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At least 991 records · Page 55Linked to original sources

bioRxiv2023

Peripheral direct current for suppression of physiologically evoked nociceptive responses at the spinal cord

Electrical neuromodulation is an established non-pharmacological treatment for chronic pain. However, existing devices using pulsatile stimulation are not suitable for all types of chronic pain. Direct current stimulation is a recently developed technology which shows better selectivity for small-diameter fibres. We investigated if this selectivity could be applied to preferentially suppress nociceptive signalling. We applied direct current to the sciatic nerve of rats and compared its effects on spinal activity produced by physiological (non-electrical) stimuli delivered to the foot. Tests were performed across models of neuropathic and inflammatory pain to further clarify potential clinical applications. We found that direct current could effectively suppress activity relating to painful stimuli in both pain models tested. These findings strongly support the use of direct current neuromodulation for chronic pain relief, and suggest that it may be effective at treating a broader range of aberrant pain conditions than existing devices.

neuroscience↗

bioRxiv2023

Photo-physiological Acclimation in Synechocystis sp. PCC 6803 Provides Insight into Growth Limitation in Underwater Spectra

Cyanobacteria play a key role in primary production in both oceans and fresh waters and hold great potential for sustainable production of a large number of commodities. During their life, cyanobacteria cells need to acclimate to a multitude of challenges, including shifts in intensity and quality of incident light. Despite our increasing understanding of metabolic regulation under various light regimes, detailed insight into fitness advantages and limitations under shifting light quality has been missing. Here, we study photo-physiological acclimation in the cyanobacterium Synechocystis sp. PCC 6803 through the whole range of photosynthetically active radiation (PAR). Using LEDs with qualitatively different narrow spectra, we describe wavelength dependence of light capture, electron transport and energy transduction to main cellular pools. In addition, we describe processes fine-tuning light capture such as state transitions and efficiency of energy transfer from phycobilisomes to photosystems. We show that growth was the most limited under blue light due to inefficient light harvesting, and that many cellular processes are tightly linked to the redox state of the PQ pool, which was the most reduced under red light. The PSI-to-PSII ratio was low under blue photons, however, it was not the main growth-limiting factor, since it was even more reduced under violet and near far-red lights, where Synechocystis grew faster compared to blue light. Our results provide insight into the spectral dependence of phototrophic growth and can provide the foundation for future studies of molecular mechanisms underlying light acclimation in cyanobacteria, leading to light optimization in controlled cultivations.

plant biology↗

bioRxiv2023

Light-exercise-induced dopaminergic and noradrenergic stimulation in the dorsal hippocampus: using a rodent physiological exercise model

Exercise activates the dorsal hippocampus, which triggers the synaptic and cellar plasticity and ultimately promotes memory formation. For decades, these benefits have been explored using demanding and stress-response-inducing exercise at moderate-to-vigorous intensities. In contrast, our translational research with animals and humans has focused on light exercise below the lactate threshold (LT), which almost anyone can safely perform with minimal stress, and found that even light exercise can stimulate hippocampal activity and enhance memory performance. Although the circuit mechanism of this boost remains unclear, arousal promotion even with light exercise implies the involvement of the ascending monoaminergic system, which is essential to modulate hippocampal activity and impact memory. To examine this hypothesis, we employed our physiological exercise model based on the LT of rats that can be applied to human and immunohistochemically assessed the neuronal activation of the dorsal hippocampal sub-regions and brainstem monoaminergic neurons. Also, we monitored the dynamics of monoamine release at the dorsal hippocampus using in vivo microdialysis. We found that even light exercise increased neuronal activity in the dorsal hippocampal sub-regions and induced noradrenaline and dopamine release. Furthermore, we found that tyrosine hydroxylase-positive neurons in the locus coeruleus (LC) and the ventral tegmental area (VTA) were activated even by light exercise and were both positively correlated with the dorsal hippocampal activation. In conclusion, our findings demonstrate that light exercise stimulates hippocampal neurons, possibly through the LC-noradrenergic and/or VTA-dopaminergic neurons. This sheds light on the circuit mechanisms responsible for hippocampal neural activation during exercise, consequently enhancing memory function. Graphical abstractOur previous research with animals and humans has demonstrated that even light exercise can boost neuronal activity in the dorsal hippocampus and improve memory. While the mechanism underlying this remains undetermined, recent studies suggest the involvement of the ascending monoaminergic system. Here, we examined this hypothesis and found a possible contribution of noradrenergic neurons in the locus coeruleus and dopaminergic neurons in the ventral tegmental area to dorsal hippocampal activation during light exercise, implying a circuit mechanism for light-exercise-enhanced memory. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/545490v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1e286e8org.highwire.dtl.DTLVardef@1070708org.highwire.dtl.DTLVardef@5ec2a6org.highwire.dtl.DTLVardef@13d2fdd_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Preserved respiratory chain capacity and physiology in mice with profoundly reduced levels of mitochondrial respirasomes

The mammalian respiratory chain complexes I, III2 and IV (CI, CIII2 and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI- CIII2-CIV), that is biochemically and structurally well documented. The role of the respirasome in bioenergetics and regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the in vivo role of the respirasome, we generated homozygous knock-in mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in the mouse, but raises questions about their alternate functions, such as relating to regulation of protein stability and prevention of age-associated protein aggregation.

molecular biology↗

bioRxiv2023

Freeze dehydration vs. supercooling in tree stems: physical and physiological modelling

Frost resistance is the major factor affecting the distribution of plant species at high latitude and elevation. The main effects of freeze-thaw cycles are damage to living cells and formation of gas embolism in tree xylem vessels. Lethal intracellular freezing can be prevented in living cells by two mechanisms: dehydration and deep supercooling. We developed a multiphysics numerical model coupling water flow, heat transfer, and phase change, considering different cell types in plant tissues, to study the dynamics and extent of cell dehydration, xylem pressure changes, and stem diameter changes in response to freezing and thawing. Results were validated using experimental data for stem diameter changes of walnut trees. The effect of cell mechanical properties was found to be negligible as long as the intracellular tension developed during dehydration was sufficiently low compared to the ice induced cryostatic suction. The model was finally used to explore the coupled effects of relevant physiological parameters (initial water and sugar content) and environmental conditions (air temperature variations) on the dynamics and extent of dehydration. It revealed configurations where cell dehydration could be sufficient to protect cells from intracellular freezing, and situations where supercooling was necessary. This model, freely available with this paper, could easily be extended to explore different anatomical structures, different species and more complex physical processes.

biophysics↗

bioRxiv2023

Complex epistatic interactions between ELF3, PRR9, and PRR7 regulates the circadian clock and plant physiology

Circadian clocks are endogenous timekeeping mechanisms that coordinate internal physiological responses with the external environment. EARLY FLOWERING3 (ELF3), PSEUDO RESPONSE REGULATOR (PRR9), and PRR7 are essential components of the plant circadian clock and facilitate entrainment of the clock to internal and external stimuli. Previous studies have highlighted a critical role for ELF3 in repressing the expression of PRR9 and PRR7. However, the functional significance of activity in regulating circadian clock dynamics and plant development is unknown. To explore this regulatory dynamic further, we firstly employed mathematical modelling to simulate the effect of the prr9/prr7 mutation on the elf3 circadian phenotype. These simulations suggested that simultaneous mutations in prr9/prr7 could rescue the elf3 circadian arrythmia. Following these simulations, we generated all Arabidopsis elf3/prr9/prr7 mutant combinations and investigated their circadian and developmental phenotypes. Although these assays could not replicate the results from the mathematical modelling, our results have revealed a complex epistatic relationship between ELF3 and PRR9/7 in regulating different aspects of plant development. ELF3 was essential for hypocotyl development under ambient and warm temperatures, while PRR9 was critical for root thermomorphogenesis. Finally, mutations in prr9 and prr7 rescued the photoperiod insensitive flowering phenotype of the elf3 mutant. Together, our results highlight the importance of investigating the genetic relationship amongst plant circadian genes.

plant biology↗

bioRxiv2023

Physiological and metabolic insights into the first cultured anaerobic representative of deep-sea Planctomycetes bacteria

Planctomycetes bacteria are ubiquitously distributed across various biospheres and play key roles in global element cycles. However, few deep-sea Planctomycetes members have been cultivated, limiting our understanding of Planctomycetes in the deep biosphere. Here, we have successfully cultured a novel strain of Planctomycetes (strain ZRK32) from a cold seep sediment, by using an enriched medium supplemented with rifampicin and different nitrogen sources. Our genomic, physiological and phylogenetic analyses indicate that strain ZRK32 is a novel species, which we propose be named: Poriferisphaera heterotrophicis. We show that strain ZRK32 replicates using a budding mode of division. Based on the combined results from growth assays and transcriptomic analyses, we found that rich nutrients, or supplementation with NO3- or NH4+ promoted the growth of strain ZRK32 by facilitating energy production through the tricarboxylic acid (TCA) cycle and the Embden-Meyerhof-Parnas (EMP) glycolysis pathway. Moreover, supplementation with NO3- or NH4+ induced strain ZRK32 to release a bacteriophage in a chronic manner, without host cell lysis. This bacteriophage then enabled strain ZRK32, and another marine bacterium that we studied, to metabolize nitrogen through the function of auxiliary metabolic genes (AMGs). Overall, these findings expand our understanding of deep-sea Planctomycetes bacteria, while highlighting their ability to metabolize nitrogen when reprogrammed by chronic viruses.

microbiology↗

bioRxiv2023

Interrogation of inter-layer neuronal connectivity via cross-region all-optical physiology with high temporal resolution

The interrogation of functional neural circuits is crucial for uncovering how the brain works during diverse behaviors. Multi-plane neurophysiological measurement systems with high temporal resolution are indispensable, especially for dissecting inter-layer functional connectivity. Here, we develop a cross-layer all-optical physiology system (CLAOP) that enables the simultaneous recording and manipulation of single-neuron activities in multiple neuronal layers, with axial intervals as large as 530 m, at high temporal resolutions. Based on spatiotemporal multiplexing, our system enables all-optical analysis with a high frame rate up to 396 Hz and minimal time delay in inter-layer imaging and photostimulation, in both the mouse cortex and hippocampus in vivo. Combined with behavioral experiments, CLAOP provides all-optical evidence linking behavioral responses to neuronal connectivity in the primary visual cortex (V1) of live mice. Furthermore, we demonstrate that CLAOP can perturb the activity response of inter-layer cortical neurons to sensory stimuli according to their functional signatures. Overall, CLAOP provides an all-optical approach for mapping inter-layer connectivity at the single-neuron level and for modifying neuronal responses in behaving animals.

neuroscience↗

bioRxiv2023

A Physiologically Based Digital Twin for Alcohol Consumption - Predicting Real-life Drinking Responses and Long-term Plasma PEth

Alcohol consumption is associated with a wide variety of preventable health complications and is a major risk factor for all-cause mortality in the age group 15-47 years. To reduce dangerous drinking behavior, eHealth applications have shown promise. A particularly interesting potential lies in the combination of eHealth apps with mathematical models. However, existing mathematical models do not consider real-life situations, such as combined intake of meals and beverages, and do not connect drinking to clinical markers, such as phosphatidylethanol (PEth). Herein, we present such a model which can simulate real-life situations and connect drinking to long-term markers. The new model can accurately describe both estimation data according to a {chi}2-test (187.0 < T{chi}2 = 226.4) and independent validation data (70.8 < T{chi}2 =93.5). The model can also be personalized using anthropometric data from a specific individual and can thus be used as a physiologically-based digital twin. This twin is also able to connect short-term consumption of alcohol to the long-term dynamics of PEth levels in the blood, a clinical biomarker of alcohol consumption. Here we illustrate how connecting short-term consumption to long-term markers allows for a new way to determine patient alcohol consumption from measured PEth levels. An additional use case of the twin could include the combined evaluation of patient-reported AUDIT forms and measured PEth levels. Finally, we integrated the new model into an eHealth application, which could help guide individual users or clinicians to help reduce dangerous drinking.

systems biology↗

bioRxiv2023

Plasticity drives geographic variation and trait coordination in blue oak drought physiology

Our ability to predict drought stress across the landscape remains limited. This uncertainty stems in part from an incomplete understanding of within-species variation in hydraulic physiology, particularly coordinated variation across multiple traits. This variation reflects genetic differentiation among populations (ecotypic variation) and phenotypic plasticity. We examined among-population differentiation in morphological and hydraulic traits in California blue oak (Quercus douglasii) using a 30 year-old common garden. We compared trait differentiation and trait-trait coordination in the garden to wild phenotypes from the original source populations. We found remarkably limited among-population differentiation in all traits in the common garden but considerable site-to-site variation in the field that could rarely be explained with site climate variables. Trait-trait relationships were also stronger in the field than in the garden, particularly links between leaf morphology, leaf hydraulic efficiency and stem hydraulic efficiency. Only four trait-trait relationship were present in both the wild and garden, but 12 of 45 relationships showed significant wild phenotypic correlations, with strong coordination among leaf and stem hydraulic efficiency apparently mediated by leaf size. Ultimately, we found limited evidence for ecotypic variation but considerable geographic in phenotypic integration in the wild, suggesting considerable acclimation potential in the face of climate change.

ecology↗

bioRxiv2023

Behavioural, physiological, and genetic drivers of coping

Animals regularly experience stressful situations, ranging from predation to social stress, yet successfully deal with them on most occasions. This adaptive mechanism, coping, reduces the adverse effects of stressors through behavioural and physiological efforts, failing to which may result in reduced fitness. However, considerable variation in coping is observed. Unlike in humans, coping is often considered a personality trait in non-human animals due to construct similarity, resulting in conceptual ambiguity. Besides, limited multidisciplinary research has rendered comprehending the drivers of coping in animals challenging. We assessed repetitive behavioural coping or coping styles (n=30), emotional arousal (n=12), and consistent inter-individual differences, i.e., personalities (n=32) of long-tailed macaques (Macaca fascicularis) using observations, ecologically relevant experiments, and infrared thermography. We finally investigated the association of coping with a Valine/Methionine polymorphism encoded by the Catechol-O-methyltransferase (COMT) gene (n=26), which is widely known for its involvement in human stress regulation. Our findings suggest that personality and the presence of the human equivalent COMT Val158Met polymorphism in long-tailed macaques are associated with emotion-focused and problem-focused coping styles. These coping styles were consistent with emotional arousal as measured with infrared thermography. We discuss these proximate drivers of coping for a better understanding of its evolution in primates.

animal behavior and cognition↗

bioRxiv2023

Accelerated physiology and increased energy expenditure in animals and humans with mitochondrial defects: A meta-analysis

Mitochondria are key energy transforming organelles in mammalian cells. However, how defects in oxidative phosphorylation (OxPhos) and other mitochondrial functions influence whole-body energy expenditure (EE) has not been rigorously studied. Cellular and organismal responses to OxPhos defects likely involve a combination of functional downregulation to conserve energy and compensatory upregulation of stress responses. If the energy cost of compensatory responses exceeds the potential energy savings of functional downregulation, as recent work suggests, the result would be an increase in total EE. To address the hypothesis that OxPhos defects increase the energetic cost of living, we performed a meta-analysis of available studies reporting EE in animal models with mitochondrial gene defects. Of all reported experimental conditions (n = 91, from 29 studies), 51% reported a >10% elevation in EE relative to control animals, compared to 11% reporting <10% reduction in EE (p<0.0001, Chi-square). Of the experimental conditions where locomotor activity was also quantified, 39% showed that OxPhos-deficient animals had elevated EE despite reduced locomotor activity, which would be expected to decrease EE. To extend this finding in humans, we re-analyzed a high-quality clinical and multi-omics dataset (Sharma et al. 2021) of mitochondrial disease patients with the m.3243A>G mutation. This analysis similarly indicates an upregulation of energetically costly physiological, immune, and metabolic parameters in people with OxPhos deficiency. These results suggest that animals and humans with mitochondrial defects must expend more energy to sustain life, a state clinically called hypermetabolism. High-quality human energetics studies are needed to understand the magnitude, mechanisms, and modifiability of hypermetabolism in mitochondrial disorders.

cell biology↗

bioRxiv2023

Anti-S-layer monoclonal antibodies impact Clostridioides difficile physiology

Clostridioides difficile (C. difficile), a gram-positive anaerobic and spore-forming bacterium, is the leading cause of nosocomial antibiotic-associated diarrhea in adults and is characterized by high levels of recurrence and mortality. Surface-layer Protein A (SlpA), the most expressed protein on bacterial surface, plays a crucial role in the early stages of infection although its role in C. difficile physiology is yet to be fully understood. Anti-S-layer antibodies have been identified in the sera of convalescent patients and correlate with improved outcome of C. difficile infection (CDI). However, the precise mechanisms of how anti-S-layer antibodies can confer protection to the host remain unknown. In this study, we report the first monoclonal antibodies (mAbs) targeting S-layer of the reference strain 630. Characterization of these mAbs unravels important roles for S-layer protein in growth, toxin secretion, and biofilm formation with, surprisingly, opposite effects of different anti-SlpA mAbs on these functions. One anti-SlpA mAb impaired C. difficile growth and restored sensitivity to lysozyme-induced lysis. These findings suggest that anti-S-layer antibody responses may include protective and detrimental effects for the host and provide important insights for designing adequate S-layer-targeting therapeutics.

microbiology↗

bioRxiv2023

Diurnal cycles drive rhythmic physiology and promote survival in facultative phototrophic bacteria

Bacteria have evolved many strategies to spare energy when nutrients become scarce. One widespread such strategy is facultative phototrophy, which helps heterotrophs supplement their energy supply using light. Our knowledge on the impact that such behaviors have on bacterial fitness and physiology is, however, still limited. Here, we study how a representative of the genus Porphyrobacter, in which aerobic anoxygenic phototrophy is ancestral, responds to different light regimes under nutrient limitation. We show that bacterial survival in stationary phase relies on functional reaction centers and varies depending on the light regime. Under dark-light alternance, our bacterial model presents a diphasic life history dependent on phototrophy: during dark phases, the cells inhibit DNA replication and part of the population lyses and releases nutrients, while subsequent light phases allow for the recovery and renewed growth of the surviving cells. We correlate these cyclic variations with a pervasive pattern of rhythmic transcription which reflects global changes in diurnal metabolic activity. Finally, we demonstrate that, compared to either a phototrophy null mutant or a bacteriochlorophyll a overproducer, the wild type strain is better adapted to natural environments, where regular dark-light cycles are interspersed with additional accidental dark episodes. Overall, our results highlight the importance of light-induced biological rhythms in a new model of aerobic anoxygenic phototroph representative of an ecologically important group of environmental bacteria.

microbiology↗

bioRxiv2023

An eco-physiological model of forest photosynthesis and transpiration under combined nitrogen and water limitation

Although the separate effects of water and nitrogen (N) limitations on forest growth are well known, the question of how to predict their combined effects remains a challenge for modeling of climate change impacts on forests. Here, we address this challenge by developing a new eco-physiological model that accounts for plasticity in stomatal conductance and leaf N concentration. Based on an optimality principle, our model determines stomatal conductance and leaf N concentration by balancing carbon uptake maximization, hydraulic risk and cost of maintaining photosynthetic capacity. We demonstrate the accuracy of the model predictions by comparing them against gross primary production estimates from eddy covariance flux measurements and sap-flow scaled canopy transpiration in a long-term fertilized and an unfertilized Scots pine (Pinus sylvestris L.) forest in northern Sweden. The model also explains the response to N fertilization as a consequence of (i) reduced carbon cost of N uptake and (ii) increased leaf area per hydraulic conductance. The results suggest that leaves optimally coordinate N concentration and stomatal conductance both on short (weekly) time scales in response to weather conditions and on longer time scales in response to soil water and N availabilities.

ecology↗

bioRxiv2023

Photoacoustic Imaging for Non-Invasive Assessment of Physiological Biomarkers of Intestinal Injury in Experimental Necrotizing Enterocolitis

BackgroundNecrotizing enterocolitis (NEC) is an often-lethal disease of the premature infants intestinal tract that is exacerbated by significant difficulties in early and accurate diagnosis. In NEC disease, the intestine often exhibits hypoperfusion and dysmotility, which contributes to advanced disease pathogenesis. However, these physiological features cannot be accurately and quantitively assessed within the current constraints of imaging modalities frequently used in the clinic (plain film X-ray and ultrasound). We have previously demonstrated the ability of photoacoustic imaging (PAI) to non-invasively and quantitively assess intestinal tissue oxygenation and motility in a healthy neonatal rat model. As a first-in-disease application, we evaluated NEC pathogenesis using PAI to assess intestinal health biomarkers in a preclinical neonatal rat experimental model of NEC. MethodsNEC was induced in neonatal rat pups from birth to 4 days old via hypertonic formula feeding, full-body hypoxic stress, and lipopolysaccharide administration to mimic bacterial colonization. Healthy breastfed (BF) controls and NEC rat pups were imaged at 2- and 4-days old. Intestinal tissue oxygen saturation was measured with PAI imaging for oxy- and deoxyhemoglobin levels. To measure intestinal motility, ultrasound and co-registered PAI cine recordings were used to capture intestinal peristalsis motion and contrast agent (indocyanine green) transit within the intestinal lumen. Additionally, both midplane two-dimensional and volumetric three-dimensional imaging acquisitions were assessed for oxygenation and motility. ResultsNEC pups showed a significant decrease of intestinal tissue oxygenation as compared to healthy BF controls at both ages (2-days old: 55.90% +/-3.77% vs 44.12% +/-7.18%; 4-days old: 56.13% +/-3.52% vs 38.86% +/-8.33%). Intestinal motility, assessed using a computational intestinal deformation analysis, demonstrated a significant reduction in the intestinal motility index in both early (2-day) and established (4-day) NEC. Extensive NEC damage was confirmed with histology and dysmotility was confirmed by small intestinal transit assay. ConclusionsThis study presents PAI as a successful emerging diagnostic imaging modality for both intestinal tissue oxygenation and intestinal motility disease hallmarks in a rat NEC model. PAI presents enormous significance and potential for fundamentally changing current clinical paradigms for detecting and monitoring intestinal pathologies in the premature infant.

bioengineering↗

bioRxiv2023

Physiological and transcriptomic characterization of cold acclimation in endodormant grapevine under different temperature regimes

It is essential for the survival of grapevines in cool climate viticultural regions that vines properly acclimate in the late fall and early winter and develop freezing tolerance. Climate change-associated abnormities in temperature during the dormant season, including oscillations between extreme cold and prolonged warmth, impacts cold acclimation and threatens the sustainability of the grape and wine industry. We conducted two experiments in controlled environment to investigate the impacts of different temperature regimes on cold acclimation ability in endodormant grapevine buds through a combination of freezing tolerance based physiological and RNA-seq based transcriptomic monitoring. Results show that the freezing tolerance of buds was not altered from field levels when exposed to stable temperatures ranging from 2 {degrees}C to 22 {degrees}C but was enhanced when exposed to temperature cycling (7{+/-}5 {degrees}C). We also characterized the transcriptomic response of endodormant buds to high and low temperatures and the potential genetic control for the maintenance of endodormancy. Several pathways that were previously reported to be responsive or functional during cold acclimation, such as the ICE-CBF-COR cascade, were not observed to play a role in the enhancement of freezing tolerance or the sensing of different temperatures, indicating our current understanding of the genetic control of cold acclimation remains a challenge when generalizing across plant species and phenological stages.

plant biology↗

bioRxiv2023

Physiological concentrations of calcium interact with alginate and extracellular DNA in the matrices of Pseudomonas aeruginosa biofilms to impede phagocytosis by neutrophils

Biofilms are communities of interacting microbes embedded in a matrix of polymer, protein, and other materials. Biofilms develop distinct mechanical characteristics that depend on their predominant matrix components. These matrix components may be produced by microbes themselves or, for infections in vivo, incorporated from the host environment. Pseudomonas aeruginosa is a human pathogen that forms robust biofilms that extensively tolerate antibiotics and effectively evade clearance by the immune system. Two of the important bacterial-produced polymers in the matrices of P. aeruginosa biofilms are alginate and extracellular DNA (eDNA), both of which are anionic and therefore have the potential to interact electrostatically with cations. Many physiological sites of infection contain significant concentrations of the calcium ion (Ca2+). In this study we investigate the structural and mechanical impacts of Ca2+ supplementation in alginate-dominated biofilms grown in vitro and we evaluate the impact of targeted enzyme treatments on clearance by immune cells. We use multiple particle tracking microrheology to evaluate the changes in biofilm viscoelasticity caused by treatment with alginate lyase and/or DNAse I. For biofilms grown without Ca2+, we correlate a decrease in relative elasticity with increased phagocytic success. However, we find that growth with Ca2+ supplementation disrupts this correlation except in the case where both enzymes are applied. This suggests that the calcium cation may be impacting the microstructure of the biofilm in non-trivial ways. Indeed, confocal laser scanning fluorescence microscopy and scanning electron microscopy reveal unique Ca2+-dependent eDNA and alginate microstructures. Our results suggest that the presence of Ca2+ drives the formation of structurally and compositionally discrete microdomains within the biofilm through electrostatic interactions with the anionic matrix components eDNA and alginate. Further, we observe that these structures serve a protective function as the dissolution of both components is required to render biofilm bacteria vulnerable to phagocytosis by neutrophils. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/563605v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@126dc6aorg.highwire.dtl.DTLVardef@50dab8org.highwire.dtl.DTLVardef@477fd6org.highwire.dtl.DTLVardef@19e8e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗