Visible Light-Mediated Enantioselective Addition of α-Aminoalkyl Radicals to Ketones Catalyzed by Chiral Oxazaborolidinium Ion.

The development of a visible light-mediated synthetic method for chiral 1,2-amino tertiary alcohols is described. In the presence of a chiral oxazaborolidinium ion catalyst and photosensitizer, the enantioselective addition of an α-aminoalkyl radical to aryl methyl ketones under visible light provides chiral 1,2-amino tertiary alcohol derivatives in high yields (up to 88%) with excellent enantioselectivities (up to 98% ee). With mechanistic studies such as radical trapping analysis, radical clock analysis, and the measurement of quantum yield, a plausible catalytic cycle is proposed.

Fingolimod phosphate inhibits astrocyte inflammatory activity in mucolipidosis IV.

Mucolipidosis IV (MLIV) is an orphan neurodevelopmental disease that causes severe neurologic dysfunction and loss of vision. Currently there is no therapy for MLIV. It is caused by loss of function of the lysosomal channel mucolipin-1, also known as TRPML1. Knockout of the Mcoln1 gene in a mouse model mirrors clinical and neuropathologic signs in humans. Using this model, we previously observed robust activation of microglia and astrocytes in early symptomatic stages of disease. Here we investigate the consequence of mucolipin-1 loss on astrocyte inflammatory activation in vivo and in vitro and apply a pharmacologic approach to restore Mcoln1-/- astrocyte homeostasis using a clinically approved immunomodulator, fingolimod. We found that Mcoln1-/- mice over-express numerous pro-inflammatory cytokines, some of which were also over-expressed in astrocyte cultures. Changes in the cytokine profile in Mcoln1-/- astrocytes are concomitant with changes in phospho-protein signaling, including activation of PI3K/Akt and MAPK pathways. Fingolimod promotes cytokine homeostasis, down-regulates signaling within the PI3K/Akt and MAPK pathways and restores the lysosomal compartment in Mcoln1-/- astrocytes. These data suggest that fingolimod is a promising candidate for preclinical evaluation in our MLIV mouse model, which, in case of success, can be rapidly translated into clinical trial.

Mice defective in interferon signaling help distinguish between primary and secondary pathological pathways in a mouse model of neuronal forms of Gaucher disease.

BACKGROUND: The type 1 interferon (IFN) response is part of the innate immune response and best known for its role in viral and bacterial infection. However, this pathway is also induced in sterile inflammation such as that which occurs in a number of neurodegenerative diseases, including neuronopathic Gaucher disease (nGD), a lysosomal storage disorder (LSD) caused by mutations in GBA. METHODS: Mice were injected with conduritol B-epoxide, an irreversible inhibitor of acid beta-glucosidase, the enzyme defective in nGD. MyTrMaSt null mice, where four adaptors of pathogen recognition receptors (PRRs) are deficient, were used to determine the role of the IFN pathway in nGD pathology. Activation of inflammatory and other pathways was analyzed by a variety of methods including RNAseq. RESULTS: Elevation in the expression of PRRs associated with the IFN response was observed in CBE-injected mice. Ablation of upstream pathways leading to IFN production had no therapeutic benefit on the lifespan of nGD mice but attenuated neuroinflammation. Primary and secondary pathological pathways (i.e., those associated or not with mouse survival) were distinguished, and a set of ~210 genes including those related to sphingolipid, cholesterol, and lipoprotein metabolism, along with a number of inflammatory pathways related to chemokines, TNF, TGF, complement, IL6, and damage-associated microglia were classified as primary pathological pathways, along with some lysosomal and neuronal genes. CONCLUSIONS: Although IFN signaling is the top elevated pathway in nGD, we demonstrate that this pathway is not related to mouse viability and is consequently defined as a secondary pathology pathway. By elimination, we defined a number of critical pathways that are directly related to brain pathology in nGD, which in addition to its usefulness in understanding pathophysiological mechanisms, may also pave the way for the development of novel therapeutic paradigms by targeting such pathways.

Absence of infiltrating peripheral myeloid cells in the brains of mouse models of lysosomal storage disorders.

Approximately 70 lysosomal storage diseases are currently known, resulting from mutations in genes encoding lysosomal enzymes and membrane proteins. Defects in lysosomal enzymes that hydrolyze sphingolipids have been relatively well studied. Gaucher disease is caused by the loss of activity of glucocerebrosidase, leading to accumulation of glucosylceramide. Gaucher disease exhibits a number of subtypes, with types 2 and 3 showing significant neuropathology. Sandhoff disease results from the defective activity of ß-hexosaminidase, leading to accumulation of ganglioside GM2. Niemann-Pick type C disease is primarily caused by the loss of activity of the lysosomal membrane protein, NPC1, leading to storage of cholesterol and sphingosine. All three disorders display significant neuropathology, accompanied by neuroinflammation. It is commonly assumed that neuroinflammation is the result of infiltration of monocyte-derived macrophages into the brain; for instance, cells resembling lipid-engorged macrophages ('Gaucher cells') have been observed in the brain of Gaucher disease patients. We now review the evidence that inflammatory macrophages are recruited into the brain in these diseases and then go on to provide some experimental data that, at least in the three mouse models tested, monocyte-derived macrophages do not appear to infiltrate the brain. Resident microglia, which are phenotypically distinct from infiltrating macrophages, are the only myeloid population present in significant numbers within the brain parenchyma in these authentic mouse models, even during the late symptomatic stages of disease when there is substantial neuroinflammation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. This article is part of the Special Issue "Lysosomal Storage Disorders".

Simple extraction method for quantification of phenothiazine residues in pork muscle using liquid chromatography-triple quadrupole tandem mass spectrometry.

In this study, an analytical method was developed for quantification of residues of the anthelmintic drug phenothiazine (PTZ) in pork muscle using liquid chromatography-tandem mass spectrometry. Muscles were extracted using 0.2% formic acid and 10 mm ammonium formate in acetonitrile, defatted and purified using n-hexane. The drug was well separated on a Waters XBridge™ C18 analytical column using a binary solvent system consisting of 0.2% formic acid and 10 mm ammonium formate in ultrapure water (A) and acetonitrile (B). Good linearity was achieved over a six-point concentration range in matrix-matched calibration with determination coefficient =0.9846. Fortified pork muscle having concentrations equivalent to and double the limit of quantification (1 ng/g) yielded recovery ranges between 100.82 and 104.03% and relative standard deviations <12%. Samples (n = 5) collected from large markets located in Seoul City tested negative for PTZ residue. In conclusion, 0.2% formic acid and ammonium formate in acetonitrile can effectively extract PTZ from pork muscle without solid-phase extraction, a step normally required for cleanup before analysis and the validated method can be used for routine analysis to ensure the quality of animal products.

Catalytic Enantioselective Synthesis of 2,5-Dihydrooxepines.

A Michael addition initiated cyclopropanation/retro-Claisen rearrangement tandem reaction was developed for the enantioselective synthesis of highly functionalized 2,5-dihydrooxepines. In the presence of a chiral oxazaborolidinium ion (COBI) catalyst, the reaction proceeds to give good yields and high enantioselectivity.

Determination of residual levels of naloxone, yohimbine, thiophanate, and altrenogest in porcine muscle using QuEChERS with liquid chromatography and triple quadrupole mass spectrometry.

A quick, easy, cheap, effective, rugged, and safe QuEChERS (method) was used for the simultaneous detection of four veterinary drug residues, namely naloxone, yohimbine, thiophanate, and altrenogest, in porcine muscle, using liquid chromatography with electrospray ionization triple quadrupole tandem mass spectrometry. Because of the unavailability of a suitable internal standard, matrix-matched calibrations were used for quantification, with determination coefficients ≥ 0.9542. The accuracy (expressed as recovery %) ranged from 60.53 to 83.25%, and the intra- and interday precisions (expressed as relative standard deviations) were <12%. The limits of quantification were 5, 0.5, 2, and 5 ng/g for naloxone, yohimbine, thiophanate, and altrenogest, respectively. Samples purchased from local markets in Seoul, Republic of Korea, revealed no traces of the target analytes. The developed method described herein is sensitive and reliable and can be applied to quantify the tested veterinary drugs in animal tissues.

Simultaneous determination of aminopyrine and antipyrine in porcine muscle, milk, and eggs using liquid chromatography with tandem mass spectrometry.

The concentrations of residual aminopyrine and antipyrine in porcine muscle, milk, and egg samples were analyzed using liquid chromatography with tandem mass spectrometry after undergoing a series of sample pretreatment steps. Owing to an ion suppression effect, matrix-matched calibrations were used for analyte quantitation with determination coefficients (R(2) ) ≥ 0.9931. The recovery rates for aminopyrine and antipyrine in various matrices at two spiking levels (5 and 10 ng/g) fell in the range of 60.96-68.87 and 61.87-66.99%, respectively. Meanwhile, the intra- and inter-day precisions (expressed as relative standard deviation) were 1.02-12.95 and 1.71-5.50%, respectively. The method's detection limit (1 ng/g) was very low, thus enabling the detection of low residue levels. The applicability of the developed method was demonstrated with actual market samples and none of the tested analytes was detected in any of the samples.

A highly efficient catalytic method for the synthesis of phosphite diesters.

In contrast to conventional methods that rely on stoichiometric activation of phosphonylating reagents, we have developed a highly efficient catalytic method for the synthesis of phosphite diesters using a readily available phosphonylation reagent and alcohols with environmentally benign Zn(ii) catalysts. Two alcohols could be introduced consecutively on the P center with release of trifluoroethanol as the sole byproduct, without any additive, under mild conditions. The products could be oxidized smoothly to access phosphate triesters. A range of alcohols, including sterically demanding and highly functionalized alcohols such as carbohydrates and nucleosides, can be applied in this reaction.

N-3 polyunsaturated fatty acid attenuates cholesterol gallstones by suppressing mucin production with a high cholesterol diet in mice.

BACKGROUND AND AIM: The increasing prevalence of cholesterol gallstone (CG) disease has become an economic burden to the healthcare system. Ursodeoxycholic acid (UDCA) is the only established medical agent used to dissolve gallstones. In investigating novel therapeutics for CG, we assessed the preventive effects of n-3 polyunsaturated fatty acids (n-3PUFA) on the formation of CG induced by feeding a lithogenic diet (LD) containing high cholesterol levels to mice. METHODS: Mice were divided into the following six groups: (A) regular diet (RD); (B) RD+n-3PUFA; (C) LD; (D) LD+n-3PUFA; (E) LD+UDCA; (F) LD+n-3PUFA+UDCA. After RD/LD feeding for 2 weeks, n-3PUFA or UDCA was administered orally and the diet maintained for 8 weeks. The levels of phospholipids and cholesterol in bile, CG formation, gallbladder wall thickness, MUC gene expression in gallbladder were analyzed. RESULTS: No stone or sludge was evident in the RD groups (Groups A, B). Mice in the n-3PUFA treatment (Groups D, F) showed significantly lower stone formation than the other LD groups (Groups C, E). The combination treatment of n-3PUFA and UDCA suppressed stone formation more than mono-therapy with n-3PUFA or UDCA. Bile phospholipid levels were significantly elevated in the Group F. Hypertrophy of the gallbladder wall was evident in mice fed LD. MUC 2, 5AC, 5B and 6 mRNA expression levels were significantly elevated in the LD-fed group, and this was suppressed by n-3PUFA with or without UDCA. CONCLUSIONS: N-3PUFA attenuated gallstone formation in mouse, through increasing the levels of bile phospholipids and suppressing bile mucin formation.

Determination of the esculetin contents of medicinal plants by liquid chromatography-tandem mass spectrometry.

We developed a LC-MS/MS method for the determination of esculetin contents in medicinal plants. The analysis was performed using multiple reaction monitoring in negative mode, and an XBridge™ C(18) column (2.1 × 100 mm, 3.5 µm) was used. Methanol and 0.1% formic acid were used for gradient analysis. The calibration curve showed good linearity (r(2) > 0.9993). The limits of detection and quantitation were 0.02 and 0.07 ng/mL, respectively. The intra-day and inter-day precisions were 1.5-6.8 and 2.0-5.3%, respectively, and the accuracy was 102.0-110.2%. The contents of esculetin in 35 different plants were determined, and Fraxini Cortex showed the highest content of esculetin (761-5475 mg/kg). In Mori Folium and Artemisiae Capillaris Herba, 5.2-21.5 and 7.0-17.6 mg/kg of esculetin were found, respectively. In other medicinal plants, no esculetin was detected, or it was present at a concentration less than 10 mg/kg. The analysis method appears to be simple, sensitive and reproducible. Contrary to expectations based on traditional medical knowledge, although Artemisiae Capillaris Herba contains a large amount of esculetin, it appears from this study that Fraxini Cortex contains a greater amount. The pharmacological effects of esculetin isolated from medicinal plants should be investigated as part of new medicines development.

Radiology-pathology correlation of endometrial carcinoma assessment on magnetic resonance imaging.

Endometrial carcinoma is the most common gynaecological cancer in developed countries. Most cases are low-volume/low-grade tumour at presentation; however, high-grade subtypes may present with locally advanced disease with higher propensity for spread outside of the pelvis. MRI has a role in local staging of the tumour and helping the clinicians in treatment decision making. This pictorial essay gives examples of endometrial carcinoma at different stages with histological correlation. It also explores the potential limitations and pitfalls of imaging in this context.

Dendropanax trifidus Sap-Mediated Suppression of Obese Mouse Body Weight and the Metabolic Changes Related with Estrogen Receptor Alpha and AMPK-ACC Pathways in Muscle Cells.

Dendropanax trifidus (DT) is a medicinal herb native to East Asia, which has been used extensively for its therapeutic properties in traditional medicine. In this study, we examined the effects of DT sap on the regulation of body weight and muscle metabolism in mice. Obese model db/db mice were administered daily with DT sap or vehicle control over a 6-week period. The effects of DT sap on muscle metabolism were studied in C2C12 muscle cells, where glycolytic and mitochondrial respiration rates were monitored. As AMP-activated protein kinase (AMPK) is a master regulator of metabolism and plays an important function as an energy sensor in muscle tissue, signaling pathways related with AMPK were also examined. We found that DT sap inhibited body weight increase in db/db, db/+, and +/+ mice over a 6-week period, while DT sap-treated muscle cells showed increased muscle metabolism and also increased phosphorylation of AMPK and Acetyl-CoA Carboxylase (ACC). Finally, we found that DT sap, which is enriched in estrogen in our previous study, significantly activates estrogen alpha receptor in a concentration-dependent manner, which can drive the activation of AMPK signaling and may be related to the muscle metabolism and weight changes observed here.

Development and validation of a liquid chromatography method with electrospray ionization tandem mass spectrometry for the determination of brotizolam residues in beef and commercial whole milk.

In this work, a liquid chromatography-tandem mass spectrometric detection technique was developed and validated for the determination of brotizolam residues in beef muscle and commercial whole milk. This procedure involves the extraction of the analyte from the samples via liquid-solid extraction, and caffeine was used as an internal standard. The analyte was successfully separated on an XTerra-C(18) column, with a mobile phase composed of 0.01% formic acid in acetonitrile and 1 mm ammonium formate-0.01% formic acid in water. The one-step extraction method evidenced good selectivity, precision (RSD = 9.87-26.47%), and the recovery of the extractable analyte was 92.61-115.98% in the matrices. The limits of quantification ranged between 0.4 and 0.5 µg/kg. The developed method is simple since it requires no additional cleanup procedures.

Enantioselective Acyloin Rearrangement of Acyclic Aldehydes Catalyzed by Chiral Oxazaborolidinium Ion.

A catalytic enantioselective acyloin rearrangement of acyclic aldehydes to synthesize highly optically active acyloin derivatives is described. In the presence of a chiral oxazaborolidinium ion catalyst, the reaction provided chiral α-hydroxy aryl ketones in high yield (up to 95%) and enantioselectivity (up to 98% ee). In addition, the enantioselective acyloin rearrangement of α,α-dialkyl-α-siloxy aldehydes produced chiral α-siloxy alkyl ketones in high yield (up to 92%) with good enantioselectivity (up to 89% ee).

Brain pathology and cerebellar purkinje cell loss in a mouse model of chronic neuronopathic Gaucher disease.

Gaucher disease (GD) is currently the focus of considerable attention due primarily to the association between the gene that causes GD (GBA) and Parkinson's disease. Mouse models exist for the systemic (type 1) and for the acute neuronopathic forms (type 2) of GD. Here we report the generation of a mouse that phenotypically models chronic neuronopathic type 3 GD. Gba-/-;Gbatg mice, which contain a Gba transgene regulated by doxycycline, accumulate moderate levels of the offending substrate in GD, glucosylceramide, and live for up to 10 months, i.e. significantly longer than mice which model type 2 GD. Gba-/-;Gbatg mice display behavioral abnormalities at ∼4 months, which deteriorate with age, along with significant neuropathology including loss of Purkinje neurons. Gene expression is altered in the brain and in isolated microglia, although the changes in gene expression are less extensive than in mice modeling type 2 disease. Finally, bone deformities are consistent with the Gba-/-;Gbatg mice being a genuine type 3 GD model. Together, the Gba-/-;Gbatg mice share pathological pathways with acute neuronopathic GD mice but also display differences that might help understand the distinct disease course and progression of type 2 and 3 patients.

Pharmacokinetic properties and antitumor efficacy of the 5-fluorouracil loaded PEG-hydrogel.

BACKGROUND: We have studied the in vitro and in vivo utility of polyethylene glycol (PEG)-hydrogels for the development of an anticancer drug 5-fluorouracil (5-FU) delivery system. METHODS: A 5-FU-loaded PEG-hydrogel was implanted subcutaneously to evaluate the drug retention time and the anticancer effect. For the pharmacokinetic study, two groups of male rats were administered either an aqueous solution of 5-FU (control group)/or a 5-FU-loaded PEG-hydrogel (treated group) at a dose of 100 mg/kg. For the pharmacodynamic study, a human non-small-cell lung adenocarcinoma (NSCLC) cell line, A549 was inoculated to male nude mice with a cell density of 3 x 10(6). Once tumors start growing, the mice were injected with 5-FU/or 5-FU-loaded PEG-hydrogel once a week for 4 weeks. The growth of the tumors was monitored by measuring the tumor volume and calculating the tumor inhibition rate (IR) over the duration of the study. RESULTS: In the pharmacokinetic study, the 5-FU-loaded PEG-hydrogel gave a mean residence time (MRT) of 8.0 h and the elimination half-life of 0.9 h; these values were 14- and 6-fold, respectively, longer than those for the free solution of 5-FU (p < 0.05). In the pharmacodynamic study, A549 tumor growth was significantly inhibited in the 5-FU-loaded PEG-hydrogel group in comparison to the untreated group beginning on Day 14 (p < 0.05-0.01). Moreover, the 5-FU-loaded PEG-hydrogel group had a significantly enhanced tumor IR (p < 0.05) compared to the free 5-FU drug treatment group. CONCLUSION: We suggest that 5-FU-loaded PEG-hydrogels could provide a useful tool for the development of an anticancer drug delivery system.

Single-step extraction followed by LC for determination of (fluoro)quinolone drug residues in muscle, eggs, and milk.

In this study, a simplified method for the extraction and determination of seven fluoroquinolone residues (danofloxacin, difloxacin, enrofloxacin, marbofloxacin, orbifloxacin, ofloxacin, and sarafloxacin) and three quinolones (oxolinic acid, flumequine, and nalidixic acid), in porcine muscle, table eggs, and commercial whole milk, which required no cleanup step, was devised. This procedure involves the extraction of analytes from the samples via liquid-phase extraction, and the subsequent quantitative determination was accomplished via LC-fluorescence detection. Analyte separation was successfully conducted on an XBridge-C(18) column, with a linear gradient mobile phase composed of acetonitrile and 0.01 M oxalic acid buffer at pH=3.5. The one-step liquid-liquid extraction method evidenced good selectivity, precision (RSDs=0.26-15.07%), and recovery of the extractable analytes, ranging from 61.12 to 115.93% in matrices. The LOQs ranged from 0.3 to 25 microg/kg. A survey of ten samples purchased from local markets was conducted, and none of the samples harbored fluoroquinolone residues. This method is an improvement over existing methodologies, since no additional cleanup was necessary.

Zinc-Catalyzed Phosphonylation of Alcohols with Alkyl Phosphites.

In the presence of a catalytic amount of either Zn(acac)2 or bis(2,2,6,6-tetramethyl-3,5-heptanedionato)zinc(II) (Zn(TMHD)2), primary, secondary, and tertiary alcohol substituents on a wide range of substrates, including acyclic and cyclic structures, carbohydrates, steroids, and amino acids, reacted with dimethyl phosphite to afford the corresponding H-phosphonate diesters in high to excellent yields.

NPC1 silent variant induces skipping of exon 11 (p.V562V) and unfolded protein response was found in a specific Niemann-Pick type C patient.

BACKGROUND: Niemann-Pick type C (NPC, MIM #257220) is a neuro-visceral disease, caused predominantly by pathogenic variants in the NPC1 gene. Here we studied patients with clinical diagnosis of NPC but inconclusive results regarding the molecular analysis. METHODS: We used a Next-Generation Sequencing (NGS)-panel followed by cDNA analysis. Latter, we used massively parallel single-cell RNA-seq (MARS-Seq) to address gene profiling changes and finally the effect of different variants on the protein and cellular levels. RESULTS: We identified novel variants and cDNA analysis allowed us to establish the functional effect of a silent variant, previously reported as a polymorphism. We demonstrated that this variant induces the skipping of exon 11 leading to a premature stop codon and identified it in NPC patients from two unrelated families. MARS-Seq analysis showed that a number of upregulated genes were related to the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in one specific patient. Also, for all analyzed variants, the NPC1 protein was partially retained in the ER. CONCLUSION: We showed that the NPC1 silent polymorphism (p.V562V) is a disease-causing variant in NPC and that the UPR is upregulated in an NPC patient.