Comparing the optimized performance metrics in this work with the estimated metrics at prescribed aspect ratio by IRDS roadmap, delay, noise delay product (NDP), power delay product (PDP), 3 doped MLGNR interconnect, respectively at 7 nm node. Increase in contact resistance leads to significant decrease in performance and increase in optimized aspect ratio of local Fecl3 doped MLGNR interconnect. Scaling down from 10 to 7 nm node results in increase of optimized aspect ratio in all levels of interconnects. Even though the performance of MLGNR degrades with scaling down but when compared to copper, the performance improves with technology scaling. Finally, this study provides circuit designers a detailed guideline for selecting an optimized aspect ratio for achieving better performance, power efficiency and reliability in doped MLGNR interconnects.In this work, aspect ratio of various intercalation doped MLGNR interconnects are optimized using a numerical approach to achieve improved performance and reliability. A numerical optimization method is presented to estimate optimized aspect ratio considering combined effects of performance, noise and reliability metrics for any arbitrary nano interconnect system. This approach is cost effective and will be extremely useful to industry for selection of aspect ratio of interconnects as it is a non-SPICE method and reduces fabrication iterations for achieving desired performance and reliability. Our numerical method suggests that by minimizing the figure of merit (i.e. Noise Delay Power Product / Breakdown Power  Their current carrying capacity has reduced resulting in poor IC performance3. Other alternative materials such as cobalt and ruthenium were proposed due to their higher EM reliability even though they have higher resistivity than copper5. Graphene nanoribbons (GNRs) have proved to have high conductivity, great electromigration reliability and superior transport properties making them more suitable as an alternative interconnect material than copper7.Copper interconnects have reached their performance limits due to high resistivity, grain boundary scattering effects and electromigration issuesMLGNRs) are preferred over single layer graphene because of their lower resistivity. However, due to inter-sheet electron hopping, it suffers from decrease in conductivity. To solve this issue, intercalation doped MLGNRs were first proposed by Xu et al.6. Sahoo et al. analyzed the crosstalk and reliability effects in MLGNR interconnects in8. Liao et al. in9 investigated high field transport in GNRs up to breakdown. In10, Jiang et al. proposed Fecl3 doped MLGNR for better performance and reliability to challenge copper as an interconnect. They showed that Fecl3 doping is very efficient for diffusion in scaled MLGNRs, is stable at room temperature and shows excellent current carrying capacity >200 MA/Cm2. But Jiang et al. did not consider the effects of via and aggressor nets which is a pressing concern in IC design. Agashiwal et al. in11 engineered a CMOS-compatible solid-phase growth technique to yield large-area multilayer graphene on dielectric (SiO) and metal (Cu) substrates and subsequently demonstrating multi-level interconnects with metal vias. Also, Fischer and his team demonstrated an ingenious method in12 to produce metallic GNRs based on the atomically precise bottom-up synthesis. These fabricational advancements have strengthened the claim of graphene nanoribbons as an effective alternative to commercial metals. Wang et al. in13 also advertised graphene nanoribbon as a promising candidate for quantum electronic applications praising its high mobility and current-carrying capability. Nishad et al. in14 optimized thickness of Lithium and AsF5 intercalated Top-Contact MLGNR (TC-MLGNR) interconnects and compared with copper and pristine interconnects. Both Jiang et al. and Nishad et al. have not shown any dimensional optimization for improved performance and reliability issues which is a concerning factor to consider for commercialization of MLGNR interconnects in near-future VLSI circuits.Multilayer Graphene Nanoribbons (AR) by minimizing delay and FOM  model as shown in Fig. The structural representation of MLGNR interconnect is shown in Fig. entclass1pt{minimaElectrical equivalent model of MLGNR interconnect system is shown in Fig. 10. 8,8.The electrical circuit of MLGNR interconnect model consists of lumped resistance, , w is the width of the MLGNR and 10, is a function of specularity parameter where, 10. P represents specularity index which is a measure of specularity of GNR edges. 6.The per unit length (p.u.l) distributed resistance of MLGNR as shown in Fig. 15.15.The p.u.l capacitance , intermediate level  and global level  interconnects are considered as 500 1 for 7 and 10 nm nodes. It is taken as 1.5 times the 1 to reduce delay and power consumption24.Aspect ratio is varied from 0.4 to 3.2 for intermediate level and 0.4 to 3.4 for global level interconnects. Width of local level  interconnects is considered as AR in AsF5, Fecl3, Lithium doped MLGNR, neutral MLGNR, cobalt and copper interconnects. Intercalated MLGNR experiences very low delay when compared to neutral MLGNR and conventional metals irrespective of interconnect length for smaller AR. The AR for intermediate level, Lithium doped MLGNR optimizes at 1.4, 1.4 and 1.2 for intermediate level and at 1.0, 1.0 and 0.8 for global level interconnects. Delay in neutral MLGNR and copper interconnect saturates for higher AR. Also it can be observed that copper and cobalt interconnects outperform all the doped MLGNRs after reaching an AR value of 1.4 and 1.8, respectively for intermediate level. Also, copper performs better than Fecl3 doped MLGNR for an AR value of 2.0 or higher for global level interconnects. AR of global level interconnects optimizes at larger value as compared to intermediate level because of higher resistance as implied by Fig. Figure AR at minimum NDP is shown in Fig. 5, Fecl3, Lithium doped MLGNR is optimized at 0.8, 0.8 and 0.6 for intermediate level and at 0.6 for global level interconnects. Intermediate level copper and cobalt cut all the doped MLGNR at AR value of 1.6 and 2, respectively. Hence, intermediate level MLGNR interconnects are more prone to noise for higher AR as compared with copper and cobalt as shown in Fig. Optimized AR increases. So, interconnects with large AR consume more power as inferred from Fig. Figure PDP is obtained at AR value of 1.0, 1.0 and 0.6 for intermediate level AsF5, Fecl3, Lithium doped MLGNR interconnects but copper and cobalt beats them at even lower AR as shown in Fig. The least AR. Breakdown power is a function of thermal healing length for intermediate level when Figure AR by minimizing the metric 5, Fecl3, Lithium doped MLGNR interconnects are obtained at 1.0, 1.0 and 0.6. In case of cobalt and copper interconnects, the metric decreases with increasing AR.The optimization of AR by minimizing this FOM for intermediate level interconnects is shown in Fig. 5, Fecl3, Lithium doped MLGNR interconnects at 0.6, 0.6 and 0.5, respectively. The FOM first decreases and then saturates with increasing AR for neutral MLGNR. It keeps decreasing with increasing AR for cobalt and copper interconnects. Doped MLGNRs are far better candidates considering an overall performance and reliability aspect specially at lower AR. Although Lithium dopant gives the highest advantage but Fecl3 is explored more in experimental literature’s. Figure 5, Fecl3, Lithium doped MLGNR interconnects are minimum at AR of 0.6, 0.6 and 0.5, respectively. And for local level it is 1.0, 1.0 and 0.6, respectively. Doped MLGNR interconnect outperforms all other candidates by having minimum FOM at all metal levels as shown in Figs. We have considered the metric 10. The comparison is shown in Table 10 for comparison.For calibration of the electrical model, we have estimated delay, energy and power consumption in k refer “” sectionentclass1pt{minimaAR estimated using our numerical model is compared with that obtained from existing works14. All the dimensional parameters are considered to be same as in respective papers for a fair comparison.Also to validate our numerical model, comparison is shown in Table NDP and PDP are calculated and compared considering the optimized AR obtained in our work and the AR prescribed by IRDS 2018 roadmap1. It can be observed that there is an insignificant advantage in intermediate level interconnect performance but when it comes to global level, a substantial improvement is registered. This improvement in global metal line becomes more valuable as the effect of via is more dominant in global metal path. Our results show improved performance in Fecl3 doped MLGNR interconnect for optimized value of AR as compared to IRDS roadmap 2015 prescribed AR. Also the improvement in FOM metric is significant indicating lower AR should be adopted considering overall performance and reliability.Table AR and FOM. Here we have varied the contact resistance from 5 25. As we can see, intermediate and global lines are not affected by it. But when it comes to local lines, increase in contact resistance leads to significant decrease in performance and increase in optimized AR. With a 75% increase in contact resistance, 3 doped MLGNR interconnect, respectively is witnessed. Optimized AR also experiences an increase of AR can be increased (which will lead to increase in number of layers and thus decrease in contact resistance). So the performance of MLGNR interconnects will not improve beyond a certain limit.Table AR value of 10 3 doped MLGNR interconnect is shown in Fig. AR value along with degradation in performance and reliability for intermediate level interconnects following IRDS trends. When compared to copper with cobalt liner (considering IRDS suggested dimensions), Improvement in FOM of Fecl3 doped MLGNR interconnect  is nm and for 10 nm, it is 3 doped MLGNR interconnect considering the FOM. Scaling down from 10 nm to 7 nm node leads to degradation in FOM in Fecl3 doped MLGNR interconnect by 2.6% and 22.6% in intermediate and global level, respectively even though we increase the AR from 0.4 to 0.6. But in case of local level, FOM is improved while scaling down by 8% if we increase the AR from 0.8 to 1.0. It is evident that with scaling there is a need of increase in AR in order to improve performance and reliability. The optimized (recommended) aspect ratio in this paper is less than 1.0. Although scaling down from 10 to 7 nm leads to an increase in optimized AR, but still it is lower as compared to IRDS suggestions (AR > 2). Jiang et al. have fabricated Fecl3 doped MLGNR interconnect with aspect ratio of 0.4 and 0.610, which strengthens our claim from manufacturing point of view.The Impact of scaling (from 10 to 7 nm node) on optimized mentclasspt{minimaAR) optimization serves to be an effective dimensional optimization technique for achieving superior performance and reliability metrics. Here, dependence of the RLC parameters on AR is discussed in order to establish relation between AR of interconnect and its delay, noise induced effects, power consumption and breakdown power.This proposed methodology provides a detailed numerical dimensional optimization procedure and is applicable to any generic nano-interconnect system. This numerical methodology can avoid costly simulators set-ups and expensive fabrication procedures for providing the dimensional design guidelines to achieve such improvement in performance. Aspect ratio ,,\documenRC product. With increasing AR, resistance decreases as shown in Fig. AR is obtained for minimum delay point. The transfer function for the crosstalk delay or noise evaluation in the victim net will be denoted as H(s). The second-order 16:16. They can be defined as a function of AR as follows,16,AR to zero, we can obtain the optimized AR at which delay is minimum:AR value at which delay is minimum. The initial guess value of AR was taken as 1 and the equation converged in less than 8 iterations giving optimized AR value of 1.418, 1.3329 and 1.213 for intermediate level AsF5, FeCl3 and Lithium doped MLGNR interconnects, respectively. Global level AsF5, FeCl3, and Lithium doped MLGNR interconnects optimized at AR of 1.06, 0.87 and 0.76, respectively.Propagation delay in an interconnect is basically a function of its AR at minimum FOM. We define Figure of Merit (FOM) as:18,19,20. Switching power dominates the equation thus is considered for further calculation for simplicity19.20. AR can be defined as:21,21. Breakdown power as a function of AR can be defined as:AR to zero, we can obtain the optimized AR at which FOM is minimum:AR value which minimizes the FOM. Here the initial guess value of AR was taken as 0.5 and the equation converged in 11 iterations giving optimized AR value of 0.633, 0.61 and 0.583 for intermediate level AsF5, FeCl3, and Lithium doped MLGNR interconnects, respectively. Global level AsF5, FeCl3, and Lithium doped MLGNR interconnects optimized at AR of 0.585, 0.579 and 0.481, respectively. And local level AsF5, FeCl3, and Lithium doped MLGNR interconnects optimized at AR of 1.08, 0.987 and 0.623, respectively.Similar approach is adopted to obtain the optimized AR of interconnects as it is a non-SPICE method. Our approach provides a detailed guideline for the Aspect ratio optimization and reduces fabricational cost to achieve high performance and reliability MLGNR interconnects by reducing iterations during fabrication process for achieving desired performance. The optimized AR of AsF5, FeCl3, Lithium doped MLGNR interconnects by minimizing delay is obtained at 1.4, 1.4 and 1.2 for intermediate level and 1.0, 1.0 and 0.8 for global level interconnects, respectively. Intermediate level, AsF5, FeCl3, Lithium doped MLGNR interconnects have an optimized AR of 1.0, 1.0 and 0.6 and global levels have an optimized AR of 0.8 at which minimum PDP is registered. 5, FeCl3, Lithium doped MLGNR interconnects, respectively. Increase in contact resistance leads to significant decrease in performance and increase in optimized AR of local FeCl3 doped MLGNR interconnect. As we scale down, the optimized AR increases with decrease in performance and reliability for intermediate and global levels. But while scaling down, increase in optimized AR leads to better FOM in local level doped MLGNR interconnect. When compared to copper (considering IRDS suggested dimensions), Improvement in FOM of FeCl3 doped MLGNR interconnect  is 3 doped MLGNR interconnect is improved by NDP by 44%, PDP by NDP, PDP, This work focuses on numerically determining optimum aspect ratio in order to improve performance, reliability and minimize noise effects and power consumption. This approach will be extremely useful to industry for selection of


NPR3NPR4 proteasome pathway. However, the functional significance of NPR1 degradation by other E3 ligases in response to ABA remains unclear. Here, we report that NPR1 is induced transcriptionally by ABA and that npr1-1 mutation results in ABA insensitivity during seed germination and seedling growth. Mutants lacking NPR1 downregulate the expression of ABA-responsive transcription factors ABA INSENSITIVE4 (ABI4) and ABA INSENSITIVE5 (ABI5), and that of their downstream targets EM6, RAB18, RD26, and RD29B. The npr1-1 mutation also affects the transcriptional activity of WRKY18, which activates WRKY60 in the presence of ABA. Furthermore, NPR1 directly interacts with and is degraded by HOS15, a substrate receptor for the DDB1-CUL4 ubiquitin E3 ligase complex. Collectively, our findings demonstrate that NPR1 acts as a positive regulator of ABA-responsive genes, whereas HOS15 promotes NPR1 degradation in a proteasome-dependent manner.The plant hormone, abscisic acid (ABA), is not only important for promoting abiotic stress responses but also plays a versatile and crucial role in plant immunity. The pathogen infection-induced dynamic accumulation of ABA mediates the degradation of non-expresser of PR genes 1 (NPR1) through the CUL3 RD29A and RD29B, under stress conditions [RD22 expression in Arabidopsis [Plants are constantly exposed to various biotic and abiotic stresses and have evolved sophisticated mechanisms to cope with harsh environmental conditions . Understnditions ,15,16. Abidopsis ,17. Sevebidopsis .Arabidopsis thaliana, cellular activities of NPR1 are regulated by intricate mechanisms. A defense-associated increase in SA levels induces NPR1 transcriptional activation [NPR3NPR4-based E3-ligase complex, and subsequent protein turnover [The transcriptional co-activator NPR1 regulates local and systemic acquired resistance (SAR) in response to SA. In tivation . Next, Stivation . Moreovetivation . Furthertivation ,23,24. Fturnover . In lineturnover . In prevturnover .Recent studies indicate significant crosstalk between SA and ABA during abiotic stress responses ,27. DuriNPR1 itself is under the transcriptional control of WKRY genes [WRKY genes have been identified as direct NPR1 targets during biotic and abiotic stress responses [WRKY18 overexpression enhances PR expression and resistance to invading pathogens [NPR1 reportedly regulates the expression of pathogenesis-related (PR) genes by binding to TGA transcription factors ,35. MoreRY genes ,38. Eighesponses . Among pesponses . WRKY18 athogens . Interesathogens . HoweverWRKY18 transcriptional activation. We observed that loss-of-function npr1-1 mutant seedlings were ABA insensitive during germination and cotyledon greening. Furthermore, compared to wild-type (Col-0), ABA-responsive genes were downregulated in npr1-1 plants. More interestingly, HOS15, a substrate receptor for the DDB1-CUL4 E3 ligase complex, interacted with and regulated NPR1 stability in an ABA-dependent manner. Taken together, our findings reveal a molecular mechanism underlying the posttranslational modification of NPR1 through HOS15 and highlight the critical role of NPR1 in regulating ABA signaling.Here, we report that NPR1 positively regulates ABA signaling via Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild-type (WT). Seeds of hos15-2, npr1-1 mutants, 35S::NPR1-GFP/npr1-1/hos15-2 and 3535S::NPR1-GFP/npr1-1 were reported by Shen et al. (2020) [abi2-2 (SALK_015166C) were obtained from ABRC. Seeds were surface-sterilized and germinated on 1/2 MS medium supplemented with 0.25% phytagel  or 1.2% agar  and 2% sucrose. Plants were grown at 23 °C under long-day conditions  in a controlled culture room.In the present study, . (2020) . Seeds oTM RT-PCR System . We performed PCR amplification using e-Taq DNA polymerase . The primers used in the RT-PCR or real-time PCR experiments are listed in We used 2 µg of total RNA extracted from seedlings using the RNeasy Plant Mini Kit , treated with DNase , for the synthesis of the first-strand cDNA using the ThermoscriptArabidopsis plants, either treated or untreated with ABA or MG132, for the Western blot assays. Total proteins were extracted and immunoblot analysis was carried out using α-GFP and α-HOS15, for GFP-tagged line and HOS15 detection, respectively. Each immunoblot was incubated with the appropriate primary antibody  for 2 h at room temperature or overnight at 4 °C. The membranes were developed using a peroxidase-conjugated secondary antibody for α-rabbit antibody  at a dilution of 1:4000. We carried out the co-immunoprecipitation studies in the tobacco transient assay by co-infiltration of tobacco (Nicotiana benthamiana) leaves with HOS15-GFP and NPR1-HA. The protein samples were extracted three days after the infiltration. For the co-immunoprecipitation studies, we extracted the total protein samples from N. benthamiana leaves and performed the pull-down assay using α-GFP, followed by immunoblotting with α-HA. Each immunoblot was incubated with the appropriate primary antibody, α-HA or α-GFP antibody, diluted at 1:2000 or 1:2000, for 2 h at room temperature or overnight at 4 °C, respectively. The membranes were developed using α-rat IgG  and α-rabbit  peroxidase-conjugated secondary antibodies, diluted at 1:1000 and 1:2000, respectively.We used 10-day-old NPR1 and HOS15 coding sequences were cloned into the gateway entry vector, TM/ZeopDONR and then sub-cloned into the destination vectors, pDEST22 or pDEST32, containing activation and binding domains, respectively. The primers used for cloning are listed in The full-length npr1-1 was isolated as a non-expresser of PR gene 1 in SA signaling, lacking the expression of PR1, PR5, and BGL2 genes in response to SA, 2,6-dichloroisonicotinic acid (INA), and avirulent bacterial pathogens [npr1-1, and 35S::NPR1-GFP/npr1-1 plants, along with the ABA-hypersensitive abi2-2 mutant as experimental control for the ABA response, were grown in the presence of exogenous ABA for one week. The npr1-1 mutant displayed an ABA-insensitive phenotype during germination  control  for 4 h to inhibit de novo protein synthesis and investigate NPR1 protein stability. When treated with CHX, the NPR1 abundance declined very rapidly in 35S::NPRI-GFP/npr1-1 plants  and chimeric pathogen-responsive reporter genes. Earlier reports have also shown that the npr1-1 point mutation results in SA-, INA- and pathogen-insensitive phenotypes during SAR induction, indicating that these inducers potentially share common signaling pathways [npr1-1 mutant showed an ABA-insensitive phenotype during early germination and post-germination seedling growth  suppressed the ABA sensitivity of hos15-2 in the double mutant (The  pathway . Intereshenotype , indicathenotype . Since nt plants . Interese mutant , indicatWRKY gene promoters [PR gene and confers resistance to invading pathogens [WRKY18 was downregulated in npr1-1, whereas that of WRKY60 was not induced at all in npr1-1 in response to ABA (NPR1 can directly associate with various romoters . Among tromoters . WRKY18 athogens . Interesathogens . The expe to ABA . These rIn addition to dehydration responses, plants also close their stomatal pores as part of their innate immunological response to keep bacteria out ,29. The WRKY18, which regulates WRKY60 and the ABA response (ABA is a major phytohormone that plays a crucial role in biotic and abiotic stress responses ,4,54. Thresponse . However


Actaea racemosa ‘AR’) are being sought to overcome the shortcomings of conventional hormonal and surgical therapies developed for the treatment of polycystic ovary syndrome (PCOS). However, AR-induced hepatotoxicity necessitates a cautionary warning to be labeled on its products as recommended by the United States Pharmacopeia, where four out of seven hepatotoxic cases in Sweden were possibly associated with black cohosh products.Complementary remedies such as the Chinese herb ‘Sheng Ma’  gene were evaluated. Additionally, holistic profiling of the AR arsenal of secondary metabolites was performed using ultra-high-performance liquid chromatography (UHPLC) coupled with quadrupole high-resolution time of flight mass spectrometry (QTOF-MS).Beneficial effects were exerted by AR in PCOS rats as antioxidant status, hormonal profile, lipid profile, glucose level, liver functions, and the induced Ki-67 expression in the granulosa, theca cell layers and interstitial stromal cells were all improved. Notably, the combination of AR with vitamin C was not only more effective in reversing the dysregulated levels of testosterone, luteinizing hormone, and mRNA level of Cyp19α1 gene in the PCOS rat, but also safer. The combination regulated both ovarian and hepatic malondialdehyde (MDA) and glutathione (GSH) levels with histological improvement observed in the liver and ovaries. In addition, the untargeted metabolomic profiling enabled the identification of 61 metabolites allocated in five major chemical classes.This study demonstrated the benefit of the combinatorial effects of AR and vitamin C in mitigating the reproductive and metabolic disorders associated with PCOS with the elimination of AR hepatotoxic risk.The online version contains supplementary material available at 10.1186/s13020-021-00444-z. Polycystic ovary syndrome (PCOS) is a complex metabolic–endocrine disorder  that affHumulus lupulus), female ginseng (Angelica sinensis), ginseng (Panax ginseng), liquorice (Glycyrrhiza glabra), black cohosh (Actaea racemosa) and kelp (Nereocystis luetkeana) were reported against PCOS − of 556.2771 and 554.261 m/z, respectively  and liver enzymes [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] were measured using commercial kits . The very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) concentration was calculated as follows:Ovaries and liver from different groups were collected and fixed in 10% neutral-buffered formalin and processed to obtain 3–4 µm paraffin-embedded sections. The sections were stained with hematoxylin and eosin (H&E) and the morphometric analysis of the ovaries was performed , 51. TheThe tissue sections were deparaffinized, rehydrated and pre-treated with 10 mM citrate buffer for antigenic retrieval. Sections were incubated overnight at 4 °C in a humidified chamber with the following primary antibody: rabbit monoclonal anti-Ki 67 antibody  in 1:25 dilution. The tissue sections were incubated with a biotinylated goat anti-rabbit and mouse antibody , streptavidin peroxidase  and 3,3′-diaminobenzidine tetrahydrochloride . The slides were counterstained with Mayer’s hematoxylin then dehydrated and mounted. The primary antibody was replaced by PBS for negative controls. The stained sections were analyzed by Leica Qwin 500 Image Analyzer . In each field, the immunolabeled dark brown areas were recorded. The percentage of positively stained area in the granulosa cell layer, theca cell layers and interstitial stromal cell layer was calculated .Total RNA was extracted from female rat ovarian tissue samples using RNeasy mini kit . The quantity and purity of the total RNA were measured by the NanoDrop spectrophotometer. The cDNA synthesis was carried out using reverse transcriptase  and oligo-dT following the manufacturer protocol. The cDNA samples were then submitted to qPCR using the following forward (5′-CTGCTGATCATGGGCCTCCT-3′) and reverse (5′-CTCCACAGGCTCGGGTTGTT-3′) primer pairs for Cyp19α1 gene. The cDNA was amplified by 40 cycles of denaturation at 95 °C for 45 s, annealing at 59 °C for 45 s and extension at 72 °C for 45 s. The 95 °C step was extended to 4 min during the first cycle. The amplicon size was confirmed by 2% agarose gel electrophoresis stained with SYBR Safe DNA gel stain (Invitrogen). The β-actin gene was furtherly amplified in the same reaction to serve as the reference gene . Each mea, b, c, d superscripts indicate statistical significance compared to the negative control group at P < 0.05 (n = 5). Data for multiple variable comparisons were analyzed by one-way analysis of variance (ANOVA) test to analyze the significant differences (P < 0.05) between groups using SPSS version 24 package for Windows.The different analytical measurements in the biological samples were carried out in triplicate and results are expressed as the mean ± SD, Metabolites were tentatively identified based on their accurate mass, isotopic distribution, fragmentation patterns, comparison with common mass libraries alongside the dictionary of natural products  and the reported literature. The untargeted metabolomic analysis enabled the putative identification of 61 compounds in both ionization modes and their structures were allocated as 13 flavonoids and glycosides, 12 fatty acids/lipids, 10 tannins, seven triterpenes, six steroids, two miscellaneous terpenoids, two carbohydrates and one phenolic acid derivative , glucose concentrations, and the activities of ALT and AST were reported in Table The negative control group showed normal ovary histology in the form of multiple follicles at different stages of development with normal granulosa, theca and interstitial stromal cell layers in addition to presence of various corpora lutea Fig. a, b. PCOFigure The livers of the negative control and vitamin C treated groups revealed a normal hepatic architecture Fig. a, e. TheReal-time PCR was implemented to evaluate the mRNA expression level of the aromatase enzyme which is a key enzyme in the steroid biosynthesis pathway. The quantitative real-time PCR analysis for the Cyp19α1 gene in ovarian tissue from different groups showed that its mRNA expression level was significantly downregulated in the PCOS group compared to the negative control. However, the groups treated with AR extract and/or vitamin C significantly upregulated the mRNA expression level for Cyp19α1 where the combination treatment was more effective Fig. .Fig. 11ERegardless the growing literature on the AR effectiveness for menopausal symptoms, definite assumptions on its exact mechanism of action cannot be deduced from clinical studies not only related to blinding issues, but also to the fact that such things require a very special study design . In the The efficacy of LTZ rat model in the induction of PCOS is well established and represents the human PCOS in various aspects , 56, 57 In LTZ model of PCOS, the conversion of androgen substrates into estrogens in the granulosa cells was blocked leading to an accumulation of androgen . As a rePrevious studies have shown that ovarian cysts were formed by apoptosis of both ovarian oocytes and granulosa cells, where Ki-67 can be used as an indicator for cellular proliferation . In our The androgen aromatization to estradiol in dominant follicles is conducted in the granulosa cells by the aromatase enzyme. Aromatase (Cyp19α1) is a key steroidogenic enzyme that separately catalyzes the conversion of testosterone to estradiol and estrone. Aromatase is encoded by the Cyp19α1 gene . IntriguThe reported elevation in testosterone and LH hormones in LTZ induced PCOS rat models , 68, 69 The hormonal disorders in PCOS are combined with metabolic disorders. High testosterone concentrations in PCOS lead to pancreatic β cell dysfunction, insulin resistance, and thus hyperglycemia and dyslipidemia , 75, in The dose of 500 mg/kg for vitamin C and 7.14 mg/kg for AR was selected based on previous studies , 85. ThiHepatic safety of AR is still a debatable issue. Although, we observed a significant increase in the ALT, AST and hepatic MDA levels alongside with a reduction in hepatic GSH activity in the AR group when compared to the negative control, vitamin C and the combination group. It was not dramatic changes as supported by the pathological finding of only mild hepatocellular necrosis with sinusoidal dilation and disorganization of hepatic cords Table a, b, 10dOne interesting finding from our study is that although vitamin C had a minor ovarian effect on PCOS, it had a protective effect when combined with AR which diminished the AR induced mild liver deterioration. The protective effect of vitamin C may be due to its scavenging effect on oxidant molecules that consequentially enhances the antioxidant capacity.Actaea racemosa and vitamin C combination to mitigate the reproductive and metabolic disorders of PCOS. The AR arsenal of secondary metabolites inhibited the androgen aromatization in the letrozole-induced PCOS rats, offsetting ovarian oxidative stress, which may be involved in the pathogenesis of PCOS, with enhanced hormonal profile, lipid profile, glucose level and liver functions. Furthermore, AR or its vitamin C combination increased Ki-67 expression in the granulosa cell layer along with a significant downregulation of Cyp19-α1 mRNA expression level. Our results of steroidogenesis modulation with a declined risk of hepatic adverse effects by AR and vitamin C combination warrant further studies of this combination in polycystic ovary syndrome.The present study demonstrated the ability of Additional file 1: Table S1. Metabolomic profiling of Actaea racemosa using UPLC–MSn, List of the identified metabolites in negative mode; structures, MSn ions and/or fragmentation tree  and the identified metabolites in positive mode; structures, MSn ions and/or fragmentation tree .


We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The −/−Uch-l1 mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1−/− compared to wild-type mice. Furthermore, cultured −/−Uch-l1 SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a  Mammalian spermatogenesis is a complex, highly regulated process of cell proliferation and differentiation sustained by spermatogonial stem cells (SSCs). These cells give rise to progenitors that undergo a finite number of synchronous cell divisions before entering meiosis as a spermatocyte . PreviouId4), paired box protein Pax-7 (Pax7), glial cell-line derived neurotrophic factors receptor alpha 1 (Gfra1) and promyelocytic leukemia zinc finger protein (Plzf). However, germ cell populations that express these markers are generally regarded as a mixed population containing SSCs among other undifferentiated spermatogonia [c-Kit neither is expressed in SSCs in vivo nor plays a role in their maintenance, but it is critical to the primordial germ cells’ movement towards the basement membrane, where they give rise to SSCs [Stra8), which is the receptor for retinoic acid and a key molecule in initiating meiosis [Clear identification of a definitive SSC has remained elusive, due to the lack of biochemical and molecular markers that unequivocally distinguish SSCs from other undifferentiated spermatogonia. Several putative SSC markers have been described, such as inhibitor of DNA binding 4 , also known as PGP9.5, is classified as a de-ubiquitinase enzyme  that is Uch-l1 gene [Uch-l1 deficient mice [The role of UCH-L1 in maintaining cell homeostasis under normal growth and oxidative stress conditions has been reported in vitro . Evidenc-l1 gene ,23,24,25-l1 gene , which p-l1 gene . UCH-L1 ent mice .Uch-l1 [Uch-l1 in all tissues [gad) mouse that had a spontaneous in-frame deletion of Uch-l1 showed a decrease in proliferating cells, tubule size and cell numbers along with an increase in apoptosis and size of the remaining cells within the seminiferous tubules at 25 weeks of age [gad testes being resistant to cryptorchid-induced injury and early apoptotic first wave, suggesting a role in BAX/BCL-2 -regulated apoptosis [To date, attempts to describe the potential function for UCH-L1 in the testis are limited to a few studies of mutant testes that either carry a spontaneous in-frame deletion of Uch-l1  or overe tissues . The tes tissues . Converss of age . Subsequpoptosis . Yet thepoptosis . Loss ofpoptosis .−/−Uch-l1 mouse model that lacks all UCH-L1 function, we found an increased number of degenerative seminiferous tubules in −/−Uchl1 compared to +/+Uch-l1, a condition that is preceded by a dysregulation of normal metabolic transitions in undifferentiated spermatogonia, as revealed by single cell RNA sequencing analysis. We further demonstrate a loss of differentiation competence of SSCs after transplantation in vivo and accelerated mitochondrial activity in vitro. These data point towards an important role of UCH-L1 in murine spermatogenesis. Loss of UCH-L1 is associated with reduced capacity of SSCs to support spermatogenesis and accompanied by increased oxidative phosphorylation (OXPHOS), mitochondrial dysfunction and metabolic perturbations. The disturbance of the tight regulation of normal metabolic transitions necessary for murine spermatogenesis associated with the loss of UCH-L1 could ultimately result in reduced fertility with age.Here we show that UCH-L1 is expressed in undifferentiated spermatogonia (SSC/progenitors) as well as in early differentiating spermatogonia. Taking advantage of the This work provides novel insight into the functional implication of UCH-L1 for the SSC metabolic homeostasis that is required for long-term metabolic plasticity and stem cell function.Uch-l1tm1Dgen , B6/SV129 and ROSA26 reporter line (B6.129S7-Gt(ROSA)26Sor/J) mice were obtained from the Jackson Laboratory. All animal experiments were carried out in compliance with the Canadian Council of Animal Care guidelines and approved by the University of Calgary’s Health Sciences Animal Care Committee .Uch-l1+/+ and Uch-l1−/− mice through magnetic-activated cell sorting  using a biotinylated rat anti-Thy1.2 mouse antibody . Cells were incubated on ice for 30 min with gentle agitation, washed twice with PBS and incubated with DynabeadsTM M-280 Streptavidin . Thy1 positive cells were resuspended in a-MEM serum-free clusters medium supplemented with GDNF (40 ng/mL), GFRa1 (300 ng/mL) and β-FGF (1 ng/mL) and cultured as previously described [Spermatogonia were isolated from 6 to 8-day old escribed ,32. Cell+/+, Uch-l1Uch-l1+/− and Uch-l1−/− mice testis tissues were collected at post-natal days 0, 7, 17, 30, 60, 90 and 120, fixed in Bouin’s solution or 4% paraformaldehyde (PFA) and embedded in paraffin. Serial paraffin-embedded sections were prepared and stained with hematoxylin and eosin (H&E) for histology examination. Immunohistochemistry was performed on PFA-fixed embedded sections following deparaffination, rehydration and heat-induced antigen retrieval steps. The sections were incubated with primary antibodies against PLZF , LIN28A , UCH-L1  and SOX9 , followed by an incubation with species-specific secondary Alexa Fluor 488, 555 or 647 antibodies. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) . At least 50 cross-sections were processed per sample from 3 tissue sections 50 µm apart. For comparison purposes, PFA-fixed archival testis sections from other mammalian species—adult human, adult buck, adult cat, adult rat, adult sheep, juvenile porcine, juvenile goat, juvenile horse and juvenile monkey—were also processed and analyzed.TM Fluorometric TUNEL system (488)  was used to detect and quantify cell apoptosis in tissue sections. The labeling was performed according to the manufacturer’s recommendations and nuclei were stained with DAPI. The assay catalytically incorporates fluorescein-12-dUTP at 3′-OH ends of DNA, which allows direct visualization of labeled DNA (in green) through fluorescence microscopy.The DeadEndTM Fixative Buffer . Cells were washed with cold PBS, permeabilized for 30 min at 4 °C with BD Perm/WashTM buffer , and blocked using PBS supplemented with 2% BSA and 0.1% NaN3 for 15 min at room temperature. Single testicular cells were stained with mouse Alexa 488-conjugated PLZF  and rat PE/Cy5-conjugated c-KIT  antibodies and their respective isotype controls, mouse Alexa 488 conjugated IgG1 kappa  and rat PE/Cy5-conjugated IgG2b kappa , at room temperature for an hour. Cells were washed twice and analysed with a BD FACSClalibur™ Cell Analyzer instrument  and FlowJo software version 10 (BD Biosciences).Single cells were collected from 17, 90 and 120-day mice testis tissue using a two-step enzymatic digestion. Briefly, tissues were digested with 6 mg/mL collagenase IV  for 15 min at 37 °C, followed by a 5-min incubation with 0.05% Trypsin-EDTA  and 10 mg/mL DNase I . Testicular cells from 90 to 120-day old mice were pelleted at 500 g for 5 min, washed twice with cold PBS and fixed for 30 min at 4 °C using BD CytofixRNA was isolated from 7, 17 and 120-day old mice testis and primary cultured cells using RNeasy Mini Kit . RNA was reverse-transcribed into cDNA using ABI High Capacity cDNA synthesis kit  according to the manufacturer’s instructions. cDNA amplification was carried out in an ABI 7500 Fast qPCR machine  using Bio-Rad Sso Fast Eva Green master mix with low Rox . The qPCR results were baseline-corrected using LinReg PCR. Primer specificity was confirmed by sequencing RT-PCR amplified products and melt curve analysis  was crossed with a ROSA26 reporter line (B6.129S7-Gt(ROSA)26Sor/J) expressing the lacZ transgene. Spermatogonia collected from Uch-l1+/+, Uch-l1−/− or heterozygous gtROSA 6 to 8-day old pups, which ubiquitously express the LacZ reporter gene, were cultured for 3 to 5 passages prior to transplantation. Approximately one million cells contained in 8 μL were transplanted into six-week old B6/SV129 mice via the efferent duct during week 6 after busulfan treatment (50 mg/kg)  as previously described [To enable the identification and quantification of cells and colonies transplanted into a germ cell-depleted recipient testis, Uch-l1escribed . Three i® BioCoat™; 354210, Corning, Glendale, AR, USA) for 30 min. A total of 15 × 106 cells was immobilized to a monolayer with centrifugation at minimal acceleration to 45 G and directly stopped with zero breaking. The cell medium was topped up to 500 μL and the plate was subsequently centrifuged with reverse plate orientation. Respiration (OCR) was measured with an OCR assay medium  supplemented with 5.56 mM glucose, 1 mM pyruvate and 4 mM glutamine. The experiment was performed with oligomycin , mitochondrial uncoupling compound carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone FCCP , respiratory chain inhibitor antimycin A  and rotenone  [4 cells/0.32 cm2).OCR assays were performed using a XF24 Seahorse flux analyzer according to the manufacturer’s instructions . Plates were treated with 250 μL (100 μg/mL) poly-D-lysine (Corningciences) . All SeaTM Single Cell 3′ v2 Chemistry library prep kit according to the manufacturer’s instructions . Samples were sequenced on an Illumina HiSeq4000 with paired end 50 base pair reads by Genome Quebec . The raw counts were subsequently processed with a 10x Genomics CellRanger pipeline using default settings, after which the outputs from the four individual samples were combined using the CellRanger aggr function to normalize the sequencing depth and produce aggregated gene-cell UMI count matrix. A total of 7874 single germ cells (spermatogonia to spermatids) was used for the downstream analysis.Testicular cells were collected via enzymatic digestion from two 8-week old mice from each genotype. Cells were stained with goat anti GFR⍺1 protein  and rat anti c-KIT protein  and subjected to fluorescence-activated cell sorting (FACS) to enrich for spermatogonia using a FACSAria III (BD Biosciences). Briefly, previous experiments have identified a population of cells based on light scatter properties that is enriched in spermatogonia . All c-K−/−Uch-l1 vs. +/+Uch-l1), Seurat V3 integration workflow was used to merge, correct for the batch effect and generate a new integrated Seurat matrix that was used for further analysis. To identify the dimensionality of the dataset, the “ElbowPlot” function was applied, and the first 15 Principal components (PCA) were selected to run the “FindNeighbors”, “FindClusters” and “RunUMAP/RunTSNE” functions with a resolution of 0.5. Once clusters were generated, the “FindMarkers” function was applied to identify differentially expressed genes (DEG) with p values lower than 0.01 and fold change of 1 or higher. The “VlnPlot”, “FeaturePlot”, and “Dimheatmap” functions were used to visualize genes of interest.The concatenated gene-cell barcode matrix was imported into Seurat3.1.0 . A pre-fhttp://www.broadinstitute.org/gsea/downloads.jsp, accessed on 24 August 2021) with default settings [http://software.broadinstitute.org/gsea/, accessed on 24 August 2021). A pre-ranked list consisting of DEGs and their corresponding fold-change values was used as an input to determine the enrichment scores for pathways. The curated gene sets used for reference were downloaded from Molecular Signatures Database (MSigDB) [“heatmap.2” function in R studio. The gene network was built with STRING [GSEA was performed with the javaGSEA application (version 4.0.3) available online (javaGSEA. settings  (http://(MSigDB) . The enrh STRING .+ standard deviation. Non-parametric t-tests (Mann–Whitney U tests) were performed when comparing two data sets and Kruskal–Wallis ANOVA for three data sets according to statistical guidelines. Significance was set at p < 0.05. For the Seahorse experiment, the results are presented as mean ± SEM, if not otherwise stated. Normality was tested with Shapiro–Wilk test ; normality was assumed if all performed tests indicated a normal distribution. Normally distributed data was analyzed with an unpaired parametric t-test, otherwise a non-parametric t-test (Mann–Whitney test) was used to compare ranks. Unequal standard deviation was accounted for with Welch’s correction. A value of p < 0.05 was considered statistically significant. GraphPad Prism 8.0 software was used for all statistical analyses.Quantitative data are displayed as mean +/+Uch-l1 mouse testes collected at different stages of development. As in other mammalian species, UCH-L1 in mice is expressed in cells located along the basement membrane of seminiferous tubules but not in further differentiating germ cells  as well as differentiating germ cells . At 120 days, a significant increase was detected in progenitor cell differentiation-associated Sall4 gene expression, associated with a significant decrease in differentiation-associated c-Kit expression in Uch-l1−/− compared to Uch-l1+/+ testes  collected from two −/−Uch-l1 and two +/+Uch-l1 mice. Seurat integrated analysis workflow was first used to merge the replicates from each genotype, correct for batch effect and perform the downstream analysis. Unsupervised, unbiased clustering projected onto t-distributed stochastic neighbor embedding (tSNE) revealed 10 and 12 cell clusters for +/+Uch-l1 . Overall, there was no difference between the two genotypes in the expression of genes that mark SSC/progenitors, differentiating and differentiated cells. However, this assessment represents only a snapshot of a single time point, which might not represent further phenotypic heterogeneity that can emerge due to UCH-L1 deficiency with aging.To capture any molecular changes that may emerge in germ cells due to the absence of ch-l1+/+ A and Uchch-l1−/− B, respecof Sycp3 ,41,42,43cMyc signaling pathways in −/−Uch-l1 SPG1, while eight gene sets were significantly upregulated in +/+Uch-l1 SPG-1, including hypoxia, p53 and cMyc signaling pathways. These data suggest the knockout of Uch-l1 caused the SSCs/progenitors (SPG1) to shift their metabolism towards an oxidative metabolism as compared to the wild-type. Interestingly, the early-differentiating cell population (SPG2) showed an abnormal increase of glycolytic and hypoxic response-related genes in −/−Uch-l1, while there was an upregulation of OXPHOS-related genes in the wild type. This indicates an imbalance of normal metabolic transition after the loss of UCH-L1 in murine germ cells.To determine whether germ cells displayed any functional change that would precede or accompany the degenerative phenotype of the seminiferous tubules, we used GSEA to identify enriched pathways whose genes were differentially expressed between cell clusters. Heat maps that display the enriched pathways for the two genotypes are shown in An enrichment of gene sets related to a higher cell proliferative state, namely G2M checkpoint, E2F_targets, and mitotic spindle, was observed in SPG3 from both genotypes. However, gene sets associated with the unfolded proteins pathway were upregulated in the late differentiating state (SPG4) in the knockout, while they were enriched early in the SPG3 population. The unfolded protein response (UPR) is a conserved cell process, induced in response to an accumulation of unfolded or misfolded proteins in the endoplasmic reticulum , which p−/−Uch-l1 genotype in one replicate (Dmrt1 (−/−Uch-l1 (as compared to +/+Uch-l1) were loaded into GSEA to search for the enriched gene sets. Here again we confirmed the upregulation of gene sets related to OXPHOS in cluster 2 of SSCs/progenitors , correct for batch effect and perform the downstream analysis. Unsupervised clustering grouped the four transcriptomes into 12 distinct clusters, as illustrated by Uniform Manifold Approximation and Projection (UMAP) A,B. As eeplicate . Comparie  was similar between genotypes , stem cell capacity (colony length) and score of a complete spermatogenesis of the transplanted cell population . Similar to the shift observed at the transcriptional level 1 gene 1 gene . This daUch-l1 knockout mouse models; however, its function in spermatogonia has not been clearly identified. We report here that the loss of UCH-L1 impacts the differentiation competence of murine SSCs in vivo. The observed disruption of metabolic homeostasis in undifferentiated spermatogonia and loss of anaerobic metabolism of SSCs after long-term cell culture indicate a role of UCH-L1 in the maintenance of low OXPHOS and protection of stemness-associated metabolism in SSCs.The role of UCH-L1 in the nervous system has become significantly better understood due to the contributions of Uch-l1 knockout mouse model to investigate morphological and functional changes associated with UCH-L1 function deficiency.UCH-L1 is highly expressed in undifferentiated spermatogonia and early differentiating spermatogonia, but not in further differentiating germ cells, and this expression pattern is phylogenetically conserved in mouse, pig and human testes ,50,51. H−/−Uch-l1 compared to +/+Uch-l1 mice at 120 days of age. Testis weight is an important reproductive trait in males that reflects both the spermatogenic capacity, as the sperm production rate positively correlates with the testis weight [−/−Uch-l1 mice remained fertile until at least 3 months of age, a reduction in confirmed matings and litters produced was observed in this study. It remains, however, plausible that the reduction in the −/−Uch-l1 mice’ ability or desire to copulate may also have resulted from progressive neurodegenerative hind limb paralysis [We report here a significant decrease in testis weight in s weight , and thes weight . Althougaralysis .−/−Uch-l1 compared to +/+Uchl-1 at 120 day of age. Considering the progressive nature of the seminiferous tubule degeneration, this phenotype becomes more pronounced in −/−Uch-l1 mice with age. Previous findings using the gad mouse model corroborate this assumption as a slight decrease in the size of seminiferous tubules was reported in 25-week old homozygous gad mice compared to those of age-matched wild-type mice [−/−Uch-l1 germinal epithelium in older animals. Uch-l1-deficient Sertoli cells also may lose their ability to provide morphological and nutritional support needed for spermatogenesis, contributing to spermatogenic failure [Of note, decreased testis weight was accompanied by a significant increase in the number of degenerative seminiferous tubules in  failure ,56.gad mouse model showed resistance to the early and massive wave of germ cell apoptosis during the pre-pubertal stage of development, a phase that is essential for functional spermatogenesis in adults. In contrast, there was no difference in cell apoptosis between −/−Uch-l1 and +/+Uch-l1 mice in the current study, either in situ or in vitro from day 7 to day 120, a timeline that captures the first as well as subsequent spermatogenic waves. The discrepancy between the two studies might be explained by a potential phenotypical difference between the two mouse models. The gad model is the result of a spontaneous congenital mutation and might possibly display additional mutations within the ubiquitin–proteasome pathway, while the model used in our study is a targeted mutant mouse model. Nonetheless, our data suggest that the progressive loss in the seminiferous tubule structure might be associated with a decrease in cell proliferation or differentiation rather than an increase in germ cell loss, since no difference in TUNEL-positive cells was apparent between the two genotypes at 90 or 120 days of age.Maintaining a normal seminiferous epithelium relies on a regulated balance between proliferation, differentiation, and apoptosis of germ cells . A potenUch-l1−/− mice as compared to +/+Uch-l1. Interestingly, these Uch-l1−/− colonies contained fewer layers of donor-derived cells, indicating that −/−Uch-l1 SSCs had a reduced capacity to regenerate the entire spermatogenic lineage. Our data showing no difference in the cell number, viability, proliferation as well as in spermatogonia-associated gene expression  between +/+Uch-l1 and −/−Uch-l1 in culture, suggest that the intrinsic loss of spermatogenic potential of −/−Uch-l1 SSCs did not affect normal SSC gene expression and viability.Long-term transplantation data support these conclusions. At 10 months post transplantation, the colonized area had expanded more in testes that received germ cells from It is becoming increasingly evident that maintaining metabolic integrity and plasticity is pivotal for SSC function ,49,57. A−/−Uch-l1 SSCs kept in culture for 21 passages (150 days) had a higher basal respiration and maximum respiration capacity as compared to +/+Uch-l1, suggesting a loss of stem cell metabolic integrity in UCH-L1-deficient SSCs. These data indicate that the loss of UCH-L1 was detrimental to metabolic homeostasis regulating SSC function, therefore causing a decrease in SSC differentiation competence, all of which was reflected in the long-term outcome after transplantation of −/−Uch-l1-derived SSCs.We show here that −/−Uch-l1 SSC/progenitors whereas genes associated with hypoxia were upregulated in +l+Uch-l1 SSC/progenitors. Upregulation of genes associated with glycolysis and hypoxia was detected in early differentiating spermatogonia of UCH-1−/−, as opposed to the physiological switch towards OXPHOS in the wild-type. Hence, UCH-L1 deficiency altered the bioenergetic equilibrium between glycolysis and OXPHOS that is necessary for spermatogonial proliferation and differentiation [Furthermore, our single-cell RNA sequencing data are consistent with a disturbed shift in cell metabolism, as an upregulation of genes associated with OXPHOS was observed in ntiation . These dntiation , which fntiation . The det−/−Uchl1 SSCs, characterized by an initial upregulation of OXHPOS-related genes followed by their decrease during differentiation, which is the inverse of the metabolic changes in the wild-type. Furthermore, mitochondrial activity also increased in −/−Uchl1 SSCs after long-term culture, while it remained low in wild-type SSCs. Similar metabolic changes have been described in neuroblastoma cells. A disruption of mitochondrial structure through the reduction of mitofusion 2 (MFN2) levels and an increased oxygen consumption rate were observed upon knockdown of UCH-L1 with shRNA within a few days [We detected metabolic dysregulation in murine few days .Although further experiments are warranted to determine the mechanism by which UCH-L1 deficiency affects germ cell metabolism, one may speculate that UCH-L1 plays a role in regulating the function of mitochondria directly via the turn-over of mitochondrial proteins or indirectly via, among others, interference with the mechanistic target of rapamycin (mTOR) or hypoxia-inducible factor-1α (HIF-1α).Stra8 expression and meiotic progression in fetal germ cells [−/−Uch-l1 spermatogonia.UCH-L1 is associated with mitochondrial quality control by regulating the expression of mitofusin-2 (MFN2), thus affecting several mitochondrial functions, especially ultrastructure in neural cells . It alsorm cells , one may−/−Uch-l1-derived SSCs in vitro and in vivo. Additional studies are required to decipher the underlying mechanism by which UCH-L1 regulates metabolism in SSCs, yet the current study highlights that stage-dependent metabolic regulation is critical for male germ cell function and reveals a unique role of UCH-L1 in this complex system.In summary, UCH-L1 plays a critical role in maintaining SSC differentiation competence in mice, as a dysregulation of normal metabolic transitions associated with SSC maintenance and differentiation occurred in


Sf9 insect cells, compared with a commercial synthetic pesticide . Three derivatives bearing a terminal benzene ring, either substituted or unsubstituted, were identified as the most potent molecules, two of them displaying higher toxicity to insect cells than CHPY. In addition, the most promising molecules were able to increase the activity of serine proteases (caspases) pivotal to apoptosis and were more toxic to insect cells than human cells. Structure-based inverted virtual screening and molecular dynamics simulations demonstrate that these molecules likely target acetylcholinesterase and/or the insect odorant-binding proteins and are able to form stable complexes with these proteins. Encapsulation assays in liposomes of DMPG and DPPC/DMPG (1:1) were performed for the most active compound, and high encapsulation efficiencies were obtained. A thermosensitive formulation was achieved with the compound release being more efficient at higher temperatures.A series of β-amino alcohols were prepared by the reaction of eugenol epoxide with aliphatic and aromatic amine nucleophiles. The synthesized compounds were fully characterized and evaluated as potential insecticides through the assessment of their biological activity against  Periplaneta americana and Drosophila melanogaster, and this was also found to be mediated via octopamine receptors. Structural changes of eugenol are known to be a useful strategy in order to improve biological activity and to obtain new analogues with reduced side effects : 254.1751; found 254.1753.To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol 2  in H2O/EtOH 2:1 (2 mL) was added octan-2-amine  and the resulting mixture was heated at 50 °C for 4 h. Then, water (2 mL) was added, and the resulting mixture extracted with EtOAc (2 mL), The organic phase was collected, dried with anhydrous MgSO4, and the solvent evaporated to afford an oil (0.202 g), which was subjected to column chromatography using DCM/MeOH as eluent of increasing polarity to give the compound 3b as a brown oil . Rf = 0.45 (MeOH/DCM 10:90). 1H-NMR δH : 6.78 , 6.72 , 6.48 , 3.92–3.89 ), 3.81 , 2.84–2.53 , 1.49 , 1.24 , 1.07 , 0.88  ppm. 13C-NMR δC : 146.69 (Ar-Cq),144.36 (Ar-Cq), 129.59 (Ar-Cq), 121.76 (Ar-C), 114.55 (Ar-C), 111.99 (Ar-C), 70.25 (CH), 69.93 (CH), 55.72 (OCH3), 51.48 (CH2), 41.32 (CH2), 35.61 (CH2), 31.68 (CH2), 29.21 (CH2), 25.77 (CH2), 22.51 (CH2), 19.17 (CH3), 13.98 (CH3) ppm.To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol 2  in H2O/EtOH 2:1 (2 mL) was added piperidine , and the resulting mixture was heated at 50 °C for 5 h. The solvent was evaporated under reduced pressure to afford compound 3c as a brown oil . Rf = 0.35 (MeOH/DCM 10:90). 1H-NMR δH : 6.83 , 6.77 , 6.67 , 4.18–4.12 ), 3.89 , 2.88–2.81 , 2.65–2.52 , 1.82–1.70  1.58–1.47  ppm. 13C-NMR δC : 146.52 (Ar-Cq), 144.31 (Ar-Cq), 129.52 (Ar-Cq), 121.80 (Ar-C), 114.27 (Ar-C), 111.80 (Ar-C), 67.01 (CH), 63.93 (CH2), 55.93 (OCH3), 54.76 (CH2), 41.30 (CH2), 24.26 (CH2), 23.05 (CH2) ppm. HRMS (ESI-TOF): calcd for C15H24NO3 [M + +H]: 266.1751, found 266.1752.To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol 2 (1 equiv) in H2O/EtOH 2:1 (2 mL) was added aniline , and the resulting mixture was heated at 50 °C for 5.5 h. Then, water (2 mL) was added, and the resulting mixture was extracted with EtOAc (2 mL). The organic phase was collected, dried with anhydrous MgSO4, and the solvent was evaporated to afford an oil (0.284 g), which was subjected to column chromatography using DCM/MeOH as eluent of increasing polarity to give compound 3d as a yellow oil . Rf = 0.7 (MeOH/DCM 5:95). 1H-NMR δH : 7.19 , 6.88 , 6.75 , 6.65 , 4.09–4.02 ), 3.87 , 3.31 , 3.10 , 2.83 ), 2.75 ) ppm. 13C-NMR δC : 147.9 (Ar-C), 146.6 (Ar-C), 144.4 (Ar-C), 129.4 (Ar-C), 129.3 (Ar-C), 122.0 (Ar-C), 118.1 (Ar-C), 114.5 (Ar-C), 113.5 (Ar-C), 111.8 (Ar-C), 71.1 (CH), 55.9 (OCH3), 49.5 (CH2), 41.2 (CH2) ppm HRMS (ESI-TOF): calcd for C16H20NO3 [M+ +H]: 274.1438; found 274.1430.To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol 2  in H2O/EtOH 2:1 (2 mL) was added 3-methoxyaniline , and the resulting mixture was heated at 50 °C for 4 h. Then, water (2 mL) was added, and the resulting mixture was extracted with DCM (2 mL). The organic phase was collected, dried with anhydrous MgSO4, and the solvent was evaporated to afford an oil (0.579 g), which was subjected to column chromatography using light petroleum/EtOAc as an eluent of increasing polarity to give compound 3e as a brown oil . Rf = 0.35 (ethyl acetate/light petroleum 1:1). 1H-NMR δH : 7.08 , 6.86 , 6.73–6.71 , 6.30 , 6.24 , 6.20–6.16 , 4.05–3.99 ), 3.84 , 3.76 , 3.27 , 3.05 , 2.83–2.70 ) ppm. 13C-NMR δC : 160.69 (Ar-Cq), 149.42 (Ar-Cq), 146.57 (Ar-Cq), 144.27 (Ar-Cq), 129.99 (Ar-Cq), 129.91 (Ar-CH), 121.85 (Ar-CH), 114.48 (Ar-CH), 111.81 (Ar-CH), 106.35 (Ar-CH), 102.95 (Ar-CH), 102.88 (Ar-CH), 71.07 (CH), 55.76 (OCH3), 54.96 (OCH3), 49.32 (CH2), 41.08 (CH2) ppm. HRMS (ESI-TOF): calcd for C14H24NO3 [M+ + H]: 304.1543, found 304.1547.To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol 2  in H2O/EtOH 2:1 (2 mL) was added 4-aminobenzonitrile , and the resulting mixture was heated at 50 °C for 37 h. Then, water (2 mL) was added, and the resulting mixture was extracted with EtOAc (2 mL), The organic phase was collected, dried with anhydrous MgSO4, and the solvent was evaporated to afford an oil (0.265 g), which was subjected to column chromatography using DCM/MeOH as an eluent of increasing polarity to give compound 3f as a dark yellow oil . Rf = 0.45 (MeOH/DCM 5:95). 1H-NMR δH : 7.39 , 6.87 , 6.72–6.69 , 6.58–6.50 , 4.07–4.01 ), 3.85 , 3.31 , 3.12 , 2.82 ), 2.73 ) ppm. 13C-NMR δC : 151.20 (Ar-Cq), 146.68 (Ar-Cq), 144.53 (Ar-Cq), 136.33 (Ar-Cq), 133.65 (Ar-CH), 128.83 (Ar-Cq), 121.88 (Ar-CH), 121.33 (Ar-CH), 114.62 (Ar-CH), 112.58 (Ar-CH), 111.72 (Ar-CH), 98.91 (Ar-CH), 70.97 (Ar-CH), 55.87 (OCH3), 48.10 (CH2), 41.20 (CH2) ppm.To a suspension of 2-methoxy-4-(oxiran-2-ilmethyl)phenol Sf9, Spodoptera frugiperda) cells were maintained as a suspension culture and cultivated in Grace’s medium with 10% FBS and 1% penicillin/streptomycin, at 28 °C with agitation. Cells were used in experiments while in the exponential phase of growth. On the other hand, HaCaT (human keratinocytes) cells were culture in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2.Insect cells  for 24 h. After this period, a commercial solution of resazurin was added (1:10), and the kinetic reaction of fluorescence increase was monitored at 560/590 nm. For HaCaT and the Sf9 cells, 30 and 60 min of incubation were used, respectively.For the assessment of viability, a resazurin-based method was used. The The Sf9 cells were cultured at the same density described above for the viability assessment. To assess the release of the stable cytosolic enzyme lactate dehydrogenase (LDH) into the media, 24 h after the incubation of cells with the molecules under study (at 100 µg/mL in Grace’s medium), 50 µL of culture media were removed to a 96-well plate. The LDH released was determined using a CytoTox 96® assay kit  according to the manufacturer’s instructions. A lysis solution (LS) was used as a positive control to generate a maximum LDH release (45 min). Absorbances were measured at 490 nm in a Multiskan GO plate reader , and results correspond to the fold increase of absorbance in treated vs. untreated cells of four independent experiments performed in duplicate.Sf9 cells were plated at the same density described for viability studies and exposed to the molecules under study for the designated time. Generally, the same method described before by some of us was used [The was used ; howeverwas used ,22. Aftewas used  and manup < 0.05.For biological assays, the Shapiro–Wilks normality test was performed in the data to ensure that it followed a normal distribution. Comparison between the means of controls and each experimental condition was performed using one-way ANOVA. Outliers were identified by the Grubbs’ test. Data were expressed as the mean ± standard deviation (SD) of at least three independent experiments. GraphPad Prism 7.0 software was used, and values were considered statistically significant with a To identify possible molecular targets of the amino alcohols derived from eugenol, an inverted virtual screening protocol was applied. A search on Scopus was performed for papers describing virtual screening (VS) studies involving targets and molecules with insecticidal activity using the keywords: “virtual screening” and “pesticides”. The selection criteria were relevance of the target and year of publication. In the 18 studies found, 23 PDB structures were identified and downloaded, enabling the creation of a structural database of putative insecticide targets. These are listed in The 23 PBD structures were prepared for docking starting using the Autodock Vina plugin for Pymol . CrystalThe docking programs/scoring functions used were GOLD  -binding pocket center, docking region radius, search efficiency, number of runs. The final optimized conditions were used for the subsequent stages. Structures for the two eugenol amino alcohol derivatives with the highest insecticide activity were prepared for docking using Datawarrior  and Open3d and 3f), which are bound to the two most promising targets identified from the inverted virtual screening study (odorant binding protein 1–3KIE and acetylcholinesterase-1QON). Since 1QON presented a gap in the structure, a homology model was generated using SWISS-MODEL [Molecular dynamics simulations were performed using the Amber18 software  for the two compounds identified from the experimental studies to have the highest insecticide activity , and the systems were placed in TIP3P water boxes with a minimum distance of 12 Å between the protein surface and the side of the box.Models for the MD simulations were prepared starting from the pose predicted for these complexes in the docking experiments during the inverted virtual screening protocol with GOLD/PLP and treated with the Leap module of AMBER . The proIn order to remove the clashes, the systems were submitted to four consecutive minimizations stages, which were followed by an equilibration and production. In the first four minimization stages, the procedure was applied to (1) water molecules 2500 steps); (2) hydrogens atoms (2500 steps); (3) side chains of all the amino acid residues (2500 steps); and (4) the full system . After the complete minimization, the systems were equilibrated by a procedure, which was divided into two stages: in the first stage, NVT ensemble, the systems were gradually heated to 298 K using a Langevin thermostat at constant volume (50 ps); in the second stage, the density of the systems was further equilibrated at 298 K (subsequent 50 ps). Finally, the productions runs were performed during 100 ns. Production was executed with an NPT ensemble at constant temperature  and pressure , with periodic boundary conditions. An integration time of 2.0 fs using the SHAKE algorithm was used to constrain all covalent bonds involving hydrogen atoms. The nonbonded interactions were cut off at 10 Å throughout the entire molecular simulation procedure. The final trajectories were analyzed in terms of RMSD to obtain confirmation that both systems were well equilibrated after the initial 10 ns. The last 90 ns of the simulation were considered for hydrogen bonding analysis, and cluster analysis of the conformations was generated. This overall procedure has been previously used with success in the treatment of several biomolecular systems 0 steps; ,55,56,573d and 3f to the odorant binding protein 1 and to acetylcholinesterase, considering a salt concentration of 0.100 mol.dm−3. In addition, the energy decomposition method was employed to estimate the contribution of all the amino acid residues for each of these binding free energies. From each MD trajectory, a total of 1400 conformations taken from the last 70 ns of simulation were considered for the MM-GBSA calculations.The Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method  was applSf9 cells, compound 3f, was encapsulated in liposomes composed of the phospholipids 1,2-dimyristoyl-sn-glycero-3-phospho- (1′-rac-glycerol)  (DMPG) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), which are either composed of DMPG (100%) or DPPC/DMPG (1:1). The liposomes  were prepared by the ethanolic injection method [−4 M) was encapsulated by co-injection with the ethanolic lipid solution. The size, polydispersity, and zeta potential of compound-loaded liposomes were measured in a Litesizer 500 Dynamic Light Scattering apparatus from Anton Paar .The most active compound against the n method  above thn method . The comThe encapsulation efficiency (EE%) was determined as previously reported  and calcb = 1 is exponential; b > 1 is sigmoid, with ascendant curvature delimited by an inflection point; and b < 1 is parabolic, displaying high initial slope and a consistent exponential character).The compound release was followed during 24 h at 20 °C and 35 °C, below and above the phase transition temperature of DMPG. The Weibull model (a distribution function) was used to study the transport mechanism involved in the compound release , being eSf9 cell line, in comparison with the corresponding precursors, in order to evaluate their application as potential natural based insecticides.A series of β-amino alcohols were prepared by reaction of eugenol epoxide with various aliphatic and aromatic amines. The obtained eugenol derivatives were subjected to biological activity evaluation in the We identified that the three derivatives bearing a terminal benzene ring, either substituted or unsubstituted, were those showing higher potency, in some cases higher than the benchmark used. We further clarified that the molecules were eliciting their effect by triggering organized cell death, and they were selective for insect cells.Inverted virtual screening studies with five independent methods suggest that these molecules display their insecticide activity most likely by targeting the insect acetylcholinesterase and/or the insect odorant binding proteins. Molecular dynamics simulations and free energy calculations confirm that these two molecules bind strongly to both targets forming very stable complexes with well-defined molecular interactions that are maintained through time.Nanoencapsulation studies allow obtaining very reasonable encapsulation efficiencies and a controlled release. Liposomes of DMPG provide a temperature-sensitive compound release, which is more effective than the DPPC/DMPG (1:1) formulation.


We demonstrate that Rab43 differentially regulates the overall surface expression of endogenous α2-AR and mAChR, as well as their signaling, in primary neurons. In parallel, Rab43 exerts distinct effects on the dendritic and postsynaptic transport of specific α2B-AR and M3 mAChR subtypes. More interestingly, the selective actions of Rab43 toward α2B-AR and M3 mAChR are neuronal cell specific and dictated by direct interaction. These data reveal novel, neuron-specific functions for Rab43 in the dendritic and postsynaptic targeting and sorting of GPCRs and imply multiple forward delivery routes for different GPCRs in neurons. Overall, this study provides important insights into regulatory mechanisms of GPCR anterograde traffic to the functional destination in neurons.G protein–coupled receptors (GPCRs) are important modulators of synaptic functions. A fundamental but poorly addressed question in neurobiology is how targeted GPCR trafficking is achieved. Rab GTPases are the master regulators of vesicle-mediated membrane trafficking, but their functions in the synaptic presentation of newly synthesized GPCRs are virtually unknown. Here, we investigate the role of Rab43,  G protein–coupled receptors (GPCRs) constitute the largest superfamily of cell surface signaling proteins and modulate a variety of physiological and pathological functions of the nervous system. Neurons are the most sophisticated cells with specialized morphology and compartmentalization in which most GPCRs are expressed in both presynaptic and postsynaptic membrane terminals where they are able to bind to respective neurotransmitters to activate cognate heterotrimeric G proteins or other signaling molecules which in turn activate downstream effectors, including ion channels, and thus control synaptic signaling and transmission , 2, 3.GPCRs are synthesized in the endoplasmic reticulum (ER). After being correctly folded and properly assembled, newly synthesized receptors can pass through the ER quality control system and export from the ER through the Golgi, along the axon and dendrites, to the synaptic compartments , 5. AlthRab GTPases are the master regulators of vesicle-mediated membrane traffic in exocytic and endocytic pathways; they regulate a number of events involved in the development of central nervous system and neuronal functioning, such as neurite morphogenesis and outgrowth, axonal and dendritic transport, synaptic vesicle fusion and elimination, neurotransmitter release, and synaptic transmission , 16, 17.2-adrenergic receptor (α2-AR) and muscarinic acetylcholine receptor (mAChR). The α2-AR and mAChR have three  and five subtypes (M1R–M5R), respectively, and all play important roles in the central and peripheral nervous systems. As compared with many other secretory Rab GTPases, the function of Rab43 is relatively poorly defined. Previous studies have shown that Rab43 is important for Golgi structure . Surprisingly, Rab43N131I expression had no significant effect on the surface expression of mAChR (B). In contrast, treatment with brefeldin A (BFA), which disrupts the Golgi structure and thus blocks the ER-to-Golgi transport, similarly inhibited the surface expression of both α2-AR and mAChR by 40 to 60% (C).As an initial approach to study the possible function of Rab43 in regulating the anterograde transport of newly synthesized GPCRs in neurons, we measured the effect of lentiviral expression of dominant-negative guanine nucleotide-deficient mutant Rab43N131I on the surface expression of endogenous α viruses , A and Bof mAChR B. In con0 to 60% C.Figure 2-AR and mAChR, we used the activation of mitogen-activated protein kinases, specifically extracellular signal–regulated kinase 1 and 2 (ERK1/2), as a readout. Although α2-AR and mAChR each have multiple subtypes which couple to distinct heterotrimeric G proteins, they are all able to activate ERK1/2. Consistent with its effects on the surface receptor expression measured in radioligand binding assays, lentiviral expression of Rab43N131I strongly inhibited ERK1/2 activation in response to stimulation with the α2-AR agonist UK14304, without altering ERK1/2 activation by the mAChR agonist oxotremorine-m (Oxo-M) (D and E). These data demonstrate that the surface expression of α2-AR and mAChR, as well as their signaling, is differentially regulated by Rab43 in neurons.To define if Rab43 could differentially affect the concomitant function of α (Oxo-M) , D and E2-AR and mAChR, specifically the α2B-AR and M3 mAChR (M3R) subtypes, in primary cultures of hippocampal neurons by confocal imaging. We first determined if expression of Rab43N131I could alter the general morphology of neurons. As accessed by the DsRed and GFP signals, expression of DsRed-tagged Rab43N131I had no obvious effect on the general morphology of hippocampal neurons compared with neurons transfected with DsRed vectors (A and B). The spine length, width, and density were very much the same in neurons expressing DsRed and Rab43N131I (B and C). Consistent with radioligand-binding data, Rab43N131I significantly reduced the dendritic expression of α2B-AR by approximately 40%, but had no effect on M3R expression at dendrites (D–F). Rab43N131I also significantly attenuated the expression of α2B-AR at the dendritic spines by about 35%, whereas M3R expression at the dendritic spines was not affected (G–I).We next determined the role of Rab43 in the dendritic and postsynaptic transport of α vectors , A and Bb43N131I , B and Cendrites , D–F. Raaffected G–I.Figu2B-AR and M3R in the hippocampal neurons. Owing to the lack to Rab43 antibodies detecting endogenous Rab43 in primary neurons, the efficiency of Rab43KO was tested by using transient expression of GFP-tagged rat Rab43 in human embryonic kidney 293 (HEK293) cells. The expression of rat Rab43 was reduced by more than 90% in cells transfected with Rab43KO plasmids targeting mouse Rab43 as compared with cells transfected with control vectors (A), suggesting that CRISPR–Cas9 KO plasmids targeting mouse Rab43 can deplete rat Rab43. In hippocampal neurons, similar to the effects of Rab43N131I, CRISPR–Cas9–mediated Rab43KO markedly inhibited the dendritic presentation of α2B-AR, but not M3R (B–D). Rab43KO by CRISPR–Cas9 also reduced the expression of α2B-AR, but not M3R, at the dendritic spines (E–G).We then studied the effect of Rab43KO by the CRISPR–Cas9 system on the dendritic and postsynaptic trafficking of α vectors A, sugges not M3R , B–D. Rac spines , E–G.Fig2B-AR or M3R, and receptor expression at postsynapses was measured by using PSD95 as a marker. Postsynaptic expression of α2B-AR was markedly reduced, whereas M3R expression remained unaffected in Rab43KO neurons as compared with neurons transfected with control vectors (H–J). These data demonstrate distinct functions of Rab43 in the forward transport of α2B-AR and M3R to the neuronal dendrites and postsynapses.To further confirm the role of Rab43 in the postsynaptic transport, hippocampal neurons were transfected with red fluorescent protein–tagged PSD95, GFP-carrying Rab43KO plasmids, and yellow fluorescent protein–tagged α vectors , H–J. Th2-AR and mAChR in human-derived neuroblastoma SHSY5Y cells and rat renal tubular epithelial NRK49F cells. Similar to the results observed from primary cultures of neurons, Rab43N131I expression inhibited the total surface expression of endogenous α2-AR in both SHSY5Y and NRK49F cells by greater than 50%, whereas Rab43N131I only attenuated the surface number of endogenous mAChR in NRK49F cells, but not in SHSY5Y cells, as measured by radioligand binding (A). In contrast, BFA treatment significantly suppressed the surface expression of α2-AR and mAChR in both cell types (B).We next compared the effects of Rab43 on the surface transport of α binding A. In conll types B.Figure 2B-AR or M3R was expressed together with venus-tagged kRas and Rab43 mutants. In addition to Rab43N131I, inactive GDP-bound Rab43T32N mutant was also used. The net BRET signal between α2B-AR and kRas was markedly inhibited by both Rab43 mutants, whereas the BRET signal between M3R and kRas was not affected by the Rab43 mutants in SHSY5Y cells (C).In bioluminescence resonance energy transfer (BRET) assays to measure receptor surface expression, Rluc-tagged α5Y cells C.2B-AR and M3R were robustly expressed at the cell surface in SHSY5Y and NRK49F cells transfected with control vectors. In Rab43N131I-expressing cells, α2B-AR was unable to transport to the cell surface and arrested in the perinuclear regions in both cell types, whereas M3R retained its ability to sufficiently move to the cell surface in SHSY5Y cells, but not in NRK49F cells (D).Confocal microscopy showed that, as expected, both α9F cells D.2B-AR and M3R. Rab43KO plasmids targeting human Rab43 were tested using GFP-tagged human Rab43 in HEK293 cells (A). Similar to Rab43N131I, Rab43KO arrested α2B-AR in the perinuclear regions in both SHSY5Y and NRK49F cells, whereas M3R was arrested only in NRK49F cells, but not in SHSY5Y cells (B).We then determined the effect of CRISPR–Cas9–mediated Rab43KO on the subcellular localization of α93 cells A. Simila5Y cells B.Figure C). Expression of human Rab43 effectively rescued the surface expression of cyan fluorescent protein (CFP)-tagged M3R in NRK49F cells expressing mouse Rab43KO plasmids (D). These data demonstrate that different actions of Rab43 on the surface transport of mAChR and specific M3R subtype are neuronal cell type specific.We then sought to determine if single-guide RNA (sgRNA)-resistant Rab43 plasmids could rescue the effect of CRISPR–Cas9–mediated Rab43KO on the surface expression of M3R. For this purpose, human Rab43 plasmids were transfected together with mouse Rab43KO plasmids in NRK49F cells. Human Rab43 expression was not affected by mouse Rab43KO plasmids C. Expresplasmids D. These 2B-AR directly interacts with Rab43 via the third intracellular loop (ICL3), to elucidate the molecular mechanisms underlying the selective regulation of α2B-AR and M3R trafficking by Rab43 in neurons and neuronal ells, we compared Rab43 interaction with the ICL3 of both receptors in glutathione-S-transferase (GST) fusion protein pull-down assays. α2B-AR and M3R possess relatively large ICL3, containing 165 and 229 residues, respectively, which were generated as GST fusion proteins. Consistent with our previous data, the α2B-AR ICL3 strongly bound Rab43. In contrast, the M3R ICL3 binding to Rab43 was negligible (A).As our previous studies have shown that αgligible A.Figure 2B-AR ICL3 (B) and measured the possible interactions between Rab43 and individual receptors in live cells in BRET assays. The net BRET signals observed between Rab43 and α2B-AR and between Rab43 and M3-2B were similar, but the net BRET signal between Rab43 and M3R was almost undetectable (C). These data suggest that the α2B-AR ICL3 can confer its interaction with Rab43 to M3R.We then generated the chimeric receptor M3-2B in which the M3R ICL3 was substituted with the α-AR ICL3 B and meatectable C. These D). Confocal microscopy showed that M3-2B was strongly expressed at the surface which was clearly impeded by Rab43N131I or Rab43KO in both SHSY5Y and NRK49F cells (E and F). The intracellularly accumulated M3-2B was extensively colocalized with calregulin, an ER marker (G and H). These data suggest that Rab43 is able to control M3-2B transport to the cell surface, specifically its export from the ER.We next determined the effect of Rab43 on M3-2B transport in cell lines and primary neurons. In contrast to M3R which is irresponsive to Rab43, the surface expression of M3-2B was inhibited by Rab43N131I and Rab43KO in SHSY5Y cells as measured by intact cell radioligand binding D. Confoc9F cells , E and FR marker , G and HA–C) and spines (D–F) was significantly inhibited by Rab43N131I and Rab43KO. Furthermore, the inhibitory effects of Rab43N131I and Rab43KO on the transport of M3-2B were similar (C and F). These data demonstrate that the α2B-AR ICL3 controls not only M3-2B interaction with Rab43 but also its Rab43-dependent trafficking.In primary hippocampal neurons, M3-2B was expressed at dendrites and spines. Similar to the results observed in cells, M3-2B expression at dendrites , A–C andd spines , D–F was similar , C and FAs the first study on the functions of the Rab family GTPases in the anterograde dendritic and synaptic delivery of GPCRs, we have focused on Rab43 which was recently demonstrated to regulate GPCR trafficking in the early secretory pathway in cell . The mos2-AR, but not mAChR, in primary neurons. By confocal microscopy to directly visualize the localization of fluorescence-tagged individual receptor subtypes, we have shown that the dendritic and postsynaptic expression of α2B-AR, but not M3R, depends on the normal function of Rab43. Such selective actions of Rab43 toward different GPCRs are also reflected by receptor-mediated signaling measured by ERK1/2 activation. These data provide direct evidence indicating novel functions for Rab43 in mediating GPCR transport to dendrites and synapses, as well as their sorting from one another in neurons. However, we cannot exclude the possibility that Rab43 may regulate the trafficking of other signaling molecules involved in receptor-mediated signal transduction pathways, which may also contribute to the observed abnormal signaling, and the role of Rab43 in the trafficking of other α2-AR and mAChR subtypes remains unknown.By using radioligand binding of intact live neurons to quantify the exact surface receptor number, we have demonstrated that Rab43 selectively regulates the total surface expression of endogenous α2-AR and specific α2B-AR subtype, but not endogenous mAChR and M3R subtype, in neuronal SHSY5Y cells. In contrast, the surface transport of both receptors requires Rab43 in non-neuronal NRK49F cells. These data suggest that the sorting function of Rab43 in the trafficking of distinct GPCRs along the biosynthetic pathways is neuronal cell specific. These data also imply that trafficking itineraries of a GPCR  may differ in neurons/neuronal cells and non-neuronal cells.Similar to the results observed in primary neurons, Rab43 affects the surface expression of endogenous α2B-AR interacts with Rab43 and its dendritic and postsynaptic transport in primary neurons and the surface transport in neuronal cells depend on Rab43. These data indicate that the Rab43-binding domain is able to effectively convert GPCR transport from a Rab43-independent pathway into a Rab43-dependent pathway and that the sorting function of Rab43 is mediated through its direct interaction with the receptors.Although GPCRs share a common structural topology, how they are sorted into distinct transport pathways is poorly understood. Our previous studies suggest that Rab43 interaction may separate GPCRs from non-GPCR plasma membrane proteins . Our rese.g., Rab43-dependent and Rab43-independent pathways) to transport from the cell body to dendrites and from the dendritic shaft to spines. It is possible that M3R transport to dendrites is mediated through lateral diffusion as suggested for most GPCRs (2B-AR uses secretory vesicles to move forward to dendrites as suggested for serotonin 1B receptor (5HT1B) , (5HT1B) , are for (5HT1B) . The den (5HT1B) , 38, 39.2-AR transport from the Golgi  and [N-methyl-3H]-scopolamine methyl chloride  were from PerkinElmer Life Sciences. All other materials were obtained as described elsewhere , the α2B-AR ICL3 fragment was amplified by PCR using primers , and the PCR products were then used to replace the M3R ICL3 by using the QuikChange site-directed mutagenesis kit (Agilent Technologies). Human Rab43 tagged with DsRed or GFP at the N-terminus was generated as described previously  as described previously  and grown on glass coverslips precoated with poly-L-lysine in Neurobasal medium supplemented with B27 and L-glutamine. Lentiviruses expressing Rab43N131I were prepared as described . To quaneviously , 66. TheHEK293 and NRK49F cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. SHSY5Y cells were cultured in F12/Minimum essential medium (V/V = 1:1) with 10% fetal bovine serum. Transient transfection of cells was carried out using Lipofectamine 2000 or 3000 reagent as described previously .The CRISPR–Cas9 Rab43KO plasmids targeting mouse or human Rab43, as well as control plasmids, were purchased from Santa Cruz Biotechnology. The Rab43KO plasmid consists of a pool of three plasmids, each encoding the Cas9 nuclease and a target-specific 20-nt sgRNA. Three sgRNA sequences in mouse Rab43 are TGCCATCGAGACCTCCGCAA, AGTCAGACCTTGCCGATTTC, and CTCACATCCTCGATCCAGTG, which correspond to the sequences TGCCATCGAGACCTCTGCCA, AGTCAGACCTTGCTGATCTC, and CACTGGATTGAGGATGTGAG in rat Rab43. The efficiency of Rab43KO plasmids was tested by using exogenously transfected GFP–Rab43 in HEK293 cells. In brief, HEK293 cells were cultured on 6-well dishes and transfected with 0.5 μg of Rab43KO plasmids. After 6 h, the cells were split at a ratio of 1:2 and cultured overnight. The cells were then transfected with 1.5 μg of Rab43KO plasmids together with 0.5 μg of GFP–Rab43 for additional 24 h. The expression of GFP–Rab43 in total cell lysates was measured by Western blotting using GFP antibodies. To determine the effect of Rab43KO on the subcellular localization of receptors, hippocampal neurons or cells were cultured on 12-well dishes and transfected with Rab43KO plasmids plus individual receptors (0.5 μg each) for 30 h before imaging. In rescue experiments, NRK49F cells were transfected with DsRed-tagged human Rab43, Rab43KO plasmids targeting mouse Rab43 and M3R–CFP (0.5 μg each) for 30 h. As Rab43KO plasmids carry GFP, transfected neurons or cells were defined by the GFP signal.2-AR and mAChR in live neurons and cells was quantified by radioligand binding using [3H]-RX821002, which binds all three α2-AR subtypes and [3H]-NMS, which binds all five mAChR subtypes as described previously . All radioligand binding assays were performed in duplicate.The surface expression of endogenous αeviously , 67, 68.Primary cortical neurons were cultured on 6-well dishes and infected with control or Rab43N131I lentiviruses for 48 h. After starvation for 48 h, the neurons were incubated with Neurobasal medium containing UK14304 at 1 μM or Oxo-M at 10 μM for 5 min. The neurons were then washed twice with cold PBS and solubilized by the addition of 100 μl of the SDS gel-loading buffer. ERK1/2 activation was determined by measuring their phosphorylation by Western blotting as described previously , 63.For image acquisition and quantification of receptor expression at dendrites and postsynapses, hippocampal neurons were fixed with 4% paraformaldehyde and 4% sucrose for 15 min and washed with PBS for three times. Images were captured with a 40× objective on a Zeiss LSM780 confocal microscope. Confocal images were analyzed and quantified with the ImageJ software . Dendritic receptor expression was measured as the dendritic area expressing individual receptors as described previously . To measFor analysis of receptor localization in SHSY5Y and NRK49F cells, the cells were grown on coverslips precoated with poly-L-lysine on 6-well dishes and transfected with 50 ng of receptors together with 150 ng of Rab43N131I or Rab43KO plasmids for 48 h. To study receptor colocalization with calregulin, the cells were fixed and permeabilized with PBS containing 0.2% Triton X-100 for 5 min. After blocking with 5% normal donkey serum for 1 h, the cells were sequentially stained with primary antibodies against calregulin (1:100 dilution) for 1 h and fluorophore-conjugated secondary antibodies (1:2000 dilution) for 1 h. Images were captured with a 63× objective on a Zeiss LSM780 confocal microscope as described previously .All data presented are available upon request from Guangyu Wu (The authors declare that they have no conflict of interest with the contents of this article.