Population Pharmacokinetics of Remdesivir and GS-441524 in Hospitalized COVID-19 Patients.

The objective of this study was to describe the population pharmacokinetics of remdesivir and GS-441524 in hospitalized coronavirus disease 2019 (COVID-19) patients. A prospective observational pharmacokinetic study was performed in non-critically ill hospitalized COVID-19 patients with hypoxemia. For evaluation of the plasma concentrations of remdesivir and its metabolite GS-441524, samples were collected on the first day of therapy. A nonlinear mixed-effects model was developed to describe the pharmacokinetics and identify potential covariates that explain variability. Alternative dosing regimens were evaluated using Monte Carlo simulations. Seventeen patients were included. Remdesivir and GS-441524 pharmacokinetics were best described by a one-compartment model. The estimated glomerular filtration rate (eGFR) on GS-441524 clearance was identified as a clinically relevant covariate. The interindividual variability in clearance and volume of distribution for both remdesivir and GS-441524 was high (remdesivir, 38.9% and 47.9%, respectively; GS-441525, 47.4% and 42.9%, respectively). The estimated elimination half-life for remdesivir was 0.48 h, and that for GS-441524 was 26.6 h. The probability of target attainment (PTA) of the in vitro 50% effective concentration (EC50) for GS-441524 in plasma can be improved by shortening the dose interval of remdesivir and thereby increasing the total daily dose (PTA, 51.4% versus 94.7%). In patients with reduced renal function, the metabolite GS-441524 accumulates. A population pharmacokinetic model for remdesivir and GS-441524 in COVID-19 patients was developed. Remdesivir showed highly variable pharmacokinetics. The elimination half-life of remdesivir in COVID-19 patients is short, and the clearance of GS-441524 is dependent on the eGFR. Alternative dosing regimens aimed at optimizing the remdesivir and GS-441524 concentrations may improve the effectiveness of remdesivir treatment in COVID-19 patients.

Antihypertensive drug concentration measurement combined with personalized feedback in resistant hypertension: a randomized controlled trial.

BACKGROUND: Adherence to antihypertensive drugs (AHDs) is crucial for controlling blood pressure (BP). We aimed to determine the effectiveness of measuring AHD concentrations using a dried blood spot (DBS) sampling method to identify nonadherence, combined with personalized feedback, in reducing resistant hypertension. METHODS: We conducted a multicenter, randomized, controlled trial (RHYME-RCT, ICTRP NTR6914) in patients with established resistant hypertension. Patients were randomized to receive either an intervention with standard of care (SoC) or SoC alone. SoC consisted of BP measurement and DBS sampling at baseline, 3 months (t3), 6 months (t6), and 12 months (t12); AHD concentrations were measured but not reported in this arm. In the intervention arm, results on AHD concentrations were discussed during a personalized feedback conversation at baseline and t3. Study endpoints included the proportion of patients with RH and AHD adherence at t12. RESULTS: Forty-nine patients were randomized to receive the intervention+SoC, and 51 were randomized to receive SoC alone. The proportion of adherent patients improved from 70.0 to 92.5% in the intervention+SoC arm ( P  = 0.008, n  = 40) and remained the same in the SoC arm (71.4%, n  = 42). The difference in adherence between the arms was statistically significant ( P  = 0.014). The prevalence of resistant hypertension decreased to 75.0% in the intervention+SoC arm ( P  < 0.001, n  = 40) and 59.5% in the SoC arm ( P  < 0.001, n  = 42) at t12; the difference between the arms was statistically nonsignificant ( P  = 0.14). CONCLUSION: Personalized feedback conversations based on DBS-derived AHD concentrations improved AHD adherence but did not reduce the prevalence of RH.

Prevalence of vitamin D deficiency among Turkish, Moroccan, Indian and sub-Sahara African populations in Europe and their countries of origin: an overview.

UNLABELLED: Vitamin D status of nonwestern immigrants in Europe was poor. Vitamin D status of nonwestern populations in their countries of origin varied, being either similar to the immigrant populations in Europe or higher than in European indigenous populations. Vitamin D concentrations in nonwestern immigrant populations should be improved. PURPOSE: The higher the latitude, the less vitamin D is produced in the skin. Most European countries are located at higher latitudes than the countries of origin of their nonwestern immigrants. Our aim was to compare the serum 25-hydroxyvitamin D (25(OH)D) concentration of nonwestern immigrant populations with those of the population in their country of origin, and the indigenous population of the country they migrated to. METHODS: We performed literature searches in the "PubMed" and "Embase" databases, restricted to 1990 and later. The search profile consisted of terms referring to vitamin D or vitamin D deficiency, prevalence or cross-sectional studies, and countries or ethnicity. Titles and abstracts were reviewed to identify studies on population-based mean serum 25(OH)D concentrations among Turkish, Moroccan, Indian, and sub-Sahara African populations in Europe, Turkey, Morocco, India, and sub-Sahara Africa. RESULTS: The vitamin D status of immigrant populations in Europe was poor compared to the indigenous European populations. The vitamin D status of studied populations in Turkey and India varied and was either similar to the immigrant populations in Europe (low) or similar to or even higher than the indigenous European populations (high). CONCLUSIONS: In addition to observed negative consequences of low serum 25(OH)D concentrations among nonwestern populations, this overview indicates that vitamin D status in nonwestern immigrant populations should be improved. The most efficacious strategy should be the subject of further study.

Sunlight exposure or vitamin D supplementation for vitamin D-deficient non-western immigrants: a randomized clinical trial.

UNLABELLED: Vitamin D deficiency is very common in non-western immigrants. In this randomized clinical trial, vitamin D 800 IU/day or 100,000 IU/3 months were compared with advised sunlight exposure. Vitamin D supplementation was more effective than advised sunlight exposure in improving vitamin D status and lowering parathyroid hormone levels. INTRODUCTION: Vitamin D deficiency (25-hydroxyvitamin D [25(OH)D] < 25 nmol/l) is common among non-western immigrants. It can be treated with vitamin D supplementation or sunlight exposure. METHODS: To determine whether the effect of vitamin D(3) supplementation (daily 800 IU or 100,000 IU/3 months) or sunlight exposure advice is similar with regard to serum 25(OH)D and parathyroid hormone (PTH) concentrations. Randomized clinical trial in 11 general practices in The Netherlands. Non-western immigrants, aged 18-65 years (n = 232) and serum 25(OH)D < 25 nmol/l were randomly assigned to supplementation (daily 800 IU or 100,000 IU/3 months) or advice for sunlight exposure for 6 months (March-September). Blood samples were collected at baseline, during treatment (3 months, 6 months), and at follow-up (12 months). Statistical analysis was performed with multilevel regression modelling. RESULTS: The intention-to-treat analysis included 211 persons. Baseline serum 25(OH)D was 22.5 ± 11.1 nmol/l. After 6 months, mean serum 25(OH)D increased to 53 nmol/l with 800 IU/day, to 50.5 nmol/l with 100,000 IU/3 months, and to 29.1 nmol/l with advised sunlight exposure (supplementation vs sunshine p < 0.001). Serum PTH decreased significantly in all groups after 3 months, more in the supplementation groups than in the advised sunlight group (p < 0.05). There was no significant effect on physical performance and functional limitations. CONCLUSION: Vitamin D supplementation is more effective than advised sunlight exposure for treating vitamin D deficiency in non-western immigrants.

Fructan as a New Carbohydrate Sink in Transgenic Potato Plants.

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plant species that are important crops. We have been studying plants for their ability to synthesize and degrade fructans to determine if this ability is advantageous. We have also been analyzing the ability to synthesize fructan in relation to other nonstructural carbohydrate storage forms like starch. To study this, we induced fructan accumulation in normally non-fructan-storing plants and analyzed the metabolic and physiological properties of such plants. The normally non-fructan-storing potato plant was modified by introducing the microbial fructosyltransferase genes so that it could accumulate fructans. Constructs were created so that the fructosyltransferase genes of either Bacillus subtilis (sacB) or Streptococcus mutans (ftf) were fused to the vacuolar targeting sequence of the yeast carboxypeptidase Y (cpy) gene. These constructs were placed under the control of the constitutive cauliflower mosaic virus 35S promoter and introduced into potato tissue. The regenerated potato plants accumulated high molecular mass (>5 [times] 106 D) fructan molecules in which the degree of polymerization of fructose units exceeded 25,000. Fructan accumulation was detected in every plant tissue tested. The fructan content in the transgenic potato plants tested varied between 1 and 30% of dry weight in leaves and 1 and 7% of dry weight in microtubers. Total nonstructural neutral carbohydrate content in leaves of soil-grown plants increased dramatically from 7% in the wild type to 35% in transgenic plants. Our results demonstrated that potato plants can be manipulated to store a foreign carbohydrate by introducing bacterial fructosyltransferase genes. This modification affected photosynthate partitioning in microtubers and leaves and increased nonstructural carbohydrate content in leaves.

High level fructan accumulation in a transgenic sugar beet.

We have transformed sugar beet into a crop that produces fructans. The gene encoding 1-sucrose:sucrose fructosyl transferase (1-SST), which was isolated from Helianthus tuberosus, was introduced into sugar beet. In H. tuberosus, 1-SST mediates the first steps in fructan synthesis through the conversion of sucrose (GF) into low molecular weight fructans GF2, GF3, and GF4. In the taproot of sugar beet transformed with the 1-sst gene, the stored sucrose is almost totally converted into low molecular weight fructans. In contrast, 1-sst expression in the leaves resulted in only low levels of fructans. Despite the storage carbohydrate having been altered, the expression of the 1-sst gene did not have any visible effect on phenotype and did not affect the growth rate of the taproot as observed under greenhouse conditions.

[Large part of The Hague children taking too little vitamin D]. / Groot deel Haagse jeugd slikt onvoldoende vitamine D.

OBJECTITVE: To investigate the extent to which 0-15-year-old children in The Hague are complying with Dutch Health Council vitamin D supplementation advice, and whether there are differences between ethnic groups. DESIGN: Cross-sectional study. METHOD: From November 2014 up to and including October 2015, The Hague organisations for youth health care (YHC) have recorded intake of vitamin D supplementation during standard consultations. YHC 0-4 recorded whether vitamin D supplementation intake was sufficient. YHC 4-18 recorded whether vitamin D supplementation was recommended for the child (aged 4 and older, in case of coloured skin or insufficient time spent outdoors) and whether this child took sufficient vitamin D supplementation. Information on ethnic origin was taken from the digital YHC records. RESULTS: A large majority of those aged 0-3 received sufficient vitamin D supplementation. Intake was unknown for most 4-year-olds. Vitamin D supplementation was recommended for half of the children aged 5 or older, but a substantial number of them received no (50%) or insufficient (18%) supplementation. Among children for whom extra vitamin D was recommended, there was little difference in vitamin D use between ethnic groups. CONCLUSION: The Dutch Health Council recommendation on vitamin D supplementation is intended to prevent vitamin D deficiencies, but there is insufficient compliance by children in The Hague, especially those aged five and older.

Vitamin D status and bone mineral density in the Chinese population: a review.

UNLABELLED: Low vitamin D status is associated with low bone mass which, in turn, is an important predictor of fracture. However, data on this relationship in non-Caucasian populations are scarce. This review shows such an association in the Chinese population in five of the 11 included studies. INTRODUCTION: In the elderly population, the serum 25-hydroxyvitamin D [25(OH)D] concentration is often inadequate. This may cause a lower bone mineral density (BMD), which is an important predictor of fracture. It is estimated that by 2050 more than half of all hip fractures worldwide will occur in Asia. However, data on the relationship between vitamin D status and BMD in a non-Caucasian population are scarce. Therefore, this study reviews the literature on the relationship between serum 25(OH)D and BMD in the Chinese population. METHODS: A search was made in PubMed, EMBASE, Web of Science and Cochrane Library (up to December 2014) to identify relevant studies using the terms vitamin D status, bone mineral density, and Chinese. RESULTS: Of the 293 studies identified, 11 fulfilled the inclusion and exclusion criteria and were analyzed. Mean serum 25(OH)D concentrations ranged from 29-82 nmol/L. In 5 of the 11 studies, an association was found between vitamin D status and BMD in the Chinese population. CONCLUSION: The evidence for a relationship between the serum 25(OH)D concentration and BMD in the middle-aged and elderly Chinese population living in Asia appears to be limited and inconsistent.

Safety and long-term effects of renal denervation: Rationale and design of the Dutch registry.

BACKGROUND: Percutaneous renal denervation (RDN) has recently been introduced as a treatment for therapy-resistant hypertension. Also, it has been suggested that RDN may be beneficial for other conditions characterised by increased sympathetic nerve activity. There are still many uncertainties with regard to efficacy, safety, predictors for success and long-term effects. To answer these important questions, we initiated a Dutch RDN registry aiming to collect data from all RDN procedures performed in the Netherlands. METHODS: The Dutch RDN registry is an ongoing investigator-initiated, prospective, multicentre cohort study. Twenty-six Dutch hospitals agreed to participate in this registry. All patients who undergo RDN, regardless of the clinical indication or device that is used, will be included. Data are currently being collected on eligibility and screening, treatment and follow-up. RESULTS: Procedures have been performed since August 2010. At present, data from 306 patients have been entered into the database. The main indication for RDN was hypertension (n = 302, 99%). Patients had a mean office blood pressure of 177/100 (±29/16) mmHg with a median use of three (range 0-8) blood pressure lowering drugs. Mean 24-hour blood pressure before RDN was 157/93 (±18/13) mmHg. RDN was performed with different devices, with the Simplicity™ catheter currently used most frequently. CONCLUSION: Here we report on the rationale and design of the Dutch RDN registry. Enrolment in this investigator-initiated study is ongoing. We present baseline characteristics of the first 306 participants.

Plant-based raw material: improved food quality for better nutrition via plant genomics.

Plants form the basis of the human food chain. Characteristics of plants are therefore crucial to the quantity and quality of human food. In this review, it is discussed how technological developments in the area of plant genomics and plant genetics help to mobilise the potential of plants to improve the quality of life of the rapidly growing world population.

Accumulation of fructose polymers in transgenic tobacco.

Fructan, a polyfructose molecule, is a storage compound in a limited number of plant species. Usually these species accumulate fructan with a low degree of polymerization (DP) and most of these plants have properties which preclude their use as a fructan source. With the eventual aim of allowing the accumulation of high DP fructans in non-fructan storing plants, we have investigated whether carbohydrate flow in the plant cell can be directed to produce this polymer. For this purpose the SacB gene from Bacillus subtilis, which encodes levansucrase, was modified and introduced into tobacco plants. Transgenic plants containing the sacB gene accumulate fructans. The size and properties of this fructan are similar to fructan produced by Bacillus subtilis, and is stable in plants. Although the level of fructan accumulation in the transgenic tobacco plants ranged from 3-8 percent of the dry weight, no levansucrase mRNA or protein could be detected in these plants. Extension of this work should permit the production of this high molecular weight biopolymer in crop plants for applications in food and non-food products.

Linkage map positions and allelic diversity of two Mal d 3 (non-specific lipid transfer protein) genes in the cultivated apple (Malus domestica).

Non-specific lipid transfer proteins (nsLTPs) of Rosaceae fruits, such as peach, apricot, cherry, plum and apple, represent major allergens for Mediterranean atopic populations. As a first step in elucidating the genetics of nsLTPs, we directed the research reported here towards identifying the number and location of nsLTP (Mal d 3) genes in the apple genome and determining their allelic diversity. PCR cloning was initially performed on two cultivars, Prima and Fiesta, parents of a core apple mapping progeny in Europe, based on two Mal d 3 sequences (AF221502 and AJ277164) in the GenBank. This resulted in the identification of two distinct sequences (representing two genes) encoding the mature nsLTP proteins. One is identical to accession AF221502 and has been named Mal d 3.01, and the other is new and has been named Mal d 3.02. Subsequent genome walking in the upstream direction and DNA polymorphism analysis revealed that these two genes are intronless and that they could be mapped on two homoeologous segments of linkage groups 12 and 4, respectively. Further cloning and sequencing of the coding and upstream region of both Mal d 3 genes in eight cultivars was performed to identify allelic variation. Assessment of the deduced nsLTP amino acid sequences gave a total of two variants at the protein level for Mal d 3.01 and three for Mal d 3.02. The consequences of our results for allergen nomenclature and the breeding of low allergenic apple cultivars are discussed.

Genomic cloning and linkage mapping of the Mal d 1 (PR-10) gene family in apple (Malus domestica).

Fresh apples can cause birch pollen-related food allergy in northern and central European populations, primarily because of the presence of Mal d 1, the major apple allergen that is cross-reactive to the homologous and sensitizing allergen Bet v 1 from birch. Apple cultivars differ significantly in their allergenicity. Knowledge of the genetic basis of these differences would direct breeding for hypoallergenic cultivars. The PCR genomic cloning and sequencing were performed on two cultivars, Prima and Fiesta, which resulted in 37 different Mal d 1 gDNA sequences. Based on the mapping of sequence-specific molecular markers, these sequences appeared to represent 18 Mal d 1 genes. Sixteen genes were located in two clusters, one cluster with seven genes on linkage group (LG) 13, and the other cluster with nine genes on the homoeologous LG 16. One gene was mapped on LG 6, and one remained unmapped. According to sequence identity, these 18 genes could be subdivided into four subfamilies. Subfamilies I-III had an intron of different size that was subfamily and gene-specific. Subfamily IV consisted of 11 intronless genes. The deduced amino acid sequence identity varied from 65% to 81% among subfamilies, from 82% to 100% among genes within a subfamily, and from 97.5% to 100% among alleles of one gene. This study provides a better understanding of the genetics of Mal d 1 and the basis for further research on the occurrence of allelic diversity among cultivars in relation to allergenicity and their biological functions.

Promoter analysis of the chalcone synthase (chsA) gene of Petunia hybrida: a 67 bp promoter region directs flower-specific expression.

In order to scan the 5' flanking region of the chalcone synthase (chs A) gene for regulatory sequences involved in directing flower-specific and UV-inducible expression, a chimaeric gene was constructed containing the chs A promoter of Petunia hybrida (V30), the chloramphenicol acetyl transferase (cat) structural sequence as a reporter gene and the chs A terminator region of Petunia hybrida (V30). This chimaeric gene and 5' end deletions thereof were introduced into Petunia plants with the help of Ti plasmid-derived plant vectors and CAT activity was measured. A 220 bp chs A promoter fragment contains cis-acting elements conferring flower-specific and UV-inducible expression. A promoter fragment from -67 to +1, although at a low level, was still able to direct flower-specific expression but could not drive UV-inducible expression in transgenic Petunia seedlings. Molecular analysis of binding of flower nuclear proteins to chs A promoter fragments by gel retardation assays showed strong specific binding to the sequences from -142 to +81. Promoter sequence comparison of chs genes from other plant species, combined with the deletion analysis and gel retardation assays, strongly suggests the involvement of the TACPyAT repeats (-59 and -52) in the regulation of organ-specificity of the chs A gene in Petunia hybrida. We also describe an in vitro organ-specific transient expression system, in which flower or purple callus protoplasts are used, that enables us to pre-screen organ-specific expression of a chimaeric reporter gene.

The TACPyAT repeats in the chalcone synthase promoter of Petunia hybrida act as a dominant negative cis-acting module in the control of organ-specific expression.

Analysis of the expression of the GUS reporter gene driven by various regions of the Petunia hybrida chalcone synthase (chsA) promoter revealed that the developmental and organ-specific expression of the chsA gene is conferred by a TATA proximal module located between -67 and -53, previously designated as the TACPyAT repeats. Histochemical analysis of GUS reporter gene expression revealed that the organ-specific 67 bp promoter fragment directs the same cell-type specificity as a 530 bp promoter, whereas additional enhancer sequences are present within the more TATA distal region. Moreover, the region between -800 and -530 is also involved in extending the cell-type specificity to the trichomes of flower organs and of young seedlings. The mechanism by which the TACPyAT repeats modulate expression during plant development was studied by analysing the expression of the GUS gene driven by chimeric promoters consisting of the CaMV 35S enhancer (domain B, -750 to -90) fused to various chsA 5' upstream sequences. Detailed enzymatic and histochemical analysis revealed that in the presence of the TACPyAT module the CaMV 35S region only enhances GUS activity in those organs in which the chsA promoter is normally active. Furthermore, this analysis shows that enhancement in the presence of the CaMV 35S domain B is accomplished by increasing the number of cell types expressing the GUS gene within the organ, rather than enhancement of the chsA cell-type-specific expression within these organs. Deletion of the TACPyAT sequences in the chimeric promoter construct completely restores the well-documented CaMV 35S domain B cell-type specificity, showing that the TACPyAT module acts as a dominant negative cis-acting element which controls both organ and developmental regulation of the chsA promoter activity.

Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility.

Inhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers, presumably because the viral CaMV 35S promoter was insufficiently expressed in cell types of this organ in which the pigments are produced. Insertion of the anther box, a homologous sequence found in other genes expressed in anthers, resulted in a modified expression pattern driven by this promoter, as monitored by the beta-glucuronidase (gus) gene. In addition to the basic CaMV 35S expression pattern in anthers, GUS activity was observed in tapetum cells when the modified promoter was fused to the gus gene. This promoter construct was subsequently used to drive an antisense chs gene in transgenic petunia, which led to the inhibition of pigment synthesis in anthers of five of 35 transformants. Transgenic plants with white anthers were male sterile due to an arrest in male gametophyte development. This finding indicated that flavonoids play an essential role in male gametophyte development.

Cloning of the fructan biosynthesis pathway of Jerusalem artichoke.

To study the regulation of fructan synthesis in plants, we isolated two full-size cDNA clones encoding the two enzymes responsible for fructan biosynthesis in Jerusalem artichoke (Helianthus tuberosus): 1-sucrose:sucrose fructosyl transferase (1-SST) and 1-fructan:fructan fructosyl transferase (1-FFT). Both enzymes have recently been purified to homogeneity from Jerusalem artichoke tubers (Koops and Jonker (1994) J.Exp.Bot.45, 1623-1631; Koops and Jonker (1996) Plant Physiol. 110, 1167-1175) and their amino acid sequences have been partially determined. Using RT-PCR and primers based on these sequences, specific fragments of the genes were amplified from tubers of Jerusalem artichoke. These fragments were used as probes to isolate the cDNAs encoding 1-SST and 1-FFT from a tuber-specific lambdal ZAP library. The deduced amino acid sequences of both cDNAs perfectly matched the sequences of the corresponding purified proteins. At the amino acid level, the cDNA sequences showed 61% homology to each other and 59% homology to tomato vacuolar invertase. Based on characteristics of the deduced amino acid sequence, the first 150 bp of both genes encode a putative vacuolar targeting signal. Southern blot hybridization revealed that both 1-SST and 1-FFT are likely to be encoded by single-copy genes. Expression studies based on RNA blot analysis showed organ-specific and developmental expression of both genes in growing tubers. Lower expression was detected in flowers and in stem. In other organs, including leaf, roots and dormant tubers, no expression could be detected. In tubers, the spatial and developmental expression correlates with the accumulation of fructans. Using the 1-sst and 1-fft cDNAs, chimeric genes were constructed driven by the CaMV 35S promoter. Analysis of transgenic petunia plants carrying these constructs showed that both cDNAs encode functional fructosyltransferase enzymes. Plants transformed with the 35S-1-sst construct accumulated the oligofructans 1-kestose (GF2), 1,1-nystose (GF3) and 1,1,1-fructosylnystose (GF4). Plants transformed with the 35S-1-fft construct did not accumulate fructans, probably because of the absence of suitable substrates for 1-FFT, i.e. fructans with a degree of polymerization > or = 3 (GF2, GF3, etc.). Nevertheless, protein extracts from these transgenic plants were able to convert GF3, when added as a substrate into fructans with a higher degree of polymerization. Progeny of crosses between a 35S-1-sst-containing plant and a 35S-1-fft-containing plant, showed accumulation of high-molecular-weight fructans in old, senescent leaves. Based on the comparison of the predicted amino acid sequences of 1-sst and 1-fft with those of other plant fructosyl transferase genes, we postulate that both plant fructan genes have evolved from plant invertase genes.