BackgroundDiabetes mellitus is a chronic illness which is becoming more prevalent in developing countries, and it is being managed mostly in hospitals or clinics in underdeveloped nations. Other strategies for treatment delivery in emerging nations must be considered as the number of diabetic patients grows. Community pharmacists are a valuable choice for diabetes care. However, only developed countries have data on community pharmacists' diabetes treatment practices.MethodologyA non-probability consecutive sampling strategy was used to gather a self-administered questionnaire from 289 community pharmacists. Six points Likert scale was employed to score current practices and pharmacists' perceived role. A response rate of 55% was attained. Characteristics associated with present behaviors and perceived roles were analyzed using Chi-square and logistic regression.ResultsThe majority of the respondents were males, 234 (81.0%). Out of 289, 229 (79.2%) were of 25-30 years of age and were pharmacists as well as qualified persons (QP) 189 (65.4%). A QP is one who has the legal authority to sell drugs to customers. The majority had <5 years of working experience as a community pharmacist, 268 (92.7%), and did not have diabetes training, 237 (82.0%). Most community pharmacies were stand-alone, 110 (38.1%), and had a single or a group of proprietors, 248 (85.8%). Open hours of most of the pharmacies were 16-20 hours per day, 202 (69.8%), and most had one pharmacist, 243 (84.1%), i.e., working as a pharmacist as well as a qualified person. Approximately 203 (70.2%) of the pharmacies had customers >2000 in a month and >100 customers purchased anti-diabetes medications per month. Only 44 (15.2%) community pharmacies had a designated room or space for patient counselling. The majority of pharmacists were also in favor of providing services other than dispensing such as counselling the patients about prescribed medicines, direction of use, use of devices for insulin administration, training on self-monitoring of glucose, and healthy lifestyle and diet practices. Pharmacy setting, ownership, patient counseling area, and the number of customers per month were key factors in the provision of diabetes services. The main obstacles identified were a lack of pharmacist availability and academic competency.ConclusionIn Rawalpindi and Islamabad, most community pharmacies only provide a basic dispensing service for diabetes patients. Most of the community pharmacists agreed to extend their duties. The expansion of pharmacist professional responsibilities would help control the rising diabetes burden. The facilitators and hurdles identified would serve as a foundation for the introduction of diabetic care in community pharmacies. Diabetes is among the most prevalent public health issues all over the world, and its prevalence is rising, especially in developing countries. Diabetes currently affects 415 million people globally, with that number estimated to rise to 642 million by 2040 . InternaMany factors such as ineffective communication, counseling and limited time-sparing factors of the healthcare workers have contributed to the failure of effective management of chronic illnesses such as diabetes . These gThe public has developed a misconception over the years that community pharmacies only sell medicines; however, the community pharmacist, as a custodian of the ailing community, can play a key role in changing this impression by providing health education about self-management, prevention of adverse effects, prevention of long-term complications, the direction of medication use, adherence, and therapeutic plan management. According to a Japanese study, community pharmacists’ consultations focusing on lifestyle changes helped patients with type II diabetes achieve better glycemic control. The patients' HbA1c levels were also observed to improve after 6 months of treatment, and the number of blood glucose-controlling medications was also reduced . So, comTherefore, the aim of the study is to evaluate existing practices and the community pharmacist's perceived role in type 2 diabetes care, as well as factors (pharmacist and community pharmacy) linked to the current practices.In Rawalpindi and Islamabad, a cross-sectional (quantitative) study was undertaken to evaluate existing practices and the community pharmacist's perceived role in type 2 diabetes care, as well as factors (pharmacist and community pharmacy) linked to the current practices. These two cities were chosen as both cities' community pharmacy procedures are comparable. Rawalpindi has a population of 22.8 million people, whereas Islamabad has a population of roughly 11.6 million . 289 comSample size and settingThe study population was all community pharmacists working in community pharmacies of Rawalpindi and Islamabad. A non-probability consecutive sampling strategy was used to choose community pharmacies. The sample size was determined from a list of community pharmacies obtained from the respective health authorities. The total number of community pharmacies in Islamabad and Rawalpindi was 550 and 600, respectively  [Data collection The frequency of providing the services was scored using a six-point Likert scale; 1 (never) to 6 (often). The perceived role of the pharmacist in current diabetic practice was scored using a six-point Likert scale; 1 (definitely no) to 6 (definitely yes) Details of community pharmacists such as gender, age, position, experience as a community pharmacist, registration year, and duration of diabetes training were collected. And details about community pharmacies such as setting, opening hours per week, ownership, opening days per week, presence of counselling area/ room, number of pharmacists per pharmacy, customers per month, customers purchasing oral anti-diabetic medications and insulin per month were also collected. Informed and verbal consent was obtained from the community pharmacists only, no other healthcare practitioners or support workers were involved in the study. The responders were verbally informed of the confidentiality of the information, and the principal investigator signed a confidentiality undertaking, after getting formal approval from the Ethical Review Committee of Al-Shifa Trust Eye Hospital, School of Public Health in Rawalpindi (Reference no: ERC-13/AST-21).A 33-question pre-tested six-point Likert scale (see Appendices) on community pharmacy-based services and pharmacists' perceived involvement in type 2 diabetes services was used. An in-person survey was conducted with the help of a questionnaire. There were four sections to the research questionnaire: (1) Community pharmacist and Community pharmacy characteristics; (2) Current practices in the treatment of type 2 diabetes; (3) consent form, and (4) pharmacists' perceived roles for type 2 diabetes patients.Data analysisStatistical Package for Social Science (SPSS) software version 26.0  and Microsoft Excel 365  were used for statistical analysis and data entry. Data entry was done according to the codes assigned to all the items in the questionnaire, while numeric variables were segregated accordingly. The characteristics of the community pharmacists and their pharmacies were summarized using descriptive statistics. Frequencies were determined for responses from Likert scales of current practice and pharmacists' perceived roles in diabetes services. Current service responses were divided into two variables indicating ‘frequent service'  versus 'less frequent service' , as well as pharmacist roles, which were also divided into two variables indicating 'agreement'  and 'disagreement' . The frequencies of these replies were determined after coding. Chi-square test of association was used to determine the significance between independent and dependent variables. At 95% confidence intervals and p<0.05, a logistic regression model was developed to evaluate the relationship between features of community pharmacy and pharmacist with the current provision of services for diabetes and the perceived role of the community pharmacist. Tables are used to present the results.Most respondents were male, 234 81.0%). Out of 289, 229 (79.2%) were of 25-30 years of age and 189 (65.4%) were pharmacists as well as qualified persons (QP). The majority of community pharmacists, 268 (92.7%), had less than 5 years of experience. Five (1.7%) pharmacists had more than 10 hours of training, 13 (4.5%) had 6-10 hours of training, 34 (11.8%) had 1-5 hours of training, and 237 (82%) had no training in diabetes care. The majority of them had no training; however, in recent times pharmacists have been highly encouraged to undergo training as the role of pharmacists is expanding due to the increased number of patients being diagnosed with diabetes. Being trained definitely had an edge over not being trained in a few detailed aspects such as instructing the patients about the interaction of food consumed and blood sugar levels, monthly refilling of anti-diabetic drugs, meal planning etc. The majority of pharmacies were stand-alone, 110 (38.1%), and were owned by a single or group of entrepreneurs, 248 (85.8%). A total of 24 (8.3%) pharmacies were owned by trained pharmacist managers. 202 (69.9%) pharmacies were open for 15-20 hours a day, and 243 (84.1%) pharmacies had only one pharmacist in each pharmacy. To evaluate pharmacist availability in each community pharmacy, a ratio of the total working hours of a pharmacist in a week to the total operating hours of a pharmacy in a week was calculated. The ratios came out were 0.0-2.0, with the majority of pharmacies having less than 1.0 (80%). Thus, most of the pharmacies did not have a pharmacist on duty during the week while they were open. As the pharmacies hired only one pharmacist and they work for approximately 40 hours a week, most of them have their day off on weekdays. Approximately 203 (70.2%) pharmacies had >2000 monthly consumers, and 218 (75.4%) had >100 diabetic patients per month. Only 44 (15.2%) pharmacies had a separate room or area dedicated to counseling. Table .0%. Out Community pharmacists reported dispensing was a prominent service, but the additional diabetic services were minimal. Patient education on pharmaceuticals, notably directions for use 221 (76.5%), directions for use of insulin devices, and storage requirements 234 (81.0%) were the only services indicated by the majority of respondents.All the community pharmacists were in favor of providing services other than dispensing and in expanding their staff. Counselling on prescribed medications like directions for use (99.7%) and use of devices for insulin administration (99.7%), training on self-monitoring of blood glucose (SMBG) (93.1%), education on healthy eating and exercise (91.3%), and strategy for prevention/treatment of long-term complications (91.3%) were the prime considerations for services other than dispensing 77.2%). Table %. Table The association of pharmacists’ characteristics and the perceived role of community pharmacists in type 2 diabetes care are shown in Table To find characteristics of community pharmacist and community pharmacy linked with current practice, logistic regression models were used. Community pharmacists who thought providing a service was part of their job were more inclined to do so. Community pharmacies with more than 50 diabetes patients purchasing insulin and diabetes medicine in a month (for education on diet) and engagement in the training of diabetes were also recognized as facilitators (for compliance monitoring services). The lack of pharmacist availability was highlighted as a barrier to efficiently providing diabetes and compliance monitoring services. Table For the management of diabetic patients, proper pharmaceutical treatment is crucial to achieving the desired outcome. Community pharmacists help in controlling blood sugar levels and enhance the quality of life by providing pharmaceutical care and prescription management services .This study highlights the community pharmacist’s existing practices and their role in type 2 diabetes care. As the survey requested input about future services, respondents are likely more enthused about them than they are about their implementation.Although dispensing was firmly established in Rawalpindi and Islamabad community pharmacies, services besides dispensing were only given to a limited level. The most regular practice here was providing basic medication information on how to use them. Also, at community pharmacies in Pakistan, prescription handling and patient management are rarely seen .Most of the research conducted in developed countries reported pharmacists were consistently counseling patients about their prescription drugs, including usage and adverse effects . HoweverCommunity pharmacists are underutilized in contrast to the situation in developed countries since they are not seen as healthcare professionals and as a result, their contribution to the healthcare system is not acknowledged When a pharmacist's responsibility included services, the regression models demonstrated that this perspective acted as a catalyst for the provision of a wide range of services for patients. Indonesian research found that pharmacists who worked in pharmacies that provided diabetes care had a much greater agreement with the service than those who worked in pharmacies that did not .The availability of pharmacists for various patients' education and monitoring activities was associated with their degree of availability. It is required by law that a pharmacist must be there throughout the operational hours of a community pharmacy . More thAdministrative barriers, insufficient implementation of rules and regulations, an insufficient number of pharmacy personnel , a pharmacist's academic expertise in terms of clinical services revolving around a patient, a deficiency of collaboration with other healthcare personnel, and a lack of knowledge about the existence and role of pharmacists are all obstacles to the expansion of pharmacy services in community settings .Weak monitoring mechanisms and law enforcement may contribute to this, making implementation dependent on the pharmacist's dedication. Because proprietors (non-pharmacist managers' owners) own the majority of pharmacies, their dedication is likely to affect this practice. According to this study, pharmacies managed by pharmacist managers had pharmacists present throughout business hours than pharmacies owned by non-pharmacist owners. Low pharmacist availability might indicate that owners are more focused on business, selling drugs in less expensive methods without the use of pharmacists. Many pharmacists would be employed only for legal reasons, which would result in reduced salaries. Pharmacists may take on other jobs due to low pay and shorter hours, causing them to be unavailable at pharmacies.Despite continuous government initiatives, and literature and policy that demand pharmacy services to be implemented, deficiency of acknowledgment of the entire breadth of pharmacy services has limited pharmacists' ability to establish themselves as skilled and acknowledged healthcare providers in Pakistan . Along wThe general public underutilizes community pharmacies, which may make it difficult for pharmacy owners to provide services like pharmacist availability. Each month, the majority of local pharmacies claimed to serve over 2000 people. Pharmacist involvement in diabetes education was found to be a useful tool for delivering teaching and monitoring duties. Pharmacists who have received diabetes training execute more diabetes-related activities than pharmacists who have not received diabetes training, according to studies conducted in Australia and Canada ,19. As aStrengths The research would provide information to enable the government and pharmacy authorities to construct community pharmacy-centered diabetes care in a developing country like Pakistan.Limitations The limitations include the availability of community pharmacists and a lack of published data on type 2 diabetes service coverage, use, or quality in Rawalpindi and IslamabadIn Rawalpindi and Islamabad, most community pharmacies only provide a basic dispensing service for diabetes patients. Most of the community pharmacists agreed to extend their duties. The expansion of pharmacist professional responsibilities would help control the rising diabetes burden. The facilitators and hurdles identified would serve as a foundation for the introduction of diabetic care in community pharmacies.


Platelet function testing is essential for the diagnosis of patients with bleeding disorders. Specifically, there is a need for a whole blood assay that is capable of analysing platelet behaviour in contact with a patient-specific autologous von Willebrand factor (vWF), under physiologically relevant conditions. The creation of surface topography capable of entrapping and uncoiling vWF for the support of subsequent platelet adhesion within the same blood sample offers a potential basis for such an assay. In this study, spin coating of polystyrene/poly (methyl methacrylate) (PS/PMMA) demixed solutions onto glass substrates in air has been used to attain surfaces with well-defined topographical features. The effect of augmenting the PS/PMMA solution with uniform 50 µm PS microspheres that can moderate the demixing process on the resultant surface features has also been investigated. The topographical features created here by spin coating under ambient air pressure conditions, rather than in nitrogen, which previous work reports, produces substrate surfaces with the ability to entrap vWF from flowing blood and facilitate platelet adhesion. The direct optical visualisation of fluorescently-labelled platelets indicates that topography resulting from inclusion of PS microspheres in the PS/PMMA spin coating solution increases the total number of platelets that adhere to the substrate surface over the period of the microfluidic assay. However, a detailed analysis of the adhesion rate, mean translocating velocity, mean translocation distance, and fraction of the stably adhered platelets measured during blood flow under arterial equivalent mechanical shear conditions indicates no significant difference for topographies created with or without inclusion of the PS microspheres. Von Willebrand’s disease (vWD) is the most common bleeding disorder, with approximately 1% of people affected globally . Types 1The role of nanotechnology and nanomaterials is increasing across a number of applications ,11. The Previous studies have reported the use of the coatings of adhesive proteins, such as collagen and fibrinogen ,18, whicIn its normal configuration, the DPFA lacks specificity, with respect to a specific patient’s vWF, due to the use of the endogenous vWF. The replacement of this externally-derived protein with a surface topography that can entrap autologous vWF from flowing whole blood, which can then support subsequent platelet adhesion from whole blood under arterial shear, has been suggested as a means of addressing this limitation of the DPFA ,29. Prev−1. Video rate image capture was used to undertake real-time dynamic tracking of platelet kinetics under standard DPFA conditions. As in previous studies, the total number of platelets that adhere to the substrate surface over the period of the microfluidic assay have been measured. In addition, a bespoke computer algorithm, developed to determine the kinetic behaviour of platelets on endogenous vWF-coated substrates, has, for the first time, been adapted for the measurement of platelet behaviour on the polymer demixed synthetic surfaces of interest here.In this study, a range of polymer demixed surfaces have been prepared from PMMA/PS and PMMA/PS + PS microsphere solutions in the air under ambient conditions and incorporated within the flow chamber of the DPFA with the resulting surfaces characterised both physically and chemically. Haematological analysis was carried out using fluorescently-labelled platelets in whole blood, from a single donor, perfused over the flow channel at a rate of 1500 sPolystyrene PS, Mw = 290,000 amu) and poly (methyl methacrylate)   were used to create a 25%PS/75%PMMA polymer blend, from which a 3% casting solution was created in chloroform . This polymer blend solution acts as the base spin coating system, which was then augmented at a 1:4 (v/v) ratio with uniform 50 µm PS microspheres . The 25PS75PMMA baseline polymer blend has been associated with producing a topography that can produce a positive platelet interaction ,29. More0,000 amuRigorously cleaned glass coverslips were placed on a vacuum chuck of a VTC-100 compact spin coater . The polymer casting solution was dispensed onto the glass slide until the surface was saturated, and the spin programme was then initiated. The spin profile encompassed an initial 2-min period, whereby speed was ramped up to 2000 RPM at a steady rate, before a 2-min ramp-down period. This instrument operates in air, under atmosphere conditions, i.e., there is no nitrogen purge present, and coated substrates are denoted by the term ‘air-spun’ substrates. Coated coverslips were removed from the chuck and stored in a clean, dry environment until required.Physical characterisation of the surfaces of air-spun coated substrates was carried out using atomic force microscopy (AFM) to determine the nature and scale of the resulting topography. Scans were acquired on a Veeco Digital Instrumentation Dimensions 3100  at a scan rate of 0.5 Hz across areas of 40 × 40 µm. Data were collected in the form of 3D visual plots and surface roughness parameters—Ra (arithmetic mean roughness) and Rq (root mean square roughness)—and line profiles generated from the midpoint, perpendicular to the xy scan direction, were also recorded (n = 5).X-ray photoelectron spectroscopy (XPS) was used for chemical characterisation of the surfaces using a Kratos Axis Ultra Delay Line Detector spectrometer system  equipped with Kα X-rays operating at an incident energy of 1486.6 eV. Wide energy survey scans (WESS) were collected at a pass energy of 160 eV and high-resolution spectra at 20 eV. Charge neutralisation was applied due to the insulating nature of the polymer thin films. This was applied via an electron gun operating at 1.95 A with a charge balance of 3.3 V. Post-acquisition, data were analysed using CasaXPS (version 2.3.19), where adventitious carbon was calibrated to 285.0 eV before peak fitting was completed for both oxygen and carbon spectra.Ethical approval for the study was obtained from the Research Ethics Committee of the Royal College of Surgeons in Ireland . All blood samples were collected in accordance with the Declaration of Helsinki . Blood w2 [−1. Optical images of the flow path were collected using an air-cooled (−80 °C) digital iXON EM+ CCD camera  operating in a video capture mode using MetaMorph  software, whilst illuminating the area with an Osram 103-W light source equipped with a fluorescein iosthiocyanate (FITC) filter set .Dynamic platelet function tests were carried out within a DPFA flow assembly, described in detail elsewhere with relevant schematics ,21,23,29On operator observation of the first platelets sticking to the surface, data collection was initiated, recording images at 19 frames per second (fps) for 1000 frames, followed by time lapse image acquisition of 120 frames at 0.5 fps. In all instances, the final frame from the time lapse image stack (120/120) was used to quantify % surface coverage using the Volocity™ software package . Platelets were masked using a “find objects by % intensity” thresholding tool using upper and lower limits of 100 and 85, respectively. Total area highlighted was then measured in pixels, with respect to the full image pixel number, and the percentage coverage was calculated.3/s), mean translocating velocity (µm/s), mean translocation distance (µm), and fraction of stably adhered platelets were measured. Datasets from the initial 1000-frame stack were analysed using an established algorithm . In shorCoatings of PS/PMMA and PS/PMMA_750 spin coated, in air, onto glass substrates were characterised by atomic force microscopy (AFM) and x-ray photoelectron spectroscopy (XPS) before being subject to haematological assessment. AFM analysis provides the physical characterisation of the surface topographical features, with p > 0.05), whilst the root mean square roughness was significantly different between substrates.No significant differences between the data for the 25PS75PMMA and 25PS75PMMA_750 surfaces are evident from As shown in Quantitative analysis of the deconvoluted C 1s and O 1s contributions to the spectral envelopes are presented in The 25PS75PMMA and 25PS75PMMA_750 substrates were subjected to haematological analysis using the DPFA protocol. Data were recorded by way of obtaining fluorescent images from the observation of the adherence of the first platelet to the surface across a total of 5 min, comprising an initial 1000-frame stack, followed by time lapse of 120 frames at lower acquisition rate, with the final frame (120/120) used for direct visual comparison of the surface coverage of the platelets.Fluorescent images for the five replicates of the equivalent air-spun 25PS75PMMA_750 substrates analysed within the DPFA assembly are provided in Platelet adhesion appears to be significantly higher for this surface, with platelets covering a large proportion of the field of view. Surface coverage values are again shown in The dynamic behaviour of platelets across the 25PS75PMMA and 25PS75PMMA_750 surfaces was assessed from the initial 1000-frame image stack to ensure that only platelet-surface interactions were analysed, in order to negate (as far as possible) platelet-platelet and thrombotic activities. p > 0.05) was evident from the number of runs performed (n = 5). These data confirm that, whilst there are clear differences on visual inspection of the images (120/120) captured, there is no statistically significant difference in the dynamic behaviour of platelets on each type of substrate topography.Whilst both the mean translocating distance and mean translocating velocity appear to slightly decrease with the addition of 50 µm spheres, both the total number of tracks and fraction of stably adhered platelets appears to increase. However, since the sample-to-sample variability was high see , no statThe surfaces created by spin coating of 25PS75PMMA and 25PS75PMMA_750 polymer demixed solutions in air under ambient conditions have been analysed with regard to the respective physical and chemical properties. Their respective ability to entrap vWF and bind platelets from flowing whole blood has been determined using a dynamic platelet function assay.The AFM results show that the topography of the 25PS75PMMA surface consists of sharply defined features (FWHM 500 nm to 1 µm) ranging from −100 to +75 nm in height. This is a significantly different topography than that created by spin coating the same PS/PMMA blend under nitrogen purge conditions, where it was found that the features were much more rounded and globular . The airXPS analysis confirms that the uppermost surface of both the air-spun 25PS75PMMA and 25PS75PMMA_750 substrates is that of PMMA only . Small dThe haematological response to the 25PS75PMMA and 25PS75PMMA_750 substrates was assessed by way of a DPFA test protocol, which has been reported previously ,21. PrevNanotopographies created from 25PS75PMMA and 25PS75PMMA_750 blends present novel substrates that facilitate a high level of platelet adhesion and, therefore, vWF binding. This is a significant improvement in the field of platelet function assay testing, as it provides an opportunity for patient-specific platelet and protein (vWF) assessment from a single, whole blood sample.−1, which then directly promotes the subsequent platelet interactions thereon. Images captured at the end of flow experiments show a substantial increase in the surface coverage of platelets on the 25PS75PMMA_750 substrate. The assessment of associated platelet dynamics on both types of substrates during the time period of the assay using a model adapted from previous studies with immobilised vWF surfaces indicates no significant difference in translocation behaviour, in terms of adhesion, velocity, or distance travelled. Whilst 25PS75PMMA substrates show a lesser number of platelets covering the surface at the end of analysis, those platelets present successfully interact with the respective topographies. The findings from this study significantly extend our knowledge of the potential use of air-spun polymer demixed substrates for dynamic platelet assay that offer patient-specific protein-platelet screening from a single blood sample. Whilst the work produced within this study addresses the research question asked, it is noted that there are associated limitations. More specifically, further research is needed to understand the accuracy of this microfluidic assay against commercially existing assays to validate its application, and the sample set needs to be widened to include different blood groups. Future works to develop this research, focusing on a collective of blood donors, are required, in order to investigate the different blood types and health conditions  and gain an insight on the robustness of this new synthetic surface as a sensor for the diagnosis of vWD and associated diseases that effect vWF-platelet interactions.Polymer demixed thin films fabricated by spin coating PMMA/PS in chloroform solutions with and without the addition of PS microspheres onto glass substrate have surface topographies capable of entrapping vWF from whole blood flowing at a rate of 75 µL/min, equivalent to an arterial shear of 1500 s


High mountainous environments are of particular interest as they play an essential role for life and human societies, while being environments which are highly vulnerable to climate change and land use intensification. Despite this, our knowledge of high mountain soils in South America and their microbial community structure is strikingly scarce, which is of more concern considering the large population that depends on the ecosystem services provided by these areas. Conversely, the Central Andes, located in the Mediterranean region of Chile, has long been studied for its singular flora, whose diversity and endemism has been attributed to the particular geological history and pronounced environmental gradients in short distances. Here, we explore soil properties and microbial community structure depending on drainage class in a well-preserved Andean valley on the lower alpine vegetation belt (~2500 m a.s.l.) at 33.5˚S. This presents an opportunity to determine changes in the overall bacterial community structure across different types of soils and their distinct layers in a soil depth profile of a highly heterogeneous environment.Five sites closely located (<1.5 km) and distributed in a well preserved Andean valley on the lower alpine vegetation belt (~2500 m a.s.l.) at 33.5˚S were selected based on a pedological approach taking into account soil types, drainage classes and horizons. We analyzed 113 soil samples using high-throughput sequencing of the 16S rRNA gene to describe bacterial abundance, taxonomic composition, and co-occurrence networks.Almost 18,427 Amplicon Sequence Variant (ASVs) affiliated to 55 phyla were detected. The bacterial community structure within the same horizons were very similar validating the pedological sampling approach. Bray-Curtis dissimilarity analysis revealed that the structure of bacterial communities in superficial horizons (topsoil) differed from those found in deep horizons (subsoil) in a site-specific manner. However, an overall closer relationship was observed between topsoil as opposed to between subsoil microbial communities. Alpha diversity of soil bacterial communities was higher in topsoil, which also showed more bacterial members interacting and with higher average connectivity compared to subsoils. Finally, abundances of specific taxa could be considered as biological markers in the transition from topsoil to subsoil horizons, like Fibrobacterota, Proteobacteria, Bacteroidota for shallower soils and Chloroflexi, Latescibacterota and Nitrospirota for deeper soils.The results indicate the importance of the soil drainage conditions for the bacterial community composition, suggesting that information of both structure and their possible ecological relationships, might be useful in clarifying the location of the edge of the topsoil-subsoil transition in mountainous environments.The online version contains supplementary material available at 10.1186/s40659-023-00445-2. Mountain soils cover 22-27% of the Earth’s total soil surface . These sAs the altitude, depth exert influences on microbial communities by shaping various habitat features. Factors such as pH, temperature, water availability, oxygen levels, and nutrient availability are known to vary with depth, thereby impacting the composition of microbial populations. For instance, soil microbial diversity has been described as decreasing with depth , 10. WhiOur study aimed to determine changes in the overall bacterial community structure across different types of mountainous soils and their distinct layers (horizons) occurring in a soil depth profile. This was carried out by describing and sampling soil profiles in five sites, reflecting five different drainage classes at four contrasting geomorphic positions: north and south facing colluvial slopes, floodplain marsh (fen) and floodplain meadow from a Mediterranean mountain environment in the central Andes (Lo Encañado floodplain) Figure A. We hyp2) tributary catchment of the Maipo River that drains towards a small oligotrophic and monomictic lake of the same name , near a small (38.8 km1 and Ck2) at the bottom, between 135 and 190 cm  and consistency (ASVs identified in at least two out of the three replicates), retaining 18,427 ASVs for posterior analyses. These ASVs encompassed 55 phyla and 695 genera across all soil profiles  followed by a MetagenomeSeq analysis and Mann–Whitney U test by profile  and eight to SS samples .When comparing all profiles in one ANCOM, thirteen phyla exhibited significant differences in abundance between TS and SS samples Figure  , located in the lower alpine vegetation belt with a cold summer mediterranean climate condition (Csc under Köppen climate classification) . Our aimThe Lo Encañado valley was carved during the Pleistocene glaciations and subsequently filled with fluvial deposits, while the valley sides are covered with a continuum of colluvial deposits. The soils found at the valley reflect a high variety of properties and associated pedogenic processes, similar to other alpine regions of the world in terms of the high diversity of soils that could be found in small areas . Small cThe contrast of drainage conditions could mostly be explained by the geomorphic position of the soil profiles, namely the topography soil forming factor described by Jenny, . AdditioWhile the effect of geographic distance has been proposed as a factor influencing soil microbial communities , there sBetween soil profiles and horizons, there are patterns of diversity that form a clear structure in the soil microbial community. This is observed when analyzing the replicates for the soil samples analyzed here, where there is a relationship of microbial diversity and abundance that remained very similar, which shows the benefit of a sampling strategy based on pedogenic horizons. It should be considered that there are biotic and abiotic factors that strongly influence the assembly of a microbial community, both at the level of diversity and abundance. For instance, the effect of soil moisture produced a gradient of richness and diversity (from higher to lower) from humid, subhumid to semiarid soils . HoweverOur data indicated that soil classified as TS, which corresponded to the most superficial horizons, were mostly shaped by different soil types in comparison to SS horizons. Both features (depth and soil type) have been used to classify TS and SS samples in studies carried out in several soil types and geographic locations , 10, 31.Alpha diversity was also lower for the deeper horizons (SS samples) of almost all profiles, a result that is displayed in many other studies using a variety of different techniques, like pyrosequencing , 41, 42,In terms of dominant taxa, Proteobacteria and Acidobacteriota were the most dominant taxa in all profiles, except in LE05, where Acidobacteriota is surpassed in relative abundance by Bacteroidota and Chloroflexi. The dominance of these phyla has been observed in a wide variety of soil types and depths in various ecosystems worldwide, indicating that they play a fundamental role in this type of ecosystems , 41, 46.Regarding changes in the bacterial distribution across the distinct layers in a soil depth profile we observed that the structure of the bacterial community displayed a specific stratification between TS and SS. At phylum taxonomic level, we found several phyla that were enriched or decreased in the SS with respect to the TS. These results provide valuable information for microbial ecology of deep soils, which remains particularly limited, especially in mountain pristine environments from Mediterranean regions. Members of Proteobacteria and Bacteroidota have shown to be dominant in TS horizons and to decrease from the surface to deeper horizons , 42, 48,Our results on bacterial diversity and composition support the idea that depth generates environmental gradients that affect soil bacterial composition and structure , 41, 57.Soils in the lower alpine vegetation belt locality of Lo Encañado show a high pedogenic variability which is closely linked to its geomorphological position in the landscape and is reflected in the drainage conditions described. This spatial pattern of soil variation is connected in a different way to the microbial communities that each soil profile hosts, as their structure is closely linked in all subsoil or topsoil horizons of the same soil profile. The spatial pattern (SS versus TS) of the soil microbial communities could not be clearly linked to soil physico-chemical properties or depth, but the similarity trends suggest that moisture availability might play an important role, as different soil developments could create long-term conditions for niche differentiation and the creation of particular microbial community patterns for each site. In fact, more bacterial members interacted within topsoils versus subsoils, showing also higher average connectivity. The composition of soil bacterial communities and the abundances of specific taxa could be considered to identify the transition from topsoil to subsoil horizons, like Fibrobacterota and Bacteroidota for shallower soils and Chloroflexi, Latescibacterota and Nitrospirota for deeper soils. The close relationship among topsoil microbial communities, despite differences in landscape position and vegetation cover, suggests that their structure is mainly influenced by shared soil surface environmental variables  given their geographic proximity. Finally, we conclude that a geomorphological and pedological approach is an adequate method to capture the microbial biodiversity in a mountain environment.The sampling area is located in the Lo Encañado valley  at ~2500 m a.s.l. in the Andes mountains in the central region of Chile  were examined in spring 2019 , electrical conductivity , soil organic matter (SOM) , micronutrients  (DTPA extraction), available nitrogen (using Kjeldahl distillation), available phosphorus (using a P-Olsen method) and available potassium (extraction with 1mol/L ammonium acetate solution at pH 7.0 and subsequent determination by atomic absorption and emission spectrophotometry), based on the methods described by Sadzawka et al. (2004) . The ana2HPO4, 1.5M NaCl, 1% (w/v) CTAB] with five grams of soil and mixed using a vortex followed by incubation at 65 °C for 2 hours with mixing every 30 minutes. Subsequently, the mixture was centrifuged at 9000g for 5 min at room temperature and the supernatant was transferred in a clean tube and added 0.5 volume of ethanol 100%. In the next step, silica columns from the DNeasy Blood & Tissue kit (Qiagen)  were used to capture DNA, following the manufacturer's recommendations from that step. The integrity of the DNA was evaluated through electrophoresis with 1% agarose gel, DNA quantification was performed through fluorometry using Qubit (Invitrogen) and DNA was stored at 4 °C until further procedures. Microbial DNA was amplified using a bacteria-specific primer set, 28 F (5′-GA GTT TGA TCM TGG CTC AG-3′) and 519R (5′-GWA TTA CCG CGG CKG CTG-3′), flanking variable regions V1-V3 of the 16S rRNA gene [www.mrdnafreesoftware.com created the file formats expected by QIIME 2 for downstream analysis.To increase the chances of obtaining DNA from soil samples, we followed the protocol described in Mandakovic et al. . BrieflyRNA gene , 76. SeqBioinformatic analysis was performed with QIIME 2 2021.11 . Raw seqAll ASVs were aligned with Mafft   . ANCOM cBacterial co-occurrence networks from topsoil and subsoil samples were generated as described by Mandakovic et al.  using geAdditional file 1: Figure S1. Location of the study sites. A. Location of the study site in the Central Andes of Chile. B. Lo Encañado Valley according to vegetation belts following distribution modelled by Luebert and Pliscoff. DEM: Digital Elevation Model.Additional file 2: Figure S2. View toward Lo Encañado valley with the typical U shape of glacial origin. Bottom left is the lake of the same name; flat valley bottom consists of a flood plain sustaining an alpine meadow. The limit between the valley bottom and lateral escarpment are filled with colluvial deposits. Soil profiles are indicated by a white arrow.Additional file 3: Figure S3. Alpha diversity and richness of TS and SS samples. Comparison of alpha diversity measures between the topsoil samplesand subsoil samples. Horizontal bars within boxes represent median. The tops and bottoms of boxes represent 75th and 25th quartiles, respectively. All outliers are plotted as individual points. * Asterisk denotes significant difference at the P ≤ 0.05 level using Kruskal-Wallistest.Additional file 4: Figure S4. Alpha diversity and richness between different sites. Comparison of alpha diversity measures between the different sites. Horizontal bars within boxes represent median. The tops and bottoms of boxes represent 75th and 25th quartiles, respectively. All outliers are plotted as individual points. *Letters denotes significant difference at the P ≤ 0.05 level using Kruskal-Wallistest.Additional file 5: Figure S5. Sampling strategy. Soil sampling procedure and nomenclature used for biological samples for a ficticious profile LEX.Additional file 6: Table S1. Physicochemical and environmental measurements. Table S2. Selected properties of the Horizons at Lo Encañado. Table S3. Community metrics including phyla, genera and OTU richness between soil profiles. Table S4. Relative abundance of bacterial phyla among profiles and horizons. Table S5. Kruskal-Wallis test for alpha diversity analysis. Table S6. Differential abundance analysis between topsoil and subsoil samples at phylum level.Additional file 7: Table S7. Differential abundance analysis between topsoil and subsoil samples at family level.


Type 2 diabetes (T2DM) and mild cognitive impairment (MCI) are common late-life physical and cognitive health conditions. Illness perceptions, an individual’s personal beliefs about the conditions, should be explored in the context of disease characteristics . This secondary analysis explored illness perceptions with a priori hypotheses about control (perceived controllability) and coherence (perceived understanding) dimensions among persons with T2DM and MCI, treating each as an exemplar of late-life physical and cognitive health conditions. We also explored whether age, education, and comorbid conditions moderate the relationships between T2DM or MCI condition groups and illness perceptions.This cross-sectional, descriptive study examined baseline data collected from 146 T2DM to 90 MCI participants in 2 independent studies. The 9-item Brief Illness Perception Questionnaire was used to identify the similarities and differences in illness perceptions among persons with T2DM and MCI. We performed hierarchical linear regression controlling for identified covariates.b = −0.943, p = .009), treatment control , and coherence , after controlling for covariates. The results suggest that persons with MCI were likely to believe that their condition is less controllable  and less understandable compared with their T2DM counterparts. Such associations remained statistically significant when the interactions were added to the models.We found that T2DM and MCI participants had significantly different illness perceptions, including perceptions of personal control (As T2DM and MCI are prevalent late-life conditions, health care professionals should consider individuals’ subjective perceptions about their conditions in the context of disease characteristics when counseling secondary prevention strategies for disease management. Further research on illness perceptions in other conditions is needed to ensure the replicability of our findings. Translational Significance: Despite the exponential increase of age-related chronic conditions, knowledge of individuals’ beliefs about their conditions  in the context of disease characteristics  remains limited. The findings of similarities and differences in illness perceptions among persons with type 2 diabetes and those with mild cognitive impairment, as examples of late-life physical and cognitive conditions, demonstrate the significance of understanding illness perceptions in the context of disease characteristics. Given that illness perceptions affect self-management behaviors, this study will accelerate the translation of research on disease management in age-related chronic conditions into clinical practice.The increasing number of older adults living with chronic conditions  has majoIndeed, T2DM and AD are among the most prevalent age-related physical and cognitive conditions in the United States . Althougillness perceptions, affect disease self-management behaviors (Common Sense Model (CSM) of Self-Regulation , (2) consequences , (3) cause , (4) timeline , (5) cure/control , (7) emotional representations , and (8) concerns  and whether the condition is understandable or confusing (coherence) to them, is paramount in developing effective interventions aiming at symptom or condition management in the context of disease characteristics.Therefore, exploring illness perceptions of T2DM and MCI, especially whether affected individuals believe their condition can be controlled or not  and illness perceptions.This cross-sectional, descriptive study used existing baseline data collected from two independent studies. For the T2DM sample, we used the HABIT Study (hereafter referred to as HABIT), designed to improve medication adherence among persons with T2DM (NIH/NINR P01 NR010949). The Return of Amyloid Imaging Scan Results (RAISR) Study (hereafter referred to as RAISR) was used for the MCI sample, which explored the decision-making process of pursuing AD biomarker testing and reactions to the scan results among persons with MCI and their care partners  were recruited from outpatient clinical practice sites within the University of Pittsburgh Medical Center Health System. Individuals were eligible if they were (a) at least 40 years of age and in treatment for T2DM; (b) taking one or more medications prescribed by a physician; (c) self-managing their medications; (d) an English speaker, and (e) had access to a telephone. persons with T2DM were excluded if they were (a) had T2DM medications managed by others, (b) unwilling to use a medication diary or electronic event monitor, and (c) participated in other intervention studies.In HABIT, persons with T2DM (N = 90) was recruited from the University of Pittsburgh Alzheimer’s Disease Research Center . Individuals were eligible if they (a) were 50 years of age or older, (b) had an MCI diagnosis through a multidisciplinary consensus meeting at the ADRC based on the guidelines of MCI classification from the National Alzheimer’s Coordinating Center and the National Institute on Aging and Alzheimer’s Association  were excluded from this analysis, so the analysis sample for persons with T2DM was 147. Care partner participants in RAISR were also excluded. This study was approved by the University of Pittsburgh.The MCI sample  rescausality item, all items, consequences, timeline, personal control, treatment control, identity, concerns, coherence, and emotional representations, were rated on an 11-point Likert scale from 0  to 10 (extremely). We excluded the causality item because the item is an open-ended question. As suggested by control and coherence items were assessed using the following questions: “How much control do you feel you have over your ‘diabetes’ [HABIT] or ‘memory or thinking difficulties’ [RAISR]?” and “How well do you feel you understand your ‘diabetes’ [HABIT] or ‘memory or thinking difficulties’ [RAISR]?” The Brief IPQ has been widely used to investigate illness perceptions across chronic conditions  reviewed and identified similarities and differences in the items of the comorbid condition questionnaires. Next, each comorbid condition item in RAISR was linked to the CRCD Comorbidity Questionnaire  because N = 147) and RAISR (N = 90) were screened using descriptive and exploratory methods separately and collectively for anomalies . The baseline data sets from both studies were pooled for analysis based on sociodemographic and clinical factors, and each dimension of illness perception.All analyses were performed using SPSS Statistics Version 28.0 . The level of statistical significance for two-sided hypothesis testing was set at less than .05. Prior to analysis, each of the data sets from HABIT  were used in our analysis.Through the data screening, we excluded one individual (0.68%) from HABIT because this person did not complete the Brief IPQ. Except for the t tests (for continuous-type variables) or chi-square tests of independence . If significant differences were found, the variables were controlled for as covariates in the main analyses. We also explored each dimension of illness perception using descriptive statistics  between persons with T2DM and those with MCI. Second, we performed a multivariate analysis of covariance (MANCOVA) to compare the set of illness perceptions simultaneously based on the eight dimensions of the Brief IPQ between persons with T2DM and those with MCI. Accordingly, hierarchical multiple linear regression analysis was conducted to examine the similarities and differences in each dimension of illness perception among persons with T2DM and those with MCI. For each dimension of illness perception, three successive linear regression models were estimated. In block one, a model with only covariates  was estimated. In block two, a primary independent variable  was added. In the final block, interaction terms between the disease group variable and targeted covariates  were added to explore the possible interactions between the disease group and targeted covariates on each dimension of illness perception. To minimize possible multicollinearity, variables of age, education, and the number of comorbid conditions were centered when creating the interaction terms.First, group comparisons were performed for all sociodemographic characteristics  by participants’ disease group (T2DM [0] or MCI [1]) using independent N = 236) included 146 persons with T2DM (61.9%) and 90 (38.1%) persons with MCI (p = .089). MCI participants were older and had more years of education, with an average of 72.5 (SD = 8.77) years of age and 16.5 (SD = 2.55) years of education, than T2DM participants who were an average of 64.4 (SD = 8.65) years of age and had 14 (SD = 2.82) years of education. Although six-tenths of persons with MCI  were women, less than half  were women in the T2DM sample. Most participants were White in both T2DM  and MCI  samples. Over half of the T2DM sample  and three-quarters of MCI sample  were married or living as married.The final pooled sample , indicating that all participants perceived their conditions as chronic rather than acute. The identity dimension was the lowest mean score in the T2DM group (mean ± SD = 4.10 ± 2.49), suggesting persons with T2DM may experience fewer disease-related symptoms. In the MCI group, the consequences dimension had the lowest mean score (mean ± SD = 4.24 ± 2.20), indicating that persons with MCI may perceive the diagnosis or symptoms of MCI had little impact on their life. The MANCOVA test was statistically significant , indicating that persons with T2DM and MCI have significantly different illness perceptions about their conditions.Results from hierarchical linear regression models assessing the differences between persons with T2DM and MCI for each dimension of illness perception considering covariates are summarized in b = −0.432, p = .027) for the perceived consequences of health conditions .Persons with T2DM and with MCI did not have a significantly different perception of the consequences of their health conditions . Howevernditions . This inb = −0.945, p = .008; b = −0.903, p = .013; A significant association between the disease group and timeline perception  and treatment control , and remained significant when the interaction terms were added to the model . These findings suggest that participants with MCI felt that their cognitive changes are not likely to be controlled by either their effort or medical treatment compared with their T2DM counterparts. We found a significant interaction between the disease group and education on treatment control .As seen in b = 0.987, p = .012; Model 2, b = 0.922, p = .020; Model 3; Participants’ disease group was the most significant predictor of the identity perception . This suggests that persons with MCI were more worried about their disease-related symptoms than those with T2DM. We found an interaction between the disease group and the number of comorbid conditions on concerns .As indicated in b = −1.265, p = .001), and this association remained significant when interactions were added to the model . The findings demonstrate that MCI participants believed they had a lower understanding of their disease than T2DM participants. Older age was significantly associated with a better perceived understanding of the disease in MCI participants  and less likely to make sense to them compared with those with T2DM. This study also revealed partial support for the exploratory aim, the moderating role of age, education, and comorbid conditions in the relationship between participants’ disease group (T2DM or MCI) and illness perceptions.The findings of lower levels of perceived control over the conditions among persons with MCI than those with T2DM are congruent with previous studies in the context of disease characteristics. For example, persons with hypertension  and thosInterestingly, in the MCI participants, more years of education were associated with a lower treatment control perception, yet this association was not significant in the T2DM participants. Information about MCI continues to evolve rapidly, especially biomarkers of ADRD for early detection and treatment. Persons with MCI who attained higher education may actively seek up-to-date information through various resources so that they may come to believe that any treatments might not be effective for their cognitive function. A meta-analysis study found that low control perceptions negatively affected psychological distress and disease state across chronic health conditions , suggestConsistent with prior investigations of physical and cognitive conditions , we obseUnlike MCI, T2DM is a relatively specific condition in the context of its diagnostic process and management of blood glucose, which may be associated with T2DM participants’ better perceived understanding of their condition or its symptoms than their MCI counterparts. Literature suggests that the extent of an individual’s perceived understanding of the disease has been associated with approaches to managing the disease or symptoms . Yet, itWe also found that individuals who were older were more likely to believe they had a better understanding of the condition than those younger among MCI participants but not among T2DM participants. Older individuals may be more exposed to cognitive impairment than younger in that cognitive changes increase with aging. However, in a nationwide study of 6,141 older adults with and without cognitive impairment , youngerIt is noteworthy that other dimensions of illness perceptions were also significantly different between persons with T2DM and those with MCI in the form of main effects or interaction effects, which was not our primary hypothesis. In particular, the interaction effects between the number of comorbid conditions and certain illness perceptions, consequences, and concerns, should be further examined. In our analysis, among persons with MCI, less comorbid conditions were associated with more worries and a higher perceived impact of MCI on life, which was the opposite finding among their T2DM counterparts. One possible explanation is that persons with MCI with less comorbid conditions may pay more attention to their cognitive changes than those with more comorbid conditions. The opposite perception among persons with T2DM may be attributable to high comorbidity risks of T2DM if not managed well. Given that older adults with multiple conditions had complex illness perceptions intertwined with other factors , illnessOur study has some limitations. First, our sample of participants with T2DM and MCI was drawn from two existing datasets, which may lead to study bias due to the mixing of different datasets. To minimize this issue, we controlled for sociodemographics  and the number of comorbid conditions which were identified as covariates. Despite the fact that the time since diagnosis and current success in disease management may be essential factors of illness perception, we could not include these variables because the parent studies did not collect the information. In addition, although some participants might have been diagnosed with both T2DM and MCI conditions, we were not able to assess how having both conditions affected the perceptions of T2DM or MCI we targeted due to the nature of the secondary analysis. Second, the use of self-reported questionnaires can be an issue. For example, responses may be influenced by social desirability; that is, participants might answer the items considered more socially accepted. Lastly, because participants had an average of five comorbid conditions, they might have multiple illness perceptions or perceptions about conditions other than T2DM or MCI, which might affect the study results  even thoNevertheless, the findings of this study provided clinical and research implications in that, to our knowledge, this is the first theory-driven study that identified similarities and differences in illness perceptions in chronic disorders in the context of disease characteristics . As T2DM and MCI are common late-life chronic conditions, our sample represents key targets for treatments to delay or stop disease progression. Assessment of unique illness perceptions depending on the disease characteristics may be an essential underpinning of patient counseling for symptom and disease management in clinical and research settings. Illness perceptions in other late-life conditions should also be explored to ensure that our findings can replicate.igad027_suppl_Supplementary_MaterialClick here for additional data file.


Diplocarpon rosae F.A. Wolf) and cercospora leaf spot of rose (Rosisphaerella rosicola Pass.), affect both the health and ornamental value of the plant. Most studies on black spot disease resistance have focused on diploid germplasm, and little work has been performed on cercospora leaf spot resistance. With the use of newly developed software tools for autopolyploid genetics, two interconnected tetraploid garden rose F1 populations (phenotyped over the course of 3 years) were used for quantitative trait locus (QTL) analysis of black spot and cercospora leaf spot resistance as well as plant defoliation. QTLs for black spot resistance were mapped to linkage groups (LGs) 1–6. QTLs for cercospora resistance and susceptibility were found in LGs 1, 4, and 5 and for defoliation in LGs 1, 3, and 5. The major locus on LG 5 for black spot resistance coincides with the previously discovered Rdr4 locus inherited from Rosa L. ‘Radbrite’ (Brite Eyes™), the common parent used in these mapping populations. This work is the first report of any QTL for cercospora resistance/susceptibility in tetraploid rose germplasm and the first report of defoliation QTL in roses. A major QTL for cercospora susceptibility coincides with the black spot resistance QTL on LG 5 (Rdr4). A major cercospora resistance QTL was found on LG 1. These populations provide a genetic resource that will further the knowledge base of rose genetics as more traits are studied. Studying more traits from these populations will allow for the stacking of various QTLs for desirable traits.Garden roses are an economically important horticultural crop worldwide, and two major fungal pathogens, black spot ( Thus, breeders are focused on the development of resistant commercial cultivars, as these cultivars require fewer fungicide applications throughout the growing season (Diplocarpon rosae F.A. Wolf) and cercospora leaf spot of rose (Rosisphaerella rosicola Pass. previously known as Cercospora rosicola Pass.) are two important fungal pathogens, both of which can cause defoliation. Black spot is ubiquitous in warm humid regions. Black spot infections cause black lesions on leaves with feathery borders that spread, causing chlorosis of the leaves around the lesion border , triploid, and tetraploid  and quantitative trait loci (QTL) for horizontal resistance -based linkage maps in tetraploid roses. Five of these maps were genotyped using the WagRhSNP 68k array , and two1 populations. This study used multiple QTL mapping software to 1) identify regions of interest affecting black spot resistance, cercospora resistance, and defoliation; 2) use QTL results to identify individuals and haplotypes useful for breeding with these populations; and 3) incorporate findings in this study with previous research to select individuals carrying multiple disease resistance loci for use in breeding.To extend our knowledge on black spot resistance, address the gap in research on cercospora resistance, and investigate the genetic factors affecting defoliation in tetraploid garden roses, we used two tetraploid garden rose F22.11 bi-parental tetraploid garden rose families, Rosa L. ‘ORAfantanov’ (Stormy Weather™) × Rosa L. ‘Radbrite’ (Brite Eyes™) (SWxBE) n = 200 and Rosa L. ‘Radbrite’ (Brite Eyes™) × Rosa L. ‘BAIgirl’ (Easy Elegance® My Girl) (BExMG) n = 157, were developed in 2016 as a joint effort between Texas A&M University Rose Breeding and Genetics Program and Weeks Roses. These populations were previously described and used for mapping rose rosette disease resistance  used for QTL mapping and phenotypic variance due to genotype, environment (each set of observations), and reps nested within the environment, using the Restricted Estimated Maximum Likelihood (REML) method in R package ASReml-R version 4.1 . Mixed mi at environment j in block k, i, j (monthly observation set), where 2.4Unexpanded young leaf tissues were collected, flash-frozen in liquid nitrogen, and stored at −80°C until DNA extraction. DNA was extracted using a cetyl trimethylammonium bromide (CTAB) protocol . Genotyphttps://www.rosaceae.org/publication_datasets).MAPpoly estimates the parental haplotypes inherited by each progeny at every map position. Thus, in the QTL mapping process, if we identify a QTL position and the parental homolog that donates the favorable allele to the trait, we are able to identify individuals with the QTL carrying this specific homolog segment. This allows the selection of individuals with multiple favorable QTL alleles and enables the efficient stacking of disease resistance QTL. Monthly, yearly, and overall BLUEs resulted in 57 QTLs that were visualized with the newly developed R Shiny application VIEWpoly . This ap2.5In addition to the bi-parental populations used for the QTL studies, another approach leveraging the interconnectedness of these two populations allowed for a joint QTL analysis. A Julia software package PolyOrigin (Version 0.5.10)  was used33.1Black spot, cercospora, and defoliation were phenotyped over 15 months (environments) over the course of 3 years. We considered each month a different environment due to varying weather conditions creating different disease pressure on the plants. Black spot ratings had a low level (4%) of phenotypic variation attributed to genotypic effects, whereas cercospora (49%) and defoliation (18%) ratings had higher amounts of variation attributed to genetic factors (A moderate positive correlation was observed between black spot and defoliation (0.52), and low negative correlations were observed between cercospora and black spot (−0.24) and between cercospora and defoliation (−0.26) , for simplicity, we present QTLs detected using BLUEs calculated from each year and across all years , and in the SWxBE population on LGs 1, 2, 4, and 5 accounting for 12%–73% of the PVE  phenotyped over the course of 3 years. The black spot meta-QTL study reported by Rdr4 or that Meta_2_5 was the same genetic factor as Rdr4, but their locations did not collocate due to mapping imprecisions. The confidence intervals of our LG 5 QTLs, qRDR.BExMG.2019-ch5, qRDR.BExMG.overall-ch5, qRDR.SWxBE.2019-ch5, and qRDR.SWxBE.overall-ch5, are near but not collinear with the estimated position of Meta_2_5. In contrast, our LG 5 QTL for black spot resistance coincides with the reported position of Rdr4 . Since the meta-QTL studies of Rdr4, our findings indicate that Meta_2_5 may be another genetic factor controlling black spot resistance further upstream of Rdr4. A gene search in the Rosa chinensis whole genome v1.0 assembly (Rdr4 (29.81–57.86 Mbp) revealed the presence of eight TIR-NBS-LRR-, one CC-NBS-LRR-, and 26 NB-ARC-related genes, all of which are related to general disease resistance mechanisms (Rdr4) is interesting, additional work is needed to better localize our QTL and to identify more suitable candidate genes in the pertinent germplasm.As discussed in the Introduction, many factors make it difficult for studying black spot disease in a landscape-like environment. However, we used multiple time points over the course of 3 years to look at the temporal progression of the disease to help overcome the uncertainty of weather conditions. Using a mixed model approach, we were able to estimate the BLUEs for each genotype that best represents the performance of each genotype while taking into account the environmental variance. A major QTL for black spot resistance was detected on LG 5 in 2019 and in the overall analysis. This QTL was localized to a region on LG 5 where the black spot resistance locus, y mapped   able to overcome the Rdr4 in our location or 2) the selection of black spot strain(s) able to overcome Rdr4 that were initially present in low frequency. One of the potential limitations of this study is the absence of a host differential set  from My Girl collocate with Meta_2_3  and overRdr4). The QTL is inherited from the same parental homolog  to create progeny that have black spot resistance without cercospora susceptibility. However, if there was only one genetic factor controlling these two traits, this association cannot be broken. In either case, further investigation will require the creation of larger mapping populations to capture more recombination breakpoints to determine if there are one or two genetic factors.Interestingly, there is a cercospora leaf spot susceptibility QTL allele that colocalized with the black spot resistance QTL on LG 5 (Rdr4) with the cercospora resistance factor on LG 1. The BExMG population has 34 individuals, and the SWxBE population has 53 individuals with both of these resistance factors. In the case of the BExMG population, we can also select individuals carrying black spot resistance factors in LG 3 and LG 5 as well as the LG 1 cercospora resistance factor (n = 22). The recent progress in developing tools for polyploids allows the rapid characterization of individuals with respect to QTL composition, which, in turn, enables the selection of genotypes that carry multiple favorable alleles for their use in subsequent breeding. One could argue that since the black spot resistance QTL on LG 5 (Rdr4) has lost effectiveness over time while the QTL for cercospora susceptibility on LG 5 is stable over time, one should avoid using black spot resistance in LG 5 in order to avoid susceptibility to cercospora leaf spot; however, while that may be the case for our specific environment, in other less humid and cooler environments (where cercospora is less of a problem), it may beneficial to have the Rdr4 black spot resistance locus. Depending on the target environment, one may choose one trait over the other. However, for our environment, having the cercospora resistance QTL on LG 1 may be more important than selecting against the cercospora susceptible QTL on LG 5. Our analysis shows that having both cercospora resistance on LG1 and cercospora leaf spot susceptibility QTL on LG5 is not as deleterious as only selecting for the susceptibility factor on LG 5 (Rdr4 (and consequently selects for cercospora susceptibility on LG 5), cercospora resistance on LG 1 should also be selected.A major QTL for cercospora resistance was also detected on LG 1 in both populations where a simplex factor from Brite Eyes lowered cercospora disease incidence. The QTL region on LG 1 spans 4.9–38.75 Mbp and has 56 TIR-NBS-LRR-, 11 CC-NBS-LRR-, 64 NB-ARC-related genes within this region along with several predicted disease resistance genes . Assuming independent segregation, 0.754 = 31.64% of the progeny in the crosses with all four QTLs would have at least all four QTLs in simplex configuration, and 0.254 = 0.39% would have all four QTLs containing two copies of the favorable allele. Tetraploid roses can tolerate some level of inbreeding; thus, intercrossing progeny identified to carry resistance QTLs for all four traits may be a viable breeding strategy. Using these discovered QTLs, we can begin to stack alleles of interest to improve disease resistance, and combined with other QTLs for aesthetic traits like low defoliation or high flowering intensity, we can create genotypes that have favorable alleles for multiple traits. However, further work needs to be performed to develop user-friendly diagnostics to track the inheritance of the QTL.The goal of studying our mapping populations was to gain insight into the genetic architecture of disease resistance and begin pyramiding resistance alleles within the context of relevant germplasm. Newly developed software tools allowed us to quickly identify individuals that carry multiple alleles of interest for use in breeding. For example, in the BExMG population, one may want to select for resistance to black spot on LG 3 and 5, cercospora resistance on LG 1, and RRD resistance previously discovered on LG 5 . The Shi5Rdr4 black spot resistance locus. We discovered a novel cercospora leaf spot resistance QTL on LG 1 and a cercospora susceptibility QTL on LG 5. However, due to the linkage between black spot resistance and cercospora susceptibility QTL on LG 5, caution is needed to use these for fungal disease resistance breeding. Defoliation QTLs were described for the first time, two of which coincided with black spot resistance QTL, one coincided with a cercospora resistance QTL, and one that did not collocate with either of the diseases. The mapping populations used in this research are currently maintained to study other traits of interest and will continue to add to the body of knowledge on tetraploid garden roses and polyploid genetics.In conclusion, we used two interconnected mapping populations to study the inheritance of resistance to black spot and cercospora leaf spot as well as defoliation. We discovered a black spot resistance QTL on LG 3 that may be the same factor as Meta_2_3 reported in diploid studies. Another black spot resistance QTL on LG 5 is believed to be the https://www.rosaceae.org/publication_datasets), accession number tfGDR1068.The data presented in the study are deposited in the Genome Database for Rosaceae repository (DB conceived the experiment. JL, OR-L, and DB prepared and edited the manuscript, and JL and EY propagated and established populations in the field. JL and HG took the phenotypic data. PK provided funding for DNA extraction. JL performed phenotypic data statistics, linkage maps, and QTL mapping. JL, DB, and OR-L worked on the interpretation of data. All authors contributed to the article and approved the submitted version.


Two different particle sizes were used at various concentrations. The impact of different CaCO3 loadings and particle sizes on the PVC foamed layer’s thermal properties were examined. Thermogravimetry (TGA and DTG) and differential scanning calorimetry (DSC) measurements were utilized to investigate the thermal properties of the PVC foamed layer and the samples have been also characterized by FTIR spectroscopy. According to the findings, the thermal stability of the foamed layer was improved with the addition of calcium carbonate. Through the higher surface area between the filler and the PVC matrix, smaller particle sizes have produced the best results. The PVC foamed layer shows also changes in FTIR spectra after adding CaCO3, and the intensity of peaks increases with decreasing CaCO3 particle size.The goal of this research is to see how the amount and particle size of calcium carbonate (CaCO Synthetic leather, also called artificial leather or coated textile, is becoming more popular as a substitute for natural leather in a variety of applications thanks to its low cost and its similar look and durability to authentic leather. The coating is increasingly becoming an important way of adding value to textiles. It aims to develop the functional properties of textiles, improve certain characteristics, and guarantee that fabrics meet parameters of performance that will not be achievable from uncoated and leathered fabrics .Polyvinyl chloride (PVC) is one of the most extensively utilized polymers in the coating industry due to its low cost, low density, fire retardancy, excellent insulation, and high mechanical and thermal properties. PVC synthetic leather is widely used in daily life –4.So far, the significance of their properties has been greatly emphasized as a result of their application in various domains such as footwear, automotive, flooring and wall coverings, handbag accessories, medical equipment, and clothing ,6. The sTheir basic components are as follows: PVC resin, a stabilizer, a plasticizer, a blowing agent, and a filler . Plastis3 has been employed to minimize its cost and enhance its melting viscosity, and mildly increase the modulus of the final product due to its small surface area and undesirable geometrical aspects [Calcium carbonate is one of the most widely used fillers for the PVC leather industry –16, conv aspects . However aspects . Particl aspects . Rigorou aspects .3 particle size and content have a significant effect on the morphological structure [Other studies have shown that CaCOtructure ,21,22, ttructure ,23,30, atructure ,21.However, no published references on the subject of the foamed layer used for PVC synthetic leather have been found.3 concentration and particle size on the thermal characteristics of the PVC synthetic leather foamed layer.This study aims to explore the functionality of calcium carbonate in the thermal property enhancement of the PVC foamed layer used for coated textiles and to provide a detailed analysis to demonstrate the impact of CaCO3 fillers, blowing agent (azodicarbonamide), kicker, transfer paper, pigment, and textile fabric were generously donated by PLASTISS company . The different calcium carbonate particle sizes (1.8 μm and 0.9 μm) were provided by SOFAP company (Sfax-Tunisia).PVC resin, Plasticizer (DINP), stabilizer, CaCOIn this study, PVC foamed layers have been developed from PVC plastisol. To make PVC plastisol, 100 parts PVC resin, 80 parts DINP, 4 parts azodicarbonamide, 2 parts Kicker, and 1.5 parts stabilizers were mixed in a mechanical stirrer. Then 25%, 50%, 75%, 100%, or 125% (by weight) of fillers were included and mixed until a consistent mixture was obtained.3), which dissipates in the plastisol. Until curing is completed, the gas must remain dissolved in the melting.The transfer coating technique has been used to create a PVC-foamed layer. The plastisol is applied to the transfer paper with a blade and the thickness is controlled simultaneously. The resultant film, called the foamed layer or internal layer, once it is dried at 200 °C for 80 s and steamed. During plasticizing, the azodicarbonamide  decomposes, generating ammonia gas .The FTIR spectra of PVC resin, represented in 3), which is characterized by the three C-O elongation modes of the carbonate groups [e groups . They ap−1.- A large and intense absorption band at 1400 cm−1.- A thin and intense band at 876 cm−1.- A thin and weak band at 714 cmThese similar absorption bands were also reported by Wen et al.  and Luo 3 with different particle sizes (0.9 μm and 1.8 μm) are shown in −1 corresponds to the C-H stretch bond [−1 suitable for The C–H aliphatic bending bond, the peak located at 1250 cm−1 is attributed to the C-H deformation bond near chlorine (Cl), the peak observed in the 1000–1100 cm−1 region corresponds to the PVC backbone chain’s C–C stretch bond. Finally, the peak detected at 620 cm−1 represents C–Cl gauche bonds, similar absorption bands have been reported by Atef et al. [The FTIR spectra of the PVC internal layer with 0% and 50% of CaCOtch bond , the peaf et al. , Lee et f et al. , and Ramf et al. .2 bending peak, with a wavenumber of approximately 1409 cm−1, is maximum in the reinforced samples, and the peak intensity increases with decreasing particle size, compared to the FTIR spectra of the reinforced foamed layer with the pure samples as a reference. Another notable difference is the presence of a very thin and sharp peak in the reinforced samples with a wavenumber of around 877 cm−1, which can be attributed to the C-O elongation modes of the carbonate groups [The CHe groups .3 foamed layers with a fine particle diameter (0.9 μm) and PVC/50% CaCO3 with a large particle diameter (1.8 μm) have revealed a linked melting peak located around 304.71 °C and 296.36 °C, respectively. It is clear that by adding CaCO3, the fusion point of the pure PVC foamed layer was raised to a higher temperature. It can be concluded that calcium carbonate improves the thermal stability of the PVC foamed layer.3’s HCl scavenger action during PVC thermal decomposition.Etienne et al. , Matthew3 due to their larger surface area, as previously reported by Liu et al. [As shown in u et al. . Calcium3 foamed layers with varying CaCO3 particle sizes. Thermal factors are listed in 3 foamed layers happens in two main stages, and two substantial weight losses can be seen [From the TGA and DTG curves illustrated in  be seen ,39.3 particle size.As also observed by Etienne et al. , a minor3 foamed layer was lower than the pure PVC foamed layer, and the internal layer filled with small particles typically has the lowest weight loss among the other samples used in this study.Moreover, the weight loss of the PVC/50% CaCO3 can ameliorate the thermal stability of the PVC foamed layer used for PVC synthetic leather, and using a small CaCO3 particle size increases the enhancement of the thermal stability. The results of the TGA and DTG analyses were in good agreement with the DSC data.We can conclude that the incorporation of CaCO3 filler on the thermal properties of the PVC foamed layer used for synthetic leather were investigated, and we have demonstrated that adding CaCO3 can ameliorate the thermal stability of the PVC foamed layer. Therefore, the melting point and the onset decomposition temperatures of the filled foamed layer increase compared with the unfilled one. Many authors attributed these findings to CaCO3’s superior ability to trap HCl gas produced during PVC decomposition. Moreover, we observed that the most important positive impact on thermal stability was detected when using a smaller particle size, which has contributed to its larger surface area that can help to consume much more HCl gases, according to several researchers.The effects of particle size and content of CaCO


Extracorporeal membrane oxygenation (ECMO) has been increasingly used for severe neonatal respiratory failure refractory to conventional treatments. This paper summarizes our operation experience of neonatal ECMO via cannulation of the internal jugular vein and carotid artery.The clinical data of 12 neonates with severe respiratory failure who underwent ECMO via the internal jugular vein and carotid artery in our hospital from January 2021 to October 2022 were collected.All neonates were successfully operated on. The size of arterial intubation was 8 F, and the size of venous intubation was 10 F. The operation time was 29 (22–40) minutes. ECMO was successfully removed in 8 neonates. Surgeons successfully reconstructed the internal jugular vein and carotid artery of these neonates. Arterial blood flow was unobstructed in 5 patients, mild stenosis was present in 2 patients, and moderate stenosis was present in 1 patient. Venous blood flow was unobstructed in 6 patients, mild stenosis was present in 1 patient, and moderate stenosis was present in 1 patient. The complications were as follows: 1 case had poor neck incision healing after ECMO removal. No complications, such as incisional bleeding, incisional infection, catheter-related blood infection, cannulation accidentally pulling away, vascular laceration, thrombosis, cerebral haemorrhage, cerebral infarction, or haemolysis, occurred in any of the patients.Cannulation of the internal jugular vein and carotid artery can quickly establish effective ECMO access for neonates with severe respiratory failure. Careful, skilled and delicate operation was essential. In addition, during the cannulation process, we should pay special attention to the position of cannulation, firm fixation and strict aseptic operation. Respiratory failure is the most common cause of neonatal death. Common causes of neonatal respiratory failure include meconium aspiration syndrome, persistent pulmonary hypertension, congenital diaphragmatic hernia and acute respiratory distress syndrome –3. With ECMO drains blood from the body to the outside through arteriovenous intubation, and then oxygenated blood is injected into the body via a pump after artificial membrane lcrng oxygenation to maintain the blood supply and oxygen supply to various organs of the body. ECMO provides respiratory and cardiac support to patients with severe cardiopulmonary failure, allowing the heart and lung to fully rest, and allowing valuable time for further treatment and recovery of cardiopulmonary function . ECMO isTwelve neonates with severe respiratory failure who underwent ECMO via the internal jugular vein and carotid artery in our hospital from January 2021 to October 2022 were enrolled. There were 9 males and 3 females with an age of 2 days (1–25 days) and a body weight of 3.4 kg (2.8–4.8) kg. The primary disease was severe pneumonia in 2 cases, persistent pulmonary hypertension combined with acute respiratory distress syndrome in 8 cases, and congenital diaphragmatic hernia in 2 cases.All patients received cannulation of ECMO bedside in the intensive care unit of cardiac surgery in our hospital. Two paediatric cardiac surgeons performed the cannulation of ECMO (the primary surgeon had more than 10 years of paediatric cardiac surgery experience). One physician and one nurse of the intensive care unit were responsible for monitoring the vital signs and ventilator status of the patients during intubation. One cardiac anaesthesiologist was responsible for the anaesthesia and intubation of the deep vein and arterial blood pressure before ECMO. One extracorporeal circulation perfusionist was responsible for the installation and work of ECMO.All patients were intubated and their breathing assisted with a ventilator. All patients were maintained under anaesthesia via midazolam 0.3 mg/kg, fentanyl 10–30 μg/kg and rocuronium 0.6 mg/kg.The patients were placed in a supine position, and the head was tilted back by padding the shoulders with cotton and turning to the left to fully expose the right neck.An incision of approximately 2 cm was made along the transverse line of the neck at 1–2 cm above the right supraclavicular fossa  and 10F cannulation . The depth of cannulation of the carotid artery was 3 cm in 9 cases and 3.5 cm in 3 cases. The depth of cannulation of the internal jugular vein was 6.5 cm in 1 case, 7 cm in 8 cases and 7.5 cm in 3 cases. The operation time was 29 (22–40) minutes.ECMO was successfully removed in 8 neonates. Finally, among them, 5 patients recovered and were discharged successfully, 2 patients died due to liver and kidney failure, and 1 patient died due to severe pneumonia. Patients who successfully underwent ECMO removal underwent reconstruction of the internal jugular vein and carotid artery. All patients underwent right cervical vascular colour Doppler ultrasound examination at 1 week after the operation. Four patients underwent neck MR angiography examination, and four patients underwent neck CT angiography examination. The situation of right common carotid artery patency was as follows. Colour Doppler ultrasound showed that the right common carotid artery of 5 patients was unobstructed. For these patients, CT showed mild stenosis in 3 patients, and MR showed mild stenosis in 2 patients. Doppler ultrasound and MR both showed mild stenosis in 2 patients. Doppler ultrasound showed mild stenosis in 1 patient, but CT showed moderate stenosis. The situation of right internal jugular vein patency was as follows. Colour Doppler ultrasound showed that the internal jugular veins of 6 patients were unobstructed. For these patients, CT showed mild stenosis in 3 patients, and MR showed mild stenosis in 3 patients. Doppler ultrasound and MR both showed mild stenosis in 1 patient. Doppler ultrasound showed mild stenosis in 1 patient, but CT showed moderate stenosis (Fig. The complications were as follows: 1 case had poor neck incision healing after ECMO removal. No complications, such as incisional bleeding, incisional infection, catheter-related blood infection, cannulation accidentally pulling away, vascular laceration, thrombosis, cerebral haemorrhage, cerebral infarction, or haemolysis, occurred in any of the patients.ECMO is an extracorporeal life support technology that can effectively replace heart and lung function. ECMO can provide cardiopulmonary support to patients with severe heart and lung failure, thereby providing valuable time for the recovery of cardiopulmonary function , 9. TherSuccessful cannulation is a necessary condition for the establishment of ECMO, and rapid establishment of suitable circulation access in a short time is the key to implementing ECMO . At the A stable flow rate is very important to the work of ECMO, and a good position of cannulation is the basis for a stable flow of ECMO , 15. If Intraoperative and postoperative complications of ECMO intubation should not be ignored . ShiftinIncisional bleeding, vascular laceration and incisional infection were common complications. It was important to be careful and use the blunt separation method to dissociate the tissue during the operation. It would not only reduce the amount of bleeding but also provide a clear position of blood vessels in the neck. When dissociating blood vessels, we should gently operate to avoid damaging the vessel wall and causing bleeding. We should also avoid excessively pulling the blood vessels; otherwise, it would result in venous contracture, which would increase the difficulty of intubation. When intubating, we should pay attention to the angle and force of the tube, and we should not intubate violently. After the cannulation entered the purse incision, the cannulation was inserted with rotating movement. At the same time, the proximal cannulation should be gently lifted to make the angle of insertion cannulation consistent with the vessel as much as possible. After successful intubation, ligating the distal end of the artery and vein, tightening the proximal end of the purse and fixing with a cannula can effectively reduce vascular bleeding at the intubation site. Intubation with ECMO is often performed at the bedside, which increases the risk of infection , 18. De DPolito A et al. found that the neurological complications of children with VA ECMO were higher than those with VV ECMO, and they considered the reason to be that the carotid artery was ligated . NeonataThis study had some limitations. First, this study was a retrospective study with the absence of a control group, which would cause biases and confounding factors. Second, the study's sample size was limited to 12 neonates, so the results may not be representative of the general neonatal population. Third, the study lacked long-term outcomes. The long-term follow-up data are currently in progress, and we will report them in the future.Cannulation of the internal jugular vein and carotid artery can quickly establish effective ECMO access for neonates with severe respiratory failure. Careful, skilled and delicate operation was essential. In addition, during the cannulation process, we should pay special attention to the position of cannulation, firm fixation and strict aseptic operation.