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bioRxiv2021

Enhancement of alphavirus replication in mammalian cells at sub-physiological temperatures

Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes mosquitoes, causing fever, rash and arthralgia in mammals. The function of the CHIKV non-structural protein 3 (nsP3) remains enigmatic. Building on previous studies (Gao et al, 2019) [1], we generated a panel of mutants in a conserved and surface-exposed cluster in the nsP3 alphavirus unique domain (AUD) and tested their replication using a sub-genomic replicon (SGR). Three of these SGR mutants replicated well in mosquito cells but poorly in mammalian cells. We observed that this difference was due to culture temperature (mosquito cells: 28{degrees}C; mammalian cells: 37{degrees}C), as the mutants exhibited no replication defect in mammalian cells grown at a sub-physiological temperature (28{degrees}C). Similar phenotypes were observed for infectious CHIKV and the closely related ONyong Nyong virus. Intriguingly, the wildtype SGR replicated more efficiently in mammalian cells at 28{degrees}C compared to 37{degrees}C. To explore the mechanism behind this difference, we focused on two known antiviral pathways: interferon-stimulated genes (ISGs) and stress granules (SGs). SGR replication was concomitant with increased expression of ISGs at 37{degrees}C, but this response was impaired at 28{degrees}C. We also observed enhanced recruitment of the SG component G3BP1 into cytoplasmic sites of viral genome replication at 28{degrees}C. These findings may have real-world implications as when a mosquito bites a mammal, the virus first infects cells in peripheral tissues which are often at sub-physiological temperatures. We propose that alphaviruses such as CHIKV have evolved mechanisms to both promote viral genome replication and concomitantly limit antiviral responses in these cells. Significance statementChikungunya virus (CHIKV) is a re-emerging arbovirus that is transmitted by Aedes mosquitos and poses epidemic threats. Arboviruses must be able to grow efficiently in both the mosquito vector and the mammalian host, which have different body temperatures. Following a mosquito bite, the first cells infected are typically in the skin and are at sub-physiological temperatures, ie lower than the internal body temperature (37{degrees}C). Here we show that CHIKV grows more efficiently in mammalian cells cultured at 28{degrees}C, compared to 37{degrees}C. We investigated the mechanism behind this increase in growth efficiency and demonstrated that at the lower temperature the production of antiviral genes was impaired. Additionally, proteins that respond to cellular stress and are needed for virus replication were more abundantly recruited to sites in the cell where the virus was replicating. We propose that CHIKV has evolved mechanisms to promote its replication in mammalian cells at sub-physiological temperatures to facilitate infection of mammals via a mosquito bite.

microbiology↗
bioRxiv2022

Physiological stress responses to non-mimetic model brood parasite eggs: leukocyte profiles and heat-shock protein Hsp70 levels

Obligate avian brood parasites lay their eggs in the nest of other bird species (hosts). Brood parasitism often imposes severe fitness costs on hosts, which selects for the evolution of effective anti-parasitic defences, such as recognition and rejection of brood parasite eggs. Glucocorticoids have been recently found to mediate host physiological and behavioural adjustments in response to brood parasite eggs; however, it remains unclear whether brood parasitism triggers a general response involving multiple physiological elements. In this study, we experimentally investigated whether a salient brood parasitic stimulus (the presence of a non-mimetic model egg in the nest) causes physiological adjustments in adult Eurasian blackbirds (Turdus merula) at immune (leukocyte profiles) and cellular (heat-shock protein Hsp70 synthesis) level. Also, we explored whether these physiological changes are mediated by variations in corticosterone levels. We found that experimental brood parasitism caused an increase in heterophils and a decrease in lymphocytes, leading to higher H/L ratios in parasitized birds. Nevertheless, we did not find trade-offs between immune function and corticosterone levels. Hsp70 synthesis was not affected by our experimental manipulation. Our findings provide evidence that brood parasite eggs trigger a general stress response in egg-rejecter hosts, including changes in cellular immune profiles.

animal behavior and cognition↗
bioRxiv2022

3D printed bioreactor enabling the physiological culture and the study of pathological processes in large vessels.

Routine cardiovascular interventions such as balloon angioplasty, result in vascular activation and remodeling, often requiring re-intervention. 2D in vitro culture models and small animal experiments have enabled the discovery of important molecular and cellular pathways involved in this process, however the clinical translation of these results is often underwhelming. There is a critical need for an ex vivo model representative of the human vascular physiology and encompassing the complexity of the vascular wall and the physical forces regulating its function. Vascular bioreactors for ex vivo culture of large vessels are viable alternatives, but their custom-made design and insufficient characterization often hinders the reproducibility of the experiments. The objective of the study was to design and validate a novel 3D printed cost-efficient and versatile perfusion system, capable of sustaining the viability and functionality of large porcine arteries for 7 days and enabling monitoring of post-injury remodeling. MultiJet Fusion 3D printing technology was used to engineer the EasyFlow insert, converting a conventional 50 ml centrifuge tube into a mini bioreactor. Porcine carotid arteries either left untreated or injured with a conventional angioplasty balloon, were cultured under pulsatile flow for up to 7 days. Pressure, heart rate, medium viscosity and shear conditions were adjusted to represent the typical arterial physiology. Tissue viability, cell activation and matrix remodeling were analyzed by immunohistochemistry, and vascular function was monitored by duplex ultrasound. Physiological blood flow conditions in the EasyFlow bioreactor preserved endothelial coverage and smooth muscle organization and extracellular matrix structure in the vessel wall, as compared to static culture. Injured arteries presented hallmarks of early remodeling, such as intimal denudation, smooth muscle cell disarray and media/adventitia activation in flow culture. Duplex ultrasound confirmed physiological hemodynamic conditions, dose-dependent vasodilator response to nitroglycerin in untreated vessels and impaired dilator response in angioplastied vessels. We here validate a low-cost, robust and reproducible system to study vascular physiopathology, laying the basis for future investigations into the pathological remodeling of blood vessels and creating a platform to test novel therapies and devices ex vivo, in a patient relevant system.

bioengineering↗
bioRxiv2022

Physiological causes and biogeographic consequences of thermal optima in the hypoxia tolerance of marine ectotherms

The minimum O2 needed to fuel the demand of aquatic animals is commonly observed to increase with temperature, driven by accelerating metabolism. However, recent measurements of critical O2 thresholds ( Pcrit) reveal more complex patterns, including those with a minimum at an inter-mediate thermal optimum. To discern the prevalence, physiological drivers, and biogeographic manifestations of such curves, we analyze new experimental and biogeographic data using a general dynamic model of aquatic water breathers. The model simulates the transfer of oxygen from ambient water, through a boundary layer and into animal tissues driven by temperature-dependent rates of metabolism, diffusive gas exchange, and ventilatory and circulatory systems with O2-protein binding. We find that a thermal optimum in Pcrit can arise even when all physiological rates increase steadily with temperature. This occurs when O2 supply at low temperatures is limited by a process that is more temperature sensitive than metabolism, but becomes limited by a less sensitive process at warmer temperatures. Analysis of species respiratory traits suggests this scenario is not uncommon in marine biota, with ventilation and circulation limiting supply under cold conditions and diffusion limiting supply at high temperatures. Using biogeographic data, we show that species with these physiological traits inhabit lowest O2 waters near the optimal temperature for hypoxia tolerance, and are restricted to higher O2 at temperatures above and below this optimum. Our results imply that O2 tolerance can decline under both cold and warm conditions, and thus may influence both poleward and equatorward species range limits. Significance StatementPhysiology shapes the ecology, biogeography, and climate responses of marine species. In aquatic ectotherms, accelerating metabolism and lowered oxygen availability generally result in increasing oxygen limitation with warming. Here we present evidence for thermal optima in hypoxia tolerance of diverse species that is explained by a dynamical model of organismal physiology. Our results indicate that this potentially widespread bidirectional pattern explains species biogeographic limits in cold and warm waters. It can be understood using a generalized Metabolic Index of O2 supply to demand, which captures the variable observed trends between temperature and species hypoxia sensitivity. Oxygen limitation of aerobic metabolism in cold water has far-reaching implications for marine biogeography and species migrations under climate change.

ecology↗
bioRxiv2022

A single dose of the catecholamine precursor Tyrosine reduces physiological arousal and decreases decision thresholds in reinforcement learning and temporal discounting

Supplementation with the catecholamine precursor L-Tyrosine might enhance cognitive performance, but overall findings are mixed. Here, we investigate the effect of a single dose of tyrosine (2g) vs. placebo on two key aspects of catecholamine-dependent decision-making: model-based reinforcement learning (2-step task) and temporal discounting, using a double-blind, placebo-controlled, within-subject design (n=28 healthy male participants). We leveraged drift diffusion models in a hierarchical Bayesian framework to jointly model participants choices and response times in both tasks. Furthermore, comprehensive autonomic monitoring (heart rate, heart rate variability, pupillometry, spontaneous eye-blink rate) was performed both pre- and post-supplementation, to explore potential physiological effects of supplementation. Across tasks, tyrosine consistently reduced participants RTs without deteriorating task-performance. Diffusion modeling linked this effect to attenuated decision-thresholds in both tasks and further revealed increased model-based control (2-step task) and (if anything) attenuated temporal discounting. On the physiological level, participants pupil dilation was predictive of the individual degree of temporal discounting. Tyrosine supplementation reduced physiological arousal as revealed by increases in pupil dilation variability and reductions in hear rate. Supplementation-related changes in physiological arousal predicted individual changes in temporal discounting. Our findings provide first evidence that tyrosine supplementation might impact psychophysiological parameters, and suggest that modeling approaches based on sequential sampling models can yield novel insights into latent cognitive processes modulated by amino-acid supplementation.

neuroscience↗
bioRxiv2022

Distinct physiological responses of Coccolithus braarudii life cycle phases to light intensity and nutrient availability

Coccolithophores feature a haplo-diplontic life cycle comprised of diploid cells producing heterococcoliths and haploid cells producing morphologically different holococcoliths. These life cycle phases of each species appear to have distinct spatial and temporal distributions in the oceans, with the heavily-calcified heterococcolithophores (HET) often more prevalent in winter and at greater depths, whilst the lightly-calcified holococcolithophores (HOL) are more abundant in summer and in shallower waters. The haplo-diplontic life cycle may therefore allow coccolithophores to expand their ecological niche, switching between life cycle phases to exploit conditions that are more favourable. However, coccolithophore life cycles remain poorly understood and fundamental information on the physiological differences between life cycle phases is required if we are to better understand the ecophysiology of coccolithophores. In this study, we have examined the physiology of HET and HOL phases of the coccolithophore Coccolithus braarudii in response to changes in light and nutrient availability. We found that the HOL phase was more tolerant to high light than the HET phase, which exhibited defects in calcification at high irradiances. The HET phase exhibited defects in coccolith formation under both nitrate (N) and phosphate (P) limitation, whilst no defects in calcification were detected in the HOL phase. The HOL phase grew to a higher cell density under P-limitation than N-limitation, whereas no difference was observed in the maximum cell density reached by the HET phase at these nutrient concentrations. HET cells grown under a light:dark cycle divided primarily in the dark and early part of the light phase, whereas HOL cells continued to divide throughout the 24 h period. The physiological differences may contribute to the distinct biogeographical distributions observed between life cycle phases, with the HOL phase potentially better adapted to high light, low nutrient regimes, such as those found in seasonally stratified surface waters. HighlightsO_LICoccolithus braarudii life cycle phases exhibit different physiological responses. C_LIO_LIThe heavily-calcified heterococcolithophores (HET) life cycle phase is more sensitive to high light. C_LIO_LIThe lightly-calcified holococcolithophores (HOL) life cycle phase may be better suited to growth under low phosphate availability. C_LI

microbiology↗
bioRxiv2022

The Root of the Red Maple Paradox: Two distinct regeneration mechanisms determine leaf morphology, physiology, and competitive success in Acer rubrum.

Red maple is one of the most abundant and wide-ranging tree species in eastern North America and has rapidly increased in abundance over the past century following forest disturbances. This species is both a site- and light-generalist and has the unique ability to regenerate in forms that represent multiple stages of forest succession. However, red maple has modest physiological traits compared to its competitors, including low maximum photosynthetic rate, low photosynthetic nitrogen-use efficiency, and low foliar nutrient content. Red maples unremarkable physiology contradicts its competitive success. To untangle this paradox, we examine red maples two distinct regeneration mechanisms: seedlings and vegetative sprouts. Red maple can regenerate from seed, but can also sprout clonally from a stump following forest disturbance. We compare the morphology, physiology, and plasticity of these two regeneration mechanisms over 24 years of forest succession using a chronosequence of regenerating forest stands. We found that sprout-origin maples grow on average 6.5x taller and 5.5x faster than seed-origin maples. Sprout-origin trees display greater leaf spectral reflectance in the near-infrared range and greater stomatal density than seed-origin trees, demonstrating vegetative sprouts low water-use efficiency. Sprout-origin trees have more robust light-capture traits, including thicker palisade mesophyll than seed-origin trees. Sprouts also have a high plasticity between upper and lower leaves in many morphological and physiological traits, while seed-origin trees exhibited much less plasticity. The two distinct regeneration mechanisms of seed-origin and sprout-origin give rise to red maple trees with dramatically different leaf traits, allowing red maples to regenerate and thrive in a range of ecological conditions. Seed-origin maples are slow-growing, late successional, and shade-tolerant. Sprout-origin maples are fast-growing, early successional, and shade intolerant. This unique bimodal regeneration strategy may explain the red maple paradox and help predict forest composition and structure following disturbance.

plant biology↗
bioRxiv2022

Organoid-based human stomach micro-physiological system to recapitulate the dynamic mucosal defense mechanism

Several stomach diseases are attributed to the dysregulation of physiological function of gastric mucosal barrier by pathogens. Gastric organoids are a promising tool to develop treatment strategies for gastric infections. However, their functional features of in vivo gastric mucosal barrier and host-microbe interactions are limited due to the lack of physiological stimuli. Herein, we describe the first human stomach micro-physiological system (hsMPS) with physiologically relevant gastric mucosal defense system based on the combination of organoid and MPS technology. A fluid flow enhanced epithelial-mesenchymal interaction in the hsMPS enables functional maturation of gastric epithelial cells, which allows for the recreation of mesh-like mucus layer containing high level of mucus protective peptides and well-developed epithelial junctional complexes. Furthermore, gastroprotection mechanisms against Helicobacter pylori are successfully demonstrated in our system. Therefore, hsMPS represents a new in vitro tool for research where gastric mucosal defense mechanism is pivotal for developing therapeutic strategies.

bioengineering↗
bioRxiv2022

Physiological trait networks enhance understanding of crop growth and water use in contrasting environments

Plant function arises from a complex network of structural and physiological traits. Explicit representation of these traits, as well as their connections with other biophysical processes, is required to advance our understanding of plant-soil-climate interactions. We used the Terrestrial Regional Ecosystem Exchange Simulator (TREES) to evaluate physiological trait networks in maize. Net primary productivity (NPP) and grain yield were simulated across five contrasting climate scenarios. Simulations achieving high NPP and grain yield in high precipitation environments featured trait networks conferring high water use strategies: deep roots, high stomatal conductance at low water potential ("risky" stomatal regulation), high xylem hydraulic conductivity, and high maximal leaf area index. In contrast, high NPP and grain yield was achieved in dry environments with low late-season precipitation via water conserving trait networks: deep roots, high embolism resistance, and low stomatal conductance at low leaf water potential ("conservative" stomatal regulation). We suggest that our approach, which allows for the simultaneous evaluation of physiological traits and their interactions (i.e., networks), has potential to improve crop growth predictions in different environments. In contrast, evaluating single traits in isolation of other coordinated traits does not appear to be an effective strategy for predicting plant performance. Summary statementOur process-based model uncovered two beneficial but contrasting trait networks for maize which can be understood by their integrated effect on water use/conservation. Modification of multiple, physiologically aligned, traits were required to bring about meaningful improvements in NPP and yield.

plant biology↗
bioRxiv2022

A Physiology Clock for Human Aging

Why people age at different rates is a fundamental, unsolved problem in biology. We created a model that predicts an individuals age from physiological traits that change with age in the large UK Biobank dataset, such as blood pressure, lung function, strength and stimulus- reaction time. The model best predicted a persons age when it heavily-weighted traits that together query multiple organ systems, arguing that most or all physiological systems (lung, heart, brain, etc.) contribute to the global phenotype of chronological age. Differences between calculated "biological" age and chronological age ({Delta}Age) appear to reflect an individuals relative youthfulness, as people predicted to be young for their age had a lower subsequent mortality rate and a higher parental age at death, even though no mortality data were used to calculate {Delta}Age. Remarkably, the effect of each year of physiological {Delta}Age on Gompertz mortality risk was equivalent to that of one chronological year. A Genome-Wide Association Study (GWAS) of {Delta}Age, and analysis of environmental factors associated with {Delta}Age identified known as well as new factors that may influence human aging, including genes involved in synapse biology and a tendency to play computer games. We identify a small number of readily measured physiological traits that together assess a persons biological age and may be used clinically to evaluate therapeutics designed to slow aging and extend healthy life.

genetics↗
bioRxiv2022

Physiological responses to drought stress and recovery reflect differences in leaf function and anatomy among grass lineages

Grasses are cosmopolitan, existing in many biome and climate types from xeric to tropical. Traits that control physiological responses to drought vary strongly among grass lineages, suggesting that tolerance strategies may differ with evolutionary history. Here, we withheld water from 12 species representing six tribes of grasses to compare how species respond to drought in different grass lineages. We measured physiological, morphological, and anatomical traits. Dominant lineages from tropical savannas, like Andropogoneae, tolerated drought due to above and belowground morphological traits (specific leaf area and root length, SLA and SRL), while temperate grasses in this study utilized conservative leaf physiology (gas exchange) and anatomy traits. Increased intrinsic water-use efficiency coincided with a larger number of stomata, resulting in greater water loss (with inherently greater carbon gain) and increased drought sensitivity. Inherent leaf and root economic strategies impacting drought response were observed in all species, resulting in either high SLA or SRL, but not both. Our results indicate that grasses subjected to severe drought were influenced by anatomical traits (e.g., number of stomata and xylem area) and similar within lineages. In addition, grasses recovered at least 50% of physiological functioning across all lineages and 92% within Andropogoneae species, illustrating how drought can influence functional responses across diverse grass lineages.

plant biology↗
bioRxiv2022

Humanized V(D)J-rearranging and TdT-expressing Mouse Vaccine Models with Physiological HIV-1 Broadly Neutralizing Antibody Precursors

Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through non-templated nucleotide additions ("N-regions") by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs V{kappa}3-20 or V{kappa}1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had non-physiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as V{kappa}1-33 and/or V{kappa}3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated V{kappa}-to-J{kappa} joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center (GC) B cell responses. V{kappa}3-20-based responses were enhanced by N-region addition, which generates V{kappa}3-20-to-J{kappa} junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs. Significance StatementMouse models that express human precursors of HIV-1 broadly neutralizing antibodies (bnAbs) are useful for evaluating vaccination strategies for eliciting such bnAbs in humans. Prior models were handicapped by non-physiological frequency and/or diversity of B lymphocytes that express the bnAb precursors. We describe a new class of mouse models in which the mice express humanized bnAb precursors at a more physiologically relevant level through developmental rearrangement of both antibody heavy and light chain gene segments that encode the precursors. The model also incorporated a human enzyme that diversifies the rearranging gene segments and promotes generation of certain variable region sequences needed for the response. This new class of mouse models should facilitate preclinical evaluation of candidate human HIV-1 vaccination strategies.

immunology↗
bioRxiv2023

Dissecting durum wheat time to anthesis into physiological traits using a QTL-based model

Fine tuning crop development is a major breeding avenue to increase crop yield and for adaptation to climate change. In this study, we used a model that integrates our current understanding of the physiology of wheat phenology to predict the development and anthesis date of a RILs population of durum wheat with genotypic parameters controlling vernalization requirement, photoperiod sensitivity, and earliness per se estimated using leaf stage, final leaf number, anthesis date data from a pot experiment with vernalized and nonvernalized treatments combined with short- and long-day length. Predictions of final leaf number and anthesis date of the QTL-based model was evaluated for the whole population of RILs in a set of independent field trials and for the two parents, which were not used to estimate the parameter values. Our novel approach reduces the number of environments, experimental costs, and the time required to obtain the required data sets to develop a QTL-based prediction of model parameters. Moreover, the use of a physiologically based model of phenology gives new insight into genotype-phenology relations for wheat. We discuss the approach we used to estimate the parameters of the model and their association with QTL and major phenology genes that collocate at QTL. HighlightWe used a modeling framework integrating our current understanding of the physiology of wheat phenology to dissect durum wheat time to anthesis into physiological traits and link them to QTL.

plant biology↗
bioRxiv2023

Revealing the chassis-effect on a broad-host-range genetic switch and its concordance with interspecies bacterial physiologies

Broad-host-range synthetic biology is an emerging frontier that aims to expand our current engineerable domain of microbial hosts for biodesign applications. As more novel species are brought to "model status", synthetic biologists are discovering that identically engineered genetic circuits can exhibit different performances depending on the organism it operates within, an observation referred to as the "chassis-effect". It remains a major challenge to uncover which genome encoded and physiological biological determinants will underpin chassis effects that govern the performance of engineered genetic devices. In this study, we compared model and novel bacterial hosts to ask whether phylogenomic relatedness or similarity in host physiology is a better predictor of toggle switch performance. This was accomplished using comparative framework based on multivariate statistical approaches to systematically demonstrate the chassis-effect and characterize the performance dynamics of a genetic toggle switch operating within six Gammaproteobacteria. Our results solidify the notion that genetic devices are significantly impacted by host-context. Furthermore, we formally determined that hosts exhibiting more similar metrics of growth and molecular physiology also exhibit more similar toggle switch performance, indicating that specific bacterial physiology underpins measurable chassis effects. The result of this study contributes to the field of broad-host-range synthetic biology by lending increased predictive power to the implementation of genetic devices in less-established microbial hosts.

synthetic biology↗
bioRxiv2023

Drug screening in human physiologic medium identifies uric acid as an inhibitor of rigosertib efficacy

The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds, including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism waste product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. Structural modelling studies suggest that uric acid interacts with the tubulin-rigosertib complex and may act as an uncompetitive inhibitor of rigosertib. These results offer a possible explanation for the failure of rigosertib in clinical trials and demonstrate the utility of physiological media to achieve in vitro results that better represent human therapeutic responses.

cancer biology↗
bioRxiv2023

Evaluation of morpho-physiological and yield-associated traits of rice (Oryza sativa L.) landraces combined with marker-assisted selection under high temperature stress and elevated atmospheric CO2 levels

Rice (Oryza sativa L.) has a tremendous domestication history and is presently used as a major cereal all over the world. In Asia, India is considered as one of the centers of origin of indica rice and has several native landraces, especially in North-Eastern India (NEI), which have the potential to cope with the negative impact of present-day climate change. The current investigation aimed to evaluate the NEI rice landraces potential under high temperatures and elevated CO2 levels in comparison with a check variety for phenological, morphological, physiological and yield-associated parameters and molecularly validated with marker-assisted genotyping. The initial experiment was carried out with 75 rice landraces to evaluate their high heat tolerance ability. Seven better-performing landraces along with the check variety (N22) were further evaluated for aforesaid traits across two years (2019 and 2020) under control (or T1) and two stress treatments - (i) mild stress or T2 [CO2 550 ppm + 4{degrees} C more than ambient temperature] and (ii) severe stress or T3 [CO2 750 ppm + 6{degrees} C more than ambient temperature] using bioreactors. In the molecular analysis, the eight selected genotypes were evaluated through 25 Start Codon Targeted (SCoT) markers. The results revealed that the mild stress (T2) had a positive impact on various morpho-physiological parameters like plant height, number of leaves, leaf area and yield parameters like spikelets panicle-1 (S/P), thousand-grain weight (TGW) and grain yield (GY). This effect could be attributed to the genotypes ability to maintain a higher photosynthetic rate and possess better tolerance ability to moderately high temperatures. However, under high-temperature conditions in T3, all genotypes exhibited a significant decrease in the studied parameters including GY. It was found that pollen traits were significantly and positively correlated to spikelet fertility% at maturity, which was further significantly associated with GY under applied stress conditions. The physiological traits including shoot biomass were evident to have a significant positive effect on yield-associated parameters like S/P, harvest index (HI), TGW and GY. Overall, two landraces Kohima special and Lisem were found to be better responsive compared to other landraces as well as the check variety N22 under stress conditions. SCoT genotyping amplified a total of 77 alleles out of which 55 were polymorphic with the PIC value ranging from 0.22 to 0.67. The investigation suggests the presence of genetic variation among the tested rice lines and further supports evidence of the closely relatedness of Kohima special and Lisem. These two are two better-performing rice landraces from North-East India based on their improving morpho-physiological parameters and yield attributes in mild and severe high temperature and elevated CO2 stress environments. The shortlisted two rice landraces can be used as valuable pre-breeding materials for future rice breeding programs to improve the stress tolerance properties, particularly to high temperatures and elevated CO2 levels under ongoing changing climatic scenarios.

plant biology↗
bioRxiv2023

Physiologic Doses of TGF-beta Improve the Composition of Engineered Articular Cartilage

For cartilage regeneration applications, transforming growth factor beta (TGF-{beta}) is conventionally administered at highly supraphysiologic doses (10-10,000 ng/mL) in an attempt to cue cells to fabricate neocartilage that matches the composition, structure, and functional properties of native hyaline cartilage. While supraphysiologic doses enhance ECM biosynthesis, they are also associated with inducing detrimental tissue features, such as fibrocartilage matrix deposition, pathologic-like chondrocyte clustering, and tissue swelling. Here we investigate the hypothesis that moderated TGF-{beta} doses (0.1-1 ng/mL), akin to those present during physiological cartilage development, can improve neocartilage composition. Variable doses of media-supplemented TGF-{beta} were administered to a model system of reduced-size cylindrical constructs (O2-O3 mm), which mitigate the TGF-{beta} spatial gradients observed in conventional-size constructs (O4-O6 mm), allowing for a novel assessment of the intrinsic effect of TGF-{beta} doses on macroscale neocartilage properties and composition. The administration of physiologic TGF-{beta} to reduced-size constructs yields neocartilage with native-matched sGAG content and mechanical properties while providing a more hyaline cartilage-like composition, marked by: 1) reduced fibrocartilage-associated type I collagen, 2) 77% reduction in the fraction of cells present in a clustered morphology, and 3) 45% reduction in the degree of tissue swelling. Physiologic TGF-{beta} appears to achieve an important balance of promoting requisite ECM biosynthesis, while mitigating hyaline cartilage compositional deficits. These results can guide the development of novel physiologic TGF-{beta}-delivering scaffolds to improve the regeneration clinical-sized neocartilage tissues.

bioengineering↗
bioRxiv2024

Between a Warm Winter and a Cold Spell: Physiological Responses to Changing Winter Climate in Amphibians

Climate change is swiftly altering environmental winter conditions, leading to significant ecological impacts such as phenological shifts in many species. As a result, animals might face physiological mismatches due to longer or earlier activity periods and are at risk of being exposed to late spring freezes. Our study points for the first time to the complex physiological challenges that amphibians face as a result of changing thermal conditions due to winter climate change. We investigated the physiological responses to a period of warmer winter days and sudden spring freeze in the common toad (Bufo bufo) by acclimating them to 4{degrees}C or 8{degrees}C for 48 h or exposing them to 4{degrees}C or -2{degrees}C for 6 h, respectively. We assessed the daily energy demands, determined body condition and cold tolerance, explored the molecular responses to freezing through hepatic tissue transcriptome analysis, and measured blood glucose levels. Toads acclimated to higher temperatures showed a higher daily energy expenditure and a reduced cold tolerance suggesting faster depletion of energy stores and the loss of winter acclimation during warmer winters. Blood sugar levels were higher in frozen toads indicating the mobilization of cryoprotective glucose with freezing which was further supported by changed patterns in proteins related to glucose metabolism. Overall, our results emphasize that increased thermal variability incurs physiological costs that may reduce energy reserves and thus affect amphibian health and survival. This might pose a serious threat to breeding adults and may have subsequent effects at the population level.

ecology↗