Plating Efficiency Research Articles

P15.02 Loss of Williams Syndrome Transcription Factor (WSTF) leads to improved temozolomide sensitivity in human glioblastoma cells irrespective of MGMT expression

Abstract BACKGROUND The prognosis for newly diagnosed adult glioblastoma multiforme (GBM) patients is poor even after standard therapy with a median survival of approximately 14–15 months. The DNA repair protein O 6 -methylguanine-DNA methyltransferase (MGMT) efficiently counteracts formation of the most lethal DNA adducts by temozolomide (TMZ) chemotherapy, and is thus associated with poor outcome in GBM patients. Williams Syndrome Transcription Factor (WSTF) has previously been suggested to regulate the DNA damage response pathway (DDR) in both an indirect (through chromatin remodeling together with SMARCA5 in the WICH complex) and direct manner (by phosphorylating H2AX at Tyr142). However, whether WSTF has any role in the development of resistance against chemotherapy through its ability to regulate the DDR in GBMs, is so far not known. In this study, we investigated whether loss of WSTF sensitizes different MGMT-proficient and -deficient GBM cell lines to TMZ treatment. MATERIAL AND METHODS We generated WSTF knockout clones from both MGMT-proficient (LN18, T98G) and -deficient GBM cell lines (U-251) using CRISPR/Cas9 gene-editing technology with lentiviral vectors. The PCR-based screening results combined with the T7 endonuclease mismatch assay for bi-allelic monoclonal knockouts were verified via sequencing and immunoblotting to identify candidate knockout clones. For each cell line, three knockout clones were chosen for further investigation. Colony formation assays were performed to determine the survival ability in response to TMZ treatment. Statistical analysis was performed using two-way ANOVA. RESULTS WSTF knockout clones showed a significant decrease in colony formation after TMZ-treatment compared to the corresponding WSTF-expressing control groups (non-target single guide RNA) (LN18: Clone 59 vs control: p= 0.0456, T98G: All three studied clones vs control: p <0.0001, U-251: Clone 7/35.1/70.2 vs control: p <0.0001/p= 0.0107/p= 0.0119). Furthermore, two out of three clones of T98G and U-251 (T98G Clone 13 and 128 vs control, p <0.0001, U-251 Clone 7 vs control, p= 0.0062; clone 70.2, p= 0.0052) showed significantly reduced plating efficiency compared to control cells. CONCLUSION WSTF is an important factor in both MGMT de- and proficient GBM cell lines for response against TMZ chemotherapy. The loss of WSTF leads to a significantly increased TMZ sensitivity in clinically relevant concentrations for all the studied cell lines. Ongoing studies are investigating the underlying mechanisms and potential alterations in the DDR pathway caused by WSTF loss.

Accelerated Electrochemical Investigation of Li Plating Efficiency as Key Parameter for Li Metal Batteries Utilizing a Scanning Droplet Cell

AbstractThe scanning droplet cell (SDC) allows for automatized electrochemical experiments leading to time‐saving and reproducible experimental conditions. Its implementation for non‐aqueous battery research is discussed, and the necessary adaptations to be operated inside an Ar‐filled glovebox in complete absence of oxygen and moisture are described. Due to the importance of the use of Li metal electrodes for next‐generation high‐energy batteries, the complex multi‐parameter optimisation of the Li plating/stripping processes are investigated by means of the SDC. In particular, the influence of pulsed Li plating protocols on the coulombic efficiency is evaluated. The results clearly show that fine tuning of the parameters of pulsed Li plating protocols, i. e. the relaxation period and Li plating duration, is required to improve Li plating efficiencies at high current densities.

Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli.

Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of β-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify among eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of β-lactams.

Physical and Biological Properties of 5-Fluorouracil Polymer-Coated Magnetite Nanographene Oxide as a New Thermosensitizer for Alternative Magnetic Hyperthermia and a Magnetic Resonance Imaging Contrast Agent: In Vitro and In Vivo Study.

This study reports a new procedure for utilizing 5-fluorouracil (5-Fu)-loaded polycaprolactone (PCL)/chitosan-covered magnetite nanographene oxide (5-Fu/SPION/NGO@PCL–LMWC) as a platform for synergistic thermo–chemotherapy. In fact, superparamagnetic iron oxide nanoparticles/nanographene oxide (SPION/NGO) nanoparticles can be coated with copolymers PCL/chitosan to attain better colloidal stability in the biological environment. Nanoparticles were synthesized and characterized for their size, surface charge, X-ray patterns, polymer content, and in vitro heat-triggered release. In vitro cytotoxic effects of nanoparticles on CT-26 cells were assessed with an MTT assay and real-time polymerase chain reaction. In vivo tumor growth inhibition was evaluated on an allograft mouse model of CT-26 cells. Tumor-bearing mice were injected with 5-Fu-loaded nanoparticles intravenously, and then, the targeted delivery was amplified using a magnetic field and finally exposed to an alternating magnetic field (AMF) (40 A/m, 13.56 MHz), during which the tumor site temperature increased to 43 °C. By using an infrared camera, we managed to heat the nanoparticles up to a constant temperature between 42.5 and 43.5 °C, with a tolerance ±0.03 °C. Finally, in vitro results showed that 5-Fu-loaded nanoparticles combined with AMF hyperthermia significantly reduced the plating efficiency of the cells (P < 0.01) and increased the Bax/Bcl-2 ratio (1.42 times, P < 0.01) compared with those achieved with each one alone. Furthermore, in vivo results demonstrated that the treatment of 5-Fu-loaded nanoparticles combined with the AMF diminished the growth of CT-26 tumor cells and increased the life span of the tumor-bearing mice (P < 0.001) by thermal energy deposition compared to that of the free 5-Fu drug. Also, the high level of accumulation of the nanoparticles within the tumor site was easily monitored with magnetic resonance imaging. It was concluded that the multifunctional magnetic nanoparticles could be used as a promising nanocarrier platform for achieving concurrent goals, drug delivery, magnetic targeting, thermal-sensitizing, cell death induction, and real-time monitoring of response to treatment.

Toward an Understanding of SEI Formation and Lithium Plating on Copper in Anode-Free Batteries.

“Anode-free” batteries present a significant advantage due to their substantially higher energy density and ease of assembly in a dry air atmosphere. However, issues involving lithium dendrite growth and low cycling Coulombic efficiencies during operation remain to be solved. Solid electrolyte interphase (SEI) formation on Cu and its effect on Li plating are studied here to understand the interplay between the Cu current collector surface chemistry and plated Li morphology. A native interphase layer (N-SEI) on the Cu current collector was observed with solid-state nuclear magnetic resonance spectroscopy (ssNMR) and electrochemical impedance spectroscopy (EIS). Cyclic voltammetry (CV) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) studies showed that the nature of the N-SEI is affected by the copper interface composition. An X-ray photoelectron spectroscopy (XPS) study identified a relationship between the applied voltage and SEI composition. In addition to the typical SEI components, the SEI contains copper oxides (CuxO) and their reduction reaction products. Parasitic electrochemical reactions were observed via in situ NMR measurements of Li plating efficiency. Scanning electron microscopy (SEM) studies revealed a correlation between the morphology of the plated Li and the SEI homogeneity, current density, and rest time in the electrolyte before plating. Via ToF-SIMS, we found that the preferential plating of Li on Cu is governed by the distribution of ionically conducting rather than electronic conducting compounds. The results together suggest strategies for mitigating dendrite formation by current collector pretreatment and controlled SEI formation during the first battery charge.

Inhibition of WHSC1 As a Promising Therapeutic Strategy in Childhood ALL

Childhood acute lymphoblastic leukemia (cALL) is the most frequent pediatric cancer, accounting for ~25% of all pediatric tumors. Nearly 20% of patients do not respond to current treatments, making cALL one of the leading cause of disease-related mortality amongst children. Moreover, sizable portion of childhood leukemia survivors develop significant long-term or late effects of treatment. This rate of refractory/relapse cases and the increased risk of chronic morbidity and early mortality among survivors highlight the pressing need for new treatment strategies in cALL. These can be developed through in-depth investigation of the genetic architecture underlying cALL. For this purpose, we performed whole exome sequencing (WES) of matched tumor-normal pair genomic DNA from 200 cALL patients to identify putative somatic driving mutations. A repertoire of 132 candidate drivers were further validated by performing a targeted knockdown functional screen in 3 pre-B ALL cell lines (NALM6, REH and 697). Through this screen, we identified WHSC1 as putative driver gene. WHSC1 is a methyltransferase that dimethylates lysine 36 on histone H3 (H3K36me2). Eight of the 200 (4%) cALL patients harboured a mutation in WHSC1 (7 pre-B and 1 pre-T ALL). In all 8 cases, the mutation led to a change at amino acid 1099 from a glutamic acid to lysine (E1099K). It has been shown that this activating mutation is associated with global increase in H3K36me2, which is accompanied by substantial changes in gene expression. Transcriptome analysis, using RNA-seq technique, of 6 cALL patients (2 with the mutation and 4 without) revealed distinctive differential expression pattern. We observed a specific enrichment in Wnt pathway genes in presence of the WHSC1 E1099K mutation (6.52 fold, P= 0.05), suggesting a stem cell signature. MCTP39 has been identified as an inhibitor of WHSC1, but has never been tested in ALL. Thus, we treated 4 pre-B ALL cell lines, 3 wild-type for WHSC1 (697, REH and Nalm6) and 1 carrying the mutation E1099K (RS411), as well as the non-leukemic human B cell precursor GM12878 cell line with variable concentrations of MCTP39 and calculated IC50. The IC50 in the leukemic cell lines 697, REH, Nalm6 and RS411 were 2.5µM, 1.7µM, 0.88µM and 0.89µM respectively, whereas it reached 25µM in the non-leukemic GM12878, suggesting a specificity for transformed cells. Although Nalm6 and RS411 had almost the same IC50 at 48h, cell growth curves showed differences in late effect (beyond 3 days), with RS411 being more sensitive to MCTP39. The latter differential effect has been confirmed by colony assay with reduction in plating efficiency of 69% and 45% in RS411 and Nalm6, respectively, when treated 48h with MCTP39 at 0.25µM. This result suggests that MCTP39 targets specifically stem/progenitor cells, particularly those that harbor the mutation WHSC1 E1099K. To confirm that the cellular effect of MCTP39 is due to WHSC1 inhibition, we assessed the impact of MCTP39 on H3K36me2 expression, using fluorometric assay and western blot. In both Nalm6 and RS411cell lines, MCTP39 led to significant decrease in H3K36me2. Then, we analyzed whether MCTP39 synergizes with doxorubicin, an anthracycline that is commonly used in cALL treatment. In both Nalm6 and RS411 cell lines, MCTP39 showed a clear synergistic effect at all concentration tested, permitting 40% reduction in doxorubicin concentration to obtain equal effect. Altogether, this study indicates that inhibition of WHSC1 is a promising treatment strategy in cALL patients, particularly in those carrying WHSC1 mutation. Further investigations are needed to recapitulate these results in vivo and to elucidate the pathways affected as result of the inhibition of H3K36me2 by MCTP39. DisclosuresNo relevant conflicts of interest to declare.

Investigating Parasitic Reactions in Anode-Free Li Metal Cells with Isothermal Microcalorimetry

Anode-free Li metal cells are one of the most appealing energy storage technologies beyond Li-ion batteries due to their superior theoretical specific and volumetric energy densities. However, long cycle life in an anode-free cell remains elusive due to difficulties reversibly plating and stripping metallic lithium. Isothermal microcalorimetry was used to study parasitic reactions in anode-free Li metal cells for electrolytes containing different Li salts. A new cycling protocol was used to measure the parasitic heat flow both on freshly plated Li surfaces and anode surfaces after stripping lithium. Existing methods were used to measure parasitic reactions occurring at high voltage. In both low- and high-voltage measurements, electrolytes containing LiDFOB had the highest parasitic heat flow compared to an electrolyte with LiPF6 salt. In contrast to previous studies of parasitic reactions in Li-ion batteries using isothermal microcalorimetry, the LiDFOB-containing electrolytes gave the longest lifetime despite having higher parasitic heat flow. This observation was attributed to decomposition of the LiDFOB salt, and subsequent formation of a favorable SEI layer that greatly improves plating and stripping efficiency. In-situ detection of parasitic heat flow with isothermal microcalorimetry techniques will be valuable for future studies of electrolyte design in anode-free Li metal cells.

A bacteriophage JN02 infecting multidrug‐resistant Shiga toxin‐producing Escherichiacoli: isolation, characterisation and application as a biocontrol agent in foods

AbstractBacteriophages are increasingly considered as novel biocontrol agents against Multidrug‐resistant (MDR) Shiga toxin‐producing Escherichia coli (STEC). In this study, a virulent phage JN02 capable of infecting MDR STEC was isolated, characterised and evaluating its efficacy in reducing MDR STEC in foods. One‐step growth and stability assay showed that JN02 had a relatively short latent period (15 min), large burst sizes (69 PFU/cell), and high physicochemical stability (temperature (30–60°C), pH (2–11), 0.1%, 1% pig bile salt, biocides and food additives (100% chloroform, 0.1% acetic acid, 1% potassium sorbate). In spot tests, 18 E. coli strains were sensitive to JN02. In efficiency of plating (EOP), 4 E. coli strains were sensitive to JN02. Electron microscopy and genome sequencing showed that JN02 belongs to the family Myoviridae. Importantly, no known virulence‐associated, lysogenic and antibiotic genes were identified in the genome of JN02. Viable cell counts of MDR STEC were significantly (P &lt; 0.05) reduced in milk (no viable counts) and beef samples (1.0–1.9 log10 CFU cm−2 reduction) when treated with JN02 at 4 °C. The results indicated that JN02 might be a new potential natural biological control agent for MDR STEC in foods.

Screening of Bacteriophage Encoded Toxic Proteins with a Next Generation Sequencing-Based Assay.

Bacteriophage vB_EcoM_fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules.

ISOLATION, CHARACTERIZATION, AND APPLICATION OF BACTERIOPHAGES AGAINST SEVERAL FOOD SPOILAGE BACTERIA: Bacillus subtilis, Bacillus cereus, AND Shewanella Putrefaciens

Introduction: Bacteriophages can be the natural alternative method to prevent food spoilage caused by bacteria. This research was conducted to isolate bacteriophages from a soil sample, retail food, and wastewater from fish and then the bacteriophages will be characterized for their activity against several food spoilage bacteria, such as Bacillus cereus, Bacillus subtilis, and Shewanella putrefaciens and will be further investigated for application as food preservation. Experimental: B. cereus (Atma Jaya culture collection), B. subtilis ATCC 6633, and S. putrefaciens ATCC 8071 were used for bacteriophage isolation. Food samples (rice, pasta, tofu, tempeh), soil samples (black soil and laterite), and wastewater sample of freshwater fish and seawater fish were used in this research. The overall process consists of eight steps: inoculum preparation, sample collection, bacteriophage isolation, purification and enrichment, titer determination, host range determination, the efficiency of plating (EOP), and bacteriophage application. Results: A total of four bacteriophages were isolated with B. cereus, B. subtilis, and S. putrefaciens as host bacteria. Bacteriophage titers observed around 109 PFU mL-1. Bacteriophages that isolated with B. cereus and B. subtilis as host bacteria tend to have high EOP with the same species bacteria. All the Bacillus phages (S1-BC, S2-BC, and S1-BS) can reduce the Bacillus species bacteria concentration for more than 90%. Conclusion: Refers to their activity, the isolated bacteriophages in this study might have a great prospect to be used as food biocontrol and also can be further tested to make a phage cocktail.

Investigation of rapid composite plating of core wire magnetized electroplated diamond wire saw

In order to shorten the time for preparing diamond wire saws and improve the efficiency of preparing diamond wire saws, this study used nickel-plated diamond particles and magnetized stainless steel core wires. In order to effectively control the density of diamond particles, based on the analysis of the mechanism of the rapid composite coating of nickel-plated diamond and magnetic core wire magnetized diamond wire saw, it proposed a dynamic model suitable for computer simulation analysis. Using the simulation analysis model, the magnetic induction intensity of the core wire, the thickness of the nickel coating on the diamond surface, the stirring speed and the composite coating time on the effect of the composite coating of the diamond wire saw were systematically analyzed, and the reasonable parameters of rapid composite plating of the diamond wire saw was got, and verify the simulation results through experiments. Experiments show that the error range of the simulation calculation is 0.78% to 8.74%, and the simulation results can be a guidance in controlling the density of diamond particles. In addition, it was found in experiments that by using core wire magnetization and nickel plating on the diamond surface, the rapid composite plating efficiency was increased by over 66%; because of the shorter composite plating time, the diameter of the finished wire saw was smaller.

Analysis of Selection Methods to Develop Novel Phage Therapy Cocktails Against Antimicrobial Resistant Clinical Isolates of Bacteria.

Antimicrobial resistance (AMR) is a major problem globally. The main bacterial organisms associated with urinary tract infection (UTI) associated sepsis are E. coli and Klebsiella along with Enterobacter species. These all have AMR strains known as ESBL (Extended Spectrum Beta-Lactamase), which are featured on the WHO priority pathogens list as “critical” for research. Bacteriophages (phages), as viruses that can infect and kill bacteria, could provide an effective tool to tackle these AMR strains. There is currently no “gold standard” for developing a phage cocktail. Here we describe a novel approach to develop an effective phage cocktail against a set of ESBL-producing E. coli and Klebsiella largely isolated from patients in United Kingdom hospitals. By comparing different measures of phage efficacy, we show which are the most robust, and suggest an efficient screening cascade that could be used to develop phage cocktails to target other AMR bacterial species. A target panel of 38 ESBL-producing clinical strains isolated from urine samples was collated and used to test phage efficacy. After an initial screening of 68 phages, six were identified and tested against these 38 strains to determine their clinical coverage and killing efficiency. To achieve this, we assessed four different methods to assess phage virulence across these bacterial isolates. These were the Direct Spot Test (DST), the Efficiency of Plating (EOP) assay, the planktonic killing assay (PKA) and the biofilm assay. The final ESBL cocktail of six phages could effectively kill 23/38 strains (61%), for Klebsiella 13/19 (68%) and for E. coli 10/19 (53%) based on the PKA data. The ESBL E. coli collection had six isolates from the prevalent UTI-associated ST131 sequence type, five of which were targeted effectively by the final cocktail. Of the four methods used to assess phage virulence, the data suggests that PKAs are as effective as the much more time-consuming EOPs and data for the two assays correlates well. This suggests that planktonic killing is a good proxy to determine which phages should be used in a cocktail. This assay when combined with the virulence index also allows “phage synergy” to inform cocktail design.

Mechanistic insight of cell anti-proliferative activity of fluoroquinolone drug-based Cu(II) complexes.

Pefloxacin-based mixed ligand Cu(II) complexes with substituted isatin of type [Cu(Isatin)(Pefloxacin)Cl] were synthesized, and characterized by EPR, mass, FT-IR, electronic spectrometry, metal content, magnetic moment, and conductance measurement. The g factors g [Formula: see text] > g [Formula: see text] > 2.0023 observed in EPR suggest a square-pyramidal environment of ligands around the copper metal. The compounds were screened for diverse biological activities. The compounds inhibit efficiently the cell proliferation of HCT 116 cancer cells. To take the insight of anticancer activity mechanism, we investigated compound-1 for further cellular assay-based biological activities like trypan blue assay, cell morphological alteration assay, colony formation assay, cell apoptosis, and cell necrosis assay. The compound-1 induced distinct morphological alteration in cells, inhibits cell viability, decreases % plating efficiency, and decreases the clonogenic ability of the HCT 116 cells. The cell death mechanism was confirmed by annexin V-FITC / PI assay and LDH release assay. The positive annexin V/PI stained cells in presence of compound-1 and the absence of a significant amount of lactate dehydrogenase suggest cell apoptosis mechanism for anticancer activity of compounds. We also screened compounds for in vitro antibacterial and cytotoxic activities. Synthesis, characterization, antibacterial, anticancer, and cytotoxicity activities of pefloxacin based Cu(II) complexes were studied. The compound -1 is more potent than standard anticancer drugs and it induced apoptosis to the HCT 116 cells.

Optimizing Protocols for Arabidopsis Shoot and Root Protoplast Cultivation.

Procedures for the direct regeneration of entire plants from a shoot and root protoplasts of Arabidopsis thaliana have been optimized. The culture media for protoplast donor-plant cultivation and protoplast culture have been adjusted for optimal plant growth, plating efficiency, and promotion of shoot regeneration. Protocols have been established for the detection of all three steps in plant regeneration: (i) chromatin relaxation and activation of auxin biosynthesis, (ii) cell cycle progression, and (iii) conversion of cell-cycle active cells to totipotent ones. The competence for cell division was detected by DNA replication events and required high cell density and high concentrations of the auxinic compound 2,4-D. Cell cycle activity and globular structure formation, with subsequent shoot induction, were detected microscopically and by labeling with fluorescent dye Rhodamine123. The qPCR results demonstrated significantly upregulated expression of the genes responsible for nuclear reorganization, auxin responses, and auxin biosynthesis during the early stage of cell reprogramming. We further optimized cell reprogramming with this protocol by applying glutathione (GSH), which increases the sensitivity of isolated mesophyll protoplasts to cell cycle activation by auxin. The developed protocol allows us to investigate the molecular mechanism of the de-differentiation of somatic plant cells.

A Novel Group of Promiscuous Podophages Infecting Diverse Gammaproteobacteria from River Communities Exhibits Dynamic Intergenus Host Adaptation.

Phages are generally described as species specific or even strain specific, implying an inherent limitation for some to be maintained and spread in diverse bacterial communities. Moreover, phage isolation and host range determination rarely consider the phage ecological context, likely biasing our notion on phage specificity. Here we isolated and characterized a novel group of six promiscuous phages, named Atoyac, existing in rivers and sewage by using a diverse collection of over 600 bacteria retrieved from the same environments as potential hosts. These podophages isolated from different regions in Mexico display a remarkably broad host range, infecting bacteria from six genera: Aeromonas, Pseudomonas, Yersinia, Hafnia, Escherichia, and Serratia Atoyac phage genomes are ∼42 kb long and highly similar to each other, but not to those currently available in genome and metagenome public databases. Detailed comparison of the phages' efficiency of plating (EOP) revealed variation among bacterial genera, implying a cost associated with infection of distant hosts, and between phages, despite their sequence similarity. We show, through experimental evolution in single or alternate hosts of different genera, that efficiency of plaque production is highly dynamic and tends toward optimization in hosts rendering low plaque formation. However, adaptation to distinct hosts differed between similar phages; whereas one phage optimized its EOP in all tested hosts, the other reduced plaque production in one host, suggesting that propagation in multiple bacteria may be key to maintain promiscuity in some viruses. Our study expands our knowledge of the virosphere and uncovers bacterium-phage interactions overlooked in natural systems.IMPORTANCE In natural environments, phages coexist and interact with a broad variety of bacteria, posing a conundrum for narrow-host-range phage maintenance in diverse communities. This context is rarely considered in the study of host-phage interactions, typically focused on narrow-host-range viruses and their infectivity in target bacteria isolated from sources distinct to where the phages were retrieved from. By studying phage-host interactions in bacteria and viruses isolated from river microbial communities, we show that novel phages with promiscuous host range encompassing multiple bacterial genera can be found in the environment. Assessment of hundreds of interactions in diverse hosts revealed that similar phages exhibit different infection efficiency and adaptation patterns. Understanding host range is fundamental in our knowledge of bacterium-phage interactions and their impact on microbial communities. The dynamic nature of phage promiscuity revealed in our study has implications in different aspects of phage research such as horizontal gene transfer or phage therapy.

LOSS OF GLUTATHIONE PEROXIDASE 1 ATTENUATES COLITIS AND IS CRITICAL IN ACTIVATING EPITHELIAL REGENERATIVE RESPONSES

Many selenium-containing “selenoproteins” function as antioxidants, and work by our lab and others has demonstrated that selenoproteins often protect against intestinal inflammatory diseases, including colitis. Glutathione peroxidase 1 (GPx1) is a ubiquitous, mitochondrial and cytosolic selenoprotein which catalyzes the reduction of hydrogen peroxide by glutathione. Previously, we determined that despite its antioxidant role, loss of GPx1 greatly reduced disease severity in the dextran sodium sulfate (DSS) colitis model. Furthermore, GPx1 loss increased baseline intestinal cell proliferation, enhanced enteroid plating efficiency, and induced expression of stem cell-associated genes, such as Lgr5.

Cover Feature: A Single‐Flow Battery with Multiphase Flow (ChemSusChem 4/2021)

Widespread adoption of redox flow batteries (RFBs) for renewable energy storage is inhibited by a relatively high cost of storage. This is due largely to typical RFBs requiring two flows, two external tanks, and expensive ion-exchange membranes. Here, we propose a potentially inexpensive Zn-Br2 RFB which is membraneless and requires only a single flow. The flow is an emulsion consisting of a continuous, Br2 -poor aqueous phase and a dispersed, Br2 -rich polybromide phase, pumped through the channel separating anode and cathode. With our prototype cell, we explore the effect of polybromide-phase volume fraction and Br2 concentration on cell performance and plating efficiencies. We demonstrate high discharge currents of up to 270 mA/cm2 , plating efficiencies up to 88 %, and dendriteless plating up to the highest Zn loading investigated of 250 mAh/cm2 . We provide mechanistic insights into cell behavior and elucidate paths towards unlocking ultra-low-cost single-flow RFBs with multiphase flow.

LOSS OF GLUTATHIONE PEROXIDASE 1 ATTENUATES COLITIS AND IS CRITICAL IN ACTIVATING EPITHELIAL REGENERATIVE RESPONSES

Abstract Many selenium-containing “selenoproteins” function as antioxidants, and work by our lab and others has demonstrated that selenoproteins often protect against intestinal inflammatory diseases, including colitis. Glutathione peroxidase 1 (GPx1) is a ubiquitous, mitochondrial and cytosolic selenoprotein which catalyzes the reduction of hydrogen peroxide by glutathione. Previously, we determined that despite its antioxidant role, loss of GPx1 greatly reduced disease severity in the dextran sodium sulfate (DSS) colitis model. Furthermore, GPx1 loss increased baseline intestinal cell proliferation, enhanced enteroid plating efficiency, and induced expression of stem cell-associated genes, such as Lgr5. Next, we aimed to determine the mechanism by which GPx1 modifies response to DSS. We observed that GPx1 is increased in colonic tissues from DSS-treated mice as compared to nontreated controls, suggesting that GPx1 may functionally contribute to intestinal injury responses. While GPx1 is expressed in both intestinal epithelial and immune cells, in situ hybridization to visualize Gpx1 identified epithelial cells as the most highly expressing cell type, with the greatest Gpx1 upregulation observed in wound-adjacent and regenerative crypts. Next, we investigated whether GPx1 loss affects stem cell function after injury. Here, we determined that both proliferation (p&amp;lt;0.01) and Lgr5 expression (p&amp;lt;0.05) were increased in the crypts of Gpx1-/- DSS-treated mice in comparison to WT controls. Similarly, organoids established from ulcerative colitis tissue displayed increased growth rates (p&amp;lt;0.01), expression of stem cell and Wnt target genes such as AXIN2 (p&amp;lt;0.0001) and LGR5 (p&amp;lt;0.01), and proliferation (p&amp;lt;0.05) following GPX1 knockdown. Together, these results indicate that GPx1 has an epithelial-cell autonomous role, and that its loss activates stem cell and proliferative responses which may both protect from intestinal injury and promote healing. Interestingly, recent research has highlighted the role of cellular metabolism in maintaining intestinal stem cell function, and GPx1 has previously been implicated in these processes. RNA-sequencing from DSS-treated mice and gene set enrichment analysis identified a positive association with oxidative phosphorylation-associated genes in Gpx1-/- mice (NES: 1.78; FDR q-val: 0.01), suggesting altered metabolism which may favor stem cell function. Further analysis of cellular metabolism using GPX1 knockdown colorectal cancer cells observed higher basal respiration (p&amp;lt;0.0001) and ATP generation (p&amp;lt;0.0001). Together, these results suggest that unlike other intestinal selenoproteins studied to date, loss of GPx1 augments stem cell injury responses to protect against intestinal inflammation, likely via augmenting epithelial regenerative responses.

Klebsiella virus UPM2146 lyses multiple drug-resistant Klebsiella pneumoniae in vitro and in vivo.

Klebsiella pneumoniae are opportunistic bacteria found in the gut. In recent years they have been associated with nosocomial infections. The increased incidence of multiple drug-resistant K. pneumoniae makes it necessary to find new alternatives to treat the disease. In this study, phage UPM2146 was isolated from a polluted lake which can lyse its host K. pneumoniae ATCC BAA-2146. Observation from TEM shows that UPM2146 belongs to Caudoviriales (Order) based on morphological appearance. Whole genome analysis of UPM2146 showed that its genome comprises 160,795 bp encoding for 214 putative open reading frames (ORFs). Phylogenetic analysis revealed that the phage belongs to Ackermannviridae (Family) under the Caudoviriales. UPM2146 produces clear plaques with high titers of 1010 PFU/ml. The phage has an adsorption period of 4 min, latent period of 20 min, rise period of 5 min, and releases approximately 20 PFU/ bacteria at Multiplicity of Infection (MOI) of 0.001. UPM2146 has a narrow host-range and can lyse 5 out of 22 K. pneumoniae isolates (22.72%) based on spot test and efficiency of plating (EOP). The zebrafish larvae model was used to test the efficacy of UPM2146 in lysing its host. Based on colony forming unit counts, UPM2146 was able to completely lyse its host at 10 hours onwards. Moreover, we show that the phage is safe to be used in the treatment against K. pneumoniae infections in the zebrafish model.

Dynamics of caspase activation upon UV induced genotoxic injury

Purpose Caspases are common mediators of cell death. Evasion of cell death including apoptosis are considered to be hallmarks of cancer. A deeper understanding of the apoptotic cascade may aid improving cancer therapies. Our aim was to characterize the progression of cell death following UV-induced genotoxic injury in a defined cell culture model. Materials and methods Hela cells were UV-irradiated with doses ranging from 0.1 to 60 mJ/cm2. Cells were counted and colony forming assays were performed with caspase inhibitors. Results In our model of HeLa cells, cells remain >90% viable until 6 hrs after UV radiation (UVR), but more than half of the cells are dead after 12 − 72 hrs after UVR. Within a dose range between 0.1 and 50 mJ/cm2, viability ranges roughly between 20 and 30%. The difference between the lowest dose applied (0.1 mJ/cm2) and the other doses applied is significant, with the exception of the next higher dose of 1 mJ/cm2. The activation of caspases precedes the cell death induction by several hrs. Caspase-9 starts to be activated at 1 hr after UVR followed by caspases 3, 6 and 7 which are fully active at 2 hrs after UVR while caspase-8 is fully active only 3 hrs after UVR. Most caspases are only weakly or not active at 0.1 mJ/cm2 after 3 hrs, but fully active at the same time point with increased radiation doses. PARP-1, a caspase substrate, is cleaved immediately after activation of the caspases. Colony formation activity of the tumor cells decreases exponentially after UVR dropping down to < 0.01% plating efficiency at a dose of 60 mJ/cm2. Interestingly, this drop in plating efficiency cannot be rescued by any of the two caspase inhibitors tested. Conclusions UV-induced cell death in this model involves the activation of apoptosis-related caspases, but this activation seems to be dispensable for the execution of cell death. Further experiments should clarify which mechanisms of cell death are really necessary for the execution of this type of cell death.