Cell Monolayers Research Articles

Construction of Nicotinamide Mononucleotide-Loaded Liposomes and Their In Vitro Transport Across the Blood–Brain Barrier

Nicotinamide mononucleotide (NMN) possesses a variety of physiological functions and has therapeutic effects on cardio-cerebral diseases, senile degenerative diseases, neurodegenerative diseases, delayed aging, etc. However, its ability to cross the blood–brain barrier (BBB) and the mechanism of its transport have not been reported. In this study, we used the immortalized hCMEC/D3 cell line to construct an in vitro monolayer cell BBB model, evaluated its ability to cross the blood–brain barrier, and explored the mechanism by carrying out transport and efflux experiments on NMN. The ability of NMN to cross the BBB was investigated by preparing NMN-loaded liposomes conjugated with ANG peptide and RVG peptide. The results showed that the transmembrane transport ability of NMN was moderate, and the transport mechanism was passive transport relying on the concentration difference. The trans-BBB ability of ANG peptide coupled with NMN could be highly significantly improved.

Trans-nasal brain delivery of anti-TB drugs by methyl-β-cyclodextrin microparticles show efficient mycobacterial clearance from central nervous system.

Central nervous system tuberculosis (CNS-TB) is the most severe extra-pulmonary manifestation of tuberculosis (TB), facing significant challenges due to the limited penetration of anti-TB drugs (ATDs) across the blood-brain barrier (BBB) and their insufficient concentrations at the site of infection. This study aimed to enhance the efficacy of ATDs by encapsulating them in methyl-β-cyclodextrin (M-β-CD) microparticles (ATD-MP) using spray drying, intended for intranasal delivery to manage CNS-TB. M-β-CD microparticles loaded with isoniazid (INH) and rifampicin (RIF) exhibited spherical shapes with slightly deflated surfaces and particle sizes of 6.24 ± 0.77 μm and 5.97 ± 0.50 μm, respectively. M-β-CD improved the permeation of ATDs through RPMI-2650 cell monolayers while reducing drug cytotoxicity. Pharmacokinetic and biodistribution analysis demonstrated that intranasal administration of ATD-MP significantly enhanced the trans-nasal brain delivery of ATDs and their distribution in the brain, achieving the minimum inhibitory concentration. In a murine model of CNS-TB, intranasal insufflation of ATD-MP for four weeks led to a significant reduction (~0.78 Log10 CFU) in mycobacterial burden in the brain compared to the untreated group (~3.60 Log10 CFU). These preclinical results underscore the potential of intranasal administration of M-β-CD microparticles as an effective therapeutic strategy for combating brain inflammation in CNS-TB.

Optimization of sulforaphane bioavailability from a glucoraphanin-rich broccoli seed extract in a model of dynamic gastric digestion and absorption by Caco-2 cell monolayers.

Broccoli is recognized for its health benefits, attributed to the high concentrations of glucoraphanin (GR). GR must be hydrolyzed by myrosinase (Myr) to form the bioactive sulforaphane (SF). The primary challenge in delivering SF in the upper gastrointestinal (GI) tract- is improving hydrolysis of GR to SF. Here, we optimized the formulation and delivery methods to improve GR conversion and SF bioavailability. We investigated whether the combination of GR-rich broccoli seed extract powder (BSE[GR]) with Myr-rich mustard seed powder (MSP[Myr]), ± ascorbic acid (AA, a co-factor of Myr), delivered as free powder or encapsulated powder, can: (i) facilitate GR hydrolysis to SF during dynamic in vitro gastric digestion and static in vitro small intestinal digestion, and (ii) increase SF bioavailability in Caco-2 cell monolayers, a model of human intestinal epithelium. Addition of exogenous Myr increased the conversion of GR to SF in free powder during small intestinal digestion, but not during gastric digestion, where Myr activity was inhibited by the acidic environment. Capsule delivery of BSE[GR]/MSP[Myr] (w/w ratio 4 : 1) resulted in a 2.5-fold higher conversion efficiency compared to free powder delivery (72.1% compared to 29.3%, respectively). AA combined with MSP[Myr] further enhanced the conversion efficiency in small intestinal digestion and the bioavailability of SF in Caco-2 cell monolayers. Bioavailability of GR as SF, SF metabolites, and GR was 74.8% in Caco-2 cell monolayers following 30 min gastric digestion and 60 min small intestinal digestion. This study highlights strategies to optimize GR bioconversion in the upper GI tract leading to enhanced SF bioavailability.

Evaluation of binding activities of a putative lipoprotein LIC_13355 of Leptospira spp.

Pathogenic Leptospira is the etiological cause of the zoonotic life-threatening infection called leptospirosis. The disease is spread worldwide with higher risk in tropical regions. Although leptospirosis represents a burden to the health of humans and animals, the pathogenic mechanisms of Leptospira infection are yet to be clarified. Leptospirosis infection is multifactorial, involving functionally redundant proteins with the capability to invade, disseminate, and escape the host's immune response. In this work, we describe a putative lipoprotein encoded by the gene LIC_13355, genome annotated as hypothetical of unknown function. The coding sequence is conserved among pathogenic Leptospira spp. with high percentage of coverage and identity. The recombinant protein, rLIC_13355, was expressed in Escherichia coli host system in its insoluble form. The circular dichroism spectrum of the refolded protein showed it containing a mixture of secondary structures. rLIC_13355 interacts with extracellular matrix (ECM) component laminin and binds plasminogen (PLG), generating plasmin (PLA), thus possibly participating during the adhesion and dissemination processes. The rLIC_13355 has the ability to interact with Ea.hy926 and HMEC-1 endothelial cells either in monolayer or suspension. The binding of rLIC_13355 with monolayer cells is dose-dependent on protein concentration. Taken together, our data suggest that this is presumably an adhesion lipoprotein that may play diverse roles in host-Leptospira interactions by mediating the interaction with host components and with endothelial cell.

Exploring the wound healing potential of Ixora coccinea and Rhododendron arboreum formulation: integrating experimental and computational approaches.

Wound healing is a complex biological process involving numerous cellular and molecular events. Ixora coccinea and Rhododendron arboreum flowers have been traditionally used for their medicinal properties, prompting an investigation into their combined effects on wound healing using both invitro and insilico approaches. Ixora and Rhododendron flowers were processed in a 1:1 ratio using an ethanolic solvent. Various concentrations of the extracts were applied to wounded mouse fibroblast cell monolayers (3T3-L1). Antioxidant potential was evaluated by DPPH and H2O2 assays, while anti-inflammatory effects were assessed using BSA and EA assays. Wound closure kinetics were monitored with image analysis software. Molecular docking studies examined interactions between active compounds and essential wound-healing proteins. The formulations inhibited ROS production at a low concentration (IC50∼1.38 μg/mL), indicating suitability for managing oxidative stress. The extracts also showed protein denaturation inhibition with an IC50 value of 14.5 μg/mL for BSA and 8.3 μg/mL for EA. In vitro, the combined extracts significantly enhanced wound closure compared to control groups, with higher concentrations (40 μg/mL) accelerating closure rates (99.66 %). Molecular docking revealed interactions between key compounds (Quercetin, Rutin) and essential wound healing proteins (MMP9, TGFβ1, IGFR), suggesting mechanisms underlying their therapeutic effects. In vitro and insilico findings suggest that Ixora and Rhododendron flower extracts promote wound closure and their interaction with key proteins in wound healing pathways, highlighting their potential therapeutic value.

Anti-Inflammatory Potential of 3-Hydroxy-β-Ionone from Moringa oleifera: Decreased Transendothelial Migration of Monocytes Through an Inflamed Human Endothelial Cell Monolayer by Inhibiting the IκB-α/NF-κB Signaling Pathway.

Moringa leaves provide numerous health benefits due to their anti-inflammatory properties. This study presents the first evidence that endothelial cell inflammation can potentially be ameliorated by moringa leaf extract. Here, we established an experimental human blood vessel cell model of inflammation using EA.hy926 cells. TNF-α was added after pre-treating the cells with crude leaf extract from Moringa oleifera Lam., a constituent fraction of the extract, and the bioactive component 3-hydroxy-β-ionone. The extract and the active ingredient significantly decreased the levels of pro-inflammatory mediators such as IL-6, IL-8, and MCP-1; decreased IκB-α and NF-κB p65 phosphorylation; and decreased the expression of VCAM-1, PECAM-1, and ICAM-1, three significant adhesion molecules. Furthermore, they attenuated THP-1 monocyte adhesion to the EA.hy926 monolayer and decreased monocyte transmigration across the monolayer. These findings suggest that 3-hydroxy-β-ionone and moringa leaf extract have anti-inflammatory properties and can be used as therapeutic agents to reduce the progression of diseases involving the inflamed endothelium by decreasing the production of inflammatory cytokines, chemokines, and adhesion molecules. This is promising for conditions such as atherosclerosis and neuroinflammation.

Unlocking Nature's Potential: Ferritin as a Universal Nanocarrier for Amplified Cancer Therapy Testing via 3D Microtissues.

In the existing development of extensive drug screening models, 3D cell cultures outshine conventional 2D monolayer cells by closely imitating the in vivo tumor microenvironment. This makes 3D culture a more physiologically relevant and convenient system in the regime of preclinical drug testing. In the nanomedicinal world, nanoconjugates as nanocarriers are largely hunted due to their capability of precisely binding to target cells and distributing essential dosages of therapeutic drugs with enhanced safety profiles. Thus, for boosted drug availability, the evolution from conventional drug treatment to combination therapies and last switching to drug carriers has gained significant progression in cancer cure. In contrast to conventional engineered nanoparticles, herein, we successfully designed biomolecule (ferritin)-based drug nanoconjugates effective both as a single drug (valproic acid-VPA) and twin-drug (valproic acid/doxorubicin-Dox) carriers, which dramatically enhance the proficiency of the tumor therapeutic modality. To question the reported adjuvant drug property of VPA, we progressed utilizing at first VPA alone as an effective yet exclusive tumor therapy when delivered via some carrier molecule, in particular protein. Subsequently, we paralleled this comprehensive investigation output to compare and test the coloading strategy of drugs and observe the synergistic and/or additive behavior of VPA in conjugation with other anticancer agents (Dox) while given via a carrier molecule. To approach this, VPA and/or Dox molecules were encapsulated into the ferritin (F) cavity using a thermosensitive synthesis method by maintaining the temperature at 60 °C. The successful encapsulation of drugs in the protein nanocage was confirmed through various characterization techniques. The F-VPA/F-VPA-Dox nanoconjugates exhibited similar morphology and structural characteristics to the hollow ferritin cage and showed significant cytotoxicity than the naked drugs when tested on physiologically relevant 3D spheroid models. Precisely, our first designed carrier nanoconjugate, i.e., F-VPA, offered more than a 3-fold increased intratumoral drug concentration than free VPA and significantly suppressed tumor growth after a single-dose treatment. However, our second modeled carrier nanoconjugate, viz. F-VPA-Dox, revealed an extended median survival period and lesser toxicity when administered at a much more effective dose (∼3-5 μM), in 3D tumor spheroid models of various cancer cell lines. All in all, importantly, ferritin nanoconjugates exhibited an enhanced tumor inhibition rate with a single-dose treatment, which further confirms the benefits of the active targeting property of these nanocarriers. Moreover, these nanocarriers also offer to deliver a significant dose of the therapeutic drug into tumor cells, alongside tremendous biocompatibility and safety profiles in numerous tumor 3D spheroid models.

Effects of the Tobacco Defensin NaD1 Against Susceptible and Resistant Strains of Candida albicans.

Today, Candida albicans is still the most common cause of both local and life-threatening systemic candidiasis. The spread of resistant fungal strains has resulted in an urgent need to search for new promising antimycotics. Here, we investigated the antifungal action of the tobacco defensin NaD1 against susceptible and resistant to azoles and echinocandins strains of C. albicans. We demonstrated that NaD1 was equally effective and fungicidal against all tested strains. The MIC and MFC values were 6.25 and 12.5 µM, respectively. We showed for the first time that NaD1 could act synergistically not only with caspofungin but also with human host defense antimicrobial peptides cathelicidin LL-37 and β-defensin-2 (HBD2) against susceptible and resistant fungal strains. Using flow cytometry, we demonstrated that NaD1 in combinations with LL-37 or HBD2 can reinforce each other by enhancing membrane disruption. Using the Caco-2 cell monolayer model, we demonstrated that NaD1 impaired the adhesion of C. albicans cells to the human epithelium. Moreover, NaD1 inhibited the formation of fungal biofilms in Sabouraud broth and less markedly in nutrient-rich RPMI-1640 medium, and enhanced the antibiofilm activity of caspofungin. Thus, we hypothesized that NaD1 might affect the development of candidiasis in vivo, including that caused by resistant fungal strains.

High resolution imaging and interpretation of three-dimensional RPE sheet structure.

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells which plays an essential role in visual function via its interaction with the adjacent neural retina. Typically hexagonal in shape and arranged in a mosaic-like pattern, RPE cells maintain a relatively uniform size and arrangement in healthy eyes. Under stress or disease conditions such as age-related macular degeneration (AMD) and other heritable vision disorders, individual RPE cell dysmorphia has been observed. This has led to investigation of potential cellular compensatory mechanisms which may be dysregulated, affecting proper barrier structure and function. A commonly observed dysmorphic trait is that of enlarged cells which appear to be multinucleated (containing more than two nuclei) when viewed in two-dimensional (2D), immunohistochemically labeled images from the apical surface perspective. One explanation for the multinucleation is that of ongoing cellular fusion which the RPE may be employing to maintain cell-to-cell contact while simultaneously conserving cellular resources in unhealthy tissue. While this may be the most likely interpretation, caution should be applied when interpreting traditional (2D) images which only use cell border outline markers in the absence of lateral markers. Here we present two examples of high-resolution confocal images which allow for three-dimensional (3D) viewing of a traditional apical border delineation marker (ZO-1) and nuclei as well as labeling of alpha catenin which can serve as a lateral cell membrane marker. We find multiple examples in two separate RPE damage models where enlarged, seemingly multinucleate, cells are in actuality not multinucleate and instead appear this way due to surrounding cell nuclei and lateral cell membrane displacement towards the central cell. When viewed from the apical surface, these nuclei appear contained by the ZO-1 border, however when viewed from multiple angles it becomes apparent that this is not the case. This approach calls for more careful analyses in future studies investigating RPE sheet dysmorphia as this could lead to potential misinterpretation of the multinucleation phenomenon and by extension, the potential underlying fusion mechanism.

Toward “Green” Vessels: Characterization of Microstructure, Mechanics, and Endothelial Cell Interaction on Three Macro‐Tubular Plants for Vascular Tissue Engineering Applications

AbstractVascular tissue engineering aims to create vessel models for in vitro research and develop vascular grafts for in vivo applications using tubular scaffolds. Natural scaffolds outperform synthetic ones due to their biocompatibility and natural microenvironment supporting cell growth. Given the importance of producing biocompatible tubular scaffolds through cost‐effective and uncomplicated processes, this study introduces nature‐derived tubular structures from three decellularized tubular plants (Water Spinach, Green Onion, and Water Horsetail) as novel alternatives. Microstructural characterization on the luminal surfaces of the plants reveals unique surface topography for each. Water Spinach is the most promising graft candidate in suturability tests besides presenting the highest elongation before rupture in tensile test. Assessment of human endothelial cells on the luminal surfaces of decellularized scaffolds shows higher expression of Ki‐67 protein and a consistent increase in cell number on water spinach and green onion scaffolds compared to tissue culture plate as a control. Focal adhesion‐related molecule Vinculin is expressed more than twice on all scaffolds compared to control, and confluent cell monolayers are formed on water spinach and green onion scaffolds, as confirmed by VE‐cadherin. This study proposes an innovative approach to use the natural structure of macro‐tubular plants for the preparation of vascular scaffolds.

Modeling development of breast cancer: from tumor microenvironment to preclinical applications.

Breast cancer is a complex disease and its progression is related not only to tumor cells but also to its microenvironment, which can not be sufficiently reflected by the traditional monolayer cell culture manner. The novel human cancer models comprising tumor microenvironment (TME), such as tumor organoids and organs-on-a-chip, has been established in recent years to help elucidate the underlying mechanisms of tumorigenesis and promote the development of cancer therapies. In this review, we first discuss the current state of breast cancer and their treatment strategies, and elucidates the complex properties of TME of breast cancer in vivo. The culture models used in breast cancer research are then summarized with insights into recent development. Finally, we also conclude by discussing the current limitations and future directions of culture models in breast cancer research for providing a preclinical reference for the precise treatment of cancer patients.

Dynamic three dimensional environment for efficient and large scale generation of smooth muscle cells from hiPSCs

BackgroundChronic ischemic limb disease often leads to amputation, which remains a significant clinical problem. Smooth-muscle cells (SMCs) are crucially involved in the development and progression of many cardiovascular diseases, but studies with primary human SMCs have been limited by a lack of availability. Here, we evaluated the efficiency of two novel protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into SMCs and assessed their potency for the treatment of ischemic limb disease.MethodshiPSCs were differentiated into SMCs via a conventional two-dimensional (2D) protocol that was conducted entirely with cell monolayers, or via two protocols that consisted of an initial five-day three-dimensional (3D) spheroid phase followed by a six-day 2D monolayer phase (3D + 2D differentiation). The 3D phases were conducted in shaker flasks on an orbital shaker (the 3D + 2D shaker protocol) or in a PBS bioreactor (the 3D + 2D bioreactor protocol). Differentiation efficiency was evaluated via the expression of SMC markers (smooth-muscle actin [SMA], smooth muscle protein 22 [SM22], and Calponin-1), and the biological activity of the differentiated hiPSC-SMCs was evaluated via in-vitro assessments of migration (scratch assay), contraction in response to the treatment with a prostaglandin H2 analog (U46619), and tube formation on Geltrex, as well as in-vivo measurements of perfusion (fluorescence angiography) and vessel density in the limbs of mice that were treated with hiPSC-SMCs after experimentally induced hind-limb ischemia (HLI).ResultsBoth 3D + 2D protocols yielded > 5.6 × 107 hiPSC-SMCs/differentiation, which was ~ nine-fold more than that produced via 2D differentiation, and flow cytometry analyses confirmed that > 98% of the 3D + 2D-differentiated hiPSC-SMCs expressed SMA, > 81% expressed SM22, and > 89% expressed Calponin-1. hiPSC-SMCs obtained via the 3D + 2D shaker protocol also displayed typical SMC-like migratory, contraction, and tube-formation activity in-vitro and significantly improved measurements of perfusion, vessel density, and SMA-positive arterial density in the ischemic limb of mouse HLI model.ConclusionsOur dynamic 3D + 2D protocols produced an exceptionally high yield of hiPSC-SMCs. Transplantation of these hiPSC-SMCs results in significantly improved recovery of ischemic limb after ischemic injury in mice.

A Quantitative Real-Time PCR Assay for Measuring Poxvirus Replication and Cell Binding.

Quantitative real-time PCR (qPCR) is a fast and reliable method to quantify viral genomes as a surrogate to titering on monolayers of cells for measuring virus replication. Whether it be for determining the number of virions released, the total number of genomes produced during infection, or the number of virions bound to a cell, qPCR assays can be adapted to quickly enumerate total viral genomes in a broad range of experiments comparing virus replication under different conditions. In addition, qPCR offers several advantages compared to plaque assays including time, linearity over 9 logs, and scalability from tens-to-hundreds of samples, depending on the qPCR machine. Here we describe a qPCR assay for quantifying vaccinia virus' dsDNA genome that can be used to determine the total number of virions produced. Furthermore, we describe a straightforward protocol for a cell-binding assay that is sensitive enough to use with small concentrations of inoculating virions. This protocol is suitable for measuring the cell-binding ability of mutations that affect virus production and infectivity.

Intracellular polarization of RNAs and proteins in the human small intestinal epithelium.

The intestinal epithelium is a polarized monolayer of cells, with an apical side facing the lumen and a basal side facing the blood stream. In mice, both proteins and mRNAs have been shown to exhibit global basal-apical polarization; however, polarization in the human intestine has not been systematically explored. Here, we employed laser-capture microdissection to isolate apical and basal epithelial segments from intestinal tissues of 8 individuals and performed RNA sequencing and mass-spectrometry proteomics. We find a substantial polarization of mRNA molecules that largely overlaps polarization patterns observed in mice. This mRNA polarization remains consistent across different zones of the intestinal villi and is generally correlated with the polarization of proteins. Our protein analysis exposes streamlined intracellular nutrient transport and processing and reveals that mitochondria and ribosomes are less polarized in humans compared to mice. Our study provides a resource for understanding human intestinal epithelial biology.

Entamoeba histolytica-induced NETs are highly cytotoxic on hepatic and colonic cells due to serine proteases and myeloperoxidase activities.

During intestinal and liver invasion by the protozoan parasite Entamoeba histolytica, extensive tissue destruction linked to large neutrophil infiltrates is observed. It has been proposed that microbicidal components of neutrophils are responsible for the damage, however, the mechanism by which they are released and act in the extracellular space remains unknown. In previous studies, we have shown that E. histolytica trophozoites induce NET formation, leading to the release of neutrophil granule content into extruded DNA. In this work, we evaluate the possible participation of NETs in the development of amoeba-associated pathology and analyze the contribution of anti-microbial components of the associated granules. E. histolytica-induced NETs were isolated and their effect on the viability and integrity of HCT 116 colonic and Hep G2 liver cultures were evaluated. The results showed that simple incubation of cell monolayers with purified NETs for 24h resulted in cell detachment and death in a dose-dependent manner. The effect was thermolabile and correlated with the amount of DNA and protein present in NETs. Pretreatment of NETs with specific inhibitors of some microbicidal components suggested that serine proteases, are mostly responsible for the damage caused by NETs on HCT 116 cells, while the MPO activity was the most related to Hep G2 cells damage. Our study also points to a very important role of DNA as a scaffold for the activity of these proteins. We show evidence of the development of NETs in amoebic liver abscesses in hamsters as a preamble to evaluate their participation in tissue damage. In conclusion, these studies demonstrate that amoebic-induced NETs have potent cytotoxic effects on target cells and, therefore, may be responsible for the intense damage associated with tissue invasion by this parasite.

Colon-Targeted Poly(ADP-ribose) Polymerase Inhibitors Synergize Therapeutic Effects of Mesalazine Against Rat Colitis Induced by 2,4-Dinitrobenzenesulfonic Acid.

Background/Objectives: In addition to oncological applications, poly(ADP-ribose) polymerase (PARP) inhibitors have potential as anti-inflammatory agents. Colon-targeted delivery of PARP inhibitors has been evaluated as a pharmaceutical strategy to enhance their safety and therapeutic efficacy against gut inflammation. Methods: Colon-targeted PARP inhibitors 5-aminoisoquinoline (5-AIQ) and 3-aminobenzamide (3-AB) were designed and synthesized by azo coupling with salicylic acid (SA), yielding 5-AIQ azo-linked with SA (AQSA) and 3-AB azo-linked with SA (ABSA). Additional conjugation of AQSA with acidic amino acids yielded glutamic acid-conjugated AQSA (AQSA-Glu) and aspartic acid-conjugated AQSA, which further increased the hydrophilicity of AQSA. Results: The distribution coefficients of PARP inhibitors were lowered by chemical modifications, which correlated well with drug permeability via the Caco-2 cell monolayer. All derivatives were effectively converted to their corresponding PARP inhibitors in the cecal contents. Compared with observations in the oral administration of PARP inhibitors, AQSA-Glu and ABSA resulted in the accumulation of much greater amounts of each PARP inhibitor in the cecum. ABSA accumulated mesalazine (5-ASA) in the cecum to a similar extent as sulfasalazine (SSZ), a colon-targeted 5-ASA prodrug. In the DNBS-induced rat colitis model, AQSA-Glu enhanced the anticolitic potency of 5-AIQ. Furthermore, ABSA was more effective against rat colitis than SSZ or AQSA-Glu, and the anticolitic effects of AQSA-Glu were augmented by combined treatment with a colon-targeted 5-ASA prodrug. In addition, the colon-targeted delivery of PARP inhibitors substantially reduced their systemic absorption. Conclusions: Colon-targeted PARP inhibitors may improve the therapeutic and toxicological properties of inhibitors and synergize the anticolitic effects of 5-ASA.

In Obesity, Esophagogastric Junction Fat Impairs Esophageal Barrier Function and Dilates Intercellular Spaces via HIF-2α.

Dilated intercellular space (DIS) in esophageal epithelium, a sign of impaired barrier function, is a characteristic finding of GERD that also is found in obese patients without GERD. We have explored molecular mechanisms whereby adipose tissue products might impair esophageal barrier integrity. We established cultures of visceral fat obtained during foregut surgery from obese and non-obese patients. Monolayer and air-liquid interface (ALI) cultures of human esophageal cells were grown with conditioned medium (CM) from fat cultures. RNA sequencing, ELISA, western blot, immunostaining, histology, and analyses of barrier function were performed; inhibitors of HIF-2α (PT2385), caspase-1 (AC-YVAD-CHO), myosin light chain (MLC) kinase (PIK), and MLC phosphatase (PIP250) were applied; blebbistatin was used to disrupt actin-myosin interactions; NAC was used to scavenge reactive oxygen species (ROS). CM from EGJ fat of obese patients (EGJ-CM-Obese) caused DIS with impaired barrier function in esophageal ALI cultures; these effects were blocked by PT2385. EGJ-CM-Obese induced ROS production that activated HIF-2α in esophageal cells. RNA sequencing analyses linked EGJ-CM-Obese-induced HIF-2α increases with innate immune response pathways. Cross-talk between HIF-2α and caspase-1 in esophageal cells led to IL-1β secretion, and IL-1β/IL-1R1 signaling caused DIS with impaired esophageal barrier function via actin-myosin interactions induced by MLC phosphorylation. We have elucidated molecular mechanisms whereby visceral fat of obese patients can impair esophageal barrier integrity by secreting substances that generate ROS, which activate HIF-2α in esophageal epithelial cells. These mechanisms could render the esophagus of obese individuals vulnerable to damage by acid and other noxious agents.

Assessment of in vitro dynamics of pathogenic environmental Acanthamoeba T4 and T9 genotypes isolated from three recreational lakes in Klang Valley, Malaysia over the HaCaT cell monolayer.

Free-living amoebae of the genus Acanthamoeba are causative agents of keratitis and amoebic encephalitis. They are widely found in various ecological environments. Therefore, the present study brings results that can help to better understand the genotypes of the environmental isolates and their pathogenicity. This study procured 26 Acanthamoeba isolates from three recreational lakes in 2022. Polymerase chain reaction amplification was performed on positive Acanthamoeba samples. The thermotolerance, osmotolerance, and cytopathogenicity in human keratinocyte (HaCaT) cells of the samples were also evaluated. The phylogenetic analysis demonstrated that 12 isolates were of genotype T4, two (T9), six (T17), four (T8), and one each from T5 and T11. The thermo- and osmotolerance assays indicated that eight Acanthamoeba samples were potentially pathogenic. Two T4 and one T9 genotype also recorded 33-, 42-, and 133-kDa serine-type proteases, respectively. The HaCaT cell monolayer revealed that three T4 and one T9 samples achieved cytopathic effects within the 50-100% range, hence significantly cytotoxic. The lactate dehydrogenase secretion results demonstrated that three (T4) and one (T9) sample exhibited exceptional toxicity (over 40%) compared to the other samples. The responses of Acanthamoeba members with similar genotypes to pathogenicity indicator assays varied considerably, rendering correlation of pathogenicity with specific genotypes challenging.

Artificial intelligence-driven prediction and validation of blood-brain barrier permeability and absorption, distribution, metabolism, excretion profiles in natural product research laboratory compounds.

Our previous research demonstrated that a large language model (LLM) based on the transformer architecture, specifically the MegaMolBART encoder with an XGBoost classifier, effectively predicts the blood-brain barrier (BBB) permeability of compounds. However, the permeability coefficients of compounds that can traverse this barrier remain unclear. Additionally, the absorption, distribution, metabolism, and excretion (ADME) characteristics of substances obtained from the Natural Product Research Laboratory (NPRL) at China Medical University Hospital (CMUH) have not yet been determined. The study aims to investigate the pharmacokinetic ADME properties and BBB permeability coefficients of NPRL compounds. A combined model using a transformer-based MegaMolBART encoder and XGBoost classifier was employed to predict BBB permeability. Machine learning (ML) tools from Discovery Studio were used to assess the ADME characteristics of the NPRL compounds. The CCK-8 assay was conducted to evaluate the cytotoxic effects of NPRL compounds on bEnd.3 brain endothelial cells after exposure to 10 μg/mL of the compounds. We assessed the permeability coefficient by subjecting bEnd.3 cell monolayers to the test compounds and measuring the permeability of FITC-dextran. There were 4956 compounds that could cross the blood-brain barrier (BBB+) and 2851 that could not (BBB-) in the B3DB dataset that was utilized for training. A total of 2461 BBB+ and 2184 BBB- compounds were used in the NPRL-CMUH dataset for testing. The permeability coefficient of temozolomide (TMZ) and 21 other BBB + compounds exceeded 10 × 10-7 cm/s. Computational analysis revealed that NPRL compounds exhibited a variety of ADME characteristics. Computer-based predictions for the NPRL of CMUH compounds regarding their capacity to traverse the BBB are verified by the findings. Artificial intelligence (AI) prediction models have effectively identified the potential ADME characteristics of various compounds.

Intestinal Cells-on-Chip for Permeability Studies.

To accurately measure permeability of compounds in the intestine, there is a need for preclinical in vitro models that accurately represent the specificity, integrity and complexity of the human small intestinal barrier. Intestine-on-chip systems hold considerable promise as testing platforms, but several characteristics still require optimization and further development. An established intestine-on-chip model for tissue explants was adopted for intestinal cell monolayer culture. A 3D-printed culture disc was designed to allow cell culture in static conditions and subsequent permeability studies in a dynamic environment. Membrane characteristics and standardized read-outs were investigated and compared to traditional permeability studies under static conditions. By starting cultures outside the chip in conventional wells plates, the new cell disc design could support accurate cell monolayer formation for both Caco-2 and human enteroids. When transferred to the chip with laminar flow, there was accurate detection of barrier integrity (FD4 and Cascade Blue) and permeability (atenolol/antipyrine). Both flow and membrane characteristics had a significant impact on permeability outcomes. This novel intestinal cell-on-chip system offers large flexibility for intestinal permeability studies, although it still requires validation with more compounds to reveal its full potential.