Compact portable higher harmonic generation microscopy for the real time assessment of unprocessed thyroid tissue.

During thyroid surgery fast and reliable intra-operative pathological feedback has the potential to avoid a two-stage procedure and significantly reduce health care costs in patients undergoing a diagnostic hemithyroidectomy (HT). We explored higher harmonic generation (HHG) microscopy, which combines second harmonic generation (SHG), third harmonic generation (THG), and multiphoton excited autofluorescence (MPEF) for this purpose. With a compact, portable HHG microscope, images of freshly excised healthy tissue, benign nodules (follicular adenoma) and malignant tissue (papillary carcinoma, follicular carcinoma and spindle cell carcinoma) were recorded. The images were generated on unprocessed tissue within minutes and show relevant morphological thyroid structures in good accordance with the histology images. The thyroid follicle architecture, cells, cell nuclei (THG), collagen organization (SHG) and the distribution of thyroglobulin and/or thyroid hormones T3 or T4 (MPEF) could be visualized. We conclude that SHG/THG/MPEF imaging is a promising tool for clinical intraoperative assessment of thyroid tissue.

Blockade of fatty acid signalling inhibits lipopolysaccharide-induced macrophage recruitment and progression of apical periodontitis.

AIM: To examine the role of palmitic acid in lipopolysaccharide (LPS)-stimulated chemotaxis of macrophages and the potential contribution of saturated fatty acid in signalling during the pathogenesis of apical periodontitis. METHODOLOGY: J774, a mouse macrophage cell line, was used in the experiments. After treatment with LPS, proteolytic maturation of sterol regulatory element-binding protein-1c (SREBP-1c) and expression of fatty acid synthase (FASN) were examined by Western analysis. Levels of palmitic acid were measured by reverse phase-high performance liquid chromatography-mass spectrometry. Knockdown of SREBP-1c and FASN was accomplished by small interfering RNA technology. Secretion of CC-chemokine ligand 2 (CCL2) and cellular chemotaxis were assessed by enzyme-linked immunosorbent assay and transwell migration assay, respectively. Sulfo-N-succinimidyl oleate (SSO) treatment was used to inhibit fatty acid signalling in vitro and also in a rat model of apical periodontitis. All data were first subjected to Levene's test. In vitro data were then analysed using ANOVA followed by Tukey's multiple comparison test. Data from animal experiments were analysed by independent t-tests. The significant level was set at 0.05. RESULTS: LPS stimulated proteolytic maturation of SREBP-1c and FASN expression in macrophages and significantly enhanced palmitic acid synthesis (P < 0.05). Knockdown of SREBP-1c attenuated LPS-enhanced FASN expression. Knockdown of FASN significantly suppressed LPS-enhanced palmitic acid synthesis (P < 0.05). LPS and exogenous palmitic acid significantly enhanced CCL2 secretion and macrophage chemotaxis (all P < 0.05). Inhibition of FASN expression significantly alleviated LPS-augmented CCL2 secretion (P < 0.05). SSO significantly suppressed CCL2 secretion and macrophage chemotaxis augmented by LPS and palmitic acid (all P < 0.05). In a rat model of induced apical periodontitis, SSO treatment significantly attenuated progression of apical periodontitis and macrophage recruitment (all P < 0.05). CONCLUSIONS: LPS/SREBP-1c/FASN/palmitic acid signalling contributed to tissue destruction caused by bacterial infection. Modulation of lipid metabolism and signalling may be helpful for the management of apical periodontitis.

Characterization of facial asymmetry in skeletal Class III malocclusion and its implications for treatment.

Jaw deviation is frequently seen in Class III patients. The aim of the study was to investigate asymmetric features of skeletal, dental and soft tissues in three types of jaw asymmetry based on our previously reported classification system. The cone-beam computed tomography (CBCT) images of 70 Class III patients were analysed. Group 1 patients showed large shift of menton and synchronous but smaller ramus deviation. The maxillomandibular complex had roll and yaw rotations to the menton-deviation side. Maxillary and dental asymmetry was obvious in transverse and vertical dimensions. Cant of occlusal plane and lip line was apparent. Group 2 patients also exhibited menton and ramus deviation to the same side but the discrepancy in ramus width was larger than menton shift. Asymmetry in Group 2 resulted from a bodily side shift of the maxillomandibular complex without obvious rotation. Group 3 patients had menton and ramus deviated in opposite directions which seemed secondary to a yaw rotation. Double-jaw surgery is generally required for Groups 1 and 3 while Group 2 patients may be successfully treated by mandibular surgery only provided that arch width discrepancy can be managed by orthodontic measures.

Simvastatin alleviates bone resorption in apical periodontitis possibly by inhibition of mitophagy-related osteoblast apoptosis.

AIM: To assess the connection between mitophagy and hypoxia-induced apoptosis in osteoblasts and whether simvastatin alleviates bone resorption in apical periodontitis through modulation of mitophagy-related apoptosis. METHODOLOGY: Hypoxia-induced generation of reactive oxygen species in mitochondria and changes in mitochondrial membrane potential were evaluated, respectively, by MitoSOX and JC-1 fluorescence dye signalling. Accumulation of mitophagy markers PTEN-induced putative kinase 1 (PINK1) and Parkin in mitochondria was examined by Western blotting and immunofluorescence microscopy. Osteoblast apoptosis was assessed by Western analysis of cleaved-poly (adenosine diphosphate ribose) polymerase (PARP). In a rat model of induced apical periodontitis, the therapeutic effect of simvastatin and its action on osteoblast mitophagy and apoptosis were examined. anova, Fisher's and Student's t-test were used for data analysis. RESULTS: Hypoxia-induced mitochondrial dysfunction and stimulated mitophagy in osteoblasts. Hypoxia also provoked apoptosis in osteoblasts and inhibition of mitophagy decreased hypoxia-augmented apoptotic activity. Simvastatin alleviated hypoxia-induced mitochondrial dysfunction, mitophagy and apoptosis. The protective action of simvastatin against apoptosis was related to its antimitophagy activity. Experiments in the rat model of induced apical periodontitis supported the laboratory findings. Simvastatin treatment mitigated periapical bone loss and reduced the activities of apoptosis and mitophagy in regional osteoblasts. CONCLUSIONS: The results suggest that modulation of osteoblast mitophagy may help diminish bone loss associated with inflammation and has potential as an auxiliary therapy for apical periodontitis.

Sirtuin 6 attenuates periapical lesion propagation by modulating hypoxia-induced chemokine (C-C motif) ligand 2 production in osteoblasts.

AIM: To investigate the attenuating effect of sirtuin 6 (SIRT6) on hypoxia-induced production of chemokine (C-C motif) ligand 2 (CCL2) by osteoblasts and the relevance of this action on the pathogenesis of periapical lesions. METHODOLOGY: Sirtuin 6 was overexpressed in MC3T3-E1 murine osteoblasts by lentivirus-mediated gene transfer. The relationship between the antiglycolytic/antioxidative activities of SIRT6 and its effect on hypoxia-induced CCL2 production were examined. Pathogenetic relevance of the actions of SIRT6 was assessed in a rat model of induced apical periodontitis. The data were analysed statistically using Student's t-test or one-way analysis of variance (anova) and then a Tukey's multiple comparison test. RESULTS: In cultured murine osteoblasts, 24-h hypoxic treatment significantly enhanced the generation of reactive oxygen species (P = 0.003), expression of lactate dehydrogenase A (LDHA) and production of lactate (P = 0.007). A reciprocal effect between hypoxia-induced redox imbalance and hypoxia-enhanced glycolysis was noted which in turn augmented the secretion of CCL2. Through its antiglycolytic and antioxidative effects, SIRT6 blocked the vicious cycle to suppress CCL2 production. In normal periapical tissues of rats, strong expression of SIRT6 and low levels of LDHA and 8-OHdG (a marker of oxidative DNA damage) were found in osteoblasts. In induced apical periodontitis, osteoblastic expression of SIRT6 was significantly suppressed (P = 0.001) which was associated with significantly elevated levels of LDHA (P = 0.003) and 8-OHdG (P = 0.004) and significantly enhanced recruitment of macrophages (P = 0.004). CONCLUSIONS: Sirtuin 6 has a therapeutic effect on periapical lesions through suppression of CCL2 synthesis. The anti-inflammatory action of SIRT6 is closely related to its regulatory activities in cellular metabolism and redox homoeostasis.

Enhancement of Thiamine Biosynthesis in Oil Palm Seedlings by Colonization of Endophytic Fungus Hendersonia toruloidea.

Thiamine, or vitamin B1 plays an indispensable role as a cofactor in crucial metabolic reactions including glycolysis, pentose phosphate pathway and the tricarboxylic acid cycle in all living organisms. Thiamine has been shown to play a role in plant adaptation toward biotic and abiotic stresses. The modulation of thiamine biosynthetic genes in oil palm seedlings was evaluated in response to root colonization by endophytic Hendersonia toruloidea. Seven-month-old oil palm seedlings were inoculated with H. toruloidea and microscopic analyses were performed to visualize the localization of endophytic H. toruloidea in oil palm roots. Transmission electron microscopy confirmed that H. toruloidea colonized cortical cells. The expression of thiamine biosynthetic genes and accumulation of total thiamine in oil palm seedlings were also evaluated. Quantitative real-time PCR was performed to measure transcript abundances of four key thiamine biosynthesis genes (THI4, THIC, TH1, and TPK) on days 1, 7, 15, and 30 in response to H. toruloidea colonization. The results showed an increase of up to 12-fold in the expression of all gene transcripts on day 1 post-inoculation. On days 7, 15, and 30 post-inoculation, the relative expression levels of these genes were shown to be downregulated. Thiamine accumulation was observed on day 7 post-colonization and subsequently decreased until day 30. This work provides the first evidence for the enhancement of thiamine biosynthesis by endophytic colonization in oil palm seedlings.

Balloon dilation for eustachian tube dysfunction: systematic review.

BACKGROUND: Eustachian tube dysfunction is a disorder for which there are limited medical and surgical treatments. Recently, eustachian tube balloon dilation has been proposed as a potential solution. METHOD: A systematic literature review was performed. Abstracts were selected for relevance, and pooled data analysis and qualitative analysis was conducted. RESULTS: Nine prospective studies, describing 713 eustachian tube balloon dilations in 474 patients (aged 18-86 years), were identified. Follow-up duration ranged from 1.5 to 18 months. Ability to perform a Valsalva manoeuvre improved from 20 to 177 out of 245 ears following eustachian tube balloon dilation and, where data were reported in terms of patient numbers, from 15 to 189 out of 210 patients. Tympanograms were classified as type A in 7 out of 141 ears pre-operatively and in 86 out of 141 ears post-operatively. CONCLUSION: Prospective case series can confirm the safety of eustachian tube balloon dilation. As a potential solution for chronic eustachian tube dysfunction, further investigations are warranted to establish a higher level of evidence of efficacy.

Antiangiogenic activity of 2-formyl-8-hydroxy-quinolinium chloride.

Tumour growth is closely related to the development of new blood vessels to supply oxygen and nutrients to cancer cells. Without the neovascular formation, tumour volumes cannot increase and undergo metastasis. Antiangiogenesis is one of the most promising approaches for antitumour therapy. The exploration of new antiangiogenic agents would be helpful in antitumour therapy. Quinoline is an aromatic nitrogen compound characterized by a double-ring structure which exhibits a benzene ring fused to pyridine at two adjacent carbon atoms. The high stability of quinoline makes it preferable in a variety of therapeutic and pharmaceutical applications, including antitumour treatment. This work is to examine the potential antiangiogenic activity of the synthetic compound 2-Formyl-8-hydroxy-quinolinium chloride. We found that 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of human umbilical vein endothelial cells in vitro. Using the diethylnitrosamine-induced hepatocarcinogenesis model, 2-Formyl-8-hydroxy-quinolinium chloride showed strong antiangiogenic activity. Furthermore, 2-Formyl-8-hydroxy-quinolinium chloride could inhibit the growth of large Hep3B xenografted tumour from the nude mice. We assume that 2-Formyl-8-hydroxy-quinolinium chloride could be a potential antiangiogenic and antitumour agent and it is worthwhile to further study its underlying working mechanism.

Mechanisms Underlying the Anti-Inflammatory Effects of Clinacanthus nutans Lindau Extracts: Inhibition of Cytokine Production and Toll-Like Receptor-4 Activation.

Clinacanthus nutans has had a long history of use in folk medicine in Malaysia and Southeast Asia; mostly in the relief of inflammatory conditions. In this study, we investigated the effects of different extracts of C. nutans upon lipopolysaccharide (LPS) induced inflammation in order to identify its mechanism of action. Extracts of leaves and stem bark of C. nutans were prepared using polar and non-polar solvents to produce four extracts, namely polar leaf extract (LP), non-polar leaf extract (LN), polar stem extract (SP), and non-polar stem extracts (SN). The extracts were standardized by determining its total phenolic and total flavonoid contents. Its anti-inflammatory effects were assessed on LPS induced nitrite release in RAW264.7 macrophages and Toll-like receptor (TLR-4) activation in TLR-4 transfected human embryonic kidney cells (HEK-Blue(TM)-hTLR4 cells). The levels of inflammatory cytokines (TNF-α, IFN-γ, IL-1ß, IL-6, IL-12p40, and IL-17) in treated RAW264.7 macrophages were quantified to verify its anti-inflammatory effects. Western blotting was used to investigate the effect of the most potent extract (LP) on TLR-4 related inflammatory proteins (p65, p38, ERK, JNK, IRF3) in RAW264.7 macrophages. All four extracts produced a significant, concentration-dependent reduction in LPS-stimulated nitric oxide, LPS-induced TLR-4 activation in HEK-Blue(TM)-hTLR4 cells and LPS-stimulated cytokines production in RAW264.7 macrophages. The most potent extract, LP, also inhibited all LPS-induced TLR-4 inflammatory proteins. These results provide a basis for understanding the mechanisms underlying the previously demonstrated anti-inflammatory activity of C. nutans extracts.

Constant strain rate compression of bovine cortical bone on the Split-Hopkinson Pressure Bar.

Cortical bone is a visco-elastic material which implies that strain rate will affect its response. Although the Split-Hopkinson Pressure Bar is an accepted technique for determining the dynamic compressive properties of cortical bone it has been shown that the strain rate of compression does not remain constant throughout the duration of a classical experiment with a uniform striker. This raises concerns as to the measurement of smeared responses. This paper presents a shaped striker technique whereby the incident pulse can be shaped to attain a constant strain rate experiment for bovine bone. Shaped strikers offer benefits such as re-usability and increased test repeatability. A comparison of the stress-strain-strain rate responses attained through classical and constant strain rate experiments shows that the shape of the stress-strain curves from conventional experiments is adversely affected in the portion where the strain rate varies. The dynamic response corridors for the two tests are similar, however the ultimate properties are affected. It is concluded that the strain rate history should be presented with dynamic stress-strain responses since the instantaneous strain rate is a likely contributor to potential constitutive models.

Non-toxic agarose/gelatin-based microencapsulation system containing gallic acid for antifungal application.

Aspergillus niger (A. niger) is a common species of Aspergillus molds. Cutaneous aspergillosis usually occurs in skin sites near intravenous injection and approximately 6% of cutaneous aspergillosis cases which do not involve burn or HIV-infected patients are caused by A. niger. Biomaterials and biopharmaceuticals produced from microparticle-based drug delivery systems have received much attention as microencapsulated drugs offer an improvement in therapeutic efficacy due to better human absorption. The frequently used crosslinker, glutaraldehyde, in gelatin-based microencapsulation systems is considered harmful to human beings. In order to tackle the potential risks, agarose has become an alternative polymer to be used with gelatin as wall matrix materials of microcapsules. In the present study, we report the eco-friendly use of an agarose/gelatin-based microencapsulation system to enhance the antifungal activity of gallic acid and reduce its potential cytotoxic effects towards human skin keratinocytes. We used optimal parameter combinations, such as an agarose/gelatin ratio of 1:1, a polymer/oil ratio of 1:60, a surfactant volume of 1% w/w and a stirring speed of 900 rpm. The minimum inhibitory concentration of microencapsulated gallic acid (62.5 µg/ml) was significantly improved when compared with that of the original drug (>750 µg/ml). The anti-A. niger activity of gallic acid -containing microcapsules was much stronger than that of the original drug. Following 48 h of treatment, skin cell survival was approximately 90% with agarose/gelatin microcapsules containing gallic acid, whereas cell viability was only 25-35% with free gallic acid. Our results demonstrate that agarose/gelatin-based microcapsules containing gallic acid may prove to be helpful in the treatment of A. niger-induced skin infections near intravenous injection sites.

Calibrating corneal material model parameters using only inflation data: an ill-posed problem.

Goldmann applanation tonometry (GAT) is a method used to estimate the intraocular pressure by measuring the indentation resistance of the cornea. A popular approach to investigate the sensitivity of GAT results to material and geometry variations is to perform numerical modelling using the finite element method, for which a calibrated material model is required. These material models are typically calibrated using experimental inflation data by solving an inverse problem. In the inverse problem, the underlying material constitutive behaviour is inferred from the measured macroscopic response (chamber pressure versus apical displacement). In this study, a biomechanically motivated elastic fibre-reinforced corneal material model is chosen. The inverse problem of calibrating the corneal material model parameters using only experimental inflation data is demonstrated to be ill-posed, with small variations in the experimental data leading to large differences in the calibrated model parameters. This can result in different groups of researchers, calibrating their material model with the same inflation test data, drawing vastly different conclusions about the effect of material parameters on GAT results. It is further demonstrated that multiple loading scenarios, such as inflation as well as bending, would be required to reliably calibrate such a corneal material model.

Microencapsulation-protected l-ascorbic acid for the application of human epithelial HaCaT cell proliferation.

l-ascorbic acid is an abundant water-soluble nutrient found in vegetables and fruits. It enhances the cell proliferation, which is helpful in wound healing process. However, it is relatively unstable and easily degraded under external environments including acidity, alkalinity, evaporation, heat, oxidization, light or moisture. Its storage remains challenged. This study reported the development of l-ascorbic acid microcapsules using the natural protein, gelatin, and the natural polysaccharide, agar, as the wall protection carrier. The physical properties including entrapment efficiency, particle size, surface morphology, chemical compositions and release profile were identified. The cell proliferation of l-ascorbic acid microcapsules was stronger than the free drug. Significant cell growth in microencapsulated l-ascorbic acid-treated human epithelial HaCaT cells was observed when compared with untreated control. Since cell proliferation and wound repair are closely related, it is believed that l-ascorbic acid microcapsules would effectively increase the potential effect of wound healing activity in human skin.

D-Glucose as a modifying agent in gelatin/collagen matrix and reservoir nanoparticles for Calendula officinalis delivery.

Gelatin/Collagen-based matrix and reservoir nanoparticles require crosslinkers to stabilize the formed nanosuspensions, considering that physical instability is the main challenge of nanoparticulate systems. The use of crosslinkers improves the physical integrity of nanoformulations under the-host environment. Aldehyde-based fixatives, such as formaldehyde and glutaraldehyde, have been widely applied to the crosslinking process of polymeric nanoparticles. However, their potential toxicity towards human beings has been demonstrated in many previous studies. In order to tackle this problem, D-glucose was used during nanoparticle formation to stabilize the gelatin/collagen-based matrix wall and reservoir wall for the deliveries of Calendula officinalis powder and oil, respectively. In addition, therapeutic selectivity between malignant and normal cells could be observed. The C. officinalis powder loaded nanoparticles significantly strengthened the anti-cancer effect towards human breast adenocarcinoma MCF7 cells and human hepatoma SKHep1 cells when compared with the free powder. On the contrary, the nanoparticles did not show significant cytotoxicity towards normal esophageal epithelial NE3 cells and human skin keratinocyte HaCaT cells. On the basis of these evidences, D-glucose modified gelatin/collagen matrix nanoparticles containing C. officinalis powder might be proposed as a safer alternative vehicle for anti-cancer treatments.

Development of ruthenium(II) complexes as topical antibiotics against methicillin resistant Staphylococcus aureus.

A series of ruthenium(II) bis(2,2'-bipyridyl) complexes containing N-phenyl-substituted diazafluorenes (Ru-C1, Ru-C6, Ru-C7 and Ru-F) was synthesized and their potential antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA) was investigated. The Ru-C7 complex showed significant improvement in both minimum inhibitory concentration (MIC, 6.25 µg mL(-1)) and minimum bactericidal concentration (MBC, 25 µg mL(-1)) towards MRSA when compared with those of methicillin (positive control) (MIC = 25 µg mL(-1) and MBC = 100 µg mL(-1)). The Ru-C7 complex possessed much stronger antibacterial effects than the Ru-C6 complex (MIC, 25 µg mL(-1), MBC, >100 µg mL(-1)). Both Ru-C6 and Ru-C7 complexes were also demonstrated to be biologically safe when tested on normal human skin keratinocytes.

Arsenic in groundwater and its influence on exposure risks through traditionally cooked rice in Prey Vêng Province, Cambodia.

Arsenic (As) contamination of communal tubewells in Prey Vêng, Cambodia, has been observed since 2000. Many of these wells exceed the WHO As in drinking water standard of 10 µg/L by a factor of 100. The aim of this study was to assess how cooking water source impacts dietary As intake in a rural community in Prey Vêng. This aim was fulfilled by (1) using geostatistical analysis techniques to examine the extent of As contaminated groundwater in Prey Vêng and identify a suitable study site, (2) conducting an on-site study in two villages to measure As content in cooked rice prepared with water collected from tubewells and locally harvested rainwater, and (3) determining the dietary intake of As from consuming this rice. Geostatistical analysis indicated that high risk tubewells (>50 µg As/L) are concentrated along the Mekong River's east bank. Participants using high risk tubewells are consuming up to 24 times more inorganic As daily than recommended by the previous FAO/WHO provisional tolerable daily intake value (2.1 µg/kg BW/day). However, As content in rice cooked in rainwater was significantly reduced, therefore, it is considered to be a safer and more sustainable option for this region.

Arecoline-stimulated placenta growth factor production in gingival epithelial cells: modulation by curcumin.

OBJECTIVE: Placenta growth factor (PlGF) is associated with the progression and prognosis of oral cancer. MATERIALS AND METHODS: This study used ELISA, quantitative polymerase chain reaction, and Western blotting to study the arecoline-stimulated (PlGF) protein or mRNA expression in human gingival epithelial S-G cells. RESULTS: Arecoline, a major areca nut alkaloid and an oral carcinogen, could stimulate PlGF protein synthesis in S-G cells in a dose- and time-dependent manner. The levels of PlGF protein secretion increased about 3.1- and 3.8-fold after 24-h exposure to 0.4 and 0.8 mM arecoline, respectively. Pretreatment with antioxidant N-acetyl-l-cysteine (NAC) and ERK inhibitor PD98059, but not NF-κB inhibitor Bay 11-7082, JNK inhibitor SP600125, p38 MAPK inhibitor SB203580, and PI3-K inhibitor LY294002, significantly reduced arecoline-induced PlGF protein synthesis. ELISA analyses demonstrated that NAC and PD98059 reduced about 43% and 38% of the arecoline-induced PlGF protein secretion, respectively. However, combined treatment with NAC and PD98059 did not show additive effect. Moreover, 10 µM curcumin and 4 mM NAC significantly inhibited arecoline-induced ERK activation. Furthermore, 10 µM curcumin completely blocked arecoline-induced PlGF mRNA expression. CONCLUSION: Arecoline-induced PlGF synthesis is probably mediated by reactive oxygen species/ERK pathways, and curcumin may be an useful agent in controlling oral carcinogenesis.