Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas.

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)-in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.

Synthesis of Cyclic Peptides in SPPS with Npb-OH Photolabile Protecting Group.

Significant efforts have been made in recent years to identify more environmentally benign and safe alternatives to side-chain protection and deprotection in solid-phase peptide synthesis (SPPS). Several protecting groups have been endorsed as suitable candidates, but finding a greener protecting group in SPPS has been challenging. Here, based on the 2-(o-nitrophenyl) propan-1-ol (Npp-OH) photolabile protecting group, a structural modification was carried out to synthesize a series of derivatives. Through experimental verification, we found that 3-(o-Nitrophenyl) butan-2-ol (Npb-OH) had a high photo-release rate, high tolerance to the key conditions of Fmoc-SPPS (20% piperidine DMF alkaline solution, and pure TFA acidic solution), and applicability as a carboxyl-protective group in aliphatic and aromatic carboxyl groups. Finally, Npb-OH was successfully applied to the synthesis of head-tail cyclic peptides and side-chain-tail cyclic peptides. Moreover, we found that Npb-OH could effectively resist diketopiperazines (DKP). The α-H of Npb-OH was found to be necessary for its photosensitivity in comparison to 3-(o-Nitrophenyl)but-3-en-2-ol (Npbe-OH) during photolysis-rate verification.

Design, Synthesis and Evaluation of 3-Substituted Coumarin Derivatives as Anti-inflammatory Agents.

Coumarin moiety has garnered momentous attention especially in the design of compounds with significant biological activities. In this work, a series of 3-substituted coumarin derivatives 6a-6l were synthesized and fully characterized. Most of the compounds could obviously inhibit the activity of cyclooxygenase-1 (COX-1) at the concentration of 10 µM. Besides, 6h and 6l exhibited highest inhibitory effects against COX-2 with inhibition rates of 33.48 and 35.71%, respectively. Detailed structure-activity relationships (SARs) were also discussed. In vivo studies, 6b, 6i and 6l could remarkably repress the xylene-induced ear swelling in mice at the dose of 20 mg/kg. Especially, 6l seemed to be the most effective compound at the dose of 10 mg/kg, displaying favorable anti-inflammatory activity comparable to indomethacin. All of these findings suggested that 6l might be utilized as a candidate for the treatment of inflammatory diseases.

Design, Synthesis and Preliminary Biological Evaluation of Benzylsulfone Coumarin Derivatives as Anti-Cancer Agents.

In this work, a series of benzylsulfone coumarin derivatives 5a-5o were synthesized and characterized. Kinase inhibitory activity assay indicated that most of the compounds showed considerable activity against PI3K. Anti-tumor activity studies of the active compounds were also carried out in vitro on the Hela, HepG2, H1299, HCT-116, and MCF-7 tumor cell lines by MTS assay. The structure-activity relationships (SARs) of these compounds were analyzed in detail. Compound 5h exhibited the most potent activities against the mentioned cell lines with IC50 values ranging from 18.12 to 32.60 µM, followed by 5m with IC50 values of 29.30-42.14 µM. Furthermore, 5h and 5m clearly retarded the migration of Hela cells in vitro. Next, an in silico molecular docking study was conducted to evaluate the binding models of 5h and 5m towards PI3Kα and PI3Kß. Collectively, the above findings suggested that compounds 5h and 5m might be promising PI3K inhibitors deserving further investigation for cancer treatment.

Design, Synthesis and Preliminary Biological Evaluation of Novel Benzyl Sulfoxide 2-Indolinone Derivatives as Anticancer Agents.

In this work, a series of novel benzyl sulfoxide 2-indolinone derivatives was designed and synthesized as potent anticancer agents. Tyrosine kinase inhibitory activity assay indicated that most of the compounds showed significant activity. The in vitro antiproliferative activity of these compounds was further investigated against five human cancer cell lines (HeLa, HepG2, MCF-7, SCC-15, and A549). Several compounds exhibited evident activities. Among them, (Z)-3-(((4-bromobenzyl)sulfinyl)methylene)indolin-2-one (6j) and (Z)-3-((benzylsulfinyl)methylene)-5-bromoindolin-2-one (6o) were found to be effective tyrosine kinase inhibitors (IC50 = 1.34 and 2.69 µM, respectively) in addition to having noteworthy antitumor potential (the average IC50 value of 6j or 6o was less than 40 µM). This class of novel derivatives has promising potential for further development as anticancer agents.

Synthesis and Evaluation of Novel Benzofuran Derivatives as Selective SIRT2 Inhibitors.

A series of benzofuran derivatives were designed and synthesized, and their inhibitory activites were measured against the SIRT1-3. The enzymatic assay showed that all the compounds showed certain anti-SIRT2 activity and selective over SIRT1 and SIRT3 with IC50 (half maximal inhibitory concentration) values at the micromolar level. The preliminary structure-activity relationships were analyzed and the binding features of compound 7e (IC50 3.81 µM) was predicted using the CDOCKER program. The results of this research could provide informative guidance for further optimizing benzofuran derivatives as potent SIRT2 inhibitors.

Arabidopsis ECERIFERUM9 involvement in cuticle formation and maintenance of plant water status.

Mutation of the ECERIFERUM9 (CER9) gene in Arabidopsis (Arabidopsis thaliana) causes elevated amounts of 18-carbon-length cutin monomers and a dramatic shift in the cuticular wax profile (especially on leaves) toward the very-long-chain free fatty acids tetracosanoic acid (C24) and hexacosanoic acid (C26). Relative to the wild type, cer9 mutants exhibit elevated cuticle membrane thickness over epidermal cells and cuticular ledges with increased occlusion of the stomatal pore. The cuticular phenotypes of cer9 are associated with delayed onset of wilting in plants experiencing water deficit, lower transpiration rates, and improved water use efficiency measured as carbon isotope discrimination. The CER9 protein thus encodes a novel determinant of plant drought tolerance-associated traits, one whose deficiency elevates cutin synthesis, redistributes wax composition, and suppresses transpiration. Map-based cloning identified CER9, and sequence analysis predicted that it encodes an E3 ubiquitin ligase homologous to yeast Doa10 (previously shown to target endoplasmic reticulum proteins for proteasomal degradation). To further elucidate CER9 function, the impact of CER9 deficiency on interactions with other genes was examined using double mutant and transcriptome analyses. For both wax and cutin, cer9 showed mostly additive effects with cer6, long-chain acyl-CoA synthetase1 (lacs1), and lacs2 and revealed its role in early steps of both wax and cutin synthetic pathways. Transcriptome analysis revealed that the cer9 mutation affected diverse cellular processes, with primary impact on genes associated with diverse stress responses. The discovery of CER9 lays new groundwork for developing novel cuticle-based strategies for improving the drought tolerance and water use efficiency of crop plants.

Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models.

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The nanobodies were collectively shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across existing VoCs; wide-ranging epitopic and mechanistic diversity and high and broad in vitro neutralization potencies. A select set of Fc-fused nanobodies showed high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a potential therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to combat multiple SARS-CoV-2 variants.

A Photolabile Carboxyl Protecting Group for Solid Phase Peptide Synthesis.

A new kind of photolabile protecting group (PLPG) for carboxyl moieties was designed and synthesized as the linker between resin and peptide. This group can be used for the protection of amino acid carboxyl groups. The peptide was synthesized on Nph (2-hydroxy-3-(2-nitrophenyl)-heptanoic acid)-derivatized resins and could be cleaved under UV exposure, thus avoiding the necessity for harsh acid-mediated resin cleavage. The PLPG has been successfully used for solid-phase synthesis of peptides.

Synthesis and antitumor effects of novel benzyl naphthyl sulfoxide/sulfone derivatives derived from Rigosertib.

In this work, a series of novel benzyl naphthyl sulfoxides/sulfones derived from Rigosertib were designed and synthesized as potential antitumor agents. The in vitro cytotoxicity against four human cancer cell lines (HeLa, MCF-7, HepG2 and SCC-15) and two normal human cell lines (HUVEC and 293T) indicated that some of the sulfones and sulfoxides possessed potent antineoplastic activity that reached nanomolar levels and relatively low toxicity to normal cells. Among them, (2-methoxy-5-((naphthalen-2-ylsulfonyl)methyl)phenyl)glycine (15b) was found to be a promising antitumor drug candidate that could significantly inhibit tumor cell migration and induce tumor cell apoptosis via the p53-Bcl-2-Bax signaling pathway at nanomolar concentrations.

Associations between insecurity and stress among Chinese university students: The mediating effects of hope and self-efficacy.

BACKGROUND: In the context of the pandemic, exploration on the association between insecurity and stress among university students is limited. The current study aims to investigate the parallel mediation role of hope and self-efficacy in the relationship between insecurity and stress among university students during the COVID-19 pandemic. METHODS: We employed a cross-sectional research design in a university by distributing questionnaires online. 5286 participants were recruited (mean age = 19.65; SD = 1.13). Items were from the Security Questionnaire, Depression Anxiety and Stress Scale-21, and the Positive Psychology Capital (Psycap) Questionnaire (PPQ). Parallel mediation analysis was performed using PROCESS macro in SPSS. RESULTS: The results indicate that insecurity predicted students' stress positively and that students with high-level perceived insecurity are more likely to perceive stress. Moreover, hope and self-efficacy mediated the relationship between insecurity and stress, indicating that hope and self-efficacy could buffer the negative effects of insecurity on stress. LIMITATIONS: This study examines the mediating model between insecurity and stress among Chinese university students. The generalizability of the findings in other regions remains to be explored. Additionally, the roles of other positive self-beliefs including optimism and resilience in relieving stress can be further explored in future research. CONCLUSIONS: This research provides direct evidence of insecurity effects on stress among university students, enriching relevant theories in the field of stress. Moreover, this research suggests that enhancing positive self-beliefs such as hope, and self-efficacy helps to relieve students' stress during COVID-19.

Grubbs Catalysts Immobilized on Merrifield Resin for Metathesis of Leaf Alcohols by using a Convenient Recycling Approach.

Three new types of heterogeneous catalysts were prepared using a facile approach by the immobilization of Grubbs catalysts on PEGylated Merrifield resin. One of the immobilized catalysts was more efficient than the free catalyst for the metathesis of leaf alcohols in conversion and selectivity and was reused repeatedly (up to 5 cycles) with only a slight loss of activity (10.5 %). The long-chain PEGylated linker provided an appropriate distance between the resin and the catalytic center so that the ruthenium catalysts acted as the free catalyst.

A Strategy To Prepare Peptide Heterodimers in the Solid Phase with an Acid-Labile Linker.

The currently synthetic methods of peptide heterodimer involve tedious synthesis and purification steps. An acid-labile traceless linker was prepared, which is highly compatible with the Fmoc strategy and could be used to prepare peptide heterodimer on resin. The linker could be cleaved concomitantly with peptide cleavage, and two model heterodimers were synthesized. The proposed synthesis procedure is simple, straightforward, and provides great convenience for preparing disulfide-linked peptide heterodimers.

A selective c-Met and Trks inhibitor Indo5 suppresses hepatocellular carcinoma growth.

BACKGROUND: Human hepatocellular carcinoma (HCC) lacks effective curative therapy and there is an urgent need to develop a novel molecular-targeted therapy for HCC. Selective tyrosine kinase inhibitors have shown promise in treating cancers including HCC. Tyrosine kinases c-Met and Trks are potential therapeutic targets of HCC and strategies to interrupt c-Met and Trks cross-signaling may result in increased effects on HCC inhibition. METHODS: The effects of Indo5 on c-Met and Trks activity were determined with in vitro kinase activity assay, cell-based signaling pathway activation, and kinases-driven cell transformation. The in vivo anti-tumor activity was determined with xenograft mice and liver orthotopic mice models. The co-expression of c-Met and TrkB in 180 pairs of HCC and adjacent normal tissues were detected using immunohistochemical staining. RESULTS: Indo5, a novel lead compound displayed biochemical potency against both c-Met and Trks with selectivity over 13 human kinases. Indo5 abrogated HGF-induced c-Met signaling activation and BDNF/NGF-induced Trks signal activation, c-Met or TrkB-mediated cell transformation and migration. Furthermore, Indo5 significantly decreased the growth of HCC cells in xenograft mice and improved the survival of mice with liver orthotopic tumors. In addition, co-expression of c-Met and TrkB in HCC patients was a predictor of poor prognosis, and combined inhibition of c-Met and TrkB exerted a synergistic suppressive effect on HCC. CONCLUSIONS: These findings indicate that Indo5 is associated with marked suppression of c-Met and Trks co-expressing HCC, supporting its clinical development as an antitumor treatment for HCC patients with co-active c-Met and Trks signaling.

Evaluation of the Biogenic Amines Formation and Degradation Abilities of Lactobacillus curvatus From Chinese Bacon.

Control of biogenic amines (BAs) is critical to guarantee the safety of fermented meat products. The aim of this study is to evaluate the BAs formation and degradation abilities of lactic acid bacteria from Chinese bacon to obtain the beneficial candidate for BAs control. Seven lactic acid bacteria were selected from the typical Chinese bacon products, identified as Lactobacillus curvatus by 16S rDNA analysis. Then, genes analysis and high-performance liquid chromatography (HPLC) analysis were performed to evaluate the BAs formation and degradation abilities of as-selected strains. All L. curvatus strains were confirmed to harbor the genes encoding the tyrosine decarboxylase and ornithine decarboxylase, and they could produce tyramine, ß-phenethylamine, putrescine, and cadaverine. In comparison, the lowest concentration of total BAs was obtained in L. curvatus G-1. Meanwhile, all L. curvatus strains were positive in amines oxidase gene analysis, and they could also degrade six common BAs, especially the L. curvatus G-1 with the highest degradation percentage (above 40%) for each BA. Furthermore, fermented meat model analysis verified that the L. curvatus G-1 could significantly reduce BAs. In conclusion, L. curvatus G-1 shows a low BAs-producing ability, as well as a high BAs-degrading ability, and this study provides a promising candidate for potential BAs control in fermented meat products.

Synthesis and radioprotective effects of novel benzyl naphthyl sulfoxide (sulfone) derivatives transformed from Ex-RAD.

In this work, a series of novel benzyl naphthyl sulfoxides (sulfones) derived from Ex-RAD were designed and synthesized as potential radioprotective agents. Some of the compounds considerably protected HUVECs against 60Co γ-irradiation, accompanied by the absence of cytotoxicity. Compared to Ex-RAD, compound 8n not only exhibited a significant protective effect on cell survival and radiation-induced DNA damage, but also remarkably enhanced the survival (100%) of mice in 30 days after being exposed to irradiation. The results suggested that some target compounds are valuable for further research as promising radioprotectors.

Neuroprotective effects of dietary supplement Kang-fu-ling against high-power microwave through antioxidant action.

Kang-fu-ling (KFL) is a polybotanical dietary supplement with antioxidant properties. This study aimed to evaluate the potential protective effects of KFL on cognitive deficit induced by high-power microwave (HPM) and the underlying mechanism for this neuroprotection. The electron spin resonance technique was employed to evaluate the free radical scavenging activity of KFL in vitro and KFL exhibited scavenging hydroxyl radical activity. KFL at doses of 0.75, 1.5 and 3 g kg(-1) and vehicle were administered orally once daily for 14 days to male Wistar rats after being exposed to 30 mW cm(-2) HPM for 15 minutes. KFL reversed HPM-induced memory loss and the histopathological changes in hippocampus of rats. In addition, KFL displayed a protective effect against HPM-induced oxidative stress and activated the nuclear factor-E2-related factor 2 (Nrf2) and its target genes in the hippocampus of rats. The Nrf2-antioxidant response element (ARE) signaling pathway may be involved in the neuroprotective effects of KFL against HPM-induced oxidative stress. In summary, the dietary supplement KFL is a promising natural complex, which ameliorates oxidative stress, with neuroprotective effects against HPM.