Olanzapine suppresses mPFC activity-norepinephrine releasing to alleviate CLOCK-enhanced cancer stemness under chronic stress.

BACKGROUND: Olanzapine (OLZ) reverses chronic stress-induced anxiety. Chronic stress promotes cancer development via abnormal neuro-endocrine activation. However, how intervention of brain-body interaction reverses chronic stress-induced tumorigenesis remains elusive. METHODS: KrasLSL-G12D/WT lung cancer model and LLC1 syngeneic tumor model were used to study the effect of OLZ on cancer stemness and anxiety-like behaviors. Cancer stemness was evaluated by qPCR, western-blotting, immunohistology staining and flow-cytometry analysis of stemness markers, and cancer stem-like function was assessed by serial dilution tumorigenesis in mice and extreme limiting dilution analysis in primary tumor cells. Anxiety-like behaviors in mice were detected by elevated plus maze and open field test. Depression-like behaviors in mice were detected by tail suspension test. Anxiety and depression states in human were assessed by Hospital Anxiety and Depression Scale (HADS). Chemo-sensitivity of lung cancer was assessed by in vivo syngeneic tumor model and in vitro CCK-8 assay in lung cancer cell lines. RESULTS: In this study, we found that OLZ reversed chronic stress-enhanced lung tumorigenesis in both KrasLSL-G12D/WT lung cancer model and LLC1 syngeneic tumor model. OLZ relieved anxiety and depression-like behaviors by suppressing neuro-activity in the mPFC and reducing norepinephrine (NE) releasing under chronic stress. NE activated ADRB2-cAMP-PKA-CREB pathway to promote CLOCK transcription, leading to cancer stem-like traits. As such, CLOCK-deficiency or OLZ reverses NE/chronic stress-induced gemcitabine (GEM) resistance in lung cancer. Of note, tumoral CLOCK expression is positively associated with stress status, serum NE level and poor prognosis in lung cancer patients. CONCLUSION: We identify a new mechanism by which OLZ ameliorates chronic stress-enhanced tumorigenesis and chemoresistance. OLZ suppresses mPFC-NE-CLOCK axis to reverse chronic stress-induced anxiety-like behaviors and lung cancer stemness. Decreased NE-releasing prevents activation of ADRB2-cAMP-PKA-CREB pathway to inhibit CLOCK transcription, thus reversing lung cancer stem-like traits and chemoresistance under chronic stress.

Deciphering the Coevolutionary Dynamics of L2 ß-Lactamases via Deep Learning.

L2 ß-lactamases, serine-based class A ß-lactamases expressed by Stenotrophomonas maltophilia, play a pivotal role in antimicrobial resistance (AMR). However, limited studies have been conducted on these important enzymes. To understand the coevolutionary dynamics of L2 ß-lactamase, innovative computational methodologies, including adaptive sampling molecular dynamics simulations, and deep learning methods (convolutional variational autoencoders and BindSiteS-CNN) explored conformational changes and correlations within the L2 ß-lactamase family together with other representative class A enzymes including SME-1 and KPC-2. This work also investigated the potential role of hydrophobic nodes and binding site residues in facilitating the functional mechanisms. The convergence of analytical approaches utilized in this effort yielded comprehensive insights into the dynamic behavior of the ß-lactamases, specifically from an evolutionary standpoint. In addition, this analysis presents a promising approach for understanding how the class A ß-lactamases evolve in response to environmental pressure and establishes a theoretical foundation for forthcoming endeavors in drug development aimed at combating AMR.

Progressively narrow the gap of PM2.5 pollution characteristics at urban and suburban sites in a megacity of Sichuan Basin, China.

Nowadays, the fine particle pollution is still severe in some megacities of China, especially in the Sichuan Basin, southwestern China. In order to understand the causes, sources, and impacts of fine particles, we collected PM2.5 samples and analyzed their chemical composition in typical months from July 2018 to May 2019 at an urban and a suburban (background) site of Chengdu, a megacity in this region. The daily average concentrations of PM2.5 ranged from 5.6-102.3 µg/m3 and 4.3-110.4 µg/m3 at each site. Secondary inorganics and organic matters were the major components in PM2.5 at both sites. The proportion of nitrate in PM2.5 has exceeded sulfate and become the primary inorganic component. SO2 was easier to transform into sulfate in urban areas because of Mn-catalytic heterogeneous reactions. In contrast, NO2 was easily converted in suburbs with high aerosol water content. Furthermore, organic carbon in urban was much greater than that in rural, other than elemental carbon. Element Cr and As were the key cancer risk drivers. The main sources of PM2.5 in urban and suburban areas were all secondary aerosols (42.9%, 32.1%), combustion (16.0%, 25.2%) and vehicle emission (15.2%, 19.2%). From clean period to pollution period, the contributions from combustion and secondary aerosols increased markedly. In addition to tightening vehicle controls, urban areas need to restrict emissions from steel smelters, and suburbs need to minimize coal and biomass combustion in autumn and winter.

Boosting the performance of molecular property prediction via graph-text alignment and multi-granularity representation enhancement.

Deep learning is playing an increasingly important role in accurate prediction of molecular properties. Prior to being processed by a deep learning model, a molecule is typically represented in the form of a text or a graph. While some methods attempt to integrate these two forms of molecular representations, the misalignment of graph and text embeddings presents a significant challenge to fuse two modalities. To solve this problem, we propose a method that aligns and fuses graph and text features in the embedding space by using contrastive loss and cross attentions. Additionally, we enhance the molecular representation by incorporating multi-granularity information of molecules on the levels of atoms, functional groups, and molecules. Extensive experiments show that our model outperforms state-of-the-art models in downstream tasks of molecular property prediction, achieving superior performance with less pretraining data. The source codes and data are available at https://github.com/zzr624663649/multimodal_molecular_property.

N-benzyl-2-methoxy-5-propargyloxybenzoamides, a new type of bleaching herbicides targeting the biosynthesis pathway of plastoquinone.

BACKGROUND: Previously, the herbicidal activity of N-benzyl-2-methoxybenzamides was discovered during a random screening program in our laboratory. The chemicals resulted in bleaching effect of newly grown leaves by interfering with the biosynthesis of ß-carotene in plant. RESULTS: A total of 28 benzamides were synthesized and subjected for the evaluation of herbicidal activity. Structure-activity relationship (SAR) showed that introducing propargyloxy group at 5-position of benzoyl-benzene ring and fluorine or methyl group at 3- or 4-position of benzyl-benzene ring is beneficial for the activity. Post-emergence herbicidal activities of compounds 406 and 412 were comparable to those of mesotrione and diflufenican. Studies on MOA showed that 406 decreased the level of both ß-carotene and plastoquinone (PQ) in treated plants. The bleaching effect in green alga caused by 406 could be reversed by supplying exogenous homogentisic acid (HGA), the precursor of plastoquinone. CONCLUSION: N-benzyl-2-methoxy-5-propargyloxybenzoamides were discovered as new candidates for bleaching herbicides. Preliminary investigation on mechanism of action (MOA) showed that the title compounds might indirectly interfere with carotenoid biosynthesis by blocking the production of PQ. © 2023 Society of Chemical Industry.

Gating interactions steer loop conformational changes in the active site of the L1 metallo-ß-lactamase.

ß-Lactam antibiotics are the most important and widely used antibacterial agents across the world. However, the widespread dissemination of ß-lactamases among pathogenic bacteria limits the efficacy of ß-lactam antibiotics. This has created a major public health crisis. The use of ß-lactamase inhibitors has proven useful in restoring the activity of ß-lactam antibiotics, yet, effective clinically approved inhibitors against class B metallo-ß-lactamases are not available. L1, a class B3 enzyme expressed by Stenotrophomonas maltophilia, is a significant contributor to the ß-lactam resistance displayed by this opportunistic pathogen. Structurally, L1 is a tetramer with two elongated loops, α3-ß7 and ß12-α5, present around the active site of each monomer. Residues in these two loops influence substrate/inhibitor binding. To study how the conformational changes of the elongated loops affect the active site in each monomer, enhanced sampling molecular dynamics simulations were performed, Markov State Models were built, and convolutional variational autoencoder-based deep learning was applied. The key identified residues (D150a, H151, P225, Y227, and R236) were mutated and the activity of the generated L1 variants was evaluated in cell-based experiments. The results demonstrate that there are extremely significant gating interactions between α3-ß7 and ß12-α5 loops. Taken together, the gating interactions with the conformational changes of the key residues play an important role in the structural remodeling of the active site. These observations offer insights into the potential for novel drug development exploiting these gating interactions.

Pyrroline-5-carboxylate reductase 1 reprograms proline metabolism to drive breast cancer stemness under psychological stress.

Cancer stem-like cells (CSCs) contribute to cancer metastasis, drug resistance and tumor relapse, yet how amino acid metabolism promotes CSC maintenance remains exclusive. Here, we identify that proline synthetase PYCR1 is critical for breast cancer stemness and tumor growth. Mechanistically, PYCR1-synthesized proline activates cGMP-PKG signaling to enhance cancer stem-like traits. Importantly, cGMP-PKG signaling mediates psychological stress-induced cancer stem-like phenotypes and tumorigenesis. Ablation of PYCR1 markedly reverses psychological stress-induced proline synthesis, cGMP-PKG signaling activation and cancer progression. Clinically, PYCR1 and cGMP-PKG signaling components are highly expressed in breast tumor specimens, conferring poor survival in breast cancer patients. Targeting proline metabolism or cGMP-PKG signaling pathway provides a potential therapeutic strategy for breast patients undergoing psychological stress. Collectively, our findings unveil that PYCR1-enhanced proline synthesis displays a critical role in maintaining breast cancer stemness.

In vitro and in vivo Activity of Phibilin Against Candida albicans.

The increase in the occurrence of antifungal-resistant Candida albicans infections necessitates more research to explore alternative effective and safe agents against this fungus. In this work, Phibilin, a new antimicrobial peptide obtained from Philomycus bilineatus and used in traditional Chinese medicine, effectively inhibits the growth and activities of C. albicans, including the clinical resistant strains. Phibilin is a fungicidal antimicrobial peptide that exhibited its antimicrobial effect against C. albicans mainly by disrupting the membrane and interacting with the DNA of the fungi. In particular, Phibilin induces the necrosis of C. albicans via the ROS-related pathway. Moreover, this antifungal compound inhibited the biofilm formation of C. albicans by preventing the development of hyphae in a dose-dependent manner. Furthermore, Phibilin and clotrimazole displayed a synergistic effect in inhibiting the growth of the fungi. In the mouse cutaneous infection model, Phibilin significantly inhibited the formation of skin abscesses and decreased the counts of C. albicans cells in the infected area. Overall, Phibilin is potentially an effective agent against skin infections caused by C. albicans.

In vitro and in vivo antibacterial activity of a lysine-rich scorpion peptide derivative.

Antimicrobial peptides are widely acknowledged as an alternative class of antimicrobial agents. In this study, a lysine-rich scorpion peptide derivative Pacavin-5K was designed, which showed an improved antibacterial spectrum, significantly higher antibacterial activity, and lower toxicity compared to the native peptide. It also showed an improved thermal and serum stability. Notably, Pacavin-5K significantly decreased the bacterial counts in the wounded region in the mouse cutaneous infection model caused by Staphylococcus aureus and Pseudomonas aeruginosa. Moreover, Pacavin-5K did not induce bacterial resistance associated with its antibacterial mechanism disrupting the membrane. Furthermore, Pacavin-5K could kill the S. aureus cells at the biofilm state. Overall, Pacavin-5K could be a potential alternative antibacterial agent against skin infection caused by S. aureus and P. aeruginosa.

Use of a stringent dimerizer-regulated gene expression system for controlled BMP2 delivery.

Gene therapy using constitutively active viral promoters to drive expression of bone morphogenetic proteins (BMPs) has been extensively evaluated as a strategy for inducing bone regeneration. However, this approach offers little control over the concentration, timing, or duration of BMP synthesis. To gain greater control over BMP kinetics, we developed a new inducible system for the controlled expression of BMP2 using a two-component transcription factor that is dimerized with rapamycin (Rap). This approach provided stringent control over BMP2 synthesis with no BMP expression detected in the uninduced state. Rapamycin or the less immunosuppressive analogue, AP21967, rapidly and reversibly induced BMP2 in a dose-dependent manner (range 0.1-10 nM). Subcutaneous implants of fibroblasts containing the Rap-inducible system in syngeneic C57BL/6 mice were highly responsive to ip Rap injection (0.1-1 mg/kg). Peak BMP2 levels were detected within 24 h of a single Rap injection and declined to undetectable levels after 8-10 days. Alternate-day Rap injections (1 mg/kg) for 6 weeks induced subcutaneous ectopic bone formation. Rap-dependent healing of a critical-sized cranial defect was also achieved using this system. This regulated BMP2 expression system will be extremely useful for examining the role of timing and sequence of BMP delivery on bone regeneration.

Gene transfer of the Runx2 transcription factor enhances osteogenic activity of bone marrow stromal cells in vitro and in vivo.

Marrow stromal cells (MSCs) have the potential to differentiate into multiple mesenchymal cell types. To harness the power of MSCs for bone regeneration, methods must be developed to direct their differentiation selectively to the osteoblast lineage. The objective of this study was to examine the feasibility of using ex vivo Runx2 gene transfer to enhance the osteogenic activity of MSCs. Primary MSCs isolated from C57BL6 mice were transduced with adenoviral vectors encoding beta-galactosidase or Runx2. Cells transduced with Ad-Runx2 expressed Runx2 protein and underwent osteoblast differentiation as measured by increases in alkaline phosphatase activity and mineralization. Time-course studies revealed that Runx2 protein was highest 1 day after transduction and declined below the limits of detection by 15 days. Osteoblast marker mRNA expression paralleled Runx2 levels. In contrast, Runx2-dependent mineralization persisted for the duration of the experiment. To assess in vivo osteogenic activity, Ad-Runx2-transduced and control MSCs were adsorbed to two different carrier scaffolds and subcutaneously implanted into C57BL6 mice. In both cases, MSCs expressing Runx2 formed substantially more bone than cells transduced with control virus. Taken together, these studies indicate that Runx2 gene transfer may be an effective route to enhance the osteogenic potential of MSCs.

Combinatorial gene therapy for bone regeneration: cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2, 4, and 7.

Bone morphogenetic proteins (BMPs) have demonstrated effectiveness as bone regeneration agents whether delivered as recombinant proteins or via gene therapy. Current gene therapy approaches use vectors expressing single BMPs. In contrast, multiple BMPs are coordinately expressed during bone development and fracture healing. Furthermore, BMPs likely exist in vivo as heterodimeric molecules having enhanced biological activity. In the present study, we test the hypothesis that gene therapy-based bone regeneration can be enhanced by expressing combinations of BMPs. For in vitro studies, mesenchymal cell lines were transduced with individual adenoviruses containing BMP2, 4, or 7 cDNA under control of a CMV promoter (AdBMP2, 4, 7) or virus combinations. Significantly, combined transduction with AdBMP2 plus AdBMP7 or AdBMP4 plus AdBMP7 resulted in a synergistic stimulation of osteoblast differentiation. This synergy is best explained by formation of BMP2/7 and 4/7 heterodimers. To test in vivo biological activity, fibroblasts were transduced with specific virus combinations and implanted into C57BL6 mice. Consistent with in vitro results, strong synergy was observed using combined AdBMP2/BMP7 treatment, which induced twofold to threefold more bone than would be predicted based on the activity of individual AdBMPs. These studies show that dramatic enhancement of osteogenesis can be achieved using gene therapy to express specific combinations of interacting regenerative molecules.

Cooperative interactions between activating transcription factor 4 and Runx2/Cbfa1 stimulate osteoblast-specific osteocalcin gene expression.

The role of ATF4 (activating transcription factor 4) in osteoblast differentiation and bone formation was recently described using ATF4-deficient mice (Yang, X., Matsuda, K., Bialek, P., Jacquot, S., Masuoka, H. C., Schinke, T., Li, L., Brancorsini, S., Sassone-Corsi, P., Townes, T. M., Hanauer, A., and Karsenty, G. (2004) Cell 117, 387-398). However, the mechanisms of ATF4 in bone cells are still not clear. In this study, we determined the molecular mechanisms through which ATF4 activates the mouse osteocalcin (Ocn) gene 2 (mOG2) expression and mOG2 promoter activity. ATF4 increased the levels of Ocn mRNA and mOG2 promoter activity in Runx2-containing osteoblasts but not in non-osteoblastic cells that lack detectable Runx2 protein. However, ATF4 increased Ocn mRNA and mOG2 promoter activity in non-osteoblastic cells when Runx2 was co-expressed. Mutational analysis of the OSE1 (ATF4-binding site) and the two OSE2s (Runx2-binding sites) in the 657-bp mOG2 promoter demonstrated that ATF4 and Runx2 activate Ocn via cooperative interactions with these sites. Pull-down assays using nuclear extracts from osteoblasts or COS-7 cells overexpressing ATF4 and Runx2 showed that both factors are present in either anti-ATF4 and anti-Runx2 immunoprecipitates. In contrast, pull-down assays using purified glutathione S-transferase fusion proteins were unable to demonstrate a direct physical interaction between ATF4 and Runx2. Thus, accessory factors are likely involved in stabilizing interactions between these two molecules. Regions within Runx2 required for ATF4 complex formation and activation were identified. Deletion analysis showed that the leucine zipper domain of ATF4 is critical for Runx2 activation. This study is the first demonstration that cooperative interactions between ATF4 and Runx2/Cbfa1 stimulate osteoblast-specific Ocn expression and suggests that this regulation may represent a novel intramolecular mechanism regulating Runx2 activity and, thereby, osteoblast differentiation and bone formation.