Curli Amyloid Fibers in Escherichia coli Biofilms: The Influence of Water Availability on their Structure and Functional Properties.

Escherichia coli biofilms consist of bacteria embedded in a self-produced matrix mainly made of protein fibers and polysaccharides. The curli amyloid fibers found in the biofilm matrix are promising versatile building blocks to design sustainable bio-sourced materials. To exploit this potential, it is crucial to understand i) how environmental cues during biofilm growth influence the molecular structure of these amyloid fibers, and ii) how this translates at higher length scales. To explore these questions, the effect of water availability during biofilm growth on the conformation and functions of curli is studied. Microscopy and spectroscopy are used to characterize the amyloid fibers purified from biofilms grown on nutritive substrates with different water contents, and micro-indentation to measure the rigidity of the respective biofilms. The purified curli amyloid fibers present differences in the yield, structure, and functional properties upon biofilm growth conditions. Fiber packing and ß-sheets content correlate with their hydrophobicity and chemical stability, and with the rigidity of the biofilms. This study highlights how E. coli biofilm growth conditions impact curli structure and functions contributing to macroscopic materials properties. These fundamental findings infer an alternative strategy to tune curli structure, which will ultimately benefit engineering hierarchical and functional curli-based materials.

Dental caries prevention in pediatric patients with molar incisor hypomineralization: a scoping review.

This scoping review aims to summarize the available evidence on strategies employed in preventing caries in patients with molar incisor hypo-mineralization (MIH). MIH refers to an enamel defect involving opacities, and sometimes post-eruptive degradation due to enamel porosity; resulting in outcomes ranging from a mild atypical caries to severe coronary destruction. A systematic review was conducted for literature in PubMed, Cochrane Library, Epistemonikos and Literatura Latinoamericana y del Caribe en Ciencias de la Salud (LILACS). The search was aimed at studies published between January 2010 and February 2022. Data were independently selected and extracted. 989 studies were found from the systematic search and 8 studies met the eligibility criteria. Most studies evaluated remineralization and cariogenic risk, both of which are crucial elements in caries prevention, as well as decreased sensitivity. The included studies investigated fluoride varnish, dental sealants, giomers, casein, and Icon as preventative methods for dental caries. Several methods for preventing dental caries in paediatric patients with MIH exist, but more research is needed to determine their effectiveness and safety. Any preventive intervention should consider the etiological aspects of the disease, the risk of caries, the type and extent of lesions, hypersensitivity level and patient's age. Collaboration between patients and carers is critical for disease diagnosis and caries prevention.

Mechanical Activation by Ball Milling as a Strategy to Prepare Highly Soluble Pharmaceutical Formulations in the Form of Co-Amorphous, Co-Crystals, or Polymorphs.

Almost half of orally administered active pharmaceutical ingredients (APIs) have low solubility, which affects their bioavailability. In the last two decades, several alternatives have been proposed to modify the crystalline structure of APIs to improve their solubility; these strategies consist of inducing supramolecular structural changes in the active pharmaceutical ingredients, such as the amorphization and preparation of co-crystals or polymorphs. Since many APIs are thermosensitive, non-thermal emerging alternative techniques, such as mechanical activation by milling, have become increasingly common as a preparation method for drug formulations. This review summarizes the recent research in preparing pharmaceutical formulations (co-amorphous, co-crystals, and polymorphs) through ball milling to enhance the physicochemical properties of active pharmaceutical ingredients. This report includes detailed experimental milling conditions (instrumentation, temperature, time, solvent, etc.), as well as solubility, bioavailability, structural, and thermal stability data. The results and description of characterization techniques to determine the structural modifications resulting from transforming a pure crystalline API into a co-crystal, polymorph, or co-amorphous system are presented. Additionally, the characterization methodologies and results of intermolecular interactions induced by mechanical activation are discussed to explain the properties of the pharmaceutical formulations obtained after the ball milling process.

Microvesicle-associated tissue factor procoagulant activity for the preoperative diagnosis of ovarian cancer.

BACKGROUND: Tissue factor (TF) is involved in tumor growth and metastasis and contributes to venous thromboembolism (VTE) in cancer, including gynecological malignancies. The diagnostic value of microvesicle-associated TF procoagulant activity (MV TF PCA) in women with suspected ovarian cancer, however, has not been studied. OBJECTIVE: To evaluate MV TF PCA as a diagnostic tool in women with an ovarian mass of unknown etiology and as a predictive biomarker for perioperative VTE. METHODS: Plasma MVs were isolated by high-speed centrifugation and analyzed for TF-specific PCA by single-stage clotting assay. In addition, plasma TF antigen and soluble P-selectin (sCD62P) were measured by ELISA. RESULTS: D-Dimer, MV TF PCA, and sCD62P, but not the tumor marker, CA-125, significantly differentiated patients with malignant (n=40) from those with benign tumors (n=15) and healthy controls (n=34). In cancer patients, only D-Dimer and CA-125 correlated with the FIGO stage. An abnormal D-dimer had the highest sensitivity for the diagnosis of cancer, while MV TF PCA above the ROC curve-derived cut-off value of 182U/mL had the highest specificity. By multivariate logistic regression analysis, addition of MV TF PCA conferred diagnostic benefit to the single variables, CA-125 (p=0.052) and D-dimer (p=0.019). Perioperative VTE occurred in 16% of cancer patients and was associated with an advanced FIGO stage, but not MV TF PCA. There was no difference in plasma TF antigen levels between study groups. CONCLUSIONS: MV TF PCA, but not plasma TF antigen, may provide valuable additional information for the diagnostic work-up of women with suspected ovarian cancer.

CD32a antibodies induce thrombocytopenia and type II hypersensitivity reactions in FCGR2A mice.

The CD32a immunoglobulin G (IgG) receptor (Fcγ receptor IIa) is a potential therapeutic target for diseases in which IgG immune complexes (ICs) mediate inflammation, such as heparin-induced thrombocytopenia, rheumatoid arthritis, and systemic lupus erythematosus. Monoclonal antibodies (mAbs) are a promising strategy for treating such diseases. However, IV.3, perhaps the best characterized CD32a-blocking mAb, was recently shown to induce anaphylaxis in immunocompromised "3KO" mice. This anaphylactic reaction required a human CD32a transgene because mice lack an equivalent of this gene. The finding that IV.3 induces anaphylaxis in CD32a-transgenic mice was surprising because IV.3 had long been thought to lack the intrinsic capacity to trigger cellular activation via CD32a. Such an anaphylactic reaction would also limit potential therapeutic applications of IV.3. In the present study, we examine the molecular mechanisms by which IV.3 induces anaphylaxis. We now report that IV.3 induces anaphylaxis in immunocompetent CD32a-transgenic "FCGR2A" mice, along with the novel finding that IV.3 and 2 other well-characterized CD32a-blocking mAbs, AT-10 and MDE-8, also induce severe thrombocytopenia in FCGR2A mice. Using recombinant variants of these same mAbs, we show that IgG "Fc" effector function is necessary for the induction of anaphylaxis and thrombocytopenia in FCGR2A mice. Variants of these mAbs lacking the capacity to activate mouse IgG receptors not only failed to induce anaphylaxis or thrombocytopenia, but also very potently protected FCGR2A mice from near lethal doses of IgG ICs. Our findings show that effector-deficient IV.3, AT-10, and MDE-8 are promising candidates for developing therapeutic mAbs to treat CD32a-mediated diseases.

Tissue factor-dependent and -independent pathways of systemic coagulation activation in acute myeloid leukemia: a single-center cohort study.

BACKGROUND: In acute myeloid leukemia (AML), disseminated intravascular coagulation (DIC) contributes to morbidity and mortality, but the underlying pathomechanisms remain incompletely understood. METHODS: We conducted a prospective study on 69 patients with newly diagnosed AML to further define the correlates of systemic coagulation activation in this hematological malignancy. Tissue factor procoagulant activity (TF PCA) of isolated peripheral blood mononuclear cells (PBMCs) and TF expression by circulating microparticles (MPs) were assessed by single-stage clotting and thrombin generation assay, respectively. Soluble plasma TF antigen and secretion of vascular endothelial growth factor (VEGF) by cultured PBMCs were measured by ELISA. Cell-free plasma DNA was quantified by staining with a fluorescent dye. RESULT: TF PCA of PBMCs was significantly increased in AML patients as compared to healthy controls. Furthermore, TF PCA was significantly associated with decompensated DIC at presentation, as defined by a plasma fibrinogen level of ≤1 g/L (n = 11). In addition to TF PCA and circulating blasts, serum lactate dehydrogenase, a surrogate marker for leukemic cell turnover, correlated with plasma D-Dimer in the total patient cohort and was significantly increased in DIC patients, suggesting a role for myeloblast apoptosis/necrosis in activation of the TF-dependent coagulation pathway. Consistently, TF-bearing plasma MPs were more frequently detected and levels of soluble TF antigen were significantly higher in DIC vs. non-DIC patients. No association was found between TF PCA expression and VEGF secretion by isolated PBMCs, but significantly increased levels of cell-free plasma DNA pointed to a contribution of the intrinsic contact pathway to systemic coagulation activation in the total patient cohort and in patients with lower TF PCA expression. While PBMC-associated TF PCA had no effect on long-term survival, DIC occurrence at presentation increased the risk of early mortality. CONCLUSION: In newly diagnosed AML, TF expression by PBMCs and shedding of TF-bearing plasma MPs are central to the pathogenesis of DIC, but additional pathways, such as DNA liberation, may contribute to systemic coagulation activation.

Assessment of anti-metastatic effects of anticoagulant and antiplatelet agents using animal models of experimental lung metastasis.

It is well established that the blood coagulation system is activated in cancer. In addition, there is considerable evidence to suggest that clotting activation plays an important role in the biology of malignant tumors, including the process of blood-borne metastasis. For many years our laboratory has used experimental models of lung metastasis to study the events that follow the introduction of procoagulant-bearing tumor cells into circulating blood. This chapter focuses on the basic methods involved in assessing the anti-metastatic effects of anticoagulants and anti-platelet agents using rodent models of experimental metastasis. In addition, it summarizes our experience with these models, which collectively suggests that intravascular coagulation and platelet activation are a necessary prelude to lung tumor formation and that interruption of coagulation pathways or platelet aggregation may be an effective anti-metastatic strategy.

Anti-CD40L immune complexes potently activate platelets in vitro and cause thrombosis in FCGR2A transgenic mice.

Anti-CD40L immunotherapy in systemic lupus erythematosus patients was associated with thromboembolism of unknown cause. We previously showed that monoclonal anti-CD40L immune complexes (ICs) activated platelets in vitro via the IgG receptor (FcgammaRIIa). In this study, we examined the prothrombotic effects of anti-CD40L ICs in vivo. Because mouse platelets lack FcgammaRIIa, we used FCGR2A transgenic mice. FCGR2A mice were injected i.v. with preformed ICs consisting of either anti-human CD40L mAb (M90) plus human CD40L, or a chimerized anti-mouse CD40L mAb (hMR1) plus mouse CD40L. ICs containing an aglycosylated form of hMR1, which does not bind FcgammaRIIa, were also injected. M90 IC caused shock and thrombocytopenia in FCGR2A but not in wild-type mice. Animals injected with hMR1 IC also experienced these effects, whereas those injected with aglycosylated-hMR1 IC did not, demonstrating that anti-CD40L IC-induced platelet activation in vivo is FcgammaRIIa-dependent. Sequential injections of individual IC components caused similar effects, suggesting that ICs were able to assemble in circulation. Analysis of IC-injected mice revealed pulmonary thrombi consisting of platelet aggregates and fibrin. Mice pretreated with a thrombin inhibitor became moderately thrombocytopenic in response to anti-CD40L ICs and had pulmonary platelet-thrombi devoid of fibrin. In conclusion, we have shown for the first time that anti-CD40L IC-induced thrombosis can be replicated in mice transgenic for FcgammaRIIa. This molecular mechanism may be important for understanding thrombosis associated with CD40L immunotherapy. The FCGR2A mouse model may also be useful for assessing the hemostatic safety of other therapeutic Abs.

Androgen regulates Cdc6 transcription through interactions between androgen receptor and E2F transcription factor in prostate cancer cells.

Androgen receptor plays a critical role in the development and maintenance of cancers in the prostate. Earlier, we have shown that Cdc6, a regulatory protein for initiation of DNA replication, is down regulated in androgen-insensitive prostate cancer cells. In this report, we studied the involvement of androgen, mediated through androgen receptor (AR) in regulation of Cdc6 expression. Our results demonstrated that androgen treatment stimulated Cdc6 expression in xenograft tumors and androgen-sensitive prostate cancer cells. We also showed that androgen treatment stimulated Cdc6 transcription through possible interaction of AR with the ARE sequence in the Cdc6 promoter and that the stimulatory effect of androgen required intact E2F binding sites in the promoter. Androgen treatment differentially altered nuclear availability of E2F1 and E2F3, and increased the amount of hypophosphorylated retinoblastoma protein (pRb) in the nucleus in a time dependent fashion. We further showed that AR interacted with E2F transcription factors in a ligand-independent manner and that ligand-bound AR was less efficient in interacting with E2F proteins. DNA-protein interaction assays indicated that androgen treatment altered binding of E2F1 to the Cdc6 promoter in prostate cancer cells. We conclude that AR regulates Cdc6 transcription through interaction with the Cdc6 promoter, and complex formation with E2F1 and E2F3 in a differential manner.

The role of tissue factor pathway inhibitor in tumor growth and metastasis.

Clotting activation occurs frequently in cancer. Tissue factor (TF), the most potent initiator of coagulation, is expressed aberrantly in many types of malignancy and is involved not only in tumor-associated hypercoagulability but also in promoting tumor angiogenesis and metastasis via coagulation-dependent and coagulation-independent (signaling) mechanisms. Tissue factor pathway inhibitor (TFPI) is the natural inhibitor of TF coagulant and signaling activities. Studies have shown that TFPI exhibits antiangiogenic and antimetastatic effects in vitro and in vivo. In animal models of experimental metastasis, both circulating and tumor cell-associated TFPI are shown to significantly reduce tumor cell-induced coagulation activation and lung metastasis. Heparins and heparin derivatives, which induce the release of TFPI from the vascular endothelium, also exhibit antitumor effects, and TFPI may contribute significantly to those effects. Indeed, a non-anticoagulant low-molecular-weight heparin with intact TFPI-releasing capacity has been shown to have significant antimetastatic effect in a similar experimental mouse model. The evidence supporting the dual inhibitory functions on TF-driven coagulation and signaling strengthen the rationale for considering TFPI as a potential anticancer agent. This article primarily summarizes the evidence for antiangiogenic and antimetastatic effects of TFPI and describes its potential mechanisms of action. The possible application of TFPI and other inhibitors of TF as potential anticancer agents is described, and information regarding potential antitumor properties of TFPI-2 (which has structural similarities to TFPI) is also included.

Expression of LIM kinase 1 is associated with reversible G1/S phase arrest, chromosomal instability and prostate cancer.

BACKGROUND: LIM kinase 1 (LIMK1), a LIM domain containing serine/threonine kinase, modulates actin dynamics through inactivation of the actin depolymerizing protein cofilin. Recent studies have indicated an important role of LIMK1 in growth and invasion of prostate and breast cancer cells; however, the molecular mechanism whereby LIMK1 induces tumor progression is unknown. In this study, we investigated the effects of ectopic expression of LIMK1 on cellular morphology, cell cycle progression and expression profile of LIMK1 in prostate tumors. RESULTS: Ectopic expression of LIMK1 in benign prostatic hyperplasia cells (BPH), which naturally express low levels of LIMK1, resulted in appearance of abnormal mitotic spindles, multiple centrosomes and smaller chromosomal masses. Furthermore, a transient G1/S phase arrest and delayed G2/M progression was observed in BPH cells expressing LIMK1. When treated with chemotherapeutic agent Taxol, no metaphase arrest was noted in these cells. We have also noted increased nuclear staining of LIMK1 in tumors with higher Gleason Scores and incidence of metastasis. CONCLUSION: Our results show that increased expression of LIMK1 results in chromosomal abnormalities, aberrant cell cycle progression and alteration of normal cellular response to microtubule stabilizing agent Taxol; and that LIMK1 expression may be associated with cancerous phenotype of the prostate.

Intervención psicológica tras los atentados del 11 de marzo en Madrid / Psychological intervention in Madrid March 11 after Terrorist Attacks

En el artículo aparece reflejado el dispositivo de coordinación y asistencia psicológica, que el Colegio Oficial de Psicólogos de Madrid, articuló para la asistencia a víctimas de los atentados terroristas del 11 de Marzo de 2004 de Madrid. Dicho dispositivo prestó asistencia a heridos, familiares de fallecidos y de heridos, población en general y personal interviniente. Las asistencias fueron realizadas en los Centros de Coordinación de Emergencias, hospitales, IFEMA, tanatorios y depósito de fallecidos, cementerios, comisarías de policía, hoteles, domicilios. Se describen los lugares y tipos de asistencia realizadas, así como las dificultades en las mismas (AU)

This paper gives detailed information on the procedure of coordination and psychological assistance provided by the Spanish Psychological Association (Colegio Oficial de Psicólogos) to assist victims of the terrorist attack that took place in Madrid on March 11 2004. Such a procedure included the assistance to injured families of the dead and injured citizens in general and rescue teams. Assistance was provided in Emergency Coordination Centers, hospitals, the zero ground, morgues, graveyards, police stations, hotels, homes, etc. A description of some of the assistance types and locations is provided, along with difficulties encountered (AU)

LIM kinase 1 is essential for the invasive growth of prostate epithelial cells: implications in prostate cancer.

Mammalian LIM kinase 1 (LIMK1) is involved in reorganization of actin cytoskeleton through inactivating phosphorylation of the ADF family protein cofilin, which depolymerizes actin filaments. Maintenance of the actin dynamics in an ordered fashion is essential for stabilization of cell shape or promotion of cell motility depending on the cell type. These are the two key phenomena that may become altered during acquisition of the metastatic phenotype by cancer cells. Here we show that LIMK1 is overexpressed in prostate tumors and in prostate cancer cell lines, that the concentration of phosphorylated cofilin is higher in metastatic prostate cancer cells, and that a partial reduction of LIMK1 altered cell proliferation by arresting cells at G2/M, changed cell shape, and abolished the invasiveness of metastatic prostate cancer cells. We also show that the ectopic expression of LIMK1 promotes acquisition of invasive phenotype by the benign prostate epithelial cells. Our data provide evidence of a novel role of LIMK1 in regulating cell division and invasive property of prostate cancer cells and indicate that the effect is not mediated by phosphorylation of cofilin. Our study correlates with the recent observations showing a metastasis-associated chromosomal gain on 7q11.2 in prostate cancer, suggesting a possible gain in LIMK1 DNA (7q11.23).

Potential involvement of extracellular signal-regulated kinase 1 and 2 in encystation of a primitive eukaryote, Giardia lamblia. Stage-specific activation and intracellular localization.

Mitogen-activated protein kinase (MAPK) pathways are major signaling systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified Giardia lamblia homologues of two members of the MAPK family ERK1 and ERK2. Functional characterization of giardial ERK1 and ERK2 revealed that both kinases were expressed in trophozoites and encysting cells as 44- and 41-kDa polypeptides, respectively, and were catalytically active. Analysis of the kinetic parameters of the recombinant proteins showed that ERK2 is approximately 5 times more efficient than ERK1 in phosphorylating myelin basic protein as a substrate, although the phosphorylating efficiency of the native ERK1 and ERK2 appeared to be the same. Immunofluorescence analysis of the subcellular localization of ERK1 and ERK2 in trophozoites showed ERK1 staining mostly in the median body and in the outer edges of the adhesive disc and ERK2 staining in the nuclei and in the caudal flagella. Our study also showed a noticeable change in the subcellular distribution of ERK2 during encystation, which became more punctate and mostly cytoplasmic, but no significant change in the ERK1 localization at any time during encystation. Interestingly, both ERK1 and ERK2 enzymes exhibited a significantly reduced kinase activity during encystation reaching a minimum at 24 h, except for an initial approximately 2.5-fold increase in the ERK1 activity at 2 h, which resumed back to the normal levels at 48 h despite no apparent change in the expression level of either one of these kinases in encysting cells. A reduced concentration of the phosphorylated ERK1 and ERK2 was also evident in these cells at 24 h. Our study suggests a functional distinction between ERK1 and ERK2 and that these kinases may play a critical role in trophozoite differentiation into cysts.

Down-regulation of Cdc6, a cell cycle regulatory gene, in prostate cancer.

CDC6 plays a critical role in regulation of the onset of DNA replication in eukaryotic cells. We have found that Cdc6 expression is down-regulated in prostate cancer as detected by semiquantitative reverse transcriptase-PCR of prostate cell lines and laser-captured microdissected prostate tissues. This result was substantiated by immunohistochemical analysis of paraffin-embedded tissue sections and immunoblot analysis of benign (BPH-1) and adenocarcinomatous prostatic cells. Furthermore, a 100-fold reduction in the transcription efficiency of the Cdc6 promoter-luciferase construct was noted in the metastatic PC3 cells compared with that in BPH-1 cells. Concentration of the E2F and Oct1 transcription factors that have putative binding sites in the Cdc6 promoter was substantially low in PC3 cells compared with BPH cells. Mutagenesis of the two E2F binding sites on the Cdc6 promoter resulted in increased promoter activity in PC3 cells owing to elimination of the negative regulation by pRb.E2F complex but not to the level of that obtained in BPH cells. We conclude that an altered interaction of transcription factors may be responsible for the down-regulation of Cdc6 transcription in PC3 cells. Our study suggests a potential use of the lack of CDC6 expression as an index of prostate cancer development.