A simple, efficient technique to make colon crypt gels for 3D models.

The pyruvate dehydrogenase multienzyme complexes are among the largest multifunctional catalytic machines in cells, catalyzing the production of acetyl CoA from pyruvate. We have previously reported the molecular architecture of an 11-MDa subcomplex comprising the 60-mer icosahedral dihydrolipoyl acetyltransferase (E2) decorated with 60 copies of the heterotetrameric (alpha(2)beta(2)) 153-kDa pyruvate decarboxylase (E1) from Bacillus stearothermophilus (Milne, J. L. S., Shi, D., Rosenthal, P. B., Sunshine, J. S., Domingo, G. J., Wu, X., Brooks, B. R., Perham, R. N., Henderson, R., and Subramaniam, S. (2002) EMBO J. 21, 5587-5598). An annular gap of approximately 90 A separates the acetyltransferase catalytic domains of the E2 from an outer shell formed of E1 tetramers. Using cryoelectron microscopy, we present here a three-dimensional reconstruction of the E2 core decorated with 60 copies of the homodimeric 100-kDa dihydrolipoyl dehydrogenase (E3). The E2E3 complex has a similar annular gap of approximately 75 A between the inner icosahedral assembly of acetyltransferase domains and the outer shell of E3 homodimers. Automated fitting of the E3 coordinates into the map suggests excellent correspondence between the density of the outer shell map and the positions of the two best fitting orientations of E3. As in the case of E1 in the E1E2 complex, the central 2-fold axis of the E3 homodimer is roughly oriented along the periphery of the shell, making the active sites of the enzyme accessible from the annular gap between the E2 core and the outer shell. The similarities in architecture of the E1E2 and E2E3 complexes indicate fundamental similarities in the mechanism of active site coupling involved in the two key stages requiring motion of the swinging lipoyl domain across the annular gap, namely the synthesis of acetyl CoA and regeneration of the dithiolane ring of the lipoyl domain.

In recent years, the field of skeletal muscle tissue engineering has experienced significant advancements, evolving from traditional two-dimensional (2D) cell cultures to increasingly sophisticated three-dimensional (3D) engineered constructs. While 2D models have provided foundational insights into muscle cell biology, emerging 3D platforms aim to better recapitulate the complex native muscle environment, including mature muscle fibers, supportive vasculature, and native-like extracellular matrix (ECM) composition. Here, we provide a comprehensive review of current in vitro skeletal muscle models, detailing their design principles, structure, and functionalities as well as the advantages and limitations inherent to each approach. We put a special emphasis on 3D engineered muscle tissues (EMTs) developed through advanced bioengineering strategies and note that design criteria such as scaffold selection, perfusion system incorporation, and co-culture with supporting cell types have significantly enhanced tissue maturity and complexity. Lastly, we explore the application of these engineered models to disease studies, highlighting models of both mendelian muscle disorders and common polygenic diseases and the potential of these platforms for drug discovery and regenerative therapies. Although an ideal in vitro model that fully recapitulates native muscular architecture, vascularization, and ECM complexity is yet to be realized, we identify current challenges and propose future directions for advancing these bioengineered systems. By integrating fundamental design criteria with emerging technologies, this review provides a roadmap for next-generation skeletal muscle models poised to deepen our understanding of muscle biology and accelerate therapeutic innovation.

Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs.

Cystic fibrosis (CF) is a frequent autosomal recessive disorder caused by mutation of a gene encoding a multifunctional transmembrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), located in the apical membrane of epithelial cells lining exocrine glands. In an attempt to get a more complete picture of the pleiotropic effects of the CFTR defect on epithelial cells and particularly on the membrane compartment, a bidimensional blue native (BN)/SDS-PAGE-based proteomic approach was used on colonic crypt samples from control and CFTR knock-out mice (cftr-/-). This approach overcomes the difficulties of membrane protein analysis by conventional two-dimensional PAGE and is able to resolve multiprotein complexes. Used here for the first time on crude membrane proteins that were extracted from murine colonic crypts, BN/SDS-PAGE allows effective separation of protein species and complexes of various origins, including mitochondria, plasma membrane, and intracellular compartments. The major statistically significant difference in protein maps obtained with samples from control and cftr-/- mice was unambiguously identified as mClCA3, a member of a family of calcium-activated chloride channels considered to be key molecules in mucus secretion by goblet cells. On the basis of this finding, we evaluated the overall expression and localization of mClCA3 in the colonic epithelium and in the lung of mice by immunoblot analysis and immunohistochemistry. We found that mClCA3 expression was significantly decreased in the colon and lung of the cftr-/- mice. In an ex vivo assay, we found that the Ca2+-dependent (carbachol-stimulated) glycoprotein secretion strongly inhibited by the calcium-activated chloride channel blocker niflumic acid (100 microm) was impaired in the distal colon of cftr-/- mice. These results support the conclusion that a ClCA-related function in the CF colon depends on CFTR expression and may be correlated with the impaired expression of mClCA3.

The probiotic effects of Lactobacillus reuteri have been speculated to partly depend on its capacity to produce the antimicrobial substance reuterin during the reduction of glycerol in the gut. In this study, the potential of this process to protect human intestinal epithelial cells against infection with Salmonella enterica serovar Typhimurium was investigated. We used a three-dimensional (3-D) organotypic model of human colonic epithelium that was previously validated and applied to study interactions between S. Typhimurium and the intestinal epithelium that lead to enteric salmonellosis. Using this model system, we show that L. reuteri protects the intestinal cells against the early stages of Salmonella infection and that this effect is significantly increased when L. reuteri is stimulated to produce reuterin from glycerol. More specifically, the reuterin-containing ferment of L. reuteri caused a reduction in Salmonella adherence and invasion (1 log unit), and intracellular survival (2 log units). In contrast, the L. reuteri ferment without reuterin stimulated growth of the intracellular Salmonella population with 1 log unit. The short-term exposure to reuterin or the reuterin-containing ferment had no observed negative impact on intestinal epithelial cell health. However, long-term exposure (24 h) induced a complete loss of cell-cell contact within the epithelial aggregates and compromised cell viability. Collectively, these results shed light on a potential role for reuterin in inhibiting Salmonella-induced intestinal infections and may support the combined application of glycerol and L. reuteri. While future in vitro and in vivo studies of reuterin on intestinal health should fine-tune our understanding of the mechanistic effects, in particular in the presence of a complex gut microbiota, this the first report of a reuterin effect on the enteric infection process in any mammalian cell type.

The capacity of the intestine to secrete fluid is dependent on the basolateral Na(+)-K(+)-2Cl(-) co-transporter (NKCC1). Given that cAMP and Ca(2+) signals promote sustained and transient episodes of fluid secretion, respectively, this study investigated the differential regulation of functional NKCC1 membrane expression in the native human colonic epithelium. Tissue sections and colonic crypts were obtained from sigmoid rectal biopsy tissue samples. Cellular location of NKCC1, Na(+)-K(+)-ATPase, M3 muscarinic acetylcholine receptor (M(3)AChR) and lysosomes was examined by immunolabelling techniques. NKCC1 activity (i.e. bumetanide-sensitive uptake), intracellular Ca(2+) and cell volume were assessed by 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), Fura-2 and differential interference contrast/calcein imaging. Unstimulated NKCC1 was expressed on basolateral membranes and exhibited a topological expression gradient, predominant at the crypt base. Cholinergic Ca(2+) signals initiated at the crypt base and spread along the crypt axis. In response, NKCC1 underwent a Ca(2+)-dependent 4 h cycle of recruitment to basolateral membranes, activation, internalization, degradation and re-expression. Internalization was prevented by the epidermal growth factor receptor kinase inhibitor tyrphostin-AG1478, and re-expression was prohibited by the protein synthesis inhibitor cylcoheximide; the lysosome inhibitor chloroquine promoted accumulation of NKCC1 vesicles. NKCC1 internalization and re-expression were accompanied by secretory volume decrease and bumetanide-sensitive regulatory volume increase, respectively. In contrast, forskolin (i.e. cAMP elevation)-stimulated NKCC1 activity was sustained, and membrane expression and cell volume remained constant. Co-stimulation with forskolin and acetylcholine promoted dramatic recruitment of NKCC1 to basolateral membranes and prolonged the cycle of co-transporter activation, internalization and re-expression. In conclusion, persistent NKCC1 activation by cAMP is constrained by a Ca(2+)-dependent cycle of co-transporter internalization, degradation and re-expression; this is a novel mechanism to limit intestinal fluid loss.

Three-dimensionally Specific Inhibition of DNA Repair-Related Genes by Activated KRAS in Colon Crypt Model

This study aimed to evaluate the potential of administering bovine colostrum to suckling and weanling piglets to modulate the growth, body composition, intestinal architecture, immunity, as well as remodeling and permeability. Twenty-four litters adjusted to 12 piglets each were selected at birth; one-half of them received liquid bovine colostrum (LC+) from d 5 to d 10, whereas the remaining litters did not receive any supplementation (LC-). Starting from d 11, all the litters received a creep feed either supplemented or not with 5% dehydrated bovine colostrum (BC+ and BC-, respectively). Animals were classified as “eaters” or “non eaters” according to their activity towards the feeders recorded by direct observation and presence of dietary marker (ferric oxide) in feces. At weaning (d 21), 16 LC+/BC+ and 16 LC-/BC- piglets (with an equal number of "eaters" and "non eaters" in each group) were selected and fed for 10 days with the same diets administered during lactation. Growth performance was recorded at d 0, 21 and 31. At d 31 bone mineral content, total body fat and lean contents were measured by dual X-ray absorptiometry; furthermore, animals were euthanized at d 31 and intestinal samples were collected to evaluate the intestinal morphology and concentration of TNFα, IL10, CXCL10, occludin, caspase 3 and proliferating cell nuclear antigen. Our results showed that LC+ and BC+ treatments increased creep feed consumption during lactation (P=0.01 and P=0.08, respectively) and affected litter weight at weaning (LC×BC interaction, P=0.04). Ten days after weaning, BC+ supplementation increased the intestinal villous to crypt ratio and reduced the occludin concentration (P<0.05). Piglets classified as creep feeding "eaters" showed greater intestinal occludin concentrations compared with "non eaters" animals (P=0.005). This study highlights the potential of bovine colostrum to increase piglet growth performance during lactation and affect the intestinal health in the peri-weaning period.

Simple SummarySalmonellosis is a dangerous disease in broilers that causes huge economic losses. We assumed that instead of antibiotics, a Bacillus-based probiotic may serve as an alternative to alleviate the negative effects of Salmonella infection. A control group with no feed additive, a positive control supplemented with a standard antibiotic and two groups that were supplemented with different strains and levels of Bacillus subtilis were the experimental animals of the present study. It was revealed that supplementation of probiotic bacteria induced similar results in terms of feed intake, body weight gain and feed efficiency in comparison with the group treated with antibiotics. In addition, the dimensions of intestinal villi were also improved in the probiotic-treated birds. As concluded from the results of the present study, probiotic bacteria could be used as an alternative to antibiotics against Salmonella in broilers. A total of 600 day-old broiler chicks (Ross 308) confirmed for the absence of Salmonella were randomly allocated to five treatments each with 10 replicates: negative control (basal diet only); positive control (basal diet) + infected with Salmonella; T1, Salmonella infected + avilamycin; T2, Salmonella infected + Bacillus subtilis (ATCC PTA-6737; 2 × 107 CFU/g) and T3, Salmonella infected + B. subtilis (DSM 172999; 1.2 × 106 CFU/g). The results revealed that feed intake (FI) and body weight (BW) were significantly (p < 0.01) lower in T1 compared to T2. The feed conversion ratio (FCR) was significantly (p < 0.01) lower in T2 and T3 compared to other treatments. Similarly, the performance efficiency factor (PEF) was also significantly (p < 0.01) higher in T2 and T3 compared to positive control. Villus height was significantly (p < 0.01) higher in T2 compared to all other treatments. However, villus width and surface area were significantly (p < 0.01) higher in T1. In conclusion, dietary supplementation with B. subtilis improved growth and intestinal health by reversing the negative effects of Salmonellosis.

Conventional cell cultures utilizing transformed or immortalized cell lines or primary human epithelial cells have played a fundamental role in furthering our understanding of Cryptosporidium infection. However, they remain inadequate with respect to their inability to emulate in vivo conditions, support long-term growth, and complete the life cycle of the parasite. Previously, we developed a 3D silk scaffold-based model using transformed human intestinal epithelial cells (IECs). This model supported C. parvum infection for up to 2weeks and resulted in completion of the life cycle of the parasite. However, transformed IECs are not representative of primary human IEC.Human intestinal enteroids (HIEs) are cultures derived from crypts that contain Lgr5+ stem cells isolated from human biopsies or surgical intestinal tissues; these established multicellular cultures can be induced to differentiate into enterocytes, enteroendocrine cells, goblet cells, Paneth cells, and tuft cells. HIEs better represent human intestinal structure and function than immortalized IEC lines. Recently, significant progress has been made in the development of technologies to culture HIEs in vitro. When grown in a 3D matrix, HIEs provide a spatial organization resembling the native human intestinal epithelium. Additionally, they can be dissociated and grown as monolayers in tissue culture plates, permeable supports or silk scaffolds that enable mechanistic studies of pathogen infections. They can also be co-cultured with other human cells such as macrophages and myofibroblasts. The HIEs grown in these novel culture systems recapitulate the physiology, the 3D architecture, and functional diversity of native intestinal epithelium and provide a powerful and promising new tool to study Cryptosporidium-host cell interactions and screen for interventions ex vivo. In this chapter, we describe the 3D silk scaffold-based model using transformed IEC co-cultured with human intestinal myofibroblasts and 2D and 3D HIE-derived models of Cryptosporidium, also co-cultured with human intestinal myofibroblasts.

Conventional cell cultures utilizing transformed or immortalized cell lines or primary human epithelial cells have played a fundamental role in furthering our understanding of Cryptosporidium infection. However, they remain inadequate with respect to their inability to emulate in vivo conditions and support long-term growth and completion of the parasite's life cycle. Previously, we developed a three-dimensional (3D) silk scaffold-based model using transformed human intestinal epithelial cells (IECs). This model supported C. parvum infection for up to 2weeks and resulted in completion of the life cycle of the parasite. However, transformed IECs are not representative of primary human IECs.Human intestinal organoids (hIOs) or human intestinal enteroids (hIEs) are 3D cultures derived from Lgr5+ stem cells isolated from the crypts of human intestinal biopsies or surgical intestinal tissues. These multicellular cultures can be induced to differentiate into enterocytes, enteroendocrine cells, goblet cells, Paneth cells, and tuft cells. hIEs better represent human intestinal structure and function than immortalized, IEC lines. Recently, significant progress has been made in the development of technologies to culture hIEs in vitro. When grown in a 3D matrix, hIEs provide a spatial organization resembling the native human intestinal epithelium. Additionally, they can be dissociated and grown as monolayers in tissue culture plates, permeable supports, or silk scaffolds that enable mechanistic studies of pathogen infections. They can also be co-cultured with other human cells such as macrophages, myofibroblasts, and immune cells. The hIEs grown in these novel culture systems recapitulate the physiology, the 3D architecture, and functional diversity of native intestinal epithelium and provide a powerful and promising new tool to study Cryptosporidium-host cell interactions and screen for interventions ex vivo. In this chapter, we describe a 3D silk scaffold-based model using transformed IECs co-cultured with human intestinal myofibroblasts as well as 2D and 3D hIE-derived models of C. parvum infection.

High-Resolution Proteomics Unravel Architecture and Molecular Diversity of Native AMPA Receptor Complexes

Core data and logs - wireline and while-drilling - were processed and integrated with a sedimentological model to provide consistent log-facies classification and petrophysical characterization as input to a 3D geological model of an off-shore deep-water turbidite reservoir. The studied reservoir belongs to a channel system, and consists of channel sands cut into background shaly deposits, and of thin beds that can be ascribed to levee and crevasse-splays. Chaotic slump deposits are also found locally. A complex fault system, related to mud-diapirs, subdivides the reservoir into hydraulically separated blocks, thus resulting in multiple hydrocarbon accumulations. Log curves from five different wells were environmentally corrected, depth-shifted and calibrated at the reservoir scale in order to ensure the overall consistency of log recordings. They were used as input to a quantitative log evaluation process that resulted in the computation of the volumes of mineralogical components (sand, silt, shale) and effective porosity along the wells. The computed curves were in turn statistically processed (cluster analysis) and compared with the sedimentological description of cores, to provide a classification of the reservoir into five log-facies, each with a well-defined sedimentological meaning. The results of NMR log interpretations and a detailed thin layer analysis carried out on high-resolution resistivity curves - available on different subsets of the studied wells - were also integrated, eventually leading to a thorough petrophysical characterization (distributions of porosity, permeability, irreducible water saturation) of each log-facies. Finally, the full-field sedimentological model - derived from 3D seismic data - and the above described petrophysical characterization provided the input for a 3D geostatistical reservoir model that was built with an object-based approach. The statistics calculated from a large number of realizations allowed a probabilistic quantification of the OHIP distribution for use as an input for future field development scenarios. Introduction Hydrocarbon production from ‘easily characterized and produced’ reservoirs has slowly declined worldwide in the last decades: as a consequence, exploration and production targets progressively shifted towards more challenging environments and/or more ‘difficult’ reservoirs. Turbidite deposits in deep and ultra-deep water offshore are a typical example. Besides their overall architectural complexity (amalgamated channels, channel-levee systems, channel-lobe systems), some of these deposits include heterolithic facies consisting of thin, cm- or mm-sized, alternating horizons of sandstone, siltstone and mudstones, which deserve accurate petrophysical characterization. This paper describes a facies reservoir characterization workflow whose objective is the integration of core data and log curves with different vertical resolutions. The workflow was applied to a deep-water turbidite reservoir. The resulting characterization proved to be a useful guide in the construction of the 3D geocellular model. In the beginning, the reservoir structural setting and the sedimentological conceptual model are presented. Next, the integrated petrophysical characterization workflow is presented: the quantitative interpretation of conventional logs, the interpretation of NMR logs, and a Thin Layer Analysis carried out on high resolution resistivity curves are first discussed; then, the log-facies classification and characterization relying on log and sedimentological interpretation is discussed. Finally, the construction of the 3D geological model is presented.

The vertebrate adult intestinal epithelium has a high self-renewal rate driven by intestinal stem cells (ISCs) in the crypts, which play central roles in maintaining intestinal integrity and homeostasis. However, the underlying mechanisms remain elusive. Here we showed that protein arginine methyltransferase 1 (PRMT1), a major arginine methyltransferase that can also function as a transcription co-activator, was highly expressed in the proliferating cells of adult mouse intestinal crypts. Intestinal epithelium-specific knockout of PRMT1, which ablates PRMT1 gene starting during embryogenesis, caused distinct, region-specific effects on small intestine and colon: increasing and decreasing the goblet cell number in the small intestinal and colonic crypts, respectively, leading to elongation of the crypts in small intestine but not colon, while increasing crypt cell proliferation in both regions. We further generated a tamoxifen-inducible intestinal epithelium-specific PRMT1 knockout mouse model and found that tamoxifen-induced knockout of PRMT1 in the adult mice resulted in the same region-specific intestinal phenotypes. Thus, our studies have for the first time revealed that the epigenetic enzyme PRMT1 has distinct, region-specific roles in the maintenance of intestinal epithelial architecture and homeostasis, although PRMT1 may influence intestinal development.

Re-evaluating the Role of Deep Crypt Secretory Cells in Intestinal Homeostasis