Cell Adhesion Assays Research Articles

1-DEOXYNOJIRIMYCIN, A GLYCOSYLATION INHIBITOR INFLUENCES THE EXPRESSION OF EXTRACELLULAR MATRIX PROTEINS IN OSTEOSARCOMA CELLS

Malignant tumour cells secrete extracellular matrix (ECM) proteins with growth factors and cytokines to promote cell motility and adhesion for metastasis. The cell-ECM adhesion sites are comprised of integrin-bound ECM proteins, which are glycoconjugates, susceptible to the aberrant glycosylation resulting in alterations of cell-ECM adhesion dynamics. Inhibition of glycosylation could decrease the rate of invasiveness in metastatic tumour cells like osteosarcoma (OS), of which the underlying mechanism is still unknown. Hence, this study aims to determine the effect of glucosidase inhibitors towards the expression of ECM proteins in metastatic OS cell lines. OS cells (MG63) were treated with 0.5 mM of 1-deoxynojirimycin (1-DNJ). Analyses such as the glycosylation assay, ECM-cell adhesion assay, scratch assay, and Western blot analysis were performed after a 24-h treatment for each respective sample. The expression level of glycosylated proteins was the highest in non-treated MG63 cells. The cell migration rate was increased in MG63 cells treated with 1-DNJ compared to the normal osteoblast cells. The cell adhesion assay results showed an increased rate of cell adhesion towards vitronectin but decreased adhesion rate towards collagen (types I, II, and IV), fibronectin, laminin, and tenascin in 1-DNJ-treated OS cells. Western blot analysis presented a high expression level of fibronectin, vitronectin, and collagen type II in both non-treated and treated groups of MG63 cells. The inhibitory effect of 1-DNJ upregulates ECM remodelling by disrupting integrin from binding to the ECM proteins, subsequently increasing the migration rate of metastatic OS cells. Keyword(s) : Extracellular matrix protein, 1-Deoxynojirimycin, integrin glycosylation, cell adhesion rate, migration assay.

Genetic Diversity and Expression of Intimin in Escherichia albertii Isolated from Humans, Animals, and Food.

Escherichia albertii (E. albertii) is an emerging diarrheagenic pathogen associated with sporadic infections and human gastroenteric outbreaks. The eae gene, which encodes intimin in the locus of enterocyte effacement (LEE) operon, contributes to the establishment of the attaching and effacing (A/E) lesion. Increasing collection of E. albertii strains from various sources has resulted in a rapid increase in the number of eae subtypes. This study systematically investigated the prevalence and genetic diversity of eae among E. albertii strains isolated from humans, animals, and food. The eae gene was present in 452/459 (98.5%) strains and 23 subtypes were identified including two novel subtypes, named eae-α11 and η3. The eae-σ subtype was the most predominant among humans, animals, and food-derived strains, while eae-γ3, τ, and α11 were unique in human-derived strains. Additionally, the LEE island was also analyzed at genomic, transcriptional, and functional levels through genomic analysis, quantitative reverse transcription PCR, and HEp-2 cell adherence assays, respectively. The eae transcript levels were variable and associated with eae subtypes. Three different adherence patterns, including localized adherence-like (LAL), diffuse adherence (DA), and detachment (DE), were observed among E. albertii strains. This study demonstrated a high diversity of functional intimin in E. albertii strains isolated from humans, animals, and food. Further in vivo and in vitro studies are warranted to better elucidate the role of intimin or LEE in different genetic backgrounds.

Differentiation-inducing factor-1 reduces lipopolysaccharide-induced vascular cell adhesion molecule-1 by suppressing mTORC1-S6K signaling in vascular endothelial cells

AimsDifferentiation-inducing factor-1 (DIF-1), a compound in Dictyostelium discoideum, exhibits anti-cancer effects by inhibiting cell proliferation and motility of various mammalian cancer cells in vitro and in vivo. In addition, DIF-1 suppresses lung colony formation in a mouse model, thus impeding cancer metastasis. However, the precise mechanism underlying its anti-metastatic effect remains unclear. In the present study, we aim to elucidate this mechanism by investigating the adhesion of circulating tumor cells to blood vessels using in vitro and in vivo systems. Main methodsMelanoma cells (1.0 × 105 cells) were injected into the tail vein of 8-week-old male C57BL/6 mice after administration of DIF-1 (300 mg/kg per day) and/or lipopolysaccharide (LPS: 2.5 mg/kg per day). To investigate cell adhesion and molecular mechanisms, cell adhesion assay, western blotting, immunofluorescence staining, and flow cytometry were performed. Key findingsIntragastric administration of DIF-1 suppressed lung colony formation. DIF-1 also substantially inhibited the adhesion of cancer cells to human umbilical vein endothelial cells. Notably, DIF-1 did not affect the expression level of adhesion-related proteins in cancer cells, but it did decrease the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells by suppressing its mRNA-to-protein translation through inhibition of mTORC1-p70 S6 kinase signaling. SignificanceDIF-1 reduced tumor cell adhesion to blood vessels by inhibiting mTORC1-S6K signaling and decreasing the expression of adhesion molecule VCAM-1 on vascular endothelial cells. These findings highlight the potential of DIF-1 as a promising compound for the development of anti-cancer drugs with anti-metastatic properties.

MiR-191-5p inhibits KLF6 to promote epithelial-mesenchymal transition in breast cancer.

MicroRNAs (miRNAs) exert certain functions in the development of several cancers and can be a potential hallmark for cancer diagnosis and prognosis. MiR-191-5p has been proven to have high expression in breast cancer (BC), while its biological role and potential regulatory mechanisms in BC remain an open issue. Bioinformatics was utilized to assay miR-191-5p level in BC tissues and predict its downstream target gene as well as the enriched signaling pathways of the target gene. qRT-PCR was carried out to assay miR-191-5p and KLF6 levels in BC cells as well as miR-191-5p level in blood-derived exosomes from BC patients. Western blot was to examine the expression of proteins linked with cell adhesion, epithelial-mesenchymal transition (EMT), and exosome markers. A dual luciferase reporter assay was utilized to verify the interaction between miR-191-5p and KLF6. Abilities of cell phenotypes of BC cells were detected by CCK8, Transwell, and cell adhesion assay, separately. Upregulated miR-191-5p expression and downregulated KLF6 expression were observed in BC cells. There was a targeting relationship between miR-191-5p and KLF6. MiR-191-5p negatively regulated KLF6 to promote EMT and malignant progression of BC cells. Additionally, we described a dramatically high level of miR-191-5p in the blood exosomes of BC patients. MiR-191-5p advances the EMT of BC by targeting KLF6, indicating that miR-191-5p and KLF6 may be new biomarkers for BC.

GMI, a Ganoderma microsporum protein, abolishes focal adhesion network to reduce cell migration and metastasis of lung cancer

BackgroundCancer metastasis is a major cause of cancer-related deaths, emphasizing the urgent need for effective therapies. Although it has been shown that GMI, a fungal protein from Ganoderma microsporum, could suppress primary tumor growth in a wide spectrum of cancer types, it is still unclear whether GMI exhibits anti-metastasis properties, particularly in lung cancers. Further investigation is needed. Aims and objectivesThe objective of this study is to investigate the potential inhibitory effects of GMI on lung cancer metastasis in vivo. Utilizing systematic and comprehensive approaches, our research aims to elucidate the underlying molecular mechanisms responsible for the anti-metastatic effects. Materials and methodsIn vitro migration and cell adhesion assays addressed the epithelial-to-mesenchymal transition (EMT)-related phenotype. Proteomic and bioinformatic analyses identified the GMI-regulated proteins and cellular responses. GMI-treated LLC1-bearing mice were analyzed using IVIS Spectrum to assess the anti-metastatic effect. Key findingsGMI inhibits EMT as well as cell migration. GMI disrupts cell adhesion and downregulates integrin, resulting in inhibition of phosphorylated FAK. GMI induces macropinocytosis and lysosome-mediated degradation of integrin αv, α5, α6 and β1. GMI downregulates Slug via inhibition of FAK activity, which in turn enhances expressions of epithelial-related markers and decreases cell mobility. Mechanistically, GMI-induced FAK inhibition engenders MDM2 expression and enhances MDM2/p21/Slug complex formation, leading to Slug degradation. GMI treatment reduces the metastatic pulmonary lesion and prolongs the survival of LLC1-bearing mice. SignificanceOur findings highlight GMI as a promising therapeutic candidate for metastatic lung cancers, offering potential avenues for further research and drug development.

Anticancer and anti-metastasis activity of 1,25 dihydroxycholecalciferols and genistein in MCF-7 and MDA-MB-231 breast cancer cell lines

A powerful steroid hormone precursor, 1,25 dihydroxycholecalciferols (1,25(OH)2D3), and dietary phytoestrogen (genistein) are essential compounds that act by binding to nuclear receptors and altering gene expression. They have many biological benefits, some of which have anticancer properties. We studied the impact of 1,25(OH)2D3 and genistein on the proliferation, progression, and metastasis of MCF-7 and MDA-MB-231 cells when they were used alone or in combination and investigated whether there was a synergistic effect between genistein and 1,25(OH)2D3. To achieve these goals, a variety of assays, including flow cytometry, cell invasion assays, cell adhesion assays, Western blotting, and RT‒PCR, were used.Our findings showed that genistein, 1,25(OH)2D3, and the two combined all effectively declined the growth of MCF-7 and MDA-MB-231 cells by arresting the cells in the G0/G1 phase and inducing an apoptotic pathway. Stimulation of apoptosis was achieved by upregulating the expression of BAX and CASP3 genes and downregulating the expression levels of BCL-2 gene. Furthermore, both compounds suppress metastasis by reducing cell adhesion and cell migration/invasion by elevating the expression level of E-cadherin and reducing the expression level of P-cadherin and N-cadherin. Additionally, both genistein and 1,25(OH)2D3 increased the expression level of ERK1 and reduced the expression levels of JNK, p38, Ras, and MEK proteins, which reduced metastasis, enhanced the response to cancer treatment, and improved overall survival. Thus, genistein and 1,25(OH)2D3 can both be considered key candidates in the search for new breast cancer treatments.

Fabrication and characterization of a hydrocolloid wound dressing functionalized with human placental derived extracellular matrix for management of skin wounds: An animal study.

Functionalization of wound dressing is one of the main approaches for promoting wound healing in skin wound management. In this study, our aim is to fabricate a bio-functionalized hydrocolloid wound dressing. The extracellular matrix (ECM) was extracted from human placental tissue. A hydrocolloid film was fabricated using Na-CMC, pectin, gelatin, styrene-isoprene-styrene adhesive, glycerol, and 0.5%-2.5% powdered ECM. A polyurethane film and a release liner were used in the hydrocolloid/ECM films. The mechanical, adhesion, swelling rate, and integrity of the films were investigated. Cell proliferation, adhesion, and migration assays, as well as, SEM and FTIR spectroscopy were also conducted. Macroscopic and microscopic evaluations of wound healing process and formation of blood vessels were conducted in mouse animal models. We successfully fabricated a three-layered ECM-functionalized hydrocolloid dressing with a water vapor transmission rate of 371 g/m2 /day and an adhesion peel strength of 176 KPa. Cellular adhesion, proliferation and migration were promoted by ECM. In the animal tests, ECM-functionalized hydrocolloids significantly improved wound closure and re-epithelialization at days 14 and 21. Also, ECM-functionalized hydrocolloids promoted the formation of hair follicles. Our findings suggest that ECM could enhance the wound healing properties of hydrocolloid wound dressings. This wound dressing could be considered for application in hard-to-heal acute wounds.

The impact of an antibody investigation algorithm emphasizing specificity on reducing potential false-positive warm autoantibody detection at a Canadian tertiary care centre.

To reduce potential false-positive warm autoantibody (WAA) by solid-phase red cell adherence assay (SPRCA), our centre implemented a new antibody investigation algorithm (AIA) by classifying cases with panreactive SPRCA but negative saline-indirect antiglobulin test as 'antibody of undetermined significance' (AUS) after excluding clinically significant antibodies. We assessed the effects of the new AIA and subsequent alloantibody formation in patients with AUS. Samples from patients with positive SPRCA screens between 1 September 2017 and 31 August 2021 were selected for the study. Frequencies of antibodies classified by the old and new AIAs were compared using Fisher's exact test. Patient demographics, transfusion history and antibody formation in cases of AUS were collected. A significant reduction in potential WAA frequencies from 127/1167 (11%) to 53/854 (6%) was observed (p < 0.001) when compared between the old and new AIAs among 2021 positive SPRCA antibody screens. While no patients with AUS later transitioned to potential WAA using the new AIA, four patients developed alloantibodies, including anti-E, anti-C, both anti-C and anti-E, and anti-Wra . A significant reduction in the frequencies of potential false-positive WAA detection at our centre was observed after implementing the new AIA, leading to less resource and phenotypically matched red blood cell (RBC) use. Some patients still developed subsequent RBC alloimmunization, so clinically relevant alloantibodies should be carefully excluded before determining AUS, taking forming or evanescent antibodies into consideration.

Synovium-Derived Mesenchymal Stem Cell-Based Scaffold-Free Fibrocartilage Engineering for Bone-Tendon Interface Healing in an Anterior Cruciate Ligament Reconstruction Model.

Current tendon and ligament reconstruction surgeries rely on scar tissue healing which differs from native bone-to-tendon interface (BTI) tissue. We aimed to engineer Synovium-derived mesenchymal stem cells (Sy-MSCs) based scaffold-free fibrocartilage constructs and investigate in vivo bone-tendon interface (BTI) healing efficacy in a rat anterior cruciate ligament (ACL) reconstruction model. Sy-MSCs were isolated from knee joint of rats. Scaffold-free sy-MSC constructs were fabricated and cultured indifferentiation media including TGF-β-only, CTGF-only, and TGF-β + CTGF. Collagenase treatment on tendon grafts was optimized to improve cell-to-graft integration. The effects of fibrocartilage differentiation and collagenase treatment on BTI integration was assessed by conducting histological staining, cell adhesion assay, and tensile testing. Finally, histological and biomechanical analyses were used to evaluate in vivo efficacy of fibrocartilage construct in a rat ACL reconstruction model. Fibrocartilage-like features were observed with in the scaffold-free sy-MSC constructs when applying TGF-β and CTGF concurrently. Fifteen minutes collagenase treatment increased cellular attachment 1.9-fold compared to the Control group without affecting tensile strength. The failure stress was highest in the Col + D + group (22.494 ± 13.74 Kpa) compared to other groups at integration analysis in vitro. The ACL Recon + FC group exhibited a significant 88% increase in estimated stiffness (p = 0.0102) compared to the ACL Recon group at the 4-week postoperative period. Scaffold-free, fibrocartilage engineering together with tendon collagenase treatment enhanced fibrocartilaginous BTI healing in ACL reconstruction.

Aldo-keto Reductase 1B10 Restrains Cell Migration, Invasion, and Adhesion of Gastric Cancer via Regulating Integrin Subunit Alpha 5.

Gastric cancer is a prevalent malignancy with unfavorable prognosis partially resulting from its high metastasis rate. Clarifying the molecular mechanism of gastric cancer occurrence and progression for improvement of therapeutic efficacy and prognosis is needed. The study tended to delineate the role and regulatory mechanism of aldo-keto reductase 1B10 (AKR1B10) in gastric cancer progression. The relationship of AKR1B10 expression with survival rate in gastric cancer was analyzed through Kaplan-Meier analysis. The mRNA levels of AKR1B10 and integrin subunit alpha 5 (ITGA5) in gastric cancer tissues and cell lines were measured by real-time quantitative polymerase chain reaction. Protein levels of AKR1B10 and integrin subunit alpha 5 were assayed via western blot. The molecular relationship between AKR1B10 and ITGA5 was analyzed by co-immunoprecipitation assay. Cell viability was assayed through Cell Counting Kit-8, invasion and migration of tumor cells was assessed through wound healing and transwell assays. Transwell assay was utilized to detect invasion. The adhesion of gastric cancer cells was detected using cell adhesion assays. The results unveiled that integrin subunit alpha 5 was upregulated, while AKR1B10 was downregulated in gastric cancer tissues and cells. Overexpressing AKR1B10 hindered gastric cancer cell proliferation, migration, invasion and adhesion. It was striking that we certified the inhibitory effect of AKR1B10 on integrin subunit alpha 5 expression and their (AKR1B10 and ITGA5)) negative relationship via bioinformatics method, real-time quantitative polymerase chain reaction, and co-immunoprecipitation assays. Via rescue experiments, it was concluded that AKR1B10 served as tumor suppressor potentially by ITGA5 expression in gastric cancer. Our results indicated that AKR1B10 inhibited migration, invasion, and adhesion of gastric cancer cells via modulation of ITGA5.

Growth of B16F10 cells is enhanced in DJ-1-deficiency pancreas

Association between cancer risk and Parkinson's disease is still debated. DJ-1, a Parkinson's disease (PD)-related gene, is encoded by PARK-7 gene and its deficiency causes early-onset PD. In our last studies, it was found that the immunosuppressive microenvironment established in DJ-1 knockout (KO) mice can enhance metastasis of melanoma cells to lungs. Therefore, we wanted to further examine whether there were some niche in other organs of DJ-1-deficiency mouse to facilitate cell growth of tumors. We used in vivo tissue-specific models of tumor growth and in vitro cellular model to verify the hypothesis. We also used protein blot assay, cell-adhesion assay and bioinformatic tools to conduct experiments. In the mouse model of subcutaneous injection, there was no difference on tumor growth between WT and DJ-1 KO mice. Moreover, the results of experimental liver metastasis by intrasplenic injection model showed that there was no difference of nodules number in both mice, but a dramatic enhancement of nodule formation and increased mucin4 levels were found in pancreas of DJ-1 KO mice. In cell cultures, we further found that B16F10 cells indeed tended to adhere well to primary DJ-1-deficiency pancreatic epithelial cells, which had higher protein levels of mucin4. Notably, a human database also showed the inverse relationship in human pancreas between DJ-1 and mucin4, and mucin4 down-regulation can reverse the enhanced cellular adhesion in DJ-1 KO pancreatic epithelial cells. These results indicated that DJ-1 KO pancreatic tissue creating an appropriate microenvironment benefited development of the cancer cells.

P‐IG‐42 | Solid‐Phase Red Cell Adherence Assay Identifies Clinically Significant Erythrocyte Antibodies Missed by Alternative Serologic Methods

P‐IG‐42 | Solid‐Phase Red Cell Adherence Assay Identifies Clinically Significant Erythrocyte Antibodies Missed by Alternative Serologic Methods

Upregulated TIMP1 facilitates and coordinates myometrial contraction by decreasing collagens and cell adhesive capacity during human labor.

Myometrial contraction is one of the key events involved in parturition. Increasing evidence suggests the importance of the extracellular matrix (ECM) in this process, in addition to the functional role of myometrial smooth muscle cells, and our previous study identified an upregulated tissue inhibitor of metalloproteinase 1 (TIMP1) in human laboring myometrium compared to nonlabor samples. This study aimed to further explore the potential role of TIMP1 in myometrial contraction. First, we confirmed increased myometrial TIMP1 levels in labor and during labor with cervical dilation using transcriptomic and proteomic analyses, followed by real-time PCR, western blotting, and immunohistochemistry. Then, a cell contraction assay was performed to verify the decreased contractility after TIMP1 knockdown in vitro. To further understand the underlying mechanism, we used RNA-sequencing analysis to reveal the upregulated genes after TIMP1 knockdown; these genes were enriched in collagen fibril organization, cell adhesion, and ECM organization. Subsequently, a human matrix metalloproteinase (MMP) array and collagen staining were performed to determine the TIMPs, MMPs and collagens in laboring and nonlabor myometrium. A real-time cell adhesion assay was used to detect cell adhesive capacity. The results showed upregulated MMP8 and MMP9, downregulated collagens, and attenuated cell adhesive capacity in laboring myometrium, while lower MMP levels and higher collagen levels and cell adhesive capacity were observed in nonlabor. Moreover, TIMP1 knockdown led to restoration of cell adhesive capacity. Together, these results indicate that upregulated TIMP1 during labor facilitates and coordinates myometrial contraction by decreasing collagen and cell adhesive capacity, which may provide effective strategies for the regulation of myometrial contraction.

Does decreasing the incubation period used in the antibody screen affect its sensitivity?

Pre-transfusion testing (PTT) encompasses a set of mandatory laboratory tests performed before red blood cell transfusion. The antibody screen, one component of PTT, commonly includes a 10-20 min incubation. The primary aim of this study was to determine if this period can be reduced when using current immunohematology methodologies. Antibody screens were performed on reagent samples using Glass or Gel-based column agglutination technologies (CAT) and a solid phase red cell adherence (SPRCA) assay, with incubation periods of 1, 5, 10 and 15 min, and 20 min (SPRCA assay only). For each method, the shortest period producing a minimum of a 1+ reaction with all reagent samples was considered optimal. The sensitivity of each assay using the optimal period was calculated after performing antibody screens on 100 patient samples. It was demonstrated that the incubation period in the SPRCA and Glass CAT systems can be reduced to 5 and 10 min, respectively, while achieving high assay sensitivity (98.9% in both). The incubation period in the Gel CAT system cannot be reduced from 15 min. Significant association between titre and reaction strength was observed for all three screening methods (p < 0.001 for both CAT methods, p = 0.041 for SPRCA). This study demonstrates that the incubation period used in the antibody screen can be reduced when using systems employing the Glass CAT and SPRCA methods, without affecting assay sensitivity. If confirmed, it could result in faster completion of PTT.

Expression of microRNA-379 reduces metastatic spread of prostate cancer.

Prostate cancer (PCa) is the most common type of cancer in males, and the metastatic form is a leading cause of death worldwide. There are currently no curative treatments for this subset of patients. To decrease the mortality of this disease, greater focus must be placed on developing therapeutics to reduce metastatic spread. We focus on dissemination to the bone since this is both the most common site of metastatic spread and associated with extreme pain and discomfort for patients. Our strategy is to exploit microRNAs (miRNAs) to disrupt the spread of primary PCa to the bone. PCa cell lines were transduced to overexpress microRNA-379 (miR-379). These transduced PCa cells were assessed using cell growth, migration, colony formation and adhesion assays. We also performed in vivo intracardiac injections to look at metastatic spread in NSG mice. A cytokine array was also performed to identify targets of miR-379 that may drive metastatic spread. PCa cells with increased levels of miR-379 showed a significant decrease in proliferation, migration, colony formation, and adhesion to bone cells in vitro. In vivo miR-379 overexpression in PC3 cells significantly decreased metastatic spread to bone and reduced levels of miR-379 were seen in patients with metastases. We identified GDF-15 to be secreted from osteoblasts when grown in conditioned media from PCa cells with reduced miR-379 levels. Taken together, our in vitro and in vivo functional assays support a role for miR-379 as a tumour suppressor. A potential mechanism is unravelled whereby miR-379 deregulation in PCa cells affects the secretion of GDF-15 from osteoblasts which in turn facilitates the metastatic establishment in bone. Our findings support the potential role of miR-379 as a therapeutic solution for prostate cancer.

Development of cobalt (Co)-doped monetites for bone regeneration.

Cobalt-doped monetite powders were synthesized by coprecipitation method under a cobalt nominal content between 2 and 20 mol % of total cation. Structural characterization of samples was performed by using X-ray diffraction (XRD), Fourier transform infrared spectroscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy. XRD results indicated that the Co-doped samples exhibited a monetite single-phase with the cell parameters and crystallite size dependent on the amount of substitutional element incorporated into the triclinic crystalline structure. Cell viability and adhesion assays using pre-osteoblastic cells showed there is no toxicity and the RTqPCR analysis showed significant differences in the expression for osteoblastic phenotype genes, showing a potential material for the bone regeneration.

Characterisation of typical enteropathogenic Escherichia coli (tEPEC) lineages and novel bfpA variants detected in Australian fruit bats (Pteropus poliocephalus)

Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhoeal disease in human infants. EPEC strains are defined by the presence of specific virulence factors including intimin (encoded by the eae gene) and bundle forming pili (Bfp). Bfp is encoded by the bfp operon and includes the bfpA gene for the major pilus subunit. By definition, Bfp are only present in typical EPEC (tEPEC), for which, humans are considered to be the only known natural host. This study detected tEPEC in faecal samples from a wild Australian fruit bat species, the grey-headed flying-fox (Pteropus poliocephalus). Whole genome sequencing of 61 E. coli isolates from flying-foxes revealed that 21.3 % (95%CI: 13 %–33 %) were tEPEC. Phylogenetic analyses showed flying-fox tEPEC shared evolutionary lineages with human EPEC, but were predominantly novel sequence types (9 of 13) and typically harboured novel bfpA variants (11 of 13). HEp-2 cell adhesion assays showed adherence to human-derived epithelial cells by all 13 flying-fox tEPEC, indicating that they all carried functional Bfp. Using an EPEC-specific duplex PCR, it was determined that tEPEC comprised 17.4 % (95%CI: 13 %–22 %) of 270 flying-fox E. coli isolates. Furthermore, a tEPEC-specific multiplex PCR detected the eae and bfpA virulence genes in 18.0 % (95%CI: 8.0 %–33.7 %) of 506 flying-fox faecal DNA samples, with occurrences ranging from 1.3 % to 87.0 % across five geographic areas sampled over a four-year period. The identification of six novel tEPEC sequence types and five novel bfpA variants suggests flying-foxes carry bat-specific tEPEC lineages. However, their close relationship with human EPEC and functional Bfp, indicates that flying-fox tEPEC have zoonotic potential and that dissemination of flying-fox tEPEC into urban environments may pose a public health risk. The consistent detection of tEPEC in flying-foxes over extensive geographical and temporal scales indicates that both wild grey-headed flying-foxes and humans should be regarded as natural tEPEC hosts.

Endogenous Galectin-1 Modulates Cell Biological Properties of Immortalized Retinal Pigment Epithelial Cells In Vitro.

In the eye, an increase in galectin-1 is associated with various chorioretinal diseases, in which retinal pigment epithelium (RPE) cells play a crucial role in disease development and progression. Since little is known about the function of endogenous galectin-1 in these cells, we developed a galectin-1-deficient immortalized RPE cell line (ARPE-19-LGALS1-/-) using a sgRNA/Cas9 all-in-one expression vector and investigated its cell biological properties. Galectin-1 deficiency was confirmed by Western blot analysis and immunocytochemistry. Cell viability and proliferation were significantly decreased in ARPE-19-LGALS1-/- cells when compared to wild-type controls. Further on, an increased attachment of galectin-1-deficient RPE cells was observed by cell adhesion assay when compared to control cells. The diminished viability and proliferation, as well as the enhanced adhesion of galectin-1-deficient ARPE-19 cells, could be blocked, at least in part, by the additional treatment with human recombinant galectin-1. In addition, a significantly reduced migration was detected in ARPE-19-LGALS1-/- cells. In comparison to control cells, galectin-1-deficient RPE cells had enhanced expression of sm-α-actin and N-cadherin, whereas expression of E-cadherin showed no significant alteration. Finally, a compensatory expression of galectin-8 mRNA was observed in ARPE-19-LGALS1-/- cells. In conclusion, in RPE cells, endogenous galectin-1 has crucial functions for various cell biological processes, including viability, proliferation, migration, adherence, and retaining the epithelial phenotype.

Perfusion and Ultrasonication Produce a Decellularized Porcine Whole-Ovary Scaffold with a Preserved Microarchitecture.

The application of decellularized scaffolds for artificial tissue reconstruction has been an approach with great therapeutic potential in regenerative medicine. Recently, biomimetic ovarian tissue reconstruction was proposed to reestablish ovarian endocrine functions. Despite many decellularization methods proposed, there is no established protocol for whole ovaries by detergent perfusion that is able to preserve tissue macro and microstructure with higher efficiency. This generated biomaterial may have the potential to be applied for other purposes beyond reproduction and be translated to other areas in the tissue engineering field. Therefore, this study aimed to establish and standardize a protocol for porcine ovaries' decellularization based on detergent perfusion and ultrasonication to obtain functional whole-ovary scaffolds. For that, porcine ovaries (n = 5) were perfused with detergents (0.5% SDS and 1% Triton X-100) and submitted to an ultrasonication bath to produce acellular scaffolds. The decellularization efficiency was evaluated by DAPI staining and total genomic DNA quantification. ECM morphological evaluation was performed by histological, immunohistochemistry, and ultrastructural analyses. ECM physico-chemical composition was evaluated using FTIR and Raman spectroscopy. A cytocompatibility and cell adhesion assay using murine fibroblasts was performed. Results showed that the proposed method was able to remove cellular components efficiently. There was no significant ECM component loss in relation to native tissue, and the scaffolds were cytocompatible and allowed cell attachment. In conclusion, the proposed decellularization protocol produced whole-ovaries scaffolds with preserved ECM composition and great potential for application in tissue engineering.

PARG Promotes Esophagus Cancer Cell Metastasis by Activation of the Wnt/β-Catenin Pathway.

Esophagus cancer (EC) is a highly malignant and metastatic cancer. Poly(ADP-ribose) glycohydrolase (PARG), a DNA replication and repair regulator, inhibits cancer cell replication defects. This study aimed to explore the role of PARG in EC. The biological behaviors were analyzed using MTT assay, Transwell assay, scratch test, cell adhesion assay, and western blot. PARG expression was detected using quantitative PCR and immunohistochemical assay. The regulation of the Wnt/β-catenin pathway was assessed using western blot. The results showed that PARG was highly expressed in EC tissues and cells. Knockdown of PARG suppressed cell viability, invasion, migration, adhesion, and epithelial-mesenchymal transition. Conversely, overexpression of PARG promoted the biological behaviors mentioned above. Moreover, overexpression of PARG promoted the activation of the Wnt/β-catenin pathway rather than the STAT and Notch pathways. XAV939, the Wnt/β-catenin pathway inhibitor, partly abolished the biological behaviors mediated by PARG overexpression. In conclusion, PARG promoted the malignant advancement of EC via activating the Wnt/β-catenin pathway. These findings suggested that PARG might be a new therapeutic target for EC.