Multiple Arabidopsis galacturonosyltransferases synthesize polymeric homogalacturonan by oligosaccharide acceptor-dependent or de novo synthesis.

Homogalacturonan (HG), the most abundant pectic glycan, functions as a cell wall structural and signaling molecule essential for plant growth, development and response to pathogens. HG exists as a component of pectic homoglycans, heteroglycans and glycoconjugates. HG is synthesized by members of the GALACTURONOSYLTRANSFERASE (GAUT) family. UDP-GalA-dependent homogalacturonan:galacturonosyltransferase (HG:GalAT) activity has previously been demonstrated for GAUTs 1, 4 and 11, as well as the GAUT1:GAUT7 complex. Here, we show that GAUTs 10, 13 and 14 are also HG:GalATs and that GAUTs 1, 10, 11, 13, 14 and 1:7 synthesize polymeric HG in vitro. Comparison of the in vitro HG:GalAT specific activities of the heterologously-expressed proteins demonstrates GAUTs 10 and 11 with the lowest, GAUT1 and GAUT13 with moderate, and GAUT14 and the GAUT1:GAUT7 complex with the highest HG:GalAT activity. GAUT13 and GAUT14 are also shown to de novo synthesize (initiate) HG synthesis in the absence of exogenous HG acceptors, an activity previously demonstrated for GAUT1:GAUT7. The rate of de novo HG synthesis by GAUT13 and GAUT14 is similar to their acceptor dependent HG synthesis, in contrast to GAUT1:GAUT7 for which de novo synthesis occurred at much lower rates than acceptor-dependent synthesis. The results suggest a unique role for de novo HG synthesis by GAUTs 13 and 14. The reducing end of GAUT13-de novo-synthesized HG has covalently attached UDP, indicating that UDP-GalA serves as both a donor and acceptor substrate during de novo HG synthesis. The functional significance of unique GAUT HG:GalAT catalytic properties in the synthesis of different pectin glycan or glycoconjugate structures is discussed.

Synthesis and process optimization of symmetric and unsymmetric barbiturates C5-coupled with 2,1-benzisoxazoles.

Benzisoxazoles represent an important pharmacophore in medicinal chemistry. Recently, an unexpected formation of symmetric 3-substituted 2,1-benzisoxazoles through reduction of 5-(2-nitrobenzylidene)barbiturates has been described. This reductive intramolecular heterocyclization probably involves a nitroso intermediary. To improve the previous reaction conditions, the nature of the reducing agent and additives, reaction time and solvents were evaluated. By applying the optimized conditions, several symmetric and unsymmetric barbiturates C5-coupled with 2,1-benzisoxazoles were prepared with an yield of 52-87%. From this set, seven compounds were novel and the unsymmetric nature of the (thio)barbituric acid moiety was explored. For that, the total synthesis, starting from the respective urea or thiourea, was successfully performed, and 11 thiobarbiturates, benzylidene barbiturate and thiobarbiturate precursors are described.

A two-phase model for the non-processive biosynthesis of homogalacturonan polysaccharides by the GAUT1:GAUT7 complex.

Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development and cell wall structure and function, and interacts with other glycans and proteoglycans in the wall. HG is synthesized by the galacturonosyltransferase (GAUT) gene family. Two members of this family, GAUT1 and GAUT7, form a heteromeric enzyme complex in Arabidopsis thaliana Here, we established a heterologous GAUT expression system in HEK293 cells and show that co-expression of recombinant GAUT1 with GAUT7 results in the production of a soluble GAUT1:GAUT7 complex that catalyzes elongation of HG products in vitro The reaction rates, progress curves, and product distributions exhibited major differences dependent upon small changes in the degree of polymerization (DP) of the oligosaccharide acceptor. GAUT1:GAUT7 displayed >45-fold increased catalytic efficiency with DP11 acceptors relative to DP7 acceptors. Although GAUT1:GAUT7 synthesized high-molecular-weight polymeric HG (>100 kDa) in a substrate concentration-dependent manner typical of distributive (nonprocessive) glycosyltransferases with DP11 acceptors, reactions primed with short-chain acceptors resulted in a bimodal product distribution of glycan products that has previously been reported as evidence for a processive model of GT elongation. As an alternative to the processive glycosyltransfer model, a two-phase distributive elongation model is proposed in which a slow phase, which includes the de novo initiation of HG and elongation of short-chain acceptors, is distinguished from a phase of rapid elongation of intermediate- and long-chain acceptors. Upon reaching a critical chain length of DP11, GAUT1:GAUT7 elongates HG to high-molecular-weight products.

Assessment of the Versius surgical robotic system for dual-field synchronous transanal total mesorectal excision (taTME) in a preclinical model: will tomorrow's surgical robots promise newfound options?

BACKGROUND: The aim of this study was to evaluate the feasibility of the Versius surgical robotic system for transanal total mesorectal excision (taTME) in a preclinical setting. METHODS: Dry laboratory and cadaveric sessions were first conducted for three experienced colorectal surgeons in order to gain familiarity with the modular surgical system and the robotic workstation. After introduction, the system was configured to allow for synchronous, totally robotic taTME in a cadaver. RESULTS: Using the modular robotic system, one surgeon performed the abdominal portion of the operation, including colonic mobilization and vascular pedicle ligation while simultaneously a second surgeon performed the transanal portion of the operation to the point of rendezvous at the peritoneal reflection, where the operation was completed cooperatively. The operation was successfully completed in 195 min demonstrating preclinical feasibility of this unique approach with an emerging robotic system. CONCLUSIONS: This is the first preclinical assessment of the Versius surgical robotic system for taTME. The ability to work simultaneously carries the theoretical advantage of reducing surgical time and thereby reducing overall operative costs. It may also allow surgeons to maintain focus on critical parts of the operation by halving the fatigue associated with long, complex cases such as taTME.

Robotic-assisted stereotactic real-time navigation: initial clinical experience and feasibility for rectal cancer surgery.

BACKGROUND: Real-time stereotactic navigation for transanal total mesorectal excision has been demonstrated to be feasible in small pilot series using laparoscopic techniques. The possibility of real-time stereotactic navigation coupled with robotics has not been previously explored in a clinical setting. METHODS: After pre-clinical assessment, and configuration of a robotic-assisted navigational system, two patients with locally advanced rectal cancer were selected for enrollment into a pilot study designed to assess the feasibility of navigation coupled with the robotic da Vinci Xi platform via TilePro interface. In one case, fluorescence-guided surgery was also used as an adjunct for structure localization, with local administration of indocyanine green into the ureters and at the tumor site. RESULTS: Each operation was successfully completed with a robotic-assisted approach; image-guided navigation provided computed accuracy of ± 4.5 to 4.6 mm. The principle limitation encountered was navigation signal dropout due to temporary loss of direct line-of-sight with the navigational system's infrared camera. Subjectively, the aid of navigation assisted the operating surgeon in identifying critical anatomical planes. The combination of fluorescence with image-guided surgery further augmented the surgeon's perception of the operative field. CONCLUSIONS: The combination of stereotactic navigation and robotic surgery is feasible, although some limitations and technical challenges were observed. For complex surgery, the addition of navigation to robotics can improve surgical precision. This will likely represent the next step in the evolution of robotics and in the development of digital surgery.

Advances in stereotactic navigation for pelvic surgery.

BACKGROUND: Stereotactic navigation could improve the quality of surgery for rectal cancer. Critical challenges related to soft tissue stereotactic pelvic navigation include the potential difference in patient anatomy between intraoperative lithotomy and preoperative supine position for imaging. The objective of this study was to determine the difference in patient anatomy, sacral tilt, and skin fiducial position between these different patient positions and to investigate the feasibility and optimal set-up for stereotactic pelvic navigation. METHODS: Four consecutive human anatomical specimens were submitted to repeated CT-scans in a supine and several degrees of lithotomy position. Patient anatomy, sacral tilt, and skin fiducial position were compared by means of an image computing platform. In two specimens, a 10-degree wedge was introduced to reduce the natural tilt of the sacrum during the shift from supine to lithotomy position. A simulation of laparoscopic and transanal surgical procedures was performed to assess the accuracy of the stereotactic navigation. RESULTS: An up-to-supracentimetric change in patient anatomy was noted between different patient positions. This observation was minimized through the application of a wedge. When switching from supine to another position, sacral retroversion occurred independent of the use of a wedge. There was considerable skin fiducial motion between different positions. Accurate stereotactic navigation was obtained with the least registration error (1.9 mm) when the position of the anatomical specimen was registered in a supine position with straight legs, without pneumoperitoneum, using a conventional CT-scan with an identical specimen positioning. CONCLUSION: The change in patient anatomy is small during the sacral tilt induced by positional changes when using a 10-degree wedge, allowing for an accurate stereotactic surgical navigation. This opens up new promising opportunities to increase the quality of surgery for rectal cancer cases where it is difficult or impossible to identify and dissect along the anatomical planes.

A step towards stereotactic navigation during pelvic surgery: 3D nerve topography.

BACKGROUND: Long-term morbidity after multimodal treatment for rectal cancer is suggested to be mainly made up by nerve-injury-related dysfunctions. Stereotactic navigation for rectal surgery was shown to be feasible and will be facilitated by highlighting structures at risk of iatrogenic damage. The aim of this study was to investigate the ability to make a 3D map of the pelvic nerves with magnetic resonance imaging (MRI). METHODS: A systematic review was performed to identify a main positional reference for each pelvic nerve and plexus. The nerves were manually delineated in 20 volunteers who were scanned with a 3-T MRI. The nerve identifiability rate and the likelihood of nerve identification correctness were determined. RESULTS: The analysis included 61 studies on pelvic nerve anatomy. A main positional reference was defined for each nerve. On MRI, the sacral nerves, the lumbosacral plexus, and the obturator nerve could be identified bilaterally in all volunteers. The sympathetic trunk could be identified in 19 of 20 volunteers bilaterally (95%). The superior hypogastric plexus, the hypogastric nerve, and the inferior hypogastric plexus could be identified bilaterally in 14 (70%), 16 (80%), and 14 (70%) of the 20 volunteers, respectively. The pudendal nerve could be identified in 17 (85%) volunteers on the right side and in 13 (65%) volunteers on the left side. The levator ani nerve could be identified in only a few volunteers. Except for the levator ani nerve, the radiologist and the anatomist agreed that the delineated nerve depicted the correct nerve in 100% of the cases. CONCLUSION: Pelvic nerves at risk of injury are usually visible on high-resolution MRI with dedicated scanning protocols. A specific knowledge of their course and its application in stereotactic navigation is suggested to improve quality of life by decreasing the likelihood of nerve injury.

Temperature- and/or pH-Responsive Surfaces with Controllable Wettability: From Parahydrophobicity to Superhydrophilicity.

Multifunctional surfaces with reversible wetting characteristics are fabricated utilizing end-anchored polymer chains on hierarchically roughened surfaces. Temperature- and/or pH-responsive surfaces are developed that exhibit reversible and controllable wettability, from the "parahydrophobic" behavior of natural plant leaves all the way to superhydrophilic properties in response to the external stimuli. For this purpose, dual scale micro/nanoroughened surfaces were prepared by laser irradiation of inorganic surfaces (Si wafers) utilizing ultrafast (femtosecond) laser pulses under a reactive gas atmosphere. End-functionalized polymer chains were anchored onto those surfaces utilizing the "grafting to" method; poly(N-isopropylacrylamide), PNIPAM, and poly(2-vinylpyridine), P2VP, were used for the formation of monofunctional as well as mixed brushes. The surfaces exhibit "parahydrophobic" behavior in the hydrophobic state (high temperature and/or high pH), with high static contact angles (∼120°) and high water adhesion (∼30° contact angle hysteresis), whereas they show superhydrophilic behavior in the hydrophilic state (low temperature and/or low pH). The surfaces were tested for their wettability under repetitive cycles and found to be stable and reproducible.

Development of an in vitro tumor spheroid culture model amenable to high-throughput testing of potential anticancer nanotherapeutics.

CONTEXT: Three-dimensional tumor spheroid cultures are a better representative of in vivo solid tumors than monolayer cultures and should be used for testing potential nanotherapeutics in vitro. OBJECTIVE: To develop techniques to test the disposition and efficacy of nanocarrier formulations in spheroids in a cost-effective manner amenable to high-throughput testing. METHODS: Spheroids were obtained using a modified liquid overlay technique in a 96-well plate. Several nanocarrier formulations were prepared and tested in the spheroid model. The disposition of the formulations in the spheroids was determined by confocal microscopy while the effect of the drug-loaded formulations was assessed in terms of the cell viability, loss of membrane integrity, induction of caspases and inhibition of growth of the spheroids. RESULTS: The surface charge of the formulations influenced the accumulation of the nanocarrier and drug in the spheroid, with the cationic formulation accumulating to the greatest extent. Also, the smallest particle size formulation, micelles, penetrated to the greatest extent in the spheroid. The iRGD tumor-penetrating peptide co-administered with unmodified liposomes exhibited both high accumulation and penetration. The effect studies revealed that the formulations that penetrated or accumulated to the highest extent in the spheroid exhibited better antitumor activity compared to the other formulations. CONCLUSION: The 96-well plate format spheroid model developed in the study can be used toward the rational selection of nanocarrier therapeutics prior to their testing in in vivo models.

Hydrophobized triphenyl phosphonium derivatives for the preparation of mitochondriotropic liposomes: choice of hydrophobic anchor influences cytotoxicity but not mitochondriotropic effect.

CONTEXT: Nanocarrier-based strategies to achieve delivery of bioactives specifically to the mitochondria are being increasingly explored due to the importance of mitochondria in critical cellular processes. OBJECTIVE: To test the ability of liposomes modified with newly synthesized triphenylphosphonium (TPP)-phospholipid conjugates and to test their use in overcoming the cytotoxicity of stearyl triphenylphosphonium (STPP)-modified liposomes when used for delivery of therapeutic molecules to the mitochondria. METHODS: TPP-phospholipid conjugates with the dioleoyl, dimyristoyl or dipalmitoyl lipid moieties were synthesized and liposomes were prepared with these conjugates in a 1 mol% ratio. The subcellular distribution of the liposomes was tested by confocal microscopy. Furthermore, the liposomes were tested for their effect on cell viability using a MTS assay, on cell membrane integrity using a lactate dehydrogenase assay and on mitochondrial membrane integrity using a modified JC-1 assay. RESULTS: The liposomes modified with the new TPP-phospholipid conjugates exhibited similar mitochondriotropism as STPP-liposomes but they were more biocompatible as compared to the STPP liposomes. While the STPP-liposomes had a destabilizing effect on cell and mitochondrial membranes, the liposomes modified with the TPP-phospholipid conjugates did not demonstrate any such effect on biomembranes. CONCLUSIONS: Using phospholipid anchors in the synthesis of TPP-lipid conjugates can provide liposomes that exhibit the same mitochondrial targeting ability as STPP but with much higher biocompatibility.

The quest for precision in transanal total mesorectal excision.

Transanal total mesorectal excision (TME) is an emerging minimally invasive approach to rectal cancer, with encouraging preliminary results. However, the new surgical anatomy of the bottom-up approach complicates surgical understanding and increases the risks of inadvertent injuries to crucial anatomical structures, including nerves. Key elements to improve the safety and stimulate interest in such a complex technique might be robotics and image guidance, to enhance the level of precision. In this editorial, some of the technologies that could be used for precision TME are outlined, in light of the experience of our Institute for Image-Guided Surgery.

GALACTURONOSYLTRANSFERASE-LIKE5 is involved in the production of Arabidopsis seed coat mucilage.

The function of a putative galacturonosyltransferase from Arabidopsis (Arabidopsis thaliana; At1g02720; GALACTURONOSYLTRANSFERASE-LIKE5 [AtGATL5]) was studied using a combination of molecular genetic, chemical, and immunological approaches. AtGATL5 is expressed in all plant tissues, with highest expression levels in siliques 7 DPA. Furthermore, its expression is positively regulated by several transcription factors that are known to regulate seed coat mucilage production. AtGATL5 is localized in both endoplasmic reticulum and Golgi, in comparison with marker proteins resident to these subcellular compartments. A transfer DNA insertion in the AtGATL5 gene generates seed coat epidermal cell defects both in mucilage synthesis and cell adhesion. Transformation of atgatl5-1 mutants with the wild-type AtGATL5 gene results in the complementation of all morphological phenotypes. Compositional analyses of the mucilage isolated from the atgatl5-1 mutant demonstrated that galacturonic acid and rhamnose contents are decreased significantly in atgatl5-1 compared with wild-type mucilage. No changes in structure were observed between soluble mucilage isolated from wild-type and mutant seeds, except that the molecular weight of the mutant mucilage increased 63% compared with that of the wild type. These data provide evidence that AtGATL5 might function in the regulation of the final size of the mucilage rhamnogalacturonan I.

Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex.

Plant cell wall pectic polysaccharides are arguably the most complex carbohydrates in nature. Progress in understanding pectin synthesis has been slow due to its complex structure and difficulties in purifying and expressing the low-abundance, Golgi membrane-bound pectin biosynthetic enzymes. Arabidopsis galacturonosyltransferase (GAUT) 1 is an α-1,4-galacturonosyltransferase (GalAT) that synthesizes homogalacturonan (HG), the most abundant pectic polysaccharide. We now show that GAUT1 functions in a protein complex with the homologous GAUT7. Surprisingly, although both GAUT1 and GAUT7 are type II membrane proteins with single N-terminal transmembrane-spanning domains, the N-terminal region of GAUT1, including the transmembrane domain, is cleaved in vivo. This raises the question of how the processed GAUT1 is retained in the Golgi, the site of HG biosynthesis. We show that the anchoring of GAUT1 in the Golgi requires association with GAUT7 to form the GAUT1:GAUT7 complex. Proteomics analyses also identified 12 additional proteins that immunoprecipitate with the GAUT1:GAUT7 complex. This study provides conclusive evidence that the GAUT1:GAUT7 complex is the catalytic core of an HG:GalAT complex and that cell wall matrix polysaccharide biosynthesis occurs via protein complexes. The processing of GAUT1 to remove its N-terminal transmembrane domain and its anchoring in the Golgi by association with GAUT7 provides an example of how specific catalytic domains of plant cell wall biosynthetic glycosyltransferases could be assembled into protein complexes to enable the synthesis of the complex and developmentally and environmentally plastic plant cell wall.

Speckle tracking echocardiography in plasma cell disorders: The role of advanced imaging in the early diagnosis of AL systemic cardiac amyloidosis.

INTRODUCTION: Amyloid light-chain amyloidosis is a rare condition characterized by the abnormal production of immunoglobulin light chain that misshape and form amyloid fibrils. Over time, these amyloid deposits can accumulate slowly, causing dysfunction in organs and tissues. Early identification is crucial to ensure optimal treatment. We aim to identify a better marker of cardiac amyloidosis, using advanced echocardiography, to improve diagnosis and the timing of available treatments. MATERIALS AND METHODS: 108 consecutive hematological patients (32, 30% female and 76, 70% male) with a plasma cell disorder referred to our Cardiological center underwent ECG, first and second-level echocardiography (Speckle Tracking) and complete biochemical profile. The best predictors of ALCA (AUC ≥ 0.8) were included in a further analysis stratified by AL score. RESULTS: At ROC analysis, the best bio-humoral predictors for the diagnosis of ALCA were Nt-pro-BNP (AUC: 0.97; p < 0.01) and Hs-Tn (AUC: 0.87; p < 0.01). Regarding echocardiography, the best diagnostic predictors were left atrial stiffness (LAS) (AUC: 0.83; p < 0.01) for the left atrium; free wall thickness for the right ventricle (AUC: 0.82; <0.01); left ventricular global longitudinal strain (LVGLS) (AUC: 0.92; p < 0.01) and LVMi (AUC 0.80; p < 0.001) for the left ventricle; and AL-score (AUC 0.83 p < 0.01). In patients with AL-SCORE < 1, LAS (AUC 0.86 vs AUC 0.79), LVGLS (AUC 0.92 vs AUC 0.86) and LV mass (AUC 0.91 vs AUC 0.72) had better diagnostic accuracy than patients with higher AL-score (AL SCORE ≥ 1). CONCLUSION: Multi-parametric imaging approach with LVGLS and LAS may be helpful for detecting early cardiac involvement in AL amyloidosis.

Determination of the Subcellular Distribution of Fluorescently Labeled Liposomes Using Confocal Microscopy.

It is being increasingly recognized that therapeutics need to be delivered to specific organelle targets within cells. Liposomes are versatile lipid-based drug delivery vehicles that can be surface modified to deliver the loaded cargo to specific subcellular locations within the cell. Hence, the development of such technology requires a means of measuring subcellular distribution by utilizing imaging techniques that can visualize and quantitate the extent of this subcellular localization. The apparent increase of resolution along the Z-axis offered by confocal microscopy makes this technique suitable for such studies. In this chapter, we will describe the application of confocal laser scanning microscopy (CLSM) to determine the subcellular distribution of fluorescently labeled mitochondriotropic liposomes.

Polymerization of the backbone of the pectic polysaccharide rhamnogalacturonan I.

Rhamnogalacturonan I (RG-I) is a major plant cell wall pectic polysaccharide defined by its repeating disaccharide backbone structure of [4)-α-D-GalA-(1,2)-α-L-Rha-(1,]. A family of RG-I:Rhamnosyltransferases (RRT) has previously been identified, but synthesis of the RG-I backbone has not been demonstrated in vitro because the identity of Rhamnogalacturonan I:Galaturonosyltransferase (RG-I:GalAT) was unknown. Here a putative glycosyltransferase, At1g28240/MUCI70, is shown to be an RG-I:GalAT. The name RGGAT1 is proposed to reflect the catalytic activity of this enzyme. When incubated together with the rhamnosyltransferase RRT4, the combined activities of RGGAT1 and RRT4 result in elongation of RG-I acceptors in vitro into a polymeric product. RGGAT1 is a member of a new GT family categorized as GT116, which does not group into existing GT-A clades and is phylogenetically distinct from the GALACTURONOSYLTRANSFERASE (GAUT) family of GalA transferases that synthesize the backbone of the pectin homogalacturonan. RGGAT1 has a predicted GT-A fold structure but employs a metal-independent catalytic mechanism that is rare among glycosyltransferases with this fold type. The identification of RGGAT1 and the 8-member Arabidopsis GT116 family provides a new avenue for studying the mechanism of RG-I synthesis and the function of RG-I in plants.

Composition and yield of non-cellulosic and cellulosic sugars in soluble and particulate fractions during consolidated bioprocessing of poplar biomass by Clostridium thermocellum.

BACKGROUND: Terrestrial plant biomass is the primary renewable carbon feedstock for enabling transition to a sustainable bioeconomy. Consolidated bioprocessing (CBP) by the cellulolytic thermophile Clostridium thermocellum offers a single step microbial platform for production of biofuels and biochemicals via simultaneous solubilization of carbohydrates from lignocellulosic biomass and conversion to products. Here, solubilization of cell wall cellulosic, hemicellulosic, and pectic polysaccharides in the liquor and solid residues generated during CBP of poplar biomass by C. thermocellum was analyzed. RESULTS: The total amount of biomass solubilized in the C. thermocellum DSM1313 fermentation platform was 5.8, 10.3, and 13.7% of milled non-pretreated poplar after 24, 48, and 120 h, respectively. These results demonstrate solubilization of 24% cellulose and 17% non-cellulosic sugars after 120 h, consistent with prior reports. The net solubilization of non-cellulosic sugars by C. thermocellum (after correcting for the uninoculated control fermentations) was 13 to 36% of arabinose (Ara), xylose (Xyl), galactose (Gal), mannose (Man), and glucose (Glc); and 15% and 3% of fucose and glucuronic acid, respectively. No rhamnose was solubilized and 71% of the galacturonic acid (GalA) was solubilized. These results indicate that C. thermocellum may be selective for the types and/or rate of solubilization of the non-cellulosic wall polymers. Xyl, Man, and Glc were found to accumulate in the fermentation liquor at levels greater than in uninoculated control fermentations, whereas Ara and Gal did not accumulate, suggesting that C. thermocellum solubilizes both hemicelluloses and pectins but utilizes them differently. After five days of fermentation, the relative amount of Rha in the solid residues increased 21% indicating that the Rha-containing polymer rhamnogalacturonan I (RG-I) was not effectively solubilized by C. thermocellum CBP, a result confirmed by immunoassays. Comparison of the sugars in the liquor versus solid residue showed that C. thermocellum solubilized hemicellulosic xylan and mannan, but did not fully utilize them, solubilized and appeared to utilize pectic homogalacturonan, and did not solubilize RG-I. CONCLUSIONS: The significant relative increase in RG-I in poplar solid residues following CBP indicates that C. thermocellum did not solubilize RG-I. These results support the hypothesis that this pectic glycan may be one barrier for efficient solubilization of poplar by C. thermocellum.