BIOANALYSIS AND BIOTRANSFORMATION OF OLIGONUCLEOTIDE THERAPEUTICS BY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY.

Since 2016, eight new oligonucleotide therapies have been approved which has led to increased interest in oligonucleotide analysis. There is a particular need for powerful bioanalytical tools to study the metabolism and biotransformation of these molecules. This review provides the background on the biological basis of these molecules as currently used in therapies. The article also reviews the current state of analytical methodology including state of the art sample preparation techniques, liquid chromatography-mass spectrometry methods, and the current limits of detection/quantitation. Finally, the article summarizes the challenges in oligonucleotide bioanalysis and provides future perspectives for this emerging field. © 2020 John Wiley & Sons Ltd.

The Effectiveness of Anti-Nerve Growth Factor Monoclonal Antibodies in the Management of Pain in Osteoarthritis of the Hip and Knee: A PRISMA Systematic Review and Meta-Analysis.

OBJECTIVE: To conduct a systematic review and meta-analysis of the efficacy of anti-nerve growth factor (NGF) monoclonal antibodies in osteoarthritis pain (hip and knee). DESIGN: Grade the evidence for anti-NGF use. METHODS: An interdisciplinary work group conducted a literature search for anti-NGF use in osteoarthritis. The systematic review was performed in accordance with methods described by the Cochrane collaboration. General inclusion criteria included all osteoarthritis trials studying any monoclonal anti-NGF antibody at any dose/phase. Excluded studies were those where participants received NSAIDs or analgesics other than anti-NGF antibodies. The Jadad Scale score was used to assess the quality of the included studies. RESULTS: Thirteen studies were included in the analysis, involving 8145 participants with a diagnosis of hip and/or knee osteoarthritis. Anti-NGF antibody treatment was associated with a significant improvement in all Western Ontario and McMaster Universities Arthritis Index (WOMAC) indices when compared to placebo. These agents were not associated with a significantly increased incidence of serious adverse events but were associated with significant increases in therapy discontinuation due to adverse events or side effects (e.g., peripheral neuropathy). CONCLUSIONS: Future randomized clinical trials are needed to characterize the overall risk-to-benefit ratio of anti-NGF antibodies in managing pain associated with OA, particularly with long-term use, in order to verify their efficacy and safety in clinical practice.

Late Development of a Large Arteriovenous Fistula with Aneurysm in an In Situ Vein Bypass Graft.

We report a case of saphenous vein bypass aneurysm and arteriovenous fistula in a 65-year-old man, 20 years after an in situ vein bypass for occlusive disease. He was found to have patent venous branches which kept the bypass open despite distal anastomotic occlusion. The saphenous vein was successfully excised without distal revascularization due to sufficient native arterial flow. This is the first reported case of aneurysmal degeneration of an in situ vein conduit with occluded distal anastomosis and patent venous side branches. Ultrasound surveillance is warranted for all bypass procedures, and early endovascular or open revisions can prevent late complications.

Molecular Basis of mRNA Cap Recognition by Influenza B Polymerase PB2 Subunit.

Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2.

Rapamycin drives selection against a pathogenic heteroplasmic mitochondrial DNA mutation.

Mitochondrial DNA (mtDNA) mutations cause a variety of mitochondrial disorders for which effective treatments are lacking. Emerging data indicate that selective mitochondrial degradation through autophagy (mitophagy) plays a critical role in mitochondrial quality control. Inhibition of mammalian target of rapamycin (mTOR) kinase activity can activate mitophagy. To test the hypothesis that enhancing mitophagy would drive selection against dysfunctional mitochondria harboring higher levels of mutations, thereby decreasing mutation levels over time, we examined the impact of rapamycin on mutation levels in a human cytoplasmic hybrid (cybrid) cell line expressing a heteroplasmic mtDNA G11778A mutation, the most common cause of Leber's hereditary optic neuropathy. Inhibition of mTORC1/S6 kinase signaling by rapamycin induced colocalization of mitochondria with autophagosomes, and resulted in a striking progressive decrease in levels of the G11778A mutation and partial restoration of ATP levels. Rapamycin-induced upregulation of mitophagy was confirmed by electron microscopic evidence of increased autophagic vacuoles containing mitochondria-like organelles. The decreased mutational burden was not due to rapamycin-induced cell death or mtDNA depletion, as there was no significant difference in cytotoxicity/apoptosis or mtDNA copy number between rapamycin and vehicle-treated cells. These data demonstrate the potential for pharmacological inhibition of mTOR kinase activity to activate mitophagy as a strategy to drive selection against a heteroplasmic mtDNA G11778A mutation and raise the exciting possibility that rapamycin may have therapeutic potential for the treatment of mitochondrial disorders associated with heteroplasmic mtDNA mutations, although further studies are needed to determine if a similar strategy will be effective for other mutations and other cell types.

Ion pair liquid chromatography method for the determination of thiamine (vitamin B1) homeostasis.

A new method for reversed phase HPLC determination of thiamine and its major in vivo phosphorylation products, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP), was developed using tetrabutylammonium hydroxide as the ion-pairing agent. The separation was performed on a Phenomenex Kinetex EVO C18 column with a gradient of a phosphate-buffered aqueous solution of the ion-pair reagent and methanol. The duty cycle for the assay was 13 min and pyrithiamine was successfully used as the internal standard for the first time in a thiamine HPLC measurement protocol. Detection of the fluorescence derivatives of the analytes as well as the IS allowed for lower detection limits in order to support biological applications in cell culture models. The linearity, sensitivity, specificity, accuracy and precision of the method were evaluated and met the requirements specified by the US Food and Drug Administration. The calibration curves proved to be linear and the method was validated over the range from 1.0-4000 nM for both cells and the media where complete recovery of the analytes was also achieved.

Phenotypic and metabolic investigation of a CSF-1R kinase receptor inhibitor (BLZ945) and its pharmacologically active metabolite.

1. 4-[2((1R,2R)-2-Hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide (BLZ945) is a small molecule inhibitor of CSF-1R kinase activity within osteoclasts designed to prevent skeletal related events in metastatic disease. Key metabolites were enzymatically and structurally characterized to understand the metabolic fate of BLZ945 and pharmacological implications. The relative intrinsic clearances for metabolites were derived from in vitro studies using human hepatocytes, microsomes and phenotyped with recombinant P450 enzymes. 2. Formation of a pharmacologically active metabolite (M9) was observed in human hepatocytes. The M9 metabolite is a structural isomer (diastereomer) of BLZ945 and is about 4-fold less potent. This isomer was enzymatically formed via P450 oxidation of the BLZ945 hydroxyl group, followed by aldo-keto reduction to the alcohol (M9). 3. Two reaction phenotyping approaches based on fractional clearances were applied to BLZ945 using hepatocytes and liver microsomes. The fraction metabolized (fm) or contribution ratio was determined for each metabolic reaction type (oxidation, glucuronidation or isomerization) as well as for each metabolite. The results quantitatively illustrate contribution ratios of the involved enzymes and pathways, e.g. the isomerization to metabolite M9 accounted for 24% intrinsic clearance in human hepatocytes. In summary, contribution ratios for the Phase I and Phase II pathways can be determined in hepatocytes.

Properties of triple shape memory composites prepared via polymerization-induced phase separation.

Research in the field of shape memory polymers has recently witnessed the introduction of increasing complexity of material response, including such phenomena as triple/multishape behavior, temperature memory, and reversible actuation. Ordinarily, such complexity in physical behaviour is achieved through comparable complexity in material composition and synthesis. Seeking to achieve a triple shape behaviour with a simple route to materials synthesis, we introduce here a method that utilizes polymerization induced phase separation (PIPS) to yield the requisite combination of microstructure and composition. Thus, two blends incorporating epoxy and poly(ε-caprolactone) were developed using commercially available reactants, one featuring a semicrystalline epoxy and the other featuring an amorphous epoxy. We show that both blends exhibited distinct transition temperatures and three modulus-temperature plateaus needed for triple shape behaviour. Despite these similarities, their physical character at room temperature is vastly different: the semicrystalline epoxy material is elastomeric and the amorphous epoxy material is highly stiff. Characterization of the triple shape behaviour revealed an ability of both systems to fix two separate deformations independently, one by PCL crystallization and a second one by epoxy crystallization or vitrification, and recover both programmed shapes separately upon heating. Given the simplicity of fabrication, we envision application as multi-shape coatings, adhesives, and films.

2-Aminothiazole based P2Y(1) antagonists as novel antiplatelet agents.

ADP receptors, P2Y1 and P2Y12 have been recognized as potential targets for antithrombotic drugs. A series of P2Y1 antagonists that contain 2-aminothiazoles as urea surrogates were discovered. Extensive SAR of the thiazole ring is described. The most potent compound 7j showed good P2Y1 binding (Ki=12nM), moderate antagonism of platelet aggregation (PA IC50=5.2µM) and acceptable PK in rats.

Discovery of nonbenzamidine factor VIIa inhibitors using a biaryl acid scaffold.

In this Letter, we describe the synthesis of several nonamidine analogs of biaryl acid factor VIIa inhibitor 1 containing weakly basic or nonbasic P1 groups. 2-Aminoisoquinoline was found to be an excellent surrogate for the benzamidine group (compound 2) wherein potent inhibition of factor VIIa is maintained relative to most other related serine proteases. In an unanticipated result, the m-benzamide P1 (compounds 21a and 21b) proved to be a viable benzamidine replacement, albeit with a 20-40 fold loss in potency against factor VIIa.

Athlete Multiple Perpetrator Rape (MPR) as Interactional and Organizational Deviance: Heuristic Insights from a Multilevel Framework.

Although athlete multiple perpetrator rape (MPR) has frequently been covered in the media, it has received more limited scholarly attention. Accordingly, I synthesize findings from multiple disciplines and integrate insights from the MPR, institutional betrayal, and organizational deviance literatures to establish a heuristic framework for understanding athlete MPR. I ultimately argue that athlete MPR is both an act of interactional deviance and an act of organizational deviance. This undertaking represents one of the only works to focus explicitly on athlete MPR. It is additionally the first to examine any form of sexual assault through an organizational deviance lens.

Investigation of correlation among safety biomarkers in serum, histopathological examination, and toxicogenomics.

This article addresses the issue of miscorrelation between hepatic injury biomarkers and histopathological findings in the drug development context. Our studies indicate that the use of toxicogenomics can aid in the drug development decision-making process associated with such miscorrelated data. BLZ945 was developed as a Colony-Stimulating Factor 1 Receptor (CSF-1R) inhibitor. Treatment of BLZ945 in rats and monkeys increased serum alanine aminotransferase (ALT) and aspartate transaminase (AST). However, liver hypertrophy was the only histopathological liver finding in rats, and there was no change in the livers of monkeys. Longer treatment of BLZ945 in rats for 6 weeks caused up to 6-fold elevation of ALT, yet hepatocyte necrosis was not detected microscopically. Toxicogenomic profiling of liver samples demonstrated that the genes associated with early response to liver injury, apoptosis/necrosis, inflammation, oxidative stress, and metabolic enzymes were upregulated. Studies are ongoing to evaluate the mechanisms underlying BL945-induced ALT and AST elevations.

Impact of Social Determinants of Health, Health Literacy, Self-perceived Risk, and Trust in the Emergency Physician on Compliance with Follow-up.

INTRODUCTION: Patients presenting to the emergency department (ED) with "low-risk" acute coronary syndrome (ACS) symptoms can be discharged with outpatient follow-up. However, follow-up compliance is low for unknown nonclinical reasons. We hypothesized that a patient's social factors, health literacy, self-perceived risk, and trust in the emergency physician may impact follow-up compliance. METHODS: This was a prospective study of a convenience sample of discharged ED patients presenting with chest pain and given a follow-up appointment prior to departing the ED. Patients were asked about social and demographic factors and to estimate their own risk for heart disease; they also completed the Short Assessment of Health Literacy-English (SAHL-E) and the Trust in Physician Scale (TiPS). RESULTS: We enrolled146 patients with a follow-up rate of 36.3%. Patients who had a low self-perceived heart disease risk (10% or less) were significantly less likely to attend follow-up than those with a higher perceived risk (23% vs 44%, P = 0.01). Other factors did not significantly predict follow-up rates. CONCLUSION: In an urban county ED, in patients who were deemed low risk for ACS and discharged, only self-perception of risk was associated with compliance with a follow-up appointment.

Potential Impact of COVID-19 on Recently Resettled Refugee Populations in the United States and Canada: Perspectives of Refugee Healthcare Providers.

Recently resettled refugee populations may be at greater risk for exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus that causes coronavirus 2019 (COVID-19), and face unique challenges in following recommendations to protect their health. Several factors place resettled refugees at elevated risk for exposure to persons with COVID-19 or increased severity of COVID-19: being more likely to experience poverty and live in crowded housing, being employed in less protected, service-sector jobs, experiencing language and health care access barriers, and having higher rates of co-morbidities. In preparing for and managing COVID-19, resettled refugees encounter similar barriers to those of other racial or ethnic minority populations, which may then be exacerbated by unique barriers experienced from being a refugee. Key recommendations for resettlement and healthcare providers include analyzing sociodemographic data about refugee patients, documenting and resolving barriers faced by refugees, developing refugee-specific outreach plans, using culturally and linguistically appropriate resources, ensuring medical interpretation availability, and leveraging virtual platforms along with nontraditional community partners to disseminate COVID-19 messaging.

Growth and Antifungal Resistance of the Pathogenic Yeast, Candida Albicans, in the Microgravity Environment of the International Space Station: An Aggregate of Multiple Flight Experiences.

This report was designed to compare spaceflight-induced cellular and physiological adaptations of Candida albicans cultured in microgravity on the International Space Station across several payloads. C. albicans is a common opportunistic fungal pathogen responsible for a variety of superficial infections as well as systemic and more severe infections in humans. Cumulatively, the propensity of this organism to be widespread through the population, the ability to produce disease in immunocompromised individuals, and the tendency to respond to environmental stress with characteristics associated with increased virulence, require a better understanding of the yeast response to microgravity for spaceflight crew safety. As such, the responses of this yeast cultivated during several missions using two in-flight culture bioreactors were analyzed and compared herein. In general, C. albicans had a slightly shorter generation time and higher growth propensity in microgravity as compared to terrestrial controls. Rates of cell filamentation differed between bioreactors, but were low and not significantly different between flight and terrestrial controls. Viable cells were retrieved and cultured, resulting in a colony morphology that was similar between cells cultivated in flight and in terrestrial control conditions, and in contrast to that previously observed in a ground-based microgravity analog system. Of importance, yeast demonstrated an increased resistance when challenged during spaceflight with the antifungal agent, amphotericin B. Similar levels of resistance were not observed when challenged with the functionally disparate antifungal drug caspofungin. In aggregate, yeast cells cultivated in microgravity demonstrated a subset of characteristics associated with virulence. In addition, and beyond the value of the specific responses of C. albicans to microgravity, this report includes an analysis of biological reproducibility across flight opportunities, compares two spaceflight hardware systems, and includes a summary of general flight and payload timelines.

Solid-phase synthesis of a library based on biphenyl-containing trypsin-like serine protease inhibitors.

The solid-phase synthesis of a library based on an unusual biphenyl-containing trypsin-like serine protease inhibitor is described. Key to this effort was the synthesis of a highly functionalized aryl boronic acid reagent which required the development of a novel and efficient method to convert a triflate to a pinacolboronate in large scale.

Prisoners' Personal Networks in the Months Preceding Prison: A Descriptive Portrayal.

This study examined personal networks of adult male prisoners ( N = 250) during a high-risk period prior to their incarceration. We present a descriptive portrait of network size, density, and relational type, and we then document the nature of ties within that network, focusing specifically on alters' criminal involvement, criminal opportunity, and reinforcement of criminal behavior. We found that prisoners' networks were large and dense, and that they were composed primarily of family and romantic partners. Most prisoners are not embedded in a personal network saturated with criminal influence before coming to prison. Yet, a small proportion are exposed to exceptionally negative influence, which, it is argued, may increase the risk of negative outcomes upon release if not addressed by evidence-based programs.

A Daily Hospital Progress Note that Increases Physician Usability of the Electronic Health Record by Facilitating a Problem-Oriented Approach to the Patient and Reducing Physician Clerical Burden.

We suggest changes in the electronic health record (EHR) in hospitalized patients to increase EHR usability by optimizing the physician's ability to approach the patient in a problem-oriented fashion and by reducing physician data entry and chart navigation. The framework for these changes is a Physician's Daily Hospital Progress Note organized into 3 sections: Subjective, Objective, and a combined Assessment and Plan section, subdivided by problem titles. The EHR would consolidate information for each problem by: 1) juxtaposing to each problem title relevant medications, key durable results, and limitations; 2) entering in the running lists under Assessment and Plan the most relevant information for that day, including abbreviated versions of relevant reports; and 3) generating a flow sheet in a problem's progress note for any key results tracked daily. To reduce physician EHR navigation, the EHR would place in the Objective section abbreviated versions of notes of other physicians, nurses, and allied health professionals as well as recent orders. The physician would enter only the analysis and plan and new information not included in the EHR. The consolidation of information for each problem would facilitate physician communication at points of transition of care including generation of a problem-oriented discharge summary.

Pyridine amides as potent and selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1.

Several series of pyridine amides were identified as selective and potent 11beta-HSD1 inhibitors. The most potent inhibitors feature 2,6- or 3,5-disubstitution on the pyridine core. Various linkers (CH(2)SO(2), CH(2)S, CH(2)O, S, O, N, bond) between the distal aryl and central pyridyl groups are tolerated, and lipophilic amide groups are generally favored. On the distal aryl group, a number of substitutions are well tolerated. A crystal structure was obtained for a complex between 11beta-HSD1 and the most potent inhibitor in this series.