The ALOG domain defines a family of plant-specific transcription factors acting during Arabidopsis flower development.

The ALOG (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 (LSH1) and Oryza G1) proteins are conserved plant-specific Transcription Factors (TFs). They play critical roles in the development of various plant organs (meristems, inflorescences, floral organs, and nodules) from bryophytes to higher flowering plants. Despite the fact that the first members of this family were originally discovered in Arabidopsis, their role in this model plant has remained poorly characterized. Moreover, how these transcriptional regulators work at the molecular level is unknown. Here, we study the redundant function of the ALOG proteins LSH1,3,4 from Arabidopsis. We uncover their role in the repression of bract development and position them within a gene regulatory network controlling this process and involving the floral regulators LEAFY, BLADE-ON-PETIOLE, and PUCHI. Next, using in vitro genome-wide studies, we identified the conserved DNA motif bound by ALOG proteins from evolutionarily distant species (the liverwort Marchantia polymorpha and the flowering plants Arabidopsis, tomato, and rice). Resolution of the crystallographic structure of the ALOG DNA-binding domain in complex with DNA revealed the domain is a four-helix bundle with a disordered NLS and a zinc ribbon insertion between helices 2 and 3. The majority of DNA interactions are mediated by specific contacts made by the third alpha helix and the NLS. Taken together, this work provides the biochemical and structural basis for DNA-binding specificity of an evolutionarily conserved TF family and reveals its role as a key player in Arabidopsis flower development.

Non-Enzymatic Phenylboronic Acid-Based Optode Membrane for Glucose Monitoring in Serums of Diabetic Patients and in the Culture Medium of Human Embryos.

Monitoring glucose levels is important not only for diabetics, but also for tracking embryonic development in human embryo culture media. In this study, an optochemical sensor (glucose-selective polymer membrane) was fabricated for the determination of glucose in serum from diabetic patients and the culture media of human embryos. The optode membranes were formulated using polyvinyl chloride (PVC) as the polymer matrix and 4',5'-dibromofluorescein octadecyl ester (ETH 7075) as the chromoionophore. The sensitivity of the optode membranes was optimized using two different plasticizers (tricresyl phosphate-TCP and nitrophenyloctyl ether-NOPE) and three ionophores (nitrophenylboronic acid-NPBA, trifluorophenyboronic acid-TFPBA, 4'-nitrobenzo-15-crown-5) and tested for glucose detection. The best optode membrane was formulated from 49.5% PVC, 49.5% TCP, 1% NPBA, and 1% ETH 7075. It showed a linear dynamic range of 10-3 M to 10-1 M, with a detection limit of 9 × 10-4 M and a response time of 2 min. The detection mechanism involves H-bonding between NPBA and glucose, which was confirmed by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR). The reaction also involves the formation of boronate esters in basic media with deprotonation of the chromoionophore (ETH 7075), leading to a decrease in UV-Vis absorbance at λmax = 530 nm. The membrane optode was used for glucose determination in synthetic culture medium, commercial embryo culture medium (GLOBAL® TOTAL® W/HEPES), and serum from normal and diabetic patients, showing good accuracy and precision of the optode.

Ionophore-modified polyaniline-based optode for the determination of hydrogen sulfite levels in beverages, wastewater, and soil.

Sulfite is a very important species, affecting human health, plant and animal life, and environmental sustainability. In this study, for the first time, an ionophore-based ion-selective optode was constructed for hydrogen sulfite determination in beverages, such as Birell® and Sprite®, water, and soil samples; instead of normal pH-chromoionophores, polyaniline film was precipitated on a glass slide and used for the transduction of the sensation mechanism. The ionophore-modified polyaniline-based optode incorporated thiourea derivative as an ionophore and tridodecyl methyl ammonium chloride as an ion-exchanger. The optode film was prepared in situ with a modified chemical polymerization method, and it was characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), and X-ray diffraction (XRD); also, FTIR spectroscopy was performed for the film before and after interaction with hydrogen sulfite for mechanism elucidation. The optode was applied in the hydrogen sulfite concentration range of 10-1 to 10-5 M with a low detection limit of 8.0 × 10-6 M and minimum interference of other interfering species, such as salicylate, iodide, and sulphide. The response mechanism was due to the ion-exchange of hydrogen sulfite with the anion exchanger, followed by the molecular recognition between thiourea ionophore and hydrogen sulfite, with concomitant redox reaction via the protonation of the polyaniline that causes a decrease in absorbance at 685 nm. The optode was applied successfully for the determination of hydrogen sulfite in real beverages, Birell® and Sprite® without any pretreatment steps. Also, it was applied successfully for the environmental monitoring of hydrogen sulfite in real wastewater and soil samples.

Single-support serial isomorphous replacement phasing.

The use of single isomorphous replacement (SIR) has become less widespread due to difficulties in sample preparation and the identification of isomorphous native and derivative data sets. Non-isomorphism becomes even more problematic in serial experiments, because it adds natural inter-crystal non-isomorphism to heavy-atom-soaking-induced non-isomorphism. Here, a method that can successfully address these issues (and indeed can benefit from differences in heavy-atom occupancy) and additionally significantly simplifies the SIR experiment is presented. A single heavy-atom soak into a microcrystalline slurry is performed, followed by automated serial data collection of partial data sets. This produces a set of data collections with a gradient of heavy-atom occupancies, which are reflected in differential merging statistics. These differences can be exploited by an optimized genetic algorithm to segregate the pool of data sets into `native' and `derivative' groups, which can then be used to successfully determine phases experimentally by SIR.

Molecularly-imprinted polymer-base bulk optode for the determination of ivabradine hydrochloride in Procoralan®.

IVH may be administered orally twice per day for treating heart failure, sinus rhythm, heart-related chest pain and angina pectoris, and its overdose may cause problems such as blurred vision, uncontrolled blood pressure, prolonged bradycardia, and others. A molecularly imprinted polymer-based bulk optode, miptode, was constructed for the determination of ivabradine hydrochloride (IVH) in its pharmaceutical preparation Procoralan®. The molecularly-imprinted polymers (MIPs) were prepared in different ratios, and MIP3 had the highest imprinting factor (1.6) as an ionophore in the miptode preparation. The miptode was prepared using weight ratios of 30% PVC polymer, 62% nitrophenyl octyl ether (NPOE) plasticizer, 6% MIP3 ionophore, 1% tetraphenyl borate derivative (TPB) ion-exchanger, and 1% ETH7075 chromoionophore; this miptode exhibited an absorbance increase at 530 nm in the concentration range of 10-2-10-5 M with a detection limit of 3.1 µM using Tris-HCl buffer of pH 7.2. The miptode was imaged using AFM, and showed the dissolution of all components except MIP particles which exhibited restricted solubility. However, the incorporation of MIP3 as an ionophore improved the selectivity coefficient over the interfering species that may exist in the pharmaceutical formulation to an extent that was not reported before; e.g. coefficients of IVH over sodium, magnesium, and glucose were improved by 5, 4 and 2 orders, when compared to the previous sensor that operated with the molecular interaction mechanism. The selectivity improvement in miptode is due to the Key-Lock fitting (host-guest molecular recognition) between the MIP particles and the template IVH molecule which is transduced with the ion-exchange process of the chromoionophore. The miptode has a response time of 1-2 minutes, and a reliable lifetime of two months. The miptode was applied successfully for the determination of IVH in the pharmaceutical preparation Procoralan® with recovery values of 89-99.8% with low standard deviations of <1.2.

Low Serum 25-Hydroxy Vitamin D (25-OHD) and Hepatic Encephalopathy in HCV-Related Liver Cirrhosis.

BACKGROUND: Patients with liver cirrhosis experience a large variety of metabolic disorders associated with more hepatic decompensation. Hepatic encephalopathy (HE) is a significant complication in liver cirrhosis patients, presenting a wide spectrum of neuropsychological symptoms. A deficiency of 25-hydroxy vitamin D (25-OHD) in the general population is associated with a loss of cognitive function, dementia, and Alzheimer's disease. Aim of the Study. Our study aims to check the relationship between low serum 25-OHD and HE in patients with HCV-related liver cirrhosis and assess its link with patient mortality. Patients and Methods. This study was observationally carried out on 100 patients with HCV-related liver cirrhosis. The patients were divided into 2 groups: Group A-included 50 HCV-related cirrhotic patients with HE, and Group B-included 50 HCV-related cirrhotic patients without HE. Assessment of disease severity using the end-stage liver disease (MELD) model and Child Turcotte Pugh (CTP) scores were done, and 25-OHD levels were measured. Comparison of vitamin D levels in different etiologies and different CTP categories was made using one-way ANOVA. Pearson's correlation between the level of vitamin D and other biomarkers was applied. RESULTS: There was a statistically significant Vitamin D level difference between the two groups. A lower level of vitamin D was observed in the HE group where the severe deficiency was 16%, while it was 6% in the other group and the moderate deficiency was 24% in HE group as compared to 10% in the other group. The insufficient vitamin D level represented 46% of the non-HE group while none of the HE group falls in this category. Vitamin D level was statistically higher in Grade 1 HE than in Grade 2 which is higher than in Grades 3 to 4. Vitamin D level was also significantly higher in those who improved from HE as compared to those who died. CONCLUSION: The lower levels of 25-OHD were associated with the higher incidence of HE in cirrhotic HCV patients. The worsening vitamin D deficiency was associated with increased severity of the liver disease, so vitamin D may be considered a prognostic factor for the severity of liver cirrhosis and high mortality rate in HE patients.

Evaluation of MicroRNA-122 as a Biomarker for Chronic Hepatitis C Infection and as a Predictor for Treatment Response to Direct-Acting Antivirals.

BACKGROUND: Treatment response to antiviral drugs is a challenging issue in patients with chronic hepatitis C virus (HCV) infection. Although microRNA-122 represents the majority of the microRNA content in hepatic tissues, few studies have evaluated its role in the treatment response, so we aimed to study its role in chronic HCV patients and in predicting the treatment response to direct-acting antivirals (DAAs). METHODS: The study included 125 chronic HCV patients (89 naïve and 36 with a prior failed peginterferon/ribavirin response) and 50 apparently healthy subjects. Complete blood count, liver function, α-fetoprotein, lipid profiles, serum creatinine, abdominal ultrasound, and FibroScan® were assessed. Viral markers, HCV antibodies, and hepatitis B surface antigen were measured by enzyme-linked fluorescent immunoassay, with quantitative estimation of HCV RNA and microRNA-122 levels by real-time PCR. RESULTS: The microRNA-122 level in HCV patients (those with a sustained virologic response 12 weeks after finishing therapy [SVR12] and non-responders) was significantly increased compared with controls and expressed more in non-responders versus SVR12 (p=0.042). ROC curve analysis of microRNA-122 for differentiating HCV patients from healthy controls revealed that a cut-off point of >1.45 had a sensitivity of 67.20%, specificity of 94.0%, AUC=0.861, and p<0.001; and for predicting response to treatment a cut-off point ≤5.66 could significantly (p=0.022) predict the occurrence of SVR, with a sensitivity of 60.34%, specificity of 66.67%, and AUC=0.729. Logistic regression analysis showed significant values for microRNA-122 in multivariate and univariate analysis for the prediction of response to DAAs. CONCLUSION: The results demonstrated the possible function of microRNA-122 as an indicative tool for distinguishing chronic HCV patients from controls and in the assessment of the therapeutic reaction to DAAs.

Molecularly Imprinted Electrochemical Sensor-Based Fe2O3@MWCNTs for Ivabradine Drug Determination in Pharmaceutical Formulation, Serum, and Urine Samples.

Ivabradine hydrochloride (IVR) is a medically important drug because of its ability to lower the heart rate. Techniques reported for IVR determination were expensive, laborious, besides being of poor selectivity. In this study, iron oxide @ carbon nanotube (Fe2O3@MWCNTs) nanocomposite and molecularly imprinted polymer (MIP) were synthesized and used in the fabrication of carbon paste electrodes (CPEs) for the potentiometric detection of IVR in biological and pharmaceutical samples. CPEs of the best sensor were formulated from graphite (41 wt%) as a carbon source, MIP (3 wt.%) as an ionophore, Fe2O3@MWCNTs (5 wt%) as a modifier, and nitrophenyl octyl ether (NPOE, 51 wt.%) as a conductive oil so-called plasticizer. The best sensor exhibits a Nernstian slope (response) of 56 mV decade-1 within the IVR concentration range from 1.0 × 10-3 M to 9.8 × 10-8 M with high selectivity against interfering species (ascorbic, maltose, glucose, lactose, dopamine, glycine) over those reported earlier. The use of Fe2O3@MWCNTs together with MIP in the electrode formulation was found to improve the limit of detection (LOD) from 630 to 98 nM along with high reversibility, a short response time of 30 s, and a good lifetime of more than 2 weeks. The sandwich membrane (SMM) method was used to quantify the H-bonding complexing strength of the MIP binding sites for IVR with Log ß ILn = 11.33. The constructed sensors were successfully applied for the IVR determination in blood serum, urine, and commercial formulations (Savapran®) with high sensitivity.

t-Butyl calixarene/Fe2O3@MWCNTs composite-based potentiometric sensor for determination of ivabradine hydrochloride in pharmaceutical formulations.

Ivabradine hydrochloride (IVB) has shown high medical importance as it is a medication for lowering the heart rate for the symptomatic chronic heart failure and symptomatic management of stable angina pectoralis. The high dose of IVB may cause severe and prolonged bradycardia, uncontrolled blood pressure, headache, and blurred vision. In this study, a highly sensitive carbon-paste electrode (CPEs) was constructed for the potentiometric determination of IVB in pharmaceutical formulations. t-Butyl calixarene (t-BCX) was used as an ionophore due to its ability to mask IVB in the cavity via multiple H-bonding at the lower rim, as estimated quantitatively by the sandwich membrane method (Log ßILn = 8.62). Besides, the use of multi-walled carbon nanotubes decorated with Fe2O3 nanoparticles (Fe2O3@MWCNTs) as an additive for the paste electrode significantly improved the detection limit of the sensor up to 36 nM, with Nernstian response of 58.9 mV decade-1 in the IVB linear dynamic range of 10-3-10-7 M in aqueous solutions. The constructed sensors showed high selectivity against interfering species that may exist in physiological fluids or pharmaceutical formulations (e.g. Na+, K+, NH4+, Ca2+, Mg2+, Ba2+, Fe3+, Co2+, Cr3+, Sr2+, glucose, lactose, maltose, glycine, dopamine, and ascorbic acid). The sensors were successfully employed for IVB determination in the pharmaceutical formulations (Savapran®).

PVC membrane, coated-wire, and carbon-paste ion-selective electrodes for potentiometric determination of galantamine hydrobromide in physiological fluids.

We report on highly-sensitive ion-selective electrodes (ISEs) for potentiometric determining of galantamine hydrobromide (GB) in physiological fluids. Galantamine hydrobromide (GB) was selected for this study due to its previous medical importance for treating Alzheimer's disease. Three different types of ISEs were investigated: PVC membrane electrode (PVCE), carbon-paste electrode (CPE), and coated-wire electrode (CWE). In the construction of these electrodes, galantaminium-reineckate (GR) ion-pair was used as a sensing species for GB in solutions. The modified carbon-paste electrode (MCPE) was prepared using graphene oxide (MCPE-GO) and sodium tetrakis (trifluoromethyl) phenyl borate (MCPE-STFPB) as ion-exchanger. The potentiometric modified CPEs (MCPE-GO and MCPE-STFPB) show an improved performance in term of Nernstian slope, selectivity, response time, and response stability compared to the unmodified CPE. The prepared electrodes PVCE, CWE, CPE, MCPE-GO and MCPE-STFPB show Nernstian slopes of 59.9, 59.5, 58.1, 58.3 and 57.0 mV/conc. decade, and detection limits of 5.0 × 10-6, 6.3 × 10-6, 8.0 × 10-6, 6.0 × 10-6 and 8.0 × 10-6 mol L-1, respectively. The prepared ISEs also show high selectivity against cations (i.e. Na+, K+, NH4+, Ca2+, Al3+, Fe3+), amino acids (i.e. glycine, L-alanine alanine), and sugars (i.e. fructose, glucose, maltose, lactose). The prepared ISEs are applicable for determining GB in spiked serums, urines, and pharmaceutical preparations, using a standard addition and a direct potentiometric method. The fast response time (<10 s), long lifetime (1-5 weeks), reversibility and stability of the measured signals facilitate the application of these sensors for routine analysis of the real samples.

Development of ionophore-based nanosphere emulsion incorporating ion-exchanger for complexometric titration of thiocyanate anion.

Ionophore-based ion-exchange nanosphere emulsion was prepared and tested for the determination of thiocyanate. The emulsified nanosphere contained the cationic additive tridodecylmethyl ammonium chloride (TDMAC), the plasticizer, and the ionophore Mn(III)-salophen or Mn(III)-salen. This emulsion was used as titrating agent for thiocyanate complexation with ionophores, which could be transduced using an ion-selective electrode (ISE) as an indicator electrode for the end point detection. The method showed no need for pH control and reliable selectivity, as thiocyanate could be determined in presence of other interfering ions with high accuracy. As well, the emulsion was stable and could be used for approximately couple of weeks. The developed emulsion could be used for the determination of thiocyanate in human saliva with standard deviation <4%. In sum, the proposed method could be used as an alternative for the argentometric titration and would open new avenues for the determination of neutral, anionic, and cationic species without necessity for water soluble ligands or pH control.

Highly selective thiocyanate optochemical sensor based on manganese(III)-salophen ionophore.

We report on the development of optochemical sensor based on Mn(III)-salophen ionophore. The sensor was prepared by embedding the ionophore in a plasticized poly (vinyl chloride) impregnated with the chromoionophore ETH7075. Optical response to thiocyanate occurred due to thiocyanate extraction into the polymer via formation of strong complex with the ionophore and simultaneous protonation of the indicator dye yielding the optical response at 545nm. The developed optochemical sensor exhibited high selectivity for thiocyanate over other anions including the most lipophilic species such as salicylate and perchlorate. For instance, the optical selectivity coefficients, logKSCN,anionopt, were as follow: ClO4-=-5.8; Sal-=-4.0; NO3-˂-6. Further, the thiocyanate optical selectivity obtained using the present optochemical sensor was greatly enhanced in comparison with that obtained using an anion-exchanger based sensor. Also, the optimized optochemical sensor exhibited micro-molar detection limit with 2min response time at pH4.5 using acetate buffer. The reversibility of the optimized sensor was poor due to strong ligation of the thiocyanate to the central Metal ion, log K=14.1, which can be overcome by soaking the optode in sodium hydroxide followed by soaking in buffer solution. The developed sensor was utilized successfully for the determination of thiocyanate in human saliva and in spiked saliva samples.

Spectrophotometric Determination of Distigmine Bromide, Cyclopentolate HCl, Diaveridine HCl and Tetrahydrozoline HCl via Charge Transfer Complex Formation with TCNQ and TCNE Reagents.

The purpose of this investigation was directed to propose sensitive, accurate and reproducible methods of analysis that can be applied to determine distigmine bromide (DTB), cyclopentolate hydrochloride (CPHC), diaveridine hydrochloride (DVHC) and tetrahydrozoline hydrochloride (THHC) drugs in pure form and pharmaceutical preparations via charge-transfer complex formation with 7,7,8,8-tetracyanoquinodimethane (TCNQ) and tetracyanoethylene (TCNE) reagents. Spectrophotometric method involve the addition a known excess of TCNQ or TCNE reagents to DTB, CPHC, DVHC and THHC drugs in acetonitrile, followed by the measurement of the absorbance of the CT complexes at the selected wavelength. The reaction stoichiometry is found to be 1:1 [drug]: [TCNQ or TCNE]. The absorbance is found to increase linearly with concentration of the drugs under investigation which is corroborated by the correlation coefficients of 0.9954-0.9981. The system obeys Beer's law for 6-400, 20-500, 1-180 and 60-560 µg mL(-1) and 80-600, 10-300, 1-60 and 80-640 µg mL(-1) for DTB, CPHC, DVHC and THHC drugs using TCNQ and TCNE reagents, respectively. The apparent molar absorptivity, sandell sensitivity, the limits of detection and quantification are also reported for the spectrophotometric method. Intra- and inter-day precision and accuracy of the method were evaluated as per ICH guidelines. The method was successfully applied to the assay of DTB, CPHC, DVHC and THHC drugs in formulations and the results were compared with those of a reference method by applying Student's t and F-tests. No interference was observed from common pharmaceutical excipients.

Development of two reference materials for all trans-retinol, retinyl palmitate, α- and γ-tocopherol in milk powder and infant formula.

Vitamins are important food constituents that can be present in almost every foodstuff. Food quality and safety depends on food surveillance by reliable quantitative analysis enabled by appropriate quality control. Certified matrix reference materials are versatile tools to support quality assurance and control. However, in the case of vitamins, which are important in various foods, there is a lack of matrix reference materials. Two certified reference materials for the determination of all-trans-retinol, retinyl palmitate, and α- and γ-tocopherol in milk powder and infant formula have been developed by the National Institute of Standards, Egypt. This article presents the preparation, characterization, homogeneity, and stability testing as well as statistical treatment of data and certified value assignment. The assignment of the certified values and associated uncertainties in the prepared natural-matrix reference materials were based on the widely used approach of combining data from independent and reliable analytical methods.