Chronic kidney disease progression to ESKD is associated with a marked increase in mortality and morbidity. Its progression is highly variable and difficult to predict.This is an observational, retrospective, single-centre study. The cohort was patients attending hospital and nephrology clinic at The Canberra Hospital from September 1996 to March 2018. Demographic data, vital signs, kidney function test, proteinuria, and serum glucose were extracted. The model was trained on the featurised time series data with XGBoost. Its performance was compared against six nephrologists and the Kidney Failure Risk Equation (KFRE).A total of 12,371 patients were included, with 2,388 were found to have an adequate density (three eGFR data points in the first 2 years) for subsequent analysis. Patients were divided into 80%/20% ratio for training and testing datasets.ML model had superior performance than nephrologist in predicting ESKD within 2 years with 93.9% accuracy, 60% sensitivity, 97.7% specificity, 75% positive predictive value. The ML model was superior in all performance metrics to the KFRE 4- and 8-variable models.eGFR and glucose were found to be highly contributing to the ESKD prediction performance.The computational predictions had higher accuracy, specificity and positive predictive value, which indicates the potential integration into clinical workflows for decision support. Chronic kidney disease (CKD) is a major cause of morbidity and mortality globally, having a reported prevalence of 11–13% . Whilst Data-driven predictive modeling is a rapidly advancing field and has been employed in a range of clinical scenarios such as opioid overdose  and acutIn this work, we describe the training of a ML model that was capable of predicting which CKD patients will progress to ESKD within 2 years. We compare the predictive power of this model against the prospective predictions of six nephrologists in a cohort of fifty CKD patients. The predictions for ESKD occurring within a 2-year period were better than the most experienced clinician. The work here shows that predictive models built with machine learning can be accurate and have a potential role in providing decision support in a clinical environment.p-value of lesser than 10−8 for distribution membership. A total of 538 outlying points were flagged, out of 869,901 data points (0.062%).The raw clinical time-series data included much manually entered information, and included erroneous extreme outliers in some variables, particularly, height, weight, standing systolic pressure, standing heart rate and protein-creatinine. These were outliers detected and removed using chi-squared outlier tests with an inclusion threshold of 99.999999%, excluding points with a Out of 12,371 patients extracted 7,565 patients recorded an eGFR value. We filtered the primary data to remove patients with fewer than three pre-ESKD eGFR measurements. This filtering step retained 4,477 patients. Primary measures of creatinine level were excluded from subsequent study due to the output predictor, eGFR, being derived from it.TS) and the Test Timepoint (TT) values were chosen as a trade-off between accuracy and the clinical usefulness of the size of the prediction window. Here, TS = 2 and TT = 8 years provided the most clinically relevant predictor, with a large amount of useful training data and relatively high accuracy. With this selection of TS, we removed patients who had reached ESKD within 2 years of commencement of their data collection. This reduced our sample size to 2,388 patients.The Training Span  of the patients formed the training set and the remaining 20% (N = 478) was used for testing. To ensure that a similar proportion of ESKD patients were randomly included in each set, a two-sample test for equality of proportions with continuity correction was performed, which confirmed that the proportions were not significantly different .To ensure that the model was trained on distinct data to the test data, individual patients were fist randomly partitioned into two groups. 80% . Four case studies with varying degrees of Comprehensive featurisation was performed: zero , 2 (only eGFR and glucose), 6 , and 19 .Data from 19 different time-series from each of the 1,910 training and 478 test patients was featurised with the tsfresh v0.12.0 python library , using tnrounds, eta, max_depth, subsample, colsample_bytree). In order to properly handle the over 8-fold class imbalance between ESKD cases (N = 263) and non-ESKD cases , the metric for tuning and evaluating performance of the predictive models was changed from accuracy to Area Under the Precision-Recall Curve, AUCPR  run had a 10-fold CV mean MCC of 0.4303 with the corresponding best hyperparameter set: nrounds = 500, eta = 0.04, max_depth = 4, subsample = 1, colsample_bytree = 0.6. SHapley Additive exPlanation (SHAP) values .A model was trained on the featurised time series data for the 1,910 training set patients with XGBoost  version tunable e, AUCPR . Upon sue, AUCPR , indeed ) values  were cal) values . Our sofWe obtained pathology and clinical records for patients attending hospital and outpatient clinics at The Canberra Hospital Department of Renal Medicine, Australian Capital Territory, Australia. This included 12,371 patients spanning 21.5 years from September 1996 to March 2018. In addition to birth year, sex and date of death, this dataset consists of 17 time-stamped clinical measures including creatinine and derived eGFR , glucose , sitting and standing blood pressure , heart rate , weight , height  and derived Body Mass Index (BMI), HbA1c , urine protein-creatinine ratio  and 24-h proteinuria (883 points). All data was observed in a normal distribution with the exception of creatinine, eGFR, glucose, HbA1c, proteinuria and urine protein/creatinine ratio . In addiOut of 2,388 patients, 1,910 were used to train the model and 478 were used as a holdout test dataset. Patients baseline characteristics are shown in , initial predictive model, including potentially interacting and confounding variables (see Methods). Throughout this analysis, we defined ESKD as the composite endpoint of an eGFR below a threshold of 10 mL/min/1.73 m2 , of 2 years to predict if the patient would reach ESKD by a test timepoint (TT) of 8 years. The initial model incorporated all 19 time series measures present in the primary data and this was augmented with full featurisation derived from these timeseries (see Methods). When predicting if ESKD would occur by 8 years follow-up, the initial model had a prediction accuracy for ESKD of 84.5% with a sensitivity and specificity of 55.8 and 88.0%, respectively.We aimed to develop a model capable of predicting whether a patient would reach ESKD and to estimate the timeframe in which this would occur. We first aggregated individual patients' clinical measurements into a single/1.73 m2  or the cinitial model, SHapley Additive exPlanation (SHAP) values (To understand the relative contribution of each feature in the ) values  were calFor the prediction of ESKD, as expected eGFR-based features  had the highest contributions, comprising six of the top 10 features . Other qoptimized model utilizing all 19 time-series measures but only comprehensively-featurised two timeseries values: that of eGFR and glucose, which were featurised each into 764 numerical features. We then only minimally-featurised the remaining 17 timeseries values . In predicting ESKD at 8 years on 2 years of patient data, this optimized model improved prediction accuracy from 84.5 to 86.2%. Sensitivity improved from 55.8 to 65.4% and specificity from 88 to 88.7%. The most predictive quantities identified from this optimized model are only slightly different   developed ESKD within 8 years. In contrast, of the 388 patients with presenting eGFR of at least 30 ml/min/1.73 m2, only 6.7% developed ESKD.To contrast optimized model performance by disease severity, we grouped patients in the test set by their eGFR at presentation  . Out of The model's accuracy in predicting ESKD in the former group was 67.8% . In contrast, the accuracy of the model in the latter group was 90.4% . These numbers highlight the sensitivity-specificity trade-off, which our single optimized model attempts to finely balance , but also suggests room for stratified ensemble models for future work.Forty-nine patients with CKD at risk of ESKD were selected as a test cohort. We considered the risk of ESKD at 2 years to be of greater clinical significance for RRT planning. Therefore, 2 years of clinical data was utilized by ML algorithm to provide a probability of reaching ESKD within the next 2 years. Six consultant nephrologists were given baseline demographics and diagnosis, medical comorbidities, proteinuria, blood pressure, and eGFR measurements over 2 years for the same cohort. The nephrologists predicted prospectively for each individual, within a 10-year window, the number of years  it would likely take for the patient to reach ESKD. The ML algorithm and clinician predictions were compared to the outcomes in the 49 patients at the 2-year mark (the time of writing). The variability in the clinician predicted ESKD date for each patient was high . As expeThe Kidney Failure Risk Equations (KFRE)  have beeLike many other clinical predictive settings, one of the main challenges in building a reliable model was its class imbalance, e.g., in the clinician test cohort, only 5 out of the 49 patients actually reached ESKD, and even a null-predictor that predicts that none would reach ESKD would achieve a misleading accuracy of 89%. Commonly, F1 score  has been used to provide a fairer measure of predictor performance, but recently the Matthews correlation coefficient has been shown  to be beF1 = 0.444, MCC = 0.408) and the better of the KFRE predictors .Revisiting our results , 4 showsoptimized model, comprising values from 19 dynamic measures including two fully-featurised time-series (eGFR and blood glucose), is able to predict the incidence of ESKD within an 8-year timeframe with an accuracy of 86.2%.The prediction of hard outcomes of CKD such as death and ESKD is often inaccurate, especially progressing toward RRT , 30. Her2 (stage 3A CKD) below which SHAP values increase sharply , a strategic initiative of the Australian National University, which aims to transform healthcare by developing new personalised health technologies and solutions in collaboration with patients, clinicians, and health care providers.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.


House dust, well known for causing allergy, contains chemicals that are harmful to fetal neurodevelopment. However, whether countermeasures for house dust allergy, such as frequent use of vacuum cleaners, frequent airing of futons, and the usage of anti-mite covers during pregnancy, are related to subsequent reduced risk of infant developmental delay remains unknown. Therefore, we examined this association by analyzing 81,106 mother-infant pairs who participated in a nationwide birth cohort in Japan. Infant developmental delays at 6 and 12 months postpartum were assessed using the Ages and Stages Questionnaire, Third Edition. A generalized linear model analysis was used to derive adjusted odds ratios (AORs) with the lowest care frequency as reference, while controlling 22 covariates. Our analysis showed that the above-mentioned cleaning measures were overall associated with a reduced risk of developmental delays, both at 6 and 12 months postpartum . Additionally, risks tended to decrease with an increase in the cleaning frequency. In conclusion, a negative dose-response association existed between these measures during pregnancy and infant developmental delay. Our results identify a potential role of frequent vacuum cleaning, airing bedding, and usage of anti-mite bedding covers in promoting intact infant development. House dust has been identified as one of the major causes of allergy. It contains several allergens such as pollen, mites, human and pet skin that mites feed on, and pet hair . ExistinBesides allergens, house dust also contains various chemicals such as lead , di, a nationwide birth cohort with >80,000 records while controlling for possible confounders such as basic characteristics and dwelling environment. We hypothesize that frequent floor and bedding cleaning activities are related to a reduced risk of developmental delay in infants.The JECS is a nationwide, government-funded birth cohort study investigating the impact of various environmental factors on children’s health and development. In the JECS, 103,062 pregnancies were registered between January 2011 and March 2014 via face-to-face recruitment at public agencies across 15 regions, including both rural and urban locations throughout Japan . The sample size was determined in advance to maintain adequate statistical power enough to detect rare diseases or conditions such as with ≤1% prevalence. The eligibility criteria for participants (expecting mothers) were as follows: (1) Residence in the study areas at the time of recruitment and expected to reside continuously in Japan for the foreseeable future; (2) expected delivery date between 1 August 2011 and mid-2014; (3) ability to comprehend the Japanese language and complete the self-administered questionnaire. Those residing outside the study areas, even if they visited the cooperating health care providers within the study areas, were excluded from the study. Follow-ups were primarily conducted via scheduled in-hospital check-ups until 1 month postpartum and via mailed letters at 6 and 12 months postpartum. Data were acquired using self-administered questionnaires or transcriptions of medical records recorded by physicians, midwives, nurses, and research coordinators.®, Third Edition (ASQ-3) , carpet on tatami, wooden flooring/tiles, carpet on wooden flooring/tiles, or other), having a pet (yes or no) [Unless otherwise specified, covariates were measured during the second or third trimester. We used the following variables as covariates for mothers: maternal age , pre-pregnancy body mass index asked during the first trimester  ,21,22, s12, ≥13) ,24, maris or no) , age of s or no)  and is s2) test. Given our large sample size and that minor differences could be easily detected, we relied not only on the p-value but also on the effect size Cramer’s V, the values being independent of the sampling size [Background differences according to the exposure variables were examined using a chi-squared  and their 95% confidence intervals (CIs). Exposure variables were: (1) Frequency of cleaning the living room floor with a vacuum cleaner, (2) frequency of cleaning the futon, (3) frequency of airing the futon, and (4) anti-mite cover use. The lowest category in each variable and non-use of anti-mite covers were set as the reference. Outcome variables were the total number of screen-positive cases (ranging from 0 to 5) each at 6 and 12 months postpartum. For example, if an infant falls in cases of communication, fine motor, and problem solving, the total number equals 3. In addition, ORs and CIs of screen-positive cases in each developmental area; (a) communication, (b) gross motor, (c) fine motor, (d) problem solving, (e) personal-social (in ASQ-3), were also calculated. The forced entry method was used to include covariates in the multivariable analysis.SAS 9.4  was used for all statistical analyses.n = 2032), number of years living in the current place of residence , physical activity , average number of hours spent outdoors , and annual household income . Each score of the five areas of the ASQ-3 had a <5.95% (max n = 4820) missing-data rate at 6 months and <10.51% (max n = 8518) at 12 months.Of the 81,106 mother-infant pairs included in the study, the missing-data rate was ≤1% for all covariates except for parity  years, mean body mass index before pregnancy was 21.2 ± 3.24 (SD), 43.3% of the mothers were primipara, 35.1% had less than 12 years of education, 39.5% had an annual household income of less than four million Japanese yen, 4.0% were current smokers, and 2.7% were current alcohol drinkers during pregnancy. The basic characteristics of mothers and dwelling environments during pregnancy are presented in The top three covariates related to each exposure were as follows: not employed (Cramer’s V = 0.297), multipara (V = 0.283), and longer hours spent outdoors (V = 0.085) for vacuum cleaning of the living room ; usage oThe frequency of each exposure measure is listed in All generalized variance inflation factors were below 2.45, and therefore, no multicollinearity was detected among any of the covariates.p-values for trend were <0.001.The mean and standard deviation of the total numbers of screen-positive cases over the five developmental areas were 0.116 ± 0.106 (SD) at 6 months and 0.222 ± 0.273 (SD) at 12 months. The numbers, crude, and adjusted ORs, according to the exposure variables, are shown in The prevalence, as well as crude and adjusted ORs for developmental delay in each developmental area at 6 and 12 months postpartum, according to the exposure variables, are presented in n > 80,000) while controlling for up to 22 potential confounders with a low level of multicollinearity. Our analyses revealed that adjusted ORs of these cleaning measures for house dust removal, the frequent use of vacuum cleaners, frequent airing of futons, and the usage of anti-mite covers during pregnancy, in screen-positive cases of developmental delay, as per the ASQ-3, were significantly lower compared to the references (which were set to the lowest frequency). This finding remained significant across five areas both at 6 and 12 months postpartum. In addition, the adjusted ORs tended to decrease with an increase in cleaning frequency. Therefore, we found a negative dose-response relationship between house dust avoidance during pregnancy and infant developmental delay.To our knowledge, this is the first study to examine the prospective association of home cleaning measures for house dust avoidance during pregnancy, including frequent vacuum cleaning, airing bedding, and usage of anti-mite bedding covers, with subsequent infant development until 1 year of age. To this end, we used a nationwide birth cohort study dataset (Fetal exposure to PAHs that are typically found in house dust is associated with decreased birth weight and smaller head circumference  as well It should be noted that conclusions regarding causality cannot be drawn from our findings. Consequently, the observed relationships do not necessarily guarantee that increasing the frequency of cleaning or airing, or introducing anti-mite covers will always prevent infants from developmental delay. In fact, the factors associated with reduced risks for developmental delay through these interventions remain unknown. Thus, further studies conducting randomized controlled trials to address causality, and in-depth studies, such as elucidating factors associated with reduced risk for developmental delay in relation to these interventions and sampling the infant and mother’s blood for known household neurotoxins, are warranted.Frequent cleaning of the floor with a vacuum cleaner and airing of the futon were strongly associated with multipara and unemployment. Mothers with young children are typically required to clean frequently, and being unemployed is virtually equal to being a full-time homemaker in Japan, this allows for daily cleaning and airing of futons. Since working mothers are less frequently at home compared to unemployed mothers, this may reduce the time that can be dedicated to cleaning. Interestingly, a history of maternal allergy was not strongly related to the frequency of these cleaning behaviors, even anti-mite cover use. Thus, the motivation for these living environment cleaning behaviors seems to be based on the natural necessity and capacity in daily life, though the cleaning tasks do not have to be performed by the mother, but by any domestic partner/husband, care taker, home helper, or older child.The study has two important strengths. First, we included data from an ongoing birth cohort with a large sample of ≥80,000 recruited on a nationwide scale between 2011 and 2014. Therefore, the sample could be regarded as representative of recent Japanese mothers. Second, although we followed expectant mothers from early pregnancy to 1 year after childbirth, a relatively high response rate was maintained. Consequently, selection bias caused by loss to follow-up was probably small.The are several limitations to this study. First, we asked only about the frequency of cleaning using vacuum cleaners or airing and did not include other means of removing house dust, such as sweeping or wiping the floor with a broom or a dust cloth. Thus, answering “almost never or never” to the question on the frequency of cleaning with a vacuum cleaner did not necessarily indicate a higher level of house dust. Second, we did not ask about manufacturer names, even though it is known that different vacuum cleaners vary in their cleaning performance, and this may have affected outcomes . Third, Our results derived from 81,106 mother-child pairs indicate a potential role of house dust avoidance, such as frequent vacuum cleaner use, frequent futon airing, and use of anti-mite covers, in promoting intact infant development. This study provides a rationale for conducting randomized controlled trials to address the effect of these measures on inhibiting developmental delay, as well as for in-depth studies elucidating the factors associated with a reduced risk of developmental delay in relation to these home cleaning measures.


Sulfur, an essential mineral element for animals, mainly exists in the form of organic sulfur-containing amino acids (SAAs), such as cystine, methionine, and cysteine, within the body. The content, form, and structure of sulfur play an important role in determining the wool fiber quality. In addition, keratin-associated proteins, one of the most crucial wool fiber components, are rich in SAAs. However, sulfur metabolism from the blood to the skin and hair follicles remains unclear. In this study, we analyzed high-sulfur protein gene and sulfur metabolism genes in the cashmere goat and explored the effects of melatonin on their expression. In total, 53 high-sulfur protein genes and 321 sulfur metabolism genes were identified. We found that high-sulfur protein genes were distributed in the 3–4 and 144M regions of chromosome 1 and the 40–41M region of chromosome 19 in goats. Moreover, all year round, allele-specific expression (ASE) is higher in the 40–41M region of chromosome 19 than in the other regions. Total of 47 high-sulfur protein genes showed interaction with transcription factors and cofactors with ASE. These transcription factors and cofactors were inhibited after melatonin implantation. The network analysis revealed that melatonin may activate the sulfur metabolism process via the regulation of the genes related to cell energy metabolism and cell cycle in the skin, which provided sufficient SAAs for wool and cashmere growth. In conclusion, our findings provide a new insight into wool growth regulation by sulfur metabolism genes and high-sulfur protein genes in cashmere goats. Cysteine is also the main component of keratinized proteins in feathers, and in birds, the demand for cysteine is fulfilled via the transsulfuration pathway. Inhibition of the transsulfuration pathway can affect hair follicle development, skin thickness, and growth in broilers; moreover, this inhibition is associated with the upregulation of hepatic cystathionine synthase and cystathionine lyase mRNAs , differentially expressed genes (DEGs), methionine, and cysteine play an important metabolic and functional role in animals. In a study, SAA supplementation was found to significantly increase wool production and sulfur concentration in wool . Moreovese mRNAs . Silva ese mRNAs .Sulfur metabolism is interwoven through various life processes, where it contributes to their vitality. Ongoing studies on the role of sulfur in goat wool fiber growth are mainly focused on sulfur’s nutritional effect, its suitable dietary level, and the nitrogen–sulfur ratio, but without much attention to the underlying molecular mechanisms. To date, the knowledge on the expression pattern of sulfur metabolism genes in cashmere goats remains limited. In this study, we hypothesize that sulfur metabolism genes interact with melatonin to regulate villus growth. Here, high-sulfur protein family genes, sulfur metabolism genes, and melatonin in cashmere goats are used as entry points to study the important genes involved in the regulation of villus growth, eventually elucidating the underlying signal transduction networks.n = 3 in each group): melatonin implantation (treatment group) and melatonin non-implantation (control group). In total, we collected 72 blood and 72 skin samples over 12 months (12 each from each goat).Six 16-month-old female cashmere goats from Hanshan, Inner Mongolia were included in this study. They were divided into two groups (2) and venous blood samples were collected and quickly frozen and stored at -80°C in liquid nitrogen. The melatonin concentration in plasma were tested  and secondary follicles (SFs) were collected in each month from four female cashmere goats from Inner Mongolia. PF samples were collected from 12 months of the year and SF samples were collected from August to February of the following year. These PFs and SFs were plucked from the side of the torso of the goats at the beginning of each month. First, the cashmere and wool were separated by careful observation, and then the cashmere and wool were quickly pulled out from the torso of the goats. Use chloroform sterilized scissors to cut the hair follicles 1 cm away from the root of the cashmere and wool. The PF and SFs were immediately frozen without any chemical solutions in liquid nitrogen for storage and transported until RNA isolation. In total, 12 PFs and 8 SFs samples were collected.g, for 10 min at 4°C. The top layer was collected after chloroform was added. A 1:1 V of isopropanol was added to each tube before being centrifuged at 12,000 g for 10 min at 4°C. The supernatants were discarded and the remaining pellets were washed with 1 ml of 75% ethanol and centrifuge at 7500 g for 5 min. All of the residual ethanol was then removed and the pellets were allowed to air dry for 4 min. The RNA samples were then redissolved in 30 μl of DEPC (Diethyl Pyrocarboanate) treated H2O and stored at –80°C. After the RNA samples were quantitated, mRNAs was enriched with oligo (DT) magnetic beads. Then, the fragment buffer was added to break the mRNA into shorter fragments. We used the mRNA as the template, single-stranded cDNA was synthesized using a randomized hexamer, followed by the synthesis of double-stranded cDNA by adding buffer, dNTPs, DNA polymerase I, and RNase H. The double-stranded cDNA was then purified using AMPure XP beads, and then the second strand of cDNA containing U was degraded using USER enzyme. Purified double-stranded cDNA was then repaired at the end, followed by the addition of a tail and connection to the sequencing connector. Next, AMPure XP beads were used to select the fragment size. Finally, the final library was obtained via PCR amplification and purification. After RNA-seq library construction, Qubit 2.0 was used for preliminary quantification, followed by library insert size detection using Agilent 2100. After the inserts met the expectations, Q-PCR was used to accurately quantify the effective concentration of the library and ensure the quality of the library. In total, 72 libraries from skin samples and 72 libraries from blood samples have been constructed.Total RNA was extracted from all blood and skin samples using TRIzol reagent (TaKaRa), according to the manufacturer’s instructions. The blood and skin samples at –80°C were thawed . 1 ml TRIzol reagent was added and thoroughly mixed. The supernatants were obtained after the samples were centrifuged at 12,000 Total RNA used for constructing an Iso-Seq library: mRNA was reverse-transcribed and then amplified using SMARTer PCR cDNA Synthesis Kit and PrimeSTAR GXL DNA Polymerase. The subsequent library construction was performed using the SMRTbell Template Prep Kit 1.0, and its library sequencing was performed on a PacBio RS II. The full-length transcriptome was analyzed using SMRTLink; it mainly included three stages: CCS acquisition, classification, and clustering. We used the Quiver algorithm in combination with the non-full-length sequences to polishing the consensus isoforms obtained through clustering and screen high-quality (HQ) and low-quality (LQ) sequences for subsequent analysis. Based on the full-length transcripts after polishing (HQ + LQ), the sequences were aligned to the reference genome (Capra hircus ARS1) by using the alignment software program Gmap . Only thARS1)  base present in any sequencing read is >50% of the base number of the read, the paired reads should be removed. We used HISAT to match the read segments to the reference genome, which was obtained from the goat reference genome and its corresponding genome annotation file in the NCBI database (ARS1) . DEGs weARS1)  with defIn the control group, 86 sulfur metabolism genes were differentially expressed in the blood and skin, whereas 4839 genes were coexpressed. A coexpression network was thus constructed using the weight gene coexpression network analysis (WGCNA)  in R pacBAM files were single-nucleotide polymorphisms (SNPs) called with samtools to generate BCF files, which were converted to VCF files using bcftools, containing genotype data for individuals. Heterozygous SNPs in an individual were filtered with the criteria that the individual’s genotype was heterozygous, the coverage was >30 in both REF and ALT reads for both individual and SNP sites in a month, and finally, high confidence expressed SNPs were obtained.P-value of ≤0.05 was considered to indicate significance.Next, for ASE, we defined a haplotype concordant with the reference genome as ref allele and its homologous chromosome as ALT allele. The probability of occurrence of each allele was calculated via binomial distribution. A corresponding To run a complete search for identifying high-sulfur genes in the goat genome, two methods were used: We first searched the high-sulfur for protein sequences based on the published high-sulfur protein sequences in human  and sheeKRTAP5, KRTAP23, and KRTAP12 subfamily. In contrast, 7, 4, 4, 14, 6, 12, 2, 5, 1, 1, 1, and 2, high-sulfur proteins were found to belong to the KRTAP1, KRTAP2, KRTAP3, KRTAP4, KRTAP9, KRTAP10, KRTAP11, KRTAP13, KRTAP15, KRTAP16, KRTAP17, and KRTAP24 subfamilies, respectively.Single-molecule long-read sequencing of hair type in cashmere goat transcriptome data was used to complement and correct the 35 high-sulfur protein gene sequences in the cashmere goat. Consequently, 35 high-sulfur protein genes with relatively complete and accurate sequences were finally obtained . To clasKRTAP10. Moreover, motifs 4, 13, 1, and 8 were conserved in KATAP9, KATAP1, KRTAP3, and KRTAP13, respectively  with coordinated amino acid expression patterns. Here, we divided the 9355 CDS (Cys ≥ 3) of the goat reference proteome into six modules. The number of CDS in each module was 1724, 6437, 621, 428, 69, and 76, and a total of 5160 genes were annotated. Of these, 52 high-sulfur protein genes were annotated in the yellow-green module, and the protein amino acid expression pattern was found to be different from that of other genes. Moreover, cysteine abundance was noted to be highest, followed by that of serine .P = 0.008; Gene expression profiling using RNA-seq and genotyping were performed on data. We identified 172771 SNPs. We further counted the number of ASE per 1M region of chromosome 19. Moreover, TBtools  was usedAfter melatonin implantation, the expression of 26 transcription cofactors and 22 transcription factors changed, all of which showed higher expression levels in the control group from August to December. Moreover, the expression pattern changed in the treatment group, with elevated expression levels in May and April .P ≤ 0.01; ELF5 (the key gene of the regulatory network) were noted to constitute a regulatory network. Pearson’s correlation analysis revealed positive regulatory relationships between the high-sulfur proteins and all the transcription factors and cofactors.In total, 47 high-sulfur protein genes were used to construct a coexpression network using Pearson’s correlation with a cutoff , with 3,715 genes upregulated in the skin and 2,612 genes upregulated in the blood. The number of upregulated DEGs in the treatment group was 3634 in the skin and 2607 in the blood.We then analyzed the changes in gene expression associated with treatment and control for different tissues (blood vs. skin). There were 6,327 DEGs in the control group  was then used to find enriched GO gene-sets upregulated in the blood and skin of the treatment and control groups. The results were generated after scoring DEGs using the Signal2Noise statistic. Only gene-sets that passed conservative significance thresholds (FDR < 25%) were selected for display in the Enrichment Map; consequently, 780 and 504 gene-sets were enriched in the skin and blood of the control group, respectively, and 824 and 504 gene-sets were enriched in the skin and blood of the treatment group, respectively. Of all the pathways enriched by the DEGs in control (blood vs. skin) and treatment (blood vs. skin) groups, 522 functional pathways of cell development regulation were strongly correlated with each other; moreover, these pathways were correlated with the WNT canonical pathway, BMP signaling pathway, cell motility, kinase activity, and muscle tissue development activity . This twTo understand the tissue-specific expression patterns of all genes, we clustered all their expression patterns (4389 genes) by using WGCNA. We identified three main gene modules, where the turquoise modules represent the tissue-specific expression patterns , includiGene set enrichment analysis  and EnriThe enriched pathways in blood appeared in most functional groups (blue nodes), indicating that the response of blood to the expression of genes with tissue-specific expression patterns was relatively strong. The immune-related pathways were significantly upregulated in the blood, whereas the oxidation-related pathways were significantly upregulated in the skin.P < 1e–20), indicating that the module obtained via WGCNA was high reliable . Many of the gene-sets upregulated only in the skin were associated with amino acid metabolism, sulfur compound biosynthesis, and transport kinase function. The tissue-specific modules were positively or negatively correlated with 86 sulfur metabolism genes, with a correlation coefficient of >9.8; moreover, the correlation coefficient of individual module was 0.99   was usedol group . Based oub genes —all of wVAT1—highly expressed specifically in skin tissue after the implantation of melatonin—is mainly involved in the oxidation–reduction process, and regulates 11 genes involved in fatty acid metabolism . There are four genes involved in cysteine and SAA metabolism . Among these, CTH interacts with PSMD12, CDO1 with ST6GALNAC2 and SLC25A4, AHCY with YWHAE and TUBA1C, and MAT1A with RAD23B. After melatonin implantation, PSMD12, ST6GALNAC2, SLC25A4, TUBA1C, and RAD23B were upregulated in the skin. The function of these genes was most related to cell energy metabolism and cell cycle.Although we constructed coexpression modules between the tissue-specific genes and sulfur metabolism genes, the specific biological function of these genes was unknown. By using Database for Annotation, Visualization and Integrated Discovery  , we perfKRTAP4 and KRTAP5 are the ultra–high-sulfur protein genes, containing eight members in total: KRTAP4-1, KRTAP4-2, KRTAP4-3, KRTAP5-1, KRTAP5-2, KRTAP5-3, KRTAP5-4, and KRTAP5-5  are higher in sheep than in cattle, horses, and dogs (P ≤ 0.01) interactions were noted between 47 high-sulfur protein genes and transcription factors and cofactors with ASE. However, the ASE of these transcription factors and cofactors was inhibited by melatonin. Thirteen genes in the interaction network were functionally associated with the functional of hair growth.Significant  and type 2 (MT2) receptors] and nuclear receptors  . MT1 and vessels . In contere goat .Based on the overall panoramic regulatory network, we concluded that melatonin affects the pathways involved in the DEG between blood and skin, and the pathways demonstrated spatially specific upregulation trends; for example, after melatonin implantation, the ErbB signaling pathway was found to be upregulated only in the blood from control cashmere goats, whereas only the Notch signaling pathway was noted to be upregulated only in the treated cashmere goat skin.In total, 53 high-sulfur protein genes and 321 sulfur metabolism genes were identified and further analyzed for their expression in the skin and blood tissue. These high-sulfur protein genes are distributed in the 3–4M and 144M regions of chromosome 1 and the 40–41M region of chromosome 19 in goats. We found that ASE in the 40–41M region of chromosome 19 was significantly higher than that in the other regions over the entire study year. In total, 47 high-sulfur protein genes were found to interact with ASE transcription factors and cofactors. The ASE transcription factors and cofactors were inhibited after melatonin implantation. We also constructed a regulatory network of tissue-specific genes and sulfur metabolism genes and found that melatonin possibly activates the sulfur metabolism process by regulating the genes related to cell energy metabolism and cell cycle in the skin. This can provide sufficient SAAs for cashmere wool growth. Our findings offer a new insight into wool growth regulation by sulfur metabolism genes and high-sulfur protein genes in cashmere goats.CRA004599 and CRA004598 that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.The datasets analyzed for this study can be found in the Genome Sequence Archive in BIG Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession numbers The animal study was reviewed and approved by the Special Committee on Scientific Research and Academic Ethics of Inner Mongolia Agricultural University [Approval No. (2020)056].YC: writing–original draft preparation and writing–reviewing and editing. WZ: supervision and project administration, and writing–reviewing and editing. YS, BL, LG, ZL, LZ, LD, and CJ: experimental sample and data analysis. CL: funding acquisition. YS: contributed as co-author. All authors contributed to the article and approved the submitted version.BL was employed by the company Nei Mongol BioNew Technology Co. Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Acupuncture is a commonly used complementary treatment for flaccid hemiplegia caused by stroke, but evidences from previous randomized trials were inconclusive. The purpose of this study was to evaluate the efficacy and safety of acupuncture in a comprehensive synthesis.  We searched literature from eight databases from their inception to December 2020. We included randomized controlled trials of acupuncture for the treatment of flaccid hemiplegia following stroke. The meta-analysis was carried out using Review Manager 5.3 and Stata 16.0. The main indicator was the Fugl-Meyer Assessment scale. The modified Barthel Index scale, Quality Of Life Assessment scale, Mini-Mental State Examination scale, Berg Balance Scale, Neurological Deficit Assessment scale, and the treatment effective rate were used to measure the secondary indicators. Adverse events from individual studies were used to determine safety. P < 0.01); acupuncture plus rehabilitation compared with rehabilitation, 9.84 ; and acupuncture plus Western medicine therapy compared with Western medicine, 16.86 , and the secondary indicators showed the same tendency.  Our search returned 7624 records, of which 27 studies involving a total of 1,293 patients fulfilled our inclusion criteria. To be noted, our results indicated that significant improvements in the scores of the primary indicator showed better clinical scores among the three groups with acupuncture than without acupuncture: acupuncture compared with rehabilitation, 13.53 (95% CI 11.65–14.41,  Acupuncture was effective and safe in the patients with flaccid hemiplegia after stroke, although there was high heterogeneity between studies. This systematic review offered a thorough assessment of the efficacy and safety of acupuncture treatment for individuals with flaccid hemiplegia following a stroke. Compared with the nonacupuncture group, the scores of the acupuncture group were significantly improved in the following aspects: motor function, ability of daily living, cognitive function, quality of daily life, neurological deficit, and the clinical effective rate, indicating that acupuncture was effective and short-term safe for patients with poststroke flaccid hemiplegia.The comprehensiveness and methodological quality of the major studies included in this evaluation have a substantial impact on the results' trustworthiness.This systematic review's protocol has been registered in PROSPERO, and it has been conducted and reported in complete compliance with PRISMA guidelines. However, selection bias may exist as we only searched the English and Chinese databases.Stroke is regarded as the leading cause of death and disability worldwide.  HemiplegAt present, the main treatments for poststroke flaccid hemiplegia include exercise rehabilitation training , noninvasive brain stimulation  and transcranial magnetic stimulation (rTMS)), and physical therapy modalities ). However, there are certain disadvantages to these treatments. In terms of unpleasant symptoms, continuous passive motion is preferable to a therapist-directed range of motion exercise . While the passive range of motion may help with brain healing, its capacity to cause significant motor alterations is debatable –6. The iAcupuncture has been practiced clinically in China for more than 3000 years and has been widely used to treat various diseases, especially in stroke rehabilitation treatment ,13. AcupDespite the numerous clinical researches on acupuncture, many professionals continue to have reservations regarding its efficacy and safety. Hence, the goal of this systematic review and meta-analysis is to determine the effectiveness and safety of acupuncture treatment for flaccid hemiplegia following stroke.The protocol of this systematic review was developed and submitted to PROSPERO, and the registration number is CRD42020180411.A systematic search was conducted in the following databases: China Biology Medicine (CBM), China National Knowledge Infrastructure (CNKI), Wan Fang Data, the Chinese Science and Technology Periodical Database (VIP), PubMed, EMBASE, the Cochrane Library, and Web of Science from the inception to December 2020. All searches were performed by a biomedical information specialist of the medical library, with an exhaustive set of search terms related to “acupuncture,” “stroke,” “randomized controlled trial,” and “flaccid hemiplegia.”Studies were included if they fulfilled the following criteria: (1) randomized controlled trials (RCTs); (2) patients were diagnosed with flaccid paralysis after stroke, regardless of the type of stroke (hemorrhagic stroke or ischemic stroke), the diseased brain area, the affected limb, gender, age, ethnicity, and nationality; and (3) acupuncture used as an intervention , regardless of the number and duration of the treatment. Studies were excluded if they did not conform to the study type  and had inadequate information.The primary outcome measure was assessed by the Fugl–Meyer Assessment (FMA) scale, which was usually used to assess motor function. The secondary outcomes included modified Barthel Index (MBI) scale, Quality of Life (QOL) Assessment scale, Mini-Mental State Examination (MMSE) scale, Berg Balance Scale (BBS), Neurological Deficit (ND) Assessment scale, or other scale data related to flaccid hemiplegia. Safety was evaluated by any recorded adverse events reported by individual study.Two independent reviewers (YT and JW) independently screened the literatures according to the predefined inclusion criteria based on the title and abstract. The two examiners then separately reviewed the full text based on the retrieved results. The included studies were cross-checked by two reviewers. Disagreements were resolved through discussion or consensus with third reviewers (WP). The research selection process is shown in the PRISMA flow chart in The two authors conducted an independent assessment of the risk of bias using the Cochrane risk-of-bias tool to assess the methodological quality of the included studies. There were seven items in total, and each item was determined to be one of the following: “low risk of bias,” “unclear risk of bias,” and “high risk of bias.” If all seven items were assessed as having a low risk of bias, the study was rated as high quality. If one or more items were assessed as having a high or unclear risk of bias, the study was rated as low quality . Continuous outcomes were evaluated using mean difference (MD) or STD mean difference (SMD), while the dichotomous outcomes were evaluated by risk ratio (RR) or odds ratio (OR). Statistical heterogeneity was assessed by A total of 7624 records were obtained from the nine databases. After removing the duplicates, 2962 articles were left. A total of 2848 articles were then excluded because they did not meet the inclusion criteria. As a result, 27 randomized controlled trials –46 were The review and meta-analysis included 27 studies involved 1,293 patients, of which 648 were in the trial group (381 men and 267 women) and 635 were in the control group (372 men and 263 women). All studies were conducted and published between 1999 and 2019. Regarding the sample size, the trials included a maximum of 146 people and a minimum of 40 people.For intervention, one study adopted acupuncture, sixteen studies adopted acupuncture combined with rehabilitation, four studies adopted electroacupuncture combined with rehabilitation, one study adopted scalp acupuncture combined with rehabilitation, one study adopted electroacupuncture, one study adopted electroacupuncture combined with Western medicine, and three studies adopted acupuncture combined with Western medicine. The detailed intervention characteristics of the included literatures are summarized and listed in In all the involved studies, seventeen studies used FMA scale to assess limb function, and eighteen studies used MBI scale to assess daily living ability. Eight studies use ND scale to assess neurological deficit. One study used MAS scale to assess spasticity. To evaluate the quality of daily life, three studies adopted the QOL scale, while two studies adopted BBS to value the balance function.P < 0.01).One study  includinP < 0.01); 32.21 ; 3.71 ; and 29.42 , respectively.One study  containiTwenty-one studies ,44–46 re(1) FMA and Subgroup Analysis of Interventions for FMA. Eleven studies [P < 0.01, I2 = 98%). studies ,39,44–46P < 0.01, I2 = 98%). Eight studies [P < 0.01, I2 = 98%); two studies [P=0.03, I2 = 97%); and one study [P < 0.01).Subgroup analysis showed that the rehabilitation combined with acupuncture regardless of its subtypes ameliorated more than the rehabilitation treatment alone in the scores on the motor function of the limbs assessed by FMA scale. After combining effect size, the MD value was 9.17 . Through sensitivity analysis, no obvious source of heterogeneity was found. After meta-regression analysis, it was found that the sample size was a source of heterogeneity. Egger's test indicated that there was no obvious publication bias (P=0.058) in After subgroup analyzing and combining data, substantial heterogeneity was observed ((2) MBI and Subgroup Analysis of Interventions for MBI. Twelve studies [P < 0.01, I2 = 96%). studies ,41,44,45P < 0.01, I2 = 94%). One study [P < 0.01, I2 = 90.5%). After sensitivity analysis, two studies [I2 = 84%, P < 0.01). Meta-regression analysis of publication year, sample size, and intervention methods were not associated with heterogeneity. Egger's test indicated that there was no obvious publication bias (P=0.964) in Subgroup analysis showed that the acupuncture combined rehabilitation group and the electroacupuncture combined rehabilitation treatment group were superior to the simple rehabilitation treatment group in improving the scores of the ability of daily living. Eleven studies ,39,41,45ne study  adopted  studies ,44 were (3) The Effective Rate. Four studies [P < 0.01, I2 = 0%). It indicated that the effective rate of acupuncture combined with rehabilitation group was higher than that of the simple rehabilitation group. studies ,35,41,45(4) ND. Four studies [P=0.93, I2 = 95%). studies ,35,39,41P=0.01, I2 = 88%).After sensitivity analysis, one study  was the Four studies ,21,29,40(1) FMA. Four studies [P < 0.01I2 = 38%). studies ,21,29,40(2) MBI and Subgroup Analysis of Interventions for MBI. Three studies [P < 0.01, I2 = 75%). studies ,21,40 usP < 0.01, I2 = 69%). One of them [P < 0.01).Two of them ,40 were  of them  was the P=0.02, I2 = 75%). After sensitivity analysis, it was found that one study [I2 = 27%, P < 0.01).After merging the subgroup analysis data, it suggested that there was still a high degree of heterogeneity (ne study  was the (3) ND and Subgroup Analysis of Interventions for ND. Three studies [P < 0.01, I2 = 90%), indicating that acupuncture combined with Western medicine was superior to Western medicine alone in reducing the neurological deficit of patients. studies ,29,40 usI2 = 63%, P=0.1). Meta-regression analysis of publication year and sample size did not find obvious sources of heterogeneity.After sensitivity analysis, it was found that Wang yu  may be t(4) The Effective Rate. Two studies [P=0.01), and the combination of acupuncture and Western medicine treatment was superior to Western medicine treatment alone in terms of effective numbers. studies ,29 took  studies  could noIn this study, we evaluated the efficacy and safety of acupuncture in the treatment of flaccid paralysis. Compared with the group without acupuncture intervention, the group with acupuncture intervention significantly improved the clinical scores, in terms of motor function, ability of daily living, cognitive function, quality of daily life, neurological deficit, and clinical effective rate, indicating that patients with flaccid hemiplegia could benefit from acupuncture.This study adopted American Heart Association/American College of Cardiology Guidelines  to evaluP < 0.01), 9.84 , and 16.86 . The results suggested that patients with flaccid hemiplegia after stroke could benefit from acupuncture. The secondary indicators also showed the same tendency.Cochrane's comments indicate that although acupuncture appears to be safe, there is no clear evidence that it was beneficial . There wCompared with three retrospective studies –51 of acIn the past studies on spastic paralysis, most studies used the Fugl–Meyer Assessment scale and the Fugl-Meyer Assessment scale as subjective evaluation indicators, and some studies used H reflex/M response as objective physiological indicators. The studies involved in this study mostly use subjective scales to evaluate the effect of acupuncture, lacking objective physiological indicators. No matter in the stage of flaccid hemiplegia or spastic paralysis after stroke, most studies used scales related to limb motor function, ability of daily living, and neurological deficits. Due to the selection of partial scales as the outcome indicators, most studies did not use Manual Muscle Test scale combined with modified Ashworth scale to assess the condition of muscle strength and muscle tension, and lacked objective physiological indexes, resulting in imperfect efficacy assessments. The three studies all searched the Chinese and English literature databases, and the Korean-related literature databases were also searched in the study of 2014 and 2015. Although we have searched a large number of different databases, due to language limitations, the databases of countries that conduct more acupuncture trials such as South Korea and Japan had not been searched, which might cause some omissions in clinical trials.P=0.058 and P=0.964, indicating that there was no obvious publication bias. Due to the limited number of trials, our findings should be treated with caution. In addition, all the evaluation indexes included in the studies were subjective judgment indexes such as the Fugl-Meyer Assessment scale and the modified Barthel Index scale, which were easily affected by the experience of clinicians and reviewers. Therefore, the existence of bias was easy to be detected.There were 3 limitations in this study. First, the included 27 studies had methodological flaws and were assessed as being of low quality. However, the reasons for its low quality should be further analyzed in detail. In most studies, the authors did not clearly state the random schemes, blind methods, allocation concealment, and other biases in the research process. Therefore, we could not judge whether the author of the article had not performed these steps, or whether they have been performed but had not been noted in the article. Researchers were unable to make specific judgments on the information due to irregular writing. Second, there was obvious heterogeneity in the scores of the Fugl-Meyer Assessment scale, the modified Barthel Index scale, and the Neurological Deficit Assessment scale. They were one of the main measurement indicators. In the subgroup analysis, the heterogeneity decreases with the intervention. At the same time, after excluding some studies through the sensitivity analysis, the heterogeneity was reduced. Meta-regression analysis found that sample size was one of the sources of heterogeneity. The diagnostic criteria of flaccid hemiplegia after stroke and the sample size were different, and acupuncture in a variety of forms, the selection of acupoints, acupuncture depth and frequency, amount of acupuncture stimulating, acupuncture operator and outcome indicators evaluation personnel qualification, and practice of the fixed number of years were different. Therefore, it might have a certain influence on the clinical efficacy and evaluation. Because the number of experimental data was not described in the study, the relevant experimental data could not be obtained after contacting the author, and the heterogeneity test of these items could not be completed. Therefore, these might be the source of heterogeneity. Third, the publication bias test was performed on groups of more than 10 studies, considering the existence of publication bias. The results of Egger's test of the Fugl-Meyer Assessment scale and the modified Barthel Index scale were Due to these limitations, the results of our study should be interpreted more cautiously. No adverse events were reported in the included 27 studies. Acupuncture treatment did not seem to cause serious adverse events and seemed to be safe in the short term, but its long-term safety was unknown, and we could not find enough evidence to support this view.Nowadays, acupuncture has been widely used to treat hemiplegia caused by stroke. When patients with stroke are in a state of hemiplegia, acupuncture can promote the rehabilitation of the patient's muscle strength and muscle tension, and improve the motor function of the limbs, which can effectively prevent the occurrence of various complications –57. HoweAccording to the available evidence, acupuncture treatment was beneficial to the recovery of patients with flaccid hemiplegia after stroke, although most of the included studies had methodological flaws. It was not yet possible to determine overall that acupuncture was better than other therapies in the treatment of flaccid hemiplegia after stroke. It is recommended that future study should be based on the CONSORT statement and the STRICTA consensus reporting plan and report the details of acupuncture treatment, such as the number of needles used in each treatment unit, specific acupuncture stimulation methods, needle insertion depth, needle response, treatment course, the qualifications of acupuncturist, evaluator, and the number of years of clinical practice, providing a standard and clear treatment plan, which will help to develop a more effective treatment prescription and evaluation system. In addition, it is necessary to expand the sample size, carry out multicenter, high-quality randomized controlled trials to confirm the conclusions, increase observation and follow-up to further clarify the long-term efficacy and safety of acupuncture treatment, and apply acupuncture to patients with flaccid hemiplegia after stroke treatment provides a more reliable clinical basis.The findings in this study suggested that acupuncture as a complementary therapy was effective and short-term safe for patients with poststroke flaccid hemiplegia. However, the methodological deficiencies in previous studies have led to the call for carefully designed larger studies to confirm the potential benefit of acupuncture for patients with poststroke flaccid hemiplegia rehabilitation in future.


Vincristine is a drug that is part of the treatment for many children with cancer. Its main side-effect is vincristine-induced peripheral neuropathy (VIPN), which often presents as tingling, pain, and lack of strength in the hands and feet. It is not yet possible to predict which children will suffer from VIPN. In this review, we report on all genetic variations that are associated with VIPN. We found that variations in genes related to vincristine transport, cell structure, hereditary nerve disease, and genes without a previously known connection to vincristine or VIPN are related to VIPN. Variations in genes involved in vincristine breakdown are not significantly associated with VIPN. In conclusion, genetic variations affect a child’s tendency to develop VIPN. In the future, this information might be used to predict the risk of VIPN and adapt treatment on this.Vincristine-induced peripheral neuropathy (VIPN) is a debilitating side-effect of vincristine. It remains a challenge to predict which patients will suffer from VIPN. Pharmacogenomics may explain an individuals’ susceptibility to side-effects. In this systematic review and meta-analysis, we describe the influence of pharmacogenomic parameters on the development of VIPN in children with cancer. PubMed, Embase and Web of Science were searched. In total, 1597 records were identified and 21 studies were included. A random-effects meta-analysis was performed for the influence of CYP3A5 expression on the development of VIPN. Single-nucleotide polymorphisms (SNPs) in transporter-, metabolism-, cytoskeleton-, and hereditary neuropathy-associated genes and SNPs in genes previously unrelated to vincristine or neuropathy were associated with VIPN. CYP3A5 expression status was not significantly associated with VIPN. The comparison and interpretation of the results of the included studies was limited due to heterogeneity in the study population, treatment protocol and assessment methods and definitions of VIPN. Independent replication is essential to validate the clinical significance of the reported associations. Future research should aim for prospective VIPN assessment in both a discovery and a replication cohort. Ultimately, the goal would be to screen patients upfront to determine optimal vincristine dosage with regards to efficacy and risk of VIPN. Vincristine is an important chemotherapeutic agent that is commonly used in treatment for pediatric cancers. It is approved by the United States Food and Drug Administration (FDA) for the treatment of acute lymphoblastic leukemia (ALL), Hodgkin and non-Hodgkin lymphoma, neuroblastoma, rhabdomyosarcoma, low-grade glioma and nephroblastoma. Furthermore, off-label uses include the treatment of Ewing sarcoma and medulloblastoma ,2. The m2 as opposed to 1.5 mg/m2) . Fu. Fu13]. Four studies assessed the influence of SNP rs924607 in CEP72 on the development of CTCAE or WHO grade 2−4 VIPN ,50,51. IGWAS or EWAS demonstrated significant associations between VIPN and eight SNPs in genes previously not associated with neuropathy, vincristine mechanism of action or metabolism . All stuGutierrez-Camino et al. found two SNPs in miRNA to be associated with VIPN, of which one miRNA could be related to the axon-guidance pathway, whereas the other could not be related to any known vincristine- or neurotoxicity-related pathway  48]..48].Several studies assessed the influence of covariates such as cumulative vincristine dosage, treatment protocol, and patient characteristics on their results, but these covariates did not have a significant influence on the reported associations . Only th2 = 50%, τ2 = 0.33). The study by Kayilioğlu et al., which could not be included in the meta-analysis due to the unavailability of appropriate data, did not find a significant association either   .This systematic review shows that pharmacogenomic parameters have a significant influence on VIPN in children with cancer and show potential for clinical relevance. Several SNPs in genes related to vincristine metabolism, hereditary neuropathy, the cytoskeleton and microtubules have been associated with VIPN. Furthermore, population-based GWAS and EWAS identified significant interactions with SNPs in genes previously unrelated to VIPN or vincristine. Our meta-analysis showed that CYP3A5 expression status was not a significant risk factor for VIPN.Several significant associations were found between SNPs in the ABC family of genes . These genes code for transmembrane proteins that mediate vincristine efflux across cell membranes; variations may thus contribute to different vincristine levels and therefore VIPN  54,55].,55.54,552) and conventional dosage (1.5 mg/m2) of vincristine during the continuation phase of treatment [Furthermore, SNPs in cytoskeleton-associated genes were associated with VIPN . Vincrisreatment . Recruitreatment ,50.In addition, we assessed the effect of CYP3A5 expression status on VIPN in a meta-analysis and found an overall pooled effect of 0.69 (95% CI 0.38−1.26) . Two stuThe comparison and interpretation of the results of the included studies is limited due to heterogeneity in the study population, treatment protocol and varying assessment methods and definitions of VIPN. Firstly, cumulative vincristine dosage varied between 6.5 and 97.5 mg across studies. Since cumulative vincristine dosage likely is an independent risk factor for VIPN, it could be of influence when establishing a relationship between a pharmacogenomic parameter and VIPN ,3. HowevIndependent replication is essential to validate the clinical significance of reported associations ,34. ThisThe strength of this systematic review and meta-analysis is the inclusion of all studies assessing the relationship between pharmacogenomic parameters and VIPN. No restrictions were applied regarding patient or study characteristics. Furthermore, we followed the PRISMA guidelines and two independent authors performed screening, data extraction and risk of bias assessment. A standardized risk of bias assessment was performed with the validated EPHPP tool . The weaFrom this systematic review, we can conclude that the following pharmacogenomic parameters have a significant influence on VIPN in children with cancer: SNPs in ABCB1, ABCC1, ABCC2, CYP3A4, GSTM1, VDR, ACTG1, CAPG, CEP72, MAPT, SYNE2, TUBB2B, SLC5A7, BAHD1, COCH, chromosome 12/chemerin, ETAA1, MRPL4, MTNR1B, NDUFAF6, TMEM215 and in three miRNAs. Our meta-analysis shows that CYP3A5 expression does not result in a heightened susceptibility of VIPN. To actualize the potential of pharmacogenomic testing, future research should prospectively assess VIPN with a sensitive measurement tool in both a discovery and replication cohort. Ultimately, the goal would be to develop an individualized protocol based on a patients’ genotype, taking all risk and protective genes into account, and subsequently give patients a dosage that limits the risk of VIPN while maintaining highest possible therapeutic efficacy. Dosage reductions or cessation of treatment, or for some patients even standardized dose capping, would no longer be necessary.


The following MeSH  terms were used: chronic myeloid leukaemia, genes and microRNAs in the title or abstract. From 806 studies retrieved from the search, only clinical studies with in-vitro experimental evidence on the target genes of the studied miRNAs in CML cells were included. Two independent reviewers independently scrutinised the titles and abstracts before examining the eligibility of studies that met the inclusion criteria. Study design, sample size, sampling type, and the molecular method used were identified for each study. The pooled miRNAs were analysed using DIANA tools, and target genes were analysed with DAVID, STRING and Cytoscape MCODE. Fourteen original research articles on miRNAs in CML were included, 26 validated downstream genes and 187 predicted target genes were analysed and clustered into 7 clusters. Through GO analysis, miRNAs’ target genes were localised throughout the cells, including the extracellular region, cytosol, and nucleus. Those genes are involved in various pathways that regulate genomic instability, proliferation, apoptosis, cell cycle, differentiation, and migration of CML cells.Chronic myeloid leukaemia is blood cancer due to a reciprocal translocation, resulting in a BCR-ABL1 oncogene. Although tyrosine kinase inhibitors have been successfully used to treat CML, there are still cases of resistance. The resistance occurred mainly due to the mutation in the tyrosine kinase domain of the BCR-ABL1 gene. However, there are still many cases with unknown causes of resistance as the etiopathology of CML are not fully understood. Thus, it is crucial to figure out the complete pathogenesis of CML, and miRNA can be one of the essential pathogeneses. The objective of this study was to systematically review the literature on miRNAs that were differentially expressed in CML cases. Their target genes and downstream genes were also explored. An electronic search was performed  BCR and ABL1 gene translocation, resulting in the Philadelphia chromosome. BCR-ABL1 fusion gene encodes an active tyrosine kinase BCR-ABL which activates several molecular pathways that cause abnormal cell adhesion, increase cell proliferation, and inhibit apoptosis. Nevertheless, tyrosine kinase plays an essential role in many signalling cascades, including biological processes such as cell growth, differentiation, metabolism, and apoptosis (Chronic myeloid leukaemia (CML) is a proliferative disorder of pluripotent stem cells. CML is linked to a specific genetic disorder involving poptosis –4.BCR-ABL1 gene. The first molecularly targeted therapy, Imatinib, is a small molecule known as tyrosine kinase inhibitor (TKI) that directly targets BCR-ABL1 tyrosine kinase activity. Despite the success of Imatinib as the front-line therapy for CML, there were reports on drug resistance that is primarily due to the presence of mutations in the BCR-ABL tyrosine kinase domain (TKD)  –7. TyrosBL1 TKIs , 8.MiRNA is a short, non-coding RNA that regulates gene expression at the post-transcriptional level. It inhibits translation by binding to the 3’untranslated (3’UTR) region of specific target mRNA. MiRNA has been linked to disease pathogenesis in CML and is known to play an essential role in tumorigenesis . MiRNA’sA comprehensive search of information was done using PubMed, Scopus, EBSCOhost MEDLINE, and Science Direct to identify relevant research publications with an unlimited starting publication date until 1st April 2021. The Medical Subject Heading (MeSH) terms like chronic myeloid leukemia, genes and microRNAs were used as the keywords in the title or abstract. The search strategy involved a combination (“AND”) of the following two sets of keywords (1): “chronic myelo* leukemia” OR CML OR “BCR*ABL*positive” and (2) mi*RNA. Synonyms for keywords were generated through MeSH terms from the Cochrane Library. Additional text terms were discovered by reviewing collected review articles. Additional references were discovered from the bibliographies of the retrieved studies.in vitro experimental evidence on the target genes of the studied miRNAs in CML cells were included in this review. Due to limited resources, only manuscripts written in English were included.Case-control and prospective observational studies with abstracts investigating the differentially expressed miRNAs on Philadelphia chromosome-positive chronic myeloid leukemia patients in chronic, accelerated, or blast phases were included. In addition, only clinical studies that have further in vitro, and in vivo studies were excluded. The review focus on the outcome of the differentially expressed miRNAs in CML patients. Therefore, studies that involved responses toward tyrosine kinase inhibitors treatment or any other intervention studies on a new treatment for CML patients were excluded from consideration. These selection criteria were used to achieve the objective of this systematic review in determining the typical miRNA expression signature in CML patients, the miRNAs target genes, and related pathways that could potentially be involved in the pathogenesis of CML.Publications that did not have primary data, such as editorials, case reports, conference proceedings, and narrative review articles, were excluded. In silico, Articles retrieved from all resources were screened in three phases. All articles with titles that did not match the inclusion criteria were excluded, and duplicates were removed in the first phase. The abstracts of the remaining articles were screened, and any articles that did not meet the inclusion criteria were excluded in the second phase. Finally, the full texts of the remaining articles were read and assessed thoroughly. Systematic reviews, meta-analyses, and articles that did not meet the inclusion criteria were excluded in this third phase. All the authors were involved in the screening, selection, and data extraction phase. Any differences in opinions were resolved by discussion between the authors. All data extraction was performed independently using a data collection form to standardize the data collection, and records on reasons for rejection were kept. Data were extracted from the studies that fulfilled inclusion criteria. Data collected from these studies include (1) author name (2), study design (3), study objective (4), study population (5), type of sample used (6), method used in gene expression analysis and experiments performed to validate their targeted genes (7), results , and (8) conclusion. The extracted details are listed in The analysis details of each report were discussed thoroughly among the reviewers to assess the quality of each study. The authors focused on the reported list of miRNAs as well as their targeted genes. Bias was excluded by adhering to the inclusion criteria. Pairs of reviewers with adequate reliability worked independently to determine the validity of each study.The miRNAs listed were further analysed using four different bioinformatics tools that include 1) DIANA-microT web server v5.0 with MiTG scores being set at more than 0.95, 2) TargetScan release 7.2 with Cumulative weighted context++ score of more than -0.5, 3) miRDB with a target score more than 90, and 4) mirDIP v5.0.2.3 with score class set at “very high”. Genes that are predicted by more than two bioinformatics tools were selected for further analysis.Two groups of analysis that include 1) a group of validated target genes and downstream genes of the miRNAs extracted from the studies and 2) a group of predicted target genes of the reported miRNAs was done. These two groups of genes were analyzed using Database for Annotation, Visualization, and Integrated Discovery (DAVID). DAVID was performed to determine the cluster of genes that displayed significant functional annotation enrichment related to CML’s pathogenesis. The contribution of genes in the pathway related to CML was based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, the Biological Biochemical Image database (BBID), BIOCARTA pathway database, and Reactome.https://string-db.org/). Results from STRING were further analyzed using Cytoscape to visualize molecular interaction networks and integrating gene expression profiles to identify clusters of protein interaction that are highly related to the pathogenesis of CML. The gene interaction relationship was downloaded in the “TSV” format file and was imported into the Cytoscape software (http://www.cytoscape.org/) for further analysis and clustering. The Cytoscape MCODE plug-in was employed to perform module analysis of the target network and protein clustering. The module selection criteria were as follows: degree cutoff = 2, node score cutoff = 0.2, node density cutoff = 0.1, K-score = 2, and max depth = 100. The list of genes in the cluster was then analyzed again using DAVID for significantly enriched ontology terms.The targeted genes were further analyzed at the protein level using protein-protein interaction network functional enrichment analysis through STRING   was used as the reference manager. Upon filtering the titles, 372 articles were identified as duplicates, and two other articles were withdrawn from publication. A total of 432 articles were retrieved for abstract reviewing. Upon screening titles and abstracts, 263 articles were removed, resulting in the selection of 159 potentially relevant articles for full-text review. However, three articles could not be retrieved as the full text are not in English. Then, 147 potentially relevant articles’ full text was thoroughly reviewed, and 130 articles were eliminated based on our inclusion and exclusion criteria. Finally, 17 articles were selected to be included in the present systematic review. All studies were original research articles published between the year 2008 to 2019. Homogeneity of the selected studies was ensured by adhering to the defined inclusion and exclusion criteria to prevent sampling bias. Notably, all the studies performed real-time polymerase chain reaction (qPCR) for miRNA expression analysis. A confirmatory method was done to validate the miRNA binding site on their selected genes. Sample sizes for each study varied from 8 to 180 samples for miRNA expression analysis. The characteristics of these studies are highlighted in via bone marrow morphology, immunology, molecular biology, and cytogenetic result, with no chemotherapy treatment before the specimen collection . A p-value of <0.05 was used as a cut-off standard. The gene listed were categorized into three functional categories of gene ontology that include biological process (BP), cellular component (CC) and molecular function (MF), as shown in A total of 26 downstream genes that were affected by the miRNAs was extracted from all the studies. The functions and pathway enrichment of these genes were analyzed using DAVID , microRNAs in cancer pathway (hsa05206), Hepatitis B (hsa05161), PI3K-Akt signalling pathway (hsa04151) and many other pathways with some directly associated with apoptosis, proliferation, and cell cycle pathways as reported in http://string-db.org), a total of 26 proteins from miRNA targets and their downstream genes were filtered into a PPI network complex, containing 22 nodes and 75 edges . A p-value of <0.05 was used as a cut-off standard. The genes listed were categorised into three functional categories of gene ontology that include biological process (BP), cellular component (CC) and molecular function (MF), as shown in The functions and pathway enrichment of predicted target genes were analysed using DAVID , focal adhesion (hsa04510), pathways in cancer (hsa05200), and many other pathways, with each pathway, involve from 13 to six predicted target genes (http://string-db.org), a total of 187 proteins from predicted target genes were filtered into a PPI network complex, containing 136 nodes and 211 edges  with PPI enrichment p-value is less than 1.0E-16. At the same time, 51 other proteins did not fall into the PPI network.Using STRING online database  consisted of 11 nodes and 45 edges  are enriched in the cytosol and nucleus. Three proteins  are enriched in the cytosol, and one protein is in the nucleus (GADD45A). MAPK8, CCND1, IRS1, GADD45A, MYC, BCL2, AKT1, IRS2, and BCL2L1 are phosphoproteins. Phosphoproteins are proteins that bind to the phosphate group that can be switched on or off. These proteins are essential in most cellular processes such as protein synthesis, cell division, signal transduction, cell growth, development, and ageing .From the KEGG pathway, MAPK8, CCND1, PIK3CA, MYC, BCL2, BAX, AKT1, MMP9, and BCL2L1, are essential genes in cancer pathways that regulate cell cycle, proliferation, apoptosis, genomic instability, and block of differentiation. At the same time, MAPK8, CCND1, PIK3CA, IRS1, GADD45A, AKT1 and IRS2 are involved in FoxO signalling pathway that can affect the regulation of cell cycle, oxidative stress resistance and DNA repair.The first dataset of those experimentally validated target genes shows a significant effect of miRNAs in CML pathogenesis. However, it is known that miRNA can target more than one target gene due to its seed sequence . Therefovia several receptor families and regulate cell’s proliferation, differentiation and migration  family member. It plays a role in the regulation of differentiation and metabolism in tissues and organs. In CML, FOXO1 could increase the activation of CML cells . Thus, iCluster 4 consisted of seven proteins, including WEE1, CCNE2, FBXW7, TDG, TET3, TET2, and TET1. Six proteins  are located in the nucleus, while four  are involved in DNA methylation. TET oncogene family that includes TET1, TET2, and TET3 plays a role in the DNA methylation process . Althougvia miR-301-5p in CML cells.Cluster 5 consisted of three proteins, including SCN9A, NAV1, and SCN2A. These proteins are involved in the voltage-gated sodium channel complex. Although there are still no CML studies on these genes, ion channel signalling mechanisms are known to be involved in cancer cells migration, invasion, and metastasis . Thus, ivia DNA methylation in CML patients is still uncertain as the findings are contradictory (via miR-30a-5p in CML is suggested.Cluster 6 consisted of seven protein-binding proteins, including KITLG, SOCS1, AGO4, TRA2B, PTEN, SRSF7, and QKI. KITLG is localised in the cytoplasm, TRA2B is in the nucleus, while SOCS1, AGO4, PTEN, SRSF7, and QKI can be found in both intracellular regions. KITLG, AGO4, PTEN are involved in phosphatidylinositol-mediated signaling that is crucial in regulating cancer cells’ survival, proliferation, invasion, and growth. SOCS1 plays an important role in regulating optimal JAK/STAT activity. However, regulation of SOCS1 adictory , 46. Thuin vitro analysis and clinical studies need to be done to validate the predicted mechanisms. Nevertheless, this review added new insight into the involvement of miRNA in CML pathogenesis for future studies.The studies included in this review only focused on their miRNA of interest and its few target genes. Therefore, this review are not able to rule out the entire networks of miRNAs and their target genes in CML. In silico analyses were done to improve the coverage of miRNA’s target genes. However, further Pathogenesis of CML at the molecular level involves a wide range of mechanisms that are still undiscovered. In this study, the function of miRNAs was found to be significant in the development of CML. The miRNA’s target genes are localised in the extracellular, cytosol and nucleus of CML cells. Thus, the importance of miRNAs cannot be denied as miRNAs are universally involved in various pathways that regulate genomic instability, proliferation, apoptosis, cell cycle, differentiation, and migration of CML cells. Therefore, from the identified miRNAs and their pathways involved in CML pathogenesis, potential new biomarkers for a better prognosis and new miRNA-based treatment for CML patients could be developed.The original contributions presented in the study are included in the article/ME collected and/or assembled data and wrote the manuscript. SS wrote, reviewed, and edited the manuscript. NA confirm the authenticity of all raw data, proofread, edited and gave approval of the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by the Fundamental Research Grant Scheme, awarded by Ministry of Higher Education of Malaysia, grant number FRGS/1/2018/SKK08/USIM/03/1.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.


Alzheimer’s disease (AD) is one of the most devastating brain disorders. Currently, there are no effective treatments to stop the disease progression and it is becoming a major public health concern. Several risk factors are involved in the progression of AD, modifying neuronal circuits and brain cognition, and eventually leading to neuronal death. Among them, obesity and type 2 diabetes mellitus (T2DM) have attracted increasing attention, since brain insulin resistance can contribute to neurodegeneration. Consequently, AD has been referred to “type 3 diabetes” and antidiabetic medications such as intranasal insulin, glitazones, metformin or liraglutide are being tested as possible alternatives. Metformin, a first line antihyperglycemic medication, is a 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activator hypothesized to act as a geroprotective agent. However, studies on its association with age-related cognitive decline have shown controversial results with positive and negative findings. In spite of this, metformin shows positive benefits such as anti-inflammatory effects, accelerated neurogenesis, strengthened memory, and prolonged life expectancy. Moreover, it has been recently demonstrated that metformin enhances synaptophysin, sirtuin-1, AMPK, and brain-derived neuronal factor (BDNF) immunoreactivity, which are essential markers of plasticity. The present review discusses the numerous studies which have explored (1) the neuropathological hallmarks of AD, (2) association of type 2 diabetes with AD, and (3) the potential therapeutic effects of metformin on AD and preclinical models. Currently, there are 44 million people dealing with dementia, being the second leading cause of death in people aged 70 and over ,2,3,4. ASince the neurodegenerative process of AD is closely related to the aging process, it has also been called the late onset AD (LOAD). LOAD lacks a clear origin, and it is mainly found in patients over 65 years old. These patients comprise 95% of AD cases, although about 1% are attributed to autosomal dominant mutations in amyloid metabolism-related genes, e.g., beta amyloid precursor protein (βAPP-chromosome 21), presenilin 1 (PS1-chromosome 14), and presenilin 2 genes (PS2-chromosome 1) [In spite AD or dementia is often related with the geriatric community, AD may also affect younger adults. Early-onset AD (EOAD) is referred to those affecting people who are diagnosed before 65 years old (between 30 and 65). As outlined by McMurtray and colleagues, early-onset dementia patients account for 20% to 34% of AD cases in most trials . Three gWhile LOAD has not a well-established etiology, two of the most relevant genes conferring a significantly risk factor are the E4 apolipoprotein allele (APOEε4) and the triggering receptor expressed on myeloid cells 2 (TREM2) ,9,10. ThType two diabetes mellitus (T2DM) and LOAD have become worldwide pandemics, with recent projections indicating that they will get worse in the coming decades. In this respect, obesity, T2DM and associated comorbidities have been described to be involved in the development of LOAD . Thus, LPreclinical research studies and clinical and epidemiological trials reported that T2DM has been related not only to the development, but also to the progression of LOAD. A retrospective study of 20 prospective clinical trials concluded that the prevalence of LOAD in patients with T2DM was 56% greater than in people without diabetes . Brain iT2DM is a widely known chronic metabolic condition characterized by high levels of blood glucose and insulin resistance ,25,26. EThe most common hypothesis recognizes Aβ as the main cause of LOAD ,47,48,49Furthermore, Aβ is linked to both neuronal oxidative stress and mitochondrial dysfunction . UnfoldeApart from the effects of Aβ oligomers at the brain, a very interesting point is the role of plasma Aβ oligomers on peripheral IR. In line with this concept, it has been reported that these oligomers also have an inhibitory effect on the peripheral insulin signaling pathway through different mechanisms mediated by oxidative stress and inflammatory responses, hence demonstrating that Aβ oligomers modify peripheral glucose metabolism through multiple ways ,52,53,54One of the characteristics of LOAD is the brain hypometabolism that is due to a decrease in glucose uptake. The drop in brain glucose levels is mainly related to a reduced glucose uptake associated with the decreased expression of glucose transporters in neurons, mainly GLUT1 and GLUT3 . TherefoChronic hyperglycemia can be responsible for the appearance of diabetic complications since it can generate glycolipotoxicity. In this way, hyperglycemia generates advanced glycation end products (AGEs) that are also a crucial link between diabetes and LOAD ,51,52,53Besides, the key enzymes glycogen synthase kinase 3β (GSK3β) and insulin degrading enzyme (IDE) are altered in bothT2DM and LOAD, being a potential link between the two diseases. GSK3β is regulated by the insulin receptor and its activation in LOAD favors the phosphorylation of tau protein and the formation of neurofibrillary tangles ,41. IDE Supporting this metabolic hypothesis of LOAD, a preclinical study with a neuron-specific human BACE1 knock-in mouse model (PLB4) conducted by Plucińska and colleagues demonstrated that the brain BACE1 overexpression by itself increased the risk of peripheral T2DM . TherefoAs we have already mentioned, tau is the other main biomarker related to the neurodegenerative process in LOAD . Tau is In this sense, brain insulin has been shown to play a key role in the regulation of tau phosphorylation through the activation of its receptor located in the brain. Indeed, as we have commented above, BIR is associated with the activation of tau kinases, including GSK3β, through the phosphatidylinositol kinase (PI3K)/protein kinase B (AKT) signaling pathway . Thus, BIn this line, Marciniak and colleagues demonstrated the role of tau as a key modulator of BIR and the insulin receptor signaling pathway, as well as the mechanisms whereby tau could modulate insulin receptor function . Hence, Likewise, tau has recently been identified as a key regulator of peripheral insulin signaling, with evidence linking tau to IR in the brain and peripheral tissues, as well as beta cell dysfunction ,62,63. T-/- mice showed an increased phosphorylation of tau through the inhibition of phosphoinositide 3 kinase (PI3K)/AKT signaling pathway [Although it is well known that insulin can modulate the phosphorylation of tau protein, its role in the regulation of the insulin receptor has been only studied in the recent years. In accordance with this idea, mice which do not express insulin receptors at the neuronal level (NIRKO mice) as well as IRS-2 pathway ,19,62,63 pathway ,19.Galega officinalis plant [Metformin is an antidiabetic drug derived from galengine, a natural product of the is plant . Metformis plant ,65,66, wis plant . Metformis plant . Furtheris plant .For the purpose of designing therapeutics or disease modifying agents for AD treatment, a wide variety of animal models have been developed to replicate the human environment of the disease ,72,73,74The transgenic mice are important models for deciphering familial AD pathology pathways. These models do not display all the anomalies found in human AD and do not duplicate the sporadic forms of AD, because they only reproduce the pathological features of AD common mutated genes . HoweverOn the other hand, many of the signature features of AD are reproduced by the injection of pharmacological or chemical agents into the brain or by the activation of lesions in specific brain regions ,77,78. FSince LOAD represents more than 95% of AD cases, associated animal models are valuable research resources for studying pathogenesis and designing experimental treatments for sporadic AD ,82,83,84According to the studies of Ditacchio and colleagues, metformin is an efficient treatment to improve insulin sensitivity, with a higher drop in blood glucose levels in the AβPP AD model . In addiFinally, Lu and colleagues demonstrated that metformin improved learning and memory performance in APP/PS1 transgenic mice, according to Morris water maze and Y-maze results . AnotherIt has been shown that metformin stops or slows the onset of dementia in adults with diabetes . In 2019A pilot study of 80 people with amnestic moderate cognitive disorder was undertaken at Columbia University in New York City from 2008 to 2012. The participants were overweight, but none of them had diabetes. They were given either 2000 mg of metformin separated into two doses or a placebo for one year. The selective reminding test (SRT) for recall and the ADAS-Cog were the primary outcomes . The secFrom 2013 to 2015 a small study at the University of Pennsylvania assessed the impact of metformin on biomarkers of AD in 20 non-diabetic individuals with moderate cognitive dysfunction or dementia related to the disease. MRI, FDG-PET, and amyloid biomarkers were used to validate the diagnosis of AD ,101. Eachttps://www.alzforum.org/therapeutics/metformin) (accessed on 23 August 2021).In February 2020, Swedish researchers began testing the impact of a year of metformin treatment plus exercise and diet on memory in 80 people with T2DM and moderate cognitive dysfunction. Recruitment, adherence, and retention rates are the primary consequences, while metabolic improvement and memory capacity are secondary measures. The research will last until December 2021  . In the Studies by Ma and colleagues showed that metformin improves the composition of the gut microbiota of obese mice. This peripheral effect could inhibit the neuroinflammatory process in the hippocampus. Likewise, this drug could prevent the deterioration of newborn neurons in the hippocampus and therefore improve the learning process and memory in obese mice. These results reinforce the hypothesis of the benefit of acting at the microbiota level to improve the cognitive process in LOAD .Cognition is one of the most complex features of the brain, and it involves perception, registration, consolidation, storage, and memory over the course of human life . Any memKodali and colleagues reported that after 10 weeks of metformin treatment, C57BL6/J mice with late middle age improved recognition memories in old age . MetformLikewise, since the hippocampus is an essential part of the brain for memory and cognition and it is widely affected in AD, the hippocampus’ neuronal activation and synaptic transmission are important for improving these functions. According to Chen and colleagues, metformin improved synapsis, memory, and cognitive deficits with disrupted hippocampal synaptic communication . This pr−1 d−1 of metformin for 18 weeks in db/db mice, which have multiple AD-like brain changes such as alterations in cognitive functions, increased phospho-tau and Aβ, as well as decreased synaptic proteins. Metformin decreased hippocampal levels of total tau, phospho-tau, and activated c-Jun-N-terminal kinase [In turn, Li and colleagues studied the effects of intraperitoneal injection of 200 mg kgl kinase . Moreovel kinase . NotwithThe theory that activating insulin receptors enhances cognition has been confirmed by clinical and preclinical trials. Hence, it is accepted that insulin improves emotional function in active and elderly people, as well as Alzheimer’s patients . InsulinLoss of synaptic activity in the AD brain can be correlated with observed cognitive deficiencies ,140,141.The transgenic APP/PS1 mice presents loss of spines as demonstrated by reduced spine density from CA1 pyramidal neurons. According to Wang and colleagues’ study, chronic metformin administration for 10 days improves synaptic defects, including surface GluA1 expression, decrease spine disappearance, and reduction in basal synaptic transmission in the hippocampus of APP/PS1 mice . FurtherAccording to Ha and colleagues, metformin possesses anti-inflammatory effects . The neu2O2, inducing the endogenous antioxidant system that includes glutathione reductase (GSH), superoxide dismutase (SOD), and catalase  ,152,153.ase (CAT ,152. It ase (CAT ,152,153.In several studies performed on mice with traumatic spinal cord injury, reactions of local inflammation along with microglia proliferation, activation, and phagocyte infiltration are used . In a deAll these data support that metformin has an antioxidant and anti-inflammatory function in different circumstances. Therefore we can conclude that, for several neurodegenerative diseases whose inflammatory pathways and oxidative stress play a role in their pathogenesis, metformin may be an effective therapeutic choice ,151,152.Chung and colleagues studied the genes and proteins whose expressions or functions were either directly or indirectly influenced by the AMPK pathway to understand the role of metformin between multiple signal pathways in neuroprotection . AMPK caInterestingly, Fatt and colleagues demonstrated a potential neurorestorative effect of metformin . They reDementia is linked to a number of co-morbid disorders in the elderly, including diabetes, asthma, dyslipidemia and cardiovascular disease, among others ,4,6. TheIn general, metformin is considered a safe and well-tolerated drug. However, the appearance of gastrointestinal adverse effects—such as diarrhea, nausea, and vomiting—has been reported . Less frMetformin, through multi-directional pathways, could be a promising candidate for prevention of not only LOAD, but also of other neurodegenerative diseases, due to beneficial effects at the central and peripheral level . Metform