Organ-on-a-Chip Approach for Accelerating Blood-Brain Barrier Nanoshuttle Discovery.

Organ-on-a-chip, which recapitulates the dynamics of in vivo vasculature, has emerged as a promising platform for studying organ-specific vascular beds. However, its practical advantages in identifying vascular-targeted drug delivery systems (DDS) over traditional in vitro models remain underexplored. This study demonstrates the reliability and efficacy of the organ-on-a-chip in screening efficient DDS by comparing its performance with that of a conventional transwell, both designed to simulate the blood-brain barrier (BBB). The BBB nanoshuttles discovered through BBB Chip-based screening demonstrated superior functionality in vivo compared to those identified using transwell methods. This enhanced effectiveness is attributed to the BBB Chip's accurate replication of the structure and dynamics of the endothelial glycocalyx, a crucial protective layer within blood vessels, especially under shear stress. This capability of the BBB Chip has enabled the identification of molecular shuttles that efficiently exploit the endothelial glycocalyx, thereby enhancing transendothelial transport efficacy. Our findings suggest that organ-on-a-chip technology holds considerable promise for advancing research in vascular-targeted DDS due to its accurate simulation of molecular transport within endothelial systems.

Mebendazole preferentially inhibits cilia formation and exerts anticancer activity by synergistically augmenting DNA damage.

The cilium is a microtubule-based organelle that plays a pivotal role in embryonic development and maintenance of physiological functions in the human body. In addition to their function as sensors that transduce diverse extracellular signals, including growth factors, fluid flow, and physical forces, cilia are intricately involved in cell cycle regulation and preservation of DNA integrity, as their formation and resorption dynamics are tightly linked to cell cycle progression. Recently, several studies have linked defects in specific ciliary proteins to the DNA damage response. However, it remains unclear whether and how primary cilia contribute to cancer development. Mebendazole (MBZ) is an anthelmintic drug with anticancer properties in some cancer cells. MBZ is continuously being tested for clinical studies, but the precise mechanism of its anticancer activities remains unknown. Here, using Xenopus laevis embryos as a model system, we discovered that MBZ significantly hinders cilia formation and induces DNA damage. Remarkably, primary cilium-bearing cancer cells exhibited heightened vulnerability to combined treatment with MBZ and conventional anticancer drugs. Our findings shed light on the specific influence of MBZ on cilia, rather than cytosolic microtubules, in triggering DNA damage, elucidating a previously unidentified mechanism underlying potential MBZ-mediated cancer therapy.

Conserved chromatin and repetitive patterns reveal slow genome evolution in frogs.

Frogs are an ecologically diverse and phylogenetically ancient group of anuran amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus, and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., arm-preserving) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding surrounded by pericentromeric LINE/L1 elements. This work explores the structure of chromosomes across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (Hi-C) data for all species. Abundant satellite repeats occupy the unusually long (~20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible associations of centromeric chromatin and of telomeres, reflecting a Rabl-like configuration. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

Early life exposure to perfluorooctanesulfonate (PFOS) impacts vital biological processes in Xenopus laevis: Integrated morphometric and transcriptomic analyses.

Perfluorooctanesulfonate (PFOS) is a ubiquitous environmental pollutant associated with increasing health concerns and environmental hazards. Toxicological analyses of PFOS exposure are hampered by large interspecies variations and limited studies on the mechanistic details of PFOS-induced toxicity. We investigated the effects of PFOS exposure on Xenopus laevis embryos based on the reported developmental effects in zebrafish. X. laevis was selected to further our understanding of interspecies variation in response to PFOS, and we built upon previous studies by including transcriptomics and an assessment of ciliogenic effects. Midblastula-stage X. laevis embryos were exposed to PFOS using the frog embryo teratogenesis assay Xenopus (FETAX). Results showed teratogenic effects of PFOS in a time- and dose-dependent manner. The morphological abnormalities of skeleton deformities, a small head, and a miscoiled gut were associated with changes in gene expression evidenced by whole-mount in situ hybridization and transcriptomics. The transcriptomic profile of PFOS-exposed embryos indicated the perturbation in the expression of genes associated with cell death, and downregulation in adenosine triphosphate (ATP) biosynthesis. Moreover, we observed the effects of PFOS exposure on cilia development as a reduction in the number of multiciliated cells and changes in the directionality and velocity of the cilia-driven flow. Collectively, these data broaden the molecular understanding of PFOS-induced developmental effects, whereby ciliary dysfunction and disrupted ATP synthesis are implicated as the probable modes of action of embryotoxicity. Furthermore, our findings present a new challenge to understand the links between PFOS-induced developmental toxicity and vital biological processes.

Convergent differentiation of multiciliated cells.

Multiciliated cells (MCCs) are epithelial cells that control body fluid flow and contribute to the clearance of pathogenic microbes and other particles from the airways, egg transport in oviducts, and circulation of cerebrospinal fluid in the central nervous system. Although MCCs have shared functions to control fluid flow via coordinated motility of multiple ciliary structures, they are found in multiple mammalian tissues originating from distinct germ layers and differentiate via distinct developmental pathways. To understand the similarities and differences of MCCs in multiple tissues, we investigated single-cell transcriptome data of nasal epithelial cells, bronchial tubes, fallopian tubes, and ependymal cells in the subventricular zone from humans and mice by cross-species data integration. Expression of cilia-associated genes was indistinguishable between these MCCs, although cell populations had unique properties by the species and tissue, demonstrating that they share the same final differentiation status for ciliary functions. We further analyzed the final differentiation step of MCCs from their distinctive progenitors and confirmed their convergent gene set expression for ciliogenesis at the final step. These results may provide new insight into understanding ciliogenesis during the developmental process.

Enrichment of Deleterious Mutated Genes Involved in Ciliary Function and Histone Modification in Brain Cancer Patient-Derived Xenograft Models.

Patient-derived xenograft (PDX) models, which can retain the characteristics of original tumors in an in vivo-mimicking environment, have been developed to identify better treatment options. However, although original tumors and xenograft tissues mostly share oncogenic mutations and global gene expression patterns, their detailed mutation profiles occasionally do not overlap, indicating that selection occurs in the xenograft environment. To understand this mutational alteration in xenografts, we established 13 PDX models derived from 11 brain tumor patients and confirmed their histopathological similarity. Surprisingly, only a limited number of somatic mutations were shared between the original tumor and xenograft tissue. By analyzing deleteriously mutated genes in tumors and xenografts, we found that previously reported brain tumor-related genes were enriched in PDX samples, demonstrating that xenografts are a valuable platform for studying brain tumors. Furthermore, mutated genes involved in cilium movement, microtubule depolymerization, and histone methylation were enriched in PDX samples compared with the original tumors. Even with the limitations of the heterogeneity of clinical lesions with a heterotropic model, our study demonstrates that PDX models can provide more information in genetic analysis using samples with high heterogeneity, such as brain tumors.

A single-cell, time-resolved profiling of Xenopus mucociliary epithelium reveals nonhierarchical model of development.

The specialized cell types of the mucociliary epithelium (MCE) lining the respiratory tract enable continuous airway clearing, with its defects leading to chronic respiratory diseases. The molecular mechanisms driving cell fate acquisition and temporal specialization during mucociliary epithelial development remain largely unknown. Here, we profile the developing Xenopus MCE from pluripotent to mature stages by single-cell transcriptomics, identifying multipotent early epithelial progenitors that execute multilineage cues before specializing into late-stage ionocytes and goblet and basal cells. Combining in silico lineage inference, in situ hybridization, and single-cell multiplexed RNA imaging, we capture the initial bifurcation into early epithelial and multiciliated progenitors and chart cell type emergence and fate progression into specialized cell types. Comparative analysis of nine airway atlases reveals an evolutionary conserved transcriptional module in ciliated cells, whereas secretory and basal types execute distinct function-specific programs across vertebrates. We uncover a continuous nonhierarchical model of MCE development alongside a data resource for understanding respiratory biology.

GPR143 controls ESCRT-dependent exosome biogenesis and promotes cancer metastasis.

Exosomes transport a variety of macromolecules and modulate intercellular communication in physiology and disease. However, the regulation mechanisms that determine exosome contents during exosome biogenesis remain poorly understood. Here, we find that GPR143, an atypical GPCR, controls the endosomal sorting complex required for the transport (ESCRT)-dependent exosome biogenesis pathway. GPR143 interacts with HRS (an ESCRT-0 Subunit) and promotes its association to cargo proteins, such as EGFR, which subsequently enables selective protein sorting into intraluminal vesicles (ILVs) in multivesicular bodies (MVBs). GPR143 is elevated in multiple cancers, and quantitative proteomic and RNA profiling of exosomes in human cancer cell lines showed that the GPR143-ESCRT pathway promotes secretion of exosomes that carry unique cargo, including integrins signaling proteins. Through gain- and loss-of-function studies in mice, we show that GPR143 promotes metastasis by secreting exosomes and increasing cancer cell motility/invasion through the integrin/FAK/Src pathway. These findings provide a mechanism for regulating the exosomal proteome and demonstrate its ability to promote cancer cell motility.

Ckb and Ybx2 interact with Ribc2 and are necessary for the ciliary beating of multi-cilia.

BACKGROUND: Motile cilia in a vertebrate are important to sustaining activities of life. Fluid flow on the apical surface of several tissues, including bronchial epithelium, ependymal epithelium, and fallopian tubules is generated by the ciliary beating of motile cilia. Multi-ciliated cells in ependymal tissue are responsible for the circulation of cerebrospinal fluid (CSF), which is essential for the development and homeostasis of the central nervous system, and airway tissues are protected from external contaminants by cilia-driven mucosal flow over the top of the airway epithelium. OBJECTIVE: A previous study reported that reduction of Ribc2 protein leads to disruption of ciliary beating in multi-ciliated cells. However, knowledge regarding the molecular function of Ribc2 is limited, thus currently available information is also limited. Therefore, we evaluated the importance of proteins involved in the interaction with Ribc2 in the process of ciliary beating. METHODS: Immunoprecipitation and mass spectrometry analysis was performed for the discovery of proteins involved in the interaction with Ribc2. Expression of the target gene was inhibited by injection of antisense morpholinos and measurement of the fluid flow on the embryonic epidermis of Xenopus was performed using fluorescent beads for examination of the ciliary beating of multi cilia. In addition, the flag-tagged protein was expressed by injection of mRNA and the changes in protein localization in the cilia were measured by immunostaining and western blot analysis for analysis of the molecular interaction between Ribc2 and Ribc2 binding proteins in multi-cilia. RESULTS: The IP/MS analysis identified Ckb and Ybx2 as Ribc2 binding proteins and our results showed that localization of both Ckb and Ybx2 occurs at the axoneme of multi-cilia on the embryonic epithelium of Xenopus laevis. In addition, our findings confirmed that knock-down of Ckb or Ybx2 resulted in abnormal ciliary beating and reduction of cilia-driven fluid flow on multi-cilia of Xenopus laevis. In addition, significantly decreased localization of Ckb or Ybx2 in the ciliary axoneme was observed in Ribc2-depleted multi-cilia. CONCLUSION: Ckb and Ybx2 are involved in the interaction with Ribc2 and are necessary for the ciliary beating of multi-cilia.

PCNB exposure during early embryogenic development induces developmental delay and teratogenicity by altering the gene expression in Xenopus laevis.

Pentachloronitrobenzene (PCNB) is an organochlorine fungicide commonly used to treat seeds against seedling infections and controlling snow mold on golf courses. PCNB has been demonstrated to be toxic to living organisms, including fish and several terrestrial organisms. However, only phenotypical deformities have been studied, and the effects of PCNB on early embryogenesis, where primary organogenesis occurs, have not been completely studied. In the current study, the developmental toxicity and teratogenicity of PCNB is evaluated by using frog embryo teratogenesis assay Xenopus (FETAX). Our results confirmed the teratogenic potential of PCNB revealing the teratogenic index of 1.29 during early embryogenesis. Morphological studies revealed tiny head, bent axis, reduced inter ocular distance, hyperpigmentation, and reduced total body lengths. Whole mount in situ hybridization and reverse transcriptase polymerase chain reaction were used to identify PCNB teratogenic effects at the gene level. The gene expression analyses revealed that PCNB was embryotoxic to the liver and heart of developing embryos. Additionally, to determine the most sensitive developmental stages to PCNB, embryos were exposed to the compound at various developmental stages, demonstrating that the most sensitive developmental stage to PCNB is primary organogenesis. Taken together, we infer that PCNB's teratogenic potential affects not just the phenotype of developing embryos but also the associated genes and involving the oxidative stress as a possible mechanism of toxicity, posing a hazard to normal embryonic growth. However, the mechanisms of teratogenesis require additional extensive investigation to be defined completely.

TRIP13 Participates in Immediate-Early Sensing of DNA Strand Breaks and ATM Signaling Amplification through MRE11.

Thyroid hormone receptor-interacting protein 13 (TRIP13) participates in various regulatory steps related to the cell cycle, such as the mitotic spindle assembly checkpoint and meiotic recombination, possibly by interacting with members of the HORMA domain protein family. Recently, it was reported that TRIP13 could regulate the choice of the DNA repair pathway, i.e., homologous recombination (HR) or nonhomologous end-joining (NHEJ). However, TRIP13 is recruited to DNA damage sites within a few seconds after damage and may therefore have another function in DNA repair other than regulation of the pathway choice. Furthermore, the depletion of TRIP13 inhibited both HR and NHEJ, suggesting that TRIP13 plays other roles besides regulation of choice between HR and NHEJ. To explore the unidentified functions of TRIP13 in the DNA damage response, we investigated its genome-wide interaction partners in the context of DNA damage using quantitative proteomics with proximity labeling. We identified MRE11 as a novel interacting partner of TRIP13. TRIP13 controlled the recruitment of MDC1 to DNA damage sites by regulating the interaction between MDC1 and the MRN complex. Consistently, TRIP13 was involved in ATM signaling amplification. Our study provides new insight into the function of TRIP13 in immediate-early DNA damage sensing and ATM signaling activation.

Regulation of RNA localization during oocyte maturation by dynamic RNA-ER association and remodeling of the ER.

Asymmetric localization of mRNAs is crucial for cell polarity and cell fate determination. By performing fractionation RNA-seq, we report here that a large number of maternal RNAs are associated with the ER in Xenopus oocytes but are released into the cytosol after oocyte maturation. We provide evidence that the majority of ER-associated RNA-binding proteins (RBPs) remain associated with the ER after oocyte maturation. However, all ER-associated RBPs analyzed exhibit reduced binding to some of their target RNAs after oocyte maturation. Our results further show that the ER is remodeled massively during oocyte maturation, leading to the formation of a widespread tubular ER network in the animal hemisphere that is required for the asymmetric localization of mRNAs in mature eggs. Thus, our findings demonstrate that dynamic regulation of RNA-ER association and remodeling of the ER are important for the asymmetric localization of RNAs during development.

Adaptive cellular response of the substantia nigra dopaminergic neurons upon age-dependent iron accumulation.

Progressive iron accumulation in the substantia nigra in the aged human brain is a major risk factor for Parkinson's disease and other neurodegenerative diseases. Heavy metals, such as iron, produce reactive oxygen species and consequently oxidative stress in cells. It is unclear, however, how neurons in the substantia nigra are protected against the age-related, excessive accumulation of iron. In this study, we examined the cellular response of the substantia nigra against age-related iron accumulation in rats of different ages. Magnetic resonance imaging confirmed the presence of iron in 6-month-old rats; in 15-month-old rats, iron accumulation significantly increased, particularly in the midbrain. Transcriptome analysis of the region, in which iron deposition was observed, revealed an increase in stress response genes in older animals. To identify the genes related to the cellular response to iron, independent of neurodevelopment, we exposed the neuroblastoma cell line SH-SY5Y to a similar quantity of iron and then analyzed their transcriptomic responses. Among various stress response pathways altered by iron overloading in the rat brain and SH-SY5Y cells, the genes associated with topologically incorrect protein responses were significantly upregulated. Knockdown of HERPUD1 and CLU in this pathway increased susceptibility to iron-induced cellular stress, thus demonstrating their roles in preventing iron overload-induced toxicity. The current study details the neuronal response to excessive iron accumulation, which is associated with age-related neurodegenerative diseases.

GJA1 depletion causes ciliary defects by affecting Rab11 trafficking to the ciliary base.

The gap junction complex functions as a transport channel across the membrane. Among gap junction subunits, gap junction protein α1 (GJA1) is the most commonly expressed subunit. A recent study showed that GJA1 is necessary for the maintenance of motile cilia; however, the molecular mechanism and function of GJA1 in ciliogenesis remain unknown. Here, we examined the functions of GJA1 during ciliogenesis in human retinal pigment epithelium-1 and Xenopus laevis embryonic multiciliated-cells. GJA1 localizes to the motile ciliary axonemes or pericentriolar regions beneath the primary cilium. GJA1 depletion caused malformation of both the primary cilium and motile cilia. Further study revealed that GJA1 depletion affected several ciliary proteins such as BBS4, CP110, and Rab11 in the pericentriolar region and basal body. Interestingly, CP110 removal from the mother centriole was significantly reduced by GJA1 depletion. Importantly, Rab11, a key regulator during ciliogenesis, was immunoprecipitated with GJA1 and GJA1 knockdown caused the mislocalization of Rab11. These findings suggest that GJA1 regulates ciliogenesis by interacting with the Rab11-Rab8 ciliary trafficking pathway.

Precision targeting tumor cells using cancer-specific InDel mutations with CRISPR-Cas9.

An ideal cancer therapeutic strategy involves the selective killing of cancer cells without affecting the surrounding normal cells. However, researchers have failed to develop such methods for achieving selective cancer cell death because of shared features between cancerous and normal cells. In this study, we have developed a therapeutic strategy called the cancer-specific insertions-deletions (InDels) attacker (CINDELA) to selectively induce cancer cell death using the CRISPR-Cas system. CINDELA utilizes a previously unexplored idea of introducing CRISPR-mediated DNA double-strand breaks (DSBs) in a cancer-specific fashion to facilitate specific cell death. In particular, CINDELA targets multiple InDels with CRISPR-Cas9 to produce many DNA DSBs that result in cancer-specific cell death. As a proof of concept, we demonstrate here that CINDELA selectively kills human cancer cell lines, xenograft human tumors in mice, patient-derived glioblastoma, and lung patient-driven xenograft tumors without affecting healthy human cells or altering mouse growth.

Quantitative Fluorescence In Situ Hybridization (FISH) of Magnetically Confined Bacteria Enables Early Detection of Human Bacteremia.

The current diagnosis of bacteremia mainly relies on blood culture, which is inadequate for the rapid and quantitative determination of most bacteria in blood. Here, a quantitative, multiplex, microfluidic fluorescence in situ hybridization method (µFISH) is developed, which enables early and rapid (3-h) diagnosis of bacteremia without the need for prior blood culture. This novel technology employs mannose-binding lectin-coated magnetic nanoparticles, which effectively opsonize a broad range of pathogens, magnetically sequestering them in a microfluidic device. Therein, µFISH probes, based on unique 16S rRNA sequences, enable the identification and quantification of sequestered pathogens both in saline and whole blood, which is more sensitive than conventional polymerase chain reaction. Using µFISH, Escherichia coli (E. coli) is detected in whole blood collected from a porcine disease model and from E. coli-infected patients. Moreover, the proportion of E. coli, relative to other bacterial levels in the blood, is accurately and rapidly determined, which is not possible using conventional diagnostic methods. Blood from E. coli-infected patients is differentiated from healthy donors' blood using cutoff values with a 0.05 significance level. Thus, µFISH is a versatile method that can be used to rapidly identify pathogens and determine their levels relative to other culturable and nonculturable bacteria in biological samples.

Augmented ERAD (ER-associated degradation) activity in chondrocytes is necessary for cartilage development and maintenance.

Chondrocytes secrete massive extracellular matrix (ECM) molecules that are produced, folded, and modified in the endoplasmic reticulum (ER). Thus, the ER-associated degradation (ERAD) complex-which removes misfolded and unfolded proteins to maintain proteostasis in the ER- plays an indispensable role in building and maintaining cartilage. Here, we examined the necessity of the ERAD complex in chondrocytes for cartilage formation and maintenance. We show that ERAD gene expression is exponentially increased during chondrogenesis, and disruption of ERAD function causes severe chondrodysplasia in developing embryos and loss of adult articular cartilage. ERAD complex malfunction also causes abnormal accumulation of cartilage ECM molecules and subsequent chondrodysplasia. ERAD gene expression is decreased in damaged cartilage from patients with osteoarthritis (OA), and disruption of ERAD function in articular cartilage leads to cartilage destruction in a mouse OA model.

Physiological Functions of Thiol Peroxidases (Gpx1 and Prdx2) during Xenopus laevis Embryonic Development.

Glutathione peroxidase 1 (Gpx1) and peroxiredoxin 2 (Prdx2) belong to the thiol peroxidase family of antioxidants, and have been studied for their antioxidant functions and roles in cancers. However, the physiological significance of Gpx1 and Prdx2 during vertebrate embryogenesis are lacking. Currently, we investigated the functional roles of Gpx1 and Prdx2 during vertebrate embryogenesis using Xenopus laevis as a vertebrate model. Our investigations revealed the zygotic nature of gpx1 having its localization in the eye region of developing embryos, whereas prdx2 exhibited a maternal nature and were localized in embryonic ventral blood islands. Furthermore, the gpx1-morphants exhibited malformed eyes with incompletely detached lenses. However, the depletion of prdx2 has not established its involvement with embryogenesis. A molecular analysis of gpx1-depleted embryos revealed the perturbed expression of a cryba1-lens-specific marker and also exhibited reactive oxygen species (ROS) accumulation in the eye regions of gpx1-morphants. Additionally, transcriptomics analysis of gpx1-knockout embryos demonstrated the involvement of Wnt, cadherin, and integrin signaling pathways in the development of malformed eyes. Conclusively, our findings indicate the association of gpx1 with a complex network of embryonic developmental pathways and ROS responses, but detailed investigation is a prerequisite in order to pinpoint the mechanistic details of these interactions.

Analysis of Porcine Model of Fecal-Induced Peritonitis Reveals the Tropism of Blood Microbiome.

Recent studies have suggested the existence of a blood microbiome in the healthy host. However, changes in the blood microbiome upon bloodstream infection are not known. Here, we analyzed the dynamics of the blood microbiome in a porcine model of polymicrobial bacteremia induced by fecal peritonitis. Surprisingly, we detected bacterial populations in the bloodstream even before the infection, and these populations were maintained over time. The native blood microbiome was notably taxonomically different from the fecal microbiome that was used to induce peritonitis, reflecting microbial tropism for the blood. Although the population composition after the infection was similar to that of the native blood microbiome, new bacterial strains entered the bloodstream upon peritonitis induction as clinical symptoms relevant to sepsis developed. This indicates that the bacteria detected in the blood before peritonitis induction were derived from the blood rather than a contamination. Comparison of the functional pathways enriched in the blood and fecal microbiomes revealed that communication and stress management pathways are essential for the survival of the blood microbiome.

A temporally resolved transcriptome for developing "Keller" explants of the Xenopus laevis dorsal marginal zone.

BACKGROUND: Explanted tissues from vertebrate embryos reliably develop in culture and have provided essential paradigms for understanding embryogenesis, from early embryological investigations of induction, to the extensive study of Xenopus animal caps, to the current studies of mammalian gastruloids. Cultured explants of the Xenopus dorsal marginal zone ("Keller" explants) serve as a central paradigm for studies of convergent extension cell movements, yet we know little about the global patterns of gene expression in these explants. RESULTS: In an effort to more thoroughly develop this important model system, we provide here a time-resolved bulk transcriptome for developing Keller explants. CONCLUSIONS: The dataset reported here provides a useful resource for those using Keller explants for studies of morphogenesis and provide genome-scale insights into the temporal patterns of gene expression in an important tissue when explanted and grown in culture.