The Influence of Homologous Arm Length on Homologous Recombination Gene Editing Efficiency Mediated by SSB/CRISPR-Cas9 in Escherichia coli.

The precise editing of genes mediated by CRISPR-Cas9 necessitates the application of donor DNA with appropriate lengths of homologous arms and fragment sizes. Our previous development, SSB/CRISPR-Cas9, has demonstrated high efficiency in homologous recombination and non-homologous end joining gene editing within bacteria. In this study, we optimized the lengths and sizes of homologous arms of the donor DNA within this system. Two sets of donor DNA constructs were generated: one set comprised donors with only 10-100 bp homologous arms, while the other set included donors with homologous arms ranging from 10-100 bp, between which was a tetracycline resistance expression cassette (1439 bp). These donor constructs were transformed into Escherichia coli MG1655 cells alongside pCas-SSB/pTargetF-lacZ. Notably, when the homologous arms ranged from 10 to 70 bp, the transformation efficiency of non-selectable donors was significantly higher than that of selectable donors. However, within the range of 10-100 bp homologous arm lengths, the homologous recombination rate of selectable donors was significantly higher than that of non-selectable donors, with the gap narrowing as the homologous arm length increased. For selectable donor DNA with homologous arm lengths of 10-60 bp, the homologous recombination rate increased linearly, reaching a plateau when the homologous arm length was between 60-100 bp. Conversely, for non-selectable donor DNA, the homologous recombination rate increased linearly with homologous arm lengths of 10-90 bp, plateauing at 90-100 bp. Editing two loci simultaneously with 100 bp homologous arms, whether selectable or non-selectable, showed no difference in transformation or homologous recombination rates. Editing three loci simultaneously with 100 bp non-selectable homologous arms resulted in a 45% homologous recombination rate. These results suggest that efficient homologous recombination gene editing mediated by SSB/CRISPR-Cas9 can be achieved using donor DNA with 90-100 bp non-selectable homologous arms or 60-100 bp selectable homologous arms.

Strategies to improve the efficiency and quality of mutant breeding using heavy-ion beam irradiation.

Heavy-ion beam irradiation (HIBI) is useful for generating new germplasm in plants and microorganisms due to its ability to induce high mutagenesis rate, broad mutagenesis spectrum, and excellent stability of mutants. However, due to the random mutagenesis and associated mutant breeding modalities, it is imperative to improve HIBI-based mutant breeding efficiency and quality. This review discusses and summarizes the findings of existing theoretical and technical studies and presents a set of tandem strategies to enable efficient and high-quality HIBI-based mutant breeding practices. These strategies: adjust the mutation-inducing techniques, regulate cellular response states, formulate high-throughput screening schemes, and apply the generated superior genetic elements to genetic engineering approaches, thereby, improving the implications and expanding the scope of HIBI-based mutant breeding. These strategies aim to improve the mutagenesis rate, screening efficiency, and utilization of positive mutations. Here, we propose a model based on the integration of these strategies that would leverage the advantages of HIBI while compensating for its present shortcomings. Owing to the unique advantages of HIBI in creating high-quality genetic resources, we believe this review will contribute toward improving HIBI-based breeding.

Characterization and the cholesterol-lowering effect of dietary fiber from fermented black rice (Oryza sativa L.).

Black rice was fermented with Neurospora crassa, after which the dietary fiber (DF) extracted from it was characterized and evaluated for its cholesterol-lowering effect in mice. The findings demonstrated that fermentation increased the level of soluble DF from 17.27% ± 0.12 to 29.69% ± 0.26 and increased the adsorption capacity of DF for water, oil, cholesterol, glucose and sodium cholate. The fermented DF had a more loose and porous structure than that extracted from unfermented rice. Additionally, feeding with DF from the fermented black rice significantly reduced body weight, lowered total cholesterol levels and improved the lipid profile in mice gavaged with a high dose (5 g per kg bw) or a low dose (2.5 g per kg·bw). ELISA showed that the hepatic expression of typical proteins and enzymes that are involved in cholesterol metabolism was regulated by the fermented rice DF, leading to reduced cholesterol production and increased cholesterol clearance. The fermented DF also modified the gut microbiota composition (e.g. Firmicutes reduced and Akkermansia increased), which promoted the production of short-chain fatty acids. In conclusion, fermentation can modify the structure and function of DF in black rice and the fermented dietary fiber has excellent cholesterol lowering effects possibly by cholesterol adsorption, cholesterol metabolism modulation, and intestinal microflora regulation.

Single-Stranded DNA-Binding Proteins Mediate DSB Repair and Effectively Improve CRISPR/Cas9 Genome Editing in Escherichia coli and Pseudomonas.

Single-stranded DNA-binding proteins (SSBs) are essential for all living organisms. Whether SSBs can repair DNA double-strand breaks (DSBs) and improve the efficiency of CRISPR/Cas9-mediated genome editing has not been determined. Here, based on a pCas/pTargetF system, we constructed pCas-SSB and pCas-T4L by replacing the λ-Red recombinases with Escherichia coli SSB and phage T4 DNA ligase in pCas, respectively. Inactivation of the E. coli lacZ gene with homologous donor dsDNA increased the gene editing efficiency of pCas-SSB/pTargetF by 21.4% compared to pCas/pTargetF. Inactivation of the E. coli lacZ gene via NHEJ increased the gene editing efficiency of pCas-SSB/pTargetF by 33.2% compared to pCas-T4L/pTargetF. Furthermore, the gene-editing efficiency of pCas-SSB/pTargetF in E. coli (ΔrecA, ΔrecBCD, ΔSSB) with or without donor dsDNA did not differ. Additionally, pCas-SSB/pTargetF with donor dsDNA successfully deleted the wp116 gene in Pseudomonas sp. UW4. These results demonstrate that E. coli SSB repairs DSBs caused by CRISPR/Cas9 and effectively improves CRISPR/Cas9 genome editing in E. coli and Pseudomonas.

Prohibitin (PHB) interacts with AKT in mitochondria to coordinately modulate sperm motility.

Prohibitin (PHB), an evolutionarily conserved mitochondrial inner membrane protein, is highly expressed in cells that require strong mitochondrial function. Recently, we demonstrated that the deletion of Phb in spermatocytes results in impaired mitochondrial function. In addition, PHB expression in the mitochondrial sheath of human sperm has a significantly negative correlation with mitochondrial reactive oxygen species levels, but a positive one with mitochondrial membrane potential and sperm motility. These results suggest that mitochondrial PHB expression plays a role in sperm motility. However, the mechanism of PHB-mediated regulation of sperm motility remains unknown. Here, we demonstrate for the first time that PHB interacts with protein kinase B (AKT) and exists in a complex with phospho-PHB (pT258) and phospho-AKT in the mitochondrial sheath of murine sperm, as determined using colocalization and coimmunoprecipitation assays. After blocking AKT activity using wortmannin (a phosphatidylinositol 3-kinase [PI3K] inhibitor), murine sperm have significantly ( P < 0.05) decreased levels of phospho-PHB (pT258) and the total and progressive motility. Furthermore, significantly ( P < 0.05) lower levels of phospho-PI3K P85 subunit α+γ (pY199 and pY467) and phospho-AKT (pS473; pT308) are found in sperm from infertile asthenospermic and oligoasthenospermic men compared with normospermic subjects, which suggest a reduced activity of the PI3K/AKT pathway in these infertile subjects. Importantly, these sperm from infertile subjects also have a significantly ( P < 0.05) lower level of phospho-PHB (pT258). Collectively, our findings suggest that the interaction of PHB with AKT in the mitochondrial sheath is critical for sperm motility, where PHB phosphorylation (pT258) level and PI3K/AKT activity are key regulatory factors.

Oligo (p-Phenylene Vinylene)/Polyisocyanopeptide Biomimetic Composite Hydrogel-Based Three-Dimensional Cell Culture System for Anticancer and Antibacterial Therapeutics.

Cells are normally cultured in 2D environment, which is usually inconsistent with the real microenvironment in vivo, and it is rarely reported that an effective cancer cell killing process occurs in a 3D network environment. Herein, a kind of new biomimetic composite hydrogel which can achieve 3D cell culture has been prepared and constructed by assembly of polyisocyanopeptide (PIC) with cationic oligo (p-phenylene vinylene) (OPV). The polymer chains of PIC can be bound and frizzled to form a 3D network when the temperature rises above the gelation temperature, followed by encapsulating the cells into biomimetic composite hydrogel. Cells grow and proliferate well in 3D composite hydrogels with excellent cell viability. When the cells undergo cancerization or microbial infection during the 3D culture, the addition of the luminol luminescence system can cause a strong bioluminescence resonance energy transfer (BRET) process to produce highly active reactive oxygen species (ROS) in 3D culture and kill the cancer cells and pathogenic microorganism effectively. Utilizing the BRET process in 3D composite biomimetic hydrogels provides an efficient antibacterial and anticancer approach in 3D culture to overcome the light-penetration limitation.

Remote-Controlling Potassium Channels in Living Cells through Photothermal Inactivation of Calmodulin.

Spatiotemporal regulation of cellular functions provides a powerful strategy for understanding underlying mechanisms of cellular bioprocesses. Here, a strategy is reported to realize the remote control of the activities of potassium channels via photothermal inactivation of calmodulin (CaM) by using reduced graphene oxide decorated with calmodulin binding peptide (rGO-P) as the transducer with near-infrared light (NIR) irradiation. Upon NIR light irradiation, the CaM/Ca2+ bound to rGO-P is inactivated by the photothermal effect of rGO-P, resulting in the incapability of binding with Ca2+ . Hence, the closed Kv10.1 channel is converted to be open in the presence of calcium in living cells. Meanwhile, the SK2 channel is induced to be closed from the open state and the Kir2.1 channel is unaffected by the intracellular inactivation of CaM. This strategy gives a noninvasive and effective approach to remotely control the activities of potassium channels, offering an alternative for the development of optogenetics.

A novel amphiphilic fluorescent probe BODIPY-O-CMC-cRGD as a biomarker and nanoparticle vector.

Fluorescent probes have been demonstrated to be promising candidates as biomarkers and biological carriers. Our study focuses on the development of a novel amphiphilic fluorescent probe with good photostability, high water solubility, excellent specificity and promising loading capability for tumor diagnosis and treatment. At first, BODIPY dye and O-carboxymethyl chitosan were prepared via a chemical reaction. Then, the prepared BODIPY dye and cRGD were bonded to O-carboxymethyl chitosan successively via an acylation reaction. Finally, we obtained the desired amphiphilic fluorescent probe: BODIPY-O-CMC-cRGD, which was based on the fluorescence resonance energy transfer (FRET) principle for selective visualization of tumors in vitro. Through a series of experiments, we found that this fluorescent probe possessed better fluorescence characteristics and tumor targeting properties. Simultaneously, by self-assembly, the amphiphilic probe encapsulated the other flexible structure of BODIPY2 and the rigid structure of porphyrin, which formed distinct nanoparticles with different particle sizes. Hence, we could observe different phagocytosis processes of the two nanoparticles in the tumor cells via the fluorescence of dyes by confocal laser scanning microscopy. Therefore, the results suggest that the fluorescent probe has advantages in tumor detection, and the constructed tumor-specific nanoparticles show high clinical potential to be utilized not only in visual and precise diagnosis but also in excellent drug delivery for tumor treatment. Henceforth, we will prepare new targeted and visualized pharmaceuticals by replacing BODIPY2 and porphyrin with antineoplastic drugs for future tumor treatment.

A Multiple-Stimulus-Responsive Biomimetic Assembly Based on a Polyisocyanopeptide and Conjugated Polymer.

An assembly was fabricated and was revealed to be a multiple-stimulus-responsive biomimetic hybrid polymer architecture. It was constructed by the hydrophobic interactions between a conjugated polyfluorene that contained 2,1,3-benzothiadiazole units (PFBT) and a tri(ethylene glycol)-functionalized polyisocyanopeptide (3OEG-PIC). The introduction of PFBT to the polyisocyanopeptide (PIC) network allowed for the incorporation of responsiveness to multiple stimuli including temperature, CO2 , carbonic anhydrase, and nonlinear mechanics, which mimics natural processes and interactions. Furthermore, the light-harvesting and signal amplification characteristics of PFBT endowed the supramolecular assembly with the essential function of fluorescence monitoring for biological processes.

Identification of Resveratrol, an Herbal Compound, as an Activator of the Calcium-Activated Chloride Channel, TMEM16A.

Calcium-activated chloride channels (CaCCs) play vital roles in a variety of physiological processes. Dysfunction of the CaCCs is implicated in many diseases. Drug discovery targeting at CaCCs has recently become possible with the determination that TMEM16A is the molecular identity of CaCCs. In this study, we demonstrated that resveratrol (RES), a Chinese traditional medicine compound, is a novel activator of TMEM16A. The yellow fluorescence protein quenching assay and measurement of intracellular calcium fluorescence intensity show that RES activates TMEM16A channels in an intracellular Ca2+-independent way. The data of inside-out patch clamp revealed that RES dose-dependently activates TMEM16A (EC50 = 47.92 ± 9.35 µM). Furthermore, RES enhanced the contractions of the ileum of guinea pigs by activating the TMEM16A channel, which indicated that RES might be a promising drug for the treatment of gastrointestinal hypomotility. As RES was able to induce TMEM16A channel activation, TMEM16A can be added to the list of RES drug targets.

Conjugated Polyelectrolyte-Based New Strategy for in Situ Detection of Carbon Dioxide.

A conjugated polymer centered on fluorene and 2,1,3-benzothia-diazole (PFBT) is prepared for sensing CO2 in situ with high sensitivity and low background. Upon introducing CO2, the weaker electrostatic repulsion and stronger hydrophobic interactions between neighboring PFBT molecules enhance the interchain contacts compared to that without CO2, leading to the energy transfer from fluorene to 2,1,3-benzothia-diazole sites and the emission color shift from blue to green, which is sensitive to sensing CO2 in atmospheric air with a content of ∼400 ppm. Importantly, PFBT is employed to monitor photosynthesis and respiration upon cycling day and night in situ.

Luminescent Lanthanide-Based Organic/Inorganic Hybrid Materials for Discrimination of Glutathione in Solution and within Hydrogels.

Glutathione (GSH) as a biothiol is an essential peptide related to various diseases. Although multiple strategies for biothiols detection have been developed, there is increasing demand for sensors that can differentiate GSH from cysteine (Cys) and homocysteine (Hcy), owing to the similar structures and thiol groups in these amino acids. Herein, we report a novel Eu3+/LAPONITE (Lap)-based organic/inorganic hybrid material for selective detection of GSH via an "off-on" process. The fluorescence of Eu(DPA)3@Lap-Tris can be quenched by Cu2+ through photoinduced electron transfer (PET). The addition of GSH into the Eu(DPA)3@Lap-Tris/Cu2+ system induces the removal of Cu2+ from Eu(DPA)3@Lap-Tris and blocks PET, resulting in the recovery of fluorescence. This proposed assay demonstrates higher selectivity toward GSH than Cys and Hcy, and showed a detection limit of 162 nM within a linear range of 0.5-30 µM. Unlike other GSH selective sensors, this platform could be formed into a hydrogel while its sensitivity was maintained. The sensitive response to GSH in serum samples makes this platform an efficient tool for biological applications because of its ease of preparation, high selectivity, good biocompatibility, and low toxicity.

Carbon Dioxide-Controlled Assembly of Water-Soluble Conjugated Polymers Catalyzed by Carbonic Anhydrase.

The CO2 -responsive and biocatalytic assembly based on conjugated polymers has been demonstrated by combining the signal amplification property of the polythiophene derivative (PTP) and the catalytic actions of carbonic anhydrase (CA). CO2 is applied as a new trigger mode to construct the smart assembly by controlling the electrostatic and hydrophobic interactions between the PTP molecules in aqueous solution, leading to the visible fluorescence changes. Importantly, the assembly transformation of PTP can be specifically and highly accelerated by CA based on the efficient catalytic activity of CA for the inter-conversion between CO2 and HCO3- , mimicking the CO2 -associated biological processes that occurred naturally in living organisms. Moreover, the PTP-based assembly can be applied for biomimetic CO2 sequestration with fluorescence monitoring in the presence of CA and calcium.

Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm.

Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase-type activity of sperm mitochondrial complex I (MCI) and PHB expression were also determined. We demonstrate that mROS and lipid peroxidation levels are significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression are significantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase-type activity levels of sperm MCI in the three groups studied suggests that the catalytic subunits of MCI in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCI associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, and it may regulate sperm motility via increases in low MMP and lipid peroxidation. This is the first report on the involvement of PHB in human sperm motility loss associated with increased generation of mROS at MCI.

Conjugated Polymer-Based Hybrid Materials for Turn-On Detection of CO2 in Plant Photosynthesis.

Detection of carbon dioxide (CO2) is of fundamental importance in diverse applications ranging from environmental analysis to agricultural production. In this work, a hybrid probe based on guanidinium-pendent oligofluorene (G-OF) and water-soluble conjugated polythiophene (PTP) has been developed for the turn on detection of CO2 with low background signal, taking advantage of the efficient fluorescence quenching of the tight aggregate of G-OF/PTP. In the presence of CO2, the electrostatic repulsion between G-OF and PTP can be effectively enhanced through protonation of the side chains, leading to the disaggregation and thus the "turn-on" fluorescence. The strategy allows for the light-up visible detection of CO2 with high sensitivity. Importantly, this system is capable of sensitively monitoring the concentration changes of CO2 in the process of the photosynthesis, which represents a concept to monitor the photosynthesis based on water-soluble conjugated polymers.

A magnetic/fluorometric bimodal sensor based on a carbon dots-MnO2 platform for glutathione detection.

A novel magnetic/fluorometric bimodal sensor was built from carbon dots (CDs) and MnO2. The resulting sensor was sensitive to glutathione (GSH), leading to apparent enhancement of magnetic resonance (MR) and fluorescence signals along with visual changes. The bimodal detection strategy is based on the decomposition of the CDs-MnO2 through a redox reaction between GSH and MnO2. This process causes the transformation from non-MR-active MnO2 to MR-active Mn(2+), and is accompanied by fluorescence restoration of CDs. Compared with a range of other CDs, the polyethylenimine (PEI) passivated CDs (denoted as pCDs) were suitable for detection due to their positive surface potential. Cross-validation between MR and fluorescence provided detailed information regarding the MnO2 reduction process, and revealed the three distinct stages of the redox process. Thus, the design of a CD-based sensor for the magnetic/fluorometric bimodal detection of GSH was emphasized for the first time. This platform showed a detection limit of 0.6 µM with a linear range of 1-200 µM in the fluorescence mode, while the MR mode exhibited a linear range of 5-200 µM and a GSH detection limit of 2.8 µM with a visible change being observed rapidly at 1 µM in the MR images. Furthermore, the introduction of the MR mode allowed the biothiols to be easily identified. The integration of CD fluorescence with an MR response was demonstrated to be promising for providing detailed information and discriminating power, and therefore extend the application of CDs in sensing and imaging.

Two Ca(2+)-Binding Sites Cooperatively Couple Together in TMEM16A Channel.

TMEM16A is the molecular basis of calcium-activated chloride channels and shows Ca(2+)-dependent gating. It is critical to understand how the Ca(2+) sensors dynamically control the gate of TMEM16A. However, the detailed mechanism by which the calcium ions bind and open the channel is still obscure. In this study, the authors confirmed that there are two Ca(2+) sensors which cooperatively couple together in TMEM16A. Our data show that mutations at both Ca(2+)-sensitive domains, E447Y and E702Q-E705Q, weaken the Ca(2+) affinity for TMEM16A channel. The EC50 for WT, E447Y, and E702Q-E705Q are 0.53 ± 0.11, 14.5 ± 0.3, and 26.5 ± 3.6 µM, respectively. The triple mutation, including both of the Ca(2+) sensors, E447Y-E702Q-E705Q, with EC50 as 55.6 ± 5.1 µM, results in much further right-shifted dose response curve than the single sensor's mutations (E447Y, E702Q-E705Q) do, which indicates that there is a cooperation between the two Ca(2+)-sensitive domains. We also found that the divalent cations, both Ca(2+) and Sr(2+), share common mechanism of gating the TMEM16A.

Liposome-mediated mycelial transformation of filamentous fungi.

Liposome-mediated transformation is common for cells with no cell wall, but has very limited usage in cells with walls, such as bacteria, fungi, and plants. In this study, we developed a procedure to introduce DNA into mycelium of filamentous fungi, Rhizopus nigricans LH 21 and Pleurotus ostreatus TD 300, by liposome-mediation but with no protoplast preparation. The DNA was transformed into R. nigricans via plasmid pEGFP-C1 and into P. ostreatus via 7.2 kb linear DNA. The mycelia were ground in 0.6 M mannitol without any grinding aids or glass powder for 15 min to make mycelial fragments suspension; the suspension was mixed with a mixture of the DNA and Lipofectamine 2000, and placed on ice for 30 min; 100 µL of the transformation solution was plated on potato dextrose agar (PDA) plate and cultivated at 28 °C for transformant screening. The plasmid and the linear DNA were confirmed to be integrated into the host chromosome, proving the success of transformation. The transformation efficiencies were similar to those of electroporation-mediated protoplast transformation (EMPT) of R. nigricans or PEG/CaCl2-mediated protoplast transformation (PMT) of P. ostreatus, respectively. The results showed that our procedure was effective, fast, and simple transformation method for filamentous fungi.

ß-Glucan synthase gene overexpression and ß-glucans overproduction in Pleurotus ostreatus using promoter swapping.

Mushroom ß-glucans are potent immunological stimulators in medicine, but their productivities are very low. In this study, we successfully improved its production by promoter engineering in Pleurotus ostreatus. The promoter for ß-1,3-glucan synthase gene (GLS) was replaced by the promoter of glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans. The homologous recombination fragment for swapping GLS promoter comprised five segments, which were fused by two rounds of combined touchdown PCR and overlap extension PCR (TD-OE PCR), and was introduced into P. ostreatus through PEG/CaCl2-mediated protoplast transformation. The transformants exhibited one to three fold higher transcription of GLS gene and produced 32% to 131% higher yield of ß-glucans than the wild type. The polysaccharide yields had a significant positive correlation to the GLS gene expression. The infrared spectra of the polysaccharides all displayed the typical absorption peaks of ß-glucans. This is the first report of successful swapping of promoters in filamentous fungi.