The Trypanosoma cruzi pleiotropic protein P21 orchestrates the intracellular retention and in-vivo parasitism control of virulent Y strain parasites.

P21 is a protein secreted by all forms of Trypanosoma cruzi (T. cruzi) with recognized biological activities determined in studies using the recombinant form of the protein. In our recent study, we found that the ablation of P21 gene decreased Y strain axenic epimastigotes multiplication and increased intracellular replication of amastigotes in HeLa cells infected with metacyclic trypomastigotes. In the present study, we investigated the effect of P21 in vitro using C2C12 cell lines infected with tissue culture-derived trypomastigotes (TCT) of wild-type and P21 knockout (TcP21-/-) Y strain, and in vivo using an experimental model of T. cruzi infection in BALB/c mice. Our in-vitro results showed a significant decrease in the host cell invasion rate by TcP21-/- parasites as measured by Giemsa staining and cell count in bright light microscope. Quantitative polymerase chain reaction (qPCR) analysis showed that TcP21-/- parasites multiplied intracellularly to a higher extent than the scrambled parasites at 72h post-infection. In addition, we observed a higher egress of TcP21-/- trypomastigotes from C2C12 cells at 144h and 168h post-infection. Mice infected with Y strain TcP21-/- trypomastigotes displayed higher systemic parasitemia, heart tissue parasite burden, and several histopathological alterations in heart tissues compared to control animals infected with scrambled parasites. Therewith, we propose that P21 is important in the host-pathogen interaction during invasion, cell multiplication, and egress, and may be part of the mechanism that controls parasitism and promotes chronic infection without patent systemic parasitemia.

Regulation of cellular and molecular markers of epithelial-mesenchymal transition by Brazilin in breast cancer cells.

Breast cancer is the most common invasive neoplasm and the leading cause of cancer death in women worldwide. The main cause of mortality in cancer patients is invasion and metastasis, where the epithelial-mesenchymal transition (EMT) is a crucial player in these processes. Pharmacological therapy has plants as its primary source, including isoflavonoids. Brazilin is an isoflavonoid isolated from Haematoxilum brasiletto that has shown antiproliferative activity in several cancer cell lines. In this study, we evaluated the effect of Brazilin on canonical markers of EMT such as E-cadherin, vimentin, Twist, and matrix metalloproteases (MMPs). By Western blot, we evaluated E-cadherin, vimentin, and Twist expression and the subcellular localization by immunofluorescence. Using gelatin zymography, we determined the levels of secretion of MMPs. We used Transwell chambers coated with matrigel to determine the in vitro invasion of breast cancer cells treated with Brazilin. Interestingly, our results show that Brazilin increases 50% in E-cadherin expression and decreases 50% in vimentin and Twist expression, MMPs, and cell invasion in triple-negative breast cancer (TNBC) MDA-MB-231 and to a lesser extend in MCF7 ER+ breast cancer cells. Together, these findings position Brazilin as a new molecule with great potential for use as complementary or alternative treatment in breast cancer therapy in the future.

Targeting the Endothelin-1 pathway to reduce invasion and chemoresistance in gallbladder cancer cells.

BACKGROUND: Gallbladder cancer (GBC) is a prevalent and deadly biliary tract carcinoma, often diagnosed at advanced stages with limited treatment options. The 5-year survival rate varies widely from 4 to 60%, mainly due to differences in disease stage detection. With only a small fraction of patients having resectable tumors and a high incidence of metastasis, advanced GBC stages are characterized by significant chemoresistance. Identification of new therapeutic targets is crucial, and recent studies have shown that the Endothelin-1 (ET-1) signaling pathway, involving ETAR and/or ETBR receptors (ETRs), plays a crucial role in promoting tumor aggressiveness in various cancer models. Blocking one or both receptors has been reported to reduce invasiveness and chemoresistance in cancers like ovarian, prostate, and colon. Furthermore, transcriptomic studies have associated ET-1 levels with late stages of GBC; however, it remains unclear whether its signaling or its inhibition has implications for its aggressiveness. Although the role of ET-1 signaling in gallbladder physiology is minimally understood, its significance in other tumor models leads us to hypothesize its involvement in GBC malignancy. RESULTS: In this study, we investigated the expression of ET-1 pathway proteins in three GBC cell lines and a primary GBC culture. Our findings demonstrated that both ETAR and ETBR receptors are expressed in GBC cells and tumor samples. Moreover, we successfully down-regulated ET-1 signaling using a non-selective ETR antagonist, Macitentan, which resulted in reduced migratory and invasive capacities of GBC cells. Additionally, Macitentan treatment chemosensitized the cells to Gemcitabine, a commonly used therapy for GBC. CONCLUSION: For the first time, we reveal the role of the ET-1 pathway in GBC cells, providing insight into the potential therapeutic targeting of its receptors to mitigate invasion and chemoresistance in this cancer with limited treatment options. These findings pave the way for further exploration of Macitentan or other ETR antagonists as potential therapeutic strategies for GBC management. In summary, our study represents a groundbreaking contribution to the field by providing the first evidence of the ET 1 pathway's pivotal role in modulating the behavior and aggressiveness of GBC cells, shedding new light on potential therapeutic targets.

Host cell cAMP-Epac-Rap1b pathway inhibition by hawthorn extract as a potential target against Trypanosoma cruzi infection.

Although the two drugs currently available for the treatment of Chagas disease, Benznidazole and Nifurtimox, have proven to be effective in the acute phase of the disease, the 60-90-day treatment leads to high toxicity and unwanted side effects, presenting, in addition, a low efficacy in the chronic phase of the disease. For this reason, new therapies that are more effective are needed. In this regard, we have recently shown that the inhibition of the Epac-Rap1b pathway suppressed the cAMP-mediated host cell invasion by Trypanosoma cruzi. Interestingly, it has been described that vitexin, a natural flavone that protects against ischemia-reperfusion damage, acts by inhibiting the expression of Epac and Rap1 proteins. Vitexin can be found in plants of the genus Crataegus spp., traditionally known as hawthorn, which are of great interest considering their highly documented use as cardio-protectors. Pre-treating cells with an extract of Crataegus oxyacantha produced levels of T. cruzi invasion comparable to the ones observed for the commercially available Epac1-specific inhibitor, ESI-09. In addition, extract-treated cells exhibited a decrease in the activation of Rap1b, suggesting that the effects of the extract would be mediated by the inhibition of the cAMP-Epac-Rap1 signaling pathway. Using HPLC-HRMS2, we could confirm the presence of vitexin, and other flavones that could act as inhibitors of Epac/Rap1b, in the extracts of C. oxyacantha. Most significantly, when cells were treated with the extract of C. oxyacantha in conjunction with Nifurtimox, an increased modulation of invasion was observed.

Biological and therapeutic implications of RKIP in Gastrointestinal Stromal Tumor (GIST): an integrated transcriptomic and proteomic analysis.

BACKGROUND: Gastrointestinal stromal tumors (GIST) represent a significant clinical challenge due to their metastatic potential and limited treatment options. Raf kinase inhibitor protein (RKIP), a suppressor of the MAPK signaling pathway, is downregulated in various cancers and acts as a metastasis suppressor. Our previous studies demonstrated low RKIP expression in GIST and its association with poor outcomes. This study aimed to expand on the previous findings and investigate the biological and therapeutic implications of RKIP loss on GIST. METHODS: To validate the RKIP prognostic significance, its expression was evaluated by immunohistochemistry in 142 bona fide GIST cases. The functional role of RKIP was evaluated in vitro, using the GIST-T1 cell line, which was knocked out for RKIP. The biological and therapeutic implications of RKIP were evaluated by invasion, migration, apoptosis, and 2D / 3D viability assays. Additionally, the transcriptome and proteome of RKIP knockout cells were determined by NanoString and mass spectrometry, respectively. RESULTS: Immunohistochemical analysis revealed the absence of RKIP in 25.3% of GIST cases, correlating with a tendency toward poor prognosis. Functional assays demonstrated that RKIP knockout increased GIST cells' invasion and migration potential by nearly 60%. Moreover, we found that RKIP knockout cells exhibited reduced responsiveness to Imatinib treatment and higher cellular viability in 2D and 3D in vitro models, as assessed by apoptosis-related protein expression. Through comprehensive genetic and proteomic profiling of RKIP knockout cells, we identified several putative RKIP-regulated proteins in GIST, such as COL3A1. CONCLUSIONS: Using a multidimensional integrative analysis, we identified, for the first time in GIST, molecules and pathways modulated by RKIP that may potentially drive metastasis and, consequently, poor prognosis in this disease.

Proliferation, Migration and Invasion of Breast Cancer Cell Lines Are Inhibited by 1,5-Disubstituted Tetrazol-1,2,3-triazole Hybrids through Interaction with p53.

The anticarcinogenic potential of a series of 1,5-disubstituted tetrazole-1,2,3-triazole hybrids (T-THs) was evaluated in the breast cancer (BC)-derived cell lines MCF-7 (ER+, PR+, and HER2-), CAMA-1 (ER+, PR+/-, and HER2-), SKBR-3 (ER+, PR+, and HER2+), and HCC1954 (ER+, PR+, and HER2+). The T-THs 7f, 7l, and 7g inhibited the proliferation of MCF-7 and CAMA-1, HCC1954, and SKBR-3 cells, respectively. The compounds with stronger effect in terms of migration and invasion inhibition were 7o, 7b, 7n, and 7k for the CAMA-1, MCF-7, HCC1954, and SKBR-3 cells respectively. Interestingly, these T-THs were the compounds with a fluorine present in their structures. To discover a possible target protein, a molecular docking analysis was performed for p53, p38, p58, and JNK1. The T-THs presented a higher affinity for p53, followed by JNK1, p58, and lastly p38. The best-predicted affinity for p53 showed interactions between the T-THs and both the DNA fragment and the protein. These results provide an opportunity for these compounds to be studied as potential drug candidates for breast cancer treatment.

The Cytoplasmic Region of SARAF Reduces Triple-Negative Breast Cancer Metastasis through the Regulation of Store-Operated Calcium Entry.

Triple-negative breast cancer has a poor prognosis and is non-responsive to first-line therapies; hence, new therapeutic strategies are needed. Enhanced store-operated Ca2+ entry (SOCE) has been widely described as a contributing factor to tumorigenic behavior in several tumor types, particularly in breast cancer cells. SOCE-associated regulatory factor (SARAF) acts as an inhibitor of the SOCE response and, therefore, can be a potential antitumor factor. Herein, we generated a C-terminal SARAF fragment to evaluate the effect of overexpression of this peptide on the malignancy of triple-negative breast cancer cell lines. Using both in vitro and in vivo approaches, we showed that overexpression of the C-terminal SARAF fragment reduced proliferation, cell migration, and the invasion of murine and human breast cancer cells by decreasing the SOCE response. Our data suggest that regulating the activity of the SOCE response via SARAF activity might constitute the basis for further alternative therapeutic strategies for triple-negative breast cancer.

MiR-495-3p regulates cell migration and invasion in papillary thyroid carcinoma.

Background: Papillary thyroid carcinoma (PTC) is the most prevalent histotype of thyroid cancer and the presence of BRAFV600E mutation in these tumors is related to the malignancy and prognosis of the disease. In recent years attention has been focused on the role of microRNAs in the biology of PTC cells, especially in their role in the modulation of pathways related to tumorigenesis. DLK1-DIO3-derived miRNAs have been shown to play important roles in tumor context and are globally downregulated in PTC. Methods: Based on a previous in silico target prediction and gene enrichment analysis, we identified miR-495-3p as the candidate with the highest tumor suppressor potential role in PTC among DLK1-DIO3-derived miRNAs. We used bioinformatics and an in vitro model of miR-495-3p overexpression to further understand the influence of this molecule on the tumorigenic processes of PTC. Results: Overexpression of miR-495-3p impaired cell migration and invasion of PTC cells harboring the BRAFV600E mutation and affected the expression of targets predicted in the bioinformatic analysis, such as TGFB2, EREG and CCND1. Conclusion: Overall, our results indicate that the loss of miR-495-3p expression during PTC development might play an important role in its progression.

Depletion of Na+/H+ Exchanger Isoform 1 Increases the Host Cell Resistance to Trypanosoma cruzi Invasion.

Na+/H+ exchanger isoform 1 (NHE1), a member of a large family of integral membrane proteins, plays a role in regulating the cortical actin cytoskeleton. Trypanosoma cruzi, the agent of Chagas disease, depends on F-actin rearrangement and lysosome mobilization to invade host cells. To determine the involvement of NHE1 in T. cruzi metacyclic trypomastigote (MT) internalization, the effect of treatment in cells with NHE1 inhibitor amiloride or of NHE1 depletion was examined in human epithelial cells. MT invasion decreased in amiloride-treated and NHE1-depleted cells. The phosphorylation profile of diverse protein kinases, whose activation is associated with remodeling of actin fibers, was analyzed in amiloride-treated and NHE1-depleted cells. In amiloride-treated cells, the phosphorylation levels of protein kinase C (PKC), focal adhesion kinase (FAK) and Akt were similar to those of untreated cells, whereas those of extracellular signal-regulated protein kinases (ERK1/2) increased. In NHE1-deficient cells, with marked alteration in the actin cytoskeleton architecture and in lysosome distribution, the levels of phospho-PKC and phospho-FAK decreased, whereas those of phospho-Akt and phospho-ERK1/2 increased. These data indicate that NHE1 plays a role in MT invasion, by maintaining the activation status of diverse protein kinases in check and preventing the inappropriate F-actin arrangement that affects lysosome distribution.

Ficus crocata leaf extracts decrease the proliferation and invasiveness of breast cancer cells.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype due to its greater invasive capacity and non-response to hormone therapy. Several species of the Ficus genus have been used as an alternative to traditional medicine against malignant diseases. Previously, leaf extracts from Ficus crocata (Miq.) Mart. ex Miq. (F. crocata) showed antiproliferative activity in vitro against breast and cervical tumor cells without having a cytotoxic effect on non-tumor cell lines. The purpose of the study was to evaluate the effect of hexane (Hex-EFc), dichloromethane (Dic-EFc), and acetone (Ace-EFc) extracts from F. crocata on the proliferative and invasive capacity of breast cancer cells MCF-7 and MDA-MB-231. Materials and methods: The phytochemical profile was carried out by gas chromatography-mass spectrometry (GC-MS). Cell proliferation, migration, and invasion were determined by MTT, wound closure, and transwell assays, respectively. MMPs activity was analyzed using gelatin zymography, and fluorescence microscopy was used to visualize F-actin distribution. Results: Hex-EFc, Dic-EFc, and Ace-EFc showed cytotoxic activity on MDA-MB-231 tumor cells and, to a lesser extent, on MCF-7 cells, without presenting cytotoxicity at the same concentrations in MCF-10A non-tumor cells. Dic-EFc and Ace-EFc (5-10 µg/mL) reduced the migration capacity of MCF-7 and MDA-MB-231 cells. Interestingly, exposure to Dic-EFc and Ace-EFc (5-10 µg/mL) inhibited the invasive ability of MDA-MB-231 cells, reducing the secretion and activity of MMP-2 and MMP-9, as well as the F-actin distribution. Conclusions: Dic-EFc and Ace-EFc at low concentrations decreased breast cancer cell proliferation and invasiveness, mainly of MDA-MB-231 cells. The above supports the potential use of compounds from leaf extracts of F. crocata in neoadjuvant therapy to reduce the progression of breast cancer tumors, mainly triple-negative tumors.

Small Extracellular Vesicles from Hypoxic Triple-Negative Breast Cancer Cells Induce Oxygen-Dependent Cell Invasion.

Hypoxia, a condition of low oxygenation frequently found in triple-negative breast tumors (TNBC), promotes extracellular vesicle (EV) secretion and favors cell invasion, a complex process in which cell morphology is altered, dynamic focal adhesion spots are created, and ECM is remodeled. Here, we investigated the invasive properties triggered by TNBC-derived hypoxic small EV (SEVh) in vitro in cells cultured under hypoxic (1% O2) and normoxic (20% O2) conditions, using phenotypical and proteomic approaches. SEVh characterization demonstrated increased protein abundance and diversity over normoxic SEV (SEVn), with enrichment in pro-invasive pathways. In normoxic cells, SEVh promotes invasive behavior through pro-migratory morphology, invadopodia development, ECM degradation, and matrix metalloprotease (MMP) secretion. The proteome profiling of 20% O2-cultured cells exposed to SEVh determined enrichment in metabolic processes and cell cycles, modulating cell health to escape apoptotic pathways. In hypoxia, SEVh was responsible for proteolytic and catabolic pathway inducement, interfering with integrin availability and gelatinase expression. Overall, our results demonstrate the importance of hypoxic signaling via SEV in tumors for the early establishment of metastasis.

The Kinetoplastid-Specific Protein TcCAL1 Plays Different Roles During In Vitro Differentiation and Host-Cell Invasion in Trypanosoma cruzi.

In the pathogen Typanosoma cruzi, the calcium ion (Ca2+) regulates key processes for parasite survival. However, the mechanisms decoding Ca2+ signals are not fully identified or understood. Here, we investigate the role of a hypothetical Ca2+-binding protein named TcCAL1 in the in vitro life cycle of T. cruzi. Results showed that the overexpression of TcCAL1 fused to a 6X histidine tag (TcCAL1-6xHis) impaired the differentiation of epimastigotes into metacyclic trypomastigotes, significantly decreasing metacyclogenesis rates. When the virulence of transgenic metacyclic trypomastigotes was explored in mammalian cell invasion assays, we found that the percentage of infection was significantly higher in Vero cells incubated with TcCAL1-6xHis-overexpressing parasites than in controls, as well as the number of intracellular amastigotes. Additionally, the percentage of Vero cells with adhered metacyclic trypomastigotes significantly increased in samples incubated with TcCAL1-6xHis-overexpressing parasites compared with controls. In contrast, the differentiation rates from metacyclic trypomastigotes to axenic amastigotes or the epimastigote proliferation in the exponential phase of growth have not been affected by TcCAL1-6xHis overexpression. Based on our findings, we speculate that TcCAL1 exerts its function by sequestering intracellular Ca2+ by its EF-hand motifs (impairing metacyclogenesis) and/or due to an unknown activity which could be amplified by the ion binding (promoting cell invasion). This work underpins the importance of studying the kinetoplastid-specific proteins with unknown functions in pathogen parasites.

Recent Advances in Host-Directed Therapies for Tuberculosis and Malaria.

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis, and malaria, caused by parasites from the Plasmodium genus, are two of the major causes of death due to infectious diseases in the world. Both diseases are treatable with drugs that have microbicidal properties against each of the etiologic agents. However, problems related to treatment compliance by patients and emergence of drug resistant microorganisms have been a major problem for combating TB and malaria. This factor is further complicated by the absence of highly effective vaccines that can prevent the infection with either M. tuberculosis or Plasmodium. However, certain host biological processes have been found to play a role in the promotion of infection or in the pathogenesis of each disease. These processes can be targeted by host-directed therapies (HDTs), which can be administered in conjunction with the standard drug treatments for each pathogen, aiming to accelerate their elimination or to minimize detrimental side effects resulting from exacerbated inflammation. In this review we discuss potential new targets for the development of HDTs revealed by recent advances in the knowledge of host-pathogen interaction biology, and present an overview of strategies that have been tested in vivo, either in experimental models or in patients.

Successful invasion of Trypanosoma cruzi trypomastigotes is dependent on host cell actin cytoskeleton.

Cellular invasion by Trypanosoma cruzi metacyclic trypomastigotes (MTs) or tissue culture trypomastigotes (TCTs) is a complex process involving host-parasite cellular and molecular interactions. Particularly, the involvement of host cell actin cytoskeleton during trypomastigote invasion is poorly investigated, and still, the results are controversial. In the present work, we compare side by side both trypomastigote forms and employ state-of-the-art live-cell imaging showing for the first time the dynamic mobilization of host cell actin cytoskeleton to MT and TCT invasion sites. Moreover, cytochalasin D, latrunculin B, and jasplakinolide-pretreated cells inhibited MT and TCT invasion. Furthermore, our results demonstrated that TCT invasion decreased in RhoA, Rac1, and Cdc-42 GTPase-depleted cells, whereas MT invasion decreased only in Cdc42-and RhoA-depleted cells. Interestingly, depletion of the three studied GTPases induced a scattered lysosomal distribution throughout the cytosol. These observations indicate that GTPase depletion is sufficient to impair parasite invasion despite the importance of lysosome spread in trypomastigote invasion. Together, our results demonstrate that the host cell actin cytoskeleton plays a direct role during TCT and MT invasion.

CD38 a biomarker and therapeutic target in non-hematopoietic tumors.

The type II transmembrane glycoprotein CD38 has recently been implicated in regulating metabolism and the pathogenesis of multiple conditions, including aging, inflammation and cancer. CD38 is overexpressed in several tumor cells and microenvironment tumoral cells, associated to migration, angiogenesis, cell invasion and progression of the disease. Thus, CD38 has been used as a progression marker for different cancer types as well as in immunotherapy. This review focuses on describing the involvement of CD38 in various non-hematopoietic cancers.

Increase in ADAR1p110 activates the canonical Wnt signaling pathway associated with aggressive phenotype in triple negative breast cancer cells.

Triple-negative breast cancer (TNBC) represents a challenge in the search for new therapeutic targets. TNBCs are aggressive and generate resistance to chemotherapy. Tumors of TNBC patients with poor prognosis present a high level of adenosine deaminase acting on RNA1 (ADAR1). We explore the connection of ADAR1 with the canonical Wnt signaling pathway and the effect of modulation of its expression in TNBC. Expression data from cell line sequencing (DepMap) and TCGA samples were downloaded and analyzed. We lentivirally generated an MDA-MB-231 breast cancer cell line that overexpress (OE) ADAR1p110 or an ADAR knockdown. Abundance of different proteins related to Wnt/ß-catenin pathway and activity of nuclear ß-catenin were analyzed by Western blot and luciferase TOP/FOP reporter assay, respectively. Cell invasion was analyzed by matrigel assay. In mice, we study the behavior of tumors generated from ADAR1p110 (OE) cells and tumor vascularization immunostaining were analyzed. ADAR1 connects to the canonical Wnt pathway in TNBC. ADAR1p110 overexpression decreased GSK-3ß, while increasing active ß-catenin. It also increased the activity of nuclear ß-catenin and increased its target levels. ADAR1 knockdown has the opposite effect. MDA-MB-231 ADAR1 (OE) cells showed increased capacity of invasion. Subsequently, we observed that tumors derived from ADAR1p110 (OE) cells showed increased invasion towards the epithelium, and increased levels of Survivin and CD-31 expressed in vascular endothelial cells. These results indicate that ADAR1 overexpression alters the expression of some key components of the canonical Wnt pathway, favoring invasion and neovascularization, possibly through activation of the ß-catenin, which suggests an unknown role of ADAR1p110 in aggressiveness of TNBC tumors.

Type-I Collagen/Collagenase Modulates the 3D Structure and Behavior of Glioblastoma Spheroid Models.

Multicellular tumor spheroids have emerged as well-structured, three-dimensional culture models that resemble and mimic the complexity of the dense and hypoxic cancer microenvironment. However, in brain tumor studies, a variety of glioblastoma multiforme (GBM) cell lines only self-assemble into loose cellular aggregates, lacking the properties of actual glioma tumors in humans. In this study, we used type-I collagen as an extracellular matrix component to promote the compaction of GBM aggregates forming tight spheroids to understand how collagen influences the properties of tumors, such as their growth, proliferation, and invasion, and collagenase to promote collagen degradation. The GBM cell lines U87MG, T98G, and A172, as well as the medulloblastoma cell line UW473, were used as standard cell lines that do not spontaneously self-assemble into spheroids, and GBM U251 was used as a self-assembling cell line. According to the findings, all cell lines formed tight spheroids at collagen concentrations higher than 15.0 µg mL-1. Collagen was distributed along the spheroid, similarly to that observed in invasive GBM tumors, and decreased cell migration with no effect on the cellular uptake of small active molecules, as demonstrated by uptake studies using the photosensitizer verteporfin. The enzymatic cleavage of collagen affected spheroid morphology and increased cell migration while maintaining cell viability. Such behaviors are relevant to the physiological models of GBM tumors and are useful for better understanding cell migration and the in vivo infiltration path, drug screening, and kinetics of progression of GBM tumors.

Genetic Profiles and Invasion Ability of Listeria monocytogenes Isolated from Bovine Carcasses in Southern Brazil.

ABSTRACT: The goals of this study were to evaluate the persistence and the virulence potential of Listeria monocytogenes isolated from beef carcasses obtained in processing facilities in the southern region of Rio Grande do Sul, Brazil, based on pulsed-field gel electrophoresis (PFGE), invasion ability in human colorectal carcinoma cells (HCT-116), internalin A (InlA) expression by Western blot, and identification of mutation points in inlA. PFGE profiles demonstrated that L. monocytogenes isolates were grouped based on their previously identified lineages and serogroups (lineage I: serogroup IIb, n = 2, and serogroup IVb, n = 5; lineage II: serogroup IIc, n = 5). Isolates with indistinguishable genetic profiles through this method were obtained from different slaughterhouses and sampling steps, with as much as a 3-year interval. Seven isolates showed high invasion ability (2.4 to 7.4%; lineage I, n = 6, and lineage II, n = 1) in HCT and expressed InlA. Five isolates showed low cell invasion ability (0.6 to 1.4%; lineage I, n = 1, and lineage II, n = 4) and did not express InlA, and two of them (lineage II, serogroup IIc) presented mutations in inlA that led to premature stop codon type 19 at position 326 (GAA → TAA). The results demonstrated that most L. monocytogenes isolates from lineage I expressed InlA and were the most invasive in HCT, indicating their high virulence potential, whereas most isolates from lineage II showed attenuated invasion because of nonexpression of InlA or the presence of premature stop codon type 19 in inlA. The obtained results demonstrated that L. monocytogenes with indistinguishable PFGE profiles can persist or be reintroduced in beef processing facilities in the studied region and that differences in their virulence potential are based on their lineages and serogroups.

Long non-coding RNA PVT1 facilitates cell migration and invasion by regulating miR-148a-3p and ROCK1 in breast cancer.

PURPOSE: Breast cancer (BC) is one of the most common malignant tumors for women. The role and potential mechanisms of long non-coding RNA plasmacytoma variant translocation 1 (lncRNA PVT1) were explored in BC cell migration and invasion. METHODS: PVT1, miR-148a-3p and Rho­associated, coiled­coil containing protein kinase 1 (ROCK1) mRNA expressions were detected using real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). The ROCK1 protein expression was detected by Western blotting. The relationship of PVT1, miR-148a-3p and ROCK1 was analyzed by Dual Luciferase activity, RNA immunoprecipitation (RIP) and Spearman correlation analysis. Cell invasion and migration were detected by Transwell assay. RESULTS: Upregulation of PVT1 and ROCK1, and downregulation of miR-148a-3p were observed in BC tissues and cell lines. According to the analysis of Dual Luciferase activity, RIP and Spearman correlation analysis, miR-148a-3p directly binds to PVT1, and ROCK1 is a target of miR-148a-3p. In addition, PVT1 regulated the cells migration and invasion by regulating miR-148a-3p and ROCK1 expression. CONCLUSION: These data demonstrated that PVT1 was upregulated and facilitated to the cell migration and invasion of BC by the regulation of miR-148a-3p and ROCK1, indicating that PVT1 may be a potential biomarker of BC diagnosis and treatment.

Shedding of Trypanosoma cruzi Surface Molecules That Regulate Host Cell Invasion Involves Phospholipase C and Increases Upon Sterol Depletion.

Metacyclic trypomastigote (MT) forms of Trypanosoma cruzi have been shown to release into medium gp82 and gp90, the stage-specific surface molecules that regulate host cell invasion, either in vesicles or in soluble form. Here, we found that during interaction of poorly invasive G strain with the host cell, gp82 and gp90 were released in vesicle-like forms, whereas no such release by highly invasive CL strain was observed. Shedding of vesicles of varying sizes by CL and G strains was visualized by scanning electron microscopy, and the protein profile of conditioned medium (CM) of the two strains was similar, but the content of gp82 and gp90 differed, with both molecules being detected in G strain as bands of high intensity in Western blotting, whereas in CL strain, they were barely detectable. Confocal images revealed a distinct distribution of gp82 and gp90 on MT surface of CL and G strains. In cell invasion assays, addition of G strain CM resulted in decreased CL strain internalization. Depletion of gp82 in G strain CM, by treatment with specific mAb-coupled magnetic beads, increased its inhibitory effect on CL strain invasion, in contrast to CM depleted in gp90. The effect of cholesterol-depleting drug methyl-ß-cyclodextrin (MßCD) on gp82 and gp90 release by MTs was also examined. G strain MTs, untreated or treated with MßCD, were incubated in serum-containing medium or in nutrient-depleted PBS++, and the CM generated under these conditions was analyzed by Western blotting. In PBS++, gp82 and gp90 were released at lower levels by untreated MTs, as compared with MßCD-treated parasites. CM from untreated and MßCD-treated G strain, generated in PBS++, inhibited CL strain internalization. Treatment of CL strain MTs with MßCD resulted in increased gp82 and gp90 shedding and in decreased host cell invasion. The involvement of phospholipase C (PLC) on gp82 and gp90 shedding was also investigated. The CM from G strain MTs pretreated with specific PLC inhibitor contained lower levels of gp82 and gp90, as compared with untreated parasites. Our results contribute to shed light on the mechanism by which T. cruzi releases surface molecules implicated in host cell invasion.