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Laccase is a lignin-degrading enzyme that is expected to move industrial applications to a greener form of biotechnology. Here, we used 2,2'-azinobis(3-ethylbenzthiazolin-6-sulfonic acid) (ABTS) as a mediator and N-benzoyl leucomethylene blue (BLMB) as a substrate to develop a fluorometric assay that we used to measure laccase activity in mushroom extracts. We then compared this novel approach to conventional absorption spectrophotometry. With this novel approach, laccase oxidizes ABTS to produce ABTS radicals that show an absorption maximum at 415 nm. The ABTS radicals oxidize BLMB to generate fluorescent methylene blue that is measured by fluorometry while absorption spectrophotometry directly measures the absorbance of the ABTS radicals at 415 nm. Under the optimal conditions, the fluorometric assay showed a linear calibration curve with limits of detection and quantification of 1.0 × 10-2 mg mL-1 and 3.2 × 10-2 mg mL-1, respectively, and those values are 1.4-fold lower than the results using conventional absorption spectrophotometry to measure ABTS radicals. Laccase activity of extracts from species of mushrooms that include eryngii and shiitake were successfully determined via both fluorometry and absorption spectrophotometry. The eryngii extract showed the highest level of activity, which was followed by the shiitake extract, but laccase activity was not observed in the shimeji extract.

ATP-sensitive potassium (KATP) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we use molecular dynamics simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, simulations suggest that K39 shows a greater preference to co-ordinate with PIP2 than with ATP. They also predict that a neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2, potentially accounting for the reduced ATP inhibition observed in electrophysiological experiments. Our work suggests that PIP2 and ATP interact allosterically to regulate KATP channel activity. (Figure presented.). Key points: The KATP channel is activated by the binding of phosphatidylinositol 4,5-bisphosphate (PIP2) lipids and inactivated by the binding of ATP. K39 has the potential to bind to both PIP2 and ATP. A mutation to this residue (K39R) results in neonatal diabetes. This study uses patch-clamp fluorometry, electrophysiology and molecular dynamics simulation. We show that PIP2 competes with ATP for K39, and this reduces channel inhibition by ATP. We show that K39R increases channel affinity to PIP2 by increasing the number of hydrogen bonds with PIP2, when compared with the wild-type K39. This therefore decreases KATP channel inhibition by ATP., QC 20230524

Det finns många biotekniska analys- och detekteringsmetoder. Metoderna används för identifiering och kvantifiering av biomarkörer. Denna studie har analyserat detekteringsmetoder i de fall där två hjärtspecifika biomarkörer används, troponin och kreatinkinas. Studien avsåg att först identifiera tillämpningsfrekvensen av detekteringsmetoder i Sverige samt internationellt. Vidare identifieras sambandet mellan avgörande parametrar i val av detekteringsmetod. Metoden gick ut på att först bestämma den mest frekventa detekteringsmetoden i Sverige med hjälp av en enkät som skickades till olika laboratorier, sedan studerades tidigare studier publicerade på olika internationella databaser. Studierna som tillämpades var på hjärtspecifika troponin och kreatinkinas för att identifiera val av detekteringsmetod, detekteringskaraktäristika och användarvänlighetsparametrar. Studiens resultat visade att nationellt finns det tre detekteringsmetoder som är de mest använda för identifiering av kreatinkinas: masspektrometri, elektrokemisk luminescence och spektrometri. Internationellt är den dominerande metoden däremot elektrokemisk luminescence. För troponin är den dominerande metoden nationellt: elektrokemisk luminescence och flödescytometri, medan internationellt: elektrokemisk luminescence. Elektrokemisk luminescence är i många fall en stark vinnare i tillämpningen. Ytterligare iakttogs korrelationskoefficienter mellan parametern för att identifiera det starkaste respektive svagaste sambandet. Avgörande parametrar i val av elektrokemisk luminescence, visar på flera samband. Elektrokemisk luminescence och kreatinkinas tilldelas en korrelationskoefficient nära ett för parametrar som volym och känslighet och en korrelationskoefficient nära minus ett för linjärt mätområde och volym, samt kostnad och minimummängd. Medan för troponin och elektrokemisk luminescence erhålls en korrelationskoefficient nära ett för parametrar som känslighet och kostnad och en koefficient nära minus ett för ko, There are many biotechnological analysis- and detection methods. The methods are used for identification and quantification of biomarkers. This study has analyzed detection methods incases where two heart-specific biomarkers are used, troponin and creatine kinase. The study was intended to first identify the application frequency of detection methods in Sweden and internationally. Then identify the relationship between crucial parameters in the choice of detection method. The method consisted of first determining the most frequent detection method in Sweden with the help of a questionnaire that was sent to different laboratories, then previous studies published on various international databases were observed. The studies applied were on topics regarding cardiac-specific troponin and creatine kinase to identify choice of detection method, detection characteristics, and ease of use parameters. The results of the study showed that nationally, the detection methods most used for creatine kinase are mass spectrometry, electrochemical luminescence, and spectrometry. Internationally, however, the dominant method is electrochemical luminescence. For troponin, on a national level the dominant methods are electrochemical luminescence and flow cytometry, while internationally: electrochemical luminescence. Electrochemical luminescence is in many cases a strong winner in application. In addition, correlation coefficients are observed between the decisive parameters for a detection method, to identify the strongest and weakest relationships. Electrochemical luminescence and creatine kinase are assigned a correlation coefficient close to one for parameters such as volume and sensitivity and a correlation coefficient when minus one for measurement range and volume, as well as cost and minimum amount. While for troponin and electrochemical luminescence, a correlation coefficient close to one is obtained for parameters such as sensitivity and cost and a coefficient close to minus o

Significance: A child with epilepsy has a previously unreported, heterozygous mutation in KCNA2, the gene encoding KV1.2 proteins. Four KV1.2 assemble into a potassium-selective channel, a protein complex at the neuronal cell surface regulating electrical signaling. KV1.2 subunits assemble with other KV1-family members to form heterotetrameric channels, contributing to neuronal potassium-channel diversity. The most striking consequence of this mutation is preventing KV1.2-subunit trafficking, i.e., their ability to reach the cell surface. Moreover, the mutation is dominant negative, as mutant subunits can assemble with wild-type KV1.2 and KV1.4, trapping them into nontrafficking heterotetramers and decreasing their functional expression. Thus, KV1-family genes’ ability to form heterotetrameric channels is a double-edged sword, rendering KV1-family members vulnerable to dominant-negative mutations in a single member gene. Abstract: We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events a

Significance: A child with epilepsy has a previously unreported, heterozygous mutation in KCNA2, the gene encoding KV1.2 proteins. Four KV1.2 assemble into a potassium-selective channel, a protein complex at the neuronal cell surface regulating electrical signaling. KV1.2 subunits assemble with other KV1-family members to form heterotetrameric channels, contributing to neuronal potassium-channel diversity. The most striking consequence of this mutation is preventing KV1.2-subunit trafficking, i.e., their ability to reach the cell surface. Moreover, the mutation is dominant negative, as mutant subunits can assemble with wild-type KV1.2 and KV1.4, trapping them into nontrafficking heterotetramers and decreasing their functional expression. Thus, KV1-family genes’ ability to form heterotetrameric channels is a double-edged sword, rendering KV1-family members vulnerable to dominant-negative mutations in a single member gene. Abstract: We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events a

Significance: A child with epilepsy has a previously unreported, heterozygous mutation in KCNA2, the gene encoding KV1.2 proteins. Four KV1.2 assemble into a potassium-selective channel, a protein complex at the neuronal cell surface regulating electrical signaling. KV1.2 subunits assemble with other KV1-family members to form heterotetrameric channels, contributing to neuronal potassium-channel diversity. The most striking consequence of this mutation is preventing KV1.2-subunit trafficking, i.e., their ability to reach the cell surface. Moreover, the mutation is dominant negative, as mutant subunits can assemble with wild-type KV1.2 and KV1.4, trapping them into nontrafficking heterotetramers and decreasing their functional expression. Thus, KV1-family genes’ ability to form heterotetrameric channels is a double-edged sword, rendering KV1-family members vulnerable to dominant-negative mutations in a single member gene. Abstract: We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events a

Det finns många biotekniska analys- och detekteringsmetoder. Metoderna används för identifiering och kvantifiering av biomarkörer. Denna studie har analyserat detekteringsmetoder i de fall där två hjärtspecifika biomarkörer används, troponin och kreatinkinas. Studien avsåg att först identifiera tillämpningsfrekvensen av detekteringsmetoder i Sverige samt internationellt. Vidare identifieras sambandet mellan avgörande parametrar i val av detekteringsmetod. Metoden gick ut på att först bestämma den mest frekventa detekteringsmetoden i Sverige med hjälp av en enkät som skickades till olika laboratorier, sedan studerades tidigare studier publicerade på olika internationella databaser. Studierna som tillämpades var på hjärtspecifika troponin och kreatinkinas för att identifiera val av detekteringsmetod, detekteringskaraktäristika och användarvänlighetsparametrar. Studiens resultat visade att nationellt finns det tre detekteringsmetoder som är de mest använda för identifiering av kreatinkinas: masspektrometri, elektrokemisk luminescence och spektrometri. Internationellt är den dominerande metoden däremot elektrokemisk luminescence. För troponin är den dominerande metoden nationellt: elektrokemisk luminescence och flödescytometri, medan internationellt: elektrokemisk luminescence. Elektrokemisk luminescence är i många fall en stark vinnare i tillämpningen. Ytterligare iakttogs korrelationskoefficienter mellan parametern för att identifiera det starkaste respektive svagaste sambandet. Avgörande parametrar i val av elektrokemisk luminescence, visar på flera samband. Elektrokemisk luminescence och kreatinkinas tilldelas en korrelationskoefficient nära ett för parametrar som volym och känslighet och en korrelationskoefficient nära minus ett för linjärt mätområde och volym, samt kostnad och minimummängd. Medan för troponin och elektrokemisk luminescence erhålls en korrelationskoefficient nära ett för parametrar som känslighet och kostnad och en koefficient nära minus ett för ko, There are many biotechnological analysis- and detection methods. The methods are used for identification and quantification of biomarkers. This study has analyzed detection methods incases where two heart-specific biomarkers are used, troponin and creatine kinase. The study was intended to first identify the application frequency of detection methods in Sweden and internationally. Then identify the relationship between crucial parameters in the choice of detection method. The method consisted of first determining the most frequent detection method in Sweden with the help of a questionnaire that was sent to different laboratories, then previous studies published on various international databases were observed. The studies applied were on topics regarding cardiac-specific troponin and creatine kinase to identify choice of detection method, detection characteristics, and ease of use parameters. The results of the study showed that nationally, the detection methods most used for creatine kinase are mass spectrometry, electrochemical luminescence, and spectrometry. Internationally, however, the dominant method is electrochemical luminescence. For troponin, on a national level the dominant methods are electrochemical luminescence and flow cytometry, while internationally: electrochemical luminescence. Electrochemical luminescence is in many cases a strong winner in application. In addition, correlation coefficients are observed between the decisive parameters for a detection method, to identify the strongest and weakest relationships. Electrochemical luminescence and creatine kinase are assigned a correlation coefficient close to one for parameters such as volume and sensitivity and a correlation coefficient when minus one for measurement range and volume, as well as cost and minimum amount. While for troponin and electrochemical luminescence, a correlation coefficient close to one is obtained for parameters such as sensitivity and cost and a coefficient close to minus o

Significance: A child with epilepsy has a previously unreported, heterozygous mutation in KCNA2, the gene encoding KV1.2 proteins. Four KV1.2 assemble into a potassium-selective channel, a protein complex at the neuronal cell surface regulating electrical signaling. KV1.2 subunits assemble with other KV1-family members to form heterotetrameric channels, contributing to neuronal potassium-channel diversity. The most striking consequence of this mutation is preventing KV1.2-subunit trafficking, i.e., their ability to reach the cell surface. Moreover, the mutation is dominant negative, as mutant subunits can assemble with wild-type KV1.2 and KV1.4, trapping them into nontrafficking heterotetramers and decreasing their functional expression. Thus, KV1-family genes’ ability to form heterotetrameric channels is a double-edged sword, rendering KV1-family members vulnerable to dominant-negative mutations in a single member gene. Abstract: We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events a

Significance: A child with epilepsy has a previously unreported, heterozygous mutation in KCNA2, the gene encoding KV1.2 proteins. Four KV1.2 assemble into a potassium-selective channel, a protein complex at the neuronal cell surface regulating electrical signaling. KV1.2 subunits assemble with other KV1-family members to form heterotetrameric channels, contributing to neuronal potassium-channel diversity. The most striking consequence of this mutation is preventing KV1.2-subunit trafficking, i.e., their ability to reach the cell surface. Moreover, the mutation is dominant negative, as mutant subunits can assemble with wild-type KV1.2 and KV1.4, trapping them into nontrafficking heterotetramers and decreasing their functional expression. Thus, KV1-family genes’ ability to form heterotetrameric channels is a double-edged sword, rendering KV1-family members vulnerable to dominant-negative mutations in a single member gene. Abstract: We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events a

The resolution of quaternary mixtures of chlorophylls a and b and pheophytins a and b has been accomplished by partial least-squares (PLS) multivariate calibration, applied to the fluorescence signals of these pigments. The total luminescence information of the compounds has been used to optimize the spectral data set to perform the calibration. After preliminary studies, a method is described in acetone media, to avoid emulsions with the olive oil samples. Different scanning paths have been selected for each method. For the simultaneous determination of the pigments in olive oil samples, a comparative study of the results found by using excitation, emission, and synchronous spectral data, as analytical signal, was performed. The excitation spectra were selected as the better analytical signals for the determination of the pigments in olive oil samples. The optimum wavelength range to record the excitation spectra (?em = 662 nm) was selected to minimize the contribution of pheophytin a and to maximize the contribution of the other pigments, which are the minor constituents in olive oil. Determination of these pigments in olive oil samples was effected from the excitation spectra of dissolutions o suitable aliquots in acetone. Recovery values from olive oil, spiked with chlorophylls a and b and pheophytins a and b, were in the ranges of 70-112, 71-111, 76-105, and 82-109%, respectively.

Numerous nanocarriers are currently being investigated as drug delivery vehicles for transporting chemotherapeutics to cancer cells. Our research group has recently synthesized a new generation of echogenic liposomes based on the concept of encapsulating one or more nanoemulsion droplets inside a liposome (called an “eLiposome”). The concept is to use a nanoemulsion droplet with a low boiling point near body temperature, thus requiring only a small acoustic nudge to vaporize the droplet from liquid to gas and break open the eLiposome, thus releasing its contents. The purpose of this note is twofold. First, we wanted to show experimentally that eLiposomes remained stable at body temperature and retained their potential to deliver drugs through the ultrasonically-activated expansion of the emulsion nanodroplet. Additionally, we examined the physical mechanism potentially involved in the release of calcein at higher temperatures. Experimental results using calcein as a model drug confirmed the eLiposome stability at physiological temperatures and suggested that heterogeneous nucleation theory was capable of capturing the general release characteristics observed in this study. Heterogeneous nucleation of gas is possibly the main mechanism at play in passive release from eLiposomes at temperatures above body temperature. More research is needed to confirm the definitive physics of the model drug’s release from these novel nanovehicles.

The edible filamentous fungi are hot candidate for future supply of functional food and feed with e.g. protein, essential amino acids, and compounds with immunostimulant activity. L-carnitine that plays a crucial role in energy metabolism represents a functional compound normally produced by Zygomycetes filamentous fungus Rhizopus oligosporus in solid-state fermentation. The present study provides the first insights on production of L-carnitine-enriched edible fungal biomass through submerged cultivation of several Ascomycetes and Zygomycetes including Aspergillus oryzae, Neurospora intermedia, Rhizopus oryzae, and Rhizopus oligosporus. A. oryzae with 3 mg L-carnitine yield per gram of fungal biomass, indicates great potential on production of this bioactive compound which is remarkably higher than the other tested fungi in this work and also previous studies. In addition to fungal strain, other factors such as cultivation time and presence of yeast extract were found to play a role. Further studies on submerged growth optimization of A. oryzae in both high-quality recipes and in medium based on low-value substrates are proposed in order to clarify its potential for production of L-carnitine-enriched fungal biomass.

The edible filamentous fungi are hot candidate for future supply of functional food and feed with e.g. protein, essential amino acids, and compounds with immunostimulant activity. L-carnitine that plays a crucial role in energy metabolism represents a functional compound normally produced by Zygomycetes filamentous fungus Rhizopus oligosporus in solid-state fermentation. The present study provides the first insights on production of L-carnitine-enriched edible fungal biomass through submerged cultivation of several Ascomycetes and Zygomycetes including Aspergillus oryzae, Neurospora intermedia, Rhizopus oryzae, and Rhizopus oligosporus. A. oryzae with 3 mg L-carnitine yield per gram of fungal biomass, indicates great potential on production of this bioactive compound which is remarkably higher than the other tested fungi in this work and also previous studies. In addition to fungal strain, other factors such as cultivation time and presence of yeast extract were found to play a role. Further studies on submerged growth optimization of A. oryzae in both high-quality recipes and in medium based on low-value substrates are proposed in order to clarify its potential for production of L-carnitine-enriched fungal biomass.

Key pointsKV 1.2 channels, encoded by the KCNA2 gene, regulate neuronal excitability by conducting K+ upon depolarization. A new KCNA2 missense variant was discovered in a patient with epilepsy, causing amino acid substitution F302L at helix S4, in the KV 1.2 voltage-sensing domain. Immunocytochemistry and flow cytometry showed that F302L does not impair KCNA2 subunit surface trafficking. Molecular dynamics simulations indicated that F302L alters the exposure of S4 residues to membrane lipids. Voltage clamp fluorometry revealed that the voltage-sensing domain of KV 1.2-F302L channels is more sensitive to depolarization. Accordingly, KV 1.2-F302L channels opened faster and at more negative potentials; however, they also exhibited enhanced inactivation: that is, F302L causes both gain- and loss-of-function effects. Coexpression of KCNA2-WT and -F302L did not fully rescue these effects. The proband's symptoms are more characteristic of patients with loss of KCNA2 function. Enhanced KV 1.2 inactivation could lead to increased synaptic release in excitatory neurons, steering neuronal circuits towards epilepsy.AbstractAn exome-based diagnostic panel in an infant with epilepsy revealed a previously unreported de novo missense variant in KCNA2, which encodes voltage-gated K+ channel KV 1.2. This variant causes substitution F302L, in helix S4 of the KV 1.2 voltage-sensing domain (VSD). F302L does not affect KCNA2 subunit membrane trafficking. However, it does alter channel functional properties, accelerating channel opening at more hyperpolarized membrane potentials, indicating gain of function. F302L also caused loss of KV 1.2 function via accelerated inactivation onset, decelerated recovery and shifted inactivation voltage dependence to more negative potentials. These effects, which are not fully rescued by coexpression of wild-type and mutant KCNA2 subunits, probably result from the enhancement of VSD function, as demonstrated by optically tracking VSD depolarization-evok

Soft microfluidic systems that capture, store, and perform biomarker analysis of microliter volumes of sweat, in situ, as it emerges from the surface of the skin, represent an emerging class of wearable technology with powerful capabilities that complement those of traditional biophysical sensing devices. Recent work establishes applications in the real-time characterization of sweat dynamics and sweat chemistry in the context of sports performance and healthcare diagnostics. This paper presents a collection of advances in biochemical sensors and microfluidic designs that support multimodal operation in the monitoring of physiological signatures directly correlated to physical and mental stresses. These wireless, battery-free, skin-interfaced devices combine lateral flow immunoassays for cortisol, fluorometric assays for glucose and ascorbic acid (vitamin C), and digital tracking of skin galvanic responses. Systematic benchtop evaluations and field studies on human subjects highlight the key features of this platform for the continuous, noninvasive monitoring of biochemical and biophysical correlates of the stress state.

Key points: KV1.2 channels, encoded by the KCNA2 gene, regulate neuronal excitability by conducting K+ upon depolarization. A new KCNA2 missense variant was discovered in a patient with epilepsy, causing amino acid substitution F302L at helix S4, in the KV1.2 voltage-sensing domain. Immunocytochemistry and flow cytometry showed that F302L does not impair KCNA2 subunit surface trafficking. Molecular dynamics simulations indicated that F302L alters the exposure of S4 residues to membrane lipids. Voltage clamp fluorometry revealed that the voltage-sensing domain of KV1.2-F302L channels is more sensitive to depolarization. Accordingly, KV1.2-F302L channels opened faster and at more negative potentials; however, they also exhibited enhanced inactivation: that is, F302L causes both gain- and loss-of-function effects. Coexpression of KCNA2-WT and -F302L did not fully rescue these effects. The proband's symptoms are more characteristic of patients with loss of KCNA2 function. Enhanced KV1.2 inactivation could lead to increased synaptic release in excitatory neurons, steering neuronal circuits towards epilepsy. Abstract: An exome-based diagnostic panel in an infant with epilepsy revealed a previously unreported de novo missense variant in KCNA2, which encodes voltage-gated K+ channel KV1.2. This variant causes substitution F302L, in helix S4 of the KV1.2 voltage-sensing domain (VSD). F302L does not affect KCNA2 subunit membrane trafficking. However, it does alter channel functional properties, accelerating channel opening at more hyperpolarized membrane potentials, indicating gain of function. F302L also caused loss of KV1.2 function via accelerated inactivation onset, decelerated recovery and shifted inactivation voltage dependence to more negative potentials. These effects, which are not fully rescued by coexpression of wild-type and mutant KCNA2 subunits, probably result from the enhancement of VSD function, as demonstrated by optically tracking VSD depolarization-evoked, QC 20201117

Pathogenic bacteria pose a serious threat to public health, and the rapid and cost-effective detection of such bacteria remains a major challenge. Herein, we present a DNAzyme-based fluorescent paper sensor for Klebsiella pneumoniae. The DNAzyme was generated by an in vitro selection technique to cleave a fluorogenic DNA-RNA chimeric substrate in the presence of K. pneumoniae. The DNAzyme was printed on a paper substrate in a 96-well format to serve as mix-and-read fluorescent assay that exhibits a limit of detection (LOD) 105 CFUs mL-1 . Evaluated with 20 strains of clinical bacterial isolates, the DNAzyme produced the desired fluorescence signal with the samples of K. pneumoniae, regardless of their source or drug resistance. The assay is simple to use, rapid, inexpensive, and avoids the complex procedures of sample preparation and equipment. We believe that this DNAzyme-based fluorescent assay has potential for practical applications to identify K. pneumoniae.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.

IntroductionVentilation system filters process recirculated indoor air along with outdoor air. This function inspires the idea of using the filter as an indoor bioaerosol sampler. While promising, there remains a need to investigate several factors that could limit the accuracy of such a sampling approach. Among the important factors are the dynamics of microbial assemblages on filter surfaces over time and the differential influence of outdoor versus recirculated indoor air.MethodsThis study collected ventilation system filter samples from an air handling unit on a regular schedule over a 21-week period and analyzed the accumulation patterns of biological particles on the filter both quantitatively (using fluorometry and qPCR) and in terms of microbial diversity (using 16S rDNA and ITS sequencing).ResultsThe quantitative result showed that total and bacterial DNA accumulated monotonically, rising to 41 ng/cm2 for total DNA and to 2.8 ng/cm2 for bacterial DNA over the 21-week period. The accumulation rate of bacterial DNA correlated with indoor occupancy level. Fungal DNA first rose to 4.0 ng/cm2 before showing a dip to 1.4 ng/cm2 between weeks 6 and 10. The dip indicated a possible artifact of this sampling approach for quantitative analysis as DNA may not be conserved on the filter over the months-long service period. The sequencing results indicate major contributions from outdoor air for fungi and from recirculated indoor air for bacteria. Despite the quantitative changes, the community structure of the microbial assemblages was stable throughout the 21-week sampling period, highlighting the robustness of this sampling method for microbial profiling.ConclusionThis study supports the use of ventilation system filters as indoor bioaerosol samplers, but with caveats: 1) an outdoor reference is required to properly understand the contribution of outdoor bioaerosols; and 2) there is a need to better understand the persistence and durability of the targeted organisms

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.

IntroductionVentilation system filters process recirculated indoor air along with outdoor air. This function inspires the idea of using the filter as an indoor bioaerosol sampler. While promising, there remains a need to investigate several factors that could limit the accuracy of such a sampling approach. Among the important factors are the dynamics of microbial assemblages on filter surfaces over time and the differential influence of outdoor versus recirculated indoor air.MethodsThis study collected ventilation system filter samples from an air handling unit on a regular schedule over a 21-week period and analyzed the accumulation patterns of biological particles on the filter both quantitatively (using fluorometry and qPCR) and in terms of microbial diversity (using 16S rDNA and ITS sequencing).ResultsThe quantitative result showed that total and bacterial DNA accumulated monotonically, rising to 41 ng/cm2 for total DNA and to 2.8 ng/cm2 for bacterial DNA over the 21-week period. The accumulation rate of bacterial DNA correlated with indoor occupancy level. Fungal DNA first rose to 4.0 ng/cm2 before showing a dip to 1.4 ng/cm2 between weeks 6 and 10. The dip indicated a possible artifact of this sampling approach for quantitative analysis as DNA may not be conserved on the filter over the months-long service period. The sequencing results indicate major contributions from outdoor air for fungi and from recirculated indoor air for bacteria. Despite the quantitative changes, the community structure of the microbial assemblages was stable throughout the 21-week sampling period, highlighting the robustness of this sampling method for microbial profiling.ConclusionThis study supports the use of ventilation system filters as indoor bioaerosol samplers, but with caveats: 1) an outdoor reference is required to properly understand the contribution of outdoor bioaerosols; and 2) there is a need to better understand the persistence and durability of the targeted organisms

Protein l-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the human pcmt1 gene, catalyzes repair of abnormal l-isoaspartyl linkages in age-damaged proteins. Pcmt1 knock-out mice exhibit a profound neuropathology and die 30-60 days postnatal from an epileptic seizure. Here we express 15 reported variants of human PIMT and characterize them with regard to their enzymatic activity, thermal stability, and propensity to aggregation. One mutation, R36C, renders PIMT completely inactive, whereas two others, A7P and I58V, exhibit activity that is 80-100% higher than wild type. G175R is highly prone to aggregation and has greatly reduced activity. R17S and R17H show markedly enhanced sensitivity to thermal denaturation. Based on previous studies of moderate PIMT variation in humans and mice, we predict that heterozygosity for R36C, G175R, R17S, and R17H will prove detrimental to cognitive function and successful aging, whereas homozygosity (if it ever occurs) will lead to severe neurological problems in the young.

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule.

Užíváním mnoha druhů léku, a vystavováním našeho těla účinkům xenobiotik vůbec, zatěžujeme tělo pochody, kterými se snaží tyto látky odbourat. Dané xenobiotikum je v organismu metabolizováno pomocí I. a II. fáze biotransformace. Cytochromy P450 patří mezi nejdůležitější detoxikační enzymy I. fáze biotransformace xenobiotik v lidském organismu. Tyto hemoproteiny se tak uplatňují společně s II. fází biotransformace na eliminaci xenobiotik z buněk a jejich celkovém odstranění z organismu. Hlavním cílem mé práce bylo optimalizovat metodický postup stanovení aktivity cytochromu 1A2 pomocí metody spektrofotometrické, spektrofluorimetrické a pomocí HPLC s UV/VIS detekcí u různých buněčných linií. Zjistili jsme, že spektrofotometrická metoda není z důvodu malé senzitivity vhodná. U metody HPLC-UV/VIS se nám také z hlediska nízké citlivosti detekce nedokázalo detekovat aktivitu CYP 1A2. Proto jsme jako nejvhodnější metodu určili metodu spektrofluorimetrickou s dobou měření aktivity v délce 1 hod. Optimalizované postupy jsme použili při analýze vzorků biologického materiálu, tj. buněčnou linii pocházející z lidských buněk hepatocytárního karcinomu HepG2, linii lidských epiteliálních ledvinných buněk proximálního tubulu HK-2 a buněčnou linii lidského plicního adenokarcinomu A549. U všech tří linií jsme se zaměřili na stanovení aktivity CYP 1A2. Až na buněčnou linii HK-2 se nám podařilo spektrofluorimetricky detekovat aktivitu CYP 1A2. S ohledem na to lze závěrem říci, že jsme zavedli a optimalizovali spektrofluorimetrickou metodu ke stanovení aktivity CYP 1A2 v podmínkách in vitro., Our organism has to metabolize a number of drugs and xenobiotics which might impair its function. The xenobiotics are metabolized through two phases of biotransformation phase I and phase II. Cytochromes P450 are likely the most important detoxification enzymes participating in phase I of xenobiotic biotransformation in human organism. These hemoproteins play an important role together with phase II processes in the elimination of xenobiotics from cells and in their total removal from the body. The main aim of my work was the optimization of a methodological approach for determination of cytochrome 1A2 activity using spectrophotometric, spectrofluorimetric methods and HPLC with UV/VIS detection in cell lines. We have found that spectrophotometric method is not suitable because of the low detection limit. In addition, we did not detect CYP 1A2 activity using HPLC-UV/VIS due to low sensitivity. Finally, we have determined the spectrofluorimetric assay as the most suitable method with 1 h duration of measurement. We used the optimized procedures of sample preparation for analysis of CYP activity in a biological material - human hepatocellular cell line HepG2, proximal tubular epithelial renal cell line HK-2 and human lung adenocarcinoma cell line A549. We focused on determination of CYP 1A2 activity in all three cell lines. Except of HK-2 cells, we were able to detect CYP 1A2 activity in both other cell lines fluorimetrically. In conclusion, we introduced and optimized the spectrofluorimetric method to determine CYP 1A2 activity in vitro., Fakulta chemicko-technologická, \par{1. Prezentace výsledků diplomové práce.\par} \par{\par{2. Diskuze k posudku vedoucího a oponenta diplomové práce.\par} \par{3. Studentka zodpověděla všechny dotazy a připomínky k DP.\par}\par}

Dystroglycan (DG) is a highly glycosylated protein complex that links the cytoskeleton with the extracellular matrix, mediating fundamental physiological functions such as mechanical stability of tissues, matrix organization and cell polarity. A crucial role in the glycosylation of the DG α subunit is played by its own N-terminal region that is required by the glycosyltransferase LARGE. Alteration in this O-glycosylation deeply impairs the high affinity binding to other extracellular matrix proteins such as laminins. Recently, three missense mutations in the gene encoding DG, mapped in the α-DG N-terminal region, were found to be responsible for hypoglycosylated states, causing congenital diseases of different severity referred as primary dystroglycanopaties.To gain insight on the molecular basis of these disorders, we investigated the crystallographic and solution structures of these pathological point mutants, namely V72I, D109N and T190M. Small Angle X-ray Scattering analysis reveals that these mutations affect the structures in solution, altering the distribution between compact and more elongated conformations. These results, supported by biochemical and biophysical assays, point to an altered structural flexibility of the mutant α-DG N-terminal region that may have repercussions on its interaction with LARGE and/or other DG-modifying enzymes, eventually reducing their catalytic efficiency.

l-Lactate is a vital biomarker for many diseases and physiological fatigue. An AIE-active fluorophore (TPE-HPro) is combined with l-lactate oxidase (LOx) to determine l-lactate in aqueous fluid. The assay shows excellent sensitivity and anti-interference performance with a limit of detection (LOD) of 5.5 μM. In addition, sensitive detection of l-lactate is achieved even in a protein-rich environment. It is proposed that quantification of l-lactate be performed at 20 or 60 min in the current method. These characteristics endow the fluorometric assay with great potential for biomedical diagnostics. © 2017 American Chemical Society.

In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule.

A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28-AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06-0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.

Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye.

Improved tools for providing specific intraoperative diagnoses could improve patient care. In neurosurgery, intraoperatively differentiating non-operative lesions such as CNS B-cell lymphoma from operative lesions can be challenging, often necessitating immunohistochemical (IHC) procedures which require up to 24-48 hours. Here, we evaluate the feasibility of generating rapid ex vivo specific labeling using a novel lymphoma-specific fluorescent switchable aptamer. Our B-cell lymphoma-specific switchable aptamer produced only low-level fluorescence in its unbound conformation and generated an 8-fold increase in fluorescence once bound to its target on CD20-positive lymphoma cells. The aptamer demonstrated strong binding to B-cell lymphoma cells within 15 minutes of incubation as observed by flow cytometry. We applied the switchable aptamer to ex vivo xenograft tissue harboring B-cell lymphoma and astrocytoma, and within one hour specific visual identification of lymphoma was routinely possible. In this proof-of-concept study in human cell culture and orthotopic xenografts, we conclude that a fluorescent switchable aptamer can provide rapid and specific labeling of B-cell lymphoma, and that developing aptamer-based labeling approaches could simplify tissue staining and drastically reduce time to histopathological diagnoses compared with IHC-based methods. We propose that switchable aptamers could enhance expeditious, accurate intraoperative decision-making.

AB Archival samples represent a significant potential for genetic studies, particularly in severe diseases with risk of lethal outcome, such as in cancer. In this pilot study, we aimed to evaluate the usability of archival bone marrow smears and biopsies for DNA extraction and purification, whole genome amplification (WGA), multiple marker analysis including 10 short tandem repeats, and finally a comprehensive genotyping of 33,683 single nucleotide polymorphisms (SNPs) with multiplexed targeted next-generation sequencing. A total of 73 samples from 21 bone marrow smears and 13 bone marrow biopsies from 18 Danish and Norwegian childhood acute lymphoblastic leukemia patients were included and compared with corresponding blood samples. Samples were grouped according to the age of sample and whether WGA was performed or not. We found that measurements of DNA concentration after DNA extraction was dependent on detection method and that spectrophotometry overestimated DNA amount compared with fluorometry. In the short tandem repeat analysis, detection rate dropped slightly with longer fragments. After WGA, this drop was more pronounced. Samples stored for 0 to 3 years showed better results compared with samples stored for 4 to 10 years. Acceptable call rates for SNPs were detected for 7 of 42 archival samples. In conclusion, archival bone marrow samples are suitable for DNA extraction and multiple marker analysis, but WGA was less successful, especially when longer fragments were analyzed. Multiple SNP analysis seems feasible, but the method has to be further optimized.

The serotonin transporter (SERT) regulates neurotransmission by the biogenic monoamine neurotransmitter serotonin (5-HT, 5-hydroxytryptamine) in the central nervous system, and drugs inhibiting SERT are widely used for the treatment of a variety of central nervous system diseases. The conformational dynamics of SERT transport function and inhibition is currently poorly understood. We used voltage-clamp fluorometry to study conformational changes in human SERT (hSERT) during 5-HT transport and inhibitor binding. Cys residues were introduced at 12 positions in hSERT to enable covalent attachment of a rhodamine-based fluorophore. Transport-associated changes in fluorescence from fluorophore-labeled hSERT expressed in Xenopus oocytes could be robustly detected at four positions in hSERT: endogenous Cys109 in the top of transmembrane domain (TM) 1b, Cys substituted for Thr323 in the top of TM6, Ala419 in the interface between TM8 and extracellular loop (EL) 4, and Leu481 in EL5. The reporter positions were used for time-resolved measurement of conformational changes during 5-HT transport and binding of cocaine and the selective serotonin reuptake inhibitors fluoxetine and escitalopram. At all reporter positions, fluorescence changes observed upon substrate application were distinctly different from those observed upon inhibitor application, with respect to relative amplitude or direction. Furthermore, escitalopram, fluoxetine, and cocaine induced a very similar pattern of fluorescent changes overall, which included movements within or around TM1b, EL4, and EL5. Taken together, our data lead us to suggest that competitive inhibitors stabilize hSERT in a state that is different from the apo outward-open conformation as well as inward-facing conformations.

Cytosine methylation is a widespread epigenetic regulation mechanism. In healthy mature cells, methylation occurs at CpG dinucleotides within promoters, where it primarily silences gene expression by modifying the binding affinity of transcription factors to the promoters. Conversely, a recent study showed that in stem cells and cancer cell precursors, methylation also occurs at non-CpG pairs and involves introns and even gene bodies. The epigenetic role of such methylations and the molecular mechanisms by which they induce gene regulation remain elusive. The topology of both physiological and aberrant non-CpG methylation patterns still has to be detailed and could be revealed by using the differential stability of the duplexes formed between site-specific oligonucleotide probes and the corresponding methylated regions of genomic DNA. Here, we present a systematic study of the thermal stability of a DNA oligonucleotide sequence as a function of the number and position of non-CpG methylation sites. The melting temperatures were determined by monitoring the fluorescence of donor-acceptor duallabelled oligonucleotides at various temperatures. An empirical model that estimates the methylationinduced variations in the standard values of hybridization entropy and enthalpy was developed.

A Master of Science thesis in Chemical Engineering by Salma Elgaili Ahmed entitled, "Dynamics of Chemotherapeutic Drug Release from Liposomes Using Low-Frequency Ultrasound," submitted in January 2015. Thesis advisor is Dr. Ghaleb Husseini. Available in both soft and hard copy., This thesis focuses on the study of liposomes; nanoparticles that can act as anticancer drug carriers, in combination with ultrasound applied as a drug trigger, with the objective of reducing the side effects caused by traditional chemotherapy. For the purpose of this thesis, PEGylated (containing poly-ethylene glycol) and non-PEGylated liposomes were used to encapsulate calcein and then release this model drug when 20- and 40-kHz ultrasound were applied at different acoustic power densities. Fluorescence techniques were used to measure the percent calcein release from these liposomes at different pH values (5.2 and 7.4). The release experiments were done offline (discontinuous) and also online (continuous measurements), where the latter was conducted to allow the modeling of the results. The size of the synthesized liposomes was determined by Dynamic Light Scattering, and found to be similar for both types of liposomes. The release results obtained offline showed that the release from non-PEGylated liposomes is higher at pH 7.4 than at pH 5.2, for all used power intensities. The release from PEGylated liposomes is similar at both pH's, except for the highest power intensity, where the release at pH 7.4 is significantly higher. Comparing the release from non-PEGylated and PEGylated liposomes at the same pH, it was observed that, at pH 5.2, there are no significant differences, but at pH 7.4 the release from non-PEGylated liposomes seems to be significantly higher. However, the results obtained from the online experiments showed no significant differences between both types of liposomes at pH 7.4. Finally, models of the release from non-PEGylated and PEGylated liposomes were built using artificial neural network (ANN)., College of Engineering, Department of Chemical Engineering

Improved tools for providing specific intraoperative diagnoses could improve patient care. In neurosurgery, intraoperatively differentiating non-operative lesions such as CNS B-cell lymphoma from operative lesions can be challenging, often necessitating immunohistochemical (IHC) procedures which require up to 24-48 hours. Here, we evaluate the feasibility of generating rapid ex vivo specific labeling using a novel lymphoma-specific fluorescent switchable aptamer. Our B-cell lymphoma-specific switchable aptamer produced only low-level fluorescence in its unbound conformation and generated an 8-fold increase in fluorescence once bound to its target on CD20-positive lymphoma cells. The aptamer demonstrated strong binding to B-cell lymphoma cells within 15 minutes of incubation as observed by flow cytometry. We applied the switchable aptamer to ex vivo xenograft tissue harboring B-cell lymphoma and astrocytoma, and within one hour specific visual identification of lymphoma was routinely possible. In this proof-of-concept study in human cell culture and orthotopic xenografts, we conclude that a fluorescent switchable aptamer can provide rapid and specific labeling of B-cell lymphoma, and that developing aptamer-based labeling approaches could simplify tissue staining and drastically reduce time to histopathological diagnoses compared with IHC-based methods. We propose that switchable aptamers could enhance expeditious, accurate intraoperative decision-making.

Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye.

Excitation-evoked Ca(2+) influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca(2+) entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I-IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of CaV1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in CaV1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.

Three screening methods-visual scoring (V), relative conductivity (C) and fluorometry (F)-were used to study the genetic variation in cold hardiness among six populations of maritime pine (Pinus pinaster Ait.) comprising both Atlantic and Mediterranean origins. Freezing damage assessments were carried out in three organs-needles, stems and buds-in two seasons, spring and autumn. We found high levels of genetic differentiation among populations for cold hardiness in autumn, but not in spring. Within populations, differences were always significant (p < 0.05) no matter which organ or screening method was used. Measuring F was the fastest and most easily replicated method to estimate cold hardiness and was as reliable as V and C for predicting the species performance. In autumn, there was a positive correlation between the damage measured in all three types of organs assessed, whereas in spring, correlation among organs was weak. We conclude that sampling date in spring has a crucial impact to detect genetic differences in maritime pine populations, whereas autumn sampling allows more stable comparisons. We also conclude that the fluorometry method provides a more efficient and stable comparison of cold hardiness in maritime pine. © 2014 Springer-Verlag Berlin Heidelberg.

PurposeTo determine whether daily irrigation with sterile saline solution during silicone hydrogel (SiH) contact lens continuous wear (CW) can mitigate increases in corneal epithelial permeability (Pdc) and reduce the risk of adverse events.MethodsIn this study, 161 non-contact lens wearers were fit with SiH contact lenses and randomized to either a treatment (n = 81) or control (n = 80) group for 30-day CW. Subjects in the treatment group irrigated every morning and whenever dryness symptoms occurred; subjects in the control group did not. Mixed effects models were employed to assess the changes in Pdc, and survival analysis methods were employed to estimate the risk for adverse events.ResultsEpithelial permeability increased over 30-day CW with SiH lenses (P = 0.001). Risk of inflammatory (odds ratio [OR] = 1.94) and lens-induced (OR = 1.90) adverse events increased with irrigation; these events also occurred sooner, on average, in the irrigation group. Although the overall risk for Asians was higher than for non-Asians, irrigation had no significant impact on risk among Asians, but significantly increased risk among non-Asians.ConclusionsThirty-day CW results in a measurable increase in epithelial permeability. Risk of inflammatory and lens-induced adverse events also increase over time during CW. Daily irrigation with sterile saline solution did not mitigate increases in epithelial permeability or decrease risk of adverse events.

PurposeTo investigate factors of ethnicity, sex, age, and diurnal variation on human corneal epithelial permeability.MethodsData of corneal epithelial permeability to sodium fluorescein (Pdc) were collected from 374 noncontact lens wearers at various times after awakening throughout the day. Mixed-effect models were developed to investigate the association between Pdc and factors of interest, including time awake (TA), age, sex, ethnicity, and interactions of these factors.ResultsTwo models evaluated the "Pdc recovery period" from awakening to 2 hours (Model 1) and the "Pdc plateau" period after TA of 2 hours (Model 2). In Model 1, Pdc declined significantly with length of awake time (P = 0.000), and showed higher Pdc with males (P = 0.098), although this sex difference was not observed after 2 hours (Model 2). Both models showed significantly higher Pdc in Asians than in non-Asians (P = 0.000) and increased Pdc with age (P = 0.048, P = 0.001).ConclusionsBaseline corneal epithelial barrier function increases after overnight sleep and varies significantly by ethnicity and age.

Current cell-based assays for determining the functional properties of high-density lipoproteins (HDL) have limitations. We report here the development of a new, robust fluorometric cell-free biochemical assay that measures HDL lipid peroxidation (HDLox) based on the oxidation of the fluorochrome Amplex Red. HDLox correlated with previously validated cell-based (r = 0.47, p<0.001) and cell-free assays (r = 0.46, p<0.001). HDLox distinguished dysfunctional HDL in established animal models of atherosclerosis and Human Immunodeficiency Virus (HIV) patients. Using an immunoaffinity method for capturing HDL, we demonstrate the utility of this novel assay for measuring HDLox in a high throughput format. Furthermore, HDLox correlated significantly with measures of cardiovascular diseases including carotid intima media thickness (r = 0.35, p<0.01) and subendocardial viability ratio (r = -0.21, p = 0.05) and physiological parameters such as metabolic and anthropometric parameters (p<0.05). In conclusion, we report the development of a new fluorometric method that offers a reproducible and rapid means for determining HDL function/quality that is suitable for high throughput implementation.

Excitation-evoked Ca(2+) influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca(2+) entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I-IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of CaV1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in CaV1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.

The Special Monitoring of Advanced Response Technologies (SMART) Protocol was developed to oversee oil dispersant application and in-situ burning during response incidents. The USCG Research and Development Center examined the use of the SMART Protocol at four major response incidents between 1999 and 2010 to identify future improvements in the areas of policy, training, and new technology. The review focused on four incidents: * M/V New Carissa Grounding, 1999 (in-situ burning) * Eugene Island Pipeline Spill, 2009 (dispersant) * T/V Krymsk Oil Spill, 2009 (dispersant) * Deep Water Horizon Blowout, 2010 (dispersant and in-situ burning) We evaluated the effectiveness of the SMART Protocol through interviews with stakeholders and other subject matter experts. Where possible, we interviewed the Federal On-Scene Coordinators and NOAA Scientific Support Coordinators who led the responses, and other parties involved in the responses. We also reviewed agency reports, and discussed equipment use and performance with SMART team personnel and vendors. This review examines SMART team activities, incident commander assessments of the protocol's effectiveness, identifies SMART Protocol capability gaps, and identifies solutions for resolving those gaps. The report recommends modifications to USCG policies, training, and technology to improve the SMART Program's future performance., The original document contains color images. Prepared in collaboration with SAIC, Mystic, CT, and Parscal Pacific, LLC, Atascadero, CA. Sponsored in part by the U.S. Department of the Interior, Bureau of Safety and Environmental Enforcement through Interagency Agreement E13PG00040 .

Current cell-based assays for determining the functional properties of high-density lipoproteins (HDL) have limitations. We report here the development of a new, robust fluorometric cell-free biochemical assay that measures HDL lipid peroxidation (HDLox) based on the oxidation of the fluorochrome Amplex Red. HDLox correlated with previously validated cell-based (r = 0.47, p<0.001) and cell-free assays (r = 0.46, p<0.001). HDLox distinguished dysfunctional HDL in established animal models of atherosclerosis and Human Immunodeficiency Virus (HIV) patients. Using an immunoaffinity method for capturing HDL, we demonstrate the utility of this novel assay for measuring HDLox in a high throughput format. Furthermore, HDLox correlated significantly with measures of cardiovascular diseases including carotid intima media thickness (r = 0.35, p<0.01) and subendocardial viability ratio (r = -0.21, p = 0.05) and physiological parameters such as metabolic and anthropometric parameters (p<0.05). In conclusion, we report the development of a new fluorometric method that offers a reproducible and rapid means for determining HDL function/quality that is suitable for high throughput implementation.

PurposeTo determine whether daily irrigation with sterile saline solution during silicone hydrogel (SiH) contact lens continuous wear (CW) can mitigate increases in corneal epithelial permeability (Pdc) and reduce the risk of adverse events.MethodsIn this study, 161 non-contact lens wearers were fit with SiH contact lenses and randomized to either a treatment (n = 81) or control (n = 80) group for 30-day CW. Subjects in the treatment group irrigated every morning and whenever dryness symptoms occurred; subjects in the control group did not. Mixed effects models were employed to assess the changes in Pdc, and survival analysis methods were employed to estimate the risk for adverse events.ResultsEpithelial permeability increased over 30-day CW with SiH lenses (P = 0.001). Risk of inflammatory (odds ratio [OR] = 1.94) and lens-induced (OR = 1.90) adverse events increased with irrigation; these events also occurred sooner, on average, in the irrigation group. Although the overall risk for Asians was higher than for non-Asians, irrigation had no significant impact on risk among Asians, but significantly increased risk among non-Asians.ConclusionsThirty-day CW results in a measurable increase in epithelial permeability. Risk of inflammatory and lens-induced adverse events also increase over time during CW. Daily irrigation with sterile saline solution did not mitigate increases in epithelial permeability or decrease risk of adverse events.

PurposeTo investigate factors of ethnicity, sex, age, and diurnal variation on human corneal epithelial permeability.MethodsData of corneal epithelial permeability to sodium fluorescein (Pdc) were collected from 374 noncontact lens wearers at various times after awakening throughout the day. Mixed-effect models were developed to investigate the association between Pdc and factors of interest, including time awake (TA), age, sex, ethnicity, and interactions of these factors.ResultsTwo models evaluated the "Pdc recovery period" from awakening to 2 hours (Model 1) and the "Pdc plateau" period after TA of 2 hours (Model 2). In Model 1, Pdc declined significantly with length of awake time (P = 0.000), and showed higher Pdc with males (P = 0.098), although this sex difference was not observed after 2 hours (Model 2). Both models showed significantly higher Pdc in Asians than in non-Asians (P = 0.000) and increased Pdc with age (P = 0.048, P = 0.001).ConclusionsBaseline corneal epithelial barrier function increases after overnight sleep and varies significantly by ethnicity and age.

PurposeTo ascertain whether a difference in the permeability of the corneal epithelium to fluorescein (Pdc) exists between Asians and non-Asians.MethodsFrom a multi-study database we extracted 632 records of baseline, open-eye Pdc measurements taken on both eyes of 176 subjects. Subjects were awake for a minimum of 4h before measurement, and were free of ocular disease and central corneal staining. Pdc was transformed by natural logarithm to better approximate normality for statistical tests.ResultsThe mean ln(Pdc) in the Asian group was significantly greater than in the non-Asian group [-2.34 ln(nm/s) vs. -2.58 ln(nm/s); p<0.001].ConclusionsCompared with non-Asians, Asians exhibited a less negative ln(Pdc), which translates to a higher Pdc and a more permeable corneal epithelium. We speculate that this may be related to anatomic differences responsible for greater eyelid tension in Asians.

PurposeTo ascertain whether a difference in the permeability of the corneal epithelium to fluorescein (Pdc) exists between Asians and non-Asians.MethodsFrom a multi-study database we extracted 632 records of baseline, open-eye Pdc measurements taken on both eyes of 176 subjects. Subjects were awake for a minimum of 4h before measurement, and were free of ocular disease and central corneal staining. Pdc was transformed by natural logarithm to better approximate normality for statistical tests.ResultsThe mean ln(Pdc) in the Asian group was significantly greater than in the non-Asian group [-2.34 ln(nm/s) vs. -2.58 ln(nm/s); p<0.001].ConclusionsCompared with non-Asians, Asians exhibited a less negative ln(Pdc), which translates to a higher Pdc and a more permeable corneal epithelium. We speculate that this may be related to anatomic differences responsible for greater eyelid tension in Asians.