Cryptosporidium parvum disrupts intestinal epithelial barrier in neonatal mice through downregulation of cell junction molecules.

BACKGROUND: Cryptosporidium spp. cause watery diarrhea in humans and animals, especially in infants and neonates. They parasitize the apical surface of the epithelial cells in the intestinal lumen. However, the pathogenesis of Cryptosporidium-induced diarrhea is not fully understood yet. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we infected C57BL/6j neonatal mice with C. parvum IIa and IId subtypes, and examined oocyst burden, pathological changes, and intestinal epithelial permeability during the infection. In addition, transcriptomic analyses were used to study the mechanism of diarrhea induced by the C. parvum IId subtype. The neonatal mice were sensitive to both C. parvum IIa and IId infection, but the IId subtype caused a wide oocyst shedding window and maintained the high oocyst burden in the mice compared with the IIa subtype. In addition, the mice infected with C. parvum IId resulted in severe intestinal damage at the peak of infection, leading to increased permeability of the epithelial barrier. The KEGG, GO and GSEA analyses revealed that the downregulation of adherens junction and cell junction molecules at 11 dpi. Meanwhile, E-cadherin, which is associated with adherens junction, was reduced at the protein level in mouse ileum at peak and late infection. CONCLUSIONS/SIGNIFICANCE: C. parvum IId infection causes more severe pathological damage than C. parvum IIa infection in neonatal mice. Furthermore, the impairment of the epithelial barrier during C. parvum IId infection results from the downregulation of intestinal junction proteins.

Association Studies on Gut and Lung Microbiomes in Patients with Lung Adenocarcinoma.

Lung adenocarcinoma (LADC) is a prevalent type of lung cancer that is associated with lung and gut microbiota. However, the interactions between these microbiota and cancer development remain unclear. In this study, a microbiome study was performed on paired fecal and bronchoalveolar lavage fluid (BALF) samples from 42 patients with LADC and 64 healthy controls using 16S rRNA gene amplicon and shotgun metagenome sequencing, aiming to correlate the lung and gut microbiota with LADC. Patients with LADC had reduced α-diversity in the gut microbiome and altered ß-diversity compared with healthy controls, and the abundances of Flavonifractor, Eggerthella, and Clostridium were higher in the gut microbiome of LADC patients. The increased abundance of microbial species, such as Flavonifractor plautii, was associated with advanced-stage LADC and a higher metastasis rate. Phylogenetically, Haemophilus parainfluenzae was the most frequently shared taxon in the lung and gut microbiota of LADC patients. Gut microbiome functional pathways involving leucine, propanoate, and fatty acids were associated with LADC progression. In conclusion, the low diversity of the gut microbiota and the presence of H. parainfluenzae in gut and lung microbiota were linked to LADC development, while an increased abundance of F. plautii and the enriched metabolic pathways could be associated with the progression of LADC.

Regulation and mechanism of Astragalus polysaccharide on ameliorating aging in Drosophila melanogaster.

Astragalus polysaccharide (APS) is a notable bioactive component of Astragalus membranaceus and has been extensively investigated for its pharmacological activities, including antioxidant, neuroprotection, and anticancer effects. However, the beneficial effects and mechanisms of APS on anti-aging diseases remain largely unknown. Here, we utilized the classic model organism Drosophila melanogaster to investigate the beneficial effects and mechanism of APS on aging-related intestinal homeostasis imbalance, sleeping disorders, and neurodegenerative diseases. The results showed that administration of APS significantly attenuated age-associated disruption of the intestinal barrier, loss of gastrointestinal acid-base balance, reduction in intestinal length, overproliferation of the intestinal stem cells (ISCs), and sleeping disorders upon aging. Furthermore, APS supplementation delayed the onset of Alzheimer's phenotypes in Aß42-induced Alzheimer's disease (AD) flies, including the extension of lifespan and the increase in motility, but without rescuing neurobehavioral deficits in the AD model of taupathy and Parkinson's disease (PD) model of Pink1 mutation. In addition, transcriptomics was used to dissect updated mechanisms of APS on anti-aging, such as JAK-STAT signaling, Toll signaling, and IMD signaling pathways. Taken together, these studies indicate that APS plays a beneficial role in modulating aging-related diseases, thereby as a potential natural drug to delay aging.

Engineering placenta-like organoids containing endogenous vascular cells from human-induced pluripotent stem cells.

The placenta is an essential organ that maintains the health of both the fetus and its mother. Understanding the development of human placenta has been hindered by the limitations of existing animal models and monolayer cell cultures. Models that can recapitulate the essential aspects of human placental multicellular components and vasculature are still lacking. Herein, we presented a new strategy to establish placenta-like organoids with vascular-like structures from human-induced pluripotent stem cells in a defined three-dimensional (3D) culture system. The resulting placenta-like tissue resembles first-trimester human placental development in terms of complex placental components and secretory function. The multicellular tissue was characterized by the inclusion of trophoblasts (cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts, and other endogenous vascular cells), which were identified by immunofluorescence, flow cytometry analyses, real-time quantitative reverse transcription polymerase chain reaction and single-cell RNA-seq. Moreover, the 3D tissue was able to secrete the placenta-specific hormone human chorionic gonadotropin ß (hCG-ß) and vascular endothelial growth factor A (VEGFA). The tissue responded to the inflammatory factor tumor necrosis factor-α (TNF-α) and VEGF receptor inhibitors. This new model system can represent the major features of placental cellular components, and function, which have not been realized in 2D monolayer cultures. The developed tissue system might open new avenues for studying normal early human placental development and its disease states.

Expression of Shh, Gli1, and Cyr61 in Gastric Cancer Predicts Overall Survival of Patients: A Retrospective Study.

OBJECTIVE: This study aimed to evaluate the expression levels of Shh, Gli1, and Cyr61 proteins in gastric cancer tissues and analyze the relationship between these three proteins and the clinicopathological factors and prognosis of patients. METHODS: This was a retrospective study. Four hundred gastric cancer tissue specimens from patients who underwent radical gastrectomy in Zhangye People's Hospital affiliated to Hexi University between February 2013 and February 2021 underwent immunohistochemical analysis. RESULTS: The positive expression rates of Shh, Gli1, and Cyr61 in gastric cancer tissues were 55.5%, 56.5%, and 64.5%, respectively. The expressions of Shh, Gli1, and Cyr61 in gastric cancer tissues were significantly correlated with tumor size, depth of invasion, and degree of differentiation (P < .05). The expression of Shh protein was positively correlated with the expression of Gli1 protein (P < .01), and the expression of Gli1 protein was positively correlated with the expression of Cyr61 protein (P < .01). Univariate and multivariate analyses showed that the expression of Shh, Gli1, and Cyr61 could predict the prognosis of patients (P < .05). Receiver operating characteristic curve analysis combined with TNM staging could better predict the three-year overall survival of patients (P < .05). CONCLUSION: Shh, Gli1, and Cyr61 proteins are significantly expressed in gastric cancer tissues and are risk factors for the prognosis of patients with gastric cancer.

Gas-Flow-Assisted Wrinkle-Free Transfer of a Centimeter-Scale Ultrathin Alumina Membrane onto Arbitrary Substrates.

The transfer of an ultrathin membrane onto arbitrary substrates is important in different practical fields. Conventional wet-transfer methods inevitably induce wrinkle defects as a result of the large contact angle of the trapped droplet between the membrane and the substrate. Here, we demonstrate a gas flow-assisted method (GFAM) to transfer centimeter (cm)-scale ultrathin membranes onto arbitrary substrates (including a curved substrate) without wrinkles. GFAM makes use of contact angle hysteresis to bulge the trapped droplet between the substrate and the ultrathin membrane and simultaneously stretch the ultrathin membrane during rapid dewetting driven by gas flow. Moreover, GFAM can be easily fulfilled by using compressed air for seconds. Compared with conventional hydrophilic treatments or organic liquid wetting, this method has no durability concern and does not change the surface nature of substrates. Taking a widely used ultrathin anodic aluminum oxide (AAO) membrane as an example, we successfully demonstrate the application of a large-area wrinkle-free ultrathin AAO membrane to defect-free ordered nanostructure array fabrication and investigate the micro-scale details of macro-scale wrinkles generated by the conventional ways. In addition, its corresponding superiority over the defective counterpart is further studied in optical sensing. This method is highly valuable for promoting the simplicity of large-area ultrathin membrane transfer in practice.

Controllable Fabrication of Composite Core-Shell Capsules at a Macroscale as Organoid Biocarriers.

The cell encapsulation technology is promising for generation of functional carriers with well-tailored structures for efficient transplantation and immunoprotection of cells/tissues. Stem cell organoids are highly potential for recapitulating the intricate architectures and functionalities of native organs and also providing an unlimited cell source for cellular replacement therapy. However, it remains challenging for loading the organoids with hundreds of micrometers size by current existing cell carriers. Herein, a simple and facile coextrusion strategy is developed for controllable fabrication of Ca-alginate/poly(ethylene imine) (Alg/PEI) macrocapsules for efficient encapsulation and cultivation of organoids. Human-induced pluripotent stem cell (hiPSC)-derived islet organoids are encapsulated in the aqueous compartments of the capsules and immunoisolated by a semipermeable Alg/PEI shell. Via electrostatic interactions, a PEI polyelectrolyte can be incorporated in the shell for restricting its swelling, thus effectively improving the stability of the capsules. The Alg/PEI macrocapsules are featured with desirable selective permeability for immunoisolation of antibodies from reaching the loaded organoids. Meanwhile, they also exhibit excellent permeability for mass transfer due to their well-defined core-shell structure. As such, the encapsulated islet organoids contain islet-specific multicellular components, with high viability and sensitive glucose-stimulated insulin secretion function. The proposed approach provides a versatile encapsulation system for tissue engineering and regenerative medicine applications.

Evaluation of hepatic drug-metabolism for glioblastoma using liver-brain chip.

Glioma is one of the most aggressive and highly fatal diseases with an extremely poor prognosis. Considering the poor clinical response to therapy in glioma, it is urgent to establish an in vitro model to facilitate the screening and assessment of anti-brain-tumor drugs. The blood-brain barrier (BBB), as well as liver metabolism plays an important role in determining the pharmacological activity of many anti-brain-tumor drugs. In this work, we designed a multi-interface liver-brain chip integrating co-culture system to assess hepatic metabolism dependent cytotoxicity of anti-brain-tumor drug in vitro. This microdevice composed of three microchannels which were separated by porous membrane and collagen. HepG2 and U87 cells were cultured in separated channels as mimics of liver and glioblastoma. Brain microvascular endothelial cells (BMECS) and cerebral astrocytes were co-cultured on collagen to mimic the brain microvascular endothelial barrier. Three common anti-tumor drugs, paclitaxel (PTX), capecitabine (CAP) and temozolomide (TMZ), were evaluated on this chip. In integrated liver-brain chip, liver enhanced the cytotoxicity of CAP on U87 cells by 30%, but having no significant effect on TMZ. The BBB decreased the cytotoxicity of PTX by 20%, while no significant effects were observed on TMZ and CAP, indicating the importance of liver metabolism and blood-brain barrier on the evaluation of anti-brain-tumor drugs. This work provides a biomimetic liver-brain model to mimic the physiological and pharmacological processes in vitro and presents a simple platform for long-term cell co-culture, drug delivery and metabolism, and real-time analysis of drug effects on brain cancer.

Appraising the Value of Serum and Serum-Derived Exosomal LncRNA-EXOC7 as a Promising Biomarker in Cervical Cancer.

BACKGROUND: The abnormally expressed long non-coding RNAs (lncRNAs) are closely related to the onset and progression of various malignant tumors. In this study, we aimed to explore the value of serum and serum-derived exosomal lncRNA-EXOC7 (long non-coding RNA exocyst complex component 7) in the diagnosis and monitoring of cervical cancer (CC). METHODS: The expression of lncRNA-EXOC7 in serum and serum-derived exosomes in CC patients was detected by quantitative real-time PCR (qRT-PCR), and the correlations between lncRNA-EXOC7 expression and clinicopathological characteristics were analyzed by Spearman's and Chi-square tests. RESULTS: Serum vesicles were successfully isolated and identified. The expression of lncRNA-EXOC7 in serum and serum-derived exosomes in CC patients was markedly elevated compared with that in healthy controls. The AUCs of serum and exosomal lncRNA-EXOC7 in distinguishing CC patients from healthy controls were 0.9388 and 0.8982, respectively. The expression of lncRNA-EXOC7 in serum and exosomes was correlated with the FIGO stage of CC (p = 0.041 or 0.010), and positively correlated with levels of CYFRA211, TPS, and SCC (all with p < 0.05). Serum and exosomal lncRNA-EXOC7 was related to the treatment and recurrence of CC; that means, it was significantly repressed after treatment and up-regulated at the time of recurrence. CONCLUSIONS: Serum and exosomal lncRNA-EXOC7 can be used as an important biomarker for the diagnosis, the evaluation of curative effect and the detection of recurrence of CC.

Modeling Pharmacokinetic Profiles for Assessment of Anti-Cancer Drug on a Microfluidic System.

The pharmacokinetic (PK) properties of drug, which include drug absorption and excretion, play an important role in determining the in vivo pharmaceutical activity. However, current in vitro systems that model PK profiles are often limited by the in vivo-like concentration profile of a drug. Herein, we present a perfused and multi-layered microfluidic chip system to model the PK profile of anti-cancer drug 5-FU in vitro. The chip device contains two layers of culture channels sandwiched by a porous membrane, which allows for drug exposure and diffusion between the two channels. The integration of upper intestine cells (Caco-2) and bottom targeted cells within the device enables the generation of loading and clearance portions of a PK curve under peristaltic flow. Fluorescein as a test molecule was initially used to generate a concentration-time curve, investigating the effects of parameters of flow rate, administration time, and initial concentration on dynamic drug concentration profiles. Furthermore, anti-cancer drug 5-FU was performed to assess its pharmaceutical activity on target cells (human lung adenocarcinoma cells or human pulmonary alveolar epithelial cells) using different drug administration regimens. A dynamic, in vivo-like 5-FU exposure refers to PK profile regimen, led to generate a lower drug concentration (dynamically fluctuate from 0 to 1 µg/mL affected by absorption) compared to the constant exposure. Moreover, the PK profile regimen alleviates the drug-induced cytotoxicity on target cells. These results demonstrate the feasibility of determining the PK profiles using this microfluidic system with in vivo-like drug administration regimens. This established system may provide a powerful platform for the prediction of drug safety and effectiveness in the pharmaceutical research.

Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins.

The current outbreak of coronavirus disease-2019 (COVID-19) poses unprecedented challenges to global health1. The new coronavirus responsible for this outbreak-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-shares high sequence identity to SARS-CoV and a bat coronavirus, RaTG132. Although bats may be the reservoir host for a variety of coronaviruses3,4, it remains unknown whether SARS-CoV-2 has additional host species. Here we show that a coronavirus, which we name pangolin-CoV, isolated from a Malayan pangolin has 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S proteins, respectively. In particular, the receptor-binding domain of the S protein of pangolin-CoV is almost identical to that of SARS-CoV-2, with one difference in a noncritical amino acid. Our comparative genomic analysis suggests that SARS-CoV-2 may have originated in the recombination of a virus similar to pangolin-CoV with one similar to RaTG13. Pangolin-CoV was detected in 17 out of the 25 Malayan pangolins that we analysed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus from pangolins that is closely related to SARS-CoV-2 suggests that these animals have the potential to act as an intermediate host of SARS-CoV-2. This newly identified coronavirus from pangolins-the most-trafficked mammal in the illegal wildlife trade-could represent a future threat to public health if wildlife trade is not effectively controlled.

Characterization of INS-15, A Metalloprotease Potentially Involved in the Invasion of Cryptosporidium parvum.

Cryptosporidium parvum is a protozoan parasite that can cause moderate-to-severe diarrhea. Insulinase-like proteases (INS) are one of the largest protein families within the small proteome of the pathogen. However, their roles in C. parvum biology remain un-elucidated. In this study, a member of the protein family, INS-15 of C. parvum encoded by cgd3_4260, was cloned, expressed and characterized to understand its function. INS-15 and its domain I were expressed in Escherichia coli and polyclonal antibodies against the domain I and one specific polypeptide were prepared in rabbits. The role of INS-15 protein in the C. parvum invasion was preliminarily studied. Recombinant INS-15 protein and its domain I were successfully expressed in E. coli, together with various degraded products. The cgd3_4260 gene had a peak expression at 2 h of in vitro C. parvum culture, while the INS-15 protein was expressed in the mid-anterior region of sporozoites and the area of merozoites opposite to the nucleus. Anti-INS-15 domain I antibodies reduced the invasion of C. parvum sporozoites by over 40%. The anterior location of INS-15 in invasion stages and partial reduction of in vitro growth indicate that INS-15 plays some roles in the invasion or early development of C. parvum.

Probing the response of lung tumor cells to inflammatory microvascular endothelial cells on fluidic microdevice.

The development of cancer depends on a complex tissue microenvironment for sustained growth, invasion, and metastasis. The extravasation of tumor cells is a critical event in tumor metastasis. However, the process and mechanism that underlie tumor cell extravasation remain unclear, which restricts the examination of many tumor processes and presents a formidable hurdle to drug development. To explore the initial steps by which lung tumor cells interact with the brain microvascular wall in the course of extravasation, we present a simple, inexpensive, and time-saving microfluidic device to mimic the inflammatory brain microvascular microenvironment and to investigate both the biochemical and mechanical causes of lung tumor cell rolling and adhesion on inflammatory endothelium to analyze the synergistic effects on tumor extravasation under fluidic shear stress conditions. Under microvascular inflammation induced by tumor necrosis factor α, the lung tumor cells (A549 cells) displayed significant adhesion activity. In addition, we found that this situation could be reversed by administration of Rho/Rho-associated protein serine/threonine kinase (ROCK) inhibitor (Y27632). We believe that this promising microdevice-based tumor adhesion and extravasation research platform can be used to study tumor behavior in an inflammatory vascular system and will make a valuable contribution for the investigation of the mechanism of tumor cell extravasation.

Assessment of cadmium-induced nephrotoxicity using a kidney-on-a-chip device.

Cadmium (Cd) is a common environmental pollutant. Its effects on human health have attracted great attention. The kidney is the organ that is the most affected by Cd exposure. Thus, it is highly desirable to develop a reliable model to evaluate Cd-induced nephrotoxicity in vitro. We present a kidney-on-a-chip with three compartmentalized culture chambers to examine Cd-induced nephrotoxicity. The culture and collection channels represent the capillary and the glomerular capsule sides of the glomerular filtration barrier, respectively. Isolated primary rat glomerular endothelial cells (GECs) were cultured on the side surface of the middle gel channel. The integrated GEC layer demonstrated the selective permeability of the renal barrier. Therefore, it was further utilized to study the nephrotoxicity induced by Cd exposure at different concentrations. Cd induced significant cytotoxicity and disrupted the expression of tight junction protein ZO-1 in a dose-dependent manner. Moreover, Cd exposure increased the permeability of the endothelial layer to large molecules, immunoglobulin G and albumin. These results facilitate the understanding of the underlying mechanism of kidney dysfunction and glomerular disease. This is the first study on Cd-induced nephrotoxicity using primary GECs in a microfluidic device. The kidney-on-a-chip device enables direct visualization and quantitative analysis of GEC responses to Cd in real time. It may provide a micro-scale platform based on the human system for nephrotoxicity testing under varying environmental exposure.

Assessment of metabolism-dependent drug efficacy and toxicity on a multilayer organs-on-a-chip.

Pharmaceutical development is greatly hindered by the poor predictive power of existing in vitro models for drug efficacy and toxicity testing. In this work, we present a new and multilayer organs-on-a-chip device that allows for the assessment of drug metabolism, and its resultant drug efficacy and cytotoxicity in different organ-specific cells simultaneously. Four cell lines representing the liver, tumor (breast cancer and lung cancer), and normal tissue (gastric cells) were cultured in the compartmentalized micro-chambers of the multilayer microdevice. We adopted the prodrug capecitabine (CAP) as a model drug. The intermediate metabolites 5'-deoxy-5-fluorocytidine (DFUR) of CAP that were metabolized from liver and its active metabolite 5-fluorouracil (5-FU) from the targeted cancer cells and normal tissue cells were identified using mass spectrometry. CAP exhibited strong cytoxicity on breast cancer and lung cancer cells, but not in normal gastric cells. Moreover, the drug-induced cytotoxicity on cells varied in various target tissues, suggesting the metabolism-dependent drug efficacy in different tissues as exisits in vivo. This in vitro model can not only allow for characterizing the dynamic metabolism of anti-cancer drugs in different tissues simultaneously, but also facilitate the assessment of drug bioactivity on various target tissues in a simple way, indicating the utility of this organs-on-chip for applications in pharmacodynamics/pharmacokinetics studies, drug efficacy and toxicity testing.

Simple Spinning of Heterogeneous Hollow Microfibers on Chip.

A novel and simple chip-based microfluidic strategy is proposed for continuously controlled spinning of desirable hollow microfibers. These fabricated fiber-shaped materials exhibit extraordinary morphological and structural complexity, as well as a heterogeneous composition. The resulting specific hollow microfibers have potential applications in numerous chemical and biomedical fields.