Immunocytochemical Assay Research Articles

Corylifol A ameliorates muscle atrophy by inhibiting TAOK1/p38-MAPK/FoxO3 pathway in cancer cachexia.

Corylifol A (CYA) is one of the main active components of Psoralea corylifolia L. CYA had been reported to have ameliorating effects on dexamethasone-induced atrophy of C2C12 mouse skeletal myotubes, but its effects on cancer cachexia were unclear. Here, we checked the influence of CYA on muscle atrophy in cancer cachexia mice and tried to clarify its mechanisms. C26 tumour-bearing mice were applied as the animal model to examine the effects of CYA in attenuating cachexia symptoms. The in vitro cell models of TNF-α-induced C2C12 myotubes or ad-mRFP-GFP-LC3B-transfected C2C12 myotubes were used to check the influence of CYA on myotube atrophy based on both ubiquitin proteasome system (UPS) and autophagy-lysosome system. The possible direct targets of CYA were searched using the biotin-streptavidin pull-down assay and then confirmed using the Microscale thermophoresis binding assay. The levels of related signal proteins in both in vitro and in vivo experiments were examined using western blotting and immunocytochemical assay. The administration of CYA prevented body weight loss and muscle wasting in C26 tumour-bearing mice without affecting tumour growth. At the end of the experiment, the body weight of mice treated with 30mg/kg of CYA (23.59±0.94g) was significantly higher than that of the C26 model group (21.66±0.56g) with P<0.05. The values of gastrocnemius muscle weight/body weight of mice treated with 15 or 30mg/kg CYA (0.53±0.02% and 0.54±0.01%, respectively) were both significantly higher than that of the C26 model group (0.45±0.01%) with P<0.01. CYA decreased both UPS-mediated protein degradation and autophagy in muscle tissues of C26 tumour-bearing mice as well as in C2C12 myotubes treated with TNF-α. The thousand-and-one amino acid kinase 1 (TAOK1) was found to be the direct binding target of CYA. CYA inhibited the activation of TAOK1 and its downstream p38-MAPK pathway thus decreased the level and nuclear location of FoxO3. siRNA knockdown of TAOK1 or regulation of the p38-MAPK pathway using activator or inhibitor could affect the ameliorating effects of CYA on myotube atrophy. CYA ameliorates cancer cachexia muscle atrophy by decreasing both UPS degradation and autophagy. The ameliorating effects of CYA on muscle atrophy might be based on its binding with TAOK1 and inhibiting the TAOK1/p38-MAPK/FoxO3 pathway.

Novel Golden Lipid Nanoparticles with Small Interference Ribonucleic Acid for Substrate Reduction Therapy in Fabry Disease.

Substrate reduction therapy (SRT) has been proposed as a new gene therapy for Fabry disease (FD) to prevent the formation of globotriaosylceramide (Gb3). Nanomedicines containing different siRNA targeted to Gb3 synthase (Gb3S) were designed. Formulation factors, such as the composition, solid lipid nanoparticles (SLNs) preparation method and the incorporation of different ligands, such as gold nanoparticles (GNs), protamine (P) and polysaccharides, were evaluated. The new siRNA-golden LNPs were efficiently internalized in an FD cell model (IMFE-1), with GNs detected in the cytoplasm and in the nucleus. Silencing efficacy (measured by RT-qPCR) depended on the final composition and method of preparation, with silencing rates up to 90% (expressed as the reduction in Gb3S-mRNA). GNs conferred a higher system efficacy and stability without compromising cell viability and hemocompatibility. Immunocytochemistry assays confirmed Gb3S silencing for at least 15 days with the most effective formulations. Overall, these results highlight the potential of the new siRNA-golden LNP system as a promising nanomedicine to address FD by specific SRT.

The journey of cardosin A in young Arabidopsis seedlings leads to evidence of a Golgi-independent pathway to the protein storage vacuole.

The aspartic proteinase cardosin A is a vacuolar enzyme found to accumulate in protein storage and lytic vacuoles in the flowers and protein bodies in the seeds of the native plant cardoon. Cardosin A was first isolated several decades ago and has since been extensively characterized, both in terms of tissue distribution and enzyme biochemistry. In the native system, several roles have been attributed to cardosin A, such as reproduction, reserve mobilization, and membrane remodeling. To participate in such diverse events, cardosin A must accumulate and travel to different compartments within the cell: protein storage vacuoles, lytic vacuoles, and the cytoplasmic membrane (and eventually outside the cell). Several studies have approached the expression of cardosin A in Arabidopsis thaliana and Nicotiana tabacum with promising results for the use of these systems to study of cardosin A trafficking. A poly-sorting mechanism has been uncovered for this protein, as two different vacuolar sorting determinants, mediating different vacuolar routes, have been described. The first is a conventional C-terminal domain, which delivers the protein to the vacuole via the Golgi, and the second is a more unconventional signal-the plant-specific insert (PSI)-that mediates a Golgi-independent route. The hypothesis that these two signals are activated according to cell needs and in organs with high metabolic activity is investigated here. An Arabidopsis line expressing cardosin A under an inducible promoter was used to understand the dynamics of cardosin A regarding vacuolar accumulation during seed germination events. Using antibodies against different regions of the protein and combining them with immunofluorescence and immunocytochemistry assays in different young seedling tissues, cardosin A was detected along the secretory pathway to the protein storage vacuole, often associated with the endoplasmic reticulum. More interestingly, upon treatment with the drug Brefeldin A, cardosin A was still detected in protein storage vacuoles, indicating that the intact protein can bypass the Golgi in this system, contrary to what was observed in N. tabacum. This study is a good starting point for further research involving the use of fluorescent fusions and exploring in more detail the relationship between cardosin A trafficking and plant development.

Magnolol as STAT3 inhibitor for treating multiple sclerosis by restricting Th17 cells

ObjectiveMultiple sclerosis (MS) is an immune disease in the central nervous system (CNS) associated with Th17 cells. Moreover, STAT3 initiates Th17 cell differentiation and IL-17A expression through facilitating RORγt in MS. Here, we reported that magnolol, isolated from Magnolia officinalis Rehd. Et Wils, was regarded as a candidate for MS treatment verified by both in vitro and in vivo studies. MethodsIn vivo, experimental autoimmune encephalomyelitis (EAE) model in mice was employed to evaluate the alleviation of magnolol on myeloencephalitis. In vitro, FACS assay was employed to evaluate the effect of magnolol on Th17 and Treg cell differentiation and IL-17A expression; network pharmacology-based study was applied to probe the involved mechanisms; western blotting, immunocytochemistry, and luciferase reporter assay was used to further confirm the regulation of magnolol on JAK/STATs signaling pathway; surface plasmon resonance (SPR) assay and molecular docking were applied to manifest affinity with STAT3 and binding sites; overexpression of STAT3 was employed to verify whether magnolol attenuates IL-17A through STAT3 signaling pathway. ResultsIn vivo, magnolol alleviated loss of body weight and severity of EAE mice; magnolol improved lesions in spinal cords and attenuated CD45 infiltration, and serum cytokines levels; correspondingly, magnolol focused on inhibiting Th17 differentiation and IL-17A expression in splenocyte of EAE mice; moreover, magnolol selectively inhibited p-STAT3(Y705) and p-STAT4(Y693) of both CD4+ and CD8+ T cells in splenocyte of EAE mice. In vitro, magnolol selectively inhibited Th17 differentiation and IL-17A expression without impact on Treg cells; network pharmacology-based study revealed that magnolol perhaps diminished Th17 cell differentiation through regulating STAT family members; western blotting further confirmed that magnolol inhibited p-JAK2(Y1007) and selectively antagonized p-STAT3(Y705) and slightly decreased p-STAT4(Y693); magnolol antagonized both STAT3 nucleus location and transcription activity; magnolol had a high affinity with STAT3 and the specific binding site perhaps to be at SH2 domain; overexpression of STAT3 resulted in failed inhibition of magnolol on IL-17A. ConclusionMagnolol selectively inhibited Th17 differentiation and cytokine expression through selectively blocking of STAT3 resulting in decreased the ratio of Th17/Treg cells for treating MS, suggesting that the potential of magnolol for treating MS as novel STAT3 inhibitor.

Osteopontin depletion in macrophages perturbs proteostasis via regulating UCHL1-UPS axis and mitochondria-mediated apoptosis.

Osteopontin (OPN; also known as SPP1), an immunomodulatory cytokine highly expressed in bone marrow-derived macrophages (BMMΦ), is known to regulate diverse cellular and molecular immune responses. We previously revealed that glatiramer acetate (GA) stimulation of BMMΦ upregulates OPN expression, promoting an anti-inflammatory, pro-healing phenotype, whereas OPN inhibition triggers a pro-inflammatory phenotype. However, the precise role of OPN in macrophage activation state is unknown. Here, we applied global proteome profiling via mass spectrometry (MS) analysis to gain a mechanistic understanding of OPN suppression versus induction in primary macrophage cultures. We analyzed protein networks and immune-related functional pathways in BMMΦ either with OPN knockout (OPNKO) or GA-mediated OPN induction compared with wild type (WT) macrophages. The most significant differentially expressed proteins (DEPs) were validated using immunocytochemistry, western blot, and immunoprecipitation assays. We identified 631 DEPs in OPNKO or GA-stimulated macrophages as compared to WT macrophages. The two topmost downregulated DEPs in OPNKO macrophages were ubiquitin C-terminal hydrolase L1 (UCHL1), a crucial component of the ubiquitin-proteasome system (UPS), and the anti-inflammatory Heme oxygenase 1 (HMOX-1), whereas GA stimulation upregulated their expression. We found that UCHL1, previously described as a neuron-specific protein, is expressed by BMMΦ and its regulation in macrophages was OPN-dependent. Moreover, UCHL1 interacted with OPN in a protein complex. The effects of GA activation on inducing UCHL1 and anti-inflammatory macrophage profiles were mediated by OPN. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome-mitochondria-mediated apoptosis (e.g., ROS, Lamp1-2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (e.g., 60S and 40S ribosomal subunits and UPS proteins). In agreement with the proteome-bioinformatics data, western blot and immunocytochemical analyses revealed that OPN deficiency perturbs protein homeostasis in macrophages-inhibiting translation and protein turnover and inducing apoptosis-whereas OPN induction by GA restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostatic balance via the regulation of protein synthesis, UCHL1-UPS axis, and mitochondria-mediated apoptotic processes, indicating its potential application in immune-based therapies.

ACE-2, TMPRSS2, and Neuropilin-1 Receptor Expression on Human Brain Astrocytes and Pericytes and SARS-CoV-2 Infection Kinetics.

Angiotensin Converting Enzyme 2 (ACE-2), Transmembrane Serine Protease 2 (TMPRSS-2) and Neuropilin-1 cellular receptors support the entry of SARS-CoV-2 into susceptible human target cells and are characterized at the molecular level. Some evidence on the expression of entry receptors at mRNA and protein levels in brain cells is available, but co-expression of these receptors and confirmatory evidence on brain cells is lacking. SARS-CoV-2 infects some brain cell types, but infection susceptibility, multiple entry receptor density, and infection kinetics are rarely reported in specific brain cell types. Highly sensitive Taqman ddPCR, flow-cytometry and immunocytochemistry assays were used to quantitate the expression of ACE-2, TMPRSS-2 and Neuropilin-1 at mRNA and protein levels on human brain-extracted pericytes and astrocytes, which are an integral part of the Blood-Brain-Barrier (BBB). Astrocytes showed moderate ACE-2 (15.9 ± 1.3%, Mean ± SD, n = 2) and TMPRSS-2 (17.6%) positive cells, and in contrast show high Neuropilin-1 (56.4 ± 39.8%, n = 4) protein expression. Whereas pericytes showed variable ACE-2 (23.1 ± 20.7%, n = 2), Neuropilin-1 (30.3 ± 7.5%, n = 4) protein expression and higher TMPRSS-2 mRNA (667.2 ± 232.3, n = 3) expression. Co-expression of multiple entry receptors on astrocytes and pericytes allows entry of SARS-CoV-2 and progression of infection. Astrocytes showed roughly four-fold more virus in culture supernatants than pericytes. SARS-CoV-2 cellular entry receptor expression and "in vitro" viral kinetics in astrocytes and pericytes may improve our understanding of viral infection "in vivo". In addition, this study may facilitate the development of novel strategies to counter the effects of SARS-CoV-2 and inhibit viral infection in brain tissues to prevent the spread and interference in neuronal functions.

Comparable Effects on Healing between Autologous Diabetic Adipose-Derived Stem Cells and Allogeneic Normal Counterparts.

Adipose-derived stem cell (ASC) therapy is considered a promising strategy for improving impaired wound healing, especially in diabetics. Although the therapeutic potential of allogeneic ASCs from healthy donors is naturally limited, that of autologous ASCs from diabetic patients is questionable. The aim of this study was to investigate the impact of diabetic ASCs in the treatment of diabetic wounds. Diabetic ASCs (DMA) and nondiabetic ASCs were isolated from db/db and C57BL/6J mice, and characterized by immunocytochemistry, proliferation, differentiation, and gene expression assays. The effects of both ASCs on healing were investigated using 36 male 10- to 12-week-old db/db mice. Wound size was measured semiweekly until day 28, and histologic and molecular analyses were performed at day 14. Both ASCs had fibroblast-like morphology and were CD44 + /CD90 + /CD34 - /CD45 - at passage 4. Compared with nondiabetic ASCs in vitro, DMA proliferative capability was restored by passage 4 ( P > 0.05). Although DMA osteogenesis was attenuated ( P < 0.01), both ASCs had similar adipogenesis and expressions of PPARγ/LPL/OCN/RUNX2 ( P > 0.05). In vivo experiments showed that, compared with phosphate-buffered saline control, both ASCs are comparable in improving wound healing ( P < 0.0001), angiogenesis ( P < 0.05), epithelial cell proliferation ( P < 0.05), and granulation tissue formation ( P < 0.0001). In both in vitro and in vivo murine models, DMAs have shown a comparable therapeutic capacity to normal ASCs in promoting diabetic wound healing by improving angiogenesis, reepithelialization, and granulation tissue formation. These results support clinical applications of autologous ASCs in diabetic wound treatments. This work has particular surgical relevance as it highlights a theoretical and clinical pathway to use diabetic patients' own ASCs to treat their wounds, bypassing any concerns of cross-host sourcing issues in regenerative medicine.

Neuroprotective Effect of a Pharmaceutical Extract of Cannabis with High Content on CBD Against Rotenone in Primary Cerebellar Granule Cell Cultures and the Relevance of Formulations.

Introduction: Preclinical research supports the benefits of pharmaceutical cannabis-based extracts for treating different medical conditions (e.g., epilepsy); however, their neuroprotective potential has not been widely investigated. Materials and Methods: Using primary cultures of cerebellar granule cells, we evaluated the neuroprotective activity of Epifractan (EPI), a cannabis-based medicinal extract containing a high level of cannabidiol (CBD), components like terpenoids and flavonoids, trace levels of Δ9-tetrahydrocannabinol, and the acid form of CBD. We determined the ability of EPI to counteract the rotenone-induced neurotoxicity by analyzing cell viability and morphology of neurons and astrocytes by immunocytochemical assays. The effect of EPI was compared with XALEX, a plant-derived and highly purified CBD formulation (XAL), and pure CBD crystals (CBD). Results: The results revealed that EPI induced a significant reduction in the rotenone-induced neurotoxicity in a wide range of concentrations without causing neurotoxicity per se. EPI showed a similar effect to XAL suggesting that no additive or synergistic interactions between individual substances present in EPI occurred. In contrast, CBD did show a different profile to EPI and XAL because a neurotoxic effect per se was observed at higher concentrations assayed. Medium-chain triglyceride oil used in EPI formulation could explain this difference. Conclusion: Our data support a neuroprotective effect of EPI that may provide neuroprotection in different neurodegenerative processes. The results highlight the role of CBD as the active component of EPI but also support the need for an appropriate formulation to dilute pharmaceutical cannabis-based products that could be critical to avoid neurotoxicity at very high doses.

Myricetin possesses the potency against SARS-CoV-2 infection through blocking viral-entry facilitators and suppressing inflammation in rats and mice

Myricetin (3,5,7-trihydroxy-2-(3,4,5-tri hydroxyphenyl)-4-benzopyrone) is a common flavonol extracted from many natural plants and Chinese herb medicines and has been demonstrated to have multiple pharmacological activities, such as anti-microbial, anti-thrombotic, neuroprotective, and anti-inflammatory effects. Previously, myricetin was reported to target Mpro and 3CL-Pro-enzymatic activity to SARS-CoV-2. However, the protective value of myricetin on SARS-Cov-2 infection through viral-entry facilitators has not yet been comprehensively understood. The aim of the current study was to evaluate the pharmacological efficacy and the mechanisms of action of myricetin against SARS-CoV-2 infection both in vitro and in vivo. The inhibitory effects of myricetin on SARS-CoV-2 infection and replication were assessed on Vero E6 cells. Molecular docking analysis and bilayer interferometry (BLI) assays, immunocytochemistry (ICC), and pseudoviruses assays were performed to evaluate the roles of myricetin in the intermolecular interaction between the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and angiotensin-converting enzyme 2 (ACE2). The anti-inflammatory potency and mechanisms of myricetin were examined in THP1 macrophages in vitro, as well as in carrageenan-induced paw edema, delayed-type hypersensitivity (DTH) induced auricle edema, and LPS-induced acute lung injury (ALI) animal models. The results showed that myricetin was able to inhibit binding between the RBD of the SARS-CoV-2 S protein and ACE2 through molecular docking analysis and BLI assay, demonstrating its potential as a viral-entry facilitator blocker. Myricetin could also significantly inhibit SASR-CoV-2 infection and replication in Vero E6 cells (EC50 55.18μM), which was further validated with pseudoviruses containing the RBD (wild-type, N501Y, N439K, Y453F) and an S1 glycoprotein mutant (S-D614G). Moreover, myricetin exhibited a marked suppressive action on the receptor-interacting serine/threonine protein kinase 1 (RIPK1)-driven inflammation and NF-kappa B signaling in THP1 macrophages. In animal model studies, myricetin notably ameliorated carrageenan-induced paw edema in rats, DTH induced auricle edema in mice, and LPS-induced ALI in mice. Our findings showed that myricetin inhibited HCoV-229E and SARS-CoV-2 replication in vitro, blocked SARS-CoV-2 virus entry facilitators and relieved inflammation through the RIPK1/NF-κB pathway, suggesting that this flavonol has the potential to be developed as a therapeutic agent against COVID-19.

In vitro evaluation of spruce xylan/MWCNTs hydrogel scaffolds for bone regeneration

In this work, hydrogels from spruce xylan (SX) mixed with functionalized multi-walled carbon nanotubes (MWCNTs) as a scaffold were evaluated for in vitro culture of osteoblastic cells is reported. Chemical conjugation of spruce xylan with tyramine (TA) was used to enable enzymatic crosslinking and obtain hydrogels with fast gelation time. The conjugate was characterized by FT-IR and NMR spectroscopies. Morphological analysis of SX-TA/MWCNTs hydrogel scaffolds were performed with Scanning Electron Microscopy (SEM); wettability properties were measured by water contact angle (WCA). Osteoblastic cells were cultured and evaluated by LIVE/DEAD®, MTS and immunocytochemical assays. The morphology and adhesion of osteoblasts on the scaffolds was analyzed by SEM. The results obtained showed that SX-TA/MWCNTs hydrogel scaffolds have a high potential to be bionanomaterials for use in bone regeneration owed to the increased levels of cell viability, proliferation, and adhesion.

The Non-Canonical Iron-Responsive Element of IRE-tvcp12 Hairpin Structure at the 3'-UTR of Trichomonas vaginalis TvCP12 mRNA That Binds TvHSP70 and TvACTN-3 Can Regulate mRNA Stability and Amount of Protein.

Trichomonas vaginalis is one of the most common sexually transmitted parasites in humans. This protozoan has high iron requirements for growth, metabolism, and virulence. However, iron concentrations also differentially modulate T. vaginalis gene expression as in the genes encoding cysteine proteinases TvCP4 and TvCP12. Our goal was to identify the regulatory mechanism mediating the upregulation of tvcp12 under iron-restricted (IR) conditions. Here, we showed by RT-PCR, Western blot, and immunocytochemistry assays that IR conditions increase mRNA stability and amount of TvCP12. RNA electrophoretic mobility shift assay (REMSA), UV cross-linking, and competition assays demonstrated that a non-canonical iron-responsive element (IRE)-like structure at the 3'-untranslated region of the tvcp12 transcript (IRE-tvcp12) specifically binds to human iron regulatory proteins (IRPs) and to atypical RNA-binding cytoplasmic proteins from IR trichomonads, such as HSP70 and α-Actinin 3. These data were confirmed by REMSA supershift and Northwestern blot assays. Thus, our findings show that a positive gene expression regulation under IR conditions occurs at the posttranscriptional level possibly through RNA-protein interactions between atypical RNA-binding proteins and non-canonical IRE-like structures at the 3'-UTR of the transcript by a parallel mechanism to the mammalian IRE/IRP system that can be applied to other iron-regulated genes of T. vaginalis.

Increased soluble E‑cadherin of spheroid formation supplemented with fetal bovine serum in colorectal cancer cells.

Cancer stem cells (CSCs) are known to be a major cause of metastasis, resistance and recurrence. Spheroid formation is one of the methods used to recruit CSCs utilizing an anchorage-independent environment in vitro. It was aimed to investigate the availability of spheroid formation culture methods in the research field of CSCs and resistance using 5-fluorouracil (5-FU)-resistant colorectal cancer cells. The wild type SNU-C5 and 5-FU-resistant SNU-C5 (SNU-C5/5-FUR) cells were cultured as usual (monolayer), and in 3-dimensional non-adhesive environments supplemented with fetal bovine serum (FBS) or growth factors, respectively. The characteristics of the spheroids were evaluated by morphometry, cell viability assay, western blotting, immunocytochemistry and enzyme-linked immunosorbent assay. Spheroid formation was induced in an environment supplemented with FBS, while SNU-C5/5-FUR cells only formed spheres in media supplemented with GFs. Sphere-formed cells showed slower cell proliferation than cells from monolayer, which coincided with an increased level of p21 and a decreased level of β-catenin. Markers for CSCs and drug resistance were not significantly changed after spheroid formation. Sphere-formed cells showed significantly increased levels of soluble E-cadherin, particularly in the environment supplemented with FBS. These results suggested that spheroid formation may be related to soluble E-cadherin, but is not related to CSCs or resistance markers.

EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells.

Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.

Neurogenic marker expression in differentiating human adipose derived adult mesenchymal stem cells.

Adipose-derived stem cells (ADSCs) are increasingly utilised in the field of neural regeneration due to their high accessibility and capacity for differentiation into neural like cells. Culturing ADSCs in the presence of various growth factors, small molecules and combinations thereof have shown promise in this regard; however, these protocols are generally complex, time-consuming and costly. The need for commercially available and chemically defined growth media/supplements is required to facilitate further developments in this area. In this study, we have examined the neural differentiation and proliferation potential of the commercially available supplements B27, CultureOne (C1) and N2 on human ADSCs (hADSCs). Through a combination of immunocytochemistry, cytokine analysis, and CNPase enzymatic assays, we provide novel insight into the neural differentiation effects of B27, C1 and N2 on hADSCs. The study found that C1 and N2 supplements initiated neural differentiation of the cells, with C1 pushing differentiation towards an oligodendrocytic lineage and N2 initiating neuronal differentiation. This suggests that C1 and N2 supplements can be used to drive neural differentiation in hADSCs. However, B27 did not show significant differentiation in the time frame in which the experiments took place and therefore is unsuitable for this purpose. These findings highlight the utility of commercially available supplements in the neural differentiation of ADSCs and may assist in establishing simpler, more affordable differentiation protocols.

Resolvin D1 attenuates mechanical allodynia after burn injury: Involvement of spinal glia, p38 mitogen-activated protein kinase, and brain-derived neurotrophic factor/tropomyosin-related kinase B signaling.

Resolvin D1 (RvD1) suppresses inflammatory, postoperative, and neuropathic pain. The present study assessed the roles and mechanisms of RvD1 in mechanical allodynia after burn injury. A rat model of burn injury was established for analyses, and RvD1 was injected intraperitoneally. Pain behavior and the expression levels of spinal dorsal horn Iba-1 (microglia marker), GFAP (astrocyte marker), p-p38 mitogen-activated protein kinase (MAPK), brain-derived neurotrophic factor (BDNF), and tropomyosin-related kinase B (TrkB) were detected by behavioral and immunocytochemical assays. The results showed that RvD1 attenuated mechanical allodynia after burn injury, prevented microglial and astroglial activation, and downregulated p-p38 MAPK in microglia and BDNF/TrkB following burn injury. Similarly, inhibition of p38 MAPK and BDNF/TrkB signaling attenuated mechanical allodynia after burn injury. In addition, inhibition of p38 MAPK prevented spinal microglial activation and downregulated BDNF/TrkB following burn injury. Furthermore, inhibition of BDNF/TrkB signaling prevented spinal microglial activation and downregulated p-p38 MAPK within spinal microglia. Taken together, this study demonstrated that RvD1 might attenuate mechanical allodynia after burn injury by inhibiting spinal cord glial activation, microglial p38 MAPK, and BDNF/TrkB signaling in the spinal dorsal horn.

A conserved RNAi molecule Ago2 involved in antiviral immunity of oyster Crassostrea gigas

Argonaute (Ago) is the core component of RNA-induced silencing complex to play a crucial role in the antiviral immunity, which always cooperates with Dicer in RNA interference (RNAi) to silence the target genes. In the present study, an Ago homologue (CgAgo2) was identified in the Pacific oyster Crassostrea gigas. There were four classical functional domains in the predicted CgAgo2 protein, including an N-terminal domain, a PAZ domain, a Mid domain, and a PIWI domain. The deduced amino acid sequence of CgAgo2 shared 63.52%–84.27% identity with other Agos. Transcriptome analysis showed that CgAgo2 was highly expressed in embryonic period and gradually decreased from blastula to gastrula. The transcripts of CgAgo2 were detectable in all the examined tissues of adult oysters, with the highest expression in haemocytes (36.61-fold of that in adductor muscle, p < 0.001). The expression level of CgAgo2 mRNA in haemocytes increased significantly at 12 h after poly (I:C) and dsRNA stimulation, which were 2.71-fold (p < 0.05) and 58.00-fold (p < 0.001) of that in the control group respectively. Immunocytochemistry assay revealed that CgAgo2 proteins were mainly distributed in the cytoplasm and nucleus of haemocytes. The interaction between the recombinant CgAgo2 protein (rCgAgo2) and cleavage protein rCgDicer was observed in vitro by BLI and pull-down assays. These results indicated that CgAgo2 participated in the antiviral immunity of oyster by functioning as a component of RNA-induced silencing complex in RNAi.

MIR503HG silencing promotes endometrial stromal cell progression and metastasis and suppresses apoptosis in adenomyosis by activating the Wnt/β‑catenin pathway via targeting miR‑191.

MIR503HG is a 786 bp long lncRNA located on chromosome Xq26.3, and it can regulate diverse cellular processes. The pathogenesis of adenomyosis (AD) is associated with endometrial stromal cells (ESCs). The present study investigated the specific role of MIR503HG in AD pathogenesis and progression using ESCs derived from the endometrium of patients with AD as a model. Expression of MIR503HG and microRNA (miR)-191 were assessed using reverse transcription-quantitative PCR. An immunocytochemistry assay was used to detect cytokeratin- or vimentin-positive ESCs. Transfections of ESCs with MIR503HG overexpression plasmid, short hairpin-MIR503HG and miR-191 inhibitor were performed. ESC viability, migration, invasion and apoptosis were evaluated using Cell Counting Kit-8, Transwell and flow cytometry assays. The association between MIR503HG and miR-191 was predicted by StarBase and confirmed using a dual-luciferase reporter assay. Expression of epithelial-mesenchymal transition-related markers (E-cadherin and N-cadherin) and Wnt/β-catenin pathway-related molecules (β-catenin) in ESCs were analyzed by western blotting. The isolated ESCs were vimentin-positive and cytokeratin-negative. MIR503HG was lowly expressed in the endometrial tissues derived from patients with AD. MIR503HG overexpression hindered ESC viability, migration and invasion while enhancing the apoptosis and downregulating miR-191 expression. MIR503HG knockdown induced the opposite effects, accompanied by downregulation of the E-cadherin expression and upregulation of N-cadherin and β-catenin levels. MIR503HG directly targeted miR-191 that was highly expressed in endometrial tissues derived from patients with AD. In ESCs, downregulation of miR-191 inhibited the viability, migration and invasion and the expression of N-cadherin and β-catenin levels while enhancing the apoptosis and E-cadherin expression in ESCs. Moreover, downregulation of miR-191 partially reversed the effect of MIR503HG knockdown. Collectively, overexpressed MIR503HG impeded the proliferation and migration of ESCs derived from endometrium of patients with AD, while promoting apoptosis via inhibition of the Wnt/β-catenin pathway via targeting miR-191.

Involvement of microRNA miR-125b in the control of porcine ovarian cell functions

The existing knowledge of the involvement of miR-125b in the control of ovarian functions is insufficient. To evaluate the role of miR-125b in the control of basic porcine ovarian granulosa cell functions, we examined the upregulation (using miR-125b mimics) and downregulation (using miR-125b inhibitor) of this miR-125b. Expression levels of miR-125b, viability, proliferation (expression and accumulation of PCNA and cyclin B1), the proportion of proliferative active cells, apoptosis (expression and accumulation of bax and caspase 3), the proportion of cells containing DNA fragmentation, steroid hormones, IGF-I, oxytocin, and prostaglandin E2 release were analysed by RT-qPCR, Trypan blue exclusion test, quantitative immunocytochemistry, XTT and TUNEL assays, and ELISA. Transfection of cells with miR-125b mimics decreased cell viability, proliferation, and the release of progesterone, testosterone, estradiol, and oxytocin, but stimulated apoptosis and prostaglandin E2 output. Transfection of cells with miR-125b inhibitor had the opposite effect. Moreover, it prevented the effects of miR-125b mimics. Our observations suggest that miR-125b is a potent physiological inhibitor of granulosa ovarian cell functions – cell cycle, apoptosis, and secretory activity.

Abstract PR018: Neuropeptide Y as a metastatic factor

Abstract Although neuropeptide Y (NPY) is known mainly as a sympathetic neurotransmitter, growing evidence indicates its role in tumor biology. Previous studies from our laboratory and other groups suggested associations of high expression of NPY and its Y5 receptor (Y5R) with invasive and metastatic phenotypes of various tumor types; yet the direct evidence for the metastatic activity was lacking. Our recent work began to fill this gap. Using Ewing sarcoma (ES) as a model of NPY-rich tumor, we identified the NPY/Y5R axis as a crucial pathway responsible for hypoxia-induced ES bone metastasis. This osseous dissemination was driven by hypoxia-dependent over-activation of the NPY/Y5R/RhoA pathway, which led to cytokinesis defects and formation of the aneuploid ES cell population with high propensity to bone metastasis. Here, we sought to determine the impact of the Y5R signaling on overall, hypoxia-independent metastatic capabilities of ES cells. To this end, a doxycycline-inducible CRIPSR/Cas9 system was used to knockout Y5R in ES orthotopic xenografts and test its impact on metastasis and cell migration assessed by a transwell assay. The frequency of the NPY5R gene modifications was determined by sequencing and confirmed by immunohistochemistry. FISH was used to identify NPY5R gene gains in ES xenografts. RhoA activity was measured by immunocytochemistry and pull-down assay. As expected, ES/Y5R-sgRNA primary tumors in doxycycline-treated mice consisted of a heterogeneous ES cell population with variable Y5R levels. In contrast, metastases developing from these xenografts were primarily initiated by ES clones with an intact NPY5R gene. The frequency of NPY5R gene modifications in metastases from the doxycycline-treated group was markedly reduced, as compared to the primary tumors. No metastases arising from ES clones lacking intact NPY5R gene were detected. Similarly, metastasis from wild type ES xenografts was associated with a selection of clones with the NPY5R gene gain that was present in a small percent of the parental cells. Subsequent in vitro assays implicated Y5R-dependent ES cell motility driven by the activation of a key cytoskeleton regulator, RhoA, as the mechanism underlying the metastatic effects of NPY. The above results, along with our previously published data, provide direct evidence for the crucial role of the NPY/Y5R axis in metastasis. We have shown that under basal conditions the NPY/Y5R/RhoA axis promotes tumor cell motility and overall cancer cell dissemination, while over-activation of this pathway under hypoxic conditions leads to genomic instability and bone metastasis. While our study focused on ES due to its high endogenous expression of NPY and its receptors, our findings may be relevant to other malignancies rich in NPY and Y5R, such as neuroblastoma, breast and prostate cancer. Given availability of the FDA approved Y5R antagonist, NPY/Y5R pathway may be a promising target for therapies preventing cancer progression. Yet, further research is required to establish effectiveness of such pharmacological interventions. Citation Format: Mina Adnani, Sung-Hyeok Hong, Susana Galli, Akanksha Mahajan, Nouran Abualsaud, Lindsay Caprio, Jason U. Tilan, Olga Rodriguez, Hingkun Wang, Christopher Albanese, Luciane R. Cavalli, Joanna Kitlinska. Neuropeptide Y as a metastatic factor [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR018.

A Novel Hemocyte-Specific Small Protein Participates in White Spot Syndrome Virus Infection via Binding to Viral Envelope Protein.

Hemocytes are essential components of the immune system against invading pathogens in shrimp. Many uncharacterized transcripts exist in hemocytes but the knowledge of them is very limited. In the present study, we identified a novel small protein from the uncharacterized transcripts in hemocytes of Litopenaeus vannamei. This transcript was specifically expressed in hemocytes and encoded a novel secretory protein, which was designated as hemocyte-specific small protein (LvHSSP). The expression level of LvHSSP was significantly up-regulated in the hemocytes of shrimp infected with white spot syndrome virus (WSSV). After knockdown of LvHSSP by RNA interference, the WSSV copy number in shrimp decreased significantly. Conversely, WSSV copy number increased in shrimp when they were infected by WSSV after incubation with recombinant LvHSSP protein. These results suggested that LvHSSP might promote viral infection in shrimp. Immunocytochemical assay showed that the recombinant LvHSSP protein was located on the membrane of hemocytes. Co-IP results showed that LvHSSP could interact with VP26, the main envelope protein of WSSV, suggesting that LvHSSP might mediate WSSV adhesion and entry into host cells by binding to viral envelope protein. Meanwhile, the total hemocyte counts were significantly decreased after LvHSSP knockdown while increased after supplementing with recombinant LvHSSP protein, supporting the idea of hemocytes as the carrier for systemic dissemination of WSSV. This study reported a novel small protein in hemocytes, which modulated the viral infection in shrimp. Our results will enrich the knowledge of invertebrate innate immunity and provide a new field in the study of hemocyte function.