diff --git "a/deduped/dedup_0088.jsonl" "b/deduped/dedup_0088.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0088.jsonl" @@ -0,0 +1,39 @@ +{"text": "O(n6)time and O(n4) space algorithm by Rivas and Eddy is currently the best available program.The general problem of RNA secondary structure prediction under the widely used thermodynamic model is known to be NP-complete when the structures considered include arbitrary pseudoknots. For restricted classes of pseudoknots, several polynomial time algorithms have been designed, where the O(n4) time and O(n2) space to predict the energetically optimal structure of an RNA sequence, possible containing such pseudoknots. Evaluation against a large collection of known pseudoknotted structures shows the adequacy of the canonization approach and our algorithm.We introduce the class of canonical simple recursive pseudoknots and present an algorithm that requires RNA pseudoknots of medium size can now be predicted reliably as well as efficiently by the new algorithm. Pseudoknots have been shown to be functionally relevant in many RNA mediated processes. Examples are the self-splicing group I introns , ribosomn into unknotted structures requires O(n3) time and O(n2) space, finding the best structure including arbitrary pseudoknots has been proved to be NP-complete O(n4) timO(n6) time and O(n4) space ]]]..........}}.This grammar is useful to judge how different an experimentally determined structure is from class csr-PK. It is not useful for programming, since it is ambiguous and does not distinguish the fine grained level of detail required in the energy model.A careful discussion is required to show that each simple recursive pseudoknot, if not canonical by itself, has (a) a canonical representative of (b) similar free energy.i' and j'. It is not canonical if one of the two helices contains bulges. However, there must be at least one pair of shorter helices without bulges at i, j with i' \u2264 i and j \u2264 j', which serves as a canonical representative, albeit with somewhat higher free energy.Rule 1 (b) affects the length of helices that are considered in forming the pseudoknot. Let there be a pseudoknot between i, k, l, j there is a canonical one with maximal helices, whose free energy is at least as low \u2013 except for the following case: The maximal helices compete with the internal structure of u, v and w. It may be possible to contrive a structure where shortening (say) helix a \u2013 a' by one base pair allows to create two pairs with new partner bases in u and v, resulting in a structure which has slightly lower energy. Still, the free energy of the canonical pseudoknot must be very similar.Rule 2 is justified by the fact that the energy model strongly favours helix extension. Clearly, for each family of pseudoknots delineated by a' or the b \u2013 b' helix.Finally, Rule 3 requires a decision where to draw the border between two helices facing each other and competing for the same bases. An arbitrary decision here can only slightly affect free energy, as the same base pairs are stacked either on the a \u2013 E(s) denote the free energy computed for structure s. Summing up, we have shown that for each simple recursive pseudoknot K, there is a canonical one C in the search space. While we cannot prove that E(C) \u2264 E(K), we have argued that this is likely, and if not, the energies will at least be close. Still, there might be another, energetically optimal canonical structure S (knotted or not) such that E(K) 25% and <75% in the parents, only two, the c. 596C>G in the exon 2 of the 4Taken into account the clinical features and the identified mutated gene, we conclude that the patient is affected by TRMA. The identified mutation was not previously described. The identification of the inactivating mutation in the family will allow prenatal diagnosis for future pregnancy in the family and for follow\u2010up and treatment of eventual new siblings. Indeed, lifelong oral treatment with thiamine at pharmacological doses (25\u2010100\u00a0mg/day) reverse anemia and delay diabetes, and, when started during the first years of life, prevent hearing defects.SLC19A2 gene. The first one was identified by direct sequencing of known genes involved in diabetes resulting in a protein truncated at the beginning of the ninth transmembrane domain.Our patient is the second Egyptian with a pathogenic mutation in the SLC19A2 inactivating mutations are diffused in the Egyptian genetic background and are different from those already retrieved worldwide. On the previous basis it appears interesting to propose the inclusion of the SLC19A2 gene in the panels of candidate genes used for the diagnosis of complex diseases associating bone marrow failure, diabetes and hearing loss and, possibly, developmental anomalies of heart and bones .Thus, it seems that The authors have no conflict of interest to declare."} +{"text": "Reducing rural\u2013urban disparities in health and health care has been a key policy goal for the Chinese government. With mental health becoming an increasingly significant public health issue in China, empirical evidence of disparities in the use of mental health services can guide steps to reduce them. We conducted this study to inform China\u2019s on-going health-care reform through examining how health insurance might reduce rural\u2013urban disparities in the utilization of mental health inpatient services in China. This retrospective study used 10\u00a0years (2005\u20132014) of hospital electronic health records from the Shandong Center for Mental Health and the DaiZhuang Psychiatric Hospital, two major psychiatric hospitals in Shandong Province. Health insurance was measured using types of health insurance and the actual reimbursement ratio (RR). Utilization of mental health inpatient services was measured by hospitalization cost, length of stay (LOS), and frequency of hospitalization. We examined rural\u2013urban disparities in the use of mental health services, as well as the role of health insurance in reducing such disparities. Hospitalization costs, LOS, and frequency of hospitalization were all found to be lower among rural than among urban inpatients. Having health insurance and benefiting from a relatively high RR were found to be significantly associated with a greater utilization of inpatient services, among both urban and rural residents. In addition, an increase in the RR was found to be significantly associated with an increase in the use of mental health services among rural patients. Consistent with the existing literature, our study suggests that increasing insurance schemes\u2019 reimbursement levels could lead to substantial increases in the use of mental health inpatient services among rural patients, and a reduction in rural\u2013urban disparities in service utilization. In order to promote mental health care and reduce rural\u2013urban disparities in its utilization in China, improving rural health insurance coverage would be a powerful policy instrument. Mental health has become an increasingly significant public health concern worldwide. Yet the utilization rates of mental health services remain low compared with those for physical health conditions , 60% of which was out-of-pocket (OOP) expenses CNHEI . MoreoveAs of 2009, severe mental illnesses were incorporated into the national public health service program of hospital electronic health records (EHRs) to examine the effects of the three main health insurance schemes on the utilization of mental health services in China. EHR data are not subject to recall bias; the actual RR was constructed to measure health insurance coverage, and is a more accurate measure than the type of health insurance.We designed a retrospective cohort study of patients hospitalized with mental illnesses to assess rural\u2013urban disparities in the utilization of mental health services, and the role of health insurance. Our study site included two major psychiatric hospitals in Shandong, China: the Shandong Center for Mental Health (SCMH) and the Daizhuang Psychiatric Hospital (DPH).Shandong is the second-most populous province in China. With more than 97 million people, its population accounts for 7.2% of the national total. In the province, 44 million people (44.99% of the population) live in rural areas, which is consistent with the national rural and urban population distribution . In total, there are 1158 general hospitals providing mental health services and 53 psychiatric hospitals in the province. SCMH is the only provincial psychiatric hospital, and DPH is one of the oldest psychiatric hospitals in the province. These two psychiatric hospitals serve almost 10% of all mental health patients every year in Shandong HFPCSD , and accOur study population was identified using patients\u2019 primary diagnosis, as recorded in the two hospitals\u2019 EHRs, which routinely record information on patients\u2019 socio-demographic characteristics ; clinical characteristics ; cost-related information ; and insurance information . A major strength of the EHR data is that they document all inpatient expenses incurred during hospitalization in a detailed, itemized, and reliable way. All data are collected and recorded by hospital registries, physician workstations, and the insurance settlement departments, with minimal recall bias. Descriptive statistics of variables are presented in \u201cAppendix 1\u201d.Figure\u00a0As discussed earlier, there are large variations in insurance coverage for mental health services, not only across different insurance programs but also within the same insurance program. Therefore, in addition to insurance type, we used actual RR in this study to measure health insurance. Compared with the conventional measures such as insurance type or policy, RR is a more accurate measure in determining how generous the health insurance is in terms of coverage . The exact equation being: RR\u00a0=\u00a0(TC-OOP)/TC\u00a0*\u00a0100%. TC represents total medical costs, and OOP represents out-of-pocket payments made by individuals for their health-care services. In our sample, the actual RR ranged from 0 to 100%.All statistical analyses were based on a pooled dataset with information from the two EHR databases on the selected sample. Descriptive analyses, the Mann\u2013Whitney U test, and multiple regressions combined with the Poisson model were employed to examine the effect of health insurance on the utilization of mental health services.Descriptive analyses were used to describe patient characteristics and use of mental health services, measured by LOS, frequency of hospitalization, and hospitalization costs. We chose to analyze the utilization of mental health inpatient services using these three measures as they are the most commonly used for inpatient resource use ; area of residence ; and admission severity . Mental health utilization is measured by hospitalization cost, LOS, and frequency of hospitalization. The insurance variable is denoted by three dummy variables: UEBMI, URBMI, and NCMS. In addition, we also measure health insurance using the reimbursement ratio (RR). The interaction terms (RR\u00a0*\u00a0Insurance) in Model (2) refers to RR\u00a0*\u00a0UEBMI, RR\u00a0*\u00a0URBMI, and RR\u00a0*\u00a0NCMS. (See \u201cAppendix 2\u201d for variable definitions.)We used the following empirical specifications to examine disparities in the utilization of mental health services:The LOS and hospitalization costs showed a seriously skewed distribution. Therefore, log transformation of these dependent variables were used in the estimations. Moreover, as a count variable, the distribution of the frequency of utilization was strongly skewed to the right. The Poisson model was used to analyze the effect of health insurance on the frequency of utilization; 18.0 SPSS software was used for the statistical analyses with an alpha level of 0.05.The descriptive statistics of our sample are presented in Table\u00a0Table\u00a0Figure\u00a0The multiple regression results for the use of mental health services, based on Models (1) and (2), are presented in Table\u00a0In China, more than 173 million people suffer from mental disorders . In order to reduce rural\u2013urban disparities in health-care utilization, the Chinese government launched the gradual merger of the URBMI and NCMS systems as of 2016, in order to establish a uniform basic medical insurance system for rural and urban residents. During this process, policy makers in China would need to consider significantly improving the insurance coverage , including increasing the RR and the annual reimbursement limit, and expanding coverage services to mental inpatients to cover the unmet need that provides a considerable amount of mental health services to people with severe mental disorders at the community level. Future studies should look at both outpatient and community mental health services. Second, patients with private health insurance were excluded from our study, given that private insurance in China is still nascent, mainly targeting high-income households and covering only about 7% of the population (Wang et al. Large rural\u2013urban disparities in the utilization of mental health services exist in China, with financial concerns being a major barrier to rural patients seeking mental health care. Health insurance coverage, particularly the reimbursement ratio, could be a powerful policy tool to influence people\u2019s health-care utilization. In order to improve access to and reduce rural\u2013urban disparities in mental health care, future health-care reform in China should consider expanding mental health coverage, particularly in rural areas, as well as making health insurance portable."} +{"text": "Nature Communications 10.1038/s41467-019-08447-z, published online 4 February 2019.Correction to: The original PDF version of this Article contained an error in Table 3, in which all chemical structures were omitted and only the numerical data was shown. This has been corrected in the PDF version of the Article. The HTML version was correct from the time of publication."} +{"text": "Field Epidemiology Training Programmes (FETPs) are functional ways of strengthening epidemiology, surveillance and outbreak response capacity in countries. However, sustainability of FETPs is a major challenge facing most countries especially in Africa. The Ghana Field Epidemiology and Laboratory Training Program (GFELTP) started in 2007 in the University of Ghana School of Public Health as a solution to gaps in the public health workforce. This paper assessed the sustainability strategies embedded in the Ghana Field Epidemiology and Laboratory Training Programme.We assessed the sustainability of GFELTP by document reviews and interviews with programme staff and stakeholders to identify sustainability structures that were in place. We grouped information into the following headings: programme structure, institutional, financial and political structures.As of July 2017, a total of 350 public health experts have been trained in both frontline and advanced courses since the programme's inception. For funding structures, the programme is funded mainly by its partners and stakeholders who are local government organisations. They provide resources for running of programme activities. Under institutional and political structures, the programme was established as a Ministry of Health/Ghana Health Service programme based in the University of Ghana. The programme steering committee which is currently chaired by the Director Public Health of Ghana Health Service, jointly ensures its implementation. Other structures of sustainability observed were involvement of stakeholders and alumni in human resource of the programme; use of stakeholders as faculty for the programme. These stakeholders include staff from University of Ghana School of Public Health, Ghana Health Service and Veterinary Service Department, World Health Organization and Centers for Disease Control and Prevention. The programme showed evidence of stable sustainability strategies in all four structures evaluated.The assessment found the GFELTP to be sustainable. The main factors that contributed to rendering it sustainable were funding, programme, institutional and political structures embedded in the programme. One remarkable sustainability element observed was the strong collaboration that existed between stakeholders of the programme who worked hand in hand to ensure the programme runs smoothly. However, more sources of funding and other essential resources need to be considered to help the programme obtain a pool of resources for carrying out its activities. Sustainability of public health programmes is of much importance since it helps in maintaining and improving existing health system structures. Field Epidemiology Training Programmes (FETPs) are described by Schneider and colleagues as functional ways of strengthening epidemiology, surveillance and outbreak response capacity in countries . The maiStudy design: the study was conducted in the GFELTP secretariat from March to June, 2017. A record review of database of residents and all programmatic documents was done. Interviews (key informant interviews (KII) were also carried out. We grouped the information collected under four main categories; programmatic, financial institutional and political structures. All components of the programme which contributed to ensuring continuous provision of the programme and its services were considered.Data collection: data collection was done with a data extraction tool which covered information on residents' demographic characteristics, and available data on the indicators of sustainability we sought to assess. An interview guide based on the indicators of sustainability we sought to assess was used for the qualitative interviews. Questions on; how the programme has been funded since its inception, who the programme stakeholders were and the roles they played, how the programme runs and political structures governing the programme and the achievements of the programme since its inception were asked. We retrieved and extracted data from a database of residents enrolled onto the program from 2007 to 2017 and examined data on resident demographic information such as gender, nationality, and place of work before enrollment into the programme and their current places of work. All programme documents including the FELTP curricula, reports on residents, programme activities, and programme support files and administrative documents of the GFELTP from its inception were reviewed and information extracted. To understand the funding of the program, we reviewed the financial records and reports on funding activities and extracted needed information. We interviewed key informants such as the Foundation Director of the programme, Past Dean-School of Public Health, Director of Public Health-Ghana Health Service, CDC, WHO, Programme Directors past and present, Field Coordinator, Programme Coordinator and administrator. These interviews were done to give a more detailed understaning of the structures the programme has in place.Data processing and analysis: we generated frequencies and proportions of residents' demographic characteristics. Recorded interviews were transcribed. We assessed sustainability by the following indicators: programme funding, programme evaluation, organisational capacity, institutional structures, political support, partnership and planning [planning ,10. Docuplanning ,12. Stakplanning ,14. Sustplanning .Programme structurehow the programme is run and its performance from 2007-2017: GFELTP runs a two-tier training programme; frontline which is a three-month course and advanced which is a two-year training course which offers an MPhil degree. As of July 2017, the frontline course had trained seven cohorts and the advanced ten cohorts.Residents trained by GFELTP: the programme has trained 350 people mainly from the Health Services with 94% (329) being Ghanaians. Residents of the programme were from all ten regions of Ghana and other national and international conferences. A total of six awards were won during these conferences (ferences .Positions occupied by Alumni of the programme: Nearly 30% (20/70) of the alumni occupy positions as Directors of Public Health Service in the Ministry of Health, Ghana Health Service, and Veterinary Services Department. Twenty percent (14/70) also head various departments in the health facilities. However, 20% (14/70) have still not been placed by the Health Service after completion of the programme . The funding of the entire programme was taken over by stakeholders when funding ceased during the fifth year. Subsequently, funding from different donors over the years have been in the form of partial support for programme activities and assistance for residents training. The Dean of the School of Public Health supports some outbreak investigations with other projects overheads and residents' self-support. Other donors who have supported the programme include; The Presidential Malaria Initiative of the USAID which supports some residents annually in conducting malaria-based researches; US President's Emergency Plan for AIDS Relief (PEPFAR); and West Africa Health Organization (WAHO). The World Health Organization (WHO) also supported the training of frontline health workers among others. Overall, most programme activities have been funded with resources provided by stakeholders.Funding of residents training: over the years, the advanced course has been funded by donors, stakeholders and residents. Stakeholders played a key role in funding of residents over the years, providing support to half (50/100) of the residents. The following were some of the ways identified: residents who were mainly supported by stakeholders paid their tuition whiles the cost of field work, outbreak investigations and attendance of conferences was borne by stakeholders of the programme. The Dean of the School of Public Health also supported some outbreak investigations done by residents with overhead costs generated from other projects running in the school. Also, the Government of Ghana granted residents study leave during the training and paid their salaries throughout the FETP training and Veterinary Service Directorate (VSD) serves as field sites for the residency training. As a competency based programme training with the one health approach, various stakeholders provide technical expertise to teach the residents. Each year, guest lecturers from partner institutions such as CDC and WHO visit the program to deliver lectures.Stakeholders and Partnership with the programme: the stakeholders of the programme include; Health Service , University of Ghana, School of Public Health and AFENET. They play an intertwined role in providing resources . These stakeholders and partners collaborate in supporting the smooth running of all activities of the programme. The programme works with both local and international partners such as CDC Ghana Country Office, WHO Ghana Country Office, WAHO and Food and Drugs Authority (FDA), among others. These organisations collaborate with the programme in implementation of its activities. Stakeholders provide various resources in the form of human resource for field supervision, outbreak investigation, teaching and mentoring of residents and provision of financial resources for running of other programme activities.Faculty of the programme: in addition to university staff on the programme, stakeholders also provide lectures in veterinary, laboratory and epidemiology in accordance with the One Health Approach. Periodically, alumni teach some didactic sessions of the courses.Alumni trained to become faculty: for continuity and availability of staff, alumni are encouraged to pursue academia so that they can take up teaching appointments offered by the programme. A total of 4 graduates have completed their upgrade from MPhil to PhD with 10 currently enrolled unto PhD programmes. These alumni offer support to the programme and other programmes in other African countries such as Liberia, Sierra Leone and Namibia through teaching and mentorship of residents.Field supervision and mentorship of residents: the programme has a strong alumni network which serves as a pool of experts for the programme. Supervision during field work/mentorship is done mainly by alumni at the districts, regional health directorate and national levels. Alumni work closely with the residents and supervise their acquisition of skills and knowledge, commonly referred to as bound volume competencies, with the assistance of Ghana Health Service staff. Some of the university faculty members periodically conduct field supervisory visits to the residents especially those from other West African countries such as Liberia and The Gambia.Political support: the programme was established under the mandate of the Ministry of Health/Ghana Health Service and is engrained in the University of Ghana system. It therefore reports to the Director General of Ghana Health Service through the Director Public Health and to the Vice Chancellor of the University of Ghana through the Dean of School of Public Health. The reporting structures were agreed to through a memorandum of understanding signed between the two stakeholders. GFELTP has a steering committee that steers the affairs of the programme. The committee is chaired by Director of Public Health, Ghana Health Service. The steering committee is made up of stakeholders and partners. This committee meets at least twice a year to plan and take major decisions for the programme.The GFELTP has been in existence since 2007. This paper sought to determine the sustainability structures embedded in the programme and other structures it has in place to ensure its smooth running. This assessment found programmatic, organizational, institutional and political sustainability strategies in the operation structures of GFELTP. The frontline training, introduced a few years ago as a means of reaching out to frontline health workers at district level; is a major sustainability strategy. This training provides frontline health workers with requisite skills to detect, monitor and manage outbreaks effectively . It creaAfter support from the initial donors ceased, the programme reached out to other organisations to build partnerships to supplement stakeholder efforts in training residents. Survival of the programme during this time was of critical concern since sustaining grant-funded programmes after funding ceases has been known as a major public health challenge . AccordiShould funds for the programme run out, the overhead cost of the programme are absorbed by the university through funds from other projects it runs. This has been one of the greatest sustainability strategies which has kept the programme running over the past years. Over the years, GFELTP has weathered the storm of financial challenges using this strategy and was saved from this major setback in its early years. This innovative idea adopted at the inception of the programme is in line with the principle that implementation and sustainability go hand in hand. Therefore, ways of ensuring the longevity of a programme need to be in place during the initial stages of implementation . ResourcLimitations: this review had a few challenges. Interviews were mainly recall of events that had happened in the past ten years. To reduce recall bias, different people were interviewed and the information obtained crosschecked for consistency with each other. Additionally, review of programme documents confirmed some of the interviews outcomes.The programme had evidence of stable sustainability strategies in all structures evaluated, namely; programme, financial, institutional and political. This assessment showed a strong collaboration between stakeholders who work hand in hand to ensure the programme runs smoothly. However, more sources of funding, broadening stakeholder partnership and other essential resources need to be considered to help the programme obtain a pool of resources for carrying out its activities.The Ghana Field Epidemiology and Laboratory Training programme trains health workers to build a resilient public health workforce in Africa;Most Field Epidemiology and Laboratory Training programmes rely mainly on donor support to run their programmes and this could have implications on the programmes if the funds run out;Sustainability of Field Epidemiology and Laboratory Training programmes is essential and needs to be looked at by all programmes.In the face of financial challenges due to the reduction in donor support, the Ghana Field Epidemiology and Laboratory Training programme was able to continue its mandate of training health workers;Aside donor support Field Epidemiology and Laboratory Training programmes can explore other alternatives of securing resources to run their programmes;The Ghana Field Epidemiology and Laboratory Training programme has innovative sustainability strategies integrated in its structure.The authors declare no competing interests."} +{"text": "R and MCF-7-TaxR compared to the parental MCF-7 breast cancer cells. Using ChIP, short-interfering RNA (siRNA) knockdown, and overexpression assays as well as Foxo1/3/4\u2212/\u2212 MEFs, we establish the endoplasmic reticulum (ER)-stress defence modulator PERK (eIF2AK3) as a direct downstream transcriptional target of FOXO3. In agreement, there is also a positive correlation between FOXO3 and PERK expression at the protein and RNA levels in breast cancer patient samples. We uncover that PERK expression is downregulated but its activity constitutively elevated in the drug-resistant cells. With this in mind, we exploit this adaptive response of low FOXO3 and PERK expression, and high PERK activity in drug-resistant breast cancer cells and show that these drug-resistant cells are specifically sensitive to PERK inhibition. In support of this finding, we show that ectopic overexpression of FOXO3 can reduce the sensitivity of the resistant cells to the PERK inhibitor GSK2606414, while the Foxo1/3/4\u2212/\u2212 MEFs expressing lower levels of PERK are more sensitive to PERK inhibition compared to wild-type MEFs. PERK inhibitor-titration and -time course experiments showed that the drug-resistant cells, which express lower expression and higher activity levels of PERK, are more sensitive to the increasing concentrations of PERK inhibitor compared to parental MCF-7 cells. Our present work thus reveals a chemotherapeutic drug-resistant cancer cell vulnerability in PERK and suggests PERK as a potential target for cancer therapy, specifically in the context of drug-resistant cancers.The major impediment to effective cancer therapy has been the development of drug resistance. The tumour suppressive transcription factor FOXO3 promotes cell cycle arrest, senescence and cell death, and mediates the cytotoxic and cytostatic functions of cancer therapeutics. In consequence, FOXO3 is often downregulated as an adaptive response in cancer and particularly in chemotherapeutic drug-resistant cells. Consistently, we find that FOXO3 expression is attenuated in the drug-resistant MCF-7-Epi Endoplasmic reticulum (ER) is a cellular organelle with a central role in maintaining proteostasis through its involvement in protein synthesis, folding, quality control and distribution. Defective proteostasis leads to the accumulation of misfolded or unfolded proteins in the ER lumen, which will cause \u2018ER stress\u2019. This cellular stress condition triggers the unfolded protein response (UPR), a highly conserved reaction, which aims to restore proteostasis. However, this survival-promoting function of the UPR can switch to a cell death-inducing mode if proteostasis is overwhelming and cannot be rectified in time. UPR is activated upon the detection of the misfolded/unfolded protein accumulation in the ER by the Heat Shock 70\u2009kDa Protein 5 (HSPA5) also known as glucose-regulated protein 78 (GRP78). UPR is mediated primarily through three signalling axes driven by three ER-transmembrane signalling proteins, IRE1\u03b1 (Inositol Requiring 1\u03b1), PERK (PKR-like ER Kinase) and ATF6\u03b1 (activating transcription factor 6\u03b1), respectively.Cells orchestrate a finely tuned balance between protein synthesis and degradation to maintain protein homoeostasis transcription factor plays a critical role in promoting cell cycle arrest, senescence and cell death, as well as mediating the cytotoxic and cytostatic functions of cancer therapeutics. FOXO3 acts downstream of the phosphatidylinositol 3-kinase-protein kinase B (PI3K-PKB/AKT) signalling pathway as a tumour suppressor, preventing the transmission of potentially oncogenic mutations and activities by negatively controlling cellular proliferation. For example, FOXO3 regulates the expression of negative cell proliferation regulators, such as p27 and BIM \u20139 but re and BIM , 10, 11. and BIM , 12. Two and BIM . Moreove and BIM . PERK ha and BIM \u201317.ER defences have been implicated in chemoresistance . TargeteR cells. Western blot analysis showed that P-PERK (T981) levels, which reflect its activity, were low in MCF-7 but high in the resistant MCF-7EpiR cells in response to epirubicin treatment -Seq study in DLD1 colon carcinoma cells [PERK promoter region in the MCF-7 and MCF-7-EpiR cell lines, with FOXO3 primers designed to recognise a region \u2013583 and \u2013794 (bp) upstream of PERK gene staining in a HER2-positive cohort of breast cancer patient samples Fig. . IHC resO3) Fig. . This fuO3) Fig. . Taken tR and MCF-7-TaxR cells. Sulforhodamine B (SRB) and clonogenic assays revealed that both MCF-7-EpiR and MCF-7-TaxR cells are significantly more sensitivity to GSK2606414 compared to the parental MCF-7 cells levels until at least 48\u2009h in MCF-7 cells. The activation of FOXO3 by GSK2606414 in MCF-7-EpiR and MCF-7-TaxR cells was mirrored by the induction of the pro-apoptotic FOXO3 target BIM in the MCF-7-EpiR and MCF-7-TaxR cells but not in the MCF-7 cells. These data further confirmed that PERK negatively regulates FOXO3 activity via AKT and suggested that MCF-7-EpiR and MCF-7-TaxR cells are more sensitive to PERK inhibition compared to MCF-7 cells, because of their lower PERK levels. Interestingly, P-PDK1 and its target P-AKT (308) did not decline following GSK2606414 treatment, suggesting PERK is unlikely to mediate AKT activation and FOXO3 phosphorylation (T32) via PDK1.Subsequent western blot results showed that treatment with a high dose (2\u2009\u03bcM) of the specific PERK inhibitor GSK2606414 caused a coordinated decline in P-PERK, P-FOXO3 and P-AKT (473) levels, suggesting that PERK inhibition by GSK2606414 induces FOXO3 activation through repressing AKT Fig. . NotablyR and MCF-7-TaxR cells as revealed by P-PERK dephosphorylation. Altogether, these results suggested that the drug-resistant cells, which present low PERK expression but high activity are more sensitive to GSK2606414.To demonstrate further that the drug-resistant cells are more sensitive to GSK2606414 because of their lower PERK levels, we next performed dose-titration experiments on the drug-sensitive and -resistant MCF-7 cells Fig. . WesternR and MCF-7-TaxR cells and treated these transfected cells with various doses of GSK2606414. RT-qPCR and western blot analysis showed that FOXO3 ectopic expression induced PERK expression, particularly after GSK2606414 treatment -stress defence modulator PERK (eIF2AK3) as a direct downstream transcriptional target of FOXO3 through its promoter. Epirubicin can impact on the phosphorylation status of FOXO3 (T32) through a number of mechanisms. First, epirubicin can activate p38 MAPK and JNK [FOXO3 has been shown to mediate the cytotoxic effects of a variety of cytotoxic chemotherapeutic drugs, including epirubicin and paclitaxel , 38\u201342. and JNK , which c and JNK . Moreove and JNK . In hereIn support of our finding, we also uncover that there is also a strong and significant correlation between FOXO3 and PERK mRNA levels in the Cancer Genome Atlas (TCGA) breast cancer patient datasets. This is likely to be due to the fact that FOXO3 regulates not only PERK expression but also its own transcription in a positive feed forward mechanism . In factFoxo1/3/4\u2212/\u2212 MEFs, which express lower PERK levels but more resistant to other conventional chemotherapeutic drugs are more sensitivity to PERK inhibition compared with their wild-type counterparts. In addition, our experiments also show that although overexpression of FOXO3 decreases the overall clonogenicity of the paclitaxel- and epirubicin-resistant cells, it increases their resistance to the PERK inhibitor.The high levels of the activated P-PERK in the drug-resistant breast cancer cell lines point to elevated UPR in response to heightened ER-stress in the drug-resistant cells. This is likely to be caused by the increased accumulation of misfolded/unfolded gene products as a result of higher metabolic rates and mutation frequencies in these drug-resistant cancer cells. This adaptive dependence on higher PERK activity to combat elevated ER-stress coupled to the intrinsically low PERK levels as a result of low expression and activity of its regulator FOXO3 in these drug-resistant breast cancer cells renders them particularly vulnerable to PERK inhibition. This notion is supported by our PERK inhibitor titration assays and time course experiments showing that PERK activity, as revealed by P-PERK (T981), is more susceptible to inhibition by increasing concentrations of GSK2606414 in the drug-resistant cells compared to MCF-7 cells because of their low PERK levels. The latter is further supported by our findings that PERK has previously been shown to be able to phosphorylate FOXO3 directly at S261, S298, S301, S303 and S311 to promote FOXO3 nuclear relocation and activation . In contIn conclusion, our study establishes PERK (eIF2AK3) as a direct downstream transcriptional target of FOXO3. Our work also identifies the adaptive response of low FOXO3 expression to boost drug survival and the overdependence on elevated PERK activity to overcome heightened ER-stress, as an acquired vulnerability of the drug-resistant cancer cells. The fact that there is a strong and significant correlation between PERK and FOXO3 expression in the breast cancer patient samples suggest that this FOXO3-PERK regulatory axis is preserved in most breast cancers. Our present work provides the explanation for the reason why drug-resistant cancer cells are prone to PERK inhibition and further suggests that PERK inhibitors provide a therapeutic opportunity for targeting cancer, in particular the drug-resistant cancer cells.The patient tissue samples came from a study consisting of 447 HER2-positive breast cancer patients who underwent surgery for primary breast cancer between 2006 and 2011, at the Department of Pathology, Asan Medical Centre, Seoul, Korea. Formalin-fixed, paraffin-embedded tissue samples from these preoperatively chemo- and radiotherapy naive patients were available for analysis as previously described . See alsR (resistant to epirubicin) [R (resistant to paclitaxel) [Foxo1/3/4\u2212/\u2212 were kind gifts from Professor Boudewijn Burgering, UMC, Utrecht, the Netherlands and have previously been described [2. The drug resistance of MCF-7-TaxR cells was maintained with 0.05\u2009\u03bcM Paclitaxel , and the MCF-7-EpiR cells in 17\u2009\u03bcM epirubicin .MCF-7, a human breast cancer cell line, originated in the American Type Culture Collection and was acquired from the Cell Culture Service, Cancer Research UK , where it was tested and authenticated. The MCF-7-Epirubicin) and MCF-litaxel) cell linlitaxel) . Mouse eescribed , 59, 60.Plasmid transfections were performed with FuGENE\u00ae HD (Promega) according to the manufacturer\u2019s protocol. For siRNA, cells were transfected with ON-TARGETplus SMARTpool siRNAs using Oligofectamine following the manufacturer\u2019s instructions. After 48\u2009h of transfection, cells were treated with epirubicin, GSK2606414 or paclitaxel for the time points indicated and collected for analysis by western blot, RT-qPCR, SRB or clonogenic assays.Kip1 (sc-528) and \u03b2-tubulin H-235; sc-9104) antibodies were purchased from Santa Cruz Biotechnology . The P-FOXO1 (Thr24)/FOXO3 (Thr32) (CST# 9464) and FOXO3 (CST#2497), PERK (CST#3192), FOXO1 (CST#9454), FOXO4 (CST #9472), Bim (CST#2933), P-AKT (S473) (CST#9271), P-AKT(T308) (CST#9275), AKT (CST#9272) P-PDK-1 (S241) (CST#3061), PDK-1 (CST#3062) and eIF2\u03b1 (CST#5324) were purchased from Cell Signaling Technology . The P-elF2\u03b1 antibody . The primary antibodies (1:1000) were detected using horseradish peroxidase-conjugated secondary antibody and visualised using the ECL detection system .Western blot was performed on whole-cell extracts as previously described using thRT-qPCR analysis was performed as described . Total RChIP analysis was performed as described . The celThe sulforhodamine B assay was used for analysing short-term cell viability in drug-treated cells following GSK2606414 treatment and/or FOXO3 overexpression and has previously been described . See alsR, MCF-7-TaxR cells were seeded into six-well plates and left overnight for adherence, after which they were treated with increasing concentrations of GSK2606414 and has previously been described [Total of 3000 MCF-7, MCF-7-Epiescribed . See alst-test was used to compare the means. For comparisons between groups of more than two unpaired values, one-way analysis of variance (ANOVA) was used. Two-way ANOVA was used between groups of two variables, and was considered significant when *P\u2009<\u20090.05\u2009s, **P\u2009<\u20090.01 and ***P\u2009<\u20090.001. ns for non-significant.The correlations between protein expression in patient samples were analysed by Chi-square and bi-variate Pearson Correlation statistical analysis using SPSS 16.0 . Other results shown are representative of three independent experiments, which were each performed in triplicate (presented as the mean\u2009\u00b1\u2009SEM). GraphPad Prism was used for statistical analysis , and two-tailed Student\u2019s Supplementary Materials and MethodsSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5Supplementary Figure S6Supplementary Figure S7Supplementary Figure S8Supplementary Figure Legends"} +{"text": "Breast cancer is the most frequent oncological disease as well as the leading cause of cancer death among women worldwide. Decline in mortality in economically strong countries is observed. This decline is mostly related to early diagnosis (an improvement in breast cancer awareness and the mammography screening program (MSP)) and a more effective treatment. In the end of 2005, MSP started in Lithuania. The main aim of this article is to evaluate the breast cancer mortality during 22 years in Lithuania, as well as changes before the start of the MSP and during its implementation, in order to assess the influence of the MSP on mortality.Analysis is based on data from the population-based Lithuanian Cancer Registry. Analysis of changes in mortality includes the period from 1998 to 2019. Age standardized mortality rates are calculated for assessment of changes. Joinpoint regression analysis is used.Applying the segmental regression model, it was found that during the study period mortality was statistically significantly decreasing by -1.1% each year. Mortality among women under the age of 50 decreased both before and during the implementation of MSP. Mortality in the target population also was already decreasing until the implementation of the program, but a significant reduction in mortality was observed in this group since 2006. Overall breast cancer mortality is decreasing in Lithuania. After the implementation of MSP the largest reduction in mortality was observed in the target population, however, it is not as pronounced as it could be with the well-organized MSP. Breast cancer (BC) is the most frequent oncological disease, also it is the leading cause of cancer death among women in 101 countries worldwide . During In Europe, BC also is the most frequently diagnosed neoplasm in women and the leading cancer site for deaths from cancer in women . The decThe primary goal of MSP is to reduce the BC mortality. MSP is a complex multistep process therefore it is very important to monitor the performance of the national BC screening program from its inception to determine how closely the benefits it achieves approach the benefits seen in the randomized trials and population demonstration projects .In the end of 2005, the MSP started in Lithuania. In a recent report on cancer screening in the EU, Lithuania was the country with the lowest participation rate (44.9% in 2014) and one of three countries, where centralized invitation through a screening registry was not implemented . DespiteThe main aim of this article is to evaluate the breast cancer mortality during 22 years in Lithuania, as well as changes before the start of MSP and during its implementation, in order to assess the influence of MSP on mortality. Analysis is based on data from the population-based Cancer Registry. The Lithuanian Cancer Registry is a population-based cancer registry that contains personal and demographic information , as well as information on the diagnosis and death (date of death and cause of death) of all cancer patients in Lithuania, where the population size is approximately 3 million residents according to the 2011 census . The priBreast Imaging Reporting and Data System) system is used, and for the evaluation of breast density, typology according to the ACR (American College of Radiology) is included [The Lithuanian BC screening program started at the end of 2005, when the order of the Lithuanian Health Ministry was issued. According to the program, the target population is defined as 50-69-year-old women. Women are referred to screening mammography by their general practitioners or gynaecologists every two years. Mammograms are obtained in two standard projections and are independently read by two radiologists. Both screen-film and digital mammography systems are present in Lithuania. For reporting of screening and additional imaging results, the BIRADS ; 70 y.o. and older, aiming to assess the influence of MSP on mortality in the target population. The period of research was divided into 2 periods: a period before MSP (1998 to 2005) and a period of MSP implementation (from 2006 to 2019). Age standardized mortality rates were calculated for assessment of changes (the direct standardization method was applied and the old European Standard Population was used). Analysis of the breast cancer mortality was carried out by using JOINPOINT software (version 4.3.1.0). Joinpoint regression analysis was used to identify points where a statistically significant change over time in the linear slope of the trend occurred. The annual percent change (APC) was calculated for the trends by means of the generalized linear model. APCs were considered statistically significant if p < 0.05. Joinpoint analysis was performed for all ages combined and age-specific rates. The graphic depiction was implemented by using Microsoft Excel software.The age standardized mortality rate in 1998 was 25.1/100 000, and in 2019 it was 21.0/100 000. During the study period, the overall breast cancer mortality was statistically decreasing by -1.1% each year . The significant decrease in mortality was observed in both younger age groups, but in the group older than 70 y.o. the nonsignificant increase by 0.2% was found . Mortality rates and their changes are presented in Before the implementation of MSP the mortality decrease was seen in women younger than 50\u00a0y.o., while the decrease in the target population was not statistically significant, and in the age group older than 70 y.o. mortality was distinctly increasing. After the implementation of MSP the mortality rates have decreased in all age groups. The largest reduction in mortality was observed in the target population. In the age group older than 70 y.o. the decrease was not statistically significant. Changes in mortality in age groups in two periods (before the start of MSP and after its implementation) are presented in Several countries started implementing national MSPs in 1980s and 1990s, based on the results of randomized trials and meta-analyses, that have shown that the breast cancer mortality can be reduced by 25-30% with mammography screening ,16. HoweIn order to assess the effectiveness of MSP implementation in the country and its impact on mortality, it is necessary to assess trends in the BC mortality in the country. Such studies evaluate the changes in mortality in the population after the introduction of MSP. For comparison, the BC mortality before and after the introduction of screening is assessed, as well as and the trends in age groups affected and unaffected by screening .Applying the segmental regression model, it was found that mortality among women under the age of 50 decreased both before and during the implementation of MSP. Mortality in the target population also was already decreasing until the implementation of the program, but since 2006 a significant reduction in mortality was observed in this group. Thus, in our country, mortality decreased both in the target population exposed to MSP and in the group of women under 50 who are not included into the program. In the group of women over the age of 70, the overall increase in mortality since 1998 was seen. Since 2006, the reduction in mortality was observed in this age group, but it was not statistically significant. Women in this age group could be indirectly exposed to MSP .One study examined trends in the BC mortality in thirty European countries from 1989 to 2006 . MortaliDetailed comparative studies of mortality trends have been performed in Spain, Italy, the Neth-erlands, Denmark and Sweden -28. In sIt is quite difficult to determine whether the implementation of MSP had an impact on the reduction of mortality in Lithuania. The fact that a decrease in mortality is also observed in young women not participating in the screening group suggests that there are other factors that contribute to the reduction in mortality. In this group of women, the reduction in mortality is mainly due to improved treatment options . The decThe decrease in mortality observed in our study among women over the age of 70 during the MSP implementation is not statistically significant and can hardly be attributed to it. These women are indirectly exposed to MSP, a reduction in mortality after some time should also be observed among them . There may be several reasons why the impact of the implemented MSP in Lithuania may not be as great as expected. It can be due to a small number of participants and a low number of early stage tumours detected during MSP, as well as a lack of unified system to ensure the most rapid and effective treatment of women diagnosed with BC during MSP. The participation rate in Lithuania seeks only a half of the target population. The cases of BC detected during MSP make only 25% of BC diagnosed in 50-69 year old women. The cases of stage I BC detected during MSP make only 49% of all tumours detected by MSP . Also, tWhile assessing the impact of MSP on mortality, mortality trends over time are usually assessed first. However, it is important to emphasize that it takes a long time for the overall statistics to reflect the impact of MSP on mortality reduction in the general population . It shouIt should also be noted that the results of trend assessments can be influenced by several factors. Women diagnosed with BC live a long time, with a five-year survival rate of 90% in developed countries . TherefoAs in most similar studies comparing the periods before and after the introduction of MSP, in our study the beginning of the second period coincided with the date of introduction of MSP in our country. Such a division of the study into periods may reduce the expected effect of MSP because its effect on population indicators takes time even in case of an effective implementation of MSP. As there are many positive developments in the treatment of BC , it becomes more difficult to interpret studies of mortality trends.It is also always difficult to accurately quantify the contribution of other factors to the reduction in mortality. Usually, countries undergo opportunistic screening for BC or pilot projects in individual regions prior to the introduction of an organized MSP at the state level, which also reduces the impact of an organized MSP on mortality changes . As our During the study period, the overall breast cancer mortality was decreasing statistically significantly. The largest decrease in mortality was observed among women younger than 50 y.o. After the implementation of MSP the largest reduction in mortality was observed in the target population, however, it is not as pronounced as it could be with the well-organized MSP. A further, more detailed research is needed in order to clarify the effects of MSP in Lithuania."} +{"text": "The HOPE Project is an ongoing RCT testing whether Senior Corps volunteering for lonely older adults (age 60+) leads to reduced loneliness and improved quality of life\u2014outcomes associated with suicide in later life. We have randomly assigned 130 participants to 12-months of volunteering or active control. We will describe the trial as well as baseline characteristics of participants that may predict non-compliance with volunteering/control. We found no difference between conditions nor demographic characteristics on non-compliance. Participants demonstrated wide variability in depression at baseline (PROMIS t-score range 38.9 to 71.4) and 18% reported suicide ideation; neither were associated with compliance (p>.20). These preliminary findings indicate that those with more severe mental health symptoms were equally willing/able to engage in volunteering as those without depression and suicide ideation. Volunteering is a highly scalable intervention (given nationwide Senior Corps infrastructure) that may function as upstream suicide prevention."} +{"text": "In comparison to male patients with coronary artery disease, female patients suffer from more comorbidities, experience symptoms of coronary artery disease differently and report poorer health-related quality of life (HRQoL) after coronary revascularization. However, there is limited data on the impact of comorbidity burden on the recovery in HRQoL in female and male patients. We investigated the impact of comorbidity burden on the change in HRQoL following coronary revascularization in female patients versus male patients. 230 patients with coronary artery disease were assessed before, and two weeks, three months and six months after coronary revascularization. Disease-specific HRQoL was measured with the Short-Form Seattle Angina Questionnaire. Physical and mental health was measured with the Short-Form Health Survey. Comorbidity burden was assessed by the total number of identified comorbidity conditions and by the Charlson comorbidity score. Linear mixed models were used to estimate the effects of time, gender and comorbidity burden on HRQoL. Whereas HRQoL improved after coronary revascularization in all patients, female patients reported poorer physical health and disease-specific HRQoL and their physical health improved more slowly than male patients. A higher comorbidity burden was related with poorer physical health and disease-specific HRQoL in male patients, but not in female patients. A higher comorbidity burden was associated with slower improvement in HRQoL for both female and male patients. Female patients reported poorer HRQoL and their physical health improved more slowly after coronary revascularization, irrespective of comorbidity burden. Higher comorbidity burden was associated with poorer physical health and disease-specific HRQoL in male patients only. Our results indicate that female and male patients recover differently after coronary revascularization. These findings highlight the importance of comorbidity- and gender-specific approaches for evaluating coronary artery disease and coronary revascularization procedures. Coronary artery disease (CAD) is the leading cause of death and disability in the Western world . The treAs CAD patients are becoming older, they are more likely to suffer from comorbidities which increases the overall disease burden and may negatively impact HRQoL \u20139. FemalThis study aims to investigate the impact of comorbidity burden on changes in HRQoL following coronary revascularization in female versus male CAD patients.All patients were recruited at the cardiology departments of the Amsterdam University Medical Centers: Academic Medical Center (AMC) and VU Medical Center (VUmc) locations. All patients were planned for coronary revascularization procedure after being discussed in the multidisciplinary \u201cheart teams\u201d. Patients were eligible if they were 18 years or older, had stable CAD and were scheduled for elective coronary artery bypass graft (CABG) or elective percutaneous coronary intervention (PCI). Patients had to have at least one comorbidity and mental health (MCS) component score. The component scores are transformed into a 0\u2013100 scale, with higher scores indicating better health.Generic HRQoL was assessed using the Short-Form Health Survey . The queDisease-specific HRQoL was assessed using the Short-Form Seattle Angina Questionnaire (SAQ) . The SAQPatients\u2019 comorbidity conditions were identified using the hospitals electronic medical records. If records were unclear, they were checked by two medical specialists. Comorbidity burden was operationalized using two methods:low (summed comorbidities = one), middle (summed comorbidities = two), and high (summed comorbidities = three or higher). The higher the category, the higher the comorbidity burden.For each patient we summed all identified comorbidity conditions and categorized them into: low (sum weighted comorbidities = zero), middle (sum weighted comorbidities = one), and high (sum weighted comorbidities = two or higher). The higher the category, the higher the comorbidity burden.The Charlson comorbidity category is based on the Charlson comorbidity score , which qAge and intervention type were included as possible confounders as they are associated with comorbidity burden and with HRQoL [th HRQoL , 7, 22.lme4 version 1.1\u201316 [We used linear mixed models to investigate if HRQoL changed over time for all patients, if there were different patterns of change in HRQoL between male and female patients, if the impact of comorbidity burden on HRQoL differed between male and female patients, and if there was a different pattern of change over time in HRQoL among patients with different levels of comorbidity burden. All analyses were adjusted for age and intervention type. Linear mixed models allow for missing HRQoL measurements and provide valid estimates without the need for imputation of data. For both the total comorbidity conditions and the Charlson comorbidity category we estimated a separate model, resulting in two estimates for each HRQoL measure. Patient\u2019s intercepts were modelled as random effects. All linear mixed models were estimated using the R-package n 1.1\u201316 .Data collection took place from September 2015 until March 2018. A total of 467 patients were approached for the study, of whom 144 patients did not respond (31%) and three patients were excluded because their coronary revascularization was delayed. From the remaining 320 patients, 57 patients were excluded because they only underwent a diagnostic procedure, 31 patients were excluded because they responded to questionnaires at only one assessment period, and two patients were excluded because no comorbidities were identified. This resulted in a final sample size of 230 in physical health , Table 2Mental health improved significantly over time for all patients. There was no difference in the pattern of change over time between male and female patients , Table 2Disease-specific HRQoL improved significantly over time for all patients. Female patients reported significantly poorer disease-specific HRQoL than male patients. However, the pattern of change in disease-specific HRQoL did not differ between female and male patients , Table 2The present study investigated the impact of comorbidity burden on the change in HRQoL in female versus male patients after coronary revascularization. The HRQoL of all patients improved after coronary revascularization. However, female patients reported poorer physical health and disease-specific HRQoL than male patients and their physical health improved more slowly after revascularization than that of male patients. Surprisingly, we found that a higher comorbidity burden was associated with poorer physical health and poorer disease-specific HRQoL in male patients but not in female patients. Lastly, a higher comorbidity burden was associated with slower improvement in disease-specific HRQoL following coronary revascularization for both female patients and male patients.While female patients reported worse physical health and disease-specific HRQoL than male patients, female patients did not differ from male patients in mental health. These results are in contrast to previous findings in both general and CAD populations where female reported poorer mental health than male , 24.We also found that the physical health of female patients recovered more slowly than that of male patients following coronary revascularization. Our findings are in line with those of Sajobi and colleagues who inveIn contrast to our findings, a previous study reported a negative impact of comorbidity on the HRQoL in both male and female patients. In this previous study they meaAlso of interest, we found that a higher comorbidity burden was accompanied with a slower improvement (change over time) in disease-specific HRQoL, whereas the rate of improvement in physical health was not related to comorbidity burden. It seems that comorbid conditions not only have worsened CAD symptoms but also have impeded recovery of disease-specific HRQoL. Interestingly, the most frequent comorbid condition in our sample was diabetes, which has been shown to be associated with lower recovery in HRQoL than in patients without diabetes .Our results indicate that HRQoL following coronary revascularization is experienced differently by female than by male patients. We also found a difference in comorbidity burden between female and male patients. Specifically, comorbidity burden was found to be more strongly associated with worse HRQoL in male patients than in female patients. Although we cannot entirely explain this finding, depression may be involved as an intermediary variable between gender and comorbidity burden . Since rWe encourage health care providers to be alert to comorbidity burden among their CAD patients who are scheduled to undergo coronary revascularization, as these patients might require extra care. Our results also emphasize the importance of gender-specific health care approaches before and after coronary revascularization. Future studies could explore whether comorbidity- and gender-specific health care approaches following coronary revascularization would increase the effectiveness of coronary revascularization. Such research is needed to ensure that CAD patients receive appropriate and patient-tailored care.This study has several limitations. We only included patients with at least one comorbidity and therefore could not contrast these patients with those without comorbidity. Further, the number of female patients was small, and accounted for only 26% of the sample, although this percentage is in line with a common CAD population. Post-hoc power analysis revealed that, with a sample size of 60 and a two-sided alpha of 0.05 we were able to detect a standardized mean difference of 0,52 between male and female patients with 80% power , 28. TheThis study has several strengths. Most patients completed all questionnaires while linear mixed models provide valid estimates of HRQoL in the presence of missing measurements. We investigated both generic and disease-specific HRQoL, rendering a comprehensive picture of HRQoL. We included consecutive patients from two major cardiology referral centres. Our study had a longitudinal design with a baseline measurement prior to the coronary revascularization and three follow-up measurements. Furthermore, the use of two measures of comorbidity burden provides robust results. Finally, comorbidity data were collected by electronic medical records and if unclear were checked by two medical specialists rather than relying on self-report measures of comorbidity.Female patients reported poorer HRQoL and their physical health improved more slowly after coronary revascularization than male patients. Higher comorbidity burden was associated with poorer physical health and disease-specific HRQoL in male patients only. Our results provide further insight that female and male patients recover differently after coronary revascularization. These findings also highlight the importance for comorbidity- and gender-specific approaches for evaluating CAD and coronary revascularization procedures.S1 Table(DOCX)Click here for additional data file.S1 Data(TXT)Click here for additional data file."} +{"text": "As such, the current study investigates whether 15dPMJ2 induces DAMP signaling in melanoma. In B16F10 cells, 15dPMJ2 caused a significant increase in the cell surface expression of calreticulin (CRT), the release of ATP and the secretion of high-mobility group box 1 (HMGB1), three molecules that serve as surrogate markers of ICD. 15dPMJ2 also stimulated the cell surface expression of the DAMP molecules, heat shock protein 70 (Hsp70) and Hsp90. In addition, the display of CRT and ATP was increased by 15dPMJ2 to a greater extent in tumorigenic compared to non-tumorigenic melanocytes. Consistent with this finding, the activation of bone marrow-derived DCs was upregulated in co-cultures with 15dPMJ2-treated tumor compared to non-tumor melanocytes. Moreover, 15dPMJ2-mediated DAMP exposure and DC activation required the electrophilic cyclopentenone double bond within the structure of 15dPMJ2 and the ER stress pathway. These results demonstrate that 15dPMJ2 is a tumor-selective inducer of DAMP signaling in melanoma.Metastatic melanoma is the most deadly skin neoplasm in the United States. Outcomes for this lethal disease have improved dramatically due to the use of both targeted and immunostimulatory drugs. Immunogenic cell death (ICD) has emerged as another approach for initiating antitumor immunity. ICD is triggered by tumor cells that display damage-associated molecular patterns (DAMPs). These DAMP molecules recruit and activate dendritic cells (DCs) that present tumor-specific antigens to T cells which eliminate neoplastic cells. Interestingly, the expression of DAMP molecules occurs in an endoplasmic reticulum (ER) stress-dependent manner. We have previously shown that ER stress was required for the cytotoxic activity of the endocannabinoid metabolite, 15-deoxy, \u0394 The immune system plays a pivotal role in preventing both the formation and survival of cancer through surveillance and elimination of abnormal cells. Tumor cells circumvent this process using different strategies including the inhibition of cytotoxic and/or the stimulation of immunosuppressive cell types . ClinicaThe endoplasmic reticulum (ER) stress pathway regulates different cellular processes including the emission of DAMP molecules , 9. ER s12,14 prostamide J2 (15dPMJ2) is a metabolic product of the endocannabinoid, arachidonoyl ethanolamide (AEA). 15dPMJ2 suppresses the growth of B16F10 melanoma tumors in C57BL/6 mice [2 is also cytotoxic towards non-melanoma skin cancer (NMSC) and colon cancer cell lines as well as primary patient melanoma cells. In addition, 15dPMJ2 induced cell death is mediated by the ER stress pathway. Hence, the goal of the current study was to determine whether 15dPMJ2 activates DAMPs in melanoma cells. Moreover, the role of ER stress in 15dPMJ2-induced DAMP signaling was explored. Our data demonstrate that 15dPMJ2 causes tumor-directed DAMP exposure and DC activation through a process that relies on the ER stress pathway.15-deoxy, \u0394L/6 mice . 15dPMJ22, B16F10 melanoma cells were treated with 15dPMJ2 and cell survival was examined. As a positive control, the cells were also treated with the prototype DAMP-ICD inducer, oxaliplatin. 15dPMJ2 and oxaliplatin significantly reduced the viability of B16F10 cells with similar levels of death occurring at a substantially lower concentration of 15dPMJ2 (5 \u03bcM) than oxaliplatin (500 \u03bcM) and colorectal cancer (HT29) cell lines . Several studies have revealed that the exposure of CRT and ATP occurs prior to apoptosis rather than as a consequence of apoptosis [2-induced DAMP exposure was investigated. Caspase 3/7 activity and phosphatidylserine exposure, two indicators of apoptosis, were initially detected after 8 hours of treatment with 15dPMJ2 (2 were pre-apoptotic events similar to other DAMP-ICD inducing agents [To verify the cytotoxicity of 15dPMJ(500 \u03bcM) . We thennd HMGB1 . Comparaliplatin and 1C. ed cells . It has liplatin and 1F. ancer HT2 cell ling agents .2 exhibited greater cytotoxicity towards tumorigenic than non-tumorigenic melanocytes [2-induced DAMP expression occurred preferentially in tumors. To examine tumor-selective DAMP induction, we utilized the B16F10 (tumorigenic) and Melan-A (non-tumorigenic) cell lines that were derived from C57BL/6 mice. In the presence of 15dPMJ2, the display of CRT and ATP was significantly elevated in tumorigenic compared to non-tumorigenic melanocytes as opposed to non-tumorigenic (HaCaT cells) keratinocytes . Hence, 15dPMJ2 causes the exposure of key DAMPs selectively in tumor cells.Our previous study showed that 15dPMJanocytes . We founanocytes , 18, we anocytes and 2B. anocytes and 2B. 2-induced DAMP exposure leads to DC activation. To evaluate DC phagocytic activity, B16F10 and Melan-A cells were labeled with the tracking dye, CMFDA, and the cells were treated with 15dPMJ2, oxaliplatin or vehicle. The labeled melanocytes were then co-incubated with na\u00efve, bone marrow-derived DCs that were extracted from C57BL/6 mice. Treatment of B16F10 cells with 15dPMJ2 or oxaliplatin stimulated its phagocytosis by DCs , however, in numerous patients, the associated immune-related adverse effects (irAE) limit their utility . ICIs diic cells . Moreovein vivo [2-induced CRT exposure, ATP secretion, and DC function. In addition, the neutral analog of 15dPMJ2, which is devoid of ER stress inducing activity [2 [2-induced DAMP exposure and death and thus, its immunogenicity. Interestingly, the requirement for ER stress in DAMP display and death also dictates whether an agent is classified as a type I or type II ICD inducer [2 caused a substantial increase in the exposure of different DAMPs including CRT, ATP, HMGB1, Hsp70 and Hsp90. In addition, notably lower concentrations of 15dPMJ2 were needed to elicit ICD than the type I inducer, oxaliplatin. This suggests that 15dPMJ2 is a tumor-selective, type II ICD inducer.According to the literature, ER stress plays a crucial role in DAMP-ICD , 9. A rein vivo . Garg anactivity , was unaivity [2 . These r inducer . Hence, 2 and neutral-15dPMJ2 were synthesized as described previously [\u00ae 3/7 assay kit, CellTiter-Glo 2.0 assay kit, and MTS reagent were purchased from Promega Life Sciences . Anti-calreticulin (Alexa Fluor 647) was from Abcam . HMGB1 ELISA kit was purchased from Novatein Biosciences . Recombinant mouse GM-CSF, 7-AAD, PE-Cy7-anti-CD80, APC-Cy7-anti-CD86, PE-Anti-Hsp90 and FITC-Anti-Hsp70 were obtained from BioLegend . APC-anti-CD11c and mouse Fc block were purchased from BD Biosciences . FITC-anti-MHC class II, anti-CRT and CellTracker Green CMFDA were purchased from Thermo Fisher Scientific . IRDye 800CW anti-rabbit and IRDye 680RD anti-mouse secondary antibodies were from LI-COR Biosciences .15dPMJeviously . GSK2606The murine melanoma cell line, B16F10, was purchased from ATCC and cultured in Dulbecco\u2019s minimal essential media containing 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 \u03bcg/ml). The murine melanocyte cell line, Melan-A, was purchased from the Bennett-Sviderskaya laboratory and cultured in RPMI1640 medium supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 \u03bcg/ml), glutamine (200 \u03bcM), and tetradecanoylphorbol acetate (200 nmol/L). The human colon cancer cell line HT29 was purchased from ATCC and cultured in McCoy's 5A medium containing 10% heat-inactivated FBS, penicillin (100 units/ml), and streptomycin (100 \u03bcg/ml). The non-tumorigenic human keratinocyte cell line, HaCaT, was purchased from Cell Line Service . The human squamous carcinoma cell line, A431, was obtained from ATCC . Both HaCaT and A431 cells were cultured in Dulbecco's minimal essential media containing 10% heat-inactivated FBS, penicillin (100 units/ml) and streptomycin (100 \u03bcg/ml).B16F10 or Melan-A cells were plated in 96-well plates and cultured for 48 hours. Serum-free media containing the listed concentration of different agents was added to the cells for the indicated period of time. MTS reagent was then added to each well and the absorbance at 495 nm was measured according to the manufacturer\u2019s instructions. Absorbance readings were acquired using the Infinite 200 Pro plate reader .Cultured cells were plated in white-walled 96-well plates and incubated for 48 hours. Serum-free media containing different concentrations of agents was added to the cells for the indicated time. Caspase-Glo 3/7 reagent was added to each well as directed by the manufacturer. The Caspase-Glo 3/7 kit measures the activity of the executioner caspases 3 and 7 using the luminogenic substrate, Z-DEVD-aminoluciferin. Luminescence was measured using the Infinite 200 Pro plate reader .2, pH 7.4) containing Annexin V and 7-AAD and then incubated at room temperature for 15 minutes. The samples were analyzed using LSRII flow cytometer and FCS Express V6 software .Apoptotic cells were detected using Annexin V Alexa Fluor 488 and the viability dye 7-AAD . Briefly, agent-treated B16F10 cells were resuspended in annexin binding buffer according to the manufacturer\u2019s instructions. Luciferin is mono-oxygenated by the luciferase enzyme in the presence of ATP producing luminescent signal. Therefore, the CellTiter-Glo 2.0 kit measures the generation of luminescent substrate. The intensity of the luminescent signal is proportional to the amount of ATP that is released into the culture medium by the cells. Luminescence was measured using the Infinite 200 Pro plate reader .Cells were cultured for 48 hours and then treated with the appropriate agents for the indicated period of time. The quantity of HMGB1 in the culture media was measured by utilizing the Mouse HMGB1 Sensitive ELISA Kit according to the manufacturer\u2019s instructions. Absorbance readings were acquired using the Infinite 200 Pro plate reader .\u00ae CLx digital fluorescence imaging system . The band intensities were quantified by using ImageJ software [Western blot analysis was conducted as described previously . Brieflysoftware .2, oxaliplatin, vehicle (0.1% DMSO) or the cells were left untreated for 2 or 4 hours. The cells were collected, washed twice with FACS Buffer and incubated for 1 hour with Alexa Fluor 647-Anti-calreticulin, PE-Anti-Hsp90, or FITC-Anti-Hsp70 antibodies diluted (1:100) in FACS buffer. The cells were washed twice with FACS buffer before conducting the analysis with the LSR II flow cytometer . The cell surface expression of CRT, Hsp70 or Hsp90 was quantified using FCS Express V6 software .B16F10 and Melan-A cells were treated with 15dPMJBMDCs were obtained from the femurs and tibias of 7\u20139 week-old C57BL/6 mice. The DCs were differentiated for 8 days using RPMI 1640 medium supplemented with 5% heat-inactivated FBS, L-glutamine (0.03%), sodium pyruvate (0.4 mM), 2-mercapthoethanol (50 \u03bcM), mGM-CSF (20 ng/ml), penicillin (100 units/ml) and streptomycin (100 \u03bcg/ml).2, oxaliplatin or vehicle (0.1% DMSO) for 24 hours. The cells were collected and then co-cultured with BMDCs (generated as described above) in a 1:1 ratio for 2 hours. The co-cultured cells were harvested, incubated with a mouse Fc block, immunostained with APC-anti-CD11c (1:100), and the data was acquired by using the LSR II flow cytometer . Data analysis was performed using FCS Express V6 software . BMDCs that phagocytosed CMFDA-labeled dead cell material were identified as CD11c+CMFDA+ double-positive cells.B16F10 and Melan-A cells were labeled with 1 \u03bcM CellTracker Green CMFDA for 30 min. The cells were then treated with 15dPMJ2, oxaliplatin or vehicle (0.1% DMSO) for 24 hours. The cells were collected and co-cultured in a 10:1 ratio with BMDCs (generated as described above) for 24 hours at 37\u00b0C. The co-cultured cells were then collected, washed once in FACS buffer and incubated with mouse Fc block for 10 minutes. The cells were then immunostained with APC-anti-CD11c (1:100), FITC-anti-MHC class II (1:500), PE-Cy7-anti-CD80 (1:100) and APC-Cy7-anti-CD86 (1:100) for 30 minutes. The expression of maturation markers; MHCII, CD86 and CD80 on the surface of CD11c+ BMDCs was analyzed using the BD LSR II flow cytometer and FCS Express 6 software.B16F10 and Melan-A cells were treated with 15dPMJAll data are representative of at least three independent experiments. The data are presented as the mean \u00b1 standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Tukey\u2019s post-hoc analysis was carried out using GraphPad Prism 5 software ."} +{"text": "Annually, millions of tons of foods are generated with the purpose to feed the growing world population. One particular eatable is orange, the production of which in 2018 was 75.54 Mt. One way to valorize the orange residue is to produce bioethanol by fermenting the reducing sugars generated from orange peel. Hence, the objective of the present work was to determine the experimental conditions to obtain the maximum yield of reducing sugars from orange peel using a diluted acid hydrolysis process. A proximate and chemical analysis of the orange peel were conducted. For the hydrolysis, two factorial designs were prepared to measure the glucose and fructose concentration with the 3,5-DNS acid method and UV-Visible spectroscopy. The factors were acid concentration, temperature and hydrolysis time. After the hydrolysis, the orange peel samples were subjected to an elemental SEM-EDS analysis. The results for the orange peel were 73.530% of moisture, 99.261% of volatiles, 0.052% of ash, 0.687% of fixed carbon, 19.801% of lignin, 69.096% of cellulose and 9.015% of hemicellulose. The highest concentration of glucose and fructose were 24.585 and 9.709 g/L, respectively. The results highlight that sugar production is increased by decreasing the acid concentration. In 2018, the United Nations Food and Agriculture Organization (F.A.O.) [The orange peel is the waste with the highest volume and ease of use in the orange industry. It is estimated that around 20% of the orange is orange peel. Therefore, there is an estimation of 15.10 Mt of orange peel generation in 2018. However, before proposing a valorization route for orange peel, the physicochemical characteristics of this waste must be known. The literature indicates that the orange peel contains 23% sugar, 22% cellulose, 25% pectins and 11% hemicellulose ,5. With In recent years, food waste valorization acquired significantly importance, whether animal or vegetable. In the case of agro-industrial waste, olive leaves have been proven to be precursors of value-added chemical compounds such as phenols . RegardiEssential oil extraction is one of the most common applications of orange peel waste. This process does not significantly transform the orange peel, it only allows the removal of the compounds that make up the essential oil such as limonene, alpha pinene, camphene, among others . NormallSome biomass residues that have been used as precursors of reducing sugars for bioethanol production are rice straw, rice husk, macaranga, bamboo, agave leaves, palm oil, wheat bran, sorghum stalk, sugarcane leaves and citronella residues, to mention a few ,18,19,20w/w) [Although it has been reported that diluted acid hydrolysis is the most appropriate for similar wastes, each author uses different temperature, time, and acid concentration values under 6% (w/w) ,26. Likew/w) ,28. TherThe chemical composition of the orange peel is mainly made up of cellulose, followed by lignin and then hemicellulose. Cellulose is the simplest natural polymer to convert into its monomers, which helps to improve the fermentation process .v/v of H2SO4, 125 \u00b0C and 120 min of operation. The previous combination forms up to 24.585 g/L of glucose and up to 9.709 g/L of fructose. It is important to note that at lower factors levels, it is possible to obtain a concentration of glucose and fructose above 10 g/L. At a pilot plant level, low levels of acid concentration are beneficial, since it prolongs the duration of the installations, and saves on reagent costs like H2SO4. Due to the above, the combination of factors at low levels is attractive [2SO4 concentration increases. Other biomass residues show increases in the concentration of reducing sugars while increasing the concentration of H2SO4 [The combination of factors that produces the greatest amount of glucose and fructose was 0.5% tractive . At the of H2SO4 , where tIn Minitab, two ANOVA tables were created, one with glucose concentration as a response variable and the other with fructose concentration as a response variable. The results of both analyses are shown in v/v. By increasing the temperature and decreasing the acid concentration, a clear increase in the glucose and fructose concentration can be observed. In the interaction between the temperature and the hydrolysis time and the temperature (B) for both designs of experiments, while for glucose the operating time (C) was also an important factor. Regarding the binary interactions, the combination of acid\u2013concentration\u2013temperature and temperature\u2013time was significant for both designs of experiments. sis time b,d a sim2SO4, 123\u2013125 \u00b0C and 1.95\u20132 h. For fructose, the ranges in which a concentration between 6\u20138 g/L can be produced are 0.5\u20130.95% H2SO4, temperature of 119\u2013125 \u00b0C and 1.7\u20132 h.The contour diagram is useful because it provides factor ranges in which a response variable is similar to the ones in the design of experiments. When combining both contour diagrams for glucose and fructose, it is observed that the region with a glucose production between 15\u201320 g/L will occur in the ranges of 0.5\u20130.75% of H2SO4. It can be seen that there is an absence of acid and NaOH. The percentage of O and C in the image was obtained without chemical modifications to the surface of the orange peel, so they are considered basal values. These values can increase or decrease depending on the type of reaction that occurs with H2SO4 and NaOH.The image in 2SO4 concentration increases in the hydrolysis, the surface contracts and becomes irregular.The images presented in 2SO4 reacts under these conditions it generated a greater quantity of reducing sugars, as a result of a greater consumption of C. In some cases, the equipment detects Al in the samples, which is attributed to parts of the orange peel that had direct contact with the container where they were placed. With respect to the surface, The operation conditions for 2SO4 contain O, there is an addition of these materials to the surface of the orange peel. In the case of C, a decrease occurs in The operating conditions of the sample in 2SO4 used was 1.5% v/v. For this EDS, it is important highlight that the average glucose and fructose productions in both hydrolysis reactions were similar at 6.560 g/L of glucose and 3.129 g/L of fructose for The operating conditions of According to proximate analysis results, thermochemical applications such as direct combustion of orange peel are not recommended due to the high moisture content. Biological applications must be carefully studied, since the amount of volatile compounds such as limonene will prevent the growth of certain microorganisms. The presence of volatile compounds is evident. Regardless of the revalorization procedure for orange peel, these compounds that comprise orange peel essential oil must be considered. As an industrial process that seeks the use of orange peel for the production of reducing sugars and bioethanol, it must consider the essential oil as a by-product to help the profitability of the process. The results of orange peel chemical composition are linked to the quantification of glucose and fructose, since cellulose degrades into these monomers when is hydrolyzed.2SO4 concentration 0.5% v/v, temperature of 125 \u00b0C and hydrolysis time 120 min. The above combination gives an average glucose and fructose concentration of 21.887 and 9.286 g/L, respectively. When increasing the H2SO4 concentration, a decrease in the sugars produced was observed. Presenting on average, at the highest levels of the factors, a production of 8.657 g/L of glucose and 3.652 g/L of fructose, up to three times lower compared to the conditions of maximum production. The glucose and fructose concentrations achieved according to the design of experiments may vary even under strict operating conditions. This is due to the fact that orange peel waste can have a different composition according to the geographical position, soil type where orange was grown and weather conditions. However, working with orange peel waste, the tendency set by the parameters of the design of experiments must be maintained, at low acid concentration, 125 \u00b0C for 2 h.Regarding the results from the design of experiments, the conditions under which the greatest amount of reducing sugars in the form of glucose and fructose are produced were\u2014HThe results of the SEM-EDS analysis yield morphological and elemental information of the surface that confirm the information obtained by the design of experiments. An important aspect to highlight is that, unlike other biomass materials such as corn, the concentration must remain at low levels, which is favorable.2SO4 concentration, due to the greater need for NaOH to maintain the pH of the hydrolysate between 4.8 and 5.2. Likewise, if the concentration of H2SO4 increases, the % of C decreases in the mixture, which reacts to form the sugars dissolved in the hydrolysate. The previous behavior is not fulfilled in the hydrolysate with the maximum factor levels, since the temperature\u2013time interaction causes a low reaction yield in the generation of reducing sugars, and that the SEM-EDS detects a % of C that was lower that its counterparts.Both the percentage of O and Na in the sample increase with increasing HFor the orange peel proximate analysis, 1 g of freshly peeled sample was weighed, which included both albedo and peel and placed inside a crucible of known mass. The crucible and the sample were placed in a muffle at 45 \u00b0C for 48 h . Once itThe chemical analysis of the orange peel began by measuring 4 g of moisture-free sample and then placing it in a thimble of known mass. Subsequently, the thimble was placed inside soxhlet extraction equipment with 200 mL of acetone as solvent and boiled for 8 h . At the For the determination of hot water extractables, 3 g of moisture-free sample of orange peel and free from acetone extractables was used. The sample was placed in a 250 mL ball flask, 200 mL of hot water was added and it was boiled in a water bath with condensation refluxed for 3 h . At the 2SO4 at 72%, stirred for 1 min at 400 rpm and for 2 h at 200 rpm. Then, the solution was placed in a 1 L Erlenmeyer flask with 560 mL of distilled water and it was boiled with condensation reflux for 4 h [The lignin content determination was carried out using 1 g of orange peel free of moisture and free of total extractables, which was mixed with 15 mL of H for 4 h . To filtFor the holocellulose content, 2 g of moisture-free orange peel that was free of total extractables was added inside an Erlenmeyer flask. Also, 150 mL of distilled water, 0.2 mL of acetic acid and 1 g of sodium chlorite were used to complete the mixture . The flaThe cellulose content determination used 2 g of sample resulting from the holocellulose determination, which was added to an Erlenmeyer flask with 10 mL of 17.5% NaOH, and allowed to rest for 5 min at 20 \u00b0C . A totalA design of experiments of the factorial design type applied to the diluted acid hydrolysis of orange peel was developed to contrast the obtained reduced sugars ,39. The 2SO4 concentration, (B) temperature, (C) hydrolysis time. A total of 3 levels were specified for factor (A) and 2 levels for factors (B) and (C), with 2 replicates per combination of variables. The amount of hydrolysis performed for this design of experiment was 24 experiments. The levels for the factors were concentration of H2SO4 0.5%, 1.0% and 1.5% all in volume percent, for the temperature 100 \u00b0C and 125 \u00b0C and for the hydrolysis time 1 and 2 h. The purpose of the factorial design is to identify which individual factors and combinations thereof are capable of significantly affecting the production of sugars. The results of the sugar production were analyzed in Minitab\u00ae where the important process variables were identified.The selected factors were\u2014(A) HOrange peel needs a pretreatment before the hydrolysis, a series of orange peel hydrodistillations were carried out to remove the essential oil. The essential oil was stored in amber vials and refrigerated at 4 \u00b0C . The ora2SO4 solution (1:30 ratio). The flask was placed in the furnace by the time of hydrolysis, and once finished, it was removed and cooled to a temperature of 30 \u00b0C. The acid pH of the hydrolysate was brought to 4.8\u20135.2 with NaOH 0.5 N, then it was filtered and stored at 4 \u00b0C [A furnace was preheated for 30 min to a temperature set in the design of experiments. At the same time, 2 g of dried orange peel was placed in a 100 mL flask, adding 60 mL of the H at 4 \u00b0C . The filFor the reducing sugars identification, the 3,5-DNS method was chosen ,44. ThisThe samples for SEM were placed on a conductive plate with carbon tape without metallic coating. A total of 6 samples were taken from 24 of the design of experiments, which had the highest amount of sugar produced. The equipment used was a JEOL JSM-6010LA analytical scanning electron microscope , with a working distance of 11 mm, acceleration voltage of 10 kV and under vacuum of 50 Pa .The general procedure of this work can be seen in 2SO4 to find the turning point, where the reducing sugars concentration starts to decrease. In addition, the effect of the volume ratio of H2SO4 and orange peel waste can be addressed, since in all the experiments it remained constant. While this methodology allows essential oil extraction from orange peel, other value-added products such as pectins can be explored before this waste is subjected to fermentation processes to obtain alcohols.Under the selected parameters of the design of experiments, the maximum concentration of glucose and fructose was achieved with an acid concentration of 0.5%, at 125 \u00b0C for 2 h. However, this does not mean that they are the best conditions to operate a hydrolysis process applied to orange peel waste. Future research could study the effect of reducing the concentration of HAlthough it is possible to modify the pretreatment of the orange peel to improve the production of sugars, the revaluation of a waste must be an integral process, which follows the principles of the biorefinery concept. That is, not just looking for punctual application for the given waste, but obtaining multiple products from the same source in such a way that can be scalable to a commercial level. Depending on the desired production scale, either at the laboratory or pilot plant level, this work provides the concentration of reducing sugars in the form of glucose and fructose, according to the 12 combinations proposed in the design of experiments."} +{"text": "After endoscopic resection (ER) of neoplasia in Barrett\u2019s esophagus (BE), it is recommended to ablate the remaining BE to minimize the risk for metachronous disease. However, we report long-term outcomes for a nationwide cohort of all patients who did not undergo ablation of the remaining BE after ER for early BE neoplasia, due to clinical reasons or performance status.Endoscopic therapy for BE neoplasia in the Netherlands is centralized in 8 expert centers with specifically trained endoscopists and pathologists. Uniformity is ensured by a joint protocol and regular group meetings. We report all patients who underwent ER for a neoplastic lesion between 2008 and 2018, without further ablation therapy. Outcomes include progression during endoscopic FU and all-cause mortality.Ninety-four patients were included with mean age 74 (\u00b1 10) years. ER was performed for low-grade dysplasia\u00a0(LGD) (10%), high-grade dysplasia (HGD) (25%), or low-risk esophageal adenocarcinoma (EAC) (65%). No additional ablation was performed for several reasons; in 73 patients (78%), the main argument was expected limited life expectancy. Median C2M5 BE persisted after ER, and during median 21 months (IQR 11\u201351) with 4 endoscopies per patient, no patient progressed to advanced cancer. Seventeen patients (18%) developed HGD/EAC: all were curatively treated endoscopically. In total, 29/73 patients (40%) with expected limited life expectancy died due to unrelated causes during FU, none of EAC.In selected patients, ER monotherapy with endoscopic surveillance of the residual BE is a valid alternative to eradication therapy with ablation. Barrett\u2019s esophagus (BE) is the most important risk factor for esophageal adenocarcinoma (EAC), which has a poor prognosis. Identifying EAC at an early stage allows for endoscopic treatment with an excellent prognosis. The first step in endoscopic treatment for BE-related neoplasia is removal of all visible lesions with endoscopic resection (ER) techniques, which serves both diagnostic and therapeutic purposes. It has been reported that the remaining flat BE that persists after ER of a neoplastic lesion has a risk of developing metachronous HGD/EAC between 15 and 30% in 3\u20135 years Although RFA therapy is highly effective for eradication of flat BE, the choice to continue with ablation requires balanced decision-making, taking into account patient\u2019s age, comorbidity, and life expectancy Although severe complications due to RFA treatment are very rare, complications do occur, most commonly esophageal strictures in up to 10\u201314% In the Netherlands, endoscopic treatment for BE is centralized in 8 Barrett Expert Centers (BECs), with a uniform treatment and follow-up protocol. Since the introduction of RFA in 2008, these centers adhered to the ER monotherapy strategy in selected patients. In the current study, we report the long-term outcomes of \u201cER monotherapy\u201d as an alternative to additional ablation therapy in patients with limited life expectancy.This study was based on the Barrett Expert Center registry (BEC registry) , which has been described in detail earlier The centers have a minimum annual case load of 10 new patients with neoplasia per year, and all new cases are registered in a database.Patients were referred to a BEC for careful work-up and staging after being diagnosed with low-grade dysplasia (LGD), HGD, or EAC. During an upper gastrointestinal endoscopy (UGE), the esophagus was carefully inspected with documentation of the Prague C&M criteria and presence of visible lesions or other abnormalities such as esophagitis or esophageal stenosis.If a visible abnormality was detected, endoscopic resection (ER) was performed for histologic staging using the ER-cap technique, multiband mucosectomy (MBM), or endoscopic submucosal dissection (ESD) per physician\u2019s discretion. Four-quadrant random biopsies were obtained from the flat BE segment according to the Seattle protocol If the ER specimen showed LGD, HGD, or low-risk (LR) EAC , a balanced decision was made between further endoscopic treatment or surveillance. In the vast majority of patients, additional ablation therapy was offered to achieve a complete eradication of the entire Barrett\u2019s segment. However, in patients with limited life expectancy, for example, due to older age and/or severe comorbidity, surveillance of the remaining BE was preferred with endoscopic intervention in case of recurrent neoplasia and/or visible lesions.All patients were prescribed bi-daily high-dose proton-pump inhibitors.FU for persisting non-dysplastic BE (NDBE)/LGD after ER consisted of yearly surveillance endoscopies in year 1 to 5, and then once per 2\u20133 years. FU was performed every 3\u20136 months for persisting HGD. The decision to stop further surveillance was made per physician\u2019s discretion in agreement with the patient.For the current study, we included all patients from the BEC registry who underwent ER monotherapy for LGD, HGD, or LR-EAC with residual flat BE before January 1, 2018.The first primary endpoint was progression to HGD/EAC in the remaining BE. For patients with remaining NDBE or LGD, detection of HGD/EAC was considered to be progression. For patients with persisting flat HGD, new EAC was progression as was a new visible lesion containing HGD. All patients were included for this analysis. This endpoint was stratified for residual grade of dysplasia.The second primary endpoint was all-cause mortality. This endpoint reflects whether the decision to prefer surveillance over ablation was justified for patients with expected limited life expectancy. Therefore, only the patients in whom the decision for ER monotherapy was based on age and/or comorbidity were included for this analysis.Secondary endpoints included symptomatic EAC and/or EAC-related death and predictors for progression. All patients were included for these analyses. We also assessed progression risk to HGD/EAC in the remaining BE among only patients who had at least 18 months of endoscopic FU.Endoscopy and pathology data were collected in standardized form in all BECs, by medical students in the final year of their degree. Additionally, all patients with endpoints and an additional 50% of the remaining patients were double-checked by dedicated research fellows . All fields were examined for missing data, unlogical values, or outliers, with data being completed or corrected where possible.The BEC registry was merged with the non-public microdata from Statistics Netherlands for date and cause of death.Continuous variables were presented as mean with standard deviation (SD) or median with interquartile range (IQR) for normally distributed or skewed data, respectively. Categorical variables were presented as numbers with percentages, and 95% confidence intervals (CI) were obtained using internal bootstrapping.Progression risks were plotted using the cumulative incidence curve (CII), taking competing risks of unrelated death into account. Annual progression rates were calculated as the number of progressors divided by the total follow-up duration in years. Predictors for progression were assessed using Cox regression and Fine and Gray competing risk analysis, the latter considered unrelated death as competing risk.Statistical analysis was performed using Rstudio for Windows (version 3.6.1) and packages: survival, survminer, cmprsk, ggplot2, and Hmisc.The Institutional Review Board of the Amsterdam University Medical Centers declared that the registry was not subject to the Medical Research Involving Human Subjects Act (\u201cwet op medisch-wetenschappelijk onderzoek met mensen\u201d in Dutch) and waived the need for formal ethical review and patient-informed consent. Patients were approached through an opt-out card with the possibility to object against participation in the registry.n = 1140), a flat BE segment remained in 1034 patients. The vast majority of these patients (91%) underwent additional ablation aimed at eradication of the entire BE segment. Ninety-four patients (9%) had ER monotherapy for LGD (n = 9), HGD (n = 23), T1a EAC (n = 47), or T1bsm1 EAC (n = 15), with remaining BE, and were included for this study.Between 2008 and 2018, a total of 1962 patients with early BE neoplasia were referred to a BEC. A visible abnormality was detected in 1395 patients (71%) and removed with ER years and ASA classification II (67%) or III/IV (23/2%), with ER performed for LGD (10%), HGD (25%), or LR-EAC (66%) , LGD , or HGD . In 11 patients (12%), no biopsies were obtained since this was considered not to change clinical decision-making.After ER for all visible abnormalities, a flat BE segment of median C2M5 remained with NDBE ; patient preference ; persistence of a small BE tongue only ; and/or complications after ER , additional ablation was not started due to age and/or comorbidity. Concomitant reasons in this group were as follows: expected poor regression after RFA due to regeneration with BE after ER (4%) Fig. .Fig. 2Ren = 11, 52%); persistence of a small BE tongue only ; expected poor regression after RFA due to BE regeneration after ER ; complications after ER ; and/or patient preference .In the remaining 21 patients (22%) in whom age and comorbidity played no role, reasons not to continue with ablation therapy were as follows: other treatment protocols (n = 10) or LR-EAC (n = 7) (Table During a median endoscopic FU of 21 months 11\u201351) with a median of 4 endoscopies 1 with a n = 7) or HGD (n = 6) or with ablation therapy for flat HGD (n = 3). A single patient who progressed from LGD to HGD had no further treatment, and the patient died shortly after due to an unrelated cause. Six progressors had developed a worse histological grade during FU, than the initial histology after baseline ER. This included baseline LGD to m-EAC in FU (n = 1), baseline HGD with m-EAC in FU (n = 4), and baseline m-EAC with sm-EAC during FU (n = 1).Sixteen out of seventeen progressors were successfully treated endoscopically, either with ER for a visible lesion containing LR-EAC .In the majority of the patients with FU < 18 months, endoscopic FU was discontinued at median 3 months (IQR 0\u20139) after ER, due to limited life expectancy. Of these 27 patients, 15 had unrelated death median 18 months after ER, whereas the remaining 12 were alive and asymptomatic at median 55 months after ER. The remaining 12/39 patients with short FU were recently treated with ER and were still under endoscopic surveillance (median 12 months).Our second aim was to asses all-cause mortality during long-term follow-up in the subgroup of patients with older age and/or comorbidity, to verify whether ER monotherapy was justified in this group of patients.As reported, in 73 patients, age and/or comorbidity played an important role in the decision not to continue with ablation therapy after ER. In 37 patients, endoscopic surveillance was stopped early at median 20 months (5\u201359) after ER and cardiovascular disease contributed the most common causes of death.Overall, 29 of 73 patients 40%) died due to unrelated causes median 28 months after ER at a median age of 80 (72\u201385) years. The remaining 44 of 73 patients were still alive at the moment of data collection median 42 months after ER. Figure 0% died dNone of the 94 patients progressed to disease stages that exceeded boundaries for curative endoscopic treatment, developed symptomatic EAC, or died from EAC.In univariable analysis, length of the residual BE was significantly associated with risk for progression during FU Table . For patWe report endoscopic and long-term all-cause mortality outcomes for all patients with ER monotherapy in the Netherlands between 2008 and 2018, to assess whether this is a justified treatment strategy in selected patients with early BE neoplasia, for example, in case of older age and/or significant comorbidity. The risk for progression to HGD or EAC was 8% per year. In all cases, progression was detected at early stages and curatively treated endoscopically. No patient developed advanced EAC, and no patient died due to EAC, even though endoscopic surveillance was stopped early in half of the patients. Overall, 40% of patients died due to EAC unrelated causes at median 28 months after ER. These data suggest that ER monotherapy with endoscopic surveillance of the residual BE is a valid alternative to prophylactic ablation therapy in selected patients.Data from the current study comport well with older studies from the pre-ablation era, reporting progression rates in remaining flat BE after ER varying from 15 during 5 years to 30% in 3 years RFA is effective and can achieve complete eradication of all BE (CE-BE) in 90\u201395% of patients. However, RFA is associated with multiple hospital visits and a risk of complications. Patients with baseline ER have the highest risk for post-RFA stenosis What is the risk for this patient to develop recurrent neoplasia, with or without ablation therapy?The decision to initiate prophylactic ablation therapy of residual BE after ER of neoplasia should be based on the answers to the following three questions:2.If this patient develops progression, what is the risk of dying from EAC?A substantial proportion of patients will never develop neoplasia in the remaining BE after ER. If the remaining BE after ER contains NDBE or LGD, the annual progression risk was only 6.4\u20136.7%. The median BE length in the current study was C2M5, and for shorter BE lengths, this annual risk will be even lower Second, the clinically relevant endpoint that should be prevented is progression to advanced, symptomatic EAC and/or EAC-related death. Most endoscopic studies define recurrent HGD or worse as an endpoint, or even recurrent LGD. Although this might be a logic endpoint for some studies, this is not the relevant endpoint that matters to a patient.3.How does the risk for EAC-related death relate to the risk of death due to other causes?Proper data describing the natural history of HGD or early EAC is lacking, but older studies report incidence rates of HGD to advanced EAC of 6.6% per patient year with 2- to 5-year duration between detection of HGD and development of advanced EAC Finally, the benefits of eradication of all BE over removal of neoplasia should be balanced against the assumed life expectancy of a patient. Differences in life expectancy would not only change the cumulative risk for recurrence as described above but would also change the curve for unrelated death in Fig What is an acceptable surveillance interval after ER monotherapy? We detected all progressors at early stages, and we found no progression within the first 18 months post-ER. This suggests that annual surveillance is an accepted strategy and that we can safely perform the first FU endoscopy 1 year after confirmation of a completely flat BE post-ER. On the other hand, in older patients with a short remaining BE segment, we may stop endoscopic FU directly after ER, based on the aforementioned considerations.Finally, if a patient has predictors for a low success chance after RFA, such as BE regeneration of the ER scar or a long BE without any squamous islands This is the first study that provides long-term FU data for an alternative treatment strategy in older patients with BE-related neoplasia. In our cohort of patients treated in a centralized setting by experienced endoscopists, this constituted 10% of the population that qualified for RFA after ER according to current guidelines. The suggested ER monotherapy strategy is advised in patients with a life expectancy of < 5\u201310 years and should be considered for a life expectancy of < 15\u201320 years. We suggest to consider and discuss this strategy in patients aged > 70 years and those with severe comorbidity.limited life expectancy, and ultra-long-term FU data therefore have no clinical consequences. This is reflected by the fact that only one-third of patients was still under endoscopic surveillance at the moment of data collection. The others had already died of other causes (one-third) or were alive after endoscopic FU was already stopped (one-third). Therefore, extended FU with a potentially higher progression rate would not have changed the long-term outcomes, mortality rates, or our conclusions and recommendations.Some limitations need to be addressed. The median duration of endoscopic FU was 21 months, while the median time to progression was 26 months, and all progressors occurred at minimal 18 months after ER. In light of this, we performed analysis that only included patients with FU over 18 months, which showed a minimally increased annual progression risk . Still, if we would have had longer endoscopic FU, the annual progression risk might potentially have increased with a peak after longer FU, suggesting that the progression risk is not constant over time but increased over the years. Unfortunately, our data are too limited for solid analysis of this aspect. On the other hand, we report results for patients with Other limitations include the low number of events to assess predictive factors, which limited us to perform univariable analysis only. The FU duration for vital status may have been too short to detect recurrent, symptomatic disease among those patients whose FU was stopped early. A total of 7 patients had no endoscopic FU and were only assessed for vital status.We are currently working on clinical prediction tools to provide individualized, evidence-based advices on optimal FU strategy after ER and/or RFA, taking account of the risk for progression and EAC-related death on the one hand, and patient age, comorbidity, and risk for unrelated death on the other. These data might help in defining the optimal strategy after ER monotherapy in the future.In conclusion, ER monotherapy with endoscopic surveillance of the residual flat BE is a valid alternative to prophylactic ablation therapy of residual BE, in selected patients."} +{"text": "IDH)1/2 genes and chromosome 1p/19q codeletion. World Health Organisation diagnosis endorses testing for 1p/19q codeletion to distinguish IDH mutant (Mut) oligodendrogliomas from astrocytomas because these gliomas require different treatments and they have different outcomes. Several methods have been used to identify 1p/19q status; however, these techniques are not routinely available and require substantial infrastructure investment. Two recent studies reported reduced immunostaining for trimethylation at lysine 27 on histone H3 (H3K27me3) in IDH Mut 1p/19q codeleted oligodendroglioma. However, the specificity of H3K27me3 immunostaining in this setting is controversial. Therefore, we developed an easy-to-implement immunohistochemical surrogate for IDH Mut glioma subclassification and evaluated a validated adult glioma cohort. We screened 145 adult glioma cases, consisting of 45 IDH Mut and 1p/19q codeleted oligodendrogliomas, 30 IDH Mut astrocytomas, 16 IDH wild-type (Wt) astrocytomas, and 54 IDH Wt glioblastomas (GBMs). We compared immunostaining with DNA sequencing and fluorescent in situ hybridization analysis and assessed differences in H3K27me3 staining between oligodendroglial and astrocytic lineages and between IDH1-R132H and non-canonical (non-R132H) IDH1/2 Mut oligodendroglioma. A loss of H3K27me3 was observed in 36/40 (90%) of IDH1-R132H Mut oligodendroglioma. In contrast, loss of H3K27me3 was never seen in IDH1-R132L or IDH2-mutated 1p/19q codeleted oligodendrogliomas. IDH Mut astrocytoma, IDH Wt astrocytoma and GBM showed preserved nuclear staining in 87%, 94%, and 91% of cases, respectively. A high recursive partitioning model predicted probability score (0.9835) indicated that the loss of H3K27me3 is frequent to IDH1-R132H Mut oligodendroglioma. Our results demonstrate H3K27me3 immunohistochemical evaluation to be a cost-effective and reliable method for defining 1p/19q codeletion along with IDH1-R132H and ATRX immunostaining, even in the absence of 1p/19q testing.Oligodendrogliomas are defined by mutation in isocitrate dehydrogenase (NADP(+)) (The online version contains supplementary material available at 10.1186/s40478-021-01194-7. TERT) promoter mutations. In contrast, astrocytoma typically exhibits Tumor Protein P53 (TP53) and ATRX Chromatin Remodeler (ATRX) mutations [The current World Health Organisation classification for CNS tumors recommends integrated diagnosis based on combined phenotypic and genotypic findings Althoughutations \u20137. From utations . Fluoresutations \u201312. HoweH3-3A) K27M-mutant GBM [Trimethylation at lysine 27 on histone H3 (H3K27me3) is an epigenetic modification that mediates gene silencing by Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2), a component of the Polycomb complex (PcG) \u201315. Losstant GBM .Although H3K27me3 has been reported to be involved in several brain tumor entities, comprehensive data about H3K27me3 in IDH Mut gliomas are controversial. Recently, Filipski et al. reportedFormalin-fixed paraffin-embedded (FFPE) glioma tissues from 145 adult patients were used, including 45 IDH Mut and 1p/19q codeleted oligodendrogliomas, 30 IDH Mut, and 16 IDH Wt astrocytoma, and 54 IDH Wt GBM. The Department of Cancer Pathology, Hokkaido University, diagnosed all cases between January 2008 and November 2020. Tissue samples were obtained from the Nakamura Memorial Hospital, Kashiwaba Neurosurgical Hospital, Sapporo Asabu Neurosurgical Hospital, Keiwakai Ebetsu Hospital, Hokkaido Neurosurgical Memorial Hospital, Sapporo Shuyukai Hospital, Shinsapporo Neurosurgical Hospital, Iwamizawa General Hospital, and Tomakomai Neurosurgical Hospital. Diagnosis was performed according to the 2016 World Health Organisation classification of Tumours of the Central Nervous System (revised 4th edition). The cases prior to 2016 that were diagnosed based on previous versions of classification were reviewed according to the new integrated diagnostic approach by three certified pathologists. Tissue and data collection was approved by and performed according to the regulations of the ethics committee of Hokkaido University Faculty of Medicine .Immunohistochemistry (IHC) was performed on 4-\u00b5m FFPE tissue sections. Heat-mediated antigen retrieval was performed in Tris/EDTA buffer (pH 9.0) at 97\u00a0\u00b0C for 20\u00a0min. The antibodies used in this study were a mouse monoclonal to anti-human IDH1-R132H , a rabbit polyclonal to anti-ATRX , a mouse monoclonal to p53 , Santa Clara, CA, USA), and a rabbit monoclonal to H3K27me3 . IHC of IDH1-R132H, ATRX, and p53 were conducted using an Autostainer Link 48, Agilent (Dako), and IHC of H3K27me3 was conducted manually according to the manufacturer's instructions. Light microscopy observation was performed for histological and immunohistochemical evaluation.All immuno-positive cases for IDH1-R132H were classified as IDH1 Mut. Negative immunostaining of ATRX in neoplastic cells in the presence of an internal positive control was considered to indicate a loss of ATRX expression. Immunohistochemistry for p53 was positive when more than 50% of tumor nuclei showed intense staining.Scoring of H3K27me3. Human colonic mucosa was used as a positive control according to the antibody datasheet. Preserved H3K27me3 in endothelial cells and immune cells served as an internal positive control. H3K27me3 immunostaining was assessed as H3K27me3-positive (nuclear retention) or -negative (nuclear loss) in a blinded manner. Complete nuclear loss or dot-like H3K27me3 staining in neoplastic cells was regarded as nuclear loss, as described previously . Each slIDH1 codon 132 and IDH2 codon 172 was performed in IDH1-R132H immuno-negative cases using an Applied Biosystems 3130 Genetic Analyzer and Sequencing Analysis Finch TV 1.4.0 software. DNA was extracted from FFPE tumor tissue using a DNA tissue extraction kit . The extracted DNA was quantified using a NanoDrop 1000 (Thermo Scientific). A fragment of 129\u00a0bp spanning the R132 codon of IDH1 was amplified using forward primer 5\u2032-CGGTCTTCAGAGAAGCCATT-3\u2032 and reverse primer 5\u2032-GCAAAATCACATTATTGCCAAC-3\u2032. Likewise, a fragment of 293\u00a0bp spanning the R172 codon of IDH2 was amplified using forward primer 5\u2032-GCTGCAGTGGGACCACTATT-3\u2032 and reverse primer 5\u2032-TGTGGCCTTGTACTGCAGAG-3\u2032.DNA sequencing of Fluorescence in situ hybridization (FISH) was performed on 3-\u00b5m thick FFPE tissue sections to assess the chromosome 1p/19q status using the Vysis 1p36/19q13 Dual Color Probe Kit as described previously . Briefly\u00aePro 15.2.0 (SAS) software . The associations among 1p/19q deletion with H3K27me3 and ATRX staining, IDH1/2 mutation, and histopathological parameters were determined using the chi-squared test/Fisher's exact test. Association with age and gender for IDH Mut gliomas and IDH Wt gliomas was determined using the chi-squared test. A partitioning model was deployed to predict H3K27me3 expression in IDH Mut 1p/19q codeleted gliomas. Hierarchical clustering based on the average intensity score was performed in R 3.6.3 (https://cran.r-project.org/) to visualize the relationship between IDH Mut 1p/19q codeleted gliomas and non-oligo gliomas (IDH Mut and Wt) with H3K27me3 staining.Statistical analysis was performed using JMPP\u2009\u2264\u20090.05). No specific differences in sex were observed among the groups. The demographics of cases are summarized in Table P\u2009\u2264\u20090.05). Retained nuclear H3K27me3 staining was observed in 94% (15/16) and 91% (49/54) of IDH Wt astrocytoma and GBM cases, respectively than patients with IDH Wt astrocytoma or GBM . The most common mutation identified in astrocytomas was IDH1-R132H . H3K27me3 has reduced in 90% (36/40) of IDH1-R132H Mut oligodendrogliomas and nuclear retention (NR). Although H3K27me3 expression was significantly different between IDH1 and IDH2 Mut 1p/19q codeleted oligodendroglioma . Therefore, H3K27me3-positive staining as a single affirmation against 1p/19q codeletion would cause misclassification of five oligodendrogliomas as astrocytomas occur predominantly in subsets of cancers and regulate central circuitry metabolism by producing the oncometabolite, 2-hydroxyglutarate (2-HG) . Lu et aImmunohistochemistry is a cost-effective and accessible technique that can be readily adapted for detecting molecular surrogates . ImmunohThe number of non-canonical IDH1/2 mutated 1p/19q codeleted oligodendrogliomas is small (n\u2009=\u20095). Thus, further investigations of the differential expression of H3K27me3 between IDH1-R132H and non-canonical IDH1/2 mutant oligodendrogliomas are required for prognostic and therapeutic application.Our study revealed that loss of H3K27me3 nuclear staining among 1p/19q codeleted oligodendrogliomas is frequent in cases harboring IDH1-R132H mutation.\u00a0We consider that H3K27me3 immunoreactivity could predict the 1p/19q codeletion status along with IDH1-R132H and ATRX immunostaining.Additional file 1: Table S1. Correlation between H3K27me3 and ATRX immunoreactivity among gliomas; Figure S1. Decision tree of recursive partitioning model starting with IDH1-R132H staining followed by ATRX and H3K27me3 staining. Blue bars correspond to IDH Mut 1p/19q codeleted oligodendrogliomas, and orange bars correspond to not IDH Mut 1p/19q codeleted gliomas. We considered IDH Mut 1p/19q codeleted oligodendroglioma as a dependent variable and immunostaining as predictors. NR nuclear retention, NL nuclear loss, Mut mutated."} +{"text": "Dietary cholesterol and palmitic acid are risk factors for cardiovascular diseases (CVDs) affecting the arteries and the heart valves. The ionizing radiation that is frequently used as an anticancer treatment promotes CVD. The specific pathophysiology of these distinct disease manifestations is poorly understood. We, therefore, studied the biological effects of these dietary lipids and their cardiac irradiation on the arteries and the heart valves in the rabbit models of CVD.Cholesterol-enriched diet led to the thickening of the aortic wall and the aortic valve leaflets, immune cell infiltration in the aorta, mitral and aortic valves, as well as aortic valve calcification. Numerous cells expressing \u03b1-smooth muscle actin were detected in both the mitral and aortic valves. Lard-enriched diet induced massive aorta and aortic valve calcification, with no detectable immune cell infiltration. The addition of cardiac irradiation to the cholesterol diet yielded more calcification and more immune cell infiltrates in the atheroma and the aortic valve than cholesterol alone. RNA sequencing (RNAseq) analyses of aorta and heart valves revealed that a cholesterol-enriched diet mainly triggered inflammation-related biological processes in the aorta, aortic and mitral valves, which was further enhanced by cardiac irradiation. Lard-enriched diet rather affected calcification- and muscle-related processes in the aorta and aortic valve, respectively. Neutrophil count and systemic levels of platelet factor 4 and ent-8-iso-15(S)-PGF2\u03b1 were identified as early biomarkers of cholesterol-induced tissue alterations, while cardiac irradiation resulted in elevated levels of circulating nucleosomes.Dietary cholesterol, palmitic acid, and cardiac irradiation combined with a cholesterol-rich diet led to the development of distinct vascular and valvular lesions and changes in the circulating biomarkers. Hence, our study highlights unprecedented specificities related to common risk factors that underlie CVD. Heart diseases are a major health burden worldwide. Coronary artery disease (CAD) is the most prevalent heart disease characterized by the build-up of atherosclerotic plaques in the artery walls, which are responsible for myocardial infarction and stroke, the two leading causes of death . AtherosValvular heart diseases (VHDs), including calcific aortic valve disease (CAVD) and degenerative mitral valve disease, are other prevalent heart diseases of the aging population . These cIn vitro data showed promising results with these inhibitors on CAVD (While dyslipidemia (lifelong exposure to high cholesterol) is a well-established risk factor for atherosclerosis and CAVD \u20136, stati on CAVD . With reDietary habits and fatty acid intake are other, less-studied risk factors for heart diseases . Fatty aBeyond dietary fats, radiation therapy to the chest, as a critical component of the treatment regimen of Hodgkin lymphoma, the lung and breast cancer, can also increase the risk of death from cardiac-related causes \u201320. RadiThis study aimed at evaluating and comparing the impact of the following: (1) two dietary fats, enriched with either cholesterol or lard, as a source of palmitic acid; (2) irradiation in combination with a cholesterol-rich diet, on the aorta and four heart valves. We also examined the interconnexion between circulating biomarkers related to inflammation, oxidative stress, platelet activation, and calcification.ad libitum. After 2 weeks of acclimation, the rabbits were randomly assigned to seven different groups and diet habituation was conducted for 2 weeks. Protocols started when the rabbits were 12 weeks old and it lasted 16 weeks. Control group received a standard chow diet. Vitamin D2 group received a chow diet with 15 days of Vitamin D2 supplementation in drinking water at the beginning of the protocol . The cholesterol-enriched diet group was treated with 0.3% of the cholesterol-enriched diet and received vitamin D2 (Vit.D2) supplementation as the Vit.D2 group. The lard-enriched diet group received 5% of the lard-enriched diet (n = 7) received a chow diet and underwent X-ray cardiac irradiation focused on the aortic valve and the thoracic aorta during the first week of the protocol using image-guided radiation on an irradiator specifically made for small animals . Irradiation combined with cholesterol-enriched diet group received 0.3% of cholesterol-enriched diet plus Vit.D2 supplementation and underwent cardiac irradiation at mid protocol . Cholesterol- and lard-enriched diets were produced by Safe (France). Bodyweight was measured on a weekly basis. Blood draws and cardiac CT were performed at baseline and at the end of every four weeks , and aortic wall calcification was visually assessed as absent or present in each of the following segments: ascending aorta, aortic cross, and the descending aorta. An extra blood draw was performed at W2. Anesthesia was induced prior to any procedure (except for body weight measurements) by intramuscular injections of droperidol (0.625 mg/kg), xylazine (5 mg/kg), and ketamine (35 mg/kg) in the hind legs. Euthanasia was conducted in the anesthetized animals by intracardiac injection of pentobarbital (200 mg/kg).All rabbit experiments conform to the European Union guidelines for the care and use of laboratory animals and were approved by the Animal Ethical Committee of the University of Li\u00e8ge (file number 1951). Seventy-three eight-week-old male New Zealand White rabbits (1.83 \u00b1 0.14 Kg) were purchased from the Charles River, France. The animals were kept in a temperature and humidity-controlled environment in an air-conditioned room with a standard rabbit chow and tap water CHT, n = was trean = 6, Vit.D2: n = 7, CHT: n = 11, Lard: n = 6, IR: n = 7, IR/CHT: n = 7). They were then embedded in paraffin wax and sectioned at 7 \u03bcm. Before histological and immunofluorescence analyses, the sections were incubated overnight at 58\u00b0C and rehydrated through xylene baths followed by a series of graded ethanol baths. Serial sections were used for the histological assessment of tissue structure on hematoxylin-eosin-stained sections, tissue calcification on alizarin red stained section, and extracellular matrix (ECM) composition on sirius red and Movat's pentachrome-strained sections. The aortic valve area was evaluated by isolating the aortic valve leaflets and measuring their area on multiple serial hematoxylin-eosin-stained sections (4 to 9 per rabbit) by using Adobe Photoshop software. For the immunofluorescence study, the sections were incubated overnight at 58\u00b0C prior to chemical rehydration. All sections were treated for 30 min at 96\u00b0C with Dako Target Retrieval solution, pH9 for antigen retrieval, then incubated for 30 min at room temperature with normal goat serum . Co-staining was conducted by sequential use of monoclonal mouse anti-rabbit RAM-11 primary antibody , TRITC-conjugated goat anti-mouse secondary antibody , and AlexaFluor 488-conjugated monoclonal mouse anti-alpha smooth muscle actin (\u03b1SMA) antibody . Nuclei were stained with 4\u2032, 6-diamidino-2-phenylindole (DAPI) . Negative controls corresponding to TRITC-conjugated goat anti-mouse secondary antibody without anti-RAM-11 primary antibody and AF488-conjugated mouse IgG2a kappa isotype control isotype were performed solution for 24 h before dehydration into 70% of ethanol solution overnight at room temperature at the end of the 16-week protocols, snap frozen in liquid nitrogen, and stored at \u221280\u00b0C until RNA extraction. Total RNA was extracted by the use of miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The integrity of the RNA sample was assessed on a Bioanalyser 2,100 with RNA 6,000 Nano and RNA 6,000 Pico chips (Agilent Technologies). The RNA libraries were prepared with 10 ng of RNA using the SMARTer\u00ae Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian according to the manufacturer's instructions. The RNA quantification and normalization were performed with the KAPA Library Quantification kit (comprising P5-P7 primers) on the ABI 7900HT system (ThermoFisher Scientific). Sequencing of the libraries was performed on a NovaSeq6000 System (Illumina) with an S4 reagent kit (300 cycles) and 1.2 nM library loading.Aorta ring and heart valves were dissected (Ctrl: https://nf-co.re/rnaseq/1.4.2), with default parameters except for -clip_R2 = 3 and -nextseq-trim = 10, in order to perform mapping, quantification, and QC, and to produce the gene count matrix. The reference genome used was Oryctolagus cuniculus OryCun2.0 with annotation from Ensembl release 99 (http://jan2020.archive.ensembl.org/index.html). Gene counts were normalized by the default method (median of ratios) in the DESeq2 package less than 0.05 and a fold-change greater than 1.5 were used to highlight a significant difference in gene expression between the two groups. Human orthologs of differentially expressed genes (DEGs) were identified by the online tool, Better Bunny were measured in 3.8% of citrated whole blood on a Cell-Dyn 3700 hematocytometer equipped with veterinary software (Abbott Laboratories). Serum aliquots were prepared after 30-min resting to allow for blood clotting followed by centrifugation at 1,700 n = 7\u201313, Vit.D2: n = 7, CHT: n = 10\u201315, Lard: n = 7\u201312, IR: n = 7, IR/CHT: n = 15).Measurements of the levels of total cholesterol, triglycerides, LDL-c, HDL-c, calcium, phosphorous, iron, albumin, AST, ALT, creatinine, and urea were performed in serum samples on an AU480 Chemistry Analyzer (Beckman Coulter). Serum levels of IL-6 and plasma [ethylenediamine tetraacetic acid (EDTA)-anticoagulated plasma] levels of DKK1 and PF4 were measured by enzyme-linked immunosorbent assays according to the manufacturer's recommendations (Cusabio). The ent-8-iso-15(S)-PGF2\u03b1 levels were measured in EDTA-plasma by the ultra-high performance liquid chromatography coupled to tandem mass spectrometry detection. Levels of circulating H3.1-nucleosomes and citrullinated histone H3R8 nucleosomes (H3R8cit-nucleosome), an epigenetic modification, were assessed by blinded Nu.Q\u00ae chemiluminescence immunoassays in EDTA-plasma ]. All tests were performed 2-sided and P-value \u2264 0.05 was considered significant. All statistical analyses were performed using SAS 9.4 .Between-treatment comparisons of all variables were performed by ANOVA and Rabbits fed for 16 weeks with lard- or cholesterol-enriched diets supplemented with Vit.D2 displayed aortic wall structural alterations that were not observed in rabbits fed with chow diet, or in those receiving the chow diet supplemented with Vit.D2 . AlizariFigure 4C).In agreement with previous studies , rabbitsAlterations of the aortic valve structure were observed in the two diet groups . In the The mitral valve was also affected in the cholesterol-enriched diet group , with ceNone of the diets caused histological modifications of the tricuspid and pulmonary valves (data not shown).Our histological analyses did not reveal any aortic histological alterations when cardiac irradiation was applied in rabbits fed with a chow diet kept for 8 weeks post-irradiation . HoweverThe addition of irradiation to the cholesterol-enriched diet induced the aortic valve structure alterations that were not observed in the irradiation-only group. Thickened aortic valve leaflets displayed more cellular infiltrates and RAM-11 positive cells than the control groups . Aortic As for mitral valves, the combination of irradiation with the cholesterol diet led to increased infiltration of RAM-11 positive cells as compared to the irradiation alone .To get insight into the biological mechanisms underlying vascular and valvular modifications, we then performed RNAseq experiments on RNA extracted from the dissected proximal aorta and all cardiac valves. The RNAseq identified 29.587 genes annotated in the rabbit genome, among which 15.135 genes could be assigned to human genes. The cholesterol-rich diet modified the expression of 1.102 (898 unique human orthologs) genes in the aorta, 612 (492) genes in the aortic valve, 281 (229) genes in the mitral valve, and only 4 (3) and 6 (6) genes in the tricuspid and pulmonary valve, respectively. The lard-rich diet yielded much less DEGs than the cholesterol-rich diet, comprising 76 (66) genes in the aorta and 40 (36) genes in the aortic valve. No DEGs were found in the three other cardiac valves in response to the lard diet.We then assessed the impact of these changes in RNA expression on biological processes in each tissue. To detect the differential effects of each diet in every tissue, both tissue-specific and common DEGs were included in these analyses.We first studied the effect of a cholesterol-rich diet on the aorta and aortic valve. Among the 612 DEGs identified in the aortic valve of rabbits from the cholesterol group, 443 DEGs were commonly affected in the aorta . These cWe next focused on the comparison between the aortic valve and mitral valve in the cholesterol-fed group. Whereas the DEGs commonly found in the mitral and aortic valves included genes involved in inflammation and cellular defense response, we surprisingly observed that the cholesterol-rich diet mainly affected lipid biosynthetic and metabolic processes as well as folic acid transport in the mitral valve . This fiWe did not identify the genes that were commonly affected in the aorta and aortic valve by the lard-rich diet. The few DEGs that were found to be specific to the aortic valve were all involved in muscle-related processes, including contraction, filament sliding, and sarcomere organization . IntriguThe addition of irradiation to a cholesterol-rich diet significantly increased the numbers of DEGs both in the aorta and the aortic valve. This was particularly true for the aortic valve. Indeed, 2,097 (1714) and 3,474 (2715) DEGs were identified in the aorta and aortic valve, respectively, which indicated that the aortic valve might be more impacted than the aorta following irradiation. The addition of irradiation to the cholesterol-rich diet modified the expression of 15 (12) additional genes in the aorta and 602 (512) genes in the aortic valve as compared to cholesterol without irradiation. Interestingly, we observed that the common DEGs identified both in the aorta and aortic valve contributed to the same biological processes as a cholesterol-rich diet alone, namely neutrophil degranulation, inflammatory, and immune responses . HoweverWe wanted to determine whether diet- and irradiation-induced structural and RNA changes could be reflected systemically. For this purpose, we measured the plasma levels of biomarkers related to some of the biological processes identified in our RNAseq dataset. These measurements were performed at baseline and after 16 weeks. We first analyzed differential blood cell counts. Neutrophil count was the only parameter to be increased in the cholesterol-treated rabbits, with or without cardiac irradiation, as compared to Ctrl rabbits . We thenWe then analyzed the levels of biomarkers involved in calcification, inflammation, oxidative stress, and platelet activation . RabbitsCholesterol-enriched diet increased the plasma levels of the specific platelet activation marker, platelet factor-4 (PF4), as well as the neutrophil count and the oxidative stress marker, ent-8-iso-15(S)-prostaglandin F2\u03b1 (ent-8-iso-15(S)-PGF2\u03b1) . CombineLard-enriched diet did not increase inflammation- or oxidative stress-related biomarkers, in accordance with large, calcified area observed in the aorta and aortic valve without the major implication of inflammatory processes as found in the RNAseq analyses.To identify possible early biomarkers of lesion development, we performed serial measurements of the neutrophil count, ent-8-iso-15(S)-PGF2\u03b1, H3.1-nucleosome, and citrullinated H3R8-nucleosome levels in cholesterol-enriched diet and irradiation combined with cholesterol-enriched diet groups. The neutrophil count increased as early as after 15 days following the dietary cholesterol intake . Levels Our study demonstrates differential effects on the vascular and valvular structures induced by cholesterol- and lard-enriched diet, as well as by combining the cholesterol diet with cardiac irradiation.via different mechanisms than a cholesterol-rich diet, leading to distinct structural lesions. Likewise, while muscle-related mechanisms are likely to be involved in the development of aortic valve alterations in response to cholesterol- and lard-enriched diet, inflammatory processes would specifically contribute to the effect of cholesterol diet on the valve.Cholesterol-rich diet not only induced well described atherosclerotic lesions but also aortic valve thickening. In addition, calcification occurred in the aortic valve leaflets. The two left-sided aortic and mitral valves displayed cellular infiltrates, including macrophages as observed by RAM-11 immunostaining, and excess ECM deposition. In contrast, a lard-rich diet caused massive aortic media calcification with no intima thickening, and the aortic valve calcified without changes in the leaflet thickness and obvious ECM remodeling. The RNAseq analyses of the aorta, aortic valve, and mitral valve revealed distinct predominant biological processes in lesion development depending on the diet. A cholesterol-rich diet mostly triggered inflammation-related processes in all three tissues. Aorta-specific biological processes were also identified that were involved in processes, such as platelet degranulation, angiogenesis, and cell migration, which are well known to contribute to atherogenesis \u201331. The The addition of cardiac irradiation to a cholesterol-enriched diet led to similar histological lesions in the aorta, the aortic valve, and the mitral valve. However, atherosclerotic plaque and aortic valve and mitral valve leaflets had higher content in RAM-11 positive cells when irradiation was combined with cholesterol-rich diet than with a cholesterol diet alone. Also, the observed increase of \u03b1SMA positive cells in the mitral valve suggests an ongoing transdifferentiation process of valvular cells. Valvular interstitial cells could transform into myofibroblasts following cardiac irradiation, which could promote valve remodeling and ECM production . These fin vitro -PGF2\u03b1, but also through histone-mediated mechanisms as indicated by the increased levels of H3.1 and citrullinated H3R8 nucleosomes. Extracellular histones have been associated with cardiac injury in patients with septic shock . They cain vitro , 42. TheSome biological processes were also specifically triggered by cardiac irradiation in the aorta and aortic valve .Platelet degranulation was found to be increased in the aorta tissue of rabbit groups fed with a cholesterol-enriched diet, combined either with cardiac irradiation or without cardiac irradiation. This result was corroborated by increased plasma levels of PF4 in both groups. Importantly, PF4 has already been associated with atherosclerosis development and could represent another biomarker of atherosclerosis and possOur study has some limitations, including that only male rabbits were included. Also, we did not detect any impact of irradiation by itself on the cardiac structures over the analyzed 8-week period. Longer time protocols should be undertaken to evaluate the sole effect of irradiation, without the influence of any diet. Also, the addition of cardiac irradiation to a lard-enriched diet would be interesting to determine whether irradiation-mediated inflammation would potentiate lard-induced lesions. Furthermore, tissue structure was only assessed by histological analyses, which did not allow for 3D visualization of the lesions, which could provide insights into their cellular and extracellular composition. Finally, RNAseq analysis was performed on complete tissues and therefore identification of specific cellular types was not possible. Single-cell RNA sequencing could bring further information regarding the impact of treatments on tissue-specific cellular type enrichment and cellular process activation or inhibition.via distinct muscle-related mechanisms. Cardiac irradiation enhanced inflammatory processes and calcification in the arteries and aortic valve. At the systemic level, markers of platelet activation, neutrophils, and specific histone modifications in nucleosomes could represent new highly valuable biomarkers for the early diagnosis of ongoing inflammation-related atherosclerosis and heart valve remodeling.Our study showed that cholesterol- and lard-enriched diets, and cardiac irradiation combined with a cholesterol-enriched diet induce the development of distinct lesions in the aorta, the aortic valve, and the mitral valve. Lard- and cholesterol-induced valvular and vascular lesions were different both in terms of histological modifications and in the underlying biological process. While cholesterol led to a global increase of inflammation-related processes, lard causes arterial and aortic valve calcification https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193060.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by Commission Ethique Animale ULi\u00e8ge, Universit\u00e9 de Li\u00e8ge, Li\u00e8ge, Belgium.CO, PL, and AN contributed to the conception and design of the study. ZJ performed the statistical analysis. ND and CO wrote the manuscript. CD'E, AH, MH, RD, M-LN, AP, DP, and ND performed the experiments. MH, JT, PDe, and PL contributed to data interpretation. FL, PC, PM, JP, PDe, and PDr performed the experiments of provided tools and expertise for setting up the animal models. All authors contributed to manuscript revision, read, and approved the submitted version.This project was supported by grants from the R\u00e9gion Wallonne and the Fonds L\u00e9on Fr\u00e9d\u00e9ricq . CO is Research Director at the National Funds for Scientific Research, Belgium (FRS-FNRS). This study was sponsored by Volition SRL. This study received funding from Belgian Volition SRL. The funder was involved in the study design and in the collection, analysis, and interpretation of the data of nucleosome plasma levels.MH and DP were employed by Belgian Volition SRL. JT was employed by Volition America. The remaining 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."} +{"text": "Medication utilization, outpatient visits, surgery, and expenditures related to CRS were assessed for older adults (>65) and compared with other age groups. As a secondary analysis, multivariable generalized linear models were utilized to compare HCE while adjusting for baseline medication utilization. Results: A total of 238,825 patients met the inclusion criteria, of which 20,927 were older adults. Older adults had the highest overall prevalence of nasal polyps (10%) and asthma (16%) among adult groups. Surgery rate was lower than other adult groups, but medication utilization was the highest. Mean overall HCE at two years was highest in older adults (USD 2545 vs. 2298 in young adults). However, HCE was highest for the young adult group after adjusting for baseline medication usage. Conclusion: Older adults had a higher rate of CRS-related co-morbidities as well as the highest CRS-related medication utilization and unadjusted two-year HCE. Although the reasons for this are unclear, possibilities include greater disease severity and preference for medical versus surgical management. HCE for CRS is expected to increase as the aging population grows.Objectives: Chronic rhinosinusitis (CRS) is a common and costly health problem in the United States. While often associated with younger adults, CRS can affect the elderly. As the aging population increases in the United States, the cost burden of CRS in older adults is important to assess. The objective of this study is to characterize healthcare resource utilization (HCRU) and healthcare expenditure (HCE) for CRS in this population. Methods: Patients meeting criteria for CRS with three years of continuous data were identified on IBM Chronic rhinosinusitis (CRS) is one of the most common chronic conditions in the United States, with an estimated prevalence of 15% and annual direct costs of up to USD 9.9 billion dollars [While CRS affects people of all ages, there is evidence that it may be more prevalent in older adults, as was reported by a population-based study from South Korea . Other sIt is estimated that 72 million Americans will be over the age of 65 by the year 2030 [\u00ae Research Databases over a five-year period (2013\u20132017). All patients (adult and pediatric) with continuous enrollment data between 1 January 2014 and 31 December 2015 were initially included. From there, patients with an index CRS diagnosis during that period were selected. Patients were considered as having CRS if CRS-related ICD diagnosis codes appeared on at least two claims on two separate dates, as described by Rudmik et al. [This study was approved by the institutional review board at Duke University (Pro00103732). This is a retrospective review of medical and pharmacy claims from the IBM Health MarketScank et al. From thiCRS-related HCRU and HCE per patient were determined for the two-year post-index period. HCRU included the following: CRS-related medication prescriptions, outpatient visits with an otolaryngologist, immunotherapy, and sinus surgery. HCEs were categorized into the following: CRS-related medications, CRS-related outpatient visits with an otolaryngologist, sinus surgery, and CRS-related overall cost. Overall cost included CRS-related medications, all inpatient and outpatient claims, office procedures, surgery, and imaging. CRS-related medications included any prescriptions in the following therapeutic classes: antibiotics, anti-inflammatory agents , adrenals , antihistamines, anticholinergics, mucolytics, sympathomimetics (decongestants), and leukotriene blockers. HCRU and HCE were then compared between four age groups: (1) pediatric (<18), (2) young adult (18\u201340), (3) middle adult (41\u201365), (4) older adult (>65).Expenditures were based on paid adjudicated claims, including deductible, coinsurance/copayment, coordination of benefits and other savings (COB), and net payment (payment received by the provider excluding patient out-of-pocket and COB). Costs were adjusted for inflation based on US Bureau of Labor Statistics indices to 2017 USD. All costs reported were after excluding patients with capitated services and outliers (<1st percentile or >99th percentile of each cost). Patients with capitated insurance plans were excluded from the cost analysis.Multivariable generalized linear models (GLM) using a gamma distribution with a log link were fit to compare the mean CRS-related costs over one-year and two-year periods between age groups, while adjusting for baseline pre-index medications. Cost ratios estimated by exponentiating the regression coefficients were reported with 95% confidence intervals (CIs). Cost ratios can be interpreted as a relative change in adjusted mean costs of one age category relative to a reference category (young adults). Regression-adjusted mean costs were calculated from the GLM to estimate the average marginal effects for each age group [There were 32,972,540 patients with continuous enrollment and complete claims data between 2014 and 2015. Of these, 238,825 unique patients met inclusion criteria. 20,927 patients were older adults. A total of 208,276 patients were included in the cost analysis after patients with capitated plans were excluded. Two-year post-index HCRU is summarized in HCE at one-year and two-years after index diagnosis is summarized in The high economic burden of CRS combined with the need for improved healthcare efficiency calls for a close examination into how limited resources are utilized from a population health perspective ,11. SuchWe hypothesized that older adult patients would have higher HCRU and HCE based on recent studies that have investigated CRS characteristics and outcomes in elderly patients. An epidemiologic study of the Korean National Health and Nutrition Examination Survey revealed a higher incidence of CRS in the elderly population . SmallerConsistent with these studies, our results suggest that older adults have more severe CRS than younger adults. The older adult group had a higher incidence of asthma and nasal polyps, and were prescribed greater numbers of antibiotics and oral steroids. Not surprisingly, this led to the highest HCE at two years. The lower surgery rate for older adults is of interest. Our data suggest that older adults are seeing the otolaryngologist at comparable rates as other groups, but may be foregoing surgery and continuing with long-term medical management because of a higher prevalence of systemic co-morbidities and greater risk with general anesthesia. This is demonstrated in the fact that HCE is roughly equivalent during the first year after CRS diagnosis, but HCE, particularly related to medications, becomes much higher during the second year after diagnosis. Our data demonstrate an inverse relationship between age, surgery rates, and HCE, supporting prior studies showing that continue medical management is often not the most cost-effective option ,13.Our regression-adjusted analysis, which adjusts for pre-index medication utilization, found that HCE was not higher in older adults. This can be explained in two ways. First, older adults may take antibiotics and anti-inflammatory medications for a host of chronic conditions, some of which may not be for CRS . Second,Because the high HCE associated with older adults resulted mainly from high medication utilization, reducing polypharmacy may be a strategy that can reduce costs . Our datClearly, HCRU and HCE are complex issues at the heart of population health science. Age is only one of many contributing factors that have recently been investigated in the literature ,18,19. HLimitations of this study are those inherent in claims-based administrative databases. Detailed clinical data are not available, and inclusion criteria do not accurately capture all relevant patients. However, such databases can provide important information for studies involving population cohorts, and the same stringent criteria used for small cohort studies are impractical to apply. It is also important to note that the Marketscan databases contain information on elderly patients with Medicare Supplemental plans, which is only a portion of all Medicare patients. Therefore, the proportion of Marketscan CRS patients in the older adult group (8.8%) is not a reflection of the true proportion in the general older adult population. However, the Marketscan databases allow for a unique opportunity to compare healthcare utilization amongst different age groups. Despite these limitations, our findings establish the presence of variations and disparities in healthcare utilization according to age. Such variations, although seemingly small per patient, can translate to billions of dollars in expenditures for the U.S. population. Future studies will need to investigate the underlying reasons for such variations, and then assess ways to reduce inefficiencies should they exist.Healthcare utilization and expenditure for CRS were substantially higher in older adults, despite the lower rates of sinus surgery. In particular, medication costs for CRS increased disproportionately in older adults during the second year after diagnosis. Greater CRS severity and preference for medical therapy over surgery may account for this discrepancy. HCE for CRS is expected to increase as the aging population grows. Additional research is needed in order to develop strategies for reducing expenditures and improving efficiency in care."} +{"text": "Background: Identifying the cardiac changes could help design measures to recover the cardiovascular system and lessen the mortality and morbidity rate. Accordingly, this cross-sectional study was performed to evaluate the echocardiography indices which are indicators of the cardiac alterations of the children with COVID19 infection.Methods: This study was performed as a cross-sectional study evaluating echocardiography indices in children infected with COVID19. Fifteen children, known cases of the COVID19, and 14 healthy children were enrolled. Evaluated parameters include left ventricle ejection fraction (LVEF), left ventricle end-diastolic diameter (LVED), mitral valve Sa (MV Sa), Tricuspid annular plane systolic excursion (TAPSE), and laboratory parameters.Results: The participants' mean age and weight were 62.8 (\u00b148.0) months and 19.95 (\u00b115.67) kg, respectively. None of the laboratory and echocardiography parameters differed between males and females, between patients with and without positive past medical history, between the patients with and without respiratory tract symptoms, and between patients with and without GI tract symptoms (P.0.05). Patients had significantly higher TAPSE (p = 0.027), although MV Sa (p = 0.01) was significantly higher among healthy children. LV EF (p = 0.425) and LVED diameter (p = 0.603) were not different significantly. None of the patients had pericardial effusion, pleural effusion, and cardiac tamponade.Conclusion: The heart can be involved during the disease course in children, even at the level of echocardiography indices. This could contribute to a worse prognosis, higher morbidity, and mortality rate, especially in patients with overt myocardial involvement. Non-classic indicators, including LVEF, may not be conclusive for cardiac involvement in non-symptomatic patients. The novel coronavirus pandemic, SARS-COV-2 COVID19), has become a significant concern due to its high mortality rate and unknown nature. Although this virus typically involves the respiratory tract, other organs are also involved with extra-pulmonary manifestations 9, has be. Better Cardiovascular involvement has been widely described in the literature, though most studies focused on adult populations. One of the most notorious cardiac manifestations is myocardial damage ; patientThis study was performed as a cross-sectional study evaluating echocardiography indices in children infected with COVID19. Fifteen children, known cases of the COVID19, and 14 healthy children were enrolled. The patients were admitted to COVID19 specific ward regarding their symptoms and undergone echocardiography based on their symptoms, including tachycardia. The healthy controls were selected from the patients referred to our clinic for non-specific non-cardiac chief complaints such as chest pain or palpitation. The COVID19 infection was considered positive if positive clinical evaluations of the COVID19 by infectious consultation existed, plus the positive chest CT results and positive reverse transcriptase-polymerase chain reaction (RT-PCR) of the nasopharyngeal swab. The COVID19 pneumonia was considered positive in the case following features that were found in the CT scan: the presence of ground-glass opacity (GGO) mainly in the peripheral and posterior lungs that did not spare the subpleural regions, consolidation, GGO with consolidation, or interlobular septal thickening . Echocart-test, Mann Whitney test, and Chi-square test were applied. A P-value below 0.05 was considered statistically significant.All data were analyzed using the SPSS version 22. Results are presented as the number (percent), mean (+ standard deviation), and mean (interquartile range). Student's This study has been approved by the Research Deputy and the Ethics Committee of Tehran University of Medical Sciences (Reference number: IR.TUMS.VCR.REC.1399.218) and conducted by the ethical standards laid down in the 1964 Declaration of Helsinki and all subsequent revisions. The informed consent form was obtained from all parents following the explanation of the goal of the study.p = 0.290), weight (p = 0.851) and gender (p = 0.139) were not different significantly between cases and controls. All patients experienced low-grade fever (<38\u00b0C); only six patients had a significant fever (over 38\u00b0C), and four of them experienced high-grade fever for more than five consecutive days, other detailed information regarding the clinical manifestations of the patients with COVID19 is shown in P = 0.05).The participants' mean age and weight were 62.8 (\u00b148.0) months and 19.95 (\u00b115.67) kg, respectively. Thirteen (44.8%) patients were female. The age (p = 0.027), although MV Sa (p = 0.01) was significantly higher among healthy children. LV EF (p = 0.425) and LVED diameter (p = 0.603) were not different significantly is characterized by high-grade fever, evidence in favor of increased inflammation in lab tests, multiorgan involvement; in addition to positive COVID19 PCR or antigen or exposure to a suspected or confirmed COVID19 patient . It is aUnfortunately, because of the critical medical situation imposed by the COVID19 crisis and the limitations of the facilities, it was not possible for all patients to undergone assessments of the additional laboratory findings associated with myocardial infarction . We learned from these findings that the heart is also involved during the disease course in children, even at echocardiography indices. This could contribute to a worse prognosis, higher morbidity, and mortality rate, especially in patients with overt myocardial involvement; furthermore, non-classic indicators, including LVEF, may not be conclusive for cardiac involvement in non-symptomatic patients. Putting all together, evaluating potential cardiac changes before and during the therapy is crucial; since it leads to taking appropriate measures to prevent further damage to the heart.There are some limitations to be acknowledged; first, the sample size was small. Furthermore, all echocardiogrphies were performed by a single cardiologist, which reduced interpersonal bias, but it has bias regarding the generalizability of the evaluated echocardiography indices. Besides, the cardiologist was not blinded, which could introduce bias.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Research Deputy and the Ethics Committee of Tehran University of Medical Sciences. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.EA and SM equally contributed to the conception and design of the research. AZ deigned the total structure of the manuscript and determined the patients should be involved. EH and AG drafted the manuscript. MK analyzed the data. MM interpreted it and reviewed and finalized the manuscript. HE and MG contributed to the acquisition. All authors critically revised the manuscript, agree to be fully accountable for ensuring the integrity and accuracy of the work, and read and approved the final 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."} +{"text": "For decades, research and policy on crime have been influenced by perspectives emphasizing that the origins of offender groups, such as chronic offenders, rest on some combination of personal traits and early-life experiences. Such explanations are incomplete, however, because they do not account for social changes that differentiate successive birth cohorts over time. Our analysis shows that arrest trajectories among cohorts separated by as little as 10 y differ markedly in ways that are not explained by individual, family, or neighborhood circumstances in childhood. Analyzing cohort differences in crime is more than an overlooked strategy; it represents a class of explanation that calls for rethinking the nature of criminal offender groups and policies predicated on early-life predictors of crime alone. This article draws on official criminal histories for multiple birth cohorts spanning a 17-y difference in birth year to study how social change can alter our understanding of influential theories and policies about criminal offender groups. Arrest histories are linked to comprehensive longitudinal measurement on over 1,000 individuals originally from Chicago. Using group-based trajectory modeling, we investigated the magnitude and type of cohort differences in trajectories of arrest over the period 1995 to 2020. Our results show that trajectory group membership varies strongly by birth cohort. Membership in the nonoffender group is nearly 15 percentage points higher for cohorts born in the mid-1990s as compared to those born in the 1980s; conversely, older cohorts are more likely to be members of adolescent-limited and chronic-offender groups. Large cohort differences in trajectory group membership persist after controlling for a wide-ranging set of demographic characteristics and early-life risk factors that vary by cohort and that prior research has identified as important influences on crime. Not only does the effect of social change on cohort differentiation persist, but its magnitude is comparable to\u2014indeed larger than\u2014differences in trajectory group membership associated with varying levels of self-control or by whether individuals grew up in high-poverty households. These results suggest that changes in the broader social environment shared by members of the same birth cohort are as powerful in shaping their trajectory group membership as classic predictors identified in prior research, a finding that carries implications for crime-control policies that rely on prediction. The finding in the early 1970s that a small group of chronic offenders accounted for the majority of all criminal offenses , scholarReflecting these high stakes, considerable interdisciplinary research focuses on explaining variation in trajectories of crime over the life course , most ofAlthough foundational theoretical perspectives in developmental and life-course studies of crime are consistent with this point , 7, the 810Social changes, such as the rise of mass incarceration, rising inequality, and the decline in violence since the mid-1990s further motivate the study of cohort differences in crime rates \u201323. CohoHow do we square the potential of social change to distinguish the life experiences of different birth cohorts with the study of criminal trajectory groups, which although central to scholarship on crime, has neglected cohort differentiation? The answer to this question remains unclear, as the concept of cohort has almost exclusively been used to study changes in aggregate crime rates or, in limited cases, average differences in crime over the life course .We advance existing research by taking advantage of the capacity of a multicohort design to study varying offender trajectory groups across cohorts. Although longstanding methodological issues in studying age, period, and cohort are important and incorporated, our contributions are primarily substantive. Developmental trajectories of crime reflect more than just people who share similar offending patterns because of their personal traits and early-life experiences. We show that social change\u2014as manifested through cohort differences in the distribution of offender trajectory groups\u2014represents a key missing piece of the puzzle: developmental trajectories also reflect the power of shared social environment to shape people\u2019s lives.We substantiate this claim empirically by drawing on 25 y of criminal history data on multiple birth cohorts that span a 17-y period. Our study includes rich measurement on over 1,000 individuals originally from Chicago, with which we investigate the degree and nature of cohort differences in trajectories of arrest over the period 1995 to 2020. We answer two main research questions. First, how does membership in offender trajectory groups, defined by arrest, vary by cohort? Studies of trajectory groups typically employ data from people born around the same time, making cohort differences virtually impossible to observe. Second, do cohort differences in trajectory group membership reflect only that cohorts differ demographically and in their level of exposure to multiple risk factors in childhood and early adolescence? Establishing whether this is the case, or whether cohort differences reflect the dynamic influence of social environment across the life course, is crucial in order to understand what cohort differences represent and what they mean for the interpretation of trajectory groups more broadly.Our results show that trajectory group membership varies strongly by birth cohort. Membership in the nonoffender group is nearly 15 percentage points higher for cohorts born in the 1990s as compared to those born in the 1980s; conversely, the older cohorts are much more likely to be members of adolescent-limited and persistent offender groups. Furthermore, these cohort differences in trajectory group membership persist even after controlling for a wide-ranging set of demographic characteristics and early-life risk factors that prior research has identified as important in predicting crime trajectories. Not only does the effect of social change on cohort differentiation persist, but its magnitude is found to be comparable to, indeed larger than, differences in trajectory group membership brought about by varying levels of self-control or by whether or not individuals grew up in households on welfare. These results demonstrate that the broader social environment shared by members of the same birth cohort is as important in shaping their trajectory group membership as classic predictors in criminological research, a finding that carries implications for crime control policies that rely on prediction.While the size of the smaller, high-rate persistent offender group varies less by cohort, we do find substantial within-group variability across cohorts in the rate of arrests per offender as well as in the types of arrests of the high-rate offenders that has been overlooked by prior research. A decomposition analysis further indicates that while about two-thirds of the overall cohort difference in offending rates between the ages of 17 and 24 y are due to differing group membership, nearly one-third is due to different arrest rates within trajectory groups, which overwhelmingly reflects differences among high-rate, persistent offenders across cohorts. A major implication is that cohort differentiation through social change not only influences which trajectory group cohort members find themselves in, but also the nature of arrest patterns given membership in a trajectory group. This finding also carries implications for predictive policies, which we discuss.The data consist of 1,057 individuals from four different age cohorts of the Project on Human Development in Chicago Neighborhoods (PHDCN). At the PHDCN\u2019s start in the mid-1990s, a representative sample of children belonging to seven age cohorts, starting with a newborn cohort and ranging up to age 18 y, was drawn from a representative sample of Chicago neighborhoods. About 35,000 households were first screened for eligible children, yielding an initial sample of over 6,200 children. Data on these children and their families were then collected during extensive in-home assessments, which included interviews with their primary caregiver. This initial wave was followed by two further waves of data collection over roughly 2.5-y intervals. Neighborhood data were also collected .In 2011 to 2013, a random sample of those last contacted at wave 3 in the 0-, 9-, 12-, and 15-y-old age cohorts was reinterviewed, labeled \u201cwave 4.\u201d Parallel to waves 1 to 3, detailed information was gathered from respondents on a wide array of topics. In addition, the caretakers of cohort 0 members were asked a battery of items measuring the behavior and circumstances of their dependent as an adolescent.SI Appendix, Section 1 for information on attrition and out-of-state migration).Arrest records starting in 1995 were collected from the Criminal History Record Information (CHRI) in Illinois and analyzed. Wave 4 respondents were matched by name and date of birth with CHRI records covering the entire state of Illinois four times\u2014in 2015, 2017, 2019, and early 2021\u2014expanding prior work by measuSI Appendix, Table S1 for further information on the data structure by cohort). To improve common support across cohorts, no data past the age of 33 y are analyzed . There are, however, arrest data on all individuals from the ages of 17 to 24 y, the prime ages for arrest, and data on 58% of respondents to age 33 y. To simplify the presentation of results, in the main analyses cohorts 9, 12, and 15 are combined into what we call the \u201colder birth\u201d cohorts who were born between 1979 and 1988 and age cohort 0, which we call the \u201cyounger birth\u201d cohorts because members were born between 1994 and 1996 (results are substantively unchanged when modeling the age cohorts separately).The four age cohorts included in wave 4 were observed at partially overlapping ages. Arrest data for cohort 0 are from age 10 to 25 y, for cohort 9 from age 10 to 34 y, for cohort 12 from age 17 to 37 y, and for cohort 15 from age 17 to 40 y. Arrest data for cohorts 12 and 15 prior to age 17 are excluded due to incomplete recordkeeping at the time . Taken together, this set of variables represent classic childhood, family, and neighborhood predictors of crime , whether the primary caregiver was employed, and the primary caregiver\u2019s relationship status. Whether parents were arrested or had trouble with the law was also measured, as was whether the primary caregiver was depressed, and the number of family members that had frequent trouble with the law, employment, getting into fights, or with school discipline. Early-life neighborhood characteristics were measured with seven tract-level variables measured when respondents were 9 y of age: the poverty rate, the unemployment rate, the fraction of households that were headed by females only, the fraction of housing that was owner-occupied, the fraction of adults with a college education, as well as the fraction of residents who were black and Hispanic. Early-life neighborhood crime exposure was measured with the tract-level homicide rate around the ages of 6 to 10 y are missing data on fewer than 10% of the control variables, with only three subjects missing data on over 50% of them; no control variable has missing data for more than 13.2% of subjects. Ten multiple imputed datasets were created to carry out the analysis that uses variables with missing data , with esP(Yi | Agei), where the random vector Yi represents individual i\u2019s longitudinal sequence of arrests defining the trajectory and the vector Agei represents individual i\u2019s age when each of those measurements is recorded. The group-based trajectory model assumes that the population distribution of trajectories arises from a finite mixture of unknown order J. The likelihood for each individual i, conditional on the number of groups J, may be written asOffender groups were identified using group-based trajectory modeling, a form of latent group analysis based on finite mixture modeling that uses maximum-likelihood estimation to jointly model groups\u2019 trajectories , 30, in \u03c0j is the probability of membership in group j, and the conditional distribution of Yi given membership in j is indexed by the unknown parameter vector \u03b2j, which among other things determines the shape of the group-specific trajectory. Typically, the trajectory is modeled with a polynomial function of age. For given j, conditional independence is assumed for the sequential realizations of the elements of Yi, yit, over the T periods of measurement. Thus, we may writewhere p(.) is the distribution of yit conditional on membership in group j and the age of individual i at time t. As our dependent variable is a count variable, arrests by age, p(*) is specified to follow the Poisson distribution. We also note that \u03c0j is specified to follow the multinomial logit function, which allows the option of generalizing the model to condition probability of trajectory group membership on baseline covariates of i.where SI Appendix, Fig. S3 for the four-group solution).To identify the best-fitting model, Bayesian information criterion statistics were compared across models with different numbers of groups and polynomial functional forms for the age trajectories. The two best-fitting models were a three-group solution and a four-group solution, each of which specified a cubic polynomial for all of the groups\u2019 trajectories. The size, shape, and level of two groups, which as described below, we call the \u201clow\u201d and \u201cmedium\u201d trajectories, remain virtually unaltered. What changes is that the four-group solution splits the \u201chigh\u201d group into two smaller groups that add no additional substantive insights. For parsimony, we focus on the three-group model in the main results . Furthermore, various tests of model adequacy support the fit of the three-group cubic model. The probability of trajectory group membership was nearly identical to the fraction of the sample assigned to each group based on the maximum posterior probability of membership . Also, the mean probability of membership in each group for subjects assigned to those groups was 0.98, 0.88, and 0.94, for the low, medium, and high groups, respectively, all of which are well above the recommended minimum level of 0.7 . For the older cohorts, probability of membership in the low and medium trajectories are, respectively, 0.74 and 0.21. In contrast, the respective probabilities for the younger cohorts are 0.88 and 0.086, a substantial absolute difference in both cases.To measure how trajectory group membership varied between the younger cohorts and the older cohorts, the model shown in P > 0.05), the difference is in the same direction as that between the low and medium group, with the younger cohorts having lower membership in the higher-rate trajectory group. Furthermore, a 1.2 percentage point difference in expected high group membership by cohort is large given what is being estimated: people who are chronic high-rate recidivists for much of their lives. For reference, 1.2% of the US population is 4 million people; were similar effect sizes applied to that population, it would represent a very large group of people who live\u2014or avoid\u2014a life of frequent criminalization due to social changes.The cohort difference in probability of membership in the high group is less pronounced. Specifically, the odds of membership in the high group as opposed to the low group are 1.575 times higher for the older versus younger cohorts. This translates into a probability of high group membership of 0.049 for the older cohorts and of 0.037 for the younger cohorts. While not statistically significant (P < 0.001). The odds of membership in the high group as opposed to the low group increase to 2.261 for the older compared to the younger cohorts, a difference that is now statistically significant at the 0.05 level (P = 0.027). Thus, the cohort differences in group membership do not reflect demographic differences or differences in risk factors. If anything, those factors partially mask the magnitude of the cohort effect .The cohort differences reported so far may not reflect the influence of shared social environment in adolescence and adulthood on trajectories of offending but instead could reflect differences in the cohorts\u2019 composition or in their exposure to risk factors that are determined earlier in life. This possibility is investigated with our main model specification that jointly conditions on five major demographic and risk factors: self-control, parental welfare (TANF) receipt, sex, race/ethnicity, and immigrant generation. The addition of these control variables does not explain or even attenuate the estimated cohort effect, but instead increases its magnitude. The older cohorts were estimated to have odds of membership in the medium group compared to the low group that are 4.167 times higher than the younger cohorts and that for membership in the high as opposed to low group is 2.638 (P = 0.035). Thus, being born into the earlier cohorts is associated with much greater risk of being classified as a moderate or severe offender, even after adjusting for demographic, compositional differences between cohorts, and a wide array of early-life criminogenic risk factors .To further test the robustness of cohort differences to the addition of covariates measuring personal circumstances, a model that adds 19 more risk factors was estimated. We are not focused on the coefficients of the large number of risk factors, many of which are correlated. It is not surprising, for example, that the association of both TANF and self-control with trajectory group membership is attenuated in this extended model. In contrast, the estimated effect sizes of being in the older cohorts as opposed to the younger cohorts are larger: the odds ratio for membership in the medium as opposed to low group is 5.124 . Put differently, within the high group, the older cohorts were arrested 34.8% more often than the younger cohorts between the peak offending ages of 17 and 24 y.P < 0.01). This large difference in drug arrest rates between cohort for the high group almost fully explains the differences in total arrests for the high group reported in Turning to the high group, arrest rates across cohorts are similar except for drug arrests, for which the arrest rate is 1.27 for the younger cohorts compared to 3.66 for the older cohorts, a difference that is statistically significant for the younger cohorts is 0.614 compared to 1.156 for the older cohorts, a gap of 0.542. If the trajectory groups of the younger cohorts had the arrest rates of the older cohorts by trajectory groups, their arrest rate would have been 0.774. That is 26% higher than it actually was. If the younger cohorts belonged to the trajectory groups in the same proportions as the older cohorts, but holding their actual arrest rates within each trajectory group constant at the observed values, then the arrest rate for the younger cohorts would have been 0.995 or 62% higher than it actually was. Thus, both differing trajectory group sizes and within-group arrest rates account for differences in the average rate of arrests between cohorts. Notably, if trajectory membership of the younger cohorts had remained at observed levels and within the low and medium trajectories arrest rates had similarly remained unchanged, with only the arrest rate of the younger cohorts in the high group increasing to that of the older cohorts\u2019 arrest rate for that group, the younger cohorts\u2019 rate would have been 0.725, or 18.2% higher than it was. This calculation confirms that the difference in the arrest rates between cohort in the small high trajectory group was an important factor in driving the overall differences in arrest rates between cohorts.Which people will go on to commit the most crimes or be arrested the most, and the rate at which they do so, cannot be known based on individual background, personal characteristics, early-life risk factors, and age alone. This result was obtained because membership in trajectory groups, as well as the nature of crimes among individuals seemingly in the same group, are both strongly influenced by the larger social environment, which itself changes over time. That is, our results indicate that arrest trajectories are outcomes that are responsive to changing historical contexts, not just to early-life circumstances or individual characteristics determined at birth or in early childhood.These findings are consistent with major developmental and life-course theories of crime, which incorporate environmental influences, including historical context , 33. CruThe implication is that greater attention in the study of crime should be given to changing historical contexts. For example, the original theoretical account of adolescent-limited offenders\u2019 behavior portrayed it as shaped by historical context , 8; thisOur findings reveal cohort differences in every trajectory group studied. While chronic offenders likely face elevated rates of early-life adversities that set them along high-rate offending trajectories, for social or psychopathological reasons, their development is still influenced to a large degree by what has yet to happen in the world around them. It follows that a fuller understanding of developmental trajectory groups demands accounting for cohort differences in social environments throughout the life course.Future research is needed to disentangle the societal-level changes that may have contributed to cohort differences in trajectory groups. For example, during this period Chicago\u2019s neighborhoods were undergoing an array of changes that may have enhanced social control while reducing opportunities for crime, such as revitalization from immigration, the mobilization of local organizations against violence, and the replacement of segregated public housing with mixed-income developments. In addition, the lives of youth in Chicago and beyond were transformed by the rise of novel technologies, ranging from smart phones to social media, as time spent engaging in unstructured socialization decreased , 34. MorOur findings also carry policy implications. For one, they suggest there are real limits to how well offender groups and offending patterns can be prospectively identified. Predicting the future risk of offending is a core component of many crime-control policies, including pervasive attempts to identify at-risk youth, as well as the recent emphasis on using machine-learning algorithms in the criminal justice system . Yet algAs illustrated by the controversy surrounding the COMPAS risk assessment instrument , much reAn important question is the extent to which gaining information on individuals\u2019 prior criminal history records\u2014the central component of criminal justice risk instruments\u2014can correct for cohort-based prediction biases. Future research should distinguish prediction biases related to the level of offending from an individual\u2019s relative ranking among cohort members. Both types of biases may be present, but the former is most relevant to criminal justice policy because the social cost of crime relates to an individual\u2019s level of offending, not from their relative ranking.Finally, our findings suggest the value of broadening crime-prevention policy, even as it relates to the highest-rate offender group, from a narrow focus on identifying individuals whose future offending patterns may be high to enhancing opportunities and structures in the larger social environment. We urge this because an individual\u2019s life is determined not only by who they are or what they have done, but also by the changing social circumstances of when they are.Supplementary File"} +{"text": "Additionally, the flexibility of CS structure due to the free protonable amino groups in the CS backbone has made it easy for the modification and functionalization of CS to be developed into a nanoparticle system with high adaptability in lung cancer treatment. In this review, the current state of chitosan nanoparticle (CNP) systems, including the advantages, challenges, and opportunities, will be discussed, followed by drug release mechanisms and mathematical kinetic models. Subsequently, various modification routes of CNP for improved and enhanced therapeutic efficacy, as well as other restrictions of conventional drug administration for lung cancer treatment, are covered.Lung cancer has been recognized as one of the most often diagnosed and perhaps most lethal cancer diseases worldwide. Conventional chemotherapy for lung cancer-related diseases has bumped into various limitations and challenges, including non-targeted drug delivery, short drug retention period, low therapeutic efficacy, and multidrug resistance (MDR). Chitosan (CS), a natural polymer derived from deacetylation of chitin, and comprised of arbitrarily distributed \u03b2-(1-4)-linked Over the last decade, the incidence and mortality rate of cancer has remained one of the leading causes of death worldwide, second to cardiovascular diseases . Based oN-deacetylation of chitin, the second most abundant natural occurring polysaccharide after cellulose [d-glucosamine and N-acetyl-d-glucosamine randomly disseminated within the polymer, enabling its cationic nature [Arising from these issues, research focus has shifted to the utilization of nanotechnology to increase treatment efficacy and increase treatment safety. Polymeric nanoparticles are a large family which can generally be divided into natural and synthetic forms. The term polymer can be defined as macromolecules that are made up of bulky repeating units arranged in a chain-like structure, presenting an assortment of alignments, structures, and characteristics ,15. Natuellulose ,23,24. Tc nature ,26,27,28c nature ,30. Howec nature ,32.The CNP-based nanoparticles in drug delivery have been utilized in various diseases treatment, including cancer-related diseases, gastrointestinal diseases, pulmonary diseases, ocular infections, and drug delivery across the blood-brain barrier ,35,36,37The study of Hassan et al. also demonstrated that CNPs promote intracellular accumulation by observing fluorescently-labeled glutamic acids (GA) with and without encapsulation by CNP under a fluorescence microscope. The results showed that the fluorescence signal was detected only in cells that were treated with with fluorescein isothiocyanate (FITC) labeled-GA (FITC-GA) encapsulated CS nanoparticles and was found mainly accumulated in the cell cytoplasm, while no fluorescence signal was detected in cells treated with free FITC-GA . Additio4) loaded CNPs showed greater therapeutic efficacy than free CuSO4 groups against osteosarcoma assessed through in vitro cytotoxicity assay, reactive oxygen species (ROS) analysis, and caspase 3 and 7 apoptotic activities analysis due to better internalization of CNP system [The utilization of nanoparticles for the encapsulation of cargos such as various therapeutic drugs or compounds and genetic materials has been reported by many researchers over the years. As shown in P system . On the P system .Escherichia coli showing its decent antimicrobial properties [Moreover, it was reported that CS and CS derivatives possessed several biological properties such as anti-oxidant ,73,74, aoperties .E. coli) and gram-positive bacteria ( gram positive. bacillus) [It has been further shown by a previous study of Al-Sherbini et al. that the development of CS/silver bionanocomposites has aided in photodegradation of organic pollutants, adsorption of heavy metals pollutants, and also showed antibacterial activities on both gram-negative (acillus) .However, apart from the advantages of CS and its derivatives in various fields as mentioned above, there are numerous challenges remaining to be resolved to improve its use in the future. One of the main concerns is about the safety of CS and its derivatives, especially in biomedical applications. Generally, CS has been categorized as relatively safe due to its biodegradability and biocompatibility properties. It has been reported that elimination of CS was low molecular weight CS can be excreted through the kidney, while the high molecular form can be renally excreted after degraded into fragments . NeverthDespite the challenges encountered by CS and its derivatives, the emergence of nanobiotechnology has opened opportunities in several fields, including agriculture, pharmaceutical, biomedicine, and the food industry . BesidesLiu et al. reported a carboxymethylated CNP system to deliver the antiepileptic drug, carbamazepine, intra-nasally, which able to bypass the blood-brain barrier so as to enhance the brain drug concentration and therapeutic efficacy. This modified CNP system had nanoparticles 218.76 \u00b1 2.41 nm in size with 80% high entrapment efficiency, and this system showed an increase in drug bioavailability and enhanced targeting properties through both in vitro and in vivo studies. . MoreoveZare et al. have also shown that DOX encapsulated by a CNP system had about 12.7 fold greater intestinal permeation than that of free DOX, which suggested enhanced drug delivery by a CNP system . On the The first introduction of controlled drug delivery was in 1952, which has progressed for more than six decades . A contrGenerally, the drug release mechanism of CNPs is divided into three different types. The first mechanism is the swelling of CS (swelling of polymer), diffusion of drug through polymeric matric or adsorbed drug, and degradation/erosion or combination of both degradation and erosion ,126,127.The diffusion-controlled drug release system was divided into monolithic- and reservoir-type, where both were generally initiated by drug permeated through the interior of the polymeric matrix and to the adjacent medium . Interesarticles ,134.The swelling of the polymer is a volumetric-growth process in which denoted by the water imbibition into the polymer until the polymer dissolves ,136. TheThe degradation and erosion release mechanism is amongst the most popular drug release mechanism as retrieval after the drug is released is not required due to the non-toxic and excretable characteristics of the delivery system . Both deBulk erosion occurs when water invades the polymer more rapidly than hydrolysis can occur . Under tMeanwhile, the occurrence of surface erosion can be initiated by both conditions were either when water invasion is slow or when hydrolysis is rapid, which starts at the exposed surface and moves downwards . PolyanhGenerally, the controlled-release mechanism can be categorized into several forms, including diffusion, swelling, and erosion, as mentioned above. Consequently, various types of nanomaterial with different physical and chemical properties and also the nature of the encapsulated drugs will be released through different routes. Therefore, it is vital to identify the release mechanism of drugs/therapeutics and ensure the release in a controlled manner. To date, various mathematical models have been utilized for the fitting of different drug release mechanisms. Among these models, zero-order, first-order, the Higuchi model, the Korsmeyer-Peppas model, and the Hixson-Crowell model have often been used as the models for the drug release fitting of nanoparticles and will be further discussed in the following sections ,1500 = initial amount of drug; Qt = cumulative amount of drug release at time \u201ct\u201d; K0 = zero-order release constant.Theoretically, the drug delivery system that has a zero-order drug release profile is the ideal system and pursued by researchers to achieve a low dosing rate with a uniformly release profile without initial burst release throughout the entire release duration . The resThis elimination of zero-order kinetics takes place at a constant rate following a linear elimination phase as the system becomes saturated, independent of the plasma concentration . Howevert is the cumulative percentage amount of drug released at time t; Q0 is the initial amount of the drug; K1 is the first-order release constant; t is time.The first-order model was first suggested by Gibaldi and Feldman in 1967 and later by Wagner in 1969 . The matIt has been commonly employed to define the absorption and/or elimination of an assortment of therapeutic agents. The elimination in first-order kinetics dependent on the concentration of only one reactant (drug) and the drug is eliminated at a constant fraction per unit of time, which also means that the elimination will increase proportionally as the plasma concentration increases, following an exponential elimination phase as the system never attains saturation . Nonethe0 is the initial amount of the drug; KH is the Higuchi constant; t is time.Next, the Higuchi model was described by Higuchi in 1961 on his study of the rate of release of ointment bases containing drugs in suspension . The matThis model appeared as one of the most famous and most frequently utilized models for the release of cargos from matrix systems. There are several important rules that must be followed in this model: (I) the initial concentration of therapeutic existed in the matrix is much greater than its solubility; (II) the diffusion of therapeutic happens only in one direction where the edge effect is negligible; (III) the thickness of the system is much greater than the size of the therapeutic; (IV) the swelling and/or dissolution of the matrix is insignificant; (V) the diffusivity of therapeutic is persistent; (VI) the perfect sink circumstances are achieved .t is the cumulative percentage amount of drug released at time t; Q0 is the initial amount of drug at time t; KHC is the Hixson-Crowell constant.On the other hand, the Hixson-Crowell model is another well-known release model that was revealed by Hixson and Crowell in 1931 upon the discovery of a collection of particles\u2019 fixed area is comparative to the cube root of its volume, while its mathematical equation is presented in Equation (5) \u221bQt \u2212 \u221bQThis model is commonly used for pharmaceutical systems such as tablets, in which the dissolution rate is equivalent to the surface of the dosage form; the surface area erodes consistently over time while the geometrical form remained unchanged. A previous study conducted by Malana and Zohra showed the in vitro release study of tramadol hydrochloric acid from chemically cross-linked ternary-polymeric hydrogels matrix tablet . The stuKP is the Korsmeyer-Peppas constant; n is the release exponent describing the drug release mechanism.Additionally, the Korsmeyer\u2013Peppas model was derived from the Power law, which is a more comprehensive model to term the drug release from a polymeric system. This model was established by Korsmeyer et al. in 1983 to describe the exponential relationship between the release of drug and the time where its mathematical equation was shown in Equation (6) Qt = KKPThis Power Law model is advantageous for the study of polymeric drug release systems, especially when the release mechanism is unfamiliar or when multiple release mechanisms are involved .This section has summarized the various drug release mechanisms of nanoparticulate drug delivery systems. The drug release of polymeric nanoparticles was categorized in diffusion, swelling, and degradation/erosion, which are characterized differently by the composition, ratio of composition, interactions between the components, and preparation methods . Apart fDespite the advantages of CNP as a great nano-carrier system, there are some limitations that exist, such as low encapsulation efficiency for poorly water-soluble compounds and low solubility in physiological pH, which limits the usage of this system ,164. ForHelicobacter pylori [The physical modification of CS nanoparticles involves physically mixing two or more polymers to create a new, improved material with altered physical characteristics, including chemical, structural, and biological properties ,168. Somr pylori . The antr pylori . On the r pylori . On the r pylori .2) and hydroxyl (-OH) groups with chemical activities in CS, numerous well-known CS derivatives have been developed by researchers through acylation, alkylation, quaternization, phosphorylation, graft copolymerization, phthaloylation, sulfonation, carboxymethylation, and carboxyalkylation of CS, as shown in N-substitution, where the reaction occurs through the amino functional group of CS [O-substitution of CS, where the reaction occurs through the hydroxyl functional group, is also a common route for chemical modification of CS [O-substitution of CS often requires protection and deprotection of primary amino groups due to the higher reactivity of amino groups than hydroxyl groups [N,O-substitution that is involved in the substitution of both the amino and hydroxyl functional groups of CS will also usually provide an amphipathic property, and at the same time, can be used to enhance the hydrophobic and hydrophilic features of CS [Subsequently, the chemical modification of CS can be achieved by modifying the primary amine groups of CS through chemical, photochemical, radiation, enzymatic grafting, and plasma-induced methods. Due to the presence of the large amount of amino CNP (FA-PEG-CNP) system for the delivery of gemcitabinein (GEM) for lung cancer treatment. The in vitro assessment revealed GEM sustained release properties of up to 10 days while their use in an in vivo tumor-bearing female Balb/c mouse model indicated the distribution of GEM delivered by FA-PEG-CNP system was significantly higher in A549 tumor compared to other organs further outlined the longer residence of GEM in the target organ . BesidesStaphylococcus aureus (MRSA), which is resistant not only to methicillin but also to many other classes of antibiotics and disinfectants, and became the main source of hospital-acquired infections [Multidrug resistance (MDR) was first discovered in bacterial strains upon the discovery of penicillin in 1928 and was followed by a huge number of antibiotics ,223. Onefections ,225. A sfections ,227,228.fections ,230,231.fections ,233. Besfections ,235. Abofections ,237. onefections ,239. Cond-\u03b1-tocopherol succinate prodrug for the enhanced loading and delivery of paclitaxel and against the MDR of tumor cells. This CNP-based system has been shown to be effective at initiating the reversal of MDR through decreasing mitochondrial membrane potential (MMP), inhibiting ATP synthesis, and suppressing P-glycoprotein (P-gp) expression, which are characteristic mechanisms of MDR [mad2 gene in the A549 cell line, which is an essential gene that is responsible for the precise chromosome segregation during mitosis and has served as an alternative in MDR lung cancer treatment. In this context, The previous study of Zhang et al. has established an \u03b1-tocopherol succinate-modified CNP system to encapsulate paclitaxel (PTX)-s of MDR . Apart fs of MDR ,242. Sins of MDR ,244. Mors of MDR . The in Present cancer therapeutic approaches have encountered various circumstances that often resulted in limited therapeutic efficacy. The underlying basis of the failure is multifactorial, comprising short retention time of drugs, non-targeted delivery of drugs, and MDR. CNPs have been proven to be a promising nano-carrier system by previous studies due to their advantageous features in different sectors, especially in the biomedical field, in which they are capable of enhancing the therapeutic efficacy and cellular uptake of several anti-cancer drugs. Noticeably, the development of a CNP-based system has relieved the disadvantages of conventional cancer disease treatment with enhanced therapeutic efficacy and reduced MDR. The CS structure endowed with high flexibility has made it possible for it to be modified and given increased functionalization, overcoming the impediments in current pharmaceutical and biomedical applications. Nonetheless, further ongoing research on CS-related nanobiotechnology should be conducted in order to improve the current medical level and to develop medications with high therapeutic efficacy so as minimize the pain that patients have to endure and at the same time maximize the efficacy of therapeutic drugs."} +{"text": "In recent decades, drug delivery systems (DDSs) based on nanotechnology have been attracting substantial interest in the pharmaceutical field, especially those developed based on natural polymers such as chitosan, cellulose, starch, collagen, gelatin, alginate and elastin. Nanomaterials based on chitosan (CS) or chitosan derivatives are broadly investigated as promising nanocarriers due to their biodegradability, good biocompatibility, non-toxicity, low immunogenicity, great versatility and beneficial biological effects. CS, either alone or as composites, are suitable substrates in the fabrication of different types of products like hydrogels, membranes, beads, porous foams, nanoparticles, in-situ gel, microparticles, sponges and nanofibers/scaffolds. Currently, the CS based nanocarriers are intensely studied as controlled and targeted drug release systems for different drugs as well as for proteins/peptides, growth factors, vaccines, small DNA (DNAs) and short interfering RNA (siRNA). This review targets the latest biomedical approaches for CS based nanocarriers such as nanoparticles (NPs) nanofibers (NFs), nanogels (NGs) and chitosan coated liposomes (LPs) and their potential applications for medical and pharmaceutical fields. The advantages and challenges of reviewed CS based nanocarriers for different routes of administration with reference to classical formulations are also emphasized. The development of polymeric drug delivery systems (DDSs) using the latest nano-technology approaches had gained scientists\u2019 attention in the pharmaceutical field, the most used polymers being those of natural origin such as chitosan, starch, cellulose, gelatin, elastin and alginate . The endChitosan as a promising natural polymer for the formulation of DDSs. Chitosan (CS) is an ideal candidate for DDSs because it is non-toxic, biocompatible, biodegradable, low imunogenic and bioadhesive. Furthermore, its structure is similar to collagen and can be used to mimic the extracellular matrix [d-glucosamine and N-acetyl-d-glucosamine, obtained by alkaline or enzymatic N-deacetylation of chitin, the second most abundant polymer in nature, after cellulose [r matrix . In addir matrix , antiacir matrix . Based or matrix . The CS r matrix ,9. CS isellulose . Chitin ellulose . The hydellulose . CS has ellulose . The posellulose .An important feature of CS is its mucoadhesive ability, which is explained by electrostatic interactions between the positively charged amino groups of polymer chain with the negatively charged mucin glycoproteins residues, rich in sialic and sulfonic acids . CS baseCS, either alone or as composites, are suitable substrates in the fabrication of different types of products like hydrogels, membranes, beads, porous foams, nanoparticles, in-situ gel, microparticles . The forChemical modifications of CS for the development of suitable CS-based DDSs. In order to improve the characteristics of CS, different derivatives were synthesized, based on the chemical reactivity of hydroxyl (primary or secondary) and primary amine groups [60Co source, safe and biocompatible membranes based CS and HEMA (2-hydroxyethyl methacrylate) were also formulated [e groups . Chemicae groups , nanofibers/nanoscaffolds (NFs), nanogels (NGs) and liposomes (LPs), targeting especially oral, transmucosal, pulmonary and transdermal administration . The advThe nanomaterials, especially NPs, NFs, NGs and LPs , due to Nanoparticles (NPs) are attractive DDSs, based on their ability to load therapeutic small drugs, peptides, ribonucleic acids and so forth, being appropriate candidates for controlled drug release at different target sites such as corneal, nasal, transdermal, intravenous and gastro-intestinal mucosa. NPs delivery systems have many advantages such as long-circulation and controlled release of drugs, improved drug solubility and stability, enhanced efficacy and reduced toxicity .Due to their smaller size (average size below 1000 nm) and increased surface area, NPs are characterized by increased selectivity and specificity to a defined target and have the capacity to cross the cell barrier by endocytosis even receptor-mediated transcytosis, which leads to improved release of molecules inside the cells . NPs canThe preparation methods of the NPs include ionic gelation, emulsion, reverse micellar method, coacervation, precipitation, nanoprecipitation and the sieving method ,18. ChitThe formation of CS-NPs is influenced by several parameters such as the concentration, the molecular weight (Mw) and the degree of deacetylation (DD) of CS, the crosslinking agent concentration, zeta potential (ZP) of NPs and so forth. It was reported that by increasing the Mw of CS there is an increase in the viscosity of the polymeric solution, obtaining particles with a larger size ,25. TherThe physical stability of the NPs in solution is measured by ZP, the extreme positive and negative values being correlated with high repulsive forces, which contribute to colloidal stability ,29,30. The most important characteristics of CS-NPs, in terms of particle size, drug loading efficiency (DLE%), drug release (DR%) profile from CS-NPs matrix and zeta potential (ZP) values as well as of CS features, are presented in The electrospun nanofiber scaffolds properties mimic the nanoscale characteristics of the native extracellular matrix (ECM) and provide a specific response, useful for the treatment of different disorders. The advantages of nanofibers are numerous, and in addition to the controlled release of drugs, these systems can improve the solubility and permeability of embedded drugs due to their porous structure and satisfactory surface/volume ratio ,34.The most common and widely used method to produce nanofibers (NFs) is the electrospinning method. This technique is very useful for preparing sub-micron or nano-scale fibers because it offers several advantages; it is a facile and a cost-effective method, it is easy to embed bioactive principles into the nanofibers, and it does not require heating during the obtaining process, which is of key importance principally for thermo-sensitive compounds . The basRegarding the polymeric solution, its viscosity and conductivity affect the diameter and the diameter distribution of NFs in a noticeable way, being crucial factors in the stretching of the charged jet . These pThe electrospinning of CS is a difficult process due to the very high viscosity of the solution in low concentration, caused by its polyelectrolytic nature . Many stRecent data revealed that many therapeutics such as anticancer, antibiotics and bio-actives were successfully loaded into CS based NFs .The most important characteristics of CS\u2013NFs, in terms of drug content (DC) and drug release (DR%) profile from CS-NFs matrix, as well as of CS features, are presented in Nanogels (NGs) are hydrogels in nanoscale, with three-dimensional polymer networks, formed through physical or chemical crosslinking, with an ability to retain a large volume of water and with the advantage of not dissolving in an aqueous environment . The CS\u20192 groups leading to the capability of forming electrostatic interactions and hydrogen bonds between the polymer chains [2 groups can also form covalent bonds in the presence of a chemical linker [2 groups at low pH contributes to the positive charge of CS and to electrostatic interactions with the components of the mucus or with the epithelial surfaces that are negatively charged. These types of interactions provide mucoadhesion properties and sustain the use of CS-NGs as mucoadhesive drug delivery systems. CS has the ability to gel itself to form NGs, without any surfactant, solvent or cross-linker, due the presence of the \u2013OH and \u2013NHr chains ,52. Thesr chains . The \u2013OHl linker . In addiThe CS-NGs can act as carriers, protecting the drugs from degradation and/or elimination. The hydrophilic nature of the CS-NGs limits their use to hydrophilic drugs delivery. The entrapment of lipophilic drugs into the polymeric matrix of CS, in the form of NGs, can take place by the binding of hydrophobic groups, such as alkyl, cholesterol, acyl, 5\u03b2-cholanic acid, cholic acid and deoxycholic acid, to the CS molecule. Furthermore, hydrophobic CS derivatives, such as carboxylate CS and CS sulfate, has an important role in conferring an amphiphilic or amphoteric character, which leads to interactions with both anionic and cationic molecules and also give the ability of self-assembling and water-solubility ,55.The most important properties of the NGs are related to the fact they can be designed as responsive NGs drug delivery . In the The most important characteristics of CS-NGs, in terms of drug content (DC)/drug loading efficiency (DLE%) and drug release (DR%) profile from CS-NGs matrix, as well as of CS features, are presented in Liposomes (LPs) are spherical vesicles formed by an inner aqueous phase protected by one or several concentric phospholipid bilayers. These nanocarriers present some specific features such as: embedding hydrophobic or hydrophilic drugs, ameliorating some properties of the incorporated drugs or protecting the degradation of the substances. LPs can also improve the penetration through tissue, the capacity of site-specific targeting and possess self-assembly property ,68,69.Among the advantages of LPs are biocompatibility, the ability to reduce the side effects of some drugs and improving the pharmacokinetics and drug target sites\u2019 bioavailability. Moreover, LPs are safe because they are similar tothe biological membranes ,68.Related to the particle size and vesicle lamellarity the LPs are unilamellar, multilamellar and multivesicular membranes. The last type is designed for the parenteral route, while the first two ones can be used for various routes, including oral. The quantity of drug embedded is affected by the vesicle bilayers size and thus the half-life circulation can be influenced .The methods by which LPs can be obtained are thin-lipid film hydration, freeze\u2013thawing, reverse-phase evaporation, dehydration\u2013rehydration, solvent injection, double emulsion, fast-extrusion, and detergent-depletion methods. Microfluidic and supercritical reverse phase evaporation techniques have been also used .The composition of LPs can modify the binding ability, the distribution, and the drug release. The most important characteristics of the liposome surface are fluidity, charge, and permeability. The liposomal surface can be changed by conjugation to polymers and/or ligands in order to design more efficient DDSs. The polymer coating can modulate the stability of LPs and so increase the bioavailability, the pharmacokinetic and pharmacodynamics profiles .CS based liposomes (CS-LPs) are often obtained by coating, which allows the change of the liposomal surface properties. It is considered that the CS coating may improve the structural rigidity, the integrity of membranes and the stability through electrostatic interactions. The increasing concentrations of CS can improve the rigidity and stability of LPs because allow the absorption of polymer chains through liposomal surface. When the concentration gets to a saturation point, this effect is maximum and after this value begins to appear some aggregations of CS chains and some not regulated tangles. Thus, it can destabilize the liposomal structure and the membrane fluidity raise .An important parameter for obtaining suitable CS-LPs is the CS/lipid ratio, by increasing this ratio, stable LPs, slightly larger in size and with enhanced storage stability and positive ZP, can be obtained . The DR N-octyl-N-arginine-CS, by grafting hydrophilic arginine groups and hydrophobic octyl groups to the amino group of CS in order to increase the bioavailability [The main disadvantage of CS coating is related with its reduced solubility at physiological pH. The formulations-based CS could also more easily release the embedded drugs, having an important swelling degree in aqueous media . For thilability . Recent lability .These studies evidenced that CS-LPs can be used as nanocarriers in various areas of drug delivery such as nasal, pulmonary, oral, ocular, transdermal and parenteral systems. These delivery systems can incorporate different drugs including peptides, proteins, hormones, anticancer and antimicrobial drugs, vaccines, genes, enzymes, and siRNA . The most important characteristics of CS-LPs, in terms of LPs size, drug loading efficiency (DLE%), drug release (DR%) profile from CS-LPs and zeta potential (ZP), as well as CS features, are presented in Oral administration or per os (p.o.) route is one of the most preferable and convenient means of drug delivery, intended to have a systemic effect. Generally, the traditional oral drug delivery systems have proven to have limited bioavailability caused by several factors, depending of the drugs\u2019 features, such as the fast gastric emptying due to low solubility of the drug in the elevated pH environment, the intense enzymatic degradation of drug at gastro-intestinal tract (GIT) or its degradation in the colon, the absorption of the drug in the upper side of the GIT or short half-life, and respectively narrow absorption window for some drugs ,83. In o2 and \u2013OH active groups of CS, changes that lead to improvement the solubility in intestinal media, the muco-adhesiveness, the drug encapsulation efficiency and the stability of CS [The release mechanism of drugs from the CS matrix consists of swelling of the polymer matrix, drug diffusion through the pores of polymeric matrix, polymer erosion and degradation. In addition, the drugs release from these formulations is dependent on the pH value, so in gastric media (pH 1.6) the release will be more then in intestinal medium (pH 6.5). It is possible to appear an initial burst release, explained through swelling capacity of the polymer and creating pores in polymer matrix, or diffusion of the drug that may exist on the polymer surface ,18. The By development of NPs, the disadvantages of oral drug administration can be overcome, because the NPs prevent the drugs enzymatic degradation and increase the GIT stability of acid-instable drugs. In addition, NPs can adhere to the GIT due to mucoadhesive properties, leading to release the drug for a longer time . To enhance the oral bioavailability of paclitaxel, a new NPs formulation based on CS cysteine and polylysine have been developed. Each component has a certain role; therefore, cysteine can increase the mucoadhesive ability and permeation effect of CS; polylysine, with positive charge, can increase the intestinal permeation of paclitaxel by strengthening the electrostatic interaction with negatively charged integrin receptors to extending the residence time. In vivo therapeutic efficacy of this formulation was performed on a Heps tumor cells mice model .Doxorubicin, an anticancer drug with a broad spectrum, has proven effective in treating some animal and human tumors. Unfortunately, this drug at a high dose causes cardiotoxicity and myelosuppression. Adimoolam et al. synthesiInsulin remains the mainstay of treatment for diabetes and obtaining insulin formulations for oral administration is a highly desired goal. Subcutaneous administration is considered awfully invasive and associated with a lot of risks and the oral bioavailability of insulin is very low due to its high molecular weight, enzymatic degradation and the molecule instability in gastric acid environment. However, the oral delivery of insulin is the preferred route for diabetics because it is easy to administer, imitates physiological insulin release, improves glucose homeostasis and avoids the displeasure of insulin regular injection. Insulin-loaded NPs were obtained via self-gelation with CS and snail mucin. This formulation proved a good NP encapsulation efficiency and a release profile over a period of 8 h . AnotherOlanzapine, a thieno-benzodiazepine derivate, is an antipsychotic from the second-generation with fewer side effects, particularly extrapyramidal symptoms. Recently, olanzapine-loaded NPs have been developed as a possibility to improve the pharmacological and pharmaco-toxicological profile of olanzapine. CS-coated- olanzapine-loaded NPs engineered for oral route by Veragten et al. showed gSeveral studies have been performed to enhance the oral absorption of enoxaparin, an anticoagulant used in various disorders. This drug has a very low oral absorption rate due to its high molecular size, negative charge, high water solubility, low absorption through intestinal wall and first pass effect. The development of solid lipid NPs loaded with enoxaparin-saturated fatty acid conjugates has proven to be a good strategy to improve the oral bioavailability of enoxaparin. Dong et al. develope2 receptor antagonist highly susceptible to microbial degradation and which has variable absorption in GIT and a half-life of only 2\u20133 h. These limitations were minimized by the formulation of a floating GRDDS consisted in tripolyphosphate (TPP)-crosslinked NFs mats based on CS and polyethylene oxide [Ranitidine hydrochloride is an Hne oxide . The in ne oxide .AnjiReddy et al. describeA novel GRDDS systems based on CS-gellan NFs loaded with resveratrol was described as a potential gastro-intestinal cancer long-term prevention and treatment therapy. The encapsulation efficiency of resveratrol in these nanofibers was 86 \u00b1 6% and the drug amount released in the gut was approximately 51%. Regarding the efficacy of this formulation in cancer therapy, studies have shown that resveratrol loaded CS-NFs have almost the same cytotoxicity against HT29 cancer cells compared to free resveratrol. The results regarding in vitro biocompatibility and cytotoxicity studies have shown that NFs formulation can be an excellent drug delivery carrier for resveratrol .NGs are considered to be efficient oral drug delivery systems that have multiple advantages: they can increase the residence time of the drug in the GI tract, protect the active substance from degradation, ensure an efficient release from the polymer matrix and increase the absorption of drugs through GI membranes . UnfortuMost studies on the oral delivery of drugs based CS-NGs refer to a local effect in gastrointestinal disorders, particularly inflammatory or tumor targets. Zhou et al. preparedFeng et al. studied Cao et al. reportedFor the development of a new system for insulin oral delivery, insulin-CS polyelectrolyte complexes associated with lecithin LPs was developed. It was revealed that the stability of insulin was increased by adding the insulin-CS polyelectrolyte complexes to lecithin liposomal dispersion. In vivo results, using a streptozotocin induced rat diabetic model, evidenced a decrease of blood glucose levels, related to subcutaneously administered insulin ,96.CS coated alendronate LPs were developed to improve the bioavailability and to decrease the gastric irritation of alendronate. These systems possessed high muco-adhesive characteristics and presented increased oral bioavailability of alendronate in rats. It was demonstrated that CS coating could increase the drug absorption and could also protect the degradation of LPs by digestive enzymes. Moreover, the liposome system could prevent the appearance of esophageal adverse effects that are related to alendronate oral administration. The results proved that the CS coated alendronate LPs is a more stable and more effective oral drug delivery system .Zhou et al. have preNew based CS-thioglycolic acid coated LPs embedded with paclitaxel and pluronic F127 were developed as stable and sustained-release drug delivery systems. The tests evidenced an increased potency of mucus adhesion, penetration through cell membranes, which lead to an extended residence time in gastro-intestinal tract. This combined drug delivery system proved to be more mucoadhesive and adherent to the intestinal mucosa in comparison with CS-thioglycolic acid coated LPs and could allow the intestinal epithelial cells passage into the systemic circulation in order to be used as nanocarrier for chemotherapy .Novel CS covered ursolic acid (UA) LPs (CS-UA-PLs) were also reported for tumor cells targeting, good, controlled drug release and low side-effects. The studies performed in vitro (HeLa cells) and in vivo demonstrated that this formulation could be used in the localized tumor treatment and to decrease the total drug dose and side-effects. In addition, the CS-LPs presented mucoadhesive features and their oral bioavailability was improved through the prolonged release of the drug at the tumor site .Some polyelectrolyte complexes formed by multilamellar vesicles (MLV) and polymer-inhibitor conjugate were synthesized. Protease inhibitor-aprotinin was covalently bound to CS. Biological evaluation included the determination of serum calcium after oral administration of calcitonin solution and two oral formulations: calcitonin embedded chitosan covered MLV or CS-aprotinin covered MLV. It was observed a more pronounced for calcitonin loaded CS-aprotinin MLV than CS coated MLV .Gradauer et al. evaluateTransmucosal administration offers multiple advantages, compared to the traditional enteral, intravenous or intramuscular routes. CS is an ideal candidate for transmucosal formulations because it has mucoadhesive properties and proved the ability to open the tight junctions between epithelial cells. Therefore, CS can promote the transport of macromolecular drugs and complex molecules such as proteins and peptides . Administration through the buccal route consists of the application of drugs in the oral area in order to absorb and penetrate them into systemic circulation. The research on buccal DDSs had drawn attention because of its advantages: the rich vascularization of the buccal area, the increase of drug bioavailability through avoiding first-pass liver metabolism and the possibility of administering vulnerable substances at the GIT .sublingual mucosa represents a portion of the oral mucosa more penetrable than palatal and buccal mucosa due to the low degree of keratinization and of small mucosal thickness (100\u2013200 \u00b5M). The very high concentration of blood and lymphatic vessels, along with its small thickness, makes it a favorable route for drugs for which rapid onset of action and immediate release are necessary [sublingual route also has some disadvantages, which consist of a small surface area for drug absorption through passive diffusion and also the continuous washout of the drug due to salivary secretions, which limits the drug residence time in the oral cavity [The ecessary . The subl cavity . The coml cavity ,105.The nasal route is another mucosal route with topical drugs administration through which a systemic or a localized effect is achieved. Most of the nasal mucosal surface (~180 cm2) is highly vascularized and the microvilli existence at the apical region of cells increases the surface area for drug absorbance [sorbance . Among tsorbance ,107. Pensorbance .Ocular drug delivery is also a favorite route of administration due to the ease of use and patient compliance. The ocular mucosa represents the layers that cover the conjunctiva and corneal surfaces and is constituted of a complex macromolecular structure of proteins, lipids, DNA and mucin [nd mucin . Ocular nd mucin . The release rate, the required drug loading and the ocular retention time of drug delivery systems depend on the bioavailability, potency and clearance of the drug at the desired site. The limitations of the loading capacity with active substances are due to the properties of the materials used and the limiting size of the absorption surface at the ocular level .co-glycolic acid or alginate), as a dual system, was also used. In vivo tests support the claim that CS contributes to corneal wound repair by enhancing keratinocyte pro healing functions, which leads to rapid onset in collagen synthesis [In the last few years, CS formulations for ocular delivery, especially NPs and LPs, have been extensively researched. CS exerts numerous beneficial properties for ocular administration, such as: high degree of biodegradability with extensive mucoadhesive characteristics and minor immune response as a result of its capacity to link Toll-like receptors. Another useful property of CS for administration to the ocular mucosa is that it can form viscous solutions through water dispersion, preventing drainage after administration. Of the CS derivatives, CS-iminothiolane represents an important category of mucoadhesive polymers that form disulphide bonds between the free thiol groups of the derived polymer and the cysteine residues present in the mucin . For impynthesis ,111. Vaginal mucosa could also be used as a transmucosal route. It is characterized by four different highly vascularized layers with ease of accessibility and which avoid the first pass effect of the drug and enable self-administration [stration ,112. Drastration .The main advantages offered by the transmucosal route compared to the traditional ones are illustrated in N-triethyl-CS for buccal delivery. This formulation protects insulin from harsh conditions in the GIT, increases its residence time on the absorption area and enhances drug penetration through the oral mucosa. Among the advantages of this formulation are a good mucoadhesion and penetration improving the effect of CS through the tiolation process. This effect occurs by creating disulfide bonds between the thiol groups of the chitosan derivative and cysteine sequences present in the mucus glycoproteins. Enhanced mucoadhesiveness can be a good method for surmounting the disadvantages of administration on the buccal mucosa such as mucus turnover, poor available area of oral mucosa for drug absorption, and the drug dilution process by the presence of saliva. An in vivo experiment developed on rabbit buccal mucosa showed excellent insulin permeation through buccal mucosa, the insulin permeability reaching 96% in 480 min [Insulin was prepared as NPs with thiolated 480 min . To incr 480 min . A mucoadhesive CS-NPs entrapping the chemotherapeutic oxaliplatin was engineered by Matos et al. for tumors\u2019 oral mucosa . The appThe nose-to-brain delivery is a new strategy to transport drug loaded NPs through the biological brain barrier. Using this strategy, pramipexole was encapsulated into CS-NPs, to deliver the drug via nose to brain for the treatment of neurological disorders, using the induced rat model of Parkinson\u2019s disease. The cumulative rate of drug penetration through the nasal mucosa for pramipexol CS-NPs was 83.03% \u00b1 3.48 after 24 h. In vivo anti-Parkinson\u2019s activity revealed the brain targeting potential of pramipexol CSNPs, as compared to the results obtained for the solution-type formulation of that drug .co-glycolide (PLGA) and CS-NPs loaded with desvenlafaxine. The results showed that this formulation enhanced levels of neurotransmitters serotonin and noradrenaline considerably compared with the depressed control. Further, it was found high levels of desvenlafaxine both in systemic circulation and in brain. Therefore, the results of this study support the claim that nose-to-brain delivery is an effective way to treat depression and other brain disorders [For enhancing the bioavailability of desvenlafaxine, an antidepressant drug, CS-NPs was developed for nose-to-brain delivery. Nowadays, antidepressants are administered mainly orally, although their therapeutic efficacy is closely related to the ability to cross the blood-brain barrier to reach the central nervous system. This new nose-to-brain drug delivery system consists of polylactide-isorders . Bhattamisra et al. preparedCarboxymethyl-CS was used for obtaining carbamazepine loaded NPs to improve its uptake to the brain via nose to brain delivery. Carboxymethyl-CS is a water-soluble derivative, which is easy to obtain and has an amphoteric character with many possible applications. Carbamazepine has the autoinduction function and an increase of its levels in the liver leads to an increase of CYP3A4 activity leading to a significant increase in drug clearance, conducting to a low half-life. This drug has a narrow therapeutic range (4\u201312 g/mL), so, fluctuations in the concentration of the drug in the blood could lead to many side effects. Following the experiments performed, it was observed that a sufficient amount of drug reached the brain through nose to brain drug delivery, leading to a higher efficacy, with decreasing dose and side effects .In a study conducted by Chhonker et al. , CS-NPs In another study, CS-NPs coated with hyaluronic acid (HA) for dexamethasone release into the eyes was developed. These CS-NPs offer, on the one hand, a sustained topical dexamethasone delivery into the eyes and, on the other hand, protect the drug from effective enzymatic degradation. HA was used to make NPs discrete, free-flowing and to improve their mucoadhesive characteristics . Another NPs formulation based on CS and HA has been developed to increase the bioavailability of erythropoietin, a neuroprotective and neuroregenerative drug, due to its biological properties, such as suppressing cellular apoptosis and reducing inflammation, oxidation and excitotoxicity ,122. EryYu et al. manufactNFs, due to its flexibility and its unique surface properties such as viscosity, mucoadhesive properties and ease of application, offer a good substitute for other mucoadhesive films or patches via buccal route administration for both local and systemic drug delivery . Fast dissolving oral films (FDOFs) based on CS/pullulan NFs were assessed as a buccal drug delivery system. Encapsulating aspirin in the FDOFs for oral mucosal release was performed in order to avoid several side effects of aspirin to the gastric mucosa: ulceration, gastric mucosal erosion, or even gastric perforation. The CS-NFs based FDOFs can achieve uniform distribution of the poorly water-soluble drug-aspirin and the result is a faster dissolution of aspirin due to hydrophilic characteristics of CS. Thermal stability and glass transition temperature of FDOFs were enhanced with the addition of CS and water solubility test showed that the FDOFs can be completely dissolved in water within 60 s . Chen et al. describeAnother substance taken into consideration for a better release and permeation across buccal mucosa ex vivo was insulin, embedded in a structure of chitosan/PEO NFs in HFP . For theAn interesting study was performed on migraine treatment with the two most commonly used drugs, sumatriptan succinate and naproxen salts, incorporated into NFs. In vitro assays performed on these drugs revealed very high dissimilarities regarding the sublingual permeability rates, meanwhile the rates of both substances were augmented several times using CS-NFs, as the drug carrier when compared to drug solutions . Because2), each of the layers with different roles. The mucoadhesive film layer will assure a good grip of the entire system to the oral/sublingual mucosa after application, while the nanofibrous reservoir stratum will function as a stockpile for dendrimers, polymeric and lipid-based NPs, virosomes, virus-like particles or LPs. The nanofibrous reservoir stratum due to its tremendously large surface area allows increased degrees of NP loading, which they can be furthermore either conversely assimilated to the surface of NFs or they can be placed in the pores between the NFs mats. In order to demonstrate this concept, a porcine model was used for the lymph-node delivery of PLGA-PEG NPs trans-/intra-mucosal. The in vivo and ex vivo pig model demonstrated the application of NFs mucoadhesive films as protecting nanoparticle reservoirs used for a controlled and sustained delivery of nanoparticles into draining lymphatic node and submucosal tissue areas. The future applications of these systems could be in the formulation of non-invasive sublingual vaccines and as well in the development of \u201cprinted vaccine technology\u201d, such as influenza and papilloma virus [Ma\u0161ek et al. describema virus .Shigella subunit antigen, N-IpaD (N-IpaD/NFs) as novel intranasal vaccine delivery biodegradable system. The antigen-containing CS nanofibrous mat was formulated by chitosan/AcOH solution electrospinning and it showed an acceptable loading capacity for proteins. The potential of this novel carrier for the nasal delivery of Shigella subunit vaccine was examined on guinea pigs. The guinea pigs intranasal immunized by administration of N-IpaD/NFs indicated elevated mucosal and serum antibody reaction when compared with those of other groups. It was also found that when subjected to wild-type S. flexneri 2a in a keratoconjunctivitis Sereny test the N-IpaD/NFs group was protected. Intranasal administration of encapsulated Shigella subunit antigen in CS-NFs considerably increased the systemic and local immune responses, compared to intramuscular or intranasal administration of soluble Shigella subunit vaccine, which means that the CS-NFs are a promising vehicle for intranasal delivery of Shigella antigens [Jahantigh et al. have preantigens .A transparent and nano-structurally stable biomimetic chitosan/collagen-hyaluronate based nanofibrous membranes as wound dressings for corneal chemical injury treatment, have been also developed . The SEMCS based electrospun NFs mats also represent useful ocular inserts for the delivery of ophthalmic drugs, as shown by Mirzaeei et al. , who devThe drugs delivery via the vaginal mucosa utilizing drug-incorporated electrospun NFs is an innovative strategy and beneficial in cases that needs a sustained local drug delivery . TraditiElectrospun CS based NFs co-loaded with doxorubicin and indocyanine green, as anticancer agents, were developed by Wang et al. . The forBased on the mucoadhesion of CS, Aminu et al. have devC. albicans together with the down-regulated Rim101 and SAP6 genes expression was improved [Nikoomanesh et al. designedimproved .A recent study combined the advantage of CS-NGs, in terms of simplicity of application and the extended remanence time with the significant permeation power of surfactant (Span 60) based nanovesicles, in designing of a new formulation of acetazolamide-nanovesicles loaded onto CS-NGs, for ocular delivery. The CS-NGs exhibited good muco-adhesion time, less irritant action and an increased and significantly prolonged release profile for acetazolamide (only 38.3% after 6 h) compared with acetazolamide-nanovesicles formulation, where the drug was released rapidly (64.9% after 6 h) .New intranasal LPs loaded with fexofenadine to treat allergic rhinitis have been developed . It was C. albicans [Various retinal diseases (including macular edema) could be conducted to permanent retinal structural damage and threatens vision. High biocompatibility, non-immu -nogenicity, corneal penetration and prolonged clearance times are the main advantages of LPs as drug carriers for ocular application. A new eye drop formulation based on CS-LPs, able to release triamcinolone acetate to the retina as non-invasive and safe drug administration system, has been developed . A good albicans .Pulmonary route has gained outstanding interest as a potential approach to obtain both local and systemic effects. The advantages of the pulmonary route include: rapid onset of action, high efficacy alongside with the lack of first pass metabolism. The fast onset of therapeutic action is due to the thin absorption barrier, high tissue vascularity and large surface area of the lungs. For inhalation drugs to reach the systemic circulation they must overcome certain impediments: the alveolar lining fluid, proteolytic degradation, lung surfactant, epithelial cells and macrophage clearance. In the last years the inclusion of drugs in nanocarriers for inhalation administration becomes an encouraging research direction in order to increase the local bioavailability and aerosol performances .CS-NPs have been vastly investigated as a carrier for lung drug delivery, due to mucoadhesive properties, a high adherence to the lung mucosa, their capacity to open the intercellular tight junctions of the lung epithelium with the improving of drug uptake. CS-NPs loaded with rifampicin, an antitubercular drug, were formulated by ionic gelation for direct targeting to the lungs, in order to increase efficacy and to scale down the side effects of rifampicin . In vitrFor lung cancer therapy, siRNA is also an attractive strategy that can be administered both intravenously and through inhalation. The bioavailability of siRNA when administered by the lung is low because of the intracellular barrier and the development of new polymer carriers of siRNA for pulmonary administration is a real challenge. Ni et al. developeHeparin, a linear anionic polysaccharide, has an anticoagulant effect, which exhibits poor bioavailability when orally administered. Currently, it is administered via the parenteral route, but the pulmonary route has drawn notable attention as a potential approach to delivering useful quantities of the anticoagulant. Heparin-loaded CS-NPs were formulated for systemic delivery upon pulmonary administration. Both the size and encapsulation efficiency of heparin loaded CS-NPs depended on the acidic or neutral media in which they were prepared. Thus, those prepared in acidic media had a size of 156 nm and encapsulation efficacy of 100% and those prepared in neutral environment had the size of 385 nm and encapsulation efficacy of 43%. The results of in vivo assay emphasized that the formulation of heparin loaded CS-NPs was efficient at delivering heparin in the bloodstream after pulmonary administration and those prepared in neutral conditions resulted in a notable prolongation of the coagulation time in comparison to the control .max 4302.52 \u00b1 234.12 \u03bcg/g), compared with conventional dry powder inhaler formulation (Cmax 3384.13 \u00b1 490.30 \u03bcg/g)) and oral drug solution (Cmax 2602.57 \u00b1 308.91 \u03bcg/g). The chronic toxicity assay in various organs also corroborated a better safety profile of CS-NPs loaded with bedaquiline [Bedaquiline is a novel anti-tuberculous (anti-TB) drug, a diaryl-quinoline compound that acts via a new mechanism by inhibiting the enzyme necessary for energy generation in bacteria (proton pump of ATP synthase) . In ordeaquiline .Rifampicin loaded chitosan-coated liposomes (CS-LPs) were developed by Zaru et al. for drugN-acetylcysteine were also prepared in order to obtain a formulation with prolonged and controlled release of the drug to the lung by inhalation. The in vitro drug release test and in vivo bio-distribution of CS-coated and uncoated LPs were also studied. A good deposition of CS-coated LPs was observed in the lung site that proved the efficacy for pulmonary drug delivery [CS-coated LPs loading delivery .The transdermal route is widely studied because it has many advantages, including boosted patient compliance, tissue targeting, increased drug release, eluding the first pass metabolism, protecting drugs from gastrointestinal enzyme or acidic environment of the stomach and so forth. A great challenge of this route of administration is the penetration of the drug through the stratum corneum, which acts as a skin barrier . TransdeAmong TDDSs, NPs take an important place based on their advantages such as increased drug encapsulation capacity of hydrophilic and hydrophobic drugs, high drug release, proper penetration across the skin barrier due to the small particle size, steady and prolonged drug release at a pre-determined rate. The three pathways for the penetration of NPs through the skin are the inter-cellular lipid route, the trans-cellular route and the follicular route. The last one seems to be the most important permeation pathway for drug delivery as a consequence of their proximity to the capillary vessels . CS thro3 and steady-state fluxes (Jss) 5.14 \u00b1 (\u03bcg/cm2\u00b7h), which means that CS-NPs has the ability to efficiently deliver curcumin through the corneum stratum [A recent study reported the encapsulation of lipophilic curcumin, well known for its anti-inflammatory, antioxidant, and antitumor effects, into CS-NPs for transdermal delivery. An in vitro experiment showed curcumin loaded CS-NPs having a high permeability coefficients (kp) 10.278 (cm/h) \u00d7 10 stratum . Al-Kass stratum have pre stratum . Pirfeni stratum .NFs, due to their multi-functional characteristics, offer great potential to improve/enhance localized drug delivery. The development of a novel NFs wound dressing with multi-functional properties, electro-activity, appropriate mechanical characteristics, antioxidant and antibacterial action in order to stimulate the wound healing process is desirable for the increasing requirements of clinical needs in the management of wound care . Recent studies report the development of electroactive NFs membranes with antioxidant and antibacterial activity, using electrospinning method of quaternized chitosan-graft-polyaniline (QCSP) and poly(\u03b5-caprolactone) (PCL) polymer solutions as wound dressing materials. The most important results were proved for QCSP15/PCL (15 wt% of QCSP in sample), which presented a balanced capacity between cell proliferation and antibacterial properties, resulting in a substantially accelerating the wound healing process in a mouse full-thickness wounds defect model in comparison with control positive sample and control negative sample (QCSP0/PCL nanofibrous membrane). The same formulation QCSP15/PCL nanofiber was also noted after histopathological analysis and immunofluorescence staining, where it exhibited more angiogenesis, granulation tissue thickness and higher collagen deposition .E. coli and S. aureus were analyzed. The in vivo biological evaluation using wound model induced on specific pathogen free grade KM mice demonstrated that CMCS-OH-30-NPs induced a smoother surface coating, a more uniform epithelial surface thickness, and a more sustained connective tissue orientation in comparison with the control. These results revealed the dual beneficial wound healing and antibacterial properties of the developed NFs and can have a great potential for improving wound management with less sequelae [Zou et al. reportedsequelae .Chamomile extract with the antioxidant, antibacterial and promoter properties of epithelial regeneration in order to develop novel biocompatible multi-layered nanofibrous patches. The composition of this nanofibrous formulation consists of 3 layers: the first outer layer of poly (\u03b5-caprolactone) (PCL), the second layer composed of hybrid nanofibers of Chamomile/carboxyethyl chitosan/polyvinyl alcohol (Chamomile/CECS/PVA) and the Chamomile loaded CECS/PVA as the third interior layer. The nanofibrous multi-layered formulation is very beneficial for wound dressings because the hydrophilic Chamomile loaded CECS/PVA nanofibrous layer allows good contact with the wound, while the hydrophobic PCL nanofibrous layer provides the strength required for the electrospinning process. The mats showed satisfactory tensile strength (8.2\u201316.03 MPa) and antioxidant characteristics against DPPH radical (6.60\u201338.01%). The antibacterial efficiency against E. coli and S. aureus demonstrated values of inhibition zones directly proportional with the concentration of the Chamomile content and a better antibacterial effect than the commercial wound dressing Ag coating. The mechanism of release from the formulated NFs was Fickian diffusion-controlled, with a sustained release of Chamomile available for 336 h. An MTT assay revealed good cell viability for all mats except one contained 30 wt% Chamomile. Based on the findings from this study, the chitosan multi-layered electrospun nanofiber formulation with a 20 wt% Chamomile content can be appropriate for wound dressings, because of its biocompatibility, antioxidant, antibacterial and mechanical properties [The incorporation of natural products with well-known anti-oxidant and antibacterial effects in the NFs electrospun structure is also one of the concerns of scientists. Such a study was recently carried out by Shokrollahiet al. , which uoperties .The recent application of CS for the production of electrospun membranes with biomedical application are depicted in ternary CS-ibuprofen-gellan NGs enhanced the ibuprofen release thru the skin by ameliorating skin penetration, skin retention, percutaneous drug delivery due to the conversion of crystalline drug into amorphous particles and due to the particle size diminution [The small size, large surface area and swelling properties makes from NGs a perfect system for the efficient delivery of drugs in healing burn wounds. El-Feky et al. reportedminution .The research of Mengoni et al. revealedLee et al. developeGene delivery opens up a promising direction in the treatment of various fatal diseases, especially cancer. Gene delivery systems are designed to target a specific cell, to enhance the delivery of genes and to provide a sustainable-release of the gene. The formulation of the gene delivery system must also ensure protection of the gene from nuclease degradation and escape capability from the lysosome, and must have low cytotoxicity and high transfection efficiency . The delsiRNA was formulated as CS-NPs to protect siRNA from the gene segmentation enzymes, from gastric breakdown and transport it to the tumor cells through an intracellular compartment . In the Recently, by chemical attachment of folate-conjugated poly(ethyleneglycol) (PEG) to glycol-CS-NGs, the nanomaterials obtained with an average size environ of 200 nm, were able to avoid the phagocytosis by macrophages due to the PEG, also having increased time to circulate in the bloodstream . The ligAnother tumor target for gene delivery is the protein that is indispensable for tumor growth and metastasis, the vascular endothelial growth factor (VEGF) . Liu et Based on the cumulative efficiency of siRNA, pH-sensitive CS liposomes for co-delivery of sorafenib (Sf) and siRNA were developed as new antitumor therapeutic systems. Tumor accumulation and antitumor activity were evaluated by the Kunming mice H22 cells-bearing tumor model. The results evidenced that this system developed a high Sf release and siRNA tumor accumulation compared to blank siRNA. A lower toxicity and an increased Sf accumulation were also noted in tumor cells .In the cancer therapy, the release of anticancer drugs precisely and specifically at a target sites is still a challenge. The design of stimuli-responsive NGs provides hope for controlling drug release at tumor tissues. The pH values differentiation between extracellular medium of the tumors (pH 6.5\u20137.2), the normal tissues and blood (pH 7.4) and the pH values of lysosomes and endosomes (pH 5.0\u20136.5) supports the research on pH-responsive NGs development. Several cancer drugs such as doxorubicin ,187, 5-fN-vinylcaprolactam), known as thermo-response polymer. The magnetic nanoparticles based on iron oxide, encapsulated into the hybrid NGs provide increase of local heat in presence of magnetic field. The in vitro drug release study demonstrated that the magnetic field produced hyperthermia correlated with significantly higher drug release (\u223c73%). In addition, the amount of DOX could be controlled by mode ON/OFF trigger of the alternating current magnetic field. DOX release was also considerably higher (\u223c58%) at pH 4.5 and above 43 \u00b0C within 1 in comparison with all other release conditions [A pH-responsive glycol CS-NGs grafted with functional 3-diethyl-aminopropyl (DEAP) groups loaded with doxorubicin (DOX) were developed. The NGs was stable at physiological pH and at slightly acidic pH condition in presence of non-small lung carcinoma A546 cells was imbalanced as a result of the DEAP protonation. DOX release was accelerated, which increased its uptake in the tumor cells . Indulek, 37 \u00b0C) .N-isopropylacrylamide) loaded with oridonin exhibited a higher antitumor activity than drug-loaded NGs without galactosylation, and the anticancer action increases in direct proportion with the number of galactose moieties of the NGs in HepG2 cells. These NGs enhance the uptake of oridonin into HepG2 cells via asialoglycoprotein receptor-mediated endocytosis [Another CS-NGs stabilized with plutoniu 127 was designed for a controlled and sustainable release of bleomycin, targeting the skin cancer. This CS-NGs showed a high entrapment efficiency for bleomycin (55%) with an insignificant release profile at normal skin cell pH but with an enhance release at moderately acidic medium (pH 5\u20136) and with a sustained release profile during 24 h . The subocytosis .Liang et al. have repPaclitaxel loaded chitosan and acylated(myristoyl and octanoyl) chitosan coated liposomal formulations were reported by Nanda et al. . It was Nanotechnology has attracted considerable interest from scientists in biomedical and pharmaceutical applications and, with the advances in biopolymer science, a large number of multifunctional nano-materials based on CS were formulated as DDSs. CS as a biocompatible, biodegradable, non-toxic polymers and with interesting biological properties plays a key role in the development process of new controlled and targeted DDSs. This article targeted the essential characteristics of CS-based nano-materials used as nanocarriers in the drug delivery medicinal area, such as oral, transmucosal, pulmonary, transdermal or targeted drug delivery. For a better understanding of the release of various active substances incorporated into the reviewed CS based formulations , a description of the particularities and advantages of the specific route of administration was made. The advantages or the draw-backs of these CS multifunctional nano-materials acting as drug delivery systems or targeted drug delivery systems were also highlighted. Despite the potential progression achieved in the nanotechnology domain, CS based nano-formulations have not yet made an efficient transition into the market, highlighting the necessity for further research to improve the existing shortcomings. There is the need to test the new developed chitosan nano-mats in suitable animal models and to make the comparison with the products that already exist in the pharmaceutical market in order to demonstrate the suitability for clinical trials. Another idea to be deepened in the future is that of scaled-up nanotechnologies for the cost-effective and industrial-scale fabrication of nano-formulations suitable for the delivery of drugs through different mucosa."} +{"text": "Artocarpus lakoocha Roxb extracts against methicillin-resistant Staphylococcus aureus (MRSA) were evaluated, with its biological properties including antioxidant activity and total phenolic content also measured. Heartwoods of A. lakoocha Roxb were extracted by solvents including water, ethanol, acetonitrile, ethyl acetate, isopropanol, and hexane. Results showed that antibacterial activity against MRSA, antioxidant activity, and total phenolic content were highest in the acetonitrile extract. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 312.5 and 625.0\u2009\u03bcg/mL, respectively. Time-killing evaluation indicated a bactericidal mode of action with IC50 values against ABTS and DPPH radicals of 2.37\u2009\u00b1\u20090.09 and 32.10\u2009\u00b1\u20090.74\u2009mg/mL, respectively, and total phenolic content 455.29\u2009\u00b1\u200918.35\u2009mg GAE/g extract. Results suggested that acetonitrile extract of A. lakoocha Roxb had good potential activity against MRSA, with promise for further development as a novel alternative drug.Antibacterial activities and mode of action of Staphylococcus aureus (MRSA) that causes high rates of morbidity and mortality [Drug-resistant bacteria are a serious problem worldwide. Some bacteria have developed the ability to survive in the presence of drugs that were designed to kill them , 2. Peoportality . In the ortality . Overuseortality , poor saortality , povertyortality , and intortality are all ortality , 2.Plants have long been recognized as valuable sources of primary human health care offering a vast diversity of secondary metabolites including alkaloids, anthraquinones, carotenoids, flavonoids, tannins, lignin, and phenolic compounds with relatively smaller quantities than primary metabolites . These bArtocarpus lakoocha Roxb is a perennial tree belonging to the Moraceae family. This traditional herbal medicine is found in tropical areas such as Thailand, Malaysia, and India [Artocarpus leaves showed antimicrobial and antioxidant activities [A. lakoocha extract showed antityrosinase activity and is used as a whitening agent in cosmetics [A. lakoocha Roxb has proven antimicrobial activity against several pathogenic bacteria but knowledge of its inhibitory effect on drug-resistant bacteria remains limited. Therefore, here, we (1) evaluated anti-MRSA activity of A. lakoocha Roxb extracts, (2) investigated the mode of action of A. lakoocha Roxb extracts against MRSA, and (3) examined the antioxidant activity and total phenolic contents of the extracts.nd India . It is cnd India . For exand India , 16, whitivities , 18. Morosmetics . A. lakoStaphylococcus aureus ATTC 43300 was used as the indicator strain. Nutrient broth and agar powder at bacteriological grade were purchased from HiMedia, India. Ethyl acetate, isopropanol, and hexane at AR grade were purchased from KemAus, Australia, while ethanol and acetonitrile at AR grade were purchased from Merck, Germany.Methicillin-resistant A. lakoocha Roxb was purchased from a Thai traditional drug store in Bangkok, Thailand. The powder was extracted by solvents with different polarity including water, ethanol, acetonitrile, ethyl acetate, isopropanol, and hexane at a ratio of 1\u2009:\u200910 (powder\u2009:\u2009solvent) via the maceration technique for 24 hours in an incubator shaker at 150\u2009rpm. The solutions were filtrated through Whatman filter papers and concentrated by a rotary evaporator. The extracts were weighed and stored in airtight bottles at 4\u00b0C for further study.Dried powder of heartwoods from A. lakoocha Roxb extracts against MRSA. Briefly, MRSA was grown in nutrient broth at 30\u00b0C for 16\u201318 hours to obtain a bacterial concentration of 108\u2009CFU/mL. Sterile discs (6\u2009mm diameter) were placed on solidified nutrient agar, covered with 100\u2009\u03bcL of MRSA. Ten microliters of A. lakoocha Roxb extracts were dropped onto the sterile discs and the nutrient agar plates were incubated at 30\u00b0C for 24 hours. Antibacterial activity was determined by measuring the inhibition zone diameter (mm). All experiments were conducted in triplicate.Agar-disc diffusion was used to screen for the antibacterial activity of A. lakoocha Roxb extracts. One hundred microliters of MRSA were added into the mixtures. The 96-well plates were incubated at 3\u2009C for 16\u201318 hours. After that, 30\u2009\u03bcL of 0.02% resazurin was added into each well and the 96-well plates were further incubated at 30\u00b0C for 16\u201318 hours to obtain the MIC. The MIC was observed as no color change of resazurin, indicating the lowest concentration of A. lakoocha Roxb extracts with inhibitory property against the indicator strain. Then, 100\u2009\u03bcL of the mixture in each well was plated on solidified nutrient agar and incubated at 30\u00b0C for 24 hours to obtain the MBC. The MBC was observed by no growth on nutrient agar, indicating that 95% inoculation was killed. The MBC was defined as the lowest concentration of A. lakoocha Roxb extract with bactericidal activity against the indicator strain. All experiments were conducted in triplicate.Minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) were determined using the resazurin microtiter assay with some modifications . One hunA. lakoocha Roxb was carried out by following the procedure of Appiah et al. with some modifications [A. lakoocha Roxb at twice the MIC was prepared and then the MRSA culture at a concentration of 106\u2009CFU/mL was added. The mixture was incubated at 30\u00b0C in an incubator shaker at 150\u2009rpm. An aliquot of 100\u2009\u03bcL of the medium was removed at time intervals of 0, 1, 2, 3, 4, 5, 6, 7, and 8 hours after innoculation. Growing cells of MRSA in nutrient broth without A. lakoocha Roxb extract were used as the control. The number of visible cells was recorded by plating on nutrient agar. All procedures were performed in triplicate and a graph of log10 (CFU/mL) was plotted against time.The time-killing curve of the acetonitrile extract from ications . The aceA. lakoocha Roxb extracts was examined by the ABTS radical cation decolorization method with some modifications [A. lakoocha Roxb extracts and ABTS cation radicals were mixed and incubated in darkness at room temperature for 30\u2009min. Absorption was measured at a wavelength of 734\u2009nm. The antioxidant effectiveness of A. lakoocha Roxb extracts was calculated using the formula:B and AbsA are the absorbances of the mixture without A. lakoocha Roxb extract and with A. lakoocha Roxb extract, respectively. Linear regression of antioxidant effectiveness was used to determine the concentration of A. lakoocha Roxb extract that could reduce the concentration of ABTS radicals by 50% (IC50).Free radical scavenging activity of ications . The ABTA. lakoocha Roxb extracts was examined by the DPPH radical method, with some modifications [A. lakoocha Roxb extracts, and then incubated in darkness at room temperature for 30\u2009min. Absorption was measured at a wavelength of 517\u2009nm. The antioxidant effectiveness of A. lakoocha Roxb extracts was calculated using the formula:B and AbsA are the absorbances of the mixture without A. lakoocha Roxb extract and with A. lakoocha Roxb extract, respectively. Linear regression of antioxidant effectiveness was used to determine the concentration of A. lakoocha Roxb extract that could reduce the concentration of DPPH radicals by 50% (IC50).Free radical scavenging activity of ications . BrieflyA. lakoocha Roxb extract was mixed with 10% Folin\u2013Ciocalteu reagent and incubated at room temperature for 10\u2009min. Then, 7.5% sodium carbonate was added and the mixture was incubated for 60\u2009min at room temperature. Absorption was evaluated at a wavelength of 765\u2009nm. Total phenolic content was determined by comparison with the gallic acid standard curve and expressed as mg gallic acid equivalent (GAE)/g extract.Total phenolic contents were determined by the Folin\u2013Ciocalteu method with some modifications . First, \u03b1\u2009=\u20090.05.Statistix version 8.0 was used for statistical analysis. Data were expressed as mean\u2009\u00b1\u2009standard deviation. Measurements with normal distribution were analyzed using one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) with a significance level of A. lakoocha Roxb extracts were different according to type of extraction solvent used. Highest yield was recorded in the water extract, with the lowest as the hexane extract with a 5-fold difference (Yields of fference .A. lakoocha Roxb extracts against MRSA are summarized in A. lakoocha Roxb extracts showed different MICs and MBCs. The acetonitrile extract exhibited highest activity against MRSA with lowest MIC and MBC values at 312.5 and 625.0\u2009\u03bcg/mL, respectively. By contrast, MRSA growth was not affected by the highest concentration of the hexane extract, which clearly indicated no anti-MRSA activity.The MICs and MBCs of A. lakoocha Roxb with the lowest MIC and MBC values, indicating highest MRSA inhibitory activity, was selected to study its mode of action. Cell viability of MRSA grew continuously over time, while a dramatic reduction in cell viability was found when MRSA was treated with the acetonitrile extract. MRSA was completely destroyed within 4\u2009hours after incubation by the acetonitrile extract (The acetonitrile extract of extract .A. lakoocha Roxb extracts and their total phenolic contents. The acetonitrile extract showed the best scavenging activity against ABTS and DPPH radicals with lowest IC50 values at 2.37\u2009\u00b1\u20090.09 and 32.10\u2009\u00b1\u20090.74\u2009mg/mL, respectively. Total phenolic content of the acetonitrile extract was also highest at 455.29\u2009\u00b1\u200918.35\u2009mg GAE/g extract. Other extracts had different antioxidant activities and total phenolic contents, with the hexane extract exhibiting lowest antioxidant activity and total phenolic content.A. lakoocha Roxb. Acetonitrile, as the optimal extraction solvent, gave the highest antibacterial activity, antioxidant activity, and total phenolic content. This finding was supported by Shanmughapriya et al. and Lima-Filho et al. who explained that the type of extraction solvent impacted the biological activities and plant-deviated constituents in crude extracts. Organic solvents usually demonstrated higher efficiency in extracting active compounds for antimicrobial activities compared with water-based methods [Results revealed that the type of solvent extraction played an important role on the isolation of bioactive compounds from methods , 25.A. lakoocha Roxb extracts, apart from the acetonitrile extract, were about 4 but the MBC/MIC ratio of the acetonitrile extract was interestingly lower than 4. This finding indicated the bactericidal effect of the acetonitrile extract of A. lakoocha Roxb against MRSA. The mode of action as bactericidal of the acetonitrile extract of A. lakoocha Roxb against MRSA was also confirmed by the time-killing assay. Within 4 hours after inoculation, MRSA growth was completely destroyed, strongly indicating that the acetonitrile extract of A. lakoocha Roxb exhibited bactericidal action against MRSA.Results of antibacterial screening by agar-disc diffusion and MIC and MBC values by the broth dilution assay concurred well, indicating the potential anti-MRSA activity of the acetonitrile extract to diffuse into agar and distinguish bactericidal and bacteriostatic effects. Modes of action for antimicrobial agents are defined as bactericidal or bacteriostatic, based on the ratio of MBC/MIC. When the MBC/MIC ratio is \u22644, this is defined as bactericidal with bacteriostatic effect at >4 , 26. In A. lakoocha Roxb exhibited anti-MRSA activity and also demonstrated potential as a good source of antioxidants and phenolic compounds. The antibacterial agents, antioxidants, and phenolic compounds available in the acetonitrile extract of A. lakoocha Roxb tended to have hydrophilic properties and preferred to dissolve in polar solvents when compared with hexane. This result was supported by several studies indicating that A. lakoocha Roxb was a very good source of antimicrobial agents, antioxidants, and phenolic compounds. Common extraction solvents used to obtain extracts with antimicrobial and antioxidant activities and phenolic content were ethanol, methanol, ethyl acetate, and acetonitrile [The acetonitrile extract of onitrile , 14\u201319.A. lakoocha Roxb occurred as a result of the synergistic manner of active components available in the acetonitrile extract. This was an advantage of using crude extract that battled pathogenic bacteria. Due to several active components, bacteria cannot easily resist against a combination of active compounds. By contrast, synthetic antibiotic usually presents antibacterial properties by causing the bacteria to develop resistant mechanisms. This explanation was supported by results from the ethanolic extract of cinnamon bark and honey [The significant anti-MRSA property and antioxidant activity of nd honey . The comnd honey . They alnd honey . The degnd honey .in vitro inhibitory activities of A. lakoocha Roxb extracts against MRSA. The acetonitrile extract showed significant anti-MRSA activity and highest antioxidant activity and total phenolic content with MIC and MBC values of 312.5 and 625.0\u2009\u03bcg/mL, respectively. Its mode of action was also defined as bactericidal. Results suggested that the acetonitrile extract of A. lakoocha Roxb showed high potential inhibitory activity against MRSA and could be further developed as a novel alternative drug.Our investigations focused on evaluating the"} +{"text": "Correction: Cellular & Molecular Biology Letters (2021) 26:46 10.1186/s11658-021-00290-9.Following publication of the original article , we haveThe affiliation group has been updated above and the original article has been corrected."} +{"text": "The purpose is to explore new cultivation modes of college students\u2019 national cognition and cultural acceptance. Deep learning (DL) technology and Educational Psychology theory are introduced, and the influence of art journal reading on college students\u2019 national cognition and cultural acceptance is analyzed under Educational Psychology. Firstly, the background of Educational Psychology, national cognition and cultural acceptance, and learning system are discussed following a literature review. The DL technology is introduced to construct the journal reading guidance system. The system can provide users with art journals and record the user habits like reading duration and preferences. Secondly, hypotheses are proposed, and a questionnaire survey is designed, with 12 specific indicators to investigate and collect research data. Finally, the collected data are analyzed. The results show that women\u2019s cognition of Chinese traditional culture, Chinese excellent revolutionary culture, and Chinese national identity is higher than that of men. By comparison, men\u2019s cognition of Chinese advanced socialist culture is higher than women\u2019s. After using the journal reading guidance system, the cognition of female college students on traditional Chinese culture is improved by 16.3%. Before and after reading art journals, the overall national cognition and cultural acceptance of Minority students are higher than that of Han students. The overall cognition of Literature and History students is higher than that of Science and Engineering students in traditional Chinese culture and China\u2019s excellent revolutionary culture and lower in advanced Chinese socialist culture and Chinese national identity. The overall cognition of college students\u2019 party members to the advanced socialist culture is higher than league members. As students read more art journals through the guidance system, their overall national cognition and cultural acceptance have increased. Therefore, reading art journals can promote college students\u2019 national cognition and cultural acceptance. A national cognition and cultural acceptance promotion system that conforms to the current situation of college students is constructed. The finding provides a reference for developing complex emotion recognition technology in human-computer interaction. The 21st century marks improved spiritual wellbeing and higher demand for the comprehensive personality of humanity. As a result, more attention has been paid to quality education . StudyinLiterature Art Studies and Hundred Schools in Arts explore literature, drama, film and television, and plastic arts. The decoration is famous in the field of arts and crafts. Also, there are Art Research, New Arts, Art Magazine, and Journal of Nanjing Arts Institute (Fine Arts and Design) in the field of the basic theory of fine arts. Journal of Beijing Dance Academy discusses dance theory. Chinese Music, Music Research, Musicology in China studies music research theory. Journal of College Chinese Traditional Opera and Theatre Arts focuses on drama research. The magazine Art and Design Research targets fashion design. Chinese Calligraphy is designed for calligraphy research. Film Art can be cited for film research. National Arts Bimonthly and Ethnic Art Studies are frequently updated for national art research . Secondly, a questionnaire survey (QS) on college students\u2019 national cognition and cultural acceptance is designed. The data on college students\u2019 national cognition and cultural acceptance under the influence of art journal reading is collected. The collected data are analyzed by Statistical Product and Service Solutions (SPSS) 25.0. Finally, the art journal reading guidance system is constructed. Innovatively, this work concretizes national cognition and cultural acceptance into four indexes to facilitate measurement. The research results have a positive effect on enhancing college students\u2019 national cognition and cultural acceptance and improving college students\u2019 national self-confidence.Mental health mainly studies people\u2019s cognition. Cognitive activities are the basis and origin of all people\u2019s psychological activities . CognitiHere, cognition is defined broadly, including individual cognitive activities, such as memory, feeling, thinking, and social cognition. Individual cognitive activities may differ greatly. The cognitive structure is also known as the archetype, which refers to an individual\u2019s existing cognitive experience . When anIn terms of individual psychological structure, people\u2019s response to external stimuli is always based on a certain psychological structure, namely, a specific experience. The analysis of the human brain\u2019s advanced psychological activities shows that memory, feeling, thinking, and perception are basic human cognitive processes. People understand the objective world, selectively accept the environmental information, and respond to various stimuli through cognition. Besides, people accept and process external information by cognition. Human cognition includes the cognition of various objects in the natural environment and the cognition of various social phenomena, people, and society. People\u2019s cognitive activities are based on previous experience and are the response to objective reality. In this case, groups and individuals with different living environments and life experiences have different cognitive styles. For example, due to geographical diversity and life experience, Chinese people may have different national cognition and cultural acceptance. People also show different psychology in national cognition and cultural acceptance. However, the core emotion brought by national cognition and cultural acceptance is the same: self-confidence. The designed learning guidance management system belongs to the learning system. The learning system can be divided into the Mobile learning (M-learning) system, personalized learning system, adaptive learning system, ubiquitous learning system, and collaborative learning system.Department of Education planned and launched the mobile education scientific research project, established in the Human-Computer Interaction (HCI) research room of Kyushu University at Berkeley. The M-learning project led by European and American countries has solved the unequal distribution of national educational resources. The project uses a minimalist counting method to promote learning information and learning experience to a large number of learners. More importantly, it allows learners to participate in learning for life. Based on the M-learning project, British researchers have analyzed the learning characteristics of some teenagers in Europe. According to the learning behavior habits of these learners, they have developed an education site that facilitates teenagers\u2019 mobile learning. To stimulate their interest in learning, researchers pay more attention to the fashionable and popular materials teenagers are interested in when selecting learning content. Domestically, research on M-learning projects has started relatively late. Peking University launched an M-learning project at the beginning of the 21st century. Domestic Internet giants have entered online education and changed the traditional education model algorithms, such as computer vision and deep reinforcement learning (DRL), are employed.The visual geometry group (VGG) is an effective deep network framework chosen to identify students\u2019 evaluations. DRL requires frequent interaction between agents and the environment. Specifically, when an agent completes an action, it gets a certain reward. To get an optimal action, the agent must consider the long-term benefits of multiple action sequences after the action to maximize the future reward. Therefore, the intelligent agent keeps trying, interacts with the environment, and gradually improves its own strategy. The problem of intelligent guidance belongs to some observable reinforcement learning problems. In some reinforcement learning models, agents can fully observe the current environmental state. This is not in line with the scenario of the proposed intelligent reading guidance system. Students\u2019 learning is adjusted through the observation of students\u2019 learning results. In other words, the proposed intelligent guidance system here is a partially observable reinforcement learning problem.The development platform of the proposed learning guidance system includes an open-source training system of MIT, the ML algorithm library Scikit-learn, the distribution version of Python: Anaconda integrating multiple scientific computing packages, and the reinforcement learning algorithm library Rllab, and the ML framework TensorFlow.Based on the previous content, this section constructs a journal reading guidance system for college students\u2019 national cognition and cultural acceptance, as shown in Here, the core of the learning guidance system: the recommendation module, is mainly introduced. The selected open-source exercise system is a learning guidance system with browser-server architecture, which interacts with users through web browsing. The browsing results of students need to be checked before recommendation. Questions will be set at the end of each chapter of the journal. The check module scores the student-submitted reports and stores the scores in the log and context data structure for the recommendation module. Then, the system will recommend the next journal based on the report evaluation. The recommendation module evaluates the students\u2019 historical information before pushing another art journal. In this part, tutor.py is modified, and the handle_recommend function is added, which calls the evaluation function based on the latest student reading records to calculate and recommend the following journal. The following paper will design a QS to compare students\u2019 national identity differences before and after using the proposed learning guidance system.A QS is designed through prediction and expert evaluation based on domestic and international research theories. The QS includes the basic situation of college students and their understanding of Chinese traditional culture, Chinese excellent revolutionary culture, and Chinese advanced socialist culture before and after reading the art journal.Here, 200 students, with 50 from each of the four grades, are selected. The specific division is shown in Here, Southeast University is selected as the study area. For more significant research results, non-art major students from the regular university who seldom read art journals are recruited. Based on the importance of online learning in teaching proposed by The QS as includesT , P-value (test result), F (variance test), and Sig. (significance). P-value > 0.05 indicates no significant data difference. 0.05 \u2265 P-value \u2265 0.01 indicates a significant data difference, and P-value < 0.01 indicate an extreme significant data difference for the publication of any potentially identifiable images or data included in this article.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication."} +{"text": "To achieve the Sustainable Development Goals, strengthening investments in health service inputs has been widely emphasized, but less attention has been paid to tackling variation in the technical efficiency of services. In this study, we estimated the technical efficiency of local public health programs for the prevention of unintentional childhood injury and explored its contribution to national trend changes and regional health disparities in Japan. Efficiency scores were estimated based on the Cobb\u2013Douglas and translog production functions using a true fixed effects model in a stochastic frontier analysis to account for unobserved time-invariant heterogeneity across prefectures. Using public data sources, we compiled panel data from 2001 to 2017 for all 47 prefectures in Japan. We treated disability-adjusted life years (DALYs) as the output, coverage rates of public health programs as inputs, and caregivers' capacity and environmental factors as constraints. To investigate the contribution of efficiency to trend changes and disparities in output, we calculated the predicted DALYs with several measures of inefficiency scores . In the translog model, mean efficiency increased from 0.62 in 2001 to 0.85 in 2017. The efficiency gaps among prefectures narrowed until 2007 and then remained constant until 2017. Holding inefficiency score constant, inputs and constraints contributed to improvements in average DALYs and widened regional gaps. Improved efficiency over the years further contributed to improvements in average DALYs. Efficiency improvement in low-output regions and stagnated improvement in high-output regions offset the trend of widening regional health disparities. Similar results were obtained with the Cobb\u2013Douglas model. Our results demonstrated that assessing the inputs, constraints, output, and technical efficiency of public health programs could provide policy leverage relevant to region-specific conditions and performance to achieve health promotion and equity. The child health burden has dramatically reduced in the last three decades. Total disability-adjusted life years (DALYs) for those aged under 20 years decreased by 46% from 1990 to 2017, dropping from 1.31 billion to 709 million. This decline was led by drastic improvements in communicable, maternal, neonatal, and nutritional diseases in low- and middle- income countries (LMICs) , suggestParental health education is known to be an effective vehicle for preventing unintentional childhood injury; health education enhances parental supervision and environmental preparedness at home in terms of using safety equipment and removing home hazards \u201311. MentThere are several potential providers of these interventions, including health care institutions, schools, and private organizations , 14\u201316. Although childhood injury mortality has declined nationally, there are considerable geographic disparities across Japan , 24. GivIn this study, we estimated the technical efficiency of public health programs aiming to prevent unintentional childhood injury in Japan, and we distinguished the contribution of technical efficiency to national trend changes and regional health disparities from those of input and constraints. Overall cost efficiency is a product of technical efficiency and allocative efficiency; here, we focused specifically on technical efficiency because it reflects the current state of technology with regard to potentially producing the maximum output attainable at each input level , 29. BecIn the following section, we briefly review previous studies that have estimated technical efficiency in health, before presenting a theoretical model of the production function of unintentional childhood injury. We relied on stochastic frontier analysis (SFA) and a true fixed effects model (TFEM) to obtain efficiency estimations, accounting for time-invariant local heterogeneity. Our estimation revealed the existence of disparities. We also showed different trajectories of improvement in technical efficiency over time, which contributed to narrowing geographic health disparities. The final section discusses several policy implications for the SDGs, including closing the health gap.health care and the production function of health and the health care labor force . In the production function of health, the output is health status, and the inputs are health care services, along with demographic characteristics, personal health habits, and environmental factors.Using the production function of health, in 2000, the World Health Organization published a league table ranking the efficiency of the health systems of 191 countries , 39, whipost-hoc medical intervention is limited. Unintentional injury is such a case. Medical care after children sustain serious injuries has limited effectiveness in terms of reducing mortality and morbidity because most trauma deaths occur before hospitalization denotes population health outcomes related to unintentional childhood injury. Y is assumed to be a function of X, which represents inputs, in addition to the constraining factors (Z). More specifically, X denotes the volume of public health programs on preventing unintentional childhood injury. Public health programs potentially modify caregivers' knowledge, attitudes, and behaviors , and the more flexible translog form, as shown in Equation (3):Y, X, and Z are the output , input , and constraining factors , respectively. Prefecture is denoted by i, year is denoted by t, and the random error term is denoted by vit. uit is the inefficiency term, which is assumed to be half-normally distributed. \u03b1i is the time-invariant prefecture-specific fixed effect, which may represent unmeasurable environmental factors such as the safety culture in communities. Y, X, and Z were natural log-transformed. Some variables were linearly transformed before being natural log-transformed for the model conversion. We assumed no time lag between input and output because we supposed that the major vehicle for reducing injury risk was parental behavioral change, which should happen in a short time , 61, 63 We also estimated technical efficiency excluding the areas damaged by the Great East Japan Earthquake .n = 799) because prefectures are the legislative units for public health policy in Japan. For the output (Y), prefecture-level numbers of DALYs by age, cause, and year were obtained from the Global Burden of Disease Study 2017 , we compiled the prefecture-level coverage rates of all public health programs related to childhood injuries for which provision is not mandatory but is strongly recommended by national laws\u2014namely, health checkups for children aged under 1 year, home visits for children aged under 1 year , health guidance at the individual or group level, and health education at the group or community level , the regional education level and unemployment rate were used as indicators of health literacy. The regional education level of the child-rearing generation was measured as the proportion of people aged 20\u201349 years in the total population who had completed tertiary education, which was derived from the 2000 and 2010 Population Censuses and allocated to 2001\u20132009 and 2010\u20132017, respectively were assessed. To gauge access to emergency medical care including specialized treatment for trauma patients, the number of tertiary emergency medical facilities per 100,000 population was obtained from the Survey of Medical Institutions and leaving the fixed effects of prefecture, inputs, and constraints as they were in Equations (2) and (3). Second, to investigate the influence of technical efficiency trends, we predicted the DALYs using the annual mean score of inefficiency . Finally, we prepared the predicted DALYs using the actual prefecture-year-specific inefficiency estimates .To investigate the contributions of inputs, constraints, and technical efficiency to average changes and disparities in DALYs, we compared the time trend of predicted DALYs using three types of inefficiency scores. First, to examine only the influences of inputs and constraining factors on DALYs, we predicted the counter-factual DALYs, holding the inefficiency score constant at its mean value for 2001 to summarize the magnitude of regional disparities in the examined variables.Because our study involved secondary analysis of anonymous data, the requirement for ethical approval and informed consent was waived under governmental-use approval and under the approval of the Institute for Health Metrics and Evaluation of the University of Washington for use of the Global Burden of Disease Study 2017 by non-commercial users.F-test in the pooled ordinary least squares model (P < 0.0001) and the Hausman test to select between the fixed effects regression model and the random-effects generalized least squares regression model (P < 0.0001), TFEM was selected to estimate technical efficiency in the Cobb\u2013Douglas and translog models. The correlations between the fixed effect terms and estimated technical efficiency by year ranged from \u22120.15 to 0.29 in the Cobb\u2013Douglas model and from \u22120.15 to 0.42 in the translog model. These correlations were not statistically significant except in 2001 in the Cobb\u2013Douglas model and in 2001, 2010, and 2012 in the translog model (P < 0.05), which suggests no or low correlations between the two estimates.Following the results of the The TFEM estimations are presented in P < 0.0001).The translog model yielded similar results to those from the Cobb\u2013Douglas model. Pearson's correlation coefficient for technical efficiency estimated from the Cobb\u2013Douglas and the translog models was 0.85 . The translog model assuming interactions between the inputs and constraining factors [Equation (4)] yielded findings that were similar to the main results [Equation (3)] . The results of the estimations excluding areas damaged by the Great East Japan Earthquake were also similar to the main results in the Cobb\u2013Douglas and translog models .In the translog model, from 2001 to 2017, the mean DALYs predicted with the mean score of inefficiency in 2001 decreased from 1135.3 to 918.6, and the CV increased from 0.04 to 0.09 , indicatCompared with the trend depicted in Our results revealed that the technical efficiency of public health programs has improved and that the disparities in efficiency across prefectures narrowed dramatically until 2007 and then remained constant until 2017. National improvements in inputs, constraints, and technical efficiency contributed to an overall reduction in unintentional childhood injury in Japan. Regional disparities in inputs and constraints widened the population health disparities, but different efficiency improvement trajectories counterbalanced this effect, offsetting the trend of widening health disparities. This study contributes to existing efficiency research by presenting a novel application of efficiency measurement to the assessment of regional health disparity in the context of public health.According to a systematic review on the technical efficiency of the overall health systems of OECD countries, Japan has consistently shown the highest efficiency, with an average value of one . In thisNevertheless, technical efficiency has improved nationally, and the disparities among prefectures narrowed dramatically from 2001 to 2007. In this study, the mean technical efficiency in the 47 prefectures increased from 0.62 in 2001 to 0.85 in 2017 in the translog model. Similar technical efficiency scores have been reported in studies on local public health sectors in the United States and China , 51. AltThe national health promotion plan \u201cHealthy Parents and Children 21,\u201d which was initiated in 2001 in Japan , is suppHowever, the narrowing trend in the efficiency gaps stagnated in 2007 and subsequently remained constant until 2017. Some prefectures even showed decreased efficiency. Although the exact reasons for these findings are unknown, local governments' prioritization of preventing unintentional injury may have been lowered because of the conflicting demands of other public health programs that were newly emphasized because of increased societal awareness , 74.For the production function of unintentional childhood injury, the coefficients of inputs were positive except for the coverage rate of health checkups, which had a negative effect. Although the frequency of health checkups is predetermined by local governments on the basis of policy rather than annual changes in health needs, areas at high risk for childhood injury may promote health staff members' efforts to follow up with children with no visits, which may have led to the negative coefficient of health checkups.Our findings suggest several policy implications. First, public health program efficiency is an important\u2014but ignored\u2014policy target for the SDGs, including closing the regional health gap. To achieve the SDGs, developing strategies based on separate assessments of inputs, constraints, and efficiency is required to identify points of intervention for individual local governments. For example, prefectures with good health outcomes because of high input and an advantageous situation in terms of constraints but low efficiency need to make efforts to improve their efficiency. In contrast, prefectures with poor health outcomes because of low input and a disadvantageous situation in terms of constraints but high efficiency need to increase their input or address their constraints. This study suggests possibilities for creating an assessment system to provide this information using practical regional health sector reports. Additionally, to advocate investments in the SDGs in LMICs, efficiency estimations can be used to produce projections of financial resource needs that are more realistic than the current estimates, which assume efficient practices or simple scenarios . Second,This study's main strength lies in its estimation of the technical efficiency of preventive public health programs using the production function of health. Our empirical model can be applied to other population health topics where preventive services may have a larger influence than curative medical care, such as dental diseases and frailty among older adults.R-squared of the ordinary least squares model regressing original DALYs on DALYs predicted with the actual inefficiency estimates was 1.0. However, the lack of randomness may be attributed to the use of imputation in the DALYs estimation, which may be one of the reasons why the standard deviation of the random error in the TFEM was quite small in this study. Although prefecture-level data on mortality from unintentional injuries are available for Japan, we adopted DALYs as the health output because the prevention programs intend to reduce not only death but also injury-related disabilities. Additionally, DALYs have advantages over mortality in that DALYs take into account random errors and deal with the garbage code of deaths. We confirmed that the estimates of efficiency using under-five mortality rate of unintentional injury . The injury incidence rate could be used as the direct output, but these data were not available. Whether re-estimation with such health outcomes would change the results should be investigated in future studies.The study also had several limitations. First, the calculation of DALYs in this public data source relied on the imputation of missing data on mortality and disease prevalence using the Bayesian meta-regression tool DisMod-MR , 75. Ourl injury are modeSecond, there are other potential inputs in the production function of unintentional childhood injury that were not investigated because of a lack of data availability\u2014namely, programs only targeting families at high risk of adverse child outcomes, prefecture- or municipality-specific parenting support services, and programs with unclear target populations . The influences of these potential inputs on DALYs, which were included in the fixed effect or inefficiency terms in this study, may be minor because the program scale and target populations are small. Estimations including these programs are needed in future studies. Additionally, public health programs and child protective services are linked, but this study focused solely on the former because the decision-making units for the two types of programs differ , 21. AltThird, if persistent inefficiency exists, it is completely absorbed in prefecture-specific heterogeneity as fixed effect terms in the TFEM. We believe that the time-variant assumption regarding efficiency in the TFEM , 61 is rFinally, differences in technical efficiency across municipalities within a single prefecture remain unknown. Although our selection of prefecture as the unit of analysis seems reasonable because it is prefectures that administer public health programs, future studies should investigate efficiency at the municipality level because the programs are operated by municipality-level centers \u201319.This study demonstrates that regional disparities exist in the technical efficiency of legally established public health programs aiming to prevent unintentional childhood injury in Japan. The assessment of inputs, constraints, output, and technical efficiency of public health programs could provide policy leverage relevant to region-specific conditions and performance, contributing to the achievement of health promotion and equity.http://ghdx.healthdata.org/, \u201cVital statistics 2001\u20132017\u201d at https://www.mhlw.go.jp/english/database/db-hw/vs01.html, \u201cPopulation Census 2000, 2005, 2010, 2015\u201d at https://www.stat.go.jp/english/data/kokusei/index.html, \u201cPopulation estimates 2001\u20132017\u201d at https://www.stat.go.jp/english/data/jinsui/index.html, \u201cReport on regional public health services and health promotion services 2001\u20132017\u201d at https://www.mhlw.go.jp/english/database/db-hss/rrphshps.html, \u201cLabour Force Survey 2001\u20132017\u201d at https://www.stat.go.jp/english/data/roudou/index.html, and \u201cSurvey of Medical Institutions 1999\u20132017\u201d at https://www.mhlw.go.jp/english/database/db-hss/smi.html.Publicly available datasets were analyzed in this study. This data can be found here: \u201cGlobal Burden of Disease Study 2017\u201d at AH was responsible for the conception and design of the study, acquisition of the data, analysis and interpretation of the data, drafting the manuscript, and obtaining funding. HK contributed to the analysis and interpretation of the data, provided technical support, and participated in the critical revision of the manuscript. HH was responsible for the conception and design of the study, analysis and interpretation of the data, critical revision of the manuscript, obtaining funding, and providing supervision. All authors approved the final version of the manuscript.This work was supported by the Institute for Health Economics and Policy for fiscal year 2019 (Wakate-kenkyusya-ikusei-kenkyu-josei), and by JSPS KAKENHI Grant Numbers JP21J10730 and JP18H04070. The funding sources had no involvement in the study design, data collection, analysis, interpretation, 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."} +{"text": "Polymeric and/or lipid platforms are promising tools for nucleic acid delivery into cells. We previously reported a lipid\u2013polymer nanocarrier, named LipoParticles, consisting of polylactic acid nanoparticles surrounded by cationic lipids, and allowing the addition of mRNA and cationic LAH4-1 peptide at their surface. Although this mRNA platform has shown promising results in vitro in terms of mRNA delivery and translation, the bulk method used to prepare LipoParticles relies on a multistep and time-consuming procedure. Here, we developed an automated process using a microfluidic system to prepare LipoParticles, and we compared it to the bulk method in terms of morphology, physicochemical properties, and ability to vectorize and deliver mRNA in vitro. LipoParticles prepared by microfluidic presented a smaller size and more regular spherical shape than bulk method ones. In addition, we showed that the total lipid content in LipoParticles was dependent on the method of preparation, influencing their ability to complex mRNA. LipoParticles decorated with two mRNA/LAHA-L1 ratios could efficiently transfect mouse DC2.4 cells except for the automated 1/5 assay. Moreover, the 1/5 mRNA/LAHA-L1 ratio drastically reduced cell toxicity observed in 1/20 ratio assays. Altogether, this study showed that homogeneous LipoParticles can be produced by microfluidics, which represents a promising platform to transport functional mRNA into cells. During the last few decades, messenger RNA (mRNA) nonviral vectors have been widely studied as a vaccine platform against infectious diseases . HoweverBenefits concerning the safety of mRNA vaccines over conventional vaccines explain the growing therapeutic interest of this new platform. First, mRNA translation occurs in the cytoplasm and does not require entry into the nucleus, unlike DNA-based vaccines. Thus, there is no risk of genomic integration ,11. SecoOne of the main approaches to the formulation of mRNA is based on its complexation by electrostatic interactions with cationic components, such as lipids and polymers. Currently, LNPs are the most promising lipid-based nanoparticles used as an mRNA delivery system, as represented in the leading mRNA vaccines against SARS-CoV-2 infection ,17. The sn-glycero-3-phosphocholine (DSPC). This study showed that LP improved the in vitro transfection efficiency of mRNA compared with cationic liposomes. Ayad et al. suggested that the presence of the polymer core increases the rigidity of nanoparticles and that this physical modification could be the reason for the higher cell transfection efficiency observed with LP [auto). We also evaluated the effect of the LipoParticles preparation method on mRNA transfection efficiency and cytotoxicity in vitro. These experiments demonstrated that LPauto produced by microfluidics presented a better homogeneity than LP, with a smaller hydrodynamic diameter and a more regular spherical shape. However, the method of preparation and the form of lipid used (preformed liposomes or lipid solution) to obtain LipoParticles impact their capacity to complex mRNA and subsequent transfection efficiency in vitro.We recently compared the in vitro transfection efficiency of mRNAs using lipid\u2013polymer hybrid nanoparticles, named LipoParticles (LP), and cationic liposomes, both decorated with cell-penetrating peptides (CPPs), LAH4-L1 . LP were with LP . The pos with LP ,35. None\u00ae (PLA nanoparticles) were purchased from Adjuvatis . Lipids and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) were purchased from Avanti Polar . LAH4-L1 peptide (KKALLAHALHLLAL-LALHLAHALKKA) was purchased from GenScript . Absolute anhydrous ethanol was purchased from Carlo Erba Reagents , sterile pyrogen-free bidistilled water OTEC\u00ae was purchased from Aguettant , and nuclease-free water was purchased from Ambion . Furthermore, 1\u00d7 and 10\u00d7 DPBS , culture medium (RPMI/glutamax and DMEM), fetal bovine serum (FBS), N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), and \u03b2-mercaptoethanol were all purchased from Gibco . Lipofectamine 2000TM transfection reagent was purchased from Invitrogen\u2122 via Thermo Scientific\u2122 .i-Particles\u00ae Dialysis cassette with a 10 kDa cutoff . All liposomes were stored at 4 \u00b0C until further use. The formation of LP was based on the stirring of PLA-NP and liposomes for 1 h at 70 \u00b0C using an orbital mixer (mLab scientific HCM 100-pro). After a centrifugation step to remove all non-adsorbed lipids in the supernatant, the pellet was resuspended in nuclease-free water. LP were also stored at 4 \u00b0C until use.The standard method of preparing LP is to incubate preformed liposomes (formulated using a microfluidic process) together with a PLA-NP aqueous dispersion . The preparation of liposomes and LP was previously described . Brieflyauto) were formulated using a NanoAssemblr\u2122 benchtop instrument equipped with a microfluidic cartridge. The aqueous phase was composed of a dispersion of PLA-NP in nuclease-free water at the same dilution used to prepare LP. The organic phase was a lipid solution of DSPC and DOTAP in a molar ratio of 15:85 in absolute ethanol at 1.65 mM. The NanoAssemblrTM benchtop equipment was set up at 40 \u00b0C with an aqueous-to-ethanol flow rate ratio of 2:1 and total flow rate of 12 mL/min. In order to remove ethanol and non-adsorbed lipids, the formulation was centrifuged at 4000\u00d7 g and 15 \u00b0C for 15 min. The supernatant was discarded, and the pellet containing LPauto was resuspended in the same volume of nuclease-free water. The LPauto dispersion was stored at 4 \u00b0C until use.Automatized LipoParticles (LPv/v) FA in hexane, B was 0.2% (v/v) FA in methanol, and C was 0.1% (v/v) FA in water. Gradient was 88% B and 12% C for 3 min, changed to 13% A, 82% B, and 5% C over 1 min, and changed back to initial solvent mixture after 6 min. The total run time was 15 min. The column temperature was 60 \u00b0C and sample temperature was 30 \u00b0C. The flow rate was 1.4 mL/min, and a sample injection volume of 20 \u00b5L was used. The electrospray ionization mass spectrometer used to detect lipids was a Waters Micromass ZQ analyzer. Nitrogen gas was used at 450 L/h, and capillary and cone voltages were set to 3 kV and 50 V. The desolvation temperature and source temperature were 220 \u00b0C and 100 \u00b0C, respectively. Quantification was carried out using SIR mode (single ion recording) in positive mode at m/z = 790.1 for DSPC and 662.0 for DOTAP. Data were processed using Waters Empower 2 software version number 6.20.00.00 .Analysis of DSPC and DOTAP was conducted using a Waters 2795 Alliance module including a quaternary pump, mobile phase degasser, autosampler, and column thermostat. Separation was carried out on a Luna C18 analytical column supplied by Phenomenex. The analytical method, derived from Zhong et al. , consistauto suspension and mixed for 2 min using a vortex and 20 s in an ultrasonic bath. The procedure was repeated twice. The mixture was centrifuged at 15,000\u00d7 g for 10 min at room temperature (RT). If necessary, the supernatant was diluted in methanol prior to injection into the column.The calibration curves of lipids were determine using diluted samples in methanol with a concentration range of 0.1 to 5 \u00b5g/mL for DSPC and 0.01 to 1 \u00b5g/mL for DOTAP. For the quantification of lipid content of LipoParticles, 200 \u00b5L of ethanol was added to 100 \u00b5L of LP or LPThe mean hydrodynamic diameter and size distribution of naked vectors and the resulting mRNA formulations were determined by dynamic light scattering (DLS) analysis at 25 \u00b0C and at a scattering angle of 173\u00b0. All zeta potentials were measured by laser Doppler velocimetry at a scattering angle of 12.5\u00b0. The apparatus used was a Zetasizer Nano ZS . All samples were prepared by a 1/50 dilution in a 0.22 \u00b5m filtered 1 mM NaCl solution. Each value given by the software was the mean of four independent measurements.auto were used at a 0.2% solid rate and deposited on carbon/Formvar-coated copper 200 mesh grids for 60 s. A negative staining consisting of 1% (w/v in water) tungsten silicate solution was then applied to the grid for 30 s, and grids were washed with distilled water for 30 s. All samples were imaged using a transmission electron microscope JEOL 1400 Flash instrument at an accelerating voltage of 120 kV. All samples were imaged at 50,000\u00d7 and 100,000\u00d7 magnification. The particle analysis of TEM images was performed using Fiji ImageJ software. The size distribution frequency of PLA-NP and LipoParticles was evaluated using 1 \u00b5m scaled images of two independent assays and more than 200 particles.LP or LPauto, Fluc mRNA was diluted at a concentration of 40 \u00b5g/mL in nuclease-free water and mixed (v/v) with LP or LPauto. Then, two volumes of LAH4-L1 peptide at a concentration of 400 \u00b5g/mL (mRNA/LAH4-L1 ratio of 1/20 w/w) or 100 \u00b5g/mL (mRNA/LAH4-L1 ratio of 1/5 w/w) were added onto the LP\u2013mRNA intermediates. Fluc mRNA was purchased from TriLink BioTechnologies .The pLbL strategy, as previously described , relies auto was evaluated using an electrophoretic mobility shift assay. To this aim, a 1% agarose gel was prepared in Tris\u2013borate\u2013EDTA (TBE) 1\u00d7 buffer, and ethidium bromide (EtBr) was added as staining. Formulations were either treated or not with heparin at RT for 30 min plus with proteinase K at 56 \u00b0C for 15 min. The gel was then loaded with samples mixed with 2\u00d7 loading dye containing 100 ng of mRNA per well. The electrophoresis was performed for 17 min at 100 V, and the stained mRNA bands were visualized on an ultraviolet transilluminator and digitalized.The complexation of mRNA onto LP and LP2 and 95% humidity. Cells were always used with a low passage number.Immortalized DC2.4 (murine bone marrow-derived dendritic cells) cells were obtained from InvivoGen . They were cultured in RPMI-1640 medium, supplemented with 10% heat-inactivated FBS, 10 mM Hepes, and 50 \u00b5M \u03b2-mercaptoethanol. Cells were cultured in a 37 \u00b0C incubator under 5% COauto. Supernatants were removed 3 h post transfection, and 100 \u03bcL of complete medium was added to each well. Finally, cells were incubated at 37 \u00b0C and 5% CO2 until the analysis 24 h later. Lipofectamine 2000TM transfection reagent was used as a positive control following the manufacturer\u2019s instructions. Non-transfected cells were used as a negative control and were labeled as \u2018untreated\u2019.One day prior to transfection, cells were seeded in a 96-well plate at a density of 20,000 cells per well. After 24 h, complete medium was removed and replaced by a 100 \u00b5L mix of formulation and medium without serum, allowing the transfection of 90 ng of Fluc mRNA or equivalent volumes of noncomplexed LP and LPv/v) to the well and left to incubate for 5 min at RT. Luminescence was then measured on a Tecan i-control Infinite M1000 (Integration time 1 s) and was determined as the mean of three replicates and three independent experiments.A luciferase assay was performed using the Bright-Glo\u2122 Luciferase Assay System according to the manufacturer\u2019s recommendations. Briefly, the luciferine substrate was added using a PrestoBlue\u2122 Assay according to the manufacturer\u2019s instructions. Briefly, 11 \u00b5L of PrestoBlue\u2122 Cell Viability Reagent was added to the wells, and plates were incubated for 20 min at 37 \u00b0C. Fluorescence was detected on a Tecan i-control Infinite M1000 instrument . Fluorescence was determined as the mean of three replicates and three independent experiments.The cytotoxicity of noncomplexed vectors and Fluc mRNA LP and LPStatistical analyses were performed using GraphPad Prism version 9.3 software . All the data are presented as the mean \u00b1 SD. Differences between groups were analyzed using one-way ANOVA for the cytotoxicity and two-way ANOVA for the transfection efficiency, both followed by Tukey\u2019s multiple comparison test. Statistical significances are indicated on the figures.auto physicochemical characteristics. Briefly, LPauto were prepared with a lipid solution at the same DSPC/DOTAP ratio and following concentrations: 0.3, 0.9, 1.5, 1.65 and 3 mM. When the lipid concentration was lower than 1.65 mM, the \u03b6 potential was inferior to +20 mV and considered as an unstable colloidal dispersion. When LPauto were synthetized with lipid solutions at 1.65 and 3 mM, no differences in hydrodynamic diameter and \u03b6 potential were observed between the two conditions (data not shown). Additionally, no difference in DLS and zeta potential of LPauto was observed when prepared at two total flow rate (TFR) conditions of the microfluidic system, 10 and 12 mL/min (data not shown). Considering these preliminary evaluations of LPauto production, the condition chosen for the microfluidic system was a TFR of 12 mL/min and the injection of an organic phase composed of a DSPC/DOTAP 15:85 (m/m) solution at 1.65 mM, which again corresponded to the lipid concentration of liposomes used to prepare LP by bulk method and, thus, enabled a fairer comparison of both LP. In both methods, LipoParticles were prepared from the same PLA-NP batch, presenting a hydrodynamic diameter of approximately 145 nm and a negative surface charge close to \u221246 mV and \u03b6 potential were first assessed using DLS and zetametry, respectively . The preauto of 0.999 for DSPC and y = 188,945,499x + 450,927 with r = 0.997 for DOTAP.In order to quantify the amount of lipid complexed to each type of LP, an HPLC\u2013MS method was developed for both DOTAP and DSPC and validated according to ICH guidelines . As showauto, the initially chosen molar ratio of 15/85 DSPC and DOTAP was maintained throughout the stages of each manufacturing process, with final proportions of 13/87 and 17/83 for LP and LPauto, respectively. However, the total concentration of lipids was found to be significantly lower (more than fourfold lower) for LPauto obtained in a single step than for LP.The results are presented in auto vectors are mainly considered for mRNA delivery, their ability to adsorb such molecules was then investigated. For this purpose, we used the previously implemented and described pLbL formulation strategy [w/w.Since LP and LPstrategy . Brieflyauto were analyzed by DLS (auto). The increase in PdI resulted from a slight heterogeneity of the formulations and may reflect the presence of particles with different sizes or some aggregates. To note, LPauto led to a more homogeneous final formulation than LP prepared using the classical method when a 1/5 w/w mRNA/LAH4-L1 ratio was used, whereas no such difference was observed between formulations from LP or LPauto at a 1/20 w/w ratio. Lastly, the zeta potential dropped in all formulations compared to unloaded LP and LPauto, suggesting an efficient immobilization of mRNA and then peptides on LP and LPauto. In addition, the surface charge varied with the amount of peptide added, while the surface charge increased by 13\u201314 mV with a 1/20 w/w ratio of mRNA/LAH4-L1 compared with the 1/5 ratio.The hydrodynamic diameter of formulations prepared with LP or LPd by DLS . The addauto to bind mRNA using the pLbL strategy, an agarose gel electrophoresis assay was performed. Regardless of the LP manufacturing process used and the mRNA/LAH4-L1 ratio, mRNA was fully complexed, as no free mRNA was observed on the gel . Furthermore, there was no significant difference between transfection realized with LP obtained via the bulk method or the microfluidic system at this ratio. However, when the amount of peptide in the formulation was decreased until an mRNA/LAH4-L1 ratio of 1/5, LPauto was significantly less efficient for mRNA transfection than conventional LP. While there was no significant difference observed between the 1/5 and 1/20 mRNA/LAH4-L1 ratio for LP, LPauto showed a reduced transfection efficiency when peptide loading in the pLbL formulation was decreased. Regarding cytotoxicity compared to LipoParticles prepared previously using the conventional bulk method (LP) [auto was performed from lipids solubilized in ethanol, Av/Ap could not be applied, as lipid vesicles or liposomes were not preformed separately. Zhang et al. [w/w) using a microfluidic chip. In order to maintain a correlation between the composition of the lipid solution and liposomes used for the preparation of LPauto and LP, respectively, we established that the microfluidic system would be loaded with a solution of DSPC/DOTAP at an equivalent molar ratio and concentration of liposomes used to prepare LP via the standard bulk method. However, the temperature used in the preparation with the microfluidic system could not be maintained at 70 \u00b0C as used for the liposome synthesis because the injection of PLA-NP lead to some precipitation and partial loss of components inside the channel of microfluidic cartridge. Then, the temperature was decreased to 40 \u00b0C to prevent the partial clogging of the system and ensure the total recovery of components and the formation of reproducible batches of LPauto. As illustrated in auto using the microfluidic system. It is important to indicate that, prior to physicochemical analyses, both LP were purified by centrifugation after synthesis in order to eliminate non-absorbed free lipids, lipid vesicles, and/or liposomes. Despite the same lipid\u2013polymer composition, LipoParticles formulated using both methods presented quite different physicochemical properties and morphology.The application of microfluidic systems has been widely used to manufacture different types of nanocarriers for the delivery of drugs and nucleic acids such as the LNP formulation used in mRNA vaccines against SARS-CoV-2 , liposomhod (LP) . In the hod (LP) . Since tg et al. demonstrauto decreased when compared to LP, indicating that the method of preparation influences the auto assembly of lipids (PLGA)\u2013lipid hybrid nanoparticles according to the order of addition of components. Using this system, they could prepare hybrid nanoparticles exhibiting only a lipid monolayer (ML-NP) or lipid bilayers (BL-NP), in which the latter required twice the amount of lipids compared to ML-NP to completely cover the PLGA core. In the case of BL-NP, liposomes were formed in the first stage prior to the injection of PLGA solution in a second stage. For ML-NP, PLGA-NP were formed in the first stage, and then lipid solution was added. Meanwhile, they used only zwitterionic lipids that exhibited a different type of interaction with PLGA compared to the electrostatic interactions between preformed PLA-NP and cationic lipids or liposomes used in our study. These results corroborate our findings, indicating that the quantity of lipid deposition in hybrid polymer\u2013lipid nanoparticles depends on the method of preparation and the form of lipids added, in solution or preformed liposomes, to interact with PLA-NP.Considering these differences, we assumed that the amount of lipid associated to LipoParticles was different when prepared using the bulk and microfluidic methods, even if the total concentration of lipids in solution or liposomes was the same for LPiposomes . Howeverg et al. showed tauto had a lower total lipid concentration than LP and, thus, a reduction in cationic lipids available for mRNA adsorption. Then, we evaluated whether LPauto had equivalent properties as an mRNA carrier, as already demonstrated for LP [auto with the same quantities of mRNA and cell-penetrating peptide, LAH4-L1, in a sequential manner as a function of the electrostatic interactions of charged components. LAH4-L1 is a CPP which is widely used for the transport and release of mRNAs into the cytoplasm. CPPs allow spontaneous interactions with anionic mRNA and lipid cell membranes; moreover, they have pH-sensitive residues that facilitate endosomal escape [auto at 1/5 and 1/20 mRNA/LAH4-L1 ratios were chosen in order to assess whether the different vectors could modify the balance between cytotoxicity and transfection efficiency of formulations. Both formulations were successfully achieved presenting a high positive zeta potential and similar dispersion homogeneity. Not surprisingly, the hydrodynamic diameter of pLbL LP was slightly higher than that of pLbL LPauto the reduced cell internalization of nanoparticles and/or (ii) the modified mRNA release and translation in vitro. To verify if there is a relationship between the physicochemical properties and in vitro activity of LipoParticles, an in-depth characterization of component organization, nanoparticle uptake pathways, and kinetics of mRNA expression needs to be considered in future studies.Lastly, we evaluated the mRNA expression and cytotoxicity in vitro of pLbL LP and pLbL LPulations . Howeverauto). These LipoParticles presented adequate physicochemical characteristics to be used as an mRNA carrier. However, the lipid shell formed was not equivalent to that obtained via the bulk method using preformed liposomes (LP). This difference could drastically reduce the amount of mRNA adsorbed onto the PLA-NP surface. Consequently, the pLbL LPauto formulation, obtained from the adsorption of mRNA and LAH4-L1 at a 1/5 (w/w) ratio using the pLbL strategy onto LPauto, exhibited reduced mRNA expression in vitro compared to pLbL LP prepared using the bulk method. In summary, our results showed that using a microfluidic system is a promising strategy for the synthesis of LipoParticles; however, additional evaluation of formulation parameters and microfluidic conditions needs to be performed to ensure the formation of a lipid shell able to adsorb a higher quantity of mRNA and improve its expression with low cytotoxicity.The present study showed that LipoParticles can be prepared in a reproducible manner through an automated microfluidic system (LP"} +{"text": "We detected no motion on any remaining ice in Venezuela. The overall ice volume in the region, 4.17\u2009\u00b1\u20090.35\u2009km3, is half of the previous best estimate of 8.11\u2009km3. These data can be used to better evaluate the status and distribution of water resources, as input for models of future glacier change, and to assess regional geohazards associated with ice-clad volcanoes.Tropical glacier melt provides valuable water to surrounding communities, but climate change is projected to cause the demise of many of these glaciers within the coming century. Understanding the future of tropical glaciers requires a detailed record of their thicknesses and volumes, which is currently lacking in the Northern Andes. We calculate present-day (2015\u20132021) ice-thicknesses for all glaciers in Colombia and Ecuador using six different methods, and combine these into multi-model ensemble mean ice thickness and volume maps. We compare our results against available field-based measurements, and show that current ice volumes in Ecuador and Colombia are 2.49\u2009\u00b1\u20090.25\u2009km These tropical glaciers represent both a valuable water resource to millions of people7 and a major source of geohazards. By storing water on multi-annual timescales, glaciers alleviate water shortages during dry seasons and in times of drought12. From a geohazards perspective, many of the Northern Andean glaciers are located on active volcanoes13. Eruptions from glaciated volcanoes commonly trigger damaging jokulhlaups (floods) and lahars (mudlflows)16. The most deadly volcanic disaster of the past century occurred in the Northern Andes, associated with volcano\u2013ice interactions during the 1985 eruption of Nevado del Ruiz, Colombia18. Finally, many tropical glaciers also hold cultural significance19, for instance those on Chimborazo20.The Northern Andes is a high-altitude mountain range in the inner tropics of Colombia, Ecuador, and Venezuela. Despite their proximity to the equator, many peaks in this region are glacierized21. This recession is particularly pronounced in tropical regions22, where glaciers may melt year-round due to the lack of temperature seasonality23. Many glaciers in the Northern Andes are forecast to disappear entirely over the course of the 21st century25. Better assessing the timing of this ice loss and forecasting its impact requires knowledge of the existing ice thickness and volume in the Northern Andes. To date, regional ice-volume assessments have focused on glacier-area change27.Climate change has driven global glacier recession over the past decades37. We validate our dataset against available field measurements of ice thickness and volume from the Northern Andes42, and compare these to previous global assessments37. For each glacier in the Northern Andes, our database includes an updated glacier-extent polygon, a 50-m-resolution gridded map of glacier-surface velocities, six different 50-m-resolution maps of ice thickness calculated using different methods, and a 50-m-resolution multi-model ensemble mean ice thickness map43. Each ice-thickness map has a complementary grid of ice-thickness uncertainty.In this study we combine remote sensing of glacier-surface velocities together with multiple ice-physics-based inversions to build a new database of present-day (2015\u20132021) ice thicknesses and volumes for all glaciers in Colombia, Ecuador, and Venezuela43 allow for an estimation of the current ice volume in Colombia, Ecuador, and Venezuela, and provide a baseline for future monitoring of volumetric ice loss. This database can support a range of future research objectives, including forecasts of glacier-mass loss and the timing of peak glacial runoff, assessments of water availability for major cities (e.g. Quito44), and improvements to regional volcanic hazard maps40.Our ice-thickness mapsWe design a workflow for calculating ice thicknesses and ice volumes in multiple ways using glacier-surface topography, glacier-surface flow speed, glacier geometry, and glacier basal shear stress Fig.\u00a0.Fig. 1St45 to identify, pre-process, and download Sentinel-2 satellite imagery46 of all ice-covered areas in Colombia, Ecuador, and Venezuela. We (i) use Randolph Glacier Inventory (RGI1) polygons to identify the glaciers, (ii) manually delete polygons for from areas that show exposed bedrock for at least part of the year, and therefore have been transformed from glaciers to seasonal snowfields, (iii) buffer polygons with a 1\u2009km margin, and (iv) merge overlapping polygons. This results in 6 extant glaciers in Ecuador , 6 glaciers or glacier zones in Colombia , and one single glacier in Venezuela .We use Google Earth Engine36. In addition to the three Sentinel-2 bands, we download the 1-arcsecond-resolution Copernicus DEM47 to provide ice surface slope for the ice-thickness inversion.We clip every available Sentinel-2 image (from 2015 to 2021) to the area of interest from each ice-cover polygon. We include only images with less than 60% cloud cover based on the cloud mask from the Sentinel-2 quality assurance (QA) band. We average three bands for use in feature tracking1. Any ice-volume calculation method using the RGI polygons37 is therefore likely to overestimate the true volume. Hence, we use a new ice-masking approach, which uses percentile analysis of satellite image timeseries to differentiate permanently glaciated regions from temporary snow cover. This script generates an ice index and a water index , and classifies pixels as ice or water when the respective index is below an optically calibrated threshold in 90% of individual images. This threshold filters out temporary snow cover but retains zones of persistent ice cover. Water pixels are merged with non-ice pixels and masked out, leaving a binary ice mask. Removing water pixels is important as these may otherwise be misclassified as ice: two recent global compilations mis-identify the El Altar crater lake as ice, and include it in their glacier volume calculations37. Due to its very high tephra cover, we manually delineate the Nevado del Ruiz glacier zone by hand using 3-m-resolution Planet DoveSat images collected on 10 and 12 February, 2020. We manually evaluate each glacier mask against low snow cover Sentinel-2 images from 2020 or 2021.We find that RGI polygons overestimate the spatial extent of the majority of glaciers in the Northern Andes, likely due to rapid glacier recession and persistent high-altitude snow cover throughout the year36. We also calculate apparent displacements over the surrounding bedrock to correct for georeferencing errors and evaluate local noise levels. We include all image pairs with a minimum temporal separation of 3 months and a maximum temporal separation of 4 years. This allows us to compile a large dataset of image pairs at each glacier, ranging from 230 pairs at Nevado del Huila to 14,198 image pairs at Sierra Nevada de Santa Marta. Calculating a large number of individual velocity maps is advantageous, as the precision of mean velocity maps improves with the number of individual velocity maps stacked48. We do not consider glacier surges, as they have not been documented in this region and the local glacier characteristics are unfavorable to their occurrence49.We use the feature-tracking toolbox GIV to calculate 50-m-resolution glacier-surface-velocity maps for each location, using a frequency-domain multi-pass image correlator36. We convert each pair-wise displacement map into a velocity map by dividing it by the temporal separation between images. We filter each velocity map by removing the value for pixels which meet any of the following criteria: the velocity exceeds the maximum velocity threshold of 100\u2009m.a\u22121, the signal to noise ratio is lower than 5, or the peak ratio is less than 1.336. These thresholds were manually selected based on local tests to exclude the majority of pixels with erroneous velocity estimates based on comparison with neighboring pixels and external datasets37. We also exclude values that differ by more than 50% from their immediate neighbours (four surrounding cells) and 200% from the mean of their larger local area (25 surrounding cells), and interpolate across these now-empty pixels using the values of the remaining ice-speed pixels. We do not filter based on flow direction because most of the region\u2019s glaciers flow radially outwards from mountain peaks. To correct for possible georeferencing errors, we subtract the median velocity over non-glacierized (stable) areas from the glacier velocity in the x and y directions for each image pair. We calculate a timeseries of velocities for each pixel, and after constructing this timeseries exclude pixels having a glacier speed in excess of 1.5 standard deviations from the mean36. Finally, we average all individual processed velocity maps into a mean velocity map covering the entire period. We crop mean velocity maps to the updated ice mask prior to inverting for ice thickness to exclude background noise over non-glacierized terrain from ice-volume calculations.For each Sentinel-2 image pair, we calculate displacements using iteratively reducing multipass template matching following the standard GIV workflow. We use the standard GIV reference window sizes of 400\u2009m, 200\u2009m, and 100\u2009m and a 50% window overlap, for a final velocity-map resolution of 50\u2009m. Displacements are evaluated to sub-pixel precision in the final pass using a Gaussian sub-pixel estimator50. We therefore use an ensemble of six different methods to calculate the thickness of all glaciers in our study area. Three of these methods use glacier surface flow speeds to invert for ice thickness36, two of these use a basal-shear-stress-based approach35, and one method uses a mass-conservation-based approach35.Previous intercomparisons of glacier-thickness calculation methods have shown that, while no single approach is clearly superior to all\u00a0others, the average of multiple different methodologies is generally more accurate than any single methodu(H) may be written as a combination of internal deformation (ud) and basal velocity :Glacier motion occurs through a combination of internal deformation, basal sliding, and subglacial sediment deformation. Ice-surface velocities H denotes velocities that are evaluated at the ice surface.Here, the ice-thickness 41. Glacial sliding requires warm-based ice and can be enhanced by water pressure51. In the tropical Northern Andes, seasonal temperature variations are low and the majority of glacier area is located in areas with a mean annual temperature below freezing23. As a consequence, basal sliding ub likely accounts for a small proportion of the total glacier surface velocity. We therefore account for basal velocity ub through a correction factor, \u03b234, which corresponds to the fraction of glacier motion derived from basal sliding.We simplify this ice-flow equation based on the characteristics of the glaciers in this study area. Field studies have not revealed extensive subglacial sediment layers, thus subglacial sediment-deformation term should be at or near zero53As a result, we directly relate internal deformation to glacier-surface velocity, and use this to compute a closed-form relationship between (unknown) ice thickness and observed ice velocity. Ice flows under its own weight, and the rate of internal deformation is a function of the ice thickness:\u03c4b is the basal shear stress, Ac is the Arrhenius creep constant, and n is Glen\u2019s flow exponent. Our use of the basal shear stress instead of the full driving stress for glacier motion comes from the shallow-ice approximation. Through this, we assume that local stresses induced by the ice are much greater than stresses induced by lateral coupling between columns of ice. Thin ice and steep slopes are characteristic of many Andean glaciers41. Both of these enhance the dominance of the basal shear stress within the full glacier driving stress, thereby supporting our use of the shallow-ice approximation.Here, \u03c4b, into measurable parameters:We expand basal shear stress, f is a shape factor accounting for lateral drag along the glacier margins54, \u03c1i is the ice density, g is gravitational acceleration, \u03b1 is the ice-surface slope angle , and H is ice-thickness. We calculate the Arrhenius creep constant based on temperature:Here, Qc\u2009=\u2009115\u2009kJ\u2009mol\u22121, R\u2009\u2248\u20090.0083145 , and T*\u2009=\u2009273 K53. We combine Eqs.\u00a0with the constants being entclass1pt{minimauH) and a digital elevation model (sin(\u03b1)).Thus, the only unknown required to solve for ice-thickness, H, is ice-surface velocity.Here, the first term contains constants and parameters and the second term contains observations obtained from GIV . This approach is closer to the original method32 but may average across multiple outlet glaciers from a single ice cap. Therefore, we also use TopoToolbox55 to divide ice caps into individual ice-drainage basins based on topographic slope. We then calculate elevation-band-averaged flow speed and slope for each elevation band within each individual ice basin (G14 b model). This approach can better honor the variable dynamics of outlet glaciers from ice caps, but may result in artificial step changes in ice thickness at the boundaries between basins.We implement this equation in three different ways, one novel to this study and two based on the work of Gantayat 57. In order to solve for this without prior knowledge of ice thickness, we iterate between ice-thickness and coupling-length calculations 5 times. In tests that we ran, three iterations were always sufficient for convergence, and applying five iterations permits a factor of safety between these tests and the present application. For the Gantayat et al.32 basin-divided and whole glacier approaches32, the coupling-length is accounted for by the elevation-band averaging.The representative length scale over which glacier surface slope is physically significant is a function of the local ice thickness. When implementing our fully distributed ice-thickness solution, we use a value of 5 times the mean ice thicknessWe may rewrite Eq.\u00a035. An empirical relationship between glacier-surface elevation range \u0394zi and basal shear stress \u03c4b is then used to compute ice thickness:This equation forms the basis for the \u2018Glacier Bed Topography\u2019 (GlabTop) approach to calculating glacier thickness\u03c4b measured in kPa and \u0394zi in km34. Similarly to the Gantayat et al.32 basin-divided and whole glacier approaches32, we implement this methods on both whole ice caps (GT2 w model) and individual ice-drainage basins (GT2 b model), and iterate between ice thickness and coupling length to calculate the final ice-thickness value.with 29, which has been applied globally35, may be written as:The conservation-of-mass approachh/\u2202t] being the change in glacier surface elevation through time, 29. Integrating Eq.\u00a0with [\u220230, equal to the glacier surface mass balance rate h/\u2202t such that:with the glacier-wide ice-flux divergence being zero. We use the apparent mass balance 35. zi represents the elevation of the cell and z0 represents the elevation of the apparent equilibrium line altitude (ELA), calculated by solving Eq.\u00a0z0 .N\u2009=\u20091000 runs for each method, randomly sampling from the probability distribution of each input parameter and higher later drag in the outlet valley glaciers53. We vary ice density uniformly between 743 and 917\u2009kg/m362, which is consistent with available ice-density profiles showing a mean ice density of around 830\u2009kg/m3\u200940. No estimate of sliding velocity currently exists for any glacier in the study area, so we vary the basal drag correction \u03b2 within a range of 0 to 0.4, corresponding to between 0 and 40% basal sliding53.Terms in Eq.\u00a0N times, and we extract the mean and standard-deviation ice thicknesses at each grid cell from this single-model ensemble. We repeat the same process for basal-shear-stress-based approaches using Eq.\u00a0For velocity-based inversions, the mean velocity for each point is derived from the process described above in Step 2, and the standard deviation is calculated from standard deviation of measured velocities over non-glaciated terrain. Equation\u00a03, and an ice density range of 830\u2013917\u2009kg/m3) to assess the spatially variable significance of these glaciers in regulating discharge62. We use TopoToolbox55 to extract a drainage network and drainage basins from the DEM, cropped to a buffered ice-mask. We define the pour points for basin delineation as the boundary of the domain, and repeat the operations for non-rectangular DEMs extending 1\u2009km, 5\u2009km, and 20\u2009km beyond each ice mask. We then sum the ice volume for each drainage basin, discarding basins containing no glaciers.We subdivide these ice masses by the river catchments into which they drain, and convert each into its water-equivalent volume .32.An ice-cap-wide ice velocity inversion, with elevation-band averaged surface slope and flow speed (G14 w model)55.An velocity inversion, with elevation-band averaged surface slope and flow speed calculated for individual glacier basins (G14 b model)35.A basal-shear-stress-based approach, with an elevation range calculated for entire ice caps (GT2 w model)55.A basal-shear-stress-based approach, with an elevation range calculated for individual glacier basins (GT2 b model)35.A conservation-of-mass-based approach, with an apparent mass balance calculated for individual glacier basins (H & F model)We calculate six suites of 1000 ice-thickness maps for each of the six methods described above :35 and M2237, which we do not include in our multi-model ensemble mean ice-thickness calculation. The accuracy and precision of each map is further discussed in the technical validation section.For each method, we calculate the mean and standard deviation from all Monte Carlo runs. We then calculate a multi-model ensemble mean ice-thickness map of each glacier as the average of the mean ice-thickness maps generated using these six methods. We provide all six individual mean ice-thickness maps alongside the multi-model ensemble mean ice-thickness map. We compare the ice-thickness maps calculated with each of our methods to two prior global compilations, F19dx and dy are the grid resolution along each axis, which in our study remains uniform, but which for the sake of generality we include within the summation.For each single-method ice-thickness map and for the multi-model ensemble mean ice-thickness map, we then calculate ice volumes as a simple area-weighted sum:Similarly, we calculate the standard deviation of the ice volume as an area-weighted sum of the individual standard deviations of ice thickness , they are of negligible importance to overall ice-volume calculations.Figure\u00a037 are provided in Table\u00a03 to 0.74\u2009\u00b1\u20090.06\u2009km3 (conservation-of-mass-based method30). Three out of six methods show a greater total volume in the El Cocuy region, although this is spread across multiple distinct ice bodies.Figures\u00a03 to 1.94\u2009\u00b1\u20090.09\u2009km3, with the velocity- and conservation-of-mass-based ice-thickness calculations clustering near the upper bound. Estimates of total ice volume in Ecuador range from 1.90\u2009\u00b1\u20090.08\u2009km3 to 2.81\u2009\u00b1\u20090.10\u2009km3. These estimates are around 50% lower than the previous best estimate of 3.30\u2009km3 for Colombia and 4.81\u2009km3 for Ecuador35) , the majority of the ice is drained eastwards for all Ecuadorian glaciers an ice core drilled to bedrock on Volc\u00e1n Chimborazo38, and (iii) multiple ice-penetrating radar lines on Volc\u00e1n Antisana42. Overall, we note that only a small number of on-site field measurements of ice thickness and volume exist in the Northern Andes. In addition, comparisons between our measurements and existing data are complicated by the rapid ice loss that has occurred during the time between the field measurements and the collection of the Sentinel-2 imagery that we use in our thickness inversions.We compare our results to data from three field studies: (i) an ice-penetrating-radar-based volume estimate for Nevado del Ruiz41. These data provide an ice-cap volume of 0.57\u2009\u00b1\u20090.20\u2009km3, calculated by extrapolating between the existing grid of IPR measurements. We calculate volumes for the Nevado del Ruiz ice cap ranging from 0.29\u2009\u00b1\u2009 0.07\u2009km3 to 0.52\u2009\u00b1\u20090.06\u2009km3, with a multi-model ensemble mean ice volume of 0.45\u2009\u00b1\u2009 0.12\u2009km3 survey of the Nevado del Ruiz ice cap was conducted in 2000km3 Fig.\u00a0. Total iume Fig.\u00a0. Farinot38. The measured depth to bedrock from this borehole is 54.4\u2009m . The mass-conservation-based approach30 and our new ice-velocity inversion exhibit the highest correlation coefficients of 0.64 and 0.55 respectively, and the multi-model ensemble mean ice thickness has a correlation coefficient of 0.43.For outlet glacier 15 nts Fig.\u00a0. Farinot30, and distributed ice-velocity inversion showing the best results. All approaches requiring division of ice caps into individual glacier basins result in artificial step changes in thickness at the boundaries between basins. We therefore recommend that studies requiring the two-dimensional pattern of glacier thickness use the ice-thickness maps produced using our new fully-distributed velocity-based inversion, while studies requiring only the glacier-wide or regional ice volume use either our new fully-distributed velocity-based inversion or the multi-model ensemble mean ice thickness maps65.The comparison presented here shows that our remotely sensed ice thicknesses and volumes are consistent with field-based data. Certain models provide a better match to the data than others, with the multi-model ensemble mean, mass-conservation-based approach3), the lateral drag factor (0.8 to 1), the basal sliding correction (0 to 0.4), and the vertical mass-balance gradients for the ablation area and the accumulation areas (0.008 to 0.01 and 0.004 to 0.006 respectively). We also vary the value of Glen\u2019s flow exponent from the constant value of 3 used in this study for investigative purposes, within a range of 2.9 to 3.1. For ease of comparison between different mean glacier volumes, we evaluate the sensitivity \u2202H as a percentage change:To further evaluate potential sources of bias within our ice-thickness dataset, we run a comprehensive sensitivity analysis on all parameters used for the ice-thickness calculation. For each parameter evaluated, we hold all other parameters constant at their mean value, vary the chosen parameter within the range used in this study Table\u00a0, and evaVi being the ice volume resulting from a specific parameter combination and Vi(ref) being a reference ice volume . Ice volume is most sensitive to temperature, ice density, and the lateral drag factor (f), with sensitivities of 5 to 10% to these parameters in isolation . A table providing the results of the sensitivity test for each individual glacier region is available in the data record43.with ion Fig.\u00a0. Ice den43. Individual ice-thickness maps are available for each glaciated area using the six individual methods described in this study, alongside the multi-model ensemble mean ice-thickness map. All data are saved in 32-bit floating-point geotiff format. The data are freely available under the Creative Commons Attribution Licence, CC BY 4.0.All ice-velocity, ice-thickness, ice-thickness-uncertainty, and basin-divided water-equivalent-volume grids may be downloaded from the Zenodo repository"} +{"text": "Neurotransmitters (NTs) with hydroxyl groups can now be identified electrochemically, utilizing a variety of electrodes and voltammetric techniques. In particular, in monoamine, the position of the hydroxyl groups might alter the sensing properties of a certain neurotransmitter. Numerous research studies using electrodes modified on their surfaces to better detect specific neurotransmitters when other interfering factors are present are reviewed to improve the precision of these measures. An investigation of the monoamine neurotransmitters at nanoscale using electrochemical methods is the primary goal of this review article. It will be used to determine which sort of electrode is ideal for this purpose. The use of carbon materials, such as graphite carbon fiber, carbon fiber micro-electrodes, glassy carbon, and 3D printed electrodes are only some of the electrodes with surface modifications that can be utilized for this purpose. Electrochemical methods for real-time detection and quantification of monoamine neurotransmitters in real samples at the nanomolar level are summarized in this paper. The chemical messengers called neurotransmitters influence a large number of physiological and psychological human bodily functions. Because they are directly related to the central nervous system (CNS), they directly handle neurophysiological exercises, such as emotions, sleep, memory, and other cognitive functions by amplifying, transferring, and converting signals through a synapse to a target cell. Defective NTs in the CNS cause a slew of diseases, including Huntington\u2019s syndrome, Parkinson\u2019s, Alzheimer\u2019s disease, autism, and epilepsy. Additionally, it can cause psychotic diseases, such as dementia, attention deficit hyperactivity disorder, schizophrenia, and depression, as well as dejection, anguish, congestive heart failure, glaucoma, arrhythmias, and sudden infant death syndrome. In 1921, German pharmacologist Otto Loewi identified the first extensively detectable neurotransmitter, acetylcholine (Ach). More than 500 NTs were found from 1921, but the actual number of these distinct NTs in humans has yet to be determined. NTs are stored in synaptic vesicles and released into the synaptic cleft and diffused across it, where they get attached to specific receptors on the post-synaptic neuron\u2019s membrane. This bonding perhaps exerts excitation (depolarization) or inhibitory (repolarization) control over the post-synaptic neuron. Immunocytochemical approaches that identify the site of the transmitter material are commonly used to determine the anatomical localization of NTs. Many transmitters, primarily neuropeptides, are co-localized, indicating that a neuron can release multiple transmitters from its synaptic terminal, according to immunocytochemical techniques. Neurotransmitters can be classified in a variety of ways. It is sufficient to divide them into monoamines, amino acids and peptides for certain classification purposes benzene-1,2-diol, belonging to the catecholamine class of NTs. Takamine was the first to isolate EP from the adrenal hormone in 1901, while Friedrich Stolz and Henry Dakin were the first to synthesize it in the laboratory. It allows the body to prepare for flight or battle by boosting blood flow to the muscles. Epinephrine is a neurotransmitter produced by the adrenal glands and is required by the neurological system. Additionally, while the existence of adrenaline is necessary for human survival, it is released from the body when a person is stressed. The effects of an adrenaline overdose on the human body include sudden numbness, slurred speech, and exacerbated breathing problems. Because of its control over the neurological system\u2019s performance, an upset in the balance of EP in the human body fluid to a level outside of 0.09\u20130.69 mg mLthus far .Norepinephrine (NE), a catecholamine NT found in the CNS of mammals and considered the most important one that regulates the circulatory system, relieves pain, detects stress, and depression, and regulates appetite. Chemically, it is 4-[(1R)-2-amino-1-hydroxyethyl]benzene-1,2-diol, and changes in NE plasma levels caused by metabolic dysfunction can result in a variety of clinical diseases. NE works on target cells by activating noradrenergic receptors on cell surfaces, resulting in increased heart rate, anxiety, bladder inhibition, and gastrointestinal motility. Hypoglycemia, anxiety, high blood pressure, paleness, and headaches are all symptoms of a disturbed NE balance in the body. As a result, measuring the amount of NE in the extracellular fluid (ECF) can be used to diagnose a variety of diseases. For the first time, modified carbon electrodes with high selectivity, stability, and sensitivity were effectively used to analyze injection samples of NE .Because of their difficult immobilization method and the instability induced by the degradation of enzymatic activity over time, enzyme-based biosensors have disadvantages. Nonenzymatic sensors, on the other hand, can easily detect electroactive NTs. Even though these NTs bio-sensors are low-cost, stable, and biocompatible for most uses, their selectivity and specificity are still under question. A large number of the biochemical genus has equivalent redox potentials in the brain, making it challenging to use nonenzymatic NTs bio-sensors, which are sensitive to these types of species . For thiCompiling these reports into a review paper is difficult because most research studies do not provide a critical comparison with previously published work. From the beginning to the present, we thoroughly presented the most valuable, promising, accurate, and successful electrochemical techniques for the sensing of these monoamines. According to us, there is no review study published that summarizes the electrochemical methods for monoamine neurotransmitters\u2019 determination.This review article gathered many reviews and research articles for NTs\u2019 detection with surface-modified electrodes (SME) based on polyelectrolytes and conducting polymers, metal and carbon nanoparticles, ionic liquids, and nanocomposites, as compared to the analytical results of different techniques. Among the best NTs\u2019 detection approaches are linear working concentration range, selectivity, sensitivity, the lower detection limits (LODs), sample prearrangement simplicity, stability of the electrode surface that has been changed, and signal intensification over bare electrodes, as well as the separation between oxidation peak potentials . The intAll four monoamines have the presence of hydroxyl groups which arOn the target cell, a neurotransmitter can have an excitatory, inhibitory, or modulatory action. The receptors that the neurotransmitter interacts with at the post-synaptic membrane determine the effect . A neuroReal-time detection of NTs is of tremendous interest in neuroscience because they perform key roles in the central nervous system and influence the severity of neurodegenerative disorders . BillionElectrochemical studies or electrochemistry is the study of various techniques to analyze the chemical reactivity using electrical simulations by detecting mainly the oxidation and reduction mechanism in a particular reaction ,17. PoteCV is, no doubt, a perfectly adaptable and versatile electroanalytical technique used for the examination of electronically active species. The fundamentals of CV consist of cycling the potential of an electrode that is immersed in a stable unstirred solution and used to measure the final resulting current. There is a setup of the three-electrode system which mainly includes working, reference, and counter electrodes. The potential of the working electrode is controlled against reference electrodes e.g., saturated calomel electrode (SCE) or Ag/AgCl electrode with Pt-wire as the counter electrode. The potential, called controlling potential, is applied across all electrodes to generate a signal considered an excitation signal, which is a linear potential scan with a triangular waveform for CV. It primarily requires a waveform generator to generate the excitation signal and a potentiostat to apply it to an electrochemical cell, as well as a current-to-voltage converter to measure the generated current, and an XY recorder or oscilloscope to display the voltammogram 21]..21].The principle of the DPV approach is to measure the difference in the rate of decay of charging and faradaic currents when the potential pulse is applied. The faradaic current is applied as a function of time during scanning. The potential is scanned with a series of pulses, while the current is monitored at the beginning and end of each pulse, which is fixed at a small amplitude while the current generates peak-shaped voltammograms . We can \u22125 cm2 s\u22121) and kinetic parameters for dopamine at the surface of the AC-CPE, such as the electron transfer coefficient (a = 0.48), apparent electron transfer rate constant (ks = 0.80 s\u22121), and catalytic reaction rate constant (kcat = 40.90 104 mol L\u22121 s\u22121), were then determined using electrochemical methods. The AC-CPE exhibited favorable electron transfer kinetics and electro-catalytic activity when it came to dopamine oxidation/reduction. The CV and DPV experiments were studied in the potential ranges of 0.5 and 1.0 V and 0.3 to + 0.7 V, respectively. Voltammograms were recorded using carbon paste electrodes that were both unmodified and AC modified. There were no oxidation and reduction peaks of the CV curve on an anodic and cathodic branch when the absence of DA at CPE and AC-CPE. Meanwhile, enhancement by one and a half times in the peak current when connected to a modified electrode shows the result that AC carbon has high electro-catalytic activity towards oxidation\u2013reduction in dopamine (DA). With a detection limit of 3.09 \u00d7 10\u22128 mol/L, DPV was effectively employed to determine DA in the linear response range of 1.0 \u00d7 10\u22127 to 1.0 \u00d7 10\u22123 mol/L erotonin . Wei et erotonin . The polon limit Table 74\u2212 was uson limit . Other non limit . The caron limit . Carbon on limit .2 NPs is performed using the gel-combustion method by combining zirconyl nitrate hexahydrate (ZNH) with glycine. To ensure the complete combustion of the materials, they were mixed in a ratio of 1:1. In a minimum amount of milli-Q-water, the ZNH and glycine as oxidizers and as fuel were mixed, respectively. The obtained transparent gel with high viscosity was transferred to a pre-heated muffle furnace at a temperature 500 \u00b0C, and the excess water from the redox mixture was evaporated. The complete combustion results in the formation of white voluminous, porous, and puffy mass used in various characterizations. The BCPE was formed by mixing graphite powder and silicone oil in an appropriate proportion of 30:70, similarly, ZrO2NPs/MCPE was prepared ZrO2 NPs with graphite powder and silicone oil in the ratio of 10:10:80 by w/w/w. The CHI-660 c model of potentiostat was used for the differential pulse and cyclic voltammetry techniques. The conservative three-electrode cell system was made up of ZMCPE as the working electrode, and saturated calomel and platinum wire as a reference electrode and a counter electrode, respectively.The synthesis of ZrO4Fe(CN)6] is uniformly used to check the electrochemical properties of ZMCPE. The cyclic voltammetry of [K4Fe(CN)6] is conducted at ZMCPE with 0.1 mM concentration with 0.005 Vs\u22121 scan rate, and 1 M KCl as a supportive electrolyte showing low signals of current. The DPV experiment of ZMCPE shows a high sensitivity for serotonin quantification in the range 10,000\u201350,000 nM, and the limit of detection is 585 nM 3\u2212/4\u2212 redox process is assigned to the redox peaks observed near about 0.2 V; on the other hand, the oxidation peak is observed around 1.0 V on the modified Au-MWCNT-PANI-RuO2 which is attributed to Au+, to Au3+, or to Au4+. This 1.0 V peak was most likely not caused by the electrode\u2019s quick electron transfer process. At the Au- MWCNT-PANI-TiO2 electrode, the redox couple of [Fe(CN)6]4\u2212/[Fe(CN)6]3\u2212 seemed reversible, with a peak current\u2019s ratio of Ipa/Ipc = 1.01 or nearly 1 for a non-irreversible process, and a peak separation of 154 mV, indicating a quick transfer of electrons. With Ipa/Ipc = 1.0, however, Au-MWCNT-PANI-RuO2 looked to be quasi reversible. The composites (MWCNT-PANI-MO) modified electrodes had a larger peak current than the other electrodes, particularly the bare gold electrode. This is attributed to the enormous surface area produced by the MWCNT and PANI, which allows for facile electrolyte transport and easy electron flow due to the reduction and oxidation reaction of the TiO2 and RuO2 NPs at the electrode. The goal of this research was to find the optimal electrode for improving electron transport qualities. The changes in CV observed2 and Au-MWCNT-PANI-TiO2 electrodes were calculated to be 0.18 nM, and 0.16 nM, respectively. The interference analysis with DPV revealed that the amino acid and EP peaks were well separated. As a result, epinephrine can be detected by using modified electrodes without being hampered by AA signals. The analytical performance of the manufactured sensors was tested for epinephrine sensing and detection in a medicinal and pharmaceutical sample and found to be satisfactory 4\u2212/3\u2212 solution(5 mM) containing 0.1 M KCl, a typical redox peak was detected. The redox peaks were significantly reduced, followed by the electrode exterior which was changed with graphene oxide to obtain graphene oxide/GCE, showing that the graphene oxide hampered electron transport. The modified L-glutamic acid electrode was made by sweeping the voltage from 0.8 to 1.7 V at 100 mV/s for ten cycles in phosphate buffer containing 5 \u00d7 10\u22123 M L-glutamic acid solution at pH 7.0, emanating in a reduction in peak current. In the CVs of L-glutamic acid-graphene/GCE, the redox peak was bigger than the redox peaks in graphene oxide/GCE. The resultant nanocomposite improved [Fe(CN)6]4\u2212/3\u2212 solution significantly, with an unambiguous redox peak between 0.2 and 0.6 V that was not evident in CV curve B.On a CHI660A electrochemical workstation, electrochemical measurements were taken . A CHI 660D electrochemistry workstation was used to perform electrochemical impedance spectroscopy (EIS) experiments . In all of the studies, the working electrode was a modified GCE, while the reference and auxiliary electrodes were a saturated calomel and a platinum wire, respectively. A reaction between L-glutamic acid and graphene produced L-glutamic acid-graphene nanocomposites. In KOH solution with a detection limit of 30 nM (S/N = 3). The analytical features of the L-glutamic acid-graphene modified electrode were compared to other epinephrine detection sensors to highlight the advantages. The outcomes are shown in The modified electrodes were utilized to evaluate the current response of epinephrine at varied doses using differential pulse voltammetry under optimum experimental conditions. It is observed that there was a linear connection between peak current and epinephrine concentrations between 1.0 \u00d7 102. The cyclic voltammetric studies of norepinephrine via modified surface electrode were conducted by using 0.01 mol/L potassium ferrocyanide and potassium ferricyanide in 0.01 mol/L phosphate buffer of 7 pH, showing the potential range of 0.4 to 1.0 V against Ag/AgCl with a scan rate of 0.1 V/s. The chronoamperometry for noradrenaline or norepinephrine sensing and detection was carried out in 0.1 mol/L PBS with a fixed potential of 0.5 V against Ag/AgCl, with a stabilization period of 100 s. The GCE was polished and layered with 0.05 \u00b5m alumina solution before being rinsed with de-ionized water and put in an ultrasonic bathtub for about 5 min in ethyl alcohol, and 10 min in a water bath, before being modified. The prepared electrode was immersed in 0.1 mol/L H2SO4 solutions after electrochemical polishing, and cyclic voltammetry was used to measure the potential range of \u22121 to 1 V against the Ag/AgCl solution. Following that method, ten milligrams of MWCNT and ten milligrams of Fco98 were suspended in two milliliters of ethanol. For 30 min, an ultrasound was used to disperse the suspension.The electrochemical analysis was proceeded using cyclic voltammetry, and chronoamperometry was carried out using an Autolab electrochemical system Galvanostat Model PGSTAT 30 with GPES software. A thermostatic electrochemical cell of 15 mL with three electrodes was used to make electrochemical measurements, 3.0 mol/L solution of Ag/AgCl/KCl), and a platinum wire was assembled as counter and reference electrodes, respectively, with a working electrode of area = 0.071 cm98 was discovered to have a precise and auspicious demand in the field of neuroscience for determining this neurotransmitter [The best composition of the electrode was 4 \u03bcL of cobalt ferrite and 10 \u03bcL of CNTs in 0.1 mol/L buffer solution of phosphate at pH 7.0. In 0.1 mol/L PBS, the electrode exhibits electrochemical activity across a broad potential range from 0.4 to 1.0 V against Ag/AgCl solution, showing excellent dynamism, electrode robustness, and stability. The original GC electrode was used to catalytically oxidize noradrenaline at +0.60 V opposite to the Ag/AgCl solution by fixing the current at 0.17 A, whereas the modified GCE containing cobalt ferrite NPs and CNTs was used with a solution of Ag/AgCl at +0.54 V with 0.23 mA of current. The concentration limit was 760 nM for the anodic peak current (Ipa) against the concentration of Noradrenaline, using the amperometry technique at the highly modified surface electrode which is linear in the concentration range from 160 to 1910 nM. In this approach, the modified electrode GC/MWCNT/Fconsmitter .\u22121 in 10 mL of double-distilled water. Before each experiment, the GCE was layered and waxed with 0.05 m alumina powder and gold sandpaper. Between each polishing process, the layered electrode was cleaned thoroughly with double-distilled water before being immersed in 50% nitric acid, ethyl alcohol, and double de-ionized water in an ultrasonic bath and dried in the air. A total of 4 \u03bcL of graphene suspension was poured onto the GCE and drying it under an IR lamp, a graphene-modified electrode (GME) was created. The redesigned electrode had an outstanding electrocatalytic impact on the oxidation of norepinephrine in phosphate buffer solution (NE). This was also utilized to determine NE, UA, and AA by CV in the presence of epinephrine. With NE concentrations ranging from 600\u2013120,000 nM and the LOD of 4000 nM, the decreased peak current showed a linear relationship. By detecting the reduced peak current in PBS pH 7.0, the modified electrode can be utilized to measure NE in the presence of UA, epinephrine, and AA. The method was successfully applied to assessing NE injection samples and has great sensitivity, selectivity, and stability [For electrochemical measurements, the electrochemical workstation CHI 660 was employed, and the graphene was characterized using the Varian660-IR at Agilent Technologies, Santa Clara, CA, USA. A well-assembled three-electrode setup with f a bare or GCE/graphene with a3 mm diameter was used as the working electrode, while platinum wire was used as a counter electrode, and an electrode containing Ag/AgCl solution was used as a reference electrode.. After preparing the nano-graphene, the graphene-modified electrode was prepared as 7 mg of graphene and was disseminated at a concentration of 0.7 mg mLtability .3 nanowires, 0.7 g of (NH4)2MoO4 was properly dissolved in 40 mL double-distilled water with continuous moderate stirring. To maintain the pH at 1.0, a high concentration of HCl was put in drop by drop. After 30 min of stirring, the reaction mixture was placed in a teflon-lined autoclave followed by the hydrothermal reactions for varying lengths of time at 180 \u00b0C temperature. To eliminate any impurities or unreacted precursors, the final product was thoroughly washed numerous times with ethyl alcohol and deionized (DI) water. The product was well dried in an oven at 80 \u00b0C for more than 3 h, further described and manufactured as an electrode for the electrochemical oxidation of NE after it was ground. A Bio-Logic VMP-300 potentiostat was used to carry out the important electrochemical experiments using a standard three-electrode system consisting of an auxiliary electrode, reference electrode, and working electrode made by using platinum wire, Ag/AgCl solution, and GCE/modified or unmodified (3 mm of diameter), respectively. The GCE was properly and gently polished with a 0.05 m alumina slurry and properly rinsed with distilled water before alteration. To remove any adsorbed alumina particles, de-ionized water, and 100% ethyl alcohol were used to wash the electrode carefully. The MoO3 NWs were dispersed in a 0.2% nafion solution in 1 mL. After that, these suspensions in a very small amount of approximately 20 \u00b5L were dropped on the pure and clean GCE, then allowed to dry at 25 \u00b0C or room temperature. After that, all electrodes were cleaned two or three times with de-ionized water before being employed in the selective recognition and binding studies. For all the electrochemical studies, the potential scanning was repeated in a range between 0.0 to 0.8 V, fixing the scan rate at 30 mV/s. The MoO3 NWs were made using a one-step hydrothermal process. The pH of the reaction medium affects the development of high-aspect-ratio nanowires during synthesis. 1-dimensional MoO3 NWs are generated in the first stage as follows:For the typical synthesis of MoO3 nanowires (NWs) were created. These MoO3 NWs were also employed as a mediatorless biosensor electrode with glassy carbon electrodes (GCEs) for the sensing and accurate detection of NE using cyclic voltammetric techniques. In the electrochemical detection of NE, the GCE/MoO3 NWs exhibited a fantastic response time of 2 s and a LOD of 0.11 nM MoO 0.11 nM . The 1D 2 NPs, assisted by cetyltrimethylammonium bromide (CTAB). Electrochemical measurements proceeded using a CH Electrochemical Workstation Instrument CHI-900 , which features an electrode system of three electrodes, including a well-assembled GCE working electrode, wire of platinum, and 3 M KCl solution of Ag/AgCl as an auxiliary and reference electrode, respectively. The measurements were carried out by using SWV in a buffer solution of Phosphate at pH 5.0, including NEP and EP at an amplitude of 50 mV, frequency of 10 Hz throughout the potential range from 0.6 to \u22120.4 V with the step potential of 5 mV.For the simultaneous sensing of epinephrine and norepinephrine, a highly selective, accurate, and sensitive electrochemical sensor was developed by using SnO2 nanoparticles of modified electrodes was examined using the SWV method using buffer solution of phosphate at 5.0 pH to observe reduction electrocatalytically. According to the figure, the curve A of Bare GCE and SnO2/GCE, the two neurotransmitters, had a relatively low current response as in curve B. By observing curve C, when the CTAB-SnO2/GCE was utilized, the combination showed two excellent, sharp, clear, accurate, and sensitive peaks corresponding to NEP and EP at 0.309 V and \u22120.045 V results in a high separation of the peak in PBS of pH 5.0 with value equals to 0.354 V. For NEP and EP, the curve of calibrations was achieved under ideal conditions throughout a large concentration range of 0.1\u2013300 and 0.1\u2013250 M, respectively, showing the lowest values of detection limits i.e., 6 nM and 10 nM. The outstanding activity and selectivity were revealed by interference experiments for the modified electrodes in the presence of interferents viz. ascorbic and uric acid. The proposed modified electrode has been auspiciously used to sense NEP and EP, simultaneously, in urine samples of human beings with satisfactory results [The behavior showed by EP and NEP in electrochemical studies at different GCE/SNO results .Comparison of various modified GCE for electrochemical detection of norepinephrine in The electrochemical analysis was studied using an Autolab-Potentiostat of Type 3 and 4.9 GPES software Utrecht, The Netherlands. The raw SWV signals observed and obtained by the electrochemical instrument were recorded in this software after being corrected using the moving average approach, with a peak width of 0.03 V and smoothed using an algorithm of the Golay and Savicky. All the voltammetric measurements were carried out with a system with three electrodes at room temperature using a 10 mL glass electrochemical Cell. The counter and reference electrodes were made of wire of platinum and a 3 mol/L solution of Ag/AgCl.Using the cyclic voltammetry approach, NE showed one clear well-defined, adsorption controlled, and irreversible oxidation peak at about +0.85 V against Ag/AgCl solution electrode in 0.1 M Britton\u2013Robinson buffer (BR) fixing pH at 2.0. On the oxidation peak current, the influence of supporting electrolyte, solution pH, and instrumental variables were optimized. In BR buffer solution using the square-wave stripping mode (SWSM), it was discovered that an excellent connection was developed between the concentration of NE and oxidation peak current in the range of 1\u2013100 \u03bcg m/L with a detection limit value of 0.254 \u03bcg m/L i.e., 1200 nM. This study effectively shows that the bare BDD electrode, a state-of-the-art electrode material, may be used as an electrochemical sensor to determine NE in pharmaceutical formulations, without the need for any chemical modifications. Furthermore, the NE concentration in the commercial pharmaceutical formulation is statistically equivalent to the NE concentration obtained using the HPLC method as the reference technique. Because it is relatively easy, economical, and quick, the combination of SWV and oxygen-terminated OT-BDD is a handy and advantageous alternative for determining NE in pharmaceutical formulations .Comparison of norepinephrine detection using various electrodes are compiled in An Autolabpotentiostat/galvanostat was used to perform the electrochemical experiments. The GPES software was used to regulate the experimental settings. At 25 \u00b0C temperature, a standard system of the three-electrode cell was employed. The reference, auxiliary, and working electrodes were 0.3 M Ag/AgCl/KCl solution, platinum-wire, and GQDs/2CBF/CPE/IL, respectively. pH measurements were taken with a Metrohm 710 pH meter. GQDs/IL/CPEs were made by using a mortar and pestle to combine graphene quantum dots (0.2 g), graphite powder (0.8 g), and ionic liquids of about 0.8 mL. After that, the paste was stuffed into the glass tube and the electrical circuit or contact was made using a Cu-wire put into the carbon paste. The samples of urine were refrigerated immediately after collection. Centrifugation of 10 mL of a sample at 2000 RPM for fifteen minutes was done. A 0.45 m filter was used to filter the supernatant. The prepared solution was then shifted to 25 mL of volumetric flask and diluted to the desired concentration with PBS at pH = 7.0. Different doses of norepinephrine and acetylcholine were used to anesthetize the diluted urine samples. The suggested process used the usual addition method to determine the content of norepinephrine and acetylcholine.CV and DPV in a phosphate buffer solution were used to examine the electrochemical behavior of norepinephrine at GQDs/IL/CPE (pH 7.0). As an electrochemical sensor, GQDs/IL/CPE showed catalytic activity in the oxidation of norepinephrine. On the modified electrode, the potential of norepinephrine oxidation was shifted to more negative potentials, and the oxidation peak current increased. Furthermore, at E = 390 mV, the GQDs/IL/CPE presents two independent oxidation signals, enabling the simultaneous study of norepinephrine and acetylcholine. The obtained data revealed a solid linear association between norepinephrine and acetylcholine oxidation peak currents and their concentrations in the range of 20\u2013400,000 nM. Finally, GQDs/IL/CPE has good accuracy and precision in determining norepinephrine (LOD = 60 nM) and acetylcholine in norepinephrine ampoule, acetylcholine ampoule, and urine samples . FajardoComparison of electrochemical norepinephrine detection at various modified electrodes is compiled in In this review, we highlighted the most recent advancements in electrochemical sensors for the detection of monoamine NTs in real samples at a nanomolar scale. Because these hydroxyl group-based monoamines are created locally in a specific region of the brain, the number of biomolecules is also present in the extracellular space. The precise quantification of NTs in vivo is difficult due to the ultra-low concentrations of NTs (nM). To overcome this problem, various kinds of modified electrodes are formed, used, and explained to determine the sensitivity of NTs. Because of their exceptional features, such as high catalytic activities, electron transfer rates, and electrical conductivity, the diverse carbon, metal/metal oxides, and polymeric nanocomposite sensing materials are incorporated into macro or microelectrodes for quick detection of NTs. The unique advantages of microelectrodes over the traditional electrochemical sensors are as they are reflected in the study of the active mechanism of electrochemical sites: the modulation strategies are more precise and controllable; they provide in situ analysis and characterization; and the microelectrode\u2019s design allows for a more efficient reduction in the theoretical model; and they allow for a more consistent confirmation of the theoretical, microscopic, and macroscopic levels. Diverse modern materials, such as carbon derivatives, metal, and polymeric nanocomposite are used as electrode materials, and they are also used as electrode modifiers. Carbon derivatives, such as graphite, graphene, fullerene, and diamond electrodes are commonly used in sensing applications. In general, carbon materials are preferred due to their wide potential window, cost-effectiveness, and biocompatible properties. Still, they have disadvantages, such as higher ET resistance when compared to metals. Metals-based modifiers are not biocompatible and are hazardous. Therefore, there is a huge scope for nanocomposite electrodes to overcome these disabilities; however, cost and tedious preparation and characterization of nanocomposites are still major concerns. These sensing materials recently received a lot of attention and give a very low limit of detection with a precise linear range. Future research will focus on developing high-throughput miniaturized probe arrays using micro/nanofabrication techniques and integrating nanostructured sensing materials to improve long-term stability, sensitivity, selectivity, and reproducibility for rapid diagnostic and point-of-care detection of NTs in real-time. We emphasized the significant progress made in the field of electrochemical sensing for in vivo neurotransmitters through this review. When it comes to diagnostic gadgets, however, there are still certain obstacles to overcome. For starters, probe size is still an issue. Implantation causes a physical change in the brain\u2019s tissue. To limit injury and inflammation, ongoing efforts to produce probe diameters comparable to interneuron lengths are being made. Furthermore, synaptic measurements will be possible due to continuous downsizing to nanoscale sizes. Second, electrochemical sensing can only detect a limited number of electroactive substances. Electrochemical sensors cannot detect peptides and proteins; thus, they cannot be measured with the same spatiotemporal resolution as smaller, more traditional neuromodulators. Modifications to electrodes using aptamers could be on the horizon for this type of research in the future. Finally, and maybe most importantly, there are no clinical measurements in humans. However, because of the tremendous progress in the field described here, we are certain that such measurements will be made possible soon."} +{"text": "The crafted electrochemical platform demonstrated excellent stability, specificity, and anti\u2010interference capability towards the sensing of dopamine.A glassy carbon electrode (GCE) has been modified by an in\u2005situ electrochemical reduction of an aryldiazonium salt generated from the reaction of 4\u2010aminobenzoic acid and sodium nitrite in acidic ethanolic solution. The as\u2010prepared phenyl carboxylic acid\u2010modified glassy carbon electrode has been, for the first time, used for the electrochemical determination of dopamine. Under optimal experimental parameters, outstanding electrocatalytic activity, high sensitivity at a LOD of 5.6\u00d710 A robust phenyl carboxylic acid\u2010modified electrode has been prepared by electrochemical reduction of a diazonium salt. The surface of the modified electrode is negatively charged, enabling not only sensitive detection of dopamine but also excellent selectivity in the presence of ascorbic acid. The process of neurotransmission is performed by neurotransmitter and it is also vital in the development and persistence of addiction.In present investigation, we modified glassy carbon electrode (GCE) by the electrochemical reduction of 4\u2010carboxyphenyldiazonium generated from the reaction of 4\u2010aminobenzoic acid with nitrous acid to make phenyl carboxylic acid\u2010modified GCE (PC\u2010GCE). The PC\u2010GCE has been explored for DA detection for the first time. Its application for selective determination of DA in the presence of AA was investigated.The GCE was modified by aryldiazonium salts obtained by the reduction of 4\u2010aminobenzoic acid in the presence of nitrous acid. The mechanism of surface modification of GCE is as follows. Firstly, 4\u2010aminobenzoic acid reacted with nitrous acid to produce 4\u2010carboxyphenyldiazonium. Then, the electrochemical reduction of electrogenerated 4\u2010carboxyphenyldiazonium produced phenyl carboxylic acid radicals on the surface of GCE. Finally, these phenyl carboxylic acid radicals reacted with glassy carbon to make PC\u2010GCE Scheme\u2005.m [Fe(CN)6]3\u2212/4\u2212. A typical redox signal of Fe(CN)63\u2212/4\u2212 was observed on the bare GCE. Contrarily, the electrochemical signal of [Fe(CN)6]3\u2212/4\u2212 was completely restrained on the PC\u2010GCE, which could be accredited to the presence of electrostatic repulsive forces between phenyl carboxylic group and [Fe(CN)6]3\u2212/4\u2212 redox couple and the blocking effect of the film.m [Fe(CN)6]3\u2212/4\u2212 were also examined as shown in Figure\u2005The designated electrode was compared with bare GCE to scrutinize the electrochemical characteristics of the designated PC\u2010GCE. Figure\u2005m AA and 1\u2005mm DA in 0.1\u2005m PBS (pH\u20057.0). All the CV measurements demonstrate quasi\u2010reversible peaks of bare and developed GCE. On modified electrode/PC\u2010GCE, a sharp oxidation peak corresponding to DA was observed whereas no redox peak corresponding to ascorbic acid was observed demonstrating the minimal oxidation of AA, whilst the DA oxidation was enhanced at the PC\u2010GCE.Figure\u2005m AA and 1\u2005mm DA at decorated PC\u2010GCE in 0.1\u2005m PBS (pH\u20057.0). DA exhibits a strong peak while AA shows a negligible peak at PC\u2010GCE, indicating good selectivity. The selectivity of the modified electrode is ascribed to \u2212COOH groups that are crafted at the surface of designated probe encompassing the attachment of the positively charged DA while resisting the adsorption of negatively charged AA molecules on the modified electrode at pH\u20057.0.Figure\u2005m DA in 0.1\u2005m PBS of diversified pH at the decorated PC\u2010GCE were recorded as illustrated in Figure\u2005\u2212 and DA moieties. Owing to the decrease in peak current of DA with the increase in pH in the range of 7.0\u20139.0, pH\u20057.0 was selected as the optimum pH value for further analysis.For the selection of a suitable pH to enhance the signal of the designated sensor, the SWVs of 1\u2005mFigures\u2005Under optimal parameters, the influence of modification time on the peak responses of DA at the decorated PC\u2010GCE is represented in Figure\u2005m PBS as depicted in Figure\u2005m with an R2 of 0.998. The LOD was estimated to be 5.6\u00d710\u22129\u2005m at a S/N of 3. We have listed a comparison of this modified electrode with other previously reported electrodes in Table\u2005m DA in 0.1\u2005m PBS of pH\u20057. The recorded RSD of the proposed methodology was estimated to be 3.1\u2009% after 50 consecutive CVs, signifying that the decorated probe had a remarkable reproducibility with an effective degree of accuracy and precision. The modified sensing platform was retained for 15\u2005days in the air to investigate the stability of the designated platform. The recorded response showed that 92.9\u2009% of the initial current was retained.Under the optimum parameters, the SWVs of PC\u2010GCE were scrutinized for different concentrations of DA in 0.1\u2005m DA which was added with a 100\u2010fold higher concentration of citric acid, glucose, and cystine, together with a 1000\u2010fold higher concentration of Na+ and K+. As depicted in Figure\u2005To examine the influence of interfering species on the prepared GC electrode, its peak responses were explored by analyzing the solution comprising 1\u2005mThe proposed sensing strategy combines high sensitivity and excellent anti\u2010sensing properties towards ascorbic acid (AA) and other electro\u2010active groups. These characteristic features make the phenyl carboxylic acid\u2010modified electrode advantageous for the selective detection of dopamine (DA) in neutral pH (7.0) with an enhanced sensitivity, a remarkable LOD and a wide linear range. Additionally, the innovative method proves to be an excellent way for the selective detection of DA in real samples.p\u2010amino phenyl carboxylic acid, and ascorbic acid (AA) were purchased from Sigma Aldrich. All other reagents were of analytical grade and were used as such. Double distilled water was used throughout the experiment.DA, m 4\u2010aminobenzoic acid in ethanol (1\u2005mL), 1.0\u2005m HCl (1\u2005mL), and 0.1\u2005m NaNO2 (0.11\u2005mL) at ambient temperature under an applied voltage of \u22120.45\u2005V.Firstly, the glassy carbon electrode/GCE was refined by using alumina slurry of 0.05\u2005\u03bcm grain size followed by rinsing with deionized water. The developed electrode was sonicated in ultra\u2010pure distilled water and ethanol for several minutes to eliminate the remains of alumina. To prepare the modified sensor, the electrode was dried at room temperature. The polished and dried GC electrode was coated in an acidic ethanolic solution comprising 10\u2005mm KCl) electrode as a reference electrode. All the electrochemical studies were scrutinized with respect to the reference electrode and all the measurements were performed at ambient temperature. The sample solution was continuously stirred during the pre\u2010concentration step using a stirrer. The sensor was immersed in 1.0\u2005mm DA solution in 0.1\u2005m PBS to maintain the pH of the solution at pH\u20057.0. The dependence of peak responses on modification time was analyzed in the range from 60\u2013480\u2005s. Furthermore, the effect of pH, accumulation potential, frequency, and deposition time on current signals of DA was scrutinized. All the electrochemical investigations including SWV and CVs at bare G.C and developed PC\u2010GCE of 1.0\u2005mm DA solution were executed under fitted parameters.The electrochemical studies including CV and Voltammetric analysis were conducted via CHI660A electrochemical workstation together with a three probe electrochemical cell consisting of modified GC (3.0\u2005mm in diameter) as indicator electrode, a Pt\u2010wire as the auxiliary electrode, and a silver/silver chloride should be addressed to the authors.Supporting InformationClick here for additional data file."} +{"text": "Variation in immune response to COVID-19 vaccines is observed among different ethnicities. We aimed to describe the reinfection rates, change in antibody titers, and adverse events among Filipinos.This is a secondary analysis of a cohort study of 307 participants within one year of having COVID-19 infection. We measured COVID-19 antibody levels at pre-determined timepoints . We monitored for COVID-19 symptoms and obtained details on COVID-19 vaccination. An adjudication committee classified the participants as probable, possible, or unlikely COVID-19 reinfection. We determined the probable reinfection rate, adverse events, and the geometric mean titer (GMT) ratio of pre- and post-vaccination antibody levels according to type and brand of COVID-19 vaccine.At the end of the follow-up period, 287 (93.5%) out of 307 study participants were fully vaccinated, 1 was partially vaccinated (0.3%), and 19 were unvaccinated (6.2%). Among the fully vaccinated participants, those given mRNA vaccines had the lowest reinfection rate , followed by viral vector vaccines . We observed the highest reinfection rate among those given inactivated virus vaccines . The reinfection rate was 8.6 cases/100 person-years for unvaccinated participants and 3.6 cases/100 person-years for partially vaccinated participants. We observed the largest rise in antibody titers among those given mRNA vaccines (GMT ratio 288.5), and the smallest rise among those given inactivated virus vaccines (GMT ratio 16.7). We observed the highest percentage of adverse events following immunization with viral vector vaccines (63.8%), followed by mRNA vaccines (62.7%), and the lowest for inactivated virus vaccines (34.7%). No serious adverse events were reported.Vaccinees given the mRNA vaccines had the lowest reinfection rate and the highest rise in antibody titers. Vaccinees given inactivated virus vaccines had the highest reinfection rate, smallest rise in antibody titers, and lowest percentage of adverse events. The small sample size and imbalanced distribution of the type of vaccines received limits the external generalizability of our results.The cohort study was registered at the Philippine Health Research Registry on December 14, 2020 (PHRR201214-003199).The online version contains supplementary material available at 10.1186/s12879-023-08743-6. The development of coronavirus disease 2019 (COVID-19) vaccines greatly altered the course of the pandemic. Vaccines prevented 14.4\u00a0million deaths globally in the first year of vaccine administration 19). The severity of their initial COVID-19 infection was mild for 170 participants (55.4%), moderate for 24 participants (7.8%), severe for 28 participants (9.1%), critical for 7 participants (2.3%), and asymptomatic for 78 participants (25.4%).After one year of follow-up, 287 (93.5%) were fully vaccinated, 1 was partially vaccinated with CoronaVac (0.3%), and 19 were unvaccinated (6.2%). There were 220 participants (71.7%) who received booster doses, of which 217 participants received one booster dose, and 3 participants received two booster doses.Among the 287 fully vaccinated participants, the most common type of primary series vaccine received was inactivated virus vaccine . There were 66 participants (23.0%) who received non-replicating viral vector vaccines, and 64 (22.3%) who received mRNA vaccines. The most common brand received was CoronaVac by Sinovac , followed by AZD1222 by Oxford/ AstraZeneca , and BNT162b2 by Pfizer/BioNTech .Among those who received a booster, 168 (76.4%) received mRNA vaccine, 39 (17.7%) received non-replicating viral vector vaccine, and 13 (5.9%) received inactivated virus vaccines. Table\u00a0The rate of probable reinfection among unvaccinated participants was 8.6 cases per 100 person-years , while the rate among partially vaccinated participants was 3.6 cases per 100 person-years . Among the fully vaccinated participants, we observed the lowest rate of reinfection among those given mRNA vaccines as primary series at 19.2 cases per 100 person-years . Participants who received inactivated virus vaccines had the highest reinfection rate at 32.7 cases per 100 person-years , followed by CoronaVac at 32.9 cases per 100 person-years . Participants who received BNT162b2 had a reinfection rate of 14.2 cases per 100 person-years . There was no identified case of probable reinfection among those given Ad26.COV2.S and inactivated Vero cell vaccines.Table\u00a0By vaccine brand, we observed the largest rise in antibody titer after primary series vaccination with BNT162b2 (GMT ratio 302.8), followed by mRNA-1273 (GMT ratio 265.5), and Sputnik V (GMT ratio 232.3). We observed the smallest rise in antibody level after primary series vaccination with inactivated Vero Cells . We observed the second lowest rise in antibody levels after primary series vaccination with CoronaVac (GMT ratio 16.9).The average interval from pre-vaccination titer determination to vaccination varied across the vaccine brands, ranging from 26.0 days (AZD1222) to 67 days (inactivated Vero Cells). The average time interval from administration of the second vaccine dose (first dose for participants who received Ad26.COV2.S) to post-vaccination titer determination ranged from 46.0 days (Ad26.COV2.S) to 93 days (inactivated Vero Cells).The GMT ratios of post- and pre-booster RBD-specific antibody titers according to the type of booster regimen is shown in Fig.\u00a0Table\u00a0By vaccine brand, we observed the highest percentage of adverse events following immunization with mRNA-1273 (74.6%), followed by Sputnik V (63.6%) and AZD1222 (60.3%). No adverse events were reported among the 2 participants who received inactivated Vero cells vaccine, while only 34.9% of those given CoronaVac reported adverse events.There were no serious adverse events reported by the study participants. We observed the highest percentage of systemic reactions among those given mRNA vaccines. Of the 295 doses of mRNA vaccines administered, 121 (41.0%) resulted in systemic reactions. Of the 162 doses of non-replicating viral vector vaccines administered, 65 (40.1%) resulted in systemic reactions. We observed the lowest percentage of systemic reactions among participants given inactivated virus vaccines. Of the 326 doses of inactivated virus vaccines administered, only 46 (14.1%) resulted in systemic reactions.The most common systemic reaction was fever for non-replicating viral vectors (27.8%) and mRNA vaccines (18.6%). The most common systemic reactions were drowsiness and headache for inactivated virus vaccines (4.9%). For all vaccine types, local reactions included pain on injection site and arm heaviness. The specific types of systemic and local reactions are summarized in Table\u00a0Considering the individual vaccine brands, we observed the highest percentage of systemic reactions among those who received mRNA-1273. Of the 130 doses of mRNA-1273 vaccines administered, 76 (58.5%) were associated with systemic reactions. This was followed by AZD1222 ; Sputnik V ; BNT162b2 ; and Ad26.COV2.S . We observed the lowest percentage of systemic reactions among those given CoronaVac, with 46 out of the 324 doses administered resulting in systemic reactions (14.2%).The most common systemic reaction was fever for mRNA-1273 (30.8%), AZD1222 (30.5%) and Sputnik V (18.2%). Body pain or myalgia was the most frequent systemic reaction for BNT162b2 (11.5%), while drowsiness and headache were the most frequent systemic reactions for CoronaVac (4.9%). Local reactions included pain on injection site and arm heaviness for CoronaVac, AZD1222, BNT162b2, and mRNA-1273, and pain on injection site for Sputnik V and Ad26.COV2.S. The specific types of systemic and local reactions are summarized in Table\u00a0We found that study participants who received mRNA vaccines as primary series had the lowest reinfection rate and the highest increase in antibody titers. Those who received inactivated virus vaccines had the highest reinfection rate and the lowest rise in antibody titers. In terms of individual brands of COVID-19 vaccine, there were no identified cases of probable reinfection among participants given inactivated Vero Cells and Ad26.COV2.S vaccines as primary series. However, these two brands had the lowest number of recipients . We observed the highest reinfection rate among vaccinees who received Sputnik V, followed by CoronaVac.We noted that the highest reinfection rate was observed among participants who received Sputnik V despite the large rise in antibody titer after primary series vaccination. This may be due to other variables that influence reinfection rates, including age, co-morbidities, employment, and exposure to COVID-19 .The reinfection rates of the unvaccinated and partially vaccinated study participants were paradoxically lower compared to those who were fully vaccinated, regardless of type of vaccine. This may be explained by the epidemiologic context in relation to the timing of vaccination, as shown in Fig.\u00a0Our findings are consistent with other studies that found that higher antibody levels were associated with a lower risk of COVID-19 infection . In our The mRNA vaccines consist of a lipid nanoparticle enveloping an mRNA molecule that encodes the viral Spike protein. This vaccine induces antigen-specific follicular helper T cell development in the germinal centers of the draining lymph nodes, which would lead to B cell activation, antibody isotype switching, affinity maturation, and formation of plasma cells and memory B cells . This meWe also observed that the GMT ratio of all types of vaccine exceeded 4. A four-fold increase in antibody titers is generally the minimum rise interpreted as an \u201cadequate\u201d antibody response . This suAmong the study participants who received booster doses, the largest GMT ratios were observed among those with inactivated virus vaccine as the primary series, likely due to the lower pre-booster titer compared to those who received viral vectors and mRNA vaccines as primary series. An inverse relationship with pre-immunization titer level and degree of humoral response has been demonstrated in other studies, where a higher pre-vaccination titer is associated with a lower rise in antibody post-vaccination .The GMT ratio was higher with heterologous boosters after inactivated virus and viral vectors primary series compared to homologous boosters. However, among those who received mRNA vaccine as primary series, the GMT ratio was higher for those given homologous boosters compared to heterologous boosters. These findings are consistent with studies in other countries reporting better immunogenicity for heterologous compared to homologous boosters for inactivated virus vaccines, and conversely, better immunogenicity for homologous boosters for mRNA vaccines , 19. TheIn this study, adverse events following immunization were more frequently reported among mRNA and viral vector vaccines compared to inactivated virus vaccines. This finding is consistent with other studies , 20. IncOur study had the following limitations. First, in the primary cohort study we conducted, we could not do laboratory confirmation of reinfection due to the unavailability of routine genomic testing for symptomatic patients. Instead, an adjudication committee determined whether reported events were probable reinfections. Hence, the reinfection rates we report in this study refer to probable reinfection rather than confirmed reinfection. Furthermore, reinfection rates in the main cohort study were probably underestimated because testing via RT-PCR or antigen test was encouraged but not provided for free for participants with symptoms consistent with COVID-19. Some symptomatic study participants refused to undergo testing. The study was also unable to detect cases of asymptomatic reinfection. Thus, the reinfection rates reported in this study are likely to be underestimated.Another limitation is that the antibody titers measured were binding antibodies, not neutralizing antibodies. Tests for neutralizing antibodies are ideal since these are the antibodies that directly interfere the binding and uptake of virus to the host cells . At the Another limitation in this study is the variation in the timing of antibody titer determination in relation to vaccination, since antibody tests were performed at fixed time points based on the initial COVID-19 infection. Means and standard deviations of the number of interval days between the antibody determination and vaccination were reported to provide appropriate context to the results.Moreover, the semi-quantitative laboratory test used in the study had an upper limit of detection of 250 U/mL. We performed 10-fold dilution according to manufacturer recommendations to increase the upper limit of detection to 2,500 U/mL. However, several results still exceeded 2,500 U/mL. We performed 100-fold and 1,000-fold dilutions to increase the upper limit of detection to 250,000 U/mL; however, the resulting values at this higher range may have diminished accuracy.Another limitation is the presence of several confounding variables that affect reinfection rate and antibody titers aside from vaccination. Due to these issues, and the small sample size of the completed cohort study, this study was designed as a descriptive study and the results are intended to be exploratory in nature. Inferential statistics was not done.Furthermore, the completed cohort study primarily aimed to determine symptoms of COVID-19 reinfection during the follow-up calls. Participants were also asked if they received the COVID-19 vaccine, and the type, brand and date of vaccination. From this recorded data, adverse events following immunization were extracted. However, this is prone to reporting bias. Although COVID-19 symptoms have several similarities as systemic adverse events following immunization, other symptoms such as rashes, flushing or local erythema which were not directly asked by the researchers may have been missed if the information was not volunteered by the study participants. Moreover, data for this study was heavily reliant on the completeness and accuracy of the data recorded in the completed cohort study.In a cohort of Filipino individuals previously infected with COVID-19, vaccination with different types and brands of COVID-19 vaccines resulted in varying reinfection rates and increase in antibody titers. We observed the lowest reinfection rate and the highest rise in antibody titers among participants who received the mRNA vaccines as primary series. We observed the highest reinfection rate, the smallest rise in antibody titers, and the lowest percentage of adverse events among participants who received inactivated virus vaccines as primary series. The external generalizability of the results is limited due to several limitations in the main cohort study, including the small sample size and imbalanced distribution in the type of vaccines received.Below is the link to the electronic supplementary material.Supplementary Material 1"} +{"text": "Renatino Canevarolo R, Pereira de Souza Melo C, Moreno Cury N, Luiz Artico LL, Ronchi Corr\u00eaa J, Tonhasca Lau Y, Sousa Mariano S, Reddy Sudalagunta P, Regina Brandalise S, Carolina de Mattos Zeri A and Andr\u00e9s Yunes J (2022) Glutathione levels are associated with methotrexate resistance in acute lymphoblastic leukemia cell lines. Front. Oncol. 12:1032336. doi: 10.3389/fonc.2022.1032336\u201d. The correct citation is \u201cCanevarolo RR, Melo CPS, Cury NM, Artic LL, Corr\u00eaa JR, Lau YT, Mariano SS, Sudalagunta PR, Brandalise SR, Zeri ACM, and Yunes JA (2022) Glutathione levels are associated with methotrexate resistance in acute lymphoblastic leukemia cell lines. Front. Oncol. 12:1032336. doi: 10.3389/fonc.2022.1032336\u201d. The publisher apologizes for this mistake.Due to a production error, the given names, and surnames of the authors in the citation were changed to \u201cThe original version of this article has been updated."} +{"text": "In the field of computer vision, hand pose estimation (HPE) has attracted significant attention from researchers, especially in the fields of human\u2013computer interaction (HCI) and virtual reality (VR). Despite advancements in 2D HPE, challenges persist due to hand dynamics and occlusions. Accurate extraction of hand features, such as edges, textures, and unique patterns, is crucial for enhancing HPE. To address these challenges, we propose SDFPoseGraphNet, a novel framework that combines the strengths of the VGG-19 architecture with spatial attention (SA), enabling a more refined extraction of deep feature maps from hand images. By incorporating the Pose Graph Model (PGM), the network adaptively processes these feature maps to provide tailored pose estimations. First Inference Module (FIM) potentials, alongside adaptively learned parameters, contribute to the PGM\u2019s final pose estimation. The SDFPoseGraphNet, with its end-to-end trainable design, optimizes across all components, ensuring enhanced precision in hand pose estimation. Our proposed model outperforms existing state-of-the-art methods, achieving an average precision of 7.49% against the Convolution Pose Machine (CPM) and 3.84% in comparison to the Adaptive Graphical Model Network (AGMN). The rapid advancement of deep neural networks has meant significant progress in hand pose estimation (HPE), a critical component in AI-driven applications such as human\u2013computer interaction (HCI) and virtual reality (VR). However, HPE remains a complex task due to the flexibility of hand joints, local similarities, and severe occlusions, including self- and object occlusion . TherefoNevertheless, 2D HPE remains a vital research direction, as it forms the foundational building block for 3D algorithms, which achieve estimation results by transposing feature maps from 2D to 3D space ,11. ReflWith the advent of DL, the Convolutional Pose Machine (CPM) emerged Attention mechanisms have significantly advanced deep learning, particularly in enhancing focus on crucial data segments. Visual attention, a key application of this concept, has shown remarkable efficacy across various domains, including image and text processing. In this study, we present the Spatial Deep Feature Pose Graph Network (SDFPoseGraphNet), building upon our previous work . This neWe introduce SDFPoseGraphNet, a novel framework that enhances the capabilities of VGG-19 by incorporating SA mechanisms; our model effectively captures spatial information from hand images, allowing VGG-19 to extract deep feature maps that capture intricate relationships among various hand joints.To address the challenge of accurate pose estimation, we incorporate a PGM into SDFPoseGraphNet. This model utilizes adaptively learned SIM parameters, derived from the deep feature maps extracted by VGG-19, to model the geometric constraints among hand joints. The adaptivity of the parameters enables personalized pose estimation, tailoring the model to the unique characteristics of each individual input image.The model combines FIM potentials and SIM parameters, which play a crucial role in the final pose estimation performed by the PGM. The inference process incorporates techniques like message passing, refining, and enhancing the accuracy of the joint predictions.Our contribution is three-fold and is stated below as follows:This article follows a structured approach with several sections. HPE is a complex task that faces several challenges, including variations in hand poses, limited depth information, and issues related to appearance and occlusion. Researchers have been actively exploring different approaches and techniques to tackle these challenges. One effective approach is the use of multi-view RGB models ,18,19,20CNNs are crucial for HPE ,27, leveThe CPM, although capable of learning strong feature maps, often encounters difficulties in capturing the geometric relationships between joints. As a result, the final predictions of joint positions can exhibit inconsistencies, which pose a significant challenge in tasks involving human pose estimation. This challenge becomes even more prominent in 2D HPE due to the higher level of articulation and the presence of self-occlusion, which further exacerbates the problem. To solve these limitations, we propose SDFPoseGraphNet, a novel framework that enhances the capabilities of VGG-19 with SA, visually depicted in K of the hand during the preliminary stage. The VGG-19 block\u2019s revert feature score can readily integrate with the proposed SDFPoseGraphNet. The final module utilizes the parameters generated by the SIM, which depict spatial constraints among the critical points of the hand. The suggested framework for HPE differentiates itself from previous frameworks by leveraging the SIM, which enables the association of the final graphical module with DCNN [The proposed model incorporates two modules called the First Inference Module (FIM) and the Second Inference Module (SIM). A final Graphical Inference Module is employed to connect the FIM and SIM sequentially. The FIM produces a provisional feature score for the keypoints ith DCNN ,14. In oK, and can be expressed as i and j denote their positions. The term The process of predicting hand poses can be formally described through a graph represented by In this context, the equation models the joint probability of hand poses by considering the interrelationships between keypoints and their positions within a graphical structure. It provides a formal representation of the predictive process used to estimate hand poses, where various components, such as the hand keypoints and the input image, are considered in the calculation of this probability. The parameter Further information and comprehensive explanations regarding each component of the proposed model SDFPoseGraphNet are introduced in the following subsections.k to yield the final attention map. To ensure that the values lie between 0 and 1, this final attention map is passed through a sigmoid activation function, which normalizes the range. These normalized values indicate the significance of each spatial location in the model.In a neural network, attention systems enhance focus on crucial portions of the input data while diminishing the importance of less relevant components. This significant approach, known as visual attention, has made substantial strides in the realm of DL research. These attention mechanisms have shown efficacy in dealing with both text and image data. To augment the existing efficiency of convolutions, numerous methods incorporating visual attention have been developed. This study proposes an innovative approach for HPE tasks by integrating the SA module with the VGG-19 model. This fusion leverages the known effectiveness of the VGG-19 architecture in feature extraction and the ability of attention mechanisms to zero in on salient spatial regions, thus creating a potent combination . These SVGG-19 produces five distinct feature maps quations .(2)Fi=sHere, C denotes a convolution operation. This series of convolutional layers reduces the channel dimension of the feature maps to a value of 128.Equation .(5)S^i=t is the target size Equation :(6)S=S^1In the initial module, the VGG-19 architecture was used up to Conv The symbol The SIM follows a similar methodology as the FIM. In particular, it upholds the most recent framework for 128 feature maps, but it generates 40 feature maps instead of 21 feature maps. The 40 channels or feature maps generated by the SIM represent information about the relationships between hand keypoints. These feature maps capture and encode information about the relative positions, distances, and interactions between different pairs of keypoints on the hand. The result produced by SIM is represented as When training the SIM, we maintain the weights of the FIM in a fixed state, effectively \u2019freezing\u2019 them. As depicted by the gray arrows in The message-passing algorithm is widely utilized in graphical model inference. It typically facilitates the effective calculation of marginal probabilities by using the sum-product operation within a graphical module. The equation for the marginal probability can be expressed in Equation .(8)pi Panoptic Hand Dataset was utilized during our study to assess the proposed model. The dataset consists of a total of 14,817 annotations that correspond to the right hand of individuals captured in images from the Panoptic Studio. The current research examines the process of HPE, as opposed to hand detection. To achieve this objective, annotated hand image patches were extracted from the initial images using a square bounding box with dimensions 2.2 times larger than the hand size. The dataset was partitioned into three subgroups using a random sampling technique as shown in The proposed model is implemented using the PyTorch framework version The mean squared error (MSE) is utilized as the loss function in the model. To prevent the diminution of the loss from reaching nominal values, the loss function is scaled by a coefficient of 35.The formulation of the loss calculation for a model involves a weighted sum of the loss function of each inference.An optimizer aims to decrease the loss function and steer the network toward improved performance by identifying optimal parameter values. Utilizing a newly derived variation of the Adam optimizer called AdamW can bolster the refinement of model optimization techniques. In contrast to its predecessor, the Adam optimizer, the AdamW algorithm effectively disentangles the weight decay component from the learning rate, allowing for individualized optimization of each component. This feature effectively addresses the issue of excessive overfitting. The outcomes reveal that the models optimized through AdamW exhibit superior generalization performance compared to those trained using other optimizers, particularly Adam. The AdamW optimizer was employed in the training of our final graphical module.Several activation functions, namely ReLU, SoftMax, and Mish, introduce nonlinear components to the neural network, allowing it to comprehend complex patterns and correlations in the data. The Mish activation function has demonstrated superior performance to alternative activation functions, primarily due to its nonlinear nature . The defThe experimental findings demonstrate that the efficacy of Mish surpasses that of widely utilized activation functions, including ReLU and SoftMax, among others, in diverse deep network architectures operating on complex datasets.k represents the number of keypoints, D represents the number of test or validation samples, and h and w represent the height and width of the sample images, respectively.We normalized the Percentage of Correct Keypoints (PCK) for our The subsequent section of this paper illustrates an exhaustive performance analysis of the proposed SDFPoseGraphNet. By undertaking a series of ablation studies, we thoroughly scrutinize the functioning of each module. Further, a comparative study is conducted between our proposed network and traditional networks employed for HPE. Finally, the predicted outcomes are made visually comprehensible to underline our results.To demonstrate qualitative results, we chose a diverse selection of images featuring different angles, complicated situations, instances of self/object occlusion, and complex backgrounds. An ablation study was carried out using the Panoptic dataset to substantiate the effectiveness of our optimization strategy. To evaluate the impact of the SA module, it was integrated into the FIM, while all other aspects were kept unchanged. As per the experimental results demonstrated in We also utilized a VGG-19 backbone model with a few extra layers and batch normalization for feature enhancement. Our analysis of each module in The presence of noise within a dataset can significantly reduce the model\u2019s performance . ConsideThis research proposed the SDFPoseGraphNet, a novel framework that combines the power of VGG-19 and spatial attention to improve hand pose estimation. By leveraging the deep feature maps extracted by VGG-19, the model captures spatial information and learns the relationships among hand joints. The incorporation of a Pose Graph Model with dynamically learned SIM parameters further enhances accuracy. FIM potentials obtained from another module contribute to pose estimation, and message-passing techniques refine joint predictions. The SDFPoseGraphNet is end-to-end trainable, allowing for joint optimization of all components, resulting in precise and reliable hand pose estimation. The present model possesses the attribute of generality, rendering its potential applicability useful in various forthcoming computer vision undertakings, such as the estimation of 3D hand poses and human poses, among others." \ No newline at end of file