Evaporation of picoliter droplets on surfaces with a range of wettabilities and thermal conductivities.

The evaporation of picoliter water and ethanol droplets generated by drop-on-demand inkjet printing was investigated on substrates with apparent contact angles between 10^{∘} and 135^{∘} and thermal conductivities between 0.25 and 149 Wm^{-1}K^{-1}. Drying times were calculated from a diffusion-limited model for droplets with both pinned and moving contact lines as a function of droplet diameter and apparent contact angle. Droplets with a moving contact line take longer to dry on hydrophilic substrates than pinned droplets. The difference in drying times between evaporative modes vanishes at large apparent contact angles. Hence similar drying times are obtained for both modes on hydrophobic substrates. The predicted drying times for glass and silicon substrates were in good quantitative agreement with experimental data, suggesting that thermal effects are negligible for substrates of these base materials. However, on a PTFE substrate which has a lower thermal conductivity more relevant to inkjet printing, evaporative cooling reduces the evaporation rate causing drying times to be underpredicted by isothermal models.

Impact of picoliter droplets on superhydrophobic surfaces with ultralow spreading ratios.

The impact of picoliter-sized water droplets on superhydrophobic CF(4) plasma fluorinated polybutadiene surfaces is investigated with high-speed imaging. Variation of the surface topography by plasmachemical modification enables the dynamics of wetting to be precisely controlled. Final spreading ratios as low as 0.63 can be achieved. A comparison of the maximum spreading ratio and droplet oscillation frequencies to models described in the literature shows that both are found to be much lower than theoretically predicted.

Palladium-103 plaque radiation therapy for macular degeneration: results of a 7 year study.

AIM: To report 7 year results of ophthalmic plaque radiotherapy for exudative macular degeneration. METHODS: In a phase I clinical trial, 30 patients (31 eyes) were treated with ophthalmic plaque irradiation for subfoveal exudative macular degeneration. Radiation was delivered to a mean 2 mm from the inner sclera (range 1.2-2.4) prescription point calculated along the central axis of the plaque. The mean prescription dose was 17.62 Gy (range 12.5-24) delivered over 34 hours (range 18-65). Early Treatment Diabetic Retinopathy Study (ETDRS) type standardised visual acuity determinations, ophthalmic examinations, and angiography were performed before and after treatment. Clinical evaluations were performed in a non-randomised and unmasked fashion. RESULTS: At 33.3 months (range 3-4), 17 of 31 (55%) eyes had lost 3 or more lines of vision on the ETDRS chart, five (16%) had improved 3 or more lines, and the remaining nine (29%) were within 2 lines of their pretreatment visual acuity measurement. Overall, 45% of patients were within or improved more than 2 lines of their initial visual acuity. Five eyes developed macular scars, eight developed subsequent neovascularisation or haemorrhage, and three progressed through therapy. Two patients were lost to follow up. The most common finding of patients followed for 6 or more months (n=18 of 29 (62%)) was regression or stabilisation of the exudative process. No radiation retinopathy, optic neuropathy, or cataracts could be attributed to irradiation. CONCLUSION: Ophthalmic plaque radiation can be used to treat exudative macular degeneration. At the dose and dose rates employed, most patients experienced decreased exudation or stabilisation of their maculas. No sight limiting radiation complications were noted during 7 year follow up. Owing to the variable natural course of this disease, a prospective randomised clinical trial should be performed to evaluate the efficacy of plaque radiation therapy for exudative macular degeneration.

Utility of current risk stratification tests for predicting major arrhythmic events after myocardial infarction.

OBJECTIVES: We surveyed the literature to estimate prediction values for five common tests for risk of major arrhythmic events (MAEs) after myocardial infarction. We then determined feasibility of a staged risk stratification using combinations of noninvasive tests, reserving an electrophysiologic study (EPS) as the final test. BACKGROUND: Improved approaches are needed for identifying those patients at highest risk for subsequent MAE and candidates for implantable cardioverter-defibrillators. METHODS: We located 44 reports for which values of MAE incidence and predictive accuracy could be inferred: signal-averaged electrocardiography; heart rate variability; severe ventricular arrhythmia on ambulatory electrocardiography; left ventricular ejection fraction; and EPS. A meta-analysis of reports used receiver-operating characteristic curves to estimate mean values for sensitivity and specificity for each test and 95% confidence limits. We then simulated a clinical situation in which risk was estimated by combining tests in three stages. RESULTS: Test sensitivities ranged from 42.8% to 62.4%; specificities from 77.4% to 85.8%. A three-stage stratification yielded a low-risk group (80.0% with a two-year MAE risk of 2.9%), a high-risk group (11.8% with a 41.4% risk) and an unstratified group (8.2% with an 8.9% risk equivalent to a two-year incidence of 7.9%). CONCLUSIONS: Sensitivities and specificities for the five tests were relatively similar. No one test was satisfactory alone for predicting risk. Combinations of tests in stages allowed us to stratify 91.8% of patients as either high-risk or low-risk. These data suggest that a large prospective study to develop a robust prediction model is feasible and desirable.

Toxicity of alpidem, a peripheral benzodiazepine receptor ligand, but not zolpidem, in rat hepatocytes: role of mitochondrial permeability transition and metabolic activation.

Whereas alpidem is hepatotoxic, zolpidem is not. Despite closely related chemical structures, alpidem, but not zolpidem, is a peripheral benzodiazepine receptor (PBR) ligand, and is also more lipophilic than zolpidem. We compared their effects in isolated rat liver mitochondria and rat hepatocytes. Zolpidem did not affect calcium-induced mitochondrial permeability transition (MPT) in mitochondria, caused little glutathione depletion in hepatocytes, and was not toxic, even at 500 microM. At 250 to 500 microM, alpidem prevented calcium-induced MPT in isolated mitochondria, but caused severe glutathione depletion in hepatocytes that was increased by 3-methylcholanthrene, a cytochrome P4501A inducer, and decreased by cystine, a glutathione precursor. Although cell calcium increased, mitochondrial cytochrome c did not translocate to the cytosol and cells died of necrosis. Cell death was prevented by cystine, but not cyclosporin A, an MPT inhibitor. At low concentrations (25-50 microM), in contrast, alpidem accelerated calcium-induced MPT in mitochondria. It did not deplete glutathione in hepatocytes, but nevertheless caused some cell death that was prevented by cyclosporin A, but not by cystine. Alpidem (10 microM) also increased the toxicity of tumor necrosis factor-alpha (1 ng/ml) in hepatocytes. In conclusion, low concentrations of alpidem increase both calcium-induced MPT in mitochondria, and TNF-alpha toxicity in cells, like other PBR ligands. Like other lipophilic protonatable amines, however, alpidem inhibits calcium-induced MPT at high concentrations. At these high concentrations, toxicity involves cytochrome P4501A-mediated metabolic activation, glutathione depletion, and increased cell calcium, without MPT involvement. In contrast, zolpidem has no mitochondrial effects, causes little glutathione depletion, and is not toxic.

Mitochondria in steatohepatitis.

For the first time in history, populations in affluent countries may concomitantly indulge in rich food and physical idleness. Various combinations of obesity, diabetes, and hypertriglyceridemia, with insulin resistance as the common feature, cause hepatic steatosis, which can trigger necroinflammation and fibrosis. Patients with "primary" steatohepatitis exhibit ultrastructural mitochondrial lesions, decreased activity of respiratory chain complexes, and have impaired ability to resynthesize ATP after a fructose challenge. Mitochondria play a major role in fat oxidation and energy production but also leak reactive oxygen species (ROS) and are the main cellular source of ROS. In patients with steatosis, mitochondrial ROS may oxidize hepatic fat deposits, as suggested in animal models. Lipid peroxidation products impair the flow of electrons along the respiratory chain, which may cause overreduction of respiratory chain components, further increasing mitochondrial ROS formation and lipid peroxidation. Another vicious circle could involve ROS-induced depletion of antioxidants, impairing ROS inactivation. Blood vitamin E is decreased in some obese children with steatohepatitis, and serum transaminases improve after vitamin E supplementation. Steatohepatitis is also caused by alcohol abuse, drugs, and other causes. In "secondary" steatohepatitis, mitochondrial ROS formation is further increased as the causative disease itself directly increases ROS or first impairs respiration, which secondarily increases mitochondrial ROS formation. This "second hit" could cause more lipid peroxidation, cytokine induction, Fas ligand induction, and fibrogenesis than in primary steatohepatitis.

Mechanisms for experimental buprenorphine hepatotoxicity: major role of mitochondrial dysfunction versus metabolic activation.

BACKGROUND/AIMS: Although sublingual buprenorphine is safely used as a substitution drug in heroin addicts, large overdoses or intravenous misuse may cause hepatitis. Buprenorphine is N-dealkylated to norbuprenorphine by CYP3A. METHODS: We investigated the mitochondrial effects and metabolic activation of buprenorphine in isolated rat liver mitochondria and microsomes, and its toxicity in isolated rat hepatocytes and treated mice. RESULTS: Whereas norbuprenorphine had few mitochondrial effects, buprenorphine (25-200 microM) concentrated in mitochondria, collapsed the membrane potential, inhibited beta-oxidation, and both uncoupled and inhibited respiration in rat liver mitochondria. Both buprenorphine and norbuprenorphine (200 microM) underwent CYP3A-mediated covalent binding to rat liver microsomal proteins and both caused moderate glutathione depletion and increased cell calcium in isolated rat hepatocytes, but only buprenorphine also depleted cell adenosine triphosphate (ATP) and caused necrotic cell death. Four hours after buprenorphine administration to mice (100 nmol/g body weight), hepatic glutathione was unchanged, while ATP was decreased and serum transaminase increased. This transaminase increase was attenuated by a CYP3A inducer and aggravated by a CYP3A inhibitor. CONCLUSIONS: Both buprenorphine and norbuprenorphine undergo metabolic activation, but only buprenorphine impairs mitochondrial respiration and ATP formation. The hepatotoxicity of high concentrations or doses of buprenorphine is mainly related to its mitochondrial effects.

Hepatitis after intravenous buprenorphine misuse in heroin addicts.

BACKGROUND: Sublingual buprenorphine is used as a substitution drug in heroin addicts. Although buprenorphine inhibits mitochondrial function at high concentrations in experimental animals, these effects should not occur after therapeutic sublingual doses, which give very low plasma concentrations. CASE REPORTS: We report four cases of former heroin addicts infected with hepatitis C virus and placed on substitution therapy with buprenorphine. These patients exhibited a marked increase in serum alanine amino transferase (30-, 37-, 13- and 50-times the upper limit of normal, respectively) after injecting buprenorphine intravenously and three of them also became jaundiced. Interruption of buprenorphine injections was associated with prompt recovery, even though two of these patients continued buprenorphine by the sublingual route. A fifth patient carrying the hepatitis C and human immunodeficiency viruses, developed jaundice and asterixis with panlobular liver necrosis and microvesicular steatosis after using sublingual buprenorphine and small doses of paracetamol and aspirin. CONCLUSIONS: Although buprenorphine hepatitis is most uncommon even after intravenous misuse, addicts placed on buprenorphine substitution should be repeatedly warned not to use it intravenously. Higher drug concentrations could trigger hepatitis in a few intravenous users, possibly those whose mitochondrial function is already impaired by viral infections and other factors.

Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice.

Extracellular ATP is implicated in numerous sensory processes ranging from the response to pain to the regulation of motility in visceral organs. The ATP receptor P2X3 is selectively expressed on small diameter sensory neurons, supporting this hypothesis. Here we show that mice deficient in P2X3 lose the rapidly desensitizing ATP-induced currents in dorsal root ganglion neurons. P2X3 deficiency also causes a reduction in the sustained ATP-induced currents in nodose ganglion neurons. P2X3-null mice have reduced pain-related behaviour in response to injection of ATP and formalin. Significantly, P2X3-null mice exhibit a marked urinary bladder hyporeflexia, characterized by decreased voiding frequency and increased bladder capacity, but normal bladder pressures. Immunohistochemical studies localize P2X3 to nerve fibres innervating the urinary bladder of wild-type mice, and show that loss of P2X3 does not alter sensory neuron innervation density. Thus, P2X3 is critical for peripheral pain responses and afferent pathways controlling urinary bladder volume reflexes. Antagonists to P2X3 may therefore have therapeutic potential in the treatment of disorders of urine storage and voiding such as overactive bladder.

Vesicular transport of newly synthesized cytochromes P4501A to the outside of rat hepatocyte plasma membranes.

Anti-cytochrome P450 (CYP)1A2 autoantibodies are found in dihydralazine-induced hepatitis, and CYPs2B and 2C have been shown to follow vesicular flow to the plasma membrane (PM). However, it is unknown whether other CYPs follow this route, whether NADPH-CYP reductase is present on the hepatocyte surface, and whether autoimmune hepatitis-inducing drugs increase PM CYPs. In this study, we determined the transmembrane topology and transport of CYPs1A in rat hepatocytes. In cultured hepatocytes, colchicine and other vesicular transport inhibitors decreased PM CYPs1A assessed by flow cytometry. Colchicine administration also decreased PM CYPs1A in vivo. Pulse chase experiments with [(35)S]methionine showed that only the newly synthesized CYP molecules are transferred to the PM, whereas microsomal CYP1A2 was stably radiolabeled for several hours. In contrast, radiolabeled CYP1A2 reached the PM and disappeared from the PM with half-lives of less than 30 min. Confocal microscopy, biotinylation, and coimmunoprecipitation experiments showed that PM CYPs1A and CYP reductase are present on the cell surface, and that the reductase is closely associated with PM CYPs. Exposure of whole cells to an anti-CYP1A1/2 antibody at 4 degrees C, before five washes and PM preparation, abolished PM CYPs1A-supported monooxygenase activity, indicating that PM CYPs are mostly located on the external surface. Dihydralazine and other CYPs1A inducers increased PM CYPs1A. In conclusion, newly synthesized CYPs1A follow vesicular flow to the outside of the PM, and NADPH-CYP reductase also is located on the hepatocyte surface. Dihydralazine administration increases PM CYP1A2, its autoimmune target.

Recurrent malignant chondroid syringoma of the foot: a case report and review of the literature.

Malignant chondroid syringoma, or mixed tumor of the skin, salivary gland type, is an uncommon neoplasm believed to originate in sweat glands. This neoplasm occurs mostly in women and is typically seen in the extremities and torso. A case of recurrent malignant chondroid syringoma of the right foot in a man aged 34 years is described with a review of pertinent literature. The surgically excised neoplasm was evaluated by routine histology, immunohistochemistry, and transmission electron microscopy. The malignant chondroid syringoma showed microscopic dermal satellite tumor nodules. Immunohistochemical staining was positive for keratin and S100 and negative for actin and p53. Ki-67 showed <10% positive staining. Ultrastructurally, the neoplasm was composed of epithelial cells with tonofilaments, cell junctions, and electron-dense amorphous keratin-like substance in the intercellular spaces. No evidence of myoepithelial differentiation was noted. Given the tumoral size, acral location, and histologic findings, the neoplasm was classified as a malignant chondroid syringoma. After reviewing the literature, it became apparent that wide surgical excision, adjuvant radiation therapy as well as patient education are critical in facilitating long-term survival.

Opening of the mitochondrial permeability transition pore causes matrix expansion and outer membrane rupture in Fas-mediated hepatic apoptosis in mice.

Although Fas stimulation has been reported to cause outer mitochondrial membrane rupture in Jurkat cells, the mechanism of this effect is debated, and it is not known if outer membrane rupture also occurs in hepatocyte mitochondria. We studied the in vivo effects of Fas stimulation on ultrastructural lesions and mitochondrial function in mice. Four hours after administration of an agonistic anti-Fas antibody (8 microg/animal), caspase activity increased 5.4-fold. Nuclear DNA showed internucleosomal fragmentation, whereas supercoiled mitochondrial DNA was replaced by circular and linear forms. Mitochondrial cytochrome c was partly released into the cytosol. Ultrastructurally, mitochondrial lesions were observed in both apoptotic hepatocytes (with nuclear chromatin condensation/fragmentation) and nonapoptotic hepatocytes (without nuclear changes). In nonapoptotic cells, outer mitochondrial membrane rupture allowed herniation of the inner membrane and matrix through the outer membrane gap. In apoptotic hepatocytes, the matrix became electron-lucent and no longer protruded through the outer membrane gap. Mitochondria clustered around the nucleus, whereas rough endoplasmic reticulum cisternae became peripheral. In liver mitochondria isolated after Fas stimulation, the membrane potential decreased, whereas basal respiration increased. Pretreatment with either z-VAD-fmk (an inhibitor of caspases) or cyclosporin A (a permeability transition inhibitor) totally or mostly prevented mitochondrial outer membrane rupture, membrane potential decrease, cytochrome c release, and apoptosis. In conclusion, in vivo Fas stimulation causes caspase activation, mitochondrial permeability transition (decreasing the membrane potential and increasing basal respiration), mitochondrial matrix expansion (as shown by matrix herniation), outer mitochondrial membrane rupture, and cytochrome c release.

Dysrhythmia hazard after hospitalization for myocardial infarction: two ECG prognostic methods compared.

We retrieved reports of heart rate variability and signal-averaged electrocardiograms (SAECG) used to predict risk of a dysrhythmic event. From each report the number of cases with and without events was extracted to establish accurate values for true positive rate (tpr = sensitivity) and false positive rate (fpr = 1 minus specificity). For all the heart rate variability reports, these values were collected and tpr values were plotted versus fpr. The (fpr,tpr) data were summarized by a meta ROC graph using the method of Moses and Shapiro. A composite weighted mean value and 95% confidence interval were also derived. A summary meta-ROC curve for the SAECG reports was similarly obtained., Meta-ROC analysis of multiple reports better summarizes the performances of different prognostic methods and allows the effect of combining tests for a larger population to be simulated.

Diode-light transillumination for ophthalmic plaque localization around juxtapapillary choroidal melanomas.

PURPOSE: An evaluation of plaque-mounted diode-light transillumination (DLT) for localization of episcleral plaques beneath juxtapapillary tumors. METHODS AND MATERIALS: Two patients scheduled for radiotherapy for juxtapapillary melanomas were offered DLT as an additional method of ophthalmic plaque localization. Plaques were constructed by affixing 4 non-heat producing, light-emitting diodes with their apertures flush with the episcleral outer surface of the plaque's rim. Bio-implantable epoxy was used to encapsulate the electronic components. Then the plaques were loaded with 103Pd seeds. After the eye-plaques were sewn to the episclera covering the base of the intraocular tumors; the diode-lights were illuminated, viewed and recorded. Photodocumentation of the relative position of the 4 lights around tumor's base was obtained in both cases. RESULTS: Digital images of plaque-mounted diode retro-transillumination were obtained. No evidence of diode-light toxicity was noted. Both tumors were found to be covered by the ophthalmic plaques. CONCLUSION: Juxtapapillary tumors are often difficult or impossible to visualize with standard transillumination techniques and have been associated with poor local control rates. We have developed plaque-mounted DLT in an effort to improve ophthalmic plaque localization. Retrobulbar transillumination was viewed by indirect ophthalmoscopy and recorded with video-imaging. This technique provides unique photographic documentation of episcleral plaque localization beneath juxtapapillary tumors.

An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice.

BACKGROUND & AIMS: Ethanol causes oxidative stress in the hepatic mitochondria of experimental animals and mitochondrial DNA deletions in alcoholics. We postulated that ethanol intoxication may cause mitochondrial DNA strand breaks. METHODS: Effects of an intragastric dose of ethanol (5 g/kg) on hepatic mitochondrial DNA levels, structure, and synthesis were determined by slot blot hybridization, Southern blot hybridization, and in vivo [3H]thymidine incorporation, respectively. RESULTS: Two hours after ethanol administration, ethane exhalation (an index of lipid peroxidation) increased by 133%, although hepatic lipids were unchanged. Mitochondrial DNA was depleted by 51%. Its supercoiled form disappeared, whereas linearized forms increased. Long polymerase chain reaction evidenced lesions blocking polymerase progress on the mitochondrial genome. Mitochondrial transcripts decreased. Subsequently, [3H]thymidine incorporation into mitochondrial DNA increased, and mitochondrial DNA levels were restored. In contrast, nuclear DNA was not fragmented and its [3H]thymidine incorporation was unchanged. Liver ultrastructure only showed inconstant mitochondrial lesions. Ethanol-induced mitochondrial DNA depletion was prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by melatonin, an antioxidant. CONCLUSIONS: After an alcoholic binge, ethanol metabolism causes oxidative stress and hepatic mitochondrial DNA degradation in mice. DNA strand breaks may be involved in the development of mitochondrial DNA deletions in alcoholics.

Identification and characterization of a myristylated and palmitylated serine/threonine protein kinase.

We report the molecular cloning and initial characterization of a novel fatty acid acylated serine/threonine protein kinase. The putative open reading frame is predicted to encode a 305 amino acid protein possessing a carboxy-terminal protein kinase domain and amino-terminal myristylation and palmitylation sites. The protein kinase has been accordingly denoted as the myristylated and palmitylated serine/threonine protein kinase (MPSK). Human and mouse MPSKs share approximately 93% identity at the amino acid level with complete retention of acylation sites. Radiation hybridization localized the human MPSK gene to chromosome 2q34-37. Northern analysis demonstrated that the human MPSK 1.7 kilobase mRNA is widely distributed. Epitope tagged human MPSK was found to be acylated by myristic acid at glycine residue 2 and by palmitic acid at cysteines 6 and/or 8. Palmitylation of MPSK in these experiments was found to require an intact myristylation site. While epitope tagged MPSK in immune complexes or purified human glutathione S transferase-MPSK was found to autophosphorylate at one or more threonine residues, the enzyme was not found to phosphorylate several other common exogenous substrates. Indeed, only PHAS-I was identified as an exogenous substrate which was found to be phosphorylated on threonine and serine residues.

Reconstitution of Btk signaling by the atypical tec family tyrosine kinases Bmx and Txk.

Bruton's tyrosine kinase (Btk) is mutated in X-linked agammaglobulinemia patients and plays an essential role in B cell receptor signal transduction. Btk is a member of the Tec family of nonreceptor protein-tyrosine kinases that includes Bmx, Itk, Tec, and Txk. Cell lines deficient for Btk are impaired in phospholipase C-gamma2 (PLCgamma2)-dependent signaling. Itk and Tec have recently been shown to reconstitute PLCgamma2-dependent signaling in Btk-deficient human cells, but it is not known whether the atypical Tec family members, Bmx and Txk, can reconstitute function. Here we reconstitute Btk-deficient DT40 B cells with Bmx and Txk to compare their function with other Tec kinases. We show that in common with Itk and Tec, Bmx reconstituted PLCgamma2-dependent responses including calcium mobilization, extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activation, and apoptosis. Txk also restored PLCgamma2/calcium signaling but, unlike other Tec kinases, functioned in a phosphatidylinositol 3-kinase-independent manner and failed to reconstitute apoptosis. These results are consistent with a common role for Tec kinases as amplifiers of PLCgamma2-dependent signal transduction, but suggest that the pleckstrin homology domain of Tec kinases, absent in Txk, is essential for apoptosis.

Mesalazine (5-aminosalicylic acid) induced chronic hepatitis.

BACKGROUND: Treatment of ulcerative colitis or Crohn's disease with sulphasalazine causes several adverse effects, including hepatitis. Sulphasalazine is cleaved by colonic bacteria into 5-aminosalicylic acid and sulphapyridine. Received wisdom was that 5-aminosalicylic acid was topically active, whereas sulphapyridine was absorbed and caused immunoallergic side effects. Mesalazine, a slow release formulation of 5-aminosalicylic acid, was expected to be a safe alternative. However, several cases of acute hepatitis have been reported. CASE REPORT: A 65 year old man had increased liver enzymes, anti-nuclear and anti-smooth muscle autoantibodies and IgG levels, and lesions of chronic hepatitis after 21 months of mesalazine treatment. Although liver dysfunction had been identified eight months earlier, simvastatin rather than mesalazine had been withdrawn, without any improvement. In contrast, liver enzyme and IgG levels became normal and autoantibodies disappeared after discontinuation of mesalazine administration. CONCLUSION: Contrary to initial expectations, mesalazine can cause most of the sulphasalazine induced adverse effects, and hepatic side effects may be almost as frequent. When liver dysfunction occurs, mesalazine administration should be discontinued to avoid the development of chronic hepatitis and liver fibrosis.