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Hepatocellular carcinoma (HCC) is a highly lethal liver cancer with late diagnosis; therefore, the identification of new early biomarkers could help reduce mortality. We determine the tissue and plasma status of five annexins during hepatocarcinogenesis by diethylnitrosamine-induced cirrhosis-HCC. We found that Anxa5 was the earliest upregulated gene at week 12 after HCC initiation, while Anxa1 and Anxa2 were upregulated in advanced HCC stages (weeks 18 and 22). Furthermore, the protein level of Annexin A1, A2, A5 and A10 was increased from the early stages. Immunofluorescence and subcellular fractionation revealed Annexin A1, A2, and A5 in the cytoplasm and nuclei of tumor cells. Notably, increased plasma levels of Annexin A5 significantly (r2 = 0.8203) correlated with Annexin A5 levels in liver tissue from week 12 and gradually increased until week 22. Using the TCGA database, we found that the expression of ANXA2 (HR = 1.7, p = 0.0046) and ANXA5 (HR = 1.8, p = 0.00077) was associated with poor survival in HCC patients. In conclusion, we have identified Annexin A1 and A5 as potentially useful early biomarkers for poor prognosis in HCC patients. [ABSTRACT FROM AUTHOR]

Globally, breast cancer is the most prevalent form of cancer in women and there is a need for alternative therapies such as plant-derived compounds with low systemic toxicity and selective toxicity to cancer cells. The aim of this study is to assess the cytotoxicity effects of 7-geranyloxycinnamic acid isolated from leaves of Melicope lunu-ankenda, a traditional medicinal plant, on the human breast cancer cell lines. Dried leaf powder was used for the preparation of different crude extracts using different solvents of increasing order of polarity. The structure of the isolated compound from the petroleum ether extract was elucidated by 1H and 13C NMR, LC-MS, and DIP−MS spectroscopy. The cytotoxic activity of the crude extract and 7-geranyloxycinnamic acid analyzed using MTT assay. Apoptotic analysis was evaluated using Annexin V-PI staining, AO/PI staining, intracellular ROS measurement, and measurement of activities of caspases 3/7, 8, and 9. Crude extracts and the isolated pure compound showed significant cytotoxicity against tested cancer cell lines. 7-geranyloxycinnamic acid was found to exert significant cytotoxic effects against breast cancer cell lines such as the MCF-7 and MDA-MB-231 cell lines. The cytotoxic effects are attributed to its ability to induce apoptosis via accumulation of ROS and activation of caspases in both breast cancer cell lines. The pure compound, 7-geranyloxycinnamic acid isolated from the leaves of M. lunu-ankenda, can exert significant cytotoxic effects against breast cancer cell lines without affecting the normal cells. [ABSTRACT FROM AUTHOR]

Aerolysin family pore-forming toxins damage the membrane, but membrane repair responses used to resist them, if any, remain controversial. Four proposed membrane repair mechanisms include toxin removal by caveolar endocytosis, clogging by annexins, microvesicle shedding catalyzed by MEK, and patch repair. Which repair mechanism aerolysin triggers is unknown. Membrane repair requires Ca2+, but it is controversial ifCa2+ flux is triggered by aerolysin. Here, we determined Ca2+ influx and repair mechanisms activated by aerolysin. In contrast to what is seen with cholesterol-dependent cytolysins (CDCs), removal of extracellular Ca2+ protected cells from aerolysin. Aerolysin triggered sustained Ca2+ influx. Intracellular Ca2+ chelation increased cell death, indicating that Ca2+-dependent repair pathways were triggered. Caveolar endocytosis failed to protect cells from aerolysin or CDCs. MEK-dependent repair did not protect against aerolysin. Aerolysin triggered slower annexin A6 membrane recruitment compared to CDCs. In contrast to what is seen with CDCs, expression of the patch repair protein dysferlin protected cells from aerolysin. We propose aerolysin triggers a Ca2+-dependent death mechanism that obscures repair, and the primary repair mechanism used to resist aerolysin is patch repair. We conclude that different classes of bacterial toxins trigger distinct repair mechanisms. [ABSTRACT FROM AUTHOR]

Centrifugation steps are regularly used for neutrophil isolation. Thereby, the influences of applied g-forces on the functionality of PMNs have hardly been analyzed and could consequently have been overlooked or led to biased results. We now hypothesize that blood PMNs—when gently isolated—can be long-lived cells and they physiologically become apoptotic rather than NETotic. Neutrophils were isolated from whole blood without centrifugation using a sedimentation enhancer (gelafundin). PMNs were analyzed via live-cell imaging for migratory activity and vitality condition by fluorescent staining. Native neutrophils showed still relevant migratory activity after more than 6 days ex vivo. The percentage of cells that were annexin V+ or PI+ increased successively with increasing ex vivo time. In addition, the characteristics of DAPI staining of gently isolated granulocytes differed markedly from those obtained by density gradient separation (DGS). We conclude that NETosis occurring after DGS is the consequence of applied g-forces and not a physiological phenomenon. Future studies on neutrophils should be performed with most native cells (applied g-time load as low as possible). [ABSTRACT FROM AUTHOR]

Dysregulated inflammatory responses are often correlated with disease severity during viral infections. Annexin A1 (AnxA1) is an endogenous pro-resolving protein that timely regulates inflammation by activating signaling pathways that culminate with the termination of response, clearance of pathogen and restoration of tissue homeostasis. Harnessing the pro-resolution actions of AnxA1 holds promise as a therapeutic strategy to control the severity of the clinical presentation of viral infections. In contrast, AnxA1 signaling might also be hijacked by viruses to promote pathogen survival and replication. Therefore, the role of AnxA1 during viral infections is complex and dynamic. In this review, we provide an in-depth view of the role of AnxA1 during viral infections, from pre-clinical to clinical studies. In addition, this review discusses the therapeutic potential for AnxA1 and AnxA1 mimetics in treating viral infections. [ABSTRACT FROM AUTHOR]

Brevilaterins, antimicrobial peptides produced by Brevibacillus laterosporus, are regarded as excellent food preservatives and are popular as antimicrobial applications. Recent research has uncovered their potent cytotoxic effects against diverse cancer cells, thereby underscoring the pressing need for more extensive and intensive investigations into this use. In this study, we explored their novel function in inducing cytotoxicity to cancer cells and systematically investigated the mechanism of action of Brevilaterin B/C (BB/BC) in vivo. Proliferation, membrane permeability, and apoptotic rate were evaluated using CCK-8 assay, LDH assay, and Annexin V-FITC/PI kits. ROS levels and mitochondrial membrane potential were detected using the fluorescent probe DCFH-DA and JC-1. Our results demonstrated that both BB and BC at concentrations of 4–6 µg/mL significantly inhibited the proliferation and migration of gastric cancer cells BGC-823. Treatment with 4 µg/mL of BB/BC rapidly increased LDH levels in the supernatant of BGC-823 cells, leading to further investigation of the mechanism of apoptosis. We found that the apoptotic rate of BGC-823 cells significantly increased upon treatment with BB/BC, demonstrating their potent induction of apoptosis. BB/BC-induced ROS production in BGC-823 cells impaired their growth and induced apoptosis, indicating a close association between apoptosis and ROS elevation. Additionally, JC-1 aggregates rapidly accumulated after treatment with 4 µg/mL of BB/BC, suggesting changes in mitochondrial membrane potential and early apoptosis. Taken together, our findings revealed that BB and BC exhibit significant anticancer effects against gastric cancer cells, highlighting the promising potential of Brevilaterins as anticancer agents. [ABSTRACT FROM AUTHOR]

Background: LOX familymembers are reported to play pivotal roles in cancer. Unlike their enzymatic activities in collagen cross-linking, their precise cancer functions are unclear. We revealed that LOXL4 is highly upregulated in breast cancer cells, and we thus sought to define an unidentified role of LOXL4 in breast cancer. Methods: We established theMDA-MB-231 sublines MDA-MB-231-LOXL4 mutCA and -LOXL4 KO, which stably overexpress mutant LOXL4 that loses its catalytic activity and genetically ablates the intrinsic LOXL4 gene, respectively. In vitro and in vivo evaluations of these cells' activities of cancer outgrowth were conducted by cell-based assays in cultures and an orthotopic xenograft model, respectively. The new target (s) of LOXL4 were explored by the MS/MS analytic approach. Results: Our in vitro results revealed that both the overexpression of mutCA and the KO of LOXL4 in cells resulted in a marked reduction of cell growth and invasion. Interestingly, the lowered cellular activities observed in the engineered cells were also reflected in the mouse model. We identified a novel binding partner of LOXL4, i.e., annexin A2. LOXL4 catalyzes cell surface annexin A2 to achieve a cross-linked multimerization of annexin A2, which in turn prevents the internalization of integrin β-1, resulting in the locking of integrin β-1 on the cell surface. These events enhance the promotion of cancer cell outgrowth. Conclusions: LOXL4 has a new role in breast cancer progression that occurs via an interaction with annexin A2 and integrin β-1 on the cell surface. [ABSTRACT FROM AUTHOR]

Objective. To examine whether density gradient centrifugation (DGC) alone or its combination with annexin V magnetic-activated cell sorting (DGC-MACS) can be used to process semen samples from infertile male patients with poor sperm quality prior to subjecting these to freeze/thaw process in order to optimize the outcomes of sperm freezing. Methods. This study enrolled sixteen patients with sperm concentration ≥ 20 × 10 6 / mL , sperm motility < 30 %, and/or <4% normal sperm morphology. Sperms were processed by DGC or DGC-MACS prior to the freeze/thaw process. Sperm motility, hyperosmotic swelling test (HOS), TUNEL test, and morphological analysis were performed before and after the freeze/thaw process. Results. The freeze/thaw process had a detrimental effect on sperm motility, viability, morphology, and DNA integrity in all three groups (RAW, DGC, and DGC + MACS groups). The DGC and DGC + MACS groups showed increased sperm motility, viability, and normal morphology following freeze/thaw than untreated frozen controls. The motility and viability were not significantly different between DGC-MACS-CPT (cryopreservation-thawing) and DGC-CPT groups. Moreover, almost no grade A or grade B sperm was observed in the DGC-MACS-CPT groups. The sperm selected by DGC or DGC + MACS showed decreased levels of sperm DNA fragmentation than RAW samples following freeze/thaw. Moreover, the sperm DNA fragmentation following freeze/thaw in the DGC-MACS-CPT group was significantly lower than that in the DGC-CPT group. Conclusions. Sperm preparation by DGC before cryopreservation improved the quality of sperm postthaw in infertile males with poor sperm quality. If the sperm quality following freeze/thaw is foreseen to be insufficient for artificial insemination with husband's sperm or in vitro fertilization, or if there is high DNA fragmentation in RAW sperm, DGC + MACS should be used prior to cryopreservation to reduce sperm DNA fragmentation and improve the quality of sperm available for intracytoplasmic sperm injection. [ABSTRACT FROM AUTHOR]

Glutamate-induced neuronal cell death plays a key role in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Some recent studies reported the potential immunomodulatory and neuroprotective properties of inhibitors of serine-threonine kinase, mTOR (mammalian target of rapamycin). However, no study was conducted about the neuroprotective potential of everolimus (EVR), a selective and potent mTOR inhibitor. Therefore, this study was planned to investigate whether EVR has protective effects against glutamate-induced toxicity in PC12 cells, which are used as model for neurons injury, and to elucidate the underlying mechanism. PC12 cells were concurrently treated with glutamate (8 mM) and EVR (0-40 nM) for 24 h. Then, the cells viability, apoptosis rate, and apoptosis-related proteins (caspase-3, bax and bcl-2) were measured using MTT, annexin V/PI and immunoblotting assays. Analyzing the protective effect of different concentrations of EVR (0-40 nM) against glutamate-induced cytotoxicity revealed a significant increase in cell viability in co-treatment regimen (p < 0.01). Also, EVR (40 nM) significantly (p < 0.01) inhibited glutamate-induced apoptosis through depressing the elevation of bax/bcl-2 ratio and expression of cleaved caspase-3, concentration depend. The results demonstrated, for the first time, that EVR could protect against glutamate-mediated PC12 cell death via inhibiting apoptosis. [ABSTRACT FROM AUTHOR]

End-stage renal disease (ESRD) is followed by alterations in adaptive immunity. The aim of this study was to evaluate B lymphocyte subtypes in ESRD patients before and after hemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). Patients and Methods. CD5, CD27, BAFF, IgM and annexin were evaluated by flow cytometry on CD19+ cells in ESRD patients (n = 40), at time of initiating HD or CAPD (T0) and 6 months later (T6). Results. A significant reduction in ESRD-T0 compared to controls was noticed for CD19+, 70.8 (46.5) vs. 171 (249), p < 0.0001, CD19+CD5−, 68.6 (43) vs. 168.9 (106), p < 0.0001, CD19+CD27−, 31.2 (22.1) vs. 59.7 (88.4), p < 0.0001, CD19+CD27+, 42.1 (63.6) vs. 84.3 (78.1), p = 0.002, CD19+BAFF+, 59.7 (37.8) vs. 127.9 (123.7), p < 0.0001 and CD19+IgM+ cells, 48.9 (42.8) vs. 112.5 (81.7) (K/μL), p < 0.0001. The ratio of early/late apoptotic B lymphocytes was reduced (16.8 (10.9) vs. 110 (25.4), p = 0.03). CD19+CD5+ cells were the only cell type with an increased proportion in ESRD-T0 patients (2.7 (3.7) vs. 0.6 (1.1), p < 0.0001). After 6 months on CAPD or HD, CD19+CD27−(%) and early apoptotic lymphocytes were reduced further. The HD patients also showed a significant increase in late apoptotic lymphocytes, from 1.2 (5.7) to 4.2 (7.2) K/mL, p = 0.02. Conclusions. B cells and most of their subtypes were significantly reduced in ESRD-T0 patients compared to controls, the only exception being CD19+CD5+ cells. Apoptotic changes were prominent in ESRD-T0 patients and were exacerbated by HD. [ABSTRACT FROM AUTHOR]

Over the past decade, the treatment of metastatic melanoma has improved significantly due to the development of innovative therapies, such as drugs that target the BRAF/MAPK kinase pathway and the PD-1 pathway. However, these therapies do not work for all patients, highlighting the need for additional research on the pathophysiology of melanoma. Paclitaxel is a chemotherapeutic agent used when first-line treatments are unsuccessful; however, its efficacy is limited. Since Krüppel-like factor 9 (KLF9) (antioxidant repressor) is downregulated in melanoma, we propose that restoring KLF9 levels may sensitize malignant melanoma to chemotherapeutic agents, such as paclitaxel. We used adenovirus overexpression and siRNA technologies to assess the role of KLF9 in mediating the response of malignant melanoma-derived cell lines RPMI-7951 and A375 to paclitaxel treatment. We found that increasing KLF9 levels potentiates the effectiveness of paclitaxel, as shown by apoptotic parameters such as decreased cell viability, pro-caspase-3 activation, increased number of annexin V-positive cells, and reduction in nuclear proliferation marker (KI67). These results suggest that KLF9 may be a potential target for improving chemotherapeutic response in melanoma. [ABSTRACT FROM AUTHOR]

Although survival in pediatric acute myeloid leukemia (AML) has increased considerably over the past decades, refractory disease and relapse rates remain high. Refractory and relapsed disease are difficult to treat, with overall survival rates less than 40– 50%. Preventing relapse should, therefore, be one of the highest priorities. Current conventional chemotherapy regimens are hard to intensify due to associated toxic complications, hence more effective therapies that do not increase toxicity are needed. A promising targeted agent is the CD33-directed antibody–drug conjugate gemtuzumab ozogamicin (GO). Because CD33 is highly expressed on leukemic cells in the majority of AML patients, GO can be useful for a broad range of patients. Better relapse-free survival (RFS) after therapy including GO has been reported in several pediatric clinical trials; however, ambiguity about the clinical value of GO in newly diagnosed children remains. Treatment with GO in de novo AML patients aged ≥ 1 month, in combination with standard chemotherapy is approved in the United States, whereas in Europe, GO is only approved for newly diagnosed patients aged ≥ 15 years. In this review, we aimed to clarify the clinical value of GO for treatment of newly diagnosed pediatric AML patients. Based on current literature, GO seems to have additional value, in terms of RFS, and acceptable toxicity when used in addition to chemotherapy during initial treatment. Moreover, in KMT2A-rearranged patients, the clinical value of GO was even more evident. Also, we addressed predictors of response, being CD33 expression and SNPs, PgP-1 and Annexin A5. The near finalized intent-to-file clinical trial in the MyeChild consortium investigates whether fractionated dosing has additional value for pediatric AML, which may pave the way for a broader application of GO in pediatric AML. [ABSTRACT FROM AUTHOR]

Anti-silencing function protein 1 homolog B (ASF1B) has been implicated in the occurrence and development of cancers. The present work explored the functional role and the expression regulation of ASF1B in pancreatic ductal adenocarcinoma (PDAC). Based on the real-time quantitative PCR (qRT-PCR) and immunohistochemistry (IHC), ASF1B was significantly upregulated in PDAC tissues. High expression of ASF1B was associated with a poor overall survival (OS) and recurrence-free survival (DFS) in the PDAC patients. ASF1B also showed a relatively higher expression in PDAC cells (AsPC-1, PANC-1) when compared with human pancreatic ductal epithelial cells (HPDFe-6). CCK8 and clone formation assay demonstrated that silencing ASF1B impaired the proliferation in PANC-1 and AsPC-1 cells, and Annexin V-PI staining showed an increased level of apoptosis upon ASF1B silencing. ASF1B silencing also suppressed the migration and invasion in PDAC cells, as revealed by Transwell assays. We further showed that miR-24-3p was downregulated in PDAC tissues and cells, which functionally interacted with ASF1B by dual-luciferase reporter assay. miR-24-3p negatively regulated ASF1B expression to modulate the malignant phenotype of PDAC cells. ASF1B shows high expression in PDAC, which promotes the malignancy and EMT process of PDAC cells. miR-24-3p is a negative regulator of ASF1B and is downregulated in PDAC cells. Our data suggest that targeting ASF1B/miR-24-3p axis may serve as an intervention strategy for the management of PDAC. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is a nasty disease with extremely high malignancy and poor prognosis. Annexin A3 (ANXA3) is a potential prognosis biomarker, displaying an excellent correlation of ANXA3 overexpression with patients' poor prognosis. Silencing the expression of ANXA3 effectively inhibits the proliferation and metastasis of TNBC, suggesting that ANXA3 can be a promising therapeutic target to treat TNBC. Herein, we report a first-in-class ANXA3-targeted small molecule (R)-SL18, which demonstrated excellent anti-proliferative and anti-invasive activities to TNBC cells. (R)-SL18 directly bound to ANXA3 and increased its ubiquitination, thereby inducing ANXA3 degradation with moderate family selectivity. Importantly, (R)-SL18 showed a safe and effective therapeutic potency in a high ANXA3-expressing TNBC patient-derived xenograft model. Furthermore, (R)-SL18 could reduce the β -catenin level, and accordingly inhibit the Wnt/ β -catenin signaling pathway in TNBC cells. Collectively, our data suggested that targeting degradation of ANXA3 by (R)-SL18 possesses the potential to treat TNBC. A potent and moderate selective ANXA3 degrader was developed and showed excellent efficacy in a TNBC patient-derived xenograft (PDX) model with high ANXA3 expression. [Display omitted] [ABSTRACT FROM AUTHOR]

As a novel nuclear factor E2‐related factor 2 (NRF2) activator, the itaconate has shown significant therapeutic potential for oxidative stress diseases. However, its role in Vohwinkel syndrome in relation to the gap junction protein beta 2 (GJB2) mutation is still unclear. This study aimed at investigating the effect of 4‐octyl itaconate (OI) on HaCaT and D66H cells and clarify its potential mechanism in vitro. The optimal concentration and treatment time of OI on HaCaT cells and D66H cells were determined by CCK‐8 and LDH experiments. The effect of OI on cell proliferation was detected by EdU staining and FACS analysis of PI, while the apoptosis was evaluated by TUNEL staining and FACS analysis of Annexin V. The ROS staining was performed, and the levels of SOD, MDA, GSH and GSH/GSSG were detected to evaluate the effect of OI on oxidative damage induced by D66H‐type mutation. CO‐IP, Western blot, immunofluorescence and qPCR analyses were employed to detect the activation of KEAP1‐NRF2‐GCLC/HO‐1 pathway by OI. Finally, sh‐NRF2 was used to confirm the activation of this pathway by OI. Results showed that OI could improve the cell viability decreased by GJB2 gene mutation by regulating the balance between cell growth and apoptosis induced by oxidative damage. Furthermore, this alleviation process was regulated by the KEAP1‐NRF2‐HO‐1/GCLC pathway. In conclusion, OI could improve the viability of HaCaT and D66H cells via regulating the KEAP1‐NRF2‐GCLC/HO‐1 pathway, which provided a wide spectrum of potential targets for effective therapeutic treatments of Vohwinkel syndrome in the clinic. [ABSTRACT FROM AUTHOR]

Perinatal brain injury following hypoxia-ischemia (HI) is characterized by high mortality rates and long-term disabilities. Previously, we demonstrated that depletion of Annexin A1, an essential mediator in BBB integrity, was associated with a temporal loss of blood-brain barrier (BBB) integrity after HI. Since the molecular and cellular mechanisms mediating the impact of HI are not fully scrutinized, we aimed to gain mechanistic insight into the dynamics of essential BBB structures following global HI in relation to ANXA1 expression. Global HI was induced in instrumented preterm ovine fetuses by transient umbilical cord occlusion (UCO) or sham occlusion (control). BBB structures were assessed at 1, 3, or 7 days post-UCO by immunohistochemical analyses of ANXA1, laminin, collagen type IV, and PDGFRβ for pericytes. Our study revealed that within 24 h after HI, cerebrovascular ANXA1 was depleted, which was followed by depletion of laminin and collagen type IV 3 days after HI. Seven days post-HI, increased pericyte coverage, laminin and collagen type IV expression were detected, indicating vascular remodeling. Our data demonstrate novel mechanistic insights into the loss of BBB integrity after HI, and effective strategies to restore BBB integrity should potentially be applied within 48 h after HI. ANXA1 has great therapeutic potential to target HI-driven brain injury. [ABSTRACT FROM AUTHOR]

Background: Head and neck cancer (HNC) is the sixth most common malignant tumor worldwide and imposes a serious economic burden on society and individuals. Annexin has been implicated in multiple functions which are essential in HNC development, including cell proliferation, apoptosis, metastasis, and invasion. This study focused on the linkage between ANXA6 variants and HNC susceptibility in Chinese people. Methods: Eight SNPs in ANXA6 from 139 HNC patients and 135 healthy controls were genotyped by the Agena MassARRAY platform. The correlation of SNPs with HNC susceptibility was evaluated using odds ratio and 95% confidence interval calculated by logistic regression using PLINK 1.9. Results: Overall analysis results demonstrated that rs4958897 was correlated with an increased HNC risk (allele: OR = 1.41, p = 0.049; dominant: OR = 1.69, p = 0.039), while rs11960458 was correlated with reduced HNC risk (OR = 0.54, p = 0.030). In age = 53, rs4958897 was related to reduce HNC risk. In males, rs11960458 (OR = 0.50, p = 0.040) and rs13185706 (OR = 0.48, p = 0.043) were protective factors for HNC, but rs4346760 was a risk factor for HNC. Moreover, rs4346760, rs4958897, and rs3762993 were also correlated with increased nasopharyngeal carcinoma risk. Conclusions: Our findings suggest that ANXA6 polymorphisms are linked to the susceptibility to HNC in the Chinese Han population, indicating that ANXA6 may serve as a potential biomarker for HNC prognosis and diagnosis. [ABSTRACT FROM AUTHOR]

Background: Cervical cancer is a common malignant gynecological disease that threatens the health of women all over the world. The abnormal expression of Annexin A5 (ANXA5) is closely related to the biological behavior of various malignant tumors, however, the relationship between ANXA5 and cervical cancer is still unclear. Therefore, the effects of low expression of ANXA5 on the proliferation, apoptosis, migration and invasion of cervical cancer cells (HeLa) and its related mechanism were explored. Methods: The cells were divided into three groups: ANXA5-si group, negative control group and blank group. RNA interference was used to suppress ANXA5 expression. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, flow cytometry and propidium iodide (PI) staining, wound healing assay and transwell assay were employed to detect cell proliferation, apoptosis, migration and invasion respectively. Meanwhile, gene expression was detected by qPCR and Western blotting. Results: ANXA5 suppression lead to the increase of proliferation, migration, invasion and the decrease of apoptosis of cervical cancer HeLa cells. Furthermore, the expression of both pPI3K and pAkt increased. Conclusion: ANXA5 might inhibit Hela cells proliferation and metastasis by regulating PI3K/Akt signal pathway. [ABSTRACT FROM AUTHOR]

Exosomes are nano-sized biomolecules that originate from the endosome, have an important role in immune modulation, and provide intercellular interaction. Exosomes have a wide variety of membrane proteins such as tetraspanins, proteins, Annexin and Rab proteins, and they are candidates for use in the treatment and diagnosis of infections, nervous system, cancer and neurodegenerative diseases. It is important for diagnosis and treatment that exosomes released from almost all body fluids can be isolated both homogeneously and in large numbers. Therefore, the purification of exosomes and obtaining specific exosomes for diagnosis is an important step in their production for therapeutic purposes. This study aimed to isolate the most and purest exosomes by using different isolation methods, ultracentrifugation, differential centrifugation-filtration and precipitation from commercial mesenchymal stem cell culture medium derived from wharton gel. Imaging, purity and size analysis of exosome characterization was determined for methods. As a result, it was observed that as a result of the isolation with the ultracentrifuge method, a much larger number of exosomes were isolated compared to both precipitation and differential centrifugationfiltration methods. According to the differential centrifugation-filtration method, it was observed that the number of exosomes was much less, but the exosomes were of the same purity as the ultracentrifuge method. Since exosomes play an active role in treating almost every disease, they provide ease of use in diagnosing diseases, and their good biodistribution, biocompatibility and low immunogenicity will also provide an advantage in the treatment phase. [ABSTRACT FROM AUTHOR]

A total of 21 novel xanthone and acridone derivatives were synthesized using the reactions of 1,2,4-triazine derivatives with 1-hydroxy-3-methoxy-10-methylacridone, 1,3-dimethoxy-, and 1,3-dihydroxanthone, followed by optional dihydrotiazine ring aromatization. The synthesized compounds were evaluated for their anticancer activity against colorectal cancer HCT116, glioblastoma A-172, breast cancer Hs578T, and human embryonic kidney HEK-293 tumor cell lines. Five compounds (7a, 7e, 9e, 14a, and 14b) displayed good in vitro antiproliferative activities against these cancer cell lines. Compounds 7a and 7e demonstrated low toxicity for normal human embryonic kidney (HEK-293) cells, which determines the possibility of further development of these compounds as anticancer agents. Annexin V assay demonstrated that compound 7e activates apoptotic mechanisms and inhibits proliferation in glioblastoma cells. [ABSTRACT FROM AUTHOR]

Quantitative surface plasmon resonance (SPR) was utilized to determine binding strength and calcium dependence of direct interactions between dysferlin and proteins likely to mediate skeletal muscle repair, interrupted in limb girdle muscular dystrophy type 2B/R2. Dysferlin canonical C2A (cC2A) and C2F/G domains directly interacted with annexin A1, calpain-3, caveolin-3, affixin, AHNAK1, syntaxin-4, and mitsugumin-53, with cC2A the primary target and C2F lesser involved, overall demonstrating positive calcium dependence. Dysferlin C2 pairings alone showed negative calcium dependence in almost all cases. Like otoferlin, dysferlin directly interacted via its carboxy terminus with FKBP8, an anti-apoptotic outer mitochondrial membrane protein, and via its C2DE domain with apoptosis-linked gene (ALG-2/PDCD6), linking anti-apoptosis with apoptosis. Confocal Z-stack immunofluorescence confirmed co-compartmentalization of PDCD6 and FKBP8 at the sarcolemmal membrane. Our evidence supports the hypothesis that prior to injury, dysferlin C2 domains self-interact and give rise to a folded, compact structure as indicated for otoferlin. With elevation of intracellular Ca2+ in injury, dysferlin would unfold and expose the cC2A domain for interaction with annexin A1, calpain-3, mitsugumin 53, affixin, and caveolin-3, and dysferlin would realign from its interactions with PDCD6 at basal calcium levels to interact strongly with FKBP8, an intramolecular rearrangement facilitating membrane repair. [ABSTRACT FROM AUTHOR]

Introduction: Long non-coding RNAs (lncRNAs) have been implicated in the initiation and progression of malignant tumor. The aim of the present study was to investigate whether LINC00336 contributes to the tumorigenesis of bladder cancer (BCa). Material and methods: LncRNA expression profiling in BCa tissues was analyzed using lncRNA microarray. Cell viability, invasion and apoptosis were detected using CCK8, Transwell and Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC) double staining, respectively. Results: LncRNA microarray and RT-qPCR revealed that LINC00336 was significantly up-regulated in BCa tissues and cell lines compared with the corresponding control group. Kaplan-Meier and multivariate regression analysis demonstrated that LINC00336 could serve as an independent risk factor for predicting the prognosis of BCa patients. In vitro experimental measurements showed that knockdown of LINC00336 had the ability to block cell proliferation and invasion, as well as to induce cell cycle arrest and apoptosis. Conclusions: These findings indicated that LINC00336 might serve as an oncogene in the initiation and progression of BCa, and LINC00336 may be a valuable and promising therapeutic target for the treatment of BCa. [ABSTRACT FROM AUTHOR]

Background: Euxanthone is a plant-based flavonoid that is mostly isolated from a Chinese medicinal plant, Polygala caudate. This study was designed to evaluate the anticancer effects of euxanthone against human medulloblastoma cells. Materials and Methods: Cell viability was evaluated by CCK-8 and EdU assays. Apoptotic cell percentage was determined by annexin V/PI assay. The mRNA expression was determined by qRT-PCR. Wound healing and transwell assays were used to assess cell migration and invasion. Results: The results revealed aberrant activation of RANK/RANKL pathway in human medulloblastoma tissues and cell lines. Euxanthone suppressed the proliferation of the D425 medulloblastoma cells with comparatively lower cytotoxic effects against the normal human cerebellar granule cells. The IC50 of euxanthone against the D425 cells was found to be 10 µM. Interestingly, silencing of receptor activator of nuclear factor kβ (RANK) could also suppress the proliferation of the D425 cells via induction of apoptosis. Nonetheless, overexpression of RANK could abolish the cytotoxic effects of euxanthone on the D425 cells. Finally, wound-heal and transwell assay showed that euxanthone suppressed the migration and invasion of the D425 medulloblastoma cells. Conclusion: Collectively, the results revealed the anticancer effects of euxanthone against human medulloblastoma cells via RANK/RANKL pathway. These results suggest the potential of euxanthone as a lead molecule in the development of chemotherapy for medulloblastoma. [ABSTRACT FROM AUTHOR]

Background: Atherosclerosis (AS), the main pathological basis of life‐threatening cardiovascular disease, is essentially caused by chronic macrophage inflammation. Overexpression of proline/serine‐rich coiled‐coil protein 1 (PSRC1) reduces macrophage inflammatory responses and delays AS development. However, the exact mechanism of PSRC1 is unclear. Methods: Proteins interacting with PSRC1 were screened by proteomics in RAW264.7 cells, followed by RT‒qPCR, immunoprecipitation and immunofluorescence to explore the specific mechanistic pathways affecting inflammation. CRISPR‒Cas9 constructs for PSRC1−/−ApoE−/−(DKO) mice and high‐fat diet‐fed ApoE−/− and DKO mice were used for AS models for in vivo experiments. Upstream transcription factors of PSRC1 were predicted by ATAC‐seq, ChIP‐seq and UCSC, and the regulatory mechanism was verified by ChIP‒qPCR and dual luciferase assays. Peripheral blood serum and monocytes were collected from coronary artery disease (CAD) patients and non‐CAD patients. Results: Increased binding of ANXA2 to PSRC1 in macrophages under oxidized low‐density lipoprotein stimulation and decreased release of ANXA2 to the extracellular compartment were observed. Knockdown of ANXA2 in AS model mice delayed AS progression. Knockdown of ANXA2 in DKO mice reversed the AS‐promoting effect of PSRC1 knockdown. Mechanistically, ANXA2 promotes STAT3 phosphorylation, which in turn promotes inflammatory responses. In addition, SP1 is a PSRC1 upstream repressive transcription factor, and the SP1 inhibitor mithramycin (Mith) elevated PSRC1 expression and exerted anti‐AS effects in AS model mice. Patients with CAD had considerably greater serum levels of ANXA2 than those without CAD, and Mith reduced the secretion of ANXA2 in peripheral blood monocytes of CAD patients. Conclusion: In macrophages, PSRC1 can interact with ANXA2 to inhibit its extracellular release and delay AS development. SP1 is an upstream transcription factor of PSRC1 and inhibits the transcription of PSRC1. The SP1 inhibitor Mith can elevate PSRC1 levels and slow AS progression while reducing ANXA2 release from monocytes in CAD patients. Mith is expected to be a new agent for AS treatment. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV) infection causes enormous economic losses to the pork industry and severe health consequences in many hosts. Annexin A2 (ANXA2) is a membrane-associated protein with various intracellular functions associated with many viral infections. However, the role of ANXA2 in alphaherpesvirus replication is still not explored. In the present study, we identified the interaction between ANXA2 and PRV US3. The deficiency of ANXA2 significantly restricted PRV proliferation. PRV infection or US3 overexpression led to ANXA2 extracellular translocation. Furthermore, we confirmed that PRV or US3 could lead to the phosphorylation of the Tyr23 ANXA2 and Tyr419 Src kinase, which was associated with the ANXA2 cell surface transposition. US3 can also bind to Src in an ANXA2-independent manner and enhance the interaction between Src and ANXA2. Additionally, inhibitors targeting ANXA2 (A2ti-1) or Src (PP2) could remarkably inhibit PRV propagation in vitro and protect mice from PRV infection in vivo. Collectively, our findings broaden our understanding of the molecular mechanisms of ANXA2 in alphaherpesvirus pathogenicity and suggest that ANXA2 is a potential therapeutic target for treating alphaherpesvirus-induced infectious diseases. IMPORTANCE PRV belongs to the alphaherpesvirus and has recently re-emerged in China, causing severe economic losses. Recent studies also indicate that PRV may pose a potential public health challenge. ANXA2 is a multifunctional calcium- and lipid-binding protein implicated in immune function, multiple human diseases, and viral infection. Herein, we found that ANXA2 was essential to PRV efficient proliferation. PRV infection resulted in the extracellular translocation of ANXA2 through phosphorylation of ANXA2 and Src. ANXA2 and Src formed a complex with PRV US3. Importantly, inhibitors targeting ANXA2 or Src prevented PRV infection in vitro and in vivo. Therefore, our studies reveal a novel strategy by which alphaherpesvirus modifies ANXA2 to promote its replication and highlight ANXA2 as a target in developing novel promising antivirus agents in viral therapy. [ABSTRACT FROM AUTHOR]

In this study, six vacuum liquid chromatography (VLC) fractions (F1–F6) of the n-BuOH extract of L. numidicum Murb. (BELN) were examined for their anticancer capacity. The composition of secondary metabolites was analyzed by LC-HRMS/MS. The antiproliferative effect against PC3 and MDA-MB-231 lines was evaluated by MTT assay. Apoptosis of PC3 cells was detected by annexin V-FITC/PI staining using a flow cytometer. The results showed that only fractions 1 and 6 inhibited PC3 and MDA-MB 231 cell proliferation in a dose-dependent manner and induced dose-dependent apoptosis of PC3 cells, evidenced by the accumulation of early and late apoptotic cells, and by the decrease in viable cells. LC-HRMS/MS profiling of fractions 1 and 6 revealed the presence of known compounds that may be responsible for the observed anticancer activity. F1 and F6 may be an excellent source of active phytochemicals for cancer treatment. [ABSTRACT FROM AUTHOR]

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an "eat-me" signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1+) derived from cancer cells compared to m/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs (annexin A1+) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes. [ABSTRACT FROM AUTHOR]

The prevalence of colon cancer (CC) is increasing at the endemic scale, which is accompanied by subsequent morbidity and mortality. Although there have been noteworthy achievements in the therapeutic strategies in recent years, the treatment of patients with CC remains a formidable task. The current study focused on to study role of biohydrogenation-derived conjugated linoleic acid (CLA) of probiotic Pediococcus pentosaceus GS4 (CLAGS4) against CC, which induced peroxisome proliferator-activated receptor gamma (PPARγ) expression in human CC HCT-116 cells. Pre-treatment with PPARγ antagonist bisphenol A diglycidyl ether has significantly reduced the inhibitory efficacy of enhanced cell viability of HCT-116 cells, suggesting the PPARγ-dependent cell death. The cancer cells treated with CLA/CLAGS4 demonstrated the reduced level of Prostaglandin E2 PGE2 in association with reduced COX-2 and 5-LOX expressions. Moreover, these consequences were found to be associated with PPARγ-dependent. Furthermore, delineation of mitochondrial dependent apoptosis with the help of molecular docking LigPlot analysis showed that CLA can bind with hexokinase-II (hHK-II) (highly expressed in cancer cells) and that this association underlies voltage dependent anionic channel to open, thereby causing mitochondrial membrane depolarization, a condition that initiates intrinsic apoptotic events. Apoptosis was further confirmed by annexin V staining and elevation of caspase 1p10 expression. Taken all together, it is deduced that, mechanistically, the upregulation of PPARγ by CLAGS4 of P. pentosaceus GS4 can alter cancer cell metabolism in association with triggering apoptosis in CC. [ABSTRACT FROM AUTHOR]

Introduction. Bisphosphonates are widely used in the treatment of osteoporosis; however, they are associated with the serious adverse event of bisphosphonate‐related osteonecrosis of the jaw (BRONJ). Aim. The aim of this study is to assess the effects of nitrogen-containing bisphosphonates (N-PHs) on the synthesis of IL-1β, TNF-α, sRANKL, cathepsin K, and annexin V in bone cells cultured in vitro. Materials and Methods. Osteoblasts and bone marrow-derived osteoclasts were cultured in vitro, subjected to treatment with alendronate, risedronate, or ibandronate at a concentration of 10−5 M for 0 to 96 h and then assayed for IL-1β, sRANKL, and TNF-α production by ELISA. Cathepsin K and Annexin V-FITC staining in osteoclasts were assessed by flow cytometry. Results. There was significant downregulation of IL-1β, sRANKL, and TNF-α in experimental osteoblasts compared to control cells, and there was upregulation of IL-1β and downregulation of RANKL and TNF-α in experimental osteoclasts. Furthermore, in osteoclasts, cathepsin K expression was downregulated at 48–72 h with alendronate treatment, while risedronate treatment resulted in upregulated annexin V expression at 48 h compared to the control treatment. Conclusion. Bisphosphonates added to bone cells inhibited osteoclastogenesis, which led to the downregulation of cathepsin K and induction of apoptosis in osteoclasts; these changes limited the capacity of bone remodelling and healing that may contribute to BRONJ induced by surgical dental procedures. [ABSTRACT FROM AUTHOR]

The molecular mechanisms of skeletal muscle adaptation to spaceflight are as yet not fully investigated and well understood. The MUSCLE BIOPSY study analyzed pre and postflight deep calf muscle biopsies (m. soleus) obtained from five male International Space Station (ISS) astronauts. Moderate rates of myofiber atrophy were found in long-duration mission (LDM) astronauts (~180 days in space) performing routine inflight exercise as countermeasure (CM) compared to a short-duration mission (SDM) astronaut (11 days in space, little or no inflight CM) for reference control. Conventional H&E scout histology showed enlarged intramuscular connective tissue gaps between myofiber groups in LDM post vs. preflight. Immunoexpression signals of extracellular matrix (ECM) molecules, collagen 4 and 6, COL4 and 6, and perlecan were reduced while matrix-metalloproteinase, MMP2, biomarker remained unchanged in LDM post vs. preflight suggesting connective tissue remodeling. Large scale proteomics (space omics) identified two canonical protein pathways associated to muscle weakness (necroptosis, GP6 signaling/COL6) in SDM and four key pathways (Fatty acid β-oxidation, integrin-linked kinase ILK, Rho A GTPase RHO, dilated cardiomyopathy signaling) explicitly in LDM. The levels of structural ECM organization proteins COL6A1/A3, fibrillin 1, FBN1, and lumican, LUM, increased in postflight SDM vs. LDM. Proteins from tricarboxylic acid, TCA cycle, mitochondrial respiratory chain, and lipid metabolism mostly recovered in LDM vs. SDM. High levels of calcium signaling proteins, ryanodine receptor 1, RyR1, calsequestrin 1/2, CASQ1/2, annexin A2, ANXA2, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) pump, ATP2A, were signatures of SDM, and decreased levels of oxidative stress peroxiredoxin 1, PRDX1, thioredoxin-dependent peroxide reductase, PRDX3, or superoxide dismutase [Mn] 2, SOD2, signatures of LDM postflight. Results help to better understand the spatiotemporal molecular adaptation of skeletal muscle and provide a large scale database of skeletal muscle from human spaceflight for the better design of effective CM protocols in future human deep space exploration. [ABSTRACT FROM AUTHOR]

Malignant melanoma, an increasingly common form of skin cancer, is a major threat to public health, especially when the disease progresses past skin lesions to the stage of advanced metastasis. Targeted drug development is an effective strategy for the treatment of malignant melanoma. In this work, a new antimelanoma tumor peptide, the lebestatin–annexin V (designated LbtA5) fusion protein, was developed and synthesized by recombinant DNA techniques. As a control, annexin V (designated ANV) was also synthesized by the same method. The fusion protein combines annexin V, which specifically recognizes and binds phosphatidylserine, with the disintegrin lebestatin (lbt), a polypeptide that specifically recognizes and binds integrin α1β1. LbtA5 was successfully prepared with good stability and high purity while retaining the dual biological activity of ANV and lbt. MTT assays demonstrated that both ANV and LbtA5 could reduce the viability of melanoma B16F10 cells, but the activity of the fusion protein LbtA5 was superior to that of ANV. The tumor volume growth was slowed in a mouse xenograft model treated with ANV and LbtA5, and the inhibitory effect of high concentrations of LbtA5 was significantly better than that of the same dose of ANV and was comparable to that of DTIC, a drug used clinically for melanoma treatment. The hematoxylin and eosin (H&E) staining test showed that ANV and LbtA5 had antitumor effects, but LbtA5 showed a stronger ability to induce melanoma necrosis in mice. Immunohistochemical experiments further showed that ANV and LbtA5 may inhibit tumor growth by inhibiting angiogenesis in tumor tissue. Fluorescence labeling experiments showed that the fusion of ANV with lbt enhanced the targeting of LbtA5 to mouse melanoma tumor tissue, and the amount of target protein in tumor tissue was significantly increased. In conclusion, effective coupling of the integrin α1β1-specific recognition molecule lbt confers stronger biological antimelanoma effects of ANV, which may be achieved by the dual effects of effective inhibition of B16F10 melanoma cell viability and inhibition of tumor tissue angiogenesis. The present study describes a new potential strategy for the application of the promising recombinant fusion protein LbtA5 in the treatment of various cancers, including malignant melanoma. [ABSTRACT FROM AUTHOR]

Annexin A1 (ANXA1) is an endogenous protein, which plays a central function in the modulation of inflammation. While the functions of ANXA1 and its exogenous peptidomimetics, N-Acetyl 2-26 ANXA1-derived peptide (ANXA1Ac2-26), in the modulation of immunological responses of neutrophils and monocytes have been investigated in detail, their effects on the modulation of platelet reactivity, haemostasis, thrombosis, and platelet-mediated inflammation remain largely unknown. Here, we demonstrate that the deletion of Anxa1 in mice upregulates the expression of its receptor, formyl peptide receptor 2/3 (Fpr2/3, orthologue of human FPR2/ALX). As a result, the addition of ANXA1Ac2-26 to platelets exerts an activatory role in platelets, as characterised by its ability to increase the levels of fibrinogen binding and the exposure of P-selectin on the surface. Moreover, ANXA1Ac2-26 increased the development of platelet-leukocyte aggregates in whole blood. The experiments carried out using a pharmacological inhibitor (WRW4) for FPR2/ALX, and platelets isolated from Fpr2/3-deficient mice ascertained that the actions of ANXA1Ac2-26 are largely mediated through Fpr2/3 in platelets. Together, this study demonstrates that in addition to its ability to modulate inflammatory responses via leukocytes, ANXA1 modulates platelet function, which may influence thrombosis, haemostasis, and platelet-mediated inflammation under various pathophysiological settings. [ABSTRACT FROM AUTHOR]

Glioblastomas are highly malignant brain tumors that derive from brain-tumor-initiating cells (BTICs) and can be subdivided into several molecular subtypes. Metformin is an antidiabetic drug currently under investigation as a potential antineoplastic agent. The effects of metformin on glucose metabolism have been extensively studied, but there are only few data on amino acid metabolism. We investigated the basic amino acid profiles of proneural and mesenchymal BTICs to explore a potential distinct utilization and biosynthesis in these subgroups. We further measured extracellular amino acid concentrations of different BTICs at baseline and after treatment with metformin. Effects of metformin on apoptosis and autophagy were determined using Western Blot, annexin V/7-AAD FACS-analyses and a vector containing the human LC3B gene fused to green fluorescent protein. The effects of metformin on BTICs were challenged in an orthotopic BTIC model. The investigated proneural BTICs showed increased activity of the serine and glycine pathway, whereas mesenchymal BTICs in our study preferably metabolized aspartate and glutamate. Metformin treatment led to increased autophagy and strong inhibition of carbon flux from glucose to amino acids in all subtypes. However, oral treatment with metformin at tolerable doses did not significantly inhibit tumor growth in vivo. In conclusion, we found distinct amino acid profiles of proneural and mesenchymal BTICs, and inhibitory effects of metformin on BTICs in vitro. However, further studies are warranted to better understand potential resistance mechanisms against metformin in vivo. [ABSTRACT FROM AUTHOR]

Host-specific plant pathogens must coordinate their life cycles with the availability of a host plant. Although this is frequently achieved through a response to specific chemical cues derived from the host plant, little is known about the molecular basis of the response to such cues and how these are used to trigger activation of the life cycle. In host-specific plant-parasitic cyst nematodes, unhatched juvenile nematodes lie dormant in the eggshell until chemical cues from a suitable host plant are detected and the hatching process is initiated. The molecular mechanisms by which hatch is linked to the presence of these chemical cues is unknown. We have identified a novel annexin-like protein that is localised to the eggshell of the potato cyst nematode Globodera rostochiensis. This annexin is unique in having a short peptide insertion that structural modelling predicts is present in one of the calcium-binding sites of this protein. Host-induced gene silencing of the annexin impacts the ability of the nematode to regulate and control permeability of the eggshell. We show that in the presence of the chemicals that induce hatching annexin lipid binding capabilities change, providing the first molecular link between a nematode eggshell protein and host-derived cues. This work demonstrates how a protein from a large family has been recruited to play a critical role in the perception of the presence of a host and provides a new potential route for control of cyst nematodes that impact global food production Author summary: Potato cyst nematodes (PCN) are pathogens of economically important solanaceous plants. Like other host-specific plant-pathogens, PCN resume their life cycle specifically in response to chemical cues from suitable host plants. Here, for the first time, we describe, an eggshell localised annexin that plays a key role in the response to the presence of secreted host chemicals. Reducing the level of this protein impacts the ability of the nematode to control permeability of the eggshell, while the lipid binding properties of the protein change specifically in the presence of host derived chemical cues, ultimately affecting hatching. This work represents the first molecular determination of how a plant-parasitic nematode responds to a host chemical. Additionally, this is the first occurrence of identification and localisation of any parasitic nematode eggshell protein. The techniques used in this research can be widely applied to other species of nematodes allowing discovery of further eggshell-associated proteins and development of a new generation of nematicides or anthelminthics. [ABSTRACT FROM AUTHOR]

Multinucleated osteoclasts, essential for skeletal remodeling in health and disease, are formed by the fusion of osteoclast precursors, where each fusion event raises their bone-resorbing activity. Here we show that the nuclear RNA chaperone, La protein has an additional function as an osteoclast fusion regulator. Monocyte-to-osteoclast differentiation starts with a drastic decrease in La levels. As fusion begins, La reappears as a low molecular weight species at the osteoclast surface, where it promotes fusion. La's role in promoting osteoclast fusion is independent of canonical La-RNA interactions and involves direct interactions between La and Annexin A5, which anchors La to transiently exposed phosphatidylserine at the surface of fusing osteoclasts. Disappearance of cell-surface La, and the return of full length La to the nuclei of mature, multinucleated osteoclasts, acts as an off switch of their fusion activity. Targeting surface La in a novel explant model of fibrous dysplasia inhibits excessive osteoclast formation characteristic of this disease, highlighting La's potential as a therapeutic target. Bone maintenance in health and disease depends on bone-resorbing osteoclasts. Whitlock et al. demonstrate that an RNA chaperon -La protein- lives a second life as a key regulator of osteoclast size and function, suggesting a new therapeutic target. [ABSTRACT FROM AUTHOR]

Antibodies to red blood cells (RBCs) may hemolyze erythrocytes via Fc-mediated phagocytosis or complement-dependent. Complement activation on RBCs can be detected by C3d-direct antiglobulin test (DAT), which is the only test in immune hematology that directly targets complement. However, a positive DAT with anti-C3d cannot distinguish between C3b-mediated extravascular hemolysis, C5b-C9-mediated intravascular hemolysis and C5b-C8-mediated eryptosis. Furthermore, DAT is not suitable to estimate the strength of hemolysis. Autoimmune hemolytic anemia (AIHA) is a rare disease that is caused by autoantibodies to red blood cells that is divided in warm AIHA and in cold agglutinin disease (CAD). The causative antibodies in CAD and sometimes in warm AIHA are from the IgM class. Depending on strength of complement activation they can induce extravascular hemolysis, intravascular hemolysis and eryptosis. We studied the three types of hemolysis by use of sera from patients with CAD under various conditions. We found that additionally to the routinely applied C3d-DAT, indirect tests for complement activity (free hemoglobin and Annexin V-binding to phosphatidylserine-exposing RBCs) should be used to determine the portion of extravascular, intravascular and eryptotic hemolysis. Eryptotic hemolysis may have a significant share in clinical relevant CAD or IgM warm AIHA, which should be considered for successful treatment. [ABSTRACT FROM AUTHOR]

Hydroxyethyl cellulose (HEC), widely known for its biocompatibility and water solubility, is a polysaccharide with potential for pharmaceutical applications. Here, we synthesized polyethylenimine2k (PEI2k)-conjugated hydroxyethyl cellulose (HECP2k) for doxorubicin/Bcl-2 siRNA co-delivery systems. HECP2ks were synthesized by reductive amination of PEI2k with periodate-oxidized HEC. The synthesis of the polymers was characterized using 1H NMR, 13C NMR, primary amine quantification, FT-IR, and GPC. Via agarose gel electrophoresis and Zeta-sizer measurement, it was found that HECP2ks condensed pDNA to positively charged and nano-sized complexes (100–300 nm, ~30 mV). The cytotoxicity of HECP2ks was low and HECP2k 10X exhibited higher transfection efficiency than PEI25k even in serum condition, showing its high serum stability from ethylene oxide side chains. Flow cytometry analysis and confocal laser microscopy observation verified the superior cellular uptake and efficient endosome escape of HECP2k 10X. HECP2k 10X also could load Dox and Bcl-2 siRNA, forming nano-particles (HECP2k 10X@Dox/siRNA). By median effect analysis and annexin V staining analysis, it was found that HECP2k 10X@Dox/siRNA complexes could cause synergistically enhanced anti-cancer effects to cancer cells via induction of apoptosis. Consequently, it was concluded that HECP2k possesses great potential as a promising Dox/Bcl-2 siRNA co-delivery carrier. [ABSTRACT FROM AUTHOR]

Objective(s): Contrast media (CM) are used for diagnostic or therapeutic intervention purposes in medicine. The main adverse reaction after the administration of CM is contrast-induced nephropathy (CIN). This complication is the third cause of renal failure after hospital treatment. The current study is designed to investigate the possible protective effect of trans-sodium crocetinate (TSC), derived from carotenoid crocetin, against sodium amidotrizoate/meglumine amidotrizoate (SAMA) induced cytotoxicity in HEK-293 cells. Materials and Methods: HEK-293 cells were incubated with different concentrations of TSC (1, 2.5, 5, 10, 25, and 50 µM, for 48 hr) and then SAMA (7 mgI/ml, for 24 hr) was added. The cell viability, intracellular ROS, and phosphatidyl serine exposure were detected by MTT assay, DCFH-DA, and annexin V-FITC/PI method, respectively. The P-ERK/ERK ratio, apoptosis (Bax/Bcl-2 ratio and cleaved caspase-3), and autophagy (LC3 II/I ratio and beclin-1) markers in cells were evaluated by the western blot method. Results: The exposure of HEK-293 cells to SAMA reduced viability, increased apoptotic cells, enhanced ROS production, and subsequently decreased P-ERK/ERK ratio. Similarly, SAMA enhanced apoptosis (Bax/Bcl-2 ratio and cleaved caspase-3) and autophagy (LC3 II/I ratio and beclin-1) markers in HEK-293 cells. The pretreatment of cells with TSC before exposure to SAMA significantly attenuated contrast-induced cytotoxicity. TSC reduced intracellular ROS production and activated the phosphorylation of ERK. In addition, TSC decreased the levels of apoptosis and autophagy proteins. Conclusion: The pretreatment of HEK-293 cells with TSC can decrease contrast-induced cytotoxicity through antioxidant effect and modulate ERK, apoptosis, and autophagy pathways. [ABSTRACT FROM AUTHOR]

Introduction: Lipid metabolism participates in various biological processes such as proliferation, apoptosis, migration, invasion, and maintenance of membrane homeostasis of prostate tumor cells. Bufadienolides, the active ingredients of Chansu, show a robust anti-proliferative effect against prostate cancer cells in vitro, but whether bufadienolides could regulate the lipid metabolism in prostate cancer has not been evaluated. Objectives: Our study explored the regulatory effects of bufadienolides on lipid metabolism in human prostate carcinoma cells (PC-3). Methods: Untargeted lipidomics and transcriptomics were combined to study the effect of different bufadienolides interventions on lipid and gene changes of PC-3 cells. The key genes related to lipid metabolism and prostate cancer development were verified by qPCR and western blotting. Results: Lipidomic analysis showed that the active bufadienolides significantly downregulated the content of long-chain lipids of PC-3 cells. Based on transcriptomic and qPCR analyses, many genes related to lipid metabolism were significantly regulated by active bufadienolides, such as ELOVL6, CYP2E1, GAL3ST1, CERS1, PLA2G10, PLD1, SPTLC3, and GPX2. Bioinformatics analysis of the Cancer Genome Atlas database and literature retrieval showed that elongation of very long-chain fatty acids protein 6 (ELOVL6) and phospholipase D1 (PLD1) might be important regulatory genes. Western blot analysis revealed that active bufadienolides could downregulate PLD1 protein levels which might promote anti-prostate cancer effect. Conclusions: All these findings support that bufadienolides might induce lipid metabolic remodeling by regulating long-chain lipids synthesis and phospholipid hydrolysis to achieve an anti-prostate cancer effect, and PLD1 would probably be the key protein. [ABSTRACT FROM AUTHOR]

Purpose: To screen the micro-ribonucleic acids (miRNAs) in the pathological processes of preeclampsia (PE), and to assess their effects on the proliferation and apoptosis of HTR-8/SVneo cells. Methods: The miRNAs in PE patients and healthy puerperae were screened out using miRNA microarray assay, and verified through low-throughput quantitative reverse transcription- polymerase chain reaction (qRT-PCR). The effects of expression level of miR-19a on proliferation and cycle of HTR-8/SVneo cells were determined using cell counting kit (CCK)-8 assay and propidium iodide (PI) staining, respectively. The influence of the expression level of miR-19a on apoptosis of HTR-8/SVneo cells was evaluated using Annexin V-fluorescein isothiocyanate (FITC)/PI staining. Subsequently, luciferase reporter gene assay, CCK-8 assay and Annexin V-FITC/PI staining were conducted, respectively. Results: Serum miR-19a was significantly decreased in PE patients, and a high level of miR-19a led to increase in the proliferation of HTR-8/SVneo cells. Decreased level of miR-19a arrested HTR-8/SVneo cell cycle at G1 phase, while increase in the miR-19a level repressed the apoptosis of HTR-8/SVneo cells. The PTEN was a direct target of miR-19a, and overexpression of miR-19a promoted the activation of Akt signaling pathway, thereby significantly decreasing the expression levels of downstream phosphorylated p21/p21 (p-p21/p21), p-p27/p27 and p-glycogen synthase kinase-3 beta (GSK3β)/GSK3β. Compared with those in cells transfected with empty vectors, the pro-proliferative and anti-apoptotic effects of miR-19a weakened in HTR-8/SVneo cell lines with overexpressed PTEN. Conclusion: MiR-19a targets PTEN in order to activate Akt signaling pathway, thereby promoting proliferation, and inhibiting apoptosis of HTR-8/SVneo cells. This provides new drug targets that can be investigated for future treatment of PE. [ABSTRACT FROM AUTHOR]

This study describes an emetic food-borne intoxication associated with a Bacillus cereus group species and the characterization of the bacterial isolates from the incident in aspects of molecular tying, genetic factors, cytotoxicity, and pathogenic mechanisms relating to emetic illness. Through the polyphasic identification approach, all seven isolates obtained from food and clinical samples were identified as Bacillus thuringiensis. According to multilocus sequence typing (MLST) analysis, intraspecific diversity was found within the B. thuringiensis isolates. Four allelic profiles were found, including two previously known STs (ST8 and ST15) and two new STs (ST2804 and ST2805). All isolates harbored gene fragments located in the cereulide synthetase (ces) gene cluster. The heat-treated culture supernatants of three emetic B. thuringiensis isolates, FC2, FC7, and FC8, caused vacuolation and exhibited toxicity to Caco-2 cells, with CC50 values of 56.57, 72.17, and 79.94 µg/mL, respectively. The flow cytometry with the Annexin V/PI assay revealed both apoptosis and necrosis mechanisms, but necrosis was the prominent mechanism that caused Caco-2 cell destruction by FC2, the most toxic isolate. [ABSTRACT FROM AUTHOR]

Annexin A1 (AnxA1) is highly secreted by neutrophils and binds to formyl peptide receptors (FPRs) to trigger anti-inflammatory effects and efferocytosis. AnxA1 is also expressed in the tumor microenvironment, being mainly attributed to cancer cells. As recruited neutrophils are player cells at the tumor sites, the role of neutrophil-derived AnxA1 in lung melanoma metastasis was investigated here. Melanoma cells and neutrophils expressing AnxA1 were detected in biopsies from primary melanoma patients, which also presented higher levels of serum AnxA1 and augmented neutrophil–lymphocyte ratio (NLR) in the blood. Lung melanoma metastatic mice (C57BL/6; i.v. injected B16F10 cells) showed neutrophilia, elevated AnxA1 serum levels, and higher labeling for AnxA1 in neutrophils than in tumor cells at the lungs with metastasis. Peritoneal neutrophils collected from naïve mice were co-cultured with B16F10 cells or employed to obtain neutrophil-conditioned medium (NCM; 18 h incubation). B16F10 cells co-cultured with neutrophils or with NCM presented higher invasion, which was abolished if B16F10 cells were previously incubated with FPR antagonists or co-cultured with AnxA1 knockout (AnxA1-/-) neutrophils. The depletion of peripheral neutrophils during lung melanoma metastasis development (anti-Gr1; i.p. every 48 h for 21 days) reduced the number of metastases and AnxA1 serum levels in mice. Our findings show that AnxA1 secreted by neutrophils favors melanoma metastasis evolution via FPR pathways, addressing AnxA1 as a potential biomarker for the detection or progression of melanoma. [ABSTRACT FROM AUTHOR]

Objectives: Excessive inflammatory responses and apoptosis are critical pathologies that contribute to sepsis-induced acute kidney injury (SI-AKI). Annexin A1 (ANXA1), a member of the calcium-dependent phospholipid-binding protein family, protects against SI-AKI through its anti-inflammatory and antiapoptotic effects, but the underlying mechanisms are still largely unknown. Methods: In vivo, SI-AKI mouse models were established via caecal ligation and puncture (CLP) and were then treated with the Ac2-26 peptide of ANXA1 (ANXA1 (Ac2-26)), WRW4 (Fpr2 antagonist) or both. In vitro, HK-2 cells were induced by lipopolysaccharide (LPS) and then treated with ANXA1 (Ac2-26), Fpr2–siRNA or both. Results: In the present study, we found that the expression levels of ANXA1 were decreased, and the expression levels of TNF-α, IL-1β, IL-6, cleaved caspase-3, cleaved caspase-8 and Bax were significantly increased, accompanied by marked kidney tissue apoptosis in vivo. Moreover, we observed that ANXA1 (Ac2-26) significantly reduced the levels of TNF-α, IL-1β and IL-6 and cleaved caspase-3, cleaved caspase-8, FADD and Bax and inhibited apoptosis in kidney tissue and HK-2 cells, accompanied by pathological damage to kidney tissue. Seven-day survival, kidney function and cell viability were significantly improved in vivo and in vitro, respectively. Furthermore, the administration of ANXA1 (Ac2-26) inhibited the CLP- or LPS-induced phosphorylation of PI3K and AKT and downregulated the level of NF-κB in vivo and in vitro. Moreover, our data demonstrate that blocking the Fpr2 receptor by the administration of WRW4 or Fpr2–siRNA reversed the abovementioned regulatory role of ANXA1, accompanied by enhanced phosphorylation of PI3K and AKT and upregulation of the level of NF-κB in vivo and in vitro. Conclusions: Taken together, this study provides evidence that the protective effect of ANXA1 (Ac2-26) on SI-AKI largely depends on the negative regulation of inflammation and apoptosis via the Fpr2 receptor. [ABSTRACT FROM AUTHOR]

A series of flavonoid derivatives bearing trimethoxyphenyl and benzimidazole were designed, synthesized and evaluated as potential anti-tumor agents. Compound 5g showed remarkable inhibitory activity against SGC-7901, HGC-27 and MCF-7 (IC50 values of 20.5 ± 5.93, 3.3 ± 2.53 and 16.6 ± 0.75 μM, respectively), and had great selectivity to cancer cells. The results of Hoechst 33258 staining and Annexin V-FITC/PI dual staining confirmed that compound 5 g induced apoptosis of the HGC-27 cells in a concentration-dependent manner. After treatment with compound 5 g, the expression of HIF-1α, VEGF and PKM2 were down-regulated, accompanied by weakened the levels of lactate. Further research revealed that compound 5 g could obviously inhibit tubulin assembly. In summary, compound 5 g possessed a promising potential for further development into anti-tumor drug candidates. [ABSTRACT FROM AUTHOR]

Introduction: Glioblastoma (GBM) has a very poor prognosis despite current treatment. We previously found cytotoxic synergy between the AURKA inhibitor alisertib and the CNS-penetrating taxane TPI 287 against GBM tumor cells in vitro. Methods: We used an orthotopic human GBM xenograft mouse model to test if TPI 287 potentiates alisertib in vivo. Western blotting, immunohistochemistry, siRNA knockdown, annexin V binding, and 3-dimensional Matrigel invasion assays were used to investigate potential mechanisms of alisertib and TPI 287 treatment interactions. Results: Alisertib + TPI 287 combination therapy significantly prolonged animal survival compared to vehicle (p = 0.011), but only marginally compared to alisertib alone. Alisertib, TPI 287, and combined alisertib + TPI 287 reduced animal tumor volume compared to vehicle-treated controls. This was statistically significant for the combination therapy at 4 weeks (p < 0.0001). Alisertib + TPI 287 treatment decreased anti-apoptotic Bcl-2 protein levels in vivo and in vitro. Expression of the pro-apoptotic protein Bak was significantly increased by combination treatment (p < 0.0001). Pro-apoptotic Bim and Bak knockdown by siRNA decreased apoptosis by alisertib + TPI 287 in GB9, GB30, and U87 cells (p = 0.0005 to 0.0381). Although alisertib and TPI 287 significantly reduced GBM cell invasion (p < 0.0001), their combination was no more effective than TPI 287 alone. Conclusions: Results suggest that apoptosis is the dominant mechanism of potentiation of GBM growth inhibition by alisertib + TPI 287, in part through effects on Bcl-2 family proteins, providing a rationale for further laboratory testing of an AURKA inhibitor plus TPI 287 as a potential therapy against GBM. [ABSTRACT FROM AUTHOR]

Small molecules have demonstrated promising results as successful alternatives to growth factors. In this study, focus was drawn to CHIR99021 and tideglusib as GSK-3 inhibitors known for their anti-inflammatory and regenerative potential. The effect of both tideglusib and CHIR99021 on the proliferation, viability, and stemness of human dental pulp stem cells (hDPSCs) was investigated to assess their possible role in regenerative dentistry. Briefly, hDPSCs were isolated from sound premolars extracted for orthodontic purposes. Cytotoxicity and proliferation assessment were performed via cell counting kit-8 followed by flow cytometric analysis of apoptotic marker ANNEXIN V. The effect of both small molecules on the stemness of hDPSCs was analyzed by qRT-PCR. Both tideglusib and CHIR99021 were proven to be safe on hDPSCs. The tideglusib concentration that resulted in higher viable cells was 100 nM, while the concentration for CHIR99021 was 5 nM. Both small molecules successfully induced cellular proliferation and demonstrated minimal expression of ANNEXIN V, indicative of the absence of cellular apoptosis and further confirming their positive effect on proliferation. Finally, both small molecules enhanced stemness markers expression as evidenced by qRT-PCR, which, again, highlighted the positive effect of both tideglusib and CHIR99021 on safely promoting the proliferation of hDPSCs while maintaining their stemness. [ABSTRACT FROM AUTHOR]

Background: Aging is associated with alterations in oxi-inflammatory-immune profile, and endothelial cell dysfunction. Indeed, increased generation of free radicals as well as immunosenescence are hallmarks of the aging process and agerelated diseases. In the past 12 years or so, data has been accumulated on fermented papaya preparation (FPP®) (Osato Research Institute, Gifu, Japan), a specific functional food with robust redox and immune regulator nutrigenomics effect. The aim of this 2-year ongoing study of which we report the first-year data, was to test FPP® in redox, endothelial, and immune markers. Methods: Study population. From a total of 106 subjects, we report the analyzed data referring to 78 clinically stable, healthy, community-dwelling males and females, aged 60 to 75 years. The study was conducted using a double-blind method with designated groups A and B to fulfill the two different treatments. The two treatments are as follows: Group A, also known as "FPP Group," was given one sachet two times per day containing 4.5g FPP®, along with one placebo capsule provided in the morning. Group B, also known as "AA Group," was given one papaya-flavored sachet two times per day, along with one antioxidant mixture capsule in the morning. Morning blood samples were collected and tested for: Ultra-sensitive c-reactive protein (a highly sensitive ELISA), inducible nitric oxide synthase (iNOS) (by Human Nitric Oxide Synthase kit), asymmetric dimethylarginine, or ADMA, (a competitive enzyme-linked immunosorbent assay), apoptosis of PBMCs (by Annexin V staining) and MOS 36-item short-form health survey (SF-36) to assess quality of life. Screening and blood tests were carried out as follows: Visit I: Day 0 - Baseline, Visit II: Day 60, or 2 months, Visit III: 6 months, Visit IV: 11 months. Results: Plasma iNOS levels were comparable among both groups at the beginning of the study. FPP®-treated subjects showed a significant increased level at Visits II and III (P<0.05 vs baseline and vs AA). ADMA values were not affected by AA supplementation whereas FPP® treatment was associated with a significant decrease beginning with observation during Visit III (P<0.05 vs baseline and vs AA administration). The FPP® intervention was associated with improvements among several domains of quality of life such as physical function, general health, and mental components (P < 0.01 vs baseline and vs AA group). There was also at significant and comparable positive effect for time on vitality shown in both AA and FPP® groups. Conclusion: Unlike with the antioxidant treatment, the FPP® intervention yielded a transient decrease of ADMA, a decrease of iNOS and lower percentage in apoptotic PBMC. These results suggest that FPP®, by a more multifaceted, subcellular mechanism, as well as non-redox modulatory properties, was beneficially effective in regulating aging markers. These mechanisms are associated with a better SF-36 profile in support of FPP® as a candidate interventional functional food for health maintenance, and specifically in middle-age/elderly subjects. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is a neuropsychiatric childhood disorder that affects social skill and language development, and is characterized by persistent stereotypic behaviors, restricted social interests, and impaired language/social skills. ASD subjects have dysregulated immune responses due to impairment in inflammatory and antioxidant signaling in immune cells, such as T cells. Thioredoxin reductase-1 (TrxR1) and thioredoxin-1 (Trx1) play a crucial role in the maintenance of redox equilibrium in several immune cells, including T cells. T-cell apoptosis plays a crucial role in the pathogenesis of several inflammatory diseases. However, it remains to be explored how the TrxR1/Trx1 redox couple affects T-cells apoptosis in ASD and typically developing control (TDC) groups. Therefore, this single-center cross-sectional study explored the expression/activity of TrxR1/Trx1, and Bcl2, 7-AAD/annexin V immunostaining in T cells of ASD (n = 25) and TDC (n = 22) groups. Further, effects of the LPS were determined on apoptosis in TDC and ASD T cells. Our data show that T cells have increased TrxR1 expression, while having decreased Trx1 expression in the ASD group. Further, TrxR enzymatic activity was also elevated in T cells of the ASD group. Furthermore, T cells of the ASD group had a decreased Bcl2 expression and an increased % of annexin V immunostaining. Treatment of T cells with LPS caused greater apoptosis in the ASD group than the TDC group, with same treatment. These data reveal that the redox couple TrxR1/Trx1 is dysregulated in T cells of ASD subjects, which is associated with decreased Bcl2 expression and increased apoptosis. This may lead to decreased survival of T cells in ASD subjects during chronic inflammation. Future studies should investigate environmental factors, such as gut dysbiosis and pollutants, that may cause abnormal immune responses in the T cells of ASD subjects due to chronic inflammation. [ABSTRACT FROM AUTHOR]