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Autophagy, an evolutionarily conserved cytoplasmic degradation system, has been implicated as a convergent mechanism in various longevity pathways. Autophagic activity decreases with age in several organisms, but the underlying mechanism is unclear. Here, we show that the expression of Rubicon, a negative regulator of autophagy, increases in aged worm, fly and mouse tissues at transcript and/or protein levels, suggesting that an age-dependent increase in Rubicon impairs autophagy over time, and thereby curtails animal healthspan. Consistent with this idea, knockdown of Rubicon extends worm and fly lifespan and ameliorates several age-associated phenotypes. Tissue-specific experiments reveal that Rubicon knockdown in neurons has the greatest effect on lifespan. Rubicon knockout mice exhibits reductions in interstitial fibrosis in kidney and reduced α-synuclein accumulation in the brain. Rubicon is suppressed in several long-lived worms and calorie restricted mice. Taken together, our results suggest that suppression of autophagic activity by Rubicon is one of signatures of aging. Autophagic activity decreases with age via unknown mechanisms. Here the authors show that expression of the negative autophagy regulator Rubicon increases with age, that its genetic ablation improves lifespan and ameliorates a number of age-associated phenotypes in invertebrates and in mouse models.

The systemic decline in autophagic activity with age impairs homeostasis in several tissues, leading to age-related diseases. A mechanistic understanding of adipocyte dysfunction with age could help to prevent age-related metabolic disorders, but the role of autophagy in aged adipocytes remains unclear. Here we show that, in contrast to other tissues, aged adipocytes upregulate autophagy due to a decline in the levels of Rubicon, a negative regulator of autophagy. Rubicon knockout in adipocytes causes fat atrophy and hepatic lipid accumulation due to reductions in the expression of adipogenic genes, which can be recovered by activation of PPARγ. SRC-1 and TIF2, coactivators of PPARγ, are degraded by autophagy in a manner that depends on their binding to GABARAP family proteins, and are significantly downregulated in Rubicon -ablated or aged adipocytes. Hence, we propose that age-dependent decline in adipose Rubicon exacerbates metabolic disorders by promoting excess autophagic degradation of SRC-1 and TIF2. Autophagic activity declines with age in several tissues and is linked to aging-associated functional decline and pathologies. Here the authors show that Rubicon, a negative regulator of autophagy, decreases in adipocytes with age, and its loss leads to adipocyte dysfunction via excess autophagic degradation of SRC-1 and TIF2.

Compelling evidence shows that brown and beige adipose tissue are protective against metabolic diseases 1 , 2 . PR domain-containing 16 (PRDM16) is a dominant activator of the biogenesis of beige adipocytes by forming a complex with transcriptional and epigenetic factors and is therefore an attractive target for improving metabolic health 3 – 8 . However, a lack of knowledge surrounding the regulation of PRDM16 protein expression hampered us from selectively targeting this transcriptional pathway. Here we identify CUL2–APPBP2 as the ubiquitin E3 ligase that determines PRDM16 protein stability by catalysing its polyubiquitination. Inhibition of CUL2–APPBP2 sufficiently extended the half-life of PRDM16 protein and promoted beige adipocyte biogenesis. By contrast, elevated CUL2–APPBP2 expression was found in aged adipose tissues and repressed adipocyte thermogenesis by degrading PRDM16 protein. Importantly, extended PRDM16 protein stability by adipocyte-specific deletion of CUL2–APPBP2 counteracted diet-induced obesity, glucose intolerance, insulin resistance and dyslipidaemia in mice. These results offer a cell-autonomous route to selectively activate the PRDM16 pathway in adipose tissues. The ubiquitin E3 ligase CUL2–APPBP2 determines PRDM16 protein stability by catalysing PRDM16 polyubiquitination in beige fat.

Purpose Since the 1980s, the detection sensitivity of mass spectrometers has increased by improving the analysis of drugs in hair. Accordingly, the number of hair strands required for the analysis has decreased. The length of the hair segment used in the analysis has also shortened. In 2016, micro-segmental hair analysis (MSA), which cuts a single hair strand at a 0.4-mm interval corresponding to a hair growth length of approximately one day, was developed. The advantage of MSA is that the analytical results provide powerful evidence of drug use in the investigation of drug-related crimes and detailed information about the mechanism of drug uptake into hair. This review article focuses on the MSA technique and its applications in forensic toxicology. Methods Multiple databases, such as SciFinder, PubMed, and Google, were utilized to collect relevant reports referring to MSA and drug analysis in hair. The experiences of our research group on the MSA were also included in this review. Results The analytical results provide a detailed drug distribution profile in a hair strand, which is useful for examining the mechanism of drug uptake into hair in detail. Additionally, the analytical method has been used for various scenarios in forensic toxicology, such as the estimation of days of drug consumption and death. Conclusions The detailed procedures are summarized so that beginners can use the analytical method in their laboratories. Moreover, some application examples are presented, and the limitations of the current analytical method and future perspectives are described.

Purpose Since the mid-2010s, lysergic acid diethylamide (LSD) analogs made for substance abuse have periodically emerged. In this case, three pieces of blotter paper labeled “1D-LSD” and presumably impregnated with this LSD analog, were seized. Several websites indicate that 1D-LSD is 1-(1,2-dimethylcyclobutane-1-carbonyl)-LSD. Because this analog is much more difficult to synthesize than previously reported LSD analogs, we doubted that the blotter paper contained 1D-LSD. Herein, we determined the structure of the absorbed compound. Methods One of the seized specimens was extracted and analyzed using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS), high-resolution mass spectrometry (HRMS), and nuclear magnetic resonance (NMR) spectroscopy to estimate the extract components. The estimated compound was then synthesized, yielding an authentic standard. The contents of the seized specimens were identified using authentic standard analysis with GC/MS, LC/MS, and NMR spectroscopy. Results Instrumental analyses confirmed the active compound to be 1-(thiophene-2-carbonyl)-LSD, which was inconsistent with the labeling on drug-infused blotter paper. Conclusion As in this case, similar blotter paper analyses should consider the possibility of a mismatch between the label and ingredient. To the authors’ knowledge, this is the first case report in which 1-(thiophene-2-carbonyl)-LSD was seized and the first seizure of an LSD analog in which an aromatic carboxylic acid had been condensed to LSD. This type of lysergamide may become prevalent in the near future, and we should remain alert for newly appearing lysergamides.

Purpose Micro-segmental hair analysis (MSA), which enables detailed measurement of the distribution of drugs in a single hair strand, is useful for examining the day of death and drug use history of a person. However, corpses are often found in severe environments, such as soil and freezers, which affect the drug contents in hair. Therefore, we examined the effects of temperature, humidity, light, and soil on drug stability in hair as a preliminary study to estimate personal profiles using MSA of corpse hair. Methods Four hay-fever medicines (fexofenadine, epinastine, cetirizine, and desloratadine) were used as model drugs to evaluate drug stability in hair. Reference hair strands consistently containing the four medicines along the hair shaft were collected from patients with hay-fever who ingested the medicines daily for 4 months. The hair strands were placed in chambers with controlled temperatures (− 30 to 60 °C) and relative humidities (ca. 18 % and > 90 %), exposed to light (sunlight and artificial lights) or buried in soil (natural soil and compost). Results Sunlight and soil greatly decomposed the hair surfaces and decreased the drug contents in hair (up to 37 %). However, all analytes were successfully detected along the hair shaft, reflecting the intake history, even when the hair was exposed to sunlight for 2 weeks and buried in the soil for 2 months. Conclusions Although the exposure to sunlight and storage in soil for long times made drug-distribution analysis difficult, MSA could be applied even to hair strands collected from corpses left in severe environments.

In postmortem examinations, the drug analysis of hair is effective for revealing drug-use history. Additionally, a method to estimate the day of death using hair was previously developed by analyzing a single hair strand segmented at 0.4-mm intervals (micro-segmental hair analysis). However, for drowned bodies, drugs in the hair may be washed out due to soaking in water for extended periods. To evaluate the possibility of measuring drug distribution in the hair of drowned bodies, drug stability in hair samples soaked in various aqueous solutions was examined. First, reference hair strands of drug users containing specific drugs consistently along the hair shaft were prepared. The participants ingested 4 hay-fever medicines (fexofenadine, epinastine, cetirizine, and loratadine) every day for approximately 4 months before hair collection. Each reference strand was divided into regions, and each region was soaked in different solutions containing various solutes for extended periods up to approximately 2 months. In solutions without divalent ions (Ca2+ and Mg2+), the drug content in the hair decreased up to approximately 5 % with increasing salt concentration and soaking time. However, the decreased drug content was negligible in solutions containing divalent ions, implying that the divalent ions prevented drugs contained in hair from washing out. As natural river and sea waters contain divalent ions, the drugs in hair were hardly washed out even when the hair was soaked for 2 months. Thus, it was concluded that drug-distribution measurements using micro-segmental analysis can also be applied to the hairs of drowned bodies.HighlightsDrug stabilities in hair soaked in various aqueous solutions were examined.Drug concentrations in hair decreased depending on solutes and soaking time.Divalent ions, such as Ca2+ and Mg2+, prevent drugs from being washed out.River and sea waters hardly affected the drug contents in hair.Micro-segmental analysis is effective even for hair of drowned bodies.

Autophagy degrades unnecessary proteins or damaged organelles to maintain cellular function. Therefore, autophagy has a preventive role against various diseases including hepatic disorders, neurodegenerative diseases, and cancer. Although autophagy in germ cells or Sertoli cells is known to be required for spermatogenesis and male fertility, it remains poorly understood how autophagy participates in spermatogenesis. We found that systemic knockout mice of Rubicon , a negative regulator of autophagy, exhibited a substantial reduction in testicular weight, spermatogenesis, and male fertility, associated with upregulation of autophagy. Rubicon -null mice also had lower levels of mRNAs of Sertoli cell–related genes in testis. Importantly, Rubicon knockout in Sertoli cells, but not in germ cells, caused a defect in spermatogenesis and germline stem cell maintenance in mice, indicating a critical role of Rubicon in Sertoli cells. In mechanistic terms, genetic loss of Rubicon promoted autophagic degradation of GATA4, a transcription factor that is essential for Sertoli cell function. Furthermore, androgen antagonists caused a significant decrease in the levels of Rubicon and GATA4 in testis, accompanied by elevated autophagy. Collectively, we propose that Rubicon promotes Sertoli cell function by preventing autophagic degradation of GATA4, and that this mechanism could be regulated by androgens.

Purpose Cannabis is regulated in many countries, and cannabis products are diversifying, which can hinder identification. Here, we report the seizure of a powder sample with a cannabis-like odor in a spice bottle labeled “nutmeg” and identification of the sample by chemical testing and cannabis DNA testing. Methods The sample was observed under a microscope, extracted with methanol, and analyzed by gas chromatography–mass spectrometry (GC–MS). The chemical profile of the seized powder was compared with that of nutmeg samples. Gas chromatography–flame ionization detection was used to estimate the total Δ 9 -tetrahydrocannabinol (Δ 9 -THC) concentration in the sample. A commercially available cannabis DNA testing kit was used to confirm the presence of cannabis plant DNA in the seized sample. Results The characteristics of cannabis in the seized powder were difficult to determine through microscopic observation alone. GC–MS analysis identified β-caryophyllene (an aromatic component of cannabis) and five cannabinoids unique to cannabis, including Δ 9 -THC. No common compounds were identified in the seized powder or nutmeg samples. The total Δ 9 -THC concentration in the sample was very high (approximately 47% by weight). Cannabis DNA testing confirmed that the seized powder contained cannabis. Conclusions The seized powder was found to be a processed product made from a finely pulverized resin-like cannabis concentrate. Our results indicate that combined chemical and DNA analysis should help identify cannabis-related samples in various forms.

[Display omitted] •Chiral SFC method for ephedrine and its stereoisomers was developed.•The method was suitable for crystalline methamphetamine including ephedrines.•Fast analysis (11min) with baseline separation was achieved.•Unsupervised multivariate analysis objectively classified seized methamphetamine. In forensic science, drug profiling is clarifying the identity of seized drugs of abuse based on their physicochemical properties and it is applied to various drugs, including crystalline methamphetamine. Impurity analysis is particularly important in drug profiling because the impurities can be a measure for speculating how the methamphetamine was synthesized in the clandestine laboratories. However, developments in scientific techniques have allowed the synthesis of high-purity, homogeneous crystalline methamphetamine, and thus new techniques to characterize methamphetamine are needed. In this study, we developed a method for chiral separation of ephedrine and its stereoisomers by supercritical fluid chromatography. Ephedrine is a common starting compound for methamphetamine synthesis. It possesses two chiral center carbon atoms and has four stereoisomers, (1R,2S)–(−)–ephedrine, (1S,2R)–(+)–ephedrine, (1S,2S)–(+)–pseudoephedrine, and (1R,2R)–(−)–pseudoephedrine. Because the stereostructure of ephedrines contained in methamphetamine seizure reflects the starting materials and the synthetic pathways, the stereoisomer ratio will provide additional information for drug profiling. The developed method achieved rapid separation of four isomers in about 11min with low limits of detection (1pg on column). Due to a switching valve connecting a chromatograph to a mass spectrometer, dense methamphetamine sample solutions containing small amount of ephedrines could be analyzed directly with a simple pre-treatment. Using multivariate analysis, 44 real samples were objectively grouped based on stereoisomer ratio. Our method is expected to improve the profiling of crystalline methamphetamine.