FAM129B is a cooperative protein that regulates adipogenesis.

FAM129B is one of Niban-like proteins described in neoplastic cells and implicated in melanoma cell invasion, but no reports have been published on FAM129B and cell differentiation. We show that FAM129B is early and transiently expressed and crucial for 3T3-F442A adipogenesis. Fam129b is expressed downstream of the early genes Cebpb, Klf4, Klf5 and Srebf1a, but upstream of Pparg2 since knockdown of Fam129b blocked Pparg2 expression and adipose differentiation. Glycogen synthase kinase 3 beta activity, a crucial kinase for adipogenesis, and the ERK1/2 are involved in FAM129B phosphorylation as part of the adipogenic program. Phosphorylated FAM129B is crucial for Pparg2 expression and the lipogenic gene expression downstream of Pparg2, and hence for adipogenesis. Fam129b knockdown reduced adipocyte cluster formation and size, regulating commitment and clonal amplification. In vivo, BAT, inguinal and epidydimal fat expressed Fam129b, suggesting a role in adipose tissue development. We conclude that FAM129B is a cooperative protein that regulates differentiation during the early stages of adipogenesis.

Comorbidities and laboratory parameters associated with SARS-CoV-2 infection severity in patients from the southeast of Mexico: a cross-sectional study.

Background. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic. Among the risk factors associated with the severity of this disease is the presence of several metabolic disorders. For this reason, the aim of this research was to identify the comorbidities and laboratory parameters among COVID-19 patients admitted to the intensive care unit (ICU), comparing the patients who required invasive mechanical ventilation (IMV) with those who did not require IMV, in order to determine the clinical characteristics associated with the COVID-19 severity. Methods. We carried out a cross-sectional study among 152 patients who were admitted to the ICU from April 1 st to July 31 st, 2021, in whom the comorbidities and laboratory parameters associated with the SARS-CoV-2 infection severity were identified. The data of these patients was grouped into two main groups: "patients who required IMV" and "patients who did not require IMV". The nonparametric Mann-Whitney U test for continuous data and the χ2 test for categorical data were used to compare the variables between both groups. Results. Of the 152 COVID-19 patients who were admitted to the ICU, 66 required IMV and 86 did not require IMV. Regarding the comorbidities found in these patients, a higher prevalence of type 2 diabetes mellitus (T2DM), hypertension and obesity was observed among patients who required IMV vs. those who did not require IMV ( p<0.05). Concerning laboratory parameters, only glucose, Interleukin 6 (IL-6), lactate dehydrogenase (LDH) and C-reactive protein (CRP) were significantly higher among patients who required IMV than in those who did not require IMV ( p<0.05). Conclusion. This study performed in a Mexican population indicates that comorbidities such as: T2DM, hypertension and obesity, as well as elevated levels of glucose, IL-6, LDH and CRP are associated with the COVID-19 severity.

Swine virome on rural backyard farms in Mexico: communities with different abundances of animal viruses and phages.

Domestic swine have been introduced by humans into a wide diversity of environments and have been bred in different production systems. This has resulted in an increased risk for the occurrence and spread of diseases. Although viromes of swine in intensive farms have been described, little is known about the virus communities in backyard production systems around the world. The aim of this study was to describe the viral diversity of 23 healthy domestic swine maintained in rural backyards in Morelos, Mexico, through collection and analysis of nasal and rectal samples. Next-generation sequencing was used to identify viruses that are present in swine. Through homology search and bioinformatic analysis of reads and their assemblies, we found that rural backyard swine have a high degree of viral diversity, different from those reported in intensive production systems or under experimental conditions. There was a higher frequency of bacteriophages and lower diversity of animal viruses than reported previously. In addition, sapoviruses, bocaparvoviruses, and mamastroviruses that had not been reported previously in our country were identified. These findings were correlated with the health status of animals, their social interactions, and the breeding/rearing environment (which differed from intensive systems), providing baseline information about viral communities in backyard swine.

Ceramide synthase 2 knockdown suppresses trophozoite growth, migration, in vitro encystment and excystment of Entamoeba invadens.

Entamoeba invadens is the protozoan which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment/excystment in vitro. Here we report that EinCerS2 knockdown promoted decrease in sphingomyelin (SM) subspecies with long-chain fatty acids (24:0) down to 50% but increase sphingolipids with short-chain fatty acids (16:0) up to three times in both trophozoites and cysts of E. invadens. EinCerS2 silencing also resulted in decreased trophozoites' movement, proliferation, cysts formation, and trophozoites hatched after excystment. By immunofluorescence assays, a polyclonal antibody against EinCerS2 detected the enzyme in the cytoplasm of E. invadens trophozoites, colocalizing with Endoplasmic Reticulum-resident cognate EiSERCA. Interestingly, EinCerS2 was redistributed close to the plasma membrane during encystation, suggesting that the generation of diacylglycerol (DAG) via synthesis of sphingolipids and the activation protein kinase C might participate in the encystment process of E. invadens.

Conserved and divergent expression dynamics during early patterning of the telencephalon in mouse and chick embryos.

The mammalian and the avian telencephalon are nearly indistinguishable at early embryonic vesicle stages but differ substantially in form and function at their adult stage. We sequenced and analyzed RNA populations present in mouse and chick during the early stages of embryonic telencephalon to understand conserved and lineage-specific developmental differences. We found approximately 3000 genes that orchestrate telencephalon development. Many chromatin-associated epigenetic and transcription regulators show high expression in both species and some show species-specific expression dynamics. Interestingly, previous studies associated them to autism, intellectual disabilities, and mental retardation supporting a causal link between their impaired functions during telencephalon development and brain dysfunction. Strikingly, the conserved up-regulated genes were differentially enriched in ontologies related to development or functions of the adult brain. Moreover, a differential enrichment of distinct repertoires of transcription factor binding motifs in their upstream promoter regions suggest a species-specific regulation of the various gene groups identified. Overall, our results reveal that the ontogenetic divergences between the mouse and chick telencephalon result from subtle differences in the regulation of common patterning signaling cascades and regulatory networks unique to each species at their very early stages of development.

The metabolic pathway of sphingolipids biosynthesis and signaling in Entamoeba histolytica.

Sphingolipids (SLs) synthesis involves a complex metabolic pathway occurring between the endoplasmic reticulum (ER) and Golgi apparatus, generating ceramide synthesis and complex lipids, respectively. Here we show that E. histolytica, apparently lacking cellular organelles (ER and Golgi apparatus), synthesizes a wide variety of sphingolipid subspecies, being particularly abundant those of long-chain fatty acids. In silico analysis showed five putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the TLC domain, a region conserved in CerS of multiple organisms. These genes are abundantly expressed in different growth phases. Silencing and overexpression of CerS C4M4U4 (the closest homolog of human CerS 2 and 3) demonstrated its involvement in the synthesis of ceramide. Additionally, we identify C4M4U4, SMS2 and PKC (α, ßII) proteins and their subcellular localization of E. histolytica, suggesting that these subcellular compartments might be involved in the biosynthesis and signaling pathway of sphingolipids, and evidencing different sphingolipid synthesis pathways in Entamoeba.

De novo synthesis of sphingolipids plays an important role during in vitro encystment of Entamoeba invadens.

Entamoeba invadens is a protozoan, which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment in vitro. Here we report for the first time the role of the de novo synthesis pathway of sphingolipids during the encystment of E. invadens. In silico analysis showed that this parasite has six putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the Lag1p motif, a region conserved in the ceramide synthases of multiple organisms, suggesting that they might be bona fide CerS. The six genes of E. invadens are differentially expressed at different time intervals in both stages trophozoite and cyst, based on the results obtained through qRT-PCR assays, the genes involved in the synthesis of sphingolipids with long-chain fatty acids CerS 2,3,4 (EIN_046610, EIN_097030, EIN_130350) have maximum points of relative expression in both stages of the E. invadens life cycle, which strongly suggest that the signaling exerted from the synthesis pathway of sphingolipids is essential for the encystment of E. invadens, since the generation of the more abundant sphingomyelin (SM) subspecies with long-chain fatty acids are fundamental for the parasite to reach its conversion from trophozoite to cyst. When myriocin was used as an inhibitor of serine palmitoyl CoA transferase (SPT), first enzyme in the de novo biosynthesis of sphingolipids, the trophozoites of E. invadens were unable to reach the encystment. Since the effect of myriocin was reversed with exogenous d-erythrosphingosine (DHS), it was demonstrated that the inhibition was specific and it was confirmed that the synthesis of sphingolipids play an essential role during the encystment process of E. invadens.

Transcriptomics and co-expression networks reveal tissue-specific responses and regulatory hubs under mild and severe drought in papaya (Carica papaya L.).

Plants respond to drought stress through the ABA dependent and independent pathways, which in turn modulate transcriptional regulatory hubs. Here, we employed Illumina RNA-Seq to analyze a total of 18 cDNA libraries from leaves, sap, and roots of papaya plants under drought stress. Reference and de novo transcriptomic analyses identified 8,549 and 6,089 drought-responsive genes and unigenes, respectively. Core sets of 6 and 34 genes were simultaneously up- or down-regulated, respectively, in all stressed samples. Moreover, GO enrichment analysis revealed that under moderate drought stress, processes related to cell cycle and DNA repair were up-regulated in leaves and sap; while responses to abiotic stress, hormone signaling, sucrose metabolism, and suberin biosynthesis were up-regulated in roots. Under severe drought stress, biological processes related to abiotic stress, hormone signaling, and oxidation-reduction were up-regulated in all tissues. Moreover, similar biological processes were commonly down-regulated in all stressed samples. Furthermore, co-expression network analysis revealed three and eight transcriptionally regulated modules in leaves and roots, respectively. Seventeen stress-related TFs were identified, potentially serving as main regulatory hubs in leaves and roots. Our findings provide insight into the molecular responses of papaya plant to drought, which could contribute to the improvement of this important tropical crop.

IL-1ß induced methylation of the estrogen receptor ERα gene correlates with EMT and chemoresistance in breast cancer cells.

Inflammation has been recently acknowledged as a key participant in the physiopathology of oncogenesis and tumor progression. The inflammatory cytokine IL-1ß has been reported to induce the expression of markers associated with malignancy in breast cancerous cells through Epithelial-Mesenchymal Transition (EMT). Aggressive breast cancer tumors classified as Triple Negative do not respond to hormonal treatment because they lack three crucial receptors, one of which is the estrogen receptor alpha (ERα). Expression of ERα is then considered a good prognostic marker for tamoxifen treatment of this type of cancer, as the binding of this drug to the receptor blocks the transcriptional activity of the latter. Although it has been suggested that inflammatory cytokines in the tumor microenvironment could regulate ERα expression, the mechanism(s) involved in this process have not yet been established. We show here that, in a cell model of breast cancer cells (6D cells), in which the inflammatory cytokine IL-1ß induces EMT by activation of the IL-1ß/IL-1RI/ß-catenin pathway, the up regulation of TWIST1 leads to methylation of the ESR1 gene promoter. This epigenetic modification produced significant decrease of the ERα receptor levels and increased resistance to tamoxifen. The direct participation of IL-1ß in these processes was validated by blockage of the cytokine-induced signaling pathway by wortmannin inactivation of the effectors PI3K/AKT. These results support our previous reports that have suggested direct participation of the inflammatory cytokine IL-1ß in the transition to malignancy of breast cancer cells.

IL-1ß induces up-regulation of BIRC3, a gene involved in chemoresistance to doxorubicin in breast cancer cells.

Epithelial to mesenchymal transition (EMT) of tumor cells facilitates their progress to metastasis. In the tumor microenvironment the inflammatory cytokine 1ß (IL-1ß) has been associated with tumor development and invasiveness. IL-1ß-induced EMT triggers the expression of markers associated with malignancy. We have recently reported that an IL-1ß-highly responsive clone (6D cells) from non-invasive MCF-7 breast cancer cells activates PI3K/Rac and IL-1RI/ß-catenin pathways that up-regulate the transcription of genes involved in an EMT-like process. However, a correlation between the EMT program induced by a pro-inflammatory environment, and the acquisition of chemoresistance has not been yet determined in these cells. In this work, we report the expression of cell survival genes after IL-1ß stimulation of 6D cells. The expression of CDKN1A, TP63, SFN and, particularly, BIRC3 was found to be up-regulated in a RNA-seq analysis and validated by qPCR. Cells stimulated with IL-1ß when challenged with doxorubicin showed resistance to the drug, whereas silencing of BIRC3 decreased viability of the cells treated with the drug. Our present results show that IL-1ß confers doxorubicin resistance to breast cancer cells, underlining the importance of an inflammatory environment in cancer malignancy.

Sequencing and de novo transcriptome assembly of Anthopleura dowii Verrill (1869), from Mexico.

Next-generation technologies for determination of genomics and transcriptomics composition have a wide range of applications. Moreover, the development of tools for big data set analysis has allowed the identification of molecules and networks involved in metabolism, evolution or behavior. By natural habitats aquatic organisms have implemented molecular strategies for survival, including the production and secretion of toxic compounds for their predators; therefore these organisms are possible sources of proteins or peptides with potential biotechnological application. In the last decade anthozoans, mainly octocorals but also sea anemones, have been proben to be a source of natural products. Members of the genus Anthopleura are one of the best known and most studied sea anemones because they are common constituents of rocky intertidal communities and show interesting ecological and biological phenomena (e.g. intraespecific competition, symbiosis, etc.); however, many aspects of these taxa remain in need to be analyzed. This work describes the transcriptome sequencing of Anthopleura dowii Verrill, 1869 (Cnidaria: Anthozoa: Actiniaria); this is the first report of this kind for these species. The data set used to construct the transcriptome has been deposited on NCBI's database. Illumina sequence reads are available under BioProject accession number PRJNA329297 and Sequence Read Archive under accession number SRP078992.

Retinoic Acid Inhibits Adipogenesis Modulating C/EBPß Phosphorylation and Down Regulating Srebf1a Expression.

Adipogenesis comprises a complex network of signaling pathways and transcriptional cascades; the GSK3ß-C/EBPß-srebf1a axis is a critical signaling pathway at early stages leading to the expression of PPARγ2, the master regulator of adipose differentiation. Previous work has demonstrated that retinoic acid inhibits adipogenesis affecting different signaling pathways. Here, we evaluated the anti-adipogenic effect of retinoic acid on the adipogenic transcriptional cascade, and the expression of adipogenic genes cebpb, srebf1a, srebf1c, pparg2, and cebpa. Our results demonstrate that retinoic acid blocks adipose differentiation during commitment, returning cells to an apparent non-committed state, since they have to be newly induced to adipose conversion after the retinoid is removed from the culture medium. Retinoic acid down regulates the expression of the adipogenic genes, srebf1a, srebf1c, pparg2, and cebpa; however, it did not down regulate the expression of cebpb, but it inhibited C/EBPß phosphorylation at Thr188, a critical step for the progression of the adipogenic program. We also found that RA inhibition of adipogenesis did not increase the expression of dlk1, the gene encoding for Pref1, a well-known anti-adipogenic factor.

The transient expression of Klf4 and Klf5 during adipogenesis depends on GSK3ß activity.

Adipogenesis is regulated by a complex cascade of transcriptional factors, among them KLF4. This factor was previously shown to be necessary for adipose differentiation. We found that GSK3ß activity was required for Klf4 and Klf5 expression during adipogenesis. In addition, retinoic acid inhibited Klf4 and Klf5 expression but not that of Cebpb. Protein synthesis inhibition showed that the transient expression of Klf4, Cebpb and Klf5 during early adipogenesis seemed to require a yet unknown protein for their repression. We also found that Klf4 forced expression in 3T3-F442A cells cultured under non-adipogenic conditions did not induce adipogenesis, nor the expression of Cebpb or Klf5, a Cebpb target gene, showing that KLF4 was not sufficient for adipose differentiation to take place. This would suggest that a more complex combination of molecular pathways not yet understood, is involved during early adipogenesis.

A surface membrane protein of Entamoeba histolytica functions as a receptor for human chemokine IL-8: its role in the attraction of trophozoites to inflammation sites.

Entamoeba histolytica trophozoites respond to the presence of IL-8, moving by chemotaxis towards the source of the chemokine. IL-8 binds to the trophozoite membrane and triggers a response that activates signaling pathways that in turn regulate actin/myosin cytoskeleton organisation to initiate migration towards the chemokine, suggesting the presence of a receptor for IL-8 in the parasite. Antibodies directed to the human IL-8 receptor (CXCR1) specifically recognised a 29 kDa protein in trophozoite membrane fractions. The same protein was immunoprecipitated by this antibody from total amebic extracts. Peptide analysis of the immunoprecipitated protein revealed a sequence with high homology to a previously identified amebic outer membrane peroxiredoxin and a motif within the third loop of human CXCR1, which is an important site for IL-8 binding and activation of signaling processes. Immunodetection assays demonstrated that the anti-human CXCR1 antibody binds to the 29 kDa protein in a different but close site to where IL-8 binds to the trophozoite surface membrane, suggesting that human and amebic receptors for this chemokine share common epitopes. In the context of the human intestinal environment, a receptor for IL-8 could be a great advantage for E. histolytica trophozoite survival, as they could reach an inflammatory milieu containing abundant nutrients. In addition, it has been suggested that the high content of accessible thiol groups of the protein and its peroxidase activity could provide protection in the oxygen rich milieu of colonic lesions, allowing trophozoite invasion of other tissues and escape from the host immune response.

A novel ß-catenin signaling pathway activated by IL-1ß leads to the onset of epithelial-mesenchymal transition in breast cancer cells.

Interleukin 1ß has been associated with tumor development, invasiveness and metastasis in various types of cancer. However, the molecular mechanisms underlying this association have not been clearly elucidated. The present study is the first to show, in breast cancer cells, that an IL-1ß/IL-1RI/ß-catenin signaling pathway induces ß-catenin accumulation due to GSK3ß inactivation by Akt phosphorylation. Translocation to the nucleus of accumulated ß-catenin and formation of the TCF/Lef/ß-catenin complex induce sequential expression of c-MYC, CCDN1, SNAIL1 and MMP2, leading to up-regulation of proliferation, migration and invasion; all of the processes shown to be required, in cancerous cells, to initiate transition from a non-invading to an invasive phenotype.

Glucocorticoid paradoxically recruits adipose progenitors and impairs lipid homeostasis and glucose transport in mature adipocytes.

Chronic treatment with glucocorticoids increases the mass of adipose tissue and promotes metabolic syndrome. However little is known about the molecular effects of dexamethasone on adipose biology. Here, we demonstrated that dexamethasone induces progenitor cells to undergo adipogenesis. In the adipogenic pathway, at least two cell types are found: cells with the susceptibility to undergo staurosporine-induced adipose conversion and cells that require both staurosporine and dexamethasone to undergo adipogenesis. Dexamethasone increased and accelerated the expression of main adipogenic genes such as pparg2, cebpa and srebf1c. Also, dexamethasone altered the phosphorylation pattern of C/EBPß, which is an important transcription factor during adipogenesis. Dexamethasone also had effect on mature adipocytes mature adipocytes causing the downregulation of some lipogenic genes, promoted a lipolysis state, and decreased the uptake of glucose. These paradoxical effects appear to explain the complexity of the action of glucocorticoids, which involves the hyperplasia of adipose cells and insulin resistance.

Toll-like receptor signaling activation by Entamoeba histolytica induces beta defensin 2 in human colonic epithelial cells: its possible role as an element of the innate immune response.

BACKGROUND: Entamoeba histolytica, a protozoan parasite of humans, produces dysenteric diarrhea, intestinal mucosa damage and extraintestinal infection. It has been proposed that the intestinal microbiota composition could be an important regulatory factor of amebic virulence and tissue invasion, particularly if pathogenic bacteria are present. Recent in vitro studies have shown that Entamoeba histolytica trophozoites induced human colonic CaCo2 cells to synthesize TLR-2 and TLR-4 and proinflammatory cytokines after binding to the amebic Gal/GalNac lectin carbohydrate recognition domain. The magnitude of the inflammatory response induced by trophozoites and the subsequent cell damage were synergized when cells had previously been exposed to pathogenic bacteria. METHODOLOGY/PRINCIPAL FINDINGS: We show here that E. histolytica activation of the classic TLR pathway in CaCo2 cells is required to induce ß defensin-2 (HBD2) mRNA expression and production of a 5-kDa cationic peptide with similar properties to the antimicrobial HBD2 expressed by CaCo2 cells exposed to enterotoxigenic Escherichia coli. The induced peptide showed capacity to permeabilize membranes of bacteria and live trophozoites. This activity was abrogated by inhibition of TLR2/4-NFκB pathway or by neutralization with an anti-HBD2 antibody. CONCLUSIONS/SIGNIFICANCE: Entamoeba histolytica trophozoites bind to human intestinal cells and induce expression of HBD2; an antimicrobial molecule with capacity to destroy pathogenic bacteria and trophozoites. HDB2's possible role as a modulator of the course of intestinal infections, particularly in mixed ameba/bacteria infections, is discussed.

Selection of a MCF-7 Breast Cancer Cell Subpopulation with High Sensitivity to IL-1ß: Characterization of and Correlation between Morphological and Molecular Changes Leading to Increased Invasiveness.

Cancer and inflammation are closely related in tumor malignancy prognosis. Breast cancer MCF-7 cells have a poor invasive phenotype, although, under IL-1ß stimulus, acquire invasive features. Cell response heterogeneity has precluded precise evaluation of the malignant transition. MCF-7A3 cells were selected for high sensitivity to IL-1ß stimulus, uniform expression of CXCR4, and stability of IL1-RI. Structural changes, colony formation ability, proliferation rate, chemotaxis, Matrigel invasion, E-cadherin mRNA expression and protein localization were determined in these cells and in MCF-7 parental cells under the stimulus of IL-1ß. Selected MCF-7A3 cells showed a uniform response to IL-1ß stimulation increasing features of invasive cells such as scattering, colony formation, proliferation, chemokinesis and invasion. Basal expression of E-cadherin mRNA was higher, and IL-1ß stimulus had no further effect at early times of cytokine exposure. Total E-cadherin levels remained unchanged in parental cells, whereas levels decreased, as MCF-7A3 cells became fibroblastoid or scattered. Triton X-100 soluble/insoluble E-cadherin ratios were highly increased in these cells, while, in MCF-7pl cells, ratios could not be correlated with morphology changes. MCF-7A3 cells uniform response to IL-1ß allowed characterization of changes induced by the cytokine that had not been assessed when using heterogeneous cell lines.

Srebf1a is a key regulator of transcriptional control for adipogenesis.

Adipogenesis is regulated by a complex cascade of transcriptional factors, but little is known about the early events that regulate the adipogenic program. Here, we report the role of the srebf1a gene in the differentiation of fibroblastic 3T3-F442A cells. We found that expression of srebf1a depended on GSK3ß activity and that GSK3ß activity was necessary for C/EBPß phosphorylation at Thr188. Knockdown of srebf1a inhibited the adipogenic program because it blocked the expression of genes encoding PPARγ2, C/EBPα, SREBP1c and even FABP4, demonstrating that SREBP1a activation is upstream of these three essential adipogenic transcription factors. Kinetic analysis during differentiation illustrated that the order of expression of adipogenic genes was the following: cebpb, srebf1a, pparg2, cebpa, srebp1c and fabp4. Our data suggest that srebf1a acts as an essential link between the GSK3ß-C/EBPß signaling axis and the beginning of the adipogenic transcriptional cascade.

Rab11 and actin cytoskeleton participate in Giardia lamblia encystation, guiding the specific vesicles to the cyst wall.

BACKGROUND: Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol. CONCLUSIONS AND SIGNIFICANCE: Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and actin may be useful as novel pharmacological targets.