Limitations in next-generation sequencing-based genotyping of breast cancer polygenic risk score loci.

Considering polygenic risk scores (PRSs) in individual risk prediction is increasingly implemented in genetic testing for hereditary breast cancer (BC) based on next-generation sequencing (NGS). To calculate individual BC risks, the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) with the inclusion of the BCAC 313 or the BRIDGES 306 BC PRS is commonly used. The PRS calculation depends on accurately reproducing the variant allele frequencies (AFs) and, consequently, the distribution of PRS values anticipated by the algorithm. Here, the 324 loci of the BCAC 313 and the BRIDGES 306 BC PRS were examined in population-specific database gnomAD and in real-world data sets of five centers of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC), to determine whether these expected AFs can be reproduced by NGS-based genotyping. Four PRS loci were non-existent in gnomAD v3.1.2 non-Finnish Europeans, further 24 loci showed noticeably deviating AFs. In real-world data, between 11 and 23 loci were reported with noticeably deviating AFs, and were shown to have effects on final risk prediction. Deviations depended on the sequencing approach, variant caller and calling mode (forced versus unforced) employed. Therefore, this study demonstrates the necessity to apply quality assurance not only in terms of sequencing coverage but also observed AFs in a sufficiently large cohort, when implementing PRSs in a routine diagnostic setting. Furthermore, future PRS design should be guided by the technical reproducibility of expected AFs across commonly used genotyping methods, especially NGS, in addition to the observed effect sizes.

Astrocytic Dagla Deletion Decreases Hedonic Feeding in Female Mice.

Introduction: Endocannabinoids and exogenous cannabinoids are potent regulators of feeding behavior and energy metabolism. Stimulating cannabinoid receptor signaling enhances appetite, particularly for energy-dense palatable foods, and promotes energy storage. To elucidate the underlying cellular mechanisms, we investigate here the potential role of astrocytic endocannabinoid 2-arachidonoylglycerol (2-AG). Astrocytes provide metabolic support for neurons and contribute to feeding regulation but the effect of astrocytic 2-AG on feeding is unknown. Materials and Methods: We generated mice lacking the 2-AG synthesizing enzyme diacylglycerol lipase alpha (Dagla) in astrocytes (GLAST-Dagla KO) and investigated hedonic feeding behavior in male and female mice. Body weight and baseline water and food intake was characterized; additionally, the mice went through milk, saccharine, and sucrose preference tests in fed and fasted states. In female mice, the estrous cycle stages were identified and plasma levels of female sex hormones were measured. Results: We found that the effects of the inducible astrocytic Dagla deletion were sex-specific. Acute milk preference was decreased in female, but not in male mice and the effect was most evident in the estrus stage of the cycle. This prompted us to investigate sex hormone profiles, which were found to be altered in GLAST-Dagla KO females. Specifically, follicle-stimulating hormone was elevated in the estrus stage, luteinizing hormone in the proestrus, and progesterone was increased in both proestrus and estrus stages of the cycle compared with controls. Conclusions: Astrocytic Dagla regulates acute hedonic appetite for palatable food in females and not in males, possibly owing to a deregulated female sex hormone profile. It is plausible that endocannabinoid production by astrocytes at least partly contributes to the greater susceptibility to overeating in females. This finding may also be important for understanding the effects of exogenous cannabinoids on sex hormone profiles.

The impact of corrosion on the adsorption of gaseous Hg0 onto the surface of steels: Implications for decommissioning in the oil and gas industry.

The rate of decommissioning of global oil and gas production facilities will accelerate over coming decades, as mature developments reach the end of use, and consumers transition towards renewable energy. Decommissioning strategies should include thorough environmental risk assessments which consider contaminants which are known to be present in oil and gas systems. Mercury (Hg) is a global pollutant that occurs naturally in oil and gas reservoirs. However, knowledge of Hg contamination in transmission pipelines and process equipment is limited. We investigated the potential for accumulation of Hg0 within production facilities, particularly those transporting gases, by considering the deposition of Hg onto steel surfaces from the gas phase. Following incubation experiments in a Hg saturated atmosphere; fresh API 5L-X65 and L80-13Cr steels were found to adsorb 1.4 × 10-5 ± 0.04 × 10-5 and 1.1 × 10-5 ± 0.04 × 10-5 g m-2, respectively, while corroded samples of the same steels adsorbed 0.12 ± 0.01 and 0.83 ± 0.02 g m-2; an increase in adsorbed mercury by four orders of magnitude. The association between surface corrosion and Hg was demonstrated by laser ablation ICPMS. The levels of Hg measured on the corroded steel surfaces indicates a potential environmental risk; therefore, mercury speciation (including the presence of ß-HgS, not considered in this study), concentrations and cleaning methods should be considered when developing oil and gas decommissioning strategies.

A homozygous p.Leu813Pro gain-of-function NLRP1 variant causes phenotypes of different severity in two siblings.

BACKGROUND: A trio exome sequencing study identified a previously unreported NLRP1 gene variant resulting in a p.Leu813Pro substitution of the LRR (leucine-rich repeats) domain of the NLRP1 protein (NACHT, LRR and PYD domains-containing protein 1). This homozygous mutation was shared by two sisters with different clinical presentation: the younger sister had generalized inflammatory nodules with keratotic plugs, clinically resembling multiple keratoacanthomas, while the older had manifestations of familial keratosis lichenoides chronica. OBJECTIVES: To analyse the consequences of this NLRP1 variant in two siblings with a different clinical spectrum of severity. METHODS: To demonstrate the pathogenicity, p.Leu813Pro was recombinantly expressed, and its effect on inflammasome assembly was assessed. Exome sequencing and RNA-Seq were performed to identify factors with potentially modifying effects on the severity of the skin manifestation between each sibling. RESULTS: The variant p.Leu813Pro triggered activation of the NLRP1 inflammasome leading to ASC (apoptosis-associated speck-like protein containing a CARD) speck formation and interleukin (IL)-1ß release. The more severely affected sister had several additional genomic variants associated with atopy and psoriasis that were not present in her sibling. IL-5 and IL-17 emerged as dominant cytokines driving prominent inflammation in the skin of the severely affected sibling. CONCLUSIONS: To the best of our knowledge, this is the first report of a NLRP1 variant that leads to a different clinical spectrum of severity within the same sibship. IL-5 and IL-17 were the main cytokines expressed in the inflammatory lesions of the severely affected patient and might be regarded as disease modifying factors, and therefore may be considered as therapeutic targets.

Pathogenic variants in the SPTLC1 gene cause hyperkeratosis lenticularis perstans.

BACKGROUND: Hyperkeratosis lenticularis perstans (HLP), also known as Flegel disease, is a rare skin disease presenting with asymptomatic small hyperkeratotic papules. The lesions often appear on the dorsal feet and lower legs, and typically develop after the fourth decade of life. A genetic basis for HLP is suspected; however, so far no gene defect linked to the development of HLP has been identified. OBJECTIVES: We aimed to identify the genetic cause of HLP. METHODS: For mutational analysis we studied a cohort of five patients with HLP using next-generation sequencing (NGS). We used DNA -extracted from fresh skin biopsies alongside ethylenediamine tetraacetic acid (EDTA) blood samples from two patients, and formalin-fixed -paraffin-embedded skin biopsy material from three patients. In addition, immunofluorescence staining of HLP lesions from four patients was investigated. RESULTS: In all samples from the five patients with HLP we identified by NGS rare variants in the SPTLC1 gene. In four patients we detected small deletions/frameshift variants and in one patient a splicing variant, predicted to disturb the splicing process. In blood samples the detected variants were heterozygous with an allele frequency of 49% and 50%, respectively. In skin biopsies the allele frequency was within the range of 46-62%. Immunofluorescence staining revealed reduced SPTLC1 protein levels in skin of patients. CONCLUSIONS: Our findings suggest that pathogenic variants in the SPTLC1 gene are the underlying genetic cause of HLP. Of note, the identified variants were either frameshift- or splicing variants probably leading to nonsense-mediated mRNA decay and thus reduced SPTLC1 protein levels. We conclude that diminished SPTLC1, the key enzyme in sphingolipid biosynthesis, leads to the development of HLP, which highlights the sphingolipid pathway as a new therapeutic target.

Osteogenic growth peptide is a potent anti-inflammatory and bone preserving hormone via cannabinoid receptor type 2.

The endocannabinoid system consists mainly of 2-arachidonoylglycerol and anandamide, as well as cannabinoid receptor type 1 and type 2 (CB2). Based on previous studies, we hypothesized that a circulating peptide previously identified as osteogenic growth peptide (OGP) maintains a bone-protective CB2 tone. We tested OGP activity in mouse models and cells, and in human osteoblasts. We show that the OGP effects on osteoblast proliferation, osteoclastogenesis, and macrophage inflammation in vitro, as well as rescue of ovariectomy-induced bone loss and prevention of ear edema in vivo are all abrogated by genetic or pharmacological ablation of CB2. We also demonstrate that OGP binds at CB2 and may act as both an agonist and positive allosteric modulator in the presence of other lipophilic agonists. In premenopausal women, OGP circulating levels significantly decline with age. In adult mice, exogenous administration of OGP completely prevented age-related bone loss. Our findings suggest that OGP attenuates age-related bone loss by maintaining a skeletal CB2 tone. Importantly, they also indicate the occurrence of an endogenous peptide that signals via CB2 receptor in health and disease.

The Importance of Extended Analysis Using Current Molecular Genetic Methods Based on the Example of a Cohort of 228 Patients with Hereditary Breast and Ovarian Cancer Syndrome.

In about 20-30% of all women with breast cancer, an increased number of cases of breast cancer can be observed in their family history. However, currently, only 5-10% of all breast cancer cases can be attributed to a pathogenic gene alteration. Molecular genetic diagnostics underwent enormous development within the last 10 years. Next-generation sequencing approaches allow increasingly extensive analyses resulting in the identification of additional candidate genes. In the present work, the germline molecular diagnostic analysis of a cohort of 228 patients with suspected hereditary breast and ovarian cancer syndrome (HBOC) was evaluated. The 27 pathogenic gene variants initially detected are listed, and their distribution in the high-risk BRCA1 and BRCA2 genes is presented in this study. In ten high-risk patients, in whom, to date, no pathogenic variant could be detected, an extended genetic analysis of previously not considered risk genes was performed. Three variants of uncertain significance and one pathogenic variant could be described. This proves the importance of extended analysis using current molecular genetic methods.

Investigation into liquisolid system processability based on the SeDeM Expert System approach.

Liquisolid systems are emerging formulation approach for poorly soluble drugs, based on adsorption/absorption of drug dispersion and obtaining free-flowing powder with good compressibility. SeDeM Expert System represents a powder processability evaluation method. It may provide additional insight into liquisolid systems critical quality attributes, but the contribution of this approach remains to be explored. The aims of this study were: pellet preparation by combination of liquisolid technology and water granulation/extrusion, evaluation of liquisolid based systems (pellets/admixtures) and investigation into the applicability of SeDeM Expert System in liquisolid systems characterization. Pellets/admixtures were prepared with microcrystalline cellulose as carrier and crospovidone/silicon dioxide as coating agent. Ibuprofen solution in polyethylene glycol 400 was used as liquid phase. After comprehensive sample characterization, experimentally obtained parameters were mathematically transformed and evaluated in the SeDeM Expert System framework. Pellets exhibited low aspect ratio and excellent flowability, despite liquid load up to 52.2%. The investigated liquisolid admixtures exhibited good flowability and faster drug dissolution than pellets. Single pellet crushing test results exhibited strong correlation with compact indentation hardness and may be used as indentation hardness predictor. SeDeM Expert System provides useful insight into liquisolid system processability and comparative evaluation and it may facilitate final solid dosage form development.

Use of Protamine in Nanopharmaceuticals-A Review.

Macromolecular biomolecules are currently dethroning classical small molecule therapeutics because of their improved targeting and delivery properties. Protamine-a small polycationic peptide-represents a promising candidate. In nature, it binds and protects DNA against degradation during spermatogenesis due to electrostatic interactions between the negatively charged DNA-phosphate backbone and the positively charged protamine. Researchers are mimicking this technique to develop innovative nanopharmaceutical drug delivery systems, incorporating protamine as a carrier for biologically active components such as DNA or RNA. The first part of this review highlights ongoing investigations in the field of protamine-associated nanotechnology, discussing the self-assembling manufacturing process and nanoparticle engineering. Immune-modulating properties of protamine are those that lead to the second key part, which is protamine in novel vaccine technologies. Protamine-based RNA delivery systems in vaccines (some belong to the new class of mRNA-vaccines) against infectious disease and their use in cancer treatment are reviewed, and we provide an update on the current state of latest developments with protamine as pharmaceutical excipient for vaccines.

Diacylglycerol lipase alpha in astrocytes is involved in maternal care and affective behaviors.

Genetic deletion of cannabinoid CB1 receptors or diacylglycerol lipase alpha (DAGLa), the main enzyme involved in the synthesis of the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG), produced profound phenotypes in animal models of depression-related behaviors. Furthermore, clinical studies have shown that antagonists of CB1 can increase the incidence and severity of major depressive episodes. However, the underlying pathomechanisms are largely unknown. In this study, we have focused on the possible involvement of astrocytes. Using the highly sensitive RNAscope technology, we show for the first time that a subpopulation of astrocytes in the adult mouse brain expresses Dagla, albeit at low levels. Targeted lipidomics revealed that astrocytic DAGLa only accounts for a minor percentage of the steady-state brain 2-AG levels and other arachidonic acid derived lipids like prostaglandins. Nevertheless, the deletion of Dagla in adult mouse astrocytes had profound behavioral consequences with significantly increased depressive-like behavioral responses and striking effects on maternal behavior, corresponding with increased levels of serum progesterone and estradiol. Our findings therefore indicate that lipids from the DAGLa metabolic axis in astrocytes play a key regulatory role in affective behaviors.

Telangiectasia-ectodermal dysplasia-brachydactyly-cardiac anomaly syndrome is caused by de novo mutations in protein kinase D1.

BACKGROUND: We describe two unrelated patients who display similar clinical features including telangiectasia, ectodermal dysplasia, brachydactyly and congenital heart disease. METHODS: We performed trio whole exome sequencing and functional analysis using in vitro kinase assays with recombinant proteins. RESULTS: We identified two different de novo mutations in protein kinase D1 (PRKD1, NM_002742.2): c.1774G>C, p.(Gly592Arg) and c.1808G>A, p.(Arg603His), one in each patient. PRKD1 (PKD1, HGNC:9407) encodes a kinase that is a member of the protein kinase D (PKD) family of serine/threonine protein kinases involved in diverse cellular processes such as cell differentiation and proliferation and cell migration as well as vesicle transport and angiogenesis. Functional analysis using in vitro kinase assays with recombinant proteins showed that the mutation c.1808G>A, p.(Arg603His) represents a gain-of-function mutation encoding an enzyme with a constitutive, lipid-independent catalytic activity. The mutation c.1774G>C, p.(Gly592Arg) in contrast shows a defect in substrate phosphorylation representing a loss-of-function mutation. CONCLUSION: The present cases represent a syndrome, which associates symptoms from several different organ systems: skin, teeth, bones and heart, caused by heterozygous de novo mutations in PRKD1 and expands the clinical spectrum of PRKD1 mutations, which have hitherto been linked to syndromic congenital heart disease and limb abnormalities.

Intestine-specific DGAT1 deficiency improves atherosclerosis in apolipoprotein E knockout mice by reducing systemic cholesterol burden.

BACKGROUND AND AIMS: Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the rate-limiting enzyme catalyzing the final step of triglyceride synthesis by esterifying a diglyceride with a fatty acid. We have previously shown that apolipoprotein E-knockout (ApoE-/-) mice lacking Dgat1 have reduced intestinal cholesterol absorption and potentiated macrophage cholesterol efflux, and consequently, exhibit attenuated atherogenesis. However, hematopoietic Dgat1 deficiency lacked beneficial effects on atherosclerosis. Due to our recent results on the critical role of intestinal Dgat1 in murine cholesterol homeostasis, we delineated whether intestinal Dgat1 deficiency regulates atherogenesis in mice. METHODS: We generated intestine-specific Dgat1-/- mice on the ApoE-/- background (iDgat1-/-ApoE-/-) and determined cholesterol homeostasis and atherosclerosis development. RESULTS: When fed a Western-type diet, iDgat1-/-ApoE-/- mice exhibited a substantial decrease in fasting plasma cholesterol content in ApoB-containing lipoproteins. Although lipid absorption was delayed, iDgat1-/-ApoE-/- mice had reduced acute and fractional cholesterol absorption coupled with an elevated fecal caloric loss. In line, increased appearance of i.v. administered [³H]cholesterol in duodena and stool of iDgat1-/-ApoE-/- animals suggested potentiated cholesterol elimination. Atherosclerotic lesions were markedly smaller with beneficial alterations in plaque composition as evidenced by reduced macrophage infiltration and necrotic core size despite unaltered collagen content, indicating improved plaque stability. CONCLUSIONS: Disruption of Dgat1 activity solely in the small intestine of ApoE-/- mice strongly decreased plasma cholesterol levels by abrogating the assimilation of dietary cholesterol, partly by reduced absorption and increased excretion. Consequently, the reduced cholesterol burden significantly attenuated atherogenesis and improved the lesion phenotype in iDgat1-/-ApoE-/- mice.

Severe metabolic alterations in liver cancer lead to ERK pathway activation and drug resistance.

BACKGROUND: The extracellular signal-regulated kinase (ERK) pathway regulates cell growth, and is hyper-activated and associated with drug resistance in hepatocellular carcinoma (HCC). Metabolic pathways are profoundly dysregulated in HCC. Whether an altered metabolic state is linked to activated ERK pathway and drug response in HCC is unaddressed. METHODS: We deprived HCC cells of glutamine to induce metabolic alterations and performed various assays, including metabolomics (with 13C-glucose isotope tracing), microarray analysis, and cell proliferation assays. Glutamine-deprived cells were also treated with kinase inhibitors (e.g. Sorafenib, Erlotinib, U0126 amongst other MEK inhibitors). We performed bioinformatics analysis and stratification of HCC tumour microarrays to determine upregulated ERK gene signatures in patients. FINDINGS: In a subset of HCC cells, the withdrawal of glutamine triggers a severe metabolic alteration and ERK phosphorylation (pERK). This is accompanied by resistance to the anti-proliferative effect of kinase inhibitors, despite pERK inhibition. High intracellular serine is a consistent feature of an altered metabolic state and contributes to pERK induction and the kinase inhibitor resistance. Blocking the ERK pathway facilitates cell proliferation by reprogramming metabolism, notably enhancing aerobic glycolysis. We have identified 24 highly expressed ERK gene signatures that their combined expression strongly indicates a dysregulated metabolic gene network in human HCC tissues. INTERPRETATION: A severely compromised metabolism lead to ERK pathway induction, and primes some HCC cells to pro-survival phenotypes upon ERK pathway blockade. Our findings offer novel insights for understanding, predicting and overcoming drug resistance in liver cancer patients. FUND: DFG, BMBF and Sino-German Cooperation Project.

Biological Activity Of miRNA-27a Using Peptide-based Drug Delivery Systems.

BACKGROUND: Endogenously expressed microRNAs (miRNAs) have attracted attention as important regulators in post-transcriptionally controlling gene expression of various physiological processes. As miRNA dysregulation is often associated with various disease patterns, such as obesity, miRNA-27a might therefore be a promising candidate for miRNA mimic replacement therapy by inhibiting adipogenic marker genes. However, application of naked nucleic acids faces some limitations concerning poor enzymatic stability, bio-membrane permeation and cellular uptake. To overcome these obstacles, the development of appropriate drug delivery systems (DDS) for miRNAs is of paramount importance. METHODS: In this work, a triple combination of atomic force microscopy (AFM), brightfield (BF) and fluorescence microscopy was used to trace the cellular adhesion of N-TER peptide-nucleic acid complexes followed by time-dependent uptake studies using confocal laser scanning microscopy (cLSM). To reveal the biological effect of miRNA-27a on adipocyte development after transfection treatment, Oil-Red-O (ORO)- staining was performed to estimate the degree of in lipid droplets accumulated ORO in mature adipocytes by using light microscopy images as well as absorbance measurements. RESULTS: The present findings demonstrated that amphipathic N-TER peptides represent a suitable DDS for miRNAs by promoting non-covalent complexation through electrostatic interactions between both components as well as cellular adhesion of the N-TER peptide - nucleic acid complexes followed by uptake across cell membranes and intracellular release of miRNAs. The anti-adipogenic effect of miRNA-27a in 3T3-L1 cells could be detected in mature adipocytes by reduced lipid droplet formation. CONCLUSION: The present DDS assembled from amphipathic N-TER peptides and miRNAs is capable of inducing the anti-adipogenic effect of miRNA-27a by reducing lipid droplet accumulation in mature adipocytes. With respect to miRNA mimic replacement therapies, this approach might provide new therapeutic strategies to prevent or treat obesity and obesity-related disorders.

Genetical, clinical, and functional analysis of a large international cohort of patients with autosomal recessive congenital ichthyosis due to mutations in NIPAL4.

Autosomal recessive congenital ichthyosis (ARCI) belongs to a heterogeneous group of disorders of keratinization. To date, 10 genes have been identified to be causative for ARCI. NIPAL4 (Nipa-Like Domain-Containing 4) is the second most commonly mutated gene in ARCI. In this study, we present a large cohort of 101 families affected with ARCI carrying mutations in NIPAL4. We identified 16 novel mutations and increase the total number of pathogenic mutations in NIPAL4 to 34. Ultrastructural analysis of biopsies from six patients showed morphological abnormalities consistent with an ARCI EM type III. One patient with a homozygous splice site mutation, which leads to a loss of NIPAL4 mRNA, showed additional ultrastructural aberrations together with a more severe clinical phenotype. Our study gives insights into the frequency of mutations, a potential hot spot for mutations, and genotype-phenotype correlations.

CB2 receptor deletion on myeloid cells enhanced mechanical allodynia in a mouse model of neuropathic pain.

Neuropathic pain can develop after nerve injury, leading to a chronic condition with spontaneous pain and hyperalgesia. Pain is typically restricted to the side of the injured nerve, but may occasionally spread to the contralateral side, a condition that is often referred to as mirror-image pain. Mechanisms leading to mirror-image pain are not completely understood, but cannabinoid CB2 receptors have been implicated. In this study, we use genetic mouse models to address the question if CB2 receptors on neurons or on microglia/macrophages are involved. First, we show that a GFP reporter protein under control of the CB2 promoter is induced upon partial sciatic nerve ligation in spinal cord, dorsal root ganglia, and highest in sciatic nerve macrophages, but not in neurons. Mice which lack CB2 receptors specifically on myeloid cells (microglia, macrophages) developed a mirror-image allodynia [treatment F1,48 = 45.69, p < 0.0001] similar to constitutive CB2 receptor knockout mice [treatment F1,70 = 92.41, p < 0.0001]. Such a phenotype was not observed after the deletion of CB2 from neurons [treatment F1,70 = 0.1315, p = 0.7180]. This behavioral pain phenotype was accompanied by an increased staining of microglia in the dorsal horn of the spinal cord, as evidenced by an enhanced Iba 1 expression [CB2KO, p = 0.0175; CB2-LysM, p = 0.0425]. Similarly, myeloid-selective knockouts showed an increased expression of the leptin receptor in the injured ipsilateral sciatic nerve, thus further supporting the notion that leptin signaling contributes to the increased neuropathic pain responses of CB2 receptor knockout mice. We conclude that CB2 receptors on microglia and macrophages, but not on neurons, modulate neuropathic pain responses.

Correlation between the solid state of lipid coating and release profile of API from hot melt coated microcapsules.

Solvent-free hot melt coating (HMC) provides a safer and more economic process compared to the conventional solvent coating techniques. However, drug release instability and the lack of fundamental understanding on it are limiting factors for application of HMC for industrial productions. In this work, we investigated glyceryl dibehenate, glyceryl monostearate and behenoyl polyoxyl-8 glyceride as HMC materials. The microstructure and solid state alteration of lipids were studied via polarized light microscopy, DSC and powder x-ray diffraction. Microcapsules of N-acetylcysteine particles were provided with these excipients and stored under long term and accelerated conditions for 3 months. The feasibility of selected lipids as HMC excipients was confirmed. The drug release from freshly coated microcapsules was dictated by microstructure, solid state and HLB of lipid coating. Alterations in the release profiles after storage under accelerated conditions were correlated with time-dependent structural alterations of selected lipids. The faster drug release from glyceryl dibehenate and behenoyl polyoxyl-8 glyceride microcapsules was correlated with a low-melting small fraction composed by mixed phases in glyceryl dibehenate and the amorphous region of polyoxyl part in behenoyl polyoxyl-8 glyceride, respectively. The slower drug release from glyceryl dibehenate after storage was explained by the transition of lipid crystals to the ß-form with dense crystalline structure. The gained information can be used to design effective tempering strategies for providing stable pharmaceutical products.

A Monoallelic Two-Hit Mechanism in PLCD1 Explains the Genetic Pathogenesis of Hereditary Trichilemmal Cyst Formation.

Trichilemmal cysts are common hair follicle-derived intradermal cysts. The trait shows an autosomal dominant mode of transmission with incomplete penetrance. Here, we describe the pathogenetic mechanism for the development of hereditary trichilemmal cysts. By whole-exome sequencing of DNA from the blood samples of 5 affected individuals and subsequent Sanger sequencing of a family cohort including 35 affected individuals, this study identified a combination of the Phospholipase C Delta 1 germline variants c.903A>G, p.(Pro301Pro) and c.1379C>T, p.(Ser460Leu) as a high-risk factor for trichilemmal cyst development. Allele-specific PCRs and cloning experiments showed that these two variants are present on the same allele. The analysis of tissue from several cysts revealed that an additional somatic Phospholipase C Delta 1 mutation on the same allele is required for cyst formation. In two different functional in vitro assays, this study showed that the protein function of the cyst-specific 1-phosphatidylinositol 4, 5-bisphosphate phosphodiesterase delta-1 protein variant is modified. This pathologic mechanism defines a monoallelic model of the two-hit mechanism proposed for tumor development and other hereditary cyst diseases.

Chronic nicotine administration restores brain region specific upregulation of oxytocin receptor binding levels in a G72 mouse model of schizophrenia.

Nicotine dependence and schizophrenia are two mental health disorders with remarkably high comorbidity. Cigarette smoking is particularly prevalent amongst schizophrenic patients and it is hypothesised to comprise a form of self-medication for relieving cognitive deficits in these patients. Emerging evidence suggests a role of the neurohypophysial peptide oxytocin in the modulation of drug addiction, as well as schizophrenia symptomology; however, the underlying mechanism remains unclear. Therefore, we sought to investigate the effects of chronic nicotine administration on oxytocin receptor (OTR) binding in the brain of a transgenic mouse model of schizophrenia that carries a bacterial artificial chromosome of the human G72/G30 locus (G72Tg). Female wild-type (WT) and heterozygous G72 transgenic CD-1 mice were treated with a chronic nicotine regimen (24 mg/kg/day, osmotic minipumps for 14 days) and quantitative autoradiographic mapping of oxytocin receptors was carried out in brains of these animals. OTR binding levels were higher in the cingulate cortex (CgCx), nucleus accumbens (Acb), and central amygdala (CeA) of saline treated G72Tg mice compared to WT control mice. Chronic nicotine administration reversed this upregulation in the CgCx and CeA. Interestingly, chronic nicotine administration induced an increase in OTR binding in the CeA of solely WT mice. These results indicate that nicotine administration normalises the dysregulated central oxytocinergic system of this mouse model of schizophrenia and may contribute towards nicotine's ability to modulate cognitive deficits which are common symptoms of schizophrenia.