A mechanism of platelet integrin αIIbß3 outside-in signaling through a novel integrin αIIb subunit-filamin-actin linkage.

The communication of talin-activated integrin αIIbß3 with the cytoskeleton (integrin outside-in signaling) is essential for platelet aggregation, wound healing, and hemostasis. Filamin, a large actin crosslinker and integrin binding partner critical for cell spreading and migration, is implicated as a key regulator of integrin outside-in signaling. However, the current dogma is that filamin, which stabilizes inactive αIIbß3, is displaced from αIIbß3 by talin to promote the integrin activation (inside-out signaling), and how filamin further functions remains unresolved. Here, we show that while associating with the inactive αIIbß3, filamin also associates with the talin-bound active αIIbß3 to mediate platelet spreading. Fluorescence resonance energy transfer-based analysis reveals that while associating with both αIIb and ß3 cytoplasmic tails (CTs) to maintain the inactive αIIbß3, filamin is spatiotemporally rearranged to associate with αIIb CT alone on activated αIIbß3. Consistently, confocal cell imaging indicates that integrin α CT-linked filamin gradually delocalizes from the ß CT-linked focal adhesion marker-vinculin likely because of the separation of integrin α/ß CTs occurring during integrin activation. High-resolution crystal and nuclear magnetic resonance structure determinations unravel that the activated integrin αIIb CT binds to filamin via a striking α-helix→ß-strand transition with a strengthened affinity that is dependent on the integrin-activating membrane environment containing enriched phosphatidylinositol 4,5-bisphosphate. These data suggest a novel integrin αIIb CT-filamin-actin linkage that promotes integrin outside-in signaling. Consistently, disruption of such linkage impairs the activation state of αIIbß3, phosphorylation of focal adhesion kinase/proto-oncogene tyrosine kinase Src, and cell migration. Together, our findings advance the fundamental understanding of integrin outside-in signaling with broad implications in blood physiology and pathology.

Optimizing biochar, vermicompost, and duckweed amendments to mitigate arsenic uptake and accumulation in rice (Oryza sativa L.) cultivated on arsenic-contaminated soil.

The accumulation of arsenic (As) in rice (Oryza sativa L.) grain poses a significant health concern in Bangladesh. To address this, we investigated the efficacy of various organic amendments and phytoremediation techniques in reducing As buildup in O. sativa. We evaluated the impact of five doses of biochar (BC; BC0.1: 0.1%, BC0.28: 0.28%, BC0.55: 0.55%, BC0.82: 0.82% and BC1.0: 1.0%, w/w), vermicompost (VC; VC1.0: 1.0%, VC1.8: 1.8%, VC3.0: 3.0%, VC4.2: 4.2% and VC5.0: 5.0%, w/w), and floating duckweed (DW; DW100: 100, DW160: 160, DW250: 250, DW340: 340 and DW400: 400 g m- 2) on O. sativa cultivated in As-contaminated soil. Employing a three-factor five-level central composite design and response surface methodology (RSM), we optimized the application rates of BC-VC-DW. Our findings revealed that As contamination in the soil negatively impacted O. sativa growth. However, the addition of BC, VC, and DW significantly enhanced plant morphological parameters, SPAD value, and grain yield per pot. Notably, a combination of moderate BC-DW and high VC (BC0.55VC5DW250) increased grain yield by 44.4% compared to the control (BC0VC0DW0). As contamination increased root, straw, and grain As levels, and oxidative stress in O. sativa leaves. However, treatment BC0.82VC4.2DW340 significantly reduced grain As (G-As) by 56%, leaf hydrogen peroxide by 71%, and malondialdehyde by 50% compared to the control. Lower doses of BC-VC-DW (BC0.28VC1.8DW160) increased antioxidant enzyme activities, while moderate to high doses resulted in a decline in these activities. Bioconcentration and translocation factors below 1 indicated limited As uptake and translocation in plant tissues. Through RSM optimization, we determined that optimal doses of BC (0.76%), VC (4.62%), and DW (290.0 g m- 2) could maximize grain yield (32.96 g pot- 1, 44% higher than control) and minimize G-As content (0.189 mg kg- 1, 54% lower than control). These findings underscore effective strategies for enhancing yield and reducing As accumulation in grains from contaminated areas, thereby ensuring agricultural productivity, human health, and long-term sustainability. Overall, our study contributes to safer food production and improved public health in As-affected regions.

The Upregulation of Integrin αDß2 (CD11d/CD18) on Inflammatory Macrophages Promotes Macrophage Retention in Vascular Lesions and Development of Atherosclerosis.

Macrophage accumulation is a critical step during development of chronic inflammation, initiating progression of many devastating diseases. Leukocyte-specific integrin αDß2 (CD11d/CD18) is dramatically upregulated on macrophages at inflammatory sites. Previously we found that CD11d overexpression on cell surfaces inhibits in vitro cell migration due to excessive adhesion. In this study, we have investigated how inflammation-mediated CD11d upregulation contributes to macrophage retention at inflammatory sites during atherogenesis. Atherosclerosis was evaluated in CD11d-/-/ApoE-/- mice after 16 wk on a Western diet. CD11d deficiency led to a marked reduction in lipid deposition in aortas and isolated macrophages. Macrophage numbers in aortic sinuses of CD11d-/- mice were reduced without affecting their apoptosis and proliferation. Adoptive transfer of fluorescently labeled wild-type and CD11d-/- monocytes into ApoE-/- mice demonstrated similar recruitment from circulation, but reduced accumulation of CD11d-/- macrophages within the aortas. Furthermore, CD11d expression was significantly upregulated on macrophages in atherosclerotic lesions and M1 macrophages in vitro. Interestingly, expression of the related ligand-sharing integrin CD11b was not altered. This difference defines their distinct roles in the regulation of macrophage migration. CD11d-deficient M1 macrophages demonstrated improved migration in a three-dimensional fibrin matrix and during resolution of peritoneal inflammation, whereas migration of CD11b-/- M1 macrophages was not affected. These results prove the contribution of high densities of CD11d to macrophage arrest during atherogenesis. Because high expression of CD11d was detected in several inflammation-dependent diseases, we suggest that CD11d/CD18 upregulation on proinflammatory macrophages may represent a common mechanism for macrophage retention at inflammatory sites, thereby promoting chronic inflammation and disease development.

Mechanisms of talin-dependent integrin signaling and crosstalk.

Cells undergo dynamic remodeling of the cytoskeleton during adhesion and migration on various extracellular matrix (ECM) substrates in response to physiological and pathological cues. The major mediators of such cellular responses are the heterodimeric adhesion receptors, the integrins. Extracellular or intracellular signals emanating from different signaling cascades cause inside-out signaling of integrins via talin, a cystokeletal protein that links integrins to the actin cytoskeleton. Various integrin subfamilies communicate with each other and growth factor receptors under diverse cellular contexts to facilitate or inhibit various integrin-mediated functions. Since talin is an essential mediator of integrin activation, much of the integrin crosstalk would therefore be influenced by talin. However, despite the existence of an extensive body of knowledge on the role of talin in integrin activation and as a stabilizer of ECM-actin linkage, information on its role in regulating inter-integrin communication is limited. This review will focus on the structure of talin, its regulation of integrin activation and discuss its potential role in integrin crosstalk. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.

HPV-type-specific response of cervical cancer cells to cisplatin after silencing replication licensing factor MCM4.

Minichoromosome maintenance (MCM) proteins play key role in cell cycle progression by licensing DNA replication only once per cell cycle. These proteins are found to be overexpressed in cervical cancer cells. In this study, we depleted MCM4, one of the MCM 2-7 complex components by RNA interference (RNAi) in four cervical cancer cell lines. The four cell lines were selected on the basis of their human papillomavirus (HPV) infection: HPV16-positive SiHa, HPV18-positive ME-180, HPV16- and HPV18-positive CaSki, and HPV-negative C-33A. The MCM4-deficient cells irrespective of their HPV status grow for several generations and maintain regular cell cycle. We did not find any evidence of augmented response to a short-term (48 h) cisplatin treatment in these MCM4-deficient cells. However, MCM4-/HPV16+ SiHa cells cannot withstand a prolonged treatment (up to 5 days) of even a sublethal dosage of cisplatin. They show increased chromosomal instability compared to their control counterparts. On the other hand, MCM4-deficient CaSki cells (both HPV16+ and 18+) remain resistant to a prolonged exposure to cisplatin. Our study indicates that cervical cancer cells may be using excess MCMs as a backup for replicative stress; however, its regulatory mechanism is dependent on the HPV status of the cells.

Kindlin-3 enhances breast cancer progression and metastasis by activating Twist-mediated angiogenesis.

The FERM domain containing protein Kindlin-3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in Kindlin-3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin-3 overexpression in a breast cancer cell line increased primary tumor growth and lung metastasis by 2.5- and 3-fold, respectively, when implanted into mice compared with cells expressing vector alone. Mechanistically, the Kindlin-3-overexpressing cells displayed a 2.2-fold increase in vascular endothelial growth factor (VEGF) secretion and enhanced ß1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor that promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production, and knockdown of ß1 integrins diminished Twist and VEGF production by Kindlin-3-overexpressing cells, while nontargeting small interfering RNA had no effect on expression of these gene products. Thus, Kindlin-3 influences breast cancer progression by influencing the crosstalk between ß1 integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis.

Histone 2B Facilitates Plasminogen-Enhanced Endothelial Migration through Protease-Activated Receptor 1 (PAR1) and Protease-Activated Receptor 2 (PAR2).

Plasminogen and its multiple receptors have been implicated in the responses of many different cell types. Among these receptors, histone 2B (H2B) has been shown to play a prominent role in macrophage responses. The contribution of H2B to plasminogen-induced endothelial migration, an event relevant to wound healing and angiogenesis, is unknown. Plasminogen enhanced the migration of endothelial cells, which was inhibited by both Protease-Activated Receptor-1 (PAR1) and 2 (PAR2) antagonists. H2B was detected on viable endothelial cells of venous and arterial origin, and an antibody to H2B that blocks plasminogen binding also inhibited the plasminogen-dependent migration by these cells. The antibody blockade was as effective as PAR1 or PAR2 antagonists in inhibiting endothelial cell migration. In pull-down experiments, H2B formed a complex with both PAR1 and PAR2 but not ß3 integrin, another receptor implicated in endothelial migration in the presence of plasminogen. H2B was found to be associated with clathrin adapator protein, AP2µ (clathrin AP2µ) and ß-arrestin2, which are central to the internationalization/signaling machinery of the PARs. These associations with PAR1-clathrin adaptor AP2µ- and PAR2-ß-arrestin2-dependent internalization/signaling pathways provide a mechanism to link plasminogen to responses such as wound healing and angiogenesis.

The role of CPT1A as a biomarker of breast cancer progression: a bioinformatic approach.

Breast cancer is the commonest malignancy of women and with its incidence on the rise, the need to identify new targets for treatment is imperative. There is a growing interest in the role of lipid metabolism in cancer. Carnitine palmitoyl-transferase-1 (CPT-1); the rate limiting step in fatty acid oxidation, has been shown to be overexpressed in a range of tumours. There are three isoforms of CPT-1; A, B and C. It is CPT-1A that has been shown to be the predominant isoform which is overexpressed in breast cancer. We performed a bioinformatic analysis using readily available online platforms to establish the prognostic and predictive effects related to CPT-1A expression. These include the KM plotter, the Human Protein Atlas, the cBioPortal, the G2O, the MethSurvand the ROC plotter. A Network analysis was performed using the Oncomine platform and signalling pathways constituting the cancer hallmarks, including immune regulation as utilised by NanoString. The epigenetic pathways were obtained from the EpiFactor website. Spearman correlations (r) to determine the relationship between CPT-1A and the immune response were obtained using the TISIDB portal. Overexpression of CPT-1A largely confers a worse prognosis and CPT-1A progressively recruits a range of pathways as breast cancer progresses. CPT-1A's interactions with cancer pathways is far wider than previously realised and includes associations with epigenetic regulation and immune evasion pathways, as well as wild-type moderate to high penetrant genes involved in hereditary breast cancer. Although CPT-1A genomic alterations are detected in 9% of breast carcinomas, both the alteration and the metagene associated with it, confers a poor prognosis. CPT-1A expression can be utilised as a biomarker of disease progression and as a potential therapeutic target.

WAVE3, an actin remodeling protein, is regulated by the metastasis suppressor microRNA, miR-31, during the invasion-metastasis cascade.

WAVE3, an actin cytoskeleton remodeling protein, is highly expressed in advanced stages of breast cancer and influences tumor cell invasion. Loss of miR-31 has been associated with cancer progression and metastasis. Here, we show that the activity of WAVE3 to promote cancer cell invasion is regulated by miR-31. An inverse correlation was demonstrated between expression levels of WAVE3 and miR-31 in invasive versus noninvasive breast cancer cell lines. miR-31 directly targeted the 3'-UTR of the WAVE3 mRNA and inhibited its expression in the invasive cancer cells, i.e., miR-31-mediated down-regulation of WAVE3 resulted in a significant reduction in the invasive phenotype of cancer cells. This relationship was specific to the loss of WAVE3 expression because re-expression of a miR-31-resistant form of WAVE3 reversed miR-31-mediated inhibition of cancer cell invasion. Furthermore, expression of miR-31 correlates inversely with breast cancer progression in humans, where an increase in expression of miR-31 target genes was observed as the tumors progressed to more aggressive forms. In conclusion, a novel mechanism for the regulation of WAVE3 expression in cancer cells has been identified, which controls the invasive properties of cancer cells. The study also identifies a critical role for WAVE3, downstream of miR-31, in the invasion-metastasis cascade.

A new species of palaemonid prawn emMacrobrachium/em emramae/em sp.nov. (Malacostraca:Decapoda:Palaemonidae) from Rupnarayana River, West Bengal, India with its molecular profiles.

A new species Macrobrachium ramae is described from Rupnarayana river, West Bengal, India along with its molecular characterization and Scanning electron microscopy. The species shares certain characters with M.gurudeve, M.jayasreei, M.kunjuramani and M.saengphani but differs remarkably from these species in the structure and shape of rostrum, telson, appendix masculina and in the size of the proximal segment of the antennular peduncle. Molecular characterization and phylogenetic analysis of M.ramae with mitochondrial COI and 16S rRNA genes reinforce the morphological conclusion and supports the view that it is a new species.

Plasminogen-induced foam cell formation by macrophages occurs through a histone 2B (H2B)-PAR1 pathway and requires integrity of clathrin-coated pits.

OBJECTIVE: Plasminogen/plasmin is a serine protease system primarily responsible for degrading fibrin within blood clots. Plasminogen mediates its functions by interacting with plasminogen receptors on the cell surface. H2B, one such plasminogen receptor, is found on the surface of several cell types including macrophages. Both basic and clinical studies support the role of plasminogen in the process of foam cell formation (FCF), a hallmark of atherosclerosis. Growing evidence also implicates serine protease-activated receptors (PARs) in atherosclerosis. These receptors are also found on macrophages, and plasmin is capable of activating PAR1 and PAR4. The goal of this study was to determine the extent of H2B's contribution to plasminogen-mediated FCF by macrophages and if PARs are involved in this process. APPROACH AND RESULTS: Treating macrophages with plasminogen increases their oxidized low-density lipoprotein uptake and plasminogen-mediated foam cell formation (Plg-FCF) significantly. The magnitude of Plg-FCF correlates with cell-surface expression of the H2B level. H2B blockade or downregulation reduces Plg-FCF, whereas its overexpression or high endogenous levels increases Plg-FCF. Modulating PAR1 level in mouse macrophages affects Plg-FCF. Activation/overexpression of PAR1 increases and its blockade/knockdown reduces this response. Confocal imaging indicates that both H2B and PAR1 colocalize with clathrin coated pits on the surface of macrophages, and reducing expression of clathrin or interfering with the clathrin-coated pits integrity reduces Plg-FCF. CONCLUSION: Our data indicate that the magnitude of Plg-FCF by macrophages is proportional to the H2B levels and demonstrate for the first time that PAR1 is involved in this process and that the integrity of clathrin-coated pits is required for the full effect of Plg-induced FCF.

Identification and characterization of multiple similar ligand-binding repeats in filamin: implication on filamin-mediated receptor clustering and cross-talk.

The actin-binding protein filamin links membrane receptors to the underlying cytoskeleton. The cytoplasmic domains of these membrane receptors have been shown to bind to various filamin immunoglobulin repeats. Notably, among 24 human filamin repeats, repeat 17 was reported to specifically bind to platelet receptor glycoprotein Ibalpha and repeat 21 to integrins. However, a complete sequence alignment of all 24 human filamin repeats reveals that repeats 17 and 21 actually belong to a distinct filamin repeat subgroup (containing repeats 4, 9, 12, 17, 19, 21, and 23) that shares a conserved ligand-binding site. Using isothermal calorimetry and NMR analyses, we show that all repeats in this subgroup can actually bind glycoprotein Ibalpha, integrins, and a cytoskeleton regulator migfilin in similar manners. These data provide a new view on the ligand specificity of the filamin repeats. They also suggest a multiple ligand binding mechanism where similar repeats within a filamin monomer may promote receptor clustering or receptor cross-talking for regulation of the cytoskeleton organization and diverse filamin-mediated cellular activities.

A green approach towards adoption of chemical reaction model on 2,5-dimethyl-2,5-di-(tert-butylperoxy)hexane decomposition by differential isoconversional kinetic analysis.

This study investigated the thermal degradation products of 2,5-dimethyl-2,5-di-(tert-butylperoxy) hexane (DBPH), by TG/GC/MS to identify runaway reaction and thermal safety parameters. It also included the determination of time to maximum rate under adiabatic conditions (TMR(ad)) and self-accelerating decomposition temperature obtained through Advanced Kinetics and Technology Solutions. The apparent activation energy (Ea) was calculated from differential isoconversional kinetic analysis method using differential scanning calorimetry experiments. The Ea value obtained by Friedman analysis is in the range of 118.0-149.0 kJ mol(-1). The TMR(ad) was 24.0 h with an apparent onset temperature of 82.4°C. This study has also established an efficient benchmark for a thermal hazard assessment of DBPH that can be applied to assure safer storage conditions.

Of Kindlins and Cancer.

Kindlins are 4.1-ezrin-ridixin-moesin (FERM) domain containing proteins. There are three kindlins in mammals, which share high sequence identity. Kindlin-1 is expressed primarily in epithelial cells, kindlin-2 is widely distributed and is particularly abundant in adherent cells, and kindlin-3 is expressed primarily in hematopoietic cells. These distributions are not exclusive; some cells express multiple kindlins, and transformed cells often exhibit aberrant expression, both in the isoforms and the levels of kindlins. Great interest in the kindlins has emerged from the recognition that they play major roles in controlling integrin function. In vitro studies, in vivo studies of mice deficient in kindlins, and studies of patients with genetic deficiencies of kindlins have clearly established that they regulate the capacity of integrins to mediate their functions. Kindlins are adaptor proteins; their function emanate from their interaction with binding partners, including the cytoplasmic tails of integrins and components of the actin cytoskeleton. The purpose of this review is to provide a brief overview of kindlin structure and function, a consideration of their binding partners, and then to focus on the relationship of each kindlin family member with cancer. In view of many correlations of kindlin expression levels and neoplasia and the known association of integrins with tumor progression and metastasis, we consider whether regulation of kindlins or their function would be attractive targets for treatment of cancer.

PI3K/AKT pathway-mediated regulation of p27(Kip1) is associated with cell cycle arrest and apoptosis in cervical cancer.

BACKGROUND: The cyclin-dependent kinase inhibitor p27(Kip1) is known to act as a putative tumor suppressor in several human cancers, including cervical cancer. Down-regulation of p27(Kip1) may occur either through transcription inhibition or through phosphorylation-dependent proteolytic degradation. As yet, the mechanism underlying p27(Kip1) down-regulation and its putative downstream effects on cervical cancer development are poorly understood. Here we assessed the expression and sub-cellular localization of p27(Kip1) and its effects on proliferation, cell cycle progression and (inhibition of) apoptosis in cervical cancer cells. METHODS: Primary cervical cancer samples (n = 70), normal cervical tissue samples (n = 30) and cervical cancer-derived cell lines (n = 8) were used to assess the expression of p27(Kip1) and AKT1 by RT-PCR, Western blotting and immunohistochemistry, respectively. The effects of the PI3K inhibitor LY294004 and the proteasome inhibitor MG132 on cervical cancer cell proliferation were investigated using a MTT assay. Apoptosis and cell cycle analyses were carried out using flow cytometry, and sub-cellular p27(Kip1) localization analyses were carried out using immunofluorescence assays. RESULTS: We observed p27(Kip1) down-regulation (p = 0.045) and AKT1 up-regulation (p = 0.046) in both the primary cervical cancer samples and the cervical cancer-derived cell lines, compared to the normal cervical tissue samples tested. Treatment of cervical cancer-derived cell lines with the PI3K inhibitor LY294002 resulted in a reduced AKT1 activity. We also observed a dose-dependent inhibition of cell viability after treatment of these cell lines with the proteasome inhibitor MG132. Treatment of the cells with LY294002 resulted in a G1 cell cycle arrest, a nuclear expression of p27(Kip1), and a cytoplasmic p27(Kip1) accumulation after subsequent treatment with MG132. Additionally, we found that the synergistic effect of MG132 and LY294002 resulted in a sub-G1 cell cycle arrest and apoptosis induction through poly (ADP-ribose) polymerase (PARP) cleavage. CONCLUSION: Our data suggest that p27(Kip1) down-regulation in cervical cancer cells is primarily regulated through PI3K/AKT-mediated proteasomal degradation. The observed synergistic effect of the MG132 and LY294002 inhibitors may form a basis for the design of novel cervical cancer therapies.