Among patients with intensive care unit-acquired weakness (ICUAW), skeletal muscle strength often decreases significantly. The present study aimed to explore the effects of testosterone propotionate on skeletal muscle using rat model of sepsis. Male SD rats were randomly divided into experimental group, model control group, sham operation group and blank control group. Rats in experimental group were given testosterone propionate two times a week, 10 mg/kg for 3 weeks. Maximal contraction force, fatigue index and cross-sectional area of the extensor digitorum longus (EDL) were measured. Myosin, IGF-1, p-AKT and p-mTOR levels in EDL were detected by Western blot. Histological changes of the testis and prostate were detected by hematoxylin and eosin staining. We found that maximal contraction force and fatigue index of EDL in experimental group were significantly higher than in model control group. Cross-sectional area of fast MHC muscle fiber of EDL in group was significantly higher than in model control group. The levels of myosin, IGF-1, p-AKT and p-mTOR of EDL in experimental group were significantly higher than in model control group. In addition, no testicle atrophy and prostate hyperplasia were detected in experimental group. In conclusion, these results suggest that testosterone propionate can significantly improve skeletal muscle strength, endurance and volume of septic rats, and the mechanism may be related to the activation of IGF-1/AKT pathway. Moreover, testosterone propionate with short duration does not cause testicular atrophy and prostate hyperplasia in septic rats. Therefore, testosterone propionate is a potential treatment for muscle malfunction in ICUAW patients. Intensive care unit-acquired weakness (ICUAW) is common in critically ill patients who have decreased muscle strength and weakened peripheral nerve excitability.  At preseTestosterone is a major male hormone applied in clinical practice in elderly men and patients with severe burns and other weaknesses to improve muscle function and quality of life . In animICUAW patients often show skeletal muscle atrophy. One major role of testosterone is to promote the synthesis of skeletal muscle protein by acting on a series of cellular signals through androgen receptors ,10. In aTherefore, the present study aimed to investigate the effect and possible mechanism of testosterone on muscle weakness in rat model with sepsis.Animal experiments were conducted at Chongqing Medical University in accordance with “Chongqing Administrative Measures on Experimental Animals” and approved by the ethics committee of Chongqing Medical University. Healthy male SD rats were purchased from the Animal Experiment Center of Chongqing Medical University (license number scxk-2017-0001) and raised in an SPF level animal house. Rats were fed standard feed at room temperature (20°C), with a circadian rhythm of 12 h of day alternated with 12 h of night.n = 8) using computer-generated numbers. The 8th day after surgery, hind limb was fixed, and a 1-ml sterile empty needle was used for vertical puncture into the muscle. Sterile cotton balls were used for compression and hemostasis after injection, and the hind limbs on both sides were injected with testosterone propionate  twice a week, 10 mg/kg for 3 weeks for rats in experimental group; rats in model control group were injected with same amount of soybean oil with the same injection method and date. Rats in sham group and blank control group (n = 8) were not injected.The rats were anesthetized with pentobarbital salt solution (40 mg/kg) via intraperitoneal injection, and then cecal ligation and perforation (CLP) was performed in experimental group and model control group to establish the animal model of sepsis. Briefly, midline laparotomy was performed, and the cecum was exteriorized and ligated distal to the ileo cecal valve without causing intestinal obstruction. Next, the cecum was perforated with a 20-gauge needle and squeezed gently to extrude a small amount of fecal contents from the punctured cecum into the abdominal cavity. The cecum was then returned to the peritoneal cavity and the incision was closed with sutures . The cec125I testosterone radioimmunoassay kit for rat .One milliliter of blood was collected from the rats by the orbital blood collection method. The serum testosterone concentration was determined by radioimmunoassay using Rats were anesthetized with pentobarbital and were placed on a mat heated to 37°C. The sciatic nerve was dissected intact through a left hip incision, and small branches of the sciatic nerve innervating the hip muscle were cut off, leaving only the anterior tibial nerve. A longitudinal incision was made in the middle of the foot, and the extensor digitalis longus (EDL) was disassociated along the tendon, while all blood vessels were preserved. The rats were placed in prone position, with the foot fixed at 90° of the ankle, and the femur fixed with clamps at an angle of approximately 100° between the femur and the tibia . The tenRats were killed by high-dose pentobarbital (100 mg/rat) and high carbon dioxide. The standard of death was loss of consciousness, respiratory arrest and electrocardiogram. EDL of the right leg was cut off and frozen at −80°C, while the middle segment of the left EDL, testicle and prostate tissue were cut off and fixed in 4% paraformaldehyde.g, 4°C for 10 min, and the supernatants were centrifuged at 100,000 × g at 4°C for 1 h. The protein concentration was determined using the Bradford Protein Assay Kit . Soluble proteins (20 μg) were used for Western blotting with primary antibodies against AKT1/2/3 , mTOR , mTOR phosphor S2481  Abcam; 1:5000), AKT1/2/3 phospho Y315 + Y316 + Y312  and fast myosin skeletal heavy chain  and goat anti rabbit IgG . Chemiluminescence was performed using a super ECL plus , and the blots were exposed to a Gel Imaging System .EDL was homogenized in ice-cold lysis buffer (pH 7.4) containing 50 mM Tris-base, 1% (v/v) Triton X-100, 0.25% (v/v) sodium desoxycholate, 150 mM NaCl, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 5 mM DTT and 1 mM PMSF. All homogenates were centrifuged at 12,000 × The frozen EDL tissues were sliced with a Cryostat Microtome . Immunohistochemical analysis of the sections was performed using fast myosin skeletal heavy chain antibody , and the staining was analyzed with Image Pro Plus 6 software. The testicle and prostate tissues were sectioned for hematoxylin and eosin (HE) staining, and observed under an optical microscope .U test was used to compare the difference. P < 0.05 was considered significant.All data were expressed as Mean ± standard deviation (SD) and analyzed by SPSS 21.0 software. For data with normal distribution, one-way ANOVA was used to compare the difference, and for data not in normal distribution, Mann–Whitney P < 0.01), and there was no significant difference between experimental group and model control group. After 3 weeks of treatment, the body weight of experimental group and model control group was still significantly lower than sham operation group and blank control group (P < 0.01), and there was no significant difference between experimental group and model control group. Three weeks after intramuscular injection of testosterone propionate, serum testosterone concentration in experimental group was significantly higher than three groups (P < 0.01, Preoperative body weight of rats among each group showed no significant difference. Body weight of rats in experimental group and model control group significantly decreased compared with sham group and blank control group on the 8th day after surgery before testosterone treatment (The maximal contractile force of EDL was significantly greater in experimental group than in model control group, while it was also significantly greater in sham operation group and blank control group than in model control group A. MoreovThe cross-sectional area of MHC fast muscle fibers of EDL was significantly larger in experimental group than in model control group, while it was also significantly larger in sham operation group and blank control group than in model control group A. MoreovTo reveal the mechanism by which testosterone improved the muscle function of EDL, we detected the expression of IGF-1 in EDL. Western blot analysis showed that IGF-1 protein level in EDL was significantly higher in experimental group than in model control group, sham operation group and blank control group A. MoreovHE staining of the testes in experimental group showed no significant atrophy compared with model control group, sham operation group and blank control group . HE staiSepsis is one of the important causes of acquired muscle weakness in either human or rats, with a high incidence and severe symptoms of ICUAW. The sepsis rat model is one of the most commonly used ICUAW animal models, and the cecal ligation perforation method can not only simulate clinical manifestations of sepsis caused by intestinal perforation and intestinal necrosis, but also adjust the severity of sepsis by controlling the length of the cecal ligation . TherefoSepsis, especially severe sepsis, can lead to a significant decrease in serum testosterone levels in either patients or experimental animals, which is currently thought to be caused by the suppression of the hypothalamic–pituitary–gonadal axis by systemic inflammatory response syndrome . In viewTestosterone propionate is an artificial testosterone preparation that has a long half-life after intramuscular injection. Testosterone propionate has been used to treat severe burns and elderly patients with frailty. The typical clinical manifestations of ICUAW include muscle weakness, muscular atrophy and decreased exercise endurance. In the present study, we found that testosterone propionate promoted maximum contraction force, fatigue index, average cross-sectional area of muscle fiber of rats with sepsis-acquired muscle weakness after short term application. These data indicate that testosterone could enhance the skeletal muscle of rats with sepsis-acquired muscle weakness in strength, endurance and volume. Moreover, the contraction time of EDL in experimental group was significantly shorter than that in model control group, indicating that skeletal muscle was more excitatory and responsive after testosterone treatment. In addition, rapid myosin degradation is one characteristic of ICUAW, and testosterone significantly increased the expression of rapid myosin in the skeletal muscle of rats with sepsis, which indicate that testosterone could improve the muscle in condition of ICUAW.IGF-1/AKT pathway is one of the main signaling pathways that regulate skeletal muscle anabolism and skeletal muscle function . IGF1 isIn summary, our study provides evidence that testosterone propionate can significantly improve skeletal muscle strength, endurance and volume of septic rats, and the mechanism may be related to the activation of IGF-1/AKT pathway, which could be further validated with AKT inhibitors. Moreover, testosterone propionate with short duration does not cause testicular atrophy and prostate hyperplasia in septic rats. However, further clinical trials are needed to validate that testosterone propionate is a potential treatment for muscle malfunction in ICUAW patients.


Morus alba). In this study, we performed whole genome bisulfite sequencing under drought stress to generate a methylated cytosines map and assessed the effects of the changes on gene expression combined with transcriptomics. We found that the percentage of methylated cytosines varied depending on the local sequence context  and external treatment . The methylation levels under DS were 8.64% higher than that of CK, and differences that were mainly due to the contribution of mCG (6.24%). Additionally, there were 3,243 different methylation and expression associated genes. In addition, methylated genes were enriched within GO subcategories including catalytic activity, cellular process, metabolic process, response to stimulus and regulation of biological process. This is the first study to comprehensively present methylation patterns in mulberry and reveal widespread DNA methylation changes in response to drought stress, which has the potential to enhance our understanding of links between DNA methylation and the modulation of gene expression in plants subjected to abiotic stresses.DNA methylation plays a significant role in many biological processes. Although some studies of DNA methylation have been performed in woody plant, none is known about the methylation patterns of mulberry ( As one of the defense mechanisms used by plants, epigenetics facilitates the genomic “plasticity” of plants and plays significant roles in growth, development and adaption to stress7. DNA methylation is a common feature of eukaryotic genomes and is one of the main types of epigenetic modifications. It plays a significant role in regulation of gene expression, genomic imprinting and transposon silencing, which has a protective effect on genomic integrity10. In plants, the DNA methylation patterns of different species are diverse. In general, methylation occurs at CG, CHG, and CHH  nucleotides of genes and transposable elements (TEs) in angiosperms11. In each of these contexts, distinct mechanisms are involved in establishing, maintaining and removing methyl groups. In the model plant Arabidopsis, which has a compact genome and rapid generation time, the first whole-genome methylome map was deciphered13. Now, many reports have documented the importance of DNA methylation in the process of plant growth and development, defense, and response abiotic stress, which includes the response to salt, drought and heavy metal exposure16. In long-term subcultures, plant growth regulator (PGR)-supplementation gradually increased global DNA methylation in Araucaria angustifolia, which compromises genomic stability and alters gene expression17. Induction of the OsMYB91 gene in rice in response to salt stress was associated with the rapid removal of DNA methylation from the promoter region of the gene and histone modification of the locus, which suggested that dynamic methylation patterns may have a role in modulating the expression of the gene18.Epigenetics is the study of heritable changes in gene expression that occur independently of DNA sequence changes20. The amplitude of drought-related transcriptional change was related to the extent to which genomic changes in DNA methylation of poplar (Populus trichocarpa) occurred, suggesting the importance of epigenetic mechanisms in the adaption of long-lived trees to local environments21. When comparing two varieties of fava bean (Vicia faba), researchers determined that DNA methylation of root tissues decreased 3.63% (from 23.43% to 19.80%) under drought stress in drought-tolerant Bachar and increased 0.66% (from 16.53% to 17.19%) in the drought-sensitive F177 variety22. Drought stress strongly induced de novo methylation (DNM) within the genome of sesame (Sesamum indicum), and most of the methylated loci were demethylated (DM) during the recovery phase23. Analysis of methylation patterns in two contrasting wheat genotypes revealed that demethylation occurred more frequently in the drought-tolerant genotype (C306) than the drought-sensitive genotype (HUW468) post-exposure to drought stress. Comparisons of different methylation-sensitive amplified polymorphism (MSAP) patterns showed that there was a percentage of polymorphic bands which occurred between tolerant and susceptible wheat genotypes (from 74.79% at anthesis to 88.89% at tillering)24. These studies revealed a direct relationship between environmental drought stress and DNA methylation changes.Drought is a major type natural disaster. With the intensification of the greenhouse effect, dry weather is predicted to occur increasingly frequently. This will likely result in huge losses with respect to industrial and agricultural production, and serious damage to the ecological environment. The plant response to drought stress is a complicated process, which involves various physiological, biochemical, and genetic responses. Among these, gene expression, metabolic networks, and DNA methylation are particularly important. Researchers have shown that drought induces changes to DNA methylation patterns in variety specific, tissue specific and stress specific waysMorus alba) is an important perennial economic tree that has a broad ecological distribution in China. Aside from its use in sericulture, mulberry has important economic and ecological value. Mulberry has a high degree of adaptability to poor environmental conditions including drought, cold, high salt, waterlogging, and heavy metal ion exposure26. Currently, the genome and the transcriptome of mulberry tree under drought stress have been determined28. The availability of high-quality reference genomes and improvements in sequencing technologies have facilitated whole-genome methylome analyses of mulberry and other plant genomes, such as the recently discovered rice DNA methylation pathways29, cytosine methylation that occurs in apple, which is associated with water deficit30, and single-base resolution methylomes of upland cotton (Gossypium hirsutum)31. However, there have been no studies that have assessed of mulberry methylome and the influence of adversity stress, such as drought stress, on mlberry methylome. Here, we generated a genome-wide, high-coverage DNA methylation maps using whole genome bisulfite sequencing (WGBS)33 in mulberry under normal and drought stressed conditions to investigate whether whole genome epigenome reprogramming occurs as a result of drought stress in mulberry. This study aimed to: (1) determine the genome-wide scale mulberry methylome level; (2) appraise changes of mulberry methylome associated with drought stress; (3) evaluate of the relationship between changes in the methylome and drought-stress associated changes in gene expression. This study has the potential to greatly enhance our understanding of the impact of level differentially methylated regions on gene expression and drought stress response, and provides a means to explore the basis of resistance to facilitate mulberry breeding.Mulberry (https://ncbi.nlm.nih.gov/subs/sra) with an accession number of PRJNA579451. Up to ~70 million sequencing reads were generated for each replicate and ~12.15 Gb clean bases representing >20-fold coverage of the NCBI reference genome were generated after filtering low quality reads, N reads and adaptor sequences. The clean rates from six mulberry samples were 87.29% to 89.96%  and 3 drought stressed samples (DS)) were sequenced using WGBS technology using Illumina Hi-Seq. 2500 platforms . Then, the corresponding methylomes were decoded and analysed. Our data have been deposited in the NCBI Sequence Read Archive (SRA)  and the external treatment provided (CK and drought). The CK-treated genome had methylation levels of ~9.95% (mCs), 33.03% (mCG), 20.63% (mCHG) and 2.86% (mCHH) for total sequenced C, CG, CHG and CHH sites, respectively, which reflected the percentage of methylation levels in the genome. Accordingly, DS-treated plants had methylation of ~10.81%, 35.09%, 20.96% and 2.98% in C, CG, CHG and CHH contexts Table  showing P ≤ 0.05). There was no obvious difference of the percentage of mCG and mCHG methylation level between CK and DS (P > 0.05). From Table P > 0.05) and the mCHG methylation level of CK and DS had significant difference (P ≤ 0.01) except for chromosomes fakechr5. This suggested that the mulberry methylation level was dynamic with environments and that mCHG methylation may be very closely correlated with drought stress.As shown in Fig. P > 0.05) in different regions of the mulberry genome between CK and DS except for mC methylation in CpGIsland (P ≤ 0.01), mCG methylation in CpGIsland (0.01 < P ≤ 0.05) and mCHG methylation in Up2k (0.01 < P ≤ 0.05).To explore the DNA methylation patterns in different regions of the mulberry genome, we analysed the methylation profiles within genes, including coding sequences and introns. In each context, and in both CK and DS genomes, methylation levels of mCG were highest and levels of mCHH methylation were lowest when the three CG, CHG and CHH types of methylation sites were compared. This was true for different genomic regions of mulberry Table . mCG metWe further evaluated the proportion of mCG, mCHG and mCHH observed relative to total mC occurring within distinct genomic elements Fig. , which cIn order to reveal the relationship between DNA methylation profiles and genes expression in detail, canonical DNA methylation profiles of the entire transcriptional units were divided into 7 distinct functional elements to study the changes of methylation level. As shown in Fig. 13. To reveal whether there was a relationship between sequence context and methylation preference in mulberry, we calculated the percentage methylation of all 9 bp sequences in which methylated cytosines were located in the fourth position using a tool named Logo Plots to explore sequence information for a methylated site or its nearby region. At the same time, the genome-wide cytosine methylation patterns were also analysed. The entropy of base was 0 as the minimum value, which indicated the four bases were evenly proportioned with 25%; the entropy of base was 2 as the maximum value, which indicated the four bases were most unevenly distributed and only a specific bases appeared, such as the fourth of C and the fifth of G. In the mulberry genome, the sequence preference around methylated sites were differed depending on mCG and non-CG context for all methylated mC sites  are stretched of DNA in a sample’s genome that have different DNA methylation patterns compared with other samples. To reveal the potential role of methylation in response to drought stress, we analysed DMRs of drought-stressed mulberry plants compared with normally grown mulberry plants. A sliding-window approach was used to identify DMRs that contained at least five CG (CHH or CHG) sites and the methylation levels from the two different samples assessed differedhttp://www.genome.jp/kegg/) pathway analysis was performed. As shown in Fig. Relative to control plants, 11,676 differentially methylated genes (DMGs) located in DMRs were identified Table , and DMGThe promoter regions of genes affect transcriptional regulation, and differentially methylated promoters may affect transcriptional expression. To characterize changes in the methylation status of promoter regions in mulberry under drought stress, we analysed differentially methylated promoters (DMPs) located within DMRs and 4,233 DMPs were identified Table , and DMPDNA methylation regulates genes in a wide variety of biological processes. To investigate potential transcriptional consequences of widespread methylation changes associated with drought stress, we performed mRNA-seq on the same normal growing conditioned and drought stressed mulberry plants that were used for methylation profiling. In total, 16010 different expression genes (DEGs) were identified between CK and DS Table , and 5,7MaMET1 under drought stress gradually ascend to the maximum with 1.93 fold (P ≤ 0.01) of the 0 day on the 5 day and then straightly reduced to the level of about 0.89 times (P ≤ 0.01) of the 0 day on the 10 day. The expression levels of MaDRM members were similar and got the highest at 5 days. MaCMT2_X3 and MaCMT3_X1 had the semblable express change and got the highest expression level at 5 days and presented the lowest level of about 0.57 times (P ≤ 0.01) of the 0 day at 10 days. The MaCMT3 and putative MaROS1 also showed highest expression level at 5 days. However, the MaDME expression gradually increased and went up to the maximum with 5.02 fold (P ≤ 0.01) of the 0 day on the 10 day. These results showed that drought stress would affect the expression pattern of genes encoding DNA methyltransferases and demethylases in mulberry.At the genome scale, we globally investigated DNA methyltransferases and demethylases encoded by methylation-related genes in mulberry, which play crucial roles in maintenance of genomic methylation Table . We founTransposable elements (TEs) can affect genome size, create mutations with insertion and excision, and affect gene expression. To characterize the methylation profiles of transposable elements in mulberry, we first identified members of TEs and members of the top 10 various classes of TEs in the mulberry genome Fig. . We founArabidopsis13, rice36, tomato37 to woody plant like apple30, poplar38 and spruce39. Here, we deciphered the mulberry methylome by WGBS and found that CK-treated genome had methylation levels of ~9.95% (mCs), 33.03% (mCG), 20.63% (mCHG) and 2.86% (mCHH) for total sequenced C, CG, CHG and CHH sites, respectively, which reflected the percentage of methylation levels in the genome. Previous studies showed that mCG methylation shows the highest levels among the various species, ranging from ~30.5% in Arabidopsis to ~92.5% in Beta vulgaris. mCHG methylation varied from ~9.3% in Eutrema salsugineum to ~81.2% in Beta vulgaris, and mCHH methylation ranged from ~1.1% in Vitis vinifera to ~18.8% in Beta vulgaris40. Among various species, mulberry had moderate methylation levels. Based on previous studies, genomic DNA methylation patterns will be changed in response to drought stress38. Accordingly, DS-treated plants had methylation of ~10.81%, 35.09%, 20.96% and 2.98% in C, CG, CHG and CHH contexts showing that methylation levels were generally up-regulated after drought stress compared with controls. We also investigated the relative proportions of mCG, mCHG and mCHH of total mC sites in mulberry, presenting ~44.28%, ~28.50% and ~27.22% methylation levels at mCG, mCHG and mCHH sequences in CK mulberry plants. And our studies were consistent with the previous methylome researches which indicated mCG methylation was highest levels among three types of methylation, and mCHG and mCHH methylation levels tended to be lower40. Notably, drought stress was associated with a slight increases in the proportions of mCG and mCHG methylation, and a decrease in mCHH methylation in mulberry. These data showed that drought stress induced methylation in two sequence contexts, including mCpG and mCHG, and displayed hyper-methylation pattern after drought stress, which was consistent with previous research41.As a specifically operative technology, WGBS has been used for ascertaining methylation patterns with singlenucleotide resolution and has been used to decipher many plant methylomes, ranging from herbaceous plants like 42. In the mulberry genome, most of the mCG methylation sites were 80–100% methylated, while mCHG sites were 20–100% methylated and mCHH sites were 20–30% range. This trend was also observed in the genomic DNA of mulberry. Similar trends were also observed in Populus trichocarpa 43 and Betula platyphylla 44. Work in Arabidopsis thaliana, however, revealed that the percentage of methylation of mCG, mCHG and mCHH were 24.60%, 6.98% and 1.70%, respectively45. These findings suggested that methylation levels dynamically change in accordance with external environmental conditions and are predominantly correlated with environmental factors.Sub-telomeric regions of the chromosomes had greater DNA methylation densities, which might be important for the control of telomere length and recombination. Furthermore, the methylation status of mCG, mCHG and mCHH varied according to the age, location and physiology of a single organism46. Other than CG methylation, mCHG and mCHH types of methylation typically do not occur within genes and are mainly found in intergenic and repeat-rich regions of the genome. Methylation within these sequence contexts plays a critical role in the silencing of transposons47. mCG methylation was highest in TTRs and down2k regions from genes and lowest in the CDSs and exons. mCHG and mCHH methylation was highest within CpG islands and lowest in the CDSs and exons. Publications have previously revealed an obvious enrichment of methylation in TEs, and 5′-UTR regions had the lowest levels of methylation in Betula platyphylla44. mCG methylation accounted for approximately half of the total mC observed, and mCHH occurred least frequently in each genomic region and under both treatment (CK and DS), which suggested that mCG sites are the most important type among the three sequence contexts examined. This was in accordance with previous findings in upland cotton31. Repeat sequences were the most highly methylated among genomic regions assessed. In maize seedlings, methylation most frequently occurred in CpG islands within the 5′ regulatory regions of genes (promoters)48. To reveal the relationship between DNA methylation profiles and genes expression levels, canonical DNA methylation profiles of the entire transcriptional units were also divided into seven distinct functional elements in castor bean seeds50. The methylation level of the gene region and its adjacent bases are one of the epigenetic regulation mechanisms of gene expression. The distribution of the mCG methylation sites in 7 distinct genomic functional elements showed strongest regularity, while mCHG and mCHH methylation sites had the some regularity.DNA methylation status varied depending on the genomic region considered, which include euchromatin and heterochromatin, repetitive sequences and coding sequences. In general, mCG methylation is observed within both genes and repetitive sequences, and is involved in the regulation of gene expression31. In the mulberry genome, the sequence preference around methylated sites were differed depending on CG and non-CG context for all methylated mC sites, and most methylation at mC sites occurred within the CAN  context. There was no significant difference between normally grown mulberry plants and those exposed to drought stress. Study in Arabidopsis thaliana suggested that mCG methylation sites were highly enriched for the sequence ACGT, and methylation of mCHG sites were depleted of upstream cytosines, but tended to contain cytosine following the methylated base13. However, there has been no obvious sequence context specificity and no correlation between sequence context and methylation preference observed in white birch44.In some eukaryotic organisms, the occurrence of cytosine methylation has been associated with its nearest sequence context51, however, the function of gene body methylation requires further study52.DMRs are used for genetic imprinting because they can be methylated in accordance with either the maternal or the paternal chromosome. The methylated allele is often, but not always, the silenced allele. Differences between the methylation patterns of parental and offspring chromosomes are considered epigenetic lesions. In total, 20,485 DMRs were identified from plants subjected to drought stress. Relative to control plants, 11,676 DMGs and 4233 DMPs located in DMRs were identified, and they were mainly assigned to pathways related to the regulation of autophagy, AMPK signaling, homologous recombination, the ras signaling pathway, MAPK signaling pathway, sphingolipid and so on. Previous research has prompted scientists to hypothesise that gene body methylation suppresses spurious transcription from cryptic promoters that might otherwise interfere with the regulation of gene expressionArabidopsis, dandelions, maize and Pyropiahaitanensis56. We identified 3243 different methylation and expression associated genes (DMEGs) that had a significant difference in methylation and expression levels in mulberry under drought stress compared to normal growing conditions, which demonstrated that there were obvious correlation between DNA methylation and gene transcription. The DMEGs were highly enriched in catalytic activity, binding, ribosome, biosynthesis of amino acids, and starch and sucrose metabolism. In this study, we found that many differentially DNA methylated regions could be induced by drought stress in mulberry plants, and some DNA methylomes regions were associated with gene expression. Similar results were also observed when Arabidopsis57 under pathogen infection and apple30 exposed to drought stress. The gene expression pattern could be affected by the changes of DNA methylation level through direct and indirect ways. In apple, the expression levels of methylation-related genes encoding DNA methyltransferases and demethylases had changed under water deficit30. The mRNA level of some methyltransferase and demethylase genes also had significant differences under drought stress in mulberry. These data will be useful for determining the impacts of differentially methylated regions on gene expression, transgenerational inheritance and stress response, and providing a means to explore the basis of resistance to facilitate mulberry breeding.Under some certain circumstances, such as abiotic and biotic stresses, gene transcription level could be altered by the changes of DNA methylation, and lead to visible phenotypes. Many studies had shown that DNA methylation played an important role in the regulation of gene expression in response to various abiotic and biotic stresses in plants including In conclusion, we provide a high-resolution and high-coverage map that shows the methylation status of individual cytosines throughout the mulberry genome in response to drought stress. We found that the overall methylation level of plants subjected to DS was 8.64% higher than that of control plants, which was mainly due to the contribution of mCG methylation sites (6.24%). Further, methylcytosine was most frequently observed at mCG sites (44.28%), and less frequently observed at mCHG and mCHH sequences . We further analysed the distribution of methylation sites throughout genomic regions and concluded that methylation tended to occur most frequently in distinct genomic elements. Our results showed the drought stress response in mulberry was probably initiated by the methylation of coding sequences and by the methylation of some promoters. Additionally, there were 3,243 DMEGs that displayed significantly different methylation and expression levels in control mulberry plants compared to those subjected to drought stress. In addition, methylated genes were highly enriched for the GO subcategories of catalytic activity, binding, membrane, cellular process, metabolic process, response to stimulus and regulation of biological process, suggesting that methylation mediates these pathways in mulberry. These data will require additional experimental data to confirm their role in the response to drought. This is the first study to comprehensively present a methylome map of the mulberry genome and reveal the occurrence of widespread DNA methylation changes in response to drought stress. The map will facilitate future studies investigating potential linkages between DNA methylation and gene expression in plants challenged with abiotic and biotic stresses.58. Mulberry cuttings were grafted to rootstocks. The grafted nurseries were planted in pots that were 35 cm in diameter that contained loam soil and one grafted plant per pot. The grafted plants were randomly grouped when new shoots had grown to 20 cm in length with two months old. The control group was well watered and irrigated daily to maintain 75–85% soil moisture that measured by soil moisture content analyser . The other group was exposed to drought stress for 5 d, 8 d and 10 d, and drought stress was conducted by withholding water to achieve 20–30% soil moisture . For the same treatment groups, equal quantities of DNA samples from different timepoints mixed together.Total genomic DNA was extracted from leaves according to a modified CTAB DNA extraction protocol31. A total amount of 5.2 μg genomic DNA spiked with 26 ng lambda DNA was fragmented by sonication using a Bioruptor  to a mean size of approximately 250 bp. This was followed by the formation of blunt ends in which dA addition to 3’-end, and adaptor ligation (in this case of methylated adaptors to protect from bisulfite conversion) was performed according to the manufacturer’s instructions. Lambda DNA was used as an unmethylated control for calculating the bisulphite conversion rate. Then, the ligated DNA was bisulfite converted using the EZ DNA Methylation-Gold kit . Different sized insert fragments were excised from the same lane of a 2% TAE agarose gel. Products were purified by using the QIAquick Gel Extraction kit  according to the manufacturer’s protocol and amplified via PCR. BS-seq library constructions and sequencing was performed on three biological replicates per sample. Finally, libraries were sequenced using the Illumina Hi-Seq 2500 platform . Three replicates from both control samples and drought stressed samples were assessed.The method of BS-seq library construction and sequencing in accordance with previous studies34 using default parameters. The reference genome was converted into a bisulphite-converted version (conversion of C to T and G to A) and then indexed using Bowtie260. Sequence reads were also transformed into fully bisulphite-converted versions (C to T and G to A converted) before reads were aligned to similarly converted versions of the genome in a directional manner. Then duplicated reads were removed and mapping results of each library were merged. Finally, we were able to calculate the mapping and bisulfite conversion rates for each sample.After sequencing data was delivered, we filtered results using SOAPnuke software  to remove adaptor sequences, contamination and low-quality reads from raw reads. Low-quality reads included two types, and the reads meet anyone of the two conditions would be removed: 1) Unknown bases were more than 10%; 2) The ratio of bases whose quality was less than 20 was over 10%. After filtering, the remaining reads were called “clean reads” and the clean data was then mapped to the reference genome using BSMAP software61, which was also equal to the mC/C ratio for each reference cytosine32. A sliding-window approach was applied for methylation level analyses. A window size of 3,000 bp and a step size of 600 bp30 was used. The sum of methylated and unmethylated read counts were calculated for each window. The methylation level of each C site reflected the fraction of methylated Cs observed, and was calculated using the following formula:Methylation levels (MLs) were determined by dividing the number of reads covering each mC site by the total reads covering that cytosine62. Given the bisulfite nonconversion rate r, the corrected ML was estimated using the following equationas:Calculated ML was further corrected by considering the bisulfite nonconversion rate according to previous studieshttp://122.228.158.106/ swDMR/), which used a sliding-window approach. The window was set to 1,000 bp and the step length was 100 bp. The Fisher test was implemented to detect DMRs35.DMRs were identified using the swDMR software  sites with a 2-fold change in MLs and Fisher test 35:To calculate the degree of difference between methyl-cytosines  of two samples while comparing MLs of DMRs from different samples CIRCOS was used according to the following formulaRm1, Rm2 represented the MLs of methyl-cytosine for CK and DS respectively, and 0.001 replaced Rm1 (or Rm2) in cases in which it equaled zero.30. Finally, libraries were sequenced using the Illumina Hi-Seq 2500 platform . Three replicates from both control samples and drought stressed samples were assessed.The mulberry plant materials used for transcriptome analysis were the same as the materials applied in methylome analysis and three biological replicates were used for transcriptome analysis. Total RNA was isolated using Trizol reagent  according to the instructions of the manufacturer. RNA quality and integrity were tested by Agilent Bioanalyzer 2100  and Nano Drop ND-1000 Spectrophotometer . A total amount of 1 μg qualified RNA was used for the RNA sample preparations and sequencing library was constructed using NEBNext Ultr RNA Library Prep Kit  for Illumina according to the manufacturer’s instructions. The library fragments were purified and enriched with AMPure XP system  to create final cDNA fragments with the size as 250–300 bp58. A total of 1.0 μg RNA was used for first-strand complementary DNA (cDNA) synthesis using M-MLV-reverse transcriptase (RTase)  according to the manufacturer’s instructions. RNA and primers were mixed with DEPC-treated water up to 17 μl and incubated at 70 °C for 10 min. Then the samples were subjected to immediate cooling on ice for more than 2 min. Then, 5 μl 5× RT-Buffer, 1.25 μl 10 mM dNTP, 0.75 μl RNase Inhibitor and 1 μl M-MLV RTase were combined in a total reaction volume of 20 μl. The synthesis was performed at 42 °C for 60 min and the inactivation of the enzyme was achieved by maintaining a temperature of 72 °C for 15 min. The resultant cDNA was used as a template for qRT-PCR and the amplification mixture volume was 20 μl per reaction. The internal housekeeping gene, β-actin, was used as a normalisation control63. The primers for targets and β-actin were added to FastStart Universal SYBR Green Master Mix kit  according to the manufacturers instructions, and 20 μl of the reaction mixture was added to each well. Reactions were performed in a LightCycler 96 Real-Time PCR System  with thermal cycling parameters that included 95 °C for 600 s followed by 45 cycles of 95 °C for 10 s, 59 °C for 10 s and 70 °C for 10 s64. The sequences of primers are listed in Table −ΔΔCt method. Standard errors and standard deviations were calculated from replicates and significance was measured via an one-way ANOVA and Duncan’s multiple test at the level of 0.01 < P ≤ 0.05 and P ≤ 0.01, respectively.At the genome scale, we globally investigated DNA methyltransferases and demethylases encoded by methylation-related genes in mulberry, which play crucial roles in maintenance of genomic methylation, and analysed expression level of methylation-related genes under drought stress. “Wansang 1” mulberry plant materials were used for qRT-PCR analysis. Plants were grown in plastic pots, and were provided the same weight of soil and irrigation conditions. One-month-old, uniformly-grown plants were subjected to drought, and leaves were harvested after 0, 1, 3, 5, 8 and 10 days. The untreated samples (watered and irrigated daily) were used as the controls. Total RNA was isolated from the leaves of control and drought-stressed plants using RNAiso plus  according to the manufacturers’ instructionshttp://www.geneontology.org/), calculating gene numbers for every term, then used hypergeometric test to find significantly enriched GO terms in the input list of DMGs, DMPs and DMEGs, based on “GO:: TermFinder” , and the method used was described as follows66:DMGs, DMPs and DMEGs were analysed for GO analysis and KEGG enrichment. GO is an international standard gene functional classification system, offers a dynamic-updated controlled vocabulary, as well as a strictly defined concept to comprehensively describe properties of genes and their products in any organism. GO enrichment analysis provided all GO terms that significantly enriched in a list of DMR related genes, comparing to a genome background, and filter the DMR-related genes that correspond to specific biological functions. Firstly, all DMR-related genes were mapped to GO terms in the database . Here, N represented the number of all genes with the KEGG annotation, n represented the number of DMGs, DMPs and DMEGs in N, M was the total number of annotated to specific pathways, and m was the number of DMGs and DMPs in M. KOBAS software was used to test the statistical significance of enrichment of DMGs, DMPs and DMEGs in KEGG pathways70.KEGG analysis was used to perform pathway enrichment analysis of DMGs, DMPs and DMEGsP ≤ 0.05 and P ≤ 0.01. Letters superscripted above the bar indicate the significant difference of the changes between differently treated groups with the level of P > 0.05 (shown as the same or no letter superscripts), 0.01 < P ≤ 0.05  and P ≤ 0.01 .The study was performed independently three times and each result shown in the figures was expressed as the mean ± standard deviation (SD). Statistical analysis was performed using Excel 2013 software  and SPSS Statistics 19.0 software  with One-Way ANOVA Duncan’s multiple range test at the level of 0.01 < Supplementary Information.


Intermittent preventive treatment of malaria in infants (IPTi) with sulfadoxine-pyrimethamine (SP) is a proven strategy to protect infants against malaria. Sierra Leone is the first country to implement IPTi nationwide. IPTi implementation was evaluated in Kambia, one of two initial pilot districts, to assess quality and coverage of IPTi services.This mixed-methods evaluation had two phases, conducted 3 (phase 1) and 15–17 months (phase 2) after IPTi implementation. Methods included: assessments of 18 health facilities (HF), including register data abstraction (phases 1 and 2); a knowledge, attitudes and practices survey with 20 health workers (HWs) in phase 1; second-generation sequencing of SP resistance markers (pre-IPTi and phase 2); and a cluster-sample household survey among caregivers of children aged 3–15 months (phase 2). IPTi and vaccination coverage from the household survey were calculated from child health cards and maternal recall and weighted for the complex sampling design. Interrupted time series analysis using a Poisson regression model was used to assess changes in malaria cases at HF before and after IPTi implementation.dhps K540E mutations increased but remained below the 50% WHO-recommended threshold for IPTi . From the household survey, 299/459 (67.4%) children ≥ 10 weeks old received the first dose of IPTi ; 274/444 (62.5%) children ≥ 14 weeks old received the second IPTi dose ; and 83/217 (36.4%) children ≥ 9 months old received the third IPTi dose (versus 52.2% for first measles vaccine dose). HF register data indicated no change in confirmed malaria cases among infants after IPTi implementation.Most HWs (19/20) interviewed had been trained on IPTi; 16/19 reported feeling well prepared to administer it. Nearly all HFs  had SP for IPTi in stock. The proportion of parasite alleles with Kambia district was able to scale up IPTi swiftly and provide necessary health systems support. The gaps between IPTi and childhood vaccine coverage need to be further investigated and addressed to optimize the success of the national IPTi programme. Despite advancements in malaria prevention and control, there were an estimated 229 million malaria cases and 409,000 malaria deaths in 2019 –9.Plasmodium falciparum enzyme dihydropteroate synthetase (dhps) gene K540E mutation) [IPTi with SP is recommended for countries  with moderate-to-high malaria endemicity where SP resistance levels are not high  . AlthougP. falciparum. Malaria remains the leading cause of both disability-adjusted life years (DALYs) and years of life lost (YLL) due to premature death in Sierra Leone [Malaria is endemic in Sierra Leone, with stable and perennial transmission throughout the country. Over 85% of malaria cases in Sierra Leone are attributed to ra Leone , with mora Leone . The Natra Leone . Despitera Leone , 12.In 2015, the NMCP formally added IPTi with SP to the 2016–2020 National Malaria Strategic Plan in recognition of the need to aggressively combat malaria. In 2017, with support and technical guidance from partners and donors, the NMCP launched IPTi within the existing EPI delivery platform for child immunization services. Implementation was rolled out in two pilot districts, Kambia and Pujehun, in March 2017 and two additional districts, Kenema and Western Area Urban, in August 2017 before national scale-up in the remaining 10 districts in three waves in 2018. An IPTi Task Force was created to guide strategic planning activities, including the development of an Implementation Field Guide, adapted from the WHO Global Malaria Programme , adaptatTrainings were designed in a cascading manner, with an initial national-level training of trainers for 40 participants, including NMCP representatives and District Health Management Teams (DHMT) from Kambia and Pujehun. This was followed by district-level trainings for up to two staff members from each health facility. Interactive training sessions were designed to teach participants about IPTi; foster and practice skills on messaging IPTi to other health workers (HWs), caregivers, and community members; reinforce appropriate dosage and delivery of SP; and demonstrate correct completion of monitoring and reporting tools. At the end of the district training, staff received the first allotment of SP stocks and materials  for their health facility.Supporting materials such as posters, fliers, job aids, monitoring and evaluation (M&E) tools, and social and behaviour change materials were also developed and distributed to all health facilities. A community education strategy that included radio spots introducing IPTi was launched prior to implementation in Kambia and Pujehun. Additionally, as part of a coordinated effort by the NMCP and the DHMTs, IPTi supportive supervision checklists were developed and implemented during supervisory visits conducted at HF to monitor IPTi implementation in the pilot districts.To inform the national scale-up of IPTi and to document lessons learned from the first, and only, national IPTi programme, an external evaluation was conducted. The evaluation assessed the quality, acceptability, and coverage of IPTi pilot activities in Kambia to provide insights into successes, challenges, and areas for improvement.A mixed-methods evaluation was conducted in Kambia with two phases of data collection. Phase 1 took place in March 2017, pre-IPTi implementation; and July 2017, three months after the launch of IPTi implementation. Phase 2 took place July–September 2018, 15–17 months after IPTi implementation.The evaluation took place in the district of Kambia, the first to implement IPTi. Kambia district is located in northern Sierra Leone bordering Guinea and has a population of more than 340,000 inhabitants, most of whom are farmers. In 2017, Kambia had 69 public health facilities, including one district-level hospital, 15 community health centres, 16 maternal and child health posts, and 37 community health posts. In Kambia, according to the 2016 Malaria Indicator Survey, 48.7% of children under 5 had slept under an ITN the previous night, 48.3% of children 6–59 months were positive for malaria via microscopy and 86.6% of women 15–49 years received two or more doses of SP in the last pregnancy . The MinData collection methods included a mix of: (a) health facility (HF) assessments; (b) a knowledge, attitudes, and practices (KAP) survey with HWs; (c) HF register data abstraction; (d) a household survey; and (e) analysis of molecular markers of SP resistance. The timing of the various data collection methods is presented in Fig. HF assessments, conducted in both phase 1 and phase 2 with purposively recruited persons in-charge, vaccinators, and laboratory/pharmacy staff, captured HF characteristics including size, patient volume, staffing, number of HWs trained in IPTi, and availability of SP, equipment, supplies, and IPTi monitoring tools. HFs were selected among those providing immunization services using stratified simple random sampling by HF type. A total of 18 HFs were included in the sample, representing a quarter of all HFs in Kambia district. The same HFs were visited in phases 1 and 2.The KAP survey was conducted in phase 1 with purposively recruited HWs who were providing IPTi to eligible infants at HF selected for the HF assessment. After obtaining written consent, trained interviewers conducted the KAP survey in English or Krio using a tablet-based survey. The knowledge domain of the questionnaire consisted of multiple-choice questions designed to assess fundamental knowledge of IPTi. The attitudes and practices domains used single-selection closed-ended questions to explore respondents’ impressions of the IPTi program and its acceptability to HWs and families, and their practices when administering IPTi to eligible infants.As part of the HF assessments in phase 1 and 2, routine data on malaria cases, and vaccine doses and IPTi doses administered were abstracted from the outpatient and EPI registers and monthly summary forms at the selected HFs. Each abstraction was conducted in both phase 1 and phase 2 and included data for the previous 13–14 months (from April 2016-June 2018).In phase 2, a household survey was conducted with caregivers of children 3–15 months old to assess perceptions, acceptability, and coverage of IPTi. Households were selected using a two-stage random selection process. At the first stage, 44 enumeration areas (EAs) were randomly selected from the 2015 Sierra Leone census list using simple random sampling, stratified by urban or rural geographic setting. Within each selected EA, all households were mapped and enumerated, generating a household listing with information on the age and sex of all household members. Then, simple random sampling was used to select 10 eligible households (at least one caregiver 18 years or older of a child aged 3–15 months) per EA. In EAs that had less than 10 eligible households, all eligible households were selected in EAs. Assuming a non-response rate of 10% and a design effect of 2 (given clustering of children within chiefdoms and unknown intracluster correlation coefficient), a sample size of 440 households was needed to have sufficient precision to determine an IPTi coverage of 50% with a ± 7-percentage point margin of error (95% CI 43–57%) among children aged 3–15 months.Survey teams visited selected eligible households, identified all eligible child(ren)’s caregiver(s), and introduced the purpose of the survey. After obtaining written informed consent, the survey team administered the household questionnaire to the caregiver(s) of all eligible children in the household in their preferred language . If the caregiver was not home at the time of the initial visit, the survey team returned to the household three times on different days. Survey questions were entered by interviewers in a tablet programmed using SurveyCTO , based on Open Data Kit platform and included questions on household characteristics; literacy and education of caregivers; awareness of health messages; health-seeking behaviours; knowledge of malaria; ownership/use of ITNs; knowledge of IPTi services; experiences with IPTi services/receipt of IPTi by eligible children; health status/recent illness or hospitalization; and child immunization history. To measure coverage of IPTi and other routine immunizations, data were abstracted from available child health cards. Similar to the approach used on the Sierra Leone Demographic and Health Surveys , when chP. falciparum dhps gene and N51I, C59R, and S108N mutations of the dhfr gene, which have been shown to confer resistance to sulfadoxine and pyrimethamine, respectively [(dhfr) gene  and the double dhps  mutations collectively form the quintuple mutation [P. falciparum treatment failure [dhps and dhfr genes to generate estimates of the prevalence of key mutations among the parasite population found in Kambia at each time point. Sequencing data was obtained using molecular inversion probes (MIP), a novel technology for high-throughput deep sequencing of Plasmodium species [dhps and ASTR [At two separate HFs in Kambia not included in the HF assessment, verbal consent was obtained from caregivers of children < 7 years and from adults ≥ 18 years presenting to the outpatient department and being tested for malaria before IPTi implementation (pre-IPTi in March 2017) and approximately one year later (post-IPTi in May 2018). Among those consenting, the same finger prick was used to conduct the malaria rapid diagnostic test as well as to collect a sample of blood on Whatman 3MM chromatography paper. These dried blood spots (DBS) from malaria-positive individuals were retained and were shipped to the Infectious Disease Epidemiology and Ecology laboratory at the University of North Carolina, USA. DNA was extracted from the DBS using Chelex extraction in pools of 10 DBS per pool and prepared for amplicon deep sequencing , 16. Seqmutation  which co failure . In addi failure . The poo species –25. Indiand ASTR . SamplesDescriptive analyses were conducted for data from the HF assessments, KAP survey, and household survey in SAS v 9.4 . For the molecular analyses, differences in the proportions of resistant alleles were compared using Fisher's exact tests using the R statistical platform v 3.6. For the household survey, sample means/proportions for measures of interest and their 95% confidence intervals were estimated and weighted for unequal sampling probabilities and non-responses and further adjusted for clustering of respondents using the ‘svy’ commands in Stata 14.0  and the Rao-Scott correction for chi-square tests . Three bInterrupted time-series analysis using a Poisson regression model and generalized estimating equations with robust variance estimation was used to analyze confirmed malaria cases among patients < 12 months based on abstracted HF register data. Covariates in the model included the total number of outpatient visits among patients < 12 months, the proportion tested for malaria, and a dummy variable for high malaria transmission season (July – October).versus 12/18 in phase 2). No change was seen in the number of HFs providing IPTi through outreach services at least once per week (8/18) or in those providing IPTi for free (18/18). Most (15/18 in phase 1 and 18/18 in phase 2) HFs reported having supervisory visits in the last six months that addressed IPTi provision.The 18 sampled HFs included 1 clinic for children under five years of age at the district hospital, 4 community health centres, 3 community health posts, and 10 maternal and child health posts. Nearly all HFs (17/18 in phase 1 and 18/18 in phase 2) had SP for IPTi in stock  aides, one community health assistant, and one immunization officer at 18 HFs. Among the 19/20 HWs trained on IPTi, most rated the didactic training provided by the DHMT as good (15/19) or excellent (3/19) quality and felt confident in providing IPTi services (16/19) as a result of training. Results of the KAP survey indicated a good understanding of IPTi among HWs. Of 20 respondents, 15 scored at least 80% on the 20-question knowledge assessment . Among identified caregivers (449), all but one were women, the mean age was 26.2 years, and 90% were married (Table Caregivers reported that most of their children (88.5%) slept under an ITN the previous night Table . Among tAmong caregivers, 67.4% reported they had heard about a “new way to prevent infants from getting malaria,” of whom 97.9% were specifically aware of a “new tablet to prevent malaria in infants.” Of these, 96.1% believed the “new malaria preventative tablet for infants” was very safe. Among the 296 caregivers who heard messages on “a new way to prevent infants from getting malaria”, the most common source of information was government clinic/hospital (84.5%), followed by community health workers (67.4%) and radio (25.4%).Child health cards were available and verified for 395/459 86.2%) of the sampled children. Vaccination status was determined based on child health card for 395 (86.2%) and caregiver report for 64 13.8%) who did not have a card. Among children ≥ 10 weeks old (n = 459), 67.4% had received the first dose of IPTi, and 80.3% received the second pentavalent dose, given at the same time); among children ≥ 14 weeks old, 62.5% had received the second dose of IPTi and 65.4% received the third pentavalent dose, given at the same time. Among children ≥ 14 weeks (n = 444), 54.9% received IPTi-1 and IPTi-2. Among children ≥ 9 months old (n = 217), 36.4% had received the third dose of IPTi and 52.2% had received the first measles vaccination, given at the same time , for the second dose was 17.4 weeks (3.4 weeks median delay), and the third dose was 9.3 months (~ 1.2 week median delay), similar to other routine vaccines co-administered with IPTi.Overall, 138 (29.2%) surveyed children did not receive any IPTi. When caregivers whose children had never received IPTi were asked for the reasons why, they most frequently cited that they did not know IPTi was required (34.7%), their child was not offered IPTi (14.4%), or their child had not visited a facility for immunizations since birth (10.5%). Other reasons cited were lack of convenient times/places for IPTi, fear of side effects, distance or inability to pay transportation and/or facility costs, and lack of SP at the facility  , nor was ITN usage among infants . However, the proportion of parasite alleles with the dhps K540E and A581G mutations marginally increased between phases 1 and 2 in samples from the two clinics in Kambia . The proportion of parasite alleles with dhfr mutations N51I, C59R, and S108N remained fixed at 100% in both phases.A total of 151 and 153 DBS from RDT-positive patients with similar gender and age distribution were collected from two HFs at pre-IPTi and post-IPTi, respectively. Figure Despite the WHO recommendations for IPTi in malaria-endemic countries where SP resistance levels are not high, Sierra Leone is the first and only country to have introduced and scaled up IPTi nationally through the primary health care system . This evAccording to HWs, IPTi was well accepted among caregivers and community leaders. A qualitative assessment in Phase 1 that included focus group discussions with community leaders, community health workers, and caregivers showed that IPTi was generally well accepted in the community and emphasized the importance of the community education strategy and the distribution of promotional materials to ensure community acceptance of IPTi prior to implementation , 28. TheWhile this evaluation indicated that Sierra Leone was able to integrate IPTi within the routine EPI program in Kambia, the results highlighted gaps in uptake, especially when compared to other vaccines given at the same visits. While the design of this evaluation did not identify the concrete reasons for these differences in coverage between IPTi and routine EPI, there are some plausible explanations. One reason could be due to staff turnover or training issues. Reasons most frequently reported among caregivers whose children had never received IPTi included not knowing it was required and that their child was not offered IPTi, among others. Most HW reported having administered IPTi to all eligible infants they saw in the past week when asked in phase 1, but it is possible that compliance might have reduced over time or that new HW might have joined after the trainings. For these reasons, refresher trainings among HWs to ensure all children who attend a routine visit receive IPTi are essential to improve future uptake of IPTi.rd IPTi dose and 50% receiving the first dose of the measles vaccine. Due to recent measles outbreaks in Sierra Leone, the Ministry of Health has supplemented routine measles immunizations with campaigns and outreach services, which could explain the large differences observed as the evaluation was not able to distinguish immunizations provided at the facility or at the community level [Another explanation could be that children are vaccinated outside of routine EPI, through catch up, when IPTi is not offered. In fact, the difference in coverage with routine vaccines were particularly concerning for the 9-month visit, with only 36% receiving the 3ty level . In factty level . These vThe success of IPTi relies on a strong EPI program. Despite high acceptance of IPTi among HWs, community leaders, and caregivers, household survey results indicated room for improvement in IPTi coverage—to close coverage gaps with other immunizations given at the same time—as well as ideally, to increase coverage of both IPTi and EPI vaccines. Given the low immunization coverages in Sierra Leone , innovatdhps K540E mutation) [IPTi is recommended in areas where levels of parasite resistance are low  , and as utation) . Howeverutation) . As the One of the strengths of this evaluation is that it was conceptualized prior to IPTi roll-out at the national level in Sierra Leone. By focusing on one of the first districts implementing IPTi, it provided important insights to improve implementation and national scale-up. As the only country to scale-up IPTi at the national level, Sierra Leone’s experience provides valuable information to other countries considering IPTi implementation.One of the limitations of this evaluation is that it was not able to identify the exact reasons why IPTi coverage was below that of routine EPI. While some plausible explanations are presented, additional research will be needed to address this coverage gap and ensure the success of the national IPTi programme. Also, the evaluation was not able to directly assess if IPTi implementation had an effect on reducing malaria incidence among children in Kambia, as previously demonstrated –9. The eAdditionally, given the modest reduction of 30% in malaria cases in well-designed trials with high coverage , the effAccording to the 2020 World Malaria Report, based on current trends, many countries will not meet the WHO Global Technical strategy’s 2020 targets to reduce malaria incidence , 40. As Additional file 1: IPTi knowledge questionnaire.Additional file 2:Interrupted time-series analysis of confirmed malaria cases among patients <12 months old from register data abstracted from health facilities in Kambia (n=17).


However, in times of global warming, restoration projects must increasingly deal with extreme events such as drought periods. Here, we evaluate the effect of the European summer drought 2018 on vegetation development and the exchange of methane (CH4) and CO2 in two rewetted minerotrophic fens (Hütelmoor—Hte and Zarnekow—Zrk) including potential carry-over effects in the post-drought year. Drought was a major stress factor for the established vegetation but also promoted the rapid spread of new vegetation, which will likely gain a lasting foothold in Zrk. Accordingly, drought increased not only respiratory CO2 losses but also photosynthetic CO2 uptake. Altogether, the drought reduced the net CO2 sink in Hte, while it stopped the persistent net CO2 emissions of Zrk. In addition, the drought reduced CH4 emissions in both fens, though this became most apparent in the post-drought year and suggests a lasting shift towards non-methanogenic organic matter decomposition. Occasional droughts can be beneficial for the restoration of the peatland carbon sink function if the newly grown vegetation increases CO2 sequestration in the long term. Nonetheless, care must be taken to prevent extensive peat decay.Peatland rewetting aims at stopping the emissions of carbon dioxide (COThis article is part of the theme issue ‘Impacts of the 2018 severe drought and heatwave in Europe: from site to continental scale'. Cumulated on a global level, drained peatlands consume 10–41% of the remaining emission budget to maintain global warming below 2°C [Peatlands constitute the largest terrestrial carbon (C) stocks and exert distinct feedback mechanisms on the climate system ,2. The nong term . Howeverethane CH in the lethane CH in the lelow 2°C ,6. In coelow 2°C .2 sequestration and C accumulation [4 [2 emissions from intense organic matter respiration [Technically, rewetting is simply feasible by shutting down the drainage infrastructure and raising the water level. However, the preceding drainage can cause irreversible changes which constrain the ecosystem functionality of the rewetted peatland and thereby impede the achievement of the desired benefits. As peat decomposition and subsidence can lower the surface level by several decimetres , rewettimulation ,11. Furtmulation . Hence, ation [4 , there apiration . Altoget4 emissions and increase peat consumption and ecosystem respiration . Furthe2 uptake . Altogets of CO2 ,20. Alth4 and net CO2 exchange  including possible carry-over effects in the year after the drought. The study was conducted in two degraded rewetted fens in Northeast Germany. Both sites feature comprehensive datasets on vegetation dynamics and GHG exchange dating back to 2014 so that the patterns occurring during the drought and the post-drought year can be compared against a profound reference record.Here, we aim to provide an integrated understanding of drought effects on vegetation development and the evolving GHG dynamics in rewetted minerotrophic fens and further assess the prospects of peatland rewetting as mitigation measure under climate change. To do so, we investigated the impact of the European summer drought 2018 on vegetation development, CH2.(a)Phragmites australis, Carex acutiformis, Bolboschoenus maritimus and Schoenoplectus tabernaemontani [Though the two study sites differ with respect to genesis and diagenetic processes, they share a common typology and land-use history which are typical for the fens of the northeastern German Plain. The Hütelmoor (Hte) is a coastal paludification fen located directly on the Baltic Sea  and created a large shallow lake surrounded by dense dominance stands of Typha latifolia.The Polder Zarnekow (Zrk) is a fen with continuous percolating groundwater flow in the Peene River valley . Peat depth partially reaches 10 m. Drainage began in the eighteenth century and was intensified between 1960 and 1990 for grassland use. Subsequent peat decomposition and surface subsidence to levels below that of the Peene River required diking and pumping, which was discontinued in the early 2000s. Starting in winter 2004/2005, the site was rewetted with water levels permanently above the ground surface. Rewetting caused an extensive dieback of the previous vegetation (mostly (b)2 and CH4 exchange rates between the atmosphere and the land surface were measured with the eddy covariance approach. This micrometeorological approach provides a continuous time series of half-hourly gas fluxes on an ecosystem scale. Though instrumentation and configuration differed slightly between both sites, each of the measurement setups is well in line with the default practice used within the eddy covariance community. Half-hourly net CO2 and CH4 fluxes were processed with the software EddyPro version 6.0.0 . The measured net ecosystem exchange of CO2 (NEE) was partitioned into the two opposed component fluxes gross ecosystem productivity  and ecosystem respiration (Reco) by modelling. Reco, the CO2 released by autotrophic and heterotrophic respiration into the atmosphere, was obtained from night-time NEE fluxes. GEP, the photosynthetic sequestration of CO2 from the atmosphere into the canopy, was then calculated from the difference between NEE and Reco. The reference record to discriminate drought effects starts in 2014 when continuous measurements were available from both sites. Details of the instrumentation setup and flux processing, including gap filling and NEE partitioning, are presented in electronic supplementary material, S1. Here, we also provide further details on the acquisition of auxiliary data including (i) meteorological data, (ii) the MODIS enhanced vegetation index (EVI), which served as a proxy for plant phenology and coverage, and (iii) the classification approach to generate vegetation maps from aerial images.CO3.(a)Weather conditions in 2018 were characterized by a combination of persistent rain deficit and high temperatures across large parts of Europe. With only 454 mm annual precipitation at both of our study sites, 2018 was among the three driest within the last 30 years. In comparison to average years, rainfall deficits in 2018 amounted to 192 mm in Hte and to 168 mm in Zrk. Lack of rain occurred primarily from spring to summer and was most pronounced in May 2018 when precipitation hardly summed up to 5 mm . 2018 was also among the warmest two of the last 30 years with mean temperatures of 9.9°C in Hte and 9.7°C in Zrk . Above-average temperatures occurred primarily during the warm season. Eventually, the combination of persistent rainfall deficit and high temperatures in 2018 culminated in a pronounced water deficit (−670 mm at Hte and −680 mm at Zrk).(b)a,b). In Hte, the water level minimum was reached in September at 0.4 m below ground. At the same time, the lowering of the water table in Zrk stopped close below ground level due to water resupply from the nearby Peene river.Both sites showed similar hydrological dynamics throughout 2018, but were to a different degree affected by drought a,b. In Hc,d). However, as suggested by the following decline in EVI, the established vegetation began to suffer from drought stress when the spatially averaged water depth fell below 10–20 cm. The second peak in EVI occurred in late August when both sites ran completely dry, and the bare peat patches were rapidly colonized by new vegetation: in Hte, the bare flats were overgrown by Tephroseris palustris and Ranunculus sceleratus, which are pioneer plants specialized to promptly spread on nutrient-rich shores of dried-out river banks [Tephroseris palustris. However, the interpretation of these results in Hte is complicated as possible carry-over effects of the previous drought are likely masked by the vegetation response to brackish water intrusion in January 2019. In Zrk, where the second EVI peak was more pronounced, it was Typha latifolia, the prevailing plant species on the spot, that transgressed previous habitat lines and thereby increased vegetation cover from 74 to 81%. Vegetation cover was still 80% in 2019, indicating that the new distribution lines of Typha latifolia persisted in the year after the drought.In both fens, the EVI peak 2018 exceeded the values of the reference records from 2014 to 2017, indicating exceptionally good growth conditions for the established vegetation in the first half of the year c,d. Howeer banks . AlthougAltogether, meteorological conditions in 2018 affected vegetation dynamics in two ways: in the first half of the year, high temperatures and radiation supply fostered exceptional growing conditions for the established vegetation until most parts of the fens fell dry and new plants, that might have been dominant before or not, took over. This pattern occurred synchronously at both study sites despite the different reference situations regarding canopy structure and species composition. Further, the massive spread of these plants is especially noteworthy as vegetation development in both sites had mostly stagnated throughout the post-rewetting period ,14. The (c)2 m−2 in Hte and Zrk . These exceeded the average annual GEP totals of the reference years 2014–2017 by 22 and 86%, respectively. Peat drought studies often report a reduction in GEP, as drought stress of plants reduces photosynthetic CO2 uptake [2 uptake was significantly higher than in reference years, as the established vegetation benefited from high radiation and high temperatures before the water finally became scarce. GEP was further boosted in the second half of the drought year, when the dried-out areas were colonized with new vegetation. In this way, very high annual GEP sums could be reached in 2018 despite drought stress of the established vegetation starting in mid growing season. In 2019, annual GEP sums in Hte decreased considerably to 2.56 kg CO2 m−2, which is probably due to the vegetation response to brackish water intrusion at the beginning of the year. Due to this event, the 2019 observations in Hte cannot be solely interpreted in relation to the previous drought period. GEP in Zrk, on the other hand, remained high in 2019, reaching annual sums of 3.39 kg m−2. This finding suggests that GEP remains at a high level if the new vegetation can permanently gain a foothold after drought.The summer drought 2018 caused new maxima in annual GEP sums of 3.58 and 3.72 kg CO and Zrk a,b. Thes2 m−2 in Hte and Zrk . This corresponded to an increase of 35 and 56%, respectively, in comparison to reference years. The rise in respiratory CO2 release in peatlands affected by drought is generally related to peat decomposition under aerobic conditions [2 m−2). Although high rates of autotrophic respiration can be expected again in 2019, the magnitude of the annual total suggests that heterotrophic respiration from the decomposition of organic matter will also remain high in the year after the drought.The warm and dry conditions in 2018 also induced new maxima in the annual Reco sums, which amounted to 3.19 and 3.88 kg CO and Zrk c,d. This2 balance amounted to −0.39 kg CO2 m−2. Hence, Hte remained a CO2 sink though net CO2 uptake was 0.18 kg m−2 lower than in previous years (e). In Zrk, photosynthetic CO2 sequestration almost doubled in 2018 and thereby offset respiratory CO2 losses by 0.03 kg CO2 m−2 (f). Indeed, net CO2 uptake was comparatively low, but the fen had previously acted as a net CO2 source with average NEE balances of +0.34 kg CO2 m−2 [2 source of 0.06 kg m−2 again, though CO2 emissions were considerably lower than in the years before the drought.In Hte, the resulting net COus years e. In Zrk CO2 m−2 f. Indeed CO2 m−2 . In 20194 sums during the drought year reached a new minimum of 53 g m−2 (f) which was 22% below the reference average. The decrease in the annual CH4 sums mainly concerned the period from August onwards, when fluxes were 60% lower than in the reference years . 2018 CH4 sums in Zrk amounted to 37 g m−2 which was 7 g below the average of reference years (g). Here, CH4 emissions from August onwards were 15% lower than in reference years. Although the annual CH4 sum was affected to varying degrees, a drought-induced reduction in CH4 emissions was expected since methanogenesis is bound to strictly anaerobic conditions [In Hte, the annual CH53 g m−2 f which wce years g. Here, nditions .4 emission was even stronger in the year after the drought, when annual sums decreased down to 9 and 18 g CH4 m−2 in Hte and Zrk, respectively. In Hte, we cannot discriminate the drought carry-over effect from a potential suppression of methanogenesis under high-sulfate supply [4 emissions observed in Zrk in 2019, however, can be fully attributed to the previous drought year. Incubation experiments have shown that the recovery of CH4 emissions following a drought can be substantially lagged due to the persistent presence of electron acceptors and/or a delayed response of the CH4-cycling microbial community [4 emissions under in situ conditions are rare.Noteworthy, the reduction in CHe supply  as it reommunity . However4 release, GEP and Reco, whereby the effect on the net CO2 budget ultimately depended on the relative change in GEP compared to Reco. In addition, the year after the drought revealed marked carry-over effects in Zrk: here, the newly formed vegetation persisted under climate-normal conditions with beneficial consequences for GEP and the net CO2 balance. Further, drought left a lasting biogeochemical imprint that shifted organic matter decomposition towards non-methanogenic pathways but might come at the expense of increased respiratory CO2 release.In both fens, the severe drought 2018 caused common trends in CH4.—Practical experience has shown that the vegetation development of rewetted peatlands and the implied achievement of the mitigation goals can stagnate for years ,11. Drou—2 sinks from the start [2 sequestration and thereby move the net CO2 budget closer towards a sink. In addition, drought can cause a lasting biogeochemical shift towards non-methanogenic decomposition pathways, though this might come at the expense of temporary peat degradation and respiratory CO2 loss. Nonetheless, occasional droughts can eventually pay off if the newly established vegetation increases CO2 sequestration in the long term. In this regard, occasional drought events can be an integral part of fen restoration though care must be taken to prevent extensive peat decay.Peatland rewetting cannot always create net COhe start . The droThe resilience of restored peatlands to global warming is critical for future prospects of climate mitigation through peatland rewetting. Here, we report on vegetation and GHG dynamics of two rewetted temperate fens affected by the European summer drought 2018 and the post-drought year. Our study indicates common response mechanisms of rewetted fens to cope with temporal water deficit and provides wider implications for the mitigation prospects of peatland rewetting under more frequent drought occurrence.


The circadian clock regulates bodily rhythms by time cues that result from the integration of genetically encoded endogenous rhythms with external cycles, most potently with the light/dark cycle. Chronic exposure to constant light in adulthood disrupts circadian system function and can induce behavioral and physiological arrhythmicity with potential clinical consequences. Since the developing nervous system is particularly vulnerable to experiences during the critical period, we hypothesized that early-life circadian disruption would negatively impact the development of the circadian clock and its adult function. Newborn rats were subjected to a constant light of 16 lux from the day of birth through until postnatal day 20, and then they were housed in conditions of L12 h (16 lux): D12 h (darkness). The circadian period was measured by locomotor activity rhythm at postnatal day 60, and the rhythmic expressions of clock genes and tissue-specific genes were detected in the suprachiasmatic nuclei, retinas, and pineal glands at postnatal days 30 and 90. Our data show that early postnatal exposure to constant light leads to a prolonged endogenous period of locomotor activity rhythm and affects the rhythmic gene expression in all studied brain structures later in life. Period 1, 2, Clock, Bmal1, Cryptochromes, Nr1d1 (encoding nuclear receptor Rev-erbα), and Rorα , with a constant wavelength of 24 h, a horizontal line fit model (H0), and cosine curve as the alternative hypothesis. Rhythmicity was proved when both tests showed significant results. When rhythmicity was confirmed by both tests, the cosine curve parameters were calculated: mesor , amplitude , and acrophase , which were compared by one-way ANOVA followed by Tukey’s multiple comparisons test. Two-way ANOVA was used with a post hoc pairwise comparison by the Sidak–Bonferroni method to compare the control and LL-exposed groups. Data are reported as the mean ± SEM of 6 to 8 animals. Two-tailed Locomotor activity measurement at P60 showed a significantly longer endogenous period in LL-reared animals compared to the light/dark (LD) group a. We alsBdnf) expression  and amplitude (p = 0.0091) compared to the control LD group  and acrophase (p = 0.0267).At P90, the clock genes pression . The twoho genes . A compaho genes  between Icer, and phase-delayed acrophase in Bdnf expression in the LL group  or, in the retina, not affected at all. This observation indicates that the other gene expression patterns perturbed at P90 may not be a direct consequence of disrupted circadian clock in every tissue. Glutamatergic neurotransmission and NMDA receptor composition undergo striking changes in the SCN early in postnatal development [The exposure of pigmented rats to constant light from birth until P20 caused a decreased amplitude and mesor of seven of eight genes in the pineal gland at P30 and three of seven genes at P90, while elopment , which celopment ,54. The elopment .Altogether, our results indicate that postnatal light experience may affect temporal patterns in behavior and gene expression and suggest implications for the control of retinal physiology and pineal gland function later in life.


Nrip1) knockout mice, and a congenic strain, B6.C3H‐Igf1. Looking at the overall data for all inbred strains, the results show no significant difference in lifespan and lifespan variation between sexes; however, considerable differences were found among and within strains. Across strains, lifespan variations of female and male mice are significantly correlated. Strikingly, between sexes, IGF1 levels correlate with the lifespan variation and maximum lifespan in different directions. Female mice with low IGF1 levels have higher variation and extended maximum lifespan. The opposite is detected in males. Compared to domesticated inbred strains, wild‐derived inbred strains have elevated lifespan variation due to increased early deaths in both sexes and extended maximum lifespan in female mice. Intriguingly, the sex differences in survival curves of inbred strains negatively associated with age of female puberty, which is significantly accelerated in domesticated inbred strains compared to wild‐derived strains. In conclusion, this study suggests that genetic factors are involved in the regulation of sexual disparities in lifespan and lifespan variation, and dissecting the mouse genome may provide novel insight into the underlying genetic mechanisms.Epidemiological studies of human longevity found two interesting features, robust advantage of female lifespan and consistent reduction of lifespan variation. To help understand the genetic aspects of these phenomena, the current study examined sex differences and variation of longevity using previously published mouse data sets including data on lifespan, age of puberty, and circulating insulin‐like growth factor 1 (IGF1) levels in 31 inbred strains, data from colonies of nuclear‐receptor‐interacting protein 1 ( Among inbred strains, the lifespan variations of female and male mice are significantly correlated, suggesting the genetic co‐regulation of lifespan variation between sexes. IGF1 levels associate with lifespan variation and maximum lifespan in a sex‐specific manner. Importantly, female mice with lower IGF1 have longer maximum lifespan, while the male mice with higher IGF1 have longer maximum lifespan.  Another evidence of greater female longevity is provided by data from the Gerontology Research Group (http://www.grg.org/Adams/Tables.htm), which shows that women comprise 90% of supercentenarians . However, little about the underlying biological mechanisms is known, not only because of the complexity, but also due to the lack of a suitable animal model for studying this phenomenon. In fact, unlike in the human, common used laboratory models, worms, fruit flies, rats and mice, show there is no robust difference in longevity between sexes . These data provided us with a unique opportunity to investigate the genetic regulation of lifespan variation—including conditions that generate sex differences in lifespan and lifespan variation—across a wide variety of well‐studied genotypes.In this paper, we used an extensive set of available data from several of our previous studies that investigated longevity‐related phenotypes in a variety of mouse strains, including inbred and genetically modified strains , as well as female and male survival curves Table . “Correcp < 0.05), plus a significant difference in either median and/or maximum lifespan (p < 0.05). Males from strains 129S1, NOD.B10, and NZW had significantly greater longevity than females. Females from strains B10 and P mice had significantly greater longevity than males. Interestingly, female MRL mice had a significantly shorter median lifespan; however, females significantly outnumbered males in the longest 10% survivorship, indicating a higher death risk after the age of the median lifespan in males than in females of MRL.The significance of the difference in maximum lifespan is determined by the combination of quantile regression and Fisher exact probability test, which takes the difference of sample size into account to avoid the potential bias caused by the inequality in sample size between groups. In this study, we defined a difference in longevity between sexes as a significant difference between survival curves .In human populations, increased lifespan expectancy is consistently accompanied with decreased variation of age at death . In males, higher IGF1 at all three time points associated with reduced MAD and the association is significant at age of 18 months . Importantly, at all three ages, the difference between sexes is significant .We previously reported that IGF1 levels negatively associated with lifespan, and identified strains with significantly higher and lower IGF1 at age of six months in both females and males . The median lifespan of female mice with low IGF1 level is significantly longer than that of the females with high IGF1 level . Surprisingly, among the 70 mice of 10% longest corrected lifespan, 52 mice belonging to seven strains are from the high IGF1 group. Only 18 mice are from the low IGF1 group. Quantile regression test shows the difference is significant (p < 0.001). These results indicate that male mice with high IGF1 have longer maximum lifespan.In males, different from the females, there is no significant difference in MAD between the high and low IGF1 groups of male mice . Although the median lifespan was not significantly different between the domesticated and wild‐derived inbred strains  deletion model, AVP is significantly delayed compared to Nrip1‐wild controls. In B6.C3H‐Igf1, a congenic strain carrying an IGF1‐increasing allele derived from C3H on B6 background, females had significantly higher IGF1 level and accelerated AVP than the parental B6 strain. In both studies, delayed AVP was associated with extended median and maximum lifespan of females, while males with the same genotypes did not show extended lifespan ; however, deletion of Nrip1, including heterozygous and homozygous deletion, reduced the difference . A comparison between female and male mice of the B6 strain, the parental strain of B6.C3H‐Igf1, showed no significant difference in the survival curves. Strikingly, in the B6.C3H‐Igf1 colony, the survival curves of female and male mice were dramatically different .To verifying this hypothesis, in this study, we examined the relationship of AVP and lifespan disparity between sexes by using two genetically engineered mouse models . Comparing the shortest‐ and longest‐lived countries, the sex difference in the lifespan expectancy at birth (LEAB) is significantly lower in the short‐lived countries, in both terms of years or the percentage of the LEAB (data not shown), indicating the sex bias increased along with the extended lifespan, which mimics the changes that have been seen in western countries in the past two centuries , while female B10 and P mice have a significantly greater lifespan than males.In an analysis of 118 survival studies of laboratory mice, Austad  Austad, . The curUnderstanding the sex inequality in lifespan is important for designing experiments, interpreting data of aging research, in which the mouse strains are involved. For instance, many embryonic stem cell lines used for generating transgenic and knockout mouse models are derived from strains within the 129S lineage Linder, , includibH2 is substituted the nonpathogenic allele from the B10 strain on the NOD background. Although NOD.B10 does not develop insulitis, cyclophosphamide‐induced diabetes, or spontaneous diabetes, it does develop autoimmunity as shown by the extensive lymphocytic infiltration in pancreas and submandibular glands  mice, alleles at multiple genetic loci, including the major histocompatibility complex (MHC), prompt the development of autoimmune diabetes. NOD.B10 is a congenic strain, in which the MHC 3.22 of the survival curve comparison showed that wild‐derived inbred strains have significantly higher similarity between sexes than that of the domesticated inbred strains, suggesting that some of the differences between sexes might have evolved during the process of domestication.The difference in lifespan between sexes may be related to the process of domestication. Most of the inbred strains were developed from mice that have been domesticated for thousands of years and raised as “fancy” mice for hundreds of years Silver, . In thisPrevious studies revealed that the processes of domesticating and inbreeding have profound effects on mice development and aging. Bronson F. H. reported that compared to wild‐caught mice, domesticated mice (not inbred) had significantly earlier onset of fertility, accompanied with greater food consumption, growth rate, and final body weight, but depressed locomotor activity Bronson, . Miller p = 0.083) with reduced difference between sexes among the inbred strains, suggesting the delayed female sexual maturation is accompanied with increased similarity between female and male longevity. Therefore, these results raise an interesting hypothesis that the alleles delaying sexual maturation may not only extend longevity but may also reduce the sexual differences of lifespan. The observations in Nrip1 knockout and B6.C3H‐Igf1 mouse colonies strongly support this hypothesis. Sexual maturation is delayed in Nrip1‐deficient females and accelerated in B6.C3H‐Igf1 females .Interestingly, as shown in Table 3.4Our previous studies suggest that IGF1 is a co‐regulator that mediates the trade‐off relationship between the age of female sexual maturation and longevity. Strains with lower IGF1 have delayed AVP and greater longevity , demonstrating the robustness of this phenomenon. Examining the survival curves and survival density plots of mice with high and low IGF1 levels helps us understand this pattern. In both sexes, mice with lower IGF1 have extended median lifespan than mice with higher IGF1. However, female mice with lower IGF1 have increased death rate at young age and longer maximum lifespan than the females with higher IGF1, mimicking the pattern seen in the comparison between wild‐derived inbred females and the domesticated‐derived inbred females. In the males, mice with lower IGF1 do not have increased death rate at young age, but have significantly shortened maximum lifespan than that of mice with higher IGF1. These results suggest that IGF1, a key component of nutrient‐sensing pathways, regulates aging and longevity in a sex‐specific manner. Evidence for sexual dimorphism in the impact of nutrient‐sensing pathways on longevity is provided also by studies of mice with deletion of genes related to insulin/IGF1 signaling (IIS) and mammalian target of rapamycin (mTOR) signaling. Heterozygous IGF1 receptor inactivation (IGF1R+/‐) and deletion of ribosomal S6 kinase 1 or insulin receptor substrate 1 genes extend longevity of female, but not male mice  resistance, in which the IGF1 level is reduced although the GH level is upregulated . The details of measuring IGF1 concentration and determining AVP were fully described in the previous publications .All other analyses were done using R software version 3.6.0 .describeBy of the package psych (Revelle, median_test, and for testing the difference in variance of lifespan, we used the function fligner_test; both functions are from the package coin (Strasser & Weber densityPlot function of the package car (Fox & Weisberg, For studying lifespan and variation in lifespan, we calculated the median longevity of the females and males of all mice combined across strains, and of the females and males per strain. We chose median instead of mean because we could not assume that the variation in lifespan was normally distributed. For the variation in lifespan, we calculated the MAD, that is, the Median Absolute Deviation, a robust metric fit for non‐normal distributions (Leys et al., Revelle, . For teset al. whereby one estimates the percentiles of a distribution using conventional quantile regression in a reduced predictive model that does not include the independent variable of interest (Redden et al., fisher.exact function of the exact2X2 package (Fay, For testing the difference in maximum lifespan, we applied the quantile regression approach, choosing the 10% longest living individuals, combined with the Fisher exact probability test as recommended by Wang et al. . To deteage Fay, . For assTo assess whether the MADs of the females and males per strain were exceptional high or low as compared to the MAD of all the females and males, we performed a permutation test. For that, we took per strain 1000 times a random sample from all females or males of the size equal to the number of females or males of that strain. For each of these samples, we calculated the MAD. This gave us the distribution of 1000 random MADs per gender per strain. Then we assessed whether the observed MAD of the females and males per strain was below the 2.5% quantile or above the 97.5% quantile of these distributions. If so, we regarded the MAD as either extremely low or extremely high (gray, respectively bold in Table lm.For calculating the slope, that is, the regression coefficient, per gender between median IGF1 level at different times of measurement and MAD of strains, we applied a linear model, including the interaction factor between median IGF1 level and sex, using the function cor.test.For the correlations between any two variables, we calculated the Pearson's R, unless the residuals were not distributed normally. In that case, we calculated Spearman's Rho with the function The authors declare that they have no conflicts of interest.RY designed and managed the study, contributed to data analysis, interpretation of results,and manuscript. CJM carried out the data analyses, contributed to results interpretation and manuscript. YZ, TE, and YS contributed to the manuscript. LLP, DEH, and AB contributed to experiment design and manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.