The missing link: ARID1B non-truncating variants causing Coffin-Siris syndrome due to protein aggregation.

ARID1B is the most frequently mutated gene in Coffin-Siris syndrome (CSS). To date, the vast majority of causative variants reported in ARID1B are truncating, leading to nonsense-mediated mRNA decay. In the absence of experimental data, only few ARID1B amino acid substitutions have been classified as pathogenic, mainly based on clinical data and their de novo occurrence, while most others are currently interpreted as variants of unknown significance. The present study substantiates the pathogenesis of ARID1B non-truncating/NMD-escaping variants located in the SMARCA4-interacting EHD2 and DNA-binding ARID domains. Overexpression assays in cell lines revealed that the majority of EHD2 variants lead to protein misfolding and formation of cytoplasmic aggresomes surrounded by vimentin cage-like structures and co-localizing with the microtubule organisation center. ARID domain variants exhibited not only aggresomes, but also nuclear aggregates, demonstrating robust pathological effects. Protein levels were not compromised, as shown by quantitative western blot analysis. In silico structural analysis predicted the exposure of amylogenic segments in both domains due to the nearby variants, likely causing this aggregation. Genome-wide transcriptome and methylation analysis in affected individuals revealed expression and methylome patterns consistent with those of the pathogenic haploinsufficiency ARID1B alterations in CSS cases. These results further support pathogenicity and indicate two approaches for disambiguation of such variants in everyday practice. The few affected individuals harbouring EHD2 non-truncating variants described to date exhibit mild CSS clinical traits. In summary, this study paves the way for the re-evaluation of previously unclear ARID1B non-truncating variants and opens a new era in CSS genetic diagnosis.

Elucidating the clinical and molecular spectrum of SMARCC2-associated NDD in a cohort of 65 affected individuals.

PURPOSE: Coffin-Siris and Nicolaides-Baraitser syndromes are recognizable neurodevelopmental disorders caused by germline variants in BAF complex subunits. The SMARCC2 BAFopathy was recently reported. Herein, we present clinical and molecular data on a large cohort. METHODS: Clinical symptoms for 41 novel and 24 previously published affected individuals were analyzed using the Human Phenotype Ontology. For genotype-phenotype correlations, molecular data were standardized and grouped into non-truncating and likely gene-disrupting (LGD) variants. Missense variant protein expression and BAF-subunit interactions were examined using 3D protein modeling, co-immunoprecipitation, and proximity-ligation assays. RESULTS: Neurodevelopmental delay with intellectual disability, muscular hypotonia, and behavioral disorders were the major manifestations. Clinical hallmarks of BAFopathies were rare. Clinical presentation differed significantly, with LGD variants being predominantly inherited and associated with mildly reduced or normal cognitive development, whereas non-truncating variants were mostly de novo and presented with severe developmental delay. These distinct manifestations and non-truncating variant clustering in functional domains suggest different pathomechanisms. In vitro testing showed decreased protein expression for N-terminal missense variants similar to LGD. CONCLUSION: This study improved SMARCC2 variant classification and identified discernible SMARCC2-associated phenotypes for LGD and non-truncating variants, which were distinct from other BAFopathies. The pathomechanism of most non-truncating variants has yet to be investigated.

Ionizable Drug Self-Associations and the Solubility Dependence on pH: Detection of Aggregates in Saturated Solutions Using Mass Spectrometry (ESI-Q-TOF-MS/MS).

It is widely accepted that solubility-pH profiles of ionizable compounds follow the Henderson-Hasselbalch equation. However, several studies point out that compounds often undergo additional processes in saturated solutions, such as sub-micellar oligomerization, micellar aggregation, or drug-buffer complexation among others, which make the experimental profiles deviate from the behavior predicted by the Henderson-Hasselbalch equation. Often, the presence of additional processes is supported by the analysis of experimental data through solubility computer programs. However, the purpose of this work is to experimentally prove the aggregation phenomena for a series of bases for which deviations from the theoretical profile have been observed. To this end, five monoprotic bases (lidocaine, maprotiline, cyproheptadine, bupivacaine, and mifepristone) susceptible to form ionic aggregates in solution have been selected, and mass spectrometry has been the technique of choice to prove the presence of aggregation. High declustering potentials have been applied to prevent aggregates from forming in the ionization source of the mass spectrometer. In addition, haloperidol has been used as a negative control since according to its profile, it is not suspected to form ionic aggregates. In all instances, except for haloperidol, the analysis of the saturated solutions revealed the presence of mixed-charged dimers (aggregates formed by a neutral molecule and a charged one) and even trimers in the case of mifepristone and bupivacaine. For lidocaine, the most soluble of the compounds, the presence of neutral aggregates was also detected. These experiments support the hypothesis that the simple Henderson-Hasselbalch equation may explain the solubility-pH behavior of certain compounds, but it can be somewhat inaccurate in describing the behavior of many other substances.

Binding thermodynamics of paromomycin, neomycin, neomycin-dinucleotide and -diPNA conjugates to bacterial and human rRNA.

Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target-drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A-site, with two well-known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin-dinucleotide and -diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin-diPNA conjugates as the best candidates for antimicrobial activity.

Molecular details of INH-C10 binding to wt KatG and Its S315T mutant.

Isoniazid (INH) is still one of the two most effective antitubercular drugs and is included in all recommended multitherapeutic regimens. Because of the increasing resistance of Mycobacterium tuberculosis to INH, mainly associated with mutations in the katG gene, new INH-based compounds have been proposed to circumvent this problem. In this work, we present a detailed comparative study of the molecular determinants of the interactions between wt KatG or its S315T mutant form and either INH or INH-C10, a new acylated INH derivative. MD simulations were used to explore the conformational space of both proteins, and results indicate that the S315T mutation did not have a significant impact on the average size of the access tunnel in the vicinity of these residues. Our simulations also indicate that the steric hindrance role assigned to Asp137 is transient and that electrostatic changes can be important in understanding the enzyme activity data of mutations in KatG. Additionally, molecular docking studies were used to determine the preferred modes of binding of the two substrates. Upon mutation, the apparently less favored docking solution for reaction became the most abundant, suggesting that S315T mutation favors less optimal binding modes. Moreover, the aliphatic tail in INH-C10 seems to bring the hydrazine group closer to the heme, thus favoring the apparent most reactive binding mode, regardless of the enzyme form. The ITC data is in agreement with our interpretation of the C10 alkyl chain role and helped to rationalize the significantly lower experimental MIC value observed for INH-C10. This compound seems to be able to counterbalance most of the conformational restrictions introduced by the mutation, which are thought to be responsible for the decrease in INH activity in the mutated strain. Therefore, INH-C10 appears to be a very promising lead compound for drug development.

Prospective evaluation of quality of life effects in patients undergoing palliative radiotherapy for brain metastases.

BACKGROUND: Recently published results of quality of life (QoL) studies indicated different outcomes of palliative radiotherapy for brain metastases. This prospective multi-center QoL study of patients with brain metastases was designed to investigate which QoL domains improve or worsen after palliative radiotherapy and which might provide prognostic information. METHODS: From 01/2007-01/2009, n=151 patients with previously untreated brain metastases were recruited at 14 centers in Germany and Austria. Most patients (82 %) received whole-brain radiotherapy. QoL was measured with the EORTC-QLQ-C15-PAL and brain module BN20 before the start of radiotherapy and after 3 months. RESULTS: At 3 months, 88/142 (62 %) survived. Nine patients were not able to be followed up. 62 patients (70.5 % of 3-month survivors) completed the second set of questionnaires. Three months after the start of radiotherapy QoL deteriorated significantly in the areas of global QoL, physical function, fatigue, nausea, pain, appetite loss, hair loss, drowsiness, motor dysfunction, communication deficit and weakness of legs. Although the use of corticosteroid at 3 months could be reduced compared to pre-treatment (63 % vs. 37 %), the score for headaches remained stable. Initial QoL at the start of treatment was better in those alive than in those deceased at 3 months, significantly for physical function, motor dysfunction and the symptom scales fatigue, pain, appetite loss and weakness of legs. In a multivariate model, lower Karnofsky performance score, higher age and higher pain ratings before radiotherapy were prognostic of 3-month survival. CONCLUSIONS: Moderate deterioration in several QoL domains was predominantly observed three months after start of palliative radiotherapy for brain metastases. Future studies will need to address the individual subjective benefit or burden from such treatment. Baseline QoL scores before palliative radiotherapy for brain metastases may contain prognostic information.

Molecular characteristics of several drugs evaluated from solvent/water partition measurements: Solvation parameters and intramolecular hydrogen bond indicator.

A wide set of well-known drugs, most of them included in the Abraham´s reference database, covering a wide variety of chemical structures and therapeutical functionalities were chosen in order to determine some molecular properties from solvent/water partition measurements. Partition data from aqueous solutions and four different solvents (n-dodecane, toluene, chloroform and n-octanol) were measured and reported. From them, Abraham´s molecular descriptors of selected compounds (A, B and S, accounting for hydrogen bond donor, hydrogen bond acceptor and dipolarity/polaritzability, respectively) were estimated. A and B values derived from the experimental measurements strongly agree with the tabulated ones showing the suitability of the used procedure to achieve reliable values for new molecules. However, obtained S values differ from those previously reported for several compounds. Moreover, values for a new indicator of the propensity to form intramolecular hydrogen bonds (Δlog Poct-tol) were estimated from the experimental data and also calculated according to both, the Abraham´s model and the molecular structures (SMD). The quality of both series of calculated descriptors was evaluated by contrast with the experimental values and satisfactory results were obtained in both instances. Thus, the Abraham´s way is useful when molecular descriptors are available but very good estimations can be achieved by SMD, which only requires the drug´s molecular structure.

BDV Syndrome: An Emerging Syndrome With Profound Obesity and Neurodevelopmental Delay Resembling Prader-Willi Syndrome.

CONTEXT: CPE encodes carboxypeptidase E, an enzyme that converts proneuropeptides and propeptide hormones to bioactive forms. It is widely expressed in the endocrine and central nervous system. To date, 4 individuals from 2 families with core clinical features including morbid obesity, neurodevelopmental delay, and hypogonadotropic hypogonadism, harboring biallelic loss-of-function (LoF) CPE variants, have been reported. OBJECTIVE: We describe 4 affected individuals from 3 unrelated consanguineous families, 2 siblings of Syrian, 1 of Egyptian, and 1 of Pakistani descent, all harboring novel homozygous CPE LoF variants. METHODS: After excluding Prader-Willi syndrome (PWS), exome sequencing was performed in both Syrian siblings. The variants identified in the other 2 individuals were reported as research variants in a large-scale exome study and in the ClinVar database. Computational modeling of all possible missense alterations allowed assessing CPE tolerance to missense variants. RESULTS: All affected individuals were severely obese with neurodevelopmental delay and other endocrine anomalies. Three individuals from 2 families shared the same CPE homozygous truncating variant c.361C > T, p.(Arg121*), while the fourth carried the c.994del, p.(Ser333Alafs*22) variant. Comparison of clinical features with previously described cases and standardization according to the Human Phenotype Ontology terms indicated a recognizable clinical phenotype, which we termed Blakemore-Durmaz-Vasileiou (BDV) syndrome. Computational analysis indicated high conservation of CPE domains and intolerance to missense changes. CONCLUSION: Biallelic truncating CPE variants are associated with BDV syndrome, a clinically recognizable monogenic recessive syndrome with childhood-onset obesity, neurodevelopmental delay, hypogonadotropic hypogonadism, and hypothyroidism. BDV syndrome resembles PWS. Our findings suggest missense variants may also be clinically relevant.

Modeling the retention of neutral compounds in gradient elution RP-HPLC by means of polarity parameter models.

A three-parameter expression, which was already employed before for the prediction of the retention time in gradient mode in reversed-phase high-performance liquid chromatography (RP-HPLC) with satisfactory results, has been tested here under a variety of gradient patterns, using methanol and acetonitrile as the organic modifiers. A wide variety of compounds have been employed as test solutes, including some complex ones used as drugs, such as hydrocortisone and estriol. Simplifications of this expression have been made by considering two- and one-parameter expressions based on the p polarity parameter model, which was successfully employed before in isocratic mode to perform predictions of retention time. The advantages that this model gives in isocratic conditions, namely simplicity and less previous experimental work, have been applied with profit in its application to gradient mode. Good correlations between the experimental retention times and the predicted ones have been obtained with both equations in most cases.

Kinetic and thermodynamic solubility values of some bioactive compounds.

Thermodynamic solubility is a decisive physicochemical property in drug development. The Chasing Equilibrium method offers an alternative to the classical procedures to measure the solubility of compounds with acid-base properties. The method is fast and yields accurate results. In this work, the solubility of several compounds including acids and bases was determined through the Chasing Equilibrium approach. A study of experimental conditions in terms of sample weight was performed to measure solubilities. The study shows that only a limited range of weights, depending on the nature and solubility of the compounds, is adequate to obtain reliable results.

A fast method for pKa determination by capillary electrophoresis.

In this article, we describe a recently developed capillary-electrophoresis method for the determination of acidity constants and compare it with other existing methods. The new method is based on the use of an internal standard (compound similar in nature and pKa value to the analyte), and offers several benefits, since it has all the advantages of capillary electrophoresis. In addition, it is very fast, because the exact measure of the pH of the separation electrolytes is not needed, and only a few electrophoretic runs are required to perform a pKa determination. The acidity constants of some monoprotic weak acids and bases were determined by this fast method, yielding a very good agreement with literature values.

Effect of temperature on the chromatographic retention of ionizable compounds. III. Modeling retention of pharmaceuticals as a function of eluent pH and column temperature in RPLC.

We propose a general simple equation for accurately predicting the retention factors of ionizable compounds upon simultaneous changes in mobile phase pH and column temperature at a given hydroorganic solvent composition. Only four independent experiments provide the input data: retention factors measured in two pH buffered mobile phases at extreme acidic and basic pH values (e. g., at least +/- 2 pH units far from the analyte pK(a)) and at two column temperatures. The equations, derived from the basic thermodynamics of the acid-base equilibria, additionally require the knowledge of the solute pK(a )and enthalpies of acid-base dissociation of both the solute and the buffer components in the hydroorganic solvent mixture. The performance of the predictive model is corroborated with the comparison between theoretical and experimental retention factors of several weak acids and bases of important pharmacological activity, in mobile phases containing different buffer solutions prepared in 25% w/w ACN in water and at several temperatures.

Application of a polarity parameter model to the separation of fat-soluble vitamins by reversed-phase HPLC.

The retention behavior of a series of fat-soluble vitamins has been established on the basis of a polarity retention model: log k = (log k)(0) + p (P(m) (N) - P(s) (N)), with p being the polarity of the solute, P(m) (N) the mobile phase polarity, and (log k)(0) and P(m) (N) two parameters for the characterization of the stationary phase. To estimate the p-values of solutes, two approaches have been considered. The first one is based on the application of a QSPR model, derived from the molecular structure of solutes and their log P(o/w), while in the second one, the p-values are obtained from several experimental measurements. The quality of prediction of both approaches has also been evaluated, with the second one giving more accurate results for the most lipophilic vitamins. This model allows establishing the best conditions to separate and determine simultaneously some fat-soluble vitamins in dairy foods.

Single-cell RNA sequencing of adult mouse testes.

Spermatogenesis is an efficient and complex system of continuous cell differentiation. Previous studies investigating the transcriptomes of different cell populations in the testis relied either on sorting cells, cell depletion, or juvenile animals where not all stages of spermatogenesis have been completed. We present single-cell RNA sequencing (scRNA-Seq) data of 2,500 cells from the testes of two 8-week-old C57Bl/6J mice. Our dataset includes all spermatogenic stages from preleptotene to condensing spermatids as well as individual spermatogonia, Sertoli and Leydig cells. The data capture the full continuity of the meiotic and postmeiotic stages of spermatogenesis, and is thus ideally suited for marker discovery, network inference and similar analyses for which temporal ordering of differentiation processes can be exploited. Furthermore, it can serve as a reference for future studies involving single-cell RNA-Seq in mice where spermatogenesis is perturbed.

Molecular interactions between warfarin and human (HSA) or bovine (BSA) serum albumin evaluated by isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE).

Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding site of human (HSA) or bovine (BSA) serum albumin have been examined in strictly controlled experimental conditions (HEPES buffer 50 mM, pH 7.4 and 25 °C) by means of isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE). Each technique is based on measurements of a different property of the biochemical system, and then the results allow a critical discussion about the suitability of each approach to estimate the drug-protein binding parameters. The strongest interaction step is properly evaluated by the three assayed approaches being the derived binding constants strongly consistent: from 4 × 104 to 7 × 104 for HSA and from 0.8 × 105 to 1.2 × 105 for BSA. Binding enthalpy variations also show consistent results: -5.4 and -5.6 Kcal/mol for HSA and -4.3 and -3.7 Kcal/mol for BSA, as measured by ITC and FS, respectively. Further high order interaction events for both albumins are detected only by FA/CE.

Critical comparison of shake-flask, potentiometric and chromatographic methods for lipophilicity evaluation (log Po/w) of neutral, acidic, basic, amphoteric, and zwitterionic drugs.

In the present study three different procedures have been compared for the determination of the lipophilicity of the unionized species (log Po/w) of neutral, acidic, basic, amphoteric, and zwitterionic drugs. Shake-flask, potentiometric and chromatographic approaches have been assayed in a set of 66 representative compounds in different phases of advanced development. An excellent equivalence has been found between log Po/w values obtained by shake-flask and potentiometry, while the chromatographic approach is less accurate but very convenient for screening purposes when a high-throughput is required. In the case of zwitterionic and amphoteric compounds, either for shake-flask and chromatographic methods, the pH has to be accurately selected in order to ensure the compound to be in its neutral form.

Effect of vinylpyrrolidone polymers on the solubility and supersaturation of drugs; a study using the Cheqsol method.

The development of methods to increase the bioavailability of drugs is of great interest, especially for those which are poorly soluble or permeable. One of the strategies to enhance the solubility (which in turn has the potential of increase bioavailability) of drugs is the use of additives in the formulation process, so that the drug can stay supersaturated in biological fluids for a period of time long enough to allow absorption. The use of polymers as pharmaceutical excipients in order to stabilize the supersaturation of drugs is common practice. In this work, the ability of different polymers of vinylpyrrolidone (K-12, K-17, K-25, K-29/32, K-90) and a copolymer of vinylpyrrolidone and vinylacetate (S-630) have been tested for their impact on the supersaturation of drugs. Sixteen drugs of different chemical nature have been selected, and analyzed using the Cheqsol method. The results of the drug alone, and of physical mixtures with the different polymers at several polymer:drug ratios have been compared in terms of supersaturation extent and duration. It has been observed that acidic compounds displayed enhanced solubility in different ways: sometimes the supersaturated state of the drug is maintained for a long time, due to the precipitation of an amorphous solid, as determined by X-ray diffraction studies; on other occasions supersaturation increases but only for a short time, compared to the drug alone, and then the drug precipitates to a crystalline form. Only a few basic drugs displayed enhanced solubility in the presence of PVP polymers, in contrast to acidic compounds.

Retention of ionisable compounds on high-performance liquid chromatography XVII. Estimation of the pH variation of aqueous buffers with the change of the methanol fraction of the mobile phase.

The use of methanol-aqueous buffer mobile phases in HPLC is a common election when performing chromatographic separations of ionisable analytes. The addition of methanol to the aqueous buffer to prepare such a mobile phase changes the buffer capacity and the pH of the solution. In the present work, the variation of these buffer properties is studied for acetic acid-acetate, phosphoric acid-dihydrogenphosphate-hydrogenphosphate, citric acid-dihydrogencitrate-hydrogencitrate-citrate, and ammonium-ammonia buffers. It is well established that the pH change of the buffers depends on the initial concentration and aqueous pH of the buffer, on the percentage of methanol added, and on the particular buffer used. The proposed equations allow the pH estimation of methanol-water buffered mobile phases up to 80% in volume of organic modifier from initial aqueous buffer pH and buffer concentration (before adding methanol) between 0.001 and 0.01 mol L(-1). From both the estimated pH values of the mobile phase and the estimated pKa of the ionisable analytes, it is possible to predict the degree of ionisation of the analytes and therefore, the interpretation of acid-base analytes behaviour in a particular methanol-water buffered mobile phase.

Determination of the chromatographic hydrophobicity index for ionisable solutes.

A model that relates the chromatographic hydrophobicity index (CHI) to the pH of the mobile phase has been tested in two of the most common high-performance liquid chromatography (HPLC) solvents: methanol and acetonitrile. A set of eight monoprotic acids and nine monoprotic bases of different chemical nature (phenols, benzoic acids, anilines, and pyridine) have been selected for the validation. The variation of CHI values with the pH of the mobile phase shows a good fit to the model for almost all compounds, regardless of their nature, and similar CHI values of the neutral form of the substances are obtained in both organic modifiers. On the contrary, higher differences are observed for the ionic form of the test solutes. The values of the other parameters obtained with the model (s, pK(a)) are discussed according to the nature of the compounds, and the variation of solvent pH and compounds pK(a) along the gradient elution.