The DNA sensor cGAS (cyclic GMP-AMP synthase) and its adaptor protein STING (Stimulator of Interferon Genes) detect the presence of cytosolic DNA as a sign of infection or damage. In cancer cells, this pathway can be activated through persistent DNA damage and chromosomal instability, which results in the formation of micronuclei and the exposure of DNA fragments to the cytosol. DNA damage from radio- or chemotherapy can further activate DNA sensing responses, which may occur in the cancer cells themselves or in stromal and immune cells in the tumour microenvironment (TME). cGAS–STING signalling results in the production of type I interferons, which have been linked to immune cell infiltration in ‘hot’ tumours that are susceptible to immunosurveillance and immunotherapy approaches. However, recent research has highlighted the complex nature of STING signalling, with tumours having developed mechanisms to evade and hijack this signalling pathway for their own benefit. In this mini-review we will explore how cGAS–STING signalling in different cells in the TME can promote both anti-tumour and pro-tumour responses. This includes the role of type I interferons and the second messenger cGAMP in the TME, and the influence of STING signalling on local immune cell populations. We examine how alternative signalling cascades downstream of STING can promote chronic interferon signalling, the activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and the production of inflammatory cytokines, which can have pro-tumour functions. An in-depth understanding of DNA sensing in different cell contexts will be required to harness the anti-tumour functions of STING signalling. Our immune system has the capacity to detect and eliminate cancer cells, despite the fact that they are derived from our own body. This is partly because cancerous and pre-cancerous cells – particularly those with a high mutational burden – express proteins that can be detected as neo-antigens by the cells of our adaptive immune system ,2. HowevCytosolic DNA is detected as pathogen-associated molecular pattern (PAMP), during viral infection for instance, but it can also serve as danger- or damage-associated molecular pattern (DAMP) when the cell’s own nuclear or mitochondrial DNA is damaged and gains access to the cytosol. Tumour cells often contain cytosolic DNA, due to persistent DNA damage and chromosomal instability (CIN), which is a hallmark of cancer ,5, and aThe DNA sensor cGAS and its adaptor protein STING constitute the key signalling axis for sensing dsDNA within the cytosol of many different cell types  15–19]..15–19]. cGAS-STING signalling is tightly regulated by post-translational modifications, including ubiquitylation with K48-, K63-, K27- and K11-linked ubiquitin chains, modification with ubiquitin-like proteins as well modification with metabolites and lipids ,47. AddiSTING can also be activated independently of cGAS, such as following membrane fusion or after uptake of cGAMP from neighbouring cells ,58. BactcGAS–STING signalling has been shown to have potent anti-cancer activities in a variety of mouse tumour models A. The prThe first potential point of activation of cGAS–STING pathway is within the cancer cells themselves following detection of cytosolic DNA from micronuclei for instance ,8. This cGAMP production and export by tumour cells can activated STING signalling in neighbouring endothelial cells, to induce further IFN-β and CXCL10 production in the TME. Activation of STING within the endothelium was also shown to be accompanied by increased expression of factors associated with T-cell adhesion to the endothelium and the infiltration of lymphocytes into the TME. The activation of STING in endothelial cells in the tumour vasculature is also thought to protect against angiogenesis and promote vascular normalisation in tumours ,88.Immune cells in the TME can respond to extracellular tumour-derived DNA and activate the DNA sensing pathway. The uptake of tumour DNA has been shown to be particularly important for activating DCs in the TME ,69,89. HActivation of STING signalling has been shown to be a feature of ‘hot’ tumours, which show infiltration of DCs and cytotoxic T cells – a pre-requisite for detection of tumour antigens by adaptive immune responses and for effective immunotherapy . Thus, tDue to of the anti-tumour effects of STING signalling, it was originally proposed that the pathway must be dysfunctional or epigenetically suppressed in cancer cells . AlthougIn addition to its potent anti-tumour activities, STING signalling has also been shown to contribute to cancer cell survival and tumour progression in some contexts. The balance of downstream STING signalling outputs may be differentially regulated in tumour cells, so that anti-tumour IFN-I and cell death responses are limited, while pro-inflammatory and pro-survival programs are promoted.While short-lived interferon responses due to acute STING stimulation have clear anti-tumour effects mediated by IFN-Is, it has been shown that low levels of chronic IFN-β signalling can promote cancer cell survival B 125–12. MechaniThe STING-dependent expression of pro-inflammatory cytokines, and particularly IL-6, can also promote inflammation-driven tumour progression. STING-deficient mice have been found to be resistant to the development of skin tumours induced by the mutagen DMBA, which drives tumorigenesis through IL-6-mediated inflammation . AnalysiThe expression of pro-inflammatory cytokines such as IL-6 is driven by the NF-kB family of transcription factors. In most cells, canonical cGAS–STING signalling induces only modest levels of NF-κB p65 activation, through signalling by TBK1 which favours the activation of the transcription factor IRF3 . HoweverAs an additional immune evasion strategy, tumour cells can also re-wire cGAS-mediated STING signalling towards increased non-canonical NF-κB activation in cancer cells through the NF-κB subunits p52 and RelB ,140. ThiIt is becoming increasingly clear that the ultimate outcome of STING signalling is also highly sensitive to cell context and differs between different cell types. For instance, while cGAMP transport to DCs can stimulate potent anti-tumour responses, its transport into astrocytes can promote brain metastasis through the production of cytokines that are beneficial for cancer cell growth and survival . Activatper se, to promote the anti-tumour functions of DNA sensing in cancer, while limiting adverse effects on tumour progression.The intense study of DNA sensing pathways in the last decade or so has revealed a crucial role of the cGAS–STING–interferon signalling axis in shaping immune responses during infection, autoinflammation and cancer. However, despite the fact that the anti-tumour role of STING signalling is well documented in mouse tumour models and multiple STING agonists have been tested in clinical trials, these approaches have not yet shown efficacy as anti-tumour agents in patients. Indeed, STING signalling has been shown to have both pro- and anti-tumour functions in cancer cells, stromal cells and the immune cells in the TME. While the activation of canonical, acute STING signalling that results in IFN-I induction has been studied in great molecular detail and clearly possess anti-tumour functions, we know much less about how low-grade, chronic STING signalling is induced in response to DNA damage. We will also need to understand how different kinds of DNA damage induced by radio- and chemotherapy regimens affect DNA sensing pathways, so that we can avoid potential pro-tumour effects. There are many open questions about how STING activation drives different cellular outcomes, from the predominant activation of IRF3 to canonical or non-canonical NF-κB activation, as well as inflammasome activation and cell death or senescence pathways. In the future, it may be possible to therapeutically influence the balance of STING signalling outputs, rather than STING activation The detection of cytosolic DNA and DNA damage results in the activation of the innate immune adaptor STING in cancer cells, immune cells or endothelial cells within the tumour microenvironment. All these cell types can contribute to the production of type I interferons which have anti-tumour functions.If the STING signalling response is evaded or diverted towards a more pro-inflammatory cytokine profile, this can instead promote the establishment of an immune-suppressive microenvironment and can aid tumour cell survival and metastasis.A deeper understanding of how different cell contexts and regulatory mechanisms influence signalling outputs downstream of STING will be important for the development of STING-targeting immunotherapies.


Our results demonstrate that the flow front stability can be improved by the gradual increase in wettability of the porous media. When the capillary number is less than 3.5 × 10−5, the viscous fingering can be successfully suppressed and the transition from unstable to stable displacement can be achieved by the wetting gradient. Moreover, under the conditions of high viscosity ratio (M > 0.01) and large porosity (Φ > 0.58), wetting gradient improves the stability of the flow front more significantly.The viscous fingering phenomenon often occurs when a low-viscosity fluid displaces a high-viscosity fluid in a homogeneous porous media, which is an undesirable displacement process in many engineering applications. The influence of wetting gradient on this process has been studied over a wide range of capillary numbers (7.5 × 10 The displacement of immiscible two-phase fluids in porous media is an important subject in natural and engineering fields, including oil exploration , carbon In order to better study the flow law of fluid in porous media, it is necessary to quantitatively analyze the results. There are several parameters for quantitative analysis, including the displacement efficiency, saturation, interface length and fractal dimension. The displacement efficiency refers to the percentage of the volume of the displaced fluid flowing out and the initial filling volume . The satM = μ1/μ2 and the capillary number Ca = μ1U/σ; here, μ1 is the viscosity of the displacing fluid. μ2 is the viscosity of the displaced fluid, U is the rate at which the displaced fluid is injected, and σ is the surface tension. The phase diagrams of M and Ca have been summarized [The flow of fluids in porous media can be divided into three types: stable displacement, capillary fingering and viscous fingering . There emmarized , which immarized  and Zhenmmarized  have alsmmarized .Zhao et al.  studied With the development of science and technology, the investigation of immiscible fluid flow in porous media is not limited to experiments, and many scholars have also tried to use numerical simulation methods to carry out corresponding research. The lattice Boltzmann method (LBM) is a mesoscopic numerical simulation method between macro and micro. Compared with macro methods, it is more convenient for dealing with a complex boundary and, at the same time, it overcomes the limitations of the micro calculation method on size. It has the advantages of the automatic capture of a two-phase interface without manual processing or flexible boundary processing, and is suitable for parallel computing . The lat−6 to 1.8 × 10−4), viscosity ratios (0.0025 to 0.04) and porosity (0.48 to 0.68). The flow results were quantitatively analyzed by means of quantitative parameters such as fractal dimension, displacement efficiency, saturation and interface length. Moreover, a phase diagram of flow stability results related to wetting gradient and capillary number was drawn.Unstable flows will create adverse effects for oil production, carbon dioxide storage and other aspects. In order to reveal the law of the unstable flow of fluid in porous media, some scholars have obtained the relevant phase diagrams through a large number of experiments. Some scholars have tried to improve the unstable flow of fluid by designing the structure of porous media. However, how a wetting gradient impacts viscous fingering remains unknown. In this paper, we used the lattice Boltzmann method to simulate the displacement process of fluids in porous media with a wetting gradient. The influence of the wetting gradient was studied over a wide range of capillary numbers  and the initial velocity field was 0 m/s. The bounce-back boundary condition was used to implement the no-slip condition on the solid walls. The inlet and outlet boundary conditions used in the current numerical simulations were velocity and pressure boundary conditions. The displaced fluid was silicon oil (the wetting phase) and the displacing fluid was water (the non-wetting phase). The parameters of these two fluids were as follows: the density and viscosity of displacing fluid were set as ρ1 = 998.3 kg/m3, μ1 = 1 mPa s. The density and viscosity of the displaced fluid were set to ρ2 = 960 kg/m3, μ2 = 200 mPa s, and the viscosity ratio was M = 0.005. The surface tension σ = 28.2 mN/m. The inlet velocity was 0.001 m/s unless otherwise specified. The wetting gradient was set as follows: Take the wetting gradient Δθ = 4° as an example, as shown in θ = 4°. The wetting gradients Δθ = 1°, Δθ = 2° and Δθ = 3° were arranged in the same way. The wetting gradient Δθ = 0° meant that the overall wettability of the porous media was uniform and the overall wetting angle was 150°.We used the computational fluid dynamics software XFlow to carry out the relevant numerical simulations. The phase field algorithm was employed for calculating the multiphase flow. The temperature type was set as isothermal. The computational domain and boundary conditions are shown in −6 s; the grid size selected for time step independence verification was 6 × 10−5 m. The total calculation time is the time required for the displacement fluid to flow to the porous media outlet. −5 m. −6 s. Thus, a grid size of 6 × 10−5 m and a time step of 1 × 10−6 s were employed in the subsequent numerical simulations.We first performed mesh independence verification and time step independence verification. The time steps selected for grid independence verification were all 1 × 10−6 and 1.4 × 10−5. In order to save computing time and resources, we simulated a part of the experimental model. The fluid parameters and the amplitude of the pore gradient decline are consistent with the parameters in the experiment. The comparisons between simulation results and experimental results are shown in We selected the experimental results of Rabbani et al.  for expe−6 in the nonuniform medium  and large porosity (Φ > 0.58), the improvement of flow front stability is more significant when adding a wetting gradient. In addition, for the porous media with a wetting gradient, decreasing the capillary number, increasing the viscosity ratio or increasing porosity can improve flow front stability and filling efficiency, which is consistent with that of homogeneous wetting media. The present findings could be helpful for the design of porous products to suppress viscous fingering, which will be of great significance for industrial applications such as composite material preparation and oil exploration.In the present study, the displacement of immiscible fluid in porous media was numerically investigated using the lattice Boltzmann method. Our results demonstrate that the displacement pattern and efficiency can be controlled by the wetting gradient. The flow front stability can be improved by setting a wetting gradient on the porous media, which is confirmed over a wide parameter range of different wetting gradients, capillary numbers, viscosity ratios and porosities. When the capillary number is less than 3.5 × 10


Structural characterization of the purely synthesized compounds was acquired by IR, 1H NMR and 13C NMR. Moreover, their photochromic and fluorescent behaviors have been investigated systematically by UV–Vis absorption and fluorescence spectra. The two probes have both high selectivity and sensitivity toward Al3+ ions in aqueous medium. The 2:1 stoichiometry between the Al3+ and probes was verified by Job’s plot. Moreover, the limits of detection (LOD) for Al3+ by L and S were 1.98 × 10−8 and 4.79 × 10−8 mol/L, respectively, which was much lower than most previously reported probes. The possible recognition mechanism was that the metal ions would complex with Schiff base probes because of the prevalence of the species optimal for complex formation, inhibiting the structural isomerization of conjugated double bonds (-C=N-), inhibiting the proton transfer process in the excited state of the molecules and resulting in changes of its color and fluorescence behavior. Furthermore, the probes will have potential applications for selectively, detecting Al3+ ions in the environmental system with high accuracy and providing a new strategy for the design and synthesis of multi-functional sensors.Two new Schiff base fluorescent probes (L and S) were designed for selectively detecting Al Howproblems ,5,6. Theproblems ,8,9. Morsessment . Therefo3+ ions, such as inductively coupled plasma atomic emission spectrometry, atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, selective electrode method, and voltammetry [3+ ions with excellent selectivity and high sensitivity. In recent years, the fluorescent probe method has been paid great attention because of its good selectivity, rapid detection, high sensitivity, simple synthesis and convenient operation [3+ ions, including hydrazone compounds [3+ ions, and the LOD was found to be 2.59 × 10−7 M [3+ ions, and this Schiff base could achieve selective detection of Al3+ over other metal ions, such as Zn2+, Hg2+, Cd2+, Pb2+, Mn2+ and so on [3+ ions [At present, there are some reported techniques for detecting Altammetry ,12,13. Hperation ,16,17,18ompounds , rhodamiompounds , quinoliompounds , naphthaompounds , quinoliompounds , and Schompounds ,25,26. Aompounds . For insnd so on . Qi et a[3+ ions .3+ in the DMSO/H2O mixed system with advantages of short recognition time, low cost and low detection limit. The synthesized procedure of probes L and S are shown in 3+ ions by using Schiff bases.Furthermore, Schiff bases have some outstanding advantages, such as higher yield, more stable structure, strong photo-physical properties and stronger fluorescence activity. Schiff base fluorescence probes, owing to hard-base donor sites with nitrogen–oxygen-rich coordination environments, not only have a more stable structure but also have properties of naked-eye recognition and fluorescence detection. Moreover, the conjugated double bonds (-C=N-) in Schiff base fluorescent probes can obtain structural isomerization, which would accelerate the non-radiative transition of the Schiff base in the excited state, further leading to the weak fluorescence of the Schiff base. After adding metal ions, the metal ions would complex with the Schiff base probes, and the protons of the donor will be removed, thereby preventing the structural isomerization of conjugated double bonds (-C=N-), inhibiting the proton transfer process in the excited state of the molecules and resulting in changes of its color and fluorescence behavior. Herein, two Schiff base fluorescent probes were successfully prepared through the one-step method. Synthesized fluorescent probes L and S can effectively recognize Al−5 mol/L) were investigated at room temperature with the following several kinds of metal ions (2.5 × 10−4 mol/L), including Al3+, Mg2+, Pb2+, Zn2+, Cu2+, Mn2+, Co2+, Cr3+, Hg2+, Cd2+, Ni2+ and Fe3+ in DMSO/H2O (v/v = 8:2), as shown in 3+ ions to the solutions containing probes L/S brought about the high fluorescence emission peak at 516 and 518 nm, which can be ascribed to coordination of probes to Al3+, further increasing its structure rigidity and inhibiting the proton transfer process in the excited state of the molecules and C=N isomerization process [3+ ions with high sensitivity. Furthermore, the color of the L/S solution changed from colorless to bright cyan fluorescence after adding Al3+ ions under irradiation of a 430/420 nm lamp, while other metal ions brought about no naked-eye fluorescence, further demonstrating the high selectivity of probes [3+ ions. Hence, it could be concluded that both probes L and S possessed excellent sensitivity and complexation ability for detecting Al3+ ions.The fluorescence emission spectra of solutions for probes L and S  into probe L and S (5.0 × 10−5 mol/L) solutions containing Mg2+, Pb2+, Zn2+, Cu2+, Mn2+, Co2+, Cr3+, Hg2+, Cd2+, Ni2+ and Fe3+ (2.5 × 10−4 mol/L), separately, shown in 3+, suggesting that the other coexistent metal ions have little effect on probes L and S as good fluorescent probes for the selective detection of Al3+ ions.To further confirm the selectivity of fluorescent probes L and S for detecting Al3+ addition on the fluorescence and ultraviolet absorption spectra of probes L and S, fluorescence and ultraviolet absorption experiments were carried out in DMSO/H2O (v:v = 8:2) solution. As illustrated in 3+ concentration (0–150 μM). Moreover, the fluorescence intensity of both probes L and S had a good linear relation between fluorescence intensity and the Al3+ concentrations from 0 to 150 μM, as shown in 3+ ions. Furthermore, detection limits of L and S were calculated to be 1.98 × 10−8 and 4.79 × 10−8 mol/L, respectively, by the formula of LOD = 3σ/K, according to the fluorescence intensity results, which were much lower than most previously reported probes for Al3+ detection [3+ ions with high sensitivity and specificity.In order to investigate the effect of Aletection ,38,39,403+ ions concentration. The absorption peak of probe L at 345 nm gradually disappeared with the increase in Al3+ concentration, and two new absorption peaks appeared at 430 and 302 nm, and absorption intensity at 430 and 302 nm constantly increased with the increase in Al3+ concentration, accompanied with no position change of the peak. The absorbance ratio (y) of probe L at 430 and 302 nm has a good linear relationship with the concentration of Al3+ in the range of 1.0 × 10−6~5.5 × 10−5 mol/L with the detection limit of 3.65 × 10−8 mol/L, as shown in 3+ concentration, and two new absorption peaks appeared at 420 and 303 nm, respectively. The absorption intensity at 420 and 303 nm constantly increased with the increase in Al3+ concentration, accompanied with no position change of the peak. The absorbance ratio (y) of probe S at 420 and 303 nm has a good linear relationship with the concentration of Al3+ in the range of 1.0 × 10−6~6.0 × 10−5 mol/L with a detection limit of 5.26 × 10−8 mol/L, as shown in 3+, with high sensitivity and strong anti-interference ability based on the analysis of UV absorption spectra.Additionally, the UV spectra of both L and S were illustrated in 3+ ions was investigated by utilizing Job’s plot, which was obtained by plotting the molar fraction vs. the changes in the emission intensity at 430 (L) and 420 nm (S), respectively. As shown in 3+/Al3+ + L/S), suggesting the formation of 2:1 stoichiometric complexations between Al3+ and L/S.The combination mode between L/S with Al3+ concentration, and UV absorption peak of probe L/S at 345/350 nm gradually disappeared with the increase in Al3+concentration, and two new absorption peaks appeared at 430/420 and 302/303 nm, respectively. This phenomenon can be explained as follows. The conjugated double bonds (-C=N-) in Schiff base fluorescent probes can obtain structural isomerization, which would accelerate the non-radiative transition of the Schiff base in the excited state, further leading to a weak fluorescence of the Schiff base. After adding Al3+ ions, the metal ions would complex with the Schiff base probes, and the protons of the donor will be removed because of the prevalence of the species optimal for complex formation, preventing structural isomerization of conjugated double bonds (-C=N-), inhibiting the proton transfer process in the excited state of the molecules and resulting in changes of its color and fluorescence behavior [3+ ion to inhibit the isomerization process. The coordination of L/S with Al3+ ions hindered the rotation around the C=N bond and prevented C=N isomerization, resulting in absorption and fluorescence enhancement [Moreover, the complexation mechanism and plausible explanation of changes in absorption and fluorescence intensity are illustrated in behavior . Simultaancement ,41. Furt3+ bonding agent—EDTA. As shown in 3+ ions. However, the fluorescence of L/S was greatly enhanced with the addition of Al3+ ions, which can be ascribed to the coordination between L/S and Al3+ ions, thus emitting bright yellow-green fluorescence. Subsequently, EDTA was added to the system, which resulted in a diminution of the fluorescence intensity at 516 and 518 nm for probes L and S, respectively, indicating regeneration of the free probe L/S. This phenomenon can be attributed to the stronger coordination reaction between Al3+ and EDTA, destroying the complexation between Al3+ and L/S. All these results could clearly indicate that both probes L and S have good reversibility in the detection of Al3+ ions, which is also important for the fabrication of devices to sense the Al3+ ions.The selective recognition of metal ions by probes mainly depends on the binding dynamics between metal ions and N, O atoms with lone pair electrons in the probe molecules. Herein, reversibility experiments were conducted to study the circulation of the probes. The reversibility of the recognition process of probes L/S was performed by adding an Al3+, i.e., 0, 0.1, 0.2, 0.3, 0.5 and 1.0 mmol/L. For probe L/S, the color of the strips was observed to change from ginger/yellow to bright yellow-green under a UV chamber by the naked eye, illustrated in 3+ in a simple and effective way.Filter paper strip experiments were performed to establish another potential application of the probes. Firstly, some thin-layer chromatography (TLC) plates were prepared and coated with probe L/S solution (1 mmol/L) and then dried in air. The prepared TLC strips interacted with various concentrations of Al1H NMR and 13C NMR spectra were tested on a Bruker 400 MHz NMR instrument with TMS as an internal standard . Ultraviolet spectra were obtained using TU-1901 Double beam UV–Visible spectrophotometer . Melting points were obtained on WRX-4 micro melting point instrument .o-Vanillin was purchased from Thain Chemical Technology Shanghai Co., Ltd. . Salicylaldehyde, o-aminophenol and p-aminoacetophenone were purchased from Shanghai McLean Biochemical Technology Co., Ltd. . Dimethyl sulfoxide was purchased from Tianjin Damao Chemical Reagent Factory . All reagents and solvents used in this study were of AR grade. All fluorescence spectra were performed on the F-380 fluorescence spectrometer . FT-IR spectra were obtained on avater-370 Fourier infrared spectrometer using KBr plates . The −1, 1625 cm−1, 1537 cm−1, 1509 cm−1, 1457 cm−1, 741 cm−1; 1H NMR , δ: 12.80 , 8.99 , 8.04 , 7.53–7.48 , 7.32–7.26 , 7.19–7.13 , 6.93 , 3.84 , 2.60 , see 13C NMR , δ: 197.43, 165.39, 152.58, 151.06, 148.42, 135.38, 130.19, 124.33, 122.03, 119.72, 119.26, 116.44, 56.36, 27.17, Firstly, p-aminoacetophenone  was dissolved in 25 mL ethanol (EtOH), which were stirred until soluble. Secondly, o-vanillin  was added into the above solution, and the mixture was refluxed for 8 h at 80 °C with continuous stirring, which then stewed for 24 h. The resulting reaction suspension was filtrated and washed by EtOH to obtain 0.4217 g (78.3%) of orange product . RMM: 270.1125 . FT-IR , δ: 12.73 , 8.97 , 8.02 , 7.69 , 7.51–7.41 , 6.99 , 2.58 , see 13C NMR  δ: 197.36, 197.34, 165.22, 160.83, 152.72, 152.70, 135.35, 134.29, 133.11, 130.16, 122.02, 119.76, 119.73, 117.16, 27.13, 27.11, see The synthesis of salicylaldehyde-p-aminoacetophenone Schiff base was similar to that of o-vanillin-p-aminoacetophenone Schiff base, except that o-vanillin  was substituted for salicylaldehyde . The resulting reaction suspension was filtered and washed with EtOH to obtain 0.3474 g (72.6%) of golden yellow product . RMM: 240.1019 . FT-IR(K−5 mol/L in 100 mL solution of DMSO/H2O (v/v = 8:2). The metal nitrate solutions were prepared as 5.0 × 10−3 mol/L in 50 mL double distilled water . Fluorescence and UV experiments were performed by gradually increasing the concentration of targeted metal ions to the probes solution to evaluate sensitivity. Moreover, the excitation wavelength for L and S was 430 and 420 nm, respectively, in the fluorescence measurements. The slit widths of both the excitation and emission were 5.0 nm. All the experiments were performed under room temperature.An analytical solution of the probe L/S was prepared as 5.0 × 103+ ions at a 1:2 ratio in DMSO/H2O (v:v = 8:2) solution, which could identify Al3+ by ultraviolet visible spectrophotometry and fluorescence with good selectivity, high sensitivity and good reversibility. The LOD of L and S for Al3+ was 1.98 × 10−8 and 4.79 × 10−8 mol/L, respectively, which was lower than most reported studies. The interaction mechanism between L/S and Al3+ was explored by Job’s plot, certifying that the protons of the donor will be removed after its coordination with Al3+ ions, thereby preventing the structural isomerization of conjugated double bonds (-C=N-), inhibiting the proton transfer process in the excited state of the molecules and resulting in changes in its color and fluorescence behavior. All of the results clearly indicated that the probes have potential applications for the detection of Al3+ ions in the environmental system and provide a new strategy for the design and synthesis of multi-functional sensors.Two new types of Schiff base probes L (o-vanillin-p-aminoacetophenone) and S  were successfully prepared by using heating reflux method with its simplicity and high yield. The probe L/S could complex with Al


Objective. Tumour budding and desmoplastic reactions in peritumoural stroma are features of the tumour microenvironment that are associated with colorectal cancer prognosis but have not been as thoroughly examined in gastric cancer. We aimed to further characterize the prognostic role of tumour budding and desmoplastic reaction in gastric adenocarcinoma with intestinal differentiation. Methods. 76 curative gastrectomy specimens were identified, excluding post-neoadjuvant cases or cases with >50% diffuse-type histology. Tumour budding was defined and graded according to the International Tumor Budding Consensus Conference recommendations and desmoplastic reaction was classified as described by Ueno et al 2017. Tumour budding and desmoplastic reaction were analyzed for associations with pathologic features and clinical outcomes. Results. Tumour budding was associated with pT (P < .001), pN (P < .004), overall stage (P < .001), LVI (P < .001) and PNI (P = .002). Desmoplastic reaction was associated with pT (P < .001), pN (P = .005), overall stage (P = .031) and PNI (P < .001), but not LVI. Survival analysis showed decreased overall survival (OS) and recurrence-free survival (RFS) for intermediate and high grade tumour budding . Immature stroma was significantly associated with RFS but not OS. Neither tumour budding nor desmoplastic reaction were independent predictors of OS or RFS on multivariate analysis in this cohort. Conclusion. Tumour budding and desmoplastic reaction were associated with known pathologic risk factors. Prognostically, tumour budding was associated with OS and RFS while desmoplastic reaction was associated with RFS only. Our data suggest that tumour budding and desmoplastic reaction have prognostic value in intestinal-type gastric adenocarcinoma. The TNM classification system (AJCC eighth edition), based on depth of invasion, nodal involvement, and distant metastasis is used in addition to other prognostic factors such as Lauren classification and lymphatic, vascular, and perineural invasion to estimate prognosis of gastric adenocarcinoma patients after resection. Despite such efforts to identify high-risk patients, survival outcomes remain heterogenous and even some early-stage patients may have poor outcomes. Additional prognostic markers would thus be useful to further stratify prognostic subgroups.Gastric adenocarcinoma is the sixth most common cancer in the world and third leading cause of death.312 Tumour budding, defined as single cells or clusters up to four cells present at the invasive front of the tumour, is thought to be a histological representation of epithelial-mesenchymal transition, which is integral to the development of a promigratory phenotype important for invasion and metastasis.1316 As such, tumour budding has been identified as an independent prognostic factor in malignant colorectal polyps and in stage II and III colorectal carcinoma. The International Tumor Budding Consensus Conference (ITBCC) established guidelines for assessing tumour budding in colorectal carcinoma, which has recently been incorporated into the College of American Pathologists’ Cancer Protocol Templates.Tumour budding and desmoplastic reaction in peritumoural stroma are features of the tumour microenvironment that have been linked to prognosis in several cancer types, most prominently colorectal cancer.612 Ueno and colleagues classified desmoplastic reaction into a three-tier system of mature, intermediate, and immature stroma, and found that intermediate and immature stroma were associated with poorer clinical outcomes in colorectal carcinoma patients compared to mature stroma.612Characteristics of desmoplastic reaction have also been shown to be associated with prognosis in colorectal carcinoma, although reporting of this feature is not currently addressed in most cancer protocols.1727 The scoring methods of tumour budding have varied across these studies, with two recent reports applying the ITBCC recommendations in their assessment of tumour budding.27 Furthermore, several of these studies include tumour budding assessment in diffuse-type gastric adenocarcinoma, which often exhibit high-grade tumour budding, and this may confound the prognostic power in patients with intestinal-type gastric adenocarcinoma. Currently, there is no consensus for the assessment of desmoplastic reaction in either colorectal carcinoma or gastric adenocarcinoma, though the three-tier system proposed by Ueno and colleagues has consistently shown independent prognostic value in stage II colorectal carcinoma across multiple cohorts. In this study we further characterized their prognostic role in a cohort limited to gastric adenocarcinoma with at least 50% intestinal differentiation using established ITBCC guidelines for tumour budding and the same criteria applied by Ueno and colleagues in colorectal carcinoma for desmoplastic reaction.The significance of tumour budding and desmoplastic reaction has not been as thoroughly examined in gastric adenocarcinoma, though several studies have suggested that they may have prognostic significance similar to that in colorectal carcinoma.In this single-institution retrospective study, we identified 76 patients with intestinal-type gastric adenocarcinoma treated with primary gastrectomy between 2000 and 2018 at Mount Sinai Hospital, Toronto, Canada. Patients undergoing non-curative surgery (i.e. debulking only) and patients who received neoadjuvant therapy were excluded. Tumours classified by Lauren criteria as “mixed-type” were included for analysis if they had at least 50% intestinal differentiation. Patients were followed up to five years while patients with less than 30 days of follow-up were excluded in survival analysis to omit poor outcomes due to surgical complications. Clinicopathological data including sex, age at diagnosis, tumour depth of invasion (pT stage), number of nodal metastases (pN stage), overall TNM stage, lymphovascular (LVI), perineural invasion (PNI), overall survival (OS) and recurrence-free survival (RFS) outcomes were collected from the electronic patient record and pathology reports. Hematoxylin and eosin (H&E) stained slides were reviewed by one of the authors, a pathology resident with interest in gastrointestinal pathology (C.P.), to assess tumour budding and desmoplastic reaction, after a training set of 20 slides were reviewed with two practicing gastrointestinal pathologists (J.C. and R.K.) with consensus. This study was approved by the institutional Research Ethics Board. Tumour budding was assessed in a single 20x objective with a 22 mm field diameter and divided by the normalization factor (1.210 based on a 22 mm eyepiece field diameter) to obtain the standardized bud count. The standardized bud count was graded into three tiers: low-grade Bd1 (0-4 buds), intermediate-grade Bd2 (5-9 buds), and high-grade Bd3 ( ≥ 10 buds). Only H&E slides were used to assess tumour budding. In cases with significant peritumoural inflammation, which may obscure assessment of buds, the hot-spot slide was reviewed collaboratively by two pathologists (J.C. and R.K.) to achieve consensus of tumour budding grade. In cases with a minor component of diffuse-type histology, tumour budding was only assessed in the areas of intestinal-type differentiation.Tumour budding grade was defined based on the ITBCC 2016 recommendations for colorectal cancer and evaluated using the hot-spot method at the invasive front of the tumour  A-D.5  In addition, stroma within the main tumour mass was also evaluated as demonstrated by Kemi et al, 2018. Cases were classified as mature , intermediate , or immature , with intermediate or immature stroma needing to fill at least one 40x field to be classified as such  and one-way ANOVA  were used to analyze associations of clinicopathological variables between tumour budding grades and desmoplastic reaction classification. Survival analysis was performed using the Kaplan-Meier method (log rank test) and Cox Regression for uni- and multivariate analyses. A p-value of 0.05 was used for determining statistical significance.The study group was comprised of 76 surgically treated patients with gastric adenocarcinoma who did not undergo neoadjuvant therapy . The medP < .001), pN stage (P = .004), overall TNM stage (P < .001), presence of LVI (P < .001) and PNI (P = .002) as shown in Assessment of tumour budding using the ITBCC method revealed 44 patients (58%) with Bd3, 16 patients (21%) with Bd2, and 16 patients (21%) with Bd1 . High-grP = .001), pN stage (P = .005), overall TNM stage (P < .031), and PNI (P < .001), but was not significantly associated with lymphovascular invasion , in keeping with the model that both tumour budding and desmoplastic reaction represent characteristics of the tumour microenvironment associated with prognosis.Desmoplastic reaction was assessed in 72 patients (after excluding 4 pT1a cases): 18 patients (25%) showed mature stroma, 20 patients (28%) showed intermediate stroma, and 34 patients (47%) showed immature stroma . IntermeP = .031, P = .014, P = .069), though there was a significant association with RFS . When tumour budding was stratified into low (Bd1) versus high (Bd2/3) by grouping intermediate and high-grade tumour budding, OS and RFS were significantly higher in Bd1 compared to Bd2/3 .Follow-up data were available for 68 patients from 30 days post-surgery for which survival analysis was performed. Median OS was 20.52 months. The OS outcome was higher in patients with Bd1 compared to Bd2 and Bd3  but not with OS  and RFS . Tumour budding, when stratified by two-tier grading, trended more towards an association with OS and RFS but was not statistically significant . When excluding 4 patients with metastatic disease, tumour budding and desmoplastic reaction remained significantly associated with RFS  but not with OS . Tumour budding, when stratified by two-tier grading in patients without metastatic disease, was significantly associated with OS and RFS . Subgroup analysis for patients without adjuvant chemotherapy showed significance with RFS for tumour budding and DR  but not for OS. When tumour budding is stratified by a two-grade (high vs. low) system it was significantly associated with both OS and RFS .In subgroup survival analysis of node-negative patients only (n = 28), tumour budding and desmoplastic reaction were not significantly associated with OS .Tumour budding and peritumoral stromal morphology are characteristics of the tumour microenvironment that play a role in cancer progression and are associated with clinical outcomes. tumour budding has been extensively studied in colorectal carcinoma where it is an established independent prognostic factor based on studies from multiple large cohorts. More recently, tumour budding has been shown to demonstrate similar prognostic power for other malignancies including esophageal, pancreatic, lung, and oral cancers. Patients with more TCD showed poorer survival outcomes. However, the assessment of TCD was hindered by the lack of objective thresholds for the four categories in this study and especially because most diffuse-type tumours were placed in the predominantly single cell category, which confounded tumour budding with Lauren classification.Neither tumour budding nor desmoplastic reaction have been extensively studied in gastric adenocarcinoma. While this may be partly due to a lower volume of gastric adenocarcinoma compared to colorectal carcinoma in most Western centres, it is likely also related to a lack of standardized guidelines for assessment of these factors in the case of desmoplastic reaction and the conceptual difficulty of how to handle diffuse-type tumours, almost all of which are Bd3 by definition, in the case of tumour budding. In 1992 Gabbert and colleagues described “tumour cell dissociation” (TCD) in gastric cancer (n = 445), and as an early precursor to the current tumour budding scoring system, categorized cases semi-quantitatively into four tiers including no TCD, few single cells, moderate single cells and predominantly single cells.1922 Three studies examined several hotspots at the invasive front to obtain an average tumour budding count,1921 while only two have assessed tumour budding in gastric adenocarcinoma using the ITBCC guidelines.27 In our current study, we applied the ITBCC guidelines in assessing tumour budding using the three-tier system and found statistically significant correlation with survival outcomes. However, our Kaplan Meier analyses showed that survival trends were similar between Bd2 and Bd3 groups. When Bd2 and Bd3 were grouped and compared against Bd1, there was an even more robust correlation with survival outcomes. These data suggest that in gastric adenocarcinoma, the distinction between low grade budding (0-4 buds) and intermediate grade budding (5-9 buds) may be a significant threshold prognostically.Several subsequent studies have categorized tumour budding in gastric cancer into high- and low-grades with varied thresholds ranging from 5 to 10 buds. Our current study consequently excluded cases that were classified as diffuse-type and mixed cases where there was less than 50% intestinal-type differentiation, while only assessing tumour budding in the intestinal-type component of mixed cases that were included.Previous studies on tumour budding in gastric adenocarcinoma have included both diffuse and intestinal type histologies. Subsequently, Kemi et al demonstrated that assessing tumour budding in diffuse-type carcinomas (n = 583) does not show significant prognostic value and is therefore not recommended. However, tumour budding was only documented as present or absent. desmoplastic reaction was categorized based on the presence of lymphoplasmacytic, myxoid and hyaline fibrotic stroma at the deepest point of invasion, although the prognostic significance of the stromal characteristics was not thoroughly discussed. A recent meta-analysis by Guo and colleagues examined data from 7 studies, which showed that high-grade tumour budding, regardless of the definition used, was associated with poorer clinical outcomes in intestinal-type gastric adenocarcinoma and with other pathological prognostic factors such as overall tumour stage, lymph node metastasis, and LVI.Du and colleagues (n = 621) evaluated tumour budding and desmoplastic reaction characteristics in early submucosal cancers. In that study, immature stroma was defined as the presence of keloid-like collagen, and it was noted that there were few tumours that contained myxoid stroma. In contrast, we found, using the criteria described by Ueno et al in colorectal carcinoma, that myxoid stroma was present in 46% of cases. This discrepancy may be due to a difference in the definition of myxoid stroma or, more likely, the minimum amount required to designate the tumour as having immature stroma, as, using the Ueno method, we required only a single full 40x field to be occupied by the most immature stromal type. Hacking and colleagues proposed a modified three-tier method of assessing desmoplastic reaction in colorectal carcinoma by using 0 , 1 , and 2 .30 This system considers the amount of myxoid stroma overall, rather than only the most immature stroma type occupying a 40x field. The authors were able to demonstrate that with this new scoring system, patients could be stratified into risk groups associated with disease-free survival. However, most data on desmoplastic reaction have used the conventional method first described by Ueno and colleagues, as we employed in this current study. The variation in criteria and occasional discrepancies in predicting prognosis between studies demonstrates the need for a standardized approach to assessing desmoplastic reaction in future studies before it can be considered for use in routine clinical practice.There are less data on stromal characteristics in gastric cancer. Kemi and colleagues (n = 583) were the first to demonstrate stromal maturity as an independent prognostic factor in gastric adenocarcinoma using a two-tier system to categorize tumours into mature and immature stroma groups. In our cohort, we found 22 cases where keloid-like collagen or myxoid areas were found within the main tumour mass but not at the invasive front. Our data suggest that assessing tumour at both locations may have a better correlation with recurrence-free survival. Kemi and colleagues have similarly assessed stroma both within the tumour mass and at the invasive front, showing similar associations with and overall survival. More direct comparative analyses between these two methods should be considered in future studies.In the current study, we examined stromal maturity both at the invasive front and within the main tumour mass and used the most immature stromal type in our analysis. Ueno and colleagues examine stromal maturity only at the invasive front.Our study adds to a growing body of literature demonstrating prognostic significance of tumour budding and desmoplastic reaction in gastric adenocarcinoma but also has several limitations. First, the sample size is smaller than other studies reviewed above, largely due to our inclusion criteria which excluded patients who received neoadjuvant therapy, which is being used increasingly commonly in gastric cancer. Eight patients were lost to follow-up, which additionally limited our statistical power due to our small sample size. We did not observe a statistically significant effect on prognosis in multivariate analyses using this limited cohort. However, we were still able to demonstrate sizeable effects for both tumour budding and desmoplastic reaction with a relatively small number of samples. This may be due to the fact that the cases were taken from a single institution, where surgical techniques, adjuvant chemotherapy decisions, and pathologic processing of resected specimens were performed in a consistent and standardized manner. Second, some cohort variables were unknown, including EBV and MSI status, which were unavailable for testing at the time of this study. Although these features are important for molecular subtyping of gastric cancers, they constitute a small proportion of gastric cancers overall and the associated statistical effect on clinical outcomes would likely be very small, if any, in our cohort, where only a few cases in each of these groups would be expected. HER2 status was available for 14 patients, with only four showing positivity and therefore the potential effect of HER2 targeted therapy could not be significantly assessed within our cohort. Third, only one observer performed tumour budding and desmoplastic reaction assessments and therefore we could not assess interobserver variability, although ITBCC consensus recommendations have been shown to have reasonable interobserver agreement in colorectal carcinoma. Assessment of desmoplastic reaction is not as well defined and further studies with larger cohorts and interobserver variability analyses would add to our current knowledge of its reproducibility.Assessment of tumour budding and desmoplastic reaction are not well studied in patients with neoadjuvant therapy and thus, these patients were excluded from this study. However, many patients with gastric cancer are now treated with neoadjuvant therapy and exploring features of the tumour microenvironment in these patients may be helpful in further stratifying risk of recurrence and mortality.Tumour budding and desmoplastic reaction have been shown to be associated with poor prognostic outcomes in colorectal carcinoma and more recently in gastric cancer. In this study of a small but well-controlled cohort of surgically resected intestinal-type gastric adenocarcinoma from a single institution, we provide further support for their prognostic value in gastric adenocarcinoma by showing that tumour budding and desmoplastic reaction were both associated with tumour stage, lymphovascular invasion, and perineural invasion. Tumour budding was associated with OS and RFS, while desmoplastic reaction was associated with RFS in univariate analyses.


ASF1B) has a tight association with the initiation and development of tumours. The expression and regulation mechanism of ASF1B in LGG were discussed.Low-grade glioma (LGG), which is the second most frequent adult brain malignancy, severely threatens patients’ health and has a high recurrence rate. Histone H3/H4 chaperone anti-silencing function 1 B . ChIP assay testified the affinity of ASF1B with TLK1. Subsequently, TLK1 was overexpressed and ASF1B expression interfered, and the functional assays were executed.ASF1B was discovered to be increased in LGG tissues and cells and indicates an unfavourable prognosis for LGG patients. ASF1B was not an independent prognostic factor for LGG. ASF1B deficiency obstructed the proliferation, cell cycle as well as metastasis of LGG cells, and induced cell death, which might be realized through the interaction with TLK1.ASF1B and TLK1 promoted the malignant progression of LGG.TLK1 interacts with ASF1B.Interference with ASF1B inhibits the proliferative, invasive and migratory capabilities and induces the cycle arrest, along with the apoptosis of LGG cells.The interaction between ASF1B and TLK1 promotes the malignant progression of LGG.The interaction between  Gliomas can be sorted into four grades in light of their malignancy . GliomasASF1) participates in DNA-related processes together with transcriptional regulation [ASF1 is composed of two subtypes, including ASF1A and ASF1B. ASF1B is closely implicated in the initiation and development of numerous tumours through regulating cell proliferation [ASF1B promoted the advancement of cervical cancer via stabilizing CDK9 [ASF1B gene [ASF1B motivated the AKT signalling to facilitate cell proliferation and migration in renal clear cell carcinoma [ASF1B on LGG have not been uncovered.Histone H3/H4 chaperone anti-silencing function 1 (gulation . ASF1 isferation . A previferation . ASF1B ping CDK9 . In renaarcinoma . ASF1B sarcinoma . In addiarcinoma . HoweverASF1B and tousled-like kinase 1(TLK1). Being a serine/threonine protein kinase, TLK1 regulated DNA replication as well as DNA repair pathways. Previous literature has shown that the knockdown of TLK1 inhibited the survival of glioblastoma polymorphic cells [TLK1 in oral squamous cell carcinoma [TLK1 was overexpressed in both cholangiocarcinoma and cholangiocarcinoma cells and silencing of TLK1 enhanced cisplatin-stimulated DNA damage [STRING and GeneMANIA databases predicted the possible interaction between ic cells . miR-16 arcinoma . TLK1 waA damage .ASF1B and TLK1 in LGG may affect the malignant progression of LGG. This paper is intended to discuss the role and regulatory mechanism of ASF1B in LGG.Therefore, it is reasonable to hypothesize that the interaction between http://gepia.cancer-pku.cn) predicted ASF1B expression in LGG tissues as well as its correlation with overall survival or disease-free survival of LGG patients [https://www.string-db.org/) [http://genemania.org/) databases predicted the correlation between ASF1B and TLK1 [GEPIA database (patients . STRING db.org/)  and Geneand TLK1 .2. The culture medium was replaced every 2 days.LGG cell lines Res186, Res259, and Hs683 cells were supplied by the Shanghai Institute of Cell Biology and Chinese Academy of Sciences . Normal human astrocytes (NHA) were provided by Shanghai Hongshun Biotechnology Co., Ltd . All cells were cultivated in DMEM medium (Sigma) with 10% FBS (Sigma) as well as 1% antibiotics (Sigma) which were placed in a humidified atmosphere at 37 °C containing 5% COvia Trizol reagent  in light of the standard protocol. With the application of Brilliant II Fast SYBR green QPCR master mix , reverse transcriptase reactions were operated. RT-qPCR was implemented with SYBR Green Master Mix  in 7500 FAST Real-Time PCR System . Comparative Ct method was employed for the calculation of mRNA expression [ASF1B forward: 5′-GCAGGATGCTGAGGAACCAT-3′, reverse: 5′-TTCAGGGTCCCAGTTGCTTC-3′; TLK1 forward: 5′-ACTGGAAGTACGGGCAGTTG-3′, reverse: 5′- CTGTGGGAGGTTTGCGTTTG-3′; GAPDH: forward: 5′-AATGGGCAGCCGTTAGGAAA-3′, reverse 5′-GCGCCCAATACGACCAAATC-3′.RNA was isolated pression . The folProteins were isolated by RIPA lysis buffer . After the subjection to 10% SDS-PAGE, the transferring of proteins to PVDF membranes was operated. Subsequently, the membranes which were impeded with 5% skim milk was cultivated with primary antibodies overnight at 4 °C, following which was the cultivation with secondary antibody conjugated with the horseradish peroxidase for 2 h. Bands were then imaged by an enhanced chemiluminescent (ECL) kit . GADPH served as a control to standardize the results. Protein bands were analysed by ImageJ software.ASF1B (sh-ASF1B) and its empty vector (sh-NC), and TLK1-specific pcDNA overexpression vector (Ov-TLK1) and its empty vector (Ov-NC) were purchased from Sangon Biotech . After cells (1 × 104cells/well) that inoculated into 6-well plates reached 70% confluence, transduction of Ov-NC (10 µl/ml) and Ov-TLK1 (10 µl/ml) or sh-ASF1B (10 µl/ml) and sh-NC (10 µl/ml) was implemented utilizing Lipofectamine 2000  [Short hairpin RNA  was adopted for the evaluation of cell viability in light of the instruction. After the supplementation of CCK-8 solution (10 μl), cells were incubated for 4 h. A microplate reader  was to analyse the OD value at 450 nm. After that, cell viability was evaluated with GraphPad Prism 7.0.Cell Counting kit-8 Following corresponding induction, Res186 cells that were plated in 10-cm plates were stained with 0.05% crystal violet solution for imaging as well as colony counting 14 days later.Flow cytometry was employed for the appraisement of the cell cycle in line with the manufacturer’s protocol. Following corresponding induction, 70% EtOH was to immobilize cells for 15 min before PBS washing. Cell pellets were suspended in 500 µl PI solution  before 40 min of cultivation at 37 °C. For flow cytometry analysis, cells were pelleted with 500 µl PBS.In situ cell death detection kit  was applied for the assessment of cell apoptotic rate.The treated cells were subjected to 10 min of fixation with 4% paraformaldehyde. Res186 cells that were placed into 6-well plates were cultivated overnight till 100% cell fusion was achieved. The initial culture medium was replaced with DMEM containing mitomycin for a further 12 h of cultivation. With the help of a pipette tip, wounds were formed, after which the images were obtained .4 cells which were collected in serum-starved DMEM. The bottom chamber was composed of medium with 10% FBS. 24 h later, residual cells in the upper chambers were discarded. After that, 0.5% crystal violet was added for 10 min of staining and 70% ethanol was utilized for immobilization. Furthermore, six fields that were selected at random were captured and observed (100× magnification).The capability of cells to invade was appraised by employing ECMatrix gel (Chemicon)-coated transwell inserts. The upper chamber was filled with 5 × 10 protocol, ChIP assay was executed. Following cross-linking with 1% formaldehyde, DNA and protein were isolated by SDS lysis buffer, and sheared via sonication. ASF1B antibody (Abcam) was used for immunoprecipitation. RT-qPCR was conducted after the purification of precipitated DNA [Based on the Chromatin immunoprecipitation ated DNA .t-test, one-way ANOVA, along with Tukey’s post hoc test was adopted for the demonstration of comparison among groups. Using Cox proportional risk regression for analysis, the risk ratio (HR) and 95% confidence interval (95% CI) were calculated. Differences were considered to be significant at p value less than 0.05. Each experiment was independently repeated three times.All data that manifested in the format of means ± SD got were analysed using SPSS 19. Student’s ASF1B had higher levels in patients suffering from LGG (ASF1B elevation was prominently concerned with the low overall survival rate of LGG patients (ASF1B expression was markedly related to low disease-free survival of LGG patients (ASF1B expression was remarkably ascended in LGG cell lines by contrast with NHA (ASF1B had the highest level in Res186 cells.GEPIA database showed that from LGG . Moreovepatients . Increaspatients . Thus, wwith NHA . Res186 ASF1B interference plasmid was constructed and its transfection efficiency was detected by RT-qPCR as well as Western blot (ASF1B#2 remarkably diminished ASF1B expression, so sh-ASF1B #2 was chosen for follow-up experiments. CCK8 results corroborated that relative to sh-NC group, cell viability in sh-ASF1B group was distinctly declined (ASF1B interference (ASF1B interference (ern blot . Sh-ASF1declined . Colony rference . It was rference .ASF1B interference (ASF1B group was dramatically strengthened by sh-ASF1B (ASF1B group were apparently lessened, while the expression of pro-apoptotic protein Bax expression was clearly aggrandized when compared to the sh-NC group.Flow cytometry showed that cell cycle arrest occurred after rference . Relativsh-ASF1B . As FiguASF1B interference (Results from wound healing  and Tranrference .ASF1B and TLK1 (TLK1 expression was noticeably fortified in Res186 cells (ASF1B and TLK1 was verified by ChIP assay (ASF1B expression significantly reduced the expression of TLK1 in Res186 cells (STRING and GeneMANIA databases uncovered potential interactions between and TLK1 . In addi86 cells . The tarIP assay . In addi86 cells .ASF1B in LGG was discussed and TLK1 overexpression plasmid was constructed, RT-qPCR, and Western blot was applied to examine the transduction efficacy (ASF1B + Ov-NC group, cell viability in sh-ASF1B + Ov-TLK1 group was evidently aggravated (ASF1B + Ov-TLK1 group was markedly increased (ASF1B + Ov-NC group (ASF1B + Ov-NC group, cell cycle arrest was reversed and apoptosis was conspicuously cut down in sh-ASF1B + Ov-TLK1 group (ASF1B + Ov-TLK1 group, while Bax expression was reduced by contrast with the sh-ASF1B + Ov-NC group. Wound healing and Transwell results uncovered that cell migration (ASF1B + Ov-TLK1 group relative to the sh-ASF1B + Ov-NC group. Western blot analysis of invasion and migration-related proteins exposed that E-cadherin expression in the sh-ASF1B + OV-TLK1 group was cut down, while N-cadherin and Snail expression were fortified by contrast with the sh-ASF1B + Ov-NC group (Next, the regulation mechanism of efficacy . CCK8 regravated . The proncreased , accompaNC group . RelativK1 group . As FiguK1 group  demonstrigration  and invaigration  were notNC group .Glioma is the most prevalent brain malignancy. LGG belongs to grade II-III glioma and is not as aggressive as high-grade glioma . HoweverASF1B in glioma tissue was significantly higher than that in normal tissue, and the increased expression of ASF1B was associated with poor prognosis of glioma patients. In this study, through GEPIA database, ASF1B was noticed to possess elevated expression in LGG tissues, and enhancive ASF1B expression had noticeable relation with unfavorable overall survival and disease-free survival rate of LGG patients. Clearly, our results are consistent with those of Zhu et al. Subsequently, ASF1B expression was also prominently increased in LGG cells. A previous study showed that ASF1B is overexpressed in multiple cancers and is closely associated with cancer survival [ASF1B in Res186 cells could remarkably halt cell proliferation, metastasis and stimulate cell cycle arrest and apoptosis in LGG in our study. ASF1B could enhance the migratory as well as invasive capabilities of lung cancer cells via the modulation of p53-mediated epithelial-mesenchymal transformation signalling pathway [ASF1B [ASF1B possessed fortified expression, and ASF1B absence could accelerate apoptosis, together with cell cycle arrest in hepatocellular carcinoma [ASF1B was also a prognostic marker for breast cancer as well as lung adenocarcinoma, and occupied an important position in the initiation and progression of tumours [ASF1B and its regulatory mechanism in LGG have not been reported. Moreover, gene expression profiles showed a significant increase in ASF1B expression following cerebral trauma injury [ASF1B as a therapeutic target for LGG or other cancers.Zhu et al.  found thsurvival . Next, i pathway . MiR-214y [ASF1B . ASF1B parcinoma . In addi tumours ,30. Howea injury . TherefoASF1B in LGG cells was deeply probed into. Potential interactions between ASF1B and TLK1 were revealed through relevant databases. Subsequently, the transcriptional regulation of TLK1 by ASF1B was confirmed through experiments in this study. It was confirmed that TLK1 mainly functioned dependent on mediating the H3/H4 histone chaperone ASF1, and ASF1B is phosphorylated by TLKs during DNA replication on its C-terminal tail residues S169 and S198 [ASF1B and TLK1 observably had a positive correlation in LGG patients and TLK1 was discovered to be distinctly overexpressed in LGG cells. It was shown that TLK1 expression was elevated in glioma tissues, and had correlation with large tumour volume and higher grade of glioma [TLK1 elevation partially reversed the impacts of ASF1B interference on cell viability, metastasis, cell cycle as well as apoptosis in LGG. The results of Ibrahim et al. [TLK1 inhibited the survival of glioblastoma polymorphic cells. Overexpression of TLK1 can significantly promote the growth, migration, and invasion of glioma cells, and inhibit cell apoptosis [Next, the regulatory mechanism of and S198 . TLK phoand S198 . So in Lf glioma . Our finm et al.  also shopoptosis . Over alin vitro cell experiments were carried out and animal experiments in vivo will be performed in the future to verify this finding.There are some disadvantages in this paper as well. First of all, only Res186 cell line was selected in our experiments and other LGG cell lines need to be chosen for validation in future experiments. Second, only ASF1B and TLK1 promotes the malignant progression of LGG, which lays a theoretical foundation for the discussion of targeted therapy for LGG.In conclusion, this paper confirms that the interaction between


Lenacapavir (LEN) is a highly potent, long-acting, first-in-class inhibitor of HIV-1 capsid protein approved for the treatment of HIV-1 infection in adults with multidrug resistance in combination with other antiretrovirals. CALIBRATE is an ongoing phase 2 study in people with HIV-1 (PWH) who are newly initiating treatment. At Week 80 (W80), subcutaneous (SC) and oral LEN, in combination with other antiretrovirals, maintained high rates of virologic suppression. In PWH initiating treatment, weight increases associated with a return to health effect have been observed. This analysis examined weight and metabolic changes to the W80 timepoint.Participants were randomized (2:2:2:1) to 1 of 4 treatment groups (TG). TG1 and TG2 both received SC LEN (927 mg) every 6 months + oral once daily (QD) emtricitabine/tenofovir alafenamide (F/TAF) for 28 weeks, after which virologically suppressed participants continued a 2-drug maintenance regimen: SC LEN (927 mg) with oral QD TAF (TG1) or oral QD bictegravir (BIC) (TG2). TG3 received oral QD LEN + F/TAF, and TG4 received oral QD BIC/F/TAF throughout. The metabolic profile of LEN was assessed from baseline to W28 and after initiating the 2-drug maintenance regimen to W80. Due to the small sample size, no statistical testing was performed.2, respectively. Weight, BMI, and fasting lipid profiles for each treatment group through W80 are presented (Table).182 participants  were randomized and dosed . Baseline median age was 29 years; 15% had baseline viral load >100,000 c/mL. Baseline median weight and body mass index (BMI) were 78.2 kg and 25.8 kg/mIn this phase 2 study of treatment-naïve PWH, treatment regimens that included SC or oral LEN in combination with other antiretroviral agents led to expected weight gain and increase in BMI, consistent with the return to health phenomenon, and were not associated with clinically relevant increases in lipids.Princy N Kumar, MD, Gilead Sciences, Inc: Grant/Research Support|Gilead Sciences, Inc: Stocks/Bonds|Johnson & Johnson: Advisor/Consultant|Johnson & Johnson: Stocks/Bonds|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Stocks/Bonds|Pfizer: Stocks/Bonds|Theratechnologies: Advisor/Consultant|Theratechnologies: Grant/Research Support|Viiv: Advisor/Consultant|Viiv: Grant/Research Support|Viiv: Stocks/Bonds Aditya H Gaur, MD, Gilead Sciences, Inc (Grant support/CTA with St. Jude): Grant/Research Support|Janssen (Grant support/CTA with St. Jude): Grant/Research Support|Viiv (Grant support/CTA with St. Jude and Serve on Pediatric Advisory Board): Board Member|Viiv (Grant support/CTA with St. Jude and Serve on Pediatric Advisory Board): Grant/Research Support Anson K Wurapa, MD, Gilead Sciences, Inc: Clinical Trial Investigator Ann M. Khalsa, MD, Gilead Sciences, Inc: Advisor/Consultant|Gilead Sciences, Inc: Grant/Research Support|Gilead Sciences, Inc: Honoraria|Glaxo Smith Kline: Advisor/Consultant|Viiv: Advisor/Consultant Cheryl L Newman, MD, Gilead Sciences, Inc: Grant/Research Support|GSK Viiv: Grant/Research Support|GSK Viiv: Honoraria|Janssen: Grant/Research Support|Merck: Grant/Research Support Gary Saunders, BSc, Gilead Sciences, Inc: Employee|Gilead Sciences, Inc: Stocks/Bonds Shan-Yu Liu, PhD, Gilead Sciences, Inc: Employee|Gilead Sciences, Inc: Stocks/Bonds Hadas Dvory-Sobol, PhD, Gilead Sciences, Inc: Employee|Gilead Sciences, Inc: Stocks/Bonds Martin Rhee, MD, Gilead Sciences, Inc: Employee|Gilead Sciences, Inc: Stocks/Bonds Samir K. Gupta, MD, Gilead Sciences: Advisor/Consultant|ViiV Healthcare: Advisor/Consultant|ViiV Healthcare: Grant/Research Support


Magnetic fluids, as smart nanomaterials, have been successfully used in sealing applications and other fields. However, the temperature of magnetic fluids in the sealing gap is a key factor affecting sealing performances, limiting their application in high-speed sealing fields. Since obtaining a direct measurement of the magnetic fluid’s temperature is difficult, due to the small clearance, accurately calculating the maximum temperature of the magnetic fluid layer in high-speed seals is crucial. Herein, a mathematical model for calculating the maximum temperature of the magnetic fluid layer was established, by using a reasonable simplification of high-speed sealing conditions, and the calculation formula was modified by studying the rheological properties of the diester-based magnetic fluid. The results suggest that the calculation of the maximum temperature is influenced by viscous dissipation, and both are related to the rheological characteristics of magnetic fluids. When the influence of rheological properties is ignored, the calculation results are not accurate for higher-velocity seals, but the calculation model applies to lower-velocity seals. When the influence of rheological properties is considered, the calculation results obtained by the corrected formula are more accurate, and they are applicable to both lower- and higher-velocity seals. This work can help us more accurately and conveniently estimate the maximum temperature of magnetic fluids in high-speed seal applications, which is of theoretical and practical research significance for determining sealing performances and thermal designs. A magnetic fluid (MF), a stable colloidal solution, is usually formed by ferromagnetic nanoparticles that are uniformly suspended in a nonmagnetic carrier fluid, utilizing a surfactant ,2. It caIn previous studies, a variety of factors affecting the performance of magnetic fluid seals have been extensively investigated. Studies on the performance of magnetic fluid low-speed seals occupy the majority of them ,6,7. TheSeveral studies have been conducted on viscous dissipation and temperature in magnetic fluid seals. As a non-contact seal, although the friction torque is generated by internal friction in the fluid, the heat accumulated during operations can be significant, due to the small gap, the low volume of magnetic fluids, and the high rotational speed. Hence, some research has focused on the two important parameters of torque friction and viscous dissipation, as they determine the temperature of magnetic fluids in the seal. Li presented analytical methods for studying the technical issues of magnetic fluid seals, such as high speeds and large diameters. It was observed that the primary heat source was viscous drag, and the viscosity of magnetic fluids changes with temperature, leading to complex heat dissipation . Cai et In addition, some experimental studies on the temperature in the sealing gap have also been carried out. Szydlo et al. conducted an experimental study on the temperature and frictional moment of magnetic fluid seals operating under vacuum conditions. It was concluded that the temperature of the seal increases with an increase in rotational speed and the number of sealing stages, both resulting in high viscous drag . BorbáthIn summary, previous studies conducted some theoretical and experimental studies on temperature and viscous dissipation in magnetic fluid seals. In terms of theoretical research, the effects of viscosity and other factors on friction torque and viscous heat were mostly studied, but the rheological properties of magnetic fluids under sealing conditions have rarely been systematically considered in viscous dissipation calculations. In terms of experimental research, most studies focused on the effect of temperature on the sealing capacity, but the temperature of magnetic fluids in the seal makes it difficult to achieve direct and accurate measurements, due to the narrow sealing gap. Therefore, the calculation, and quantification, of temperature in magnetic fluid seals is still incomplete, especially with respect to studying the maximum temperature of magnetic fluids in high-speed seals. This article establishes a calculation model of the maximum temperature of magnetic fluid layers in the sealing gap, by simplifying high-speed sealing conditions. The rheological properties of magnetic fluid under the simulation conditions of high-speed seals are measured, and the formula for calculating the viscous dissipation and MF temperature is subsequently modified. The maximum temperature of diester-based magnetic fluids is calculated accurately, under the condition of high-speed seals without cooling. This theoretical calculation can help to guide the application of magnetic fluids in high-speed sealing fields conveniently and quickly, such as judging the performance of magnetic fluids, sealing capacity, the cooling conditions of the seal, etc. We anticipate that our study can lay theoretical and practical foundations for promoting the research of magnetic fluid high-speed seals, which have great potential applications in military and aerospace and sealing other high-end equipment, in the future.3O4-diester magnetic fluid studied herein was prepared via chemical co-precipitation by our group. The sample was successfully applied in commercial conditions and placed stably for more than three years without visible particle agglomeration. The TEM image of Fe3O4 nanoparticles was observed by the JEM-2100F transmission electron microscope. The FeIn this study, the magnetic fluid’s flow in a high-speed seal clearance is simplified into an incompressible stable flow, in a two-dimensional plate model. In the actual working condition, the sealing gap is narrow, and the magnetic fluid is wrapped by the shaft, pole pieces, and shell; thus, the temperature and heat transfer process of magnetic fluids in the clearance cannot be measured directly. Therefore, the following reasonable assumptions and simplifications were introduced, to calculate the temperature of the magnetic fluid layer in high-speed seals: (1) completely axisymmetric; (2) ignoring the heat transfer along the axial direction, and only the radial heat transfer is considered; (3) the height of the magnetic fluid layer is far smaller than the radius of the shaft, so it can be considered as a flat infinite plate model; (4) the characteristic length of the fluid layer is much larger than its height, and the temperature of the magnetic fluid in the central layer is considered in engineering calculations. A schematic diagram of the calculation model herein is shown in Here, The following governing equations are used to study the flow and heat transfer of magnetic fluid in the above-defined model.For a steady state, the velocity distribution at a certain fluid layer height can be approximately regarded as the following linear relationship:For a given rotating speed, viscous dissipation is determined when the viscosity of a fluid is regarded as a constant, and it can be calculated by the following formula.Therefore, the energy in Equation (2) can be expressed as follows.The following dimensionless parameters are introduced to transform the above Formula (5) into the dimensionless form:Substituting dimensionless parameters (6)–(8) into Formula (5), the dimensionless expression of the temperature field can be obtained.By carrying out the double integration of Equation (9), the general solution of dimensionless temperature is obtained:Under the condition of high-speed sealing, it is considered that the heat flux generated by viscous dissipation is constant throughout the boundary between the rotating shaft’s surface and the magnetic fluid, and the upper boundary between the magnetic fluid and the pole piece surface maintains a constant temperature. Therefore, the following boundary conditions were applied to solve Equation (10):By substituting boundary condition (11) into Equation (10), the following integral constants can be obtained:The dimensionless temperature distribution expression of the magnetic fluid layer is further obtained:According to dimensionless parameter expressions (6)–(8), the dimensional expression of the magnetic fluid’s temperature distribution is as follows:We take a derivative of Formula (15) and obtain the maximum dimensionless temperature when We further obtain the following equation:By substituting the above Formula (18) into Formula (16), the maximum temperature of the magnetic fluid layer in the high-speed seal can be calculated as follows:−1) to allow the viscosity to reach high shear-limiting viscosities.Viscosity is one of the important physical parameters of MF, and it is affected by magnetic fields, shear rate, and temperature. To accurately obtain the viscosity of MF under the sealing conditions, an Anton Paar MCR302 rotational rheometer was used, to measure the rheological properties of MF in a simulated seal environment. The relationship between the shear stress and shear rate was measured at room temperature, as shown in −1, as indicated in Magnetic fluids produce magnetic viscous effects under a magnetic field, and the variation in viscosity with respect to the magnetic field was measured experimentally at a shear rate of 1000 s−1. As depicted in Based on the results of Viscosity is the root cause of thermal dissipation within the magnetic fluid seal, and the temperature increase generated by viscous frictional dissipation is the key reason for the degradation, or even failure, of high-speed magnetic fluid seals. At the starting operation of high-speed seals, the magnetic fluid’s temperature was lower and its viscosity was greater, resulting in higher dissipation power due to viscous friction, which caused the temperature increase in the seal gap. When magnetic fluid temperature increases, viscosity decreases and the dissipation power decreases. Therefore, viscosity, dissipation power, and temperature interact and influence each other. In order to accurately calculate the thermal dissipation power during high-speed seal operations, the viscosity–temperature relationship of magnetic fluids is taken into account. Based on the study of rheological performances in The basic form of magnetohydrodynamic equations is similar to that of conventional fluids, with the exception that magnetic fluid is subjected to an additional magnetic force in the presence of magnetic fields. In high-speed magnetic fluid seal conditions, the following assumptions are made: (1) Magnetic fluid is a homogeneous incompressible Newtonian fluid; (2) it achieved a stable state, By integrating Equation (22), and then substituting the boundary conditions in (24), the analytical solution of the tangential velocity distribution in the l indicates the total axial length of the actual contact between the magnetic fluid and shaft.The subjected viscous shear stress of the magnetic fluid in the sealing gap is expressed as follows:The viscous resistance torque leads to the generation of frictional dissipation power; thus, its expression can be described as follows.From Equation (28), in addition to the influence of the seal’s structural parameters and rotating shaft speed, the viscosity of the magnetic fluid is an important factor affecting the dissipation power.Based on the results and analysis in Substituting the above equation into Equation (9) yields the modified formula for calculating the viscous frictional dissipation power of high-speed diester-based magnetic fluid seals.The following high-speed sealing conditions were taken as an example for calculating the maximum temperature of the diester-based magnetic fluid layer. The radius of the rotating shaft was 24 mm, the rotating speed was 1000–8000 rpm, the sealing clearance was 0.1 mm, and the initial boundary temperature was 20 °C. When the rheological properties of magnetic fluids are ignored, the viscosity of the magnetic fluid can be considered as a constant under the sealing conditions, and then the heat flux is expressed as follows.According to the above equation, the heat flux applied to the sealing gap due to viscous dissipation at different linear velocities was calculated, as depicted in In order to verify the correctness of the calculation model in According to the previous study, described in T in the equation denotes the average integration temperature, and it is obtained by the following equation:When performing viscosity calculations for diester-based magnetic fluids, based on viscosity–temperature expression (29), under the characteristics of sealing conditions, The calculated average integration temperature at different velocities was substituted into Equation (29) to obtain the corresponding viscosity, in order to further obtain the corrected heat flux and the corrected calculation of the magnetic fluid’s maximum temperature. As shown in As stated above, the formula for calculating the maximum temperature of the diester-based magnetic fluid layer in high-speed seals, was corrected by considering the rheological properties of the magnetic fluid as follows:In the present work, a mathematical model for calculating the maximum temperature of the magnetic fluid layer in the sealing gap was established, based on the simplified high-speed sealing conditions. By analyzing the factors affecting the accuracy of the calculation model, the rheological characteristics of the magnetic fluid were measured and the viscous dissipation formula was modified. Thus, a modified calculation formula of the maximum temperature of magnetic fluids in the seals was obtained. The main conclusions from the calculation results are as follows.Under high-speed sealing conditions, the temperature of the magnetic fluid in the sealing gap is mainly influenced by the viscous dissipation generated by viscosity, while viscosity is also influenced by the temperature. When the effect of the rheological properties of magnetic fluid on viscous dissipation is ignored, this calculation model applies to low-speed seals, and the calculation results are not accurate at high-speed seals. When the effect of the rheological properties of magnetic fluids on viscous dissipation is considered, this calculation model applies to both low- and high-speed seals, and the calculation results are more accurate. The correction formula for calculating the maximum temperature of the magnetic fluid is obtained based on the viscosity–temperature function of the magnetic fluid under high-speed sealing conditions. The correction formula applies to predicting the maximum temperature of magnetic fluid in the seal, without cooling, in the range of 0–20 m/s linear velocity. The accuracy of the temperature calculation in the higher linear velocity range needs to be further verified. Although the calculation model simplifies and ignores some influencing factors, and the calculation results have some deviations from the actual values, this calculation method is simple and convenient. Within the allowable range of error, the modified calculation model described in this paper can provide theoretical and practical reference values for quickly understanding and judging the performance of magnetic fluid seals and guiding engineering applications. In the future, we will further improve the modeling and solving methods of the temperature calculation based on this work, to obtain more accurate calculation results and wider applications.


NAR Genomics and Bioinformatics, Volume 3, Issue 2, June 2021, lqab054, https://doi.org/10.1093/nargab/lqab054In the originally published version of this manuscript, the following grant was inadvertently omitted from the Funding section:‘NIH Grant R01CA228272’.These details have been corrected only in this correction notice to preserve the published version of record.


This study aimed to characterise epithelial cell adhesion molecule (EpCAM) expression patterns in colorectal carcinomas (CRC) from Nigerian patients, its association with E-cadherin and tumour characteristics, to forecast patient selection for anti-EpCAM therapy among whom no data existed previously.p ≤ 0.05 regarded as statistically significant.Tissue microarray blocks of formalin-fixed and paraffin-embedded CRC tissues, with their non-cancer margins of resection, were sectioned and stained with EpCAM and E-cadherin primary antibodies. Scoring for antibody staining was done semiquantitatively by combining staining proportion and intensity. The outcome was correlated with patient age, gender and tumour histological parameters with p < 0.001). Also, EpCAM overexpression was significantly associated with reduced E-cadherin (p < 0.035) expression in tumour cells. Tumour extent within the gut wall was equal (50% each) for early and late pT stages among EpCAM overexpressing tumours but two-thirds (8/12) of cases expressing E-cadherin had later pT stage paradoxically, while distant metastasis was negligible among tumours bearing both markers. Also, tumours overexpressing EpCAM had significant association with tumour-associated lymphocytes (p < 0.02 each).Sixty-three carcinoma tissues had staining status for the two markers and were included in this study. Of these, 36 (57.1%) showed positive EpCAM expression (immunoscore ≥3) out of which 83% (30/36 positive cases) were overexpressed (combined immunoscore ≥4) while 12 (19%) tissues were positive for E-cadherin. Non-tumour margins of resection tissues showed less EpCAM positivity in 24% (6/25) of histospots. The difference in staining between tumour and non-tumour margin tissues with EpCAM was significant (CRC in this study preferentially overexpress EpCAM over E-cadherin whose strong cell-cell contact inhibitory role is weakened even when expressed, resulting in further local tumour spread. This, and the observed immune response, supports targeted therapy among eligible patients. Colorectal carcinoma (CRC) is the third most common cancer globally after female breast cancer and lung cancer . WhereasThis was a retrospective descriptive study of CRC resections diagnosed over a 10-year period (January 2008–December 2017) conducted at a University Hospital in Southwestern Nigeria. All patients that had suitable formalin fixed paraffin-embedded (FFPE) cancer tissues at the time of the study were included in the study. Cases with missing FFPE tissue blocks, or unrepresentative/inadequate tissues within the paraffin wax, were excluded. Haematoxylin and eosin-stained slides of the tumour tissues were reviewed to document tumour histology, grade, stage, tumour-associated lymphocytes (TAL) status, lymphovascular invasion and perineural invasion , 13 in aThe Kononen TMA technique was used in the construction of the TMA blocks in this study . H&E staSections (4 μm thick) from the TMA tissue blocks were cut and mounted onto electrostatically-charged slides. Deparaffinisation was by xylene treatment. Antigen retrieval was done using the heat treatment method with incubation in 0.01% buffered Ethylenediaminetetraacetic acid (EDTA) for 5 minutes at 95°C. To block the activities of endogenous peroxidase, the tissue slides were incubated for five minutes in 1% hydrogen peroxidase and subsequently washed in phosphate-buffered saline.The indirect immunoperoxidase staining technique was adopted for the immunohistochemical staining of the tissues. Monoclonal primary antibodies were obtained from Santa Cruz Biotechnology, Inc. Bergheimer Str.89-2, 69115 Heidelberg Germany with catalogue numbers as follows: Ep-CAM, sc-25308; and E-cadherin, sc-56527. Colour development was visualised by incubating the specimen in a hydrogen peroxide-diaminobenzidine mixture mixed in 0.1 M Tris-HCL PH 7.6.Antigen expression was defined as specific when a staining signal was present on the tumour cell membrane. Similar to the Allred score in the evaluation of oestrogen receptor positivity, EpCAM expression was evaluated by calculating a total immunostaining score (TIS) as the product of a proportion score (PS) and an intensity score (IS). The PS describes the estimated fraction of positively stained tumour cells  . The IS Immunostaining intensity and proportion of cellular staining was determined semiquantitatively: the intensity score represents the average staining intensity  . The proSeventy-three (67%) of the 109 histospots had representative tumour tissues fit for evaluation of EpCAM antibody expression, and 65 (60%) histospots for E-cadherin. The others were unevaluable due to the insufficient number of epithelial tumour cells, extensive necrosis, poor tumour preservation or loss of tissue on the TMA. Likewise, the normal (margin of resection) colonic tissue recipient paraffin blocks yielded evaluable 25 (61%) out of 41 histospots created.Thereafter, we identified 63 histospots that when sectioned and stained immunohistochemically on different slides, had adequate tumour cells that could each be evaluated for the presence or lack of EpCAM and E-cadherin expression simultaneously and these were included in the final analysis.t-test statistics. The level of statistical significance was set at ≤0.05.Derived data were analysed statistically using Statistical Package for the Social Sciences version 20. Descriptive statistics was used to determine the proportion of each categorical variable, mean and median of continuous variables. Chi-square test was used to determine association between categorical variables. Differences in means of age were determined using student Of the 63 cases, there were 33 females and 30 males giving a female-male ratio of 1.1:1. The mean age of the patients was 56.6 ± 18 years. p =0.035). The positive and negative EpCAM and E-cadherin immunostains are shown in Positive EpCAM expression  was observed in 36 (57.1%) of the cases. Normal colon tissue showed positive EpCAM expression in 6 (24%) of the 25 histospot specimens. Among the EpCAM-positive tumours, 83.3% (30/36 positive cases) showed marker overexpression . Twenty of the negative cases had no immunostaining while the remaining seven had total immunoscore of 2 (five cases) and score of 1 (two cases). E-cadherin expression occurred in greater proportion among non-tumour-bearing colon tissues being present in 15 (60%) out of 25 tissues from the margins of resection. Tumour tissues had positive expression in 12 (19%) of tumours. Half of the positive tumours showed moderate expression (aggregate immunoscore 3–4) whilst the other half had strong E-cadherin expression (aggregate immunoscore 5–6). There was a significant association between EpCAM and E-cadherin expression in these tumour cells . There was a marginally significant association between E-cadherin positivity and tumour differentiation into low and high grade (p = 0.050).Chi-square test statistic results of the association between the immunomarkers and clinicopathological parameters are shown in Since its discovery, EpCAM has come to be associated with a plethora of roles, including cell adhesion, modulating other adhesion molecules, intracellular signalling, stemness in circulating tumour cells and as a drug target , 18. Excet al [et al [et al [et al [et al [et al [et al [Patients whose tumours express EpCAM might benefit from anti-EpCAM targeted therapy. Overall, the present study found lower positive EpCAM expression compared to the finding of 94% (97/110) by Spizzo et al  among thl [et al , in a stl [et al , reportel [et al , used onl [et al , and thel [et al . Besidesl [et al , had earl [et al .et al [et al [Patient age and gender in this study showed no difference in the proportion of EpCAM expression. This finding is similar to the study by Kim et al . Contrarl [et al , tumour l [et al , 25. Thil [et al .et al [Tumour histology in CRC often reflects the tumour grade (differentiation). Neither tumour histology nor tumour grade was significantly associated with EpCAM expression in the present study contrary to the study by Went et al , which fet al , 25. Yetet al . We thinExisting data on the prognostic role of EpCAM is equivocal, some suggesting a dual role as a tumour suppressor and an oncogene , 26. To This study showed an association between TAL and EpCAM overexpression, that was not seen with E-cadherin but studies describing the association between EpCAM expression and TAL are uncommon thereby making comparison difficult whereas the presence of TIL, , has been associated with better survival outcomes in CRC patients . It is kA limitation of this study was the drawback of the use of the TMA technique limiting tumour tissue representation on the histospots and loss of some histospots by lifting during tissue processing. This reduces the number of cases included in the final data analysis. Other authors have observed similar effects , 20. A sWe have shown in this study that the majority of CRC in our population preferentially overexpress EpCAM over E-cadherin and this favours local tumour progression by conferring more invasive properties to the tumour cells even among E-cadherin-expressing tumours. EpCAM-bearing tumours are also more likely to attract TALs whose role needs to be ascertained. Further studies involving a larger sample volume are required to test these findings and amass data that could determine interventions to mitigate the present precarious state of the disease in our region.The authors declare that they have no conflict of interest to declare with regard to this study.None.This study was reviewed and approved by the University of Ibadan/University College Hospital Ibadan Ethics and Review Board with approval number UI/EC/17/0190.USE, GOO and OJO conceived the study. USE and MIM prepared the TMA FFPE tissue blocks. All the authors reviewed the H&E and immunohistochemistry slides and interpreted the data. All the authors contributed in writing and editing the manuscript. All the authors gave approval to the final draft for submission.


Influenza point of care (POC) testing can support rapid influenza detection and outbreak management in nursing homes (NHs). We hypothesize cost-free on-site access to influenza POC tests increases POC test use and influenza detection. A current study positions us to evaluate this hypothesis.A prospective cohort of U.S. NHs enrolled in a comparative effectiveness trial of baloxavir versus oseltamivir for outbreak management through 2 influenza seasons, 2020-2022 (NCT05012189). We compared study NHs provided with influenza POC tests for use when clinically indicated to non-study facilities participating in weekly National Healthcare Safety Network (NHSN) reporting for occurrence of influenza outbreaks. For study facilities, we received reports upon influenza detection and directly surveyed them for POC use and influenza incidence. We compared study NHs and other NHSN NHs for reported influenza case rates.We recruited 586 facilities with an average of 89 long-stay residents and 120 Medicare certified beds which reported 185 new influenza activity in 159 facilities. Study test use frequency was reported (N=250), mean use was 28 tests per facility whether or not influenza was detected. Upon influenza detection, 52% of NHs initiated antiviral treatment or chemoprophylaxis on 1 or more residents. Study NHs reported 201 cases with a cumulative incidence of 3.7 cases per 1000 resident days versus 2,124 cases (1.7 per 1000) in non-participating NHs in the same period. Non-participating U.S. NHs had fewer beds (76 long-stay residents), 106 Medicare certified beds and less than half the rate of outbreak detection from study facilities.NHs that participated in our prospective study reported influenza disproportionately more than other NHs reporting to NHSN. We interpret that the availability of easy-to-use and freely accessible rapid POC influenza tests positions NHs to detect better and manage influenza outbreaks. The ease of access to POC on-site tests may play a significant role and become a best practice approach in this positive outcome to improve influenza detection and support early intervention.Yasin Abdul, MD, Genentech: Grant/Research Support Ed Davidson, PharmD, MPH, Genentech: Grant/Research Support Kevin McConeghy, PharmD, Genentech: Grant/Research Support|Pfizer: Grant/Research Support|Sanofi-Pasteur: Grant/Research Support|Seqirus: Grant/Research Support Kaley Hayes, PharmD, PhD, Sanofi Aventis: Grant/Research Support Lisa Han, MPH, Genentech: Grant/Research Support Melissa LaMantia, MA, Genentech: Grant/Research Support Elie Saade, MD, Envision Pharma: Speaker, Presenter|Johnson and Johnson: Speaker, Travel, Lodging|Protein Sciences Corp: Grant/Research Support|Sanofi Pasteur: Speaker, Travel, Lodging David Canaday, MD, Pfizer: Grant/Research Support Stefan Gravenstein, MD, MPH, CDC: Grant/Research Support|Genentech: Advisor/Consultant|Genentech: Grant/Research Support|GSK: Advisor/Consultant|GSK: Honoraria|Janssen: Advisor/Consultant|Janssen: Honoraria|NIH: Grant/Research Support|Pfizer: Grant/Research Support|Pfizer: Honoraria|Sanofi: Advisor/Consultant|Sanofi: Grant/Research Support|Sanofi: Honoraria|Seqirus: Grant/Research Support|Seqirus: Honoraria