Genome editing of FTR42 improves zebrafish survival against virus infection by enhancing IFN immunity.

The development of CRISPR-Cas9 technology introduces an efficient tool for precise engineering of fish genomes. With a short reproduction cycle, zebrafish infection mode can be referenced as antiviral breeding researches in aquaculture fish. Previously we identified a crucian carp-specific gene ftrca1 as an inhibitor of interferon response in vitro. Here, we demonstrate that genome editing of zebrafish ftr42, a homolog of ftrca1, generates a zebrafish mutant (ftr42lof/lof) with an improved resistance to SVCV infection. Zebrafish ftr42 acts as a virus-induced E3 ligase and downregulates IFN antiviral response by facilitating TBK1 protein degradation and also IRF7 mRNA decay. Genome editing results in loss of function of zebrafish ftr42, which enables zebrafish to have enhanced interferon response, thus improving zebrafish survival against virus infection. Our results suggest that fine-tuning fish IFN innate immunity through genome editing of negative regulators can genetically improve viral resistance in fish.

In vitro evolution of diagnostic antibodies targeting native antigens in plasma by sandwich flow cytometry.

Monoclonal antibodies (mAbs) that recognize and bind to specific antigens (Ags) have a wide range of applications in research, therapy, and diagnostics. However, many of these antibodies cannot bind well to the native Ags. In this study, based on the Chinese hamster ovary (CHO) cell display platform developed previously in our lab, we reported a novel artificial evolution procedure to improve the affinity of mAb against the native Ag directly using the plasma samples without purification of the native Ag. In this procedure, a pair of antibodies able to bind the Ag in sandwich manner are first confirmed (Ab1/Ab2) and the antibody (Ab) to be affinity-improved (Ab1) is displayed on CHO cells for Ab mutation. Then the cells were detected and sorted with flow cytometry in the form of Ab1-Ag-fluorescence labeled Ab2, which we named sandwich flow cytometry. Here, we used soluble isoform of suppression of tumorigenicity 2 (sST2) protein as model Ag, carried out "sandwich" maturation directly using the plasma samples containing the native sST2 protein and optimized a pair of antibodies with significantly improved sensitivity in the detection of the native sST2 in plasma. This method could be very useful in optimization of the diagnostic Ab pairs working in a "sandwich" manner if more antibodies were also successfully affinity-matured with this method.

A zebrafish NLRX1 isoform downregulates fish IFN responses by targeting the adaptor STING.

In mammals, NLRX1 is a unique member of the nucleotide-binding domain and leucine-rich repeat (NLR) family showing an ability to negatively regulate IFN antiviral immunity. Intron-containing genes, including NLRX1, have more than one transcript due to alternative splicing; however, little is known about the function of its splicing variants. Here, we identified a transcript variant of NLRX1 in zebrafish (Danio rerio), termed NLRX1-tv4, as a negative regulator of fish IFN response. Zebrafish NLRX1-tv4 was slightly induced by viral infection, with an expression pattern similar to the full-length NLRX1. Despite the lack of an N-terminal domain that exists in the full-length NLRX1, overexpression of NLRX1-tv4 still impaired fish IFN antiviral response and promoted viral replication in fish cells, similar to the full-length NLRX1. Mechanistically, NLRX1-tv4 targeted STING for proteasome-dependent protein degradation by recruiting an E3 ubiquitin ligase RNF5 to drive the K48-linked ubiquitination, eventually downregulating the IFN antiviral response. Mapping of NLRX1-tv4 domains showed that its N-terminal and C-terminal regions exhibited a similar potential to inhibit STING-mediated IFN antiviral response. Our findings reveal that like the full-length NLRX1, zebrafish NLRX-tv4 functions as an inhibitor to shape fish IFN antiviral response.IMPORTANCEIn this study, we demonstrate that a transcript variant of zebrafish NLRX1, termed NLRX1-tv4, downregulates fish IFN response and promotes virus replication by targeting STING for protein degradation and impairing the interaction of STING and TBK1 and that its N- and C-terminus exhibit a similar inhibitory potential. Our results are helpful in clarifying the current contradictory understanding of structure and function of vertebrate NLRX1s.

Study on Antibacterial Activity and Mechanism of Improved Dian Dao San Against Cutibacterium acnes (C. acnes).

Purpose: The hyperproliferation of C. acnes has long been regarded as a primary etiological factor in the development of acne vulgaris (AV). Antibiotics targeting C. acnes have been the mainstay in AV treatment. Meanwhile, C. acnes has developed resistance to numerous antibiotics. IDDS, as traditional Chinese medicine, exhibits potent antibacterial activity against C. acnes. However, the mechanism of IDDS against C. acnes remains unclear. Methods: In this study, we conducted a systematic investigation in vitro to determine the minimal bactericidal concentration (MBC) and time-kill curves. The MBC and time-kill curves were assessed by quantifying Colony Forming Units countsIn order to establish an in vivo rat ear model of acne, a single intradermal injection of 100µL C. acnes suspension was administered, and oleic acid was applied to the right ear pinna for a duration of 14 days. The intervention involved the utilization of IDDS medications. Additionally, the levels of inflammatory mediators tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) were assessed using respective ELISA kits, while Hematoxylin and eosin (HE) staining was employed to visualize the rat ear model. The antimicrobial mechanism was investigated through the analysis of mRNA levels using real-time, quantitative PCR. ELISA analysis was performed according to the protocols outlined for energy metabolism and antioxidant system. Results: Our research has demonstrated that IDDS possesses antibacterial activity against C. acnes both in vitro and in vivo. The mechanisms underlying these effects involve energy metabolism and antioxidant systems. Conclusion: The data has provided further insights into the mechanism of IDDS against C. acnes, which establishes a robust foundation for the clinical application of IDDS.

LINC01354 enhances the metastatic ability of gastric cancer cells by adjusting miR-153-5p/CADM2 expression.

LINC01354 is a long non-coding RNA (lncRNA) highly expressed in gastric cancer (GC). However, studies have shown that it plays a critical role in the progression of other tumors. This study attempts to uncover the role of LINC01354 in GC. LINC01354 expression in GC tissues and cell lines was assessed using qRT-PCR. Subsequently, LINC01354 knockdown and overexpression were induced in GC cells, and epithelial-mesenchymal transition (EMT) progression was detected. A dual-luciferase reporter assay was used to assess the relation between LINC01354, miR-153-5p, and CADM2. Finally, the metastatic ability of GC cells was assessed by Transwell and wound healing assays. LINC01354 expression was abnormally elevated in cancerous tissues and GC cells, and LINC01354 knockdown suppressed EMT progression, migration, and invasion of GC cells. Transfection of miR-153-5p mimics inhibited the expression of CADM2 by banding to the 3'UTR region, while LINC01354 promoted CADM2 expression by blocking miR-153-5p. The fluorescence experiment indicated that CADM2 is directly regulated by LINC01354/miR-153-5p. Overexpression of LINC01354 promoted EMT progression, migration, and invasion of GC cells, which could be absolutely reversed by co-expression of miR-153-5p. Our research demonstrates that LINC01354 has an important function in the EMT progression of GC cells. LINC01354 promotes GC cell migration and invasion by adjusting miR-153-5p/CADM2 expression.

Evaluation of changes in M. aeruginosa growth and microcystin production under phosphorus starvation via transcriptomic surveys.

Phosphorus (P) is an important nutrient for the growth and metabolism of algae. Although P typically limits the growth of algae, little is known regarding the molecular response of Microcystis aeruginosa under P starvation. The transcriptomic and physiological responses of Microcystis aeruginosa to P starvation were investigated in this study. P starvation affected the growth, photosynthesis, and Microcystin (MC) production of Microcystis aeruginosa and triggered cellular P-stress responses for 7 days. In terms of physiology, P starvation inhibited the growth and MC production, while the slight promotion of photosynthesis in Microcystis aeruginosa compared to P-replete. For transcriptome, the down-regulation of genes related to MC production controlled by mcy genes and ribosome metabolism (17 genes encoding ribosomal proteins) was observed while transport genes (sphX and pstSAC) were significantly upregulated. In addition, some other genes are related to photosynthesis and the use of other forms of P displayed increases or decreases in transcripts abundance. These results suggested that the limitation of P had a diverse performance on aspects of growth and metabolism in M. aeruginosa and obviously enhanced the ability to adapt to the P stress environment. They provide a comprehensive understanding of the P physiology of Microcystis aeruginosa and theoretical support for eutrophication.

The application of adenine deaminase in antibody affinity maturation.

Previously, we established a platform for antibody/protein affinity maturation based on CHO cell display. The gene of interest was mutated by activation-induced cytidine deaminase (AID), and then, a mutation library mainly containing G/C to A/T conversion was obtained by simply proliferating cells. However, the AID-induced G/C to A/T conversion limits the diversity space of the mutation library. In contrast to AID, adenine deaminase (ADA) can convert A/T to G/C. In this study, we demonstrated that ADA could efficiently induce random A/T to G/C mutations on the target gene in the CHO cell display and could be applied in affinity maturation. Our data also showed that more mutant types were obtained through the combined use of AID and ADA, thus offering an opportunity to acquire new mutants offering higher affinities than those obtained by only using AID. Examples presented in this study showed that ADA contributed to the improvement of antibody affinity either with or without AID in CHO display. KEY POINTS: • ADA is able to induce random mutations on antibody gene in mammalian cells. • ADA induces mutations on A/T bases to compensate AID which can induce mutation on G/C. • Combination of AID and ADA can increase mutation types and maturation efficiencies.

Antibacterial Mechanism of Patrinia scabiosaefolia Against Methicillin Resistant Staphylococcus epidermidis.

Purpose: Staphylococcus epidermidis has become one of the most common causes of septicemia. Meanwhile, S. epidermidis has acquired resistance to many antibiotics. Among these, methicillin-resistant S. epidermidis (MRSE) were frequently isolated. Similar to methicillin resistant Staphylococcus aureus (MRSA), they also exhibited multi-resistance, which presented a danger to human health. Patrinia scabiosaefolia as traditional Chinese medicine had strong antibacterial activity against MRSE. However, the mechanism of P. scabiosaefolia against MRSE is not clear. Methods: Here, the morphology of cell wall and cell membrane, production of ß-lactamase and PBP2, energy metabolism, antioxidant system were systematically studied. Results: The data showed that P. scabiosaefolia damaged the cell wall and membrane. In addition, ß-lactamase, energy metabolism and antioxidant system were involved in mechanisms of P. scabiosaefolia against MRSE. Conclusion: These observations provided new understanding of P. scabiosaefolia against MRSE to control MRSE infections.

Zebrafish HERC7c Acts as an Inhibitor of Fish IFN Response.

In humans, four small HERCs (HERC3-6) exhibit differential degrees of antiviral activity toward HIV-1. Recently we revealed a novel member HERC7 of small HERCs exclusively in non-mammalian vertebrates and varied copies of herc7 genes in distinct fish species, raising a question of what is the exact role for a certain fish herc7 gene. Here, a total of four herc7 genes (named HERC7a-d sequentially) are identified in the zebrafish genome. They are transcriptionally induced by a viral infection, and detailed promoter analyses indicate that zebrafish herc7c is a typical interferon (IFN)-stimulated gene. Overexpression of zebrafish HERC7c promotes SVCV (spring viremia of carp virus) replication in fish cells and concomitantly downregulates cellular IFN response. Mechanistically, zebrafish HERC7c targets STING, MAVS, and IRF7 for protein degradation, thus impairing cellular IFN response. Whereas the recently-identified crucian carp HERC7 has an E3 ligase activity for the conjugation of both ubiquitin and ISG15, zebrafish HERC7c only displays the potential to transfer ubiquitin. Considering the necessity for timely regulation of IFN expression during viral infection, these results together suggest that zebrafish HERC7c is a negative regulator of fish IFN antiviral response.

Exploring the active ingredients and potential mechanisms of action of sinomenium acutum in the treatment of rheumatoid arthritis based on systems biology and network pharmacology.

Objective: To investigate and predict the targets and signaling pathways of sinomenium acutum (SA) in the treatment of rheumatoid arthritis (RA) through systems biology and network pharmacology, and to elucidate its possible mechanisms of action. Methods: We screened the active ingredients and corresponding target proteins of SA in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Traditional Chinese Medicines Integrated Database (TCMID) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN); and obtained the targets of rheumatoid arthritis diseases in a database of gene-disease associations (DisGeNET), Online Mendelian Inheritance in Man (OMIM) database. The two targets were mapped by Venn diagram and the intersection was taken. The intersecting targets were used to construct protein-protein interaction (PPI) network maps in the String database, and Metascape was used for Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Finally, the molecular docking technique was applied to validate and further clarify the core target of SA for the treatment of rheumatoid arthritis. Results: A total of six active ingredients and 217 potential targets were obtained after screening; 2,752 rheumatoid arthritis-related targets and 66 targets common to RA and SA. GO function and KEGG pathway enrichment analysis yielded 751 GO function entries (652 GO biological processes, 59 GO molecular functions and 40 GO cellular components) and 77 KEGG signaling pathways. It mainly involves pathways related to neural activity ligand-receptor interaction pathways, cancer pathways, calcium signaling channels, Th17 cell differentiation and others, which are mainly classified into four categories, including regulation of immunity, anti-inflammation, regulation of cell growth and apoptosis, and signaling. The molecular docking results showed that the binding energy of PTGS2, CASP3, JUN and PPARG to the key components beta-sitosterol, 16-epi-Isositsirikine, Sinomenine and Stepholidine were ≤ -6.5 kcal/mol, suggesting the existence of molecular binding sites. Conclusion: SA acts on key targets such as PTGS2, CASP3, JUN, and PPARG to modulate signaling pathways such as neural activity ligand-receptor interaction, cancer, calcium ion, NF-κB, and Th17 cell differentiation to regulate immunity, anti-inflammation, modulation of cell cycle, bone metabolism, and signaling for the treatment of RA. It was also confirmed that the treatment of RA with SA has multi-component, multi-target, multi-pathway and multi-mechanism characteristics.

Yellow catfish RIO kinases (RIOKs) negatively regulate fish interferon-mediated antiviral response.

In mammals, right open reading frame kinases (RIOKs) are initially reported to participate in cancer cell proliferation, apoptosis, migration and invasion, and recently they have been related to host immune response. Little is known about the homologs of RIOKs in fish. In the current study, we cloned three homologous genes of RIOK family in yellow catfish (Pelteobagrus fulvidraco), termed Pfriok1, Pfriok2 and Pfriok3. Pfriok1, Pfriok2 and Pfriok3 were constitutively expressed at relatively high levels in yellow catfish tissues, and their mRNA levels were not changed under viral infection. Individual overexpression of PfRIOK1, PfRIOK2 and PfRIOK3 attenuated fish interferon (IFN) response, thereby promoting viral replication in fish cells. Mechanistically, yellow catfish RIOK proteins downregulated fish IFN response through attenuating TBK1 protein levels in cytoplasm. Our findings suggest that yellow catfish RIOK1, RIOK2 and RIOK3 are involved in downregulating fish IFN antiviral response.

Transcriptome analysis of changes in M. aeruginosa growth and microcystin production under low concentrations of ethinyl estradiol.

Ethinyl estradiol (EE2) is a synthetic environmental estrogen with considerable estrogenic activity. It has been found to consequently pose a significant threat to the aquatic ecosystem. Harmful algal blooms are a major aquatic ecological issue. However, the relationship between EE2 and cyanobacterial bloom is mainly unknown. In this study, the physiological and molecular responses of Microcystis aeruginosa to EE2 exposure were investigated. A low level of EE2 (0.02 µg/L) significantly enhanced the growth of algal cells (P < 0.05), whereas higher concentrations of EE2 (0.2-200 µg/L) inhibited it. EE2 at doses ranging from 0.02 to 200 µg/L promoted the production of microcystins (MCs), with genes mcyABD playing a key role in the regulation of MC synthesis. The alterations of chlorophyll-a, carotenoid, and phycocyanin contents caused by EE2 showed the same trend as cell growth. At the molecular level, 200 µg/L EE2 significantly down-regulated genes in photosynthetic pigment synthesis, light harvesting, electron transfer, NADPH, and ATP generation. High concentrations of EE2 caused oxidative damage to algal cells on the 4th d. After 12d exposure, although there was no significant change in superoxide dismutase (SOD) content and no damage observed in membrane lipids, genes related to SOD and glutathione were changed. In addition, due to the down-regulation of pckA, PK, gltA, nrtA, pstS, etc., carbon fixation, glycolysis, TCA cycle, nitrogen and phosphorus metabolism were hindered by EE2 (200 µg/L). Gene fabG in fatty acid biosynthesis was significantly up-regulated, promoting energy storage in cells. These findings provide important clues to elucidate the effects and mechanisms of cyanobacterial blooms triggered by EE2 and help to effectively prevent and control cyanobacterial blooms.

Design of A-D-A-Type Organic Third-Order Nonlinear Optical Materials Based on Benzodithiophene: A DFT Study.

The acceptor-donor-acceptor (A-D-A) type conjugated organic molecule has been widely applied in the organic optoelectronics field. A total of Nine compounds (1-9) were designed under the A-D-A framework, with the electron donor benzodithiophene as the core and dicyanomethylene as the acceptor moiety, modifying the benzodithiophene with the phenyl, naphthyl, and difluorinated phenyl groups. The conjugation length can be changed by introducing a thiophene π-conjugated bridge. The geometric structures, electronic structure, excited state properties, aromaticity, and the static- and frequency-dependent second hyperpolarizabilities were investigated by employing high-precision density functional theory (DFT) calculations with an aug-cc-pVDZ basis set. As a result, the three compounds with the longest conjugation length exhibit a smaller energy gap (Egap), larger UV-vis absorption coefficient, and response range, which are the three strongest third-order nonlinear optical (NLO) response properties in this work. This work systematically explored the connection between molecular structure and NLO response, which provides a rational design strategy for high-performance organic NLO materials.

Simultaneous Maturation of Single Chain Antibody Stability and Affinity by CHO Cell Display.

Antibody stability and affinity are two important features of its applications in therapy and diagnosis. Antibody display technologies such as yeast and bacterial displays have been successfully used for improving both affinity and stability. Although mammalian cell display has also been utilized for maturing antibody affinity, it has not been applied for improving antibody stability. Previously, we developed a Chinese hamster ovary (CHO) cell display platform in which activation-induced cytidine deaminase (AID) was used to induce antibody mutation, and antibody affinity was successfully matured using the platform. In the current study, we developed thermo-resistant (TR) CHO cells for the purpose of maturing both antibody stability and affinity. We cultured TR CHO cells displaying an antibody mutant library and labeled them at temperatures above 41 °C, enriching cells that displayed antibody mutants with both the highest affinities and the highest display levels. To evaluate our system, we chose three antibodies to improve their affinities and stabilities. We succeeded in simultaneously improving both affinities and stabilities of all three antibodies. Of note, we obtained an anti-TNFα antibody mutant with a Tm (dissolution temperature) value 12 °C higher and affinity 160-fold greater than the parent antibody after two rounds of cell proliferation and flow cytometric sorting. By using CHO cells with its advantages in protein folding, post-translational modifications, and code usage, this procedure is likely to be widely used in maturing antibodies and other proteins in the future.

Zebrafish MARCH8 downregulates fish IFN response by targeting MITA and TBK1 for protein degradation.

Recent studies have related the membrane-associated RING-CH-type finger (MARCH) family proteins to host innate immune response. Zebrafish (Danio rerio) MARCH8 is reported to target SVCV glycoprotein for degradation; however, little is known about whether fish MARCH8 is involved in innate interferon (IFN) response. In this study, zebrafish march8 was significantly induced by SVCV infection. Overexpression of MARCH8 diminished fish IFN-mediated antiviral response, thus promoting the replication of SVCV and GCRV in fish cells. Mechanistically, MARCH8 interacts with and degrades MITA and TBK1 proteins to inhibit IFN response. Moreover, MARCH8 has an E3 ligase activity and enhances MITA and TBK1 polyubiquitination. Our findings reveal a mechanism whereby zebrafish MARCH8 downregulates fish IFN response and facilitates viral replication by targeting MITA and TBK1 for protein degradation.

Promoter Binding and Nuclear Retention Features of Zebrafish IRF Family Members in IFN Response.

Interferon regulatory factors (IRFs) constitute a family of transcription factors that synchronize interferon (IFN) antiviral response through translocating to nucleus and binding to the promoters of IFN and IFN-stimulated genes (ISGs). Fish contain 11 IRF members; however, whether or how fish IRF family genes function in IFN response remains limited. Herein, we determine the regulatory roles of 11 zebrafish IRF family members in IFN response relevant to their subcellular localization and promoter binding. Zebrafish IRF family members display three patterns of constitutive localization, only in nucleus (IRF1/2/9/11), only in cytoplasm (IRF3/5/7), and largely in nucleus with small amounts in cytoplasm (IRF4b/6/8/10). DNA pull-down assays confirm that all zebrafish IRF proteins are capable to bind fish IFN promoters, albeit to various degrees, thus regulating IFN gene transcription as activators (IRF1/3/5/6/7/8/9/11) or repressors (IRF2/4b/10). Further characterization of distinct IFN gene activation reveals that IRF1/3/5/6/7/8/9/11 efficiently stimulate zebrafish IFNφ1 expression, and IRF1/7/11 are responsible for zebrafish IFNφ3 expression. Two conserved basic residues within the helix α3 of DNA binding domains (DBDs) contribute to constitutive or inducible nuclear import for all zebrafish IRF family members and DNA binding for most members, thereby enabling them to function as transcription factors. Our results reveal a conserved and general mechanism that specifies zebrafish IRF family proteins to nuclear import and DNA binding, thereby regulating fish IFN response.

Comparative Proteomic Analysis Reveals Antibacterial Mechanism of Patrinia scabiosaefolia Against Methicillin Resistant Staphylococcus epidermidis.

Purpose: As a kind of opportunist pathogen, Staphylococcus epidermidis (MRSE) can cause nosocomial infections and easily evolve into resistant bacteria. Among these, methicillin-resistant Staphylococcus epidermidis (MRSE) exhibit significantly higher rates. Our previous study showed that Patrinia scabiosaefolia (PS) possessed strong antibacterial activity against MRSE. However, the mechanism of PS against MRSE is not clear. Methods: Here, a tandem mass tag-based (TMT) proteomic analysis was performed to elucidate the potential mechanism of PS against MRSE. We compared the differential expression proteins of MRSE under PS stress. Results: Based on a fold change of >1.2 or < 1/1.2 (with p value set at <0.05), a total of 248 proteins (128 up-regulated proteins, 120 down-regulated proteins) were identified. Bioinformatic analysis showed that proteins including arginine deiminase (arcA), ornithine carbamoyltransferase (arcB) and carbamate kinase (arcC), serine-tRNA ligase (serS), phenylalanine-tRNA ligase beta and subunit (pheT), DltD (dlt), d-alanyl carrier protein (dlt), accumulation-associated protein (SasG), serine-aspartate repeat-containing protein C (SdrC) and hemin transport system permease protein HrtB (VraG) played important roles in mechanism of PS against MRSE. Conclusion: In summary, these results indicated that arginine deiminase pathway (ADI) pathway, protein synthesis, cell wall synthesis, biofilm formation and uptake of iron were related to mechanisms of PS against MRSE. Our findings provide an insight into the the mechanism of PS against MRSE, and may be valuable in offering new targets to develop more anti-MRSE drugs.

Proteomic Analysis of the Antibacterial Effect of Improved Dian Dao San against Propionibacterium acnes.

Propionibacterium acnes (P. acnes) is a major pathogen of acne vulgaris. The traditional Chinese medicine (TCM) compound prescription, Dian Dao San (DDS), is effective for treating P. acnes. Previous clinical work by our team demonstrated that improved Dian Dao San (IDDS) has better antibacterial effects. However, the mechanism of IDDS inhibition of P. acnes is still unknown. Hence, the isobaric tags for relative and absolute quantitation (iTRAQ) technology was applied to explore the antibacterial mechanism of IDDS against P. acnes. Our results suggested that the antibacterial mechanism of IDDS was related to the glycolytic pathway. gap, pgk, and tpiA enzymes were found to be potential target proteins in the bacterial glycolytic pathway as an antibacterial mechanism of inhibition. In addition, SEM and TEM analyses revealed that IDDS may destruct bacterial plasma membrane and cell wall. The results provide a reliable, direct, and scientific theoretical basis for wide application of IDDS.

Overlooked environmental risks deriving from aqueous transformation of bisphenol alternatives: Integration of chemical and toxicological insights.

Owing to the widespread prevalence and ecotoxicity of bisphenol alternatives such as bisphenol S, bisphenol F, and bisphenol AF, the past decade has witnessed the publication of a remarkable number of studies related to their transformation and remediation in natural waters. However, the reactivity, removal efficiency, transformation products (TPs), and mechanisms of such emerging pollutants by different treatment processes have not been well elucidated. Particularly, the transformation-driven environmental risks have been mostly overlooked. Therefore, we present a review to address these issues from chemical and toxicological viewpoints. Four degradation systems can be largely classified as catalytic persulfate (PS) oxidation, non-catalytic oxidation, photolysis and photocatalysis, and biodegradation. It was found that bisphenol alternatives possess distinct reactivities with different oxidizing species, with the highest performance for hydroxyl radicals. All systems exhibit superior elimination efficiency for these compounds. The inadequate mineralization suggests the formation of recalcitrant TPs, from which the overall reaction pathways are proposed. The combined experimental and in silico analysis indicates that many TPs have developmental toxicity, endocrine-disrupting effects, and genotoxicity. Notably, catalytic PS systems and non-catalytic oxidation result in the formation of coupling products as well as halogenated TPs with higher acute and chronic toxicity and lower biodegradability than the parent compounds. In contrast, photolysis and photocatalysis generate hydroxylated and bond-cleavage TPs with less toxicity. Overall, this review highlights the secondary environmental risks from the transformation of bisphenol alternatives by conventional and emerging treatment processes. Finally, future perspectives are recommended to address the knowledge gaps of these contaminants in aquatic ecosystems.

High efficiency CHO cell display-based antibody maturation.

Previously, we developed a CHO cell display-based antibody maturation procedure in which an antibody (or other protein) gene of interest was induced to mutate by activation-induced cytidine deaminase (AID) and then form a library by simply proliferating the CHO cells in culture. In this study, we further improved the efficiency of this maturation system by reengineering AID, and optimizing the nucleic acid sequence of the target antibody gene and AID gene as well as the protocol for AID gene transfection. These changes have increased both the mutation rate and the number of mutation type of antibody genes by more than 10 fold, and greatly improved the maturation efficiency of antibody/other proteins.