Repurposing SGLT2 inhibitors: Treatment of renal proximal tubulopathy in Fanconi-Bickel syndrome with empagliflozin.

Renal proximal tubulopathy in Fanconi-Bickel syndrome is caused by impaired basolateral glucose transport via GLUT2 and consequently, intracellular accumulation of glucose and glycogen. SGLT2 inhibitors act on apical glucose reabsorption of renal proximal tubular cells. The purpose of this study was to retrospectively describe the first experiences with repurposing the SGLT2 inhibitor empagliflozin to treat the generalized tubulopathy in Fanconi-Bickel syndrome. A case series was conducted of seven persons from five families (five males, two females; three children, who were 14y5m, 2y9m, and 1y6m old) with genetically confirmed Fanconi-Bickel syndrome, off-label treated with empagliflozin. Median (range) age at start of empagliflozin was 27 years (1y6m - 61y) and duration of follow-up under empagliflozin treatment was 169 days (57-344). Under empagliflozin (up to 25 mg/d), biochemical parameters of tubular cell integrity (urinary N-acetyl-glucosaminidase) and/or tubular functions (including urinary α1-microglobulin) improved in all persons with Fanconi-Bickel syndrome, albeit to varying degrees. Clinically, supplementations (i.e., phosphate, alkali, carnitine, and alfacalcidol) could be completely discontinued in the three children, whereas results in the four adult patients were more variable and not as significant. Empagliflozin was well-tolerated and no symptomatic hypoglycemia was observed. In conclusion, SGLT2 inhibitors such as empagliflozin shift the metabolic block in Fanconi-Bickel syndrome, that is, they intervene specifically in the underlying pathophysiology and can thus attenuate renal proximal tubulopathy, especially when started in early childhood.

The relation between dietary polysaccharide intake and urinary excretion of tetraglucoside.

The urinary metabolite tetraglucoside (Glc4) is a potential biomarker for hepatic glycogen storage diseases (GSDs). Glc4 is believed to reflect body glycogen content and/or turnover. However, dietary polysaccharide intake may influence Glc4 excretion, potentially limiting the utility of Glc4 as a monitoring biomarker in hepatic GSDs. We aimed to investigate the association of dietary polysaccharide intake with Glc4 excretion. Urinary Glc4 excretion (mmol/mmol creatinine and mmol/24 h) was analyzed using a validated LC-MS/MS method. Data was analyzed from 65 kidney transplant recipients and 58 healthy kidney donors in the TransplantLines cohort study. Spearman's correlation and multivariable linear regression analyses were performed. In the multivariable analysis, dry lean body mass (DLBM), dietary polysaccharide intake, transplantation status, age, sex, and glycated hemoglobin (HbA1c) served as independent variables. Daily variation was examined in 21 healthy individuals of urinary Glc4 excretion in 2-h collections over a 24-h period. Mixed generalized additive models were built to study the association of prior polysaccharide intake with Glc4 excretion. No (univariate) associations were found between polysaccharide intake and Glc4 excretion. However, a significant interaction between DLBM and polysaccharide on 24 h Glc4 excretion was observed in the multivariate analysis. Glc4 excretion throughout the day exhibited no relationship to prior polysaccharide intake. Our findings suggest an indirect effect of polysaccharide intake on Glc4 excretion, potentially due to changes in muscle glycogen content and/or turnover. We have found no evidence that dietary polysaccharides under normal intakes increase urinary Glc4 directly.

Fasting and meal-stimulated serum C-peptide in long-standing type 1 diabetes mellitus.

AIMS: This study aims to evaluate the stability of C-peptide over time and to compare fasting C-peptide and C-peptide response after mixed-meal tolerance test (MMTT) at T90 or T120 with C-peptide area under the curve (AUC) in long-standing type 1 diabetes. METHODS: We included 607 type 1 diabetes individuals with diabetes duration >5 years. C-peptide concentrations (ultrasensitive assay) were collected in the fasting state, and in a subpopulation after MMTT (T0, just prior to, T30-T60-T90-T120, 30-120 min after ingestion of mixed-meal) (n = 168). Fasting C-peptide concentrations (in n = 535) at Year 0 and Year 1 were compared. The clinical determinants associated with residual C-peptide secretion and the correspondence of C-peptide at MMTT T90 / T120 and total AUC were assessed. RESULTS: A total of 153 participants (25%) had detectable fasting serum C-peptide (i.e ≥ 3.8 pmol/L). Fasting C-peptide was significantly lower at Year 1 (p < 0.001, effect size = -0.16). Participants with higher fasting C-peptide had a higher age at diagnosis and shorter disease duration and were less frequently insulin pump users. Overall, 109 of 168 (65%) participants had both non-detectable fasting and post-meal serum C-peptide concentrations. The T90 and T120 C-peptide values at MMTT were concordant with total AUC. In 17 (10%) individuals, C-peptide was only detectable at MMTT and not in the fasting state. CONCLUSIONS: Stimulated C-peptide was detectable in an additional 10% of individuals compared with fasting in individuals with >5 years of diabetes duration. T90 and T120 MMTT measurements showed good concordance with the MMTT total AUC. Overall, there was a decrease of C-peptide at 1-year follow-up.

How low is really low? Comparison of two C-peptide assays to establish residual C-peptide production in type 1 diabetes.

INTRODUCTION: C-peptide is an important marker to assess residual insulin production in individuals with type 1 diabetes (T1D). The accuracy and detection limits of C-peptide assays are important to detect C-peptide microsecretion and to reliably observe changes over time in these people. We compared and verified two commercially available assays able to measure C-peptide in the picomolar range. METHODS: The ultrasensitive Mercodia enzyme-linked immunosorbent C-peptide assay (ELISA) was compared with the Beckman immunoradiometric assay (IRMA) for C-peptide, assessing reproducibility (coefficient of variation [CV]), limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ). RESULTS: For both assays within-run and between-run variation were high at the low (around the detection limit) C-peptide concentration range, with CVs of around 40%. LoB values for the ultrasensitive ELISA and the IRMA were 1.3 and 0.16 pmol/L respectively. LoD values were 2.4 and 0.54 pmol/L respectively. LoQ values were 9.7 and 3.8 pmol/L respectively. Only the IRMA met the specifications claimed by the manufacturer. CONCLUSIONS: The IRMA provided the lowest threshold for quantification of serum C-peptide. LoQ of commercially available assays should be established in-house before applying them in research studies and clinical trials in which low C-peptide levels have clinical or scientific relevance.

Chest CT for triage during COVID-19 on the emergency department: myth or truth?

PURPOSE: We aimed to investigate the diagnostic performance of chest CT compared with first RT-PCR results in adult patients suspected of COVID-19 infection in an ED setting. We also constructed a predictive machine learning model based on chest CT and additional data to improve the diagnostic accuracy of chest CT. METHODS: This study's cohort consisted of 319 patients who underwent chest CT and RT-PCR testing at the ED. Patient characteristics, demographics, symptoms, vital signs, laboratory tests, and chest CT results (CO-RADS) were collected. With first RT-PCR as reference standard, the diagnostic performance of chest CT using the CO-RADS score was assessed. Additionally, a predictive machine learning model was constructed using logistic regression. RESULTS: Chest CT, with first RT-PCR as a reference, had a sensitivity, specificity, PPV, and NPV of 90.2%, 88.2%, 84.5%, and 92.7%, respectively. The prediction model with CO-RADS, ferritin, leucocyte count, CK, days of complaints, and diarrhea as predictors had a sensitivity, specificity, PPV, and NPV of 89.3%, 93.4%, 90.8%, and 92.3%, respectively. CONCLUSION: Chest CT, using the CO-RADS scoring system, is a sensitive and specific method that can aid in the diagnosis of COVID-19, especially if RT-PCR tests are scarce during an outbreak. Combining a predictive machine learning model could further improve the accuracy of diagnostic chest CT for COVID-19. Further candidate predictors should be analyzed to improve our model. However, RT-PCR should remain the primary standard of testing as up to 9% of RT-PCR positive patients are not diagnosed by chest CT or our machine learning model.

Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate.

Liquid-liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membrane-free compartments by phase separation.

Associative Interactions in Crowded Solutions of Biopolymers Counteract Depletion Effects.

The cytosol of Escherichia coli is an extremely crowded environment, containing high concentrations of biopolymers which occupy 20-30% of the available volume. Such conditions are expected to yield depletion forces, which strongly promote macromolecular complexation. However, crowded macromolecule solutions, like the cytosol, are very prone to nonspecific associative interactions that can potentially counteract depletion. It remains unclear how the cytosol balances these opposing interactions. We used a FRET-based probe to systematically study depletion in vitro in different crowded environments, including a cytosolic mimic, E. coli lysate. We also studied bundle formation of FtsZ protofilaments under identical crowded conditions as a probe for depletion interactions at much larger overlap volumes of the probe molecule. The FRET probe showed a more compact conformation in synthetic crowding agents, suggesting strong depletion interactions. However, depletion was completely negated in cell lysate and other protein crowding agents, where the FRET probe even occupied slightly more volume. In contrast, bundle formation of FtsZ protofilaments proceeded as readily in E. coli lysate and other protein solutions as in synthetic crowding agents. Our experimental results and model suggest that, in crowded biopolymer solutions, associative interactions counterbalance depletion forces for small macromolecules. Furthermore, the net effects of macromolecular crowding will be dependent on both the size of the macromolecule and its associative interactions with the crowded background.

Alcohol use disorder and muscle weakness: Original study of the effect of vitamin D supplementation in ambulatory participants with alcohol use disorder.

INTRODUCTION: Chronic alcohol-related myopathy presents with proximal muscle weakness. We studied the effect of vitamin D supplementation on muscle weakness in adults with alcohol use disorder. METHOD: The study was a randomized controlled trial. Participants were community-dwelling adults with alcohol use disorder. Participants allocated to VIDIO, vitamin D intensive outreach, received bimonthly oral doses of 50,000-100,000 IU cholecalciferol for 12 months. Participants allocated to CAU, care as usual, received prescriptions of once-a-day tablets containing 800 IU cholecalciferol and 500 mg calcium carbonate. Data included demographic variables, laboratory tests, alcohol use, and rating scales of help-seeking and support. Main outcomes were the participants' quadriceps maximum voluntary contractions (qMVC) and serum-25(OH)vitamin D concentrations, 25(OH)D. RESULTS: In 66 participants, sex ratio 50/16, mean age 51 years, alcohol use was a median of 52 [IQR 24-95] drinks per week. Baseline qMVC values were 77% (SD 29%) of reference values. Laboratory tests were available in 44/66 participants: baseline 25(OH)D concentrations were 39.4 (SD 23.7) nmol/L. Thirty-one participants with 25(OH)D concentrations <50 nmol/L received either VIDIO or CAU and improved in qMVC, respectively, with a mean of 51 (p < 0.05) and 62 N (no p value because of loss of follow-up) after one year of treatment. Vitamin D status increased with a mean of +56.1 and + 37.4 nmol/L, respectively, in VIDIO and CAU. CONCLUSION: The qMVC values improved during vitamin supplementation in adults with vitamin D deficiency and alcohol use disorder. Despite higher 25(OH)D concentrations in VIDIO, in terms of muscle health no advice could be given in favor of one vitamin strategy over the other.

Impact of Phenylketonuria on the Serum Metabolome and Plasma Lipidome: A Study in Early-Treated Patients.

BACKGROUND: Data suggest that metabolites, other than blood phenylalanine (Phe), better and independently predict clinical outcomes in patients with phenylketonuria (PKU). METHODS: To find new biomarkers, we compared the results of untargeted lipidomics and metabolomics in treated adult PKU patients to those of matched controls. Samples (lipidomics in EDTA-plasma (22 PKU and 22 controls) and metabolomics in serum (35 PKU and 20 controls)) were analyzed using ultra-high-performance liquid chromatography and high-resolution mass spectrometry. Data were subjected to multivariate (PCA, OPLS-DA) and univariate (Mann-Whitney U test, p < 0.05) analyses. RESULTS: Levels of 33 (of 20,443) lipid features and 56 (of 5885) metabolite features differed statistically between PKU patients and controls. For lipidomics, findings include higher glycerolipids, glycerophospholipids, and sphingolipids species. Significantly lower values were found for sterols and glycerophospholipids species. Seven features had unknown identities. Total triglyceride content was higher. Higher Phe and Phe catabolites, tryptophan derivatives, pantothenic acid, and dipeptides were observed for metabolomics. Ornithine levels were lower. Twenty-six metabolite features were not annotated. CONCLUSIONS: This study provides insight into the metabolic phenotype of PKU patients. Additional studies are required to establish whether the observed changes result from PKU itself, diet, and/or an unknown reason.

Hydrazine synthase, a unique phylomarker with which to study the presence and biodiversity of anammox bacteria.

Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the biogeochemical cycling of nitrogen. They derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. Several methods have been used to detect the presence and activity of anammox bacteria in the environment, including 16S rRNA gene-based approaches. The use of the 16S rRNA gene to study biodiversity has the disadvantage that it is not directly related to the physiology of the target organism and that current primers do not completely capture the anammox diversity. Here we report the development of PCR primer sets targeting a subunit of the hydrazine synthase (hzsA), which represents a unique phylogenetic marker for anammox bacteria. The tested primers were able to retrieve hzsA gene sequences from anammox enrichment cultures, full-scale anammox wastewater treatment systems, and a variety of freshwater and marine environmental samples, covering all known anammox genera.

Polycyclic aromatic hydrocarbons as plausible prebiotic membrane components.

Aromatic molecules delivered to the young Earth during the heavy bombardment phase in the early history of our solar system were likely to be among the most abundant and stable organic compounds available. The Aromatic World hypothesis suggests that aromatic molecules might function as container elements, energy transduction elements and templating genetic components for early life forms. To investigate the possible role of aromatic molecules as container elements, we incorporated different polycyclic aromatic hydrocarbons (PAH) in the membranes of fatty acid vesicles. The goal was to determine whether PAH could function as a stabilizing agent, similar to the role that cholesterol plays in membranes today. We studied vesicle size distribution, critical vesicle concentration and permeability of the bilayers using C(6)-C(10) fatty acids mixed with amphiphilic PAH derivatives such as 1-hydroxypyrene, 9-anthracene carboxylic acid and 1,4 chrysene quinone. Dynamic Light Scattering (DLS) spectroscopy was used to measure the size distribution of vesicles and incorporation of PAH species was established by phase-contrast and epifluorescence microscopy. We employed conductimetric titration to determine the minimal concentration at which fatty acids could form stable vesicles in the presence of PAHs. We found that oxidized PAH derivatives can be incorporated into decanoic acid (DA) vesicle bilayers in mole ratios up to 1:10 (PAH:DA). Vesicle size distribution and critical vesicle concentration were largely unaffected by PAH incorporation, but 1-hydroxypyrene and 9-anthracene carboxylic acid lowered the permeability of fatty acid bilayers to small solutes up to 4-fold. These data represent the first indication of a cholesterol-like stabilizing effect of oxidized PAH derivatives in a simulated prebiotic membrane.

Which C-peptide assay do you use? Increasing need for describing C-peptide assay performance.

Many C-peptide assays are commercially available for research and routine use. However, not all assays yield consistent results, especially in the low concentration ranges. We searched the literature describing C-peptide measurements to assess which assays are mainly used in the diabetes research field and if they are specified. Percentages of publications on C-peptide measurements in type 1 diabetes (T1D), type 2 diabetes (T2D) and other forms of diabetes were 32%, 54% and 14%, respectively. In only 54% of the publications the used assay was specified. Information on detection limit, measurement range and variation was provided in 12%, 2% and 11% of publications, respectively. In 22% of all publications no C-peptides concentrations were mentioned. This may be a problem especially for T1D research, where measuring very low levels of C-peptide is becoming increasingly important and concordance between assays is low.

Impact of Thyroglobulin and Thyroglobulin Antibody Assay Performance on the Differential Classification of DTC Patients.

CONTEXT: Measurements of thyroglobulin (Tg) and Tg antibodies are crucial in the follow-up of treated differentiated thyroid cancer (DTC) patients. Interassay differences may significantly impact follow-up. OBJECTIVE: The aim of this multicenter study was to explore the impact of Tg and Tg antibody assay performance on the differential classification of DTC patients, as described in national and international guidelines. DESIGN: Four commonly used Tg and Tg antibody assays were technically compared to reflect possible effects on patients with DTC follow-up. Storage stability at different storage temperatures was also investigated for LIAISON® and Kryptor assays, as this is an underexposed topic in current literature. RESULTS: B.R.A.H.M.S. assays yield approximately 50% lower Tg values over the whole range compared to the DiaSorin and Roche assays investigated. These differences between assays may result in potential misclassification in up to 7% of patients if fixed cutoffs (eg, 1 ng/mL) are applied. Poor correlation was also observed between the Tg antibody assays when the method-specific upper limits of normal are used as cutoffs. Storage of Tg and Tg antibodies was possible for 3 to 4 weeks at -20°C and -80°C. Calibration of the assays, however, was found to be crucial for stable results over time. CONCLUSIONS: Technical aspects of Tg and Tg antibody assays, including interassay differences, calibration and standardization, and cutoff values, may have a significant clinical impact on the follow-up of DTC patients.

[Vitamin B6 deficiency caused by the use of levodopa]. / Vitamine B6-deficiëntie bij gebruik van levodopa.

BACKGROUND: There are indications that the use of levodopa/carbidopa can cause symptomatic vitamin B6 deficiency. However, this has only been described for patients who used the product in the form of an intestinal gel. CASE DESCRIPTION: We are describing 3 patients who developed vitamin B6 deficiency while using levodopa/carbidopa in tablet form. All 3 patients developed symptoms consistent with peripheral axonal polyneuropathy. CONCLUSION: The cases of our patients are providing support for the idea that oral use of levodopa/carbidopa can cause symptomatic vitamin B6 deficiency.

Dissipative adaptation in driven self-assembly leading to self-dividing fibrils.

Out-of-equilibrium self-assembly of proteins such as actin and tubulin is a key regulatory process controlling cell shape, motion and division. The design of functional nanosystems based on dissipative self-assembly has proven to be remarkably difficult due to a complete lack of control over the spatial and temporal characteristics of the assembly process. Here, we show the dissipative self-assembly of FtsZ protein (a bacterial homologue of tubulin) within coacervate droplets. More specifically, we show how such barrier-free compartments govern the local availability of the energy-rich building block guanosine triphosphate, yielding highly dynamic fibrils. The increased flux of FtsZ monomers at the tips of the fibrils results in localized FtsZ assembly, elongation of the coacervate compartments, followed by division of the fibrils into two. We rationalize the directional growth and division of the fibrils using dissipative reaction-diffusion kinetics and capillary action of the filaments as main inputs. The principle presented here, in which open compartments are used to modulate the rates of dissipative self-assembly by restricting the absorption of energy from the environment, may provide a general route to dissipatively adapting nanosystems exhibiting life-like behaviour.

Macromolecular crowding creates heterogeneous environments of gene expression in picolitre droplets.

Understanding the dynamics of complex enzymatic reactions in highly crowded small volumes is crucial for the development of synthetic minimal cells. Compartmentalized biochemical reactions in cell-sized containers exhibit a degree of randomness due to the small number of molecules involved. However, it is unknown how the physical environment contributes to the stochastic nature of multistep enzymatic processes. Here, we present a robust method to quantify gene expression noise in vitro using droplet microfluidics. We study the changes in stochasticity in the cell-free gene expression of two genes compartmentalized within droplets as a function of DNA copy number and macromolecular crowding. We find that decreased diffusion caused by a crowded environment leads to the spontaneous formation of heterogeneous microenvironments of mRNA as local production rates exceed the diffusion rates of macromolecules. This heterogeneity leads to a higher probability of the molecular machinery staying in the same microenvironment, directly increasing the system's stochasticity.

Rational design of functional and tunable oscillating enzymatic networks.

Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks.

Sliding over the blocks in enzyme-free RNA copying--one-pot primer extension in ice.

Template-directed polymerization of RNA in the absence of enzymes is the basis for an information transfer in the 'RNA-world' hypothesis and in novel nucleic acid based technology. Previous investigations established that only cytidine rich strands are efficient templates in bulk aqueous solutions while a few specific sequences completely block the extension of hybridized primers. We show that a eutectic water/ice system can support Pb(2+)/Mg(2+)-ion catalyzed extension of a primer across such sequences, i.e. AA, AU and AG, in a one-pot synthesis. Using mixtures of imidazole activated nucleotide 5'-monophosphates, the two first "blocking" residues could be passed during template-directed polymerization, i.e., formation of triply extended products containing a high fraction of faithful copies was demonstrated. Across the AG sequence, a mismatch sequence was formed in similar amounts to the correct product due to U·G wobble pairing. Thus, the template-directed extension occurs both across pyrimidine and purine rich sequences and insertions of pyrimidines did not inhibit the subsequent insertions. Products were mainly formed with 2'-5'-phosphodiester linkages, however, the abundance of 3'-5'-linkages was higher than previously reported for pyrimidine insertions. When enzyme-free, template-directed RNA polymerization is performed in a eutectic water ice environment, various intrinsic reaction limitations observed in bulk solution can then be overcome.