Clinical Characterization of the Persistent COVID-19 Symptoms: A Descriptive Observational Study in Primary Care.

INTRODUCTION: Evidence and clinical experience suggest that there are a range of signs of health issues that affect patients who have recovered from acute COVID-19 infection. This condition is commonly referred to as "persistent COVID-19," which is not connected with the severity of the disease. We have identified the prevalence and clinical-epidemiological characteristics of patients with COVID-19 and persistent symptoms treated in primary care centers. This is a descriptive observational study conducted between December 2020 and May 2022, the data were collected from digitized medical records and interviewing 1542 individuals with laboratory-confirmed SARS-CoV-2 infection. These patients were clinically followed for up to 1 year, based on the prevalence of symptoms. When stratifying by the risk of developing persistent COVID-19, 37.09% of the patients exhibited risk factors, with age (over 60 years) and cardiovascular risk factors predominating. The obtained prevalence of persistent COVID-19 at 90 days was 12.39%, with a slight predominance in females (55%) and a mean age of 45.8 years. The most affected systems were the cardiovascular, respiratory, and psychoneurological systems, with predominant symptoms of fatigue (41.88%), dyspnea (32.46%), and headache (14.66%), among others. The average duration of persistent symptoms was 178 days, equivalent to 6 months. In conclusion, over 10% of patients who recover from acute SARS-CoV-2 infection developed long-term consequences. OBJECTIVE: To measure the prevalence and clinical-epidemiological characteristics of individuals diagnosed with COVID-19 and persistent symptoms treated in primary care centers in Área Sanitaria Sur de Córdoba (Spain) between December 2020 and May 2022. METHODS: A retrospective observational study of the population of Área Sanitaria Sur de Córdoba was developed between December 2020 and May 2022. The scope of the study was 1542 individuals, and the prevalence of patients diagnosed with COVID-19 and persistent symptoms was determined based on laboratory-confirmed cases, randomly selected from adults who had progressed beyond the acute phase of the disease. All data were managed by the Área Sanitaria Sur de Córdoba (Spain). RESULTS: The risk factor of exhibiting one or more risk factors associated with developing persistent COVID-19 is 37.09%. The obtained prevalence of persistent COVID-19 at 90 days is 12.39%, it is higher in females (55%) than males and the mean age was 45.8 years. The most affected systems were the cardiovascular, respiratory, and psychoneurological systems, with predominant symptoms of fatigue (41.88%), dyspnea (32.46%), and headache (14.66%), among others. CONCLUSIONS: Results confirm that more than 10% of individuals recovering from acute SARS-CoV-2 infection showed long-term consequences and the observed persistent symptom duration was 178 days on average.

Hispanic mothers' accounts of vaccinating their adolescent children against HPV: features of the clinic visit.

ABSTRACTObjective: Despite the widespread availability of the human papillomavirus (HPV) vaccine in the US, rates of vaccination among Hispanic adolescents lag behind those of other recommended vaccines. Understanding what happens during the HPV vaccination visit should provide important insight into communication between health care providers and Hispanic mothers and identifies areas where communication can be improved. As such, this qualitative study explored Hispanic mothers' experiences during their adolescent child's HPV vaccination visit.Design: Fifty-one participants completed individual interviews. Transcripts were analyzed using a conventional content analysis approach to identify emergent categories or themes.Results: We identified three features of the HPV vaccination visit including: the primary reason for the visit, the type of counseling the mother received about the vaccine and the type of HPV vaccine recommendation received. Most mothers reported that their child was vaccinated against HPV at a routine well-child visit. Some mothers reported that they received in-depth counseling about the vaccine, while others received brief or no counseling from the provider. Mothers also reported receiving either a strong recommendation to vaccinate, a recommendation to vaccinate that emphasized her choice, or no recommendation to vaccinate.Conclusion: Most Hispanic mothers report that they received counseling and a recommendation from their adolescent child's health care provider before vaccinating. However, most of the mothers first heard about the HPV vaccine at the vaccination visit. Mothers who had previously heard about the vaccine outside of the clinic, reported making an appointment specifically for their child to be vaccinated against HPV. Together, these findings indicate a need to raise awareness of the vaccine and to promote HPV vaccination more strongly in this population. Education efforts should target mothers in community settings, in addition to clinic settings in order to increase awareness and vaccination in this population.

The cAMP effectors PKA and Epac activate endothelial NO synthase through PI3K/Akt pathway in human endothelial cells.

3',5'-Cyclic adenosine monophosphate (cAMP) exerts an endothelium-dependent vasorelaxant action by stimulating endothelial NO synthase (eNOS) activity, and the subsequent NO release, through cAMP protein kinase (PKA) and exchange protein directly activated by cAMP (Epac) activation in endothelial cells. Here, we have investigated the mechanism by which the cAMP-Epac/PKA pathway activates eNOS. cAMP-elevating agents (forskolin and dibutyryl-cAMP) and the joint activation of PKA (6-Bnz-cAMP) and Epac (8-pCPT-2'-O-Me-cAMP) increased cytoplasmic Ca2+ concentration ([Ca2+]c) in ≤30% of fura-2-loaded isolated human umbilical vein endothelial cells (HUVEC). However, these drugs did not modify [Ca2+]c in fluo-4-loaded HUVEC monolayers. In DAF-2-loaded HUVEC monolayers, forskolin, PKA and Epac activators significantly increased NO release, and the forskolin effect was reduced by inhibition of PKA (Rp-cAMPs), Epac (ESI-09), eNOS (L-NAME) or phosphoinositide 3-kinase (PI3K; LY-294,002). On the other hand, inhibition of CaMKII (KN-93), AMPK (Compound C), or total absence of Ca2+, was without effect. In Western blot experiments, Serine 1177 phosphorylated-eNOS was significantly increased in HUVEC by cAMP-elevating agents and PKA or Epac activators. In isolated rat aortic rings LY-294,002, but not KN-93 or Compound C, significantly reduced the vasorelaxant effects of forskolin in the presence of endothelium. Our results suggest that Epac and PKA activate eNOS via Ser 1177 phosphorylation by activating the PI3K/Akt pathway, and independently of AMPK or CaMKII activation or [Ca2+]c increase. This action explains, in part, the endothelium-dependent vasorelaxant effect of cAMP.

The microRNA-7-mediated reduction in EPAC-1 contributes to vascular endothelial permeability and eNOS uncoupling in murine experimental retinopathy.

AIMS: To investigate the consequences of oxidative stress and hypoxia on EPAC-1 expression during retinopathy. METHODS: Oxygen-induced retinopathy was induced in mice and EPAC-1 expression investigated by immunofluorescence. In silico analyses were used to identify a link between EPAC-1 expression and microRNA-7-5p in endothelial cells and confirmed by western blot analyses on cells expressing microRNA-7-5p. In vitro, endothelial cells were either incubated at 2% oxygen or transfected with microRNA-7-5p, and the effects of these treatments on EPAC-1 expression, endothelial hyperpermeability and NO production were assessed. In the Ins2Akita mouse model, levels of EPAC-1 expression as well as microRNA-7-5p were assessed by qPCR. Endothelial nitric oxide synthase was assessed by immunoblotting in the Ins2Akita model. RESULTS: Hypoxia induces the expression of microRNA-7-5p that translationally inhibits the expression of EPAC-1 in endothelial cells, resulting in hyperpermeability and the loss of eNOS activity. Activation of EPAC-1 by the cAMP analogue 8-pCPT-2'-O-Me-cAMP reduced the sensitivity of EPAC-1 to oxidative stress and restored the endothelial permeability to baseline levels. Additionally, 8-pCPT-2'-O-Me-cAMP rescued eNOS activity and NO production. In mouse models of retinopathy, i.e., oxygen-induced retinopathy and the spontaneous diabetic heterozygous Ins2Akita mice, EPAC-1 levels are decreased which is associated with an increase in microRNA-7-5p expression and reduced eNOS activity. CONCLUSION/INTERPRETATION: In retinopathy, EPAC-1 expression is decreased in a microRNA-7-mediated manner, contributing to endothelial dysfunction. Pharmacological activation of remnant EPAC-1 rescues endothelial function. Collectively, these data indicate that EPAC-1 resembles an efficacious and druggable target molecule for the amelioration of (diabetic) retinopathy.

Activation of PKA and Epac proteins by cyclic AMP depletes intracellular calcium stores and reduces calcium availability for vasoconstriction.

AIMS: We investigated the implication of PKA and Epac proteins in the endothelium-independent vasorelaxant effects of cyclic AMP (cAMP). MAIN METHODS: Cytosolic Ca(2+) concentration ([Ca(2+)]c) was measured by fura-2 imaging in rat aortic smooth muscle cells (RASMC). Contraction-relaxation experiments were performed in rat aortic rings deprived of endothelium. KEY FINDINGS: In extracellular Ca(2+)-free solution, cAMP-elevating agents induced an increase in [Ca(2+)]c in RASMC that was reproduced by PKA and Epac activation and reduced after depletion of intracellular Ca(2+) reservoirs. Arginine-vasopressin (AVP)-evoked increase of [Ca(2+)]c and store-operated Ca(2+) entry (SOCE) were inhibited by cAMP-elevating agents, PKA or Epac activation in these cells. In aortic rings, the contractions induced by phenylephrine in absence of extracellular Ca(2+) were inhibited by cAMP-elevating agents, PKA or Epac activation. In these conditions, reintroduction of Ca(2+) induced a contraction that was inhibited by cAMP-elevating agents, an effect reduced by PKA inhibition and reproduced by PKA or Epac activators. SIGNIFICANCE: Our results suggest that increased cAMP depletes intracellular, thapsigargin-sensitive Ca(2+) stores through activation of PKA and Epac in RASMC, thus reducing the amount of Ca(2+) released by IP3-generating agonists during the contraction of rat aorta. cAMP rise also inhibits the contraction induced by depletion of intracellular Ca(2+), an effect mediated by reduction of SOCE after PKA or Epac activation. Both effects participate in the cAMP-induced endothelium-independent vasorelaxation.

Insight into the functional and structural properties of 3-arylcoumarin as an interesting scaffold in monoamine oxidase B inhibition.

The design, synthesis, pharmacological evaluation, and theoretical studies of a new series of halogenated 3-arylcoumarins were carried out with the aim of finding new structural and biological features. This series displays several alkyl, hydroxy, halogen, and/or alkoxy groups in both benzene rings of the 3-arylcoumarin scaffold. Most of the compounds studied show high affinity and selectivity for the human monoamine oxidase B (hMAO-B) isoenzyme, with IC50 values in the low nanomolar and picomolar range. Most of the evaluated compounds display higher MAO-B inhibitory activity and selectivity than selegiline (the reference compound). Coumarin 12 (3-(3-bromophenyl)-6-methylcoumarin) is the most active compound (IC50 =134 pM), being 140-fold more active than selegiline and showing the highest specificity for hMAO-B. To better understand the structure-activity relationships, docking experiments were carried out on human monoamine oxidase (A and B) structures. Finally, the prediction of passive blood-brain partitioning, based on in silico derived physicochemical descriptors, was performed.

PKA and Epac activation mediates cAMP-induced vasorelaxation by increasing endothelial NO production.

Vascular relaxation induced by 3',5'-cyclic adenosine monophosphate (cAMP) is both endothelium-dependent and endothelium-independent, although the underlying signaling pathways are not fully understood. Aiming to uncover potential mechanisms, we performed contraction-relaxation experiments on endothelium-denuded and intact rat aorta rings and measured NO levels in isolated human endothelial cells using single cell fluorescence imaging. The vasorelaxant effect of forskolin, an adenylyl cyclase activator, was decreased after selective inhibitor of protein kinase A (PKA), a cAMP-activated kinase, or L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, only in intact aortic rings. Both selective activation of PKA with 6-Bnz-cAMP and exchange protein directly activated by cAMP (Epac) with 8-pCPT-2'-O-Me-cAMP significantly relaxed phenylephrine-induced contractions. The vasorelaxant effect of the Epac activator, but not that of the PKA activator, was reduced by endothelium removal. Forskolin, dibutyryl cAMP (a cAMP analogue), 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP increased NO levels in endothelial cells and the forskolin effect was significantly inhibited by inactivation of both Epac and PKA, and eNOS inhibition. Our results indicate that the endothelium-dependent component of forskolin/cAMP-induced vasorelaxation is partially mediated by an increase in endothelial NO release due to an enhanced eNOS activity through PKA and Epac activation in endothelial cells.

Trans-resveratrol simultaneously increases cytoplasmic Ca(2+) levels and nitric oxide release in human endothelial cells.

SCOPE: The aim of this study was to investigate whether the dietary polyphenol trans-resveratrol (t-Resv) increases [Ca(2+)](c) in endothelial cells, leading to a simultaneous augmentation of nitric oxide (NO) biosynthesis. METHODS AND RESULTS: We have separately and simultaneously measured [Ca(2+)](c) and NO in human endothelial cells using the Ca(2+) indicator fura-2 and the NO-sensitive fluorescent probe 4,5-diaminofluorescein. In ∼30% of cells, t-Resv (30 µM) induced an increase in [Ca(2+)](c) with a transient as well as sustained component and a simultaneous increase in NO biosynthesis. This effect was reduced by non-selective Ca(2+) channel blockers, inhibition of intracellular Ca(2+) release, inhibition of endothelial nitric oxide synthase (eNOS) and, to a lesser extent, inhibition of extracellular signal-regulated kinase 1/2 (ERK 1/2) or 5' adenosine monophosphate-activated protein kinase (AMPK). t-Resv did not modify in vitro eNOS activity, suggesting that the observed stimulation of NO generation proceeds via mobilisation of Ca(2+) and not through direct effects on eNOS. CONCLUSION: We therefore show, for the first time, that t-Resv induces a concentration-dependent, simultaneous increase in [Ca(2+)](c) and NO biosynthesis that could be linked to its endothelium-dependent vasorelaxant effect. Under the assumption that t-Resv exhibits similar behaviour in human blood vessels in vivo, the pharmacological properties described here may contribute to the beneficial cardiovascular effects of this polyphenol by improving endothelial function.

Synthesis and vasorelaxant and platelet antiaggregatory activities of a new series of 6-halo-3-phenylcoumarins.

A series of 6-halo-3-hydroxyphenylcoumarins (resveratrol-coumarins hybrid derivatives) was synthesized in good yields by a Perkin reaction followed by hydrolysis. The new compounds were evaluated for their vasorelaxant activity in intact rat aorta rings pre-contracted with phenylephrine (PE), as well as for their inhibitory effects on platelet aggregation induced by thrombin in washed human platelets. These compounds concentration-dependently relaxed vascular smooth muscle and some of them showed a platelet antiaggregatory activity that was up to thirty times higher than that shown by trans-resveratrol and some other previously synthesized derivatives.