Continuous Culture Research Articles

Grazing resistance developed in Escherichia coli K-12 during coexistence with a bacterivorous protist.

A development of grazing resistance in Escherichia coli K-12 was examined in the presence of a bacterivorous protist, Spumella sp. TGKK2. Two transformants were generated from E. coli K12 for grazing experiments. One was E. coli K-12-TGF, which possesses tetracycline resistance and green fluorescence. The other was E. coli K-12-KRF with kanamycin resistance and red fluorescence. These strains can be selectively colonized on antibiotic-containing agar media and further confirmed by their fluorescent colors. First, we added protist-untouched E. coli K-12-KRF to protist-touched residual E. coli K-12-TGF that had been attacked by Spumella sp. TGKK2 in a batch test. Then the survivability of the respective strains was investigated. Consequently, E. coli K-12-KRF was predated preferentially. On the other hand, E. coli K-12-TGF in the same tube was less predated, indicating some grazing resistance. Similar phenomena were observed when the conditions of these two strains of bacteria were reversed. Also, a continuous culture device supplied with a glucose-containing medium as a substrate was operated. The device connected two complete mixed reactors in series. E. coli K-12-TGF was cultivated in the first reactor, and then grown E. coli K-12-TGF was predated by Spumella sp. TGKK2 in the second reactor. The effluent in the second reactor containing residual E. coli K-12-TGF and Spumella sp. TGKK2 was supplemented with batch-cultured E. coli K-12-KRF. Consequently, it was confirmed that bach-cultured E. coli K-12-KRF never exposed to protist was predated preferentially. These findings reveal that E. coli K12 acquires some predation resistance through coexistence with the bacterivorous protist.

Towards Optimisation of Microalgae Cultivation through Monitoring and Control in Membrane Photobioreactor Systems

This research lays a foundation for optimised membrane photobioreactor performance and introduces novel control parameters crucial for advancing microalgae cultivation techniques and promoting environmental sustainability. Particularly, this study presents an innovative solids retention time (SRT) controller designed for a pilot-scale membrane photobioreactor. Employing a fuzzy-logic knowledge-based approach, this controller uses the first derivative of pH data dynamics (pH′) as an input variable, directly correlated with nitrogen recovery rate and biomass productivity when normalised by average light irradiance (I2). Through a feedback mechanism, it regulates daily SRT variations, ensuring stable reactor operation, optimal volatile suspended solids concentration, efficient nitrogen removal, and enhanced biomass productivity. Normalised nitrogen recovery rate, considering solar light irradiance and volatile suspended solids concentration, increased by 51% compared to previous studies employing fixed SRT and hydraulic retention time (HRT). Combining this SRT controller with a previously studied HRT controller could potentially amplify biomass productivity efficiency. In addition, controlling or not controlling the HRT and SRT are assessed in terms of filtration performance and GHG emissions. Finally, a new dissolved-oxygen-based parameter shows promise for continuous microalgae culture control.

Effects of Energy Feed Sources in Hanwoo Cattle Diets on Fermentation and Microbial Amino Acids Composition in a Rumen Simulation Continuous Culture System

Effects of Energy Feed Sources in Hanwoo Cattle Diets on Fermentation and Microbial Amino Acids Composition in a Rumen Simulation Continuous Culture System

Effects of monensin and cashew nut-shell extract on bacterial community composition in a dual-flow continuous culture system.

The objective of this study was to evaluate the effects of including monensin and two doses of CNSE in a high producing dairy cow diet on ruminal bacterial communities. A dual-flow continuous culture system was used in a replicated 4 × 4 Latin Square design. A basal diet was formulated to meet the requirements of a cow producing 45kg of milk per d (17% crude protein and 27% starch). There were four experimental treatments: the basal diet without any feed additive (CON), 2.5 μM monensin (MON), 100ppm CNSE granule (CNSE100), and 200ppm CNSE granule (CNSE200). Samples were collected from the fluid and solid effluents at 3, 6, and 9h after feeding; a composite of all time points was made for each fermenter within their respective fractions. Bacterial community composition was analyzed by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform. Treatment responses for bacterial community structure were analyzed with the PERMANOVA test run with the R Vegan package. Treatment responses for correlations were analyzed with the CORR procedure of SAS. Orthogonal contrasts were used to test the effects of (1) ADD (CON vs. MON, CNSE100, and CNSE200); (2) MCN (MON vs. CNSE100 and CNSE200); and (3) DOSE (CNSE100 vs. CNSE200). Significance was declared at P ≤ 0.05. We observed that the relative abundance of Sharpea (P < 0.01), Mailhella (P = 0.05), Ruminococcus (P = 0.03), Eubacterium (P = 0.01), and Coprococcus (P < 0.01) from the liquid fraction and the relative abundance of Ruminococcus (P = 0.03) and Catonella (P = 0.02) from the solid fraction decreased, while the relative abundance of Syntrophococcus (P = 0.02) increased in response to MON when compared to CNSE treatments. Our results demonstrate that CNSE and monensin have similar effects on the major ruminal bacterial genera, while some differences were observed in some minor genera. Overall, the tested additives would affect the ruminal fermentation in a similar pattern.

Biological properties of foot-and-mouth disease virus А 2205/G-IV strain

According to the World Organisation for Animal Health, foot-and-mouth disease (FMD) is regularly reported in domestic and wild cloven-hoofed animals in Africa. G-I, G-IV, G-VI, G-VII, ASIA/Iran-05 genetic lineages of serotype А FMD virus are considered to be the most widespread on the African continent. Given the close economic and trade relations maintained by the Russian Federation with the countries of North Africa, of particular interest for us is studying the FMD virus of serotype A G-IV genetic lineage, which has been responsible for the infection outbreaks in the naturally susceptible animal population of the said region every year since 2012, and there is a risk of introduction of this virus genotype in to the Russian Federation. Therefore, the issues of FMD introduction risk assessment and timely diagnosis are relevant for the Veterinary Service of Russia. FMD virus A 2205/G-IV strain tested for its biological and antigenic properties in cell cultures and naturally susceptible animals (cattle and pigs) was adapted for its reproduction in initially trypsinized porcine kidney (PK) cell culture, continuous monolayer cell cultures (IB-RS-2, PSGK-30, YaDK-04, ВНК-21) by five serial passages. The virus was considered to be adapted when 90–95% cytopathic effect developed within 14–19 hours after the cell culture infection. The virus adapted to the cell cultures was tested for its infectivity with microtitration in IB-RS-2 cell culture. The virus strain tested for vaccine matching with microneutralization test (MNT) demonstrated significant difference from production A/Turkey/06, А22 No. 550/Azerbaijan/64, A22/Iraq/64, A/Iran/97, A No. 2155/Zabaikalsky/2013, A No. 2166/Krasnodarsky/2013, A No. 2269/ARRIAH/2015 strains of FMD virus.

Acid adaptation alters Streptococcus mutans drug susceptibility profile

The ability of Streptococcus mutans, the primary causative agent of dental caries, to generate and tolerate an acidic environment is a leading factor in its capacity to outcompete other organisms within the oral cavity. The acid tolerance response (ATR) involves coordination of multiple pathways to upregulate macromolecule repair, maintenance of cytosolic pH, and restructuring the membrane. In this study, we asked if the changes associated with adaptation to a low pH environment would result in detectable changes to drug susceptibility. Accordingly, we screened using the Selleck library of 853 FDA approved drugs for agents that exhibit antimicrobial activity toward planktonic cultures of S. mutans or disrupt biofilm formation in growth medium titrated to pH 5 or in the presence of hydrogen peroxide. Results revealed that a total of 182 compounds displayed antimicrobial properties toward the test strain in at least one screening condition. Our findings provide evidence that the S. mutans ATR is characterized by a concerted shift in drug susceptibility. The presence of an intact ATR, associated with growth in continuous culture at pH 5, leads to increased sensitivity to 22 compounds and decreased sensitivity to a separate set of 20 compounds. Disruption of the ATR in a mutant lacking the fabM gene resulted in a susceptibility profile that was distinct from the result of our results. This suggests that loss of unsaturated fatty acids produces a physiological change that is not only measurable, but perhaps more significant than the process of ATR. Additional tests revealed that S. mutans exhibits a distinct drug susceptibility profile compared to a closely related, non-cariogenic bacterium, S. gordonii. Overall, shifts in S. mutans susceptibility patterns based on pH of growth medium or type of culture (batch vs. continuous culture), indicate that the ATR can be tracked using changes in drug susceptibility profile. Furthermore, our results provide clues that compounds could be useful in treatment of dental caries as well as providing molecular tools to study shifts in physiology of disease-associated bacteria.

Exclusive D-lactate-isomer production during a reactor-microbiome conversion of lactose-rich waste by controlling pH and temperature

Lactate is among the top-ten-biobased products. It occurs naturally as D- or L-isomer and as a racemic mixture (DL-lactate). Generally, lactate with a high optical purity is more valuable. In searching for suitable renewable feedstocks for lactate production, unutilized organic waste streams are increasingly coming into focus. Here, we investigated acid whey, which is a lactose-rich byproduct of yogurt production, that represents a considerable environmental footprint for the dairy industry. We investigated the steering of the lactate-isomer composition in a continuous and open culture system (HRT = 0.6 d) at different pH values (pH 5.0 vs. pH 6.5) and process temperatures (38°C to 50°C). The process startup was achieved by autoinoculation. At a pH of 5.0 and a temperature of 47°C-50°C, exclusive D-lactate production occurred because of the dominance of Lactobacillus spp. (> 95% of relative abundance). The highest volumetric D-lactate production rate of 722 ± 94.6 mmol C L−1 d−1 (0.90 ± 0.12 g L−1 h−1), yielding 0.93 ± 0.15 mmol C mmol C−1, was achieved at a pH of 5.0 and a temperature of 44°C (n = 18). At a pH of 6.5 and a temperature of 44°C, we found a mixture of DL-lactate (average D-to-L-lactate production rate ratio of 1.69 ± 0.90), which correlated with a high abundance of Streptococcus spp. and Enterococcus spp. However, exclusive L-lactate production could not be achieved. Our results show that for the continuous conversion of lactose-rich dairy waste streams, the pH was a critical process parameter to control the yield of lactate isomers by influencing the composition of the microbiota. In contrast, temperature adjustments allowed the improvement of bioprocess kinetics.

Chlortetracycline Concentration Impact on Salmonella Typhimurium Sustainability in the Presence of Porcine Gastrointestinal Tract Bacteria Maintained in Continuous Culture.

Concern exists that the continued use of antibiotics in animal feeds may lead to an increased prevalence of resistant bacteria within the host animal's gastrointestinal tract. To evaluate the effect of chlortetracycline on the persistence of Salmonella enterica serotype Typhimurium within a diverse population of porcine cecal bacteria, we cultured a mixed population of cecal bacteria without or with added chlortetracycline. When grown at a 24 h vessel turnover rate, chlortetracycline-susceptible S. Typhimurium exhibited more than 2.5 times faster (p < 0.05) disappearance rates than theoretically expected (0.301 log10 colony-forming unit/mL per day) but did not differ whether treated or not with 55 mg of chlortetracycline/L. Chlortetracycline-resistant S. Typhimurium was not recovered from any of these cultures. When the mixed cultures were inoculated with a chlortetracycline-resistant S. Typhimurium, rates of disappearance were nearly two times slower (p < 0.05) than those observed earlier with chlortetracycline-susceptible S. Typhimurium, and cultures persisted at >2 log10 colony-forming units/mL for up to 14 days of treatment with 110 mg of chlortetracycline/L. Under the conditions of this study, chlortetracycline-resistant S. Typhimurium was competitively enabled to persist longer within the mixed populations of porcine gut bacteria than chlortetracycline-susceptible S. Typhimurium, regardless of the presence or absence of added chlortetracycline.

Glycerol and glucose co‐fermentation by 1,3‐propanediol producer Limosilactobacillus reuteri: A kinetic study of batch and continuous cultivations

AbstractThe conversion of glycerol into 1,3‐propanediol (1,3‐PDO) has gained significant attention as a sustainable pathway within the biodiesel production chain. Limosilactobacillus reuteri, a bacterium capable of efficiently converting glycerol to 1,3‐PDO, holds promise for this application. In industrial biotechnology, the development of kinetic models is important for bioreactor design, process analysis and scalability, supporting the advancement of biorefineries and the bioeconomy. This study focuses on investigating the kinetics of co‐fermentation of glucose and glycerol by L. reuteri, utilizing data from previous batch culture experiments. Three different models were evaluated for these batch runs, and the parameters were adjusted using the Levenberg–Marquardt algorithm in conjunction with an integration method. The results from these previous batch experiments indicate that pH did not significantly influence the specific growth rate of the bacterium. However, as expected, reducing the temperature from 37 to 30°C resulted in a 60% reduction in the growth rate. Moreover, continuous co‐fermentation experiments were conducted, and a simple kinetic model was proposed. The advantage of the continuous process is that the productivity of 1,3‐PDO is 3.5 times higher compared with co‐fermentation in batch mode. Notably, this study represents the first attempt to model continuous co‐fermentation under a range of dilution rates (0.25–0.9 h−1), identifying a dilution rate of 0.5 h−1 as the optimal condition to produce 1,3‐PDO, considering productivity and substrate conversion. The findings of this research contribute valuable insights for future investigations on the viability of 1,3‐PDO production from glycerol, particularly within the context of waste valorization.

The Continuing Education of The University Librarians in Hebei Province

This paper examines the landscape of continuing education for university librarians in Hebei Province, emphasizing best practices and challenges encountered in the process. Within the context of rapidly evolving academic and technological landscapes, continuing education stands as a cornerstone for librarians' growth and proficiency. The study analyzes existing literature, incorporating insights from experts and practitioners in the field. Key challenges identified include resource constraints, tailoring education to professional needs, securing institutional support, technological readiness, and sustaining engagement. Recommendations encompass strategic resource allocation, customized education approaches, enhanced technological integration, and the cultivation of a continuous learning culture. This study advocates for proactive measures to navigate these challenges and optimize continuing education strategies, ensuring librarians are well-equipped to meet the evolving demands of the academic environment.

Selecting for a high lipid accumulating microalgae culture by dual growth limitation in a continuous bioreactor

A dual-growth-limited continuous operated bioreactor (chemostat) was used to enhance lipid accumulation in an enrichment culture of microalgae. The light intensity and nitrogen concentration where both limiting factors resulting in high lipid accumulation in the mixed culture. Both conditions of light and nitrogen excess and deficiency were tested. Strategies to selectively enrich for a phototrophic lipid-storing community, based on the use of different nitrogen sources (ammonium vs. nitrate) and vitamin B supplementation in the growth medium, were evaluated. The dual limitation of both nitrogen and light enhanced the accumulation of storage compounds. Ammoniacal nitrogen was the preferred nitrogen source. Vitamin B supplementation led to a doubling of the lipid productivity. The availability of vitamins played a key role in selecting an efficient lipid-storing community, primarily consisting of Trebouxiophyceae (with an 82 % relative abundance among eukaryotic microorganisms). The obtained lipid volumetric productivity (387 mg L−1 d−1) was among the highest reported in literature for microalgae bioreactors. Lipid production by the microalgae enrichment surpassed the efficiencies reported for continuous microalgae pure cultures, highlighting the benefits of mixed-culture photo-biotechnologies for fuels and food ingredients in the circular economy.

Programmatic assessment in Medical Education – Review

A programmatic assessment is a systematic process by which we evaluate our processes, services, and outcomes through a systematic approach with the intention of enhancing our quality and performance. With the help of a structured approach, we will be able to gather, analyze, and interpret data in order to determine strengths and weaknesses within our company's operations. As part of the organization's assessment program, a variety of methodologies will be used, including surveys, performance metrics, and peer reviews, to provide an understanding of the effectiveness of the organization as a whole. In this review, the primary objectives are to identify the benefits, the approach to their implementation, as well as areas for improvement, set measurable goals, develop strategic changes, and identify the challenges associated with implementing programmatic assessments in medical education. Through the adoption of a continuous improvement culture, this assessment program will have the ability to empower our organization to adapt to evolving challenges as well as provide better services to our stakeholders.

TMT-based quantitative proteomics reveals the nutritional and stress resistance functions of anaerobic fungi in yak rumen during passage at different time intervals

ObjectivesAnaerobic fungi are critical for nutrient digestion in the yak rumen. Although studies have reported the effects of passage at different time intervals on the community structure of yak rumen anaerobic fungi, it is unknown whether passage culture at different time intervals affects the microbial proteins of rumen anaerobic fungi and their functions. MethodsMycelium was obtained using the anaerobic continuous batch culture (CBC) of yak rumen fluid at intervals of 3 d, 5 d and 7 d. Quantitative analysis of fungal proteins and functional analysis was performed using tandem mass tagging (TMT) and bioinformatics. ResultsA total of 56 differential proteins (DPs) were found in 5 d vs. 3 d and 7 d vs. 3 d. Gene ontology (GO) enrichment indicated that the up-regulated proteins were mainly involved in biological regulation, cellular process, metabolic process, macromolecular complex, membrane, cell part, organelle, binding, catalytic activity and transporter activity. The downregulated proteins were mainly enriched in metabolic process, cell part, binding and catalytic activity. Furthermore, the downregulated proteins in 7 d vs. 3 d were related to membrane and organelle. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results indicated that DPs were enriched in 14 pathways in 5 d vs. 3 d and 7 d vs. 3 d, mainly including terpenoid backbone biosynthesis, alaine, aspartate and glutamate metabolism, arginine biosynthesis, hypotaurine, cyanoamino acid, glutathione, β-alanine, pyrimidine, purine, galactose and propanate metabolism, steroid biosynthesis, ribosome biogenesis in eukaryotes and aminoacyl tRNA biosynthesis. The DPs were enriched in only 2 pathways in 5 d vs 3 d, lysine biosynthesis and cysteine and methionine metabolism. N-glycan biosynthesis and retinol metabolism are only found in the metabolism of DPs in 7 d vs 3 d. ConclusionsYak rumen anaerobic fungal proteins are involved in nutrition and stress tolerance during passage at different time intervals.

Thermococcus thermotolerans sp. nov., a hyperthermophilic archaeon isolated from a chimney in the Southwest Indian Ocean.

An anaerobic hyperthermophilic archaeon was isolated from a black smoker chimney with a snail attachment at a water depth of 2 739 m in the Southwest Indian Ocean. The sample was taken from the chimney exterior wall. The enrichment was conducted under a continuous culture with temperature fluctuation of 80-130 °C over 24 h for 42 days at 28 MPa. The isolation was performed at 90 °C at 0.1 MPa. Cells of the isolated strain 813A4T were irregular cocci. Strain 813A4T grew at 60-94 °C (optimal growth at 85 °C) at 0.1 MPa, and growth was detected at up to 99 °C at 28 MPa. At 85 °C, the strain was able to grow at pressures ranging from 0.1 to 110 MPa (optimal pressure, 0.1-40 MPa). At 85 °C, the cells of 813A4T grew at pH 5.5-9 (optimal, pH 7.0) and a NaCl concentration of 1.0-4.0 % (w/v; optimum concentration, 2.5 % NaCl). Strain 813A4T utilized yeast extract, tryptone and peptone as single carbon sources for growth. Elemental sulphur stimulated its growth. The G+C content of the complete genome was 53.48 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 813A4T belonged to the genus Thermococcus, with the highest sequence similarity to Thermococcus barossii SHCK-94T (99.73 %). The average nucleotide identity between strains 813A4T and SHCK-94T was 82.56 %. All these data indicated that strain 813A4T should be classified as representing a novel species of the genus Thermococcus, for which Thermococcus thermotolerans sp. nov. is proposed. The type strain is 813A4T (=JCM 39367T=MCCC M28628T).

Simultaneous medium chain carboxylic acids and 1,3-propanediol production in a bioaugmented lactate-based chain elongation induced with glycerol

The objective was to investigate the impact of the bioaugmentation on chain elongation process using glycerol, lactate and lactose as substrates in an open culture fermentation. In the batch trials the highest selectivity for chain elongation product, i.e. caproate, was observed in trials inoculated with co-culture of Megasphaera elsdenii and Eubacterium limosum grown on glycerol (28.6%), and in non-bioaugmented open culture run on lactose + lactate (14.8%). The results showed that E. limosum, out of two bioaugmented strains, was able to survive in the open culture. A continuous open culture fermentation of glycerol led to caproate and 1,3-propanediol (1,3-PDO) formation, while lactate addition led to 1,3-PDO and short chain carboxylates production. Moving the process into batch mode triggered even-carbon chain elongation. Presence of E. limosum promoted odd-carbon chain elongation and valerate production. Imaging flow cytometry combined with machine learning enabled the discrimination of Eubacterium cells from other microbial strains during the process.

Abstract B164: ASTX029 is a dual mechanism ERK1/2 inhibitor with activity in models of MAPK-inhibitor resistance

Abstract Clinical efficacy of RAF, MEK1/2 (MEK) and KRASG12C inhibitors has been demonstrated in MAPK-activated cancers. However, response is often short-lived due to resistance mechanisms, which commonly confer reactivation of ERK1/2 (ERK). As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for overcoming resistance to upstream inhibition. ASTX029 is a potent and selective dual-mechanism ERK inhibitor. Due to its distinctive ERK-binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK by MEK. It is currently in Phase 1/2 trial in patients with advanced solid tumors (NCT03520075). Here we demonstrate the potent activity of ASTX029 in models of acquired MAPK inhibitor resistance. Two MAPK inhibitor-resistant models were generated by continuous culture of A375 (BRAFV600E -mutant melanoma) cells in the presence of the BRAF inhibitor vemurafenib (A375R) or by introduction of an NRASQ61K mutation (A375-NRASQ61K). The A375-NRASQ61K and A375R cell lines were resistant to vemurafenib in cell proliferation assays (IC50 &amp;gt;3 µM and &amp;gt;10 µM respectively, compared to IC50 110 nM in parental A375 cells). They were also less sensitive to the MEK inhibitor selumetinib (IC50 270 nM and 1000 nM, respectively, compared to IC50 43 nM in parental A375). Both models were highly sensitive to ASTX029, with proliferation assay IC50 values of 12 nM (A375-NRASQ61K) and 7.2 nM (A375R), values which were not significantly different to the ASTX029 IC50 of parental A375. ASTX029 inhibited MAPK signalling in both models, whereas vemurafenib and selumetinib had no effect. Treatment of A375R tumour-bearing mice with 50 mg/kg bid vemurafenib (which causes significant tumor growth inhibition in parental A375 xenografts), did not result in significant tumor growth inhibition. In contrast, 75 mg/kg qd ASTX029 conferred significant anti-tumor activity (P&amp;lt; 0.001). The acquisition of MEK mutations is a known resistance mechanism to RAF, MEK and KRASG12C inhibitors. We performed a CRISPR-tiling screen on MEK1 and identified mutations that conferred resistance to MEK inhibitors. The identified MEK1 mutations either caused MEK activation (indicated by elevated pERK levels) or were predicted to prevent MEK inhibitor binding (supported by MEK1 structural analysis). All mutations conferred resistance to selumetinib (7- to 200-fold increase in cell proliferation IC50 values relative to parental cells). Mutations predicted to prevent MEK inhibitor binding were sensitive to vemurafenib and the pan-RAF inhibitor LY3009120, whereas those that caused MEK1 activation were resistant (5- to 10-fold increase in cell proliferation IC50 relative to parental cells). However, they were all sensitive to ASTX029 (IC50 values of 4 to 17 nM). We conclude that the MAPK inhibitor resistance mechanisms described did not confer resistance to ASTX029. These data highlight the therapeutic potential of ASTX029 for the treatment of cancers, which have acquired resistance to inhibitors of upstream components of the MAPK pathway. Citation Format: Zhiqiang Zhang, Christopher Hindley, Andrea Biondo, Nicola Wallis, John Lyons, Joanne Munck. ASTX029 is a dual mechanism ERK1/2 inhibitor with activity in models of MAPK-inhibitor resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B164.

Abstract B064: Single-cell RNA sequencing reveals tumorigenic trajectories of mismatch repair deficient cells

Abstract Mismatch repair (MMR) deficiency is commonly found in tumors with high mutational burden. Although the mutational signatures of these deficiencies are determined, the initial trajectories these cells follow to reach a tumorigenic state with high mutational load have not been studied due to lack of isogenic model systems. For this reason, we generated three isogenic MMR deficient cell lines from a normally untransformed cell line with few genomic abnormalities and low mutational burden. After continued culturing of these MMR-deficient cell lines for several months, we profiled them by single cell RNA sequencing using the 10X Chromium platform. Next, we identified different clusters of cells in MMR deficient cell lines that acquire tumorigenic properties. In these clusters, we identified gene expression signatures that are responsible from the tumorigenic properties of these clusters of cells. After isolation of the single clones that bear these gene expression signatures, we characterized how MMR deficiency can give rise to tumors with particular gene expression trajectories. In addition, we performed CRISPR/Cas9-based genome-wide screens to understand the vulnerabilities of these clusters of cells. Altogether, our results shed light on the early steps of tumorigenesis in MMR deficiency. Citation Format: Alexandra Vitor, Salvatore Benfatto, Frank Zenke, Sebastian Waszak, Balca Mardin. Single-cell RNA sequencing reveals tumorigenic trajectories of mismatch repair deficient cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B064.

Analysis of full nitrification performance and optimization of reaction properties using N and O isotope fractionation

Isotopic fractionation properties have been successfully applied to identify the distribution and fate of nitrogen in ecosystems, revealing the dynamic response of N and O elements during nitrogen transport and transformation. However, only a few studies used the dual isotope technology in activated sludge treatment of domestic wastewater and many aspects of the process are unclear. Here, we use the dual isotope techniques to increase the understanding of the substrates required for nitrification reactions, nitrification performance, and process operation. Mixed sludge was successfully enriched with nitrifying bacteria in a continuous culture, and three dissolved oxygen (DO; 0.2–0.4, 3–4, and 7–8 mg/L) and three temperature levels (18 ± 1, 25 ± 1, and 33±1 °C) were tested for efficiency of nitrate nitrogen accumulation. Both δ15NNO3 and δ18ONO3 showed a gradual increase with an increase in DO or temperature, the increase in DO slowed down the fractionation effect of isotopes, and the increase in temperature reduced the variability in N and O utilization. The slope of δ15NNO3:δ18ONO3 gradually approached 1 with the increase in DO (<7 mg/L) or in temperature, and the optimal range of DO and temperature were accurately judged to strengthen the denitrification performance of nitrifying bacteria. δ18OH2O was successfully taken up to form NO2−-N and NO3−-N with 74 and 91% replacement rates, respectively, indicating that DO and H2O jointly completed the formation of nitrate nitrogen during the long nitrification process. In summary, the in situ dual isotope technology can help optimize the influence of environmental factors on nitrification performance to guide the long-term stable operation of nitrification reactions in sludge treatment and provide a reliable basis for complex activated sludge nitrification systems.

Production of poly‐gamma‐glutamic acid and spore formation in Bacillus velezensis 83 are affected by acidic growing conditions and glucose uptake rate

AbstractBACKGROUNDBacillus velezensis 83 is a biocontrol agent and plant growth promoting bacteria that produces antifungal compounds (surfactin and bacilomycin D), plant growth stimulation metabolites such as acetoin and butanediol and the polymer poly‐gamma‐glutamic acid (γ‐PGA), which is essential for bacterial plant colonization. Bacillus sporulation is an important trait in order to obtain long shelf‐life formulations for biocontrol purposes. γ‐PGA production and sporulation are closed‐related processes controlled by glucose availability and modulated by quorum sensing. High spore production (and its recovery) in B. velezensis 83 cultures is limited by the high viscosity oxygen‐limited broths resulting from γ‐PGA accumulation. This work studied the influence of acidic growing conditions and glucose specific uptake rate on growth, quorum sensing‐related metabolites production and sporulation of B. velezensis 83.RESULTSIn batch cultures developed at pH = 5, no bacterial sporulation was detected and quorum sensing related metabolites as γ‐PGA (not detected) and surfactin and bacillomycin were significantly decreased. Instead, acetoin and butanediol were the main products. In contrast, in continuous cultures developed at pH = 5, spores were detected at D = 0.06 h−1. The results indicate that sporulation at pH = 5 is not inhibited by affectations on quorum sensing signals but could be due to a low availability/transport of energy sources (i.e., glucose or acetoin/butanediol) or an affectation on Rap‐Phr system.CONCLUSIONThe findings in this study allow to establish a two‐stage process for Bacillus velezensis 83 spore production: bacterial growth at pH = 5 and sporulation at pH = 6.8. © 2023 Society of Chemical Industry (SCI).

Features of SARS-CoV-2 Replication in Various Types of Reptilian and Fish Cell Cultures.

SARS-CoV-2 can enter the environment from the feces of COVID-19 patients and virus carriers through untreated sewage. The virus has shown the ability to adapt to a wide range of hosts, so the question of the possible involvement of aquafauna and animals of coastal ecosystems in maintaining its circulation remains open. the aim of this work was to study the tropism of SARS-CoV-2 for cells of freshwater fish and reptiles, including those associated with aquatic and coastal ecosystems, and the effect of ambient temperature on this process. In a continuous cell culture FHM (fathead minnow) and diploid fibroblasts CGIB (silver carp), SARS-CoV-2 replication was not maintained at either 25 °C or 29 °C. At 29 °C, the continuous cell culture TH-1 (eastern box turtle) showed high susceptibility to SARS-CoV-2, comparable to Vero E6 (development of virus-induced cytopathic effect (CPE) and an infectious titer of 7.5 ± 0.17 log10 TCID50/mL on day 3 after infection), and primary fibroblasts CNI (Nile crocodile embryo) showed moderate susceptibility (no CPE, infectious titer 4.52 ± 0.14 log10 TCID50/mL on day 5 after infection). At 25 °C, SARS-CoV-2 infection did not develop in TH-1 and CNI. our results show the ability of SARS-CoV-2 to effectively replicate without adaptation in the cells of certain reptile species when the ambient temperature rises.