Simple and User-Friendly Methodology for Crystal Water Determination by Quantitative Proton NMR Spectroscopy in Deuterium Oxide.

In drug research and development, knowledge of the precise structure of an active ingredient is crucial. However, it is equally important to know the water content of the drug molecule, particularly the number of crystal waters present in its structure. Such knowledge ensures the avoidance of drug dosage and formulation errors since the number of water molecules affects the physicochemical and pharmaceutical properties of the molecule. Several methods have been used for crystal water measurements of organic compounds, of which thermogravimetry and crystallography may be the most common ones. To the best of our knowledge, solution-state NMR spectroscopy has not been used for crystal water determination in deuterium oxide. Quantitative NMR (qNMR) method will be presented in the paper with a comparison of single-crystal X-ray diffraction and thermogravimetric analysis results. The qNMR method for water content measurement is straightforward, reproducible, and accurate, including measurement of 1H NMR spectrum before and after the addition of the analyte compound, and the result can be calculated after integration of the reference compound, analyte, and HDO signals using the given equation. In practical terms, there is no need for weighing the samples under study, which makes it simple and is a clear advantage to the current determination methods. In addition, the crystal structures of two model bisphosphonates used herein are reported: that of monopotassium etidronate dihydrate and monosodium zoledronate trihydrate.

Transition in endocrinology: predictors of drop-out of a heterogeneous population on a long-term follow-up.

PURPOSE: To evaluate: (1) clinical and epidemiological characteristics of outpatients transitioned from Pediatrics Endocrine (PED) to Adult Endocrine Department (AED) in a tertiary center; (2) transition process features, and predictors of drop-out. METHODS: Demographic, clinical, and transition features of 170 consecutive patients with pediatric onset of chronic endocrine or metabolic disease (excluded type 1 diabetes) who transitioned from PED to AED (2007-2020) were retrospective evaluated. RESULTS: The age at transition was 18.4 ± 4 years (F:M = 1.2: 1), and mean follow-up 2.8 years. The population was heterogeneous; the most (69.4%) was affected by one, 24.1% by two or more endocrine diseases, 6.5% were followed as part of a cancer survivor's surveillance protocol. The comorbidity burden was high (37, 20.6, and 11.2% of patients had 2, 3, 4, or more diseases). The number of visits was associated with the number of endocrine diseases and the type of them. Adherent subjects had a higher number of comorbidities. Thyroid disorders and more than one comorbidity predicted the adherence to follow-up. Having performed one visit only was predictive of drop-out, regardless of the pathology at diagnosis. CONCLUSION: This is the first study that analyzed a specific transition plan for chronic endocrine diseases on long-term follow-up. The proposed "one-size-fits-all model" is inadequate in responding to the needs of patients. A structured transition plan is an emerging cornerstone.

Real-Time On-Site Multielement Analysis of Environmental Waters with a Portable X-ray Fluorescence (pXRF) System.

Strict regulations are in place to control the effluents of mining sites and other industries. Heavy metal contamination of aquatic systems caused by leakages is difficult to mitigate as it takes time to detect and localize the leak. Dynamic sampling would drastically reduce the time to locate leakages and allow faster actions to reduce the impact on the environment. The present study introduces a novel portable multielement water analysis system to simultaneously measure Mn, Ni, Cu, Zn, Pb, and U in water samples from natural sources within 15 min from the sampling. The metals are preconcentrated from a 10 mL water sample into a nanoporous filter based on bisphosphonate-modified thermally carbonized porous silicon. The metals can be conveniently analyzed from the filter with a portable XRF analyzer in field conditions. The system was empirically calibrated for a lake water matrix with neutral pH and low alkaline metal concentration. A strong correlation between the XRF intensities and the ICP-MS results was obtained in a concentration range from 50 to 10 000 µg/L. With a DPO-2000C XRF analyzer, the detection limits were 103, 86, 92, 35, 44, and 43 µg/L for Mn, Ni, Cu, Zn, Pb, and U, respectively. The corresponding values with X-MET8000 Expert Geo were 137, 46, 62, 38, 29, and 54. The system was successfully validated with simulated multielement lake water samples and piloted in field conditions. The system provides an efficient way to monitor metals in environmental waters in cases where quick on-site results are needed.

Synthesis of the biologically important dideuterium-labelled adenosine triphosphate analogue ApppI(d 2).

The chemical synthesis of the dideuterium-labelled ATP analogue 1-adenosin-5'-yl-3-(3-methylbut-3-en-1,1-d 2-1-ol) triphosphoric acid diester (ApppI(d 2)) is described. ApppI has been reported to be an important mevalonate pathway metabolite, induced by nitrogen-containing bisphosphonates used for the treatment of several diseases related to the calcium metabolism, of which osteoporosis is the most well-known. The availability of ApppI(d 2) opens possibilities to quantitative measurements of ApppI in biological samples by mass spectrometry. The synthesized target compound ApppI(d 2) was purified by high-performance counter current chromatography and characterized by 1H, 13C, and 31P NMR spectroscopy as well as high-resolution mass spectrometry.

A strategic framework for the development and enhancement of safety culture in the artificial tanning sector in Greece.

Herein, a strategic framework for the development and enhancement of safety culture in the artificial tanning sector in Greece is presented. This framework has been designed and promoted by the competent national regulatory authority, which is the Greek Atomic Energy Commission (EEAE). The aim is to ensure a common understanding regarding ultraviolet radiation (UVR) and artificial tanning among artificial tanning professionals, stakeholders and the general public. The strategic framework is founded on the recommendations of international organizations, EU requirements and the relevant technical standards. It comprises three autonomous but interconnected components: (A) a sunbed operators' e-training course and certification process; (B) a code of practice addressed individually to business owners, sunbed operators and sunbed users; (C) communication strategies aiming to raise awareness regarding UVR and artificial tanning among all interested parties. The artificial tanning safety culture framework presented here is the policy option undertaken by the EEAE and is embedded in the upcoming legislation and regulations for the provision of artificial tanning services in Greece. EEAE considers that the structure of the artificial tanning safety culture strategic framework will serve as a guide for the development and promotion of relevant strategic safety culture frameworks for the provision of aesthetic/wellness services utilising other non-ionising radiation sources, which do not currently exist.

Synthesis of medronic acid monoesters and their purification by high-performance countercurrent chromatography or by hydroxyapatite.

We achieved the synthesis of important medronic acid monoalkyl esters via the dealkylation of mixed trimethyl monoalkyl esters of medronic acid. Two methods were developed for the purification of medronic acid monoesters: 1) small scale (10-20 mg) purification by using hydroxyapatite and 2) large scale (tested up to 140 mg) purification by high-performance countercurrent chromatography (HPCCC).

Two strategies for the synthesis of the biologically important ATP analogue ApppI, at a multi-milligram scale.

Two strategies for the synthesis of the ATP (adenosine triphosphate) analogue ApppI [1-adenosin-5'-yl 3-(3-methylbut-3-enyl)triphosphoric acid diester] (1) are described. ApppI is an active metabolite of the mevalonate pathway and thus is of major biological significance. Chemically synthezised ApppI was purified by using triethylammonium bicarbonate as the counter ion in ion-pair chromatography and characterized by (1)H, (13)C, (31)P NMR and MS spectroscopical methods.

Synthesis of triple-bond-containing 1-hydroxy-1,1-bisphosphonic acid derivatives to be used as precursors in "click" chemistry: two examples.

The synthesis of novel (ω-alkynyl-1-hydroxy-1,1-diyl)bisphosphonic acid tetramethyl esters (1a-c), their P,P'-dimethyl esters (2a-c), and two trimethyl ester derivatives (3a and 3b) is reported. The prepared compounds can be attached to many kinds of molecules containing azide (-N3) functionalities using a "click" chemistry approach. As an example, bisphosphonate trimethyl ester 3a and P,P'-dimethyl ester 2b were attached to triethylene glycol to form triethylene glycol-bisphosphonate conjugates 4 and 5 as model compounds for further studies in, for example, nanoparticle targeting.

Vitamin D levels in a paediatric population of normal weight and obese subjects.

BACKGROUND: Vitamin D exerts pleiotropic effects and few studies are available in the Italian population. AIM: Aim of our study was to evaluate vitamin D status in children living in Northern Italy. METHODS: We studied vitamin D levels in a population of 113 normal weight (NW) and 444 obese (OB) children, prepubertal and pubertal. We considered vitamin D levels >20 ng/ml as normal, but also measured percentage of children with vitamin D levels higher than a cutoff of 30 ng/ml. RESULTS: 68.2 % of NW children showed normal levels of vitamin D, while 31.8 % showed a clear vitamin D deficiency. Only 28.3 % showed vitamin D levels higher than 30 ng/ml. Obese children showed 55.6 % of subjects with normal levels of vitamin D and 44.4 % of subjects a status of vitamin D deficiency. Only the 18.9 % showed vitamin D levels higher than 30 ng/ml. Mean vitamin D levels in NW children (27.3 ± 1.2 ng/ml) were higher than in OB children (21.8 ± 0.6 ng/ml). No differences have been found between prepubertal and pubertal children in terms of vitamin D levels. CONCLUSIONS: Our paediatric population demonstrates a low percentage of vitamin D sufficiency. In particular, obese children show only 19 % of subjects with normal levels while almost half of this population shows a clear deficiency. Further studies are needed to support these results and to evaluate the possible metabolic consequences.

Differentiation of species of the genus Saccharomyces using biomolecular fingerprinting methods.

The genus Saccharomyces comprises very closely related species. This high degree of relationship makes a simple identification and differentiation of strains difficult since these species are hardly discriminable by their morphological and physiological features. A sequence analysis of ribosomal DNA and the corresponding internal transcribed spacers can only rarely be successfully applied. In this study, we proved the applicability of a novel DNA fingerprinting method, the SAPD-PCR (specifically amplified polymorphic DNA) and of MALDI-TOF-MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry) fingerprinting with the MALDI Biotyper for the differentiation of species belonging to the genus Saccharomyces. It was possible with SAPD-PCR to create specific banding patterns for all Saccharomyces species. Different strains of the same species produced nearly the same banding patterns. Specific and reproducible reference spectra could be generated for each of the strains with the MALDI Biotyper. Therefore, SAPD-PCR and MALDI-TOF-MS can be fast and reliable tools to identify these related Saccharomyces species which are applied in many biotechnological processes.

Comparison of two classifications of metabolic syndrome in the pediatric population and the impact of cholesterol.

BACKGROUND: To establish the rate of agreement in predicting metabolic syndrome (MS) in different pediatric classifications using percentiles or fixed cut-offs, as well as exploring the influence of cholesterol. SUBJECTS AND METHODS: Cross-sectional study in a tertiary care center. Nine hundred and twenty-three obese children and adolescents were evaluated for metabolic characteristics, cholesterol levels, the agreement rate and prevalence of MS across age subgroups with pediatric National Cholesterol Education Program/ Adult Treatment Panel III (NCEP-ATP III) and International Diabetes Federation (IDF) classifications. RESULTS: The overall prevalence of MS was 36.2% and 56.7% with NCEPATP III and IDF. The overall concordance was fair (k: 0.269), with substantial values observed only in children older than 10 (k: 0.708) and 16 yr (0.694). Concordant subjects for both classifications, ≤6 yr, had higher triglycerides, blood pressure (p<0.05) and lower HDL-cholesterol (p<0.0001), with respect to those found to be discordant. Concordant subjects ranging 6-10 yr had all parameters higher than those discordant for IDF (p<0.01) and insulin resistance (p<0.05) than those discordant for NCEP-ATP III. Concordant subjects ≥10 yr presented more altered parameters than those included only in NCEP-ATP III (p<0.05). Overt glucose alterations were uncommon (7.4%; confidence interval 95% 0.1-14.9%), although glucose was modestly higher in MS subjects (p<0.01). Total and LDL-cholesterol was lower in subjects with MS than in those without (p<0.05), and in concordant rather than discordant subjects (p<0.05). CONCLUSIONS: Classifications of MS do not identify the same pediatric population. Subjects who satisfied any classification were the most compromised. Lipid alterations were precocious in the youngest. Obese youths with MS presented lower total and LDL-cholesterol.

Fast identification of wine related lactic acid bacteria by multiplex PCR.

The microflora of must and wine consists of yeasts, acetic acid bacteria and lactic acid bacteria (LAB). The latter group plays an important role for wine quality. The malolactic fermentation carried out by LAB leads to deacidification and stabilisation of wines. Nevertheless, LAB are often associated with wine spoilage. They are mainly responsible for the formation of biogenic amines. Furthermore, some strains produce exopolysaccharide slimes, acetic acid, diacetyl and other off-flavours. In this context a better monitoring of the vinification process is crucial to improve wine quality. Moreover, a lot of biodiversity studies would also profit from a fast and reliable identification method. In this study, we propose a species-specific multiplex PCR system for a rapid and simultaneous detection of 13 LAB species, frequently occurring in must or wine: Lactobacillus brevis, Lb. buchneri, Lb. curvatus, Lb. hilgardii, Lb. plantarum, Leuconostoc mesenteroides, Oenococcus oeni, Pediococcus acidilactici, P. damnosus, P. inopinatus, P. parvulus, P. pentosaceus and Weissella paramesenteroides.

Rescaling invariance and anomalous energy transport in a small vertical column of grains.

It is well known that energy dissipation and finite size can deeply affect the dynamics of granular matter, often making usual hydrodynamic approaches problematic. Here we report on the experimental investigation of a small model system, made of ten beads constrained into a 1D geometry by a narrow vertical pipe and shaken at the base by a piston excited by a periodic wave. Recording the beads motion with a high frame rate camera allows to investigate in detail the microscopic dynamics and test hydrodynamic and kinetic models. Varying the energy, we explore different regimes from fully fluidized to the edge of condensation, observing good hydrodynamic behavior down to the edge of fluidization, despite the small system size. Density and temperature fields for different system energies can be collapsed by suitable space and time rescaling, and the expected constitutive equation holds very well when the particle diameter is considered. At the same time, the balance between dissipated and fed energy is not well described by commonly adopted dependence due to the up-down symmetry breaking. Our observations, supported by the measured particle velocity distributions, show a different phenomenological temperature dependence, which yields equation solutions in agreement with experimental results.

Structural Diversity in Antiosteolytic Bisphosphonates: Deciphering Structure-Activity Trends in Ultra Long Controlled Release Phenomena.

Bisphosphonate (BP)-based treatments have been extensively prescribed for bone-related conditions, particularly for osteoporosis. Their low bioavailability creates the need for prescribed dosage increase to reach therapeutic levels but generates a plethora of undesirable side effects. A viable approach to alleviating these issues is to design and exploit controlled release strategies. Herein, the controlled release profiles of 15 structurally characterized BPs (actual drugs and structural analogs) were thoroughly studied from tablets containing three (cellulose, lactose, and silica) or two (cellulose, and silica) excipients in human stomach-simulated pH conditions. The BPs were of two types, alkyl-BPs and amino-BPs. Alkyl-BPs included four derivatives of etidronate (acid, disodium, tetra-sodium, and monopotassium forms), medronic acid, and three analogs of etidronate, in which the -CH3 group was replaced by the moieties -H, -CH2CH2CH3, and -CH2CH2CH2CH2CH3. Amino-BPs included the commercial drugs pamidronate, alendronate, neridronate, and ibandronate, as well as three analog compounds. Release curves were constructed based on data taken from 1H NMR peak integration and were expressed as "% BP release" vs time. The controlled release profiles (initial release rate, plateau value, etc.) were correlated with certain structural features (number of hydrogen and metal-oxygen bonds), showing that the molecular and crystal lattice features of each BP profoundly influence its release characteristics. It was concluded that for all BPs, in general, the initial rate became lower as the total number of lattice interactions increased. For the alkyl-BPs elongation of the alkyl side chain seems to decelerate the release. Amino-BPs, in general, show slower release than the alkyl-BPs. No adverse effects of alkyl- and amino-BP drugs on NIH3T3 cell viability were noted.

Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels.

BACKGROUND: Ghrelin circulates in blood as acylated (AG) and unacylated (UAG) ghrelin. The physiological role of the two forms is poorly understood, in particular in childhood. Aim of the study was to evaluate the AG and UAG levels in obese and normal weight (NW) children, pre-pubertal and pubertal, and their relationship with insulin, leptin and adiponectin levels. SUBJECTS AND METHODS: A population based study in which AG, UAG, leptin, adiponectin, glucose, insulin, testosterone or estradiol levels, insulinemic indexes were evaluated in 82 NW and 58 obese (OB) children. RESULTS: Both ghrelin forms in NW were higher (AG, p<0.02; UAG, p<0.0001) than in OB subjects, with similar ratio AG/UAG . While no differences were observed for gender, puberty AG (p<0.01) and UAG (p<0.0001) levels were higher in pre-pubertal than pubertal NW and OB subjects. Adiponectin levels in NW subjects were higher (p<0.001), while leptin and insulin levels were lower (p<0.0001) than in OB subjects. NW children showed homeostasis model assessment (HOMA) and HOMAß indices lower than OB children (p<0.0001) with a higher a quantitative insulin sensitivity check index (p<0.0001). AG and UAG levels correlated to each other (p<0.0001), each showing a negative correlation to age, height, weight and body mass index. Both forms, but more strongly UAG, correlated with adiponectin, leptin, and insulin. CONCLUSIONS: OB children show lower levels of both AG and UAG when compared to NW subjects, with lower levels during puberty. These results demonstrate a peculiar strong relationship between UAG levels and metabolic parameters in the pediatric population, suggesting a role for UAG in metabolic functions.

[Urethroplasty with lateral pedunculated flap for complicated hypospadias]. / Uretroplastia con colgajo lateral pediculado para las hipospadias complicadas.

UNLABELLED: With the modern techniques, we can resolve almost the totallity of hypospadias. But there are patients with recurrent fistulas associated to uretral stenosis that can finish all the surgical possibilities. MATERIAL AND METHODS: Retrospective review. Lateral based flap uretroplasty consists in the exposition of the uretra, to reconstruct in one-stage the original neourethra with the lateral skin of the penis. RESULTS: From 2008, we have correct 5 patients with a mean age of 12,1 years (9-15) and a weight of 55,34 kg (22-98 kg). All of them were previously corrected 3 to 7 times, with recurrent fistulas. At the office, stenosis urethral was verified. Surgical correction was made in 90-110 minutes and results were positive in all patients, correcting the stenosis after a follow-up of more than a year. Only the oldest two patients had present minimally glans fistula. CONCLUSIONS: Lateral based flap urethroplasty is a useful technique for the correction of the complicated fistulas, allowing the correction of the fistulas and the stenosis of the urethra.

Preparation of mixed trialkyl alkylcarbonate derivatives of etidronic acid via an unusual route.

A method to prepare four (3a-d) trialkyl alkylcarbonate esters of etidronate from P,P'-dimethyl etidronate and alkyl chloroformate was developed by utilizing unexpected demethylation and decarboxylation reactions. The reaction with the sterically more hindered isobutyl chloroformate at a lower temperature (90 °C) produced the P,P'-diester (2) as a stable intermediate product. A possible reaction mechanism is discussed to explain these methyl substitutions. These unusual reactions also clarify why it is difficult to prepare alkylcarbonate prodrugs from bisphosphonates. The compounds prepared were analysed by spectroscopic techniques.

Crystal structure of ADAMTS13 CUB domains reveals their role in global latency.

ADAMTS13 is a plasma metalloprotease that is essential for the regulation of von Willebrand factor (VWF) function, mediator of platelet recruitment to sites of blood vessel damage. ADAMTS13 function is dynamically regulated by structural changes induced by VWF binding that convert it from a latent to active conformation. ADAMTS13 global latency is manifest by the interaction of its C-terminal CUB1-2 domains with its central Spacer domain. We resolved the crystal structure of the ADAMTS13 CUB1-2 domains revealing a previously unreported configuration for the tandem CUB domains. Docking simulations between the CUB1-2 domains with the Spacer domain in combination with enzyme kinetic functional characterization of ADAMTS13 CUB domain mutants enabled the mapping of the CUB1-2 domain site that binds the Spacer domain. Together, these data reveal the molecular basis of the ADAMTS13 Spacer-CUB interaction and the control of ADAMTS13 global latency.

Insulin resistance is a risk factor for high blood pressure regardless of body size and fat distribution in obese children.

BACKGROUND AND AIM: The prevalence of children with hypertension is increasing, especially in obese children. This study was to assess the relationship between blood pressure, indexes of adiposity, body fat distribution and insulin resistance. SAMPLE: 1044 children (M/F: 484/560; aged 6-11 years). Anthropometry and blood pressure were measured and fasting blood samples were tested for triacylglycerol, total cholesterol, HDL cholesterol, glucose, insulin and ALT. The prevalence of high blood pressure in overweight males and females was 14.3 and 6.4%, respectively (chi(2)=16.73, p<0.001) and in obese it was 40.4 and 32.8%, respectively (chi(2)=5.56, p<0.001). High blood pressure increased progressively with BMI z-score categories (chi(2)=67.99, p<0.001) as well as with waist/height ratio (W/Hr) categories (chi(2)=23.51, p<0.001). Hypertensive subject had significantly higher insulin (15.6+/-9.8 vs 11.9+/-7.2, p<0.001 and 20.63+/-14.7 vs 15.26+/-9.8, p<0.001 in males and females respectively) and HOMA(IR) (3.23+/-2.1 vs 2.42+/-1.49, p<0.001 and 4.12+/-2.87 vs 3.07+/-1.98, p<0.001 in males and in females, respectively) than non-hypertensive ones. Among metabolic and cardiovascular risk factors, HOMA(IR) was the only variable able to predict high blood pressure in obese boys and girls, in addition to BMI or body fat distribution (waist, W/Hr). The highest HOMA(IR) category was the most important predicting factor of high blood pressure in overweight and obese children in addition to body size or body fat distribution. CONCLUSIONS: Blood pressure is associated with the degree of overweight and the indices of body fat distribution. Insulin resistance is an independent additional risk factor for hypertension.

Breakdown of Scaling and Friction Weakening in Intermittent Granular Flow.

Many materials are produced, processed and stored as grains, while granularity of matter can be crucial in triggering potentially catastrophic geological events like landslides, avalanches and earthquakes. The response of grain assemblies to shear stress is therefore of utmost relevance to both human and natural environment. At low shear rate a granular system flows intermittently by distinct avalanches. In such state the avalanche velocity in time is expected to follow a symmetrical and universal average behavior, whose dependence on the slip size reduces to a scale factor. Analyzing data from long lasting experiments, we observe a breakdown of this scaling: While in short slips velocity shows indeed a self-similar and symmetric profile, it does not in long slips. The investigation of frictional response in these different regimes evidences that this breakdown can be traced back to the onset of a friction weakening, which is of dynamical origin and can amplify instabilities exactly in this critical state, the most frequent state for natural hazards.