Modeling the Mechanisms by Which Coexisting Biomolecular RNA-Protein Condensates Form.

Liquid-liquid phase separation is an emerging mechanism for intracellular organization. This work presents a mathematical model to examine molecular mechanisms that yield phase-separated droplets composed of different RNA-protein complexes. Using a Cahn-Hilliard diffuse interface model with a Flory-Huggins free energy scheme, we explore how multiple (here two, for simplicity) protein-RNA complexes (species) can establish a heterogeneous droplet field where droplets with single or multiple species phase separate and evolve during coarsening. We show that the complex-complex de-mixing energy tunes whether the complexes co-exist or form distinct droplets, while the transient binding kinetics dictate both the timescale of droplet formation and whether distinct species phase separate into droplets simultaneously or sequentially. For specific energetics and kinetics, a field of droplets driven by the formation of only one protein-RNA complex will emerge. Slowly, the other droplet species will accumulate inside the preformed droplets of the other species, allowing them to occupy the same droplet space. Alternatively, unfavorable species mixing creates a parasitic relationship: the slow-to-form protein-RNA complex will accumulate at the surface of a competing droplet species, siphoning off the free protein as it is released. Once this competing protein-RNA complex has sufficiently accumulated on the droplet surface, it can form a new droplet that is capable of sharing an interface with the first complex droplet but is not capable of mixing. These results give insights into a wide range of phase-separation scenarios and heterogeneous droplets that coexist but do not mix within the nucleus and the cytoplasm of cells.

A minimal physiologically based pharmacokinetic model that predicts anti-PEG IgG-mediated clearance of PEGylated drugs in human and mouse.

Circulating antibodies that specifically bind polyethylene glycol (PEG), a polymer routinely used in protein and nanoparticle therapeutics, have been associated with reduced efficacy and increased adverse reactions to some PEGylated therapeutics. In addition to acute induction of anti-PEG antibodies (APA) by PEGylated drugs, typically low but detectable levels of APA are also found in up to 70% of the general population. Despite the broad implications of APA, the dynamics of APA-mediated clearance of PEGylated drugs, and why many patients continue to respond to PEGylated drugs despite the presence of pre-existing APA, remains not well understood. Here, we developed a minimal physiologically based pharmacokinetic (mPBPK) model that incorporates various properties of APA and PEGylated drugs. Our mPBPK model reproduced clinical PK data of APA-mediated accelerated blood clearance of pegloticase, as well as APA-dependent elimination of PEGyated liposomes in mice. Our model predicts that the prolonged circulation of PEGylated drugs will be compromised only at APA concentrations greater than ~500 ng/mL, providing a quantitative explanation to why the effects of APA on PEGylated treatments appear to be limited in most patients. This mPBPK model is readily adaptable to other PEGylated drugs and particles to predict the precise levels of APA that could render them ineffective, providing a powerful tool to support the development and interpretation of preclinical and clinical studies of various PEGylated therapeutics.

Nonlinear signatures of entangled polymer solutions in active microbead rheology.

We present experimental data and numerical modeling of a nonlinear phenomenon in active magnetic microbead rheology that appears to be common to entangled polymer solutions (EPS). Dynamic experiments in a modest range of magnetic forces show: 1. a short-lived high viscosity plateau, followed by 2. a bead acceleration phase with a sharp drop in apparent viscosity, and 3. a terminal steady state that we show resides on the shear-thinning slope of the steady-state flow curve from cone and plate data. This latter feature implies a new protocol to access the nonlinear steady-state flow curve for many biological EPS only available in microliter-scale volumes. We solve the moment-closure form of the Rolie-Poly kinetic model for EPS hydrodynamics, together with a decoupling approximation that obviates the need for a full 3D flow solver, and show that the model qualitatively reproduces the dynamic experimental sequence above. In this way, we explain the phenomenon in terms of entangled polymer physics, and show how the nonlinear event (acceleration and termination on the shear-thinning response curve) is tunable by the interplay between molecular-scale mechanisms (relaxation via reptation and chain retraction) and magnetic force controls. The experimental conditions mimic movement of cilia tips, bacteria, and sperm in mucus barriers, implying a physiological relevance of the phenomenon, and compelling further development of the fully coupled, 3D flow-microstructure model to achieve quantitative accuracy.

[A new possible phenotype of carbimazole embryopathy: A case report]. / Un nouveau phénotype possible d'embryopathie au carbimazole : à propos d'une observation.

Many disorders have been described in infants exposed to carbimazole during the first weeks of pregnancy. The most common of them are congenital aplasia cutis, choanal atresia and esophageal atresia. Rather unspecific dysmorphic features and developmental delay have also been reported. This set of congenital malformations suggests the existence of a phenotype of carbimazole embryopathy. To date, about 30 cases have been reported. We report on a new case of pregnancy accidentally conducted under carbimazole which gave birth to a newborn presenting with a hypertrophic pyloric stenosis associated with hiatus hernia and tracheomalacia. These anomalies have been associated with other malformations already identified in children exposed in utero to carbimazole such as scalp defects, retrognathia and gothic palate. As no relation between propylthiouracil and congenital malformations has yet been described, this drug seems highly preferable for pregnant women presenting with hyperthyroidism during the 1st trimester of their pregnancy.

21-Hydroxylase deficiency: an exemplary model of the contribution of molecular biology in the understanding and management of the disease.

Congenital adrenal hyperplasia (CAH) is a family of autosomal recessive disorders caused by mutations in genes encoding the enzymes involved in one of the various steps of adrenal steroid synthesis. Steroid 21-hydroxylase deficiency (21-OHD) is responsible for over 95% of the 5 forms of CAH, and results due to enzymatic defect owing to mutation in the CYP21 gene. The disease has two major clinical presentations. The "classical" form is severe, and divided into a salt wasting (SW) and simple virilizing (SV) subgroups. In both, affected female fetuses undergo virilization of the external genitalia prenatally and present at birth with sexual ambiguity. In addition, in both sexes infants with SW CAH are at risk of life-threatening adrenal crisis without treatment. This is why it is so important to make a diagnosis and to counsel the families. The diagnosis is easy by measuring the plasma levels of 17-hydroxyprogesterone (17-OHP) in antenatal (amniotic fluid), or perinatal samples (peripheral blood). Confirmation by molecular genetic analysis is advised. The second form of 21-OHD is called "non classical" because the presentation is much less severe and the onset of clinical expression occurs long after birth, often in the peripubertal period, as non-specific symptoms of hyperandrogeny. The unambiguous diagnosis of the latter requires a simple short ACTH test, with the measurement of 17-OHP at 60 min. In both forms, the mutations on the gene CYP21 responsible for the disease are now well known and can be identified by molecular biology techniques. There is a good correlation between phenotypes and genotypes, due to variable amount of the 21-hydroxylase-enzyme activity left (null to 50-60%). SW, SV and NC forms are associated with distinct mutations or combination of mutations. Nowadays, by combining hormonal and molecular tests, it is possible to predict the clinical form of the disease in a given family in the context of a prenatal diagnosis, which can lead to a prenatal treatment. Therefore, 21-OHD genotyping also appears essential for a new approach of genetic counseling, prediction of clinical form after postnatal screening and to define the post-ACTH 17-OHP values indicating the cut-off lines between NC, heterozygote and normal subjects.

[Adrenal hypoplasia congenita: four new cases in children]. / Hypoplasie congénitale des surrénales : à propos de quatre observations.

Adrenal hypoplasia congenita (AHC) is an extremely uncommon disease of early onset. This condition can be lethal in the absence of adapted treatment. Some of these diseases are related to changes in the gene DAX1 that encodes a member of the superfamily of hormone nuclear receptors. It is a transcriptional repressor that is central in the morphogenesis of the adrenals and the gonadic differentiation. Here we report on four cases of X- linked AHC. In the first two familial cases, mutations were identified and mothers were heterozygotes. Abnormally low levels of estriol were evidenced during the pregnancy leading to an early diagnosis and adapted care of the affected male neonates. These children are doing well with a 21-and 20 months follow-up with hormone replacement at the present time. The two last cases corresponded to a contiguous gene syndrome associating AHC to glycerol-kinase deficiency that was revealed respectively at six days and seven years of age by acute adrenal insufficiency.

Sex hormone-binding globulin during puberty in normal and hyperandrogenic girls.

Human sex hormone-binding globulin (SHBG) regulates the cellular bioavailability of SHBG-bound steroid hormones. Subtle decreases in plasma SHBG levels during puberty have a perceptible effect on the androgen-estrogen balance. This SHBG decrease is more pronounced in girls with premature pubarche who are at risk to develop functional ovarian hyperandrogenism as well as insulin resistance syndrome. Insulin is a potent inhibitor of SHBG production in the liver, and there is now evidence that SHBG is a marker of hyperinsulinemia and insulin resistance that can be associated in both obese and non-obese patients with polycystic ovary syndrome. Therefore, low SHBG could be a useful tool for identifying presymptomatic individuals with diabetes mellitus type 2 including those with androgen disorders.

Brain processing of visual sexual stimuli in human males.

Despite its critical sociobiological importance, the brain processing of visual sexual stimuli has not been characterized precisely in human beings. We used Positron Emission Tomography (PET) to investigate responses of regional cerebral blood flow (rCBF) in nine healthy males presented with visual sexual stimuli of graded intensity. Statistical Parametric Mapping was used to locate brain regions whose activation was associated with the presentation of the sexual stimuli and was correlated with markers of sexual arousal. The claustrum, a region whose function had been unclear, displayed one of the highest activations. Additionally, activations were recorded in paralimbic areas (anterior cingulate gyrus, orbito-frontal cortex), in the striatum (head of caudate nucleus, putamen), and in the posterior hypothalamus. By contrast, decreased rCBF was observed in several temporal areas. Based on these results, we propose a model of the brain processes mediating the cognitive, emotional, motivational, and autonomic components of human male sexual arousal.

Exact banded patterns from a Doi-Marrucci-Greco model of nematic liquid crystal polymers.

Elements of pattern formation in nematic liquid crystal polymers are presented using the Doi-Marrucci-Greco (DMG) moment-averaged theory. The theory yields a full tensor orientation equation, accounting for excluded-volume and distortional elasticity potentials, with rotational molecular diffusion. Spinodal decomposition associated with unstable homogeneous phases is described first by way of an exact solution of the linearized DMG model. A variety of uniaxial and biaxial banded spatial patterns are then explicitly constructed from the DMG model. Exact solutions are given that possess order parameter spatial variations as well as solutions whose banded intensity patterns arise from sinuous director heterogeneity. These constructions pose as analytical models for banded structures observed during and after cessation of simple shear or elongation.

17alpha-hydroxylase/17,20-lyase deficiency as a model to study enzymatic activity regulation: role of phosphorylation.

Cytochrome P450 17alpha-hydroxylase (CYP17) is a single gene-encoded protein with two activities: 17alpha-hydroxylase and 17,20-lyase. The two catalytic activities are differentially regulated in health and disease. We took advantage of naturally occurring human mutations to understand the molecular bases of this differential regulation. We identified eight novel mutations in the CYP17 gene, different in nature and spread throughout the gene. As posttranslational modifications appear to be important for activity control, we investigated the phosphorylation state of wild-type and mutant CYP17 proteins. Although phospholabeled protein was seen when the wild-type and most mutant proteins were expressed, no phosphorylation was detected for the F417C mutant. F417C is the only 17,20-lyase deficiency case confirmed at the molecular level and represents the first phosphorylation CYP17-deficient mutant. In search of the physiological agents involved in this process, the effect of cAMP was tested on activity and phosphorylation state of our mutant CYP17 proteins. cAMP stimulates activity and phosphorylation in all cases, except in the F417C and R35L mutants. The lack of response to the physiological second messenger might explain the different phenotypes. The F417C mutant protein, which is already shown to be associated with the lack of electron transfer, provides for the first time a link between the electron transfer system and the phosphorylation state of the CYP17 enzyme in the control of 17,20-lyase activity.

New insight into the molecular basis of 3beta-hydroxysteroid dehydrogenase deficiency: identification of eight mutations in the HSD3B2 gene eleven patients from seven new families and comparison of the functional properties of twenty-five mutant enzymes.

Classical 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3betaHSD) deficiency is a form of congenital adrenal hyperplasia that impairs steroidogenesis in both the adrenals and gonads resulting from mutations in the HSD3B2 gene and causing various degrees of salt-wasting in both sexes and incomplete masculinization of the external genitalia in genetic males. To identify the molecular lesion(s) in the HSD3B2 gene in the 11 patients from the seven new families suffering from classical 3betaHSD deficiency, the complete nucleotide sequence of the whole coding region and exon-intron splicing boundaries of this gene was determined by direct sequencing. Five of these families were referred to Morel's molecular diagnostics laboratory in France, whereas the two other families were investigated by Peter's group in Germany. Functional characterization studies were performed by Simard's group in Canada. Following transient expression in 293 cells of each of the mutant recombinant proteins generated by site-directed mutagenesis, the effect of the 25 mutations on enzyme activity was assessed by incubating intact cells in culture with 10 nM [14C]-DHEA as substrate. The stability of the mutant proteins has been investigated using a combination of Northern and Western blot analyses, as well as an in vitro transcription/translation assay using rabbit reticulocyte lysates. The present report describes the identification of 8 mutations, in seven new families with individuals suffering from classical 3betaHSD deficiency, thus increasing the number of known HSD3B2 mutations involved in this autosomal recessive disorder to 31 (1 splicing, 1 in-frame deletion, 3 nonsense, 4 frameshift and 22 missense mutations). In addition to the mutations reported here in these new families, we have also investigated for the first time the functional significance of previously reported missense mutations and or sequence variants namely, A82T, A167V, L173R, L205P, S213G and K216E, P222H, T259M, and T259R, which have not previously been functionally characterized. Furthermore, their effects have been compared with those of the 10 previously reported mutant enzymes to provide a more consistent and comprehensive study. The present results are in accordance with the prediction that no functional 3betaHSD type 2 isoenzyme is expressed in the adrenals and gonads of the patients suffering from a severe salt-wasting form of CAH due to classical 3betaHSD deficiency. Whereas the nonsalt-losing form also results from missense mutation(s) in the HSD3B2 gene, which cause an incomplete loss in enzyme activity, thus leaving sufficient enzymatic activity to prevent salt wasting. The functional data described in the present study concerning the sequence variants A167V, S213G, K216E and L236S, which were detected with premature pubarche or hyperandrogenic adolescent girls suspected to be affected from nonclassical 3betaHSD deficiency, coupled with the previous studies reporting that no mutations were found in both HSD3B1 and/or HSD3B2 genes in such patients strongly support the conclusion that this disorder does not result from a mutant 3betaHSD isoenzyme. The present study provides biochemical evidence supporting the involvement of a new molecular mechanism in classical 3betaHSD deficiency involving protein instability and further illustrates the complexity of the genotype-phenotype relationships of this disease, in addition to providing further valuable information concerning the structure-function relationships of the 3betaHSD superfamily.

Neuroanatomical correlates of visually evoked sexual arousal in human males.

Brain areas activated in human male sexual behavior have not been characterized precisely. For the first time, positron emission tomography (PET) was used to identify the brain areas activated in healthy males experiencing visually evoked sexual arousal. Eight male subjects underwent six measurements of regional brain activity following the administration of [15O]H2O as they viewed three categories of film clips: sexually explicit clips, emotionally neutral control clips, and humorous control clips inducing positive but nonsexual emotions. Statistical Parametric Mapping was used to identify brain regions demonstrating an increased activity associated with the sexual response to the visual stimulus. Visually evoked sexual arousal was characterized by a threefold pattern of activation: the bilateral activation of the inferior temporal cortex, a visual association area; the activation of the right insula and right inferior frontal cortex, which are two paralimbic areas relating highly processed sensory information with motivational states; and the activation of the left anterior cingulate cortex, another paralimbic area known to control autonomic and neuroendocrine functions. Activation of some of these areas was positively correlated with plasma testosterone levels. Although this study should be considered preliminary, it identified brain regions whose activation was correlated with visually evoked sexual arousal in males.

Evaluation of gonadal function in 107 intersex patients by means of serum antimüllerian hormone measurement.

Fetal male sexual differentiation is driven by two testicular hormones: testosterone (synthesized by interstitial Leydig cells) and antimüllerian hormone (AMH; produced by Sertoli cells present in the seminiferous tubules). Intersex states result either from gonadal dysgenesis, in which both Leydig and Sertoli cell populations are affected, or from impaired secretion or action of either testosterone or AMH. Until now, only Leydig cell function has been assessed in children with ambiguous genitalia, by means of testosterone assay. To determine whether serum AMH would help in the diagnosis of intersex conditions, we assayed serum AMH levels in 107 patients with ambiguous genitalia of various etiologies. In XY patients, AMH was low when the intersex condition was caused by abnormal testicular determination (including pure and partial gonadal dysgenesis) but was normal or elevated in patients with impaired testosterone secretion, whereas serum testosterone was low in both groups. AMH was also elevated during the first year of life and at puberty in intersex states caused by androgen insensitivity. In 46,XX patients with a normal male phenotype or ambiguous genitalia, in whom the diagnosis of female pseudohermaphroditism had been excluded, serum AMH levels higher than 75 pmol/L were indicative of the presence of testicular tissue and correlated with the mass of functional testicular parenchyma. In conclusion, serum AMH determination is a powerful tool to assess Sertoli cell function in children with intersex states, and it helps to distinguish between defects of male sexual differentiation caused by abnormal testicular determination and those resulting from isolated impairment of testosterone secretion or action.

Receptors for anti-müllerian hormone on Leydig cells are responsible for its effects on steroidogenesis and cell differentiation.

Strong overexpression of anti-Müllerian hormone (AMH) in transgenic mice leads to incomplete fetal virilization and decreased serum testosterone in the adult. Conversely, AMH-deficient mice exhibit Leydig cell hyperplasia. To probe the mechanism of action of AMH on Leydig cell steroidogenesis, we have studied the expression of mRNA for steroidogenic proteins in vivo and in vitro and performed a morphometric analysis of testicular tissue in mice overexpressing the hormone. We show that overexpression of AMH in male transgenic mice blocks the differentiation of Leydig cell precursors. Expression of steroidogenic protein mRNAs, mainly cytochrome P450 17 alpha-hydroxylase/C17-20 lyase (P450c17), is decreased in transgenic mice overexpressing AMH and in AMH-treated purified Leydig cells. In contrast, transgenic mice in whom the AMH locus has been disrupted show increase expression of P450c17. In vitro, but not in vivo, AMH also decreases the expression of the luteinizing hormone receptor. The effect of AMH is explained by the presence of its receptor on Leydig cells. Our results provide insight into the action of AMH as a negative modulator of Leydig cell differentiation and function.

[Adrenal enzyme deficiency]. / Déficits enzymatiques surrénaux.

The defect in 21-hydroxylase is responsible for 95% of the congenital adrenal hyperplasia. It is a recessive autosomic genetic disease with two major clinical presentation. The classical presentation is severe and frequent. It associates an adrenal deficiency with sexual ambiguity in the girl. The diagnosis is easy by measuring the plasma level of 17 OH progesterone in antenatal, perinatal samples. The non classical presentation are less severe and are clinically expressed later during the life by an hyperandrogenic syndrome. The diagnosis requires an ACTH test. In any cases, the responsible mutations on the gene CYP21 are now well known and could be identified easily by molecular biology technics. By combinating hormonal and molecular tests, it is now possible to predict the clinical form of the disease in the context of a prenatal diagnosis, which can lead to a prenatal treatment.