Brief Report: Decreased Bone Health in Children with Autism Spectrum Disorder and Avoidant Restrictive Food Intake Disorder.

PURPOSE: Children with autism spectrum disorder (ASD) and food selectivity are at increased risk for nutritional deficiencies which could affect bone health. METHODS: We report on four male patients with ASD and avoidant restrictive food intake disorder (ARFID) with significant bone conditions including rickets, vertebral compression fractures, osteopenia, and slipped capital femoral epiphyses. RESULTS: Each patient was at risk for at least one nutritional deficiency. Two out of four patients had deficiencies in Vitamins A, B12, E, and zinc. Calcium and Vitamin D deficiency were noted in all four. Two out of four patients with Vitamin D deficiency developed rickets. CONCLUSION: Provisional evidence suggests that children with ASD and ARFID are at elevated risk for serious adverse bone health outcomes.

Simultaneous inhibition of endocytic recycling and lysosomal fusion sensitizes cells and tissues to oligonucleotide therapeutics.

Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.

Structures of annexin A2-PS DNA complexes show dominance of hydrophobic interactions in phosphorothioate binding.

The introduction of phosphorothioate (PS) linkages to the backbone of therapeutic nucleic acids substantially increases their stability and potency. It also affects their interactions with cellular proteins, but the molecular mechanisms that underlie this effect are poorly understood. Here, we report structural and biochemical studies of interactions between annexin A2, a protein that does not possess any known canonical DNA binding domains, and phosphorothioate-modified antisense oligonucleotides. We show that a unique mode of hydrophobic interactions between a sulfur atom of the phosphorothioate group and lysine and arginine residues account for the enhanced affinity of modified nucleic acid for the protein. Our results demonstrate that this mechanism of interaction is observed not only for nucleic acid-binding proteins but can also account for the association of PS oligonucleotides with other proteins. Using the anomalous diffraction of sulfur, we showed that preference for phosphorothioate stereoisomers is determined by the hydrophobic environment around the PS linkage that comes not only from protein but also from additional structural features within the ASO such as 5-Me groups on cytosine nucleobases.

Diving into the vertical dimension of elasmobranch movement ecology.

Knowledge of the three-dimensional movement patterns of elasmobranchs is vital to understand their ecological roles and exposure to anthropogenic pressures. To date, comparative studies among species at global scales have mostly focused on horizontal movements. Our study addresses the knowledge gap of vertical movements by compiling the first global synthesis of vertical habitat use by elasmobranchs from data obtained by deployment of 989 biotelemetry tags on 38 elasmobranch species. Elasmobranchs displayed high intra- and interspecific variability in vertical movement patterns. Substantial vertical overlap was observed for many epipelagic elasmobranchs, indicating an increased likelihood to display spatial overlap, biologically interact, and share similar risk to anthropogenic threats that vary on a vertical gradient. We highlight the critical next steps toward incorporating vertical movement into global management and monitoring strategies for elasmobranchs, emphasizing the need to address geographic and taxonomic biases in deployments and to concurrently consider both horizontal and vertical movements.

Towards next generation antisense oligonucleotides: mesylphosphoramidate modification improves therapeutic index and duration of effect of gapmer antisense oligonucleotides.

The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.

Auto-deconvolution and molecular networking of gas chromatography-mass spectrometry data.

We engineered a machine learning approach, MSHub, to enable auto-deconvolution of gas chromatography-mass spectrometry (GC-MS) data. We then designed workflows to enable the community to store, process, share, annotate, compare and perform molecular networking of GC-MS data within the Global Natural Product Social (GNPS) Molecular Networking analysis platform. MSHub/GNPS performs auto-deconvolution of compound fragmentation patterns via unsupervised non-negative matrix factorization and quantifies the reproducibility of fragmentation patterns across samples.

The Unexpected Base-Pairing Behavior of Cyanuric Acid in RNA and Ribose versus Cyanuric Acid Induced Helicene Assembly of Nucleic Acids: Implications for the Pre-RNA Paradigm.

The cyanuric acid (CA) heterocycle forms supramolecular structures with adenine nucleobases/nucleosides and oligonucleotides, leading to speculation that they can act as forerunners to RNA. Herein, the assembly behavior of RNA containing CA and CA-ribose nucleoside was studied. Contrary to previous reports, CA in RNA and the CA-ribonucleoside resulted in destabilization of supramolecular assemblies, which led to a reevaluation of the CA-adenine hexameric rosette structure. An unprecedented noncovalent supramolecular helicene structure is proposed to account for the striking difference in behavior, which has implications for novel paradigms for reorganizing the structures of nucleic acids, the synthesis of long helicenes, and pre-RNA world paradigms. The results caution against extrapolating the self-assembly behavior of individual heterocycles from the level of monomers to oligomers because the base-paring properties of (non-)canonical nucleobases are impacted by the type of oligomeric backbone to which they are attached.

Heterologous Expression of Cryptomaldamide in a Cyanobacterial Host.

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, nonribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drug leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide; however, high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.

Origins of the Increased Affinity of Phosphorothioate-Modified Therapeutic Nucleic Acids for Proteins.

The phosphorothioate backbone modification (PS) is one of the most widely used chemical modifications for enhancing the drug-like properties of nucleic acid-based drugs, including antisense oligonucleotides (ASOs). PS-modified nucleic acid therapeutics show improved metabolic stability from nuclease-mediated degradation and exhibit enhanced interactions with plasma, cell-surface, and intracellular proteins, which facilitates their tissue distribution and cellular uptake in animals. However, little is known about the structural basis of the interactions of PS nucleic acids with proteins. Here, we report a crystal structure of the DNA-binding domain of a model ASO-binding protein PC4, in complex with a full PS 2'-OMe DNA gapmer ASO. To our knowledge this is the first structure of a complex between a protein and fully PS nucleic acid. Each PC4 dimer comprises two DNA-binding interfaces. In the structure one interface binds the 5'-terminal 2'-OMe PS flank of the ASO, while the other interface binds the regular PS DNA central part in the opposite polarity. As a result, the ASO forms a hairpin-like structure. ASO binding also induces the formation of a dimer of dimers of PC4, which is stabilized by base pairing between homologous regions of the ASOs bound by each dimer of PC4. The protein interacts with the PS nucleic acid through a network of electrostatic and hydrophobic interactions, which provides insights into the origins for the enhanced affinity of PS for proteins. The importance of these contacts was further confirmed in a NanoBRET binding assay using a Nano luciferase tagged PC4 acting as the BRET donor, to a fluorescently conjugated ASO acting as the BRET acceptor. Overall, our results provide insights into the molecular forces that govern the interactions of PS ASOs with cellular proteins and provide a potential model for how these interactions can template protein-protein interactions causative of cellular toxicity.

Nanopore Sequencing of an Expanded Genetic Alphabet Reveals High-Fidelity Replication of a Predominantly Hydrophobic Unnatural Base Pair.

Unnatural base pairs (UBPs) have been developed and used for a variety of in vitro applications as well as for the engineering of semisynthetic organisms (SSOs) that store and retrieve increased information. However, these applications are limited by the availability of methods to rapidly and accurately determine the sequence of unnatural DNA. Here we report the development and application of the MspA nanopore to sequence DNA containing the dTPT3-dNaM UBP. Analysis of two sequence contexts reveals that DNA containing the UBP is replicated with an efficiency and fidelity similar to that of natural DNA and sufficient for use as the basis of an SSO that produces proteins with noncanonical amino acids.

"Like a Normal Person Again": A Qualitative Analysis of the Impact of Headache Surgery.

BACKGROUND: Outcomes after migraine surgery have been previously assessed using quantitative measurements, including the migraine headache index. Qualitative methodologies offer the ability to analyze patients' perceptions and pain experience, and may point to changes in domains not captured by quantitative instruments. The purpose of this study was to characterize individual patients' experiences with migraines and to analyze how patients' experience of headaches changes in relation to surgery. METHODS: Patients who previously underwent migraine surgery performed by a single surgeon participated in semistructured interviews at least 1 year after surgery. Purposive sampling was used to recruit patients [n = 15 (73 percent female)]. Interviews were transcribed verbatim. A multidisciplinary team with backgrounds in surgery, pain management, medicine, and health services research coded and analyzed transcripts. RESULTS: Participants reported improvements in one or more domains of pain following surgery, and changes in medication use and effectiveness. Even in individuals with persistent pain postoperatively, surgery appeared to facilitate an improvement in headache self-efficacy, including an ability to participate in daily activities. Migraineurs frequently described a new degree of control over at least one aspect of their pain. CONCLUSIONS: Migraine surgery appears to positively impact patients' lives in ways that support and expand on previously published outcomes. Patients report benefiting from surgery in ways that are not currently captured in commonly used metrics. This study's findings support the need for more specific patient-reported outcome measures to help clinicians and patients understand the impact of surgery and which outcomes matter most to patients.

Periprocedural Considerations for Patients with Gastric Electrical Stimulators.

OBJECTIVES: Gastric electrical stimulation (GES) is a technology that uses neurostimulation for the modulation of gastric activity. In clinical practice, the most commonly encountered form of GES is high frequency GES. GES devices are typically used for the treatment of refractory gastroparesis, although they have also been investigated for obesity management and the treatment of refractory gastroesophageal reflux disease. Just as many patients with chronic diseases require surgery, patients with an implanted GES device may encounter the need for periprocedural care. Therefore, the purpose of this review is to address the special needs of patients with an implanted GES device. MATERIALS AND METHODS: A systematic computerized search of the literature was performed to consolidate existing knowledge on GES management in the periprocedural setting. Duplicate results were eliminated, and results were further narrowed based on title and abstract. All articles with possible relevance were then reviewed in full. Manufacturer information including pamphlets and websites were also reviewed. RESULTS: A total of 1201 articles were identified for initial review, and 33 met inclusion criteria. CONCLUSIONS: Available data suggests GES is a technology with increasing prevalence. When patients with an implanted GES device present for periprocedural care, the anesthesia staff must consider the device when planning for the procedure. Topics addressed include general anesthetic considerations, nerve localization, radiation exposure, electrocautery, diathermy, emergency external defibrillation, and MRI compatibility.

Combined Effects of Acute Temperature Change and Elevated pCO2 on the Metabolic Rates and Hypoxia Tolerances of Clearnose Skate (Rostaraja eglanteria), Summer Flounder (Paralichthys dentatus), and Thorny Skate (Amblyraja radiata).

Understanding how rising temperatures, ocean acidification, and hypoxia affect the performance of coastal fishes is essential to predicting species-specific responses to climate change. Although a population's habitat influences physiological performance, little work has explicitly examined the multi-stressor responses of species from habitats differing in natural variability. Here, clearnose skate (Rostaraja eglanteria) and summer flounder (Paralichthys dentatus) from mid-Atlantic estuaries, and thorny skate (Amblyraja radiata) from the Gulf of Maine, were acutely exposed to current and projected temperatures (20, 24, or 28 °C; 22 or 30 °C; and 9, 13, or 15 °C, respectively) and acidification conditions (pH 7.8 or 7.4). We tested metabolic rates and hypoxia tolerance using intermittent-flow respirometry. All three species exhibited increases in standard metabolic rate under an 8 °C temperature increase (Q10 of 1.71, 1.07, and 2.56, respectively), although this was most pronounced in the thorny skate. At the lowest test temperature and under the low pH treatment, all three species exhibited significant increases in standard metabolic rate (44-105%; p < 0.05) and decreases in hypoxia tolerance (60-84% increases in critical oxygen pressure; p < 0.05). This study demonstrates the interactive effects of increasing temperature and changing ocean carbonate chemistry are species-specific, the implications of which should be considered within the context of habitat.

Optimization of Replication, Transcription, and Translation in a Semi-Synthetic Organism.

Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.

Perioperative and Periprocedural Care of Patients With Intrathecal Pump Therapy.

INTRODUCTION: There are an ever-increasing number of patients who have implanted devices for targeted delivery of drug therapy to the intrathecal space for the management of spasticity or chronic pain. This leads to a growing number of people with implanted pumps presenting for procedures and surgeries, yet there is a paucity of consolidated information available to describe the appropriate precautions and patient management during this period. METHODS: This was a systematic review to provide a summary of existing literature on intrathecal drug delivery system (IDDS) management in the perioperative and procedural period, and to highlight additional areas that require further research. Topics addressed include the time surrounding magnetic resonance imaging, defibrillation, radiation therapy, high output ultrasound, lithotripsy, ablation, diathermy, electroconvulsive therapy, and the perioperative period, all of which have their own specific considerations. RESULTS: A total of 42 articles met criteria to be included in this review. Inclusion criteria were English language, and that the article was primarily focused on the perioperative or periprocedural management of IDDSs. Exclusion criteria included commentaries, surveys, published abstracts, or articles that did not discuss the perioperative or periprocedural care of IDDS. CONCLUSION: Our article outlined perioperative considerations when dealing with a patient with intrathecal pump undergoing surgical or imaging modality.

Synthesis of Fluorescence Turn-On DNA Hybridization Probe Using the DEA tC 2'-Deoxycytidine Analog.

DEA tC is a tricyclic 2'-deoxycytidine analog that can be incorporated into oligonucleotides by solid-phase synthesis and that exhibits a large fluorescence enhancement when correctly base-paired with a guanine base in a DNA-DNA duplex. The synthesis of DEA tC begins with 5-amino-2-methylbenzothiazole and provides the DEA tC nucleobase analog over five synthetic steps. This nucleobase analog is then silylated using N,O-bis(trimethylsilyl)acetamide and conjugated to Hoffer's chlorosugar to provide the protected DEA tC nucleoside in good yield. Following protective-group removal and chromatographic isolation of the ß-anomer, dimethoxytritylation and phosphoramidite synthesis offer the monomer for solid-phase DNA synthesis. Solid-phase DNA synthesis conditions using extended coupling of the DEA tC amidite and a short deprotection time are employed to maximize efficiency. By following the protocols described in this unit, the DEA tC fluorescent probe can be synthesized and can be incorporated into any desired synthetic DNA oligonucleotide. © 2018 by John Wiley & Sons, Inc.

Naloxone Academic Detailing: Role of Community Outreach Teaching.

PURPOSE OF REVIEW: Testing the efficacy of academic detailing in improving the practice of prescribing naloxone for patients on high-dose opioids. RECENT FINDINGS: Academic detailing has been identified as an effective method for improving health care practices through focused community education. We found that academic detailing is an effective method in improving health care providers' knowledge about the importance of prescribing naloxone for patients on high-dose opioids. We also found that prescribers prescribed more naloxone after our education program. This study reflects the importance of education and academic detailing in resolving health problems. Academic detailing can provide effective preventive tools that can reduce the incidence of health problems we encounter.

Heterogeneous Pyrophosphate-Linked DNA-Oligonucleotides: Aversion to DNA but Affinity for RNA.

Pyrophosphate linkages are important in extant biology and are hypothesized to have played a role in prebiotic chemistry and in the origination of oligonucleotides. Inspired by pyrophosphate as backbones of primordial oligomers, DNA oligomers with varying amounts of pyrophosphate inserts (ppDNA) were synthesized and investigated for their base-pairing properties. As expected, pyrophosphate inserts into the backbone compromised the thermal stability of ppDNA-DNA duplexes. In contrast, the ppDNA-RNA duplex exhibited, remarkably, duplex stability, even with accumulation of pyrophosphate linkages. This seems to be a consequence of an increase in the diameter of the double-helix with eight-bond-repeat units, and higher inclination of the base-pair axis with respect to the backbone in RNA (A-form), compared with that in DNA (B-form). These results suggest that pyrophosphate-linked oligonucleotides could harbor functional capabilities with implications for their roles in the origins of life and chemical biology.

Emerging Drugs for the Treatment of Cancer Pain: A Review of Patent Literature in 2014.

BACKGROUND: Pain in oncology patients may be related to underlying disease process, as well as a side effect of chemotherapy and radiation. More than half of all patients with a diagnosis of cancer experience pain, which is often poorly controlled. As such, the treatment of pain in oncology patients demonstrates a significant need to develop new treatment options. OBJECTIVE: This review summarizes 35 patents on the treatment of pain among cancer patients. METHOD: A thorough literature review of all patents filed in 2014 was conducted using PubMed and http://ep.espacenet.com/ as sources identify new treatment options for cancer pain. RESULTS: Our literature search returned 35 patents filed in 2014 related to the treatment of pain in cancer patients. CONCLUSION: In 2014, a number of patents were filed for the treatment of pain among oncology patients. The great variety of chemical compounds presented indicates that there is a great variety in current research for cancer-related pain. The development of novel treatment options will hopefully lead to more effective therapies in the treatment of cancer-related pain.