Trametinib ameliorates aging-associated gut pathology in Drosophila females by reducing Pol III activity in intestinal stem cells.

Pharmacological therapies are promising interventions to slow down aging and reduce multimorbidity in the elderly. Studies in animal models are the first step toward translation of candidate molecules into human therapies, as they aim to elucidate the molecular pathways, cellular mechanisms, and tissue pathologies involved in the anti-aging effects. Trametinib, an allosteric inhibitor of MEK within the Ras/MAPK (Ras/Mitogen-Activated Protein Kinase) pathway and currently used as an anti-cancer treatment, emerged as a geroprotector candidate because it extended lifespan in the fruit fly Drosophila melanogaster. Here, we confirm that trametinib consistently and robustly extends female lifespan, and reduces intestinal stem cell (ISC) proliferation, tumor formation, tissue dysplasia, and barrier disruption in guts in aged flies. In contrast, pro-longevity effects of trametinib are weak and inconsistent in males, and it does not influence gut homeostasis. Inhibition of the Ras/MAPK pathway specifically in ISCs is sufficient to partially recapitulate the effects of trametinib. Moreover, in ISCs, trametinib decreases the activity of the RNA polymerase III (Pol III), a conserved enzyme synthesizing transfer RNAs and other short, non-coding RNAs, and whose inhibition also extends lifespan and reduces gut pathology. Finally, we show that the pro-longevity effect of trametinib in ISCs is partially mediated by Maf1, a repressor of Pol III, suggesting a life-limiting Ras/MAPK-Maf1-Pol III axis in these cells. The mechanism of action described in this work paves the way for further studies on the anti-aging effects of trametinib in mammals and shows its potential for clinical application in humans.

Transcranial Doppler Ultrasonography detection on cerebrovascular flow for evaluating neonatal hypoxic-ischemic encephalopathy modeling.

Objective: This study aimed to investigate the feasibility of Transcranial Doppler Ultrasonography (TCD) in evaluating neonatal hypoxic-ischemic encephalopathy (NHIE) modeling through monitoring the alteration of cerebrovascular flow in neonatal hypoxic-ischemic (HI) rats. Methods: Postnatal 7-day-old Sprague Dawley (SD) rats were divided into the control group, HI group, and hypoxia (H) group. TCD was applied to assess the changes of cerebral blood vessels, cerebrovascular flow velocity, and heart rate (HR) in sagittal and coronal sections at 1, 2, 3, and 7 days after the operation. For accuracy, cerebral infarct of rats was examined by 2,3,5-Triphenyl tetrazolium chloride (TTC) staining and Nissl staining to simultaneously verify the establishment of NHIE modeling. Results: Coronal and sagittal TCD scans revealed obvious alteration of cerebrovascular flow in main cerebral vessels. Obvious cerebrovascular back-flow was observed in anterior cerebral artery (ACA), basilar artery (BA), middle cerebral artery (MCA) of HI rats, along with accelerated cerebrovascular flows in the left internal carotid artery (ICA-L) and BA, decreased flows in right internal carotid artery (ICA-R) relative to those in the H and control groups. The alterations of cerebral blood flows in neonatal HI rats indicated successful ligation of right common carotid artery. Besides, TTC staining further validated the cerebral infarct was indeed caused due to ligation-induced insufficient blood supply. Damage to nervous tissues was also revealed by Nissl staining. Conclusion: Cerebral blood flow assessment by TCD in neonatal HI rats contributed to cerebrovascular abnormalities observed in a real-time and non-invasive way. The present study elicits the potentials to utilize TCD as an effective means for monitoring the progression of injury as well as NHIE modeling. The abnormal appearance of cerebral blood flow is also beneficial to the early warning and effective detection in clinical practice.

Ageing-associated changes in transcriptional elongation influence longevity.

Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing1-4. However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin-IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms5 and flies6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures.

The First-Water-Layer Evolution at the Graphene/Water Interface under Different Electro-Modulated Hydrophilic Conditions Observed by Suspended/Supported Field-Effect-Device Architectures.

Interfacial water molecules affect carrier transportation within graphene and related applications. Without proper tools, however, most of the previous works focus on simulation modeling rather than experimental validation. To overcome this obstacle, a series of graphene field-effect transistors (GFETs) with suspended (substrate-free, SF) and supported (oxide-supported, OS) configurations are developed to investigate the graphene-water interface under different hydrophilic conditions. With deionized water environments, in our experiments, the electrical transportation behaviors of the graphene mainly originate from the evolution of the interfacial water-molecule arrangement. Also, these current-voltage behaviors can be used to elucidate the first-water layer at the graphene-water interface. For SF-GFET, our experimental results show positive hysteresis in electrical transportation. These imply highly ordered interfacial water molecules with a separated-ionic distributed structure. For OS-GFET, on the contrary, the negative hysteresis shows the formation of the hydrogen-bond interaction between the interfacial water layer and the SiO2 substrate under the graphene. This interaction further promotes current conduction through the graphene/water interface. In addition, the net current-voltage relationship also indicates the energy required to change the orientation of the first-layer water molecules during electro-potential change. Therefore, our work gives an insight into graphene-water interfacial evolution with field-effect modulation. Furthermore, this experimental architecture also paves the way for investigating 2D solid-liquid interfacial features.

Jian-Pi-Yi-Shen decoction inhibits mitochondria-dependent granulosa cell apoptosis in a rat model of POF.

As a widely applied traditional Chinese medicine (TCM), Jian-Pi-Yi-Shen (JPYS) decoction maybe applied in curing premature ovarian failure (POF) besides chronic kidney disease (CKD). In vivo experiments, 40 female SD (8-week-old) rats were randomized into four groups, namely, control group (negative control), POF model group, JPYS treatment group, and triptorelin treatment group (positive control). JPYS group was treated with JPYS decoction (oral, 11 g/kg) for 60 days, and the triptorelin group was treated with triptorelin (injection, 1.5 mg/kg) for 10 days before the administration of cyclophosphamide (CTX) (50 mg/kg body weight) to establish POF model. We examined apoptosis, mitochondrial function, and target gene (ASK1/JNK pathway and mitochondrial fusion/fission) expression. In vitro experiments, the KGN human granulosa cell line was used. Cells were pretreated with CTX (20, 40, and 60 µg/mL) for 24 h, followed by JPYS-containing serum (2, 4, and 8 %) for 24 h. Thereafter, these cells were employed to assess apoptosis, mitochondrial function, and target gene levels of protein and mRNA. In vivo, JPYS alleviated injury and suppressed apoptosis in POF rats. In addition, JPYS improved ovarian function. JPYS inhibit apoptosis of granulosa cells through improving mitochondrial function by activating ASK1/JNK pathway. In vitro, JPYS inhibited KGN cell apoptosis through inhibited ASK1/JNK pathway and improved mitochondrial function. The effects of GS-49977 were similar to those of JPYS. During POF, mitochondrial dysfunction occurs in the ovary and leads to granulosa cell apoptosis. JPYS decoction improves mitochondrial function and alleviates apoptosis through ASK1/JNK pathway.

Review-Hysteresis in Carbon Nano-Structure Field Effect Transistor.

In recent decades, the research of nano-structure devices (e.g., carbon nanotube and graphene) has experienced rapid growth. These materials have supreme electronic, thermal, optical and mechanical properties and have received widespread concern in different fields. It is worth noting that gate hysteresis behavior of field effect transistors can always be found in ambient conditions, which may influence the transmission appearance. Many researchers have put forward various views on this question. Here, we summarize and discuss the mechanisms behind hysteresis, different influencing factors and improvement methods which help decrease or eliminate unevenness and asymmetry.

Long-lasting geroprotection from brief rapamycin treatment in early adulthood by persistently increased intestinal autophagy.

The licensed drug rapamycin has potential to be repurposed for geroprotection. A key challenge is to avoid adverse side effects from continuous dosing. Here we show that geroprotective effects of chronic rapamycin treatment can be obtained with a brief pulse of the drug in early adulthood in female Drosophila and mice. In Drosophila, a brief, early rapamycin treatment of adults extended lifespan and attenuated age-related decline in the intestine to the same degree as lifelong dosing. Lasting memory of earlier treatment was mediated by elevated autophagy in intestinal enterocytes, accompanied by increased levels of intestinal LManV and lysozyme. Brief elevation of autophagy in early adulthood itself induced a long-term increase in autophagy. In mice, a 3-month, early treatment also induced a memory effect, with maintenance similar to chronic treatment, of lysozyme distribution, Man2B1 level in intestinal crypts, Paneth cell architecture and gut barrier function, even 6 months after rapamycin was withdrawn.

Sexual identity of enterocytes regulates autophagy to determine intestinal health, lifespan and responses to rapamycin.

Pharmacological attenuation of mTOR presents a promising route for delay of age-related disease. Here we show that treatment of Drosophila with the mTOR inhibitor rapamycin extends lifespan in females, but not in males. Female-specific, age-related gut pathology is markedly slowed by rapamycin treatment, mediated by increased autophagy. Treatment increases enterocyte autophagy in females, via the H3/H4 histone-Bchs axis, whereas males show high basal levels of enterocyte autophagy that are not increased by rapamycin feeding. Enterocyte sexual identity, determined by transformerFemale expression, dictates sexually dimorphic cell size, H3/H4-Bchs expression, basal rates of autophagy, fecundity, intestinal homeostasis and lifespan extension in response to rapamycin. Dimorphism in autophagy is conserved in mice, where intestine, brown adipose tissue and muscle exhibit sex differences in autophagy and response to rapamycin. This study highlights tissue sex as a determining factor in the regulation of metabolic processes by mTOR and the efficacy of mTOR-targeted, anti-aging drug treatments.

A TORC1-histone axis regulates chromatin organisation and non-canonical induction of autophagy to ameliorate ageing.

Age-related changes to histone levels are seen in many species. However, it is unclear whether changes to histone expression could be exploited to ameliorate the effects of ageing in multicellular organisms. Here we show that inhibition of mTORC1 by the lifespan-extending drug rapamycin increases expression of histones H3 and H4 post-transcriptionally through eIF3-mediated translation. Elevated expression of H3/H4 in intestinal enterocytes in Drosophila alters chromatin organisation, induces intestinal autophagy through transcriptional regulation, and prevents age-related decline in the intestine. Importantly, it also mediates rapamycin-induced longevity and intestinal health. Histones H3/H4 regulate expression of an autophagy cargo adaptor Bchs (WDFY3 in mammals), increased expression of which in enterocytes mediates increased H3/H4-dependent healthy longevity. In mice, rapamycin treatment increases expression of histone proteins and Wdfy3 transcription, and alters chromatin organisation in the small intestine, suggesting that the mTORC1-histone axis is at least partially conserved in mammals and may offer new targets for anti-ageing interventions.

Electrochemically Enabled Double C-H Activation of Amides: Chemoselective Synthesis of Polycyclic Isoquinolinones.

We developed an electrochemically enabled dehydrogenative annulation reaction of amides and alkynes for the synthesis of antitumor polycyclic isoquinolinones through a double C-H activation route. No external oxidant is required in this reaction, and electricity is used for Ru catalyst circulation. The most remarkable feature of this reaction is the effective improvement of product regioselectivity under mild electrolytic conditions in comparison with previously set strong oxidant conditions.

Global metabolomic analyses of the hemolymph and brain during the initiation, maintenance, and termination of pupal diapause in the cotton bollworm, Helicoverpa armigera.

A strategy known as diapause (developmental arrest) has evolved in insects to increase their survival rate under harsh environmental conditions. Diapause causes a dramatic reduction in the metabolic rate and drastically extends lifespan. However, little is known about the mechanisms underlying the metabolic changes involved. Using gas chromatography-mass spectrometry, we compared the changes in the metabolite levels in the brain and hemolymph of nondiapause- and diapause-destined cotton bollworm, Helicoverpa armigera, during the initiation, maintenance, and termination of pupal diapause. A total of 55 metabolites in the hemolymph and 52 metabolites in the brain were detected. Of these metabolites, 21 and 12 metabolite levels were altered in the diapause pupal hemolymph and brain, respectively. During diapause initiation and maintenance, the number of metabolites with increased levels in the hemolymph of the diapausing pupae is far greater than the number in the nondiapause pupae. These increased metabolites function as an energy source, metabolic intermediates, and cryoprotectants. The number of metabolites with decreased levels in the brain of diapausing pupae is far greater than the number in the nondiapause pupae. Low metabolite levels are likely to directly or indirectly repress the brain metabolic activity. During diapause termination, most of the metabolite levels in the hemolymph of the diapausing pupae rapidly decrease because they function as energy and metabolic sources that promote pupa-adult development. In conclusion, the metabolites with altered levels in the hemolymph and brain serve as energy and metabolic resources and help to maintain a low brain metabolic activity during diapause.

Proteomic and metabolomic profiles of larval hemolymph associated with diapause in the cotton bollworm, Helicoverpa armigera.

BACKGROUND: Diapause is programmed developmental arrest coupled with the depression of metabolic activity and the enhancement of stress resistance. Pupal diapause is induced by environmental signals and is prepared during the prediapause phase. In the cotton bollworm, Helicoverpa armigera, the prediapause phase, which contains two sub-phases, diapause induction and preparation, occurs in the larval stage. Here, we performed parallel proteomic and metabolomic analyses on H. armigera larval hemolymph during the prediapause phase. RESULTS: By two-dimensional electrophoresis, 37 proteins were shown to be differentially expressed in diapause-destined larvae. Of these proteins, 28 were successfully identified by MALDI-TOF/TOF mass spectrometry. Moreover, a total of 22 altered metabolites were found in diapause-destined larval hemolymph by GC-MS analysis, and the levels of 17 metabolites were elevated and 5 were decreased. CONCLUSIONS: The proteins and metabolites with significantly altered levels play different roles in diapause-destined larvae, including diapause induction, metabolic storage, immune response, stress tolerance, and others. Because hemolymph circulates through the whole body of an insect, these differences found in diapause-destined larvae most likely correspond to upstream endocrine signals and would further influence other organ/tissue activities to determine the insect's fact: diapause or development.

Polycomb repressive complex 2 (PRC2) protein ESC regulates insect developmental timing by mediating H3K27me3 and activating prothoracicotropic hormone gene expression.

The decision made by insects to develop into adults or halt development (enter diapause and prolong lifespan) is commonly based on environmental signals that provide reliable predictors of future seasons of adversity. For example, the short day lengths of early autumn accurately foretell the advent of winter, but little is known about the molecular mechanisms that preside over the hormonal events dictating whether the insect proceeds with development or enters diapause. In Helicoverpa armigera we show that day length affects H3K27me3 by affecting polycomb repressive complex 2 (PRC2) protein extra sex comb (ESC) and regulates the prothoracicotropic hormone (PTTH) gene, thus directly influencing developmental timing. ESC expression in brains of developing (nondiapause) pupae is higher than in brains from diapausing pupae. High ESC expression is localized in two pairs of PTTH neurosecretory cells, and H3K27me3 recruits on the PTTH promoter. Double strand ESC and PRC2 inhibitor (DzNep) treatment in vitro show that ESC triggers PTTH promoter activity, which in turn depends on PRC2 methyltransferase activity. Injection of DzNep into pupae programmed for development reduces the H3K27me3 mark and PTTH gene expression, thereby delaying development. Although ESC is best known as a transcriptional repressor, our results show that ESC prompts development and metamorphosis. We believe this is the first report showing that the PRC2 complex functions as an activator and that a low level of H3K27me3 can prolong lifespan (i.e. induce diapause) by controlling PTTH gene expression in insects.

A regulatory pathway, ecdysone-transcription factor relish-cathepsin L, is involved in insect fat body dissociation.

Insect fat body is the organ for intermediary metabolism, comparable to vertebrate liver and adipose tissue. Larval fat body is disintegrated to individual fat body cells and then adult fat body is remodeled at the pupal stage. However, little is known about the dissociation mechanism. We find that the moth Helicoverpa armigera cathepsin L (Har-CL) is expressed heavily in the fat body and is released from fat body cells into the extracellular matrix. The inhibitor and RNAi experiments demonstrate that Har-CL functions in the fat body dissociation in H. armigera. Further, a nuclear protein is identified to be transcription factor Har-Relish, which was found in insect immune response and specifically binds to the promoter of Har-CL gene to regulate its activity. Har-Relish also responds to the steroid hormone ecdysone. Thus, the dissociation of the larval fat body is involved in the hormone (ecdysone)-transcription factor (Relish)-target gene (cathepsin L) regulatory pathway.

Cross-talk between the fat body and brain regulates insect developmental arrest.

Developmental arrest, a critical component of the life cycle in animals as diverse as nematodes (dauer state), insects (diapause), and vertebrates (hibernation), results in dramatic depression of the metabolic rate and a profound extension in longevity. Although many details of the hormonal systems controlling developmental arrest are well-known, we know little about the interactions between metabolic events and the hormones controlling the arrested state. Here, we show that diapause is regulated by an interplay between blood-borne metabolites and regulatory centers within the brain. Gene expression in the fat body, the insect equivalent of the liver, is strongly suppressed during diapause, resulting in low levels of tricarboxylic acid (TCA) intermediates circulating within the blood, and at diapause termination, the fat body becomes activated, releasing an abundance of TCA intermediates that act on the brain to stimulate synthesis of regulatory peptides that prompt production of the insect growth hormone ecdysone. This model is supported by our success in breaking diapause by injecting a mixture of TCA intermediates and upstream metabolites. The results underscore the importance of cross-talk between the brain and fat body as a regulator of diapause and suggest that the TCA cycle may be a checkpoint for regulating different forms of animal dormancy.

Integrated proteomic and metabolomic analysis of larval brain associated with diapause induction and preparation in the cotton bollworm, Helicoverpa armigera.

Diapause is a developmental arrest that allows an organism to survive unfavorable environmental conditions and is induced by environmental signals at a certain sensitive developmental stage. In Helicoverpa armigera, the larval brain receives the environmental signals for diapause induction and then regulates diapause entry at the pupal stage. Here, combined proteomic and metabolomic differential display analysis was performed on the H. armigera larval brain. Using two-dimensional electrophoresis, it was found that 22 proteins were increased and 27 proteins were decreased in the diapause-destined larval brain, 37 of which were successfully identified by MALDI-TOF/TOF mass spectrometry. RT-PCR and Western blot analyses showed that the expression levels of the differentially expressed proteins were consistent with the 2-DE results. Furthermore, a total of 49 metabolites were identified in the larval brain by GC-MS analysis, including 4 metabolites at high concentrations and 14 metabolites at low concentrations. The results gave us a clue to understand the governing molecular events of the prediapause phase. Those differences that exist in the induction phase of diapause-destined individuals are probably relevant to a special memory mechanism for photoperiodic information storage, and those differences that exist in the preparation phase are likely to regulate accumulation of specific energy reserves in diapause-destined individuals.

Proteomic and phosphoproteomic analysis at diapause initiation in the cotton bollworm, Helicoverpa armigera.

Diapause is a period of developmental arrest that allows a species to adapt to unfavorable conditions. Many insect species reduce metabolic activity and then enter diapause at a certain stage in their life cycles. The cotton bollworm, Helicoverpa armigera, will be destined for pupal diapause when larvae are reared under short daylengths and low temperature. The brain is an important organ for diapause decision, and some signaling molecules from the brain of diapause-destined individuals are released into the hemolymph to regulate diapause. In this study, we performed 2-D gel-based comparative proteomic and phosphoproteomic analyses to search for differentially expressed proteins between nondiapause- and diapause-destined pupal brains. A total of 79 proteins and 23 phosphoproteins showed significant differences between these two groups, and 41 proteins and 10 phosphoproteins were identified by MALDI-TOF/TOF MS. Further, gene expression patterns in diapause- and nondiapause-destined pupal brains were confirmed by RT-PCR or Western blot analysis. These differentially expressed proteins act in the metabolic change, stress response, and signal transduction pathways at early pupal stage for diapause initiation. Thus, these identified proteins may depress metabolism in diapause-destined pupae to lead the insect to enter developmental arrest.