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A major problem after tendon injury is adhesion formation to the surrounding tissue leading to a limited range of motion. A viable strategy to reduce adhesion extent is the use of physical barriers that limit the contact between the tendon and the adjacent tissue. The purpose of this study was to fabricate an electrospun bilayered tube of hyaluronic acid/polyethylene oxide (HA/PEO) and biodegradable DegraPol® (DP) to improve the anti-adhesive effect of the implant in a rabbit Achilles tendon full laceration model compared to a pure DP tube. Additionally, the attachment of rabbit tenocytes on pure DP and HA/PEO containing scaffolds was tested and Scanning Electron Microscopy, Fourier-transform Infrared Spectroscopy, Differential Scanning Calorimetry, Water Contact Angle measurements, and testing of mechanical properties were used to characterize the scaffolds. In vivo assessment after three weeks showed that the implant containing a second HA/PEO layer significantly reduced adhesion extent reaching levels comparable to native tendons, compared with a pure DP implant that reduced adhesion formation only by 20 %. Tenocytes were able to attach to and migrate into every scaffold, but cell number was reduced over two weeks. Implants containing HA/PEO showed better mechanical properties than pure DP tubes and with the ability to entirely reduce adhesion extent makes this implant a promising candidate for clinical application in tendon repair., Competing Interests: Declaration of competing interest The authors declare that they do not have any competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests., Objectives: To determine how diminished myosin activity affects platelet functions, including and beyond clot contraction., Methods: Using the myosin IIA-specific pharmacologic inhibitor blebbistatin, we modulated myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition by interrogating distinct platelet functions at each stage of thrombus formation using a range of complementary assays., Results: Partial myosin IIA inhibition neither affected platelet von Willebrand factor interactions under arterial shear nor platelet spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting platelet spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion, and platelet aggregation downstream of glycoprotein (GP)VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by adenosine diphosphate or arachidonic acid was unaffected., Conclusion: Our findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling., (© 2024 The Author(s).)

Transglutaminase 2 (TG2) plays a vital role in stabilizing extracellular matrix (ECM) proteins through enzymatic crosslinking during tissue growth, repair, and inflammation. TG2 also binds non-covalently to fibronectin (FN), an essential component of the ECM, facilitating cell adhesion, migration, proliferation, and survival. However, the interaction between TG2 and fibrillar FN remains poorly understood, as most studies have focused on soluble or surface-adsorbed FN or FN fragments, which differ in their conformations from insoluble FN fibers. Using a well-established in vitro FN fiber stretch assay, we discovered that the binding of a crosslinking enzyme to ECM fibers is mechano-regulated. TG2 binding to FN is tuned by the mechanical tension of FN fibers, whereby TG2 predominantly co-localizes to low-tension FN fibers, while fiber stretching reduces their affinity for TG2. This mechano-regulated binding relies on the proximity between the N-terminal β-sandwich and C-terminal β-barrels of TG2. Crosslinking mass spectrometry (XL-MS) revealed a novel TG2-FN synergy site within TG2's C-terminal β-barrels that interacts with FN regions located outside of the canonical gelatin binding domain, specifically FNI 2 and FNIII 14-15 . Combining XL-MS distance restraints with molecular docking revealed the mechano-regulated binding mechanism between TG2 and modules FNI 7-9 by which mechanical forces regulate TG2-FN interactions. This highlights a previously unrecognized role of TG2 as a tension sensor for FN fibers. This novel interaction mechanism has significant implications in physiology and mechanobiology, including how forces regulate cell adhesion, spreading, migration, phenotype modulation, depending on the tensional state of ECM fibers. Data are available via ProteomeXchange with identifier PXD043976., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier B.V.)

Tracks rich in matrix and cells, as described in several cancer types, have immunosuppressive functions and separate tumor nests and stroma, yet their origin is unknown. Immunostainings of cryosections from mouse breast tumors show that these tracks are bordered by an endothelial-like basement membrane, filled with fibers of collagen adjacent to tenascin-C (TNC) and low-tension fibronectin (Fn) fibers. While present in early-stage tumors and maturing with time, tracks still form under TNC KO conditions, however, host (not tumor cell)-derived TNC is important for track maturation. Tumor infiltrating leukocytes (mostly M2 macrophages and CD8+ T cells) are retained in tracks of early-stage tumors. Following track maturation, retained tumor infiltrating leukocyte (TIL) numbers get reduced and more CD8+ TIL enter the tumor nests in the absence of TNC. As these tracks are enriched with platelets and fibrinogen and have a demarcating endothelial-like basement membrane often adjacent to endothelial cells, this suggests a role of blood vessels in the formation of these tracks. The Fn fiber tension probe FnBPA5 colocalizes with TNC and immune cells in the tracks and shows decreased binding in tracks lacking TNC. Consequently, FnBPA5 can serve as probe for tumor matrix tracks that have immune suppressive properties., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Spermatogonial stem cells (SSCs) originate from gonocytes that differentiate from primordial germ cells (PGCs). In the developing mouse testis, expression of the gene LIM homeobox 1 (Lhx1) marks the most undifferentiated SSCs, which has not yet been reported for spermatogonia-like cells generated in vitro. Previously, it was shown that a chemical intervention in male mouse embryonic stem (ES) cells in serum culture, including Sirtuin 1 (SIRT1) inhibitor Ex-527, DNA methyltransferase (DNMT) inhibitor RG-108 and electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with molecular markers of PGC to gonocyte differentiation. Here, we report the in vitro differentiation of male mouse ES cells, cultured under dual chemical inhibition of GSK3β and MEK (2i) with leukemia inhibitory factor (LIF) (2iL) and serum, into cells with spermatogonia-like morphology (CSMs) and population-averaged expression of spermatogonia-specific genes by removal of 2iL and a specific schedule of twice daily partial medium replacement. Combination of this new protocol with the previously reported chemical intervention increased population-averaged gene expression of Lhx1 in the resulting CSMs. Furthermore, we detected single CSMs with strong nuclear LHX1/5 protein signal only in the chemical intervention group. We propose that further investigation of CSMs may provide new insights into male germline development., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Victim-offender contact has been studied extensively in prisons, but research on contact between victims and mentally disordered offenders in forensic mental health settings is lacking. Therefore, an exploratory study was conducted on contact between victims and offenders in four Dutch forensic psychiatric hospitals. These offenders have committed serious (sexually) violent offenses, for which they could not be held fully responsible due to severe psychopathology. During the mandatory treatment, it is possible for offenders and their victims to engage in contact with each other if both parties agree to this. To explore the conditions under which this contact is suitable, we interviewed 35 social workers about their experiences in 57 cases from four Dutch forensic psychiatric hospitals. Findings demonstrated that, according to the social workers, no type of offense or psychopathology were obvious exclusion criteria for victim-offender contact. Social workers described offenders' problem awareness, stable psychiatric condition, and ability to keep to agreements as important factors that enable victim-offender contact. Implications and suggestions for future research are provided., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Previous research has shown that female persistent offenders have multiple psychiatric and psychosocial problems, such as substance use disorders, other mental disorders, financial problems and housing problems. The present study examined recidivism and predictors of recidivism in a sample of 74 Dutch female high level persistent offenders who had been subjected to a special court order for persistent offenders, called ISD [Inrichting Stelselmatige Daders]. The criminal records were studied to gain insight in the criminal charges against women after release from the ISD. Results showed that 43% of the persistent female offenders had registered justice contacts within one year after release, of which the majority concerned non-violent property offences. However, the number of offences was found to be significantly reduced after their release. No offence-related, demographic, substance-related, psychiatric or personal history characteristics were found to be predictive for general recidivism., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study examined if a macro-, meso-, and micro outcome measurement instrument that constitutes the evaluation stage of a Dutch forensic psychiatric outcome monitor, the Hoeven Outcome Monitor (HOM), can provide a first step towards a more evidence based groundwork in forensic mental health. General, serious, very serious, special, and tbs meriting recidivism during treatment, after treatment, and overall were charted for forensic psychiatric patients discharged from a Dutch forensic psychiatric centre between 1999 and 2008 (N=164). Re-conviction data were obtained from the official Criminal Records System, and the mean follow-up time was 116.2months. First, the results showed that the macro-measurements provide comparative outcome measures to generate insight into the overall effectiveness of forensic psychiatric treatment. Second, the meso-measurements yielded clinically relevant treatment outcome data for all discharged patients to generate a complete view of treatment effectiveness. Finally, the micro-measurements allowed access to detailed patient and treatment effectiveness assessments that provides the empirical foundation to conduct aetiological research into the prediction and control of high-risk behaviour. Thus, an outcome measurement instrument in line with Evidence Based Medicine and best practice guidelines was designed that provides an empirically sound evaluation framework for treatment effectiveness, and an impetus for the development of effective interventions to generate an evidence based groundwork in forensic mental health., (Copyright © 2017. Published by Elsevier Ltd.)

Despite of the progress made to engineer structured microtissues such as BioMEMS and 3D bioprinting, little control exists how microtissues transform as they mature, as the misbalance between cell-generated forces and the strength of cell-cell and cell-substrate contacts can result in unintended tissue deformations and ruptures. To develop a quantitative perspective on how cellular contractility, scaffold curvature and cell-substrate adhesion control such rupture processes, human aortic smooth muscle cells were grown on glass substrates with submillimeter semichannels. We quantified cell sheet detachment from 3D confocal image stacks as a function of channel curvature and cell sheet tension by adding different amounts of Blebbistatin and TGF-β to inhibit or enhance cell contractility, respectively. We found that both higher curvature and higher contractility increased the detachment probability. Variations of the adhesive strength of the protein coating on the substrate revealed that the rupture plane was localized along the substrate-extracellular matrix interface for non-covalently adsorbed adhesion proteins, while the collagen-integrin interface ruptured when collagen I was covalently crosslinked to the substrate. Finally, a simple mechanical model is introduced that quantitatively explains how the tuning of substrate curvature, cell sheet contractility and adhesive strength can be used as tunable parameters as summarized in a first semi-quantitative phase diagram. These parameters can thus be exploited to either inhibit or purposefully induce a collective detachment of sheet-like microtissues for the use in tissue engineering and regenerative therapies., Statement of Significance: Despite of the significant progress in 3D tissue fabrication technologies at the microscale, there is still no quantitative model that can predict if cells seeded on a 3D structure maintain the imposed geometry while they form a continuous microtissue. Especially, detachment or loss of shape control of growing tissue is a major concern when designing 3D-structured scaffolds. Utilizing semi-cylindrical channels and vascular smooth muscle cells, we characterized how geometrical and mechanical parameters such as curvature of the substrate, cellular contractility, or protein-substrate adhesion strength tune the catastrophic detachment of microtissue. Observed results were rationalized by a theoretical model. The phase diagram showing how unintended tissue detachment progresses would help in designing of mechanically-balanced 3D scaffolds in future tissue engineering applications., (Copyright © 2016. Published by Elsevier Ltd.)

Enzymatically-induced degradation of bovine serum albumin (BSA) by serine proteases (trypsin and α-chymotrypsin) in various concentrations was monitored by means of Fourier transform infrared (FT-IR) and ultraviolet circular dichroism (UV-CD) spectroscopy. In this study, the applicability of both spectroscopies to monitor the proteolysis process in real time has been proven, by tracking the spectral changes together with secondary structure analysis of BSA as proteolysis proceeds. On the basis of the FTIR spectra and the changes in the amide I band region, we suggest the progression of proteolysis process via conversion of α-helices (1654 cm(-1)) into unordered structures and an increase in the concentration of free carboxylates (absorption of 1593 and 1402 cm(-1)). For the first time, the correlation between the degree of hydrolysis and the concentration of carboxylic groups measured by FTIR spectroscopy was revealed as well. The far UV-CD spectra together with their secondary structure analysis suggest that the α-helical content decreases concomitant with an increase in the unordered structure. Both spectroscopic techniques also demonstrate that there are similar but less spectral changes of BSA for the trypsin attack than for α-chymotrypsin although the substrate/enzyme ratio is taken the same., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To comply with the need for a more evidence based risk assessment and management in forensic mental health, an outcome monitor is being developed in the Dutch forensic psychiatric centre Van der Hoeven Kliniek in Utrecht, the Hoeven Outcome Monitor (HOM). Conform evidence based medicine (EBM) guidelines, the HOM is subdivided into three consecutive stages, (1) the evaluation stage, (2) the aetiology stage and (3) the implementation stage. In this article an account is provided for the design of the evaluation stage. To account for predicaments in previous research that pertain to a lack of uniformity and disregard of specific context- and patient-related characteristics, a macro-, meso- and micro-treatment evaluation instrument is developed. This instrument provides for the first step to build an evidence base for specific interventions and treatments in forensic psychiatry., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Early wound healing is associated with fibroblasts assembling a provisional fibronectin-rich extracellular matrix (ECM), which is subsequently remodeled and interlaced by type I collagen. This exposes fibroblasts to time-variant sets of matrices during different stages of wound healing. Our goal was thus to gain insight into the ECM-driven functional regulation of human foreskin fibroblasts (HFFs) being either anchored to a fibronectin (Fn) or to a collagen-decorated matrix, in the absence or presence of cyclic mechanical strain. While the cells reoriented in response to the onset of uniaxial cyclic strain, cells assembled exogenously added Fn with a preferential Fn-fiber alignment along their new orientation. Exposure of HFFs to exogenous Fn resulted in an increase in matrix metalloproteinase (MMP) expression levels, i.e. MMP-15 (RT-qPCR), and MMP-9 activity (zymography), while subsequent exposure to collagen slightly reduced MMP-15 expression and MMP-9 activity compared to Fn-exposure alone. Cyclic strain upregulated Fn fibrillogenesis and actin stress fiber formation, but had comparatively little effect on MMP activity. We thus propose that the appearance of collagen might start to steer HFFs towards homeostasis, as it decreased both MMP secretion and the tension of Fn matrix fibrils as assessed by Fluorescence Resonance Energy Transfer. These results suggest that HFFs might have a high ECM remodeling or repair capacity in contact with Fn alone (early event), which is reduced in the presence of Col1 (later event), thereby down-tuning HFF activity, a processes which would be required in a tissue repair process to finally reach tissue homeostasis., (Copyright © 2014. Published by Elsevier B.V.)

Objectives: To evaluate CD24/CD44/CD47 cancer stem cell marker expressions in bladder cancer (BCa) and provide data on their prognostic significance for clinical outcome in patients undergoing radical cystectomy (RC)., Material and Methods: Primary BCa tissue was used for xenograft studies. A tissue microarray was prepared using specimens from a cohort of 132 patients. All patients underwent RC for urothelial BCa between 2001 and 2010. Expression of CD24, CD44, and CD47 was examined in primary samples and xenografts by fluorescence-activated cell sorting. Populations of CD24(low)- and CD24(high)-expressing cells were sorted and evaluated for tumorigenicity in vivo. Tissue microarray was analyzed for CD24/CD44 staining intensity and tumor-specific vs. stromal cell staining. Associations with BCa survival, BCa stage, and lymph node status were evaluated by univariate and multivariate analyses., Results: CD24 and CD44/CD47 expressions mark distinct cell populations within the normal urothelium as well as in BCa. CD24(high/low) expression was not sufficient to characterize CD24 as a BCa-initiating marker in in vivo primary xenotransplants. CD24 and CD44 expressions correlated with lower cancer-specific survival in patients. However, multivariate analyses of CD24 or CD44 did not demonstrate significantly increased hazards for cancer-specific death if analyzed together with stage, grade, and nodal status of patients., Conclusions: Cancer stem cell markers CD24/CD44/CD47 are differentially expressed in cells of urothelial BCa in patients undergoing RC and influence cancer-specific survival of patients. Further evaluation of CD24/CD44/CD47 protein expression could be of high therapeutic value in BCa. However, both CD24 and CD44 expressions cannot be regarded as independent prognostic parameters for patients undergoing RC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Tamoxifen and raloxifene reduce the risk of breast cancer in women at elevated risk of disease, but the duration of the effect is unknown. We assessed the effectiveness of selective oestrogen receptor modulators (SERMs) on breast cancer incidence., Methods: We did a meta-analysis with individual participant data from nine prevention trials comparing four selective oestrogen receptor modulators (SERMs; tamoxifen, raloxifene, arzoxifene, and lasofoxifene) with placebo, or in one study with tamoxifen. Our primary endpoint was incidence of all breast cancer (including ductal carcinoma in situ) during a 10 year follow-up period. Analysis was by intention to treat., Results: We analysed data for 83,399 women with 306,617 women-years of follow-up. Median follow-up was 65 months (IQR 54-93). Overall, we noted a 38% reduction (hazard ratio [HR] 0·62, 95% CI 0·56-0·69) in breast cancer incidence, and 42 women would need to be treated to prevent one breast cancer event in the first 10 years of follow-up. The reduction was larger in the first 5 years of follow-up than in years 5-10 (42%, HR 0·58, 0·51-0·66; p<0·0001 vs 25%, 0·75, 0·61-0·93; p=0·007), but we noted no heterogeneity between time periods. Thromboembolic events were significantly increased with all SERMs (odds ratio 1·73, 95% CI 1·47-2·05; p<0·0001). We recorded a significant reduction of 34% in vertebral fractures (0·66, 0·59-0·73), but only a small effect for non-vertebral fractures (0·93, 0·87-0·99)., Interpretation: For all SERMs, incidence of invasive oestrogen (ER)-positive breast cancer was reduced both during treatment and for at least 5 years after completion. Similar to other preventive interventions, careful consideration of risks and benefits is needed to identify women who are most likely to benefit from these drugs., Funding: Cancer Research UK., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Electrospun grafts have been widely investigated for vascular graft replacement due to their ease and compatibility with many natural and synthetic polymers. Here, the effect of the processing parameters on the scaffold's architecture and subsequent reactions of partially heparinized blood triggered by contacting these topographies were studied. Degrapol® (DP) and poly(lactic-co-glycolic acid) (PLGA) electrospun fibrous scaffolds were characterized with regard to fiber diameter, pore area and scaffold roughness. The study showed that electrospinning parameters greatly affect fiber diameter together with pore dimension and overall scaffold roughness. Coagulation cascade activation, early platelet adhesion and activation were analyzed after 2h of exposure of blood to the biomaterials. While no differences were found between DP and PLGA with similar topographies, the blood reactions were observed to be dependent on the fiber diameter and scaffold roughness. Scaffolds composed of thin fibers (diameter <1μm) triggered very low coagulation and almost no platelets adhered. On the other hand, scaffolds with a bigger fiber diameter (2-3μm) triggered higher thrombin formation and more platelets adhered. The highest platelet adhesion and activations rates as well as coagulation cascade activation were found in blood incubated in contact with the scaffolds produced with the biggest fiber diameter (5μm). These findings indicate that electrospun grafts with small fiber diameter (<1μm) could perform better with reduced early thrombogenicity due to lower platelet adhesion and lower activation of platelets and coagulation cascade., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Fourier transform infrared (FT-IR)- and UV-circular dichroism (UV-CD) spectroscopy have been used to study real-time proteolytic digestion of β-lactoglobulin (β-LG) and β-casein (β-CN) by trypsin at various substrate/enzyme ratios in D(2)O-buffer at 37°C. Both techniques confirm that protein substrate looses its secondary structure upon conversion to the peptide fragments. This perturbation alters the backbone of the protein chain resulting in conformational changes and degrading of the intact protein. Precisely, the most significant spectral changes which arise from digestion take place in the amide I and amide II regions. The FT-IR spectra for the degraded β-LG show a decrease around 1634 cm(-1), suggesting a decrease of β-sheet structure in the course of hydrolysis. Similarly, the intensity around the 1654 cm(-1) band decreases for β-CN digested by trypsin, indicating a reduction in the α-helical part. On the other hand, the intensity around ∼1594 cm(-1) and ∼1406 cm(-1) increases upon enzymatic breakdown of both substrates, suggesting an increase in the antisymmetric and symmetric stretching modes of free carboxylates, respectively, as released digestion products. Observation of further H/D exchange in the course of digestion manifests the structural opening of the buried groups and accessibility to the core of the substrate. On the basis of the UV-CD spectra recorded for β-LG and β-CN digested by trypsin, the unordered structure increases concomitant with a decrease in the remaining structure, thus, revealing breakdown of the intact protein into smaller fragments. This model study in a closed reaction system may serve as a basis for the much more complex digestion processes in an open reaction system such as the stomach., (Copyright © 2011 Elsevier B.V. All rights reserved.)

RUNX1/ETO, the fusion protein resulting from the chromosomal translocation t(8;21), is one of the most frequent translocation products in acute myeloid leukemia. Several in vitro and in vivo studies have shown that the homo-tetramerization domain of ETO, the nervy homology region 2 (NHR2), is essential for RUNX1/ETO oncogenic activity. We analyzed the energetic contribution of individual amino acids within the NHR2 to RUNX1/ETO dimer-tetramer transition and found a clustered area of 5 distinct amino acids with strong contribution to the stability of tetramers. Substitution of these amino acids abolishes tetramer formation without affecting dimer formation. Similar to RUNX1/ETO monomers, dimers failed to bind efficiently to DNA and to alter expression of RUNX1-dependent genes. RUNX1/ETO dimers do not block myeloid differentiation, are unable to enhance the self-renewal capacity of hematopoietic progenitors, and fail to induce leukemia in a murine transplantation model. Our data reveal the existence of an essential structural motif (hot spot) at the NHR2 dimer-tetramer interface, suitable for a molecular intervention in t(8;21) leukemias.

The growth of natural biominerals is often tightly regulated by surface adsorption and subsequent incorporation of proteins into the crystal structure. Understanding how macromolecules intercalate into inorganic crystal lattices and how incorporation affects protein structure is crucial to learning how to engineer biomimetic materials with advanced properties, yet knowledge about the molecular-level interactions between organic guests and inorganic hosts remains sparse. Here we have used fluorescence resonance energy transfer (FRET) to probe conformational changes of a macromolecule as it adsorbs to, and becomes incorporated within, a biomineral crystal. Calcium oxalate monohydrate (COM) was used as a model due to its large size and kinetic stability under a wide range of pH values. Since the conformation of the extracellular matrix protein fibronectin (Fn) is highly sensitive to local ion concentrations, major conformational changes can be observed by FRET, as Fn senses and responds to varying local ionic conditions. When transferred from a physiological buffer to a supersaturated solution, Fn's crossed-over dimeric arms separate, indicating a weakening of the electrostatic interactions which otherwise stabilize the compact conformation of the protein. Fn returns to a more compact state when binding to the flat (-101) surface of the crystal, suggesting that Fn might sense a zone of ion depletion right at the interface of the growing crystal. As the crystal begins to grow around the absorbed protein, the dimeric Fn arms separate again, potentially driven by interactions with the newly formed charged step edges forming around it during the embedding process. FRET thus reveals for the first time how local changes in the electrostatic environment during the growth of a biomineral can cause major alterations in protein conformation. The insights derived using FRET and atomic force microscopy (AFM) could stimulate novel ways to tailor and tune the properties of organic-inorganic composites by exploiting dynamically changing electrostatic guest-host interactions.

Many aspects of cellular motility and mechanics are cyclic in nature such as the extension and retraction of lamellipodia or filopodia. Inherent to the cycles of extension and retraction that test the environment is the production of mechano-chemical signals that can alter long-term cell behavior, transcription patterns, and cell fate. We are just starting to define such cycles in several aspects of cell motility, including periodic contractions, integrin cycles of binding and release as well as the normal oscillations in motile activity. Cycles of local cell contraction and release are directly coupled to cycles of stressing and releasing extracellular contacts (matrix or cells) as well as cytoplasmic mechanotransducers. Stretching can alter external physical properties or sites exposed by matrix molecules as well as internal networks; thus, cell contractions can cause a secondary wave of mechano-regulated outside-in and internal cell signal changes. In some cases, the integration of both external and internal signals in space and time can stimulate a change in cell state from quiescence to growth or differentiation. In this review we will develop the basic concept of the mechano-chemical cycles and the ways in which they can be described and understood.

Gaboxadol, a selective extra synaptic GABA(A) receptor agonist, has been in clinical development for the treatment of insomnia. Development of tolerance to therapeutic effects (e.g. hypnotic and anticonvulsant and sedative) and withdrawal symptoms (e.g. REM sleep rebound and reduced seizure threshold) upon treatment discontinuation is reported for GABA(A) receptor allosteric modulators acting via the benzodiazepine binding site, e.g. zolpidem and indiplon. We conducted a head to head comparison in rats of the hypnotic (sleep EEG after 21 daily doses and 24 and 48 h after the last dose) and seizure threshold modifying (bicuculline assay 24 h after 28 daily doses) effects of gaboxadol and benzodiazepine ligands. Furthermore, we investigated in further details a previously reported apparent rapid development of tolerance to gaboxadol's effects in a rat rotarod motor coordination assay and related this effect to CNS exposure levels and in vitro potency at extra synaptic GABA(A) receptors. Sleep EEG studies demonstrated lack of tolerance and withdrawal effects after 28 daily doses with gaboxadol, whereas zolpidem produced both tolerance and withdrawal effects under a similar dosing regimen. Daily dosing with gaboxadol, zolpidem or indiplon for 28 days and acute discontinuation of treatment left the threshold to bicuculline-induced seizures unchanged. The rapidly attenuated effect of repeated gaboxadol dosing was confirmed in the rotarod model. However, re-challenge of gaboxadol insensitive animals with gaboxadol produced a maximum response, ruling out that receptor desensitisation accounts for these effects. By comparing CNS exposure at rotarod responses and concentration response relation at cloned GABA(A) receptors expressed in Xenopus oocytes it appears that the decline in response in the rotarod model coincides with the steep part of the concentration response curve for gaboxadol at extra synaptic GABA(A) receptors. In conclusion, rat sleep EEG repeated dose studies of gaboxadol confirm a hypnotic-like profile and no withdrawal effects, whereas tolerance and withdrawal effects were shown with zolpidem. Withdrawal from gaboxadol, zolpidem and indiplon did not affect the seizure threshold to bicuculline. Gaboxadol's apparent rapid development of tolerance in the rotarod assay appears to be kinetically determined.

In response to growing needs for quantitative biochemical and cellular assays that address whether the extracellular matrix (ECM) acts as a mechanochemical signal converter to co-regulate cellular mechanotransduction processes, a new assay is presented where plasma fibronectin fibers are manually deposited onto elastic sheets, while force-induced changes in protein conformation are monitored by fluorescence resonance energy transfer (FRET). Fully relaxed assay fibers can be stretched at least 5-6 fold, which involves Fn domain unfolding, before the fibers break. In native fibroblast ECM, this full range of stretch-regulated conformations coexists in every field of view confirming that the assay fibers are physiologically relevant model systems. Since alterations of protein function will directly correlate with their extension in response to force, the FRET vs. strain curves presented herein enable the mapping of fibronectin strain distributions in 2D and 3D cell cultures with high spatial resolution. Finally, cryptic sites for fibronectin's N-terminal 70-kD fragment were found to be exposed at relatively low strain, demonstrating the assay's potential to analyze stretch-regulated protein-protein interactions.

Experimental studies have indicated that FN-III modules undergo reversible unfolding as a mechanism of elasticity. The unfolding of FN-III modules, including the cell-binding FN-III(10) module, has further been suggested to be functionally relevant by exposing buried cryptic sites or modulating cell binding. While steered molecular dynamics (SMD) simulations have provided one tool to investigate this process, computational requirements so far have limited detailed analysis to the early stages of unfolding. Here, we use an extended periodic box to probe the unfolding of FN-III(10) for extensions longer than 60A. Up to three plateaus, corresponding to three metastable intermediates, were observed in the extension-time profile from SMD stretching of FN-III(10). The first and second plateaus correspond to a twisted and an aligned state prior to unraveling FN-III(10) beta-strands. The third plateau, at an extension of approximately 100A, follows unraveling of FN-III(10) A and B-strands and precedes breaking of inter-strand hydrogen bonds between F and G-strands. The simulations revealed three forced unfolding pathways of FN-III(10), one of which is preferentially selected under physiological conditions. Implications for fibronectin fibrillogenesis are discussed.

The synergy site on fibronectin's FN-III(9) module, located approximately 32 A away from the RGD-loop on FN-III(10), greatly enhances integrin alpha(5)beta(1) mediated cell binding. Since fibronectin is exposed to mechanical forces acting on the extracellular matrix in vivo, we used steered molecular dynamics to study how mechanical stretching of FN-III(9-10) affects the relative distance between these two synergistic sites. Our simulations predict the existence of an intermediate state prior to unfolding. In this state, the synergy-RGD distance is increased from 32 A to approximately 55 A, while the conformations of both sites remain unperturbed. This distance is too large for both sites to co-bind the same receptor, as indicated by experiments that confirm that increasing the length of the linker chain between FN-III(9) and FN-III(10) reduces alpha(5)beta(1) binding. Our simulations thus suggest that increased alpha(5)beta(1)-binding attributed to the synergy site, along with the associated downstream cell-signaling events, can be turned off mechanically by stretching FN-III(9-10) into this intermediate state. The potential physiological implications are discussed.

At the time of the release of the findings of the Breast Cancer Prevention Trial (BCPT), early interim results from two smaller European studies were also released. These smaller studies included one from the Royal Marsden (RM) Hospital in London, England, and another from the Italian National Cancer Institute. Since then, there has been much discussion about the relevance of the interim findings from the European studies and the likely reason for the failure of these studies to find a treatment effect. In some instances, the discussion has been incomplete or inconsistent with the observations from the trial. This has caused some confusion regarding the likely differences among the three studies of breast cancer prevention with tamoxifen. Recently, investigators from the RM study have published their interpretation of the reasons for the negative findings from the European studies. The discussions of the RM investigators are reviewed and used as a basis to illustrate some misconceptions regarding key differences in trial design and implementation among the BCPT and the European trials. The investigator discussions are also used to illustrate the significance of performing an appropriate benefit/risk assessment to identify women who would likely have a net beneficial effect when using tamoxifen to reduce the risk of breast cancer occurrence. Differences in terms of the characteristics of the study populations resulting in inadequate statistical power is the most likely reason for the failure to detect treatment differences in the European trials. Possible confounding due to the use of hormone replacement therapy is another reason that must be considered. Also, benefit/risk analysis indicates that tamoxifen has substantial public health potential as an approach to reduce breast cancer incidence and the physical and mental morbidity associated with this disease. The drug cannot be used indiscriminately due to the potential side effects, but benefit/risk assessment methodology can be used to identify substantial numbers of women in whom treatment would provide a net beneficial effect.

The streptavidin two-dimensional (2D) crystallization model has served as a paradigm for molecular self-assembly at interfaces. We have developed quantitative Brewster angle microscopy for the in situ measurement of spatially resolved relative protein surface densities. This allows investigation of both the thermodynamics and morphologies of 2D crystal growth. For crystal structure analysis, we employ TEM on grown crystals transferred to solid substrates. Comparison of results between commercially available streptavidin, recombinant streptavidin, and site-directed streptavidin mutants has provided insight into the protein protein and protein-lipid interactions that underlie 2D crystallization.

We compared health behavior and attitudes of older and younger women toward breast cancer screening by using data from the 1987 Texas Breast Screening Project, a community-based, low-cost screening program sponsored by the American Cancer Society, Texas Division. Because age is an important risk factor for breast cancer, the women were categorized into three age groups: 55 to 64 years old, 65 to 74 years old, and 75 years and older. Approximately 67% of the women 75 years and older, 64% of women in the 65 to 74 age group, and 59% of the younger (55 to 64) women had never had mammography. Moreover, only 14% of the women in both the 65 to 74 and 75+ age groups and 19% of the younger women reported having two or more mammographic examinations. Fewer older women (65 to 74 and 75+ age groups) reported having recent clinical breast examination. There were no differences among the age groups in the factors that attracted the women to participate in this screening program; in the three age groups, the most influential factors to participate were media publicity and lower mammography costs. Women in all age groups reported a lack of physician referral and cost as reasons for not previously participating in mammography screening. This study shows that older women are underscreened. Educational programs about the benefits of early detection should target older women because they are at increased risk for breast cancer.

Public health educational campaigns can attract large numbers of one-time participants, but the impact on subsequent behavior remains unstudied. The American Cancer Society Texas Division, Inc. sponsored a statewide $50.00 mammography screening project in early 1987. More than 64,000 mammograms were completed at 306 centers; 37,000 screenees answered a 31-item questionnaire. Attitudes toward screening were assessed, and screening history was recorded. Eighteen months after the project, a follow-up questionnaire was sent to 1000 screenees; 411 women returned the questionnaires. In the year following the project, 51% of the women 50 years and older reported having a subsequent mammogram. Among the women in this group who had never had a mammogram prior to 1987, 42% had screening mammography repeated in the following year. These data show that media-based public education projects can be effective mechanisms for improving and maintaining compliance with mammography screening recommendations.

Tongue thrust or reverse swallowing refers to the condition in which portions of the tongue rest or press against the teeth. In very severe cases the result can be dental malocclusion and/or speech difficulties. The present investigation evaluates the efficacy of cueing and positive practice in increasing the frequency of correct tongue position using a modified single subject withdrawal design with a 21 year old college student. The data indicate that the frequency of correct tongue positioning increased with the introduction of treatment. Follow-up data obtained 30 months post-treatment indicate that positive effects were being maintained.