Nitroblue Tetrazolium Chloride Research Articles

A combinatorial approach toward fabrication of surface-adsorbed metal nanoparticles for investigation of an enzyme reaction

We are presenting a combinatorial approach for the preparation of surface-adsorbed metal nanoparticles for investigation of biological phenomena. Metal nanoparticles, in particular noble metal nanoparticles, have become increasingly recognized for their importance in studies of biological systems. An intense near-field associated with these particles can be exploited to improve the performance of various spectroscopic techniques. In this paper, we combine the metal nanoparticle with a colorimetric enzyme reaction and explore spectral changes of the metal nanoparticle dependent on the extent of the reaction, alkaline phosphatase acting on nitro-blue tetrazolium chloride. Changing the shape of the gold particle has a clear effect on the extent of the spectral shift. Furthermore, we demonstrate that the method we employ for preparation of metal particles, vacuum evaporation of a metal on a monolayer of surface-adsorbed monodisperse polystyrene or silica nanospheres, is supremely suited for a combinatorial approach. By depositing two different metals on nanospheres as multi-layers while varying the ratio as well as absolute thicknesses, it is possible to produce a large variety of metal particles in parallel. We show the potential of such a combinatorial approach for rapidly producing metal nanoparticles of choice.

Histological comparison of two different fractional photothermolysis devices operating at 1,550 nm

There are a wide variety of fractional resurfacing devices that are available and it is important to understand the tissue effect of different devices at different parameters to ensure a well-controlled treatment. Thus, we have chosen to characterize and compare two different fractional laser devices, the Fraxel SR750 and SR1500 (re:store) (Solta Medical, Hayward, CA). While the SR750 has a fixed focus spot diameter, the SR1500 features an internally controlled zoom optic allowing for an adjustable spot size. Exposures were performed in vitro on human skin samples at 37 degrees C. The exposures were performed for the SR750 at pulse energies between 6 and 40 mJ at 125 MTZ/cm(2) with up to 20 passes, and for the SR1500 between 6 and 100 mJ, at Treatment Level 7 and 8 passes. The skin samples then were processed for serial frozen sectioning, stained with Nitro-Blue-Tetrazolium-Chloride (NBTC) and lesion depth and width was determined. Mean lesion depth was significantly greater for lesions treated with the SR1500 laser compared to the SR750 at pulse energies of 6, 10, 30, and 40 mJ (P<0.001) with a borderline difference at 20 mJ. Mean lesion width was comparable for energies up to 20 mJ and relatively increased for the SR1500 for higher energies. The depth-to-width ratio (DWR) was in general higher for the SR1500, reaching significance at 6, 10, and 40 mJ. We have characterized the lesion depth and width for the for two different Fractional Photothermolysis devices (SR750 vs. SR1500). The device with the adjustable spot size (SR1500 or Fraxel re:store) provides generally deeper lesions at the same energy level. It remains to be shown whether increased lesion depth improves efficacy for certain clinical applications.

Ambivalent effects of compound C (dorsomorphin) on inflammatory response in LPS-stimulated rat primary microglial cultures

It was proven that compound C displays beneficial effects in models of inflammatory-induced anemia, ischemic stroke, and fibrodysplasia ossificans progressiva. Compound C influence on microglia, playing a major role in neuroinflammation, has not been evaluated yet. The aim of the present study was to determine the effect of compound C on cytokine release, NO, and reactive oxygen species (ROS) production. The rat microglial cultures were obtained by shaking the primary mixed glial cultures. Cytokine and nitrite concentrations were assayed using ELISA kits. ROS were assayed with nitroblue tetrazolium chloride. AMPK activity was assayed using the SAMS peptide. The expression of arginase I, NF-kappaB p65, and hypoxia-inducible factor-1 alpha (HIF-1 alpha) was evaluated using Western blot. Compound C displayed ambivalent effect depending on microglia basal activity. It up-regulated the release of TNF alpha and NO production and increased the expression of arginase I in non-stimulated microglia. However, compound C down-regulated IL-1 beta, IL-6 and TNF alpha release, NO, ROS production, and AMPK activity, diminished NF-kappaB and HIF-1 alpha expression, as well as increased arginase I expression in lipopolysaccharide (LPS)-stimulated microglia. Compound C did not affect iNOS expression and IL-10 and TGF-beta release in non-stimulated and LPS-stimulated microglia. The observed alterations in the release or production of inflammatory mediators may be explained by the changes in NF-kappaB, HIF-1 alpha, and arginase I expression and 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide values in response to LPS, whereas the basis for the compound C effect on non-stimulated microglia remains to be investigated.

Kindlin-1 Is Required for RhoGTPase-Mediated Lamellipodia Formation in Keratinocytes

Kindlin-1 is an epithelial-specific member of the novel kindlin protein family, which are regulators of integrin functions. Mutations in the gene that encodes Kindlin-1, FERMT1 (KIND1), cause the Kindler syndrome (KS), a human disorder characterized by mucocutaneous fragility, progressive skin atrophy, ulcerative colitis, photosensitivity, and propensity to skin cancer. Our previous studies indicated that loss of kindlin-1 resulted in abnormalities associated with integrin functions, such as adhesion, proliferation, polarization, and motility of epidermal cells. Here, we disclosed novel FERMT1 mutations in KS and used them, in combination with small-interfering RNA, protein, and imaging studies, to uncover new functions for kindlin-1 in keratinocytes and to discern the molecular pathology of KS. We show that kindlin-1 forms molecular complexes with beta1 integrin, alpha-actinin, migfilin, and focal adhesion kinase and regulates cell shape and migration by controlling lamellipodia formation. Kindlin-1 governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin. Loss of these kindlin-1 functions forms the biological basis for the epithelial cell fragility and atrophy in the pathology of KS.

A Cobalt-Based Tetrazolium Salts Reduction Test To Assay Polyphenols

A novel assay was developed to measure the capacity of polyphenols to chelate cobalt(II) by using the reduction of the tetrazolium salts, NBT (nitroblue tetrazolium chloride), MTT (methylthiazolyldiphenyl-tetrazolium bromide), and XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) to formazan products. The reduction of the salts is initiated by a cocktail comprised of cobalt(II), H(2)O(2), and selenium(IV), which generates hydroxyl radical, peroxide, and superoxide ions. However, because cobalt(II) could not be replaced either by Fe(II), Mn(II), or Cu(II), the classical Fenton transitional metals, it indicates that cobalt is the key player in the tetrazolium salt reduction. Micromolar concentrations of a large variety of antioxidant polyphenols and minute amounts of fruit beverages rich in polyphenols can readily chelate cobalt, resulting in the inhibition of the reduction of tetrazolium salt to formazan, in a dose-dependent manner. However, this method is unsuitable to measure low molecular weight antioxidants such as ascorbate, uric acid, and vitamin E since these have no chelating properties for cobalt(II). The newly described tetrazolium reduction method is as sensitive as the ferric ion reducing antioxidant power, 2,2-diphenyl-2-picrylhydrazyl hydrate, and the luminol-dependent chemiluminescence antioxidant assays. The practical advantages of using the newly described method to quantify polyphenol levels from various sources are briefly discussed.

Treatment of Resistant Port-Wine Stains with a Pulsed Dual Wavelength 595 and 1064 nm Laser: A Histochemical Evaluation of the Vessel Wall Destruction and Selectivity

Pulsed dye laser (PDL) is the current treatment of choice for port-wine stains (PWS), but 25-50% of treated lesions do not demonstrate a significant improvement. Hybrid lasers may improve treatment efficacy, especially those using the synergies between PDL and Nd:YAG 1064 nm laser. The objectives of this study were to assess vessel wall damage and epidermal sparing after a dual wavelength treatment with the two lasers, using different laser parameters. Post-treatment biopsies, after using a laser platform that allows sequential pulses of PDL and Nd:YAG 1064 nm lasers, were performed in five patients with PWS resistant to PDL. The biopsies were stained with nitroblue-tetrazolium chloride (NBTC), using enzymatic activity that stops immediately after cell death and allows a better identification of viable cells. Five patients with PWS and a median age of 33 years were enrolled in this study. Selectivity and efficacy was observed with this dual wavelength approach, with the best results observed with PDL pulses shorter than 10 ms, use of the 10 mm spot, and a second pass with PDL only. Histochemical studies with NBTC stain can help the laser surgeon establish the best treatment parameters and understand some of the unwanted side effects. The dual wavelength used in this study showed efficacy, but better assessment of treatment parameters, such as the delay between the two lasers, is needed to avoid side effects.

Effects of non‐invasive, 1,210 nm laser exposure on adipose tissue: Results of a human pilot study

Laser radiation (1,210 nm) has been previously shown to be capable of selective photothermolysis of adipose tissue in vitro when applied non-invasively. The objective of this pilot study was to evaluate the in vivo effects of this laser in human subjects. Twenty-four adult subjects were exposed non-invasively on the abdomen to a 1,210 nm laser at fluences of 70, 80, and 90 J/cm(2), with a 10 mm spot size, 5 seconds pre-cooling, and 3 seconds exposure duration delivered with parallel contact cooling. There was an impairment of the skin-cooling device during the study. Exposure and control sites were biopsied at either 1-3 days or 4-7 weeks. Tissue was processed for nitroblue tetrazolium chloride (NBTC) staining, a marker for thermal damage, and hematoxylin and eosin (H&E) staining. Laser exposures were painful, requiring local anesthesia in most subjects, but otherwise well tolerated. At 1-3 days after exposure, there was a fluence-dependent loss of NBTC staining in the fat and dermis. In 2 of 14 subjects (2 of 42 exposure sites) evaluated at 1-3 days after exposure, epidermal damage was noted within a small portion of the test site, likely due to impaired contact cooling. At 4-7 weeks, lipomembranous changes of the fat were seen in 89% of test sites and 33% of control sites. This in vivo study shows histologic evidence of laser-induced damage of fat. With further development, this might become a useful treatment for disorders involving the fat and/or lower dermis.

Signal intensities of radiolabeled cRNA probes used alone or in combination with non-isotopic in situ hybridization histochemistry

This study addressed the question of whether radioactive hybridization signal intensities are reduced in combined isotopic and non-isotopic double in situ hybridization (DISH) compared with those in single in situ hybridization (ISH). Non-isotopic digoxigenin (Dig)-labeled hybrids were detected using an alkaline phosphatase (AP) enzymatic reaction which results in nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP)-salt precipitation that could shield S 35-radiation from penetrating to the surface. Sections were plastic coated of with 2% parlodion to prevent a chemical reaction between AP and developer during processing of the photosensitive emulsion, which could further reduce radioactive hybridization signal detection by autoradiography. We used DISH with a hybridization cocktail of radioactive S 35- and Dig-labeled GAD67 cRNA probes. In order to avoid competition for the same complementary sequence, the probes were directed towards different sequences of the glutamic acid decarboxylase (GAD67) mRNA, resulting in co-detection of isotopic and non-isotopic hybrids in close to 100% of GAD67 positive cells. Quantitation of autoradiograms showed that there was no reduction of autoradiographic signal intensity from S 35-labeled hybrids in the presence of Dig-labeled hybrids. Plastic coating of single or dual hybridized sections did not reduce the radioactive signal intensity. When mRNAs for nicotinic acetylcholine receptor (nAChR) subunits were detected with subunit specific S 35-labeled cRNA probes in GAD67 hippocampal interneurons the total numbers of nAChR subunit expressing cells remained the same in single or double hybridized sections even for low abundant mRNAs. Together, these results indicate that combined radioactive and non-radioactive DISH does not interfere with the detection of the radiation signal from the S 35-labeled hybrids, and neither specificity nor sensitivity is compromised.

Pilot clinical study of a novel minimally invasive bipolar microneedle radiofrequency device

Noninvasive bipolar and monopolar radiofrequency (RF) deep dermal heating devices have previously been described. A novel minimally invasive RF device employing a bipolar microneedle electrode system is introduced and its resultant thermal effects on human skin in vivo were characterized for the first time. An investigational 35 W RF device was configured to operate in bipolar mode delivering energy directly within the dermis using 5 microneedle electrode pairs with real-time feedback of tissue temperature for treatment control. Superficial cooling was achieved using a Peltier device. A range of pulse durations between 1 and 25 seconds, and lesion temperatures between 60 and 80 degrees C were tested in vivo on 15 human subjects. Thermal effects were assessed histologically using either hematoxylin & eosin (H&E) or nitroblue-tetrazoliumchloride (NBTC) staining. Treatment effects and adverse events were also monitored clinically. The investigational bipolar RF device delivered controlled heating within dermal tissue. Histological staining with H&E revealed the presence of zones of denatured collagen within the reticular dermis. Lesions were generated at preselected temperatures between 60 and 80 degrees C. Fractional lesions separated by zones of sparing as well as contiguous lesion patterns were demonstrated. Histological staining with H&E and NBTC revealed sparing of adnexal structures and adipose tissue. No major adverse events were observed. A novel fractional RF device utilizing a minimally invasive bipolar microneedle delivery system for the treatment of human tissue was developed. Treatment of 15 human subjects illustrated the controlled creation of dermally located thermal coagulation zones, herein known as radiofrequency thermal zones. We discovered that varying the pulse length allowed for fractional sparing of dermal tissue. To our knowledge, this is the first report to describe use of a direct real-time temperature and impedance feedback system to control energy delivery during deep dermal heating.

The effects of multiple passes on the epidermal thermal damage pattern in nonablative fractional resurfacing

Nonablative fractional resurfacing is a concept of cutaneous re-modeling whereby laser-induced microscopic treatment zones (MTZs) are surrounded by normal viable tissue. Such thermal damage pattern with a small diameter of individual lesions allows fast re-epithelialization with minimal side effects. The purpose of this in vitro study was to determine the fraction of thermal injury per unit surface area (fill factor) and lesion size in relation to pulse energy and number of passes. Full thickness abdominal skin samples were exposed ex vivo to the Fraxel SR 750 laser (Reliant Technologies, Mountain View, CA). One set of exposures was performed for pulse energies in the range of 8 to 40 mJ for a single pass at 250 MTZ/cm(2). A second set of exposures was performed at 10 mJ with number of passes from 1 to 30. The thermal damage pattern was assessed by incubation of epidermal sheets with NitroBlueTetrazoliumChloride (NBTC) stain. Size of individual MTZ and fill factor were determined by image analysis (ImageJ, NIH, Bethesda, MD) of digital micrographs. Width of the thermal injury zone was directly related to the pulse energy used. The fill factor did not have a uniform relationship with the number of passes. Due to the stochastic placement of individual MTZs, even for greater number of passes, some residual undamaged tissue was found. Due to formation of thermal damage clusters, defined as overlapping individual MTZs, the size of the resulting clustering lesions which we defined as microscopic treatment cluster (MTC) increased linearly as a function of the number of passes. We have described the fill factor as it relates to the number of passes and have demonstrated that the average size of individual lesions depends on the number of passes. Clustering of MTZs lead to the development of MTC, the average size of which increased with the number of passes. The clinical implications of these findings are contingent on further studies.

Isoliquiritigenin induces monocytic differentiation of HL-60 cells

It has been proven that isoliquiritigenin could inhibit the proliferation of some kinds of cancer cell lines and has a strong antioxidative activity. The purpose of this study is to investigate whether the antioxidant isoliquiritigenin affects the proliferation and redifferentiation in HL-60 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) colorimetric method and trypan blue staining were used to measure cell proliferentiation and survival. The morphological changes, nitroblue tetrazolium chloride (NBT) reductive activity, and the CD11b and CD14 surface antigens were used as the biomarkers of redifferentiation of HL-60 cells. The intracellular reactive oxygen species (iROS) level was detected by a fluorescent probe, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). Isoliquiritigenin (ISL) inhibited the cell proliferation and decreased the iROS levels in a dose-dependent manner, while the treatment did not increase the lethality rate. After 72 h treatment with 10 μg/ml ISL, a typical differentiated morphology was observed in HL-60 cells, including the decrease of karyoplasmic ratio and the increase of kidney-shape nuclear cells. The positive rate (%) of CD11b (26.4 ± 3.90 vs 7.70 ± 1.04, P < 0.01) and CD14 (20.4 ± 2.30 vs 2.63 ± 0.133, P < 0.01) cells increased significantly. The NBT reductive activity increased 2.3-fold as compared to that of the control group. As an antioxidant, ISL decreased the iROS formation in a dose-dependent manner. All the results indicate that the antioxidant ISL is able to induce the monocytic differentiation in leukemia cells. ISL has the potential as a drug to cure leukemia with fewer side effects.

Intense Focused Ultrasound: Evaluation of a New Treatment Modality for Precise Microcoagulation within the Skin

Focused ultrasound can produce thermal and/or mechanical effects deep within tissue. We investigated the capability of intense focused ultrasound to induce precise and predictable subepidermal thermal damage in human skin. Postmortem human skin samples were exposed to a range of focused ultrasound pulses, using a prototype device (Ulthera Inc.) emitting up to 45 W at 7.5 MHz with a nominal focal distance of 4.2 mm from the transducer membrane. Exposure pulse duration ranged from 50 to 200 ms. Thermal damage was confirmed by light microscopy using a nitroblue tetrazolium chloride assay, as well as by loss of collagen birefringence in frozen sections. Results were compared with a computational model of intense ultrasound propagation and heating in tissue. Depth and extent of thermal damage were determined by treatment exposure parameters (source power, exposure time, and focal depth). It was possible to create individual and highly confined lesions or thermal damage up to a depth of 4 mm within the dermis. Thermal lesions typically had an inverted cone shape. A precise pattern of individual lesions was achieved in the deep dermis by applying the probe sequentially at different exposure locations. Intense focused ultrasound can be used as a noninvasive method for spatially confined heating and coagulation within the skin or its underlying structures. These findings have a significant potential for the development of novel, noninvasive treatment devices in dermatology.

Selective transcutaneous delivery of energy to porcine soft tissues using intense ultrasound (IUS)

Various energy delivery systems have been utilized to treat superficial rhytids in the aging face. The Intense Ultrasound System (IUS) is a novel modality capable of transcutaneously delivering controlled thermal energy at various depths while sparing the overlying tissues. The purpose of this feasibility study was to evaluate the response of porcine tissues to various IUS energy source conditions. Further evaluation was performed of the built-in imaging capabilities of the device. Simulations were performed on ex vivo porcine tissues to estimate the thermal dose distribution in tissues after IUS exposures to determine the unique source settings that would produce thermal injury zones (TIZs) at given depths. Exposures were performed at escalating power settings and different exposure times (in the range of 1-7.6 J) using three IUS handpieces with unique frequencies and focal depths. Ultrasound imaging was performed before and after IUS exposures to detect changes in tissue consistency. Porcine tissues were examined using nitro-blue tetrazolium chloride (NBTC) staining sensitive for thermal lesions, both grossly and histologically. The dimensions and depth of the TIZs were measured from digital photographs and compared. IUS can reliably achieve discrete, TIZ at various depths within tissue without surface disruption. Changes in the TIZ dimensions and shape were observed as source settings were varied. As the source energy was increased, the thermal lesions became larger by growing proximally towards the tissue surface. Maximum lesion depth closely approximated the pre-set focal depth of a given handpiece. Ultrasound imaging detected well-demarcated TIZ at depths within the porcine muscle tissue. This study demonstrates the response of porcine tissue to various energy dose levels of Intense Ultrasound. Further study, especially on human facial tissue, is necessary in order to understand the utility of this modality in treating the aging face and potentially, other cosmetic applications.

The Chaotrope-soluble Glycoprotein GP2 Is a Precursor of the Insoluble Glycoprotein Framework of the Chlamydomonas Cell Wall

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists of an insoluble, hydroxyproline-rich glycoprotein framework and several chaotrope-soluble, hydroxyproline-containing glycoproteins. Up to now, there have been no data concerning the amino acid sequences of the hydroxyproline-containing polypeptides of the insoluble wall fraction. Matrix-assisted laser desorption ionization time-of-flight analyses of peptides released from the insoluble cell wall fraction by trypsin treatment revealed the presence of 14 peptide fragments that could be attributed to non-glycosylated domains of the chaotrope-soluble cell wall glycoprotein GP2. However, these peptides cover only 15% of the GP2 polypeptide backbone. Considerably more information concerning the presence of GP2 in the insoluble cell wall fraction was obtained by an immunochemical approach. For this purpose, 407 overlapping pentadecapeptides covering the whole known amino acid sequence of GP2 were chemically synthesized and probed with a polyclonal antibody raised against the deglycosylated, insoluble cell wall fraction. This particular antibody reacted with 297 of the 407 GP2-derived peptides. The peptides that were recognized by this antibody are distributed over the whole known GP2 sequence. The epitopes recognized by polyclonal antibodies raised against the 64- and 45-kDa constituents purified from the deglycosylation products of the insoluble cell wall fraction are also distributed over the whole GP2 backbone, although the corresponding antigens are considerably smaller than GP2. The significance of the latter results for the structure of the insoluble cell wall fraction is discussed.

New tetrazolium method for phosphatase assay using ascorbic acid 2-phosphate as a substrate

A new method to assay alkaline and acid phosphatases assay using ascorbic acid 2-phosphate (AsA-P) and ditetrazolium salt nitroblue tetrazolium chloride (NBT) was developed. AsA-P is hydrolyzed in the presence of phosphatase to yield ascorbic acid. In turn, the ascorbic acid reduces NBT directly or indirectly, opening the tetrazole ring to produce an insoluble formazan as a colored precipitate. The proposed method for alkaline phosphatase was compared with a conventional method in which 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is used in combination with NBT in the dot blots of a dilution series of β-lactoglobulin. AsA-P reduced NBT more effectively than BCIP in the presence of alkaline phosphatase. AsA-P could be also used as the chromogenic substrate for an acid phosphatase assay in the presence of phenazinium methylsulfate and NBT.

Clinical Pilot Study of Intense Ultrasound Therapy to Deep Dermal Facial Skin and Subcutaneous Tissues

To evaluate the clinical safety of intense ultrasound in the treatment of the dermis and subcutaneous tissues of the face and neck in terms of skin inflammation, pain, adverse events, and histologic features. In an open-label, phase 1 study, patients scheduled to undergo a rhytidectomy were enrolled into immediate (face-lift surgery within 24 hours of intense ultrasound treatment) and delayed (face-lift surgery 4-12 weeks after treatment) treatment groups. Intense ultrasound treatments were performed as a series of several linear exposures delivered 1.5 to 2.0 mm apart with the use of 1 of 3 available handpieces with different focal depths. Subject pain ratings and standardized digital photographs were obtained at uniform points. Photographs were blindly rated for inflammation. Histologic evaluation of treated tissues was performed with nitroblue tetrazolium chloride viability stain. Fifteen subjects with a mean +/- SD age of 53 +/- 7 years were enrolled. Seven subjects were nonrandomly assigned to the immediate group and 8 were in the delayed group. On histologic examination, thermal injury zones were consistently identified in the dermis at exposure levels greater than 0.5 J as focal areas of denatured collagen. At this threshold level or above, most patient exposures were associated with transient superficial skin erythema and slight to mild discomfort on a standardized pain scale. No other adverse effects were noted in any case. Thermal injury zones were produced in the expected linear pattern and were consistent in size and depth from zone to zone. Increasing source power did not increase the depth of the epicenter of the thermal injury zone. Epidermis was spared in all cases. In this first clinical study of intense ultrasound therapy to facial tissues, the intense ultrasound system allowed for the safe and well-tolerated placement of targeted, precise, and consistent thermal injury zones in the dermis and subcutaneous tissues with sparing of the epidermis.

Selective Creation of Thermal Injury Zones in the Superficial Musculoaponeurotic System Using Intense Ultrasound Therapy

To transcutaneously deliver intense ultrasound (IUS) energy to target the facial superficial musculoaponeurotic system (SMAS), to produce discrete thermal injury zones (TIZs) in the SMAS, and to demonstrate the relative sparing of adjacent nontargeted layers superficial and deep to the SMAS layer. In 6 unfixed human cadaveric specimens, the SMAS layer was visualized and targeted using the ultrasound imaging component of the IUS device. Using 2 IUS handpieces, 202 exposure lines were delivered bilaterally in multiple facial regions by varying combinations of power and exposure time (0.5-8.0 J). Tissue was then excised and examined grossly and histologically for evidence of thermal injury using nitroblue tetrazolium chloride viability stain. Reproducible TIZs were produced selectively in the SMAS at depths of up to 7.8 mm, and sparing of surrounding tissue including the epidermis. Higher energy settings and high-density exposure line pattern produced a greater degree of tissue shrinkage. In human cadaveric facial tissue, IUS can noninvasively target and selectively produce TIZs of reproducible location, size, and geometry in the SMAS layer. The ability to produce focused thermal collagen denaturation in the SMAS to induce shrinkage and tissue tightening has not been previously reported and has significant implications for aesthetic facial rejuvenation.

The effects of pulse energy variations on the dimensions of microscopic thermal treatment zones in nonablative fractional resurfacing

We examined the effects of pulse energy variations on the dimensions of microscopic thermal injury zones (MTZs) created on human skin ex vivo and in vivo using nonablative fractional resurfacing. A Fraxel SR laser system emitting at 1,550 nm provided an array of microscopic spots at variable densities. Pulse energies ranging from 4.5 to 40 mJ were tested on human abdominal skin ex vivo and in vivo. Tissue sections were stained with hematoxylin and eosin (H&E) or nitro blue tetrazolium chloride (NBTC) and MTZ dimensions were determined. Ex vivo and in vivo results were compared. Dosimetry analyses were made for the surface treatment coverage calculation as a function of pulse energy and collagen coagulation based on H&E stain or cell necrotic zone based on NBTC stain. Each MTZ was identified by histological detection of a distinct region of loss of tissue birefringence and hyalinization, representing collagen denaturation and cell necrosis within the irradiated field immediately, 1, 3, and 7 days after treatment. At high pulse energies, the MTZ depth could exceed 1 mm and width approached 200 microm as assessed by H&E. NBTC staining revealed viable interlesional tissue. In general, no statistically significant difference was found between in vivo and ex vivo depth and width measurements. The Fraxel SR laser system delivers pulses across a wide range of density and energy levels. We determined that increases in pulse energy led to increases in MTZ depth and width without compromising the structure or viability of interlesional tissue.

Antioxidant and free radical scavenging activities of phenols from onion ( Allium cepa)

Four (red, violet, white and green) varieties of Allium cepa were studied for their total phenolic contents (TPC), antioxidant (AOA) and free radical scavenging activities (FRSA). The TPC varied from 4.6 to 74.1 mg/g GAE, AOA varied from 13.6% to 84.1% and FRSA showed wide range in terms of IC 50 (inhibitory concentration) from 0.1 to 15.2 mg/ml, EC 50 (efficient concentration) from 4.3 to 660.8 mg/mg and ARP (antiradical power) from 0.15 to 23.2. The outer dry layers of red and violet varieties showed better inhibition of lipid peroxidation assayed by ammonium thiocyanate than α-tocopherol. The non-site-specific inhibition of hydroxyl radical induced deoxyribose degradation was also higher in the outer dry layers of red and violet varieties than in their middle and inner layers. The outer layers were also potential inhibitors of nitroblue tetrazolium chloride (NBT) reduction caused by superoxide anions. On the other hand the ferrous ion chelating capacity of the red and violet varieties was highest in the inner layers. Specific phenolic composition performed through HPLC and LC–MS/MS showed the presence of gallic acid, ferulic acid, protocatechuic acid, quercetin, and kaempferol. The unutilised outer layers of the red variety were a rich source of quercetin (5110 μg/g) with high AOA, FRSA and also showed significant protection of DNA damage caused by free radicals.

Hyphal Invasion of Candida albicans Inhibits the Expression of Human β-Defensins in Experimental Oral Candidiasis

Oral epithelium reacts to microbial challenges by eliciting a defensive response that includes the production of antimicrobial peptides. This study investigated the expression of human beta-defensins-1, 2, and 3 in reconstituted human oral epithelia during experimental oral infections with six different Candida species, and a wild-type C. albicans isolate and five of its mutants. The expression of defensins was induced after 12 hours infection with the wild-type C. albicans, but this response was not seen for the noninvasive hyphal mutants nor the secreted aspartyl proteinase mutants. Furthermore, defensin expression was not detected after 48 hours in epithelia infected with either C. albicans wild-type isolate or its invasive hyphal and proteinase mutants. Most of the non-albicans Candida were capable of inducing the expression of defensins in epithelia after 24 or 48 hours of infection. These Candida-host interaction patterns suggest that the oral epithelia possess mechanisms for sensing the early invasion of C. albicans through recognition of the presence of hyphae and proteinases of Candida and respond to the insult by producing antimicrobial peptides. This hyphal-invasion-dependent inhibition of defensin expression in oral epithelium that undermines the host surveillance system represents a hitherto undescribed novel pathogenic mechanism of C. albicans.