Background: Patients who are on hemodialysis (HD) and are waiting for kidney transplantation encounter various psychological issues.Objective: The current research aimed to compare the effectiveness of regular nursing care with that of nursing care coupled with dedicated psychological counseling in patients who were on HD before they underwent kidney transplantation.Methods: Baseline data were collected 1 month before kidney transplantation in patients of both the control  and intervention  groups. The Mental Status Scale in Non-Psychiatric Settings (MSSNS) was administered to assess and record the psychological status. Clinicodemographic and end-stage renal disease (ESRD)-related details, including duration of dialysis, causes for ESRD, the number of dialysis sessions performed before transplantation, and MSSNS scores, were recorded and compared between the groups.Results: A total of 37 patients were enrolled, including 19 in the control group and 18 in the intervention group. The number of dialysis sessions performed before transplantation was 143  and 227.5 , and dialysis duration was 20.4 ± 14.5 and 14.4 ± 12.1 months in the intervention and control groups, respectively. There was no significant difference in baseline negative emotions between the two groups (P > 0.05). The psychological intervention group reported significantly lower anxiety, depression, anger, and loneliness scores than the control group (P < 0.05).Conclusion: Psychological counseling before kidney transplantation in patients on HD could reduce their negative emotions and enhance mental well-being. Kidney transplantation is a primary therapeutic option for patients with end-stage renal disease (ESRD). Patients who are on hemodialysis (HD) and are waiting for kidney transplantation encounter various psychological issues, which often get unnoticed or under-recognized by the treating clinicians and the family of patients. Anxiety and depression are known to be prevalent among patients with ESRD, particularly among females and older patients .Hitschfeld et al. reported that lung transplant recipients with a low score in the “Psychosocial Assessment of Candidates for Transplant Scale” could reflect weak or borderline psychosocial candidacy and develop a higher likelihood of mortality . Such fiFor patients with ESRD, especially for those undergoing living related donor kidney transplantation, the primary causes of psychological irregularities include long-term HD, suffering from the disease, heavy economic burden, and the very concern about surgery . The negKidney transplantation is a critical life-saving incident, but it also constitutes reflective psychological, relational, and social changes for the patient, donor, and their families. Self-orientation, knowledge on the transplantation surgery, and support from family and society are critical factors to influence the surgery outcomes, which can eventually alter the psychological stability of the patient. Therefore, an adequate pre-transplant psychosocial assessment provides a deeper understanding of the patients' psychological health status and helps to develop a suitable customized post-transplant psychotherapy plan for coping strategies .There is a significant gap in the evidence available on the impact of psychological interventions in patients with ESRD. Most of the psychological interventions would not include psychological consoling , 11. ThePatients from the Second Hospital of Anhui Medical University, Hefei, Anhui Province, China who were on HD and received routine nursing care  and nursing care coupled with dedicated psychological counseling  before undergoing kidney transplantation were recruited by a non-randomized controlled intervention design. The inclusion criteria were: (1) ≥18 years old, (2) clinically confirmed ESRD, (3) required allogeneic kidney transplantation, (4) patients who are actively receiving HD, (5) otherwise stable vital signs and physical conditions to tolerate transplantation surgery, and (6) clear consciousness. The exclusion criteria were: (1) severe infections and other pre-surgery complications; (2) history of mental disorders, neuropathy, and traumatic brain injury; (3) hearing and language disorders; (4) history of drug and alcohol dependence; and (5) severe heart, brain, lung, and other organ damages. The study protocol was thoroughly reviewed and approved by the Second Hospital of Anhui Medical University Ethics Committee, and written informed consent was obtained from all patients before enrollment.The control (2011–2015) group received routine nursing, regular dialysis , dietary guidance, and routine clinical care. The control group did not do any intervention treatment and only did a questionnaire survey after the follow-up. On the other hand, the intervention (2015–2018) group received dedicated psychological counseling in addition to the treatments that the control group received.Approximately a month before kidney transplantation, the “Mental Status Scale in Non-Psychiatric Settings” (MSSNS) was administered to patients of both the groups to assess and record the psychological status.This scale contained four key dimensions: anxiety (13 items), depression (10 items), anger (eight items), and loneliness (seven items), which are rated by a 5-point Likert scale (0–4 points). The total score of 38–152 points had different ranges for these dimensions: 13–52 points for anxiety, 10–40 points for depression, 8–32 points for anger, and 7–28 points for loneliness. A higher score indicated significant negative emotions. The Cronbach coefficient α of the scale was 0.933, which had good reliability and validity.Trained nurses (two nurses supervised by a head nurse) formed the psychological counseling team. Based on the scores obtained from the MSSNS scale, the psychological problems of the patients in the intervention group were analyzed to understand the causes. Group discussions were arranged to develop weekly psychological counseling programs, and each lasted 20–30 min during the HD treatment.To be specific, the intervention depended on the personalized customization of patients. Patients with psychological problems were screened out according to the MSSNS scale and scored which part  goes higher. Then, the nurses pointed out the reason that caused the negative mood of the patients by communicating with them and adopted corresponding nursing on the basis of that. For example, if the patient felt lonely, during the dialysis treatment, more companionship and communication were provided by the nurses. Moreover, the nurses also reminded the family members to pay attention to the patient's mood swings and give more care and companionship.Clinicodemographic and ESRD-related details, such as gender, age, marital status, educational level, occupation, duration of dialysis, causes for ESRD, number of dialysis sessions performed before transplantation, and baseline MSSNS scores, were recorded 1 month before transplantation for all patients, and the scored dimensions of the MSSNS were compared between the groups. After 1 month of treatment (till the day before surgery), all patients were recorded the MSSNS scores again.t-test was used, whereas the comparison between the groups was performed by independent samples t-test. P < 0.05 was considered statistically significant.SPSS 20.0 software  was used for statistical analyses. Continuous data were tested with the Kolmogorov–Smirnov test for normal distribution. Normally distributed continuous data were expressed as mean ± standard deviation (SD), and non-normally distributed continuous data were expressed as median (range). The counts were expressed as percentage and compared using the chi-square test. The measurement data were expressed as mean ± SD. For comparison within groups before and after treatment, paired samples P > 0.05) , and the mean ages were 35.3 ± 6.7 and 40.4 ± 12.0 years, in both the groups, respectively. The sociodemographic features of the two groups were comparable ( > 0.05) .The primary causes of ESRD for kidney transplantation did not differ significantly between the groups. The median number of dialysis sessions performed before transplantation was 143  and 227.5 , and dialysis duration was 20.4 ± 14.5 and 14.4 ± 12.1 months in the control and intervention groups, respectively .P = 0.530), anxiety (P = 0.089), depression (P = 0.841), anger (P = 0.389), and loneliness scores (P = 0.344) in baseline. After intervention, the absolute values of total MSSNS score, anxiety, depression, anger, and loneliness scores in the intervention group were statistically lower than those in the control group . The absolute scores difference between before and post-intervention in the intervention group was greater than that in the control group (P < 0.001) as shown in There were no significant differences in the control and intervention groups on the absolute values of total MSSNS score (The present study involved the comparison of nursing alone and nursing with psychological counseling of patients who were on HD before kidney transplantation. The results suggested that well-planned psychological counseling of patients would ease the negative emotions in this patient population and result in mental well-being.In recent years, with the advances in medical technology, kidney transplantation has become the treatment of choice of patients with ESRD. The incidence of uremia in China is gradually increasing, and the number of patients undergoing living, related, donor kidney transplantation is on the rise. Although kidney transplantation can effectively prolong the life of patients, the trauma and drug therapy associated with the surgery may induce tolerability issues in few patients. The cost of kidney transplantation is expensive in most settings, and the other crucial factors, such as postoperative adverse reactions, complications, and possible rejection due to immunosuppression, may adversely affect the physical and psychological health of patients . EspeciaSince June 2015, this department had performed psychological interventions to patients who were on HD before kidney transplantation. The overall psychological states for the treatment group after the intervention, including anxiety, depression, anger, and loneliness scores, were significantly lower than those in the control group, indicating that comprehensive psychological interventions could improve the mental well-being and ease out the psychological issues of patients before kidney transplantation. Patients undergoing kidney transplantation lack subjective initiative in self-management. When rejection or complications occur at a later point in time or when donors have physical and mental reactions, the patients develop distinct negative emotions of anxiety, tension, guilt, and self-accusation. Therefore, preoperative psychological interventions should be conducted to mobilize their initiative for self-management.Fu and Coyte reported in a large cohort retrospective study of data from 5,922 kidney transplant recipients from deceased donors that predialysis psychosocial conditions were associated with the increased relative risk of post-transplant death . PatientHence, education and counseling on mental health should be provided to patients timely before surgery, and professional nurses should help patients to establish an excellent psychological defense mechanism and change negative emotions into positive psychological experiences. Previous studies have also revealed that correct and positive nursing interventions can significantly improve the quality of nursing and eliminate the patient's negative psychological state. In this study, the preoperative dialysis plans were developed per the patient-specific conditions, including hypotension, coronary heart disease, and heart failure, which could significantly improve the success rate of kidney transplantation. Adequate dialysis has always been the most basic and essential aspect of ESRD treatment, which could prolong the survival time. It can provide a good preparatory state and a stable health condition before surgery, which would ultimately improve the QoL.In general, the nursing diagnoses and interventions seem limited in the care of kidney transplant recipients. A holistic approach to treatment and management offers physical, psychological, and social well-being of the patients . In someIn order to improve mental health, it is imperative to evaluate the depression and stress-coping types at a very early stage, maybe in the dialysis period itself. A well-developed educational intervention supported by need-based counseling would benefit these patients . HD patiThere are some limitations to this study. This is a single-center study with a small sample. Furthermore, the patients in this study were enrolled in a non-random way, which would result in the bias to interfere with the evaluation of effectiveness. Therefore, a large sample size and multi-center randomized controlled trial is needed in the future.In conclusion, the psychological intervention in patients with ESRD before kidney transplantation could have a positive impact on mental well-being and favorable outcomes postoperatively by improving their negative emotions.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 study protocol was thoroughly reviewed and approved by The Second Hospital of Anhui Medical University Ethics Committee. The patients/participants provided their written informed consent to participate in this study.QZ designed and implemented this research. SZ was responsible for the writing and revision of the paper. RY participated in the data collection and processing. All authors contributed to the article and approved the submitted version.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.


Coccidioidomycosis is a deep-seated fungal infection that occurs exclusively in semiarid areas in the Americas. In Brazil, coccidioidomycosis occurs exclusively in rural areas in the northeast region and affects counties that are hit by recurrent droughts, poverty and economic stagnation. Since 1978, approximately 136 cases of the disease have been reported in Brazil, according to scientific publications. However, a lack of governmental epidemiological data as well as a similarity to tuberculosis have led scientists and experts to assume that a greater number of cases occur in the country, which are not diagnosed and/or reported. In this review, general characteristics of coccidioidomycosis are presented, followed by a description of the main clinical and epidemiological data of cases in Brazil. The purpose of this article is to discuss the inclusion of coccidioidomycosis in the list of neglected tropical diseases. We believe that the adoption of coccidioidomycosis as a neglected tropical disease will enable the creation of an effective epidemiological surveillance system and the development of feasible public health solutions for its control in vulnerable populations. Coccidioides immitis and C. posadasii, which present subtle phenotypic differences, such as conidium size [C. immitis occurs in southern California and, recently, in eastern Washington [C. posadasii occurs mainly in Nevada, Arizona, Texas, Utah, and Latin America [Coccidioidomycosis (CM) is a fungal infection endemic within arid and semiarid areas of the Western Hemisphere located between 40° N and 40° S latitudes. CM is caused by the two sibling species ium size  and therium size , but caushington , C. posa America ,4.According to the epidemiological data available, the US has the highest incidence rates of the disease among endemic countries , and theCoccidioides antigens [Coccidioides antigens revealed up to 64% positive reactions in the Mexican population [CM cases outside the US have also been reported. The second country in number of CM cases is Mexico, where disease prevalence is comparable to that of endemic regions in the US . Mexicanantigens . Recent pulation . In addipulation ,8.In addition to humans, CM has also been reported in nonhuman primates as well as in dogs, equine, camelids, and even in bats ,12,13,14Coccidioides spp. parasitic structures. The cycle continues unless endospores return to the soil, where they undergo morphological shifts and convert to their mycelial saprobic stage [Infection in both humans and nonhumans begins with the inhalation of aerosolized arthroconidia released by the mycelial phase (infective form) after soil disturbance. Inhaled arthroconidia may escape from phagocytosis and form a spherule (parasitic form), as a result of intricate genetic and morphological shifts. Spherules internally develop endospores, as a consequence of mitotic nuclear division, and then endospores are released after cell wall rupture. Each endospore is likely to form another spherule, resulting in an exponential increase in ic stage ,15.Coccidioides species are well-recognized soil inhabitants, their recovery from natural sources is a difficult task. Coccidioides spp. have been isolated from alkaline soil in endemic areas [Coccidioides spp. as endozoans of small mammals, instead of primary soil fungal species [Coccidioides may live as spherules in granulomas without causing apparent disease in these mammals. When the host dies, spherules are then released from granulomas and convert into hyphae. Using dead animals as growth substrate, hyphae produce arthroconidia that may be inhaled by other small mammals, allowing the fungal life cycle to be sustained [Although ic areas ,18,19,20ic areas ,22,23. A species . Coccidiustained .CM clinical manifestations are highly variable. It is estimated that 60% of patients present asymptomatic infection that can be detected only by serological or skin tests.Primary respiratory signs and symptoms of CM infection may be indistinguishable from common bacterial pneumonia and commonly involve cough, dyspnea, thoracic pain, fever, arthralgia, myalgia, and fatigue, occurring up to three weeks after arthoconidia inhalation ,26,27. RExtrapulmonary disease develops in fewer than 5% of immunocompetent patients and may involve different organs and tissues . Skin isAs the clinical signs and symptoms described above are not pathognomonic, a well-performed anamnesis, consisting of a history of living or travelling to endemic regions, is extremely important for early clinical suspicion of this infection ,31. AlthCoccidioides spp. are those from respiratory secretions, such as sputum, bronchoalveolar lavage, and endobronchial and transbronchial biopsies. However, the fungus may also be isolated from other sites, such as urine, blood, bone marrow, skin biopsies, and cerebrospinal fluid [Coccidioides species requires biological safety level three facilities, which are not available in most clinical laboratories in Latin American countries [The gold standard for CM diagnosis is direct visualization of spherules or culture isolation of the pathogen from clinical specimens . The mosal fluid ,41. Micrountries .Coccidioides antigens can be detected in urine, EDTA-treated serum, cerebrospinal fluid, bronchoalveolar lavage, and other biological fluids [Serological tests based on detection of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies are also performed for CM diagnosis ,25. Testl fluids .Coccidioides spp., once it enables the detection of fungal DNA in contaminated samples and clinical samples that yielded negative cultures. In addition, molecular diagnosis eliminates the risk of handling infecting filamentous cultures of the pathogen [Coccidioides: ITS  region of ribosomal DNA [Ag2/PRA) [Coccidioides specific-antigen (CSA) [Coccidioides spp. [Molecular techniques based on polymerase chain reaction (PCR) and DNA hybridization may also be useful for the diagnosis of infections caused by pathogen ,44,45. Tomal DNA ,47; antien (CSA) ; and seqen (CSA) . Furtherdes spp. ,51.Most cases of CM are self-limited, not requiring therapeutic intervention. In cases of mild forms of the disease, patients can receive clinical and radiological follow-up in an outpatient regimen. In severe clinical conditions, chronic and disseminated diseases require specific drug approach and/or surgery. Amphotericin B is often used in patients with respiratory failure and other severe manifestations. Since the 1980s, azole derivatives have emerged as an important therapeutic arsenal. Fluconazole and itraconazole are used in sequential or combined treatment, especially in chronic forms of the disease. The duration of therapy with azoles often varies from months to years, and for some patients, life-long suppressive therapy may be necessary in order to avoid relapses . Few cliCoccidioides spp. [Coccidioides spp. In addition, the patient showed a positive reaction to a coccidioidin skin test. Ten years later, CM was detected again in semi-arid Brazil. A 74-year-old male patient from Jaguaribara, in Ceará State (5°39′29″ S–38°37′12″ W), who had been suffering from progressive dysphonia in the last six months without pulmonary symptoms, had a presumptive diagnosis of carcinoma, but histology showed Coccidioides spherules [The first case of CM reported in Brazil occurred in 1978 , in a 28des spp. . The secdes spp. , in a 35pherules .Coccidioides spp.; patients also showed positivity to spherulin skin tests [The international scientific community turned its attention to CM in Brazil after an outbreak occurred in Oeiras, Piauí State (07°01′30″ S–42°07′51″ W). In August 1991, three individuals and eight of their dogs who went on an armadillo hunt developed severe pneumonia; three dogs died a few days later. Mycological diagnosis was achieved after direct examination and culture of sputum samples. Inoculation of the same sputum samples in mice assured in vivo positive results for in tests . Three yin tests .In 1998, Brazil was included in the international list of countries with endemic areas of the disease , with zoIn Brazil, CM occurs exclusively in the northeast region, where approximately 136 cases of the disease have been reported since 1978: 94 cases in the state of Piauí ; six casThe cases of CM reported in Brazil share several clinical and epidemiological characteristics: all patients were from the same biogeographic origin (semi-arid region), were of the same gender , had ages varying between 11 and 82 years, although most of them were between 25 and 45 years old, and near all participated in armadillo hunts for at least two weeks before becoming ill. All cases started between the months of September and January, during the dry season in that region. Only two patients had chronic disease, while 134 patients had acute pulmonary CM. Cutaneous manifestations were described in 32 patients and included hypersensitivity reactions, such as erythema nodosum and erythema multiforme . One patDasypus novemcinctus armadillos  in the ecology of C. posadasii in Brazil was initially studied by Eulalio et al. [C. posadasii strains were obtained after direct cultivation and/or animal inoculation of soil samples collected in or nearby armadillo burrows in Ceará State [C. posadasii-related DNA in soil samples from armadillo burrows in Piauí State.The role of o et al. , who deso et al. ,65,66,67rá State  and Piaurá State . Additiorá State  detectedC. posadasii can be found in Brazil are not known in detail. Only a single study details the granulometric and mineralogic properties of positive soil samples for C. posadasii [The physical and chemical characteristics of the soils where osadasii . AuthorsIn Brazil, CM is considered a non-notifiable disease, which compromises understanding its impact in the country and hinders the establishment of surveillance and control measures. Failures in recording epidemiological data are common, and consequently, information on the disease is incomplete. As a consequence, data provided by the Brazilian government show divergences from scientific publications, which may indicate that the former probably includes other diseases, such as paracoccidioidomycosis.For instance, the search for deaths caused by CM between 1996 and 2017 in the Notifiable Diseases Information System from SUS revealed 48 events, distributed as follows: five in the north region, 22 in the northeast region, nine in the southeast region, 11 in the south region, and one in the midwest region. However, these governmental data are incoherent and unreliable, since the disease occurs exclusively in the northeast region. In turn, data obtained by the Department of Information Technology of the Brazilian Unified Health System showed that 829 cases of CM occurred in the country in 2011, a remarkably high number of cases, considering the scientific notification in that period. Data provided by the Hospital Admissions Information System from SUS show 400 cases of CM per year between 2000 and 2007, most of which were reported in the south and southeast regions . Once again, government data seem to mistakenly report other infections as CM, since the south and southeast regions of Brazil do not present the adequate biological conditions for the occurrence of the disease.Coccidioides spp., respectively, has been used in the US since the 1940s. So far, only two tests of this nature have been performed in Brazil. In 1991, Wanke et al. [Among the most important strategies for epidemiological investigation in populations exposed to fungi is intradermal testing. This approach, which uses spherulin or coccidioidin, structural antigens obtained from the parasitic and saprophytic phases of e et al.  performee et al.  carried This scenario suggests that, although the disease has been occurring for over three decades in northeastern Brazil, and despite the efforts made by local research groups, government authorities responsible for epidemiological surveillance continue to ignore the existence of the disease in the region.2, or about 18% of the Brazilian territory, with more than 53 million inhabitants, or almost 25% of the Brazilian population. Inside NEB there is a vast area classified as semi-arid, located between 3° to 18° S and 35° to 46° W. This semi-arid region occupies 982,563.3 km2, with nearly 22,600,000 inhabitants, or almost 12% of Brazilian population, distributed among 1262 municipalities. The area of NEB comprises the most populous semiarid region of the world [Northeastern Brazil (NEB) is a region located between 2.5° to 16.1° S and 34.8° to 46° W and encompasses the States of Maranhão, Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Sergipe, Alagoas, and Bahia. It corresponds to an area of approximately 1,540,000 kmhe world .2), followed by the Savanna/Cerrado (29.4%), Atlantic Forest (10.7%), and Amazon (7.4%). The Caatinga occurs only in NEB, and it is formed by xerophytic plants, mostly deciduous trees and shrubs, and annual therophytic herbs [The predominant biome in NEB is the Caatinga Phytogeographic Domain  have daily earnings below US $1.90 and are regarded as people living in extreme poverty by the World Bank . ConsideSuch economic and social inequalities have made NEB’s population historically vulnerable to many infectious diseases. Local authorities face bureaucratic problems in hiring human resources, and a shortage of physicians is a recurrent problem in rural areas . MoreoveNeglected tropical diseases (NTDs) are a diverse group of infectious diseases primarily associated with poverty and limited access to healthcare systems and education that affect more than one billion people in rural or peri-urban areas in low- and middle-income countries in Africa, Asia, and Latin America . As theyAccording to the WHO , NTDs arBrazil accounts for the largest number of cases of dengue fever, leprosy, schistosomiasis, Chagas disease, and leishmaniasis in the Americas, most of which occur in areas with low socioeconomic indexes, such as NEB. Approximately 10,000 NTD-related deaths are recorded in Brazil annually, and it was estimated that, in 2016, NTDs caused 475,410 disability-adjusted life-years (DALYs) in the country . In BrazFungal infections are also prevalent in Brazil and are estimated to affect more than 3.8 million people ; in someAt the present time, the coronavirus disease 2019 (COVID-19) pandemic is a demanding health problem faced by more than 180 counties . The SevA review of CM cases in Brazil suggests the disease fulfills the WHO criteria for NTD: (1) The disease occurs in rural areas of NEB, affecting mainly socially and economically vulnerable people. Our experience shows that patients often do not find adequate treatment in their local cities, being forced to travel long distances to more advanced centers in search of hospitalization. (2) Logistical difficulties and economic limitations occasionally have led to discontinuation of treatment by the patient, himself. The impact of early treatment interruption on the health and quality of life of these patients is unknown. (3) Government authorities have little control over the disease, which leads to systematic failures in prevention, treatment, detection, and reporting of cases. Misdiagnosis of CM as tuberculosis may occur, since the latter is endemic and has a high incidence in Brazil ,101, whiOn the other hand, CM has been considered a neglected disease in the US . In thatThe recognition of CM as a neglected disease in Latin America will allow for the establishment of measures to define its prevalence in Brazil. With this information, a risk assessment and epidemiological surveillance system can be created, allowing for the establishment of specific prophylactic measures in high-risk populations as well as the development of health education strategies. Taken together, these strategies may reduce the environmental risks of acquiring CM in areas already impacted by poverty and the high incidence of other infectious diseases.CM is an important underreported disease that has been occurring in Brazil for over three decades, but has been overlooked by public health authorities. The disease occurs in areas extremely vulnerable to climate change and in poor populations facing economic and social challenges. The recent economic crisis afflicting the country as well as the present COVID-19 pandemic has worsened access to health services by local populations, especially those living in rural areas. The data presented here suggest that the CM fits the definition of an NTD. The inclusion of CM in the WHO NTD list could support efforts for the prevention and control of the disease, especially in economically vulnerable populations. The Brazilian government needs to establish public policies for the prevention and control of this disease as well as to allocate a greater amount of financial resources in scientific research to expand the knowledge on the impact of CM in the country.


Traditional Chinese medicine (TCM) has gained increasing acceptance and popularity by the global community. The current study aimed to investigate self-reported evaluations of learning TCM and opinions about TCM courses among undergraduate international students majoring in conventional medicine at a university in China.n = 157). The course employed a student-centered, multiform learning model. Demographic data and self-reported evaluations of TCM learning background and TCM learning course were obtained to conduct the analysis.A cross-sectional survey was conducted at the Zhejiang University School of Medicine. A self-reported questionnaire was administered to international undergraduates who were enrolled in the TCM course during the 2018 and 2019 academic years (p = 0.025). After the course, 77.2% of students agreed that the course had brought about many benefits, 86.4% were satisfied with the course content, and 77.3% were satisfied with the teaching method. Students expressed their willingness to further learn acupuncture and to obtain more skilled practice through more visualized learning methods.A total of 133 students responded to the questionnaire. Among the respondents, 21.0% had some TCM-related knowledge, and 51.1% were interested in learning TCM before the course. Ninety-six students (85.7%) were from Asia. Students from Thailand showed significantly more interest in learning TCM than did students from other Asian countries (The majority of the international students we surveyed agreed that the TCM course improved their interest in and understanding of TCM. It is thus suggested that TCM education should be directed toward students’ learning barriers and needs.The online version contains supplementary material available at 10.1186/s12906-020-03174-1. Traditional Chinese medicine (TCM) education is an important component of biomedical education in China . The ChiWith many international students coming to China to learn medicine, Chinese medical institutions have attached importance to the internationalization of their education system. There are 52 medical schools that offer an English Bachelor of Medicine and Surgery degree, which is a medical education program specifically designed for international students who wish to pursue a medical bachelor’s degree in China , 10. TheZhejiang University is one of China’s higher education institutions. It currently ranks among the top 10 universities on the Chinese mainland  and withThe cross-sectional survey was conducted at Zhejiang University, Hangzhou, China. In the 2018–2019 academic year, the course titled “Basic Traditional Chinese Medicine” was reformed from the traditional teacher-centered lecture-based learning (LBL) method to a student-centered, multiform learning model. The general objective of this course is to introduce the fundamental theories and skills practice of TCM to undergraduate international students.n = 80 and n = 77, respectively, for a total of 157) were enrolled in this study. Informed consent and a voluntary questionnaire were administered to the participants during the last class.Two consecutive cohorts of students attending the “Basic Traditional Chinese Medicine” course in 2018 and 2019  of the six-year MBBS program. It covers 32 lessons, for a total of 1440 mins completed in eight teaching weeks, including 24 theoretical lessons and 8 practical lessons. The theoretical lessons consist of six parts: an introduction to TCM and traditional Chinese philosophy; TCM etiology and pathomechanism; TCM diagnostics; TCM therapeutic principles and methods; TCM life nurturing; and modern research on TCM. The practical lessons are interspersed and coordinated with theoretical lessons and are designed to help the students practice the skills of acupuncture, moxibustion, massage, cupping, the identification of Chinese herbs, the four diagnostic methods of TCM, and TCM methods of life nurturing such as Taiji. Beyond the 32 lessons, the course also includes clinical practice at the Department of Chinese Integrative Medicine, Women’s Hospital, School of Medicine, Zhejiang University, China. The course employs various teaching methods, including LBL, flipped classroom, problem-based learning (PBL), team-based learning (TBL), case-based learning (CBL), skill practice, experiential teaching and visualized teaching. One noticeable feature is the application of the flipped classroom. In brief, teachers determine the learning arrangements and lecture about theoretical knowledge, while students are divided into groups and encouraged to watch videos or consult literature beyond the class boundaries to consolidate or self-learn TCM-related knowledge. The classroom then becomes a place where presentations and discussions are performed by students for half the class time. Interaction is increased by the use of the question-and-answer mode between teachers and students. Students are graded in light of their attendance, class assignments, class presentation and final essays.An anonymous, self-administered questionnaire . Statistical analysis was performed using IBM SPSS . Descriptive information for variables was provided as the mean ± standard deviation (SD) or n (%), according to the nature of the variable. Variables were compared using one-way analysis of variance (ANOVA) at a statistical significance level of 0.05.A total of 133 students responded to the questionnaire out of the 157 students who were enrolled in the “Basic Traditional Chinese Medicine” course during the 2018 and 2019 academic years. The response rate was 84.7%. The number of respondents for each question of the questionnaire is shown in Fig. As shown in Table p = 0.025).As most students were from Asian countries, with India and Thailand being in the majority, the mean score of the self-reported evaluations of students from India, Thailand, other Asian countries and non-Asian countries were compared (shown in Table α) was 0.826, indicating that this subscale of the questionnaire (7 items) had good internal consistency. The Cronbach’s α values measured in 2018 and 2019 were both higher than 0.8, indicating that the reliability was stable across time.We assessed the validity of 7 single-choice questions from the questionnaire that focused on the students’ self-reported evaluations of the TCM course. As shown in Table Before the course, 50.9 and 43.9% of the students’ impressions of TCM were focused on its practicability and mysterious nature, respectively Fig. a; 63.9 aIn this study, even though the undergraduate international students we surveyed were majoring in conventional medicine, a few students had basic prior knowledge of TCM, and half of them showed interest in TCM. After taking the TCM course, the majority of students agreed that the course had improved their interest, perception and understanding toward TCM, and they were satisfied with the course content and the teaching method. Students also expressed their willingness to further study acupuncture and obtain more skill practices in regard to acupuncture. Suggestions from students regarding the teaching methods included more skilled practice and more visualized learning methods.TCM has gained increasing acceptance and popularity within the global community because of its alignment with biomedical models of professionalization, education, research and practice , 16. AmoThe respondents of this questionnaire-based study were mainly from Asia, among which more were from India and Thailand than from other countries. As students come from different ethnic groups and nations with a variety of cultural backgrounds and modes of thought , they haTCM education has been made a mandatory part of the medical education in most medical universities in China. Although challenges are perceived as persisting with regard to TCM education for international students in medical universities or schools in China , 25, 26,When international students were asked about their impression of TCM before the course, 50.9% agreed that it seemed practicable, and 43.9% considered it mysterious. A few students (21.0% of the respondents) reported that they had some prior TCM knowledge, and half of them (51.1% of the respondents) showed an interest in learning TCM. We investigated whether the course had positive impacts, and the majority of the students self-reported an improved perception, interest and understanding of TCM and that they had obtained benefits from the course. They also expressed satisfaction with the course. However, some students disagreed that the course had improved their interest in and understanding of TCM . The reason for this outcome may be because the course is mainly theoretical and the theory of TCM itself is challenging. Moreover, similar to previous reports , the intThis study has several limitations. First, although students from two academic years participated in the study, the sample size was small. The study was conducted at a single university, and the majority of the international students surveyed in this study were from Asian countries; thus, the responses did not reflect all international medical students in China. Second, the validity of the questionnaire has not been fully determined, and the self-administered questionnaire might lead to potential information bias from the participants. Third, we could not recruit a control group for the study due to the feasibility, and we also failed to have a pre- and post- evaluation to determine the effects of the educational intervention. Accordingly, in the future, we would consider a pretest/posttest study design on a continuous basis and across a larger sample of students to investigate the effects of the TCM course.In conclusion, international undergraduates majoring in conventional medicine in China seem to have a basic prior knowledge of and interest in TCM, especially in regard to acupuncture. The majority of the international students we surveyed agreed that the TCM course improved their interest in and understanding of TCM. They also showed satisfaction with the TCM course, which employed a student-centered, multiform learning model. It is suggested that TCM education reform should be directed toward students’ learning barriers and needs.Additional file 1. Title: Questionnaire for the “Basic Traditional Chinese Medicine” course. Description: Not applicable.


Lolium perenne pollen genes by RNAseq, in two wild populations with different levels of air pollution. The objective is to search for proteins that are expressed differentially in both situations and to establish a relationship with increased allergenic capacity. Two populations of L. perenne  have been studied in two consecutive years, under the rationale that overexpressed genes in Madrid, with higher levels of NO2 and SO2, could be a cause for their greater allergenic capacity. Heat shock proteins (HSP), glycoside hydrolases, proteins with leucin-rich repeat motifs, and proteins with EF-HAND motifs were consistently overexpressed in Madrid pollen in the two years studied. Interestingly, some genes were overexpressed only in one of the years studied, such as pectinesterases in the first year, and lipid transfer proteins (LTPs) and thaumatin in the second. Despite the fact that the potential of all these proteins in relation to possible allergies has been reported, this is the first time they are cited as possible allergens of L. perenne. The results found can contribute decisively to the knowledge of the allergens of L. perenne and their relationship with atmospheric pollution, and to the development of much more effective vaccines.The relationship between air pollution and the allergenic capacity of pollen is widely accepted, with allergenicity being directly related to air pollution. To our knowledge, this is the first study comparing the differential expression of  Most immunoglobulin E (IgE)-mediated allergies are caused by the plant’s allergens, which may cause different symptoms such as rhinoconjunctivitis, edema, urticarial, asthma, and anaphylaxis . Nowaday2O2 concentration, and enhanced NADPH oxidase activity in pollen. These two factors (H2O2 concentration and NADPH oxidase) are considered as very relevant in the increase of the allergenic capacity of pollen [The relationship between air pollution and the increased allergenic capacity of pollen is a widely accepted fact ,9,10,11.f pollen .Poa pratensis (Kentucky bluegrass), Phleum pratense, and L. perenne . [Pollen from different types of plants has been found to trigger allergic reactions . Up to eegrass). . The polegrass). ,17,18. L. perenne have been described and it is assumed that they are always present in pollen of this species, regardless of the place of collection. This is certainly one of the problems, since pollen used in skin tests rarely comes from the place where the patient lives. Furthermore, it is necessary to investigate new allergens, since in many cases vaccines do not have the desired effect, probably because they are not being manufactured with the appropriate allergens .On the other hand, according to clinical data collected by allergists consultations through the usual skin tests, the true extent of the problem is not evidenced, as there are many more cases of pollen allergies. To date, six pollen allergens of Plants have an inducible adaptative metabolism known as secondary metabolism, through which they are able to adapt to both biotic and abiotic stresses. This metabolism is capable of synthesizing a plethora of molecules of different nature that only appear under stress . The besPollen used in this work was collected from two cities in central Spain , near heavy traffic areas in both cases. However, pollution levels, especially nitrogen oxides and sulfur oxides, were higher in Madrid throughout the plant’s vegetative and reproductive period . The polL. perenne. In order to achieve these objectives, the gene differential expression of pollen from both localities was studied using the RNAseq technique, in two consecutive years, placing a special emphasis on those genes that were overexpressed in the Madrid pollen, and that could be responsible for its greater allergenic capacity. Once the relationship of air pollution with a greater allergenic capacity of pollen had been demonstrated, the objective of this work was to identify overexpressed genes in the high pollution area (Madrid) as compared to the low pollution area  by means of a transcriptomic analysis, with the secondary aim to find new potential allergens in the pollen of L. perenne from Ciudad Real and three from Madrid.To construct a transcriptome database, six mRNA libraries were generated in each collection moment (May 2017 and 2018) by Illumina sequencing, three from each population of p adjust value were not obtained in the normalized expression analysis. A total of 115,895 genes were available for the genetic expression analysis, of which 107,599 had a p adjust value over 0.05. When the pollen of the two cities was compared, the expression pattern showed that in 2017, 366 genes were common to both cities (expression without significant differences), and 2443 genes were significantly overexpressed in the pollen from Madrid, while 5487 genes were significantly overexpressed in Ciudad Real , and 7568 genes were significantly overexpressed in the pollen from Madrid, while 3823 genes were significantly overexpressed in Ciudad Real , and two lipid transport proteins (LTPs). All of them have been described as allergens.In Ciudad Real, overexpressed genes were related to the primary metabolism, none of which was involved in routes of secondary adaptive metabolism by which potential allergenic molecules were synthesized. Conversely, among the genes overexpressed in the Madrid pollen some were involved in secondary metabolism, especially in 2018. In 2017, genes involved in the shikimic acid pathway, in the phenylpropanoid pathway, and heat shock proteins of family 20 (HSP 20) were overexpressed. In 2018, almost all overexpressed genes were related to secondary metabolism. There were 14 isoforms of the heat shock protein of 20 family, two isoforms of allergen 1 from The GO analysis  shows thAmong the upregulated genes in Madrid samples, ten showed isoforms in which overexpression was different depending on the year ; the numNowadays, allergic diseases have become a pandemic health problem. Among them, pollen allergies are considered the most important . Some stSome studies have shown the existence of an in situ allergic response in patients with negative skin prick test (SPT) results and undetectable IgE in the serum . This clhttp://www.allergen.org/search.php?allergensource=Lolium+perenne) establishes six allergens in Lolium perenne . The first three are expansins, proteins specialized in pollination, with its role being to weaken the cell wall during the development of the pollen tube.The International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee , were overexpressed in Madrid samples, but they were by no means the most overexpressed. The most overexpressed genes in the Madrid pollen in the two years studied were heat shock proteins (HSP), specifically with a molecular weight of 20, 70, and 90  genes have also been detected to be overexpressed in pollen from Madrid in the two years studied. LRR are repeated sequences present in a number of proteins with diverse functions and cellular locations. These repeated sequences are usually involved in protein-protein interactions. LRR domains are composed of beta-alpha units that form curved horseshoe structures with a parallel beta sheet on the concave side and mostly helical elements on the convex side. LRR domains are often flanked by cysteine rich domains ,36. Nowa2+ and chelate the cytosolic calcium to regulate calcium homeostasis [Alnus glutinosa (Aln g 4), Brassica napus , Chenopodium album (Che a 3), Olea europaea (Ole e 3 and Ole e 8), etc. However, there is no reference to L. perenne.Genes of proteins with EF-HAND motifs were also overexpressed in the Madrid pollen in the two years studied. The EF-hand-containing proteins actively bind to Caeostasis . The majSalsola kali pollen was shown to be a major allergen [We have found that some genes overexpressed in Madrid in the first or second year of study, but not in both. During the first year, genes that code for pectinesterases (seven isoforms) were overexpressed. These enzymes play an important role in the cell wall metabolism during fruit ripening . Sal k 1allergen .n = 7), pollen of trees and weeds (n = 9), fruits (n = 18), nuts and seeds (n = 4), as well as latex (n = 1) [Cupressus arizonica (Cup a 3) [L. perenne pollen neither for LPTs nor for TLPs. During the second year of study, seven genes that code for isoenzymes of lipid transfer proteins (LTPs) and six isoenzymes of thaumatin were overexpressed in Madrid. Both are described as pathogenesis-related (PR) proteins and have a reputation for their allergenic capacity. LPTs are from family 14 (PR 14) and thaumatins belong to family 5 (PR 5). To date, the International Union of Immunological Societies Allergen Nomenclature Subcommittee has reported 39 allergenic LTPs from vegetables ( (n = 1) . ThaumatCup a 3) . There iL. perenne plants growing in natural conditions in Ciudad Real and Madrid cities was used for the experiments. Pollen was collected from plants in the maximum pollen production period (mid of May 2017 and 2018). Three populations of L. perenne separated between 50 and 100 m were selected. Plants of each population were harvested and each one constituted a replicate. Pollen was recollected by the company Iberpolen S.L. . Pollen from Before the RNA library assembly, ribosomal RNA was removed. This was performed with the Ribo-Zero rRNA kit removal kit. The TruSeq Stranded Total RNA library Prep kit was used to generate the libraries of RNA. First of all, 2 μg of total RNA (RIN > 9) libraries, were sequenced using a HiSeq2500 instrument . Sequenced readings were paired-end with a length of 101 bp reading performed in six samples . The estimated coverage was around 59 million reads per sample (one lane). Library generation and RNA sequencing was done at Sistemas Genómicos S.L.  following the manufacturer’s instructions.Lolium perenne” ASM173568v1 genome provided by the NCBI database using the Tophat2 2.1.0 algorithm [p-value (Padj) corrected by FDR ≤ 0.05 to avoid the identification of false positives across the differential expression data.The FastQC v0.11.4 tool was used to check the quality control of the raw data. Then, the raw paired-end reads were mapped against the “lgorithm . Insuffilgorithm  and Picalgorithm . This melgorithm . Finallylgorithm . The DEGlgorithm . By appllgorithm , we idenhttp://wego.genomics.org.cn/.The gene category enrichment analysis was performed by comparing the differentially expressed genes to the Uniprot database by using Blastx and setting an e-value of 0.01 and a minimum of 40% of the protein length/transcript ratio. With the obtained terms, an over representation test was performed using an in-house R Script developed at Sistemas Genómicos . The graphical plotting of DEGs distribution within GO categories was performed at L. perenne. These proteins have to be synthesized by heterologous cloning before their allergenicity can be checked with skin tests. In the case of positive responses, our contribution to the knowledge of allergens of L. perenne and their relationship with atmospheric pollution would be confirmed. On the other hand, this would contribute to the development of much more effective vaccines, probably solving the problem of an allergic response in patients with a negative skin prick test (SPT).In conclusion, the results obtained in this work can be very useful, since we have described genes that code for some overexpressed proteins in conditions of higher air pollution with potential allergenic capacity for the first time in


The relationships between family history, sex, age at onset, and migraine occurrence have been documented. However, the associations between these factors across different sexes and subgroups of patients have yet to be elucidated. This study evaluated the association between family history and migraine in male and female patients experiencing episodic and chronic migraine with and without aura.This cross-sectional, case–control study included 299 headache-free controls and 885 patients receiving outpatient treatment for migraine. Participants were classified into episodic (1–14 days/month) and chronic (≥15 days/month) migraine groups.P < 0.001) in male patients, particularly in male patients without aura ; it was less frequently observed  in female patients with aura. Family history was correlated with an earlier age at onset , particularly in patients without aura , who were women .Positive family history was significantly more frequently observed in the episodic group than in the chronic group (49.5% vs. 26%; Different patterns of association between family history and migraine can be observed between men and women. A positive family history of migraine is correlated with an earlier age at onset, particularly among female patients without aura. Migraine is a prevalent headache disorder worldwide, affecting up to 12% of the population , 4. MostCACNA1A, ATP1A2, and SCN1A [Similar to a number of neurological diseases, such as Parkinson’s disease , amyotrond SCN1A –16. Howend SCN1A , and epind SCN1A , 18, 19.nd SCN1A . The abond SCN1A , therebynd SCN1A .To evaluate the heritability of common forms of migraine, Genizi et al. recently investigated the association between family history and pediatric migraineurs with aura (MA). They found that these patients are more likely to have a family history of migraine than pediatric migraineurs without aura (MO) . The samAlthough the genetic basis is still unknown, the findings of the aforementioned studies highlight the heritability of migraine, revealing differences in inheritance patterns between MA and MO and differences in age at onset between generations. However, a key limitation of these studies is the method of patient grouping, with groupings based on presentation of aura and family history. The relationships between sex, migraine frequency, and interaction effects between all these factors have not been evaluated. We hypothesize that there may be a different pattern of association between family history, age at onset, and sex in migraineurs with (MA) and without aura (MO). We hypothesized that differences may also exist across individuals who experience episodic versus chronic migraines.The aim of the present study was to evaluate whether different familial patterns and age at onset exist between migraineurs. To this end, patients were divided into groups according to factors such as sex, frequency, and presentation of aura.This observational cross-sectional study adopted and met the requirements of the STROBE  statement.This cross-sectional controlled study included a cohort of 1184 participants between the age of 20 and 60 years. 299 healthy, headache-free controls were volunteers recruited from the community, and 885 were participants undergoing outpatient monitoring in a headache clinic at the Department of Neurology of Tri-Service General Hospital (TSGH) between November 2015 and April 2018. The study protocol was approved by the Institutional Review Board of TSGH (TSGH-IRB-1-105-05-006), and all the participants provided informed written consent prior to enrollment. Patients suffering from migraine, both with and without aura, were enrolled. After excluding patients with concomitant primary and secondary headache disorders, those with insufficient data to determine migraine frequency, and those lacking information on clinical characteristics, a total of 885 patients were included in the final analysis.Each participant completed a screening questionnaire and was subsequently interviewed directly by a board-certified neurologist and headache specialist (FCY); diagnoses were based on criteria defined in the International Classification of Headache Disorders, 3rd edition . Thus, pPatients in the episodic and chronic migraine groups were further divided into four groups based on sex and the presentation of aura. Of the 885 participants with migraine, 291 (32.9%) experienced aura and 594 did not experience aura. The enrolment process is illustrated in Participants were checked for any history of physical, cognitive, or degenerative diseases of the central nervous system or severe head injuries with loss of consciousness. All participants underwent detailed neurologic examinations as well as Doppler ultrasound examination by an experienced radiologist to screen for any possible underlying vascular disorders including cervical artery dissection that might cause secondary headache and mimic migraine , 27. TheInformation on the family history and age at onset of headaches was collected using a structured questionnaire. Participants were then interviewed by a board-certified neurologist and headache specialist (FCY). The presentation of migraine and accompanying symptoms was clarified through an interview. The participants’ family members who were present and reported to have migraine were also interviewed by FCY. Information regarding headache in other relatives who were not present was obtained second-hand from those who were present. Patients were assigned to the positive-family-history group only if at least one of their first-degree relatives had migraine-like headaches; other types of headache and secondary causes were excluded. The structured questionnaire comprised the Beck Depression Inventory-II , the HosThe normality of continuous variables was tested using a Kolmogorov-Smirnov test. However, none of the normality tests met the assumption due to the relatively large sample size of this study. According to the normality plot  and the standard deviation values relative to means, the distribution of these continuous variables was not far from normal (data not shown). Data for continuous and categorical variables are expressed as mean ± standard deviation and frequency and proportion, respectively. Differences in continuous and categorical variables between the study groups  were tested using one-way analysis of variance and chi-squared test, respectively. Pairwise comparisons were performed using the Bonferroni adjustment, only where an overall test was significant. The proportions of patients with a migraine family history in the study groups were compared using the chi-squared test and Bonferroni-adjusted multiple comparison.t-test, and the analysis was further stratified by aura and sex. Finally, to investigate the association between baseline characteristics and risks of migraine, we performed a multivariable logistic regression analysis. With the assumption of a mean age at onset of 20 years in the patients with a family history of migraine and 22 years in patients without a family history of migraine, a standard deviation of 10 years, and an alpha level of 5%, a minimum sample size of 686 patients with migraine was required to achieve a power of 80%. A two-sided P value of <0.05 was considered to be statistically significant. No adjustment for multiple testing (multiplicity) was carried out in this study to avoid low statistical power. Statistical analyses were conducted using SPSS 22 .Age at onset of migraine in patients with and without family history was compared using the Student P = 0.021). The proportion of smoking was higher in the two migraine groups than that in the control group (P = 0.015). No significant difference was observed regarding sex, age, and alcohol and coffee consumption between the three study groups. The scores for anxiety, depression, and sleep quality were worse for the chronic migraine group than for the episodic migraine and control groups (P < 0.001). The proportion of family history of migraine was higher in the migraine groups (51.8% and 52.1% in the episodic and chronic migraine groups) than that in the control group. No significant difference was observed between the episodic and chronic migraine groups.P < 0.05; P = 0.003). However, in female patients with aura, the proportion of family history was more frequent in the chronic migraine group than in the episodic migraine group ; however, this phenomenon was observed only in patients without aura (P = 0.002) but not in male patients without aura (out aura . Furtherout aura .P = 0.002). The age of onset in male participants was 21 years and 22.1 years for those with and without a family history of migraine, respectively, and was not statistically significant (P = 0.313). In contrast, the age of onset in female participants was 20.6 years and 23.1 years for those with and without a family history of migraine, respectively, and was statistically significant (P = 0.002). The age difference was more clinically significant in females (2.5 years) than in males (1.1 years). After further stratification by aura and sex, this phenomenon was observed only in patients without aura and female patients without aura.In the present study, patients with migraine had a higher proportion of first-degree relatives with migraine than the controls; nevertheless, no significant differences were observed between the episodic and chronic migraine groups. However, after stratifying by sex, a difference in family history and aura presentation emerged in that the prevalence of a positive family history was higher in male patients without aura in the episodic group. Conversely, the prevalence of a positive family history was higher in female patients with aura in the chronic migraine group. Based on similar stratification strategy, we also evaluated the age at onset of migraine and categorized the participants into groups based on sex, presentation of aura, and family history. We observed that the age at onset of migraine was earlier in patients with family history than in those without , aphasic (18%), and motor (6%) symptoms . This stIn conclusion, the results of the present study reveal a different pattern of association between family history and migraine in men and women. Moreover, the results demonstrate that a positive family history correlates with an earlier age at onset, particularly among female patients without aura. Thus, this study highlights the associations between migraine prevalence, aura, age at onset, family history, and sex. Future studies should investigate these factors in more detail, clarifying the effects of paternal versus maternal relationships and how different forms of migraine in first-degree relatives influence migraine risk.S1 Fig(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.


Citrus and its relatives. The gain and loss of pigmentation is intriguing and is inherited variously among species. Methods: Citrus germplasm was used to investigate the anthocyanin pigmentation of tissues never before considered, including stamen, style and stigma, and of young leaves, petals, rind and flesh of 28 genotypes belonging to 14 species. Citrus genotypes encompassed citron, lemon, sweet orange, lime, and Citrus relatives included Microcitrus, Murraya, and Severinia. A relative qRT-PCR analysis was carried out on the structural and regulatory genes: phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3′-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), uridine diphosphate glucose flavonoid glucosyl-transferase (UFGT), glutathione S-transferase (GST), Ruby and Noemi. Image analysis and a genomic approach were employed to evaluate how the red pigmentation is inherited among tissues and species. Results: Pigmentation of young leaves and petals is specific to citron and its hybrids. Ruby controls the pigmentation of petals, but not of leaves. The red color of the rind and flesh is a trait that particularly characterizes a diversity of sweet oranges, citron hybrids and Citrus relatives. Color expression depends on external factors and also on developmental stage. The coloration of stamen and style is citron-specific, while a red stigma is exclusive to Moro orange and its hybrids. Conclusion: It is hypothesized that there is a relationship among Citrus species and genes controlling anthocyanin pigmentation.Background: Anthocyanin pigmentation characterizes a number of tissues of  Citrus genus. A recent study [Citrus species, hypothesizing that they developed sometime from the late Miocene (10.4–10.5 million years ago) to the early Pliocene (5.3–5.4 million years ago). It is thought that in addition to mandarin (Citrus reticulata), pummelo (Citrus maxima), and citron (Citrus medica), which are defined as the ‘primary’ species from which most cultivated Citrus have originated, another two species are crucial to the origin of limes and calamondin, namely, Citrus micrantha (belonging to the subgenus Papeda) and Fortunella japonica  [Sweet oranges, lemons, limes, grapefruits, and pummelos are all economically important species belonging to the nt study  describekumquat) .Citrus species and their relatives, such as Microcitrus australasica spp., Severinia spp., Citrus latipes, and Citrus hystrix, exhibit significant variability in anthocyanin pigmentation among their different tissues, especially in their leaves and petals. The accumulation of anthocyanins in the mature fruits is a particular feature that characterizes the flesh and the rind of ‘blood’ oranges  and occurs exclusively in the rind of ancient pummelos [pummelos ,4.Anthocyanins are water-soluble pigments, and represent the largest class of the flavonoid family. They are responsible for the red to purple color of plant tissues. The different hues are the result of vacuolar pH, co-pigmentation with other polyphenols, and the presence of ionic complexes . AnthocyCitrus, as in most other species, light also regulates pigmentation [Environmental conditions play a crucial role in controlling anthocyanin pigmentation. In the various blood orange cultivars, the anthocyanin accumulation is cold-dependent. Storage conditions in the range 4 to 9 °C have been shown to increase the transcription levels of the associated biosynthetic and regulatory genes, as well as increasing the anthocyanin content ,11,12,13entation .Citrus, it has been demonstrated that the insertion of a Copia-like Long Terminal Repeat (LTR) retrotransposon, Tcs1, promotes the expression of Ruby, the MYB-like gene that, together with Noemi (bHLH-like) and a WD40 repeat, control the anthocyanin trait in the pigmented orange fruits [Citrus [Citrus fruits [Citrus and its wild relatives [Two categories of genes are necessary for anthocyanin production: structural genes, encoding for enzymes directly involved in anthocyanin biosynthesis, and the complex WMBW made up of transcription factors , that control the tissue-specific expression of the structural genes. These have all been studied extensively . The mose fruits ,19. In as fruits . Additioelatives .Citrus genus, where red and purple colors are present in the young leaves and flowers.A number of studies have focused on tissue pigmentation of the sweet orange cultivars, especially that of the flesh ,24,25,26C. medica, C. sinensis, C. limon, C. limonia, C. aurantifolia, C. latifolia, C. meyeri, C. celebica, C. latipes, C. hystrix, Microcitrus australasica, Murraya paniculata, Severina disticha, and S. buxifolia). The genotypes were chosen based on the variability of pigmentation in a number of their tissues. This study aims to elucidate the phenotypic and genotypic variability of red–purple pigmentation. It suggests possible inheritance traits in each tissue across these species. The results of the study advance our understanding of the control of anthocyanin expression, and provides a foundation for further cultivar improvement and for the breeding of novel genotypes. Furthermore, it proposes new phenotypic markers, such as a purple stigma, that can be used to trace breeding materials.The present work employs transcriptional analysis to investigate the main structural genes  and regulatory genes . Here, these are evaluated in a set of tissues including: young leaves, petals, stamens, styles, stigmas, flesh/juice, and rind. Some of these have not been evaluated previously in 28 genotypes belonging to 14 species  subgenus Papeda and related genera. From one to three negative controls (no anthocyanin pigmentation) for each tissue were also selected, depending on the variability of the pigmentation and the availability of the tissues and genotypes for each group. The main traits and the origins of the accessions are described in The 28 accessions selected for our study belong to 14 species. They were gathered in four groups in consideration of their parentage: (1) sweet orange and its hybrids, (2) citron and its hybrids, (3) other M. australasica and Faustrime), and (3) 100 mg of young leaves, petals, stamens, styles, and stigmas, previously homogenized for 30 s at 30 rpm using the Tissuelyser II (Qiagen). One volume of extraction buffer  SDS), one volume of phenol, and 0.02 volume of β-mercaptoethanol were added to the sample. After incubation at 50 °C for 5 min, samples were centrifuged at 4000 rpm at 4 °C for 15 min. Two cycles of centrifugation were carried out, adding to the upper aqueous phase one volume of chloroform:isoamyl alcohol . RNA was precipitated, adding one-half volume of 6 M LiCl to the upper phase at −20 °C overnight. After centrifugation at 8500 rpm for 40 min, the precipitated RNA was washed with 70% (v/v) ethanol and centrifuged at 7500 rpm for 20 min. The total RNA was resuspended in 50 μL of RNase-free water. The qualities and the quantities were evaluated using a Nanodrop 1000 spectrophotometer (Thermo Scientific) and by gel electrophoresis (agarose 0.8% in TAE 1×). The quality was considered optimal for values of 260/280 between 1.80 and 2.0.Total RNA was extracted from: (1) 3 mL of filtered juice, (2) 2 g of rind and pulp (exclusively for DNAse treatment was carried out by adding to 40 μL of RNA 1× of RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), 0.1 M of DTT (Invitrogen), 5× Buffer, 1× of DNAse in a final volume of 50 μL. Samples were incubated at 37 °C for 30 min and purified using RNA Cleanup protocol (Qiagen), according to the manufacturer’s protocol. cDNA synthesis was carried out as previously described  using thRelative quantification qRT-PCR was carried out using the ABI 7300 Real Time PCR System (Applied Biosystems). The PCR mixture  contained 7.5 μL Power SYBR Green PCR Master mix (Applied Biosystems), 0.1 mM of each gene-specific forward and reverse primer and 100 ng of the cDNA sample, according to the manufacturer’s protocol. The following standard thermal profile was used for all PCRs: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Three technical replicates were assayed and a no-template negative control was routinely included in each plate. Data analyses were carried out using the standard curve method. For each gene of each tissue the calibrator was represented by the sample with the lowest ratio of ‘EF mean and gene mean’ and the qRT-PCR results are indicated as ‘mRNA fold-increase’ with respect to the calibrator.Primers were designed using Primer 3 software , preferaC. sinensis genome [Citrus genome collection [C. maxima, C. medica, C. reticulata, and S. buxifolia, representing the parents or the species themselves that we considered in the present study. Moreover, we also blasted against the C. ichangensis genome, a Papeda like C. hystrix and C. latipes. Finally, we also included the blast against Fortunella hindsii which is close enough to the Citrus species, although it belongs to another genus [To ensure the evaluation of genes corresponding as closely as possible to each accession, all primers were first designed on the s genome . Then a s genome  was carrllection ,38. Specer genus . The CLCer genus  was usedM. australasica genome (SRR6188442 SRA) was downloaded in FASTQ format using the “FASTQ-dump” tool of the SRA toolkit [C. maxima reference genome [The  toolkit  from NCB SRR61884 SRA was e genome  by Stampe genome , which ie genome  was usede SRR61882 SRA wase genome , and thee genome .Photographs of the petals were taken against a green background under artificial light sources . As a rep-values ≤ 0.05.Correlations among gene expressions were evaluated as mRNA-fold increases for each tissue using the Pearson method and data were considered discriminant for t-test, while the samples that were not normally distributed were tested using the Wilcoxon signed-rank test [A qualitative test was used to compare the significant differences between pigmented and non-pigmented samples for all tissues, except young leaves, stamens and styles because only one negative control was collected. The normal distribution of the expression data, based on the mRNA-fold increase values, was tested using the Shapiro-Wilk method . The meaank test .p-values were applied to all genes and all accessions evaluated for each tissue, except for the stamens, styles, and stigmas because of the small number of accessions (from 4 to 6). The analyses were done using the default setting by ‘pvclust’ R package [Two different Ward hierarchical clustering analyses with bootstrapped  package .Principal component analysis (PCA) was carried out on all genes and the accessions of all tissues  by the ‘prcomp’ function .Partial least square regression (PLS) was carried out on all the genes of the accessions, where petals were analyzed by the ‘plsr’ function of ‘the pls’ package  and valiAll statistical analyses were carried out in R ambience .M. australasica. Methanolic extracts were obtained by adding 2 g of M. australasica flesh to 50 mL of 80% acidified methanol (0.5% HCl), stored under agitation in the dark for 3 h, and then centrifuged at 8000 rpm for 40 min. Total anthocyanins were expressed as milligrams per liter (mg/L) of cyanidin 3 glucoside.Anthocyanin quantification was carried out spectrophotometrically at 520 nm of absorbance (Varian UV-Vis spectrophotometer mod. Cary 100 Scan) according to the differential pH method . VolumesM. australasica flesh and, similarly, with 20 mL of hexane/acetone/ethanol solution (50:25:25) mixed with 10 mL of orange juice. Lycopene content, expressed in milligrams per liter (mg/L), was determined using the molar extinction coefficient of E1%1 cm = 3450 [Lycopene quantification was carried out spectrophotometrically at 503 nm of absorbance  by extram = 3450 .M. paniculata genome has been released but is not yet available (Unpublished data-“CREA-OFA jointly IGA-TS De novo sequencing of M. paniculata-ORPRAMed project”). The genome of M. australasica is available but it has been aligned against the C. maxima genome resulting in an average coverage of 38 ×, covering 269 Mb of the total 302 Mb of the reference genome.The genomes of all accessions considered here are not totally or publicly available. The Generally, we observed that anthocyanin biosynthesis is highly conserved in all the species we considered. This finding is supported by the high sequence homology for all structural genes.M. paniculata and S. buxifolia, in which the primers worked correctly, even though very few nucleotidic and aminoacidic mutations were reported in the primer(s); the second relates to genes with null expressions even though no mutations were reported in the primer sequences.Two considerations deserve mention: the first relates to Murraya spp. is known for its ornamental value  and for the uses of their leaves as spices (M. koeniji). From a phylogenetic point of view, the relationship between the Murraya and Citrus genera is controversial, even though the Murraya genus is considered quite distant from Citrus. Recently some authors [M. paniculata is more closely related to Citrus than previously thought. However, this suggestion has not yet been fully accepted [M. paniculata genome. This leads us to hypothesize that the brilliant red color of the fruit may not be due to anthocyanins [M. paniculata contain coumarin and coumurrayin [ authors ,64 have accepted . This isocyanins , as alsomurrayin , in addimurrayin .Severinia buxifolia and S. disticha represent unusual accessions, because S. disticha, in particular, accumulates anthocyanins in both mature fruits and young leaves [S. buxifolia is associated with the unusual control of anthocyanins, being due to Ruby2 [M. australasica, and C. ichangensis, demonstrating very close conservation . In othThe expression data indicate that most EBGs do not usually show differences between pigmented and non-pigmented samples . NeverthIn our study, around 85% of the variance in the pigmentation of young leaves is explained by two main components a. Pure cC. maxima as a parent [The PCA shows that ‘Meyer’ is the accession that diverges most from the others a,b. Presa parent . There aC. micrantha [M. australasica , only those species with citron and C. micrantha as parents are characterized by purple young leaves, not considering their related genera. We suggest that species-specific transcription factors could be responsible for the inheritance of the trait for purple pigmentation in the young leaves. Such is demonstrated for the strategic role of Ruby2 exclusively in the leaves of S. buxifolia but not in its (purple) fruit [The regulatory genes Ruby and Noemi induce a divergence between ‘Mexican lime’ and ‘Lima rossa corrugata’, on the one hand, and ‘Buddha’s hand’ and ‘Diamante’ on the other a,b. The icrantha ,85,86. Tralasica . Ruby iss leaves . Phenotye) fruit . In Rosae) fruit .Citrus, the anthocyanin pigmentations of young leaves and of petals generally characterize the same species. Indeed, it is unusual to find accessions with purple young leaves and white flowers, or vice versa. Despite this, our expression analyses in association with image analyses, indicate that the pigmentation of petals is under the control of a complex genetic machine.In C. celebica’ represent the sample with the highest expression of PAL. ‘Zagara bianca’, whose petals are completely white, show an expression level 47-fold higher than of the purple petalled ‘Incomparabile’. Similarly, the EBGs are not much different between the pigmented and non-pigmented samples. In fact, for CHS, CHI and F3H, although more highly expressed in C. celebica than in ‘Zagara Bianca’ (about a 45-fold difference), F3H is still highly expressed in comparison with several of the pigmented accessions. Surprisingly, the LBG expressions are not very different between pigmented and non-pigmented samples.By browsing our expression results for petals, no differences are observed between pigmented accessions and ‘Zagara bianca’ . Even thC. celebica (C. celebica can be ascribed to its origins: C. celebica derives from C. micrantha (similar to ‘Mexican lime’), even though it is sited in the opposite direction  are characterized by having pigmented fruits only when they are very small, the pigmentation then decreases, disappearing altogether as the fruit enlarges. We hypothesize that the separation of ‘Incomparabile’ and ‘Pink fleshed’ from all the other accessions can be ascribed to stage of development and maturity, very precocious compared to the mature fruits of all of the other accessions.The separation between pigmented and non-pigmented rinds is clearly seen in the PCA, where all negative controls are near the origin of the plot a. Around The PCA a and the The PCA c also sund light . The fruCitrus species, being exclusive to a subset of sweet orange varieties including Moro, Tarocco, and Sanguinello and of hybrids with one of these as a parent. Even though pummelo represents one of the parents of the sweet orange, where a very ancient and original variety has been reported to be externally pigmented, the pigmentation of the flesh is specific only to sweet orange. The red color of the flesh of M. australasica var. sanguinea represents another example of inner fruit pigmentation, but different from sweet orange. In fact, the fruits of M. australasica are characterized by a homogeneous distribution of pigmentation in the flesh, independent of ripening. In sweet orange the pigmentation of the flesh increases during the fruit ripening. This is the first time that a genetic overview has been conducted on a subset of accessions, that include sweet orange varieties with different levels of pigmentation, and other Citrus species.Flesh pigmentation is a very unusual trait among Expression analysis of the genes involved in the early steps of biosynthesis shows no particular difference between the pigmented and non-pigmented samples . The expM. australasica and ‘Tarocco Lempso’ (both highly pigmented), even though the expression level of ANS in ‘Vaniglia sanguigno’ is very low. The expression levels of UFGT in ‘Vaniglia sanguigno’ and ‘Moro’ juice are very similar (about 30-fold increases). Lastly, the expression of GST in ‘Vaniglia sanguigno’ is also very similar to that in ‘Tarocco Lempso’ and M. australasica. Anthocyanin and lycopene quantifications in ‘Vaniglia sanguigno’ show that the red pigmentation could be due to lycopene . Less rFrom a statistical point of view, the qualitative difference between the pigmented and non-pigmented accessions is due to PAL and Ruby , the majOverall, more than 86% of the variance among the samples is explained by the first two principal components a. In parM. australasica var sanguinea is clearly far from the other samples, because it belongs to a different genus. It is interesting to observe that the collocation of ‘Vaniglia sanguigno’ and ‘M. australasica’, on opposite extremes of the second dimension, is presumably due to the strategic role played by Noemi, the bHLH transcription factor that, together with Ruby, controls anthocyanin pigmentation [Citrus fruits low acid [M. australasica represents the most acidic sample among those considered, with its citric acid content higher than that of the other Citrus fruits [Microcitrus is commonly known as ‘lemon cavial’ due to the spherical shape of vesicle juice and to the high acidity typical of lemon. The interconnection between anthocyanin pigmentation and the low acid trait has been reported [From a genetic point of view, the PCA a and theentation . The peclow acid . The prilow acid . The qRTs fruits . Microcireported , demonstreported ,19.Drosophila wings whose pigmentation contributes to the separation of species [Pigmentation represents a suitable model for studying how new patterns are generated along the evolution of species, as previously demonstrated in  species , or in m species . In plan species  and to a species . The dev species . Therefo species . Moreove species .Citrus, the color of the flowers represents an impressive trait, mostly for accessions of interest for their ornamental value. However, little is known about the pigmentation of stamens, styles, and stigmas, either from a phenotypic or from a genetic point of view. To the best our knowledge, this is the first time the different tissues of flowers have been characterized in Citrus from a genetic point of view, evaluating the role of anthocyanin’s biosynthetic and regulatory genes. Citrus species generally have perfect flowers comprising an androecium , made up of 20–40 stamens topped by white or yellow anthers containing the pollen. Meanwhile, the gynoecium  comprises a pistil including (from bottom to top) an ovary, a style, and a stigma.Even though red flowers have evolved repeatedly in plants, as well as in M. australasica (a one-fold increase) - the former non-pigmented and the latter highly colored. As for the other tissues, LBGs (except UFGT for the style) showed preferential high expressions for all pigmented samples compared to negligible expressions in the negative controls, ‘Zagara bianca’ and ‘Navel’, independent of the expression level . These results suggest a putative relationship between Ruby and Noemi, as observed exclusively for the rind  and LBGs are highly expressed in ‘Moro’ and ‘Sunred’ compared to the negative controls ‘Navel’, ‘Mexican lime’, and ‘Tahiti’ . The stiCitrus the red pigmentation of the stamens, styles, and stigmas is very interesting. The first phenotypic observation is that there is no correspondence or relationship with one another. The pigmentation of stamens is almost exclusively in citron and M. australasica and we found this to be very stable and independent of environmental conditions. The purple pigmentation of the styles is most frequent among citron and lemons but it is highly dependent on light. Interestingly, flowers, with abnormal development characterized by a protrusion of the style, show an unconventional blood pigmentation  has been inherited by citron, even though not stably, as observed for young leaves and petals. Not all accessions with pigmented leaves and petals show colored styles and stamens.Among entation . GeneralIn contrast, the pigmentation of the stigmas is an exception and is genotype-dependent. The pigmentation of the stigmas is unique and exclusive to ‘Moro’ and ‘Sunred’. The stigmas of the other pigmented and non-pigmented sweet orange varieties showed no phenotypic pigmentation . The pigCitrus and related genera, a wide selection of pigmented tissues has been characterized genetically and phenotypically. A hypothesis is proposed regarding the genetic inheritance of anthocyanin pigmentation in leaves, petals, flesh, rind, stamens, styles, and stigmas . Ruby represents the MYB transcription factor that controls the pigmentation of petals, but not of leaves.The pigmentation in young leaves and petals depends on citron. In species different from Citrus, but stably characterizes blood oranges. The control of the variability in the flesh and rind represents one of the main traits sought by consumers and producers, and one of the focuses of breeding programs all over the world.The pigmentation of fruit tissues has been gained and lost frequently through the history of Microcitrus for stamens.The pigmentation of stamens and styles has given rise to the citron parent, but this trait is genetically less stable than in leaves and petals. Ruby and Noemi also control the pigmentation of stamens, in species different from citron and its hybrids, such as in The pigmentation of the stigma is Moro-dependent, the only genotype-dependent trait, representing a new strategic phenotypic marker. The study of light and cold-dependent control of stigma pigmentation is similar to that known in fruits, and is currently under evaluation.For the first time in  stigmas . We findCitrus genome will help identify the specific genes forming the WMBW complex that, under various rearrangements, may provide a punctilious genetic basis to explain the generation of new patterns of pigmentation in all species.A more thorough investigation of the promoter regions, as well as of transcription factors other than from Ruby and Noemi, is need if we are to better explain which mutations and molecular mechanisms dominate in the control of pigmentation in all tissues. Fuller understanding of the


Myocardial infarction is the most common cause of death all over the world. There are many studies in predicting mortality. The aim of this study was to determine the effectiveness of hematologic parameters measured at the moment of admission to the emergency room in predicting in-hospital mortality and to determine cutoff values of strongly predictive values. T values were recorded as cardiac markers. For statistical analysis, “SPSS for Windows version 21” package program was used.  A total of 681 patients over 18 years of age, whose date could be obtained, were included in the study. From the hemogram parameters, white blood cells (WBC), red cell distribution width (RDW), mean platelet volume (MPV), and neutrophils-to-lymphocytes ratio (NLR) values were determined and recorded. CK-MB and high-sensitive troponin n = 426) of the patients were male, and 37.4% (n = 426) of the patients were male, and 37.4% ( 62.6% ( According to the hemogram results which were acquired with a simple and cheap method, we found that WBC and especially NLR values obtained with a simple method can be used as powerful predictors. Cardiovascular diseases are some of the most important causes of mortality and morbidity in society. These diseases may present as stable coronary artery disease (CAD) or acute coronary syndromes. Non-ST-elevation myocardial infarction (NSTEMI) is a presentation in the acute coronary syndrome spectrum and accounts for 61% of myocardial infarction . For thiAcute coronary syndromes are triggered by the rupture of atherosclerotic plaques in the coronary arteries. The occurring situation starts a thrombotic process. When the artery lumen is partially obstructed, unstable angina or NSTEMI appears, while the artery lumen is completely blocked, STEMI appears. The relationship between acute myocardial infarction (AMI) and inflammation has been expressed in various epidemiological and clinical investigations –6. AmongLiterature review suggests that many publications including NSTEMI patients mention the predictive and prognostic significance of hematologic parameters –11. HoweThe aim of this study was to determine the effectiveness of hematologic parameters measured at the moment of admission to the emergency room in predicting in-hospital mortality and to determine cutoff values of strongly predictive values.The study began with approval of the ethics committee . A total of 1917 patients hospitalized to our hospital's coronary care unit with a NSTEMI diagnosis between 01 January, 2013, and 31 December, 2016, were scanned retrospectively and documented from the hospital automation system. Patients' ages, sex, laboratory results, angiography results, and in-hospital outcomes (living and dying) were recorded. A total of 681 patients over 18 years of age, whose data could be obtained, were included in the study. A total of 1236 patients were excluded from the study, who had missing information in their file, were under the age of 18, referred to another center, refused treatment, or who were identified as a NSTEMI diagnosis as the final diagnosis. Additionally, while NSTEMI-related deaths were accepted as inclusion criteria, other death-related diseases  were excluded from the study.T) measurements of patients included in the study were performed via venous blood sample taken during the initial admission to the emergency room. From the hemogram parameters, white blood cells (WBC), red cell distribution width RDW, mean platelet volume (MPV), and NLR values were determined and recorded. CK-MB and high-sensitive troponin T values were recorded as cardiac markers. The venous blood sample taken during the emergency room admission of the patients was used. Complete blood count measurements were made using the Sysmex XN 10  automatic measurement device. CK-MB and high sensitivity troponin T measurements were made using the Cobas 6000  automatic measurement device.Hemogram and cardiac marker  curve analysis and area under the curve (AUC) calculations of hematologic parameters and cardiac markers were used to calculate their effectiveness in predicting mortality. The cutoff values of strong predictor parameters were recorded with the most appropriate specificity and sensitivity ratio.For statistical analysis, “SPSS for Windows version 21” package program was used. Descriptive statistical methods (mean and standard deviation) and Student's T values of the patients are shown in 681 patients were included in the study. The WBC, RDW, MPV, NLR, platelet, CK-MB, and troponin n = 426) were male, and 37.4% (n = 255) were female (n = 19) were exitus in the hospital (group 1), 97.2% of the patients (n = 662) were discharged from the hospital (group 2). All of the group 1 patients consisted of NSTEMI-related patients who died. Age, WBC, NLR, and high-sensitive troponin T values were significantly higher in patients with exitus than the other group (p < 0.05 for each of them)  .When the efficacy of hematologic parameters and cardiac markers in predicting mortality was examined by ROC analysis, NLR was found to be the strongest predictor . As a result of the analysis, when the cutoff value of NLR was taken as 3.625, it was found that it had 84.2% sensitivity and 66.3% specificity. It was found that the WBC value came in the second place after NLR as a strong predictor of mortality . As a result of the analysis, it was determined that when the cutoff value for WBC was taken as 10870, it had 68.4% sensitivity and 62.5% specificity , Table 4The hemogram is requested for almost every critically ill patient who is assessed in the emergency room. It is a fast-paced, simple to evaluate, and a very basic laboratory test. The NLR value can also be calculated easily from the obtained hemogram results. According to the literature data, it is suggested that many parameters can be used in the evaluation of hemogram for short- and long-term evaluation of mortality of acute myocardial infarction patients. Clinical and meta-analysis studies are available that indicate that NLR and WBC may be independent factors in determining mortality , 10, 12.Advanced age is a known risk factor for coronary artery disease. Bajari and Tak studied 400 patients with acute coronary syndrome, 163 of whom had NSTEMI and unstable angina clinic. In this group, mortality for the period of initial admission to 6 months was examined.  The meanAge over 45 for men and over 55 for women is a strong risk factor for coronary heart disease . This prThe relationship between myocardial infarction and inflammation has been known for over 80 years . EvidencDetermining the number of peripheral leukocytes is a common and inexpensive method to assess the presence of any inflammation.  AccordinNLR is not affected by risk factors such as hypertension, diabetes mellitus, smoking, obesity, and hyperlipidemia . It is aIn a study conducted by Bajari and colleagues on 435 patients, NLR values higher than 5.25 were found to be strongly predictive of in-hospital mortality. In this study, isolated NSTEMI patients were not evaluated . In a siIncreased leukocyte count in the peripheral vasculature is a prominent sign which puts microvascular reperfusion at risk . There iNSTEMI is still a condition with very high mortality despite advancing treatment options and angiographic interventions. The use of predictors for the prediction of mortality for these patients is of great importance for faster implementation of treatment modalities. According to the hemogram results which were acquired with a simple and cheap method, we found that WBC and especially NLR values obtained with a simple method can be used as powerful predictors. We believe that the cutoff values we acquired will aid the literature and research with large series, which are multicenter, prospective, randomised and where NSTEMI patients are analysed isolated.