A Gelatin/Alginate Double Network Hydrogel Nerve Guidance Conduit Fabricated by a Chemical-Free Gamma Radiation for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) are widely developed using various materials for the functional repair of injured or diseased peripheral nerves. Especially, hydrogels are considered highly suitable for the fabrication of NGCs due to their beneficial tissue-mimicking characteristics (e.g., high water content, softness, and porosity). However, the practical applications of hydrogel-based NGCs are hindered due to their poor mechanical properties and complicated fabrication processes. To bridge this gap, a novel double-network (DN) hydrogel using alginate and gelatin by a two-step crosslinking process involving chemical-free gamma irradiation and ionic crosslinking, is developed. DN hydrogels (1% alginate and 15% gelatin), crosslinked with 30 kGy gamma irradiation and barium ions, exhibit substantially improved mechanical properties, including tensile strength, elastic modulus, and fracture stain, compared to single network (SN) gelatin hydrogels. Additionally, the DN hydrogel NGC exhibits excellent kink resistance, mechanical stability to successive compression, suture retention, and enzymatic degradability. In vivo studies with a sciatic defect rat model indicate substantially improved nerve function recovery with the DN hydrogel NGC compared to SN gelatin and commercial silicone NGCs, as confirm footprint analysis, electromyography, and muscle weight measurement. Histological examination reveals that, in the DN NGC group, the expression of Schwann cell and neuronal markers, myelin sheath, and exon diameter are superior to the other controls. Furthermore, the DN NGC group demonstrates increased muscle fiber formation and reduced fibrotic scarring. These findings suggest that the mechanically robust, degradable, and biocompatible DN hydrogel NGC can serve as a novel platform for peripheral nerve regeneration and other biomedical applications, such as implantable tissue constructs.

High energy density in artificial heterostructures through relaxation time modulation.

Electrostatic capacitors are foundational components of advanced electronics and high-power electrical systems owing to their ultrafast charging-discharging capability. Ferroelectric materials offer high maximum polarization, but high remnant polarization has hindered their effective deployment in energy storage applications. Previous methodologies have encountered problems because of the deteriorated crystallinity of the ferroelectric materials. We introduce an approach to control the relaxation time using two-dimensional (2D) materials while minimizing energy loss by using 2D/3D/2D heterostructures and preserving the crystallinity of ferroelectric 3D materials. Using this approach, we were able to achieve an energy density of 191.7 joules per cubic centimeter with an efficiency greater than 90%. This precise control over relaxation time holds promise for a wide array of applications and has the potential to accelerate the development of highly efficient energy storage systems.

Laminin-Augmented Decellularized Extracellular Matrix Ameliorating Neural Differentiation and Neuroinflammation in Human Mini-Brains.

Non-neural extracellular matrix (ECM) has limited application in humanized physiological neural modeling due to insufficient brain-specificity and safety concerns. Although brain-derived ECM contains enriched neural components, certain essential components are partially lost during the decellularization process, necessitating augmentation. Here, it is demonstrated that the laminin-augmented porcine brain-decellularized ECM (P-BdECM) is xenogeneic factor-depleted as well as favorable for the regulation of human neurons, astrocytes, and microglia. P-BdECM composition is comparable to human BdECM regarding brain-specificity through the matrisome and gene ontology-biological process analysis. As augmenting strategy, laminin 111 supplement promotes neural function by synergic effect with laminin 521 in P-BdECM. Annexin A1(ANXA1) and Peroxiredoxin(PRDX) in P-BdECM stabilized microglial and astrocytic behavior under normal while promoting active neuroinflammation in response to neuropathological factors. Further, supplementation of the brain-specific molecule to non-neural matrix also ameliorated glial cell inflammation as in P-BdECM. In conclusion, P-BdECM-augmentation strategy can be used to recapitulate humanized pathophysiological cerebral environments for neurological study.

Circular RNA circSMAD4 regulates cardiac fibrosis by targeting miR-671-5p and FGFR2 in cardiac fibroblasts.

Heart failure is a leading cause of death and is often accompanied by activation of quiescent cardiac myofibroblasts, which results in cardiac fibrosis. In this study, we aimed to identify novel circular RNAs that regulate cardiac fibrosis. We applied transverse aortic constriction (TAC) for 1, 4, and 8 weeks in mice. RNA sequencing datasets were obtained from cardiac fibroblasts isolated by use of a Langendorff apparatus and then further processed by use of selection criteria such as differential expression and conservation in species. CircSMAD4 was upregulated by TAC in mice or by transforming growth factor (TGF)-ß1 in primarily cultured human cardiac fibroblasts. Delivery of si-circSMAD4 attenuated myofibroblast activation and cardiac fibrosis in mice treated with isoproterenol (ISP). si-circSmad4 significantly reduced cardiac fibrosis and remodeling at 8 weeks. Mechanistically, circSMAD4 acted as a sponge against the microRNA miR-671-5p in a sequence-specific manner. miR-671-5p was downregulated during myofibroblast activation and its mimic form attenuated cardiac fibrosis. miR-671-5p mimic destabilized fibroblast growth factor receptor 2 (FGFR2) mRNA in a sequence-specific manner and interfered with the fibrotic action of FGFR2. The circSMAD4-miR-671-5p-FGFR2 pathway is involved in the differentiation of cardiac myofibroblasts and thereby the development of cardiac fibrosis.

Reversible Mg-Metal Batteries Enabled by a Ga-Rich Protective Layer through One-Step Interface Engineering.

Practical applications of Mg-metal batteries (MMBs) have been plagued by a critical bottleneck─the formation of a native oxide layer on the Mg-metal interface─which inevitably limits the use of conventional nontoxic electrolytes. The major aim of this work was to propose a simple and effective way to reversibly operate MMBs in combination with Mg(TFSI)2-diglyme electrolyte by forming a Ga-rich protective layer on the Mg metal (GPL@Mg). Mg metal was carefully reacted with a GaCl3 solution to trigger a galvanic replacement reaction between Ga3+ and Mg, resulting in the layering of a stable and ion-conducting Ga-rich protective film while preventing the formation of a native insulating layer. Various characterization tools were applied to analyze GPL@Mg, and it was demonstrated to contain inorganic-rich compounds (MgCO3, Mg(OH)2, MgCl2, Ga2O3, GaCl3, and MgO) roughly in a double-layered structure. The artificial GPL on Mg was effective in greatly reducing the high polarization for Mg plating and stripping in diglyme-based electrolyte, and the stable cycling was maintained for over 200 h. The one-step process suggested in this work offers insights into exploring a cost-effective approach to cover the Mg-metal surface with an ion-conducting artificial layer, which will help to practically advance MMBs.

Aptamer Nanoconstructs Crossing Human Blood-Brain Barrier Discovered via Microphysiological System-Based SELEX Technology.

Blood-brain barrier (BBB) remains one of the critical challenges in developing neurological therapeutics. Short single-stranded DNA/RNA nucleotides forming a three-dimensional structure, called aptamers, have received increasing attention as BBB shuttles for efficient brain drug delivery owing to their practical advantages over Trojan horse antibodies or peptides. Aptamers are typically obtained by combinatorial chemical technology, termed Systemic Evolution of Ligands by EXponential Enrichment (SELEX), against purified targets, living cells, or animal models. However, identifying reliable BBB-penetrating aptamers that perform efficiently under human physiological conditions has been challenging because of the poor physiological relevance in the conventional SELEX process. Here, we report a human BBB shuttle aptamer (hBS) identified using a human microphysiological system (MPS)-based SELEX (MPS-SELEX) method. A two-channel MPS lined with human brain microvascular endothelial cells (BMECs) interfaced with astrocytes and pericytes, recapitulating high-level barrier function of in vivo BBB, was exploited as a screening platform. The MPS-SELEX procedure enabled robust function-based screening of the hBS candidates, which was not achievable in traditional in vitro BBB models. The identified aptamer (hBS01) through five-round of MPS-SELEX exhibited high capability to transport protein cargoes across the human BBB via clathrin-mediated endocytosis and enhanced uptake efficiency in BMECs and brain cells. The enhanced targeting specificity of hBS01 was further validated both in vitro and in vivo, confirming its powerful brain accumulation efficiency. These findings demonstrate that MPS-SELEX has potential in the discovery of aptamers with high target specificity that can be widely utilized to boost the development of drug delivery strategies.

Leveraging microfluidic dielectrophoresis to distinguish compositional variations of lipopolysaccharide in E. coli.

Lipopolysaccharide (LPS) is the unique feature that composes the outer leaflet of the Gram-negative bacterial cell envelope. Variations in LPS structures affect a number of physiological processes, including outer membrane permeability, antimicrobial resistance, recognition by the host immune system, biofilm formation, and interbacterial competition. Rapid characterization of LPS properties is crucial for studying the relationship between these LPS structural changes and bacterial physiology. However, current assessments of LPS structures require LPS extraction and purification followed by cumbersome proteomic analysis. This paper demonstrates one of the first high-throughput and non-invasive strategies to directly distinguish Escherichia coli with different LPS structures. Using a combination of three-dimensional insulator-based dielectrophoresis (3DiDEP) and cell tracking in a linear electrokinetics assay, we elucidate the effect of structural changes in E. coli LPS oligosaccharides on electrokinetic mobility and polarizability. We show that our platform is sufficiently sensitive to detect LPS structural variations at the molecular level. To correlate electrokinetic properties of LPS with the outer membrane permeability, we further examined effects of LPS structural variations on bacterial susceptibility to colistin, an antibiotic known to disrupt the outer membrane by targeting LPS. Our results suggest that microfluidic electrokinetic platforms employing 3DiDEP can be a useful tool for isolating and selecting bacteria based on their LPS glycoforms. Future iterations of these platforms could be leveraged for rapid profiling of pathogens based on their surface LPS structural identity.

Objective analysis of facial bone fracture CT images using curvature measurement in a surface mesh model.

The human facial skeleton consists of multiple segments and causes difficulty during analytic processes. We developed image analysis software to quantify the amount of injury and validate the smooth curvature of the surface after facial bone reduction surgery. Three-dimensional computed tomography images of facial bone were obtained from 40 patients who had undergone open reduction surgery to treat unilateral zygomaticomaxillary fractures. Analytic software was developed based on the discrete curvature of a triangular mesh model. The discrete curvature values were compared before and after surgery using two regions of interest. For the inferior orbital rim, the weighted average of curvature changed from 0.543 ± 0.034 to 0.458 ± 0.042. For the anterior maxilla, the weighted average of curvature changed from 0.596 ± 0.02 to 0.481 ± 0.031, showing a significant decrement (P < 0.05). The curvature was further compared with the unaffected side using the Bray-Curtis similarity index (BCSI). The BCSI of the inferior orbital rim changed from 0.802 ± 0.041 to 0.904 ± 0.015, and that for the anterior maxilla changed from 0.797 ± 0.029 to 0.84 ± 0.025, demonstrating increased similarity (P < 0.05). In computational biology, adequate analytic software is crucial. The newly developed software demonstrated significant differentiation between pre- and postoperative curvature values. Modification of formulas and software will lead to further advancements.

Lack of evidence that fibrillin1 regulates bone morphogenetic protein 4 activity in kidney or lung.

BACKGROUND: The Bone morphogenetic protein 4 (BMP4) precursor protein is cleaved at two sites to generate an active ligand and inactive prodomain. The ligand and prodomain form a noncovalent complex following the first cleavage, but dissociate after the second cleavage. Transient formation of this complex is essential to generate a stable ligand. Fibrillins (FBNs) bind to the prodomains of BMPs, and can regulate the activity of some ligands. Whether FBNs regulate BMP4 activity is unknown. RESULTS: Mice heterozygous for a null allele of Bmp4 showed incompletely penetrant kidney defects and females showed increased mortality between postnatal day 6 and 8. Removal of one copy of Fbn1 did not rescue or enhance kidney defects or lethality. The lungs of Fbn1+/- females had enlarged airspaces that were unchanged in Bmp4+/- ;Fbn1+/- mice. Additionally, removal of one or both alleles of Fbn1 had no effect on steady state levels of BMP4 ligand or on BMP activity in postnatal lungs. CONCLUSIONS: These findings do not support the hypothesis that FBN1 plays a role in promoting BMP4 ligand stability or signaling, nor do they support the alternative hypothesis that FBN1 sequesters BMP4 in a latent form, as is the case for other BMP family members.

Injectable Conductive Hydrogels with Tunable Degradability as Novel Implantable Bioelectrodes.

Bioelectrodes have been developed to efficiently mediate electrical signals of biological systems as stimulators and recording devices. Recently, conductive hydrogels have garnered great attention as emerging materials for bioelectrode applications because they can permit intimate/conformal contact with living tissues and tissue-like softness. However, administration and control over the in vivo lifetime of bioelectrodes remain challenges. Here, injectable conductive hydrogels (ICHs) with tunable degradability as implantable bioelectrodes are developed. ICHs were constructed via thiol-ene reactions using poly(ethylene glycol)-tetrathiol and thiol-functionalized reduced graphene oxide with either hydrolyzable poly(ethylene glycol)-diacrylate or stable poly(ethylene glycol)-dimaleimide, the resultant hydrogels of which are degradable and nondegradable, respectively. The ICH electrodes had conductivities of 21-22 mS cm-1 and Young's moduli of 15-17 kPa, and showed excellent cell and tissue compatibility. The hydrolyzable conductive hydrogels disappeared 3 days after in vivo administration, while the stable conductive hydrogels maintained their shapes for up to 7 days. Our proof-of-concept studies reveal that electromyography signals with significantly improved sensitivity from rats could be obtained from the injected ICH electrodes compared to skin electrodes and injected nonconductive hydrogel electrodes. The ICHs, offering convenience in use, controllable degradation and excellent signal transmission, will have great potential to develop various bioelectronics devices.

An agonistic anti-Tie2 antibody suppresses the normal-to-tumor vascular transition in the glioblastoma invasion zone.

Tumor progression is intimately associated with the vasculature, as tumor proliferation induces angiogenesis and tumor cells metastasize to distant organs via blood vessels. However, whether tumor invasion is associated with blood vessels remains unknown. As glioblastoma (GBM) is featured by aggressive invasion and vascular abnormalities, we characterized the onset of vascular remodeling in the diffuse tumor infiltrating zone by establishing new spontaneous GBM models with robust invasion capacity. Normal brain vessels underwent a gradual transition to severely impaired tumor vessels at the GBM periphery over several days. Increasing vasodilation from the tumor periphery to the tumor core was also found in human GBM. The levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) showed a spatial correlation with the extent of vascular abnormalities spanning the tumor-invading zone. Blockade of VEGFR2 suppressed vascular remodeling at the tumor periphery, confirming the role of VEGF-VEGFR2 signaling in the invasion-associated vascular transition. As angiopoietin-2 (ANGPT2) was expressed in only a portion of the central tumor vessels, we developed a ligand-independent tunica interna endothelial cell kinase 2 (Tie2)-activating antibody that can result in Tie2 phosphorylation in vivo. This agonistic anti-Tie2 antibody effectively normalized the vasculature in both the tumor periphery and tumor center, similar to the effects of VEGFR2 blockade. Mechanistically, this antibody-based Tie2 activation induced VE-PTP-mediated VEGFR2 dephosphorylation in vivo. Thus, our study reveals that the normal-to-tumor vascular transition is spatiotemporally associated with GBM invasion and may be controlled by Tie2 activation via a novel mechanism of action.

ANGPTL4 stabilizes atherosclerotic plaques and modulates the phenotypic transition of vascular smooth muscle cells through KLF4 downregulation.

Atherosclerosis, the leading cause of death, is a vascular disease of chronic inflammation. We recently showed that angiopoietin-like 4 (ANGPTL4) promotes cardiac repair by suppressing pathological inflammation. Given the fundamental contribution of inflammation to atherosclerosis, we assessed the role of ANGPTL4 in the development of atherosclerosis and determined whether ANGPTL4 regulates atherosclerotic plaque stability. We injected ANGPTL4 protein twice a week into atherosclerotic Apoe-/- mice and analyzed the atherosclerotic lesion size, inflammation, and plaque stability. In atherosclerotic mice, ANGPTL4 reduced atherosclerotic plaque size and vascular inflammation. In the atherosclerotic lesions and fibrous caps, the number of α-SMA(+), SM22α(+), and SM-MHC(+) cells was higher, while the number of CD68(+) and Mac2(+) cells was lower in the ANGPTL4 group. Most importantly, the fibrous cap was significantly thicker in the ANGPTL4 group than in the control group. Smooth muscle cells (SMCs) isolated from atherosclerotic aortas showed significantly increased expression of CD68 and Krüppel-like factor 4 (KLF4), a modulator of the vascular SMC phenotype, along with downregulation of α-SMA, and these changes were attenuated by ANGPTL4 treatment. Furthermore, ANGPTL4 reduced TNFα-induced NADPH oxidase 1 (NOX1), a major source of reactive oxygen species, resulting in the attenuation of KLF4-mediated SMC phenotypic changes. We showed that acute myocardial infarction (AMI) patients with higher levels of ANGPTL4 had fewer vascular events than AMI patients with lower levels of ANGPTL4 (p < 0.05). Our results reveal that ANGPTL4 treatment inhibits atherogenesis and suggest that targeting vascular stability and inflammation may serve as a novel therapeutic strategy to prevent and treat atherosclerosis. Even more importantly, ANGPTL4 treatment inhibited the phenotypic changes of SMCs into macrophage-like cells by downregulating NOX1 activation of KLF4, leading to the formation of more stable plaques.

In Situ Mesopore Formation in SiOx Nanoparticles by Chemically Reinforced Heterointerface and Use of Chemical Prelithiation for Highly Reversible Lithium-Ion Battery Anode.

SiOx is a promising next-generation anode material for lithium-ion batteries. However, its commercial adoption faces challenges such as low electrical conductivity, large volume expansion during cycling, and low initial Coulombic efficiency. Herein, to overcome these limitations, an eco-friendly in situ methodology for synthesizing carbon-containing mesoporous SiOx nanoparticles wrapped in another carbon layers is developed. The chemical reactions of vinyl-terminated silanes are designed to be confined inside the cationic surfactant-derived emulsion droplets. The polyvinylpyrrolidone-based chemical functionalization of organically modified SiO2 nanoparticles leads to excellent dispersion stability and allows for intact hybridization with graphene oxide sheets. The formation of a chemically reinforced heterointerface enables the spontaneous generation of mesopores inside the thermally reduced SiOx nanoparticles. The resulting mesoporous SiOx -based nanocomposite anodes exhibit superior cycling stability (≈100% after 500 cycles at 0.5 A g-1 ) and rate capability (554 mAh g-1 at 2 A g-1 ), elucidating characteristic synergetic effects in mesoporous SiOx -based nanocomposite anodes. The practical commercialization potential with a significant enhancement in initial Coulombic efficiency through a chemical prelithiation reaction is also presented. The full cell employing the prelithiated anode demonstrated more than 2 times higher Coulombic efficiency and discharge capacity compared to the full cell with a pristine anode.

Deep learning-based diagnosis of stifle joint diseases in dogs.

In this retrospective, analytical study, we developed a deep learning-based diagnostic model that can be applied to canine stifle joint diseases and compared its accuracy with that achieved by veterinarians to verify its potential as a reliable diagnostic method. A total of 2382 radiographs of the canine stifle joint from cooperative animal hospitals were included in a dataset. Stifle joint regions were extracted from the original images using the faster region-based convolutional neural network (R-CNN) model, and the object detection accuracy was evaluated. Four radiographic findings: patellar deviation, drawer sign, osteophyte formation, and joint effusion, were observed in the stifle joint and used to train a residual network (ResNet) classification model. Implant and growth plate groups were analyzed to compare the classification accuracy against the total dataset. All deep learning-based classification models achieved target accuracies exceeding 80%, which is comparable to or slightly less than those achieved by veterinarians. However, in the case of drawer signs, further research is necessary to improve the low sensitivity of the model. When the implant group was excluded, the classification accuracy significantly improved, indicating that the implant acted as a distraction. These results indicate that deep learning-based diagnoses can be expected to become useful diagnostic models in veterinary medicine.

Regulating Na Electrodeposition by Sodiophilic Grafting onto Porosity-Gradient Gel Polymer Electrolytes for Dendrite-Free Sodium Metal Batteries.

Sodium metal batteries have been emerging as promising candidates for post-Li battery systems owing to the natural abundance, low costs, and high energy density of Na metal. However, exploiting an Na metal anode is accompanied by uncontrolled Na electrodeposition, particularly concerning dendrite growth, hampering practical Na metal battery applications. Herein, we propose sodiophilic gel polymer electrolytes with a porosity-gradient Janus structure to alleviate Na dendrite growth. Tethering only 1.1 mol % sodiophilic poly(ethylene glycol) to poly(vinylidene fluoride-co-hexafluoropropylene) suppresses Na dendrites by regulating homogeneous Na+ distribution, which relies on molecular-level coordination between Na+ and the sodiophilic functional groups. By exploiting the porosity-gradient Janus structure, we have demonstrated that regular porosity and well-defined morphology of polymer electrolytes, particularly at the Na/electrolyte interface, significantly impact dendrite growth. This study provides new insights into the rational design of Na dendrite-suppressing polymer electrolytes, primarily focusing on the ion-regulating ability achieved by surface engineering.

Characterization of Exosomes and Exosomal RNAs Isolated from Post-Mortem Body Fluids for Molecular Forensic Diagnosis.

Exosomes have been mainly studied for their potential applications in biomarker detection and drug delivery for diagnosis and treatment. However, in the field of forensic research, the potential value of exosomes derived from post-mortem body fluids has not been investigated to date. Here, we isolated the exosomes and exosomal RNAs from post-mortem body fluids, including cardiac blood, pericardial fluid, and urine. We also compared commercial exosome isolation kits to determine the optimal method for post-mortem exosome isolation. Transmission electron microscopy (TEM), the Agilent bioanalyzer system, and western blotting were used to evaluate the efficiencies of alternative isolation methods and the characteristics of isolated exosomes. There were no significant differences between exosomes obtained from post-mortem and ante-mortem body fluids in the expression of exosome surface markers or morphology. The exosomes were well-preserved even under simulated post-mortem conditions. Among the isolation procedures tested, the membrane affinity column-based method was the most suitable for post-mortem exosomal RNA isolation. These results suggest that exosomes are well-preserved in post-mortem body fluids and could be utilized for forensic diagnosis.

Characterizing chemical signaling between engineered "microbial sentinels" in porous microplates.

Living materials combine a material scaffold, that is often porous, with engineered cells that perform sensing, computing, and biosynthetic tasks. Designing such systems is difficult because little is known regarding signaling transport parameters in the material. Here, the development of a porous microplate is presented. Hydrogel barriers between wells have a porosity of 60% and a tortuosity factor of 1.6, allowing molecular diffusion between wells. The permeability of dyes, antibiotics, inducers, and quorum signals between wells were characterized. A "sentinel" strain was constructed by introducing orthogonal sensors into the genome of Escherichia coli MG1655 for IPTG, anhydrotetracycline, L-arabinose, and four quorum signals. The strain's response to inducer diffusion through the wells was quantified up to 14 mm, and quorum and antibacterial signaling were measured over 16 h. Signaling distance is dictated by hydrogel adsorption, quantified using a linear finite element model that yields adsorption coefficients from 0 to 0.1 mol m-3 . Parameters derived herein will aid the design of living materials for pathogen remediation, computation, and self-organizing biofilms.

Metastable hexagonal close-packed palladium hydride in liquid cell TEM.

Metastable phases-kinetically favoured structures-are ubiquitous in nature1,2. Rather than forming thermodynamically stable ground-state structures, crystals grown from high-energy precursors often initially adopt metastable structures depending on the initial conditions, such as temperature, pressure or crystal size1,3,4. As the crystals grow further, they typically undergo a series of transformations from metastable phases to lower-energy and ultimately energetically stable phases1,3,4. Metastable phases sometimes exhibit superior physicochemical properties and, hence, the discovery and synthesis of new metastable phases are promising avenues for innovations in materials science1,5. However, the search for metastable materials has mainly been heuristic, performed on the basis of experiences, intuition or even speculative predictions, namely 'rules of thumb'. This limitation necessitates the advent of a new paradigm to discover new metastable phases based on rational design. Such a design rule is embodied in the discovery of a metastable hexagonal close-packed (hcp) palladium hydride (PdHx) synthesized in a liquid cell transmission electron microscope. The metastable hcp structure is stabilized through a unique interplay between the precursor concentrations in the solution: a sufficient supply of hydrogen (H) favours the hcp structure on the subnanometre scale, and an insufficient supply of Pd inhibits further growth and subsequent transition towards the thermodynamically stable face-centred cubic structure. These findings provide thermodynamic insights into metastability engineering strategies that can be deployed to discover new metastable phases.

Bacterial response to spatial gradients of algal-derived nutrients in a porous microplate.

Photosynthetic microalgae are responsible for 50% of the global atmospheric CO2 fixation into organic matter and hold potential as a renewable bioenergy source. Their metabolic interactions with the surrounding microbial community (the algal microbiome) play critical roles in carbon cycling, but due to methodological limitations, it has been challenging to examine how community development is influenced by spatial proximity to their algal host. Here we introduce a copolymer-based porous microplate to co-culture algae and bacteria, where metabolites are constantly exchanged between the microorganisms while maintaining physical separation. In the microplate, we found that the diatom Phaeodactylum tricornutum accumulated to cell abundances ~20 fold higher than under normal batch conditions due to constant replenishment of nutrients through the porous structure. We also demonstrate that algal-associated bacteria, both single isolates and complex communities, responded to inorganic nutrients away from their host as well as organic nutrients originating from the algae in a spatially predictable manner. These experimental findings coupled with a mathematical model suggest that host proximity and algal culture growth phase impact bacterial community development in a taxon-specific manner through organic and inorganic nutrient availability. Our novel system presents a useful tool to investigate universal metabolic interactions between microbes in aquatic ecosystems.