3D Cellular Architecture Research Articles

Rapid and Controllable Multilayer Cell Sheet Assembly via Biodegradable Nanochannel Membranes

AbstractThe ability to precisely arrange and control the assembly of diverse cell types into intricate 3D structures remains a critical challenge in tissue engineering. Herein, a versatile and programmable 3D cell sheet assembly is described technology by developing a biodegradable nanochannel (BNC) membrane to fulfill this unmet need. This membrane, hierarchically assembled from 2D nanomaterial aggregates, exhibits both exceptional fluid permeability and rapid biodegradation under physiological conditions. The unique properties of the BNC membrane enable precise spatial and temporal control over cell assembly, facilitating the creation of complex 3D cellular architectures. The BNC membrane is integrated with a programmable negative‐pressure‐based cell assembly strategy to form single and multicellular 3D sheets in a highly controllable manner. To demonstrate the feasibility and translatability of this technology in the field of tissue engineering approaches to screen stem cell‐derived therapeutics with “core–shell” macrophage‐fibroblast multicellular patterns and treat murine diabetic skin wounds via scaffold‐free 3D adipose‐derived mesenchymal stem cell (ADMSC) sheets are devised. In summary, the results demonstrate that the BNC membrane‐based 3D cell sheet assembly approach significantly advances current tissue engineering capabilities, offering substantial potential for both regenerative medicine applications and the development of physiologically relevant disease models.

Topological analysis of 3D digital ovules identifies cellular patterns associated with ovule shape diversity.

Tissue morphogenesis remains poorly understood. In plants, a central problem is how the 3D cellular architecture of a developing organ contributes to its final shape. We address this question through a comparative analysis of ovule morphogenesis, taking advantage of the diversity in ovule shape across angiosperms. Here, we provide a 3D digital atlas of Cardamine hirsuta ovule development at single cell resolution and compare it with an equivalent atlas of Arabidopsis thaliana. We introduce nerve-based topological analysis as a tool for unbiased detection of differences in cellular architectures and corroborate identified topological differences between two homologous tissues by comparative morphometrics and visual inspection. We find that differences in topology, cell volume variation and tissue growth patterns in the sheet-like integuments and the bulbous chalaza are associated with differences in ovule curvature. In contrast, the radialized conical ovule primordia and nucelli exhibit similar shapes, despite differences in internal cellular topology and tissue growth patterns. Our results support the notion that the structural organization of a tissue is associated with its susceptibility to shape changes during evolutionary shifts in 3D cellular architecture.

Correlative cryo-soft X-ray tomography and cryo-structured illumination microscopy reveal changes to lysosomes in amyloid-β-treated neurons

Protein misfolding is common to neurodegenerative diseases (NDs) including Alzheimer's disease (AD), which is partly characterized by the self-assembly and accumulation of amyloid-beta in the brain. Lysosomes are a critical component of the proteostasis network required to degrade and recycle material from outside and within the cell and impaired proteostatic mechanisms have been implicated in NDs. We have previously established that toxic amyloid-beta oligomers are endocytosed, accumulate in lysosomes, and disrupt the endo-lysosomal system in neurons. Here, we use pioneering correlative cryo-structured illumination microscopy and cryo-soft X-ray tomography imaging techniques to reconstruct 3D cellular architecture in the native state revealing reduced X-ray density in lysosomes and increased carbon dense vesicles in oligomer treated neurons compared with untreated cells. This work provides unprecedented visual information on the changes to neuronal lysosomes inflicted by amyloid beta oligomers using advanced methods in structural cell biology.

Customizable 3D printed perfusion bioreactor for the engineering of stem cell microenvironments.

Faithful modeling of tissues and organs requires the development of systems reflecting their dynamic 3D cellular architecture and organization. Current technologies suffer from a lack of design flexibility and complex prototyping, preventing their broad adoption by the scientific community. To make 3D cell culture more available and adaptable we here describe the use of the fused deposition modeling (FDM) technology to rapid-prototype 3D printed perfusion bioreactors. Our 3D printed bioreactors are made of polylactic acid resulting in reusable systems customizable in size and shape. Following design confirmation, our bioreactors were biologically validated for the culture of human mesenchymal stromal cells under perfusion for up to 2weeks on collagen scaffolds. Microenvironments of various size/volume (6-12mm in diameter) could be engineered, by modulating the 3D printed bioreactor design. Metabolic assay and confocal microscopy confirmed the homogenous mesenchymal cell distribution throughout the material pores. The resulting human microenvironments were further exploited for the maintenance of human hematopoietic stem cells. Following 1 week of stromal coculture, we report the recapitulation of 3D interactions between the mesenchymal and hematopoietic fractions, associated with a phenotypic expansion of the blood stem cell populations.Our data confirm that perfusion bioreactors fit for cell culture can be generated using a 3D printing technology and exploited for the 3D modeling of complex stem cell systems. Our approach opens the gates for a more faithful investigation of cellular processes in relation to a dynamic 3D microenvironment.

Multimodal imaging shows fibrosis architecture and action potential dispersion are predictors of arrhythmic risk in spontaneous hypertensive rats.

Hypertensive heart disease (HHD) increases risk of ventricular tachycardia (VT) and ventricular fibrillation (VF). The roles of structural vs. electrophysiological remodelling and age vs. disease progression are not fully understood. This cross‐sectional study of cardiac alterations through HHD investigates mechanistic contributions to VT/VF risk. Risk was electrically assessed in Langendorff‐perfused, spontaneously hypertensive rat hearts at 6, 12 and 18 months, and paced optical membrane voltage maps were acquired from the left ventricular (LV) free wall epicardium. Distributions of LV patchy fibrosis and 3D cellular architecture in representative anterior LV mid‐wall regions were quantified from macroscopic and microscopic fluorescence images of optically cleared tissue. Imaging showed increased fibrosis from 6 months, particularly in the inner LV free wall. Myocyte cross‐section increased at 12 months, while inter‐myocyte connections reduced markedly with fibrosis. Conduction velocity decreased from 12 months, especially transverse to the myofibre direction, with rate‐dependent anisotropy at 12 and 18 months, but not earlier. Action potential duration (APD) increased when clustered by age, as did APD dispersion at 12 and 18 months. Among 10 structural, functional and age variables, the most reliably linked were VT/VF risk, general LV fibrosis, a measure quantifying patchy fibrosis, and non‐age clustered APD dispersion. VT/VF risk related to a quantified measure of patchy fibrosis, but age did not factor strongly. The findings are consistent with the notion that VT/VF risk is associated with rate‐dependent repolarization heterogeneity caused by structural remodelling and reduced lateral electrical coupling between LV myocytes, providing a substrate for heterogeneous intramural activation as HHD progresses. Key points There is heightened arrhythmic risk with progression of hypertensive heart disease.Risk is related to increasing left ventricular fibrosis, but the nature of this relationship has not been quantified.This study is a novel systematic characterization of changes in active electrical properties and fibrotic remodelling during progression of hypertensive heart disease in a well‐established animal disease model.Arrhythmic risk is predicted by several left ventricular measures, in particular fibrosis quantity and structure, and epicardial action potential duration dispersion.Age alone is not a good predictor of risk.An improved understanding of links between arrhythmic risk and fibrotic architectures in progressive hypertensive heart disease aids better interpretation of late gadolinium‐enhanced cardiac magnetic resonance imaging and electrical mapping signals.

One- and Two-Photon Phototherapeutic Effects of RuII Polypyridine Complexes in the Hypoxic Centre of Large Multicellular Tumor Spheroids and Tumor-Bearing Mice*.

During the last decades, photodynamic therapy (PDT), an approved medical technique, has received increasing attention to treat certain types of cancer. Despite recent improvements, the treatment of large tumors remains a major clinical challenge due to the low ability of the photosensitizer (PS) to penetrate a 3D cellular architecture and the low oxygen concentrations present in the tumor center. To mimic the conditions found in clinical tumors, exceptionally large 3D multicellular tumor spheroids (MCTSs) with a diameter of 800 μm were used in this work to test a series of new RuII polypyridine complexes as one-photon and two-photon PSs. These metal complexes were found to fully penetrate the 3D cellular architecture and to generate singlet oxygen in the hypoxic center upon light irradiation. While having no observed dark toxicity, the lead compound of this study showed an impressive phototoxicity upon clinically relevant one-photon (595 nm) or two-photon (800 nm) excitation with a full eradication of the hypoxic center of the MCTSs. Importantly, this efficacy was also demonstrated on mice bearing an adenocarcinomic human alveolar basal epithelial tumor.

Construction of higher-order cellular microstructures by a self-wrapping co-culture strategy using a redox-responsive hydrogel

In this report, a strategy for constructing three-dimensional (3D) cellular architectures comprising viable cells is presented. The strategy uses a redox-responsive hydrogel that degrades under mild reductive conditions, and a confluent monolayer of cells (i.e., cell sheet) cultured on the hydrogel surface peels off and self-folds to wrap other cells. As a proof-of-concept, the self-folding of fibroblast cell sheet was triggered by immersion in aqueous cysteine, and this folding process was controlled by the cysteine concentration. Such folding enabled the wrapping of human hepatocellular carcinoma (HepG2) spheroids, human umbilical vein endothelial cells and collagen beads, and this process improved cell viability, the secretion of metabolites and the proliferation rate of the HepG2 cells when compared with a two-dimensional culture under the same conditions. A key concept of this study is the ability to interact with other neighbouring cells, providing a new, simple and fast method to generate higher-order cellular aggregates wherein different types of cellular components are added. We designated the method of using a cell sheet to wrap another cellular aggregate the ‘cellular Furoshiki’. The simple self-wrapping Furoshiki technique provides an alternative approach to co-culture cells by microplate-based systems, especially for constructing heterogeneous 3D cellular microstructures.

Graphene-based 3D lightweight cellular structures: Synthesis and applications

Ultralight three-dimensional (3D) cellular architectures are both a challenge and an opportunity for those academic and industrial applications involved with high performance material design. The challenges have been accessed by employing a variety of materials. Accordingly, graphene has shown greatest potential by virtue of its unique chemical, thermal, electronic, and mechanical properties for applications ranging from structural materials to energy storage/conversion devices. In this review, we highlight recent efforts and advances made in the implementation of graphene-based 3D cellular architectures, especially focusing on the ultralight density region (<10 mg·cm−3). First, we reviewed the synthetic approaches for generating graphene-based 3D lightweight cellular structures according to the criteria of closed-cellular structures (CCSs) or open-cellular structures (OCSs) whether cell faces between constituent unit cells exist or not, respectively. These structural differences could lead to discerning physical properties, such as in mass transport across pores, heat/electron transfer through graphene framework, and various modes in mechanical deformation. We then collectively introduce recent achievements for representative applications utilizing different types of cellular structures such as flexible electronics, energy storage/conversion systems, fluid absorption, mechanical dampers, and sensors. Finally, we highlight the future perspectives of graphene-based lightweight cellular structures to surpass existing technological challenges.

Microfluidic Synthesis of Carbon Nanotube-Networked Solid-Shelled Bubbles.

Carbon nanotubes (CNTs) have attracted considerable attention because of their high electrical conductivity and outstanding mechanical properties. As such, there have been numerous attempts to form CNTs into diverse structures for use in a wide range of applications. However, the intrinsic high aspect ratios of CNTs and resulting deformability have prevented the fabrication of sophisticated CNT-based structures, especially for three-dimensional (3D) cellular architectures. To challenge this limitation, we present a novel method to fabricate a 3D CNT cellular network from the assembly of microfluidically synthesized CNT-shelled microbubbles. We successfully generated stable spherical CNT-shelled bubbles with excellent size and shape uniformity by precisely controlling bubble dimensions by varying microfluidic variables. We also developed a fundamental understanding of the bubble stability, which allowed us to suppress shrinkage-induced deformation. The synthesized CNT-shelled bubbles were assembled into a 3D close-packed structure, followed by treatment with thermal reduction to induce interfacial bonding and transformation into a closed cellular network structure. Overall, this work provides a new strategy of assembling 1D nanomaterials as the building blocks for well-regulated 3D closed cellular architectures with improved structural or physical properties.

3D Cellular Architecture Modulates Tyrosine Kinase Activity, Thereby Switching CD95-Mediated Apoptosis to Survival

The death receptor CD95 is expressed in every cancer cell, thus providing a promising tool to target cancer. Activation of CD95 can, however, lead to apoptosis or proliferation. Yet the molecular determinants of CD95's mode of action remain unclear. Here, we identify an optimal distance between CD95Ligand molecules that enables specific clustering of receptor-ligand pairs, leadingto efficient CD95 activation. Surprisingly, efficientCD95 activation leads to apoptosis in cancer cells invitro and increased tumor growth invivo. We show that allowing a 3D aggregation of cancercells invitro switches the apoptotic response to proliferation. Indeed, we demonstrate that the absence or presence of cell-cell contactsdictates the cell response to CD95. Cellcontacts increase global levels of phosphorylated tyrosines, including CD95's tyrosine. A tyrosine-to-alanine CD95 mutant blocks proliferation in cellsin contact. Our study sheds light intothe regulatory mechanism of CD95 activation that canbe further explored for anti-cancer therapies.

Effects of substrate patterning on cellular spheroid growth and dynamics measured by gradient light interference microscopy (GLIM).

The development of three-dimensional (3D) cellular architectures during development and pathological processes involves intricate migratory patterns that are modulated by genetics and the surrounding microenvironment. The substrate composition of cell cultures has been demonstrated to influence growth, proliferation and migration in 2D. Here, we study the growth and dynamics of mouse embryonic fibroblast cultures patterned in a tissue sheet which then exhibits 3D growth. Using gradient light interference microscopy (GLIM), a label-free quantitative phase imaging approach, we explored the influence of geometry on cell growth patterns and rotational dynamics. We apply, for the first time to our knowledge, dispersion-relation phase spectroscopy (DPS) in polar coordinates to generate the radial and rotational cell mass-transport. Our data show that cells cultured on engineered substrates undergo rotational transport in a radially independent manner and exhibit faster vertical growth than the control, unpatterned cells. The use of GLIM and polar DPS provides a novel quantitative approach to studying the effects of spatially patterned substrates on cell motility and growth.

Mechanical properties of 3D double-U auxetic structures

Auxetic materials or structures, especially 3D cellular lattice architectures with negative Poisson's ratio (NPR) have attracted great attention due to their unprecedented mechanical behaviors and promising applications in recent years. Many 3D auxetic architected cellular materials have been manufactured and characterized with polymers and metals using additive manufacturing technology as well as some fibre reinforced polymers (FRP) composites via assembly method, however metal 3D printed auxetic structures with high-quality are rarely reported and FRP composites using assembly method contain a variety of defects which result in a knock-down effect on mechanical properties. Auxetic structures made from polymers are normally of low stiffness and strength and FRP composites are of low toughness and impact resistance, which causes limitations on their structural and functional applications. If rationally designed structures can be made from high-performance metal materials the specific stiffness and strength of which are much higher than polymers, their specific stiffness and strength can be significantly increased which will make them more suitable for various potential applications. This paper presents novel 3D double-U hierarchical structures (DUHs) based on double-V hierarchical structures (DVHs) with tunable Poisson's ratio and Young's modulus by tailoring the geometry parameters. Experimental, numerical and theoretical results show that DUHs have smooth geometry that can reduce manufacturing damage and stress concentration under elastic loading. Another advantage of DUHs is that the curved configurations have enhanced auxetic behavior and higher static collapse stress than DVHs under crushing. The advanced 3D printing technology and those excellent mechanical properties of 3D DUHs meet the requirements of structural and functional applications.

335-LB: Towards Generating Spatiotemporal Multiscale Models of Pancreatic ß Cells

To characterize biology, we must consider how molecular machines and organelles within a cell interact and traffic, similar to the importance of understanding traffic patterns in major cities. We use an imaging across scales approach to understand architectural changes that occur in pancreatic β-cells during insulin secretion. β-cells produce and release insulin in response to glucose and are therefore a target for diabetes therapies. Diabetes is a worldwide problem affecting hundreds of millions of people with increasing patient numbers every year. To acquire cellular architectural information, we are integrating data from cryo-electron tomography, soft x-ray tomography (SXT), and fluorescence microscopy. We used multiple glucose and GPCR agonist concentrations to investigate the cellular organization changes induced during insulin vesicle biogenesis and trafficking. The SXT method allows for investigating the 3D cellular architecture and is an ideal method for investigating cell-to-cell variabilities. Additionally, we are using machine learning methods for image analysis and autosegmentation which increased the number of conditions and samples we can analyze rapidly. Furthermore, we can use the information from cryo-ET and SXT along with with live cell imaging to assemble a data-driven, dynamic model of insulin granules within pancreatic β-cells. Furthermore, the SXT method provides a unique opportunity for comparing cell-to-cell heterogeneity and will be a useful technique for comparing differences between rodent and human primary cell architectures. The integration of data from different time and spatial scales represents a sophisticated convergence of our understanding of cellular structure and function, which will revolutionize biological discovery, open new dimensions of research, and accelerate advancements in healthcare. Disclosure K.L. White: None. J. Singla: None. R.C. Stevens: Board Member; Self; BirdRock Bio, Danaher Corp. Funding University of Southern California

Mesoscale Architecture of Beta Cells Upon Glucose and Ex-4 Stimulation

To understand biology we must consider how molecular machines and organelles within a cell interact and traffic, similar to the importance of understanding traffic patterns in major cities. We use an imaging across scales approach to understand architectural changes that occur in pancreatic β-cells during insulin secretion. β-cells produce and release insulin in response to glucose and are therefore a target for diabetes therapies. Diabetes is a worldwide problem affecting hundreds of millions of people with increasing patient numbers every year. To acquire cellular architectural information, we are integrating data from cryo-electron tomography, soft x-ray tomography (SXT), and fluorescence microscopy. We imaged INS-1E cells (rat pancreatic β-cell line) under multiple glucose and GPCR agonist concentrations to investigate the cellular organization changes induced during insulin vesicle biogenesis and trafficking. The SXT method allows for investigating the 3D cellular architecture and is an ideal method for investigating cell-to-cell variabilities. Additionally, we are using machine learning methods for image analysis and autosegmentation which increased the number of conditions and samples we can analyze rapidly. Furthermore, we can use the information from cryo-ET and SXT along with with live cell imaging to assemble a data-driven, dynamic model of insulin granules within pancreatic β-cells. The integration of data from different time and spatial scales represents a sophisticated convergence of our understanding of cellular structure and function, which will revolutionize biological discovery, open new dimensions of research, and accelerate advancements in healthcare.

Beyond 5G With UAVs: Foundations of a 3D Wireless Cellular Network

In this paper, a novel concept of three-dimensional (3D) cellular networks, that integrate drone base stations (drone-BS) and cellular-connected drone users (drone-UEs), is introduced. For this new 3D cellular architecture, a novel framework for network planning for drone-BSs and latency-minimal cell association for drone-UEs is proposed. For network planning, a tractable method for drone-BSs’ deployment based on the notion of truncated octahedron shapes is proposed, which ensures full coverage for a given space with a minimum number of drone-BSs. In addition, to characterize frequency planning in such 3D wireless networks, an analytical expression for the feasible integer frequency reuse factors is derived. Subsequently, an optimal 3D cell association scheme is developed for which the drone-UEs’ latency, considering transmission, computation, and backhaul delays, is minimized. To this end, first, the spatial distribution of the drone-UEs is estimated using a kernel density estimation method, and the parameters of the estimator are obtained using a cross-validation method. Then, according to the spatial distribution of drone-UEs and the locations of drone-BSs, the latency-minimal 3D cell association for drone-UEs is derived by exploiting tools from an optimal transport theory. The simulation results show that the proposed approach reduces the latency of drone-UEs compared with the classical cell association approach that uses a signal-to-interference-plus-noise ratio (SINR) criterion. In particular, the proposed approach yields a reduction of up to 46% in the average latency compared with the SINR-based association. The results also show that the proposed latency-optimal cell association improves the spectral efficiency of a 3D wireless cellular network of drones.

3D Cellular Architecture Affects MicroRNA and Protein Cargo of Extracellular Vesicles.

The success of malignant tumors is conditioned by the intercellular communication between tumor cells and their microenvironment, with extracellular vesicles (EVs) acting as main mediators. While the value of 3D conditions to study tumor cells is well established, the impact of cellular architecture on EV content and function is not investigated yet. Here, a recently developed 3D cell culture microwell array is adapted for EV production and a comprehensive comparative analysis of biochemical features, RNA and proteomic profiles of EVs secreted by 2D vs 3D cultures of gastric cancer cells, is performed. 3D cultures are significantly more efficient in producing EVs than 2D cultures. Global upregulation of microRNAs and downregulation of proteins in 3D are observed, indicating their dynamic coregulation in response to cellular architecture, with the ADP‐ribosylation factor 6 signaling pathway significantly downregulated in 3D EVs. The data strengthen the biological relevance of cellular architecture for production and cargo of EVs.

Use of ionic liquid for X-ray micro-CT specimen preparation of imbibed seeds.

X-ray micro-CT is one of the most useful techniques to examine 3D cellular architecture inside dry seeds. However, the examination of imbibed seeds is difficult because immersion in water causes a decline in the image quality. Here, we examined the use of ionic liquids for specimen preparation of chemically fixed imbibed seeds of Arabidopsis. We found that treatment with high concentrations of ionic liquids after osmium tetroxide fixation helped not only to prevent the structural damage caused by seed shrinkage, but also to preserve the image quality. Under these conditions, the cellular architecture of seeds was also well maintained.

Graphene-Bridged Multifunctional Flexible Fiber Supercapacitor with High Energy Density.

Portable fiber supercapacitors with high-energy storage capacity are in great demand to cater for the rapid development of flexible and deformable electronic devices. Hence, we employed a 3D cellular copper foam (CF) combined with the graphene sheets (GSs) as the support matrix to bridge the active material with nickel fiber (NF) current collector, significantly increasing surface area and decreasing the interface resistance. In comparison to the active material directly growing onto the NF in the absence of CF and GSs, our rationally designed architecture achieved a joint improvement in both capacity (0.217 mAh cm-2/1729.413 mF cm-2, 1200% enhancement) and rate capability (87.1% from 1 to 20 mA cm-2, 286% improvement), which has never been achieved before with other fiber supercapacitors. The in situ scanning electron microscope (SEM) microcompression test demonstrated its superior mechanical recoverability for the first time. Importantly, the assembled flexible and wearable device presented a superior energy density of 109.6 μWh cm-2 at a power density of 749.5 μW cm-2, and the device successfully coupled with a flexible strain sensor, solar cell, and nanogenerator. This rational design should shed light on the manufacturing of 3D cellular architectures as microcurrent collectors to realize high energy density for fiber-based energy storage devices.

3D Printing of Carbides Using Renewable Resources

Here we present our initial results for fabrication of 3D cellular architectures of tungsten carbide by 3D printing. We 3D printed cellular architectures of a biopolymer gel composite that consisted of iota-carrageenan and chitin as carbon precursors and tungsten tri-oxide as the tungsten source. The 3D printed gel composite was dried to obtain a 3D structure of biopolymer xerogel. Heat treatment of the xerogel structures resulted in 3D cellular architectures of porous tungsten carbide (WC). Significant shrinkage occurred during the drying and heat treatment process. The X-ray diffraction pattern confirmed that WC was formed after heat treatment. Scanning electron microscopy showed that the WC formed here featured a porous network of particle agglomerates. Ongoing work includes compressive testing to determine the Young’s modulus of samples. A design of experiments will be implemented to develop a relationship between mechanical behavior and the geometry of the cellular architecture. Additionally, shrinkage, material solvent, printing substrate and filament diameter require further study to control rheology and increase precision during fabrication.

Unveiling 3D physicochemical changes of sugarcane bagasse during sequential acid/alkali pretreatments by synchrotron phase-contrast imaging

Several pretreatment strategies focus on the removal of a significant part of lignin from plant cell walls, as it is considered the main barrier to the process of deconstructing biomass to simple sugars by hydrolytic enzymes. The ability to chemically differentiate and spatially locate lignins across cell walls provides an important contribution to the effort to improve these processes. Here, we present a novel approach using synchrotron phase-contrast tomography to probe the physicochemical features of plant cell walls. In addition to the 3D cellular architecture, the distribution of dense packed lignin–carbohydrate complexes (or, simply dense lignin) over larger regions and within cell walls has been successfully provided. We examined in particular the effect of the sequential H2SO4 and NaOH pretreatment on sugacane bagasse. Our results revealed that aggregates of dense lignin form elongated 3D structures in cell corners (CC) following the orientation of the sclerenchyma fibers; a remarkable result in the light of the conventional perception that lignin particles are discrete and roughly spherical. After the acid pretreatment, a considerable fraction of the dense lignin primarily located in CCs was removed; and it was further dissolved with the subsequent alkali treatment. Unexpectedly, the two-step pretreatment did not contribute to the cellulose accessibility in terms of available surface area and porosity. This study provides new insights into the underlying mechanisms of biomass deconstruction by pretreatments; and the approach established here can be extended to other systems relevant to the bioenergy and biotechnology arenas.