Target Screening Research Articles

Toxicokinetics and analytical toxicology of the phenmetrazine-derived new psychoactive substance 3,4-methylenedioxyphenmetrazine studied by means of in vitro systems.

Compounds derived from known drugs are usually brought on the new psychoactive substance (NPS) market without any previous toxicological risk assessment. The European Union Drugs Agency issued an EU early notification for 3,4-methylenedioxyphenmetrazine (MDPM) in 2024. It is structurally related to the stimulants amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), and phenmetrazine and expected to have similar effects. So far, no scientific reports are available describing its toxicokinetic and analytical profile. This study aimed to provide such data to allow a thorough risk assessment and to ease its analytical detectability in forensic and clinical toxicology and doping control. Data reported include the in vitro plasma protein binding, the in vitro half-life and in vitro metabolism of MDPM by human liver microsomes and S9 fraction (pHLS9) and by HepaRG cells. A monooxygenase mapping and the in vitro cytochrome P450 inhibition of MDPM was elucidated. Results showed that HepaRG cells and pHLS9 formed the same MDPM metabolites via demethylenation and O-methylation and that MDPM has a low plasma protein binding and is a low-turnover drug. Monooxygenase mapping revealed that the demethylenation was exclusively CYP2D6-mediated. MDPM showed strong inhibition of CYP2D6 and moderate inhibition of CYP1A2 and CYP3A4. Polymorphisms or the simultaneous intake of substances that are also CYP2D6 substrates can have a considerable impact on the toxicity of MDPM. Based on in vitro data, the demethylenyl-methyl metabolite of MDPM and the parent compound are recommended as analytical urine screening targets.

Machine learning enables high-throughput, low-replicate screening for novel anti-seizure targets and compounds using combined movement and calcium fluorescence in larval zebrafish

Machine learning enables high-throughput, low-replicate screening for novel anti-seizure targets and compounds using combined movement and calcium fluorescence in larval zebrafish

Identification and regulation of a novel leptin receptor-linked enhancer during zebrafish ventricle regeneration.

Vertebrates vary greatly in their abilities to regenerate injured hearts. Zebrafish possess a remarkable capacity for cardiac regeneration, making them an excellent model for regeneration research. Recent studies have reported the activation and underlying regulatory mechanisms of leptin b (lepb) and the leptin b-linked enhancer (LEN) in injured hearts. However, the regenerative response activity of the leptin receptor (lepr) and its regulatory mechanisms still warrant further exploration. We identified a novel lepr-linked enhancer (leprEnh) and generated a stable transgenic zebrafish line for validation. We also employed a genetic ventricle ablation system to elucidate the mechanisms governing its activation. Immunofluorescence, in situ hybridization and confocal imaging of larvae treated with various inhibitors during ventricle regeneration were performed. Our results revealed that both lepr expression and leprEnh-directed EGFP fluorescence were weakly expressed in the ventricle during early heart development but displayed a sharp increase after ventricle ablation. Strong injury response activity was also observed in the atrium. Furthermore, the regeneration-responsive activity was attenuated by hemodynamic force alteration and was modulated by Notch, ErbB2 and BMP signaling pathways. Our study sheds light on the regulation of lepr and leprEnh during heart regeneration and provide a basis for screening for novel therapeutic targets for myocardial infarction.

Cell membrane biomimetic magnetic fluorescent bifunctional nanoplatform for drug lead discovery.

Biomimetic nanoplatforms based on membrane coating strategies have received increasing attention in the field of medical research. However, it cannot perform biomedical imaging screening, which is essential for real-time identification. As a rich source of new drug discovery, traditional Chinese medicine (TCM) has made important contributions to the treatment of many diseases. Therefore, a magnetic fluorescent bifunctional nanomaterial was developed for screening the active ingredients in Fuzi. The magnetic fluorescence nanoparticles Fe3O4@GO@RhB synthesized by one-step synthesis showed low toxicity, excellent fluorescence properties and fast magnetic separation. After coating the magnetic nanoparticle with cell membrane, the screening procedure was optimized with positive drug verapamil hydrochloride. Under the optimal conditions, the active ingredients in aconite were screened, and the three active ingredients were identified as benzoyl-mesaconine, benzoyl-aconine and benzoyl-hypacoitine by UPLC-MS/MS. In addition, in situ imaging technique was used to further verify the proliferative activity of the screened active ingredients. The magnetic fluorescence bifunctional nanoplatform for cell membrane bionic screening combined with in situ imaging technology established in this paper not only can rapidly and effectively target screening and separation of active ingredients in TCM, but also provides a new platform for bioimaging screening.

Abstract TP399: Modulation of cytotoxic T cells by Poly-Glu/Tyr attenuates neuroinflammation after ischemic stroke

After ischemic stroke (IS), the cytotoxic T (Cyto T) cells infusing to the infarct site enhance the neuroinflammation. Previous studies have shown that increasing the proportion of regulatory T (Treg) cells relative to Cyto T cells can alleviate inflammatory conditions. Poly-Glu/Tyr polypeptide (Poly-YE; P-YE) was reported as a one of the key immune modulators to just modulate T cell balance. Therefore, we focused on screening target protein that affect T cell balance by P-YE. To screen target protein, we carried out protein microarray and showed that P-YE strongly bound to neutrophil cytosolic factor (NCF1) which is a one part of NOX complex. In the inflammatory response, activated NCF1 is known to bind to Moesin (MSN) to migrate forward to the membrane and assemble into the NOX complex. Also, recent study showed that unbound MSN to NCF1 induces FOXP3 expression in helper T cells. Based on this data, we hypothesized that 'NCF1+ P-YE' binding leads to the inhibition of NOX complex assembly and the conversion of Cyto T cells to Treg cells in neuroinflammation. P-YE loaded on Nanoparticles (Np P-YE; 3ug/mL) was treated to primary Cyto T (CD8+ T) cell isolated from the spleen of 8-week-old C57BL/6 male mouse. These cells were cultured in Oxygen-Glucose Deprivation-conditioned microglia medium (OGD-conditioned medium; CM) to mimic exposure to neuroinflammation. The results showed that Np P-YE fused to the cell membrane and entered to cytosol of CD8+ T cell. In the CM group treated with P-YE, superoxide was remarkably decreased and CD8+ T cells expressed FOXP3. In Co-IP, we confirmed that the binding of NCF1 and P-YE liberated MSN. Also, Proteome cytokine array showed that P-YE treatment group reduced cytokines compared with non-treatment group. In tMCAO modeling mice injected with P-YE (1mg/kg), the infarct area decreased and the pro-inflammatory phenotype of microglia was reduced. Finally, through behavior tests, the mice injected P-YE after tMCAO surgery showed improvement in stereotyped behavior and weakness due to ischemic damage. Taken together, NCF1 bound to P-YE can’t form NOX complex and MSN dissociated from NCF1 may lead to FOXP3 expression. Therefore, P-YE binds to NCF1 under neuroinflammatory conditions, converting some CD8+ T cells into CD8+FOXP3+ cells, thereby inhibiting ROS production and exerting anti-inflammatory effects.

Temporal Association Cortex Gates Sound-Evoked Arousal from NREM Sleep.

Sound-evoked wakefulness from sleep is crucial in daily life, yet its neural mechanisms remain poorly understood. It is found that CaMKIIα+ neurons in the temporal association cortex (TeA) of mice are not essential for natural awakening from sleep. However, optogenetic activation of these neurons reliably induces wakefulness from non-rapid eye movement (NREM) sleep but not from rapid eye movement (REM) sleep. In vivo electrophysiological and calcium recordings further demonstrated that TeA neurons are monotonically tuned to sound intensity but not frequency. More importantly, it is found that the activity of CaMKIIα+ neurons in TeA can gate sound-evoked arousal from NREM sleep, which is further confirmed by optogenetic manipulations. Further investigation reveals that the baseline excitability of TeA CaMKIIα+ neurons and the delta oscillations in the electroencephalogramare particularly important in regulating the evoked activity of TeA neurons. Anatomical and functional screening of downstream targets of TeA reveals that excitatory projections from TeA glutamatergic neurons to glutamatergic neurons in the basolateral/lateral amygdala are critical for modulating sound-evoked arousal from NREM sleep. These findings uncover a top-down regulatory circuit that selectively governs sound-evoked arousal from NREM sleep, with the TeA functioning as a key connecting cortex to subcortical regions.

Dedenser: A Python Package for Clustering and Downsampling Chemical Libraries.

The screening of chemical libraries is an essential starting point in the drug discovery process. While some researchers desire a more thorough screening of drug targets against a narrower scope of molecules, it is not uncommon for diverse screening sets to be favored during the early stages of drug discovery. However, a cost burden is associated with the screening of molecules, with potential drawbacks if particular areas of chemical space are needlessly overrepresented. To facilitate triaged sampling of chemical libraries and other collections of molecules, we have developed Dedenser, a tool for the downsampling of chemical clusters. Dedenser functions by reducing the membership of clusters within chemical point clouds while maintaining the initial topology or distribution in chemical space. Dedenser is a Python package that utilizes Hierarchical Density-Based Spatial Clustering of Applications with Noise to first identify clusters present in 3D chemical point clouds and then downsamples by applying Poisson disk sampling to clusters based on either their volume or density in chemical space. A command line interface tool and graphic user interface are available with Dedenser, which allow for the generation of chemical point clouds, using Mordred for QSAR descriptor calculations and uniform manifold approximation and projection for 3D embedding, as well as visualization. We hope that Dedenser will serve the community by enabling quick access to reduced collections of molecules that are representative of larger sets and selecting even distributions of molecules within clusters rather than single representative molecules from clusters. All code for Dedenser is open source and available at https://github.com/MSDLLCpapers/dedenser.

Incorporating Transformation Products for an Integrated Assessment of Antibiotic Pollution and Risks in Surface Water.

The widespread presence of antibiotics in aquatic ecosystems is a global challenge, yet the occurrence and risks associated with their transformation products (TPs) remain poorly understood. This study investigated the occurrence and potential risks of antibiotics and their TPs in water along the Chaobai River in Beijing. We used high-resolution mass spectrometry and an integrated target, suspect, and nontarget screening approach to identify 21 parent antibiotics and 78 TPs among 90 water samples, with the majority from macrolides and sulfonamides. Notably, target quantification and machine-learning-assisted semiquantification revealed that the cumulative concentrations of TPs were higher than the cumulative concentrations of parent compounds, with average contributions of TPs ranging between 50.7 and 63.7%. Most downstream water samples were largely influenced by domestic sewage, as indicated by the significantly higher concentrations and proportions of TPs, as well as the greater diversity in their composition profiles compared to upstream and reservoir samples. Moreover, of the 78 TPs, 26.9, 67.9, and 6.4% exhibited greater persistence, mobility, or toxicity than their parent antibiotics, respectively. Sixteen macrolide TPs presented both greater ecological risks to aquatic organisms and higher resistance selection risks than their parent antibiotics. TPs contributed substantially to the overall antibiotic-related risks by an average of between 31.2 and 54.1%. This study highlights the occurrence of antibiotic TPs in river water, underscoring the need to consider TPs in comprehensive risk assessments of antibiotics.

P-1185. Targeting and Universal Congenital Cytomegalovirus Screening in a Neonatal Intensive Care Unit

Abstract Background Congenital cytomegalovirus (cCMV) infection, confirmed by urine or saliva CMV PCR within 3 weeks of life, affects 0.5 % of live births in the United States. This number varies in different populations, and it is affected by socio-demographic characteristics. In Neonatal Intensive Care Units (NICUs), higher prevalence has been reported in preterm and low birth weight infants, but timing of screening remains a challenge without universal screening because only 10% of cCMV are symptomatic. Regardless of symptoms, infected infants are at increased risk of adverse outcomes such as hearing loss. Thus, we sought to determine the incidence and associated characteristics of cCMV infection using targeting and subsequently universal screening in our NICU population Methods From Feb 1, 2023 to Apr 30, 2024, infants who were admitted at Tampa General Hospital (TGH) NICU and had a CMV PCR testing performed during admission were followed prospectively. Before Feb 28, 2024, cCMV screening was performed only in infants who were symptomatic (IUGR, microcephaly, thrombocytopenia) or failed hearing screen. After that, all infants born preterm < 33 weeks gestational age (GA) and all infants with anticipated NICU stay > 3 weeks were screened for cCMV at admission. Characteristics such as GA, birth weight, and race and ethnicity are recorded in a RedCap database. Descriptive statistics were used Results A total of 715 infants were screened for CMV while hospitalized in the TGH NICU during the study period. Most of the infants (68.2%) were screened after failing newborn hearing screen and only four (0.6%) were screened because of their symptoms. The characteristics of the infants with positive and negative saliva or urine CMV PCR are described in Table 1. Only three (0.4%) infants tested positive for CMV and were confirmed to have cCMV. Two infants with cCMV infection were screened because they were symptomatic Conclusion At TGH NICU, the incidence of cCMV using targeting and universal screening was comparable with the U.S. general population and lower than reported in other NICUs. We plan to perform association when statistically feasible, and compare incidence of cCMV between the targeting and universal screening periods Disclosures Claudia M. Espinosa, MD, MSc, AstraZeneca: Grant/Research Support|Clinetics: Grant/Research Support|Enanta: Grant/Research Support|Gilead: Advisor/Consultant|Jansen & Jansen: Advisor/Consultant|Kentucky Health Rural Association: Honoraria|Medimmune: Grant/Research Support|Melinta: Grant/Research Support|Merck: Grant/Research Support|Sanofi: Advisor/Consultant|Sanofi: Honoraria

Investigation of Pyrrole Analogues Inhibition on Mpro by Spectroscopic Analysis, Molecular Docking and Enzyme Activity.

The main protease (Mpro) is a cysteine enzyme and represents a vital target for antiviral drug screening. In this work, Twenty-five pyrrole derivatives were synthesized and screened by enzyme activity experiments. Results indicate that six pyrrole derivatives can bind to Mpro and have inhibitory effect on Mpro. The binding mode involves a static quenching process. Among them, 1d exhibits the highest binding affinity. The interactions between pyrrole derivatives and Mpro are investigated by spectra and molecular docking. The interaction between 1d and Mpro is spontaneous and enthalpy-driven. Hydrogen bonding is identified as the primary binding force for 1d. Furthermore, nitro groups are important for the binding between pyrrole analogues and Mpro. This study elucidates the mechanism of interactions and provides direction and valuable insights for developing novel Mpro inhibitors.

Finding a needle in a haystack: functional screening for novel targets in cancer immunology and immunotherapies.

Genome-wide functional genetic screening has been widely used in the biomedicine field, which makes it possible to find a needle in a haystack at the genetic level. In cancer research, gene mutations are closely related to tumor development, metastasis, and recurrence, and the use of state-of-the-art powerful screening technologies, such as clustered regularly interspaced short palindromic repeat (CRISPR), to search for the most critical genes or coding products provides us with a new possibility to further refine the cancer mapping and provide new possibilities for the treatment of cancer patients. The use of CRISPR screening for the most critical genes or coding products has further refined the cancer atlas and provided new possibilities for the treatment of cancer patients. Immunotherapy, as a highly promising cancer treatment method, has been widely validated in the clinic, but it could only meet the needs of a small proportion of cancer patients. Finding new immunotherapy targets is the key to the future of tumor immunotherapy. Here, we revisit the application of functional screening in cancer immunology from different perspectives, from the selection of diverse in vitro and in vivo screening models to the screening of potential immune checkpoints and potentiating genes for CAR-T cells. The data will offer fresh therapeutic clues for cancer patients.

TDDA: A Targeted Data-Dependent Acquisition Mode for Rapid Screening of Targets in Complex Matrices.

Miniaturized mass spectrometers offer significant potential for in situ analysis due to their high specificity and portability. In traditional data-dependent acquisition (DDA) mode, precursor ions for tandem analysis are selected based on the full-scan mass spectrum. However, in situ applications often require the direct analysis of complex samples without extensive sample pretreatment, making them susceptible to chemical noise that can result in false negatives. To address this challenge, we propose a targeted data-dependent acquisition (tDDA) mode that substantially improves the accurate detection of target compounds in complex matrices. Unlike conventional DDA, the tDDA method eliminates reliance on the full-scan mass spectrum, where signals of interest are often obscured by matrix effects. This approach leverages sine amplitude modulation of sinusoidal frequency modulated (SAM-SFM) waveforms technology, which enables the real-time generation of isolated waveforms, allowing tDDA to achieve parallel, high-speed screening. Additionally, targeted automatic gain control (AGC) technology enhances the detection of low-concentration analytes, further reducing the false-negative rate. The tDDA mode was successfully integrated and validated on a modified "Brick" miniaturized ion trap mass spectrometer. Experimental results demonstrated its capability to detect low concentrations of illicit drugs spiked in blood and saliva samples, highlighting its potential for effective in situ screening.

Early markers of literacy and numeracy in Chinese children: The roles of counting and rapid automatized naming.

This study investigated the relationships between counting, Rapid Automatized Naming (RAN), and reading and arithmetic abilities in Chinese children at different developmental stages. Study 1 examined 51 kindergarteners (mean age 5.43 years) for character reading accuracy and arithmetic accuracy before formal schooling. Study 2 extended the investigation to 158 primary school children in Grades 1-3, assessing word and sentence reading fluency, arithmetic fluency, and associated cognitive factors. The findings from both studies revealed that RAN significantly predicts reading abilities across all age groups, with its importance increasing in older children. Counting was a crucial predictor for arithmetic skills, especially in early grades. While counting correlated with reading abilities in Chinese, it did not explain unique variance beyond RAN and phonological awareness. These results highlight differences across age groups in cognitive and academic skills, emphasizing the increasing role of RAN in both reading and arithmetic fluency as children progress through primary school. The study underscores the need for linguistically sensitive frameworks in education and suggests potential targets for early screening and intervention to enhance academic outcomes in Chinese children.

Causal association between circulating α-Klotho levels and venous thromboembolism: a two-sample Mendelian randomization study

Backgroundα-Klotho may involve in the occurrence and development of venous thromboembolism (VTE). However, the underlying relationship between circulating α-Klotho levels and VTE is still unclear.MethodsThis two-sample Mendelian Randomization (MR) study aims to explore the causal associations of circulating α-Klotho levels with different types of venous thromboembolism. Data of exposure and outcomes were extracted from the genome-wide association study (GWAS) of the MRC Integrative Epidemiology Unit (MRC-IEU). The fixed inverse variance weighted (IVW), MR-Egger, MR-Robust Adjusted Profile Score (RAPS) and the weighted-median methods were utilized to investigate the causal associations of circulating α-Klotho levels with different types of VTE. The effect size was expressed as odds ratios (ORs) and 95% confidence intervals (CIs), and the False Discovery Rate (FDR) test was used for correction. The MR scatter plot and leave-one-out test were used for sensitivity analysis. In addition, reverse causal associations were assessed.ResultsIVW estimates suggested that an elevated circulating α-Klotho level was associated with lower odds of deep vein thrombosis (DVT) of lower extremities (OR = 0.992, 95%CI: 0.986–0.998, P = 0.0074), pulmonary embolism (PE) (OR = 0.474, 95%CI: 0.255–0.881, P = 0.0183), and DVT of lower extremities combined with PE (OR = 0.984, 95%CI: 0.971–0.997, P = 0.0175). However, after the FDR correction, only negatively causal association between circulating α-Klotho level and increased odds of lower-extremity DVT was statistically significant (FDR P = 0.0296). Also, there were no reverse causal associations between the circulating α-Klotho levels and different types of VTE (all P > 0.05). Additionally, both the MR scatter plots and leave-one-out test results showed that these causal associations were relatively robust.ConclusionAn elevated circulating α-Klotho levels was associated with lower risk of DVT of lower extremities, PE, and DVT of lower extremities combined with PE, indicating α-Klotho has the potential to act as a target for early screening or treatment for VTE. However, the specific mechanism that α-Klotho influencing the occurrence of VTE still needed further exploration.Graphical

Elevated SREBP1 accelerates the initiation and growth of pancreatic cancer by targeting SOX9

Pancreatic cancer is a lethal disease with an insidious onset, and little is known about its early molecular events. Here, we found that the sterol regulatory element-binding protein 1 (SREBP1) expression is gradually upregulated during the initiation of pancreatic cancer. Through in vitro 3D culture of pancreatic acinar cells and experiments in LSL-KrasG12D/+;Pdx1-Cre (KC) mice, we found that pharmacological inhibition of SREBP1 suppressed pancreatic tumorigenesis. In vitro, either knockdown or pharmacological inhibition of SREBP1 suppressed tumor proliferation but SREBP1 overexpression promoted tumor proliferation. In LSL-KrasG12D/+;Trp53fl/+;Pdx1-Cre (KPC) mice, we confirmed the tumor-promoting role of SREBP1 in pancreatic cancer progression. Mechanistically, we revealed SOX9 as a downstream target of SREPB1. SREBP1 inhibition decreased SOX9 expression in both acinar cells and pancreatic cancer cells. Indeed, we identified SREBP1 binding sites in the SOX9 promoter region and reported that SOX9 is transcriptionally regulated by SREBP1. Taken together, our findings demonstrate that SREBP1/SOX9 inhibition suppresses pancreatic cancer initiation and growth, suggesting that SREBP1 could serve as a potential target for cancer screening and treatment.

Identifying MTHFD1 and LGALS4 as Potential Therapeutic Targets in Prostate Cancer Through Multi-Omics Mendelian Randomization Analysis.

Background: Prostate cancer remains one of the leading causes of cancer-related mortality in men worldwide. The treatment of it is currently based on surgical removal, radiotherapy, and hormone therapy. It is crucial to improve therapeutic prospects for the diagnosis and treatment of prostate cancer via drug target screening. Methods: We integrated eQTL data from the eQTLGen Consortium and pQTL data from UK Biobank Proteome Plasma Proteins (UKB-PPP) and deCODE health datasets. MR analyses (SMR, heterogeneity in dependent instruments (HEIDI), IVW, Wald ratio, weighted median, and MR-Egger) were used to screen candidate genes associated with prostate adenocarcinoma (PRAD) risk. Candidate genes were further verified through TCGA-based gene expression profile, survival analysis, and immune microenvironment evaluations. TIDE analysis was utilized to investigate gene immunotherapy response. Single-cell RNA sequencing data from the GSE176031 dataset were used to investigate the gene expression patterns. The Drug Bank, Therapeutic Target Database and Drug Signatures Database were utilized to predict targeted drugs for candidate genes. Results: MTHFD1 and LGALS4 were identified as promising therapeutic targets for PRAD, with evidence provided at multi-omics levels. LGALS4 was predominantly expressed in malignant cells and was correlated with enhanced immune checkpoint pathways, increased TIDE scores, and immunotherapy resistance. In contrast, MTHFD1was expressed in both tumor and microenvironmental cells and was associated with poor survival. Drug target prediction suggested that there are no currently approved drugs specifically targeting MTHFD1 and LGALS4. Conclusions: Our study identified MTHFD1 and LGALS4 as potential preventive targets for PRAD. However, future experiments are warranted to assess the utility and effectiveness of these candidate proteins.

Unraveling the Synergistic Neuroprotective Mechanism of Natural Drug Candidates Targeting TRPV1 and TRPM8 on an Ischemic Stroke.

The development of multitargeted drugs is urgent for ischemic stroke. TRPV1 and TRPM8 are important targets of ischemic stroke. Previous drug candidate screening has identified that muscone, l-borneol, and ferulic acid may target TRPV1 and TRPM8 for ischemic stroke. However, the mechanisms of these drug candidates on targets were ill-informed. Therefore, firstly, a tongue-tissue biosensor was constructed. It explored the activation or inhibition mechanisms of drug candidates targeting TRPV1 and TRPM8 in a near-physiological environment. It was found that muscone could specifically inhibit TRPM8 and selectively activate TRPV1, while l-borneol exhibited the opposite effect. It suggested a synergistic network between these two drug candidates. Furthermore, more selective protein biosensors were developed to delve deeper into the synergistic mechanisms. A strong synergistic effect of muscone and l-borneol was proved. Molecular docking revealed that the synergistic effect was caused by different action sites, respectively. Subsequently, the synergistic effect of muscone and l-borneol was further confirmed by hypoxic nerve injury models of Caenorhabditis elegans (C. elegans) and antithrombus and anti-ischemic models of zebrafish. Ultimately, through nontargeted metabolomics, it was found that muscone and l-borneol mainly regulated Ca2+ concentration and energy metabolism by pathways such as purine and amino acid metabolisms. In conclusion, this research identified critical targets and synergistic drug candidates for multitarget neuroprotection of ischemic stroke. In addition, it has systemically demonstrated the feasibility of the integration of tissue/protein biosensors and metabolomics for the research and development of multitarget drugs. Compared to other screening and validation methods for drugs and targets, the biosensors we developed not only achieved higher sensitivity and specificity in complex physiological environments, ensuring a wider detection range, but also greatly saved biological samples. Simultaneously, they could be extended to other complex systems, such as biomarker screening in clinical samples and exosomes isolated from stem cells.

The Role of de novo and Ultra-Rare Variants in Hirschsprung Disease (HSCR): Extended Gene Discovery for Risk Profiling of Patients.

Hirschsprung disease (HSCR) is a rare neurodevelopmental disorder caused by disrupted migration and proliferation of enteric neural crest cells during enteric nervous system development. Genetic studies suggest a complex etiology involving both rare and common variants, but the contribution of ultra-rare pathogenic variants (PAs) remains poorly understood. We perform whole-exome sequencing (WES) on 301 HSCR probands and 109 family trios, employing advanced statistical methods and gene prioritization strategies to identify genes carrying de novo and ultra-rare coding pathogenic variants. Multiple study designs, including case-control, de novo mutation analysis and joint test, are used to detect associated genes. Candidate genes are further prioritized based on their biological and functional relevance to disease associated tissues and onset period (i.e., human embryonic colon). We identify 19 risk genes enriched with ultra-rare coding pathogenic variants in HSCR probands, including four known genes ( RET , EDNRB , ZEB2 , SOX10 ) and 15 novel candidates (e.g., COLQ , NES , FAT3 ) functioning in neural proliferation and neuromuscular synaptic development. These genes account for 17.5% of the population-attributable risk (PAR), with novel candidates contributing 6.5%. Notably, a positive correlation between pathogenic mutational burden and disease severity is observed. Female cases exhibit at least 42% higher ultra-rare pathogenic variant burden than males (P = 0.05). This first-ever genome-wide screen of ultra-rare variants in a large, phenotypically diverse HSCR cohort highlights the substantial contribution of ultra-rare pathogenic variants to the disease risk and phenotypic variability. These findings enhance our understanding of the genetic architecture of HSCR and provide potential targets for genetic screening and personalized interventions.

MLC1 alteration in iPSCs give rise to disease-like cellular vacuolation phenotype in the astrocyte lineage.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare and progressive neurodegenerative disorder involving the white matter, is not adequately recapitulated by current disease models. Somatic cell reprogramming, along with advancements in genome engineering, may allow the establishment of in-vitro human models of MLC for disease modeling and drug screening. In this study, we utilized cellular reprogramming and gene-editing techniques to develop induced pluripotent stem cell (iPSC) models of MLC to recapitulate the cellular context of the classical MLC-impacted nervous system. Somatic cell reprogramming of peripheral patient-derived blood mononuclear cells (PBMCs) was used to develop iPSC models of MLC. CRISPR-Cas9 system-based genome engineering was also utilized to create the MLC1 knockout model of the disease. Directed differentiation of iPSCs to neural stem cells (NSCs) and astrocytes was performed in a 2D cell culture format, followed by various cellular and molecular biology approaches, to characterize the disease model. MLC iPSCs established by somatic cell reprogramming and genome engineering were well characterized for pluripotency. iPSCs were subsequently differentiated to disease-relevant cell types: neural stem cells (NSCs) and astrocytes. RNA sequencing profiling of MLC NSCs revealed a set of differentially expressed genes related to neurological disorders and epilepsy, a common clinical finding within MLC disease. This gene set can serve as a target for drug screening for the development of a potential therapeutic for this disease. Upon differentiation to the more disease relevant cell type-astrocytes, MLC-characteristic vacuoles were clearly observed, which were distinctly absent from controls. This emergence recapitulated a distinguishing phenotypic marker of the disease. Through the creation and analyses of iPSC models of MLC, our work addresses a critical need for relevant cellular models of MLC for use in both disease modeling and drug screening assays. Further investigation can utilize MLC iPSC models, as well as generated transcriptomic data sets and analyses, to identify potential therapeutic interventions for this debilitating disease.

Isolation, cytotoxicity evaluation, and molecular docking of 3,4,3’-tri-O-methylflavellagic acid from Anogeissus leiocarpus (DC.) Guill. & Perr. root

Cancer kills about 10 million people every year. Medicinal plants remain a major source in the global search for anticancer drugs. In this study, 3,4,3’-tri-O-methylflavellagic acid (MFA) was isolated from the methanol root extract of Anogeissus leiocarpus. The structure was determined by 1D- and 2D-NMR data. The cytotoxic effects of MFA were evaluated against human breast (MCF-7), colorectal (Caco-2), and cervical (HeLa) cancer cell lines using the 3-[4,5-dimethylthiazole-2-yl] 3,5-diphenyltetrazolium bromide assay. A multi-protein target screening via molecular docking was conducted against ten cancer-related proteins, and ADMET properties were evaluated. MFA exhibited the most potent activity against Caco-2 (IC50: 46.75 ± 13.00 µM). Molecular docking analysis showed that MFA had a strong binding affinity for the colchicine-binding site of αβ-tubulin and polo-like kinase-1 (binding energies: −8.5 and −8.4 kcal/mol, respectively). MFA also satisfied the Lipinski’s Rule of Five. MFA could, therefore, potentially serve as a scaffold for developing new anticancer molecules.