Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery.

Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors in vivo. Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.

Precision Medicines for Retinal Lipid Metabolism-Related Pathologies.

Oxidation of lipids and lipoproteins contributes to inflammation processes that promote the development of eye diseases. This is a consequence of metabolism dysregulation; for instance, that of the dysfunctional peroxisomal lipid metabolism. Dysfunction of lipid peroxidation is a critical factor in oxidative stress that causes ROS-induced cell damage. Targeting the lipid metabolism to treat ocular diseases is an interesting and effective approach that is now being considered. Indeed, among ocular structures, retina is a fundamental tissue that shows high metabolism. Lipids and glucose are fuel substrates for photoreceptor mitochondria; therefore, retina is rich in lipids, especially phospholipids and cholesterol. The imbalance in cholesterol homeostasis and lipid accumulation in the human Bruch's membrane are processes related to ocular diseases, such as AMD. In fact, preclinical tests are being performed in mice models with AMD, making this area a promising field. Nanotechnology, on the other hand, offers the opportunity to develop site-specific drug delivery systems to ocular tissues for the treatment of eye diseases. Specially, biodegradable nanoparticles constitute an interesting approach to treating metabolic eye-related pathologies. Among several drug delivery systems, lipid nanoparticles show attractive properties, e.g., no toxicological risk, easy scale-up and increased bioavailability of the loaded active compounds. This review analyses the mechanisms involved in ocular dyslipidemia, as well as their ocular manifestations. Moreover, active compounds as well as drug delivery systems which aim to target retinal lipid metabolism-related diseases are thoroughly discussed.

Cell penetrating peptides-functionalized Licochalcone-A-loaded PLGA nanoparticles for ocular inflammatory diseases: Evaluation of in vitro anti-proliferative effects, stabilization by freeze-drying and characterization of an in-situ forming gel.

Licochalcone-A (Lico-A) PLGA NPs functionalized with cell penetrating peptides B6 and Tet-1 are proposed for the treatment of ocular anti-inflammatory diseases. In this work, we report the in vitro biocompatibility of cell penetrating peptides-functionalized Lico-A-loaded PLGA NPs in Caco-2 cell lines revealing a non-cytotoxic profile, and their anti-inflammatory activity against RAW 264.7 cell lines. Given the risk of hydrolysis of the liquid suspensions, freeze-drying was carried out testing different cryoprotectants (e.g., disaccharides, alcohols, and oligosaccharide-derived sugar alcohol) to prevent particle aggregation and mitigate physical stress. As the purpose is the topical eye instillation of the nanoparticles, to reduce precorneal wash-out, increase residence time and thus Lico-A bioavailability, an in-situ forming gel based on poloxamer 407 containing Lico-A loaded PLGA nanoparticles functionalized with B6 and Tet-1 for ocular administration has been developed. Developed formulations remain in a flowing semi-liquid state under non-physiological conditions and transformed into a semi-solid state under ocular temperature conditions (35 °C), which is beneficial for ocular administration. The pH, viscosity, texture parameters and gelation temperature results met the requirements for ophthalmic formulations. The gel has characteristics of viscoelasticity, suitable mechanical and mucoadhesive performance which facilitate its uniform distribution over the conjunctiva surface. In conclusion, we anticipate the potential clinical significance of our developed product provided that a synergistic effect is achieved by combining the high anti-inflammatory activity of Lico-A delivered by PLGA NPs with B6 and Tet-1 for site-specific targeting in the eye, using an in-situ forming gel.

Argentine Guidelines for the Prevention and Treatment of Glucocorticoid-Induced Osteoporosis in Postmenopausal Women and Men Aged 50 Years and Older.

OBJECTIVE: The aim of this study was to provide an evidence-based framework to guide health care professionals treating patients under glucocorticoid (GC) therapy and develop guidelines for the prevention and treatment of glucocorticoid-induced osteoporosis (GIO) in postmenopausal women and men aged ≥50 years. METHODS: An expert panel on bone diseases designed a series of clinically meaningful questions following the PICO (Population, Intervention, Comparator, and Outcome) structure. Using GRADE (Grading of Recommendations Assessment, Development, and Evaluation) methodology, we made a systematic literature review, extracted and summarized the effect estimates, and graded the quality of the evidence. The expert panel voted each PICO question and made recommendations after reaching an agreement of at least 70%. RESULTS: Seventeen recommendations (9 strong and 8 conditional) and 8 general principles were developed for postmenopausal women and men aged ≥50 years under GC treatment. Bone mineral density (BMD), occurrence of fragility fractures, probability of fracture at 10 years by Fracture Risk Assessment Tool, and other screening factors for low BMD are recommended for patient evaluation and stratification according to fragility fracture risk. The treatment of patients under GC therapy should include counseling on lifestyle habits and strict control of comorbidities. The goal of GIO treatment is the nonoccurrence of new fragility fractures as well as to increase or maintain BMD in certain clinical situations. This was considered for the therapeutic approach in different clinical scenarios. CONCLUSIONS: This GIO guideline provides evidence-based guidance for health care providers treating patients.

Human Metapneumovirus Infections during COVID-19 Pandemic, Spain.

We describe an unusual outbreak of respiratory infections caused by human metapneumovirus in children during the sixth wave of COVID-19 in Spain, associated with the Omicron variant. Patients in this outbreak were older than usual and showed more hypoxia and pneumonia, longer length of stay, and greater need for intensive care.

Efficacy of nirsevimab against respiratory syncytial virus lower respiratory tract infections in preterm and term infants, and pharmacokinetic extrapolation to infants with congenital heart disease and chronic lung disease: a pooled analysis of randomised controlled trials.

BACKGROUND: In a phase 2b trial and the phase 3 MELODY trial, nirsevimab, an extended half-life, monoclonal antibody against respiratory syncytial virus (RSV), protected healthy infants born preterm or at full term against medically attended RSV lower respiratory tract infection (LRTI). In the MEDLEY phase 2-3 trial in infants at higher risk for severe RSV infection, nirsevimab showed a similar safety profile to that of palivizumab. The aim of the current analysis was to assess the efficacy of nirsevimab using a weight-banded dosing regimen in infants born between 29 weeks gestational age and full term. METHODS: Infants enrolled in the phase 2b and MELODY trials were randomised (2:1) to receive a single intramuscular injection of nirsevimab (infants weighing <5 kg received 50 mg; those weighing ≥5 kg received 100 mg) or placebo before the RSV season. Infants in MEDLEY were randomised (2:1) to receive one dose of nirsevimab (infants weighing <5 kg received 50 mg; those weighing ≥5 kg received 100 mg) followed by four monthly placebo doses, or five once-a-month intramuscular doses of palivizumab. We report a prespecified pooled efficacy analysis assessing the weight-banded dosing regimen proposed on the basis of the phase 2b and MELODY trials, in addition to extrapolated efficacy in infants with chronic lung disease, congenital heart disease, or extreme preterm birth (<29 weeks' gestational age) based on pharmacokinetic data from the phase 2-3 MEDLEY safety trial. For the pooled efficacy analysis, the primary endpoint was incidence of medically attended RSV LRTI through 150 days post-dose. The secondary efficacy endpoint was number of admissions to hospital for medically attended RSV LRTI. The incidence of very severe RSV LRTI was an exploratory endpoint, defined as cases of hospital admission for medically attended RSV LRTI that required supplemental oxygen or intravenous fluids. We also did a prespecified exploratory analysis of medically attended LRTI of any cause (in the investigator's judgement) and hospital admission for respiratory illness of any cause (defined as any upper respiratory tract infection or LRTI leading to hospital admission). Post hoc exploratory analyses of outpatient visits and antibiotic use were also done. Nirsevimab serum concentrations in MEDLEY were assessed using population pharmacokinetic methods and the pooled data from the phase 2b and MELODY trials. An exposure target was defined on the basis of an exposure-response analysis. To successfully demonstrate extrapolation, more than 80% of infants in MEDLEY had to achieve serum nirsevimab exposures at or above the predicted efficacious target. FINDINGS: Overall, 2350 infants (1564 in the nirsevimab group and 786 in the placebo group) in the phase 2b and MELODY trials were included in the pooled analysis. Nirsevimab showed efficacy versus placebo with respect to the primary endpoint of medically attended RSV LRTI (19 [1%] nirsevimab recipients vs 51 [6%] placebo recipients; relative risk reduction [RRR] 79·5% [95% CI 65·9-87·7]). Consistent efficacy was shown for additional endpoints of RSV LRTI hospital admission (nine [1%] nirsevimab recipients vs 21 [3%] placebo recipients; 77·3% [50·3-89·7]) and very severe RSV (five [<1%] vs 18 [2%]; 86·0% [62·5-94·8]). Nirsevimab recipients had fewer hospital admissions for any-cause respiratory illness (RRR 43·8% [18·8-61·1]), any-cause medically attended LRTI (35·4% [21·5-46·9]), LRTI outpatient visits (41·9% [25·7-54·6]), and antibiotic prescriptions (23·6% [3·8-39·3]). Among infants with chronic lung disease, congenital heart disease, or extreme preterm birth in MEDLEY, nirsevimab serum exposures were similar to those found in the pooled data; exposures were above the target in more than 80% of the overall MEDLEY trial population (94%), including infants with chronic lung disease (94%) or congenital heart disease (80%) and those born extremely preterm (94%). INTERPRETATION: A single dose of nirsevimab protected healthy infants born at term or preterm from medically attended RSV LRTI, associated hospital admission, and severe RSV. Pharmacokinetic data support efficacy extrapolation to infants with chronic lung disease, congenital heart disease, or extreme prematurity. Together, these data suggest that nirsevimab has the potential to change the landscape of infant RSV disease by reducing a major cause of infant morbidity and the consequent burden on caregivers, clinicians, and health-care providers. FUNDING: AstraZeneca and Sanofi.

Therapeutic Approaches for Age-Related Macular Degeneration.

Damage to the retinal pigment epithelium, Bruch's membrane and/or tissues underlying macula is known to increase the risk of age-related macular degeneration (AMD). AMD is commonly categorized in two distinct types, namely, the nonexudative (dry form) and the exudative (wet form). Currently, there is no ideal treatment available for AMD. Recommended standard treatments are based on the use of vascular endothelial growth factor (VEGF), with the disadvantage of requiring repeated intravitreal injections which hinder patient's compliance to the therapy. In recent years, several synthetic and natural active compounds have been proposed as innovative therapeutic strategies against this disease. There is a growing interest in the development of formulations based on nanotechnology because of its important role in the management of posterior eye segment disorders, without the use of intravitreal injections, and furthermore, with the potential to prolong drug release and thus reduce adverse effects. In the same way, 3D bioprinting constitutes an alternative to regeneration therapies for the human retina to restore its functions. The application of 3D bioprinting may change the current and future perspectives of the treatment of patients with AMD, especially those who do not respond to conventional treatment. To monitor the progress of AMD treatment and disease, retinal images are used. In this work, we revised the recent challenges encountered in the treatment of different forms of AMD, innovative nanoformulations, 3D bioprinting, and techniques to monitor the progress.

Functional Alterations Involved in Increased Bleeding in Hereditary Hemorrhagic Telangiectasia Mouse Models.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal-dominant genetic disorder involving defects in two predominant genes known as endoglin (ENG; HHT-1) and activin receptor-like kinase 1 (ACVRL1/ALK1; HHT-2). It is characterized by mucocutaneous telangiectases that, due to their fragility, frequently break causing recurrent epistaxis and gastrointestinal bleeding. Because of the severity of hemorrhages, the study of the hemostasis involved in these vascular ruptures is critical to find therapies for this disease. Our results demonstrate that HHT patients with high bleeding, as determined by a high Epistaxis Severity Score (ESS), do not have prolonged clotting times or alterations in clotting factors. Considering that coagulation is only one of the processes involved in hemostasis, the main objective of this study was to investigate the overall mechanisms of hemostasis in HHT-1 (Eng +/-) and HHT-2 (Alk1 +/-) mouse models, which do not show HHT vascular phenotypes in the meaning of spontaneous bleeding. In Eng +/- mice, the results of in vivo and in vitro assays suggest deficient platelet-endothelium interactions that impair a robust and stable thrombus formation. Consequently, the thrombus could be torn off and dragged by the mechanical force exerted by the bloodstream, leading to the reappearance of hemorrhages. In Alk1 +/- mice, an overactivation of the fibrinolysis system was observed. These results support the idea that endoglin and Alk1 haploinsufficiency leads to a common phenotype of impaired hemostasis, but through different mechanisms. This contribution opens new therapeutic approaches to HHT patients' epistaxis.

Customized cationic nanoemulsions loading triamcinolone acetonide for corneal neovascularization secondary to inflammatory processes.

Customized cationic oil-in-water nanoemulsions (NEs) have been produced to improve the bioavailability of poorly water-soluble drugs, such as triamcinolone acetonide (TA). TA is a synthetic glucocorticoid with anti-inflammatory and antiangiogenic therapeutic properties and it is widely used as an effective treatment in ocular disorders. In this work, TA-NEs were characterized using two different custom-made cationic surfactants, showing a high positive surface charge favouring corneal penetration and a particle size below 300 nm. Both TA-NE formulations demonstrated to be stable at 4 °C during the first months of storage. Furthermore, TA-NEs were able to produce antiangiogenic effects in chicken membranes. The TA-NEs safety profile was evaluated using in vitro and in vivo ocular tolerance tests. Out of the two formulations, the one showing no irritant effects was screened in vivo demonstrating capacity to ameliorate ocular inflammation in New Zealand rabbits significantly, specially to reduce the risk of ocular inflammation processes, with antiangiogenic activity, and can therefore be exploited as a suitable formulation to avoid inflammatory reactions upon ocular surgical procedures, such as cataracts.

Permeability, anti-inflammatory and anti-VEGF profiles of steroidal-loaded cationic nanoemulsions in retinal pigment epithelial cells under oxidative stress.

Age-related macular degeneration (AMD) is defined as a degenerative, progressive and multifactorial disorder that affects the macula with a complex etiology. The retinal pigment epithelium is a monolayer of cells that has the function to separate the surface of the choroid from the neural retina that is involved in the signal transduction leading to vision. The blood-aqueous barrier and the blood retinal barrier limit the permeation of drugs into the retina and thereby reducing their efficacy. Triamcinolone acetonide (TA) is widely used as anti-inflammatory and immunomodulatory drug that promotes the inhibition of the inflammatory processes. The factors that stimulate or inhibit angiogenesis in AMD create a local balance that is responsible for the growth of sub-retinal neovascularization. In AMD, the main angiogenic stimulus is the vascular endothelial growth factor (VEGF). In this work, nanoemulsions with cationic surfactants (mono- and dicationic DABCO and quinuclidine) were produced to deliver TA, and were found to reduce the production of tumor necrosis factor alpha (TNF-α), which stimulates the choroidal neovascularization development by upregulating the VEGF production, and consequently decreased the VEGF levels. Our results support the potential use of mono- and dicationic DABCO and quinuclidine-based cationic nanoemulsions for the delivery of TA in the treatment of AMD.

Physicochemical and biopharmaceutical aspects influencing skin permeation and role of SLN and NLC for skin drug delivery.

The skin is a complex and multifunctional organ, in which the static versus dynamic balance is responsible for its constant adaptation to variations in the external environment that is continuously exposed. One of the most important functions of the skin is its ability to act as a protective barrier, against the entry of foreign substances and against the excessive loss of endogenous material. Human skin imposes physical, chemical and biological limitations on all types of permeating agents that can cross the epithelial barrier. For a molecule to be passively permeated through the skin, it must have properties, such as dimensions, molecular weight, pKa and hydrophilic-lipophilic gradient, appropriate to the anatomy and physiology of the skin. These requirements have limited the number of commercially available products for dermal and transdermal administration of drugs. To understand the mechanisms involved in the drug permeation process through the skin, the approach should be multidisciplinary in order to overcome biological and pharmacotechnical barriers. The study of the mechanisms involved in the permeation process, and the ways to control it, can make this route of drug administration cease to be a constant promise and become a reality. In this work, we address the physicochemical and biopharmaceutical aspects encountered in the pathway of drugs through the skin, and the potential added value of using solid lipid nanoparticles (SLN) and nanostructured lipid vectors (NLC) to drug permeation/penetration through this route. The technology and architecture for obtaining lipid nanoparticles are described in detail, namely the composition, production methods and the ability to release pharmacologically active substances, as well as the application of these systems in the vectorization of various pharmacologically active substances for dermal and transdermal applications. The characteristics of these systems in terms of dermal application are addressed, such as biocompatibility, occlusion, hydration, emollience and the penetration of pharmacologically active substances. The advantages of using these systems over conventional formulations are described and explored from a pharmaceutical point of view.

Development and Characterization of Nanoemulsions for Ophthalmic Applications: Role of Cationic Surfactants.

The eye is a very complex organ comprising several physiological and physical barriers that compromise drug absorption into deeper layers. Nanoemulsions are promising delivery systems to be used in ocular drug delivery due to their innumerous advantages, such as high retention time onto the site of application and the modified release profile of loaded drugs, thereby contributing to increasing the bioavailability of drugs for the treatment of eye diseases, in particular those affecting the posterior segment. In this review, we address the main factors that govern the development of a suitable nanoemulsion formulation for eye administration to increase the patient's compliance to the treatment. Appropriate lipid composition and type of surfactants (with a special emphasis on cationic compounds) are discussed, together with manufacturing techniques and characterization methods that are instrumental for the development of appropriate ophthalmic nanoemulsions.

DABCO-Customized Nanoemulsions: Characterization, Cell Viability and Genotoxicity in Retinal Pigmented Epithelium and Microglia Cells.

Quaternary derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO) and of quinuclidine surfactants were used to develop oil-in-water nanoemulsions with the purpose of selecting the best long-term stable nanoemulsion for the ocular administration of triamcinolone acetonide (TA). The combination of the best physicochemical properties (i.e., mean droplet size, polydispersity index, zeta potential, osmolality, viscoelastic properties, surface tension) was considered, together with the cell viability assays in ARPE-19 and HMC3 cell lines. Surfactants with cationic properties have been used to tailor the nanoemulsions' surface for site-specific delivery of drugs to the ocular structure for the delivery of TA. They are tailored for the eye because they have cationic properties that interact with the anionic surface of the eye.

Mono- and Dicationic DABCO/Quinuclidine Composed Nanomaterials for the Loading of Steroidal Drug: 32 Factorial Design and Physicochemical Characterization.

Oil-in-water nanoemulsions (NEs) are considered a suitable nanotechnological approach to improve the eye-related bioavailability of lipophilic drugs. The potential of cationic NEs is prominent due to the electrostatic interaction that occurs between the positively charged droplets with the negatively charged mucins present in the tear film. This interaction offers prolonged NEs residence at the ocular surface, increasing the drug absorption. Triamcinolone acetonide (TA) is one of the first pharmacologic strategies applied as an intravitreal injection in the treatment of age-related macular degeneration (AMD). Newly synthesized quaternary derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO) and quinuclidine surfactants have been screened with the purpose to select the best compound to formulate long-term stable NEs that combine the best physicochemical properties for the loading of TA intended for ocular administration.

Psoriasis: From Pathogenesis to Pharmacological and Nano-Technological-Based Therapeutics.

Research in the pathogenesis of inflammatory skin diseases, such as skin dermatitis and psoriasis, has experienced some relevant breakthroughs in recent years. The understanding of age-related factors, gender, and genetic predisposition of these multifactorial diseases has been instrumental for the development of new pharmacological and technological treatment approaches. In this review, we discuss the molecular mechanisms behind the pathological features of psoriasis, also addressing the currently available treatments and novel therapies that are under clinical trials. Innovative therapies developed over the last 10 years have been researched. In this area, advantages of nanotechnological approaches to provide an effective drug concentration in the disease site are highlighted, together with microneedles as innovative candidates for drug delivery systems in psoriasis and other inflammatory chronic skin diseases.

Viruses that affect Argentinian honey bees (Apis mellifera).

Beekeeping is a widespread activity in Argentina, mainly producing honey that has gained both national and international recognition. There are more than 3,000,000 hives in the country, mainly concentrated in Buenos Aires Province (approximately 1,000,000 hives). In recent decades, worrying rates of hive loss have been observed in many countries around the world. In Latin America, the estimated loss of hives is between 13% (Peru and Ecuador) and 53% (Chile). Argentina had annual losses of 34% for the period of October 1, 2016 to October 1, 2017. The causes of these losses are not clear but probably involve multiple stressors that can act simultaneously. One of the main causes of loss of bee colonies worldwide is infestation by the ectoparasitic mite Varroa destructor in combination with viral infections. To date, 10 viruses have been detected that affect honey bees (Apis mellifera) in Argentina. Of these, deformed wing virus, sacbrood virus, acute bee paralysis virus, chronic bee paralysis virus, and Israeli acute bee paralysis can be transmitted by mites. Deformed wing virus and the AIK complex are the viruses most often associated with loss of hives worldwide. Considering that bee viruses have been detected in Argentina in several hymenopteran and non-hymenopteran insects, these hosts could act as important natural reservoirs for viruses and play an important role in their dispersal in the environment. Further studies to investigate the different mechanisms by which viruses spread in the environment will enable us to develop various strategies for the control of infected colonies and the spread of viruses in the habitat where they are found.

Nano-engineering of ketorolac tromethamine platforms for ocular treatment of inflammatory disorders.

Aim: The development and optimization of Ketorolac tromethamine-loaded polylactic-co-glycolic acid nanoparticles (KT-NPs) for the treatment of inflammatory processes of the eye. Materials & methods: KT-NPs were developed by factorial design and characterized by assessing their physicochemical properties. Biopharmaceutical behavior studies, ocular tolerance, anti-inflammatory efficacy and bioavailability tests were performed on pigs. Results: Optimized KT-NPs of 112 nm, narrow distribution with encapsulation efficiency near 100% were obtained. KT release followed the Weibull model and there was significantly greater retention in the cornea and sclera than in the commercial reference. KT-NPs showed no signs of ocular irritancy and similar anti-inflammatory efficacy to the commercial reference. Conclusion: KT-NPs were a suitable alternative for the treatment of inflammatory disorders of the anterior and posterior segments of the eye as an alternative to conventional topical formulations.