Unveiling the Binding between the Armadillo-Repeat Domain of Plakophilin 1 and the Intrinsically Disordered Transcriptional Repressor RYBP.

Plakophilin 1 (PKP1), a member of the p120ctn subfamily of the armadillo (ARM)-repeat-containing proteins, is an important structural component of cell-cell adhesion scaffolds although it can also be ubiquitously found in the cytoplasm and the nucleus. RYBP (RING 1A and YY1 binding protein) is a multifunctional intrinsically disordered protein (IDP) best described as a transcriptional regulator. Both proteins are involved in the development and metastasis of several types of tumors. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with RYBP by using in cellulo methods, namely immunofluorescence (IF) and proximity ligation assay (PLA), and in vitro biophysical techniques, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), and isothermal titration calorimetry (ITC). We also characterized the binding of the two proteins by using in silico experiments. Our results showed that there was binding in tumor and non-tumoral cell lines. Binding in vitro between the two proteins was also monitored and found to occur with a dissociation constant in the low micromolar range (~10 µM). Finally, in silico experiments provided additional information on the possible structure of the binding complex, especially on the binding ARM-PKP1 hot-spot. Our findings suggest that RYBP might be a rescuer of the high expression of PKP1 in tumors, where it could decrease the epithelial-mesenchymal transition in some cancer cells.

The intrinsically disordered, epigenetic factor RYBP binds to the citrullinating enzyme PADI4 in cancer cells.

RYBP (Ring1 and YY 1 binding protein) is a multifunctional, intrinsically disordered protein (IDP), best described as a transcriptional regulator. It exhibits a ubiquitin-binding functionality, binds to other transcription factors, and has a key role during embryonic development. RYBP, which folds upon binding to DNA, has a Zn-finger domain at its N-terminal region. By contrast, PADI4 is a well-folded protein and it is one the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. As both proteins intervene in signaling pathways related to cancer development and are found in the same localizations within the cell, we hypothesized they may interact. We observed their association in the nucleus and cytosol in several cancer cell lines, by using immunofluorescence (IF) and proximity ligation assays (PLAs). Binding also occurred in vitro, as measured by isothermal titration calorimetry (ITC) and fluorescence, with a low micromolar affinity (~1 µM). AlphaFold2-multimer (AF2) results indicate that PADI4's catalytic domain interacts with the Arg53 of RYBP docking into its active site. As RYBP sensitizes cells to PARP (Poly (ADP-ribose) polymerase) inhibitors, we applied them in combination with an enzymatic inhibitor of PADI4 observing a change in cell proliferation, and the hampering of the interaction of both proteins. This study unveils for the first time the possible citrullination of an IDP, and suggests that this new interaction, whether it involves or not citrullination of RYBP, might have implications in cancer development and progression.

Direct Oral FXa Inhibitors Binding to Human Serum Albumin: Spectroscopic, Calorimetric, and Computational Studies.

Direct FXa inhibitors are an important class of bioactive molecules (rivaroxaban, apixaban, edoxaban, and betrixaban) applied for thromboprophylaxis in diverse cardiovascular pathologies. The interaction of active compounds with human serum albumin (HSA), the most abundant protein in blood plasma, is a key research area and provides crucial information about drugs' pharmacokinetics and pharmacodynamic properties. This research focuses on the study of the interactions between HSA and four commercially available direct oral FXa inhibitors, applying methodologies including steady-state and time-resolved fluorescence, isothermal titration calorimetry (ITC), and molecular dynamics. The HSA complexation of FXa inhibitors was found to occur via static quenching, and the complex formation in the ground states affects the fluorescence of HSA, with a moderate binding constant of 104 M-1. However, the ITC studies reported significantly different binding constants (103 M-1) compared with the results obtained through spectrophotometric methods. The suspected binding mode is supported by molecular dynamics simulations, where the predominant interactions were hydrogen bonds and hydrophobic interactions (mainly π-π stacking interactions between the phenyl ring of FXa inhibitors and the indole moiety of Trp214). Finally, the possible implications of the obtained results regarding pathologies such as hypoalbuminemia are briefly discussed.

Mercury ions impact the kinetic and thermal stabilities of human lens γ-crystallins via direct metal-protein interactions.

Loss of metal homeostasis may be involved in several age-related diseases, such as cataracts. Cataracts are caused by the aggregation of lens proteins into light-scattering high molecular weight complexes that impair vision. Environmental exposure to heavy metals, such as mercury, is a risk factor for cataract development. Indeed, mercury ions induce the non-amyloid aggregation of human γC- and γS crystallins, while human γD-crystallin is not sensitive to this metal. Using Differential Scanning Calorimetry (DSC), we evaluate the impact of mercury ions on the kinetic stability of the three most abundant human γ-crystallins. The metal/crystallin interactions were characterized using Isothermal Titration Calorimetry (ITC). Human γD-crystallins exhibited kinetic stabilization due to the presence of mercury ions, despite its thermal stability being decreased. In contrast, human γC- and γS-crystallins are both, thermally and kinetically destabilized by this metal, consistent with their sensitivity to mercury-induced aggregation. The interaction of human γ-crystallins with mercury ions is highly exothermic and complex, since the protein interacts with the metal at more than three sites. The isolated domains of human γ-D and its variant with the H22Q mutation were also studied, revealing the importance of these regions in the mercury-induced stabilization by a direct metal-protein interaction.

Relationship between level CPAP titration, anthropometric variables, and drug-induced sleep endoscopy DISE.

INTRODUCTION: Subjects with palatal obstruction alone vs. multilevel obstruction on DISE had better outcomes after palate surgery. We asked ourselves if the therapeutic level positive airway pressure (PAP) titration could predict the level of airway obstruction and its complexity. PURPOSE: The aim of this study was to identify possible relationships between therapeutic level of positive airway pressure initial titration and levels of collapse in drug-induced sleep endoscopy (DISE). A secondary objective was to establish the relationship the other variables and DISE. METHODS: We analyzed retrospective clinical histories between March 2020 to March 2022 of 37 patients with polysomnography or cardiorespiratory polygraphy studies and PAP initial titration who were taken to drug-induced sleep endoscopy. Sleep study data, anthropometric variables, and patterns of airway collapse during DISE were analyzed with PAP initial titration levels. RESULTS: Most of the patients with complex collapse had concentric velum collapse (p < 0.006). A significant association was found between the apnea-hypopnea index (AHI) and oropharyngeal collapse; (p < 0.0030) and finally we demonstrated relationship between neck circumference and gender with epiglottis collapse (p < 0.046), (p < 0.037), respectively. CONCLUSIONS: Our findings show a strong relationship between that complex collapses and concentric velum collapse; patients with greater oropharyngeal collapse have a higher mean AHI. Patients without epiglottic collapse have a higher mean neck circumference. An association between mean pressure initial titration and complex collapse could not be established.

Transpyloric Feeding is Associated With Improved Oxygenation Compared With Gastric Feeding Among Nonintubated Extremely Low Birth Weight Infants.

OBJECTIVE: To study the impact of transpyloric (TP) feed initiation on short-term oxygenation and manual oxygen blender titration among extremely low birth weight infants. STUDY DESIGN: This retrospective study evaluated several measures of oxygenation among extremely low birth weight infants receiving positive pressure respiratory support for 96 hours before and after TP tube placement in a single neonatal intensive care unit during the years 2017- 2020. The measures included the achieved oxygen saturation (SpO2), the baseline fraction of inspired oxygen (FiO2), the SpO2/FiO2 ratio, the number and severity of hypoxemic episodes and the frequency of manual oxygen titrations (titration index) and were analyzed using an interrupted time series regression approach. RESULTS: A total of 56 infants were evaluated. No significant differences were observed in any oxygenation measures during TP vs gastric feeding among 14 intubated infants. However, among 42 nonintubated patients, significant improvements were observed in the median SpO2/FiO2 ratios (P = .001), median titration index (P = .05), median number of hypoxemic episodes (P = .02), and median severity of hypoxemic episodes (P = .008) after TP tube placement. CONCLUSIONS: The transition from gastric to TP tube feeding was temporally associated with acute improvement in oxygenation for nonintubated infants, but not for intubated infants.

Clinical and polysomnographic predictors of suboptimal auto-adjusting CPAP titration in adult OSA patients: a single-center study.

PURPOSE: To examine potential clinical, demographic, anthropometric, and polysomnographic predictors of successful auto-adjusting continuous positive airway pressure (CPAP) titration for treatment of obstructive sleep apnea (OSA). METHODS: This cross-sectional study was conducted in adults diagnosed with moderate-to-severe OSA (baseline apnea-hypopnea index [AHI] ≥ 15.0/h), who underwent auto-adjusting CPAP titration (S9 or S10 AutoSet ResMed®) in a sleep laboratory setting while wearing a nasal or pillow mask. Participants were then grouped into two groups: optimal CPAP titration (residual AHI < 5.0/h) or suboptimal CPAP titration (residual AHI ≥ 5.0/h). Multivariate logistic regression analysis was used to assess possible independent predictive factors for suboptimal CPAP titration. RESULTS: A total of 1222 adults consisting of 874 subjects with optimal CPAP titration (71.5%) and 348 subjects with suboptimal CPAP titration (28.5%) were evaluated. Multivariate analysis resulted in a model with an adequate calibration (Hosmer-Lemeshow chi-square-test: 7.088; p = 0.527), with male sex, higher values of baseline AHI, therapeutic pressure (95th percentile), and mask leak (95th percentile) emerging as significant and independent predictors for suboptimal CPAP titration: adjusted odds ratio (OR): 1.456 (95% confidence interval [CI] 1.076-1.971; p = 0.015), OR: 1.009 (95% CI 1.002-1.016; p = 0.013), OR: 1.281 (95% CI 1.206-1.361; p < 0.001), and 1.035 (1.026-1.043; p < 0.001), respectively. CONCLUSIONS: In a large cohort of adults undergoing auto-adjusting CPAP titration due to moderate-to-severe OSA, male sex, increased values of baseline AHI, pressure requirements, and mask leak were significant predictors for less than optimal CPAP titration.

Acid-base potentiometric titration using a stainless steel electrode without oxidative treatment.

An AISI 304 stainless steel laminar electrode without oxidative treatment was investigated for the potentiometric titration of hydrochloric acid with sodium hydroxide. The proposed electrode was obtained from metalworking cuttings. Scanning electron microscopy, energy dispersive X-ray spectroscopy, X-ray fluorescence spectroscopy and X-ray diffraction were used to study the surface morphology and chemical composition of the electrode. The electrode showed a sensitivity of 59.18 ± 0.37 mV/pH, which was reproducible under intermediate conditions. Potentiometric titration showed a curve with deviations from pH 9.5 with respect to the glass electrode. However, this did not affect the quantification as the jumps of the curves coincided. The endpoint was 9.25 mL for both electrodes and the hydrochloric acid concentration was 0.0845 mol/L, with a deviation of 0.0004 mol/L from the standard concentration of 0.0841 mol/L. The nonartificially oxidised electrode did not show any crystalline oxide phases, whereas after oxidation it showed semicrystalline phases of iron and chromium oxides and increased the crystallinity of the steel. Despite the low content of surface oxides, stainless steel electrodes can give a Nernstian response to pH, depending on the surface characteristics of the material. This leads to the need to calibrate any electrode prior to oxidative treatment to rule out a Nernstian response without surface modification.

Determination of Water Content in Direct Resin Composites Using Coulometric Karl Fischer Titration.

This study evaluated the water content and sorption of direct composites over 60 days using coulometric Karl Fischer titration (KFT). Plate-shaped specimens (10 × 10 × 1 mm3 of thickness) were built up using the composites Clearfil Majesty Posterior (CM), Grandio SO (GS), and Filtek Supreme XT (FS). Water contents were determined in non-stored specimens (control) or after storage in distilled water for up to 60 days (n = 5). The amount of water transferred from the specimens heated at 200 °C (isothermal mode) was measured in the Coulometer. The water content of non-stored specimens ranged from 0.28 to 1.69 wt% (5.6 to 31.2 µg/mm3) for GS and FS, respectively. The highest values of water sorption were observed for FS (25.3 µg/mm3 after 60 days). GS and CM showed similar water sorption after 60 days (≈9 µg/mm3), but an ultimate higher water content was observed for CM (0.9 wt%; 22.0 µg/mm3) than GS (0.7 wt%; 14.8 µg/mm3). Except for CM, no significant water sorption was observed between 21 and 60 days of storage. Since all composites presented some base water content, water sorption data alone do not account for the ultimate water content in direct resin-based composites.

Comparing CPAP masks during initial titration for Obstructive Sleep Apnea Syndrome: one-year experience

Abstract Objective: Continuous Positive Airway Pressure (CPAP) is the gold standard treatment for OSA. A wide variety of masks are used during CPAP therapy. The aim of the study is to compare polysomnography outcomes during titration for different types of masks (oronasal, nasal and pillow) and assess the impact on PAP titration. Methods: We retrospectively analyzed all CPAP titration polysomnography for one year. Anthropometric data and baseline apnea severity were documented. Results: A total of 497 patients were evaluated. Nasal masks were used in 82.3% (n = 409), pillow in 14.1% (n = 70) and oronasal in 3.6% (n = 18). There was no difference according to body mass index and baseline apnea-hypopnea index among the groups. The oronasal group was older and had higher titrated CPAP pressure, higher residual apnea-hypopnea index, lower sleep efficiency, more superficial N1 sleep and longer wake time after sleep onset. All other polysomnography outcomes were similar among the groups. The mean CPAP level was 11.6 ± 2.1 cm H2O for the oronasal mask, 10.1 ± 2.1 cm H2O for the nasal mask and 9.8 ± 2.2 cm H2O for the pillow. The residual apnea-hypopnea index was 10.4 ± 7.9 for the oronasal mask, 5.49 ± 5.34 events/h for the nasal mask and 4.98 ± 5.48 events/h for the pillow. The baseline apnea-hypopnea index was correlated with of a higher CPAP pressure for all the groups (p< 0.001 for the nasal group, p = 0.001 for the pillow group and p = 0.049 for the oronasal group). Body mass index and residual AHI were correlated with of a higher CPAP pressure for the nasal and pillow groups only (p < 0.001). Conclusion: The interface can have a significant impact on the effectiveness of PAP titration. Patients with oronasal masks have higher CPAP pressure, higher residual apnea-hypopnea index, lower sleep efficiency and higher wake time after sleep onset. Oronasal masks should not be recommended as the first choice for apnea patients. Level of evidence: Level 3.

Oxygenation Factors Associated with Retinopathy of Prematurity in Infants of Extremely Low Birth Weight.

OBJECTIVE: To study characteristics of oxygenation during the first 2 postnatal months and correlation with the occurrence and severity of retinopathy of prematurity (ROP) among infants of extremely low birth weight. STUDY DESIGN: This retrospective study analyzed the incidence and severity of hyperoxemia and hypoxemia while on respiratory support with or without supplemental oxygen among infants of extremely low birth weight (birth weight <1000 g) admitted to the neonatal intensive care unit during 2016-2020. The findings were correlated with the occurrence and severity of ROP after adjusting for baseline covariates. RESULTS: After adjusting for differences in baseline demographic and clinical features, the group with severe ROP was exposed to greater fraction of inspired oxygen (FiO2) (P = .001) and experienced more frequent FiO2 titration adjustments (P = .001) compared with the group without ROP. Ambient air hyperoxemia occurred more frequently in the group without ROP (P = .003), and iatrogenic hyperoxemia occurred more frequently in the group with severe ROP (P = .046). There were no differences in the severity of ambient and iatrogenic hyperoxemia in the study population. The group with severe ROP demonstrated more hypoxemic episodes (P = .01) and longer time spent in the severe hypoxemic range (P = .005) compared with the group without ROP. CONCLUSIONS: Severe ROP is associated with greater FiO2 exposure, increased iatrogenic hyperoxemia, decreased ambient air hyperoxemia, and increased hypoxemia in infants of extremely low birth weight despite a greater frequency of FiO2 titration. This study illustrates the need for automated closed loop FiO2 delivery systems to further optimize oxygen saturation targeting in this high-risk population.

Suplemento alimentar de vitamina C: fiscalização durante o período de adequação à nova legislação / Vitamin C dietary supplement: inspection during adequacy period of new legislation

Os suplementos à base de vitamina C são amplamente consumidos pela população, evidenciando a importância da fiscalização desses produtos. Em julho de 2018, a ANVISA (Agência Nacional de Vigilância Sanitária) publicou as novas regulamentações sobre suplementos alimentares, estabelecendo-se o prazo de 60 meses para as empresas se adequarem a essas normas. O presente trabalho teve como objetivo determinar os teores de vitamina C, comparar os valores analisados com os declarados na informação nutricional e avaliar os dizeres de rotulagem em produtos à base dessa vitamina. Os teores de vitamina C foram determinados por titulação potencio métrica e as análises de rotulagem foram realizadas com base nas legislações da ANVISA em doze amostras de suplementos colhidos pelas Vigilâncias Sanitárias do Estado de São Paulo. Os teores de vitamina C avaliados estavam de acordo com os declarados na informação nutricional do rótulo, com exceção de uma amostra que apresentou teor abaixo do valor declarado. Em relação aos dizeres de rotulagem, sete amostras (58%) apresentaram um ou mais itens em desacordo com a legislação, mostrando a necessidade de monitoramento constante desse tipo de produto. O trabalho representa um estudo preliminar de avaliação dos suplementos no período de adequação às novas regulamentações (AU).

Vitamin C-based supplements are widely consumed by the population, highlighting the importance of monitoring these products. In July 2018, the ANVISA (National Health Surveillance Agency) published new regulations for dietary supplements,setting a 60 months deadline for companies to comply with these standards.The objective of the present work was to evaluate the contents of vitamin C, and compare the analyzed/real values with those reported on the nutrition facts label, and to evaluate the labeling of vitamin supplements. Vitamin C contents were determined by potentiometric titration, and labeling analysis were performed based on ANVISA legislation in twelve samples of supplements collected by the Sanitary Surveillance of the State of São Paulo.The contents of vitamin C were in accordance with those declared in the nutritional information on the label, with the exception of one sample that presented content below the declared value. Regarding the labeling analysis, seven samples (58%) presented one or more items in disagreement with the legislation, showing the need for constant monitoring of this type of product.The work represents a preliminary study to evaluate the supplements in the adequacy period of new regulations (AU).

Exploration of interaction between angiotensin I-converting enzyme (ACE) and the inhibitory peptide from Wakame (Undaria pinnatifida).

The interaction between angiotensin I-converting enzyme (ACE) and the inhibitory peptide KNFL from Wakame was explored using isothermal titration calorimetry, multiple spectroscopic techniques and molecular dynamics simulations, and an inhibition model was established based on free energy binding theory. The experiments revealed that the binding of KNFL to ACE was a spontaneous exothermic process driven by enthalpy and entropy and occurred via multiple binding sites to form stable complexes. The complexes may be formed through multiple steps of inducing fit and conformational selection. The peptide KNFL had a fluorescence quenching effect on ACE and its addition not only affected the microenvironment around the ACE Trp and Tyr residues, but also increased the diameter and altered the conformation of ACE. This study should prove useful for improving our understanding of the mechanism of ACE inhibitory peptides.

Ocorrência de sorogrupos/sorovares de Leptospira spp. em cães com suspeita clínica de leptospirose em Santa Maria ­ RS / Occurrence of serogroups/serovars of Leptospira spp. in dogs with clinical suspicion of leptospirosis in Santa Maria ­ RS

A leptospirose é uma zoonose de distribuição mundial que pode acometer cães e ser altamente letal para a espécie. No Brasil, tal enfermidade tem caráter endêmico na espécie canina e configura-se como um sério problema de saúde pública. Objetivou-se verificar a ocorrência dos sorogrupos/sorovares de Leptospira spp. que mais acometem cães com suspeita clínica de leptospirose na cidade de Santa Maria ­ RS, Brasil, analisando sua titulação de anticorpos. No estudo, utilizou-se os laudos dos exames de soroaglutinação microscópica (SAM) para leptospirose de 218 cães provenientes da cidade de Santa Maria ­ RS durante o período de janeiro de 2015 a dezembro de 2019. Todos os laudos foram emitidos pelo Laboratório de Leptospirose (LabLepto - UFSM). Das 218 amostras de soro processadas nos cinco anos, 101 (46,33%) resultaram positivas para, pelo menos, um sorogrupo/sorovar testado. Este estudo demonstrou maior ocorrência do sorogrupo Icterohaemorrhagiae (53,37%) contemplado pelas sorovares Copenhageni e Icterohaemorrhagiae, com 49 (30,06%) e 38 (23,31%) soros reagentes, respectivamente. As titulações variaram de 100 a 6400, sendo que a mais recorrente encontrada nas amostras deste estudo foi 100, representando 47,85% dos títulos de anticorpos. Os dados obtidos neste estudo são de grande valia para o conhecimento dos sorogrupos/sorovares circulantes na região e sua epidemiologia podendo, inclusive, auxiliar para futuras formulações vacinais considerando sorogrupos/sorovares mais frequentemente detectados, contribuindo, assim, com a saúde pública.

Leptospirosis is a worldwide distribution zoonosis that can affect dogs and be highly lethal for the species. In Brazil, this disease is endemic in the canine species and represents a serious public health problem. The aim of this study was to verify the occurrence of Leptospira spp. that most affect dogs with clinical suspicion of leptospirosis in the city of Santa Maria ­ RS, Brazil, analyzing their antibody titers. In the study, we used the reports of microscopic agglutination tests (MAS) for leptospirosis in 218 dogs from the city of Santa Maria - RS during the period from January 2015 to December 2019. All reports were issued by the Leptospirosis Laboratory (LabLepto - UFSM). Of the 218 serum samples processed over the five years, 101 (46.33%) were positive for at least one serogroup/serovar tested. This study showed a higher occurrence of serogroup Icterohaemorrhagiae (53.37%) covered by serovars Copenhageni and Icterohaemorrhagiae, with 49 (30.06%) and 38 (23.31%) reagent sera, respectively. The titers ranged from 100 to 6400, and the most recurrent found in the samples in this study was 100, representing 47.85% of the antibody titers. The data obtained in this study are of great value for understanding the serogroups/serovars circulating in the region and their epidemiology, and may even contribute to future vaccine formulations considering the most frequently detected serogroups/serovars, thus contributing to public health.

Batch and continuous adsorption of Cu(II) and Zn(II) ions from aqueous solution on bi-functionalized sugarcane-based biosorbent.

A new one-pot synthesis method optimized by a 23 experimental design was developed to prepare a biosorbent, sugarcane bagasse cellulose succinate pyromellitate (SBSPy), for the removal of Cu(II) and Zn(II) from single-component aqueous solutions, in batch and continuous modes. The bi-functionalization of the biosorbent with ligands of different chemical structures increased its selectivity, improving its performance for removing pollutants from contaminated water. The succinate moiety favored Cu(II) adsorption, while the pyromellitate moiety favored Zn(II) adsorption. Sugarcane bagasse (SB) and SBSPy were characterized using several techniques. Analysis by 13C Multi-CP SS NMR and FTIR revealed the best order of addition of each anhydride that maximized the chemical modification of SB. The maximum adsorption capacities of SBSPy for Cu(II) and Zn(II), in batch mode, were 1.19 and 0.95 mmol g-1, respectively. Homogeneous surface diffusion, intraparticle diffusion, and Boyd models were used to determine the steps involved in the adsorption process. Isothermal titration calorimetry was used to assess changes in enthalpy of adsorption as a function of SBSPy surface coverage. Fixed-bed column adsorption of Cu(II) and Zn(II) was performed in three cycles, showing that SBSPy has potential to be used in water treatment. Breakthrough curves were well fitted by the Thomas and Bohart-Adams models.

Virus Propagation and Plaque Assay for Dengue Virus.

This chapter describes the methods of propagation and titration for DENV-1 to -4, which are required for most of the experiments using live viruses. DENV does not reach titers as high as those of other viruses or as high as desired for their use in biological assays. Although DENV grows in many different cell lines derived from both vertebrate and invertebrate cells, the most common cell lines used for virus isolation and propagation are mosquito cells C6/36 from Aedes albopictus. Amongst several methods for virus quantification, plaque assay stands out as being very practical and inexpensive. This technique is carried out essentially to estimate virion density in a particular material, being extremely important when evaluating the effect of an antiviral treatment or antibody neutralization capacity, for example. In this assay, viral particles are serially diluted and added onto confluent cell monolayers immersed in a semisolid medium, which is responsible for limiting virus spread throughout the culture. Therefore, regarding the medium consistency, once a virion successfully infects a cell, the newly produced particles can only infect neighboring cells, eventually leading them to death. This phenomenon can be observed as round gaps or plaques in the culture after staining live cells with a crystal violet solution. Then, plaques are counted and used to determine plaque-forming units per milliliter. Here, we describe a protocol established by our group for dengue virus (DENV) titration with porcine kidney (PS) cells.

Mathematical Description of the Enzymatic Activity of Proteins with Ionizable Groups Exhibiting Deviations from the Henderson-Hasselbalch Equation.

The ionization equilibrium implied in the calculation of the specific activity is classically described through the Henderson-Hasselbalch equation. An extension for the description of anomalous ionization profiles using the Hill equation is presented in this communication. The proposed framework was applied to the description of the specific enzymatic activity curve as a function of pH of five enzymes presenting different ionization states in their active site. The developed equation improves the description of relative enzymatic curves that deviate from the bell curve predicted by the application of the Henderson-Hasselbalch equation, regardless of the ionization scheme related to the active site.

Comparing CPAP masks during initial titration for Obstructive Sleep Apnea Syndrome: one-year experience.

OBJECTIVE: Continuous Positive Airway Pressure (CPAP) is the gold standard treatment for OSA. A wide variety of masks are used during CPAP therapy. The aim of the study is to compare polysomnography outcomes during titration for different types of masks (oronasal, nasal and pillow) and assess the impact on PAP titration. METHODS: We retrospectively analyzed all CPAP titration polysomnography for one year. Anthropometric data and baseline apnea severity were documented. RESULTS: A total of 497 patients were evaluated. Nasal masks were used in 82.3% (n = 409), pillow in 14.1% (n = 70) and oronasal in 3.6% (n = 18). There was no difference according to body mass index and baseline apnea-hypopnea index among the groups. The oronasal group was older and had higher titrated CPAP pressure, higher residual apnea-hypopnea index, lower sleep efficiency, more superficial N1 sleep and longer wake time after sleep onset. All other polysomnography outcomes were similar among the groups. The mean CPAP level was 11.6 ±â€¯2.1 cm H2O for the oronasal mask, 10.1 ±â€¯2.1 cm H2O for the nasal mask and 9.8 ±â€¯2.2 cm H2O for the pillow. The residual apnea-hypopnea index was 10.4 ±â€¯7.9 for the oronasal mask, 5.49 ±â€¯5.34 events/h for the nasal mask and 4.98 ±â€¯5.48 events/h for the pillow. The baseline apnea-hypopnea index was correlated with of a higher CPAP pressure for all the groups (p < 0.001 for the nasal group, p = 0.001 for the pillow group and p = 0.049 for the oronasal group). Body mass index and residual AHI were correlated with of a higher CPAP pressure for the nasal and pillow groups only (p < 0.001). CONCLUSION: The interface can have a significant impact on the effectiveness of PAP titration. Patients with oronasal masks have higher CPAP pressure, higher residual apnea-hypopnea index, lower sleep efficiency and higher wake time after sleep onset. Oronasal masks should not be recommended as the first choice for apnea patients. LEVEL OF EVIDENCE: Level 3.

A method for characterizing the thermal stability and antimicrobial binding to Lipopolysaccharides of Gram-negative isogenic mutant strains.

Isothermal Titration Calorimetry (ITC) is widely employed to assess antimicrobial affinity for lipopolysaccharide (LPS); nevertheless, experiments are usually limited to commercially available-LPS chemotypes. Herein we show a method that uses Differential Scanning Calorimetry (DSC) to characterize homogeneity artificial vesicles of LPS (LPS-V) extracted from isogenic mutant bacterial strains before analyzing the antimicrobial binding by ITC. This method allows us to characterize the differences in the Polymyxin-B binding and gel to crystalline liquid (ß↔α) phase profiles of LPS-V made of LPS extracted from Escherichia coli isogenic mutant strains for the LPS biosynthesis pathway, allowing us to obtain the comparable data required for new antimicrobial discovery. A method for:•Obtaining LPS vesicles from isogenic mutant bacterial strains.•Characterize artificial LPS vesicles homogeneity.•Characterize antimicrobial binding to LPS.