Premature ovarian failure (POF) can be found in 1% of women at the age of 35-40, mostly due to unknown causes. PI3K-Akt signaling is associated with both ovarian function and growth of primordial follicles. In thisstudy, we examined the effects of autologous The longitudinal prospective observational study included 50 patients (between 30 and 50years) with a diagnosis of POF and infertility. This multicenter study was performed at Jevremova Special Hospital inBelgrade, Saint James Hospital , and Remedica Skoplje Hospital, between 2015 and 2018. All patients wentthrough numerous laboratory testings, including hormonal status. The autologous bone marrow mesenchymal stemcells (BMSCs) and growth factors were used in combination for activation of ovarian tissue before its re-transplantation. The software package SPSS 20.0 was used for statistical analysis of the results. Differences in follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), and progesterone (PG) hormone concentrations before and after 3, 6, and 12 months post-transplantation were tested in correlationwith the volume of transplanted ovarian tissue. A significant correlation (P=0.029) was found between the change inE2 level after 3 months and the volume of re-transplanted tissues. Also after re-transplantation, 64% of the patientshad follicles resulting in aspiration of oocytes in 25% of positive women with follicles.The SEGOVA method could potentially solve many human reproductive problems in the future due tothe large number of patients diagnosed with POF, as well as the possibility of delaying menopause, thus improvingthe quality of life and general health (Registration number: NCT04009473). Female gametes are extremely sensitive cells in thehuman body, which decrease in number drasticallyfrom fetal period to the prepuberty when they countabout 300 000. Their number continues to decreasein life, so that the residual pool in women over 35years of age is about 25 000 oocytes . Early fFollicles show initial growth but end with atresia. Thosewhich continue to grow and develop further become preantral, and when they achieve 3-6 layers of granulosacells, cell mobilization from the ovary stroma begins, and the cells gradually group in the outer sides of the basalmembrane. This event simply does not occur if the oocyte does not secrete biosignals, mainly the differentiatinggrowth factor 9 (GDF-9) . Peptide hormones of granulosa cells are importantparacrine modulators of theca cells, and also affect thefunctions of gonadotropic pituitary cells. These effectsin the middle of the follicular phase lead to the selectionof a dominant follicle and formation of a preovulationfollicle. Antimüllerian hormone (AMH), with its paracrine action, inhibits the FSH effect, and together with thepituitary gland, reaches its maximum production on theseventh day of the follicular phase, selectively inhibitsthe release of FSH and leads to atresia of the competitivefollicle . From 300,000 follicles at the beginning of puberty,450 of them achieve the ovulation phase, while about660 follicles are consumed for one ovulation. In eachovary we normally find 5-15 antral follicles called antralfollicular count (AFC), which is correlated with both thelevel of AMH and the concentration of inhibitors B, inparticular  growth factors. This novel approach involves application of bonemarrow stem cells (BMSC) that can be readily obtained and amplified in large quantitiesin vitro. BMSCs are a good choice for transplantation in patients withPOF. BMSCs migrate to the impaired ovary and together with cytokines that haveantiapoptotic, antifibrotic, anti-inflammatory and immunoregulatory effects improve ovarianfunction . The study was performed at Jevremova Special Hospital in Belgrade, Sant James Hospital inMalta, and Remedica Skoplje Hospital, between 2015 and2018. The criteria for patient involvement included the following: signed consent form, women over 18 years of age,primary or secondary amenorrhea for at least 3 months,AMH hormone values <0.42 ng/ml and FSH >20 IU/L,and/or failure of previous attempts at assisted reproductive techniques due to limited ovarian response (less than 4oocytes obtained), normal karyotype 46, XX, presence ofat least one ovary, normal anti-microsomal antibodies, andproper thyroid function as evidenced by normal serum levels of thyroid stimulating hormone (TSH). The exclusioncriteria included: currently pregnant or breastfeeding, history or evidence of existing gynecological malignancy, thepresence of adnexal masses indicating the need for furtherevaluation, mental health disorder, active substance abuseor addiction, existence of contraindication to laparoscopicsurgery and/or general anesthesia in the past two weeks, useof the following medicines: oral or systemic corticosteroids, hormones ,Danazol, anticoagulants, herbal or botanical supplementswith possible hormonal effects, type I or type II diabetesmellitus or if they are receiving antidiabetic medicines,known significant anemia (hemoglobin <8 g/dL), deep veinthrombosis and/or pulmonary embolus, cerebrovasculardisease, presence of heart and liver disease , andpresence of kidney disease (defined as blood urea> 30 mg/dL or serum creatinine> 1.6 mg/dL). In addition, patientswith positive history of cancer, in case of any positive tumor markers, or with structural changes on the ovaries,would be immediately excluded from the procedure. However, no such patients applied for this study.Before the procedure, patients had to have balanced nutritional status, optimized trace elements and antioxidantsthrough proper nutrition and supplements, as well as optimized physical activity (exercise plan).All patients went through the following laboratory testing:1.;2.Karyotype;3.Microbiological analysis of vaginal and cervicalswabs with antibiogram, cervical swabs on Ureoplasm, Mycoplasma and Chlamydia) - not older than3 months;4.Kg Rh D factor, total blood cell count, blood glucose,urea, creatinine, K, Na, NaHCO, proteinogram, coagulogram;5.Serological analysis: HIV, HBSAg, HCV;6.Lung RTG, findings and opinion of internal medicinedoctors for general anesthesia;7.in vitrofertilization (IVF) cycles.3D ultrasound ,personal and family history, length of infertility, number of All patients answered questions about menstruation,parity, the prevalence of POF in the family, education,and possible pre-amenorrhea stress, and a quality of lifequestionnaire. In addition, all women diagnosed withPOF included in this study had normal results for thefollowing: blood count, glucose, and lipoprotein profile,prolactin, testosterone, androstenedione, DHEA sulfate,17-OH progesterone, thyroxine, TSH, parathyroid hormone, adrenocorticotropic hormone and cortisol. Alltested patients had negative test results for anti-ovarianantibodies, anti-cardiolipin antibodies, anti-thyroglobulin antibodies, anti-microsomal antibodies, and hada normal karyotype. Sonographic examination of thepelvis in all patients showed that there were no pathological changes in the ovarian or follicular activities. Allwomen included in this study signed a written consentapproved by the Ethic Committee of Jevremova SpecialHospital for Gyneclogy in Belgrade, Serbia (number:63/2015).The SEGOVA is a group of procedures and methodsdesigned by Professor Aleksandar Ljubić to improvewomen’s general and reproductive health by restoringovarian function and improving the quality of life. TheSEGOVA approach has several important advantagesover other in vitro ovarian activation approaches1. in vitro activation.First, conservative minimally invasive surgery (ovarian cortex laparoscopic biopsy).This approach permits re-transplantation to the same site, from which the tissue was takenfor 2. Autologous platelet-rich plasma (PLRP) growthfactors are used instead of chemical stimulation ofgenetic pathways.3. in vitro activated ovariantissue is re-transplanted through ultrasound control. Second laparoscopic surgery is avoided, 1. Laparoscopic ovary tissue biopsy ;2. Under laboratory conditions, micro-fragmentation inhibits a group of HIPPO genes thatadversely affect follicular growth.3. Autologous activation is then performed by thegrowth factors of the AKT group of genes that positively influence the growth and development of thefollicles;4. Bone marrow biopsy and MSC processing;5. Transplantation of activated ovarian tissue under thecontrol of 3D doppler ultrasound into the ovary;6. Bone marrow stem cell implantation into ultrasound-controlled ovarian tissue;7. Activated tissue has an effect on the cells, whichtriggers the function of hormone production, follicular growth, and the activation and differentiationof oocytes. The hormonal status of the patients werealways assessed on days 2-5 of menstrual cycle.The procedure of surgical resection of the ovariancortex is performed using a modified "single port" laparoscopic technique with an entrance through the umbilicus. After an incision of about 2 cm in the umbilicalzone, three 5 mm portals will be placed, establishing anintra-abdominal pressure between 10 and 12 mm Hg. A5 mm diameter laparoscope is then introduced, as wellas auxiliary trocars. In some cases, and as an aid to thetechnique, a uterine manipulator is introduced transvaginally. After visualization of the ovaries, the cortex isfixed with the help of adequate instruments and cut withanother instrument (scissors). Hemostasis control, portremoval and wound closure are performed subsequently.2 . Inaddition, under ultrasound monitoring with a 3D color ultrasound (GE Voluson 730 Pro), atransvaginal puncture of both ovaries is performed under general anesthesia, with a 30 cmlong 16 gauge needle. After incubation, fragmented ovarian tissue with PLRP is injectedinto the subcortical region of the right and left ovaries .Further, the obtained ovarian cortices are chopped by multiple cuttings with a scalpelno. 25 to fragments that are smaller than 1×1 mm. The tissue prepared in this way isweighed using an analytical balance and placed in a petri dish, where it is washed withgamete buffer (Cook). After washing, tissue samples are transferred to PRP medium andfurther activated by autologous thrombin medium (Sigma-Aldrich). The sample prepared inthis way is incubated for 48 hours at a temperature of 37°C and in 5% COBone marrow cells are taken from the tibia or iliacbone. The biopsy is performed under general or localanesthesia. A small incision (7 mm) is made with a special needle to penetrate the periosteum. A volume of100 to 150 ml of bone marrow is aspirated and centrifuged in a particularly automated Angel system-Arthrex under sterile conditions. After extraction, thesamples were centrifuged to separate the acellular partand the erythrocytes which were discarded from thenucleated cells. Nucleated cells were further used fortreatment. After treatment, 3-5 mL of bone marrow aspirate concentrate (BMAC) is obtained. Bone marrowconcentrate contains a significant number of stem cells,leukocytes, platelets, erythrocytes, hematopoietic stemcells (HSCs), and MSCs. Each sample was analyzed by flow cytometry using specific markers: CD34, CD146,CD90, CD45, CD105, CD73, CD133, Stro-1. Cell viability and the total nucleated cell count (TNCs), wereassessed using 7AAD. The ovarian tissue is microfragmented, incubated and activated by autologousgrowth factors. After incubation, the prepared tissue ismixed with BMAC and re- transplanted via intraovarian injection.The platelet-rich plasma (PRP) procedure involvesdrawing blood from patients (104 ml) with a specialdouble syringe, which is then left to be centrifuged atthe appropriate number of revolutions over a period oftime to separate plasma-rich growth factors. A separatesyringe enriched with platelets is then drawn with another syringe and used for further treatment. The wholeblood will be processed using the Angel separationsystem . Separation of PRP from lowplatelet plasma is performed in a closed system - in afully automated machine designed for this purpose. Thismachine can process from 40 ml to 180 ml of bloodtaken from the patient. Under sterile conditions, usingthe Angel system, 18 times higher the platelet concentration in PRP than the baseline measured value in thepatient can be obtained. In our case, the optimal concentration is between 6 and 8 times more concentrated thanin patient’s blood, which can be determined and adjustedon the machine. For the PRP procedure and laboratoryanalysis, a whole blood sample will be taken from thecubital vein and mixed with Anticoagulant Acid CitrateDextrose (ACD) in a ratio of 7: 1. Two 60 mL syringeswill be taken and filled with up to 60 mL blood, makinga total volume of 104 mL whole blood and 16 mL ACD.The second phase, or laboratory phase, involves the application of complex technology, which uses specialseparators and systems, divides and filters certain cells,prepares them and activates the growth factors in them.At the end of this procedure, we get between 5-7 mL ofactive plasma containing a large amount of growth factors, including PDGF, TGF-β, VEGF, EGF, and manyothers. In the next step, a volume of PRP product (5 mL)is mixed with the prepared ovarian fragments. Immediately before administration, PRP is activated by autologous thrombin in a 10: 1 ratio. The maximum timeinterval from activation to the end of instillation in theovary is 45 seconds. Activation is performed for eachovary separately. The changes in hormone levels in serum of the patients were evaluated 3, 6 and 12 months after the procedure. The effects of hormonal changes on ovarian reproductive function  were monitoredusing ultrasound and based on the outcome of IVF procedures.Since the observed sample size of the included women was relatively small, and the data on the level ofobserved hormones, the volume of ovarian cortex autografted and the volume of the BMAC of the patientswere not homogeneous, different methods of nonparametric statistics were used for data analysis, depending on the specific goals of the study. The data wasprocessed in the statistical package SPSS 20.0. Toinvestigate the changes in the levels of the hormonesFSH, LH, progesterone (PG) and estradiol (E2), theamounts detected prior to the intervention, as well as3, 6 and 12 months post-intervention, were comparedusing Wilcoxon’s rank test. Namely, the following nullhypotheses have been tested: median levels of a particular hormone detected before the intervention donot differ from the median hormone levels at 3 monthspost-intervention, 6 months post-intervention and 12months post-intervention. The alternative hypothesisis that differences among hormone medians measuredat different time points do exist. The hypotheses arebeing tested using Z statistics, based on the data difference directions, but also the relative strength of thesedifferences. This approach represents one of the mostuseful non-parametric methods. The test statistic Z isapproximately normally distributed with an arithmeticmean equal to zero and a variance equal to one, and hasthe form of:where W is a statistic representing a smaller sum ofrankings with one characterw is the arithmetic mean of the W statistics and is calculated according tothe formula:μw represents the standard deviation of the statistics W and is obtainedaccording to the formula:a σs measures the dependence between the rankings of two data sets and isobtained according to the formula:In order to investigate the effects of the volume of re-transplanted activated ovariantissue on changes in hormone values of FSH, LH, PG and E2, Spearman’s coefficient ofcorrelation of the rank was used. The Spearman’s rank correlation coefficientri is the difference between the rankings of two data sets. Certainly,after calculating the value of the Spearman’s rank correlation coefficient, it isnecessary to check the statistical significance. Testing is done by checking if the nullhypothesis coefficient is not statistically significant (H0 :ρs =0)against the alternative hypothesis that it is statistically significant (H0:ρs ≠0). The test statistics formula used for this purpose is:where dThe follow-up of hormones FSH, LH, E2 and PGincluded evaluation of the changes in values of theobserved hormones before the intervention and at three,six, and twelve months post-intervention. When observing the pre-intervention period, ourresults showed that 50% of the women had a FSHhormone value ranging from 28.10 to 86. 50 IU/mL,while the median value of this hormone was 46.50 IU/mL. These values were obtained on the basis of a sampleof 47 women. Three months after re-transplantation, the medianFSH level was raised (2.68%) compared to the preintervention period and amounted to 47.75 IU/mL, with50% of the women ranging between 24.28 and 86.50IU/mL. These values were obtained from 19 women at 3months following the intervention.Three months after re-transplantation, the medianFSH level was raised (2.68%) compared to the preintervention period and amounted to 47.75 IU/mL, with50% of the women ranging between 24.28 and 86.50IU/mL. These values were obtained from 19 women at 3months following the intervention.For the period of 6 months after intervention, themedian FSH hormone value was 34.49 IU/mL, and 50%of the women had a FSH hormone value between 15.80and 63.50 IU/mL. These values were obtained from23 women. At 6 months post-intervention, we noticedthat both median values were decreased by 25.82%compared to this value prior to the intervention.The last follow-up measurement was done at 12 monthspost-intervention, when the mean FSH hormone valuewas 35.36 IU/mL , while 50% of the women had a FSHlevel ranging from 21.86 to 70.08 IU/mL. Due to the loss of the follow-up data in some of the patients at 12months after the intervention, the results were obtainedon a sample size of 14 women. The median values ofthe FSH hormone before intervention and at 3, 6 and 12months after the procedure, are shown in Figure 1. Before the intervention the median value of the hormoneLH was 24.80 mIU/mL, based on the measurement ofthe hormone value in 45 women. The median LH valuewas slightly decreased (7.29%) after 3 months and was22.99 mIU/mL. At 6 months post-intervention it furtherdecreased to 18.60 mIU/mL median value (25% decreasecompared to before the intervention). After twelve monthsthe median value was 20.19 mIU/mL, which is 18.58%reduction compared to the value prior to the intervention.These values were obtained from sample sizes of 20, 21,and 16 at 3, 6 and 12 months, respectively. We can alsoobserve the change in the borderline interval values ofhormone LH, resulting from 50% of the women. Beforethe interventions the boundaries of this interval rangedfrom 15.22 to 46.56 mIU/mL, while 3 months after theintervention the boundaries were 9.23 to 41.73 mIU/mL,from which we can conclude that the interval of variationof these data has decreased. Six months post-intervention,the boundaries ranged from 11.00 to 32.29 mIU/mL,while at 12 months post-intervention, these values were10.03 to 37.24 mIU/mL .The third hormone observed in this study, E2, had a preintervention median value of 41.55 pg/mL, while 50% ofthe women had the value of this hormone in the rangeof 13.21 to 103.00 pg/mL. These values were obtainedfrom a sample of 42 women who were evaluated forthis hormone before intervention. From the observed42 women, 24 were measured for E2 at 3 months post-intervention, when the median value of 52.38 pg/mL was recorded (26.6% increase compared to pre-intervention).The range of E2 hormone value was from 21.34 to 151.60pg/mL. At 6 months following the intervention, the medianvalue of the observed hormone was decreased to 36.00 pg/mL , from a sample of 22 women. The increase in themedian value of E2 was recorded at 12 months post-intervention, when it was 45.10 pg/mL . This value wasobtained from a sample of 19 women due to the loss of thefollow-up measurements for this hormone . The hormone analysis also included follow-upmeasurements of PG levels in the subjects. Prior to theintervention, this hormone was measured in 32 womenand its median value was 0.32 ng/mL. Half of the observedwomen had the value of this hormone in the interval from0.09 to 0.89 ng/mL. After 3 months, the median PG valuewas 0.28 ng/mL , while the intervals ranged from 0.14to 0.73 ng/mL. These values were obtained from a sampleof 12 women. At 6 months post-intervention, a decreasewas observed in the median value of progesterone,being at 0.14 ng/mL (56.25% reduction compared topre-intervention) based on the result of 13 women. Theborderline intervals were, however, almost unchangedand ranged from 0.05 to 0.76 ng/mL. At 12 months post-intervention, based on the values measured from 11women, the calculated PG median value was 0.28 ng/mL , with 50% of the women having thevalue of PG in the range from 0.05 to 0.58 ng/mL . The first aim of the study was to examine thedifferences in the levels of four different hormonesand comparing their levels before and at 3, 6 and 12months after ovarian intervention. Consequently, foreach of the individual hormones, a null hypothesis hasbeen tested that the hormone levels prior to and afterthe intervention are not different. This hypothesis wastested against the alternative hypothesis that differencesin hormone levels do exist. Our results indicated thatdifferences observed in FSH hormone levels in 19women prior to the intervention compared to 3 months post-intervention are not statistically significant, sincethe value of Z statistics is -0.283 with a correspondingP value of 0.777. However, when the FSH hormonevalues were analyzed in 21 women 6 months after theintervention, it was noted that there were statisticallysignificant differences in hormone levels compared tothe pre-intervention values, with Z statistics havinga value of -2.091, and the corresponding P value as(0.037). At 12 months post-intervention the level of thehormone observed in a sample of 13 women showedan insignificant difference compared to the level of thehormone before the intervention, with the P value of0.196 with Z statistics -1.293 (data not shown).With regards to the hormone LH, the results showthat in 18 women there is not a significant differencebetween hormone levels at 3 months post-interventioncompared to the pre-intervention LH levels, since Z isequal to -1.285 with a correspondinge P value of 0.199.Subsequently, the level of this hormone was measuredand compared at 6 and 12 months post-intervention.After 6 months, 20 women were evaluated for the levelof hormone LH in the blood and the results showedthat there were no statistically significant differencesin relation to its pre-intervention levels . The results for 15women who were assessed for the level of this hormoneat 12 months post-intervention were Z=-1.590 with aP value of 0.112, which was not significantly differentcompared to the pre-intervention measurements, at thesignificance level α = 0.10 (data not shown). The blood levels of E2 at 3 and 6 months post-intervention were measured in a sample of 21 women,displaying no statistically significant differences in thelevels of this hormone before and after the intervention.The Z value of the statistics for comparing thedifferences between E2 levels before and at 3 monthsafter intervention -1,195 with the corresponding Pvalue of 0,232, which clearly indicates that significantdifferences do not exist, confirming the null hypothesis.After 6 months, the result of statistical analysis is thesame [Z=-0.408 (P=0.683)], showing no significantdifferences between the levels before and after theintervention. After 12 months, 15 women were reevaluated for the E2 hormone and statistical analysisindicated that there were no significant differences inhormone levels before the intervention and 12 monthsafter the intervention. The hormone PG was measuredin a much smaller sample of women compared tothe previously analyzed hormones; 8 women wereevaluated at 3 months after the intervention, 10 womenat 6 months after the intervention, and 7 women at 12months post-intervention. It could be noticed that thedifferences in hormone levels prior to the interventionand 3 months after the intervention were not statisticallysignificant, because the Z statistics was -0.845, andthe corresponding P value was 0.398, therefore thesefindings suggested that the null hypothesis was correct.A null hypothesis was also adopted that there was no difference between the levels of hormone PG before and6 months post-intervention, since the P value (0.735)that was corresponding to the Z statistics (0.338) wasgreater than α=0.10. When it comes to the differences inhormone levels of PG prior to and 12 months after theintervention, it is also concluded that these differencesare not statistically significant due to a corresponding Pvalue of 0.500, which is again greater than the level ofsignificance of 10%.There are several factors affecting graft function afterovarian re-transplantation. The ovarian reserve in most ofthe patients re-transplanted with ovarian tissue is usuallylow due to the limited primordial follicles, so most of thepregnancies are conceived within the first 12 months afterauto-transplantation. However, the main disadvantage isthe limited number of available tissue fragments to betransplanted, due to ovary size. One of the aims of this study was to examine whether thevolume of the autografted ovarian cortex affects the endocrinefunction of the ovary. The differences in the concentrationsof the hormones FSH, LH, E2 and PG before and at 3, 6 and12 months after transplantation were tested for correlationwith the volume of the transplanted ovarian tissue (data notshown). Another parameter tested for a correlation betweenthe differences in the concentrations of the hormones FSH,LH, E2 and PG before and at 3, 6 and 12 months post-transplantation, was the TNC from BMAC . OuraimClose monitoring of ovarian function wasconducted after re-transplantation, including repeatedmeasurements of FSH and E2, PG and LH levels at3, 6 and 12 months post-intervention, and continuoussonographic evaluations during 12 months. AMHlevels were not good predictors of graft function, sothey were not measured after transplantation.Repeated ultrasound examinations were performedin patients by a single physician to clearly monitorfollicle development from each ovary separately.Table 2 presents a summary of the total results ofultrasonography for all 50 patients. During the 12months period after the re-transplantation 64% ofpatients had presence of a follicles. Attempts to perform oocyte retrieval resulted in aspiratedoocytes in 25% of the follicle-positive women . Fertilization rate of theaspirated oocytes was 75%, resulting in embryos in 12%of the women out of the total number of patients. Embryotransfers were performed in 66.6% of embryo-positivewomen , while 50%of embryo-positive women had vitrified embryos . Two patients spontaneouslyconceived after transplantation, while one pregnancy wasconceived with IVF, resulting in the birth of twin babies.in vitro ovarianactivation using stem cells and autologous growth factors on reproductive and endocrinefunctions in patients with ovarian insufficiency. The results of the current studyindicate that the SEGOVA procedure can play a potentially important role in addressing theproblem of infertility in patients with impaired or lost ovarian function, as well asimproving endocrine ovarian function, which affects a woman’s overall health and qualityof life. With regards to the recent published data in the literature, the results of ourstudy make important contribution to today’s scientific findings in the field of femalereproduction, and open novel possibilities in treatment of female infertility. In this study, we examined the effects of autologous The profound socioeconomic changes in our societyhave increasingly caused women to delay the decision tostart a family. Today, this factor leads to a major problemin the reproductive field, since female age is a decisivecause of infertility, it is common knowledge that as awoman gets older, both the quality and the number of hereggs available decrease. Therefore, many centers aroundthe world are trying to address this problem by usingdifferent strategies to preserve fertility at an earlier age byfor instance vitrification of the oocyte or using differenttherapeutic alternatives, allowing the patients to enjoymotherhood that would have been impossible in the pastwith low follicular reserve. The widespread use of oocyte donation is also asolution, but this is only a partial solution because manycouples have serious difficulties in accepting such ideas.Therefore, preventive and therapeutic measures shouldbe implemented. Preventive measures should addresstwo relevant phenomena associated with ovarian aging: decreased follicles and decreased quality of oocytescontained within these follicles. Accordingly, ovarianaging has become a key challenge for reproductivemedicine, as the ovaries change chronologically beforeother organs, causing fertility decline in the thirties,leading to ovarian fibrosis and complete loss of ovarianfunction in the early fifties -30. AdvaDue to the importance of aging in infertility, over the last10 years, new research has emerged aimed at developingmethods for rejuvenating oocytes by repairing geneticdamage or introducing new sources of energy . Anotherin vitro cultures increase the risk of cells losing some of theirspecific regenerative properties or accumulating chromosomal aberrations, which is alsoone of the reasons that we transferred BMSCs to the ovaries on the third day of theprocedure, shortly after they were isolated from the bone marrow. To circumvent thisconcern, it was proposed in our study (SEGOVA) to use minimally invasive protocols, wherebone marrow stem cells are not incubated under in vitro conditions and are rather underthe control of ultrasound. These cells are then applied to ovarian tissue together within vitro activated and incubated ovarian tissue with autologous growthfactors. Here we have been able to regenerate the ovary in women with impaired or lostovarian function. Based on this, we sought to evaluate the effects of autologous in vitroactivation of ovaries following the transfer of growth factors and stem cells (SEGOVA) tothe ovarian reserve in women with very poor prognosis. This novel study showed that SEGOVAimproves ovarian reserve biomarkers and reproduction results, leading to the developmentof more follicles and oocytes after ovarian stimulation. This technique allowed forspontaneous pregnancies in women with POF diagnosis. In short, ovarian rejuvenation is adifficult task because age is characterized by a number of significant changes, includinggenomic instability, telomeric shortening, mitochondrial dysfunction and epigeneticchanges  to be laparoscopically removed and treatedoutside the body and then laparoscopically reimplanted near its fallopian tubes.Cord blood, amniotic membrane, menstrual blood, adipose tissue and endometrial tissue areconsidered to be possible sources of MSCs with promising results for several degenerativediseases within and outside the reproductive system , 21, 22.changes . Addressin vitro ovarian activation and re-transplantation of the activatedtissue, a woman undergoes ovulation stimulation and undergoes IVF procedures. Folliculargrowth was observed in eight women, all of whom had signs of retained follicles beforetransplantation. These eight patients underwent ovulation stimulation, with five womendeveloping mature eggs for IVF. The oocytes were fertilized with sperm from a malepartner, and the resulting embryos were frozen and then transferred to the uterus. Duringthe study, one patient underwent embryo transfer of one embryo, but failed to becomepregnant, for another patient successful embryo transfer and pregnancy were achieved, butthat pregnancy ended with a miscarriage (missed ab). The third patient underwent embryotransfer of two embryos, and had a successful pregnancy that resulted in the birth of ahealthy boy. The remaining two women were preparing for embryo transfer and undergoingadditional egg collection cycles. Since some of the patients had unsuccessful IVF orintracytoplasmic sperm injection (ICSI) attempts in the past, and on the other hand, someof them who had ovarian insufficiency did not have any IVF attempts before the SEGOVAprocedure, the results of our study are very promising. The main limitation of thisprospective clinical study is that we are not able to have a control group of femalessince the laparoscopic treatment would be required for isolation of ovarian tissue and itsplacebo treatment. However, at the same time avoiding the second laparoscopy andperforming simple re-implantation of the activated ovarian tissue by ovarian aspirationneedle under control of transvaginal ultrasound is a less invasive method, as performed inthe past. A future perspective would be in exploring ways to control Hippo and phosphataseand tensin homolog (PTEN) pathways with drugs, without in vitro ovarianactivation.After in vivo. When stem cells areisolated from native tissue and grown and incubated in substrates, they rapidly lose theirrole and function that they originally had. In addition, they may have a shorter lifespandue to overexpansion in vitro. Furthermore, cellular DNA becomes unstableduring long-term culture. Such hostincorporated cells lead to low cell survival rates andpoor outcomes in growth, localization, differentiation, and paracrine effects. SEGOVAprogram overcomes these problems by performing autologous stem cell therapy withoutincubation. Within the study population, we showed that the hormone levels were different6 months after the intervention, and it was noted that there were statisticallysignificant differences among participants with respect to the level of the same hormonebefore the intervention.SEGOVA ovarian rejuvenation procedure is unique because it uses the minimally invasiveprocedure of LPSC NOS surgery for taking a segment of the ovarian cortex for in vitroactivation. Success criterion for this study was regaining the hormonal function in femalesubjects, activation of dormant follicles, promotion of antral follicle growth anddevelopment to mature oocytes. Another advantage of this method is that thetransplantation of the activated tissue is performed under ultrasound control and notthrough laparoscopy. In SEGOVA PRP process there are also special systems and machines forseparating certain cell lines, allowing to increase the concentration of desired cells(growth factors derived from them) up to 18 times the initial concentration. This approachis different from most other PRP ovarian therapies. While autologous BMSC transplantationcan have a positive effect on patients with POF, allogeneic BMSC transplantation in womenwith POF can cause transplant rejection with further complications and consequences.SEGOVA acts on the intracellular signaling system and BMSC transplantation is withoutprevious culturing and incubation in a way to save the original stem cell niche. The maindifficulty with stem cell therapy is to maintain cell viability, cell properties and cellfunction before and after implantation


Diabetes is prevalent worldwide including hospitalized patients with heart failure with reduced ejection fraction (HFrEF). This retrospective study investigated the association of diabetes with in-hospital adverse events in patients with HFrEF.We analyzed data from electronic medical records of patients hospitalized with HFrEF in West China Hospital of Sichuan University from January 1, 2011, to September 30, 2018. Propensity score matching balances the baseline characteristics between patients with and without diabetes. Logistic and Poisson regressions investigated the association of diabetes with risks of intubation, cardiogenic shock, acute kidney injury (AKI), intensive care unit (ICU) admission and death during hospitalization, and length of ICU and hospital stay in the matched cases.P<0.001), cardiogenic shock , AKI at any stage , ICU admission , and death  during hospitalization. Patients with diabetes had longer ICU  and hospital stay  than those without diabetes. There were potential subgroup effects by age and by hypertension, and CKD status on the association of diabetes with risk of AKI at any stage; and subgroup effects by sex and CKD status on the association of diabetes with risk of intubation. The increase in length of hospital stay was larger in patients without hypertension than those with hypertension.Among 6,022 eligible patients , 1,930 patient pairs with and without diabetes were included by propensity score matching. Patients with diabetes had a significantly increased risk of intubation (odds ratio [OR], 2.69; 95% confidence interval [CI], 2.25–3.22; Among patients with HFrEF, those with diabetes have a worse prognosis, including a higher risk of in-hospital intubation, cardiogenic shock, AKI, ICU admission and death during hospitalization, and longer ICU and hospital stay. Diabetes is a common comorbidity of heart failure (HF) and is present in more than 40% of patients with HF, representing an increasing disease burden worldwide –6. HF wiControversial reports debate whether diabetes is associated with poorer in-hospital prognosis of hospitalized patients with HFrEF . Althougvia the Clinical Research and Exploration System of West China Hospital, Sichuan University. We included patients 1) aged ≥18 years; 2) with a length of hospitalization stay >2 days; 3) with all echocardiography LVEF values <40% during hospitalization; 4) with available records on prescription and discharge diagnosis; and 5) with available laboratory data  and vital signs .This retrospective cohort study enrolled Chinese patients hospitalized with HFrEF in the West China Hospital of Sichuan University between January 1, 2011, and September 30, 2018, based on electronic medical records (EMRs) which could be accessed , the dates and records of resuscitation from the prescription records, vital signs , height, and weight from the nursing records, laboratory test results from the laboratory information records , and LVEF from the echocardiography reading. For patients with more than one hospitalization, we extracted the data pertaining to the last hospitalization. Where multiple values were available for a given parameter at the hospitalization, we used the earliest value to describe the characteristics of patients. The estimated glomerular rate filtration (eGFR) was calculated according to the chronic kidney disease epidemiology collaboration (CKD-EPI) formula  codes, admission department (Department of Cardiology/others), admission date, calendar year of admission date, discharge date, and patient status on discharge. We also collected prescription information  formula . The Cha formula , 24 was Patients were identified as having diabetes if they had at least one laboratory record of fasting glucose >7.0 mmol/L, 2-hour blood glucose after 75 g glucose challenge >11.1 mmol/L, random glucose >11.1 mmol/L, or glycated hemoglobin A1c (HbA1c) >6.5% during hospitalization, or a discharge diagnosis of ICD-10 codes E10–E14 . Others The following adverse events during hospitalization were identified:intubation: if a patient required an invasive ventilator.cardiogenic shock: if a patient required positive inotropic drugs such as intravenous milrinone, dobutamine, or noradrenaline for emergency use.AKI: identified adopting from a previous report  with theICU admission: if a patient was admitted to the medical ICU.death: if there was a death record or resuscitation records within one calendar day before voluntary discharge.length of stay in ICU: the total days of a patient treated in medical ICU.length of stay in hospital: the total days of a patient treated in the hospital.The follow-up time started on the date of admission to hospital and ended on when the date of a given outcome or discharge comes first.t-test or the Mann–Whitney U test. Categorical variables were described as frequencies (percentages) and compared using the Chi-square test.Baseline characteristics are presented using summary statistics, including mean ± standard deviation (SD) for continuous variables with normal distribution by the Kolmogorov–Smirnov test (P ≥0.001), and median  if not normal, compared between groups using the two-sided Student’s . The standardized differences in covariates between groups before and after PS matching (PSM) were calculated, and covariates with a standardized difference >0.10 after matching were considered sub-optimally matched.Propensity scores (PS) were used to estimate the probability of having diabetes using a multivariable logistic regression model, conditional upon age, sex, heart rate, eGFR, +/− ischemic heart disease (IHD), systolic BP, LVEF, CCI, calendar year of admission date, and admission department (Department of Cardiology versus others) . Each pa2. We calculated the E-value to quantify the minimum magnitude of potential unmeasured confounders that could explain the observed association between diabetes and each clinical outcome (We estimated the average treatment effect on the treated (ATT) based on each PS-matched sample. We assessed the association of diabetes with each dichotomous adverse event during hospitalization using Logistic regression and reported odds ratios (ORs) and 95% confidence intervals (CIs). We applied generalized Poisson regressions to estimate the time ratio of the length of ICU stay and hospitalization stay between patients with and without diabetes, and then estimated the excess length of ICU stay and hospital stay as well as its 95% CI in patients with diabetes. We also examined the impact of diabetes on in-hospital adverse events in subgroups stratified by sex, age, +/− HD, +/− hypertension, and +/− CKD, which was defined as eGFR <60 mL/min/1.73 m outcome .ggplot2     and figum, 2016)  and forey, 2020) . A two-sP<0.001) and were more likely to have IHD . Compared with patients without diabetes, those with diabetes had a significantly higher median (P<0.001) N-terminal pro-B type natriuretic peptide , blood glucose (7.7 mmol/L vs 5.4 mmol/L), HbA1c (7.2 mmol/L vs 5.8 mmol/L), and CCI (1.0 vs 0.0), but lower eGFR  . The overlap in PS between groups pre- and post-PSM is shown in .As shown in P<0.001), cardiogenic shock , AKI in any stage , ICU admission , and death  during hospitalization. Patients with diabetes also had a higher incidence risk of AKI stage 2 or 3  and AKI stage 3  during hospitalization . The observed associations between diabetes and the outcomes of intubation, cardiogenic shock, AKI in any stage, AKI stage 2 or stage 3, AKI stage 3, ICU admission, and death could be explained by the existence of unmeasured confounding, with ORs over 2.66, 2.19, 1.91, 1.76, 1.97, 2.09, and 7.97, respectively . The excess median length of ICU and hospital stay in patients with diabetes was 1.47 days  and 2.20 days , respectively .After PSM, diabetes was found to be significantly associated with higher risks of intubation , without hypertension , or without CKD . Similar subgroup effects by age, +/− hypertension, and +/− CKD were found for the association of diabetes with risks of AKI stage 2 or stage 3 and AKI stage 3, as well as the subgroup effect by +/− IHD, shown in P<0.05) or without IHD , shown in P<0.001), shown in In the present study, approximately one-third of patients hospitalized with HFrEF had diabetes, and diabetes had a negative impact on short-term prognosis before discharge, increasing the risks of adverse events of respiratory, cardiovascular, kidney systems as well as the ICU admission and death during hospitalization, and extending the length of ICU and hospital stays. Our findings indicate there is a clinical need for increased focus on the respiratory, cardiovascular, and kidney systems of patients with both diabetes and HFrEF during hospitalization and for appropriate management to prevent in-hospital death.In the present study, patients hospitalized with HFrEF who also had diabetes experienced longer hospitalization stay and ICU stay as well as a higher admission rate to ICU, in line with previous findings , 34, 35,Our findings also expand the understanding of the impact of diabetes on short-term adverse clinical outcomes in patients hospitalized with HFrEF. We observed a higher rate of AKI among HFrEF patients with diabetes, consistent with previous findings –39. It iStrengths of our study included the selection of patients hospitalized with HFrEF as the study population, including those in ICUs, which enabled investigation of the association of diabetes with a short-term prognosis of HFrEF. Second, we analyzed five patient-important outcomes covering cardiovascular, kidney, and respiratory systems to comprehensively evaluate the clinical prognosis of HFrEF. Furthermore, we adjusted for the calendar year to improve the covariate balance when applying PSM, which enhanced the power of our study. We calculated the E-value of the primary analyses to evaluate the strength of the association between diabetes and each adverse event. Our study also had some limitations, including the single-center design and the inability to conclude any causation between diabetes and the adverse outcomes.The short-term prognosis for patients with HFrEF is worse in those with diabetes as comorbidity, with this population being more likely to receive intubation, develop new-onset AKI or cardiogenic shock, be admitted to ICU, die during hospitalization, and have a longer ICU stay or hospital stay. Clinicians need to monitor the patient’s condition more closely in those with both diabetes and HFrEF, who may face additional in-hospital risks than those without diabetes.Lisheyu@gmail.com.The datasets presented in this article are not readily available because the individual-level data was restricted in storage and utilized within the informatic server of West China Hospital. Any public use and transaction were prohibited to protect the privacy of the patients. Requests to access the datasets should be directed to This study was approved by the ethics committee of West China Hospital, Sichuan University (No. 2019-472). Patient consent was waived for this retrospective study of data from electronic medical records.All authors made substantial contributions to the conception and design of the work, the acquisition, analysis, and interpretation of data, and the drafting or critical revision of the article for important intellectual content. All authors contributed to the article and approved the submitted version.SYL received grants from the Sichuan Science and Technology Program (grant number 2019YFH0150) and the 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (grant number ZYGD18022 and 2020HXF011), National Natural Science Foundation of China (grant number 21534008), and the Chief Scientist Office Project (reference number CGA/19/10).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


DPYD) gene may reduce DPD function, the main enzyme responsible for the metabolism of fluoropyrimidines. This leads to drug accumulation and to an increased risk of toxicity. Routine genotyping of this gene, which usually includes DPYD *HapB3, *2A, *13 and c.2846A > T (D949V) variants, helps predict approximately 20–30% of toxicity cases. For DPD intermediate (IM) or poor (PM) metabolizers, a dose adjustment or drug switch is warranted to avoid toxicity, respectively. Societies such as the Spanish Society of Pharmacogenetics and Pharmacogenomics (SEFF), the Dutch Pharmacogenetics Working Group (DPWG) or the Clinical Pharmacogenetics Implementation Consortium (CPIC) and regulatory agencies  already recommend DPYD routine genotyping. However, the predictive capacity of genotyping is currently still limited. This can be explained by the presence of unknown polymorphisms affecting the function of the enzyme. In this case-control work, 11 cases of severe fluoropyrimidine toxicity in patients who did not carry any of the four variants mentioned above were matched with 22 controls, who did not develop toxicity and did not carry any variant. The DPYD exome was sequenced (Sanger) in search of potentially pathogenic mutations. DPYD rs367619008 , rs200643089  and rs76387818  increased the percentage of explained toxicities to 38–48%. Moreover, there was an intronic variant considered potentially pathogenic: rs944174134 (c.322-63G > A). Further studies are needed to confirm its clinical relevance. The remaining variants were considered non-pathogenic.Among cancer patients treated with fluoropyrimidines, 10–40% develop severe toxicity. Polymorphism of the dihydropyrimidine dehydrogenase ( DPYD gene, metabolizes 5-FU to dihydro-5-fluorouracil (DHFU). Dihydropyrimidinase (DHP) acts on this compound, cleaving the pyrimidine ring and producing 5-fluoro-ureidopropionic acid (FUPA). FUPA is metabolized to α-fluoro-β-alanine (FBAL) by the β-ureido-propionase enzyme (BUP1), and FBAL is excreted in the urine. The DPD enzyme is the limiting factor in this pathway that regulates the cytosolic accumulation of 5-FU . y 20–30% . ConsistDPYD genotypes consistent with the development of toxicities. The first case carried the rs367619008  variant (in heterozygosis). In Europeans, this variant shows a frequency of <0.01% [Among the 11 studied cases, two showed f <0.01% . The lysf <0.01% . Three sf <0.01% ,23,24, wThe second case showed the rs200643089  and rs76387818  variants. The first variant shows a frequency of 0.003% in the global population; the second has a frequency of 3.2% in Europeans. To our knowledge, only one study previously analyzed the first variant, present in a patient with toxicity and, therefore, proposed to cause an alteration of DPD function by being in close proximity to the 5-FU binding site . In the Two other intronic variants were associated with the development of toxicity; however, due to the nature of the variants  and the literature support, our conclusions are less compelling. The first of them is c.322-63G > A (rs944174134), which was present in heterozygosis in one case exclusively. To the best of our knowledge, this is the first work that finds this variant in the context of fluoropyrimidine treatment and relates it to the development of toxicity. The second one was c.1740 + 39C > T (rs2786783), which was more prevalent in cases than in controls. Previous works evaluated this variant, showing controversial findings: in two of them, this variant, along with other variants, was related to the development of toxicity ,29, whilDPYD core variants only prevents 20–30% of toxicities, since, among the remaining 70–80%, this tool has no predictive power for two reasons: first and foremost, because most of these toxicities are not related to DPD activity/DPYD polymorphism, and second, because some of them may be related to unknown polymorphisms. Therefore, among the eleven cases, nine (82%) belong to the first group and two to the second (18%). Considering that at least two of them exhibited pathogenic or potentially pathogenic variants , the percentage of toxicities explained would have been 38–48% if rs367619008, rs200643089 and rs76387818 had been genotyped prospectively.Overall, as mentioned earlier, the prospective genotyping of The remaining variants were considered non-pathogenic, based on the literature evidence and the results obtained, since their presence in the controls helps to rule out that they cause lower DPD enzyme activity. These include all the variants identified which were defined as normal function variants by CPIC. The variants rs2297595, rs3790387, rs2811202 and rs56293913 appeared together in four controls, all of them in heterozygosity. They are in linkage disequilibrium, appearing together in 11.21% of the Iberian population, thus probably defining a previously unidentified allele . None ofThe main limitation of this study is its retrospective nature and the reduced sample size. Hence, our findings and the interpretation of them should be considered with caution; confirmatory studies are required, preferably prospective and with large sample sizesDPYD: *HapB3, *2A, *13 and c.2846A > T (D949V). In this work, rs367619008 , rs200643089  and rs76387818  variants increased the percentage of the explained toxicities to 38–48%. Further studies are warranted to confirm the clinical relevance of the intronic variants. The remaining variants were considered non-pathogenic, including the identified allele formed by the combination of rs2297595, rs3790387, rs2811202 and rs56293913 variants.Only 20–30% of the cases of toxicity in patients prescribed fluoropyrimidines can be explained by the four basic variants of


Acute myeloid leukemia (AML) is the most common leukemia among the adult population and accounts for about 80% of all cases. Despite advancements in therapeutic regimens, the prognosis remains very poor, especially in the elderly population. Selinexor is a first-in-class, oral, small molecule Exportin-1 inhibitor that is being developed for the treatment of a variety of cancers, including AML. The efficacy and safety issues of selinexor in the treatment of AML are still the focus of attention. Therefore, we conducted a meta-analysis to evaluate the efficacy and safety of selinexor in the treatment of AML.According to the search strategy, regardless of publication date or language, randomized controlled trials of selinexor for AML will be retrieved from 8 databases. First of all, the literature was screened according to the eligibility criteria, and use the Cochrane Collaboration's tool to assess the quality of the included literature. Then, using Rev Man 5.3 and STATA 14.2 software for traditional meta-analysis. Finally, the evaluation of the quality of the evidence and the strength of the recommendations will adopt the Grading of Recommendations, Assessment, Development, and Evaluation method.This study will evaluate the efficacy and safety of selinexor for AML, thereby providing more evidence support for clinical decision-making in AML.Our research will provide more references for the clinical medication of patients with AML. While 10% to 40% of younger AML patients are primarily refractory to AML induction therapy, the number is considerably higher for patients above 60 years (40%–60%). The cure rates have increased up to 15% in patients older than 60 years and about 40% in patients below 60 years of age only. So the prognosis remains very poor in the AML population. Still, the mainstay of treatment is chemotherapy, which has remained mostly unchanged over the past 4 decades. Therefore, extending the survival period of patients with AML, reducing the mortality rate, and improving the prognosis are key issues that need to be resolved.Acute myeloid leukemia (AML) is a clinically heterogeneous disease characterized by accumulation and expansion of immature myeloid cells in the bone marrow and peripheral blood, with consequent failure of normal hematopoiesis. XPO1 is the major nuclear exporter for tumor suppressor proteins , growth regulators, and oncoprotein mRNAs , and is overexpressed in a variety of cancer cell types.–7 Selinexor has orphan drug status in the USA and EU for AML, and is currently being evaluated in clinical trials in patients with AML. Therefore, this study proposes a systematic review program to evaluate the efficacy and safety of selinexor on AML, and to provide sufficient basis for further guidance of clinical medication, so as to avoid unnecessary traps.Selinexor is a first-in-class, oral, small molecule inhibitor of Exportin-1 (XPO1), also known as chromosome maintenance protein 1, being developed for the treatment of cancer.22.1Our protocol has been registered on the International Platform of Registered Systematic Review and Meta-Analysis Protocols (INPLASY). The number was INPLASY2021100056 (URL = 10.37766/inplasy2021.10.0056). We will be based on the Preferred Reporting Items for Systematic Review and Meta Analysis Protocols (PRISMA-P), and strictly follow the requirements and conduct.2.2We will conduct a literature search from the following electronic databases: PubMed, EMBASE, the Cochrane Library, Web of Science, CNKI, Wan-fang Data, Chinese Biomedical Literature Database, Chinese Scientific Journal Database. There are no restrictions on publication date and language. In addition, the references listed in each included article are also manually searched.Retrieve the databases by combining subject words with random words. Appropriate adjustments will be made according to the grammatical rules of different databases to ensure the completeness and comprehensiveness of the search. We will firstly conduct a pre-search, and discuss the problems encountered in the search process with the team. After confirming that there are no problems, we will conduct a formal literature search. Taking PubMed as an example, the retrieval strategy is shown in Table 2.3We will formulate the inclusion and exclusion criteria for this study based on the PICOS principles.2.3.1Patients with AML diagnosed by WHO criteria diagnosis of AML will be included. There are no restrictions on nationality, age, sex, or race. Patients with severe liver and kidney, or other uncontrolled systemic diseases are excluded.2.3.2–12The recommended phase II dose (RP2D) of selinexor was 60 mg twice weekly in the treatment group. The control group was only given routine chemotherapy, or the same dose of placebo was given on the basis of routine chemotherapy. Routine chemotherapy regimens mainly include cytarabine plus idarubicin, high-dose cytarabine and mitoxantrone, decitabine, cytarabine, etoposide and mitoxantrone, cytarabine, cladribine and granulocyte-colony stimulating factor, cytarabine and fudarabine, etc.2.3.3The primary outcomes include ORR, CR, Cri, and DOR; the secondary outcomes include decrease in bone marrow blasts from baseline, quality of life, etc; the safety indicators include thrombocytopenia, fatigue, nausea, anemia, decreased appetite, decreased weight, diarrhea, vomiting, hyponatremia, neutropenia, leukopenia, constipation, and other adverse events.2.3.4Randomized controlled trials (RCTs) will be included in this study irrespective of language or publication category. Animal trials, review article, and studies with incorrect RCT designs will be excluded.2.4Use Endnote X9.0 software to manage literature. After searching literature based on the above steps, import them into endnote software for literature screening. First, 2 independent researchers will conduct a preliminary literature screening based on the titles and abstracts of the included literature to eliminate duplicate and non-RCTs. Then read the full text of the remaining literature according to the previously designed principles of eligibility criteria, and finally determine the appropriate literature. When 2 researchers disagree, a third researcher will resolve it. The specific screening process is shown in Figure According to the Cochrane Handbook for Systematic Reviews of Interventions, 2 researchers independently extracted and recorded the required information from all the included literature. When 2 researchers disagree, they will discuss to reach an agreement, otherwise they will work with the third researcher to resolve. The required information mainly includes the author, publication time, study design, participants number and demographic characteristics , treatment status , and outcomes. If any of the above information in the included literature is incomplete, we will contact the corresponding author via email to obtain the required data.2.5 The results of the quality assessment will be completed using software Review Manager 5.3.Two reviewers will independently assess the quality of the included literature according to the Cochrane Collaboration's tool for RCTs. If there is a disagreement between 2 reviewers, the third reviewer resolves the issue. According to Cochrane Handbook V.5.2.0, characteristics of each item will be evaluated in 3 categories: low, unclear, and high.2.62.6.1P < .05 is considered statistically significant. The heterogeneity among the included literature will be assessed using the Q test method and I2 statistic method. When the Q statistic corresponds to P ≤ .10 or I2 > 50%, it indicates that there is heterogeneity among the included literature, and assess the effect size by the random effect; on the contrary, a fixed effect model is used.We will execute Rev Man 5.3 and STATA 14.2 software for traditional meta-analysis. For dichotomous data, we will calculate a summary estimate with 95% confidence interval odds ratio value; for continuous data, we will calculate a summary estimate of standardized mean difference with 95% confidence interval, and 2.6.2Taking into account the issue of heterogeneity, we will conduct a subgroup analysis based on the specific circumstances of the included literature. If there is a problem of heterogeneity, we will conduct a subgroup analysis of age, sex, and interventions.2.6.3This systematic review will use the method of eliminating each study one by one for sensitivity analysis. If the effective indicators of selinexor in the treatment of AML have not changed significantly, it indicates that the study is robustness. On the contrary, it is not robustness. According to the specific situation, low-quality research is excluded.2.6.4Publication biases will be assessed by a funnel plot for meta-analysis and quantified by the Egger method. It should be noted that if the number of included literature is ≥10, it is appropriate to use a funnel plot to assess potential publication bias. However, if the included literature is <10, it may affect the overall test power because the included number is too small, and it is difficult to accurately evaluate the symmetry of the funnel plot.2.7The Grading of Recommendations, Assessment, Development, and Evaluation used to assess the quality of evidence. The quality of evidence is divided into 4 levels from 5 aspects, namely high, medium, low, and very low.2.8This study is a systematic review, it does not involve medical ethics and patients’ informed consent. This study will publish the results of meta-analysis in journal papers.3 the prognosis of relapsed or refractory AML remains poor. With high relapse rates and few targeted therapeutic options, there is a need to develop novel solutions for the treatment of AML.AML is a form of cancer that is characterized by infiltration of the bone marrow, blood, and other tissues by proliferative, clonal, abnormally differentiated. In spite of advances, including allogeneic stem cell transplantation and a growing molecular tailoring of treatment toward driver genes such as FLT3, Selective inhibition of nuclear export by selinexor results in accumulation of tumor suppressor proteins in the nucleus, decreased levels of oncoproteins, cell cycle arrest, and apoptosis of cancer cells, while sparing normal cells.–7 Selinexor has been shown to be effective in many clinical trials of AML. Selinexor plus cytarabine plus idarubicin produced an ORR of 55% (n = 42) in patients with relapsed or refractory AML in a phase II trial (NCT0224909). Perhaps it can bring hope to the treatment of AML.Selinexor selectively inhibits XPO1 by forming a slowly reversible covalent bond with cysteine 528 in the cargo binding pocket of XPO1.Although this systematic review has many advantages, such as statistical power and accuracy of effect size estimation, subgroup analysis of factors affecting treatment effects, a more objective evaluation of evidence and a more accurate and objective evaluation of effect indicators evaluation. However, it also has some problems. For example, this study is an evaluation of published literature, and there may be problems such as unscientific and non-strict RCT design, resulting in uneven quality of literature research, which affects the credibility of this study. Or the research results included in the literature have false negatives and false positives. In any case, this systematic review can provide reliable results for AML, and provide strong evidence for the significant advantages of selinexor in the treatment of AML.Conceptualization: Liming Yu, Siyuan Cui.Data curation: Xuewei Yin.Formal analysis: Yuping Si.Funding acquisition: Siyuan Cui, Liming Yu.Methodology: Yan Wang, Jingyi Wang.Project administration: Siyuan Cui.Writing – original draft: Liming Yu, Siyuan Cui.Writing – review & editing: Liming Yu, Xuewei Yin.


Diamond-Blackfan Anemia (DBA) is an inherited rare disease characterized with severe pure red cell aplasia, and it is caused by the defective ribosome biogenesis stemming from the impairment of ribosomal proteins. Among all DBA-associated ribosomal proteins, RPS19 affects most patients and carries most DBA mutations. Revealing how these mutations lead to the impairment of RPS19 is highly demanded for understanding the pathogenesis of DBA, but a systematic study is currently lacking. In this work, based on the complex structure of human ribosome, we comprehensively studied the structural basis of DBA mutations of RPS19 by using computational methods. Main structure elements and five conserved surface patches involved in RPS19-18S rRNA interaction were identified. We further revealed that DBA mutations would destabilize RPS19 through disrupting the hydrophobic core or breaking the helix, or perturb the RPS19-18S rRNA interaction through destroying hydrogen bonds, introducing steric hindrance effect, or altering surface electrostatic property at the interface. Moreover, we trained a machine-learning model to predict the pathogenicity of all possible RPS19 mutations. Our work has laid a foundation for revealing the pathogenesis of DBA from the structural perspective. Diamond-Blackfan Anemia  is an inherited rare pure red blood cell aplasia (∼5 to 7 per million birth)  characteIn DBA, almost all linked genetic lesions come from genes encoding ribosomal proteins (RP) , whose hDBA is mainly inherited in an autosomal dominant manner and caused by loss-of-function mutations . All DBARPS19 have affected the most majority of DBA patients (∼25%)  . As an is (∼25%) . During s (∼25%) .With the continuous efforts that have been put in genetic screening and clinical studies of DBA, a considerable number of pathogenic RPS19 mutations have been cataloged , accountRPS19 gene have been studied experimentally (P. abyssi), which shares 36% sequence identity with human RPS19, and then analyzed 16 missense DBA mutations based on it. These mutations were classified according to the impact on protein folding or on surface properties, and this binary classification has been applied to interpret pathogenic mutations in others’ work , several human ribosome structures have been determined and have thus created opportunities of further direct analyses . Second, we revealed that RPS19 interacts with 18S rRNA in the mature SSU through five conserved surface patches. Third, we identified several specific approaches through which DBA mutations destabilize the protein structure or affect interactions, and thereby lead to defected RPS19. Last, based on the understandings of the structural basis of DBA mutations, we trained a support vector machine (SVM) model to predict the pathogenicity of all possible missense mutations of RPS19, which will be valuable in future studies when new mutations are identified.An in-depth understanding of structure is the basis of studying mutations in a structural context, so we investigated the structure of human RPS19 in detail. Its structures both in free state and in the SSU complex were inspected in order to obtain an accurate description of its secondary structure, hydrophobic core, and interaction with other molecules.The recent determined Cryo-EM structure of human ribosome can be used as a starting point to assign the secondary structure of RPS19 . ConsideTwo IDRs have been roughly proposed based on the archaea RPS19 structure previously . Here weThrough analyzing the interaction interface of RPS19 in the complex of ribosome SSU, we found that RPS19 mainly interacts with 18S rRNA, RPS16, and RPS18 . The larAfter mapping the conservation score to the solvent-excluded molecular surface by specific color scheme , we furtPreviously, p = 8.9E-5, one-tailed Mann-Whitney U test, We have curated 51 DBA missense mutations  and 30 nOverall, DBA mutations are more likely to occur at positively charged, hydrophobic or small residues . ArgininOne striking feature is that a larger number of residues  have been mutated into Proline (Pro) , a well-Based on the analyses concerning the residue types, we can roughly infer that DBA mutations may mainly disrupt RPS19 from two perspectives: protein folding stability and interaction, as other studies have done  or reviep = 2.27E-4, one-tailed Mann-Whitney U test, To investigate to what extent the mutations affect stability of the protein structure, we calculated the change in free energy (ΔΔG) of folding of RPS19 caused by mutations using FoldX, a protein design algorithm that uses an empirical force field . We founp = 2.17E-5, one-tailed Mann-Whitney U test). Further restricting in the 30 DBA mutations with ΔΔG > 1 kcal/mol shows even smaller rSASA values (median: 0.061). We checked the residue types of these 30 mutations, and found two major classes: 13 are mutated from typical hydrophobic residues  to other types with 22.96% hydrophobicity decreased on average (We further investigated the approaches by which DBA mutations decrease the structure stability. The first is the destruction of hydrophobic core. We compared the relative solvent accessible surface area (rSASA) between DBA and neutral mutations , and fou average , and 7 o average .3), to larger Arg and Glu (volume = 173.4 and 138.4 Å3) would pose significant steric hindrance effect with 188.52 and 130.28% volume increase, respectively. Moreover, Arg and Glu have charged side chains with high hydrophilicity, which will also disturb the formation of the hydrophobic core during protein folding. It should be these two effects that cause Gly127Arg and Gly127Glu to result in the largest structural destabilization with ΔΔG = 18.72 and 17.12 kcal/mol, respectively  to build the RPS19-specific prediction tool. After further curation, 29 DBA mutations (positive samples) and 30 neutral ones (negative samples) were selected from the mutation dataset as training data. We then extracted the features based on our understandings of the structural basis of DBA mutations. In total, 18 candidate features were extracted for each mutation concerning the interaction with rRNA, structural stability, conservation, etc. After feature selection, 8 features were finally selected  .Based on fivefold cross-validation, we identified the best hyper-parameters , and 26 of the 29 DBA mutations can be correctly predicted in the cross-validation, similar to other well-known tools . At the Finally, we re-trained the predictor, namely RPS19-SVM, on all the training data with the best hyper-parameters, and utilized it to predict the pathogenicity for all possible missense mutations of RPS19 . The resAs ExAC has been upgraded to gnomAD later , we retrMoreover, the heatmap based on the pathogenicity scores  also conIn this work, we conducted a systematic study aiming at revealing the structural basis of DBA mutations at RPS19. Our study illustrated that DBA mutations would disrupt the hydrophobic core related to structural stability or perturb the interactions between RPS19 and 18S rRNA through two or three mechanisms, respectively. Based on these, we further trained an RPS19-specific predictor and predicted the pathogenicity of all possible RPS19 mutations. Logically, the RPS19 molecules bearing DBA mutations would thus be subject to faster degradation or incapability of assembling into the ribosome SSU, resulting in insufficient ribosome quantities and finally DBA symptoms.Compared with a previous study , our worIt should be admitted that our analyses have some shortcomings yet. First, ribosomopathies, including DBA, often manifest in a tissue-specific way, with which two hypotheses have been proposed: the specialized ribosome hypothesis and the ribosome concentration hypothesis . Our worSecond, in addition to being the indispensable component of ribosome SSU, RPS19 also plays essential roles in many other pathways, including pre-rRNA processing, exportation of SSU precursors from nucleus to cytoplasm, and conformation maturation of 18S rRNA . Our worThird, we only analyzed the structural stability and interactions of RPS19, but the mutations may act from other aspects, such as subcellular localization. Ribosome precursors are first formed in the nucleoli  and thenIt should be noted that the approaches we proposed cannot cover all the DBA mutations. Some residue substitutions are highly likely to disrupt the protein structure, such as Ala17Pro, Ala20Pro, Ala57Pro, Ala58Pro, and Leu64Pro, as they have introduced proline into helical regions. But the free energy changes calculated by FoldX did not support this. Considering that previous studies have suggested that FoldX calculation for mutations containing Pro needs to be optimized , the ΔΔGExcept for RPS19, DBA may also stem from defects at other RPs. In our perspective, further studies on the DBA mutations in all related RPs in the future would provide a more comprehensive picture of the pathogenesis mechanisms, which may also shed light on the pathogenesis of other ribosomopathies. Some DBA mutations in other RPs may share similar structural basis as those in RPS19. For example, an N2-Q22 deletion variant of RPS24 is known to cause DBA . We checThe RPS19 structure was extracted from the coordinates of human ribosome SSU complex (PDB ID: 6G5H) , and wasBefore production simulation, energy minimization was performed to relax possible atom collisions, and the system was equilibrated for 2 ns with the final temperature reaching 300.0 K. The production simulation was conducted in an NPT ensemble and lasted 1,000 ns. A timestep of 2 fs was used in both equilibration and production simulation.The conformations were saved in the trajectory file with an interval of 1 ps, and were analyzed using CPPTRAJ . Conform1; The interaction interface of RPS19 in the ribosome complex was identified by using PDBePISA  with default parameters .The Consurf scores and electrostatic potential were rendered at the protein’s solvent-excluded molecular surface with different color schemes by using UCSF Chimera .The effect of a mutation on protein structural stability was measured by the change of folding free energy (ΔΔG), and was calculated by using the FoldX package . In detaThe solvent accessible surface area (SASA) of each residue was calculated by DSSP . The relThe neutral mutations collected from ExAC were treated as negative samples. The positive samples were selected from the DBA mutations if they met any one of these criteria: (1) identified in more than one patient, (2) explicitly annotated as “Pathogenic” or “Likely pathogenic” in ClinVar, and (3) experimentally confirmed to affect the physiological function of RPS19 .Based on our analyses of DBA mutations, 18 features in four categories were extracted for describing each mutation. The details of these features are listed in The feature selection and the SVM hyper-parameter searching were conducted by using the scikit-learn (version: 0.21.3) package in Python. In detail, all the possible feature combinations with at least 5 features were enumerated, and the hyper-parameters C and γ were enumerated from 0.001, 0.01, 0.1, 1, and 100. For each combination of features and hyper-parameters, fivefold cross-validation was run on the training dataset containing the positive and negative samples. When the maximum cross-validation MCC was reached, the optimal feature combination and optimal hyper-parameters were obtained accordingly. The resulted optimal hyper-parameters and features were then utilized to train the RPS19-SVM predictor on all the training data. The predicted results of PMut , MutPredThe original contributions presented in the study are included in the article/KA, Z-QY, and Y-DW conceived the study. KA, J-BZ, YX, and Z-QY collected the data and performed the computational analysis. KA and Z-QY initially drafted the manuscript. Z-QY, Y-DW, WH, and TW revised and proved the manuscript. Z-QY and Y-DW supervised the whole work. 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.


Ants are an important insect group that includes a considerable number of species. Along with this diversity in species, they also exhibit a wide variation in chromosome numbers, from 1 up to 60 chromosomes. DNA molecules can be counted in a specific stage of the cell life cycle and quantified. These DNA molecules are very tightly packed together with several proteins and are called chromosomes. Each species shows a specific number of chromosomes with different shapes and sizes, as well as different quantities of DNA. We can use such information  as morphological attributes to study evolution at the species level. In this study, we describe new karyotypes of several ant species. In addition, from previous studies, we have compiled all the available information regarding the chromosome number and DNA quantity in fungus-farming ant cells. Different processes, called rearrangements, can change chromosomes over time, producing new character states. Such states can be tracked, along with the species and groups of similar species, using their relationships to identify patterns. We use DNA sequences to reconstruct the relationships of fungus-farming ant species (molecular phylogeny). By comparing such phylogeny with the chromosome number and DNA quantity, we discuss the evolution of chromosomes and DNA quantity (or genome size), and the potential limits to these features across fungus-farming ants. Myrmecia croslandi, while some have as many as 60 chromosomes, as in the males of the giant Neotropical ant Dinoponera lucida. Fungus-growing ants are a diverse group in the Neotropical ant fauna, engaged in a symbiotic relationship with a basidiomycete fungus, and are widely distributed from Nearctic to Neotropical regions. Despite their importance, new chromosome counts are scarcely reported, and the marked variation in chromosome number across species has been poorly studied under phylogenetic and genome evolutionary contexts. Here, we present the results of the cytogenetic examination of fungus-farming ants and compile the cytogenetic characteristics and genome size of the species studied to date to draw insights regarding the evolutionary paths of karyotype changes and diversity. These data are coupled with a fossil-calibrated phylogenetic tree to discuss the mode and tempo of chromosomal shifting, considering whether there is an upper limit for chromosome number and genome size in ants, using fungus-farming ants as a model study. We recognize that karyotypes are generally quite variable across fungus-farming ant phylogeny, mostly between genera, and are more numerically conservative within genera. A low chromosome number, between 10 and 12 chromosomes, seems to present a notable long-term evolutionary stasis (intermediate evolutionary stasis) in fungus-farming ants. All the genome size values were inside a limited spectrum below 1 pg. Eventual departures in genome size occurred with regard to the mean of 0.38 pg, indicating that there is a genome, and likely a chromosome, number upper limit.Ants are an important insect group that exhibits considerable diversity in chromosome numbers. Some species show only one chromosome, as in the males of the Australian bulldog ant  Althougiewed in ,3). Thisiewed in ,5). Despo n = 27 .Detailed karyotype descriptions , chromosome counts, and their comparative analyses are important independent tools for use in taxonomy and in understanding chromosome evolution, particularly when relying on the phylogenetic tree . The advBeyond the number of DNA molecules, estimating the genome content of cell nuclei has contributed to our understanding of genomes through the tree of life. Recently, the nuclear content of approximately 100 ant species was estimated using flow cytometry , and nowApterostigma madidiense , Mycocepurus goeldii , Sericomyrmex parvulus , Cyphomyrmex transversus , and Myrmicocrypta sp., based on karyomorphometric analysis. We added these karyotypes to the available data on fungus-farming ants in the Ant Chromosome Database , depicting the heterochromatic banding pattern and chromosome morphology. We further compiled the genome size information available for the species. All these data were scattered through a time-calibrated phylogeny to evaluate the mode and tempo of chromosomal evolution in fungus-farming ants. We evaluate and discuss our results with regard to chromosome number, morphology, and genome size variation under a phylogenetic framework with a focus on the question: Is there an upper limit of chromosome number for fungus-farming ants? Our intention was not to determine a specific upper number, but to discuss and propose an indication for a limit, considering the GS estimates of ants and the outcomes of the minimum interaction theory. We further discuss the importance of cytogenetic studies.In this study, we first describe five new karyotypes of fungus-farming ants: A. madidiense from Cachoeira do Campo, Minas Gerais (MG); S. parvulus from Marliéria, Minas Gerais (MG) ; C. transversus and Myrmicocripta sp. from Cabo Frio, Rio de Janeiro (RJ) ; and M. goeldii from Morro dos Conventos, Araranguá–Santa Catarina (SC) . The sample collection was authorized by the ICMBio . All colonies were excavated and transferred to the Laboratório de Genética Evolutiva e de Populações of the Universidade Federal de Ouro Preto–MG and kept according to the protocols described by Cardoso et al. [Colonies were sampled from distinct Brazilian states: o et al. . The mair = L/S), and the proportional contribution of each chromosome length (RL) in relation to the total length of all chromosomes (TL × 100/∑TL). The distribution pattern of heterochromatin was obtained using the BSG  banding technique following the method described by Sumner [2.The chromosome slides were obtained from 20 individuals following the protocol described by Imai et al. , with smy Sumner , with mowww.ants.ufop.br, accessed on 30 November 2021 [n), haploid chromosome number (n), and karyotype or karyotypic formulae. The karyotype provides the number of each type of chromosome, whether a chromosome is m, sm, st, t, or acrocentric (a). All the data, including the new counts and karyotypes described here, are presented in The Ant Chromosome Database (ACdb)  from GenBank, which comprised the dataset assembled by Cristiano et al. [To put the cytogenetic data in a phylogenetic context focusing on fungus-farming genera, coupling the phylogenetic relationship of the species with the karyotype information, we retrieved sequences of five nuclear genes . The heterochromatic pattern of the species studied here are summarized in The metaphases of  2n = 8, , 2 K = 6 2n = 8, . The met2n = 28, , 2 K = 22n = 28, . The met2n = 42, , 2 K = 22n = 42, . Some in2n = 42, . The met2n = 24, , 2 K = 22n = 24, . The met 2n = 50 , 2 K = 3Apterostigma spp. (0.61–0.81 pg) and C. transversus (0.50 pg). Spreading the cytogenetic and GS data across the calibrated phylogeny that was generated, we detected wide chromosomal and genome size variability among the species of fungus-farming ants (n > 15) are restricted to some lineages within the genera. Nevertheless, even higher chromosome numbers (n > 25) are rare and restricted to lineages of Neoattini  show a higher number of positive heterochromatic blocks, including chromosomes with a heterochromatic long arm  that, imes  up to 27 haploid chromosomes in Mycetarotes parallelus . The diploid chromosome numbers are, in general, variable in fungus-farming ants, as is the genome size , the Mycetomollerius clade, and the Trachymyrmex clade are estimated at approximately ~19 Mya, ~24.8 Mya, and ~16.8 Mya, respectively , and the chromosome number is indeed consistent within each genus  and show a great distribution of heterochromatic regions, as revealed by the C-banding [Mycetophylax simplex n = 18 (n > 15) also shows a much higher number of C-banded heterochromatic regions compared with the sister species studied cytogenetically [Considering the phylogenetic relationship of fungus-farming species, the general slight variation in genome size across lineages, with few exceptions, shows that a low chromosome number between 10 to 12 haploid chromosome and genome size near to 0.38 pg see , should tion see . These ition see . In facttion see . As an ech genus . However-banding . Similarn ≤ 15, as observed in Mycetomollerius and Cyphomyrmex , but unexpectedly low karyotypes, such as n < 15, are found in the anciently divergent clade called Paleoattina, which comprises Mycocepurus, Myrmicocripta, and Aptersotigma (n = 25 or n = 19 (n > 15), are scattered in the clade comprising recently divergent lineages such as Sericomyrmex and Acromyrmex. The cytogenetic data plotted in the phylogeny reveals that the karyotype n = 10, or a number close to that (n = 11 and 12), is the most common karyotype across fungus-farming ant genera, showing that the karyotype of the lineages is around the average or the most frequent number. Thus, we do not discard that the minimum interaction theory may be a driving force behind karyotype evolution in ants; however, it seems that other forces may regulate chromosome number by imposing a general common karyotype number, such as n < 15, in fungus-farming ants. A few exceptions where the karyotype reaches higher chromosome numbers appear to overcome the barrier likely caused by the expansion of repetitive genomic elements [Accordingly, in the course of evolution, chromosomes will increase in number and reduce in size. Taking the cytogenetic data available to date, phylogenetic relationships, and the time of divergence studied here, a higher chromosome number would be expected in ancient lineages than in recent lineages. The higher chromosome number known is observed in rsotigma . Furtherelements . This iselements ,54  atlanticus (n = circa 223) [Several hypotheses have been invoked and tested to explain the tight genome size of ants: the developmental strategy (holometabolous) and metabolic rate have been suggested as constraints against genome size expansion, as well as the eusociality ,59. Simirca 223) . HoweverIn this paper, we evaluated the currently available cytogenetic information on fungus-farming ants, a distinguishable group of neotropical fauna. From the data studied to date, it is possible to make some assumptions regarding chromosome numbers, karyotype evolution, and genome size variation. Among all the ant species, the highest amount of cytogenetic data available is on the Myrmicinae, specifically fungus-farming ants. However, considering the diversity of ants and their ecological importance, we have only superficially touched on a much deeper compendium of knowledge when we consider that less than five percent of ants has been studied cytogenetically thus far . In our cox 1 or COI) sequences for further identification ; secondion (see ), in the


NAD(P)H:quinone acceptor oxidoreductase-1 (NQO1) is a ubiquitous flavin adenine dinucleotide-dependent flavoprotein that promotes obligatory two-electron reductions of quinones, quinonimines, nitroaromatics, and azo dyes. NQO1 is a multifunctional antioxidant enzyme whose expression and deletion are linked to reduced and increased oxidative stress susceptibilities. NQO1 acts as both a tumor suppressor and tumor promoter; thus, the inhibition of NQO1 results in less tumor burden. In addition, the high expression of NQO1 is associated with a shorter survival time of cancer patients. Inhibiting NQO1 also enables certain anticancer agents to evade the detoxification process. In this study, a series of phytobioactives were screened based on their chemical classes such as coumarins, flavonoids, and triterpenoids for their action on NQO1. The in silico evaluations were conducted using PyRx virtual screening tools, where the flavone compound, Orientin showed a better binding affinity score of −8.18 when compared with standard inhibitor Dicumarol with favorable ADME properties. An MD simulation study found that the Orientin binding to NQO1 away from the substrate-binding site induces a potential conformational change in the substrate-binding site, thereby inhibiting substrate accessibility towards the FAD-binding domain. Furthermore, with this computational approach we are offering a scope for validation of the new therapeutic components for their in vitro and in vivo efficacy against NQO1. NQO1 is one of the two main quinone reductases in the mammalian system. The paralog of cytosolic protein NQO1 is NQO2. NQO1 was isolated in the late 1950s, identified in rat liver, and named DT-diaphorase (DTD) by Lars Ernster. It is highly inducible in rat liver by azo dyes and polycyclic aromatic hydrocarbons regulated by the Keap1/Nrf2/ARE pathway and plays various roles in cellular stress response, including oxidative stress . UtiliziNQO1 is a promising target for cancer detection, selective anticancer therapy, and provides multiple layers of protection to cells against carcinogenesis. NQO1 is involved in shielding cells from a range of harmful compounds, including quinones and other reactive oxygen species (ROS). Apart from this, it also plays a significant role in p53 stabilization . This enIn cancer cells, it is over expressed, encouraging cell proliferation, malignant transformation, and drug resistance. Hence NQO1 acts as both a cancer suppressor and tumor promoter  11]..11].The high amount of NQO1 in cancer cells is thought to help them cope up with oxidative stress, just as they do in healthy cells. Studies of transcription factor Nrf2, which controls NQO1 expression, have partially supported this hypothesis. Gain-of-function mutations in Nrf2 or loss-of-function mutations in Keep over-activate Nrf2 in many cancers, including lung, pancreas, liver, gall bladder, and ovarian cancers . Nrf2 geAyurveda, the Indian system of medicine, is one of the earliest systems of medical practice in health management and has played a vital role in providing health care service to human civilization from its inception. Medicinal plants are still the essential source of modern medicine. Additionally, the literature reveals that Indian traditional plants have high potent phytobioactive compounds for developing new drugs ,16. The The proposed study was primarily focused on quinones, but the chemical classes like flavones, curcumin, and coumarins were previously reported as the potent inhibitors for NQO1. The flavonoids are identified as a potent inhibitor for NQO1 through competitive inhibition of NAD(P)H ,21,22. TThe inhibitors of NQO1 have shown promising outcomes in certain aspects where dicoumarol, a competitive inhibitor of NQO1, potentiates cisplatin-induced apoptosis in p53 wild-type urogenital cancer cell lines . Meanwhill lines . Based oMedicinal plants rich in quinones, flavones, and terpenoids were noted and their phytoconstituents were shortlisted accordingly. A list of biologically phytobioactives is depicted in http://www.arguslab.com/, (accessed on 11 August 2021)) was used to improve the geometric augmentation of the ligands. The two-dimensional and three-dimensional structures of prepared ligands are shown in The selected ligands were downloaded from the NCBI, USA—PubChem chemistry database in three-dimensional form. The molecular docking software Argus Lab version 4.0.1  was retrieved from the Protein Data Bank (PDB). Furthermore, the non-standard amino acids bound to the downloaded NQO1 PDB file protein were removed or modified using Pymol software .http://sts.bioe.uic.edu/in, (accessed on 11 August 2021)).The binding pockets of NQO1(2F1O) protein-ligand binding were predicted using the in silico tool CASTp ). The Ramachandran plot was used to validate and evaluate the refined protein structures of NQO1, revealing that the energetically allowed protein structure of 2F1O regions for backbone dihedral angles toward amino acid residues were found. RAMPAGE was used to build the plots. The PROCHECK RAMPAGE results for the NQO1 revealed that its structure is stable, and the results are shown in The NQO1 (2F1O) protein structure was refined using the Mod Refiner server for protein refinement (The prix version 0.8 open access docking program was used for the better understanding of molecular interaction and the docking between the NQO1 protein and phytobioactives (ligands) were uploaded. The PDB format NQO1 protein was selected, the ligands were uploaded, and the grid box was marked to shield the active site residues, ready to be preferred binding residues to achieve the maximum orientation with the lowest binding affinity  values.The Ligplot+2.2 and the BIOVIA Discovery Studio Visualizer were used to visualize the docked conformation of the ligand against the NQO1 protein. The interactions like hydrogen bonds, hydrophobic interaction, and bond length were visualized using this program, including the NQO1 protein and a variety of plant-derived compounds with some naturally occurring quinones. The 3D Visualization was done with the BIOVIA Discovery Studio Visualizer, while the 2D interaction was done with Ligplot+2.2 ,27,28,29+ ion  and Cl−  for NQO1 was used. This step automatically neutralizes the charge of the system during the study of the protein dynamics when the ligand is competing at the binding site , (accessed on 15 August 2021) by using the entire MMFF94 force field to perform molecular mechanics calculations [The molecular energy, electronic density, and orbital energies of a particular system, including the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO) were determined using the Kohn-Sham (KS) approach ,34,35,36ulations ,46,47,48ulations  was consulations ,51,52 onulations ,65,66,67ulations  and the ulations  were conulations  in conneThe NQO1 protein structure validation was examined using RAMPAGE. The initial validation was 92.4% of residues in a favorable region, and thus the protein could be subjected to further docking.The consequences of this study can be deciphered from Molecular docking is a commonly used approach in drug discovery for drug discovery with little or no adverse effects. It is a two-step process that begins with geometrical optimization of the ligand and target biomolecule. The conformations in the receptor-identified active region are then ranked according to their score, which varies from one software to the next depending on the algorithms used to develop them. Among the selected compounds, Orientin was found to be more potent against NQO1. All the listed compounds in Using the Molecular Docking (in silico) technique, we analyzed the interaction between the NQO1 protein and a ligand molecule. Dicumarol is known as a potent (standard) inhibitory molecule for NQO1. The Dicumarol A moleculTo study the protein dynamics when the Orientin binds to NQO1, MDS studies were carried out to analyze the changes in protein during simulation for the docked protein-ligand complex. The Desmond equilibrates the system to achieve a stable conformation if the initial structure was energetically unstable. Hence in the present study, we performed MDS for docked complexes of Orientin and NQO1 . The pro to 50ns . The gra to 50ns . Thus, MThe calculated global reactivity descriptors ,41,42,43Because global hardness is a direct measure of electron density deformation and chemical reactivity, which is related to the HOMO-LUMO gap, it can be seen that Orientin will be the most reactive ligand, with Dicumarol and Melatonin being very similar in reactivity, and Trimethadione will be the least reactive of all the ligands considered. The electrodonating power EDP is more important than its electroaccepting EAP counterpart for all the ligands, which can be explained in terms of their molecular structures. However, when the values of EDP and EAP for each molecule are compared, it can be deduced that the Orientin and Dicumarol ligands will have considerably different reactivity than the other ligands. The electrophilicity ironment . Accordist case) ,73,74 anLocal reactivity descriptors have been developed in addition to global reactivity descriptors to gain a sense of the changes in chemical reactivity across the atoms inside the molecule. The Fukui functions ,38,39 anAlthough there is some overlap between the different sections inside the ligands, these graphical representations allow for a clear distinction between the locations within the molecules where the Dual Descriptor will be bigger or smaller than zero, signaling chemical reactivity differences.In summary, the phytoactive compounds such as Orientin, Trimethadione, Alliin, and some other potent plant-derived molecules from a range of chemical classes has shown better interaction towards the NQO1 protein when compared to the standard NQO1 inhibitor Dicumarol. Understanding the complex mechanism and functioning of NQO1 in cancer studies reveals that this protein exhibits as both tumor suppressor and tumor promoter under different tools. The molecular docking and molecular dynamics studies revealed that the phytobioactive, Orientin, exhibited higher interaction with NQO1. The Orientin is a flavone molecule with an anticancer property that also includes anti-inflammation, neuroprotective, radiation protective, vasodilation, cardioprotective, antiviral, antibacterial, antidepressant-like antiadipogenesis, antinociceptive, and various antioxidant properties . The res