Molecular mechanisms of embryonic tail development in the self-fertilizing mangrove killifish Kryptolebias marmoratus.

Using the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.

One-Pot Direct Mechanochemical Silicon Replacement of Sodium Fluorosilicate into Sodium Fluorozirconate and Functionalization of Graphite for Enhanced Sodium-Ion Storage.

Efficient sodium ion storage in graphite is as yet unattainable, because of the thermodynamic instability of sodium ion intercalates-graphite compounds. In this work, sodium fluorozirconate (Na3ZrF7, SFZ) functionalized graphite (SFZ-G) is designed and prepared by the in situ mechanochemical silicon (Si) replacement of sodium fluorosilicate (Na2SiF6, SFS) and functionalization of graphite at the same time. During the mechanochemical process, the atomic Si in SFS is directly replaced by atomic zirconium (Zr) from the zirconium oxide (ZrO2) balls and container in the presence of graphite, forming SFZ-G. The resulting SFZ-G, working as an anode material for sodium ion storage, shows a significantly enhanced capacity of 418.7 mAh g-1 at 0.1 C-rate, compared to pristine graphite (35 mAh g-1) and simply ball-milled graphite (BM-G, 200 mAh g-1). In addition, the SFZ-G exhibits stable sodium-ion storage performance with 86% of its initial capacity retention after 1000 cycles at 2.0 C-rate.

Revisiting the impact of genomic hot spots: C12orf35 locus as a hot spot and engineering target.

Traditional Chinese hamster ovary (CHO) cell line development is based on random integration (RI) of transgene that causes clonal variation and subsequent large-scale clone screening. Therefore, site-specific integration (SSI) of transgenes into genomic hot spots has recently emerged as an alternative method for cell line development. However, the specific mechanisms underlying hot spot site formation remain unclear. In this study, we aimed to generate landing pad (LP) cell lines via the RI of transgenes encoding fluorescent reporter proteins flanked by recombination sites to facilitate recombinase-mediated cassette exchange. The RI-based LP cell line expressing high reporter levels with spontaneous C12orf35 locus deletion exhibited similar reporter fluorescent protein levels compared to targeted integrants with an identical reporter LP construct at the CHO genome hot spot, the C12orf35 locus. Additionally, Resf1, a C12orf35 locus gene, knockout (KO) in the RI-based LP cell line with conserved C12orf35 increased reporter expression levels, comparable to those in cell lines with C12orf35 locus disruption. These results indicate that the effect of SSI into the C12orf35 locus, a genomic hot spot, on high-level transgene expression was caused by C12orf35 disruption. In contrast to C12orf35 KO, KO at other well-known hot spot sites at specific loci of genes, including Fer1L4, Hprt1, Adgrl4, Clcc1, Dop1b, and Ddc, did not increase transgene expression. Overall, our findings suggest that C12orf35 is a promising engineering target and a hot spot for SSI-based cell line development.

Changes in Induced-Antipredation Defense Traits and Transcriptome Regulations of Daphnia magna in Response to 5-HT1A Receptor Antagonist.

The serotonin signaling system plays a crucial role in regulating the ontogeny of crustaceans. Here, we describe the effects of different concentrations of the 5-hydroxytryptamine 1A receptor antagonist (WAY-100635) on the induced antipredation (Rhodeus ocellatus as the predator), morphological, behavioral, and life-history defenses of Daphnia magna and use transcriptomics to analyze the underlying molecular mechanisms. Our results indicate that exposure to WAY-100635 leads to changes in the expression of different defensive traits in D. magna when faced with fish predation risks. Specifically, as the length of exposure to WAY-100635 increases, high concentrations of WAY-100635 inhibit defensive responses associated with morphological and reproductive activities but promote the immediate negative phototactic behavioral defense of D. magna. This change is related to the underlying mechanism through which WAY-100635 interferes with gene expression of G-protein-coupled GABA receptors by affecting GABBR1 but promotes serotonin receptor signaling and ecdysteroid signaling pathways. In addition, we also find for the first time that fish kairomone can significantly activate the HIF-1α signaling pathway, which may lead to an increase in the rate of immediate movement. These results can help assess the potential impacts of serotonin-disrupting psychotropic drugs on zooplankton in aquatic ecosystems.

Non-negligible Toxicity to Fish in the Early Life Stages Triggered by Aqueous Leachate of Takeaway Plastic Containers.

Ordering takeout is a growing social phenomenon and may raise public health concerns. However, the associated health risk of compounds leaching from plastic packaging is unknown due to the lack of chemical and toxicity data. In this study, 20 chemical candidates were tentatively identified in the environmentally relevant leachate from plastic containers through the nontargeted chemical analysis. Three main components with high responses and/or predicted toxicity were further verified and quantified, namely, 3,5-di-tert-butyl-4-hydroxycinnamic acid (BHC), 2,4-di-tert-butylphenol (2,4-DTBP), and 9-octadecenamide (oleamide). The toxicity to zebrafish larvae of BHC, a degradation product of a widely used antioxidant Irganox 1010, was quite similar to that of the whole plastic leachate. In the same manner, RNA-seq-based ingenuity analysis showed that the affected canonical pathways of zebrafish larvae were quite comparable between BHC and the whole plastic leachate, i.e., highly relevant to neurological disease, metabolic disease, and even behavioral disorder. Longer-term exposure (35 days) did not cause any effect on adult zebrafish but led to decreased hatching rate and obvious neurotoxicity in zebrafish offspring. Collectively, this study strongly suggests that plastic containers can leach out a suite of compounds causing non-negligible impacts on the early stages of fish via direct or parental exposure.

Polystyrene nanoplastic and engine oil synergistically intensify toxicity in Nile tilapia, Oreochromis niloticus : Polystyrene nanoplastic and engine oil toxicity in Nile tilapia.

Polystyrene nanoplastic (PS-NPs) and Engine oil (EO) pose multiple ecotoxic effects with increasing threat to fish ecosystems. The current study investigated the toxicity of 15 days exposure to PS-NPs and / or EO to explore their combined synergistic effects on Nile tilapia, Oreochromis niloticus (O. niloticus). Hematobiochemical parameters, proinflammatory cytokines, and oxidative stress biomarkers as well as histological alterations were evaluated. The experimental design contained 120 acclimated Nile tilapia distributed into four groups, control, PS-NPs (5 mg/L), EO (1%) and their combination (PS-NPs + EO). After 15-days of exposure, blood and tissue samples were collected from all fish experimental groups. Results indicated that Nile tilapia exposed to PS-NPs and / or EO revealed a significant decrease in almost all the measured hematological parameters in comparison to the control, whereas WBCs and lymphocyte counts were significantly increased in the combined group only. Results clarified that the combined PS-NPs + EO group showed the maximum decrease in RBCs, Hb, MCH and MCHC, and showed the maximum significant rise in interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) in comparison to all other exposed groups. Meanwhile, total antioxidant capacity (TAC) showed a significant (p < 0.05) decline only in the combination group, whereas reduced glutathione (GSH) showed a significant decline in all exposed groups in comparison to the control. Both malondialdehyde (MDA) and aspartate aminotransferase (AST) showed a significant elevation only in the combination group. Uric acid showed the maximum elevation in the combination group than all other groups, whereas creatinine showed significant elevation in the EO and combination group when compared to the control. Furthermore, the present experiment proved that exposure to these toxicants either individually or in combination is accompanied by pronounced histomorpholgical damage characterized by severe necrosis and hemorrhage of the vital organs of Nile tilapia, additionally extensively inflammatory conditions with leucocytes infiltration. We concluded that combination exposure to both PS-NPs and EO caused severe anemia, extreme inflammatory response, oxidative stress, and lipid peroxidation effects, thus they can synergize with each other to intensify toxicity in fish.

Hyaluronic acid impacts hematological endpoints and spleen histological features in African catfish (Clarias gariepinus).

Since its identification in the vitreous humour of the eye and laboratory biosynthesis, hyaluronic acid (HA) has been a vital component in several pharmaceutical, nutritional, medicinal, and cosmetic uses. However, little is known about its potential toxicological impacts on aquatic inhabitants. Herein, we investigated the hematological response of Clarias gariepinus to nominal doses of HA. To achieve this objective, 72 adult fish were randomly and evenly distributed into four groups: control, low-dose (0.5 mg/l HA), medium-dose (10 mg/l HA), and high-dose (100 mg/l HA) groups for two weeks each during both the exposure and recovery periods. The findings confirmed presence of anemia, neutrophilia, leucopoenia, lymphopenia, and eosinophilia at the end of exposure to HA. In addition, poikilocytosis and a variety of cytomorphological disturbances were observed. Dose-dependent histological alterations in spleen morphology were observed in the exposed groups. After HA removal from the aquarium for 2 weeks, the groups exposed to the two highest doses still exhibited a notable decline in red blood cell count, hemoglobin concentration, mean corpuscular hemoglobin concentration, and an increase in mean corpuscular volume. Additionally, there was a significant rise in neutrophils, eosinophils, cell alterations, and nuclear abnormalities percentages, along with a decrease in monocytes, coupled with a dose-dependent decrease in lymphocytes. Furthermore, only the highest dose of HA in the recovered groups continued to cause a significant increase in white blood cells. White blood cells remained lower, and the proportion of apoptotic RBCs remained higher in the high-dose group. The persistence of most of the haematological and histological disorders even after recovery period indicates a failure of physiological compensatory mechanisms to overcome the HA-associated problems or insufficient duration of recovery. Thus, these findings encourage the inclusion of this new hazardous agent in the biomonitoring program and provide a specific pattern of hematological profile in HA-challenged fish. Further experiments are highly warranted to explore other toxicological hazards of HA using dose/time window protocols.

Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening.

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO-K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.

Endogenous BiP reporter system for simultaneous identification of ER stress and antibody production in Chinese hamster ovary cells.

As the biopharmaceutical industry expands, improving the production of therapeutic proteins using Chinese hamster ovary (CHO) cells is important. However, excessive and complicated protein production causes protein misfolding and triggers endoplasmic reticulum (ER) stress. When ER stress occurs, cells mediate the unfolded protein response (UPR) pathway to restore protein homeostasis and folding capacity of the ER. However, when the cells fail to control prolonged ER stress, UPR induces apoptosis. Therefore, monitoring the degree of UPR is required to achieve high productivity and the desired quality. In this study, we developed a fluorescence-based UPR monitoring system for CHO cells. We integrated mGFP into endogenous HSPA5 encoding BiP, a major ER chaperone and the primary ER stress activation sensor, using CRISPR/Cas9-mediated targeted integration. The mGFP expression level changed according to the ER stress induced by chemical treatment and batch culture in the engineered cell line. Using this monitoring system, we demonstrated that host cells and recombinant CHO cell lines with different mean fluorescence intensities (MFI; basal expression levels of BiP) possess a distinct capacity for stress culture conditions induced by recombinant protein production. Antibody-producing recombinant CHO cell lines were generated using site-specific integration based on host cells equipped with the BiP reporter system. Targeted integrants showed a strong correlation between productivity and MFI, reflecting the potential of this monitoring system as a screening readout for high producers. Taken together, these data demonstrate the utility of the endogenous BiP reporter system for the detection of real-time dynamic changes in endogenous UPR and its potential for applications in recombinant protein production during CHO cell line development.

Recombinase-mediated cassette exchange-based screening of a CRISPR/Cas9 library for enhanced recombinant protein production in human embryonic kidney cells: Improving resistance to hyperosmotic stress.

Targeted engineering of mammalian cells has been widely attempted to ensure the efficient production of therapeutic proteins with proper quality during bioprocesses. However, the identification of novel targets for cell engineering is labor-intensive and has not yet been fully substantiated. Here, we established a CRISPR/Cas9 library screening platform in human embryonic kidney (HEK293) cells based on guide RNA integration mediated by recombinase-mediated cassette exchange (RMCE) to interrogate gene function in a high-throughput manner. This platform was further advanced using a nuclear localization signal-tagged recombinase that increased RMCE efficiency by 4.8-fold. Using this platform, we identified putative target genes, such as CDK8, GAS2L1, and GSPT1, and their perturbation confers resistance to hyperosmotic stress that inhibits cell growth and induces apoptosis. Knockout of these genes in monoclonal antibody (mAb)-producing recombinant HEK293 (rHEK293) cells enhanced resistance to hyperosmotic stress-induced apoptosis, resulting in enhanced mAb production. In particular, GSPT1-knockout yielded 2.3-fold increase in maximum mAb concentration in fed-batch culture where hyperosmotic stress naturally occurs due to nutrient feeding. Taken together, this streamlined screening platform allows the identification of novel targets associated with hyperosmotic stress, enabling the development of stress-resistant cells producing recombinant proteins.

Improving the secretory capacity of CHO producer cells: The effect of controlled Blimp1 expression, a master transcription factor for plasma cells.

With the advent of novel therapeutic proteins with complex structures, cellular bottlenecks in secretory pathways have hampered the high-yield production of difficult-to-express (DTE) proteins in CHO cells. To mitigate their limited secretory capacity, recombinant CHO (rCHO) cells were engineered to express Blimp1, a master regulator orchestrating B cell differentiation into professional secretory plasma cells, using the streamlined CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform. The expression of Blimp1α or Blimp1ß in rCHO cells producing DTE recombinant human bone morphogenetic protein-4 (rhBMP-4) increased specific rhBMP-4 productivity (qrhBMP-4). However, since Blimp1α expression suppressed cell growth more significantly than Blimp1ß expression, only Blimp1ß expression enhanced rhBMP-4 yield. In serum-free suspension culture, Blimp1ß expression significantly increased the rhBMP-4 concentration (>3-fold) and qrhBMP-4 (>4-fold) without significant increase in hBMP-4 transcript levels. In addition, Blimp1ß expression facilitated mature rhBMP-4 secretion by active proteolytic cleavage in the secretory pathway. Transcriptomic profiling (RNA-seq) revealed global changes in gene expression patterns that promote protein processing in secretory organelles. In-depth integrative analysis of the current RNA-seq data, public epigenome/RNA-seq data, and in silico analysis identified 45 potential key regulators of Blimp1 that are consistently up- or down-regulated in Blimp1ß expressing rCHO cells and plasma cells. Blimp1ß expression also enhanced the production of easy-to-express monoclonal antibodies (mAbs) and modulated the expression of key regulators in rCHO cells producing mAb. Taken together, the results show that controlled expression of Blimp1ß improves the production capacity of rCHO cells by regulating secretory machinery and suggest new opportunities for engineering promising targets that are resting in CHO cells.

Identification and characterization of homeobox gene clusters in harpacticoid and calanoid copepods.

In this study, we have identified the entire complement of typical homeobox (Hox) genes (Lab, Pb, Dfd, Scr, Antp, Ubx, Abd-A, and Abd-B) in harpacticoid and calanoid copepods and compared them with the cyclopoid copepod Paracyclopina nana. The harpacticoid copepods Tigriopus japonicus and Tigriopus kingsejongensis have seven Hox genes (Lab, Dfd, Scr, Antp, Ubx, Abd-A, and Abd-B) and the Pb and Ftz genes are also present in the cyclopoid copepod P. nana. In the Hox gene cluster of the calanoid copepod Eurytemora affinis, all the Hox genes were present linearly in the genome but the Antp gene was duplicated. Of the three representative copepods, the P. nana Hox gene cluster was the most compact due to its small genome size. The Hox gene expression profile patterns in the three representative copepods were stage-specific. The Lab, Dfd, Scr, Pb, Ftz, and Hox3 genes showed a high expression in early developmental stages but Antp, Ubx, Abd-A, and Abd-B genes were mostly expressed in later developmental stages, implying that these Hox genes may be closely associated with the development of segment identity during early development.

Multi-biomarkers approach to assess the toxicity of novel insecticide (Voliam flexi®) on Clarias gariepinus: From behavior to immunotoxicity.

This study was conducted to determine for the first time the immunological, histopathological, histochemical, and ultrastructural changes; hematological and biochemical alterations; and poikilocytosis induced in Clarias gariepinus by Voliam flexi® 40% WG (thiamethoxam + chlorantraniliprole). Beside control fish, juvenile C. gariepinus were subjected to three sublethal concentrations of Voliam flexi® (43.5, 87.5, and 175 mg/L) for 15 days. Voliam flexi® induced immunotoxic impairments in C. gariepinus, such as a decrease in some immunity variables (lysozyme and phagocyte activity, immunoglobulin concentration, and nitro blue tetrazolium level). It also caused an extreme increase in the levels of primary cytokines (interleukin-1ß and IL-6), compared with the control. The toxic effects of Voliam flexi® increased gradually with the increasing concentrations tested. Histological examination of the liver demonstrated necrosis, vacuolated hepatocytes (fatty deposition), melanomacrophage centers, foci of inflammatory cells, congested and dilated blood sinusoids, hepatic degeneration, fibrosis increment (Sirius Red stain), and glycogen depletion, as well as cytopathological alterations. We conclude that the toxic effects of Voliam flexi® must be restricted or prevented by using control mechanisms in aquatic systems.

Environmentally relevant concentrations of microplastics modulated the immune response and swimming activity, and impaired the development of marine medaka Oryzias melastigma larvae.

Microplastics (MPs), due to their impacts on the ecosystem and their integration into the food web either through trophic transfer or ingestion directly from the ambient environment, are an emerging class of environmental contaminants posing a great threat to marine organisms. Most reports on the toxic effects of MPs exclusively focus on bioaccumulation, oxidative stress, pathological damage, and metabolic disturbance in fish. However, the collected information on fish immunity in response to MPs is poorly defined. In particular, little is known regarding mucosal immunity and the role of mucins. In this study, marine medaka (Oryzias melastigma) larvae were exposed to 6.0 µm beads of polystyrene microplastics (PS-MPs) at three environmentally relevant concentrations (102 particles/L, 104 particles/L, and 106 particles/L) for 14 days. The experiment was carried out to explore the developmental and behavioural indices, the transcriptional profiles of mucins, pro-inflammatory, immune, metabolism and antioxidant responses related genes, as well as the accumulation of PS-MPs in larvae. The results revealed that PS-MPs were observed in the gastrointestinal tract, with a concentration- and exposure time-dependent manner. No significant difference in the larval mortality was found between the treatment groups and the control, whereas the body length of larvae demonstrated a significant reduction at 106 particles/L on 14 days post-hatching. The swimming behaviour of the larvae became hyperactive under exposure to 104 and 106 particles/L PS-MPs. In addition, PS-MP exposure significantly up-regulated the mucin gene transcriptional levels of muc7-like and muc13-like, however down-regulated the mucin gene expression levels of heg1, muc2, muc5AC-like and muc13. The immune- and inflammation and metabolism-relevant genes (jak, stat-3, il-6, il-1ß, tnf-а, ccl-11, nf-κb, and sod) were significantly induced by PS-MPs at 104 and 106 particles/L compared to the control. Taken together, this study suggests that PS-MPs induced inflammation response and might obstruct the immune functions and retarded the growth of the marine medaka larvae even at environmentally relevant concentrations.

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes.

Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism.

Hydrophobic Surface Coating Can Reduce Toxicity of Zinc Oxide Nanoparticles to the Marine Copepod Tigriopus japonicus.

Coated zinc oxide nanoparticles (ZnO-NPs) are more commonly applied in commercial products but current risk assessments mostly focus on bare ZnO-NPs. To investigate the impacts of surface coatings, this study examined acute and chronic toxicities of six chemicals, including bare ZnO-NPs, ZnO-NPs with three silane coatings of different hydrophobicity, zinc oxide bulk particles (ZnO-BKs), and zinc ions (Zn-IONs), toward a marine copepod, Tigriopus japonicus. In acute tests, bare ZnO-NPs and hydrophobic ZnO-NPs were less toxic than hydrophilic ZnO-NPs. Analyses of the copepod's antioxidant gene expression suggested that such differences were governed by hydrodynamic size and ion dissolution of the particles, which affected zinc bioaccumulation in copepods. Conversely, all test particles, except the least toxic hydrophobic ZnO-NPs, shared similar chronic toxicity as Zn-IONs because they mostly dissolved into zinc ions at low test concentrations. The metadata analysis, together with our test results, further suggested that the toxicity of coated metal-associated nanoparticles could be predicted by the hydrophobicity and density of their surface coatings. This study evidenced the influence of surface coatings on the physicochemical properties, toxicity, and toxic mechanisms of ZnO-NPs and provided insights into the toxicity prediction of coated nanoparticles from their coating properties to improve their future risk assessment and management.

Microplastics habituated with biofilm change decabrominated diphenyl ether degradation products and thyroid endocrine toxicity.

Microplastics (MPs) are rapidly colonized by microbial biofilms in a natural aquatic environment, and the nature of the microbial community and type of MP can result in different degradation products of organic pollutants. Here, we quantified the degradation products of a ubiquitously detected pollutant, decabrominated diphenyl ether (BDE-209), under both light-only and biota conditions and in the absence or presence of three kinds of MPs, styrofoam polystyrene, hard polyamide, and polypropylene film. The results showed that the BDE-209 concentration increased by 0.7-2.8 fold in the presence of MPs, probably due to the "sustained release" desorption effect. Under light-only conditions, the penta- and hexa-BDE concentrations in the presence of styrofoam or hard MPs were significantly reduced, which can be deemed a beneficial effect. However, when biota were present, the debromination products increased with the addition of MPs, particularly in the presence of styrofoam MPs. These products caused a 1.7-fold upregulation in triiodothyronine content and a 5.9-fold upregulation of thyroid stimulating hormone ß expression in zebrafish larvae. The increase in debromination products could be attributed to the distinct high abundance of the bacteria Chloroflexi, Proteobacteria, and Basidiomycotina on styrofoam MPs that can participate in pollutant degradation. Collectively, our results indicate that MPs can alter the degradation pathways of BDE-209 and increase the toxicity to the endocrine system and the thyroid in aquatic organisms.

Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering.

The impact of the pandemic declaration on public awareness and behavior: Focusing on COVID-19 google searches.

The unprecedented outbreaks of epidemics such as the coronavirus has caused major socio-economic changes. To analyze public risk awareness and behavior in response to the outbreak of epidemic diseases, this study focuses on RSV (Relative Search Volume) provided by Google Trends. This study uses the social big data provided by Google RSV to investigate how the WHO's pandemic declaration affected public awareness and behavior. 37 OECD countries were analyzed and clustered according to the degree of reaction to the declaration, and the United States, France and Germany were selected for comparative study. The results of this study statistically confirmed that the pandemic declaration increased public awareness and had the effect of increasing searches for information on COVID-19 by more than 20%. In addition, this rapid rise in RSV also reflected interest in the COVID-19 test and had the effect of inducing individuals to be tested, which helped identify new cases. The significance of this study is that it provided the theoretical foundation for using RSV and its implications to understand and strategically utilize public awareness and behavior in situations where the WHO and governments must launch policies in response to the outbreak of new infectious diseases such as COVID-19.

Optimized CRISPR/Cas9 strategy for homology-directed multiple targeted integration of transgenes in CHO cells.

Site-specific integration has emerged as a promising strategy for precise Chinese hamster ovary (CHO) cell line engineering and predictable cell line development (CLD). CRISPR/Cas9 with the homology-directed repair (HDR) pathway enables precise integration of transgenes into target genomic sites. However, inherent recalcitrance to HDR-mediated targeted integration (TI) of transgenes results in low targeting efficiency, thus requiring a selection process to find a targeted integrant in CHO cells. Here, we explored several parameters that influence the targeting efficiency using a promoter-trap-based single- or double-knock-in (KI) monitoring system. A simple change in the donor template design by the addition of single-guide RNA recognition sequences strongly increased KI efficiency (2.9-36.0 fold), depending on integration sites and cell culture mode, compared to conventional circular donor plasmids. Furthermore, sequential and simultaneous KI strategies enabled us to obtain populations with ~1-4% of double-KI cells without additional enrichment procedures. Thus, this simple optimized strategy not only allows efficient CRISPR/Cas9-mediated TI in CHO cells but also paves the way for the applicability of multiplexed KIs in one experimental step without the need for sequential and independent CHO-CLD procedures.