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Background: Exercise and vitamin D can improve bone density by reducing bone loss. Growth factors such as IGF-I and IGFBP-3 are appeared to increase bone turnover in response to mechanical load, and free radicals attenuate the release of these growth factors. Objectives: We assessed the effect of concurrent aerobic training and cholecalciferol administration along with hydrogen peroxide injection on the levels of IGF-I and IGFBP-3 expression in the bone tissue of rats. Methods: Thirty-six adult Wistar rats were randomized into six groups (n = 6), including healthy control, sham, hydrogen peroxide (H2O2), H2O2 + aerobic training, H2O2 + cholecalciferol, and H2O2 + aerobic training + cholecalciferol. The rats were intraperitoneally administered with one mmol/kg. Body weight (BW) of H2O2 three times a week on even days and 0.5 µg/kg.bw of cholecalciferol daily. Aerobic training (at a speed of 4 - 20 m/min, for 20 - 60 minutes) was performed five days/w for eight weeks. The expression of IGF-I and IGFBP-3 was measured by real-time (RT)-PCR. Data were analyzed using the independent t-test, two-way ANOVA (exercise × vitamin D), and Bonferroni’s post-hoc test in SPSS 26 at the significance level of P ≤ 0.05. Results: The results showed that H2O2 significantly reduced the gene expressions of IGF-I (P = 0.001) and IGFBP-3 (P = 0.001) in the bone tissue. Also, exercise and vitamin D augmented the expression of IGF-I (P = 0.008) and IGFBP-3 (P = 0.0001) as post-hoc analysis showed that aerobic training had the greatest effect on the expression of IGF-I and IGFBP-3 (P < 0.05). In addition, the amplifying effects of aerobic training and cholecalciferol on the gene expressions of IGF-I and IGFBP-3 were also remarkable (P < 0.1). Conclusions: The mechanical load created by aerobic training exerted the greatest augmenting effect on the gene expression of IGF-I and IGFBP-3. Moreover, the interactive effect of aerobic training and cholecalciferol was also significant. [ABSTRACT FROM AUTHOR]

Broadband UV-visible femtosecond transient absorption spectroscopy and steady-state integrated fluorescence were used to study the excited state dynamics of 7-dehydrocholesterol (provitamin D3, DHC) in solution following excitation at 266 nm. The major results from these experiments are: (1) The excited state absorption spectrum is broad and structureless spanning the visible from 400 to 800 nm. (2) The state responsible for the excited state absorption is the initially excited state. Fluorescence from this state has a quantum yield of ∼2.5 × 10-4 in room temperature solution. (3) The decay of the excited state absorption is biexponential, with a fast component of ∼0.4-0.65 ps and a slow component 1.0-1.8 ps depending on the solvent. The spectral profiles of the two components are similar, with the fast component redshifted with respect to the slow component. The relative amplitudes of the fast and slow components are influenced by the solvent. These data are discussed in the context of sequential and parallel models for the excited state internal conversion from the optically excited 11B state. Although both models are possible, the more likely explanation is fast bifurcation between two excited state geometries leading to parallel decay channels. The relative yield of each conformation is dependent on details of the potential energy surface. Models for the temperature dependence of the excited state decay yield an intrinsic activation barrier of ∼2 kJ/mol for internal conversion and ring opening. This model for the excited state behavior of DHC suggests new experiments to further understand the photochemistry and perhaps control the excited state pathways with optical pulse shaping. [ABSTRACT FROM AUTHOR]

X-linked dominant Conradi-Hünermann syndrome (CDPX2; MIM 302960) is one of a group of disorders with aberrant punctate calcification in cartilage, or chondrodysplasia punctata (CDP). This is most prominent around the vertebral column, pelvis and long bones in CPDX2. Additionally, CDPX2 patients may have asymmetric rhizomesomelia, sectorial cataracts, patchy alopecia, ichthyosis and atrophoderma. The phenotype in CDPX2 females ranges from stillborn to mildly affected individuals identified in adulthood. CDPX2 is presumed lethal in males, although a few affected males have been reported. We found increased 8(9)-cholestenol and 8-dehydrocholesterol in tissue samples from seven female probands with CDPX2 (ref. 4). This pattern of accumulated cholesterol intermediates suggested a deficiency of 3beta-hydroxysteroid-delta8,delta7-isomerase (sterol-delta8-isomerase), which catalyses an intermediate step in the conversion of lanosterol to cholesterol. A candidate gene encoding a sterol-delta8-isomerase (EBP) has been identified and mapped to Xp11.22-p11.23 (refs 5,6). Using SSCP analysis and sequencing of genomic DNA, we found EBP mutations in all probands. We confirmed the functional significance of two missense alleles by expressing them in a sterol-delta8-isomerase-deficient yeast strain. Our results indicate that defects in sterol-delta8-isomerase cause CDPX2 and suggest a role for sterols in bone development.

X-linked dominant Conradi-Hünermann syndrome (CDPX2; MIM 302960) is one of a group of disorders with aberrant punctate calcification in cartilage, or chondrodysplasia punctata (CDP). This is most prominent around the vertebral column, pelvis and long bones in CPDX2. Additionally, CDPX2 patients may have asymmetric rhizomesomelia, sectorial cataracts, patchy alopecia, ichthyosis and atrophoderma. The phenotype in CDPX2 females ranges from stillborn to mildly affected individuals identified in adulthood. CDPX2 is presumed lethal in males, although a few affected males have been reported. We found increased 8(9)-cholestenol and 8-dehydrocholesterol in tissue samples from seven female probands with CDPX2 (ref. 4). This pattern of accumulated cholesterol intermediates suggested a deficiency of 3beta-hydroxysteroid-delta8,delta7-isomerase (sterol-delta8-isomerase), which catalyses an intermediate step in the conversion of lanosterol to cholesterol. A candidate gene encoding a sterol-delta8-isomerase (EBP) has been identified and mapped to Xp11.22-p11.23 (refs 5,6). Using SSCP analysis and sequencing of genomic DNA, we found EBP mutations in all probands. We confirmed the functional significance of two missense alleles by expressing them in a sterol-delta8-isomerase-deficient yeast strain. Our results indicate that defects in sterol-delta8-isomerase cause CDPX2 and suggest a role for sterols in bone development.

Vitamin D is important in multiple health conditions. Vitamin D deficiency is prevalent globally even with exposure to adequate sunlight. Reduction in provitamin D3 (7-dehydrocholesterol, 7-DHC) is an important cause of vitamin D3 deficiency. Vitamin supplementation, food fortification, and use of probiotics are some approaches to reduce vitamin D3 deficiency. This study investigates plausibility of 7-DHC biosynthesis through dietary prebiotics supplementation. Furthermore, it reports mechanistic details and constraints for the biosynthesis using flux balance analysis (FBA) simulations. The FBA simulations using co-metabolism models comprising human host and a resident bacterium (Faecalibacterium prausnitzii or Bacteroides thetaiotamicron) indicated increased flux of 7-DHC with short-chain fructooligosaccharide (scFOS) or inulin supplementation. We observed around 2-fold increase in flux compared to the baseline. Biosynthesis of 7-DHC was primarily modulated through acetate, pyruvate and lactate secreted by the bacterium. We observed diverse mechanisms and dose dependent responses. We extended this assessment to 119 resident bacteria and investigated the metabolites profiles with prebiotics supplementation. In summary, the current study suggests the potential use of applying prebiotics in enhancing 7-DHC biosynthesis. Furthermore, performance of the different gut bacteria with prebiotic supplementation for secreted metabolites profile is reported. These results may be useful to design future clinical studies. [ABSTRACT FROM AUTHOR]

Abstract Aim Opioid-induced bowel syndromes deteriorate patients' quality of life and may lead to nonadherence to opioid schedule and under-treatment of pain. The objective was to characterize the pharmacological profile of 17-cyclopropylmethyl-6,7-didehydro-4,5α-epoxy-6′-ethoxycarbonyl-3,14-dihydroxyindolo [2′,3′-6,7]morphinan (TAN-452), a novel peripherally acting opioid receptor antagonist. Main methods The in vitro binding affinity for the μ-, δ-, and κ-opioid receptors (MOR, DOR, and KOR) and the inhibition of [35S]GTPγS binding were examined using membrane preparations from recombinant human (h) MOR, DOR, or KOR expressing cell. In vivo assays were performed to determine the inhibitory effect of TAN-452 on morphine-induced emesis (anti-emetic activity) in ferrets, morphine-induced inhibition of small intestinal transit (anti-constipation activity) in rats, and morphine-induced antinociception (anti-analgesic activity) in rats. A pharmacokinetic study was also performed. Key findings The binding affinities of TAN-452 (K i) were 36.56 ± 1.48 nM, 0.47 ± 0.09 nM, and 5.31 ± 1.80 nM for hMOR, hDOR, and hKOR, respectively. Its antagonistic activities (K b) were 9.43 ± 0.58 nM, 0.21 ± 0.06 nM, and 7.18 ± 0.75 nM, respectively. The ED 50 values (95% Confidence interval) were <1.0 mg/kg p.o. and <0.3 mg/kg s.c. for anti-emetic activity, 9.45 (4.09, 47.79) mg/kg p.o. and 0.52 (0.10, 1.08) mg/kg s.c. for anti-constipation activity, and >300 mg/kg p.o. and >30 mg/kg s.c. for anti-analgesic activity. The pharmacokinetic study demonstrated low brain penetrability of TAN-452. Significance TAN-452 is a peripherally acting opioid receptor antagonist with selectivity for DOR that attenuates morphine-induced side effects, such as nausea, vomiting, and constipation, without affecting pain control. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of 7-dehydrocholesterol loaded cyclodextrin (7-DHCLC) and cholesterol loaded cyclodextrin (CLC) with the addition of different glycerol (G) rates on bull semen cryopreservation. Semen samples of three bulls were pooled and divided into seven groups; control group (C) and the other six were extended with standard Tris extender including different G rates (3, 6 and 9%) with CLC or 7-DHCLC to reach 1.5 mg/120x106. Extended samples were frozen in 0.25 mL straws. After thawing, motion characteristics and motility, viability, acrosome integrity, mitochondrial activation, functional membrane integrity and abnormal spermatozoa rates were assessed. The highest membrane integrity, viability and total motility were detected in 7-DHCLC with 9% of G group (P<0.001). The lowest progressive motility was detected in 3% G groups (P<0.001). 7-DHCLC with 9% G had higher mitochondrial activity compared with 3% G groups. In addition, no statistical difference was observed between groups in terms of acrosome integrity and abnormal spermatozoa rates (P>0.05). According to these results, 7-DHCLC with 9% G addition maintained some spermatological parameters better than other groups after thawing in bull semen thus, it was concluded that 7-DHCLC could be used instead of CLC in bull semen cryopreservation. [ABSTRACT FROM AUTHOR]

The germacranolide 1- epi -tatridin B has been isolated from the aerial parts of L aurus novocanariensis . We have observed that the identification of this lactone and its epimer tetradin B in the scientific literature is confusing and contradictory. We have therefore studied this issue clarifying errors and contributing to the structural elucidation of other related products. Moreover, we have isolated from this plant a lactone with an 1,5-ether bridge, previously obtained from Austrolabium candidum . We have now named it austroliolide, reassigned its 13 C NMR spectrum and compared its structure with that of badgerin. In addition, we have also isolated from L. novocanariensis the known germacranolides artemorin, costunolide, tatridin A, tulirinol and verlotorin, the eudesmanolides β-cyclopyrethrosin, 1β-hydroxy-arbusculin A, magnoliaolide and reynosin, and the guaianolide dehydrocostus lactone. [ABSTRACT FROM AUTHOR]

While antipsychotic medications provide important relief from debilitating psychotic symptoms, they also have significant adverse side effects, which might have relevant impact on human health. Several research studies, including ours, have shown that commonly used antipsychotics such as haloperidol and aripiprazole affect cholesterol biosynthesis at the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol. This transformation is promoted by the enzyme DHCR7 and its inhibition causes increases in plasma and tissue levels of 7-DHC. The inhibition of this enzymatic step by mutations in the Dhcr7 gene leads to Smith-Lemli-Opitz syndrome, a devastating human condition that can be replicated in rats by small molecule inhibitors of DHCR7. The fact that two compounds, brexpiprazole and cariprazine, that were recently approved by the FDA have substructural elements in common with the DHCR7 inhibitor aripiprazole, prompted us to evaluate the effect of brexpiprazole and cariprazine on cholesterol biosynthesis. We report that cariprazine affects levels of 7-DHC and cholesterol in cell culture incubations at concentrations as low as 5 nM. Furthermore, a common metabolite of cariprazine and aripiprazole, 2,3-(dichlorophenyl) piperazine, inhibits DHCR7 activity at concentrations comparable to those of the potent teratogen AY9944. The cell culture experiments were corroborated in mice in studies showing that treatment with cariprazine elevated 7-DHC in brain and serum. The consequences of sterol inhibition by antipsychotics in the developing nervous system and the safety of their use during pregnancy remains to be established. [ABSTRACT FROM AUTHOR]

Objective: The aim was to investigate whether vitamin D supplementation, combined with a hypocaloric diet, could have an independent effect on insulin sensitivity in subjects with both overweight and hypovitaminosis D. Changes from baseline in anthropometric parameters, body composition, glucose tolerance, and insulin secretion were considered as secondary outcomes.Methods: Eighteen volunteers who were nondiabetic and vitamin D deficient and had BMI > 25 kg/m2 were randomized (1:1) in a double-blind manner to a hypocaloric diet + either oral cholecalciferol at 25,000 IU/wk or placebo for 3 months. Hyperinsulinemic-euglycemic clamp to measure insulin sensitivity was performed at baseline and after intervention.Results: Body weight in both groups decreased significantly (-7.5% in the vitamin D group and -10% in the placebo group; P < 0.05 for both), with no between-group differences. Serum 25-hydroxyvitamin D levels in the vitamin D group increased considerably (from 36.7 ± 13.2 nmol/L to 74.8 ± 18.7 nmol/L; P < 0.001). Insulin sensitivity in the vitamin D group improved (from 4.6 ± 2.0 to 6.9 ± 3.3 mg·kg-1 ·min-1 ; P < 0.001), whereas no changes were observed in the placebo group (from 4.9 ± 1.1 to 5.1 ± 0.3 mg·kg-1 ·min-1 ; P = 0.84).Conclusions: Cholecalciferol supplementation, combined with a weight loss program, significantly improves insulin sensitivity in healthy subjects with obesity and might represent a personalized approach for insulin-resistant subjects with obesity. [ABSTRACT FROM AUTHOR]

Vitamin D is one of the main groups of sterols; playing an important role in phospho-calcic metabolism. The conversion of 7-dehydrocholesterol to pre- vitamin D3 in the skin, through solar ultraviolet B radiation, is the main source of vitamin D. Since lupus patients are usually photosensitive, the risk of developing vitamin D deficiency in is high in this population. Although evidences showed the connotation between systemic lupus erythematosus (SLE) and vitamin D through which SLE can lead to lower vitamin D levels, it is also important to consider the possibility that vitamin D deficiency may have a causative role in SLE etiology. This paper analyzes existing data from various studies to highlight the role of vitamin D deficiency in SLE occurrence and aggravation and the probable efficacy of vitamin D supplementation on SLE patients. We searched 'Science Direct' and 'Pub Med' using 'Vitamin D' and 'SLE' for finding the studies focusing on the association between vitamin D deficiency and SLE incidence and consequences. Evidences show that vitamin D plays an important role in the pathogenesis and progression of SLE and vitamin D supplementation seems to ameliorate inflammatory and hemostatic markers; so, can improve clinical subsequent. [ABSTRACT FROM AUTHOR]

7-Dehydrocholesterol peroxide (5α,8α-epidioxycholest-6-ene-3β-ol, CEP) and its acetate and hemisuccinate derivatives were synthesized and isolated for the first time, which exhibit improved anticancer activity and selectivity over ergosterol peroxide (5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol, EEP), showing potential as new chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Loss-of-function mutations in the enzyme 7-dehydrocholesterol reductase are responsible for the Smith-Lemli-Opitz syndrome, in which 7-dehydrocholesterol (7-DHC) levels are markedly increased in the plasma and tissues of patients. This increase in 7-DHC is probably associated with the painful and itchy photosensitivity reported by the majority of patients with Smith-Lemli-Opitz syndrome. To identify the molecular targets involved in the activation and photosensitization of primary afferents by 7-DHC, we focused on TRPA1 and TRPV1, two ion channels expressed in nociceptive nerve endings and previously shown to respond to ultraviolet and visible light under pathophysiological circumstances. Recombinant human TRPA1 is activated and photosensitized in the presence of 7-DHC. Prolonged preexposure to 7-DHC causes more pronounced photosensitization, and while TRPV1 contributes less to the acute effect, it too becomes highly photosensitive upon preincubation with 7-DHC for 1 to 15 hours. Dorsal root ganglion neurons in primary culture display acute sensitivity to 7-DHC in the dark and also light-evoked responses in the presence of 7-DHC, which are exclusively dependent on TRPA1 and TRPV1. Similarly, prolonged exposure of mouse dorsal root ganglion neurons to 7-DHC renders these cells photosensitive in a largely TRPA1- and TRPV1-dependent manner. Single-fiber recordings in mouse skin-nerve preparations demonstrate violet light-evoked activation and a sensitization to 7-DHC exposure. Vice versa, 7-DHC pretreatment of the isolated trachea leads to a TRPA1- and TRPV1-dependent increase of the light-induced calcitonin gene-related peptide release. Taken together, our results implicate TRPA1 and TRPV1 channels as potential pharmacological targets to address the 7-DHC-induced hypersensitivity to light in patients. [ABSTRACT FROM AUTHOR]

The enzyme Δ 24 -dehydrocholesterol reductase (DHCR24) catalyzes the reduction of the Δ 24 -double bond in the side chain of cholesterol precursors. Recent biochemical investigations fuel the hope that inhibition of DHCR24, resulting in an accumulation of desmosterol, can open new therapeutic options for treating hepatitis C virus infections, certain forms of cancer and atherosclerosis. In turn, there is a high need for selective, potent and non-toxic inhibitors of DHCR24. Previous reports as well as our re-evaluation showed that established DHCR24 inhibitors are not suitable for this purpose. Based on the lathosterol-derived amide MGI-21 (IC 50 823 nM for inhibition of overall cholesterol biosynthesis in HL-60 cells) we performed a systematic variation of the side chain functionality and identified the steroidal 3,22-diols 29 and 30 , as well as several esters thereof, as extremely potent (IC 50 < 5 nM), selective, and non-toxic DHCR24 inhibitors. In mice, diester 27 (SH-42) led to a significant increase in plasma desmosterol levels. The new inhibitors described here are valuable tools for investigating the therapeutic potential of DHCR24 inhibition. [ABSTRACT FROM AUTHOR]

Rationale: Patients with chronic rhinosinusitis with nasal polyps (CRSwNP) have been shown to be vitamin D3 (VD3) deficient, which is associated with more severe disease and increased polyp size. To gain mechanistic insights into these observational studies, we examined the impact of VD3 deficiency on inflammation and VD3 metabolism in an Aspergillus fumigatus (Af) mouse model of chronic rhinosinusitis (Af-CRS). Methods: Balb/c mice were fed control or VD3 deficient diet for 4 weeks. Mice were then sensitized with intraperitoneal Af, and one week later given Af intranasally every three days for four weeks while being maintained on control or VD3 deficient diet. Airway function, sinonasal immune cell infiltrate and sinonasal VD3 metabolism profiles were then examined. Results: Mice with VD3 deficiency had increased Penh and sRaw values as compared to controls as well as exacerbated changes in sRaw when coupled with Af-CRS. As compared to controls, VD3 deficient and Af-CRS mice had reduced sinonasal 1α-hydroxylase and the active VD3 metabolite, 1,25(OH)2D3. Differential analysis of nasal lavage samples showed that VD3 deficiency alone and in combination with Af-CRS profoundly upregulated eosinophil, neutrophil and lymphocyte numbers. VD3 deficiency exacerbated increases in monocyte-derived dendritic cell (DC) associated with Af-CRS. Conversely, T-regulatory cells were decreased in both Af-CRS mice and VD3 deficient mice, though coupling VD3 deficiency with Af-CRS did not exacerbate CD4 or T-regulatory cells numbers. Lastly, VD3 deficiency had a modifying or exacerbating impact on nasal lavage levels of IFN-γ, IL-6, IL-10 and TNF-α, but had no impact on IL-17A. Conclusions: VD3 deficiency causes changes in sinonasal immunity, which in many ways mirrors the changes observed in Af-CRS mice, while selectively exacerbating inflammation. Furthermore, both VD3 deficiency and Af-CRS were associated with altered sinonasal VD3 metabolism causing reductions in local levels of the active VD3 metabolite, 1,25(OH)2D3, even with adequate circulating levels. [ABSTRACT FROM AUTHOR]

In this study, we analyzed 5α(H)-stanol/Δ 5 -sterol ratios in offshore marine sediments using offline tetramethylammonium hydroxide thermochemolysis (TMAH) gas chromatography–mass spectrometry (GC–MS). Data were used to reconstruct redox events recorded in a southern California marine sediment core (Ocean Drilling Program, Leg167, Hole 1017E). The 24-nordehydrocholestanol/24-nordehydrocholesterol and diatomstanol/diatomsterol ratios clearly recorded oxygen minimum zone (OMZ) fluctuations in southern California during the last 40 kyr. In particular, two ratios corresponding to Marine Isotope Stage 3 (MIS3) varied significantly at the OMZ, exhibiting millennial-scale fluctuations related to Dansgaard-Oeschger shifts of warming and cooling intervals. In contrast, other ratios (cholestanol/cholesterol, campestanol/campesterol, sitostanol/sitosterol, and 22-dehydrocholestanol/22-dehydrocholesterol) showed no significant trends, possibly reflecting the influence of stanol-creating organisms. Our results show that 5α(H)-stanol/Δ 5 -sterol ratios obtained using the TMAH GC–MS method are useable tracers of water column and surface sediment redox conditions in southern California marine sediments. [ABSTRACT FROM AUTHOR]

Cholesterol metabolism is vital for brain function. Previous work in cultured cells has shown that a number of psychotropic drugs inhibit the activity of 7-dehydrocholesterol reductase (DHCR7), an enzyme that catalyzes the final steps in cholesterol biosynthesis. This leads to the accumulation of 7-dehydrocholesterol (7DHC), a molecule that gives rise to oxysterols, vitamin D, and atypical neurosteroids. We examined levels of cholesterol and the cholesterol precursors desmosterol, lanosterol, 7DHC and its isomer 8-dehydrocholesterol (8DHC), in blood samples of 123 psychiatric patients on various antipsychotic and antidepressant drugs, and 85 healthy controls, to see if the observations in cell lines hold true for patients as well. Three drugs, aripiprazole, haloperidol and trazodone increased circulating 7DHC and 8DHC levels, while five other drugs, clozapine, escitalopram/citalopram, lamotrigine, olanzapine, and risperidone, did not. Studies in rat brain verified that haloperidol dose-dependently increased 7DHC and 8DHC levels, while clozapine had no effect. We conclude that further studies should investigate the role of 7DHC and 8DHC metabolites, such as oxysterols, vitamin D, and atypical neurosteroids, in the deleterious and therapeutic effects of psychotropic drugs. Finally, we recommend that drugs that increase 7DHC levels should not be prescribed during pregnancy, as children born with DHCR7 deficiency have multiple congenital malformations. [ABSTRACT FROM AUTHOR]

Vitamin D3 (VitD3) has several beneficial effects on many metabolic pathways such as immunity system, bone development. The aim of the study, encapsulation of VitD3 with solid lipids, determine encapsulation efficiency and biocompatibility of nanoparticles. Therefore, VitD3-loaded solid lipid nanoparticles (SLNPs) were developed by optimising ratios of VitD3, stearic acid, beeswax and sodium dodecyl sulphate (SDS). Thermal stability, degradation profile, crystallinity rate, encapsulation efficiency and release profile of SLNPs were determined. Cytotoxicity of SLNPs on HaCaT, L929 and HUVEC cells were investigated. Negatively charged and VitD3-loaded nanoparticles with diameters between 30 and 60 nm were obtained. SLNPs containing up to 5.1 mg VitD3 per 10 mg powder samples were obtained. Cell proliferations were stimulated after exposure with VitD3-loaded SLNPs. Besides, inflammatory response after exposure to VitD3-loaded SLNPs was evaluated via determining IL10 and TNF-alpha levels on THP-1 cells. According to the results, no inflammatory response was observed. [ABSTRACT FROM PUBLISHER]

The study was conducted to determine the effect of 7-dehydrocholesterol loaded cyclodextrin (7-DHCLC) and cholesterol-loaded cyclodextrin (CLC) on changes of bull sperm motility during short term storage (+4°C). Collected ejaculates were pooled and divided into 7 groups, as following one control (C); three 7-DHCLC and the other three CLC concentrations. Diluted semen samples were transferred and stored at +4°C and sperm motility was analysed in determined intervals during three days storage period with computer aided sperm analyse (CASA) system. Motility decreased in all groups; however, 7-DHCLC and CLC groups motility decreased in the next two days gradually. But, CLC 1.5 mg/120x106 group were identified as the best groups for protecting motility in short time preservation. In conclusion, adding different rates of 7-DHCLC and CLC to semen extender preserved motility for three days in bull semen stored at +4°C. [ABSTRACT FROM AUTHOR]

Biodegradable polymer based novel drug delivery systems brought a considerable attention in enhancing the therapeutic efficacy and bioavailability of various drugs. 14-deoxy 11, 12-didehydro andrographolide (poorlywater soluble compound) loaded polycaprolactone (nano-DDA)was synthesized using the solvent evaporation technique. Nano-DDAwas characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS) studies. Fourier Transform InfraRed Spectroscopy (FTIR) was used to investigate the structural interaction between the drug and the polymer. Functional characterization of the formulation was determined using drug content, cellular uptake and in vitro drug release. 2-deoxy-D-[1-³H] glucose uptake assaywas carried out to assess the antidiabetic potential of nano-DDA in L6myotubes. The nano-DDA displayed spherical shape with a smooth surface (252.898 nm diameter), zeta potential, encapsulation and loading efficiencies of -38.9 mV, 91.98 ± 0.13% and 15.09 ± 0.18% respectively. No structural alteration between the drug and the polymerwas evidenced (FTIR analysis). Confocal microscopy studies with rhodamine 123 loaded polycaprolactone nanoparticles (Rh123-PCL NPs) revealed the internalization of Rh123-PCL NPs in a time dependent manner in L6myoblasts.A dose dependent increase in glucose uptakewas observed for nano-DDA with a maximal uptake of 108.54 ± 1.42% at 100 nM on L6 myotubes, thereby proving its anti-diabetic efficacy.A biphasic pattern of in vitro drug release demonstrated an initial burst release at 24 h followed by a sustained release for up to 11 days. To conclude, our results revealed that nano-DDA formulation can be a potent candidate for antidiabetic drug delivery. [ABSTRACT FROM AUTHOR]

Objectives: To engineer Yarrowia lipolytica for improving the heterologous production of campesterol (a key precursor to manufacture pharmaceutical steroids). Results: By screening 7-dehydrocholesterol reductase (DHCR7) from diverse species, DHCR7 from Danio rerio was the best candidate for campesterol synthesis. Overexpression of ACL (ATP: citrate lyase) or POX2 (peroxisome acyl-CoA oxidase 2) were key to improving campesterol production. The highest yield of campesterol was 942 mg/l was with the strain overexpressing POX2 in a 5 l bioreactor via high cell density fermentation process with a restricted supply of carbon sourc, sunflower seed oil. Conclusions: A promising platform to synthesize downstream steroid drugs was established. Efficient approaches were provided to improve the production of desired molecules in Y. lipolytica with high oil utilization efficiency. [ABSTRACT FROM AUTHOR]

Problems arising during treatment of tuberculosis are well known, therefore studies of Mycobacterium drug molecular targets are an area of particular importance. Members of the cytochrome P450 family (CYP) may belong to potential candidates for drug targets being involved in metabolism of biologically important molecules in the host organism. CYP124 of Mycobacterium tuberculosis (MTCYP124) catalyzes ω-hydroxylation of methyl-branched lipids. The data obtained in the present study indicate that this enzyme can also oxidize provitamin D3 (7-dehydrocholesterol) and vitamin D3. We found that the final product is different from 1α- and 25-hydroxyvitamin D3, so we propose that MTCYP124 is involved in alternative pathway for metabolism of vitamin D3. [ABSTRACT FROM AUTHOR]

Studies on the determinants of vitamin D status have tended to concentrate on input – exposure to ultraviolet B radiation and the limited sources in food. Yet, vitamin D status, determined by circulating concentrations of 25-hydroxyvitamin D (25(OH)D), can vary quite markedly in groups of people with apparently similar inputs of vitamin D. There are small effects of polymorphisms in the genes for key proteins involved in vitamin D production and metabolism, including 7-dehydrocholesterol reductase, which converts 7-dehydrocholesterol, the precursor of vitamin D, to cholesterol, CYP2R1, the main 25-hydroxylase of vitamin D, GC, coding for the vitamin D binding protein which transports 25(OH)D and other metabolites in blood and CYP24A1, which 24-hydroxylates both 25(OH)D and the hormone, 1,25-dihydroxyvitamin D. 25(OH)D has a highly variable half-life in blood. There is evidence that the half-life of 25(OH)D is affected by calcium intake and some therapeutic agents. Fat tissue seems to serve as a sink for the parent vitamin D, which is released mainly when there are reductions in adiposity. Some evidence is presented to support the proposal that skeletal muscle provides a substantial site of sequestration of 25(OH)D, protecting this metabolite from degradation by the liver, which may help to explain why exercise, not just outdoors, is usually associated with better vitamin D status. [ABSTRACT FROM AUTHOR]

Biocatalysis is a fundamental concept in biotechnology. The topic integrates knowledge of several disciplines; therefore, it was included in the course 'design and optimization of biological systems' which is offered in the biochemistry curricula. We selected the ciliate tetrahymena as an example of a eukaryotic system with potential for the biotransformation of sterol metabolites of industrial interest; in particular, we focused on the conversion of cholesterol to provitamin D3. The students work with wild type and recombinant strains and learn how sterol pathways could be modified to obtain diverse sterol moieties. During the course the students identify and measure the concentration of sterols. They also search for related genes by bioinformatic analysis. Additionally, the students compare biotransformation rates, growing the ciliate in plate and in a bioreactor. Finally, they use fluorescence microscopy to localize an enzyme involved in biotransformation. The last day each team makes an oral presentation, explaining the results obtained and responds to a series of key questions posed by the teachers, which determine the final mark. In our experience, this course enables undergraduate students to become acquainted with the principles of biocatalysis as well as with standard and modern techniques, through a simple and robust laboratory exercise, using a biological system for the conversion of valuable pharmaceutical moieties. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):105-114, 2017. [ABSTRACT FROM AUTHOR]

Cholesterol is essential for survival, but too much or too little can cause disease. Thus, cholesterol levels must be kept within close margins. 7-dehydrocholesterol reductase (DHCR7) is a terminal enzyme of cholesterol synthesis, and is essential for embryonic development. Largely, DHCR7 research is associated with the developmental disease Smith-Lemli-Opitz syndrome, which is caused by mutations in the DHCR7 gene. However, little is known about what regulates DHCR7 activity. Here we provide evidence that phosphorylation plays a role in controlling DHCR7 activity, which may provide a means to divert flux from cholesterol synthesis to vitamin D production. DHCR7 activity was significantly decreased when we used pharmacological inhibitors against two important kinases, AMP-activated protein kinase and protein kinase A. Moreover, mutating a known phosphorylated residue, S14, also decreased DHCR7 activity. Thus, we demonstrate that phosphorylation modulates DHCR7 activity in cells, and contributes to the overall synthesis of cholesterol, and probably vitamin D. [ABSTRACT FROM AUTHOR]

Introduction Due to the high prevalence of vitamin D deficiency, strategies are needed to improve vitamin D status. Food components can affect vitamin D metabolism and have to be considered when estimating the efficacy of vitamin D supplements. 7-dehydrocholesterol (7-DHC) occurs naturally in food, but its impact on vitamin D metabolism has not yet been examined. Methods Three groups of male C57BL/6 mice ( n = 12 per group) were placed on a diet that contained 0, 2.5 or 5 mg 7-DHC per kg diet over a period of 6 weeks. Vitamin D and other sterols in the serum, skin, liver and kidney were quantified by LC–MS/MS. The relative mRNA abundance of hepatic genes encoding vitamin D hydroxylation enzymes and transporters was analyzed by real-time RT-PCR. Results We found a substantial dose-dependent increase of non-hydroxylated vitamin D 3 in the liver and kidney of mice fed a diet containing 7-DHC. The vitamin D 3 content in the liver was 2.80 ± 0.61 pmol/g, 7.34 ± 4.28 pmol/g and 12.9 ± 3.58 pmol/g in groups that received 0, 2.5 and 5 mg/kg 7-DHC, respectively. In the kidney, the vitamin D 3 content of these groups was 1.78 ± 1.17 pmol/g, 3.55 ± 1.06 and 6.36 ± 2.29 pmol/g, respectively. The serum and tissue concentrations of 25-hydroxyvitamin D 3 (25(OH)D 3 ) remained unaffected by 7-DHC. The relative mRNA data provided no plausible mechanism for the observed effects of 7-DHC on vitamin D 3 . All groups of mice had similar concentrations of cholesterol, desmosterol and 7-DHC in their serum and tissues. Conclusion The current findings provide the first evidence that dietary 7-DHC seems to affect vitamin D metabolism. The underlying mechanism remains elusive and needs further investigation. [ABSTRACT FROM AUTHOR]

The conversion of 7-dehydrocholesterol to cholesterol, the final step of cholesterol synthesis in the Kandutsch-Russell pathway, is catalyzed by the enzyme 7-dehydrocholesterol reductase (DHCR7). Homozygous or compound heterozygous mutations in DHCR7 lead to the developmental disease Smith-Lemli-Opitz syndrome, which can also result in fetal mortality, highlighting the importance of this enzyme in human development and survival. Besides serving as a substrate for DHCR7, 7-dehydrocholesterol is also a precursor of vitamin D via the action of ultraviolet light on the skin. Thus, DHCR7 exerts complex biological effects, involved in both cholesterol and vitamin D production. Indeed, we argue that DHCR7 can act as a switch between cholesterol and vitamin D synthesis. This review summarizes current knowledge about the critical enzyme DHCR7, highlighting recent findings regarding its structure, transcriptional and post-transcriptional regulation, and its links to vitamin D synthesis. Greater understanding about DHCR7 function, regulation and its place within cellular metabolism will provide important insights into its biological roles. [ABSTRACT FROM AUTHOR]

Our prior discovery of a novel biexponential photochemical ring-opening in 7-dehydrocholesterol (DHC) to previtamin D3 [Tang J. Chem. Phys. 2011, 134, 104503 ] is further explored with ultrafast transient absorption spectroscopy, and the results are compared with recently reported high-level theoretical calculations. Three types of experiments are reported. First, variation of the excitation wavelength from 297 to 266 nm leaves the excited state dynamics unaffected. The biexponential decay of the excited state absorption is independent of excitation wavelength with time constants of 0.57 ± 0.06 and 1.88 ± 0.09 ps, in excellent agreement with the results reported earlier (0.56 ± 0.06 and 1.81 ± 0.15 ps) following excitation at 266 nm. Second, variation of the chirp of the excitation pulse influences the relative amplitude of the fast and slow decay components but has no influence on the photoproduct yield. Third, a 545 nm pulse delayed by 0.64 ps with respect to the initial 266 nm pulse was used to perturb the "slow" population and probe the influence of additional electronic or vibrational energy on the reaction process. The results show ultrafast internal conversion Sn → S1 on a ca. 150 fs time scale but no subsequent effect on the reaction dynamics. The experiments reported here are consistent with the recent state averaged complete active space self-consistent field ab initio multiple spawning (SA-CASSCF-AIMS) calculations of Snyder et al. [J. Phys. Chem. Lett. 2016, 7, 2444] that assign the biexponential decay to nonequilibrium dynamics related to the opening and closing motion of the cyclohexadiene ring moiety on the excited state surface. These new experiments support the model prediction that the biexponential dynamics does not involve multiple minima and demonstrate the direction for new experimental designs to manipulate the product yields and pathways. [ABSTRACT FROM AUTHOR]

In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions. [ABSTRACT FROM AUTHOR]

Although antiretroviral agents have been used successfully in suppressing viral production, they have also been associated with a number of side effects. The antiretroviral toxic neuropathy induces debilitating and extremely difficult to treat pain syndromes that often lead to discontinuation of antiretroviral therapy. Due to the critical need for the identification of novel therapeutic targets to improve antiretroviral neuropathic pain management, we investigated the role of the JNK signalling pathway in the mechanism of antiretroviral painful neuropathy. Mice were exposed to zalcitabine (2′,3′-dideoxycytidine, ddC) and stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T) that induced a persistent mechanical allodynia and a transient cold allodynia. Treatment with the JNK blocker SP600125 before antiretroviral administration abolished mechanical hypersensitivity with no effect on thermal response. A robust spinal JNK overphosphorylation was observed on post-injection day 1 and 3, along with a JNK-dependent increase in p-c-Jun and ATF3 protein levels. Co-immunoprecipitation experiments showed the presence of a heterodimeric complex between ATF3 and c-Jun indicating that these transcription factors can act together to regulate transcription through heterodimerization. A rise in BDNF and caspase-3 protein levels was detected on day 1 and BDNF sequestration prevented both caspase-3 and p-JNK increase. These data suggest that BDNF plays a role in the early stages of ddC-induced allodynia by promoting apoptotic events and the activation of a hypernociceptive JNK-mediated pathway. We illustrated the activation of a BDNF-mediated JNK pathway involved in the early events responsible for the promotion of neuropathic pain, leading to a better knowledge of the mechanisms involved in the antiretroviral neuropathy. Summary JNK blockade prevents antiretroviral-induced pain hypersensitivity. This may represent a potential prophylactic treatment of neuropathic pain to improve antiretroviral tolerability. [ABSTRACT FROM AUTHOR]

A small library of pharmacologically active compounds (the NIH Clinical Collection) was assayed in Neuro2a cells to determine their effect on the last step in the biosynthesis of cholesterol, the transformation of 7-dehydrocholesterol (7-DHC) to cholesterol promoted by 7-dehydrocholesterol reductase, DHCR7. Of some 727 compounds in the NIH Clinical Collection, over 30 compounds significantly increased 7-DHC in Neuro2a cells when assayed at 1 μM. Active compounds that increased 7-DHC with a Z-score of +3 or greater generally gave rise to modest decreases in desmosterol and increases in lanosterol levels. Among the most active compounds identified in the library were the antipsychotic, antidepressant, and anxiolytic compounds that included perospirone, nefazodone, haloperidol, aripiprazole, trazodone, and buspirone. Fluoxetine and risperidone were also active at 1 μM, and another 10 compounds in this class of pharmaceuticals were identified in the screen at concentrations of 10 μM. Increased levels of 7-DHC are associated with Smith-Lemli-Opitz syndrome (SLOS), a human condition that results from a mutation in the gene that encodes DHCR7. The SLOS phenotype includes neurological deficits and congenital malformations, and it is linked to a higher incidence of autism spectrum disorder. The significance of the current study is that it identifies common pharmacological compounds that may induce a biochemical presentation similar to SLOS. Little is known about the side effects of elevated 7-DHC postdevelopmentally, and the elevated 7-DHC that results from exposure to these compounds may also be a confounder in the diagnosis of SLOS. [ABSTRACT FROM AUTHOR]

Cholesterol is detrimental to human health in excess but is also essential for normal embryogenesis. Hence, enzymes involved in its synthesis possess many layers of regulation to achieve balanced cholesterol levels. 7-Dehydrocholesterol reductase (DHCR7) is the terminal enzyme of cholesterol synthesis in the Kandutsch-Russell pathway, converting 7-dehydrocholesterol (7DHC) to cholesterol. In the absence of functional DHCR7, accumulation of 7DHC and a lack of cholesterol production leads to the devastating developmental disorder, Smith-Lemli-Opitz syndrome. This study identifies that statin treatment can ameliorate the low DHCR7 expression seen with common Smith-Lemli-Opitz syndrome mutations. Furthermore, we show that wild-type DHCR7 is also relatively labile. In an example of end-product inhibition, cholesterol accelerates the proteasomal degradation of DHCR7, resulting in decreased protein levels and activity. The loss of enzymatic activity results in the accumulation of the substrate 7DHC, which leads to an increased production of vitamin D. Thus, these findings highlight DHCR7 as an important regulatory switch between cholesterol and vitamin D synthesis. [ABSTRACT FROM AUTHOR]

Tissue accumulation of 7-dehydrocholesterol (7DHC) is a hallmark of Smith-Lemli-Opitz Syndrome (SLOS), a human inborn error of the cholesterol (CHOL) synthesis pathway. Retinal 7DHC-derived oxysterol formation occurs in the AY9944-induced rat model of SLOS, which exhibits a retinal degeneration characterized by selective loss of photoreceptors and associated functional deficits, Müller cell hypertrophy, and engorgement of the retinal pigment epithelium (RPE) with phagocytic inclusions. We evaluated the relative effects of four 7DHC-derived oxysterols on three retina-derived cell types in culture, with respect to changes in cellular morphology and viability. 661W (photoreceptor-derived) cells, rMC-1 (Müller glia-derived) cells, and normal diploid monkey RPE (mRPE) cells were incubated for 24 h with dose ranges of either 7-ketocholesterol (7kCHOL), 5,9-endoperoxy-cholest-7-en-3β,6α-diol (EPCD), 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), or 4β-hydroxy-7-dehydrocholesterol (4HDHC); CHOL served as a negative control (same dose range), along with appropriate vehicle controls, while staurosporine (Stsp) was used as a positive cytotoxic control. For 661W cells, the rank order of oxysterol potency was: EPCD > 7kCHOL >> DHCEO > 4HDHC ≈ CHOL. EC 50 values were higher for confluent vs . subconfluent cultures. 661W cells exhibited much higher sensitivity to EPCD and 7kCHOL than either rMC-1 or mRPE cells, with the latter being the most robust when challenged, either at confluence or in sub-confluent cultures. When tested on rMC-1 and mRPE cells, EPCD was again an order of magnitude more potent than 7kCHOL in compromising cellular viability. Hence, 7DHC-derived oxysterols elicit differential cytotoxicity that is dose-, cell type-, and cell density-dependent. These results are consistent with the observed progressive, photoreceptor-specific retinal degeneration in the rat SLOS model, and support the hypothesis that 7DHC-derived oxysterols are causally linked to that retinal degeneration as well as to SLOS. [ABSTRACT FROM AUTHOR]

The polyhydroxylated derivatives of 6-keto,7-dehydrocholesterol (ecdysone, ecdysteroids, Ecd) are natural compounds widely distributed in plants. They exhibit strong anabolic, vitamin D-like, pharmacological effects in vertebrate animals and in the human body. In the larval stages of insects, injections of pure Ecd cause serious pathophysiological, “hyperecdysonic” syndromes associated with neuromuscular paralysis, premature cuticular apolysis and complete inhibition of ecdysis. Ecds do not penetrate insect cuticle. For this reason, all previous attempts to induce ecdysone responses by topical applications of Ecd failed. In this work, we tried to induce the topical effects of Ecd by preparation of more lipophilic complexes, with 2 or 4 molecules of 20-hydroxyecdysone (E20) attached to a relatively large nucleus of the porphyrin. The resulting porphyrin-E20 complexes (ecdysogens) have been subjected to standardised assays for ecdysone activity in the ligatured larvae (“ dauerlarvae ”) of the greater waxmoth ( Galleria mellonella ). Similarly like the free E20 alone, porphyrin-E20 complexes had no effect when applied on the body surface or administered in the larval diet. When injected, however, they exhibited delayed effects, but the adverse (“hyperecdysonic”) pathophysiological syndromes were reduced or abolished. It is concluded, therefore, that the replacement of pathophysiological, precocious or “hyperecdysonic” moults by the larval-pupal transformation, was due to successive metabolic liberation of the biologically active, free E20 from the porphyrin-E20 complex. The biological status of Ecd does not agree with their definition as the prothoracic gland (PG) hormone of insects, nor with the assumptions about a growth hormone of plants. A possibility that the most important status of Ecd may depend on the pharmacological properties of a sterolic D 6 vitamin has been discussed. [ABSTRACT FROM AUTHOR]

Three hand-reared, 50-53 day-old, red-legged seriema ( Cariama cristata) chicks were evaluated for acute lameness and reluctance to ambulate. Two of the 3 chicks presented with angular limb deformities of the proximal tarsometatarsi and external rotation of the legs. Radiographs demonstrated decreased opacity of the long bone of the legs, with poorly delineated cortices and deviation of the proximal tarsometarsi. Serum concentrations of 25-hydroxycholecalciferol revealed all 3 chicks were deficient in vitamin D3 at presentation. The chicks were administered injectable vitamin D3 (cholecalciferol), oral vitamin D3, and an ultraviolet B (UV-B) light was placed in their enclosure. Elastic, therapeutic taping was used to correct angular limb deformities present in 2 of the 3 chicks. Taping was continued until the angular limb deformities were corrected and lameness resolved. Hypovitaminosis D is a common cause of metabolic bone disease in captive avian species. Cholecalciferol administration, UV-B light supplementation, and elastic, therapeutic taping were effective treatments for osteodystrophy and secondary angular limb deformities due to hypovitaminosis D. This multifaceted treatment may be useful in other long-legged juvenile birds with similar clinical signs. [ABSTRACT FROM AUTHOR]

7-Dehydrocholesterol, an immediate metabolic predecessor of cholesterol, can accumulate in tissues due to some metabolic abnormalities, causing an array of symptoms known as Smith-Lemli-Opitz syndrome. Enrichment of cellular membranes with 7-dehydrocholesterol interferes with normal cell-signaling processes, which involve interaction between rafts and formation of the so-called signaling platforms. In model membranes, cholesterol-based ordered domains usually merge upon contact. According to our experimental data, ordered domains in the model systems where cholesterol is substituted for 7-dehydrocholesterol never merge on the time scale of the experiment, but clusterize into necklace-like aggregates. We attribute such different dynamical behavior to altered properties of the domain boundary. In the framework of thickness mismatch model, we analyzed changes of interaction energy profiles of two approaching domains caused by substitution of cholesterol by 7-dehydrocholesterol. The energy barrier for domain merger is shown to increase notably, with simultaneous appearance of another distinct local energy minimum. Such energy profile is in perfect qualitative agreement with the experimental observations. The observed change of domain dynamics can impair proper interaction between cellular rafts underlying pathologies associated with deviations in cholesterol metabolism. [ABSTRACT FROM AUTHOR]

Well-established cell culture models were combined with new analytical methods to assess the effects of small molecules on the cholesterol biosynthesis pathway. The analytical protocol, which is based on sterol derivation with the dienolphile PTAD, was found to be reliable for the analysis of 7-DHC and desmosterol. The PTAD method was applied to the screening of a small library of pharmacologically active substances, and the effect of compounds on the cholesterol pathway was determined. Of some 727 compounds, over 30 compounds decreased 7-DHC in Dhcr7-deficient Neuro2a cells. The examination of chemical structures of active molecules in the screen grouped the compounds into distinct categories. In addition to statins, our screen found that SERMs, antifungals, and several antipsychotic medications reduced levels of 7-DHC. The activities of selected compounds were verified in human fibroblasts derived from Smith-Lemli-Opitz syndrome (SLOS) patients and linked to specific transformations in the cholesterol biosynthesis pathway. [ABSTRACT FROM AUTHOR]

Sunscreens protect the skin against erythemal radiation (Eer). But at the same time they reduce the effective radiation dose (EVD) responsible for the formation of previtamin D in the skin. The paper describes a calculation method for optimizing the ratio EVD/Eer behind sunscreens e.g. with SPF 5, 15 and 30 respectively. Taking into account that a majority of people in industrialized countries suffer from a shortage in vitamin D even in summer time, the ratio Evd/Eer is a new and important criterion for the quality of sunscreens. Furthermore the exposure time tvd needed per day for forming the equivalent of the recommended amount of 2000 IU of vitamin D per day for skin type 2 is estimated when sunscreens with different filter compositions are used. In vitro experiments show a significant increase of the conversion of 7-dehydrocholesterol (7-DHC) to previtamin D when exposed to artificial solar radiation behind an experimental sunscreen optimized for previtamin D production compared to a commercial sunscreen having the same SPF. [ABSTRACT FROM AUTHOR]

In the current work the ability of Bacillus megaterium to take up hydrophobic substrates and efficiently express eukaryotic membrane proteins was utilized for establishing a CYP27A1-based biocatalyst. The human mitochondrial cytochrome P450CYP27A1 was co-expressed with its redox partners adrenodoxin reductase (Adr) and adrenodoxin (Adx). CYP27A1 could be localized at the cell’s polyhydroxybutyrate (PHB) granules, carbon storage serving organelle-like vesicles that can take up cholesterol, resulting in bioreactor-like structures in B. megaterium . The resulting whole cell system allowed the efficient biotechnological conversion of the CYP27A1 substrates cholesterol, 7-dehydrocholesterol (7-DHC) and vitamin D3. After 48 h, nearly 100% of cholesterol was metabolized producing a final concentration of 113.14 mg/l 27-hydroxycholesterol (27-HC). Moreover, 70% of vitamin D3 was converted into 25-hydroxyvitamin D3 (25-OH-D3) with a final concentration of 80.81 mg/l. Also more than 97% of 7-DHC were found to be metabolized into two products, corresponding to 26/27-hydroxy-7-dehydrocholesterol (P1) and 25-hydroxy-7-dehydrocholesterol (P2). To our knowledge this is the first CYP27A1-based whole-cell system, allowing the efficient and low-cost production of pharmaceutically interesting metabolites of this enzyme from relatively cheap substrates. [ABSTRACT FROM AUTHOR]

Campesterol is an important precursor for many sterol drugs, e.g. progesterone and hydrocortisone. In order to produce campesterol in Yarrowia lipolytica, C-22 desaturase encoding gene ERG5 was disrupted and the heterologous 7-dehydrocholesterol reductase (DHCR7) encoding gene was constitutively expressed. The codon-optimized DHCR7 from Rallus norvegicus, Oryza saliva and Xenapus laevis were explored and the strain with the gene DHCR7 from X. laevis achieved the highest titer of campesterol due to D409 in substrate binding sites. In presence of glucose as the carbon source, higher biomass conversion yield and product yield were achieved in shake flask compared to that using glycerol and sunflower seed oil. Nevertheless, better cell growth rate was observed in medium with sunflower seed oil as the sole carbon source. Through high cell density fed-batch fermentation under carbon source restriction strategy, a titer of 453±24.7 mg/L campesterol was achieved with sunflower seed oil as the carbon source, which is the highest reported microbial titer known. Our study has greatly enhanced campesterol accumulation in Y. lipolytica, providing new insight into producing complex and desired molecules in microbes. [ABSTRACT FROM AUTHOR]

Vitamin D metabolites are essential for bone regeneration and mineral homeostasis. The vitamin D precursor 7-dehydrocholesterol can be used after UV irradiation to locally produce active vitamin D by osteoblastic cells. Furthermore, UV-irradiated 7-dehydrocholesterol is a biocompatible coating for titanium implants with positive effects on osteoblast differentiation. In this study, we examined the impact of titanium implants surfaces coated with UV-irradiated 7-dehydrocholesterol on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. First, the synthesis of cholecalciferol (D3) was achieved through the incubation of the UV-activated 7-dehydrocholesterol coating for 48 h at 23℃. Further, we investigated in vitro the biocompatibility of this coating in human umbilical cord mesenchymal stem cells and its potential to enhance their differentiation towards the osteogenic lineage. Human umbilical cord mesenchymal stem cells cultured onto UV-irradiated 7-dehydrocholesterol-coated titanium implants surfaces, combined with osteogenic supplements, upregulated the gene expression of several osteogenic markers and showed higher alkaline phosphatase activity and calcein blue staining, suggesting increased mineralization. Thus, our results show that the use of UV irradiation on 7-dehydrocholesterol -treated titanium implants surfaces generates a bioactive coating that promotes the osteogenic differentiation of human umbilical cord mesenchymal stem cells, with regenerative potential for improving osseointegration in titanium-based bone anchored implants. [ABSTRACT FROM AUTHOR]

To investigate whether novel pathways of vitamin D3 (D3) and 7-dehydrocholesterol (7DHC) metabolism initiated by CYP11A1 and previously characterized in vitro, occur in vivo, we analyzed samples of human serum and epidermis, and pig adrenals for the presence of intermediates and products of these pathways. We extracted human epidermis from 13 individuals and sera from 13 individuals and analyzed them by LC/qTOF-MS alongside the corresponding standards. Pig adrenal glands were also analyzed for these steroids and secosteroids. Epidermal, serum and adrenal samples showed the presence of D3 hydroxy-derivatives corresponding to 20(OH)D3, 22(OH)D3, 25(OH)D3, 1,25(OH)2D3, 20,22(OH)2D3, 20,23(OH)2D3, 20,24(OH)2D3, 20,25(OH)2D3, 20,26(OH)2D3, 1,20,23(OH)3D3 and 17,20,23(OH)3D3, plus 1,20(OH)2D3 which was detectable only in the epidermis. Serum concentrations of 20(OH)D3 and 22(OH)D3 were only 30- and 15-fold lower than 25(OH) D3, respectively, and at levels above those required for biological activity as measured in vitro. We also detected 1,20,24(OH)3D3, 1,20,25(OH)3D3 and 1,20,26(OH)3D3 in the adrenals. Products of CYP11A1 action on 7DHC, namely 22(OH)7DHC, 20,22(OH)27DHC and 7-dehydropregnenolone were also detected in serum, epidermis and the adrenal. Thus, we have detected novel CYP11A1-derived secosteroids in the skin, serum and adrenal gland and based on their concentrations and biological activity suggest that they act as hormones in vivo. [ABSTRACT FROM AUTHOR]

We performed differential scanning calorimetric (DSC) and Fourier transform infrared (FTIR) spectroscopic studies of the effects of cholesterol (CHOL), 7-dehydrocholeterol (7DHC) and desmosterol (DES) on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes. 7DHC and DES are the immediate biosynthetic precursors of CHOL in the Kandutch–Russell and Bloch pathways and 7DHC and DES differ in structure from CHOL only by the presence of an additional double bond at C7 of ring B or C24 of the alkyl side chain, respectively. Our DSC results indicate that the incorporation of all three sterols produces comparable decreases in the temperature of the pretransition of DPPC, but CHOL decreases its cooperativity and enthalpy more strongly than 7DHC and especially DES. These findings indicate that all three sterols decrease the thermal stability of gel phase DPPC bilayers but that 7DHC and especially DES are less miscible in them. However, the incorporation of CHOL and DES produce comparable increases in the temperature of the broad component of the main phase transition of DPPC while 7DHC decreases it, but again CHOL produces greater decreases in its cooperativity and enthalpy then 7DHC and especially DES. These results indicate that CHOL and DES stabilize the sterol-rich domains of fluid DPPC bilayers, but that 7DHC and especially DES are less miscible in them. Our FTIR spectroscopic results indicate that CHOL increases the rotational conformational order of fluid DPPC bilayers to a somewhat and markedly greater degree than DES and 7DHC, respectively, consistent with our DSC findings. Our spectroscopic results also indicate that although all three sterols produce comparable degrees of H-bonding (hydration) of the DPPC ester carbonyl groups in fluid bilayers, CHOL is again found to be fully soluble in gel state DPPC bilayers at low temperatures, whereas 7DHC and especially DES are not. In general, we find that 7DHC and DES incorporation produce considerably different effects on DPPC bilayer membranes. In particular, the presence of an additional double bond at C7 or C24 produces a marked reduction in the ability of 7DHC to order fluid DPPC bilayers and in the miscibility of DES in such bilayers, respectively. These different effects may be the biophysical basis for the reduction of these double bonds in the last steps of CHOL biosynthesis, and for the deleterious biological effects of the accumulation of these sterols in vivo. [ABSTRACT FROM AUTHOR]

Pathogenic variants in the DHCR7 gene cause Smith-Lemli-Opitz syndrome (SLOS), a defect of cholesterol biosynthesis resulting in an autosomal recessive congenital metabolic malformation disorder. In approximately 4% of patients, the second mutation remains unidentified. In this study, 12 SLOS patients diagnosed clinically and/or by elevated 7-dehydrocholesterol (7-DHC) have been investigated by customized multiplex ligation-dependent probe amplification (MLPA) analysis, because only one DHCR7 sequence variant has been detected. Two unrelated patients of this cohort carry different large deletions in the DHCR7 gene. One patient showed a deletion of exons 3-6. The second patient has a deletion of exons 1 and 2 (non-coding) and lacks the major part of the promoter. These two patients show typical clinical and biochemical phenotypes of SLOS. Second disease-causing mutations are p.(Arg352Trp) and p.(Thr93Met), respectively. Deletion breakpoints were characterized successfully in both cases. Such large deletions are rare in the DHCR7 gene but will resolve some of the patients in whom a second mutation has not been detected. [ABSTRACT FROM AUTHOR]

Background Cholecalciferol (vitamin D 3 ), produced in the skin by UVB irradiation (290–315 nm) of 7-dehydrocholesterol, is metabolized in the liver into 25-hydroxyvitamin D [25(OH)D] which is a major circulating metabolite. Aim To examine changes in serum concentrations of cholecalciferol and its metabolites after UVB exposure of different skin areas. Methods 21 healthy Caucasians (skin type II and III, aged 23–47 years) were exposed to broadband UVB (290–320 nm) and randomized to either exposure to one minimal erythema dose given as a single dose, or a suberythemic dose given for 3 subsequent days. The following areas were exposed: face and back of hands, upper half of the body and the whole body, respectively. Serum cholecalciferol and 25(OH)D were measured immediately before start and 24 h after the 1st and last exposure, respectively. Results Subjects with whole body exposure had an average S-cholecalciferol increase per dose unit of 0.18 ng ml −1 mJ −1 cm 2 , 0.95 CI: (0.16, 0.20), upper body treatment 0.13 ng ml −1 mJ −1 cm 2 , 0.95 CI: (0.10, 0.15) and face and hands exposure 0.013 ng ml −1 mJ −1 cm 2 , 0.95 CI: (−0.012, 0.037). The increase in cholecalciferol correlated positively to the UVB dose and skin erythema and negatively to body mass index (BMI) when controlling for other factors. Exposure of face and hands induces smaller cholecalciferol production in comparison with exposure of larger skin areas. Conclusion Size of the exposed skin area, UVB dose, skin erythema and BMI were the major determinants for serum levels of skin synthesized cholecalciferol. Exposure of hands and face induces smaller cholecalciferol production in comparison with exposure of larger skin areas. [ABSTRACT FROM AUTHOR]

Smith-Lemli-Opitz syndrome (SLOS) is an inherited metabolic disease in the cholesterol biosynthesis pathway which is characterised by accumulation of 7- and 8-dehydrocholesterol and by reduced cholesterol concentrations in all tissues and body fluids. With this study, we developed a new, rapid, robust and high-throughput tandem mass spectrometric method as routine application for the selective SLOS screening and therapy monitoring in serum and dried blood. After protein precipitation of 10 μL serum or 4.7 mm dried blood spot, the sum of 7- and 8-dehydrocholesterol (DHC) was analysed by rapid chromatography combined with tandem mass spectrometry. Method comparison with GC-MS was performed for 46 serum samples. A comparison between serum and corresponding dried blood spots for DHC and cholesterol was performed with 40 samples from SLOS patients. Concentrations of DHC and cholesterol were analysed in 2 dried blood samples from newborns with SLOS and 100 unaffected newborns. Intra- and inter-assay variabilities ranged between 3.7 and 17.7 % for serum and dried blood spots. Significant correlations between the new LC-MS/MS method and GC-MS were determined for DHC ( r = 0.937, p < 0.001) and for cholesterol ( r = 0.946, p < 0.001). Significant coefficients of correlation between serum and dried blood spot samples above 0.8 were calculated for both analytes. A cut-off value of 5.95 for the ratio of DHC/cholesterol (multiplied by 1000) was found to distinguish newborns diagnosed with SLOS from normal newborns in a retrospective analysis after 5 years. The developed method enables a rapid quantification of the sum parameter 7- and 8-DHC in newborns and SLOS patients under therapy in serum as well as dried blood spot samples. [ABSTRACT FROM AUTHOR]

Background: The aim of this preliminary study was to assess the safety and efficacy of high-dose oral vitamin D3 supplementation during pregnancy in women with multiple sclerosis (MS) in Isfahan, Iran. Methods: In a single center open-label randomized, controlled clinical Phase I/II pilot study, 15 pregnant women with confirmed MS with low serum 25-hydroxyvitamin D (25(OH)D) levels were randomly allocated to receive either 50,000 IU/week vitamin D3 or routine care from 12 to 16 weeks of gestation till delivery. The main outcome measures were mean change in serum 25(OH)D levels, expanded disability status scale (EDSS) score, and number of relapse events during pregnancy and within 6 months after delivery. Results: Average serum 25(OH)D level at the end of trial in vitamin D3 supplemented group was higher than routine care group (33.7 ng/mL vs. 14.6 ng/ml, P < 0.050). In vitamin D3 group, the mean EDSS did not changed 6 months after delivery (P > 0.050), whereas in routine care group, the mean EDSS increased from 1.3 (0.4) to 1.7 (0.6) (P < 0.070). Women in vitamin D3 group appeared to have fewer relapse events during pregnancy and within 6 months after delivery. No significant adverse events occurred. Conclusion: Adding high dose vitamin D3 supplementation during pregnancy to routine care of women with MS had significant effect on the serum 25(OH)D levels, EDSS and number of relapse events during pregnancy and within 6 months after delivery. [ABSTRACT FROM AUTHOR]

Profiling and imaging of cholesterol and its precursors by mass spectrometry (MS) are important in a number of cholesterol biosynthesis disorders, such as in Smith-Lemli-Opitz syndrome (SLOS), where 7-dehydrocholesterol (7-DHC) is accumulated in affected individuals. SLOS is caused by defects in the enzyme that reduces 7-DHC to cholesterol. However, analysis of sterols is challenging because these hydrophobic olefins are difficult to ionize for MS detection. We report here sputtered silver matrix-assisted laser desorption/ionization (MALDI)-ion mobility-MS (IM-MS) analysis of cholesterol and 7-DHC. In comparison with liquid-based AgNO and colloidal Ag nanoparticle (AgNP), sputtered silver NP (10-25 nm) provided the lowest limits-of-detection based on the silver coordinated [cholesterol + Ag] and [7-DHC + Ag] signals while minimizing dehydrogenation products ([M + Ag-2H]). When analyzing human fibroblasts that were directly grown on poly-L-lysine-coated ITO glass plates with this technique, in situ, the 7-DHC/cholesterol ratios for both control and SLOS human fibroblasts are readily obtained. The m/z of 491 (specific for [7-DHC + Ag]) and 495 (specific for [cholesterol + Ag]) were subsequently imaged using MALDI-IM-MS. MS images were co-registered with optical images of the cells for metabolic ratio determination. From these comparisons, ratios of 7-DHC/cholesterol for SLOS human fibroblasts are distinctly higher than in control human fibroblasts. Thus, this strategy demonstrates the utility for diagnosing/assaying the severity of cholesterol biosynthesis disorders in vitro. Graphical Abstract [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]