The analyses were carried out by atomic absorption spectrometry, ICP-MS, spectrophotometric, spectrometric, gravimetric, flamephotometric, titrimetric methods, potentiometric, and conductometric determination. The data are useful for comparative analysis of the hydrochemical characteristics of Lake Onego and other large lakes, and they also allow to assess the water quality of the lake as a whole and in its particular individual areas.The present dataset contains the chemical parameters of Lake Onego water. The data were obtained in different seasons covering the period 2019-2021. The concentration of Na The database can also be applied to produce and verify mathematical models of chemical substances circulation in large lakes.1The dataset contains information on the water chemical composition of Lake Onego in different seasons of 2019-2021. Water samples were collected using research vessels in spring, summer, and autumn. In winter, water samples were collected from the ice surface. The location of the sampling sites is presented in 22.123, a mean depth of 30 m, a maximum depth – 120 m, and a surface area – 9720 km2Lake Onego is a large boreal lake situated in the north-western part of the Russian Federation and belongs to the Baltic Sea basin. Having a catchment area of 53100 km3/year 3/year), and storm runoff from Petrozavodsk are sources of contamination in the Petrozavodsk Bay 3/year 3/year) The largest bays of Lake Onego are Petrozavodsk, Kondopoga, and Povenets, situated in the northern part of the lake. These bays are experiencing significant natural and anthropogenic influences. The River Shuya discharge (3.00 km2.2in situ using the CastAway probe (USA). Water samples were collected in clean plastic vials. To determine the total phosphorus and iron content, water was collected in 250 mL polyethylene vials and then preserved with 4N H2SO4 solution . For Na+, Cl−, color, and TSS, determination subsamples were filtered with 0.45 µm membrane filters . Subsamples for heavy metal analysis were collected in 100 mL polyethylene flasks and instantly acidified with concentrated HNO3 . The samples were stored in the dark at 4 °C before the analysis.Water samples were collected in autumn 2019, spring and summer 2020 at Lake Onego (35 stations in its different regions), including the lake outflow (Svir' River) and in the estuarial areas of the rivers Shuya, Vodla, Andoma, and Vytegra, from RVs “Ecolog” and “Poseidon”. In winter 2021, samples were collected in the Kondopoga, Petrozavodsk bays and in the pelagic part of the Lake at station C3 from the ice surface. To determine most of the chemical characteristics, samples were collected at a depth of 0.5 m below the surface and a hight 1 m above the bottom with a Niskin Bottle. For heavy metal analysis, samples were collected in the same location and at the same depth with a polytetrafluoroethylene bathometer. The water depth and temperature were measured at each station Chemical analyses were performed by corresponding methods  in the L2.3Quality analysis and quality control procedures were implemented according to Mikhail Zobkov: Conceptualization, Methodology, Investigation, Writing - Review & Editing, Visualization, Supervision, Funding acquisition. Maria Zobkova: Investigation, Resources, Writing - Review & Editing. Natalia Galakhina: Data Curation, Writing – Original Draft. Tatiana Efremova: Investigation, Resources. Natalya Efremenko: Validation, Resources, Data acquisition. Natalia Kulik: Resources, Data acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Implementing a community scorecard approach may help increase utilization of maternal, neonatal, and child health services in primary health care facilities. The results of our study show the importance of engaging both the community and health workers to measure and continuously improve health care processes and improve the health system performance. The average community scorecard measurements on health workforce behavior toward patients, availability of services, patient waiting time, facility infrastructure, ambulance service, and cleanliness and safety of the health facility significantly improved over 1 year at primary health care units.Of the 10 key maternal neonatal and child health performance indicators measured, 9 were found to improve as a result of implementing the community scorecard approach.The implementation of a community scorecard approach enhances a culture of social accountability, transparency, and engagement of citizens in planning, implementing, and evaluating maternal, neonatal, and child health services. In addition, it improves the negotiation capacities and involvement of both community members and health workers, resulting in increased availability and utilization of health service.Program managers and development partners should continue their support for the government- led social accountability interventions to ensure the sustainability of improvements in maternal, neonatal, and child health outcomes. Social accountability approaches, such as the community scorecard (CSC), can improve the performance of health systems in low-income countries by providing a mechanism for obtaining and incorporating community input. This longitudinal study assessed the effects of CSCs implemented by primary health care units (PHCUs) on health system performance in Ethiopia.This study used a pre-post design and was conducted from October 2018 to September 2019 in 159 PHCUs in 31 districts in Amhara and Southern, Nations, Nationalities and Peoples' regional states. The data were extracted from a routine health information management system database at baseline, midterm, and endline stages over 12 months for statistical analysis. The effects of implementing CSCs on health system performance were evaluated against selected key performance indicators (KPIs).P=.001). Post hoc analysis with Wilcoxon signed-rank tests was conducted with a Bonferroni correction and the results showed higher CSC and KPI values from baseline to midterm and from midterm to endline (P<.017).The CSC measurement results were based on input from 38,556 community representatives. The mean CSC score with standard deviation (±SD) was 60.8%±12.5%, 66.3%±10.8%, and 70.6%±10.0% at baseline, midterm, and endline, respectively. The mean KPI score was 54.9%±17.4%, 61.9%±15.1%, and 67.6%±14.6% at baseline, midterm, and endline, respectively. The average CSC and KPI values were positively correlated (r>0.37). Using a nonparametric Friedman's test, we found a statistically significant difference in CSC and KPI scores at baseline, midterm, and endline (The use of CSCs in Ethiopia contributed to the health system's performance in terms of maternal and child health services. The responsiveness of health workers and utilization of basic health services by community members were found to increase significantly with CSC use. We recommend continued implementation of the CSC intervention at PHCUs. The national implementation guide dictates that community groups  work together to make basic health services equitable, effective, efficient, transparent, responsive, and accountable.–,The exercise of developing a national strategy led to the development of a theory of change. The goal of implementing CSCs is to contribute to the prevention of maternal and child deaths.The U.S. Agency for International Development (USAID) Transform: Primary Health Care is a bilateral project, implemented by a consortium of international and local development partners, in collaboration with communities and the public health sector in 4 regional states in Ethiopia.The primary health care system of Ethiopia is the foundation of the country's 3-tier health system. It is composed of a district hospital and 3 or 4 health centers, each overseeing about 5 health posts. A PHCU includes 1 health center and 5 satellite health posts, serving an average of 25,000 people.To date, to our knowledge, no studies have assessed the effects of CSCs on health system performance in Ethiopia in terms of service utilization using longitudinal data. Despite the shortage of rigorous evidence on the inputs, process, and outcomes of implementing CSCs in Ethiopia, wide use of CSCs by the public health sector for performance improvement has continued based on the global evidence. Therefore, the results of this longitudinal pre-post interventional study present the effect of CSCs on maternal and child service performance in 2 administrative zones of Ethiopia.To our knowledge, no studies have assessed the effects of CSCs on health system performance in Ethiopia in terms of service utilization using longitudinal data.This study was conducted in South Wollo and Kembata Tembaro administrative zones of Amhara and SNNP regional states, respectively. The USAID Transform: Primary Health Care project provides technical, financial, and other resource support to 91 districts in Amhara and 84 in SNNP, as well as to districts in other regional states.Ethiopia has a 3-tier health care delivery system. Level 1 is the district (woreda) level, composed of primary hospitals that cover 60,000–100,000 people, health centers serving 15,000–25,000 people, and their satellite health posts covering 3,000–5,000 people, connected to each other through a referral system. The primary hospitals, health centers, and health posts form PHCUs. Districts are subdivided into kebeles (villages), the lowest administrative units. Level 2 includes general hospitals covering 1–1.5 million people. Level 3 includes specialized hospitals covering 3.5–5 million people.In the last 3 decades, the country has expanded access to primary health care through 17,187 health posts; 7,245 private health facilities; 3,724 health centers; and 266 hospitals. In addition, more than 151,053 health professionals are serving communities.To institutionalize accountability and transparency as a tool for performance management in the health system in both Amhara and SNNP regional states of Ethiopia, 632 participants attended the CSC training of trainers, including 155 from district health offices and 477 from PHCUs. A 3-day classroom theoretical orientation and practical sessions were facilitated in July 2018 at the respective capital cities of the regional states. The CSC orientations were given to 4,053 client councils within the targeted 159 health centers in South Wollo and Kembata Tembaro administrative zones in August 2018.From September 2018 to December 2019, USAID Transform: Primary Health Care provided technical, financial, and other resource support to its targeted districts. Some forms of this support were providing team-based strategic problem-solving trainings for health care providers with the formation of performance improvement and quality improvement projects, enhancing the capacity of performance management team members through offering the use of data for decision-making trainings, providing performance improvement subgrant funding for primary health care entities, and organizing and facilitating community-facility interface meetings.In addition, the project's staff and experts from zone health departments provided follow-up visits and on-site coaching for all PHCUs and district health offices on a quarterly basis. During facility visits, the coaching team facilitated the exploration of CSC measurements with feedback and gave opportunities for PHCU management staff to systematically analyze the root causes of any issues, propose prioritized solutions, and develop doable action plans. In addition, the coaches revised the concepts of social accountability, provided feedback on performance, and supported the client councils and health care providers. The coaches then submitted a copy of agreed measurement reports, identified gaps, and developed action plans with district health offices and to zone health departments. Baseline data were extracted from the period of October 10–20, 2018. At 6 and 12 months after the intervention periods, secondary data were collected during April 10–20, 2019, as midterm assessments, and during October 10–22, 2019, as endline measurements by the data collection teams.For this study, the investigators used a longitudinal pre-post interventional study design.The study participants were identified from the Amhara and SNNP regional states in Ethiopia. Two administrative zones where the project had provided technical and other resource support for 12 months or more were purposively selected. All 31 districts (woredas) and 159 PHCUs were included in selected zone administrations. Documents and records from each primary health care entity were reviewed at the 3 points of the assessment.The required routine health management information system data on CSC and on maternal and child health services were extracted from 159 PHCUs' RHIMS database. To ensure data completeness, accuracy, consistency, and reliability, 8 data managers and 2 supervisors were trained for 3 days. The training covered the objective of the pre-post interventional study, ethical issues, quantitative data extraction methods, and piloting all tools and ethical principles. During the real data collection, all investigators actively monitored completeness and consistency of data on a daily basis. Data were extracted using structured and pretested forms.Dependent variables: The average measures, from 0% to 100% on 10 key performance indicators (KPIs) were reviewed and collated: (1) contraceptive acceptance rate, (2) syphilis screening among antenatal care (ANC) clients, (3) skilled delivery services coverage, (4) postnatal care coverage, (5) full immunization coverage, (6) under 2 years growth monitoring coverage, (7) proportion of available essential or tracer drugs, (8) proportion of clean and safe health facility standards met, (9) proportion of available laboratory and diagnostic services, and (10) patient flow and service organization  compassionate, respectful, and caring health workforce; (2) patient waiting time; (3) availability of services, biomedical equipment, and pharmaceutical supplies; (4) health facility infrastructure; (5) ambulance service and management; and (6) clean and safe health facility, reported as an average score from a 5-point Likert scale measurement (Supplement 1), that is, 5=very good to 1=very low. The overall summary of CSCs was reported as a percentage from 0% to 100.0% on 3 occasionsP-value of <.017, indicating presence of a significant difference between CSCs and KPIs at baseline, midterm, and endline measurements.The PHCU scores of CSCs and KPIs were reviewed for completeness and consistency. Quantitative data analysis methods were used, which included descriptive statistic frequencies, mean, median, interquartile ranges, and standard deviations. To determine the presence of a linear relationship between baseline, midterm, and endline scores, a Pearson Product-Moment Correlation technique was employed. After checking the assumption of the nonparametric test, we used the FriedmanThe ethical clearances of this study were granted by the JSI Institutional Review Board (IRB), and the Amhara Public Health Institute and SNNP Regional State Health Bureaus' IRBs.The research protocol of this pre-post interventional study was granted certification from the Amhara Public Health Institute (reference number: M/T/SH/D/03/435) and the SNNP Regional State Health Bureau Research and Ethics Review Committees (reference number NS 12/36/22). In addition, the study protocol was reviewed at the JSI Research & Training Institute, Inc. IRB, which determined that this activity was exempt from human subjects' oversight (reference number IRB no. 19-16E). To maintain the confidentiality of collected data, anonymity was maintained throughout the research process.Permission to use data from zone health departments and PHCUs were obtained through formal written requests. Informed individual written consent was taken from each study subject. Both quantitative and qualitative data were collected in aggregate forms. All PHCUs were encouraged to use the data for evidence-based decision making and to be responsive to the demands of rights holders. The summary of CSCs and KPIs were submitted to district health offices, zone health departments, and regional health bureaus at the 3 points of the assessment. Throughout the research process, the investigators maintained national and international ethical principles.In this study, a total of 31 districts from 2 regional states of Ethiopia were enrolled. 2(2)=252.642, P=.000. Post hoc analysis using the Wilcoxon signed-rank tests was conducted with a Bonferroni correction applied, resulting in a significance level set at P<.017. Median (interquartile range) CSC measurements at baseline, midterm, and endline were 60.4 (52.9–70.3), 66.9 (58.3–73.9), and 71.0 (61.1–77.6), respectively. There were statistically significant positive differences between CSC at midterm and baseline ; endline and baseline , and endline and midterm . Therefore, it can be concluded that long-term community engagement elicits a significant rise in perceived CSC measurement values.At baseline, the mean score of CSCs was 60.8; at midterm, it was 66.3; and at endline, it was 70.6. A statistically significant difference was found in CSC ratings at baseline, midterm, and endline measurements, χ2(2)=267.142, p=0.001. Post hoc analysis with Wilcoxon signed-rank tests was conducted with a Bonferroni correction applied, resulting in a significance level set at P<.017. Median (interquartile range) KPI measurements at baseline, midterm, and endline were 53.6 (42.0–68.5), 60.1 (51.6–74.5), and 66.3 (56.7–80.0), respectively. There were statistically significant positive differences between KPI at midterm and baseline , endline and baseline , and endline and midterm . Therefore, it can be concluded that long-term community engagement brings a significant increase in KPI measurement values.At baseline, the mean score of KPIs was 54.94; at midterm, it rose to 61.93; and at endline, it increased even further to 67.61. A statistically significant difference was found in KPIs at baseline, midterm and endline measurements, χThis longitudinal, pre-post CSC interventional study in 31 districts and 159 PHCUs documented the establishment of 579 client councils and the participation of 38,556 community members. The support of the project, which included capacity building of health workers and community members, as well as provision of job aids, reporting forms, and performance improvement subgrants, helped the PHCUs to increase their performance. The baseline, midterm, and endline data showed statistically significant ongoing increases. The results revealed positive correlations between the CSC average values and the average KPI achievements. Therefore, it can be concluded that building the capacity of community members and health workers on the implementation of social accountability interventions can help to improve service availability and utilization of primary health care services.The support of the project, including health worker capacity building, job aids, and reporting forms, helped the PHCUs to increase their performance.The measurement results of CSCs were positively correlated and had statistically significant higher differences, which may have occurred due to the responsiveness of the health system to the needs and demands of community members in terms of health worker behavior, availability of services, and improvements in supplies, infrastructure, and waiting time. This finding was in line with an article by Ho et al.Supplement 2). This finding was in line with Gullo et al.,Maternal and child health service utilization increased, specifically for skilled delivery (from 45.3% to 57.3%), postnatal care services (from 57.3% to 70.7.0%), syphilis screening (from 63.4% to 80.2%), full immunization for infants under 1 year (from 82.3% to 85.9%), and growth monitoring for children under 3 years (from 27.0% to 31.8%) . These r,The implementation of CSCs at the PHCU level was aligned with the performance measurement and enhancing accountability, transparency, and engagement of public and civil societies and duty holders (citizens) to achieve the strategic objectives of the Ethiopian Health Sector Transformation Plan (HSTP 2015-2020) of the Ethiopian government.This longitudinal pre-post interventional study revealed that the implemented CSC intervention, which is a nationally recommended citizen measurement, engagement, and accountability tool, helps health workers and community members to contribute to the improvement of the performance of the health system.This study has some limitations. The main limitation is related to the pre-post study design; unlike a randomized study design, it is difficult to conclude the causal association between the CSC intervention and the KPI improvements with a pre-post design. The other limitation of this study is the lack of literature on government-led large-scale implementation of CSC. Hence, this study uses results from projects implemented by civic society organizations in limited areas. As the study targeted 2 zone administrations, before generalizing the findings, the context should be noted.Based on this study's results, implementing CSCs as a tool to enhance accountability, transparency, and community engagement could help to contribute to improvements in the performance of the health system regarding maternal and child health services. Ensuring continuity of the implementation of the CSC intervention by orienting client councils on the principles of CSCs, providing reporting forms and job aids, and providing subgrants for PHCUs is recommended. In addition, a qualitative study to document the process of CSC implementation and experiences of community representatives is recommended.


Hyaluronic acid, together with collagen, vitamins or plant extracts, is a part of many cosmetic and food preparations. For example, this polysaccharide is used in formulation of many food supplements due to its protective effects on human health. In this work, the screening of the chemical composition of three chosen dietary supplements  containing hyaluronic acid was carried out using Fourier-transform infrared spectroscopy. Because of the low amount of analyte in all these samples, it was isolated or concentrated prior to the analysis using a suitable sequential fractionation protocol. Individual isolation procedures were established for each sample based on their declared composition. Firstly, the major components such as collagen or vitamins were removed to obtain polysaccharide fractions by the enzymatic treatment and/or washing out with the appropriate solvents. In some cases, the water insoluble part was removed from the rest dissolved in water. Then, hyaluronic acid was precipitated with copper(II) cations and thus separated from the other polysaccharides. Finally, the analyte was identified in the enriched fractions by the characteristic vibrational bands. The amount of hyaluronic acid in the purified fractions was determined in three ways: gravimetrically, spectrophotometrically, and using isotachophoresis. The combination of the appropriate preparative and analytical steps led to the successful evaluation of chemical composition, finding and quantification of hyaluronic acid in all the studied samples. Hyaluronic acid is the only non-sulfated glycosaminoglycan with unique physicochemical and biological properties [N-acetyl groups promote interaction with cell membranes and hydrophobic regions of membrane proteins, which is important for ensuring cell motility [Hyaluronic acid is an acidic polysaccharide of animal and bacterial origin that serves various physiological purposes, for example, in connective tissues of vertebrates and bacterial capsules ,2,3,4. Ioperties . It is aoperties ,7,8. Eveoperties ,10,11,12motility . Thus, tmotility ,15,16.Hyaluronic acid is involved in protective and other physiological processes, including healing of wound and burns ,19,20,21Determination of hyaluronic acid in food supplements is difficult due to the low content of this analyte, as well as the presence of many other components such as proteins (collagen), other polysaccharides, vitamins or plant extracts. Moreover, these substances are often present in larger quantities than the target compound, which further complicates the analysis. Hyaluronic acid has been determined as intact polymeric chains in food supplements and medicinal preparations by separation methods including capillary zone electrophoresis (CZE), isotachophoresis (ITP) and high-performance liquid chromatography (HPLC) ,32,33,34The disadvantage of FT-IR spectroscopy compared to spectrophotometry and separation methods such as HPLC or CZE is its low sensitivity, so it is well suited only for the determination of major components. Therefore, in order to detect hyaluronic acid in dietary supplements, it is necessary to concentrate it through suitable purification steps. The water-insoluble part can be easily separated from the aqueous solution of the remaining compounds, small molecules can be washed out using suitable solvents, and the proteins must be pre-hydrolyzed with proteases, and then the resulting peptides must be removed in the same way as other small molecules. A difficult situation can arise if, as a result of all the above stages, hyaluronic acid remains in a mixture with other polysaccharides that can be added together with it to the formulation, for example, with starch or starch modification products. In this case, precipitation with metal counter cations can be used to separate hyaluronic acid from neutral polysaccharides.β-d-glucuronic acid units, while the N-acetamide group of the N-acetyl-β-d-glucosamine units is not involved in coordination. Therefore, as in the case of pectins, copper(II) cations are able to crosslink hyaluronic acid macromolecules through carboxylate groups and therefore precipitate copper(II) hyaluronate from a solution in which neutral macromolecules remain. Precipitation of copper(II) hyaluronate occurs under neutral and weak alkaline conditions, while under weakly acidic conditions, the complex is stable in solution [Multivalent metal cations are known as effective cross-linkers for biopolymers including anionic polysaccharides . When trsolution , possiblsolution .This work is devoted to the screening analysis of chemical composition, identification and quantification of hyaluronic acid in three dietary supplements of different dosage forms  and composition declared by manufacturers using a combination of suitable purification steps, including precipitation with copper(II) cations, and FT-IR spectroscopy of the fractions obtained. For each sample, individual isolation procedures for analyte enrichment were chosen in relation to sample composition and analyte content in raw material. Quantification was carried out gravimetrically by the mass of the final fractions identified by FT-IR as hyaluronic acid, as well as by spectrophotometry and isotachophoresis. This approach is suitable for the detailed characterization of food supplements and similar samples containing hyaluronic acid together with an excess of other components .w/w.The purpose of the study was to identify the hyaluronic acid in three samples of dietary supplements with different composition obtained from the manufacturers and/or from the domestic market . ExpecteHyaluronic acid, glycine, 2-hydroxyethylcellulose, pepsin, copper(II) chloride dihydrate Citric acid Ethanol 96%, ethyl acetate, hexane 99%, sodium hydroxide, hydrochloric acid, sodium carbonate, sodium hydrogen carbonate Potassium bromide for IR spectroscopy Following chemicals were used in this work:2CO3 and 50 g of NaHCO3 in 1 L of distilled water [A carbonate-bicarbonate buffer was prepared by dissolving 129 g of Naed water . All cheDue to the low amount of the analyte in all food supplements, the appropriate isolation procedures were made to obtain fractions rich of hyaluronic acid. For each supplement sample, a specific isolation procedure was developed based on a combination of extraction and precipitation steps. An individual hyaluronic acid isolation procedure was chosen for each sample in relation to the sample composition and analyte content. The presence of target polysaccharide in the enriched fractions was confirmed using FT-IR spectroscopy. Isolation procedures for supplements 1–3 are illustrated in −1 HCl in 80% ethanol) and then again with 96% ethanol. The ethanolic suspensions were stirred on a magnetic stirrer for 30 min. Then, the mixture was filtered through filter paper and the solids assigned as fraction 1.1 were dried on a watch glass. The dried solids were dissolved in 50 mL of 0.01 mol L−1 hydrochloric acid, and 4 g of pepsin was added to the solution. Next, the solution was stirred for 30 min on a magnetic stirrer C-MAG HS7 , and after dissolving the enzyme; it was placed in a thermostat BT 120  at 40 °C overnight. The enzyme was then denatured by heating the solution at 90 °C for 10–15 min on magnetic stirrer and filtrated. The solution was cooled and solids were precipitated with an excess of 96% ethanol (3: 1 v/v) and then filtered off. The resulting precipitate (0.65 g) was air dried and designated as fraction 1.2. The dried precipitate was ground in a powder mortar and then dissolved in 20 mL of distilled water. A small amount of copper(II) chloride was added with stirring. The pH value was adjusted to 7.0 by adding a small volume of 0.1 mol L−1 sodium hydroxide solution. The resulting precipitate, designated as fraction 1.3, was centrifuged and washed with acidified ethanol to remove copper(II) cations that were detected by visual decolonization of the precipitate. Finally, the precipitate was washed with 96% ethanol to pH ~7.0. The supernatant was precipitated with excess ethanol and the resulting precipitate, i.e., fraction 1.4, was washed in the same way as fraction 1.3.The powder sample was weighed (13.2 g) and successively washed with 96% ethanol, acidified ethanol (0.1 mol LThe sample (30 tablets) was weighed and then washed with 96% ethanol, acidified ethanol and then again with 96% ethanol yielding fraction 2.1. Cellulose was separated from the other polysaccharides by suspending this fraction in 150 mL of distilled water and heating to 80 °C. The mixture was filtered through filter paper. The solids, assigned as fraction 2.2, contained cellulose, which is insoluble in water, while the filtrate contained water-soluble polysaccharides including hyaluronic acid. The filtrate was orange due to the presence of β-carotene, which was then removed by washing with ethyl acetate, and a small amount of copper(II) chloride was added to the clear solution with stirring. The pH was adjusted to 7.0 by adding of sodium hydroxide. The resulting precipitate, i.e., fraction 2.3, containing hyaluronic acid, was washed with acidified ethanol and further with 96% ethanol to remove copper(II) cations from the sample. The neutral polysaccharides were precipitated from supernatant with ethanol excess, and the resulting precipitate, i.e., fraction 2.4, was washed with 96% ethanol.−1 sodium hydroxide. The precipitate formed was washed with acidified ethanol and further with 96% ethanol to remove copper(II) cations and then centrifuged. These solids containing hyaluronic acid were assigned as fraction 3.3. The supernatant was treated with excess ethanol, but no precipitate was obtained; therefore, the sample did not contain neutral polysaccharides.Firstly, the shells of 10 capsules were removed, and the internal liquid content (emulsion) was poured onto a watch glass. Then, the emulsion (3.4 g) was decomposed and decolorized by successive washing with hexane, ethyl acetate and 96% ethanol. As a result, insoluble powdered fraction 3.1 was obtained. The presence of insoluble minerals such as zinc(II) oxide has been declared by the manufacturer see . To remo−1) of the supplements and isolated fractions were recorded in KBr pellets by Nicolet 6700 FT-IR spectrometer using Omnic 8.0 software ; 64 scans were accumulated with a spectral resolution of 2.0 cm−1. The spectra were smoothed by 11 points, baseline corrected, and exported in ASCII format to Origin 6.0  software for preparation of graphs. The position of shoulders was determined using the 2nd derivative algorithm.FT-IR spectra  ,58. The −1) in mixture with carbonate buffer (1: 4 v/v) were used for calibration. For quantitative determination, a standard solution of hyaluronic acid was used.The content of hyaluronic acid in dietary supplements was determined by photometry according to the method which has been proposed for the determination of pectin , with soBecause of the low amount of analyte in the samples, it was decided to isolate hyaluronic acid from food supplements and subsequently confirm its presence in the enriched fractions by FT-IR spectroscopy. The first step was the removal of major components such as collagen and low-molecular substances, in particular various vitamins, to obtain polysaccharide fractions for further purification. Enzymatic hydrolysis by pepsin led to removal of already partially hydrolyzed collagen. The low-molecular components were washed out by suitable organic solvents. Then, hyaluronic acid was separated from the other polysaccharides present in the fractions, including cellulose and maltodextrins. Soluble polysaccharides, including hyaluronic acid, were separated from cellulose by dissolving in water. Finally, hyaluronic acid was precipitated with copper(II) cations and thus separated from other maltodextrins. The presence of hyaluronic acid was confirmed by FT-IR spectroscopy based on the assignment of the band’s characteristic for this polysaccharide and comparison with the literature. The combination of the described preparative and analytical steps and methods led to the successful finding of hyaluronic acid in all the studied samples.The chosen procedure for the isolation of hyaluronic acid from the starting material should meet two basic requirements. First, the process should be as efficient as possible, along with minimal losses during the isolation process. If the sample contains low molecular weight hyaluronic acid or its fragments (oligosaccharides), which can dissolve in dilute aqueous ethanol, there is a possibility of analyte loss. To prevent this, precipitation with excess ethanol should be replaced by a combination of dialysis through suitable membranes and lyophilization. Second, there should be no structural degradation of the polysaccharide during the isolation process. It is essential that the sample does not decompose during operation by chemicals or enzymes used to remove unwanted components.w/w) of each sample and their expected composition. Supplement 1 contained mainly biopolymer components  in addition to hyaluronic acid. Enzymatic hydrolysis with pepsin was chosen to remove collagen. Supplements 2 and 3 contained a large amount of low molecular weight substances that had to be removed first.The next step was to separate the individual polysaccharides based on their solubility or ability to form complexes with copper(II) cations. Thus, hyaluronic acid was separated from cellulose and maltodextrins. Fractions 1.3 and 2.3, which allegedly contained hyaluronic acid, were determined in the case of supplement 2 to less than a tenth of a percent. On the contrary, the respective fraction 3.3 of supplement 3 (capsule content) had a content in the order of units of percent, which corresponds to a larger amount of this polysaccharide in the sample, as declared by the manufacturer see . It was −1 indicate the presence of proteins [−1 originated mainly from COC, CO and CC stretching vibrations in carbohydrates which are typical for starch and its oligomers dextrins [l-methionine (~46 % w/w) and l-ascorbic acid (~46% w/w), respectively, as it has been declared by manufacturers (l-methionine at 2917 cm−1 (CH2 symmetric stretching), 1613 and 1512 cm−1 (NH3+ bending), 1584 and 1409 cm−1 (COO– stretching), 1447 and 1352 cm−1 (CH2 bending), 1318 cm−1 (SCH and CH3 bending), 1275 and 1243 cm−1 (CCH and SCH bending), 1027 (CC stretching), 543 and 418 cm−1 (CCO and CCN bending) as well as many other bands of this amino acid were found in the FT-IR spectrum of supplement 2 [l-ascorbic acid at 3525–3215 cm−1 (OH stretching), 1673 cm−1 (C=C stretching), 1660, 1457 and 1246 cm−1 (CH and CH2 deformation modes), 1364, 1321 and 1276 cm−1 (in-plane ring deformation), 1142–821 cm−1 (CO and CC stretching), 756 (in-plane OH twisting), 722 and 685 cm−1 (C=O deformation modes) and skeletal ring vibrations at 629–448 cm−1 [−1 indicates stretching C=O vibrations of organic acids and esters, and together with other bands at 2925, 2855, 1466 and 721 cm−1 (vibration of CH2 groups) confirmed the presence of fats [−1 were assigned to the stretching vibrations of OH and NH bonds. However, characteristic bands of hyaluronic acid were not pronounced in FT-IR spectra of all raw supplements 1–3 because of low amounts of this polysaccharide.The FT-IR spectra of food supplements 1, 2 and 3 where the presence of hyaluronic acid was declared are shown in proteins ,60, i.e.dextrins ,62. By cacturers . Indeed,lement 2 ,64. Simi448 cm−1  were fou−1 (amide I and amide II vibrations) significantly decreased [−1 region were much more intense. These results confirm that as a result of enzymatic hydrolysis, the collagen content decreased, and at the same time, the proportion of polysaccharides, mainly maltodextrins, increased. In the FT-IR spectrum of fraction 1.3 obtained by precipitation with copper(II), bands at 1738, 1617 and 1560 cm−1 indicate the presence of carboxyl and amide groups of hyaluronic acid [−1 (stretching vibrations of CO and CC), as well as weaker bands of skeletal vibrations in the region of 930–445 cm−1 [The FT-IR spectra of fractions 1.1–1.4 isolated from food supplement 1 are shown in ecreased ,60. In cnic acid ,40. On t445 cm−1 .l-methionine and other low molecular weight substances. The spectrum contains a number of bands at 3346, 2902, 1432, 1373, 1337, 1319, 1165, 1113, 1059, 1032, 897, 710, 668, 617, 560, 519 and 436 cm−1 characteristics of cellulose [The FT-IR spectra of fractions 2.1–2.4 isolated from additive 2 are shown in ellulose ,68. Thisl-ascorbic acid, fats and other low molecular weight components, and various bands in the areas 1800–1500 cm−1 and 1200-950 cm−1 indicated the presence of hyaluronic acid, but there are a number of other bands. The water-insoluble part of 3.2 had a very different FT-IR spectrum. This fraction probably contained insoluble inorganic substances . Finally, fraction 3.3 was precipitated with copper(II) cations and then purified. This fraction contained mainly hyaluronic acid, which was confirmed by a number of characteristic bands, including vibrations of amide and carboxyl groups  in three measurements were less than 3%, indicating good reliability for determination of hyaluronic acid in food supplements. These values were similar to those obtained previously for the determination of pectin [Since fractions 1.3, 2.3 and 3.3 were identified as hyaluronic acid by FT-IR, their relative masses were used to quantify this analyte gravimetrically in supplements 1, 2 and 3, respectively. Moreover, the level of hyaluronic acids in the supplements was determined by spectrophotometry and isotachophoresis. The isotachopherograms of the standard solution of hyaluronic acid and the purified fraction of supplement 1 are shown in f pectin  and for f pectin ,58. In af pectin ,70,71, wf pectin ,51,52,53The aim of this work was to identify hyaluronic acid in three food supplements that differ in dosage form and composition. For each sample, a specific isolation procedure for the isolation and concentration of the analyte was proposed. The first step was to remove low molecular weight substances by washing the samples with suitable solvents. The obtained biopolymeric fractions, in addition to hyaluronic acid itself, contained proteins  as well as various oligo- or polysaccharides such as maltodextrins, starch, and cellulose. The second step was to remove proteins by enzymatic hydrolysis. This step has proven to be very effective in removing collagen that was initially partially hydrolyzed. The separation of cellulose or other water-insoluble components was achieved by dissolving in hot water followed by filtration. Then, hyaluronic acid was precipitated by the addition of copper(II) cations, which form insoluble complexes with anionic polysaccharides. Neutral polysaccharides such as starch and maltodextrins do not form such complexes and remain in solution. The effectiveness of each of these steps was confirmed by Fourier-transform infrared spectroscopy, which is suitable for the structural analysis of organic and some inorganic compounds. The conclusion of this work is the confirmation of the presence of hyaluronic acid in all of the analyzed samples, and the content of this analyte was determined by three analytical methods that gave similar results. On the example of three food additives, the effectiveness of the selected preparative and analytical procedures was demonstrated in assessing the composition of the obtained fractions and quantitative estimation of hyaluronic acid. The approach discussed in this paper can be used to analyze various dietary supplements, as well as medicinal and cosmetic preparations containing hyaluronic acid, for example, as an active substance for the regeneration of cartilage tissue, skin softening or wound healing.


Background: This study aimed to analyse the effects of carbohydrate (CHO) intake on inflammatory markers, comfort, and clinical outcomes in elderly patients undergoing open radical prostatectomy.Methods: Patients aged ≥65 years who underwent open radical prostatectomy were randomly divided into CHO, drinking water, and fasting groups. A total of 90 patients were enrolled in this study . Patients in the CHO group were given 800 and 400 ml of carbohydrates 8 and 2–3 h before surgery, respectively. Patients in the placebo group were given 800 and 400 ml of water 8 and 2–3 h before surgery, respectively. Patients in the fasting group did not consume any liquids. The main result is levels of inflammation markers. Secondary results included cellular immunity, comfort, body weight, grip index, and clinical results.Results: Compared with the fasting group, the CHO group exhibited a decrease in interleukin 6 (IL-6) levels on days 1 and 7 , IL-8 levels on day 1 (274.61 pg/mL) and tumour necrosis factor (TNF) levels on days 1, 3, and 7 . The placebo group exhibited a decrease in IL-8 (390.26 pg/mL) and TNF levels (13.99 pg/mL) on day 1. Compared with the placebo group, the CHO group exhibited a decrease in IL-6 levels on day 1 and TNF levels on day 3. In the CHO and placebo groups, the thirst and hunger scores decreased on the morning of surgery.Conclusion: Preoperative CHO and drinking water are associated with decreased levels of IL-6, IL-8, and TNF. CHO and water can also reduce thirst and hunger scores. Therefore, we recommend that patients without contraindications should be given 200–400 ml of fluid 2–3 h before surgery, preferably CHO.Clinical Trial Registration:http://www.chictr.org.cn/edit.aspx?pid=21783&htm=4; ChiCTR-INR-17012867. Patients who undergo radical prostatectomy are generally at an advanced age with multiple comorbidities. Surgical trauma generally leads to a longer recovery time; therefore, accelerated rehabilitation is required. Owing to the popularity of enhanced recovery after surgery (ERAS) , 2, admiMajor open abdominal or pelvic surgery has a higher incidence of postoperative adverse events such as cardiopulmonary insufficiency, pain, thromboembolic complications, and infection. The main reason for such complications is the stress response caused by surgical trauma, followed by a relatively high-level demand for a patient's immunity and energy reserve. The relatively high-level demand for a patient's organ function is considered to be mediated by endocrine and metabolic changes caused by trauma.The levels of C-reactive protein (CRP) and cytokines are closely related to immune reaction, inflammatory response and the extent of the inflamed tissue. Interleukin (IL-6) levels are associated with the incidence of postoperative complications and are one of the predictors of the incidence of adverse events postoperatively.We hypothesise that drinking fluids before surgery can improve the immune function of patients after surgery, and the level of certain important inflammatory factors has increased. The level of inflammatory factors has a certain warning effect on the outcome of patients . Therefore, this study hypothesises that drinking liquids before surgery can improve the outcome of patients by regulating inflammatory factors.2; the American Society of Anesthesiologists (ASA) physical status I-III and normal heart, lung, liver, kidney, and blood coagulation function. Oral anticoagulants were discontinued 5–7 days before the operation. The exclusion criteria were as follows: age <65 years, inability to drink transparent liquid or allergy, gastrointestinal emptying disorder or obstruction, diabetes, liver cirrhosis, severe cardiac and renal insufficiency, corticosteroid administration at a dose more than 5 mg/day, and ASA physical status IV. The trial was approved by the Ethics Committee of the Shanghai Tenth People's Hospital, and all patients signed a written informed consent form before participating in the study.Patients who underwent open radical resection of prostate cancer in the Shanghai Tenth People's Hospital were selected for this study. The inclusion criteria were as follows: elective radical resection of prostate cancer; age ranging from 65 to 85 years; body mass index (BMI) ranging from 17.0 to 32.0 kg/mAll patients were randomly divided into the following three groups: CHO, water (placebo group) and routine water abstinence groups (fasting group). The patients were divided as follows: According to the required sample size of 120 patients, 120 two-digit random number series were generated using a random number table. The order of the remainder obtained by dividing the two-digit random number series by 3 was the order in which the patients were randomly divided into three groups. Eventually, the grouping scheme was kept in a sealed envelope. The patients were assigned to the three respective groups based on the grouping scheme. Both patients and researchers were unaware of fluid distribution in patients. Fluid was given to patients by a person who knew the distribution of CHO and placebo water and was not involved in the study.Patients who met the criteria were selected and randomly assigned to the CHO, water (placebo group), and routine water abstinence (fasting group) groups according to the envelope clue. CHO  and placebo products were produced by Jiangsu Zhengda Fenghai Pharmaceutical Co., Ltd., and both products had the same outer packaging. After completing data entry and database locking, the company revealed the product code to the researchers. The study design is demonstrated in All three groups were banned from solid food at least 6 h before surgery. From 07:00 PM to 12:00 AM on the evening before surgery, patients in the CHO group were given 800 mL of a CHO drink . On the day of surgery, patients in the CHO group consumed ~400 mL of Su Qian 2–3 h before the scheduled induction of anaesthesia, with an interval of more than 20 min. Patients in the placebo group were given the same amount of seasoning water at the same time points , which had the same taste and appearance as the CHO drink. In the fasting group, no fluid was given to patients preoperatively. To ensure smooth implementation of the experiment, the patients were scheduled for the first operation on the day of surgery. All operations were performed by the same group of experienced urological surgeons.All patients received the same general anaesthesia regimen, with sufentanil at a dose of 0.25–0.5 μg/kg, propofol at a dose of 1.5–2 mg/kg and cisatracurium benzenesulfonate at a dose of 0.2 mg/kg. After endotracheal intubation, sevoflurane inhalation was used to maintain anaesthesia with end-expiratory sevoflurane volume fraction of 0.9–1.2 minimum alveolar concentration, remifentanil at a dose of 2–5 μg/kg/h was used to induce analgesia, and cis-atracurium besylate at a dose of 4–10 mg/h was used to maintain muscle relaxation. During surgery, fluid infusion was guided based on blood pressure, heart rate, bleeding, and urine volume. Ringer's lactate solution and hydroxyethyl starch were used as supplements, crystal fluid: Colloidal fluid = 3/1 and appropriate adjuvant vasoactive drugs were also used.2) <32 mmHg (<4.3 kPa), white blood cell count >12 × 10 ∧9/L or <4 × 10 ∧9/L or immature cell count >10% and changes in the consciousness level.After surgery, the patients were encouraged to sit by the bedside or get out of the bed as soon as their health conditions permitted. If there was no nausea and vomiting, the patients were asked to drink water and eat as soon as possible. Infection is defined as the presence of sepsis, which can be diagnosed as follows: body temperature >38°C or <36°C, heart rate >90 beats/min, systolic blood pressure ≤ 100 mmHg, respiratory rate >22 beats min or partial pressure of carbon dioxide , and CRP and cellular immunity. Venous blood samples were collected at the same time point on days 1, 3, and 7 postoperatively. In addition, comfort and grip strength of the patients were measured at the same time point preoperatively and on days 1, 3, and 7 postoperatively. Comfort was measured using a 100-mm visual analogue scale (VAS)  based onOutcome indicators included the following:Main outcome indicators: levels of inflammatory markers ;Secondary outcomes indicators: cellular immunity level , comfort , the index of grip strength of body mass (grip strength [kg]/body weight [kg] × 100%) and clinical outcomes .P ≤ 0.05 indicated that the difference was statistically significant. IBMSPSS 20.0 was used for statistical analysis, and the statistical graphs were plotted using GraphPad Prism version 8.3.0.Measurement data conforming to normal distribution were represented by mean (standard deviation); non-normal distribution was represented by median , and count data were represented by the rate of adoption (%) or composition ratio (%). Standardised differences were used to evaluate the chief demographic and clinical characteristics among different groups, and the maximum value of standardised differences between two groups compared in pairs was used as the evaluation index. The measurement data were used for repeated measures analysis of variance with adjusted covariates including Age, BMI, AT, Fluid, Blood Loss, and ASA. If the difference between the treatment groups and interaction between the repetitive (time) and treatment factors were statistically significant, multiple comparisons of the treatment factors were performed according to the measurement time points (Bonferroni method). F = 0.746, P = 0.471) among the groups. Statistical differences were observed in IL-10 levels at different time points preoperatively and postoperatively . No statistically significant differences were observed in IL-10 levels among the three groups .The results of repeated measures analysis of variance revealed no interaction between IL-10 levels and measurement time points (P < 0.01). No difference was observed in the weight grip index among the three groups.Indicators for the clinical evaluation of immune function include inflammatory markers   and cellThis study revealed that the levels of inflammatory markers in the placebo and CHO groups were lower than those of the fasting group; the levels were especially lower in the CHO group. Compared with the fasting group, the CHO group exhibited a decrease in TNF levels on days 1, 3, and 7 postoperatively, IL-6 levels on days 1 and 7 postoperatively and IL-8 levels on day 1 postoperatively. Compared with the fasting group, the placebo group exhibited a decrease in IL-8 and TNF levels on the first postoperative day. The levels of three major inflammatory factors  were significantly reduced on the first postoperative day, indicating that CHO was closely associated with decreased levels of inflammatory markers. The levels of two inflammatory factors (IL-8 and TNF) in the placebo group were also significantly reduced on the first postoperative day, indicating that drinking water was also associated with the reduction of inflammatory factors. Compared with the placebo group, the CHO group only exhibited a decrease in IL-6 levels on the first postoperative day and TNF levels on the third postoperative day, indicating that CHO did not offer many advantages to reduce the levels of inflammatory factors. Therefore, preoperative consumption of a certain amount of liquid, whether CHO, sweet water, or other clear liquids, exerts similar effects on postoperative inflammation indicators. Su Qian is an energy-rich CHO beverage, whereas water is a transparent liquid without energy-rich nutrients; the difference between Sugan and water is that their sugar and energy contents are 1 and 0, respectively. Sugar and energy may not play an important role in regulating the level of inflammatory markers, and a certain amount of fluid intake preoperatively may exert significant effects on postoperative results. Compared with water deprivation, preoperative intake of a certain amount of fluid can significantly reduce the levels of inflammatory markers in the body.Anti-inflammatory cytokines are immunoregulatory molecules that control the pro-inflammatory cytokine response. They interact with specific cytokine inhibitors and soluble cytokine receptors to regulate the human immune response. Their physiological role in inflammation and pathological role in systemic inflammatory states are increasingly being recognised. Major anti-inflammatory cytokines include IL-1 receptor antagonist, IL-4, IL-10, IL-11, and IL-13. Of all anti-inflammatory cytokines, IL-10 exhibits potent anti-inflammatory properties, repressing the expression of inflammatory cytokines such as TNF-α, IL-6 and IL-1 by activated macrophages. In addition, IL-10 can upregulate endogenous anti-cytokines and downregulate pro-inflammatory cytokine receptors. Therefore, it can counter-regulate the production and function of pro-inflammatory cytokines at multiple levels .In this study, no statistical difference was observed in IL-10 levels among the three groups, indicating that the effect of preoperative liquid intake was weaker on anti-inflammatory factors such as IL-10 than that on pro-inflammatory factors such as IL-6, IL-8, and TNF.Although significant differences were observed in the levels of IL-6, IL-8, and TNF among the three groups, no difference was observed in the levels of cellular immunity indicators and the incidence of postoperative infection among the three groups; therefore, the clinical significance of CHO administration could not be determined. Because several factors affect the incidence of postoperative infection, preoperative fluid intake may not be a key factor in reducing the incidence of postoperative infections.Several studies  have demClinical outcomes among the three groups were not significantly different. Preoperative liquid intake did not play an important role because several factors affect the clinical outcome of patients. For example, the time to get out of bed is affected by factors such as medical staff education, medical cognition update, fear of getting out of bed, postoperative pain, and weakness. Some studies  have repThis quality study has some limitations. The sample size was small, and the level of inflammatory markers was not necessarily associated with the incidence of infection. Therefore, it is necessary to further investigate the influence of CHO or clear liquid on the inflammatory markers and clinical outcomes of elderly patients undergoing major surgery.Preoperative CHO and drinking water are associated with decreased levels of IL-6, IL-8, and TNF. CHO and water can also reduce thirst and hunger scores. Therefore, we recommend that patients without contraindications should be given 200–400 ml of fluid 2–3 h before surgery, preferably CHO.The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the ethical standards of Shanghai Tenth People's Hospital and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.ZH planned and implemented the experiment and wrote the article. JL retouched, revised, and contributed to the article. FW supervised the experiment and supported it with financial support. All authors contributed to the article and approved the submitted version.This study was funded by the Shanghai Science Committee Foundation  and the Natural Science Foundation of China .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Few studies have investigated the correlation between maternal polyunsaturated fatty acids (PUFAs) and telomeres in offspring, and the underlying influential mechanisms. In this study, we assessed the associations of maternal PUFAs with telomere length (TL) and DNA methylation of the telomerase reverse transcriptase (TERT) promoter in the cord blood and the placenta. A total of 274 pregnant women and their newborn babies were enrolled in this study. Maternal blood before delivery, the cord blood, and the placenta at birth were collected. Fatty acids in maternal erythrocytes and cord blood cells were measured by gas chromatography (GC). TL in the cord blood and the placenta was determined using real-time quantitative PCR (qPCR) by calculating the product ratio of telomeric DNA to the single-copy gene β-globin. The TERT promoter methylation was analyzed by DNA bisulfite sequencing. The associations of maternal fatty acids with TL were analyzed by univariate and multivariate regression. We found that low concentrations of docosapentaenoci acid  and total n-3 PUFAs, adrenic acid , and osbond acid  and high concentrations of linoleic acid  in maternal erythrocytes were associated with the shortened TL in cord blood cells , and that low concentrations of cord blood docosahexaenoic acid  were related to the shortened TL in cord blood cells. Differently, high concentrations of α-linolenic acid , eicosatrienoic acid , DHA, and γ-linoleic acid  in maternal erythrocytes were associated with the shortened TL in the placenta . Further examination demonstrated that the concentrations of DHA and total n-3 PUFAs in maternal erythrocytes had positive associations with DNA methylation of the TERT promoter in the cord blood instead of the placenta. These data suggest that maternal PUFAs are closely correlated to infant TL and the TERT promoter methylation, which are differently affected by maternal n-3 PUFAs between the cord blood and the placenta. Therefore, keeping higher levels of maternal n-3 PUFAs during pregnancy may help to maintain TL in the offspring, which is beneficial to long-term health. Two  in life , 4. It h in life , 6. Mean in life .Telomeres, the specialized DNA-protein structures located at the end of eukaryotic chromosomes, are composed of non-coding double-stranded repeats of guanine-rich tandem DNA sequences and shelterin protein structures , which fTelomere length is strictly regulated in the placenta and embryo by telomerase, playing a crucial role in fetal development . It has This study was based on a mother–child cohort in the Department of Obstetrics and Gynecology of Fuxing Hospital, Capital Medical University. Pregnant women from the Chinese Han population aged 22–35 years in the third trimester were recruited to participate in the study between July 2020 and June 2021. Women who had metabolic, endocrine, and hereditary disorders, and diseases treated with drugs were excluded. Finally, out of a total of 290 pregnant women, 274 pregnant women and their newborn babies were enrolled in the study. This study has been registered on the Clinical Trials.gov (No: ChiCTR-OCH-14004900) and approved by the Institutional Review Board and Committee on Human Research at Fuxing Hospital, Capital Medical University (2020FXHEC-KSKY005). The written informed consent from all women was obtained after oral and written explanation of this study.All pregnant women were invited to complete a general form to obtain the information  from themselves and their husbands, and a semiquantitative food frequency questionnaire to obtain food intake and supplementation of nutrients, including DHA during the whole period of pregnancy. The gestational age, gender, birth weight, and the length of newborns were collected from medical records. Maternal venous blood samples were collected in EDTA anticoagulant tubes before delivery. Plasma and blood cells were fractionated by centrifugation , exactly at 15 min after blood withdrawal. During delivery, the amnion of the placenta was removed, and a biopsy of 2 cm × 2 cm × 2 cm was cut out nearly 5 cm from the umbilical cord insertion and 1.0–1.5 cm below the fetal membrane, so as to obtain homogenous samples from the placental villous parenchyma. Then, the residual maternal blood in the placenta was removed by washing with sterile phosphate buffer saline. The babies' umbilical vein blood was collected with EDTA as an anticoagulant in the labor room or operation theater immediately after delivery by trained midwives, and processed in the same way as the maternal blood samples. The separated cord blood cells consisted of complex varieties of cells, including stem cells, leukocytes, and erythrocytes. All samples including placenta, plasma, and blood cells were stored at −80°C until analysis.2CO3 solution was added to the tube, mixed on a vortex, and centrifuged, and the clear n-hexane top layer containing FAMEs was transferred to a GC autosampler vial for analysis. FAMEs were analyzed on an Agilent 6,890 N GC system, and the quantity of fatty acids was expressed as the percentage (%) (wt/wt) of total fatty acids as described in our previous studies  based on fatty acid methyl esters (FAMEs), which were prepared according to a modified method of Lepage , 36. Bri studies , 38. TheCtt−ΔCts)−− and an Animal Tissue DNA kit , respectively. TL was determined using the real-time qPCR on a CFX96 Touch TM Real-Time PCR Detection System (Bio-Rad) by measuring the ratio of telomeric DNA product to the single-copy gene product (T/S ratio), which was calculated by 2TCACC-3′ , 39.http://www.ncbi.nlm.nih.gov) have been examined with binding sites for myeloid-specific zinc finger protein 2 (MZF2). Bisulfite sequencing was used to assess DNA methylation of the TERT promoter. Genomic DNA was given bisulfite modification with the EZ DNA Methylation TM kit . Then, nested PCR was conducted on the methyl-modified DNA with special primers, and the products were sequenced directly. Specific primers for the TERT promoter were as follows: F, 5′- TTTGAGAATTTGTAAAGAGAAATGA-3′; inner R, 5′-AATATAAAAACCCTAAAAACAAATAC-3′; and outer R, 5′-AAAAAAACCATAATATAAAAACCCT-3′  −746 to −445 (positions are given relative to the transcription start site) within nts 1254,147–1253,148  38). Th. Th38). ACCCT-3′ .p < 0.05 was considered to be statistically significant.All analyses were performed with SPSS version 20.0. The Kolmogorov–Smirnov test was used to evaluate whether the data are normally distributed. Variables conforming to normal distribution were presented as mean and SD. The characteristics for parents  and newborns , outcome variables, and fatty acid concentrations were descriptively analyzed. The linear regression analysis was conducted to explore the univariate associations between individual fatty acid concentrations in maternal erythrocytes and TL in the cord blood cells and the placenta. For each fatty acid, the continuous model and the categorical model were used. The continuous model included the SD score (SDS) as continuous concentrations of fatty acids, in which fatty acid concentrations in maternal erythrocytes were independent variables and TL were dependent variables. The categorical model included quintiles as categorical concentrations of fatty acids. In the categorical model, quintiles were performed as a measure of maternal fatty acid concentration with the middle quintile as a reference to determine the potential non-linearity of the association , and the2) was in the normal range, whereas paternal BMI (25.45 ± 3.67 kg/m2) was located in the range of overweight based on Chinese population standards. Almost all mothers during pregnancy were given folic acid supplementation for the purpose of infant neural tube defect prevention. Dietary records showed that 52.19% of mothers received DHA supplementation during pregnancy, who had higher erythrocyte DHA concentrations than those with no DHA supplementation (p < 0.05), with a shorter TL in boys than in girls.The characteristics of mothers and their newborn babies are shown in entation . The newNo differences were shown in TL between the umbilical cord blood (0.99 ± 0.08) and the placenta (0.97 ± 0.59). Maternal erythrocyte n-3 PUFA concentrations with quintile distributions are presented in The associations between maternal erythrocyte PUFA concentrations and TL in the placenta are shown in As shown in As shown in In the current study, after the adjustment for relevant variables, lower concentrations of maternal erythrocyte GLA, ADA, OA, DPA, and total n-3 PUFAs, and higher concentrations of LA, total n-6 PUFAs, and higher n-6/n-3 PUFA ratio were associated with the shortened TL in cord blood cells. Further analysis demonstrated a positive correlation between cord blood TL and DHA concentrations in cord blood cells instead of maternal erythrocytes, implying the effect of maternal erythrocyte n-3 PUFAs on cord blood TL might be associated with increased DHA levels in the fetus. Whereas, in the placenta, higher concentrations of maternal erythrocyte GLA, LNA, EA, and DHA were associated with the shortened TL. So far, the associations of the maternal PUFA status with offspring TL have been reported in only two studies, showing no correlations between maternal n-3 PUFAs and TL in newborns and children , 31, whiBeing different from the Paleolithic diet, modern westernized diets are deficient in n-3 PUFAs and have excessive amounts of n-6 PUFAs, resulting in a high n-6/n-3 ratio, which has been shown to promote the pathogenesis of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases , 48. TheInterestingly, we found that maternal LA, total n-6 PUFAs, and n-6/n-3 PUFA ratio were negatively associated with cord blood TL, whereas GLA, ADA, and OA belonging to n-6 series were positively associated. The reason for this to happen may be involved in their effects to inhibit inflammation, which is a risk factor for TL shortening. For example, GLA is converted into DGLA and forms a substrate for cyclooxygenase enzyme that fights inflammation, being in contrast to LA and AA, which tend to promote inflammation . Also, i12 are inversely associated with placental TL, whereas 25 (OH) D3 is positively associated, and no associations were observed with cord blood TL  and GLA with TL in the placenta, which was in contrast to the results in the cord blood. This might be attributable to the differences in lifespan, functions, and sensitivity to maternal nutrition between the placenta and cord blood , 33. In blood TL .The TERT expression represents the rate-limiting step in telomerase expression and activity and plays a key role in maintaining TL during cellular aging, immortalization, and transformation . DNA metOur data suggest that a higher intake of n-3 PUFAs during pregnancy may help to maintain TL in the offspring, which is beneficial for long-term health. Some limitations of our study should be addressed. Previous studies have demonstrated the associations between TL and chronic diseases, including cardiovascular diseases and obesity. Unfortunately, we only examined TL in the cord blood cells and the placenta with no data on TL after birth available, and thus its association with chronic diseases in later life could not be investigated in the current study. Meanwhile, we did not determine single-nucleotide polymorphisms in the fatty acid desaturase, which has strong effects on levels of long-chain PUFAs. Thus, this genetic factor should be included in analyzing the association of PUFAs with TL in the future.In summary, low concentrations of maternal n-3 PUFAs and some of n-6 PUFAs , and high concentrations of LA and high n-6/n-3 PUFA ratio were associated with the shortened TL in cord blood cells; low concentrations of cord blood DHA were related to the shortened cord blood TL. Whereas, in the placenta, higher concentrations of either maternal n-3 PUFAs  or GLA were associated with the shortened TL. Furthermore, maternal DHA and total n-3 PUFAs had a positive association with the TERT promoter DNA methylation in the cord blood instead of the placenta. Therefore, maternal PUFAs are closely correlated to infant TL and the TERT promoter methylation. Different effects of maternal n-3 PUFAs on TL and the TERT promoter methylation between the cord blood and the placenta are suggested.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Institutional Review Board and Committee on Human Research at Fu Xing Hospital, Capital Medical University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.XL performed the experiment, analyzed the data, and prepared the manuscript. QS performed the experiment. XF participated in designing the research and analyzed leukocyte TL. HC, NC, and YZ performed questionnaires and sample collection. KQ designed the research and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Research Funds of Reform and Development Budget from Beijing Municipal Science and Technology Commission (2020-bjsekyjs to KQ), the National Key R&D Program of China , and Beijing Xicheng District Talent Project - top team (202048). The funders had no role in study design, data collection and analysis, interpretation of data, decision to publish, or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ObjectiveWith the aging of the global population, an increase in the proportion of elderly patients presenting with traumatic brain injury (TBI) is expected. This population presents several distinctive characteristics that impact management and outcome of TBI, such as comorbidities, frailty, and preinjury use of medications - specially antiplatelets and anticoagulants. The purpose of this study was to assess the general characteristics and prognostic factors of elderly patients with TBI that were surgically managed at a single institution.MethodsThe authors performed a retrospective review of all elderly patients (age ≥ 65 years) with a history of TBI that underwent cranial neurosurgical procedures at their institution, between 2015 and 2019. Clinical characteristics, laboratory tests, and radiological scans, as well as surgeries, performed, outcome, and prognostic factors were analyzed, comprising 133 consecutive cases overall.ResultsThe mean age of patients was 76.6 ± 7.3 years, ranging from 65 years to 97 years. There was a predominance of males (71.4%) and the most frequent mechanism of injury was fall (80.4%). Mild TBI comprised 57.1% of the cases, followed by severe TBI in 25.6%. Frequent signs and symptoms were impaired consciousness (69.9%), focal motor deficits (32.3%), and gait disturbances (12.8%). The majority had reported comorbidities upon admission (79.7%), with cardiac disease (79.2%) and diabetes (24.5%) as the most frequent. Preinjury anticoagulation was reported in 18.8% and use of antiplatelet drugs in 17.3%. The most common finding in the head CT was chronic subdural hematoma (48.1%), followed by acute subdural hematoma (37.6%). Coagulation was found to be altered in 12.8% of the patients. The most common neurosurgical procedure performed was trephination for hematoma evacuation (56.3%), followed by craniotomy (21.2%). Blood product transfusion was needed in 61.7% of the patients. Overall mortality was 42.1%, with the majority in the first month after admission (83.9%). Unfavorable outcome  at discharge was identified in 73% of the patients. Identified prognostic factors were TBI severity, absent pupillary reactivity, acute intracranial bleeding on head CT, basal cisterns obliteration, altered coagulation status, and need for blood transfusion.ConclusionsTBI severity, pupillary reactivity, coagulation status, need for blood products transfusion and acute bleeding, as well as basal cisterns obliteration found in head CT, are factors that influenced the outcome in this series of elderly patients with TBI that need surgical management. It is paramount to observe the particularities of this population in this context, to optimize outcomes, avoid complications and ultimately generate awareness focused on prevention. It is currently well-known that the global population is aging, with estimates from the United Nations that one in six people in the world will be over 65 years by 2050 . In the Traumatic brain injury (TBI) has three peak incidences, occurring mainly among children, young adults, and the elderly . TBI aloUnlike their younger counterparts, the most common TBI manifestations in the elderly are contusions and subdural and intracerebral hemorrhage. The physiological changes that occur in this population explain the differences in the patterns of brain injury - reduced brain volume associated with increased subdural space, fragile walls of cerebral vessels - especially bridging veins - and adherence of the dura-mater to the inner surface of the cranium . In addiThe goal of this study was to analyze the clinical characteristics and prognostic factors of a consecutive series of elderly patients that underwent neurosurgical procedures at our institution following TBI.Population studiedThe authors performed a retrospective review of records and radiological scans of all elderly patients (age ≥ 65 years upon admission) with a diagnosis of TBI and who underwent any TBI-related neurosurgical procedure at the University Hospital of Ribeirão Preto Medical School, São Paulo, Brazil, a level I regional trauma center, between 2015 and 2019. Patients with radiological findings of chronic subdural hematoma, but no confirmed history of trauma were excluded from this study.All acute trauma patients were evaluated according to the Advanced Trauma Life Support guidelines of the American College of Surgeons, and further radiological and neurosurgical assessments were employed whenever deemed necessary by the admitting team. The remaining patients, for which there were no acute injuries , were admitted to the neurosurgical ward for observation.Data were obtained from electronic medical charts and included demographic information, mechanism of injury, known comorbidities, status of anticoagulation and platelet antiaggregation, prescription drugs, clinical signs, and symptoms upon admission - including pupillary reaction - associated lesions, head computed tomography findings, laboratory results, surgeries performed, length of stay, complications, need for blood transfusion, outcomes  and mortality. Outcome was classified in favorable (GOS = 5) and unfavorable (GOS <5).The serum lactate and base deficit levels on admission were categorized as described by Callaway et al., for their known role as prognostic biomarkers; the lactate level was classified as normal (0 to 2.4 mmol/L), moderately elevated (2.5 to 4.0 mmol/L), or severely elevated (>4.0 mmol/L) and base deficit was classified as normal (> 0 mEq/L), moderate (0 to -6 mEq/L) or severe (< -6 mEq/L) . CoagulaInstitutional approval for this study was obtained from the Ethics Committee of the University Hospital of Ribeirão Preto Medical School - University of São Paulo.Statistical analysisGraphPad Prism 8  was used for the statistical analyses. Descriptive data were presented as means (with standard deviation), median - when applicable, and proportions. Univariate analysis was performed with Fisher’s exact test for categorical variables, except for admission Glasgow Coma Scale, lactate level and base deficit, which were analyzed with chi-square. Odds-ratio (OR) with 95% confidence intervals (CI) were calculated. Statistical significance was assumed when ɑ = 0.05.A total of 133 elderly patients who matched the including criteria were identified. Table 3/mm3. Upon admission, approximately fifty percent of patients (63 patients) had normal serum lactate levels and 63% had moderate base deficit (62 patients). Coagulation status was measured by prothrombin time and presented as per the INR, with a mean of 1.59 ± 1.7 (range 0.86-11.4) and was considered altered (INR ≥ 2.0) in approximately 12.8% of patients, including two patients with an uncoagulable status. Fibrinogen was measured in thirty patients and was considered reduced (< 180 mg/dL) in five patients (16.7%).Table A total of 151 surgical procedures were performed for this series of patients. Table Finally, clinical outcome and analysis of prognostic factors are reported in Table The main mechanisms of injury in this series were fall 80.4%), motor vehicle collision (11.3%) - pedestrian and vehicle occupant - and assault (1.5%), but no gun-related injuries. However, there was a predominance of fall over the remaining mechanisms, when comparing with other reported series (30-51% of trauma cases in this age group) that account for elderly patients with TBI in general - surgical and conservative cases 0.4%, mot,15. ThisClinical presentation was variable, however, 69.9% of the patients had an impaired consciousness upon admission, followed by focal motor deficits and gait disturbances. This raises the importance to alert caretakers and family members for changes in behavior, focal weakness, and gait disturbances, signs that may be overlooked and mistakenly attributed to physiological consequences of aging, leading to underreporting and failure to seek medical attention promptly . Also, aA large study assessing the influence of preexisting medical conditions in elderly trauma patients identified 33% of patients with at least one comorbidity and it was associated with an increased risk of death . In thisThe role of polypharmacy  spanning from inappropriate use of prescribed medicines, as well as self-medicating without an indication, in this age group is associated with the high prevalence of comorbidities .Preinjury use of anticoagulant or antiplatelet drugs is an example of a worrisome issue in this population, due to the higher risk of complications and mortality following low energy traumas, such as falls. Besides the prompt identification of the coagulation status of the elderly patient with TBI, is the need for urgent reversal of anticoagulation or antiplatelet agents, when a TBI is suspected, in order to reduce the risk of mortality or unfavorable outcome -24. AlthThe high mortality and predominant proportion of unfavorable outcomes in this series underscore the importance of the already reported specific protocols for the management of elderly patients with TBI. Protocols spanning from hospital admission, inpatient care - if possible, in a dedicated unit or early admission to an intensive care unit - and transfer to a rehabilitation facility or discharge with an integral follow-up -30. AlsoLimitationsThe major limitation of this study is that it only comprises elderly patients with TBI that underwent any neurosurgical procedure following admission. This limitation is due to the nature of the database used - the registry of all neurosurgical procedures of our institution. Thus, it is expected that the results will be impacted towards worse clinical presentations, higher mortality, and unfavorable outcomes. Also, patients that had a favorable outcome upon hospital discharged, were commonly discharged from the neurosurgery outpatient clinic if no complications or limitations were detected, thus reducing the data available for outcome measures at 60 days, six months, and one-year post-admission. Another limitation may be that not all the patients had been tested for fibrinogen, serum lactate, and base deficit levels upon admission - in our institution only the severe and acute trauma patients have the blood gases and fibrinogen tested, thus reducing the statistical power of the analysis of these factors.The ongoing transition in the epidemiology of TBI following the aging of the global population highlights the necessity of special attention to the peculiarities of this group, with an emphasis on the physiological changes, comorbidities, and use of multiple medications as factors that impact the outcome, obviating the need to raise awareness towards TBI prevention in this particular group. Factors that affect prognosis in the population studied herein are TBI severity, pupillary reactivity, bleeding type as found in head CT, basal cisterns obliteration, anticoagulation status, and transfusion of blood products.


IDHmut gliomas are also less aggressive than their IDHwt counterparts, but still usually recur after treatment and are ultimately fatal; breakthrough seizures are often a harbinger of such recurrences. Here, we report an extremely unusual case where a low-grade glioma only had IDHmut and did not recur or cause breakthrough seizures until 22 years after original diagnosis, without a progression in grade. Yet, seizures manifested at both the original presentation and recurrence. This case raises questions about the nature of IDHmut and its natural course when secondary TP53/ATRX or TERT/1p/19q alterations do not develop.Gliomas with mutations in isocitrate dehydrogenase 1 or 2 . Because of this sensitive location, surgical management was limited to a biopsy, but this biopsy did not yield a firm diagnosis and was deemed non-diagnostic (outside pathology not available). Soon after that biopsy, the patient had her first focal seizure, characterized by clonic movements of the left arm and face. She was initially started on phenobarbital, and carbamazepine was subsequently added due to continued focal motor seizures of the left hand. Later that year, the patient underwent a craniotomy and a second biopsy, again at an outside institution, which generated a pathologic diagnosis of diffuse astrocytoma, WHO grade 2. Adjuvant therapy included 30 fractions of radiation, but no chemotherapy. She was weaned off phenobarbital, continued carbamazepine monotherapy, and remained seizure-free on that regimen for 22 years, with stable MRIs during that entire time.Twenty-two years after her first seizure, the patient (now 59) experienced a focal seizure that was characterized by clonic movements of the left arm and face lasting approximately 15 minutes. An MRI showed an ill-defined lesion in the right frontal perirolandic area with equivocal contrast enhancement . The patAfter medication adjustments stabilized her seizures, the patient underwent another biopsy to determine whether the astrocytoma had recurred and progressed.The newest biopsy from the right motor strip, 22 years after initial disease presentation, showed mildly hypercellular gray matter with extensive vascular hyalinization, the latter attributable to the remote history of radiotherapy . AlthougMGMT promoter methylation. No other molecular alterations were detected, including no mutations in ATRX, TP53, or TERT promoter, and no copy number alterations on chromosomal microarray.Next-generation sequencing of the newest biopsy confirmed the IDH1 R132H mutation with a 7% variant allelic fraction, as well as positive mut gliomas were published in 2008 and 2009.2 Now, the classification of adult-type diffuse gliomas is based on the presence or absence of IDHmut (and 1p/19q codeletion).3 IDHmut, TERTwt, 1p/19q-intact gliomas are astrocytomas, and the vast majority of IDHmut astrocytomas contain mutations in TP53 and/or ATRX.5 In our analysis of 240 IDHmut, TERTwt, 1p/19q-intact astrocytomas in The Cancer Genome Atlas (TCGA) via GlioVis,6 only four (1.7%) were wild-type for both TP53 and ATRX. All four were histologically grade 2, all occurred in 20- to 30-year-old patients , and all four patients had survived at least 17 months after diagnosis, with no recorded deaths in the database.Molecular diagnostics has revolutionized the workup and classification of central nervous system tumors, allowing for more accuracy than by evaluation of histological findings alone. The original papers describing IDHmut appears to be the first alteration to occur in IDHmut astrocytomas, with TP53 and/or ATRX arising later.8 In the current case, the initial tumor was diagnosed by microscopy as a grade 2 astrocytoma, about 10 years before the discovery of IDHmut in gliomas. Although the original tumor tissue was not available, it seems reasonable to assume that IDHmut was present at that time. While IDHmut gliomas are less aggressive compared to IDHwt glioblastomas, IDHmut gliomas are ultimately fatal in the overwhelming majority of cases. Even in the least aggressive subtype—IDHmut, 1p/19q-codeleted oligodendrogliomas—survival beyond 15 years is uncommon. In the current case, after 22 years, the recurrent tumor cells were difficult to detect by microscopy, and by molecular testing, those cells only had IDHmut. This suggests that IDHmut by itself acts in a relatively indolent manner, and that most cases coming to clinical attention do so because they acquire additional TP53 and ATRX alterations (or TERT promoter mutations and 1p/19q codeletion), thereby accelerating tumor proliferation and evolution. Indeed, the long cell processes highlighted by IDH1 R132H IHC  was low compared to what is typical for most glioma patients. Our prior work, and that of others, consistently showed that IDHmut in glioma, with a similarly rare extended patient survival. This also illustrates the fidelity of breakthrough seizures as a marker of glioma recurrence, even in such a prolonged disease course.In summary, this case highlights an extremely rare instance of isolated IDH


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