Calidris alpina, trapped during migration at a stopover site on the southern Baltic Sea shore. Males were consistently in a slightly worse condition than females, likely due to male-biased parental investment in brood rearing. Annual productivity indices were positively correlated with the respective condition indices of breeders from the Eurasian Arctic, indicating that in ‘good’ years, despite great effort spent on reproduction, breeders leave the breeding grounds in better condition. The pattern did not hold for 1992: productivity was low, but the average condition of adults during migration was the highest noted over the decade. We suggest that the delayed effect of the Mount Pinatubo eruption in the Philippines in 1991, could be responsible for the unexpected high condition of Arctic breeders in 1992. High population-level average condition, coupled with the low productivity could stem from severe weather caused by the volcano eruption a year before and strong predation pressure, which in turn lead to a reduced investment in reproduction. The importance of large-scale episodic phenomena, like this volcano eruption, may blur the statistical associations of wildlife with local environmental drivers.Weather and predation constitute the two main factors affecting the breeding success of those Arctic waders whose productivity is highly variable over the years. We tested whether reproductive success is associated with the post-breeding condition of adults, in which in ‘good’ years  the condition of breeders and their productivity is high. To verify this hypothesis, we used a 10-year dataset comprising 20,792 dunlins  Reproduction constitutes one of the biggest energetic efforts in birds’ annual cycle . ArrivalCalidris alpina as an example.The body condition of birds during migration, at their arrival at the breeding grounds, during egg-laying and chick rearing periods has been well studied , 17, 18.Despite both sexes performing brood care in dunlin, there is a male-biased effort in chick rearing. Females are fully involved only at the incubation stage  andMost Calidrid species do not attempt breeding when one year old (their second year of life) and they generally stay at the wintering grounds or nearby. The dunlin represents an exception: birds in their second year of life (hereafter ‘immatures’) undertake northward migration and attempt to breed , 25, 26.alpina, centralis and schinzii [alpina occupies the western Eurasian Arctic, while centralis breeds in the more eastern part of the range. The schinzii subspecies’ breeding range is restricted to lower latitudes, mostly around the Baltic Sea [Three out of nine recognized dunlin subspecies  occur atschinzii , 34, 35.ltic Sea . Migratiltic Sea . TherefoThe proportion of first-year birds recorded during migration or at the winter quarters represents an approximate measure of breeding success in a given year , 36. In In the present work, we investigate this relationship in the dunlin, a long-distance migrant, wader species breeding in the high Arctic. We used a 10-year dataset of dunlins, trapped at the stopover site during their southward migration, to test if the condition of breeders correlates with productivity. Additionally, we attempt to identify factors responsible for the variation in condition, by answering the following questions: (i) do males have a poorer post-breeding condition, due to their higher investment in raising broods; (ii) are immatures in worse condition compared to older birds due to being unexperienced; and (iii) is there any effect of origin–i.e., do ‘western’ and ‘eastern’ birds differ in condition, in line with the distance passed during migration.th July and 25th September (exact dates of the beginning and ending of trapping differed by 1–3 days). Birds were removed from the traps by professional ornithologists and volunteers at two-hour intervals each day (from 05:00 to 23:00 CET) and transported to the ringing station located outside the feeding areas, at a distance of up to some hundred meters from the trapping area. Each bird was measured, weighted, ringed and released immediately after processing. A set of measurements were taken, including body mass (to the nearest 1 g), wing length , tarsus length  and bill length . All these measurements showed some sexual dimorphism [We used data of migrant dunlins trapped at the stopover site in the Vistula Mouth, Poland  between 1990 and 2000. The year 1997 was excluded because a flood during the migration peak in July resulted in most birds being missed, as trapping had to cease. Birds were stopping over to feed on flat, sandy beaches and annually trapped with walk-in traps between the 12morphism , but we morphism , followimorphism , see alsmorphism ). Immatumorphism , 49, 50.morphism , 49, 51–morphism . Juvenilmorphism , 53. In morphism ) and at morphism , PacificBecause body mass is size-dependent, we used residual body mass from regression of body mass on bill length as an index of body condition at the individual level. This index is commonly used in waders , 58, whit-statistics, P-values and predictions (in line with [We used additive linear mixed effects models (GAMMs) with normal error distribution for their flexibility in capturing non-linear patterns via smoothers and where the optimal amount of smoothing is obtained via cross-validation , 60. Theine with ).We first fitted the global model with age, sex, origin and their interactions as fixed factors and the two smoothers (one estimated for ‘western’ and another one for ‘eastern’ birds). After fitting the global model, using the mgcv R-Package , we procedf, between 6.31 and 6.33, Across the entire dataset, in non-juveniles, the mean yearly body condition varied significantly over the years , with thThe pattern across years was similar in both ‘western’ and ‘eastern’ birds, with the best condition in 1992–1993 and the poorest in 1996 . Males hYearly productivity indices varied strongly, from 0.12 in 1996 to 0.75 in 1993 . Estimat1992 was the outlying year , with the best condition (estimated group means between 1.31 and 2.12) and the second-lowest productivity (0.20) over the decade, implying that the pattern observed for the whole period does not hold .There is a great linkage between all stages of the annual cycle, especially in migratory animals. Recent ecological studies recognize this phenomenon as carry-over effects: events that occur in one season (or stage), influence fitness at both the individual and population level in the following season (or stage) , 67. ThiOur results confirm the assumption that male-biased effort in chick rearing period affects the males’ body condition in dunlin. The differences noted were not large, but consistent and highly significant. In all years, males were in poorer condition than females, irrespective of age and origin. Despite the fact that females incur the undoubtedly high costs of egg laying, males apparently face higher energy expenditures resulting from nest and chicks’ defence after female desertion.We found it surprising that there was little support for the age effect. Immature dunlins undertake breeding in their second year of life , 25, 26 alpina breeding range  annually migrate through the region in lower numbers than their western counterparts [centralis breeding range remains unclear [alpina/centralis contact zone [centralis at the southern Baltic during southward migration is likely [alpina range or may represent westernmost centralis. ‘Western’ dunlins are either alpina or–to a minor extent, given its small and declining population—schinzii. The difference in body condition observed between ‘eastern’ and ‘western’ birds may thus be a consequence of the difference in the distance covered between their breeding and wintering grounds. Our results indicate that ‘eastern’ birds had worse body condition than their ‘western’ counterparts. Dunlins breeding further east cover longer distances during their southward migration [Dunlin subspecies overlap in morphological characters, which makes it difficult to determine the origin of migrant birds observed at stopover sites and wintering grounds , 75. Theterparts , 35, 77. unclear , 79 and act zone , but stis likely , 34, 35.s likely . Therefos likely , 79. It The results indicate that there is a strong positive relationship between the condition of dunlins during the post-breeding migration time and productivity in a given year. In years with higher productivity, the condition of breeders was also higher, with the exception of 1992.Our findings suggest that in spite of the high energetic costs of reproduction, high breeding output may be achieved without significant reduction of adults’ body condition in favourable years. It has been well documented in waders that production of juveniles is strongly influenced by feeding conditions during the pre-fledging period  and summA potential caveat that could limit our conclusions include the fact that some juveniles might migrate through the Baltic after the trapping period at our stopover site  and thusOur dataset included the year 1992, when an aerosol cloud originating from the 1991 eruption of Mount Pinatubo in the Phillipines led to severe weather conditions and heavily affected avian reproduction over most of the Arctic . In consAlopex lagopus) in low rodent years and wader productivity in a given year is associated with the phase of the rodent cycle [Microtus sp.) in 1992 and the high predation rate on birds’ clutches additionally affected Arctic-nesting waterfowl and the breeding success of waders (including dunlin) to a large degree (estimates of breeding success ranged from 0% to 10%) [Arenaria interpres, grey phalaropes Phalaropus fulicarius and white-rumped sandpipers Calidris fuscicollis) in 1992. From the dunlin capture data at our study site (1990–2000) we conclude that southward migration in 1992 was on the same dates as in remaining years, indicating no earlier migration to winter quarters. Gunnarsson et al. [Limosa limosa islandica and whimbrel Numenius phaeopus productivity was reduced to almost zero after two volcanoes erupted in 2010 and 2011 in southern Iceland, but during the subsequent years productivity substantially increased. These findings suggest that unpredictable phenomena, which occur rarely, may significantly affect the performance of animal populations even at large distances–as in the case of the 1991 eruption. If missed, some patterns that hold may appear ‘insignificant’ or easily go undetected.The unusually good condition coupled with low productivity in 1992, clearly outstanding from the overall pattern, could be caused by the cumulative effects of multiple factors. Low numbers of juvenile dunlins were also reported at other stopover sites in Europe, both during the autumn migration in 1992 and the spring migration in 1993 . Probablnt cycle , 86, 87. to 10%) . These e to 10%) , 85 but  to 10%) , who repn et al.  also repS1 Fig(TIF)Click here for additional data file.S2 FigThe outlying year 1992 is marked with red. Symbols represent mean productivity and mean condition index for one year, error bars– 95% confidence intervals for both productivity and condition indices. Regression lines  are drawn to illustrate relationship for nine years, after excluding 1992.(TIF)Click here for additional data file.S1 File(TXT)Click here for additional data file.


Limb amputation in axolotls was performed to obtain data demonstrating that a chemical agonist of Wnt (int-related protein)/β-catenin signalling can have a role in axolotl limb regeneration   Value of Data•The data showed that Wnt/β-catenin signalling was active throughout the limb regeneration process.•It showed that the Wnt agonist activates Wnt/β-catenin signalling.•This data can be used to determine that Wnt agonist has effects during axolotl limb regeneration.•It can be used to determine that Wnt agonist is able to activate β-catenin signalling in Leydig cells.•This data can be used to determine that Wnt agonist is able to induce limb malformations during limb regeneration.1Data in Data in Data in Data in 22.1Ambystoma mexicanum). One forelimb was amputated at mid-zeugopod after they were anesthetised with 0.05% Tricaine . The animals were placed in the solution with the Wnt agonist  on different days after amputation. The vehicle, dimethyl sulfoxide (DMSO) was used as control. Limbs were amputated at the base of the arm and collected at the end of the experiment.All experiments were performed on 5–6.5 cm axolotls  and sectioned into 7 µm for tissue staining. They were stained with the Masson Trichrome technique. For skeleton staining, limbs were fixed in 95% ethanol and permeabilized overnight with acetone. Later, collected limbs were stained in an Alcian blue/Alizarin red solution for three days and were cleared in 1% KOH/20% glycerol and stored in 50% ethanol/50% glycerol.2.3Active-β-catenin  and 1:500 anti-rabbit Alexa 488  was evaluated. Fluorescent immunostainings were observed under laser confocal system . Anti-β-catenin immunohistochemistry was performed in cryosections of 40 µm.2.4c-myc forward primer 5′-TGACCCTTCAGTGGTCTTCC-3′c-myc reverse primer 5′-CGCCTCTTGTCGTTCTCTTC-3′axin2 forward primer 5′-GAGTCTGACGCTTGGACACT-3′axin2 reverse primer 5′-AGAAACTCGGTGAGTGGCATT-3′RNA expression detected RT-PCR using RNA collected from eight 12-day post-amputated axolotl limb skins, from animals treated either with the Wnt agonist or with DMSO. We used the following primers:ef1α primers and PCR conditions were obtained from The A luciferase assay and RKO (colon cancer cell line) cell culture were performed to determine the efficiency of the drug to activate the Wnt/β-catenin signalling as described previously


It is challenging to reinforce and toughen thermoset epoxy resins. We describe a slurry-compounding technique to transfer a uniform dispersion of chitin nanowhiskers (CW) in ethanol into an epoxy matrix. The incorporation of the hydrophilic CW reinforces the oil-soluble diglycidyl ether of bisphenol A (DGEBA). The resultant CW/epoxy bionanocomposites were transparent and showed considerably enhanced thermal and mechanical properties with tensile strength, modulus, toughness, and elongation at break being increased by 49%, 16%, 457%, and 250%, with only 2.5 wt.% CW. This improvement in strength and toughness is rare for thermoset epoxy/rigid nanofiller systems. We hypothesize that CW with many free amine groups could function not only as a nanofiller but also as a macromolecular polyamine hardener that participates in epoxy curing. The strong covalent interaction between the filler and the matrix allowed for efficient load transfer across the interfaces, which accounted for the greater strength and toughness. Because of increased attention to ecological safety and green/renewable materials, natural fibers as bio-fillers/reinforcement materials for composites are utilized widely in industry and academia –3. ChitiPrior to the addition of CW into polymer matrices, raw CW need to be separated into uniform CW bundles with diameters as small as possible . EffectiToughness is the ability of any material to deform plastically and to absorb energy during fracturing . To tougThe polymer matrix used is epoxy DER 332 (Dow Plastics), diglycidyl ether of bisphenol A (DGEBA), with an epoxide equivalent weight of 171–175 g/equiv, and density of 1.16g/mL. Chitin from shrimp shell in powder was obtained from Sigma-Aldrich in practical grade. The di-amine curing agent is Ethacure LC-100 from Albemarle Corp., which is a mixture of two diethyltoluenediamine (DETDA) isomers . All other chemical agents and solvents were purchased from Sigma-Aldrich and used as received.CW was synthesized following the procedures reported previously with some modification . The rawCW/epoxy bionanocomposites were prepared via slurry compounding. For example, to prepare epoxy nanocomposite with 1 wt% CW, 1g of CW in 1ml ethanol suspension was added into 78.4 g of DGEBA in a beaker and the mixture was heated at 60°C under stirring until no bubble came out. During the mixing process, the CW/DGEBA mixture turned from a little turbid to white transparent, indicating CW being well mixed with epoxy. Then 20.6 g amine hardener 100-LC  was added into the CW/DGEBA and homogenized at 2000 rpm for 30 min. Last, the three component CW/DGEBA/LC-100 mixture was degassed under vacuum at 80°C for 1h and cured at 100°C for 2h and at 200°C for 5h. The curing condition was carefully determined with the help of rheometer to make sure the completion of the process. This slurry compounding method is efficient and cost-effective in terms of time and amounts of solvents required for the preparation and thus eco-friendly and economically. Three CW/epoxy nanocomposites were prepared and denoted as CW/Epoxy1, CW/Epoxy2 and CW/Epoxy3 at CW loadings of 0.5, 1, 2.5 wt%.-1. Before FTIR testing, CW was washed with ethanol and water three times each, then dried at 60°C for 24 h. CW/epoxy nanocomposites were evaluated in ATR-FTIR mode.FTIR spectra were acquired with a Perkin-Elmer 2000 spectrometer operated in the absorbance mode over a scan range of 4000 to 400 cmXRD patterns were obtained using a Bruker X-ray diffractometer  in reflection mode. Test was carried out using nickel filtered Cu Kα radiation (λ = 0.15418 nm) under a voltage of 40 kV and a current of 40 mA.The fracture surface of the CW/epoxy bionanocomposite samples after tensile test was observed with a field emission-scanning electron microscopy (Agilent 8500 FE-SEM) without metal sputter coating.High resolution TEM micrograph was taken with a Philips CM300 at 300 kV. A drop of CW/Ethanol solution was deposited on a 200 mesh copper grid and vacuum dried before observation.Thermal stability of CW/epoxy nanocomposites was evaluated with a thermo-gravimetric analyzer (TA instrument Q500) at a heating rate of 10°C/min from room temperature to 800°C under air. The degradation temperatures of the nanocomposites were determined as the minimum point of the derivative weight versus temperature.Tg) was assigned as the maximum of the tan δ.Dynamic mechanical properties were examined with a TA Instruments DMA 2980 operated in the single cantilever bending mode at an oscillation frequency of 1.0 Hz and a scanning rate of 3°C/min. The samples for DMA tests were cut into rectangular bars having dimensions (L×W×T) of 35 mm × 5 mm × 3 mm. A minimum of 3 specimens of each composition were tested. The glass transition temperature (Tensile properties were determined using the Instron 5569 testing machine according to the ASTM standard D638-03 at a speed of 1mm/min at room temperature. The specimens were cut into dog-bone shape with dimensions (L×W×T) of 55 mm× 10 mm × 3 mm. A minimum of 8 specimens of each composition were tested, and the mean vale and standard deviation were calculated.Data were expressed as mean ± standard deviation (SD). Student t test or two-way analysis of variance (ANOVA) was employed to analyze the difference between groups. A p-value < 0.05 was considered statistically significant. *p < 0.05 versus control group.Inspired by a study on clay/epoxy nanocomposites , a slurrA schematic illustration of the slurry-compounding process is shown in Clearly, the dispersion and maximum loading of CW depends on several parameters, such as the compatibility of the CW and the epoxy, the solvent polarity, and the intensity of mixing, the mixing entropy of all the curing components and the kinetics of the cross-linking reaction, which need to be investigated with more sophisticated methods and instruments. In this study, the slurry-compounding technique uses only water and ethanol to fabricate oil-soluble DEGBA bionanocomposites based on CW. This is a green approach and one of the novelties of this study in terms of cost and the properties of the final product.110 is the maximum intensity of the (110) lattice diffraction and Iam is the intensity of amorphous diffraction in the same units at 2θ = 16. Thus, the crystalline degree of α-CW prepared in this study is ca. 91%.Acid hydrolysis of raw chitin produces α-chitin nanocrystals or nanowhiskers. In -1, belonging to OH and NH of primary amine, respectively. The absorption peak at 1665 cm-1 is commonly assigned to the stretching of the C = O group hydrogen bonded to N-H of the adjacent intra-sheet chain. The absorption peak at 1631 cm-1 is attributed to a specific hydrogen bond of C = O in the hydroxyl methyl group of the next chitin residue [-1 originates from the amide II band for chitin [DA) was calculated from:1655 and A3455 are the absorbance ratios at 1665 cm-1 and 3455 cm-1, respectively, proposed by Baxter et al. [In  residue . The absr chitin . A compar et al. , and was-1, epoxy rings usually show absorptions at 904, 833, and 744 cm-1[-1[-1 belonging to bonded primary amine of CW [-1 [-1 and its increased intensity with the addition of CW [ 744 cm-1, the int4 cm-1[-1, as showne of CW  disappeaf CW [-1 , which cf CW [-1 . Other ton of CW . Then itE’) and tan δ for the neat epoxy resin and CW bionanocomposites. At room temperature, the E’ of the epoxy increases with CW content, indicating more rigid nanocomposites formed upon the incorporation of CW. The peak position of tan δ corresponds to the glass transition temperature (Tg). Clearly, the Tg of neat epoxy resin increases with increasing CW content, due to both the steric hindrance of the filler to the epoxy molecules and the interactions between them. The symmetric shape of tan δ is indicative of complete curing of neat epoxy and all CW/epoxy nanocomposites. In the temperature range of 100–170°C, a rubbery plateau region of E’ exists for all samples, implying a tough network structure in all epoxy nanocomposites. On the other hand, the heterogeneity and cross-linking density of the network could be qualitatively assessed with a peak factor, which is defined as the full width at half maximum of the tan δ peak divided by its height [s height . ObviousE), the yield strength (σy), the tensile strength at maximum elongation (σmax), the strain at break (εb), and the toughness; representative tensile testing curves are shown in In this study, acid-hydrolyzed chitin with an appropriate number of amine groups in epoxy functions not only as a strengthening filler but also as a polyamine hardener, as evidenced by FITR measurements. Most studies on CW/polymer nanocomposite systems have shown that the addition of the high crystalline CW led to stiffer materials , 34, butSimultaneously reinforcing and toughening thermoset epoxy resins are challenging due to the brittleness of the epoxy resins. CW-reinforced thermoset epoxy was prepared by a modified slurry-compounding method, which used low amounts of solvents and demonstrated the feasibility and cost-saving nature of this method. The epoxy/CW nanocomposites showed superior thermal and mechanical properties compared with neat epoxy. The mechanism underlying this substantial enhancement in thermo- and mechanical properties is that CWs with free amine functionality could participate in epoxy curing as a macromolecular hardener. The resulting covalent network structure formed between CW and epoxy could enhance load transfer across the matrix/filler interface. This strategy for the choice of the filler and its modification may inspire material scientists to design versatile hardeners to prepare epoxy composites with desired properties.


Multiple myeloma is the second most frequent hematological malignancy in the western world and remains incurable, predominantly due to acquired drug resistance and disease relapse. The highly conserved Wnt signal transduction pathway, which plays a key role in regulating cellular processes of proliferation, differentiation, migration, and stem cell self-renewal, is associated with multiple aspects of disease. Bone homeostasis is severely disturbed by Wnt antagonists that are secreted by the malignant plasma cells in the bone marrow. In the vast majority of patients, this results in osteolytic bone disease, which is associated with bone pain and pathological fractures and was reported to facilitate disease progression. More recently, cumulative evidence also indicates the importance of intrinsic Wnt signaling in the survival of multiple myeloma cells. However, Wnt pathway-activating gene mutations could not be identified. The search for factors or processes responsible for Wnt pathway activation currently focuses on aberrant ligand levels in the bone marrow microenvironment, increased expression of Wnt transcriptional co-factors and associated micro-RNAs, and disturbed epigenetics and post-translational modification processes. Furthermore, Wnt pathway activation is associated with acquired cell adhesion-mediated resistance of multiple myeloma cells to conventional drug therapies, including doxorubicin and lenalidomide. In this review, we present an overview of the relevance of Wnt signaling in multiple myeloma and highlight the Wnt pathway as a potential therapeutic target for this disease. Multiple myeloma (MM) is a neoplastic disorder that is characterized by the infiltration and clonal proliferation of antibody-secreting post-germinal center plasma cells (PCs) in the bone marrow (BM). It is the second most frequent hematological malignancy, accounting for approximately 10% of all hematological cancers, and particularly affects the elderly with a median age at diagnosis of 69 years . MM is tThe Wnt signal transduction pathway is a highly evolutionary conserved and pleiotropic cell-to-cell communication route that regulates cellular processes including proliferation, differentiation, and fate determination. During embryogenesis, it functions as a concentration-dependent and long-range morphogen in establishing body axis formation . In matuThe relevance of Wnt signaling in MM was first acknowledged for its regulating role in bone homeostasis. Wnt signaling tightly controls the balance between bone-forming osteoblasts and bone-resorbing osteoclasts, both by direct and indirect mechanisms . MalignaIn recent years, more emphasis was put on the biological significance of active Wnt signaling intrinsic in MM malignant PCs. Human multiple myeloma cell lines (HMCLs) were found to express multiple Wnt receptors, co-receptors, and downstream pathway mediators that stabilized in response to ligand-mediated pathway activation . FurtherIn addition to canonical β-catenin-mediated transcriptional effects, Wnt signaling can also affect cell morphology, migration, and adhesion in a more direct way via the non-canonical Wnt/planar cell polarity (PCP) pathway. By acting on the small GTPases that regulate cytoskeleton formation, Wnt signaling can increase adhesion of malignant PCs to BM stromal cells and thereby enhance drug resistance . The acqHere, we present a complete overview of the relevance of Wnt signaling in MM. We summarize the role of Wnt signaling in MM bone disease, discuss the relevant studies performed in the context of intrinsic Wnt signaling in MM pathogenesis, both focusing on the biological significance as well as mechanisms responsible for signaling activation, and highlight the role of Wnt signaling in drug resistance and its potential as a therapeutic target in the treatment of MM.Xenopus laevis XWnt8 [The Wnt family currently consists of 19 secreted lipid-modified glycoproteins that can signal through one or more of the Wnt signal transduction pathways and can do so in a paracrine or autocrine fashion. They exhibit unique expression patterns and developmental functions, but were previously classified based on their ability to induce transformation of the epithelial cell line C57MG. Highly transforming members are thought to signal mainly through the β-catenin-dependent canonical Wnt pathway and include Wnt1, Wnt2, Wnt3, and Wnt3a, while the non-transforming members Wnt4, Wnt5a, Wnt5b, and Wnt7b were reported to have no effects on transformation and are likely to signal towards the non-canonical Wnt pathways. Several additional Wnts including Wnt6 and Wnt7a do not belong to these categories and are classified as intermediate transforming members, resulting in weak morphological changes . There iis XWnt8 . Human Wis XWnt8 . In the is XWnt8 . Additiois XWnt8 . Since Wis XWnt8 .CCND1 (encoding cyclinD1) and MYC and the survival molecule BIRC5, which translates into the inhibitor-of-apoptosis family member survivin. Survivin is overexpressed in a subset of MM patients and is correlated with multidrug resistance [AXIN2, a structural and functional homolog of Axin1, is transcribed upon β-catenin-LEF/TCF mediated transcription and thereby initiates a negative feedback loop upon canonical Wnt pathway activation [The canonical Wnt signaling pathway all revolves around the transcriptional co-activator β-catenin. When the Wnt pathway is inactive, continuously synthesized β-catenin is eliminated by a cytosolic destruction complex consisting of the scaffold proteins APC and Axin1 and the kinases GSK3 and CK1 Fig. . This desistance . Also, Ativation .Fig. 1Sc2+ pathway, similar recruitment of Dvl results in activation of heterotrimeric G-proteins, which ultimately induce a calcium flux into the cytosol  of Wnts present, as well as the receptor and co-receptor it engages with. Following the classification of Wnt ligands also Fzd receptors were divided into separate groups based on their ability to activate either canonical or non-canonical Wnt pathways upon overexpression . However studies . Many ad studies . Express studies . Further studies . In addi studies . The bes studies . All theMM cells preferentially home to the BM as their host organ, which is a very active site of Wnt signaling. The BM is a soft, well vascularized and highly spatially organized tissue that harbors a diverse cellular content consisting of bone-forming osteoblasts and bone-resorbing osteoclasts, BM adipocytes, endothelial cells, BM stromal cells, mesenchymal stem cells (MSC), and HSC . The latMM cells interrupt Wnt signaling in the BM and thereby severely disturb bone homeostasis. Essentially, bone metabolism is controlled by three cell types: osteoblasts, osteocytes, and osteoclasts. Osteoblasts are derived from pluripotent MSCs, which also produce fibroblasts, chondrocytes, myoblasts, and adipocytes. Wnt signaling has a major impact on this differentiation process by directing MSC differentiation away from the chrondrocytic and adipocytic lineages, towards differentiation into osteoblasts . AdditioOsteocytes comprise approximately 95% of all cells present in the bone matrix and are the only cell type identified in bone that possess a significantly active intrinsic canonical Wnt signaling pathway . Within Osteoclasts are the natural counterpart of osteoblasts and facilitate the process of bone remodeling by resorbing bone tissue. They do so by secreting lytic enzymes that degrade the bone matrix . OsteoclIn healthy human adults, bone remodeling is executed to replenish old bone, strengthen bone tissue in response to physical stress, or at sites of injury. Osteoclasts resorb the compromised bone tissue, after which adjacent cells, and presumably also tissue specific macrophages called osteomacs, clean the affected surface. The osteoclasts then recruit and stimulate osteoblasts to form new bone tissue in order to fill the created space. To balance bone resorption with equal bone formation, osteoblast and osteoclast activity is matched, which is referred to as coupling , AXIN1, or the tumor suppressor APC, as is normally observed in canonical Wnt-driven tumors like colon cancer [With previous studies taken into account, one could state that canonical Wnt signaling is of importance during many of the major proliferative stages in the development and activation of B cells: HSC self-renewal, population expansion of developing B lymphocytes, and PC generation upon antigenic stimulation of mature B cells. From this perspective, (re)activation of the canonical Wnt pathway in MM cells, which evidently have an active cell cycle, seems intuitive. In addition, aberrant Wnt pathway activation is very often implicated in cancer. This accounts for solid tumors, as well as different types of leukemia , 19. Indn cancer .BCL9 gene, which is located on the frequently amplified 1q chromosomal region, was first identified in B cell acute lymphoblastic leukemia as a result of the t translocation [First studies indicated a role for BCL9 in the activation of intrinsic Wnt signaling in MM. The location . The nuclocation . Mani etlocation . A follolocation .Several studies also suggested a role for dysregulated post-translational modification of Wnt pathway mediators in aberrant intrinsic Wnt pathway activation in MM cells. In 2015, Huang et al. reported that β-catenin is subject to sumoylation . PreviouAberrant epigenetics were suggested to be responsible for intrinsic Wnt pathway activation in MM cells by Chim et al., who analyzed DNA methylation of CpG islands, indicative of transcriptional repression and subsequent gene silencing, in the promoter regions of the Wnt antagonists WIF1, Dkk3, APC and sFRP1, sFRP2, sFRP4, and sFRP5. Of the 50 MM patients enrolled in this study, 42% showed abnormal methylation of at least one of these genes. Within this group, more than two thirds of the patients even showed methylation of two or more inhibitors. This is in contrast to healthy PC control samples, in which no methylation of any of these genes was detected .Very recently, van Andel et al. proposed two additional mechanisms for Wnt/β-catenin activation in MM cells. The first hypothesis concerns the loss of the tumor suppressor deubiquitinating enzyme CYLD, which is a known negative regulator of NFκB, TGFβ, and Notch signaling –105. A pBesides the controversial mechanism that is responsible for activation of the canonical Wnt pathway in MM cells, there are some additional observations that remain unclear. Although Derksen et al. showed that MM PCs have an aberrantly active Wnt pathway due to β-catenin overexpression, signal transduction in response to Wnt ligands remained functional. Additional stimulation by Wnt3a-conditioned medium, purified Wnt3a, treatment with LiCl, or ectopic expression of constitutively active β-catenin, all resulted in a further two - to fourfold increase in proliferation of HMCLs . MM cellThe major topic for discussion, however, concerns the biological consequences of intrinsic canonical Wnt signaling in MM. In 2003, Qiang et al. reported that Wnt pathway stimulation of HMCLs by Wnt3a-conditioned medium resulted in activation of the canonical Wnt pathway. This was demonstrated by increased levels and activation of Dvl2 and Dvl3, cellular stabilization of transcriptionally active β-catenin and an approximate sixfold increase in Wnt activity using aTopFlash reporter assay that is indicative of TCF/LEF-mediated transcription. However, they did not report an effect on the level of cell proliferation. Instead, major morphological changes were observed: the MM cells acquired a fibroblast-like phenotype, started to become adherent and developed filopodia-like protrusions . This waSince all previous observations are based on in vitro experiments, several independent research groups used genetically engineered mouse models to gain more insight into the in vivo mechanism of action. Both Yaccoby et al. and Qiang et al. reported a reduction in MM tumor growth upon forced activation of the Wnt signaling pathway. Yaccoby et al. used the SCID-rab model and, upon transplantation with primary MM cells, treated these mice with antibodies to neutralize the canonical Wnt inhibitor Dkk1. This resulted in a general reduction of MM tumor burden . In a slAlthough the additional use of in vivo mouse models could not elucidate the exact molecular mechanism of Wnt signaling or its relevance for tumor growth, these studies, when combined with in vitro data, did reveal an interesting trend. Extra stimulation of the Wnt pathway did not confer a growth advantage for MM per se. However, almost complete termination of canonical Wnt signaling, either by knockdown of β-catenin or disruption of the transcriptionally active β-catenin-TCF/LEF complex, induced MM cell apoptosis and significantly reduced tumor growth. This is similar to what was observed for Wnt signaling in HSC biology and could be the direct result of essential growth factor deprivation., this process is studied by the TopFlash reporter assay, in which the cells are transfected with a promoter-reporter construct that contains multiple Wnt responsive elements (WRE) followed by the sequence encoding the luciferase enzyme. After addition of substrate, the amount of measurable product formed by the enzyme reflects the level of Wnt/β-catenin transcriptional activity. Some studies additionally analyze expression levels of up to three Wnt target genes, often including MYC, CD44 and CCND1. Although these are all direct Wnt target genes, MYC and CD44 are both a-specific, since their activity is incorporated in and influenced by many other cell signaling pathways [CCND1 is not an ideal candidate, since upregulation of this gene is very often observed, either due to gene duplication or chromosomal translocations involving the cyclin-D locus [LEF1 gene is however quite distinct from the TCF genes. Whereas all TCFs are able to produce an “E” tail isoform at their carboxyl terminus, the LEF1 gene does not encode such a tail at all. This “E” tail, which encodes a DNA-binding domain and a region that facilitates interaction with p300, was shown to be essential for the regulation of multiple Wnt target genes. The TopFlash reporter, with the multimerized WREs, is however so optimally constructed that reporter activation is tail-independent [The ultimate purpose of the canonical Wnt/β-catenin pathway is to activate transcription of Wnt target genes. In vitropathways , 116. Ad-D locus . When Wn-D locus . One expependent . This coependent . Anotherependent . Since tBesides the possible effects of Wnt signaling on MM cell proliferation and subsequent MM disease progression, two studies suggest a role for Wnt in cell adhesion-mediated drug resistance (CAM-DR) of MM. Qiang et al. already reported that Wnt ligands can induce migration and invasion of MM cells by activating the non-canonical Wnt pathway mediators RhoA and PKC . In 2007It seems counterintuitive that MM cells require an active intrinsic canonical Wnt pathway to maintain cell survival and tumor growth, but at the same time also upregulate the canonical Wnt inhibitor Dkk1. However, Dkk1 was found to be a direct gene target of canonical Wnt signaling as well and could therefore function in a negative feedback loop, like is established for Axin2 . In thisAlthough Wnt signaling is extensively studied and its role in malignancies is well established, biologicals targeting this cascade have not yet entered the standard-of-care procedures. This is primarily due to concerns about drug safety, as Wnt signaling is required for stem cell maintenance and tissue homeostasis during the complete course of adult life . HoweverDespite the aforementioned challenges, many compounds that modulate Wnt pathway activity have been identified and developed in the past decade and some have now made it to the first phases of clinical trials. The compounds LGK974 and ETC-159, also referred to as ETC-1922159, are small molecules that block the secretion of Wnt ligands by inhibition of porcupine. This membrane-bound O-acetyltransferase is required for Wnt palmitoylation, which is an essential step in ligand secretion . Both drFor the antibody-based inhibitor OPM-54F28 the first dose-escalating phase Ia clinical trial in solid tumors has been completed in 2016. OPM-54F28 consists of the cysteine-rich domain of Fzd8 fused to the IgG1 Fc region and thereby acts as a decoy receptor for extracellular Wnt ligands. Also for this compound, efficacy was validated in a Wnt-sensitive murine cancer model and additional in vivo data showed synergy in combination with the chemotherapeutic agent gemcitabine. Furthermore, a reduction in the cancer stem cell population was reported . CurrentSeveral monoclonal antibody therapies that directly target the membrane-bound receptor complex have also been developed. OMP-18R5, also referred to as vantictumab, binds to a conserved epitope of the canonical Fzd1, Fzd2, Fzd5, Fzd7, and Fzd8 receptors. Binding studies showed that this blocks the binding of Wnt3a to the extracellular domain of Fzd5, which subsequently leads to inhibition of downstream Wnt/β-catenin signaling. Treatment of human solid tumor xenografts in mice showed a clear reduction in tumor growth in a subset of the tumors and also reported significant synergistic effects in combination with standard chemotherapeutic agents, including taxol and gemcitabine . Three pMost compounds developed to inhibit canonical Wnt signaling function by interfering with the downstream β-catenin-LEF/TCF complex . One of PTPRK(e1)-RSPO3(e2) fusion. Although clear sensitivity to the Porcupine inhibitor LGK974 was observed in vitro and in vivo in a murine xenograft model, long-term treatment led to the emergence of a resistant population. This resistant population was characterized by two novel frame-shift deletions in AXIN1, resulting in protein loss [It is yet too early to determine which strategy of inhibiting Wnt signaling in cancer patients will be most successful and targeting the Wnt signaling pathway for the treatment of MM is still at the preclinical stage. Multiple studies have been published in the last years suggesting a range of molecules that inhibit Wnt signaling in MM, either as a single agent or in combination with currently used therapies. An example is the β-catenin inhibitor BC2059 that was shown to induce apoptosis of HMCLs, even in the presence of the protective feeder cell line HS5. Apoptosis of malignant PCs from relapsed and/or refractory MM patients induced by BC2059 was reported to be synergistically enhanced when combined with the proteasome inhibitor Bortezomib. Furthermore, NOD/SCID/gamma mice transplanted with HMCL U266 intravenously, that were treated with BC2059 showed an impaired tumor burden and an increased survival when compared to the vehicle control . In addiein loss . AlthougThe Wnt signaling pathway is implicated in multiple aspects of MM disease Fig. . By secr


Mineral dust provenances are closely related to the orogenic processes which may have distinct Hf-Nd isotopic signatures. Here we report the clay-sized (<2 μm) Hf-Nd isotope data from Asian dust sources to better constrain the source and transport dynamics of dust deposition in the North Pacific. Our results show that there is a more positive radiogenic Hf isotopic composition with clay-sized fractions than the corresponding bulk sample and a decoupling of the Hf-Nd couplets in the clay formation during the weathering process. The clay-sized Hf-Nd isotopic compositions of the desert samples from the Sino-Korean-Tarim Craton (SKTC) are different from those of the Gobi and deserts from the Central Asian Orogeny Belt (CAOB) due to varying tectonic and weathering controls. The Hf-Nd isotopic compositions of dust in the North Pacific central province (NPC) match closely with those from the Taklimakan, Badain Jaran and adjacent Tengger deserts, implying that the NPC dust was mainly transported from these potential sources by the westerly jet. Our study indicates that dusts from the CAOB Gobi deserts either didn't arrive in NPC or were quantitatively insignificant, but they were likely transported to the North Pacific margin province (NPM) by East Asian winter monsoon. Our objectives are to address the following questions. 1) What controls the Hf-Nd isotopic composition of clay-sized fractions within desert sands? 2) What are the general characteristics of Asian dust and how do those characteristics differ from other dust sources? 3) What are implications for the source and transport pathway of eolian dusts in the North Pacific Ocean?Nd values ranging from −17.30 to 0.98 (mean = −8.40) and εHf from −5.94 to 4.63 (mean = −0.97). The Mongolian Gobi is more radiogenic in Nd and Hf isotope compositions which ranges from −5.99 to −2.67  and from −2.56 to 3.68 , respectively. It is clear that the clay-sized fractions have higher radiogenic Hf isotopic composition than silt-to-sand silicate fractions (>2 μm) (see Nd (εNd = −5.4) of <2 μm fraction in sample BT-46 are similar to those of <75 μm fractions (εNd = −5.6)Hf of >2 μm fractions of BT-46 have εHf values ranging from −21.01 to −6.72 (see μm fraction<2  = 0.1) of the <2 μm fraction is much higher than that (Lu/Hfμm fraction>2  = 0.05) of the >2 μm fraction (Sampling sites of desert sands are situated on the Sino-Korean-Tarim Craton941 μm) see . The εNd6.72 see . The Sm/6.72 see . Howeverfraction .Hf = −2.58 to 3.68  and εNd = −10.19 to −0.98 .The CAOB, including the Mongolian Gobi, Gubanunggut Deserts, the Hunlun Buir sandy land, and the west Horqin sandy land, with εHf from −5.94 to 1.20  and εNd from −17.30 to −6.24 .The STKC, including the Taklimakan, Qaidam, Badaim Jaran, Tengger and Mu Us Deserts, and the east Hobq sandy land, with εThe clay-sized Hf-Nd isotopic compositions are relatively consistent for samples taken from individual deserts  and samp8Hf – εNd value within CAOB indicates that the clay-sized fraction is generated from CAOB juvenile continental crust, which was formed by the collision between the Siberian Plate and the southern blocks during the early stages of the orogeny about 1.0 Ga and continued to about 250 MaHf – εNd values controlled by CAOB are higher than SKTC terrane, whereas the variability of both εHf and εNd in the CAOB are smaller than the corresponding SKTC εHf and εNd. There are two obvious end-members easily discerned from present clay-sized εHf – εNd compositions shown in Hf and εNd values, especially the samples from the Mu Us and Hobq Deserts. The isotopic regions are consistent relative to the clay-sized εHf – εNd values from deserts of the same geologic setting, suggesting that not all the isotopic differences are caused entirely by the heterogeneity of material at their source. Geologically, blocks and/or cratons formed the Chinese continent through multiple collisions and aggregation42The Hf-Nd isotopic signatures of the Chinese and Mongolia Gobi Deserts are consistent with previous Sr-Nd isotopic observations, suggesting that the isotopic composition of deserts is closely related to the tectonic setting of the surrounding mountains41Hf – εNd values. In fact, the sand sediments in the southern Horqin sandy land appears to be transported directly from the northern mountainous margin of North China Craton by riversIt is noteworthy that samples D17 and Nmy-8 are SKTC end members whereas the other Horqin samples belong to CAOB end members. These are the few exceptions to the geographic distribution of the Hf-Nd isotopic composition, although both D17 and Nmy-8 are from the southern-most edge of Horqin sandy land which is located in the SKTC . One posHf = (0.45 ± 0.04) × εNd + 2.81 ± 0.35 , because the clay fractions are too fine (<2 μm) to contain any zircon. However, the clay array is above and underscored by the zircon-free sediment arrayHf – εNd relationships because the clay-sized fractions contain relatively more radiogenic Hf than fine-grained sediments (zircon-free sediment). During the weathering process, clay minerals incorporate and/or adsorb the incongruent released radiogenic Hf to form the decoupling of the clay-sized Hf and Nd isotopic compositions, which are determined by both the weathering regime and source provenance.Regression of all the clay-sized data yields a clay array: εThis Hf-Nd isotopic decoupling is attributed to the different Goldschmidt behavior of Hf and Nd during weathering. Hf is both similar to REE and Zr, whereas Nd is one of the REE. REE barely fractionate during weathering and have been used for studying the provenance of detrital sediments. More specifically, ratios such as REE ratio and Nd isotopic compositions of weathered material, are considered to represent the compositions of source rocks8The decoupling of clay-sized Hf-Nd isotopic compositions may explain the different Hf-Nd correlation patterns between SKTC and CAOB. It is noted that the SKTC clay-sized array has a higher radiogenic Hf isotopic composition for its corresponding Nd isotopic composition than the CAOB array. The Hf isotope signatures from SKTC also show more scatter. This is attributable to the SKTC being older and thereby containing less radiogenic Nd than CAOB as demonstrated above. The clay reservoir from the SKTC had more time to produce more radiogenic Hf isotopes than that from the CAOB. This further suggests that the Hf isotope fractionation between clay and crustal arrays is larger than the older the source rocks are.The Chinese deserts and Mongolia Gobi are major Asian dust sources on a hemispheric scale931505152Nd > −10.9 and −16.2 > εHf > −4.5, which encompassed the range observed for the Neogene time series of Ocean Drilling Program (DSDP) 885/886 and LL44-GPC3 L44-GPC3 . It was Hf = 0.78εNd + 5.66) is very close to our clay-sized array  885/886 and LL44-GPC3, the source of Neogene dust in NPC may come from SKTC. Our clay-sized Hf-Nd isotopic signals from major Asian Gobi/sandy deserts indicate that sources and dispersal patterns of dust deposits in the NPC and NPM are spatially different  overnight and then a decarbonation step was carried out using excess 1M acetic acid for 10 hours in order to eliminate the influence of secondary carbonate on Hf isotopic composition. The samples were subsequently rinsed at least three times with MilliQ water to completely remove major ions and soluble salts. Different fractions were extracted by sieving the ultrasonically dispersed samples in mesh with MilliQ water, and the <2 μm particles were separated based on the Stokes' Law and then were recovered by centrifuging4 mixture in a steel jacketed autoclaves at 180 ~ 200°C for 72 hours4 at 110 ~ 140°C for 72 hours. Second, the purification for Hf and Nd with ion chromatography. The Hf analysis used a modified version of the method of Yang et al (2010)4 mixture and separating them by chromatographic extraction through an cation exchange resin was analyzed to provide a calibration value for the standard: 176Hf/177Hf = 0.282161 ± 0.000004 . The JNDi-1 Nd standard gave an average value of 143Nd/144Nd = 0.512118 ± 0.000005 (50 ppb), which is similar to the referenced value of 143Nd/144Nd = 0.512115 ± 000007 146Nd/144Nd ratio of 0.7219 and 179Hf/177Hf ratio of 0.7325. The external reproducibility of the 176Hf/177Hf and 143Nd/144Nd ratios was estimated from repeated measurements of JMC 475 and JNDi-1 Nd standard solutions every tenth sample. The relative standard deviations are better than 9 × 10−6. Epsilon Hf and Nd values were calculated using chondritic values of 176Hf/177Hf = 0.282785 and 143Nd/144Nd = 0.512630Hf and εNd were processed and yielded an external reproducibility of better than ± 0.1 (2σ) for εNd and ± 0.1 (2σ) for εHf.The mass spectrometric analyses were performed in Class 1000 clean laboratories. The JMC-475 Hf standardSupplementary information


Liver cancer is the second leading cause of cancer-related death worldwide. The high frequency of recurrence and metastasis is the main reason for poor prognosis. Liver cancer stem cells (CSCs) have unlimited self-renewal, differentiation, and tumor-regenerating capacities. The maintenance of CSCs may account for the refractory features of liver cancer. Despite extensive investigations, the underlying regulatory mechanisms of liver CSCs remain elusive. miRNA and lncRNA, two major classes of the ncRNA family, can exert important roles in various biological processes, and their diverse regulatory mechanisms in CSC maintenance have acquired increasing attention. However, to the best of our knowledge, there is a lack of reviews summarizing these findings. Therefore, we systematically recapitulated the latest studies on miRNAs and lncRNAs in sustaining liver CSCs. Moreover, we highlighted the potential clinical application of these dysregulated ncRNAs as novel diagnostic and prognostic biomarkers and therapeutic targets. This review not only sheds new light to fully understand liver CSCs but also provides valuable clues on targeting ncRNAs to block or eradicate CSCs in cancer treatment. Despitecascades . Though With the development of whole genome and transcriptome sequencing technologies, numerous noncoding RNAs (ncRNAs) without protein-coding potential have been identified . ncRNAs MiRNAs, with a length of 19~25 nucleotides, usually function as negative regulators to repress gene expression via base-pair complementation with the 3′-UTR of target genes. Currently, many miRNAs' alterations have been documented to participate in liver CSC regulation via various targets, as listed in β-catenin signaling pathway has been shown to play an important role in regulating stem cell and tumorigenic properties in liver cancer. Inhibition of the Wnt pathway has also been shown to be effective in eliminating CSC-like features [β-catenin from the Axin/APC/GSK3β destructive complex. Then, β-catenin accumulates and is translocated into the nucleus to form the β-catenin/LEF/TCF transcriptional complex, which initiates the transcription of downstream genes to regulate liver CSC maintenance [β-catenin cascade to regulate liver CSCs.The Wnt/features , 23. Whentenance . Several+ liver CSCs. These miRNAs can directly bind to the 3′-UTR of CDX2, GATA6, and NLK mRNAs and inhibit their expression. CDX2 and GATA6 are the transcriptional regulators for hepatic differentiation, and NLK functions as the inhibitor of Wnt/β-catenin signaling. Therefore, miR-181 can enhance the self-renewal ability of EpCAM+ liver CSCs and maintain the undifferentiated state simultaneously [β expression, leading to the nuclear accumulation and activation of β-catenin, which promotes stem cell-like phenotypes in CD133+ liver CSCs. Moreover, the important CSC transcriptional factor OCT4 can directly bind to the miR-1246 promoter to increase its expression [+133+ in liver cancer cells via inhibiting FZD4 expression to inactivate the Wnt/β-catenin signaling cascade [β-catenin pathway. For example, miRNA-200a can directly repress β-catenin; miRNA-214 can directly target β-catenin and indirectly inhibit β-catenin through EZH2 together; miR-612 can indirectly decrease the nuclear accumulation of β-catenin, and let-7a can deplete TCF4 [The miR-181 family is critical for maintaining the “stemness” of EpCAManeously . In addi cascade . Li et a cascade , 27. Theete TCF4 –31.PTEN is a well-known tumor suppressor that serves as the natural inhibitor of PI3K to negatively regulate AKT. MiR-25 can directly target PTEN to stimulate the PI3K/AKT pathway, which enables liver CSCs to resist apoptosis . MiR-21 + liver CSCs to increase stem cell-associated gene expression, self-renewal, chemotherapeutic resistance, and tumorigenicity capacities. They found that miR-429 can inhibit the expression of PBBP4 by binding to its 3′-UTR, which promotes the transcription activity of E2F1 on OCT4. Successively, the increased OCT4 is recruited to the EpCAM promoter and enhances its expression to strengthen liver CSCs' properties [+ liver CSCs [α2δ1 may serve as a novel liver CSC marker. miR-424, miR-222, miR-200b, and let-7c are downregulated in α2δ1+ liver CSCs, which synergistically play important roles in the acquisition and maintenance of liver CSC properties. These researchers found that low expression levels of the four miRNAs can increase the expression of PBX3, which can activate critical genes for liver CSCs, including CACNA2D1, EpCAM, SOX2, SALL2, NOTCH3, and WNT10A [miR-612 can target and decrease the expression of SP1, which is an important transcriptional activator of the stemness-factor, Nanog. Through silencing SP1 to reduce Nanog expression, miR-612 can shrink the number and size of liver CSCs . OCT4 isoperties . It was operties . Surfaceoperties , 60. CD2operties . CD133 ioperties . miR-142ver CSCs . Han et d WNT10A .+ liver CSC characteristics, and the downregulation of miR-122 plays an important role in the abnormal metabolic process. Downregulation of miR-122 leads to the upregulated expression of its direct target, pyruvate dehydrogenase kinase 4 (PDK4). PDK4 can stimulate glycolysis and further increase the stemness gene expression and spheroid formation capacity in CD133+ liver CSCs [Metabolic reprogramming is one of the most common cancer hallmarks. For example, the famous “Warburg effect” indicates that cancer cells prefer to elevate glycolysis and lactate production even in the presence of oxygen . Song etver CSCs .+ liver CSCs, can repress TP53INP1 expression by directly targeting its 3′-UTR of TP53INP1, which helps CSCs to enhance proliferation, resist the chemotherapeutic drug, doxorubicin, and increase the expression of a series of stem cell-associated genes, including β-catenin, Notch-1, and Nestin [+ liver CSCs [+ cells [β is an antimitogenic cytokine that becomes oncogenic in advanced tumors [β pathway [TP53INP1 is a tumor suppressor and has antiproliferative and proapoptotic activities , 65. miRd Nestin . miR-155d Nestin . KIT, a ver CSCs . MAP3K8 ver CSCs . miR-150[+ cells . miR-148[+ cells . miR-137[+ cells . TGF-β id tumors . The res pathway .In contrast to miRNAs, lncRNAs have versatile mechanisms to control gene expression at both the transcriptional and posttranscriptional levels. lncRNAs can serve as a scaffold to recruit transcriptional factors within the promoter region to affect gene expression. lncRNAs can directly bind to DNA and RNA via a complementary sequence to impact transcriptional initiation or RNA stability. Additionally, lncRNA can modulate the posttranslational modification of proteins. Numerous lncRNAs have been demonstrated to be deregulated in cancers and exert critical roles in cancer development, such as malignant proliferation, metastasis, invasion, antiapoptosis, therapeutic resistance, and CSC formation. Recently, accumulating studies have focused on the regulation of lncRNAs in liver CSCs, as listed in + CD133+ cell population. Among these dysregulated lncRNAs, lncTCF7 and lnc-β-Catm are the most upregulated lncRNAs, which play critical roles in driving CSC self-renewal and tumor propagation via activating the Wnt/β-catenin pathway [β-Catm can strengthen β-catenin protein stability via posttranscriptional modification to activate the Wnt signaling pathway. lnc-β-Catm is located on chromosome 1q, which frequently occurs as copy-number amplification in liver cancer cells. lnc-β-Catm is highly expressed in liver CSCs and positively correlated with tumor aggressiveness and poor prognosis in liver cancer. lnc-β-Catm functions as a scaffold to associate β-catenin and the methyltransferase EZH2 in liver CSCs. Successively, EZH2 methylates β-catenin at Lys49, which abolishes its polyubiquitination and protects β-catenin from proteasomal degradation. β-Catenin subsequently forms a complex with TCF and acts as a cotranscriptional factor to initiate Wnt signaling. The lnc-β-Catm and EZH2-dependent β-catenin stabilization is required for oncosphere formation in vitro and increases tumorigenic cell frequency in vivo [β-catenin and stimulating the Wnt pathway. lncRNA-DANCR is dramatically overexpressed in liver CSCs and acts as an independent predictor for poor prognostic outcome in liver cancer. Instead of forming a complex with proteins, DANCR interacts with the β-catenin mRNA transcript within its 3′UTR that harbors miR-214 and miR-320a binding sites. The competitive occupation enables DANCR to block miRNA-mediated β-catenin depletion and raises the β-catenin reservoir to elevate its downstream AXIN2, NOTUM, and OAT levels. Through derepressing β-catenin and eliciting Wnt signaling, DANCR enhances the expansion and maintenance of liver CSCs [CD133 and CD13 are two widely used liver CSC surface markers, and the double-positive cell fraction exhibits obvious CSC properties, including a strong self-renewal capacity and chemical drug resistance. The transcriptome microarray analysis identified many differentially expressed lncRNAs in the CD13 pathway , 47. lnc pathway . Unlike +, CD24+, or OV6+ liver CSCs, has a tumor suppressive function. A stem cell signaling PCR array revealed that lnc-DILC can modulate the JAK2/STAT3 cascade. A gain in lnc-DILC expression decreases the activated phospho-STAT3 protein levels, attenuates STAT3 nuclear translocation, and represses the transcriptional activity of STAT3-responsive elements. Consistently, increased phospho-STAT3 is observed in lnc-DILC-silenced spheroid-formed xenografts. Mechanistic studies have revealed that lnc-DILC binds to the IL-6 promoter and inhibits its transcription, while IL-6 is a critical cytokine in the activation of the JAK2/STAT3 axis. Of note, IL-6 can be induced by the inflammatory factors, TNF-α and IL-1β, and the inflammatory microenvironment is a major aspect for liver cancer progression. In CSC spheroids, the downregulation of lnc-DILC also enhances TNF-α and IL-1β-induced IL-6 expression. These findings suggest that lnc-DILC can coordinate the crosstalk between inflammatory signaling and the autocrine IL-6/STAT3 pathway to promote liver CSC expansion [+ liver cancer cell fraction and CSC spheroids, and lncSox4 is essential for liver CSC self-renewal and tumorigenic capacity. Mechanistically, lncSox4 interacts with STAT3 and recruits it to the Sox4 promoter region, inducing H3K4me3 and H3K27ac modification to drive the Sox4 promoter activation and augments Sox4 expression. The lncSox4/STAT3-dependent Sox4 expression exerts an indispensable function in sustaining liver CSC propagation, which may serve as an important target for CSC eradication [Apart from the Wnt pathway, the IL-6/STAT3 signaling cascade plays important roles in regulating liver CSC maintenance as well. lncRNA DILC and lncSox4 have been established to control CSC features via the STAT3 pathway in liver cancer. lnc-DILC, which is downregulated in EpCAMxpansion . Throughdication .β-catenin promoter structure to drive the expression of these oncogenes [+/CD44+/CD24+/EpCAM+ liver CSCs. Mechanistically, CUDR interacts with cyclin D1 to increase lncRNA H19 expression and enhance the association between TERT and TERC, thereby promoting telomerase activity and prolonging telomere length. Additionally, with the help of CTCF, the CUDR-cyclin D1 complex is recruited to the C-Myc gene promoter region to increase C-Myc expression. Synergistically, the excessive TERT and C-Myc expression account for liver cancer stem cell proliferation [Wu et al. found that lncRNA HULC and MALAT1 are dramatically upregulated in liver cancer cells. When MALAT1 is cooverexpressed with HULC in liver CSCs, the two lncRNAs can cooperate to promote liver CSC growth via the telomere repeat-binding factor 2 (TRF2). MALAT1 and HULC coexpression facilitates the TRF2 promoter and enhancer to form a loop, recruiting P300, RNA pol II, and CREPT into the loop, which enhances TRF2 expression and its phosphorylation and SUMOylation. Then, the excessive TRF2 forms a complex with HULC and MALAT1 on the telomeric region to protect telomeres from degradation. As a result, telomerase activity and microsatellite instability (MSI) are obviously induced in the liver CSCs . CUDR isncogenes . Furtherferation .+ CD133+ transcriptome microarray and is required for liver CSCs to maintain the self-renewal potential and initiate tumorigenicity. lncBRM can facilitate BRG1-embedded BAF complex formation and recruit the complex to the YAP1 promoter to trigger YAP1 transcription in a KLF4-dependent manner, thereby driving liver CSC properties [+ CD133+ CSCs and induces liver CSC proliferation. The lncCAMTA1 transcription orientation is antisense to the tumor suppressive gene CAMTA1, and their expression is negatively correlated in liver cancer samples. lncCAMTA1 physically binds to the CAMTA1 promoter and mediates a repressive chromatin structure to decrease CAMTA1 expression. The lncCAMTA1-dependent CAMTA1 downregulation accounts for liver CSC properties [+ liver CSC population. LncICR can form an RNA duplex with the ICAM-1 transcript via their complementary sequence, which increases the stability of ICAM-1 mRNA and augments its expression to maintain the liver CSC feature [lncBRM is another upregulated lncRNA identified in the CD13operties . lncRNA-operties . Ding etoperties . ICAM-1 operties . LncICR  feature . Lu et a feature .α treatment in liver cancer [miRNA and lncRNA are two major classes of ncRNAs. Previous studies have demonstrated that miRNAs and lncRNAs are stable in body fluids, and they can be easily and noninvasively accessible. Some of the miRNAs and lncRNAs have tissue- or disease-specific expression patterns. Therefore, miRNAs and lncRNAs have been recognized to be ideal biomarkers for early diagnosis, prognostic prediction, and therapeutic evaluation in cancer , 70. Forr cancer . The pror cancer . Additior cancer  and antir cancer . Given tβ-catenin pathway, like miR-148, miR-1246, miR-200a, lncRNA TCF-7, lncRNA β-Ctam, and so on , OMP54F28 (soluble FZD decoy receptor), and PRI-724 (inhibitor of TCF-CBP interaction) [β-catenin activity is essential to sustain normal cell survival. How to precisely regulate Wnt/β-catenin is a great conundrum. Targeting these regulatory miRNAs and lncRNAs may be an alternative approach to accomplish precise modulation of Wnt/β-catenin activation.In this review, we emphasized the effects of ncRNAs on signaling pathways, finding that many miRNAs or lncRNAs control liver CSC properties by targeting different components of the Wnt/nd so on . Recentlraction) . HoweverIn conclusion, we overview the multiple functions and diverse mechanisms of miRNAs and lncRNAs in liver CSCs and highlight their potential clinical applications as novel diagnostic and prognostic biomarkers and therapeutic targets. Our review provides new insights to understand liver CSCs and delineates new clues to develop a ncRNA-based therapeutic strategy for liver cancer.


Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancy. Semaphorin 3F (SEMA3F) is highly conserved but present at a lower level in various cancers than in healthy tissues. While it has been reported that SEMA3F is involved in cancer cell proliferation, migration and invasion, its function in OSCC remains unknown..The expression of SEMA3F in OSCC tissues and OSCC-derived cells was analyzed using qRT-PCR and western blotting. Using SAS and HSC2 cells, we also monitored the effect of SEMA3F on OSCC cell proliferation, migration and invasion using MTT, colony formation and transwell assays. The function of SEMA3F in OSCC tumor formation was also assessed in vivo.SEMA3F was significantly downregulated in OSCC tissues and OSCC-derived cells. SEMA3F shows growth inhibitory activity in SAS and HSC2 cells and may act as a tumor suppressor. It can inhibit the migration and invasion potential of OSCC cells. Our results also demonstrate that SEMA3F can suppress the growth of OSCC cells in vivoThis study revealed that SEMA3F plays a role as a tumor suppressor in OSCC cell proliferation, migration and invasion. Our finding provides new insight into the progression of OSCC. Therapeutically, SEMA3F has some potential as a target for OSCC treatment, given sufficient future research. Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity , 2. AccoDespite considerable advances in the treatment of OSCC over the past two decades, the understanding of its pathogenesis is limited , 10. RecSemaphorin 3F (SEMA3F), was first identified as a repulsive factor with a role in axonal guidance and neuronal development, where it modulates cell polarization and migration . It has The expression of SEMA3F in cancer cells inhibits tumor growth, invasion and metastasis –25. It h. These findings provide new insight into the mechanism of OSCC pathogenesis.In this study, we tested SEMA3F expression in 16 OSCC tissues and 4 OSCC-derived cell lines via qRT-PCR, and found that SEMA3F was downregulated in OSCC tissues and OSCC-derived cell lines. We then validated that SEMA3F suppressed OSCC cell growth, invasion and migration. Our data further demonstrated that SEMA3F inhibited OSCC tumor formation in vivoTissues were obtained from 30 OSCC patients at the Tianjin First Center Hospital. Written informed consent was obtained from all patients and the study was approved by the ethics committees of the Tianjin First Center Hospital.The OSCC tissues were embedded with paraffin and sectioned. The sections were subjected to a routine three-step immunohistochemical (IHC) staining procedure. The slides were incubated with rabbit anti-SEMA3F antibody  and anti-Ki67 antibody  at 4 °C overnight. Then, the slides were incubated with horseradish peroxidase-labelled secondary antibody for 30 min at room temperature. The chromogen 3,3′-diaminobenzidine tetrachloride  was used as a substrate.The cell nucleus was dyed with Harris hematoxylin solution. Levels of SEMA3F expression were determined based on the extent and intensity of staining with the scoring using this scale: negative for <5%, weak for 5–25%, mid for 25–50%, distinct for 50–75%, and strong for ≥75%. The staining intensity and stained area percentage were multiplied to produce a weighted score. Three independent evaluators assessed the scoring.2.We used 4 human OSCC-derived cell lines: SAS, Ca9–22, HSC2 and HSC4. One normal oral keratinocyte strain was obtained from a patient who had undergone dental surgery. This served as the control. The patient provided written informed consent prior to the start of the study. The cell lines were cultured in DMEM (Thermo-Fisher) with 10% fetal bovine serum (FBS) plus 100 IU/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere with 5% COThe CDS sequence of SEMA3F was obtained via PCR using specific primers. The PCR products were cloned into the pcDNA3.1(+) vector. The plasmid was named pcDNA-SEMA3F. The primers were: SEMA3F forward: 5’-CTAGCTAGCATGCTTGTCGCCGGTC-3′; SEMA3F reverse: 5’-CTAGTCTAGATCATGTGTCCGGAG-3′.Transfection of cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Briefly, cells were seeded in 6-well plates at 30–40% confluence 24 h prior to transfection. pcDNA-SEMA3F (2 μg per well) and the control were used for each transfection.For stable transfection, HSC2 cells were selected in G418 (800 mg/ml). Individual G418-resistant colonies were isolated after 15 days of culture. Cells were treated with G418 (200 mg/ml) and maintained in culture until needed for subcutaneous inoculation into mice.3 cells/well). Next, the cells were incubated with 25 μl of MTT  at 37 °C for 4 h, the supernatants were removed, and 150 μl methylsulfoxide  was added to each well. The absorbance value (OD) of each well was measured at 490 nm.The MTT assay was performed daily over 3 days to evaluate cell proliferation. First, cells were transfected with plasmids (control or pcDNA-SEMA3F). After 24 h, cells were seeded into 96-well plates (3 × 105 cells per well) were seeded in 6-well plates and transfected with pcDNA-SEMA3F (2 μg per well) or the control for 24 h. The medium was refreshed every 3 days. After 2–3 weeks of culture, the colonies were fixed with methanol, stained with 1.25% crystal violet and counted under a light microscope. All experiments were performed three times and the average results were calculated.For the colony formation assay, cells . Then, 1 μg of the RNA was converted to cDNA using Revert Acid Reverse Transcriptase (Fermentas). Real-time PCR was conducted using a Sigma-Aldrich FastStart Universal SYBR Green Master (ROX) Kit according to the manufacturer’s instructions. Double-stranded DNA specific expression was tested by the comparative Ct method using 2-ΔΔCt. The sequence of the primers used were: SEMA3F forward: 5’-CTCTGGGCTTCCCTACTGAC-3′; reverse: 5’-CACTCGCCGTTGACATCC-3′; GAPDH forward: 5’-ATCACCATCTTCCAGGAGCG A-3′; reverse: 5’-CCTTCTCCATGGTGGTGAAGAC-3′. Experiments were performed in triplicate.Cellular proteins were extracted in RIPA buffer (Biomed) after transfection for 48 h. Proteins were separated by gel electrophoresis and transferred to membranes, which were then incubated with primary antibodies. The concentrations and sources of the antibodies were: rabbit polyclonal anti-NRP2 , rabbit polyclonal anti-SEMA3F  and mouse monoclonal antihuman anti-GAPDH . Treatment with secondary antibodies diluted in PBST was done at room temperature for 1 h. Membranes were washed in PBST and the bound antibody was detected using an enhanced chemiluminescence system. The experiments were repeated three times.4 cells/insert in DMEM. The lower chamber contained DMEM (600 μl) with 10% FBS. The migrated cells were fixed with 4% paraformaldehyde in PBS and stained in 0.5% crystal violet. The membranes were mounted on a microscope slide. Migrated cells were counted and photographed. The percentage of migrated cells was calculated. The migration index was expressed relative to the control cells. All the experiments were carried out three times and the results were expressed as the means ± SD.Cells were transfected with pcDNA-SEMA3F and the control. After 48 h, the cells were seeded on the upper chamber of an 8-μm pore size transwell insert (Costar) at 2.5 × 105 cells/ml in 400 μl of medium containing 0.5% FBS. Medium with 10% FBS was added to the lower chamber. Following 24 h incubation at 37 °C with 5% CO2, the invading cells were fixed in 100% methanol and stained with 0.5% crystal violet. Photographs were taken randomly for at least four fields of each membrane. The number of invading cells was expressed as the average number of cells per microscopic field over four fields.Cell invasion assays were performed in 24-well plates using transwell chambers  were coated with matrigel. The cells were transfected and then seeded in the upper chamber at a density of 2 × 102)/2. All animals were treated according to the guidelines established by the National Institutes of Health Guide (NIH).A total of twelve nude mice  were randomly divided into two groups. HSC2 cells stably transfected with pcDNA-SEMA3F and control were inoculated subcutaneously into the flanks of nude mice. The length and width of tumors were taken with Vernier calipers every five days, and the mice were euthanized after thirty days. The volume of the implanted tumor was calculated using the formula: volume =  and one normal oral keratinocyte strains (control) using qRT-PCR Fig. . The expThese results show that SEMA3F was significantly downregulated in OSCC cells compared with control cells at both the mRNA and protein levels. This suggests that SEMA3F may be a tumor suppressor in OSCC.To detect the effect of SEMA3F on OSCC cell growth, we cloned the SEMA3F CDS and transfected it into the human OSCC cell lines SAS and HSC2. Increased expression of SEMA3F upon transfection was confirmed using qRT-PCR and western blot Fig. . As demoWe attempted to identify the effect of SEMA3F on OSCC cell migration and invasion according to the potent capacity of OSCC to invade locally and form distant metastases. We used the transwell migration assay for this experiment. Our results showed that the rate of migration for SAS and HSC2 cells transfected with pcDNA-SEMA3F was significantly lower than for control transfected cells Fig. . FurtherTo explore whether changes in SEMA3F expression could influence the growth of OSCC tumors in vivo, two groups of nude mice were subcutaneously injected with HSC2 cells stably transfected with pcDNA-SEMA3F and control plasmids. Tumor formation and weight were assessed in these two groups. Our results showed that the ectopic expression of SEMA3F inhibited OSCC tumorigenesis in vivo Fig. . The aveWe also tested the expression levels of SEMA3F in the tumors from mice using the IHC assay. Our data revealed that SEMA3F levels were higher in pcDNA-SEMA3F-transfected HSC2 cells group than in the control group Fig. . In addiRecent study of SEMA3F has shown that it can play crucial roles in the regulation of cell proliferation, migration and invasion , 27, 28.Firstly, we tested the expression of SEMA3F in 30 pair of OSCC tissues and adjacent normal tissues. Our data show that SEMA3F is significantly downregulated in OSCC tissues compared with adjacent normal tissues. The relative expression of SEMA3F in 4 human OSCC-derived cell lines  was also significantly downregulated compared with normal control cells.Zhang et al. demonstrated that the expression level of SEMA3F is downregulated in OSCC cancer tissues . Our resIt has been reported that NRP2 is a receptor of SEMA3F, so we tested the expression levels of NRP2 and SEMA3F in OSCC cells. We found that the NRP2 protein level was upregulated but that the SEMA3F protein level was downregulated. This suggests that there would be a SEMA3F/NRP2 axis pathway during the progression of OSCC.Functional studies were required to clarify the role of SEMA3F in the development of OSCC cells. We tested the function of SEMA3F, as a putative tumor suppressor, in SAS and HSC2 cell lines. Overexpression of SEMA3F in SAS and HSC2 cells shows a significant growth-suppressing effect through the inhibition of cell proliferation and colony formation.It has been reported that SEMA3F suppressed the migration and invasion of several cancers –32. To b. Our data confirmed that Ki-67, a marker of proliferation, was induced in the restoration of the SEMA3F group. Thus, we conclude that SEMA3F is able to suppress the proliferation of OSCC in vivo.In addition, we observed that overexpression of SEMA3F could significantly decrease the OSCC tumor volume and weight in vivoIn summary, our data show that SEMA3F is significantly downregulated in OSCC tissues and OSCC-derived cells. Our study is the first to demonstrate that SEMA3F inhibits OSCC cell growth, migration and invasion in vitro and in vivo. This means that SEMA3F may be a potential biomarker for OSCC diagnosis and could serve as a new target for OSCC therapy in the future.We show that the SEMA3F plays an important role in OSCC cell proliferation, migration and invasion. Our finding is definitely important for studies on the development of OSCC, since it may provide new insight into the role of SEMA3F in OSCC cancer progression and be helpful in the development of further targeted drugs for OSCC cancer treatment.