Arabidopsis VILLIN5 bundles actin filaments using a novel mechanism.

Being a bona fide actin bundler, Arabidopsis villin5 (VLN5) plays a crucial role in regulating actin stability and organization within pollen tubes. Despite its significance, the precise mechanism through which VLN5 bundles actin filaments has remained elusive. Through meticulous deletion analysis, we have unveiled that the link between gelsolin repeat 6 (G6) and the headpiece domain (VHP), rather than VHP itself, is indispensable for VLN5-mediated actin bundling. Further refinement of this region has pinpointed a critical sequence spanning from Val763 to Ser823, essential for VLN5's actin-bundling activity. Notably, the absence of Val763-Ser823 in VLN5 results in decreased filamentous decoration within pollen tubes and a diminished ability to rescue actin bundling defects in vln2vln5 mutant pollen tubes compared to intact VLN5. Moreover, our findings highlight that the Val763-Ser823 sequence harbors a binding site for F-actin, suggesting that this linker-based F-actin binding site, in conjunction with the F-actin binding site localized in G1-G6, enables a single VLN5 to concurrently bind to two adjacent actin filaments. Therefore, our study unveils a novel mechanism by which VLN5 bundles actin filaments.

Genome-Wide Analysis of VILLIN Gene Family Associated with Stress Responses in Cotton (Gossypium spp.).

The VILLIN (VLN) protein plays a crucial role in regulating the actin cytoskeleton, which is involved in numerous developmental processes, and is crucial for plant responses to both biotic and abiotic factors. Although various plants have been studied to understand the VLN gene family and its potential functions, there has been limited exploration of VLN genes in Gossypium and fiber crops. In the present study, we characterized 94 VLNs from Gossypium species and 101 VLNs from related higher plants such as Oryza sativa and Zea mays and some fungal, algal, and animal species. By combining these VLN sequences with other Gossypium spp., we classified the VLN gene family into three distinct groups, based on their phylogenetic relationships. A more in-depth examination of Gossypium hirsutum VLNs revealed that 14 GhVLNs were distributed across 12 of the 26 chromosomes. These genes exhibit specific structures and protein motifs corresponding to their respective groups. GhVLN promoters are enriched with cis-elements related to abiotic stress responses, hormonal signals, and developmental processes. Notably, a significant number of cis-elements were associated with the light responses. Additionally, our analysis of gene-expression patterns indicated that most GhVLNs were expressed in various tissues, with certain members exhibiting particularly high expression levels in sepals, stems, and tori, as well as in stress responses. The present study potentially provides fundamental insights into the VLN gene family and could serve as a valuable reference for further elucidating the diverse functions of VLN genes in cotton.

Villin controls the formation and enlargement of punctate actin foci in pollen tubes.

Self-incompatibility (SI) in the poppy Papaver rhoeas triggers dramatic alterations in actin within pollen tubes. However, how these actin alterations are mechanistically achieved remains largely unexplored. Here, we used treatment with the Ca2+ ionophore A23187 to mimic the SI-induced elevation in cytosolic Ca2+ and trigger formation of the distinctive F-actin foci. Live-cell imaging revealed that this remodeling involves F-actin fragmentation and depolymerization, accompanied by the rapid formation of punctate actin foci and subsequent increase in their size. We established that actin foci are generated and enlarged from crosslinking of fragmented actin filament structures. Moreover, we show that villins associate with actin structures and are involved in this actin reorganization process. Notably, we demonstrate that Arabidopsis VILLIN5 promotes actin depolymerization and formation of actin foci by fragmenting actin filaments, and controlling the enlargement of actin foci via bundling of actin filaments. Our study thus uncovers important novel insights about the molecular players and mechanisms involved in forming the distinctive actin foci in pollen tubes.

Atypical chemokine receptor 3 induces colorectal tumorigenesis in mice by promoting ß-arrestin-NOLC1-fibrillarin-dependent rRNA biogenesis.

Atypical chemokine receptor 3 (ACKR3) has emerged as a key player in various biological processes. Its atypical "intercepting receptor" properties have established ACKR3 as the major regulator in the pathophysiological processes in many diseases. In this study, we investigated the role of ACKR3 activation in promoting colorectal tumorigenesis. We showed that ACKR3 expression levels were significantly increased in human colon cancer tissues, and high levels of ACKR3 predicted the increased severity of cancer. In Villin-ACKR3 transgenic mice with a high expression level of CKR3 in their intestinal epithelial cells, administration of AOM/DSS induced more severe colorectal tumorigenesis than their WT littermates. Cancer cells of Villin-ACKR3 transgenic mice were characterised by the nuclear ß-arrestin-1 (ß-arr1)-activated perturbation of rRNA biogenesis. In HCT116 cells, cotreatment with CXCL12 and AMD3100 selectively activated ACKR3 and induced nuclear translocation of ß-arr1, leading to an interaction of ß-arr1 with nucleolar and coiled-body phosphoprotein 1 (NOLC1). NOLC1, as the phosphorylated protein, further interacted with fibrillarin, a conserved nucleolar methyltransferase responsible for ribosomal RNA methylation in the nucleolus, thereby increasing the methylation in histone H2A and promoting rRNA transcription in ribosome biogenesis. In conclusion, ACKR3 promotes colorectal tumorigenesis through the perturbation of rRNA biogenesis by the ß-arr1-induced interaction of NOLC1 with fibrillarin.

Targeted deletion of keratin 8 in intestinal epithelial cells disrupts tissue integrity and predisposes to tumorigenesis in the colon.

Keratin 8 (K8) is the main intestinal epithelial intermediate filament protein with proposed roles for colonic epithelial cell integrity. Here, we used mice lacking K8 in intestinal epithelial cells (floxed K8 and Villin-Cre1000 and Villin-CreERt2) to investigate the cell-specific roles of intestinal epithelial K8 for colonocyte function and pathologies. Intestinal epithelial K8 deletion decreased K8 partner proteins, K18-K20, 75-95%, and the remaining keratin filaments were located at the colonocyte apical regions with type II K7, which decreased 30%. 2-Deoxy-2-[18F]-fluoroglucose positron emission tomography in vivo imaging identified a metabolic phenotype in the lower gut of the conditional K8 knockouts. These mice developed intestinal barrier leakiness, mild diarrhea, and epithelial damage, especially in the proximal colon. Mice exhibited shifted differentiation from enterocytes to goblet cells, displayed longer crypts and an increased number of Ki67 + transit-amplifying cells in the colon. Significant proproliferative and regenerative signaling occurred in the IL-22, STAT3, and pRb pathways, with minor effects on inflammatory parameters, which, however, increased in aging mice. Importantly, colonocyte K8 deletion induced a dramatically increased sensitivity to azoxymethane-induced tumorigenesis. In conclusion, intestinal epithelial K8 plays a significant role in colonocyte epithelial integrity maintenance, proliferation regulation and tumor suppression.

Microscopic interpretation of folding ϕ-values using the transition path ensemble.

All-atom molecular dynamics simulations now allow us to create movies of proteins folding and unfolding. However, it is difficult to assess the accuracy of the folding mechanisms observed because experiments cannot yet directly resolve events occurring along the transition paths between unfolded and folded states. Protein folding ϕ-values provide residue-resolved information about folding mechanisms by comparing the effects of mutations on folding rates and stability, but determining ϕ-values by separately simulating mutant proteins would be computationally demanding and prone to large statistical errors. Here we use transition path theory to develop a method for computing ϕ-values directly from the transition path ensemble, without the need for additional simulations. This path-based approach uses the full transition path information available from equilibrium folding and unfolding trajectories, or from transition path sampling, and does not require identification of folding transition states. Applying our approach to a set of simulations of 10 small proteins by Shaw and coworkers [Lindorff-Larsen K, Piana S, Dror RO, Shaw DE (2011) Science 334(6055):517-520; Piana S, Lindorff-Larsen K, Shaw DE (2011) Biophys J100(9):L47-L49; and Piana S, Lindorff-Larsen K, Shaw DE (2013) Proc Natl Acad Sci USA 110(15):5915-5920], we find good agreement with experiments in most cases where data are available. We can further resolve the contributions to fractional ϕ-values coming from partial contact formation versus transition path heterogeneity. Although in some cases, there is substantial heterogeneity of folding mechanism, in others, such as Ubiquitin, the mechanism is strongly conserved.

Unrolr: Structural analysis of protein conformations using stochastic proximity embedding.

Molecular dynamics (MD) simulations are widely used to explore the conformational space of biological macromolecules. Advances in hardware, as well as in methods, make the generation of large and complex MD datasets much more common. Although different clustering and dimensionality reduction methods have been applied to MD simulations, there remains a need for improved strategies that handle nonlinear data and/or can be applied to very large datasets. We present an original implementation of the pivot-based version of the stochastic proximity embedding method aimed at large MD datasets using the dihedral distance as a metric. The advantages of the algorithm in terms of data storage and computational efficiency are presented, as well as the implementation realized. Application and testing through the analysis of a 200 ns accelerated MD simulation of a 35-residue villin headpiece is discussed. Analysis of the simulation shows the promise of this method to organize large conformational ensembles. © 2018 Wiley Periodicals, Inc.

GhVLN4 is involved in cell elongation via regulation of actin organization.

MAIN CONCLUSION: GhVLN4 exhibited activity of cross-linking actin filaments into bundles. Overexpression of GhVLN4 increased the abundance of thick actin bundles and resulted in longer cell phenotypes. Actin bundle is a dynamic, higher-order cytoskeleton structure that is essential for cell expansion. Villin is one of the major proteins responsible for crosslinking actin filaments into bundles. However, this kind of actin binding protein has rarely been investigated in cotton. In the present work, a cotton villin gene was molecularly cloned from Upland cotton and denominated as GhVLN4. This gene was more highly expressed in fiber-bearing wild-type cotton TM-1 (Texas Marker-1) than in Ligon lintless-1 mutant (Li-1). Biochemical analysis combined with subcellular localization revealed that GhVLN4 is an actin-binding protein performing actin filament bundling activity in vitro. In line with these findings, a greater abundance of thick actin filament bundles were observed in GhVLN4-overexpressing transgenic plants compared with those in wild-type control. Moreover, ectopic expression of GhVLN4 significantly enhanced the cell length-width ratio of Schizosaccharomyces pombe yeast and increased the length of various Arabidopsis cells, including root cells, root hairs and pollen tubes. Taken together, our results demonstrate that GhVLN4 is involved in the generation of actin filament bundles, suggesting that GhVLN4 may play important roles in regulating plant cell morphogenesis and expansion by remodeling actin cytoskeleton.

The expression of VILL protein is hypoosmotic-dependent in the lamellar gill ionocytes of Otocephala teleost fish, Chanos chanos.

Milkfish, a species within the primitive teleost lineage Otocephala, can survive in water conditions ranging from hypo- to hyper-saline. This study explored the effects of environmental salinity on apical morphologies of ionocytes and the expression of villin homologs in the gills of milkfish acclimated to either seawater (SW) or fresh water (FW). Scanning electron microscopy revealed that the ionocytes in the gill filaments of SW and FW milkfish, respectively, cellular apical morphologies were hole-type and squint-type. The flat-type ionocytes were observed in the gill lamellae of FW milkfish. Furthermore, apical surfaces of some lamellar ionocytes exhibited microvilli. Villin 1 is a microvilli marker expressed in the epithelial cells of various vertebrates. In the phylogenetic tree of villin 1 homologs, primitive teleosts exhibit villin 1-like (VILL) and villin 1 proteins. Two mRNA sequences, villin 1 and VILL, were identified from the milkfish transcriptome by next generation sequencing. Low but constant expression of villin 1 (gene and protein) was observed in the gills for both SW and FW fish. VILL gene and protein expression levels in the gills were higher in FW fish, compared to SW fish. Double immunofluorescence staining demonstrated that VILL protein was present in some lamellar ionocytes of FW milkfish, but not in the filament ionocytes of either FW or SW milkfish. Taken together, these findings indicated that the VILL expression of ionocytes is hypoosmotic-dependent. The VILL might be involved in the formation of microvilli in the lamellar ionocytes for hyperosmoregulation of the milkfish.

Colon cancer with micropapillary carcinoma component: a clinopathologic study of 9 cases.

Recently, colon cancer with micropapillary pattern (MPP) has been identified. MPP is defined as tight tufts surrounded by cleft-like space and lacking true fibrovascular cores and showing reverse polarity. In this article, we studied nine cases of colon cancer with MPP. MPP usually accounted for a minor component in total tumour volume, which ranged from 3 to 40% with a mean percentage of 19.2%. Associated histological subtype showed moderately differentiated tubular adenocarcinoma in all cases. The reverse polarity of villin (9/9, diffuse) in MPP component was superior to that of CA125 (5/9, focal) or CD10 (5/9, diffuse 2/9, focal 3/9). In clinicopathological indicators such as sex, tumour location, tumour depth, lymphovascular invasion, lymph node metastasis, or pathological stage and clinical behaviour, there was statistically no difference between the MPP group and the non-MPP group of the colon. In conclusion, colon cancer with MPP is characterised by frequent association with moderately differentiated tubular adenocarcinoma as a minor component. Villin immunohistochemistry is useful in the detection of reverse polarity of MPP of colon cancer.

Protein Stability and Unfolding Following Glycine Radical Formation.

Glycine (Gly) residues are particularly susceptible to hydrogen abstraction; which results in the formation of the capto-dative stabilized Cα-centered Gly radical (GLR) on the protein backbone. We examined the effect of GLR formation on the structure of the Trp cage; tryptophan zipper; and the villin headpiece; three fast-folding and stable miniproteins; using all-atom (OPLS-AA) molecular dynamics simulations. Radicalization changes the conformation of the GLR residue and affects both neighboring residues but did not affect the stability of the Trp zipper. The stability of helices away from the radical center in villin were also affected by radicalization; and GLR in place of Gly15 caused the Trp cage to unfold within 1 µs. These results provide new evidence on the destabilizing effects of protein oxidation by reactive oxygen species.

Towards fast, rigorous and efficient conformational sampling of biomolecules: Advances in accelerated molecular dynamics.

BACKGROUND: Accelerated molecular dynamics (aMD) has been proven to be a powerful biasing method for enhanced sampling of biomolecular conformations on general-purpose computational platforms. Biologically important long timescale events that are beyond the reach of standard molecular dynamics can be accessed without losing the detailed atomistic description of the system in aMD. Over other biasing methods, aMD offers the advantages of tuning the level of acceleration to access the desired timescale without any advance knowledge of the reaction coordinate. SCOPE OF REVIEW: Recent advances in the implementation of aMD and its applications to small peptides and biological macromolecules are reviewed here along with a brief account of all the aMD variants introduced in the last decade. MAJOR CONCLUSIONS: In comparison to the original implementation of aMD, the recent variant in which all the rotatable dihedral angles are accelerated (RaMD) exhibits faster convergence rates and significant improvement in statistical accuracy of retrieved thermodynamic properties. RaMD in conjunction with accelerating diffusive degrees of freedom, i.e. dual boosting, has been rigorously tested for the most difficult conformational sampling problem, protein folding. It has been shown that RaMD with dual boosting is capable of efficiently sampling multiple folding and unfolding events in small fast folding proteins. GENERAL SIGNIFICANCE: RaMD with the dual boost approach opens exciting possibilities for sampling multiple timescales in biomolecules. While equilibrium properties can be recovered satisfactorily from aMD-based methods, directly obtaining dynamics and kinetic rates for larger systems presents a future challenge. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

The effect of microbial challenge on the intestinal proteome of broiler chickens.

BACKGROUND: In poultry production intestinal health and function is paramount to achieving efficient feed utilisation and growth. Uncovering the localised molecular mechanisms that occur during the early and important periods of growth that allow birds to grow optimally is important for this species. The exposure of young chicks to used litter from older flocks, containing mixed microbial populations, is a widely utilised model in poultry research. It rarely causes mortality but effects an immunogenic stimulation sufficient enough to cause reduced and uneven growth that is reflective of a challenging growing environment. METHODS: A mixed microbial challenge was delivered as used litter containing Campylobacter jejuni and coccidial oocysts to 120 male Ross 308 broiler chicks, randomly divided into two groups: control and challenged. On day 12, 15, 18 and 22 (pre- and 3, 6 and 10 days post-addition of the used litter) the proximal jejunum was recovered from 6 replicates per group and differentially abundant proteins identified between groups and over time using 2D DiGE. RESULTS: The abundance of cytoskeletal proteins of the chicken small intestinal proteome, particularly actin and actin associated proteins, increased over time in both challenged and control birds. Villin-1, an actin associated anti-apoptotic protein, was reduced in abundance in the challenged birds indicating that many of the changes in cytoskeletal protein abundance in the challenged birds were as a result of an increased rate of apoptosis. A number of heat shock proteins decreased in abundance over time in the intestine and this was more pronounced in the challenged birds. CONCLUSIONS: The small intestinal proteome sampled from 12 to 22 days of age showed considerable developmental change, comparable to other species indicating that many of the changes in protein abundance in the small intestine are conserved among vertebrates. Identifying and distinguishing the changes in proteins abundance and molecular pathways that occur as a result of normal growth from those that occur as a result of a challenging microbial environment is important in this major food producing animal.

Tissue specific loss of A-type lamins in the gastrointestinal epithelium can enhance polyp size.

The nuclear lamina, comprised of the A and B-type lamins, is important in maintaining nuclear shape and in regulating key nuclear functions such as chromatin organization and transcription. Deletion of the A-type lamins results in genome instability and many cancers show altered levels of A-type lamin expression. Loss of function mutations in the mouse Lmna gene result in early postnatal lethality, usually within 3-5 weeks of birth making an analysis of the role of lamins in carcinogenesis difficult. To circumvent early lethality, and determine the role of the A-type lamins in specific tissues in older mice we derived a conditional allele of Lmna(FL/FL) (floxed). Lmna(FL/FL) was specifically deleted in the gastrointestinal (GI) epithelium by crossing the Lmna(FL/FL) mice with Villin-Cre mice. Mice lacking Lmna in the GI are overtly normal with no effects on overall growth, longevity or GI morphology. On a GI specific sensitized (Apc(Min/+)) background, polyp numbers are unchanged, but polyp size is slightly increased, and only in the duodenum. Our findings reveal that although A-type lamins are dispensable in the postnatal GI epithelium, loss of Lmna under malignant conditions may, to a limited extent, enhance polyp size indicating that A-type lamins may regulate cell proliferation in the transformed GI epithelium.

Disease specificity of anti-tryptophan hydroxylase-1 and anti-AIE-75 autoantibodies in APECED and IPEX syndrome.

Autoantibodies to autoimmune enteropathy-related 75 kDa antigen (AIE-75) and villin are disease markers of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome which is characterized by a peripheral tolerance defect. On the other hand, anti-tryptophan hydroxylase-1 (TPH-1) antibodies are detected in autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy (APECED), a central tolerance defect, especially when complicated with gastrointestinal dysfunction. However, to date, anti-AIE-75 and anti-villin antibodies or anti-TPH-1 antibodies have not been tested in APECED or IPEX syndrome, respectively. In the present study, we confirmed the disease specificity of both anti-AIE-75 and anti-TPH-1, although anti-villin antibodies were detected in some patients with APECED. Our observation suggests that immunotolerance to AIE-75 depends on the peripheral mechanism, whereas the tolerance to TPH-1 depends on the central mechanisms.

Plant villins: versatile actin regulatory proteins.

Regulation of actin dynamics is a central theme in cell biology that is important for different aspects of cell physiology. Villin, a member of the villin/gelsolin/fragmin superfamily of proteins, is an important regulator of actin. Villins contain six gelsolin homology domains (G1-G6) and an extra headpiece domain. In contrast to their mammalian counterparts, plant villins are expressed widely, implying that plant villins play a more general role in regulating actin dynamics. Some plant villins have a defined role in modifying actin dynamics in the pollen tube; most of their in vivo activities remain to be ascertained. Recently, our understanding of the functions and mechanisms of action for plant villins has progressed rapidly, primarily due to the advent of Arabidopsis thaliana genetic approaches and imaging capabilities that can visualize actin dynamics at the single filament level in vitro and in living plant cells. In this review, we focus on discussing the biochemical activities and modes of regulation of plant villins. Here, we present current understanding of the functions of plant villins. Finally, we highlight some of the key unanswered questions regarding the functions and regulation of plant villins for future research.

Cdc42 coordinates proliferation, polarity, migration, and differentiation of small intestinal epithelial cells in mice.

BACKGROUND & AIMS: Cdc42 is a Rho GTPase that regulates diverse cellular functions, including proliferation, differentiation, migration, and polarity. In the intestinal epithelium, a balance among these events maintains homeostasis. We used genetic techniques to investigate the role of Cdc42 in intestinal homeostasis and its mechanisms. METHODS: We disrupted Cdc42 specifically in intestinal epithelial cells by creating Cdc42flox/flox-villin-Cre+ and Cdc42flox/flox-Rosa26-CreER+ mice. We collected intestinal and other tissues, and analyzed their cellular, molecular, morphologic, and physiologic features, compared with the respective heterozygous mice. RESULTS: In all mutant mice studied, the intestinal epithelium had gross hyperplasia, crypt enlargement, microvilli inclusion, and abnormal epithelial permeability. Cdc42 deficiency resulted in defective Paneth cell differentiation and localization without affecting the differentiation of other cell lineages. In mutant intestinal crypts, proliferating stem and progenitor cells increased, compared with control mice, resulting in increased crypt depth. Cdc42 deficiency increased migration of stem and progenitor cells along the villi, caused a mild defect in the apical junction orientation, and impaired intestinal epithelium polarity, which can contribute to the observed defective intestinal permeability. The intestinal epithelium of the Cdc42flox/flox-villin-Cre+ and Cdc42flox/flox-Rosa26-CreER+ mice appeared similar to that of patients with microvillus inclusion disease. In the digestive track, loss of Cdc42 also resulted in crypt hyperplasia in the colon, but not the stomach. CONCLUSIONS: Cdc42 regulates proliferation, polarity, migration, and differentiation of intestinal epithelial cells in mice and maintains intestine epithelial barrier and homeostasis. Defects in Cdc42 signaling could be associated with microvillus inclusion disease.

Prediction of villin expression and tumor behavior in colorectal cancer.

BACKGROUND: Colorectal cancer is one of the most common cancers and the leading cause of cancer-related deaths worldwide. The incidence is gradually increasing, and the mortality and recurrence rates of the disease remain high. METHODS: This study was conducted as a cross-sectional study using tissue samples of 106 patients who underwent surgery at Sina Hospital from 2021 to 2022. After histopathological examination and identification of the pathological features of the tumor, the samples were subjected to immunohistochemical staining using a monoclonal antibody against villin. RESULTS: In this study, we observed a significant association between villin expression and tumor depth, as well as a correlation between villin expression and tumor location (colon or rectum). However, no association was found between villin expression and the number of affected lymph nodes and age, sex, tumor grade, and size. Furthermore, there was no significant association between villin expression and tumor vascular or neural invasion. CONCLUSION: The extent of local invasion and metastasis are important factors in disease progression and can lead to treatment failure. Therefore, new biomarkers are needed to identify patients at risk of local and distant metastases and to enable appropriate treatment of patients.

The utility of the lineage specific immunohistochemical stains SATB2, CDX2, and villin, and the mucin glycoproteins MUC2, MUC5AC, and MUC6 to distinguish pulmonary invasive mucinous adenocarcinoma from metastatic colorectal carcinoma.

CONTEXT: The lungs are a common site of tumor metastasis. While morphology and immunophenotype can help differentiate primary from metastatic tumors, distinguishing pulmonary invasive mucinous adenocarcinoma (PIMA) from metastatic colorectal adenocarcinoma (CRC) may occasionally be challenging due to overlapping morphological and immunohistochemical features. Lineage-specific markers such as CDX2, TTF-1, and napsin A are helpful with pulmonary non-mucinous adenocarcinoma (PNMA), however they are non-specific and insensitive when applied to PIMA. SATB2 is a newer marker that distinguishes CRC from upper gastrointestinal and pancreaticobiliary tumors; its utility in distinguishing CRC from PIMA has not been fully elucidated. OBJECTIVE: To evaluate the performance of lineage-specific and mucin glycoprotein immunostains in distinguishing PIMA and CRC. DESIGN: We stained tissue microarrays comprising 34 PNMA, 31 PIMA, and 32 CRC with CK7, CK20, SATB2, CDX2, villin, TTF-1, napsin A, and gel-forming mucins MUC2, MUC5AC, and MUC6. RESULTS: PIMA showed significant (>50% of cells) expression of SATB2 (6%), CDX2 (6%), villin (74%), TTF-1 (13%), and napsin A (23%). However, significant CK7 expression was seen in nearly all PIMA (30/31) and none of the metastatic CRC. CONCLUSION: Our results suggest that CK7 remains one of the most useful markers for distinguishing primary PIMA from metastatic CRC. Expression of the mucin glycoproteins MUC5AC and MUC6 and lack of expression of MUC2 favored a diagnosis of PIMA, but expression of these markers was too heterogeneous to be of clinical utility. To our knowledge this is the only study comparing the immunohistochemical profile of PIMA and metastatic CRC in lung metastasectomy specimens.

Low dietary carbohydrate induces structural alterations in enterocytes of the chicken ileum.

We investigated the role of dietary carbohydrates in the maintenance of the enterocyte microvillar structure in the chicken ileum. Male chickens were divided into the control and three experimental groups, and the experimental groups were fed diets containing 50%, 25%, and 0% carbohydrates of the control diet. The structural alterations in enterocytes were examined using transmission electron microscopy and immunofluorescent techniques for ß-actin and villin. Glucagon-like peptide (GLP)-2 and proglucagon mRNA were detected by immunohistochemistry and in situ hybridization, respectively. Fragmentation and wide gap spaces were frequently observed in the microvilli of the 25% and 0% groups. The length, width, and density of microvilli were also decreased in the experimental groups. The experimental groups had shorter terminal web extensions, and there were substantial changes in the mitochondrial density between the control and experimental groups. Intensities of ß-actin and villin immunofluorescence observed on the apical surface of enterocytes were lower in the 0% group. The frequency of GLP-2-immunoreactive and proglucagon mRNA-expressing cells decreased with declining dietary carbohydrate levels. This study revealed that dietary carbohydrates contribute to the structural maintenance of enterocyte microvilli in the chicken ileum. The data from immunohistochemistry and in situ hybridization assays suggest the participation of GLP-2 in this maintenance system.