Centella asiatica (L.) Urban contains two ursane-type triterpene saponins, asiaticoside and madecassoside, as major secondary metabolites. In order to select candidate genes encoding UDP-glucosyltransferases (UGTs) involved in asiaticoside biosynthesis, we performed transcriptomic analysis of leaves elicited by methyl jasmonate (MeJA). Among the unigenes, 120 isotigs and 13 singletons of unique sequences were annotated as UGTs, including 37 putative full-length cDNAs, and 15 of the putative UGT genes were named according to the UGT committee nomenclature protocols. One of them, UGT73AH1, was characterized by heterologous expression in Escherichia coli BL21 (DE3) cells. After induction with IPTG, a total protein extract was assayed with UDP-glucose and asiatic acid. UPLC-QTOF/MS analysis showed that UGT73AH1 catalyzes the glycosylation of asiatic acid to its monoglucoside. It remains unclear whether glycosylation occurs on the triterpene C-2α, C-3β, C-23, or C-28 position. However, it is very likely that UGT73AH1 glucosylates the C-28 position, because only C-28 bears a glucose moiety in the final pathway product of asiatic acid, while C-2α, C-3β, and C-23 remain un-conjugated. Many plant secondary metabolites have characteristic carbon skeletons linked to one or more sugar moieties, and such glycosylation strongly affects the solubility, biochemical properties, subcellular localization, and biological function of the natural products . TypicalBupleurum falcatum, and soyasaponins from Glycine max. Plants produce a large variety of triterpene saponins, with molecular structures featuring an alicyclic triterpene backbone linked through glucosidic bonds to one or more sugar moieties . More thUGT genes involved in triterpenoid biosynthesis has seen relatively little progress over the last decades, likely because all plant genomes encode numerous UGTs homologs, making it difficult to select and characterize candidate genes [Arabidopsis thaliana and G. max genomes contain 115 and 242 UGT genes, respectively [UGT genes involved in the biosynthesis of particular triterpene saponins. In particular, the UGT73K1 and UGT71G1 genes from Medicago truncatula, UGT73P2 and UGT91H4 from G. max, UGT74M1 from Saponaria vaccaria, UGT73C10 and UGT73C11 from Barbarea vulgaris, and UGT74H6 from Avena strigosa, were shown to be involved in glycosylation of oleanane saponins [UGT73AD1, encoding a UGT responsible for forming ursane-type triterpene saponins has been identified to date [The functional characterization of te genes . For exaectively ,9,10,11.saponins ,14,15,16 to date , and it Centella asiatica (L.) Urban contains mainly ursane-type triterpene saponins, the most prominent ones being madecassoside and asiaticoside [C. asiatica, which have traditionally been used in various pharmaceutical applications. It is now firmly established that the major bioactivities of C. asiatica leaf extracts are due to these saponins, including memory improvement, wound and vein healing, antihistaminic, antiulcer and antilepsory treatments, as an antidepressant, and as antibacterial, antifungal, and antioxidant agents [The medicinal plant ticoside . Both mat agents ,20,21.C. asiatica. However, other UGT genes have not been characterized, to date.Asiaticoside and madecassoside have identical sugar chains (glucose-glucose-rhamnose) bonded to their carboxyl groups . TherefoC. asiatica. To this end, we selected UGT candidate genes from transcriptomic data of elicited C. asiatica leaves, and characterized them by heterologous expression.Therefore, the goal of the present study was to isolate and characterize the glycosyl transferase(s) responsible for the formation of asiaticoside triterpene saponins in C. asiatica leaves was analyzed, glycosyltransferase unigenes were selected based on phyologeny and expression patterns, and the resulting candidate genes were biochemically characterized using heterologous expression in Escherichia coli and product analysis with UPLC-QTOF/MS.The goal of the present study was to identify one or more glycosyl transferases involved in asiaticoside formation using an omics approach. In particular, the transcriptome of MeJA-elicited C. asiatica leaves yielded a total of 1,282,298 raw reads of 415.4 bps on average. After deleting adaptor sequences and eliminating reads < 100 bps, 40,064 unique sequences comprising 31,050 isotigs and 9014 singletons were identified and annotated, based on sequence similarity with complete sequences in the Swiss-Prot, TAIR (The Arabidopsis Information Resource), NCBI non-redundant protein (Nr), and NCBI non-redundant nucleotide (Nt) databases (−3).Transcriptome analysis of elicited atabases . A totalC. asiatica transcriptome dataset contained 133 unique sequences annotated as UGTs based on BLAST similarity, including 120 isotigs and 13 singletons, and 37 putative full-length cDNAs  belonged to the UGT73 subfamily, which comprises the UGT73K1, UGT73P2, and UGT73C10 genes encoding triterpene glucosyltransferases of M. truncatula, G. max, and Barbarea vulgaris, respectively (C. asiatica UGTs (UGT74AH1 and UGT74AG2) belonged to the UGT74 family, which comprises the UGT74M1 and UGT74H6 genes known to be involved in saponin formation in S. vaccaria and Avena strigosa, respectively. All six C. asiatica UGT73/74 genes had high EST representation and RPKM (Reads per Kilobase per Milion mapped reads) values (Six of the ectively . On the ) values , and werC. asiatica genes encoding enzymes along the biosynthetic pathway from farnesyl diphosphate to triterpene glycosides. We therefore hypothesized that UGTs involved in asiaticoside and/or madecassoside formation should also be relatively highly expressed upon MeJA induction. Hence, in order to select strong candidates of UGT, we used qRT-PCR to compare the expression levels of the six UGT candidate genes in C. asiatica hairy root cultures elicited by 0.1 mM MeJA for 24 h . However, the proteins were found mainly in inclusion bodies, and all four UGT genes therefore had to be re-cloned into vectors with stop codons to enhance their solubility [E. coli strains, BL21 and BL21 (DE3), however, no soluble protein products were observed for UGT73A21 and UGT73A22 + and m/z 651.453 (Asiatic acid + Glc + H)+ , reflecting the presence of both a hexose moiety and asiatic acid  regio-specificity of the enzymes remains to be determined. C. asiatica [To date, 33 oleanane- and ursane-type saponins have been reported in asiatica , all of C. asiatica were established from node segments as previously described by Kim et al. [Whole plant cultures of m et al. . Plants C. asiatica were sequenced using the Genome sequencer FLX , and the raw data were processed with the Roche GS FLX software  at quality score threshold 40 and quality-trimmed with Lucy software  [http://compbio.dfci.harvard.edu). The resulting sequence reads were assembled de novo using the software package Newbler (a) set to extend low-depth overlaps. The assembled unique transcripts were first compared to the GenBank database using the BLASTN algorithm with an E value cut-off of 10−3, to identify and remove ribosomal RNA sequences. The remaining sequences, putatively encoding proteins, were searched against the Arabidopsis protein database at The Arabidopsis Information Resource , the Swiss-Prot protein database , and the NCBI non-redundant protein (Nr) database  using the BLASTX algorithm with an E value cut-off of 10−3. All the resulting C. asiatica high-quality reads were deposited at NCBI, and can be accessed in the Sequence Read Achieve (SRA) database under project accession number SRX1050304.Total RNA samples of ge.net/)  and SeqCp values in multiple tests. Unigenes were considered differentially expressed when the RPKM of treated sample and control displayed a more than twofold change, with p less than 10−3.Expression levels of all isotigs were calculated using the RPKM (Reads Per Kilobase per Milion mapped reads) method , with thThe protein sequences were aligned with the CLUSTALW program and phylogenetic trees constructed using the MEGA5.2 software  using thC. asiatica treated with MeJA for 0, 12, 24, 48, or 72 h cDNAs were used to harvest total RNA as described by Kim et al. [Ct) value for each reaction was obtained, and the differences were calculated using the delta-delta-Ct method, with the β-actin gene (GenBank accession No. JK517508) as internal control. The fold change in transcript levels for each gene is presented as the mean and standard error of three independent experiments.Hairy roots of m et al. . First-sE. coli expression clone was constructed using the entry vector and a destination vector pET62-DEST  following the protocol provided. After confirmation of sequences, the plasmids were used for E. coli transformation.Full-length cDNAs of target UGT were cloned using the Gateway system with specific primers, based on sequences from the transcriptome data analysis. Sequence information for the putative UGT genes has been deposited in GenBank with accession numbers . To minimize inclusion bodies of recombinant proteins, complete open reading frames (ORFs) with stop codon were used . The ORFd-galactoside (IPTG) was added to a final concentration of 0.5 mM, and incubation was continued at 20 °C for 16 h. Finally, cells were collected by centrifugation at 15,000× g for 5 min, suspended in extraction buffer , and then disrupted by sonication for three times 10 s. After cell debris was removed by centrifugation at 15,000× g, the supernatant was checked for the presence of the expressed protein using 10% SDS-PAGE. The total protein extract was used for in vitro assays, performed by incubating 1 mg protein in 1.5 mL 50 mM Tris-HCl, pH 8.5, containing 5 mM uridine-5′-diphosphoglucose and 0.5 mM asiatic acid, at 35 °C for 4 h. Finally, 1.5 mL butanol was added to stop the reaction and extract products, and the organic layer was collected, and the solvent evaporated under nitrogen gas. The resulting residue was re-dissolved in 200 µL of methanol, and stored at 4 °C for chemical analysis.Single colonies of BL21 (DE3) cells harboring the pET-UGT73AH1 or pET-UGT74AH1 vectors were cultured at 37 °C in liquid lysogeny broth (LB) medium containing 50 µg/mL ampicillin to an optical density of 0.5 at 600 nm. Then, the cultures were shifted to 28 °C, and isopropyl thio-β-m/z 556.276 (ESI+), m/z 554.262 (ESI−)). Data were collected between m/z 100 and m/z 2000. UPLC was performed using an ACQUITY H-Class UPLC  according to the protocol reported by Lee et al.  with an C. asiatica leaves. UPLC-TOF/MS analysis of in vitro assays showed that UGT73AH1 produces asiatic acid monohexoside. We thus demonstrated that the UGT73AH1 protein catalyzes the glycosylation of a ursane-type triterpene, now enabling further investigations into the genetic engineering of triterpene saponin biosynthesis. As part of these studies, it will be interesting to test the relative activity of the enzyme on other substrates, including madecassic acid as well as oleanane backbone isomers of both asiatic and madecassic acids.Four candidate genes of UGT were selected from transcriptome data based sequence comparisons, and based on their expression patterns in MeJA-elicited


Daily moderate exercise (DME) and stress management are underemphasized in the care of patients with lupus nephritis (LN) due to a poor comprehensive understanding of their potential roles in controlling the inflammatory response. To investigate these effects on murine LN, disease progression was monitored with either DME or social disruption stress (SDR) induction in NZM2410/J mice, which spontaneously develop severe, early-onset LN. SDR of previously established social hierarchies was performed daily for 6 days and DME consisted of treadmill walking (8.5 m/min for 45 min/day). SDR significantly enhanced kidney disease when compared to age-matched, randomly selected control counterparts, as measured by histopathological analysis of H&E staining and immunohistochemistry for complement component 3 (C3) and IgG complex deposition. Conversely, while 88% of non-exercised mice displayed significant renal damage by 43 weeks of age, this was reduced to 45% with exercise. DME also reduced histopathology in kidney tissue and significantly decreased deposits of C3 and IgG complexes. Further examination of renal infiltrates revealed a macrophage-mediated inflammatory response that was significantly induced with SDR and suppressed with DME, which also correlated with expression of inflammatory mediators. Specifically, SDR induced IL-6, TNF-α, IL-1β, and MCP-1, while DME suppressed IL-6, TNF-α, IL-10, CXCL1, and anti-dsDNA autoantibodies. These data demonstrate that psychological stressors and DME have significant, but opposing effects on the chronic inflammation associated with LN; thus identifying and characterizing stress reduction and a daily regimen of physical activity as potential adjunct therapies to complement pharmacological intervention in the management of autoimmune disorders, including LN. Lupus nephritis (LN), a debilitating clinical manifestation of systemic lupus erythematous (SLE) contributing to disease morbidity, affects more than 50% of adult and pediatric patients with lupus  levels were measured biweekly and weights were recorded weekly. As clinical indicators of kidney damage, experimental removal criteria was defined by a threshold of 20% weight loss and a BUN level above 50 mg/dL.Female and male NZM2410/J and wild-type C57BL/6 control mice were obtained from The Jackson Laboratories . Mouse maintenance and protocols were approved by the Institutional Animal Care and Use Committee at The Ohio State University Wexner Medical Center (OSUWMC). The animal facility was maintained at 22–23°C and 30–50% relative humidity with a 12-h light/dark cycle; chow and water were supplied n = 9 to demonstrate repeatability. Control NZM2410/J mice were housed in the same location and handled similarly under identical standard of care conditions, but not exposed aggressors.The SDR protocol was carried out as previously described . Control mice were handled similarly, but not exercised on the treadmill. The primary endpoint of the exercise arm of the study was mortality; when mice displayed signs of terminal renal disease , kidney tissue and serum were collected. To demonstrate repeatability, experiments were performed twice in cohorts of Serum was collected biweekly using the MaxDiscovery Blood Urea Nitrogen Enzymatic Assay Kit  according to manufacturer's protocol. Blood was obtained via submandibular bleeds and serum was isolated by centrifugation after blood clotting. Absorbance values were determined using the Dynex MRX-TC Revelation microplate reader/colorimeter . Results were exported to Microsoft excel (v2013) for analysis.Blood was obtained via submandibular bleeding or axillary vessel incision at the time of sacrifice. Cytokine analysis on collected serum was done using electrochemiluminescence detection  per manufacturer's protocol. Anti-dsDNA ELISAs were performed following manufacturer's instruction using the Mouse Anti-dsDNA kit . Serum from SDR experimental endpoint was compared with a randomly selected, age-matched control (non-stressed) NZM2410/J mouse. For analysis of DME, relative changes in expression were analyzed over a 2 month period of bi-weekly measurements. MCP-1 ELISAs were performed using Mouse CCL2 (MCP-1) ELISA Ready-SET-Go!  according to manufacturer's protocol.Mouse tissues were dissected for paraffin processing according to previously established protocol  using the 10x objective of a bright-field light microscope. Scoring criteria were defined as: 0 = within normal limits ; 1 = minimal nephritis ; 2 = mild nephritis ; 3 = moderate nephritis ; and 4 = marked nephritis .Slides were digitally scanned using the Aperio ScanScope XT eSlide capture device  and analyzed using Aperio Digital Image Analysis software (v9.1) to measure pixel intensities according to previously described methods  using Graph Pad Prism 6. Statistical analysis for two groups was performed using unpaired, two-tailed nonparametric Mann-Whitney U test or unpaired, two-tailed parametric Student's To examine the effects of psychosocial stressors on chronic autoimmune-mediated inflammation, NZM2410/J mice were subjected to the SDR model as previously described . Additional IHC staining was done to further investigate the inflammatory components found in the renal tissues. Specifically, immune complexes and C3 protein were measured because of the strong association with renal injury in LN; inflammatory pathology of LN is characterized by activation of the compliment cascade, which leads to deposition of C3 fragments on target cells. IHC analysis showed that SDR resulted in over 50-fold more IgG complex deposition  to determine the influence of exercise on LN disease progression. Control mice were handled similarly, but not exercised on the treadmill. When mice displayed signs of terminal renal disease , kidney tissue and serum were collected for analysis.p < 0.05; Figure p < 0.05) in the non-exercised (11 mg/dL ± 4.3) mice at 32 weeks of age when compared to mice that were exercised (2.4 mg/dL ± 1.2). To further confirm changes in inflammatory pathology, IHC for C3 protein and antibody complexes of IgG were quantified. Exercised mice showed over 10-fold reduction in positive staining for IgG antibodies , IL-6 , TNF-α , IL-10 , and CXCL1  relative to wild-type mice , IL-6 , TNF-α , IL-10 , and CXCL1   for comparison to age-matched wild-type controls (32–35 weeks) and younger NZM2410/J mice (20–25 weeks) not yet meeting these criteria. While serum levels of IFN-γ, IL-2, IL-4, and IL-12 did not display a difference between experimental groups (data not shown), expression of IL-5, IL-6, TNF-α, IL-10, CXCL1, and IL-1β was elevated in the NZM2410/J mice displaying signs of severe kidney damage , TNF-α by 1.4-fold , and IL-1β by 3.4-fold ; IL-10, CXCL1, and anti-dsDNA autoantibody levels did not change significantly and IL-5 expression was downregulated , IL-6 by 3.1-fold , CXCL1 by 1.45-fold , TNF-α by 1.62-fold , and anti-dsDNA autoantibodies by 7.2-fold , while no significant changes were observed with IL-5 or IL-1β production  and macrophages were detected primarily in the interstitial regions -1 promotes macrophage-mediated inflammation and its association to renal pathology in autoimmunity is specifically evidenced by elevated levels in the urine of patients with LN : influence of daily moderate exercise and stress reduction by Tai Chi in lupus,” active SLE patients were enrolled into a daily Tai Chi program, which emphasized moderate exercise levels with meditative breathing to provide daily physical activity and stress reduction (unpublished observations). Our preliminary data indicate that daily Tai Chi can potentially be an effective adjunct therapy to compliment current pharmacological interventions. Future investigations will expand SMILE study enrollment for a large randomized trial and further examine the molecular pathways regulated by SDR and DME in mice. Studies to better characterize these pathways may be especially advantageous in patients where behavioral modifications to alter psychological stress and physical activity levels may not be feasible.Animal subjects: This study was carried out in accordance with the recommendations of the University Laboratory Animal Resources at The Ohio State University Wexner Medical Center. The protocol was approved by the Institutional Animal Care and Use Committee.Conceived and designed the experiments of the study: SA, JH, MB, SAg, NP, JS, WJ, and NY; Data collection and analysis: SA, MB, JH, BB, and NY; Performed experiments: SA, MB, JH, KJ, and NY; Edited manuscript: SA, JH, MB, KJ, GV, LW, MY, WW, SAr, SAg, BB, NP, JS, NS, WJ, and NY; Statistical assessments: SA, BB, and NY; Wrote manuscript: SA and NY; Contributed reagents/materials/analysis tools: BB, SAg, and WJ; Made substantial, direct and intellectual contribution to the work, and approved it for publication: SA, JH, MB, KJ, GV, LW, MY, WW, SAr, SAg, BB, NP, JS, NS, WJ, and NY.The CCTS was supported by Award Number Grant UL1TR001070 from the National Center for Advancing Translational Science. The CPMP was supported by grant P30 CA016058 from the National Cancer Institute. This study was also supported directly by OSUWMC funds. Additionally, NY and NS have research support from Ironwood Pharmaceuticals examining the role of exercise in inflammation and The Ohio State University is supported in part by a grant from the Lupus Research Alliance, which indirectly supported these research endeavors. The authors declare that they have funding from Ironwood Pharmaceuticals to explore the immunomodulatory effects of exercise, which indirectly contributed to this research. The funder was not involved in the study design or collection, analysis, or interpretation of the data.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. The reviewer AB and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Brassica rapa var. parachinensis) is reported. The expression of BrWRKY65 was up-regulated following leaf chlorophyll degradation and yellowing during postharvest senescence. Subcellular localization and transcriptional activation assays showed that BrWRKY65 was localized in the nucleus and exhibited trans-activation ability. Further electrophoretic mobility shift assay (EMSA) and transient expression analysis clearly revealed that BrWRKY65 directly bound to the W-box motifs in the promoters of three senescence-associated genes (SAGs) such as BrNYC1 and BrSGR1 associated with chlorophyll degradation, and BrDIN1, and subsequently activated their expressions. These findings demonstrate that BrWRKY65 may be positively associated with postharvest leaf senescence, at least partially, by the direct activation of SAGs. Taken together, these findings provide new insights into the transcriptional regulatory mechanism of postharvest leaf senescence in Chinese flowering cabbage.Plant-specific WRKY transcription factors (TFs) have been implicated to function as regulators of leaf senescence, but their association with postharvest leaf senescence of economically important leafy vegetables, is poorly understood. In this work, the characterization of a Group IIe WRKY TF, BrWRKY65, from Chinese flowering cabbage ( As the final phase of leaf development, senescence is an important biological process accompanying the cessation of photosynthesis, chlorophyll breakdown, leaf proteins degradation, amino acids removal, as well as the mobilization of nitrogen, carbon and other minerals ,2,3,4. ASAGs) [During senescence, leaves undergo a series of programmed molecular changes mediated by senescence-associated genes (SAGs) ,5,9, whiSAGs) ,11,12. TSAGs) ,13. In pSAGs) ,11,14.Arabidopsis thaliana and rice, respectively [WRKY gene is identified from sweet potato [Arabidopsis thaliana wrky53 mutant displays delayed leaf senescence, while its over-expression results in precocious senescence, demonstrating that WRKY53 is positively involved in leaf senescence [Arabidopsis thaliana [WRKYs form a major family of plant-specific TFs, with more than 70 and 100 members in model species ectively ,16. WRKYectively ,16. The t potato , and subt potato . WRKYs hnescence ,20. Besithaliana ,11,12,21thaliana ,23,24, athaliana . To dateBrassica rapa L. comprises many economically important worldwide cultivated leafy vegetables, such as Chinese cabbage (B. rapa ssp. pekinensis), bok choy (B. rapa ssp. chinensis), and Chinese flowering cabbage/choy sum (B. rapa ssp parachinensis) [SAGs genes related to postharvest leaf senescence of leafy vegetables, and established storage technologies that delay leaf senescence [SAGs genes including BrNYC1 and BrSGR2 related to chlorophyll degradation, and BrDIN1.The inensis) . These linensis) . Howevernescence ,29,30,31Leaf yellowing due to a rapid breakdown of chlorophyll is an obvious symptom of senescence in cabbages ,30. As sBrassica rapa plants such as Chinese cabbage [WRKY gene (NP_001306245.1) showed increased expression during postharvest cabbage leaf senescence. The full-length of this WRKY was cloned and homology search indicated that it exhibited the highest identity with AtWRKY65 (85%); so it was designated as BrWRKY65. The ORF of BrWRKY65 is 786 bp in length and encodes a polypeptide of 262 amino acids. The calculated molecular weight and pI of BrWRKY65 is 29 kDa and 5.41 respectively. Multiple alignments of BrWRKY65 revealed that it possesses a highly conserved WRKY domain, with WRKYGQK sequence, which is the defining character of WRKY proteins [Plant-specific WRKY TFs play a central role in regulating leaf senescence ,9,12,33. cabbage ,35, and  cabbage , the mecproteins , as wellAccording to the number of WRKY domains and structure of the zinc-finger motif, WRKY TFs are clustered into three major groups (I-III). Group II can be further divided into five subfamilies (IIa-e) ,16. A phAs TFs, WRKYs are usually nuclear proteins and possess transcriptional activity ,37,38,39BrWRKY65 transcript level increased during senescence, reaching about 4.2- and 3.8-fold of the initial level at day seven and nine, respectively (DIN) genes are induced in dark-adapted and senescing leaves [SAG genes in our RNA-seq transcriptome database, and three SAGs such as BrNYC1 (NON-YELLOW COLORING1/Chl b reductase) (XP_009139641.1) and BrSGR1 (STAY-GREEN1) (XP_018512704.1) associated with chlorophyll degradation, and BrDIN1 (XP_009150416.1) were selected. Their expressions were also analyzed. As can be seen in BrWRKY65, the expression of BrNYC1, BrSGR1, and BrDIN1 were all up-regulated during cabbage postharvest leaf senescence.To further confirm the possible association of BrWRKY65 with cabbage postharvest leaf senescence, its expression was examined by qRT-PCR. Consistent with its trend in our RNA-seq database, ectively . In addig leaves ,45, whiccis-element W-box with a core sequence (C/T)TGAC(C/T) in the promoter [BrNYC1, BrSGR1, and BrDIN1, and found that they all contain putative W-box motifs . Purified recombinant glutathione S-transferase (GST)-BrWRKY65 fusion protein was successfully obtained via prokaryotic expression  assays will be needed to confirm the bindings of BrWRKY65 to BrNYC1, BrSGR1, and BrDIN1 in vivo.Numerous studies reveal that WRKY TFs control their target gene expressions by binding to the typical promoter ,33,39,46x motifs . In addinescence . Thus, ipression . As expex probes . The mobx probes , indicatBrNYC1, BrSGR1, and BrDIN1 by BrWRKY65 was further determined through transient dual-luciferase assays in tobacco leaves. The BrNYC1, BrSGR1, and BrDIN1 promoters with 1331, 533, and 1229 bp, respectively, were fused with the LUC reporter. CaMV 35S-driving REN reporter in the same vector was used as an internal control to normalize the expression of each reporter  [NYE1 , NYC1, and PAO (pheophorbide a oxygenase) via directly binding to their promoters, leading to accelerated chlorophyll degradation during leaf senescence, seed or fruit degreening. Intriguingly, Arabidopsis ETHYLENE INSENSITIVE3 (EIN3) and ORE1/NAC2 establish a network controlling ethylene-induced leaf senescence via directly targeting NYE1, NYC1, and PAO [Arabidopsis are found to speed ABA-mediated chlorophyll degradation and leaf senescence by the trans-activation of NYE1, NYE2, NYC1, PAO, SAG12, and SAG29 [Since BrWRKY65 is a transcription activator C, the trreporter A. BrWRKYreporter A. The renescence . Accordi3 (ABI3) , ABI5, E3 (ABI3) , phytoch3 (ABI3) , MYC2/3/3 (ABI3) , ANAC0723 (ABI3) , and ERF3 (ABI3) , are rep and PAO . More rend SAG29 . CollectBrassica rapa var. parachinensis) were obtained from a local commercial vegetable farm near Guangzhou, southern China. After pre-cooling, the harvested cabbages were transported to the lab immediately. Only cabbages with no mechanical damage and uniform appearance were chosen. The selected cabbages were placed into plastic baskets (20 per box) packing with 0.04 mm thickness polyethylene perforated plastic bags. The packed cabbages were then stored in the incubators at 15 °C. Samples were taken at 0, 5, 7, and 9 days of storage, and the second leaf from the bottom of the cabbages was collected. The collected leaves were sliced into small pieces, frozen in liquid nitrogen and stored at −80 °C until analysis.Chinese flowering cabbages  was applied to synthesize cDNAs, following the manufacturer’s instructions. Total RNA was extracted from Chinese flowering cabbage leaves using the RNeasy Mini kit  following the manufacturer’s protocol. The quality and integrity of total RNA was monitored by running ~1 μg in a formamide denaturing gel, and using a spectrophotometer to confirm total RNA quantity. A PrimeScriptchiifu in BRAD (http://brassicadb.org/brad/), a WRKY showing high sequence homology to Arabidopsis thaliana WRKY65, named BrWRKY65 (NP_001306245.1), was found to be up-regulated during postharvest leaf senescence. BrWRKY65 was cloned, sequenced (primers are listed in pI) and mass value for BrWRKY65 protein were predicted at the online website (http://web.expasy.org/compute_pi/). Multiple alignments were performed using CLUSTALW (version 1.83) and GeneDoc softwares. The phylogenetic tree of WRKY proteins was constructed using the MEGA5.0 software with the Neighbor–Joining method.Based on our RNA-seq database and genome of Chinese cabbage ® qPCR Master Mix Kit  on a Bio-Rad CFX96 Real-Time PCR System. Data were normalized to reference gene EF-1-α (GO479260) [Ct) value corresponding to the reference gene. Primers used for qRT-PCR are listed in Quantitative RT-PCR (qRT-PCR) was conducted as previously described . PCR reaO479260) . All qRTAgrobacterium tumefaciens strain GV3101 using Gene PulserXcellTM Electroporation Systems . The Agrobacterium harboring pEAQ-BrWRKY65-GFP was injected into the abaxial side of 4- to 6-week-old tobacco (Nicotiana benthamiana) leaves using a 1-mL syringe without a needle as described previously [The complete Open Reading Frame (ORF) of BrWRKY65 was amplified and inserted into the pEAQ-GFP vector  with nested PCR (specific primers are listed in http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was employed to predict the conserved cis-element motifs presented in promoters. Genomic DNA was prepared from Chinese flowering cabbage leaves using the CTAB-based methods . The proEscherichia coli strain BM Rosetta (DE3). GST-BrWRKY65 protein expression was induced by 1mM isopropyl thio-β-d-galactoside (IPTG) at 30 °C for 6 h, and the recombinant fusion protein was purified using Glutathione-Superflow Resin  according to the manufacturer’s protocol.The GST-BrWRKY65 expression vector was constructed with pGEX-4T-1 , Beijing, China) and then transformed into BrNYC1, BrSGR2, and BrDIN1 were labeled with biotin at the 5’ end. An EMSA was performed essentially using the LightShift Chemiluminescent EMSA Kit  as our previous studies described [The fragments of ~60 bp containing putative WRKY binding region in the promoters of escribed ,60. PuriBrNYC1, BrSGR2, and BrDIN1 promoters, these promoters were inserted into pGreenII0800-LUC vector, while BrWRKY65 was cloned into the pGreenII 62-SK vector as the effector [The transcriptional activity of BrWRKY65 was assayed using the dual-luciferase transient expression system in tobacco leaves. The reporter vector was modified from the pGreenII 0800-LUC vector . The fireffector . PrimersThe constructed reporter and effector plasmids were transiently co-expressed in tobacco leaves as described above. After 48 h of infiltration, dual-luciferase assay kit (Promega (Beijing) Biotech Co., Ltd., Beijing, China) was adopted to detect LUC and REN luciferase activity on a Luminoskan Ascent Microplate Luminometer  according to the manufacturer’s instructions. The transcriptional activity of BrWRKY65, and trans-activation of BrWRKY65 to each promoter was indicated by the LUC to REN ratio. At least six independent repeats were measured for each pair.t-test.Experiments were performed according to a complete randomized design. Data in figures are expressed as the mean ± standard errors (S.E.) of at least three independent replicates. The data were statistically analyzed by applying Student’s SAG genes, and activates their expressions. Overall, our work provides novel information about the transcriptional regulation of leaf senescence in commercially important leafy vegetables, such as Chinese flowering cabbage.In conclusion, a postharvest leaf senescence-inducible transcriptional activator, BrWRKY65, from Chinese flowering cabbage is isolated and characterized. Furthermore, BrWRKY65 binds to the promoters of three


Pseudoalteromonas tetraodonis and Pseudoalteromonas lipolytica strains, isolated from the gut of the sea cucumber Apostichopus japonicus, to provide a useful means for facilitating the study of antibacterial, bacteriolytic, agarolytic, and algicidal activities of marine Pseudoalteromonas species.We present here the complete genome sequences of two newly isolated  Pseudoalteromonas belongs to the Gammaproteobacteria class, with 38 recognized species so far. Pseudoalteromonas spp. are heterotrophic Gram-negative flagellated bacteria commonly found in various ocean habitats, including the deep sea, polar sea, and other extreme marine habitats .The genus habitats –3. Alongtivities . Pseudoaodotoxin , and antorphosis . Here, wP. tetraodonis and P. lipolytica, respectively, resulting in 294- and 267-fold coverage of the genomes, respectively. The raw reads were preprocessed and trimmed using the Trimmomatic tool  prepared by Accel-NGS 2S PCR-free library kit , according to the manufacturer’s protocol. A total of 7,155,540 and 7,760,584 raw reads were generated from Pdenovo2 , and SPAPdenovo2 , under dPdenovo2 . The finPdenovo2 . The resP. tetraodonis CSB01KR genome contains 3,567 genes, including 3,479 protein-coding sequences classified in 456 subsystems, 5 5S rRNAs, 5 short subunit (SSU) rRNAs, 3 long subunit (LSU) rRNAs, and 75 tRNAs. In the P. lipolytica CSB02KR genome, 3,984 genes that consist of 3,904 protein-coding sequences classified in 487 subsystems, 2 5S rRNAs, 1 SSU rRNA, 2 LSU rRNAs, and 75 tRNAs were annotated. We believe that the full-genome sequences of the two Pseudoalteromonas strains expand the repertoire of genomic information for the genus Pseudoalteromonas and will provide important insights into their role within microbial communities.Automatic gene annotation was performed using the program Rapid Annotations using Subsystems Technology  . The annP. tetraodonis and P. lipolytica were deposited at GenBank with accession numbers MBMS01000001 to MBMS01000049 and MBSZ01000001 to MBSZ01000049, respectively.The raw data of the two strains have been deposited in the NCBI database under the BioProject accession number PRJNA328511. The assembled draft genomes of


Yersinia pestis isolates belonged to group q3, different from the previous outbreak. During a pneumonic plague outbreak in Moramanga, Madagascar, we identified 4 confirmed, 1 presumptive, and 9 suspected plague case-patients. Human-to-human transmission among close contacts was high (reproductive number 1.44) and the case fatality rate was 71%. Phylogenetic analysis showed that the  Yersinia pestis, is a fleaborne disease responsible for the death of tens of millions of persons throughout history  displayed symptoms on August 17, 2015, while in a remote area in Antsahatsianarina hamlet, Tsiazompody village, Ampasipotsy Gara commune, within the Moramanga district of Madagascar  Table. C,Before illness onset, case-patients 2–14 had social or spatial contact with a symptomatic person who could be traced back to case-patient 1. Two of the deceased case-patients (2 and 10) were visitors to Antsahatsianarina who had returned home to their respective communities Ambatoharanana and Ambilona , potentiY. pestis infection, we performed bacteria culture on sputum or organ puncture (Y. pestis (2 by culture and 2 by seroconversion) and 1/5 was presumptive for Y. pestis infection. No samples were collected from the other 9  persons, who were thus considered suspected plague case-patients  was 1.44, and the transmission rate was 0.41 susceptibles/day (More men (n = 10) than women (n = 4) were infected. Median patient age was 22.5 (range 15–80) years, and the overall case-fatality rate was 71%. The most common signs and symptoms were fever, dyspnea, chest pain, and cough (100% of patients); blood-stained sputum (93%); chills (86%); and headache (71%). The average duration from onset of full-blown disease to death was 1.9 days, and the average infectious period was 3.5 days. Basic reproductive number (Rhttp://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2010:276:0033:0079:EN:PDF) and tested by using an F1 antigen rapid diagnostic test  reported no disease present in their household. Taken together, the epidemiologic data suggest that case-patient 1’s hamlet (Antsahatsianarina) was the probable outbreak epicenter. Y. pestis q3 phylogenetic subgroup within group I (node k). The matched genetic grouping between the 2 human samples is consistent with human-to-human transmission. The matched genetic grouping among all 3 samples is consistent with Y. pestis in the initial case-patient originating from the environment rather than another human. We compared these phylogenetic data with data from 4 archived isolates (human and rodent) obtained in 2000, 2002, and 2003 from the same area. The archived isolates were also assigned to group I (node k), but unlike the isolates from this study, they were assigned to the q2 subgroup  have been recorded in Moramanga, suggesting multiple genotypes are persisting within this region (Using published single-nucleotide polymorphisms (Despite Madagascar having an effective surveillance system, plague control remains a public health challenge. Pneumonic plague is rare but persists as a threat in Madagascar, where poor healthcare systems and traditional burial practices promote these outbreaks. Our findings provide additional understanding of pneumonic plague transmission patterns, argues for continued public education, and informs authorities about effective outbreak response practices.Numbers of seropositive, seronegative, and unsampled contacts of plague case-patients among the total population per site in Moramanga, Madagascar in 2015.


DNA methylation is a major epigenetic mark with important roles in genetic regulation. Methylated cytosines are found primarily at CpG dinucleotides, but are also found at non-CpG sites . The general functions of CpG and non-CpG methylation include gene silencing or activation depending on the methylated regions. CpG and non-CpG methylation are found throughout the whole genome, including repetitive sequences, enhancers, promoters, and gene bodies. Interestingly, however, non-CpG methylation is restricted to specific cell types, such as pluripotent stem cells, oocytes, neurons, and glial cells. Thus, accumulation of methylation at non-CpG sites and CpG sites in neurons seems to be involved in development and disease etiology. Here, we provide an overview of CpG and non-CpG methylation and their roles in neurological diseases. Epigenetic marks include a variety of gene regulatory events, such as chromatin structure remodeling, histone modifications, DNA methylation, and small noncoding RNAs, that do not entail changes in the DNA sequence . Gene exIn 1975, two independent research groups suggested that the methylation of cytosine might play a pivotal role as an epigenetic mark in animals ,4. DNA mDNA methylation is also found at sites other than CpG sequences. This type of methylation is referred to as non-CpG methylation, and includes methylation at cytosines followed by adenine, thymine, or another cytosine B. Non-CpOn the other hand, 5mC can also be demethylated via either passive or active DNA demethylation. Passive demethylation is carried out by DNA synthesis without de novo methylation, by which 5mC is diluted following replication. Active demethylation entails the modification of the cytosine base via deamination and/or oxidation  Figure C. DeaminIn this review, we will provide an overview of CpG and non-CpG methylation and their functions in brain cells, and neurodevelopmental and neurodegenerative diseases.Dnmt1 alleles is 62.2% in wild-type mouse ES cells, 66% in mouse liver, and 81.6% in mouse lung [Epigenetic marks have important roles as regulators of gene expression, which is basically regulated by histone modifications and DNA methylation . DNA metuse lung . In humause lung .Most 5mCs are found in repetitive sequences, gene bodies, and intergenic regions ,17,21,22DNMTs are enzymes involved in the transfer of methyl groups to cytosines in DNA. Briefly, methyl groups from the cofactor S-adenosylmethionine are transferred to the 5th carbons of cytosines by DNMTs . Dnmt1 iperoxisome proliferator-activated receptor γ coactivator 1α (PGC-1 α) gene is more methylated at non-CpG sites than in healthy controls. This results in the downregulation of PGC-1 α [In mammals, DNA methylation at CpG dinucleotides is critical for cellular development and differentiation ,41. Five PGC-1 α . A corre PGC-1 α . Other r PGC-1 α ,49,50.Recent studies have revealed that non-CpG methylation is enriched in ES cells ,8, inducAmong the different types of non-CpG methylation , methylation is most common at CpA sites. For instance, in human iPS cells, 5mCs are found in approximately 68.31%, 7.81%, 1.99%, and 1.05% of CpG, CpA, CpT, and CpC sites, respectively . In addiDnmt1 knockout mouse ES cells were shown to retain non-CpG methylation patterns. Compared to Dnmt1 knockout mice, Dnmt3l knockout mice have significantly lower levels of CpA methylation in their prospermatogonia [Dnmt3a and Dnmt3b double knockout ES cells, where Dnmt3l is associated with both Dnmt3a and Dnmt3b expression, have much lower CpA methylation levels [In mammals, DNA methylation in somatic cells is carried out by DNMT1, DNMT3a, DNMT3b, and DNMT3L ,17,38. Natogonia . Furthern levels ,57. CollAccording to previous studies, DNA methylation has critical roles in epigenetic regulation of brain development and neurogenesis . DNA met−/NeuN+), GABAergic interneurons (SOX6−/NeuN+), and glial cells (SOX6−/NeuN−) separately. This genome-wide analysis indicated that the average level of DNA methylation at CpG sites was higher (about 3–4%) in GABAergic interneurons than in glutamatergic neurons and glial cells. DNA hydroxymethyl cytosine, which is an intermediate molecule on the 5mC demethylation pathway, is also higher in glutamatergic neurons than in GABAergic interneurons.Different neuronal subtypes also have differences in non-CpG methylation patterns in the human brain . The mamIn the in vivo DNA methylome of the adult mouse dentate gyrus, neurons have about 75% CpG methylation and 25% non-CpG methylation . The nonmethyl-CpG binding protein 2 (MeCP2) gene is especially highly expressed in the brain and some other tissues, such as lung and spleen. Similar to 5mCs in CpGs, non-CpG methylation can also be recognized by MeCP2 [MeCP2 expression is higher in neurons than in glia [MeCP2 lead to neurological diseases, such as Rett syndrome (RTT), in humans.Transcriptional repression of non-CpG methylation may be mediated by the interaction between DNA-binding proteins and DNA regulatory elements . The metby MeCP2 . In brai in glia ,68, and  in glia ,70. As M in glia ,72. MeCP in glia ,72. Muta2+- and 2-oxoglutarate-dependent dioxygenase TETs [The formation of 5hmC, a 5mC demethylation intermediate, is catalyzed by Fease TETs ,14. In mase TETs ,74,75,76ase TETs ,78,79,80ase TETs . Mature ase TETs . Human Ease TETs . In addiase TETs . AlthougGenome-wide studies suggest that 5hmC is enriched in gene bodies, promoters, and distal regulatory regions. Importantly, 5hmC levels are increased and enriched preferentially in gene bodies of neuron-related genes during neuronal differentiation . The levInterestingly, MeCP2, a 5mC-binding protein, is also bind to the 5hmC. However, high-affinity binding of MeCP2 is specific to the CpA hydroxymethylation (5hmCpA), but not to the 5hmCpG, 5hmCpC, and 5hmCpT . MeCP2 bAlthough 5hmC is likely to be a stable epigenetic marker and is abundant in the adult brain, the exact mechanistic involvement of 5hmC with neurodevelopmental and neurodegenerative diseases remains unclear. However, many researchers have suggested a potential role of 5hmC in neurological diseases, such as Rett syndrome, autism, Huntington’s disease (HD), and Alzheimer’s disease (AD) . Two subDNA methylation has been suggested to play an important role in brain function, especially in memory formation ,94. ThusDNA methylation plays an important role in gene regulation. It is thus not strange that some diseases are caused by abnormal DNA methylation . Mutatiointerleukin-1β (IL-1β) and IL-6 in AD, and found that methylation was increased during the first stages of AD and was decreased thereafter. They also found that the expression of complement C3a receptor 1 (C3ar1) is increased during late-onset AD progression. Only IL-1β had a pattern of DNA methylation that correlated with gene expression levels. It was little correlation between DNA methylation and gene expression levels for IL-6 and C3ar1 [Gene-specific DNA methylation alterations are thought to be related to AD and AD animal models . AD is cnd C3ar1 . Thus, ithromboxane A2 receptor (TBXA2R), sorbin and SH3 domain containing 3 (SORBS3), and spectrin β4 (SPTBN4). These data suggest that AD may be caused by aberrant DNA methylation in genes associated with cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) activation (in TBXA2R), the axon initial segment (in SPTBN4), and synapse formation (in SORBS3).Sanchez-Mut et al. analyzed 12 distinct brain regions to identify aberrant DNA methylation changes in AD mouse models. These patterns were also shown to be present in patients with AD . They idIntriguingly, a recent report has suggested that there is a relationship between altered mitochondrial DNA (mtDNA) methylation and AD . Blanch The association between 5hmC and AD is still controversial. Some studies suggest that there is a decrease in 5hmC levels in hippocampal regions of patients with AD ,106, whiMeCP2 [brain-derived neurotrophic factor (Bdnf), [FK506 binding protein 5 [distal-less homeobox 5 (Dlx5) [Dlx6 [ubiquitin-protein ligase E3A [serum/glucocorticoid regulated kinase 1 [MeCP2 is located on the X chromosome, RTT arises mostly in the females and is rare males [MeCP2 gene is mutated before birth, but RTT features appear 6–18 months after birth. Although the reason for the delayed onset of RTT symptoms remains unclear, it may be related to the interaction between MeCP2 and non-CpG methylation [Bdnf, which is a key gene for brain development [Rett syndrome is an X-linked neurodevelopmental disorder that usually occurs in girls and has a prevalence of 1 in 10,000 to 15,000 ,110. It MeCP2 , which i (Bdnf), ,111, FK5rotein 5 , distal-5 (Dlx5) , Dlx6 [15) [Dlx6 , ubiquitgase E3A , and serkinase 1 ,63,112. re males . The MeChylation . Chen etelopment . It is ielopment . MeCP2 belopment . These felopment , the MeCFMR1 , which is highly expressed in neurons. FMRP (encoded by FMR1) binds to target mRNA and inhibits translation, which leads to regulation of the formation of dendrites and synapses [FXS is a common hereditary neurodevelopmental disorder associated with the X-linked gene synapses ,119. Thesynapses . Patientsynapses .FMR1 gene, which results in histone deacetylation and DNA hypermethylation. These DNA changes then lead to the transcriptional silencing of the FMR1 gene [FMR1 gene [The major cause of FXS is the abnormal expansion of CGG repeats in the 5’ untranslated region of the MR1 gene ,119. NorMR1 gene . InteresMR1 gene ,124,125.MR1 gene . These gsynuclein-α  from peripheral blood obtained from patients with PD patients indicates hypomethylation of SNCA in PD [SNCA.PD is universal common neurodegenerative movement disorder ,128. TheCA in PD . In addiWnt, are more hypermethylated in PD brain than in normal brain [Wnt, forkhead box C1, neurogenin 2, sprouty RTK signaling antagonist 1, and catenin beta 1) also have reduced protein expression in midbrain DA neurons. Consistent with this finding, SNCA levels have been shown to be correlated with those of Dnmt1 PD brain [Wnt and other neurogenesis-involved genes may be involved in the progression of pathogenesis in PD. An inflammatory cytokine, tumor necrosis factor alpha (TNF-α), also appears to be linked to the loss of DA neurons in patients with PD [TNF-α promoter, which increases TNF-α expression, may increase susceptibility to TNF-α-mediated inflammation, which may then be followed by apoptosis in neuronal cells. Suppression of TNF-α may be mediated by DNA methylation at critical CpG sites of TNF-α promoters, which modulate the AP2 and specificity protein 1 transcription factors [Global DNA methylation analysis in a number of patients with PD has revealed that multiple genes involved in neurogenesis, including al brain . These hPD brain . These r with PD . Hypomet factors .Similar to AD, PD is associated with mtDNA methylation. However, in contrast to AD (increased DNA methylation), the D-loop region (both CpG and non-CpG sites) of mitochondrial DNA in the substantia nigra has a loss of methylation in patients with PD when compared to control individuals . It remaHtt) gene, which is located on chromosome 4p16.3 [Htt gene commonly carries a CAG repeat length of 17–20. Repeat lengths over 35 are considered pathogenic [Htt in different HD models and in human HD brain. [Bdnf, which is a gene that is downregulated in HD. This indicates that mutant Htt may induce transcriptional dysregulation of genes implicated in HD pathogenesis.HD is an autosomal dominant neurodegenerative disorder characterized by constant movement and cognitive disability . HD is ce 4p16.3 . The Httthogenic . DNA metthogenic . SeveralD brain. ,141,142.D brain. . IncreasAdora2a , whose expression is reduced in patients with HD, is regulated by 5hmC levels. This indicates that 5hmC may be involved in the progression of HD by controlling gene expression [Wang et al. have suggested that the global loss of 5hmC in brain tissue may be a novel epigenetic marker for HD . They fopression .C9orf72 gene [Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that involves motor neuron degeneration in the brain, brainstem, and spinal cord ,145,146.f72 gene ,154. Thef72 gene . Howeverf72 gene ,157,158.Research on epigenetic modifications, including DNA methylation, and their effects on neurological disorders is a rapidly growing field . In factUnderstanding neurological diseases using the epigenetic approach is as yet in its beginning stages. It is thus not easy to obtain reproducible and reliable data regarding DNA methylation from patients with neurological diseases. This is due to the inaccessibility of brain tissue from patients and normal controls. Interestingly, comparison studies using brain tissue and blood have shown that methylation patterns in the brain are very similar to those found in blood . Therefo


MicroRNAs (miRNAs) are small noncoding RNAs that modulate the cellular transcriptome at the post-transcriptional level. miRNA plays important roles in different disease manifestation, including type 2 diabetes mellitus (T2DM). Many studies have characterized the changes of miRNAs in T2DM, a complex systematic disease; however, few studies have integrated these findings and explored the functional effects of the dysregulated miRNAs identified. To investigate the involvement of miRNAs in T2DM, we obtained and analyzed all relevant studies published prior to 18 October 2016 from various literature databases. From 59 independent studies that met the inclusion criteria, we identified 158 dysregulated miRNAs in seven different major sample types. To understand the functional impact of these deregulated miRNAs, we performed targets prediction and pathway enrichment analysis. Results from our analysis suggested that the altered miRNAs are involved in the core processes associated with T2DM, such as carbohydrate and lipid metabolisms, insulin signaling pathway and the adipocytokine signaling pathway. This systematic survey of dysregulated miRNAs provides molecular insights on the effect of deregulated miRNAs in different tissues during the development of diabetes. Some of these miRNAs and their mRNA targets may have diagnostic and/or therapeutic utilities in T2DM. Diabetes mellitus has become a global epidemic. It affects roughly 8.3% of the population, with 90% of these patients suffering from type 2 diabetes mellitus (T2DM) . T2DM iswww.mirbase.org). It has been estimated that more than 60% of protein coding transcripts are regulated by one or more miRNAs [MicroRNAs (miRNAs) are 17–21 nt regulatory RNAs that suppress the translation and stability of mRNA through imperfect base pairing in the 3′ untranslated region of its mRNA targets . There ae miRNAs . A numbee miRNAs . SystemaMany studies have characterized the changes and involvement of miRNA in different cell types and organs during the development of T2DM ,20,21,22n = 405); thesis or conference reports/abstracts (n = 268); unrelated studies (n = 1973); non-English or Chinese publications (n = 18). After detailed evaluation, another 71 studies were removed due to not using human samples (n = 52), being T1DM studies (n = 16) and studies without specify T1 or T2DM (n = 3). At the end, the results from 59 published studies were retrieved from public databases, which include a total of 2671 T2DM patients and 2573 healthy controls . The dataset compared adipose tissues gene expression differences between women with type 2 diabetes with age and BMI matched normal women. The omental biopsies samples were from five diabetic and five control females undergoing cholecystectomy. All subjects used in the study were older than 50 years with BMI values greater than 30. In both pancreatic islet and adipose tissues, the dysregulated miRNAs target several well-known T2DM-associated genes. For example, the adiponectin receptor 2 (ADIPOR2) in the adipocytokine pathway is targeted by miR-375 and miR-136-5p in pancreatic islet and miR-146a in adipose tissues. The key enzymes in lipid biosynthesis and fatty acid degradation, acyl-coA synthetase long-chain family members , are also targeted by multiple miRNAs, including miR-17-5p, -130b-3p, -134-5p and -181a-5p in adipose tissue and miR-7-5p, -369-3p, -495-3p and -655-3p in islets.Based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway map and affected miRNAs from each sample type, we constructed miRNA-mRNA interaction networks for three key T2DM-associated pathways, T2DM signaling, insulin signaling and adipocytokine pathways in pancreatic islet  and adipEven though the etiology of T2DM is yet to be fully understood, insulin resistance and pancreatic β-cell dysfunction are the two major causes for various T2DM-associated clinical phenotypes , and thePDPK1 (3-phosphoinositide-dependent protein kinase 1) expression [IRS1, IRS2, AKT1, PPARA, MAPK9, MAPK10, STAT3, PPKAG2, ACSL3 and ACSL4, which are important genes involved in insulin signaling and type 2 diabetes pathways [It is well-known that hyperglycemia, hyperlipidemia and islet β cell dysfunction are associated with the onset of T2DM . Our anapression , an impopression . In addipathways ,90 , including PPARG, also known as PPARγ, and adipocyte-enriched genes , SOCS1, SOCS3, GRB2, INSR and PPARG), to regulate many aspects of the lipid and glucose metabolisms [PPARG [SLC2A4 (GLUT4) translocation to the cell membrane. In addition, miR-130b-3p, -132-3p, -181a-5p, -199a-5p and -221-3p can directly target PPKAs (AMP-activated protein kinase) to affect energy metabolism in various signal transduction pathways, including adipocytokine and insulin signaling pathways [Adipose tissues play a key role in maintaining the homeostasis of the body’s energy metabolism by releasing an array of hormones and cytokines: adipocytokines. The current study revealed that the dysregulated miRNAs play important roles in glucose, as well as lipid metabolism  29,50,9,950,92. abolisms ,93. Somes [PPARG ,95. miR-pathways ,93 , a transcriptional coactivator, interacts with PPARgamma (PPARG) to regulate genes involved in energy metabolism and is a target of mouse miR-375, but not human. Therefore, in the current study, we only selected studies from human patient samples in order to investigate the functional involvement of miRNAs in human T2DM pathology. These findings may provide a more accurate and more realistic view of the effect of miRNAs in diabetes.There are a number of studies investigating the relationship of miRNA and diabetes based on animal models and cell line systems ,102,103.www.mirgator.kobic.re.kr/) [mpd.bioinf.uni-sb.de/) [Some of the miRNA-mRNA interactions in .re.kr/)  and miRP-sb.de/) . All of In the past few years, there have been a number of reports revealing disease-associated concentration changes of specific miRNA in circulation, such as miR-375 in patients with T2DM  31,34,3,75. HoweIn conclusion, we present a systematic analysis by integrating various T2DM-related miRNA datasets from the literature. These dysregulated miRNAs affect various metabolic and signal transduction pathways associated with T2DM and may play important roles in the development of diabetes. The pathway analysis results also provide possible links to kidney and cardiovascular complications associated the T2DM. However, many other factors, both endogenous and exogenous, may influence the spectrum of miRNA in tissues and circulation. Recent findings suggest that diet, a key component in T2DM disease management, affects the spectra of miRNA in various tissues ,110. Forhttp://www.cnki.net), Wanfang database (available online: http://www.wanfangdata.com), VIP database  and Google Scholar. The search was conducted by using combinations of the following key words: “miRNA” or “microRNA” and “diabetes” or “type 2 diabetes”. The online search was accompanied by checking cited references from the articles for potentially eligible reports. References of identified studies that met the inclusion criteria have been assessed by two independent reviewers. To ensure that relevant studies were not missed, searches in Gene Expression Omnibus  and Array Express (available online: www.ebi.ac.uk/arrayexpress) repositories were also performed.We scanned all relevant studies published prior to 18 October 2016 through a comprehensive search of records in PubMed, The Cochrane Library, the Chinese National Knowledge Infrastructure (CNKI) database  full text of original experimental articles based on miRNA expression profiling between diabetic patients and normal individuals; (2) studies based on human samples; and (3) original articles published in a language of either English or Chinese. The main exclusion criteria were as follows: (1) meta-analyses, series, abstract, commentary, review, letters and editorial; (2) duplicate data; (3) studies that compared different stages of diabetes, but did not include normal samples; and (4) studies based on cell lines or animal models.Data were extracted by two independent reviewers from all publications that met the inclusion criteria. The lists of miRNAs with statistically-significant expression changes were extracted from the publications. All miRNA names were standardized according to miRBase Version 21 . Any disWe assessed the quality of the data using the QUADAS2  checklist. The categories in the scoring system used for assessing study quality are summarized in http://mirsystem.cgm.ntu.edu.tw/index.php) web server, which provides information from seven miRNA target prediction algorithms, DIANA-microT, miRanda, mirBridge, PicTar, PITA, RNA22 and TargetScan, and two experimentally-validated miRNA target databases, TarBase and miRecords [http://david.abcc.ncifcrf.gov/home.jsp). Cytoscape (available online: http://www.cytoscape.org/) and KEGG pathway maps (available online: http://www.genome.jp/kegg/pathway.html) were used to generate miRNA-mRNA interaction networks. We also included the mRNA expression level changes from visceral adipose tissue expression profiling data from the public domain (GSE16415).To identify the functional involvement of miRNAs in the progression of T2DM, we used miRNA target prediction algorithms and information from validated miRNA target databases to generate the list of putative miRNA targets. The list of putative interacting targets for the miRNAs was generated using the miRSystem (available online: iRecords . To incr


TUFM, SPI1, APOB48R, etc.). Also 3 WHR associated genes were detected . The significant association between Chr16p11 and BMI was observed by GSEA . The TGCTGCT, MIR-15A, MIR-16, MIR-15B, MIR-195, MIR-424, and MIR-497  gene set appeared to be linked with WHR. Our results provide novel clues for the genetic mechanism studies of obesity. This study also illustrated the good performance of SMR for susceptibility gene mapping.To identify novel susceptibility genes and gene sets for obesity, we conducted a genomewide expression association analysis of obesity via integrating genomewide association study (GWAS) and expression quantitative trait loci (eQTLs) data. GWAS summary data of body mass index (BMI) and waist-to-hip ratio (WHR) was driven from a published study, totally involving 339,224 individuals. The eQTLs dataset  was obtained from eQTLs meta-analysis of 5,311 subjects. Integrative analysis of GWAS and eQTLs data was conducted by SMR software. The SMR single gene analysis results were further subjected to gene set enrichment analysis (GSEA) for identifying obesity associated gene sets. A total of 13,311 annotated gene sets were analyzed in this study. SMR single gene analysis identified 20 BMI associated genes ( CREBRF was associated with increased risk of obesity while being associated with decreased risk of type 2 diabetes in 3000 Samoans [Obesity is a complex health issue affecting millions of people around the world, causing serious health problems and a substantial economic burden in the developed country , 2. It h Samoans . Associa Samoans . Another Samoans . HoweverGenomewide association study (GWAS) contributes greatly to the susceptibility gene mapping of human complex diseases. Despite the great power of GWAS, it has several limitations. For instance, due to strict genomewide significance thresholds, GWAS usually focuses on the most significant loci and ignores the rest with moderate and modest phenotypic effects , 12. WitExpression quantitative trait loci (eQTLs) are generally noncoding loci that influence the levels of gene transcripts . With thRecently, summary data-based Mendelian randomization (SMR) was proposed . SMR resIn this study, utilizing a recently published eQTLs annotation dataset, SMR was first applied to large scale GWAS summary data for screening novel genes, the expression levels of which were associated with obesity. To reveal the biological significance of identified genes, we also extended the SMR approach and conducted a genomewide gene set expression association analysis of obesity. The SMR single gene summary data was subjected to gene set enrichment analysis (GSEA) to identify obesity associated biological gene sets. The main objective of our study is to reveal obesity associated novel genes and gene sets on expression levels.n = 236,231) and 43 with Metabochip results . European-descent genotype imputation was conducted using the HapMap CEU reference panel, while the African American and Hispanic genotype imputation was conducted using HapMap CEU + YRI + CHB + JPT reference panel [Large scale GWAS meta-analysis summary data of body mass index (BMI) and waist-to-hip ratio (WHR) was used here . Brieflyce panel . The invce panel . Detailece panel .p values < 9.28 × 10−6 (0.05/5390) after Bonferroni correction.The GWAS meta-analysis summary datasets of BMI and WHR were subjected to SMR analysis . The eQThttp://software.broadinstitute.org/gsea/msigdb/index.jsp). It contained a total of 13,311 annotated gene sets [p value and FDR adjusted p value of each gene set [p values < 0.05.The SMR gene-level summary data of BMI and WHR were analyzed by GSEA for gene set association analysis . The latene sets . 5,000 pp value = 3.16 × 10−22), SPI1 , and APOB48R . For WHR, SMR detected 3 significant genes, including CPEB4 , WARS2 , and L3MBTL3 .A total of 5,390 genes with both GWAS summary and eQTLs data were analyzed by SMR in this study. After strict Bonferroni correction, SMR identified 20 BMI associated genes , such asp value = 1 × 10−3) gene set and NITROGEN_COMPOUND_METABOLIC_PROCESS  were significantly associated with BMI. For WHR, the TGCTGCT, MIR-15A, MIR-16, MIR-15B, MIR-195, MIR-424, MIR-497  gene set showed significant association signal.We found that the Chr16p11 . The biological functional studies of WARS2 and L3MBTL3 are limited. To the best of our knowledge, little effort has been paid to investigate the possible impact of WARS2 and L3MBTL3 on the development of obesity. Further studies are warranted to confirm our findings and to reveal the potential mechanism of identified genes involved in the development of obesity. and WHR . AdditioSeveral significant gene sets were identified via gene set enrichment analysis, including chr16p11 and NITROGEN_COMPOUND_METABOLIC_PROCESS for BMI and TGCTGCT, MIR-15A, MIR-16, MIR-15B, MIR-195, MIR-424, and MIR-497 for WHR. The Chr16p11 gene set consists of 212 genes, some of which are associated with obesity, including SH2B1, APOBR, SULT1A1, SULT1A2, and TUFM. Volckmar et al. identified variants in APOBR and SH2B1 which are associated with extreme obesity in the chromosomal region chr16p11.2 . It was The TGCTGCT, MIR-15A, MIR-16, MIR-15B, MIR-195, MIR-424, and MIR-497 gene set mainly comprises miR-15 microRNA precursor family, including MIR-15A, MIR-15B, MIR-16, MIR-195, and MIR-497. Dong et al. found that MIR-15A and MIR-15B could promote adipogenesis in porcine preadipocyte . Our resThe significant genes identified by SMR were also significant in the original GWAS, which illustrated the good performance of SMR for susceptibility gene mapping. SMR is capable of integrating GWAS summaries and eQTLs annotation information to detect the genes, whose expression levels are associated with complex diseases. The SMR results were consistent with that of previous functional studies of obesity and highlighted several genes for obesity. Therefore, our study results suggested that the genes identified by SMR contributed to the development of obesity through abnormal expression of the genes. Furthermore, we extended SMR to gene set enrichment analysis for obesity, and finally several significant gene sets associated with BMI and WHR were identified, providing novel clues on the genetic mechanism of obesity. That is exactly the extension and innovation of our study to SMR analysis.In summary, this study explored the genetic basis of obesity via integrating analysis of GWAS and eQTLs data utilizing SMR approach. We identified multiple novel genes and gene sets, which were significantly associated with BMI or WHR. Our results provide new clues for clarifying the genetic mechanism of obesity. This study also illustrated the good performance of SMR and extended it to gene set association analysis for complex diseases.


Dickeya fangzhongdai DSM 101947T was isolated from pear trees (Pyrus pyrifolia) in Zhejiang Province, China, and is the causal agent of bleeding canker, a devastating disease of pear trees. Here, we provide the complete genome sequence of this bacterium, which has a 5,027,163-bp-long genome, including 4,375 protein-coding genes and 100 RNA genes. This strain possesses a number of genes encoding virulence factors of pathogens. Dickeya fangzhongdai is the causal agent of bleeding canker, a pathogen that uniquely affects wood plants in seven species of Dickeya . This resulted in 148 contigs, with an N50 value of 111,372 bp, N90 value of 36,275 bp, and genome size of 5,027,163 bp at 150× coverage. The assembled DSM 101947T genome revealed a single circular chromosome and an overall G+C content of 56.76%. A total of 4,375 protein-coding genes were predicted in the genome, and 78 tRNAs and 22 rRNAs were found in the chromosome. In addition, DSM 101947T has 1 small plasmid of 5,284 bp containing 5 protein-coding genes.The genomic DNA was isolated using the QIAamp DNA minikit , according to the manufacturer’s instructions. DNA quality was checked using a NanoDrop spectrophotometer (Thermo Scientific) and quantitated using a Qubit 2.0 fluorometer (Life Technologies) and Agilent 2100 Bioanalyzer . The whole-genome sequence of strain DSM 101947Dickeya fangzhongdai DSM 101947T has been deposited in GenBank under the accession numbers CP025003(chromosome) and CP025004 (plasmid).The complete genome sequence of


Squamous cell carcinoma antigens 1 and 2 , members of the ovalbumin serpin (ov-serpin)/clade B serpin family, were originally discovered as tumor-specific antigens and are used as tumor markers for various kinds of squamous cell carcinomas. Recently, our understanding of the underlying mechanisms of how SCCA1/2 enhance tumor growth has greatly increased. Moreover, it has been shown that SCCA1/2 are involved in the pathogenesis of several inflammatory diseases: asthma, psoriasis, and atopic dermatitis (AD). IL-22 and IL-17, signature cytokines of type 17 inflammation, as well as IL-4 and IL-13, signature cytokines of type 2 inflammation, both of which are positively correlated with the pathogenesis of psoriasis and allergic diseases, respectively, can induce expression of SCCA1/2 in airway epithelial cells and/or keratinocytes, leading to high expression of SCCA1/2 in these diseases. Based on these findings, several trials have been performed to examine the potential of applying SCCA1/2 to biomarkers for these diseases. The findings show that SCCA2 is useful to aid diagnosis, estimate clinical severity and disease type, and assess responses to treatment in psoriasis and AD. These results suggest that SCCA2 has emerged as a novel biomarker for skin inflammatory diseases. Dermatophagoides pteronyssinus 1 (Der p 1) and Dermatophagoides farinae 1 (Der f 1). SCCA molecules are used as tumor markers for various kind of squamous cell carcinomas—esophagus, lung, head and neck, anal canal, and uterine cervix—reflecting tumor stage, tumor size, stromal invasion, lymph-vascular space status, and lymph node status . Th. Th18]. Der p 1, and mast cell chymase, whereas Serpin b3b can inhibit cathepsin L and cathepsin G, but not Der p 1 or mast cell chymase. This contrasts with the characteristics of SCCA1 and SCCA2; these two serpin molecules complement target proteases. The distributions of Serpinb3a and Serpinb3b were also different; Serpinb3a was ubiquitously expressed, whereas Serpinb3b was expressed only in keratinocytes, as far as we surveyed. Moreover, neither IL-4 nor IL-13 could induce expression of Serpinb3a and Serpinb3b, and these molecules were constitutively expressed in keratinocytes. These results suggest that the mouse orthologs of SCCA1 and SCCA2 would exhibit pathophysiological roles similar to, but to some extent different from, human orthologs. It is known that in rodents, orthologs of human ov-serpins are well conserved, expanding the repertoire of several orthologs including SERPINB3 . With reUteroglobin/clara cell 10 kDa protein (CC10) is a steroid-inducible, multifunctional, secreted protein with potent anti-inflammatory and immunomodulatory protein . UteroglUsing Serpinb3a-deficient mice, Sivaprasad et al. directly examined the roles of Serpinb3a in the pathogenesis of airway allergic inflammation . DeficieIn 1998, the National Institutes of Health Biomarkers Definitions Working Group defined a biomarker as a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention . The typp < 0.005). When we compared the SCCA levels in the exacerbation stage and in the convalescence stage in a small population, SCCA levels tended to be higher in the exacerbation stage  than in the convalescence stage , although statistical significance was not reached (p = 0.09). Nishi et al. expanded this study, examining a larger population . In. In63]. n = 123, median: 2.7 ng/mL, interquartile range: 1.25–7.75 ng/mL, vs. n = 25, median: 0.70 ng/mL, interquartile range: 0.40–0.80 ng/mL, p < 0.0001), but comparable with AD patients . All three types of psoriasis—psoriasis vulgaris, psoriasis arthritis, and pustular psoriasis—showed higher SCCA levels  than healthy controls, with no statistically significant difference among these three groups. The area under the receiver operating characteristic (ROC) curve (AUC) between psoriasis patients and healthy volunteers was 0.877, giving us high specificity (77.3%) and sensitivity (92%) with the cut-off value of 1.05 ng/mL. Serum SCCA2 levels were significantly correlated with severity of psoriasis, using Psoriasis Area Severity Index (PASI) scores . Moreover, SCCA2 levels were well correlated with serum IL-22 (p < 0.05), but not IL-17A or IL-36γ, consistent with the finding that IL-22 is the most dominant inducer of SCCA2. Based on these findings, SCCA2 has emerged as a useful biomarker indicating the severity of psoriasis.Based on the knowledge that SCCA molecules are highly expressed in psoriatic skin and that SCCAs are downstream molecules of IL-22/IL-17 tightly correlated with pathogenesis of psoriasis, we explored the usefulness of SCCA2 as a biomarker for psoriasis . We chosAD is a chronic or relapsing, inflammatory skin disease characterized by persistent pruritus and recurrent eczema . It is wSeveral comprehensive analyses to examine the gene expression profile of AD patients showed that expression of both SCCA1 and SCCA2 was upregulated in lesional AD skin compared with skin derived from normal subjects ,73. MoreAspergillus fumigatus, these mice showed decreases in transepidermal water loss, skin lesion score , and epidermal thickness, compared with wild-type mice. Moreover, upregulated expression of various pro-inflammatory genes including S100A8 was impaired in Aspergillus fumigatus-exposed Serpinb3a-deficinet mice. Knockdown of both SCCA1/2 in human keratinocytes also downregulated S1008 expression. These findings using Serpinb3a-deficient mice support the pro-inflammatory effects of SCCA molecules in the pathogenesis of AD as well as of asthma.Using Serpinb3a-deficient mice, Sivaprasad et al. examined the role of SCCA in the pathogenesis of AD . When SeIn the initial studies to analyze SCCA levels in AD patients, these levels were found to be upregulated, tightly correlated with clinical severity ,78. The After we established ELISA systems to distinguish SCCA1 and SCCA2, we explored the usefulness of measuring SCCA2 in both adult and pediatric AD patients because in vitro analyses had shown that SCCA2, compared with SCCA1, is dominantly expressed by IL-4 or IL-13 in keratinocytes ,25,76. WIn the past decade, our understanding of the functions of SCCA molecules has grown. With it, the usefulness of the SCCA molecules, particularly SCCA2, for treating inflammatory skin diseases psoriasis and AD has emerged. However, several questions remain. Although the underlying mechanism of how SCCA molecules enhance tumor growth has been well clarified, it is still obscure how SCCA molecules enhance inflammation in vivo, as shown in several mouse models. Moreover, many molecularly targeted drugs for psoriasis and AD—TNF, IL-12/IL-23p40 subunit, IL-23p19 subunit, IL-17A, IL-17RA, and IL-4Rα—have been developed. Diagnostics to estimate the efficacy of these drugs, so-called “companion diagnostics,” are needed. Moreover, diagnostics to monitor the efficacy of these drugs and to access the time points to stop these drugs are still to be developed. It will be of particular interest to examine whether SCCA2 has such abilities. Further research aiming at these points should be undertaken.


For instance, the mitochondrial oxidative phosphorylation (OXPHOS) ensures a supply of adenosine triphosphate (ATP), but is also the main source of potentially harmful levels of reactive oxygen species (ROS). Moreover, mitochondrial function is strongly linked to mitochondrial Ca2+ homeostasis and mitochondrial shape, which undergo various alterations during aging. Since mitochondria play such a critical role in an organism’s process of aging, they also offer promising targets for manipulation of senescent cellular functions. Accordingly, interventions delaying the onset of age-associated disorders involve the manipulation of mitochondrial function, including caloric restriction (CR) or exercise, as well as drugs, such as metformin, aspirin, and polyphenols. In this review, we discuss mitochondria’s role in and impact on cellular aging and their potential to serve as a target for therapeutic interventions against age-related cellular dysfunction.Senescence is related to the loss of cellular homeostasis and functions, which leads to a progressive decline in physiological ability and to aging-associated diseases. Since mitochondria are essential to energy supply, cell differentiation, cell cycle control, intracellular signaling and Ca In a rapidly aging society, new treatment options for age-related disorders and diseases will be increasingly important ,2. Conse2+ homeostasis, as well as in endoplasmic reticulum (ER)-mitochondria crosstalk, could reduce the adaptive capacity of cells to withstand stress and increase their vulnerability to age-related diseases [As adenosine triphosphate (ATP)-producing power plants of the cell, mitochondria are in a unique position to influence an organism’s aging process. Recent reports suggest that mitochondrial function is linked to age-associated biphasic alterations in metabolic activity, including an increase and afterwards progressive decrease in mitochondrial function . In addidiseases . Consequdiseases  or exercdiseases , as welldiseases ,6, targediseases . Neverthdiseases , the rigNotably, impaired mitochondrial functions were reported to cause accelerated aging that affects primarily organs with high levels of energy demand, such as the brain, the heart, the skeletal muscle, as well as liver and kidney . The criThis review provides a summary of present knowledge about mitochondria’s unique role in the process of aging and the development of age-related disorders and diseases. Furthermore, the potential of mitochondria to serve as targets for therapeutic interventions against age-related diseases is highlighted.As descendants of α-proteobacteria, mitochondria are double-membraned and equipped with their own circular genome. After 1.5 billion years of incorporation into the eukaryotic cell, mitochondria are well-integrated and irreplaceable cellular compartments . While tThe protein complexes of the mitochondrial respiration chain are located at the cristae . These i2+ uptake from the IMS to the mitochondrial matrix, a complex machinery of proteins is located at the IMM [2+ uptake is linked to Ca2+ mobilization from the biggest internal Ca2+ store, the ER [2+ [2+ entry (SOCE). In this process low Ca2+ levels in the ER cause oligomerization and, finally, a conformational change of the Ca2+ sensing stromal interaction molecule-1 (STIM1), which then activates the plasma membrane Ca2+ channel (ORAI1) through protein-protein interactions, causing Ca2+ entry [2+ elevations are sensed by mitochondria, which take up Ca2+ to buffer cytosolic Ca2+ levels in distinct regions of the cytosol, so-called microdomains, and thereby control the activity of Ca2+-dependent enzymes [2+ to the ER [+/Ca2+ exchanger (NCX) and the plasma membrane Ca2+ ATPase (PMCA) also regulate cytosolic Ca2+ levels by extrusion of Ca2+ [To ensure and control mitochondrial Ca the IMM . Mitocho, the ER , or the e ER [2+ . The clo2+ entry . Cytosol enzymes  and ion  enzymes . Mitochoo the ER . Moreove of Ca2+ . 2+ uptake, including the pore-forming mitochondrial calcium uniporter (MCU) [2+-sensing proteins mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial Ca2+ uptake 2 (MICU2), which control mitochondrial Ca2+ homeostasis, to MCU [2+ uptake, including mitochondrial calcium uniporter regulator 1 (MCUR1) [2+ uptake [A finely-tuned complex of proteins ensures and controls the mitochondrial Caer (MCU) ,45, whicer (MCU) . Moreoveer (MCU)  links th, to MCU . In addi (MCUR1) , the EF- (MCUR1)  as well  (MCUR1) . Recentl+ uptake .2+ ions control the activity of the tricarboxylic acid (TCA) cycle by Ca2+-dependent dehydrogenases [2), donate electrons to the electron transport chain (ECT). Electrons are accepted at the NADH dehydrogenase (complex I) and cytochrome bc1 complex (complex III) and are shuttled via succinate dehydrogenase (complex II), ubiquinone, cytochrome bc1 complex (complex III), and cytochrome c to cytochrome c oxidase (complex IV), where O2 is reduced to H2O. The electron transport through complex I to complex IV is coupled to proton pumping from the mitochondrial matrix to the IMS. The electrochemical gradient produced is used for the proton transport from the IMS into the matrix through ATP synthase (complex V), causing a release of energy and phosphorylation of adenosine diphosphate (ADP) to ATP [2O2 [2O2 may diffuse from the mitochondria to the cytosol, in the matrix of mitochondria several enzymes, like peroxiredoxins (PRDX) 3 and 5, catalase, and glutathione peroxidases 1 and 4 reduce H2O2 to H2O [2O2 is also linked to the NAD and NADP systems and is a key signaling molecule [2O2 by overexpression of PRDX3 improved glucose tolerance in mice, potentially via inhibition of glycogen synthase kinase-3. Moreover, cells from animals overexpressing PRDX3 had increased resistance to stress-induced cell death and apoptosis [Mclk1+/− mutant mice that produce higher levels of ROS than wild-type mice, had an enhanced immune reactivity and slowed aging progression [Caenorhabditis elegans (C. elegans) [In the mitochondrial matrix, Caogenases ,54. The ) to ATP . If elec) to ATP . To neutATP [2O2 . While a2 to H2O . Notablymolecule . Hence, poptosis . High lepoptosis . In contpoptosis . Interesgression . Moreoveelegans) .c into the cytosol through a mechanism yet to be described [c binds to apoptotic protease activating factor 1 (APAF1) and ATP, forming the apoptosome complex, which cleaves pro-caspase 9 into its active form caspase-9, and thereby activates effector caspases [While mitochondria ensure cell viability by ATP production, they also execute programmed cell death . In respescribed . By deaccaspases . During caspases . Since all of these mitochondrial tasks are highly complex and need to be well-balanced and controlled, numerous targets may be affected by aging or impacted by dysregulation of the process of aging.2+ uptake and homeostasis undergoes remarkable changes. For instance, the MCU channel is oxidized by ROS, resulting in increased MCU channel activity, which, in turn, further elevates mitochondrial ROS levels and increases the risk for mitochondrial Ca2+ overload-induced cell death [2+ levels, while store-operated Ca2+ entry was inhibited via downregulation of STIM1 and ORAI1 in these aging neurons [2+ accumulation by inositol 1,4,5-trisphosphate receptor type 2 (ITPR2)-triggered Ca2+ release from the ER and mitochondrial Ca2+ uptake via MCU in human mammary epithelial cells (HMEC). In turn, loss of ITPR2 and MCU counteracted the development of senescence [2+ overload, as well as high levels of ROS promotes cell death by collapse of the mitochondrial membrane potential and opening of the mitochondrial permeability transition pore (mPTP) [2+, NAD+, and glutathione into the cytosol was shown to disrupt cellular homeostasis and to increase oxidative damage [2+ flux in response to cytosolic Ca2+ was reported to decrease progressively in C. elegans during aging [2+ uptake was demonstrated in mice lacking Fus1, a small mitochondrial protein regulating mitochondrial Ca2+ homeostasis. Loss of Fus1 caused inefficient accumulation of Ca2+ in mitochondria and decreased respiratory reserve capacity, resulting in the decreased lifespan of mice with knockout of Fus1 [2+ homeostasis during aging is a tightrope walk between meeting mitochondria’s demand for Ca2+ [2+ overload [During the process of aging, the protein machinery controlling mitochondrial Call death . Moreove neurons . In linenescence . Mitochoe (mPTP) . Moreovee (mPTP)  might fue (mPTP) . Releasee damage  and possng aging . The imp of Fus1 . These pfor Ca2+  and trigoverload .+/NADH ratio at middle-age (15–16 years) and elevation in increased age (28–32 years) [2+ levels mirrored those in energy production—initially elevated [2+ overload-induced damage [1FO ATP synthase activity and defective F1FO complex coupling, which may be caused by the mitochondrial protein cyclophilin D [Aging was typically associated with decreased mitochondrial metabolism . In appa2 years) . Moreoveelevated ,69 beford damage —it seemsd age 28–2 years [d damage . In thisd damage . Anotherd damage . Interesd damage . It seemd damage . In contd damage . Consistphilin D . Moreovephilin D . In summmt) was suggested as a possible mechanism to tolerate mtDNA deletions to a certain extent [mt is a protective transcriptional program from the cells to sense and respond to mitochondrial dysfunction by inducing mitochondrial protein homeostasis and regeneration of defective mitochondria [C. elegans strain, containing wild-type mtDNA and mtDNA lacking four essential genes, UPRmt was constitutively activated by dysfunctional OXPHOS. Moreover, even deleterious mtDNA was maintained by UPRmt, possibly in an attempt to recover OXPHOS activity by promoting mitochondrial biogenesis [C. elegans that OXPHOS dysfunction induces the activating transcription factor associated with stress (ATFS1), which binds directly to the OXPHOS gene promotors in the nuclear and mitochondrial genomes and thereby promotes OXPHOS recovery during the UPRmt [C. elegans and even rescued UPRmt signaling in ATFS1 deficient worms [mt on the process of aging was demonstrated in worms, in which UPRmt delayed aging and promoted life span via ATFS1 [mt was also shown to prolong the lifespan of Drosophila melanogaster by activation of the forkhead protein O (FOXO), which induces a set of chaperones implicated in lifespan extension [Mutations of mitochondrial DNA (mtDNA) are estimated to be 10- to 17-fold higher than in the nuclear genome . In linen extent . UPRmt ichondria . In a heogenesis . It was he UPRmt . In mammnt worms . Furtheria ATFS1 . Recentlxtension .mt in nematodes, ATFS1, controls mitochondrial biogenesis to compensate for loss of OXPHOS activity [mt activation during aging [C. elegans and linked to age-related decline in so-called mitophagy [mitochondria-on-a-string (MOAS), characterized by thin segments of mitochondria mixed with large ones, a phenotype already described for neurodegenerative diseases, was found and linked to incomplete mitochondrial fission due to dysregulation of the fission machinery [D. melanogaster. A shift towards accumulation of dysfunctional elongated mitochondria was thereby prevented and health, as well as lifespan, improved significantly [D. melanogaster (10–12 weeks), mitochondria showed cristae vacuolization and the distribution of functional proteins such as ATP synthase was hampered [D. melanogaster, characterized by the lack of well-developed cristae and cristae junctions and positively correlating with the rate of mitochondrial respiration [The main transcription factor of UPRactivity . Consequng aging . Indeed,itophagy . After mitophagy . For effitophagy . Notablyachinery . Short-tficantly . Notablyficantly . Neverthficantly . Consistficantly . Howeverficantly . Moreoveficantly . Acute dficantly . In longhampered . Anotherpiration . In contpiration . In cardpiration . In yeaspiration . In summC. elegans [2+ retention capacity was enhanced strongly by CR in comparison to ad libitum diet, offering protection from ischemia/reperfusion damage, a condition frequently observed in age [2+ and enhanced tolerance against excitotoxicity in rat brain mitochondria [+ levels [2 ratio at baseline benefited from CR in regard to muscle mitochondrial ATP synthesis rates and coupling [Caloric restriction: From  elegans  to prima elegans , caloric elegans ,115. The elegans . While r elegans . CR also elegans . Consist elegans . The effd in age . Additiochondria . In yeaschondria . Moreovechondria . AMPK wa+ levels  and to i+ levels . Indeed,+ levels . Neverthcoupling . These c2+ homeostasis has also been shown to be affected during aging, revealing downregulation of MCU in aged mice [2+ uptake and in mitochondrial shape by nine weeks of physical training or neuromuscular electrical stimulation. Improved muscle function and structure by both types of training were linked to increased protein levels of MCU. Interestingly, electron microscopy revealed remodeling of mitochondrial shape in muscles trained by electrical stimulation, which was potentially mediated by an increased expression of cristae structure-shaping protein OPA1 [Exercise: In a number of species, age-related muscle loss is linked to impaired mitochondrial biogenesis and function, as well as to decreased metabolic capacity . Prolifeged mice . Analysiein OPA1 . Interesein OPA1 .Polyphenols: Polyphenols, such as resveratrol (RSV) or green tea polyphenols, were shown to extend lifespan in Saccharomyces cerevisiae [C. elegans [D. melanogaster [French paradox, was hypothesized to be due to frequent consumption of RSV-rich red wine [French paradox has to be reevaluated.revisiae , C. eleg elegans , D. melanogaster , fish [1nogaster , and micnogaster . Moreovered wine . Clinicared wine . The lowred wine , as wellred wine . Howeverred wine . Thus, t2+ levels and activation of the CaMKKβ-AMPK pathway [+ levels [2O2 production, whereas the ubiquinone derivative, MitoQ, an electron scavenger preventing mitochondrial ROS formation [2O2 levels under this condition [Nevertheless, RSV was found to mimic features of CR, such as glucagon and catecholamine release, boosting cyclic adenosine monophosphate production, which, in turn, activates cell-type dependent effectors and leads to elevated Ca pathway . Enhance+ levels . Other r+ levels ,141, cau+ levels  and alte+ levels . Moreove+ levels . Low lev+ levels  as well + levels . Neverth+ levels . For insormation , could nondition .Antioxidants: In addition to MitoQ, the Murphy lab has synthesized several other mitochondria-targeted antioxidants by covalently attaching lipophilic triphenylphosphonium cations to endogenous antioxidants, including α-tocopherol (MitoVitE) and the synthetic spin trap compound α-phenyl-tert-butylnitrone (MitoPBN) [MitoPBN) . FurtherMitoPBN) . Due to MitoPBN) . In rat MitoPBN) . FurtherMitoPBN) . In addiMitoPBN) . MitoQ wMitoPBN) . MoreoveMitoPBN) , was sucMitoPBN)  and is cMitoPBN) . All of C. elegans [Nevertheless, the relevance of antioxidants in preventing age-related cellular dysfunction by the reduction of oxidative stress was challenged by the finding that small levels of ROS provoke a vaccination-like stimulus, thus causing development of antioxidant defense mechanisms and a positive impact on lifespan . Consequ elegans . The dif elegans . In anim elegans . In cont elegans . In the  elegans . Aspirin: Long-term usage of acetylsalicylic acid (Aspirin®), a nonsteroidal anti-inflammatory drug, was shown to slightly increase the lifespan of worms [C. elegans, aspirin treatment increased the expression of lipid hydrolysis and fatty acid β-oxidation-related genes, which activated DAF-12 and DAF-16, the worm orthologue to FOXO, and extended lifespan [of worms , flies [of worms , and mamof worms . After oof worms . This meof worms . Additiolifespan . Neverthlifespan  and potelifespan ,166, thelifespan .Omega-3 fatty acids: Treatment with ω-3 fatty acid α-linolenic acid (ALA) increased lifespan of C. elegans via the activation of the transcription factors NHR-49, the worm orthologue of human PPARα, and SKN1, the worm orthologue of human NRF2. NHR-49-activated genes are involved in the β-oxidation of free fatty acids, promoting the generation of energy via mitochondrial respiration and NRF2-induced mitochondrial biogenesis [ogenesis . In the ogenesis . Consistogenesis . NeverthMetformin: Metformin, a biguanide used for diabetes prevention and management for 60 years, was reported to display greatly beneficial effects on aging in cellular and animal models, as well as in pilot human studies, far beyond that expected from improving glycemic control [C. elegans [ control . Despite control , possibl control . That me control . Moreove elegans . As a ca elegans  and inhi elegans . Interes elegans . Consequ elegans , causingSenolytics: Senolytic drugs were reported to induce apoptosis of senescent cells, thus preventing the local and systemic pathogenic effects of senescent cells conveyed by the release of proinflammatory cytokines and chemokines, tissue-damaging proteases and inhibition of stem and progenitor cell function [function . By hostfunction . Accordifunction . The canfunction  and to ifunction . Navitocfunction . Moreovefunction . Therefo2+ handling [The process of aging evokes various alterations in mitochondrial Cahandling , mitochohandling , mitochohandling , as wellhandling , which ahandling . Initialhandling , exercishandling , and druhandling , antioxihandling , metformhandling , ω-3 fathandling , aspirinhandling , and senhandling , specifihandling . Consequently, it is of major importance to further investigate the molecular processes behind the role of mitochondria in aging, as well as their potential to serve as targets for therapeutic interventions.