Ultrasound clinical references of the healthy toenail unit.

The toenail unit, commonly called the nail, is one of the most frequently examined and treated structures in clinical podiatry. Ultrasound is a standard clinical technique because it is a noninvasive, painless, and rapid diagnostic tool. The main objective of this study was to obtain morphometric data of the healthy toenail unit by ultrasound for clinical application. The nails of 76 participants (152 hallux nails; 38 men, 38 women, average age 26.83 ± 12.20) were examined using a VINNO E35 ultrasound system and an X6-16L linear probe with a frequency of 18 MHz. Five ultrasound measures of the healthy toenail unit were obtained, of which only the distance from the center of the distal phalange to the nail plate varied with age, sex, weight, and foot (p-values ≤ 0.050). The other four parameters were less influenced by the variables analyzed, except sex, which influenced nearly all (p-values ≤ 0.050). In one of these variables, indications of significance were observed (p-values = 0.060), with greater distances in the men than in the women, except for nail plate curvature, which showed a higher value. The other variables studied did not influence the parameters analyzed. High-frequency ultrasound can be used to examine the healthy toenail unit and define anthropometric reference measurements that can be used for more accurate and comparative diagnosis.

Improvement of Early Outcomes in Type A Acute Aortic Syndrome After an Aorta Code Implementation.

BACKGROUND: Reduction of variability through process reengineering can improve surgical results for patients with type A acute aortic syndrome. We compare short-term results before and after implementation of an Aorta Code for patients with type A acute aortic syndrome who underwent surgery. METHODS: The Aorta Code was implemented in a 5-hospital healthcare network in 2019. This critical pathway was based on a simple diagnostic algorithm, ongoing training, immediate patient transfer, and treatment by an expert multidisciplinary team. We retrospectively compared all patients operated on in our center before (2005-2018) and after (January 2019 to February 2023) its implementation. RESULTS: One hundred two and 70 patients underwent surgery in the precode and code periods, respectively. In the code period the number of patients operated on per year increased (from 7.3 to 16.8), and the median elapsed time until diagnosis (6.5 hours vs 4.2 hours), transfer (4 hours vs 2.2 hours), and operating room (2.7 hours vs 1.8 hours) were significantly shorter (P < .05). Aortic root repair and total arch replacement were more frequent (66.7% vs 82.9% [P = .003] and 20.6% vs 40% [P = .001]). Cardiopulmonary bypass and ischemia times were also shorter (179.7 minutes vs 148.2 minutes [P = .001] and 105 minutes vs 91.2 minutes [P = .022]). Incidence of prolonged mechanical ventilation (53.9% vs 34.3%, P = .011), major stroke (17.7% vs 7.1%, P = .047), and 30-day mortality (27.5% vs 7.1%, P = .001) decreased significantly. CONCLUSIONS: An Aorta Code can be successfully implemented by using a standardized protocol within a hospital network. The number of cases increased; time to diagnosis, transfer, and operating room were reduced; and 30- day mortality significantly decreased.

Implementation of clinical assistants in a pediatric oncology department: An impact analysis.

Bureaucratic and administrative tasks associated with health care provision have historically fallen on health care professionals, which is one among the factors contributing to low job satisfaction and lower productivity. Incorporating new professional roles that help to better respond to the needs of both patients and professionals can increase the quality and efficiency of service provision. This article aims to evaluate the impact of the clinical assistant's introduction in the Sant Joan de Déu Barcelona Children's Hospital's pediatric oncology department, in terms of (i) displacement of activity loads carried out by this new professional role and the consequent time freed up for physicians, (ii) physicians' satisfaction and (iii) efficiency of the new care model. This is an observational and retrospective study using administrative data based on the type of activity performed by clinical assistants and the measurement of the time freed up in favor of the physicians. The potential skill mix productivity increase, survey of physicians' satisfaction, and reduction in costs with the new model was analyzed. During the first year of its implementation in the pediatric oncology department, clinical assistants have performed 13,553 requests (69% of the total), representing a total saving of 266.83 hours or 6.67 workweeks of 40 hours. They performed 74% of outpatient surgical requests in the oncology department, 87% of day hospital requests and 54% of total requests in the outpatient consultations area. Physicians are overall satisfied with the new role and think they can use the time gained to do other things such as research or improving the quality of care. The role change allows reducing the cost per request by 56% in relation to the conventional model. In conclusion, the introduction of clinical assistants in the oncology department could be efficient to the extent that it displaces a significant part of the bureaucratic and administrative tasks previously performed by health care professionals and thus enables to reduce the cost of these processes. This delegation allows them to work more closely to the maximum of their competences and the physicians to have more time for higher added value clinical tasks and increase professional satisfaction.

Perieccrine Coil Elastosis: An Unusual and Previously Unrecognized Aging-Related Phenomenon.

ABSTRACT: Elastic fibers are present as a thin line around the normal secretory coil of eccrine and apocrine glands, although they are virtually imperceptible with hematoxylin-eosin staining. Skin aging is a consequence of intrinsic and extrinsic factors, and glycation and ultraviolet irradiation are involved in this process favoring elastosis. We report an unusual and prominent perieccrine elastosis on the left temple in the vicinity of a basal cell carcinoma in a 78-year old man with type 2 diabetes, dyslipidemia, and hypertension. Very thick multilamellar and tortuous elastic fibers surrounded the eccrine coils. This increased amount of elastic fibers was confirmed by orcein staining as well as amyloid-P and lysozyme immunostaining. Perieccrine coil elastosis is a very unusual phenomenon that to the best of our knowledge has not been reported. Similar to dermal actinic elastosis, the presence of perieccrine coil elastosis in a skin cancer microenvironment might hypothetically promote tumor growth because of the release of elastin-derived peptides.

Evolución del uso en los tratamientos de las verrugas plantares / Evolution of use in the treatments of plantar warts

Objetivo: Las verrugas plantares son lesiones cutáneas causadas por el virus del papiloma humano, teniendo una prevalencia en torno al 12 % en la población general. Sus tratamientos han evolucionado a lo largo de los años, por lo cual el objetivo principal del estudio fue conocer cómo ha evolucionado el uso de los distintos tratamientos físicos y químicos para verrugas plantares en España. Material y métodos: Se realizó un estudio longitudinal mediante una encuesta en dos periodos de tiempo diferentes, a través de un formulario de Google-Forms con 17 preguntas, con el fin de recopilar datos de podólogos en activo que tuvieran experiencia clínica en el tratamiento de verrugas plantares y que trabajasen en España. Se obtuvo un total de 889 respuestas procedentes de los 17 colegios de podólogos de España en ambos periodos. Resultados: Los resultados indicaron un aumento en el uso del láser (6.1 %), pasando a ser tratamiento de primera elección en el segundo periodo, y un aumento en el uso de cantaridina (8.0 %). Además, se mostró una disminución en el uso de crioterapia (14.5 %), ácido monocloroacético/tricloroacético (13.0 %) y ácido nítrico (2.3 %), aunque este último se mantiene como tratamiento de primera elección en ambos periodos. Conclusiones: La aparición de nuevos tratamientos menos invasivos y menos dolorosos, como el láser, provoca que otros tratamientos se vean desplazados, entre los que se encuentran algunos ácidos.(AU)

Aims: Plantar warts are skin lesions caused by the human papilloma virus, with a prevalence of around 12 % in the general population. Its treatment has evolved over the years, so the main objective of the study is to find out how the use of different physical and chemical treatments for plantar warts in Spain has evolved through a longitudinal study. Material and methods: A survey was carried out through a Google-Forms form with 17 questions to collect data from active podiatrist clinicians, who had clinical experience in the treatment of plantar warts and who work in Spain. Results: A total of 889 responses were obtained from the 17 podiatry associations in Spain in both periods. The results indicated an increase in the use of the laser (6.1 %), becoming the treatment of first choice in the second period, and an increase in the use of cantharidin (8 %). In addition, a decrease in the use of cryotherapy (14.5 %), monochloroacetic/trichloroacetic acid (13 %) and nitric acid (2.3 %) is shown, although the latter remains as a first-choice treatment in both periods. Conclusions: The use of the different treatments against plantar warts has been modified over a period of 7 years, increasing in physical treatments such as laser and decreasing chemical treatments such as cryotherapy or monochloroacetic/trichloroacetic acid. The appearance of new treatments, less invasive and less painful, causes other treatments to be displaced.(AU)

Comparative Analysis of Type I Keratin Expression By Nail Consistency: An Immunohistochemistry Study.

The nail plate is one of the essential structures of the nail apparatus and is highly keratinized, making it difficult to handle this tissue experimentally. Different types of nail consistency were identified by applying distal pressure to the nail plate. To analyze the relationship between the keratins expressed in the nail plate and nail consistency, we chose a sample of 32 adult individuals (age 49.81±3.21 y) with the same number of each sex, who had a similar percentage of nail consistency types (56.25% hard consistency nails and 43.75% soft consistency nails). Immunohistochemical analyses showed that hard consistency nails contain more keratin 17 than soft consistency nails (P=0.026). These novel results allow nail consistency to be defined by the differential expression of keratins in the nail plate, and have potential clinical implications for the diagnosis of possible nail disorders and/or systemic disease.

Meta-analysis of the antifungal activities of three essential oils as alternative therapies in dermatophytosis infections.

AIMS: This work examines the available scientific evidence about the efficiency of essential oils (EO) as an alternative therapy to traditional treatment of fungal infections, including onychomycosis, assessing the effect of the three EO most frequently studied for their antifungal activity (thyme, cinnamon and tea tree EO) against three causative agents of fungal diseases in humans: Trichophyton rubrum, Trichophyton mentagrophytes complex and Candida albicans. METHODS AND RESULTS: The PRISMA statement protocol was followed to conduct a bibliographical search and 54 articles that met all the inclusion criteria were retrieved. Differences were observed in the MIC and MFC values depending on the micro-organism strain and the EO used. The lowest MIC were observed with Cinnamomum zeylanicum EO (0.013-1120 µl ml-1 ) against the three micro-organisms. For MFC, the lowest value was found for Thymus vulgaris EO (4.2 µl ml-1 ) against Trichophyton rubrum. CONCLUSIONS: The antifungal effects of EO could be a very promising solution to overcome the therapeutic shortcomings of antimycotic medication. More experiments are needed to examine the properties of these oils to devise effective and nonaggressive therapies for treatment of dermatophytosis. SIGNIFICANCE AND IMPACT OF STUDY: The results indicate that EO remain good candidates for future treatments and could provide a solution for failed medications and/or adverse reactions to current pharmacological treatments.

Differences in Hygiene Habits among Children Aged 8 to 11 Years by Type of Schooling.

Personal hygiene is one of the basic activities in the care of our body. Parents are responsible for their children's hygiene to prevent infections and keep them healthy. However, children must acquire hygiene habits correctly and independently. This study examines the sociodemographic profile, hygiene habits and knowledge, and level of autonomy of children who are starting to perform their personal care autonomously to identify the areas in which their habits could be improved. A descriptive cross-sectional study was conducted concerning 125 children aged 8-11 years attending schools in northern Extremadura, Spain. The children were surveyed with the HICORIN® questionnaire and the resulting data were statistically processed with SPSS 22.0 (IBM, Armonk, NY, USA). The majority of participating children required help to perform personal hygiene activities. Children in preferential schooling (PS) require less help than children in mainstream schooling (MS) but have less knowledge about personal hygiene. Different habits were observed in the frequency and time of day for performing personal hygiene between groups (p-values < 0.005). In general, more than 80% of children aged 8 to 11 years are not autonomous in some aspect of their personal hygiene, and they are not all familiar with personal hygiene. Because of this, it is necessary to conduct theory and practical workshops with children who must acquire correct personal hygiene habits autonomously to prevent infection and promote health.

Effects of pollutants and microplastics ingestion on oxidative stress and monoaminergic activity of seabream brains.

Nowadays, microplastics (MPs) and adsorbed pollutants are considered a global thread to marine ecosystems. This study describes the effects of pollutants and MPs ingestion on fish brains through the assessment of oxidative stress biomarkers and monoaminergic neurotransmitters using gilthead seabream (Sparus aurata) as fish model. Juveniles were experimentally exposed to three different dietary treatments for 90 days: Control treatment (C) consisted of standard feed; Virgin treatment (V) contained feed enriched with 10% of MPs; and Exposed treatment (E) consisted of feed with 10% of MPs that were exposed to seawater in an anthropogenically impacted area for 2 months in order to enrich the plastic with the pollutants within the water column. Sampling was made at the start of the experiment (T0), at the end of the dietary treatments (T90) and after a posterior detoxification period of 30 days (T120). Results evidenced that a MPs and pollutants enriched diet increases the activity of some of the oxidative stress biomarkers (e.g. CAT and GST), and it was shown for the first time alterations on dopaminergic and serotonergic system activity on seabream brains, indicating potential neurofunctional effects associated to MPs and pollutants ingestion. In addition, results showed a tendency to recover enzymatic and brain monoaminergic neurotransmitter levels after a 30-day detoxification period. In conclusion, MPs and pollutants exposure for 90 days induced oxidative stress and changes on monoaminergic activity in the brain of S. aurata.

Overcoming Resistance to Immunotherapy in Advanced Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (CSCC) is the second most frequent cancer in humans, and is now responsible for as many deaths as melanoma. Immunotherapy has changed the therapeutic landscape of advanced CSCC after the FDA approval of anti-PD1 molecules for the treatment of locally advanced and metastatic CSCC. However, roughly 50% of patients will not respond to this systemic treatment and even those who do respond can develop resistance over time. The etiologies of primary and secondary resistance to immunotherapy involve changes in the neoplastic cells and the tumor microenvironment. Indirect modulation of immune system activation with new therapies, such as vaccines, oncolytic viruses, and new immunotherapeutic agents, and direct modulation of tumor immunogenicity using other systemic treatments or radiotherapy are now under evaluation in combined regimens. The identification of predictors of response is an important area of research. In this review, we focus on the features associated with the response to immunotherapy, and the evaluation of combination treatments and new molecules, a more thorough knowledge of which is likely to improve the survival of patients with advanced CSCC.

Acute Lung Injury Biomarkers in the Prediction of COVID-19 Severity: Total Thiol, Ferritin and Lactate Dehydrogenase.

SARS-CoV-2 (COVID-19) patients who develop acute respiratory distress syndrome (ARDS) can suffer acute lung injury, or even death. Early identification of severe disease is essential in order to control COVID-19 and improve prognosis. Oxidative stress (OS) appears to play an important role in COVID-19 pathogenesis; we therefore conceived a study of the potential discriminative ability of serum biomarkers in patients with ARDS and those with mild to moderate disease (non-ARDS). 60 subjects were enrolled in a single-centre, prospective cohort study of consecutively admitted patients: 29 ARDS/31 non-ARDS. Blood samples were drawn and marker levels analysed by spectrophotometry and immunoassay techniques. C-reactive protein (CRP), lactate dehydrogenase (LDH), and ferritin were significantly higher in ARDS versus non-ARDS cases at hospital admission. Leukocytes, LDH, ferritin, interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) were also significantly elevated in ARDS compared to non-ARDS patients during the hospital stay. Total thiol (TT) was found to be significantly lower in ARDS. Conversely, D-dimer, matrix metalloproteinase-9 (MMP-9) and advanced glycosylated end products (AGE) were elevated. Leukocytes, LDH, CRP, ferritin and IL-6 were found to be significantly higher in non-survivors. However, lymphocyte, tumour necrosis factor beta (TGF-ß), and TT were lower. In summary, our results support the potential value of TT, ferritin and LDH as prognostic biomarkers for ARDS development in COVID-19 patients, distinguishing non-ARDS from ARDS (AUCs = 0.92; 0.91; 0.89) in a fast and cost-effective manner. These oxidative/inflammatory parameters appear to play an important role in COVID-19 monitoring and can be used in the clinical management of patients.

A facility and community-based assessment of scabies in rural Malawi.

BACKGROUND: Scabies is a neglected tropical disease of the skin, causing severe itching, stigmatizing skin lesions and systemic complications. Since 2015, the DerMalawi project provide an integrated skin diseases clinics and Tele-dermatology care in Malawi. Clinic based data suggested a progressive increase in scabies cases observed. To better identify and treat individuals with scabies in the region, we shifted from a clinic-based model to a community based outreach programme. METHODOLOGY/PRINCIPAL FINDINGS: From May 2015, DerMalawi project provide integrated skin diseases and Tele-dermatological care in the Nkhotakota and Salima health districts in Malawi. Demographic and clinical data of all patients personally attended are recorded. Due to a progressive increase in the number of cases of scabies the project shifted to a community-based outreach programme. For the community outreach activities, we conducted three visits between 2018 to 2019 and undertook screening in schools and villages of Alinafe Hospital catchment area. Treatment was offered for all the cases and school or household contacts. Scabies increased from 2.9% to 39.2% of all cases seen by the DerMalawi project at clinics between 2015 to 2018. During the community-based activities approximately 50% of the population was assessed in each of three visits. The prevalence of scabies was similar in the first two rounds, 15.4% (2392) at the first visit and 17.2% at the second visit. The prevalence of scabies appeared to be lower (2.4%) at the third visit. The prevalence of impetigo appeared unchanged and was 6.7% at the first visit and 5.2% at the final visit. CONCLUSIONS/SIGNIFICANCE: Prevalence of scabies in our setting was very high suggesting that scabies is a major public health problem in parts of Malawi. Further work is required to more accurately assess the burden of disease and develop appropriate public health strategies for its control.

Predictive Model of Nail Consistency Using Scanning Electron Microscopy with Energy-Dispersive X-Ray.

The nail plate is made up of tightly packed keratin-rich cells. Factors such as the special distribution of the intermediate filaments in each layer (dorsal, intermediate, and ventral), the relative thickness of the layers, and their chemical composition define the characteristics of each nail. The main objective of this study is to determine nail consistency by calculating a predictive model based on elemental composition analysis using scanning electron microscopy. Nail consistency was determined in 57 participants (29 women and 28 men) in two age groups (young people and adults). Elemental composition was analysed in each layer using scanning SEM-EDS, and nail plate thickness was measured by image analysis. A total of 12 elements were detected in nail plates, of which carbon, nitrogen, phosphorus, sulphur, and calcium showed significant differences between layers (p-values ≤ 0.01). The level of calcium in the dorsal layer was the main predictive variable in calculating the predictive model of consistency, with 75.4% correctly classified cases. Elemental analysis in each layer of the nail plate by SEM-EDS can be used to develop a predictive model of nail consistency that will help health professionals to objectively determine nail consistency.

Tau phosphorylation by glycogen synthase kinase 3ß modulates enzyme acetylcholinesterase expression.

In Alzheimer's disease (AD), the enzyme acetylcholinesterase (AChE) co-localizes with hyperphosphorylated tau (P-tau) within neurofibrillary tangles. Having demonstrated that AChE expression is increased in the transgenic mouse model of tau Tg-VLW, here we examined whether modulating phosphorylated tau levels by over-expressing wild-type human tau and glycogen synthase kinase-3ß (GSK3ß) influences AChE expression. In SH-SY5Y neuroblastoma cells expressing higher levels of P-tau, AChE activity and protein increased by (20% ± 2%) and (440% ± 150%), respectively. Western blots and qPCR assays showed that this increment mostly corresponded to the cholinergic ACHE-T variant, for which the protein and transcript levels increased ~60% and ~23%, respectively. Moreover, in SH-SY5Y cells differentiated into neurons by exposure to retinoic acid (10 µM), over-expression of GSK3ß and tau provokes an imbalance in cholinergic activity with a decrease in the neurotransmitter acetylcholine in the cell (45 ± 10%). Finally, we obtained cerebrospinal fluid (CSF) from AD patients enrolled on a clinical trial of tideglusib, an irreversible GSK3ß inhibitor. In CSF of patients that received a placebo, there was an increase in AChE activity (35 ± 16%) respect to basal levels, probably because of their treatment with AChE inhibitors. However, this increase was not observed in tideglusib-treated patients. Moreover, CSF levels of P-tau at the beginning measured by commercially ELISA kits correlated with AChE activity. In conclusion, this study shows that P-tau can modulate AChE expression and it suggests that AChE may possibly increase in the initial phases of AD.

Prevalence of foot disorders according to chronic kidney disease stage.

BACKGROUND: Chronic kidney disease (CKD) is a public health disease that affects 15.1% of the adult population. Although a high prevalence (94.1%) of skin disorders has been detected in people on haemodialysis or with advanced CKD, few studies have analysed foot disorders at initial CKD stages. OBJECTIVES: To analyse the prevalence of foot disorders according to CKD stage. PARTICIPANTS: A total of 209 people with a mean age of 73.2 ± 13.8 years (52.0% women) in the nephrology department of Virgen del Puerto Hospital, Plasencia (Spain) were examined from January 2018 to April 2019. MEASUREMENTS: CKD stages were determined by nephrologists according to the Kidney Disease Improving Global Outcomes Guideline. An expert podiatrist identified foot disorders. Data were statistically treated with the IBM SPSS Statistics. Comparisons between variables were analysed by the χ2  test or Fisher's exact test, with a significance level of less than 5%. RESULTS: The prevalence of foot disorders was high for skin disorders (97.6% dermatopathies and 66.0% keratopathies), nail disorders (98.5% onychopathies) and toe deformities (97.1%). People at initial and intermediate stages presented more keratopathies (hyperkeratosis at G1 and G3a and pinch callus at G3a). Stage G1 showed fewer changes in nail colour and half and half nails. Stage G4 showed more claw toes and hematoma and stage G5 more Beau's lines, changes in skin colour, hematomas and thin shiny skin. CONCLUSIONS: The high prevalence of foot disorders detected in people with CKD requires specific and personalised professional care to relieve symptoms and avoid complications, helping to improve the quality of life of people with this condition.

Occupational Safety and Health Training for Undergraduates Nursing Students: A Spanish Pilot.

Most of blood borne and airborne pathogens are highly contagious, harmful and have prevalence among healthcare workers. In this group, healthcare students, especially nursing undergraduates, have even higher risk to be exposed and suffered a contagious accident. One of the main pillars to prevent exposure to such pathogens and decrease accidents seems to be through education. A prospective observational educational research focused on quantifying the students' knowledge, and prevention culture was carried out. The educational approach based on the development of a technological tool, its integration in the students' education, and posterior assessment. The Chi-square, ANOVA, Kruskal-Wallis, Man-Whitney U, and Spearman correlations were used to determine the effect of such educational methodology. The results, previous to the integration of the educational approach, showed differences between the elementary and proficient knowledge and correct procedure in each academic year (p < 0.05), being the best year the third academic year. The mean of elementary knowledge among second year students after the inclusion of the educational methodology improved for 2017/2018 with a mean of 7.5 (1.11) and in 2018/2019 with 7.87 (1.34). This study argued that the educational approach proposed could improve the prevention culture and knowledge among students and future healthcare professionals.

Tuberculosis and Other Airborne Microbes in Occupational Health and Safety.

Airborne pathogens and non-malignant infectious diseases such as tuberculosis are highly contagious and can have severe effects on healthcare workers. The symptoms of these diseases take time to manifest, which can prevent workers from noticing that they have been exposed until symptoms appear. The current paper sought to assess the occupational safety and preventative measures taken in laboratories in Spain, and to compare these measures with those reported by other studies worldwide. A cross-sectional study of workers (35-50 years old) was conducted using a web survey (N = 30), and a bibliometric analysis was carried out in the Scopus database (92 documents were selected). The occupational safety and health measures were inadequate, according to the opinions of the workers. The training (p < 0.01), the amount of work (p < 0.05), and how the workers followed their protocols (p < 0.001) were linked to incidents and exposure to airborne pathogens. The most significant previous publication was a report (848 citations) stating that the previous variables linked to exposure are vital for prevention. Most works focused on countries like the U.S.A. (p = 0.009) were reviews, with a limited number of studies focused on occupational safety.

Bibliometric Study of Technology and Occupational Health in Healthcare Sector: A Worldwide Trend to the Future.

Since the eighties, technological tools have modified how people interact in their environment. At the same time, occupational safety and health measures have been widely applied. The European Agency for Safety and Health at Work considers that information and communication technologies are the main methods to achieve the goals proposed to improve working life and the dissemination of good practices. The principal objective was to determine the trends of publications focused on these technologies and occupational safety in the healthcare sector during the last 30 years. A bibliometric study was carried out. The 1021 documents showed an increased trend per country, especially for the United States (p < 0.001) and year (p < 0.001). The citations per year showed significant differences between citations of articles published before 2007 (p < 0.001). The year was also linked to the increase or decrease of articles (72.2%) and reviews (14.9%) (p < 0.001). The analysis of journal co-citations also showed that the main journals (such as Infection Control and Hospital Epidemiology) were linked to other important journals and had a major part in the clusters formed. All these findings were discussed in the manuscript and conclusions were drawn.