The increased risk of fracture has been associated with weight loss in patients with diabetes or chronic kidney disease. However, the relationship between weight changes over time and fracture risk in patients with diabetic kidney disease is still unknown.A total number of 78,922 patients with diabetic kidney disease, aged ≥ 40 years, were selected using the Korean National Health Insurance Service database, between 2009 and 2012. They were followed up until the end of 2018. Weight change was defined as the difference in body weight from the index year to 2 years later. Weight changes were then divided into five categories, ranging from weight loss of ≥10% to weight gain of ≥10%.Fractures were identified in 9,847 patients with diabetic kidney disease, over a median follow-up of 5.2 years. The risk of composite fracture of the vertebral, hip, or other sites increased as the weight change increased. Specifically, patients with ≥10% weight loss  and ≥10% weight gain  showed a higher HR compared to those with ≤ 5% weight change after adjusting for several confounding factors. Higher HR of vertebral and hip fractures was also seen with increased weight loss or gain. In particular, patients with ≥10% weight loss showed the highest HR for hip fractures .Both weight loss and weight gain increase the risk of fracture in patients with diabetic kidney disease. Therefore, patients with diabetic kidney disease who experience weight changes should be made aware of the risk of fracture. The increasing prevalence of diabetic kidney disease (DKD) parallels the dramatic worldwide rise in the occurrence of diabetes , 2. The Bone fractures are concerning events that have devastating health consequences. Although factors such as age, sex, comorbidities, and poor health status play a role, it is the fracture event itself that is responsible for an increased mortality risk , 6. DeclIn addition to the traditional fracture risk factors, such as age, body mass index (BMI), comorbid conditions, fracture and fall history, and bone mineral density (BMD), a recent study showed that weight change is associated with increased fracture incidence in postmenopausal women . MoreoveOur objective was therefore to investigate the association between loss or gain of body weight (BW) over 2 years and the subsequent incidence of overall fracture. Furthermore, we aimed to determine whether weight change would increase the incidence of specific fractures classified by vertebral, hip, and other sites in DKD patients selected from a nationwide cohort.We used the national health insurance claims database established by the Korean National Health Insurance Service (NHIS), in our study. This database includes data on all claims provided by the NHIS and Medical Aid programs. The Korean NHIS database represents the entire South Korean population and the details of this database have been previously described , 16. KorWe initially identified 2,746,079 patients with diabetes, who between 2009 and 2012 underwent a health check-up. Of these, we excluded the following patients from our study: patients who did not undergo a repeat health check-up after 2 years, those without a history of CKD, those with malignancy, those with a history of fracture before the index date, patients < 40 years of age (fractures are rare in this subgroup), and patients who had any health examination data missing. From these vast data results, we finally included 78,922 patients with DKD in our study. The date of the last health check-up was set as the index date. The selection of study participants is presented in a detailed flowchart in Weight change was calculated over a 2-year interval based on the weight difference between the first and the second health check-up (expressed as a percentage). Based on a previous study , patient2  codes  and a history of antihypertensive drug use, or a recorded systolic blood pressure (BP) ≥ 140 mmHg, or diastolic BP ≥ 90 mmHg. Diabetes mellitus was defined as a previous clinical diagnosis (ICD-10-CM codes E11–14) and a treatment history of diabetes or a recorded fasting serum glucose level ≥ 126 mg/dL. CKD was calculated using the Modification of Diet in Renal Disease formula and was defined as an estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2 . DyslipiThe incidence of fracture was the study endpoint. Vertebral fracture was defined as more than two claims per year of the following ICD-10-CM codes: S22.0, S32.0, S32.1, M48.4, and M48.5, while hip fracture was defined as claims of the following diagnostic codes: S72.0, S72.1, and S72.2 at admission. -Other fractures were defined as more than two claims per year of the following ICD-10-CM codes: S42.0, S42.2, S42.3, S52.5, S52.6, S82.3, S82.5, and S82.6. Lastly, composite fracture of the vertebral, hip, or other sites was defined as any-fracture. We excluded traumatic fractures in this study.2), the use of insulin, and the number of oral hypoglycemic agents (≥3) used. Interaction terms were added to test for effect modification across subgroups . All data analyses were conducted using the SAS software , and P < 0.05 was considered statistically significant.Data are presented as mean ± standard deviation for continuous variables and numbers with proportions for categorical variables. Non-normally distributed variables are presented as geometric means . Chi-square test or analysis of variance was used to test inter-group differences, as appropriate. The incidence rate of fractures is presented per 1,000 person-years. Multivariable Cox proportional hazard regression analysis was used to estimate the hazard ratios (HRs) and 95% CIs of weight change associated fracture risk after adjusting for age, sex, smoking, alcohol consumption, regular exercise, low-income status, use of insulin, number of oral hypoglycemic agents, duration of diabetes, previous history of hypertension, dyslipidemia and ESRD, and previous BW. Sensitivity analyses were performed after excluding patients who had fractures within 1-year of follow-up. Furthermore, we also performed analyses to identify whether there was an association between the risk of fractures and weight change over 4-years between health check-ups. Subgroup analyses were performed to assess the effect modification on the risk of fractures in DKD patients based on age (<65 and ≥65-years groups), sex, history of hypertension, duration of diabetes (<5 and ≥5-year groups), the presence of proteinuria, BMI , the adjusted HRs for any-fracture increased to 1.100  and 1.286  in participants with 5–10% weight loss and ≥10% weight loss, respectively. Similarly, participants with ≥10% weight gain demonstrated a significantly increased risk of any-fracture compared to participants with stable weight during the period of observation (adjusted HRs [95% CIs]: 1.198 [1.080–1.330]).Subsequent evaluation of the risk of vertebral, hip, or other site fractures according to weight changes using the multivariable Cox analysis revealed that participants with ≥10% weight loss had the highest risk of vertebral and hip fractures relative to the reference group , similar to the any-fracture risk. Moreover, participants with ≥10% weight gain also showed an increased risk of vertebral and hip fractures. However, weight changes did not significantly correlate with the risk of fracture at any other sites.To account for the possibility of reverse causation, a sensitivity analysis was performed, and participants who had fractures within 1-year of follow-up were excluded . Even af2). In contrast, in patients with obesity (BMI ≥ 25 kg/m2), the ≥10% weight gain group had a higher HR, but the weight loss group had no significant association with any-fracture risk.The association between weight change and the risk of any-fracture was explored, using subgroup analyses, after stratification by age, BMI, sex, diabetes duration, presence of proteinuria, history of hypertension, number of oral hypoglycemic agents, and insulin use . In all In this nationwide population-based cohort study, we found that DKD patients with ≥10% weight loss over 2-years had a 29% higher risk of any-fracture, 30% higher risk of vertebral fracture, and 74% higher risk of hip fracture compared to patients with < 5% weight change. Similarly, DKD patients with ≥10% weight gain had a 20% higher risk of any-fracture, 22% higher risk of vertebral fracture, and 36% higher risk of hip fracture compared to patients those who had a stable BW. Moreover, compared to the stable weight group, ≥10% weight gain was associated with an increased risk of any-fracture in patients with hypertension or obesity as well as in patients with diabetes for > 5 years. To the best of our knowledge, this is the first study to report the association of a 2-year weight loss or gain with the risk of fracture at various anatomical sites in patients with DKD, using annual health check-up data.Previous general population studies have reported that a weight loss of ≥5% or 10% is associated with hip fracture in both postmenopausal women and men aged ≥ 50 , 19–22. 2 and fracture risk in the general population  and performed in accordance with the ethical standards of the committee responsible for human experimentation and the Helsinki Declaration of 1975, as revised in 2013. The Ethics Committee waived the requirement of written informed consent for participation.CSK, KH, and SWK conceived and designed the study, and were responsible for the acquisition, analysis, and interpretation of the data. CSK, BK, and KH contributed to the statistical analysis. CSK and SWK contributed to the acquisition of funding for the study and drafted the manuscript. HSC, EHB, SKM, and SWK supervised the study. All authors critically revised the manuscript and approved the final version of the manuscript.This work was supported by the National Research Foundation of Korea (NRF) funded by the Korea Government (MSIT) (NRF-2019R1A2C2086276), by Basic Science Research Program through the of NRF funded by the Ministry of Education (NRF-2018R1D1A1B07042999), and by grants (BCRI22080 and BCRI21023) from Chonnam National University Hospital Biomedical Research Institute.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Background: Aplastic anemia is a rare and life-threatening condition, seldomly witnessed concomitantly with Chagas disease. We aim to discuss the management of these patients under risk of chronic Chagas disease reactivation (CDR), a severe condition with a high morbimortality that occurs in chronic Chagas disease patients under immunosuppression. Case reports: Trypanosoma cruzi (T. cruzi) parasitemia was monitored in three patients for 4–58 months by conventional PCR (cPCR), quantitative PCR (qPCR), microhematocrit/buffy coat, blood culture, and/or xenodiagnosis. One patient received antiparasitic treatment (benznidazole) and the other received allopurinol. Although parasitemia was controlled during and after benznidazole treatment at 300 mg/d for 51 days, in one patient, hematologic parameters worsened continuously before, during, and after treatment. Allopurinol led only to the temporary suppression of T. cruzi parasitemia in the second patient, but after danazol and hematological improvement, parasitemia became undetectable until the end of monitoring. Discussion and Conclusion: Unexpected undetectable or low parasitemia by cPCR/qPCR was reported. We show that the monitoring of parasitemia by qPCR and the use of preemptive therapy when the parasitemia was positive proved to be beneficial to our patients. As a result of the toxicity of more effective antiparasitics, shorter regimens of benznidazole or less toxic drugs in preemptive therapy are options that deserve future studies. Acquired aplastic anemia (AA) is a rare, life-threatening condition characterized by cytopenia and bone marrow (BM) failure, caused by a physical, chemical, or biological factors . Bone maRecovery after treatment with anti-thymocyte globulin (ATG), cyclosporin A, or anti-lymphocyte globulin, suggests that autoimmunity may be involved in the pathogenesis of AA. Immune dysfunction is expressed by an altered number of lymphocyte subsets, increased effects of cytotoxic lymphocytes on hematopoietic stem and progenitor cells (HSPC), and low percentages of NK cells. In parallel, genetic background and intrinsic deficiencies of the HSPC, evidenced by somatic mutations such as shortened telomeres and viral infection, may also be associated with AA pathogenesis ,5.Although Chagas disease is endemic in Latin America, aplastic anemia in patients bearing chronic Chagas disease has rarely been reported . The immT. cruzi and is acquired through vector and maternal–fetal transmissions, blood product transfusions, organ transplantations, contaminated food, and accidents with infected biological material [Chagas disease is a neglected tropical disease affecting approximately six million people in Latin America, and is now globally widespread due to the immigration of infected people from Latin America to other continents . It is cmaterial . After cmaterial . In thismaterial . Chronic Chagas disease patients under immunosuppression and/or immune dysfunction caused by aplastic anemia are at risk for CDR characterized by severe morbimortality ,10. T. cruzi infection. For allogeneic HSCT, infected donors are not accepted, except in emergencies, where no other non-T. cruzi infected donor is available. Prospective monitoring of parasitemia plays a key role in its control and in reducing the risk of CDR in immunosuppressed patients either with HIV infection [Screening for Chagas disease is mandatory in patients living in endemic regions, for those born from infected mothers, or for those that received multiple blood or derivatives transfusions. Two serological tests with different methodologies confirm the diagnosis of nfection ,12, autonfection , or undenfection ,15.In this scenario of AA associated with chronic CD, the evolution of both diseases and the risk of CDR is not known, representing challenges for patients’ follow-up. An additional issue to address is the myelotoxicity and other adverse events of benznidazole and nifurtimox, both recommended for antiparasitic treatment  and compT. cruzi parasitemia monitoring in the management of three AA patients with chronic Chagas disease, aiming to analyze the role of qPCR and preemptive therapy to prevent CDR. This work describes the role of T. cruzi was monitored by cPCR using the S35/S36 primer pair and qPCR for the microsatellite sequence TCZ3/TCZ4, as previously described [The inclusion criterion for Chagas disease was reactivity by two different serologies (ELISA and Indirect Immunofluorescence) . Parasitescribed ; blood cescribed . Chagas escribed . This study was approved by the Ethics and Research Committee of the Hospital das Clínicas da Faculdade de Medicina, University of São Paulo, Brazil (protocol number 095/1995); and the inclusion of retrospectively collected data was approved by the Ethics and Research Committee of the Department of Infectious and Parasitic Diseases of the Faculdade de Medicina, University of São Paulo, under protocol 003/22, ensuring that the confidentiality of patients’ identity was maintained. All data were collected anonymously from medical records.Patient 1T. cruzi parasitemia monitoring for 28 months.Male, 55 years old, from the state of Alagoas, Brazil, with no previous antiparasitic treatment. Underwent Previous arterial hypertension and Chagas heart disease with a pacemaker due to total atrioventricular block and ventricular dysfunction.3; platelets 8000/mm3; total leukocytes 2980/mm3 (neutrophils 760/mm3). Bone marrow biopsy (30 January 2019) showed global hypocellularity (10%). A diagnosis of severe aplastic anemia was made, and cyclosporine use began in February.On 29 January 2019, the patient was admitted to the emergency room with symptoms of presyncope, gingival bleeding, and bruising. Hb = 4.4 g/dL; reticulocytes, 7000/mmT. cruzi parasitemia was performed. Positive cPCR and qPCR below detection levels were observed on 27 March 2019 .On 30 April 2019, the complete blood count (CBC) showed Hb 8.4 g/dL; platelets 5000/mmThe patient’s evaluation on the eighth day of benznidazole revealed a good response; on the 15th day, cPCR was negative, as well as on the 30th day . On day 3; leukocytes 1210/mm3 . On 27 May 2019, BCC showed Hb 5.6 g/dL; Ht 16.1%; 1000 platelets/mmDuring the period from 6 February 2019 to 11 March 2020, he received 200 mg/day of cyclosporine, tranexamic acid, losartan, atenolol, multiple red blood cell transfusions, and platelet concentrates by apheresis.ATG infusion was prescribed from 29 May 2019 to 2 June 2019, and prednisone prescription followed the protocol.On 10 December 2019, a new bone marrow biopsy was performed and showed no improvement in cellularity. Cyclosporine was suspended on 11 March 2020, and Alemtuzumab was introduced from 2 March 2020 to 6 March 2020, with total doses of 100 mg.Direct examination of parasitemia was persistently negative during 26 months of follow-up and after ATG; cPCR was continuously negative after the treatment, and qPCR was negative on 21 July 2021 .He underwent courses of antibiotics associated with neutropenia and multiple transfusions for symptomatic anemia and cutaneous–mucosal bleeding associated with thrombocytopenia. Hospitalization on 5 July 2021, for febrile neutropenia due to pulmonary infection, with clinical improvement.3, and platelets from 8000–47,000/mm3.Hematological examinations from this period up until 30 July 2021 revealed Hb from 5.1-7.2 g/dL, leukocytes from 160–280/mmOn 31 July 2021, he evolved with a refractory septic shock of abdominal focus (neutropenic enterocolitis) and died on 2 August 2021.Patient 2T. cruzi parasitemia monitoring for 4 months and 20 days.Female, 47 years old, born in Bolivia. Underwent She came to Brazil for treatment for aplastic anemia diagnosed in 2014 and was previously treated in Bolivia. She had Chagas disease with non-typical cardiac alteration , morbid obesity, and arterial hypertension in treatment.On 20 May 2016, she was hospitalized with pancytopenia, symptomatic anemia, and thrombocytopenia with gingival and vaginal bleeding. She was admitted again by febrile neutropenia 1.5 months later, which resolved after clinical treatment. Mycophenolate mofetil (360 mg 12/12 h) was reintroduced and she required multiple transfusions.T. cruzi parasitemia was undetectable by both molecular methods and direct microscopy for CDR diagnosis in this period  after performing an aspirate and BM biopsy that confirmed AA. On 15 July 2016, ATG was administered for five days at a dose of 2.5 mg/kg/day, and fter ATG .Four months later, under the daily use of cyclosporine, no hematological response was observed. Cyclosporine was suspended after a pulmonary infection associated with febrile neutropenia. On 1 February 2017, without an HLA-matched donor or improvement in peripheral blood after seven months of ATG, danazol was introduced at a dose of 400 mg/day.Ten days later, she was admitted to the emergency unit due to septic shock of abdominal focus/enterocolitis, evolving to death on 13 February 2017.There was never a detection of positive parasitemia during the 140 days of parasitemia monitoring, so antiparasitic treatment was not administered.Patient 3T. cruzi parasitemia monitoring for 58 months.Female, 63 years old, born in Bolivia, living in Brazil since 2011. Underwent Comorbidities: chronic hepatitis B (tenofovir 300 mg/d), HA , and indeterminate form of Chagas Disease; denied antiparasitic treatment.On September 2011, she had fatigue and epistaxis. Bone marrow aspiration and biopsy showed a global hypocellularity, diagnosed with bone marrow aplasia.3; leukocytes 2000/mm3 . On 9 November 2011, CBC showed Hb 7.6 g/dL; platelets 18,000/mmUpon diagnosis of severe aplastic anemia, she started treatment on 14 December 2011, at the Hematology Unit with 200 mg/day of cyclosporine, with no response at three months and partial response at six months.T. cruzi were found (Positive cPCR and qPCR (9.32 parasites Eq/mL) and negative direct microscopic examination, with no reactivation symptoms, were shown three months after the initiation of cyclosporine; then, negative cPCR and qPCR below the detection level, and positive blood culture were found . Preemptre found .In the absence of reactivation criteria, antiparasitic therapy with a lower risk of myelotoxicity was considered. The risk–benefit of benznidazole was evaluated, as well as the possibility of treatment with posaconazole, which was currently unavailable, and allopurinol was opted for as an alternative drug.On 9 January 2013, cyclosporine was replaced due to an adverse effect, and danazol was introduced at a dose of 300 mg/day.Allopurinol was started on 23 May 2013, at a dose of 300 mg/day for the first 14 days, then reduced to 200 mg/day (dose adjusted for creatinine clearance), completing a total of 38 days (non-complete treatment). After treatment, parasitemia occurred .On 9 April 2014, a complete response to danazol was observed and was withdrawn after one year of a plateau response. Danazol was suspended on 18 March 2015. The patient was stable, without infectious complications and bleeding or symptomatic anemia.3; leukocytes 4150/mm3 . On 28 March 2014, CBC showed Hb 11.3 g/dL; platelets 200,000/mmDuring the period of stability of the underlying disease, she evolved with positive cPCR and undetectable qPCR for 15 months, but in the final 14 months of follow-up, cPCR reverted to negative and qPCR remained undetectable .T. cruzi growth in synergism with TNFα, through nitrite oxide secretion [Two patients showed severe aplastic anemia and received an aggressive immunosuppressive regimen , consequently presenting a deficiency of T lymphocytes and macrophage functions. One patient with moderate aplastic anemia received cyclosporine and danazol. The crucial role of CD4+, macrophage activation, and IFNγ secretion to kill the parasites has been shown ,17,18. Iecretion .3. In addition, positive parasitemia does not seem to depend on the absolute number of granulocytes or monocytes.As seen in 3 concomitantly with a low number of neutrophils and platelets under different drugs. The unexpected undetectable and/or low levels of parasitemia in our three patients raised questions concerning the control of parasite growth. For example, P2 never had detectable parasitemia in the presence of a low/normal number of lymphocytes/mm3 and negative parasitemia. Unfortunately, lymphocyte or macrophage functions, as well as lymphocyte subsets or cytokines were not analyzed, except in P1, with low levels of CD4 (CD4/CD8 = 0.6 and CD4/CD8 = 0.18) concomitantly with < 100 lymphocytes/mm3 [T. cruzi, and other intracellular infections have rarely been described in AA patients [T. cruzi immunity, other expressions of immune responses, such as an innate response, could control parasite growth and need to be better understood.As already described, before immunosuppressive treatment, AA patients may have normal to hyperactive immune systems and intact intertegumentary and mucosal barriers , but the3 ,21,22, bpatients ,20. ConsAnother question to be addressed is the choice for antiparasitic treatment, either efficient but myelotoxic drugs (benznidazole and nifurtimox) , or the Benznidazole was prescribed to P1 before ATG treatment as a result of positive parasitemia and was interrupted earlier than the classic period of 60 days. Decreasing Hb, leucocytes, and platelets were seen during antiparasitic therapy; however, besides possible myelotoxicity, the severe natural evolution of AA needs to be considered. From the beginning of antiparasitic therapy to 28 months later, parasitemia was negative, even though ATG, cyclosporine, corticosteroid, and alemtuzumab had been prescribed. According to guidelines, a regimen of 5 mg/kg/day of BNZ for 60 days is mandatory to treat CDR and acute Chagas disease [For P3, a less severe AA case with a low number of parasites (Eq/mL), allopurinol was prescribed as an alternative antiparasitic therapy. Temporary suppression of parasitemia was observed during allopurinol, but PCR returned to positive 18 months later, after the end of this therapy . RemarkaT. cruzi infected AA patient [T. cruzi uninfected donor, had GVHD and CDR after the transplant, and recovered after 60 days of benznidazole treatment [Our three cases added new information to the previously reported  patient . This paFor the management of AA patients, we should consider, on the one hand, the intensity of hypocellularity and the myelotoxicity of antiparasitic drugs, and, on the other hand, the low parasitemia in our severe AA patients and the report of CDR in one case after a regimen of severe immunosuppression after transplantation .In addition, it is necessary to keep in mind that the antiparasitic treatment aims to either cure , decrease parasitemia, or interfere with the evolution of Chagas disease .Patients with parasitemia (cPCR or blood culture/xenodiagnosis) under a highly immunosuppressive regime (ATG or HSCT transplantation) or only high parasitemia ≥ 100 parasites Eq/mL by qPCR under another immunosuppressive regimen. In addition, because of the known myelotoxicity of more efficient antiparasitic drugs, a shorter regimen of benznidazole could be an option based on promising studies , at 300 Patients with a low number of parasites (Eq/mL) by qPCR, parasitemia by cPCR, or any parasitological enrichment method persistently positive under less aggressive therapy, should be carefully considered as benznidazole poses a greater risk of granulocytopenia worsening in patients whose parasitemia is under control. For this situation, a safer option is represented by less toxic schemes such as intermittent benznidazole, whose preliminary results need to be confirmed , or alloConsidering these facts, preemptive therapy seems to be a more reliable recommendation for AA patients without CDR in different situations:T. cruzi parasitemia, continue to be challenges to be addressed in other studies.Alongside the search for new effective and less toxic drugs or drug combinations for antiparasitic treatment, the role of different cells and innate immunity, as well as the role of immunosuppressive drugs such as danazol in the control of T. cruzi-HIV or other immunosuppressive conditions [T. cruzi parasitemia, shorter and less toxic schemes are options to be prescribed under strict control of myelotoxicity and parasitemia, which are deserving of future studies.In summary, we observed that the monitoring of parasitemia by cPCR/qPCR was proven to be a useful tool for the introduction of a preemptive antiparasitic therapy in two patients, who had a conversion of parasitemia from positive to negative temporarily or until the end of follow-up. The same application of molecular methods has been previously reported, in the coinfection nditions ,13,14,15nditions . Further


In conclusion, our findings suggest that healthy organizational practices promoted worker engagement and decreased worker burnout during the COVID-19 pandemic, contributing to the postulates of the HERO model. In addition, we were able to visualize a similar scenario, which showed that burnout during a pandemic decreases when worker engagement mediates the relationship with HOP.The COVID-19 pandemic has substantially impacted mental health—workers at institutions are not exempt. In our research, from positive organizational psychology, specifically from the healthy and resilient organization (HERO) model, we analyzed the relationship between healthy organizational practices–engagement and workers’ burnout, and evaluated the mediation role of engagement between healthy organizational practices and worker burnout levels during the COVID-19 pandemic, through structural equation models of a cross-sectional survey-based study. We collected data from a sample of 594 Chilean workers. Our results of the correlations and structural equations demonstrate the relationship between PHOs with engagement (β = 0.51;  Will COVID-19 have a substantial impact on the mental health of humanity? This is a question that Izaguirre-Torres and Siche ,3,4,5,6.Grover et al.  noted thThe confinement/isolation of people has been the main measure taken by governments to stop the spread of COVID-19, which, according to Fiorillo et al. , has proOrganizations and companies are not exempt. Salanova  noted thEngagement is one variable that expresses well-being and health in workers . In addiVarious investigations show that managerial practices that organizations implement are fundamental to generate engagement in workers ,24,25,26The healthy and resilient organization (HERO) model is a management model that proposes the development of planned–systematic practices and resources at organizations, which will generate positive work environments and have positive impacts on the well-being and health of employees; this in turn will promote good individual and team performances, implying healthy organizational results. The model proposes a group of healthy organizational practices (HOP). HOP influence worker engagement  as well The COVID-19 pandemic has led to (and will continue to lead to) mental health problems . The folIn this research, we analyzed the relationship between healthy organizational practices–engagement and workers’ burnout; we evaluated the mediation role of engagement between healthy organizational practices and worker burnout levels during the COVID-19 pandemic.Although our approach was from positive organizational psychology, we could not extract ourselves from reality; therefore, we integrated burnout among the variables of this research due to its presence in workers during the COVID-19 pandemic ,31,32.Since the beginning of the millennium, positive psychology has greatly developed in the organizational field ,34 and iFrom this approach, organizations must develop management practices that promote good health and the well-being of their employees, which will imply good individual and team performances. Thus, these management practices will impact the financial health of the company and its results .p < 0.01). Furthermore, by applying the structural equation model, a positive and significant relationship between HOP and engagement  was found.Using the HERO-based model , Acosta Due to its relevance, the theoretical support in the HERO model, and its results, we defined the work of this research  as the iHypothesis 0 (H0).The relationship between the perception of healthy organizational practices and employee engagement was positive and significant during the COVID-19 pandemic.For Wright and McMahan  (p. 298)Research ,23,40,41r between 0.17 and 0.23, p < 0.01) and dedication , and negatively with cynicism . However, only social support correlated with burnout .Schaufeli et al.  conducter between 0.20 and 0.36, p < 0.001); and negatively and significantly related to burnout . However, when they applied linear regressions, they only found significant results when organizational practices predicted the dimension “dedication” .Matziani et al.  found thSimilar to research by Schaufeli et al.  and MatzHypothesis 1 (H1).The relationship between the perception of healthy organizational practices and employee burnout was negative and significant during the COVID-19 pandemic.Engagement is a positive motivational state of vigor, dedication, and absorption , which iBurnout syndrome is a state of emotional exhaustion, cynicism, and low personal fulfillment ,45. TherBakker et al.  note thaParker, Bindl, and Strauss  reviewedp < 0.001). These results provided empirical evidence that a worker who experiences engagement with their job is less likely to present burnout [Through applying structural equation modeling, Hussein  conducte burnout ,51 .Engagement acts as a mediational variable for the perception of healthy organizational practices to decrease burnout.In this cross-sectional survey-based study, we carried out self-administered surveys through an online questionnaire between October and December 2020, after seven months of operation under confinement conditions in Ñuble, Chile (the first confined city to lockdown in Chile). Through convenience sampling in the stage, 594 surveys were obtained from different labor sectors: education (46.10%), health (20.02%), commerce (12.50%), industry (8.90%), and other items (12.30%).In order to participate in the study, the workers had to be over 18 years of age, and, at the time of answering the questionnaire, formally work with signed contracts. Self-employed workers were not included.The online questionnaire was sent via email to workers at different companies, after contacting representatives of the institutions, who delivered the database of workers who met the inclusion criteria.Due to the conditions generated by the pandemic, it was not possible to contact all the companies in the Ñuble region, Chile.Healthy organizational practices. The statements proposed by the HERO model [HOP = 0.94).RO model  were extRO model . Nine stEngagement. Engagement was evaluated with the Utrecht Work Engagement Scale .le (UWES ): vigor Burnout. The Maslach Burnout Inventory General Survey . (MBI-GS ) was use  ; and the root mean square approximation error index (RMSEA), which allows analyzing the residuals, where the values that suppose a good fit in terms of residuals are those less than 0.08. In addition, the following indicators were used: comparative fit index (CFI), which corresponds to a standardization of the Bentler index and is recommended for use instead of chi2, together with RMSEA for samples over one hundred cases. A good fit is considered at values above 0.90; the non-normed fit index (NFI) or the Tucker–Lewis index (TLI), for which values equal to or greater than 0.90 are expected [The estimation method used in this study was the one with maximum likelihood because it allowed recovering weak factors in the context of analyzing confirmatory models with n > 100 , at the n > 100  that wasexpected . One indexpected . These lexpected , equal texpected ,59.All analyses were performed using JASP 0.16 software .The distribution by gender was 65.50% women and 34.50% men. According to age, 12.00% were under 24 years old, 30.60% were between 24 and 30 years old, 25.10% between 31 and 39 years old, 17.50% between 40 and 49 years old, 12.00% between 50 and 59 years old and 2.70% over 60 years old. Regarding the educational level, 2.00% corresponded to people with basic educational levels, 19.40% were people with complete middle educational levels, 44.90% studied at higher-level technical training centers, 30.80 % corresponded to people with professional studies, and 2.50% had postgraduate studies.The means, standard deviations, and correlations between the study variables are presented in The correlations are as expected according to the literature reviewed, i.e., positive and significant for all healthy organizational practices (HOP) with engagement, and negative for all HOP with burnout. The effects regarding the correlations between HOP and engagement were medium and the correlations between HOP and burnout were small. Nevertheless, professional inefficiency has a poor or null correlation with HOP.As shown in According to The objectives of this work were to analyze the relationship between healthy organizational practices with worker engagement and burnout and to study the mediation of engagement between healthy organizational practices and worker burnout during the COVID-19 pandemic. Recent research revealed the importance of work engagement for the well-being of workers and good performance ,62. MoreAlthough the hypothesized models present similar adjustments with acceptable solutions, we consider them advanced according to their developments concerning the existing literature, for the following reasons: model 0, which evaluates the impact of HOP on engagement, presents an explanatory capacity of the latter of 25%, which confirms hypothesis 0, although its explanatory level is lower compared to the 40% that is explained in its original study . HoweverIn general terms, these results support several recent investigations ,71,72 thAmong the limitations of the present study is that we did not include the influence of age and sex in the models analyzed. These two variables are relevant because evidence indicates that engagement is higher in women than in men and higher in people over 40 years old ,44, whicWe consider it essential to point out that confirmatory analyses using structural equation models allowed us to explicitly specify the relationships between variables and evaluate their impacts on dependent variables, which contributed to the understanding of phenomena, such as the one studied here. However, and as indicated in the literature , we undeOur findings suggest that healthy organizational practices are related to worker engagement and burnout during the COVID-19 pandemic, promoting engagement and reducing burnout, contributing to the postulates of the HERO model, developed in various investigations ,23.Our results allowed us to visualize a similar scenario, but in the same line, showing that burnout in times of a pandemic decreases when worker engagement mediates its relationship with healthy organizational practices.These results strengthen the postulates of the scientific movement of positive psychology, especially those of positive organizational psychology. Positive organizational psychology establishes the relevance of the strengths of people  and positive institution orientation  so that people and organizations flourish and can face the various events of life (such as the COVID-19 pandemic) with proactivity and optimism, to survive them, and emerge stronger, seeing opportunities where others see catastrophe.


Hypercalcemia is a rare metabolic disorder in the pediatric population, with several differential diagnoses that resemble hematologic malignancies. In cases of severe hypercalcemia, therapeutic strategies other than hyperhydration, such as the use of bisphosphonates, have been described.We present the case of a previously healthy 12-year-old boy who was admitted to the emergency department due to fatigue, hypo-responsiveness, and progressively worsening poor appetite for the previous 19 days. Initial laboratory tests revealed severe hypercalcemia , hyperphosphatemia, elevated creatinine, and hyperuricemia. Management with hyperhydration and xanthine oxidase inhibitor  was provided. The patient was transferred to the pediatric intensive care unit where treatment with furosemide, systemic corticosteroid, and zoledronic acid was started. Metabolic, infectious, renal, and endocrinological causes were excluded. Follow-up paraclinical studies showed a progressive hematologic involvement with heterogeneous hypochromic microcytic anemia, thrombocytopenia, and elevated lactic dehydrogenase. Bone marrow aspiration and biopsy were performed, which confirmed the diagnosis of B-precursor acute lymphoblastic leukemia. Hypercalcemia was resolved 72 h after the application of bisphosphonates.Hypercalcemia as an oncological metabolic emergency in the onset of acute lymphoblastic leukemia is uncommon in children. The use of intravenous bisphosphonates is an effective therapy in the early resolution of the condition. We present the case of a 12-year-old patient with malignant hypercalcemia who responded favorably to the use of a single dose of bisphosphonates. Hypercalcemia is a rare metabolic disorder in the pediatric population. There are different associated etiologies  . MalignaA 12-year-old male patient, with a non-relevant medical history, consulted for 19 days of progressive hypo-responsiveness and hyporexia, with clinical decline and emesis, in the last days before admission. The mother reported a loss of 5 kg in the patient in the last 15 days. At the initial consultation, they reported severe hypercalcemia of 19 mg/dl (>14.5 mg/dl), hyperphosphatemia (6.6 mg/dl), elevated creatinine (1.45 mg/dl) as well as hyperuricemia (9.3 mg/dl) . An x-ra2. Management with furosemide was added, and given the severe levels of hypercalcemia, methylprednisolone and bisphosphonate (a single dose of zoledronic acid 0.05 mg/kg/IV) were added.Upon admission to our institution, the patient presented normal vital signs and appropriate anthropometry for his age. Mucocutaneous paleness was the only abnormal finding. Hyperhydration was continued and management with a single dose of rasburicase was indicated. A hypercalcemic crisis with kidney involvement secondary to volume depletion was confirmed, with acute kidney injury RIFLE I and glomerular filtration rate (GFR) at 48.8 ml/min/1.73 mStudies were performed for possible infectious, metabolic, renal, endocrinological, and neoplastic etiologies associated with acquired hypercalcemia . A maligEndocrinological studies were expanded; parathyroid hormone (PTH) levels were found to be in the suboptimal range, calcitriol was normal, and 25-hydroxyvitamin D3 levels were optimal. As the patient had no history of drug use to justify serum calcium levels and had not presented any state of prolonged immobilization, severe PTH-independent hypercalcemia was diagnosed. Among secondary causes, thyroid function disorders were ruled out by normal thyroid ultrasound. Normal alkaline phosphatase levels and reported imaging studies made the bone origin of hypercalcemia less likely.Regarding the nephrological study, the abdominal ultrasound was not suggestive of nephrocalcinosis, as evidenced by the hypercalciuria , which was justified by the severity of the hypercalcemia, making a tubular reabsorption disorder unlikely.There were no clinical or paraclinical signs of infection; the elevated level of C-reactive protein (9 mg/dl) was justified by acute inflammation of unknown etiology. The patient persisted with anemia and elevation of indirect bilirubin, ruling out hemolytic etiologies. A subsequent paraclinical follow-up showed thrombocytopenia in addition to heterogeneous hypochromic microcytic anemia. The control blood count reported 7% immature cells, which had not previously been discovered. Lactate dehydrogenase (LDH) levels remained high (453 U/L). Suspicion of the lymphoproliferative syndrome was considered; a biopsy and a bone marrow aspirate were indicated on the third day of hospital admission.2. He never developed any additional complications beyond acute renal failure secondary to the hypercalcemic crisis. He completed treatment with allopurinol for 7 days, achieving normal uric acid levels from the second day of admission and his calcium levels normalized 72 h after the application of zoledronic acid, subsequently requiring replacement due to hypocalcemia without additional severe adverse reactions  1 status. Translocations , BCR/ABL , and 46 XY leukemic karyotype were all ruled out, and therapy according to the Acute Lymphoblastic Leukaemia Intercontinental 2009 protocol was subsequently started. The patient continued multidisciplinary management and his renal function normalized on the fourth day of admission, reaching a GFR of 71.17 ml/min/1.73 meactions .We present an unusual debut of a hematologic malignancy and the efficacy of a non-common therapy in the pediatric population, such as the use of bisphosphonates. Hypercalcemia is a rare metabolic disorder in children. Its etiology in the pediatric population is different from adults and includes immobilization, granulomatous disorders , hyperparathyroidism, hypervitaminosis D and/or A, endocrine disorders , and medications  among others , 4. Hype3, and hypercalcemia at the time of diagnosis, as it presented itself in our case  is defined as a hypercalcemic crisis, which is typically symptomatic and represents a medical emergency. CNS dysfunction or cardiovascular alterations are, together with kidney failure, the main causes of mortality in these patients, forcing immediate corrective measures to be taken for the restoration of homeostasis and the correct functioning of the compromised organs .During the first clinical approach, it is important to determine whether it is a PTH-dependent or PTH-independent cause. When hypercalcemia is independent of PTH, primary (genetic) and secondary causes such as malignancy should be studied , 10. TheTreatment of hypercalcemia depends on the underlying disease . The urgThe excretion of urinary calcium can be increased by inhibiting sodium reabsorption from the proximal tubule and the Henle loop, thus reducing passive calcium reabsorption . ProximaGlucocorticoids inhibit the conversion of 25OHD to its active metabolite, 1,25 (OH) 2D, decrease intestinal calcium absorption, and may be useful in hematologic malignancy . CalcitoIV bisphosphonates, such as pamidronate and zoledronic acid, are approved only for adults by the US Food and Drug Administration . They prZoledronic acid is considered more potent, effective, and convenient than pamidronate. Zoledronic acid in a single dose of 4 mg as an IV infusion over at least 15 min is recommended for the initial treatment of malignant hypercalcemia . HoweverAlthough the number of reports of its use in patients with leukemia is limited , the preHypercalcemia is an oncological metabolic emergency in the onset of acute lymphoblastic leukemia and an uncommon presenting sign in children. In addition to management with rehydration, it has been established that the use of IV bisphosphonates is an effective therapy in the early resolution of the condition. Despite the poor evidence on the dosage of zoledronic acid in children, we consider that it could represent an effective alternative in management. We present the case of a 12-year-old patient with malignant hypercalcemia who responded favorably and quickly to the use of zoledronic acid at a dose of 0.5 mg/kg in a single dose.


Citrus sunki, whereas, Tuju is a kind of red orange with vermilion peel, small fruit and high sugar content, and closely clustered with Citrus erythrosa. The phytochemical analysis showed that many nutrition and antioxidant related differentially accumulated metabolites (DAMs) were detected in the peel and pulp of Zangju and Tuju fruits. Furthermore, it was found that the relative abundance of some key flavonoids and phenolic acids, such as tangeritin, sinensetin, diosmetin, nobiletin, and sinapic acid in the peel and pulp of Zangju and Tuju were higher than that in sour range Daidai and satsuma mandarin. Additionally, Zangju pulp and Tuju peel showed the strongest ferric reducing/antioxidant power (FRAP) activity, whereas, Tuju peel and pulp showed the strongest DPPH and ABTS free radical scavenging activities, respectively. Moreover, both the antioxidant activities of peel and pulp were significantly correlated with the contents of total phenols, total flavonoids or ascorbic acid. These results indicate that the two local citrus varieties have certain nutritional and medicinal value and potential beneficial effects on human health. Our findings will also provide an important theoretical basis for further conservation, development and medicinal utilization of Zangju and Tuju.Citrus is widely grown all over the world, and citrus fruits have long been recognized for their nutritional and medical value for human health. However, some local citrus varieties with potentially important value are still elusive. In the current study, we elucidated the biological characteristics, phylogenetic and phytochemical profiling, antioxidants and antioxidant activities of the two local citrus varieties, namely Zangju and Tuju. The physiological and phylogenetic analysis showed that Zangju fruit has the characteristics of wrinkled skin, higher acidity, and phylogenetically closest to sour mandarin  Citrus belong to the family Rutaceae and are considered as one of the largest fruit plant species broadly dispersed in the temperate, tropical, and subtropical areas with potential socio-economic influence , while mandarin (Citrus reticulata) was rich in hesperidin , improve digestive function, and prevent cardiovascular diseases, inflammation, diabetes, and neurological diseases –20. Diffsperidin , 28, 29.speridin , 28. Thesperidin . AdditioCitrus, and many important citrus genotypes originated from here , Tuju (Chunan county of Zhejiang province), Daidai, satsuma mandarin trees were divided into peel  and pulp (segment epidermis and juice vesicle). Five similar fruits were collected as one repeat, three repeats for each group. For each variety, peel and pulp of more than five fruits collected from different trees were sampled as one repeat and frozen in liquid nitrogen for metabolites extraction and subsequent RNA extractions, three repeats for each group.–1) was measured using a NanoDrop spectrophotometer, and fragmentation was achieved using sonication, and integrity was evaluated using 0.8% agarose gel. Sequencing was performed using an Illumina NovaSeq 6000 platform  with PE250 based on Sequencing by Synthesis (SBS) technology. Except for Zangju and Tuju, the chloroplast genomic sequences of the other 20 citrus varieties and 2 close genus were downloaded from NCBI is used for and manually annotated for phylogenetic tree analysis. MAFFTv.5  of the supernatant was carefully removed to the tube and dried in a vacuum concentrator. An aliquot (160 μl) of the extract  was added to the dried metabolites for resolution. After vortexing for 30 s being treated with ultrasound for 10 min in an ice water bath, the solution was centrifuged at 4°C and 12,000 × g for 15 min. A sample (120 μl) of the supernatant was transferred to a 2 ml injection flask and mixed with 10 μl from each sample for the QC sample analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for metabolites annotation and enrichment analysis.Samples of the peel and pulp of mature fruits of Zangju, Tuju, Daidai, and satsuma mandarin stored at −80°C were used for metabolites extraction. Metabolome analysis was performed by Biomarker Technologies Co., Ltd.  using non-target liquid chromatography–mass spectrometry  to identify differences in the metabolite profile among treatments. The LC/MS system for metabolomics analysis is composed of Waters Acquity I-Class PLUS ultra-high performance liquid tandem Waters Xevo G2-XS QT of high resolution mass spectrometer. The column used is purchased from Waters Acquity UPLC HSS T3 column (1.8 μm 2.1 × 100 mm). The experimental methods were as follows. The sample (50 mg) was added to 1 ml extract buffer containing an internal standard , swirled for 30 s. Then mixture was treated with ultrasound for 10 min in an ice water bath and standing for 1 h at −20°C. The homogenate was centrifuged at 4°C and 12,000 × g for 10 min. Total phenols content was determined using the Folin-Denis method described previously with some modifications . Gallic acid was used as a standard and total phenols was expressed as mg GAE/g FW extract.To measure the total phenols in peel and pulp, 100 mg of freeze-dried flesh samples was homogenized with 1.5 ml of 60% aqueous ethanol and vibrated for 2 h under sonication, then centrifuged at 25°C and 12,000 × ications . The sam2 were added to a 50 μl extract in a volumetric flask, and the mixture was kept for 6 min under dark at room temperature. Then, 30 μl of 10% Al (NO3)3 was added to the mixture and incubated for 6 min again. At last, 105 μl of 1 M NaOH was added. After incubating for 15 min at room temperature, the absorbance was measured at 510 nm. Results were expressed as mg of rutin equivalents (RE) per gram of FW (mg RE/g FW) for the total flavonoids content.Total flavonoids content was determined according to the method described by Kim et al. . After e3-ethanol (w/v) for 60 min at 30°C. The ascorbic acid content was calculated at 534 nm and expressed in mg⋅g–1 FW.Measurement of ascorbic acid content was performed according to Chiaiese et al. . Fresh s3⋅6H2O solution (20 mM) and 20 ml TPTZ solution (10 in 40 mM HCl). Then 3.80 ml FRAP reagent was added to 200 μl of peel or pulp extract. After 30 min at room temperature, the absorbance of extracts and reference substances Trolox was detected at a wavelength of 590 nm by NanoDrop 2000C . For DPPH assay, 3.50 ml of DPPH was added into 500 μl ethanol extracts. After 30 min for darkness, the absorbance was detected at wavelength of 517 nm. For ABTS assay, fruit extracts (40 μl) were allowed to react with 3.90 ml of the ABTS radical solution under dark conditions for 10 min, and then the absorbance at 734 nm was measured. The results of ABTS, DPPH, and FRAP were represented as μmol of Trolox equivalent per gram fresh weight (μmol Trolox/g FW) for peels and pulps. Three repeats were performed for one sample.Ferric reducing/antioxidant power, DPPH  and ABTS  assays were conducted by the method in a previous report . FRAP reP < 0.05). The Pearson correlation analysis was also performed by SPSS 22.0 at P < 0.05 for determination of the correlations among the contents of total phenols, total flavonoids, and vitamin C and the antioxidant activities of citrus peel and pulp, respectively.All tests were conducted in triplicate. Statistical analysis was performed using IBM SPSS Statistics 22.0. Significant differences of the contents of total phenols, total flavonoids, ascorbic acid, and the antioxidants activities of FRAPs, DPPH and ABTS in peel and pulp of the four different citrus varieties were calculated. Tukey’s test was performed by using one-way analysis of variance (ANOVA) at the 5% level (Citrus sunki Hort. ex Sakurai cv. Zangju), also known as Dengrongzhoupigan, is mainly distributed in Derong county of Ganzi Tibetan Autonomous Prefecture of Sichuan province, with a longitude of 99° 30′ 15″ and a latitude of 28° 61′ 27″. The planting area of Zangju in the county is about 1,000 μ, mainly distributed in Bendu and Guxue towns. The biological characteristics of Zangju are as follows (Zangju ( follows : trees oCitrus erythrosa Hort. ex Tanaka cv. Tuju), is mainly distributed in Jiukeng town, Chunan county of Zhejiang province, with a longitude of 118° 38′ 42″ and a latitude of 29° 38′ 15″. There is little amount of cultivation of Tuju in the local area, and it has been developed and utilized in recent years to process tangerine peel, small green tangerine, tea wine, etc. Hence, the cultivation of Tuju has been gradually expanded. The biological characteristics of Tuju are as follows  contained the higher amounts of flavonoids and medicinal components than other citrus species according , Tuju (TG), Daidai (DD), and satsuma mandarin (WZMG), we performed widely targeted UPLC-MS/MS-based non-targeted metabolite profiling of these four species. The satsuma mandarin WZMG, the most widely cultivated variety in China  and DD fccording . Hence, ccording .r2 > 0.7), whereas peel and pulp samples showed significant differences  , revealiferences . As showferences , 25, it ferences .A total of 1,042 differentially accumulated metabolites (DAMs) were detected in the peel , and 985In order to explore the metabolite function of TG and ZG fruits, we further analyzed the KEGG enrichment pathway of the DAMs. The results showed that, compared with the peel and pulp of DD and WZMG, the DAMs in TG and ZG fruit were mainly enriched in ascorbate and aldarate metabolism, tyrosine metabolism, flavonoid biosynthesis, isoflavonoid biosynthesis, biosynthesis of alkaloids derived from terpenoid and polyketide, flavone and flavonol biosynthesis, biosynthesis of phenylpropanoids, phenylalanine metabolism, pyruvate metabolism, tryptophan metabolism, starch and sucrose metabolism, biosynthesis of terpenoids and steroids, xylene degradation, and citrate cycle (TCA cycle) pathway . AccordiThe antioxidant activity and medicinal value of citrus fruits are mainly related to flavonoids, phenolic acids, and vitamin C , 21. BasImportantly, besides the polyphenols listed in Citrus peel is a rich source of naturally occurring antioxidants . AntioxiMoreover, the TG pulp contained the highest content of total phenols of 1.64 mg GAE/g FW , followePhenolic compounds, including flavonoids and phenolic acids, are known to be responsible for antioxidant activity of different fruits, and fruits with higher total phenolic content generally showed stronger antioxidant capacity . The antFurther, FRAP activity of the four citrus varieties pulp ranged from 4.39 to 7.98 μmol Trolox/g FW, where ZG Pulp showed the strongest FRAP activity, followed in the order TG pulp > DD pulp > WZMG pulp . The DPPRecently, few studies concluded that total antioxidant activity of citrus fruits are mainly attributed to phenolic compounds and vitamin C, though there is disagreement as to which compound is the major contributor . To idenr = 0.873, P < 0.01), followed by the vitamin C content  and total flavonoid content . The correlation between DPPH value and vitamin C content was the strongest , followed by the correlation with total flavonoids , but there was no significant correlation with total phenols content. However, ABTS value in peel showed no significant correlation with the content of total flavonoids, total phenols, and vitamin C. For pulp, FRAP of the four citrus varieties had the strongest correlation with the content of total flavonoids , but displayed no significant correlation with the content of total phenols and vitamin C. DPPH value of pulp also had the strongest correlation with the content of total flavonoids , followed by the content of vitamin C , but had no significant correlation with the content of total phenols. However, ABTS value of pulp showed the strongest significant correlation with total phenols content . These results suggest that the main antioxidant components in citrus fruits are phenols, flavonoids, and vitamin C, which is consistent with previous reports  and Tuju (TG), were systematically analyzed for the first time. We found that ZG fruit grown in Sichuan province has a little acidic fruit flavor and phylogenetically closest relationship with arieties . These tCitrus fruits are highly nutritious, containing many primary metabolites, which are important sources of essential nutrients, and secondary metabolites, which form an excellent source of bioactive substances and exhibit potent health-promoting effects . ChemicaIn general, citrus fruits are considered as one of the natural resources of antioxidants, which contain an appreciable amount of ascorbic acid, flavonoids, and phenols compounds –56. ThesIn the present study, we found that both FRAP value of the peels and pulp of the four citrus varieties displayed the strongest correlation with the total phenols content and total flavonoids, respectively, and this observation is in accordance with the previous reports that a positive correlation between the polyphenols contents and the antioxidant activities of different citrus germplasms . HoweverOur results show that the two local citrus varieties, ZG and TG, are rich in antioxidant related secondary metabolites and have strong antioxidant activities. Based on these results, we proposed that fruits of ZG and TG have certain medicinal value. Moreover, our findings will provide an important theoretical basis for the conservation and utilization of these two local citrus germplasm resources. Further research is needed to develop and utilize the medicinal value of these two local varieties to improve the economic benefits of citrus growers.C. reticulata, which had the closest phylogenetic relationship with sour orange C. sunki. Tuju is a kind of red orange with vermilion peel, small fruit and high sugar content, which is closely clustered with C. erythrosa. Phytochemical metabolites analysis showed that the relative content of some flavonoids and phenolic acid, such as tangeritin, sinensetin, diosmetin, nobiletin, and sinapic acid in the peel and pulp of both Zangju and Tuju were higher than that in sour range Daidai and satsuma mandarin. The contents of total flavonoids, total phenols, antioxidant activity (FRAP), DPPH and ABTS free radical scavenging capacity of peels of Zangju and Tuju were significantly higher than that in pulp. Compared with Daidai and satsuma mandarin, Zangju pulp and Tuju peel showed the strongest FRAP activity, and Tuju peel and pulp had the highest DPPH and ABTS value, respectively. Moreover, both the antioxidant activity in peel and pulp were significantly correlated with the contents of total phenols, total flavonoids, and ascorbic acid. The results suggested that Zangju and Tuju are rich in key antioxidants and have stronger antioxidant activity, indicating that they may have certain medicinal value and potential beneficial effects on human health.In the present study, the biological, phylogenetic characteristics, and phytochemical profiles, antioxidants contents and antioxidant activities of two local citrus varieties, ZG and TG, were systematically demonstrated for the first time. The results showed that Zangju fruit had the characteristics of wrinkled skin and acidity, and grouped with The original contributions presented in this study are included in the article/LS, JX, and FK conceived and designed the experiments. JX, ZN, and JS collected the fruits. LS, JX, ZN, JS, and XH analyzed the fruit characteristics and quality. LS, JX, LW, and ZN performed the experiments of antioxidant activities. LS, LW, and N analyzed the data and elaborated the figures. LS wrote the manuscript. LS and N revised the manuscript. FK supervised the project. All authors read and approved the final manuscript.


STAT3, CEBPB, SPI1, and regulatory mechanisms identified more accurately than with single-omics but also immunotherapy targeting central TFs to drugs was found to be significantly different between patients. Thus, in addition to providing new insights into the potential regulatory mechanisms and pathogenic genes of AD microglia, this approach can assist clinicians in making the most rational treatment plans for patients with different risks; it also has significant implications for identifying AD immunotherapy targets and targeting microglia-associated immune drugs.Microglia, the major immune cells in the brain, mediate neuroinflammation, increased oxidative stress, and impaired neurotransmission in Alzheimer’s disease (AD), in which most AD risk genes are highly expressed. In microglia, due to the limitations of current single-omics data analysis, risk genes, the regulatory mechanisms, the mechanisms of action of immune responses and the exploration of drug targets for AD immunotherapy are still unclear. Therefore, we proposed a method to integrate multi-omics data based on the construction of gene regulatory networks (GRN), by combining weighted gene co-expression network analysis (WGCNA) with single-cell regulatory network inference and clustering (SCENIC). This enables snRNA-seq data and bulkRNA-seq data to obtain data on the deeper intermolecular regulatory relationships, related genes, and the molecular mechanisms of immune-cell action. In our approach, not only were central transcription factors (TF)  Alzheimer’s disease (AD) is a fatal neurodegenerative disease  with a tIn the last decade, the development of RNA-seq data has contributed greatly to the understanding of AD; however, given the random variation of gene expression in single cells, missing data, and technical differences, sometimes the signal-to-noise ratio of snRNA-seq data is even lower than that of bulkRNA-seq data. In addition, it is also difficult to distinguish between direct and indirect regulations, based on the snRNA-seq data only. Therefore, to overcome these problems and to improve the expression of gene regulatory network (GRN) inference, the integration of other data is considered to be a modified approach ,6. CompaThere are many tools used to build gene networks, including PIDC, ACTION, and SINCERA. SCENIC tops the list in terms of accuracy and biological significance and is currently the tool of choice for building GRNs. Current approaches to integrate multi-omics in AD include the intra-brain combined with peripheral blood, ATAC-seq combined with snRNA-seq data, finding differential genes, and mapping them to another kind of data. However, these integrated multi-omics are more likely to incorporate gene expression than gene activity, much less GRN.STAT3, CEBPB, and SPI1, which were significant in the regulatory mechanism of microglia and have been confirmed to play critical roles in the pathogenesis of AD. Moreover, by modeling the prognoses of these central TFs and assessing the sensitivity to drug treatment of AD patients with different risks, the experiments showed that the sensitivity of some immune drugs was significantly different for high- and low-risk patients. In this study, more accurate potential key genes and their regulatory mechanisms in microglia were identified by constructing GRN through multi-omics analysis of bulkRNA-seq data and snRNA-seq data, which are helpful for understanding more systematically the regulatory relationships of microglia in AD.In this paper, we not only propose a method to combine bulkRNA-seq data with snRNA-seq data but also construct GRN modules using single-cell regulatory network inference and clustering (SCENIC) on the basis of both data, filtering the central regulators enriched by both data, which can effectively combine both histological data and improve the utilization of sample information. In the bulkRNA-seq data, we combine WGCNA with SCENIC to construct GRN modules after co-expression network analysis. GRN mapping is depicted for co-expression modules to explore the AD regulatory mechanisms and pathogenic genes within the modules. This can greatly integrate the advantages of both, and it makes up for the shortcoming that WGCNA can only explore inter-module interactions and the sample size of SCENIC cannot be too large. The central regulators of both data were found separately by using PPI; the central regulators of both data were assumed to intersect and were identified, such as In this study, the pre-processing of multi-omics datasets was the first stage, the bilateral Wilcoxon rank sum test was used for snRNA-seq data to screen out AD-associated microglia subpopulations and established GRN to identify regulators by SCENIC; for the bulkRNA-seq data, WGCNA combined with SCENIC were applied to identify the AD microglia-associated modules and establish GRN to determine the regulators. Secondly, those regulators that were identified by both data used PPI to determine the key genes; key genes were selected by taking their intersection. Finally, key genes were used to construct prognostic models and assess drug sensitivity. The experimental flow of this study is shown in The snRNA-seq data are widely used in GRN to describe regulatory relationships in cells. There are many tools available, such as PIDC, ACTION, SINCERA, etc., but SCENIC is considered to be superior to other algorithms in the face of massive snRNA-seq data processing. Moreover, GENIE3, as the most reproducible algorithm, has been used in SCENIC, and the RcisTarget database is used in SCENIC to remove indirect targets lacking enriched motifs . TherefoFirstly, co-expression modules between TFs and target genes were constructed using GENIE3. Secondly, for each co-expression module, the information is imported into the RcisTarget database to identify potential targets that are significantly enriched in transcription factor binding motifs. Finally, the regulator activity in the cells is scored using AUcell, thereby generating a binary activity matrix.j, and jf only makes use of the gene j’s direct regulators expression in j. Recovering the regulatory links pointing to gene j are thus equal to the gene of the expression of finding the predictable gene, j [Each p-feature selection task, which is the way in which the GENIE3 method views network inference problems, aims to retrieve the regulators for a specific gene. The method adopts the premise that each gene’s expression levels rely on those of other genes under the same circumstances and was first designed to work with steady-state data . The for gene, j .Here is a demonstration of how the GENIE3 procedure operates.j = 1 to p:For j for learning samples.Generation of input-output pairs of gene fj from i,j (i ≠ j), i = 1,...,p, using the feature-ranking technique, except for gene j [i, j as the weight i → j of the supervisory link.We learn r gene j . We use It should be noted that inputting genes as In this paper, the GRNs of microglia and a subpopulation of microglia were constructed using SCENIC to find the key regulators within each GRN network.The current SCENIC was constructed with default parameters, using the Rcistarget database   with conFor bulkRNA-seq data, the construction of gene co-expression networks enables us to understand the similarity of expression between genes in the disease process and, thus, to obtain information about abnormal cellular roles, functions, and their impact on the pathogenesis of the disease. One of the most widely used methods for gene co-expression network construction is WGCNA , which iBulkRNA-seq data was analyzed by WGCNA modularity, then a soft threshold was calculated, and a scaling-free topology model was constructed. The Pearson correlation coefficient (CC) was >0.7 and data were selected with a threshold of 6. After weight-based filtering, the key modules were finally identified . SCENIC https://string-db.org accessed on 1 February 2022) [For the snRNA-seq data, a number of TFs with high activity and specificity were detected in the microglia subpopulations after the GRN was established using SCENIC. To identify the key factors for these specific TFs, PPI networks were used. PPI action networks were generated using the STRING program (ry 2022) . The detSimilarly, for bulkRNA-seq data, after extracting the TFs of microglia-related modules using WGCNA and SCENIC, the PPI network was also used to identify the 6 top-ranked key genes. The PPI network determines key genes, based on centrality metrics .To identify the key genes more accurately, we took the intersection of key genes from both data sets and obtained the final key genes.p < 0.05 were then placed in the LASSO Cox regression analysis. Regression analysis was performed with 1000 iterations of the glmnet [After the identification of key TFs, AD risk profiles were developed using the univariate and least absolute shrinkage and selection operator (LASSO) Cox regression models. The set identified as syn 8691134 was used as the training set and syn 4009614 was used as the validation set, to identify the effect of prognostic AD key genes on overall survival (OS). Genes with a value of e glmnet  R packagSince testing the effects of drugs on different patients can enable physicians to provide an accurate clinical judgment, the prognostic model was used in our study to forecast the half-maximal inhibitory concentration (IC50) of chemotherapeutic medicines in high- and low-risk patients and to determine the sensitivity of various individuals. Here, the pRRophetic package  in R wasThis was performed to ensure the effectiveness of the prognostic models constructed by these genes and to infer whether these drugs are meaningful for the treatment of patients.The running environment for this experiment is R, version 4.1.2 (released 1 November 2021). The specific R package versions are shown in https://www.synapse.org/#!Synapse:syn22079621/) (accessed on 1 February 2022). The snRNA-seq data analysis was performed on prefrontal cortex (PFC) nuclei that were isolated from postmortem human tissue derived from AD and healthy individuals (74–90+ years) . The dataset was preprocessed to remove those cells that did not fit the analysis, such as dead cells. The data were preprocessed using the Seurat package; we eliminated those cells with over 5000 or under 200 unique feature counts. By keeping cells with under 5% of mitochondrial readings, dead cells were excluded (percentage mt < 5%). Ultimately, 60,137 cells were eligible for the subsequent analysis. To reduce the effect of data imbalance on sequencing depth, the data overall were positively distributed. The data were normalized using the Sctransform function in the Seurat package, instead of using the traditional NormalizeData, ScaleData and FindVariableFeatures preprocessing functions. In total, 6000 highly variable genes (HVGs) were selected [The snRNA-seq dataset used in this study was downloaded from Synapse  modular optimization. K nearest neighbors is calculated and SNN graphs are constructed. Then the modularity function is optimized to determine the cell clusters. The resolution was chosen to be 0.5. In total, 60,137 cell nuclei were clustered, based on 6000 HVGs .https://www.synapse.org/#!Synapse:syn22079621/) (accessed on 1 February 2022). Bulk RNA-seq data were derived from subjects enrolled in the Religious Orders Study (ROS) or the Rush Memory and Aging Project (MAP), while the dorsolateral prefrontal cortex (DLPFC) data, derived from 90 subjects , were used for the downstream analysis. To eliminate data noise, in this paper, the COMBAT function inside the SVA [The Bulk RNA-seq dataset used in this study was downloaded from . There were also 34 cell clusters .GFAP and SLC1A2; EX for SYT1; inhibitory neurons for GRIP1; MG for LRMDA and DOCK8; ODC for MBP, MOBP, and ST18; OPC for LHFPL3 and TNR) (Cell types were annotated using the previous marker genes  (ASC forand TNR) a,b.LAMP5/LINC00507 for the EX1 cluster, RORB for the EX2 cluster, RORB/THEMIS for the EX3 cluster, FEZF2 for the EX4 cluster, NRGN for the EX5 cluster, VIP for the INH1 cluster, PVALB for the INH2 cluster, SST for the INH3 cluster, and LAMP5 for the INH4 cluster). Similarly, we sought to annotate our glial subpopulations, isolating our astrocyte clusters according to GFAP expression . Our microglia clusters are annotated based on the expression of homeostatic and activation markers  [Cell subpopulation annotations for the above results were performed according to the literature . The anncluster) . A totalThe extensive oligodendrocyte annotation is in agreement with the literature ; this paTo pick out certain cell subpopulations associated with AD for specific study, after snRNA-seq data clustering and the identification of cell types, disease cell clusters were screened for subsequent analysis using a proportional analysis with a two-sided Wilcoxon rank-sum test .−3), while the ASC4 cluster significantly decreased (FDR = 3.52 × 10−3). In addition, the proportion of the MG1 cluster also increased with disease (FDR = 2.43 × 10−2). Although the results for the MG3 cluster were also significant, the number of cells involved in the MG3 cluster was too small, so the MG1 cluster was chosen as the disease cell subpopulation.Examination of the composition of each cluster revealed that several clusters were significantly over- or under-represented in AD compared to controls c. The ASp-value of <0.005, and p. adjust of <0.05, yielding 178 disease differential genes. Then, the disease ontology (DO) enrichment analysis of the DEGs in the MG1 cluster by Metascape (https://metascape.org) (accessed on 1 February 2022)  d revealeETV5, XRC44, SMARCA4, ELF2, CEBPB, STAT3, SREBF2, etc.Considering that regulators play a very important role in AD, the GRN of microglia was further constructed using SCENIC. Some of the more active TFs were listed , such asETS2, NFATC2, CUX1, TCF4, YY1, CEBPB, STAT3, RB1, TAF1, BACH1, SPI1, RUNX1, and MITF seem to be mostly activated in the MG1 cluster. EP300 and ELK3 are mainly activated in the MG2 cluster. SF1, BCL6, CHD2, and CLF2 are mainly activated in the MG3 cluster. Several TFs are also more active in all microglia subpopulations, for example, ETV5, ETS2, etc.  was defined, based on Jensen-Shannon scattering. Specific regulators with the highest RSS values were selected and their functional properties were further examined . ThroughETS2, NFATC2, CUX1, TCF4, YY1, CEBPB, STAT3, RB1, TAF1, BACH1, SPI1, RUNX1, and MITF are significantly upregulated and NFIC is significantly downregulated in the MG1 cluster ,  cluster a. NotablNR3C1, KLF8, ATF6, CEBPB, and STAT3 are highly specific for AD. MLXIPL, NFIC, FOSP2, FOXO1, and ARID3A are highly specific for healthy samples.From our results, we can see that a good overview of well-characterized cell-type regulators is provided, and for each cell type, a small number of regulators with very high specificity scores were identified. These conserved and specific TFs can play different roles in the various cell subtypes, which may be related to heterogeneity within the microglia subpopulations.SPP1, CD163, CX3CR1, ETS1, CEBPB, STAT3, SPI1, NFATC2, DOCK8, RUNX1, ETV6, etc. Surprisingly, these genes were also enriched in the MG1 cluster in snRNA-seq data.After preprocessing the bulkRNA-seq data, we performed WGCNA analysis on the data and divided these genes into multiple modules a. The blIn addition, to extract and verify the modules with specificity in AD/Con, their correlations were calculated using Pearson correlation coefficients. The degree of correlation between all modules, including the black module, and the clinical traits was also explored. The different modules have different expression patterns and correlations with AD or normal samples. Among them, black (Control/AD: −0.3/0.3), orange (Control/AD: −0.17/0.17), and grey (Control/AD: −0.19/0.19) modules have a relatively high correlation. The black module was found to show the most positive correlation with AD and the most negative correlation with the normal sample, as we expected b.Therefore, we performed DO enrichment analysis on the black module to verify the correlations between it and the disease. The results showed that the black module revealed significant correlations between the Alzheimer’s disease pathway and the tauopathy pathway c. The abIRF1, STAT3, SPI1, ELK1, EP300, ETS1, etc. Many regulators in the black module overlapped with the regulators of the MG1 cluster. The activity heat map of the regulators in the black module is shown in After constructing the co-expression module, the regulatory relationship between the genes within the module is not clear. Therefore, SCENIC analysis was performed on the co-expressed genes in the black module to construct the GRN. Many regulators were found to be enriched, such as To identify more accurate biomarkers or key genes, after identifying the specific regulators of MG1 clusters in snRNA-seq data and the regulators of the black module in the bulkRNA-seq data, the PPI network was constructed for each of them to screen the central genes and take the intersection.CEBPB, SPI1, STAT3, CUX1, YY1, and RB1 were identified as the central TFs of the pathological microglia cluster (MG1 cluster).In the MG1 cluster of snRNA-seq data, we created a PPI network graph via STRING b and useLEF1, CEBPB, SPI1, SOX2, ETV6, STAT3, ETS1, TCF7L2, GATA2, STAT5A, RUNX1, and BCL6 were obtained as the central top TFs.Similarly, in the black module of bulkRNA-seq data, PPI networks were constructed for all regulators a Supple. The TFsSTAT3, CEBPB, and SPI1 were enriched in both, while many of the target genes that they regulated were similar.These TFs in the black module intersected with specific TFs in the MG1 cluster; we found that only CEBPB and STAT3 were not only specific in the MG1 cluster but are also specific regulators and disease-associated differential genes that are significantly upregulated in AD .Surprisingly, STAT3, CEBPB, and SPI1 in the MG1 cluster, while STAT3, among which PLEKHH1, NEAT1, STAT3, CPEB4 USP15, and SLC1A are DEGs in the MG1 cluster. When STAT3 is the target gene, it is regulated by several high-confidence TFs . All three central TFs regulate BCL6, but the role that BCL6 plays in AD is currently unknown.In the MG1 cluster, there are many target genes regulated by CEBPB is a core TF, although it regulates few high-confidence target genes; it regulates SPP1, which, in turn, is a marker gene of the MG1 cluster. This implies that CEBPB affects SPP1 and will promote or suppress the inflammatory response.IRF1, STAT3, SPI1, ELK1, EP300, and ETS1, etc. The regulated target genes were also related to those in MG1 cluster, such as CEBPB and BCL6, etc. Given that there were too many regulators, we only selected the top 100 regulators of weights and enrichment scores of the three central TFs for visualization. We likewise found that BCL6 was regulated by three central TFs at the same time. In addition to the three central TFs, CEBPB, STAT3, and SPI1; we also found that BCL6 was regulated by the three central TFs, and there was no doubt that BCL6 plays a crucial role in AD.From these two results, we can see that the GRN constructed in the black module and MG1 cluster obtained similar regulatory results . Many TFSTAT3, CEBPB, SPI1). To assess whether the obtained key TFs were reasonable or associated with AD, we reviewed known AD-related genes from the NCBI official website and found that these specific key TFs were significantly associated with AD. Our study successfully demonstrated that the method in this paper can accurately identify AD-related key TFs, and proved the accuracy and plausibility of our experiment.In summary, we combined multi-omics data to build the GRN and found high confidence TFs identified in both data simultaneously . The 1-year survival AUC values, 3-year survival AUC values, and 5-year survival AUC values for the training set ROC curves were 0.530, 0.585, and 0.753, respectively  in high- and low-risk patients and found that between the high- and low-risk groups of AD patients, the IC50 values were significantly different .p < 0.05). Gefitinib may be used to treat low-risk individuals more successfully, as can be observed.For example, one drug, Gefitinib, had a higher IC50 in high-risk patients  of the MG1 cluster. Therefore, CEBPB affects SPP1 to show very high specificity in the MG1 cluster and AD. SPP1 is a marker of neuroinflammation, a chemoattractant, and an adhesion protein that is involved in wound healing [ healing .CEBPB is directly associated with the progression of human AD, with elevated levels of the protein appearing in the brains of AD patients, compared to healthy controls. CEBPB is highly expressed in macrophages, microglia, and also plays a key role in identifying their activation status [These TFs regulate the genes essential for microglia activation; the promoter and enhancer regions of several cytokines and pro-inflammatory genes include binding sites for these TFs. n status .STAT3 is also a regulator of disease cell-cluster specificity, the AD-specific regulator, the DEG of AD, is significantly upregulated in AD and is regulated by SPI1 in the black module. STAT3 is associated with neurodegenerative diseases, wherein neuroinflammation increases the production of pro-inflammatory cytokines and the hyperactivation of microglia. The release of cytokines, which is linked to neuroinflammation in AD, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, is influenced by STAT3 phosphorylation [rylation .CEBPB promoter, STAT3 functions as a TF for CEBPB as well [STAT3 is present in neurons, the vascular endothelium, astrocytes, and microglia. In response to cytokines, intercellular mediators, and growth factors, it is activated by the phosphorylation of Janus kinase (JAK), which has a variety of biological effects, including cell proliferation, differentiation, and death [By connecting to the distal portion of the  as well ,26. STATnd death ,27,28.STAT3 is a canonical inducer of gliosis; the deficiency of STAT3 not only affects the classical hallmarks of AD pathology but also has a strong positive effect on brain network dysfunction. In the black module, STAT3 shows the highest activity with its target genes and may have a key role in the severity of AD. It has been documented that rats lacking STAT3 exhibit significant spatial learning and memory preservation at relatively late stages of the disease. Importantly, these positive behaviors suggest that novel drugs identified by targeting STAT3 or via increased gliosis are candidates for clinical AD trials in the future. STAT3 has been nominated in the literature as an AD drug target; the inhibitory agents of STAT3 reduce neuroinflammation, tau phosphorylation, and the endogenous production of Aβ42 [STAT3, the drug Gefitinib was found to be the drug of choice regarding STAT3. It has a significant effect on the inhibition of STAT3. of Aβ42 . In subsSPI1 is not a DEG of AD, it was established that SPI1 is a disease-specific TF by establishing the GRN in the MG1 cluster. SPI1 also shows very high activity in the black module. The AD gene network’s major hub, PU.1, a TF encoded by the SPI1 gene, is strongly linked to AD pathogenesis. Immune cells, macrophages, and microglia, which have a common embryonic ancestor, all produce SPI1 at high levels [SPI1 is a key TF that regulates AD-related genes in the inflammatory response of primary human microglia and microglia [SPI1 levels lead not only to transcription of AD-related inflammatory genes but also to the dysregulation of genes related to immune response and interferon signaling.Although h levels . SPI1 isicroglia . IncreasBCL6 is simultaneously regulated by these three central TFs, and in the literature [BCL6 is a key gene associated with AD immunity. Although the specific role played by BCL6 in AD microglia is not yet clear, there is no doubt that BCL6 is likely to serve as a new potential target for AD.Both in the MG1 cluster and the black module, terature , it was NFIC is significantly downregulated in MG1, and NFIC is located on chromosome 19p13.3. NFIC is associated with TGF-β, and TGF-β is a key signaling factor in innate immunity that is associated with APOE pathogenesis in AD. NFIC does not appear to be required for normal brain development and it is a functionally unknown transcription factor, the exact cause of which requires further study [In MG1, er study ,34.Another contribution of our study is that the prognostic characteristics of these central TFs are not only reliable for predicted prognosis but also enable the estimation of drug-treatment response in AD patients, which may provide important clinical implications for guiding customized anti-microglia therapy in AD patients and may also have significant implications for targeting microglia-related drugs in the future. Prognostic modeling of central TFs to determine drug sensitivity identified differences in a range of drugs, including Gefitinib, Bosutinib, Camptothecin, Cisplatin, Cyclopamine, DMOG, and Docetaxe, in high- and low-risk patients, which are relevant to AD treatment.BACE1 is a potential target for the treatment of AD, and Gefitinib is a potential lead compound for BACE1 [or BACE1 . Epidermor BACE1 . GefitinThere is a small limitation in this paper, in that only the snRNA-seq data and bulkRNA-seq data in the brain were combined, while the blood was not considered. Only the GRN in the microglia was explored, while other cell types were little explored. However, more data, such as spatial transcriptome data and brain imaging data, can subsequently be combined to investigate the rest of the glial cells in a deeper way.In addition, we have another limitation, in that our data are from only the prefrontal cortex; although the prefrontal cortex is very beneficial for studying AD, it shows some lopsidedness and limitations. It would be desirable to study data from multiple brain cortexes, such as the prefrontal and hippocampus, in future studies. Therefore, combining blood samples with samples from multiple brain regions would be a great step forward for future studies.STAT3, SPI1, and CEBPB were identified as the central TFs of microglia in the AD brain. Based on the AD datasets and the related literature, the roles of the extracted TFs in AD were obtained and confirmed. These central TFs are diagnostic markers and therapeutic targets in AD. CEBPB promotes amyloid beta pathology and cognitive dysfunction and will regulate target genes regarding inflammation. STAT3 promotes glial cell production and exacerbates the incidence of AD. SPI1 regulates primary human microglia and microglial cell inflammatory responses in AD. Prognostic models and drug sensitivity experiments were constructed by these three central TFs, and drugs such as Gefitinib were found to have different sensitivities in different patients. Therefore, the proposed method to construct GRN by the bulkRNA-seq data and snRNA-seq data of multi-omics analysis efficiently provide a greater understanding of microglial regulatory relationships and key genes. This will play an important role in the future prediction of targeting gene-formulating drugs and immune drugs, which will assist clinicians to make more rational and accurate treatment plans.Exploring potential targets has been limited by single histology data when judging key genes by cellular expression, which is far from sufficient. In addition, considering that the bulkRNA-seq data is relatively sparse, snRNA-seq data sometimes show less signal-to-noise ratio than bulkRNA-seq data, while the regulatory mechanisms and risk genes in microglia are not yet fully identified. Single-omics data provide limited information to address complex scientific questions, and a single view is insufficient to capture the full picture of cellular heterogeneity. In this paper, we proposed an approach to integrate multi-omics data based on GRN and combined WGCNA with SCENIC. By using a PPI and centrality index approach,