Extraction and characterization of fiber from the flower stalk of Sansevieria cylindrica.

A number of natural fibers are being proposed for use in composite materials, especially those extracted from local plants, especially those able to grow spontaneously as they are cost-efficient and have unexplored potential. Sansevieria cylindrica, within the Asparagaceae (previously Agavacae) family, has recently been considered for application in polymer and rubber matrix composites. However, its characterization and even the sorting out of technical fiber from the stem remains scarce, with little available data, as is often the case when the fabrication of textiles is not involved. In this study, Sansevieria cylindrica fibers were separated down to the dimensions of a filament at an 8-15 micron diameter from the stem of the plant, then characterized physically and chemically, using Fourier transform infrared spectroscopy (FTIR), morphologically by scanning electron microscopy (SEM), as well as their thermal degradation, by thermogravimetric analysis (TGA). Their crystallinity surface roughness was measured by X-ray diffraction (XRD) and atomic force microscopy (AFM), respectively. The results indicate over 70% cellulose fibers content with a very high crystallinity (92%) and small crystallite size (1.45 nm), which suggests a low water absorption, with thermal degradation peaking at 294°C. Despite this, due to the significant porosity of the cellular structure, the density of 1.06 g cm-3 is quite low for a mainly cellulose fiber. Roughness measurements indicate that the porosities and foamy structure result in a highly negative skewness (-3.953), in the presence of deep valleys, which may contribute to an effective relation with a covering resin.

Network of epistatic interactions in an enzyme active site revealed by large-scale deep mutational scanning.

Cooperative interactions between amino acids are critical for protein function. A genetic reflection of cooperativity is epistasis, which is when a change in the amino acid at one position changes the sequence requirements at another position. To assess epistasis within an enzyme active site, we utilized CTX-M ß-lactamase as a model system. CTX-M hydrolyzes ß-lactam antibiotics to provide antibiotic resistance, allowing a simple functional selection for rapid sorting of modified enzymes. We created all pairwise mutations across 17 active site positions in the ß-lactamase enzyme and quantitated the function of variants against two ß-lactam antibiotics using next-generation sequencing. Context-dependent sequence requirements were determined by comparing the antibiotic resistance function of double mutations across the CTX-M active site to their predicted function based on the constituent single mutations, revealing both positive epistasis (synergistic interactions) and negative epistasis (antagonistic interactions) between amino acid substitutions. The resulting trends demonstrate that positive epistasis is present throughout the active site, that epistasis between residues is mediated through substrate interactions, and that residues more tolerant to substitutions serve as generic compensators which are responsible for many cases of positive epistasis. Additionally, we show that a key catalytic residue (Glu166) is amenable to compensatory mutations, and we characterize one such double mutant (E166Y/N170G) that acts by an altered catalytic mechanism. These findings shed light on the unique biochemical factors that drive epistasis within an enzyme active site and will inform enzyme engineering efforts by bridging the gap between amino acid sequence and catalytic function.

Exploiting the Carboxylate-Binding Pocket of ß-Lactamase Enzymes Using a Focused DNA-Encoded Chemical Library.

ß-Lactamase enzymes hydrolyze and thereby provide bacterial resistance to the important ß-lactam class of antibiotics. The OXA-48 and NDM-1 ß-lactamases cause resistance to the last-resort ß-lactams, carbapenems, leading to a serious public health threat. Here, we utilized DNA-encoded chemical library (DECL) technology to discover novel ß-lactamase inhibitors. We exploited the ß-lactamase enzyme-substrate binding interactions and created a DECL targeting the carboxylate-binding pocket present in all ß-lactamases. A library of 106 compounds, each containing a carboxylic acid or a tetrazole as an enzyme recognition element, was designed, constructed, and used to identify OXA-48 and NDM-1 inhibitors with micromolar to nanomolar potency. Further optimization led to NDM-1 inhibitors with increased potencies and biological activities. This work demonstrates that the carboxylate-binding pocket-targeting DECL, designed based on substrate binding information, aids in inhibitor identification and led to the discovery of novel non-ß-lactam pharmacophores for the development of ß-lactamase inhibitors for enzymes of different structural and mechanistic classes.

Ultrahigh Resolution Lipid Mass Spectrometry Imaging of High-Grade Serous Ovarian Cancer Mouse Models.

No effective screening tools for ovarian cancer (OC) exist, making it one of the deadliest cancers among women. Considering little is known about the detailed progression and metastasis mechanism of OC at a molecular level, it is crucial to gain more insights on how metabolic and signaling alterations accompany its development. Herein, we present a comprehensive study using ultra-high-resolution Fourier transform ion cyclotron resonance matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to investigate the spatial distribution and alterations of lipids in ovarian tissues collected from double knockout (n = 4) and a triple mutant mouse models (n = 4) of high-grade serous ovarian cancer (HGSC). Lipids belonging to a total of 15 different classes were annotated and their abundance changes compared to those in healthy mouse reproductive tissue (n = 4), mapping onto major lipid pathways involved in OC progression. From intermediate-stage OC to advanced HGSC, we provide a direct visualization of lipid distributions and their biological links to inflammatory response, cellular stress, cell proliferation, and other processes. We also show the ability to distinguish tumors at different stages from healthy tissues via a number of highly specific lipid biomarkers, providing targets for future panels that could be useful in diagnosis.

Experimental investigation and multi-objective optimization of eco-friendly near-dry electrical discharge machining of shape memory alloy using Cu/SiC/Gr composite electrode.

The near-dry electrical discharge machining processes have been conducted using air-mist or gas mist as a dielectric fluid to minimize the environmental impacts. In this article, near-dry electrical discharge machining (NDEDM) experiments have been performed to improve machining performance using an oxygen-mist dielectric fluid, a copper composite electrode, and Cu-Al-Be polycrystalline shape memory alloy (SMA) work materials. The copper composite electrode is made up of 12 wt% silicon carbide and 9 wt% graphite particles. The oxygen-mist pressure (Op), pulse on time (Ton), spark current (Ip), gap voltage (Gv), and flow rate of mixed water (Fr) were used as process parameters, and the material removal rate (MRR), tool wear rate (TWR), and surface roughness (SR) were used as performance characteristics. The global optimal alternative solution has been predicted by the PROMETHEE-II (Preference Ranking Organization METhod for Enrichment Evaluations-II) optimization technique. The best combinations of process parameters have been used to examine the microstructure of composite tools and SMA-machined surfaces by scanning electron microscopy (SEM) analysis. The best global optimum settings (oP: 9 bar, Ip: 60 µs, Ip: 12 A, Gv: 40 V, and Fr: 12 ml/min) are predicted to attain optimum machining performance (MRR: 39.049 g/min, TWR: 1.586 g/min, and SR: 1.78 µm). The tool wear rate of the NDEDM process has been significantly reduced by the copper composite electrode due to increasing microhardness, wear resistance, and melting point. When compared to the pure copper electrode tool, the MRR of NDEDM is improved to 21.91%, while the TWR and SR are reduced to 46.66% and 35.02%, respectively.

Machine Learning Reveals Lipidome Remodeling Dynamics in a Mouse Model of Ovarian Cancer.

Ovarian cancer (OC) is one of the deadliest cancers affecting the female reproductive system. It may present little or no symptoms at the early stages and typically unspecific symptoms at later stages. High-grade serous ovarian cancer (HGSC) is the subtype responsible for most ovarian cancer deaths. However, very little is known about the metabolic course of this disease, particularly in its early stages. In this longitudinal study, we examined the temporal course of serum lipidome changes using a robust HGSC mouse model and machine learning data analysis. Early progression of HGSC was marked by increased levels of phosphatidylcholines and phosphatidylethanolamines. In contrast, later stages featured more diverse lipid alterations, including fatty acids and their derivatives, triglycerides, ceramides, hexosylceramides, sphingomyelins, lysophosphatidylcholines, and phosphatidylinositols. These alterations underscored unique perturbations in cell membrane stability, proliferation, and survival during cancer development and progression, offering potential targets for early detection and prognosis of human ovarian cancer.

Discovery of Highly Potent and BMPR2-Selective Kinase Inhibitors Using DNA-Encoded Chemical Library Screening.

The discovery of monokinase-selective inhibitors for patients is challenging because the 500+ kinases encoded by the human genome share highly conserved catalytic domains. Until now, no selective inhibitors unique for a single transforming growth factor ß (TGFß) family transmembrane receptor kinase, including bone morphogenetic protein receptor type 2 (BMPR2), have been reported. This dearth of receptor-specific kinase inhibitors hinders therapeutic options for skeletal defects and cancer as a result of an overactivated BMP signaling pathway. By screening 4.17 billion "unbiased" and "kinase-biased" DNA-encoded chemical library molecules, we identified hits CDD-1115 and CDD-1431, respectively, that were low-nanomolar selective kinase inhibitors of BMPR2. Structure-activity relationship studies addressed metabolic lability and high-molecular-weight issues, resulting in potent and BMPR2-selective inhibitor analogs CDD-1281 (IC50 = 1.2 nM) and CDD-1653 (IC50 = 2.8 nM), respectively. Our work demonstrates that DNA-encoded chemistry technology (DEC-Tec) is reliable for identifying novel first-in-class, highly potent, and selective kinase inhibitors.

Machine Learning Reveals Lipidome Remodeling Dynamics in a Mouse Model of Ovarian Cancer.

Ovarian cancer (OC) is one of the deadliest cancers affecting the female reproductive system. It may present little or no symptoms at the early stages, and typically unspecific symptoms at later stages. High-grade serous ovarian cancer (HGSC) is the subtype responsible for most ovarian cancer deaths. However, very little is known about the metabolic course of this disease, particularly in its early stages. In this longitudinal study, we examined the temporal course of serum lipidome changes using a robust HGSC mouse model and machine learning data analysis. Early progression of HGSC was marked by increased levels of phosphatidylcholines and phosphatidylethanolamines. In contrast, later stages featured more diverse lipids alterations, including fatty acids and their derivatives, triglycerides, ceramides, hexosylceramides, sphingomyelins, lysophosphatidylcholines, and phosphatidylinositols. These alterations underscored unique perturbations in cell membrane stability, proliferation, and survival during cancer development and progression, offering potential targets for early detection and prognosis of human ovarian cancer. Teaser: Time-resolved lipidome remodeling in an ovarian cancer model is studied through lipidomics and machine learning.

Advancing ASMS with LC-MS/MS for the discovery of novel PDCL2 ligands from DNA-encoded chemical library selections.

BACKGROUND: A safe, effective, and reversible nonhormonal male contraceptive drug is greatly needed for male contraception as well as for circumventing the side effects of female hormonal contraceptives. Phosducin-like 2 (PDCL2) is a testis-specific phosphoprotein in mice and humans. We recently found that male PDCL2 knockout mice are sterile due to globozoospermia caused by impaired sperm head formation, indicating that PDCL2 is a potential target for male contraception. Herein, our study for the first time developed a biophysical assay for PDCL2 allowing us to screen a series of small molecules, to study structure-activity relationships, and to discover two PDCL2 binders with novel chemical structure. OBJECTIVE: To identify a PDCL2 ligand for therapeutic male contraception, we performed DNA-encoded chemical library (DECL) screening and off-DNA hit validation using a unique affinity selection mass spectrometry (ASMS) biophysical profiling strategy. MATERIALS AND METHODS: We employed the screening process of DECL, which contains billions of chemically unique DNA-barcoded compounds generated through individual sequences of reactions and different combinations of functionalized building blocks. The structures of the PDCL2 binders are proposed based on the sequencing analysis of the DNA barcode attached to each individual DECL compound. The proposed structure is synthesized through multistep reactions. To confirm and determine binding affinity between the DECL identified molecules and PDCL2, we developed an ASMS assay that incorporates liquid chromatography with tandem mass spectrometry (LC-MS/MS). RESULTS: After a screening process of PDCL2 with DECLs containing >440 billion compounds, we identified a series of hits. The selected compounds were synthesized as off-DNA small molecules, characterized by spectroscopy data, and subjected to our ASMS/LC-MS/MS binding assay. By this assay, we discovered two novel compounds, which showed good binding affinity for PDCL2 in comparison to other molecules generated in our laboratory and which were further confirmed by a thermal shift assay. DISCUSSION AND CONCLUSION AND RELEVANCE: With the ASMS/LC-MS/MS assay developed in this paper, we successfully discovered a PDCL2 ligand that warrants further development as a male contraceptive.

Ultrahigh resolution lipid mass spectrometry imaging of high-grade serous ovarian cancer mouse models.

No effective screening tools for ovarian cancer (OC) exist, making it one of the deadliest cancers among women. Considering that little is known about the detailed progression and metastasis mechanism of OC at a molecular level, it is crucial to gain more insights into how metabolic and signaling alterations accompany its development. Herein, we present a comprehensive study using ultra-high-resolution Fourier transform ion cyclotron resonance matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to investigate the spatial distribution and alterations of lipids in ovarian tissues collected from double knockout (n = 4) and triple mutant mouse models (n = 4) of high-grade serous ovarian cancer (HGSOC). Lipids belonging to a total of 15 different classes were annotated and their abundance changes were compared to those in healthy mouse reproductive tissue (n = 4), mapping onto major lipid pathways involved in OC progression. From intermediate-stage OC to advanced HGSC, we provide direct visualization of lipid distributions and their biological links to inflammatory response, cellular stress, cell proliferation, and other processes. We also show the ability to distinguish tumors at different stages from healthy tissues via a number of highly specific lipid biomarkers, providing targets for future panels that could be useful in diagnosis.

An active site loop toggles between conformations to control antibiotic hydrolysis and inhibition potency for CTX-M ß-lactamase drug-resistance enzymes.

ß-lactamases inactivate ß-lactam antibiotics leading to drug resistance. Consequently, inhibitors of ß-lactamases can combat this resistance, and the ß-lactamase inhibitory protein (BLIP) is a naturally occurring inhibitor. The widespread CTX-M-14 and CTX-M-15 ß-lactamases have an 83% sequence identity. In this study, we show that BLIP weakly inhibits CTX-M-14 but potently inhibits CTX-M-15. The structure of the BLIP/CTX-M-15 complex reveals that binding is associated with a conformational change of an active site loop of ß-lactamase. Surprisingly, the loop structure in the complex is similar to that in a drug-resistant variant (N106S) of CTX-M-14. We hypothesized that the pre-established favorable loop conformation of the N106S mutant would facilitate binding. The N106S substitution results in a ~100- and 10-fold increase in BLIP inhibition potency for CTX-M-14 and CTX-M-15, respectively. Thus, this indicates that an active site loop in ß-lactamase toggles between conformations that control antibiotic hydrolysis and inhibitor susceptibility. These findings highlight the role of accessible active site conformations in controlling enzyme activity and inhibitor susceptibility as well as the influence of mutations in selectively stabilizing discrete conformations.

Multiobjective Optimization of Fabrication Parameters of Jute Fiber/Polyester Composites with Egg Shell Powder and Nanoclay Filler.

In the present study, a model is presented to optimize the fabrication parameters of natural fiber reinforced polyester matrix composites with dual fillers. In particular, jute fiber mat was chosen as reinforcement and eggshell powder (ESP) and montmorillonite nanoclay (NC) were selected as fillers. The weight per square meter (GSM) of the fiber, the weight percentage of ESP and NC have been chosen as independent variables and the influence of these variables on tensile, flexural and impact strength of the composite has been inspected. The permutations of the different combinations of factors are intended to accomplish higher interfacial strength with the lowest possible number of tested specimens. The experiments were designed by the Taguchi strategy and a novel multi-objective optimization technique named COPRAS (COmplex PRoportional ASsessment of alternatives) was used to determine the optimal parameter combinations. Affirmation tests were performed with the optimal parameter settings and the mechanical properties were evaluated and compared. Experimental results show that fiber GSM and eggshell powder content are significant variables that improve mechanical strength, while the nanoclay appears less important.

Identifying Oxacillinase-48 Carbapenemase Inhibitors Using DNA-Encoded Chemical Libraries.

Bacterial resistance to ß-lactam antibiotics is largely mediated by ß-lactamases, which catalyze the hydrolysis of these drugs and continue to emerge in response to antibiotic use. ß-Lactamases that hydrolyze the last resort carbapenem class of ß-lactam antibiotics (carbapenemases) are a growing global health threat. Inhibitors have been developed to prevent ß-lactamase-mediated hydrolysis and restore the efficacy of these antibiotics. However, there are few inhibitors available for problematic carbapenemases such as oxacillinase-48 (OXA-48). A DNA-encoded chemical library approach was used to rapidly screen for compounds that bind and potentially inhibit OXA-48. Using this approach, a hit compound, CDD-97, was identified with submicromolar potency (Ki = 0.53 ± 0.08 µM) against OXA-48. X-ray crystallography showed that CDD-97 binds noncovalently in the active site of OXA-48. Synthesis and testing of derivatives of CDD-97 revealed structure-activity relationships and informed the design of a compound with a 2-fold increase in potency. CDD-97, however, synergizes poorly with ß-lactam antibiotics to inhibit the growth of bacteria expressing OXA-48 due to poor accumulation into E. coli. Despite the low in vivo activity, CDD-97 provides new insights into OXA-48 inhibition and demonstrates the potential of using DNA-encoded chemistry technology to rapidly identify ß-lactamase binders and to study ß-lactamase inhibition, leading to clinically useful inhibitors.

Homogeneous and Functional Group Tolerant Ring-Closing Metathesis for DNA-Encoded Chemical Libraries.

Reaction heterogeneity, poor pH control, and catalyst decomposition in the ring-closing metathesis (RCM) of DNA-chemical conjugates lead to poor yields of the cyclized products. Herein we address these issues with a RCM reaction system that includes a novel aqueous solvent combination to enable reaction homogeneity, an acidic buffer system which masks traditionally problematic functional groups, and a decomposition-resistant catalyst which maximizes conversion to the cyclized product. Additionally, we provide a systematic study of the substrate scope of the on-DNA RCM reaction, a demonstration of its applicability to a single-substrate DNA-encoded chemical library that includes sequencing analysis, and the first successful stapling of an unprotected on-DNA [i, i+4] peptide.

The genomic landscape of estrogen receptor α binding sites in mouse mammary gland.

Estrogen receptor α (ERα) is the major driving transcription factor in the mammary gland development as well as breast cancer initiation and progression. However, the genomic landscape of ERα binding sites in the normal mouse mammary gland has not been completely elucidated. Here, we mapped genome-wide ERα binding events by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in the mouse mammary gland in response to estradiol. We identified 6237 high confidence ERα binding sites in two biological replicates and showed that many of these were located at distal enhancer regions. Furthermore, we discovered 3686 unique genes in the mouse genome that recruit ER in response to estradiol. Interrogation of ER-DNA binding sites in ER-positive luminal epithelial cells showed that the ERE, PAX2, SF1, and AP1 motifs were highly enriched at distal enhancer regions. In addition, comprehensive transcriptome analysis by RNA-seq revealed that 493 genes are differentially regulated by acute treatment with estradiol in the mouse mammary gland in vivo. Through integration of RNA-seq and ERα ChIP-seq data, we uncovered a novel ERα targetome in mouse mammary epithelial cells. Taken together, our study has identified the genomic landscape of ERα binding events in mouse mammary epithelial cells. Furthermore, our study also highlights the cis-regulatory elements and cofactors that are involved in estrogen signaling and may contribute to ductal elongation in the normal mouse mammary gland.

Palladium-Catalyzed Hydroxycarbonylation of (Hetero)aryl Halides for DNA-Encoded Chemical Library Synthesis.

A strategy for DNA-compatible, palladium-catalyzed hydroxycarbonylation of (hetero)aryl halides on DNA-chemical conjugates has been developed. This method generally provided the corresponding carboxylic acids in moderate to very good conversions for (hetero)aryl iodides and bromides, and in poor to moderate conversions for (hetero)aryl chlorides. These conditions were further validated by application within a DNA-encoded chemical library synthesis and subsequent discovery of enriched features from the library in selection experiments against two protein targets.

A Mild, DNA-Compatible Nitro Reduction Using B2(OH)4.

A hypodiboric acid system for the reduction of nitro groups on DNA-chemical conjugates has been developed. This transformation provided good to excellent yields of the reduced amine product for a variety of functionalized aromatic, heterocyclic, and aliphatic nitro compounds. DNA tolerance to reaction conditions, extension to decigram scale reductions, successful use in a DNA-encoded chemical library synthesis, and subsequent target selection are also described.

Reprogramming of the estrogen responsive transcriptome contributes to tamoxifen-dependent protection against tumorigenesis in the p53 null mammary epithelial cells.

The tumor suppressor gene p53 is frequently mutated in human breast cancer and is a marker for poor prognosis and resistance to chemotherapy. Transplantation of p53 null mouse mammary epithelium into syngeneic wild-type mice leads to normal mammary gland development followed by spontaneous mammary tumors that recapitulate many of the phenotypic, molecular and genetic features of human breast cancer. Transient exposure of p53 null mice to the anti-estrogen, tamoxifen leads to sustained and robust protection against tumor development. However the mechanism underlying this anti-tumor activity remains poorly understood. Here we demonstrate that transient exposure to tamoxifen leads to a reduction in mammary ductal side-branching and epithelial cell proliferation after tamoxifen withdrawal. Global gene expression analysis showed that transient tamoxifen exposure leads to persistent changes in the expression of a subset of estrogen regulated gene signatures in mammary epithelial cells (MECs). Among these was the protein tyrosine phosphatase, non-receptor type 5 (Ptpn5). We show that Ptpn5 is a novel tamoxifen regulated target gene which is upregulated in MECs after transient tamoxifen exposure and displays tumor suppressor activity in human breast cancer cells. Further, PTPN5 expression is strongly associated with good clinical outcome in tamoxifen treated human breast cancer patients suggesting that PTPN5 may represent a novel biomarker of tamoxifen response in human breast cancer.

Stimulatory effect of insulin on theca-interstitial cell proliferation and cell cycle regulatory proteins through MTORC1 dependent pathway.

The present study examined the effect of insulin-mediated activation of the mammalian target of rapamycin complex 1 (MTORC1) signaling network on the proliferation of primary culture of theca-interstitial (T-I) cells. Our results show that insulin treatment increased proliferation of the T-I cells through the MTORC1-dependent signaling pathway by increasing cell cycle regulatory proteins. Inhibition of ERK1/2 signaling caused partial reduction of insulin-induced phosphorylation of RPS6KB1 and RPS6 whereas inhibition of PI3-kinase signaling completely blocked the insulin response. Pharmacological inhibition of MTORC1 with rapamycin abrogated the insulin-induced phosphorylation of EIF4EBP1, RPS6KB1 and its downstream effector, RPS6. These results were further confirmed by demonstrating that knockdown of Mtor using siRNA reduced the insulin-stimulated MTORC1 signaling. Furthermore, insulin-stimulated T-I cell proliferation and the expression of cell cycle regulatory proteins CDK4, CCND3 and PCNA were also blocked by rapamycin. Taken together, the present studies show that insulin stimulates cell proliferation and cell cycle regulatory proteins in T-I cells via activation of the MTORC1 signaling pathway.

Luteinizing hormone/human chorionic gonadotropin-mediated activation of mTORC1 signaling is required for androgen synthesis by theca-interstitial cells.

LH triggers the biosynthesis of androgens in the theca-interstitial (T-I) cells of ovary through the activation of a cAMP-dependent pathway. We have previously shown that LH/human chorionic gonadotropin (hCG) activates mammalian target of rapamycin complex 1 (mTORC1) signaling network, leading to cell proliferation. In the present study, we provide evidence that the LH/hCG-mediated activation of the mTORC1 signaling cascade is involved in the regulation of steroidogenic enzymes in androgen biosynthesis. Treatment with LH/hCG increased the expression of downstream targets of mTORC1, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E as well as steroidogenic enzymes. LH/hCG-mediated stimulation of the steroidogenic enzyme mRNA was blocked by the mTORC1 inhibitor, rapamycin. This inhibitory effect was selective because rapamycin failed to block hCG-mediated increase in the expression of Star mRNA levels. Furthermore, pharmacological targeting of mTORC1 with rapamycin also blocked LH/hCG- or forskolin-induced expression of cAMP response element-binding protein (CREB) and steroidogenic enzymes (P450 side-chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase type 1, and 17α-hydroxylase/17,20 lyase) but produced no effect on steroidogenic acute regulatory protein levels. These results were further confirmed by demonstrating that the knockdown of mTOR using small interfering RNA selectively abrogated the LH/hCG-induced increase in steroidogenic enzyme expression, without affecting steroidogenic acute regulatory protein expression. LH/hCG-stimulated androgen production was also blocked by rapamycin. Furthermore, the pharmacological inhibition of mTORC1 or ribosomal protein S6 kinase 1 signaling prevented the LH/hCG-induced phosphorylation of CREB. Chromatin immunoprecipitation assays revealed the association of CREB with the proximal promoter of the Cyp17a1 gene in response to hCG, and this association was reduced by rapamycin treatment. Taken together, our findings show for the first time that LH/hCG-mediated activation of androgen biosynthesis is regulated by the mTORC1 signaling pathway in T-I cells.