Post transition metal substituted Keggin POMs as thin film chemiresistive sensors for H2O and CO2 detection

Chemiresitive sensing allows the affordable and facile detection of small molecules such as H2O and CO2. Herein, we report a novel class of earth abundant post transition metal substituted Keggin polyoxometalates (POMs) for chemiresistive sensing applications, with conductivies up to 0.01 S cm-1 under 100% CO2 and 65% Relative Humidity (RH)

Development and characterisation of acoustofluidic devices using detachable electrodes made from PCB.

Acoustofluidics has been increasingly applied in biology, medicine and chemistry due to its versatility in manipulating fluids, cells and nano-/micro-particles. In this paper, we develop a novel and simple technology to fabricate a surface acoustic wave (SAW)-based acoustofluidic device by clamping electrodes made using a printed circuit board (PCB) with a piezoelectric substrate. The PCB-based SAW (PCB-SAW) device is systematically characterised and benchmarked with a SAW device made using the conventional photolithography process with the same specifications. Microparticle manipulations such as streaming in droplets and patterning in microchannels were demonstrated in the PCB-SAW device. In addition, the PCB-SAW device was applied as an acoustic tweezer to pattern lung cancer cells to form three or four traces inside the microchannel in a controllable manner. Cell viability of ∼97% was achieved after acoustic manipulation using the PCB-SAW device, which proved its ability as a suitable tool for acoustophoretic applications.The authors would gratefully acknowledge the financial support from EPSRC (EP/P002803/1, EP/P018998/1), EPSRC IAA, Welcome Trust, Global Challenges Research Fund (GCRF), the Royal Society (IEC\NSFC\170142, IE161019), and the Natural Science Foundation of China (NSFC) (Grant No. 51811530310)

Plasmacytoid dendritic cells orchestrate innate and adaptive anti-tumor immunity induced by oncolytic coxsackievirus A21

Background: The oncolytic virus, coxsackievirus A21 (CVA21), has shown promise as a single agent in several clinical trials and is now being tested in combination with immune checkpoint blockade. Combination therapies offer the best chance of disease control; however, the design of successful combination strategies requires a deeper understanding of the mechanisms underpinning CVA21 efficacy, in particular, the role of CVA21 anti-tumor immunity. Therefore, this study aimed to examine the ability of CVA21 to induce human anti-tumor immunity, and identify the cellular mechanism responsible. Methods: This study utilized peripheral blood mononuclear cells from i) healthy donors, ii) Acute Myeloid Leukemia (AML) patients, and iii) patients taking part in the STORM clinical trial, who received intravenous CVA21; patients receiving intravenous CVA21 were consented separately in accordance with local institutional ethics review and approval. Collectively, these blood samples were used to characterize the development of innate and adaptive anti-tumor immune responses following CVA21 treatment. Results: An Initial characterization of peripheral blood mononuclear cells, collected from cancer patients following intravenous infusion of CVA21, confirmed that CVA21 activated immune effector cells in patients. Next, using hematological disease models which were sensitive (Multiple Myeloma; MM) or resistant (AML) to CVA21-direct oncolysis, we demonstrated that CVA21 stimulated potent anti-tumor immune responses, including: 1) cytokine-mediated bystander killing; 2) enhanced natural killer cell-mediated cellular cytotoxicity; and 3) priming of tumor-specific cytotoxic T lymphocytes, with specificity towards known tumor-associated antigens. Importantly, immune-mediated killing of both MM and AML, despite AML cells being resistant to CVA21-direct oncolysis, was observed. Upon further examination of the cellular mechanisms responsible for CVA21-induced anti-tumor immunity we have identified the importance of type I IFN for NK cell activation, and demonstrated that both ICAM-1 and plasmacytoid dendritic cells were key mediators of this response. Conclusion: This work supports the development of CVA21 as an immunotherapeutic agent for the treatment of both AML and MM. Additionally, the data presented provides an important insight into the mechanisms of CVA21-mediated immunotherapy to aid the development of clinical biomarkers to predict response and rationalize future drug combinations

Reovirus exerts potent oncolytic effects in head and neck cancer cell lines that are independent of signalling in the EGFR pathway

Background: reovirus exploits aberrant signalling downstream of Ras to mediate tumor-specific oncolysis. Since ~90% squamous cell carcinomas of the head and neck (SCCHN) over-express EGFR and SCCHN cell lines are sensitive to oncolytic reovirus, we conducted a detailed analysis of the effects of reovirus in 15 head and neck cancer cell lines. Both pre- and post-entry events were studied in an attempt to define biomarkers predictive of sensitivity/resistance to reovirus. In particular, we analysed the role of EGFR/Ras signalling in determining virus-mediated cytotoxicity in SCCHN. Methods: to test whether EGFR pathway activity was predictive of increased sensitivity to reovirus, correlative analyses between reoviral IC50 by MTT assay and EGFR levels by western blot and FACS were conducted. Inhibition or stimulation of EGFR signalling were analysed for their effect on reoviral oncolysis by MTT assay, and viral growth by TCID50 assay. We next analysed the effects of inhibiting signalling downstream of Ras, by specific inhibitors of p38MAPK, PI3-K or MEK, on reoviral killing examined by MTT assay. The role of PKR in reoviral killing was also determined by blockade of PKR using 2-aminopurine and assaying for cell survival by MTT assay. The apoptotic response of SCCHN to reovirus was examined by western blot analysis of caspase 3 cleavage. Results: correlative analyses between reoviral sensitivity and EGFR levels revealed no association. Intermediate sub-viral and core particles showed the same infectivity/cytotoxicity as intact reovirus. Therefore, sensitivity was not determined by cell entry. In 4 cell lines, oncolysis and viral growth were both unaffected by inhibition or stimulation of EGFR signalling. Inhibition of signalling downstream of Ras did not abrogate reoviral oncolysis and, in addition, modulation of PKR using 2-aminopurine did not alter reovirus sensitivity in resistant cell lines. Caspase 3 cleavage was not detected in infected cells and oncolysis was observed in pan-caspase inhibited cells. Conclusions: in summary, reovirus is potently oncolytic in a broad panel of SCCHN cell lines. Attempts to define sensitivity/resistance by analysis of the EGFR/Ras/MAPK pathway have failed to provide a clear predictive biomarker of response. Further analysis of material from in vitro and clinical studies is ongoing in an attempt to shed further light on this issue

Ribosome Distribution in HeLa Cells during the Cell Cycle

In this study, we employed a surface-specific antibody against the large ribosome subunit to investigate the distribution of ribosomes in cells during the cell cycle. The antibody, anti-L7n, was raised against an expansion segment (ES) peptide from the large subunit ribosomal protein L7, and its ribosome-surface specificity was evident from the positive immuno-reactivity of ribosome particles and the detection of 60 S immune-complex formation by an immuno-electron microscopy. Using immunofluorescent staining, we have microscopically revealed that ribosomes are dispersed in the cytoplasm of cells throughout all phases of the cell cycle, except at the G2 phase where ribosomes show a tendency to gather toward the nuclear envelope. The finding in G2 cells was confirmed by electron microscopy using a morphometric assay and paired t test. Furthermore, further observations have shown that ribosomes are not distributed immune-fluorescently with nuclear envelope markers including the nuclear pore complex, the integral membrane protein gp210, the inner membrane protein lamin B2, and the endoplasm reticulum membrane during cell division we propose that the mechanism associated with ribosome segregation into daughter cells could be independent of the processes of disassembly and reassembly of the nuclear envelope

Nanoparticle vesicle encoding for imaging and tracking cell populations.

For phenotypic behavior to be understood in the context of cell lineage and local environment, properties of individual cells must be measured relative to population-wide traits. However, the inability to accurately identify, track and measure thousands of single cells via high-throughput microscopy has impeded dynamic studies of cell populations. We demonstrate unique labeling of cells, driven by the heterogeneous random uptake of fluorescent nanoparticles of different emission colors. By sequentially exposing a cell population to different particles, we generated a large number of unique digital codes, which corresponded to the cell-specific number of nanoparticle-loaded vesicles and were visible within a given fluorescence channel. When three colors are used, the assay can self-generate over 17,000 individual codes identifiable using a typical fluorescence microscope. The color-codes provided immediate visualization of cell identity and allowed us to track human cells with a success rate of 78% across image frames separated by 8 h

Integrated Proteomic and Metabolomic Analysis of an Artificial Microbial Community for Two-Step Production of Vitamin C

An artificial microbial community consisted of Ketogulonicigenium vulgare and Bacillus megaterium has been used in industry to produce 2-keto-gulonic acid (2-KGA), the precursor of vitamin C. During the mix culture fermentation process, sporulation and cell lysis of B. megaterium can be observed. In order to investigate how these phenomena correlate with 2-KGA production, and to explore how two species interact with each other during the fermentation process, an integrated time-series proteomic and metabolomic analysis was applied to the system. The study quantitatively identified approximate 100 metabolites and 258 proteins. Principal Component Analysis of all the metabolites identified showed that glutamic acid, 5-oxo-proline, L-sorbose, 2-KGA, 2, 6-dipicolinic acid and tyrosine were potential biomarkers to distinguish the different time-series samples. Interestingly, most of these metabolites were closely correlated with the sporulation process of B. megaterium. Together with several sporulation-relevant proteins identified, the results pointed to the possibility that Bacillus sporulation process might be important part of the microbial interaction. After sporulation, cell lysis of B. megaterium was observed in the co-culture system. The proteomic results showed that proteins combating against intracellular reactive oxygen stress (ROS), and proteins involved in pentose phosphate pathway, L-sorbose pathway, tricarboxylic acid cycle and amino acids metabolism were up-regulated when the cell lysis of B. megaterium occurred. The cell lysis might supply purine substrates needed for K. vulgare growth. These discoveries showed B. megaterium provided key elements necessary for K. vulgare to grow better and produce more 2-KGA. The study represents the first attempt to decipher 2-KGA-producing microbial communities using quantitative systems biology analysis

Probing the Behaviors of Gold Nanorods in Metastatic Breast Cancer Cells Based on UV-vis-NIR Absorption Spectroscopy

In this work, behaviors of positively-charged AuNRs in a highly metastatic tumor cell line MDA-MB-231 are examined based on UV-vis-NIR absorption spectroscopy in combination with inductively coupled plasma mass spectrometry (ICP-MS), transmission electron microscopy (TEM) and dark-field microscopic observation. It is found that characteristic surface plasmon resonance (SPR) peaks of AuNRs can be detected using spectroscopic method within living cells that have taken up AuNRs. The peak area of transverse SPR band is shown to be proportionally related to the amount of AuNRs in the cells determined with ICP-MS, which suggests a facile and real time quantification method for AuNRs in living cells. The shape of longitudinal SPR band in UV-vis-NIR spectrum reflects the aggregation state of AuNRs in the cells during the incubation period, which is proved by TEM and microscopic observations. Experimental results reveal that AuNRs are internalized by the cells rapidly; the accumulation, distribution and aggregation of AuNRs in the cells compartments are time and dose dependent. The established spectroscopic analysis method can not only monitor the behaviors of AuNRs in living cells but may also be helpful in choosing the optimum laser stimulation wavelength for anti-tumor thermotherapy

Enhanced tonic GABAA inhibition in typical absence epilepsy

The cellular mechanisms underlying typical absence seizures, which characterize various idiopathic generalized epilepsies, are not fully understood, but impaired GABAergic inhibition remains an attractive hypothesis. In contrast, we show here that extrasynaptic GABAA receptor–dependent ‘tonic’ inhibition is increased in thalamocortical neurons from diverse genetic and pharmacological models of absence seizures. Increased tonic inhibition is due to compromised GABA uptake by the GABA transporter GAT–1 in the genetic models tested, and GAT–1 is critical in governing seizure genesis. Extrasynaptic GABAA receptors are a requirement for seizures in two of the best characterized models of absence epilepsy, and the selective activation of thalamic extrasynaptic GABAA receptors is sufficient to elicit both electrographic and behavioural correlates of seizures in normal animals. These results identify an apparently common cellular pathology in typical absence seizures that may have epileptogenic significance, and highlight novel therapeutic targets for the treatment of absence epilepsy.peer-reviewe

Growth, cell division and sporulation in mycobacteria

Bacteria have the ability to adapt to different growth conditions and to survive in various environments. They have also the capacity to enter into dormant states and some bacteria form spores when exposed to stresses such as starvation and oxygen deprivation. Sporulation has been demonstrated in a number of different bacteria but Mycobacterium spp. have been considered to be non-sporulating bacteria. We recently provided evidence that Mycobacterium marinum and likely also Mycobacterium bovis bacillus Calmette–Guérin can form spores. Mycobacterial spores were detected in old cultures and our findings suggest that sporulation might be an adaptation of lifestyle for mycobacteria under stress. Here we will discuss our current understanding of growth, cell division, and sporulation in mycobacteria