Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

Raman-based dynamic feeding strategies using real-time glucose concentration monitoring system during adalimumab producing CHO cell cultivation

The use of Process Analytical Technology tools coupled with chemometrics has been shown great potential for better understanding and control of mammalian cell cultivations through real-time process monitoring. In-line Raman spectroscopy was utilized to determine the glucose concentration of the complex bioreactor culture medium ensuring real-time information for our process control system. This work demonstrates a simple and fast method to achieve a robust partial least squares calibration model under laboratory conditions in an early phase of the development utilizing shake flask and bioreactor cultures. Two types of dynamic feeding strategies were accomplished where the multi-component feed medium additions were controlled manually and automatically based on the Raman monitored glucose concentration. The impact of these dynamic feedings was also investigated and compared to the traditional bolus feeding strategy on cellular metabolism, cell growth, productivity, and binding activity of the antibody product. Both manual and automated dynamic feeding strategies were successfully applied to maintain the glucose concentration within a narrower and lower concentration range. Thus, besides glucose, the glutamate was also limited at low level leading to reduced production of inhibitory metabolites, such as lactate and ammonia. Consequently, these feeding control strategies enabled to provide beneficial cultivation environment for the cells. In both experiments, higher cell growth and prolonged viable cell cultivation were achieved which in turn led to increased antibody product concentration compared to the reference bolus feeding cultivation.     

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

The rate of rotation of the rotor in the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or steady parts of the enzyme, is estimated in native vacuolar membrane vesicles from Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are formed spontaneously after exposing purified yeast vacuoles to osmotic shock. The fraction of total ATPase activity originating from the V-ATPase is determined by using the potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during ten min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit of the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data are incompatible with models assuming multiple binding sites), to the inhibitor titration curve determines the concentration of the enzyme. Combining this with the known ATP/rotation stoichiometry of the V-ATPase and the assayed concentration of inorganic phosphate liberated by the V-ATPase, leads to an average rate of ~10 Hz for full 360° rotation (and a range of 6–32 Hz, considering the ± standard deviation of the enzyme concentration), which, from the time-dependence of the activity, extrapolates to ~14 Hz (8–48 Hz) at the beginning of the reaction. These are lower-limit estimates. To our knowledge, this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and is not fixed to a solid support; instead it is functioning in its native membrane environment.     

The Burden of Provider-Initiated Preterm Birth and Associated Factors: Evidence from the Brazilian Multicenter Study on Preterm Birth (EMIP)

Background

About 15 million children are born under 37 weeks of gestation worldwide. Prematurity is the leading cause of neonatal deaths and short/long term morbidities, entailing consequences not only for the individual, but also their family, health agencies, facilities and all community. The provider-initiated preterm birth is currently one of the most important obstetric conditions related to preterm births, particularly in middle and high income countries, thus decreasing the need for therapeutic preterm birth is essential to reduce global prematurity. Therefore detailed knowledge on the factors associated with provider-initiated preterm birth is essential for the efforts to reduce preterm birth rates and its consequences. In this current analysis we aimed to assess the proportion of provider-initiated (pi-PTB) among preterm births in Brazil and identify associated factors.

Methods and Findings

This is an analysis of a multicenter cross-sectional study with a nested case-control component called Brazilian Multicenter Study on Preterm Birth (EMIP). EMIP was conducted in 20 referral obstetric hospitals located in the three most populated of the five Brazilian regions. We analysed data of women with pi-PTB, defined as childbirth occurring at less than 37 weeks, medically indicated for maternal/fetal compromise or both; and women with term birth, childbirth at or after 37 weeks. Maternal, sociodemographic, obstetric, prenatal care, delivery, and postnatal characteristics were assessed as possible factors associated with pi-PTB, compared to term births. The overall prevalence of preterm births was 12.3%. Of these, approximately one-third of cases were initiated by the provider. Hypertensive disorders, placental abruption, and diabetes were the main maternal conditions leading to pi-PTB. Caesarean section was the most common mode of delivery. Chronic hypertension (OR 7.47; 95%CI 4.02–13.88), preeclampsia/eclampsia/HELLP syndrome (OR 15.35; 6.57–35.88), multiple pregnancy (OR 12.49; 4.86–32.05), and chronic diabetes (OR 5.24; 2.68–10.25) were the most significant factors independently associated with pi-PTB.

Conclusions

pi-PTB is responsible for about one-third of all preterm births, requiring special attention. The decision-making process relative to the choice of provider-initiated birth is complex, and many factors should be elucidated to improve strategies for its prevention, including evidence-based guidelines on proper management of the corresponding clinical conditions.     

Nutrient Requirement and Growth of a Synechococcus Species Isolated from Lake Balaton

A pico sized Synechococcus species isolated from Lake Balaton was studied in batch and continuous cultures. This picocyanobacterium had a pH optimum at 8.5 and a temperature optimum at 28-30°C. The I_k value for growth was 52 μEinstein m⁻² S⁻¹, the maximum growth rate 2.27 d⁻¹, the half saturation Constant of growth 1.2 μg PO₄-P I⁻¹ and the minimal cell quota 1.74 nig P g dry weight⁻¹. The dry weight of cells showed a minimum, the chlorophyll-a/biomass ratio a maximum as a function of growth rate. Above the quota of 3.4 fg P Cell⁻¹ significant amounts of non-reactive dissolved Phosphorous were released.     

Recognition of lumenal prion protein aggregates by post-ER quality control mechanisms is mediated by the preoctarepeat region of PrP

Prion diseases are fatal transmissible neurodegenerative disorders linked to an aberrant conformation of the cellular prion protein (PrP(c)). We have shown previously that the chemical compound suramin induced aggregation of fully matured PrP(c) in post-ER compartments, thereby, activating a post-ER quality control mechanism and preventing cell surface localization of PrP by intracellular re-routing of aggregated PrP from the Golgi/TGN directly to lysosomes. Of note, drug-induced PrP aggregates were not toxic and could easily be degraded by neuronal cells. Here, we focused on determining the PrP domains mediating these effects. Using PrP deletion mutants we show that intracellular re-routing but not aggregation depends on the N-terminal PrP (aa 23-90) and, more precisely, on the preoctarepeat domain (aa 23-50). Fusion of the PrP N-terminus to the GPI-anchored protein Thy-1 did not cause aggregation or re-routing of the chimeric protein, indicating that the N-terminus is only active in re-routing when prion protein aggregation occurs. Insertion of a region with a comparable primary structure contained in the PrP paralogue prnd/doppel (aa 27-50) into N-terminally deleted PrP re-established the re-routing phenotype. Our data reveal an important role for the conserved preoctarepeat region of PrP, namely controlling the intracellular trafficking of misfolded PrP.     

Gender-specific growth patterns for stature,sitting height and limbs length in Croatian children and youth (3 to 18 years of age)

In a cross-sectional study of growth, 5,155 children (2,591 females, 2,564 males) from the town of Zagreb (Croatia) were measured. Four traits of linear dimensionality (stature, sitting height, arm and leg lengths) were studied in the age span of 3 to 18 years. A significant average annual increase of all four anthropometric parameters were observed up to 14 and 15 years of age in girls and 16 years of age in boys, showing that girls had a shorter growing period. In the prepubertal period until 9 years of age, gender differences were negligible. At the age of 10, boys were overgrown by girls in all parameters due to the earlier onset of puberty in girls. The growth gains for girls, when compared with those for boys, show a different pattern across variables. The female growth advantage remained in a two years period for the limbs length, but in a three year period for stature and the longest, for 4 years, for sitting height. The male predominance in size had an onset at the age of 13 for the limbs and in the age of 14 for stature and sitting height. The patterns of sexual dimorphism in stature and sitting height during growing years are similar to those observed in other populations of Europe. Growth of Croatian children and youth is very similar to that of the tallest European populations.