Sex Determining Region Y-Box 12 Promotes Proliferation, Migration and Invasion of Osteosarcoma Cells by Activating β-Catenin/T Cell Factor Axis

Objectives: This study aims to clarify the role of sex determining region Y-box 12 (SOX12) in accelerating the proliferative, migratory and invasive abilities of osteosarcoma (OS) via β-catenin/TCF axis. Materials and Methods: SOX12 levels in human osteosarcoma cell lines and human fetal osteoblastic cell line were determined by RT-qPCR. The proliferation rates of osteosarcoma cells were both determined by CCK-8 assay and EdU staining. In addition, osteosarcoma cell migration and migration were determined by wound healing assay and trans-well assay, respectively. TOPFlash/FOPFlash reporter activity assay and western blot assay were simultaneously performed for the detection of β-catenin/TCF axis. Results: SOX12 was elevated in osteosarcoma cell lines, developing the critical role in proliferation, migration and invasion of osteosarcoma cells. The β-catenin/TCF pathway was activated in osteosarcoma. SOX12 overexpression exerted promotive effects on activation of β-catenin/TCF pathway and SOX12 knockdown showed the opposite effects. Conclusions: SOX12 accelerates proliferation, migration and invasion of osteosarcoma cells by activating β-catenin/TCF axis, thus stimulating the progression of OS.

Interaction between Macrophages and Human Mesenchymal Stromal Cells Derived from Bone Marrow and Wharton’s Jelly—A Comparative Study

Despite intensive clinical research on the use of mesenchymal stromal cells (MSCs), further basic research in this field is still required. Herein, we compared human bone marrow MSCs (BM-MSCs, n = 6) and Wharton’s jelly MSCs (WJ-MSCs, n = 6) in their ability to interact with human primary macrophages. Evaluation of secretory potential revealed that under pro-inflammatory stimulation, WJ-MSCs secreted significantly more IL-6 than BM-MSCs (2-fold). This difference did not translate into the effect of MSCs on macrophages: both types of MSCs significantly directed M1-like macrophages toward the M2 phenotype (based on CD206 expression) to a similar extent. This observation was consistent both in flow cytometry analysis and immunocytochemical assessment. The effect of MSCs on macrophages was sustained when IL-6 signaling was blocked with Tocilizumab. Macrophages, regardless of polarization status, enhanced chemotaxis of both BM-MSCs and WJ-MSCs (p < 0.01; trans-well assay), with WJ-MSCs being significantly more responsive to M1-derived chemotactic signals than BM-MSCs. Furthermore, WJ-MSCs increased their motility (scratch assay) when exposed to macrophage-conditioned medium while BM-MSCs did not. These results indicate that although both BM-MSCs and WJ-MSCs have the ability to reciprocally interact with macrophages, the source of MSCs could slightly but significantly modify the response under clinical settings.

A Genetic Platform for Functionally Profiling Odorant Receptors Ex Vivo Using Olfactory Cilia.

The molecular basis for odor perception in humans remains a black box as odorant receptors (ORs) are notoriously difficult to study outside of their native environment. Efforts toward OR expression and functional profiling in heterologous systems have been met with limited success due to poor efficiency of cell surface expression and consequently reduced G-protein signal amplification. We previously reported a genetic strategy in mice to increase the number of sensory neurons expressing specific ORs, which transforms the 10 million neurons of the mouse nose into a bioreactor producing large quantities of fully functional OR protein. We now describe the isolation of cilia from these bioreactors for two human ORs. Cilia are known to contain all components of the olfactory signal transduction machinery and can be placed into an ex vivo well-plate assay to rapidly measure robust, reproducible odor-specific responses. Our OR1A1 and OR5AN1 isolated cilia reveal 10-100fold more sensitivity than existing assays. Tissue from a single animal produces up to 4,000 384-well assay wells, and isolated olfactory cilia can be stored frozen and thus preserved for long term usage. This pipeline offers a sensitive, highly scalable ex vivo odor screening platform that opens the door for decoding human olfaction.

Impact of bacterial microcompartment-dependent ethanolamine and propanediol metabolism on Listeria monocytogenes interactions with Caco-2 cells

Bacterial microcompartment (BMC) dependent ethanolamine (eut) and propanediol utilization (pdu) has recently been shown to stimulate anaerobic growth of Listeria monocytogenes. This metabolic repertoire conceivably contributes to the competitive fitness of L. monocytogenes in the human gastrointestinal (GI) tract, where these compounds become available following phospholipid degradation and mucus-derived rhamnose metabolism by commensal microbiota. Previous transcriptomics and mutant studies of eut and pdu L. monocytogenes suggested a possible role of eut and pdu BMC metabolism in transmission in foods and pathogenicity, but data on a potential role of L. monocytogenes interaction with human cells is currently absent. First, we ask which cellular systems are expressed in the activation of eut and pdu BMC metabolism and the extent to which these systems are conserved between the states. We find common and unique systems related to metabolic shifts, stress and virulence factors. Next, we hypothesize that these common and unique activated cellular systems contribute to a role in the interaction of L. monocytogenes interaction with human cells. We present evidence that metabolically primed L. monocytogenes with active eut and pdu BMCs, as confirmed by metabolic analysis, transmission electron microscopy and proteomics, show significantly enhanced translocation efficacy compared to non-induced cells in a trans-well assay using Caco-2 cells, while adhesion and invasion capacity was similar. Taken together, our results provide insights into the possible key cellular players that drive translocation efficacy upon eut and pdu BMC activation.

A short report on the tumor suppressor role of BIRC7 in pancreatic cancer

<p>Pancreatic cancer (PC) is accepted to be an aggressive malignancy among all type of cancers due to its poor prognosis and high cancer-led mortality ratio mostly affecting male community in older age. Multiple genes are involved in PC initiation, progression and metastasis including K-RAS, CDKN2A, p53, SMAD4. Baculoviral IAP repeat containing 7 (BIRC7) commonly known as Livin, an inhibitor of apoptosis protein (IAP) involved in the inhibition of cell death via apoptosis by preventing caspase activity through various approaches. The biological role of BIRC7 was previously identified in multiple cancers but ill investigated in PC. In this study, we investigate the function role of BIRC7 in PC. Multiple phenotypic tests including wound healing assay, CCK8 assay, trans-well assay and colony formation assay was run to rule out BIRC7 gene effect on PC genesis. We for the first time indicated that, overexpression of BIRC7 significantly reduced the proliferation, development, progression and metastasis of PANC-1 cell <em>in vitro</em>. Therefore, we anticipated that BIRC7 gene is a suppressor gene and might be a suitable candidate gene for therapeutic purposes in PC.</p>

Interleukin-1β-induced matrix metalloproteinase-3 via ERK1/2 pathway to promote mesenchymal stem cell migration

Human umbilical cord Wharton’s jelly derived mesenchymal stem cells (hUCMSCs), a source of cell therapy, have received a great deal of attention due to their homing or migrating ability in response to signals emanating from damaged sites. It has been found that IL-1β possesses the ability to induce the expression of matrix metalloproteinase-3 (MMP-3) in bone marrow MSCs. MMP-3 is involved in cell migration in various types of cells, including glioblastoma, vascular smooth muscle, and adult neural progenitor cells. In this study, we proposed that IL-1β influences hUCMSCs migration involving MMP-3. The expression level of MMP-3 in IL-1β-induced hUCMSCs was verified using cDNA microarray analysis, quantitative real-time PCR, ELISA and Western blot. Wound-healing and trans-well assay were used to investigate the cell migration and invasion ability of IL-1β-treated hUCMSCs. In addition, we pre-treated hUCMSCs with interleukin-1 receptor antagonist, MMP-3 inhibitors (ALX-260-165, UK 356618), or transfected with MMP-3 siRNA to confirm the role of MMP3 in IL-1β-induced cell migration. Our results showed that IL-1β induced MMP-3 expression is related to the migration of hUCMSCs. Moreover, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) inhibitor U0126, p38 inhibitor SB205380, JNK inhibitor SP600125 and Akt inhibitor GSK 690693 decreased IL-1β-induced MMP-3 mRNA and protein expression. The migration and invasion ability analyses showed that these inhibitors attenuated the IL-1β-induced migration and invasion ability of hUCMSCs. In conclusion, we have found that IL-1β induces the expression of MMP-3 through ERK1/2, JNK, p38 MAPK and Akt signaling pathways to enhance the migration of hUCMSCs. These results provide further understanding of the mechanisms in IL-1β-induced hUCMSCs migration to injury sites.

Dihydroartemisinin Shows Promising Effects in the Treatment of Experimental Autoimmune Encephalomyelitis and Maintains Inflammatory Homeostasis by Targeting AXL in Microglia

Background: During EAE progression, the endogenous mechanisms mediating nervous autoimmune inflammation balance, as represented by AXL, were proved to be pathologically disturbed, immune balance and axon repair. Therapeutically, by activating AXL signaling, the inflammatory rebalance from promotion to resolution has attracted increasing attention and showed advantages in autoimmune disease treatment. Previous studies implied that DHA had potential effects in treating autoimmune diseases. However, the detailed mechanisms in inflammation regulation, especially in CNS, remain unclear.Methods: C57BL/6 mice were immunized with MOG35-55 and treated daily with DHA. Then clinical scores, pathology, and ethology features of EAE were assessed through histological staining (H&E, LFB staining), TEM and gait analysis. Moreover, DHA-responsive cells and genes were screened by 10x Genomics. The immunological responses to DHA were measured by flow cytometry and fluorescence microscope in BV2 cells. The concentrations and bio-activities of chemokines were respectively evaluated through ELISA and trans-well assay. Results: After DHA treatment, the clinical scores and body weight were significantly improved. Histologically, mice showed slighter spinal cord lesion, less inflammatory cuffs. By using gait analysis, DHA obviously improved physical coordination. 10x Genomics demonstrated that DHA selectively upregulated AXL expression in microglia. Immunologically, by enhancing AXL signaling, the phagocytic and chemotactic potential of microglia and the Treg differentiation followed by upregulating PDL1 were significantly influenced by DHA. Conversely, specific blocking of AXL by SGI7079 was sufficient to reverse above-mentioned functions. Molecularly, DHA specifically rebalanced the overactivated inflammation through STAT1:SOCS3: AXL: IFNAR pathway. Conclusions: The present study highlighted the central role of AXL signaling in DHA mediated inflammatory transition.

A comparison of the inhibitory activities of Lactobacillus and Bifidobacterium against Penicillium expansum and an analysis of potential antifungal metabolites

ABSTRACT The infection of fruits by Penicillium expansum (P. expansum) do not only cause economic loss but also potentially endanger human health, especially because few biocontrol agents against this fungus have been well studied yet. In this work, to verity the antifungal activity against P. expansum of 22 Bifidobacterium and 44 Lactobacillus, dual-culture overlay assay, microtiter plate well assay and agar spot assay were successively performed. One of the strain, Bifidobacterium adolescentis (B. adolescentis) CCFM1108 exhibited the most potent inhibition ability among all tested strains. Additionally, we showed that multiple antifungal compounds produced by tested strain synergistically inhibit the growth of P. expansum, including lactic acid, acetic acid, 3-phenyllactic acid and p-hydroxyphenyllactic acid. Those active compounds mentioned were detected in the cell-free supernatant and characterized by metabolomics analysis using GC-MS. Correspondingly, B. adolescentis CCFM1108 supernatant disrupted plasma membrane integrity of the P. expansum mycelial and drastically reduced patulin production in P. expansum. The inhibitive effects of B. adolescentis CCFM1108 were also confirmed with three other P. expansum strains. The active inhibitory properties of Bifidobacterium strains, especially B. adolescentis CCFM1108, indicate that B. adolescentis can be potentially used as a novel bioagent to prevent or delay fungal spoilage on fruit.

Implementation and evaluation of a novel real-time multiplex assay for SARS-CoV-2: In-field learnings from a clinical microbiology laboratory

ABSTRACTThe unprecedented scale of testing required to effectively control the coronavirus disease (COVID-19) pandemic has necessitated urgent implementation of rapid testing in clinical microbiology laboratories. To date, there are limited data available on the analytical performance of emerging commercially available assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and integration of these assays into laboratory workflows. Here, we performed a prospective validation study of a commercially available assay, the AusDiagnostics Coronavirus Typing (8-well) assay. Respiratory tract samples for SARS-CoV-2 testing were collected between 1st March and 25th March 2020. All positive samples and a random subset of negative samples were sent to a reference laboratory for confirmation. In total, 2,673 samples were analyzed using the Coronavirus Typing assay. The predominant sample type was a combined nasopharyngeal/throat swab (2,640/2,673; 98.8%). Fifty-four patients were positive for SARS-CoV-2 (0.02%) using the Coronavirus Typing assay; 53/54 (98.1%) positive results and 621/621 (100%) negative results were concordant with the reference laboratory. Compared to the reference standard, sensitivity of the Coronavirus Typing assay for SARS-CoV-2 was 100% [95% CI 93.2%-100%], specificity 99.8% [95% CI 99.1%-100%], positive predictive value 98.1% (95% CI 90.2%-99.7%] and negative predictive value 100% [95% CI 99.4%-100%]. In many countries, standard regulatory requirements for the introduction of new assays have been replaced by emergency authorizations and it is critical that laboratories share their post-market validation experiences, as the consequences of widespread introduction of a sub-optimal assay for SARS-CoV-2 are profound. Here, we share our in-field experience, and encourage other laboratories to follow suit.

Pre-clinical studies of honey-related synthetic peptides in malignancy control

Backgound: The challenge with malignancy control approaches, although of all traditional methods of treatment like surgery, chemotherapy, radiotherapy, immunotherapy etc. makes more stress upon the scientific community to look for a solution for this challenge even far from the traditional ways. Methods: A growth inhibitory assay was performed. Samples of peptide A and peptide F, 5 mg each, were dissolved in 500 μL Dulbecco’s Phosphate-Buffered Saline to prepare the stock solution, which was added into the wells directly. A 1% trifluoroacetic acid (TFA) solution was used as the control. (a) Cells were placed into a 96-well assay plate at a density of 5000 and 10000 cells/well for adherent and suspension cell lines, respectively, with a 50 μL complete cell culture medium. (b) Stock solutions (10 μL) of the compounds were added into the wells. (c) For data analysis, 10 μL 1% TFA solution was used as control. (d) Complete medium of 40 μL was added into the wells to reach a total volume of 100 μL. (e) The plate was incubated at 37℃ for 72 hours. (f) CellTiter-Glo assay mix solution (50 μL) was added to each well and mixed gently at room temperature for 10 minutes; subsequently, the luminescence was read using PHERAstar Plus (Molecular Devices). Results: Apparent inhibitory effects of those peptides were noticed in 17 of 18 tumour cell lines. Similar effects were noted in an in vivo study using the U87MG tumour mouse model. Conclusion: This study has introduced new synthetic peptides derived from honey proteins, which may help in cancer control through direct apoptotic action not limited to only a certain tumour cell line. Patent: WO/2014/040605A1, US/2015/0291663A1, and CN105283198A.