphological and phagocytotic properties, and compared with their parental cell line THP-1 across 5-, 10-, 15-, 20- and 25-day post-differentiation. Subsequently, cytotoxic effect of Doxorubicin at 0.5, 1, 2 and 4μM on both cells were investigated with CCK-SK viability assay, and apoptotic effect investigated with cell cycle analysis. 
Our results revealed that in terms of size and morphology, both THP-1 and differentiated monocytic cells remained comparable up to 25 days post-differentiation. Within the same duration, their phagocytotic rates also exhibited no significant differences (p>0.05) when investigated with carboxylate-modified red fluorescent latex beads. However, after 24h Doxorubicin treatment, the IC50 value determined for THP-1 cells was 0.59µM, but the IC50 value for differentiated monocytic cells could not be determined; suggesting that they were significantly (p<0.05) less responsive to Doxorubicin. Upon similar treatment conditions, 92.5±3.9% of THP-1 cells entered late apoptosis stage. However, for differentiated monocytic cells, only 0.3±0.2% entered late apoptosis stage, 37.2±1.5% entered necrosis stage and 62.5±1.6% remained in viable stage. Our results showed that transgenes integration was causing monocytic cells to be resistant to Doxorubicin despite the fact the they should only affect haematopoietic differentiation. In addition, the apoptotic effect of Doxorubicin had also been switched to necrotic effect. More interesting, transgene integration did not seem to alter the morphology and phagocytosis of monocytic cells. We postulate that transgenes Oct3/4, Sox2 and c-Myc interfered with the intercalation of Doxorubicin into the DNA, subsequently altering downstream pathways thereby disrupting cell death via apoptosis. In conclusion, further investigations are warranted to determine the mechanism of reprogramming transgene-induced drug resistance in monocytic cells derived from AML-M5-iPSC.


poster

Engaging our students and supporting them to develop agency in assessment and feedback is important for driving effective self-regulation (Sadler, 2010). We will showcase several novel initiatives we have introduced in the School of Biological Sciences at the University of Bristol, including a student partnership project to co-create an assessment and feedback portfolio (AFP) with interactive assessment landscapes; converting our AFP from a voluntary activity to a summative assessment to ‘force’ our final year UG students to reflect and act on their feedback; introducing a regular Biological Sciences ‘feedback café’ to promote dialogic feedback, co-creating a new programme of assessments for a Biological Sciences professional placement year and a scaffolding our peer-assisted study scheme (PASS) around assessments.

Empowering student agency in assessment and feedback in Biological Sciences.

Scientific discovery relies on high-quality research that is transparent, reproducible, and available for outside scrutiny. Unfortunately, the current reward system often selects for poor quality research by incentivising practices that favour sensationalism, shortcuts, and novelty rather than quality and rigour. Globally, the annual science budget wasted on irreproducible research is estimated in the tens of billions of dollars. Such alarming levels of irreproducibility are not solely due to issues of poor statistical practices, confirmation bias, and selective reporting - various forms of misconduct play large roles. Regardless of the reasons for which misconduct occurs, scientists have a moral and ethical obligation to report it when observed. Here, we present guidance for researchers who suspect or discover scientific misconduct, ranging from the initial stages of fact checking to the later stages of formal investigations. We use our own experiences and lessons learned from conversations with other scientists who have had the unfortunate experience of witnessing fraud. We confirm that whistleblowing is one example of the self-correcting nature of science.

Scientific misconduct - advice on being a whistle-blower

The continual progression of climate change is creating intense challenges for food producers, as delivering adequate crop yield and quality to a growing population is becoming increasingly difficult. Incremental increases in atmospheric CO2 concentrations unpredictably affect crop physiology and biochemical profiles, leading to inconsistent crop yields and nutritional quality. Of course, eCO2 will not occur in isolation and, when coupled with high temperatures, may further deteriorate crop nutrient content. Research suggests that plants using the rare C2 carbon concentrating mechanism may tolerate warm temperature better than plants using the very common C3 photosynthetic pathway. C2 photosynthesis uses a glycine shuttle to effectively concentrate and reclaim carbon released from photorespiration, ultimately boosting rates of photosynthesis under warm and dry environments that promote high rates of photorespiration. Although C3 plants are predicted to fix more carbon under eCO2, the warm temperatures that accompany climate change should disadvantage C3 plants. eCO2 also influence plant biochemistry and consequently nutrition. The carbon dilution effect effectively reduces nutrient and protein contents, which translates into lower quality crops. How the nutritive properties of C2 plants will respond to eCO2 has never been investigated. Using diverse cultivars of wild rocket (Diplotaxis tenuifolia), the only crop in commercial food production that uses C2 photosynthesis, we examine the combined effects of temperature and eCO2 on the nutritional profile of this salad crop.


Understanding nutritional response to eCO2 under a range of temperatures in wild rocket (Diplotaxis tenuifolia) 


Seed aging is a process that seed gradually loses its ability to germinate. Improper storage conditions can diminish seed vigor, adversely affecting seed germination and seedling establishment. Therefore, understanding the seed-aging process and exploring strategies to enhance seed-aging resistance are paramount. In this study, we observed that seed aging during storage leads to a decline in seed vigor and can coincide with the accumulation of hydrogen peroxide (H2O2) in the radicle, resulting in compromised or uneven germination and asynchronous seedling emergence. We identified the abscisic acid (ABA) catabolism gene, abscisic acid 8’-hydroxylase 2 (OsABA8ox2), as significantly induced by aging treatment. Interestingly, transgenic seeds overexpressing OsABA8ox2 exhibited reduced seed vigor, while gene knockout enhanced seed vigor, suggesting its role as a negative regulator. Similarly, seeds pretreated with ABA or diphenyleneiodonium chloride (DPI, an H2O2 inhibitor) showed increased resistance to aging, with more robust early seedling establishment. Both OsABA8ox2 mutant seeds and seeds pretreated with ABA or DPI displayed lower H2O2 content during aging treatment. Overall, our findings indicate that ABA mitigates rice seed aging by reducing H2O2 accumulation in the radicle. This study offers valuable germplasm resources and presents a novel approach to enhancing seed resistance against aging.

ABA Inhibits Rice Seed Aging by Reducing H2O2 Accumulation in the Radicle of Seeds

Despite the evidence of the effect of topography on forest systems at the landscape level, only a few reports are available regarding the impact of varied topography on physiology and tree growth within catchment areas of the mountain ecosystem. The topographic influence of topoclimate and catchment hydrology plays a significant role in shaping forest feedback to future climate conditions and shapes the structure and function of the forest. The studies to model the forest response to environmental alterations comprise landscape-level processes but generally exclude finer-scale dynamics, which may be similarly significant to the ecosystem functions of the forest. The present study revealed aspect-regulated finer-scale variations in leaf ecophysiology. During our study, these variations were evidenced by increased photosynthesis of Q. semecarpifolia, growing in higher light, warmer, and drier conditions of the south (S) aspect relative to the contrasting north (N) aspect of the same catchment, in the subalpine forest of the central Himalaya. In general, the seasonality and aspect-driven light environments both posed remarkable changes in the leaf ecophysiology (i.e., leaf gas exchange and plant water relation). The effect of variation in the light environment was more pronounced and affected the overall productivity and leaf ecophysiology of the plants more severely, seasonally as well as diurnally. It has also been realized throughout the study that photosynthesis and other leaf gas exchange parameters were highly variable and highly influenced by microclimatic environmental factors like air temperature, soil moisture, relative humidity, light quality, intensity, and duration. Therefore, while predicting the gross primary productivity, it needs to take more site-specific, time-specific (diurnal), and season-specific data to accurately and precisely account for forest productivity. Our study also suggests that brown oak interspecifically adapts by adjusting its physiological traits against several abiotic stresses to maintain its higher productivity.


Influence of Topography on Variation and Intra-specific adaptations in Leaf Ecophysiology of Quercus Semecarpifolia Forest of High Altitudinal Central Himalaya

Abstract: Clonal propagation is a widely utilised technique in forestry and horticulture and holds significant promise for conserving desired traits within specific species. Successful adventitious root development from cutting stems bases is essential and depends on environmental and physiological factors among which plant hormones play a crucial role. This study investigates the effect of auxin and jasmonic acid on cutting adventitious root development, both independently and in interaction, across three distinct peas cultivars: ‘Meteor’, ‘RTP’, and ‘Torsdag’. Our findings reveal cultivar-specific responses to auxin and jasmonic acid in a dosage dependent manner. Moreover, our study reveals the hormone dosage is directly related to the speed of adventitious root development. By further exploring the site and timing of hormone interaction, we aim to elucidate the physiological mechanisms underlying successful root formation. These findings have significant implications for tree production through cutting propagation, offering insights that can optimise hormone treatments, improve efficiency, and promote sustainability in forestry and horticulture practices. Ultimately, our research contributes to advancing the understanding of hormonal regulation in clonal propagation, paving the way for more efficient and scalable tree production methods.

Rooting Buddies: role of plant hormone crosstalk in cutting propagation

Hyperspectral imaging (HSI) is a a cornerstone of non-destructive plant phenotyping. With the growing interest in field phenotyping systems,
Photon System Instruments (PSI) has developed the mobile PlantScreenTM Field Phenotyping System, which integrates range of imaging sensors
including the HySpec VNIR Camera and PSI RGB camera modules.
In a pilot collaborative project with CATRIN/UPOL/CRI, we applied hyperspectral VNIR and RGB field-based imaging to non-destructively characterize
the specific metabolite profile in Calendula sp. flowers of 8 different cultivars. Here, we present the pipeline for processing hyperspectral and RGB
data, including validation based on reflectance spectra from a hand-held Polypen spectrometer (PSI) and integration with metabolomic data from
Liquid Chromatography/Mass Spectrometry (LC/MS).

Sunlit Insights: Transferring Hyperspectral Imaging from Lab to Field

Improving our understanding of plant photosynthetic acclimation to environmental fluctuations is a crucial aspect of developing crop varieties adapted to future climatic conditions. Here we propose a theoretical model of photosynthetic protein turnover that explains photosynthetic acclimation as the accumulated effect of light and temperature on the synthesis and degradation of photosynthetic proteins in three functional pools: carboxylation, electron transport, and light harvesting. However, parameterizing this model across a wide panel of winter wheat varieties is challenging due to the number of genotype-specific parameters. Consequently, we propose hyperspectral phenotyping as a high-throughput approach for indirect assessment of parameters such as maximum carboxylation rate (Vcmax), maximum electron transport rate (Jmax), and chlorophyll concentration. In order to determine the individual effect of light and temperature fluctuations, we are conducting a series of experiments growing 60 winter wheat genotypes covering the recent German breeding history in a controlled environment with different combinations of varying and constant conditions. These experiments aim to characterize the three protein pool dynamics through periodic measurements of pigment content, chlorophyll fluorescence and gas exchange; and establish correlations between this ground truth and hyperspectral data, which will be instrumental in understanding how breeding has affected the photosynthetic acclimation.

Characterizing the individual effect of fluctuating light and temperature on source acclimation on 60 winter wheat genotypes

Nitrogen (N) is essential for agriculture and its excessive use causes severe environmental damage. Improving nitrogen use efficiency (NUE) is crucial for achieving more productive crops under conditions of limited N availability. To the best of our knowledge, this is the first study aiming to integrate the morphophysiological responses, transcriptomics and proteomic profiles of leaves and roots of popcorn to understand NUE. For this, two NUE-contrasting inbred lines P2 (high NUE) L80 (low NUE), were cultivated under high (100% of N supply) and low (10% of N supply) N conditions. Morphological and physiological traits such as photochemical quenching (qP), non-photochemical quenching (NPQ), quantum yield of photosystem II (ΦPSII), and potential photosynthesis (Apot) were evaluated. P2 showed more pronounced vegetative growth under low N, as well as higher values of qP, NPQ, ΦPSII and Apot. Comparative transcriptomics and proteomics analysis revealed that more than 215 differentially accumulated proteins (DAPs) and 430 differentially expressed genes (DEGs) were involved in the response to low N in both lines. In leaves, DAPs and DEGs involved in the response to oxidative stress, energy metabolism, and photosynthesis represented the main differences between P2 and L80. In roots, the mechanisms of nitrate transport, ammonium assimilation, and amino acid metabolism seem to have contributed to the improved NUE in P2. This study provides valuable insights into the molecular mechanisms underlying NUE and opens avenues for molecular breeding aimed at selecting superior genotypes for the development of a more sustainable agriculture.

Combined transcriptomics and proteomics profiling reveals mechanisms underlying nitrogen use efficiency in popcorn (Zea mays var. everta)

High tomato (Solanum lycopersicum) sugar content, redness of color, and firm texture are associated with a prominence of rich flavor, directly influencing consumer preferences. One major challenge of tomato improvement is to deliver superior flavor in the context of high yield and long postharvest shelf-life. TAM-SP18-157 line is extraordinarily firm resulting in long shelf-life fruit that conserves quality characterized by lack of locule gel caused by a single recessive gene mapped to chromosome 10. As opposed to long shelf-life tomatoes with impaired ripening hormone emissions, ethylene production is high in TAM-SP18-157. Tomato line TAM-SP18-157 was crossed with locule gel carrying line TAM-SP18-501. Fruits from TAM-SP18-157 lacking locule gel were 43.75% and 9.41% more firm than fruits having gel from TAM-SP18-501 and F1, respectively. Furthermore, fruits lacking locule gel were still very firm up to for 21 days (0.8 KgF), while fruits with gel were softer at that time point (0.6 KgF). Similar trend was observed for Brix, were fruits lacking gel had higher soluble solid content than fruit with gel with intermediate levels in the F1 hybrid. Fruit acidity content was lower in fruits lacking locule gel, while fruits from the F1 cross had higher acidity content as compared with the two parental lines. Together, result indicate that locule gel plays an important role in postharvest life and quality. Therefore, tomato line TAM-SP18-157 can be used to improve those characteristics by breeding for locule gel content in tomato fruits.

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Empowering student agency in assessment and feedback in Biological Sciences.

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Integration of reprogramming transgenes into AML-M5-iPSC caused differentiated monocytic cells to be resistant to Doxorubicin