Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili.

Type IV pili (Tfp), which are key virulence factors in many bacterial pathogens, define a large group of multipurpose filamentous nanomachines widespread in Bacteria and Archaea. Tfp biogenesis is a complex multistep process, which relies on macromolecular assemblies composed of 15 conserved proteins in model gram-negative species. To improve our limited understanding of the molecular mechanisms of filament assembly, we have used a synthetic biology approach to reconstitute, in a nonnative heterologous host, a minimal machinery capable of building Tfp. Here we show that eight synthetic genes are sufficient to promote filament assembly and that the corresponding proteins form a macromolecular complex at the cytoplasmic membrane, which we have purified and characterized biochemically. Our results contribute to a better mechanistic understanding of the assembly of remarkable dynamic filaments nearly ubiquitous in prokaryotes.

Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The result of the GAA deficiency is a ubiquitous lysosomal and non-lysosomal accumulation of glycogen. The most affected tissues are heart, skeletal muscle, liver, and the nervous system. Replacement therapy with the currently approved enzyme relies on M6P-mediated endocytosis. However, therapeutic outcomes still leave room for improvement, especially with regard to skeletal muscles. We tested the uptake, activity, and effect on glucose metabolism of a non-phosphorylated recombinant human GAA produced in moss (moss-GAA). Three variants of moss-GAA differing in glycosylation pattern have been analyzed: two with terminal mannose residues in a paucimannosidic (Man3) or high-mannose (Man 5) configuration and one with terminal N-acetylglucosamine residues (GnGn). Compared to alglucosidase alfa the moss-GAA GnGn variant showed increased uptake in differentiated myotubes. Moreover, incubation of immortalized muscle cells of Gaa-/- mice with moss-GAA GnGn led to similarly efficient clearance of accumulated glycogen as with alglucosidase alfa. These initial data suggest that M6P-residues might not always be necessary for the cellular uptake in enzyme replacement therapy (ERT) and indicate the potential of moss-GAA GnGn as novel alternative drug for targeting skeletal muscle in Pompe patients.

Pharmacokinetics, pharmacodynamics, and safety of moss-aGalactosidase A in patients with Fabry disease.

Moss-aGalactosidase A (moss-aGal) is a moss-derived version of human α-galactosidase developed for enzyme replacement therapy in patients with Fabry disease. It exhibits a homogenous N-glycosylation profile with >90% mannose-terminated glycans. In contrast to mammalian cell produced α-galactosidase, moss-aGal does not rely on mannose-6-phosphate receptor mediated endocytosis but targets the mannose receptor for tissue uptake. We conducted a phase 1 clinical trial with moss-aGal in six patients with confirmed diagnosis of Fabry disease during a 28-day schedule. All patients received a single dose of 0.2 mg/kg moss-aGal by i.v.-infusion. Primary endpoints of the trial were safety and pharmacokinetics; secondary endpoints were pharmacodynamics by analyzing urine and plasma Gb3 and lyso-Gb3 concentrations. In all patients, the administered single dose was well tolerated. No safety issues were observed. Pharmacokinetic data revealed a stable nonlinear profile with a short plasma half-life of moss-aGal of 14 minutes. After one single dose of moss-aGal, urinary Gb3 concentrations decreased up to 23% 7 days and up to 60% 28 days post-dose. Plasma concentrations of lyso-Gb3 decreased by 3.8% and of Gb3 by 11% 28 days post-dose. These data reveal that a single dose of moss-aGal was safe, well tolerated, and led to a prolonged reduction of Gb3 excretion. As previously shown, moss-aGal is taken up via the mannose receptor, which is expressed on macrophages but also on endothelial and kidney cells. Thus, these data indicate that moss-aGal may target kidney cells. After these promising results, phase 2/3 clinical trials are in preparation.

Screening Endoscopy Contributes to Relevant Modifications of Therapeutic Regimen in Children With Intestinal Failure.

OBJECTIVE: The role of endoscopy in the diagnostic workup of children with intestinal failure (IF) is not well defined. It is unclear whether endoscopies should be performed as a screening procedure or only upon manifestation of symptoms. The aim of this study is to evaluate, whether performing screening endoscopy contributes to clinical management in children with IF. METHODS: Comparative retrospective case series study in children with IF (mean age 70.4 months ± 58.4 months) presenting for diagnostic workup in a single intestinal rehabilitation center. Endoscopies were performed either as a screening procedure (Group 1, n = 45) or as indicated by symptoms of gastrointestinal dysfunction (Group 2, n = 11). RESULTS: A total of 92 endoscopies (56 esophagogastroduodenoscopies; 12 enteroscopies; 24 colonoscopies) were performed in 56 children. IF etiology included short bowel syndrome (n = 37), motility disorder (n = 16), and mucosal enteropathy (n = 3). Comparing Group 1 with Group 2 abnormal endoscopic findings were detected in 66.7% versus 81.8%. Findings led to adaptation of therapeutic regimes in 64.7% versus 85.7%. We detected a higher rate of erosive and ulcerative gastritis and duodenitis, enteritis, and colitis in Group 1 compared to Group 2. CONCLUSIONS: Endoscopy in children with IF frequently reveals abnormal findings, leading to recommendations for treatment adaptation in the majority of cases, irrespective of whether endoscopy has been performed as a screening procedure or as indicated by symptoms. Using endoscopy as a screening tool may improve both, the detection of gastrointestinal pathology and the clinical management of children with IF.

High Incidence of Proteinuria in Children With Chronic Intestinal Failure Under Long-term Parenteral Nutrition.

Long-term home parenteral nutrition (LTPN) in children with chronic intestinal failure (CIF) is associated with renal complications such as fluid and electrolyte imbalances, nephrocalcinosis, nephrolithiasis, and chronic kidney disease (CKD). The etiology of CIF-associated nephropathy is multifactorial. The aim of this study was to evaluate renal involvement under LTPN. In this study, 50 patients with CIF, median age 4.2 years (1.4-9.3; 23 girls) were included. Kidney involvement was a frequent finding in this study. CKD was present in 30% of patients without correlation with LTPN duration. Glomerular proteinuria was found in the majority of patients (76%), an important long-term risk factor for the progression of CKD. Increased urinary α-1 microglobulin was significantly associated with duration (years) of LTPN and was increased in patients with CKD.

Moss-Produced, Glycosylation-Optimized Human Factor H for Therapeutic Application in Complement Disorders.

Genetic defects in complement regulatory proteins can lead to severe renal diseases, including atypical hemolytic uremic syndrome and C3 glomerulopathies, and age-related macular degeneration. The majority of the mutations found in patients with these diseases affect the glycoprotein complement factor H, the main regulator of the alternative pathway of complement activation. Therapeutic options are limited, and novel treatments, specifically those targeting alternative pathway activation, are highly desirable. Substitution with biologically active factor H could potentially treat a variety of diseases that involve increased alternative pathway activation, but no therapeutic factor H is commercially available. We recently reported the expression of full-length recombinant factor H in moss (Physcomitrella patens). Here, we present the production of an improved moss-derived recombinant human factor H devoid of potentially immunogenic plant-specific sugar residues on protein N-glycans, yielding approximately 1 mg purified moss-derived human factor H per liter of initial P. patens culture after a multistep purification process. This glycosylation-optimized factor H showed full in vitro complement regulatory activity similar to that of plasma-derived factor H and efficiently blocked LPS-induced alternative pathway activation and hemolysis induced by sera from patients with atypical hemolytic uremic syndrome. Furthermore, injection of moss-derived factor H reduced C3 deposition and increased serum C3 levels in a murine model of C3 glomerulopathy. Thus, we consider moss-produced recombinant human factor H a promising pharmaceutical product for therapeutic intervention in patients suffering from complement dysregulation.

Surgical treatment of children with total colonic aganglionosis: functional and metabolic long-term outcome.

BACKGROUND: Total colonic aganglionosis (TCA) is a rare variant of Hirschsprung's disease occurring in 3-10% of the cases. Only few studies reported the long-term clinical and metabolic outcomes of patients with TCA. The aim of this study was to evaluate the functional and metabolic long-term outcomes of children undergoing surgical treatment for TCA. METHODS: A 15-year retrospective study was performed. Blood chemistry tests and stool analysis performed at the last follow-up visit were recorded. Height and weight development were assessed using the corresponding percentiles for age. Faecal continence and quality of life were evaluated using a detailed questionnaire. RESULTS: Eleven patients were included in the study. The median age at surgery was 6 months (range: 3-72 months). After histological confirmation, all patients underwent a total colectomy. Ileoanal anastomosis (n = 6), ileorectal anastomosis (n = 1), J-pouch (n = 1) and Duhamel procedure (n = 3) were performed. Temporary ileostomy was closed after a median of 8 weeks in 10/11 patients. After a median follow-up of 78 months (range: 27-199 months), all evaluated patients were continent. Height and weight were appropriate for age in only 5 patients. Vitamin B12 and folic acid serum levels were normal in all examined patients. Ten patients had normal hemoglobin serum levels. Seven patients had low transferrin saturation in serum. Hemoccult tests were negative in all examined patients. Despite complex postoperative courses in some cases, patients and parents showed good overall satisfaction in terms of quality of life. CONCLUSION: The majority of patients reported a good quality of life. This can result from the adaptation of the patients to certain disease states. The failure to thrive seems to be related with the extent of aganglionosis. The inclusion of these patients in interdisciplinary long-term follow-up care, in which pediatric surgeons, gastroenterologists, and dieticians are involved, is essential.

Mannose receptor-mediated delivery of moss-made α-galactosidase A efficiently corrects enzyme deficiency in Fabry mice.

Enzyme replacement therapy (ERT) is an effective treatment for several lysosomal storage disorders (LSDs). Intravenously infused enzymes are taken up by tissues through either the mannose 6-phosphate receptor (M6PR) or the mannose receptor (MR). It is generally believed that M6PR-mediated endocytosis is a key mechanism for ERT in treating LSDs that affect the non-macrophage cells of visceral organs. However, the therapeutic efficacy of MR-mediated delivery of mannose-terminated enzymes in these diseases has not been fully evaluated. We tested the effectiveness of a non-phosphorylated α-galactosidase A produced from moss (referred to as moss-aGal) in vitro and in a mouse model of Fabry disease. Endocytosis of moss-aGal was MR-dependent. Compared to agalsidase alfa, a phosphorylated form of α-galactosidase A, moss-aGal was more preferentially targeted to the kidney. Cellular localization of moss-aGal and agalsidase alfa in the heart and kidney was essentially identical. A single injection of moss-aGal led to clearance of accumulated substrate in the heart and kidney to an extent comparable to that achieved by agalsidase alfa. This study suggested that mannose-terminated enzymes may be sufficiently effective for some LSDs in which non-macrophage cells are affected, and that M6P residues may not always be a prerequisite for ERT as previously considered.

Bacterial sensor kinases: diversity in the recognition of environmental signals.

Bacteria sense and respond to a wide range of physical and chemical signals. Central to sensing and responding to these signals are two-component systems, which have a sensor histidine kinase (SK) and a response regulator (RR) as basic components. Here we review the different molecular mechanisms by which these signals are integrated and modulate the phosphorylation state of SKs. Apart from the basic mechanism, which consists of signal recognition by the SK that leads to an alteration of its autokinase activity and subsequently a change in the RR phosphorylation state, a variety of alternative modes have evolved. The biochemical data available on SKs, particularly their molecular interactions with signals, nucleotides, and their cognate RRs, are also reviewed.

Molecular mechanism of bacterial type 1 and P pili assembly.

The formation of adhesive surface structures called pili or fimbriae ('bacterial hair') is an important contributor towards bacterial pathogenicity and persistence. To fight often chronic or recurrent bacterial infections such as urinary tract infections, it is necessary to understand the molecular mechanism of the nanomachines assembling such pili. Here, we focus on the so far best-known pilus assembly machinery: the chaperone-usher pathway producing the type 1 and P pili, and highlight the most recently acquired structural knowledge. First, we describe the subunits' structure and the molecular role of the periplasmic chaperone. Second, we focus on the outer-membrane usher structure and the catalytic mechanism of usher-mediated pilus biogenesis. Finally, we describe how the detailed understanding of the chaperone-usher pathway at a molecular level has paved the way for the design of a new generation of bacterial inhibitors called 'pilicides'.

An ancient light-harvesting protein is critical for the regulation of algal photosynthesis.

Light is necessary for photosynthesis, but its absorption by pigment molecules such as chlorophyll can cause severe oxidative damage and result in cell death. The excess absorption of light energy by photosynthetic pigments has led to the evolution of protective mechanisms that operate on the timescale of seconds to minutes and involve feedback-regulated de-excitation of chlorophyll molecules in photosystem II (qE). Despite the significant contribution of eukaryotic algae to global primary production, little is known about their qE mechanism, in contrast to that in flowering plants. Here we show that a qE-deficient mutant of the unicellular green alga Chlamydomonas reinhardtii, npq4, lacks two of the three genes encoding LHCSR (formerly called LI818). This protein is an ancient member of the light-harvesting complex superfamily, and orthologues are found throughout photosynthetic eukaryote taxa, except in red algae and vascular plants. The qE capacity of Chlamydomonas is dependent on environmental conditions and is inducible by growth under high light conditions. We show that the fitness of the npq4 mutant in a shifting light environment is reduced compared to wild-type cells, demonstrating that LHCSR is required for survival in a dynamic light environment. Thus, these data indicate that plants and algae use different proteins to dissipate harmful excess light energy and protect the photosynthetic apparatus from damage.

The chloroplast calcium sensor CAS is required for photoacclimation in Chlamydomonas reinhardtii.

The plant-specific calcium binding protein CAS (calcium sensor) has been localized in chloroplast thylakoid membranes of vascular plants and green algae. To elucidate the function of CAS in Chlamydomonas reinhardtii, we generated and analyzed eight independent CAS knockdown C. reinhardtii lines (cas-kd). Upon transfer to high-light (HL) growth conditions, cas-kd lines were unable to properly induce the expression of LHCSR3 protein that is crucial for nonphotochemical quenching. Prolonged exposure to HL revealed a severe light sensitivity of cas-kd lines and caused diminished activity and recovery of photosystem II (PSII). Remarkably, the induction of LHCSR3, the growth of cas-kd lines under HL, and the performance of PSII were fully rescued by increasing the calcium concentration in the growth media. Moreover, perturbing cellular Ca(2+) homeostasis by application of the calmodulin antagonist W7 or the G-protein activator mastoparan impaired the induction of LHCSR3 expression in a concentration-dependent manner. Our findings demonstrate that CAS and Ca(2+) are critically involved in the regulation of the HL response and particularly in the control of LHCSR3 expression.

Moss-produced human complement factor H with modified glycans has an extended half-life and improved biological activity.

Most drugs that target the complement system are designed to inhibit the complement pathway at either the proximal or terminal levels. The use of a natural complement regulator such as factor H (FH) could provide a superior treatment option by restoring the balance of an overactive complement system while preserving its normal physiological functions. Until now, the systemic treatment of complement-associated disorders with FH has been deemed unfeasible, primarily due to high production costs, risks related to FH purified from donors' blood, and the challenging expression of recombinant FH in different host systems. We recently demonstrated that a moss-based expression system can produce high yields of properly folded, fully functional, recombinant FH. However, the half-life of the initial variant (CPV-101) was relatively short. Here we show that the same polypeptide with modified glycosylation (CPV-104) achieves a pharmacokinetic profile comparable to that of native FH derived from human serum. The treatment of FH-deficient mice with CPV-104 significantly improved important efficacy parameters such as the normalization of serum C3 levels and the rapid degradation of C3 deposits in the kidney compared to treatment with CPV-101. Furthermore, CPV-104 showed comparable functionality to serum-derived FH in vitro, as well as similar performance in ex vivo assays involving samples from patients with atypical hemolytic uremic syndrome, C3 glomerulopathy and paroxysomal nocturnal hematuria. CPV-104 - the human FH analog expressed in moss - will therefore allow the treatment of complement-associated human diseases by rebalancing instead of inhibiting the complement cascade.

Vascular rehabilitation in children with chronic intestinal failure reduces the risk of central-line associated bloodstream infections and catheter replacements.

BACKGROUND & AIMS: Children with chronic intestinal failure (IF) require a long-term central venous catheter (CVC) for provision of parenteral nutrition. Vascular, mechanical and infectious complications such as central line-associated bloodstream infections (CLABSI) may lead to progressive loss of venous access sites. Handling and management of CVCs therefore play an important role. Our vascular rehabilitation concept (VRC) is a core component of our intestinal rehabilitation program (IRP) and consists of an education program, optimization of skin care, catheter fixation and lock solution, and the use of hybrid technique for catheter placement. Aim of this study is to analyse the effectiveness of our VRC on CLABSI rates and need for CVC replacements. METHODS: Retrospective analysis of all children treated in our IRP that were followed up between 2018 and 2023. RESULTS: A total of 117 children with chronic IF could be included for analysis of 248864 catheter days (CD). 91 patients were referred from other hospitals (127117 CD before and 89359 CD after entry into our IRP). Children receiving primary care at our IRP (32388 CD) showed a significantly lower CLABSI and line replacement rate than patients referred from external centers (p < 0.001). After entering our IRP, CLABSI rates and need for CVC replacements per 1000 CD decreased significantly in referred patients: 1.19 to 0.26 and 1.77 to 0.59, respectively (p < 0.001). CONCLUSION: Management of paediatric chronic IF patients by an IRP with a vascular rehabilitation concept significantly lowers the rate of CLABSI episodes and the need for catheter replacements.

Composition and structure of photosystem I in the moss Physcomitrella patens.

Recently, bryophytes, which diverged from the ancestor of seed plants more than 400 million years ago, came into focus in photosynthesis research as they can provide valuable insights into the evolution of photosynthetic complexes during the adaptation to terrestrial life. This study isolated intact photosystem I (PSI) with its associated light-harvesting complex (LHCI) from the moss Physcomitrella patens and characterized its structure, polypeptide composition, and light-harvesting function using electron microscopy, mass spectrometry, biochemical, and physiological methods. It became evident that Physcomitrella possesses a strikingly high number of isoforms for the different PSI core subunits as well as LHCI proteins. It was demonstrated that all these different subunit isoforms are expressed at the protein level and are incorporated into functional PSI-LHCI complexes. Furthermore, in contrast to previous reports, it was demonstrated that Physcomitrella assembles a light-harvesting complex consisting of four light-harvesting proteins forming a higher-plant-like PSI superstructure.

The Harpers THMC flow bench: A triaxial multi-reactor setup for the investigation of long-term coupled thermo-hydro-mechanical-chemical fluid-rock interaction.

The scientific analysis and interpretation of coupled thermo-hydro-mechanical-chemical (THMC) processes in rocks requires complex and diverse instrumentation. In this study, we introduce the "Harpers THMC Flow Bench," a multi-cell, flow-through reactor system that allows long-term testing on rock plugs and powdered samples. The setup consists of four small triaxial cells that can hold confining and pore pressure of up to 20 MPa and an axial load of up to 300 MPa. Axial deformation of the samples is measured with linear variable differential transducers. The cells can be heated to 90 °C, and effluents (gas, water, and brine) can be sampled for compositional analysis. An additional Hastelloy-autoclave enables fluid mixing and saturation with gas prior to injection into the samples. Each cell can be operated individually, allowing independent experiments over long testing periods. The sample holders were designed such that they are transparent for X-rays during X-ray computer tomography, minimizing sample handling effects on the imaging results. To demonstrate examples of the capabilities of the flow bench, we present case studies on Carnmenellis granite (Cornwall, UK) and Castlegate sandstone (Utah, USA) samples. Permeability measurements are shown using fractured granite undergoing periodic loading of effective pressure. To demonstrate chemical measurement capabilities, we used deionized water to leach elements from granite powders. We then analyzed effluent compositions using inductively coupled plasma optical emission spectroscopy. Finally, we conducted a strength test and a cyclic differential stress test on sandstone to demonstrate the mechanical testing capabilities of the setup.

Pore-scale visualization of hydrogen storage in a sandstone at subsurface pressure and temperature conditions: Trapping, dissolution and wettability.

HYPOTHESIS: Underground hydrogen (H2) storage is a potentially viable solution for large-scale cyclic H2 storage; however, the behavior of H2 at subsurface pressure and temperature conditions is poorly known. This work investigates if the pore-scale displacement processes in H2-brine systems in a porous sandstone can be sufficiently well defined to enable effective and economic storage operations. In particular, this study investigates trapping, dissolution, and wettability of H2-brine systems at the pore-scale, at conditions that are realistic for subsurface H2 storage. EXPERIMENTS: We have performed in situ X-ray imaging during a flow experiment to investigate pore-scale processes during H2 injection and displacement in a brine saturated Bentheimer sandstone sample at temperature and pressure conditions representative of underground reservoirs. Two injection schemes were followed for imbibition: displacement of H2 with H2-equilibrated brine and with non-H2-equilibrated brine. The results from the two cycles were compared with each other. FINDINGS: The sandstone was found to be wetting to the brine and non-wetting to H2 after both displacement cycles, with average contact angles of 54° and 53° for H2-equilibrated and non-H2-equilibrated brine respectively. We also found a higher recovery of H2 (43.1%) when displaced with non-H2-equilibrated brine compared to that of H2-equilibrated brine (31.6%), indicating potential dissolution of H2 in the unequilibrated imbibing brine at reservoir conditions. Our results suggest that underground H2 storage may indeed be a suitable strategy for energy storage, but considerable further research is needed to fully comprehend the pore-scale interactions at reservoir conditions.

Comparison of efficacy between subcutaneous and intravenous application of moss-aGal in the mouse model of Fabry disease.

Fabry disease (FD, OMIM 301500) is a rare X-linked inherited lysosomal storage disorder associated with reduced activities of α-galactosidase A (aGal, EC 3.2.1.22). The current standard of care for FD is based on enzyme replacement therapy (ERT), in which a recombinantly produced version of αGal is intravenously (iv) applied to Fabry patients in biweekly intervals. Though the iv application is clinically efficacious, periodical infusions are inconvenient, time- and resource-consuming and they negatively impact the patients' quality of life. Subcutaneous (sc) injection, in contrast, is an established route of administration for treatment of chronic conditions. It opens the beneficial option of self-administration, thereby improving patients' quality of life and at the same time reducing treatment costs. We have previously shown that Moss-α-Galactosidase (moss-aGal), recombinantly produced in the moss Physcomitrium patens, is efficient in degrading accumulated Gb3 in target organs of murine model of FD and in the phase I clinical study, we obtained first efficacy evidence in human patients following single iv infusion. Here, we tested the efficacy of subcutaneous administration of moss-aGal and compared it with the results observed following iv infusion in Fabry mice. The obtained findings demonstrate that subcutaneously applied moss-aGal is correctly transported to target organs and efficacious in degrading Gb3 deposits there and thus suggest the possibility of using this route of administration for therapy of Fabry disease.

The structure and function of eukaryotic photosystem I.

Eukaryotic photosystem I consists of two functional moieties: the photosystem I core, harboring the components for the light-driven charge separation and the subsequent electron transfer, and the peripheral light-harvesting complex (LHCI). While the photosystem I-core remained highly conserved throughout the evolution, with the exception of the oxidizing side of photosystem I, the LHCI complex shows a high degree of variability in size, subunits composition and bound pigments, which is due to the large variety of different habitats photosynthetic organisms dwell in. Besides summarizing the most current knowledge on the photosystem I-core structure, we will discuss the composition and structure of the LHCI complex from different eukaryotic organisms, both from the red and the green clade. Furthermore, mechanistic insights into electron transfer between the donor and acceptor side of photosystem I and its soluble electron transfer carrier proteins will be given. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.

X-ray diffraction study of K- and Ca-exchanged montmorillonites in CO2 atmospheres.

Powder X-ray diffraction shows that K- and Ca-exchanged montmorillonites swell upon interacting with CO(2) at ambient temperatures, depending on their initial hydration state. K-exchanged montmorillonite swells rapidly to a maximum d(001) of ∼12.2 Å. In contrast, Ca-exchanged montmorillonite swells more slowly, but reaches a maximum d(001) of ∼15.1 Å. Reaction kinetics differ significantly between the K- and Ca-exchanged montmorillonite complexes. Expansion of K-exchanged montmorillonite samples was rapid, occurring on time scales of tens of minutes or less. The Ca-exchanged montmorillonite samples continued to expand over periods up to 42 h. Aging of both K- and Ca-exchanged montmorillonite complexes at elevated CO(2) pressure for 1-2 days resulted in greater stability when CO(2) pressure was released. The observed intercalation reactions have important consequences for carbon sequestration: (1) CO(2) absorption by swelling clays may represent a significant pathway for storage of CO(2). (2) The swelling of smectites under CO(2) pressure may have a significant impact on the permeability of caprock formations.