New roles for the de-ubiquitylating enzyme OTUD4 in an RNA-protein network and RNA granules.

Mechanisms that regulate the formation of membrane-less cellular organelles, such as neuronal RNA granules and stress granules, have gained increasing attention over the past years. These granules consist of RNA and a plethora of RNA-binding proteins. Mutations in RNA-binding proteins have been found in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). By performing pulldown experiments and subsequent mass spectrometry on mouse brain lysates, we discovered that the de-ubiquitylating enzyme OTU domain-containing protein 4 (OTUD4) unexpectedly is part of a complex network of multiple RNA-binding proteins, including core stress granule factors, such as FMRP (also known as FMR1), SMN1, G3BP1 and TIA1. We show that OTUD4 binds RNA, and that several of its interactions with RNA-binding proteins are RNA dependent. OTUD4 is part of neuronal RNA transport granules in rat hippocampal neurons under physiological conditions, whereas upon cellular stress, OTUD4 is recruited to cytoplasmic stress granules. Knockdown of OTUD4 in HeLa cells resulted in defects in stress granule formation and led to apoptotic cell death. Together, we characterize OTUD4 as a new RNA-binding protein with a suggested function in regulation of translation.

Gdf-15 deficiency does not alter vulnerability of nigrostriatal dopaminergic system in MPTP-intoxicated mice.

Growth/differentiation factor-15 (Gdf-15) is a member of the transforming growth factor-ß (Tgf-ß) superfamily and has been shown to be a potent neurotrophic factor for midbrain dopaminergic (DAergic) neurons both in vitro and in vivo. Gdf-15 has also been shown to be involved in inflammatory processes. The aim of this study was to identify the role of endogenous Gdf-15 in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease (PD) by comparing Gdf-15 (+/+) and Gdf-15 (-/-) mice. At 4 days and 14 days post-MPTP administration, both Gdf-15 (+/+) and Gdf-15 (-/-) mice showed a similar decline in DAergic neuron numbers and in striatal dopamine (DA) levels. This was followed by a comparable restorative phase at 90 days and 120 days, indicating that the absence of Gdf-15 does not affect the susceptibility or the recovery capacity of the nigrostriatal system after MPTP administration. The MPTP-induced microglial and astrocytic response was not significantly altered between the two genotypes. However, pro-inflammatory and anti-inflammatory cytokine profiling revealed the differential expression of markers in Gdf-15 (+/+) and Gdf-15 (-/-) mice after MPTP administration. Thus, the MPTP mouse model fails to uncover a major role of endogenous Gdf-15 in the protection of MPTP-lesioned nigrostriatal DAergic neurons, in contrast to its capacity to protect the 6-hydroxydopamine-intoxicated nigrostriatal system.

From petals to healing: consolidated network pharmacology and molecular docking investigations of the mechanisms underpinning Rhododendron arboreum flower's anti-NAFLD effects.

Rhododendron arboreum: Sm., also known as Burans is traditionally used as an anti-inflammatory, anti-diabetic, hepatoprotective, adaptogenic, and anti-oxidative agent. It has been used since ancient times in Indian traditional medicine for various liver disorders. However, the exact mechanism behind its activity against NAFLD is not known. The aim of the present study is to investigate the molecular mechanism of Rhododendron arboreum flower (RAF) in the treatment of NAFLD using network pharmacology and molecular docking methods. Bioactives were also predicted for their drug-likeness score, probable side effects and ADMET profile. Protein-protein interaction (PPI) data was obtained using the STRING platform. For the visualisation of GO analysis, a bioinformatics server was employed. Through molecular docking, the binding affinity between potential targets and active compounds were assessed. A total of five active compounds of RAF and 30 target proteins were selected. The targets with higher degrees were identified through the PPI network. GO analysis indicated that the NAFLD treatment with RAF primarily entails a response to the fatty acid biosynthetic process, lipid metabolic process, regulation of cell death, regulation of stress response, and cellular response to a chemical stimulus. Molecular docking and molecular dynamic simulation exhibited that rutin has best binding affinity among active compounds and selected targets as indicated by the binding energy, RMSD, and RMSF data. The findings comprehensively elucidated toxicity data, potential targets of bioactives and molecular mechanisms of RAF against NAFLD, providing a promising novel strategy for future research on NAFLD treatment.

In silico and network pharmacology analysis of fucosterol: a potent anticancer bioactive compound against HCC.

The Fucaceae family of marine brown algae includes Ascophyllum nodosum. Fucosterol (FSL) is a unique bioactive component that was identified through GC-MS analysis of the hydroalcoholic extract of A. nodosum. Fucosterol's mechanism of action towards hepatocellular cancer was clarified using network pharmacology and docking study techniques. The probable target gene of FSL has been predicted using the TargetNet and SwissTargetPred databases. GeneCards and the DisGNet database were used to check the targeted genes of FSL. By using the web programme Venny 2.1, the overlaps of FSL and HCC disease demonstrated that 18 genes (1.3%) were obtained as targeted genes Via the STRING database, a protein-protein interaction (PPI) network with 18 common target genes was constructed. With the aid of CytoNCA, hub genes were screened using the Cytoscape software, and the targets' hub genes were exported into the ShinyGo online tool for study of KEGG and gene ontology enrichment. Using the software AutoDock, a hub gene molecular docking study was performed. Ten genes, including AR, CYP19A1, ESR1, ESR2, TNF, PPARA, PPARG, HMGCR, SRC, and IGF1R, were obtained. The 10 targeted hubs docked with FSL successfully. The active components FSL of ASD, the FSL, are engaged in fatty liver disease, cancer pathways, and other signalling pathways, which could prove beneficial for the management of HCC.

Network pharmacology-based anti-colorectal cancer activity of piperlonguminine in the ethanolic root extract of Piper longum L.

Colorectal cancer (CRC) has the second highest incidence and fatality rates of any malignancy, at 10.2 and 9.2%, respectively. Plants and plants-based products for thousands of years have been utilized to treat cancer along with other associated health issues. Alkaloids are a valuable class of chemical compounds with great potential as new medicine possibilities. Piper longum Linn contains various types of alkaloids. In this research, the ethanolic root extract of P. longum (EREPL) is the subject of study based on network pharmacology. Two alkaloids were chosen from the gas chromatography mass spectrometry (GC-MS) analysis. However, only piperlonguminine received preference because it adhered to Lipinski's rule and depicted no toxicity. Web tools which are available online, like, Swiss ADME, pkCSMand ProTox-II were used to evaluate the pharmacokinetics and physiochemical properties of piperlonguminine. The database that SwissTargetPrediction and TCMSP maintain contains the targets for piperlonguminine. Using DisGeNET, GeneCards and Open Targets Platform databases, we were able to identify targets of CRC. The top four hub genes identified by Cytoscape are SRC, MTOR, EZH2, and MAPK3. The participation of hub genes in colorectal cancer-related pathways was examined using the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database. The colorectal cancer pathway, the ErbB signaling pathway and the mTOR signaling pathway emerged to be important. Our findings show that the hub genes are involved in the aforementioned pathways for tumor growth, which calls for their downregulation. Additionally, piperlonguminine has the potential to become a successful medicine in the future for the treatment of CRC.

Network pharmacology of apigeniflavan: a novel bioactive compound of Trema orientalis Linn. in the treatment of pancreatic cancer through bioinformatics approaches.

Pancreatic cancer is the seventh most prevalent cause of mortality globally. Since time immemorial, plant-derived products have been in use as therapeutic agents due to the existence of biologically active molecules called secondary metabolites. Flavonoids obtained from plants participate in cell cycle arrest, induce autophagy and apoptosis, and decrease oxidative stress in pancreatic cancer. The present study involves network pharmacology-based study of the methanolic leaf extract of Trema orientalis (MLETO) Linn. From the high-resolution mass spectrometry (HRMS) analysis, 21 nucleated flavonoids were screened out, of which only apigeniflavan was selected for further studies because it followed Lipinski's rule and showed no toxicity. The pharmacokinetics and physiochemical characteristics of apigeniflavan were performed using the online web servers pkCSM, Swiss ADME, and ProTox-II. This is the first in silico study to report the efficiency of apigeniflavan in pancreatic cancer treatment. The targets of apigeniflavan were fetched from SwissTargetPrediction database. The targets of pancreatic cancer were retrieved from DisGeNET and GeneCards. The protein-protein interaction of the common genes using Cytoscape yielded the top five hub genes: KDR, VEGFA, AKT1, SRC, and ESR1. Upon molecular docking, the lowest binding energies corresponded to best docking score which indicated the highest protein-ligand affinity. Kyoto Encyclopaedia of Genes and Genomes (KEGG) database was employed to see the involvement of hub genes in pathways related to pancreatic cancer. The following, pancreatic cancer pathway, MAPK, VEGF, PI3K-Akt, and ErbB signaling pathways, were found to be significant. Our results indicate the involvement of the hub genes in tumor growth, invasion and proliferation in the above-mentioned pathways, and therefore necessitating their downregulation. Moreover, apigeniflavan can flourish as a promising drug for the treatment of pancreatic cancer in future.

Network pharmacology-based anti-pancreatic cancer potential of kaempferol and catechin of Trema orientalis L. through computational approach.

In pancreatic cancer, healthy cells in the pancreas begin to malfunction and proliferate out of control. According to our conventional knowledge, many plants contain several novel bioactive compounds, having pharmaceutical applications for the treatment of disease like pancreatic cancer. The methanolic fraction of fruit extract of Trema orientalis L. (MFETO) was analysed through HRMS. In this in silico study, pharmacokinetic and physicochemical properties of the identified flavonoids from MFETO were screened out by ADMET analysis. Kaempferol and catechin followed Lipinski rules and showed no toxicity in Protox II. Targets of these compounds were taken from SwissTarget prediction and TCMSP whilst targets for pancreatic cancer were taken from GeneCards and DisGeNET databases. The protein-protein interaction (PPI) network of common genes was generated through STRING and then exported to the Cytoscape to get top 5 hub genes (AKT1, SRC, EGFR, TNF, and CASP3). The interaction between compounds and hub genes was analysed using molecular docking, and high binding affinity between them can be visualised by Biovia discovery studio visualizer. Our study shows that, five hub genes related to pancreatic cancer play an important role in tumour growth induction, invasion and migration. Kaempferol effectively check cell migration by inhibiting ERK1/2, EGFR-related SRC, and AKT pathways by scavenging ROS whilst catechin inhibited TNFα-induced activation and cell cycle arrest at G1 and G2/M phases by induction of apoptosis of malignant cells. Kaempferol and catechin containing MFETO can be used for formulation of potent drugs for pancreatic cancer treatment in future.

Tissue morphology influences the temporal program of human brain organoid development.

Progression through fate decisions determines cellular composition and tissue architecture, but how that same architecture may impact cell fate is less clear. We took advantage of organoids as a tractable model to interrogate this interaction of form and fate. Screening methodological variations revealed that common protocol adjustments impacted various aspects of morphology, from macrostructure to tissue architecture. We examined the impact of morphological perturbations on cell fate through integrated single nuclear RNA sequencing (snRNA-seq) and spatial transcriptomics. Regardless of the specific protocol, organoids with more complex morphology better mimicked in vivo human fetal brain development. Organoids with perturbed tissue architecture displayed aberrant temporal progression, with cells being intermingled in both space and time. Finally, encapsulation to impart a simplified morphology led to disrupted tissue cytoarchitecture and a similar abnormal maturational timing. These data demonstrate that cells of the developing brain require proper spatial coordinates to undergo correct temporal progression.