The functions of Sertoli cells in spermatogenesis have attracted much more attention recently. Normal spermatogenesis depends on Sertoli cells, mainly due to their influence on nutrient supply, maintenance of cell junctions, and support for germ cells’ mitosis and meiosis. Accumulating evidence in the past decade has highlighted the dominant functions of the MAPK, AMPK, and TGF-β/Smad signaling pathways during spermatogenesis. Among these pathways, the MAPK signaling pathway regulates dynamics of tight junctions and adherens junctions, proliferation and meiosis of germ cells, proliferation and lactate production of Sertoli cells; the AMPK and the TGF-β/Smad signaling pathways both affect dynamics of tight junctions and adherens junctions, as well as the proliferation of Sertoli cells. The AMPK signaling pathway also regulates lactate supply. These signaling pathways combine to form a complex regulatory network for spermatogenesis. In testicular tumors or infertile patients, the activities of these signaling pathways in Sertoli cells are abnormal. Clarifying the mechanisms of signaling pathways in Sertoli cells on spermatogenesis provides new insights into the physiological functions of Sertoli cells in male reproduction, and also serves as a pre-requisite to identify potential therapeutic targets in abnormal spermatogenesis including testicular tumor and male infertility. Sertoli cells support, nourish, and protect spermatogenic cells via various signal pathways.The TGF-β/Smad, AMPK, and MAPK signaling pathways in Sertoli cells support spermatogenesis via regulating cell junction dynamics, proliferation of Sertoli cells and germ cells, and lactate supply for spermatids.Activity of the TGF-β/Smad, AMPK, and MAPK signaling pathways in Sertoli cells turns abnormal in non-obstructive azoospermia and patients with testicular cancer.Which pathway plays a decisive role when the TGF-β/Smad, AMPK, and MAPK signaling pathways in Sertoli cells regulate the same process?What are the detailed molecular downstream mechanisms and interactions of proteins involved in the pathways mediating physiological functions of Sertoli cells?What is the key role of the TGF-β/Smad, AMPK, and MAPK signaling pathways in tumorigenesis and infertility?Is it possible to identify specific pathways and related proteins as diagnostic and therapeutic targets for testicular cancer and male infertility?1. In this process, spermatogonial stem cells (SSCs) are triggered to produce spermatogonia, which will transform to spermatocytes, spermatids, and finally mature spermatozoa3. The migration of germ cells (GCs) and the release of spermatozoa require timely cell junctions disassemble and reassemble between Sertoli cells-Sertoli cells (SCs-SCs) and SCs-GCs5. Such adherens junctions (AJs) are named as ectoplasmic specializations (ES) in the testis, and are divided into the basal ES at the SCs-SCs interface and the apical ES at the SCs-spermatids interface6. In the mammalian testis, the basal ES, desmosomes, gap junctions, and tight junctions (TJs) between SCs form the blood–testis barrier (BTB)7. The TJs at the BTB are constituted of various tight junctional proteins, including the claudin (CLDN) family, junctional adhesion molecule (JAM) family, etc. , which will bind to actin via the zonula occludens-1, -2 and -3  in SCs9. At stage VII-VIII of the epithelial cycle, the preleptotene and leptotene spermatocytes must move through the BTB and enter the adluminal compartment10. Most researchers focused on the synchronization of spermatogenesis currently, but few of them have addressed the issue from the perspective of Sertoli cells17.Spermatogenesis is a significant physiological process of sperm production in the epithelium of the seminiferous tubules18. Throughout mammalian spermatogenesis, SCs provide morphogenetic support via cell–cell interactions and also biochemical components via secreting lactate, cytokines, and hormones20. Apart from the mechanical and nutritional support, SCs also form an immune-protective environment to protect germ cells via the BTB24. At the end of spermatogenesis, AJs between GCs and SCs allow SCs endocytosis of the elongated spermatids’ cytoplasm, and finally morphologically shape the spermatids25. Therefore, SCs are considered as nurse like cells to support spermatogenesis26.Sertoli cells are the only somatic cells in the seminiferous epithelium28. Until now, numerous signaling pathways have been found in Sertoli cells, including the androgen-signaling pathway30, the AMP-activated protein kinase (AMPK) signaling pathway31, the follicle stimulating hormone (FSH)/adenylate cyclase/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway32, the Hippo signaling pathway34, the intergrin mediated signaling pathway35, the Janus kinase/signal transducer and activator of transcription signaling pathway37, the mitogen-activated protein kinases (MAPK) signaling pathway44, the nuclear factor kappa B signaling pathway46, the nitric oxide/soluble guanylyl cyclase/cyclic guanosine monophosphate/protein kinase G signaling pathway48, the Notch signaling pathway49, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3k)/AKT serine/threonine kinase (Akt) signaling pathway51, the Sonic Hedgehog signaling pathway53, the transforming growth factor-β (TGF-β)/Smad signaling pathway54, and the Wnt signaling pathway56  superfamily contains activin, inhibin, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), and TGF-β homodimeric proteinsges Fig. .Fig. 2Sc66. Consistent with this, Itman et al. found that activin-induced nuclear accumulation of Smad2 and Smad3 in post mitotic mouse SCs, and also screened out the activin target genes Gja1 and Serpina5 via microarray analysis. These two genes encode connexin 43 and serine protease inhibitor, respectively, which are required for SCs maturation54. In Smad3−/− mouse, delayed SCs differentiation and decreased testis size were accompanied by an inhibition of androgen receptor and Smad2 expression67. Uncovering the link between Smad2/3 and their downstream network may make us benefit in studying the effects of SCs proliferation on sperm output.Precisely regulated Smad2/3 signaling is required for SCs to differentiate from the proliferating state to a differentiated state. Type IIA activin receptor exhibits high-expression transiently in rat SCs at stage VII-IX69. BMP4 was observed to increase Smad1/5 phosphorylation and to enhance proliferation of human SCs, but administration of noggin, the BMP4 antagonist, showed conversely inhibitory effects. When Hai et al. knocked down BMP4 in human SCs, fibroblast growth factor-2 (FGF-2) and SCF production was also suppressed70. However, the contribution of Smad1/5 pathway against the ID2/3 pathway in BMP4-induced SCs proliferation enhancement was not evaluated in their research. This problem also exists in the study of BMP6, particularly, whether the Smad2/3 signaling pathway directly mediates the BMP6-induced proliferation, and increased levels of SCF and Glial cell-derived neurotrophic factor (GDNF) in human SCs71.BMP4 and BMP6 promote proliferation and DNA synthesis of human SCs via an autocrine pathway73. Both TGF-β2 and TGF-β3 downregulate the expression of JAM-B via the Smad3 signaling pathway. Wang and Lui discovered that in mouse SCs, TGF-β2 served as an anti-expression factor of JAM-B at the pre-transcriptional level. TGF-β2 increases phosphorylation level of Smad3, which would compete with the transcription factors Sp1 and Sp3 for the TG interacting factor (TGIF) motif, and ultimately repress the JAM-B transcription74. However, TGF-β3 treatment can decrease the JAM-B protein level at a post-translational way in mouse SCs. The degradation of JAM-B can be relieved upon administration of proteasome inhibitors, including MG-132 and lactacystin. This process requires Smad3/4 activation. If Smad3 and Smad4 are knocked down, TGF-β3-induced JAM-B degradation will be inhibited in turn75. Consequently, TGF-β2 and TGF-β3 may establish a precise-regulating network for disassembly and assembly of the BTB via the Smad signaling pathway.TGF-βs and GDF9 participate in the regulation of TJs and AJs dynamics. Given that the transition and relocation of spermatocytes through the BTB require coordinated disassembly and reassembly of cell junctions, timely regulation of JAM-B expression is crucial for migration of GCsCLDN11 expression in mouse TM4 cell lines76. As a component of TJ, CLDN11 is crucial for spermatocyte migration through the BTB into the adluminal compartment79. When the Smad signaling pathway is stimulated in TM4 cells, Smad3/4 binds to the GATA/NF-Y motif in CLDN11 promoter. Quantity of the complex formed in this way will be decreased upon anti-Smad3 antibody treatment in the electrophoretic mobility shift assay. Ulteriorly, the binding complex can recruit histone deacetylase 1 and co-repressor mSin3A. Thus, transactivation of GATA and CREB, as well as the activity of the promoter in CLDN11 gene were inhibited76.Apart from JAM-B, Smad3 also supports preleptotene spermatocytes translocation by decreasing 80. GDF9 receptor ALK5 and Smad2/3 were highly detected in adult alpaca and cat SCs82. Here, we suggest that further experimental investigations should focus on whether GDF9 regulates TJs via the GDF9/Smad2/3 signaling pathway.Few researches have addressed the issue on the GDF9/Smad signaling pathway, but Nicholls et al. did detect disruption of the inter-Sertoli TJ permeability barrier after adding recombinant GDF9 in mouse SCs cultures83. The AMPK signaling pathway in SCs has been found to regulate energy metabolism, junctional complex stability, and proliferation84. Once the balance is disrupted, the microenvironment of testis and the quality of sperm will be affected. For example, in α1AMPK globally knocked out mouse, spermatozoa showed abnormal head, curved sheaths, and impaired mobility85. When α1AMPK is conditionally knocked out in mouse SCs, the mutant mice still showed an abnormal phenotype, including thin head spermatozoa, reduced expression of junctional proteins , and deregulation of energy homeostasis86. These findings support the contribution of the AMPK signaling pathway in SCs during spermatogenesis  regulate glucose metabolism via limiting substrate transmembrane transport, while monocarboxylate transporters (MCTs) control lactate transport and supply to GCs, both of which contribute to adjust lactate production in SCs90.Lactate is a preferring energy source of spermatocytes and spermatids, the majority of which is provided by Sertoli cells91. Glucose deprivation in rat SCs will induce activation of the AMPK and p38 MAPK signaling pathway, increase the mRNA level of GLUT1 and maintain the uptake of glucose. Such adaptation ensures or rescues lactate production even in the absence of glucose94. Adenosine and its analog AICAR were also proven to promote lactate secretion from rat SCs via AMPK activation, while mechanism of AIACR regulation is illustrated more integrally91. AICAR can increase lactate production via the AMPK-induced glucose intake in rat SCs, at least through increase in GLUT1 protein level and MCT4 mRNA level, and decrease in MCT1 and GLUT3 mRNA levels95. Overall, adaptation to the environment and response to those signal molecules via the AMPK signaling pathway in SCs will thus stabilize an appropriate lactate supply for GCs energy demand.In response to various signaling factors and environmental conditions, the AMPK signaling pathway in SCs serves as a key regulator in providing lactate for energy metabolism of GCs and maintaining spermatogenesis91. Also, heat stress can cause dysfunction of TJs in porcine testis reversibly via Ca2+/calmodulin-dependent protein kinase kinase B (CaMKKB) induced inhibition of the AMPK signaling pathway. Yang et al. treated SCs from 3-week-old piglets at 43 °C for 0.5 h, and such hyperthermia inhibited the AMPK signaling pathway to inhibit expression of CLDN11, JAMA, occludin, especially ZO-1 in porcine SCs96.The AMPK signaling pathway maintains junctional complex stabilization in testis. It has been shown that activation of AMPK by adenosine stabilizes ZO-1 on rat SCs membranes, and the AMPK inhibitor compound C can decline adenosine affected ZO-197. Based on this phenomenon observed in bortezomib exposure, we suggest that the detailed mechanisms of the normal situation are also worth studying.As for AJs, the relationship has been clarified between the 26S proteasome inhibitor bortezomib, the AMPK signaling pathway and AJs among SCs and GCs in mouse. Bortezomib can induce AMPK activation and then antagonize Akt and extracellular signal-regulated kinase (ERK) signaling pathway in mouse SCs. As a consequence, AJs impairment, immature GCs desquamation and sperm quantity reduction are followed98. AMPK activation potentially leads to detention of rat SCs proliferation at least partially by inhibition of mTORC1 and stimulation of cyclin-dependent kinase inhibitors expression. Moreover, lower activity of mTORC1 was due to accumulation of phosphorylated Raptor99. Consequently, SCs mitotic activity, which is stimulated by FSH and mediated by the PI3K/Akt signaling pathway, is counteracted by the AMPK signaling pathway. Similarly, the activated AMPK signaling pathway also mediated 17β-estradiol inhibition on boar SCs proliferation, which would be abolished by compound C treatment. Zhang et al. administrated 10 μM of 17β-estradiol on boar SCs and observed inhibition of miR-1285 expression59. Recently, they clarified that miR-1285 can downregulate α2AMPK mRNA and protein level. 17β-estradiol treatment retains AMPK activity by maintaining α2AMPK31. As for the downstream effect, the phosphorylated AMPK increases the expression of the cyclin-dependent kinase inhibitor p27 (p27) and tumor suppressor p53 (p53), but inhibits the protein level of phosphorylated mTOR and S-phase kinase-associated protein 2 (Skp2)100. This regulatory network ultimately leads to reduction of SCs number and sperm production in boars.Apart from regulating AJs integrity, SCs proliferation inhibition is also mediated by the AMPK signaling pathway1). According to the microarray data, a majority of MAPK pathway-related genes exist in immature rat SCs, which shows the existence of the MAPK signaling cascades in SCs101 , ERK, and p38 MAPK MAPK14). The isoforms and distribution of JNKs, ERKs and p38 MAPKs present in mammalian SCs have been summarized. In rat testis, (phosphorylated) ERK1/22, (phosphorylated) JNK1/2, (phosphorylated) p38 MAPK are located in SCs, while ERK7, JNK3 are investigated in testis  promotion of JAM-B transcription in rat SCs. Activated p38 MAPK phosphorylated the ETS domain transcription factor (Elk-1). Phosphorylation allows Elk-1 to bind on TGIF and proximal Sp1 (pSp1) + E2F motifs. Such interaction will increase Sp1 and NRSF trans-activated 103. Overexpression of TGF-β3 in primary rat SCs can magnify above damage effect in vitro, with occludin, N-cadherin, and ZO-1 decline104. In CdCl2-induced adult rat BTB damage, a specific p38 MAPK activity inhibitor SB202190 can blocked loss of ZO-1 and occludin, and thus abolish the damage of TGB-β3 on the AJ and TJ barrier function. It strengthens the physiological importance of the TGF-β3/p38 MAPK signaling pathway in AJ and TJ dynamics105.Moreover, TGF‐β3 has been found to perturb TJs barrier assembly in the p38 MAPK signaling pathway via a transient increase in phosphorylated p38-MAPK instead of overall p38 MAPK104. As for TNF-α, it binds to TNFR1 and/or TNFR2 on rat SCs membrane and activates only p38 MAPK without ERK, downregulating occludin and ZO-1 expression transiently to allow relocation of preleptotene and leptotene spermatocytes crossing the BTB and differentiation of them to pachytene spermatocytes106.The event differs when the p38 MAPK signaling pathway and/or the ERK signaling pathway regulate cell junctions upon TGF‐β3 and tumor necrosis factor α (TNF-α) treatment. After adapter CD2-associated protein (CD2AP) binds to TGF-β3 and TGF-β receptor I (TGFβRI) complex, the BTB integrity remains normal, but SCs-GCs adhesion is disrupted reversibly via the activated ERK-signaling pathway in rat SCs. However, when both TAK1-binding protein 1 (TAB1) and CD2AP bind to TGFβR I, the p38 MAPK and ERK signaling pathway are both activated. Not only occludin, ZO-1 at the BTB, but also cadherins at the apical ES and BTB decreases, which leads to disruption of SCs-GCs adhesion and the BTB as well6. After transfected with pCI-neo/ICAM-1 plasmids, the rat SCs overexpressed ICAM-1 with increase of transepithelial electrical resistance and enhancement of TJs barrier function107. TNF-α stimulation on JNK is related to ICAM-1. After secreting from round spermatids, TNF-α binds to the p55 receptors (TNFR1) on mouse SCs membrane, activates the JNK signaling pathway and thus increases ICAM-1 expression109. Further studies need to focus on whether ICAM-1 overexpression can stabilize TJ dynamics in vivo upon TNF-α-activated JNK pathway.Recent evidences strongly support that the JNK signaling pathway contributes to the BTB function and GCs migration. Intercellular adhesion molecule-1 (ICAM-1) is the constitution of BTB and a pivotal regulator in BTB dynamics, which is co-localized with occludin and N-cadherin2-induced BTB disruptive effects in adult rats, which is just contrary to the p38 MAPK signaling pathway. During CdCl2-induced BTB disruption, the JNK signaling pathway leads to α2-macroglobulin (α2-MG) expression, which is a protease inhibitor localized at the SCs-SCs and SCs-GCs interface110. Wong et al. used the protein kinase inhibitor 6-dimethylaminopurine which can downregulate α2-MG protein level to examine its effect111. After 6-dimethylaminopurine pretreatment before CdCl2 administration in rat, they observed losing of GCs and flaking of the most seminiferous epithelium in the basement membrane111. These evidences reveal the importance of α2-MG in inhibiting unwanted proteolysis and maintaining TJs and AJs integrity in defending the CdCl2-induced BTB disruption.Furthermore, the JNK signaling pathway will also reduce CdCl112. Hirai et al. investigated that overexpression of FGF-4 in mouse SCs inhibited apoptosis of GCs due to mild hyperthermia. They injected mice with recombinant FGF-4 adenovirus and then treat them at 43 °C for 15 min after 5 days. Dissection of testis showed fewer sperm count and less testicular weight in response to mild heat treatment than that of control, along with the increase of phosphorylation level of the ERK1/2 in mouse SCs and GCs. It indicates the potential mechanism that FGF-4 prevents GCs from apoptosis and promotes GCs survival via triggering the ERK signaling pathway in SCs and GCs114.Different from the JNK and p38 MAPK signaling pathways, the ERK signaling pathway directly regulates apoptosis, mitosis, and meiosis progression of GCs. In situ hybridization of mouse testis and primary cell culture have confirmed that fibroblast growth factor-4 (FGF-4) expresses only in Sertoli cells throughout the spermatogenic cycle115.Furthermore, meiosis of spermatocytes depends on activation of the ERK signaling pathway in co-culture of SCs and pachytene spermatocytes. Godet et al. detected the phosphorylated ERK1/2 in such co-culture. After pre-treatment of MEK1/2 inhibitor U0126, the number of pachytene spermatocytes and secondary spermatocytes declined. But no similar phenomenon emerged in pachytene spermatocytes culture upon U0126 treatment. These different phenomena emphasize the determination of the ERK signaling pathway in SCs for spermatocytes meiosis116. GDNF expression in mouse SCs can be upregulated via the cAMP/PKA signaling pathway and the Wnt/β-catenin signaling pathway118, but be downregulated by the Notch signaling pathway122. Mouse SCs also use the ERK signaling pathway for regulating GNDF expression and thus influencing SSCs niches. During the self-renewal phase of mouse SSCs, the level of GDNF in SCs rises with the activation trend of ERK 1/2 in SCs, which preserves the undifferentiated state of SSCs123.GDNF has been identified as a paracrine factor to promote proliferation and migration, but prevents differentiation of SSCs via binding onto the RET/GFRα1 co-receptors and activating of Ras/ERK1/2 signaling pathway in SSCs124. Similar trends of phosphorylated ERK were also detected in normal mice, though without FSH treatment in vitro, where phosphorylation level of ERK increased until puberty, followed by a decrease during adulthood in wild type mice125.FSH decides the states of the ERK signaling pathway at a stage-dependent manner in SCs, with each stage activated or inhibited. At 5 days after birth, FSH treatment on isolated rat SCs stimulated MEK-1 activation, and then increased phosphorylation and nucleic relocation level of ERK1/2, the former of which can be eliminated by pre-incubation of SCs with MEK-1 inhibitor PD98059. This way, the expression of cyclin D1 (CCND1) and proliferation rate of the neonatal SCs are promoted. However, SCs maturation stage displays an opposite effect of FSH on the ERK signaling pathway. At 19 days after birth, FSH treatment turns to inhibit the ERK signaling pathway in rat SCs, leaving number of S-phase SCs and protein level of CCND1 less sensitive to FSH stimulation126. We have addressed the changes in phosphorylated ERK levels is consistent with the proliferation of SCs, so that the periodical rising and falling of the ERK signaling pathway activation are probably closely linked with numbers of SCs and differentiated GCs during testicular development.Furthermore, ouabain, which is a mammal adrenal gland cortex-produced endogenous cardiotonic steroid, can induce CCND1 expression and primary rat SCs proliferation accompanied with activation of the ERK signaling pathway127. Galardo et al. further analyzed the intrinsic molecular mechanism behind these results128. There is a CRE-like sequence on the promoter of the transferrin encoding gene and a consensus CRE sequence on the promoter of the LDH A gene in rat131. Treating rat SCs cultures with FGF-2 could increase phosphorylated CREB level, while PD98059 incubation inhibited FGF-2 stimulation on phosphorylated CREB, LDH A, and transferrin uprising level128. So CREB may act as the target of ERK1/2 signaling to regulate iron and lactate supplies in SCs.FGF-2 utilizes the ERK signaling pathway to regulate transferrin secretion and lactate dehydrogenase (LDH) activity in rat SCs, thus influencing iron and lactate supplies for GCs, respectively. Incubation of rat SCs with U0126 or PD98059 both blocked phosphorylated-ERK-induced transferrin secretion and LDH catalytic activity137. We had discussed of the TGF-β/Smad, AMPK, and MAPK signaling pathways in SCs to regulate normal spermatogenesis. There are also clinical studies which revealed the relevance of the three pathways and abnormal spermatogenesis.In patients with testicular tumor or infertility, abnormal activity of signaling pathways was observed, including the Wnt signaling pathway, the PI3k/Akt signaling pathway, etc139. From 1990 to 2010, the number of infertile couples increased globally, and 48.5 million couples worldwide were disturbed140. Among them, approximately 20–70% of cases are owing to the male factor, and at least 30 million men worldwide being diagnosed with infertility according to statistic research in 2015141. Abnormal quality and insufficient quantity of sperm are the primary causes of male infertility, most of which are clinically manifested as oligozoospermia, asthenozoospermia, teratospermia, or azoospermia142. Azoospermia is classified as obstructive azoospermia and nonobstructive azoospermia143, the latter of which is a major course for male infertility and affects 10–15% of infertile men144. The microarray analysis on testicular biopsy samples from azoospermic men detected over-activation of the MAPK signaling pathway in SCs145. For azoospermic patients, Sertoli cell-only syndrome affects 26.3–57.8% of them, whose testicular histology biopsies shows no germ cells and only Sertoli cells in the seminiferous tubules146. In testicular biopsies from nonobstructive azoospermia patients with Sertoli cell-only syndrome, BMP4, TGF-β receptor II (TGFβRII), and Smad2 are more highly expressed70. As for the AMPK signaling pathway, studies in SCs of infertile humans are insufficient. However, number of pups per litter in SC-α1AMPK-cKO mice did decrease by 25%, accompanied with disturbed cell junction dynamics86. Thus, for the purpose of elucidating the molecular basis and developing therapeutic options for azoospermia therapy, the causes of azoospermia deserve more attention in the future, especially from the perspective of the TGF-β/Smad, AMPK, and MAPK signaling pathways in SCs150.Infertility is an emerging worldwide public health issue151. In the year of 2018, the worldwide estimated number of new cases of testicular cancer at all ages reached 71,105 according to International Agency for Research on Cancer152. Testicular tumors can be classified into germ cell tumors, sex cord–stromal tumors, mixed germ cell/sex cord–stromal tumors, and lymphomas153. Sex cord-stromal tumors consist of Sertoli cell tumors, Leydig cell tumors, granulosa cell tumors, and unclassified tumors154. Testicular cancer development is potentially linked with the TGF-β/Smad signaling pathway, especially when the BMP signaling SMADs (BR-SMADs) participate. Smad4, the Co-Smad in the TGF-β/Smad signaling pathway, may serve as a key mediator in Leydig cell adenomas. When Smad4 was conditionally knocked out in mouse Sertoli cells and Leydig cells, 87.5% of the mutant mice exhibited Leydig cell adenomas at 56–62 weeks of age64. After the BR-SMADs  in mice SCs is deleted via tissue-specific ablation, all male Smad1/Smad5 KO mice (14 samples) developed Sertoli-Leydig tumors after 28 weeks of age with 100% metastases to lymph and peritonea, implicating the role of the BR-SMAD signaling pathway as a tumor suppressor in testis155.Testicular cancer is a common malignancy which can cause infertility and death in men156. For instance, FSH suppresses Sertoli cell tumor progression during the 1st or 2nd week after birth, which is the first wave of spermatogenesis in inhibin α-KO mice157. Since these signaling molecules are often involved in multiple signaling pathways in SCs or regulated by various signaling pathways159, identifying the determining signaling pathway that controls abnormal spermatogenesis is the first step to study the causes of abnormal spermatogenesis, progression of testicular cancer, and infertility160. These basic researches may facilitate diagnostics and therapeutics for testicular cancer and infertility, as well as development of targeted drugs, and all these advances will reduce cancer mortality and infertility morbidity in the future.In present, the relationship between signaling pathways, infertility and tumorigenesis in SCs still remains unknown. However, various hormones, cytokines or proteins have been indicated to express differently in SCs if abnormal spermatogenesis occurs


The enhanced expression of LOX-1 in atherosclerotic aorta was also dramatically decreased by administration of UA. Taken together, these results suggested that UA, with anti-atherosclerotic activity through inhibition of LOX-1 mediated by ROS/NF-κB signaling pathways, may become a valuable vascular protective candidate for the treatment of atherosclerosis.Atherosclerosis, a chronic inflammatory disease, is a major contributor to cardiovascular diseases. Ursolic acid (UA) is a phytonutrient with widely biological effects including anti-oxidative, anti-inflammatory, and so on. At present, the effect of UA on atherosclerosis and the mechanism of action are still obscure. This study focused on investigating the effects of UA on atherosclerosis both in vivo and in vitro. We first selected LOX-1 as our target, which was reckoned as a new promising receptor for treating atherosclerosis. The evaluation in vitro suggested that UA significantly decreased endothelial LOX-1 expression induced by LPS both in mRNA and protein levels. Pre-treatment of UA also inhibited TLR4/MyD88 signaling activated by LPS. Moreover, UA reduced ROS production and suppressed the activation of NF-κB stimulated by LPS. Particularly, the evaluation in vivo further verified the conclusion obtained in vitro. In ApoE Atherosclerosis, a chronic inflammatory disease of the arterial wall, is a major cause of morbidity and mortality worldwide and is marked by the formation of atherosclerotic plaques . One criTLR4-MyD88 pathway leads to activation of the transcription factor NF-κB, thereby influencing inflammatory responses. Oxidative stress is characterized by an increased production of free oxygen radicals . NumerouFructus Ligustrum lucidum [Forsythiae fructus [−/− mice in vivo; and related mechanisms such as LOX-1 expression in endothelial cells. Our findings may supply a new sight for illustrating role of UA, which benefits the development of the anti-athrogenic drug.Ursolic acid (UA) is a pentacyclic triterpenoid found in many herbs and spices like rosemary and thyme, especially in valuable Chinese medicinal herbs such as  lucidum  and Fors fructus . It show fructus , anti-in fructus , and oth fructus ,22. Howe fructus  reportedFirstly, the cytotoxic effect of UA on HUVECs was determined by MTT assay. Results showed that UA was cytotoxic to HUVECs at 50 μM . To miniTLR4/MyD88 signal pathway is involved in LPS-induced inflammation . As expeROS is one key signaling molecule involved in inflammation, and NF-κB pathway plays an essential role in LPS-induced LOX-1 expression in HUVECs . In this−/− mice. UA administration significantly reduced plaque sizes as well as shrank necrotic core areas , LPS , 5-(6)-carboxy-2′, 7′-dichlorodihydrofluorescein diacetate (DCFH2-DA), and BAY11-7082 were purchased from Sigma Aldrich . Antibodies for TLR4, MyD88 were purchased from Santa Cruz Biotechnology . Antibodies for NF-κB p65, GAPDH, and Histone H3 were purchased from Cell Signaling Technology . Anti-LOX-1 antibody was obtained from R&D . The BCA protein kits were purchased from Thermo Fisher . SiRNAs for TLR4, MyD88 were purchased from Gene Pharma Company . Oil Red O Staining Kit and HE Staining Kit were obtained from Nanjing Jiancheng Bioengineering Research Institute .UA (purity > 98%) was purchased from Chengdu Herbpurify Co. Ltd. . Hoechst 33342, 2. Before passaging cells, issue culture flasks, 96-well plates, and 6-well plates were pre-coated with 0.1% gelatin. All assays were conducted using low cell passage cells (2–5 passages).Human umbilical vein endothelial cells (HUVECs)  were cultured in Vascular Cell Basal Medium with Endothelial Cell Growth Kit-BBE at 37 °C in a humidified atmosphere of 5% CO−/− mice (6–8 weeks old) on C57BL/6J background and age-matched wild-type C57BL/6J controls were purchased from Beijing HFK Bioscience Co., Ltd. . Mice were housed in SPF-grade animal facilities with a 12 h light/dark cycle at 23 °C (±2 °C). All animal procedures follow the NIH guide for the Care and Use of Laboratory Animals . Starting from 6 weeks, the mice were fed with a HCD  for 12 weeks. All ApoE−/− mice were dosed daily via intragastric gavage with 100 mg·kg-1·day−1 UA and 25 mg·kg−1·day−1 simvastatin dissolved in 0.5% CMC-Na or administered 0.5% CMC-Na alone (vehicle control) (n = 8 per group).Male ApoE4 cells/well) were seeded on glass slides in 96-well plates. After LPS treatment (with or without UA pretreatment), the slides were fixed with 4% PFA for 30 min. Then, the slides were permeabilized with PBS-T (containing 0.1% Triton x-100 in PBS solution) and blocked with PBS-B (containing 4% BSA in PBS solution). After being incubated with primary antibody (1:1000) and secondary antibody (1:5000), cells were stained with Hoechst 33342 in dark for 5 min. The protein location and expression were observed with IN Cell Analyzer 2000 .Cells  at room temperature for 2 h, membranes were incubated with specific primary antibodies and secondary antibodies. The protein-antibody complexes were detected by ECL Advanced Western Blot Detection Kit. The intensity of the band was quantitated with Quantity One software (Bio-Rad).6/well) were seeded in 6-well plate overnight. 100 pM siRNA was diluted in 100 μL Opti-MEM reduced serum medium in each well and then mixed gently. Similarly, 5 μL of Lipofectamine TM 2000 was diluted in 100 μL of Opti-MEM reduced serum medium and mixed gently. After incubation for 5 min at room temperature, the diluted siRNA and diluted lipofectamine  were mixed gently and incubated for another 20 min at room temperature. Further, siRNA-lipofectamine complex (200 μL) was added to each well. After incubation for 4 h, the complexes were discarded and cells were cultured with completed medium.Gene silencing experiment of TLR4, MyD88 with siRNA was performed according to our previous report . Briefly® qPCR Master Mix . The primer sequences used in this experiment are listed for LOX-1: 5′-TGGGAAAAGAGCCAAGAGAA-3′ (forward), 5′- TAAGTGGGGCATCAAAGGA’ (reverse); anf for GAPDH: 5′-AGAAGGCTGGGGCTCATTTG-3′ (forward), 5′-AGGGGCCATCCACAGTCTTC-3′ (reverse). The levels of LOX-1 expression were determined by normalizing to GAPDH expression.The mRNA expression of LOX-1 was determined with real-time PCR as our previous report . Briefly.To evaluate plaque extension, frozen sections of the aortic sinus (8 mm) were stained using Oil Red O, hematoxylin, and eosin (H&E), respectively. Related experiments were performed following the method of Paigen et al. .−/− mice were randomly allocated to groups and equal group sizes were obtained (n = 8 per group). Data were expressed as the means ± SD. The differences between groups were analyzed using Prism 5.0 , and the statistical analysis was performed by analysis of variance (one-way ANOVA) followed by Student Newman–Keuls test.Twenty four ApoEIn summary, this study showed that natural product-UA inhibited NF-κB-mediated LOX-1 expression both in vivo and in vitro through ROS generation. In view of the key roles of LOX-1 in the pathogenesis of atherosclerosis, inhibition of LOX-1 contributed to the anti-atherosclerotic effect of UA. Our study demonstrates that UA showed the effect of ameliorating atherosclerosis and put forward the potential mechanism. It supplies a new sight to illustrate the mechanism of action of UA for attenuates atherosclerosis in mice.


Brazil has at least 800,000 HTLV-1 infected individuals. HTLV-1 can be transmitted via sexual intercourse, contact with blood and from mother to child, mainly by breastfeeding. Treatments for the high morbidity/mortality associated diseases (ATL and HAM/TSP) are limited, therefore, infection prevention is of utmost importance. However, antenatal screening is not routinely performed in Brazil. A lack of data regarding the number of individuals infected via breastfeeding impairs the development of government policies. The objective is to estimate the number of HTLV-1 infections occurring annually due to mother to child transmission (MTCT) in Brazil, nationally and regionally.To estimate HTLV-1 MTCT in Brazil the following variables are modelled: number of births, prevalence of HTLV-1 infection in pregnant women, breastfeeding duration rate and transmission risk according to breastfeeding period. The number of cases of HAM/TSP and ATL attributable to MTCT are also estimated.In 2008, there were 2,934,828 live births in Brazil. HTLV prevalence in pregnant women in Brazil ranges between 0.1–1.05% by region. An estimated 16,548 HTLV-1 infected women are pregnant each year. According to the breastfeeding pattern and HTLV-1 prevalence of each region there are an estimated 3,024 new cases of HTLV-1 infection due to MTCT annually of which 2,610 are preventable through infant feeding advice. These 3,024 transmissions will result in 120–604 cases of ATL and 8–272 of HAM/TSP. North-East region comprises the high number of MTCT cases, followed by South-East.A high number of new HTLV-1 infections due to MTCT occur every year in Brazil. Antenatal screening and avoiding breastfeeding are essential to prevent subsequent development of HTLV-1-associated diseases. The human T-cell lymphotropic virus type 1 (HTLV-1) causes lifelong infection in humans. Although most infected individuals remain asymptomatic HTLV-1 causes severe disease in at least 5% of carriers. In the present study we estimate the number of HTLV-1 infections that occur annually due to mother to child transmission (MTCT) in Brazil and within its regions. The high number of estimated cases due to MTCT  which will give rise in adult life to HTLV-1associated diseases suggest the importance of performing HTLV-1 antenatal screening in Brazil to reduce the dissemination of this infection and to thus prevent a blood cancer to which most individuals succumb within 8 months of presentation and in others a disabling disease of the spinal cord. The human T-cell lymphotropic virus type 1 (HTLV-1) infects at least 5–10 million individuals throughout the world. Brazil has approximately 800,000 carriers . The lifHTLV-1 infection may be detected using screening tests such as enzyme-linked immunosorbent assay (ELISA). However, confirmatory and typing tests are mandatory to conclude the diagnosis. Western blot (WB) and the HTLV-1 proviral DNA detection by polymerase chain reaction (PCR) are considered confirmatory tests –6.To reduce HTLV-1 transmission in Brazil, since 1993 blood donations are tested before transfusion. HoweverSeveral studies have demonstrated that a reduction in the number of mothers breast-feeding and a shortening of the breast-feeding period decreased the MTCT by about 80% ,11. ThisLack of data regarding the number of individuals infected via breastfeeding impairs the development and implementation of government policies. In 2016, for example, in Brazil, there were 87 new diagnoses of HIV infection reported in children under 5 years. HoweverOur aim is to estimate the number of HTLV-1 infections that occur annually due to mother to child transmission (MTCT) in Brazil.For the estimation of the number of HTLV-1 cases due to MTCT per year in Brazil, the following variables were used: number of births in Brazil and its regions, prevalence of HTLV-1 infection in pregnant women in the country, breastfeeding duration rate and transmission risk according to breastfeeding period. We also estimated the number of HAM/TSP and ATL cases due to MTCT according to the lifetime risk of each disease. The number of births in Brazil and the breastfeeding duration rate were acquired from Health Ministry of Brazil data.To determine the prevalence of HTLV-1 infection in pregnant women in Brazil, a literature review was performed according to recommendations established by Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). The seaThe following inclusion criteria were: 1) studied population includes Brazilian pregnant women; 2) it provides measures of prevalence; 3) studies used confirmatory tests for HTLV-1 infection, such as WB or molecular assays, such as PCR. The exclusion criteria were: 1) studies that did not provide prevalence; 2) studied population was not pregnant women; 3) pregnant women were not from Brazil; 4) the study performed only screening test for HTLV infection.For this systematic review no protocol was registered, and no quality scoring system was applied.Data regarding the number of analysed samples, year of study publication, HTLV-1 test method, Brazilian state, prevalence of HTLV in pregnant women were extracted from the selected studies.In 2008, a large study regarding the prevalence of breastfeeding within Brazil was performed by Brazilian Health Ministry. In this study, the prevalence of breastfeeding was determined from 30 days until 1 year after birth and it included analysis per country region and by Brazilian state . These dTo estimate HTLV-1 MTCT cases in Brazil we used the methodology described in Malik et al. 2018. The HTLV-1 transmission risk according to breastfeeding period assumed were those reported by Takezaki and colleagues . A line In a similar process, by retrieving published data on breastfeeding rates against time for each region of Brazil  Table 1Table 1, By multiplying the number of breastfeeding women in three-month periods from 0 to 3 years, midpoint viral transmission rate for the respective period and the seroprevalence of HTLV-1 amongst pregnant women in the population ; the numFor completeness, a 2.5 percent risk of mother to child transmission from sources other than breastfeeding is incorporated in the analysis . This waHAM/TSP and ATL development rate comprises the proportion of individuals who develop those diseases following HTLV-1 infection. The lifetime risk of HAM varies between 0.25% to 3%. However, in Brazil, some studies showed a high risk for HAM/TSP onset . The incThe systematic review regarding HTLV prevalence in pregnant woman from Brazil identified 565 studies. In 2008, there were 2,934,828 births in Brazil. A summary of the findings for each region of Brazil is presented in Regarding HTLV associated diseases, supposing 4–20% and 3% (0.25–9%) the risk of ATL and HAM/TSP development respectively, 120–604 ATL and 91 cases (8–272) HAM/TSP cases should occur in the future due to MTCT each year.HTLV-1 infection and its associated diseases remain neglected . While AStrongyloides infection results in an increased rate of MTCT (31%) [To estimate the number of new cases of HTLV infections due to MTCT in Brazil we used the transmission risk rates observed in Japan as they are very robust. A recent paper from Brazil, obtained very similar rates of those that were used, confirming that they are suitable for this study. Different factors, such as HTLV proviral load and coinfections can influence the MTCT risk. For example, in Peru, CT (31%) ,38. EarlCT (31%) , where tCT (31%)  was condThe present analysis indicates that a large number of new cases of HTLV infections due to mother to child transmission in Brazil occur each year. When comparing with other diseases that are currently included in prenatal and neonatal screening, such as HIV, the size of the problem becomes more evident. Beside this, we also calculate that antenatal screening to identify infected mothers who would be advised regarding the risk of transmission and associated diseases and potentially to avoid or limit breastfeeding, would lead to the prevention of 104–522 cases of ATL and 6–235 cases of HAM/TSP which result from vertical transmission each year . These diseases present high morbidity and mortality. However, even apparently asymptomatic infection has a negative impact on the life of HTLV-1 seropositive patients. Decreased quality of life, increased incidence of depression and anxiety among asymptomatic individuals diagnosed with HTLV infection has been demonstrated and are not necessarily a consequence of being informed of the diagnosis –41. In BIn this study, to estimate the number of individuals that will develop clinical manifestations resulting from HTLV infection we used the range described in the literature. The risk of HAM/TSP development after vertical transmission is not known. However, there are many reports that HAM/TSP can occur after mother to child transmission –48. AlthAntenatal screening would also detect, and with the same intervention could also prevent mother to child transmission of, HTLV-2. This virus type was not included in the study, since the data regarding its prevalence are scarce, especially in pregnant women.It is also worth noting that, preventing the occurrence of a single new case, it is possible to inhibit the transmission chain of this individual, blocking virus maintenance for generations and intra-family dissemination, a fact commonly observed in HTLV infection ,9,52–54.In Japan, a country with a high HTLV prevalence, a nationwide MTCT prevention program was implemented in 2011. To prevent the vertical transmission, the HTLV-1 seropositive pregnant women are recommended to use three feeding methods: formula feeding, short term breast-feeding (up to 3 months), and feeding with thawed frozen milk. It is noteworthy that infection may occur in the mother after the antenatal screening therefore, the period in which the screening is made may be relevant and it perhaps it should be performed later into the pregnancy. However, the cost of this would be much higher than adding HTLV-1 to the existing screening process. Incident maternal infection during the breast-feeding period has not be considered in this analysis.Antenatal screening implementation is an important tool to prevent HTLV dissemination. In 1987, the Nagasaki Prefecture in Japan, implemented the ATLL Prevention Program Nagasaki, and the avoidance of breastfeeding by HTLV infected mother resulted in an important reduction of HTLV-1 MTCT from 20.3% to 2.5%, confirming the importance of antenatal screening in disease control ,56. SimiIt is important to highlight that avoidance of breastfeeding may be harmful, mainly in low-income regions, as early weaning could result in an increased infant mortality and in the number of diarrheal diseases events . SeroposAlthough antenatal HTLV-1 screening is performed in some Brazilian’s cities/states, such as Salvador, the city with the highest reported HTLV-1 prevalence, it is not included among the tests that are currently offered for pregnant woman by the Brazilian health system.Finally, this study showed that the non-implementation of the test in the antenatal routine performed by SUS cannot be justified on the grounds of scarcity of infected individuals, since the national neonatal screening program screens for diseases such as, cystic fibrosis and phenylketonuria that affect approximately 1,250 and 1,225 inhabitants in Brazil respectively ,60.Therefore, we conclude that HTLV-1/2 antenatal screening should be implemented in the routine evaluation of pregnant women from Brazil that is offered by the national health system in order to ensure that which is established in the Brazilian Constitution: health as a universal right.


Constitutive expression of the chemokine Mcp1 in mouse cardiomyocytes creates a model of inflammatory cardiomyopathy, with death from heart failure at age 7–8 months. A critical pathogenic role has previously been proposed for induced oxidative stress, involving NADPH oxidase activation. To test this idea, we exposed the mice to elevated oxygen levels. Against expectation, this prevented, rather than accelerated, the ultrastructural and functional signs of heart failure. This result suggests that the immune signaling initiated by Mcp1 leads instead to the inhibition of cellular oxygen usage, for which mitochondrial respiration is an obvious target. To address this hypothesis, we combined the Mcp1 model with xenotopic expression of the alternative oxidase (AOX), which provides a sink for electrons blocked from passage to oxygen via respiratory complexes III and IV. Ubiquitous AOX expression provided only a minor delay to cardiac functional deterioration and did not prevent the induction of markers of cardiac and metabolic remodeling considered a hallmark of the model. Moreover, cardiomyocyte-specific AOX expression resulted in exacerbation of Mcp1-induced heart failure, and failed to rescue a second cardiomyopathy model directly involving loss of cIV. Our findings imply that mitochondrial involvement in the pathology of inflammatory cardiomyopathy is multifaceted and complex. Its immediate effect is to promote the infiltration of immune cells, principally monocytes, and facilitate their activation as phagocytes able to attack pathogens and remove debris. Although the exact sequence of events is not fully understood, secondary signaling from immune cells promotes repair, stress-resistance and tissue remodeling, as appropriate. In the case of a cardiac infarct resulting from localized ischemia, the responses induced in this manner by Mcp1 have been shown to facilitate the survival and repair of the surrounding tissue, as well as revascularization and scar formation3.Monocyte chemoattractant protein 1 (Mcp1), a small cytokine of the chemokine family, is expressed and secreted by pathogen-infected or damaged cells, to activate appropriate immune responses (see relevant reviewsMcp1 in cardiomyocytes in the young (8–12 week-old) mouse was shown to protect against some of the consequences of myocardial infarct, notably by limiting the extent of scar formation and adverse tissue remodeling, in specific pathological paradigms5. However, in other experiments Mcp1 was found to be detrimental to recovery after a cardiac ischemic episode, based on the phenotypic improvement brought about by expressing a truncated, dominant-negative version of human MCP16. Mcp1 and other cytokines have been implicated in human heart failure7, where their chronic activation leads to an inflammatory crisis, with generalized remodeling that eventually compromises contractile function.Accordingly, constitutive over-expression of Mcp1 in adult cardiomyocytes (referred to hereafter as ‘the Mcp1 mouse’) shows progressive phenotypic deterioration, involving myocardial infiltration by monocytes that fail to differentiate properly, leading to an inflammatory crisis that is ultimately fatal8. The Mcp1 mouse is therefore an animal model for several different aspects of cardiac pathology, including ischemic preconditioning5, inflammatory cardiomyopathy and chronic heart failure due to adverse remodeling of the myocardium. The initial phase of tissue remodeling can be considered to be beneficial, following tissue damage due to ischemia or infection. This process is envisaged to switch metabolism at the borders of a damaged area of the myocardium to a lower energy, more glycolytic state, preserving tissue integrity during repair. On the other hand, chronic inflammatory signaling initiated by Mcp1 results in adverse remodeling of the entire myocardium. Studies in a cardiac myoblast cell-line have also suggested a direct effect of Mcp1 on cardiomyocytes, mediated by Mcp1-inducible protein (MCPIP), which elicits the activation of NADPH oxidase at the cell membrane, promoting autophagy and eventual cell death due to the resulting excessive ROS production9.The mouse line constitutively overexpressing In order to test whether such an oxidative crisis can explain the pathology of the Mcp1 mouse, we exposed the mice to a hyperoxic regime, which would be predicted to synergize with NADPH oxidase activation. Against expectation, this treatment did not accelerate the inflammatory crisis, but instead conferred protection against it. The result suggested that the inflammatory crisis involved inhibition of the major oxygen-using machinery of the cell, namely mitochondrial oxidative phosphorylation (OXPHOS).Ciona intestinalis10. AOX is absent from mammals but is found in most other taxa. It serves as a by-pass of the terminal steps of respiratory electron transfer from ubiquinol (CoQ) to oxygen, catalyzed by OXPHOS complex III  and cIV (cytochrome c oxidase). In contrast to cIII and cIV, AOX is a non-proton-motive enzyme, for which reason its engagement also leads to a substantial drop in ATP yield. Importantly, AOX only becomes active under conditions where the reduction state of its primary substrate, ubiquinol, surpasses a certain threshold, typically with around 30–40% of it accumulating in the reduced form13. Thus, AOX is only engaged when the standard respiratory chain is inhibited or overloaded downstream of ubiquinol reduction, and thus its expression does not generate a global ‘short-circuit’ in mitochondrial energy metabolism.To test the involvement of OXPHOS, we made use of a mouse model expressing the alternative oxidase (AOX) from 14. Nevertheless, in the face of specific pathological stresses, such as exposure to cyanide, a poison targeted on cIV14, AOX-expressing mice show spectacular resistance. They also resist the cardiotoxicity of a point mutation in an assembly factor for cIII15, and the lethal inflammatory crisis induced by lipopolysaccharide16. These two findings have obvious potential relevance to the Mcp1 mouse: the first because a cardiomyopathy was rescued, the second because AOX was able to attenuate macrophage activation due to respiratory chain inhibition and succinate overload. A similar process should be driven by chronic Mcp1 expression in the heart, which AOX should be able to alleviate by the same mechanism.In accordance with this, mice expressing AOX under the strong, ubiquitously active CAG promoter are viable, and exhibit essentially no physiological differences from wild-type mice when reared under standard, non-stressful conditions10, which expresses AOX ubiquitously, and a second model that we constructed, enabling us to express AOX specifically in cardiomyocytes. This allowed us to assess separately whether AOX is able to overcome the lethal phenotype induced by directly crippling cIV in cardiomyocytes. Our findings, presented below, indicate that any role of mitochondrial dysfunction in the inflammatory crisis provoked by chronic Mcp1 overexpression is primarily in cells other than cardiomyocytes.To address the issue of cell-type specificity we made use of two murine AOX models: the one already describedIn the Mcp1-overexpressing heart, tissue remodeling was marked by a progressive disruption of the tissue, involving degradation of the intracellular actomyosin network Fig. , disorga16. Furthermore, since OXPHOS is the major producer of cellular ATP, its inhibition in cardiomyocytes could also be postulated to be part of a machinery that switches metabolism to a tissue-protective, more glycolytic state. This would result in adverse remodeling of the entire tissue, if left unchecked.The effect of hyperoxia suggests a completely different explanation for the phenotype produced by Mcp1 overexpression, namely that Mcp1 provokes the inhibition of the major oxygen-dependent pathway of cells, mitochondrial respiration. Respiratory inhibition at or beyond the level of cIII has already been implicated in macrophage activation10 , we constructed a second mouse line (‘SNAPf-AOX’) containing a version of the AOX transgene placed downstream of a floxed copy of the coding sequence of the SNAP-tag peptide17. To prevent readthrough expression of AOX, the SNAP-tag cassette included three, reiterated copies of the poly(A) addition sequence from SV40 . Expression of the SNAP-tag peptide was verified in hemizygous SNAPf-AOX mice in all tissues tested by Western blot . To complement the use of the AOX mouse described aboveV40 Fig. . The entlot Fig. , whilst lot Fig. . Crossinlot Fig. . Note th18. However, cardio AOX in combination with Mcp1 produced a clear acceleration of the lethal phenotype, with double-expressor mice dying between 16–25 weeks of age (blue trace). Cardiac parameters followed a similar pattern. Whilst mean diastolic and systolic volumes in Mcp1-overexpressing mice showed only minor deviations from the controls . In the case of CoQ10 /CoQ10(ox) that was almost restored to that of wild-type  monocytes and other immune cells, as well as fibroblasts, endothelial cells and neighboring cardiomyocytes. This complexity undoubtedly contributes to the apparently contradictory or biphasic nature of the Mcp1 response reported in the literature, which promotes vascularization and limits scar formation after an ischemic episodeult Figs . Althouga priori reason to invoke a direct effect on cardiomyocytes. Our findings with AOX strongly imply that at least one other cell-type is involved in inducing the remodeling process leading to contractile failure, for which macrophages are the obvious candidates. To pin down whether Mcp1 has any direct effect upon cardiomyocytes would require further genetic modeling, such as cell-specific knockout of the Mcp1 receptor CCR2.Note that our data do not address the question of the cell-type(s) upon which Mcp1 itself acts. The canonical targets of all chemokines are immune cells, so there is no 24, including some directing myocardial expression25. It can also be background-dependent22. Myh6-cre appears to be efficient26, but there is some reported mosaicism in the timing of its expression27 that may be relevant. However, even if present, metabolic heterogeneity of cardiomyocytes may not have a major functional effect28.There remain other possible explanations for the phenotypic difference between global AOX and cardiomyocyte-specific AOX expression in the Mcp1 mouse, which should be considered. One is that Myh6-cre is not sufficiently expressed in all cells to produce a uniform genomic rearrangement. In SNAPf-AOX mice also expressing Myh6-cre, some residual SNAP-tag expression was seen in heart Fig. , which i29. In contrast, Myh6-cre is only expressed when the adult-type myosin heavy chain 6 would normally start to replace the fetal-type myosin heavy chain 7. Thus, AOX would be expressed in cardiomyocytes only from late fetal stages on, which may conflict with the epigenetic programming of metabolism set earlier in development.Another possibility is that constitutive AOX expression from even before fertilization enables energy metabolism to be epigenetically adapted to its presence30. We inferred that an unknown signal emanating directly from cIII or cIV must impose such a regulation on cI, rather than simply the interruption of respiratory electron flow to oxygen. Introducing a by-pass of cI via the alternative NADH dehydrogenase Ndi1 from yeast was able to overcome this blockade30. However, implementing both by-passes simultaneously in the Mcp1 model in vivo is very unlikely to succeed, since it would completely short-circuit mitochondrial ATP production. Although it can work in cultured cells, it failed in an OXPHOS disease model in Drosophila31, instead producing lethality. To test it in the Mcp1 mouse we would need first to elucidate its mechanistic basis in cultured cells, then target the relevant molecular machinery. The action of Mcp1 is evidently complex, and will require much deeper knowledge of macrophage as well as cardiomyocyte biology, before its roles in cardiac disease can be properly understood.Although a key finding of the current study, it is difficult to follow up on the observation that hyperoxia counteracts heart failure in the Mcp1 mouse, without recourse to the kinds of genetic models described here. However, combining it with cell-specific activation of AOX, deletion of CCR2 and manipulation of other oxygen-sensitive enzymes and cytokines should now be a useful approach, helping to define where it acts and by what mechanism. One attractive idea is that Mcp1 induces a hypoxia-like response, for example by HIF activation, and this is counteracted by high oxygen levels, but not by AOX. In principle, this should be testable. Another possibility, consistent with this idea and with our observation that Mcp1 does provoke a clear respiratory defect Fig. , is thatMyh6 (Myhca) promoter32, here described as ‘Mcp1 mice’, were supplied by Pappachan Kolattukudy, University of Central Florida. Cox10fl/fl mice33 were supplied by Carlos Moraes, University of Miami. Mice expressing Myh6-cre (Stock No: 011038) and ACTB-cre (Stock No: 019099) were purchased from Jackson Laboratories. For the initial immunohistochemistry and hyperoxia experiments, Mcp1 mice were maintained in the original background (FVB) in which they were supplied, using non-transgenic FVB littermates as controls. For all other experiments, Mcp1, Cox10fl/fl, Myh6-cre and ACTB-cre mice were backcrossed over ≥8 generations to C57Bl6/JOlaHsd (Envigo), in which the previously described AOXRosa26 strain10 was also maintained. SNAPf-AOX mice, containing a floxed SNAP-tag expression cassette upstream of the AOX coding region as described in this paper, were created and backcrossed to strain C57Bl6/JOlaHsd using essentially the same methods as described previously for the AOXRosa26 mice10. All animals were maintained and treated according to the regulations of the competent animal welfare agencies, and all of the experimental protocols were approved according to the following permits: for experiments conducted in Germany, Regierungspräsidium Darmstadt  and for experiments conducted in Finland, ELLA . Mice were housed in a humidity and temperature-regulated animal facility with food and water provided ad libitum, and were euthanized at the end of the experiment by cervical dislocation, in some cases with prior exposure to CO2. Humane end points were followed as described in the animal permits but, in addition, signs of prolonged immobility and/or consistent body shivers were considered as indicators of severe stress and the mice were duly terminated. Except where stated, experiments used males only, in order to minimize the total number of animals required. To test the effects of hyperoxia, mice were transferred at 12 weeks of age to an 85% O2 environment in a closed chamber, alternating at 24 h intervals with ambient air34 , or by echocardiography using a Vevo 2100 imager10, as indicated in the relevant figures. Body weight measurement and endurance (treadmill) experiments were performed as previously described10, except that, for the treadmill procedure, the total distance run was recorded instead of time, although the experimental setup was otherwise identical.Cardiac function was assessed by MRI10, with the primer sets indicated in Table All animals used in the experiments were PCR-genotyped for all relevant loci, using crude ear-punch DNA as described previouslyen bloc in half-saturated uranyl acetate, dehydrated in an ascending ethanol series and finally embedded in Agar 100 (Agar Scientific Ltd). Ultrathin sections (60–70 nm) were cut using an Ultracut E ultramicrotome (Leica-Reichert) and examined by transmission electron microscopy . Digital images were captured with a slow-scan 2 K CCD camera . For each animal, images were generated from 2–3 sections from each of 2 heart-tissue blocks, using 3 individual animals of each genotype. An alternative procedure used in some experiments involved fixation in 3% glutaraldehyde buffered with 0.1 M sodium cacodylate, embedding in Epon medium (Sigma-Aldrich) and staining with uranyl acetate and lead citrate. These samples were viewed and photographed in a Philips CM 10 electron microscope.A small piece of left ventricle (from mice of both sexes) was excised and stored at 4 °C in a buffer containing 1.5% glutaraldehyde, 1.5% formaldehyde, 0.15 M HEPES/KOH, pH 7.4 until embedding and sectioning. For epoxy resin embedding, the samples were post-fixed in 1% osmium tetroxide, stained 2 until cryo-sectioning. Sections were stained for LC3 , and counterstained for DNA with DAPI (Bioquest #17507) and for F-actin with phalloidin (Sigma-Aldrich #P5282). Sections were imaged using a Leica SP2 laser-scanning confocal microscope.Tissue samples were snap-frozen and stored in liquid Ngmax for 15 min at 4 °C. The upper (aqueous) phase was collected and RNA precipitated with an equal volume of isopropanol, followed by centrifugation at 12,000 gmax for 15 min at 4 °C. The pellet was washed with 70% ethanol, air dried, resuspended in 20 μl nuclease-free water and quantified by NanoDrop spectrophotometry (ThermoFisher Scientific). Samples were DNase treated and reverse transcribed using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (ThermoFisher Scientific), following the manufacturer’s recommended procedure. The final 20 μl reaction volume was diluted 1:10 with nuclease-free water and 5 μl aliquots were used for qRTPCR, using the primers listed in Table Rps18 gene, then to the wild-type control, and finally used as exponents to generate 2−ddCT.Total RNA was extracted from left ventricle tissue using bead homogenization in 1 ml (>10 volumes) of TRIzol reagent (Ambion). After incubation for 10 min at room temperature, samples were gently extracted with 0.2 volumes of chloroform and centrifuged at 12,000 10. 10 μg protein aliquots were electrophoresed on 4–20% TGX stain-free gels (Bio-Rad) and transferred to PVDF membranes using Trans-Blot® TurboTM PVDF Transfer pack (Bio-Rad). Primary antibodies and their dilutions were as specified previously10, plus the following: porin , OXPHOS cocktail , AOX , HSP60 , GAPDH  and LC3b . Secondary antibodies and their dilutions were: goat anti-mouse IgG  and goat anti-rabbit IgG . Blot images have been cropped, rotated and framed where appropriate, and optimized for contrast and brightness, with reprobings of the same gel or different regions of the same gel shown as distinct image panels separated by white spacers. No other manipulations were introduced.Protein isolation and Western blotting were conducted essentially as described previously10. Flux values [pmol O2/(s × ml)] obtained from the traces were normalized to the amount of mitochondrial protein. The activity of AOX , as well that of cytochrome c oxidase in all samples, was verified, even though these measurements are not shown in the figures.Mitochondria were isolated from heart and used for respirometry, essentially as described previously36. CoQ detection was performed by a Coulochem III ESA electrochemical detector linked to the HPLC system, and conditioning cell was set up before the injection valve in order to maintain the oxidation state of the sample analytes. For redox status analysis, oxidized and reduced peaks of both CoQ9 and CoQ10 were quantified and their ratio calculated. Commercial CoQ6 was used as an internal standard in all runs.The ratio of reduced to oxidized CoQ was determined by a rapid lipid extraction method. 10 to 15 mg of heart tissue (both sexes) was homogenized with a manual micro-pestle in 20 mM potassium phosphate buffer, pH 7.5. Debris was removed by maximum speed centrifugation at 4 °C. 100 μl of sample was mixed by vortexing with 330 μl of n-propanol in the presence of 0.5 mM β-mercaptoethanol. After a further centrifugation at maximum speed to remove any remaining debris the clean lipid extracts were injected into a reverse-phase Beckmann 166 HPLC system, equipped with a C18 column  and column oven set up at 40 °C. The mobile phase, flow rate and gradient settings were as described previouslyad libitum was maintained. Mice (both sexes) were anesthetized with 4% isoflurane before injecting either U-13C6 labeled  or 12C6  glucose solution (2 g/kg bodyweight). 15 min after injection, mice were sacrificed by cervical dislocation and a piece of left ventricle tissue was collected and snap-frozen in liquid nitrogen. All samples were stored at −80 °C until processing.Food was withdrawn 4–6 h before the experiment, whilst water supply Frozen heart tissues were homogenized in 1 ml cold (−80 °C) 80% methanol on dry ice. After clarification by centrifugation for 5 min at 4 °C, the supernatant was transferred to an Eppendorf tube. The remaining pellet was resuspended in RIPA protein lysis buffer and protein concentration was determined (by the BCA method). A fraction of the supernatant corresponding to 5 μg protein from each sample was transferred to a glass vial and 5 nmol norvaline was added as an internal control. Metabolite extracts were dried using an EZ-2 Elite evaporator (SP Scientific) and stored at −80 °C. Samples were analyzed with an UltiMate 3000RSLC HPLC (Thermo Scientific) coupled to a Q Exactive mass spectrometer (Thermo Scientific). After resuspension in 50% acetonitrile, one-tenth of each sample was loaded onto a Luna NH2  column. Separation was achieved using (A) 5 mM ammonium acetate (pH 9.9) and (B) acetonitrile at a flowrate of 200 μl/min. A gradient from 15% to 95% of (A) over 18 min was utilized, followed by an isocratic step at 95% (A) for 9 min and re-equilibration to the initial 15% (A) for 7 min. The Q Exactive was run with polarity switching (+3.50 kV/−3.50 kV) in full-scan mode with an m/z range of 65–975. Metabolites were quantified with TraceFinder 4.1 (ThermoFisher Scientific) using accurate mass measurements (≤3 ppm) and expected retention times established with pure standards. Data analysis, including principal component analysis and hierarchical clustering was performed using in-house scripts in the statistical language R. Statistical differences were determined by one-way ANOVA testing.post hoc Tukey HSD (multiple comparisons) test or, where, two factors were considered, by two-way ANOVA, followed by post hoc Tukey HSD (multiple comparisons) test where interaction was detected or where more than two levels were considered for a given significant factor. Survival curves were compared by the log-rank (Mantel-Cox) and Gehan-Brewslow-Wilcoxon tests, all using GraphPad Prism software. The significance of progeny-class numbers obtained in Cox10 crosses was assessed by the chi-squared test with Yates’ correction for continuity, implemented using an online tool37. Statistical analyses presented in Supplementary Tables give exact p values, which are generally quoted in figures and legends using a conventional *, **, *** nomenclature to denote appropriate thresholds.Data groups were compared by one-way ANOVA, with Mcp1 supplementaryS1S2S3S4S5S6S7S8


Temporomandibular joint osteoarthritis (TMJ-OA), mainly exhibit extracellular matrix loss and condylar cartilage degradation, is the most common chronic and degenerative maxillofacial osteoarthritis; however, no efficient therapy for TMJ-OA exists due to the poor understanding of its pathological progression. MicroRNA (miR)-140-5p is a novel non-coding microRNAs (miRNAs) that expressed in osteoarthritis specifically. To investigate the molecular mechanisms of miR-140-5p in TMJ-OA, primary mandibular condylar chondrocytes (MCCs) from C57BL/6N mice were treated with interleukins (IL)-1β or transfected with miR-140-5p mimics or inhibitors, respectively. The expression of matrix metallopeptidase (MMP)-13, miR-140-5p, nuclear factor (NF)-kB, Smad3 and transforming growth factor (TGF)-β3 were examined by western blotting or quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The interaction between the potential binding sequence of miR-140-5p and the 3′-untranslated region (3′UTR) of Smad3 mRNA was testified by dual-luciferase assay. Small Interfering RNA of Smad3 (Si-Smad3) was utilized to further identify the role of Smad3 mediated by miR-140-5p. The data showed MMP13, miR-140-5p and NF-kB increased significantly in response to IL-1β inflammatory response in MCCs, meanwhile, Smad3 and TGF-β3 reduced markedly. Moreover, transfection of miR-140-5p mimics significantly suppressed the expression of Smad3 and TGF-β3 in MCCs, while miR-140-5p inhibitors acted in a converse manner. As the luciferase reporter of Smad3 mRNA observed active interaction with miR-140-5p, Smad3 was identified as a direct target of miR-140-5p. Additionally, the expression of TGF-β3 was regulated upon the activation of Smad3. Together, these data suggested that miR-140-5p may play a role in regulating mandibular condylar cartilage homeostasis and potentially serve as a novel prognostic factor of TMJ-OA-like pathology. Temporomandibular joint osteoarthritis is a common condition in temporomandibular joint disorder (TMD). Approximately 40% of TMD patients presented TMJ-OA . Mainly in vitro showed lower levels of phosphorylated p38, phosphorylated JNK, and beta-catenin  are novel non-coding RNAs that regulate target genes negatively. Approximately 1/3 of human mRNAs can be regulated by miRNAs, which demonstrated that miRNAs played essential roles in controlling gene expression . MiRNAs Recent studies showed that the TGF-β superfamily play essential roles in almost all aspect of development, from the generation of germ cells to organ formation, and into postnatal life . TGF-β3 C57BL/6N mice  were obtained from Model Animal Research Center of Kunming Medical University . This study was carried out in accordance with the recommendations of Chinese Academy of Sciences. Mice mandibular condylar cartilage were cut into tiny fragments, then digested with 0.25% trypsin  for 25 min at 37°C and 0.2% collagenase  for 45min at 37°C. Followed by dissociation and filtration, cells were centrifuged at 1200 rpm for 5min. Primary MCCs were cultured according to previous method . FreshlymiR-140-5p mimics, miR-140-5p inhibitors, and their corresponding negative controls were designed and obtained from RiboBio . Cultured and plated in 6-well plates (∼50% confluence), MCCs were starved for 12 h, followed by 48 h transfection of miR-140-5p mimics or inhibitors, then treated with IL-1β for 24 h, respectively. MCCs transfected with microRNA mimics or inhibitors of miR-140-5p at a concentration of 50 nM by using riboFECTTM CP Transfection Kit (166T)  according to the manufacturer’s protocol.v/v) methanol/50% (v/v) acetone for 5 min. Subsequently, cells were rinsed in 1 × DPBS  for 3 times, each time 5 min, followed by incubation with rabbit anti-Smad3  at 4°C overnight. FITC-labeled goat anti-rabbit IgG  were used as secondary antibodies and incubate for 60 min. Cell nucleus were counterstained with DAPI  for 10 min. Coverslips were mounted in fluorescent mounting medium . Finally, the fluorescence micrograph were captured and analyzed by confocal laser scanning microscope system .Mandibular condylar chondrocytes were cultured on 18-mm glass coverslips  and stained for single immunofluorescence staining. Fixed in 50%  or mutant-type (MUT) 3′URT of Smad3, which containing the predicted binding sequences or mutant binding sites, was cloned into pmiR-RB-REPORTTM luciferase vector  by XhoI/NotI restriction sites, respectively. The coding sequences of 3′UTR of Smad3 for PCR-amplified primers were as follows: 5′-TATGTTGGCTGGAAAACCACAA-3′. After amplification, the PCR products were cloned into the vector for dual-luciferase assay. At a concentration of 1.5 × 104, cells (293T) in a 96-well plate were transfected with miR-140-5p or negative control by LipofectamineTM 3000  and incubated for 48 h. Finally, luciferase activity was measured posttransfection using Dual-Glo® Luciferase Assay System . The activity ratio of each group was normalized to the corresponding Renilla luciferase activity.miRTarBaseMandibular condylar chondrocytes were transfected with small interference RNA of mouse Smad3 (Si-Smad3) to suppress the Smad3 expression. According to the protocol, the transfection were performed by riboFECTTM CP Transfection Kit (166T) . After starved for 12 h, MCCs were followed by 48 h transfection of Si-Smad3.® Super Total RNA Extraction Kit  then reversed transcribed into cDNA by A5000 GoScriptTM Reverse Transcription System . Then miRNAs were reverse transcribed into cDNA with stem-loop primers by using miDETECT A TrackTM miRNA qRT-PCR Starter Kit . According to the manufacturer’s instructions, qRT-PCR was performed in a CFX96 system  using SybrGreen qPCR Mastermix . β-actin and small nuclear RNA U6 were used as internal controls to normalized gene expressions. The thermal cycling condition was 95°C for 10 min, followed by 40 cycles of 95°C for 30 s and 60°C for 30 s. The relative expression of miR-140-5p to U6, and relative expression of mRNA to β-actin were measured by using the comparative 2-ΔΔct method . GraphPad Prism 5.0 or SPSS 17.0 were performed to statistical analyses. Student’s t-test was conducted when two groups compared. One-way ANOVA followed by Bonferroni post hoc tests was used for multiple groups comparisons. The statistically significant difference considered when P values < 0.05.All data in the present study were represented as mean ± standard deviation (SD). Each group was repeated three times  for 24 h. The effects of IL-1β were examined in Figure In order to established TMJ-OA model To further elucidate the role of miR-140-5p in TMJ-OA like damage, specific miRNA mimics and inhibitors of mouse miR-140-5p were used for transfection experiment. MCCs were transfected with miR-140-5p mimics, or treated with miR-140-5p inhibitors for 48 h, respectively, followed with or without stimulation of IL-1β for 24 h. As showed in Figure To investigate whether miR-140-5p regulates the expression of Smad3 directly, we identified a potential miR-140-5p binding sequence in the 3′-untranslated region (3′UTR) of Smad3 Figure  by miRNATo identify the signaling pathway of Smad3 in mediating the downregulation of TGF-β3 was induced by miR-140-5p, we utilized small interfering RNA of Smad3 (Si-Smad3) for further transfection experiment. MCCs were co-transfected with or without Si-Smad3 in the presence or absence of miR-140-5p. Cells were harvested after 48 h transfection. As western blotting results showed in Figure in vitro.As widely accepted, IL-1β is a key mediator in accelerating the activity of MMPs . MMP13, Previous study reported that TGF-β is an essential initiator of chondrogenesis. Inhibition of TGF-β caused a reduction of extracellular matrix and resulted in OA . Smad3 pThe present study exhibit an increased expression of miR-140-5p was found in MCCs induced by IL-1β. MiR-140-5p, originated from miR-140, was reported to be expressed in cartilage particularly. As a key transcription factor in mediating TGF-β signaling pathway, Smad3 was observed an inversely relationship to miR-140-5-p. Next, we used dual-luciferase constructs and reporter assay to identify whether miR-140-5p inhibits Smad3 expression directly. Dual-luciferase assay demonstrated that miR-140-5p interacted with predicted binding sequence in the 3′UTR of Smad3, which consistent with At last, we provided evidence to demonstrate miR-140-5p regulated the expression of TGF-β3 through Smad3. To further investigation, we utilized small interfering RNA of mouse Smad3 (Si-Smad3) to co-transfected with miR-140-5p in MCCs. Compared with negative control, respectively, both of Si-Smad3 and miR-140-5p mimics exhibit an efficiently reduction of TGF-β3. Interestingly, co-transfection of miR-140-5p mimics and Si-Smad3 exhibit a more efficient reduction of TGF-β3 protein expression. It suggested that TGF-β3 may regulated by miR-140-5p in multiple ways, including Smad3 pathway. Novel evidences demonstrating that miR-140-3p negatively controlled TGF-β3, which proved to be a direct target of miR-140-3p . That’s Taken together, the present study provided evidence that miR-140-5p regulated TMJ-OA pathogenesis through TGF-β/Smad signaling pathway and might be a potential predictive factor for TMJ-OA.According to the recommendations of Chinese Academy of Sciences, the protocol of this study was approved by the Animal Experimental Ethics Committee of Kunming Medical University.WL and SL designed and developed the research. SZ, HY, CZ, QK, and JD conducted the experiments, and collected and analyzed the data. YX and YD wrote and drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Feeding of fresh cassava root to animals is restricted because it contains hydrocyanic acid at a high level, which is the origin for poisoning. High levels of hydrocyanic acid from fresh cassava root could be detoxified by sulfur addition to become nontoxic to cattle. The addition of 2% sulfur in a fermented total mixed ration containing fresh cassava root and ensiling for 7 days could improve dry matter digestibility, efficiency of microbial protein synthesis, and concentrations of total volatile fatty acid, propionic acid, and blood thiocyanate. p < 0.01). Bacterial populations were significantly different in the FTMR containing sulfur at 2% and 7 days of ensiling. Furthermore, microbial crude protein and efficiency of microbial protein synthesis were higher in the FTMR containing 2% sulfur and 7 days of ensiling (p < 0.01). Thus, high levels of hydrocyanic acid from fresh cassava root could be detoxified by a sulfur addition with an ensiling process to become nontoxic to cattle.The influence of sulfur included in fermented total mixed ration (FTMR) containing fresh cassava root on rumen characteristics, microbial protein synthesis, and blood metabolites in cattle was evaluated. Four Thai native beef cattle were randomly assigned according to a 2 × 2 factorial in a 4 × 4 Latin square design, and dietary treatments were as follows: factor A included a level of sulfur at 1% and 2% in total mixed ration, and factor B featured ensiling times at zero and 7 days. Digestibility of dry matter was increased when FTMR was supplemented with 2% sulfur. Blood thiocyanate increased by 69.5% when ensiling time was 7 days compared to no ensiling ( Cassava root is the main carbohydrate source produced for ruminant consumption in the tropical zone, particularly Thailand. The starchy root is produced generally for human consumption or as a carbohydrate source for animal livestock feed . Fresh cLactobacillus buchneri and fermenting for 90 day, and revealed an increased feed intake and growth rate in sheep when compared to those fed the nonfermented feed. In addition, Vasupen et al. [Fermented total mixed ration (FTMR) is a basic process to potentially maintain nutrient utilization, extend preservation, and reduce antinutritive substances in feeds . FTMR isn et al.  indicateRuminants receiving HCN from crops could eliminate it by enzymes such as rhodanese and β mercapto pyruvate sulfur transferase, which are excreted from rumen microbes . RhodaneIt was hypothesized that sulfur addition and the ensiling method could improve feed utilization and rumen fermentation efficiency. Thus, the goal of this work was to study the influence of sulfur doses included in FTMR containing fresh cassava root on nutrient intake, rumen characteristics, microbial protein synthesis, and blood metabolites in Thai native breeds of cattle.All cattle were handled per the guidelines of the Committee of Animal Experimentation on Animal Welfare for Experimental Animals of Khon Kaen University (record no. ACUC-KKU 32/61), and all experiments were performed under the Ethics of Animal Experimentation of the National Research Council of Thailand.Four Thai native beef cattle with an initial BW of 90 ± 5.0 kg were randomly assigned according to a 2 × 2 factorial in a 4 × 4 Latin square design, and dietary treatments were as follows: factor A included a level of sulfur supplementation at 1% and 2% in total mixed ration (TMR), and factor B featured ensiling times at 0 and 7 day. The optimum sulfur level and ensiling time were selected from our previous study [Fresh cassava root was collected from the Khon Kaen province areas of Thailand and then was chopped into chip form to go through a sieve (1 cm) by a machine (Yasothon Thailand). The FTMR was a rice straw chop with the concentrate mixed by machine and then adjusted with water to a moisture content of 55%, and lactic acid bacteria were not inoculated to the mixture before ensiling. FTMR then went through ensiling under anaerobic conditions in a plastic, sealed container to analyze ingredient and chemical composition . SamplesDuring the last 7 day of each period, samples of FTMR, feces, and urine were taken by using a total collection method, as animals were on metabolism crates to study nutrient digestibility and nitrogen balance. FTMR and feces were dried at 60 °C for 48 h then ground to pass through a 1 mm screen  and used for chemical analysis. The diets and feces were chemically analyzed for dry matter (DM), ash and crude protein (CP), and acid detergent fiber (ADF) . NeutralX mmol/day) corresponding to the purine derivatives excreted (Y mmol/day) was estimated based on the relationship derived by Chen and Gomes [Y = 0.85X + (0.385W0.75), where Y is the excretion of purine derivatives (mmol/day), X the microbial purines absorbed (mmol/day), and W is body weight of the animal (g/kg BW0.75).Nitrogen (N) was analyzed from the urine samples, and N utilization was calculated according to the equation described by the AOAC . Allantond Gomes : Y = 0.8X is the absorption of purine derivatives in mmol/day, which followed the assumptions calculated by Chen and Gomes [The supplies of microbial crude protein (MCP) in grams per day were calculated as follows:nd Gomes . The dig2SO4) at a 1:9 ratio, centrifuged at 16,000× g for 15 min, and then analyzed for ammonia nitrogen (NH3-N) , while total volatile fatty acid (VFA) and VFA profiles were performed using HPLC ; ETL Testing Laboratory, Inc., Cortland, NY, USA) according to Samuel et al. [Ten milliliters of blood was sampled from the animals’ jugular vein at 0 and 4 h after feeding on the 21st day of each period to analyze BUN and blood thiocyanate, as described by Lambert et al. . Approxil et al. . One milliliter of ruminal fluid was mixed with 9 mL of formalin solution, and then its population of bacteria and protozoa was counted using a microscope with a hemocytometer . Blood metabolites and rumen characteristics were expressed as mean values.ijkY is the observation for cattle j receiving diet i in period k; μ is the overall mean; iM is the effect of the various doses sulfur ; jE is the effect of the different ensiling times ; kA is the effect of the animal ; Pl is the effect of the period ; and ijkε is the residual effect. Results were presented as mean values with the standard error of the means. Differences between treatment means were tested by Duncan’s new multiple range test, and differences amongst means were considered statistically significant at p < 0.05.Data appropriate for the 2 × 2 factorial in a 4 × 4 Latin square design were analyzed using a Generalized Linear Model (GLM) procedure . The model is as follows:The chemical compositions of the treatments are shown in The HCN concentrations of FTMR supplementations at 1% and 2% sulfur were reduced 99.3% to 99.5% when compared to the fresh cassava root. Furthermore, reduction of the HCN concentration, even without the ensiling process, could be due to the preparation of the TMR process by machine, which may generate high temperatures, thereby resulting in lower HCN. Furthermore, FTMR supplementation at 2% sulfur and 7 d ensiling time was lowest when compared with other groups. Sulfur is an essential mineral for cell synthesis and maintenance of the cellular metabolisms of microbes in the rumen . In addip > 0.05) between sulfur levels and ensiling times on all parameters. Total FTMR intakes were similar among feed groups and ranged from 100.8 to 109.1 g/kg BW0.75. The levels of sulfur supplementation did not differ in the intake of nutrients and energy (p > 0.05). Differences in ensiling times did not affect nutrient digestibility. Nevertheless, the digestibility of DM was increased when supplemented at 2% sulfur (p < 0.05) in the FTMR, which was similar to the report by Supapong and Cherdthong [The DM intake and nutrient digestibility of FTMR in animals are shown in erdthong . Similarerdthong , who foup > 0.05), which ranged from 6.9 to 7.1 and 38.7 °C to 39.1 °C, respectively. Ruminal pH and temperature values for all FTMR were in the suitable range for microbial synthesis and activity to ferment the feed in the rumen [3-N values showed no interactions between sulfur levels and ensiling times. Ruminal NH3-N was significantly lowest at 15.2 mg/dL when supplemented with sulfur at a 2% level (p < 0.02). This could be due to ruminal microorganisms using NH3-N as material for cell synthesis and carbohydrate as an energy source. Thus, the utilization of NH3-N with the provided sulfur for microbial protein synthesis might be expected [The feeding of FTMR containing fresh cassava root did not change ruminal pH and temperature (he rumen . The conexpected . p > 0.05), and BUN concentration was in the optimal range for balanced protein utilization in the animals. This could be because ruminal NH3-N is highly beneficial for microbial protein synthesis, along with carbohydrates from fresh cassava root and sulfur, which thereby resulted in an unaltered BUN level. This result was related to nitrogen utilization and microbial protein synthesis. The concentration of blood thiocyanate were not altered by the levels of sulfur and ensiling times (p > 0.05). Blood thiocyanate increased by 69.5% with an ensiling time of 7 day compared to no ensiling (p < 0.01), possibly because the ensiling time can provide high-temperature conditions and acid from microbial activities to detoxify the HCN in FTMR. In addition, the ensiling time could change the structure of sulfur to sulfate and sulfide, which combines with thiosulfate to modulate to thiocyanate [BUN was not altered with the dietary treatments . Bacterial populations were significantly different with an FTMR inclusion of sulfur at 2% and 7 day of ensiling, which increased by 5.7% when compared with the FTMR inclusion of 1% sulfur and no ensiling (p < 0.01). This may be because ensiling time provides suitable fermentable energy for ruminal microbial synthesis and can stimulate feed digestion, as found in previous studies [The sulfur levels and ensiling time did not affect the protozoal concentration  and ranged from 76.7 to 79.5 g/day and 23.8 to 25.8 g/day, respectively. Total N intakes were similar in all treatments, as all Thai native beef cattle were fed the diet ad libitum. Therefore, all animals consumed the same amount of total dry matter intake (DMI), which showed no difference on total N intake. The results on purine absorption were significantly different among the groups, which showed increase  in the steers supplemented with FTMR ensiled for 7 day and had no effect on sulfur levels (p > 0.05). Furthermore, MCP and the efficiency of microbial protein synthesis (EMPS) were different among treatments, and higher values were found in the groups supplemented with FTMR that contained 2% sulfur and was ensiled for 7 day, while the lowest was found in the non-ensiling FTMR diet (p < 0.01). Similarly, Khosravi et al. [i et al.  revealedi et al. . It can p < 0.05) (p < 0.01). A diet with an exceedingly high level of sulfur (2% of the DM) with an ensiling time of 7 day increased total VFA and propionic acid concentrations. This could be because ensiling time improves the amount of available carbohydrates, which are rapidly fermented into total VFA and propionate in the rumen. In addition, a long ensiling time could provide more carbohydrates and result in high total VFA and propionic acid concentrations in the rumen [The proportions of total VFA, propionic acid, and butyric acid were significantly different among FTMR diets ( < 0.05) . The amohe rumen . Furtherhe rumen . Similarhe rumen  revealedhe rumen  found thThe addition of 2% sulfur in FTMR containing fresh cassava root and ensiling for 7 days could improve dry matter digestibility, efficiency of microbial protein synthesis, and concentrations of total volatile fatty acid, propionic acid, and blood thiocyanate. In addition, the high levels of HCN from fresh cassava root could be detoxified by sulfur addition with an ensiling process to become nontoxic to cattle. However, these findings should be further investigated regarding milk production to elucidate the effect of FTMR on animal milk production.