Development and Assessment of Herpes Simplex Virus Type 1 (HSV-1) Amplicon Vectors with Sensory Neuron-Selective Promoters.

BACKGROUND: Neurogenic detrusor overactivity (NDO) is a severe pathological condition characterized by involuntary detrusor contractions leading to urine leakage. This condition is frequent after spinal cord injury (SCI). Gene therapy for NDO requires the development of vectors that express therapeutic transgenes driven by sensory neuron-specific promoters. The aim of this study was to develop and assess tools for the characterization of sensory neuron-specific promoters in dorsal root ganglia (DRG) neurons after transduction with herpes simplex virus type 1 (HSV-1)-based amplicon defective vectors. METHODS: The HSV-1 vector genome encoded two independent transcription cassettes: one expressed firefly luciferase (FLuc) driven by different promoters' candidates (rTRPV1, rASIC3, rCGRP, or hCGRP), and the other expressed a reporter gene driven by an invariable promoter. The strength and selectivity of promoters was assessed in organotypic cultures of explanted adult DRG, or sympathetic and parasympathetic ganglia from control and SCI rats. RESULTS: The rCGRP promoter induced selective expression in the DRG of normal rats. The rTRPV-1 promoter, which did not display selective activity in control rats, induced selective expression in DRG explanted from SCI rats. CONCLUSIONS: This study provides a methodology to assess sensory neuron-specific promoters, opening new perspectives for future gene therapy for NDO.

Hippocampal synaptic and membrane function in the DBA/2J-mdx mouse model of Duchenne muscular dystrophy.

Dystrophin deficiency is associated with alterations in cell physiology. The functional consequences of dystrophin deficiency are particularly severe for muscle physiology, as observed in Duchenne muscle dystrophy (DMD). DMD is caused by the absence of a 427 kDa isoform of dystrophin. However, in addition to muscular dystrophy symptoms, DMD is frequently associated with memory and attention deficits and epilepsy. While this may be associated with a role for dystrophin in neuronal physiology, it is not clear what neuronal alterations are linked with DMD. Our work shows that CA1 pyramidal neurons from DBA/2J-mdx mice have increased afterhyperpolarization compared to WT controls. All the other electrotonic and electrogenic membrane properties were unaffected by this genotype. Finally, basal synaptic transmission, short-term and long-term synaptic plasticity at Schaffer collateral to CA1 glutamatergic synapses were unchanged between mdx and WT controls. These data show that the excitatory component of hippocampal activity is largely preserved in DBA/2J-mdx mice. Further studies, extending the investigation to the inhibitory GABAergic function, may provide a more complete picture of the functional, network alterations underlying impaired cognition in DMD. In addition, the investigation of changes in neuronal single conductance biophysical properties associated with this genotype, is required to identify the functional alterations associated with dystrophin deficiency and clarify its role in neuronal function.

Local myostatin inhibition improves skeletal muscle glucose uptake in insulin-resistant high-fat diet-fed mice.

Myostatin inhibition is thought to improve whole body insulin sensitivity and mitigate the development of insulin resistance in models of obesity. However, although myostatin is known to be a major regulator of skeletal muscle mass, the direct effects of myostatin inhibition in muscle on glucose uptake and the mechanisms that may underlie this are still unclear. We investigated the effect of local myostatin inhibition by adeno-associated virus-mediated overexpression of the myostatin propeptide on insulin-stimulated skeletal muscle glucose disposal in chow-fed or high fat diet-fed mice and evaluated the molecular pathways that might mediate this. We found that myostatin inhibition improved glucose disposal in obese high fat diet-fed mice alongside the induction of muscle hypertrophy but did not have an impact in chow-fed mice. This improvement was not associated with greater glucose transporter or peroxisome proliferator-activated receptor-γ coactivator-1α expression or 5' AMP-activated protein kinase activation as previously suggested. Instead, transcriptomic analysis suggested that the improvement in glucose disposal was associated with significant enrichment in genes involved in fatty acid metabolism and translation of mitochondrial genes. Thus, myostatin inhibition improves muscle insulin-stimulated glucose disposal in obese high fat diet-fed mice independent of muscle hypertrophy, potentially involving previously unidentified pathways.

Engineered botulinum neurotoxins as new therapeutics.

Botulinum neurotoxins (BoNTs) cause flaccid paralysis by inhibiting neurotransmission at cholinergic nerve terminals. Each BoNT consists of three domains that are essential for toxicity: the binding domain, the translocation domain, and the catalytic light-chain domain. BoNT modular architecture is associated with a multistep mechanism that culminates in the intracellular proteolysis of SNARE (soluble N-ethylmaleimide-sensitive-fusion-protein attachment protein receptor) proteins, which prevents synaptic vesicle exocytosis. As the most toxic proteins known, BoNTs have been extensively studied and are used as pharmaceutical agents to treat an increasing variety of disorders. This review summarizes the level of sophistication reached in BoNT engineering and highlights the diversity of approaches taken to utilize the modularity of the toxin. Improved efficiency and applicability have been achieved by direct mutagenesis and interserotype domain rearrangement. The scope of BoNT activity has been extended to nonneuronal cells and offers the basis for novel biomolecules in the treatment of secretion disorders.

Identification of Acetomycin as an Antifungal Agent Produced by Termite Gut-Associated Streptomycetes against Pyrrhoderma noxium.

Plant fungal pathogen Pyrrhoderma noxium is responsible for the destructive and invasive disease of brown root rot currently affecting the city of Brisbane, Australia. In order to address this issue, environmentally friendly and safe alternatives to chemical control are preferred due to the city's public setting. Antifungal natural products are ideal candidates as biological control alternatives and can be detected through investigating the metabolomes of microbial symbionts. Within this study, an NMR-based metabolomics approach was applied to fermentation extracts obtained from 15 termite gut-associated streptomycetes. By analysing the NMR spectra, six of the extracts which displayed similar chemical profiles exhibited antifungal activity against the P. noxium pathogen. The major compound within these extracts was identified as acetomycin using NMR and X-ray crystallography analyses. This is the first reporting of acetomycin as a potential natural product fungicide, particularly as an antifungal agent against P. noxium. Inhibitory activity was also found against other important fungal crop pathogens, including Aspergillus niger, Botrytis cinerea, and Alteranaria alternata. Further experimentation using a woodblock test found inhibitory activity on the growth of the P. noxium pathogen for up to 3 weeks and a significant difference in the integrity of the woodblocks when conducting compression strength tests after 6 weeks. Therefore, acetomycin may be used as a biological control agent and natural product fungicide against P. noxium.

In vitro biological control of Pyrrhoderma noxium using volatile compounds produced by termite gut-associated streptomycetes.

Introduction: Pyrrhoderma noxium is a plant pathogen that causes economic losses in agricultural and forestry industries, including significant destruction to amenity trees within the city of Brisbane in Australia. Use of chemical control agents are restricted in public areas, there is therefore an urgent need to investigate biological control approaches. Members of the phylum Actinomycetota, commonly known as actinomycetes, are known for their industrially important secondary metabolites including antifungal agents. They have proven to be ideal candidates to produce environmentally friendly compounds including the volatile organic compounds (VOCs) which can be used as biofumigants. Methods: Different Streptomyces species (n=15) previously isolated from the guts of termites and stored in the University of the Sunshine Coast'sMicrobial Library were tested for their antifungal VOCs against Pyrrhoderma noxium. Results: Fourteen of them were found to display inhibition (39.39-100%) to the mycelial development of the pathogen. Strongest antifungal activity displaying isolates USC-592, USC-595, USC-6910 and USC-6928 against the pathogen were selected for further investigations. Their VOCs were also found to have plant growth promotional activity observed for Arabidopsis thaliana with an increase of root length (22-36%) and shoot length (26-57%). The chlorophyll content of the test plant had a slight increase of 11.8% as well. Identified VOCs included geosmin, 2-methylisoborneol, 2-methylbutyrate, methylene cyclopentane, ß-pinene, dimethyl disulfide, ethyl isovalerate, methoxyphenyl-oxime and α-pinene. Additionally, all 15 Streptomyces isolates were found to produce siderophores and indole acetic acid as well as the enzyme chitinase which is known to break down the fungal cell wall. Discussion: Findings indicate that termite gut-associated streptomycetes might be used to control Pyrrhoderma noxium by utilizing their wide range of inhibitory mechanisms.

Untargeted MS-Based Metabolomic Analysis of Termite Gut-Associated Streptomycetes with Antifungal Activity against Pyrrhoderma noxium.

Pyrrhoderma noxium is a plant fungal pathogen that induces the disease of brown root rot in a large variety of tree species. It is currently infecting many of the amenity trees within Brisbane City of Queensland, Australia. Steering away from harmful chemical fungicides, biological control agents offer environmentally friendly alternatives. Streptomycetes are known for their production of novel bioactive secondary metabolites with biocontrol potential, particularly, streptomycete symbionts isolated from unique ecological niches. In this study, 37 termite gut-associated actinomycete isolates were identified using molecular methods and screened against P. noxium. A majority of the isolates belonged to the genus Streptomyces, and 15 isolates exhibited strong antifungal activity with up to 98.5% mycelial inhibition of the fungal pathogen. MS/MS molecular networking analysis of the isolates' fermentation extracts revealed several chemical classes with polyketides being among the most abundant. Most of the metabolites, however, did not have matches to the GNPS database, indicating potential novel antifungal compounds in the active extracts obtained from the isolates. Pathway enrichment and overrepresentation analyses revealed pathways relating to polyketide antibiotic production, among other antibiotic pathways, further confirming the biosynthetic potential of the termite gut-associated streptomycetes with biocontrol potential against P. noxium.

Molecular and phenotypic characterization of a mouse model of oculopharyngeal muscular dystrophy reveals severe muscular atrophy restricted to fast glycolytic fibres.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by ptosis, dysphagia and proximal limb weakness. Autosomal-dominant OPMD is caused by a short (GCG)(8-13) expansions within the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres. In order to gain insight into the different physiological processes affected in OPMD muscles, we have used a transgenic mouse model of OPMD (A17.1) and performed transcriptomic studies combined with a detailed phenotypic characterization of this model at three time points. The transcriptomic analysis revealed a massive gene deregulation in the A17.1 mice, among which we identified a significant deregulation of pathways associated with muscle atrophy. Using a mathematical model for progression, we have identified that one-third of the progressive genes were also associated with muscle atrophy. Functional and histological analysis of the skeletal muscle of this mouse model confirmed a severe and progressive muscular atrophy associated with a reduction in muscle strength. Moreover, muscle atrophy in the A17.1 mice was restricted to fast glycolytic fibres, containing a large number of intranuclear inclusions (INIs). The soleus muscle and, in particular, oxidative fibres were spared, even though they contained INIs albeit to a lesser degree. These results demonstrate a fibre-type specificity of muscle atrophy in this OPMD model. This study improves our understanding of the biological pathways modified in OPMD to identify potential biomarkers and new therapeutic targets.

Antisense-induced myostatin exon skipping leads to muscle hypertrophy in mice following octa-guanidine morpholino oligomer treatment.

Myostatin is a negative regulator of muscle mass, and several strategies are being developed to knockdown its expression to improve muscle-wasting conditions. Strategies using antimyostatin-blocking antibodies, inhibitory-binding partners, signal transduction blockers, and RNA interference system (RNAi)-based knockdown have yielded promising results and increased muscle mass in experimental animals. These approaches have, however, a number of disadvantages such as transient effects or adverse immune complications. We report here the use of antisense oligonucleotides (AOs) to manipulate myostatin pre-mRNA splicing and knockdown myostatin expression. Both 2'O-methyl phosphorothioate RNA (2'OMePS) and phosphorodiamidate morpholino oligomers (PMO) led to efficient exon skipping in vitro and in vivo and knockdown of myostatin at the transcript level. The substantial myostatin exon skipping observed after systemic injection of Vivo-PMO into normal mice led to a significant increase in soleus muscle mass as compared to the controls injected with normal saline suggesting that this approach could be feasible to ameliorate muscle-wasting pathologies.

Chronic systemic therapy with low-dose morpholino oligomers ameliorates the pathology and normalizes locomotor behavior in mdx mice.

The administration of antisense oligonucleotides (AOs) to skip one or more exons in mutated forms of the DMD gene and so restore the reading frame of the transcript is one of the most promising approaches to treat Duchenne muscular dystrophy (DMD). At present, preclinical studies demonstrating the efficacy and safety of long-term AO administration have not been conducted. Furthermore, it is essential to determine the minimal effective dose and frequency of administration. In this study, two different low doses (LDs) of phosphorodiamidate morpholino oligomer (PMO) designed to skip the mutated exon 23 in the mdx dystrophic mouse were administered for up to 12 months. Mice treated for 50 weeks showed a substantial dose-related amelioration of the pathology, particularly in the diaphragm. Moreover, the generalized physical activity was profoundly enhanced compared to untreated mdx mice showing that widespread, albeit partial, dystrophin expression restores the normal activity in mdx mice. Our results show for the first time that a chronic long-term administration of LDs of unmodified PMO, equivalent to doses in use in DMD boys, is safe, significantly ameliorates the muscular dystrophic phenotype and improves the activity of dystrophin-deficient mice, thus encouraging the further clinical translation of this approach in humans.

Pharmacologic Management of Mycobacterium chimaera Infections: A Primer for Clinicians.

Mycobacterium chimaera, a member of the Mycobacterium avium complex, can cause infections in individuals after open heart surgery due to contaminated heater-cooler units. The diagnosis can be challenging, as the incubation period can be quite variable, and symptoms are nonspecific. In addition to aggressive surgical management, combination pharmacologic therapy is the cornerstone of therapy, which should consist of a macrolide, a rifamycin, ethambutol, and amikacin. Multiple second-line agents may be utilized in the setting of intolerances or toxicities. In vitro susceptibility of these agents is similar to activity against other species in the Mycobacterium avium complex. Drug-drug interactions are frequently encountered, as many individuals have chronic medical comorbidities and are prescribed medications that interact with the first-line agents used to treat M. chimaera. Recognition of these drug-drug interactions and appropriate management are essential for optimizing treatment outcomes.

Testing the Biocontrol Ability of a Trichoderma-Streptomycetes Consortium against Pyrrhoderma noxium (Corner) L.W. Zhou and Y.C. Dai in Soil.

The Brown root rot pathogen Pyrrhoderma noxium (Corner) L.W. Zhou and Y.C. Dai is known to infect a large number of culturally and economically important plant species across the world. Although chemical control measures have been effective in managing this pathogen, their adverse effects on the ecosystem have limited their use. The use of biological control agents (BCAs) thus is generally accepted as an environmentally friendly way of managing various pathogens. Testing various consortia of the BCAs with different antagonistic mechanisms may even provide better disease protection than the use of a single BCA against aggressive plant pathogens such as the P. noxium. In the presented study, the wood decay experiment and the pot trial confirmed that the consortium of Trichoderma strains (#5029 and 5001) and streptomycetes (#USC-6914 and #USC-595-B) used was effective in protecting wood decay and plant disease caused by P. noxium. Among the treatments, complete elimination of the pathogen was observed when the BCAs were applied as a consortium. In addition, the BCAs used in this study promoted the plant growth. Therefore, Trichoderma and streptomycetes consortium could be used as a potential biocontrol measure to manage P. noxium infections in the field over the application of hazardous chemical control measures.

Assessing the Various Antagonistic Mechanisms of Trichoderma Strains against the Brown Root Rot Pathogen Pyrrhoderma noxium Infecting Heritage Fig Trees.

A wide range of phytopathogenic fungi exist causing various plant diseases, which can lead to devastating economic, environmental, and social impacts on a global scale. One such fungus is Pyrrhoderma noxium, causing brown root rot disease in over 200 plant species of a variety of life forms mostly in the tropical and subtropical regions of the globe. The aim of this study was to discover the antagonistic abilities of two Trichoderma strains (#5001 and #5029) found to be closely related to Trichoderma reesei against P. noxium. The mycoparasitic mechanism of these Trichoderma strains against P. noxium involved coiling around the hyphae of the pathogen and producing appressorium like structures. Furthermore, a gene expression study identified an induced expression of the biological control activity associated genes in Trichoderma strains during the interaction with the pathogen. In addition, volatile and diffusible antifungal compounds produced by the Trichoderma strains were also effective in inhibiting the growth of the pathogen. The ability to produce Indole-3-acetic acid (IAA), siderophores and the volatile compounds related to plant growth promotion were also identified as added benefits to the performance of these Trichoderma strains as biological control agents. Overall, these results show promise for the possibility of using the Trichoderma strains as potential biological control agents to protect P. noxium infected trees as well as preventing new infections.

Anthrax toxins regulate pain signaling and can deliver molecular cargoes into ANTXR2+ DRG sensory neurons.

Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.

Development of antisense-mediated myostatin knockdown for the treatment of insulin resistance.

Myostatin is a negative regulator of muscle mass and its inhibition represents a promising strategy for the treatment of muscle disorders and type 2 diabetes. However, there is currently no clinically effective myostatin inhibitor, and therefore novel methods are required. We evaluated the use of antisense phosphorodiamidate morpholino oligomers (PMO) to reduce myostatin expression in skeletal muscle and measured their effects on muscle mass and glucose uptake. C57/Bl6 mice received intramuscular or intravenous injections of anti-myostatin PMOs. Repeated intramuscular administration lead to a reduction in myostatin transcript levels (~ 20-40%), and an increase in muscle mass in chow and high-fat diet (HFD)-fed mice, but insulin-stimulated glucose uptake was reduced in PMO-treated muscles of HFD-fed mice. Five weekly intravenous administrations of 100 nmol PMO did not reduce myostatin expression, and therefore had no significant physiological effects. Unexpectedly, exon skipping levels were higher after intramuscular administration of PMO in HFD- than chow-fed mice. These results suggest that a modest PMO-induced reduction in myostatin transcript levels is sufficient to induce an increase in muscle mass, but that a greater degree of inhibition may be required to improve muscle glucose uptake.

Comments on Immunogenicity Associated with Botulinum Toxin Treatment. Toxins 2019, 11, 491.

In contrast to the prevailing arguments presented in the current review, the incidence of neutralising antibody (NAb) formation is not a significant issue for any of the present type A therapeutic botulinum neurotoxin (BoNT) products. Furthermore, clinical non-responsiveness is poorly correlated with the presence of NAbs. The overriding evidence supports the view that the rate of NAb formation is low, does not differ significantly between the different type A BoNT products and that it is not the major factor in clinical response. BoNT products are highly effective and important therapies for the treatment of a variety of neurological and non-neurological conditions.

Care of the Renal Transplant Patient.

Renal transplant has become a mainstay of treatment of patients with chronic renal failure. With improving survival outcomes, primary care physicians should be informed of the nuances that come with the ongoing care of this population and feel empowered to take part in the multidisciplinary care of these patients. This article provides an overview of the renal transplant process from initial evaluation through surgery and then focuses on long-term issues that renal transplant patients face in the primary care setting.

Duration of Symptom Relief Between Injections for AbobotulinumtoxinA (Dysport®) in Spastic Paresis and Cervical Dystonia: Comparison of Evidence From Clinical Studies.

Background: Botulinum toxin-A is a well-established treatment for adult and pediatric spastic paresis and cervical dystonia. While guidelines and approved labels indicate that treatment should not occur more frequently than every 12 weeks, studies and real-world evidence show that the timing of symptom recurrence between treatments may vary. Methods: We report retreatment criteria and response duration (retreatment intervals) from four pivotal, double-blind, placebo-controlled studies with open-label extensions involving patients treated with abobotulinumtoxinA (aboBoNTA) for upper limb (NCT01313299) or lower limb (NCT01249404) spastic paresis in adults, lower limb spastic paresis in children (NCT01249417), and cervical dystonia in adults (NCT00257660). We review results in light of recently available preclinical data. Results: In spastic paresis, 24.0-36.9% of upper limb patients treated with aboBoNTA and 20.1-32.0% of lower limb patients did not require retreatment before 16 weeks. Moreover, 72.8-93.8% of aboBoNTA-treated pediatric patients with lower limb spastic paresis did not require retreatment before 16 weeks (17.7-54.0% did not require retreatment before 28 weeks). In aboBoNTA-treated patients with cervical dystonia, 72.6-81.5% did not require retreatment before 16 weeks. Conclusion: AboBoNTA, when dosed as recommended, offers symptom relief beyond 12 weeks to many patients with spastic paresis and cervical dystonia. From recently available preclinical research, the amount of active neurotoxin administered with aboBoNTA might be a factor in explaining this long duration of response.

Frontline perspective on credentialing and privileging of ambulatory care pharmacists.

OBJECTIVES: To present the current state of, and frontline advice on, the implementation of successful credentialing and privileging processes for practicing pharmacists in the United States. METHODS: The American Society of Health-System Pharmacists (ASHP) Section Advisory Group on Compensation and Practice Sustainability surveyed ambulatory care pharmacists via ASHP Connect about the status, structure and oversight of their ambulatory care clinical practice sites with credentialed and privileged (C&P) pharmacists. KEY FINDINGS: Over 80% of survey respondents identified themselves as a C&P pharmacist, and over 90% indicated it is 'Important' or 'Very Important' for pharmacists to be C&P. Qualitative survey responses indicated the most important considerations for establishing or expanding a credentialing and privileging process for ambulatory care pharmacists were 'don't re-create the wheel', 'establish a physician champion and/or obtain leadership buy-in', 'be persistent and patient', 'develop a guidance document' and 'work within existing processes'. CONCLUSIONS: Starting a credentialing and privileging process is critical in preparation for, or response to, provider status recognition of pharmacists in the United States. When used with existing guidance documents on credentialing and privileging, 'front line' advice from practicing pharmacists can help promote expanded roles for pharmacists within healthcare systems.

Onset and Duration of AbobotulinumtoxinA for Aesthetic Use in the Upper face: A Systematic Literature Review.

OBJECTIVE: We sought to analyze the current literature regarding time to onset and duration of effect of abobotulinumtoxinA (aboBoNT-A, Dysport®/Azzalure®) for upper facial aesthetic indications. METHODS: We conducted a systematic review of literature databases (PubMed/MEDLINE, Embase, Cochrane Library, and Google Scholar) to identify English-language publications relevant to: population (patients with aesthetic indications [including glabellar lines and wrinkles]); interventions (aboBoNT-A); comparators (no restrictions); outcomes (efficacy, including onset of action and duration of effect); and settings (clinical). A manual search of review paper bibliographies was performed. Structured data extraction was used to enable interstudy analysis. RESULTS: Overall, 42 original research papers relevant to aboBoNT-A onset and/or duration were identified. All 24 studies assessing efficacy within one week post-injection demonstrated some response at the first time point assessed, and all 37 studies assessing duration showed some response after 12 weeks. Although methodologies for assessing onset and duration differed, when outcomes were refined by reported mean/median, at least 50 percent of patients responding to treatment, or significance versus placebo or baseline at a given time point, onset was most often reported within 2 to 3 days (7 studies), and as early as 24 hours (2 studies). Duration was most often reported as four months (18 studies), although four studies provided evidence that aboBoNT-A efficacy was maintained at five months and three studies at or after six months post-injection. CONCLUSION: This review indicates that aboBoNT-A has a median onset of efficacy of 2 to 3 days and a longer duration of action (3-6 months across studies) than the current labelled minimum treatment interval (12 weeks).