The mechanism of fluidity improvement of cement slurry by graphene oxide: a study on nanofriction.

Graphene oxide (GO) as a nano-reinforcing material has received extensive attention in cement composite materials. This paper employed molecular dynamics to simulate the friction process of calcium silicate hydrate (CSH) particles in the presence of double-sided and single-sided GCOOH (graphene oxide with a -COOH functional group, covering 10% of the surface). The investigation uncovered the lubricating effects of bifacial and unifacial GCOOH on the CSH interface. The findings indicate that the interfacial friction among CSH particles follows the sequence of double-sided GCOOH > pure CSH > single-sided GCOOH. In the double-sided GCOOH system, a greater external force is needed on the opposing side to alter the interaction with water molecules, calcium ions, and silica-oxygen tetrahedra, thereby enhancing friction. In contrast, the majority of the carboxyl groups on the single-sided GCOOH surface are strongly adsorbed onto the CSH surface, facilitating the entry of additional water molecules into the interlayer. Conversely, the unmodified side of the GCOOH has lower interactions with water molecules, hence improving its lubricating properties.

Molecular Insight into the Pozzolanic Reaction of Metakaolin and Calcium Hydroxide.

The reaction mechanism of the pozzolanic reaction of metakaolin (MK) from the atomic point of view has not yet been explored. To explain the process and mechanism of the pozzolanic reaction from the atomic point of view, molecular insight into the pozzolanic reaction of MK and calcium hydroxide (CH) was analyzed through the reaction molecular dynamics (MD) simulation. The results show that the pozzolanic reaction of MK and CH can be essentially regarded as the CH decomposition and penetration into MK. Also, the structure evolution after the pozzolanic reaction shows that the water molecules cannot penetrate the MK structure till the participation of Ca2+ and OH- ions of CH. The Ca2+ and OH- ions have strong interaction with MK and drill into the MK structure, followed by the destruction of a part of the MK structure and water penetration. The final structure of CH removed by MK can be regarded as the precursor of the CASH gel structure.

Nitric Oxide Synthases in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by severe joint damage and disability. However, the specific mechanism of RA has not been thoroughly clarified over the past decade. Nitric oxide (NO), a kind of gas messenger molecule with many molecular targets, is demonstrated to have significant roles in histopathology and homeostasis. Three nitric oxide synthases (NOS) are related to producing NO and regulating the generation of NO. Based on the latest studies, NOS/NO signaling pathways play a key role in the pathogenesis of RA. Overproduction of NO can induce the generation and release of inflammatory cytokines and act as free radical gas to accumulate and trigger oxidative stress, which can involve in the pathogenesis of RA. Therefore, targeting NOS and its upstream and downstream signaling pathways may be an effective approach to managing RA. This review clearly summarizes the NOS/NO signaling pathway, the pathological changes of RA, the involvement of NOS/NO in RA pathogenesis and the conventional and novel drugs based on NOS/NO signaling pathways that are still in clinical trials and have good therapeutic potential in recent years, with an aim to provide a theoretical basis for further exploration of the role of NOS/NO in the pathogenesis, prevention and treatment of RA.

Metronomic capecitabine as adjuvant therapy in locoregionally advanced nasopharyngeal carcinoma: a multicentre, open-label, parallel-group, randomised, controlled, phase 3 trial.

BACKGROUND: Patients with locoregionally advanced nasopharyngeal carcinoma have a high risk of disease relapse, despite a high proportion of patients attaining complete clinical remission after receiving standard-of-care treatment (ie, definitive concurrent chemoradiotherapy with or without induction chemotherapy). Additional adjuvant therapies are needed to further reduce the risk of recurrence and death. However, the benefit of adjuvant chemotherapy for nasopharyngeal carcinoma remains controversial, highlighting the need for more effective adjuvant treatment options. METHODS: This multicentre, open-label, parallel-group, randomised, controlled, phase 3 trial was done at 14 hospitals in China. Patients (aged 18-65 years) with histologically confirmed, high-risk locoregionally advanced nasopharyngeal carcinoma (stage III-IVA, excluding T3-4N0 and T3N1 disease), no locoregional disease or distant metastasis after definitive chemoradiotherapy, an Eastern Cooperative Oncology Group performance status of 0 or 1, sufficient haematological, renal, and hepatic function, and who had received their final radiotherapy dose 12-16 weeks before randomisation, were randomly assigned (1:1) to receive either oral metronomic capecitabine (650 mg/m2 body surface area twice daily for 1 year; metronomic capecitabine group) or observation (standard therapy group). Randomisation was done with a computer-generated sequence (block size of four), stratified by trial centre and receipt of induction chemotherapy (yes or no). The primary endpoint was failure-free survival, defined as the time from randomisation to disease recurrence (distant metastasis or locoregional recurrence) or death due to any cause, in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of capecitabine or who had commenced observation. This trial is registered with ClinicalTrials.gov, NCT02958111. FINDINGS: Between Jan 25, 2017, and Oct 25, 2018, 675 patients were screened, of whom 406 were enrolled and randomly assigned to the metronomic capecitabine group (n=204) or to the standard therapy group (n=202). After a median follow-up of 38 months (IQR 33-42), there were 29 (14%) events of recurrence or death in the metronomic capecitabine group and 53 (26%) events of recurrence or death in the standard therapy group. Failure-free survival at 3 years was significantly higher in the metronomic capecitabine group (85·3% [95% CI 80·4-90·6]) than in the standard therapy group (75·7% [69·9-81·9]), with a stratified hazard ratio of 0·50 (95% CI 0·32-0·79; p=0·0023). Grade 3 adverse events were reported in 35 (17%) of 201 patients in the metronomic capecitabine group and in 11 (6%) of 200 patients in the standard therapy group; hand-foot syndrome was the most common adverse event related to capecitabine (18 [9%] patients had grade 3 hand-foot syndrome). One (<1%) patient in the metronomic capecitabine group had grade 4 neutropenia. No treatment-related deaths were reported in either group. INTERPRETATION: The addition of metronomic adjuvant capecitabine to chemoradiotherapy significantly improved failure-free survival in patients with high-risk locoregionally advanced nasopharyngeal carcinoma, with a manageable safety profile. These results support a potential role for metronomic chemotherapy as an adjuvant therapy in the treatment of nasopharyngeal carcinoma. FUNDING: The National Natural Science Foundation of China, the Key-Area Research and Development Program of Guangdong Province, the Natural Science Foundation of Guangdong Province, the Innovation Team Development Plan of the Ministry of Education, and the Overseas Expertise Introduction Project for Discipline Innovation. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Molecular Insights into the Reaction Process of Alkali-Activated Metakaolin by Sodium Hydroxide.

When metakaolin (MK) is alkalized with an alkaline activator, it depolymerizes under the action of the alkali. However, the process of MK alkalinization is still unrevealed. Here, we supplied a molecular insight into the process of MK alkalinization through reaction molecular dynamics (MD) simulation. The structure, dynamics, and process of MK alkalinization are systematically investigated. The results showed that the layered structure of MK was destroyed and the silicates in MK were dissolved by sodium hydroxide solution during the alkalinization reaction of MK. The aluminates in MK are not dissolved, indicating that aluminates are more stable than silicates. Moreover, the equilibrium structures of MK with H2O and MK with NaOH solution show that only when both sodium hydroxide and water are involved in the alkalinization reaction, the silicates in MK undergo depolymerization. Also, the observed final state of MK alkalinization can be recognized as the precursor of alkali-activated materials (AAMs).

Roles of autophagy in rheumatoid arthritis.

Autophagy, a vital mechanism restricted in tissues, exerts its cytoprotective role through the degradation mechanism of damaged or aging organelles, harmful protein aggregates and intracellular pathogens, followed by energy furnishment. However, dysfunctional autophagy is associated with the development of autoimmune diseases such as rheumatoid arthritis (RA). In pathological conditions, autophagy may be involved in the maturation, survival and proliferation of various immune and non-immune cells and plays a key role in the pathogenesis of RA. Furthermore, autophagy appears to be involved in the citrullination of T lymphocytes and the presentation of citrullinated peptides, which are presented to T lymphocytes via the major histocompatibility complex, causing immune responses and chronic inflammation, as well as bone and cartilage destruction associated with apoptosis resistance of RA fibroblast-like synoviocyte (RAFLS) and osteoclastogenesis. In this review, we have summarised the roles of autophagy in the pathogenesis of RA including citrullination, immune tolerance break, osteoclastogenesis, RA FLS cell dysplasia, apoptosis resistance, together with the therapeutic potentials of autophagy regulators.

Role of Cholinergic Signaling in Alzheimer's Disease.

Acetylcholine, a neurotransmitter secreted by cholinergic neurons, is involved in signal transduction related to memory and learning ability. Alzheimer's disease (AD), a progressive and commonly diagnosed neurodegenerative disease, is characterized by memory and cognitive decline and behavioral disorders. The pathogenesis of AD is complex and remains unclear, being affected by various factors. The cholinergic hypothesis is the earliest theory about the pathogenesis of AD. Cholinergic atrophy and cognitive decline are accelerated in age-related neurodegenerative diseases such as AD. In addition, abnormal central cholinergic changes can also induce abnormal phosphorylation of ttau protein, nerve cell inflammation, cell apoptosis, and other pathological phenomena, but the exact mechanism of action is still unclear. Due to the complex and unclear pathogenesis, effective methods to prevent and treat AD are unavailable, and research to explore novel therapeutic drugs is various and active in the world. This review summaries the role of cholinergic signaling and the correlation between the cholinergic signaling pathway with other risk factors in AD and provides the latest research about the efficient therapeutic drugs and treatment of AD.

Potent antitumor effect of T cells armed with anti-GD2 bispecific antibody.

BACKGROUND: Humanized 3F8-bispecific antibody (hu3F8-BsAb) using the IgG(L)-scFv format (where scFv is single-chain variable fragment), where the anti-CD3 huOKT3 scFv is fused with the carboxyl end of the hu3F8 light chain, has potent antitumor cytotoxicity against GD2(+) tumors. To overcome the insufficient number and function of T cells in cancer patients, they can be rejuvenated and expanded ex vivo before arming with hu3F8-BsAb for adoptive transfer, potentially reducing toxic side effects from direct BsAb administration. PROCEDURE: T cells from normal volunteers were expanded and activated ex vivo using CD3/CD28 beads for 8 days. Activated T cells (ATCs) were harvested and co-incubated with a Good Manufacturing Practice grade hu3F8-BsAb at room temperature for 20 min. These armed ATCs were tested for cytotoxicity in vitro and in vivo against human GD2(+) cell lines and patient-derived xenografts in BALB-Rag2-/- IL-2R-γc-KO mice. RESULTS: Hu3F8-BsAb armed ATCs showed robust antigen-specific tumor cytotoxicity against GD2(+) tumors in vitro. In vivo, T cells armed with hu3F8-BsAb were highly cytotoxic against GD2(+) melanoma and neuroblastoma xenografts in mice, accompanied by T-cell infiltration without significant side effects. Only zeptomole (10-21 ) quantities of BsAb per T cell was required for maximal antitumor effects. Tumor response was a function of T-cell dose. CONCLUSION: BsAb armed T cells may have clinical utility as the next generation of cytotherapy combined with recombinant BsAb against human tumors for both adult and pediatrics, if autologous T cells can be activated and expanded ex vivo.

Role of prostaglandins in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterised by systemic and chronic synovitis that lead to joint destruction, pain, and many complications. Treatments only relieve certain symptoms, but do not cure RA completely. Prostaglandins (PGs) are lipid signalling molecules and released in the early phase of RA. Increasing evidences have shown five main contribution of PGs to the different stages and symptoms of RA. First, PGs maintain the autoimmune response and immune-system inflammation by modulating the differentiation, maturation, and cytokine production of immune cells. Second, PGs are beneficial for leukocyte infiltration, synovial hyperplasia, and angiogenesis to promote synovitis. Third, PGs are involved in cartilage degradation and bone resorption. Fourth, PGs are important mediators of joint-pain regulation. Finally, in the late stage of RA inflammation, PGs play a part in joint protection. Those findings suggest that PGs are potential therapy targets for RA. This review highlights recent advances in the RA development caused by PGs, and provides recommendations for future research directions.

Ideal physical activity in association with incident ankylosing spondylitis: a community-based, prospective cohort study.

OBJECTIVES: Only limited risk factors for ankylosing spondylitis (AS) have been identified to date. Therefore, we aimed to explore whether cardiovascular health (CVH) behaviours and factors are associated with the risk of developing AS. METHODS: Patients with incident AS were identified in cohorts from two ongoing prospective studies. Assessments were made of the association of AS with individual baseline cardiovascular health lifestyle behaviours (including smoking status, body mass index, physical activity and diet) and cardiovascular health factors (including total cholesterol levels, blood pressure levels and fasting plasma glucose levels), and with a cardiovascular health metric determined by the number of ideal behaviours and factors. Cox regression analysis was used for the estimation of hazard ratios (HRs) for AS. RESULTS: Among 124,303 participants, incident AS was identified in 53 individuals within the 8 years of follow-up. For participants with ideal physical activity (>80 min/week) the HR was 0.21 (95% CI 0.05-0.89) compared with participants without ideal physical activity after adjusting for potential confounders. No signi cant risk of developing AS was associated with baseline smoking, diet, body mass index, blood pressure, fasting blood glucose or total cholesterol status, nor did cardiovascular health metrics. CONCLUSIONS: Adherence to ideal physical activity may reduce the risk of developing AS.

The Roles of Nitric Oxide Synthase/Nitric Oxide Pathway in the Pathology of Vascular Dementia and Related Therapeutic Approaches.

Vascular dementia (VaD) is the second most common form of dementia worldwide. It is caused by cerebrovascular disease, and patients often show severe impairments of advanced cognitive abilities. Nitric oxide synthase (NOS) and nitric oxide (NO) play vital roles in the pathogenesis of VaD. The functions of NO are determined by its concentration and bioavailability, which are regulated by NOS activity. The activities of different NOS subtypes in the brain are partitioned. Pathologically, endothelial NOS is inactivated, which causes insufficient NO production and aggravates oxidative stress before inducing cerebrovascular endothelial dysfunction, while neuronal NOS is overactive and can produce excessive NO to cause neurotoxicity. Meanwhile, inflammation stimulates the massive expression of inducible NOS, which also produces excessive NO and then induces neuroinflammation. The vicious circle of these kinds of damage having impacts on each other finally leads to VaD. This review summarizes the roles of the NOS/NO pathway in the pathology of VaD and also proposes some potential therapeutic methods that target this pathway in the hope of inspiring novel ideas for VaD therapeutic approaches.

Nomogram predicting survival as a selection criterion for postmastectomy radiotherapy in patients with T1 to T2 breast cancer with 1 to 3 positive lymph nodes.

BACKGROUND: The role of postmastectomy radiotherapy (PMRT) in women with pT1-T2N1 breast cancer is controversial. The authors developed a nomogram that was predictive for overall survival (OS) and identified patients who derived no benefit from PMRT. METHODS: The authors retrospectively evaluated 4869 patients with pT1-T2N1 breast cancer who were treated with mastectomy between 2000 and 2014 in 11 Chinese hospitals. Rates of locoregional recurrence and distant metastasis were calculated using competing risk analysis, and disease-free survival and OS rates were calculated using the Kaplan-Meier method. Based on the risk factors identified from Cox regression analysis in 3298 unirradiated patients, a nomogram predicting OS was developed. The benefit of PMRT was evaluated in different risk groups stratified by the nomogram model. RESULTS: After a median follow-up of 65.9 months, the 5-year OS, disease-free survival, locoregional recurrence, and distant metastasis rates were 93.3%, 84.3%, 5.2%, and 8.3%, respectively. A total of 1571 patients (32.3%) underwent PMRT. On multivariable analyses, PMRT was found to increase OS significantly (hazard ratio, 0.61; P = .002). An OS prediction nomogram evaluated the effect of age; tumor location; tumor size; positive lymph node ratio; estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status; and treatment with trastuzumab. Based on nomogram scores, the entire patient cohort was classified into 3 risk groups. PMRT significantly improved the OS of patients in the intermediate-risk (P < .001) and high-risk groups (P = .004), but not in the low-risk group (P = .728). CONCLUSIONS: The authors developed a nomogram that is predictive of OS among women with pT1-T2N1 breast cancer after mastectomy. This nomogram may help to select a subgroup of patients with a good prognosis who will not benefit from PMRT.

An N-Acetylgalactosamino Dendron-Clearing Agent for High-Therapeutic-Index DOTA-Hapten Pretargeted Radioimmunotherapy.

Clearing agents (CAs) can rapidly remove nonlocalized targeting biomolecules from circulation for hepatic catabolism, thereby enhancing the therapeutic index (TI), especially for blood (marrow), of the subsequently administered radioisotope in any multistep pretargeting strategy. Herein we describe the synthesis and in vivo evaluation of a fully synthetic glycodendrimer-based CA for DOTA-based pretargeted radioimmunotherapy (DOTA-PRIT). The novel dendron-CA consists of a nonradioactive yttrium-DOTA-Bn molecule attached via a linker to a glycodendron displaying 16 terminal α-thio-N-acetylgalactosamine (α-SGalNAc) units (CCA α-16-DOTA-Y3+; molecular weight: 9059 Da). Pretargeting [177Lu]LuDOTA-Bn with CCA α-16-DOTA-Y3+ to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [177Lu]LuDOTA-Bn for blood, tumor, liver, spleen, and kidneys of 11.7, 468, 9.97, 5.49, and 13.3 cGy/MBq, respectively. Tumor-to-normal tissues absorbed-dose ratios (i.e., TIs) ranged from 40 (e.g., for blood and kidney) to about 550 for stomach.

LTB4-induced anti-apoptosis and infiltration of neutrophils in rheumatoid arthritis.

Rheumatoid arthritis (RA) is the most common autoimmune disease, resulting in synovitis, joint pain and stiffness, even deformity and disability. The interactions between leukotriene B4 (LTB4) and neutrophils in RA progression have not been elucidated in detail. Our review focuses on the correlation of LTB4 and neutrophils in the development of RA especially in terms of infiltration and delayed life span of neutrophils. In this article, the roles of LTB4 in the anti-apoptosis of neutrophils will be detailed, which is achieved by suppressed pro-apoptotic Bax and up-regulated anti-apoptotic Mcl-1, and several key molecules, as well as signalling pathways and factors relevant to the enhancement of LTB4 production and functions. The mechanisms of LTB4-induced anti-apoptosis and infiltration of neutrophils provide more potential targets in the treatment of RA and recent therapeutic strategies are also discussed.

TZAP plays an inhibitory role in the self-renewal of porcine mesenchymal stromal cells and is implicated the regulation of premature senescence via the p53 pathway.

BACKGROUND: Mesenchymal stromal cells (MSCs) were originally characterized by the ability to differentiate into different mesenchymal lineages in vitro, and their immunomodulatory and trophic functions have recently aroused significant interest in the application of MSCs in cell-based regenerative medicine. However, a major problem in clinical practice is the replicative senescence of MSCs, which limits the cell proliferation potential of MSCs after large-scale expansion. Telomeric zinc finger-associated protein (TZAP), a novel specific telomere-binding protein, was recently found to stimulate telomere trimming and prevent excessive telomere elongation. The aim of this study was to elucidate the role of TZAP in regulating MSCs senescence, differentiation and proliferation. METHOD: Primary porcine mesenchymal stromal cells (pMSCs) were isolated from the bone marrow of Tibet minipigs by a noninvasive method in combination with frequent medium changes (FMCs). The deterioration of the pMSCs' proliferation capacity and their resultant entry into senescence were analyzed by using CCK8 and EdU incorporation assays, SA-ß-gal staining and comparisons of the expression levels of cellular senescence markers (p16INK14 and p21) in pMSC cell lines with TZAP overexpression or knockout. The effects of TZAP overexpression or knockout on the differentiation potential of pMSCs were assessed by alizarin red S staining after osteogenic induction or by oil red O staining after adipogenic induction. The effect of TZAP overexpression and the involvement of the p53 signaling pathway were evaluated by detecting changes in ARF, MDM2, P53 and P21 protein levels in pMSCs. RESULTS: TZAP levels were significantly elevated in late-passage pMSCs compared to those in early-passage pMSCs. We also observed significantly increased levels of the senescence markers p16INK4A and p21. Overexpression of TZAP reduced the differentiation potential of the cells, leading to premature senescence in early-passage pMSCs, while knockout of TZAP led to the opposite phenotype in late-passage pMSCs. Furthermore, overexpression of TZAP activated the P53 pathway (ARF-MDM2-P53-P21WAF/CDKN1A) in vitro. TZAP also downregulated the expression levels of PPARγ and Cebpα, two key modulators of adipogenesis. CONCLUSIONS: This study demonstrates that the level of TZAP is closely related to differentiation potential in pMSCs and affects cellular senescence outcomes via the p53 pathway. Therefore, attenuation of intracellular TZAP levels could be a new strategy for improving the efficiency of pMSCs in cell therapy and tissue engineering applications.

Roles of eNOS in atherosclerosis treatment.

BACKGROUND: Atherosclerosis (AS) is the main pathogeny of coronary heart disease, cerebral infarction and peripheral vascular disease. Endothelial dysfunction is one of the important pathogenesis of AS. As an important endothelium-derived relaxation factor, nitric oxide (NO) plays a role in cardiovascular protection and anti-AS function; but in the pathological state, endothelial nitric oxide synthase (eNOS) disorder causes an abnormal production of NO, which may damage endothelial function and trigger AS. This review summarized the research progresses in the treatment strategies for AS based on correcting the disordered eNOS/ NO signaling pathway. MAIN BODY: According to the topic, select the search terms 'atherosclerosis,' 'nitric oxide,' 'eNOS,' 'treatment,' 'management,' 'medication,' 'maintenance,' 'remission'. Using these terms, a structured literature search via multiple electronic databases was performed for the most recent trial evidence in recent years. We read and analyze these literatures carefully, classified these literatures according to their content, and then summarized and outlined the common main points in these classified literatures. Finally, literature data were organized to discuss these main points logically. We found that both aberrant expression and dysfunction of eNOS are closely related to AS development, and some new treatment strategies aimed at eNOS have been proposed, including upregulation of eNOS expression and inhibition of eNOS uncoupling. The former one is mainly related to inflammatory inhibition and protection of the PKB-eNOS signaling pathway; whereas the latter one is associated with the addition of the L-arginine substrate of eNOS, arginase inhibition, and the supplement of tetrahydrobiopterin, which can elevate no level. CONCLUSIONS: eNOS can be an important target for prevention and treatment of AS, and eNOS drugs may be another potent class of effective therapeutic treatment for AS following traditional lipid-lowering, anti-platelet, vasodilator drugs. But applying these experimental results to clinic treatment still requires further studies and development of biotechnology.

Pain and bone damage in rheumatoid arthritis: role of leukotriene B4.

Rheumatoid arthritis is a chronic autoimmune disease characterised by unbearable joint pain as well as bone and cartilage destruction. Although RA development is greatly controlled, the pain and bone damage failed to be relieved and managed. Leukotriene B4 (LTB4) has been proved to play an essential role in the induction of pain and bone damage. The nerve injury of RA can promote the production of LTB4, which act on their receptors, leading to the increased release of pro-inflammatory cytokines and ROS to reduce neuron viability and pain threshold. Moreover, LTB4-BLT1 activation can also increase intracellular calcium concentration and neuron excitability as well as NF-κB pathway activation, which further promote the production of MMP-9 and CXC3R-1. The mutual promotion between LTB4 and neutrophil accumulation accelerates the release of TNF-α and IL-ß, which enhance both peripheral and central nerve system sensitisation. LTB4 also involve in TrpV1 channel activation and modulation of P2X3 receptor activation. All above mechanisms contribute to the development of RA pain. IL-23, cPLA2 and PI3K increase the production of CD11b+Gr1high myeloid subtype and calcium concentration, which promote the production of LTB4 and further accelerate IL-17 and TNF activation as well as calcium influx to conduce to osteoclastogenesis, resulting in aggregated bone damage. Our review is the first to conclude the signalling pathways and associated molecules in LTB4-induced pain and bone damage.

Gambogic Acid Efficiently Kills Stem-Like Colorectal Cancer Cells by Upregulating ZFP36 Expression.

BACKGROUND/AIMS: Gambogic acid (GA), the main active compound of Gamboge hanburyi, has been reported to be a potential novel antitumor drug. Whether GA inhibits putative cancer stem cells (CSCs), which are considered to be the major cause of cancer treatment failure, remains largely unknown. This study investigated whether GA inhibits the CSCs of colorectal cancer (CRC) and its possible mechanisms. METHODS: We performed CCK8 and tumor sphere formation assays, percentage analysis of both side population and CD133+CD44+ cells, and the detection of stem cells markers, in order to assess the role of GA in inhibiting the stem celllike features of CRC. An mRNA microarray was performed to identify the downstream gene affected by GA and rescue assays were performed to further clarify whether the downstream gene is involved in the GA induced decrease of the stem cell-like CRC population. CRC cells were engineered with a CSC detector vector encoding GFP and luciferase (Luc) under the control of the Nanog promoter, which were utilized to investigate the effect of GA on putative CSC in human tumor xenograft-bearing mice using in vivo bioluminescence imaging. RESULTS: Our results showed that GA significantly reduced tumor sphere formation and the percentages of side population and CD133+CD44+ cells, while also decreasing the expression of stemness and EMT-associated markers in CRC cells in vitro. GA killed stem-like CRC cells by upregulating the expression of ZFP36, which is dependent on the inactivation of the EGFR/ ERK signaling pathway. GFP+ cells harboring the PNanog-GFP-T2A-Luc transgene exhibited CSC characteristics. The in vivo results showed that GA significantly inhibited tumor growth in nude mice, accompanied by a remarkable reduction in the putative CSC number, based on whole-body bioluminescence imaging. CONCLUSION: These findings suggest that GA significantly inhibits putative CSCs of CRC both in vitro and in vivo by inhibiting the activation of the EGFR/ ERK/ZFP36 signaling pathway and may be an effective drug candidate for anticancer therapies.

Evaluation of Next-Generation Sequencing for the Diagnosis of Infections of the Central Nervous System Caused by the Neurotropic Herpes Viruses: A Pilot Study.

BACKGROUND: There are sparse and limited studies on small sample size reporting the application of next-generation sequencing (NGS) in the detection of central nervous system (CNS) viral infections. We assessed the diagnostic performance of NGS of cerebrospinal fluid (CSF) for predicting viral infections of the CNS caused by the neurotropic herpes viruses in a pilot population. MATERIALS AND METHODS: We prospectively collected CSF samples from 24 patients with CNS viral infection from April 2017 to October 2018. Of the 24 patients, 19 patients were infected with herpes simplex virus 1 (HSV-1), 1 patient with HSV-2, and 4 patients with varicella-zoster virus (VZV). All CSF samples were screened for viral DNA using NGS technologies to detect viral CNS infections. RESULTS: Of the 24 patients with confirmed viral CNS infection caused by the neurotropic herpes viruses, 10 (10/24, 41.67%) patients exhibited positive NGS results. With the help of NGS, HSV-1 DNA was detected in the CSF of 6 patients (6/19; 31.58%). HSV-2 DNA was detected in 1 patient (1/1; 100%) and VZV DNA was detected in 3 patients (3/4; 75%). The positive rate of virus detected by NGS decreased with time. The positive rates of NGS of CSF in the first, second, and third weeks were 54.5% (6/11), 44.4% (4/9), and 0% (0/4), respectively. CONCLUSIONS: NGS method is a promising pathogen detection tool for identifying viral CNS infections. It should be recommended to sequence viral DNA of CSF in the early stage of CNS viral infections.

Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8.

Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy.