Facilely Fabricated Single-Site Ptδ+-O(OH)x- Species Associated with Alkali on Zirconia Exhibiting Superior Catalytic Oxidation Reactivity.

Fabrication of robust isolated atom catalysts has been a research hotspot in the environment catalysis field for the removal of various contaminants, but there are still challenges in improving the reactivity and stability. Herein, through facile doping alkali metals in Pt catalyst on zirconia (Pt-Na/ZrO2), the atomically dispersed Ptδ+-O(OH)x- associated with alkali metal via oxygen bridge was successfully fabricated. This novel catalyst presented remarkably higher CO and hydrocarbon (HCs: C3H8, C7H8, C3H6, and CH4) oxidation activity than its counterpart (Pt/ZrO2). Systematically direct and solid evidence from experiments and density functional theory calculations demonstrated that the fabricated electron-rich Ptδ+-O(OH)x- related to Na species rather than the original Ptδ+-O(OH)x-, serving as the catalytically active species, can readily react with CO adsorbed on Ptδ+ to produce CO2 with significantly decreasing energy barrier in the rate-determining step from 1.97 to 0.93 eV. Additionally, owing to the strongly adsorbed and activated water by Na species, those fabricated single-site Ptδ+-O(OH)x- linked by Na species could be easily regenerated during the oxidation reaction, thus considerably boosting its oxidation reactivity and durability. Such facile construction of the alkali ion-linked active hydroxyl group was also realized by Li and K modification which could guide to the design of efficient catalysts for the removal of CO and HCs from industrial exhaust.

Biodiversity and Bioprospecting of Fungal Endophytes from Houttuynia cordata Thunb. as a Potential Antibacterial and Anticancer Agent.

Endophytic fungi are considered a new source of bioactive compounds that have important applications in agriculture and medicine. This study aims to investigate the biodiversity and potential of endophytic fungi isolated from Houttuynia cordata Thunb. as antimicrobials and anticancer agents. Out of ten isolated endophytes, four species have never been reported to be associated with H. cordata: Ceratobasidium sp., Cladosporium sp., Phomopsis sp., and Fusarium sp. The antibacterial activity assay revealed that the ethyl acetate extract of Ceratobasidium sp. HCS-3 possessed most potent antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, its cytotoxic activity test showed the promising anticancer activity on lung cancer A549, osteosarcoma MG-63, and cervical cancer HeLa cells with IC50 of 4.55 ±1.16, 32.14 ±2.78, and 1.54 ±0.66 ppm, respectively. Furthermore, metabolite profiling identified 66 compounds suggesting that benzoic acid, farnesol, and cyclopeptides may contribute to the antibacterial activity, while 4-methoxycinnamic acid may have anticancer potential.

Dynamic changes in glymphatic function in reversible cerebral vasoconstriction syndrome.

BACKGROUND: The pathophysiology of the reversible cerebral vasoconstriction syndrome (RCVS) remains enigmatic and the role of glymphatics in RCVS pathophysiology has not been evaluated. We aimed to investigate RCVS glymphatic dynamics and its clinical correlates. METHODS: We prospectively evaluated the glymphatic function in RCVS patients, with RCVS subjects and healthy controls (HCs) recruited between August 2020 and November 2023, by calculating diffusion-tensor imaging along the perivascular space (DTI-ALPS) index under a 3-T MRI. Clinical and vascular (transcranial color-coded duplex sonography) investigations were conducted in RCVS subjects. RCVS participants were separated into acute (≤ 30 days) and remission (≥ 90 days) groups by disease onset to MRI interval. The time-trend, acute stage and longitudinal analyses of the DTI-ALPS index were conducted. Correlations between DTI-ALPS index and vascular and clinical parameters were performed. Bonferroni correction was applied to vascular investigations (q = 0.05/11). RESULTS: A total of 138 RCVS patients (mean age, 46.8 years ± 11.8; 128 women) and 42 HCs (mean age, 46.0 years ± 4.5; 35 women) were evaluated. Acute RCVS demonstrated lower DTI-ALPS index than HCs (p < 0.001) and remission RCVS (p < 0.001). A continuously increasing DTI-ALPS trend after disease onset was demonstrated. The DTI-ALPS was lower when the internal carotid arteries resistance index and six-item Headache Impact test scores were higher. In contrast, during 50-100 days after disease onset, the DTI-ALPS index was higher when the middle cerebral artery flow velocity was higher. CONCLUSIONS: Glymphatic function in patients with RCVS exhibited a unique dynamic evolution that was temporally coupled to different vascular indices and headache-related disabilities along the disease course. These findings may provide novel insights into the complex interactions between glymphatic transport, vasomotor control and pain modulation.

Astroglial gliotransmitters released via Cx43 hemichannels regulate NMDAR-dependent transmission and short-term fear memory in the basolateral amygdala.

Astrocytes release gliotransmitters via connexin 43 (Cx43) hemichannels into neighboring synapses, which can modulate synaptic activity and are necessary for fear memory consolidation. However, the gliotransmitters released, and their mechanisms of action remain elusive. Here, we report that fear conditioning training elevated Cx43 hemichannel activity in astrocytes from the basolateral amygdala (BLA). The selective blockade of Cx43 hemichannels by microinfusion of TAT-Cx43L2 peptide into the BLA induced memory deficits 1 and 24 h after training, without affecting learning. The memory impairments were prevented by the co-injection of glutamate and D-serine, but not by the injection of either alone, suggesting a role for NMDA receptors (NMDAR). The incubation with TAT-Cx43L2 decreased NMDAR-mediated currents in BLA slices, effect that was also prevented by the addition of glutamate and D-serine. NMDARs in primary neuronal cultures were unaffected by TAT-Cx43L2, ruling out direct effects of the peptide on NMDARs. Finally, we show that D-serine permeates through purified Cx43 hemichannels reconstituted in liposomes. We propose that the release of glutamate and D-serine from astrocytes through Cx43 hemichannels is necessary for the activation of post-synaptic NMDARs during training, to allow for the formation of short-term and subsequent long-term memory, but not for learning per se.

P2X7 receptors and pannexin1 hemichannels shape presynaptic transmission.

Over the last decades, since the discovery of ATP as a transmitter, accumulating evidence has been reported about the role of this nucleotide and purinergic receptors, in particular P2X7 receptors, in the modulation of synaptic strength and plasticity. Purinergic signaling has emerged as a crucial player in orchestrating the molecular interaction between the components of the tripartite synapse, and much progress has been made in how this neuron-glia interaction impacts neuronal physiology under basal and pathological conditions. On the other hand, pannexin1 hemichannels, which are functionally linked to P2X7 receptors, have appeared more recently as important modulators of excitatory synaptic function and plasticity under diverse contexts. In this review, we will discuss the contribution of ATP, P2X7 receptors, and pannexin hemichannels to the modulation of presynaptic strength and its impact on motor function, sensory processing, synaptic plasticity, and neuroglial communication, with special focus on the P2X7 receptor/pannexin hemichannel interplay. We also address major hypotheses about the role of this interaction in physiological and pathological circumstances.

Random Telegraph Noise Degradation Caused by Hot Carrier Injection in a 0.8 µm-Pitch 8.3Mpixel Stacked CMOS Image Sensor.

In this work, the degradation of the random telegraph noise (RTN) and the threshold voltage (Vt) shift of an 8.3Mpixel stacked CMOS image sensor (CIS) under hot carrier injection (HCI) stress are investigated. We report for the first time the significant statistical differences between these two device aging phenomena. The Vt shift is relatively uniform among all the devices and gradually evolves over time. By contrast, the RTN degradation is evidently abrupt and random in nature and only happens to a small percentage of devices. The generation of new RTN traps by HCI during times of stress is demonstrated both statistically and on the individual device level. An improved method is developed to identify RTN devices with degenerate amplitude histograms.

High Throughput 3D Cell Migration Assay Using Micropillar/Microwell Chips.

The 3D cell migration assay was developed for the evaluation of drugs that inhibit cell migration using high throughput methods. Wound-healing assays have commonly been used for cell migration assays. However, these assays have limitations in mimicking the in vivo microenvironment of the tumor and measuring cell viability for evaluation of cell migration inhibition without cell toxicity. As an attempt to manage these limitations, cells were encapsulated with Matrigel on the surface of the pillar, and an analysis of the morphology of cells attached to the pillar through Matrigel was performed for the measurement of cell migration. The micropillar/microwell chips contained 532 pillars and wells, which measure the migration and viability of cells by analyzing the roundness and size of the cells, respectively. Cells seeded in Matrigel have a spherical form. Over time, cells migrate through the Matrigel and attach to the surface of the pillar. Cells that have migrated and adhered have a diffused shape that is different from the initial spherical shape. Based on our analysis of the roundness of the cells, we were able to distinguish between the diffuse and spherical shapes. Cells in Matrigel on the pillar that were treated with migration-inhibiting drugs did not move to the surface of the pillar and remained in spherical forms. During the conduct of experiments, 70 drugs were tested in single chips and migration-inhibiting drugs without cell toxicity were identified. Conventional migration assays were performed using transwell for verification of the four main migration-inhibiting drugs found on the chip.

Modeling the impact of potentially harmful elements on the groundwater quality of a mining area (Nigeria) by integrating NSFWQI, HERisk code, and HCs.

With excess potentially harmful elements (PHEs), drinking water is marked unsuitable and could pose some health risks when ingested or absorbed by humans. Different age groups are exposed to varied risk levels of PHEs. Analyzing the health risks of PHEs for several age groups could provide detailed insights for effective water resources management. No known study in Ameka Pb-Zn mine province (Nigeria) investigated the health risks of PHEs in water resources for several age groups. Therefore, in this paper, the carcinogenic and non-carcinogenic health risks (due to ingestion and dermal contact) of PHEs in groundwater resources of this area were investigated for nine age groups. To achieve its aim, this study integrated novel HERisk code, NSFWQI (national sanitation foundation water quality index), and hierarchical clusters (HCs) in modeling the groundwater quality. Standard elemental composition analysis revealed that the groundwater is polluted with PHEs. The NSFWQI indicated that 15% of the analyzed water samples have moderate water quality whereas 85% are unsuitable for drinking. The HERisk code, which considered nine age groups (1 to < 2 years, 2 to < 3 years, 3 to < 6 years, 6 to < 11 years, 11 to < 16 years, 16 to < 18 years, 18 to < 21 years, 21 to < 65 years, and > 65 years), revealed that all the samples pose high chronic and cancer risks to all the age groups due to oral ingestion. However, it was realized that age groups 1 to < 16 and > 65 are posed with higher risks than age groups 18 to < 65. Overall, it was realized that all the age groups are far more exposed to ingest or absorb Se, Co, Cd, Se, As, Ni, and Pb than Cu, Fe, and Zn. Nevertheless, the health risks due to dermal absorption are far lower than the risks due to oral ingestion. Conclusively, children and aging people are more predisposed to the health threats than middle-aged populations. HCs and geospatial maps aided the spatiotemporal analysis of the groundwater quality.

Differences in susceptibility of HT-29 and A549 cells to statin-induced toxicity: An investigation using high content screening.

Statins are a group of hydroxymethylglutaryl coenzyme A reductase inhibitors that are used in the treatment of cardiovascular diseases. However, statins have been found to be cytotoxic, and many unexpected side effects have been reported in clinical applications. The susceptibilities of different cell lines toward statins are diverse, and the mechanisms of cytotoxicity remain unknown. Therefore, the present study aimed to investigate differences in the susceptibility to and mechanisms of statin-induced cytotoxicity in two cell lines, HT-29 and A549, using a high content screening-based multiparametric toxicity assay panel. We found that the two cell types exhibited differing susceptibilities to the cytotoxic effects of the different statins. Additionally, the cytotoxicity was inconsistent between different statins in the two cell lines. Four statins with strong cytotoxicity decreased the viability of HT-29 cells via the mitochondrial pathway, as evidenced by decreased mitochondrial membrane potential, and elevated mitochondrial mass, calcium release and cell apoptosis, and reactive oxygen species. In contrast, these four statins only induced a decrease in the mitochondrial membrane potential in A549 cells. The above results provide an objective reason for future evaluations of cytotoxic differences in cell types and the underlying mechanisms of cytotoxicity in different statins, and provide a good scientific basis for further research on countermeasures against statin-induced cell injuries.

Robust Adaptive HCS MPPT Algorithm-Based Wind Generation System Using Model Reference Adaptive Control.

To treat the stochastic wind nature, it is required to attain all available power from the wind energy conversion system (WECS). Therefore, several maximum power point tracking (MPPT) techniques are utilized. Among them, hill-climbing search (HCS) techniques are widely implemented owing to their various features. Regarding current HCS techniques, the rotor speed is mainly perturbed using predefined constants or objective functions, which makes the selection of step sizes a multifaceted task. These limitations are directly reflected in the overall dynamic WECS performance such as tracking speed, power fluctuations, and system efficiency. To deal with the challenges of the existing HCS techniques, this paper proposes a new adaptive HCS (AD-HCS) technique with self-adjustable step size using model reference adaptive control (MRAC) based on the PID controller. Firstly, the mechanical power fluctuations are detected, then the MRAC continuously optimizes the PID gains so as to generate an appropriate dynamic step size until harvesting the maximum power point (MPP) under the optimal tracking conditions. Looking specifically at the simulation results, the proposed AD-HCS technique exhibits low oscillations around the MPP and a small settling time. Moreover, WECS efficiency is increased by 5% and 2% compared to the conventional and recent HCS techniques, respectively. Finally, the studied system is confirmed over a 1.5 MW, gird-tied, double-fed induction generator (DFIG) WECS using MATLAB/Simulink.

Transcriptome-Wide Identification and Quantification of Caffeoylquinic Acid Biosynthesis Pathway and Prediction of Its Putative BAHDs Gene Complex in A. spathulifolius.

The phenylpropanoid pathway is a major secondary metabolite pathway that helps plants overcome biotic and abiotic stress and produces various byproducts that promote human health. Its byproduct caffeoylquinic acid is a soluble phenolic compound present in many angiosperms. Hydroxycinnamate-CoA shikimate/quinate transferase is a significant enzyme that plays a role in accumulating CQA biosynthesis. This study analyzed transcriptome-wide identification of the phenylpropanoid to caffeoylquinic acid biosynthesis candidate genes in A. spathulifolius flowers and leaves. Transcriptomic analyses of the flowers and leaves showed a differential expression of the PPP and CQA biosynthesis regulated unigenes. An analysis of PPP-captive unigenes revealed a major duplication in the following genes: PAL, 120 unigenes in leaves and 76 in flowers; C3'H, 169 unigenes in leaves and 140 in flowers; 4CL, 41 unigenes in leaves and 27 in flowers; and C4H, 12 unigenes in leaves and 4 in flowers. The phylogenetic analysis revealed 82 BAHDs superfamily members in leaves and 72 in flowers, among which five unigenes encode for HQT and three for HCT. The three HQT are common to both leaves and flowers, whereas the two HQT were specialized for leaves. The pattern of HQT synthesis was upregulated in flowers, whereas HCT was expressed strongly in the leaves of A. spathulifolius. Overall, 4CL, C4H, and HQT are expressed strongly in flowers and CAA and HCT show more expression in leaves. As a result, the quantification of HQT and HCT indicates that CQA biosynthesis is more abundant in the flowers and synthesis of caffeic acid in the leaves of A. spathulifolius.

Screening of Natural Extracts for Inhibitors against Japanese Encephalitis Virus Infection.

The mosquito-borne Japanese encephalitis virus (JEV) causes serious illness worldwide that is associated with high morbidity and mortality. Currently, there are no effective drugs approved for the treatment of JEV infection. Drug-repurposing screening is an alternative approach to discover potential antiviral agents. In this study, high-content screening (HCS) of a natural extracts library was performed, and two hit FDA-approved Na+/K+-ATPase inhibitors, ouabain and digoxin, were identified as having robust efficiency against JEV infection with the selectivity indexes over 1,000. The results indicated that ouabain and digoxin blocked the JEV infection at the replication stage by targeting the Na+/K+-ATPase. Furthermore, it was proven that ouabain significantly reduced the morbidity and mortality caused by JEV in a BALB/c mouse model. This work demonstrated that Na+/K+-ATPase could serve as the target of treatment of JEV infection, and ouabain has the potential to be developed as an effective anti-JEV drug.

Trust in the Israeli Healthcare System Among Arabs, Jewish Immigrants, and Non-immigrants.

BACKGROUND: Previous studies have shown that minorities and immigrants have low levels of trust in healthcare systems (HCSys), which might present a barrier in access to and utilization of healthcare services. We compared the levels of trust in Israel's HCSys among the Arab minority, immigrant Jews, and non-immigrant Jew sand draw on the integrative model of organizational trust to explore factors that can explain differences in the trust level within and between the study groups. METHOD: We obtained cross-sectional census data from the 2017 Social Survey of the Israel Central Bureau of Statistics. We studied levels of trust based on a survey question: "Do you have trust in the HCSys?" We used logistic regression models to compare levels of trust in HCSys among the study groups, adjusting for components of the integrative model: characteristics (sex, age, education, religiosity, and healthcare service use), abilities (private health insurance ownership), integrity (discrimination, trusting the justice system and government), and perceived risk (self-reported health). RESULTS: Our findings revealed that Arabs (odds ratio (OR) = 4.20, 95% confidence intervals (CI) = 4.17, 4.23) and immigrant Jews (OR = 2.54, 95% CI = 2.51, 2.58) had more trust in the HCSys compared to non-immigrant Jews, even after adjusting for all the component variables. Different components of the integrative model explained trust in each population group. CONCLUSION: Minority and immigrant groups had greater trust in the HCSys compared to the non-immigrant group. These findings may indicate different expectations with respect to patient-caregiver relations and HCSys utilization and raise questions regarding access to HCS and quality of care among minority and immigrant groups.

Molecular profiling of the biphasic components of hepatic carcinosarcoma by the use of targeted next-generation sequencing.

AIMS: To better understand the tumourogenesis and molecular features of hepatic carcinosarcoma (HCS). METHODS AND RESULTS: We selected 13 cases of HCS, including the clinicopathological and immunohistochemical features, and analysed the molecular alterations in separately microdissected carcinomatous and sarcomatous components in eight cases by using targeted next-generation sequencing with a panel of 329 cancer-related genes. As a result, transitional areas were observed between the two components of HCS in all cases. Concordance and overlap in genetic alterations were identified in the two histological components of the eight HCS patients, indicating the clonal relatedness of the two tumour components. The most common gene alterations found in both components were TP53 (75%, 6/8) and NF1/2 (38%, 3/8) mutations and VEGFA amplification (25%, 2/8), which may be strongly associated with HCS tumorigenesis. Unique mutations and amplifications found only in one component were also identified. Amplifications involving MET (38%, n = 3/8) and PDGFRA (25%, n = 2/8) were present only in the sarcomatous components, whereas mutation affecting ERBB4 (25%, n = 2/8) and amplifications of CCND1 and FGF3/4/19 (38%, n = 3/8) were present only in the carcinomatous components, indicating their involvement in the clonal evolution of HCS. Furthermore, multiple potential therapeutic targets were identified for HCS. CONCLUSIONS: Our findings indicate that HCS could have been of monoclonal origin, and that the diverse clonal evolution might be driven by special molecular alterations in each tumour component. Our results also identify multiple therapeutic targets of HCS, which are valuable for the personalised treatment of HCS.

UK vs US physician decision-making in the treatment of haemophilia.

INTRODUCTION: Patient-physician shared decision-making (SDM) has become increasingly seen as having a positive effect on management of chronic diseases. However, little is known of the factors that encourage SDM or how effective it may be at improving health outcomes or how cost-effective it is. AIM: To investigate the uses and applications of patient physician-SDM in the management of haemophilia and the influence of healthcare systems in the United States and the United Kingdom. METHODS: This was a qualitative study based on interviews with treatment experts in the United States and United Kingdom. A grounded theory approach was used to analyse the data from the transcribed interviews and themes that emerged as related to the decision influencers. Twelve physicians from each country were interviewed by the author. RESULTS: Treatment guidelines were viewed as having only limited applicability because of the lack of universal best options in haemophilia. The US physicians in the sample appeared to be more influenced by patient preferences than physicians in the UK, who instead tended to follow policies and standards of care more closely. Physicians in both countries commented that many of their patents had become highly knowledgeable of their bleeding disorder. US physicians were sometimes limited by insurance company policies but also reported that they were often successful in appealing insurance decisions. CONCLUSION: The research suggests that there are different influences on decision-making between healthcare systems; patients and overarching healthcare systems play a major role in how physicians treat haemophilia.

Review of high-content screening applications in toxicology.

High-content screening (HCS) technology combining automated microscopy and quantitative image analysis can address biological questions in academia and the pharmaceutical industry. Various HCS experimental applications have been utilized in the research field of in vitro toxicology. In this review, we describe several HCS application approaches used for studying the mechanism of compound toxicity, highlight some challenges faced in the toxicological community, and discuss the future directions of HCS in regards to new models, new reagents, data management, and informatics. Many specialized areas of toxicology including developmental toxicity, genotoxicity, developmental neurotoxicity/neurotoxicity, hepatotoxicity, cardiotoxicity, and nephrotoxicity will be examined. In addition, several newly developed cellular assay models including induced pluripotent stem cells (iPSCs), three-dimensional (3D) cell models, and tissues-on-a-chip will be discussed. New genome-editing technologies (e.g., CRISPR/Cas9), data analyzing tools for imaging, and coupling with high-content assays will be reviewed. Finally, the applications of machine learning to image processing will be explored. These new HCS approaches offer a huge step forward in dissecting biological processes, developing drugs, and making toxicology studies easier.

Establishment and characterization of a cell line HCS1220 from human liver metastasis of colon cancer.

BACKGROUND: To establish one primary cell line of human liver metastasis of colon cancer. METHODS: HCS1220 cell line was derived from one liver metastasis of colon cancer patient's resected tumor sample. The characterization of the cell line was defined by karyotype analysis, short tandem repeat (STR) analysis and mycoplasma contamination. Subcutaneous injection 1 × 106 cells to four BALB/c nude mice, the viable tumors were developed and diagnosed (H&E staining). The expression of biomarkers CK20 and CDX2 for colon cancer were determined by immunocytochemistry assay. RESULTS: HCS1220 cell line can grow stably and continuously passage. During the grow process, the contact loss in the growth process and superimposed growth, which could be defined as proliferation of malignant tumor. Chromosome analysis revealed the cells derived from human female. The cells were not contaminated by mycoplasma. By immunohistochemistry, the cell line was proven to express the biomarkers of colon cancer CK20 and CDX2, while a-fetoprotein, hep-1 and glypican-3 were stained negative, which demonstrated that the HCS1220 cell line originating from the intestinal tissue. CONCLUSIONS: HCS1220 cell line has the characteristics of primary human liver metastasis of colon cancer. The results of STR have genetically showed that cell line is original, which can provided cell materials for research in vitro and can also help for establishing the mechanism model of liver metastasis of colon cancer and preparing, screening and evaluating anti-tumor drugs.

RNA fluorescence in situ hybridization for high-content screening.

Single molecule RNA imaging using fluorescent in situ hybridization (FISH) can provide quantitative information on mRNA abundance and localization in a single cell. There is now a growing interest in screening for modifiers of RNA abundance and/or localization. For instance, microsatellite expansion within RNA can lead to toxic gain-of-function via mislocalization of these transcripts into RNA aggregate and sequestration of RNA-binding proteins. Screening for inhibitors of these RNA aggregate can be performed by high-throughput RNA FISH. Here we describe detailed methods to perform single molecule RNA FISH in multiwell plates for high-content screening (HCS) microscopy. We include protocols adapted for HCS with either standard RNA FISH with fluorescent oligonucleotide probes or the recent single molecule inexpensive FISH (smiFISH). Recommendations for success in HCS microscopy with high magnification objectives are discussed.

Accelerating glioblastoma drug discovery: Convergence of patient-derived models, genome editing and phenotypic screening.

Patients diagnosed with glioblastoma (GBM) continue to face a bleak prognosis. It is critical that new effective therapeutic strategies are developed. GBM stem cells have molecular hallmarks of neural stem and progenitor cells and it is possible to propagate both non-transformed normal neural stem cells and GBM stem cells, in defined, feeder-free, adherent culture. These primary stem cell lines provide an experimental model that is ideally suited to cell-based drug discovery or genetic screens in order to identify tumour-specific vulnerabilities. For many solid tumours, including GBM, the genetic disruptions that drive tumour initiation and growth have now been catalogued. CRISPR/Cas-based genome editing technologies have recently emerged, transforming our ability to functionally annotate the human genome. Genome editing opens prospects for engineering precise genetic changes in normal and GBM-derived neural stem cells, which will provide more defined and reliable genetic models, with critical matched pairs of isogenic cell lines. Generation of more complex alleles such as knock in tags or fluorescent reporters is also now possible. These new cellular models can be deployed in cell-based phenotypic drug discovery (PDD). Here we discuss the convergence of these advanced technologies (iPS cells, neural stem cell culture, genome editing and high content phenotypic screening) and how they herald a new era in human cellular genetics that should have a major impact in accelerating glioblastoma drug discovery.

Identification of a 3-Alkylpyridinium Compound from the Red Sea Sponge Amphimedon chloros with In Vitro Inhibitory Activity against the West Nile Virus NS3 Protease.

Viruses are underrepresented as targets in pharmacological screening efforts, given the difficulties of devising suitable cell-based and biochemical assays. In this study we found that a pre-fractionated organic extract of the Red Sea sponge Amphimedon chloros was able to inhibit the West Nile Virus NS3 protease (WNV NS3). Using liquid chromatography⁻mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the identity of the bioactive compound was determined as a 3-alkylpyridinium with m/z = 190.16. Diffusion Ordered Spectroscopy (DOSY) NMR and NMR relaxation rate analysis suggest that the bioactive compound forms oligomers of up to 35 kDa. We observed that at 9.4 µg/mL there was up to 40⁻70% inhibitory activity on WNV NS3 protease in orthogonal biochemical assays for solid phase extracts (SPE) of A. chloros. However, the LC-MS purified fragment was effective at inhibiting the protease up to 95% at an approximate amount of 2 µg/mL with negligible cytotoxicity to HeLa cells based on a High-Content Screening (HCS) cytological profiling strategy. To date, 3-alkylpyridinium type natural products have not been reported to show antiviral activity since the first characterization of halitoxin, or 3-alkylpyridinium, in 1978. This study provides the first account of a 3-alkylpyridinium complex that exhibits a proposed antiviral activity by inhibiting the NS3 protease. We suggest that the here-described compound can be further modified to increase its stability and tested in a cell-based assay to explore its full potential as a potential novel antiviral capable of inhibiting WNV replication.