New Pyridyl and Dihydroisoquinoline Alkaloids Isolated from the Chevron Nemertean Amphiporus angulatus.

Nemertean worms contain toxins that are used to paralyze their prey and to deter potential predators. Hoplonemerteans often contain pyridyl alkaloids like anabaseine that act through nicotinic acetylcholine receptors and crustacean chemoreceptors. The chemical reactivity of anabaseine, the first nemertean alkaloid to be identified, has been exploited to make drug candidates selective for alpha7 subtype nAChRs. GTS-21, a drug candidate based on the anabaseine scaffold, has pro-cognitive and anti-inflammatory actions in animal models. The circumpolar chevron hoplonemertean Amphiporus angulatus contains a multitude of pyridyl compounds with neurotoxic, anti-feeding, and anti-fouling activities. Here, we report the isolation and structural identification of five new compounds, doubling the number of pyridyl alkaloids known to occur in this species. One compound is an isomer of the tobacco alkaloid anatabine, another is a unique dihydroisoquinoline, and three are analogs of the tetrapyridyl nemertelline. The structural characteristics of these ten compounds suggest several possible pathways for their biosynthesis.

Müllerian Agenesis in a patient with Rubinstein-Taybi Syndrome: A Case Series and Review of the Overlapping Developmental Biologic Pathways.

BACKGROUND: Rubinstein-Taybi syndrome (RSTS) is a multi-system neurodevelopmental condition caused by deficiency of CREBBP (16p13.3) or EP300 (22q13.2). Müllerian agenesis, or Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, is defined as congenital agenesis of the uterus, cervix, and upper vagina without a definite genetic cause. INDEX CASE AND CASE SERIES: We present a 14-year-old female with RSTS type 1 (CREBBP, c.4395-2A>C) and MRKH, the first documented in the literature. Following presentation to Gynecology for anticipatory guidance regarding future menstrual suppression and follow-up of previously diagnosed labial adhesions, exam under anesthesia revealed a single urogenital opening with cystoscopy demonstrating a normal urethra and bladder. Laboratory evaluation was consistent with peripubertal female gonadotropins and estradiol, 46,XX karyotype, and normal microarray, and a pelvic MRI confirmed Müllerian agenesis. Given this case, we assessed our cohort of females with RSTS and found that 4 of 12 individuals also had Müllerian anomalies. CONCLUSION: Gynecologic evaluation should be a part of medical care for females with RSTS, particularly in individuals with delayed menarche or abnormal menstrual history, on the basis of the observed association between RSTS and Müllerian anomalies in this case series. Although several candidate genes and copy number variants are associated with MRKH, no candidate genes in close proximity to the 16p13.3 region have been identified to explain both RSTS and MRKH in the index patient. Due to the regulatory nature of CREBBP during embryonic development, we theorize that CREBBP may play a role in the migration of Müllerian structures during embryogenesis.

Discovery of the Nicotinic Receptor Toxin Anabaseine in a Polystiliferan Nemertean.

Nemerteans (also called Nemertines) are a phylum of predominantly marine worms that use toxins to capture prey and to defend themselves against predators. Hoplonemerteans have a proboscis armed with one or more stylets used in prey capture and are taxonomically divided into Order Monostilifera, whose members possess a single large proboscis stylet, and Order Polystilifera, whose members have multiple small stylets. Many monostiliferans contain alkaloidal toxins, including anabaseine, that stimulate and then desensitize nicotinic acetylcholine receptors that are present in all animals. These compounds also interact with pyridyl chemoreceptors in crustaceans, reducing predation and larval settlement. Anabaseine has been a lead compound in the design of alpha7 nicotinic acetylcholine receptor agonists like GTS-21 (also called DMXBA) to treat disorders of cognition such as Alzheimer's disease and schizophrenia. These drug candidates also display anti-inflammatory activities of potential medical importance. Most polystiliferans live deep in open oceans and are relatively inaccessible. We fortunately obtained two live specimens of a large benthic polystiliferan, Paradrepanophorus crassus (Pc), from the coast of Spain. MS and NMR analyses of the Ehrlich's reagent derivative allowed identification of anabaseine. A spectrophotometric assay for anabaseine, also based on its reaction with Ehrlich's reagent, revealed high concentrations of anabaseine in the body and proboscis. Apparently, the biosynthetic mechanism for producing anabaseine was acquired early in the evolution of the Hoplonemertea, before the monostiliferan-polystiliferan divergence.

Characterization of a rare mosaic unbalanced translocation of t(3;12) in a patient with neurodevelopmental disorders.

BACKGROUND: Unbalanced translocations may be de novo or inherited from one parent carrying the balanced form and are usually present in all cells. Mosaic unbalanced translocations are extremely rare with a highly variable phenotype depending on the tissue distribution and level of mosaicism. Mosaicism for structural chromosomal abnormalities is clinically challenging for diagnosis and counseling due to the limitation of technical platforms and complex mechanisms, respectively. Here we report a case with a tremendously rare maternally-derived mosaic unbalanced translocation of t(3;12), and we illustrate the unreported complicated mechanism using single nucleotide polymorphism (SNP) array, fluorescence in situ hybridization (FISH), and chromosome analyses. CASE PRESENTATION: An 18-year-old female with a history of microcephaly, pervasive developmental disorder, intellectual disability, sensory integration disorder, gastroparesis, and hypotonia presented to our genetics clinic. She had negative karyotype by parental report but no other genetic testing performed previously. SNP microarray analysis revealed a complex genotype including 8.4 Mb terminal mosaic duplication on chromosome 3 (3p26.3->3p26.1) with the distal 5.7 Mb involving two parental haplotypes and the proximal 2.7 Mb involving three parental haplotypes, and a 6.1 Mb terminal mosaic deletion on chromosome 12 (12p13.33->12p13.31) with no evidence for a second haplotype. Adjacent to the mosaic deletion is an interstitial mosaic copy-neutral region of homozygosity (1.9 Mb, 12p13.31). The mother of this individual was confirmed by chromosome analysis and FISH that she carries a balanced translocation, t(3;12)(p26.1;p13.31). CONCLUSION: Taken together, the proband, when at the stage of a zygote, likely carried the derivative chromosome 12 from this translocation, and a postzygotic mitotic recombination event occurred between the normal paternal chromosome 12 and maternal derivative chromosome 12 to "correct" the partial 3p trisomy and partial deletion of 12p. To the best of our knowledge, it is the first time to report the mechanism utilizing a combined cytogenetic and cytogenomic approach, and we believe it expands our knowledge of mosaic structural chromosomal disorders and provides new insight into clinical management and genetic counseling.

Post translational modifications of Trifolitoxin: a blue fluorescent peptide antibiotic.

Trifolitoxin (TFX, C41H63N15O15S) is a selective, ribosomally-synthesized, post-translationally modified, peptide antibiotic, produced by Rhizobium leguminosarum bv. trifolii T24. TFX specifically inhibits α-proteobacteria, including the plant symbiont Rhizobium spp., the plant pathogen Agrobacterium spp. and the animal pathogen Brucella abortus. TFX-producing strains prevent legume root nodulation by TFX-sensitive rhizobia. TFX has been isolated as a pair of geometric isomers, TFX1 and TFX2, which are derived from the biologically inactive primary amino acid sequence: Asp-Ile-Gly-Gly-Ser-Arg-Gln-Gly-Cys-Val-Ala. Gly-Cys is present as a thiazoline ring and the Arg-Gln-Gly sequence is extensively modified to a UV absorbing, blue fluorescent chromophore. The chromophore consists of a conjugated, 5-membered heterocyclic ring and side chain of modified glutamine.

XY Gonadal Dysgenesis in a Phenotypic Female Identified by Direct-to-Consumer Genetic Testing.

We report a 16-year-old phenotypic female with 46,XY complete gonadal dysgenesis and metastatic dysgerminoma, unexpectedly discovered through direct-to-consumer (DTC) commercial genetic testing. This case underscores the importance of timely interdisciplinary care, including psychosocial intervention and consideration of gonadectomy, to optimize outcomes for individuals with differences of sex development. Her unique presentation highlights the implications of DTC genetic testing in a new diagnostic era and informs general pediatricians as well as specialists of nongenetic services about the value, capabilities, and limitations of DTC testing.

The effectiveness of combined lower limb strengthening and whole-body vibration, compared to strengthening alone, for improving patient-centred outcomes in adults with COPD: A systematic review.

BACKGROUND: People with chronic obstructive pulmonary disease (COPD) experience various impairments, reducing quality of life (QoL). Rehabilitation that does not elicit dyspnoea, such as strength training, is recommended to improve patient outcomes. OBJECTIVES: To systematically review evidence for the effectiveness of lower limb strengthening combined with whole-body vibration training (WBVT), versus lower limb strengthening alone, in adults with COPD for improving lower limb muscle strength, functional exercise capacity (FEC), pulmonary function and QoL. METHOD: Eight databases were searched (inception-May 2019). Only randomised controlled trials (RCTs) with PEDro scores ≥ 5/10 were included. Heterogeneity rendered meta-analyses inappropriate; thus data were synthesised narratively. RESULTS: Five RCTs (mean PEDro score: 5.8/10) were included. Only one RCT showed a significant difference for leg press peak force (kg) at 12 weeks (p = 0.001), favouring WBVT. FEC improved significantly (p < 0.05) in favour of WBVT at 3 and 12 weeks. Combined training was not more effective for short-term (≤ 12 weeks) improvements in pulmonary function or QoL. CONCLUSION: Level II evidence suggests that combining strengthening with WBVT has significant beneficial short-term effects on FEC in adults with COPD. Results are limited by the small number of studies and small sample sizes. Combined WBVT and strengthening was not more effective than strengthening alone for improving lower limb muscle strength, pulmonary function and QoL. CLINICAL IMPLICATIONS: Combining low (6-10 Hz) to moderate (24-26 Hz) frequency WBVT with strengthening may be a more effective modality to improve FEC than strengthening alone, should resources allow.

Nitrogen starvation-induced accumulation of triacylglycerol in the green algae: evidence for a role for ROC40, a transcription factor involved in circadian rhythm.

Microalgal triacylglycerol (TAG), a promising source of biofuel, is induced upon nitrogen starvation (-N), but the proteins and genes involved in this process are poorly known. We performed isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics to identify Chlorella proteins with modulated expression under short-term -N. Out of 1736 soluble proteins and 2187 membrane-associated proteins identified, 288 and 56, respectively, were differentially expressed under -N. Gene expression analysis on select genes confirmed the same direction of mRNA modulation for most proteins. The MYB-related transcription factor ROC40 was the most induced protein, with a 9.6-fold increase upon -N. In a previously generated Chlamydomonas mutant, gravimetric measurements of crude total lipids revealed that roc40 was impaired in its ability to increase the accumulation of TAG upon -N, and this phenotype was complemented when wild-type Roc40 was expressed. Results from radiotracer experiments were consistent with the roc40 mutant being comparable to the wild type in recycling membrane lipids to TAG but being impaired in additional de novo synthesis of TAG during -N stress. In this study we provide evidence to support the hypothesis that transcription factor ROC40 has a role in -N-induced lipid accumulation, and uncover multiple previously unknown proteins modulated by short-term -N in green algae.

Predictive value of fetal lung volume in prenatally diagnosed skeletal dysplasia.

OBJECTIVE: Pulmonary hypoplasia is a major cause of death in lethal skeletal dysplasias. We hypothesize that in fetuses with prenatally diagnosed skeletal dysplasia, comparison of observed-to-expected (O/E) lung volume will help predict lethality. STUDY DESIGN: We conducted a retrospective chart review of patients referred for evaluation of suspected fetal skeletal anomalies. Twenty-three pregnancies were identified with confirmed fetal diagnosis of skeletal dysplasia for which fetal magnetic resonance imaging (MRI) was performed between 21 and 38 weeks of gestation and ultrasound biometry data were available. Femur length to abdominal circumference ratio (FL/AC) and O/E lung volumes were calculated. The association between O/E lung volume, FL/AC, and lethality was measured using logistic regression. RESULTS: Lethality was significantly associated with O/E lung volume (p = 0.002) and FL/AC (p = 0.0476). Analysis with receiver-operating characteristic curves suggested that O/E lung volume of 47.9% or FL/AC of 0.124 could be useful clinical cutoffs in the prediction of lethality. CONCLUSION: In fetuses with skeletal dysplasia, fetal MRI-derived O/E lung volume was predictive of lethality. When evaluating a fetal skeletal dysplasia, fetal MRI may be considered in cases for which ultrasound-based lethality prediction is ambiguous or uncertain in order to provide families with the most complete and accurate information.

Conversion of membrane lipid acyl groups to triacylglycerol and formation of lipid bodies upon nitrogen starvation in biofuel green algae Chlorella UTEX29.

Algal lipids are ideal biofuel sources. Our objective was to determine the contributors to triacylglycerol (TAG) accumulation and lipid body formation in Chlorella UTEX29 under nitrogen (N) deprivation. A fivefold increase in intracellular lipids following N starvation for 24 h confirmed the oleaginous characteristics of UTEX29. Ultrastructural studies revealed increased number of lipid bodies and decreased starch granules in N-starved cells compared to N-replete cells. Lipid bodies were observed as early as 3 h after N removal and plastids collapsed after 48 h of stress. Moreover, the identification of intracellular pyrenoids and differences in the expected nutritional requirements for Chlorella protothecoides (as UTEX29 is currently classified) led us to conduct a phylogenetic study using 18S and actin cDNA sequences. This indicated UTEX29 to be more phylogenetically related to Chlorella vulgaris. To investigate the fate of different lipids after N starvation, radiolabeling using ¹4C-acetate was used. A significant decrease in ¹4C-galactolipids and phospholipids matched the increase in ¹4C-TAG starting at 3 h of N starvation, consistent with acyl groups from structural lipids as sources for TAG under N starvation. These results have important implications for the identification of key steps controlling oil accumulation in N-starved biofuel algae and demonstrate membrane recycling during lipid body formation.

Polyphenol oxidase inhibitor from blue mussel (Mytilus edulis) extract.

UNLABELLED: Enzymatic browning remains a problem for the fruit and vegetable industry, especially new emerging markets like pre-cuts. A crude inhibitor from blue mussel (Mytilus edulis) showed broad inhibition for apple (58%), mushroom (32%), and potato (44%) polyphenol oxidase (PPO) and was further characterized. Inhibition increased as the concentration of inhibitor increased in the reaction mixture eventually leveling off at a maximum inhibition of 92% for apple PPO. The inhibitor was capable of bleaching the brown color formed in the reaction mixture with apple PPO. Identification of the inhibitor by mass spectrometry and high-performance liquid chromatography revealed it to be hypotaurine (C2 H7 NO2 S). Hypotaurine and other sulfinic acid analogs (methane and benzene sulfinic acids) showed very good inhibition for apple PPO at various concentrations with the highest inhibition occurring at 500 µM for hypotaurine (89%), methane sulfinic acid (100%), and benzene sulfinic acid (100%). PRACTICAL APPLICATION: An inhibitor found in the expressed liquid from blue mussel shows very good inhibition on enzymatic browning. Since this enzyme is responsible for losses to the fruit and vegetable industry, natural inhibitors that prevent browning would be valuable. Finding alternative chemistries that inhibit browning and understanding their mode of action would be beneficial to the fruit and vegetable industries and their segments such as pre-cuts, juices, and so on. Inhibitors from products ingested by consumers are more acceptable as natural ingredients.

Bilirubin present in diverse angiosperms.

BACKGROUND AND AIMS: Bilirubin is an orange-yellow tetrapyrrole produced from the breakdown of heme by mammals and some other vertebrates. Plants, algae and cyanobacteria synthesize molecules similar to bilirubin, including the protein-bound bilins and phytochromobilin which harvest or sense light. Recently, we discovered bilirubin in the arils of Strelitzia nicolai, the White Bird of Paradise Tree, which was the first example of this molecule in a higher plant. Subsequently, we identified bilirubin in both the arils and the flowers of Strelitzia reginae, the Bird of Paradise Flower. In the arils of both species, bilirubin is present as the primary pigment, and thus functions to produce colour. Previously, no tetrapyrroles were known to generate display colour in plants. We were therefore interested in determining whether bilirubin is broadly distributed in the plant kingdom and whether it contributes to colour in other species. METHODOLOGY: In this paper, we use HPLC/UV and HPLC/UV/electrospray ionization-tandem mass spectrometry (HPLC/UV/ESI-MS/MS) to search for bilirubin in 10 species across diverse angiosperm lineages. PRINCIPAL RESULTS: Bilirubin was present in eight species from the orders Zingiberales, Arecales and Myrtales, but only contributed to colour in species within the Strelitziaceae. CONCLUSIONS: The wide distribution of bilirubin in angiosperms indicates the need to re-assess some metabolic details of an important and universal biosynthetic pathway in plants, and further explore its evolutionary history and function. Although colour production was limited to the Strelitziaceae in this study, further sampling may indicate otherwise.

Benzotriazole-assisted solid-phase assembly of Leu-enkephalin, amyloid beta segment 34-42, and other "difficult" peptide sequences.

Microwave-assisted solid-phase syntheses of six "difficult" peptides, H-VVSVV-NH(2) (3), H-VVVSVV-NH(2) (4), H-VIVIG-OH (5), H-TVTVTV-NH(2) (6), H-VKDGYI-NH(2) (7), and H-VKDVYI-NH(2) (8), were achieved utilizing N-(Fmoc-alpha-aminoacyl)benzotriazoles. Extension to the syntheses of Leu-enkephalin (9) and amyloid-beta (34-42) (10) demonstrates that this strategy comprises an efficient route to new and known "difficult" peptides.

Fluorescent labeling of peptides on solid phase.

N(alpha)-Fmoc-N(epsilon)-[(7-methoxycoumarin-4-yl)acetyl]-L-lysine (N(alpha)-Fmoc-L-Lys(Mca)-OH) 3 is conveniently prepared by benzotriazole methodology (52% over two steps). N-Acylbenzotriazoles Mca-Bt 2, N(alpha)-Fmoc-L-Lys(Mca)-Bt 4, coumarin-3-ylcarbonyl (Cc)-Bt 5, N(alpha)-Fmoc-L-Lys(Cc)-Bt 7 and N(alpha)-(Cc)-L-Lys(Fmoc)-Bt 9 enable the efficient microwave enhanced solid-phase fluorescent labeling of peptides.

Microwave-assisted solid-phase peptide synthesis utilizing N-Fmoc-protected (alpha-aminoacyl)benzotriazoles.

A novel microwave-assisted solid-phase peptide synthesis utilizing N-Fmoc-protected(alpha-aminoacyl)benzotriazoles was applied in the preparation of tri-, tetra-, penta-, hexa-, and heptapeptides in 71% average crude yield.

A comparison of single-dose caudal clonidine, morphine, or hydromorphone combined with ropivacaine in pediatric patients undergoing ureteral reimplantation.

BACKGROUND: Caudal blockade is a common technique for pediatric postoperative analgesia. While safe and effective, caudal opioids are associated with troublesome side effects. Caudal clonidine may offer significant analgesic benefits. We prospectively compared the analgesic, side effect, and rehabilitation profiles of caudal clonidine, hydromorphone, or morphine in a group of 60 pediatric patients undergoing ureteral reimplantation. METHODS: Patients aged 6 mo to 6 yr were evenly and randomly enrolled in a double-blind manner. Patients received a single caudal dose of 2 mcg/kg of clonidine, 10 mcg/kg of hydromorphone, or 50 mcg/kg of morphine, combined with 1.0 mL/kg of 0.2% ropivacaine with epinephrine. After sevoflurane in oxygen/air anesthesia, all subjects received proxy nurse-controlled analgesia with morphine. Postoperative pain intensity, use of IV morphine, and side effects were assessed during the first 24 h. Oral intake and discharge home were recorded. RESULTS: Caudal clonidine resulted in less postoperative nausea and vomiting (P = 0.01) and pruritus (P = 0.007) than did caudal hydromorphone or caudal morphine. Caudal morphine produced more sustained initial analgesia than did caudal clonidine (P = 0.02). No difference was observed in pain scores, total morphine use, time to first oral intake or discharge home. No postoperative respiratory depression, excessive sedation, hypotension, or bradycardia was identified. CONCLUSIONS: Although caudal morphine may result in more sustained initial analgesia, caudal clonidine combined with nurse-controlled analgesia appears to provide comparable analgesia with fewer side effects. Based on these results, the use of caudal clonidine may be superior to caudal opioids after pediatric ureteral reimplantation.

Degradation and effects of the potential mosquito larvicides methazolamide and acetazolamide in sheepshead minnow (Cyprinodon variegatus).

To test for environmental persistence in order to determine the potential of carbonic anhydrase inhibitors as larvicides, the decomposition and degradation of samples containing methazolamide (MTZ) and acetazolamide (ACZ) in aqueous solution were monitored under different conditions. Additionally, nontarget species impact was assessed in an acute toxicity test using sheepshead minnow (Cyprinodon variegatus). The fish were exposed for 120 h to 10(-3) and 10(-4) M each compound in replicate seawater tanks. In the high-MTZ treatment, all fish died within 48 h, while mortality in the low-MTZ treatment was 27% at 120 h. In the high-ACZ treatment mortality reached 83% at 120 h. We observed no mortality for the lowest dose of ACZ. Tissue samples were collected from the fish to investigate absorption of the compounds. In the gills, MTZ concentrations were around 40 microg g(-1) and ACZ reached concentrations up to 80 microg g(-1). Liver concentrations were low for MTZ probably due to metabolism.

Identification of ellagic acid conjugates and other polyphenolics in muscadine grapes by HPLC-ESI-MS.

Ellagic acid, ellagic acid glycosides, and ellagitannins found in various fruits and nuts, including muscadine grape, are reported to have potential health-promoting benefits and antioxidant properties. This study isolated and identified several ellagic acid derivatives present in muscadine grapes and determined their relative antioxidant properties (AOX). Compounds were extracted from grape skins and pulp using methanol, and the solvent was evaporated. Isolates were dissolved in citric acid buffer (pH 3.5) and absorbed onto C18 cartridges. Nonretained polyphenolics were collected separately and again partitioned from Sephadex LH-20, whereas retained polyphenolics were first eluted with ethyl acetate followed by methanol. Ellagic acid derivatives were identified on the basis of UV and mass spectra, and the presence of ellagitannins was confirmed by a significant increase in free ellagic acid with HPLC followed by acid hydrolysis. Muscadine grapes contained phenolic acids, flavonols, anthocyanins, ellagic acid, and numerous ellagic acid derivatives. AOX varied in the order ethyl acetate > methanol > C18 nonretained fractions; each correlated to both total phenolics (r = 0.90) and total ellagic acid (r = 0.99) contents. Results of this study revealed previously unidentified ellagic acid derivatives in muscadine grapes.