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Peripheral neuropathy and associated factors in patients with type 2 diabetes mellitus in Vietnam: A cross-sectional study Diabetes peripheral neuropathy (DPN) is the most common complication associated with diabetes mellitus. We aimed to establish the characteristic and associated factors of DPN in Vietnam. In total, 140 patients with type 2 diabetes mellitus (T2DM) were screened and enrolled consecutively over a six month period at the endocrinology department in 103 Military hospital. Questionnaires, demographic details, clinical examination, and blood tests were used to collect data from all patients. DPN was evaluated using diabetes neuropathy symptom score and nerve conduction study. Potential factors related to DPN were explored including body mass index, years with disease ( Disclosure N. T. T. Trang: None. T. Fong: None. T. D. Do: None.

Macrophages would engulf circulating oxidized (ox)- low-density lipoprotein and form lipid droplet-laden foam cells. Macrophage foam cells are considered an important therapeutic target of atherosc...

Abnormal lipolysis is correlated with metabolic syndrome. Caffeic acid phenethyl ester (CAPE), a natural product from honeybee hives, has been reported to improve metabolic syndrome. However, the effects of CAPE on lipolysis and perilipin-1 (the major lipid droplet-associated protein) in mature adipocytes were not clarified. In this study, mature adipocytes were isolated from the epididymal fat pads of male rats and incubated with CAPE to estimate lipolysis by measuring glycerol release. It was found that the basal lipolysis was inhibited by CAPE in a dose- and time-dependent manner. The lipid droplet-associated perilipin-1 and phosphorylated peroxisome proliferator-activated receptor (PPAR) gamma levels increased following CAPE treatment by Western blot analysis. Moreover, a specific PPAR-gamma inhibitor (T0070907) could partly reverse the effect of CAPE on basal lipolysis. Furthermore, treatment of adipocytes with dibutyryl-cAMP (db-cAMP) or isoproterenol (ISO) increased lipolysis, but the drug-induced lipolysis was abrogated by combination treatment with CAPE. The lipid droplet-associated perilipin-1 level was also decreased in the drug-induced groups but increased when combined treatment with CAPE. In conclusion, our results revealed that a decrease in basal lipolysis and an increase in lipid droplet-associated perilipin-1 levels induced by CAPE may be involved in the regulation of lipid metabolism through activation of PPAR-gamma in mature adipocytes.

Background: The percentage of female medical students has been significant elevating worldwide. The demographic shift is expected to influence the proportion of male versus female surgeons soon. The objective of this study was to evaluate the gender differences in the acquisition of robotic suturing skills. Methods: We compared the robotic suturing performance between 39 male and 19 female medical students. We separated the training into two parts: phase I, involving virtual reality (VR) robotic simulation, and phase II, involving robotic dry-laboratory simulation training. Participants first conducted step-by-step exercises on the VR robotic simulator and then the robotic skin-suturing pad using the da Vinci robot. Results: The metric analysis of the VR task “suture sponge” showed that female students required less time (difference: −170.7 seconds, 95% CI: −247.4 to −94.0) and had fewer errors (error difference: −50, 95% CI: −74.2 to −25.8) to complete the suture sponge exercise compared to male students. Moreover, female students completed more stitches than male students (differences in mean stitch achieved: .35; 95% CI: .06 to .65). However, there was no difference in the quality scores of stitches by gender (p = 0.85). Conclusion: Female medical students performed better in the VR task of suture spongy and achieved more stitches than male students with the da Vinci system despite no difference in robotic suture quality by gender. Because this is the first study comparing gender performance on a robotic platform, further studies are required to investigate if different training approaches will affect the performance by gender. Keywords: Robotic surgery, Simulation, Gender difference, Virtual reality, Medical education

Myofascial pelvic pain (MFPP) of pelvic floor muscles is a common cause of chronic pelvic pain (CPP). The pathological mechanisms and treatments of MFPP are complex and still unclear until now. The levator ani muscle (LAM) is the major pelvic floor muscle. The purpose of this study was to examine the fascia and attachment of LAM through the electromyogram (EMG) and cadaver dissection. Electrophysiological stimulation of the obturator fascia above the arcus tendinous levator ani (ATLA) could trigger contraction and electrophysiological changes in LAM insertion. The LAM of embalmed adult cadavers was examined especially in the area above the ATLA. Some skeletal muscle fibers were found above the ATLA within the obturator fascia and were confirmed by Masson’s trichrome section staining. Our electromyography (EMG) and anatomical data implied that the attachment of LAM aponeurosis extended beyond ATLA to the inferior border of the superior ramus of the pubic bone. The new discovered attachment of LAM could provide a reference position for clinical diagnosis and treatment of MFPP or CPP.

Platelets play a crucial role in cardiovascular disorders (CVDs); thus, development of a therapeutic target that prevents platelet activation reduces CVDs. Pterostilbene (PTE) has several remarkable pharmacological activities, including anticancer and neuroprotection. Herein, we examined the inhibitory mechanisms of PTE in human platelets and its role in the prevention of vascular thrombosis in mice. At very low concentrations (1-5 μmol/L), PTE strongly inhibited collagen-induced platelet aggregation, but it did not have significant effects against thrombin and 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin (U46619). PTE markedly reduced P-selectin expression on isolated α-granules by a novel microchip. Moreover, PTE inhibited adenosine triphosphate (ATP) release, intracellular ([Ca2+]i) mobilization (resting, 216.6 ± 14.0 nmol/L; collagen-activated platelets, 396.5 ± 25.7 nmol/L; 2.5 μmol/L PTE, 259.4 ± 8.8 nmol/L; 5 μmol/L PTE, 231.8 ± 9.7 nmol/L), phospholipase C (PLC)γ2/protein kinase C (PKC), Akt, and mitogen-activated protein kinase (MAPK) phosphorylation. Neither 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) nor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reversed platelet aggregation inhibited by PTE. PTE did not affect vasodilator-stimulated phosphoprotein phosphorylation. In mice, PTE obviously reduced the mortality (from 100 to 37.5%) associated with acute pulmonary thromboembolism without increasing the bleeding time. Thus, PTE could be used to prevent CVDs.

Columbianadin (CBN), a natural coumarin isolated from Angelica decursiva, is reported to have numerous biological activities, including anticancer and platelet aggregation inhibiting properties. Here, we investigated CBN’s anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated RAW 264.7 cell activation and deciphered the signaling process, which could be targeted by CBN as part of the mechanisms. Using a mouse model of LPS-induced acute liver inflammation, the CBN effects were examined by distinct histologic methods using trichrome, reticulin, and Weigert’s resorcin fuchsin staining. The result showed that CBN decreased LPS-induced expressions of TNF-α, IL-1β, and iNOS and NO production in RAW 264.7 cells and mouse liver. CBN inhibited LPS-induced ERK and JNK phosphorylation, increased IκBα levels, and inhibited NF-κB p65 phosphorylation and its nuclear translocation. Application of inhibitors for ERK (PD98059) and JNK (SP600125) abolished the LPS-induced effect on NF-κB p65 phosphorylation, which indicated that ERK and JNK signaling pathways were involved in CBN-mediated inhibition of NF-κB activation. Treatment with CBN decreased hydroxyl radical (•OH) generation and increased HO-1 expression in RAW 264.7 cells. Furthermore, LPS-induced liver injury, as indicated by elevated serum levels of liver marker enzymes (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) and histopathological alterations, were reversed by CBN. This work demonstrates the utility of CBN against LPS-induced inflammation, liver injury, and oxidative stress by targeting JNK/ERK and NF-κB signaling pathways.

Poor oral hygiene (POH) is associated with oral squamous cell carcinoma (OSCC). Oral microbes often proliferate due to POH. Array data show that LDOC1 plays a role in immunity against pathogens. We investigated whether LDOC1 regulates the production of oral microbe-induced IL-1&beta, an oncogenic proinflammatory cytokine in OSCC. We demonstrated the presence of Candida albicans (CA) in 11.3% of OSCC tissues (n = 80). CA and the oral bacterium Fusobacterium nucleatum stimulate higher levels of IL-1&beta, secretion by LDOC1-deficient OSCC cells than by LDOC1-expressing oral cells. CA SC5314 increased OSCC incidence in 4-NQO (a synthetic tobacco carcinogen) and arecoline-cotreated mice. Loss and gain of LDOC1 function significantly increased and decreased, respectively, CA SC5314-induced IL-1&beta, production in oral and OSCC cell lines. Mechanistic studies showed that LDOC1 deficiency increased active phosphorylated Akt upon CA SC5314 stimulation and subsequent inhibitory phosphorylation of GSK-3&beta, S9 by activated Akt. PI3K and Akt inhibitors and expression of the constitutively active mutant GSK-3&beta, S9A significantly reduced the CA SC5314-stimulated IL-1&beta, production in LDOC1-deficient cells. These results indicate that the PI3K/Akt/pGSK-3&beta, signaling pathway contributes to LDOC1-mediated inhibition of oral microbe-induced IL-1&beta, production, suggesting that LDOC1 may determine the pathogenic role of oral microbes in POH-associated OSCC.

Macrophage foam cells play a central role in the initiation and progression of atherosclerosis. The present study investigates the effect of YC-1, a synthetic benzylindazole compound, on lipid metabolism in macrophage-derived foam cells. Foam cell formation was induced by incubating culturcd RAW264,7 macrophages with oleic acid (OA) or cobalt chloride (CoCI2) (a hypoxia mimetic), Morphological observation showed several Nile red-positive lipid droplets in the cytoplasm of OA-treated RAW264.7 macrophages, Pretreatment with 60 μM YC-1 could inhibit lipid accumulation. Co-incubation with OA and YC-1 also attenuated lipid accumulation due to the decrease in surface area of lipid droplets. Post-treatment with YC-1 induced lipolysis and free fatty acid release in lipid-laden macrophages in both dose- and time-dependent manners. In addition, ODQ, a soluble guanynyl cyclasc (sGC) inhibitor, could not reverse the inhibitory effect of YC-1 on lipid accumulation, or block the lipolytic effect of YC-1 in lipid-laden macrophages. Neither another nitric oxide-independent sGC activator (BAY 41-2272) nor dibutyryl cGMP (db-cGMP) could mimic the effects of YC-1. Both intracellular sGC activity and the cGMP level remained unchanged following YC-1 incubation. These results suggested that the mechanism of YC-1-mediated lipid metabolism was via an sGC/cGMP-independent signal transduction pathway in RAW264.7 macrophages. In addition, YC-1 significantly attenuatced CoCl2-induced lipid droplets accumulation. Our results demonstrated that YC-1 could mediate lipid metabolism in macro phage and reduce foam cell formation, and it could be regarded as a potential drug for the prevention or therapy of atherosclerosis.

Ultrastructural observation of biological specimens or nanogranules usually requires the use of electron microscopy. Electron microscopy takes a lot of time, requires many steps, and uses many chemicals, which may affect the native state of biological specimens. A novel microchip (K-kit) was used as a specimen kit for in situ imaging of human platelet granules in an aqueous solution using a transmission electron microscope. This microchip enabled us to observe the native human platelet granules very quickly and easily. The protocols included blood collection, platelet purification, platelet granule isolation, sample loading into this microchip, and then observation by a transmission electron microscope. In addition, these granules could still remain in aqueous solution, and only a very small amount of the sample was required for observation and analysis. We used this microchip to identify the native platelet granules by negative staining. Furthermore, we used this microchip to perform immunoelectron microscopy and successfully label &alpha, granules of platelets with the anti-P-selectin antibody. These results demonstrate that the novel microchip can provide researchers with faster and better choices when using a transmission electron microscope to examine nanogranules of biological specimens in aqueous conditions.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are a well-known and novel class of oral antihyperglycaemic drugs. DPP-4 inhibition facilitates ulcer healing in patients with diabetes. However, the actual mechanisms, which are independent of lower blood glucose levels, are still unknown. Therefore, the aim of this study was to analyse the effect of the DPP-4 inhibitor sitagliptin on wound healing through a glucose-independent pathway. In this study, DPP-4 inhibitors facilitate keratinocyte differentiation and the proliferation, increase blood flow in the cutaneous of wounds in healthy C57BL/6 mice. Additionally, the administration of the DPP-4 inhibitor ameliorates wound healing and enhances adiponectin expression in healthy C57BL/6 mice. Taken together, our results reveal a protective role for the DPP-4 inhibitor sitagliptin in wound healing by regulating adiponectin and phospho-eNOS levels in keratinocytes. Based on these results, the DPP-4 inhibitor may have therapeutic potential for healing wounds through a diabetes-independent mechanism.

Hsu, CC, Fong, TH, Chang, HM, Su, B, Chi, CP, Kan, NW, and Hsu, MC. Low second-to-fourth digit ratio has high explosive power? A prepubertal study. J Strength Cond Res 32(7): 2091-2095, 2018-A recent study reported that lower limb explosive power had no correlation with the index finger: ring finger (2D:4D) ratio. However, many studies hypothesized that a lower 2D:4D ratio (reflecting a relative higher testosterone exposure) predicts higher physical fitness. The aim of this study was to replicate the study of explosive power and the 2D:4D ratio in a sample of Taiwanese children. A total of 541 Taiwanese prepubertal children (257 girls and 284 boys aged 9-10 years) participated in this study. This study analyzed the relationship between the 2D:4D ratio and explosive power. Explosive power of the lower limbs was assessed using the standing long jump (SLJ) test. The lengths of the second and fourth fingers of the right hand were measured to calculate the 2D:4D ratio. The SLJ length was correlated with the 2D:4D ratios (r = -0.144, p = 0.015) in boys. After controlling for age and the body mass index, this correlation remained significant (r = -0.134, p = 0.024). For girls, 2D:4D ratios were not significantly correlated with SLJ scores. These results indicate that the SLJ distance was negatively correlated with the 2D:4D ratio in boys, but not in girls. These findings might suggest that prenatal testosterone exposure is negatively correlated with the explosive power in men, but not in women.

Lycopene is the most abundant carotenoid in tomatoes, which has been identified to have the properties of anti-inflammation in addition to the capability to inhibit the expression of adhesion molecules. Intercellular adhesion molecules play a critical role in the pathogenesis of psoriasis. Here, we report that the topical use of a lycopene decreased imiquimod (IMQ)-induced psoriasis-like inflammatory responses, the progress of which was based on adhesion molecules. In vitro analysis showed that lycopene decreased keratinocyte and monocyte adhesion. Evidence suggests that intercellular adhesion molecule-1 (ICAM-1) is a main mediator of psoriasis pathogenesis. Therefore, it will be interesting to investigate the factors that contribute to the lycopene-mediated inhibition of ICAM-1 expression in psoriasis. We expect that lycopene will with potential value in the treatment of psoriasis.

Ruthenium metal complex has been shown to exert several chemical and biological activities. A series of three novel ruthenium derivatives (TQ 1, 2 and 4) were synthesized to evaluate the anti-inflammatory and hepatoprotective activities in lipopolysaccharide (LPS)-stimulated macrophages and mice liver injury. The hydroxyl radical (OH°) scavenging activity of these derivatives has also been evaluated. The results revealed that among the tested compounds, TQ-4 effectively attenuated LPS-induced abnormal alteration in liver histoarchistructure via reducing alanine transaminase (ALT) and aspartate transaminase (AST). This compound exhibited significant inhibition of inflammatory cytokines (TNF-α and IL-1β), inflammatory enzyme (iNOS), the component of NF-κB signaling pathway (p65) and JNK phosphorylation in LPS-induced mice liver tissues. In vitro results showed that TQ-4 had the best inhibition of NO production and iNOS expression in LPS-induced RAW 264.7 cells. Mechanistic approach indicated that TQ-4 inhibited the LPS-induced JNK phosphorylation, IκBα degradation, NF-κB p65 phosphorylation and its nuclear translocation, and hydroxyl radical (OH°) productions in RAW 264.7 cells. However, the compounds TQ-1 and 2 had no effects in this study. TQ-4 also inhibited LPS-induced OH° production. This study reveals the protective effect of TQ-4 against LPS-induced acute liver injury, inflammation, and oxidative reaction by destructing JNK/NF-κB signaling pathways. The result of this study may infer that TQ-4 might be a promising ruthenium metal derivative and/or therapeutic agent for treating liver injury.

Several studies have compared molecular components between red and white skeletal muscles in mammals. However, mammalian skeletal muscles are composed of mixed types of muscle fibers. In the current study, we analyzed and compared the distributions of titin, lipid, phosphate ions, and fatty acid levels in red and white muscles using a fish model (Tilapia), which is rich in red and white muscles, and these are well separated. Oil-red O staining showed that red muscle had more-abundant lipids than did white muscle. A time-of-flight secondary-ion mass spectrometric (TOF-SIMS) analysis revealed that red muscle possessed high levels of palmitic acid and oleic acid, but white muscle contained more phosphate ions. Moreover, elastica-van Gieson (EVG) and Mito-Tracker green FM staining showed that collagen and elastic fibers were highly, respectively, distributed in connective tissues and mitochondria in red muscle. An electron micrographic analysis indicated that red muscle had a relatively higher number of mitochondria and longer sarcomere lengths and Z-line widths, while myofibril diameters were thicker in white muscle. Myofibrillar proteins separated by SDS-PAGE showed that the major giant protein, titin, was highly expressed in white muscle than in red muscle. Furthermore, ratios of titin to myosin heavy chain (MHC) (titin/MHC) were about 1.3 times higher in white muscle than red muscle. We postulated that white muscle is fit for short and strong contractile performance due to high levels of titin and condensed sarcomeres, whereas red muscle is fit for low intensity and long-lasting activity due to high levels of lipids and mitochondria and long sarcomeres.

The sliding filament model of the sarcomere was developed more than half a century ago. This model, consisting only of thin and thick filaments, has been efficacious in elucidating many, but not all, features of skeletal muscle. Work during the 1980s revealed the existence of two additional filaments: the giant filamentous proteins titin and nebulin. Nebulin, a giant myofibrillar protein, acts as a protein ruler to maintain the lattice arrays of thin filaments and plays a role in signal transduction and contractile regulation. However, the change of nebulin and its effect on thin filaments in denervation-induced atrophic muscle remains unclear. The purpose of this study is to examine the content and pattern of nebulin, myosin heavy chain (MHC), actin, and titin in innervated and denervated tibialis anterior (TA) muscles of rats using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), densitometry and electron microscopic (EM) analyses. The results revealed that denervation induced muscle atrophy is accompanied by decreased nebulin content in a time-dependent manner. For instant, the levels of nebulin in denervated muscles were markedly (P < 0.05) decreased, about 24.6% and 40.2% in comparison with innervated muscle after denervation of 28 and 56 days, respectively. The nebulin/MHC, nebulin/actin, and nebulin/titin ratios were decreased, suggesting a concomitant reduction of nebulin in denervated muscle. Moreover, a western blotting assay proved that nebulin declined faster than titin on 28 and 56 days of denervated muscle. In addition, EM study revealed that the disturbed arrangements of myofilaments and a disorganized contractile apparatus were also observed in denervated muscle. Overall, the present study provides evidence that nebulin is more sensitive to the effect of denervation than MHC, actin, and titin. Nebulin decline indeed resulted in disintegrate of thin filaments and shortening of sarcomeres.

The objective of the current study was to investigate the effects of Ca(2+) levels on myofibril alignment during zebrafish embryogenesis. To investigate how altered cytoplasmic Ca(2+) levels affect myofibril alignment, we exposed zebrafish embryos to 2-aminothoxyldiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor inhibitor that reduces cytosolic Ca(2+) levels) and caffeine (a ryanodine receptor activator that enhances cytosolic Ca(2+) levels). The results demonstrated that the most evident changes in zebrafish embryos treated with 2-APB were shorter body length, curved trunk and malformed somite boundary. In contrast, such malformed phenotypes were evident neither in untreated controls nor in caffeine-treated embryos. Subtle morphological changes, including changes in muscle fibers, F-actin and ultrastructures were easily observed by staining with specific monoclonal antibodies (F59 and α-laminin), fluorescent probes (phalloidin) and by transmission electron microscopy. Our data suggested that: (1) the exposure to 2-APB and/or caffeine led to myofibril misalignment; (2) 2-APB-treated embryos displayed split and short myofibril phenotypes, whereas muscle fibers from caffeine-treated embryos were twisted and wavy; and (3) zebrafish embryos co-exposed to 2-APB and caffeine resulted in normal myofibril alignment. In conclusion, we proposed that cytosolic Ca(2+) is important for myogenesis, particularly for myofibril alignment.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system, and it also appears in peripheral tissues. Platelets are anuclear blood cells that play a central role in hemostatic processes. Although platelets possess a GABA uptake system, the functional activity of GABA in platelets has remained unclear. We determined that GABA is abundantly distributed in the platelets at a concentration of approximately 1.03 ng/10(6) cells. GABA (0.5 μM) specifically inhibited collagen-induced platelet activation accompanied by [Ca(2+)]i mobilization, phospholipase Cγ2, protein kinase C, Akt phosphorylation, and hydroxyl radical formation. In addition, GABA interfered with fluorescein isothiocyanate-collagen binding to platelet membranes and produced a concentration-dependent shift in the collagen concentration-response curve and a Schild plot slope of -0.96 ± 0.11, indicating competitive inhibition. Platelet activation induced by convulxin, a glycoprotein VI agonist, was inhibited by GABA, whereas activation induced by the integrin α(2)β(1) agonist, aggretin, was not. Immunoprecipitation and surface plasmon resonance revealed that GABA binds directly to glycoprotein VI in human platelets with equilibrium dissociation (binding) constant (K(D)) of 41.4 nM. The closure time of whole blood and the occlusion time of platelet plug formation were significantly prolonged by GABA in vivo. In this study, GABA is a specific inhibitor of collagen glycoprotein VI and may be involved in an endogenous negative feedback mechanism for platelet activation. Thus, GABA may represent a potential target for the development of novel interventions for the treatment of cardiovascular diseases associated with platelet activation, such as stroke and myocardial infarction.GABA is abundantly distributed in the platelets. GABA inhibited platelet activation stimulated by convulxin. GABA directly associated with glycoprotein VI in platelet membrane. GABA prolonged the closure time of whole blood and the occlusion time of platelet plug formation in vivo.

The aim of this study was to investigate the in vivo toxicities of some novel synthetic chalcones. Chalcone and four chalcone analogues 1a-d were evaluated using zebrafish embryos following antibody staining to visualize their morphological changes and muscle fiber alignment. Results showed that embryos treated with 3'-hydroxychalcone (compound 1b) displayed a high percentage of muscle defects (96.6%), especially myofibril misalignment. Ultrastructural analysis revealed that compound 1b-treated embryos displayed many muscle defect phenotypes, including breakage and collapse of myofibrils, reduced cell numbers, and disorganized thick (myosin) and thin (actin) filaments. Taken together, our results provide in vivo evidence of the myotoxic effects of the synthesized chalcone analogues on developing zebrafish embryos.

In this study, we analyzed the key parameters of modified transcutaneous lower blepharoplasty based on multidisciplinary principles (biochemical findings and biophysical wrinkling theory). A total of 408 female patients received our subciliary lower blepharoplasty between March 2002 and January 2010. The severity of the eyebags (dynamic wrinkle numbers and prolapse) was evaluated through preoperative and postoperative photography, whereas the excised lower eyelid skin specimens from 56 patients were investigated with hematoxylin and eosin staining. The modified techniques produced significant improvements in the severity of eyebags in all age groups (P < 0.001). Poor surgical outcome was found to correlate significantly with preoperative dynamic wrinkle numbers (P < 0.001). Age, dynamic wrinkle numbers, and prolapse correlated significantly with dermal fiber density (P = 0.004, 0.000, and 0.000, respectively) but not epidermal, rete ridge, and dermal thickness or the number of rete ridges. In conclusion, modified transcutaneous lower blepharoplasty provides significant improvement to dynamic wrinkles and prolapse in the eyebags. Periorbital aging progressively disturbs the dermal compactness (fiber density) until the structure can no longer hold its integrity at the critical age (around the age of 40).

Background 3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) is a potential anticancer drug that may activate soluble guanylyl cyclase (sGC) and increase the level of cyclic guanosine monophosphate (cGMP). The aim of this study was to explore the effects of YC-1 on lipid droplet accumulation and foam cell formation in macrophages. Results Human-oxidized low density lipoprotein (ox-LDL) was used to induce accumulation of lipid droplets in a murine macrophage cell line, RAW 264.7. Oil red O staining showed that treatment with 20 μM YC-1 for 24 h increased the area of intracellular lipid droplets in macrophages. The results of high content screening (HCS) with the AdipoRed™ assay further revealed that YC-1 enhanced ox-LDL-induced foam cell formation. This was evidenced by an increase in the total area of lipid droplets and the mean fluorescence intensity per cell. Inhibition of cGMP-dependent protein kinase (PKG) using KT5823 significantly reduced YC-1-enhanced lipid droplet formation in ox-LDL-induced macrophage foam cells. Conclusion YC-1 induces lipid droplet formation in macrophages, possibly through the sGC/cGMP/PKG signaling pathway. This chemical should be tested with caution in future clinical trials.

STUDY DESIGN Experimental, controlled trial, animal study. OBJECTIVE To assess autophagy expression after rat spinal cord injury (SCI) and investigate the effect of methylprednisolone treatment on autophagy. SUMMARY OF BACKGROUND DATA Although it is evident that SCI induces necrosis and apoptosis, its relationship to autophagy is uncertain. Autophagy is implicated in various pathological states in the nervous system, such as neurodegenerative diseases, cerebral ischemia, and traumatic brain injury. Up to now, no autophagy expression was evidenced by transmission electronic microscope (TEM) and the autophagy marker, microtubule-associated protein light chain 3 (LC3) in neural tissue after SCI. METHODS Sixty-six Sprague-Dawley rats were used for the experimental procedure. In the SCI group, laminectomy at T9 were performed, followed by impactor contusion of the spinal cord. In the sham group, only a laminectomy was performed without contusion. We used Western blot to analyze LC3 at 2 hours, 4 hours, 1 day, 3 days, and 7 days after SCI. We also investigated the effect of methylprednisolone on autophagy expression of contused spinal cord. Cellular localization and ultrastructural changes after spinal cord injury were compared with those sham-operated rats using immunofluorescent double labeling and TEM, respectively. Data from the Western blot were analyzed using a nonparametric Kruskal-Wallis test with P < 0.05 being considered significant. RESULTS We detected significantly elevated level of LC3 2 hours after SCI, and then the level declined until 1 week after SCI. Methylprednisolone decreased LC3 expression at 2 hours after SCI. LC3 positive cells were colocalized with neuronal nuclei, but not with glial fibrillary acidic protein. The existence of autophagy and progress of autophagic cell death after SCI were confirmed by TEM. CONCLUSION Through observing the enhanced autophagy expression in neurons soon after contusion injury and the inhibitive effect of methylprednisolone treatment, this study demonstrates the characteristics of autophagy expression after SCI and suggests that autophagic cell death may play a role in neuronal death after spinal cord trauma.

Subperiosteal placement of nasal implants has been performed widely for dorsal nasal augmentation in Asia. The authors introduced anatomical and biomechanical studies to investigate the influence of the periosteum on the subperiosteal technique.Nasal periosteum on 20 cadavers was investigated by hematoxylin and eosin staining and biomechanical methods. Nasal profiles (n = 160) and motility testing (n = 1317) were used to analyze patients who underwent augmentation rhinoplasty between 2003 and 2008. An alternative high-split subperiosteal technique was developed for higher placement of the nasal implant.In the cadaver study, the authors discovered that the periosteum and bone were bound together by Sharpey's fibers below the nasion (1.3 +/- 0.6 mm) that penetrated into the nasofrontal suture. The nasal periosteum above the nasofrontal suture (1.77 +/- 0.19 mm) was thicker than the one below the suture (0.83 +/- 0.15 mm) (p0.001). Men had relatively thicker periosteum (1.41 +/- 0.49 mm) compared with women (1.18 +/- 0.5 mm), and the difference was statistically significant (p0.001). Motility testing for clinical cases revealed that there was a higher percentage of the mobilized implant in women than in men (p = 0.042). When the subperiosteal technique was compared with the subcutaneous technique, analysis of the Poisson ratio for the periosteum (0.33 +/- 0.02) indicated greater strength than in the subcutaneous tissue (0.45 +/- 0.02). The alternative high-split subperiosteal technique significantly shortened the radix-to-glabella distance, increased the radix height, and made the nasofrontal angle more obtuse than the conventional subperiosteal technique (p0.001, p0.001, and p = 0.02, respectively).Analysis of the nasal periosteum provided scientific support for using the subperiosteal technique in dorsal nasal augmentation.

Contour asymmetry of the legs is a major aesthetic concern among Asian women. This study enrolled 60 patients with asymmetric calf hypertrophy, defined as a differential calf circumference exceeding 0.6 cm. Differential selective neurectomy techniques, which depend on the sizes of the small and large calves, were performed exactly via a 1-cm popliteal wound. The pre- and postoperative mean differences between the larger and smaller calf circumferences at the 1-year follow-up consultation were 1.38 +/- 0.65 and 0.42 +/- 0.38 cm, respectively (p0.01). The reduction was significantly greater in the leg that was initially larger. The procedure was effective in reducing circumference discrepancies so that leg contours were more balanced. All the patients were able to ambulate normally within 5 months after the procedure without disability. The authors posit that differential neurectomy is a safe and reliable technique for the correction of asymmetric calf hypertrophy, with minimal morbidities.

Nanosized iron oxide particles exhibit osteogenic and radiopaque properties. Thus, iron oxide (Fe3O4) nanoparticles were incorporated into a biodegradable polymer (poly-L-lactic acid, PLLA) to fabricate a composite bone screw. This multifunctional, 3D printable bone screw was detectable on X-ray examination. In this study, mechanical tests including three-point bending and ultimate tensile strength were conducted to evaluate the optimal ratio of iron oxide nanoparticles in the PLLA composite. Both injection molding and 3D printing techniques were used to fabricate the PLLA bone screws with and without the iron oxide nanoparticles. The fabricated screws were implanted into the femoral condyles of New Zealand White rabbits. Bone blocks containing the PLLA screws were resected 2 and 4 weeks after surgery. Histologic examination of the surrounding bone and the radiopacity of the iron-oxide-containing PLLA screws were evaluated. Our results indicated that addition of iron oxide nanoparticles at 30% significantly decreased the ultimate tensile stress properties of the PLLA screws. The screws with 20% iron oxide exhibited strong radiopacity compared to the screws fabricated without the iron oxide nanoparticles. Four weeks after surgery, the average bone volume of the iron oxide PLLA composite screws was significantly greater than that of PLLA screws without iron oxide. These findings suggested that biodegradable and X-ray detectable PLLA bone screws can be produced by incorporation of 20% iron oxide nanoparticles. Furthermore, these screws had significantly greater osteogenic capability than the PLLA screws without iron oxide.

Summary Although penile venous surgery has almost been abandoned and the venous factor eliminated as a contributing factor to erectile dysfunction, new concepts of erection-related veins has recently been described and reported in literature. We sought to conduct a haemodynamic study on human cadavers in order to elucidate to what extent penile veins act in erection, and to explore the possible role of erection-related veins as an important contributor to impotence. From November 2002 to December 2003, seven fresh human cadavers of men who had no sexual activity for at least 6 months prior to death, and in whom the penis was intact were used for this study. Infusion cavernosometry was carried out with an induction flow of 150 mL/min before and after the erection-related veins were removed. A rigid erection was attained in all subjects, lasting significantly longer (p = 0.043) after removal of erection-related veins. Similarly, there were significant differences in the maintenance flow (p = 0.043), Tmax (p = 0.043), Vmax (p = 0.043), and pressure loss (p = 0.043). In cadaveric penises, a rigid erection could be maintained in spite of the fact that the low flow rate of 21 mL/min is much lower than the average arterial perfusion rate observed in cases of arterial insufficiency. We therefore concluded that penile veins may play a significant role in attaining sufficient erection, and further research is required to study this possible clinical implication.

Titin, an elastic and giant myofibrillar protein, is responsible for generating passive tension and maintaining sarcomere structure in striated muscles. Several studies have reported attenuation of passive tension and disorganization of sarcomere in atrophic muscles, but the changes of titin have not been investigated after denervation. For this purpose, we used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunofluorescent staining to examine titin in innervated and denervated tibialis anterior (TA) muscles of the rat. With increasing denervation time, we found a greater loss of titin than myosin heavy chain (MHC) and actin contents in atrophic TA muscle. The ratios of titin/MHC and titin/actin gradually decreased following denervation. In contrast, ratios of MHC/actin in the denervated groups showed no significant differences with the controls even at 56 days postdenervation. The ultrastructure of myofibrils also showed disturbed arrangements of myofilaments and a disorganized contractile apparatus in denervated muscle. Immunofluorescent staining displayed translocation of the titin epitope from the Z-line to the I-band, suggesting that the apparent cleavage of titin occurred near the Z-line region during the atrophying process. Our study provides evidence that titin is more sensitive to degradation than MHC and actin after denervation. Moreover, the titin decline results in the loss of titin-based sarcomeric integrity in atrophic muscle.

1. The aim of this study was to identify the presence of matrix metalloproteinase-9 (MMP-9) in human platelets and systematically examine its inhibitory mechanisms of platelet activation. 2. In this study, we report on an efficient method for the quantitative analysis of pro-MMP-9 in human platelets using capillary zone electrophoresis (CZE). To elucidate subcellular localization of MMP-9 in human platelets, we investigated intraplatelet MMP-9 by immunogold labeling and visualized it using electron microscopy. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium. 3. MMP-9-gold labeling was observed on the plasma membrane, alpha-granules, open canalicular system, and within the cytoplasma both in resting and activated platelets. Furthermore, activated MMP-9 concentration-dependently (15-90 ng ml(-1)) inhibited platelet aggregation stimulated by agonists. Activated MMP-9 (21 and 90 ng ml(-1)) inhibited phosphoinositide breakdown, intracellular Ca(2+) mobilization, and thromboxane A(2) formation in human platelets stimulated by collagen (1 microg ml(-1)). In addition, activated MMP-9 (21 and 90 ng ml(-1)) significantly increased the formation of nitric oxide/cyclic GMP. 4. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12, 13-dibutyrate (PDBu) (60 nm). This phosphorylation was markedly inhibited by activated MMP-9 (21 and 90 ng ml(-1)). Activated MMP-9 (1 microg g(-1)) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules. 5. These results indicate that the antiplatelet activity of activated MMP-9 may be involved in the following pathways. (1) Activated MMP-9 may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown, protein kinase C activation, and thromboxane A(2) formation, thereby leading to inhibition of intracellular Ca(2+) mobilization. (2) Activated MMP-9 also activated the formation of nitric oxide/cyclic GMP, resulting in inhibition of platelet aggregation. These results strongly indicate that MMP-9 is a potent inhibitor of aggregation. It may play an important role as a negative feedback regulator during platelet activation.

In the present study, the signal pathways involved in NO formation and iNOS expression in RAW 264.7 macrophages stimulated by LTA were investigated. We also compared the relative inhibitory activities and mechanisms of PMC, a novel potent antioxidant of alpha-tocopherol derivatives, with those of YC-1, an sGC activator, on the induction of iNOS expression by LTA in cultured macrophages in vitro and LTA-induced hypotension in vivo. LTA induced concentration (0.1-50 microg/mL)- and time (4-24 hr)-dependent increases in nitrite (an indicator of NO biosynthesis) in macrophages. Both PMC (50 microM) and YC-1 (10 microM) inhibited NO production, iNOS protein, mRNA expression, and IkappaBalpha degradation upon stimulation by LTA (20 microg/mL) in macrophages. On the other hand, PMC (50 microM) almost completely suppressed JNK/SAPK activation, whereas YC-1 (10 microM) only partially inhibited its activation in LTA-stimulated macrophages. Moreover, PMC (10 mg/kg, i.v.) and YC-1 (5 mg/kg, i.v.) significantly inhibited the fall in MAP stimulated by LTA (10 mg/kg, i.v.) in rats. In conclusion, we demonstrate that YC-1 shows more-potent activity than PMC at abrogating the expression of iNOS in macrophages in vitro and reversing delayed hypotension in rats with endotoxic shock stimulated by LTA. The inhibitory mechanisms of PMC may be due to its antioxidative properties, with a resulting influence on JNK/SAPK and NF-kappaB activations. YC-1 may be mediated by increasing cyclic GMP, followed by, at least partly, inhibition of JNK/SAPK and NF-kappaB activations, thereby leading to inhibition of iNOS expression.

In this study, magnesium sulphate dose-dependently (0.6-3.0 mmol/l) inhibited platelet aggregation in human platelets stimulated by agonists. Furthermore, magnesium sulphate (3.0 mmol/l) markedly interfered with the binding of fluorescein isothiocanate-triflavin to the glycoprotein (GP)IIb/IIIa complex in platelets stimulated by collagen. Magnesium sulphate (1.5 and 3.0 mmol/l) also inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. Magnesium sulphate (3.0 mmol/l) significantly inhibited thromboxane A2 formation stimulated by collagen in platelets. Moreover, magnesium sulphate (1.5 and 3.0 mmol/l) obviously increased the fluorescence of platelet membranes tagged with diphenylhexatriene. In addition, magnesium sulphate (1.5 and 3.0 mmol/l) increased the formation of cyclic adenosine monophosphate (AMP) in platelets. Phosphorylation of a protein of Mr 47 000 (P47) was markedly inhibited by magnesium sulphate (1.5 mmol/l). In conclusion, the antiplatelet activity of magnesium sulphate may involve the following two pathways. (1) Magnesium sulphate may initially induce membrane fluidity changes with resulting interference of fibrinogen binding to the GPIIb/IIIa complex, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca2+ mobilization and phosphorylation of P47. (2) Magnesium sulphate might also trigger the formation of cyclic AM, ultimately resulting in inhibition of the phosphorylation of P47 and intracellular Ca+2 mobilization.

Perilipin and ADRP, located on the surface of intracellular lipid droplets, are proposed to be involved in adipocyte lipid metabolism. The aim of the present study was to investigate the effect of PKA and PKC activities on the distribution of perilipin and ADRP in primary cultured adrenal cells, and the role of ERK in PMA- and calphostin C-induced steroidogenesis. Immunofluorescence staining indicated that in addition to p160, a capsular protein of steroidogenic lipid droplets, perilipin and ADRP were localized on the lipid droplet surface. Stimuli such as activation of PKA by db cAMP or inhibition of PKC by calphostin C, which increase corticosterone synthesis in various magnitudes, caused detachment of p160 and perilipin, but not ADRP, from the lipid droplet surface. Activation of PKC by PMA induced increase in corticosterone synthesis, however, it did not affect the distribution of perilipin, p160, or ADRP on the lipid droplet surface, suggesting the presence of mechanisms for promoting sterodiogensis other than causing detachment of lipid droplet surface proteins. We further demonstrated that ERK pathway was involved in PMA-induced steroidogenesis, since PD98059, specific inhibitor of MEK, blocked the increases in steroidogenesis and phosphorylation of ERK caused by PMA, but not by cAMP-PKA. These data indicate that p160, perilipin, and ADRP were all located on the lipid droplet surface in rat adrenal cells. On the basis of its non-responsiveness to lipolytic stimulation, ADRP may be a structural protein of the lipid droplet surface, whereas their immediate response to lipolytic stimuli suggest that perilipin and p160 are functional proteins. PKC regulates adrenal steroidogenesis through ERK cascade, whereas PKA pathway does not involve ERK.

Previous studies demonstrated that static magnetic fields (SMF) were effective in down-regulating the expression of lipopolysaccharide (LPS)-induced inflammatory cytokines. The aim of this study was to provide histological evidence of SMF attenuating LPS-induced multiple organ failure (MOF).In this study, BALB/cByJNarl (5 weeks, weighing 20-25 g) mice were chosen as test subjects. The tested animals were challenged with 50 mg/kg LPS after they were exposed to a continuous SMF for 2 h. The survival rate and pathological changes in lungs, kidneys, and livers of the LPS- challenged mice were examined with and without SMF treatment. In addition, the effects of SMF exposure on body temperature control of the LPS-challenged mice were monitored.Our results showed that at 30 h the survival rate of LPS-challenged mice increased 3.6-fold (p0.05). In addition, 6 h after LPS injection, the average body temperature of SMF-exposed mice was 1.07°C lower than that of unexposed animals. Tissue biopsies demonstrated that SMF exposure reduced damage to the lungs, livers, and kidneys in the LPS-challenged mice.SMF show potential as a viable prophylactic alternative for controlling LPS-induced MOF.

We have found that the antibody A2, a marker for the capsule of steroidogenic lipid droplets, reacts with an intermediate filament-associated protein, P200, in 3T3-L1 preadipocytes. Supporting evidence came from the colocalization pattern of P200 with vimentin in double label experiments. The association of P200 with vimentin was further confirmed by its copurification with vimentin after high salt extraction and colocalization of these two proteins in high salt-extracted and vinblastine-treated cells. In preadipocytes this protein was distributed on the vimentin filament network. At the early stage of adipose conversion, this protein was found to encircle nascent lipid droplets ranging from 0.1 to 0.2 micron, accompanied with a decreased distribution on the vimentin filament system. This infers a possible translocation of P200 from the vimentin filaments to the droplet surface. Meanwhile, the vimentin filaments remained in a normal distribution in the cytoplasm and were apparently not associated with the nascent droplet. The association of vimentin filaments to droplet surfaces became prominent in lipid droplets larger than 0.2 micron, forming a typical vimentin cage. Immunogold staining also confirmed the translocation of P200 immunoreactivity from the droplet surface to the vimentin cage. The relocation of P200 from the cytoplasmic vimentin filaments to the droplet surface prior to the formation of the vimentin cage, as well as the reorganization of this protein in the vimentin cage, suggests a stabilizing role in the lipid droplet formation and an inducing function of this protein in the formation of the vimentin cage.

In a previous study, we used a monoclonal antibody, A2, to demonstrate the presence of the lipid droplet-specific capsule in adrenocortical cells and the stimulation of steroid secretion with adrenocorticotrophic hormone (ACTH), resulting in the detachment of this capsule from the droplet surface into the cytosol. To investigate the signaling pathway for this event, we tested the role of adenylate cyclase, cAMP, and protein kinases A and C (PKA and PKC) in this response. ACTH-induced decapsulation of lipid droplets was blocked by either adenylate cyclase inhibitor or PKA inhibitor and stimulated by Bt2cAMP. We conclude that the signaling mechanism involved in lipid droplet decapsulation is the cascade consisting of adenylate cyclase activation, cAMP elevation, and subsequent PKA activation. Furthermore, the cytosolic detached capsular protein was able to relocate to the lipid droplet surface on cessation of ACTH or Bt2cAMP stimulation. In addition to PKA-mediated decapsulation, inhibition of PKC by calphostin C alone was enough to induce decapsulation, a process that was independent of PKA activity, whereas activation of PKC could prevent Bt2cAMP-induced decapsulation. A cAMP radioimmunoassay also confirmed that ACTH caused a marked increase in intracellular levels of cAMP, while PMA or calphostin C caused no significant changes. We conclude that PKA and PKC are reciprocally operated to regulate the decapsulation of lipid droplets, the same mechanism adopted in steroidogenesis. A time-course study also indicates that decapsulation of lipid droplets was accompanied by detectable changes in the size and the area of lipid droplets upon the stimulation of Bt2cAMP or calphostin C, implying a possible coupling between the capsule detachment and steroidogenesis.

We report the presence of a novel filament system in goldfish xanthophores using a monoclonal antibody (A2) made against 40-70 kD proteins derived from cytoskeletal preparations. On Western blots, this antibody recognized a 45 kD protein in xanthophore cell extracts. In cells with dispersed pigment, immunofluorescence staining of xanthophores revealed a uniform distribution of A2-reactive filaments. In cells with aggregated pigment, these filaments assumed a distinctively radial orientation, such that filaments emanated from the central pigment mass (CPM). At the electron microscopic level, immunogold labeling identified a filament system with a diameter of 7 nm. Overall, the cellular distribution of A2-reactive filaments was distinctly different from that of the other known components of the cytoskeleton, such as intermediate filaments, actin filaments, and microtubules. A2-reactive filaments also appeared resistant to agents known to perturb the cytoskeleton such as cytochalasin B, which depolymerized the actin filaments. When xanthophores were treated with vinblastine, shown to depolymerize microtubules and induce the collapse of intermediate filaments (vimentin and keratin) in other cell types, no effect on the A2 filament distribution was observed. On the other hand, treatment with calyculin A, a phosphatase inhibitor, converted A2 filaments into wavy bundles, the effect of which was completely reversible by the removal of the drug from culture medium. These novel properties of A2 filaments, together with their reorganization in response to pigment translocation suggest that A2 filaments might play a yet unidentified role in intracellular organelle transport in these cells. Cell Motil. Cytoskeleton 36:216–227, 1997. © 1997 Wiley-Liss, Inc.

The objective of the current study was to investigate the effects of Ca(2+) levels on myofibril alignment during zebrafish embryogenesis. To investigate how altered cytoplasmic Ca(2+) levels affect myofibril alignment, we exposed zebrafish embryos to 2-aminothoxyldiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor inhibitor that reduces cytosolic Ca(2+) levels) and caffeine (a ryanodine receptor activator that enhances cytosolic Ca(2+) levels). The results demonstrated that the most evident changes in zebrafish embryos treated with 2-APB were shorter body length, curved trunk and malformed somite boundary. In contrast, such malformed phenotypes were evident neither in untreated controls nor in caffeine-treated embryos. Subtle morphological changes, including changes in muscle fibers, F-actin and ultrastructures were easily observed by staining with specific monoclonal antibodies (F59 and α-laminin), fluorescent probes (phalloidin) and by transmission electron microscopy. Our data suggested that: (1) the exposure to 2-APB and/or caffeine led to myofibril misalignment; (2) 2-APB-treated embryos displayed split and short myofibril phenotypes, whereas muscle fibers from caffeine-treated embryos were twisted and wavy; and (3) zebrafish embryos co-exposed to 2-APB and caffeine resulted in normal myofibril alignment. In conclusion, we proposed that cytosolic Ca(2+) is important for myogenesis, particularly for myofibril alignment.

Ageing dynamically disrupts the multilayered supporting components of the skin that are held together by cell adhesion molecules (CAMs). Skin specimens from 33 female Chinese patients undergoing lower blepharoplasty were divided into three age groups and examined by haematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and Elastica-van Gieson (EVG) stains, western blotting, surface electron microscopy (SEM) and biomechanical tension analysis. The SEM density (skin surface topology) showed a negative linear relationship with age. The triangular pattern of the skin surface in the younger group gradually broke down into quadrangular and irregular patterns in the older group. Collagens and elastic fibres in the dermis showed anisotropy and decreased density in the older groups compared with the younger group, especially in the papillary dermis. Anisotropy means that physical properties differ according to the direction of measurement. E-cadherin and integrin αv (whose functions are to bind epidermal and dermal elements respectively) increased and decreased, respectively, in the oldest group. Skin resilience decreased significantly in this group under repetitive stress. In conclusion, a loss of skin surface textures, integrin αv expressions, epidermal-dermal connections and dermal compactness led to the multilayered structure of the skin becoming separated. This in turn decreased resilience during ageing. These findings may therefore explain why aged skins cannot tolerate repetitive facial expressions, and why this action produces further dynamic wrinkles.