Optical tweezers for probing the interactions of ZnO and Ag nanoparticles with E. coli.

The antibacterial effect of nanoparticles is mainly studied on the ensembles of the bacteria. In contrast, the optical tweezer technique allows the investigation of similar effects on individual bacterium. E. coli is a self-propelled micro-swimmer and ATP-driven active microorganism. In this work, an optical tweezer is employed to examine the mechanical properties of E. coli incubated with ZnO and Ag nanoparticles (NP) in the growth medium. ZnO and Ag NP with a concentration of 10 µg/ml were dispersed in growth medium during active log-growth phase of E. coli. This E. coli-NP incubation is further continued for 12 h. The E. coli after incubation for 2 h, 6 h and 12 h were separately studied by the optical tweezer for their mechanical property. The IR laser (λ = 975 nm; power = 100 mW) was used for trapping the individual cells and estimated trapping force, trapping stiffness and corner frequency. The optical trapping force on E. coli incubated in nanoparticle suspension shows linear decreases with incubation time. This work brings the importance of optical trapping force measurement in probing the antibacterial stress due to nanoparticles on the individual bacterium.

Probing Effect of 6 MeV Electron Beam Irradiation on Haemoglobin Protein Using Spectroscopic Techniques.

In this work, we study the effect of 6 MeV electron beam irradiation on the physicochemical properties of lyophilized Human Haemoglobin A (HbA). Electron beams generated from Race Track Microtron accelerator with energy 6 MeV were used to irradiate HbA at fluences of 5 × 1014 e-/cm2 and 10 × 1014 e-/cm2. Pristine and electron beam irradiated HbA were characterized using UV-visible and Fourier transform infrared spectroscopy (FTIR) spectroscopy. The interfacial tension of the aqueous solutions of HbA are also analysed by pendant drop method. Absorbance intensity, % transmittance and interfacial tension decrease with fluence. The peak position of the Soret band (λsoret = 404 nm) remains unaffected by the fluences. FTIR spectroscopy confirms the changes in the secondary structure of the haemoglobin. In the amide band I, the percentage of α-helix reduced from 8% to 1%, and an increase in ß-sheet (19% to 29%) and ß helix (6.3% to 15%) is observed. Interfacial tension decreases from 46.0 mN/m and 44.0 mN/m with increase in irradiation dose. These finding provides realistic guideline for biological cells exposure to electron beam radiation doses.

Synthesis of α3ß4 Nicotinic Acetylcholine Receptor Modulators Derived from Aristoquinoline That Reduce Reinstatement of Cocaine-Seeking Behavior.

Growing evidence suggests that inhibition of the α3ß4 nicotinic acetylcholine receptor (nAChR) represents a promising therapeutic strategy to treat cocaine use disorder. Recently, aristoquinoline (1), an alkaloid from Aristotelia chilensis, was identified as an α3ß4-selective nAChR inhibitor. Here, we prepared 22 derivatives of 1 and evaluated their ability to inhibit the α3ß4 nAChR. These studies revealed structure-activity trends and several compounds with increased potency compared to 1 with few off-target liabilities. Additional mechanistic studies indicated that these compounds inhibit the α3ß4 nAChR noncompetitively, but do not act as channel blockers, suggesting they are negative allosteric modulators. Finally, using a cocaine-primed reinstatement paradigm, we demonstrated that 1 significantly attenuates drug-seeking behavior in an animal model of cocaine relapse. The results from these studies further support a role for the α3ß4 nAChR in the addictive properties of cocaine and highlight the possible utility of aristoquinoline derivatives in treating cocaine use disorder.

Performance assessment and economic analysis of a human Liver-Chip for predictive toxicology.

BACKGROUND: Conventional preclinical models often miss drug toxicities, meaning the harm these drugs pose to humans is only realized in clinical trials or when they make it to market. This has caused the pharmaceutical industry to waste considerable time and resources developing drugs destined to fail. Organ-on-a-Chip technology has the potential improve success in drug development pipelines, as it can recapitulate organ-level pathophysiology and clinical responses; however, systematic and quantitative evaluations of Organ-Chips' predictive value have not yet been reported. METHODS: 870 Liver-Chips were analyzed to determine their ability to predict drug-induced liver injury caused by small molecules identified as benchmarks by the Innovation and Quality consortium, who has published guidelines defining criteria for qualifying preclinical models. An economic analysis was also performed to measure the value Liver-Chips could offer if they were broadly adopted in supporting toxicity-related decisions as part of preclinical development workflows. RESULTS: Here, we show that the Liver-Chip met the qualification guidelines across a blinded set of 27 known hepatotoxic and non-toxic drugs with a sensitivity of 87% and a specificity of 100%. We also show that this level of performance could generate over $3 billion annually for the pharmaceutical industry through increased small-molecule R&D productivity. CONCLUSIONS: The results of this study show how incorporating predictive Organ-Chips into drug development workflows could substantially improve drug discovery and development, allowing manufacturers to bring safer, more effective medicines to market in less time and at lower costs.

Drug development is lengthy and costly, as it relies on laboratory models that fail to predict human reactions to potential drugs. Because of this, toxic drugs sometimes go on to harm humans when they reach clinical trials or once they are in the marketplace. Organ-on-a-Chip technology involves growing cells on small devices to mimic organs of the body, such as the liver. Organ-Chips could potentially help identify toxicities earlier, but there is limited research into how well they predict these effects compared to conventional models. In this study, we analyzed 870 Liver-Chips to determine how well they predict drug-induced liver injury, a common cause of drug failure, and found that Liver-Chips outperformed conventional models. These results suggest that widespread acceptance of Organ-Chips could decrease drug attrition, help minimize harm to patients, and generate billions in revenue for the pharmaceutical industry.

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed. Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions. Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure for the in vitro modeling of interferon gamma (IFNγ)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

A Novel Microphysiological Colon Platform to Decipher Mechanisms Driving Human Intestinal Permeability.

BACKGROUND & AIMS: The limited availability of organoid systems that mimic the molecular signatures and architecture of human intestinal epithelium has been an impediment to allowing them to be harnessed for the development of therapeutics as well as physiological insights. We developed a microphysiological Organ-on-Chip (Emulate, Inc, Boston, MA) platform designed to mimic properties of human intestinal epithelium leading to insights into barrier integrity. METHODS: We combined the human biopsy-derived leucine-rich repeat-containing G-protein-coupled receptor 5-positive organoids and Organ-on-Chip technologies to establish a micro-engineered human Colon Intestine-Chip (Emulate, Inc, Boston, MA). We characterized the proximity of the model to human tissue and organoids maintained in suspension by RNA sequencing analysis, and their differentiation to intestinal epithelial cells on the Colon Intestine-Chip under variable conditions. Furthermore, organoids from different donors were evaluated to understand variability in the system. Our system was applied to understanding the epithelial barrier and characterizing mechanisms driving the cytokine-induced barrier disruption. RESULTS: Our data highlight the importance of the endothelium and the in vivo tissue-relevant dynamic microenvironment in the Colon Intestine-Chip in the establishment of a tight monolayer of differentiated, polarized, organoid-derived intestinal epithelial cells. We confirmed the effect of interferon-γ on the colonic barrier and identified reorganization of apical junctional complexes, and induction of apoptosis in the intestinal epithelial cells as mediating mechanisms. We show that in the human Colon Intestine-Chip exposure to interleukin 22 induces disruption of the barrier, unlike its described protective role in experimental colitis in mice. CONCLUSIONS: We developed a human Colon Intestine-Chip platform and showed its value in the characterization of the mechanism of action of interleukin 22 in the human epithelial barrier. This system can be used to elucidate, in a time- and challenge-dependent manner, the mechanism driving the development of leaky gut in human beings and to identify associated biomarkers.

Modeling alcohol-associated liver disease in a human Liver-Chip.

Alcohol-associated liver disease (ALD) is a global health issue and leads to progressive liver injury, comorbidities, and increased mortality. Human-relevant preclinical models of ALD are urgently needed. Here, we leverage a triculture human Liver-Chip with biomimetic hepatic sinusoids and bile canaliculi to model ALD employing human-relevant blood alcohol concentrations (BACs) and multimodal profiling of clinically relevant endpoints. Our Liver-Chip recapitulates established ALD markers in response to 48 h of exposure to ethanol, including lipid accumulation and oxidative stress, in a concentration-dependent manner and supports the study of secondary insults, such as high blood endotoxin levels. We show that remodeling of the bile canalicular network can provide an in vitro quantitative readout of alcoholic liver toxicity. In summary, we report the development of a human ALD Liver-Chip as a powerful platform for modeling alcohol-induced liver injury with the potential for direct translation to clinical research and evaluation of patient-specific responses.

Investigating alterations in the cellular envelope of Staphylococcus aureus in simulated microgravity using a random positioning machine.

Continuous rotation of liquid bacterial culture in random positioning machine (RPM) causes formation of a colloidal bacterial culture in the culture tube, due to lack of sedimentation and convection. Interestingly, similar colloidal bacterial cultures can also be seen in suspended bacterial cultures in a spaceflight environment. Thus, as a consequence of no sedimentation, an alteration in the microenvironment of each bacterial cell in simulated microgravity is introduced, compared to the bacterial culture grown in normal gravity wherein they sediment slowly at the bottom of the culture tube. Apparently, a bacterial cell can sense changes in its environment through various receptors and sensors present at its surface, thus it can be speculated that this change in its microenvironment might induce changes in its cell wall and cell surface properties. In our study, changes in growth kinetics, cell wall constitution using FTIR (Fourier Transform Infrared Spectroscopy), cell surface hydrophobicity, autoaggregation ability and antibiotic susceptibility of Staphylococcus aureus NCIM 2079 strain, in simulated microgravity (using RPM) was studied in detail. Noteworthy alterations in its growth kinetics, cell wall constitution, cell surface hydrophobicity, autoaggregation ability and antibiotic susceptibility especially to Erythromycin and Clindamycin were observed. Our data suggests that microgravity may cause alterations in the cellular envelope of planktonic S.aureus cultures.

Comparative studies on measurement of membrane potential of bacterial cells treated with ZnO nanoparticles by Spectrofluorometry, fluorescence microscopy and flowcytometry.

Many methods are developed to assess antimicrobial action of ZnO nanoparticles (NPs). A large number of methods associated with the use of fluorescent probes are developed, including Spectrofluorometry, fluorescence microscopy, and cytometry. In this study, flowcytometry, Spectrofluorometry and fluorescent microscopy was used to measure membrane potential variation of E. coli and S. aureus cells treated with two different sizes of zinc oxide (ZnO) NPs and were compared with conventional methods. In order to estimate change in membrane potential, E. coli and S. aureus cells were treated with iopnophore agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) and membrane potential was evaluated using fluorescent probe 3,3'-Diethyloxacarbocyanine, iodide (DIOC2(3)). All the three methods showed similar results and among these Spectrofluorometry was easy to use and inexpensive to assess the viability of bacterial cells via their membrane potential.

Duodenum Intestine-Chip for preclinical drug assessment in a human relevant model.

Induction of intestinal drug metabolizing enzymes can complicate the development of new drugs, owing to the potential to cause drug-drug interactions (DDIs) leading to changes in pharmacokinetics, safety and efficacy. The development of a human-relevant model of the adult intestine that accurately predicts CYP450 induction could help address this challenge as species differences preclude extrapolation from animals. Here, we combined organoids and Organs-on-Chips technology to create a human Duodenum Intestine-Chip that emulates intestinal tissue architecture and functions, that are relevant for the study of drug transport, metabolism, and DDI. Duodenum Intestine-Chip demonstrates the polarized cell architecture, intestinal barrier function, presence of specialized cell subpopulations, and in vivo relevant expression, localization, and function of major intestinal drug transporters. Notably, in comparison to Caco-2, it displays improved CYP3A4 expression and induction capability. This model could enable improved in vitro to in vivo extrapolation for better predictions of human pharmacokinetics and risk of DDIs.

Reproducing human and cross-species drug toxicities using a Liver-Chip.

Nonclinical rodent and nonrodent toxicity models used to support clinical trials of candidate drugs may produce discordant results or fail to predict complications in humans, contributing to drug failures in the clinic. Here, we applied microengineered Organs-on-Chips technology to design a rat, dog, and human Liver-Chip containing species-specific primary hepatocytes interfaced with liver sinusoidal endothelial cells, with or without Kupffer cells and hepatic stellate cells, cultured under physiological fluid flow. The Liver-Chip detected diverse phenotypes of liver toxicity, including hepatocellular injury, steatosis, cholestasis, and fibrosis, and species-specific toxicities when treated with tool compounds. A multispecies Liver-Chip may provide a useful platform for prediction of liver toxicity and inform human relevance of liver toxicities detected in animal studies to better determine safety and human risk.

Introducing an automated high content confocal imaging approach for Organs-on-Chips.

Organ-Chips are micro-engineered systems that aim to recapitulate the organ microenvironment. Implementation of Organ-Chips within the pharmaceutical industry aims to improve the probability of success of drugs reaching late stage clinical trial by generating models for drug discovery that are of human origin and have disease relevance. We are adopting the use of Organ-Chips for enhancing pre-clinical efficacy and toxicity evaluation and prediction. Whilst capturing cellular phenotype via imaging in response to drug exposure is a useful readout in these models, application has been limited due to difficulties in imaging the chips at scale. Here we created an end-to-end, automated workflow to capture and analyse confocal images of multicellular Organ-Chips to assess detailed cellular phenotype across large batches of chips. By automating this process, we not only reduced acquisition time, but we also minimised process variability and user bias. This enabled us to establish, for the first time, a framework of statistical best practice for Organ-Chip imaging, creating the capability of using Organ-Chips and imaging for routine testing in drug discovery applications that rely on quantitative image data for decision making. We tested our approach using benzbromarone, whose mechanism of toxicity has been linked to mitochondrial damage with subsequent induction of apoptosis and necrosis, and staurosporine, a tool inducer of apoptosis. We also applied this workflow to assess the hepatotoxic effect of an active AstraZeneca drug candidate illustrating its applicability in drug safety assessment beyond testing tool compounds. Finally, we have demonstrated that this approach could be adapted to Organ-Chips of different shapes and sizes through application to a Kidney-Chip.

In Situ Tissue Regeneration of Renal Tissue Induced by Collagen Hydrogel Injection.

Host stem/progenitor cells can be mobilized and recruited to a target location using biomaterials, and these cells may be used for in situ tissue regeneration. The objective of this study was to investigate whether host biologic resources could be used to regenerate renal tissue in situ. Collagen hydrogel was injected into the kidneys of normal mice, and rat kidneys that had sustained ischemia/reperfusion injury. After injection, the kidneys of both animal models were examined up to 4 weeks for host tissue response. The infiltrating host cells present within the injection regions expressed renal stem/progenitor cell markers, PAX-2, CD24, and CD133, as well as mesenchymal stem cell marker, CD44. The regenerated renal structures were identified by immunohistochemistry for renal cell specific markers, including synaptopodin and CD31 for glomeruli and cytokeratin and neprilysin for tubules. Quantitatively, the number of glomeruli found in the injected regions was significantly higher when compared to normal regions of renal cortex. This phenomenon occurred in normal and ischemic injured kidneys. Furthermore, the renal function after ischemia/reperfusion injury was recovered after collagen hydrogel injection. These results demonstrate that introduction of biomaterials into the kidney is able to facilitate the regeneration of glomerular and tubular structures in normal and injured kidneys. Such an approach has the potential to become a simple and effective treatment for patients with renal failure. Stem Cells Translational Medicine 2018;7:241-250.

Ellagic Acid Enhances Apoptotic Sensitivity of Breast Cancer Cells to γ-Radiation.

Herbal polyphenols have gained increased significance because of the promises they hold in the prevention and treatment of cancer. There exists an enormous opportunity for the screening and valuation of natural dietary compounds in the development of an effective chemopreventive drug and radiosensitizer that may be of practical use for patients undergoing cancer therapy. This study describes the effect of the flavonoid ellagic acid (EA) on gamma-irradiated human breast cancer MCF-7 cells in vitro when administered alone or in combination with radiation. It was interesting to find the radioprotective effect of EA on NIH3T3, which is a normal cell line. Irradiation of breast tumor cells in the presence of EA (10 µM) to doses of 2 and 4-Gy gamma radiation produced a marked synergistic tumor cytotoxicity while it was found to aid recovery from the radiation damage to NIH3T3 cells. When cells were given a combined treatment of EA and radiation, the cell death increased to 21.7% and 20.7% in the 2 and 4-Gy-treated cells respectively, significantly (P < 0.05) reducing the capacity of MCF-7 cells to form colonies. Even at 24 h, 38 foci/cell were observed in samples that were given the combined treatment, suggesting the cells' inability in repairing the damage. Also, increased apoptosis in EA+ 2Gy (50%) and EA+ 4 Gy (62%)-treated cells was observed in the the sub-G1 phase of the cell cycle. A 6.2-fold decrease in the mitochondrial membrane potential was observed in the combined treatment of EA and IR that facilitated the upregulation of pro-apopttotic Bax and downregulation of Bcl-2, pushing the MCF-7 cells to undergo an apoptotic cell death. It is suggested that EA may be a potential drug adjuvant for improving cancer radiotherapy by increasing tumor toxicity and reducing the normal cell damage caused by irradiation.

Monte Carlo based investigations of electron contamination from telecobalt unit head in build up region and its impact on surface dose.

A Telecobalt unit has wide range of applications in cancer treatments and is used widely in many countries all around the world. Estimation of surface dose in Cobalt-60 teletherapy machine becomes important since clinically useful photon beam consist of contaminated electrons during the patient treatment. EGSnrc along with the BEAMnrc user code was used to model the Theratron 780E telecobalt unit. Central axis depth dose profiles including surface doses have been estimated for the field sizes of 0×0, 6×6, 10×10, 15×15, 20×20, 25×25, 30×30cm2 and at Source-to-surface distance (SSD) of 60 and 80cm. Surface dose was measured experimentally by the Gafchromic RTQA2 films and are in good agreement with the simulation results. The central axis depth dose data are compared with the data available from the British Journal of Radiology report no. 25. Contribution of contaminated electrons has also been calculated using Monte Carlo simulation by the different parts of the Cobalt-60 head for different field size and SSD's. Moreover, depth dose curve in zero area field size is calculated by extrapolation method and compared with the already published data. They are found in good agreement.

Biosurfactant/s from Lactobacilli species: Properties, challenges and potential biomedical applications.

Lactic acid bacteria are generally believed to have positive roles in maintaining good health and immune system in humans. A number of Lactobacilli spp. are known to produce important metabolites, among which biosurfactants in particular have shown antimicrobial activity against several pathogens in the intestinal tract and female urogenital tract partly through interfering with biofilm formation and adhesion to the epithelial cells surfaces. Around 46 reports are documented on biosurfactant production from Lactobacillus spp. of which six can be broadly classified as cell free biosurfactant and 40 as cell associated biosurfactants and only approximately 50% of those have reported on the structural composition which, in order of occurrence were mainly proteinaceous, glycolipidic, glycoproteins, or glycolipopeptides in nature. Due to the proteinaceous nature, most biosurfactant produced by strains of Lactobacillus are generally believed to be surlactin type with high potential toward impeding pathogens adherence. Researchers have recently focused on the anti-adhesive and antibiofilm properties of Lactobacilli-derived biosurfactants. This review briefly discusses the significance of Lactobacilli-derived biosurfactants and their potential applications in various fields. In addition, we highlight the exceptional prospects and challenges in fermentation economics of Lactobacillus spp.-derived biosurfactants' production processes.

Hierarchical nanostructures of Au@ZnO: antibacterial and antibiofilm agent.

The perpetual use of antibiotics against pathogens inadvertently altered their genes that have translated into an unprecedented resistance in microorganisms in the twenty-first century. Many researchers have formulated bactericidal and bacteriostatic inorganic nanoparticle-based antiseptics that may be linked to broad-spectrum activity and far lower propensity to induce microbial resistance than organic-based antibiotics. Based on this line, herein, we present observations on microbial abatement using gold-based zinc oxide nanostructures (Au@ZnO) which are synthesized using hydrothermal route. Inhibition of microbial growth and biofilm using Au@ZnO is a unique feature of our study. Furthermore, this study evinces antimicrobial and antibiofilm mechanisms of photo-eradiated Au@ZnO by disruption of cellular functions and biofilms via reactive oxygen species (ROS)-dependent generation of superoxide anion radical. The present study is significant as it introduces novel functionalities to Au@ZnO in the biomedical field which can be extended to other species of microbial pathogens.

Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity.

How the direction of axon guidance is determined is not understood. In Caenorhabditis elegans the UNC-40 (DCC) receptor mediates a response to the UNC-6 (netrin) guidance cue that directs HSN axon development. UNC-40 becomes asymmetrically localized within the HSN neuron to the site of axon outgrowth. Here we provide experimental evidence that the direction of guidance can be explained by the stochastic fluctuations of UNC-40 asymmetric outgrowth activity. We find that the UNC-5 (UNC5) receptor and the cytoskeletal binding protein UNC-53 (NAV2) regulate the induction of UNC-40 localization by UNC-6. If UNC-40 localization is induced without UNC-6 by using an unc-53 mutation, the direction of UNC-40 localization undergoes random fluctuations. Random walk models describe the path made by a succession of randomly directed movement. This model was experimentally tested using mutations that affect Wnt/PCP signaling. These mutations inhibit UNC-40 localization in the anterior and posterior directions. As the axon forms in Wnt/PCP mutants, the direction of UNC-40 localization randomly fluctuates; it can localize in either the anterior, posterior, or ventral direction. Consistent with a biased random walk, over time the axon will develop ventrally in response to UNC-6, even though at a discrete time UNC-40 localization and outgrowth can be observed anterior or posterior. Also, axon formation is slower in the mutants than in wild-type animals. This is also consistent with a random walk since this model predicts that the mean square displacement (msd) will increase only linearly with time, whereas the msd increases quadratically with time for straight-line motion.