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The ability to adapt to osmotically diverse and fluctuating environments is critical to the survival and resilience of bacteria that colonize the human gut and urinary tract. Environmental stress often provides cross-protection against other challenges and increases antibiotic tolerance of bacteria. Thus, it is critical to understand how E. coli and other microbes survive and adapt to stress conditions. The osmotically inducible protein Y (OsmY) is significantly upregulated in response to hypertonicity. Yet its function remains unknown for decades. We determined the solution structure and dynamics of OsmY by nuclear magnetic resonance spectroscopy, which revealed that the two Bacterial OsmY and Nodulation (BON) domains of the protein are flexibly linked under low- and high-salinity conditions. In-cell solid-state NMR further indicates that there are no gross structural changes in OsmY as a function of osmotic stress. Using cryo-electron and super-resolution fluorescence microscopy, we show that OsmY attenuates plasmolysis-induced structural changes in E. coli and improves the time to growth resumption after osmotic upshift. Structure-guided mutational and functional studies demonstrate that exposed hydrophobic residues in the BON1 domain are critical for the function of OsmY. We find no evidence for membrane interaction of the BON domains of OsmY, contrary to current assumptions. Instead, at high ionic strength, we observe an interaction with the water channel, AqpZ. Thus, OsmY does not play a simple structural role in E. coli but may influence a cascade of osmoregulatory functions of the cell., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Continuous assessment of the impact of SARS-CoV-2 on the host at the cell-type level is crucial for understanding key mechanisms involved in host defense responses to viral infection. We investigated host response to ancestral-strain and Alpha-variant SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from younger adults (26-32 Y) and older children (12-14 Y) using single-cell RNA-sequencing. Ciliated and secretory-ciliated cells formed the majority of highly infected cell-types, with the latter derived from ciliated lineages. Strong innate immune responses were observed across lowly infected and uninfected bystander cells and heightened in Alpha-infection. Alpha highly infected cells showed increased expression of protein-refolding genes compared with ancestral-strain-infected cells in children. Furthermore, oxidative phosphorylation-related genes were down-regulated in bystander cells versus infected and mock-control cells, underscoring the importance of these biological functions for viral replication. Overall, this study highlights the complexity of cell-type-, age- and viral strain-dependent host epithelial responses to SARS-CoV-2., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

The recurrence of colorectal liver metastasis (CRLM) following liver resection is common; approximately 40% of patients will experience tumor recurrence post-surgery. Renin-angiotensin inhibitors (RASis) have been shown to attenuate the growth and progression of CRLM in pre-clinical models following liver resection. This study examined the efficacy of the RASi captopril on patient-derived colorectal liver metastasis organoids. Patient-derived organoids (PDOs) were established using fresh samples of colorectal liver metastasis from appropriately consented patients undergoing liver resection. To mimic the regenerating liver post-CRLM liver resection, PDOs were cultured under hepatocyte regeneration conditions in vitro. CRLM PDOs were established from three patients' parent tissue. CRLM PDOs and parent tissue expressed markers of colorectal cancer, CDX2 and CK20, consistently. Furthermore, CRLM PDOs treated with captopril showed a dose dependent reduction in their expansion in vitro. In conclusion, CRLM PDOs recapitulate in vivo disease and displayed a dose-dependent response to treatment with captopril. RASis may be an additional viable treatment for patients with CRLM.

The width of the periplasmic space of Gram-negative bacteria is only about 25-30 nm along the long axis of the cell, which affects free diffusion of (macro)molecules. We have performed single-particle displacement measurements and diffusion simulation studies to determine the impact of confinement on the apparent mobility of proteins in the periplasm of Escherichia coli. The diffusion of a reporter protein and of OsmY, an osmotically regulated periplasmic protein, is characterized by a fast and slow component regardless of the osmotic conditions. The diffusion coefficient of the fast fraction increases upon osmotic upshift, in agreement with a decrease in macromolecular crowding of the periplasm, but the mobility of the slow (immobile) fraction is not affected by the osmotic stress. We observe that the confinement created by the inner and outer membranes results in a lower apparent diffusion coefficient, but this can only partially explain the slow component of diffusion in the particle displacement measurements, suggesting that a fraction of the proteins is hindered in its mobility by large periplasmic structures. Using particle-based simulations, we have determined the confinement effect on the apparent diffusion coefficient of the particles for geometries akin the periplasmic space of Gram-negative bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The intrahepatic cholangiocyte organoids (ICOs) model was evaluated for host differences in hepatitis B virus (HBV) infection, cellular responses, antiviral and immunomodulator responses. Twelve ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterized using histology, confocal, and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma-derived HBV (genotype B and C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT-PCR (RNA, covalently closed circular DNA [cccDNA], extracellular DNA) and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon-stimulated genes [ISG]) to interferon-α and viral mimic (PolyI:C) were assessed. ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well-differentiated ICOs using spinoculation, with time and donor-dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg and HBsAg. Donor-dependent drug responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon-α and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Human ICOs support HBV infection and replication with donor-dependent variation in viral dynamics and cellular responses. These features can be utilized for the development of personalized drug testing platform for antivirals., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Intrahepatic cholangiocyte organoids (ICOs) model was evaluated for host differences in hepatitis B virus (HBV) infection, cellular responses, antiviral, and immunomodulator responses. Twelve ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterized using histology, confocal, and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma derived HBV (genotype B & C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT-PCR (RNA, cccDNA, extracellular DNA), and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon-stimulated genes [ISG]) to interferon-α and viral mimic (PolyI:C) were assessed. ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well-differentiated ICOs using spinoculation, with time and donor dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg, and HBsAg. Donor dependent drug-responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon-α and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Human ICOs support HBV infection and replication with donor dependent variation in viral dynamics and cellular responses. These features can be utilized for development of personalized drug testing platform for antivirals., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors. Multiple clinical trials have been undertaken for this target in the past 15 years; however, we are still to see market approval of the first liver-targeted adeno-associated virus (AAV)-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically and clinically predictive preclinical models. To this end, this study reports findings of a functional evaluation of 6 AAV vectors in 12 preclinical models of the human liver, with the aim to uncover which combination of models is the most relevant for the identification of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

The human pathogen Listeria monocytogenes can cope with severe environmental challenges, for which the high molecular weight stressosome complex acts as the sensing hub in a complicated signal transduction pathway. Here, we show the dynamics and functional roles of the stressosome protein RsbR1 and its paralogue, the blue-light receptor RsbL, using photo-activated localization microscopy combined with single-particle tracking and single-molecule displacement mapping and supported by physiological studies. In live cells, RsbR1 is present in multiple states: in protomers with RsbS, large clusters of stressosome complexes, and in connection with the plasma membrane via Prli42. RsbL diffuses freely in the cytoplasm but forms clusters upon exposure to light. The clustering of RsbL is independent of the presence of Prli42. Our work provides a comprehensive view of the spatial organization and intracellular dynamics of the stressosome proteins in L. monocytogenes, which paves the way towards uncovering the stress-sensing mechanism of this signal transduction pathway., (© 2023. The Author(s).)

Background & Aims: Necroptosis is a highly inflammatory mode of cell death that has been implicated in causing hepatic injury including steatohepatitis/ nonalcoholic steatohepatitis (NASH); however, the evidence supporting these claims has been controversial. A comprehensive, fundamental understanding of cell death pathways involved in liver disease critically underpins rational strategies for therapeutic intervention. We sought to define the role and relevance of necroptosis in liver pathology., Methods: Several animal models of human liver pathology, including diet-induced steatohepatitis in male mice and diverse infections in both male and female mice, were used to dissect the relevance of necroptosis in liver pathobiology. We applied necroptotic stimuli to primary mouse and human hepatocytes to measure their susceptibility to necroptosis. Paired liver biospecimens from patients with NASH, before and after intervention, were analyzed. DNA methylation sequencing was also performed to investigate the epigenetic regulation of RIPK3 expression in primary human and mouse hepatocytes., Results: Identical infection kinetics and pathologic outcomes were observed in mice deficient in an essential necroptotic effector protein, MLKL, compared with control animals. Mice lacking MLKL were indistinguishable from wild-type mice when fed a high-fat diet to induce NASH. Under all conditions tested, we were unable to induce necroptosis in hepatocytes. We confirmed that a critical activator of necroptosis, RIPK3, was epigenetically silenced in mouse and human primary hepatocytes and rendered them unable to undergo necroptosis., Conclusions: We have provided compelling evidence that necroptosis is disabled in hepatocytes during homeostasis and in the pathologic conditions tested in this study., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The biological removal of antibiotic residue in the environment has earned great interest. This study presented the biodegradation of amoxicillin (AMX) using B. cereus C1 isolated from the catfish pond sludge in Vietnam. This AMX-degrading bacterial strain grew well in the range of temperatures between 25ΟC and 40ΟC under aerobic condition. In a culture medium containing nitrogen source of NH4Cl (1 g.L-1) alone, the bacterium showed a AMX degradation ability of 54%. The AMX degradation ability of this bacterial strain was the highest level of 94% in the culture medium with 1.5 g.L-1 of NH4Cl and 3 g.L-1 of glucose. B. cereus C1 exhibited a great antibiotic degradation capability on high AMX concentration of 250 μg.mL-1 of AMX with AMX removal efficiency of 84% in 16 h of cultivation., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors.)

(1) Liver regeneration following partial hepatectomy for colorectal liver metastasis (CRLM) has been linked to tumour recurrence. Inhibition of the renin−angiotensin system (RASi) attenuates CRLM growth in the non-regenerating liver. This study investigates whether RASi exerts an antitumour effect within the regenerating liver following partial hepatectomy for CRLM and examines RASi-induced changes in the tumour immune microenvironment; (2) CRLM in mice was induced via intrasplenic injection of mouse colorectal tumour cells, followed by splenectomy on Day 0. Mice were treated with RASi captopril (250 mg/kg/day), or saline (control) from Day 4 to Day 16 (endpoint) and underwent 70% partial hepatectomy on Day 7. Liver and tumour samples were characterised by flow cytometry and immunofluorescence; (3) captopril treatment reduced tumour burden in mice following partial hepatectomy (p < 0.01). Captopril treatment reduced populations of myeloid-derived suppressor cells (MDSCs) (CD11b+Ly6CHi p < 0.05, CD11b+Ly6CLo p < 0.01) and increased PD-1 expression on infiltrating hepatic tissue-resident memory (TRM)-like CD8+ (p < 0.001) and double-negative (CD4-CD8-; p < 0.001) T cells; (4) RASi reduced CRLM growth in the regenerating liver and altered immune cell composition by reducing populations of immunosuppressive MDSCs and boosting populations of PD-1+ hepatic TRMs. Thus, RASi should be explored as an adjunct therapy for patients undergoing partial hepatectomy for CRLM.

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air-liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID 50 , while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

A major unmet clinical need is a therapeutic capable of removing hepatitis B virus (HBV) genome from the liver of infected individuals to reduce their risk of developing liver cancer. A strategy to deliver such a therapy could utilize the ability to target and promote apoptosis of infected hepatocytes. Presently there is no clinically relevant strategy that has been shown to effectively remove persistent episomal covalently closed circular HBV DNA (cccDNA) from the nucleus of hepatocytes. We used linearized single genome length HBV DNA of various genotypes to establish a cccDNA-like reservoir in immunocompetent mice and showed that clinical-stage orally administered drugs that antagonize the function of cellular inhibitor of apoptosis proteins can eliminate HBV replication and episomal HBV genome in the liver. Primary human liver organoid models were used to confirm the clinical relevance of these results. This study underscores a clinically tenable strategy for the potential elimination of chronic HBV reservoirs in patients.

Cell cultures have been successfully used to study hepatitis C virus (HCV) for many years. However, most work has been done using traditional, 2-dimensional (2D) cell cultures (cells grown as a monolayer in growth flasks or dishes). Studies have shown that when cells are grown suspended in an extra-cellular-matrix-like material, they develop into spherical, 'organoid' arrangements of cells (3D growth) that display distinct differences in morphological and functional characteristics compared to 2D cell cultures. In liver organoids, one key difference is the development of clearly differentiated apical and basolateral surfaces separated and maintained by cellular tight junctions. This phenomenon, termed polarity, is vital to normal barrier function of hepatocytes in vivo. It has also been shown that viruses, and virus-like particles, interact very differently with cells derived from 2D as compared to 3D cell cultures, bringing into question the usefulness of 2D cell cultures to study virus-host cell interactions. Here, we investigate differences in cellular architecture as a function of cell culture system, using confocal scanning laser microscopy, and determine differences in binding interactions between HCV virus-like particles (VLPs) and their cognate receptors in the different cell culture systems using atomic force microscopy (AFM). We generated organoid cultures that were polarized, as determined by localization of key apical and basolateral markers. We found that, while uptake of HCV VLPs by both 2D and 3D Huh7 cells was observed by flow cytometry, binding interactions between HCV VLPs and cells were measurable by AFM only on polarized cells. The work presented here adds to the growing body of research suggesting that polarized cell systems are more suitable for the study of HCV infection and dynamics than non-polarized systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

(1) Background: Recent clinical and experimental data suggests that the liver's regenerative response following partial hepatectomy can stimulate tumor recurrence in the liver remnant. The Wnt/β-catenin pathway plays important roles in both colorectal cancer carcinogenesis and liver regeneration. Studies have shown that the Wnt/β-catenin pathway regulates multiple renin-angiotensin system (RAS) genes, whilst RAS inhibition (RASi) reduces tumor burden and progression. This study explores whether RASi attenuates features of tumor progression in the regenerating liver post-hepatectomy by modulating Wnt/β-catenin signaling. (2) Methods: Male CBA mice underwent CRLM induction, followed one week later by 70% partial hepatectomy. Mice were treated daily with captopril, a RASi, at 250 mg/kg/day or vehicle control from experimental Day 4. Tumor and liver samples were analyzed for RAS and Wnt signaling markers using qRT-PCR and immunohistochemistry. (3) Results: Treatment with captopril reduced the expression of down-stream Wnt target genes, including a significant reduction in both c-myc and cyclin-D1, despite activating Wnt signaling. This was a tumor-specific response that was not elicited in corresponding liver samples. (4) Conclusions: We report for the first time decreased c-myc expression in colorectal tumors following RASi treatment in vivo. Decreased c-myc expression was accompanied by an attenuated invasive phenotype, despite increased Wnt signaling.

Background: Germline pathogenic variants in the cadherin-1 (CDH1) gene cause a predisposition to hereditary diffuse gastric cancer (HDGC). We report an HDGC case in Vietnam and identify a novel mutation in the CDH1 gene., Case Presentation: A 28-year-old Vietnamese man was diagnosed with HDGC and a novel mutation at c.639G>A. All exons of CDH1 were sequenced in his pedigree, which revealed the c.639G>A mutation in the proband, his father, and uncle. The patient refused treatment and died 4 months after diagnosis. Endoscopic surveillance of the father and the uncle showed structural abnormalities in the father., Conclusion: In cases of HDGC, identification of the CDH1 gene mutation is very important for better counseling and more effective strategies to prevent the development of diseases, such as prophylactic gastrectomy for family members with genetic mutations.

Protein mobility in the cytoplasm is essential for cellular functions, and slow diffusion may limit the rates of biochemical reactions in the living cell. Here, we determined the apparent lateral diffusion coefficient ( D L ) of GFP in Listeria monocytogenes as a function of osmotic stress, temperature, and media composition. We find that D L is much less affected by hyperosmotic stress in L. monocytogenes than under similar conditions in Lactococcus lactis and Escherichia coli . We find a temperature optimum for protein diffusion in L. monocytogenes at 30°C, which deviates from predicted trends from the generalized Stokes-Einstein equation under dilute conditions and suggests that the structure of the cytoplasm and macromolecular crowding vary as a function of temperature. The turgor pressure of L. monocytogenes is comparable to other Gram-positive bacteria like Bacillus subtilis and L. lactis but higher in a knockout strain lacking the stress-inducible sigma factor SigB. We discuss these findings in the context of how L. monocytogenes survives during environmental transmission and interaction with the human host., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tran, Prabha, Iyer, O’Byrne, Abee and Poolman.)

Frizzled 7 activates β-catenin-dependent and β-catenin-independent Wnt signalling pathways, is highly conserved through evolution from the ancient phylum hydra to man, plays essential roles in stem cells, tissue homeostasis and regeneration in the adult, and is upregulated in diverse cancers. Much of what is known about the core components of the Wnt signalling pathways was derived from studying the function of Frizzled 7 orthologues in the development of lower organism. As we interrogate Frizzled 7 signalling and function for therapeutic targeting in cancer, it is timely to revisit lower organisms to gain insight into the context dependent and dynamic nature of Wnt signalling for effective drug design., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

An emerging theme for Wnt-addicted cancers is that the pathway is regulated at multiple steps via various mechanisms. Infection with hepatitis B virus (HBV) is a major risk factor for liver cancer, as is deregulated Wnt signaling, however, the interaction between these two causes is poorly understood. To investigate this interaction, we screened the effect of the various HBV proteins for their effect on Wnt/β-catenin signaling and identified the pre-core protein p22 as a novel and potent activator of TCF/β-catenin transcription. The effect of p22 on TCF/β-catenin transcription was dose dependent and inhibited by dominant-negative TCF4. HBV p22 activated synthetic and native Wnt target gene promoter reporters, and TCF/β-catenin target gene expression in vivo. Importantly, HBV p22 activated Wnt signaling on its own and in addition to Wnt or β-catenin induced Wnt signaling. Furthermore, HBV p22 elevated TCF/β-catenin transcription above constitutive activation in colon cancer cells due to mutations in downstream genes of the Wnt pathway, namely APC and CTNNB1 . Collectively, our data identifies a previously unappreciated role for the HBV pre-core protein p22 in elevating Wnt signaling. Understanding the molecular mechanisms of p22 activity will provide insight into how Wnt signaling is fine-tuned in cancer.

Influenza A viruses are often present in environmental and clinical samples at concentrations below the limit of detection (LOD) of molecular diagnostics. Here we report an integrated microfluidic preconcentration and nucleic amplification system (μFPNAS) which enables both preconcentration of influenza A virus H1N1 (H1N1) and amplification of its viral RNA, thereby lowering LOD for H1N1. H1N1 virus particles were first magnetically preconcentrated using magnetic nanoparticles conjugated with an antibody specific for the virus. Their isolated RNA was amplified to cDNA through thermocycling in a trapezoidal chamber of the μFPNAS. A detection limit as low as 100 TCID50 (50% tissue culture infective dose) in saliva can be obtained within 2 hours. These results suggest that the LOD of molecular diagnostics for virus can be lowered by systematically combining immunomagnetic separation and reverse transcriptase-polymerase chain reaction (RT-PCR) in one microfluidic device.

Background: Hepatitis B virus (HBV) is the leading cause of liver cancer, but the mechanisms by which HBV causes liver cancer are poorly understood and chemotherapeutic strategies to cure liver cancer are not available. A better understanding of how HBV requisitions cellular components in the liver will identify novel therapeutic targets for HBV associated hepatocellular carcinoma (HCC)., Main Body: The development of HCC involves deregulation in several cellular signalling pathways including Wnt/FZD/β-catenin, PI3K/Akt/mTOR, IRS1/IGF, and Ras/Raf/MAPK. HBV is known to dysregulate several hepatocyte pathways and cell cycle regulation resulting in HCC development. A number of these HBV induced changes are also mediated through the Wnt/FZD/β-catenin pathway. The lack of a suitable human liver model for the study of HBV has hampered research into understanding pathogenesis of HBV. Primary human hepatocytes provide one option; however, these cells are prone to losing their hepatic functionality and their ability to support HBV replication. Another approach involves induced-pluripotent stem (iPS) cell-derived hepatocytes. However, iPS technology relies on retroviruses or lentiviruses for effective gene delivery and pose the risk of activating a range of oncogenes. Liver organoids developed from patient-derived liver tissues provide a significant advance in HCC research. Liver organoids retain the characteristics of their original tissue, undergo unlimited expansion, can be differentiated into mature hepatocytes and are susceptible to natural infection with HBV., Conclusion: By utilizing new ex vivo techniques like liver organoids it will become possible to develop improved and personalized therapeutic approaches that will improve HCC outcomes and potentially lead to a cure for HBV.

The discovery of Lgr5 as a marker of adult stem cells meant that stem cell populations could be purified and studied in isolation. Importantly, when cultured under the appropriate conditions these stem cells form organoids in tissue culture that retain many features of the tissue of origin. The organoid cultures are accessible to genetic and biochemical manipulation, bridging the gap between in vivo mouse models and conventional tissue culture. Here we describe robust protocols to establish organoids from gastrointestinal tissues (stomach, intestine, liver) and Cre-recombinase mediated gene manipulation in vitro.

This study reports an advanced approach to effectively generate hollow fibers in a triple-flow polydimethylsiloxane (PDMS) microfluidic device based on the gelation of alginate induced with CaCl 2 inside a coaxial flow system. Two PDMS replicas with a semi-cylindrical microchannel were assembled to obtain a complete microchannel with a circular cross-section, which allowed the formation of mild and continuous coaxial flows for the fabrication of hollow fibers without employing complex glass microcapillaries. Mineral oil was introduced into the central flow to serve as an inert space inside the Ca-alginate wall. This was used to maintain the consistent formation of the hollow core of the microfiber and to easily transport fluid through the lumen structure in subsequent applications. The hollow fibers exhibited characteristics such as flexibility while showing robust mechanical strength, high permeability, and biocompatibility, and were used as scaffolds for the attachment and proliferation of human umbilical vein endothelial cells (HUVECs) to mimic a blood vessel. The fully covered HUVEC fibers were further integrated into a neurovascular system and co-cultured with astrocytes forming an on-chip blood brain barrier (BBB) platform. The use of this neurovascular model for drug testing will pave the way for developing or synthesizing a new drug that can cross the BBB in the human brain.

Nanoporous anodic aluminum oxide internalized with gold nanoparticles was utilized as an integrated platform miniaturized for consecutively performing on-chip PCR and downstream detection of the amplified product of a 183 bp eaeA gene fragment from Escherichia coli O157:H7 using surface-enhanced Raman scattering (SERS).

The Wnt pathway is at the heart of organoid technology, which is set to revolutionize the cancer field. We can now predetermine a patient's response to any given anticancer therapy by exposing tumor organoids established from the patient's own tumor. This cutting-edge biomedical platform translates to patients being treated with the correct drug at the correct dose from the outset, a truly personalized and precise medical approach. A high throughput drug screen on organoids also allows drugs to be tested in limitless combinations. More recently, the tumor cells that are resistant to the therapy given to a patient were selected in culture using the patient's organoids. The resistant tumor organoids were then screened empirically to identify drugs that will kill the resistant cells. This information allows diagnosis in real-time to either prevent tumor recurrence or effectively treat the recurring tumor. Furthermore, the ability to culture stem cell-derived epithelium as organoids has enabled us to begin to understand how a stem cell becomes a cancer cell or to pin-point the genetic alteration that underlies a given genetic syndrome. Here we summarize these advances and the central role of Wnt signaling, and identify the next challenges for organoid technology., (Copyright © 2018 Elsevier Inc. All rights reserved.)

There is growing interest in rapid microbial pre-concentration methods to lower the detection limit of bacterial pathogens of low abundance in samples. Here, we report an integrated microfluidic PCR system that enables bacterial cells of interest in samples to be concentrated prior to PCR. It consists of two major compartments: a preconcentration chamber for the immunomagnetic separation of bacterial cells, and a PCR chamber for the DNA amplification of the concentrated cells. We demonstrate the feasibility of the system for the detection of microbial pathogens by preconcentrating the human pathogen Escherichia coli O157:H7, and also amplifying its DNA. The detection limit of E. coli O157:H7 in the PCR system is 1 × 10 3  CFU (colony forming unit)/mL. On-chip processing steps, including preconcentration and PCR steps, take less than two hours. Our system can serve as a rapid, specific, and quantitative platform for the detection of microbial pathogens in samples of large volume.

Here, we introduce a simple and fast method for bonding a poly(dimethylsiloxane) (PDMS) silicone elastomer to different plastics. In this technique, surface modification and subsequent bonding processes are performed at room temperature. Furthermore, only one chemical is needed, and no surface oxidation step is necessary prior to bonding. This bonding method is particularly suitable for encapsulating biomolecules that are sensitive to external stimuli, such as heat or plasma treatment, and for embedding fracturable materials prior to the bonding step. Microchannel-fabricated PDMS was first oxidized by plasma treatment and reacted with aminosilane by forming strong siloxane bonds (Si-O-Si) at room temperature. Without the surface oxidation of the amine-terminated PDMS and plastic, the two heterogeneous substrates were brought into intimate physical contact and left at room temperature. Subsequently, aminolysis occurred, leading to the generation of a permanent seal via the formation of robust urethane bonds after only 5 min of assembling. Using this method, large-area (10 × 10 cm) bonding was successfully realized. The surface was characterized by contact angle measurements and X-ray photoelectron spectroscopy (XPS) analyses, and the bonding strength was analyzed by performing peel, delamination, leak, and burst tests. The bond strength of the PDMS-polycarbonate (PC) assembly was approximately 409 ± 6.6 kPa, and the assembly withstood the injection of a tremendous amount of liquid with the per-minute injection volume exceeding 2000 times its total internal volume. The thermal stability of the bonded microdevice was confirmed by performing a chamber-type multiplex polymerase chain reaction (PCR) of two major foodborne pathogens - Escherichia coli O157:H7 and Salmonella typhimurium - and assessing the possibility for on-site direct detection of PCR amplicons. This bonding method demonstrated high potential for the stable construction of closed microfluidic systems socketed with biomolecule-immobilized surfaces such as DNA, antibody, enzyme, peptide, and protein microarrays.

Background: Glucocorticosteroids are effective in the treatment of allergic rhinitis, a disease characterized by a variety of symptoms, e.g. rhinorrhea and itching. The time course of symptomatic relief for allergic rhinitis by steroids has not been examined in detail to date, although the onset of steroid action is one of the main discriminations between genomic and nongenomic actions of steroids. We therefore investigated the time course of subjective and objective measures of nasal affection after steroid administration in patients with allergic rhinitis following specific allergen challenge., Methods: Six female and 18 male volunteers (median age 26 years) with a history of allergic rhinitis but currently free of symptoms were included in this randomized, placebo-controlled, double-blind, three-period crossover study. A single dose of either betamethasone (60 mg), methylprednisolone (400 mg) or placebo was given intravenously, 5 min after intranasal allergen provocation. After 10, 20, 60, 150 and 240 min, nasal itching and nasal obstruction were assessed using a standardized visual analogue scale. In addition, nasal airflow was measured by anterior rhinomanometry., Results: Nasal itching was markedly reduced following either of the two steroids within 10 min after administration of study drug. Itching was depressed by 38% following betamethasone (P<0.05) and by 18% following methylprednisolone (P=0.07) compared with placebo. Nasal airflow and nasal obstruction were not significantly altered by steroids during the first 2 h of the study. However, after 150 min, nasal airflow was 21% rsp. 19% higher after methylprednisolone and betamethasone (P<0.05) compared with placebo. After 240 min, nasal airflow was increased by 20% following betamethasone (P<0.05) and by 19% following methylprednisolone. Nasal obstruction was also beneficially affected by both steroids 150 and 240 min after administration compared with placebo (P<0.05 for both time points following betamethasone)., Conclusion: This study for the first time shows rapid in vivo effects of external glucocorticosteroids in humans. Itching, a pathophysiologically complex sensation, is favourably influenced by steroids within 10 min, therefore presumably via nongenomic mechanisms. Though no detailed mechanisms can be derived from this study, steroid interaction with receptors in the central nervous system may play an important role in mediating this effect.

Previous studies have revealed beneficial in vitro effects of progesterone on sperm function. The aim of this pilot study was to prove if orally given micronized progesterone leads to elevations in progesterone and/or 17alpha-hydroxyprogesterone levels in seminal plasma, since higher seminal plasma levels of these hormones could possibly have a beneficial effect on sperm function as seen in in vitro investigations. Multiple application of micronized progesterone given over 4 days (daily dose 400 mg) to 6 healthy subjects resulted in elevated seminal plasma levels of progesterone (10.90 +/- 9.02 nmol/l vs. 1.43 +/- 0.56 nmol/l, p = 0.04) and 17alpha-hydroxyprogesterone (3.09 +/- 1.72 nmol/l vs. 1.62 +/- 1.26 nmol/l, p = 0.04) whereas no significant difference could be found in testosterone levels (34.82 +/- 13.00 vs. 30.91 +/- 8.56 nmol/l, p = 0.43). In contrast, androstendione levels in seminal plasma were reduced (2.68 1.28 nmol/l vs. 3.65 +/- 1.36 nmol/l, p = 0.01). Although micronized progesterone is rapidly metabolized, oral application resulted in pronounced elevations of progesterone and 17alpha-hydroxyprogesterone in seminal plasma. Further studies will show if oral application of micronized progesterone can induce beneficial effects on sperm function such as those seen in in vitro investigations.