Low Nucleic Acid Research Articles

Is in-situ burning an acceptable mitigation option after a major oil spill? Impact on marine plankton

Major oil spills can impose a significant environmental hazard on the marine ecosystem, and a promising mitigation measure is in-situ oil burning (ISB). However, our knowledge of the impact of the burned residues and soot deposition on the marine ecosystem is still limited. We investigated the effects of burned oil residue and soot deposition on the marine plankton communities of the oligotrophic Eastern Mediterranean Sea with a mesocosm experiment. Three triplicated treatments were tested: (1) Iranian crude oil was added and burned (Burned treatment); (2) soot was collected and deposited with artificial rain (Soot); and (3) a non-contaminated Control. Results revealed that Low Nucleic Acid heterotrophic bacteria, Synechococcus spp., and pigmented pico-nano Eukaryotes (pnEuk) were negatively affected in the Burned and Soot treatments. Viruses, heterotrophic pnEuk and ciliates (in Soot) were crucial for controlling the High Nucleic Acid bacteria. Ciliates and most dinoflagellates showed a negative response to the burned residues but were less affected or were even favored when exposed to soot. Our results show that ISB affected the structure and dynamics of the plankton food web through burned residues and soot depositions. However, since the effects appeared at least three days after the ignition, ISB could be combined with subsequent burned residue collection to minimize its impact on the pelagic ecosystem.

CHAMP: A Centrifugal Microfluidics-Based CRISPR/Cas12b-Combined Real-Time LAMP One-Pot Method for Mycoplasma pneumoniae Infection Diagnosis.

The Mycoplasma pneumoniae outbreak poses health risks to community residents. However, it still has limitations for current clinical diagnostic methods (qPCR nucleic acid assay or IgM immunoassay), including specialized handling, expensive equipment, prolonged turnaround time, and false positives and negatives, highlighting the need to improve clinical diagnostic methods. Herein, we present a novel centrifugal microfluidics-based method for rapidly diagnosing M. pneumoniae infections (CHAMP system). This user-friendly method combines CRISPR/Cas12b and real-time loop-mediated isothermal amplification (LAMP) in a one-pot reaction, offering high sensitivity, specificity, and simplicity for methodology. By adding fully automated nucleic acid magnetic bead-extracted samples to a prepackaged centrifugal microfluidics chip, 48 samples can be automated tested simultaneously within 15 to 60 min at 60 °C. 427 clinical nasopharyngeal swab specimens were used for validation, demonstrating good positive and negative predictive values and good diagnostic sensitivity, specificity, and significant time savings. This method is particularly suitable for detecting low nucleic acid copies of M. pneumoniae samples.

Comparing Conventional and Modern Methods for The Phycocyanin Extraction from Spirullina sp

Spirulina platensis, a blue-green algae abundant in tropical regions, is rich in minerals, vitamins, fibers, and pigments, with low nucleic acid content. It has unique chromoproteins called phycobiliproteins, notably phycocyanin, used in various applications. This study aims to optimize phycocyanin extraction using different solvents (distilled water and sodium phosphate buffer pH 6.7) and methods (freeze-thaw and sonication). Spirulina platensis biomass was extracted in both solvents, then some of them was freeze for 24 and 48 hours followed by thawing overnight. The other was sonicated for 2.5 minutes, 50 Hz then soaked for 1, 2, and 3 hours. All of the samples were centrifuged at 6000 rpm for 10 minutes and the absorbance was measured using a UV-Vis spectrophotometer at wavelengths of 280, 620, and 650 nm. with freeze-thawing for 48 hours yielded the highest phycocyanin concentration (0.55%), with a yield of 11.07 and purity of 0.21. Sonication improved phycocyanin concentration, yield, and purity significantly, yielding 1.108, 25.85, and 0.26, respectively.

Metagenomics analysis reveals effects of salinity fluctuation on diversity and ecological functions of high and low nucleic acid content bacteria

Salinity is a critical environmental factor in marine ecosystems and has complex and wide-ranging biological effects. However, the effects of changing salinity on diversity and ecological functions of high nucleic acid (HNA) and low nucleic acid (LNA) bacteria are not well understood. In this study, we used 16S rRNA sequencing and metagenomic sequencing analysis to reveal the response of HNA and LNA bacterial communities and their ecological functions to salinity, which was decreased from 26 ‰ to 16 ‰. The results showed that salinity changes had significant effects on the community composition of HNA and LNA bacteria. Among LNA bacteria, 14 classes showed a significant correlation between relative abundance and salinity. Salinity changes can lead to the transfer of some bacteria from HNA bacteria to LNA bacteria. In the network topology relationship, the complexity of the network between HNA and LNA bacterial communities gradually decreased with decreased salinity. The abundance of some carbon and nitrogen cycling genes in HNA and LNA bacteria varied with salinity. Overall, this study demonstrates the effects of salinity on diversity and ecological functions and suggests the importance of salinity in regulating HNA and LNA bacterial communities and functions.

Adaptive strategies of high and low nucleic acid prokaryotes in response to declining resource availability and selective grazing by protozoa

Prokaryotes play a fundamental role in global ocean biogeochemical cycles. However, how the abundance and metabolic activity of ecologically distinct subgroups (i.e., high nucleic acid (HNA) and low nucleic acid (LNA) cells), and their regulating factors, change in response to changing marine environmental conditions remains poorly understood. Here, we delved into the time-evolving dynamic responses of the HNA and LNA prokaryotic subgroups to declining resource availability and selective grazing by protozoa by conducting a 73-day incubation experiment in a large-volume (117,000 L) macrocosm that facilitates community-level exploration. We found that the metabolic activity of the HNA subgroup was higher than that of the LNA subgroup when the macrocosm was resource replete but that the HNA subgroup declined more rapidly than the LNA subgroup as the resources became increasingly scarce, leading to a steadily increasing contribution of LNA cells to prokaryotic activity. Meanwhile, as resources in the macrocosm became limited, protozoan grazing preference shifted from the HNA to the LNA subgroup and the contributions of the LNA subgroup to the carbon flow within the macrocosm increased. The findings highlight the resilience of LNA cells in resource-limited environments, illuminate the critical role of selective grazing by protozoa in balancing distinct prokaryotic subgroups under changing resource conditions, and demonstrate the complex and adaptive interactions between protozoa and prokaryotes across diverse environmental contexts.

Endogenous EWSR1 Exists in Two Visual Modalities That Reflect Its Associations with Nucleic Acids and Concentration at Sites of Active Transcription.

EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

Contrasting functions of ATP hydrolysis by MDA5 and LGP2 in viral RNA sensing

Cytosolic long dsRNA, among the most potent proinflammatory signals, is recognized by melanoma differentiation-associated protein 5 (MDA5). MDA5 binds dsRNA cooperatively forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-β responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. Consecutive rounds of ATP hydrolysis amplify the filament footprint, displacing tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling identifies an internally bound MDA5-LGP2-RNA complex, with the LGP2 C-terminal tail forming the key contacts with MDA5. These contacts are specifically required for NTP-dependent internal RNA binding. We conclude that NTPase-dependent binding of LGP2 to internal dsRNA sites complements NTPase-independent binding to dsRNA ends, via distinct binding modes, to increase the number and signaling output of MDA5-dsRNA complexes.

Predicting Anaerobic Membrane Bioreactor Performance Using Flow-Cytometry-Derived High and Low Nucleic Acid Content Cells.

Having a tool to monitor the microbial abundances rapidly and to utilize the data to predict the reactor performance would facilitate the operation of an anaerobic membrane bioreactor (AnMBR). This study aims to achieve the aforementioned scenario by developing a linear regression model that incorporates a time-lagging mode. The model uses low nucleic acid (LNA) cell numbers and the ratio of high nucleic acid (HNA) to LNA cells as an input data set. First, the model was trained using data sets obtained from a 35 L pilot-scale AnMBR. The model was able to predict the chemical oxygen demand (COD) removal efficiency and methane production 3.5 days in advance. Subsequent validation of the model using flow cytometry (FCM)-derived data (at time t - 3.5 days) obtained from another biologically independent reactor did not exhibit any substantial difference between predicted and actual measurements of reactor performance at time t. Further cell sorting, 16S rRNA gene sequencing, and correlation analysis partly attributed this accurate prediction to HNA genera (e.g., Anaerovibrio and unclassified Bacteroidales) and LNA genera (e.g., Achromobacter, Ochrobactrum, and unclassified Anaerolineae). In summary, our findings suggest that HNA and LNA cell routine enumeration, along with the trained model, can derive a fast approach to predict the AnMBR performance.

Isolasi, Identifikasi, dan Produksi Miselia Rhizopus sp. Berkadar Asam Nukleat Rendah untuk Pengembangan Mikoprotein

Fungi, including Rhizopus sp., are food sources that commonly contain high nucleic acid levels. Therefore, the nucleic acid content must be reduced to achieve health standard requirements. This study aimed to isolate, identify, and produce Rhizopus sp. mycelium containing low nucleic acid. The Rhizopus spp. were isolated from tempeh collected from 12 different areas in Indonesia. Fungal identification was conducted based on morphological characteristics. The fungal isolates were selected based on mycelial growth and spore production on PDA. Biomass production of mycelium was carried out in potato extract and soybean extract media obtained from 200 g/L and 333.3 g/L, respectively. In each medium, 6 sugar levels were added, namely 0, 2, 3, 4, 5, and 6 g/L. Mycelium nucleic acid content reduction was achieved by heat treatment at 50°C and 60°C for 15 minutes and measured by a spectrophotometer at 260 nm. Fifty-eight isolates that were identified into 3 species were obtained in this experiment: R. oryzae, R. stolonifera, and R. microsporus. R. Microsporus had higher mycelium biomass and lower spore number than the other species. R. Microsporus produced a higher mycelium biomass in the soybean extract medium with 5 g/L additional sugar. The nucleic acid content of the 50°C heat-treated mycelium was 1.82% and 1.73% at 65°C. These values fulfilled the standard of mycelial nucleic acid content permitted in food by the USDA.
 
 Keywords: morphology, Rhizopus microspores, spore, soybean extract, tempeh

Stimulus-Responsive Nanodelivery and Release Systems for Cancer Gene Therapy: Efficacy Improvement Strategies.

Introduction of exogenous genes into target cells to overcome various tumor diseases caused by genetic defects or abnormalities and gene therapy, a new treatment method, provides a promising strategy for tumor treatment. Over the past decade, gene therapy has made exciting progress; however, it still faces the challenge of low nucleic acid delivery and release efficiencies. The emergence of nonviral vectors, primarily nanodelivery and release systems (NDRS), has resulted in a historic breakthrough in the application of gene therapy. NDRS, especially stimulus-responsive NDRS that can respond in a timely manner to changes in the internal and external microenvironment (eg, low pH, high concentration of glutathione/reactive oxygen species, overexpressed enzymes, temperature, light, ultrasound, and magnetic field), has shown excellent loading and release advantages in the precision and efficiency of tumor gene therapy and has been widely applied. The only disadvantage is that poor transfection efficiency limits the in-depth application of gene therapy in clinical practice, owing to the presence of biological barriers in the body. Therefore, this review first introduces the development history of gene therapy, the current obstacles faced by gene delivery, strategies to overcome these obstacles, and conventional vectors, and then focuses on the latest research progress in various stimulus-responsive NDRS for improving gene delivery efficiency. Finally, the future challenges and prospects that stimulus-responsive NDRS may face in clinical application and transformation are discussed to provide references for enhancing in-depth research on tumor gene therapy.

Spatial and seasonal variability of picoplankton abundance and growth rates in the southern Bay of Biscay

Autotrophic and heterotrophic picoplankton play fundamental roles in marine food webs and biogeochemical cycles. However, their growth responses have seldom been jointly assessed, including many temperate regions such as the Bay of Biscay. There, previous studies have shown their relevance in carbon fluxes. We describe here the spatio-temporal variability of the abundances and growth rates of the picoplanktonic groups routinely distinguished by flow cytometry (Synechococcus and Prochlorococcus cyanobacteria, two groups of differently sized picoeukaryotes and two groups of heterotrophic bacteria distinguished by their relative nucleic acid content) in the central Cantabrian Sea (S Bay of Biscay). To that end, from February to December 2021 we collected surface water on 5 occasions from 6 stations distributed along the S Bay of Biscay (6-3°W) and incubated it after removing protistan grazers in order to determine their dynamics along the seasonal cycle as well as the inshore-offshore and the west-east gradients. Seasonal variations in initial and maximum abundances generally matched previous knowledge of the region but growth rates were more variable, with Prochlorococcus and high nucleic acid (HNA) bacteria showing the maximum values (up to 2 d−1) while negative growth was observed in one third of Synechococcus incubations. Temporal differences generally overrode differences along the inshore-offshore gradient in trophic status while in situ and maximum abundances of most of the groups generally decreased towards the east following the increase in stratification and lower nutrient availability. Responses to stratification suggest Prochlorococcus and low nucleic acid (LNA) cells may prevail among autotrophic and heterotrophic bacteria, respectively, in a warmer ocean.

Low and high nucleic acid content bacteria play discrepant roles in response to various carbon supply modes.

Planktonic bacteria can be grouped into 'high nucleic acid content (HNA) bacteria' and 'low nucleic acid content (LNA) bacteria.' Nutrient input modes vary in environments, causing nutrient availability heterogeneity. We incubated them with equal amounts of total glucose added in a continuous/pulsed mode. The pulse-treated LNA bacteria exhibited twice the cell abundance and four times the viability of the continuous-treated LNA, while HNA did not show an adaptation to pulsed treatment. In structural equation modelling, LNA bacteria had higher path coefficients than HNA, between growth and carbon-saving metabolic pathways, intracellular ATP and the inorganic energy storage polymer, polyphosphate, indicating their low-cost growth, and flexible energy storage and utilisation. After incubation, the pulse-treated LNA bacteria contained more proteins and polysaccharides (0.00064, 0.0012 ng cell-1 ) than the continuous-treated LNA (0.00014, 0.00014 ng cell-1 ), conferring endurance and rapid response to pulses. Compared to LNA, HNA keystone taxa had stronger correlations with the primary glucose metabolism step, glycolysis, and occupied leading positions to explain the random forest model. They are essential to introduce glucose into the element cycling of the whole community under both treatments. Our work outlines a systematic bacterial response to carbon input.

Associations of ITPA gene polymorphisms with outcomes among chronic hepatitis C patients treated with Peg-interferon/ribavirin: A 5-year follow-up study.

To investigate associations between inosine triphosphatase (ITPA) gene polymorphisms and long-term outcomes among chronic hepatitis C (CHC) patients in Northeast China treated with Peg-interferon (IFN)/ribavirin (RBV). CHC patients who received Peg-IFN-2a/RBV treatment during between 2011 and 2013 at 5 hepatitis centers in Northeast China were enrolled. ITPA single nucleotide polymorphisms rs1127354 and rs7270101 from all patients were detected and their associations with 5-year outcomes were analyzed. A total of 635 patients, including 421 infected with hepatitis C virus (HCV) genotype 1 and 214 infected with non-genotype 1 were included. No significant differences were observed in the distribution frequencies of ITPA rs1127354 variants and ITPase activity between patients with HCV genotype 1 and non-genotype 1. In patients who received more than 80% of the planned RBV dose, the 5-year virological response rate and the improvement in liver fibrosis were higher in those with ITPA rs1127354 non-CC with ITPase activity <25% compared with these outcomes in patients with ITPA rs1127354 CC with 100% ITPase activity. Multiple regression analysis revealed that HCV genotype non-1, low baseline HCV ribose nucleic acid (RNA) levels (≤4 × 105 IU/mL), interleukin-28B rs12979860 CC genotype, low baseline liver fibrosis (Fibroscan 0-2), and ITPA rs1127354 non-CC genotype were independent predictors for a high long-term virological response rate, whereas interleukin-28B rs12979860 CC genotype, ITPA rs1127354 non-CC genotype, and low baseline liver fibrosis were independent predictors for improvement of liver fibrosis. ITPA rs1127354 polymorphisms is predictors of long-term outcomes in CHC patients treated with Peg-IFN/RBV.

Influence of phycospheric bacterioplankton disruption or removal on algae growth and survival

Phycospheric bacteria play a crucial role in the survival of microalgae. However, the potential of using the growth regulation and community structure modulation of phycospheric bacteria to prevent the occurrence of blooms is yet to be verified. The phycospheric bacterioplankton of Cyclotella sp. can be categorized into HNA (high nucleic acid) bacteria and LNA (low nucleic acid) bacteria. 16S rRNA sequencing showed that the HNA bacteria exhibited higher α-diversity compared to the LNA bacteria, and the microbial community composition also exhibited variations. Metagenomic sequencing further indicated the distinct ecological functions between HNA and LNA bacteria. Furthermore, the study showcased the restorative capacity of the phycospheric bacterioplankton. Biomass analysis revealed that the recovery of phycospheric bacterioplankton positively influenced the microalgae growth, thus affirming the significance of phycospheric bacterioplankton to microalgae. The community structure of phycospheric bacterioplankton demonstrated a notable decrease in the abundance of restored LNA core bacteria. Additionally, the restored phycospheric bacterioplankton exhibited a more complex co-occurrence network structure, resulting in decreased resistance and sensitivity of microalgae to adverse environments. The presence of phycospheric bacterioplankton provides a protective shield for microalgae, and thus destabilizing or removing phycospheric bacterioplankton may effectively inhibit growth of microalgae.

Roles of high/low nucleic acid bacteria in flocs and probing their dynamic migrations with respirogram

Bacterial migration is crucial for the stability of activated sludge but rarely reported. The static distribution was explored by changes in bacteria concentration with extracellular polymeric substances (EPS) extractions. Next, denitrification and aeration were conducted as normal running conditions for examining the bacterial migration between floc-attached and dispersed growth. Above observations were further explored by conducting copper ion (Cu2+) shock as an extreme running condition. After extracting EPS, low nucleic acid (LNA) bacteria migrated from the sludge to the supernatant primarily, and high nucleic acid (HNA) bacteria remained in the residual sludge, suggesting that HNA bacteria mainly distributed inside the sludge while LNA bacteria outside the sludge. During the denitrification process, LNA bacteria migrated out of flocs, which increased by 6.94 × 106 events/mL in the supernatant. During the feast phase of aeration, LNA bacteria grew attached to flocs, causing the increased flocs diameter from 45.60 to 47.40 μm. During the following aerobic famine phase, LNA bacteria grew dispersedly, but HNA bacteria remained unchanged. However, a further severe famine phase drove HNA bacteria to be dispersed, breaking flocs with the decreased diameter from 48.10 to 46.50 μm. When the Cu2+ shock was employed, LNA and HNA bacteria increased but the LNA/HNA ratio decreased in the supernatant, indicating more HNA bacteria migrating to the dispersed phase. From a structural perspective, HNA bacteria distributed inside the sludge and functioned as the backbone of flocs, undertaking the maintenance of flocs stability primarily; while LNA bacteria distributed outside the sludge and functioned as filling materials, having a secondary influence on flocs stability. These processes were also probed by respirogram exactly, correlating the system-scale measurement and microscale migrations and providing an early warning signal under abnormal circumstances. The processed HNA-backbone theory is promising for regulating the stability of activated sludge based on bacterial migrations.

A novel fluorinated polyethyleneimine with microRNA-942-5p-sponges polyplex gene delivery system for non-small-cell lung cancer therapy

Gene delivery for non-small-cell lung cancer treatment has been a challenge due to low nucleic acid binding ability, cell-wall barrier, and high cytotoxicity. Cationic polymers, such as the traditional “golden standard” polyethyleneimine (PEI) 25 kDa have emerged as a promising carrier for non-coding RNA delivery. However, the high cytotoxicity associated with its high molecular weight has limited its application in gene delivery. To address this limitation, herein, we designed a novel delivery system using fluorine-modified polyethyleneimine (PEI) 1.8 kDa for microRNA-942-5p-sponges non-coding RNA delivery. Compared to PEI 25 kDa, this novel gene delivery system demonstrated an approximately six-fold enhancement in endocytosis capability and maintain a higher cell viability. In vivo studies also showed good biosafety and anti-tumor effects, attribute to the positive charge of PEI and the hydrophobic and oleophobic properties of the fluorine-modified group. This study provides an effective gene delivery system for non-small-cell lung cancer treatment.

Tracking the early signals of crude oil in seawater and plankton after a major oil spill in the Red Sea

Understanding the immediate impacts of oil spills is essential to recognizing their long-term consequences on the marine environment. In this study, we traced the early (within one week) signals of crude oil in seawater and plankton after a major oil spill in October 2019 in the Red Sea. At the time of sampling, the plume had moved eastward, but we detected significant signs of incorporation of oil carbon into the dissolved organic carbon pool, resulting in a 10–20% increase in the ultraviolet (UV) absorption coefficient (a254) of chromophoric dissolved organic matter (CDOM), elevated oil fluorescence emissions, and depletion of the carbon isotope composition (δ13C) of the seawater. The abundance of the picophytoplankton Synechococcus was not affected, but the proportion of low nucleic acid (LNA) bacteria was significantly higher. Moreover, specific bacterial genera (Alcanivorax, Salinisphaera, and Oleibacter) were enriched in the seawater microbiome. Metagenome-assembled genomes (MAGs) suggested that such bacteria presented pathways for growing on oil hydrocarbons. Traces of polycyclic aromatic hydrocarbons (PAHs) were also detected in zooplankton tissues, revealing the rapid entry of oil pollutants into the pelagic food web. Our study emphasizes the early signs of short-lived spills as an important aspect of the prediction of long-term impacts of marine oil spills.

Abstract 1469: Visualization of endogenous EWSR1 in Ewing sarcoma cells reveals a regular nucleoplasmic organization that includes concentration at regions of active transcription

Abstract EWSR1, a member of the FET protein family, contains low complexity and nucleic acid binding domains and regulates transcription. Biochemical and depletion studies have demonstrated that EWSR1 regulates basal transcription, and that in Ewing sarcoma (EWS) cells expressing the fusion oncoprotein EWSR1::FLI1, EWSR1 may contribute to the deregulation of gene expression observed in this cancer. To further study EWSR1’s function in Ewing sarcoma, we generated reporter cell lines to visualize and quantify its endogenous expression within EWS cells. To generate EWSR1-fluorescent reporter A673 and TC-32 EWS cell lines, we used CRISPR-Cas9 and sgRNAs targeting the 5’ end of the first exon of EWSR1 and a donor plasmid consisting of the mNeonGreen (mNG) reporter gene, a G4S3 linker, and EWSR1 homology arm templates. Upon validation of successfully modified clones (A673 and TC-32), we used confocal microscopy to confirm EWSR1’s localization to the nucleoplasm. Also, consistent with previous analysis of EWSR1’s protein-protein interactions, we confirmed using immunoprecipitation that the mNG-EWSR1 protein retains its interaction with EWSR1::FLI1 and RNA polymerase II (RNA pol II) - total and phosphorylated (pS5 and pS2). FRAP analysis (Zeiss Airyscan) showed that in cells, EWSR1 exists in two fractions, one that rapidly exchanges (A673 T½: 9.47 secs; TC-32 T½: 6.13 secs) and one that remains bound for a longer duration (A673 T½: 92.15 secs; TC-32; T½: 52.63 secs). We next used structured illumination microscopy to interrogate EWSR1’s spatial organization in EWS cells at a high resolution (~100 nm). First, we determined that EWSR1’s organization within the nucleoplasm is regular as measured by a signal-to-signal distance of ~300 nm in both cell lines (A673 cells 297±6.71 nm; TC-32 cells 304±8.59 nm). Analysis of EWSR1’s spatial organization relative to nucleic acids and proteins that function in gene expression indicates that EWSR1 partially co-localizes with DNA (visualized using DAPI), Histone 3, BRD4 and MED1 (visualized using immunofluorescence - IF). In contrast, we observed EWSR1 colocalizes completely with nascent RNA (visualized using Click-IT RNA Alexa Fluor 488, Invitrogen) (Manders overlap coefficient (MOC) A673: 0.96±0.005; TC-32: 0.97±0.005). Furthermore, we detected over 90% colocalization of concentrated EWSR1 signals (defined by specific fluorescent intensities) with RNA pol II (IF) in states associated with transcriptional initiation (pS5) (MOC A673: 0.94±0.011; TC-32: 0.93±0.02) and elongation (pS2) (MOC A673: 0.95±0.02; TC-32: 0.97±0.06). These studies will aid analysis of changes in EWSR1’s localization within the nucleus and relative to proteins that regulate gene expression following inhibition of transcription, including the application of compounds that disrupt EWSR1::FLI1’s transcriptional activity. Citation Format: Soumya Sundara Rajan, Vernon Ebegboni, Tamara L. Jones, Patrick Pichling, Katelyn R. Ludwig, Raj Chari, Michael Kruhlak, Jadranka Loncarek, Natasha J. Caplen. Visualization of endogenous EWSR1 in Ewing sarcoma cells reveals a regular nucleoplasmic organization that includes concentration at regions of active transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1469.

Sustainable Bioconversion of Glycerine Pitch into the Novel Wickerhamomyces anomalus Bio-preservative

Background: Yeasts with high protein content are used as single-cell proteins due to their nutritional values and rare pathogenicity. However, the nucleic acid of the yeasts must be removed before consumption to avoid hazards to health. Wickerhamomyces anomalus is an authorized bio-preservative with promising antifungal activity and safety. However, its high protein content associ-ated with high nucleic acid content results in high nitrogen content that imposes additional down-stream processing costs due to the nucleic acid removal step required to prevent uric acid precipitation which leads to various health concerns. Objectives: The objectives were to (i) isolate a novel Wickerhamomyces anomalus strain with low nitrogen content and (ii) to optimize the production of W. anomalus biomass through response surface methodology (RSM). Methods: The novel Wickerhamomyces sp. USMAST-TP1 with low nitrogen content was isolated from fermented food and its biomass was optimized through RSM. Results: Wickerhamomyces sp. USMAST-TP1 showed promising tolerance to glycerine pitch with a higher composition of growth-inhibiting impurities where its biomass was not negatively affected by excessive glycerine pitch supply. Upon optimization through RSM, 11.4 g/L biomass harboring pro-tein content of 13% was achieved. Conclusion: The &gt;17-fold lower nitrogen content of 2% indicated low nucleic acid content compared to common W. anomalus strains, thus the costly nucleic acid purification steps can be excluded, easing applications in agro-food industries.

Microbial Community Abundance and Metabolism Close to the Ice-Water Interface of the Blomstrandbreen Glacier (Kongsfjorden, Svalbard): A Sampling Survey Using an Unmanned Autonomous Vehicle

Polar marine environments host a complex assemblage of cold-adapted auto- and heterotrophic microorganisms that affect water biogeochemistry and ecosystem functions. However, due to logistical difficulties, remote regions like those in close proximity to glaciers have received little attention, resulting in a paucity of microbiological data. To fill these gaps and obtain novel insights into microbial structure and function in Arctic regions, a survey of microbial communities in an area close to the Blomstrandbreen glacier in Kongsfjorden (Svalbard Archipelago; Arctic Ocean) was carried out during an early summer period. An Unmanned Autonomous Vehicle designed to safely obtain seawater samples from offshore-glacier transects (PROTEUS, Portable RObotic Technology for Unmanned Surveys) was equipped with an automatic remotely-controlled water multi-sampler so that it could sample just beneath the glacier, where access from the sea is difficult and dangerous. The samples were analysed by image analysis for the abundance of total prokaryotes, viable and respiring cells, their morphological traits and biomass; by flow cytometry for autotrophic and prokaryotic cells (with high and low nucleic acid contents) as well as virus-like particle counts; by BIOLOG ECOPLATES for potential community metabolism; and by fluorimetry for potential enzymatic activity rates on organic polymers. Contextually, the main physical and chemical (temperature, salinity, pH, dissolved oxygen and nutrients) parameters were detected. Altogether, besides the PROTEUS vehicle’s suitability for collecting samples from otherwise inaccessible sites, the multivariate analysis of the overall dataset allowed the identification of three main sub-regions differently affected by the haline gradient (close to the glacier) or terrigenous inputs coming from the coast. A complex microbiological scenario was depicted by different patterns of microbial abundance and metabolism among the transects, suggesting that ice melting and Atlantic water inflow differently supported microbial growth.