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Abstract Background The nucleotide-binding oligomeric domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is believed to be a key mediator of neuroinflammation and subsequent secondary brain injury induced by ischemic stroke. However, the role and underlying mechanism of the NLRP3 inflammasome in neonates with hypoxic-ischemic encephalopathy (HIE) are still unclear. Methods The protein expressions of the NLRP3 inflammasome including NLRP3, cysteinyl aspartate specific proteinase-1 (caspase-1) and interleukin-1β (IL-1β), the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor (AMPAR) subunit, and the ATPase valosin-containing protein (VCP/p97), were determined by Western blotting. The interaction between p97 and AMPA glutamate receptor 1 (GluA1) was determined by co-immunoprecipitation. The histopathological level of hypoxic-ischemic brain damage (HIBD) was determined by triphenyltetrazolium chloride (TTC) staining. Polymerase chain reaction (PCR) and Western blotting were used to confirm the genotype of the knockout mice. Motor functions, including myodynamia and coordination, were evaluated by using grasping and rotarod tests. Hippocampus-dependent spatial cognitive function was measured by using the Morris-water maze (MWM). Results We reported that the NLRP3 inflammasome signaling pathway, such as NLRP3, caspase-1 and IL-1β, was activated in rats with HIBD and oxygen-glucose deprivation (OGD)-treated cultured primary neurons. Further studies showed that the protein level of the AMPAR GluA1 subunit on the hippocampal postsynaptic membrane was significantly decreased in rats with HIBD, and it could be restored to control levels after treatment with the specific caspase-1 inhibitor AC-YVAD-CMK. Similarly, in vitro studies showed that OGD reduced GluA1 protein levels on the plasma membrane in cultured primary neurons, whereas AC-YVAD-CMK treatment restored this reduction. Importantly, we showed that OGD treatment obviously enhanced the interaction between p97 and GluA1, while AC-YVAD-CMK treatment promoted the dissociation of p97 from the GluA1 complex and consequently facilitated the localization of GluA1 on the plasma membrane of cultured primary neurons. Finally, we reported that the deficits in motor function, learning and memory in animals with HIBD, were ameliorated by pharmacological intervention or genetic ablation of caspase-1. Conclusion Inhibiting the NLRP3 inflammasome signaling pathway promotes neurological recovery in animals with HIBD by increasing p97-mediated surface GluA1 expression, thereby providing new insight into HIE therapy.

Abstract Fair and timely delivery of supplies plays a critical role under major public health emergencies. In this paper, aiming at fairness and timeliness, an optimization model of open vehicle routing problem for medical supplies distribution is established considering the urgency of the demand. We adopt a differential evolutionary algorithm with fast non-dominated solution sorting to solve the proposed model, obtaining an approximate Pareto optimal solution set. Through the comparison of algorithms, the results showed that the differential evolutionary algorithm with non-dominated sorting is superior with a shorter runtime and more diverse solutions, while the epsilon constraint method has more accurate solutions. In the case verification, the quality of the solutions of both algorithms was within the acceptable range, but the runtime of the epsilon constraint method was too long to be applicable. The results can provide theoretical suggestions and practical guidance for decision-makers in emergency supplies distribution.

Background: Cognitive impairment is a serious nonmotor symptom in patients with Parkinson's disease (PD). Currently, there are few studies investigating the relationship of serum markers and retinal structural changes with cognitive function in PD. Objective: To investigate the relationship between retinal structural changes, serum high mobility group box-1 (HMGB1) levels and cognitive function and motor symptoms in PD patients. Methods: Eighty-nine participants, including 47 PD patients and 42 healthy subjects, were enrolled. PD patients were divided into Parkinson's disease with normal cognitive (PD-NC), Parkinson's disease with mild cognitive impairment (PD-MCI), and Parkinson's disease with dementia (PDD) groups. The motor and nonmotor symptoms of PD patients were evaluated with clinical scale. Serum HMGB1 levels were detected by enzyme-linked immunosorbent assay (ELISA), and ganglion cell-inner plexiform layer complex (GCIPL) thickness changes in the macula were quantitatively analyzed by swept source optical coherence tomography (SS-OCT) in all patients. Results: Compared with the control group, the macular GCIPL (t = −2.308, P = 0.023) was thinner and serum HMGB1 (z = −2.285, P = 0.022) was increased in PD patients. Macular GCIPL thickness in patients with PD-MCI and PDD were significantly lower than that in PD-NC patients, but there were no significant difference between the PD-MCI and PDD groups. Serum HMGB1 levels in patients with PD-MCI and PDD were significantly higher than those in PD-NC patients, and serum HMGB1 levels in PDD patients were higher than those in PD-MCI patients. Correlation analysis showed that serum HMGB1 levels in PD patients were positively correlated with disease duration, HY stage, UPDRS-I score, UPDRS-III score, and UPDRS total score and negatively correlated with MOCA score. Macular GCIPL thickness was negatively correlated with HY stage and positively correlated with MOCA score, and macular GCIPL thickness was negatively correlated with serum HMGB1 level. Logistic regression analysis showed that elevated serum HMGB1 level, thinner macular GCIPL thickness, and higher HY stage were independent risk factors for Parkinson's disease with cognitive impairment (PD-CI). The areas under the receiver operating characteristic curve (AUC) for the serum HMGB1 level and macular GCIPL thickness-based diagnosis of PD-MCI, PDD and PD-CI based on in patients with PD were 0.786 and 0.825, 0.915 and 0.856, 0.852 and 0.841, respectively. The AUC for the diagnosis of PD-MCI, PDD and PD-CI with serum HMGB1 level and GCIPL thickness combined were 0.869, 0.967 and 0.916, respectively. Conclusion: The macular GCIPL thickness and serum HMGB1 level are potential markers of cognitive impairment in PD patients, and their combination can significantly improve the accuracy of the diagnosis of cognitive impairment in PD.

Porcine epidemic diarrhea virus (PEDV) is one of the critical pathogens causing diarrhea in piglets and has caused huge economic losses to the swine industry in worldwide. However, there is currently no effective therapeutic medication available for the treatment of PEDV. Natural compounds are a hot topic for researching and screening antiviral lead compounds due to their abundant sources, varied activities, and low toxicity. In this study, a total of 6 compounds from different plant sources were selected for in vitro anti-PEDV screening, including chrysin, naringenin, soy isoflavone, glycyrrhetinic acid, oleanolic acid, and geniposide. Then two active compounds, chrysin and naringenin, were further evaluated on PEDV infected cells at different stage. And the anti-PEDV mechanism was analyzed by molecule docking and molecular dynamics. The results showed that both chrysin and naringenin showed the most significant anti-PEDV activity by increasing the cell viability and decreasing the virus copy number. Both natural compounds could inhibit viral titer, mRNA and protein levels in the prophylactic and post-viral entry stages of PEDV infection. Furthermore, chrysin and naringenin mainly interacted with viral replicase proteins such as 3CLpro and PLP-2 through hydrogen bonds and hydrophobic forces. The complexes formed by chrysin and naringenin with the two PEDV replication proteases had high stability. These results suggested that chrysin and naringenin may exert antiviral effects by interacting with the virus 3CLpro protein or PLP2 protein, thereby affecting their role in the formation of PEDV non-structural proteins or interfering with virus replication. This study lays the foundation for developing chrysin and naringenin as novel anti-PEDV therapeutic drugs.

BackgroundBladder cancer (BLCA) is a common urinary system malignancy with a significant morbidity and death rate worldwide. Non-muscle invasive BLCA accounts for over 75% of all BLCA cases. The imbalance of tumor metabolic pathways is associated with tumor formation and proliferation. Pyrimidine metabolism (PyM) is a complex enzyme network that incorporates nucleoside salvage, de novo nucleotide synthesis, and catalytic pyrimidine degradation. Metabolic reprogramming is linked to clinical prognosis in several types of cancer. However, the role of pyrimidine metabolism Genes (PyMGs) in the BLCA-fighting process remains poorly understood.MethodsPredictive PyMGs were quantified in BLCA samples from the TCGA and GEO datasets. TCGA and GEO provided information on stemness indices (mRNAsi), gene mutations, CNV, TMB, and corresponding clinical features. The prediction model was built using Lasso regression. Co-expression analysis was conducted to investigate the relationship between gene expression and PyM.ResultsPyMGs were overexpressed in the high-risk sample in the absence of other clinical symptoms, demonstrating their predictive potential for BLCA outcome. Immunological and tumor-related pathways were identified in the high-risk group by GSWA. Immune function and m6a gene expression varied significantly between the risk groups. In BLCA patients, DSG1, C6orf15, SOST, SPRR2A, SERPINB7, MYBPH, and KRT1 may participate in the oncology process. Immunological function and m6a gene expression differed significantly between the two groups. The prognostic model, CNVs, single nucleotide polymorphism (SNP), and drug sensitivity all showed significant gene connections.ConclusionsBLCA-associated PyMGs are available to provide guidance in the prognostic and immunological setting and give evidence for the formulation of PyM-related molecularly targeted treatments. PyMGs and their interactions with immune cells in BLCA may serve as therapeutic targets.

Abstract Neonatal hypoxic-ischemic encephalopathy (HIE) induced by perinatal asphyxia is a major cause of neurological disability among infants. Dihydroartemisinin (DHA), derived from artemisinin, well known as an anti-malarial medicine, was proved to be able to inhibit oxidative stress and inflammation. However, whether those functions of DHA play roles in hypoxic-ischemic brain damage (HIBD), an animal model of HIE in patient which also been observed to have oxidative stress and inflammation, is unknown. In this study, we demonstrated that the DHA treatment on newborn rats significantly relieved the neuron loss and motor and cognitive impairment caused by HIBD. One of the underlying mechanisms is that DHA enhanced the anti-oxidant capacity of HIBD rats by up-regulating the total antioxidant capacity (T-AOC), gluathione reductase (GR) and catalase (CAT) while down regulating the pro-oxidative substances including hydrogen peroxide (H2O2), total nitric oxide synthase (T-NOS) and inducible nitric oxide synthase (iNOS). Thus, our study illustrated that DHA could alleviate the damage of brains and improve the cognitive and motor function of HIBD rats by inhibiting oxidative stress, provided an opportunity to interrogate potential therapeutics for affected HIE patients.

After experiencing the COVID-19 pandemic, employees' health and well-being become a priority for firms. Organizational health-oriented strategies assist them in coping with health-related crises. Based on the social exchange theory, the present study attempts to determine the role of organizational health-oriented strategies in promoting employees' job performance. This study hypothesized that the organizations' health-oriented strategies positively correlate with employees' job performance. This study also assessed the mediating role of employees' psychological wellbeing and trust and moderating role of perceived medical mistrust. For the empirical examination, data of the present study was gathered from the textile sector in China. This study analyzed data through partial least square structural equation modeling (PLS-SEM). For this purpose, Smart-PLS software was used. The outcomes revealed that organizational health-oriented strategies positively enhance the employees' psychological wellbeing, trust, and job performance. Moreover, the results revealed that employees' psychological wellbeing and trust positively mediate the proposed relationships. This study found that perceived medical mistrust moderates the relationship between employees' psychological wellbeing and job performance. However, the findings revealed that perceived medical mistrust does not moderate the relationship between employees' trust and job performance. In addition, the present study's findings provide insights to the firms about the importance of health-oriented strategies. Moreover, this study's findings also serve the literature by providing important theoretical and practical implications.

Water conservancy personnel usually need to know the water level by water gauge images in real-time and with an expected accuracy. However, accurately recognizing the water level from water gauge images is still a complex problem. This article proposes a composite method applied in the Wuyuan City, Jiangxi Province, in China. This method can detect water gauge areas and number areas from complex and changeable scenes, accurately detect the water level line from various water gauges, and finally, obtain the accurate water level value. Firstly, FCOS is improved by fusing a contextual adjustment module to meet the requirements of edge computing and ensure considerable detection accuracy. Secondly, to deal with scenes with indistinct water level features, we also apply the contextual adjustment module for Deeplabv3+ to segment the water gauge area above the water surface. Then, the area can be used to obtain the position of the water level line. Finally, the results of the previous two steps are combined to calculate the water level value. Detailed experiments prove that this method solves the problem of water level recognition in complex hydrological scenes. Furthermore, the recognition error of the water level by this method is less than 1 cm, proving it is capable of being applied in real river scenes.

Bisphenols (BPs) are a class of endocrine disruptors widely existing in the environment. They have a great impact on human health owing to their environmental endocrine disrupting effects, chronic toxicity, neurotoxicity, cytotoxicity and genetic toxicity. In this paper, an on-line packed fiber solid phase extraction (PFSPE) coupling with column-switching HPLC-FLD determination method was developed for the determination of eight BPs in drinking water. The poly (dibenzo-18-crown-6-ether)/polystyrene composite nanofibers (PDB18C6/PS) were prepared by electrospinning and used as an adsorbent for the on-line PFSPE column. The on-line PFSPE-HPLC equipment contained a dual ternary pump and a switching valve to enable enrichment, purification, and analysis directly in the system. The results showed that the proposed on-line PFSPE-HPLC-FLD method realized the simultaneous separation and detection of eight BPs: BPF, BPE, BPA, BPB, BPAF, BPAP, BPC and BPZ. The curves of the target analytes were prepared with good correlation coefficient values (r2 > 0.998) in the range of 50–1000 pg/mL. The limit of detection (S/N = 3) was 20 pg/mL, the limit of quantitation (S/N = 10) is 50 pg/mL. The recoveries of eight BPs were 94.8–127.3%, and the intra-day precisions (RSD) were less than 10%. The PFSPE column made of the PDB18C6/PS composite nanofibers has stable properties and can be reused at least 200 times. In the detection of drinking water samples, BPZ was detected in nearly 80% of drinking water samples, and BPA, BPAP, BPF and BPAF were also detected in some water samples. This high level of integration and automation was achieved in pretreatment of eight BPs from water samples. The proposed simple, rapid, and practical method has been successfully applied to the detection of eight BPs in drinking water, which can provide powerful technical support for drinking water quality and safety monitoring.

Two different pretreatment approaches have been used for the enrichment and separation of biogenic monoamines and metabolites in plasma for high performance liquid chromatography (HPLC) determination. The first approach, based on on-line packed-fiber solid-phase extraction (PFSPE) coupled with HPLC, allows for the simultaneous detection of epinephrine (E), norepinephrine (NE), dopamine (DA), 3-methoxyl epinephrine (MN), norepinephrine (NMN), 3-methoxytyramine (3-MT), and 5-hydroxytryptamin (5-HT). Using this developed on-line PFSPE–HPLC method, the limit of detections (LODs) of the seven analytes ranged from 1 ng/mL (NMN and MN) to 2 ng/mL (NE, E, DA, 3-MT and 5-HT). The reportable ranges were 5–300 ng/mL for NE and DA, 5–100 ng/mL for E, and 5–200 ng/mL for NMN, MN, 3-MT and 5-HT. The off-line PFSPE–HPLC was employed in the second approach and could provide simultaneous detection of NE, E, DA, NMN, and MN. The linearity was verified in the range of 0.5–20 ng/mL (NE, E, and DA) and 20–250 ng/mL (NMN and MN). The LODs of the five analytes ranged from 0.2 ng/mL (NE, E, and DA) to 5 ng/mL (NMN and MN). This study verified the possibility of using nanofibers as an adsorbent in an on-line PFSPE–HPLC system for the determination of biogenic monoamines and their metabolites in human plasma. Compared with the off-line PFSPE approach, the on-line PFSPE method deserves attention mainly due to its greener character, derived from the automation of the process and high-throughput with less operators’ handling.

With the wide application of mobile healthcare systems, the total amount of healthcare data is ever increasing rapidly as users interact with healthcare service providers frequently. This leads to a challenging task to manage healthcare data. Existing work mainly pay attention to centralized and blockchain-based mechanisms. But they cannot adapt to the increasing amount of global healthcare data and suffer from complex application challenges, respectively. Decentralized and collaborative data management assisted by edge computing exhibits major advantages in improving overall system performance. We present a secure and efficient data management system named as EdgeCare for mobile healthcare systems. Local authorities are established to schedule edge servers for processing healthcare data and facilitating data trading. A hierarchical architecture with collaboration is designed for feasible implementation of EdgeCare. After that, we investigate secure data uploading and sharing in the system. We use an electronic medical record to show how healthcare data is processed with security considerations. We also conduct the Stackelberg game-based optimization algorithm to approach the optimal incentive mechanism for a data collector and users in the fair decentralized data trading. The numerical results with security analysis are provided to demonstrate that EdgeCare offers effective solutions to protect healthcare data, and support efficient data trading.

In data center networks, MultiPath TCP (MPTCP) obtains both higher network utilization and fairer allocation of capacity by exploring multiple paths simultaneously. However, MPTCP experiences more queue oscillation in switch buffer with the increasing of the number of subflows. When using explicit congestion notification as the congestion indication to track the queue length in switch buffer, it is difficult for MPTCP to capture the accurate congestion state, resulting in wrong behavior in congestion control. Therefore, we propose an enhanced MPTCP protocol, namely, advanced MPTCP (AMP), which adjusts the time granularity of the congestion detection and control under a different number of subflows. The test results show that compared with Linked Increases Algorithm and eXplicit MultiPath, AMP achieves lower latency for small flows and higher throughput for large flows.

Every metal and metallurgical industry is associated with the generation of wastewater, influencing the living and non-living environment, which is alarming to environmentalists. The strict regulations about the dismissal of acid and metal into the environment and the increasing emphasis on the recycling/reuse of these effluents after proper remedy have focused the research community’s curiosity in developing distinctive approaches for the recovery of acid and metals from industrial wastewaters. This study reports the synthesis of UiO-66-(COOH)2 using dual ligand in water as a green solvent. Then, the prepared MOF nanoparticles were introduced into the DMAM quaternized QPPO matrix through a straightforward blending approach. Four defect-free UiO-66-(COOH)2/QPPO MMMs were prepared with four different MOF structures. The BET characterization of UiO-66-(COOH)2 nanoparticles with a highly crystalline structure and sub-nanometer pore size (~7 Å) was confirmed by XRD. Because of the introduction of MOF nanoparticles with an electrostatic interaction and pore size screening effect, a separation coefficient (SHCl/FeCl2) of 565 and UHCl of 0.0089 m·h−1 for U-C(60)/QPPO were perceived when the loading dosage of the MOF content was 10 wt%. The obtained results showed that the prepared defect-free MOF membrane has broad prospects in acid recovery applications.

Background: Puffball is a common Chinese medicine of edible and medicinal fungus. It promotes hemostasis and muscle regeneration. Recent studies have shown that puffball has a limiting effect on pain and inflammation, but the specific mechanism behind this action remains unclear. This study aimed to reveal the pharmacological mechanism of the anti-inflammatory effect of puffball using the network pharmacology method. Methods: TCMSP and SwissTargetPrediction were used to analyze and predict the active ingredients and corresponding targets in puffball. The DisGeNET and GeneCards databases were searched to obtain inflammation-related targets, and the PPI network of the cross-genes of puffball ingredients and diseases was constructed through STRING and Cytoscape. The Metascape database was used for GO function enrichment analysis of co-presence genes and KEGG signal pathway enrichment analysis to construct an “ingredient-target-pathway” network and analyze its mechanism of action. Meanwhile, molecular docking was used to verify the ability of active ingredients to bind to key targets. Results: A total of 6 inflammation-related active ingredients were screened from the 32 candidate compounds of puffball, and 236 targets that correspond to ingredients and 328 inflammation-related targets were collected. In summary, the β-sitosterol and melanin components in puffball can act on signal transducers and activators of transcription (STAT3) and epidermal growth factor receptor (EGFR) targets and are mainly differentiated through Th17 cells signal pathway and cancer-related pathway to play an anti-inflammatory effect. Conclusion: This study has successfully predicted the active ingredients and potential targets of Puffball's anti-inflammatory effects, which helped to systematically elucidate its mechanism of action. The network pharmacology method can provide new insights for the research and development of edible and medicinal fungi.

Abstract In this paper, we consider a massive MIMO-enabled multi-way amplify-and-forward relay network with channel aging, where multiple users mutually exchange information via an intermediate relay equipped with massive antennas. For this system, we propose an energy-efficient power allocation scheme for the optimization of energy efficiency (EE). Specifically, we firstly derive accurate closed-form expressions of the system sum rate with aged channel state information (CSI) and predicted CSI. Secondly, based on the derived analytical results, a unified power allocation optimization problem with aged/predicted CSI is formulated for maximizing the system EE. To solve this challenging problem, the successive convex approximation technique is invoked to transform the original optimization problem into a tractable concave fractional programming problem. Then, Dinkelbach’s algorithm and Lagrangian dual method are adopted to find the optimal solution. In addition, to strike a balance between the computational complexity and the optimality, the EE maximization problem using the equal power allocation scheme is solved by extreme value theorem, leading to a closed-form optimal solution. Numerical results demonstrate the accuracy of our analytical results and the effectiveness of the proposed algorithms. Moreover, the impact of several important system parameters on the system performance achieved by the proposed algorithms is also illustrated.

To achieve the maximum network energy efficiency (EE) and guarantee the fairness of EE among cognitive users (CUs), respectively, in the massive multiple-input multiple-output cognitive radio network, we investigate two power optimization problems: network EE optimization problem (NEP) and fair EE optimization problem (FEP) under a practical power consumption model. Because of the fractional nature of EE and the interference, both NEP and FEP are non-convex and NP-hard. To tackle these issues, we propose two energy-efficient power control algorithms, in which we decompose NEP/FEP into two steps, and solve them with an alternating iterative optimization scheme. Specifically, in the first step, for an initial transmit power, the maximum network EE/fair EE is achieved by the bisection method based on fractional programming; then, with the achieved EE, in the second step, the adapted optimal transmit power can be obtained by an efficient iterative algorithm based on sequential convex programming. These two steps are performed alternately until the stop conditions are reached. Numerical results confirm the fast convergence of these proposed algorithms and demonstrate their effectiveness with high network EE and well fairness of EE among CUs. Furthermore, it is illustrated that, under a practical power consumption model, more cognitive base station antennas would cause some loss of network EE but bring some improvements on the network spectral efficiency (SE), whereas higher circuit power consumption would reduce the network EE but only slightly affect the network SE.

This paper proposes a roadside unit (RSU)-coordinated synchronous multi-channel medium access control (MAC) scheme for vehicular ad hoc networks (VANETs). The proposed scheme allows the on board unit (OBU) to reserve service channels (SCHs) on the control channel (CCH) during almost the whole synchronous interval and supports simultaneous transmissions on different SCHs. It enhances the performance of VANETs and decreases the CCH congestion. Moreover, we use RSU to record the rendezvous information and broadcast it to the OBUs. This method avoids the multi-channel hidden terminal problem. An analytical model is developed to evaluate the aggregate throughput on SCHs. This model considers the following factors: non-safety message transmission probability in each synchronous interval, average size of the non-safety message, and the number of OBUs. Furthermore, the requirement of obtaining the maximum throughput is computed. Simulation results are provided to validate the analytical model and to demonstrate the improvement in throughput. The results indicate that the proposed scheme can provide higher aggregate throughput than that of the vehicular enhanced multi-channel MAC and IEEE 1609.4, and better performance in CCH congestion control especially under high network load conditions.

A new distance-aware broadcasting algorithm was proposed to enhance the propagation distance in the latency time of safety-related message broadcasting. The IEEE 802.11p standard states that if the medium is detected as idle, a station would defer its transmission within a backoff time to avoid collisions with other stations. The backoff times follow uniform distribution over [0, CW ]. In this way, fairness among all the stations can be guaranteed. However, propagation distance was ignored and in safety-related message broadcasting fairness is not the most important issue. In the proposed algorithm, the lengths of backoff times are generated from a nonuniform distribution. They are related with the distances between the source station and its forwarding stations. The farthest forwarding station has the highest probability to forward messages. Performance of the proposed algorithm is analyzed by using a 2D Markov chain. Analytical and simulation results demonstrate that the proposed algorithm can enhance the performance of safety-related message broadcasting in terms of propagation distance, which is reflected by the successful transmission probability. The proposed algorithm does not need additional waiting time, RTS/CTS, and ACK, therefore having better compatibility with the IEEE 802.11p standard than earlier distance-aware algorithms.

BACKGROUND:Achaete scute-like 2 (Ascl2), a basic helix-loop-helix (bHLH) transcription factor, controls the fate of intestinal stem cells. However, the role of Ascl2 in colon cancer progenitor cells remains unknown. The cell line HT-29 (47.5-95% of CD133(+) population) and LS174T (0.45% of CD133(+) population) were chosen for functional evaluation of Ascl2 in colon cancer progenitor cells after gene knockdown by RNA interference. METHODOLOGY/PRINCIPAL FINDINGS:Immunohistochemistry demonstrated that Ascl2 was significantly increased in colorectal adenocarcinomas. Downregulation of Ascl2 using RNA interference in cultured colonic adenocarcinoma HT-29 and LS174T cells reduced cellular proliferation, colony-forming ability, invasion and migration in vitro, and resulted in the growth arrest of tumor xenografts in vivo. The Ascl2 protein level in CD133(+) HT-29 cells was significantly higher than in CD133(-) HT-29 cells. Ascl2 blockade via shRNA interference in HT-29 cells (shRNA-Ascl2/HT-29 cells) resulted in 26.2% of cells staining CD133(+) compared with 54.7% in control shRNA-Ctr/HT-29 cells. The levels of 'stemness' associated genes, such as CD133, Sox2, Oct4, Lgr5, Bmi1, and C-myc, were significantly decreased in shRNA-Ascl2/HT-29 and shRNA-Ascl2/LS174T cells in vitro as well as in the corresponding tumor xenograft (CD133 was not performed in shRNA-Ascl2/LS174T cells). The shRNA-Ascl2/HT-29 cells had inhibited abilities to form tumorspheres compared with control. The microRNA (miRNAs) microarrays, identified 26 up-regulated miRNAs and 58 down-regulated miRNAs in shRNA-Ascl2/HT-29 cells. Expression levels of let-7b, miRNA-124, miRNA-125b, miRNA-17, miRNA-20a and miRNA-302b, involved in the regulation of 'stemness', were quantified with qPCR, which confirmed their identities. Restoration of miRNA-302b, via its mimic, led to the restoration of shRNA-Ascl2/HT-29 'stemness' characteristics, including tumorsphere formation and 'stemness' associated genes levels, and the recovery of cellular behaviors, including colony-forming ability, invasion and migration in vitro. CONCLUSIONS/SIGNIFICANCE:Ascl2 may be a potential target for the inhibition of colon cancer progenitor cells, and functions through a miR-302b-related mechanism.

The secreted proteins from goblet cells compose the intestinal mucus. The aims of this study were to determine how they exist in two intestinal mucus layers.The intestinal mucosa was fixed with Carnoy solution and immunostained. Mucus from the loose layer, the firm layer was gently suctioned or scraped, respectively, lysed in SDS sample buffer with or without DTT, then subjected to the western blotting of rTFF3, rIgGFcγBP or rMuc2. The non-reduced or reduced soluble mucus samples in RIPA buffer were co-immunoprecipitated to investigate their possible interactions. Polyclonal antibodies for rTFF3, the rIgGFcγBP C-terminal domain and the rMuc2 C-terminal domain confirmed their localization in the mucus layer and in the mucus collected from the rat intestinal loose layer or firm layer in both western blot and immunoprecipitation experiments. A complex of rTFF3, which was approximately 250 kDa, and a monomer of 6 kDa were present in both layers of the intestinal mucus; rIgGFcγBP was present in the complex (250-280 kDa) under non-reducing conditions, but shifted to 164 kDa under reducing conditions in both of the layers. rMuc2 was found mainly in a complex of 214-270 kDa under non-reducing conditions, but it shifted to 140 kDa under reducing conditions. The co-immunoprecipitation experiments showed that binding occurs among rTFF3, rIgGFcγBP and rMuc2 in the RIPA buffer soluble intestinal mucus. Blocking the covalent interaction by 100 mM DTT in the RIPA buffer soluble intestinal mucus disassociated their binding.Rat goblet cell-secreted TFF3, IgGFcγBP and Muc2, existing in the two intestinal mucus layers, are bound together by covalent interactions in the soluble fraction of intestinal mucus and form heteropolymers to be one of the biochemical mechanisms of composing the net-like structure of mucus.