Abstract
Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized ...that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.
Abstract
We determined the effect of attaching palmitate, tocopherol or cholesterol to PS ASOs and their effects on plasma protein binding and on enhancing ASO potency in the muscle of rodents and ...monkeys. We found that cholesterol ASO conjugates showed 5-fold potency enhancement in the muscle of rodents relative to unconjugated ASOs. However, they were toxic in mice and as a result were not evaluated in the monkey. In contrast, palmitate and tocopherol-conjugated ASOs showed enhanced potency in the skeletal muscle of rodents and modest enhancements in potency in the monkey. Analysis of the plasma-protein binding profiles of the ASO-conjugates by size-exclusion chromatography revealed distinct and species-specific differences in their association with plasma proteins which likely rationalizes their behavior in animals. Overall, our data suggest that modulating binding to plasma proteins can influence ASO activity and distribution to extra-hepatic tissues in a species-dependent manner and sets the stage to identify other strategies to enhance ASO potency in muscle tissues.
The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler ...path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5′ phosphate to achieve activity in vivo, and we developed a metabolically stable 5′-(E)-vinylphosphonate (5′-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.
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► Chemically modified single-stranded siRNAs silence gene expression in animals ► ss-siRNAs require 5′ phosphate and association with AGO2 for gene silencing ► Passenger strand is dispensable for potent RNAi-mediated gene silencing ► Single-stranded siRNAs provide an alternate option for RNAi therapeutics
Chemically modified RNA oligonucleotides load into Ago2-containing RISC complexes to effect RNA silencing without the need for a passenger strand. These single-stranded siRNAs can be applied directly in animals and thus offer new alternatives for developing therapeutics and diagnostics.
Conjugation of antisense oligonucleotide (ASO) with a variety of distinct lipophilic moieties like fatty acids and cholesterol increases ASO accumulation and activity in multiple tissues. While lipid ...conjugation increases tissue exposure in mice and reduces excretion of ASO in urine, histological review of skeletal and cardiac muscle indicates that the increased tissue accumulation of lipid conjugated ASO is isolated to the interstitium. Administration of palmitic acid-conjugated ASO (Palm-ASO) in mice results in a rapid and substantial accumulation in the interstitium of muscle tissue followed by relatively rapid clearance and only slight increases in intracellular accumulation in myocytes. We propose a model whereby increased affinity for lipid particles, albumin, and other plasma proteins by lipid-conjugation facilitates ASO transport across endothelial barriers into tissue interstitium. However, this increased affinity for lipid particles and plasma proteins also facilitates the transport of ASO from the interstitium to the lymph and back into circulation. The cumulative effect is only a slight (∼2-fold) increase in tissue accumulation and similar increase in ASO activity. To support this proposal, we demonstrate that the activity of lipid conjugated ASO was reduced in two mouse models with defects in endothelial transport of macromolecules: caveolin-1 knockout (Cav1-/-) and FcRn knockout (FcRn-/-).
Both serotonergic signalling disruption and systemic inflammation have been associated with the pathogenesis of Alzheimer's disease (AD). The common denominator linking the two is the catabolism of ...the essential amino acid, tryptophan. Metabolism via tryptophan hydroxylase results in serotonin synthesis, whilst metabolism via indoleamine 2,3-dioxygenase (IDO) results in kynurenine and its downstream derivatives. IDO is reported to be activated in times of host systemic inflammation and therefore is thought to influence both pathways. To investigate metabolic alterations in AD, a large-scale metabolic phenotyping study was conducted on both urine and serum samples collected from a multi-centre clinical cohort, consisting of individuals clinically diagnosed with AD, mild cognitive impairment (MCI) and age-matched controls.
Metabolic phenotyping was applied to both urine (n = 560) and serum (n = 354) from the European-wide AddNeuroMed/Dementia Case Register (DCR) biobank repositories. Metabolite data were subsequently interrogated for inter-group differences; influence of gender and age; comparisons between two subgroups of MCI - versus those who remained cognitively stable at follow-up visits (sMCI); and those who underwent further cognitive decline (cMCI); and the impact of selective serotonin reuptake inhibitor (SSRI) medication on metabolite concentrations.
Results revealed significantly lower metabolite concentrations of tryptophan pathway metabolites in the AD group: serotonin (urine, serum), 5-hydroxyindoleacetic acid (urine), kynurenine (serum), kynurenic acid (urine), tryptophan (urine, serum), xanthurenic acid (urine, serum), and kynurenine/tryptophan ratio (urine). For each listed metabolite, a decreasing trend in concentrations was observed in-line with clinical diagnosis: control > MCI > AD. There were no significant differences in the two MCI subgroups whilst SSRI medication status influenced observations in serum, but not urine.
Urine and serum serotonin concentrations were found to be significantly lower in AD compared with controls, suggesting the bioavailability of the neurotransmitter may be altered in the disease. A significant increase in the kynurenine/tryptophan ratio suggests that this may be a result of a shift to the kynurenine metabolic route due to increased IDO activity, potentially as a result of systemic inflammation. Modulation of the pathways could help improve serotonin bioavailability and signalling in AD patients.
In females, ovarian estradiol modulates kisspeptin (Kiss-1) synthesis to act as an obligatory regulator of downstream gonadotropin release in vivo, via stimulation of GnRH neurons. Changes in the ...ovarian condition are relayed to the neuroendocrine hypothalamus via two sexually dimorphic Kiss-1 populations, located in the anteroventral periventricular (AVPV) and arcuate nuclei, conveying estradiol-positive and -negative feedback, respectively. To elucidate how differential responsiveness to estradiol is mediated in these populations, we generated two kisspeptin-secreting cell lines from an adult kiss1-green fluorescent protein (GFP) female mouse. These lines recapitulate in vivo responsiveness to estradiol, with KTaV-3 (AVPV) cells demonstrating significantly increased kiss1 expression under high physiological estradiol exposure, whereas KTaR-1 (arcuate) cells exhibit kiss1 suppression after lower estradiol exposure. Baseline expression of estrogen receptor-α (esr1) differs significantly between KTaV-3 and KTaR-1 cells, with KTaR-1 cells demonstrating higher basal expression of esr1. Estradiol stimulation of kiss1 expression in KTaV-3 cells is modulated in a dose-dependent manner up to 25.0 pM, with less responsiveness observed at higher doses (>50.0 pM). In contrast, KTaR-1 kiss1 attenuates at lower estradiol doses (2.0–5.0 pM), returning to baseline levels at 25.0 pM and greater. Furthermore, the expression of the core clock genes bmal1 and per2 show normal rhythms in KTaV-3 cells, regardless of estradiol treatment. Conversely, KTaR-1 antiphasic transcription of bmal1 and per2 is phase delayed by low estradiol treatment. Strikingly, estradiol induces circadian rhythms of kiss1 expression only in KTaV-3 cells. Further exploration into estradiol responsiveness will reveal mechanisms responsible for the differential expression pattern demonstrated in vivo between these cell types.
► The review explores circadian influences on hypothalamic and pituitary endocrine function. ► Endocrine influence on the suprachiasmatic nucleus and endogenous cellular oscillators is covered. ► The ...reproductive axis is explored as an example of convergence of endocrine and circadian factors. ► More research is needed into mechanisms underlying circadian regulation of endocrine functions.
Recent strides in circadian biology over the last several decades have allowed researchers new insight into how molecular circadian clocks influence the broader physiology of mammals. Elucidation of transcriptional feedback loops at the heart of endogenous circadian clocks has allowed for a deeper analysis of how timed cellular programs exert effects on multiple endocrine axes. While the full understanding of endogenous clocks is currently incomplete, recent work has re-evaluated prior findings with a new understanding of the involvement of these cellular oscillators, and how they may play a role in constructing rhythmic hormone synthesis, secretion, reception, and metabolism. This review addresses current research into how multiple circadian clocks in the hypothalamus and pituitary receive photic information from oscillators within the hypothalamic suprachiasmatic nucleus (SCN), and how resultant hypophysiotropic and pituitary hormone release is then temporally gated to produce an optimal result at the cognate target tissue. Special emphasis is placed not only on neural communication among the SCN and other hypothalamic nuclei, but also how endogenous clocks within the endocrine hypothalamus and pituitary may modulate local hormone synthesis and secretion in response to SCN cues. Through evaluation of a larger body of research into the impact of circadian biology on endocrinology, we can develop a greater appreciation into the importance of timing in endocrine systems, and how understanding of these endogenous rhythms can aid in constructing appropriate therapeutic treatments for a variety of endocrinopathies.
CD6 is a transmembrane protein with an extracellular region containing three scavenger receptor cysteine rich (SRCR) domains. The membrane proximal domain of CD6 binds the N-terminal immunoglobulin ...superfamily (IgSF) domain of another cell surface receptor, CD166, which also engages in homophilic interactions. CD6 expression is mainly restricted to T cells, and the interaction between CD6 and CD166 regulates T-cell activation. We have solved the X-ray crystal structures of the three SRCR domains of CD6 and two N-terminal domains of CD166. This first structure of consecutive SRCR domains reveals a nonlinear organization. We characterized the binding sites on CD6 and CD166 and showed that a SNP in CD6 causes glycosylation that hinders the CD6/CD166 interaction. Native mass spectrometry analysis showed that there is competition between the heterophilic and homophilic interactions. These data give insight into how interactions of consecutive SRCR domains are perturbed by SNPs and potential therapeutic reagents.
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•First structure of consecutive scavenger receptor cysteine rich domains in CD6•Structure of the two N-terminal domains of CD166 which is the ligand for CD6•Mapping binding sites on CD6 and CD166•Insight into how CD6 and its interactions are perturbed by polymorphisms and mAbs
Chappell et al. present structures of the T-cell surface receptor, CD6, the first of consecutive scavenger receptor cysteine rich domains and its ligand, CD166. The structures give insight into how CD6 and its interactions are perturbed by competition between homophilic and heterophilic interactions, SNPs, and mAbs.
Summary
Background
The Province of Ontario maintains a registry of racehorse deaths occurring within 60 days of a race or trial entry that provides insight into mortality rates and costs of ...competition.
Objectives
To characterise and quantify mortality and identify breed differences.
Study design
Retrospective annualised cohort study.
Methods
The Ontario Death Registry for 2003–2015, containing 1713 cases, was audited and information on the relationship between death and official work added. Race and trial data from industry performance databases were used to determine mortality rates according to breed, year, age, sex and circumstances of death.
Results
Breed differences in mortality rate and individual risk were found. Thoroughbreds (Tb) had the greatest exercise‐associated mortality (EAM) rate and risk by all measures (2.27 deaths/1000 race starts, 0.95–1.0% annual individual risk), followed by Quarter horses (Qh, 1.49, 0.60–0.69%). Rate and risk were lowest for Standardbreds (Sb, 0.28, 0.23–0.24%). Nonexercise annual individual risk was highest for the Sb (0.45%, vs. Tb 0.33%, and Qh 0.32%). Pattern and type of EAM mirrored the characteristics of competitive activity in each industry, with high Tb and Qh mortality being associated with exercise and involving musculoskeletal injuries, dying suddenly and accidents. Low Sb EAM reflected the more extensive nature of training preparation and racing for this breed.
Main limitations
Available data provided no information on morbidity, mortality beyond the 60‐day horizon or for horses not racing. Numbers for the Qh were low.
Conclusions
Race‐intensity exercise is clearly hazardous for horses, with hazards varying widely between breeds and showing parallels with industry cultural and management norms. Breed differences provide insights concerning strategies that could reduce mortality, while improving welfare and reducing costs of participation. For all breeds, musculoskeletal injury was the major contributing cause of mortality.
A voluntary upper respiratory biosurveillance program in the USA received 9740 nasal swab submissions during the years 2008-2021 from 333 veterinarians and veterinary clinics. The nasal swabs were ...submitted for qPCR testing for six common upper respiratory pathogens:equine influenza virus (EIV), equine herpesvirus-1 (EHV-1), equine herpesvirus-4 (EHV-4),
subspecies
(
), equine rhinitis A virus (ERAV), and equine rhinitis B virus (ERBV). Additional testing was performed for equine gamma herpesvirus-2 (EHV-2) and equine gamma herpesvirus-5 (EHV-5) and the results are reported. Basic frequency statistics and multivariate logistic regression models were utilized to determine the associations between risk factors and EIV positivity. The EIV qPCR-positivity rate was 9.9%. Equids less than 9 years of age with a recent history of travel and seasonal occurrence in winter and spring were the most common population that were qPCR positive for EIV. This ongoing biosurveillance program emphasizes the need for molecular testing for pathogen identification, which is critical for decisions associated with therapeutics and biosecurity intervention for health management and vaccine evaluations and development.
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