The response of osteoblasts to nanocrystalline silicon-substituted hydroxyapatite thin films

Magnetron co-sputtering has been employed to fabricate thin nanocrystalline coatings of silicon-substituted hydroxyapatite (SiHA) of different Si compositions: 0.8 wt%, 2.2 wt%, and 4.9 wt%. A human osteoblast-like (HOB) cell model was used to study the long-term interaction between the HOB cells and coatings in vitro. Results showed that the number of cells growing on all coated titanium (Ti) samples were statistically significantly higher than on uncoated Ti. In addition, HOB cells growing on all SiHA surfaces displayed enhanced cell spreading, with extensive extracellular matrix synthesis. DNA staining revealed normal phenotype nuclear morphology for HOB cells, with several dense chromosomes surrounded by a periphery of intact nuclear membrane. Furthermore, immunofluorescent staining indicated that cells showed improved adhesion on the coated surfaces with increasing Si content, developing mature cytoskeletons with numerous distinct and well-defined actin stress fibres in the cell membranes. Results also demonstrated that the bone mineralisation process was greatest in the presence of the highest Si level (4.9 wt%). However, at very early culturing time point, cells did not attach so readily on the surface of this coating due to rapid dissolution. Thus, this work suggests that a Si content of 2.2 wt% may be the optimum loading to improve the bioactive property of HA thin films.     

Value of a molecular screening program to support clinical trial enrollment in Asian cancer patients: The Integrated Molecular Analysis of Cancer (IMAC) Study

The value of precision oncology initiatives in Asian contexts remains unresolved. Here, we review the institutional implementation of prospective molecular screening to facilitate accrual of patients into biomarker‐driven clinical trials, and to explore the mutational landscape of advanced tumors occurring in a prospective cohort of Asian patients (n = 396) with diverse cancer types. Next‐generation sequencing (NGS) and routine clinicopathological assays, such as immunohistochemistry, copy number analysis and in situ hybridization tests, were performed on tumor samples. Actionable biomarker results were used to identify eligibility for early‐phase, biomarker‐driven clinical trials. Overall, NGS was successful in 365 of 396 patients (92%), achieving a mean depth of 1,943× and coverage uniformity of 96%. The median turnaround time from sample receipt to return of genomic results was 26.0 days (IQR, 19.0–39.0 days). Reportable mutations were found in 300 of 365 patients (82%). Ninety‐one percent of patients at study enrollment indicated consent to receive incidental findings and willingness to undergo genetic counseling if required. The most commonly mutated oncogenes included KRAS (19%), PIK3CA (16%), EGFR (5%), BRAF (3%) and KIT (3%); while the most frequently mutated tumor suppressor genes included TP53 (40%), SMARCB1 (12%), APC (8%), PTEN (6%) and SMAD4 (5%). Among 23 patients enrolled in genotype‐matched trials, median progression‐free survival was 2.9 months (IQR, 1.5–4.0 months). Nine of 20 evaluable patients (45%; 95% CI, 23.1–68.5%) derived clinical benefit, including 3 partial responses and 6 with stable disease lasting ≥ 8 weeks.     

Growth monitoring: testing the new guidelines

OBJECTIVE: To assess the impact of recent guidelines from the UK joint working party of child health surveillance recommending that all children be measured at age 5 and again between 7 and 9 years of age to determine how many normal school age children are likely to be referred for specialist assessment. METHODS: The longitudinal data of 486 children measured by school nurses in a community setting were examined and compared with measurements made in a research setting by a single, skilled observer. MAIN OUTCOME MEASURES: Number of children identified as having abnormal stature (< 0.4th or > 99.6th centile) and abnormal growth rate height standard deviation score (HSDS) change > 0.67). RESULTS: The community survey identified seven (1.4%) children as having abnormal stature (four short, three tall), 11 (2.3%) were identified as "slow growing", and nine (1.9%) increased their HSDS by more than 0.67. These results were comparable to data collected in ideal research conditions. CONCLUSIONS: Following the recommendations would not result in an excess number of inappropriate referrals. However, this study highlights several unresolved issues such as interobserver variability and time interval between measurements. A large scale prospective study should be considered to establish realistic and cost-effective criteria before implementation of a national screening programme.     

Vulvovaginal candidosis: contemporary challenges and the future of prophylactic and therapeutic approaches

Vulvovaginal candidosis (VVC) is a common gynaecological disorder that is delineated by the inflammation of vaginal wall and it is caused by the opportunistic fungal pathogen Candida species. In fact, three out of every four women will experience at least one occasion of VVC during some point in their lives. Although uncomplicated VVC is relatively harmless, the complicated VVC such as recurrent attack often creates restlessness and depression in the patients, thus greatly affects their quality of life. Managements of VVC are usually associated with the use of antimycotic suppositories, topical cream or oral agents. These antimycotic agents are either available over‐the‐counter or prescribed by the clinicians. In recent decades, the rise of clinical challenges such as the increased prevalence of resistant Candida strains, recurrent VVC infection and adverse effects of multidrug interactions have necessitated the development of novel therapeutic or prophylactic options to combat the complicated VVC in the future. In this review, we discuss the current antimycotic treatments available for Candida vaginitis and the problems that exist in these seemingly effective treatments. Besides, we attempt to contemplate some of the future and prospective strategies surrounding the development of alternative therapeutic and prophylactic options in treating and preventing complicated VVC respectively.     

Functional MRI registration with tissue‐specific patch‐based functional correlation tensors

Population studies of brain function with resting‐state functional magnetic resonance imaging (rs‐fMRI) rely on accurate intersubject registration of functional areas. This is typically achieved through registration using high‐resolution structural images with more spatial details and better tissue contrast. However, accumulating evidence has suggested that such strategy cannot align functional regions well because functional areas are not necessarily consistent with anatomical structures. To alleviate this problem, a number of registration algorithms based directly on rs‐fMRI data have been developed, most of which utilize functional connectivity (FC) features for registration. However, most of these methods usually extract functional features only from the thin and highly curved cortical grey matter (GM), posing great challenges to accurate estimation of whole‐brain deformation fields. In this article, we demonstrate that additional useful functional features can also be extracted from the whole brain, not restricted to the GM, particularly the white‐matter (WM), for improving the overall functional registration. Specifically, we quantify local anisotropic correlation patterns of the blood oxygenation level‐dependent (BOLD) signals using tissue‐specific patch‐based functional correlation tensors (ts‐PFCTs) in both GM and WM. Functional registration is then performed by integrating the features from different tissues using the multi‐channel large deformation diffeomorphic metric mapping (mLDDMM) algorithm. Experimental results show that our method achieves superior functional registration performance, compared with conventional registration methods.     

Modulation of gastrointestinal function by MuDelta,a mixed μ opioid receptor agonist/ μ opioid receptor antagonist

BACKGROUND & PURPOSE

Loperamide is a selective µ opioid receptor agonist acting locally in the gastrointestinal (GI) tract as an effective anti-diarrhoeal but can cause constipation. We tested whether modulating µ opioid receptor agonism with δ opioid receptor antagonism, by combining reference compounds or using a novel compound (‘MuDelta’), could normalize GI motility without constipation.

EXPERIMENTAL APPROACH

MuDelta was characterized in vitro as a potent µ opioid receptor agonist and high-affinity δ opioid receptor antagonist. Reference compounds, MuDelta and loperamide were assessed in the following ex vivo and in vivo experiments: guinea pig intestinal smooth muscle contractility, mouse intestinal epithelial ion transport and upper GI tract transit, entire GI transit or faecal output in novel environment stressed mice, or four weeks after intracolonic mustard oil (post-inflammatory). Colonic δ opioid receptor immunoreactivity was quantified.

KEY RESULTS

δ Opioid receptor antagonism opposed µ opioid receptor agonist inhibition of intestinal contractility and motility. MuDelta reduced intestinal contractility and inhibited neurogenically-mediated secretion. Very low plasma levels of MuDelta were detected after oral administration. Stress up-regulated δ opioid receptor expression in colonic epithelial cells. In stressed mice, MuDelta normalized GI transit and faecal output to control levels over a wide dose range, whereas loperamide had a narrow dose range. MuDelta and loperamide reduced upper GI transit in the post-inflammatory model.

CONCLUSIONS AND IMPLICATIONS

MuDelta normalizes, but does not prevent, perturbed GI transit over a wide dose-range in mice. These data support the subsequent assessment of MuDelta in a clinical phase II trial in patients with diarrhoea-predominant irritable bowel syndrome.     

CENTS: Cortical enhanced neonatal tissue segmentation

The acquisition of high‐quality magnetic resonance (MR) images of neonatal brains is largely hampered by their characteristically small head size and insufficient tissue contrast. As a result, subsequent image processing and analysis, especially brain tissue segmentation, are often affected. To overcome this problem, a dedicated phased array neonatal head coil is utilized to improve MR image quality by augmenting signal‐to‐noise ratio and spatial resolution without lengthening data acquisition time. In addition, a specialized hybrid atlas‐based tissue segmentation algorithm is developed for the delineation of fine structures in the acquired neonatal brain MR images. The proposed tissue segmentation method first enhances the sheet‐like cortical gray matter (GM) structures in the to‐be‐segmented neonatal image with a Hessian filter for generation of a cortical GM confidence map. A neonatal population atlas is then generated by averaging the presegmented images of a population, weighted by their cortical GM similarity with respect to the to‐be‐segmented image. Finally, the neonatal population atlas is combined with the GM confidence map, and the resulting enhanced tissue probability maps for each tissue form a hybrid atlas is used for atlas‐based segmentation. Various experiments are conducted to compare the segmentations of the proposed method with manual segmentation (on both images acquired with a dedicated phased array coil and a conventional volume coil), as well as with the segmentations of two population‐atlas‐based methods. Results show the proposed method is capable of segmenting the neonatal brain with the best accuracy, and also preserving the most structural details in the cortical regions. Hum Brain Mapp, 2011. © 2010 Wiley‐Liss, Inc.     

Macrophage Matrix Metalloproteinase-9 Mediates Epithelial-Mesenchymal Transition in Vitro in Murine Renal Tubular Cells

As a rich source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages are well-placed to play an important role in renal fibrosis. However, the exact underlying mechanisms and the extent of macrophage involvement are unclear. Tubular cell epithelial−mesenchymal transition (EMT) is an important contributor to renal fibrosis and MMPs to induction of tubular cell EMT. The aim of this study was to investigate the contribution of macrophages and MMPs to induction of tubular cell EMT. The murine C1.1 tubular epithelial cell line and primary tubular epithelial cells were cultured in activated macrophage-conditioned medium (AMCM) derived from lipopolysaccharide-activated J774 macrophages. MMP-9, but not MMP-2 activity was detected in AMCM. AMCM-induced tubular cell EMT in C1.1 cells was inhibited by broad-spectrum MMP inhibitor (GM6001), MMP-2/9 inhibitor, and in AMCM after MMP-9 removal by monoclonal Ab against MMP-9. AMCM-induced EMT in primary tubular epithelial cells was inhibited by MMP-2/9 inhibitor. MMP-9 induced tubular cell EMT in both C1.1 cells and primary tubular epithelial cells. Furthermore, MMP-9 induced tubular cell EMT in C1.1 cells to an extent similar to transforming growth factor-β. Transforming growth factor-β-induced tubular cell EMT in C1.1 cells was inhibited by MMP-2/9 inhibitor. Our in vitro study provides evidence that MMPs, specifically MMP-9, secreted by effector macrophages can induce tubular cell EMT and thereby contribute to renal fibrosis.Interstitial macrophage infiltration is a hallmark of all progressive renal diseases regardless of the initial cause of the injury.^1,2 Macrophages have long been known to play an important role in renal fibrosis,³ which is a central component of the final common pathway leading to renal failure. Previous studies have demonstrated a close association between macrophage infiltrate and excessive extracellular matrix protein accumulation in diseased human kidney as well as in experimental models.^4–6 In addition, the number of infiltrating macrophages has been shown to correlate well with the number of myofibroblasts,^7,8 the effector cells responsible for secretion of extracellular matrix proteins. A recent study revealed that blockade of macrophage recruitment in obstructive renal injury resulted in a reduction in renal fibrosis via tubular cell epithelial−mesenchymal transition (EMT),⁹ which has been recognized as an important source of myofibroblasts in renal fibrosis. However, the exact mechanism underlying the contribution of macrophages to renal fibrosis via tubular cell EMT remains undefined. As a major source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages may be major determinants of the outcome of renal fibrosis.Tubular cell EMT is a process by which tubular epithelial cells lose their epithelial characteristics and acquire a mesenchymal phenotype. This process has been recognized as one of several pathways contributing to the myofibroblast population in renal fibrosis.¹⁰ Despite emerging and conflicting evidence about the relative importance of various sources of myofibroblasts,^11,12 it is generally accepted that tubular cell EMT plays an important role in renal fibrosis. Since the concept of tubular cell EMT was first proposed, numerous studies have provided evidence for tubular cell EMT in various experimental models as well as in human biopsies.¹⁰ Furthermore, the importance of tubular cell EMT has been demonstrated by Iwano et al¹³ using transgenic mice and direct genetic tagging of tubular epithelial cells to show that more than a third of myofibroblasts in kidneys with unilateral ureteral obstruction are derived from tubular epithelial cells via tubular cell EMT. Moreover, blockade of tubular EMT has been shown to attenuate renal fibrosis in obstructive nephropathy.¹⁴ However, some controversy remains as to whether tubular cell EMT plays a consistent role in other experimental models, and its exact contribution in renal fibrosis is yet to be established.Although pro-fibrogenic growth factors are well known as inducers of tubular cell EMT, cumulative evidence suggests an important role for MMPs. Traditionally, MMPs were thought to be antifibrogenic due to their ability to degrade extracellular matrix proteins, yet MMPs—in particular MMP-2 and MMP-9—have been recognized as promoters of tubular cell EMT via basement membrane disruption. In fact, induction of tubular cell EMT in vitro¹⁵ and in vivo¹⁴ has been shown to be associated with increased expression of MMP-2 and MMP-9. Earlier studies have demonstrated that tubular epithelial cells undergoing mesenchymal transition are closely associated with damaged tubular basement membrane and that complete transition requires tubular basement membrane damage.¹⁶ Later studies have shown directly that MMPs can disrupt basement membrane integrity; loss of MMP-9 expression lead to preservation of basement membrane integrity and inhibition of tubular cell EMT in obstructed kidney of tissue type plasminogen activator knockout mice.¹⁴ Despite this evidence supporting induction of tubular cell EMT by MMPs, the precise contribution of MMPs may have been underestimated. In cancer research, MMPs are well known to directly induce EMT in tumor cells of epithelial origin and to promote tumor progression via basement membrane disruption.¹⁷ MMP-2 has been shown consistently to be necessary and sufficient to induce tubular cell EMT in a rat tubular epithelial cell line (NRK52e).¹⁸ In addition, recent studies from our laboratory have demonstrated that MMP-3 and MMP-9 are also capable of inducing tubular cell EMT in NRK52e cells via the disruption of the cell adhesion molecule E-cadherin. Finally, the fact that transforming growth factor (TGF)-β-induced tubular cell EMT in NRK52e was inhibited by a broad spectrum MMP inhibitor suggests a primary role of MMP in TGF-β-induced tubular cell EMT.¹⁹ Together, these data suggest that MMPs from macrophages may play a major role in induction of tubular cell EMT. Therefore the aim of this study was to investigate the contribution of macrophages and their secreted MMPs to the induction of tubular cell EMT.     

Matrix metalloproteinases contribute to kidney fibrosis in chronic kidney diseases

Matrix metalloproteinases (MMPs) are members of the neutral proteinase family. They were previously thought to be anti-fibrotic because of their ability to degrade and remodel of extracellular matrix. However, recent studies have shown that MMPs are implicated in initiation and progression of kidney fibrosis through tubular cell epithelial–mesenchymal transition (EMT) as well as activation of resident fibroblasts, endothelial-mesenchymal transition (EndoMT) and pericyte-myofibroblast transdifferentiation. Interstitial macrophage infiltration has also been shown to correlate with the severity of kidney fibrosis in various chronic kidney diseases. MMPs secreted by macrophages, especially MMP-9, has been shown by us to be profibrotic by induction of tubular cells EMT. EMT is mainly induced by transforming growth factor-β (TGF-β). However, MMP-9 was found by us and others to be up-regulated by TGF-β1 in kidney tubular epithelial cells and secreted by activated macrophages, resulting in EMT and ultimately kidney fibrosis. Therefore, MMP-9 may serve as a potential therapeutic target to prevent kidney fibrosis in chronic kidney disease. This review, by a particular focus on EMT, seeks to provide a comprehensive understanding of MMPs, especially MMP-9, in kidney fibrosis.