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Abstract Chinese hamster ovary (CHO) cells play a pivotal role in the production of recombinant therapeutics. In the present study, we conducted a genome-scale pooled CRISPR knockout (KO) screening using a virus-free, recombinase-mediated cassette exchange-based platform in CHO-K1 host and CHO-K1 derived recombinant cells. Genome-wide guide RNA (gRNA) amplicon sequencing data were generated from cell libraries, as well as short- and long-term KO libraries, and validated through phenotypic assessment and gRNA read count distribution. Additionally, we obtained gRNA amplicon sequencing data from the highly productive recombinant cell populations. By analyzing these datasets, essential genes involved in cell fitness as well as functional target genes associated with therapeutic protein production can be identified. Collectively, our next-generation sequencing datasets, derived from a robust and reliable CRISPR screening method, provide valuable insights into CHO genomic functions, advancing the development of next-generation CHO factories.

High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties.

The mineralization of organic matter at the sediment plays a crucial role in ecosystem functioning by facilitating the biogeochemical cycling of carbon and nutrients. This process not only supports nutrient availability for primary production but also regulates the long-term storage of carbon within sediments. To understand the biogeochemical processes associated with organic matter mineralization and nutrient regeneration, we estimated total and diffusive sediment oxygen uptake rates, benthic nutrient fluxes, and organic carbon (OC) budgets at four sites in the semi-enclosed Jinhae Bay (JB). The total oxygen uptake (TOU) rates ranged from 38.4 to 49.6 mmol O2 m–2 d–1, and diffusive oxygen uptake (DOU) rates ranged from 12.3 ± 1.8 to 15.1 ± 1.4 mmol O2 m–2 d–1. The average ratio of TOU : DOU ranged from 3.12 to 3.28 over JB, which suggests significant benthic faunal activities in JB sediments. The vertical flux of organic carbon ranged from 45.5 ± 7.0 to 93.0 ± 25.3 mmol C m-2 d–1, and mainly consisted of biodeposits associated with aquaculture activities. The burial flux into the sediment ranged from 3.96 ± 1.00 to 7.17 ± 1.64 mmol C m–2 d–1, and burial efficiencies were 4.25 to 15.8%, which indicated that deposited organic carbon was either mineralized in surface sediment before burial or laterally transferred by resuspension. The benthic nutrient fluxes at four sites ranged from 1.50 to 2.07 mmol m–2 d–1 for nitrogen, from 0.02 to 0.05 mmol m–2 d–1 for phosphate, and from 6.72 to 9.11 mmol m–2 d–1 for silicate. The benthic nitrogen and phosphate fluxes accounted for 82.1 to 149% and 23.1 to 57.6%, respectively, of the required levels for primary production in the water column. Our results suggest that OC oxidation in the JB sediment may significantly contribute to the biogeochemical OC cycles and tight benthic–pelagic coupling associated with nutrient regeneration.

Vascular tissue engineering faces significant challenges in creating in vitro vascular disease models, implantable vascular grafts, and vascularized tissue/organ constructs due to limitations in manufacturing precision, structural complexity, replicating the composited architecture, and mimicking the mechanical properties of natural vessels. Light-based 3D bioprinting, leveraging the unique advantages of light including high resolution, rapid curing, multi-material adaptability, and tunable photochemistry, offers transformative solutions to these obstacles. With the emergence of diverse light-based 3D bioprinting techniques and innovative strategies, the advances in vascular tissue engineering have been significantly accelerated. This review provides an overview of the human vascular system and its physiological functions, followed by an in-depth discussion of advancements in light-based 3D bioprinting, including light-dominated and light-assisted techniques. We explore the application of these technologies in vascular tissue engineering for creating in vitro vascular disease models recapitulating key pathological features, implantable blood vessel grafts, and tissue analogs with the integration of capillary-like vasculatures. Finally, we provide readers with insights into the future perspectives of light-based 3D bioprinting to revolutionize vascular tissue engineering.

Abstract 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.

Abstract 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-1β (IL-1β), and interleukin-6 (IL-6) in comparison to all other exposed groups. Meanwhile, total antioxidant capacity (TAC) showed a significant (p

Abstract We measured dissolved organic carbon (DOC) and total dissolved amino acid (TDAA) in seawater and sediment porewater of the Ulleung Basin in the East/Japan Sea. The DOC and TDAA concentrations were 1.1‐ and 1.4‐fold higher in the euphotic zone, and 11‐ and 43‐fold higher in sediment porewater, respectively, than those in the deep ocean. Consequently, in the deep ocean, TDAA and DOC input fluxes from porewater were 2‐ and 0.4‐fold of those from the euphotic zone, respectively. This larger contribution of benthic flux for TDAA and its shorter residence time in the benthic boundary layer (BBL) (1.3 ± 0.9 yr) seem to result in steep TDAA increases in the BBL, although DOC concentrations remained relatively uniform throughout the entire deep ocean. AA‐derived indices also show enhanced bioavailability of dissolved organic matter in the BBL. Benthic inputs seem to supply a significant amount of bioavailable TDAA to the deep ocean, fueling microbial activity.

Abstract Cerebral vessels are composed of highly complex structures that facilitate blood perfusion necessary for meeting the high energy demands of the brain. Their geometrical complexities alter the biophysical behavior of circulating tumor cells in the brain, thereby influencing brain metastasis. However, recapitulation of the native cerebrovascular microenvironment that shows continuities between vascular geometry and metastatic cancer development has not been accomplished. Here, we apply an in-bath 3D triaxial bioprinting technique and a brain-specific hybrid bioink containing an ionically crosslinkable hydrogel to generate a mature three-layered cerebrovascular conduit with varying curvatures to investigate the physical and molecular mechanisms of cancer extravasation in vitro. We show that more tumor cells adhere at larger vascular curvature regions, suggesting that prolongation of tumor residence time under low velocity and wall shear stress accelerates the molecular signatures of metastatic potential, including endothelial barrier disruption, epithelial–mesenchymal transition, inflammatory response, and tumorigenesis. These findings provide insights into the underlying mechanisms driving brain metastases and facilitate future advances in pharmaceutical and medical research.

Abstract Human skin is an organ located in the outermost part of the body; thus, it frequently exhibits visible signs of physiological health. Ethical concerns and genetic differences in conventional animal studies have increased the need for alternative in vitro platforms that mimic the structural and functional hallmarks of natural skin. Despite significant advances in in vitro skin modeling over the past few decades, different reproducible biofabrication strategies are required to reproduce the pathological features of diseased human skin compared to those used for healthy-skin models. To explain human skin modeling with pathological hallmarks, we first summarize the structural and functional characteristics of healthy human skin. We then provide an extensive overview of how to recreate diseased human skin models in vitro, including models for wounded, diabetic, skin-cancer, atopic, and other pathological skin types. We conclude with an outlook on diseased-skin modeling and its technical perspective for the further development of skin engineering.

Abstract Potassium oxide (K2O) is used as a promotor in industrial ammonia synthesis, although metallic potassium (K) is better in theory. The reason K2O is used is because metallic K, which volatilizes around 400 °C, separates from the catalyst in the harsh ammonia synthesis conditions of the Haber-Bosch process. To maximize the efficiency of ammonia synthesis, using metallic K with low temperature reaction below 400 °C is prerequisite. Here, we synthesize ammonia using metallic K and Fe as a catalyst via mechanochemical process near ambient conditions (45 °C, 1 bar). The final ammonia concentration reaches as high as 94.5 vol%, which was extraordinarily higher than that of the Haber-Bosch process (25.0 vol%, 450 °C, 200 bar) and our previous work (82.5 vol%, 45 °C, 1 bar).

The temperature–size rule (TSR) has been consistently observed in numerous studies, showing that ectotherms reared at higher temperatures experience accelerated growth during the juvenile stage and ultimately reach smaller sizes and younger ages at maturity. One explanation for this response is that it occurs when the effect of temperature on mortality, including predation, outweighs its effect on food intake and metabolism. While several studies have found that the latter effect is close to the expected result based on the Q10 = 2 assumption, confirmation of this hypothesis requires evidence that the effect of temperature on mortality exceeds the Q10 = 2 threshold. To test this hypothesis, we conducted experiments with two fish species: rudd and Malabar danio. We examined the capture rate, which serves as a proxy for mortality, as well as the standard metabolic rate (SMR) and several parameters characterising the mobility of the fish and their planktonic prey (Daphnia) at different temperatures. The results strongly supported our hypothesis, as the capture rate increased significantly more than expected based on the Q10 = 2 assumption, especially for the danio. This substantial effect cannot be attributed solely to the thermal sensitivity of the SMR, as the Q10 for the SMR was only around 2. The most likely explanation seems to be a much more pronounced increase in the fish’s mobility and resulting reaction field volume compared to its planktonic prey at elevated temperatures. This increased mobility leads to an improved attack rate by the fish, which exceeds the prediction made by the Q10 = 2 assumption. This mechanism may explain not only the TSR pattern in zooplankton, but also their reduced mean body size and density at population and community levels at elevated temperatures, and may hypothetically be observed at other predator–prey interfaces.

Abstract CRISPR/Cas9‐mediated targeted gene integration (TI) has been used to generate recombinant mammalian cell lines with predictable transgene expression. Identifying genomic hot spots that render high and stable transgene expression and knock‐in (KI) efficiency is critical for fully implementing TI‐mediated cell line development (CLD); however, such identification is cumbersome. In this study, we developed an artificial KI construct that can be used as a hot spot at different genomic loci. The ubiquitous chromatin opening element (UCOE) was employed because of its ability to open chromatin and enable stable and site‐independent transgene expression. UCOE KI cassettes were randomly integrated into CHO‐K1 and HEK293T cells, followed by TI of enhanced green fluorescent protein (EGFP) onto the artificial UCOE KI site. The CHO‐K1 random pool harboring 5′2.2A2UCOE‐CMV displayed a significant increase in EGFP expression level and KI efficiency compared with that of the control without UCOE. In addition, 5′2.2A2UCOE‐CMV showed improved Cas9 accessibility in the HEK293T genome, leading to an increase in indel frequency and homology‐independent KI. Overall, this assessment revealed the potential of UCOE KI constructs as artificial integration sites in streamlining the screening of high‐production targeted integrants by mitigating the selection of genomic hot spots.

In this study, we propose a lightweight and low-latency advanced encryption standard (AES) accelerator. Instead of being connected to the bus through its own slave wrapper, the proposed AES accelerator is located within the slave wrapper of the static random-access memory (SRAM) and is directly attached to the SRAM. Hence, the AES accelerator can directly access data in the SRAM and share SRAM space for storing expanded keys, resulting in no time for transferring input and output data, no resource usage for storing keys, and no power wastage for repeated key expansion. The proposed AES accelerator has a latency of 53 clock cycles per encryption/decryption process and has a gate count of 2912 when synthesized using 28 nm process technology. The latency is similar to that of another AES accelerator with the same 32-bit data path; however, the size of the proposed accelerator is 46.0% smaller. Furthermore, compared with other AES accelerators with 8-bit data path, the proposed AES accelerator has a 3.0–22.0 times smaller latency with a slightly larger area.

This study assessed the impact of intensive human activities on organic matter (OM) and heavy metal cycles in Shihwa Lake, South Korea. Sediment oxygen demand (SOD), benthic nutrient flux (BNF), and benthic heavy metal flux were estimated using in situ benthic chambers. The combined analysis of sediment trap and SOD showed that the vertical supply of OM was a major controlling factor for benthic respiration. The BNF accounted for 35–144% and 32–184% of the N and P required, respectively, for primary production (PP) in the water column. The higher SOD may have also accelerated the release of Mn, Fe, Co, and Ni from the sediment. Benthic fluxes of Cr, As, Cd, Pb, Cu, and Zn were highest near the industrial complex, with ranges of 1.3 ± 0.9, 6.4 ± 4.9, 0.2 ± 0.1, 0.5 ± 0.4, 7.7 ± 1.4, and 452 ± 133 μmol m−2 d−1, respectively. Mn, Fe, Co, As, Pb, Ni, and Cu contributed more than 10% of the sediment to the current standing stock at Shihwa Lake.

Using common carp as a model, we assessed the effects of polyethylene (PE) plastics on the brain. We measured activity of acetylcholinesterase (AChE), monoamine oxidase (MAO), and the content of nitric oxide (NO) in carp brain following exposure to 100 mg/L of either macroplastics (MaP), microplastics (MPs), or nanoplastic (NPs) for 15 days compared to an unexposed group. Following exposure, each biochemical biomarker was reduced 30–40%, with a higher magnitude of change corresponding to the smaller size of the particles (NPs > MPs > MaPs). In the carp tectum, exposure for 15 days to plastic particles caused varying degrees of necrosis, fibrosis, changes in blood capillaries, tissue detachment, edema, degenerated connective tissues, and necrosis in large cerebellar neurons and ganglion cells. In the carp retina, there was evidence for necrosis, degeneration, vacuolation, and curvature in the inner layer. Here we provide evidence that exposure to plastic particles can be associated with neurotoxicity in common carp.

Site-specific integration has emerged as a promising strategy for streamlined and predictable Chinese hamster ovary (CHO) cell line development (CLD). However, the low specific productivity of the targeted integrants limits their practical application. In this study, we developed a hybrid CLD platform combining site-specific integration of a transgene and dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification to generate high-producing recombinant CHO cell lines. We used the CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform to integrate the DHFR expression cassette and transgene landing pad into a CHO genomic hot spot, C12orf35 locus, of DHFR-knockout CHO-K1 host cell lines. When subjected to various MTX concentrations up to 1 μM, EGFP-expressing targeted integrants showed a 3.6-fold increase in EGFP expression in the presence of 200 nM MTX, accompanied by an increase in the DHFR and EGFP copy number. A single-step 200 nM MTX amplification increased the specific monoclonal antibody (mAb) productivity (qmAb) of recombinant mAb-producing targeted integrants by 2.8-folds, reaching a qmAb of 9.1–11.0 pg/cell/day. Fluorescence in situ hybridization analysis showed colocalization of DHFR and mAb sequences at the intended chromosomal locations without clear amplified arrays of signals. Most MTX-amplified targeted integrants sustained recombinant mAb production during long-term culture in the absence of MTX, supporting stable gene expression in the amplified cell lines. Our study provides a new CLD platform that increases the productivity of targeted integrants by amplifying the transgene copies.

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-1β, 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.

We investigated changes in heterotrophic bacterial metabolic activities and associated carbon cycles in response to a change in dominant phytoplankton communities during two contrasting environmental conditions in austral summer in the Amundsen Sea polynya (ASP), Antarctica: the closed polynya condition in 2014 (ANA04) and the open polynya condition in 2016 (ANA06). In ANA04, Phaeocystis antarctica predominated phytoplankton biomass, comprising 78% of total phytoplankton carbon biomass, whereas diatoms and Dictyocha speculum accounted for 45% and 48% of total phytoplankton carbon biomass, respectively, in ANA06. Bacterial production (BP) showed a significant positive correlation with only chlorophyll-a (Chl-a, rho = 0.66, p < 0.001) in P. antarctica-dominated ANA04, whereas there were significant positive relationships of BP with various organic carbon pools, such as chromophoric dissolved organic matter (CDOM, rho = 0.84, p < 0.001), Chl-a (rho = 0.59, p < 0.001), and dissolved organic carbon (DOC, rho = 0.51, p = 0.001), in ANA06 when diatoms and D. speculum co-dominated. These results indicate that BP depended more on DOC directly released from P. antarctica in ANA04, but was supported by DOC derived from various food web processes in the diatom-dominated system in ANA06. The BP to primary production (BP : PP) ratio was three-fold higher in P. antarctica-dominated ANA04 (BP: PP = 0.09), than in diatom- and D. speculum-co-dominated ANA06 (BP : PP = 0.03). These results suggested that the microbial loop is more significant in Phaeocystis-dominated conditions than in diatom-dominated conditions. In addition, the decreases in BP : PP ratio and bacterial respiration with increasing diatom proportion in the surface mixed layer indicated that the change from P. antarctica to diatom predominance enhanced biological carbon pump function by increasing particulate organic carbon export efficiency. Consequently, our results suggest that bacterial metabolic response to shifts in phytoplankton communities could ultimately affect larger-scale ecological and biogeochemical processes in the water column of the ASP.

Although, the effects of 4-non-ylphenol (4-NP) on fish’s reproductive hormones were assessed in several studies using adult models, however, the effect of this endocrine disruptor on immature fish’s reproductive hormones was not addressed commonly. We aimed to study the apoptosis induction, hematotoxicity, reproductive toxicity, and the recovery associated with 4-NP exposure in juvenile African catfish [Clarias garepinus) using some hormones [17β-estradiol (E2), testosterone (T), follicle-stimulating hormone (FSH), and luteinizing hormone (LH)] and gonad histology as biomarkers. The toxic effects of 4-NP have been studied in many animal models, but there is still limited knowledge about the dose-dependent damage caused by 4-NP exposure in juvenile Clarias gariepinus. A healthy juvenile C. gariepinus was categorized into four groups (n = 3/group; three replicates in each group). The first group was the control, and the other three groups were subjected to 4-NP concentrations as 0.1, 0.2, and 0.3 mg/L for 15 days; they were left for a recovery period of another 15 days. The reproductive hormones of C. gariepinus exposed to 4-NP for 15 days exhibited significant variations between the treatment groups and the control (P < 0.05), which were evident in E2 and T-values, whereas FSH, LH, total protein, and lipid peroxidation values showed non-significant differences among all groups at P < 0.05. Such a situation referred to the fact that the 15-day recovery period was insufficient to remove the impacts of 4-NP doses in concern. The trend of dose-dependent increase/decrease was recorded for T, E2, FSH, and LH. The histopathological alterations of 4-NP treated in gonad tissues were recorded in juvenile C. gariepinus, reflecting their sensitivity to 4-NP estrogenic-like effects. Overall, our results investigate that recovery has improved the reproductive toxicity caused by 4-NP in juvenile C. garepinus. Significant variations between the treated groups and the control group (P < 0.05) were evident in hematological parameters except for hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). The parameters exhibiting significance decreased with such increased doses [red blood cells (RBCs), hematocrit (Hct), and white blood cells (WBCs)]. Similar patterns of significant variations toward the increase or decrease were recorded following the 15-day recovery period. Apoptotic frequency in erythrocytes and brain cells has increased significantly with increased 4-NP exposure, indicating that 4-NP caused cytotoxic effects, such as apoptosis in a dose-dependent manner. However, these cellular alterations greatly decreased after the 15-day recovery period.

The complete mitochondrial genomes of Brachionus manjavacas German strain were 10,721 bp (mitochondrial DNA I) and 12,274 bp (mitochondrial DNA II) in size, while the complete mitochondrial genomes of B. manjavacas Australian strain were 10,889 bp (mitochondrial DNA I) and 12,443 bp (mitochondrial DNA II) in size. Of 12 protein-coding genes (PCGs), 99.6% of amino acid sequences were identical between the two strains. Of 12 PCGs of both B. manjavacas strains, three genes (ND1, ATP6, and ND5) had incomplete stop codon T. Furthermore, ATA was the start codon for ND4, ND5, and CO3 genes, whereas that for other PCGs was ATG in both strains. The base compositions of 12 PCGs in the B. manjavacas strains were similar, indicating that the mitochondrial genome of the two strains was structurally conserved over evolution. The gene structure and its orientation of 12 PGCs of B. manjavacas strains were identical, as shown in other marine Brachionus rotifers and the freshwater Brachionus rotifers, while the freshwater rotifer B. calyciflorus had an additional cytochrome b gene in the mitochondrial DNA I.

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 β 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.

Medaka (Oryzias sp.) is an important fish species in ecotoxicology and considered as a model species due to its biological features including small body size and short generation time. Since Japanese medaka Oryzias latipes is a freshwater species with access to an excellent genome resource, the marine medaka Oryzias melastigma is also applicable for the marine ecotoxicology. In genome era, a high-density genetic linkage map is a very useful resource in genomic research, providing a means for comparative genomic analysis and verification of de novo genome assembly. In this study, we developed a high-density genetic linkage map for O. melastigma using restriction-site associated DNA sequencing (RAD-seq). The genetic map consisted of 24 linkage groups with 2,481 single nucleotide polymorphism (SNP) markers. The total map length was 1,784 cM with an average marker space of 0.72 cM. The genetic map was integrated with the reference-assisted chromosome assembly (RACA) of O. melastigma, which anchored 90.7% of the assembled sequence onto the linkage map. The values of complete Benchmarking Universal Single-Copy Orthologs were similar to RACA assembly but N50 (23.74 Mb; total genome length 779.4 Mb; gap 5.29%) increased to 29.99 Mb (total genome length 778.7 Mb; gap 5.2%). Using MapQTL analysis with SNP markers, we identified a major quantitative trait locus for sex traits on the Om10. The integration of the genetic map with the reference genome of marine medaka will serve as a good resource for studies in molecular toxicology, genomics, CRISPR/Cas9, and epigenetics.

Summary: Pooled CRISPR screens have been widely applied to mammalian and other organisms to elucidate the interplay between genes and phenotypes of interest. The most popular method for delivering the CRISPR components into mammalian cells is lentivirus based. However, because lentivirus is not always an option, virus-free protocols are starting to emerge. Here, we demonstrate an improved virus-free, genome-wide CRISPR screening platform for Chinese hamster ovary cells with 75,488 gRNAs targeting 15,028 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad, resulting in a very high percentage of single gRNA insertions and minimal clonal variation. Using this platform, we perform a negative selection screen on cell proliferation that identifies 1,980 genes that affect proliferation and a positive selection screen on the toxic endoplasmic reticulum stress inducer, tunicamycin, that identifies 77 gene knockouts that improve survivability. Motivation: Although lentivirus-based delivery of genome-wide CRISPR screen components has proven successful, there are situations in, e.g., industry and hospitals where working with live viruses is difficult or simply not an option. For those situations we have developed an alternative to virus-based, genome-wide CRISPR screens that retains compatibility with the software tools developed for analyzing the results, takes a similar amount of time, and offers improved signal-to-noise ratio.

Copepods represent an interesting alternative or a complement live food to brine shrimps and rotifers commonly used in aquaculture. They constitute the natural prey of many fish species and therefore do not require a potential nutritional enrichment. But an optimization of the microalgal diets used to feed copepods is essential to improve their mass culture. This study examined the effects of seven microalgal diets, namely single-species diets of Rhodomonas salina (R), Tisochrysis lutea (T), and Pavlova lutheri (=Diacronema lutheri) (P), two-species diets (R + T, T + P, and R + P), and a three-species diet (R + T + P), on the fatty acid and monosaccharide composition of the cyclopoid copepod Paracyclopina nana as well as its reproductive investment. Experiments were run during 15 days in 10-L beakers; starting with nauplii collected from a large 300-L batch culture. Copepods fatty acid contents were studied, particularly the relative amounts of docosahexaenoic acid (DHA) and eicosa-pentaenoic acid (EPA). The R + T, R, and T diets induced the highest total fatty acid amount in copepods. R + T and R also generated the lowest DHA/EPA ratios in copepods due to high EPA contents. The highest value of total monosaccharides was found in copepods fed with R + T + P. Diets R + T and R induced the greatest prosome volumes and clutch volumes in ovigerous females. Both prosome volume and clutch volume in P. nana ovigerous females were correlated to the individual EPA amount. The results demonstrated that all diets including R. salina enhanced the productivity of P. nana in mass culture, particularly when combined with T. lutea. R. salina, and T. lutea induced complementary fatty acid and monosaccharide profiles, confirming that R + T represents the best microalgae combination for productive culture of P. nana. Conversely, P. lutheri did not enhance the nutritional profile nor the fecundity of P. nana in the culture. This study is the first to demonstrate that R. salina is a suitable microalga for productive mass culture of P. nana for use as live food in aquaculture.

Background: The purpose of this article was to evaluate the effect of paracervical block before ultrasound-guided high-intensity-focused ultrasound treatment of adenomyosis and uterine fibroids. Methods: This retrospective analysis examined 2173 women who received HIFU treatment for uterine fibroids and adenomyosis, among them 311 patients with paracervical block and 1862 patients without paracervical block, from February 4, 2010 to March 8, 2019. We assessed treatment time, ablation time, treatment energy and volume reduction. Paracervical block (10 cc of 2% lidocaine diluted with epinephrine 1:1 million) was performed before treatment. Results: When paracervical block was performed, the total treatment time was estimated to be 0.886 times (expected (–0.121)) which was statistically significantly shorter (p < 0.0001), ablation time was statistically significantly shorter by 0.853 times (expected (–0.159)) (p < 0.0001), and the total energy was statistically significantly smaller by 0.891 times (expected (–0.115)) (p = 0.0003). There was no significant difference in volume change between the group with and without paracervical block. However, total treatment time, ablation time, and treatment energy were all statistically significantly lower in the group treated with paracervical block. Conclusions: Paracervical block before USgHIFU treatment was a cost-effective method because it helped reduce the total treatment time, ablation time, and total energy.

We investigated the photosynthetic characteristics of the crustose coralline alga Pneophyllum fragile (Corallinales, Rhodophyta) according to elevated water temperature and irradiance on the coast of Jeju in 2018. P. fragile was cultured under different temperature (11 °C, 21 °C, 26 °C, and 31 °C) and irradiance (0–1250 μmol photon m−2 s−1) conditions. Oxygen (O2) concentrations at the P. fragile mat–water interface (MWI) were measured using an O2 microsensor. At the MWI, the diffusive boundary layer thicknesses ranged from 200 to 400 μm. The O2 concentrations at the mat surface increased in response to increasing irradiance, and reached 344% air saturation. The maximum photosynthesis capacity (Pmax) and respiration rate in the dark (Rd) at 31 °C were about 3 times higher than those recorded at 11 °C. The compensation irradiance (Ec) and saturation irradiance (Ek) increased with increasing water temperature. The Pmax, Rd, and Ec were statistically correlated with temperature (p < 0.05). The Ek increased up to 833 μmol photon m−2 s−1 at 31 °C and exhibited a strong dependence on irradiance at high temperatures. The adaptability of P. fragile to high temperatures and strong irradiance was distinct from that observed for coralline algae in other temperate waters.

We measured oxygen (O2) fluxes in two major shallow subtidal benthic habitats (kelp bed (KB) and bare rock (BR) covered with crustose coralline algae) of Dokdo islet in the East Sea by applying noninvasive in-situ aquatic eddy covariance (AEC). The AEC device allows time series measurements (~24 h) of three-dimensional velocity (u, v, and w components) and high-resolution dissolved O2. This allows estimation of O2 exchange flux via benthic habitats. Local flow rates and irradiance levels were found to be major factors controlling O2 exchange flux in the rocky habitats. Gross primary production rates tended to be significantly higher in KB (163 mmol O2 m−2 d−1) than in BR (51 mmol O2 m−2 d−1). The net ecosystem metabolisms were assessed as opposite types, with 8 mmol O2 m−2 d−1 in KB (autotrophy) and –12 mmol O2 m−2 d−1 in BR (heterotrophy). Our results indicate that kelp beds are important for organic carbon cycling in rocky coastal waters and that AEC application to macroalgae habitats is a useful assessment approach.

We characterized the biogeochemical organic carbon (Corg) cycles in the surface sediment layer of the Ulleung Basin (UB) of the East Sea. The total oxygen uptake (TOU) rate and the diffusive oxygen uptake (DOU) rate of the sediment were measured using an autonomous in situ benthic lander equipped with a benthic chamber (KIOST BelcII) and a microprofiler (KIOST BelpII). The TOU rate was in the range of 1.51 to 1.93 mmol O2 m−2 d−1, about double the DOU rate. The high TOU/DOU ratio implies that the benthic biological activity in the upper sediment layer is one of the important factors controlling benthic remineralization. The in situ oxygen exposure time was about 20 days, which is comparable to the values of other continental margin sediments. The sedimentary Corg oxidation rates ranged from 6.4 to 6.5 g C m−2 yr−1, which accounted for ~2% of the primary production in UB. The Corg burial fluxes ranged from 3.14 ± 0.12 to 3.48 ± 0.60 g C m−2 yr−1, corresponding to more than 30% of the deposited Corg buried into the inactive sediment deep layer.

The complete mitochondrial genome was sequenced from the calanoid copepod Eurytemora affinis. The sequenced total genome size was 18,553 bp. The mitochondrial genome of E. affinis has 13 protein-coding genes (PCGs), two rRNAs, and 22 tRNAs. Of 13 PCGs, ND1, ND5, and ATP6 genes had incomplete stop codons TA–, T—, and TA–, respectively. Furthermore, the stop codons of the remaining eleven PCGs were TAG or TAA while the start codon of 13 PCGs was ATG (Cytb, ATP8, ATP6, and CO3 genes), ATT (CO1, ND2, ND3, ND4L, ND5, and ND6 genes), and ATA (ND1, ND4, and CO2 genes), respectively. The ratio of A + T and G + C nucleotides of 13 PCGs of E. affinis mitogenome showed 63.9% and 36.1%, respectively while those ratio of the entire sequences were 65.5% and 34.5%, respectively.

The two complete mitochondrial genomes were sequenced from the Netherlands strain of the freshwater monogonont rotifer Brachionus calyciflorus. The mitochondrial genome sequences were 27,698 bp and 9,906 bp in size, respectively. The gene order and contents of the two B. calyciflorus strains were mostly identical to one another, except for the additional identification and translocation of several tRNAs in mitochondrial DNA I and II. Of 13 protein-coding genes (PCGs), three genes (ND1, ND5, and ND3) had incomplete stop codons. Furthermore, the start codon of ND2, CO2, and CO3 and ND4 genes was ATT, GTG, and ATA, respectively, while the start codon of other PCGs was ATG. The base composition of 13 PCGs of B. calyciflorus (the Netherlands strain) mitogenome showed 31.1% for A, 37.6% for T, 16.5% for C, and 14.8% for G, respectively.

Abstract Background The mangrove killifish Kryptolebias marmoratus is the only vertebrate that reproduces by self-fertilizing and is an important model species in genetics and marine ecotoxicology. Using whole-genome and transcriptome sequences, we identified all members of the cytochrome P450 (CYP) family in this model teleost and compared them with those of other teleosts. Results A total of 74 cytochrome P450 genes and one pseudogene were identified in K. marmoratus. Phylogenetic analysis indicated that the CYP genes in clan 2 were most expanded, while synteny analysis with other species showed orthologous relationships of CYP subfamilies among teleosts. In addition to the CYP2K expansions, five tandem duplicated gene copies of CYP5A were observed. These features were unique to K. marmoratus. Conclusions These results shed a light on CYP gene evolution, particularly the co-localized CYP2K, CYP5A, and CYP46A subfamilies in fish. Future studies of CYP expression could identify specific endogenous and exogenous environmental factors that triggered the evolution of tandem CYP duplication in K. marmoratus.

The complete mitochondrial genome was sequenced from the marine water flea Diaphanosoma celebensis. The sequenced mitochondrial genome size was 17,060 bp, possessing identical gene order of 13 protein-coding genes (PGCs) to those of the congeneric freshwater species Diaphanosoma dubium in the genus Diaphanosoma. The mitochondrial genome of D. celebensis had 13 PGCs, two rRNAs, and 22 tRNAs. Of 13 PGCs, three genes (CO3, ND3, and ND4) had incomplete stop codons. Furthermore, the stop codons of the remaining ten PGCs were TAA (for CO1, ATP8, ATP6, ND5, ND6, and ND1) and TAG (for NL4L, Cytb, and ND2). The second and third base composition of codon on 9 PCGs on the L strand in D. celebensis mitogenome showed an anti-G bias (11.0% and 15.0%), respectively.

The two complete mitochondrial genomes were sequenced from the freshwater monogonont rotifer Brachionus angularis. The mitochondrial genome sequences were 10,764 bp (mitochondrial DNA I) and 12,238 bp (mitochondrial DNA II) in size, respectively. The gene structure and its orientation of 12 protein-coding genes (PCGs) of complete mitochondrial genomes of B. angularis was identical to those shown in other marine rotifers and the freshwater rotifer Brachionus rubens, but was different from the freshwater rotifer Brachionus calyciflorus. Of 12 PCGs, one gene (ND5) had incomplete stop codon. Furthermore, the start codon for CO1, ND4L, ND5, and CO2 was GTG, while the start codon for ND3 and other PCGs was ATA and ATG, respectively. The base composition of 12 PCGs in B. angularis mitogenome showed 20.4% for A, 47.3% for T, 17.5% for C, and 14.8% for G, respectively. The mitochondrial genome A + T base composition (67.7%) of 12 PCGs was higher than G + C (32.3%), while the complete mitochondrial genome A + T base composition (66.3%) was higher than G + C (33.7%).

The complete mitochondrial genome was sequenced from the marine monogonont rotifer Proales similis. The size of mitochondrial genome sequences was 16,819 bp and all the genes were unidirectional on the same strand. Of 12 protein-coding genes (PCGs), two genes (ND5 and ND2) had incomplete stop codons TG (for ND5) and T (for ND2). Furthermore, the start codon of all the genes was ATG, while the stop codons were TAA, TAG, and TGA. The base composition of 12 PCGs in P. similis mitogenome showed 20.8% for A, 47.0% for T, 17.4% for C, and 14.8% for G, respectively. The mitochondrial genome A + T base composition (67.8%) of 12 PCGs was higher than G + C (32.2%), while the complete mitochondrial genome A + T base composition (67.2%) was higher than G + C (32.8%).

The complete mitochondrial genome was sequenced from the NIES strain of the freshwater water flea Daphnia magna. The sequenced mitochondrial genome size of the NIES strain of D. magna was 17,432 bp, possessing identical gene order and the contents to those of the congeneric species Daphnia galeata in the genus Daphnia, but 16S and 12S rRNA genes was in opposite direction compared to other daphnid, while ND2 gene was also often found in opposite direction in the genus Daphnia. Overall, the mitochondrial genome of D. magna NIES strain has 13 protein-coding genes (PGCs), two rRNAs, and 22 tRNAs. Of 13 PGCs, four genes (CO1, CO2, ND5, and ND4) had incomplete stop codons. Furthermore, the stop codons of the remaining nine PGCs were TAG and TAA, while the start codons of CO1, ND6, and ND3 genes were ATT, ATT, and ATC, respectively. The base composition of D. magna NIES strain mitogenome shows an anti-G and –T bias (16.5% and 16.0%) on the L strand, respectively.

Mild hypothermia has been widely used to enhance transgene expression and improve the cellular productivity of mammalian cells. This study investigated mild hypothermia-responsive exogenous promoters in human embryonic kidney 293 (HEK293) cells using site-specific integration of various promoter sequences, including CMV, EF1α, SV40, and TK promoters, into the well-known genomic safe harbor site, AAVS1. EGFP expression driven by the CMV promoter increased up to 1.5-fold at 32 °C versus 37 °C under stable expression, while others showed no hypothermic response. Integration of short CMV variants revealed that the CMV-enhancer region is responsible for the positive hypothermic response. CMV-enhancer-specific transcription factors (TFs) were then predicted through in silico analysis and RNA-sequencing analysis, resulting in the selection of one TF, NKX3-1. At 37 °C, overexpression of NKX3-1 in recombinant HEK293 cells expressing EGFP through the CMV promoter (CMV-EGFP) increased EGFP expression up to 1.6-fold, compared with that in CMV-EGFP, the expression level of which was comparable to that of CMV-EGFP at 32 °C. Taken together, this work demonstrates promoter-dependent hypothermia responses in HEK293 cells and emphasizes interactions between endogenous TFs and promoter sequences.

Sediment oxygen demand (SOD) and benthic nutrient fluxes (BNFs) were measured using an in situ benthic chamber at a fish farm (FF), oyster farm (OF), and controls (FF-C and OF-C) to assess the impact of aquaculture activities on organic carbon (OC) and nutrients cycles in coastal waters of Korea. The SOD at FF and OF ranged from 60 ± 2 to 157 ± 3 mmol m−2 d−1 and from 77 ± 14 to 84 ± 16 mmol m−2 d−1, respectively, more than five times those of the control sites. The SOD at farm sites is highly correlated with fish stock and food input, suggesting that excess feed input is an important control factor for OC remineralization. The combined analysis of sediment trap and SOD indicates that most of the deposited OC oxidized in the sediment and/or was laterally transported by the current before being buried in the sediment. The benthic nutrient fluxes at farms ranged from 5.45 to 8.95 mmol N m−2 d−1 for nitrogen and from 0.51 to 1.67 mmol P m−2 d−1 for phosphate, respectively, accounting for 37–270% and 52–804% of the N and P required for primary production in the water column. These results indicate that aquaculture farming may profoundly impact biogeochemical cycles in coastal waters.

The two complete mitochondrial genomes were sequenced from the freshwater monogonont rotifer Brachionus rubens. The genome sequences were 12,041 bp and 13,793 bp in size, and the gene order and contents were identical to those of the freshwater rotifer B. rubens China, but were different in three tRNA-Arg, tRNA-Ile, and tRNA-Leu between both B. rubens mitochondrial genomes, while B. calyciflorus had peculiar gene order in mitochondrial DNA I. Of 12 protein-coding genes (PCGs), one gene (ND5) had incomplete stop codons. Furthermore, the start codon of ND4 and CO2 gene was ATT, while the start codon of other PCGs was ATG. The base composition of 12 PCGs in B. rubens mitogenome showed 22.5% for A, 46.5% for T, 16.3% for C, and 14.7% for G, respectively.

The two complete mitochondrial genomes were sequenced from the euryhaline monogonont rotifer Brachionus paranguensis. The mitochondrial genome sequences were 11,603 bp and 12,901 bp in size, and the gene order of 12 protein-coding genes (PCGs) were identical to those of the marine rotifer Brachionus plicatilis, but the positions of some tRNAs (e.g. tRNA-Ile, tRNA-Leu[TTA], tRNA-Phe, and tRNA-Leu[CTC]) of mitochondrial DNA I were different between B. paranguensis and B. plicatilis mitochondrial genomes. Of 12 PCGs, four genes (ND1, ATPase 6, ND5, and ND3) had incomplete stop codons. Furthermore, the start codon of ND4, ND5, and CO3 genes was ATT, while the start codon of other PCGs was ATG. The base composition of 12 PCGs in B. paranguensis mitochondrial genomes was 26.6% for A, 43.0% for T, 17.65% for C, and 12.75% for G, respectively.