Respiratory syncytial virus incidence and typing in the last six seasons in the north of Spain (Asturias). Genetic characterization during the SARS-CoV-2 pandemic.

Respiratory syncytial virus is associated with lower respiratory tract infections. As several types and genotypes can circulate at the same time, genomic characterization is important for timely epidemiological control and treatment measures. In the last 6 seasons (2017-2023), 191 236 nasopharyngeal swabs were processed for respiratory viruses to determine the etiology of acute respiratory infections, describe the incidence and distribution of RSV types and enrich the data of epidemiological molecular studies on RSV in Spain. The incidence of RSV reached 7% in the pre-pandemic season. RSV was most frequent in children under 5 years of age (12.6%), but was also significant in those over 70 years of age (5.63%). The measures taken to control SARS-CoV-2 infection were useful for RSV control and the incidence decreased to 1.8%, but caused a change in the types. Pre-pandemic, the majority circulating types were RSV-B/RSV-B/RSV-A and in the pandemic it was RSV-B/RSV-B. In the last season, RSV-B and RSV-A were detected in the same proportion. Genetic characterization showed three new clades. This has been taken into account to understand the epidemiology as well as the development of therapeutic and preventive strategies.

Effect of Melatonin on Herpesvirus Type 1 Replication.

Acute HSV-1 infection is associated with mild symptoms, such as fever and lesions of the mouth, face and skin. This phase is followed by a latency period before reactivation, which is associated with symptoms ranging from ulcers to encephalitis. Despite available anti-HSV-1 drugs, the development of new antiviral agents is sought due to the presence of resistant viruses. Melatonin, a molecule secreted by the pineal gland, has been shown to be an antioxidant, inducer of antioxidant enzymes, and regulator of various biological processes. Clinical trials have explored its therapeutic utility in conditions including infections. This study focuses on melatonin's role in HSV-1 replication and the underlying mechanisms. Melatonin was found to decrease the synthesis of HSV-1 proteins in infected Vero cells measured by immunofluorescence, indicating an inhibition of HSV-1 replication. Additionally, it regulates the activities of antioxidant enzymes and affects proteasome activity. Melatonin activates the unfolded protein response (UPR) and autophagy and suppresses apoptosis in HSV-1-infected cells. In summary, melatonin demonstrates an inhibitory role in HSV-1 replication by modulating various cellular responses, suggesting its potential utility in the treatment of viral infections.

Melatonin Alleviates Liver Mitochondrial Dysfunction in Leptin-Deficient Mice.

Despite efforts to elucidate the cellular adaptations induced by obesity, cellular bioenergetics is currently considered a crucial target. New strategies to delay the onset of the hazardous adaptations induced by obesity are needed. Therefore, we evaluated the effects of 4 weeks of melatonin treatment on mitochondrial function and lipid metabolism in the livers of leptin-deficient mice. Our results revealed that the absence of leptin increased lipid storage in the liver and induced significant mitochondrial alterations, which were ultimately responsible for defective ATP production and reactive oxygen species overproduction. Moreover, leptin deficiency promoted mitochondrial biogenesis, fusion, and outer membrane permeabilization. Melatonin treatment reduced the bioenergetic deficit found in ob/ob mice, alleviating some mitochondrial alterations in the electron transport chain machinery, biogenesis, dynamics, respiration, ATP production, and mitochondrial outer membrane permeabilization. Given the role of melatonin in maintaining mitochondrial homeostasis, it could be used as a therapeutic agent against adipogenic steatosis.

NLRP3 Contributes to Sarcopenia Associated to Dependency Recapitulating Inflammatory-Associated Muscle Degeneration.

Sarcopenia, a complex and debilitating condition characterized by progressive deterioration of skeletal muscle, is the primary cause of age-associated disability and significantly impacts healthspan in elderly patients. Despite its prevalence among the aging population, the underlying molecular mechanisms are still under investigation. The NLRP3 inflammasome is crucial in the innate immune response and has a significant impact on diseases related to inflammation and aging. Here, we investigated the expression of the NLRP3 inflammasome pathway and pro-inflammatory cytokines in skeletal muscle and peripheral blood of dependent and independent patients who underwent hip surgery. Patients were categorized into independent and dependent individuals based on their Barthel Index. The expression of NLRP3 inflammasome components was significantly upregulated in sarcopenic muscle from dependent patients, accompanied by higher levels of Caspase-1, IL-1ß and IL-6. Among older dependent individuals with sarcopenia, there was a significant increase in the MYH3/MYH2 ratio, indicating a transcriptional shift in expression from mature to developmental myosin isoforms. Creatine kinase levels and senescence markers were also higher in dependent patients, altogether resembling dystrophic diseases and indicating muscle degeneration. In summary, we present evidence for the involvement of the NLRP3/ASC/NEK7/Caspase-1 inflammasome pathway with activation of pro-inflammatory SASP in the outcome of sarcopenia in the elderly.

Benefits of the Neurogenic Potential of Melatonin for Treating Neurological and Neuropsychiatric Disorders.

There are several neurological diseases under which processes related to adult brain neurogenesis, such cell proliferation, neural differentiation and neuronal maturation, are affected. Melatonin can exert a relevant benefit for treating neurological disorders, given its well-known antioxidant and anti-inflammatory properties as well as its pro-survival effects. In addition, melatonin is able to modulate cell proliferation and neural differentiation processes in neural stem/progenitor cells while improving neuronal maturation of neural precursor cells and newly created postmitotic neurons. Thus, melatonin shows relevant pro-neurogenic properties that may have benefits for neurological conditions associated with impairments in adult brain neurogenesis. For instance, the anti-aging properties of melatonin seem to be linked to its neurogenic properties. Modulation of neurogenesis by melatonin is beneficial under conditions of stress, anxiety and depression as well as for the ischemic brain or after a brain stroke. Pro-neurogenic actions of melatonin may also be beneficial for treating dementias, after a traumatic brain injury, and under conditions of epilepsy, schizophrenia and amyotrophic lateral sclerosis. Melatonin may represent a pro-neurogenic treatment effective for retarding the progression of neuropathology associated with Down syndrome. Finally, more studies are necessary to elucidate the benefits of melatonin treatments under brain disorders related to impairments in glucose and insulin homeostasis.

Enhanced Detection of Viral RNA Species Using FokI-Assisted Digestion of DNA Duplexes and DNA/RNA Hybrids.

The accurate detection of nucleic acids from certain biological pathogens is critical for the diagnosis of human diseases. However, amplified detection of RNA molecules from a complex sample by direct detection of RNA/DNA hybrids remains a challenge. Here, we show that type IIS endonuclease FokI is able to digest DNA duplexes and DNA/RNA hybrids when assisted by a dumbbell-like fluorescent sensing oligonucleotide. As proof of concept, we designed a battery of sensing oligonucleotides against specific regions of the SARS-CoV-2 genome and interrogated the role of FokI relaxation as a potential nicking enzyme for fluorescence signal amplification. FokI-assisted digestion of SARS-CoV-2 probes increases the detection signal of ssDNA and RNA molecules and decreases the limit of detection more than 3.5-fold as compared to conventional molecular beacon approaches. This cleavage reaction is highly specific to its target molecules, and no detection of other highly related B-coronaviruses was observed in the presence of complex RNA mixtures. In addition, the FokI-assisted reaction has a high multiplexing potential, as the combined detection of different viral RNAs, including different SARS-CoV-2 variants, was achieved in the presence of multiple combinations of fluorophores and sensing oligonucleotides. When combined with isothermal rolling circle amplification technologies, FokI-assisted digestion reduced the detection time of SARS-CoV-2 in COVID-19-positive human samples with adequate sensitivity and specificity compared to conventional reverse transcription polymerase chain reaction approaches, highlighting the potential of FokI-assisted signal amplification as a valuable sensing mechanism for the detection of human pathogens.

FURIN gene variants (rs6224/rs4702) as potential markers of death and cardiovascular traits in severe COVID-19.

Furin is a protease that plays a key role in the infection cycle of SARS-CoV-2 by cleaving the viral proteins during the virus particle assembly. In addition, Furin regulates several physiological processes related to cardio-metabolic traits. DNA variants in the FURIN gene are candidates to regulate the risk of developing these traits as well as the susceptibility to severe COVID-19. We genotyped two functional FURIN variants (rs6224/rs4702) in 428 COVID-19 patients in the intensive care unit. The association with death (N = 106) and hypertension, diabetes, and hyperlipidaemia was statistically evaluated. The risk of death was associated with age, hypertension, and hypercholesterolemia. The two FURIN alleles linked to higher expression (rs6224 T and rs4702 A) were significantly increased in the death cases (odds ratio= 1.40 and 1.43). Homozygosis for the two high expression genotypes (rs6224 TT and rs4702 AA) and for the T-A haplotype was associated with an increased risk of hypercholesterolemia. In the multiple logistic regression both, hypercholesterolemia and the TT + AA genotype were significantly associated with death. In conclusion, besides its association with hypercholesterolemia, FURIN variants might be independent risk factors for the risk of death among COVID-19 patients.

Chronic Treatment with Melatonin Improves Hippocampal Neurogenesis in the Aged Brain and Under Neurodegeneration.

Adult hippocampal neurogenesis is altered during aging and under different neuropsychiatric and neurodegenerative diseases. Melatonin shows neurogenic and neuroprotective properties during aging and neuropathological conditions. In this study, we evaluated the effects of chronic treatment with melatonin on different markers of neurodegeneration and hippocampal neurogenesis using immunohistochemistry in the aged and neurodegenerative brains of SAMP8 mice, which is an animal model of accelerated senescence that mimics aging-related Alzheimer's pathology. Neurodegenerative processes observed in the brains of aged SAMP8 mice at 10 months of age include the presence of damaged neurons, disorganization in the layers of the brain cortex, alterations in neural processes and the length of neuronal prolongations and ß-amyloid accumulation in the cortex and hippocampus. This neurodegeneration may be associated with neurogenic responses in the hippocampal dentate gyrus of these mice, since we observed a neurogenic niche of neural stem and progenitor/precursors cells in the hippocampus of SAMP8 mice. However, hippocampal neurogenesis seems to be compromised due to alterations in the cell survival, migration and/or neuronal maturation of neural precursor cells due to the neurodegeneration levels in these mice. Chronic treatment with melatonin for 9 months decreased these neurodegenerative processes and the neurodegeneration-induced neurogenic response. Noticeably, melatonin also induced recovery in the functionality of adult hippocampal neurogenesis in aged SAMP8 mice.

Neuroinvasion of influenza A/H3N2: a fatal case in an immunocompetent adult.

Acute necrotizing encephalopathy (ANE) is a severe neurologic complication caused by influenza virus that has been infrequently reported in adult population. The diagnosis is made on epidemiological, clinical, and neuroimaging suspicion, but is rarely confirmed by microbiological findings in samples from the central nervous system (CNS), thus making it difficult to define the mechanism of pathogenesis of influenza-associated encephalitis/encephalopathies (IAE). We report a microbiologically documented case of ANE caused by influenza A/H3N2, in a previously healthy adult patient infected during a flu epidemic in Asturias (Spain). Direct viral invasion of the CNS was demonstrated with the isolation of the virus in a brain biopsy.

Therapeutic potential of melatonin related to its role as an autophagy regulator: A review.

There are several pathologies, syndromes, and physiological processes in which autophagy is involved. This process of self-digestion that cells trigger as a survival mechanism is complex and tightly regulated, according to the homeostatic conditions of the organ. However, in all cases, its relationship with oxidative stress alterations is evident, following a pathway that suggests endoplasmic reticulum stress and/or mitochondrial changes. There is accumulating evidence of the beneficial role that melatonin has in the regulation and restoration of damaged autophagic processes. In this review, we focus on major physiological changes such as aging and essential pathologies including cancer, neurodegenerative diseases, viral infections and obesity, and document the essential role of melatonin in the regulation of autophagy in each of these different situations.

Human respiratory syncytial virus load normalized by cell quantification as predictor of acute respiratory tract infection.

Human respiratory syncytial virus (HRSV) is a common cause of respiratory infections. The main objective is to analyze the prediction ability of viral load of HRSV normalized by cell number in respiratory symptoms. A prospective, descriptive, and analytical study was performed. From 7307 respiratory samples processed between December 2014 to April 2016, 1019 HRSV-positive samples, were included in this study. Low respiratory tract infection was present in 729 patients (71.54%). Normalized HRSV load was calculated by quantification of HRSV genome and human ß-globin gene and expressed as log10 copies/1000 cells. HRSV mean loads were 4.09 ± 2.08 and 4.82 ± 2.09 log10 copies/1000 cells in the 549 pharyngeal and 470 nasopharyngeal samples, respectively (P < 0.001). The viral mean load was 4.81 ± 1.98 log10 copies/1000 cells for patients under the age of 4-year-old (P < 0.001). The viral mean loads were 4.51 ± 2.04 cells in patients with low respiratory tract infection and 4.22 ± 2.28 log10 copies/1000 cells with upper respiratory tract infection or febrile syndrome (P < 0.05). A possible cut off value to predict LRTI evolution was tentatively established. Normalization of viral load by cell number in the samples is essential to ensure an optimal virological molecular diagnosis avoiding that the quality of samples affects the results. A high viral load can be a useful marker to predict disease progression.

Detection and quantification of Merkel cell polyomavirus. Analysis of Merkel cell carcinoma cases from 1977 to 2015.

This study investigates the presence of Merkel cell polyomavirus (MCPyV) in skin lesions of patients with Merkel cell carcinoma (MCC). MCPyV was quantified using quantitative Real-Time-PCR (qRT-PCR) in 34 paraffinized MCC samples (resected/biopsied) originally taken between 1977 and 2015, and six non-MCC samples. In 31 (91.2%) MCC-individuals, MCPyV was detected. No virus was observed in any non-MCC tumor. Average age at diagnosis was 78.2 ± 9.35 (55-97) years for women (n = 19) and 69.5 ± 14.7 (45-91) for men (n = 15) (P = 0.04). MCC tumor location, known in 25 cases, was: 11 (44%) in the head region, 6 (24%) in upper limbs, 4 (16%) in lower limbs, and 4 (16%) in the trunk. All but one patient had received some sort of treatment: 15 (45.45%) underwent both radio and chemotherapy, 13 (39.39%) only surgery, 2 (6.06%) surgery, plus radio and chemotherapy, 2 (6.06%) surgery and chemotherapy, and 1 (3.03%) only radiotherapy. Follow up data were available for 21/34 patients: recurrence was recorded for 4 (19.04%), and metastasis for 13 (61.9%). Recorded data showed that 10 men and 5 women (total 44.1%) died during follow up, 7 (46.7%) of them within 2 years of diagnosis. Viral load was 5.8 ± 1.4 log copies/105 cells (3.1-8.6), independent of any variable. MCPyV was very frequent in MCC. It was principally associated with head and limb tumors, it more commonly affected men, who in this study were, on average, younger than women, and had high rates of recurrence and mortality. The amplification techniques described here are easily applied and suitable for detecting the presence of MCPyV virus in MCC.

Melatonin reduces endoplasmic reticulum stress and autophagy in liver of leptin-deficient mice.

The sedentary lifestyle of modern society along with the high intake of energetic food has made obesity a current worldwide health problem. Despite great efforts to study the obesity and its related diseases, the mechanisms underlying the development of these diseases are not well understood. Therefore, identifying novel strategies to slow the progression of these diseases is urgently needed. Experimental observations indicate that melatonin has an important role in energy metabolism and cell signalling; thus, the use of this molecule may counteract the pathologies of obesity. In this study, wild-type and obese (ob/ob) mice received daily intraperitoneal injections of melatonin at a dose of 500 µg/kg body weight for 4 weeks, and the livers of these mice were used to evaluate the oxidative stress status, proteolytic (autophagy and proteasome) activity, unfolded protein response, inflammation and insulin signalling. Our results show, for the first time, that melatonin could significantly reduce endoplasmic reticulum stress in leptin-deficient obese animals and ameliorate several symptoms that characterize this disease. Our study supports the potential of melatonin as a therapeutic treatment for the most common type of obesity and its liver-associated disorders.

Melatonin as a Potential Agent in the Treatment of Sarcopenia.

Considering the increased speed at which the world population is aging, sarcopenia could become an epidemic in this century. This condition currently has no means of prevention or treatment. Melatonin is a highly effective and ubiquitously acting antioxidant and free radical scavenger that is normally produced in all organisms. This molecule has been implicated in a huge number of biological processes, from anticonvulsant properties in children to protective effects on the lung in chronic obstructive pulmonary disease. In this review, we summarize the data which suggest that melatonin may be beneficial in attenuating, reducing or preventing each of the symptoms that characterize sarcopenia. The findings are not limited to sarcopenia, but also apply to osteoporosis-related sarcopenia and to age-related neuromuscular junction dysfunction. Since melatonin has a high safety profile and is drastically reduced in advanced age, its potential utility in the treatment of sarcopenic patients and related dysfunctions should be considered.

Melatonin: an ancient molecule that makes oxygen metabolically tolerable.

Melatonin is remarkably functionally diverse with actions as a free radical scavenger and antioxidant, circadian rhythm regulator, anti-inflammatory and immunoregulating molecule, and as an oncostatic agent. We hypothesize that the initial and primary function of melatonin in photosynthetic cyanobacteria, which appeared on Earth 3.5-3.2 billion years ago, was as an antioxidant. The evolution of melatonin as an antioxidant by this organism was necessary as photosynthesis is associated with the generation of toxic-free radicals. The other secondary functions of melatonin came about much later in evolution. We also surmise that mitochondria and chloroplasts may be primary sites of melatonin synthesis in all eukaryotic cells that possess these organelles. This prediction is made on the basis that mitochondria and chloroplasts of eukaryotes developed from purple nonsulfur bacteria (which also produce melatonin) and cyanobacteria when they were engulfed by early eukaryotes. Thus, we speculate that the melatonin-synthesizing actions of the engulfed bacteria were retained when these organelles became mitochondria and chloroplasts, respectively. That mitochondria are likely sites of melatonin formation is supported by the observation that this organelle contains high levels of melatonin that are not impacted by blood melatonin concentrations. Melatonin has a remarkable array of means by which it thwarts oxidative damage. It, as well as its metabolites, is differentially effective in scavenging a variety of reactive oxygen and reactive nitrogen species. Moreover, melatonin and its metabolites modulate a large number of antioxidative and pro-oxidative enzymes, leading to a reduction in oxidative damage. The actions of melatonin on radical metabolizing/producing enzymes may be mediated by the Keap1-Nrf2-ARE pathway. Beyond its direct free radical scavenging and indirect antioxidant effects, melatonin has a variety of physiological and metabolic advantages that may enhance its ability to limit oxidative stress.

Role of parC Mutations at Position 84 on High-Level Delafloxacin Resistance in Methicillin-Resistant Staphylococcus aureus.

High-level delafloxacin-resistant (H-L DLX-R) Staphylococcus aureus isolates (minimum inhibitory concentration ≥1 mg/L) associated with mutations affecting position 84 of ParC have emerged. We aimed to elucidate the role of these mutations as a mechanism of H-L DLX resistance in methicillin-resistant S. aureus (MRSA) isolates recovered from blood cultures. Susceptibility to DLX was determined in 75 MRSA isolates by E-test, and an rt-PCR was developed to detect mutations affecting position 84 of ParC to screen a further 185 MRSA isolates. The genomes of 48 isolates, including all DLX-R isolates or with alterations at position 84, and also a subset of DLX-susceptible isolates were analyzed. Among the 75 isolates studied, 77.34% were DLX-susceptible and only 4 H-L DLX-R isolates were found. Seven (3.8%) isolates with alterations at position 84 of ParC were detected by rt-PCR. Genomic analysis showed that 89.9% (8/9) of isolates with the substitution E84K/G in ParC, together with other mutations in gyrA and parC, were H-L DLX-R. However, the E84K substitution in ParC alone or with other alterations was found in two isolates without H-L DLX-R. Alterations at position 84 of ParC are rare but play a key role in H-L DLX resistance in MRSA but only when other alterations in GyrA are present.

Prognostic impact of muscle ultrasound-guided diagnosis of sarcopenia in older adults with severe aortic stenosis.

BACKGROUND: Muscle ultrasound is increasingly popular thanks to its advantages over other techniques. However, its usefulness in the diagnosis of sarcopenia in older adults with aortic stenosis (AS) has not been studied to date. OBJECTIVES: to analyze the prevalence of sarcopenia using muscle ultrasound and its impact on the health outcomes in older patients with AS. METHODS: The single-center FRESAS (FRailty-Evaluation-in-Severe-Aortic-Stenosis) registry was used to study patients over 75 years with severe AS susceptible to valve replacement. Sarcopenia was suspected in those individuals with diminished grip strength, and the diagnosis was confirmed in the presence of reduced ultrasound quadriceps muscle thickness, following the recommendations of the EWGSOP2 (European-Working-Group-on-Sarcopenia-in-Older-People). The primary composite endpoint was urgent hospital admission and mortality of cardiac cause 6 months after the diagnosis. RESULTS: Of the 150 patients studied, 55.3% were females, and only 17.3% were frail; the mean age was 83.4 years. Sarcopenia was diagnosed in 42 patients (28%). The overall survival rate at 6 months was 92%. The primary endpoint was recorded in 23.2% of the cases and was more frequent in the sarcopenic patients (33.3%) than in the non-sarcopenic individuals (17.6%) (p = 0.01). The regression analysis found that sarcopenia was associated with an increased risk of the primary endpoint (HR: 2.25; 95% CI 1.19-4.45; p = 0.02), adjusting for potential confounding factors. CONCLUSIONS: The incidence of serious cardiac complications in older patients with sarcopenia and severe AS is significant. The present study describes a noninvasive, ultrasound-guided diagnostic technique that may prove efficient in its predictive capacity.

Beneficial actions of melatonin in the management of viral infections: a new use for this "molecular handyman"?

Melatonin (N-acetyl-5-methoxytryptamine) is a multifunctional signaling molecule that has a variety of important functions. Numerous clinical trials have examined the therapeutic usefulness of melatonin in different fields of medicine. Clinical trials have shown that melatonin is efficient in preventing cell damage under acute (sepsis, asphyxia in newborns) and chronic states (metabolic and neurodegenerative diseases, cancer, inflammation, aging). The beneficial effects of melatonin can be explained by its properties as a potent antioxidant and antioxidant enzyme inducer, a regulator of apoptosis and a stimulator of immune functions. These effects support the use of melatonin in viral infections, which are often associated with inflammatory injury and increases in oxidative stress. In fact, melatonin has been used recently to treat several viral infections, which are summarized in this review. The role of melatonin in infections is also discussed herein.

Phenotypic and Genotypic Antimicrobial Susceptibility Testing of Chlamydia trachomatis Isolates from Patients with Persistent or Clinical Treatment Failure in Spain.

The aim of this multicentre project (seven hospitals across the Spanish National Health Service) was to study the phenotypic and genotypic susceptibility of C. trachomatis to the main antimicrobials used (macrolides, doxycycline, and quinolones) in isolates from patients with clinical treatment failure in whom reinfection had been ruled out. During 2018-2019, 73 clinical isolates were selected. Sixty-nine clinical specimens were inoculated onto confluent McCoy cell monolayers for phenotypic susceptibility testing. The minimum inhibitory concentration for azithromycin and doxycycline was defined as the lowest concentration associated with an at least 95% reduction in inclusion-forming units after one passage in the presence of the antibiotic compared to the initial inoculum for each strain (control). Sequencing analysis was performed for the genotypic detection of resistance to macrolides, analysing mutations in the 23S rRNA gene (at positions 2057, 2058, 2059, and 2611), and quinolones, analysing a fragment of the gyrA gene, and searching for the G248T mutation (Ser83->Ile). For tetracyclines, in-house RT-PCR was used to test for the tet(C) gene. The phenotypic susceptibility testing was successful for 10 isolates. All the isolates had minimum inhibitory concentrations for azithromycin ≤ 0.125 mg/L and for doxycycline ≤ 0.064 mg/L and were considered sensitive. Of the 73 strains studied, no mutations were found at positions T2611C or G248T of the gyrA gene. We successfully sequenced 66 isolates. No macrolide resistance-associated mutations were found at positions 2057, 2058, 2059, or T2611C. None of the isolates carried the tet(C) gene. We found no evidence for genomic resistance in this large, clinically relevant dataset.